Pubmed Result

1. J Econ Entomol. 2018 Feb 27. doi: 10.1093/jee/toy017.
[Epub ahead of print]
Host Plant Selection by the Wheat Bug, Nysius huttoni (Hemiptera: Lygaeidae) on a
Range of Potential Trap Plant Species.
Tiwari S(1)(2), Dickinson N(3), Saville DJ(1)(4), Wratten SD(1).
Author information:
(1)Bio-Protection Research Centre, Lincoln University, Lincoln, New Zealand.
(2)Department of Entomology, Agriculture and Forestry University, Bharatpur,
Nepal.
(3)Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, New
Zealand.
(4)Saville Statistical Consulting Limited, Lincoln, New Zealand.
The wheat bug, Nysius huttoni L. is an endemic New Zealand pest. The seedlings of
forage brassicas are highly susceptible to direct feeding damage by this insect,
and this can reduce plant establishment. Prophylactic use of pesticides is the
usual practice for N. huttoni management. These practices have been linked to
environmental pollution, biodiversity loss, and pollinator population declines in
brassicas and other crops. Habitat management of the bug utilizing potential trap
crops can be a better option for its management. A series of choice, no-choice,
and paired-choice tests were conducted in a controlled-temperature room to
evaluate the pest's preferences on seedlings of eight plant species. Kale plants
(Brassica oleracea) were used as a potentially susceptible control, and seven
non-kale plants were compared with kale as potential trap-plant species. These
were: Lobularia maritima (L.) Desvaux (alyssum), Triticum aestivum L. (wheat),
Phacelia tanacetifolia Bentham (phacelia), Fagopyrum esculentum Moench
(buckwheat), Coriandrum sativum L. (coriander), Trifolium repens L. (white
clover), and Medicago sativa L. (lucerne). In choice tests, wheat was the most
suitable followed by alyssum, buckwheat, and phacelia, all significantly more
favored than kale. In no-choice tests, alyssum was significantly more favored
than kale and the other plant species except wheat and phacelia. First feeding
damage was recorded on alyssum in both the above test conditions. For
paired-choice tests including kale, wheat, and alyssum were significantly more
suitable than brassica. These findings are important for developing
agro-ecological management strategies. Alyssum followed by wheat were the most
suitable trap plants for N. huttoni. These two plant species can be deployed in
and around brassica fields either independently or as in a multiple trap-cropping
system to reduce bug damage, minimizing or avoiding pesticides, and delivering a
range of ecosystem services.
DOI: 10.1093/jee/toy017
PMID: 29506032
2. Sci Total Environ. 2018 Feb 26;630:570-577. doi:

10.1016/j.scitotenv.2018.02.252.
Phytoextraction of rhenium by lucerne (Medicago sativa) and erect milkvetch

(Astragalus adsurgens) from alkaline soils amended with coal fly ash.
He H(1), Dong Z(2), Pang J(3), Wu GL(1), Zheng J(1), Zhang X(1).
Author information:
(1)State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau,
Institute of Soil and Water Conservation, Northwest A&F University, Yangling,
Shaanxi 712100, China; Institute of Soil and Water Conservation, Chinese Academy
of Sciences and Ministry of Water Resources, Yangling, Shaanxi 712100, China.
Electronic address: dongzhigang@nwsuaf.edu.cn.
(3)School of Agriculture and Environment, and Institute of Agriculture, The
University of Western Australia, Crawley, WA 6009, Australia.
Coal fly ash (CFA) is an industrial waste generated in huge amounts worldwide,
and the management of CFA has become an environmental concern. Recovery of
valuable metals from CFA is one of the beneficial reuse options of CFA. Rhenium
(Re) is one of the rarest metals in the Earth's crust and one of the most
expensive metals of strategic significance in the world market. A CFA at the
Jungar Thermal Power Plant, Inner Mongolia, China, contains more Re than two
alkaline soils in the surrounding region. Pot experiments were undertaken to grow
lucerne (Medicago sativa) and erect milkvetch (Astragalus adsurgens) in a
loessial soil and an aeolian sandy soil amended with different rates (5%, 10%,
20%, and 40%) of CFA. The results show that plant growth was considerably
enhanced and Re concentration in plants was significantly increased when CFA was
applied to the alkaline soils at rates of ≤20%; while in some cases plant growth
was also markedly enhanced by the 40% CFA treatment, which increased plant Re
concentration the most of all treatments. Both lucerne and erect milkvetch showed
potential for phytoextracting Re from CFA-amended alkaline soils. Using CFA for
soil amendment not only offers a potential solution for the waste disposal
problem of CFA, but the phytoextraction of Re by both lucerne and erect milkvetch
may also bring an economic profit in the future.
Copyright © 2018 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.scitotenv.2018.02.252
PMID: 29494967
3. Planta. 2018 Feb 28. doi: 10.1007/s00425-018-2866-1. [Epub ahead of print]
Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa.
Gao R(1), Feyissa BA(1)(2), Croft M(1), Hannoufa A(3)(4).
Author information:
(1)Agriculture and Agri-Food Canada, London, ON, Canada.
(2)Department of Biology, University of Western Ontario, London, ON, Canada.
Abdelali.Hannoufa@agr.gc.ca.
MAIN CONCLUSION: The CRISPR/Cas9 technique was successfully used to edit the
genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa).
RNA-guided genome engineering using Clustered Regularly Interspersed Short
Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in
plants. Successful application and validation of the CRISPR technique in a
multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to
major advances in the improvement of this crop. We used CRISPR/Cas9 technique to
mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because
of the complex features of the alfalfa genome, we first used droplet digital PCR
(ddPCR) for high-throughput screening of large populations of CRISPR-modified
plants. Based on the results of genome editing rates obtained from the ddPCR
screening, plants with relatively high rates were subjected to further analysis
by restriction enzyme digestion/PCR amplification analyses. PCR products
encompassing the respective small guided RNA target locus were then sub-cloned
and sequenced to verify genome editing. In summary, we successfully applied the
CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some
insights into opportunities to apply this technology in future alfalfa breeding.
The overall efficiency in the polyploid alfalfa genome was lower compared to
other less-complex plant genomes. Further refinement of the CRISPR technology
system will thus be required for more efficient genome editing in this plant.
DOI: 10.1007/s00425-018-2866-1
PMID: 29492697
4. J Dairy Sci. 2018 Feb 14. pii: S0022-0302(18)30135-8. doi:

10.3168/jds.2017-13926. [Epub ahead of print]
The effect of alfalfa (Medicago sativa) silage chop length and inclusion rate
within a total mixed ration on the ability of lactating dairy cows to cope with a
short-term feed withholding and refeeding challenge.
Thomson AL(1), Humphries DJ(1), Crompton LA(1), Reynolds CK(2).
Author information:
(1)Sustainable Agriculture and Food Systems Research Division, Centre for Dairy
Research, School of Agriculture, Policy and Development, University of Reading,
Whiteknights Road, Reading, RG6 6AR, UK.
(2)Sustainable Agriculture and Food Systems Research Division, Centre for Dairy
Research, School of Agriculture, Policy and Development, University of Reading,
Whiteknights Road, Reading, RG6 6AR, UK. Electronic address:
c.k.reynolds@reading.ac.uk.
The objectives of the study were (1) to test whether 6 h of feed deprivation
followed by refeeding induces an acidosis challenge in dairy cattle and (2) to
quantify the acidosis challenge mitigation potential of increased alfalfa silage
concentration in the diet. Alfalfa silage constituted either 25 or 75% of forage
dry matter (DM) replacing corn silage [low (LA) or high alfalfa (HA)] and was
chopped to either 14 or 19 mm theoretical length [short (S) or long (L)]. Dietary
treatments LAS, LAL, HAS, or HAL were offered to 4 rumen-cannulated Holstein
dairy cattle (161 d in milk; 5th or 6th parity) in a 4 × 4 Latin square design
study with 21-d periods. Starch concentration was 69 g/kg of DM higher for LA
diets than HA diets. Feed was withheld for 6 h followed by ad libitum refeeding
on d 18 of each period. Measurements of DM intake, milk yield and composition,
rumen pH, and eating and rumination behavior were taken on 1 baseline day, the
challenge day, and 2 further recovery days. After refeeding, rumen pH was reduced
in cows fed LA diets but not HA diets. Feeding LAL resulted in the greatest
subclinical acidosis risk (pH <5.8 for 355 min on the first recovery day).
Animals fed LA produced 4.4 L less milk on the challenge day in comparison to
baseline. It was concluded that short-term feed deprivation detrimentally
affected rumen health and milk yield in dairy cattle normally fed ad libitum, but
had no effect on DM intake or milk composition. Feeding alfalfa silage in place
of corn silage mitigated acidosis risk due to interrupted feed supply, likely due
to a combination of lower starch concentration in HA diets, greater effective
fiber concentration, and higher buffering capacity of alfalfa relative to corn
silage.
Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc.

All rights reserved.
DOI: 10.3168/jds.2017-13926
PMID: 29454691
5. BMC Plant Biol. 2018 Feb 15;18(1):35. doi: 10.1186/s12870-018-1250-4.
Comparative analysis of alfalfa (Medicago sativa L.) leaf transcriptomes reveals

genotype-specific salt tolerance mechanisms.
Lei Y(1)(2), Xu Y(2), Hettenhausen C(2), Lu C(2), Shen G(2), Zhang C(2), Li J(2),
Song J(2), Lin H(3), Wu J(4).
Author information:
(1)Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment,
College of Life Science, State Key Laboratory of Hydraulics and Mountain River
Engineering, Sichuan University, Chengdu, 610000, China.
(2)Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for
Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, China.
Engineering, Sichuan University, Chengdu, 610000, China. hhlin@scu.edu.cn.
Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, China. wujianqiang@mail.kib.ac.cn.
BACKGROUND: Soil salinity is an important factor affecting growth, development,

and productivity of almost all land plants, including the forage crop alfalfa
(Medicago sativa). However, little is known about how alfalfa responds and adapts
to salt stress, particularly among different salt-tolerant cultivars.
RESULTS: Among seven alfalfa cultivars, we found that Zhongmu-1 (ZM) is
relatively salt-tolerant and Xingjiang Daye (XJ) is salt-sensitive. Compared to
XJ, ZM showed slower growth under low-salt conditions, but exhibited stronger
tolerance to salt stress. RNA-seq analysis revealed 2237 and 1125 differentially
expressed genes (DEGs) between ZM and XJ in the presence and absence of salt
stress, among which many genes are involved in stress-related pathways. After
salt treatment, compared with the controls, the number of DEGs in XJ (19373) was
about four times of that in ZM (4833). We also detected specific differential
gene expression patterns: In response to salt stress, compared with XJ, ZM
maintained relatively more stable expression levels of genes related to the ROS
and Ca2+ pathways, phytohormone biosynthesis, and Na+/K+ transport. Notably,
several salt resistance-associated genes always showed greater levels of
expression in ZM than in XJ, including a transcription factor. Consistent with
the suppression of plant growth resulting from salt stress, the expression of
numerous photosynthesis- and growth hormone-related genes decreased more
dramatically in XJ than in ZM. By contrast, the expression levels of
photosynthetic genes were lower in ZM under low-salt conditions.
CONCLUSIONS: Compared with XJ, ZM is a salt-tolerant alfalfa cultivar possessing
specific regulatory mechanisms conferring exceptional salt tolerance, likely by
maintaining high transcript levels of abiotic and biotic stress
resistance-related genes. Our results suggest that maintaining this specific
physiological status and/or plant adaptation to salt stress most likely arises by
inhibition of plant growth in ZM through plant hormone interactions. This study
identifies new candidate genes that may regulate alfalfa tolerance to salt stress
and increases the understanding of the genetic basis for salt tolerance.
DOI: 10.1186/s12870-018-1250-4
PMCID: PMC5815232
PMID: 29448940
6. Electron Physician. 2017 Nov 25;9(11):5826-5833. doi: 10.19082/5826. eCollection
2017 Nov.
A review of effective herbal medicines in controlling menopausal symptoms.
Kargozar R(1), Azizi H(2), Salari R(3).
Author information:
(1)Ph.D. student of Persian Medicine, Department of Persian Medicine, School of
Persian and Complementary Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran.
(2)M.D., Assistant Professor, Department of Chinese and Complementary Medicine,
School of Persian and Complementary Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran.
(3)Ph.D. of Drug Control, Assistant Professor, Department of Clinical Persian
Pharmacy, School of Persian and Complementary Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran.
Background: Acute menopausal syndrome especially hot flashes, is one of the most
common gynecological problems during menopause. Due to the side effects of
hormone therapy, herbal and complementary medicines are always of immense
interest to people in the treatment and management of the symptoms and
complications of menopause.
Objective: The aim of this study was to investigate the mechanisms and effects of
medicinal plants employed in the treatment of menopausal symptoms.
Methods: This review article was carried out by examining clinical trial studies
between the period of 1994 and 2016. The keywords, which include menopause,
climacteric, hot flushes, flashes, herb and phytoestrogens were used to search
for herbal medicines used in clinical trials for the treatment of menopausal
symptoms using databases such as PubMed, Medline, Scopus, Google scholar, SID and
Magiran.
Results: The results of the study showed that the medicinal plants, which include
Sage herb (Salvia officinalis), Lemon balm (Melissa officinalis), Valerina
officinalis, Black cohosh (Cimicifuga racemosa), Fenugreek (Trigonella
foenum-graecum), Black cumin (Nigella sativa), Vitex (Vitex agnus-castus), Fennel
(Foeniculum vulgare), Evening primrose (Oenothera biennis), Ginkgo biloba,
Alfalfa (Medicago sativa), Hypericum perforatum, Panax ginseng, Pimpinella
anisum, Licorice (Glycyrrhiza glabra), Passiflora incarnata, Red clover
(Trifolium pratense), and Glycine soja were effective in the treatment of acute
menopausal syndrome with different mechanisms.
Conclusion: Medicinal plants can play an imperative role in the treatment of
acute menopausal syndrome; however, further studies are required to buttress
their efficacy in the treatment of acute menopausal syndrome.
DOI: 10.19082/5826
PMCID: PMC5783135
PMID: 29403626
Conflict of interest statement: Conflict of Interest: There is no conflict of

interest to be declared.
7. J Anim Sci. 2018 Feb 1. doi: 10.1093/jas/skx061. [Epub ahead of print]
Voluntary intake and digestibility by sheep of alfalfa ensiled at different

moisture concentrations following fertilization with dairy slurry.
Clark JK(1), Coffey KP(1), Coblentz WK(2), Shanks BC(3), Caldwell JD(4), Muck
RE(5), Philipp D(1), Borchardt MA(2), Rhein RT(1), Jokela WE(2), Backes EA(4),
Bertram MG(6), Smith WB(7).
Author information:
(1)University of Arkansas Division of Agriculture, Department of Animal Science,
Fayetteville, AR.
(2)USDA, ARS, Dairy Forage Research Center, Marshfield, WI.
(3)Lincoln University, Department of Agriculture and Environmental Sciences,
Jefferson City, MO.
(4)Purina Animal Nutrition Center, Gray Summit, MO.
(5)USDA, ARS, Dairy Forage Research Center, Madison, WI.
(6)Superintendent, University of Wisconsin Arlington Agricultural Research
Station, Arlington, WI.
(7)Tarleton St. Univ.; Dept. of Animal & Veterinary Technology, Stephenville, TX.
Dairy slurry is used commonly as an animal-sourced fertilizer in agronomic

production. However, residual effects of slurry application on intake and
digestibility of alfalfa (Medicago sativa L.) silage from subsequent harvests are
not well known. The objective of this study was to determine if moisture
concentration of alfalfa silage and timing of dairy slurry application relative
to subsequent harvest affected intake and digestibility by sheep. Katahdin
crossbred ewes (n = 18; 48 ± 5.3 kg) in mid-gestation were stratified by BW and
allocated randomly in each of 2 periods to 1 of 6 treatments arranged in a 2 × 3
factorial arrangement. Treatments consisted of recommended (RM; 46.8%) or low
(LM; 39.7%) moisture at baling after either no slurry application (NS), slurry
application to stubble immediately after removal of the previous cutting (S0), or
slurry application 14 d after removal of the previous cutting (S14). Silages were
chopped through a commercial straw chopper, packed into plastic trash cans, and
then offered to ewes within 4 d of chopping. Period 1 of the intake and digestion
study consisted of a 14-d adaptation followed by a 7-d fecal collection period.
Period 2 followed period 1 after a 4-d rest and consisted of an 11-d adaptation
followed by 7 d of fecal collection. Ewes were housed individually in 1.4 × 4.3-m
pens equipped with rubber mat flooring. Feces were swept from the floor twice
daily, weighed, and dried at 50°C. Ewes had ad libitum access to water and were
offered chopped silage for a minimum of 10% refusal (DM). Blood samples were
collected immediately prior to feeding, and 4 and 8 h after feeding on the day
prior to the end of each period. Organic matter intake (g/kg BW) and OM
digestibility tended (P < 0.10) to be, and digestible OMI (g/kg BW) was reduced
by slurry application. Lymphocytes (% of total white blood cells) were greater (P
< 0.05) from LM vs. RM and from NS vs. S0 and S14. Red blood cell concentrations
were greater (P < 0.05) from S14 vs. S0 and from S0 and S14 vs. NS. Serum urea N
concentrations did not differ (P > 0.17) across treatments. Therefore, moisture
concentration of alfalfa silage within the range used in this study may not
affect voluntary intake or digestibility, but slurry application may have an
effect on digestible OM intake. Also, moisture concentration of alfalfa silage
and time of dairy slurry application may affect specific blood hemograms.
© The Author(s) 2018. Published by Oxford University Press on behalf of American

Society of Animal Science. All rights reserved. For permissions, please email:
journals.permissions@oup.com.
DOI: 10.1093/jas/skx061
PMID: 29401268
8. BMC Genomics. 2018 Feb 2;19(1):116. doi: 10.1186/s12864-018-4495-2.

Comparative transcriptomic analysis of resistant and susceptible alfalfa
cultivars (Medicago sativa L.) after thrips infestation.
Tu X(1), Liu Z(2), Zhang Z(3).
Author information:
(1)State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute
of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193,
People's Republic of China.
(2)Institute of Agro-Resourse and Environment, Hebei Academy of Agriculture and
Forestry Sciences, Shijiazhuang, 050051, People's Republic of China.
People's Republic of China. zhangzehua@caas.cn.
BACKGROUND: Plant breeding for resistance to agricultural pests is an essential

element in the development of integrated crop management systems; however, the
molecular and genetic mechanisms underlying resistance are poorly understood. In
this pilot study, a transcriptomic analysis of a resistant (R) vs. a susceptible
(S) variety of alfalfa, with (+T) or without (-T) thrips (= 4 treatments) was
conducted, 'GN-1' (China) was defined as the resistant cultivar, and 'WL323'
(America) was defined as the susceptible cultivar.
RESULTS: A total of 970 mRNAs were differentially expressed, of which 129 up- and
191 down-regulated genes were identified in the R + T/R-T plants, while 413 up-
and 237 down-regulated genes were identified in the S + T/S-T plants. KEGG
analysis mapped 33 and 80 differentially expressed genes to 11 and 14
substantially enriched pathways for GN-1 and WL323, respectively. Five shared
pathways were linked to plant resistance traits, including beta-Alanine
metabolism, fatty acid degradation, chloroalkane and chloroalkene degradation,
flavonoid biosynthesis, and phenylalanine metabolism.
CONCLUSIONS: Results indicated both thrips resistant and susceptible alfalfa
cultivars can regulate gene expression in the salicylic acid (SA) and flavonoid
biosynthesis pathways to induce defensive genes and protein expression (e.g.
polyphenol oxidase, protease inhibitor), which enhances plant defence capacity.
DOI: 10.1186/s12864-018-4495-2
PMCID: PMC5797364
PMID: 29394889
9. Sci Rep. 2018 Jan 26;8(1):1695. doi: 10.1038/s41598-018-20091-z.
Targeted and Untargeted Metabolic Profiling of Wild Grassland Plants identifies

Antibiotic and Anthelmintic Compounds Targeting Pathogen Physiology, Metabolism
and Reproduction.
French KE(1), Harvey J(2), McCullagh JSO(3).
Author information:
(1)Department of Plant Sciences, University of Oxford, South Parks Road, Oxford,
OX1 3RB, UK. katherine.french@plants.ox.ac.uk.
(2)Department of Chemistry, University of Oxford, Mansfield Road, Oxford, OX1
3TA, UK.
(3)Department of Chemistry, University of Oxford, Mansfield Road, Oxford, OX1
3TA, UK. james.mccullagh@chem.ox.ac.uk.
Plants traditionally used by farmers to manage livestock ailments could reduce

reliance on synthetic antibiotics and anthelmintics but in many cases their
chemical composition is unknown. As a case study, we analyzed the metabolite
profiles of 17 plant species and 45 biomass samples from agricultural grasslands
in England using targeted and untargeted metabolite profiling by
liquid-chromatography mass spectrometry. We identified a range of plant secondary
metabolites, including 32 compounds with known antimicrobial/anthelmintic
properties which varied considerably across the different plant samples. These
compounds have been shown previously to target multiple aspects of pathogen
physiology and metabolism in vitro and in vivo, including inhibition of quorum
sensing in bacteria and egg viability in nematodes. The most abundant bioactive
compounds were benzoic acid, myricetin, p-coumaric acid, rhamnetin, and
rosmarinic acid. Four wild plants (Filipendula ulmaria (L.) Maxim., Prunella
vulgaris L., Centuarea nigra L., and Rhinanthus minor L.) and two forage legumes
(Medicago sativa L., Trifolium hybridium L.) contained high levels of these
compounds. Forage samples from native high-diversity grasslands had a greater
abundance of medicinal compounds than samples from agriculturally improved
grasslands. Incorporating plants with antibiotic/anthelmintic compounds into
livestock feeds may reduce global drug-resistance and preserve the efficacy of
last-resort drugs.
DOI: 10.1038/s41598-018-20091-z
PMCID: PMC5786025
PMID: 29374230
10. Environ Sci Technol. 2018 Feb 20;52(4):2217-2224. doi: 10.1021/acs.est.7b05667.

Epub 2018 Feb 5.
Coupling between Nitrogen Fixation and Tetrachlorobiphenyl Dechlorination in a

Rhizobium-Legume Symbiosis.
Wang X(1)(2), Teng Y(1), Tu C(3), Luo Y(1)(3), Greening C(4), Zhang N(1), Dai
S(1)(2), Ren W(1), Zhao L(1), Li Z(1).
Author information:
(1)Key Laboratory of Soil Environment and Pollution Remediation, Institute of
Soil Science , Chinese Academy of Sciences, Nanjing 210008, P.R. China.
(2)University of Chinese Academy of Sciences , Beijing 100049, P.R. China.
(3)Key Laboratory of Coastal Environmental Processes and Ecological Remediation,
Yantai Institute of Coastal Zone Research , Chinese Academy of Sciences, Yantai
264003, P.R. China.
(4)School of Biological Sciences, Monash University , Clayton, Victoria 3800,
Australia.
Legume-rhizobium symbioses have the potential to remediate soils contaminated

with chlorinated organic compounds. Here, the model symbiosis between Medicago
sativa and Sinorhizobium meliloti was used to explore the relationships between
symbiotic nitrogen fixation and transformation of tetrachlorobiphenyl PCB 77
within this association. 45-day-old seedlings in vermiculite were pretreated with
5 mg L-1 PCB 77 for 5 days. In PCB-supplemented nodules, addition of the
nitrogenase enhancer molybdate significantly stimulated dechlorination by
7.2-fold and reduced tissue accumulation of PCB 77 (roots by 96% and nodules by
93%). Conversely, dechlorination decreased in plants exposed to a nitrogenase
inhibitor (nitrate) or harboring nitrogenase-deficient symbionts (nifA mutant) by
29% and 72%, respectively. A range of dechlorinated products (biphenyl,
methylbiphenyls, hydroxylbiphenyls, and trichlorobiphenyl derivatives) were
detected within nodules and roots under nitrogen-fixing conditions. Levels of
nitrogenase-derived hydrogen and leghemoglobin expression correlated positively
with nodular dechlorination rates, suggesting a more reducing environment
promotes PCB dechlorination. Our findings demonstrate for the first time that
symbiotic nitrogen fixation acts as a driving force for tetrachlorobiphenyl
dechlorination. In turn, this opens new possibilities for using rhizobia to
enhance phytoremediation of halogenated organic compounds.
DOI: 10.1021/acs.est.7b05667
PMID: 29363956
11. J Bacteriol. 2018 Jan 22. pii: JB.00622-17. doi: 10.1128/JB.00622-17. [Epub
ahead
of print]
OxyR-dependent Transcription Response of Sinorhizobium meliloti to Oxidative

Stress.
Lehman AP(1), Long SR(2).
Author information:
(1)Department of Biology, Stanford University, Stanford CA 94305.
(2)Department of Biology, Stanford University, Stanford CA 94305
SRL@stanford.edu.
Reactive oxygen species such as peroxides play an important role in plant

development, cell wall maturation, and defense responses. During nodulation with
host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H2O2 in
infection threads and developing nodules (1). S. meliloti likely also experience
oxidative stress, from both internal and external sources, during life in the
soil. Here, we present microarray transcription data of S. meliloti wild type
cells compared to a mutant deficient in the key oxidative regulatory protein
OxyR, each in response to H2O2 treatment. Several alternative sigma factor genes
are upregulated in the response to H2O2: the stress sigma gene rpoE2 shows
OxyR-dependent induction by H2O2, while rpoH1 expression is induced by H2O2
irrespective of oxyR genotype. The activity of the RpoE2 sigma factor in turn
causes increased expression of two more sigma factor genes, rpoE5 and rpoH2
Strains with deletions of rpoH1 showed improved survival in H2O2 as well as
increased oxyR and total catalase expression. These results imply that ΔrpoH1
strains are primed to deal with oxidative stress. This work presents a global
view of S. meliloti gene expression changes, and regulation of those changes, in
response to H2O2IMPORTANCE Like all aerobic organisms, the symbiotic
nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress
throughout its complex lifecycle. This study describes the global transcriptional
changes S. meliloti makes in response to H2O2, and the roles of the
transcriptional regulator OxyR and the sigma factor RpoH1 in regulating those
changes. By understanding the complex regulatory response of S. meliloti to
oxidative stress, we may further understand the role that reactive oxygen species
play as both a stressor as well as a potential signal during symbiosis.
Copyright © 2018 American Society for Microbiology.
DOI: 10.1128/JB.00622-17
PMID: 29358497
12. Sci Total Environ. 2018 Jan 19;626:776-784. doi:

10.1016/j.scitotenv.2018.01.063.
Soybean supplementation increases the resilience of microbial and nematode

communities in soil to extreme rainfall in an agroforestry system.
Sun F(1), Pan K(2), Li Z(1), Wang S(1), Tariq A(1), Olatunji OA(1), Sun X(3),
Zhang L(3), Shi W(4), Wu X(3).
Author information:
(1)CAS Key Laboratory of Mountain Ecological Restoration and Bioresource
Utilization & Ecological Restoration Biodiversity Conservation Key Laboratory of
Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041, People's Republic of China; University of Chinese Academy of
Sciences, Beijing 100039, People's Republic of China.
Chengdu 610041, People's Republic of China. Electronic address: pankw@cib.ac.cn.
Chengdu 610041, People's Republic of China.
(4)Chongqing Key Laboratory of Karst Environment, School of Geographical
Sciences, Southwest University, Chongqing 400715, People's Republic of China.
Electronic address: shiweiyu@swu.edu.cn.
A current challenge for ecological research in agriculture is to identify ways in

which to improve the resilience of the soil food web to extreme climate events,
such as severe rainfall. Plant species composition influence soil biota
communities differently, which might affect the recovery of soil food web after
extreme rainfall. We compared the effects of rainfall stress up on the soil
microbial food web in three planting systems: a monoculture of the focal species
Zanthoxylum bungeanum and mixed cultures of Z. bungeanum and Medicago sativa or
Z. bungeanum and Glycine max. We tested the effect of the presence of a legume on
the recovery of trophic interactions between microorganisms and nematodes after
extreme rainfall. Our results indicated that all chemical properties of the soil
recovered to control levels (normal rainfall) in the three planting systems
45 days after exposure to extreme rain. However, on day 45, the bulk microbial
community differed from controls in the monoculture treatment, but not in the two
mixed planting treatments. The nematode community did not fully recover in the
monoculture or Z. bungeanum and M. sativa treatments, while nematode populations
in the combined Z. bungeanum and G. max treatment were indistinguishable from
controls. G. max performed better than M. sativa in terms of increasing the
resilience of microbial and nematode communities to extreme rainfall. Soil
microbial biomass and nematode density were positively correlated with the
available carbon and nitrogen content in soil, demonstrating a link between soil
health and biological properties. This study demonstrated that certain leguminous
plants can stabilize the soil food web via interactions with soil biota
communities after extreme rainfall.
DOI: 10.1016/j.scitotenv.2018.01.063
PMID: 29358146
13. Pak J Pharm Sci. 2018 Jan;31(1):57-67.
Toxic effect of common poisonous plants of district Bannu, Khyber Pakhtunkhwa,

Pakistan.
Khan RU(1), Mehmood S(1), Khan SU(1).

Author information:
(1)Department of Botany, University of Science & Technology Bannu, KPK, Pakistan.
The present paper was a part of Ph.D research work, conducted during the year
2014, in which 87 poisonous plants belonging to 54 genera, were collected,
documented and preserved in the herbarium of Bannu, Department of Botany UST,
Bannu Khyber Pakhtunkwa Pakistan. The plants were identified botanically,
arranged alphabetically along with their Latin name, family name, common name,
poisonous parts, toxicity, affects, toxin and their effects. Aim of the study was
to induce awareness in the local people of district Bannu about the poisonous
effects of the commonly used plants. Data about poisonous effect were collected
from the local experienced and mostly old age people through questionnaire. Some
information were collected from a number of veterinary texts and literature. The
most important plants genera studied in the area were Brassica 6 species
(11.11%), Lathyrus 5 spp (9.26%), Astragalus, Euphorbia and Prunus were with 4
spp (7.40%). Datura, Jatropha, Ranunculus, Solanum and Sorghum were with 3 spp
(5.56%) while Allium, Amaranthus, Chenopodium, Melilotus and Taxus were with 2
spp (3.70%). These 15 genera contribute 48 species (55.17 %) while the remaining
39 genera have single species each and contribute 44.83% to the total poisonous
flora of the research area. Other important poisonous plants were Anagallis
arvensis L., Cannabis sativa, Datura stramonium L., D. metel L., Euphorbia
species, Heliotropium europaeum, Ipomoea tricolor, Jatropha curcas, Lolium
temulentum L., Malus domestica, Mangifera indica L., Medicago sativa L.,
Melilotus alba Desr., M. officinalis (L.) Lam., Mirabilis jalapa L., Narcissus
tazetta, Nicotiana tabacum L., Sorghum halepense (L) Pers., and Xanthium
strumarium. It was concluded that the local population had poor knowledge about
the poisonous effect of the plants and the present research work was anticipated
for use by health care professionals, veterinarians, farmers, homeowners, as well
as botanically curious individuals.
PMID: 29348085
14. BMC Plant Biol. 2018 Jan 15;18(1):13. doi: 10.1186/s12870-017-1226-9.
Influence of drought stress on afalfa yields and nutritional composition.
Liu Y(1), Wu Q(1), Ge G(1), Han G(1), Jia Y(2)(3)(4).
Author information:
(1)Key Laboratory of Forage Cultivation, Processing and High Efficient
Utilization, Ministry of Agriculture, Erdos Street 29, Saihan District, Hohhot,
010019, China.
(2)Key Laboratory of Forage Cultivation, Processing and High Efficient
Utilization, Ministry of Agriculture, Erdos Street 29, Saihan District, Hohhot,
010019, China. 610480018@qq.com.
(3)Laboratory of Grassland Resources, Ministry of Education, Erdos Street 29,
Saihan District, Hohhot, 010019, China. 610480018@qq.com.
(4)College of Grassland, Resources and Environment, Inner Mongolia Agricultural
University, Hohhot, 010019, China. 610480018@qq.com.
BACKGROUND: It is predicted that climate change may increase the risk of local
droughts, with severe consequences for agricultural practices.
METHODS: Here we report the influence of drought on alfalfa yields and
nutritional composition, based on artificially induced drought conditions during
two field experiments. Two types of alfalfa cultivars were compared, Gold Queen
and Suntory. The severity and timing of drought periods were varied, and the crop
was harvested either early during flowering, or late at full bloom.
RESULTS: The obtained dry mass yields of Gold Queen were higher than Suntory, and
the first was also more resistant to drought. Early harvest resulted in higher
yields. Decreases in yields due to water shortage were observed with both
cultivars, and the fraction of crude protein (CP) decreased as a result of
drought stress; this fraction was higher in Gold Queen than in Suntory and higher
in early harvest compared to late harvest. Severe drought late in spring had the
highest effect on CP content. The fraction of fibre, split up into neutral
detergent fibre (NDF) and acid detergent fibre (ADF) increased as a result of
drought and was lower in early compared to late harvested plants. Suntory alfalfa
produced higher fibre fractions than Gold Queen. The fraction of water-soluble
carbohydrates (WSC) was least affected by drought. It was consistently higher in
Gold Queen compared to Suntory alfalfa, and late harvest resulted in higher WSC
content.
CONCLUSIONS: In combination, these results suggest that the nutritive value of
alfalfa will likely decrease after a period of drought. These effects can be
partly overcome by choosing the Gold Queen cultivar over Suntory, by targeted
irrigation, in particular in late spring, and by harvesting at an earlier time.
DOI: 10.1186/s12870-017-1226-9
PMCID: PMC5769550
PMID: 29334916
15. Front Plant Sci. 2017 Dec 13;8:2125. doi: 10.3389/fpls.2017.02125. eCollection
2017.
Over-Expression of Arabidopsis EDT1 Gene Confers Drought Tolerance in Alfalfa

(Medicago sativa L.).
Zheng G(1)(2)(3), Fan C(1)(3), Di S(1), Wang X(2), Xiang C(4), Pang Y(1)(2).
Author information:
(1)Key Laboratory of Plant Resources and Beijing Botanical Garden, Institute of
Botany, Chinese Academy of Sciences, Beijing, China.
(2)Institute of Animal Science, Chinese Academy of Agricultural Sciences,
Beijing, China.
(3)University of Chinese Academy of Sciences, Beijing, China.
(4)School of Life Sciences, University of Science and Technology of China, Hefei,
China.
Alfalfa (Medicago sativa L.) is an important legume forage crop with great
economic value. However, as the growth of alfalfa is seriously affected by an
inadequate supply of water, drought is probably the major abiotic environmental
factor that most severely affects alfalfa production worldwide. In an effort to
enhance alfalfa drought tolerance, we transformed the Arabidopsis Enhanced
Drought Tolerance 1 (AtEDT1) gene into alfalfa via Agrobacterium-mediated
transformation. Compared with wild type plants, drought stress treatment resulted
in higher survival rates and biomass, but reduced water loss rates in the
transgenic plants. Furthermore, transgenic alfalfa plants had increased stomatal
size, but reduced stomatal density, and these stomatal changes contributed
greatly to reduced water loss from leaves. Importantly, transgenic alfalfa plants
exhibited larger root systems with larger root lengths, root weight, and root
diameters than wild type plants. The transgenic alfalfa plants had reduced
membrane permeability and malondialdehyde content, but higher soluble sugar and
proline content, higher superoxide dismutase activity, higher chlorophyll
content, enhanced expression of drought-responsive genes, as compared with wild
type plants. Notably, transgenic alfalfa plants grew better in a 2-year field
trial and showed enhanced growth performance with increased biomass yield. All of
our morphological, physiological, and molecular analyses demonstrated that the
ectopic expression of AtEDT1 improved growth and enhanced drought tolerance in
alfalfa. Our study provides alfalfa germplasm for use in forage improvement
programs, and may help to increase alfalfa production in arid lands.
DOI: 10.3389/fpls.2017.02125
PMCID: PMC5733502
PMID: 29326737
16. Ann Bot. 2018 Jan 9. doi: 10.1093/aob/mcx207. [Epub ahead of print]
Hydrogen peroxide acts downstream of melatonin to induce lateral root formation.
Chen Z(1), Gu Q(1), Yu X(1), Huang L(2), Xu S(3), Wang R(3), Shen W(2), Shen
W(1).
Author information:
(1)College of Life Sciences, Laboratory Center of Life Sciences, Nanjing
Agricultural University, Nanjing, P.R. China.
(2)College of Sciences, Nanjing Agricultural University, Nanjing, P.R. China.
(3)Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing,
P.R. China.
Background and Aims: Although several studies have confirmed the beneficial roles
of exogenous melatonin in lateral root (LR) formation, the molecular mechanism is
still elusive. Here, the role of hydrogen peroxide (H2O2) in the induction of LR
formation triggered by melatonin was investigated.
Methods: Alfalfa (Medicago sativa 'Biaogan') and transgenic Arabidopsis seedlings
were treated with or without melatonin, diphenyleneiodonium (DPI, NADPH oxidase
inhibitor), N,N'-dimethylthiourea (DMTU, H2O2 scavenger), alone or combined.
Then, H2O2 content was determined with 2',7'-dichlorofluorescein diacetate
(H2DCFDA)-dependent fluorescence and spectrophotography. Transcript levels of
cell cycle regulatory genes were analysed by real-time reverse transcription-PCR.
Key Results: Application of exogenous melatonin not only increased endogenous
H2O2 content but also induced LR formation in alfalfa seedlings. Consistently,
melatonin-induced LR primordia exhibited an accelerated response. These inducible
responses were significantly blocked when DPI or DMTU was applied. Compared with
the wild-type, transgenic Arabidopsis plants overexpressing alfalfa MsSNAT (a
melatonin synthesis gene) increased H2O2 accumulation and thereafter LR
formation, both of which were blocked by DPI or DMTU. Similarly,
melatonin-modulated expression of marker genes responsible for LR formation,
including MsCDKB1;1, MsCDKB2;1, AtCDKB1;1 and AtCDKB2;1, was obviously impaired
by the removal of H2O2 in both alfalfa and transgenic Arabidopsis plants.
Conclusions: Pharmacological and genetic evidence revealed that endogenous
melatonin-triggered LR formation was H2O2-dependent.
DOI: 10.1093/aob/mcx207
PMID: 29325001
17. Sci Rep. 2018 Jan 10;8(1):357. doi: 10.1038/s41598-017-18348-0.
Profiling of metabolome and bacterial community dynamics in ensiled Medicago

sativa inoculated without or with Lactobacillus plantarum or Lactobacillus
buchneri.
Guo XS(1)(2), Ke WC(3)(4), Ding WR(3)(4), Ding LM(3)(4), Xu DM(3)(4), Wang WW(3),
Zhang P(3)(4), Yang FY(5).
Author information:
(1)State Key Laboratory of Grassland and Agro-ecosystems, School of Life
Sciences, Lanzhou University, Lanzhou, 730000, PR China. guoxsh07@lzu.edu.cn.
(2)Probiotics and Biological Feed Research Center, Lanzhou University, Lanzhou,
730000, PR China. guoxsh07@lzu.edu.cn.
(3)State Key Laboratory of Grassland and Agro-ecosystems, School of Life
Sciences, Lanzhou University, Lanzhou, 730000, PR China.
(4)Probiotics and Biological Feed Research Center, Lanzhou University, Lanzhou,
730000, PR China.
(5)Institute of Grassland Science, College of Animal Science and Technology,
China Agricultural University, Beijing, 100193, PR China. yfuyu@126.com.
Using gas chromatography mass spectrometry and the PacBio single molecule with
real-time sequencing technology (SMRT), we analyzed the detailed metabolomic
profiles and microbial community dynamics involved in ensiled Medicago sativa
(alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or
heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances
and 102 different metabolites were present in ensiled alfalfa. Inoculation of L.
buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric
acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable
down-accumulation in cadaverine and succinic acid were observed in L.
plantarum-inoculated silages. Completely different microbial flora and their
successions during ensiling were observed in the control and two types of
inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters
the microbial composition dynamics of the ensiled forage in very different
manners. Our study demonstrates that metabolomic profiling analysis provides a
deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed
the microbial composition and its succession during the ensiling process at the
species level. This provides information regarding the microbial processes
underlying silage formation and may contribute to target-based regulation methods
to achieve high-quality silage production.
DOI: 10.1038/s41598-017-18348-0
PMCID: PMC5762819
PMID: 29321642
18. Front Microbiol. 2017 Dec 14;8:2466. doi: 10.3389/fmicb.2017.02466. eCollection

2017.
Improved Drought Stress Response in Alfalfa Plants Nodulated by an IAA

Over-producing Rhizobium Strain.
Defez R(1), Andreozzi A(1), Dickinson M(2), Charlton A(2), Tadini L(3), Pesaresi
P(4), Bianco C(1).
Author information:
(1)Institute of Biosciences and BioResources - National Research Council, Naples,
Italy.
(2)Fera Science Ltd., National Agri-Food Innovation Campus, Sand Hutton, United
Kingdom.
(3)Dipartimento di Bioscienze, Università degli Studi di Milano, Milan, Italy.
(4)Dipartimento di Scienze Agrarie e Forestali, Università degli Studi di Milano,
Milan, Italy.
The drought-stress response in plant involves the cross-talk between abscisic

acid (ABA) and other phytohormones, such as jasmonates and ethylene. The auxin
indole-3-acetic acid (IAA) plays an integral part in plant adaptation to drought
stress. Investigation was made to see how the main auxin IAA interacted with
other plant hormones under water stress, applied through two different growth
conditions (solid and hydroponic). Medicago sativa plants nodulated by the
Ensifer meliloti wild type 1021 (Ms-1021) and its IAA-overproducing RD64
derivative strains (Ms-RD64) were subjected to drought stress, comparing their
response. When the expression of nifH gene and the activity of the nitrogenase
enzyme were measured after stress treatments, Ms-RD64 plants recorded a
significantly weaker damage. These results were correlated with a lower biomass
reduction, and a higher Rubisco protein level measured for the Ms-RD64-stressed
plants as compared to the Ms-1021-stressed ones. It has been verified that the
stress response observed for Ms-RD64-stressed plants was related to the
production of greater amount of low-molecular-weight osmolytes, such as proline
and pinitol, measured in these plants. For the Ms-RD64 plants the immunoblotting
analysis of thylakoid membrane proteins showed that some of the photosystem
proteins increased after the stress. An increased non-photochemical quenching
after the stress was also observed for these plants. The reduced wilting signs
observed for these plants were also connected to the significant down-regulation
of the MtAA03 gene involved in the ABA biosynthesis, and with the unchanged
expression of the two genes (Mt-2g006330 and Mt-8g095330) of ABA signaling. When
the expression level of the ethylene-signaling genes was evaluated by qPCR
analysis no significant alteration of the key positive regulators was recorded
for Ms-RD64-stressed plants. Coherently, these plants accumulated 40% less
ethylene as compared to Ms-1021-stressed ones. The results presented herein
indicate that the variations in endogenous IAA levels, triggered by the
overproduction of rhizobial IAA inside root nodules, positively affected drought
stress response in nodulated alfalfa plants.
DOI: 10.3389/fmicb.2017.02466
PMCID: PMC5735143
PMID: 29312178
19. Ecol Evol. 2017 Nov 12;7(24):10916-10929. doi: 10.1002/ece3.3607. eCollection

2017 Dec.
Salinity tolerance of three competing rangeland plant species: Studies in

hydroponic culture.
Sagers JK(1), Waldron BL(2), Creech JE(1), Mott IW(2), Bugbee B(1).
Author information:
(1)Plants, Soils, and Climate DepartmentUtah State UniversityLoganUTUSA.
(2)Forage and Range Research LaboratoryUSDA Agricultural Research
ServiceLoganUTUSA.
Halogeton (Halogeton glomeratus) is an invasive species that displaces Gardner's

saltbush (Atriplex gardneri) on saline rangelands, whereas, forage kochia (Bassia
prostrata) potentially can rehabilitate these ecosystems. Salinity tolerance has
been hypothesized as the predominant factor affecting frequency of these species.
This study compared relative salinity tolerance of these species, and tall
wheatgrass (Thinopyrum ponticum) and alfalfa (Medicago sativa). Plants were
evaluated in hydroponics, eliminating the confounding effects of drought, for
28 days at 0, 150, 200, 300, 400, 600, and 800 mmol/L NaCl. Survival, growth, and
ion accumulation were determined. Alfalfa and tall wheatgrass shoot mass were
reduced to 32% of the control at 150 mmol/L. Forage kochia survived to
600 mmol/L, but mass was reduced at all salinity levels. Halogeton and Gardner's
saltbush increased or maintained shoot mass up to 400 mmol/L. Furthermore, both
actively accumulated sodium in shoots, indicating that Na+ was the principle ion
in osmotic adjustment, whereas, forage kochia exhibited passive (linear) Na+
accumulation as salinity increased. This study confirmed the halophytic nature of
these three species, but, moreover, discovered that Gardner's saltbush was as
saline tolerant as halogeton, whereas, forage kochia was less tolerant.
Therefore, factors other than salinity tolerance drive these species'
differential persistence in saline-desert ecosystems.
DOI: 10.1002/ece3.3607
PMCID: PMC5743640
PMID: 29299269
20. J Biotechnol. 2018 Feb 10;267:55-62. doi: 10.1016/j.jbiotec.2017.12.020. Epub

2017 Dec 29.
Isolation, taxonomic analysis, and phenotypic characterization of bacterial

endophytes present in alfalfa (Medicago sativa) seeds.
López JL(1), Alvarez F(1), Príncipe A(2), Salas ME(1), Lozano MJ(1), Draghi
WO(1), Jofré E(2), Lagares A(3).
Author information:
(1)Instituto de Biotecnología y Biología Molecular-CONICET CCT-La Plata,
Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad
Nacional de La Plata, La Plata, Argentina.
(2)Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad
Nacional de Río Cuarto, Ruta 36-Km 601-5800, Río Cuarto-Córdoba, Argentina.
Nacional de La Plata, La Plata, Argentina. Electronic address:
lagares@biol.unlp.edu.ar.
A growing body of evidence has reinforced the central role of microbiomes in the
life of sound multicellular eukaryotes, thus more properly described as true
holobionts. Though soil was considered a main source of plant microbiomes, seeds
have been shown to be endophytically colonized by microorganisms thus
representing natural carriers of a selected microbial inoculum to the young
seedlings. In this work we have investigated the type of culturable endophytic
bacteria that are carried within surface-sterilized alfalfa seeds. MALDI-TOF
analysis revealed the presence of bacteria that belonged to 40 separate genera,
distributed within four taxa (Proteobacteria, Actinobacteria, Firmicutes, and
Bacteroidetes). Nonsymbiotic members of the Rhizobiaceae family were also found.
The evaluation of nine different in-vitro biochemical activities demonstrated
isolates with complex combinations of traits that, upon a
Principal-Component-Analysis, could be classified into four phenotypic groups.
That isolates from nearly half of the genera identified had been able to colonize
alfalfa plants grown under axenic conditions was remarkable. Further analyses
should be addressed to investigating the colonization mechanisms of the alfalfa
seeds, the evolutionary significance of the alfalfa-seed endophytes, and also how
after germination the seed microbiome competes with spermospheric and
rhizospheric soil bacteria to colonize newly emerging seedlings.
DOI: 10.1016/j.jbiotec.2017.12.020
PMID: 29292130
21. Microb Ecol. 2017 Dec 30. doi: 10.1007/s00248-017-1133-5. [Epub ahead of print]
Endosymbionts Differentially Alter Exploratory Probing Behavior of a

Nonpersistent Plant Virus Vector.
Angelella G(1)(2), Nalam V(3), Nachappa P(3), White J(4), Kaplan I(5).
Author information:
(1)Department Entomology, Purdue University, 901 West State St., Lafayette, IN,
47907, USA. ginama@vt.edu.
(2)Department Horticulture, Virginia Tech University, 33446 Research Dr.,
Painter, VA, 23420, USA. ginama@vt.edu.
(3)Department Biology, Indiana University-Purdue University Fort Wayne, 2101 E.
Coliseum Blvd., Fort Wayne, IN, 46805, USA.
(4)Department Entomology, University of Kentucky, S-225 Agricultural Science
Center N, Lexington, KY, 40546, USA.
(5)Department Entomology, Purdue University, 901 West State St., Lafayette, IN,
47907, USA.
Insect endosymbionts (hereafter, symbionts) can modify plant virus epidemiology

by changing the physiology or behavior of vectors, but their role in
nonpersistent virus pathosystems remains uninvestigated. Unlike propagative and
circulative viruses, nonpersistent plant virus transmission occurs via transient
contamination of mouthparts, making direct interaction between symbiont and virus
unlikely. Nonpersistent virus transmission occurs during exploratory
intracellular punctures with styletiform mouthparts when vectors assess potential
host-plant quality prior to phloem feeding. Therefore, we used an electrical
penetration graph (EPG) to evaluate plant probing of the cowpea aphid, Aphis
craccivora Koch, an important vector of cucurbit viruses, in the presence and
absence of two facultative, intracellular symbionts. We tested four isolines of
A. craccivora: two isolines were from a clone from black locust (Robinia
pseudoacacia L.), one infected with Arsenophonus sp. and one cured, and two
derived from a clone from alfalfa (Medicago sativa L.), one infected with
Hamiltonella defensa and one cured. We quantified exploratory intracellular
punctures, indicated by a waveform potential drop recorded by the EPG, initiation
speed and frequency within the initial 15 min on healthy and watermelon mosaic
virus-infected pumpkins. Symbiont associations differentially modified
exploratory intracellular puncture frequency by aphids, with H. defensa-infected
aphids exhibiting depressed probing, and Arsenophonus-infected aphids an
increased frequency of probing. Further, there was greater overall aphid probing
on virus-infected plants, suggesting that viruses manipulate their vectors to
enhance acquisition-transmission rates, independent of symbiont infection. These
results suggest facultative symbionts differentially affect plant-host
exploration behaviors and potentially nonpersistent virus transmission by
vectors.
DOI: 10.1007/s00248-017-1133-5
PMID: 29290035
22. G3 (Bethesda). 2018 Feb 2;8(2):461-468. doi: 10.1534/g3.117.300099.
Selection Mapping Identifies Loci Underpinning Autumn Dormancy in Alfalfa

(Medicago sativa).
Munjal G(1)(2), Hao J(1)(2), Teuber LR(1), Brummer EC(3)(2).
Author information:
(1)Department of Plant Sciences, University of California-Davis, California
95616.
(2)Plant Breeding Center, University of California-Davis, California 95616.
(3)Department of Plant Sciences, University of California-Davis, California 95616
ecbrummer@ucdavis.edu.
Autumn dormancy in alfalfa (Medicago sativa) is associated with agronomically

important traits including regrowth rate, maturity, and winter survival.
Historical recurrent selection experiments have been able to manipulate the
dormancy response. We hypothesized that artificial selection for dormancy
phenotypes in these experiments had altered allele frequencies of
dormancy-related genes. Here, we follow this hypothesis and analyze allele
frequency changes using genome-wide polymorphisms in the pre- and postselection
populations from one historical selection experiment. We screened the nondormant
cultivar CUF 101 and populations developed by three cycles of recurrent
phenotypic selection for taller and shorter plants in autumn with markers derived
from genotyping-by-sequencing (GBS). We validated the robustness of our
GBS-derived allele frequency estimates using an empirical approach. Our results
suggest that selection mapping is a powerful means of identifying genomic regions
associated with traits, and that it can be exploited to provide regions on which
to focus further mapping and cloning projects.
Copyright © 2018 Munjal et al.
DOI: 10.1534/g3.117.300099
PMID: 29255116
23. J Sep Sci. 2018 Mar;41(5):1118-1128. doi: 10.1002/jssc.201701147. Epub 2018 Jan
16.
Determination of sugars and cyclitols isolated from various morphological parts

of Medicago sativa L.
Al-Suod H(1)(2), Ratiu IA(2)(3), Ligor M(1), Ligor T(1)(2), Buszewski B(1)(2).
Author information:
(1)Department of Environmental Chemistry and Bioanalytics, Faculty of Chemistry,
Nicolaus Copernicus University, Toruń, Poland.
(2)Interdisciplinary Centre of Modern Technologies, Nicolaus Copernicus
University, Toruń, Poland.
(3)Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering,
Cluj-Napoca, Romania.
Plant research interest has increased all over the world, and a large body of
evidence has been collected to show the huge potential of medicinal plants in
various disease treatments. Medicago sativa L., known as alfalfa, is a rich
source of biologically active components and secondary metabolites and was
frequently used from the ancient times both as fodder crop and as a traditional
medicine in the treatment of various diseases. Cyclitols, naturally occurring in
this plant, have a particular interest for us due to their significant
anti-diabetic, antioxidant, anti-inflammatory, and anti-cancer properties. In the
present study we revealed the isolation, the identification, and the
quantification of some cyclitols and sugars extracted from different
morphological parts of alfalfa plant. Soxhlet extraction and solid phase
extraction were used as extraction and purification methods, while for the
analyses derivatization followed by gas chromatography with mass spectrometry was
involved. The obtained results showed significant differences in the quantities
of cyclitols and sugars found in the investigated morphological parts, ranging
between 0.02 and 13.86 mg/g of plant in case of cyclitols, and in the range of
0.09 and 40.09 mg/g of plant for sugars. However, roots have the richest part of
cyclitols and sugars in contrast to the leaves.
© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/jssc.201701147
PMID: 29250921
24. PLoS One. 2017 Dec 15;12(12):e0189781. doi: 10.1371/journal.pone.0189781.

eCollection 2017.
Resistant and susceptible responses in alfalfa (Medicago sativa) to bacterial

stem blight caused by Pseudomonas syringae pv. syringae.
Nemchinov LG(1), Shao J(1), Lee MN(1), Postnikova OA(1), Samac DA(2).
Author information:
(1)USDA-ARS, Molecular Plant Pathology Laboratory, Beltsville, Maryland, United
States of America.
(2)USDA-ARS, Plant Science Research Unit, St. Paul, Minnesota, United States of
America.
Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common

disease of alfalfa (Medicago sativa L). Little is known about host-pathogen
interactions and host defense mechanisms. Here, individual resistant and
susceptible plants were selected from cultivars Maverick and ZG9830 and used for
transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate
PssALF3. Bioinformatic analysis revealed a number of differentially expressed
genes (DEGs) in resistant and susceptible genotypes. Although resistant plants
from each cultivar produced a hypersensitive response, transcriptome analyses
indicated that they respond differently at the molecular level. The number of
DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick,
suggesting that ZG9830 plants had a more rapid effector triggered immune
response. Unique up-regulated genes in resistant ZG9830 plants included genes
encoding putative nematode resistance HSPRO2-like proteins, orthologs for the
rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking
both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick
plants had an over-representation of R genes in the CC-NBS-LRR family including
two genes for atypical CCR domains and a putative ortholog of the Arabidopsis
RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY
family transcription factors and expression of genes involved in protein
phosphorylation, regulation of transcription, defense response including
synthesis of isoflavonoids, and oxidation-reduction processes. These results will
further the identification of mechanisms involved in resistance to facilitate
selection of parent populations and development of commercial varieties.
DOI: 10.1371/journal.pone.0189781
PMCID: PMC5731681
PMID: 29244864 [Indexed for MEDLINE]
25. Interdiscip Sci. 2017 Dec 14. doi: 10.1007/s12539-017-0230-y. [Epub ahead of
print]
Genome-Wide Assessment of Polygalacturonases-Like (PGL) Genes of Medicago

truncatula, Sorghum bicolor, Vitis vinifera and Oryza sativa Using Comparative
Genomics Approach.
Anand G(1), Yadav S(1), Tanveer A(1), Nasim J(1), Singh NK(1)(2), Dubey AK(1),
Yadav D(3).
Author information:
(1)Department of Biotechnology, D.D.U Gorakhpur University, Gorakhpur, Uttar
Pradesh, 273 009, India.
(2)Department of Biotechnology, Indian Institute of Technology Madras, Chennai,
600 036, India.
(3)Department of Biotechnology, D.D.U Gorakhpur University, Gorakhpur, Uttar
Pradesh, 273 009, India. dinesh_yad@rediffmail.com.
The polygalacturonases (PG) is one of the important members of pectin-degrading

glycoside hydrolases of the family GH28. In plants, PG represents multigene
families associated with diverse processes. In the present study, an attempt has
been made to investigate the diversity of PG genes among monocots and dicots with
respect to phylogeny, gene duplication and subcellular localization to get an
insight into the evolutionary and functional attributes. The genome-wide
assessment of Medicago truncatula, Vitis vinifera Sorghum bicolor, and Oryza
sativa L. ssp. japonica genomes revealed 53, 49, 38 and 35 PG-like (PGL) genes,
respectively. The predominance of glyco_hydro_28 domain, hydrophilic nature and
genes with multiple introns were uniformly observed. The subcellular localization
showed the presence of signal sequences targeting the secretory pathways. The
phylogenetic tree constructed marked uniformity with three distinct clusters for
each plant irrespective of the variability in the genome sizes. The site-specific
selection pressure analysis based on K a/K s values showed predominance of
purifying selection pressures among different groups identified in these plants.
The functional divergence analysis revealed significant site-specific selective
constraints. Results of site-specific selective pressure analysis throw light on
the functional diversity of PGs in various plant processes and hence its
constitutive nature. These findings are further strengthened by functional
divergence analysis which reveals functionally diverse groups in all the four
species representing monocots and dicots. The outcome of the present work could
be utilized for deciphering the novel functions of PGs in plants.
DOI: 10.1007/s12539-017-0230-y
PMID: 29243204
26. Plant Cell. 2018 Jan;30(1):15-36. doi: 10.1105/tpc.17.00581. Epub 2017 Dec 11.
Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell
Types Reveals Common Gene Regulatory Principles and New Control Modules.
Maher KA(1)(2), Bajic M(1)(3), Kajala K(4), Reynoso M(5), Pauluzzi G(5), West
DA(6), Zumstein K(6), Woodhouse M(6), Bubb K(7), Dorrity MW(7), Queitsch C(7),
Bailey-Serres J(5), Sinha N(6), Brady SM(4), Deal RB(8).
Author information:
(1)Department of Biology, Emory University, Atlanta, Georgia 30322.
(2)Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory
University, Atlanta, Georgia 30322.
(3)Graduate Program in Genetics and Molecular Biology, Emory University, Atlanta,
Georgia 30322.
(4)Department of Plant Biology and Genome Center, University of California,
Davis, California 95616.
(5)Center for Plant Cell Biology, Botany and Plant Sciences Department,
University of California, Riverside, California 92521.
(6)Department of Plant Biology, University of California, Davis, California
95616.
(7)University of Washington, School of Medicine, Department of Genome Sciences,
Seattle, Washington 98195.
(8)Department of Biology, Emory University, Atlanta, Georgia 30322
roger.deal@emory.edu.
The transcriptional regulatory structure of plant genomes remains poorly defined

relative to animals. It is unclear how many cis-regulatory elements exist, where
these elements lie relative to promoters, and how these features are conserved
across plant species. We employed the assay for transposase-accessible chromatin
(ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula,
Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and
transcription factor (TF) binding sites across each genome. Despite 10-fold
variation in intergenic space among species, the majority of open chromatin
regions lie within 3 kb upstream of a transcription start site in all species. We
find a common set of four TFs that appear to regulate conserved gene sets in the
root tips of all four species, suggesting that TF-gene networks are generally
conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair
cell types revealed extensive similarity as well as many cell-type-specific
differences. Analyzing TF binding sites in differentially accessible regions
identified a MYB-driven regulatory module unique to the hair cell, which appears
to control both cell fate regulators and abiotic stress responses. Our analyses
revealed common regulatory principles among species and shed light on the
mechanisms producing cell-type-specific transcriptomes during development.
© 2018 American Society of Plant Biologists. All rights reserved.
DOI: 10.1105/tpc.17.00581
PMCID: PMC5810565 [Available on 2019-01-01]
PMID: 29229750
27. J Proteomics. 2018 Feb 20;173:126-138. doi: 10.1016/j.jprot.2017.11.029. Epub

2017 Dec 8.
Proteomics reveals key proteins participating in growth difference between fall

dormant and non-dormant alfalfa in terminal buds.
Du H(1), Shi Y(1), Li D(1), Fan W(2), Wang Y(1), Wang G(1), Wang C(3).
Author information:
(1)College of Animal Science and Veterinary Medicine, Henan Agricultural
University, Zhengzhou, Henan 450002, China.
(2)School of Animal Science and Technology, Henan University of Science and
Technology, Luoyang, Henan 471023, China.
University, Zhengzhou, Henan 450002, China. Electronic address:
wangcz@henau.edu.cn.
To explore the molecular mechanism of growth differences between fall dormant

(FD) and non-FD alfalfa, we conducted iTRAQ-based quantitative proteomics on
terminal buds of Maverick (FD) and Cuf101 (non-FD) cultivars, identified
differential abundance protein species (DAPS) and verified expression profiling
of certain corresponding mRNA by qRT-PCR. A total of 3872 protein species were
annotated. Of the 90 DAPS, 56 and 34 were respectively up- and down-accumulated
in Maverick, compared to Cuf101. They were grouped into 35 functional categories
and enriched in seven pathways. Of which, auxin polar transport was up-regulated,
while phenylpropanoid biosynthesis, pyruvate metabolism and transportation,
vitamin B1 synthesis process and flavonoid biosynthesis were down-regulated in
Maverick, comparing with Cuf101. In Maverick, mRNA abundances of l-asparaginase,
chalcone and stilbene synthase family protein, cinnamyl alcohol
dehydrogenase-like protein, thiazole biosynthetic enzyme, pyruvate dehydrogenase
E1 beta subunit, and aldo/keto reductase family oxidoreductase were significantly
lower at FD than at other stages, and lower than in Cuf101. We also observed
opposite mRNA profiles of thiazole biosynthetic enzyme, chalcone and stilbene
synthase family protein, pyruvate dehydrogenase E1 beta subunit in both cultivars
from summer to autumn. Our results suggest that these DAPS could play important
roles in growth difference between FD and non-FD alfalfa.BIOLOGICAL SIGNIFICANCE:
Up to now, as far as we know, currently the proteins related with the growth
differences between FD and non-FD alfalfa cultivars in autumn have not yet been
identified in terminal buds. This study identified the protein species expressed
in alfalfa terminal buds, selected differentially abundant protein species in
terminal buds between Maverick (FD) and Cuf101 (non-FD) cultivars in autumn and
identified the important protein species participated in the growth differences.
This study lays a foundation for further investigation of the molecular mechanism
of the growth differences between FD and non-FD alfalfa and the cultivation of
advanced alfalfa cultivars.
DOI: 10.1016/j.jprot.2017.11.029
PMID: 29229487
28. Georgian Med News. 2017 Nov;(272):133-137.
[STUDY OF SOME PLANT SEED LIPIDS GROWING IN GEORGIA].
[Article in Russian]
Kikalishvili B(1), Gorgaslidze N(1), Sulakvelidze T(1), Malania M(1), Urushadze

N(1), Turabelidze D(1).
Author information:
(1)Tbilisi State Medical University, I. Kutateladze Institute of
Pharmacochemistry, Georgia.
The purpose of the research was to study seed lipids of cultivated plants:
Medicago sativa, Ruta graveolens L., Linum usitatissimum L., which are growing on
experimental field of I. Kutateladze Institute of Pharmacochemistry. From the
above-mentioned plant seeds was obtained the sum of neutral and polar lipids, the
classes were determined qualitatively. Using standard methods were determined
some physico-chemical constant of oils. Fatty acids was established qualitatively
and was determined quantitatively using Gas chromatography and High performance
liquid chromatography methods. As a result of research in the seed oil of
Medicago Sativa was revealed high percentage content of Oktadekadien - 9,12 fatty
acid. Oktadekadien -9 dominates in the oil obtained from the seeds of Ruta
graveolens L. and Oktadekantrien - 9,12,15 dominates in the oil obtained from the
seeds of Linum usitatissimum L. Phospholipids were established qualitatively and
using spectrophotometric method were determined In the sum of polar lipids: (λ
820 nm). The oils which are obtained from above-mentioned plant seeds contains
biologically active compounds, which gives the opportunity to use these oils as
in medical practice, also in cosmetology.
PMID: 29227272

eCollection 2017.
Screening and identification of key genes regulating fall dormancy in alfalfa

leaves.
Du H(1), Shi Y(1), Li D(1), Fan W(2), Wang G(1), Wang C(1).
Author information:
University, Henan Key Laboratory of Innovation and Utilization of Grassland
Resources, Zhengzhou, Henan, China.
(2)School of Animal Science and Technology, Henan University of Science and
Technology, Luoyang, Henan, China.
Fall dormancy (FD) determines the adaptation of an alfalfa variety and affects
alfalfa production and quality. However, the molecular mechanism underlying FD
remains poorly understood. Here, 44 genes regulating FD were identified by
comparison of the transcriptomes from leaves of Maverick (fall-dormant alfalfa)
and CUF101(non-fall-dormant), during FD and non-FD and were classified them
depending on their function. The transcription of IAA-amino acid hydrolase
ILR1-like 1, abscisic acid receptor PYL8, and monogalactosyldiacylglycerol
synthase-3 in Maverick leaves was regulated by daylength and temperature, and the
transcription of the abscisic acid receptor PYL8 was mainly affected by
daylength. The changes in the expression of these genes and the abundance of
their messenger RNA (mRNA) in Maverick leaves differed from those in CUF101
leaves, as evidenced by the correlation analysis of their mRNA abundance profiles
obtained from April to October. The present findings suggested that these genes
are involved in regulating FD in alfalfa.
PMCID: PMC5718555
30. Glob Chang Biol. 2018 Apr;24(4):1762-1770. doi: 10.1111/gcb.14009. Epub 2017
Dec
22.
Nitrogen-rich microbial products provide new organo-mineral associations for the

stabilization of soil organic matter.
Kopittke PM(1), Hernandez-Soriano MC(1), Dalal RC(1), Finn D(1), Menzies NW(1),
Hoeschen C(2), Mueller CW(2).
Author information:
(1)School of Agriculture and Food Sciences, The University of Queensland, St.
Lucia, Qld, Australia.
(2)Lehrstuhl für Bodenkunde, Technische Universität München, Freising, Germany.
Understanding the cycling of C and N in soils is important for maintaining soil

fertility while also decreasing greenhouse gas emissions, but much remains
unknown about how organic matter (OM) is stabilized in soils. We used nano-scale
secondary ion mass spectrometry (NanoSIMS) to investigate the changes in C and N
in a Vertisol and an Alfisol incubated for 365 days with 13 C and 15 N pulse
labeled lucerne (Medicago sativa L.) to discriminate new inputs of OM from the
existing soil OM. We found that almost all OM within the free stable
microaggregates of the soil was associated with mineral particles, emphasizing
the importance of organo-mineral interactions for the stabilization of C. Of
particular importance, it was also found that 15 N-rich microbial products
originating from decomposition often sorbed directly to mineral surfaces not
previously associated with OM. Thus, we have shown that N-rich microbial products
preferentially attach to distinct areas of mineral surfaces compared to
C-dominated moieties, demonstrating the ability of soils to store additional OM
in newly formed organo-mineral associations on previously OM-free mineral
surfaces. Furthermore, differences in 15 N enrichment were observed between the
Vertisol and Alfisol presumably due to differences in mineralogy
(smectite-dominated compared to kaolinite-dominated), demonstrating the
importance of mineralogy in regulating the sorption of microbial products.
Overall, our findings have important implications for the fundamental
understanding of OM cycling in soils, including the immobilization and storage of
N-rich compounds derived from microbial decomposition and subsequent N
mineralization to sustain plant growth.
© 2017 John Wiley & Sons Ltd.
DOI: 10.1111/gcb.14009
PMID: 29211318
31. Zoo Biol. 2018 Jan;37(1):23-34. doi: 10.1002/zoo.21393. Epub 2017 Dec 6.
Behavioral and hormonal responses to the availability of forage material in

Western lowland gorillas (Gorilla gorilla gorilla).
Fuller G(1), Murray A(1), Thueme M(1), McGuire M(2), Vonk J(2), Allard S(1).
Author information:
(1)Center for Zoo Animal Welfare and Ethics, Detroit Zoological Society, Royal
Oak, Michigan.
(2)Department of Psychology, Oakland University, Rochester, Michigan.
We investigated how forage material affects indicators of welfare in three male

Western lowland gorillas (Gorilla gorilla gorilla) at the Detroit Zoo. In
addition to their maintenance diet and enrichment foods, the gorillas generally
received forage material four times a week. From this baseline, we systematically
manipulated how much forage material the group received on a weekly basis, with
either daily or bi (twice)-weekly presentation of browse (mulberry, Morus sp.) or
alfalfa hay. We collected behavioral data (60 hr per gorilla) and measured fecal
glucocorticoid metabolites (FGM). Mixed models indicated that the presence of
forage material significantly increased time feeding (F2,351 = 9.58, p < 0.001),
and decreased rates of noncontact aggression (F2,351 = 3.69, p = 0.03), and
regurgitation and reingestion (F2,353 = 4.70, p = 0.01). Regurgitation and
reingestion were never observed during the condition when forage material was
provided daily. When forage material was provided, time spent feeding was similar
across gorillas, compared to a disproportionately greater amount of time spent
feeding by the dominant individual when forage material was absent. Providing
forage material in addition to the regular diet likely created more opportunities
for equitable feeding for the subordinate gorillas. FGM concentrations did not
vary based on the presence or type of forage material available and, instead,
likely reflected group social dynamics. In general, alfalfa and mulberry had
similar impacts on behavior, indicating that alfalfa can be an adequate
behavioral substitute during times when browse is less readily available for
gorillas housed in seasonally variable climates.
© 2017 Wiley Periodicals, Inc.
DOI: 10.1002/zoo.21393
32. Arch Virol. 2018 Mar;163(3):799-803. doi: 10.1007/s00705-017-3673-x. Epub 2017

Dec 5.
Genome characterization of an Argentinean isolate of alfalfa leaf curl virus.
Bejerman N(1)(2), Trucco V(3), de Breuil S(3)(4), Pardina PR(3), Lenardon

S(3)(5), Giolitti F(3).
Author information:
(1)IPAVE-CIAP-INTA, Av. 11 de Septiembre 4755, 5020, Córdoba, Argentina.
nicobejerman@gmail.com.
(2)Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy
Cruz 2290 (1425 FBQ) CABA, Buenos Aires, Argentina. nicobejerman@gmail.com.
(3)IPAVE-CIAP-INTA, Av. 11 de Septiembre 4755, 5020, Córdoba, Argentina.
(4)Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy
Cruz 2290 (1425 FBQ) CABA, Buenos Aires, Argentina.
(5)Facultad de Agronomía y Veterinaria (FAV), Universidad Nacional de Río Cuarto
(UNRC), Enlaces Rutas 8 y 36, 5800, Río Cuarto, Córdoba, Argentina.
We investigated the molecular characteristics of an Argentinean isolate of

alfalfa leaf curl virus (ALCV-Arg), a virus of the genus Capulavirus in the
family Geminiviridae that was isolated from alfalfa plants showing dwarfism. The
genome was found to be 2,750 nucleotides in length. In pairwise comparisons, this
ALCV isolate shared 83.2% to 92.6% sequence identity with European ALCV isolates.
Sequence comparisons and phylogenetic analysis showed that this isolate combines
features of strains A and B of ALCV. Recombination analysis showed that ALCV-Arg
is a recombinant isolate that was generated by intraspecific recombination
between ALCV strains A and B. The results of this study not only show that
ALCV-Arg is unique because it combines features of strains A and B but also show
that ALCV naturally infects this forage crop on the American continent.
DOI: 10.1007/s00705-017-3673-x
33. Micron. 2018 Feb;105:70-81. doi: 10.1016/j.micron.2017.10.007. Epub 2017 Oct

18.
NaCl - Changes stem morphology, anatomy and phloem structure in Lucerne (Medicago
sativa cv. Gabès): Comparison of upper and lower internodes.
Nja RB(1), Merceron B(2), Faucher M(3), Fleurat-Lessard P(4), Béré E(5).
Author information:
(1)Université de Carthage, Faculté des Sciences de Bizerte, Tunisie et Université
de Limoges, Faculté des Sciences et techniques, 87100 Limoges Cedex, France.
Electronic address: bennjariheb@gmail.com.
(2)Université de Poitiers, Image UP, Service de Microscopie Electronique et
Photonique, Pôle Biologie Santé, 1 rue Georges Bonnet, TSA 51106, Poitiers Cedex
9, France. Electronic address: b.merceron@univ-poitiers.fr.
(3)Université de Poitiers, Campus Sciences, Laboratoire EBI, UMR CNRS 7267,
Equipe SEVE, Bâtiment B31, 3, Rue Jacques Fort, TSA 51106, 86022, Poitiers Cedex
9, France. Electronic address: mireille.faucher@univ-poitiers.fr.
(4)Université de Poitiers, Campus Sciences, Laboratoire EBI, UMR CNRS 7267,
Equipe SEVE, Bâtiment B31, 3, Rue Jacques Fort, TSA 51106, 86022, Poitiers Cedex
9, France. Electronic address: pfleur24@gmail.com.
(5)Université de Poitiers, Campus Sciences, Image UP, Service de Microscopie
Electronique et Photonique, Pôle Biologie Santé, Bâtiment B36 BP 633, 1, Rue
Georges Bonnet, TSA 51106, 86022 Poitiers Cedex 9, France. Electronic address:
ebere@univ-poitiers.fr.
In M. sativa cv. Gabès plants treated with 150mM NaCl, the height of the stem is
decreased and the internode number, length and diameter are reduced. This
depressive effect on growth, but also on photosynthetic activity and water
balance, is accompanied by structural changes. In the upper internodes, NaCl
treatment increases cambium development, so that the vascular ring is initiated
earlier than in controls. In the lower internodes, the number of lignified phloem
fibers is increased by NaCl, and their wall thickness is augmented, compared to
controls; in the phloem complex, the nacreous layer is enlarged, the number of
internal wall ingrowths is increased, but companion cells are damaged. In the
treated lower internodes, few vessels occur in the secondary xylem, which is by
contrast rich in lignified fibers and in wide vessels grouped in the metaxylem
area; protoxylem parenchyma and adjacent pith are also lignified. In addition, in
treated lower internodes, starch grains are less abundant than in controls, and
this variation might be related to the decrease of photosynthesis. When taken
together, qualitative and quantitative results indicate that the saline stress
has a marked morpho-anatomical impact on the M. sativa Gabès stem. In particular,
variations of secondary derivative distribution, increased wall thickening,
lignification of phloem and xylem fibers and damage in the phloem complex are
NaCl-induced responses, and are more expressed in the lower than in the upper
internodes. The reinforcement of the stem lignified vasculature is thus a
positive response to stress, but it has a negative impact on the quality of the
forage.
Copyright © 2017 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.micron.2017.10.007
PMID: 29190502
34. Bull Environ Contam Toxicol. 2018 Feb;100(2):280-285. doi:

10.1007/s00128-017-2211-6. Epub 2017 Nov 29.
Hydrocarbon Degradation and Lead Solubility in a Soil Polluted with Lead and Used
Motor Oil Treated by Composting and Phytoremediation.
Escobar-Alvarado LF(1), Vaca-Mier M(1), López R(1), Rojas-Valencia MN(2).
Author information:
(1)Metropolitan Autonomous University, Azcapotzalco, 02200, Mexico City, Mexico.
(2)Institute of Engineering, National Autonomous University of Mexico, 04510,
Mexico City, Mexico. nrov@pumas.iingen.unam.mx.
Used lubricant oils and metals can be common soil pollutants in abandoned sites.
When soil is contaminated with various hazardous wastes, the efficiency of
biological treatments could be affected. The purpose of this work was to
investigate the effect of combining phytoremediation and composting on the
efficiency of hydrocarbon degradation and lead solubility in a soil contaminated
with 31,823 mg/kg of total petroleum hydrocarbon (TPH) from used motor oil and
8260 mg/kg of lead. Mexican cactus (Opuntia ficus indica) and yard trimmings were
added in the composting process, and lucerne (Medicago sativa) was used in the
phytoremediation process. After a 9 week composting process, only 13% of the
initial TPH concentration was removed. The following 20 week phytoremediation
process removed 48% of TPH. The highest TPH degradation percentage (66%), was
observed in the experiment with phytoremediation only. This work demonstrates
sustainable technologies, such as biological treatments, represent low-cost
options for remediation; however, they are not frequently used because they
require long periods of time for success.
DOI: 10.1007/s00128-017-2211-6
PMID: 29188328
35. Int J Genomics. 2017;2017:2763259. doi: 10.1155/2017/2763259. Epub 2017 Oct 18.
Identification and Characterization of the Diverse Stress-Responsive R2R3-RMYB

Transcription Factor from Hibiscus sabdariffa L.
Mohamed BB(1)(2), Aftab B(1), Sarwar MB(1), Rashid B(1), Ahmad Z(1), Hassan S(1),
Husnain T(1).
Author information:
(1)Center of Excellence in Molecular Biology, University of the Punjab, 87-West
Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan.
(2)National Center for Research (NCR), Ministry of Sciences and
Telecommunications, Khartoum, Sudan.
Various regulatory proteins play a fundamental role to manage the healthy plant
growth under stress conditions. Differential display reverse transcriptase PCR
and random amplification of cDNA ends (RACE) was used to explore the osmotic
stress-responsive transcripts. We identified and characterized the salt
stress-responsive R2R3 type RMYB transcription factor from Hibiscus sabdariffa
which has an open reading frame of 690 bp, encoding 229 long chain amino acids.
In silico analysis confirmed the conserved R2 and R3 domain as well as an NLS-1
localization site. The deduced amino acids of RMYB shared 83, 81, 80, 79, 72, 71,
and 66% homology with Arabidopsis thaliana, Glycine max, Oryza sativa, Zea maize,
Malus domestica, Populus tremula × Populus alba, and Medicago sativa specific MYB
family, respectively. We observed the gene upregulation in stem, leaf, and root
tissue in response to abiotic stress. Furthermore, RMYB gene was cloned into
plant expression vector under CaMV35S promoter and transformed to Gossypium
hirsutum: a local cotton cultivar. Overexpression of RMYB was observed in
transgenic plants under abiotic stresses which further suggests its regulatory
role in response to stressful conditions. The RMYB transcription
factor-overexpressing in transgenic cotton plants may be used as potential agent
for the development of stress tolerant crop cultivars.
DOI: 10.1155/2017/2763259
PMCID: PMC5664376
PMID: 29181384
36. J Agric Food Chem. 2017 Dec 20;65(50):10859-10867. doi:

10.1021/acs.jafc.7b04173.
Epub 2017 Dec 7.
Dietary Alfalfa and Calcium Salts of Long-Chain Fatty Acids Alter Protein
Utilization, Microbial Populations, and Plasma Fatty Acid Profile in Holstein
Freemartin Heifers.
He Y(1), Qiu Q(1), Shao T(1), Niu W(1), Xia C(1), Wang H(1), Li Q(1), Gao Z(1),
Yu Z(1), Su H(1), Cao B(1).
Author information:
(1)State Key Laboratory of Animal Nutrition, College of Animal Science and
Technology, China Agricultural University , 100193 Beijing, China.
This study presented the effects of alfalfa and calcium salts of long-chain fatty
acids (CSFA) on feed intake, apparent digestibility, rumen fermentation,
microbial community, plasma biochemical parameters, and fatty acid profile in
Holstein freemartin heifers. Eight Holstein freemartin heifers were randomly
divided into a 4 × 4 Latin Square experiment with 2 × 2 factorial diets, with or
without alfalfa or CSFA. Dietary supplementation of CSFA significantly increased
the apparent digestibility of dry matter, crude protein, neutral detergent fiber,
organic matter, and significantly reduced N retention (P < 0.05). CSFA increased
the concentration of ammonia nitrogen in the ruminal fluid (P < 0.05), but
alfalfa increased the concentration of valerate and isovalerate (P < 0.05). CSFA
increased the concentration of ammonia nitrogen and the relative population of
Streptococcus bovis in the rumen (P < 0.05) and inhibited the relative population
of Ruminococcus flavefaciens, methanogens, and protozoa (P < 0.05). Alfalfa
instead of Leymus chinensis increased the relative population of Butyrivibrio
fibrisolvens and Ruminobacter amylophilus in the rumen (P < 0.05) and reduced the
relative population of the Ruminococcus albus and Megasphaera elsdenii (P <
0.05). Supplemental CSFA increased the concentration of cholesterol and
low-density lipoprotein cholesterol in the plasma (P < 0.05). And it also altered
the composition of fatty acids in the plasma, which was expressed in reducing
saturated fatty acid (ΣSFA) ratio and C14-C17 fatty acids proportion except C16:0
(P < 0.05) and increasing the proportion of polyunsaturated fatty acid (ΣPUFA)
and unsaturated fatty acid (ΣUFA) (P < 0.05). The results showed that alfalfa and
CSFA had interaction effect on the apparent digestibility of ether extracts,
plasma triglyceride concentration, isobutyrate concentration, and Ruminococcus
albus relative abundance in the rumen. It was concluded that alfalfa substituting
Leymus chinensis did not change the apparent digestibility of nutrients in the
final stage of fattening Holstein freemartin heifers, while CSFA increased the
cholesterol and the proportion of unsaturated fatty acids in plasma. Alfalfa and
CSFA had mutual interaction effect on fat digestion and plasma triglycerides.
DOI: 10.1021/acs.jafc.7b04173
37. Front Plant Sci. 2017 Oct 30;8:1870. doi: 10.3389/fpls.2017.01870. eCollection
2017.
Alfalfa Root Growth Rate Correlates with Progression of Microtubules during

Mitosis and Cytokinesis as Revealed by Environmental Light-Sheet Microscopy.
Vyplelová P(1), Ovečka M(1), Šamaj J(1).
Author information:
(1)Department of Cell Biology, Centre of the Region Haná for Biotechnological and
Agricultural Research, Palacký University Olomouc, Olomouc, Czechia.
Cell division and expansion are two fundamental biological processes supporting
indeterminate root growth and development of plants. Quantitative evaluations of
cell divisions related to root growth analyses have been performed in several
model crop and non-crop plant species, but not in important legume plant Medicago
sativa. Light-sheet fluorescence microscopy (LSFM) is an advanced imaging
technique widely used in animal developmental biology, providing efficient fast
optical sectioning under physiological conditions with considerably reduced
phototoxicity and photobleaching. Long-term 4D imaging of living plants offers
advantages for developmental cell biology not available in other microscopy
approaches. Recently, LSFM was implemented in plant developmental biology
studies, however, it is largely restricted to the model plant Arabidopsis
thaliana. Cellular and subcellular events in crop species and robust plant
samples have not been studied by this method yet. Therefore we performed LSFM
long-term live imaging of growing root tips of transgenic alfalfa plants
expressing the fluorescent molecular marker for the microtubule-binding domain
(GFP-MBD), in order to study dynamic patterns of microtubule arrays during
mitotic cell division. Quantitative evaluations of cell division progress in the
two root tissues (epidermis and cortex) clearly indicate that root growth rate is
correlated with duration of cell division in alfalfa roots. Our results favor
non-invasive environmental LSFM as one of the most suitable methods for
qualitative and quantitative cellular and developmental imaging of living
transgenic legume crops.
DOI: 10.3389/fpls.2017.01870
PMCID: PMC5670501
PMID: 29163595
38. Appl Environ Microbiol. 2018 Jan 17;84(3). pii: e01937-17. doi:
10.1128/AEM.01937-17. Print 2018 Feb 1.
Sinorhizobium meliloti Glutathione Reductase Is Required for both Redox

Homeostasis and Symbiosis.
Tang G(1)(2), Li N(1), Liu Y(1), Yu L(1), Yan J(1), Luo L(3).
Author information:
(1)Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Plant
Science Center, Shanghai University, Shanghai, China.
(2)School of Communication and Information Engineering, Shanghai University,
Shanghai, China.
Science Center, Shanghai University, Shanghai, China liluo@shu.edu.cn.
Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants

in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago
sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione
(GSH) or oxidized glutathione (GSSG), and its content is regulated by two
pathways in S. meliloti The first pathway is the de novo synthesis of glutathione
from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by
γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The
second pathway is the recycling of GSSG via glutathione reductase (GR). However,
whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic
interactions with alfalfa remains unknown. In this study, a plasmid insertion
mutation of the S. melilotigor gene, which encodes GR, was constructed, and the
mutant exhibited delayed alfalfa nodulation, with 75% reduction in
nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of
GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective
growth in rich broth and minimal broth and was more sensitive to the oxidants
H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1,
katA, and katB was induced in the mutant. These findings reveal that the
recycling of glutathione is important for S. meliloti to maintain redox
homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant
glutathione is regulated by its synthetase and reductase in cells. In the
symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is
essential for alfalfa nodulation and nitrogen fixation. In this study, we
observed that the recycling of glutathione from GSSG not only was required for
redox homeostasis and oxidative stress protection in S. meliloti cells but also
contributed to alfalfa nodule development and competition capacity. Our findings
demonstrate that the recycling of glutathione plays a key role in nitrogen
fixation symbiosis.

DOI: 10.1128/AEM.01937-17
PMID: 29150514
39. Plant Mol Biol. 2018 Jan;96(1-2):119-133. doi: 10.1007/s11103-017-0683-8. Epub

2017 Nov 17.
SPL13 regulates shoot branching and flowering time in Medicago sativa.
Gao R(1), Gruber MY(2), Amyot L(1), Hannoufa A(3).
Author information:
(1)Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 4T3,
Canada.
(2)Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2,
Canada.
Canada. Abdelali.Hannoufa@agr.gc.ca.
KEY MESSAGE: Our results show SPL13 plays a crucial role in regulating vegetative
and reproductive development in Medicago sativa L. (alfalfa), and that MYB112 is
targeted and downregulated by SPL13 in alfalfa. We previously showed that
transgenic Medicago sativa (alfalfa) plants overexpressing microRNA156 (miR156)
show a bushy phenotype, reduced internodal length, delayed flowering time, and
enhanced biomass yield. In alfalfa, transcripts of seven SQUAMOSA-PROMOTER
BINDING PROTEIN-LIKE (SPL) transcription factors, including SPL13, are targeted
for cleavage by miR156. Thus, association of each target SPL gene to a trait or
set of traits is essential for developing molecular markers for alfalfa breeding.
In this study, we investigated SPL13 function using SPL13 overexpression and
silenced alfalfa plants. Severe growth retardation, distorted branches and
up-curled leaves were observed in miR156-impervious 35S::SPL13m over-expression
plants. In contrast, more lateral branches and delayed flowering time were
observed in SPL13 silenced plants. SPL13 transcripts were predominantly present
in the plant meristems, indicating that SPL13 is involved in regulating shoot
branch development. Accordingly, the shoot branching-related CAROTENOID CLEAVAGE
DIOXYGENASE 8 gene was found to be significantly downregulated in SPL13 RNAi
silencing plants. A R2R3-MYB gene MYB112 was also identified as being directly
silenced by SPL13 based on Next Generation Sequencing-mediated transcriptome
analysis and chromatin immunoprecipitation assays, suggesting that MYB112 may be
involved in regulating alfalfa vegetative growth.
DOI: 10.1007/s11103-017-0683-8
40. Environ Microbiol. 2017 Dec;19(12):5130-5145. doi: 10.1111/1462-2920.13992.

Epub
2017 Nov 29.
Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti

under free-living and symbiotic oxidative stress.
Tang G(1)(2), Xing S(1), Wang S(1), Yu L(1), Li X(3), Staehelin C(4), Yang M(5),
Luo L(1).
Author information:
Science Center, Shanghai University, Shanghai, 200444, China.
(2)School of Communication & Information Engineering, Shanghai University,
Shanghai, 200444, China.
(3)Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular
Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
(4)State Key Laboratory of Biocontrol and Guangdong Key Laboratory of
Bioresources, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006,
China.
(5)College of Animal Science & Technology, China-Australia Joint-Laboratory for
Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for
Animal Health Inspection & Internet Technology, Zhejiang A&F University,
Zhejiang, Lin'an, 311300, China.
The development of legume nitrogen-fixing nodules is regulated by reactive oxygen

species (ROS) produced by symbionts. Several regulators from Rhizobium are
involved in ROS sensing. In a previous study, we found that Sinorhizobium
meliloti LsrB regulates lipopolysaccharide production and is associated with H2
O2 accumulation in alfalfa (Medicago sativa) nodules. However, its underlying
regulatory mechanism remains unclear. Here, we report that the cysteine residues
in LsrB are required for adaptation to oxidative stress, gene expression, alfalfa
nodulation and nitrogen fixation. Moreover, LsrB directly activated the
transcription of lrp3 and gshA (encoding γ-glutamylcysteine synthetase,
responsible for glutathione synthesis) and this regulation required the cysteine
(Cys) residues in the LsrB substrate-binding domain. The Cys residues could sense
oxidative stress via the formation of intermolecular disulfide bonds, generating
LsrB dimers and LsrB-DNA complexes. Among the Cys residues, C238 is a positive
regulatory site for the induction of downstream genes, whereas C146 and C275 play
negative roles in the process. The lsrB mutants with Cys-to-Ser substitutions
displayed altered phenotypes in respect to their adaptation to oxidative stress,
nodulation and nitrogen fixation-related plant growth. Our findings demonstrate
that S. meliloti LsrB modulates alfalfa nodule development by directly regulating
downstream gene expression via a post-translational strategy.
© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.
DOI: 10.1111/1462-2920.13992
PMID: 29124841
41. FEMS Microbiol Lett. 2017 Dec 15;364(23). doi: 10.1093/femsle/fnx233.
Overproduction of Sinorhizobium meliloti ArgC (N-acetyl-gamma-glutamyl phosphate

reductase) promotes growth delay and inefficient nodules.
Vargas-Lagunas C, Mora Y, Díaz R, Martínez-Batallar G, Girard L, Encarnación S,

Peralta H, Mora J.
argC encodes N-acetyl-gamma-glutamyl phosphate reductase, the enzyme that

catalyzes the high-energy-consuming third step in the arginine synthesis pathway.
A comparative analysis revealed two translation start sites in argC from
Sinorhizobium meliloti. To determine whether both protein versions are
synthesized in the organism and their functional role, we obtained genetic
constructs with one (1S) or two (2S) start sites, with promoters of low (pspeB)
or high (plac) transcriptional rate. The constructs were transferred to the S.
meliloti 1021 derivative argC mutant strain. Both protein versions were found in
the free-living proteomes, but only ArgC 1S showed post-translational
modification. Expression levels from argC 1S were five times higher than those of
2S, when transcribed by plac, and in concordance, its protein activity was 3-fold
greater. The overexpression of both versions under plac delayed cellular growth.
Inoculation of Medicago sativa plants with the S. meliloti strain harboring the
argC 1S under plac induced nodulation but not nitrogen fixation. However, the
strain with the argC 2S under the same promoter had a positive phenotype.
Overproduction of ArgC protein for the synthesis of arginine induced
physiological and symbiotic effects.
© FEMS 2017. All rights reserved. For permissions, please e-mail:

DOI: 10.1093/femsle/fnx233
42. Genet Mol Biol. 2017 Oct-Dec;40(4):824-833. doi: 10.1590/1678-4685-GMB-2017-

0053.
Epub 2017 Nov 6.
Transcriptome sequencing analysis of alfalfa reveals CBF genes potentially

playing important roles in response to freezing stress.
Shu Y(1), Li W(1), Zhao J(1), Zhang S(1), Xu H(1), Liu Y(1), Guo C(1).
Author information:
(1)College of Life Science and Technology, Harbin Normal University, Harbin
Heilongjiang, China.
Alfalfa (Medicago sativa L.) is an important perennial forage, with high

nutritional value, which is widely grown in the world. Because of low freezing
tolerance, its distribution and production are threatened and limited by winter
weather. To understand the complex regulation mechanisms of freezing tolerance in
alfalfa, we performed transcriptome sequencing analysis under cold (4 °C) and
freezing (-8 °C) stresses. More than 66 million reads were generated, and we
identified 5767 transcripts differentially expressed in response to cold and/or
freezing stresses. These results showed that these genes were mainly classified
as response to stress, transcription regulation, hormone signaling pathway,
antioxidant, nodule morphogenesis, etc., implying their important roles in
response to cold and freezing stresses. Furthermore, nine CBF transcripts
differentially expressed were homologous to CBF genes of Mt-FTQTL6 site,
conferring freezing tolerance in M. truncatula, which indicated that a genetic
mechanism controlling freezing tolerance was conservative between M. truncatula
and M. sativa. In summary, this transcriptome dataset highlighted the gene
regulation response to cold and/or freezing stresses in alfalfa, which provides a
valuable resource for future identification and functional analysis of candidate
genes in determining freezing tolerance.
DOI: 10.1590/1678-4685-GMB-2017-0053
PMCID: PMC5738619
PMID: 29111565
43. Trop Anim Health Prod. 2018 Mar;50(3):531-536. doi: 10.1007/s11250-017-1464-8.

Epub 2017 Nov 7.
Herbage intake of dairy cows in mixed sequential grazing with breeding ewes as
followers.
Jiménez-Rosales JD(1), Améndola-Massiotti RD(2), Burgueño-Ferreira JA(3),

Ramírez-Valverde R(1), Topete-Pelayo P(4), Huerta-Bravo M(1).
Author information:
(1)Programa de Posgrado en Producción Animal, Universidad Autónoma Chapingo,
Carretera México - Texcoco km 38.5, CP 56230, Chapingo, Estado de México, Mexico.
(2)Programa de Posgrado en Producción Animal, Universidad Autónoma Chapingo,
Carretera México - Texcoco km 38.5, CP 56230, Chapingo, Estado de México, Mexico.
r_amendola@yahoo.com.
(3)Unidad de Biometría y Estadísticas, Laboratorio de Informática Aplicada a los
Cultivos, Centro Internacional de Mejoramiento de Maíz y Trigo, Apdo. Postal
6-641, México, D.F, Mexico.
(4)Departamento de Zootecnia, Universidad Autónoma Chapingo, Carretera México -
Texcoco km 38.5, CP 56230, Chapingo, Estado de México, Mexico.
This study aimed to evaluate the hypothesis that mixed sequential grazing of
dairy cows and breeding ewes is beneficial. During the seasons of spring-summer
2013 and autumn-winter 2013-2014, 12 (spring-summer) and 16 (autumn-winter)
Holstein Friesian cows and 24 gestating (spring-summer) and lactating
(autumn-winter) Pelibuey ewes grazed on six (spring-summer) and nine
(autumn-winter) paddocks of alfalfa and orchard grass mixed pastures. The
treatments "single species cow grazing" (CowG) and "mixed sequential grazing with
ewes as followers of cows" (MixG) were evaluated, under a completely randomized
design with two replicates per paddock. Herbage mass on offer (HO) and residual
herbage mass (RH) were estimated by cutting samples. The estimate of herbage
intake (HI) of cows was based on the use of internal and external markers; the
apparent HI of ewes was calculated as the difference between HO (RH of cows) and
RH. Even though HO was higher in CowG, the HI of cows was higher in MixG during
spring-summer and similar in both treatments during autumn-winter, implying that
in MixG the effects on the cows HI of higher alfalfa proportion and herbage
accumulation rate evolving from lower residual herbage mass in the previous cycle
counteracted that of a higher HO in CowG. The HI of ewes was sufficient to enable
satisfactory performance as breeding ewes. Thus, the benefits of mixed sequential
grazing arose from higher herbage accumulation, positive changes in botanical
composition, and the achievement of sheep production without negative effects on
the herbage intake of cows.
DOI: 10.1007/s11250-017-1464-8
PMID: 29110238
44. J Anim Sci. 2017 Oct;95(10):4421-4429. doi: 10.2527/jas2017.1856.
Grazing management of mixed alfalfa bermudagrass pastures.
Beck PA, Sims MB, Kegley EB, Hubbell D, Hess T, Galyen W, Butler TJ, Rogers JK,
Jennings J.
Rotational grazing management is commonly recommended to improve persistence of

legumes interseeded into bermudagrass, but impacts on animal performance are
poorly understood. Steers (n = 365, BW = 249 ± 22.9 kg) grazed mixed alfalfa
(Medicago sativa L.)/bermudagrass (Cynodon dactylon [L.] Pers.) pastures (n = 10;
1.6-ha) with either continuous grazing (CONT) or rotational grazing (ROT)
management over 3 yr. Initial stocking rate was set at 6.25 steers/ha. When
forage allowance of CONT became limiting in mid-July each year, 2 randomly
selected steers were removed from all pastures to maintain equal stocking rates
in CONT and ROT. Rotational grazing pastures were divided into 8 paddocks with
3-d grazing and 21-d rest between grazing events. Initial, final, and interim
(28-d) BW were collected following a 16-h removal from feed and water. Data were
analyzed as a repeated measures experiment with completely random design using
the mixed procedure of SAS (SAS Inst. Inc., Cary, NC) using pasture within
treatment by year in the random statement. Across years, steer BW did not differ
(P ≥ 0.47) at the midpoint or end of the grazing season. Daily BW gains during
the early summer period tended (P = 0.10) to be 0.11 kg greater for CONT than ROT
in yr 1, but did not differ (P ≥ 0.24) in yr 2 and 3. In the late summer, ADG was
0.15 kg less (P < 0.01) for CONT than ROT in yr 1, were not different (P = 0.23)
in yr 2, and were 0.29 kg less (P < 0.01) for CONT than ROT in yr 3. Over the
entire experimental period, ADG did not differ (P ≥ 0.67) in yr 1 or 2, but
tended (P = 0.09) to be 0.10 kg greater for ROT than CONT in yr 3. Forage mass of
ROT was greater (P < 0.01) than CONT throughout the grazing season. Because
stocking rates were maintained at the same level, ROT pastures provided greater
(P < 0.01) forage allowance during the grazing season than CONT explaining
differences in ADG between treatments. Alfalfa stand percentage did not differ (P
= 0.79) at the beginning of the experiment, but was less (P ≤ 0.03) for CONT than
ROT at all other sampling dates. These data indicate that at equal stocking
rates, ROT can maintain greater alfalfa persistence, forage nutritive quality,
and forage allowance compared with continuous grazing and provided increased
animal performance during the late summer when the alfalfa stand was reduced in
CONT pastures.
DOI: 10.2527/jas2017.1856
PMID: 29108051
45. Appl Soil Ecol. 2017 Nov;120:35-43. doi: 10.1016/j.apsoil.2017.06.030.
Is there sufficient Ensifer and Rhizobium species diversity in UK farmland soils

to support red clover (Trifolium pratense), white clover (T. repens), lucerne
(Medicago sativa) and black medic (M. lupulina)?
Roberts R(1), Jackson RW(2), Mauchline TH(3), Hirsch PR(3), Shaw LJ(4), Döring
TF(5), Jones HE(1).
Author information:
(1)School of Agriculture, Policy and Development, University of Reading, RG6 6AR,
UK.
(2)School of Biological Sciences, Knight Building, University of Reading, RG6
6AJ, UK.
(3)Rothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, UK.
(4)Soil Research Centre, School of Archaeology, Geography and Environmental
Science, University of Reading, RG6 6AB, UK.
(5)Department of Agronomy and Crop Science, Faculty of Life Sciences, Humboldt
Universität zu Berlin, Albrecht-Thaer-Weg 5, 14195 Berlin, Germany.
Rhizobia play important roles in agriculture owing to their ability to fix

nitrogen through a symbiosis with legumes. The specificity of rhizobia-legume
associations means that underused legume species may depend on seed inoculation
with their rhizobial partners. For black medic (Medicago lupulina) and lucerne
(Medicago sativa) little is known about the natural prevalence of their rhizobial
partner Ensifer meliloti in UK soils, so that the need for inoculating them is
unclear. We analysed the site-dependence of rhizobial seed inoculation effects on
the subsequent ability of rhizobial communities to form symbioses with four
legume species (Medicago lupulina, M. sativa, Trifolium repens and T. pratense).
At ten organic farms across the UK, a species-diverse legume based mixture (LBM)
which included these four species was grown. The LBM seed was inoculated with a
mix of commercial inocula specific for clover and lucerne. At each site, soil
from the LBM treatment was compared to the soil sampled prior to the sowing of
the LBM (the control). From each site and each of the two treatments, a
suspension of soils was applied to seedlings of the four legume species and grown
in axenic conditions for six weeks. Root nodules were counted and their rhizobia
isolated. PCR and sequencing of a fragment of the gyrB gene from rhizobial
isolates allowed identification of strains. The number of nodules on each of the
four legume species was significantly increased when inoculated with soil from
the LBM treatment compared to the control. Both the proportion of plants forming
nodules and the number of nodules formed varied significantly by site, with sites
significantly affecting the Medicago species but not the Trifolium species. These
differences in nodulation were broadly reflected in plant biomass where site and
treatment interacted; at some sites there was a significant advantage from
inoculation with the commercial inoculum but not at others. In particular, this
study has demonstrated the commercial merit of inoculation of lucerne with
compatible rhizobia.
DOI: 10.1016/j.apsoil.2017.06.030
PMCID: PMC5637928
PMID: 29104370
46. Proteome Sci. 2017 Oct 30;15:19. doi: 10.1186/s12953-017-0127-z. eCollection

2017.
Proteomic analysis of early salt stress responsive proteins in alfalfa roots and
shoots.
Xiong J(#)(1), Sun Y(#)(2), Yang Q(3), Tian H(1), Zhang H(1), Liu Y(1), Chen
M(1).
Author information:
(1)Hubei Key Laboratory of Animal Embryo and Molecular Breeding, Institute of
Animal and Veterinary Science, Hubei Academy of Agricultural Science, Yaoyuan 1,
Hongshan, Wuhan, Hubei 430017 China.
(2)Institute of Grassland Science, China Agricultural University, 2 West Road,
Yuan Ming Yuan, Beijing, 100193 China.
(3)Institute of Animal Science, Chinese Academy of Agricultural Science, West
Road 2, Yuan Ming Yuan, Beijing, 100193 China.
(#)Contributed equally
Background: Alfalfa (Medicago sativa) is the most extensively cultivated forage

legume in the world, and salinity stress is the most problematic environmental
factors limiting alfalfa production. To evaluate alfalfa tissue variations in
response to salt stress, comparative physiological and proteomic analyses were
made of salt responses in the roots and shoots of the alfalfa.
Method: A two-dimensional gel electrophoresis (2-DE)-based proteomic technique
was employed to identify the differentially abundant proteins (DAPs) from
salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No
1 cultivar, which was subjected to a range of salt stress concentrations for
9 days. In parallel, REL, MAD and H2O2 contents, and the activities of
antioxidant enzymes of shoots and roots were determinand.
Result: Twenty-seven spots in the shoots and 36 spots in the roots that exhibited
showed significant abundance variations were identified by MALDI-TOF-TOF MS.
These DAPs are mainly involved in the biological processes of photosynthesis,
stress and defense, carbohydrate and energy metabolism, second metabolism,
protein metabolism, transcriptional regulation, cell wall and cytoskeleton
metabolism, ion transpor, signal transduction. In parallel, physiological data
were correlated well with our proteomic results. It is worth emphasizing that
some novel salt-responsive proteins were identified, such as CP12,
pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin
2'-O-methyltransferase. qRT-PCR was used to study the gene expression levels of
the four above-mentioned proteins; four patterns are consistent with those of
induced protein.
Conclusion: The primary mechanisms underlying the ability of alfalfa seedlings to
tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary
metabolism, and ion transport. And it also suggests that the different tissues
responded to salt-stress in different ways.
DOI: 10.1186/s12953-017-0127-z
PMCID: PMC5663070
PMID: 29093645
47. J Bacteriol. 2017 Dec 20;200(2). pii: e00436-17. doi: 10.1128/JB.00436-17.

Print
2018 Jan 15.
Characterization of Mutations That Affect the Nonoxidative Pentose Phosphate

Pathway in Sinorhizobium meliloti.
Hawkins JP(1), Ordonez PA(1), Oresnik IJ(2).
Author information:
(1)Department of Microbiology, University of Manitoba, Winnipeg, Manitoba,
Canada.
(2)Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
ivan.oresnik@umanitoba.ca.
Sinorhizobium meliloti is a Gram-negative alphaproteobacterium that can enter

into a symbiotic relationship with Medicago sativa and Medicago truncatula
Previous work determined that a mutation in the tkt2 gene, which encodes a
putative transketolase, could prevent medium acidification associated with a
mutant strain unable to metabolize galactose. Since the pentose phosphate pathway
in S. meliloti is not well studied, strains carrying mutations in either tkt2 and
tal, which encodes a putative transaldolase, were characterized. Carbon
metabolism phenotypes revealed that both mutants were impaired in growth on
erythritol and ribose. This phenotype was more pronounced for the tkt2 mutant
strain, which also displayed auxotrophy for aromatic amino acids. Changes in
pentose phosphate pathway metabolite concentrations were also consistent with a
mutation in either tkt2 or tal The concentrations of metabolites in central
carbon metabolism were also found to shift dramatically in strains carrying a
tkt2 mutation. While the concentrations of proteins involved in central carbon
metabolism did not change significantly under any conditions, the levels of those
associated with iron acquisition increased in the wild-type strain with
erythritol induction. These proteins were not detected in either mutant,
resulting in less observable rhizobactin production in the tkt2 mutant. While
both mutants were impaired in succinoglycan synthesis, only the tkt2 mutant
strain was unable to establish symbiosis with alfalfa. These results suggest that
tkt2 and tal play central roles in regulating the carbon flow necessary for
carbon metabolism and the establishment of symbiosis.IMPORTANCESinorhizobium
meliloti is a model organism for the study of plant-microbe interactions and
metabolism, especially because it effects nitrogen fixation. The ability to
derive the energy necessary for nitrogen fixation is dependent on an organism's
ability to metabolize carbon efficiently. The pentose phosphate pathway is
central in the interconversion of hexoses and pentoses. This study characterizes
the key enzymes of the nonoxidative branch of the pentose phosphate pathway by
using defined genetic mutations and shows the effects the mutations have on the
metabolite profile and on physiological processes such as the biosynthesis of
exopolysaccharide, as well as the ability to regulate iron acquisition.
DOI: 10.1128/JB.00436-17
48. Sci Total Environ. 2018 Mar 15;618:753-764. doi:

10.1016/j.scitotenv.2017.08.143.
Epub 2017 Oct 18.
Pilot scale aided-phytoremediation of a co-contaminated soil.
Marchand C(1), Mench M(2), Jani Y(3), Kaczala F(3), Notini P(3), Hijri M(4),
Hogland W(3).
Author information:
(1)Institut de Recherche en Biologie Végétale (IRBV), Université de Montréal,
4101 Rue Sherbrooke Est, Montréal, Québec H1X 2B2, Canada; Department of Biology
& Environmental Sciences, Linnaeus University, Landgången 3, Kalmar, SE -391 82,
Sweden. Electronic address: charlotte.marchand@lnu.se.
(2)BIOGECO, INRA, Univ. Bordeaux, 33615 Pessac, France.
(3)Department of Biology & Environmental Sciences, Linnaeus University,
Landgången 3, Kalmar, SE -391 82, Sweden.
(4)Institut de Recherche en Biologie Végétale (IRBV), Université de Montréal,
4101 Rue Sherbrooke Est, Montréal, Québec H1X 2B2, Canada.
A pilot scale experiment was conducted to investigate the aided-phytoextraction

of metals and the aided-phytodegradation of petroleum hydrocarbons (PHC) in a
co-contaminated soil. First, this soil was amended with compost (10% w/w) and
assembled into piles (Unp-10%C). Then, a phyto-cap of Medicago sativa L. either
in monoculture (MS-10%C) or co-cropped with Helianthus annuus L. as companion
planting (MSHA-10%C) was sown on the topsoil. Physico-chemical parameters and
contaminants in the soil and its leachates were measured at the beginning and the
end of the first growth season (after five months). In parallel, residual soil
ecotoxicity was assessed using the plant species Lepidium sativum L. and the
earthworm Eisenia fetida Savigny, 1826, while the leachate ecotoxicity was
assessed using Lemna minor L. After 5months, PH C10-C40, PAH-L, PAH-M PAH-H, Pb
and Cu concentrations in the MS-10%C soil were significantly reduced as compared
to the Unp-10%C soil. Metal uptake by alfalfa was low but their translocation to
shoots was high for Mn, Cr, Co and Zn (transfer factor (TF) >1), except for Cu
and Pb. Alfalfa in monoculture reduced electrical conductivity, total organic C
and Cu concentration in the leachate while pH and dissolved oxygen increased.
Alfalfa co-planting with sunflower did not affect the extraction of inorganic
contaminants from the soil, the PAH (M and H) degradation and was less efficient
for PH C10-C40 and PAH-L as compared to alfalfa monoculture. The co-planting
reduced shoot and root Pb concentrations. The residual soil ecotoxicity after
5months showed a positive effect of co-planting on L. sativum shoot dry weight
(DW) yield. However, high contaminant concentrations in soil and leachate still
inhibited the L. sativum root DW yield, earthworm development, and L. minor
growth rate.
DOI: 10.1016/j.scitotenv.2017.08.143
PMID: 29054647
49. Int J Phytoremediation. 2017 Oct 20:0. doi: 10.1080/15226514.2017.1365339.

[Epub
ahead of print]
Lead phytoextraction from printed circuit computer boards by Lolium perenne L.
and Medicago sativa L.
Díaz Martínez ME(1), Argumedo-Delira R(1)(2), Sánchez-Viveros G(1), Alarcón A(3),

Trejo-Téllez LI(3).
Author information:
(1)a Posgrado en Ciencias Agropecuarias, Facultad de Ciencias Agrícolas,
Universidad Veracruzana. Circuito Gonzalo Aguirre Beltrán s/n , 91000 , Xalapa ,
Veracruz , México.
(2)b Unidad de Servicios de Apoyo en Resolución Analítica (SARA), Universidad
Veracruzana. Luis Castelazo Ayala s/n, Col. Industrial Animas , 91190 , Xalapa ,
Veracruz , México.
(3)c Posgrado de Edafología. Colegio de Postgraduados. Carretera México-Texcoco
km 36.5. Montecillo 56230 , Estado de México.
This work assessed the ability of Lolium perenne and Medicago sativa for
extracting lead (Pb) from particulate printed circuit computer boards (PCB) mixed
in sand with the following concentrations: 0.5, 1.0 and 1.5 g of PCB, and
including a control treatment without PCB. The PCB were obtained from computers,
and grinded in two particle sizes: 0.0594 mm (PCB1) and 0.0706 mm (PCB2). The PCB
particle sizes at their corresponding concentrations were applied to L. perenne
and M. sativa by using three experimental assays. In assay II, PCB2 affected the
biomass production for both plants. For assay III, the PCB1 increased the biomass
of M. sativa (236.5%) and L. perenne (142.2%) when applying either 0.5 or 1.0 g,
respectively. In regards to phytoextraction, assay I showed the highest
Pb-extraction by roots of L. perenne (4.7%) when exposed to 1.5 g of PCB1. At
assay I, L. perenne showed a Pb-bioconcentration factor higher than 1.0 when
growing at 0.5 g of PCB1, and when HNO3 was used as digestion solution; moreover,
in assay III both plants showed a Pb-translocation factor higher than 1.0.
Therefore, Lolium perenne and Medicago sativa are able to recover Pb from
electronic wastes (PCB).
DOI: 10.1080/15226514.2017.1365339
PMID: 29053342
50. Plant Cell Environ. 2017 Oct 19. doi: 10.1111/pce.13093. [Epub ahead of print]
Metabolic flux towards the (iso)flavonoid pathway in lignin modified alfalfa

lines induces resistance against Fusarium oxysporum f. sp. medicaginis.
Gill US(1), Uppalapati SR(1), Gallego-Giraldo L(1), Ishiga Y(1), Dixon RA(1),
Mysore KS(1).
Author information:
(1)Noble Research Institute, LLC, Ardmore, OK, 73401, USA.
Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with

increased availability of cell wall polysaccharides in plant cells. We tested
transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase
(CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium
wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant
to this disease. Transcriptomic and metabolomic analyses indicated that the
improved disease resistance against Fusarium wilt is due to increased
accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some
(iso)flavonoids and their pathway intermediate compounds showed strong
accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp.
medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and
7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp.
medicaginis. These results suggested that the increased accumulation and/or
shift/spillover of flux towards the (iso)flavonoid pathway in CCoAOMT
downregulated plants is associated with induced disease resistance.
DOI: 10.1111/pce.13093
PMID: 29047109
51. Sci Total Environ. 2018 Mar 15;618:1314-1323. doi:

10.1016/j.scitotenv.2017.09.252. Epub 2017 Oct 16.
Influence of different revegetation choices on plant community and soil

development nine years after initial planting on a reclaimed coal gob pile in the
Shanxi mining area, China.
Li S(1), Liber K(2).
Author information:
(1)Institute of Loess Plateau, Shanxi University, Taiyuan 030006, PR China.
(2)Institute of Loess Plateau, Shanxi University, Taiyuan 030006, PR China;
Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada.
Electronic address: karsten.liber@usask.ca.
In order to identify suitable pioneer plant species for future revegetation of

coal gob piles, a field survey was conducted to assess the success of different
revegetation species and their influence on soil development nine years after
initial planting on a reclaimed coal gob pile in the Yangquan mining area of
Shanxi province, China. Data were analyzed using a quantitative classification
method (TWINSPAN), ordination techniques (DCA and DCCA), Principal Component
Analysis (PCA) and a geo-accumulation index (Igeo). The results revealed that the
low levels of soil moisture and available N were the major limiting factors
affecting plant community development on the coal gob pile, not metal toxicity.
The plant communities that developed had significantly improved the topsoil
(0-10cm) quality on the reclaimed coal gob pile nine years after initial planting
(p<0.05), but the degree of improvement varied greatly with different planted
species. Revegetation types comprised of the planted leguminous species, Medicago
sativa and Amorpha fruticosa, and the non-leguminous species, Populus tomentosa
and Salix babylonica had the best ecological restoration effects on the reclaimed
coal gob pile. Revegetation using these species is therefore recommended for
future reclamation of abandoned coal gob piles in this region.
DOI: 10.1016/j.scitotenv.2017.09.252
PMID: 29046231
52. Plant Cell Environ. 2017 Oct 18. doi: 10.1111/pce.13090. [Epub ahead of print]
Molecular improvement of alfalfa for enhanced productivity and adaptability in a

changing environment.
Singer SD(1), Hannoufa A(2), Acharya S(1).
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre,
Lethbridge, Alberta, T1J 4B1, Canada.
(2)Agriculture and Agri-Food Canada, London Research and Development Centre,
London, Ontario, N5V 4T3, Canada.
Due to an expanding world population and increased buying power, the demand for
ruminant products such as meat and milk is expected to grow substantially in
coming years, and high levels of forage crop production will therefore be a
necessity. Unfortunately, urbanization of agricultural land, intensive
agricultural practices, and climate change are all predicted to limit crop
production in the future, which means that the development of forage cultivars
with improved productivity and adaptability will be essential. Because alfalfa
(Medicago sativa L.) is one of the most widely cultivated perennial forage crops,
it has been the target of much research in this field. In this review, we discuss
progress that has been made towards the improvement of productivity, abiotic
stress tolerance, and nutrient-use efficiency, as well as disease and pest
resistance, in alfalfa using biotechnological techniques. Furthermore, we
consider possible future priorities and avenues for attaining further
enhancements in this crop as a means of contributing to the realization of food
security in a changing environment.
DOI: 10.1111/pce.13090
PMID: 29044610
53. J Anim Sci. 2017 Sep;95(9):3905-3913. doi: 10.2527/jas2017.1709.
Sheep from low-methane-yield selection lines created on alfalfa pellets also have
lower methane yield under pastoral farming conditions.
Jonker A, Hickey S, Pinares-Patiño C, McEwan J, Olinga S, Díaz A, Molano G,

MacLean S, Sandoval E, Harland R, Birch D, Bryson B, Knowler K, Rowe S.
Selection lines of sheep with low and high CH yield (g/kg DMI; CH/DMI) are being
developed on the basis of feeding pelleted alfalfa hay at 2.0 times maintenance
ME requirements in respiration chambers, but their divergence under predominant
grazing conditions, as in New Zealand, is not known. The objectives of this study
were to determine CH emissions and rumen fermentation characteristics in sheep
from low and high CH/DMI selection lines while grazing pasture. Two grazing
experiments were conducted with 42 selection line ewes in March 2013 (Exp. 1) and
98 selection line progeny ewe hoggets in October/November 2014 (Exp. 2), with CH
emissions estimated by the SF tracer technique and DMI estimated by titanium
oxide in combination with natural long-chain -alkanes. Total daily CH production
(g/d) was similar between high and low CH/DMI selection line sheep in Exp. 1 and
lower for low CH/DMI progeny compared with high CH/DMI progeny in Exp. 2 ( <
0.05). The CH/DMI tended to be 20% lower for low CH/DMI line sheep compared with
high CH/DMI selection line sheep in Exp. 1 ( < 0.10) and was 15% lower for the
low CH/DMI line in Exp. 2 ( < 0.01). Total VFA concentration and concentrations
(m) of acetate, butyrate, and isobutyrate plus isovalerate were lower ( < 0.05)
for low CH/DMI line sheep compared with high CH/DMI selection line sheep in both
experiments. The current study indicates that differences in CH/DMI and VFA
concentrations in selection line sheep, previously established on alfalfa
pellets, are also present to a similar magnitude when grazing pasture.
DOI: 10.2527/jas2017.1709
54. Gene. 2018 Jan 5;638:7-12. doi: 10.1016/j.gene.2017.09.069. Epub 2017 Sep 30.
Identification of novel RNA viruses in alfalfa (Medicago sativa): an

Alphapartitivirus, a Deltapartitivirus, and a Marafivirus.
Kim H(1), Park D(1), Hahn Y(2).
Author information:
(1)Department of Life Science, Research Center for Biomolecules and Biosystems,
Chung-Ang University, Seoul 06974, South Korea.
(2)Department of Life Science, Research Center for Biomolecules and Biosystems,
Chung-Ang University, Seoul 06974, South Korea. Electronic address:
hahny@cau.ac.kr.
Genomic RNA molecules of plant RNA viruses are often co-isolated with the host
RNAs, and their sequences can be detected in plant transcriptome datasets. Here,
an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA
viruses were identified, which were named Medicago sativa alphapartitivirus 1
(MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa
marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and
MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively,
with their closest virus species. Sequence similarity and phylogenetic analyses
indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong
to the genus Alphapartitivirus of the family Partitiviridae, the genus
Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the
family Tymoviridae, respectively. The bioinformatics procedure applied in this
study may facilitate the identification of novel RNA viruses from plant
transcriptome data.
DOI: 10.1016/j.gene.2017.09.069
55. Plant Sci. 2017 Nov;264:122-128. doi: 10.1016/j.plantsci.2017.09.003. Epub 2017

Sep 12.
Effect of photoperiod prior to cold acclimation on freezing tolerance and

carbohydrate metabolism in alfalfa (Medicago sativa L.).
Bertrand A(1), Bipfubusa M(2), Claessens A(2), Rocher S(2), Castonguay Y(2).
Author information:
(1)Quebec Research and Development Centre, Agriculture and Agri-Food Canada,
Québec, QC, G1V 2J3, Canada. Electronic address: Annick.bertrand@agr.gc.ca.
(2)Quebec Research and Development Centre, Agriculture and Agri-Food Canada,
Québec, QC, G1V 2J3, Canada.
Cold acclimation proceeds sequentially in response to decreases in photoperiod

and temperature. This study aimed at assessing the impact of photoperiod prior to
cold acclimation on freezing tolerance and related biochemical and molecular
responses in two alfalfa cultivars. The fall dormant cultivar Evolution and
semi-dormant cultivar 6010 were grown in growth chambers under different
photoperiods (8, 10, 12, 14 or 16h) prior to cold acclimation. Freezing tolerance
was evaluated as well as carbohydrate concentrations, levels of transcripts
encoding enzymes of carbohydrate metabolism as well as a K-3dehydrin, before and
after cold acclimation. The fall dormant cultivar Evolution had a better freezing
tolerance than the semi-dormant cultivar 6010. The effect of photoperiod prior to
cold acclimation on the level of freezing tolerance differed between the two
cultivars: an 8h-photoperiod induced the highest level of freezing tolerance in
Evolution and the lowest in 6010. In Evolution, the 8h-induced superior freezing
tolerance was associated with higher concentration of raffinose-family
oligosaccharides (RFO). The transcript levels of sucrose synthase (SuSy)
decreased whereas those of sucrose phosphatase synthase (SPS) and galactinol
synthase (GaS) increased in response to cold acclimation in both cultivars. Our
results indicate that RFO metabolism could be involved in short
photoperiod-induced freezing tolerance in dormant alfalfa cultivars.
Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.
DOI: 10.1016/j.plantsci.2017.09.003
PMID: 28969792
56. Ecotoxicol Environ Saf. 2018 Jan;147:861-871. doi:

10.1016/j.ecoenv.2017.09.054.
Epub 2017 Oct 10.
Methane alleviates alfalfa cadmium toxicity via decreasing cadmium accumulation

and reestablishing glutathione homeostasis.
Gu Q(1), Chen Z(1), Cui W(1), Zhang Y(1), Hu H(2), Yu X(1), Wang Q(1), Shen W(3).
Author information:
Agricultural University, Nanjing 210095, China.
(2)Institute of Agricultural Products Processing, Jiangsu Academy of Agricultural
Sciences, Nanjing 210014, China.
Agricultural University, Nanjing 210095, China. Electronic address:
wbshenh@njau.edu.cn.
Although methane (CH4) generation triggered by some environmental stimuli,

displays the protective response against oxidative stress in plants, whether and
how CH4 regulates plant tolerance against cadmium stress is largely unknown.
Here, we discovered that cadmium (Cd) stimulated the production of CH4 in alfalfa
root tissues. The pretreatment with exogenous CH4 could alleviate seedling growth
inhibition. Less amounts of Cd accumulation was also observed. Consistently, in
comparison with Cd stress alone, miR159 transcript was down-regulated by CH4, and
expression levels of its target gene ABC transporter was increased. By contrast,
miR167 transcript was up-regulated, showing a relatively negative correlation
with its target gene Nramp6. Meanwhile, Cd-triggered redox imbalance was improved
by CH4, evidenced by the reduced lipid peroxidation and hydrogen peroxide
accumulation, as well as the induction of representative antioxidant genes.
Further results showed that Cd-triggered decrease of the ratio of
reduced/oxidized (homo)glutathione was rescued by CH4. Additionally,
CH4-triggered alleviation of seedling growth was sensitive to a selective
inhibitor of glutathione biosynthesis. Overall, above results revealed that
CH4-alleviated Cd accumulation at least partially, required the modulation of
heavy metal transporters via miR159 and miR167. Finally, the role of glutathione
homeostasis elicited by CH4 was preliminarily suggested.
Copyright © 2017 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ecoenv.2017.09.054
PMID: 28968939
57. Chemosphere. 2017 Dec;189:517-524. doi: 10.1016/j.chemosphere.2017.09.091. Epub
2017 Sep 19.
Rhizoremediation of a dioxin-like PCB polluted soil by alfalfa: Dynamic

characterization at temporal and spatial scale.
Tu C(1), Ma L(2), Guo P(1), Song F(1), Teng Y(2), Zhang H(1), Luo Y(3).
Author information:
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
264003, China.
Soil Science, Chinese Academy of Sciences, Nanjing 210008, China.
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
264003, China; Key Laboratory of Soil Environment and Pollution Remediation,
Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China.
Electronic address: ymluo@yic.ac.cn.
This study investigates the temporal and spacial dissipation dynamics of a

dioxin-like polychlorinated biphenyl (PCB 77) in the rhizosphere of alfalfa. A
three-chamber rhizobox was designed to compare the PCB 77 dissipation efficiency
in the rhizosphere, near-rhizosphere, and far-rhizosphere zones.
Culture-independent techniques, including quantitative PCR (qPCR), Biolog-ECO
plate, and denatured gradient gel electrophoresis (DGGE) were employed to
investigate the variation of bacterial quantity, metabolic diversity and
community structure in the alfalfa-rhizobium symbiosis rhizosphere at different
rhizoremediation stages. PCB dissipation rates in different rhizosphere zones
were in the order: rhizosphere (90.9%) > near-rhizosphere
(80.5%) > far-rhizosphere (31.7%). The number of the bacterial 16S rRNA gene
copies in the rhizosphere zone in the polluted treatment reached the highest
value of all the treatments. Microbial metabolic diversity, as indicated by
average well color development (AWCD) in both rhizosphere and near-rhizosphere
zones, had recovered from the PCB 77 pollution. The soil bacterial community
diversity improved greatly in the rhizosphere of alfalfa, with some new species
appeared in the rhizosphere and near-rhizosphere zones. In conclusion, the
dissipation of PCB 77, the quantity of total soil bacteria, soil microbial
metabolic diversity, and soil microbial community structure were significantly
improved in rhizosphere and near-rhizosphere zones of alfalfa.
DOI: 10.1016/j.chemosphere.2017.09.091
58. Int J Phytoremediation. 2017 Sep 26:0. doi: 10.1080/15226514.2017.1374342.

[Epub
ahead of print]
Trace element transfer from two contaminated soil series to Medicago sativa and
one of its herbivores, Spodoptera exigua.
Myriam G(1), Lilian M(1)(2), Marie F(3), Michel M(1), Bastien C(1).
Author information:
(1)a BIOGECO, INRA, Univ. Bordeaux , Cestas , France.
(2)b Department of Biodiversity Conservation and Ecosystem Restoration , Pyrenean
Institute of Ecology (IPE), Spanish National Research Council (CSIC) , Jaca
(Huesca) , Spain.
(3)c DGIMI , INRA, Université Montpellier , Montpellier , France.
Alfalfa was cultivated in two potted soil series obtained from two sandy soils
contaminated by Cu (SM) and metal(loids)/PAH (CD). Shoot production was monitored
for 8 weeks. Then, larvae of Spodoptera exigua were reared on alfalfa of both
soil series for eight days. A biotest (using Phaseolus vulgaris) was used to
assess the soil phytotoxicity. Increasing soil contamination reduced P. vulgaris
growth, but alfalfa growth was only reduced on the SM soil series. Exposure to
the SM soil was mirrored by shoot Cu and Cr concentrations of alfalfa
(respectively, in mg kg -1 DW, Cu and Cr ranged from 11.9 and 0.4 in the CTRL
soil to 98.5 and 1.2 in the SM one). Exposure to the CD soil series was mirrored
by shoot Zn concentrations (i.e. 48 to 91.6 mg kg-1 DW). Internal metal(loid)
concentrations of S. exigua remained generally steady across both soil series
(respectively Cd 0.05-0.16, Cr 0.5-3.3, Cu 5.8-98.5, Ni 0.6-1.6, Pb 0.4-1.3 and
Zn 57-337 mg kg-1 DW), and most of the associated transfer factors were lower
than 1. Here, due to the excluder phenotype of alfalfa across our TE
contamination gradients, S. exigua could cope with high total metal(loid)
concentration in both contaminated soils.
DOI: 10.1080/15226514.2017.1374342
PMID: 28949765
59. Plant Biotechnol J. 2017 Sep 21. doi: 10.1111/pbi.12841. [Epub ahead of print]
From model to crop: functional characterization of SPL8 in M. truncatula led to

genetic improvement of biomass yield and abiotic stress tolerance in alfalfa.
Gou J(1)(2), Debnath S(1), Sun L(1), Flanagan A(1)(2), Tang Y(1)(2), Jiang Q(1),
Wen J(1), Wang ZY(1)(2).
Author information:
(1)Noble Research Institute, Ardmore, OK, USA.
(2)BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, TN,
USA.
Biomass yield, salt tolerance and drought tolerance are important targets for
alfalfa (Medicago sativa L.) improvement. Medicago truncatula has been developed
into a model plant for alfalfa and other legumes. By screening a Tnt1
retrotransposon-tagged M. truncatula mutant population, we identified three
mutants with enhanced branching. Branch development determines shoot architecture
which affects important plant functions such as light acquisition, resource use
and ultimately impacts biomass production. Molecular analyses revealed that the
mutations were caused by Tnt1 insertions in the SQUAMOSA PROMOTER BINDING
PROTEIN-LIKE 8 (SPL8) gene. The M. truncatula spl8 mutants had increased biomass
yield, while overexpression of SPL8 in M. truncatula suppressed branching and
reduced biomass yield. Scanning electron microscopy (SEM) analysis showed that
SPL8 inhibited branching by directly suppressing axillary bud formation. Based on
the M. truncatula SPL8 sequence, alfalfa SPL8 (MsSPL8) was cloned and transgenic
alfalfa plants were produced. MsSPL8 down-regulated or up-regulated alfalfa
plants exhibited similar phenotypes to the M. truncatula mutants or
overexpression lines, respectively. Specifically, the MsSPL8 down-regulated
alfalfa plants showed up to 43% increase in biomass yield in the first harvest.
The impact was even more prominent in the second harvest, with up to 86% increase
in biomass production compared to the control. Furthermore, down-regulation of
MsSPL8 led to enhanced salt and drought tolerance in transgenic alfalfa. Results
from this research offer a valuable approach to simultaneously improve biomass
production and abiotic stress tolerance in legumes.
© 2017 The Authors. Plant Biotechnology Journal published by Society for

Experimental Biology and The Association of Applied Biologists and John Wiley &
Sons Ltd.
DOI: 10.1111/pbi.12841
PMID: 28941083
60. Chem Res Toxicol. 2017 Oct 16;30(10):1835-1846. doi:

10.1021/acs.chemrestox.7b00166. Epub 2017 Oct 4.
Detoxification of Atrazine by Low Molecular Weight Thiols in Alfalfa (Medicago

sativa).
Zhang JJ(1)(2), Xu JY(1), Lu FF(1), Jin SF(1), Yang H(1).
Author information:
(1)Jiangsu Key Laboratory of Pesticide Science, College of Sciences, Nanjing
Agricultural University , Nanjing 210095, China.
(2)College of Plant Protection, Henan Agricultural University , Zhengzhou 450002,
China.
Low molecular weight (LMW) thiols in higher plants are a group of sulfur-rich
nonprotein compounds and play primary and multiple roles in cellular redox
homeostasis, enzyme activities, and xenobiotics detoxification. This study
focused on identifying thiols-related protein genes from the legume alfalfa
exposed to the herbicide atrazine (ATZ) residues in environment. Using
high-throughput RNA-sequencing, a set of ATZ-responsive thiols-related protein
genes highly up-regulated and differentially expressed in alfalfa was identified.
Most of the differentially expressed genes (DEGs) were involved in regulation of
biotic and abiotic stress responses. By analyzing the genes involved in
thiols-mediated redox homeostasis, we found that many of them were
thiols-synthetic enzymes such as γ-glutamylcysteine synthase (γECS),
homoglutathione synthetase (hGSHS), and glutathione synthetase (GSHS). Using
liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we further
characterized a group of ATZ-thiols conjugates, which are the detoxified forms of
ATZ in plants. Cysteine S-conjugate ATZ-HCl+Cys was the most important metabolite
detected by MS. Several other ATZ-conjugates were also examined as ATZ-detoxified
metabolites. Such results were validated by characterizing their analogs in rice.
Our data showed that some conjugates under ATZ stress were detected in both
plants, indicating that some detoxified mechanisms and pathways can be shared by
the two plant species. Overall, these results indicate that LMW thiols play
critical roles in detoxification of ATZ in the plants.
DOI: 10.1021/acs.chemrestox.7b00166
61. J Food Sci Technol. 2017 Aug;54(9):2661-2669. doi: 10.1007/s13197-017-2703-8.

Epub 2017 May 23.
Perennial legumes as a source of ingredients for healthy food: proximate, mineral

and phytoestrogen composition and antibacterial activity.
Butkutė B(1), Padarauskas A(2), Cesevičienė J(1), Pavilonis A(3), Taujenis L(2),
Lemežienė N(4).
Author information:
(1)Chemical Research Laboratory, Institute of Agriculture, Lithuanian Research
Centre for Agriculture and Forestry, 58344 Akademija, Kėdainiai District,
Lithuania.
(2)Department of Analytical and Environmental Chemistry, Vilnius University,
03225 Vilnius, Lithuania.
(3)Institute of Microbiology and Virology, Lithuanian University of Health
Sciences, 44307 Kaunas, Lithuania.
(4)Department of Grass Breeding, Institute of Agriculture, Lithuanian Research
Centre for Agriculture and Forestry, 58344 Akademija, Kėdainiai District,
Lithuania.
ABSTRACT: Perennial legumes have been used as edible or medicinal plants since
ancient times. The focus of the current study are perennial legumes-Trifolium
pratense L., T. medium L., Medicago sativa L., M. lupulina L., Onobrychis
viciifolia Scop., Astragalus glycyphyllos L. and A. cicer L.-of branching stage
as a potential source of value-added ingredients for healthy food. Freeze-dried
samples were analysed for proximal composition, mineral, isoflavone and
coumestrol contents as well as for antimicrobial activity. Legumes were
protein-rich (23.0/100 g on average). Mineral contents in 100 g of plant dry
matter averaged: K 2.64 g, Ca 1.81 g, Mg 0.475 g, P 0.324 g, Zn 2.76 mg and Fe
37.8 mg. According to the total amount of phytoestrogens, the species ranked as
follows: T. medium (34.4 mg/g) ≫ T. pratense ≫ O. viciifolia ≥ M. sativa = A.
cicer = M. lupulina ≥ A. glycyphyllos (0.207 mg/g). Extracts of legumes,
especially that of O. viciifolia, exhibited noticeable potency to inhibit the
growth of Gram-positive and Gram-negative bacteria. Perennial legumes of
branching stage can be used as protein, mineral and phytoestrogen rich source for
food ingredients and supplements.
GRAPHICAL ABSTRACT:
DOI: 10.1007/s13197-017-2703-8
PMID: 28928506
62. Appl Environ Microbiol. 2017 Dec 15;84(1). pii: e01561-17. doi:
10.1128/AEM.01561-17. Print 2018 Jan 1.
Succinate Transport Is Not Essential for Symbiotic Nitrogen Fixation by

Sinorhizobium meliloti or Rhizobium leguminosarum.
Mitsch MJ(1), diCenzo GC(1), Cowie A(1), Finan TM(2).
Author information:
(1)Department of Biology, McMaster University, Hamilton, Ontario, Canada.
(2)Department of Biology, McMaster University, Hamilton, Ontario, Canada
finan@mcmaster.ca.
Symbiotic nitrogen fixation (SNF) is an energetically expensive process performed

by bacteria during endosymbiotic relationships with plants. The bacteria require
the plant to provide a carbon source for the generation of reductant to power
SNF. While C4-dicarboxylates (succinate, fumarate, and malate) appear to be the
primary, if not sole, carbon source provided to the bacteria, the contribution of
each C4-dicarboxylate is not known. We address this issue using genetic and
systems-level analyses. Expression of a malate-specific transporter (MaeP) in
Sinorhizobium meliloti Rm1021 dct mutants unable to transport C4-dicarboxylates
resulted in malate import rates of up to 30% that of the wild type. This was
sufficient to support SNF with Medicago sativa, with acetylene reduction rates of
up to 50% those of plants inoculated with wild-type S. melilotiRhizobium
leguminosarum bv. viciae 3841 dct mutants unable to transport C4-dicarboxylates
but expressing the maeP transporter had strong symbiotic properties, with Pisum
sativum plants inoculated with these strains appearing similar to plants
inoculated with wild-type R. leguminosarum This was despite malate transport
rates by the mutant bacteroids being 10% those of the wild type. An
RNA-sequencing analysis of the combined P. sativum-R. leguminosarum nodule
transcriptome was performed to identify systems-level adaptations in response to
the inability of the bacteria to import succinate or fumarate. Few
transcriptional changes, with no obvious pattern, were detected. Overall, these
data illustrated that succinate and fumarate are not essential for SNF and that,
at least in specific symbioses, l-malate is likely the primary C4-dicarboxylate
provided to the bacterium.IMPORTANCE Symbiotic nitrogen fixation (SNF) is an
economically and ecologically important biological process that allows plants to
grow in nitrogen-poor soils without the need to apply nitrogen-based fertilizers.
Much research has been dedicated to this topic to understand this process and to
eventually manipulate it for agricultural gains. The work presented in this
article provides new insights into the metabolic integration of the plant and
bacterial partners. It is shown that malate is the only carbon source that needs
to be available to the bacterium to support SNF and that, at least in some
symbioses, malate, and not other C4-dicarboxylates, is likely the primary carbon
provided to the bacterium. This work extends our knowledge of the minimal
metabolic capabilities the bacterium requires to successfully perform SNF and may
be useful in further studies aiming to optimize this process through synthetic
biology approaches. The work describes an engineering approach to investigate a
metabolic process that occurs between a eukaryotic host and its prokaryotic
endosymbiont.
DOI: 10.1128/AEM.01561-17
PMID: 28916561
63. Int J Mol Sci. 2017 Sep 12;18(9). pii: E1951. doi: 10.3390/ijms18091951.
An Insight into T-DNA Integration Events in Medicago sativa.
Nicolia A(1), Ferradini N(2), Veronesi F(3), Rosellini D(4).
Author information:
(1)Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università degli
Studi di Perugia, 06121 Perugia, Italy. alessandro.nicolia@crea.gov.it.
Studi di Perugia, 06121 Perugia, Italy. nicoletta.ferradini@gmail.com.
Studi di Perugia, 06121 Perugia, Italy. fabio.veronesi@unipg.it.
Studi di Perugia, 06121 Perugia, Italy. daniele.rosellini@unipg.it.
The molecular mechanisms of transferred DNA (T-DNA) integration into the plant
genome are still not completely understood. A large number of integration events
have been analyzed in different species, shedding light on the molecular
mechanisms involved, and on the frequent transfer of vector sequences outside the
T-DNA borders, the so-called vector backbone (VB) sequences. In this work, we
characterized 46 transgenic alfalfa (Medicago sativa L.) plants (events),
generated in previous works, for the presence of VB tracts, and sequenced several
T-DNA/genomic DNA (gDNA) junctions. We observed that about 29% of the transgenic
events contained VB sequences, within the range reported in other species.
Sequence analysis of the T-DNA/gDNA junctions evidenced larger deletions at LBs
compared to RBs and insertions probably originated by different integration
mechanisms. Overall, our findings in alfalfa are consistent with those in other
plant species. This work extends the knowledge on the molecular events of T-DNA
integration and can help to design better transformation protocols for alfalfa.
DOI: 10.3390/ijms18091951
PMCID: PMC5618600
PMID: 28895894
Conflict of interest statement: The authors declare no conflict of interest.
64. Environ Sci Pollut Res Int. 2017 Nov;24(31):24438-24446. doi:

10.1007/s11356-017-0085-0. Epub 2017 Sep 11.
The responses of two native plant species to soil petroleum contamination in the
Yellow River Delta, China.
Xie W(1), Zhang Y(2), Li R(2)(3), Yang H(2), Wu T(2), Zhao L(2), Lu Z(2)(3).
Author information:
(1)School of Biotechnology, Key Laboratory of Instrumental Analysis of Binzhou
City, Shandong Provincial Key Laboratory of Eco-Environmental Science for Yellow
River Delta, Binzhou University, Binzhou, 256603, China. xwjeric@hotmail.com.
(2)School of Biotechnology, Key Laboratory of Instrumental Analysis of Binzhou
City, Shandong Provincial Key Laboratory of Eco-Environmental Science for Yellow
River Delta, Binzhou University, Binzhou, 256603, China.
(3)Institute of Restoration Ecology, China University of Mining and Technology,
Beijing, 100083, China.
Petroleum contamination is a significant environmental problem in the Yellow

River Delta. The responses of two native salt-tolerant plant species, alfalfa
(Medicago sativa) and bristle grass (Setaria uiridis Beauv), to soil petroleum
contamination were investigated at five levels between 0 and 2.0% (w/w). Results
showed that the total, aboveground and underground plant biomasses of both
species were significantly reduced by petroleum contamination (p < 0.05), with
the inhibition enhanced with increased petroleum levels. However, the emergence
rate of bristle grass was promoted by petroleum contamination. Following 100 days
of exposure, the number of soil petroleum degraders increased greatly, with a
trend of initial increase followed by a decrease at 1.5% contamination or higher.
Compared to bulk soils, bacteria-degrading alkanes, total hydrocarbons and PAHs
in alfalfa rhizosphere soils increased by 1.33-4.18-, 0.85-3.01- and
4.12-12.75-fold, respectively, with an increase of 2.80-10.00-, 4.42-14.44- and
7.30-26.00-fold in bristle grass rhizosphere soils, respectively. The greatest
number of petroleum degraders in bristle grass rhizosphere soils resulted in the
highest petroleum degradation rate. Bristle grass may be the optimal species for
petroleum remediation in the studied area.
DOI: 10.1007/s11356-017-0085-0
PMID: 28895014
65. PeerJ. 2017 Sep 6;5:e3775. doi: 10.7717/peerj.3775. eCollection 2017.
Divergence of compost extract and bio-organic manure effects on lucerne plant and
soil.
Ren H(1), Hu J(1), Hu Y(1), Yang G(1), Zhang Y(1)(2).
Author information:
(1)College of Agro-grassland Science, Nanjing Agricultural University, Nanjing,
China.
(2)Department of Grassland Science, China Agricultural University, Beijing,
China.
AIM: Application of organic materials into agricultural systems enhances plant

growth and yields, and improves soil fertility and structure. This study aimed to
examine the effects of "compost extract (CE)", a soil conditioner, and
bio-organic manure (BOM) on the growth of lucerne (Medicago sativa), and compare
the efficiency between BOM (including numbers of microorganisms) and CE
(including no added microorganisms).
METHOD: A greenhouse experiment was conducted with four soil amendment treatments
(control, BOM, CE and CEBOM), and was arranged in a completely randomized design
with 10 replicates for each treatment. Plant biomass, nutritive value and
rhizobia efficacy as well as soil characteristics were monitored.
RESULT: CE rather than BOM application showed a positive effect on plant growth
and soil properties when compared with the control. Lucerne nodulation responded
equally to CE addition and rhizobium inoculation. CE alone and in combination
with BOM significantly increased plant growth and soil microbial activities and
improved soil structure. The synergistic effects of CE and BOM indicate that
applying CE and BOM together could increase their efficiency, leading to higher
economic returns and improved soil health. However, CE alone is more effective
for legume growth since nodulation was suppressed by nitrogen input from BOM. CE
had a higher efficiency than BOM for enriching soil indigenous microorganisms
instead of adding microorganisms and favouring plant nodulation.
DOI: 10.7717/peerj.3775
PMCID: PMC5591637
PMID: 28894647
Conflict of interest statement: The authors declare there are no competing

interests.
66. Sci Total Environ. 2018 Jan 1;610-611:1428-1438. doi:

10.1016/j.scitotenv.2017.08.196. Epub 2017 Aug 30.
Complementarity of three distinctive phytoremediation crops for multiple-trace

element contaminated soil.
Desjardins D(1), Brereton NJB(2), Marchand L(3), Brisson J(1), Pitre FE(4),
Labrecque M(4).
Author information:
(1)Institut de recherche en biologie végétale, Université de Montréal, 4101
Sherbrooke Est, Montréal, QC H1X 2B2, Canada.
Sherbrooke Est, Montréal, QC H1X 2B2, Canada. Electronic address:
nicholas.brereton@umontreal.ca.
(3)INRA, UMR 1202 BIOGECO, 69 route d'Arcachon, FR-33612 Cestas cedex, France;
University of Bordeaux, UMR 1202 BIOGECO, Bat B2, Allée Geoffroy St-Hilaire,
CS50023, FR-33615 Pessac cedex, France.
Sherbrooke Est, Montréal, QC H1X 2B2, Canada; Montreal Botanical Garden, 4101
Sherbrooke Est, Montréal, QC H1X 2B2, Canada.
Trace element (TE) contaminated land represents an important risk to the
environment and to human health worldwide. These soils usually contain a variety
of TEs which can be a challenge for plant-based remediation options. As
individual plant species often possess a limited range of TE remediation
abilities, functional complementarity principles could be of value for
remediation of soil contaminated by multiple TEs using assemblages of species.
Monocultures and polycultures of Festuca arundinacea, Medicago sativa and Salix
miyabeana were grown for 4months in aged-polluted soil contaminated by Ag, As,
Cd, Cr, Cu, Pb, Se and Zn. Above and belowground biomass yields, root surface
area (RSA) and TE tissue concentrations were recorded. In monoculture, the
greatest aboveground biomass was produced by S. miyabeana (S), the greatest
belowground biomass was from M. sativa (M) and F. arundinacea (F) produced the
highest RSA. The polycultures of F+M, F+S and F+M+S produced among the highest
values across all three traits. F. arundinacea monoculture and its combination
with S. miyabeana (F+S) accumulated the highest amounts of total TEs in
belowground tissues, whereas the most effective combination (or monoculture) for
aboveground extraction yields varied depending on the TE considered. The crops
demonstrated complementarity in their biomass allocation patterns as well as
facilitative interactions. When considering contamination with a particular TE,
the best phytomanagement approach could include a specific monoculture option;
however, when above and belowground biomass allocation patterns, TE-remediation
abilities as well as nitrogen accessibility are considered, co-cropping all three
species (F+M+S) was the most robust scenario for remediation of multiple-TE
contaminated land. By more effectively addressing a diversity of TE, species
assemblage approaches could represent an important advancement towards enabling
the use of plants to address contaminated-land issues worldwide.
Copyright © 2017. Published by Elsevier B.V.
DOI: 10.1016/j.scitotenv.2017.08.196
PMID: 28873664
67. Food Chem. 2018 Jan 15;239:679-687. doi: 10.1016/j.foodchem.2017.07.004. Epub

2017 Jul 3.
Exploitation of alfalfa seed (Medicago sativa L.) flour into gluten-free rice
cookies: Nutritional, antioxidant and quality characteristics.
Giuberti G(1), Rocchetti G(2), Sigolo S(2), Fortunati P(2), Lucini L(3), Gallo
A(2).
Author information:
(1)Institute of Food Science and Nutrition, Faculty of Agriculture, Food and
Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense
84, 29121 Piacenza, Italy. Electronic address: gianluca.giuberti@unicatt.it.
(2)Institute of Food Science and Nutrition, Faculty of Agriculture, Food and
Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense
84, 29121 Piacenza, Italy.
(3)Institute of Environmental and Agricultural Chemistry, Faculty of Agriculture,
Food and Environmental Sciences, Università Cattolica del Sacro Cuore, Via Emilia
Parmense 84, 29121 Piacenza, Italy.
In an effort to increase the nutritional value of common gluten-free (GF)

cereal-based foods, GF cookies using alfalfa seed flour (ASF), at different
substitution levels to common rice flour (0% as control, 15%, 30% and 45% w/w),
were produced. Crude protein, total dietary fibre, total polyunsaturated, total
n-3 and n-6 fatty acid contents increased linearly (p<0.05) by raising the
substitution levels of rice with ASF. The hardness, the total phenolic content,
the in vitro antioxidant capacity and the resistant starch increased linearly
(p<0.05), whereas the starch hydrolysis index decreased linearly (p<0.05) by
raising the substitution levels of rice flour with ASF. Despite the fact that
ASF-substituted GF cookies had inferior sensory attributes compared to the
control, the score given by the panellists remained at fairly good levels for all
tested parameters, showing acceptability of the substituted GF cookies.
DOI: 10.1016/j.foodchem.2017.07.004
68. Plant J. 2017 Nov;92(4):596-610. doi: 10.1111/tpj.13704. Epub 2017 Oct 16.
A distinct class of vesicles derived from the trans-Golgi mediates secretion of

xylogalacturonan in the root border cell.
Wang P(1), Chen X(2), Goldbeck C(3), Chung E(2), Kang BH(1).
Author information:
(1)Cellular and Molecular Biology Program, State Key Laboratory of
Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong,
New Territories, Hong Kong, China.
(2)Department of Mathematics, The Chinese University of Hong Kong, New
Territories, Hong Kong, China.
(3)Department of Mathematics, University of California, Santa Barbara, CA, 93106,
USA.
Root border cells lie on the surface of the root cap and secrete massive amounts
of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the
border cells have hypertrophied margins, reflecting elevated biosynthetic
activity to produce the polysaccharide components of the mucilage. To investigate
the three-dimensional structures and macromolecular compositions of these Golgi
stacks, we examined high-pressure frozen/freeze-substituted alfalfa root cap
cells with electron microscopy/tomography. Golgi stacks in border cells and
peripheral cells, precursor cells of border cells, displayed similar
morphological features, such as proliferation of trans cisternae and swelling of
the trans cisternae and trans-Golgi network (TGN) compartments. These swollen
margins give rise to two types of vesicles larger than other
Golgi-associated vesicles. Margins of trans-Golgi cisternae accumulate the LM8
xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles
(LVs) after release from the Golgi. Epitopes for xyloglucan (XG),
polygalacturonic acid/rhamnogalacturonan-I (PGA/RG-I) are detected in the
trans-most cisternae and TGN compartments. LVs produced from TGN compartments
(TGN-LVs) stained lighter than LVs and contained the cell wall polysaccharide
epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma
membrane only in border cells, whereas TGN-LVs containing the XG and PGA/RG-I
epitopes fuse with the plasma membrane of both peripheral cells and border cells.
Taken together, these results indicate that XGA is secreted by a novel type of
secretory vesicles derived from trans-Golgi cisternae. Furthermore, we simulated
the collapse in the central domain of the trans-cisternae accompanying
polysaccharide synthesis with a mathematical model.
© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.
DOI: 10.1111/tpj.13704
PMID: 28865155
69. Arch Microbiol. 2018 Jan;200(1):137-145. doi: 10.1007/s00203-017-1427-5. Epub
2017 Sep 1.
Fecal microbiota of lambs fed purple prairie clover (Dalea purpurea Vent.) and
alfalfa (Medicago sativa).
Huang Q(1)(2)(3), Holman DB(2), Alexander T(2), Hu T(3), Jin L(2), Xu Z(2),
McAllister TA(2), Acharya S(2), Zhao G(1), Wang Y(4).
Author information:
(1)College of Animal Science and Technology, Yangzhou University, Yangzhou,
225009, China.
(2)Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000,
Lethbridge, AB, T1J 4B1, Canada.
(3)College of Animal Science and Technology, Northwest A&F University, Yangling,
712100, China.
(4)Lethbridge Research Centre, Agriculture and Agri-Food Canada, P.O. Box 3000,
Lethbridge, AB, T1J 4B1, Canada. yuxi.wang@agr.gc.ca.
The present study assessed the effect of purple prairie clover (PPC) and PPC
condensed tannins (CT) on the fecal microbiota of lambs using high-throughput 16S
rRNA gene pyrosequencing. A total of 18 individual lambs were randomly divided
into three groups and fed either green chop alfalfa (Alf), a 40:60 (DM basis;
Mix) mixture of Alf and PPC, or Mix supplemented with polyethylene glycol (Mix-P)
for 18 days. Fecal samples were collected on days 13 through 18 using digital
rectal retrieval. The DNA of fecal samples was extracted and the microbial 16S
rRNA gene amplicons were sequenced using 454 pyrosequencing. Regardless of diet,
the bacterial community was dominated by Firmicutes and Bacteroidetes with many
sequences unclassified at the genus level. Forage type and CT had no effect on
the fecal microbial composition at the phylum level or on α-diversity. Compared
to the Alf diet, the Mix diet reduced the relative abundance of Akkermansia
(P = 0.03) and Asteroleplasma (P = 0.05). Fecal microbial populations in Alf and
Mix-P clustered separately from each other when assessed using unweighted UniFrac
(P < 0.05). These results indicate that PPC CT up to 36 g/kg DM in the diet had
no major effect on fecal microbial flora at the phyla level and exerted only
minor effects on the genera composition of fecal microbiota in lambs.
DOI: 10.1007/s00203-017-1427-5
PMID: 28864945
70. Sci Total Environ. 2018 Jan 1;610-611:1271-1280. doi:

10.1016/j.scitotenv.2017.08.186. Epub 2017 Aug 30.
Using alternative forage species to reduce emissions of the greenhouse gas

nitrous oxide from cattle urine deposited onto soil.
Luo J(1), Balvert SF(2), Wise B(3), Welten B(3), Ledgard SF(3), de Klein CAM(4),
Lindsey S(3), Judge A(3).
Author information:
(1)AgResearch Limited, Ruakura Research Centre, Private Bag 3123, Hamilton 3240,
New Zealand. Electronic address: Jiafa.luo@agresearch.co.nz.
New Zealand; University of Waikato, Faculty of Science and Engineering, Private
Bag 3105, Hamilton 3240, New Zealand.
New Zealand.
(4)AgResearch Limited, Invermay Research Centre, Private Bag 50034, Mosgiel 9053,
New Zealand.
Grazed pastures are a major contributor to emissions of the greenhouse gas

nitrous oxide (N2O), and urine deposition from grazing animals is the main source
of the emissions. Incorporating alternative forages into grazing systems could be
an approach for reducing N2O emissions through mechanisms such as release of
biological nitrification inhibitors from roots and increased root depth. Field
plot and lysimeter (intact soil column) trials were conducted in a free draining
Horotiu silt loam soil to test whether two alternative forage species, plantain
(Plantago lanceolate L.) and lucerne (Medicago sativa L.), could reduce N2O
emissions relative to traditional pasture species, white clover (Trifolium repens
L.) and perennial ryegrass (Lolium perenne L.). The amounts of N2O emitted from
the soil below each forage species, which all received the same cow urine at the
same rates, was measured using an established static chamber method. Total N2O
emissions from the plantain, lucerne and perennial ryegrass controls (without
urine application) were generally very low, but emissions from the white clover
control were significantly higher. When urine was applied in autumn or winter N2O
emissions from plantain were lower compared with those from perennial ryegrass or
white clover, but this difference was not found when urine was applied in summer.
Lucerne had lower emissions in winter but not in other seasons. Incorporation of
plantain into grazed pasture could be an approach to reduce N2O emissions.
However, further work is required to understand the mechanisms for the reduced
emissions and the effects of environmental conditions in different seasons.
DOI: 10.1016/j.scitotenv.2017.08.186
PMID: 28851147
71. Can J Microbiol. 2017 Nov;63(11):881-893. doi: 10.1139/cjm-2017-0124. Epub 2017

Aug 25.
Rhizosphere effect is stronger than PAH concentration on shaping spatial

bacterial assemblages along centimetre-scale depth gradients.
Bourceret A(1)(2), Leyval C(1)(2), Thomas F(1)(2), Cébron A(1)(2).
Author information:
(1)a CNRS, LIEC UMR7360, Faculté des Sciences et Technologies, boulevard des
Aiguillettes, B.P. 70239, 54506 Vandoeuvre-lès-Nancy, France.
(2)b Université de Lorraine, LIEC UMR7360, Faculté des Sciences et Technologies,
boulevard des Aiguillettes, B.P. 70239, 54506 Vandoeuvre-lès-Nancy, France.
At centimetre scale, soil bacterial assemblages are shaped by both abiotic

(edaphic characteristics and pollutants) and biotic parameters. In a rhizobox
experiment carried out on planted industrial soil contaminated with polycyclic
aromatic hydrocarbons (PAHs), we previously showed that pollution was distributed
randomly with hot and cold spots. Therefore, in the present study, we
investigated the effect of this patchy PAH distribution on the bacterial
community assemblage and compared it with that of root depth gradients found in
the rhizosphere of either alfalfa or ryegrass. Sequencing of 16S rRNA amplicons
revealed a higher bacterial diversity in ryegrass rhizosphere and enrichment in
specific taxa by the 2 plant species. Indeed, Bacteroidetes, Firmicutes, and
Gammaproteobacteria were globally favored in alfalfa, whereas Acidimicrobiia,
Chloroflexi, Alpha-, and Betaproteobacteria were globally favored in ryegrass
rhizosphere. The presence of alfalfa created depth gradients of root biomass,
carbohydrate, and pH, and actually shaped the bacterial assemblage, favoring
Actinobacteria near the surface and Gemmatimonadetes and Proteobacteria at
greater depths. Contrarily, the bacterial assemblage was homogeneous all along
depths of the ryegrass root system. With both plant species, the PAH content and
random distribution had no significant effect on bacterial assemblage. Globally,
at centimeter scale, bacterial community assemblages were mostly shaped by soil
physical and chemical depth gradients induced by root growth but not by patchy
PAH content.
DOI: 10.1139/cjm-2017-0124
72. An Acad Bras Cienc. 2017 Jul-Sep;89(3):1801-1813. doi:

10.1590/0001-3765201720170005. Epub 2017 Aug 17.
Selection of alfalfa genotypes for resistance to the foliar pathogen Curvularia

geniculata.
Ávila MR(1), Dall'agnol M(1), Martinelli JA(2), Silva GBPD(2), Bremm C(3), Nunes
T(1).
Author information:
(1)Deapartment of Forage Plants and Agrometeorology, Federal University of Rio
Grande do Sul, Av. Bento Gonçalves, 7712, 91501-970 Porto Alegre, RS, Brazil.
(2)Department of Plant Pathology, Federal University of Rio Grande do Sul, Av.
Bento Gonçalves, 7712, 91501-970 Porto Alegre, RS, Brazil.
(3)State Foundation for Agricultural Research, Rua Gonçalves Dias, 570, 90130-060
Porto Alegre, RS, Brazil.
Foliar diseases impose severe restrictions on the persistence and productivity of

Medicago sativa, both of which may be increased by developing disease resistant
and more competitive genotypes that can improve pasture quality. We found
Curvularia geniculata as the principal alfalfa foliar pathogen in the Brazilian
state of Rio Grande do Sul (RS). Growth chamber experiments evaluated the
resistance of alfalfa genotypes 'E1C4', 'CPPSul', 'ABT 805' and 'CUF-101' to C.
geniculata as compared the control 'Crioula' genotype. These genotypes were also
evaluated in field trials at a sea level site in Eldorado do Sul in central RS
and at two sites £200 m above sea level, one in Bagé municipality in south west
RS and the other at a farm near the town of Alto Feliz in north east RS. Plants
were spray-inoculated with 1.6 x 106 ml-1 of C. geniculata spores and visually
evaluated for leaf damage 14 days later. The C. geniculata infection rates varied
from zero to 100%. Alfalfa persistence and forage mean dry mass (DM) production
at the Eldorado site were measured during different seasons from November 2013 to
January 2015 by calculating the incidence of invasive plants and morphologically
separating leaves from stems and calculating both leaf and stem DM. Data were
analysed using mixed statistical models. The best results for persistence and
forage DM were shown by the 'CPPSul' genotypes (DM = 16,600 kg ha-1) and
'Crioula' (DM = 15,750 kg ha-1). These two genotypes will be used for subsequent
investigations and selection cycles.
DOI: 10.1590/0001-3765201720170005
PMID: 28832724
73. J Dairy Res. 2017 Aug;84(3):240-247. doi: 10.1017/S002202991700036X.
Replacing wheat with canola meal and maize grain in the diet of lactating dairy
cows: Feed intake, milk production and cow condition responses.
McDonnell RP(1), Staines MV(1).
Author information:
(1)Department of Agriculture and Food Western Australia,Verschuer
Place,Bunbury,WA 6230,Australia.
This research paper describes the effect of partially replacing wheat with maize
grain and canola meal on milk production and body condition changes in early
lactation Holstein-Friesian dairy cows consuming a grass silage-based diet over
an 83-d period. Two groups of 39 cows were stratified for age, parity, historical
milk yield and days in milk (DIM), and offered one of two treatment diets. The
first treatment (CON) reflected a typical diet used by Western Australian dairy
producers in summer and comprised (kg DM/cow per d); 8 kg of annual ryegrass
silage, 6 kg of crushed wheat (provided once daily in a mixed ration), 3·6 kg of
crushed lupins (provided in the milking parlour in two daily portions) and ad
libitum lucerne haylage. The second treatment diet (COMP) was identical except
the 6 kg of crushed wheat was replaced by 6 kg of a more complex concentrate mix
(27% crushed wheat, 34% maize grain and 37% canola meal). Lucerne haylage was
provided independently in the paddock to all cows, and no pasture was available
throughout the experiment. The COMP group had a greater mean overall daily intake
(22·5 vs 20·4 kg DM/cow) and a higher energy corrected milk (ECM) yield (29·2 vs
27·1 kg/cow; P = 0·047) than the CON cows. The difference in overall intake was
caused by a higher daily intake of lucerne haylage in COMP cows (4·5 vs 2·3 kg
DM/cow). The CON group had a higher concentration of milk fat (42·1 vs 39·3 g/kg;
P = 0·029) than COMP cows. Milk protein yield was greater in COMP cows (P <
0·021); however, milk fat yield was unaffected by treatment. It is concluded that
partially replacing wheat with canola meal and maize grain in a grass
silage-based diet increases voluntary DMI of conserved forage and consequently
yields of ECM and milk protein.
DOI: 10.1017/S002202991700036X
74. J Mol Evol. 2017 Oct;85(3-4):79-83. doi: 10.1007/s00239-017-9808-6. Epub 2017

Aug
21.
Microevolution Rather than Large Genome Divergence Determines the Effectiveness

of Legume-Rhizobia Symbiotic Interaction Under Field Conditions.
Jozefkowicz C(1)(2), Brambilla S(2), Frare R(1)(2), Stritzler M(1)(2), Puente

M(3), Piccinetti C(3), Soto G(1)(2), Ayub N(4)(5).
Author information:
(1)Consejo Nacional de Investigaciones Científicas y Técnicas, CABA, Buenos
Aires, Argentina.
(2)Instituto de Genética Ewald A. Favret (INTA), De los Reseros S/N, Castelar
C25(1712), Buenos Aires, Argentina.
(3)Instituto de Microbiología y Zoología Agrícola (INTA), Buenos Aires,
Argentina.
(4)Consejo Nacional de Investigaciones Científicas y Técnicas, CABA, Buenos
Aires, Argentina. nicoayub@gmail.com.
(5)Instituto de Genética Ewald A. Favret (INTA), De los Reseros S/N, Castelar
C25(1712), Buenos Aires, Argentina. nicoayub@gmail.com.
Despite the vast screening for natural nitrogen-fixing isolates by public and
private consortia, no significant progresses in the production of improved
nitrogen-fixing inoculants for alfalfa production have been made in the last
years. Here, we present a comprehensive characterization of the nitrogen-fixing
strain Ensifer meliloti B399 (originally named Rhizobium meliloti 102F34),
probably the inoculant most widely used in alfalfa production since the 1960s.
Complete nucleotide sequence and genome analysis of strain B399 showed that the
three replicons present in this commercial strain and the model bacterium Ensifer
meliloti 1021 are extremely similar to each other in terms of nucleotide identity
and synteny conservation. In contrast to that observed in B399-treated plants,
inoculation of plants with strain 1021 did not improve nitrogen content in
different alfalfa cultivars under field conditions, suggesting that a small
genomic divergence can drastically impact on the symbiotic phenotype. Therefore,
in addition to the traditional screening of natural nitrogen-fixing isolates, the
genome engineering of model strains could be an attractive strategy to improve
nitrogen fixation in legume crops.
DOI: 10.1007/s00239-017-9808-6
75. Chemosphere. 2017 Nov;186:743-748. doi: 10.1016/j.chemosphere.2017.08.056. Epub

2017 Aug 11.
Photosynthesis of alfalfa (Medicago sativa) in response to landfill leachate

contamination.
Yang L(1), Sun T(2), Liu Y(2), Guo H(2), Lv L(2), Zhang J(2), Liu C(2).
Author information:
(1)School of Resources and Environmental Engineering, Wuhan University of
Technology, 430070 Wuhan, PR China. Electronic address: yanglie612@163.com.
(2)School of Resources and Environmental Engineering, Wuhan University of
Technology, 430070 Wuhan, PR China.
Thousands of unlined landfills and open dumpsites have put great threat on the
security of soil and ground water due to leachate leakage. Alfalfa is believed
potential as a phytoremediation plant for leachate contamination based on strong
root system and the excellent capacity of removing various kinds of pollutants. A
lab-scale investigation was conducted to figure out the sensitiveness of alfalfa
photosynthesis in response to leachate contamination. The results demonstrated
that both of the maximum photosynthetic efficiency (Fv/Fm) and net photosynthetic
rate (Pn) were slightly inhibited in the high-dosage group. Based on statistical
analysis, higher sensitivity of Pn to leaching parameters than Fv/Fm was
observed. There were significant correlations between most of leaching parameters
(pH, ammonium and COD) and Pn with correlation coefficients of 0.530, -0.580
and -0.578 (p < 0.01), respectively. Therefore, Pn is potential for acting as an
effective indicator for staple leaching characteristics of leachate contaminated
soils.
76. Biometals. 2017 Oct;30(5):719-732. doi: 10.1007/s10534-017-0040-z. Epub 2017

Aug
15.
Methane enhances aluminum resistance in alfalfa seedlings by reducing aluminum

accumulation and reestablishing redox homeostasis.
Cui W(1), Cao H(1), Yao P(1), Pan J(1), Gu Q(1), Xu S(2), Wang R(2), Ouyang Z(3),
Wang Q(4), Shen W(5).
Author information:
Agricultural University, Nanjing, 210095, China.
(2)Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing,
210014, China.
(3)Shuigu Environmental Protection Technological Company Ltd, Shanghai, 200437,
China.
Agricultural University, Nanjing, 210095, China. wangqy@njau.edu.cn.
Agricultural University, Nanjing, 210095, China. wbshenh@njau.edu.cn.
Methane (CH4) is emerging as a candidate of signal molecule recently. However,

whether or how CH4 enhances plant adaptation to aluminum (Al)-contaminated
environment is still unknown. In this report, the physiological roles and
possible molecular mechanisms of CH4 in the modulation of Al toxicity in alfalfa
seedlings were characterized. Our results showed that, CH4 pretreatment could
alleviate Al-induced seedling growth inhibition and redox imbalance. The
defensive effects of CH4 against Al toxicity including the remission of
Al-induced root elongation inhibition, nutrient disorder, and relative
electrolyte leakage. Moreover, contents of organic acids, including citrate,
malate, and oxalate, were increased by CH4. These results were paralleled by the
findings of CH4 regulated organic acids metabolism and transport genes, citrate
synthase, malate dehydrogenase, aluminum-activated malate transporter, and
aluminum activated citrate transporter. Consistently, Al accumulation in seedling
roots was decreased after CH4 treatment. In addition, Al-induced oxidative stress
was also alleviated by CH4, through the regulation of the activities of
anti-oxidative enzymes, such as ascorbate peroxidase, superoxide dismutase, and
peroxidase, as well as their corresponding transcripts. Our data clearly
suggested that CH4 alleviates Al toxicity by reducing Al accumulation in organic
acid-dependent fashion, and reestablishing redox homeostasis.
DOI: 10.1007/s10534-017-0040-z
PMID: 28812165
77. J Anim Sci. 2017 Aug;95(8):3654-3665. doi: 10.2527/jas.2016.1129.
Effects of dietary tannins on total and extractable nutrients from manure.
Halvorson JJ, Kronberg SL, Hagerman AE.
The effects of condensed tannins on N dynamics in ruminants have been a topic of

research for some time, but much less work has focused on their impacts on other
nutrients in manure. A 4 × 4 Latin square trial was used to determine if intake
of sericea lespedeza (; SL; a condensed tannin source), at 0, 10, 20, or 40% of
the diet (as-fed basis), would affect concentrations of nutrients in manure and
patterns of total excretion when offered with alfalfa (; ALF) to sheep. With SL
additions, average daily manure production increased linearly ( ≤ 0.01), from 40
to 50% of the diet mass. The concentrations of total C, total N, soluble P, total
and soluble Na, total and soluble S, total and soluble Mn, and total and soluble
B in feces increased ( ≤ 0.05) while soluble N, total Ca, total and soluble Mg,
soluble Zn, total and soluble Fe, total and soluble Cu decreased ( ≤ 0.02). Total
P, total and soluble K, soluble Ca, and total Zn were less affected ( > 0.05).
Comparing diets containing 0 to 40% SL, average daily outputs of total C, total
N, soluble P, soluble K, total and soluble Na, and total Mn increased linearly (
≤ 0.01) by 42.0, 71.2, 93.3, 45.2, 111, 148, and 52.4 percentage points,
respectively. Total K, total and soluble S, soluble Mn, and total and soluble B
increased quadratically ( ≤ 0.02) by 26.1, 52.3, 26.7, 147, 100, and 19.5
percentage points, respectively. Conversely, outputs of soluble Zn and total Fe
decreased linearly ( ≤ 0.01), by -51.5 and -24.8 percentage points, while total
Ca, total and soluble Mg, soluble Fe, and soluble Cu decreased quadratically ( ≤
0.05) by -15.7, -12.3, -40.0, -89.9, and -60.3 percentage points, respectively.
Outputs of soluble N, total P, soluble Ca, total Zn, and total Cu remained
unchanged ( ≥ 0.14). Ratios of manure outputs to feed inputs for C, N, K, and B
increased ( ≤ 0.02) but those for P and Mg were unchanged ( ≥ 0.10). Ratios of
soluble to total manure outputs (S:O) increased ( ≤ 0.01) for P, Ca, Na, Mn;
decreased ( ≤ 0.05) for N, S, Mg, Zn, Fe, Cu, and B; and were unaffected by
treatment ( ≤ 0.16) for K. Decreasing S:O ratios are consistent with the
formation of complexes that adsorb these nutrients to insoluble fiber fractions
of manure and could thus affect mineralization rates. This study suggests that
dietary tannins, found in forages like SL, can alter the concentrations, total
excretion rates and throughput efficiency of nutrients in manure.
DOI: 10.2527/jas.2016.1129
78. J AOAC Int. 2017 Nov 1;100(6):1681-1693. doi: 10.5740/jaoacint.17-0234. Epub

2017
Jul 13.
Comparison of Various Extraction Techniques of Medicago sativa: Yield,

Antioxidant Activity, and Content of Phytochemical Constituents.
Krakowska A(1), Rafińska K(1), Walczak J(1), Kowalkowski T(1), Buszewski B(1).
Author information:
(1)Nicolaus Copernicus University, Faculty of Chemistry, Department of
Environmental Chemistry and Bioanalytics, Gagarina 7 St, 87-100 Toruń, Poland;
Nicolaus Copernicus University, Interdisciplinary Centre of Modern Technologies,
Wileńska 4, 87-100 Toruń, Poland.
Medicago sativa L. (M. sativa) is a source of many valuable secondary

metabolites. Extraction yield and the concentration of phenolics, flavonoids, and
saponins, as well as antioxidant potential were determined in extracts from
different parts of M. sativa obtained using extraction methods such as
maceration, ultrasound-assisted extraction (UAE), accelerated solvent extraction
(ASE), and supercritical fluid extraction (SFE). The concentrations of the listed
groups of compounds were spectrophotometrically determined and confirmed by
HPLC-MS. The results showed that ASE of flowers with 70% ethanol (EtOH) provided
the highest yield of extraction (47.5 ± 4.0%), whereas the lowest yield was
obtained in stems (4.0 ± 0.2%). The 70% EtOH extract from flowers showed the
highest phenolic content [48.4 ± 4.6 mg gallic acid equivalents/g dry matter
(DM)], as well as the highest antioxidant activity. The highest total flavonoid
content (139.0 ± 7.1 mg rutin equivalents/g DM) was observed in the extract from
leaves obtained through SFE. This extract was also especially rich in saponins
[622.2 ± 30.3 mg oleanolic acid equivalents (OAE)/g DM]. However, the lowest
compound content was observed in maceration extracts from stems (54.6 ± 27.0 mg
OAE/g DM). The results suggest that EtOH extracts from alfalfa flowers and SFE
extracts from M. satvia leaves, especially, may serve as potential sources of
natural antioxidants for nutraceuticals, food additives, and cosmetic
ingredients.
DOI: 10.5740/jaoacint.17-0234
PMID: 28791945
79. J Med Food. 2017 Oct;20(10):1039-1046. doi: 10.1089/jmf.2017.0046. Epub 2017

Jul
27.
Nutraceutical Potential of New Alfalfa (Medicago sativa) Ingredients for Beverage

Preparations.
Soto-Zarazúa MG(1)(2), Bah M(1), Costa ASG(2), Rodrigues F(2), Pimentel FB(2),
Rojas-Molina I(1), Rojas A(1), Oliveira MBPP(2).
Author information:
(1)1 Faculty of Chemistry, Autonomous University of Querétaro, Cerro de las
Campanas, Querétaro, Qro., Mexico .
(2)2 LAQV/REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy,
University of Porto , Porto, Portugal .
Alfalfa (Medicago sativa) has been extensively used as animal feed, due to its
fiber, protein, minerals, and vitamins, being also a useful source of phenolic
compounds with potential therapeutic benefits. Nevertheless, its potential use as
human ingredient is scarce. The aim of this work was to assess the nutritional
composition, amino acid profile, and antioxidant capacity (AOC) of freeze-dried
juice (FDJ) and fibrous residual material (RM), two new alfalfa-derived products
(Adps) recently launched as ingredients for beverage preparations. Results
demonstrated a high content of proteins (23-30 g/100 g FDJ and 13-17 g/100 g RM),
crude fiber (29 g/100 g RM), and minerals (such as sodium, calcium, iron, and
zinc). No significant difference was found in caloric content (4 kcal/g).
Essential and nonessential amino acids were quantified in both Adps being leucine
and lysine the most abundant. Total phenolic and total flavonoid contents (TPC
and TFC, respectively) and their changes along the different harvesting periods
of the year were also examined. FDJ presented the highest TPC in May (19 mg
gallic acid equivalents/g dry weight [dw]), while in October TFC had the maximum
value (4 mg catechin equivalents/g dw). Both products exhibited an interesting
AOC by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power
assays. This study reports the nutraceutical potential of two new types of Adps.
DOI: 10.1089/jmf.2017.0046
80. Anticancer Agents Med Chem. 2017 Nov 24;17(11):1508-1518. doi:

10.2174/1871520617666170727152805.
Activity of Saponins from Medicago species Against HeLa and MCF-7 Cell Lines and
their Capacity to Potentiate Cisplatin Effect.
Avato P(1), Migoni D(2), Argentieri M(1), Fanizzi FP(2), Tava A(3).
Author information:
(1)Dipartimento di Farmacia- Scienze del Farmaco, Via Orabona 4, 70125 Bari,
Italy.
(2)DISTEBA, Universita del Salento, Via Monteroni, 73100 Lecce, Italy.
(3)Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria -
Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (CREA-FLC),
viale Piacenza 29, 26900 Lodi, Italy.
BACKGROUND: Saponins from Medicago species display several biological activities,
among them apoptotic effects against plant cells have been evidenced. In
contrast, their cytotoxic and antitumor activity against animal cells have not
been studied in great details.
OBJECTIVE: To explore the cytotoxic properties of saponin from Medicago species
against animal cells and their effect in combination with the antitumoral drug
cisplatin.
METHOD: Cytotoxic activity of saponin mixtures from M. arabica (tops and roots),
M. arborea (tops) and M. sativa (tops, roots and seeds) and related prosapogenins
from M. arborea and M. sativa (tops) against HeLa and MCF-7 cell lines is
described. In addition, cytotoxicity of soyasaponin I and purified saponins (1-8)
of hederagenin, medicagenic and zanhic acid is also presented. Combination
experiments with cisplatin have been also conducted.
RESULTS: Saponins from M. arabica tops and roots (mainly monodesmosides of
hederagenin and bayogenin) were the most effective to reduce proliferation of
HeLa and MCF-7 cell lines. Among the purified saponins, the most cytotoxic was
saponin 1, 3-O-ß-D-glucopyranosyl(1→2)-α-L-arabinopyranosyl hederagenin. When
saponins, derived prosapogenins and pure saponins were used in combination with
cisplatin, they all, to different extent, were able to potentiate cisplatin
activity against HeLa cells but not against MCF-7 cell lines. Moreover uptake of
cisplatin in these cell lines was significantly reduced.
CONCLUSION: Overall results showed that specific molecular types of saponins
(hederagenin glycosides) have potential as anti-cancer agents or as leads for
anti-cancer agents. Moreover saponins from Medicago species have evidenced
interesting properties to mediate cisplatin effects in tumor cell lines.
Copyright© Bentham Science Publishers; For any queries, please email at

epub@benthamscience.org.
DOI: 10.2174/1871520617666170727152805
81. Front Plant Sci. 2017 Jul 11;8:1208. doi: 10.3389/fpls.2017.01208. eCollection
2017.
Metabolomic Analysis of Alfalfa (Medicago sativa L.) Root-Symbiotic Rhizobia

Responses under Alkali Stress.
Song T(1), Xu H(2), Sun N(2), Jiang L(2), Tian P(2), Yong Y(2), Yang W(2), Cai
H(2), Cui G(1).
Author information:
(1)College of Animal Sciences and Technology, Northeast Agricultural
UniversityHarbin, China.
(2)College of Life Sciences, Northeast Agricultural UniversityHarbin, China.
Alkaline salts (e.g., NaHCO3 and Na2CO3) causes more severe morphological and
physiological damage to plants than neutral salts (e.g., NaCl and Na2SO4) due to
differences in pH. The mechanism by which plants respond to alkali stress is not
fully understood, especially in plants having symbotic relationships such as
alfalfa (Medicago sativa L.). Therefore, a study was designed to evaluate the
metabolic response of the root-nodule symbiosis in alfalfa under alkali stress
using comparative metabolomics. Rhizobium-nodulized (RI group) and non-nodulized
(NI group) alfalfa roots were treated with 200 mmol/L NaHCO3 and, roots samples
were analyzed for malondialdehydyde (MDA), proline, glutathione (GSH), superoxide
dismutase (SOD), and peroxidase (POD) content. Additionally, metabolite profiling
was conducted using gas chromatography combined with time-of-flight mass
spectrometry (GC/TOF-MS). Phenotypically, the RI alfalfa exhibited a greater
resistance to alkali stress than the NI plants examined. Physiological analysis
and metabolic profiling revealed that RI plants accumulated more antioxidants
(SOD, POD, GSH), osmolytes (sugar, glycols, proline), organic acids (succinic
acid, fumaric acid, and alpha-ketoglutaric acid), and metabolites that are
involved in nitrogen fixation. Our pairwise metabolomics comparisons revealed
that RI alfalfa plants exhibited a distinct metabolic profile associated with
alkali putative tolerance relative to NI alfalfa plants. Data provide new
information about the relationship between non-nodulized, rhizobium-nodulized
alfalfa and alkali resistance.
DOI: 10.3389/fpls.2017.01208
PMCID: PMC5504246
PMID: 28744296
82. Front Microbiol. 2017 Jul 6;8:1236. doi: 10.3389/fmicb.2017.01236. eCollection

2017.
Transcriptomic Insight in the Control of Legume Root Secondary Infection by the

Sinorhizobium meliloti Transcriptional Regulator Clr.
Zou L(1), Gastebois A(1), Mathieu-Demazière C(1), Sorroche F(1), Masson-Boivin

C(1), Batut J(1), Garnerone AM(1).
Author information:
(1)LIPM, Université de Toulouse, INRA, CNRSCastanet-Tolosan, France.
The cAMP-dependent transcriptional regulator Clr of Sinorhizobium meliloti

regulates the overall number of infection events on Medicago roots by a so-far
unknown mechanism requiring smc02178, a Clr-target gene of unknown function. In
order to shed light on the mode of action of Clr on infection and potentially
reveal additional biological functions for Clr, we inventoried genomic Clr target
genes by transcriptome profiling. We have found that Clr positively controls the
synthesis of cAMP-dependent succinoglycan as well as the expression of genes
involved in the synthesis of a so-far unknown polysaccharide compound. In
addition, Clr activated expression of 24 genes of unknown function in addition to
smc02178. Genes negatively controlled by Clr were mainly involved in swimming
motility and chemotaxis. Functional characterization of two novel Clr-activated
genes of unknown function, smb20495 and smc02177, showed that their expression
was activated by the same plant signal as smc02178 ex planta. In planta, however,
symbiotic expression of smc02177 proved independent of clr. Both smc02177 and
smb20495 genes were strictly required for the control of secondary infection on
M. sativa. None of the three smc02177, smc02178 and smb20495 genes were needed
for plant signal perception. Altogether this work provides a refined view of the
cAMP-dependent Clr regulon of S. meliloti. We specifically discuss the possible
roles of smc02177, smc02178, smb20495 genes and other Clr-controlled genes in the
control of secondary infection of Medicago roots.
DOI: 10.3389/fmicb.2017.01236
PMCID: PMC5498481
PMID: 28729859
83. Asian-Australas J Anim Sci. 2018 Feb;31(2):270-277. doi: 10.5713/ajas.17.0284.

Epub 2017 Jun 27.
Effects of dietary alfalfa flavonoids on the performance, meat quality and lipid
oxidation of growing rabbits.
Dabbou S(1), Gasco L(2)(3), Rotolo L(2), Pozzo L(4), Tong JM(5), Dong XF(5),
Rubiolo P(6), Schiavone A(1)(3), Gai F(3).
Author information:
(1)Department of Veterinary Sciences, University of Turin, 10095 Grugliasco,
Italy.
(2)Department of Agricultural, Forest and Food Sciences, University of Turin,
10095 Grugliasco, Italy.
(3)Institute of Science of Food Production, National Research Council, 10095
Grugliasco, Italy.
(4)Institute of Agricultural Biology and Biotechnology, National Research
Council, 56124 Pisa, Italy.
(5)Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing
100193, China.
(6)Department of Drug Science and Technology, University of Turin, 10125 Torino,
Italy.
OBJECTIVE: The present experiment has tested the effect of dietary alfalfa
flavonoids (AAF) supplementation on the productive performances, carcass
characteristics, meat quality and lipid oxidation of growing rabbits.
METHODS: One hundred and sixty crossbred rabbits (42 days old) were divided into
four groups of forty animals each and were fed either a control diet (AAF0) or an
AAF0 diet supplemented with 400, 800, or 1,200 mg of AAF/kg per diet (AAF4, AAF8,
and AAF12, respectively) from weaning to slaughtering (102 days old). Performance
data were recorded over a period of 60 days. At the end of the trial, 12 rabbits
were slaughtered per group, and the carcass characteristics were recorded.
Moreover, the plasma, liver and dorsal muscles were sampled from 12
rabbits/group, and were analyzed for lipid oxidation.
RESULTS: No significant differences were recorded for the performance, carcass
characteristics and meat quality traits except for lightness parameter that was
lower in the control group. Dietary AAF supplementation significantly (p<0.01)
affected the malondialdehyde (MDA) levels of the frozen meat in a dose-related
manner, with the lowest value (0.24 mg MDA/kg fresh meat) recorded in the AAF12
group samples.
CONCLUSION: These findings indicated that the dietary inclusion of AAF in rabbit
diets improved muscle oxidation stability with no adverse effects on the growth
performance of the animals even if a slight impact on meat lightness color
parameter was recorded.
DOI: 10.5713/ajas.17.0284
PMCID: PMC5767510
PMID: 28728357
84. J Anim Sci. 2017 Jul;95(7):2859-2870. doi: 10.2527/jas.2017.1502.
Effects of intravenous lipopolysaccharide administration on feed intake, ruminal

forage degradability, and liquid parameters and physiological responses in beef
cattle.
Lippolis KD, Cooke RF, Schubach KM, Marques RS, Bohnert DW.
This experiment compared DMI, ruminal forage degradability, and liquid parameters
as well as physiological responses in beef cattle receiving a lipopolysaccharide
(LPS) challenge or not. Eight ruminally cannulated Angus × Hereford steers (485 ±
16 kg BW) were housed in individual pens on d -7, ranked by BW, and allocated to
1 of 2 treatments administered on d 0: 1) an intravenous (i.v.) bolus dose (0.5
μg/kg of BW, diluted in 5 mL of 0.9% sterile saline) of bacterial LPS ( 0111:B4)
or 2) a 5-mL i.v. injection of 0.9% sterile saline (CON). Steers had free-choice
access to mixed alfalfa-grass hay, water, and a commercial vitamin + mineral mix
during the experiment (d -7 to 6). Hay DMI was evaluated daily from d -5 to 6.
Immediately prior to treatment administration (h 0), polyester bags containing 4
g of ground dietary hay (DM basis) were immersed into the rumen of each steer and
incubated for 0, 4, 8, 12, 24, 36, and 48 h for DM and NDF degradability
evaluation. Steers were also intraruminally pulse-dosed with 5 g of Co-EDTA
immediately prior to treatment administration, and rumen fluid samples were
collected at 0, 2, 4, 6, 8, 12, 16, and 24 h for ruminal liquid volume and
dilution rate calculations. Blood was collected every 2 h from -2 to 8 h, every 4
h from 8 to 16 h, every 12 h from 24 to 72 h, and every 24 h from 96 to 144 h
relative to treatment administration. Values obtained before treatment
administration were used as a covariate within each respective analysis. Steers
receiving LPS had less ( ≤ 0.03) DMI on d 0 and 1 compared with CON steers.
Steers receiving LPS had reduced ( ≤ 0.05) rumen liquid volume and dilution rate
as well as ruminal disappearance rate and effective degradability of DM and NDF
compared with CON steers. Steers receiving LPS had greater ( ≤ 0.05) plasma tumor
necrosis factor α at 2 h, greater plasma haptoglobin from 24 to 72 h, greater
plasma cortisol from 12 to 16 h, greater serum NEFA from 6 to 48 h, greater
plasma insulin and glucose at 2 h, reduced plasma glucose from 4 to 12 h, greater
plasma cholecystokinin at 16 h, and greater plasma leptin concentrations at 8,
12, 16, 36, 48, and 60 h after treatment administration compared with CON steers.
Hence, LPS administration transiently reduced DMI in steers via physiological
reactions that modulate gastrointestinal motility and satiety centers in the
central nervous system, in addition to potential host-microbiome endocrine
interactions that impaired ruminal hay DM and NDF degradability.
DOI: 10.2527/jas.2017.1502
85. J Anim Sci. 2017 Jun;95(6):2408-2420. doi: 10.2527/jas.2017.1377.
Effects of feeding pregnant beef cows selenium-enriched alfalfa hay on selenium

status and antibody titers in their newborn calves.
Wallace LG, Bobe G, Vorachek WR, Dolan BP, Estill CT, Pirelli GJ, Hall JA.
In newborn dairy calves, it has been demonstrated that supranutritional maternal

and colostral Se supplementation using Se yeast or sodium selenite, respectively,
improves passive transfer of IgG. In beef cattle, agronomic biofortification with
Se is a more practical alternative for Se supplementation, whereby the Se
concentration of hay is increased through the use of Se-containing fertilizer
amendments. It has been previously demonstrated that agronomic Se
biofortification is an effective strategy to improve immunity and performance in
Se-replete weaned beef calves. The objective of this experiment was to determine
the effects of feeding beef cows Se-enriched alfalfa () hay during the last 8 to
12 wk of gestation on passive transfer of antibodies to calves. At 10 wk ± 16 d
before calving, 45 cows were assigned to 1 of 3 treatment groups with 3 pens (5
cows/pen) per treatment: Control cows were fed non-Se-fortified alfalfa hay plus
a mineral supplement containing 120 mg/kg Se from sodium selenite, Med-Se cows
were fed alfalfa hay fertilized with 45.0 g Se/ha as sodium selenate, and High-Se
cows were fed alfalfa hay fertilized with 89.9 g Se/ha as sodium selenate; both
the Med-Se and the High-Se groups received mineral supplement without added Se.
Colostrum and whole blood (WB) were collected from cows at calving, and WB was
collected from calves within 2 h of calving and at 12, 24, 36, and 48 h of age.
Concentrations of IgG1 and J-5 antibody in cow colostrum and calf serum were
quantified using ELISA procedures. Selenium concentrations linearly increased in
WB ( < 0.001) and colostrum ( < 0.001) of cows and in WB of newborn calves ( <
0.001) with increasing Se concentration in alfalfa hay. Colostrum concentrations
of IgG1 ( = 0.03) were increased in cows fed Se-biofortified alfalfa hay, but J-5
antibody ( = 0.43) concentrations were not. Calf serum IgG1 ( = 0.43) and J-5
antibody ( = 0.44) concentrations during the first 48 h of age were not affected
by prior Se treatment of cows. These data suggest that feeding Se-biofortified
alfalfa hay promotes the accumulation of Se and antibodies in colostrum but does
not affect short-term serum antibody concentrations in calves.
DOI: 10.2527/jas.2017.1377
86. J Anim Sci. 2017 Jun;95(6):2558-2575. doi: 10.2527/jas.2017.1449.
Supplementation of glycerol or fructose via drinking water to grazing lambs on

tissue glycogen level and lipogenesis.
Volpi-Lagreca G, Duckett SK.
Lambs ( = 18; 40.1 ± 7.4 kg BW) were used to assess supplementation of glycerol
or fructose via drinking water on growth, tissue glycogen levels, postmortem
glycolysis, and lipogenesis. Lambs were blocked by BW and allocated to alfalfa
paddocks (2 lambs/paddock and 3 paddocks/treatment). Each paddock within a block
was assigned randomly to drinking water treatments for 30 d: 1) control (CON), 2)
120 g fructose/L of drinking water (FRU), or 3) 120 g glycerol/L of drinking
water (GLY). Lambs grazed alfalfa with free access to water treatments for 28 d
and then were fasted in indoor pens for a final 2 d with access to only water
treatments. Data were analyzed using the MIXED procedure of SAS with water
treatment and time (when appropriate) in the model. During the 28-d grazing
period, ADG was greater ( < 0.05) for GLY than for CON or FRU. During the 2-d
fasting period, BW shrink was lower ( < 0.05) for GLY compared with CON or FRU.
Hot carcass weight was greater ( < 0.05) for GLY than for FRU. The interaction
for glycogen content × postmortem time was significant ( = 0.003) in LM and
semitendinosus (ST) muscles. Glycogen content in the LM was greater ( < 0.05) for
GLY at 2 and 3 h and for FRU at 1 h postmortem compared with CON. Glycogen
content in ST did not differ between treatments ( > 0.05). Liver glycogen content
was over 14-fold greater ( < 0.05) for GLY compared with FRU or CON. Liver free
glucose was greater ( < 0.05) for GLY than for CON, whereas liver lipid content
was higher ( < 0.05) for CON than for GLY. Supplementation with GLY increased ( <
0.05) odd-chain fatty acids in LM, subcutaneous fat (SQ), and the liver. Stearic
acid (C18:0) concentrations were reduced in LM ( = 0.064) and subcutaneous
adipose tissue (SQ; = 0.018), whereas oleic acid (C18:1 -9) concentration tended
to be increased ( = 0.066) in SQ with FRU and GLY. Linolenic acid (C18:3 -3) was
reduced ( = 0.031) and all long-chain -3 fatty acid (eicosapentaenoic acid,
docosapentaenoic acid, and docosahexaenoic acid) concentrations were increased (
< 0.05) with FRU and GLY compared with CON. Glycerol supplementation upregulated
( < 0.05) stearoyl-CoA desaturate () and fatty acid synthase () mRNA by over
40-fold in the SQ and 5-fold in the liver. Glycerol supplementation also
upregulated ( < 0.05) glucose transporters and glycogen branching enzyme in the
liver. Overall, glycerol supplementation improved growth, reduced BW shrink
during fasting, increased glycogen content in muscle and the liver, and
stimulated de novo lipogenesis.
DOI: 10.2527/jas.2017.1449
87. J Anim Sci. 2017 May;95(5):2156-2167. doi: 10.2527/jas.2017.1403.
Potential for improving fiber digestion in the rumen of cattle (Bos taurus)
through microbial inoculation from bison (Bison bison): In situ fiber
degradation.
Griffith C, Ribeiro GO, Oba M, McAllister TA, Beauchemin KA.
The objective of this experiment was to determine if partial replacement of

cattle rumen contents with those from bison would increase in situ ruminal fiber
degradation of various forages. The second objective was to examine individual
variation among cattle in their ability to degrade forage and their responses to
inoculation. In situ degradation of barley straw, canola straw, alfalfa hay, and
timothy hay was measured in 16 ruminally cannulated heifers fed a barley
straw-based diet before and after inoculation with combined rumen contents from
32 bison (performed twice, 14 d apart). Each feed was incubated in the rumen of
each heifer for 0, 4, 8, 12, 24, 48, 96, and 120 h, and the degradation
parameters were determined as washout fraction (), potentially degradable
fraction (), rate of digestion of fraction (), and total potentially degradable
fraction (). The of barley straw decreased ( = 0.04) after inoculation, whereas
fraction of NDF increased ( = 0.03) and fraction of NDF and ADF decreased ( ≤
0.02) by inoculation. In contrast, of alfalfa hay NDF and ADF decreased ( =
0.002) after inoculation, but fraction of NDF and ADF ( ≤ 0.02) increased. There
were no major effects ( > 0.06) of inoculation on the fiber degradation of
timothy hay or canola straw. The differential response between barley straw and
alfalfa hay may have occurred because the cattle were previously adapted to a
barley straw diet, whereas the bison were fed barley silage and oats. Some
animals consistently ranked higher or lower for or across at least 3 of the 4
feeds incubated, but the rankings changed after inoculation. In conclusion,
inoculation of cattle with bison rumen contents failed to improve degradation of
fiber from barley straw, canola straw, or timothy hay in cattle well adapted to a
barley straw diet, although there were small improvements in the extent of
degradation of fiber from alfalfa hay. Cattle varied both in their ability to
degrade various forages and in their responses to inoculation with bison rumen
contents.
DOI: 10.2527/jas.2017.1403
88. PLoS One. 2017 Jul 18;12(7):e0181589. doi: 10.1371/journal.pone.0181589.

eCollection 2017.
Salt-tolerant and -sensitive alfalfa (Medicago sativa) cultivars have large

variations in defense responses to the lepidopteran insect Spodoptera litura
under normal and salt stress condition.
Lei Y(1)(2)(3), Liu Q(2), Hettenhausen C(3), Cao G(3), Tan Q(3), Zhao W(3), Lin
H(1), Wu J(3).
Author information:
Engineering, Sichuan University, Chengdu, China.
(2)Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization
& Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan
Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu,
China.
Wild Plant Resources, Kunming institute of Botany, Chinese Academy of Sciences,
Kunming, China.
In nature, plants are often exposed to multiple stress factors at the same time.
Yet, little is known about how plants modulate their physiology to counteract
simultaneous abiotic and biotic stresses, such as soil salinity and insect
herbivory. In this study, insect performance bioassays, phytohormone
measurements, quantification of transcripts, and protein determination were
employed to study the phenotypic variations of two alfalfa (Medicago sativa)
cultivars in response to insect Spodoptera litura feeding under normal and salt
stress condition. When being cultivated in normal soil, the salt-tolerant alfalfa
cultivar Zhongmu-1 exhibited lower insect resistance than did the salt-sensitive
cultivar Xinjiang Daye. Under salinity stress, the defense responses of Xinjiang
Daye were repressed, whereas Zhongmu-1 did not show changes in resistance levels.
It is likely that salinity influenced the resistance of Xinjiang Daye through
suppressing the accumulation of jasmonic acid-isoleucine (JA-Ile), which is the
bioactive hormone inducing herbivore defense responses, leading to attenuated
trypsin proteinase inhibitor (TPI) activity. Furthermore, exogenous ABA
supplementation suppressed the insect herbivory-induced JA/JA-Ile accumulation
and levels of JAR1 (jasmonate resistant 1) and TPI, and further decreased the
resistance of Xinjiang Daye, whereas Zhongmu-1 showed very little response to the
increased ABA level. We propose a mechanism, in which high levels of abscisic
acid induced by salt treatment may affect the expression levels of JAR1 and
consequently decrease JA-Ile accumulation and thus partly suppress the defense of
Xinjiang Daye against insects under salt stress. This study provides new insight
into the mechanism by which alfalfa responds to concurrent abiotic and biotic
stresses.
PMCID: PMC5515460
89. Front Plant Sci. 2017 Jun 29;8:1152. doi: 10.3389/fpls.2017.01152. eCollection
2017.
Identification of Single-Nucleotide Polymorphic Loci Associated with Biomass

Yield under Water Deficit in Alfalfa (Medicago sativa L.) Using Genome-Wide
Sequencing and Association Mapping.
Yu LX(1).
Author information:
(1)United States Department of Agriculture-Agricultural Research Service, Plant
Germplasm Introduction Testing and ResearchProsser, WA, United States.
Alfalfa is a worldwide grown forage crop and is important due to its high biomass
production and nutritional value. However, the production of alfalfa is
challenged by adverse environmental factors such as drought and other stresses.
Developing drought resistance alfalfa is an important breeding target for
enhancing alfalfa productivity in arid and semi-arid regions. In the present
study, we used genotyping-by-sequencing and genome-wide association to identify
marker loci associated with biomass yield under drought in the field in a panel
of diverse germplasm of alfalfa. A total of 28 markers at 22 genetic loci were
associated with yield under water deficit, whereas only four markers associated
with the same trait under well-watered condition. Comparisons of marker-trait
associations between water deficit and well-watered conditions showed
non-similarity except one. Most of the markers were identical across harvest
periods within the treatment, although different levels of significance were
found among the three harvests. The loci associated with biomass yield under
water deficit located throughout all chromosomes in the alfalfa genome agreed
with previous reports. Our results suggest that biomass yield under drought is a
complex quantitative trait with polygenic inheritance and may involve a different
mechanism compared to that of non-stress. BLAST searches of the flanking
sequences of the associated loci against DNA databases revealed several
stress-responsive genes linked to the drought resistance loci, including
leucine-rich repeat receptor-like kinase, B3 DNA-binding domain protein,
translation initiation factor IF2, and phospholipase-like protein. With further
investigation, those markers closely linked to drought resistance can be used for
MAS to accelerate the development of new alfalfa cultivars with improved
resistance to drought and other abiotic stresses.
DOI: 10.3389/fpls.2017.01152
PMCID: PMC5489703
PMID: 28706532
90. Plants (Basel). 2017 Jul 11;6(3). pii: E29. doi: 10.3390/plants6030029.
Alfalfa Responses to Gypsum Application Measured Using Undisturbed Soil Columns.
Tirado-Corbalá R(1)(2), Slater BK(3), Dick WA(4), Barker D(5).
Author information:
(1)School of Environment and Natural Resources, The Ohio State University,
Columbus, OH 43210, USA. rebecca.tirado@upr.edu.
(2)Agro-Environmental Science Department, University of Puerto Rico, Mayagüez,
Puerto Rico, 00681, USA. rebecca.tirado@upr.edu.
Columbus, OH 43210, USA. slater.39@osu.edu.
(4)School of Environment and Natural Resources, The Ohio State University, 1680
Madison Avenue, Wooster, OH 44691, USA. dick.5@osu.edu.
(5)Department of Horticulture and Crop Science, The Ohio State University,
Columbus, OH 43210, USA. barker.169@osu.edu.
Gypsum is an excellent source of Ca and S, both of which are required for crop
growth. Large amounts of by-product gypsum [Flue gas desulfurization
gypsum-(FGDG)] are produced from coal combustion in the United States, but only
4% is used for agricultural purposes. The objective of this study was to evaluate
the effects of (1) untreated, (2) short-term (4-year annual applications of
gypsum totaling 6720 kg ha-1), and (3) long-term (12-year annual applications of
gypsum totaling 20,200 kg ha-1) on alfalfa (Medicago sativa L.) growth and
nutrient uptake, and gypsum movement through soil. The study was conducted in a
greenhouse using undisturbed soil columns of two non-sodic soils (Celina silt
loam and Brookston loam). Aboveground growth of alfalfa was not affected by
gypsum treatments when compared with untreated (p > 0.05). Total root biomass
(0-75 cm) for both soils series was significantly increased by gypsum application
(p = 0.04), however, increased root growth was restricted to 0-10 cm depth. Soil
and plant analyses indicated no unfavorable environmental impact from of the
4-year and 12-year annual application of FGDG. We concluded that under sufficient
water supply, by-product gypsum is a viable source of Ca and S for land
application that might benefit alfalfa root growth, but has less effect on
aboveground alfalfa biomass production. Undisturbed soil columns were a useful
adaptation of the lysimeter method that allowed detailed measurements of alfalfa
nutrient uptake, root biomass, and yield and nutrient movement in soil.
DOI: 10.3390/plants6030029
PMCID: PMC5620585
PMID: 28696383

91. Ecol Evol. 2017 May 30;7(13):4907-4918. doi: 10.1002/ece3.3028. eCollection
2017
Jul.
Combining field experiments and predictive models to assess potential for

increased plant diversity to climate-proof intensive agriculture.
Mason NWH(1), Palmer DJ(1), Romera A(2), Waugh D(2), Mudge PL(1).
Author information:
(1)Landcare ResearchHamiltonNew Zealand.
(2)DairyNZHamiltonNew Zealand.
Agricultural production systems face increasing threats from more frequent and
extreme weather fluctuations associated with global climate change. While there
is mounting evidence that increased plant community diversity can reduce the
variability of ecosystem functions (such as primary productivity) in the face of
environmental fluctuation, there has been little work testing whether this is
true for intensively managed agricultural systems. Using statistical modeling
techniques to fit environment-productivity relationships offers an efficient
means of leveraging hard-won experimental data to compare the potential
variability of different mixtures across a wide range of environmental contexts.
We used data from two multiyear field experiments to fit
climate-soil-productivity models for two pasture mixtures under intensive
grazing-one composed of two drought-sensitive species (standard), and an
eight-species mixture including several drought-resistant species (complex). We
then used these models to undertake a scoping study estimating the mean and
coefficient of variation (CV) of annual productivity for long-term climate data
covering all New Zealand on soils with low, medium, or high water-holding
capacity. Our results suggest that the complex mixture is likely to have
consistently lower CV in productivity, irrespective of soil type or climate
regime. Predicted differences in mean annual productivity between mixtures were
strongly influenced by soil type and were closely linked to mean annual soil
water availability across all soil types. Differences in the CV of productivity
were only strongly related to interannual variance in water availability for the
lowest water-holding capacity soil. Our results show that there is considerable
scope for mixtures including drought-tolerant species to enhance certainty in
intensive pastoral systems. This provides justification for investing resources
in a large-scale distributed experiment involving many sites under different
environmental contexts to confirm these findings.
DOI: 10.1002/ece3.3028
PMCID: PMC5496536
PMID: 28690818
92. J Dairy Sci. 2017 Sep;100(9):7127-7138. doi: 10.3168/jds.2017-12914. Epub 2017

Jul 6.
Effects of replacing maize silage with lucerne silage and lucerne silage chop
length on rumen function and milk fatty acid composition.
Thomson AL(1), Humphries DJ(1), Kliem KE(1), Dittmann MT(1), Reynolds CK(2).
Author information:
(1)Centre for Dairy Research, School of Agriculture, Policy and Development,
University of Reading, PO Box 237, Earley Gate, Reading, RG6 6AR, United Kingdom.
(2)Centre for Dairy Research, School of Agriculture, Policy and Development,
University of Reading, PO Box 237, Earley Gate, Reading, RG6 6AR, United Kingdom.
Electronic address: c.k.reynolds@reading.ac.uk.
The objective of this study was to investigate whether higher lucerne (Medicago
sativa; alfalfa) silage inclusion rate and longer lucerne chop length improves
rumen function through increased provision of physically effective fiber, when
included in a maize and lucerne silage-based total mixed ration. Diets were
formulated to contain a 50:50 forage:concentrate ratio [dry matter (DM) basis]
and be isonitrogenous and contain equal levels of neutral detergent fiber (320
g/kg). The forage portion of the offered diets was composed of maize and lucerne
silage DM in proportions (wt/wt) of either 25:75 (high lucerne; HL) or 75:25 (low
lucerne; LL). Second-cut lucerne was harvested and conserved as silage at either
a long (L) or a short (S) chop length (geometric mean particle lengths of 9.0 and
14.3 mm, respectively). These variables were combined in a 2 × 2 factorial
arrangement to give 4 treatments (HLL, HLS, LLL, LLS), which were fed in a 4 × 4
Latin square design study to 4 rumen-cannulated, multiparous, Holstein dairy cows
in mid lactation. Effects on DM intake, chewing behavior, rumen volatile fatty
acid concentration, rumen pH, rumen and fecal particle size, milk production, and
milk fatty acid profile were measured. Longer chop length increased rumination
times per kilogram of DM intake (+2.8 min/kg) relative to the S chop length, with
HLL diets resulting in the most rumination chews. Rumen concentrations of total
volatile fatty acids, acetate, and n-valerate were higher for the HLS diet than
the other 3 diets, whereas rumen propionate concentration was lowest for the HLL
diet. Physically effective fiber (particles >4 mm) percentage in the rumen mat
was increased when L chop length was fed regardless of lucerne inclusion rate. No
effect of treatment was observed for milk yield, although milk protein
concentration was increased by L for the LL diet (+1.6 g/kg) and decreased by L
for the HLL diet (-1.4 g/kg). Milk fat concentrations of total cis-18:1 (+3.7
g/100 g of fatty acids) and 18:3 n-3 (+0.2 g/100 g of fatty acids) were greater
with HL. In conclusion, longer lucerne silage chop length increased time spent
ruminating per kilogram of DM intake, but had no effect on rumen pH in the
present study. Increasing dietary lucerne inclusion rate had no effects on
rumination activity or rumen pH, but decreased the ratio of n-6:n-3
polyunsaturated fatty acid concentrations in milk fat.

DOI: 10.3168/jds.2017-12914
PMID: 28690065
93. PLoS One. 2017 Jul 7;12(7):e0180424. doi: 10.1371/journal.pone.0180424.

eCollection 2017.
Plant-fed versus chemicals-fed rhizobacteria of Lucerne: Plant-only teabags

culture media not only increase culturability of rhizobacteria but also recover a
previously uncultured Lysobacter sp., Novosphingobium sp. and Pedobacter sp.
Hegazi NA(1), Sarhan MS(1), Fayez M(1), Patz S(2), Murphy BR(3), Ruppel S(2).
Author information:
(1)Environmental Studies and Research Unit (ESRU), Department of Microbiology,
Faculty of Agriculture, Cairo University, Giza, Egypt.
(2)Leibniz Institute of Vegetable and Ornamental Crops Großbeeren/ Erfurt e.V.
(IGZ), Großbeeren, Germany.
(3)Department of Botany, School of Natural Sciences, Trinity College Dublin,
Dublin, Ireland.
In an effort to axenically culture the previously uncultivable populations of the
rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags
culture media to mimic the nutritional matrix available in the rhizosphere. Here,
we show that culture media prepared from Lucerne powder teabags substantially
increased the cultivability of Lucerne rhizobacteria compared with a standard
nutrient agar, where we found that the cultivable populations significantly
increased by up to 60% of the total bacterial numbers as estimated by
Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of
16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis
(PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct
composition and separation of bacterial populations recovered on the plant-only
teabags culture media than those developed on a standard nutrient agar. Further,
the new plant medium gave preference to the micro-symbiont Sinorhizobium
meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most
closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The
present study may encourage other researchers to consider moving from the
well-established standard culture media to the challenging new plant-only culture
media. Such a move may reveal previously hidden members of rhizobacteria, and
help to further explore their potential environmental impacts.
PMCID: PMC5501534
94. Saudi J Biol Sci. 2017 Jul;24(5):1051-1055. doi: 10.1016/j.sjbs.2016.12.009.

Epub
2016 Dec 23.
Alfalfa (Medicago sativa L.) seed yield in relation to phosphorus fertilization

and honeybee pollination.
Al-Kahtani SN(1), Taha EA(1)(2), Al-Abdulsalam M(1).
Author information:
(1)Department of Arid Land Agriculture, College of Agriculture and Food Sciences,
King Faisal University, Al-Ahsa, Saudi Arabia.
(2)Department of Economic Entomology, College of Agriculture, Kafrelsheikh
University, Kafrelsheikh, Egypt.
This investigation was conducted at the Agricultural and Veterinary Training and
Research Station, King Faisal University, Al-Ahsa, Saudi Arabia, during the
alfalfa growing season in 2014. The study aimed to evaluate the impact of
phosphorus fertilization and honeybee pollination on alfalfa seed production. The
experiment was divided into 9 treatments of open pollination, honeybee
pollination, and non-pollination with three different levels (0, 300 or 600 kg
P2O5/ha/year) of triple super phosphate. All vegetative growth attributes of
Hassawi alfalfa were significantly higher in the non-insect pollination plots,
while the yield and yield component traits were significantly higher with either
open pollination or honeybee pollination in parallel with the increasing level of
phosphorus fertilizer up to 600 kg P2O5/ha/year in light salt-affected loamy sand
soils. There was no seed yield in Hassawi alfalfa without insect pollination.
Therefore, placing honeybee colonies near the fields of Hassawi alfalfa and
adding 600 kg P2O5/ha/year can increase seed production.
DOI: 10.1016/j.sjbs.2016.12.009
PMCID: PMC5478283
PMID: 28663703
95. Cell Biol Int. 2017 Jun 27. doi: 10.1002/cbin.10810. [Epub ahead of print]
Protein phosphatases potentially associated with regulation of microtubules,

their spatial structure reconstruction and analysis.
Samofalova DO(1), Karpov PA(1), Raevsky AV(1), Blume YB(1).
Author information:
(1)Institute of Food Biotechnology and Genomics, Natl. Academy of Sci. of
Ukraine, Osipovskogo str. 2a, Kyiv, 04123, Ukraine.
According to the sequence and profile comparison with known catalytic domains,
where identified protein phosphatases potentially involved in regulation of
microtubule dynamics and structure from Arabidopsis thaliana, Nicotiana tabacum,
Medicago sativa, Oryza sativa subsp. japonica, Zea mays, and Triticum aestivum.
Selected proteins were related to classical non-receptor,
serine/threonine-specific and dual protein phosphatases. By application of
template structures of human protein phosphatases, it was performed homology
modelling of the catalytic domains of 17 plant protein phosphatases. Based on the
results of the structural alignment, molecular dynamics, and conservatism in
positions of functionally importance, it was confirmed homology of selected plant
proteins and known protein phosphatases regulating structure and dynamics of
microtubules.
© 2017 International Federation for Cell Biology.
DOI: 10.1002/cbin.10810
PMID: 28653783
96. Chemosphere. 2017 Oct;184:892-899. doi: 10.1016/j.chemosphere.2017.06.081. Epub

2017 Jun 19.
Linking potential nitrification rates, nitrogen cycling genes and soil properties
after remediating the agricultural soil contaminated with heavy metal and
fungicide.
Zhang M(1), Bai SH(2), Tang L(3), Zhang Y(4), Teng Y(5), Xu Z(6).
Author information:
(1)Environmental Futures Research Institute, School of Natural Sciences, Griffith
University, Brisbane, Queensland, 4111, Australia; Key Laboratory of Soil
Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy
of Sciences, Nanjing, 210008, China. Electronic address: manyunzhang@126.com.
University, Brisbane, Queensland, 4111, Australia; GeneCology Research Centre,
Faculty of Science, Health, Education and Engineering, University of the Sunshine
Coast, Maroochydore DC, Queensland, 4558, Australia.
University, Brisbane, Queensland, 4111, Australia; University of Chinese Academy
of Sciences, Beijing, 100049, China.
University, Brisbane, Queensland, 4111, Australia.
Soil Science, Chinese Academy of Sciences, Nanjing, 210008, China. Electronic
address: yteng@issas.ac.cn.
University, Brisbane, Queensland, 4111, Australia. Electronic address:
zhihong.xu@griffith.edu.au.
Apart from the contaminant removal, the remediation of agricultural soil should
also pay more attention to soil nutrient retention and biogeochemical cycling.
This study aimed to evaluate changes of soil properties, potential nitrification
rates (PNRs), and functional gene abundances and link their relationships after
remediating co-contaminated agricultural soil with Medicago sativa L. (alfalfa)
planting, alone or together with biochar application. Compared with the control
(CK), alfalfa planting, alone or together with biochar application, could
significantly increase soil organic matter (SOM) contents and discrepantly affect
soil pH values. The PNRs of the amended treatments were significantly higher than
that of the CK. Moreover, alfalfa plantings also enhanced the abundances of
functional genes related to soil nitrification and denitrification, with the sole
exception of nosZ gene. Stepwise regression analysis revealed that the PNRs were
best described by the gene abundance ratios of AOB amoA/nifH and nirS gene
abundances. Compared with the CK, alfalfa planting, alone or with biochar
application, could restore nitrogen cycling in the co-contaminated agricultural
soil and enhance the PNRs via decreasing contaminant bio-availabilities and
increasing SOM contents and gene abundance ratios of AOB amoA/nifH.
97. Environ Microbiol. 2017 Sep;19(9):3423-3438. doi: 10.1111/1462-2920.13820. Epub

2017 Jul 13.
Specificity traits consistent with legume-rhizobia coevolution displayed by

Ensifer meliloti rhizosphere colonization.
Salas ME(1), Lozano MJ(1), López JL(1), Draghi WO(1), Serrania J(2), Torres
Tejerizo GA(1), Albicoro FJ(1), Nilsson JF(1), Pistorio M(1), Del Papa MF(1),
Parisi G(3), Becker A(2), Lagares A(1).
Author information:
(2)LOEWE Center for Synthetic Microbiology and Faculty of Biology, Philipps
University, Marburg, Germany.
(3)Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal,
Argentina.
Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to

fix atmospheric nitrogen. The chemical communication between roots and rhizobia
begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we
performed a genome-wide screening for Ensifer meliloti genes that participate in
colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca.
6,000 mutants indicated that genes relevant for rhizosphere colonization account
for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally
located, pointing to the relevance and ancestral origin of the bacterial ability
to colonize plant roots. The identified genes were related to metabolic
functions, transcription, signal transduction, and motility/chemotaxis among
other categories; with several ORFs of yet-unknown function. Most remarkably, we
identified a subset of genes that impacted more severely the colonization of the
roots of alfalfa than of pea. Further analyses using other plant species revealed
that such early differential phenotype could be extended to other members of the
Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae
tribes. The results suggest that consolidation of E. meliloti into its current
symbiotic state should have occurred in a rhizobacterium that had already been
adapted to rhizospheres of the Trifoliae tribe.
DOI: 10.1111/1462-2920.13820
98. Trop Anim Health Prod. 2017 Aug;49(6):1309-1316. doi: 10.1007/s11250-017-1330-

8.
Epub 2017 Jun 12.
Replacement of alfalfa hay (Medicago sativa L.) with subabul (Leucaena

leucocephala) leaf meal in diets of Najdi goats: effect on digestion activity of
rumen microorganisms.
Mohammadabadi T(1), Jolazadeh A(2).
Author information:
(1)Department of Animal Science, Ramin Agriculture and Natural Resource
University of Khouzestan, Mollasani, Khouzestan, 63517-73637, Iran.
t.mohammadabadi.t@gmail.com.
(2)Department of Animal Science, Ramin Agriculture and Natural Resource
University of Khouzestan, Mollasani, Khouzestan, 63517-73637, Iran.
This study investigated the effect of replacing alfalfa hay by subabul leaf meal
(SLM) on digestion, fermentation parameters and rumen bacteria and fungi activity
of Najdi goats. Six Najdi goats (150 ± 15 days of age and initial body weight of
35 ± 1.1 kg) were randomly assigned to one of two dietary treatments in a
balanced completely randomized design (three goats per treatment) for 56 days.
Experimental treatments included alfalfa hay as control group and diet containing
SLM (SLM replacing alfalfa hay at 50% level). Bacterial and fungi activity and
rumen fermentation parameters of animals fed experimental diets were determined.
Dry matter disappearance (DMD) was unaffected by replacing SLM with alfalfa hay
for both rumen bacteria and fungi in different incubation times, except for 48 h
of incubation in specific culture medium of mixed rumen bacteria, which decreased
for SLM group (P > 0.05). NDF disappearance (NDFD) and ADF disappearance (ADFD)
after 24 and 48 h of incubation in specific culture medium of mixed rumen
bacteria was not affected by experimental diets (P > 0.05). However, 72 h after
incubation, NDFD in SLM treatment decreased (P > 0.05). Gas production parameters
of rumen bacteria were similar for both experimental diets, but partitioning
factor (PF), efficiency microbial biomass production (EMBP), microbial protein
production (MP), and organic matter truly digested (OMTD) decreased (p < 0.05) in
specific culture medium of mixed rumen fungi for SLM diet. There was significant
decrease in total protozoa population for diet containing SLM (p < 0.05). Results
indicated that replacement of alfalfa hay by SLM had no major effect on rumen
microorganisms' activity of Najdi goats, so it may be used as an alternative for
alfalfa (at 50% level) in susceptible areas.
DOI: 10.1007/s11250-017-1330-8
99. Eur J Soil Sci. 2017 May;68(3):305-316. doi: 10.1111/ejss.12415. Epub 2017 Mar
21.
Changes in soil organic matter over 70 years in continuous arable and ley-arable
rotations on a sandy loam soil in England.
Johnston AE(1), Poulton PR(1), Coleman K(1), Macdonald AJ(1), White RP(2).
Author information:
(1)Department of Sustainable Soils and Grassland SystemsRothamsted ResearchWest
CommonHarpendenAL5 2JQUK.
(2)Department of Computational and Systems BiologyRothamsted ResearchWest
CommonHarpendenAL5 2JQUK.
The sequestration in soil of organic carbon (SOC) derived from atmospheric carbon
dioxide (CO2) by replacing arable crops with leys, has been measured over
70 years on a sandy loam soil. The experiment was designed initially to test the
effect of leys on the yields of arable crops. A 3-year grazed grass with clover
(grass + clover) ley in a 5-year rotation with arable crops increased percentage
organic carbon (%OC) in the top 25 cm of the soil from 0.98 to 1.23 in 28 years,
but with little further increase during the next 40 years with all-grass leys
given fertilizer nitrogen (N). In this second period, OC inputs were balanced by
losses, suggesting that about 1.3% OC might be near the equilibrium content for
this rotation. Including 3-year lucerne (Medicago sativa) leys had little effect
on %OC over 28 years, but after changing to grass + clover leys, %OC increased to
1.24 during the next 40 years. Eight-year leys (all grass with N or
grass + clover) in 10-year rotations with arable crops were started in the 1970s,
and after three rotations %OC had increased to ca. 1.40 in 2000-2009. Over
70 years, %OC declined from 0.98 to 0.94 in an all-arable rotation with mainly
cereals and to 0.82 with more root crops. Applications of 38 t ha-1 farmyard
manure (FYM) every fifth year increased %OC by 0.13% by the mid-1960s when
applications ceased. Soil treated with FYM still contained 0.10% more OC in
2000-2009. Changes in the amount of OC have been modelled with RothC-26.3 and
estimated inputs of C for selected rotations. Little of the OC input during the
70 years has been retained; most was retained in the grazed ley rotation, but
9 t ha-1 only of a total input of 189 t ha-1. In other rotations more than 98% of
the total OC input was lost. Despite large losses of C, annual increases in OC of
4‰ are possible on this soil type with the inclusion of grass or grass + clover
leys or the application of FYM, but only for a limited period. Such increases in
SOC might help to limit increases in atmospheric CO2.HIGHLIGHTS: Can leys
sequester significant amounts of atmospheric CO 2 in SOM and contribute to the 4‰
initiative?Changes in the percentage and amount of OC were measured and modelled
over 70 years and OC losses estimated.Three-year grass or grass + clover leys
increased %OC, but only to an equilibrium level that was then maintained.Despite
large losses, sequestering CO 2-C at 4‰ year-1 by growing grass or grass + clover
leys is possible.
DOI: 10.1111/ejss.12415
PMCID: PMC5439491
PMID: 28603450
100. Water Air Soil Pollut. 2017;228(6):215. doi: 10.1007/s11270-017-3399-0. Epub

2017
May 25.
Study of Heavy Metal Accumulation and Residual Toxicity in Soil Saturated with
Phosphate Processing Wastewater.
Mekki A(1), Sayadi S(1).

Author information:
(1)Laboratory of Environmental Bioprocesses, Center of Biotechnology of Sfax, AUF
(PER-LBP), BP: 1177, 3018 Sfax, Tunisia.
The effects of phosphate processing wastewater (PPWW) on heavy metal accumulation

in a Mediterranean soil (Tunisia, North Africa) were investigated. Moreover, the
residual toxicities of PPWW-irrigated soils extracts were assessed. Results
showed that heavy metal accumulation was significantly higher in PPWW-irrigated
soil extracts than in control soil. The heavy metal accumulation increased over
time in treated soil samples and their average values followed the following
order: Iron (Fe 252.72 mg l-1) > Zinc (Zn 152.95 mg l-1) > Lead (Pb
128.35 mg l-1) > Copper (Cu 116.82 mg l-1) > Cadmium (Cd 58.03 mg l-1). The
residual microtoxicity and phytotoxicity of the various treated soil samples
extracts were evaluated by monitoring the bioluminescence inhibition (BI %) of
Vibrio ficheri and the measurement of the germination indexes (GI %) of Lepidium
sativum and Medicago sativa seeds. The results showed an important increase of
residual toxicities of PPWW-treated soil extracts over time.
DOI: 10.1007/s11270-017-3399-0
PMCID: PMC5443876
PMID: 28603317
101. Mol Biol Rep. 2017 Jul;44(3):261-272. doi: 10.1007/s11033-017-4104-5. Epub

2017
Jun 9.
Comparative proteomic analysis of alfalfa revealed new salt and drought

stress-related factors involved in seed germination.
Ma Q(1)(2), Kang J(1)(3), Long R(3), Zhang T(3), Xiong J(4), Zhang K(1), Wang
T(1), Yang Q(3), Sun Y(5).
Author information:
(1)College of Animal Science and Technology, China Agricultural University,
100193, Beijing, China.
(2)School of Chemistry and Chemical Engineering, Ningxia University, 750021,
Yinchuan, China.
(3)Institute of Animal Sciences, Chinese Academy of Agricultural Sciences,
100193, Beijing, China.
(4)Institute of Animal Science, Hubei Academy of Agricultural Sciences, 430064,
Wuhan, China.
100193, Beijing, China. 02008@cau.edu.cn.
Salinity and drought are two major environmental factors that limit the growth
and yield of many forage crops in semi-arid and arid regions. Alfalfa (Medicago
sativa L.) is one of the most important forage crops in many countries. We aim to
investigate the molecular mechanisms of alfalfa in response to salt and drought
stresses in this study. Physiological and proteomic analyses were applied to
examine the Zhongmu NO.3 alfalfa seed germination stage with 200 mM NaCl and
180 g·L-1 polyethylene glycol (PEG) treatments. The germination ability of the
seed and the accumulation of osmotic solutes were quite different between the
NaCl and PEG treatments. More than 800 protein spots were detected by proteomics
technology on two-dimensional electrophoresis (2-DE) gels. The abundance of
twenty-eight proteins were decreased or increased after salt and drought stress.
Seventeen of these proteins were identified and classified into six functional
categories through mass spectrometry (MS). The six groups involved in salt- and
PEG-mediated stress included defense response, energy metabolism, protein
synthesis and degradation, oxidative stress, carbohydrate metabolism-associated
proteins, and unknown proteins. We discovered that some proteins related to
carbohydrate metabolism and energy production increased in abundance under salt-
and PEG-mediated drought stress. This demonstrates a common mechanism of energy
consumption during abiotic stresses. Further study of these proteins with unknown
function will provide insights into the molecular mechanisms of abiotic stress
and the discovery of new candidate markers.
DOI: 10.1007/s11033-017-4104-5
102. Front Plant Sci. 2017 May 24;8:853. doi: 10.3389/fpls.2017.00853. eCollection
2017.
Genome-Wide Association Mapping of Loci Associated with Plant Growth and Forage
Production under Salt Stress in Alfalfa (Medicago sativa L.).
Liu XP(1)(2), Yu LX(1).
Author information:
Germplasm Introduction and Testing ResearchProsser, WA, United States.
(2)College of Animal Science and Veterinary Medicine, Heilongjiang Bayi
Agricultural UniversityDaqing, China.
Salinity tolerance is highly desirable to sustain alfalfa production in marginal

lands that have been rendered saline. In this study, we used a diverse panel of
198 alfalfa accessions for mapping loci associated with plant growth and forage
production under salt stress using genome-wide association studies (GWAS). The
plants were genotyped using genotyping-by-sequencing (GBS). A greenhouse
procedure was used for phenotyping four agronomic and physiological traits
affected by salt stress, including dry weight (DW), plant height (PH), leaf
chlorophyll content (LCC), and stomatal conductance (SC). For each trait, a
stress susceptibility index (SSI) was used to evaluate plant performance under
stressed and non-stressed conditions. Marker-trait association identified a total
of 42 markers significantly associated with salt tolerance. They were located on
all chromosomes except chromosome 2 based on the alignment of their flanking
sequences to the reference genome (Medicago truncatula). Of those identified, 13
were associated with multiple traits. Several loci identified in the present
study were also identified in previous reports. BLAST search revealed that 19
putative candidate genes linked to 24 significant markers. Among them, B3
DNA-binding protein, Thiaminepyrophosphokinase and IQ calmodulin-binding motif
protein were identified among multiple traits in the present and previous
studies. With further investigation, these markers and candidates would be useful
for developing markers for marker-assisted selection in breeding programs to
improve alfalfa cultivars with enhanced tolerance to salt stress.
DOI: 10.3389/fpls.2017.00853
PMCID: PMC5442208
PMID: 28596776
103. PLoS One. 2017 Jun 8;12(6):e0179215. doi: 10.1371/journal.pone.0179215.

eCollection 2017.
Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal
microbiota compared with healthy controls.
Hall JA(1), Isaiah A(2), Estill CT(3)(4), Pirelli GJ(4), Suchodolski JS(2).
Author information:
(1)Department of Biomedical Sciences, College of Veterinary Medicine, Oregon
State University, Corvallis, Oregon, United States of America.
(2)Gastrointestinal Laboratory, College of Veterinary Medicine, Department of
Small Animal Clinical Sciences, Texas A&M University, College Station, Texas,
United States of America.
(3)Department of Clinical Sciences, College of Veterinary Medicine, Oregon State
University, Corvallis, Oregon, United States of America.
(4)Department of Animal and Rangeland Sciences, College of Agricultural Sciences,
Oregon State University, Corvallis, Oregon, United States of America.
Selenium (Se) is an essential trace mineral important for immune function and
overall health of cattle. The nasopharyngeal microbiota in cattle plays an
important role in overall respiratory health, especially when stresses associated
with weaning, transport, and adaptation to a feedlot affect the normal
respiratory defenses. Recent evidence suggests that cattle diagnosed with bovine
respiratory disease complex have significantly less bacterial diversity. The
objective of this study was to determine whether feeding weaned beef calves
Se-enriched alfalfa (Medicago sativa) hay for 9 weeks in a preconditioning
program prior to entering the feedlot alters nasal microbiota. Recently weaned
beef calves (n = 45) were blocked by sex and body weight, randomly assigned to 3
treatment groups with 3 pens of 5 calves per treatment group, and fed an alfalfa
hay based diet for 9 weeks. Alfalfa hay was harvested from fields fertilized with
sodium selenate at a rate of 0, 45.0 or 89.9 g Se/ha. Blood samples were
collected biweekly and analyzed for whole-blood Se concentrations. Nasal swabs
were collected during week 9 from one or two calves from each pen (total n = 16).
Calculated Se intake from dietary sources was 3.0, 15.6, and 32.2 mg Se/head/day
for calves consuming alfalfa hay with Se concentrations of 0.34 to 2.42 and 5.17
mg Se/kg dry matter, respectively. Whole-blood Se concentrations after 8 weeks of
feeding Se-fertilized alfalfa hay were dependent upon Se-application rates (0,
45.0, or 89.9 g Se/ha) and were 155, 345, and 504 ng/mL (PLinear < 0.0001).
Microbial DNA was extracted from nasal swabs and amplified and sequenced. Alpha
rarefaction curves comparing the species richness (observed OTUs) and overall
diversity (Chao1, Observed OTU, and Shannon index) between calves fed
selenium-biofortified alfalfa hay compared with control calves showed that
Se-supplementation tended to be associated with an enriched nasal microbiota.
ANOSIM of unweighted UniFrac distances showed that calves fed high
Se-biofortified alfalfa hay clustered separately when compared with control
calves in the PCoA plot (R = 0.216, P = 0.04). The bacterial orders
Lactobacillales and Flavobacteriales were increased in healthy control calves
compared with Clostridiales and Bacteroidales being increased in calves fed
Se-biofortified alfalfa hay. Although there were strong trends, no significant
differences were noted for any of the bacterial taxa. Based upon these findings,
we suggest that weaned beef calves fed Se-biofortified hay tend to have an
enriched nasal microbiota. Feeding Se-biofortified alfalfa hay to weaned beef
calves prior to entering the feedlot is a strategy for increasing nasopharyngeal
microbial diversity.
PMCID: PMC5464631
104. Plant Physiol. 2017 Aug;174(4):2445-2456. doi: 10.1104/pp.17.00278. Epub 2017

Jun
7.
ROOT DETERMINED NODULATION1 Is Required for M. truncatula CLE12, But Not CLE13,
Peptide Signaling through the SUNN Receptor Kinase.
Kassaw T(1), Nowak S(1), Schnabel E(1), Frugoli J(2).
Author information:
(1)Department of Genetics and Biochemistry, Clemson University, Clemson, South
Carolina 29630.
(2)Department of Genetics and Biochemistry, Clemson University, Clemson, South
Carolina 29630 jfrugol@clemson.edu.
The combinatorial interaction of a receptor kinase and a modified CLE peptide is

involved in several developmental processes in plants, including autoregulation
of nodulation (AON), which allows legumes to limit the number of root nodules
formed based on available nitrogen and previous rhizobial colonization. Evidence
supports the modification of CLE peptides by enzymes of the hydroxyproline
O-arabinosyltransferase (HPAT/RDN) family. Here, we show by grafting and genetic
analysis in Medicago truncatula that, in the AON pathway, RDN1, functioning in
the root, acts upstream of the receptor kinase SUNN, functioning in the shoot. As
expected for a glycosyltransferase, we found that RDN1 and RDN2 proteins are
localized to the Golgi, as was shown previously for AtHPAT1. Using composite
plants with transgenic hairy roots, we show that RDN1 and RDN2 orthologs from
dicots as well as a related RDN gene from rice (Oryza sativa) can rescue the
phenotype of rdn1-2 when expressed constitutively, but the less related MtRDN3
cannot. The timing of the induction of MtCLE12 and MtCLE13 peptide genes
(negative regulators of AON) in nodulating roots is not altered by the mutation
of RDN1 or SUNN, although expression levels are higher. Plants with transgenic
roots constitutively expressing MtCLE12 require both RDN1 and SUNN to prevent
nodule formation, while plants constitutively expressing MtCLE13 require only
SUNN, suggesting that the two CLEs have different requirements for function.
Combined with previous work, these data support a model in which RDN1
arabinosylates MtCLE12, and this modification is necessary for the transport
and/or reception of the AON signal by the SUNN kinase.
© 2017 American Society of Plant Biologists. All Rights Reserved.
DOI: 10.1104/pp.17.00278
PMCID: PMC5543944
105. J Sci Food Agric. 2018 Jan;98(1):328-335. doi: 10.1002/jsfa.8475. Epub 2017
Aug
2.
Effects of four short-chain fatty acids or salts on fermentation characteristics

and aerobic stability of alfalfa (Medicago sativa L.) silage.
Yuan XJ(1), Wen AY(1)(2), Wang J(1)(3), Desta ST(1), Dong ZH(1), Shao T(1).
Author information:
(1)Institute of Ensiling and Processing of Grass, Nanjing Agricultural
University, Nanjing, China.
(2)College of Animal Science, Anhui Science and Technology University, Feng Yang,
China.
(3)Institute of Tropical Agriculture and Forestry collage, Hainan University,
Haikou, China.
BACKGROUND: The objective of the present study was to evaluate the effects of
four chemicals on the fermentation quality and aerobic stability of alfalfa
(Medicago sativa L.) silage. Wilted alfalfa was ensiled without additive
(control), or with formic acid (FA), potassium diformate (KDF), sodium diacetate
(SDA) or calcium propionate (CAP).
RESULTS: After 60 days of ensiling, the pH values in FA, KDF and SDA silages were
lower (P < 0.05) compared to that of control and CAP silages, and chemicals (P <
0.05) decreased butyric acid and ammonia N concentrations and populations of
aerobic bacteria and yeasts compared to the control. The SDA and CAP silages had
a higher (P < 0.05) lactic acid bacteria content compared to the FA and KDF
silages. The SDA and CAP silages had higher (P < 0.05) acetic and propionic acid
contents compared to the other silages, respectively. The ammonia N
concentrations in the FA and KDF silages were lower compared to the other silages
during the first 5 days of aerobic exposure, and then increased sharply to 105
and 100 g kg-1 total N, respectively, which was higher (P < 0.05) than that of
the SDA and CAP silages on day 9 of aerobic exposure. Yeasts and aerobic bacteria
counts in SDA silage slowly increased and remained at lower levels compared to
the other silages after 7 days of aerobic exposure.
CONCLUSION: Additives prolonged the aerobic stability duration compared to the
control, and the SDA and CAP silages remained stable for more than 216 h,
followed by the KDF and FA silages (202 and 196 h, respectively). © 2017 Society
of Chemical Industry.
© 2017 Society of Chemical Industry.
DOI: 10.1002/jsfa.8475
106. BMC Plant Biol. 2017 Jun 6;17(1):97. doi: 10.1186/s12870-017-1047-x.
Identification of genetic loci associated with crude protein and mineral

concentrations in alfalfa (Medicago sativa) using association mapping.
Jia C(1), Wu X(2), Chen M(1), Wang Y(2), Liu X(1), Gong P(1), Xu Q(3), Wang X(1),
Gao H(1), Wang Z(4).
Author information:
(2)Institute of Animal Husbandry and Veterinary Science, Shanxi Academy of
Agricultural Sciences, Taiyuan, 030032, China.
(3)College of Animal Science and Veterinary Medicine, Shanxi Agricultural
University, Taigu, 030801, China.
Beijing, 100193, China. wangzan@caas.cn.
BACKGROUND: Alfalfa (Medicago sativa) is one of the most important legume forage
species in China and many other countries of the world. It provides a quality
source of proteins and minerals to animals. Genetic underpinnings for these
important traits, however, are elusive. An alfalfa (M. sativa) association
mapping study for six traits, namely crude protein (CP), rumen undegraded protein
(RUP), and four mineral elements (Ca, K, Mg and P), was conducted in three
consecutive years using a large collection encompassing 336 genotypes genotyped
with 85 simple sequence repeat (SSR) markers.
RESULTS: All the traits were significantly influenced by genotype, environment,
and genotype × environment interaction. Eight-five significant associations
(P < 0.005) were identified. Among these, five associations with Ca were
repeatedly observed and six co-localized associations were identified.
CONCLUSIONS: The identified marker alleles significantly associated with the
traits provided important information for understanding genetic controls of
alfalfa quality. The markers could be used in assisting selection for the
individual traits in breeding populations for developing new alfalfa cultivars.
DOI: 10.1186/s12870-017-1047-x
PMCID: PMC5460482
107. Acta Crystallogr F Struct Biol Commun. 2017 Jun 1;73(Pt 6):315-320. doi:
10.1107/S2053230X17006057. Epub 2017 May 11.
Crystal structure of Rv1220c, a SAM-dependent O-methyltransferase from

Mycobacterium tuberculosis.
Yan Q(1), Shaw N(1), Qian L(1), Jiang D(1).
Author information:
(1)College of Life Science, Nankai University, Weijin Road, Nankai District,
Tianjin City 300071, People's Republic of China.
Rv1220c from Mycobacterium tuberculosis is annotated as an O-methyltransferase

(MtbOMT). Currently, no structural information is available for this protein.
Here, the crystal structure of MtbOMT refined to 2.0 Å resolution is described.
The structure reveals the presence of a methyltransferase fold and shows clear
electron density for one molecule of S-adenosylmethionine (SAM), which was
apparently bound by the protein during its production in Escherichia coli.
Although the overall structure of MtbOMT resembles the structures of
O-methyltransferases from Cornybacterium glutamicum, Coxiella burnetti and Alfa
alfa, differences are observed in the residues that make up the active site.
Notably, substitution of Asp by His164 seems to abrogate metal binding by MtbOMT.
A putative catalytic His-Asp pair located in the vicinity of SAM is absolutely
conserved in MtbOMT homologues from all species of Mycobacterium, suggesting a
conserved function for this protein.
DOI: 10.1107/S2053230X17006057
108. Mycorrhiza. 2017 Jun 1. doi: 10.1007/s00572-017-0779-7. [Epub ahead of print]
An in vivo whole-plant experimental system for the analysis of gene expression in

extraradical mycorrhizal mycelium.
Pepe A(1), Sbrana C(2), Ferrol N(3), Giovannetti M(4).
Author information:
(1)Department of Agriculture, Food and Environment, University of Pisa, Via del
Borghetto 80, 56124, Pisa, Italy.
(2)CNR, Institute of Agricultural Biology and Biotechnology, UOS Pisa, Via del
Borghetto 80, 56124, Pisa, Italy.
(3)Departamento de Microbiologia del Suelo y Sistemas Simbioticos, Estacion
Experimental del Zaidin, CSIC, Profesor Albareda 1, 18008, Granada, Spain.
Borghetto 80, 56124, Pisa, Italy. manuela.giovannetti@unipi.it.
Arbuscular mycorrhizal fungi (AMF) establish beneficial mutualistic symbioses

with land plants, receiving carbon in exchange for mineral nutrients absorbed by
the extraradical mycelium (ERM). With the aim of obtaining in vivo produced ERM
for gene expression analyses, a whole-plant bi-dimensional experimental system
was devised and tested with three host plants and three fungal symbionts. In such
a system, Funneliformis mosseae in symbiosis with Cichorium intybus var.
foliosum, Lactuca sativa, and Medicago sativa produced ERM whose lengths ranged
from 9.8 ± 0.8 to 20.8 ± 1.2 m per plant. Since ERM produced in symbiosis with C.
intybus showed the highest values for the different structural parameters
assessed, this host was used to test the whole-plant system with F. mosseae,
Rhizoglomus irregulare, and Funneliformis coronatus. The whole-plant system
yielded 1-7 mg of ERM fresh biomass per plant per harvest, and continued
producing new ERM for 6 months. Variable amounts of high-quality and intact total
RNA, ranging from 15 to 65 μg RNA/mg ERM fresh weight, were extracted from the
ERM of the three AMF isolates. Ammonium transporter gene expression was
successfully determined in the cDNAs obtained from ERM of the three fungal
symbionts by RT-qPCR using gene-specific primers designed on available (R.
irregulare) and new (F. mosseae and F. coronatus) ammonium transporter gene
sequences. The whole-plant experimental system represents a useful research tool
for large production and easy collection of ERM for morphological, physiological,
and biochemical analyses, suitable for a wide variety of AMF species, for a
virtually limitless range of host plants and for studies involving diverse
symbiotic interactions.
DOI: 10.1007/s00572-017-0779-7
PMID: 28573458
109. PLoS One. 2017 May 30;12(5):e0178222. doi: 10.1371/journal.pone.0178222.

eCollection 2017.
Alfalfa virus S, a new species in the family Alphaflexiviridae.
Nemchinov LG(1), Grinstead SC(2), Mollov DS(2).
Author information:
(1)USDA-ARS, Molecular Plant Pathology Laboratory, Beltsville, Maryland, United
States of America.
(2)USDA-ARS, National Germplasm Recourses Laboratory, Beltsville, Maryland,
A new species of the family Alphaflexiviridae provisionally named alfalfa virus S

(AVS) was discovered in alfalfa samples originating from Sudan. A complete
nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding
the 3' poly(A) tail was determined by high throughput sequencing (HTS) on an
Illumina platform. NCBI BLAST searches revealed that the virus shares the
greatest degree of sequence identity with members of the family
Alphaflexiviridae, genus Allexivirus. The AVS genome contains six
computationally-predicted open reading frames (ORF) encoding viral replication
protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown
38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for
allexiviruses, and coat protein (CP). AVS lacks a clear 3' proximal ORF that
encodes a nucleic acid-binding protein typical for allexiviruses. The identity of
the virus was confirmed by RT-PCR with primers derived from the HTS-generated
sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and
Western blot analysis with antibodies produced against a peptide derived from the
CP sequence. Transmission electron microscopic observations of the infected
tissues showed the presence of filamentous particles similar to allexiviruses in
their length and appearance. To the best of our knowledge, this is the first
report on the identification of a putative allexivirus in alfalfa (Medicago
sativa). The genome sequence of AVS has been deposited in NCBI GenBank on
03/02/2016 as accession № KY696659.
PMCID: PMC5448757
110. Plant Sci. 2017 Aug;261:28-37. doi: 10.1016/j.plantsci.2017.05.001. Epub 2017

May
10.
Melatonin confers plant tolerance against cadmium stress via the decrease of
cadmium accumulation and reestablishment of microRNA-mediated redox homeostasis.
Gu Q(1), Chen Z(1), Yu X(1), Cui W(1), Pan J(1), Zhao G(1), Xu S(2), Wang R(2),
Shen W(3).
Author information:
Agricultural University, Nanjing, Jiangsu, 210095, China.
(2)Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing
210014, China.
Agricultural University, Nanjing, Jiangsu, 210095, China. Electronic address:
wbshenh@njau.edu.cn.
Although melatonin-alleviated cadmium (Cd) toxicity both in animals and plants

have been well studied, little is known about its regulatory mechanisms in
plants. Here, we discovered that Cd stress stimulated the production of
endogenous melatonin in alfalfa seedling root tissues. The pretreatment with
exogenous melatonin not only increased melatonin content, but also alleviated
Cd-induced seedling growth inhibition. The melatonin-rich transgenic Arabidopsis
plants overexpressing alfalfa SNAT (a melatonin synthetic gene) exhibited more
tolerance than wild-type plants under Cd conditions. Cd content was also reduced
in root tissues. In comparison with Cd stress alone, ABC transporter and PCR2
transcripts in alfalfa seedlings, PDR8 and HMA4 in Arabidopsis, were up-regulated
by melatonin. By contrast, Nramp6 transcripts were down-regulated. Changes in
above transporters were correlated with the less accumulation of Cd. Additionally
Cd-triggered redox imbalance was improved by melatonin. These could be supported
by the changes of the Cu/Zn Superoxide Dismutase gene regulated by miR398a and
miR398b. Histochemical staining, laser scanning confocal microscope, and H2O2
contents analyses showed the similar tendencies. Taking together, we clearly
suggested that melatonin enhanced Cd tolerance via decreasing cadmium
accumulation and reestablishing the microRNAs-mediated redox homeostasis.
DOI: 10.1016/j.plantsci.2017.05.001
111. Transgenic Res. 2017 Aug;26(4):541-557. doi: 10.1007/s11248-017-0024-3. Epub

2017
May 25.
MsmiR156 affects global gene expression and promotes root regenerative capacity
and nitrogen fixation activity in alfalfa.
Aung B(1)(2), Gao R(1), Gruber MY(3), Yuan ZC(1), Sumarah M(1), Hannoufa A(4)(5).
Author information:
Canada.
(2)Department of Biology Department, University of Western Ontario, 1151 Richmond
Street, London, ON, N6A 5B7, Canada.
(3)Agriculture and Agri-Food Canada, 106 Science Place, Saskatoon, SK, S7N 0X2,
Canada.
(5)Department of Biology Department, University of Western Ontario, 1151 Richmond
Street, London, ON, N6A 5B7, Canada. Abdelali.Hannoufa@agr.gc.ca.
MicroRNA156 (miR156) regulates a network of downstream genes to affect plant

growth and development. We previously generated alfalfa (Medicago sativa) plants
that overexpress homologous miR156 (MsmiR156OE), and identified three of its SPL
target genes. These plants exhibited increased vegetative yield, delayed
flowering and longer roots. In this study, we aimed to elucidate the effect of
miR156 on the root system, including effect on nodulation and nitrogen fixation.
We found that MsmiR156 overexpression increases root regeneration capacity in
alfalfa, but with little effect on root biomass at the early stages of root
development. MsmiR156 also promotes nitrogen fixation activity by upregulating
expression of nitrogenase-related genes FixK, NifA and RpoH in roots inoculated
with Sinorrhizobium meliloti. Furthermore, we conducted transcriptomics analysis
of MsmiR156OE alfalfa roots and identified differentially expressed genes
belonging to 132 different functional categories, including plant cell wall
organization, peptidyl-hypusine synthesis, and response to water stress.
Expression analysis also revealed miR156 effects on genes involved in nodulation,
root development and phytohormone biosynthesis. The present findings suggest that
miR156 regulates root development and nitrogen fixation activity. Taken together,
these findings highlight the important role that miR156 may play as a tool in the
biotechnological improvement of alfalfa, and potentially other crops.
DOI: 10.1007/s11248-017-0024-3
PMID: 28547343
2017.
Genotyping-by-Sequencing and Its Exploitation for Forage and Cool-Season Grain

Legume Breeding.
Annicchiarico P(1), Nazzicari N(1), Wei Y(1)(2), Pecetti L(1), Brummer EC(2).
Author information:
(1)Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie (FLC),
CREALodi, Italy.
(2)Plant Breeding Center, Department of Plant Sciences, University of California,
Davis, DavisCA, USA.
Genotyping-by-Sequencing (GBS) may drastically reduce genotyping costs compared

with single nucleotide polymorphism (SNP) array platforms. However, it may
require optimization for specific crops to maximize the number of available
markers. Exploiting GBS-generated markers may require optimization, too (e.g., to
cope with missing data). This study aimed (i) to compare elements of GBS
protocols on legume species that differ for genome size, ploidy, and breeding
system, and (ii) to show successful applications and challenges of GBS data on
legume species. Preliminary work on alfalfa and Medicago truncatula suggested the
greater interest of ApeKI over PstI:MspI DNA digestion. We compared KAPA and NEB
Taq polymerases in combination with primer extensions that were progressively
more selective on restriction sites, and found greater number of polymorphic SNP
loci in pea, white lupin and diploid alfalfa when adopting KAPA with a
non-selective primer. This protocol displayed a slight advantage also for
tetraploid alfalfa (where SNP calling requires higher read depth). KAPA offered
the further advantage of more uniform amplification than NEB over fragment sizes
and GC contents. The number of GBS-generated polymorphic markers exceeded 6,500
in two tetraploid alfalfa reference populations and a world collection of lupin
genotypes, and 2,000 in different sets of pea or lupin recombinant inbred lines.
The predictive ability of GBS-based genomic selection was influenced by the
genotype missing data threshold and imputation, as well as by the genomic
selection model, with the best model depending on traits and data sets. We
devised a simple method for comparing phenotypic vs. genomic selection in terms
of predicted yield gain per year for same evaluation costs, whose application to
preliminary data for alfalfa and pea in a hypothetical selection scenario for
each crop indicated a distinct advantage of genomic selection.
DOI: 10.3389/fpls.2017.00679
PMCID: PMC5423274
PMID: 28536584
113. Am J Bot. 2017 May;104(5):772-781. doi: 10.3732/ajb.1600405. Epub 2017 May 21.
Floral traits influencing plant attractiveness to three bee species: Consequences

for plant reproductive success.
Bauer AA(1), Clayton MK(2), Brunet J(3)(4).
Author information:
(1)Department of Entomology, 1630 Linden Drive, University of Wisconsin, Madison,
Wisconsin 53706, USA.
(2)Department of Statistics, 1300 University Avenue, University of Wisconsin,
Madison, Wisconsin 53706, USA.
(3)Department of Entomology, 1630 Linden Drive, University of Wisconsin, Madison,
Wisconsin 53706, USA jbrunet@wisc.edu.
(4)USDA-Agricultural Research Service, Vegetable Crop Research Unit, 1630 Linden
Drive, Madison, Wisconsin 53706, USA.
PREMISE OF THE STUDY: The ability to attract pollinators is crucial to plants

that rely on insects for pollination. We contrasted the roles of floral display
size and flower color in attracting three bee species and determined the
relationships between plant attractiveness (number of pollinator visits) and seed
set for each bee species.
METHODS: We recorded pollinator visits to plants, measured plant traits, and
quantified plant reproductive success. A zero-inflated Poisson regression model
indicated plant traits associated with pollinator attraction. It identified
traits that increased the number of bee visits and traits that increased the
probability of a plant not receiving any visits. Different components of floral
display size were examined and two models of flower color contrasted.
Relationships between plant attractiveness and seed set were determined using
regression analyses.
KEY RESULTS: Plants with more racemes received more bee visits from all three bee
species. Plants with few racemes were more likely not to receive any bee visits.
The role of flower color varied with bee species and was influenced by the choice
of the flower color model. Increasing bee visits increased seed set for all three
bee species, with the steepest slope for leafcutting bees, followed by bumble
bees, and finally honey bees.
CONCLUSIONS: Floral display size influenced pollinator attraction more
consistently than flower color. The same plant traits affected the probability of
not being visited and the number of pollinator visits received. The impact of
plant attractiveness on female reproductive success varied, together with
pollinator effectiveness, by pollinator species.
© 2017 Bauer et al. Published by the Botanical Society of America. This work is
licensed under a Creative Commons public domain license (CC0 1.0).
DOI: 10.3732/ajb.1600405
114. Int J Mol Sci. 2017 May 22;18(5). pii: E1105. doi: 10.3390/ijms18051105.
The Occurrence, Biosynthesis, and Molecular Structure of Proanthocyanidins and

Their Effects on Legume Forage Protein Precipitation, Digestion and Absorption in
the Ruminant Digestive Tract.
Jonker A(1)(2), Yu P(3).
Author information:
(1)Department of Animal and Poultry Science, College of Agriculture and
Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8,
Canada. arjan.jonker@agresearch.co.nz.
(2)Grasslands Research Centre, AgResearch Ltd., Tennent Drive, Private Bag 11008,
Palmerston North 4442, New Zealand. arjan.jonker@agresearch.co.nz.
Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8,
Canada. peiqiang.yu@usask.ca.
Forages grown in temperate regions, such as alfalfa (Medicago sativa L.) and
white clover (Trefolium repens L.), typically have a high nutritional value when
fed to ruminants. Their high protein content and degradation rate result,
however, in poor utilization of protein from the forage resulting in excessive
excretion of nitrogen into the environment by the animal. Proanthocyanindins
(also known as condensed tannins) found in some forage legumes such as birdsfoot
trefoil (Lotus corniculatus L.), bind to dietary protein and can improve protein
utilization in the animal. This review will focus on (1) the occurrence of
proanthocyanidins; (2) biosynthesis and structure of proanthocyanidins; (3)
effects of proanthocyanidins on protein metabolism; (4) protein precipitating
capacity of proanthocyanidins and their effects on true intestinal protein
adsorption by ruminants; and (5) effect on animal health, animal performance and
environmental emissions.
DOI: 10.3390/ijms18051105
PMCID: PMC5455013
PMID: 28531145
115. J Dairy Sci. 2017 Jul;100(7):5293-5304. doi: 10.3168/jds.2016-12404. Epub 2017

May 17.
Nutritive value and fermentation characteristics of alfalfa-mixed grass forage

wrapped with minimal stretch film layers and stored for different lengths of
time.
Coblentz WK(1), Ogden RK(2), Akins MS(3), Chow EA(4).

Author information:
(1)USDA-ARS, US Dairy Forage Research Center, Marshfield, WI 54449. Electronic
address: wayne.coblentz@ars.usda.gov.
(2)USDA-ARS, US Dairy Forage Research Center, Marshfield, WI 54449.
(3)Department of Dairy Science, University of Wisconsin, Madison 53706.
(4)Kuraray America Inc., Pasadena, TX 77507.
A key aspect of managing baled silages is to quickly achieve and then rigorously
maintain anaerobic conditions within the silage mass. The concept of inserting an
O2-limiting barrier (OB) into plastic commercial silage wraps has been evaluated
previously, yielding mixed or inconclusive results. Our objective for this study
was to maximize the challenge to a commercial polyethylene bale wrap, or the
identical wrap containing an OB, by using minimal plastic (4 layers), and then
extending storage periods as long as 357 d. Forty-eight 1.2 × 1.2-m large-round
bales of alfalfa (Medicago sativa L.) and mixed grass forage (66.3 ± 8.66%
alfalfa; DM basis) were made at 2 moisture concentrations [47.5 (ideal) or 36.1%
(dry)], wrapped with 4 layers of plastic containing an OB or no OB, and then
stored for 99, 243, or 357 d. After storage, yeast counts within the 0.15-m deep
surface layer were not affected by treatment (mean = 5.85 log10 cfu/g); mold
counts could not be analyzed statistically because 26 bales were nondetectable at
a 3.00 log10 cfu/g detection limit, but means among detectable counts were
numerically similar for OB (4.74 log10 cfu/g) and no OB (4.77 log10 cfu/g).
Fermentation characteristics were most affected by initial bale moisture,
resulting in a more acidic final pH for ideal compared with dry bales (5.52 vs.
6.00). This was facilitated by greater concentrations of total fermentation acids
(3.80 vs. 1.45% of dry matter), lactic acid (2.24 vs. 0.71% of dry matter), and
acetic acid (1.07 vs. 0.64% of dry matter) within ideal compared with dry
silages. Plastic wrap type had no effect on final concentrations of any
fermentation product. During fermentation and storage, we noted greater change in
concentrations of fiber components and whole-plant ash within the 0.15-m deep
surface layer than in the bale core, and these changes always differed
statistically from 0 (no change) based on pre-ensiled baseline concentrations.
Overall, concentrations of water-soluble carbohydrates were reduced (mean = 2.3
percentage units) during fermentation and storage, which resulted (indirectly) in
increased concentrations of fiber components and crude protein, as well as an
overall energy cost of 2.2 percentage units of total digestible nutrient. It
remains unclear under what conditions an OB plastic wrap will consistently
benefit the fermentation and preservation of baled silages.

DOI: 10.3168/jds.2016-12404
PMID: 28527804
116. Environ Sci Pollut Res Int. 2017 Jun;24(17):15196-15201. doi:

10.1007/s11356-017-9137-8. Epub 2017 May 12.
Effects of Se on the growth, tolerance, and antioxidative systems of three

alfalfa cultivars.
Dai H(1), Jia G(2).
Author information:
(1)Bio-resources Key Laboratory of Shaanxi Province, Shaanxi University of
Technology, Hanzhong, Shaanxi, 723001, People's Republic of China.
daihp72@aliyun.com.
(2)College of Science, Northwest Agriculture & Forestry University, Yangling,
Shaanxi, 712100, People's Republic of China.
The aim of this study was to evaluate the selenium (Se) tolerance and exploit the
physiological mechanisms in roots, stem, and leaves of three alfalfa cultivars to
different concentrations of Se for 60 days. Among three cultivars, Medicago
sativa ssp. displayed the highest Se concentrations in tissues, the largest Se
amount in aerial parts, the highest bioconcentration factor (BCF) in aerial
parts, and translocation factor (TF) under Se stress. Under Se stress, induced
O2•- production in roots and leaves and increases in free proline and activities
of antioxidative enzymes were observed in M. sativa ssp. Based on the above
results, it is concluded that M. sativa ssp. is superior to the other two
cultivars for Se phytoremediation, and its well-coordinated physiological changes
under Se stress confer the great Se tolerance of this cultivar.
DOI: 10.1007/s11356-017-9137-8
117. Antonie Van Leeuwenhoek. 2017 Aug;110(8):1087-1103. doi:

10.1007/s10482-017-0883-x. Epub 2017 May 12.
Diversity and plant growth promoting properties of rhizobia isolated from root
nodules of Ononis arvensis.
Wdowiak-Wróbel S(1), Marek-Kozaczuk M(2), Kalita M(2), Karaś M(2), Wójcik M(2),
Małek W(2).
Author information:
(1)Department of Genetics and Microbiology, Maria Curie -Skłodowska University,
Akademicka 19 St., 20-033, Lublin, Poland. s.wdowiak@poczta.umcs.lublin.pl.
(2)Department of Genetics and Microbiology, Maria Curie -Skłodowska University,
Akademicka 19 St., 20-033, Lublin, Poland.
This is the first report describing isolates from root nodules of Ononis arvensis
(field restharrow). The aim of this investigation was to describe the diversity,
phylogeny, and plant growth promoting features of microsymbionts of O. arvensis,
i.e., a legume plant growing in different places of the southern part of Poland.
Twenty-nine bacterial isolates were characterized in terms of their phenotypic
properties, genome fingerprinting, and comparative analysis of their 16S rRNA,
nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies,
the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium
and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of
all the isolates tested exhibited the highest similarities to the corresponding
gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in
the genomes of rhizobia specific for O. arvensis implies that these bacteria may
promote the growth and development of their host plant in stress conditions. The
isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction,
all of them (except three) exhibited DNA fingerprints specific only for them. Our
studies showed that restharrow isolates formed effective symbiotic interactions
with their native host (O. arvensis) and Ononis spinosa but not with Trifolium
repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis
species and not with Lotus corniculatus, representing the tribe Loteae.
DOI: 10.1007/s10482-017-0883-x
PMCID: PMC5511607
PMID: 28500544
118. J Food Sci. 2017 Jun;82(6):1461-1466. doi: 10.1111/1750-3841.13722. Epub 2017
May
10.
Attachment and Biofilm Formation by Selected Strains of Salmonella enterica and

Entrohemorrhagic Escherichia coli of Fresh Produce Origin.
Han R(1), Klu YAK(1), Chen J(1).
Author information:
(1)Dept. of Food Science and Technology, The Univ. of Georgia, 1109 Experiment
Street, Griffin, GA 30223, U.S.A.
This study compared the abilities of selected Salmonella enterica and

enterohemorrhagic Escherichia coli (EHEC) strains of fresh produce origin to form
biofilms on polystyrene surface and to attach to alfalfa and bean sprouts. Each
of the 7 S. enterica and 4 EHEC inocula (2 mL; 107 CFU/mL) was placed in 6
different broths in 24-well polystyrene tissue culture plates at 28 °C for 1 to 7
d. Developed biofilms were quantified using the crystal violet binding assay. In
a separate experiment, alfalfa and mung bean sprouts (5 g) were exposed to 25 mL
inocula (107 CFU/mL) of S. enterica or EHEC at 22 °C for 2 h with shaking at 40
rpm. Contaminated sprouts were thoroughly rinsed and homogenized in 0.1% peptone
water, and bacteria attached to sprouts were enumerated. Biofilm mass accumulated
on polystyrene surface increased with incubation time (P < 0.05). Among the
microbiological media used, LB no salt (NaCl) broth better supported biofilm
development (P < 0.05). Two EHEC strains formed more biofilms than the Salmonella
and other two EHEC strains (P < 0.05). However, more Salmonella cells (5.66 log
CFU/g) attached to sprouts than EHEC cells (3.46 log CFU/g). Both Salmonella and
EHEC attached in higher numbers to mung bean, than alfalfa, sprouts (P < 0.05).
© 2017 Institute of Food Technologists®.
DOI: 10.1111/1750-3841.13722
119. Animal. 2017 Dec;11(12):2175-2183. doi: 10.1017/S1751731117000933. Epub 2017

May
8.
Supplementing high-quality fresh forage to growing lambs fed a total mixed ration
diet led to higher intake without altering nutrient utilization.
Pérez-Ruchel A(1), Repetto JL(2), Cajarville C(1).
Author information:
(1)1Departamento de Nutrición,Instituto de Producción Animal,Facultad de
Veterinaria,Universidad de la República,Ruta 1 km 42.500,San José 80100,Uruguay.
(2)2Departamento de Bovinos,Instituto de Producción Animal, Facultad de
Veterinaria,Universidad de la República,Ruta 1 km 42.500,San José 80100,Uruguay.
The effect of supplementing high-quality fresh forage, mainly based on alfalfa,

to growing lambs fed with decreasing levels of total mixed ration (TMR) was
studied on intake, digestion and ruminal environment. In total, 24 catheterized
lambs (25.2±3.67 kg) housed in individual metabolism cages were assigned to one
of four treatment diets: 'TMR100': TMR offered ad libitum; 'TMR75' and 'TMR50':
TMR at a level of 0.75 and 0.50 of potential intake, respectively, complemented
with fresh forage without restriction; 'TMR0': only fresh forage ad libitum. The
feeding behavior, nutrient intake and digestibility, kinetics of passage and
rumen environment were evaluated. As the level of TMR in the diet decreased,
lambs increased the forage intake and spent more time eating and ruminating, less
time resting and demonstrated a higher rate of intake. Those changes resulted in
a higher nutrient intake of dry matter, organic matter, nitrogen, NDF and ADF,
but a slightly lower organic matter digestibility, while no differences were
detected in the output rate of particles. As a consequence, with the decrease of
TMR and increase of forage intake, the ingested energy increased. Higher ruminal
pH and NH3-N concentrations were observed for lower levels of TMR in the diet.
The total volatile fatty acids, acetate and propionate concentrations presented a
quadratic response. Total volatile fatty acids and acetate concentrations were
higher and propionate concentration was lower in lambs consuming mixed diets
(TMR50 and TMR75). We concluded that the inclusion of high-quality fresh forage
in a combined diet with TMR in lambs had positive effects on nutrient intake
without negative consequences on digestion and rumen environment.
DOI: 10.1017/S1751731117000933
120. Environ Entomol. 2017 Jun 1;46(3):565-578. doi: 10.1093/ee/nvx070.
Using Publicly Available Data to Quantify Plant-Pollinator Interactions and

Evaluate Conservation Seeding Mixes in the Northern Great Plains.
Otto CRV(1), O'Dell S(1), Bryant RB(2)(3), Euliss NH Jr(1), Bush RM(4), Smart
MD(1).
Author information:
(1)U.S. Geological Survey Northern Prairie Wildlife Research Center, Jamestown,
ND 58401 (cotto@usgs.gov; sam.odell@gmail.com; Russ.Bryant@humboldt.edu;
eulissfamilyinnd@gmail.com; msmart@usgs.gov).
(2)U.S. Geological Survey Northern Prairie Wildlife Research Center, Jamestown,
ND 58401 ( cotto@usgs.gov ; sam.odell@gmail.com ; Russ.Bryant@humboldt.edu ;
eulissfamilyinnd@gmail.com ; msmart@usgs.gov ).
(3)College of Natural Resources and Sciences, Humboldt State University, 1 Harpst
St, Arcata, CA 95521.
(4)Pheasants Forever, Inc. 1166, Wahl Street, Dickinson, ND 58601
(rbush@pheasantsforever.org).
Concern over declining pollinators has led to multiple conservation initiatives

for improving forage for bees in agroecosystems. Using data available through the
Pollinator Library (npwrc.usgs.gov/pollinator/), we summarize plant-pollinator
interaction data collected from 2012-2015 on lands managed by the U.S. Fish and
Wildlife Service and private lands enrolled in U.S. Department of Agriculture
conservation programs in eastern North Dakota (ND). Furthermore, we demonstrate
how plant-pollinator interaction data from the Pollinator Library and seed cost
information can be used to evaluate hypothetical seeding mixes for pollinator
habitat enhancements. We summarize records of 314 wild bee and 849 honey bee
(Apis mellifera L.) interactions detected on 63 different plant species. The wild
bee observations consisted of 46 species, 15 genera, and 5 families. Over 54% of
all wild bee observations were represented by three genera-Bombus, Lassioglossum,
and Melissodes. The most commonly visited forbs by wild bees were Monarda
fistulosa, Sonchus arvensis, and Zizia aurea. The most commonly visited forbs by
A. mellifera were Cirsium arvense, Melilotus officinalis, and Medicago sativa.
Among all interactions, 13% of A. mellifera and 77% of wild bee observations were
made on plants native to ND. Our seed mix evaluation shows that mixes may often
need to be tailored to meet the unique needs of wild bees and managed honey bees
in agricultural landscapes. Our evaluation also demonstrates the importance of
incorporating both biologic and economic information when attempting to design
cost-effective seeding mixes for supporting pollinators in a critically important
part of the United States.
Published by Oxford University Press on behalf of Entomological Society of

America 2017. This work is written by US Government employees and is in the
public domain in the US.
DOI: 10.1093/ee/nvx070
121. Onderstepoort J Vet Res. 2017 Apr 11;84(1):e1-e7. doi:

10.4102/ojvr.v84i1.1259.
The use of <i>Lespedeza cuneata</i> for natural control of gastrointestinal

nematodes in Merino sheep.
Van Zyl EA(1), Botha FS, Eloff KJN, Msuntsha PP, Oosthuizen PA, Stevens C.
Author information:
(1)Dundee Research Station, KZN Department of Agriculture and Rural Development.
eavzyl@gmail.com.
Lespedeza cuneata (poorman's lucerne; sericea lespedeza), a tannin-rich perennial

legume, was offered as hay to dry Merino ewes in a confined feeding experiment to
evaluate the effect on the level of gastrointestinal parasite infection in sheep.
Medicago sativa (a low tannin containing perennial legume) was used as the
control treatment. Parameters faecal egg count (FEC), FAMACHA© scores and rectal
temperatures were used. FECs were substantially lower (p = 0.05) in the Lespedeza
group after 35 days, together with a trend of higher rectal temperatures,
compared with the Medicago group. Although non-significant (p > 0.05), the
higher rectal temperatures suggested a lower level of anaemia in the sheep on the
Lespedeza ration and, therefore, a lower parasite-worm burden. However, FAMACHA
© scores showed no significant (p > 0.05) differences between treatments

despite the differences in FEC that were recorded, indicating that host
homeostasis was possibly mediated by improved nutrition as a result of the high
protein content of both experimental diets.
PMID: 28470083

eCollection 2017.
Breaking continuous potato cropping with legumes improves soil microbial

communities, enzyme activities and tuber yield.
Qin S(1), Yeboah S(2), Cao L(1), Zhang J(1), Shi S(3), Liu Y(1).
Author information:
(1)College of Horticulture, Gansu Agricultural University, Lanzhou, P.R. China.
(2)CSIR-Crops Research Institute, Kumasi, Ghana.
(3)Key Laboratory of Grassland Ecology System, Ministry of Education (Gansu
Agricultural University), Lanzhou, P.R. China.
This study was conducted to explore the changes in soil microbial populations,
enzyme activity, and tuber yield under the rotation sequences of Potato-Common
vetch (P-C), Potato-Black medic (P-B) and Potato-Longdong alfalfa (P-L) in a
semi-arid area of China. The study also determined the effects of continuous
potato cropping (without legumes) on the above mentioned soil properties and
yield. The number of bacteria increased significantly (p < 0.05) under P-B
rotation by 78%, 85% and 83% in the 2, 4 and 7-year continuous cropping soils,
respectively compared to P-C rotation. The highest fungi/bacteria ratio was found
in P-C (0.218), followed by P-L (0.184) and then P-B (0.137) rotation over the
different cropping years. In the continuous potato cropping soils, the greatest
fungi/bacteria ratio was recorded in the 4-year (0.4067) and 7-year (0.4238)
cropping soils and these were significantly higher than 1-year (0.3041), 2-year
(0.2545) and 3-year (0.3030) cropping soils. Generally, actinomycetes numbers
followed the trend P-L>P-C>P-B. The P-L rotation increased aerobic azotobacters
in 2-year (by 26% and 18%) and 4-year (40% and 21%) continuous cropping soils
compared to P-C and P-B rotation, respectively. Generally, the highest urease and
alkaline phosphate activity, respectively, were observed in P-C (55.77 mg g-1)
and (27.71 mg g-1), followed by P-B (50.72 mg mg-1) and (25.64 mg g-1) and then
P-L (41.61 mg g-1) and (23.26 mg g-1) rotation. Soil urease, alkaline phosphatase
and hydrogen peroxidase activities decreased with increasing years of continuous
potato cropping. On average, the P-B rotation significantly increased (p <0.05)
tuber yield by 19% and 18%, compared to P-C and P-L rotation respectively. P-L
rotation also increased potato tuber yield compared to P-C, but the effect was
lesser relative to P-B rotation. These results suggest that adopting
potato-legume rotation system has the potential to improve soil biology
environment, alleviate continuous cropping obstacle and increase potato tuber
yield in semi-arid region.
PMCID: PMC5413038
123. Meat Sci. 2017 Aug;130:69-80. doi: 10.1016/j.meatsci.2017.03.011. Epub 2017

Apr
8.
Addition of an extract of lucerne (Medicago sativa L.) to cattle diets - Effects

on fatty acid profile, meat quality and eating quality of the M. longissimus
muscle.
MacKintosh SB(1), Richardson I(2), Kim EJ(1), Dannenberger D(3), Coulmier D(4),
Scollan ND(5).
Author information:
(1)Institute of Biological, Environmental and Rural Sciences, Aberystwyth
University, Aberystwyth SY23 3EB, UK.
(2)Division of Farm Animal Science, Department of Clinical Veterinary Science,
University of Bristol, Langford BS40 5DU, UK.
(3)Leibniz Institute for Farm Animal Biology, Institute of Muscle Biology and
Growth, 18196 Dummerstorf, Wilhelm-Stahl-Allee 2, Germany.
(4)Desialis, Complexe Agricole Mont Bernard, 51007 Chalons en Champagne, France.
(5)Institute for Global Food Security, Queens University Belfast, Stranmillis
Road, Belfast BT9 5AG, UK. Electronic address: nigel.scollan@qub.ac.uk.
There is considerable interest in enhancing beneficial fatty acids, particularly

18:3n-3, conjugated linoleic acid and long chain n-3 fatty acids such as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in beef to provide
benefits to human health. Here, the potential to enhance these fatty acids by
feeding a n-3 polyunsaturated fatty acid (PUFA)-rich plant extract (PX) from
lucerne is presented. Cattle (n=8/diet) were assigned to the following finishing
diets: straw and concentrate (S-CC); straw and concentrate containing 25% PX and
2000IUvitaminE/kg PX-concentrate (S-PXC); grass silage (GS); GS plus 75gPX/kg dry
matter intake (DMI) (GS-LPX) or GS plus 150g PX/kg DMI (GS-HPX). Addition of PX
to concentrates or GS increased the n-3 fatty acid content of loin muscle and had
favourable effects on the n-6:n-3 PUFA ratio (P<0.05), without detrimental
effects on loin muscle sensory characteristics.
Copyright © 2017. Published by Elsevier Ltd.
DOI: 10.1016/j.meatsci.2017.03.011
124. BMC Evol Biol. 2017 Apr 21;17(1):104. doi: 10.1186/s12862-017-0951-x.
Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred

from comparative cytogenetic analysis.
Yu F(1)(2), Wang H(1), Zhao Y(1)(2), Liu R(1)(2), Dou Q(3), Dong J(4), Wang T(4).
Author information:
(1)Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest
Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810008, China.
(2)University of Chinese Academy of Sciences, Beijing, 100049, China.
Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810008, China.
douqw@nwipb.cas.cn.
(4)State Key Laboratory of Agrobiotechnology, College of Biological Sciences,
China Agricultural University, Beijing, 100193, China.
BACKGROUND: Polyploidy plays an important role in the adaptation and speciation

of plants. The alteration of karyotype is a significant event during polyploidy
formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and
tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded
as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the
autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is
still in doubt. In this study, detailed comparative cytogenetic analysis between
diploid to tetraploid species, as well as genomic affinity across different
species in the M. sativa complex, were conducted based on comparative mapping of
11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ
hybridization (FISH) technique.
RESULTS: FISH patterns of the repeats in diploid subspecies caerulea were highly
similar to those in tetraploid subspecies sativa. Distinctly different FISH
patterns were first observed in diploid ssp. falcata, with only centromeric
hybridizations using centromeric and multiple region repeats and a few
subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies
falcata was unexpectedly found to possess a highly variable karyotype, which
agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of
chromosome-doubling process of diploid ssp. caerulea showed that chromosome
changes have occurred during polyploidization process.
CONCLUSIONS: The comparative cytogenetic results provide reliable evidence that
diploid subspecies caerulea is the direct progenitor of tetraploid subspecies
sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial
diploidization by the progressive accumulation of chromosome structural
rearrangements during evolution. However, the tetraploid subspecies falcata is
far from a simple autopolyploid from diploid subspecies falcata although no
obvious morphological change was observed between these two subspecies.
DOI: 10.1186/s12862-017-0951-x
PMCID: PMC5399346
125. Int J Environ Res Public Health. 2017 Apr 14;14(4). pii: E418. doi:
10.3390/ijerph14040418.
Comparison of Biogenic Amines and Mycotoxins in Alfalfa and Red Clover Fodder
Depending on Additives.
Skladanka J(1), Adam V(2), Zitka O(3), Mlejnkova V(4), Kalhotka L(5), Horky P(6),
Konecna K(7), Hodulikova L(8), Knotova D(9), Balabanova M(10), Slama P(11),
Skarpa P(12).
Author information:
(1)Faculty of AgriSciences, Mendel University in Brno, Zemedelska 1, 61300 Brno,
Czech Republic. jiri.skladanka@mendelu.cz.
Czech Republic. vojtech.adam@mendelu.cz.
Czech Republic. ondrej.zitka@mendelu.cz.
Czech Republic. veronika.mlejnkova@mendelu.cz.
Czech Republic. libor.kalhotka@mendelu.cz.
Czech Republic. pavel.horky@mendelu.cz.
(7)Research Institute for Fodder Crops, Ltd. Troubsko, Zahradni 1, 66441
Troubsko, Czech Republic. konecna@vupt.cz.
Czech Republic. xhodulik@node.mendelu.cz.
(9)Research Institute for Fodder Crops, Ltd. Troubsko, Zahradni 1, 66441
Troubsko, Czech Republic. knotova@vupt.cz.
Czech Republic. marie.balabanova@mendelu.cz.
Czech Republic. petr.slama@mendelu.cz.
Czech Republic. petr.skarpa@mendelu.cz.
In the production of fermented feed, each crop can be contaminated with a variety
of microorganisms that may produce natural pollutants. Biogenic amines,
mycotoxins, and undesirable organic acids can decrease health feed safety. The
aim of this study was to compare the counts of microorganisms, levels of biogenic
amines, and the mycotoxins in forage legumes, and also to compare the occurrence
of microorganisms and levels of mycotoxins in green fodder and subsequently
produced silage and the influence of additives on the content of natural harmful
substances in silage. The experimental plot was located in Troubsko and Vatín, in
the Czech Republic. Two varieties of Medicago sativa and one variety of Trifolium
pratense were compared. Green fodder and subsequently produced silage reaching up
to 23% of dry matter were evaluated and prepared using a bio-enzymatic additive
and a chemical additive. Green fodder of Medicago sativa was more contaminated by
Enterococci than Trifolium pratense fodder. The obvious difference was determined
by the quality of silage leachate. The silage prepared from Medicago sativa
fodder was more contaminated with butyric acid. Fungi were present in higher
counts in the anaerobic environment of green fodder and contaminated it with
zearalenone and deoxynivalenol. Lower counts of fungi were found in silage,
although the zearalenone content did not change. Lower content of deoxynivalenol
was detected in silage, compared with green fodder. Silages treated with a
chemical additive were found not to contain butyric acid. Lower ethanol content
was determined, and the tendency to reduce the risk of biogenic amines occurrence
was evident. The additives proved to have no influence on the content of
mycotoxins.
DOI: 10.3390/ijerph14040418
PMCID: PMC5409619
126. J Environ Manage. 2017 Jul 15;197:428-439. doi: 10.1016/j.jenvman.2017.04.028.

Epub 2017 Apr 12.
Impacts of coal fly ash on plant growth and accumulation of essential nutrients
and trace elements by alfalfa (Medicago sativa) grown in a loessial soil.
He H(1), Dong Z(2), Peng Q(3), Wang X(3), Fan C(4), Zhang X(2).
Author information:
Electronic address: honghuahe@ms.iswc.ac.cn.
(3)Institute of Soil and Water Conservation, Chinese Academy of Sciences and
Ministry of Water Resources, Yangling, Shaanxi 712100, China; University of
Chinese Academy of Sciences, Beijing 100049, China.
Shaanxi 712100, China.
Coal fly ash (CFA) is a problematic solid waste all over the world. One distinct
beneficial reuse of CFA is its utilization in land application as a soil
amendment. A pot experiment was carried out to assess the feasibility of using
CFA to improve plant growth and increase the supply of plant-essential elements
and selenium (Se) of a loessial soil for agricultural purpose. Plants of alfalfa
(Medicago sativa) were grown in a loessial soil amended with different rates (5%,
10%, 20% and 40%) of CFA for two years and subjected to four successive cuttings.
Dry mass of shoots and roots, concentrations of plant-essential elements and Se
in plants were measured. Shoot dry mass and root dry mass were always
significantly increased by 5%, 10% and 20% CFA treatments, and by 40% CFA
treatment in all harvests except the first one. The CFA had a higher supply of
exchangeable phosphorus (P), magnesium (Mg), copper (Cu), zinc (Zn), molybdenum
(Mo), and Se than the loessial soil. Shoot P, calcium (Ca), Mg, Mo, boron (B),
and Se concentrations were generally markedly increased, but shoot potassium (K),
Cu, and Zn concentrations were generally reduced. The CFA can be a promising
source of some essential elements and Se for plants grown in the loessial soil,
and an application rate of not higher than 5% should be safe for agricultural
purpose without causing plant toxicity symptoms in the studied loessial soil and
similar soils. Field trials will be carried out to confirm the results of the pot
experiment.
DOI: 10.1016/j.jenvman.2017.04.028
127. Environ Microbiol. 2017 Jul;19(7):2661-2680. doi: 10.1111/1462-2920.13757.

Epub
2017 May 11.
A conserved α-proteobacterial small RNA contributes to osmoadaptation and

symbiotic efficiency of rhizobia on legume roots.
Robledo M(1), Peregrina A(1), Millán V(1), García-Tomsig NI(1), Torres-Quesada

O(1), Mateos PF(2), Becker A(3), Jiménez-Zurdo JI(1).
Author information:
(1)Grupo de Ecología Genética de la Rizosfera, Estación Experimental del Zaidín,
CSIC, 18008 Granada, Spain.
(2)Departamento de Microbiología y Genética and CIALE, Edificio Departamental,
Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain.
(3)LOEWE Center for Synthetic Microbiology and Faculty of Biology,
Philipps-Universität Marburg, 35043 Marburg, Germany.
Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive
responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we
provide primary insights into the function and activity mechanism of the
Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA).
Northern blot probing and transcription tracking with fluorescent
promoter-reporter fusions unveiled high nfeR1 expression in response to salt
stress and throughout the symbiotic interaction. The strength and differential
regulation of nfeR1 transcription are conferred by a motif, which is conserved in
nfeR1 promoter regions in α-proteobacteria. NfeR1 loss-of-function compromised
osmoadaptation of free-living bacteria, whilst causing misregulation of
salt-responsive genes related to stress adaptation, osmolytes catabolism and
membrane trafficking. Nodulation tests revealed that lack of NfeR1 affected
competitiveness, infectivity, nodule development and symbiotic efficiency of S.
meliloti on alfalfa roots. Comparative computer predictions and a genetic
reporter assay evidenced a redundant role of three identical NfeR1 unpaired anti
Shine-Dalgarno motifs for targeting and downregulation of translation of multiple
mRNAs from transporter genes. Our data provide genetic evidence of the
hyperosmotic conditions of the endosymbiotic compartments. NfeR1-mediated gene
regulation in response to this cue could contribute to coordinate nutrient uptake
with the metabolic reprogramming concomitant to symbiotic transitions.
DOI: 10.1111/1462-2920.13757
128. Food Microbiol. 2017 Aug;65:221-230. doi: 10.1016/j.fm.2017.03.005. Epub 2017

Mar
14.
Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and

non-pasteurized fresh fruit-vegetable juices including the presence of
Cronobacter spp.
Berthold-Pluta A(1), Garbowska M(2), Stefańska I(3), Pluta A(4).
Author information:
(1)Division of Milk Biotechnology, Department of Biotechnology, Microbiology and
Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences -
SGGW, Nowoursynowska 159C St, 02-787 Warsaw, Poland. Electronic address:
anna.berthold@wp.pl.
(2)Prof. Wacław Dąbrowski Institute of Agricultural and Food Biotechnology,
Inter-Department Problem Group for Dairy Industries, Rakowiecka 36 St, 02-532
Warsaw, Poland.
(3)Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw
University of Life Sciences - SGGW, Ciszewskiego 8 St, 02-787 Warsaw, Poland.
(4)Division of Milk Biotechnology, Department of Biotechnology, Microbiology and
Food Evaluation, Faculty of Food Sciences, Warsaw University of Life Sciences -
SGGW, Nowoursynowska 159C St, 02-787 Warsaw, Poland.
Bacteria of the genus Cronobacter are emerging food-borne pathogens. Foods

contaminated with Cronobacter spp. may pose a risk to infants or adults with
suppressed immunity. This study was aimed at determining the microbiological
quality of ready-to-eat (RTE) plant-origin food products available on the Polish
market with special emphasis on the prevalence of Cronobacter genus bacteria.
Analyses were carried out on 60 samples of commercial RTE type plant-origin food
products, including: leaf vegetables (20 samples), sprouts (20 samples) and
non-pasteurized vegetable, fruit and fruit-vegetable juices (20 samples). All
samples were determined for the total count of aerobic mesophilic bacteria (TAMB)
and for the presence of Cronobacter spp. The isolates of Cronobacter spp. were
subjected to genetic identification and differentiation by 16S rDNA sequencing,
PCR-RFLP analysis and RAPD-PCR and evaluation of antibiotic susceptibility by the
disk diffusion assay. The TAMB count in samples of lettuces, sprouts and
non-pasteurized fruit, vegetable and fruit-vegetable juices was in the range of
5.6-7.6, 6.7-8.4 and 2.9-7.7 log CFU g-1, respectively. The presence of
Cronobacter spp. was detected in 21 (35%) samples of the products, including in 6
(30%) samples of leaf vegetables (rucola, lamb's lettuce, endive escarola and
leaf vegetables mix) and in 15 (75%) samples of sprouts (alfalfa, broccoli, small
radish, lentil, sunflower, leek and sprout mix). No presence of Cronobacter spp.
was detected in the analyzed samples of non-pasteurized fruit, vegetable and
fruit-vegetable juices. The 21 strains of Cronobacter spp. isolated from leaf
vegetable and sprouts included: 13 strains of C. sakazakii, 4 strains of
C. muytjensii, 2 strains of C. turicensis, one strain of C. malonaticus and one
strain of C. condimenti. All isolated C. sakazakii, C. muytjensii, C. turicensis
and C. malonaticus strains were sensitive to ampicillin, cefepime,
chloramphenicol, gentamycin, streptomycin, tetracycline, ciprofloxacin and
cotrimoxazol, whereas the C. condimenti isolate showed intermediate resistance to
streptomycin and cotrimoxazole.
DOI: 10.1016/j.fm.2017.03.005
129. Mol Plant Microbe Interact. 2017 Apr;30(4):312-324. doi:

10.1094/MPMI-02-17-0030-R. Epub 2017 Apr 10.
Heterologous Complementation Reveals a Specialized Activity for BacA in the

Medicago-Sinorhizobium meliloti Symbiosis.
diCenzo GC(1), Zamani M(1), Ludwig HN(1), Finan TM(1).
Author information:
(1)Department of Biology, McMaster University, 1280 Main St. W., Hamilton,
Ontario L8S 4K1, Canada.
The bacterium Sinorhizobium meliloti Rm2011 forms N2-fixing root nodules on
alfalfa and other leguminous plants. The pSymB chromid contains a 110-kb region
(the ETR region) showing high synteny to a chromosomally located region in
Sinorhizobium fredii NGR234 and related rhizobia. We recently introduced the ETR
region from S. fredii NGR234 into the S. meliloti chromosome. Here, we report
that, unexpectedly, the S. fredii NGR234 ETR region did not complement deletion
of the S. meliloti ETR region in symbiosis with Medicago sativa. This phenotype
was due to the bacA gene of NGR234 not being functionally interchangeable with
the S. meliloti bacA gene during M. sativa symbiosis. Further analysis revealed
that, whereas bacA genes from S. fredii or Rhizobium leguminosarum bv. viciae
3841 failed to complement the Fix- phenotype of a S. meliloti bacA mutant with M.
sativa, they allowed for further developmental progression prior to a loss of
viability. In contrast, with Melilotus alba, bacA from S. fredii and R.
leguminosarum supported N2 fixation by a S. meliloti bacA mutant. Additionally,
the S. meliloti bacA gene can support N2 fixation of a R. leguminosarum bacA
mutant during symbiosis with Pisum sativum. A phylogeny of BacA proteins
illustrated that S. meliloti BacA has rapidly diverged from most rhizobia and has
converged toward the sequence of pathogenic genera Brucella and Escherichia.
These data suggest that the S. meliloti BacA has evolved toward a specific
interaction with Medicago and highlights the limitations of using a single model
system for the study of complex biological topics.
DOI: 10.1094/MPMI-02-17-0030-R
PMID: 28398123
130. Front Plant Sci. 2017 Mar 27;8:390. doi: 10.3389/fpls.2017.00390. eCollection
2017.
Arbuscular Mycorrhiza Stimulates Biological Nitrogen Fixation in Two Medicago

spp. through Improved Phosphorus Acquisition.
Püschel D(1), Janoušková M(2), Voříšková A(2), Gryndlerová H(3), Vosátka M(2),
Jansa J(3).
Author information:
(1)Department of Mycorrhizal Symbioses, Institute of Botany, Czech Academy of
SciencesPrůhonice, Czechia; Laboratory of Fungal Biology, Institute of
Microbiology, Czech Academy of SciencesPrague, Czechia.
(2)Department of Mycorrhizal Symbioses, Institute of Botany, Czech Academy of
Sciences Průhonice, Czechia.
(3)Laboratory of Fungal Biology, Institute of Microbiology, Czech Academy of
Sciences Prague, Czechia.
Legumes establish root symbioses with rhizobia that provide plants with nitrogen
(N) through biological N fixation (BNF), as well as with arbuscular mycorrhizal
(AM) fungi that mediate improved plant phosphorus (P) uptake. Such complex
relationships complicate our understanding of nutrient acquisition by legumes and
how they reward their symbiotic partners with carbon along gradients of
environmental conditions. In order to disentangle the interplay between BNF and
AM symbioses in two Medicago species (Medicago truncatula and M. sativa) along a
P-fertilization gradient, we conducted a pot experiment where the
rhizobia-treated plants were either inoculated or not inoculated with AM fungus
Rhizophagus irregularis 'PH5' and grown in two nutrient-poor substrates subjected
to one of three different P-supply levels. Throughout the experiment, all plants
were fertilized with 15N-enriched liquid N-fertilizer to allow for assessment of
BNF efficiency in terms of the fraction of N in the plants derived from the BNF
(%NBNF). We hypothesized (1) higher %NBNF coinciding with higher P supply, and
(2) higher %NBNF in mycorrhizal as compared to non-mycorrhizal plants under P
deficiency due to mycorrhiza-mediated improvement in P nutrition. We found a
strongly positive correlation between total plant P content and %NBNF, clearly
documenting the importance of plant P nutrition for BNF efficiency. The AM
symbiosis generally improved P uptake by plants and considerably stimulated the
efficiency of BNF under low P availability (below 10 mg kg-1 water extractable
P). Under high P availability (above 10 mg kg-1 water extractable P), the AM
symbiosis brought no further benefits to the plants with respect to P nutrition
even as the effects of P availability on N acquisition via BNF were further
modulated by the environmental context (plant and substrate combinations). As a
response to elevated P availability in the substrate, the extent of root length
colonization by AM fungi was reduced, the turning points occurring at about 8 and
10 mg kg-1 water extractable P for M. sativa and M. truncatula, respectively. Our
results indicated competition for limited C resource between the two kinds of
microsymbionts and thus degradation of AM symbiotic functioning under ample P
supply.
DOI: 10.3389/fpls.2017.00390
PMCID: PMC5366336
PMID: 28396674
131. 3 Biotech. 2017 May;7(1):8. doi: 10.1007/s13205-017-0612-1. Epub 2017 Apr 8.
Population and function analysis of cultivable bacteria associated with spores of

arbuscular mycorrhizal fungus Gigaspora margarita.
Long L(1), Lin Q(2), Yao Q(3), Zhu H(4).
Author information:
(1)College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037,
China. longlk602@njfu.edu.cn.
(2)Nanjing Institute for the Comprehensive Utilization of Wild Plants, Nanjing,
210042, China.
(3)College of Horticulture, South China Agricultural University, Guangzhou,
510642, China.
(4)Guangdong Institute of Microbiology, Guangdong Provincial Key Laboratory of
Microbial Culture Collection and Application, Guangdong Open Laboratory of
Applied Microbiology, State Key Laboratory of Applied Microbiology South China,
Guangzhou, 510070, China. zhuhh@gdim.cn.
This study was aimed to investigate the diversity and function of bacterial
population associated with Gigaspora margarita spores. The fungus was propagated
in sterilized sand/soil pots using alfalfa (Medicago sativa), grain sorghum
(Sorghum bicolor), or maize (Zea mays) as host plants, or in sterilized
vermiculite pots using alfalfa as host plants, respectively. Bacteria were
isolated from the new-formed spores using diluted plate method, and typical
bacterial isolates were identified according to 16S rRNA gene phylogenetic
analysis. Total 43 bacterial isolates affiliated to three phyla and 23 genera
were obtained. The spore-associated bacterial communities were obviously
different among the four source spores, suggesting that plant species or
substrates could influence the bacterial population. Bacillus and Streptomyces
were most frequently associated with the fungal spores. Function analysis of
these bacteria by plate tests, it was found that about 30.2% isolates stimulated
the spore germination, five out of seven tested isolates improved the hyphal
growth, total 57.5% of the tested isolates solubilized phosphorus at different
levels, 15% isolates degraded chitin, and a few isolates suppressed the growth of
Escherichia coli or Staphylococcus aureus. In pot experiment, three bacterial
isolates (belonging to Curtobacterium, Ensifer, or Bacillus, respectively)
displayed improvement effect on alfalfa growth and/or the colonization of roots
by G. margarita.
DOI: 10.1007/s13205-017-0612-1
PMCID: PMC5385182
PMID: 28391471
132. PLoS One. 2017 Apr 7;12(4):e0175307. doi: 10.1371/journal.pone.0175307.

eCollection 2017.
Transcriptomic and physiological analyses of Medicago sativa L. roots in response

to lead stress.
Xu B(1), Wang Y(2), Zhang S(1), Guo Q(1), Jin Y(1), Chen J(1), Gao Y(1), Ma H(1).
Author information:
(1)College of Animal Science and Technology, Jilin Agricultural University,
Changchun, Jilin, China.
(2)Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences,
Gongzhuling, Jilin, China.
Lead (Pb) is one of the nonessential and toxic metals that threaten the
environment and human health. Medicago sativa L. is a legume with high salt
tolerance and high biomass production. It is not only a globally important forage
crop but is also an ideal plant for phytoremediation. However, the biological and
molecular mechanisms that respond to heavy metals are still not well defined in
M. sativa. In this study, de novo and strand-specific RNA-sequencing was
performed to identify genes involved in the Pb stress response in M. sativa
roots. A total of 415,350 unigenes were obtained from the assembled cDNA
libraries, among which 5,416 were identified as significantly differentially
expressed genes (DEGs) (false discovery rate < 0.005) between cDNA libraries from
control and Pb-treated plants. Gene ontology and Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway enrichment analyses of the DEGs showed they mainly
clustered with terms associated with binding, transport, membranes, and the
pathways related to signal and energy metabolism. Moreover, a number of candidate
genes included antioxidant enzymes, metal transporters, and transcription factors
involved in heavy metal response were upregulated under Pb stress. Quantitative
real-time PCR(qRT-PCR) validation of the expression patterns of 10 randomly
selected candidate DEGs were consistent with the transcriptome analysis results.
Thus, this study offers new information towards the investigation of biological
changes and molecular mechanisms related to Pb stress response in plants.
PMCID: PMC5384761
133. J Anim Sci. 2017 Mar;95(3):1325-1334. doi: 10.2527/jas.2016.1157.
Influence of forage source and forage inclusion level on growth performance,

feeding behavior, and carcass characteristics in finishing steers.
Swanson KC, Carlson ZE, Ruch MC, Gilbery TC, Underdahl SR, Keomanivong FE, Bauer
ML, Islas A.
Two experiments were conducted to determine the effects of forage source (Exp. 1)
and forage inclusion level (Exp. 2) in finishing diets on growth performance and
feeding behavior. In Exp. 1, sixty-four steers (394 ± 3.6 kg BW) were allotted by
BW to 3 pens. Within each pen, steers were assigned randomly to 1 of 4 dietary
treatments containing different forage sources: 1) alfalfa hay, 2) corn silage,
3) wheat straw, and 4) corn stover. Alfalfa hay was provided at 10% of the diet
DM and the other forage sources were offered to provide the same percentage of
NDF from forage. In Exp. 2, forty-four steers (451 ± 4.6 kg BW) were used in a
completely randomized design and were fed dry-rolled corn-based diets containing
a mixture of hay and corn silage as the forage source at 5%, 10%, 15%, or 20%
forage (DM basis). Intake and feeding behavior traits were calculated from data
generated by the Insentec feeding system. In Exp. 1, final BW, ADG, and G:F did
not differ between treatments. Dry matter intake and feeding behavior traits
responded differently depending on week (interaction ≤ 0.04) of the experiment
with DMI generally greater in steers fed alfalfa or corn silage early in the
experiment and time per meal generally greater and eating rate slower in steers
fed diets containing wheat straw or corn stover. In Exp. 2, ADG and G:F decreased
linearly ( < 0.001) with increasing forage inclusion. Quadratic effects ( ≤
0.002) were observed for eating time (per visit, meal, and d) and DMI (per visit,
meal, and min) with eating time greatest in the 10% forage treatment and DMI the
least in the 20% forage treatment. Dry matter intake per d responded differently
depending on week (interaction = 0.01) with some weeks exhibiting linear and
other quadratic effects that were primarily the result of the largest decrease in
DMI in the 20% forage inclusion treatment. These data indicate that growth
performance was not influenced by forage source, when fed at a similar NDF
inclusion level, but was negatively impacted by increasing forage inclusion in
high-concentrate finishing diets. However, both forage source and inclusion level
impacted feeding behavior as cattle consuming bulkier forages (wheat straw or
corn stover) or at greater inclusion levels typically had a slower eating rate
and took longer to consume a meal. Additionally, DMI may decrease at greater
forage inclusion levels (> 15%).
DOI: 10.2527/jas.2016.1157
134. J Environ Sci (China). 2017 Mar;53:217-223. doi: 10.1016/j.jes.2016.04.019.

Epub
2016 Jul 2.
Uptake and translocation of sulfamethazine by alfalfa grown under hydroponic

conditions.
Kurwadkar S(1), Struckhoff G(2), Pugh K(3), Singh O(4).
Author information:
(1)Department of Civil and Environmental Engineering, California State
University, Fullerton, CA 92831, USA. Electronic address:
skurwadkar@fullerton.edu.
(2)Department of Civil and Environmental Engineering, California State
University, Fullerton, CA 92831, USA.
(3)IDS Engineering Group, Houston, TX 77040, USA.
(4)Division of Biological and Health Sciences, University of Pittsburgh,
Bradford, PA 16701, USA.
Antibiotics are routinely used in intensive animal agriculture operations

collectively known as Concentrated Animal Feed Operations (CAFO) which include
dairy, poultry and swine farms. Wastewater generated by CAFOs often contains low
levels of antibiotics and is typically managed in an anaerobic lagoon. The
objective of this research is to investigate the uptake and fate of aqueous
sulfamethazine (SMN) antibiotic by alfalfa (Medicago sativa) grass grown under
hydroponic conditions. Uptake studies were conducted using hydroponically grown
alfalfa in a commercially available nutrient solution supplemented with 10mg/L of
SMN antibiotic. Analysis of alfalfa sap, root zone, middle one-third, and top
portion of the foliage showed varying uptake rate and translocation of SMN. The
highest average amount of SMN (8.58μg/kg) was detected in the root zone, followed
by the top portion (1.89μg/kg), middle one-third (1.30μg/kg), and sap (0.38μg/kg)
samples, indicating a clear distribution of SMN within the sampled regions. The
ultraviolet (UV) spectra of parent SMN and translocated SMN identified in
different parts of the plant present the possibility of metabolization during the
uptake process. Uptake of SMN using alfalfa grown under hydroponic conditions has
potential as a promising remediation technology for removal of similar
antibiotics from wastewater lagoons.
DOI: 10.1016/j.jes.2016.04.019
135. J Appl Microbiol. 2017 Jun;122(6):1456-1470. doi: 10.1111/jam.13456. Epub 2017

May 3.
Dynamics of microbial community during ensiling direct-cut alfalfa with and

without LAB inoculant and sugar.
Zheng ML(1), Niu DZ(1), Jiang D(1), Zuo SS(1), Xu CC(1).
Author information:
(1)College of Engineering, China Agricultural University, Beijing, China.
AIM: To gain deeper insights into the clostridial community dynamics and chemical
transformations during the ensiling of alfalfa.
METHODS AND RESULTS: Direct-cut alfalfa silage (with the dry matter content of
240 g kg-1 ) was prepared with or without the addition of a lactic acid bacterial
inoculant and sucrose. Silages were sampled at 0, 3, 7, 14, 28 and 56 days after
ensiling and their bacterial community was determined using high-throughput
sequencing with a special focus on the clostridial community. A clostridial
fermentation occurred in the control silage, with high contents of acetic acid,
butyric acid and ammonia nitrogen and Clostridia counts; while the inoculated
silage was well preserved, with low pH and high lactic acid content. Lactic acid
bacteria dominated the bacterial community during the ensiling process. In the
control silage, Weissella confusa, Lactobacillus brevis, Enterococcus mundtii and
Pediococcus acidilactici were identified at the beginning of the fermentation.
Thereafter, W. confusa, Lactobacillus helsingborgensis and Bifidobacterium
asteroides appeared and quickly prevailed. In the inoculated silage,
Lactobacillus plantarum dominated the whole ensiling process. The genus
Clostridium dominated the clostridial community, and was depressed with the
inoculated treatment. Clostridium perfringens, Garciella sp. and Clostridium
baratii were the main initiators of the clostridial fermentation of the control
silage, while Clostridium tyrobutyricum became the most abundant Clostridia with
prolonged ensiling. Overall in the inoculated silage, little changes in the
clostridial community were observed throughout the ensiling period. Treating
alfalfa silage with a homolactic acid-based bacterial inoculant prevented a
clostridial fermentation resulting in more efficient fermentation.
CONCLUSION: Distinct changes in the bacterial community with a special focus on
the clostridial community were associated with the development of the clostridial
fermentation during the ensiling of alfalfa.
SIGNIFICANCE AND IMPACT OF THE STUDY: High-throughput sequencing based on a novel
Clostridia-specific primer set proved a potentially useful tool to study the
clostridial community dynamics, and could aid to elucidate the mechanism by which
the clostridial fermentation develops during the ensiling of alfalfa.
© 2017 The Society for Applied Microbiology.
DOI: 10.1111/jam.13456
136. J Biotechnol. 2017 Jun 10;251:14-20. doi: 10.1016/j.jbiotec.2017.03.023. Epub

2017 Mar 28.
Plant tissue cultures as sources of new ene- and ketoreductase activities.
Magallanes-Noguera C(1), Cecati FM(1), Mascotti ML(2), Reta GF(1), Agostini E(3),
Orden AA(4), Kurina-Sanz M(5).
Author information:
(1)INTEQUI-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad
Nacional de San Luis, Chacabuco y Pedernera, CP 5700 San Luis, Argentina.
(2)IMIBIO-CONICET, Facultad de Química, Bioquímica y Farmacia, Universidad
Nacional de San Luis, Chacabuco y Pedernera, Argentina.
(3)Departamento de Biología Molecular, FCEFQN, Universidad Nacional de Río
Cuarto, Ruta 36 Km 601, CP 5800 Río Cuarto, Córdoba, Argentina.
Electronic address: alejandroorden@gmail.com.
Electronic address: marcelakurina@gmail.com.
While many redox enzymes are nowadays available for synthetic applications, the
toolbox of ene-reductases is still limited. Consequently, the screening for these
enzymes from diverse sources in the search of new biocatalyst suitable for green
chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa
and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were
selected due to their ability to hydrogenate the CC double bond of the model
substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more
preference toward highly activated molecules such as nitrostyrene and maleimide
rather than the classical substrates of the well-known Old Yellow Enzymes,
resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When
the three biocatalytic systems were applied in the reduction of chalcones, T.
absinthioides showed high chemoselectivity toward the CC double bond whereas the
other two demonstrated abilities to biohydrogenate the CC double bounds and the
carbonyl groups in a sequential fashion.
DOI: 10.1016/j.jbiotec.2017.03.023
137. Int J Mol Sci. 2017 Mar 20;18(3). pii: E664. doi: 10.3390/ijms18030664.
Gene-Transformation-Induced Changes in Chemical Functional Group Features and

Molecular Structure Conformation in Alfalfa Plants Co-Expressing Lc-bHLH and
C1-MYB Transcriptive Flavanoid Regulatory Genes: Effects of Single-Gene and
Two-Gene Insertion.
Heendeniya RG(1), Yu P(2).

Author information:
Bioresources, University of Saskatchewan, Saskatoon, SK S7N5A8, Canada.
rgv863@mail.usask.ca.
Bioresources, University of Saskatchewan, Saskatoon, SK S7N5A8, Canada.
peiqiang.yu@usask.ca.
Alfalfa (Medicago sativa L.) genotypes transformed with Lc-bHLH and Lc

transcription genes were developed with the intention of stimulating
proanthocyanidin synthesis in the aerial parts of the plant. To our knowledge,
there are no studies on the effect of single-gene and two-gene transformation on
chemical functional groups and molecular structure changes in these plants. The
objective of this study was to use advanced molecular spectroscopy with
multivariate chemometrics to determine chemical functional group intensity and
molecular structure changes in alfalfa plants when co-expressing Lc-bHLH and
C1-MYB transcriptive flavanoid regulatory genes in comparison with non-transgenic
(NT) and AC Grazeland (ACGL) genotypes. The results showed that compared to NT
genotype, the presence of double genes (Lc and C1) increased ratios of both the
area and peak height of protein structural Amide I/II and the height ratio of
α-helix to β-sheet. In carbohydrate-related spectral analysis, the double
gene-transformed alfalfa genotypes exhibited lower peak heights at 1370, 1240,
1153, and 1020 cm-1 compared to the NT genotype. Furthermore, the effect of
double gene transformation on carbohydrate molecular structure was clearly
revealed in the principal component analysis of the spectra. In conclusion,
single or double transformation of Lc and C1 genes resulted in changing
functional groups and molecular structure related to proteins and carbohydrates
compared to the NT alfalfa genotype. The current study provided molecular
structural information on the transgenic alfalfa plants and provided an insight
into the impact of transgenes on protein and carbohydrate properties and their
molecular structure's changes.
DOI: 10.3390/ijms18030664
PMCID: PMC5372676
138. J Plant Res. 2017 Jul;130(4):647-658. doi: 10.1007/s10265-017-0921-9. Epub

2017
Mar 22.
Allelopathic interference of alfalfa (Medicago sativa L.) genotypes to annual

ryegrass (Lolium rigidum).
Zubair HM(1)(2), Pratley JE(3)(4), Sandral GA(4)(5), Humphries A(6).
Author information:
(1)School of Agricultural and Wine Sciences, Faculty of Science, Charles Sturt
University, Building No 286, Boorooma Street, Wagga Wagga, NSW, 2650, Australia.
zubair.sau@gmail.com.
(2)Graham Centre for Agricultural Innovation (an alliance between Charles Sturt
University and NSW Department of Primary Industries), Wagga Wagga, NSW, 2650,
Australia. zubair.sau@gmail.com.
(3)School of Agricultural and Wine Sciences, Faculty of Science, Charles Sturt
University, Building No 286, Boorooma Street, Wagga Wagga, NSW, 2650, Australia.
(4)Graham Centre for Agricultural Innovation (an alliance between Charles Sturt
University and NSW Department of Primary Industries), Wagga Wagga, NSW, 2650,
Australia.
(5)Department of Primary Industries, Wagga Wagga Agricultural Institute, Wagga
Wagga, NSW, 2650, Australia.
(6)South Australian Research and Development Institute, Waite Campus, Adelaide,
SA, 5001, Australia.
Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for
allelopathic impact using annual ryegrass (Lolium rigidum) as the target species
in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and
40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM).
Alfalfa genotypes displayed a range of allelopathic interference in ryegrass
seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased
in response to increasing density of alfalfa seedlings. At the lowest density,
Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index
was calculated to rank each alfalfa genotype. Reduction in seed germination of
annual ryegrass occurred in the presence of several alfalfa genotypes including
Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using
Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes.
Variation in chemical compounds was found between alfalfa root extracts and
exudates and also between genotypes. Further individual compound assessments and
quantitative study at greater chemical concentrations are needed to clarify the
allelopathic activity. Considerable genetic variation exists among alfalfa
genotypes for allelopathic activity creating the opportunity for its use in weed
suppression through selection.
DOI: 10.1007/s10265-017-0921-9
139. Plant Sci. 2017 May;258:122-136. doi: 10.1016/j.plantsci.2017.01.018. Epub

2017
Feb 3.
MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by

silencing SPL13.
Arshad M(1), Feyissa BA(2), Amyot L(3), Aung B(4), Hannoufa A(5).
Author information:
(1)Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5 V
4T3, Canada. Electronic address: Muhammad.Arshad@agr.gc.ca.
4T3, Canada; Biology Department, University of Western Ontario, 1151 Richmond
Street, London, Ontario N6A 5B7, Canada. Electronic address:
Biruk.Feyissa@agr.gc.ca.
4T3, Canada. Electronic address: Lisa.Amyot@agr.gc.ca.
4T3, Canada. Electronic address: banyae.ong@gmail.com.
4T3, Canada; Biology Department, University of Western Ontario, 1151 Richmond
Street, London, Ontario N6A 5B7, Canada. Electronic address:
Alfalfa (Medicago sativa) is an important forage crop that is often grown in

areas that frequently experience drought and water shortage. MicroRNA156 (miR156)
is an emerging tool for improving various traits in plants. We tested the role of
miR156d in drought response of alfalfa, and observed a significant improvement in
drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared
to the wild type control (WT). In addition to higher survival and reduced water
loss, miR156OE genotypes also maintained higher stomatal conductance compared to
WT during drought stress. Furthermore, we observed an enhanced accumulation of
compatible solute (proline) and increased levels of abscisic acid (ABA) and
antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced
expression of miR156-targeted SPL13 showed reduced water loss and enhanced
stomatal conductance, chlorophyll content and photosynthetic assimilation.
Several genes known to be involved in drought tolerance were differentially
expressed in leaf and root of miR156 overexpression plants. Taken together, our
findings reveal that miR156 improves drought tolerance in alfalfa at least
partially by silencing SPL13.
DOI: 10.1016/j.plantsci.2017.01.018
140. J Plant Physiol. 2017 Jun;213:23-29. doi: 10.1016/j.jplph.2017.02.011. Epub

2017
Mar 1.
Asparagus decline: Autotoxicity and autotoxic compounds in asparagus rhizomes.
Kato-Noguchi H(1), Nakamura K(2), Ohno O(3), Suenaga K(3), Okuda N(2).
Author information:
(1)Department of Applied Biological Science, Faculty of Agriculture, Kagawa
University, Miki, Kagawa 761-0795, Japan. Electronic address:
hisashi@ag.kagawa-u.ac.jp.
University, Miki, Kagawa 761-0795, Japan.
(3)Department of Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku, Yokohama 223-8522, Japan.
Asparagus (Asparagus officinalis L.) is a perennial vegetable, but its crop

productivity and quality decrease gradually. One possible reason for "asparagus
decline" is thought to be the autotoxicity of asparagus. However, the autotoxic
property of asparagus rhizomes remains unknown. The objective of this study was
to determine the potential role of rhizomes in the autotoxicity of asparagus. An
aqueous methanol extract of asparagus rhizomes inhibited the growth of asparagus
seedlings and six other test plants in a concentration-dependent manners: garden
cress (Lepidum sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa
L.), ryegrass (Lolium multiflorum Lam.), timothy (Phleum pratense L.) and
barnyardgrass (Echinochloa crus-galli (L.) Beauv.). These results suggest that
asparagus rhizomes contain autotoxic compounds. The extract was purified through
several chromatographic steps with monitoring the autotoxic activity, and
p-coumaric acid and iso-agatharesinol were isolated. These compounds inhibited
the shoot and root growth of asparagus and two other test plants, garden cress
and ryegrass, at concentrations higher than 0.1mM. The concentrations required
for 50% inhibition of the root and shoot growth of these test plants ranged from
0.36 to 0.85mM and 0.41-1.22mM for p-coumaric acid and iso-agatharesinol,
respectively. Therefore, these compounds may contribute to the autotoxicity
caused by asparagus rhizomes and may be involved in "asparagus decline".
Copyright © 2017 Elsevier GmbH. All rights reserved.
DOI: 10.1016/j.jplph.2017.02.011
141. Sci Total Environ. 2017 Aug 15;592:68-77. doi:
10.1016/j.scitotenv.2017.03.066.
Epub 2017 Mar 14.
Rhizobial symbiosis alleviates polychlorinated biphenyls-induced systematic

oxidative stress via brassinosteroids signaling in alfalfa.
Wang X(1), Teng Y(2), Zhang N(3), Christie P(3), Li Z(3), Luo Y(3), Wang J(4).
Author information:
Soil Science, Chinese Academy of Sciences, Nanjing 210008, China; University of
Chinese Academy of Sciences, Beijing 100049, China.
Soil Science, Chinese Academy of Sciences, Nanjing 210008, China. Electronic
address: yteng@issas.ac.cn.
(4)Chongqing Research Academy of Environmental Sciences, Chongqing 401147, China.
The role of symbiotic rhizobia in the alleviation of polychlorinated biphenyl

(PCB)-induced phytotoxicity in alfalfa and the brassinosteroid (BR) hormone
signaling involved were investigated during phytoremediation. The association
between alfalfa and Sinorhizobium meliloti was adopted as a remediation model.
Phytotoxicity due to PCB 77 (3,3',4,4'-tetrachlorobiphenyl) exerted adverse
impacts on plant performance (biomass accumulation and photosynthesis) and
elicited cellular oxidative stress (overproduction of reactive oxygen species,
lipid peroxidation, and cell necrosis) which was largely attenuated by
pre-inoculation with S. meliloti strain NM. The protective role may have been
achieved as a result of strengthening of basic antioxidant defense before stress
as evidenced by the augmented activity and gene expression of antioxidative
enzymes (peroxidase, glutathione reductase, superoxide dismutase, catalase, and
ascorbate peroxidase) of both leaves and roots. In nodulated seedlings peroxidase
showed additive increased activity following PCB exposure but the activities of
the other four enzymes tended to remain stable after stress. Furthermore,
application of strain NM and brassinolide both triggered the accumulation of
endogenous BRs and the antioxidant network, while pre-treatment of seedlings with
a biosynthetic inhibitor of BRs, brassinazole, abolished the rhizobia-induced
activation of detoxification responses towards PCB. These observations indicate
that association with S. meliloti NM enhanced the systemic antioxidant defenses
of alfalfa to detoxify PCB, at least in part, via BR-dependent signaling
pathways. These results contribute to our knowledge of the 'logistic role' played
by rhizobia in assisting the phytoremediation of PCB-contaminated soils and
suggest an optimum manipulation strategy for bioremediation.
DOI: 10.1016/j.scitotenv.2017.03.066
PMID: 28314132
142. Ecotoxicol Environ Saf. 2017 Jun;140:264-270. doi:

10.1016/j.ecoenv.2017.02.053.
Epub 2017 Mar 8.
Comprehensive analysis of degradation and accumulation of ametryn in soils and in

wheat, maize, ryegrass and alfalfa plants.
Liu Y(1), Ma LY(2), Lu YC(3), Jiang SS(1), Wu HJ(1), Yang H(4).
Author information:
(1)Jiangsu Key Laboratory of Pesticide Science, College of Science, Nanjing
Agricultural University, Nanjing 210095, China; State & Local Joint Engineering
Research Center of Green Pesticide Invention and Application, Nanjing
Agricultural University, Nanjing 210095, China; College of Food Science and Light
Industry, Nanjing Tech University, Nanjing 211800, China.
hongyang@njau.edu.cn.
Ametryn is a selective herbicide belonging to the triazine family and widely used
for killing annual grasses or weeds in China and other parts of the world.
However, reports on its environmental risk assessment with regard to soil and
crop contamination are limited. In this study, accumulation of ametryn in wheat,
maize, ryegrass and alfalfa crops along with ametryn residues in the soil planted
with the plants were comparatively investigated. Soil enzyme activities and low
molecular weight organic acids (LMWOAs), as well as antioxidant and degradation
enzyme activities in plant tissues were measured. The maximum accumulation of
ametryn was found in shoots and roots of wheat and alfalfa. Ryegrass had the
maximum ametryn translocation factor (TF) from roots to shoots, with more than
three times over the other crops. The ametryn residue in ryegrass-planted soil
was much lower than that in soil planted with others. The residual content of
ametryn in crop-planted soils was ordered as rhizosphere soil<bulk
soil<non-rhizosphere soil<control (without plants). Activities of catalase (CAT),
glutathione S-transferase (GST) and laccase (LAC) in ametryn-exposed ryegrass
were significant higher than those in non-ametryn exposed ryegrass. The maximum
activities of CAT in ryegrass shoot and root were increased by 6.16- and
28.84-fold over the control, respectively. Exudation of organic acids in the crop
was induced by ametryn and contributed a lot to the degradation of the herbicide.
Thus, ryegrass was shown to have a relatively strong ability to remove ametryn
from ametryn-contaminated soil and its plant tissues as well.
DOI: 10.1016/j.ecoenv.2017.02.053
143. Integr Zool. 2017 Sep;12(5):409-420. doi: 10.1111/1749-4877.12263.
Turning in mid-air allows aphids that flee the plant to avoid reaching the risky
ground.
Meresman Y(1), Ben-Ari M(1), Inbar M(1).
Author information:
(1)Department of Evolutionary and Environmental Biology, University of Haifa,
Haifa, Israel.
When forced to drop from the plant, flightless arboreal insects can avoid
reaching the risky ground by maneuvering their body through the air. When
wingless pea aphids (Acyrthosiphon pisum) are threatened by natural enemies, they
often drop off their host plant while assuming a stereotypic posture that rotates
them in mid-air, aligning them with their feet pointing downwards. This position
may increase their chances of re-clinging onto lower plant parts and avoid facing
the dangers on the ground, although its effectiveness in realistic field
conditions has not been tested. We performed both laboratory and outdoor
experiments, in which we dropped aphids upon host plants to quantify clinging
success in plants with different characteristics such as height and leaf size.
Live aphids had twofold higher clinging rates than dead ones, indicating that
clinging success is indeed affected by the active aerial-righting of dropping
aphids. The ability to cling was positively dependent on the plants' foliage
cover as viewed in vertical direction from above. Therefore, we released aphids
in commercial alfalfa (Medicago sativa) fields with varying plant heights and
foliage cover and induced them to drop. Most (up to 75%) of the aphids avoided
reaching the ground in taller plants (65 cm), and 17% in shorter plants (21 cm),
demonstrating the efficiency of the aphids' response in averting risks: both
those of an approaching enemy on the plant and the plethora of new risks on the
ground. Evidently, even in complex field environment, the aerial-righting
mechanism can substantially reduce the possible risks following escape from a
predator.
© 2017 International Society of Zoological Sciences, Institute of Zoology/Chinese

Academy of Sciences and John Wiley & Sons Australia, Ltd.
DOI: 10.1111/1749-4877.12263
144. J Dairy Sci. 2017 May;100(5):3548-3562. doi: 10.3168/jds.2016-12073. Epub 2017

Mar 2.
Replacing alfalfa silage with tannin-containing birdsfoot trefoil silage in total

mixed rations for lactating dairy cows.
Broderick GA(1), Grabber JH(2), Muck RE(2), Hymes-Fecht UC(2).
Author information:
(1)Agricultural Research Service, USDA, US Dairy Forage Research Center, Madison,
WI 53706. Electronic address: gbroderi@wisc.edu.
WI 53706.
Two lactation trials were conducted comparing the feeding value of silages made
from birdsfoot trefoil (BFT, Lotus corniculatus L.) that had been selected for
low (BFTL), medium (BFTM), and high (BFTH) levels of condensed tannins (CT) to an
alfalfa silage (AS) when fed as the principal forage in total mixed rations.
Diets also included corn silage, high-moisture shelled corn, soybean meal, soy
hulls, and supplemental fat. In trial 1, 32 lactating Holstein cows were blocked
by days in milk, assigned to treatment sequences in 8 balanced 4 × 4 Latin
squares, and fed 50% dietary dry matter from AS or 1 of 3 BFT silages containing
0.6, 1.2, or 1.7% CT. Diets averaged 17.5 to 19.5% crude protein and 26% neutral
detergent fiber on a dry matter basis. Data were collected over the last 2 wk of
each 4-wk period. Intakes were 1.3 to 2.8 kg of dry matter/d greater on BFT than
on AS and cows gained 0.5 kg of body weight/d on BFT diets while losing 0.14 kg
of body weight/d on the AS diet; this resulted in greater milk per dry matter
intake (DMI) on AS. Linear effects indicated true protein yield and milk urea
nitrogen declined with increasing CT concentration and quadratic effects
indicated DMI, energy-corrected milk, and fat yield were increased at
intermediate CT concentration. True protein yield and apparent N-efficiency were
greater, and milk urea nitrogen lower, on all BFT diets than on AS. In trial 2,
50 lactating Holstein cows were fed a covariate AS diet for 2 wk and then blocked
by parity and days in milk and randomly assigned to 1 of 5 diets that were fed
continuously for 12 wk. Diets contained (dry matter basis) 48% AS, 16% AS plus
32% of 1 of 3 BFT silages with 0.5, 0.8, or 1.5% CT, or 48% of an equal mixture
of each BFT silage. Diets averaged 16.5% crude protein and 30% neutral detergent
fiber. Intake and milk yield tended to be lower on AS than BFT, but body weight
gains averaged 0.6 kg/d on all diets. Cows fed any of the BFT silages had reduced
milk urea nitrogen and ruminal ammonia and reduced urinary N excretion. Feeding
the BFT mixture reduced concentrations of milk true protein and milk urea
nitrogen and depressed apparent nutrient digestibility. Among diets containing
the individual BFT silages, linear reductions in DMI and yield of milk, fat, true
protein, lactose, and SNF were observed with increasing CT concentration. By
contrast, a previous trial with the same BFT populations showed that substituting
BFTH silage containing 1.6% CT for AS in rations containing 60% silage dry matter
had no effect on intake, increased yield of milk, energy-corrected milk and milk
components, elevated protein use-efficiency, but with a more modest reduction in
milk urea nitrogen and urinary N excretion. Silage analyses suggested that the
inconsistent responses among trials were related to growth environment or
ensiling effects that altered tannin-protein interactions in BFT silage.
Differences in diet formulation among trials may have also influenced responses.
Results from the current and previous trials indicate further work is needed to
identify optimum tannin levels in forages.

DOI: 10.3168/jds.2016-12073
145. PLoS One. 2017 Mar 1;12(3):e0171994. doi: 10.1371/journal.pone.0171994.

eCollection 2017.
Residual soil nitrate content and profitability of five cropping systems in

northwest Iowa.
De Haan RL(1), Schuiteman MA(2), Vos RJ(3).
Author information:
(1)Environmental Studies Department, Dordt College, Sioux Center, Iowa, United
States of America.
(2)AJS Farms, Sioux Center, Iowa, United States of America.
(3)Agriculture Department, Dordt College, Sioux Center, Iowa, United States of
America.
Many communities in the Midwestern United States obtain their drinking water from
shallow alluvial wells that are vulnerable to contamination by NO3-N from the
surrounding agricultural landscape. The objective of this research was to assess
cropping systems with the potential to produce a reasonable return for farmers
while simultaneously reducing the risk of NO3-N movement into these shallow
aquifers. From 2009 to 2013 we conducted a field experiment in northwest Iowa in
which we evaluated five cropping systems for residual (late fall) soil NO3-N
content and profitability. Soil samples were taken annually from the top 30 cm of
the soil profile in June and August, and from the top 180 cm in November (late
fall). The November samples were divided into 30 cm increments for analysis.
Average residual NO3-N content in the top 180 cm of the soil profile following
the 2010 to 2013 cropping years was 134 kg ha-1 for continuous maize (Zea mays
L.) with a cereal rye (Secale cereale L.) cover crop, 18 kg ha-1 for perennial
grass, 60 kg ha-1 for a three year oat (Avena sativa L.)-alfalfa (Medicago sativa
L.)-maize rotation, 85 kg ha-1 for a two year oat/red clover (Trifolium pratense
L.)-maize rotation, and 90 kg ha-1 for a three year soybean (Glycine max (L.)
Merr.)-winter wheat (Triticum aestivum L.)-maize rotation. However, residual
NO3-N in the 90 to 180 cm increment of the soil profile was not significantly
higher in the oat-alfalfa-maize cropping system than the perennial grass system.
For 2010 to 2013, average profit ($ ha-1 yr-1) was 531 for continuous corn, 347
for soybean-winter wheat-maize, 264 for oat-alfalfa-maize, 140 for oat/red
clover-maize, and -384 (loss) for perennial grass. Considering both residual soil
NO3-N and profitability data, the oat-alfalfa-maize rotation performed the best
in this setting. However, given current economic pressures widespread adoption is
likely to require changes in public policy.
PMCID: PMC5332022
146. J Pineal Res. 2017 May;62(4). doi: 10.1111/jpi.12401. Epub 2017 Mar 21.
Melatonin systemically ameliorates drought stress-induced damage in Medicago

sativa plants by modulating nitro-oxidative homeostasis and proline metabolism.
Antoniou C(1), Chatzimichail G(1), Xenofontos R(1), Pavlou JJ(1), Panagiotou

E(1), Christou A(2), Fotopoulos V(1).
Author information:
(1)Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus
University of Technology, Lemesos, Cyprus.
(2)Agricultural Research Institute, Ministry of Agriculture, Rural Development
and Natural Recourses, Nicosia, Cyprus.
Recent reports have uncovered the multifunctional role of melatonin in plant

physiological responses under optimal and suboptimal environmental conditions. In
this study, we explored whether melatonin pretreatment could provoke priming
effects in alfalfa (Medicago sativa L.) plants subsequently exposed to prolonged
drought stress (7 days), by withholding watering. Results revealed that the
rhizospheric application of melatonin (10 μmol L-1 ) remarkably enhanced the
drought tolerance of alfalfa plants, as evidenced by the observed plant tolerant
phenotype, as well as by the higher levels of chlorophyll fluorescence and
stomatal conductance, compared with nontreated drought-stressed plants. In
addition, lower levels of lipid peroxidation (MDA content) as well as of both H2
O2 and NO contents in primed compared with nonprimed stressed plants suggest that
melatonin pretreatment resulted in the systemic mitigation of drought-induced
nitro-oxidative stress. Nitro-oxidative homeostasis was achieved by melatonin
through the regulation of reactive oxygen (SOD, GR, CAT, APX) and nitrogen
species (NR, NADHde) metabolic enzymes at the enzymatic and/or transcript level.
Moreover, melatonin pretreatment resulted in the limitation of cellular redox
disruption through the regulation of the mRNA levels of antioxidant and
redox-related components (ADH, AOX, GST7, GST17), as well via osmoprotection
through the regulation of proline homeostasis, at both the enzymatic (P5CS) and
gene expression level (P5CS, P5CR). Overall, novel results highlight the
importance of melatonin as a promising priming agent for the enhancement of plant
tolerance to drought conditions through the regulation of nitro-oxidative and
osmoprotective homeostasis.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
DOI: 10.1111/jpi.12401
147. Front Plant Sci. 2017 Feb 7;8:89. doi: 10.3389/fpls.2017.00089. eCollection
2017.
The Impact of Genotyping-by-Sequencing Pipelines on SNP Discovery and

Identification of Markers Associated with Verticillium Wilt Resistance in
Autotetraploid Alfalfa (Medicago sativa L.).
Yu LX(1), Zheng P(2), Bhamidimarri S(3), Liu XP(1), Main D(2).
Author information:
(1)Plant Germplasm Introduction and Testing Research, United States Department of
Agriculture-Agricultural Research Service, Prosser WA, USA.
(2)Department of Horticulture, Washington State University, Pullman WA, USA.
(3)S&W Seed Company, Arlington WI, USA.
Verticillium wilt (VW) of alfalfa is a soilborne disease causing severe yield

loss in alfalfa. To identify molecular markers associated with VW resistance, we
used an integrated framework of genome-wide association study (GWAS) with
high-throughput genotyping by sequencing (GBS) to identify loci associated with
VW resistance in an F1 full-sib alfalfa population. Phenotyping was performed
using manual inoculation of the pathogen to cloned plants of each individual and
disease severity was scored using a standard scale. Genotyping was done by GBS,
followed by genotype calling using three bioinformatics pipelines including the
TASSEL-GBS pipeline (TASSEL), the Universal Network Enabled Analysis Kit (UNEAK),
and the haplotype-based FreeBayes pipeline (FreeBayes). The resulting numbers of
SNPs, marker density, minor allele frequency (MAF) and heterozygosity were
compared among the pipelines. The TASSEL pipeline generated more markers with the
highest density and MAF, whereas the highest heterozygosity was obtained by the
UNEAK pipeline. The FreeBayes pipeline generated tetraploid genotypes, with the
least number of markers. SNP markers generated from each pipeline were used
independently for marker-trait association. Markers significantly associated with
VW resistance identified by each pipeline were compared. Similar marker loci were
found on chromosomes 5, 6, and 7, whereas different loci on chromosome 1, 2, 3,
and 4 were identified by different pipelines. Most significant markers were
located on chromosome 6 and they were identified by all three pipelines. Of those
identified, several loci were linked to known genes whose functions are involved
in the plants' resistance to pathogens. Further investigation on these loci and
their linked genes would provide insight into understanding molecular mechanisms
of VW resistance in alfalfa. Functional markers closely linked to the resistance
loci would be useful for MAS to improve alfalfa cultivars with enhanced
resistance to the disease.
DOI: 10.3389/fpls.2017.00089
PMCID: PMC5293825
PMID: 28223988
148. J Dairy Sci. 2017 Apr;100(4):2672-2681. doi: 10.3168/jds.2016-11645. Epub 2017

Feb 16.
Replacing alfalfa hay with dry corn gluten feed and Chinese wild rye grass:
Effects on rumen fermentation, rumen microbial protein synthesis, and lactation
performance in lactating dairy cows.
Hao XY(1), Gao H(1), Wang XY(1), Zhang GN(1), Zhang YG(2).
Author information:
(1)College of Animal Science and Technology, Northeast Agricultural University,
Harbin, 150030, China.
Harbin, 150030, China. Electronic address: zhangyonggen@sina.com.
This experiment was conducted to investigate nutrient digestibility, rumen

microbial protein synthesis, and lactation performance when a portion of alfalfa
was replaced with combinations of dry corn gluten feed (DCGF) and Chinese wild
rye grass in the diet of lactating cows. Six multiparous and 3 primiparous
Chinese Holsteins were arranged in a replicated 3 × 3 Latin square experiment for
21-d periods. The animals were fed 1 of 3 treatment diets during each period: (1)
0% DCGF (0DCGF); (2) 6.5% DCGF (7DCGF); and (3) 11% DCGF (11DCGF). Diets were
isonitrogenous, and a portion of alfalfa hay was replaced with DCGF and Chinese
wild rye grass, with similar concentrate mixtures and corn silage contents. The
dry matter intake was greater for 11DCGF (21.9 kg/d) than for 0DCGF (20.7 kg/d)
or 7DCGF (21.2 kg/d). The treatment diets did not result in difference in milk
production, fat and lactose concentration, or yield. Compared with 0DCGF, the
ration containing 11% DCGF improved the milk protein concentration. Dry matter
and neutral detergent fiber digestibility was greater for 7DCGF (62.7% and 45.6%)
and 11DCGF (63.1% and 47.2%) than for 0DCGF (59.4% and 42.3%), and the nitrogen
digestibility was similar for the 3 treatments. The concentration of rumen
volatile fatty acids was higher in cows fed the 11DCGF diet than in those fed the
0DCGF diet, with no difference between the 7DCGF and 11DCGF diets. The estimated
microbial crude protein yield was greater for the 11DCGF diet (1985.1 g/d) than
for the 0DCGF diet (1745.0 g/d), with no difference between the 0DCGF and 7DCGF
diets. Thus, it appears that feeding DCGF and Chinese wild rye grass in
combination can effectively replace a portion of alfalfa hay in the rations of
lactating dairy cows.

DOI: 10.3168/jds.2016-11645
149. Animal. 2017 Sep;11(9):1464-1471. doi: 10.1017/S1751731117000209. Epub 2017

Feb
20.
Nutritive value and anthelmintic effect of sainfoin pellets fed to experimentally

infected growing rabbits.
Legendre H(1), Hoste H(2), Gidenne T(1).
Author information:
(1)1GenPhySE,Université de Toulouse,INRA,INPT,ENVT,31326 Castanet Tolosan,France.
(2)2UMR 1225 IHAP INRA/ENVT,23 Chemin des Capelles,31076 Toulouse,France.
Alternative strategies to synthetic chemical drugs are needed in livestock and

are a key issue in organic farming today. This study aimed at examining the
potentialities of sainfoin, a legume rich in condensed tannins, as a
nutraceutical that combines nutritive and antiparasitic effects in rabbits. To
test the effect of infection with a helminth (I: infected groups; NI: not
infected groups) and the effect of substituting 40% of the alfalfa in a control
diet (C) with sainfoin (diet S), four groups of 16 weaned rabbits were arranged
according to a 2×2 bifactorial design. The sainfoin diet differed from the
control by its tannin concentration (1.8% v. 1.0% tannic acid equivalent) and its
ADL concentration (84 v. 43 g/kg). For each diet, 16 rabbits were infected with
2125 third-stage larvae of Trichostrongylus colubriformis. Growth, feed intake,
feed conversion ratio and nematode faecal egg counts (FECs) were controlled for 6
weeks. A digestibility trial was performed. After necropsy, adult worms and eggs
in utero per female were counted and egg-hatching rate calculated. Growth tended
to be lower for S groups than for C groups (38.2 v. 39.5 g/day; P=0.06). Feed
intake was higher for S groups compared with C groups (+5.2 g dry matter/day;
P<0.01), as was the feed conversion ratio (3.2 v. 2.9; P<0.001), probably in
relation to the dietary ADL level. Protein digestibility was reduced in S groups
compared with C groups (-6.0 points; P<0.001), probably associated with the
effect of the tannin concentration. Digestibility of hemicelluloses was reduced
in infected rabbits compared with non-infected ones (-5 points; P=0.01). Using
the substitution method, the digestible energy of dehydrated sainfoin pellets
used as raw material was calculated at 11.12 MJ/kg and digestible proteins at 110
g/kg. The infection did not produce any clinical signs of digestive disorders. No
differences were observed according to the diet, neither in the number of adult
worms (972; P=0.50), the number of eggs in utero per female (14; P=0.95), nor FEC
(400 eggs/g; P=0.57). In contrast, the rate of faecal egg hatching in the S group
tended to be lower than in the control (58.3% v. 85.2%; P=0.08). In conclusion,
sainfoin seems to fit nutritive requirements for rabbits, supplies a large
quantity of fibre and particularly lignins, and limits the development of
nematode eggs in faeces.
DOI: 10.1017/S1751731117000209
150. PLoS One. 2017 Feb 16;12(2):e0171990. doi: 10.1371/journal.pone.0171990.

eCollection 2017.
Didehydrophenylalanine, an abundant modification in the beta subunit of plant

polygalacturonases.
Sergeant K(1), Printz B(1)(2), Gutsch A(1)(3), Behr M(1)(2), Renaut J(1), Hausman
JF(1).
Author information:
(1)Luxembourg Institute of Science and Technology (LIST), Environmental Research
and Innovation (ERIN) department, Esch-sur-Alzette, Luxembourg.
(2)Université catholique de Louvain, Earth and Life Institute Agronomy, Groupe de
Recherche en Physiologie Végétale Louvain-la-Neuve, Belgium.
(3)University of Hasselt, Centre for Environmental Sciences, Environmental
Biology, Diepenbeek, Belgium.
The structure and the activity of proteins are often regulated by transient or
stable posttranslational modifications (PTM). Different from well-known,
abundant modifications such as phosphorylation and glycosylation some
modifications are limited to one or a few proteins across a broad range of
related species. Although few examples of the latter type are known, the
evolutionary conservation of these modifications and the enzymes responsible for
their synthesis suggest an important physiological role. Here, the first
observation of a new, fold-directing PTM is described. During the analysis of
alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine
residues in the repeated tetrapeptide FxxY of the beta-subunit of
polygalacturonase. This modular protein is known to be involved in developmental
and stress-responsive processes. The presence of this modification was confirmed
using in-house and external datasets acquired by different commonly used
techniques in proteome studies. Based on these analyses it was found that all
identified phenylalanine residues in the sequence FxxY of this protein were
modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible
identification of ΔPhe in different species arguments that substantiate the
fold-determining role of ΔPhe are given.
PMCID: PMC5313189
151. J Econ Entomol. 2017 Feb 1;110(1):6-12. doi: 10.1093/jee/tow243.
Migratory Bee Hive Transportation Contributes Insignificantly to Transgenic

Pollen Movement Between Spatially Isolated Alfalfa Seed Fields.
Boyle NK(1), Kesoju SR(2), Greene SL(3), Martin RC(4), Walsh DB(2).
Author information:
(1)USDA-ARS Pollinating Insects Research Unit, North Logan, UT, USA.
(2)Department of Entomology, Washington State University, Prosser, WA, USA.
(3)USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins,
CO, USA.
(4)USDA-ARS National Forage Seed Production Research Center, Corvallis, OR, USA.
Contracted commercial beekeeping operations provide an essential pollination

service to many agricultural systems worldwide. Increased use of genetically
engineered crops in agriculture has raised concerns over pollinator-mediated gene
flow between transgenic and conventional agricultural varieties. This study
evaluated whether contracted migratory beekeeping practices influence transgenic
pollen flow among spatially isolated alfalfa fields. Twelve honey bee (Apis
mellifera L.) colonies were permitted to forage on transgenic alfalfa blossoms
for 1 wk in Touchet, WA. The hives were then transported 112 km to caged
conventional alfalfa plots following one and two nights of isolation (8 and 32 h,
respectively) from the transgenic source. Alfalfa seed harvested from the
conventional plots was assessed for the presence of the transgene using a new
seedling germination assay. We found that 8 h of isolation from a transgenic
alfalfa source virtually eliminated the incidence of cross-pollination between
the two varieties.
DOI: 10.1093/jee/tow243
152. Micron. 2017 Apr;95:23-30. doi: 10.1016/j.micron.2017.01.004. Epub 2017 Jan

19.
Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by

fluorescence microscopy.
Alves WS(1), Manoel EA(2), Santos NS(3), Nunes RO(3), Domiciano GC(3), Soares
MR(3).
Author information:
(1)Departamento de Bioquímica, Instituto de Química, UFRJ-Centro de Tecnologia,
Bloco A, Cidade Universitária, Cep: 21.941-909, Rio de Janeiro, Brazil; Programa
Químico de Petróleo e Biocombustíveis PRH-01, Instituto de Química, UFRJ-Centro
de Tecnologia, Bloco A, Cidade Universitária, Cep: 21.941-909, Rio de Janeiro,
Brazil. Electronic address: Wilber.sa@hotmail.com.
(2)Departamento de Biotecnologia Farmacêutica, Faculdade de Farmácia,
UFRJ-Avenida Carlos Chagas Filho, 373, Centro de Ciências da Saúde, Bloco K,
Cidade Universitária, Cep: 21.941-902, Rio de Janeiro, Brazil.
(3)Departamento de Bioquímica, Instituto de Química, UFRJ-Centro de Tecnologia,
Bloco A, Cidade Universitária, Cep: 21.941-909, Rio de Janeiro, Brazil.
Green technologies, such as phytoremediation, are effective for removing organic

pollutants derived from oil and oil products, including polycyclic aromatic
hydrocarbons (PAHs). Given the increasing popularity of these sustainable
remediation techniques, methods based on fluorescence microscopy and multiphoton
microscopy for the environmental monitoring of such pollutants have emerged in
recent decades as effective tools for phytoremediation studies aimed at
understanding the fate of these contaminants in plants. However, little is known
about the cellular and molecular mechanisms involved in PAH uptake, responses and
degradation by plants. Thus, the present study aimed to detect the location of
pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in
shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially
contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then
collected and observed under a fluorescence microscope to detect the presence of
PAHs in various tissues. One important finding of the present study was intense
fluorescence in the glandular secreting trichomes (GSTs) of plants grown in
contaminated soil. These trichomes, with a previously unknown function, may be
sites of PAH conjugation and degradation.
DOI: 10.1016/j.micron.2017.01.004
PMID: 28178583
153. Can J Microbiol. 2017 Apr;63(4):278-286. doi: 10.1139/cjm-2016-0369. Epub 2016

Nov 30.
The effects of Pantoea sp. strain Y4-4 on alfalfa in the remediation of

heavy-metal-contaminated soil, and auxiliary impacts of plant residues on the
remediation of saline-alkali soils.
Li S(1), Wang J(1), Gao N(1), Liu L(2), Chen Y(1)(3).
Author information:
(1)a College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu,
(2)b Jiangsu Provincial Academy of Agricultural Sciences, Nanjing, Jiangsu,
(3)c Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource,
National Joint Local Engineering Research Center for Rural Land Resources Use and
Consolidation, Nanjing, Jiangsu, People's Republic of China.
The plant-growth-promoting rhizobacterium (PGPR) Y4-4 was isolated from plant

rhizosphere soil and identified as Pantoea sp. by 16S rRNA sequence analysis. The
effects of strain Y4-4 on alfalfa grown in heavy-metals-contaminated soil was
investigated using a pot experiment. In a Cu-rich environment, the shoot dry mass
and total dry mass of plants inoculated with strain Y4-4 increased by 22.6% and
21%, and Cu accumulation increased by 15%. In a Pb-Zn-rich environment, the shoot
dry mass and total dry mass of plants inoculated with strain Y4-4 increased by
23.4% and 22%, and Zn accumulation increased by 30.3%. In addition, the salt
tolerance and biomass of wheat seedlings could be improved by applying strain
Y4-4 mixed with plant residue as a result of the Cu-rich plant residues providing
copper nutrition to wheat. This study offers an efficient PGPR with strong salt
tolerance and a safe strategy for the post-treatment of plant residue.
DOI: 10.1139/cjm-2016-0369
154. Sci Rep. 2017 Feb 7;7:42026. doi: 10.1038/srep42026.
Assessing of the contributions of pod photosynthesis to carbon acquisition of

seed in alfalfa (Medicago sativa L.).
Zhang W(1), Mao P(2), Li Y(3), Wang M(2), Xia F(2), Wang H(2).
Author information:
(1)Key Laboratory of Eco-Environment-Related Polymer Materials, Ministry of
Education of China, Key Laboratory of Gansu Polymer Materials, College of
Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070,
China.
(2)Institute of Grassland Science, China Agricultural University, Beijing 100193,
China.
(3)Dryland Farming Institute, Hebei Academy of Agricultural and Forestry
Sciences, Hengshui 053000, China.
The distribution of carbon from a branch setting pod in alfalfa was investigated
during the seed development of seeds to determine the relative contribution of
pod and leaf photoassimilates to the total C balance and to investigate the
partitioning of these photoassimilates to other plant organs. A 13Clabeling
procedure was used to label C photoassimilates of pods and leaves in alfalfa, and
the Δ13C values of a pod, leaves, a section of stem and roots were measured
during seed development on day 10, 15, 20 and 25 after labeling of the pod. The
results showed that the alfalfa pod had photosynthetic capacity early in the
development of seeds, and that pod photosynthesis could provide carbon to alfalfa
organs including seeds, pods, leaves, stems and roots, in addition to leaf
photosynthesis. Photosynthesis in the pod affected the total C balance of the
alfalfa branch with the redistribution of a portion of pod assimilates to other
plant organs. The assimilated 13C of the pod was used for the growth requirements
of plant seeds and pods. The requirements for assimilated C came primarily from
the young pod in early seed development, with later requirements provided
primarily from the leaf.
DOI: 10.1038/srep42026
PMCID: PMC5294575
PMID: 28169330
Conflict of interest statement: The authors declare no competing financial

interests.
155. Plant Pathol J. 2017 Feb;33(1):43-52. doi: 10.5423/PPJ.OA.06.2016.0143. Epub

2017
Feb 1.
Lucerne transient streak virus; a Recently Detected Virus Infecting Alfafa

(Medicago sativa) in Central Saudi Arabia.
Raza A(1), Al-Shahwan IM(1), Abdalla OA(1), Al-Saleh MA(1), Amer MA(1).
Author information:
(1)Department of Plant Protection, College of Food and Agricultural Sciences,
King Saud University, Riyadh 11451, Saudi Arabia.
A survey was conducted to determine the status of Lucerne transient streak virus
(LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh,
Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and
seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of
these samples were positive to LTSV. Two isolates of LTSV from each region were
selected for molecular studies. RT-PCR confirmed the presence of LTSV in the
selected samples using a specific primer pair. Percentage identity and homology
tree comparisons revealed that all Saudi isolates were more closely related to
each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%)
and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV
with ten other sobemoviruses based on the coat protein gene sequences confirmed
the distant relationship between them. Eleven out of fourteen plant species used
in host range study were positive to LTSV. This is the first time to document
that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and
Lathyrus sativus are new host plant species for LTSV and that N. occidentalis
being a good propagative host for it.
DOI: 10.5423/PPJ.OA.06.2016.0143
PMCID: PMC5291397
PMID: 28167887
156. Proteomics. 2017 Apr;17(8). doi: 10.1002/pmic.201600449. Epub 2017 Mar 13.
Cell wall proteome analysis of Arabidopsis thaliana mature stems.
Duruflé H(1), Clemente HS(1), Balliau T(2), Zivy M(2), Dunand C(1), Jamet E(1).
Author information:
(1)Laboratoire de Recherche en Sciences Végétales, CNRS, UPS, Université de
Toulouse, Auzeville, Castanet Tolosan, France.
(2)PAPPSO, GQE - Le Moulon, INRA, Univ. Paris-Sud, CNRS, AgroParisTech,
Université Paris-Saclay, Gif-sur-Yvette, France.
Plant stems carry flowers necessary for species propagation and need to be
adapted to mechanical disturbance and environmental factors. The stem cell walls
are different from other organs and can modify their rigidity or viscoelastic
properties for the integrity and the robustness required to withstand mechanical
impacts and environmental stresses. Plant cell wall is composed of complex
polysaccharide networks also containing cell wall proteins (CWPs) crucial to
perceive and limit the environmental effects. The CWPs are fundamental players in
cell wall remodeling processes, and today, only 86 have been identified from the
mature stems of the model plant Arabidopsis thaliana. With a destructive method,
this study has enlarged its coverage to 302 CWPs. This new proteome is mainly
composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6%
oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins
with interacting domains with proteins or polysaccharides. Compared to stem cell
wall proteomes already available (Brachypodium distachyon, Sacharum officinarum,
Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher
proportion of proteins acting on polysaccharides and of proteases, but a lower
proportion of oxido-reductases.
DOI: 10.1002/pmic.201600449
157. J Plant Res. 2017 Mar;130(2):387-396. doi: 10.1007/s10265-017-0905-9. Epub

2017
Feb 1.
Molecular cloning and functional analysis of the drought tolerance gene MsHSP70
from alfalfa (Medicago sativa L.).
Li Z(1), Long R(1), Zhang T(1), Wang Z(1), Zhang F(1), Yang Q(1), Kang J(2), Sun
Y(3).
Author information:
Beijing, 100193, People's Republic of China.
Beijing, 100193, People's Republic of China. kangjmei@126.com.
(3)College of Animal Science and Technology, China Agriculture University,
Beijing, 100193, People's Republic of China. cts-china@cau.edu.cn.
Heat shock proteins (HSPs) are a ubiquitously expressed class of protective

proteins that play a key role in plant response to stressful conditions. This
study aimed to characterize and investigate the function of an HSP gene in
alfalfa (Medicago sativa). MsHSP70, which contains a 2028-bp open reading frame,
was identified through homology cloning. MsHSP70 shares high sequence identity
(94.47%) with HSP70 from Medicago truncatula. Expression analysis of MsHSP70 in
alfalfa organs revealed a relatively higher expression level in aerial organs
such as flowers, stems and leaves than in roots. MsHSP70 was induced by heat
shock, abscisic acid (ABA) and hydrogen peroxide. Transgenic Arabidopsis
seedlings overexpressing MsHSP70 were hyposensitive to polyethylene glycol (PEG)
and ABA treatments, suggesting that exogenous expression of MsHSP70 enhanced
Arabidopsis tolerance to these stresses. Examination of physiological indexes
related to drought and ABA stress demonstrated that in comparison with
non-transgenic plants, T3 transgenic Arabidopsis plants had an increased proline
content, higher superoxide dismutase (SOD) activity, and decreased
malondialdehyde (MDA) content. Furthermore, higher relative water content (RWC)
was detected in transgenic plants compared with non-transgenic plants under
drought stress. These findings clearly indicate that molecular manipulation of
MsHSP70 in plants can have substantial effects on stress tolerance.
DOI: 10.1007/s10265-017-0905-9
158. Int J Mol Sci. 2017 Jan 29;18(2). pii: E298. doi: 10.3390/ijms18020298.
The Use of Gene Modification and Advanced Molecular Structure Analyses towards
Improving Alfalfa Forage.
Lei Y(1), Hannoufa A(2), Yu P(3).
Author information:
Bioresources, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada.
yal263@mail.usask.ca.
(2)Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3,
Canada. abdelali.hannoufa@agr.gc.ca.
Bioresources, University of Saskatchewan, Saskatoon, SK S7N 5A8, Canada.
Alfalfa is one of the most important legume forage crops in the world. In spite
of its agronomic and nutritive advantages, alfalfa has some limitations in the
usage of pasture forage and hay supplement. High rapid degradation of protein in
alfalfa poses a risk of rumen bloat to ruminants which could cause huge economic
losses for farmers. Coupled with the relatively high lignin content, which
impedes the degradation of carbohydrate in rumen, alfalfa has unbalanced and
asynchronous degradation ratio of nitrogen to carbohydrate (N/CHO) in rumen.
Genetic engineering approaches have been used to manipulate the expression of
genes involved in important metabolic pathways for the purpose of improving the
nutritive value, forage yield, and the ability to resist abiotic stress. Such
gene modification could bring molecular structural changes in alfalfa that are
detectable by advanced structural analytical techniques. These structural
analyses have been employed in assessing alfalfa forage characteristics, allowing
for rapid, convenient and cost-effective analysis of alfalfa forage quality. In
this article, we review two major obstacles facing alfalfa utilization, namely
poor protein utilization and relatively high lignin content, and highlight
genetic studies that were performed to overcome these drawbacks, as well as to
introduce other improvements to alfalfa quality. We also review the use of
advanced molecular structural analysis in the assessment of alfalfa forage for
its potential usage in quality selection in alfalfa breeding.
DOI: 10.3390/ijms18020298
PMCID: PMC5343834
159. Mol Ecol. 2017 Mar;26(6):1641-1651. doi: 10.1111/mec.14027. Epub 2017 Feb 23.
Two cultivated legume plants reveal the enrichment process of the microbiome in
the rhizocompartments.
Xiao X(1), Chen W(1), Zong L(1), Yang J(1), Jiao S(1), Lin Y(1), Wang E(2), Wei
G(1).
Author information:
(1)State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life
Sciences, Northwest A&F University, Yangling, Shaanxi, 712100, China.
(2)Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas,
Instituto Politécnico Nacional, 11340, México, D.F., Mexico.
The microbiomes of rhizocompartments (nodule endophytes, root endophytes,

rhizosphere and root zone) in soya bean and alfalfa were analysed using
high-throughput sequencing to investigate the interactions among legume species,
microorganisms and soil types. A clear hierarchical filtration of microbiota by
plants was observed in the four rhizocompartments - the nodule endosphere, root
endosphere, rhizosphere and root zone - as demonstrated by significant variations
in the composition of the microbial community in the different compartments. The
rhizosphere and root zone microbial communities were largely influenced by soil
type, and the nodule and root endophytes were primarily determined by plant
species. Diverse microbes inhabited the root nodule endosphere, and the
corresponding dominant symbiotic rhizobia belonged to Ensifer for alfalfa and
Ensifer-Bradyrhizobium for soya bean. The nonsymbiotic nodule endophytes were
mainly Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The
variation in root microbial communities was also affected by the plant growth
stage. In summary, this study demonstrated that the enrichment process of nodule
endophytes follows a hierarchical filtration and that the bacterial communities
in nodule endophytes vary according to the plant species.

DOI: 10.1111/mec.14027
160. Appl Environ Microbiol. 2017 Mar 17;83(7). pii: e03170-16. doi:
10.1128/AEM.03170-16. Print 2017 Apr 1.
Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia

coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds.
Cui Y(1), Walcott R(2), Chen J(3).
Author information:
(1)Department of Food Science and Technology, The University of Georgia, Griffin,
Georgia, USA.
(2)Department of Plant Pathology, The University of Georgia, Athens, Georgia,
USA.
(3)Department of Food Science and Technology, The University of Georgia, Griffin,
Georgia, USA jchen@uga.edu.
Vegetable seeds have the potential to disseminate and transmit foodborne

bacterial pathogens. This study was undertaken to assess the abilities of
selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to
attach to fungicide-treated versus untreated, and intact versus mechanically
damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized
seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C
for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized
water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C.
The seeds were then vortexed vigorously for 1 min, and pathogen populations in
seed rinse water and soaking buffer were determined using a standard plate count
assay. In general, the Salmonella cells had higher attachment ratios than the
EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached
Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In
contrast, individual fenugreek seeds had more attached pathogen cells, followed
by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC
cells attached to mechanically damaged seeds than to intact seeds (P < 0.05).
Although, on average, significantly more Salmonella and EHEC cells were recovered
from untreated than fungicide-treated seeds (P < 0.05), fungicide treatment did
not significantly affect the attachment of individual bacterial strains to
vegetable seeds (P > 0.05), with a few exceptions. This study fills gaps in the
current body of literature and helps explain bacterial interactions with
vegetable seeds with differing surface characteristics.IMPORTANCE Vegetable
seeds, specifically sprout seeds, have the potential to disseminate and transmit
foodborne bacterial pathogens. This study investigated the interaction between
two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds
with differing surface characteristics. This research helps understand whether
seed surface structure, integrity, and fungicide treatment affect the interaction
between bacterial cells and vegetable seeds.
DOI: 10.1128/AEM.03170-16
PMCID: PMC5359487
161. Genome Announc. 2017 Jan 26;5(4). pii: e01513-16. doi: 10.1128/genomeA.01513-
16.
Genome Sequence of the Symbiotic Type Strain Rhizobium tibeticum CCBAU85039T.
Torres Tejerizo G(1)(2), Wibberg D(3), Winkler A(3), Ormeño-Orrillo E(4),

Martínez-Romero E(5), Niehaus K(3), Pühler A(3), Kalinowski J(3), Lagares A(2),
Schlüter A(3), Pistorio M(2).
Author information:
(1)CeBiTec, Bielefeld University, Genome Research of Industrial Microorganisms,
Bielefeld, Germany gtorrest@cebitec.uni-bielefeld.de.
(2)IBBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La Plata,
(3)CeBiTec, Bielefeld University, Genome Research of Industrial Microorganisms,
Bielefeld, Germany.
(4)Laboratorio de Ecología Microbiana y Biotecnología, Departamento de Biología,
Facultad de Ciencias, Universidad Nacional Agraria La Molina, Lima, Peru.
(5)Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México,
Cuernavaca, Morelos, Mexico.
Rhizobium tibeticum was originally isolated from root nodules of Trigonella

archiducis-nicolai grown in Tibet, China. This species is also able to nodulate
Medicago sativa and Phaseolus vulgaris The whole-genome sequence of the type
strain, R. tibeticum CCBAU85039T, is reported in this study.
Copyright © 2017 Torres Tejerizo et al.
DOI: 10.1128/genomeA.01513-16
PMCID: PMC5270700
PMID: 28126941
162. Int J Phytoremediation. 2017 Aug 3;19(8):732-738. doi:

10.1080/15226514.2017.1284749.
Polychlorinated biphenyls in alfalfa: Accumulation, sorption and speciation in

different plant parts.
Teng Y(1), Sun X(1), Zhu L(1), Christie P(1), Luo Y(1)(2).
Author information:
(1)a Key Laboratory of Soil Environment and Pollution Remediation , Institute of
Soil Science, Chinese Academy of Sciences , Nanjing , China.
(2)b Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences ,
Yantai , China.
The accumulation, chemical speciation and distribution of polychlorinated

biphenyls (PCBs) were investigated in various parts of alfalfa. Moreover, the
adsorption characteristics for PCB 28 by alfalfa and the influencing factors of
the adsorption characteristics were studied. There were different degrees of PCB
accumulation in alfalfa roots, root nodules and shoots. The decreasing order of
the accumulation of PCBs in plant tissues was root nodules > roots > shoots, and
the decreasing order of the total PCB contents was roots > shoots > root nodules,
indicating that the roots were the main sink for PCB accumulation. There were
three modes of PCB speciation in alfalfa roots and root nodules, comprising
strong sorption (78%) and weak sorption (19%) on tissue surfaces and absorption
within tissues (2%). The adsorption isotherms of PCB 28 indicate that the
adsorption capacities of root nodules and shoots were both significantly higher
than that of the roots. Both lipids and carbohydrates, and especially lipids,
affected the PCB adsorption capacities of the tissues. These results may help in
the elucidation of the mechanisms of sorption and accumulation of PCBs in the
plants and their main influencing factors and thus contribute to the development
of phytoremediation technologies for PCB-contaminated soils.
DOI: 10.1080/15226514.2017.1284749
163. Environ Sci Pollut Res Int. 2017 Mar;24(8):7565-7571. doi:

10.1007/s11356-017-8466-y. Epub 2017 Jan 23.
Removal of phenanthrene in contaminated soil by combination of alfalfa, white-rot

fungus, and earthworms.
Deng S(1), Zeng D(2).
Author information:
(1)School of Resource and Environmental Engineering, Wuhan University of
Technology, Wuhan, 430070, China.
(2)School of Resource and Environmental Engineering, Wuhan University of
Technology, Wuhan, 430070, China. df5152@163.com.
The aim of this study was to investigate the removal of phenanthrene by

combination of alfalfa, white-rot fungus, and earthworms in soil. A 60-day
experiment was conducted. Inoculation with earthworms and/or white-rot fungus
increased alfalfa biomass and phenanthrene accumulation in alfalfa. However,
inoculations of alfalfa and white-rot fungus can significantly decrease the
accumulation of phenanthrene in earthworms. The removal rates for phenanthrene in
soil were 33, 48, 66, 74, 85, and 93% under treatments control, only earthworms,
only alfalfa, earthworms + alfalfa, alfalfa + white-rot fungus, and alfalfa +
earthworms + white-rot fungus, respectively. The present study demonstrated that
the combination of alfalfa, earthworms, and white-rot fungus is an effective way
to remove phenanthrene in the soil. The removal is mainly via stimulating both
microbial development and soil enzyme activity.
DOI: 10.1007/s11356-017-8466-y
164. Asian-Australas J Anim Sci. 2017 Jun;30(6):804-810. doi: 10.5713/ajas.16.0773.

Epub 2016 Dec 26.
Effects of sodium diacetate on the fermentation profile, chemical composition and

aerobic stability of alfalfa silage.
Yuan X(1), Wen A(1)(2), Desta ST(1), Wang J(1), Shao T(1).
Author information:
University, Nanjing 210095, China.
(2)College of Animal Science, Anhui Science and Technology University, Fengyang
233100, China.
OBJECTIVE: The objective of this study was to evaluate the effect of sodium
diacetate (SDA) on fermentation profile, chemical composition and aerobic
stability of alfalfa (Medicago sativa L.) silage.
METHODS: Fresh alfalfa was ensiled with various concentrations of SDA (0, 3, 5,
7, and 9 g/kg of fresh forage). After 60 days of the ensiling, the samples were
collected to examine the fermentative quality, chemical composition and aerobic
stability.
RESULTS: The application of SDA significantly (p<0.05) decreased silage pH with
the lowest value in silage with 7 g/kg of SDA. The proliferations of
enterobacteria, yeasts, molds and clostridia were inhibited by SDA, resulted in
lower ethanol, propionic and butyric acid concentrations and dry matter loss in
SDA treated silages than control. The increasing SDA linearly decreased free
amino acid N (p<0.001), ammonia N (p = 0.018) and non-protein N (p<0.001), while
linearly increased water soluble carbohydrate (p<0.001) and peptide N (p<0.001).
It is speculated that SDA accelerated the shift from homofermentative to
heterofermentative lactic acid bacteria during the silage fermentation, indicated
by lower lactic acid production in SDA-9 than SDA-7 silages after 60 days of
ensiling. Alfalfa silages treated with SDA at 7 g/kg had highest Flieg's point
and remained stable more than 9 d during aerobic exposure under humid and hot
conditions in southern China.
CONCLUSION: SDA may be used as an additive for alfalfa silages at a level of 7
g/kg.
DOI: 10.5713/ajas.16.0773
PMCID: PMC5411843
PMID: 28111451
165. Meat Sci. 2017 May;127:21-29. doi: 10.1016/j.meatsci.2016.12.013. Epub 2016

Dec
29.
Effect of supplementation with linseed or a blend of aromatic spices and time on

feed on fatty acid composition, meat quality and consumer liking of meat from
lambs fed dehydrated alfalfa or corn.
Realini CE(1), Bianchi G(2), Bentancur O(3), Garibotto G(2).
Author information:
(1)Ruminant Production Group, Facultad de Ciencias, CENUR Noroeste, Gral. Rivera
1350, Salto, Uruguay. Electronic address: carolina.realini@unorte.edu.uy.
(2)Department of Animal Production, Estación Experimental "Dr. Mario A.
Cassinoni", Facultad de Agronomía, Ruta 3 km 363.500, CP: 60000 Paysandú,
Uruguay.
(3)Biometrics Department of Statistics and Computing, Estación Experimental "Dr.
Mario A. Cassinoni", Facultad de Agronomía, Ruta 3 km 363.500, CP: 60000
Paysandú, Uruguay.
Cross-bred lambs (n=72) were fed finishing diets using a factorial arrangement of
treatments: BASAL DIET (alfalfa pellets or corn), SUPPLEMENT (none, linseed or
aromatic spices), TIME ON FEED (41 or 83days). Carcass and meat quality traits,
fatty acid composition, color stability and consumer liking were determined.
Feeding alfalfa improved sensory ratings and fatty acid composition of lamb.
However, corn or longer alfalfa feeding would be recommended if heavier and
fatter carcasses are sought. Consumer liking and fatty acid composition of lamb
were improved with addition of spices and linseed, respectively. But additional
antioxidant strategies should be considered to delay meat color deterioration
during storage if lambs are fed corn-linseed for 83days. Although alfalfa basal
diet and linseed supplementation improved fatty acid composition, feeding the
basal diets for at least 41days resulted in low n-3 fatty acid concentrations in
muscle.
DOI: 10.1016/j.meatsci.2016.12.013

10.1007/s11356-017-8459-x. Epub 2017 Jan 20.
Dissipation and phytoremediation of polycyclic aromatic hydrocarbons in freshly

spiked and long-term field-contaminated soils.
Wei R(1), Ni J(2), Li X(1), Chen W(1), Yang Y(1).
Author information:
(1)Key Laboratory of Humid Subtropical Eco-geographical Process, Ministry of
Education, College of Geographical Sciences, Fujian Normal University, Fuzhou,
350007, China.
(2)Key Laboratory of Humid Subtropical Eco-geographical Process, Ministry of
Education, College of Geographical Sciences, Fujian Normal University, Fuzhou,
350007, China. nijz@fjnu.edu.cn.
Pot experiments were used to compare the dissipation and phytoremediation effect
of alfalfa (Medicago sativa L.) for polycyclic aromatic hydrocarbons (PAHs) in a
freshly spiked soil and two field-contaminated soils with different soil organic
carbon (SOC) contents (Anthrosols, 1.41% SOC; Phaeozems, 8.51% SOC). In spiked
soils, the dissipation rates of phenanthrene and pyrene were greater than 99.5
and 94.3%, respectively, in planted treatments and 95.0 and 84.5%, respectively,
in unplanted treatments. In field-contaminated Anthrosols, there were limited but
significant reductions of 10.2 and 15.4% of total PAHs in unplanted and planted
treatments, respectively. In field-contaminated Phaeozems, there were no
significant reductions of total PAHs in either unplanted or planted treatments. A
phytoremediation effect was observed for the spiked soils and the Anthrosols, but
not for the Phaeozems. The results indicated that laboratory tests with spiked
soils cannot reflect the real state of field-contaminated soils. Phytoremediation
efficiency of PAHs in field-contaminated soils was mainly determined by the
content of SOC. Phytoremediation alone has no effect on the removal of PAHs in
field-contaminated soils with high SOC content.
DOI: 10.1007/s11356-017-8459-x
167. Bioresour Technol. 2017 Apr;229:69-77. doi: 10.1016/j.biortech.2017.01.012.

Epub
2017 Jan 12.
Effect of sole or combined administration of nitrate and 3-nitro-1-propionic acid

on fermentation and Salmonella survivability in alfalfa-fed rumen cultures in
vitro.
Correa AC(1), Trachsel J(2), Allen HK(2), Corral-Luna A(3), Gutierrez-Bañuelos

H(4), Ochoa-Garcia PA(3), Ruiz-Barrera O(3), Hume ME(5), Callaway TR(5), Harvey
RB(5), Beier RC(5), Anderson RC(6), Nisbet DJ(5).
Author information:
(1)USDA/ARS, Southern Plains Agricultural Research Center, Food and Feed Safety
Research Unit, 2881 F&B Road, College Station, TX 77845, USA; Facultad de
Zootecnia y Ecología, Universidad Autónoma de Chihuahua, Periférico Francisco R.
Almada km 1, Chihuahua, Chihuahua 31453, Mexico.
(2)USDA/ARS, National Animal Disease Center, Food Safety and Enteric Pathogens
Research Unit, 1920 Dayton Ave, Ames, IA 50010, USA.
(3)Facultad de Zootecnia y Ecología, Universidad Autónoma de Chihuahua,
Periférico Francisco R. Almada km 1, Chihuahua, Chihuahua 31453, Mexico.
(4)Unidad Academica de Medicina Veterinaria y Zootecnia, Universidad Autonoma de,
Zacatecas, Zacatecas 98500, Mexico.
Research Unit, 2881 F&B Road, College Station, TX 77845, USA.
Research Unit, 2881 F&B Road, College Station, TX 77845, USA. Electronic address:
robin.anderson@ars.usda.gov.
Ruminal methanogenesis is a digestive inefficiency resulting in the loss of

dietary energy consumed by the host and contributing to environmental methane
emission. Nitrate is being investigated as a feed supplement to reduce rumen
methane emissions but safety and efficacy concerns persist. To assess potential
synergies of co-administering sub-toxic amounts of nitrate and
3-nitro-1-propionate (NPA) on fermentation and Salmonella survivability with an
alfalfa-based diet, ruminal microbes were cultured with additions of 8 or 16mM
nitrate, 4 or 12mM NPA or their combinations. All treatments decreased
methanogenesis compared to untreated controls but volatile fatty acid production
and fermentation of hexose were also decreased. Nitrate was converted to nitrite,
which accumulated to levels inhibitory to digestion. Salmonella populations were
enriched in nitrate only-treated cultures but not in cultures co- or solely
treated with NPA. These results reveal a need for dose optimization to safely
reduce methane production with forage-based diets.
Published by Elsevier Ltd.
DOI: 10.1016/j.biortech.2017.01.012
168. PLoS One. 2017 Jan 18;12(1):e0170179. doi: 10.1371/journal.pone.0170179.

eCollection 2017.
Molybdate in Rhizobial Seed-Coat Formulations Improves the Production and

Nodulation of Alfalfa.
Zhou J(1), Deng B(1), Zhang Y(1), Cobb AB(2), Zhang Z(3).
Author information:
(1)Department of Grassland Science, College of Animal Science & Technology, China
Agricultural University, Beijing, China.
(2)Oklahoma State University, Stillwater, Oklahoma, United States of America.
(3)Institute of Agricultural Resources and Regional Planning, Chinese Academy of
Agricultural Science, Beijing, China.
Rhizobia-legume symbiosis is the most well researched biological nitrogen

fixation system. Coating legume seeds with rhizobia is now a recognized practical
measure for improving the production of legume corp. However, the efficacy of
some commercial rhizobia inoculants cannot be guaranteed in China due to the low
rate of live rhizobia in these products. A greenhouse experiment was conducted to
assess the effects of different rhizobial inoculant formulations on alfalfa
productivity and nitrogen fixation. Two rhizobia strains, (ACCC17631 and
ACCC17676), that are effective partners with alfalfa variety Zhongmu No. 1 were
assessed with different concentrations of ammonium molybdate in seed-coat
formulations with two different coating adhesives. Our study showed that the
growth, nodulation, and nitrogen fixation ability of the plants inoculated with
the ACCC17631 rhizobial strain were greatest when the ammonium molybdate
application was0.2% of the formulation. An ammonium molybdate concentration of
0.1% was most beneficial to the growth of the plants inoculated with the
ACCC17676 rhizobial strain. The sodium carboxymethyl cellulose and sodium
alginate, used as coating adhesives, did not have a significant effect on alfalfa
biomass and nitrogen fixation. However, the addition of skimmed milk to the
adhesive improved nitrogenase activity. These results demonstrate that a new
rhizobial seed-coat formulation benefitted alfalfa nodulation and yield.
PMCID: PMC5242510
Conflict of interest statement: The authors have declared that no competing

interests exist.
169. J Appl Microbiol. 2017 Apr;122(4):940-952. doi: 10.1111/jam.13401. Epub 2017

Feb
27.
Isolation and characterization of Rhizobium sp. strain YS-1r that degrades lignin
in plant biomass.
Jackson CA(1), Couger MB(1), Prabhakaran M(1), Ramachandriya KD(2), Canaan P(3),
Fathepure BZ(1).
Author information:
(1)Department of Microbiology and Molecular Genetics, Oklahoma State University,
Stillwater, OK, USA.
(2)Department of Biosystems and Agricultural Engineering, Oklahoma State
University, Stillwater, OK, USA.
(3)Department of Biochemistry and Molecular Biology, Oklahoma State University,
Stillwater, OK, USA.
AIMS: The aim of this work was to isolate novel lignin-degrading organisms.
METHODS AND RESULTS: Several pure cultures of bacteria that degrade lignin were
isolated from bacterial consortia developed from decaying biomass. Among the
isolates, Rhizobium sp. strain YS-1r (closest relative of Rhizobium petrolearium
strain SL-1) was explored for its lignin-degrading ability. Microcosm studies
showed that strain YS-1r was able to degrade a variety of lignin monomers, dimers
and also native lignin in switchgrass and alfalfa. The isolate demonstrated
lignin peroxidase (LiP) activity when grown on alkali lignin, p-anisoin,
switchgrass or alfalfa, and only negligible activity was measured in
glucose-grown cells suggesting inducible nature of the LiP activity. Analysis of
the strain YS-1r genome revealed the presence of a variety of genes that code for
various lignin-oxidizing, H2 O2 -producing as well as polysaccharide-hydrolysing
enzymes.
CONCLUSIONS: This study shows both the genomic and physiological capability of
bacteria in the genus Rhizobium to metabolize lignin and lignin-like compounds.
This is the first detailed report on the lignocellulose-degrading ability of a
Rhizobium species and thus this study expands the role of alpha-proteobacteria in
the degradation of lignin.
SIGNIFICANCE AND IMPACT OF THE STUDY: The organism's ability to degrade lignin is
significant since Rhizobia are widespread in soil, water and plant rhizospheres
and some fix atmospheric nitrogen and also have the ability to degrade aromatic
hydrocarbons.
DOI: 10.1111/jam.13401
170. Biometals. 2017 Feb;30(1):97-111. doi: 10.1007/s10534-017-9989-x. Epub 2017

Jan
13.
Methane alleviates copper-induced seed germination inhibition and oxidative

stress in Medicago sativa.
Samma MK(1), Zhou H(1), Cui W(1), Zhu K(1), Zhang J(1), Shen W(2).
Author information:
Agricultural University, Nanjing, 210095, China.
Agricultural University, Nanjing, 210095, China. wbshenh@njau.edu.cn.
Recent results discovered the protective roles of methane (CH4) against oxidative
stress in animals. However, the possible physiological roles of CH4 in plants are
still unknown. By using physiological, histochemical and molecular approaches,
the beneficial role of CH4 in germinating alfalfa seeds upon copper (Cu) stress
was evaluated. Endogenous production of CH4 was significantly increased in
Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH4. The pretreatment
with CH4 significantly alleviated the inhibition of seed germination and seedling
growth induced by Cu stress. Cu accumulation was obviously blocked as well.
Meanwhile, α/β amylase activities and sugar contents were increased, all of which
were consistent with the alleviation of seed germination inhibition triggered by
CH4. The Cu-triggered oxidative stress was also mitigated, which was confirmed by
the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma
membrane integrity in CH4-pretreated alfalfa seedlings. The results of
antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase
(SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic
activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD),
indicated that CH4 reestablished cellular redox homeostasis. Further, Cu-induced
proline accumulation was partly impaired by CH4, which was supported by the
alternation of proline metabolism. Together, these results indicated that CH4
performs an advantageous effect on the alleviation of seed germination inhibition
caused by Cu stress, and reestablishment of redox homeostasis mainly via
increasing antioxidant defence.
DOI: 10.1007/s10534-017-9989-x
171. Genes Genomics. 2017;39(1):111-119. doi: 10.1007/s13258-016-0481-8. Epub 2016

Oct
21.
Reconstruction of a composite comparative map composed of ten legume genomes.
Lee C(1), Yu D(2)(3), Choi HK(#)(2), Kim RW(#)(4).
Author information:
(1)Department of Medical Bioscience, Graduate School, Dong-A University,
Nakdong-Daero 550-Beongil 37, Saha-Gu, Busan, 49315 Republic of Korea.
(2)Department of Molecular Genetics, College of Natural Resources and Life
Science, Dong-A University, Nakdong-Daero 550-Beongil 37, Saha-Gu, Busan, 49315
Republic of Korea.
(3)SeqGenesis Inc., Gajeongbuk-Ro 96, Yuseong-Gu, Daejeon, 34111 Republic of
Korea.
(4)Korea Bioinformation Center, Korea Research Institute of Bioscience &
Biotechnology, Gwahak-Ro 125 Yuseong-Gu, Daejeon, 34141 Republic of Korea.
The Fabaceae (legume family) is the third largest and the second of agricultural
importance among flowering plant groups. In this study, we report the
reconstruction of a composite comparative map composed of ten legume genomes,
including seven species from the galegoid clade (Medicago truncatula, Medicago
sativa, Lens culinaris, Pisum sativum, Lotus japonicus, Cicer arietinum, Vicia
faba) and three species from the phaseoloid clade (Vigna radiata, Phaseolus
vulgaris, Glycine max). To accomplish this comparison, a total of 209
cross-species gene-derived markers were employed. The comparative analysis
resulted in a single extensive genetic/genomic network composed of 93 chromosomes
or linkage groups, from which 110 synteny blocks and other evolutionary events
(e.g., 13 inversions) were identified. This comparative map also allowed us to
deduce several large scale evolutionary events, such as chromosome
fusion/fission, with which might explain differences in chromosome numbers among
compared species or between the two clades. As a result, useful properties of
cross-species genic markers were re-verified as an efficient tool for
cross-species translation of genomic information, and similar approaches,
combined with a high throughput bioinformatic marker design program, should be
effective for applying the knowledge of trait-associated genes to other important
crop species for breeding purposes. Here, we provide a basic comparative
framework for the ten legume species, and expect to be usefully applied towards
the crop improvement in legume breeding.
DOI: 10.1007/s13258-016-0481-8
PMCID: PMC5196017
PMID: 28090266
Conflict of interest statement: Chaeyoung Lee, Dongwoon Yu, Hong-Kyu Choi, Ryan
W. Kim declare that they have no conflict of interest. Ethical approval This
article does not contain any studies with human subjects or animals performed by
any of the authors.
172. Ann Bot. 2017 Mar 1;119(5):901-914. doi: 10.1093/aob/mcw259.
Evolution and structural diversification of Nictaba-like lectin genes in food

crops with a focus on soybean (Glycine max).
Van Holle S(1), Rougé P(2), Van Damme EJM(1).
Author information:
(1)Laboratory of Biochemistry and Glycobiology, Department of Molecular
Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium.
(2)UMR 152 PHARMA-DEV, Université de Toulouse, IRD, UPS, Chemin des Maraîchers
35, 31400 Toulouse, France.
Background and Aims: The Nictaba family groups all proteins that show homology to
Nictaba, the tobacco lectin. So far, Nictaba and an Arabidopsis thaliana
homologue have been shown to be implicated in the plant stress response. The
availability of more than 50 sequenced plant genomes provided the opportunity for
a genome-wide identification of Nictaba -like genes in 15 species, representing
members of the Fabaceae, Poaceae, Solanaceae, Musaceae, Arecaceae, Malvaceae and
Rubiaceae. Additionally, phylogenetic relationships between the different species
were explored. Furthermore, this study included domain organization analysis,
searching for orthologous genes in the legume family and transcript profiling of
the Nictaba -like lectin genes in soybean.
Methods: Using a combination of BLASTp, InterPro analysis and hidden Markov
models, the genomes of Medicago truncatula , Cicer arietinum , Lotus japonicus ,
Glycine max , Cajanus cajan , Phaseolus vulgaris , Theobroma cacao , Solanum
lycopersicum , Solanum tuberosum , Coffea canephora , Oryza sativa , Zea mays,
Sorghum bicolor , Musa acuminata and Elaeis guineensis were searched for Nictaba
-like genes. Phylogenetic analysis was performed using RAxML and additional
protein domains in the Nictaba-like sequences were identified using InterPro.
Expression analysis of the soybean Nictaba -like genes was investigated using
microarray data.
Key Results: Nictaba -like genes were identified in all studied species and
analysis of the duplication events demonstrated that both tandem and segmental
duplication contributed to the expansion of the Nictaba gene family in
angiosperms. The single-domain Nictaba protein and the multi-domain F-box Nictaba
architectures are ubiquitous among all analysed species and microarray analysis
revealed differential expression patterns for all soybean Nictaba-like genes.
Conclusions: Taken together, the comparative genomics data contributes to our
understanding of the Nictaba -like gene family in species for which the
occurrence of Nictaba domains had not yet been investigated. Given the ubiquitous
nature of these genes, they have probably acquired new functions over time and
are expected to take on various roles in plant development and defence.
DOI: 10.1093/aob/mcw259
PMCID: PMC5379587
173. Sci Rep. 2017 Jan 13;7:40625. doi: 10.1038/srep40625.
P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E

biosynthesis and abscisic acid-mediated seed germination.
Jiang J(1), Chen Z(2), Ban L(3), Wu Y(1), Huang J(1)(3), Chu J(4), Fang S(4),
Wang Z(1), Gao H(1), Wang X(1).
Author information:
Beijing 100193, China.
(2)National Animal Husbandry Service, Ministry of Agriculture, Beijing 100125,
China.
(4)National Centre for Plant Gene Research, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.
P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved

in the biosynthesis of vitamin E, and is characterized by catalyzing the
conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an
HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was
expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl,
abscisic acid and salicylic acid were shown to significantly induce MsHPPD
expression, especially in the cotyledons and root tissues. Overexpression of
MsHPPD was found to significantly increase the level of β-tocotrienol and the
total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic
Arabidopsis seeds exhibited an accelerated germination time, compared with
wild-type seeds under normal conditions, as well as under NaCl and ABA
treatments. Meanwhile, the expression level of several genes associated with ABA
biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3
and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic
lines, as well as the total free ABA content. Taken together, these results
demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway,
but also plays a critical role in seed germination via affecting ABA biosynthesis
and signaling.
DOI: 10.1038/srep40625
PMCID: PMC5233959
PMID: 28084442
174. Am J Bot. 2017 Jan;104(1):62-71. doi: 10.3732/ajb.1600354. Epub 2017 Jan 12.
Five species, many genotypes, broad phenotypic diversity: When agronomy meets
functional ecology.
Prieto I(1), Litrico I(2), Violle C(3), Barre P(4).
Author information:
(1)CNRS, CEFE UMR 5175, Université de Montpellier-Université Paul Valéry-EPHE,
1919 Route de Mende 34293 Montpellier Cedex 5, France philippe.barre@inra.fr
iprietoaguilar@gmail.com.
(2)INRA, URP3F, RD 150, site du chêne, BP 86006 86600 Lusignan, France.
(3)CNRS, CEFE UMR 5175, Université de Montpellier-Université Paul Valéry-EPHE,
1919 Route de Mende 34293 Montpellier Cedex 5, France.
(4)INRA, URP3F, RD 150, site du chêne, BP 86006 86600 Lusignan, France
philippe.barre@inra.fr iprietoaguilar@gmail.com.
PREMISE OF THE STUDY: Current ecological theory can provide insight into the
causes and impacts of plant domestication. However, just how domestication has
impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes
and 35 cultivars from five grassland species to explore how selection drives
functional trait coordination and genetic differentiation.
METHODS: We quantified the extent of genetic diversity among different sets of
functional traits and determined how much genetic diversity has been generated
within populations of natural ecotypes and selected cultivars.
KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster
growth rates) and had larger and thinner leaves (greater SLA). We found large
(average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth
rate) genetic variability. The relative extent of genetic variability was greater
for whole-plant than for organ-level traits. This pattern was consistent within
ecotypes and within cultivars. However, ecotypes presented greater ITV
variability.
CONCLUSIONS: The results indicated that genetic diversity is large in
domesticated species with contrasting levels of heritability among functional
traits and that selection for high yield has led to indirect selection of some
associated leaf traits. These findings open the way to define which target traits
should be the focus in selection programs, especially in the context of
community-level selection.
© 2017 Botanical Society of America.
DOI: 10.3732/ajb.1600354
175. Acta Sci Pol Technol Aliment. 2016 Jul-Sep;15(3):333-338. doi:

10.17306/J.AFS.2016.3.32.
Legume seeds and cereal grains' capacity to accumulate iron while sprouting in
order to obtain food fortificant.
Zielińska-Dawidziak M(1), Staniek H(2), Król E(2), Piasecka-Kwiatkowska D(1),

Twardowski T(3).
Author information:
(1)Department of Food Biochemistry and Analysis, Poznań University of Life
Sciences, Poland.
(2)Department of Human Nutrition and Hygiene, Poznań University of Life Sciences,
Poland.
(3)Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland.
BACKGROUND: Prepared sprouts, after culturing in a medium with an increased iron

concentration, could become a beneficial food iron fortificant. However, the
efficient iron accumulation depends on the plants genus, species and/or
varieties. The aim of the study was to indicate the seeds or grains which
accumulate iron most efficiently during the sprouting process.
METHODS: Alfalfa, lentil, lupine and soybean seeds as well as wheat grains were
sprouted in abiotic stress conditions induced by the excess of iron(II) in
culture media. The tolerance of these plants to iron concentration and its
accumulation in the material obtained (with FAAS method) were analyzed.
RESULTS: The smallest tolerance was noted for lentil seeds and wheat grains.
Other plants developed in 25 mM solution of FeSO4. The highest accumulation of
iron was observed in alfalfa sprouts. However, lupine and soybean seeds are the
most recommended raw material for the production of the sprouts on an industrial
scale.

eCollection 2017.
Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago

sativa) Forage Quality Traits.
Biazzi E(1), Nazzicari N(1), Pecetti L(1), Brummer EC(2), Palmonari A(3), Tava
A(1), Annicchiarico P(1).
Author information:
(1)Council for Agricultural Research and Economics-Research Centre for Fodder
Crops and Dairy Productions (CREA-FLC), Lodi, Italy.
(2)Plant Breeding Center, Department of Plant Sciences, University of California,
Davis, CA, United States of America.
(3)Department of Veterinary Medicine, University of Bologna, Bologna, Italy.
Genetic progress for forage quality has been poor in alfalfa (Medicago sativa
L.), the most-grown forage legume worldwide. This study aimed at exploring
opportunities for marker-assisted selection (MAS) and genomic selection of forage
quality traits based on breeding values of parent plants. Some 154 genotypes from
a broadly-based reference population were genotyped by genotyping-by-sequencing
(GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein,
neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem
NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib
progenies in three growing conditions (summer harvest, full irrigation; summer
harvest, suspended irrigation; autumn harvest). Trait-marker analyses were
performed on progeny values averaged over conditions, owing to modest germplasm ×
condition interaction. Genomic selection exploited 11,450 polymorphic SNP
markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a
genome-wide association study (GWAS). GWAS confirmed the polygenic control of
quality traits and, in agreement with phenotypic correlations, indicated
substantially different genetic control of a given trait in stems and leaves. It
detected several SNPs in different annotated genes that were highly linked to
stem protein content. Also, it identified a small genomic region on chromosome 8
with high concentration of annotated genes associated with leaf ADL, including
one gene probably involved in the lignin pathway. Three genomic selection models,
i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar
prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were
moderate (0.3-0.4) for stem NDFD and leaf protein content, modest for leaf ADL
and NDFD, and low to very low for the other traits. Along with previous results
for the same germplasm set, this study indicates that GBS data can be exploited
to improve both quality traits (by genomic selection or MAS) and forage yield.
PMCID: PMC5222375

interests exist.
177. Environ Entomol. 2017 Feb 1;46(1):68-74. doi: 10.1093/ee/nvw150.
The Effects of Bean Leafroll Virus on Life History Traits and Host Selection
Behavior of Specialized Pea Aphid (Acyrthosiphon pisum, Hemiptera: Aphididae)
Genotypes.
Davis TS(1), Wu Y(2), Eigenbrode SD(2).
Author information:
(1)Forest & Rangeland Stewardship, Colorado State University, CO
(seth.davis@colostate.edu).
(2)Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, ID
(ywu@uidaho.edu; sanforde@uidaho.edu).
Intraspecific specialization by insect herbivores on different host plant species

contributes to the formation of genetically distinct "host races," but the
effects of plant virus infection on interactions between specialized herbivores
and their host plants have barely been investigated. Using three genetically and
phenotypically divergent pea aphid clones (Acyrthosiphon pisum L.) adapted to
either pea (Pisum sativum L.) or alfalfa (Medicago sativa L.), we tested how
infection of these hosts by an insect-borne phytovirus (Bean leafroll virus;
BLRV) affects aphid performance and preference. Four important findings emerged:
1) mean aphid survival rate and intrinsic rate of population growth (Rm) were
increased by 15% and 14%, respectively, for aphids feeding on plants infected
with BLRV; 2) 34% of variance in survival rate was attributable to clone × host
plant interactions; 3) a three-way aphid clone × host plant species × virus
treatment significantly affected intrinsic rates of population growth; and 4)
each clone exhibited a preference for either pea or alfalfa when choosing between
noninfected host plants, but for two of the three clones tested these preferences
were modestly reduced when selecting among virus-infected host plants. Our
studies show that colonizing BLRV-infected hosts increased A. pisum survival and
rates of population growth, confirming that the virus benefits A. pisum. BLRV
transmission affected aphid discrimination of host plant species in a
genotype-specific fashion, and we detected three unique "virus-association
phenotypes," with potential consequences for patterns of host plant use by aphid
populations and crop virus epidemiology.
© The Authors 2016. Published by Oxford University Press on behalf of

Entomological Society of America. All rights reserved. For Permissions, please
email: journals.permissions@oup.com.
DOI: 10.1093/ee/nvw150
178. Genome. 2017 Feb;60(2):104-127. doi: 10.1139/gen-2016-0111. Epub 2016 Oct 7.
Transcript analysis in two alfalfa salt tolerance selected breeding populations

relative to a non-tolerant population.
Gruber MY(1)(2), Xia J(2), Yu M(1), Steppuhn H(3), Wall K(3), Messer D(3), Sharpe
AG(4), Acharya SN(5), Wishart DS(2)(6), Johnson D(7), Miller DR(7), Taheri A(1).
Author information:
(1)a Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science
Place, Saskatoon, SK S7J 0X2, Canada.
(2)b Department of Computing Science, University of Alberta, 2-21 Athabasca Hall,
Edmonton, AB T6G 2R3, Canada.
(3)c Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-Food
Canada, P.O. Box 1030, Swift Current, SK S9H 3X2, Canada.
(4)d National Research Council, 110 Gymnasium Pl., Saskatoon, SK S7N 0W9, Canada.
(5)e AAFC Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 -
1st Avenue S., Lethbridge, AB T1J 4B1, Canada.
(6)f Department of Biological Sciences, University of Alberta, 11455 Saskatchewan
Drive, Edmonton, AB T6G 2R3, Canada.
(7)g Alforex Seeds, an affiliate of Dow AgroSciences, N4505 CTH M, West Salem, WI
54669, USA.
With the growing limitations on arable land, alfalfa (a widely cultivated,

low-input forage) is now being selected to extend cultivation into saline lands
for low-cost biofeedstock purposes. Here, minerals and transcriptome profiles
were compared between two new salinity-tolerant North American alfalfa breeding
populations and a more salinity-sensitive western Canadian alfalfa population
grown under hydroponic saline conditions. All three populations accumulated
two-fold higher sodium in roots than shoots as a function of increased electrical
conductivity. At least 50% of differentially expressed genes (p < 0.05) were
down-regulated in the salt-sensitive population growing under high salinity,
while expression remained unchanged in the saline-tolerant populations. In
particular, most reduction in transcript levels in the salt-sensitive population
was observed in genes specifying cell wall structural components, lipids,
secondary metabolism, auxin and ethylene hormones, development, transport,
signalling, heat shock, proteolysis, pathogenesis-response, abiotic stress, RNA
processing, and protein metabolism. Transcript diversity for transcription
factors, protein modification, and protein degradation genes was also more
strongly affected in salt-tolerant CW064027 than in salt-tolerant Bridgeview and
salt-sensitive Rangelander, while both saline-tolerant populations showed more
substantial up-regulation in redox-related genes and B-ZIP transcripts. The
report highlights the first use of bulked genotypes as replicated samples to
compare the transcriptomes of obligate out-cross breeding populations in alfalfa.
DOI: 10.1139/gen-2016-0111
179. Anal Chem. 2017 Jan 17;89(2):1117-1122. doi: 10.1021/acs.analchem.6b03101.
Epub
2016 Dec 22.
Direct Raman Spectroscopic Measurements of Biological Nitrogen Fixation under

Natural Conditions: An Analytical Approach for Studying Nitrogenase Activity.
Jochum T(1), Fastnacht A(2), Trumbore SE(2), Popp J(1)(3), Frosch T(1)(3).
Author information:
(1)Leibniz Institute of Photonic Technology , 07745 Jena, Germany.
(2)Max Planck Institute for Biogeochemistry , 07745 Jena, Germany.
(3)Institute of Physical Chemistry and Abbe Center of Photonics , 07745 Jena,
Germany.
Biological N2 fixation is a major input of bioavailable nitrogen, which

represents the most frequent factor limiting the agricultural production
throughout the world. Especially, the symbiotic association between legumes and
Rhizobium bacteria can provide substantial amounts of nitrogen (N) and reduce the
need for industrial fertilizers. Despite its importance in the global N cycle,
rates of biological nitrogen fixation have proven difficult to quantify. In this
work, we propose and demonstrate a simple analytical approach to measure
biological N2 fixation rates directly without a proxy or isotopic labeling. We
determined a mean N2 fixation rate of 78 ± 5 μmol N2 (g dry weight nodule)-1 h-1
of a Medicago sativa-Rhizobium consortium by continuously analyzing the amount of
atmospheric N2 in static environmental chambers with Raman gas spectroscopy. By
simultaneously analyzing the CO2 uptake and photosynthetic plant activity, we
think that a minimum CO2 mixing ratio might be needed for natural N2 fixation and
only used the time interval above this minimum CO2 mixing ratio for N2 fixation
rate calculations. The proposed approach relies only on noninvasive measurements
of the gas phase and, given its simplicity, indicates the potential to estimate
biological nitrogen fixation of legume symbioses not only in laboratory
experiments. The same methods can presumably also be used to detect N2 fluxes by
denitrification from ecosystems to the atmosphere.
DOI: 10.1021/acs.analchem.6b03101
PMID: 28043118
180. Chemosphere. 2017 Mar;171:476-484. doi: 10.1016/j.chemosphere.2016.12.093.

Epub
2016 Dec 22.
Comparison of arbuscular mycorrhizal fungal effects on the heavy metal uptake of

a host and a non-host plant species in contact with extraradical mycelial
network.
Mnasri M(1), Janoušková M(2), Rydlová J(2), Abdelly C(3), Ghnaya T(4).
Author information:
(1)Laboratoire des Plantes Extremophiles (LPE), Centre de Biotechnologies de la
Technopole de Borj Cedria, BP 901, Hammam Lif, 2050, Tunisia; Université de Tunis
El Manar, Tunisia; Institute of Botany, The Czech Academy of Sciences, Zámek 1,
252 43, Průhonice, Czechia.
(2)Institute of Botany, The Czech Academy of Sciences, Zámek 1, 252 43,
Průhonice, Czechia.
Technopole de Borj Cedria, BP 901, Hammam Lif, 2050, Tunisia.
Technopole de Borj Cedria, BP 901, Hammam Lif, 2050, Tunisia. Electronic address:
Tahar.ghnaya@gmail.com.
The effects of inoculation with an arbuscular mycorrhizal (AM) fungus on Cd and

Ni tolerance and uptake in Medicago sativa, an AM host, and Sesuvium
portulacastrum, a non-host plant, were investigated in a greenhouse experiment.
The plants were cultivated in sterilized sand in a two-compartmented system,
which prevented root competition but enabled colonization of the whole substrate
by AM fungal extraradical mycelium. M. sativa was either left non-inoculated or
inoculated with the AM fungus Rhizophagus irregularis, and both plants were
either cultivated without heavy metal (HM) addition or supplied with cadmium (Cd)
or nickel (Ni), each in two doses. Additional pots with singly cultivated plants
were established to control for the effect of the co-cultivation. AM
significantly enhanced the growth of M. sativa and substantially increased its
uptake of both HMs. The roots of S. portulacastrum became colonized by AM fungal
hyphae and vesicles. The presence of the AM fungus in the cultivation system
tended to increase the HM uptake of S. portulacastrum, but the effect was less
consistent and pronounced than that in M. sativa. We conclude that AM fungal
mycelium radiating from M. sativa did not negatively affect the growth and HM
uptake of S. portulacastrum. On the contrary, we hypothesize that it stimulated
the absorption and translocation of Cd and Ni in the non-host species. Thus, our
results suggest that AM fungal mycelium radiating from mycorrhizal plants does
not decrease the HM uptake of non-host plants, many of which are considered
promising candidate plants for phytoremediation.
2016.
Modulation of Legume Defense Signaling Pathways by Native and Non-native Pea

Aphid Clones.
Sanchez-Arcos C(1), Reichelt M(1), Gershenzon J(1), Kunert G(1).
Author information:
(1)Department of Biochemistry, Max Planck Institute for Chemical Ecology Jena,
Germany.
The pea aphid (Acyrthosiphon pisum) is a complex of at least 15 genetically

different host races that are native to specific legume plants, but can all
develop on the universal host plant Vicia faba. Despite much research, it is
still unclear why pea aphid host races (biotypes) are able to colonize their
native hosts while other host races are not. All aphids penetrate the plant and
salivate into plant cells when they test plant suitability. Thus plants might
react differently to the various pea aphid host races. To find out whether legume
species vary in their defense responses to different pea aphid host races, we
measured the amounts of salicylic acid (SA), the jasmonic acid-isoleucine
conjugate (JA-Ile), other jasmonate precursors and derivatives, and abscisic acid
(ABA) in four different species (Medicago sativa, Trifolium pratense, Pisum
sativum, V. faba) after infestation by native and non-native pea aphid clones of
various host races. Additionally, we assessed the performance of the clones on
the four plant species. On M. sativa and T. pratense, non-native clones that were
barely able to survive or reproduce, triggered a strong SA and JA-Ile response,
whereas infestation with native clones led to lower levels of both phytohormones.
On P. sativum, non-native clones, which survived or reproduced to a certain
extent, induced fluctuating SA and JA-Ile levels, whereas the native clone
triggered only a weak SA and JA-Ile response. On the universal host V. faba all
aphid clones triggered only low SA levels initially, but induced clone-specific
patterns of SA and JA-Ile later on. The levels of the active JA-Ile conjugate and
of the other JA-pathway metabolites measured showed in many cases similar
patterns, suggesting that the reduction in JA signaling was due to an effect
upstream of OPDA. ABA levels were downregulated in all aphid clone-plant
combinations and were therefore probably not decisive factors for aphid-plant
compatibility. Our results suggest that A. pisum clones manipulate plant-defense
signaling to their own advantage, and perform better on their native hosts due to
their ability to modulate the SA- and JA-defense signaling pathways.
DOI: 10.3389/fpls.2016.01872
PMCID: PMC5156717
PMID: 28018405
182. Plant Cell Physiol. 2017 Jan 1;58(1):e3. doi: 10.1093/pcp/pcw199.
CATchUP: A Web Database for Spatiotemporally Regulated Genes.
Nakamura Y(1), Kudo T(1), Terashima S(1), Saito M(1), Nambara E(2), Yano K(1).
Author information:
(1)Bioinformatics Laboratory, School of Agriculture, Meiji University,
Higashi-mita, Tama-ku, Kawasaki, Kanagawa, Japan.
(2)Department of Cell & Systems Biology, University of Toronto, Willcocks Street,
Toronto, Ontario, Canada.
For proper control of biological activity, some key genes are highly expressed in
a particular spatiotemporal domain. Mining of such spatiotemporally expressed
genes using large-scale gene expression data derived from a broad range of
experimental sources facilitates our understanding of genome-scale functional
gene networks. However, comprehensive information on spatiotemporally expressed
genes is lacking in plants. To collect such information, we devised a new index,
Δdmax, which is the maximum difference in relative gene expression levels between
sample runs which are neighboring when sorted by the levels. Employing this
index, we comprehensively evaluated transcripts using large-scale RNA sequencing
(RNA-Seq) data stored in the Sequence Read Archive for eight plant species:
Arabidopsis thaliana (Arabidopsis), Solanum lycopersicum (tomato), Solanum
tuberosum (potato), Oryza sativa (rice), Sorghum bicolor (sorghum), Vitis
vinifera (grape), Medicago truncatula (Medicago), and Glycine max (soybean).
Based on the frequency distribution of the Δdmax values, approximately 70,000
transcripts showing 0.3 or larger Δdmax values were extracted for the eight
species. Information on these genes including the Δdmax values, functional
annotations, conservation among species, and experimental conditions where the
genes show high expression levels is provided in a new database, CATchUP
(http://plantomics.mind.meiji.ac.jp/CATchUP). The CATchUP database assists in
identifying genes specifically expressed under particular conditions with
powerful search functions and an intuitive graphical user interface.
© The Author 2016. Published by Oxford University Press on behalf of Japanese

Society of Plant Physiologists. All rights reserved. For permissions, please
DOI: 10.1093/pcp/pcw199
183. J Hazard Mater. 2017 Mar 15;326:165-176. doi: 10.1016/j.jhazmat.2016.12.033.
Epub
2016 Dec 19.
Effects of untreated and treated wastewater at the morphological, physiological

and biochemical levels on seed germination and development of sorghum (Sorghum
bicolor (L.) Moench), alfalfa (Medicago sativa L.) and fescue (Festuca
arundinacea Schreb.).
Rekik I(1), Chaabane Z(2), Missaoui A(2), Bouket AC(3), Luptakova L(4), Elleuch
A(2), Belbahri L(5).
Author information:
(1)Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, University of
Sfax, Tunisia; NextBiotech, 98 Rue Ali Belhouane, 3030 Agareb, Tunisia.
(2)Laboratory of Plant Biotechnology, Faculty of Sciences of Sfax, University of
Sfax, Tunisia.
(3)NextBiotech, 98 Rue Ali Belhouane, 3030 Agareb, Tunisia; Graduate School of
Life and Environmental Sciences, Osaka Prefecture University, Osaka, Sakai,
Japan.
(4)NextBiotech, 98 Rue Ali Belhouane, 3030 Agareb, Tunisia; University of
Veterinary Med. and Pharmacy, Institute of Biology, Zoology and Radiobiology,
Department of Biology and Genetics, Komenského 73, 04181 Kosice, Slovakia.
(5)NextBiotech, 98 Rue Ali Belhouane, 3030 Agareb, Tunisia; Laboratory of Soil
Biodiversity, University of Neuchatel, 11 Rue Emile Argand, CH-2000, Neuchatel,
Switzerland. Electronic address: lassaad.belbahri@unine.ch.
Wastewater reuse in agriculture may help mitigate water scarcity. This may be
reached if high quality treatments removing harmful pollutants are applied. The
aim of the present study was to compare the effect of untreated (UTW) and treated
wastewater (TW) on germination and seedlings development of alfalfa (Medicago
sativa L.), fescue (Festuca arundinacea Schreb.) and sorghum (Sorghum bicolor
(L.) Moench). UTW presented high turbidity (130 NTU), chemical and biological
oxygen demand (COD, 719mgL-1, BOD5, 291mgL-1) and metal concentrations. These
levels caused mortality (18% for fescue), decreased germination speed in seeds
(37.5% for alfalfa) and reductions of root and stem length in seedlings (80% and
22% respectively for alfalfa). Adverse effects on seeds germination were
reflected at the biochemical level by increased H2O2 levels (6 times for sorghum
after 5days) and by increased Malondialdehyde (MDA) levels (more than 600 times
for sorghum roots) during seedlings development. When TW was used, these
parameters were close to control seeds ones. They were also dependent on plant
species and developmental stage. Therefore, for efficient reclaimed wastewater
reuse in irrigation, suitable crops, displaying wide tolerance to toxic contents
during germination and later seedling development stages have to be selected.
DOI: 10.1016/j.jhazmat.2016.12.033
PMID: 28013160
184. J Proteomics. 2017 Jan 30;152:109-120. doi: 10.1016/j.jprot.2016.10.013. Epub

2016 Oct 28.
Proteomic analysis provides insights into the molecular bases of hydrogen

gas-induced cadmium resistance in Medicago sativa.
Dai C(1), Cui W(2), Pan J(2), Xie Y(2), Wang J(3), Shen W(4).
Author information:
(1)College of Life Sciences, Nanjing Agricultural University, Nanjing 210095,
China; Laboratory Center of Life Sciences, Nanjing Agricultural University,
Nanjing 210095, China.
China.
(3)Laboratory Center of Life Sciences, Nanjing Agricultural University, Nanjing
210095, China.
China. Electronic address: wbshenh@njau.edu.cn.
Recently, molecular hydrogen (H2) has emerged as a bio-regulator both in animals

and plants. Normally, functions of endogenous generated H2 could be mimicked by
exogenously applied hydrogen-rich water (HRW) or hydrogen-rich saline
(particularly in animals). Although alfalfa seedlings showed more cadmium (Cd)
resistance after the administration with HRW, corresponding molecular mechanism
is still elusive. To address this gap, iTRAQ-based quantitative proteomics was
used. The results showed that a total of 2377 proteins were identified with <1%
FDR, and 1254 protein abundance perturbations were confidently assessed. Total of
248 significant differential proteins were identified in Cd- and/or HRW-treated
samples. Furthermore, 92 proteins from the 248 proteins were selected for further
bioinformatics analysis. Interestingly, results indicated that they were
classified into seven categories: defense and response to stress, sulfur compound
metabolic process, amino acid and protein metabolic process, carbohydrate and
energy metabolic process, secondary metabolic process, oxidation-reduction
process, and metal ion homeostasis. In addition, the protein expression patterns
were consistent with the results of decreased lipid peroxidation, increased
non-protein thiols abundance, as well as iron and zinc content. These suggest
that HRW alleviates Cd toxicity mainly by decreasing oxidative damage, enhancing
sulfur compound metabolic process, and maintaining nutrient element
homeostasis.BIOLOGICAL SIGNIFICANCE: Contamination of soils by Cd has become a
potential concern to crops. Medicago sativa is a widely used forage around the
world. Recently, hydrogen gas (H2) was suggested as a candidate of signal
molecule, and found to effectively attenuate Cd-induced damage in alfalfa
seedlings. However, the underlying molecular mechanism still needs to be further
elucidated. In the present work, an iTRAQ-based quantitative proteomics was
firstly carried out, and the results revealed the main molecular targets and
metabolic processes associated with Cd resistance conferred by H2. This study may
expand our understanding of hydrogen gas-medicated heavy metal tolerance in
plants.
DOI: 10.1016/j.jprot.2016.10.013

eCollection 2016.
Identification of Alfalfa Leaf Diseases Using Image Recognition Technology.
Qin F(1), Liu D(2), Sun B(3), Ruan L(1), Ma Z(1), Wang H(1).
Author information:
(1)Department of Plant Pathology, China Agricultural University, Beijing, China.
(2)College of Agriculture and Forestry Science and Technology, Hebei North
University, Zhangjiakou, Hebei Province, China.
(3)Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Common leaf spot (caused by Pseudopeziza medicaginis), rust (caused by Uromyces

striatus), Leptosphaerulina leaf spot (caused by Leptosphaerulina briosiana) and
Cercospora leaf spot (caused by Cercospora medicaginis) are the four common types
of alfalfa leaf diseases. Timely and accurate diagnoses of these diseases are
critical for disease management, alfalfa quality control and the healthy
development of the alfalfa industry. In this study, the identification and
diagnosis of the four types of alfalfa leaf diseases were investigated using
pattern recognition algorithms based on image-processing technology. A sub-image
with one or multiple typical lesions was obtained by artificial cutting from each
acquired digital disease image. Then the sub-images were segmented using twelve
lesion segmentation methods integrated with clustering algorithms (including
K_means clustering, fuzzy C-means clustering and K_median clustering) and
supervised classification algorithms (including logistic regression analysis,
Naive Bayes algorithm, classification and regression tree, and linear
discriminant analysis). After a comprehensive comparison, the segmentation method
integrating the K_median clustering algorithm and linear discriminant analysis
was chosen to obtain lesion images. After the lesion segmentation using this
method, a total of 129 texture, color and shape features were extracted from the
lesion images. Based on the features selected using three methods (ReliefF, 1R
and correlation-based feature selection), disease recognition models were built
using three supervised learning methods, including the random forest, support
vector machine (SVM) and K-nearest neighbor methods. A comparison of the
recognition results of the models was conducted. The results showed that when the
ReliefF method was used for feature selection, the SVM model built with the most
important 45 features (selected from a total of 129 features) was the optimal
model. For this SVM model, the recognition accuracies of the training set and the
testing set were 97.64% and 94.74%, respectively. Semi-supervised models for
disease recognition were built based on the 45 effective features that were used
for building the optimal SVM model. For the optimal semi-supervised models built
with three ratios of labeled to unlabeled samples in the training set, the
recognition accuracies of the training set and the testing set were both
approximately 80%. The results indicated that image recognition of the four
alfalfa leaf diseases can be implemented with high accuracy. This study provides
a feasible solution for lesion image segmentation and image recognition of
alfalfa leaf disease.
PMCID: PMC5158033

interests exist.
186. Mol Phylogenet Evol. 2017 Feb;107:367-381. doi: 10.1016/j.ympev.2016.11.020.

Epub
2016 Dec 2.
A cryptic species produced by autopolyploidy and subsequent introgression

involving Medicago prostrata (Fabaceae).
Eriksson JS(1), Blanco-Pastor JL(2), Sousa F(2), Bertrand YJK(2), Pfeil BE(2).
Author information:
(1)Department of Biological and Environmental Sciences, University of Gothenburg,
Box 461, 40530 Gothenburg, Sweden. Electronic address:
jonna.eriksson@bioenv.gu.se.
(2)Department of Biological and Environmental Sciences, University of Gothenburg,
Box 461, 40530 Gothenburg, Sweden.
Although hybridisation through genome duplication is well known, hybridisation

without genome duplication (homoploid hybrid speciation, HHS) is not. Few
well-documented cases have been reported. A possible instance of HHS in Medicago
prostrata Jacq. was suggested previously, based on only two genes and one
individual. We tested whether this species was formed through HHS by sampling
eight nuclear loci and 22 individuals, with additional individuals from related
species, using gene capture and Illumina sequencing. Phylogenetic inference and
coalescent simulations were performed to infer the causes of gene tree
incongruence. We found no evidence that phylogenetic differences among M.
prostrata individuals were the result of HHS. Instead, an autopolyploid origin of
tetraploids with introgression from tetraploids of the M. sativa complex is
likely. We argue that tetraploid M. prostrata individuals constitute a new
species, characterised by a partially non-overlapping distribution and
distinctive alleles (from the M. sativa complex). No gene flow from tetraploid to
diploid M. prostrata is apparent, suggesting partial reproductive isolation.
Thus, speciation via autopolyploidy appears to have been reinforced by
introgression. This raises the intriguing possibility that introgressed alleles
may be responsible for the increased range exploited by tetraploid M. prostrata
with respect to that of the diploids.
DOI: 10.1016/j.ympev.2016.11.020
187. J Anim Sci. 2016 Nov;94(11):4771-4780. doi: 10.2527/jas.2016-0603.
An insufficient glucose supply causes reduced lactose synthesis in lactating

dairy cows fed rice straw instead of alfalfa hay.
Wang B, Zhao FQ, Zhang BX, Liu JX.
The objective of the present study was to investigate the nutrient availability
for milk production in the mammary gland of lactating cows fed different
forage-based diets. The 3 diets contained 30% corn stover (CS), 30% rice straw
(RS), or 23% alfalfa hay plus 7% Chinese wild rye hay (AH) as a forage source.
All diets contained 15% of DM as corn silage and 55% of DM as concentrate. The
percentage of milk lactose was always lower in the RS-fed cows than in the cows
fed AH or CS during the 12-wk feeding trial ( < 0.01). Ruminal propionate
concentrations were lower in the RS group than in the AH group ( = 0.03). The
ratio of insulin to glucagon in the mammary venous plasma was greater in the AH
group than in the CS or RS group ( = 0.04). The abundance of the pyruvate
carboxylase mRNA in the liver was lower in the RS group than in the AH or CS
group ( = 0.04), and the abundance of mitochondrial phosphoenolpyruvate
carboxykinase, IGF-1 receptor, and phosphofructokinase-liver,
phosphofructokinase-muscle, and phosphofructokinase-platelet mRNA in the liver
were lower in the RS group than in the AH group ( < 0.05). The mammary glucose
uptake was greater in the AH-fed cows than in the CS- or RS-fed cows ( = 0.02).
The mRNA abundance of the glucose transporters in the mammary gland was similar
among the 3 treatments. The mRNA abundance of α-lactalbumin in the mammary gland
of the cows fed RS tended to be greater compared with that of the cows fed AH or
CS. The milk potassium concentration was greater in the cows fed RS than those
fed AH or CS ( < 0.01). In summary, the insufficient ruminal propionate
concentrations in the cows fed RS were associated with lower gluconeogenesis in
the liver, resulting in the shortage of glucose supply for mammary utilization.
DOI: 10.2527/jas.2016-0603
188. J Anim Sci. 2016 Oct;94(10):4326-4337. doi: 10.2527/jas.2016-0646.
Enteric methane and carbon dioxide emissions measured using respiration chambers,
the sulfur hexafluoride tracer technique, and a GreenFeed head-chamber system
from beef heifers fed alfalfa silage at three allowances and four feeding
frequencies.
Jonker A, Molano G, Antwi C, Waghorn GC.
The objective of this study was to determine methane (CH) and carbon dioxide (CO)
emissions from 8 beef heifers (approximately 20 mo of age and 382 ± 24.3 kg BW)
measured by respiration chambers and the sulfur hexafluoride (SF) tracer
technique and a mobile head-chamber, spot-sampling system (GreenFeed; C-Lock
Inc., Rapid City, SD) when fed alfalfa silage at 3 feeding levels and 4 feeding
frequencies. Feeding frequency may affect CH yield (g/kg DMI), and measurement
systems (such as GreenFeed or SF) are needed to obtain accurate estimates of CH
emissions from individual cattle under grazing where new pasture is provided once
or twice daily. The Hereford × Friesian heifers were used in 5 consecutive
periods (P1 to P5) of 14 d with CH and CO emissions measured with the SF
technique in wk 1 (5-6 d), with chambers in wk 2 (2 d), and with the GreenFeed
system when not in chambers (8 d) of each period. Alfalfa silage was restricted
to 6, 8, 8, and 8 kg DM/d in P1, P2, P3, and P4, respectively, and provided ad
libitum (10.9-12.2 kg DM/d) in P5. Silage was fed in 2, 2, 3, and 4 meals per day
in P1, P2, P3, and P4, respectively, and was continuously available (refilled
twice daily) in P5. Methane production increased from 141 to 265 g/d as DMI
doubled ( < 0.001), but average CH yields measured in respiration chambers (24.5
g/kg DMI) and by the SF technique (22.8 g CH/kg DMI) and the GreenFeed system
(26.2 g/kg DMI) were unaffected by feeding management ( = 0.6 for chambers and SF
and = 0.06 for GreenFeed). The CH yields estimated by the GreenFeed system did
not differ from CH yields estimated by the chambers in P1, P2, P3, and P5 but
were greater ( < 0.02) than CH yields estimated by the SF technique in P2, P3,
P4, and P5. Yields of CO (g/kg DMI) decreased with increasing DMI ( < 0.04) and
CO production (g/d) increased from 5,293 to 9,167 g/d as DMI increased ( <
0.001). In general, the SF technique and the GreenFeed system provided means for
CH yield that were not different from those of respiration chambers, and CH
yields (g/kg DMI) were unaffected by DMI level or feeding frequency.
DOI: 10.2527/jas.2016-0646
One-hour portable chamber methane measurements are repeatable and provide useful
information on feed intake and efficiency.
Robinson DL, Cameron M, Donaldson AJ, Dominik S, Oddy VH.
Feed intake (FI), live weight (LW), and ADG were recorded over 31 d in ninety-six
12-month-old ewes (progeny of 4 sires) given ad libitum access to chaffed
lucerne/cereal hay. Methane (CH) and CO emissions of each ewe were measured for
40 to 60 min in portable accumulation chambers (PAC) and in respiration chambers
(RC) over 22 h. Testing in RC increased the variability of FI on the test day and
depressed the amount eaten from an average of 1,384 to 1,062 g/d; FI depression
increased by 0.63 ± 0.24 percentage points for every kilogram of additional LW.
Repeatabilities of PAC measurements were 0.76 (CH) and 0.81 (CO). After adjusting
for LW and ADG, repeatabilities were 0.47 (PAC CH) and 0.43 (PAC CO). Daily FI
measurements had similar repeatability (0.76 before and 0.42 after adjustment for
LW and ADG). The PAC measurements were highly correlated with mean 31-d FI ( =
0.81 for both CH and CO). After adjustment for LW and ADG, PAC measurements were
moderately correlated with residual feed intake (RFI; = 0.37 for CH, 0.31 for
CO). The CH:CO ratio was also significantly correlated with mean 31-d FI ( =
0.52). After most of the ewes had given birth and raised lambs, repeat PAC
measurements were available for 91 of the ewes at 2 years of age (with ad libitum
access to the same feed). Correlations with the 2012 PAC measurements were 0.64
(CH) and 0.75 (CO). After adjusting 2014 PAC measurements for LW, correlations
with RFI in 2012 were 0.34 (CH) and 0.33 (CO), with a clear relationship between
sire means for RFI in 2012 and PAC CH adjusted for LW in 2014. These results
suggest that PAC tests under similar feeding conditions are repeatable over an
extended time period and can provide useful information on FI and feed efficiency
as well as methane emissions. Analyses of RC measurements might need to consider
FI depression.
DOI: 10.2527/jas.2016-0620
190. Sci Rep. 2016 Nov 25;6:37746. doi: 10.1038/srep37746.
Expanding and exploring the diversity of phytoplasmas from lucerne (Medicago

sativa).
Gopurenko D(1), Fletcher MJ(2), Liu J(2)(3)(4), Gurr GM(2)(3)(4).
Author information:
(1)Graham Centre for Agricultural Innovation (Charles Sturt University &NSW
Department of Primary Industries), NSW Department of Primary Industries, Wagga
Wagga Agricultural Institute, Pine Gully Rd, Wagga Wagga, NSW 2650, Australia.
(2)Graham Centre for Agricultural Innovation (Charles Sturt University &NSW
Department of Primary Industries), PO Box 883, Orange, NSW 2800, Australia.
(3)State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,
Fujian Agriculture and Forestry University, Fuzhou 350002, China.
(4)Institute of Applied Ecology, Fujian Agriculture &Forestry University, Fuzhou
350002, China.
Phytoplasmas are a group of insect-vectored bacteria responsible for disease in

many plant species worldwide. Among the crop species affected is the economically
valuable forage species lucerne. Here we provide comprehensive molecular evidence
for infection in multiple lucerne plants by a phytoplasma not previously known
from this plant species. This phytoplasma had a >99% genetic similarity to an
unclassified 16S rRNA subgroup previously reported as Stylosanthes little leaf
from Stylosanthes spp. and was genetically and symptomatically distinct from a
co-occurring but less common 16SrIIA group phytoplasma. Neighbour-joining
analyses with publicly available sequence data confirmed the presence of two
distinct phytoplasma lineages in the plant population. No PCR detections were
made among 38 individuals of 12 co-occurring weed species. Sequence analysis
revealed that all nine PCR detections from among 106 individuals of five
Hemiptera insect species from the site, three of which had previously been
reported as likely vectors, were false positives. This study demonstrates the
importance of sequencing to complement PCR detection and avoid potentially
inaccurate conclusions regarding vectors, highlights that sampling over a wide
spatio-temporal scale is important for vector and alternative host studies, and
extends to eight the number of phytoplasma 16 Sr groups known from lucerne.
DOI: 10.1038/srep37746
PMCID: PMC5123570
PMID: 27886229
191. Chemosphere. 2017 Feb;169:224-232. doi: 10.1016/j.chemosphere.2016.11.027.

Epub
2016 Nov 20.
Peroxidases from root exudates of Medicago sativa and Sorghum bicolor: Catalytic
properties and involvement in PAH degradation.
Dubrovskaya E, Pozdnyakova N, Golubev S, Muratova A, Grinev V, Bondarenkova A,

Turkovskaya O.
Peroxidases from root exudates of sorghum (Sorghum bicolor L. Moench) and alfalfa
(Medicago sativa L.) were purified and characterized, and their ability to
oxidize native PAHs and PAH-derivatives was evaluated. The obtained data confirm
that peroxidases are involved in the rhizosphere degradation of PAHs.
Nondenaturing PAGE showed that the peroxidases of both plants were represented by
a range of isoforms/isoenzymes (five to eight). Minor forms were lost during
further purification, and as a result, the major anionic form from alfalfa root
exudates and the major cationic form from those of sorghum were obtained. Both
electrophoretically homogeneous peroxidases were monomeric proteins with a
molecular weight of about 46-48 kDa. The pH optima and the main catalytic
constants for the test substrates were determined. On the basis of their
molecular and catalytic properties, the obtained enzymes were found to be typical
plant peroxidases. Derivatives of PAHs and potential products of their microbial
degradation (9-phenanthrol and 9,10-phenanthrenequinone), unlike the parent PAH
(phenanthrene), inhibited the catalytic activity of the peroxidases, possibly
indicating greater availability of the enzymes' active centers to these
substances. Peroxidase-catalyzed decreases in the concentrations of a number of
PAHs and their derivatives were observed. Sorghum peroxidase oxidized anthracene
and phenanthrene, while alfalfa peroxidase oxidized only phenanthrene.
1-Hydroxy-2-naphthoic acid was best oxidized by peroxidase of alfalfa. However,
quinone derivatives of PAHs were unavailable to sorghum peroxidase, but were
oxidized by alfalfa peroxidase. These results indicate that the major peroxidases
from root exudates of alfalfa and sorghum can have a role in the rhizosphere
degradation of PAHs.
The response of the soil microbial food web to extreme rainfall under different
plant systems.
Sun F(1)(2), Pan K(1), Tariq A(1)(2), Zhang L(1), Sun X(1), Li Z(1)(2), Wang
S(1)(2), Xiong Q(1)(2), Song D(1)(2), Olatunji OA(1)(2).
Author information:
Utilization &Ecological Restoration Biodiversity Conservation Key Laboratory of
Chengdu 610041, People's Republic of China.
(2)University of Chinese Academy of Sciences, Beijing, 100039, People's Republic
of China.
An agroforestry experiment was conducted that involved four planting systems:

monoculture of the focal species Zanthoxylum bungeanum and mixed cultures of Z.
bungeanum and Capsicum annuum, Z. bungeanum and Medicago sativa and Z. bungeanum
and Glycine max. Soil microbial food web (microorganisms and nematodes) was
investigated under manipulated extreme rainfall in the four planting systems to
assess whether presence of neighbor species alleviated the magnitude of extreme
rainfall on nutrient uptake of the focal species by increasing the stability of
soil food web. Our results indicate that in the focal species and G. max mixed
culture, leaf nitrogen contents of the focal species were higher than in the
monoculture and in the other mixed cultures under extreme rainfall. This result
was mainly due to the significant increase under extreme rainfall of G. max
species root biomass, resulting in enhanced microbial resistance and subsequent
net nitrogen mineralization rate and leaf nitrogen uptake for the focal species.
Differences in functional traits of neighbors had additive effects and led to a
marked divergence of soil food-web resistance and nutrient uptake of the focal
species. Climate change can indirectly alleviate focal species via its influence
on their neighbors.
DOI: 10.1038/srep37662
PMCID: PMC5118748
PMID: 27874081
193. Environ Sci Pollut Res Int. 2017 Jan;24(3):3117-3130. doi:

10.1007/s11356-016-8025-y. Epub 2016 Nov 17.
Comparison of the phytoremediation potentials of Medicago falcata L. And Medicago

sativa L. in aged oil-sludge-contaminated soil.
Panchenko L(1), Muratova A(2), Turkovskaya O(2).
Author information:
(1)Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian
Academy of Sciences, 13 Prospekt Entuziastov, Saratov, 410049, Russia.
lvpanchenko56@yandex.ru.
Academy of Sciences, 13 Prospekt Entuziastov, Saratov, 410049, Russia.
Thirteen-year monitoring of the vegetation growing in the industrial and adjacent

areas of an oil refinery showed the prevalence of yellow medick (Medicago falcata
L.) over other plant species, including alfalfa (Medicago sativa L.). A
comparative field study of the two Medicago species established that yellow
medick and alfalfa exhibited similar resistance to soil petroleum hydrocarbons
and that the pollutant concentration in their rhizosphere was 30% lower than that
in the surrounding bulk soil. In laboratory pot experiments, yellow medick
reduced the contaminant content by 18% owing to the degradation of the major
heavy oil fractions, such as paraffins, naphthenes, and alcohol and benzene tars;
and it was more successful than alfalfa. Both species were equally effective in
stimulating the total number of soil microorganisms, but the number of
hydrocarbon-oxidizing microorganisms, including polycyclic aromatic hydrocarbon
degraders, was larger in the root zone of alfalfa. In turn, yellow medick
provided a favorable balance of available nitrogen. Both Medicago species equally
stimulated the dehydrogenase and peroxidase activities of the soil, and yellow
medick increased the activity of soil polyphenol oxidase but reduced the activity
of catalase. The root tissue activity of catalase, ascorbate oxidase, and
tyrosinase was grater in alfalfa than in yellow medick. The peroxidase activity
of plant roots was similar in both species, but nondenaturing polyacrylamide gel
electrophoresis showed some differences in the peroxidase profiles of the root
extracts of alfalfa and yellow medick. Overall, this study suggests that the
phytoremediation potentials of yellow medick and alfalfa are similar, with some
differences.
DOI: 10.1007/s11356-016-8025-y
194. J Environ Radioact. 2017 Feb;167:201-210. doi: 10.1016/j.jenvrad.2016.10.022.

Epub 2016 Nov 11.
Tracing the source of sedimentary organic carbon in the Loess Plateau of China:
An integrated elemental ratio, stable carbon signatures, and radioactive isotopes
approach.
Liu C(1), Dong Y(2), Li Z(3), Chang X(4), Nie X(5), Liu L(4), Xiao H(4), Bashir
H(5).
Author information:
Institute of Soil and Water Conservation, Chinese Academy of Sciences, Yangling,
Shanxi, 712100, PR China; College of Environmental Science and Engineering, Hunan
University, Changsha 410082, PR China; Key Laboratory of Environmental Biology
and Pollution Control (Hunan University), Ministry of Education, Changsha 410082,
PR China.
Shanxi, 712100, PR China; Tianshui Soil and Water Conservation Experimental
Station, Yellow River Conservancy Commission, Tianshui, Gansu Province, 741000,
PR China. Electronic address: hwtsdyt@163.com.
Shanxi, 712100, PR China; College of Environmental Science and Engineering, Hunan
University, Changsha 410082, PR China; Key Laboratory of Environmental Biology
and Pollution Control (Hunan University), Ministry of Education, Changsha 410082,
PR China. Electronic address: lizw@hnu.edu.cn.
Shanxi, 712100, PR China.
(5)College of Environmental Science and Engineering, Hunan University, Changsha
410082, PR China; Key Laboratory of Environmental Biology and Pollution Control
(Hunan University), Ministry of Education, Changsha 410082, PR China.
Soil erosion, which will induce the redistribution of soil and associated soil
organic carbon (SOC) on the Earth's surface, is of critically importance for
biogeochemical cycling of essential elements and terrestrial carbon
sequestration. Despite the importance of soil erosion, surprisingly few studies
have evaluated the sources of eroded carbon (C). This study used natural
abundance levels of the stable isotope signature (13C) and radioactive isotopes
(137Cs and 210Pbex), along with elements ratio (C/N) based on a two end member
mixing model to qualitatively and quantitatively identify the sources of
sedimentary OC retained by check dam in the Qiaozigou small watershed in the
Loess Plateau, China. Sediment profiles (0-200 cm) captured at natural
depositional area of the basin was compared to possible source materials, which
included: superficial Loess mineral soils (0-20 cm) from three land use types
[i.e., grassland (Medicago sativa), forestland (Robinia pseudoacacia.), shrubland
(Prunus sibirica), and gully land (Loess parent material.)]. The results
demonstrated that SOC in sediments showed significantly negative correlation with
pH (P < 0.01), and positive correlation with soil water content (SWC) (P < 0.05).
The sedimentary OC was not derived from grasslands or gullies. Forestland and
shrubland were two main sources of eroded organic carbon within the surface
sediment (0-60 cm deep), except for that in the 20-40 cm soil layer.
Radionuclides analyses also implied that the surface sediments retained by
check-dams mainly originated from soils of forestland and shrubland. Results of
the two end-member mixing model demonstrated that more than 50% SOC (mean
probability estimate (MPE) 50.13% via 13C and 60.53% via C/N) in surface sediment
(0-20 cm deep) derived from forestland, whereas subsurface sedimentary SOC
(20-200 cm) mainly resulted from shrubland (MPE > 50%). Although uncertainties on
the sources of SOC in deep soils exist, the soil organic δ13C and C/N is still an
effective indicator for sources of sedimentary organic carbon in the deposition
zone in the short term (<10 years).
DOI: 10.1016/j.jenvrad.2016.10.022
195. Front Plant Sci. 2016 Oct 27;7:1598. eCollection 2016.
Assessment of Stress Tolerance, Productivity, and Forage Quality in T1 Transgenic

Alfalfa Co-overexpressing ZxNHX and ZxVP1-1 from Zygophyllum xanthoxylum.
Kang P(1), Bao AK(1), Kumar T(1), Pan YQ(1), Bao Z(1), Wang F(1), Wang SM(1).
Author information:
(1)State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral
Agriculture Science and Technology, Lanzhou University Lanzhou, China.
Salinization, desertification, and soil nutrient deprivation are threatening the

production of alfalfa (Medicago sativa L.) in northern China. We have previously
generated T0 transgenic alfalfa co-overexpressing Zygophyllum xanthoxylum ZxNHX
and ZxVP1-1 genes with enhanced salt and drought tolerance. To further develop
this excellent breeding material into the new forage cultivar, stress tolerance,
productivity, and forage quality of T1 transgenic alfalfa (GM) were assessed in
this study. The GM inherited the traits of salt and drought tolerance from T0
generation. Most importantly, co-overexpression of ZxNHX and ZxVP1-1 enhanced the
tolerance to Pi deficiency in GM, which was associated with more Pi accumulation
in plants. Meanwhile, T1 transgenic alfalfa developed a larger root system with
increased root size, root dry weight and root/shoot ratio, which may be one
important reason for the improvement of phosphorus nutrition and high biomass
accumulation in GM under various conditions. GM also accumulated more crude
protein, crude fiber, crude fat, and crude ash than wild-type (WT) plants,
especially under stress conditions and in the field. More interestingly, the
crude fat contents sharply dropped in WT (by 66-74%), whereas showed no change or
decreased less in GM, when subjected to salinity, drought or low-Pi. Our results
indicate that T1 transgenic alfalfa co-overexpressing ZxNHX and ZxVP1-1 shows
stronger stress tolerance, higher productivity and better forage quality. This
study provides a solid foundation for creating the alfalfa cultivars with high
yield, good quality and wide adaptability on saline, dry, and nutrient-deprived
marginal lands of northern China.
DOI: 10.3389/fpls.2016.01598
PMCID: PMC5081344
PMID: 27833624
Increase phosphorus availability from the use of alfalfa (Medicago sativa L)

green manure in rice (Oryza sativa L.) agroecosystem.
Gao X(1)(2), Shi D(3), Lv A(1), Wang S(1), Yuan S(1), Zhou P(1), An Y(1)(4).
Author information:
(1)School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai
200240, P. R. China.
(2)Research Center for Karst Wetland Ecology, Guizhou Minzu University, Guiyang
550025, P. R. China.
(3)School of Biology Science, Heze University, Heze 274015, P. R. China.
(4)Key Laboratory of Urban Agriculture (South), Ministry of Agriculture,
Shanghai, P. R. China.
Alfalfa is a good green manure source, but its effect on rice growth has not been
fully elucidated. Two green manure species, alfalfa and broad bean (Vicia faba
L.), and two N fertilizer levels, alone or combination, were applied to a rice
field. The results indicated that alfalfa had more pronounced effects on
increasing soil labile phosphorus (P) fractions (including NaHCO3-Pi, NaOH-Pi), P
uptake and soil enzyme activities (dehydrogenase, urease, acid phosphatase and
β-glucosidase) than broad bean and N fertilizer. The transformation of NaHCO3-Po
to labile P regulated by alfalfa played a significant direct and indirect effect
on grain yield. Although a much lower N input from alfalfa addition, a similar
grain yield with N fertilizer treatment was achieved, and the integration of
alfalfa with N fertilizer produced the highest grain yield and P availability,
which was associated with the highest urease, acid phosphatase and β-glucosidase
activity in soil. These results indicate that alfalfa green manure had a great
ability of increasing grain yield through enhancing P availability in rice paddy,
which could give us a way to reduce N fertilizer application by enhancing P
availability.
DOI: 10.1038/srep36981
PMCID: PMC5105083
PMID: 27833163
197. J Environ Manage. 2017 Jan 15;186(Pt 2):240-252. doi:

10.1016/j.jenvman.2016.06.050. Epub 2016 Nov 4.
Bioassessment of heavy metals in the surface soil layer of an opencast mine aimed
for its rehabilitation.
Ličina V(1), Akšić MF(2), Tomić Z(2), Trajković I(2), Antić Mladenović S(2),
Marjanović M(2), Rinklebe J(3).
Author information:
(1)University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080, Belgrade,
Zemun, Serbia. Electronic address: licina@agrif.bg.ac.rs.
(2)University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080, Belgrade,
Zemun, Serbia.
(3)University of Wuppertal, Soil- and Groundwater-Management, Pauluskirchstraße
7, 42285, Wuppertal, Germany. Electronic address: rinklebe@uni-wuppertal.de.
The contemporary reclamation method in an opencast coal mine closure comprises

the use of the preserved surface soil layer (SSL) before mining, and can be
directly returned to the areas being rehabilitated. The present study emphasizes
a risk in the use of such a SSL in mine rehabilitation due to the possible
excessive amount of heavy metals which usually derives from a metal-rich sediment
or fluvial character of overburden material. This indication was approved by the
bioassessment of cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead
(Pb), nickel (Ni), and zinc (Zn) in root and aerial parts of maize (Zea mays),
alfalfa (Medicago sativa), sweet clover (Melilotus officinalis), wheat (Triticum
aestivum), barley (Hordeum sativum), white clover (Trifolium repens), pasture
(Poales sp.), tomato (Solanum lycopersicum), potato (Solanum tuberosum) and
carrot (Daucus carota) grown on SSL in the opencast mine area. The fluvial layers
of the investigated mine SSL revealed the excessive existence of Ni and Cr,
probably of geogenic origin, according to the X-ray diffraction (XRD) which
detected Ni- and Cr-bearing minerals in soil fractions. In addition, the highest
residual fraction of these two heavy metals, obtained by sequential extraction
analyses, together with all other tested soil parameters, supported this
assumption. Nevertheless, the accumulations of Cr in tomato fruit (2.93 mg kg-1),
potato tuber (5.89 mg kg-1) and carrot root (7.35 mg kg-1) grown on the
investigated SSL were found to exceed a critical level of this element for human
nutrition. However, despite the evident excess of Ni in the investigated SSL, a
similar trend was not found in edible part of plants. The transfer and mobility
of the investigated metals was evaluated using the accumulation factor (AF < 1.0)
where the root were the preferential organ for the storage of heavy metals. This
investigation could bring an important input for its acceptability of use in soil
restoration after mining for food/fodder production, or it could indicate the
potential risks of the presence of heavy metals regarding its possible use in
improving the human surrounding.
DOI: 10.1016/j.jenvman.2016.06.050
198. Methods Mol Biol. 2017;1524:267-285.
Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein

(MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell
Suspension Culture.
Ayaydin F(1), Kotogány E(2), Ábrahám E(3), Horváth GV(4).
Author information:
(1)Laboratory of Cellular Imaging, Biological Research Center, Hungarian Academy
of Sciences, Szeged, Hungary.
(2)Flow Cytometer and Cell Sorter Laboratory, Biological Research Center,
Hungarian Academy of Sciences, Szeged, Hungary.
(3)Laboratory of Plant Genomics, Biological Research Center, Hungarian Academy of
Sciences, Szeged, Hungary.
(4)Laboratory of Molecular Regulators of Plant Growth, Biological Research
Center, Hungarian Academy of Sciences, 62 Temesvári krt, Szeged, 6726, Hungary.
hvg@brc.hu.
Deepening our knowledge on the regulation of the plant cell division cycle
depends on techniques that allow for the enrichment of cell populations in
defined cell cycle phases. Synchronization of cell division can be achieved using
different plant tissues; however, well-established cell suspension cultures
provide large amount of biological sample for further analyses. Here, we describe
the methodology of the establishment, propagation, and analysis of a Medicago
sativa suspension culture that can be used for efficient synchronization of the
cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for
the estimation of cell fraction that enters DNA synthesis phase of the cell cycle
and we also demonstrate the changes in the phosphorylation level of Medicago
sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.
DOI: 10.1007/978-1-4939-6603-5_17
199. Planta. 2017 Feb;245(2):397-408. doi: 10.1007/s00425-016-2613-4. Epub 2016 Nov

2.
Identification of bean hydroxycinnamoyl-CoA:tetrahydroxyhexanedioate

hydroxycinnamoyl transferase (HHHT): use of transgenic alfalfa to determine
acceptor substrate specificity.
Sullivan ML(1).
Author information:
(1)US Department of Agriculture, Agricultural Research Service, US Dairy Forage
Research Center, 1925 Linden Drive, Madison, WI, 53706, USA.
michael.sullivan@ars.usda.gov.
MAIN CONCLUSION: Transgenic alfalfa ( Medicago sativa L.) provides a useful

reverse genetics platform to elucidate acceptor substrate specificity for
uncharacterized BAHD family hydroxycinnamoyl-CoA hydroxycinnamoyl transferases.
Tissues of many plant species accumulate hydroxycinnamoyl derivatives, often
esters, thought to serve in protection against biotic and abiotic stresses. In
many cases, these specialized metabolites are produced by BAHD family
hydroxycinnamoyl-CoA hydroxycinnamoyl transferases (HCTs). Bean (Phaseolus
vulgaris) leaves contain both hydroxycinnamoyl-malate esters and an HCT activity
capable of making them. In seeking to identify this HCT from bean, we identified
a gene whose predicted protein showed a high degree of sequence similarity (75%)
to the Trifolium pratense (red clover) enzyme that carries out this reaction. The
encoded bean protein, however, failed to carry out the malate transfer reaction
when expressed in Escherichia coli. Expression of the gene in alfalfa (Medicago
sativa) resulted in accumulation of several new hydroxycinnamates not present in
nontransformed alfalfa, many of which corresponded to phenolics present in bean.
Using accurate mass and UV absorption spectral data, we identified the acceptor
substrate for this HCT as tetrahydroxyhexanedioic acids and demonstrated this
predicted transferase activity with the E. coli-expressed protein. This finding
adds to the growing number of BAHD family HCTs that have been characterized with
respect to substrate specificity. Such data, combined with primary sequence and
protein structural data will allow for a better understanding of the
structure/function relationships of these enzymes and may eventually aid the
rational design of such enzymes for altered substrate specificities.
Additionally, expression of HCTs of unknown substrate specificity in alfalfa and
characterization of the resulting accumulated novel metabolites could be a useful
approach to characterizing putative BAHD HCT enzymes.
DOI: 10.1007/s00425-016-2613-4
200. Plant J. 2016 Dec;88(6):1046-1057. doi: 10.1111/tpj.13315. Epub 2016 Nov 3.
Tricin-lignins: occurrence and quantitation of tricin in relation to phylogeny.
Lan W(1)(2), Rencoret J(3), Lu F(1)(4)(5), Karlen SD(1)(4), Smith BG(6), Harris
PJ(7), Del Río JC(3), Ralph J(1)(2)(4).
Author information:
(1)DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute,
University of Wisconsin, Madison, WI, USA.
(2)Department of Biological System Engineering, University of Wisconsin, Madison,
WI, USA.
(3)Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS), CSIC,
Avenida de la Reina Mercedes, 10, 41012, Seville, Spain.
(4)Department of Biochemistry, University of Wisconsin, Madison, WI, USA.
(5)State Key Laboratory of Pulp and Paper Engineering, South China University of
Technology, Guangzhou, China.
(6)School of Chemical Sciences, The University of Auckland, Auckland, New
Zealand.
(7)School of Biological Sciences, The University of Auckland, Auckland, New
Zealand.
Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a
flavone, was recently established as an authentic monomer in grass lignification
that likely functions as a nucleation site. It is linked onto lignin as an aryl
alkyl ether by radical coupling with monolignols or their acylated analogs.
However, the level of tricin that incorporates into lignin remains unclear.
Herein, three lignin characterization methods: acidolysis; thioacidolysis; and
derivatization followed by reductive cleavage; were applied to quantitatively
assess the amount of lignin-integrated tricin. Their efficiencies at cleaving the
tricin-(4'-O-β)-ether bonds and the degradation of tricin under the corresponding
reaction conditions were evaluated. A hexadeuterated tricin analog was
synthesized as an internal standard for accurate quantitation purposes.
Thioacidolysis proved to be the most efficient method, liberating more than 91%
of the tricin with little degradation. A survey of different seed-plant species
for the occurrence and content of tricin showed that it is widely distributed in
the lignin from species in the family Poaceae (order Poales). Tricin occurs at
low levels in some commelinid monocotyledon families outside the Poaceae, such as
the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the
non-commelinid monocotyledon family Orchidaceae (Orchidales). One eudicotyledon
was found to have tricin (Medicago sativa, Fabaceae). The content of
lignin-integrated tricin is much higher than the extractable tricin level in all
cases. Lignins, including waste lignin streams from biomass processing, could
therefore provide a large and alternative source of this valuable flavone,
reducing the costs, and encouraging studies into its application beyond its
current roles.
© 2016 The Authors. The Plant Journal published by Society for Experimental
Biology and John Wiley & Sons Ltd.
DOI: 10.1111/tpj.13315
201. J Agric Food Chem. 2016 Nov 2;64(43):8138-8145. Epub 2016 Oct 21.
Characterization of Chalcones from Medicago sativa L. and Their Hypolipidemic and

Antiangiogenic Activities.
Ma QG(1), Li T(1), Wei RR(2), Liu WM(1), Sang ZP(1), Song ZW(1).
Author information:
(1)College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University
, Nanyang, Henan 473061, People's Republic of China.
(2)Department of Pharmacology, College of Pharmacy, China Pharmaceutical
University , Nanjing, Jiangsu 210009, People's Republic of China.
Medicago sativa L. is the most important cultivated herbage, known as "the king
of forage" and "feed queen", in the world. A total of 8 new chalcones (1-8), and
12 known chalcones (9-20) were isolated from the aerial parts of M. sativa for
the first time. Their structures were identified by extensive spectral data and
references. The hypolipidemic and antiangiogenic activities of compounds (1-20)
were evaluated for the first time. Compounds 3, 4, 11, 12, and 20 (10 μM)
exhibited significant hypolipidemic activities by measuring the triglyceride
content in HepG2 cells, with simvastatin as the positive control. Moreover,
compounds 6, 8, 18, and 19 exhibited moderate antiangiogenic activities, which
inhibited vascular-endothelial-growth-factor-induced human umbilical vein
endothelial cell proliferation in vitro, with IC50 values of 13.86 ± 0.43, 15.53
± 0.19, 39.52 ± 0.24, and 45.04 ± 0.51 μM, respectively. These research results
may guide the search for new natural products with hypolipidemic and
antiangiogenic attributes.
DOI: 10.1021/acs.jafc.6b03883
202. Regul Toxicol Pharmacol. 2016 Nov;81:39-46. doi: 10.1016/j.yrtph.2016.07.010.

Epub 2016 Jul 18.
Antioxidant effects of alfalfa can improve iron oxide nanoparticle damage: Invivo
and invitro studies.
Sadeghi L(1), Tanwir F(2), Yousefi Babadi V(3).
Author information:
(1)Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares
University, Tehran, Iran. Electronic address: l.sadeghi@modares.ac.ir.
(2)Department of Odontology, Karolinska Institutet, Stockholm, Sweden.
(3)Department of Physiology, Payam Noor University of Iran, Iran. Electronic
address: v.yoosefi@gmail.com.
Medicago sativa Linn. or alfalfa (Leguminosae) has been used traditionally as an

effective cure for CNS, heart and metabolic disorders and digestive aids. Alfalfa
is a resistant plant against stress due to small antioxidant molecules and
enzymes. Our previous work demonstrated that 100 μg/ml of 50 nm size Fe2O3-NP
causes harsh oxidative stress in HepG2 cells and 100 mg/kg of same nanoparticle
causes extreme damage in rat's liver. Therefore it can be used as a useful model
for invivo and invitro studies of oxidative stress. This study assessed the
effects of two concentration of alfalfa on the mentioned invivo and invitro
damage. Our results showed alfalfa reduced reactive oxygen species (ROS)
production and enhanced reduced glutathione (GSH) that cause reduction of DNA
fragmentation and prevent apoptosis pathway so improve viability of the cells.
Results also showed alfalfa decreased hepatic enzymes penetrating and lipid
peroxidation in rat's liver. Note that Fe2O3-NP potentially has widespread
biological application but its usage is limited due to bio incompatibility. A
suitable antioxidant compound that reduce nanoparticle side effects can be used
as an effective adjuvant with iron oxide nanoparticle (and may be other
nanomaterials) in biological applications.
Copyright Â© 2016 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.yrtph.2016.07.010
203. Aust Vet J. 2016 Nov;94(11):433-434. doi: 10.1111/avj.12509.
Acute hepatic necrosis and death in a subadult southern white rhinoceros

(Ceratotherium simum) associated with exposure to sterigmatocystin in forage
contaminated with Aspergillus nidulans.
Bryant BR(1), Campbell M(2), Sangster C(3).
Author information:
(1)Taronga Western Plains Zoo, Obley Road, Dubbo, New South Wales 2830,
Australia. bryantbenn@gmail.com.
(2)Taronga Western Plains Zoo, Obley Road, Dubbo, New South Wales 2830,
Australia.
(3)Taronga Zoo, Mosman, NSW, Australia.
A young male southern white rhinoceros (Ceratotherium simum), which was resident
in a zoo as part of a multi-rhinoceros group, died suddenly. Necropsy and
histopathological findings supported a diagnosis of death from acute hepatic
necrosis. The microscopic distribution of liver lesions was suggestive of
hepatotoxicosis. Further investigation revealed potential exposure to a
mycotoxin, sterigmatocystin, present in spoiled lucerne hay contaminated with
Aspergillus nidulans. It was concluded that mycotoxicosis was the likely cause of
the hepatic necrosis and death in this animal.
© 2016 Australian Veterinary Association.
DOI: 10.1111/avj.12509
204. Int J Food Microbiol. 2016 Dec 5;238:222-232. doi:

10.1016/j.ijfoodmicro.2016.09.006. Epub 2016 Sep 13.
Plasma inactivation of microorganisms on sprout seeds in a dielectric barrier

discharge.
Butscher D(1), Van Loon H(2), Waskow A(2), Rudolf von Rohr P(1), Schuppler M(3).
Author information:
(1)ETH Zurich, Institute of Process Engineering, Sonneggstrasse 3, 8092 Zurich,
Switzerland.
(2)ETH Zurich, Institute of Process Engineering, Sonneggstrasse 3, 8092 Zurich,
Switzerland; ETH Zurich, Institute of Food Science and Nutrition,
Schmelzbergstrasse 7, 8092 Zurich, Switzerland.
(3)ETH Zurich, Institute of Food Science and Nutrition, Schmelzbergstrasse 7,
8092 Zurich, Switzerland. Electronic address: markus.schuppler@hest.ethz.ch.
Fresh produce is frequently contaminated by microorganisms, which may lead to

spoilage or even pose a threat to human health. In particular sprouts are
considered to be among the most risky foods sold at retail since they are grown
in an environment practically ideal for growth of bacteria and usually consumed
raw. Because heat treatment has a detrimental effect on the germination abilities
of sprout seeds, alternative treatment technologies need to be developed for
microbial inactivation purposes. In this study, non-thermal plasma
decontamination of sprout seeds is evaluated as a promising option to enhance
food safety while maintaining the seed germination capabilities. In detail,
investigations focus on understanding the efficiency of non-thermal plasma
inactivation of microorganisms as influenced by the type of microbial
contamination, substrate surface properties and moisture content, as well as
variations in the power input to the plasma device. To evaluate the impact of
these parameters, we studied the reduction of native microbiota or artificially
applied E. coli on alfalfa, onion, radish and cress seeds exposed to non-thermal
plasma in an atmospheric pressure pulsed dielectric barrier discharge streamed
with argon. Plasma treatment resulted in a maximum reduction of 3.4 logarithmic
units for E. coli on cress seeds. A major challenge in plasma decontamination of
granular food products turned out to be the complex surface topology, where the
rough surface with cracks and crevices can shield microorganisms from
plasma-generated reactive species, thus reducing the treatment efficiency.
However, improvement of the inactivation efficiency was possible by optimizing
substrate characteristics such as the moisture level and by tuning the power
supply settings (voltage, frequency) to increase the production of reactive
species. While the germination ability of alfalfa seeds was considerably
decreased by harsh plasma treatment, enhanced germination was observed under mild
conditions. In conclusion, the results from this study indicate that cold plasma
treatment represents a promising technology for inactivation of bacteria on seeds
used for sprout production while preserving their germination properties.
DOI: 10.1016/j.ijfoodmicro.2016.09.006
205. PLoS One. 2016 Oct 20;11(10):e0165018. doi: 10.1371/journal.pone.0165018.

eCollection 2016.
Impacts of Cropping Systems on Aggregates Associated Organic Carbon and Nitrogen

in a Semiarid Highland Agroecosystem.
Chu J(1), Zhang T(1), Chang W(1), Zhang D(1), Zulfiqar S(1), Fu A(1), Hao Y(1).
Author information:
(1)Key Laboratory of Resource Biology and Biotechnology in Western China
(Northwest University), Ministry of Education, Xi'an, Shaanxi, 710069, China.
The effect of cropping system on the distribution of organic carbon (OC) and
nitrogen (N) in soil aggregates has not been well addressed, which is important
for understanding the sequestration of OC and N in agricultural soils. We
analyzed the distribution of OC and N associated with soil aggregates in three
unfertilized cropping systems in a 27-year field experiment: continuously cropped
alfalfa, continuously cropped wheat and a legume-grain rotation. The objectives
were to understand the effect of cropping system on the distribution of OC and N
in aggregates and to examine the relationships between the changes in OC and N
stocks in total soils and in aggregates. The cropping systems increased the
stocks of OC and N in total soils (0-40 cm) at mean rates of 15.6 g OC m-2 yr-1
and 1.2 g N m-2 yr-1 relative to a fallow control. The continuous cropping of
alfalfa produced the largest increases at the 0-20 cm depth. The OC and N stocks
in total soils were significantly correlated with the changes in the >0.053 mm
aggregates. 27-year of cropping increased OC stocks in the >0.053 mm size class
of aggregates and N stocks in the >0.25 mm size class but decreased OC stocks in
the <0.053 mm size class and N stocks in the <0.25 mm size class. The increases
in OC and N stocks in these aggregates accounted for 99.5 and 98.7% of the total
increases, respectively, in the continuous alfalfa system. The increases in the
OC and N stocks associated with the >0.25 mm aggregate size class accounted for
more than 97% of the total increases in the continuous wheat and the legume-grain
rotation systems. These results suggested that long-term cropping has the
potential to sequester OC and N in soils and that the increases in soil OC and N
stocks were mainly due to increases associated with aggregates >0.053 mm.
PMCID: PMC5072639

interests exist.
206. Front Plant Sci. 2016 Oct 4;7:1502. eCollection 2016.
Proteomic Identification of Differentially Expressed Proteins during Alfalfa

(Medicago sativa L.) Flower Development.
Chen L(1), Chen Q(2), Zhu Y(3), Hou L(3), Mao P(3).
Author information:
(1)Beijing Key Laboratory of Grassland Science, Forage Seed Lab, China
Agricultural UniversityBeijing, China; Chifeng Academy of Agricultural and Animal
SciencesChifeng, China.
(2)Chengdu Municipal Development and Reform Commission Chengdu, China.
(3)Beijing Key Laboratory of Grassland Science, Forage Seed Lab, China
Agricultural University Beijing, China.
Flower development, pollination, and fertilization are important stages in the

sexual reproduction process of plants; they are also critical steps in the
control of seed formation and development. During alfalfa (Medicago sativa L.)
seed production, some distinct phenomena such as a low seed setting ratio,
serious flower falling, and seed abortion commonly occur. However, the causes of
these phenomena are complicated and largely unknown. An understanding of the
mechanisms that regulate alfalfa flowering is important in order to increase seed
yield. Hence, proteomic technology was used to analyze changes in protein
expression during the stages of alfalfa flower development. Flower samples were
collected at pre-pollination (S1), pollination (S2), and the post-pollination
senescence period (S3). Twenty-four differentially expressed proteins were
successfully identified, including 17 down-regulated in pollinated flowers, one
up-regulated in pollinated and senesced flowers, and six up-regulated in senesced
flowers. The largest proportions of the identified proteins were involved in
metabolism, signal transduction, defense response, oxidation reduction, cell
death, and programmed cell death (PCD). Their expression profiles demonstrated
that energy metabolism, carbohydrate metabolism, and amino acid metabolism
provided the nutrient foundation for pollination in alfalfa. Furthermore, there
were three proteins involved in multiple metabolic pathways: dual specificity
kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone
oxidoreductase-like protein. Expression patterns of these proteins indicated that
MAPK cascades regulated multiple processes, such as signal transduction, stress
response, and cell death. PCD also played an important role in the alfalfa flower
developmental process, and regulated both pollination and flower senescence. The
current study sheds some light on protein expression profiles during alfalfa
flower development and contributes to the understanding of the basic molecular
mechanisms during the alfalfa flowering process. These results may offer insight
into potential strategies for improving seed yield, quality, and stress tolerance
in alfalfa.
DOI: 10.3389/fpls.2016.01502
PMCID: PMC5047909
PMID: 27757120
207. Plant Physiol Biochem. 2016 Nov;108:344-352. doi:
10.1016/j.plaphy.2016.08.002.
Epub 2016 Aug 3.
Rhizobial strains exert a major effect on the amino acid composition of alfalfa
nodules under NaCl stress.
Bertrand A(1), Bipfubusa M(2), Dhont C(3), Chalifour FP(4), Drouin P(5),
Beauchamp CJ(6).
Author information:
(1)Soils and Crops Research and Development Centre, Agriculture and Agri-Food
Canada, Québec, G1V 2J3, QC, Canada. Electronic address:
Annick.Bertrand@agr.gc.ca.
(2)Departement de phytologie, 2425 rue de l'agriculture, Université Laval,
Québec, G1V 0A6, QC, Canada. Electronic address: marie.bipfubusa.1@ulaval.ca.
Québec, G1V 0A6, QC, Canada. Electronic address: Catherine.dhont.1@ulaval.ca.
Québec, G1V 0A6, QC, Canada. Electronic address:
Francois-p.chalifour@fsaa.ulaval.ca.
(5)Université du Québec en Abitibi-Témiscamingue (UQAT), Rouyn-Noranda, J9X 5E4,
QC, Canada. Electronic address: pdrouin@lallemand.com.
Québec, G1V 0A6, QC, Canada. Electronic address:
Chantal.Beauchamp@fsaa.ulaval.ca.
Specific amino acids have protective functions in plants under stress conditions.
This study assessed the effects of rhizobial strains on the amino acid
composition in alfalfa under salt stress. Two alfalfa cultivars (Medicago sativa
L. cv Apica and salt-tolerant cv Halo) in association with two Sinorhizobium
meliloti strains with contrasting growth under salt stress (strain A2 and
salt-tolerant strain Rm1521) were exposed to different levels of NaCl (0, 20, 40,
80 or 160 mM NaCl) under controlled conditions. We compared root and shoot
biomasses, as well as root:shoot ratio for each association under these
conditions as indicators of the salt tolerance of the symbiosis. Amino acid
concentrations were analyzed in nodules, leaves and roots. The total
concentration of free amino acids in nodules was mostly rhizobial-strain
dependent while in leaves and roots it was mostly responsive to salt stress. For
both cultivars, total and individual concentrations of amino acids including
asparagine, proline, glutamine, aspartate, glutamate, γ-aminobutyric acid (GABA),
histidine and ornithine were higher in Rm1521 nodules than in A2 nodules.
Conversely, lysine and methionine were more abundant in A2 nodules than in Rm1521
nodules. Proline, glutamine, arginine, GABA and histidine substantially
accumulated in salt-stressed nodules, suggesting an enhanced production of amino
acids associated with osmoregulation, N storage or energy metabolism to
counteract salt stress. Combining the salt-tolerant strain Rm1521 and the
salt-tolerant cultivar Halo enhanced the root:shoot ratios and amino acid
concentrations involved in plant protection which could be in part responsible
for the enhancement of salt tolerance in alfalfa.
Crown Copyright © 2016. Published by Elsevier Masson SAS. All rights reserved.
DOI: 10.1016/j.plaphy.2016.08.002
208. J Sci Food Agric. 2017 Jul;97(9):2759-2766. doi: 10.1002/jsfa.8103. Epub 2016
Nov
23.
Effects of four short-chain fatty acids or salts on the dynamics of nitrogen

transformations and intrinsic protease activity of alfalfa silage.
Yuan X(1), Wen A(1)(2), Desta ST(1), Dong Z(1), Shao T(1).
Author information:
University, Wei gang 1, Nanjing, 210095, China.
(2)College of Animal Science, Anhui Science and Technology University, Feng yang,
233100, China.
BACKGROUND: Short-chain fatty salts have been widely used as food and forage
preservatives because of their antimicrobial properties. This study evaluated the
effects of four chemical compounds with antimicrobial properties on nitrogen
transformations and intrinsic protease activity of alfalfa silage.
RESULTS: Potassium diformate (PD) and formic acid (FA) rapidly reduced silage pH.
Silages treated with sodium diacetate (SD) and calcium propionate (CAP) had
higher final peptide N concentrations than other silage. The free amino acid N
contents in PD and FA treated silages were lower than other silages at all
intervals of ensilage. The ammonia N concentrations in FA and PD silages were the
lowest, followed by SD and CAP silages. As ensiling progressed, the
aminopeptidase activity was completely lost by day 5 for FA and PD silages and
inactive by day 7 for SD silage, while it remained active after day 7 for control
and CAP silage. The carboxypeptidase activities in FA and PD silages were already
reduced below 50% by day 1 of ensiling.
CONCLUSION: Potassium diformate was as effective as formic acid in depressing the
proteolysis, while sodium diacetate and calcium propionate were inferior to
formic acid in protecting alfalfa proteins from being hydrolysed. © 2016 Society
of Chemical Industry.
DOI: 10.1002/jsfa.8103
209. Mol Microbiol. 2017 Jan;103(2):333-346. doi: 10.1111/mmi.13561. Epub 2016 Nov
11.
Sinorhizobium meliloti chemotaxis to quaternary ammonium compounds is mediated by

the chemoreceptor McpX.
Webb BA(1), Karl Compton K(1), Castañeda Saldaña R(1), Arapov TD(1), Keith Ray
W(2), Helm RF(2), Scharf BE(1).
Author information:
(1)Department of Biological Sciences, Life Sciences I, Virginia Tech, Blacksburg,
VA, 24061, USA.
(2)Department of Biochemistry, Life Sciences I, Virginia Tech, Blacksburg, VA,
24061, USA.
The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host

plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are
exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards
betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type
displayed a positive response to all QACs. Using LC-MS, we determined that each
germinating alfalfa seed exuded QACs in the nanogram range. Compared to the
closely related nonhost species, spotted medic (Medicago arabica), unique
profiles were released. Further assessments of single chemoreceptor deletion
strains revealed that an mcpX deletion strain displayed little to no response to
these compounds. Differential scanning fluorimetry showed interaction of the
isolated periplasmic region of McpX (McpXPR and McpX34-306 ) with QACs.
Isothermal titration calorimetry experiments revealed tight binding to McpXPR
with dissociation constants (Kd ) in the nanomolar range for choline and glycine
betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the
millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to
plant-derived QACs adds another role to this group of compounds, which are known
to serve as nutrient sources, osmoprotectants and cell-to-cell signalling
molecules. This is the first report of a chemoreceptor that mediates QACs taxis
through direct binding.
DOI: 10.1111/mmi.13561
210. Physiol Mol Biol Plants. 2016 Jul;22(3):321-330. Epub 2016 Aug 24.
Improvement of in vitro embryo maturation, plantlet regeneration and

transformation efficiency from alfalfa (Medicago sativa L.) somatic embryos using
Cuscuta campestris extract.
Amini M(1), Deljou A(1), Nabiabad HS(2).
Author information:
(1)Department of Biotechnology, College of Agriculture, Bu-Ali Sina University,
Hamadan, Iran.
(2)Department of Medicinal Plant Production, Nahavand University, Nahavand, Iran.
Developmental deficiency of somatic embryos and regeneration to plantlets,

especially in the case of transformation, are major problems of somatic embryo
regeneration in alfalfa. One of the ways to overcome these problems is the use of
natural plant regulators and nutrients in the culture medium of somatic embryos.
For investigating the influence of Cuscuta campestris extract on the efficiency
of plant regeneration and transformation, chimeric tissue type plasminogen
activator was transferred to explants using Agrobacterium tumefaciens, and
transgenic plants were recovered using medium supplemented with different
concentration of the extract. Transgenic plants were analyzed by PCR and RT-PCR.
Somatic embryos of Medicago sativa L. developed into plantlets at high frequency
level (52 %) in the maturation medium supplemented with 50 mg 1-1C. campestris
extract as compared to the medium without extract (26 %). Transformation
efficiency was 29.3 and 15.2 % for medium supplemented with dodder extract and
without the extract, respectively. HPLC and GC/MS analysis of the extract
indicated high level of ABA and some compounds such as Phytol, which can affect
the somatic embryo maturation. The antibacterial assay showed that the extract
was effective against some strains of A. tumefaciens. These results have provided
a scientific basis for using of C. campestris extract as a good natural source of
antimicrobial agents and plant growth regulator as well, that can be used in
tissue culture of transgenic plants.
DOI: 10.1007/s12298-016-0374-y
PMCID: PMC5039162
PMID: 27729718
Conflict of interest statement: The authors declare that they have no conflict of
interest.
211. Environ Microbiol. 2017 Jan;19(1):218-236. doi: 10.1111/1462-2920.13570. Epub

2016 Dec 12.
A putative 3-hydroxyisobutyryl-CoA hydrolase is required for efficient symbiotic

nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234.
Zamani M(1), diCenzo GC(1), Milunovic B(1), Finan TM(1).
Author information:
Ontario, Canada, L8S 4K1.
We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti
is a novel symbiotic gene required for full N2 -fixation. Deletion of smb20752
resulted in lower nitrogenase activity and smaller nodules without impacting
overall nodule morphology. Orthologs of smb20752 were present in all alpha and
beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A
ngr_b20860 mutant formed Fix- determinate nodules that developed normally to a
late stage of the symbiosis on the host plants Macroptilium atropurpureum and
Vigna unguiculata. However an early symbiotic defect was evident during symbiosis
with Leucaena leucocephala, producing Fix- indeterminate nodules. The smb20752
and ngr_b20860 genes encode putative 3-hydroxyisobutyryl-CoA (HIB-CoA)
hydrolases. HIB-CoA hydrolases are required for l-valine catabolism and appear to
prevent the accumulation of toxic metabolic intermediates, particularly
methacrylyl-CoA. Evidence presented here and elsewhere (Curson et al., , PLoS ONE
9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic
toxicity, are required for l-valine metabolism, and play an undefined role in
3-hydroxybutyrate catabolism. We present evidence that the symbiotic defect of
the HIB-CoA hydrolase mutants is independent of the inability to catabolize
l-valine and suggest it relates to the toxicity resulting from metabolism of
other compounds possibly related to 3-hydroxybutyric acid.
DOI: 10.1111/1462-2920.13570
212. Appl Microbiol Biotechnol. 2017 Feb;101(4):1385-1394. doi:

10.1007/s00253-016-7900-2. Epub 2016 Oct 10.
Comparative microbiota assessment of wilted Italian ryegrass, whole crop corn,

and wilted alfalfa silage using denaturing gradient gel electrophoresis and
next-generation sequencing.
Ni K(1)(2), Minh TT(1), Tu TT(1), Tsuruta T(1), Pang H(2), Nishino N(3).
Author information:
(1)Department of Animal Science, Graduate School of Environmental and Life
Science, Okayama University, Okayama, 700-8530, Japan.
(2)Henan Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou
University, Zhengzhou, China.
(3)Department of Animal Science, Graduate School of Environmental and Life
Science, Okayama University, Okayama, 700-8530, Japan. j1oufeed@okayama-u.ac.jp.
The microbiota of pre-ensiled crop and silage were examined using denaturing
gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Wilted
Italian ryegrass (IR), whole crop corn (WC), and wilted alfalfa (AL) silages
stored for 2 months were examined. All silages contained lactic acid as a
predominant fermentation product. Across the three crop species, DGGE detected 36
and 28 bands, and NGS identified 253 and 259 genera in the pre-ensiled crops and
silages, respectively. The NGS demonstrated that, although lactic acid bacteria
(LAB) became prevalent in all silages after 2 months of storage, the major groups
were different between crops: Leuconostoc spp. and Pediococcus spp. for IR
silage, Lactobacillus spp. for WC silage, and Enterococcus spp. for AL silage.
The predominant silage LAB genera were also detected by DGGE, but the presence of
diverse non-LAB species in pre-ensiled crops was far better detected by NGS.
Likewise, good survival of Agrobacterium spp., Methylobacterium spp., and
Sphingomonas spp. in IR and AL silages was demonstrated by NGS. The diversity of
the microbiota described by principal coordinate analysis was similar between
DGGE and NGS. Our finding that analysis of pre-ensiled crop microbiota did not
help predict silage microbiota was true for both DGGE and NGS.
DOI: 10.1007/s00253-016-7900-2
213. Plant Physiol Biochem. 2016 Dec;109:199-208. doi:

10.1016/j.plaphy.2016.09.020.
Epub 2016 Oct 1.
Overexpressing Arabidopsis ABF3 increases tolerance to multiple abiotic stresses

and reduces leaf size in alfalfa.
Wang Z(1), Su G(1), Li M(1), Ke Q(2), Kim SY(3), Li H(1), Huang J(1), Xu B(1),
Deng XP(1), Kwak SS(4).
Author information:
Institute of Soil and Water Conservation, Northwest A&F University, Yangling, PR
China.
(2)Plant Systems Engineering Research Center, Korea Research Institute of
Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea.
(3)Department of Biotechnology, Chonnam National University, Gwangju, Republic of
Korea.
Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea. Electronic
address: sskwak@kribb.re.kr.
Arabidopsis ABSCISIC ACID-RESPONSIVE ELEMENT-BINDING FACTOR 3 (ABF3), a bZIP

transcription factor, plays an important role in regulating multiple stress
responses in plants. Overexpressing AtABF3 increases tolerance to various
stresses in several plant species. Alfalfa (Medicago sativa L.), one of the most
important perennial forage crops worldwide, has high yields, high nutritional
value, and good palatability and is widely distributed in irrigated and semi-arid
regions throughout the world. However, drought and salt stress pose major
constraints to alfalfa production. In this study, we developed transgenic alfalfa
plants (cv. Xinjiang Daye) expressing AtABF3 under the control of the sweetpotato
oxidative stress-inducible SWPA2 promoter (referred to as SAF plants) via
Agrobacterium tumefaciens-mediated transformation. After drought stress
treatment, we selected two transgenic lines with high expression of AtABF3, SAF5
and SAF6, for further characterization. Under normal conditions, SAF plants
showed smaller leaf size compared to non-transgenic (NT) plants, while no other
morphological changes were observed. Moreover, SAF plants exhibited enhanced
drought stress tolerance and better growth under drought stress treatment, which
was accompanied by a reduced transpiration rate and lower reactive oxygen species
contents. In addition, SAF plants showed an increased tolerance to salt and
oxidative stress. Therefore, these transgenic AtABF3 alfalfa plants might be
useful for breeding forage crops with enhanced tolerance to environmental stress
for use in sustainable agriculture on marginal lands.
Copyright Â© 2016 Elsevier Masson SAS. All rights reserved.
DOI: 10.1016/j.plaphy.2016.09.020
214. Chemosphere. 2017 Jan;167:204-211. doi: 10.1016/j.chemosphere.2016.09.145.

Epub
2016 Oct 6.
Nitrogen fertilizer enhances growth and nutrient uptake of Medicago sativa

inoculated with Glomus tortuosum grown in Cd-contaminated acidic soil.
Liu M(1), Sun J(1), Li Y(1), Xiao Y(2).
Author information:
210095, PR China.
210095, PR China. Electronic address: xiaoyan@njau.edu.cn.
This study aimed to explore whether nitrogen availability could influence

mycorrhizal function and their associations with host plants in Cd-contaminated
acidic soils or not. A greenhouse pot experiment was conducted to assess the
effects of mycorrhizal inoculation (non-mycorrhizal inoculation (NM), Glomus
aggregatum (Ga), G. tortuosum (Gt) and G. versiforme (Gv)) and inorganic N
amendment on the growth, nutrient and Cd uptake of Medicago sativa grown in
Cd-contaminated acidic soils (10 mg Cd kg-1 soil). AMF inoculations significantly
increased the shoot and total biomass and decreased the shoot Cd concentration in
comparison to plants uninoculated. N addition increased markedly concentration
and content of N and decreased those of P in plants at all inoculation
treatments. Shoot K, Na and Mg concentration in plants inoculated with Ga and Gv
were decreased by N addition, whereas shoot K, Na, Ca and Mg concentration in
plants inoculated with Gt were not negatively affected. It was observed that N
addition only increased mycorrhizal colonization, shoot biomass, shoot K, Ca and
Mg content of plants inoculated with Gt. Irrespective of N addition, plants with
Gt inoculation got the maximum shoot and root P concentration and content, as
well as P/Cd concentration molar ratio among all inoculation treatment. Neither
AMF nor N fertilizer contributed to the decrease of soil exchangeable Cd and
increase of soil pH. These results suggested that N fertilizer only elevated
plant performance of alfalfa with Gt inoculation grown in acidic soil, by
diluting Cd concentration and alleviating of nutrient deficiency, especially P.
215. J Agric Food Chem. 2016 Oct 5. [Epub ahead of print]

Oxytetracycline Toxicity and its Effect on Phytoremediation by Sedum
Plumbizincicola and Medicago Sativa in Metal Contaminated Soil.
Ma T, Zhou L, Chen L', Li Z, Wu L, Christie P, Luo Y.
Excessive use of antibiotics potentially threatens human health, agricultural

production and soil phytoremediation. This arouses concern over the potential
adverse effects of a commonly used antibiotic, oxytetracycline (OTC), on plants
used for soil remediation and possible stimulation of antibiotic resistance genes
in soils. A greenhouse experiment was conducted to investigate different rates
(0, 1, 5, and 25 mg kg-1) and frequencies (one single high and daily low
application) of OTC addition to soil on phytoremediation of a heavy metal
contaminated soil by Sedum plumbizincicola and/or Medicago sativa (alfalfa).
After 90 days both Cd and Zn were substantially removed by phytoextraction into
S. plumbizincicola shoots especially at the high OTC (25 mg kg-1) treatment which
also led to inhibition of anti-oxidative enzyme activities in both plant species.
Soil microbial activity decreased significantly with the addition of OTC and this
was ameliorated by planting alfalfa and S. plumbizincicola together. OTC at < 5
mg kg-1 increased the biomass of both plant species but the frequency of OTC
addition had no effect on the rate of metal removal. Alfalfa exhibited greater
detoxification ability and effectiveness in soil microbial activity promotion
than S. plumbizincicola with intercropping. Phytoremediation by alfalfa and S.
plumbizincicola in association can both promote the removal of heavy metals and
also alleviate the toxic effects of pollutants to plants and soil microbes even
at relatively high soil OTC concentrations.
DOI: 10.1021/acs.jafc.6b02140
PMID: 27704817
216. J Anim Sci. 2016 Aug;94(8):3464-3475.
Methane emissions changed nonlinearly with graded substitution of alfalfa silage

with corn silage and corn grain in the diet of sheep and relation with rumen
fermentation characteristics in vivo and in vitro.
Jonker A, Lowe K, Kittelmann S, Janssen PH, Ledgard S, Pacheco D.
Feeding grain and corn silage have been proposed as practices to reduce enteric
methane (CH) emissions per unit of intake from ruminants, but the inclusion level
required in the diet is normally not specified. The objectives of the current
study were to determine the CH emission factor (g/kg DMI) of sheep fed alfalfa
silage substituted with increasing levels of corn silage or corn grain at a fixed
DMI level (2% of BW) and determine its relationship with rumen fermentation
characteristics and microbial community composition and with in vitro
fermentation characteristics of the same diets incubated using a standard
laboratory method. Romney ewe hoggets (approximately 14 mo old; = 64) were
randomly allocated to 8 dietary treatments, which included chaffed alfalfa silage
alone or substituted with either 25, 50, 75 or 100% corn silage or 25, 50 or 65%
rolled corn grain on a DM basis. After acclimatization to the diet, DMI and CH
emissions were measured from individual sheep for 2 consecutive days in
open-circuit respiration chambers and a rumen sample was collected at 3 h after
feeding. The same diets were also incubated in an automated in vitro gas
production system for 48 h using rumen liquid of fistulated nonlactating dairy
cows grazing pasture. Increasing the substitution of alfalfa silage with corn
silage or corn grain in the diet of sheep resulted in a quadratic response ( <
0.01) in CH emissions per unit of DMI (CH/DMI) with either supplement. For both
supplements, CH/DMI increased in mixtures of up to 50% supplement inclusion and
then decreased with greater supplement inclusion, especially with corn grain
inclusion, but the level did not fall below that for 100% alfalfa silage. The
ratio of acetate + butyrate to propionate + valerate and the propionate
proportion alone in rumen liquid were the strongest single predictors for CH/DMI
in the overall data set and explained 37.1 and 32.5%, respectively, of the
variation in CH/DMI. Methanogens of (21.1% of total methanogens; = 0.247) and
(10.7% of total methanogens; = -0.411) clades had weak to moderate correlations
with in vivo CH/DMI. There was a weak quadratic relationship ( < 0.35) between in
vivo CH/DMI and the in vitro parameters of gas and CH production and total VFA,
whereas there was a moderate relationship ( = -0.50) between in vivo CH/estimated
rumen degradable carbohydrates and in vitro CH/DM. In conclusion, CH/DMI changed
in a nonlinear fashion with increasing supplement inclusion in the alfalfa forage
diet when fed at 2% of BW to sheep; however, implications on predicting its
influence on greenhouse gas emissions per unit of animal product, for whole farm
emissions in life cycle analysis or total national emissions in the national
inventories, should be determined.
DOI: 10.2527/jas.2015-9912
217. J Dairy Sci. 2016 Dec;99(12):9668-9681. doi: 10.3168/jds.2016-11398. Epub 2016

Sep 28.
Characterization and comparison of the temporal dynamics of ruminal bacterial

microbiota colonizing rice straw and alfalfa hay within ruminants.
Liu J(1), Zhang M(1), Xue C(1), Zhu W(1), Mao S(2).
Author information:
(1)Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health,
College of Animal Science and Technology, Nanjing Agricultural University,
Nanjing 210095, China.
(2)Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health,
College of Animal Science and Technology, Nanjing Agricultural University,
Nanjing 210095, China. Electronic address: maoshengyong@163.com.
Three ruminally cannulated Holstein cows were used to characterize the dynamics
of bacterial colonization of rice straw and alfalfa hay and to assess the
differences in the composition and inferred gene function of the colonized
microbiota between these 2 forages. Nonincubated (0h) rice straw and alfalfa hay
samples and residues in nylon bags incubated for 0.5, 2, 6, 16, and 48h were
analyzed for dry matter and were used for DNA extraction and MiSeq (Illumina
Inc., San Diego, CA) sequencing of the 16S rRNA gene. The microbial communities
that colonized the air-dried and nonincubated (0h) rice straw and alfalfa hay
were both dominated by members of the Proteobacteria (contributing toward 70.47%
of the 16S RNA reads generated). In situ incubation of the 2 forages revealed
major shifts in the community composition: Proteobacteria were replaced within
30min by members belonging to the Bacteroidetes and Firmicutes, contributing
toward 51.9 and 36.6% of the 16S rRNA reads generated, respectively. A second
significant shift was observed after 6h of rumen incubation, when members of the
Spirochaetes and Fibrobacteria phyla became abundant in the forage-adherent
community. During the first 30min of rumen incubation, ~20.7 and 36.1% of the
rice straw and alfalfa hay, respectively, were degraded, whereas little biomass
degradation occurred between 30min and 2h after the rice straw or alfalfa hay was
placed in the rumen. Significant differences were noted in attached bacterial
community structure between the 2 forage groups, and the abundances of dominant
genera Anaeroplasma, Butyrivibrio, Fibrobacter, and Prevotella were affected by
the forage types. Real-time PCR results showed that the 16S rRNA copies of total
bacteria attached to these 2 forages were affected by the forage types and
incubation time, and higher numbers of attached bacterial 16S rRNA were observed
in the alfalfa hay samples than in the rice straw from 0.5 to 16h of incubation.
The metagenomes predicted by phylogenetic investigation of communities by
reconstruction of unobserved states (PICRUSt) revealed that the forage types
significantly affected 21 metabolic pathways identified in the Kyoto Encyclopedia
of Genes and Genomes, and 33 were significantly changed over time. Collectively,
our results reveal a difference in the dynamics of bacterial colonization and the
inferred gene function of microbiota associated with rice straw and alfalfa hay
within the rumen. These findings are of great importance for the targeted
improvement of forage nutrient use efficiency in ruminants.

DOI: 10.3168/jds.2016-11398
218. Pharmacol Rev. 2016 Oct;68(4):1026-1073.
Botanicals and Their Bioactive Phytochemicals for Women's Health.
Dietz BM(1), Hajirahimkhan A(1), Dunlap TL(1), Bolton JL(2).
Author information:
(1)University of Illinois at Chicago/National Institutes of Health Center for
Botanical Dietary Supplements, Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago,
Illinois.
(2)University of Illinois at Chicago/National Institutes of Health Center for
Botanical Dietary Supplements, Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago,
Illinois judy.bolton@uic.edu.
Botanical dietary supplements are increasingly popular for women's health,

particularly for older women. The specific botanicals women take vary as a
function of age. Younger women will use botanicals for urinary tract infections,
especially Vaccinium macrocarpon (cranberry), where there is evidence for
efficacy. Botanical dietary supplements for premenstrual syndrome (PMS) are less
commonly used, and rigorous clinical trials have not been done. Some examples
include Vitex agnus-castus (chasteberry), Angelica sinensis (dong quai), Viburnum
opulus/prunifolium (cramp bark and black haw), and Zingiber officinale (ginger).
Pregnant women have also used ginger for relief from nausea. Natural
galactagogues for lactating women include Trigonella foenum-graecum (fenugreek)
and Silybum marianum (milk thistle); however, rigorous safety and efficacy
studies are lacking. Older women suffering menopausal symptoms are increasingly
likely to use botanicals, especially since the Women's Health Initiative showed
an increased risk for breast cancer associated with traditional hormone therapy.
Serotonergic mechanisms similar to antidepressants have been proposed for
Actaea/Cimicifuga racemosa (black cohosh) and Valeriana officinalis (valerian).
Plant extracts with estrogenic activities for menopausal symptom relief include
Glycine max (soy), Trifolium pratense (red clover), Pueraria lobata (kudzu),
Humulus lupulus (hops), Glycyrrhiza species (licorice), Rheum rhaponticum
(rhubarb), Vitex agnus-castus (chasteberry), Linum usitatissimum (flaxseed),
Epimedium species (herba Epimedii, horny goat weed), and Medicago sativa
(alfalfa). Some of the estrogenic botanicals have also been shown to have
protective effects against osteoporosis. Several of these botanicals could have
additional breast cancer preventive effects linked to hormonal, chemical,
inflammatory, and/or epigenetic pathways. Finally, although botanicals are
perceived as natural safe remedies, it is important for women and their
healthcare providers to realize that they have not been rigorously tested for
potential toxic effects and/or drug/botanical interactions. Understanding the
mechanism of action of these supplements used for women's health will ultimately
lead to standardized botanical products with higher efficacy, safety, and
chemopreventive properties.
Copyright © 2016 by The Author(s).
DOI: 10.1124/pr.115.010843
PMCID: PMC5050441
PMID: 27677719
219. Theor Appl Genet. 2017 Feb;130(2):261-268. doi: 10.1007/s00122-016-2782-3.

Epub
2016 Sep 23.
Identification of loci controlling forage yield and nutritive value in diploid

alfalfa using GBS-GWAS.
Sakiroglu M(1), Brummer EC(2).
Author information:
(1)Bioengineering Department, Kafkas University, 36100, Kars, Turkey.
msakiroglu@kafkas.edu.tr.
(2)Department of Plant Sciences, Plant Breeding Center, The University of
California, Davis, CA, 95616, USA.
KEY MESSAGE: We attempted to identify genomic regions controlling forage yield

and nutritive value in alfalfa. Several candidate genes and associated genetic
markers were identified that could potentially be useful for alfalfa breeding to
more efficiently develop improved cultivars. Alfalfa is one of the most widely
cultivated forage legumes worldwide and improving alfalfa forage yield and
nutritive value is a major global breeding goal. Genotyping-by-sequencing (GBS)
provides cost-effective molecular marker genotyping for genome-wide association
studies (GWAS). Using more than 15,000 genome-wide single nucleotide
polymorphisms (SNP) identified from GBS, we conducted a GWAS to investigate
forage yield and nutritive value-related traits. We have detected a number of
associations for all the traits evaluated and a number of associations detected
were located on the Medicago truncatula genome. The SNP in a coding region of a
cell wall biosynthesis gene was associated with several cell wall-related traits,
and we suggest that it may be the causative polymorphism. Two other SNPs residing
in meristematic development and early growth genes were found to associate with
the total biomass yield. None of the SNPs associated with regrowth after harvest
or with spring regrowth were mapped to the M. truncatula genome, possibly
reflecting the fact that M. truncatula is an annual species related to alfalfa
that typically has limited ability to regrow. The alleles we identify with the
major impact on forage yield and nutritive value can be rapidly incorporated into
our breeding program.
DOI: 10.1007/s00122-016-2782-3
220. Int J Syst Evol Microbiol. 2016 Dec;66(12):5294-5300. doi:

10.1099/ijsem.0.001510. Epub 2016 Sep 19.
Ensifer alkalisoli sp. nov. isolated from root nodules of Sesbania cannabina
grown in saline-alkaline soils.
Li Y(1), Yan J(2), Yu B(1), Wang ET(3), Li X(1), Yan H(4), Liu W(1), Xie Z(1).
Author information:
(1)1Key Laboratory of Coastal Biology and Utilization, Yantai Institute of
Coastal Zone Research, Chinese Academy of Sciences, 264003 Yantai, PR China.
(2)2Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography
and Agroecology, Chinese Academy of Sciences, Harbin, PR China.
(3)3Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas,
Instituto Politécnico Nacional, 11340 Mexico City, D.F., México.
(4)4State Key Laboratory of Agro-Biotechnology, College of Biological Sciences
and Rhizobium Research Center, China Agricultural University, Beijing, PR China.
A group of Sesbania cannabina rhizobia belonging to four recA genotypes of a

novel group was further characterized in comparison with the related Ensifer
species. They showed 98.2 to 99.9 % similarities among themselves and 92.9 to
93.3 % similarities with the most related strain Ensifer sojae CCBAU 05684T in
multilocus sequence analysis of recA, atpD and glnII. The genome average
nucleotide identity values between representative strain YIC4027T and the type
strains of its closely related species were 81.6 to 88.9 %. Identical symbiotic
gene (nodA, nodC and nifH) sequences highly similar with those in other
Sesbania-nodulating strains (Rhizobium sp. SIN-1, Neorhizobium huautlense S02T,
Ensifer saheli ORS609T and Rhizobium sp. IRBG74) were detected. The
representative strain YIC4027T could form effective nodules on its original host
Sesbaniacannabina, but not on Sophora flavescens, Trifolium repens, Glycine max,
Glycyrrhiza uralensis, Phaseolus vulgaris or Medicago sativa. The use of
lactulose as sole carbon source, possession of C13 : 0 2-OH, C13 : 1 at 12-13,
C15 : 1 iso ω9c, C17 : 0 anteiso and C18 : 0 iso and absence of C14 : 0 anteiso,
C15 : 0 anteiso and C18 : 0 3-OH in fatty acids distinguished the strain YIC4027T
from the type strains of its closely related species. Based on all the analyses
mentioned above, we propose a novel species Ensifer alkalisoli sp. nov. and
designate YIC4027T (=HAMBI 3655T=LMG 29286T) as the type strain. The genome size
of YIC4027T is 5.97 Mbp, comprising 5588 predicted genes, and the DNA G+C content
is 62.2 mol%.
DOI: 10.1099/ijsem.0.001510
221. Anim Sci J. 2017 Apr;88(4):633-642. doi: 10.1111/asj.12689. Epub 2016 Sep 9.
Interactive effect of inoculant and dried jujube powder on the fermentation

quality and nitrogen fraction of alfalfa silage.
Tian J(1), Li Z(1), Yu Z(1), Zhang Q(1), Li X(1).
Author information:
Beijing, China.
The interactive effect of inoculants and dried jujube powder (DJP) on the
fermentation and nitrogen fraction (PA, PB1, PB2, PB3 and PC fractions) of
alfalfa silage was investigated. Three of the Lactobacillus plantarum inoculants
(LP1, LP2 or LP3) were used. The DJP was added at rates of 0, 3, 6, 9, 12 or 15%
of the whole fresh forage. The combination of DJP and inoculants decreased the pH
value and ammonia nitrogen content and increased the PC portion. As the DJP ratio
increased, there was a peak in lactic acid : acetic acid ratio (12% of DJP ratio)
and PB2 fraction (9% of DJP ratio) while the PA content decreased linearly. The
LP1 and LP2 had the highest lactic acid content. Inoculants decreased the PB1
portion of true protein. The LP1 treated silage had the highest acetic acid
content with the lowest lactic acid : acetic acid ratio and had lower PB3 and PC
and higher PB2 than LP2 or LP3 treated silages. The result showed that the
application of DJP or inoculants have positive effect on the fermentation,
nutrition and N fraction value in the high moisture alfalfa silages, and the
combination of DJP and inoculants preserves best.
© 2016 Japanese Society of Animal Science.
DOI: 10.1111/asj.12689
222. PeerJ. 2016 Aug 18;4:e2365. doi: 10.7717/peerj.2365. eCollection 2016.
Agricultural land-use change in a Mexican oligotrophic desert depletes ecosystem

stability.
Hernández-Becerra N(1), Tapia-Torres Y(2), Beltrán-Paz O(1), Blaz J(3), Souza

V(3), García-Oliva F(1).
Author information:
(1)Laboratorio de biogeoquímica de suelos, Instituto de Investigaciones en
Ecosistemas y Sustentabilidad, UNAM , Morelia , Michoacán , Mexico.
(2)ENES Unidad Morelia, Universidad Nacional Autónoma de México , Morelia ,
Michoacán , Mexico.
(3)Instituto de Ecología, Universidad Nacional Autónoma de México , Mexico.
BACKGROUND: Global demand for food has led to increased land-use change,
particularly in dry land ecosystems, which has caused several environmental
problems due to the soil degradation. In the Cuatro Cienegas Basin (CCB), alfalfa
production irrigated by flooding impacts strongly on the soil.
METHODS: In order to analyze the effect of such agricultural land-use change on
soil nutrient dynamics and soil bacterial community composition, this work
examined an agricultural gradient within the CCB which was comprised of a native
desert grassland, a plot currently cultivated with alfalfa and a former
agricultural field that had been abandoned for over 30 years. For each site, we
analyzed C, N and P dynamic fractions, the activity of the enzyme phosphatase and
the bacterial composition obtained using 16S rRNA clone libraries.
RESULTS: The results showed that the cultivated site presented a greater
availability of water and dissolved organic carbon, these conditions promoted
mineralization processes mediated by heterotrophic microorganisms, while the
abandoned land was limited by water and dissolved organic nitrogen. The low
amount of dissolved organic matter promoted nitrification, which is mediated by
autotrophic microorganisms. The microbial N immobilization process and specific
phosphatase activity were both favored in the native grassland. As expected,
differences in bacterial taxonomical composition were observed among sites. The
abandoned site exhibited similar compositions than native grassland, while the
cultivated site differed.
DISCUSSION: The results suggest that the transformation of native grassland into
agricultural land induces drastic changes in soil nutrient dynamics as well as in
the bacterial community. However, with the absence of agricultural practices,
some of the soil characteristics analyzed slowly recovers their natural state.
DOI: 10.7717/peerj.2365
PMCID: PMC4994104
PMID: 27602304
Conflict of interest statement: Valeria Souza is an Academic Editor for PeerJ.
223. J Anim Physiol Anim Nutr (Berl). 2017 Jun;101(3):466-474. doi:

10.1111/jpn.12563.
Epub 2016 Sep 7.
Chemical composition of alfalfa silage with waste date and its feeding effect on
ruminal fermentation characteristics and microbial protein synthesis in sheep.
Rajabi R(1), Tahmasbi R(1), Dayani O(1), Khezri A(1).
Author information:
(1)Department of Animal Science, Faculty of Agriculture, Shahid Bahonar
University of Kerman, Kerman, Iran.
This study was conducted to evaluate the effect of feeding ensiled alfalfa with
waste date on ruminal fermentation characteristics, microbial protein synthesis,
protozoa population and blood parameters in sheep. Eight rams were used in a
2 × 2 change over design. Each experimental period consisted of 21 days including
16 days for adaptation and 5 days for sampling. For ensiling, fresh alfalfa
(Medicago sativa L.) with different levels of waste date (Phoenix dactylifera L.)
were mixed together and ensiled in 100-l containers for 45 days. Chemical
composition of silages such as dry matter (DM), crude protein, NH3 -N, organic
matter, NDF, ADF and pH were determined. Then, it was used as 30% (DM basis) in
diets. The experimental diets were as follows: (i) control (diet containing
alfalfa silage without waste date), (ii) diet containing alfalfa silage with 5 g
waste date/95 g DM, (iii) diet containing alfalfa silage with 10 g waste
date/90 g DM, and (iv) diet containing alfalfa silage with 15 g waste date/85 g
DM. The results of this experiment showed that adding waste date to alfalfa
during ensiling, improved silage quality, DM and energy level. Total protozoa
population and all of holotrich, cellulolytic and entodinia in rumen fluid were
increased linearly by increasing the level of waste date. Nitrogen (N) intake,
urinary N excretion and nitrogen retention were affected by dietary treatments.
Also, allantoin, uric acid, total purine derivatives, microbial protein
synthesis, cholesterol level and blood urea nitrogen were significantly
different. In conclusion, direct ensilage of alfalfa can be attained by mixing
15 g waste date/85 g DM and positive associative effects such as increased
metabolizable energy (ME) and silage quality occurred.
Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.
DOI: 10.1111/jpn.12563
224. Animal. 2017 Apr;11(4):608-615. doi: 10.1017/S1751731116001798. Epub 2016 Aug

30.
High-concentrate diets based on forages harvested at different maturity stages

affect ruminal synthesis of B vitamins in lactating dairy cows.
Castagnino DS(1), Kammes KL(2), Allen MS(2), Gervais R(3), Chouinard PY(3),
Girard CL(1).
Author information:
(1)1Agriculture and Agri-Food Canada,Sherbrooke Research and Development
Centre,2000 College,Sherbrooke,QC,CanadaJ1M 0C8.
(2)3Department of Animal Science,Michigan State University,East Lansing,MI
48824-1225,USA.
(3)2Département des sciences animales,Université Laval,2425 rue de
l'Agriculture,Québec,QC,CanadaG1V 0A6.
Effects of plant maturity on apparent ruminal synthesis and post-ruminal supply

of B vitamins were evaluated in two feeding trials. Diets containing alfalfa
(Trial 1) or orchardgrass (Trial 2) silages harvested either (1) early cut, less
mature (EC) or (2) late cut, more mature (LC) as the sole forage were offered to
ruminally and duodenally cannulated lactating Holstein cows in crossover design
experiments. In Trial 1, conducted with 16 cows (569±43 kg of empty BW (ruminal
content removed) and 43.7±8.6 kg/day of 3.5% fat-corrected milk yield; mean±SD)
in two 17-day treatment periods, both diets provided ~22% forage NDF and 27%
total NDF, and the forage-to-concentrate ratios were 53 : 47 and 42 : 58 for EC
and LC, respectively. In Trial 2, conducted with 13 cows (588±55 kg of empty BW
and 43.7±7.7 kg/day of 3.5% fat-corrected milk yield; mean±SD) in two 18-day
treatment periods, both diets provided ~25% forage NDF and 31% total NDF; the
forage-to-concentrate ratios were 58 : 42 and 46 : 54 for EC and LC,
respectively. Thiamin, riboflavin, niacin, vitamin B6, folates and vitamin B12
were measured in feed and duodenal content. Apparent ruminal synthesis was
calculated as the duodenal flow minus the intake. Diets based on EC alfalfa
decreased the amounts of thiamin, niacin and folates reaching the duodenum,
whereas diets based on EC orchardgrass increased riboflavin duodenal flow. Daily
apparent ruminal synthesis of thiamin, riboflavin, niacin and vitamin B6 were
correlated negatively with their intake, suggesting a microbial regulation of
their concentration in the rumen. Vitamin B12 apparent ruminal synthesis was
correlated negatively with total volatile fatty acids concentration, but
positively with ruminal pH and microbial N duodenal flow.
DOI: 10.1017/S1751731116001798
225. Biochem Genet. 2017 Feb;55(1):63-76. doi: 10.1007/s10528-016-9772-7. Epub 2016

Aug 27.
Evaluating Agronomic Performance and Investigating Molecular Structure of Drought

and Heat Tolerant Wild Alfalfa (Medicago sativa L.) Collection from the
Southeastern Turkey.
Basbag M(1), Aydin A(2), Sakiroglu M(3).
Author information:
(1)Department of Field Crops, Faculty of Agriculture, Dicle University, 21280,
Diyarbakir, Turkey.
(2)Department of Field Crops, Faculty of Agriculture and Natural Sciences, Recep
Tayyip Erdogan University, 53300, Rize, Turkey.
(3)Department of Bioengineering, Kafkas University, 36100, Kars, Turkey.
msakiroglu@kafkas.edu.tr.
Drought is a major stress factor for agricultural production including alfalfa

production. One way to counterbalance the yield losses is the introgression of
drought tolerant germplasm into breeding programs. As an effort to exploit such
germplasm, 16 individual plants were selected from the Southeastern Turkey from
their natural habitat and clonally propagated in field trials with an ultimate
goal to use the germplasm as parents for releasing a synthetic cultivar. Forage
yield and forage quality traits were evaluated and molecular genetic diversity
among genotypes were determined using inter simple sequence repeat markers.
Genotypes showed a variation from growth habit to yield and quality traits
indicating sufficient phenotypic variation for diverse breeding efforts (for
grazing or harvesting) and long term selection schemes. A large amount of genetic
variation was observed even with a limited number of marker and genotypes.
However, no pattern of spatial genetic structure was observed for the scale of
the study when genetic variation is linked to the geographic origin. We conclude
that ex situ natural variation provides a wealth of germplasm that could be
incorporated into breeding programs aiming to improve drought tolerance. We also
suggest an extensive collection of seeds/plant tissue from unique plants with
desirable traits rather than putting more efforts to create a spatial germplasm
sampling efforts in narrow regions.
DOI: 10.1007/s10528-016-9772-7
226. Genet Mol Biol. 2016 Oct-Dec;39(4):616-628. doi: 10.1590/1678-4685-GMB-2015-

0228.
Epub 2016 Jul 25.
Molecular cloning and characterization of a novel freezing-inducible DREB1/CBF

transcription factor gene in boreal plant Iceland poppy (Papaver nudicaule).
Huang Z(1), He J(1), Zhong XJ(1), Guo HD(1), Jin SH(1), Li X(1), Sun LX(1).
Author information:
(1)College of Landscape Architecture, Sichuan Agricultural University, Wenjiang,
Sichuan, China.
DREB1 of the AP2/ERF superfamily plays a key role in the regulation of plant
response to low temperatures. In this study, a novel DREB1/CBF transcription
factor, PnDREB1, was isolated from Iceland poppy (Papaver nudicaule), a plant
adaptive to low temperature environments. It is homologous to the known DREB1s of
Arabidopsis and other plant species. It also shares similar 3D structure, and
conserved and functionally important motifs with DREB1s of Arabidopsis. The
phylogenetic analysis indicated that the AP2 domain of PnDREB1 is similar to
those of Glycine max, Medicago truncatula, and M. sativa. PnDREB1 is
constitutively expressed in diverse tissues and is increased in roots. qPCR
analyses indicated that PnDREB1 is significantly induced by freezing treatment as
well as by abscissic acid. The expression levels induced by freezing treatment
were higher in the variety with higher degree of freezing tolerance. These
results suggested that PnDREB1 is a novel and functional DREB1 transcription
factor involved in freezing response and possibly in other abiotic stresses.
Furthermore, the freezing-induction could be suppressed by exogenous gibberellins
acid, indicating that PnDREB1 might play some role in the GA signaling
transduction pathway. This study provides a basis for better understanding the
roles of DREB1 in adaption of Iceland poppy to low temperatures.
DOI: 10.1590/1678-4685-GMB-2015-0228
PMCID: PMC5127145
PMID: 27560992
227. Environ Sci Pollut Res Int. 2016 Nov;23(22):22633-22646. Epub 2016 Aug 24.
Assessment of nitrogen losses through nitrous oxide from abattoir

wastewater-irrigated soils.
Matheyarasu R(1), Seshadri B(2)(3), Bolan NS(2)(3), Naidu R(2)(3).
Author information:
(1)Future Industries Institute, University of South Australia, Mawson Lakes,
Adelaide, South Australia, 5095, Australia. matry020@mymail.unisa.edu.au.
(2)Global Centre for Environmental Remediation, The University of Newcastle,
Callaghan, Adelaide, New South Wales, 2308, Australia.
(3)Cooperative Research Centre for Contamination Assessment and Remediation of
the Environment, P.O. Box 486, Salisbury, Adelaide, South Australia, 5106,
Australia.
The land disposal of waste and wastewater is a major source of N2O emission. This
is due to the presence of high concentrations of nitrogen (N) and carbon in the
waste. Abattoir wastewater contains 186 mg/L of N and 30.4 mg/L of P. The
equivalent of 3 kg of abattoir wastewater-irrigated soil was sieved and taken in
a 4-L plastic container. Abattoir wastewater was used for irrigating the plants
at the rates of 50 and 100 % field capacity (FC). Four crop species were used
with no crop serving as a control. Nitrous oxide emission was monitored using a
closed chamber technique. The chamber was placed inside the plastic container,
and N2O emission was measured for 7 days after the planting. A syringe and
pre-evacuated vial were used for collecting the gas samples; a fresh and clean
syringe was used each time to avoid cross-contamination. The collected gas
samples were injected into a gas chromatography device immediately after each
sampling to analyse the concentration of N2O from different treatments. The
overall N2O emission was compared for all the crops under two different abattoir
wastewater treatment rates (50 and 100 % FC). Under 100 % FC (wastewater
irrigation), among the four species grown in the abattoir wastewater-irrigated
soil, Medicago sativa (23 mg/pot), Sinapis alba (21 mg/pot), Zea mays (20 mg/pot)
and Helianthus annuus (20 mg/pot) showed higher N2O emission compared to the 50 %
treatments-M. sativa (17 mg/pot), S. alba (17 mg/pot), Z. mays (18 mg/pot) and H.
annuus (18 mg/pot). Similarly, pots with plants have shown 15 % less emission
than the pots without plants. Similar trends of N2O emission flux were observed
between the irrigation period (4-week period) for 50 % FC and 100 % FC. Under the
100 % FC loading rate treatments, the highest N2O emission was in the following
order: week 1 > week 4 > week 3 > week 2. On the other hand, under the 50 % FC
loading rate treatments, the highest N2O emission was recorded in the first few
weeks and in the following order: week 1 > week 2 > week 3 > week > 4. Since N2O
is a greenhouse gas with high global warming potential, its emission from
wastewater irrigation is likely to impact global climate change. Therefore, it is
important to examine the effects of abattoir wastewater irrigation on soil for
N2O emission potential.
DOI: 10.1007/s11356-016-7438-y
PMCID: PMC5101283
228. Front Plant Sci. 2016 Aug 9;7:1171. doi: 10.3389/fpls.2016.01171. eCollection
2016.
Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for

Studying the Salivary Protein Functions in the Pea Aphid Complex.
Guy E(1), Boulain H(1), Aigu Y(1), Le Pennec C(1), Chawki K(1), Morlière S(1),
Schädel K(2), Kunert G(2), Simon JC(1), Sugio A(1).
Author information:
(1)INRA, UMR1349, Institute of Genetics, Environment and Plant Protection Le
Rheu, France.
(2)Department of Biochemistry, Max Planck Institute for Chemical Ecology Jena,
Germany.
Erratum in
Front Plant Sci. 2017 Jan 10;7:2046.
Aphids are piercing-sucking insect pests and feed on phloem sap. During feeding,
aphids inject a battery of salivary proteins into host plant. Some of these
proteins function like effectors of microbial pathogens and influence the outcome
of plant-aphid interactions. The pea aphid (Acyrthosiphon pisum) is the model
aphid and encompasses multiple biotypes each specialized to one or a few legume
species, providing an opportunity to investigate the underlying mechanisms of the
compatibility between plants and aphid biotypes. We aim to identify the aphid
factors that determine the compatibility with host plants, hence involved in the
host plant specialization process, and hypothesize that salivary proteins are one
of those factors. Agrobacterium-mediated transient gene expression is a powerful
tool to perform functional analyses of effector (salivary) proteins in plants.
However, the tool was not established for the legume species that A. pisum feeds
on. Thus, we decided to optimize the method for legume plants to facilitate the
functional analyses of A. pisum salivary proteins. We screened a range of
cultivars of pea (Pisum sativum) and alfalfa (Medicago sativa). None of the M.
sativa cultivars was suitable for agroinfiltration under the tested conditions;
however, we established a protocol for efficient transient gene expression in two
cultivars of P. sativum, ZP1109 and ZP1130, using A. tumefaciens AGL-1 strain and
the pEAQ-HT-DEST1 vector. We confirmed that the genes are expressed from 3 to 10
days post-infiltration and that aphid lines of the pea adapted biotype fed and
reproduced on these two cultivars while lines of alfalfa and clover biotypes did
not. Thus, the pea biotype recognizes these two cultivars as typical pea plants.
By using a combination of ZP1109 and an A. pisum line, we defined an
agroinfiltration procedure to examine the effect of in planta expression of
selected salivary proteins on A. pisum fitness and demonstrated that transient
expression of one candidate salivary gene increased the fecundity of the aphids.
This result confirms that the agroinfiltration can be used to perform functional
analyses of salivary proteins in P. sativum and consequently to study the
molecular mechanisms underlying host specialization in the pea aphid complex.
DOI: 10.3389/fpls.2016.01171
PMCID: PMC4977312
PMID: 27555856
229. BMC Genomics. 2016 Aug 19;17(1):658. doi: 10.1186/s12864-016-3014-6.
Comparative transcriptome investigation of global gene expression changes caused

by miR156 overexpression in Medicago sativa.
Gao R(1), Austin RS(1)(2), Amyot L(1), Hannoufa A(3)(4).
Author information:
Canada.
(2)Department of Biology, University of Western Ontario, 151 Richmond Street,
London, ON, N6A 5B7, Canada.
(4)Department of Biology, University of Western Ontario, 151 Richmond Street,
London, ON, N6A 5B7, Canada. Abdelali.Hannoufa@agr.gc.ca.
BACKGROUND: Medicago sativa (alfalfa) is a low-input forage and potential

bioenergy crop, and improving its yield and quality has always been a focus of
the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a
precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group.
These plants (miR156OE) showed enhanced biomass yield, reduced internodal length,
increased shoot branching and trichome density, and a delay in flowering time.
Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6,
MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in
alfalfa.
RESULTS: To further illustrate the molecular mechanisms underlying the effects of
miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next
Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads
were obtained from our available sequenced samples. A total of 160,472
transcripts were generated using Trinity de novo assembly and 4,985 significantly
differentially expressed genes were detected in miR156OE plants A11a and A17
using the Medicago truncatula genome as reference. A total of 17 genes (including
upregulated, downregulated, and unchanged) were selected for quantitative
real-time PCR (qRT-PCR) validation, which showed that gene expression levels were
largely consistent between qRT-PCR and RNA-Seq data. In addition to the
established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3,
MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants.
These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII,
which have been reported to be targeted by miR156 in Arabidopsis thaliana. The
gene ontology terms characterized electron transporter, starch synthase activity,
sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual
reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the
phenotypes of miR156OE alfalfa plants.
CONCLUSIONS: This is the first report of changes in global gene expression in
response to miR156 overexpression in alfalfa. The discovered miR156-targeted SPL
genes belonging to different clades indicate miR156 plays fundamental and
multifunctional roles in regulating alfalfa plant development.
DOI: 10.1186/s12864-016-3014-6
PMCID: PMC4992203
230. Environ Sci Pollut Res Int. 2016 Oct;23(20):20131-20150. Epub 2016 Aug 19.
Remediation of metalliferous mines, revegetation challenges and emerging

prospects in semi-arid and arid conditions.
Nirola R(1)(2), Megharaj M(3), Beecham S(4), Aryal R(4), Thavamani P(3),
Vankateswarlu K(5), Saint C(4).
Author information:
(1)Future Industries Institute, Division of Information Technology, Engineering
and the Environment, University of South Australia, Adelaide, SA, 5095,
Australia. Ramkrishna.nirola@mymail.unisa.edu.au.
(2)Natural & Built Environments Research Centre, Division of Information
Technology, Engineering and the Environment, University of South Australia,
Adelaide, SA, 5095, Australia. Ramkrishna.nirola@mymail.unisa.edu.au.
(3)Global Centre for Environmental Remediation (GCER), University of Newcastle,
ATC Building, Callaghan, Newcastle, NSW, 2308, Australia.
(4)Natural & Built Environments Research Centre, Division of Information
Technology, Engineering and the Environment, University of South Australia,
Adelaide, SA, 5095, Australia.
(5)Faculty of Life Sciences, Sri Krishnadevaraya University, Anantapur, 515055,
India.
Understanding plant behaviour in polluted soils is critical for the sustainable

remediation of metal-polluted sites including abandoned mines. Post-operational
and abandoned metal mines particularly in semi-arid and arid zones are one of the
major sources of pollution by soil erosion or plant hyperaccumulation bringing
ecological impacts. We have selected from the literature 157 species belonging to
50 families to present a global overview of 'plants under action' against heavy
metal pollution. Generally, all species of plants that are drought, salt and
metal tolerant are candidates of interest to deal with harsh environmental
conditions, particularly at semi-arid and arid mine sites. Pioneer metallophytes
namely Atriplex nummularia, Atriplex semibaccata, Salsola kali, Phragmites
australis and Medicago sativa, representing the taxonomic orders Caryophyllales,
Poales and Fabales are evaluated in terms of phytoremediation in this review.
Phytoremediation processes, microbial and algal bioremediation, the use and
implication of tissue culture and biotechnology are critically examined. Overall,
an integration of available remediation plant-based technologies, referred to
here as 'integrated remediation technology,' is proposed to be one of the
possible ways ahead to effectively address problems of toxic heavy metal
pollution. Graphical abstract Integrated remediation technology (IRT) in
metal-contaminated semi-arid and arid conditions. The hexagonal red line
represents an IRT concept based on remediation decisions by combination of plants
and microbial processes.
DOI: 10.1007/s11356-016-7372-z
231. J Agric Food Chem. 2016 Sep 14;64(36):6856-68. doi: 10.1021/acs.jafc.6b02018.

Epub 2016 Sep 6.
Impact of Brassica and Lucerne Finishing Feeds and Intramuscular Fat on Lamb
Eating Quality and Flavor. A Cross-Cultural Study Using Chinese and Non-Chinese
Australian Consumers.
Frank D(1), Watkins P(2), Ball A(3), Krishnamurthy R(1), Piyasiri U(1), Sewell
J(4), Ortuño J(1), Stark J(5), Warner R(6).
Author information:
(1)Commonwealth Scientific Industrial Research Organisation (CSIRO) , 11 Julius
Avenue, North Ryde, NSW 2113, Australia.
(2)Commonwealth Scientific Industrial Research Organisation (CSIRO) , 671 Sneydes
Road, Werribee, VIC 3030, Australia.
(3)Meat & Livestock Australia (MLA) , Level 1, 40 Mount Street, North Sydney, NSW
2060, Australia.
(4)PGG Wrightson Seeds (Australia) Leigh Creek Research Station , 4 Blackswamp
Road, Leigh Creek, VIC 3052, Australia.
(5)Commonwealth Scientific Industrial Research Organisation (CSIRO) , 39 Kessels
Road, Coopers Plains, QLD 4108, Australia.
(6)Faculty of Veterinary and Agricultural Science, The University of Melbourne ,
Royal Parade, Parkville, VIC 3010, Australia.
Use of forage brassicas (Brassica napus) and lucerne (alfalfa; Medicago sativa)
as ruminant feeds has been linked to unacceptable flavors in sheepmeat. Lambs
from low and high intramuscular fat sires were allocated to one of four finishing
feeds-perennial ryegrass (Lolium perenne), lucerne, and two brassica forages-for
a 6 week period. Grilled loins (Longissimus thoracis et lumborum) were subjected
to chemical and sensory analysis by a trained panel and also evaluated by
non-Chinese and Chinese background Australian consumers. Consumer liking was
similar for both groups, and liking was highest for the brassica- and
lucerne-finished lamb, especially from high intramuscular fat sires. No evidence
of a distinctive lucerne- or brassica-induced flavor taint was measured by the
trained panel or gas chromatography-mass spectrometry-olfactometry. The diets
influenced the composition of lipids and branched-chain fatty acids in the
subcutaneous fat, and the concentration of total branched-chain fatty acids was
positively correlated with flavor and overall liking. Significantly higher levels
of key aroma volatiles were measured in the higher fat samples.
DOI: 10.1021/acs.jafc.6b02018
2016.
Role of Silicon Counteracting Cadmium Toxicity in Alfalfa (Medicago sativa L.).
Kabir AH(1), Hossain MM(1), Khatun MA(1), Mandal A(2), Haider SA(1).
Author information:
(1)Plant and Crop Physiology Laboratory, Department of Botany, University of
Rajshahi Rajshahi, Bangladesh.
(2)System Biology Research Center, School of Bioscience, University of Skövde
Skövde, Sweden.
Cadmium (Cd) is one of the most phytotoxic elements causing an agricultural

problem and human health hazards. This work investigates whether and how silicon
(Si) ameliorates Cd toxicity in Alfalfa. The addition of Si in Cd-stressed plants
caused significant improvement in morpho-physiological features as well as total
protein and membrane stability, indicating that Si does have critical roles in Cd
detoxification in Alfalfa. Furthermore, Si supplementation in Cd-stressed plants
showed a significant decrease in Cd and Fe concentrations in both roots and
shoots compared with Cd-stressed plants, revealing that Si-mediated tolerance to
Cd stress is associated with Cd inhibition in Alfalfa. Results also showed no
significant changes in the expression of two metal chelators [MsPCS1
(phytochelatin synthase) and MsMT2 (metallothionein)] and PC (phytochelatin)
accumulation, indicating that there may be no metal sequestration or change in
metal sequestration following Si application under Cd stress in Alfalfa. We
further performed a targeted study on the effect of Si on Fe uptake mechanisms.
We observed the consistent reduction in Fe reductase activity, expression of
Fe-related genes [MsIRT1 (Fe transporter), MsNramp1 (metal transporter) and
OsFRO1 (ferric chelate reductase] and Fe chelators (citrate and malate) by Si
application to Cd stress in roots of Alfalfa. These results support that limiting
Fe uptake through the down-regulation of Fe acquisition mechanisms confers
Si-mediated alleviation of Cd toxicity in Alfalfa. Finally, an increase of
catalase, ascorbate peroxidase, and superoxide dismutase activities along with
elevated methionine and proline subjected to Si application might play roles, at
least in part, to reduce H2O2 and to provide antioxidant defense against Cd
stress in Alfalfa. The study shows evidence of the effect of Si on alleviating Cd
toxicity in Alfalfa and can be further extended for phytoremediation of Cd
toxicity in plants.
DOI: 10.3389/fpls.2016.01117
PMCID: PMC4961700
PMID: 27512401
233. Int J Syst Evol Microbiol. 2016 Nov;66(11):4451-4457. doi:

10.1099/ijsem.0.001373. Epub 2016 Aug 3.
Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa
(Medicago sativa L).
Torres Tejerizo G(1), Rogel MA(2), Ormeño-Orrillo E(2), Althabegoiti MJ(3),
Nilsson JF(3), Niehaus K(4), Schlüter A(4), Pühler A(4), Del Papa MF(3), Lagares
A(3), Martínez-Romero E(2), Pistorio M(3).
Author information:
(1)1IBBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La
Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Calles 47 y 115 (1900) La Plata, Argentina
2CeBiTec, Bielefeld Universität, Bielefeld, Germany.
(2)3Centro de Ciencias Genómicas Av. Universidad 1001, Col. Chamilpa. 62210
Cuernavaca, Universidad Nacional Autónoma de México, Morelos, Mexico.
(3)1IBBM (Instituto de Biotecnología y Biología Molecular), CCT-CONICET-La
Plata, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Calles 47 y 115 (1900) La Plata, Argentina.
(4)2CeBiTec, Bielefeld Universität, Bielefeld, Germany.
Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root
nodules of alfalfa, grown in acid soils from Argentina and the USA. These two
strains, which shared the same plasmid pattern, lipopolysaccharide profile,
insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting
pattern, were different from reference strains representing species of the genus
Rhizobium with validly published names. On the basis of previously reported data
and from new DNA-DNA hybridization results, phenotypic characterization and
phylogenetic analyses, strains LPU83T and Or191 can be considered to be
representatives of a novel species of the genus Rhizobium, for which the name
Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is
LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is
available.
DOI: 10.1099/ijsem.0.001373
234. Phytochemistry. 2016 Sep;129:14-23. doi: 10.1016/j.phytochem.2016.07.007. Epub

2016 Jul 25.
Stress tolerance of transgenic barley accumulating the alfalfa aldose reductase

in the cytoplasm and the chloroplast.
Nagy B(1), Majer P(1), Mihály R(2), Pauk J(2), Horváth GV(3).
Author information:
(1)Institute of Plant Biology, Biological Research Center, Hungarian Academy of
Sciences, Temesvári krt. 62, 6726, Szeged, Hungary.
(2)Cereal Research Non-Profit Company, Alsó kikötő sor 9, 6726, Szeged, Hungary.
Sciences, Temesvári krt. 62, 6726, Szeged, Hungary. Electronic address:
horvath.gabor@brc.mta.hu.
Barley represents one of the major crops grown worldwide; its genetic
transformation provides an important tool for the improvement of crop quality and
tolerance to environmental stress factors. Biotic and abiotic stresses produce
reactive oxygen species in the plant cells that can directly oxidize the cellular
components including lipid membranes; resulting in lipid peroxidation and
subsequently the accumulation of reactive carbonyl compounds. In order to protect
barley plants from the effects of stress-produced reactive carbonyls, an
Agrobacterium-mediated transformation was carried out using the Medicago sativa
aldose reductase (MsALR) gene. In certain transgenic lines the produced MsALR
enzyme was targeted to the chloroplasts to evaluate its protective effect in
these organelles. The dual fluorescent protein-based method was used for the
evaluation of tolerance of young seedlings to diverse stresses; our results
demonstrated that this technique could be reliably applied for the detection of
cellular stress in a variety of conditions. The chlorophyll and carotenoid
content measurements also supported the results of the fluorescent protein-based
method and the stress-protective effect of the MsALR enzyme. Targeting of MsALR
into the chloroplast has also resulted in increased stress tolerance, similarly
to the observed effect of the cytosolic MsALR accumulation. The results of the
DsRed/GFP fluorescent protein-based method indicated that both the cytosol and
chloroplast accumulation of MsALR can increase the abiotic stress tolerance of
transgenic barley lines.
DOI: 10.1016/j.phytochem.2016.07.007
235. Trop Anim Health Prod. 2016 Oct;48(7):1427-33. doi: 10.1007/s11250-016-1111-9.

Epub 2016 Jul 26.
Effect of partial replacement of alfalfa hay with Moringa species leaves on milk
yield and composition of Najdi ewes.
Babiker EE(1), Al Juhaimi F(2), Ghafoor K(2), Mohamed HE(3), Abdoun KA(4).
Author information:
(1)Food Science and Nutrition Department, College of Food and Agricultural
Sciences, King Saud University, P. O. Box 2460, Riyadh, 11451, Saudi Arabia.
elfadilbabiker@yahoo.com.
(2)Food Science and Nutrition Department, College of Food and Agricultural
Sciences, King Saud University, P. O. Box 2460, Riyadh, 11451, Saudi Arabia.
(3)Department of Physiology, Faculty of Medicine, AlBaha University, Al-Baha,
Saudi Arabia.
(4)Animal Production Department, College of Food and Agricultural Sciences, King
Saud University, P. O. Box 2460, Riyadh, 11451, Saudi Arabia.
The present study was carried out to investigate changes in milk yield and
composition of Najdi ewes fed 25 % Moringa oleifera (MOD) or Moringa peregrina
(MPD) leaf diets as a supplement to alfalfa hay diet (AHD). Thirty ewes (average
55 kg, 2 years old) were randomly sorted into three experimental groups with 10
animals each and were fed for a 6-week period with these diets (AHD, MOD, or
MPD). Diets dry matter, crude protein, and crude fiber were comparable, while
fat, nitrogen-free extract (NFE), neutral detergent fiber (NDF), acid detergent
fiber (ADF), metabolizable energy (ME), total phenolic, and antioxidant activity
varied (p ≤ 0.05) between the diets. Feeding ewes with MOD increased (p ≤ 0.05)
the milk yield compared to those fed AHD while milk composition was similar
(p ≤ 0.05) between treatments. The concentration of malondialdehyde (MDA) in the
ewes' milk and serum was lower (p ≤ 0.05) for MOD, while the total antioxidant
capacity, catalase activity, and vitamin C contents were increased (p ≤ 0.05).
The serum cholesterol and glucose of the ewes were lower (p ≤ 0.05) for those fed
MOD. Moringa diets increased (p ≤ 0.05) average daily weight gain of lambs
compared to those fed alfalfa diets. The results obtained showed that the
inclusion of Moringa, especially M. oleifera, in the diet of Najdi ewes can
improve milk yield and quality.
DOI: 10.1007/s11250-016-1111-9
2016.
De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline

Stress.
An YM(1), Song LL(1), Liu YR(1), Shu YJ(1), Guo CH(1).
Author information:
(1)State Key Laboratory of Molecular Genetics, Harbin Normal University Harbin,
China.
Saline-alkaline stress, caused by high levels of harmful carbonate salts and high
soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a
widely cultivated perennial forage legume with some tolerance to biotic and
abiotic stresses, especially to saline-alkaline stress. To elucidate the
mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome
analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0
day (control), 1 day (short-term treatment), and 7 days (long-term treatment)
using ion torrent sequencing technology. A transcriptome database dataset of
53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially
expressed in the short-term and long-term treatment, respectively. Gene ontology
analysis revealed 14 highly enriched pathways and demonstrated the differential
response of metabolic pathways between the short-term and long-term treatment.
The expression levels of 109 and 96 transcription factors were significantly
altered significantly after 1 day and 7 days of treatment, respectively. Specific
responses of peroxidase, flavonoids, and the light pathway component indicated
that the antioxidant capacity was one of the central mechanisms of
saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially
expressed genes examined by real time PCR, the expression levels of eight genes,
including inositol transporter, DNA binding protein, raffinose synthase,
ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans
glucosylase, and a NAC transcription factor, exhibited different patterns in
response to saline and alkaline stress. The expression levels of the NAC
transcription factor and glutathione S-transferase were altered significantly
under saline stress and saline-alkaline stress; they were upregulated under
saline-alkaline stress and downregulated under salt stress. Physiology assays
showed an increased concentration of reactive oxygen species and malondialdehyde
and a decreased content of chlorophyll, indicating that anti-oxidation and
detoxification play an important role in response to saline-alkaline stress.
Overall, the transcriptome analysis provided novel insights into the
saline-alkaline stress tolerance response mechanisms in alfalfa.
DOI: 10.3389/fpls.2016.00931
PMCID: PMC4931813
PMID: 27458463
237. BMC Microbiol. 2016 Jul 26;16(1):163. doi: 10.1186/s12866-016-0780-x.
Loss of malic enzymes leads to metabolic imbalance and altered levels of

trehalose and putrescine in the bacterium Sinorhizobium meliloti.
Zhang Y(1)(2), Smallbone LA(1), diCenzo GC(1), Morton R(1), Finan TM(3).
Author information:
(1)Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON,
L8S 4K1, Canada.
(2)College of Fisheries and Life Science, Shanghai Ocean University, Shanghai,
China.
(3)Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON,
L8S 4K1, Canada. finan@mcmaster.ca.
BACKGROUND: Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle

intermediate malate to the glycolytic end-product pyruvate and are well
positioned to regulate metabolic flux in central carbon metabolism. Despite the
wide distribution of these enzymes, their biological roles are unclear in part
because the reaction catalyzed by these enzymes can be by-passed by other
pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a
NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help
understand the role of these enzymes, we investigated growth, metabolomic, and
transcriptional consequences resulting from loss of these enzymes in free-living
cells.
RESULTS: Loss of DME, TME, or both enzymes had no effect on growth with the
glycolytic substrate, glucose. In contrast, the dme mutants, but not tme, grew
slowly on the gluconeogenic substrate succinate and this slow growth was further
reduced upon the addition of glucose. The dme mutant strains incubated with
succinate accumulated trehalose and hexose sugar phosphates, secreted malate, and
relative to wild-type, these cells had moderately increased transcription of
genes involved in gluconeogenesis and pathways that divert metabolites away from
the TCA cycle. While tme mutant cells grew at the same rate as wild-type on
succinate, they accumulated the compatible solute putrescine.
CONCLUSIONS: NAD(P)-malic enzyme (DME) of S. meliloti is required for efficient
metabolism of succinate via the TCA cycle. In dme mutants utilizing succinate,
malate accumulates and is excreted and these cells appear to increase metabolite
flow via gluconeogenesis with a resulting increase in the levels of
hexose-6-phosphates and trehalose. For cells utilizing succinate, TME activity
alone appeared to be insufficient to produce the levels of pyruvate required for
efficient TCA cycle metabolism. Putrescine was found to accumulate in tme cells
growing with succinate, and whether this is related to altered levels of NADPH
requires further investigation.
DOI: 10.1186/s12866-016-0780-x
PMCID: PMC4960864
238. Plant Cell Rep. 2016 Sep;35(9):1987-90. doi: 10.1007/s00299-016-2026-7. Epub

2016
Jul 22.
Minimizing the time and cost of production of transgenic alfalfa libraries using
the highly efficient completely sequenced vector pPZP200BAR.
Jozefkowicz C(1)(2), Bottero E(2), Pascuan C(1)(2), Pagano E(2), Ayub ND(1)(2),
Soto G(3)(4).
Author information:
(1)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos
Aires, Argentina.
(2)Instituto de Genética Ewald A. Favret (CICVyA-INTA), De los Reseros S/N,
Castelar C25 (1712), Buenos Aires, Argentina.
(3)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos
Aires, Argentina. soto.gabrielacinthia@inta.gob.ar.
(4)Instituto de Genética Ewald A. Favret (CICVyA-INTA), De los Reseros S/N,
Castelar C25 (1712), Buenos Aires, Argentina. soto.gabrielacinthia@inta.gob.ar.
Alfalfa is the most important forage legume worldwide. However, similar to other
minor forage crops, it is usually harvested along with weeds, which decrease its
nutrient quality and thus reduce its high value in the market. In addition, weeds
reduce alfalfa yield by about 50 %. Although weeds are the limiting factor for
alfalfa production, little progress has been made in the incorporation of
herbicide-tolerant traits into commercial alfalfa. This is partially due to the
high times and costs needed for the production of vast numbers of transgenic
alfalfa events as an empirical approach to bypass the random transgenic silencing
and for the identification of an event with optimal transgene expression. In this
focus article, we report the complete sequence of pPZP200BAR and the extremely
high efficiency of this binary vector in alfalfa transformation, opening the way
for rapid and inexpensive production of transgenic events for alfalfa improvement
public programs.
DOI: 10.1007/s00299-016-2026-7
2016.
Genome-Wide Association Study Identifies Loci for Salt Tolerance during

Germination in Autotetraploid Alfalfa (Medicago sativa L.) Using
Genotyping-by-Sequencing.
Yu LX(1), Liu X(1), Boge W(1), Liu XP(1).
Author information:
(1)Plant Germplasm Introduction Testing and Research, United States Department of
Agriculture-Agricultural Research Service Prosser, WA, USA.
Salinity is one of major abiotic stresses limiting alfalfa (Medicago sativa L.)
production in the arid and semi-arid regions in US and other counties. In this
study, we used a diverse panel of alfalfa accessions previously described by
Zhang et al. (2015) to identify molecular markers associated with salt tolerance
during germination using genome-wide association study (GWAS) and
genotyping-by-sequencing (GBS). Phenotyping was done by germinating alfalfa seeds
under different levels of salt stress. Phenotypic data of adjusted germination
rates and SNP markers generated by GBS were used for marker-trait association.
Thirty six markers were significantly associated with salt tolerance in at least
one level of salt treatments. Alignment of sequence tags to the Medicago
truncatula genome revealed genetic locations of the markers on all chromosomes
except chromosome 3. Most significant markers were found on chromosomes 1, 2, and
4. BLAST search using the flanking sequences of significant markers identified 14
putative candidate genes linked to 23 significant markers. Most of them were
repeatedly identified in two or three salt treatments. Several loci identified in
the present study had similar genetic locations to the reported QTL associated
with salt tolerance in M. truncatula. A locus identified on chromosome 6 by this
study overlapped with that by drought in our previous study. To our knowledge,
this is the first report on mapping loci associated with salt tolerance during
germination in autotetraploid alfalfa. Further investigation on these loci and
their linked genes would provide insight into understanding molecular mechanisms
by which salt and drought stresses affect alfalfa growth. Functional markers
closely linked to the resistance loci would be useful for MAS to improve alfalfa
cultivars with enhanced resistance to drought and salt stresses.
DOI: 10.3389/fpls.2016.00956
PMCID: PMC4923157
PMID: 27446182
2016.
PlantAPA: A Portal for Visualization and Analysis of Alternative Polyadenylation

in Plants.
Wu X(1), Zhang Y(1), Li QQ(2).
Author information:
(1)Department of Automation, Xiamen University Xiamen, China.
(2)Key Laboratory of the Ministry of Education for Coastal and Wetland
Ecosystems, College of the Environment and Ecology, Xiamen UniversityXiamen,
China; Graduate College of Biomedical Sciences, Western University of Health
SciencesPomona, CA, USA.
Alternative polyadenylation (APA) is an important layer of gene regulation that

produces mRNAs that have different 3' ends and/or encode diverse protein
isoforms. Up to 70% of annotated genes in plants undergo APA. Increasing numbers
of poly(A) sites collected in various plant species demand new methods and tools
to access and mine these data. We have created an open-access web service called
PlantAPA (http://bmi.xmu.edu.cn/plantapa) to visualize and analyze genome-wide
poly(A) sites in plants. PlantAPA provides various interactive and dynamic
graphics and seamlessly integrates a genome browser that can profile
heterogeneous cleavage sites and quantify expression patterns of poly(A) sites
across different conditions. Particularly, through PlantAPA, users can analyze
poly(A) sites in extended 3' UTR regions, intergenic regions, and ambiguous
regions owing to alternative transcription or RNA processing. In addition, it
also provides tools for analyzing poly(A) site selections, 3' UTR lengthening or
shortening, non-canonical APA site switching, and differential gene expression
between conditions, making it more powerful for the study of APA-mediated gene
expression regulation. More importantly, PlantAPA offers a bioinformatics
pipeline that allows users to upload their own short reads or ESTs for poly(A)
site extraction, enabling users to further explore poly(A) site selection using
stored PlantAPA poly(A) sites together with their own poly(A) site datasets. To
date, PlantAPA hosts the largest database of APA sites in plants, including Oryza
sativa, Arabidopsis thaliana, Medicago truncatula, and Chlamydomonas reinhardtii.
As a user-friendly web service, PlantAPA will be a valuable addition to the
community of biologists studying APA mechanisms and gene expression regulation in
plants.
DOI: 10.3389/fpls.2016.00889
PMCID: PMC4914594
PMID: 27446120
241. Water Air Soil Pollut. 2016;227:253. Epub 2016 Jul 5.
Abattoir Wastewater Irrigation Increases the Availability of Nutrients and

Influences on Plant Growth and Development.
Matheyarasu R(1), Bolan NS(2), Naidu R(3).
Author information:
(1)Future Industries Institute, University of South Australia, Mawson Lakes,
South Australia 5095 Australia.
(2)Global Centre for Environmental Remediation, The University of Newcastle,
Callaghan, New South Wales 2308 Australia.
(3)Cooperative Research Centre for Contamination Assessment and Remediation of
the Environment, P.O. Box 486, Salisbury, South Australia 5106 Australia.
This study evaluated the effects of abattoir wastewater irrigation on plant

growth and development. The soils used in this study were collected from Primo
Smallgoods Abattoir (Port Wakefield, South Australia) at different sites such as
currently irrigated (CI), currently not irrigated (CNI) and soil outside the
irrigation area as control (CTRL). A completely randomised block design was
employed for the plant growth experiment, where four crops (Pennisetum purpureum,
Medicago sativa, Sinapis alba and Helianthus annuus) were grown separately on
three different soils (CI, CNI and CTRL) in plastic pots. Two types of water (tap
water and wastewater) and two loadings were applied throughout the planting
period based on the field capacity (FC 100 and 150 %). The overall dry matter
yield was compared between the soils and treatments. Under wastewater irrigation,
among the four species grown in the CI soil, P. purpureum (171 g) and H. annuus
(151 g) showed high biomass yields, followed by S. alba (115 g) and M. sativa
(31 g). The plants grown under tap water showed about 70 % lower yields compared
to the abattoir wastewater irrigation (AWW). Similar trends in the biomass yields
were observed for CNI and CTRL soils under the two water treatments, with the
biomass yields in the following order CI > CNI > CTRL soils. The results confirm
the beneficial effects of AWW at the greenhouse level. However, a proper cropping
pattern and wastewater irrigation management plan is essential to utilise the
nutrients available in the wastewater-irrigated land treatment sites. The
increase in fertility is evident from the effects of wastewater on biomass growth
and also the abundance of nutrients accumulated in plants. A mass balance
calculation on the applied, residual and the plant-accumulated nutrients over a
few cropping periods will help us in understanding the nutrient cycling processes
involved in the abattoir-irrigated land treatment sites, which will serve as an
effective tool for the environmental management.
DOI: 10.1007/s11270-016-2947-3
PMCID: PMC4932140
PMID: 27440946
242. Sci Rep. 2016 Jul 20;6:30079. doi: 10.1038/srep30079.
Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is

mediated by interrupting auxin transport and accumulation in roots.
Wang S(1), Ren X(1), Huang B(2), Wang G(3), Zhou P(1), An Y(1)(4).
Author information:
(1)School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai
200240, P. R. China.
(2)Department of Plant Biology and Pathology, Rutgers, The State University of
New Jersey, New Jersey, NJ 08901, USA.
(3)Instrumental Analysis Centre of Shanghai Jiao Tong University, Shanghai Jiao
Tong University, Shanghai 200240, P. R. China.
(4)Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, Shanghai
201101, China.
The objective of this study was to investigate Al(3+)-induced IAA transport,

distribution, and the relation of these two processes to Al(3+)-inhibition of
root growth in alfalfa. Alfalfa seedlings with or without apical buds were
exposed to 0 or 100 μM AlCl3 and were foliar sprayed with water or 6 mg L(-1)
IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell
shapes, altered cell structure, and a shorter length of the meristematic zone in
root tips. Aluminium stress significantly decreased the IAA concentration in
apical buds and root tips. The distribution of IAA fluorescence signals in root
tips was disturbed, and the IAA transportation from shoot base to root tip was
inhibited. The highest intensity of fluorescence signals was detected in the
apical meristematic zone. Exogenous application of IAA markedly alleviated the
Al(3+)-induced inhibition of root growth by increasing IAA accumulation and
recovering the damaged cell structure in root tips. In addition, Al(3+) stress
up-regulated expression of AUX1 and PIN2 genes. These results indicate that
Al(3+)-induced reduction of root growth could be associated with the inhibitions
of IAA synthesis in apical buds and IAA transportation in roots, as well as the
imbalance of IAA distribution in root tips.
DOI: 10.1038/srep30079
PMCID: PMC4951802
PMID: 27435109
243. Biotechnol Lett. 2016 Nov;38(11):1893-1901. Epub 2016 Jul 7.
Recombinant protein, AlnA, combined with transgenic alfalfa remediates

polychlorinated biphenyl-contaminated soils: efficiency and rhizosphere microbial
community response.
Ren H(1), Su Y(1), Zhang J(2), Pan H(2), Chen B(2)(3), Wang Y(4)(5).
Author information:
(1)Key Laboratory of Groud Water Resources and Environment of the Ministry of
Education, College of Environment and Resources, Jilin University, 2519 Jiefang
Road, Changchun, 130021, Jilin, People's Republic of China.
(2)College of Plant Sciences, Jilin University, 5333, Xi'an Road, Changchun,
130062, Jilin, People's Republic of China.
(3)Research Institute of Agricultural Resources and Environment, Key Laboratory
of Plant Nutrition and Agro-Environment in Northeast Region, Jilin Academy of
Agricultural Sciences, MOA, Changchun, Jilin, People's Republic of China.
Road, Changchun, 130021, Jilin, People's Republic of China. wang197911@163.com.
(5)College of Plant Sciences, Jilin University, 5333, Xi'an Road, Changchun,
130062, Jilin, People's Republic of China. wang197911@163.com.
OBJECTIVE: To investigate the remediation efficiency of polychlorinated biphenyl

(PCB)-contaminated soils by the combination of a bioemulsifying protein, AlnA,
and alfalfa expressing bphC.
RESULT: The combination of AlnA and transgenic alfalfa promoted PCB soil
remediation through the pot experiments. The removal rates of tri-PCBs (PCB
16/PCB 32 and PCB 31/PCB 28) and tetra-PCB (PCB 49) in transgenic alfalfa/AlnA
treatment were 3.6-, 1.1-, and 2-fold higher than in transgenic alfalfa treatment
alone. Analysis of gene copy number revealed that the PCB-degrading gene, bphC,
of Pseudomonas-like bacterial populations in transgenic alfalfa/AlnA treatment
increased 1.5-fold compared with that of unplanted soils. Bacterial community
Illumina sequencing showed Pseudomonas, Arthrobacter, and Sphingomonas positively
correlated with the removal rates of PCBs.
CONCLUSIONS: PCB removal was unrelated to bacterial community diversity but
positively correlated with their specific degraders and bphC gene expression.
DOI: 10.1007/s10529-016-2169-1
244. Genet Mol Res. 2016 Jul 14;15(2). doi: 10.4238/gmr.15028323.

Comparative sequence and expression analysis of tapetum specific male sterility
related genes in Medicago truncatula.
Shao LH(1), Zheng XW(1), Yi DX(1), Li C(1).
Author information:
(1)Institute of Animal Sciences, the Chinese Academy of Agricultural Sciences,
Beijing, China.
Heterosis, or enhancement through outbreeding, is one of the most promising

approaches for increasing crop yield. Male sterility (MS), which promotes
heterosis, has been widely applied in hybrid crop production. Medicago truncatula
is a model legume species and is closely related to M. sativa, an important
legume forage plant. Although the molecular mechanisms of MS in M. truncatula and
M. sativa remain unclear, several studies of MS have been conducted in
Arabidopsis thaliana. Previous research has shown that MS is associated with the
destruction of tapetal cell layers. Disruption of tapetum developmental processes
may result in pollen abortion. In an effort to identify genes useful for breeding
in M. sativa, we identified MS related genes in M. truncatula using BLAST and
homology to A. thaliana genes. In this study, we identified 63 tapetum specific
male sterility (TSMS) related genes. The length of TSMS genes varied from 225 to
3747 bp. We identified 15 conserved domains and 8 cis-elements associated with
TSMS related genes. Analysis of the phylogenetic relationships among these genes
allowed them to be classified into three groups, MtTsms A, MtTsms B, and MtTsms
C. Expression analyses revealed that these genes may be involved in developmental
processes and response to abiotic stress.
DOI: 10.4238/gmr.15028323
245. Guang Pu Xue Yu Guang Pu Fen Xi. 2016 Mar;36(3):702-5.
[Determination of Hard Rate of Alfalfa (Medicago sativa L.) Seeds with Near
Infrared Spectroscopy].
[Article in Chinese]
Wang XX, Chen LL, Zhang YW, Mao PS.
Alfalfa (Medicago sativa L.) is the most commonly grown forage crop due to its
better quality characteristics and high adaptability in China. However, there was
20%-80% hard seeds in alfalfa which could not be identified easily from non hard
seeds which would cause the loss of seed utilization value and plant production.
This experiment was designed for 121 samples of alfalfa. Seeds were collected
according to different regions, harvested year and varieties. 31 samples were
artificial matched as hard rates ranging from 20% to 80% to establish a model for
hard seed rate by near infrared spectroscopy (NIRS) with Partial Least Square
(PLS). The objective of this study was to establish a model and to estimate the
efficiency of NIRS for determining hard rate of alfalfa seeds. The results showed
that the correlation coefficient (R2(cal)) of calibration model was 0.981 6, root
mean square error of cross validation (RMSECV) was 5.32, and the ratio of
prediction to deviation (RPD) was 3.58. The forecast model in this experiment
presented the satisfied precision. The proposed method using NIRS technology is
feasible for identification and classification of hard seed in alfalfa. A new
method, as nondestructive testing of hard seed rate, was provided to theoretical
basis for fast nondestructive detection of hard seed rates in alfalfa.
2016.
Mixed Nodule Infection in Sinorhizobium meliloti-Medicago sativa Symbiosis

Suggest the Presence of Cheating Behavior.
Checcucci A(1), Azzarello E(2), Bazzicalupo M(1), Galardini M(3), Lagomarsino

A(4), Mancuso S(2), Marti L(2), Marzano MC(2), Mocali S(4), Squartini A(5),
Zanardo M(5), Mengoni A(1).
Author information:
(1)Department of Biology, University of Florence Florence, Italy.
(2)Department of Agri-Food Production and Environmental Science, University of
Florence Florence, Italy.
(3)European Molecular Biology Laboratory - European Bioinformatics Institute,
Wellcome Trust Genome Campus Cambridge, UK.
(4)Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria,
Centro di Ricerca per l'Agrobiologia e la Pedologia Florence, Italy.
(5)Department of Agronomy, Food, Natural Resources, Animals and the Environment,
University of Padua Padova, Italy.
In the symbiosis between rhizobia and legumes, host plants can form symbiotic
root nodules with multiple rhizobial strains, potentially showing different
symbiotic performances in nitrogen fixation. Here, we investigated the presence
of mixed nodules, containing rhizobia with different degrees of mutualisms, and
evaluate their relative fitness in the Sinorhizobium meliloti-Medicago sativa
model symbiosis. We used three S. meliloti strains, the mutualist strains Rm1021
and BL225C and the non-mutualist AK83. We performed competition experiments
involving both in vitro and in vivo symbiotic assays with M. sativa host plants.
We show the occurrence of a high number (from 27 to 100%) of mixed nodules with
no negative effect on both nitrogen fixation and plant growth. The estimation of
the relative fitness as non-mutualist/mutualist ratios in single nodules shows
that in some nodules the non-mutualist strain efficiently colonized root nodules
along with the mutualist ones. In conclusion, we can support the hypothesis that
in S. meliloti-M. sativa symbiosis mixed nodules are formed and allow
non-mutualist or less-mutualist bacterial partners to be less or not sanctioned
by the host plant, hence allowing a potential form of cheating behavior to be
present in the nitrogen fixing symbiosis.
DOI: 10.3389/fpls.2016.00835
PMCID: PMC4904023
PMID: 27379128
247. J Sci Food Agric. 2017 Mar;97(4):1349-1358. doi: 10.1002/jsfa.7873. Epub 2016
Aug
4.
Anatomy, nutritional value and cell wall chemical analysis of foliage leaves of
Guadua chacoensis (Poaceae, Bambusoideae, Bambuseae), a promising source of
forage.
Panizzo CC(1), Fernández PV(2)(3), Colombatto D(4), Ciancia M(2)(3), Vega AS(1).
Author information:
(1)Cátedra de Botánica General, Departamento de Rcursos Naturales y Ambiente,
Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
(2)Cátedra de Química de Biomoléculas, Departamento de Biología Aplicada y
Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires,
Argentina.
(3)CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón
2, 1428 Buenos Aires, Argentina.
(4)Cátedra de Bovinos de Carne, Departamento de Producción Animal, Facultad de
Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
BACKGROUND: The present study combines morphological and anatomical studies, cell
wall chemical composition analysis, as well as assessment of the nutritional
value of Guadua chacoensis foliage leaves.
RESULTS: Foliage leaves of G. chacoensis are a promising source of forage
because: (a) as a native woody bamboo, it is adapted to and helps maintain
environmental conditions in America; (b) leaf anatomical studies exhibit
discontinuous sclerenchyma, scarcely developed, while pilose indumentum, silica
cells, prickles and hooks are also scarce; (c) it has a high protein content,
similar to that of Medicago sativa, while other nutritional parameters are
similar to those of common forages; and (d) glucuronoarabinoxylan, the major
extracted polysaccharide, has one-third of the 4-linked β-d-xylopyranosyl units
of the backbone substituted mainly with α-l-arabinofuranose as single stubs or
non-reducing end of short chains, but also 5-linked α-l-arabinofuranose units,
terminal β-d-xylopyranose and d-galactopyranose units, as well as α-d-glucuronic
acid residues and small amounts of its 4-O-methylated derivative.
CONCLUSION: These results constitute the first report on this species, and as
culms are utilized in constructions and crafts, the remaining leaves, when used
as forage, constitute a byproduct that allows an additional income opportunity. ©
2016 Society of Chemical Industry.
DOI: 10.1002/jsfa.7873
248. J Food Prot. 2016 Jul;79(7):1115-26. doi: 10.4315/0362-028X.JFP-15-397.
Inactivation of Salmonella on Sprouting Seeds Using a Spontaneous Carvacrol

Nanoemulsion Acidified with Organic Acids.
Landry KS(1), Komaiko J(1), Wong DE(1), Xu T(1), McClements DJ(1), McLandsborough
L(2).
Author information:
(1)Department of Food Science, University of Massachusetts, Amherst,
Massachusetts 01003, USA.
(2)Department of Food Science, University of Massachusetts, Amherst,
Massachusetts 01003, USA. lm@foodsci.umass.edu.
Over the past decade, demand has increased for natural, minimally processed
produce, including sprout-based products. Sanitization with 20,000 ppm of calcium
hypochlorite is currently recommended for all sprouting seeds before germination
to limit sprout-related foodborne outbreaks. A potentially promising disinfectant
as an alternative to calcium hypochlorite is acidified spontaneous essential oil
nanoemulsions. In this study, the efficacy of an acidified carvacrol nanoemulsion
was tested against mung beans and broccoli seeds artificially contaminated with a
Salmonella enterica Enteritidis cocktail (ATCC BAA-709, ATCC BAA-711, and ATCC
BAA-1045). Treatments were performed by soaking inoculated seeds in acidified (50
mM acetic or levulinic acid) carvacrol nanoemulsions (4,000 or 8,000 ppm) for 30
or 60 min. After treatment, the number of surviving cells was determined via
plate counts and/or the most probable number (MPN) approach. Treatment for 30 min
successfully reduced Salmonella Enteritidis by 4 log CFU/g on mung beans (from an
initial contamination level of 4.2 to 4.6 log CFU/g) and by 2 log CFU/g on
broccoli seeds (from an initial contamination level of 2.4 to 2.6 log CFU/g) to
below our detection limit (≤3 MPN/g). Treated seeds were sprouted and tested for
the presence of pathogens and sprout yield. The final sprout product had no
detectable pathogens, and total sprout yield was not influenced by any treatment.
DOI: 10.4315/0362-028X.JFP-15-397
249. Anim Sci J. 2017 Mar;88(3):456-462. doi: 10.1111/asj.12637. Epub 2016 Jun 27.
Effects of chlorpyrifos and chlorantraniliprole on fermentation quality of

alfalfa (Medicago sativa L.) silage inoculated with or without Lactobacillus
plantarum LP.
Zhang Q(1), Yu Z(1), Wang X(1), Na R(2).
Author information:
Beijing, China.
(2)College of Ecology and Environment Science, Inner Mongolia Agricultural
University, Hohhot, China.
The effects of pesticides and Lactobacillus plantarum (LP) on fermentation

quality of alfalfa (Medicago sativa L.) silage were investigated. Chlorpyrifos
and chlorantraniliprole were sprayed on the surface of alfalfa plants at 658.6
and 45.0 g active ingredient/ha, respectively. Alfalfa plants were harvested on
day 5 post-application and ensiled with or without LP. Chlorpyrifos and
chlorantraniliprole decreased the yeast count of alfalfa material (P < 0.05).
Both pesticides increased the butyric acid content of alfalfa silage (P < 0.001).
Chlorpyrifos increased pH and decreased lactic acid, acetic acid and short-chain
fatty acid contents (P < 0.05). LP decreased pH and butyric acid content, and
increased lactic acid and short-chain fatty acid contents of alfalfa silage
treated with pesticides (P < 0.05). LP increased the concentration of
chlorpyrifos residue in alfalfa silage (P < 0.05). Chlorpyrifos and
chlorantraniliprole affected the microbial communities of the material before
ensiling, especially coliform bacteria and yeast; the two pesticide residues were
reduced after the fermentation of alfalfa silage and affected the fermentation
process, whereas LP improved the fermentation quality of pesticides-contaminated
alfalfa silage and slowed down the dissipation of chlorpyrifos.
DOI: 10.1111/asj.12637
250. Sci Rep. 2016 Jun 24;6:28358. doi: 10.1038/srep28358.
Assessing quality of Medicago sativa silage by monitoring bacterial composition

with single molecule, real-time sequencing technology and various physiological
parameters.
Bao W(1), Mi Z(1), Xu H(1), Zheng Y(1), Kwok LY(1), Zhang H(1), Zhang W(1).
Author information:
(1)Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education,
Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China.
The present study applied the PacBio single molecule, real-time sequencing
technology (SMRT) in evaluating the quality of silage production. Specifically,
we produced four types of Medicago sativa silages by using four different lactic
acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the
changes in pH, organic acids (including butyric acid, the ratio of acetic
acid/lactic acid, γ-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic
acid), mycotoxins, and bacterial microbiota during silage fermentation. Our
results showed that the use of the additives was beneficial to the silage
fermentation by enhancing a general pH and mycotoxin reduction, while increasing
the organic acids content. By SMRT analysis of the microbial composition in eight
silage samples, we found that the bacterial species number and relative
abundances shifted apparently after fermentation. Such changes were specific to
the LAB species in the additives. Particularly, Bacillus megaterium was the
initial dominant species in the raw materials; and after the fermentation
process, Pediococcus acidilactici and Lactobacillus plantarum became the most
prevalent species, both of which were intrinsically present in the LAB additives.
Our data have demonstrated that the SMRT sequencing platform is applicable in
assessing the quality of silage.
DOI: 10.1038/srep28358
PMCID: PMC4920031
251. J Exp Bot. 2017 Apr 1;68(8):1851-1872. doi: 10.1093/jxb/erw239.
Flooding tolerance of forage legumes.
Striker GG(1)(2), Colmer TD(2)(3).
Author information:
(1)IFEVA, Universidad de Buenos Aires, CONICET, Facultad de Agronomía, Avenida
San Martín 4453, CPA 1417, DSE Buenos Aires, Argentina.
(2)School of Plant Biology, Faculty of Science, The University of Western
Australia, 35 Stirling Hwy, Crawley WA 6009, Australia.
(3)The UWA Institute of Agriculture, The University of Western Australia, 35
Stirling Hwy, Crawley WA 6009, Australia.
We review waterlogging and submergence tolerances of forage (pasture) legumes.

Growth reductions from waterlogging in perennial species ranged from >50% for
Medicago sativa and Trifolium pratense to <25% for Lotus corniculatus, L. tenuis,
and T. fragiferum. For annual species, waterlogging reduced Medicago truncatula
by ~50%, whereas Melilotus siculus and T. michelianum were not reduced. Tolerant
species have higher root porosity (gas-filled volume in tissues) owing to
aerenchyma formation. Plant dry mass (waterlogged relative to control) had a
positive (hyperbolic) relationship to root porosity across eight species.
Metabolism in hypoxic roots was influenced by internal aeration. Sugars
accumulate in M. sativa due to growth inhibition from limited respiration and low
energy in roots of low porosity (i.e. 4.5%). In contrast, L. corniculatus, with
higher root porosity (i.e. 17.2%) and O2 supply allowing respiration, maintained
growth better and sugars did not accumulate. Tolerant legumes form nodules, and
internal O2 diffusion along roots can sustain metabolism, including N2 fixation,
in submerged nodules. Shoot physiology depends on species tolerance. In M.
sativa, photosynthesis soon declines and in the longer term (>10 d) leaves suffer
chlorophyll degradation, damage, and N, P, and K deficiencies. In tolerant L.
corniculatus and L. tenuis, photosynthesis is maintained longer, shoot N is less
affected, and shoot P can even increase during waterlogging. Species also differ
in tolerance of partial and complete shoot submergence. Gaps in knowledge include
anoxia tolerance of roots, N2 fixation during field waterlogging, and
identification of traits conferring the ability to recover after water subsides.
© The Author 2016. Published by Oxford University Press on behalf of the Society
for Experimental Biology. All rights reserved. For permissions, please email:
DOI: 10.1093/jxb/erw239
252. Sci Rep. 2016 Jun 21;6:28435. doi: 10.1038/srep28435.
Roots of forbs sense climate fluctuations in the semi-arid Loess Plateau:

Herb-chronology based analysis.
Shi S(1)(2), Li Z(1), Wang H(1)(2), von Arx G(3), Lü Y(1), Wu X(1), Wang X(4),
Liu G(1), Fu B(1).
Author information:
(1)State Key Laboratory of Urban and Regional Ecology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
(2)University of the Chinese Academy of Sciences, Beijing 100049, China.
(3)Swiss Federal Institute for Forest, Snow and Landscape Research WSL,
Birmensdorf 8903, Switzerland.
(4)College of Forestry, Northeast Forestry University, Harbin 150040, China.
Growth of herbaceous plants responds sensitively and rapidly to climate

variability. Yet, little is known regarding how climate warming influences the
growth of herbaceous plants, particularly in semi-arid sites. This contrasts with
widely reported tree growth decline and even mortality in response to severe
water deficits due to climate warming around the world. Here, we use the
relatively novel approach of herb-chronology to analyze the correlation between
climatic factors and annual ring width in the root xylem of two perennial forb
species (Medicago sativa, Potentilla chinensis) in the Loess Plateau of China. We
show that warming-induced water deficit has a significant negative effect on the
growth of herbaceous plants in the Loess Plateau. Our results indicate that the
growth of forbs responds rapidly and sensitively to drought variability, implying
that water availability plays a dominant role in regulating the growth of
herbaceous plants in semi-arid areas. If warming and drying in the Loess Plateau
continue in the future, further affects the growth of herbaceous plants,
potentially driving regional changes in the relationship between herbaceous
vegetation and climate.
DOI: 10.1038/srep28435
PMCID: PMC4914992
PMID: 27323906
253. Genet Mol Res. 2016 Jun 3;15(2). doi: 10.4238/gmr.15027968.
Altered callose deposition during embryo sac formation of multi-pistil mutant

(mp1) in Medicago sativa.
Zhou HC(1), Jin L(2), Li J(1), Wang XJ(2).
Author information:
(1)School of Pastoral Agriculture Science and Technology, Lanzhou University,
Lanzhou, China.
(2)Natural History Research Center, Shanghai Natural History Museum, Shanghai
Science & Technology Museum, Shanghai, China.
Whether callose deposition is the cause or result of ovule sterility in Medicago

sativa remains controversial, because it is unclear when and where changes in
callose deposition and dissolution occur during fertile and sterile embryo sac
formation. Here, alfalfa spontaneous multi-pistil mutant (mp1) and wild-type
plants were used to compare the dynamics of callose deposition during embryo sac
formation using microscopy. The results showed that both mutant and wild-type
plants experienced megasporogenesis and megagametogenesis, and there was no
significant difference during megasporogenesis. In contrast to the wild-type
plants, in which the mature embryo sac was observed after three continuous cycles
of mitosis, functional megaspores of mutant plants developed abnormally after the
second round of mitosis, leading to degeneration of synergid, central, and
antipodal cells. Callose deposition in both mutant and wild-type plants was first
observed in the walls of megasporocytes, and then in the megaspore tetrad walls.
After meiosis, the callose wall began to degrade as the functional megaspore
underwent mitosis, and almost no callose was observed in the mature embryo sac in
wild-type plants. However, callose deposition was observed in mp1 plants around
the synergid, and increased with the development of the embryo sac, and was
mainly deposited at the micropylar end. Our results indicate that synergid,
central, and antipodal cells, which are surrounded by callose, may degrade owing
to lack of nutrition. Callose accumulation around the synergid and at the
micropylar end may hinder signals required for the pollen tube to enter the
embryo sac, leading to abortion.
DOI: 10.4238/gmr.15027968
254. Genet Mol Res. 2016 May 23;15(2). doi: 10.4238/gmr.15028002.
Expression of an alfalfa (Medicago sativa L.) peroxidase gene in transgenic

Arabidopsis thaliana enhances resistance to NaCl and H2O2.
Teng K(1), Xiao GZ(2), Guo WE(1), Yuan JB(1), Li J(3), Chao YH(1), Han LB(1).
Author information:
(1)Turfgrass Research Institute, College of Forestry, Beijing Forestry
University, Beijing, China.
(2)The College of Horticulture and Garden, Yangtze University, Jingzhou, China.
(3)Institute of Grassland Research, Chinese Academy of Agricultural Sciences,
Hohhot, China.
Peroxidases (PODs) are enzymes that play important roles in catalyzing the
reduction of H2O2 and the oxidation of various substrates. They function in many
different and important biological processes, such as defense mechanisms, immune
responses, and pathogeny. The POD genes have been cloned and identified in many
plants, but their function in alfalfa (Medicago sativa L.) is not known, to date.
Based on the POD gene sequence (GenBank accession No. L36157.1), we cloned the
POD gene in alfalfa, which was named MsPOD. MsPOD expression increased with
increasing H2O2. The gene was expressed in all of the tissues, including the
roots, stems, leaves, and flowers, particularly in stems and leaves under
light/dark conditions. A subcellular analysis showed that MsPOD was localized
outside the cells. Transgenic Arabidopsis with MsPOD exhibited increased
resistance to H2O2 and NaCl. Moreover, POD activity in the transgenic plants was
significantly higher than that in wild-type Arabidopsis. These results show that
MsPOD plays an important role in resistance to H2O2 and NaCl.
DOI: 10.4238/gmr.15028002
255. BMC Vet Res. 2016 Jun 14;12(1):110. doi: 10.1186/s12917-016-0733-5.
Effects of two alfalfa preparations with different particle sizes on the gastric
mucosa in weanlings: alfalfa chaff versus alfalfa pellets.
Vondran S(1), Venner M(2), Vervuert I(3).
Author information:
(1)Institute of Animal Nutrition, Nutrition Diseases and Dietetics, Faculty of
Veterinary Medicine, University of Leipzig, D-04103, Leipzig, Germany.
(2)Equine Veterinary Clinic, D-38162, Destedt, Germany.
Veterinary Medicine, University of Leipzig, D-04103, Leipzig, Germany.
ingrid.vervuert@vetmed.uni-leipzig.de.
BACKGROUND: Feeding alfalfa hay is often recommended for its buffering

components, like protein and calcium, to prevent lesions of the gastric mucosa in
horses. Until now, there has been no information regarding the influence of
alfalfa particle size on the gastric mucosa. The aim of this study was to
investigate the effects of feeding two alfalfa preparations with different
particle sizes (alfalfa chaff vs alfalfa pellets) in comparison with grass hay on
the gastric mucosa in weanling horses. We hypothesized that feeding a high
proportion of fine alfalfa particles would negatively impact gastric mucosa and
that feeding long alfalfa chaff would improve gastric mucosal health in
weanlings.
RESULTS: Before weaning, the prevalence of gastric mucosa lesions (one or more
lesions considering all locations in the stomach) was 84.3 %; at 14 days after
weaning, it was almost 100 %. Before and after weaning, most of the lesions were
found at the greater curvature of the squamous mucosa and at the lesser
curvature. After weaning, gastric mucosal lesions at the pylorus were
significantly more severe in the group fed alfalfa chaff (p = 0.002). In the
other regions, no differences related to the feeding regimes were observed.
CONCLUSIONS: Feeding alfalfa failed to improve gastric mucosal lesion scores in
weanlings. Furthermore, foals fed alfalfa chaff had higher lesion scores at the
pylorus. Alfalfa leaves contain a superior protein source and high amounts of
calcium and magnesium, providing extra nutritional advantages in growing horses.
At this time, either traditional grass hay rations or grass hay with alfalfa
pellets can be recommended.
DOI: 10.1186/s12917-016-0733-5
PMCID: PMC4908680

May 10.
Overexpression of Medicago sativa TMT elevates the α-tocopherol content in

Arabidopsis seeds, alfalfa leaves, and delays dark-induced leaf senescence.
Jiang J(1), Jia H(2), Feng G(3), Wang Z(1), Li J(4), Gao H(5), Wang X(6).
Author information:
100193, China.
100193, China; Present address: Animal Husbandry and Veterinary institute, Shanxi
Academy of Agricultural Sciences, Taiyuan 030032, China.
100193, China; Present address: Department of Grassland Science, College of
Animal Science and Technology, Sichuan Agricultural University, Wenjiang 611130,
China.
100193, China; Present address: Institute of Grassland Research, Chinese Academy
of Agricultural Sciences, Huhhot 010020,China.
100193, China. Electronic address: gaohongwen@263.net.
100193, China. Electronic address: wangxuemin@caas.cn.
Alfalfa (Medicago sativa L.) is a major forage legume for livestock and a target
for improving their dietary quality. Vitamin E is an essential vitamin that
animals must obtain from their diet for proper growth and development.
γ-tocopherol methyltransferase (γ-TMT), which catalyzes the conversion of δ- and
γ-tocopherols (or tocotrienols) to β- and α-tocopherols (or tocotrienols),
respectively, is the final enzyme involved in the vitamin E biosynthetic pathway.
The overexpression of M. sativa L.'s γ-TMT (MsTMT) increased the α-tocopherol
content 10-15 fold above that of wild type Arabidopsis seeds without altering the
total content of vitamin E. Additionally, in response to osmotic stress, the
biomass and the expression levels of several osmotic marker genes were
significantly higher in the transgenic lines compared with wild type.
Overexpression of MsTMT in alfalfa led to a modest, albeit significant, increase
in α-tocopherol in leaves and was also responsible for a delayed leaf senescence
phenotype. Additionally, the crude protein content was increased, while the acid
and neutral detergent fiber contents were unchanged in these transgenic lines.
Thus, increased α-tocopherol content occurred in transgenic alfalfa without
compromising the nutritional qualities. The targeted metabolic engineering of
vitamin E biosynthesis through MsTMT overexpression provides a promising approach
to improve the α-tocopherol content of forage crops.
Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights
reserved.
DOI: 10.1016/j.plantsci.2016.05.004

eCollection 2016.
Assessing the Spatial Variability of Alfalfa Yield Using Satellite Imagery and
Ground-Based Data.
Kayad AG(1)(2)(3), Al-Gaadi KA(1)(2), Tola E(2), Madugundu R(2), Zeyada AM(2),
Kalaitzidis C(4).
Author information:
(1)Department of Agricultural Engineering, College of Food and Agriculture
Sciences, King Saud University, Riyadh, Saudi Arabia.
(2)Precision Agriculture Research Chair, King Saud University, Riyadh, Saudi
Arabia.
(3)Agricultural Engineering Research Institute (AEnRI), Agricultural Research
Centre, Giza, Egypt.
(4)Department of Geoinformation in Environmental Management, Mediterranean
Agronomic Institute of Chania (MAICh), Chania, Greece.
Understanding the temporal and spatial variability in a crop yield is viewed as

one of the key steps in the implementation of precision agriculture practices.
Therefore, a study on a center pivot irrigated 23.5 ha field in Saudi Arabia was
conducted to assess the variability in alfalfa yield using Landsat-8 imagery and
a hay yield monitor data. In addition, the study was designed to also explore the
potential of predicting the alfalfa yield using vegetation indices. A calibrated
yield monitor mounted on a large rectangular hay baler was used to measure the
actual alfalfa yield for four alfalfa harvests performed in the period from
October 2013 to May 2014. A total of 18 Landsat-8 images, representing different
crop growth stages, were used to derive different vegetation indices (VIs). Data
from the yield monitor was used to generate yield maps, which illustrated a
definite spatial variation in alfalfa yield across the experimental field for the
four studied harvests as indicated by the high spatial correlation values (0.75
to 0.97) and the low P-values (4.7E-103 to 8.9E-27). The yield monitor-measured
alfalfa actual yield was compared to the predicted yield form the Vis. Results of
the study showed that there was a correlation between actual and predicted yield.
The highest correlations were observed between actual yield and the predicted
using NIR reflectance, SAVI and NDVI with maximum correlation coefficients of
0.69, 0.68 and 0.63, respectively.
PMCID: PMC4900617
258. Funct Integr Genomics. 2016 Sep;16(5):495-511. doi: 10.1007/s10142-016-0500-5.

Epub 2016 Jun 7.
Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown

buds acclimated to freezing stress.
Song L(1), Jiang L(1), Chen Y(1), Shu Y(1), Bai Y(1), Guo C(2).
Author information:
(1)Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang
Province, College of Life Science and Technology, Harbin Normal University,
Harbin, 150025, Heilongjiang, China.
Harbin, 150025, Heilongjiang, China. kaku_2008@163.com.
Medicago sativa L. (alfalfa) 'Zhaodong' is an important forage legume that can

safely survive in northern China where winter temperatures reach as low as
-30 °C. Survival of alfalfa following freezing stress depends on the amount and
revival ability of crown buds. In order to investigate the molecular mechanisms
of frost tolerance in alfalfa, we used transcriptome sequencing technology and
bioinformatics strategies to analyze crown buds of field-grown alfalfa during
winter. We statistically identified a total of 5605 differentially expressed
genes (DEGs) involved in freezing stress including 1900 upregulated and 3705
downregulated DEGs. We validated 36 candidate DEGs using qPCR to confirm the
accuracy of the RNA-seq data. Unlike other recent studies, this study employed
alfalfa plants grown in the natural environment. Our results indicate that not
only the CBF orthologs but also membrane proteins, hormone signal transduction
pathways, and ubiquitin-mediated proteolysis pathways indicate the presence of a
special freezing adaptation mechanism in alfalfa. The antioxidant defense system
may rapidly confer freezing tolerance to alfalfa. Importantly, biosynthesis of
secondary metabolites and phenylalanine metabolism, which is of potential
importance in coordinating freezing tolerance with growth and development, were
downregulated in subzero temperatures. The adaptive mechanism for frost tolerance
is a complex multigenic process that is not well understood. This systematic
analysis provided an in-depth view of stress tolerance mechanisms in alfalfa.
DOI: 10.1007/s10142-016-0500-5
259. Environ Entomol. 2016 Aug;45(4):865-72. doi: 10.1093/ee/nvw066. Epub 2016 Jun
6.
The Effects of Crop Intensification on the Diversity of Native Pollinator

Communities.
Mogren CL(1), Rand TA(2), Fausti SW(3), Lundgren JG(4).
Author information:
(1)USDA-ARS, North Central Agricultural Research Laboratory, 2923 Medary Ave.,
Brookings, SD 57006 (cmogren@gmail.com; jgl.entomology@gmail.com) Current
Address: Louisiana State University AgCenter, Entomology Department, 404 Life
Sciences, Baton Rouge, LA 70803 cmogren@gmail.com.
(2)USDA-ARS, Northern Plains Agricultural Research Laboratory, 1500 North Central
Ave., Sidney, MT 59270 (tatyana.rand@ars.usda.gov).
(3)Economics Department, South Dakota State University, Brookings, SD 57007
(scott.fausti@sdstate.edu).
(4)USDA-ARS, North Central Agricultural Research Laboratory, 2923 Medary Ave.,
Brookings, SD 57006 (cmogren@gmail.com; jgl.entomology@gmail.com) Current
Address: Ecdysis Foundation, 46958 188th St, Estelline, SD 57234.
Increases in agricultural conversion are leading to declines in native grasslands

and natural resources critical for beneficial insects. However, little is known
regarding how these changes affect pollinator diversity. Land use types were
categorized within 300 m and 3 km radii of pollinator sampling locations in
Brookings County, SD. Pollinator abundance and species richness were regressed on
the proportion of the landscape dedicated to row crops, grass and pasture, forage
crops, small grains, and aquatic habitats using variance components modeling. Row
crops had a negative effect on bee abundance at 300 m, after fixed effects
modeling accounted for outliers skewing this relationship. At 3 km, corn
positively affected bee abundance and richness, while soybean acreage decreased
species richness. The landscape matrix of outlying sites consisted of large
monocultured areas with few alternative habitat types available, leading to
inflated populations of Melissodes and Halictidae. Syrphids had a positive
parabolic relationship between diversity and row crops, indicating potential for
competitive exclusion from intermediate landscapes. Unlike other studies,
landscape diversity within 300 m was not found to significantly benefit
pollinator diversity. Within especially agriculturally developed areas of the
region, high abundances of pollinators suggest selection for a few dominant
species. There was no effect of forage crops or aquatic habitats on pollinator
diversity, indicating that less highly managed areas still represent degraded
habitat within the landscape. Incorporating pollinator-friendly crops at the farm
level throughout the region is likely to enhance pollinator diversity by
lessening the negative effects of large monocultures.
DOI: 10.1093/ee/nvw066
260. FEMS Microbiol Ecol. 2016 Aug;92(8). pii: fiw118. doi: 10.1093/femsec/fiw118.
Epub 2016 Jun 5.
Rhizobium leguminosarum symbiovar trifolii, Ensifer numidicus and Mesorhizobium

amorphae symbiovar ciceri (or Mesorhizobium loti) are new endosymbiotic bacteria
of Lens culinaris Medik.
Sami D(1), Mokhtar R(2), Peter M(3), Mohamed M(1).
Author information:
(1)Research Unit Biodiversity & Valorization of Arid Areas Bioressources, Faculty
of Sciences of Gabès, Erriadh-Zrig, Gabes 6072, Tunisia.
(2)Research Unit Biodiversity & Valorization of Arid Areas Bioressources, Faculty
of Sciences of Gabès, Erriadh-Zrig, Gabes 6072, Tunisia rejili@dbi.udel.edu.
(3)Institute for IntegrativeBiology of the Cell, Centre National de la Recherche
Scientifique, Avenue de la Terrasse Bât. 34, 91198 Gif-sur-Yvette, France.
A total of 142 rhizobial bacteria were isolated from root nodules of Lens
culinaris Medik endemic to Tunisia and they belonged to the species Rhizobium
leguminosarum, and for the first time to Ensifer and Mesorhizobium, genera never
previously described as microsymbionts of lentil. Phenotypically, our results
indicate that L. culinaris Medik strains showed heterogenic responses to the
different phenotypic features and they effectively nodulated their original host.
Based on the concatenation of the 16S rRNA with relevant housekeeping genes
(glnA, recA, dnaK), rhizobia that nodulate lentil belonged almost exclusively to
the known R. leguminosarum sv. viciae. Interestingly, R. leguminosarum sv.
trifolii, Ensifer numidicus (10 isolates) and Mesorhizobium amorphae (or M. loti)
(9 isolates) isolates species, not considered, up to now, as a natural symbiont
of lentil are reported. The E. numidicus and M. amorphae (or M. loti) strains
induced fixing nodules on Medicago sativa and Cicer arietinum host plants,
respectively. Symbiotic gene phylogenies showed that the E. numidicus, new
symbiont of lentil, markedly diverged from strains of R. leguminosarum, the usual
symbionts of lentil, and converged to the symbiovar meliloti so far described
within E. meliloti Indeed, the nodC and nodA genes from the M. amorphae showed
more than 99% similarity with respect to those from M. mediterraneum, the common
chickpea nodulating species, and would be included in the new infrasubspecific
division named M. amorphae symbiovar ciceri, or to M. loti, related to the
strains able to effectively nodulate C. arietinum host plant. On the basis of
these data, R. leguminosarum sv. trifolii (type strain LBg3 (T)), M. loti or M.
amorphae sv. ciceri (type strain LB4 (T)) and E. numidicus (type strain LBi2 (T))
are proposed as new symbionts of L. culinaris Medik.

DOI: 10.1093/femsec/fiw118
261. Ann Bot. 2016 Jun 1. pii: mcw073. [Epub ahead of print]
Root adaptations to soils with low fertility and aluminium toxicity.
Rao IM(1), Miles JW(2), Beebe SE(2), Horst WJ(3).
Author information:
(1)Centro Internacional de Agricultura Tropical (CIAT), A. A. 6713, Cali,
Colombia and i.rao@cgiar.org.
(2)Centro Internacional de Agricultura Tropical (CIAT), A. A. 6713, Cali,
Colombia and.
(3)Leibniz University of Hannover, Herrenhaeuser Str. 2, D-30419 Hannover,
Germany.
BACKGROUND: Plants depend on their root systems to acquire the water and
nutrients necessary for their survival in nature, and for their yield and
nutritional quality in agriculture. Root systems are complex and a variety of
root phenes have been identified as contributors to adaptation to soils with low
fertility and aluminium (Al) toxicity. Phenotypic characterization of root
adaptations to infertile soils is enabling plant breeders to develop improved
cultivars that not only yield more, but also contribute to yield stability and
nutritional security in the face of climate variability.
SCOPE: In this review the adaptive responses of root systems to soils with low
fertility and Al toxicity are described. After a brief introduction, the purpose
and focus of the review are outlined. This is followed by a description of the
adaptive responses of roots to low supply of mineral nutrients [with an emphasis
on low availability of nitrogen (N) and phosphorus (P) and on toxic levels of
Al]. We describe progress in developing germplasm adapted to soils with low
fertility or Al toxicity using selected examples from ongoing breeding programmes
on food (maize, common bean) and forage/feed (Brachiaria spp.) crops. A number of
root architectural, morphological, anatomical and metabolic phenes contribute to
the superior performance and yield on soils with low fertility and Al toxicity.
Major advances have been made in identifying root phenes in improving adaptation
to low N (maize), low P (common bean) or high Al [maize, common bean, species and
hybrids of brachiariagrass, bulbous canarygrass (Phalaris aquatica) and lucerne
(Medicago sativa)].
CONCLUSIONS: Advanced root phenotyping tools will allow dissection of root
responses into specific root phenes that will aid both conventional and molecular
breeders to develop superior cultivars. These new cultivars will play a key role
in sustainable intensification of crop-livestock systems, particularly in
smallholder systems of the tropics. Development of these new cultivars adapted to
soils with low fertility and Al toxicity is needed to improve global food and
nutritional security and environmental sustainability.
© The Author 2016. Published by Oxford University Press on behalf of the Annals
of Botany Company.
DOI: 10.1093/aob/mcw073
PMCID: PMC5055624
PMID: 27255099
2016.
Clarification on Host Range of Didymella pinodes the Causal Agent of Pea

Ascochyta Blight.
Barilli E(1), Cobos MJ(1), Rubiales D(1).
Author information:
(1)Institute for Sustainable Agriculture CSIC, Córdoba, Spain.
Didymella pinodes is the principal causal agent of ascochyta blight, one of the
most important fungal diseases of pea (Pisum sativum) worldwide. Understanding
its host specificity has crucial implications in epidemiology and management;
however, this has not been clearly delineated yet. In this study we attempt to
clarify the host range of D. pinodes and to compare it with that of other close
Didymella spp. D. pinodes was very virulent on pea accessions, although
differences in virulence were identified among isolates. On the contrary, studied
isolates of D. fabae, D. rabiei, and D. lentil showed a reduced ability to infect
pea not causing macroscopically visible symptoms on any of the pea accessions
tested. D. pinodes isolates were also infective to some extend on almost all
species tested including species such as Hedysarum coronarium, Lathyrus sativus,
Lupinus albus, Medicago spp., Trifolium spp., Trigonella foenum-graecum, and
Vicia articulata which were not mentioned before as hosts of D. pinodes. On the
contrary, D. lentil and D. rabiei were more specific, infecting only lentil and
chickpea, respectively. D. fabae was intermediate, infecting mainly faba bean,
but also slightly other species such as Glycine max, Phaseolus vulgaris,
Trifolium spp., Vicia sativa, and V. articulata. DNA sequence analysis of the
nuclear ribosomal internal transcribed spacer region (ITS) was performed to
confirm identity of the isolates studies and to determine phylogenetic
relationship among the Didymella species, revealing the presence of two clearly
distinct clades. Clade one was represented by two supported subclusters including
D. fabae isolates as well as D. rabiei with D. lentil isolates. Clade two was the
largest and included all the D. pinodes isolates as well as Phoma medicaginis
var. pinodella. Genetic distance between D. pinodes and the other Didymella spp.
isolates was not correlated with overall differences in pathogenicity. Based on
evidences presented here, D. pinodes is not specialized on pea and its host range
is larger than that of D. fabae, D. lentil, and D. rabiei. This has relevant
implications in epidemiology and control as these species might act as
alternative hosts for D. pinodes.
DOI: 10.3389/fpls.2016.00592
PMCID: PMC4865514
PMID: 27242812
263. J Appl Microbiol. 2016 Aug;121(2):333-40. doi: 10.1111/jam.13103.
In vitro ruminal fermentation of treated alfalfa silage using ruminal inocula

from high and low feed-efficient lactating cows.
Contreras-Govea FE(1), Muck RE(2), Weimer PJ(2), Hymes-Fecht UC(2).
Author information:
(1)Department of Dairy Science, University of Wisconsin-Madison, Madison, WI,
USA.
(2)USDA-Dairy Forage Research Center, Madison, WI, USA.
AIMS: To assess the effect of two additives on alfalfa silage and on in vitro
ruminal fermentation when using ruminal inocula from high feed-efficient (HE) and
low feed-efficient (LE) lactating cows.
METHODS AND RESULTS: First- and second-cut alfalfa was harvested at 40% bloom
stage, treated with control (no additive), Lactobacillus plantarum (LP) or formic
acid (Formic), ensiled in 1·0 l minisilos, and fermented for 60 days. Fermented
alfalfa was incubated in vitro for 24 h using ruminal inoculum from HE and LE
lactating cows. The pH was lower in alfalfa silage treated with LP and Formic,
and produced lower ammonia-N than did the control. In vitro true dry matter
digestibility (IVTDMD) was higher with ruminal inoculum from HE than LE cows, but
there was no consistent effect of treated alfalfa on microbial biomass yield and
in vitro volatile fatty acids.
CONCLUSIONS: The IVTDMD was numerically greater with ruminal inoculum from higher
feed-efficient cows although statistical significance was only demonstrated with
the first-cut alfalfa. However, treated alfalfa silage did not show the effect
expected on in vitro microbial biomass yield.
SIGNIFICANCE AND IMPACT OF THE STUDY: The feed efficiency of cows used as a
source of ruminal inocula may affect IVTDMD and be a source of variation across
in vitro runs. Differences in ruminal fermentation between cows of different feed
efficiency could help to explain differences in milk yield and other parameters
of dairy cattle performance.
Published 2016. This article is a U.S. Government work and is in the public
domain in the USA.
DOI: 10.1111/jam.13103
264. J Dairy Sci. 2016 Aug;99(8):6229-6236. doi: 10.3168/jds.2016-11274. Epub 2016

May
26.
Particle length of silages affects apparent ruminal synthesis of B vitamins in

Castagnino DS(1), Kammes KL(2), Allen MS(2), Gervais R(3), Chouinard PY(3),
Girard CL(4).
Author information:
(1)Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre,
2000 College, Sherbrooke, Québec, Canada, J1M 0C8; Département des sciences
animales, Université Laval, 2425 rue de l'Agriculture, Québec, Québec, Canada,
G1V 0A6.
(2)Department of Animal Science, Michigan State University, East Lansing
48824-1225.
(3)Département des sciences animales, Université Laval, 2425 rue de
l'Agriculture, Québec, Québec, Canada, G1V 0A6.
(4)Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre,
2000 College, Sherbrooke, Québec, Canada, J1M 0C8. Electronic address:
Christiane.Girard@agr.gc.ca.
Effects of particle length of silages on apparent ruminal synthesis (ARS) and

postruminal supply of B vitamins were evaluated in 2 feeding trials. Diets
containing alfalfa (trial 1) or orchardgrass (trial 2) silages, chopped to either
19mm (long cut, LC) or 10mm (short cut, SC) theoretical particle length, as the
sole forage were offered to ruminally and duodenally cannulated lactating
Holstein cows in crossover design experiments. Forages chopped to a theoretical
particle length of 19 and 10mm had mean particles sizes of 14.1 and 8.1mm,
respectively, in trial 1, and 15.3 and 11.3mm, respectively, in trial 2. Trial 1
was conducted with 13 multiparous cows in two 19-d treatment periods; both diets
contained approximately 20% forage neutral detergent fiber (NDF), 25% total NDF,
and forage-to-concentrate ratios were approximately 47:53. Trial 2 was conducted
with 15 cows in two 18-d treatment periods; both diets contained approximately
23% forage NDF, 28% total NDF, and had a forage-to-concentrate ratio of 50:50.
Thiamine, riboflavin, niacin, vitamin B6, folates, and vitamin B12 were measured
in feed and duodenal content. Daily ARS was calculated as the duodenal flow minus
the intake. In trial 1, daily intake of individual B vitamins was increased with
the LC diet, but ARS of thiamine, riboflavin, vitamin B6, and folates was
reduced. In trial 2, except for folates, intakes of the other B vitamins were
decreased with the LC diets, whereas ARS of riboflavin, niacin, and vitamin B6
was increased. Daily ARS of thiamine, riboflavin, niacin, and vitamin B6 were
correlated negatively with their intake, suggesting that ruminal bacteria reduced
their synthesis when dietary supply increased. Microbial activity could have also
reduced degradation of thiamine, riboflavin, and niacin, which is supported by
(1) the negative correlation between ARS of these vitamins and ruminal pH or
microbial N duodenal flow; and (2) the positive correlation between ARS and
ruminal concentrations of volatile fatty acids. Folate ARS followed the opposite
correlation pattern. Nevertheless, in spite of differences in intake and ARS,
with both forages, decreasing particle length of silages had limited effects on
the amounts of B vitamins reaching the sites of absorption in the small intestine
of dairy cows.

DOI: 10.3168/jds.2016-11274
The Role of Proanthocyanidins Complex in Structure and Nutrition Interaction in

Alfalfa Forage.
Jonker A(1)(2), Yu P(3).
Author information:
(1)College of Agriculture and Bioresources, University of Saskatchewan, 51 Campus
Drive, Saskatoon, SK S7N 5A8, Canada. arjan.jonker@agresearch.co.nz.
(2)Current address: Grasslands Research Centre, AgResearch Ltd., Tennent Drive,
Private Bag 11008, Palmerston North 4442, New Zealand.
arjan.jonker@agresearch.co.nz.
Drive, Saskatoon, SK S7N 5A8, Canada. peiqiang.yu@usask.ca.
Alfalfa (Medicago sativa L.) is one of the main forages grown in the world.
Alfalfa is a winter hardy, drought tolerant, N-fixing legume with a good
longevity, high yield, high nutrient levels, high digestibility, unique
structural to non-structural components ratio, high dry matter intake, and high
animal productivity per hectare. However, its main limitation is its excessively
rapid initial rate of protein degradation in the rumen, which results in pasture
bloat and inefficient use of protein with consequent excessive excretions of
nitrogen into the environment. Proanthocyanidins are secondary plant metabolites
that can bind with protein and thereby reduce the rate and extent of ruminal
protein degradation. However, these secondary metabolites do not accumulate in
alfalfa. This review aims to firstly describe the events involved in the rapid
release of protein from alfalfa and its effect on ruminant nutrition,
environmental pollution, and pasture bloat; secondly, to describe occurrence,
structure, functions and benefits of moderate amounts of proanthocyanidin; and
finally, to describe the development of alfalfa which accumulates moderate
amounts of proanthocyanidins. The emphasis of this review focuses on the role of
proanthocyanidins compounds in structure and nutrition interaction in ruminant
livestock systems.
DOI: 10.3390/ijms17050793
PMCID: PMC4881609
266. Planta Med. 2017 Jan;83(1-02):40-50. doi: 10.1055/s-0042-107357. Epub 2016 May
24.
Therapeutic Potential of Hydroxypropyl-β-Cyclodextrin-Based Extract of Medicago

sativa in the Treatment of Mucopolysaccharidoses.
Fumić B(1), Končić MZ(1), Jug M(2).
Author information:
(1)Department of Pharmacognosy, Faculty of Pharmacy and Biochemistry, University
of Zagreb, Zagreb, Croatia.
(2)Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry,
University of Zagreb, Zagreb, Croatia.
Mucopolysaccharidoses are inherited metabolic disorders resulting in the

dysfunction of enzymes involved in the degradation of glycosaminoglycans, leading
to severe clinical symptoms and a significantly shortened life span of patients.
Flavonoids are recognized as glycosaminoglycan metabolism modulators, able to
correct glycosaminoglycan cell storage. Therefore, the aim of this work was the
development of an efficient and eco-friendly extraction process of phytochemicals
from Medicago sativa by simultaneous use of ultrasound extraction and
hydroxypropyl-β-cyclodextrin complexation, and investigation of the potential of
such an extract as a glycosaminoglycan metabolism modulator. The Box-Behnken
design and response surface methodology were used in order to optimize the
extraction process, considering hydroxypropyl-β-cyclodextrin concentration,
ultrasonic power, and extraction time as the key parameters. The dependent
variables included total phenolicand total flavonoid content, DPPH radical
scavenging activity, and Fe2+ chelating activity, due to the importance of
oxidative stress in the pathology of mucopolysaccharidoses. The developed
technology using hydroxypropyl-β-cyclodextrin led to more selective flavonoid
extraction from M. sativa than obtained either by the use of water or ethanol.
The lyophilization of extracts resulted in products with high radical scavenging
activity, suitable for further use. The application of 20 mM
hydroxypropyl-β-cyclodextrin solution, 432 W ultrasonic power, and an extraction
time of 45 min resulted in an extract with both the highest total flavonoid
content and the lowest radical scavenging activity IC50. This extract reduced the
levels of glycosaminoglycans in skin fibroblasts of mucopolysaccharidose III
patient in a dose-dependent manner. At concentrations of 3 and 6 µg/mL, the
observed levels of glycosaminoglycans were reduced by 41.2 and 51.1 %,
respectively, clearly demonstrating the validity of the selected approach.
Georg Thieme Verlag KG Stuttgart · New York.
DOI: 10.1055/s-0042-107357
267. Int J Phytoremediation. 2016 Nov;18(11):1136-47. doi:

10.1080/15226514.2016.1186594.
Effect of Medicago sativa L. and compost on organic and inorganic pollutant

removal from a mixed contaminated soil and risk assessment using ecotoxicological
tests.
Marchand C(1), Hogland W(2), Kaczala F(2), Jani Y(2), Marchand L(3), Augustsson
A(2), Hijri M(1).
Author information:
(1)a Institut de Recherche en Biologie Végétale (IRBV), Université de Montréal ,
Montréal , QC , Canada.
(2)b Department of Biology and Environmental Sciences , Linnaeus University ,
Kalmar , Sweden.
(3)c INRA, UMR 1202 BIOGECO , Cestas cedex , France.
Several Gentle Remediation Options (GRO), e.g., plant-based options

(phytoremediation), singly and combined with soil amendments, can be
simultaneously efficient for degrading organic pollutants and either stabilizing
or extracting trace elements (TEs). Here, a 5-month greenhouse trial was
performed to test the efficiency of Medicago sativa L., singly and combined with
a compost addition (30% w/w), to treat soils contaminated by petroleum
hydrocarbons (PHC), Co and Pb collected at an auto scrap yard. After 5 months,
total soil Pb significantly decreased in the compost-amended soil planted with M.
sativa, but not total soil Co. Compost incorporation into the soil promoted PHC
degradation, M. sativa growth and survival, and shoot Pb concentrations [3.8 mg
kg(-1) dry weight (DW)]. Residual risk assessment after the phytoremediation
trial showed a positive effect of compost amendment on plant growth and earthworm
development. The O2 uptake by soil microorganisms was lower in the
compost-amended soil, suggesting a decrease in microbial activity. This study
underlined the benefits of the phytoremediation option based on M. sativa
cultivation and compost amendment for remediating PHC- and Pb-contaminated soils.
DOI: 10.1080/15226514.2016.1186594
268. Sci Rep. 2016 May 23;6:26586. doi: 10.1038/srep26586.
Contribution of the pod wall to seed grain filling in alfalfa.
Wang H(1)(2), Hou L(1)(2), Wang M(1)(2), Mao P(1)(2).
Author information:
(1)Forage Seed Laboratory, China Agricultural University, Beijing 100193, P. R.
China.
(2)Beijing Key Laboratory of Grassland Science, Beijing 100193, P. R. China.
Three genotypes of alfalfa viz. Medicago sativa (Zhongmu No. 1, Zhongmu No. 2)
and M. varia (Caoyuan No. 3) grown in the filed were investigated for the
contribution of pod wall and leaves by shading all pods and leaves on July 15, 20
and 25, respectively. Date was recorded for total pod weight (TPW), pod wall
weight (PWW), seed weight per pod (SWP), seed number per pod (SNP) and single
seed weight (SSW) of one-coil and two-coil spiral pods. TPW, SNP, PWW and SWP
were reduced by shading all leaves or pods, whereas SSW was not significantly
affected. The relative photosynthetic contribution of pod wall to SWP was
25.6-48.1% in three genotypes on July 15. The pod wall in one-coil spiral pods
generated a greater relative contribution to the TPW and SWP than in two-coil
spiral pods. In the last stage (July 25), the relative photosynthetic
contribution of leaves to SWP sharply decreased, whereas the relative
photosynthetic contribution of pod wall to SWP was stable in the late stage (July
20 and 25). In conclusion, the pod wall of alfalfa could carry out photosynthesis
and the pod wall played an important role in pod filling at the late growth
stage.
DOI: 10.1038/srep26586
PMCID: PMC4876384
PMID: 27210048
269. J Sci Food Agric. 2017 Feb;97(3):882-888. doi: 10.1002/jsfa.7810. Epub 2016
Jun
17.
Extraction, composition, and functional properties of dried alfalfa (Medicago

sativa L.) leaf protein.
Hojilla-Evangelista MP(1), Selling GW(1), Hatfield R(2), Digman M(2).
Author information:
(1)Plant Polymer Research Unit, National Center for Agricultural Utilization
Research (NCAUR), USDA Agricultural Research Service (ARS), 1815 N. University
St, Peoria, IL, 61604, USA.
(2)United States Dairy Forage Research Center, USDA ARS, 1925 Linden Drive West,
Madison, WI, 53706, USA.
BACKGROUND: Alfalfa is considered a potential feedstock for biofuels; co-products

with value-added uses would enhance process viability. This work evaluated dried
alfalfa leaves for protein production and describes the functional properties of
the protein.
RESULTS: Dried alfalfa leaves contained 260 g kg-1 dry basis (DB) crude protein,
with albumins being the major fraction (260 g kg-1 of total protein). Alkali
solubilization for 2 h at 50 °C, acid precipitation, dialysis, and freeze-drying
produced a protein concentrate (600 g kg-1 DB crude protein). Alfalfa leaf
protein concentrate showed moderate solubility (maximum 500 g kg-1 soluble
protein from pH 5.5 to 10), excellent emulsifying properties (activity 158-219 m2
g-1 protein, stability 17-49 min) and minimal loss of solubility during heating
at pH ≥ 7.0.
CONCLUSIONS: It is technically feasible to extract protein with desirable
emulsifying and heat stability properties from dried alfalfa leaves; however, the
dried form may not be a practical starting material for protein production, given
the difficulty of achieving high yields and high-purity protein product. © 2016
Society of Chemical Industry.
DOI: 10.1002/jsfa.7810
270. Mol Biol Rep. 2016 Aug;43(8):815-26. doi: 10.1007/s11033-016-4008-9. Epub 2016
May 19.
Molecular cloning and characterization of the MsHSP17.7 gene from Medicago sativa
L.
Li ZY(1), Long RC(1), Zhang TJ(1), Yang QC(1), Kang JM(2).
Author information:
Beijing, 100193, People's Republic of China. kangjmei@126.com.
Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial
roles in plant development and adaptation to stress, and the aim of this study is
to characterize the HSP gene in alfalfa. Here we isolated a small heat shock
protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7
contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The
amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1
(83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis
revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat
shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR
indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and
drought stress. Prokaryotic expression indicated that the salt and peroxide
tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis
plants overexpressing MsHSP17.7 exhibited increased root length of transgenic
Arabidopsis lines under salt stress compared to the wild-type line. The
malondialdehyde (MDA) levels in the transgenic lines were significantly lower
than in wild-type, although proline levels were similar between transgenic and
wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative
stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might
play a key role in response to high salinity stress.
DOI: 10.1007/s11033-016-4008-9
PMCID: PMC4947596
Gene-Silencing-Induced Changes in Carbohydrate Conformation in Relation to

Bioenergy Value and Carbohydrate Subfractions in Modeled Plant (Medicago sativa)
with Down-Regulation of HB12 and TT8 Transcription Factors.
Li X(1), Hannoufa A(2), Zhang Y(3), Yu P(4).
Author information:
Harbin 150030, China. youlixinxin@163.com.
(2)Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3,
Canada. abdelali.hannoufa@agr.gc.ca.
Harbin 150030, China. zhangyonggen@sina.com.
Drive, Saskatoon, SK S7N 5A8, Canada. peiqiang.yu@usask.ca.
Gene silencing with RNA interference (RNAi) technology may be capable of

modifying internal structure at a molecular level. This structural modification
could affect biofunctions in terms of biodegradation, biochemical metabolism, and
bioactive compound availability. The objectives of this study were to (1) Detect
gene silencing-induced changes in carbohydrate molecular structure in an alfalfa
forage (Medicago sativa spp. sativa: alfalfa) with down-regulation of genes that
encode transcription factors TT8 and HB12; (2) Determine gene silencing-induced
changes in nutrient bioutilization and bioavailability in the alfalfa forage
(Medicago sativa); and (3) Quantify the correlation between gene
silencing-induced molecular structure changes and the nutrient bioutilization and
bioavailability in animals of ruminants. The experimental treatments included: T1
= Non-transgenic and no-gene silenced alfalfa forage (code "NT"); T2 = HB12-RNAi
forage with HB12 gene down regulation (code "HB12"); T3 = TT8-RNAi forage with
TT8 gene down regulation (code "TT8"). The HB12 and TT8 gene silencing-induced
molecular structure changes were determined by non-invasive and non-destructive
advanced molecular spectroscopy in a middle infrared radiation region that
focused on structural, non-structural and total carbohydrate compounds. The
nutrient bioutilization and bioavailability of the modified forage were
determined using NRC-2001 system in terms of total digestive nutrient (TDN),
truly digestible fiber (tdNDF), non-fiber carbohydrate (tdNDF), fatty acid
(tdFA), crude protein (tdCP) and bioenergy profiles (digestible energy,
metabolizable energy, net energy) for ruminants. The carbohydrate subfractions
were evaluated using the updated CNCPS 6.0 system. The results showed that gene
silencing significantly affected tdNFC (42.3 (NT) vs. 38.7 (HB12) vs. 37.4% Dry
Matter (TT8); p = 0.016) and tdCP (20.8 (NT) vs. 19.4 (HB12) vs. 22.3% DM (TT8);
p = 0.009). The gene-silencing also affected carbohydrate CA4 (7.4 (NT) vs. 4.2
(HB12) and 4.4% carbohydrate (CHO) (TT8), p = 0.063) and CB1 fractions (5.3 (NT)
vs. 2.0 (HB12) and 2.6% CHO (TT8), p = 0.006). The correlation study showed that
the structural CHO functional group peak area intensity at ca. 1315 cm(-1) was
significantly correlated to the TDN1x (r = -0.83, p = 0.042) and the tdNFC (r =
-0.83, p = 0.042), the structural CHO functional group height intensity at ca.
1370 cm(-1) was significantly correlated to the tdNDF (r = -0.87, p = 0.025). The
A_Non-stCHO to A_StCHO ratio and A_Non-stCHO to A_CHO ratio were significantly
correlated to the tdFA (r = 0.83-0.91, p < 0.05). As to carbohydrate fractions,
both CA4 and CB1 correlated with carbohydrate spectral intensity of the H_1415
and the H_1315 (p = 0.039; p = 0.059, respectively), CB3 tended to correlate with
the H_1150, H_1100 and H_1025 (p < 0.10). In conclusion, RNAi-mediated silencing
of HB12 and TT8 modified not only inherent CHO molecular structure but also the
biofunctions. The CHO molecular structure changes induced by RNAi gene silencing
were associated with biofunctions in terms of the carbohydrate subfractions and
nutrient digestion.
DOI: 10.3390/ijms17050720
PMCID: PMC4881542

May
11.
Short communication: The effects of dry matter and length of storage on the
composition and nutritive value of alfalfa silage.
Santos MC(1), Kung L Jr(2).
Author information:
(1)Department of Animal and Food Sciences, University of Delaware, Newark 19716.
(2)Department of Animal and Food Sciences, University of Delaware, Newark 19716.
Electronic address: lksilage@udel.edu.
During the ensiling of feeds, various processes result in chemical changes that
can affect their ultimate nutritive value at feed out. The primary objective of
this study was to evaluate the effect of prolonged ensiling times on potential
changes in in vitro digestibility of neutral detergent fiber (NDF-D) of alfalfa
ensiled at about 33% [low dry matter (DM), LDM] or 45% (high DM, HDM) whole-plant
DM. Alfalfa from the same field (direct chopped or wilted) was chopped with a
conventional forage harvester set for a theoretical length of cut of 0.95 cm and
ensiled in mini silos for 45, 180, 270, and 360 d. Fresh forages and silages were
analyzed for nutrient content, fermentation end-products, and 30-h NDF-D. The pH
of the fresh forages ranged from 6.1 to 6.2 and decreased to approximately 4.7
and 4.3 in HDM and LDM silages, respectively. Production of acids and alcohols
were less in HDM compared with LDM as expected. Concentrations of soluble protein
and NH3-N also increased with time of storage as expected but soluble protein was
greater, whereas NH3-N was lower in HDM compared with LDM silage. The effect of
length of storage and DM on hemicellulose and NDF concentrations were very small,
whereas DM content at harvest tended to slightly increase the concentration of
acid detergent fiber in HDM compared with LDM up to 270 d of storage. The NDF-D
was greater in fresh forage compared with corresponding silages. However, time of
storage between 45 and 360 d had no effect on the NDF-D of alfalfa silage,
regardless of DM concentration at ensiling.

DOI: 10.3168/jds.2016-10866

May
11.
Effects of partial replacement of corn and alfalfa silage with tall fescue hay on
total-tract digestibility and lactation performance in dairy cows.
Bender RW(1), Lopes F(1), Cook DE(1), Combs DK(2).
Author information:
(1)Department of Dairy Science, University of Wisconsin-Madison, Madison 53706.
Electronic address: dkcombs@wisc.edu.
Our objective was to evaluate the effects of replacing either corn or alfalfa
silage with tall fescue hay on total-tract neutral detergent fiber (NDF)
digestibility and lactation performance in dairy cows. Twenty-four primiparous
(75±35 d in milk) and 40 multiparous (68±19 d in milk) Holstein cows were blocked
by parity and randomly assigned to 1 of 4 treatment groups in a pen equipped with
32 feeding gates to record intake by cow. Each gate was randomly assigned to 1
treatment group; thus, each cow had access to all 8 gates within the respective
treatment and cow was the experimental unit. Treatments were formulated to
replace either corn silage (CS) or alfalfa silage (AS) with tall fescue hay (TF)
as follows (DM basis): 33% AS and 67% CS (control; 33AS67CS), 60% TF and 40% AS
(60TF40AS), 60% TF and 40% CS (60TF40CS), and 33% TF and 67% CS (33TF67CS). The
experiment was a 7-wk continuous lactation trial with a 2-wk covariate period.
Milk production did not differ among treatments and averaged 40.4 kg/d. Fat yield
and concentration and protein yield and concentration did not differ among
treatments and averaged 1.58 kg/d, 3.94%, 1.28 kg/d, and 3.15%, respectively. Dry
matter intake was greater for 33AS67CS (24.5 kg/d) compared with 60TF40CS (22.1
kg/d) and 33TF67CS (22.7 kg/d), and tended to be greater than 60TF40AS (23.2
kg/d). In vivo total-tract dry matter digestibility did not differ among
treatments and averaged 66.2%. In vivo total-tract NDF digestibility was lower
for 33AS67CS (37.8%) compared with 60TF40AS (44.4%) and 33TF67CS (45.3%), and
similar to 60TF40CS (42.4%). In vivo total-tract NDF digestibility and an
estimate of in situ total-tract NDF digestibility were similar between techniques
across all treatment diets (42.3 vs. 42.6%, respectively). Inclusion of tall
fescue grass hay increased the total-tract NDF digestibility of the diet and has
the potential to replace corn silage and alfalfa silage and maintain milk
production if economically feasible based on current market prices.

DOI: 10.3168/jds.2015-10222
274. AoB Plants. 2016 Jul 11;8. pii: plw031. doi: 10.1093/aobpla/plw031. Print
2016.
Seed germination of seven desert plants and implications for vegetation

restoration.
Lai L(1), Chen L(2), Jiang L(1), Zhou J(1), Zheng Y(3), Shimizu H(4).
Author information:
(1)Key Laboratory of Resource Plants, Beijing Botanical Garden, West China
Subalpine Botanical Garden, Institute of Botany, Chinese Academy of Sciences,
Xiangshan, Beijing 100093, China.
(2)Institute of Applied Ecology, Chinese Academy of Sciences, Wenhua Road,
Shenyang 110016, China.
(3)Key Laboratory of Resource Plants, Beijing Botanical Garden, West China
Subalpine Botanical Garden, Institute of Botany, Chinese Academy of Sciences,
Xiangshan, Beijing 100093, China zhengyr@ibcas.ac.cn.
(4)National Institute for Environmental Studies, Tsukuba 305-8506, Japan.
Germination cues reflect the conditions under which a species is likely to

succeed in recruitment. Therefore, knowledge of the seed germination
characteristics of key plant species in desertified areas is essential for
restoration. The aims of this study were to evaluate the seed germination
responses of seven native species, and to explore the implications for vegetation
restoration. Seeds of seven desert species were sown in Petri dishes and
subjected to various temperature and light conditions. The seeds germinated well
at day/night temperatures of 25/15 °C and 30/20 °C but poorly at 35/25 °C. Seeds
germinated best in the dark, and final germination percentages of all species
were strongly inhibited at a photon irradiance of 1000 µmol m(-2) s(-1) Based on
these results and the environmental conditions of their natural habitat,
Agropyron cristatum and Artemisia halodendron are best adapted to shifting sand
dunes: Elymus dahuricus, Caragana korshinskii and C. microphylla for semi-fixed
sand dunes: and Medicago sativa and Melilotus suaveolen for fixed sand dunes. If
seeds are sown in early May, they will likely be buried in sand, and the
precipitation and temperature conditions will be suitable for seedling survival.
Published by Oxford University Press on behalf of the Annals of Botany Company.
DOI: 10.1093/aobpla/plw031
PMCID: PMC4940479
PMID: 27179541
275. AoB Plants. 2016 Jul 11;8. pii: plw035. doi: 10.1093/aobpla/plw035. Print
2016.
Intraspecific variation in thermal acclimation of photosynthesis across a range

of temperatures in a perennial crop.
Zaka S(1), Frak E(1), Julier B(1), Gastal F(1), Louarn G(2).
Author information:
(1)INRA, UR4 URP3F, BP6, F86600 Lusignan, France.
(2)INRA, UR4 URP3F, BP6, F86600 Lusignan, France gaetan.louarn@lusignan.inra.fr.
Interest in the thermal acclimation of photosynthesis has been stimulated by the

increasing relevance of climate change. However, little is known about
intra-specific variations in thermal acclimation and its potential for breeding.
In this article, we examined the difference in thermal acclimation between
alfalfa (Medicago sativa) cultivars originating from contrasting origins, and
sought to analyze the mechanisms in play. A series of experiments was carried out
at seven growth temperatures between 5 and 35 °C using four cultivars from
temperate and Mediterranean origin. Leaf traits, the photosynthetic rate at 25 °C
(A400 (25)), the photosynthetic rate at optimal temperature (A400 (opt)), the
thermal optimum of photosynthesis (Topt), and the photosynthetic parameters from
the Farqhuar model were determined. Irrespective of cultivar origin, a clear
shift in the temperature responses of photosynthesis was observed as a function
of growth temperature, affecting thermal optimum of photosynthesis,
photosynthetic rate at optimal temperature and photosynthetic rate at 25 °C. For
both cultivars, Topt values increased linearly in leaves grown between 5 and
35 °C. Relative homeostasis of A400 (25) and A400 (opt) was found between 10 °C
and 30 °C growth temperatures, but sharp declines were recorded at 5 and 35 °C.
This homeostasis was achieved in part through modifications to leaf nitrogen
content, which increased at extreme temperatures. Significant changes were also
recorded regarding nitrogen partitioning in the photosynthetic apparatus and in
the temperature dependence of photosynthetic parameters. The cultivars differed
only in terms of the temperature response of photosynthetic parameters, with
Mediterranean genotypes displaying a greater sensitivity of the maximum rate of
Rubisco carboxylation to elevated temperatures. It was concluded that
intra-specific variations in the temperature acclimation of photosynthesis exist
among alfalfa cultivars, but that Mediterranean genotypes presented no evidence
of superior performance at high temperatures.
DOI: 10.1093/aobpla/plw035
PMCID: PMC4940478
PMID: 27178065
276. J Agric Food Chem. 2016 Jun 1;64(21):4255-63. doi: 10.1021/acs.jafc.6b01111.

Epub
2016 May 19.
Phytotoxic Potential of Secondary Metabolites and Semisynthetic Compounds from

Endophytic Fungus Xylaria feejeensis Strain SM3e-1b Isolated from Sapium
macrocarpum.
García-Méndez MC, Macías-Ruvalcaba NA(1), Lappe-Oliveras P(2), Hernández-Ortega

S, Macías-Rubalcava ML.
Author information:
(1)Facultad de Quıımica, Departamento de Fisicoquıımica, Universidad Nacional
Autónoma de México (UNAM), Ciudad Universitaria , Coyoacán, México, D.F. 04510,
México.
(2)Instituto de Biologııa, Departamento de Botánica, Universidad Nacional
Autónoma de México (UNAM), Ciudad Universitaria , Coyoacán, México, D.F. 04510,
México.
Bioactivity-directed fractionation of the combined culture medium and mycelium

extract of the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from
Sapium macrocarpum, led to the isolation of three known natural products:
(4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-enone or coriloxine,
1; 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 2; and
2,6-dihydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione or fumiquinone B,
3. This is the first report of compound 3 being isolated from this species.
Additionally, four new derivatives of coriloxine were prepared:
(4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, 4;
6-hydroxy-5-methyl-3-(methylamino)cyclohexa-2,5- diene-1,4-dione, 5;
(4R,5R,6R)-4,5-dihydroxy-3-methoxy-5-methyl-6-(phenylamino)cyclohex-2-enone, 6;
and 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 7.
X-ray analysis allowed us to unambiguously determine the structures and absolute
configuration of semisynthetic derivatives 4, 5, and 6. The phytotoxic activity
of the three isolated natural products and the coriloxine derivatives is
reported. Germination of the seed, root growth, and oxygen uptake of the
seedlings of Trifolium pratense, Medicago sativa, Panicum miliaceum, and
Amaranthus hypochondriacus were significantly inhibited by all of the tested
compounds. In general, they were more effective inhibiting root elongation than
suppressing the germination and seedling oxygen uptake processes as shown by
their IC50 values.
DOI: 10.1021/acs.jafc.6b01111
277. Int J Phytoremediation. 2016 Oct 2;18(10):1014-21. doi:

10.1080/15226514.2016.1183570.
A field study on heavy metals phytoattenuation potential of monocropping and

intercropping of maize and/or legumes in weakly alkaline soils.
Zhu S(1), Ma X(1), Guo R(1), Ai S(1), Liu B(1), Zhang W(1), Zhang Y(1).
Author information:
(1)a Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental
Pollution, School of Life Sciences, Lanzhou University , Lanzhou , China.
The study focused on the phytoattenuation effects of monocropping and

intercropping of maize (Zea mays) and/or legumes on Cu, Zn, Pb, and Cd in weakly
alkaline soils. Nine growth stages of monocropping maize were chosen to study the
dynamic process of extraction of heavy metals. The total content of heavy metals
extracted by the aerial part of monocropped maize increased in a sigmoidal
pattern over the effective accumulative temperature. The biggest biomass, highest
extraction content, and lowest heavy metals bioaccumulation level occurred at
physiological maturity. Among the different planting patterns, including
monocropping and intercropping of maize and/or soybean (Glycine max), pea (Pisum
sativum), and alfalfa (Medicago sativa), the extraction efficiency of Cu, Zn, Pb,
and Cd varied greatly. Only intercropping of maize and soybean yielded relatively
higher extraction efficiency for the four metals with no significant difference
in the total biomass. Moreover, the heavy metals concentrations in dry biomass
from all the planting patterns in the present study were within China's national
legal thresholds for fodder use. Therefore, slightly polluted alkaline soils can
be safely used through monocropping and intercropping of maize and/or legumes for
a range of purposes. In particular, this study indicated that intercropping
improves soil ecosystems polluted by heavy metals compared with monocropping.
DOI: 10.1080/15226514.2016.1183570
278. J Appl Microbiol. 2016 Aug;121(2):380-400. doi: 10.1111/jam.13174.
Phytotoxic and antimicrobial activity of volatile and semi-volatile organic

compounds from the endophyte Hypoxylon anthochroum strain Blaci isolated from
Bursera lancifolia (Burseraceae).
Ulloa-Benítez Á(1), Medina-Romero YM(1), Sánchez-Fernández RE(1), Lappe-Oliveras

P(2), Roque-Flores G(1), Duarte Lisci G(3), Herrera Suárez T(2), Macías-Rubalcava
ML(1).
Author information:
(1)Departamento de Productos Naturales, Instituto de Química, Universidad
Nacional Autónoma de México (UNAM), Ciudad Universitaria, Delegación Coyoacán,
México, DF, México.
(2)Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma
de México (UNAM), Ciudad Universitaria, Delegación Coyoacán, México, DF, México.
(3)Facultad de Química, Unidad de Servicios de Apoyo a la Investigación,
Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Delegación
Coyoacán, México, DF, México.
AIMS: To evaluate the phytotoxic, antifungal and antioomycete activity; and,

determine the chemical composition of the volatile organic compounds (VOCs) and
semi-volatile metabolites produced by the endophyte Hypoxylon anthochroum strain
Blaci isolated from Bursera lancifolia.
METHODS AND RESULTS: Based on its macro- and micro-morphological features, the
strain Blaci was identified as Nodulisporium sp.; partial analysis of its
ITS1-5.8-ITS2 ribosomal gene sequence revealed the identity of the teleomorphic
stage of the fungus as H. anthochroum. Phytotoxic and antimicrobial activities of
VOCs, and culture medium and mycelium organic extracts from H. anthochroum Blaci
were determined by simple and multiple antagonism bioassays, and gas phase and
agar dilution bioassays respectively. The volatile and semi-volatile metabolites
were identified by gas chromatography-mass spectrometry. VOCs from a 5-day
H. anthochroum strain Blaci culture caused the inhibition of seed germination,
root elongation and seedling respiration on Amaranthus hypochondriacus, Panicum
miliaceum, Trifolium pratense and Medicago sativa. In addition, extracts,
phenylethyl alcohol and eucalyptol main compounds present in the VOCs and extract
displayed a high phytotoxic activity, inhibiting the three physiological
processes on the four test plants in a concentration-dependent manner.
CONCLUSIONS: The results revealed that H. anthochroum strain Blaci produces a
mixture of VOCs. These VOCs showed a strong phytotoxic activity on seed
germination, root elongation, and seedling respiration of four plants and
slightly affected the growth of phytopathogenic fungi and oomycetes. Also, the
culture medium and mycelium extracts of H. anthochroum showed a high phytotoxic
activity on the four test plants and, generally, the culture medium extract was
more phytotoxic than the mycelium extracts.
SIGNIFICANCE AND IMPACT OF THE STUDY: This work firstly reports the phytotoxic
activity of volatile and semi-volatile compounds produced by the endophyte
H. anthochroum strain Blaci on seed germination, root elongation, and seedling
respiration of four different plants; consequently, these compounds could be
useful in biocontrol of weeds and plant pathogens.
Journal of Applied Microbiology © 2016 The Society for Applied Microbiology.
DOI: 10.1111/jam.13174
279. Pestic Biochem Physiol. 2016 Jun;130:17-21. doi: 10.1016/j.pestbp.2015.12.005.

Epub 2015 Dec 18.
Antioxidant responses in soybean and alfalfa plants grown in DDTs contaminated

soils: Useful variables for selecting plants for soil phytoremediation?
Mitton FM(1), Ribas Ferreira JL(2), Gonzalez M(3), Miglioranza KSB(4), Monserrat
JM(2).
Author information:
(1)Laboratorio de Ecotoxicología y Contaminación Ambiental, Instituto de
Investigaciones, Marinas y Costeras (IIMyC), Facultad de Ciencias Exactas y
Naturales, Universidad Nacional de Mar del Plata (UNMdP) - Consejo Nacional de
Investigaciones, Científicas y Técnicas (CONICET), Dean Funes 3350, Mar del Plata
7600, Argentina. Electronic address: fmmitton@mdp.edu.ar.
(2)Universidade Federal de Rio Grande - FURG, Instituto de Ciências Biológicas
(ICB), Rio Grande do Sul, Brazil.
7600, Argentina.
7600, Argentina. Electronic address: kmiglior@mdp.edu.ar.
Phytoremediation is a low-cost alternative technology based on the use of plants

to remove pollutants from the environment. Persistent organic pollutants such as
DDTs with a long half-life in soils are attractive candidates for remediation.
This study aimed to determine the potential of antioxidant response use in the
evaluation of plants' tolerance for selecting species in phytoremediation
purposes. Alfalfa and soybean plants were grown in DDT contaminated soils. After
60days, growth, protein content, antioxidant capacity, GST activity,
concentration of proteic and non-proteic thiol groups, chlorophyll content and
carotenoid content were measured in plant tissues. Results showed no effect on
alfalfa or soybean photosynthetic pigments but different responses in the protein
content, antioxidant capacity, GST activity and thiol groups on roots, stems and
leaves, indicating that DDTs affected both species. Soybean showed higher
susceptibility than alfalfa plants due to the lower antioxidant capacity and GST
activity in leaves, in spite of having the lowest DDT accumulation. This study
provides new insights into the role of oxidative stress as an important component
of the plant's response to DDT exposure.
DOI: 10.1016/j.pestbp.2015.12.005
280. Anim Sci J. 2017 Jan;88(1):99-106. doi: 10.1111/asj.12599. Epub 2016 May 5.
Effects of different source additives and wilt conditions on the pH value,

aerobic stability, and carbohydrate and protein fractions of alfalfa silage.
Tao L(1), Zhou H(2), Zhang N(1), Si B(1), Tu Y(1), Ma T(1), Diao Q(1).
Author information:
(1)Key Laboratory of Feed Biotechnology, Ministry of Agriculture/Feed Research
Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
China Agricultural University, Beijing, China.
To improve the silage quality and reduce the silage additive cost, the present
experiment was designed to evaluate the potential of applying the fermented juice
of epiphytic lactic acid bacteria (FJLB) as an additive in alfalfa silage. The
effects of FJLB on the fermentation quality, carbohydrate and protein fractions,
and aerobic stability of alfalfa silage wilted under five different conditions
were investigated and compared with commercial lactic acid bacteria (CLAB) and
the control. The FJLB application decreased the pH value, the volatile fatty
acids and non-protein nitrogen content, and the loss of sugar by 9.9%, 22.9%,
19.6% and 9.6%, respectively; it increased the lactic acid concentration by 29.5%
and the aerobic stability by 17 h in comparison to the control. The FJLB
application also decreased the pH value (4.44 vs. 4.66) and volatile fatty acid
content (38.32 vs. 44.82) and increased the lactic acid concentration (68.99 vs.
63.29) in comparison to the CLAB-treated silage. However, the FJLB treatment had
lower aerobic stability (254 h vs. 274 h) than the CLAB treatment. The FJLB
application improved silage quality in comparison to the control; in addition,
its effect as a fermentation stimulant may be comparable to or even better than
CLAB.
DOI: 10.1111/asj.12599
281. Toxicol Rep. 2016 May 5;3:473-480. doi: 10.1016/j.toxrep.2016.05.003.

eCollection
2016.
Evaluation of toxicity of biorational insecticides against larvae of the alfalfa

weevil.
Reddy GVP(1), Antwi FB(1), Shrestha G(1), Kuriwada T(2).
Author information:
(1)Montana State University, Western Triangle Agricultural Research Center, 9546
Old Shelby Rd., P.O. Box 656, Conrad, MT 59425, USA.
(2)Kagoshima University, Faculty of Education, Korimoto 1-20-6, Kagoshima,
890-0065, Japan.
The alfalfa weevil, Hypera postica (Coleoptera: Curculionidae), is a major pest

of alfalfa Medicago sativa L. (Fabaceae). While H. postica usually causes the
most damage before the first cutting, in summer of 2015 damaging levels of the
pest persisted in Montana well after the first harvest of alfalfa. Although
conventional insecticides can control H. postica, these chemicals have adverse
effects on non-target organisms including pollinators and natural enemy insects.
In this context, use of biorational insecticides would be the best alternative
options, as they are known to pose less risk to non-target organisms. We
therefore examined the six commercially available biorational insecticides
against H. postica under laboratory condition: Mycotrol® ESO (Beauveria bassiana
GHA), Aza-Direct® (Azadirachtin), Met52® EC (Metarhizium brunneum F52), Xpectro
OD® (B. bassiana GHA + pyrethrins), Xpulse OD® (B. bassiana GHA + Azadirachtin)
and Entrust WP® (spinosad 80%). Concentrations of 0.1, 0.5, 1.0, and 2.0 times
the lowest labelled rates were tested for all products. However, in the case of
Entrust WP, additional concentrations of 0.001 and 0.01 times the lowest label
rate were also assessed. Mortality rates were determined at 1-9 days post
treatment. Based on lethal concentrations and relative potencies, this study
clearly showed that Entrust was the most effective, causing 100% mortality within
3 days after treatment among all the tested materials. With regard to other
biorational, Xpectro was the second most effective insecticide followed by
Xpulse, Aza-Direct, Met52, and Mycotrol. Our results strongly suggested that
these biorational insecticides could potentially be applied for H. postica
control.
DOI: 10.1016/j.toxrep.2016.05.003
PMCID: PMC5615908
PMID: 28959570
282. J Environ Manage. 2016 Jul 15;177:169-76. doi: 10.1016/j.jenvman.2016.04.030.

Epub 2016 Apr 18.
Heated blends of phosphate waste: Microstructure characterization, effects of

processing factors and use as a phosphorus source for alfalfa growth.
Loutou M(1), Hajjaji M(2), Mansori M(3), Favotto C(4), Hakkou R(3).
Author information:
(1)LPCME, URAC20, Université Cadi Ayyad, Faculté des Sciences Semlalia, Bd.
Prince My Abdellah, B.P. 2390, 40001 Marrakech, Morocco; LCME, Université Cadi
Ayyad, Faculté des Sciences et Techniques, Av. A. Khattabi, B.P. 549, 40000
Marrakech, Morocco; IM2NP, Institut des Matériaux Microélectronique Nanosciences
de Provence, Université du Sud Toulon-Var, Bât. R e B.P. 20132, 83 957 La Garde
Cedex, France.
(2)LPCME, URAC20, Université Cadi Ayyad, Faculté des Sciences Semlalia, Bd.
Prince My Abdellah, B.P. 2390, 40001 Marrakech, Morocco. Electronic address:
hajjaji@uca.ma.
(3)LCME, Université Cadi Ayyad, Faculté des Sciences et Techniques, Av. A.
Khattabi, B.P. 549, 40000 Marrakech, Morocco.
(4)IM2NP, Institut des Matériaux Microélectronique Nanosciences de Provence,
Université du Sud Toulon-Var, Bât. R e B.P. 20132, 83 957 La Garde Cedex, France.
Microstructure of expandable lightweight aggregates (LWAs), which was composed of

phosphate waste (PW), cement kiln dust (CKD) and raw clay (RC) was investigated,
and the effects of processing factors (temperature, waste content, soaking time)
on their physical properties were quantified by using response surface
methodology (RSM). The potential use of LWAs as a phosphorus source was assessed
through the use of seeds of alfalfa. It was found that the main minerals of the
waste, namely carbonates and fluorapatite, were involved in the formation of
labradorite/anorthite and melt respectively. Stability of mullite- the main
constituent of CKD- was sensitive to the melt content. The assemblage of the
identified phases was discussed based on the CaO-SiO2-Al2O3 phase diagram. The
results of RSM showed that the change of compressive strength, firing shrinkage
and water absorption of LWAs versus processing factors was well described with a
polynomial model and the weights of the effects of the factors increased in the
following order: sintering temperature > waste content (in the case of
PW-RC) > soaking time. On the other hand, it was found that due to the release of
phosphorus by soil-embedded pellets, the growth of alfalfa plants improved, and
the rate enhanced in this order: PW-RC > PW-CKD > PW-CKD-RC. The absorbed
quantity of phosphorus (0.12%) was still lower than the common uptake amount.
DOI: 10.1016/j.jenvman.2016.04.030
283. Ecotoxicol Environ Saf. 2016 Aug;130:103-12. doi:

10.1016/j.ecoenv.2016.04.009.
Epub 2016 Apr 18.
Identification of transcriptome involved in atrazine detoxification and

degradation in alfalfa (Medicago sativa) exposed to realistic environmental
contamination.
Zhang JJ(1), Lu YC(1), Zhang SH(2), Lu FF(1), Yang H(3).
Author information:
hongyang@njau.edu.cn.
Plants are constantly exposed to a variety of toxic compounds (or xenobiotics)

such as pesticides (or herbicides). Atrazine (ATZ) as herbicide has become one of
the environmental contaminants due to its intensive use during crop production.
Plants have evolved strategies to cope with the adverse impact of ATZ. However,
the mechanism for ATZ degradation and detoxification in plants is largely
unknown. Here we employed a global RNA-sequencing (RNA-Seq) strategy to dissect
transcriptome variation in alfalfa (Medicago sativa) exposed to ATZ. Four
libraries were constructed including Root-ATZ (root control, ATZ-free),
Shoot-ATZ, Root+ATZ (root treated with ATZ) and Shoot+ATZ. Hierarchical
clustering was performed to display the expression patterns for all
differentially expressed genes (DEGs) under ATZ exposure. Transcripts involved in
ATZ detoxification, stress responses (e.g. oxidation and reduction, conjugation
and hydrolytic reactions), and regulations of cysteine biosynthesis were
identified. Several genes encoding glycosyltransferases, glutathione
S-transferases or ABC transporters were up-regulated notably. Also, many other
genes involved in oxidation-reduction, conjugation, and hydrolysis for herbicide
degradation were differentially expressed. These results suggest that ATZ in
alfalfa can be detoxified or degraded through different pathways. The expression
patterns of some DEGs by high-throughput sequencing were well confirmed by
qRT-PCR. Our results not only highlight the transcriptional complexity in alfalfa
exposed to ATZ but represent a major improvement for analyzing transcriptional
changes on a large scale as well.
DOI: 10.1016/j.ecoenv.2016.04.009
284. Chem Biodivers. 2016 May;13(5):549-54. doi: 10.1002/cbdv.201500175.
A Potent Phytotoxic Substance in Aglaia odorata Lour.
Kato-Noguchi H(1), Suzuki M(2), Noguchi K(2), Ohno O(3), Suenaga K(3),
Laosinwattana C(4).
Author information:
University, Miki, Kagawa, 761-0795, Japan. hisashi@ag.kagawa-u.ac.jp.
University, Miki, Kagawa, 761-0795, Japan.
(3)Department of Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku, Yokohama, 223-8522, Japan.
(4)Department of Plant Production Technology, Faculty of Agricultural Technology,
King Mongkut's Institute of Technology Ladkrabang, Bangkok, 10520, Thailand.
Aglaia odorata Lour. (Meliaceae) was found to have very strong allelopathic
activity and a bioherbicide PORGANIC(™) was developed from its leaf extracts.
However, the phytotoxic substances causing the strong allelopathic activity of
the plants have not yet been determined. Therefore, we investigated allelopathic
properties and phytotoxic substances in A. odorata. Aqueous EtOH extracts of
A. odorata leaves inhibited root and shoot growth of garden cress (Lepidum
sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), timothy (Phleum
pratense), ryegrass (Lolium multiflorum), and Echinochloa crus-galli with the
extract concentration-dependent manner. The extracts were then purified and a
major phytotoxic substance with allelopathic activity was isolated and identified
by spectral data as rocaglaol. Rocaglaol inhibited the growth of garden cress and
E. crus-galli at concentrations > 0.3 and 0.03 μm, respectively. The
concentrations required for 50% inhibition ranged from 0.09 to 2.5 μm. The
inhibitory activity of rocaglaol on the weed species, E. crus-galli, was much
greater than that of abscisic acid. These results suggest that rocaglaol may be a
major contributor to the allelopathic effect of A. odorata and bioherbicide
PORGANIC(™) .
© 2016 Verlag Helvetica Chimica Acta AG, Zürich.
DOI: 10.1002/cbdv.201500175
285. New Phytol. 2016 May;210(3):1022-32. doi: 10.1111/nph.13838. Epub 2016 Jan 27.
Carbon and phosphorus exchange may enable cooperation between an arbuscular

mycorrhizal fungus and a phosphate-solubilizing bacterium.
Zhang L(1), Xu M(2), Liu Y(3), Zhang F(1), Hodge A(4), Feng G(1).
Author information:
(1)College of Resources and Environmental Sciences, Research Center for
Resources, the Environment and Food Safety, China Agricultural University,
(2)Institute of Agricultural Resources and Regional Planning, Chinese Academy of
Agricultural Sciences, Beijing, 100081, China.
(3)College of Life Sciences, Zhejiang University, Hangzhou, 310058, China.
(4)Department of Biology, University of York, Wentworth Way, York, YO10 5DD, UK.
Arbuscular mycorrhizal fungi (AMF) transfer plant photosynthate underground which

can stimulate soil microbial growth. In this study, we examined whether there was
a potential link between carbon (C) release from an AMF and phosphorus (P)
availability via a phosphate-solubilizing bacterium (PSB). We investigated the
outcome of the interaction between the AMF and the PSB by conducting a microcosm
and two Petri plate experiments. An in vitro culture experiment was also
conducted to determine the direct impact of AMF hyphal exudates on growth of the
PSB. The AMF released substantial C to the environment, triggering PSB growth and
activity. In return, the PSB enhanced mineralization of organic P, increasing P
availability for the AMF. When soil available P was low, the PSB competed with
the AMF for P, and its activity was not stimulated by the fungus. When additional
P was added to increase soil available P, the PSB enhanced AMF hyphal growth, and
PSB activity was also stimulated by the fungus. Our results suggest that an AMF
and a free-living PSB interacted to the benefit of each other by providing the C
or P that the other microorganism required, but these interactions depended upon
background P availability.
© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
DOI: 10.1111/nph.13838
286. Zhongguo Zhong Yao Za Zhi. 2015 Nov;40(21):4155-9.
[Allelopathic effects of extracts from tuberous roots of Aconitum carmichaeli on

three pasture grasses].
Jiao YJ, Wang YQ, Yuan L.
The tuberous roots of Aconitum carmichaeli are largely used in traditional

Chinese medicine and widely grown in Jiangyou, Sichuan, China. During the growth
process, this medicinal plant releases a large amount of allelochemicals into
soil, which retard the growth and development of near and late crops. Therefore,
a pure culture experiment was thus carried out by seed soaking to study the
allelopathic effects of extracts from tuberous roots of A. carmichaeli (ETR) on
the seed germination and young seedling growth of Lolium perenne, Trifolium
repens, and Medicago sativa, the late pasture grasses after cultivation of A.
carmichaeli. The results showed that three pasture grasses varied significantly
in seed germination and young seedling growth in response to ETR concentrations.
Seed germination of M. sativa was stimulated by low ERT concentration (0.01 x
g(-1)), while all of pasture grass seeds germinated poorly in solution with 1.00
g x L(-1). Seed soaking with 1.00 g x L(-1) also inhibited significantly the
growth of pasture young seedlings, with M. sativa showing the highest seedling
height reduction of 42.05% in seeding height, followed by T. repens (40.21%) and
L. perenne with about 11%. Cultivation of L. perenne could thus be beneficial to
increase whole land productivity in A. carmichaeli-pasture grass cropping
systems. In addition, hydrolysis of protein, starch, and inositol phosphates was
blocked and free amino acids, soluble sugars and phosphorus were decreased in
seeds by seed soaking with ETR, which could be one of the reason for the
inhibition of seed germination. There was a significant reduction in root vigor,
nitrate reductase, and chlorophyll after the seed treatment with ETR, indicating
the suppression of nutrient uptake, nitrate assimilation, and photosynthesis by
allelopathic chemicals in ETR, which could lead to the slow growth rate of
pasture grass seedlings.
287. Arch Virol. 2016 Jul;161(7):2029-32. doi: 10.1007/s00705-016-2854-3. Epub 2016

Apr 11.
Complete genome sequence of a new enamovirus from Argentina infecting alfalfa

plants showing dwarfism symptoms.
Bejerman N(1)(2), Giolitti F(3), Trucco V(3), de Breuil S(3)(4), Dietzgen RG(5),
Lenardon S(3).
Author information:
(1)IPAVE-CIAP-INTA, Av. 11 de Septiembre 4755, Córdoba, 5020, Argentina.
nicobejerman@gmail.com.
(2)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy
Cruz 2290 (1425 FBQ) CABA, Buenos Aires, Argentina. nicobejerman@gmail.com.
(3)IPAVE-CIAP-INTA, Av. 11 de Septiembre 4755, Córdoba, 5020, Argentina.
(4)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy
Cruz 2290 (1425 FBQ) CABA, Buenos Aires, Argentina.
(5)Queensland Alliance for Agriculture and Food Innovation, The University of
Queensland, St Lucia, QLD, 4072, Australia.
Alfalfa dwarf disease, probably caused by synergistic interactions of mixed virus

infections, is a major and emergent disease that threatens alfalfa production in
Argentina. Deep sequencing of diseased alfalfa plant samples from the central
region of Argentina resulted in the identification of a new virus genome
resembling enamoviruses in sequence and genome structure. Phylogenetic analysis
suggests that it is a new member of the genus Enamovirus, family Luteoviridae.
The virus is tentatively named "alfalfa enamovirus 1" (AEV-1). The availability
of the AEV-1 genome sequence will make it possible to assess the genetic
variability of this virus and to construct an infectious clone to investigate its
role in alfalfa dwarfism disease.
DOI: 10.1007/s00705-016-2854-3
2016.
Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago

sativa and Medicago truncatula in Response to Salt Stress.
Long R(1), Li M(2), Zhang T(1), Kang J(1), Sun Y(2), Cong L(1), Gao Y(1), Liu
F(3), Yang Q(1).
Author information:
(1)Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
Beijing, China.
(2)Department of Grass and Forage Science, College of Animal Science and
Technology, China Agricultural University Beijing, China.
(3)Institute of Pratacultural Science, Heilongjiang Academy of Agricultural
Sciences Haerbin, China.
Salt stress is an important abiotic stress that causes decreased crop yields.
Root growth and plant activities are affected by salt stress through the actions
of specific genes that help roots adapt to adverse environmental conditions. For
a more comprehensive understanding of proteins affected by salinity, we used
two-dimensional gel electrophoresis and mass spectrometry to characterize the
proteome-level changes associated with salt stress response in Medicago sativa
cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological
and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than
Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly
affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The
tandem mass spectrometry analysis of the differentially accumulated proteins
resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong
A17 roots, respectively. Function analyses indicated molecule binding and
catalytic activity were the two primary functional categories. These proteins
have known functions in various molecular processes, including defense against
oxidative stress, metabolism, photosynthesis, protein synthesis and processing,
and signal transduction. The transcript levels of four identified proteins were
determined by quantitative reverse transcription polymerase chain reaction. Our
results indicate that some of the identified proteins may play key roles in salt
stress tolerance.
DOI: 10.3389/fpls.2016.00424
PMCID: PMC4814493
PMID: 27066057
289. J Anim Sci. 2016 Mar;94(3):1141-8. doi: 10.2527/jas.2015-0111.
Effect of different alfalfa hay levels on growth performance, rumen fermentation,

and structural growth of Holstein dairy calves.
Nemati M, Amanlou H, Khorvash M, Mirzaei M, Moshiri B, Ghaffari MH.
Introducing forage in dairy calf diets during the preweaning period stimulates
development of rumen capacity and function and gut wall integrity. It was
hypothesized that calves fed alfalfa hay (AH) at greater levels (up to 25% of DM)
would result in an increase in starter diet intake and growth performance. The
objective of this study was to determine the effects of increasing levels of
supplemental AH on growth performance, rumen fermentation, blood metabolites, and
feeding behavior in intact male dairy calves. For this purpose, 45 Holstein male
calves (3 d of age and 41 ± 2.5 kg of BW) were randomly assigned to 1 of the
following 3 starter diets differing in their forage level: 1) diet without AH
(control), 2) diet with 12.5% AH on a DM basis, and 3) diet with 25% AH on a DM
basis. Supplementation of AH in the starter diets linearly increased total DMI
and ADG during the postweaning ( < 0.01) and overall ( = 0.02) periods. Alfalfa
hay supplementation linearly increased ( < 0.01) ruminal pH on d 35 and 70 of the
study. The concentrations of total VFA ( = 0.85), acetate ( = 0.74), propionate (
= 0.93), and butyrate ( = 0.91) were not affected by treatments. Furthermore,
forage supplementation tended (linearly, = 0.08) to increase the acetate to
propionate ratio compared with the control calves. Blood β-hydroxybutyrate (BHBA)
concentrations in the control calves were less ( = 0.03) than in the AH-fed
calves on d 70 of the study. Among all the measured variables of skeletal growth,
supplementation of AH in the starter diets linearly increased heart girth ( =
0.05) and abdominal girth ( < 0.01) on d 70 of the study. These results indicate
that the feed intake, feeding behavior, rumen fermentation parameters, and blood
BHBA concentration may be affected by rations differing in AH level so that
providing calves with 25% AH can improve calf performance.
DOI: 10.2527/jas.2015-0111
290. J Anim Sci. 2016 Feb;94(2):845-57. doi: 10.2527/jas.2015-9790.
Interaction between a tannin-containing legume and endophyte-infected tall fescue

seed on lambs' feeding behavior and physiology.
Villalba JJ, Spackman C, Goff BM, Klotz JL, Griggs T, MacAdam JW.
It was hypothesized that a tannin-rich legume such as sainfoin attenuates the

negative postingestive effects of ergot alkaloids in tall fescue. Thirty-two
4-mo-old lambs were individually penned and randomly assigned to a 2 × 2
factorial arrangement with 2 legume species, sainfoin (SAN; 2.9% condensed
tannins) or cicer milkvetch (CIC; without tannins) and a mixed ration containing
tall fescue seed (50:30:20 seed:beet pulp:alfalfa) with 2 levels of endophyte
infection (endophyte-infected tall fescue seed [E+; 3,150 ug/L ergovaline] or
endophyte-free tall fescue seed [E-]). For a 10-d baseline period, half of the
lambs were fed SAN and half were fed CIC and all lambs had ad libitum amounts of
E-. In an ensuing 10-d experimental period, the protocol was the same except half
of the lambs fed SAN or CIC received E+ instead of E-. Subsequently, all lambs
could choose between their respective legume and seed-containing ration and
between E+ and E-. Finally, an in vitro radial diffusion assay was conducted to
determine whether tannins isolated from SAN would bind to alkaloids isolated from
E+. All groups consumed similar amounts of E- during baseline period ( > 0.10),
but lambs ate more E- than E+ during the experimental period ( < 0.05) and lambs
offered SAN ate more E+ than lambs offered CIC ( < 0.05). Groups fed E- during
the baseline and experimental periods had similar rectal temperatures ( > 0.10),
but lambs fed E+ had lower rectal temperatures per gram of feed ingested when
supplemented with SAN than with CIC ( < 0.05). Lambs fed E+ had greater
concentrations of hemoglobin and more red blood cells than lambs fed E- ( <
0.05), but plasmatic concentrations of cortisol and prolactin did not differ
among treatments ( > 0.10). All lambs preferred their treatment ration over their
treatment legume, but lambs in the SAN and E+ treatment ate more legume + ration
than lambs in the CIC and E+ (CIC-E+; < 0.05) treatment. All lambs preferred E-
over E+, but lambs in the CIC-E+ treatment ate the least amount of E+ ( < 0.05).
Binding of isolated SAN tannins to protein was reduced by the E+ isolate ( <
0.05), suggesting a tannin-alkaloid complexation but only from tannins extracted
from SAN fed early in the experimental period. In summary, SAN supplementation
increased intake of and preference for E+ and reduced rectal temperatures
relative to CIC supplementation. Our results suggest that these effects were
mediated by the condensed tannins in SAN through alkaloid inactivation, an
interaction that declined with plant maturity.
DOI: 10.2527/jas.2015-9790
291. J Food Prot. 2016 Apr;79(4):574-81. doi: 10.4315/0362-028X.JFP-15-440.
Adaptation and Validation of E-Probe Diagnostic Nucleic Acid Analysis for

Detection of Escherichia coli O157:H7 in Metagenomic Data from Complex Food
Matrices.
Blagden T(1), Schneider W(2), Melcher U(3), Daniels J(4), Fletcher J(4).
Author information:
(1)Department of Entomology and Plant Pathology, 127 NRC, Oklahoma State
University, Stillwater, Oklahoma 74078, USA. trenna.bladgen@okstate.edu.
(2)U.S. Department of Agriculture, Agricultural Research Service, Room 139,
Building 1301, 1301 Ditto Avenue, Fort Detrick, Maryland 21702, USA.
(3)Department of Biochemistry and Molecular Biology, 246 NRC, Oklahoma State
University, Stillwater, Oklahoma 74078, USA.
(4)Department of Entomology and Plant Pathology, 127 NRC, Oklahoma State
University, Stillwater, Oklahoma 74078, USA.
The Centers for Disease Control and Prevention recently emphasized the need for
enhanced technologies to use in investigations of outbreaks of foodborne
illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA)
was adapted and validated as a tool for the rapid, effective identification and
characterization of multiple pathogens in a food matrix. In EDNA, unassembled
next generation sequencing data sets from food sample metagenomes are queried
using pathogen-specific sequences known as electronic probes (e-probes). In this
study, the query of mock sequence databases demonstrated the potential of EDNA
for the detection of foodborne pathogens. The method was then validated using
next generation sequencing data sets created by sequencing the metagenome of
alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the
negative control sample indicated the need for additional filtration of the
e-probes to enhance specificity. There was no significant difference in the
ability of an e-probe to detect the target pathogen based upon the length of the
probe set oligonucleotides. The results from the queries of the sample database
using E. coli e-probe sets were significantly different from those obtained using
random decoy probe sets and exhibited 100% precision. The results support the use
of EDNA as a rapid response methodology in foodborne outbreaks and investigations
for establishing comprehensive microbial profiles of complex food samples.
DOI: 10.4315/0362-028X.JFP-15-440
2016.
Above-Belowground Herbivore Interactions in Mixed Plant Communities Are

Influenced by Altered Precipitation Patterns.
Ryalls JM(1), Moore BD(1), Riegler M(1), Johnson SN(1).
Author information:
(1)Hawkesbury Institute for the Environment, Western Sydney University Richmond,
NSW, Australia.
Root- and shoot-feeding herbivores have the capacity to influence one another by
modifying the chemistry of the shared host plant. This can alter rates of
nutrient mineralization and uptake by neighboring plants and influence
plant-plant competition, particularly in mixtures combining grasses and legumes.
Root herbivory-induced exudation of nitrogen (N) from legume roots, for example,
may increase N acquisition by co-occurring grasses, with knock-on effects on
grassland community composition. Little is known about how climate change may
affect these interactions, but an important and timely question is how will
grass-legume mixtures respond in a future with an increasing reliance on legume N
mineralization in terrestrial ecosystems. Using a model grass-legume mixture,
this study investigated how simultaneous attack on lucerne (Medicago sativa) by
belowground weevils (Sitona discoideus) and aboveground aphids (Acyrthosiphon
pisum) affected a neighboring grass (Phalaris aquatica) when subjected to
drought, ambient, and elevated precipitation. Feeding on rhizobial nodules by
weevil larvae enhanced soil water retention under ambient and elevated
precipitation, but only when aphids were absent. While drought decreased
nodulation and root N content in lucerne, grass root and shoot chemistry were
unaffected by changes in precipitation. However, plant communities containing
weevils but not aphids showed increased grass height and N concentrations, most
likely associated with the transfer of N from weevil-attacked lucerne plants
containing more nodules and higher root N concentrations compared with
insect-free plants. Drought decreased aphid abundance by 54% but increased total
and some specific amino acid concentrations (glycine, lysine, methionine,
tyrosine, cysteine, histidine, arginine, aspartate, and glutamate), suggesting
that aphid declines were being driven by other facets of drought (e.g., reduced
phloem hydraulics). The presence of weevil larvae belowground decreased aphid
numbers by 30%, likely associated with a significant reduction in proline in
weevil-treated lucerne plants. This study demonstrates how predicted changes to
precipitation patterns and indirect interactions between herbivores can alter the
outcome of competition between N-fixing legumes and non-N-fixing grasses, with
important implications for plant community structure and productivity.
DOI: 10.3389/fpls.2016.00345
PMCID: PMC4804199
PMID: 27047522
2016.
Association Mapping for Fiber-Related Traits and Digestibility in Alfalfa

(Medicago sativa).
Wang Z(1), Qiang H(1), Zhao H(2), Xu R(3), Zhang Z(1), Gao H(1), Wang X(1), Liu
G(2), Zhang Y(3).
Author information:
(1)Institute of Animal Sciences, Chinese Academy of Agriculture Sciences Beijing,
China.
(2)Institute of Dry Farming, Hebei Academy of Agriculture and Forestry Sciences
Hengshui, China.
(3)Department of Grassland Science, China Agricultural University Beijing, China.
Association mapping is a powerful approach for exploring the molecular genetic

basis of complex quantitative traits. An alfalfa (Medicago sativa) association
panel comprised of 336 genotypes from 75 alfalfa accessions represented by four
to eight genotypes for each accession. Each genotype was genotyped using 85
simple sequence repeat (SSR) markers and phenotyped for five fiber-related traits
in four different environments. A model-based structure analysis was used to
group all genotypes into two groups. Most of the genotypes have a low relative
kinship (<0.3), suggesting population stratification not be an issue for
association analysis. Generally, the Q + K model exhibited the best performance
to eliminate the false associated positives. In total, 124 marker-trait
associations were predicted (p < 0.005). Among these, eight associations were
predicted in two environments repeatedly and 20 markers were predicted to be
associated with multiple traits. These trait-associated markers will greatly help
marker-assisted breeding programs to improve fiber-related quality traits in
alfalfa.
DOI: 10.3389/fpls.2016.00331
PMCID: PMC4797558
PMID: 27047512
294. Vet J. 2016 May;211:32-8. doi: 10.1016/j.tvjl.2016.03.004. Epub 2016 Mar 17.
Alfalfa hay induced primary photosensitization in horses.
Puschner B(1), Chen X(2), Read D(3), Affolter VK(4).
Author information:
(1)Department of Molecular Biosciences, School of Veterinary Medicine, University
of California, 1089 Veterinary Medicine Drive, Davis, CA 95616, USA. Electronic
address: bpuschner@ucdavis.edu.
(2)Department of Molecular Biosciences, School of Veterinary Medicine, University
of California, 1089 Veterinary Medicine Drive, Davis, CA 95616, USA.
(3)California Animal Health and Food Safety Laboratory System, San Bernadino
Branch, 105 W. Central Avenue, San Bernardino, CA 92408, USA.
(4)Department of Pathology, Microbiology, Immunology, School of Veterinary
Medicine, University of California, Davis, One Shields Avenue, Davis, CA 95616,
USA.
Photosensitization, also known as photodermatitis, occurs when phototoxic or

photoactive substances accumulate in the skin and interact with sunlight to
result in an often severe, crusting, itching or painful dermatitis in unpigmented
and/or lightly haired areas of the skin. Primary photosensitization, caused by
direct ingestion of photosensitizing agents, has been reported anecdotally in
horses after ingestion of alfalfa hay. Between 2004 and 2014, several large
outbreaks of primary photosensitization in horses fed primarily alfalfa hay were
investigated in California. Alfalfa hay samples were collected and carefully
examined for the presence of known photosensitizing plants and pesticide residues
but none were identified. Select hay samples were evaluated for unusual fungal
infestation and for phototoxicity assay using a specific Candida albicans assay;
results were negative. In the 2004 outbreak, a feeding study was conducted with
three horses exclusively fed alfalfa hay that was suspected to have caused the
outbreak. Two weeks after ingestion of alfalfa hay, two horses developed several
lesions in non-pigmented skin characterized as chronic ulcerative and necrotizing
dermatitis with superficial vasculitis, which was consistent with
photosensitization. In the 2014 outbreak, seven different implicated alfalfa hay
samples were analyzed for chlorophyll a and b, and pheophorbide a. These
compounds had been suspected to play a role in alfalfa-induced primary
photosensitization. The chlorophyll contents ranged from 0.90 to 2.30 mg/g in the
alfalfa hay samples, compared to 1.37 and 2.94 mg/g in locally grown alfalfa and
orchard grass hay. The pheophorbide a levels ranged from 3.36 to 89.87 µg/g in
alfalfa samples compared to 81.39 and 42.33 µg/g in control alfalfa and orchard
grass hay samples. These findings eliminate chlorophyll a, chlorophyll b, and
pheophorbide a as possible causes for alfalfa-hay induced primary
photosensitization.
DOI: 10.1016/j.tvjl.2016.03.004
295. J Dairy Sci. 2016 Jun;99(6):4427-4436. doi: 10.3168/jds.2015-10766. Epub 2016

Mar
31.
Control of Escherichia coli O157:H7 in contaminated alfalfa silage: Effects of

silage additives.
Ogunade IM(1), Kim DH(2), Jiang Y(1), Weinberg ZG(3), Jeong KC(1), Adesogan
AT(4).
Author information:
(1)Department of Animal Sciences, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville 32608.
University of Florida, Gainesville 32608; Division of Applied Life Science
(BK21Plus, Institute of Agriculture and Life Science), Gyeongsang National
University, Jinju 660-701, South Korea.
(3)Department of Food Safety and Quality, Agricultural Research Organization, The
Volcani Center, Bet Dagan, Israel 50250.
University of Florida, Gainesville 32608. Electronic address: adesogan@ufl.edu.
This study was conducted to examine if adding microbial inoculants or propionic

acid to alfalfa silages contaminated with Escherichia coli O157:H7 would inhibit
the growth of the pathogen during or after ensiling. Alfalfa forage was harvested
at the early bloom stage, wilted to a dry matter concentration of 54%, chopped to
19-mm lengths, and ensiled after treatment with one of the following: (1)
distilled water (control); (2) 1×10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and
1×10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1×10(6) cfu/g of
Lactobacillus buchneri (EC+LB); and (5) EC and 2.2g/kg of propionic acid (EC+PA).
Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, 16,
and 100d and analyzed for EC counts, pH, and organic acids. In addition, samples
from d 100 were analyzed for chemical composition, ammonia-N, counts of yeasts
and molds, and aerobic stability. Escherichia coli O157:H7 was detected in all
silages until d 7, but by d 16 it was not detected in those treated with EC+LB
and EC+LP, though it was still detected in EC and EC+PA silages. However, by d
100, the pathogen was not detected in any silage. The rate of pH decrease to 5.0
was fastest for the EC+LP silage (7d), followed by the EC+LB silage (16d).
Nevertheless, all silages had attained a pH of or less than 5.0 by d 100. The
rapid decrease in pH in EC+LP and EC+LB silages was observed due to higher
lactate and acetate concentrations, respectively, relative to the other silages
during the early fermentation phase (d 3-16). Propionic acid was only detected in
the EC+PA silage. Yeast counts were lowest in EC+LB and EC+PA silages. Subsamples
of all d-100 silages were reinoculated with 1×10(5) cfu/g of EC immediately after
silo opening. When the pathogen was subsequently enumerated after 168h of aerobic
exposure, it was not detected in silages treated with EC+PA, EC+LB, or EC+LP,
which all had pH values less than 5.0. Whereas the EC silage had a pH value of
5.4 and 2.3 log cfu/g of the pathogen. Certain bacterial inoculants can hasten
the inhibition of E. coli O157:H7 during ensiling, such as propionic acid, and
they can also prevent its growth on silage contaminated with the pathogen after
ensiling.

DOI: 10.3168/jds.2015-10766
296. Virology. 2016 Jun;493:142-53. doi: 10.1016/j.virol.2016.03.016. Epub 2016 Mar

31.
Molecular characterization and prevalence of two capulaviruses: Alfalfa leaf curl

virus from France and Euphorbia caput-medusae latent virus from South Africa.
Bernardo P(1), Muhire B(2), François S(3), Deshoux M(1), Hartnady P(2), Farkas
K(4), Kraberger S(4), Filloux D(1), Fernandez E(1), Galzi S(1), Ferdinand R(1),
Granier M(1), Marais A(5), Monge Blasco P(6), Candresse T(5), Escriu F(7),
Varsani A(8), Harkins GW(9), Martin DP(2), Roumagnac P(10).
Author information:
(1)CIRAD-INRA-SupAgro, UMR BGPI, Campus International de Montferrier-Baillarguet,
Montpellier Cedex-5, France.
(2)Computational Biology Group, Institute of Infectious Disease and Molecular
Medicine, Faculty of Health Sciences, University of Cape Town, Observatory, South
Africa.
(3)CIRAD-INRA-SupAgro, UMR BGPI, Campus International de Montferrier-Baillarguet,
Montpellier Cedex-5, France; INRA, UMR 1333, DGIMI, Montpellier, France;
CNRS-IRD-UM1-UM2, UMR 5290, MIVEGEC, Avenue Agropolis, Montpellier, France.
(4)School of Biological Sciences and Biomolecular Interaction Centre, University
of Canterbury, Private Bag 4800, Christchurch, New Zealand.
(5)INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon Cedex,
France; Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie,
Villenave d'Ornon Cedex, France.
(6)Unidad de Sanidad Vegetal, Centro de Investigacion y Tecnologıa
Agroalimentaria de Aragon (CITA), Av. Montañana 930, 50059 Zaragoza, Spain.
(7)Unidad de Sanidad Vegetal, Centro de Investigacion y Tecnologıa
Agroalimentaria de Aragon (CITA), Av. Montañana 930, 50059 Zaragoza, Spain;
Unidad de Sanidad Vegetal, Instituto Agroalimentario de Aragón IA2 (CITA -
Universidad de Zaragoza), Av. Montañana 930, 50059 Zaragoza, Spain.
(8)School of Biological Sciences and Biomolecular Interaction Centre, University
of Canterbury, Private Bag 4800, Christchurch, New Zealand; Department of Plant
Pathology and Emerging Pathogens Institute, University of Florida, Gainesville,
USA; Structural Biology Research Unit, Department of Clinical Laboratory
Sciences, University of Cape Town, Observatory, South Africa.
(9)South African National Bioinformatics Institute, MRC Unit for Bioinformatics
Capacity Development, University of the Western Cape, Cape Town, South Africa.
(10)CIRAD-INRA-SupAgro, UMR BGPI, Campus International de
Montferrier-Baillarguet, Montpellier Cedex-5, France. Electronic address:
philippe.roumagnac@cirad.fr.
Little is known about the prevalence, diversity, evolutionary processes, genomic

structures and population dynamics of viruses in the divergent geminivirus
lineage known as the capulaviruses. We determined and analyzed full genome
sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf
curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV
isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia
caput-medusae, severe alfalfa disease symptoms were associated with ALCV in
southern France. The prevalence of both viruses exceeded 10% in their respective
hosts. Besides using patterns of detectable negative selection to identify ORFs
that are probably functionally expressed, we show that ALCV and EcmLV both
display evidence of inter-species recombination and biologically functional
genomic secondary structures. Finally, we show that whereas the EcmLV populations
likely experience restricted geographical dispersion, ALCV is probably freely
moving across the French Mediterranean region.
DOI: 10.1016/j.virol.2016.03.016
297. Sci Total Environ. 2016 Jul 1;557-558:652-64. doi:

10.1016/j.scitotenv.2016.03.054. Epub 2016 Mar 31.
Stress-related phenomena and detoxification mechanisms induced by common

pharmaceuticals in alfalfa (Medicago sativa L.) plants.
Christou A(1), Antoniou C(2), Christodoulou C(2), Hapeshi E(3), Stavrou I(3),
Michael C(3), Fatta-Kassinos D(4), Fotopoulos V(5).
Author information:
(1)Agricultural Research Institute, P.O. Box 22016, 1516 Nicosia, Cyprus.
University of Technology, 3603 Lemesos, Cyprus.
(3)NIREAS-International Water Research Center, University of Cyprus, P.O. Box
20537, 1678 Nicosia, Cyprus.
(4)Department of Civil and Environmental Engineering, University of Cyprus, P.O.
Box 20537, 1678 Nicosia, Cyprus; NIREAS-International Water Research Center,
University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus.
University of Technology, 3603 Lemesos, Cyprus. Electronic address:
vassilis.fotopoulos@cut.ac.cy.
Pharmaceutically active compounds (PhACs) have been recently shown to exert

phytotoxic effects. The present study explores the uptake, systemic
translocation, and abiotic stress responses and detoxification mechanisms induced
by the exposure of alfalfa plants grown in sand under greenhouse conditions to
four common, individually applied PhACs (10μgL(-1)) (diclofenac,
sulfamethoxazole, trimethoprim, 17a-ethinylestradiol) and their mixture. Stress
physiology markers (lipid peroxidation, proline, H2O2 and NO content, antioxidant
activity assays) and gene expression levels of key plant detoxification
components (including glutathione S-transferases, GST7, GST17; superoxide
dismutases, CuZnSOD, FeSOD; proton pump, H(+)-ATP, and cytochrome c oxidase,
CytcOx), were evaluated. PhACs were detected in significantly higher
concentrations in roots compared with leaves. Stress related effects, manifested
via membrane lipid peroxidation and oxidative burst, were local (roots) rather
than systemic (leaves), and exacerbated when the tested PhACs were applied in
mixture. Systemic accumulation of H2O2 in leaves suggests its involvement in
signal transduction and detoxification responses. Increased antioxidant enzymatic
activities, as well as upregulated transcript levels of GST7, GST17, H(+)-ATPase
and CytcOx, propose their role in the detoxification of the selected PhACs in
plants. The current findings provide novel biochemical and molecular evidence
highlighting the studied PhACs as an emerging abiotic stress factor, and point
the need for further research on wastewater flows under natural agricultural
environments.
DOI: 10.1016/j.scitotenv.2016.03.054
298. Microbiology. 2016 Jun;162(6):979-88. doi: 10.1099/mic.0.000284. Epub 2016 Mar

24.
Expanding the regulatory network that controls nitrogen fixation in Sinorhizobium

meliloti: elucidating the role of the two-component system hFixL-FxkR.
Reyes-González A(1), Talbi C(2), Rodríguez S(2), Rivera P(2), Zamorano-Sánchez

D(3), Girard L(2).
Author information:
(1)1 Programa de Dinámica Genómica, Centro de Ciencias Genómicas, Universidad
Nacional Autónoma de México, Cuernavaca, Morelos, Mexico 2 Instituto de
Investigaciones Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos, Mexico.
(2)1 Programa de Dinámica Genómica, Centro de Ciencias Genómicas, Universidad
Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
(3)3 Department of Microbiology and Environmental Toxicology, University of
California, Santa Cruz, USA.
In Sinorhizobium meliloti, nitrogen fixation is regulated in response to oxygen

concentration through the FixL-FixJ two-component system (TCS). Besides this
conserved TCS, the field isolate SM11 also encodes the hFixL-FxkR TCS, which is
responsible for the microoxic response in Rhizobium etli. Through genetic and
physiological assays, we evaluated the role of the hFixL-FxkR TCS in S. meliloti
SM11. Our results revealed that this regulatory system activates the expression
of a fixKf orthologue (fixKa), in response to low oxygen concentration. Null
mutations in either hFixL or FxkR promote upregulation of fixK1, a direct target
of FixJ. Furthermore, the absence of this TCS translates into higher nitrogen
fixation values as well as higher expression of fixN1 in nodules. Individual
mutations in each of the fixK-like regulators encoded in the S. meliloti SM11
genome do not completely restrict fixN1 or fixN2 expression, pointing towards
redundancy among these regulators. Both copies of fixN are necessary to achieve
optimal levels of nitrogen fixation. This work provides evidence that the
hFixL-FxkR TCS is activated in response to low oxygen concentration in S.
meliloti SM11 and that it negatively regulates the expression of fixK1, fixN1 and
nitrogen fixation.
DOI: 10.1099/mic.0.000284
299. FEMS Microbiol Lett. 2016 May;363(9). pii: fnw069. doi: 10.1093/femsle/fnw069.
Epub 2016 Mar 23.
Expression of the Sinorhizobium meliloti small RNA gene mmgR is controlled by the
nitrogen source.
Ceizel Borella G(1), Lagares A Jr(1), Valverde C(2).
Author information:
(1)Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el
Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes,
Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina.
(2)Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el
Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes,
Roque Sáenz Peña 352, Bernal B1876BXD, Buenos Aires, Argentina
cvalver@unq.edu.ar.
Small non-coding regulatory RNAs (sRNAs) are key players in post-transcriptional

regulation of gene expression. Hundreds of sRNAs have been identified in
Sinorhizobium meliloti, but their biological function remains unknown for most of
them. In this study, we characterized the expression pattern of the gene encoding
the 77-nt sRNA MmgR in S. meliloti strain 2011. A chromosomal transcriptional
reporter fusion (PmmgR-gfp) showed that the mmgR promoter is active along
different stages of the interaction with alfalfa roots. In pure cultures,
PmmgR-gfp activity paralleled the sRNA abundance indicating that mmgR expression
is primarily controlled at the level of transcriptional initiation. PmmgR-gfp
activity was higher during growth in rhizobial defined medium (RDM) than in TY
medium. Furthermore, PmmgR-gfp was induced at 60 min after shifting growing cells
from TY to RDM medium, i.e. shorter than the cell doubling time. In defined RDM
medium containing NO3 (-), both PmmgR-gfp and MmgR level were repressed by the
addition of tryptone or single amino acids, suggesting that mmgR expression
depends on the cellular nitrogen (N) status. In silico analysis failed to detect
conserved motifs upstream the promoter RNA polymerase binding site, but revealed
a strongly conserved motif centered at -28 that may be linked to the observed
regulatory pattern by the N source.

DOI: 10.1093/femsle/fnw069
300. Lett Appl Microbiol. 2016 May;62(5):379-85. doi: 10.1111/lam.12566.
A multiplex PCR assay for the detection and quantification of Sclerotinia

sclerotiorum and Botrytis cinerea.
Reich JD(1)(2), Alexander TW(2), Chatterton S(2).

Author information:
(1)University of Lethbridge, Lethbridge, AB, Canada.
(2)Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada,
Lethbridge, AB, Canada.
Traditional culture methods for identifying the plant fungal pathogens

Sclerotinia sclerotiorum (Lib.) de Bary and Botrytis cinerea Pers.:Fr. are slow
and laborious. The goal of this study was to develop a multiplex real-time PCR
(qPCR) assay to detect and quantify DNA from S. sclerotiorum and B. cinerea. A
primer set (SsIGS_5) for S. sclerotiorum was designed that targeted the
intergenic spacer (IGS) regions of the ribosomal DNA. Addition of a probe to the
assay increased its specificity: when the primer/probe set was tested against 21
fungal species (35 strains), amplification was detected from all S. sclerotiorum
strains and no other species. For qPCR, the SsIGS_5 primer and probe set
exhibited a linear range from 7·0 ng to 0·07 pg target DNA (R(2) = 0·99).
SsIGS_5 was then multiplexed with a previously published primer/probe set for
B. cinerea to develop a high-throughput method for the detection and
quantification of DNA from both pathogens. When multiplexed, the sensitivity and
specificity of both assays were not different from individual qPCR reactions. The
multiplex assay is currently being used to detect and quantify S. sclerotiorum
and B. cinerea DNA from aerosol samples collected in commercial seed alfalfa
fields.SIGNIFICANCE AND IMPACT OF THE STUDY: A primer and probe set for the
quantification of Sclerotinia sclerotiorum DNA in a PCR assay was developed. The
probe-based nature of this assay signifies an improvement over previous assays
for this species by allowing multiplex reactions while maintaining high
sensitivity. The primer/probe set was used in a multiplex real-time PCR assay for
the quantification of S. sclerotiorum and Botrytis cinerea DNA, enabling rapid
analysis of environmental samples. In crops susceptible to both pathogens, this
multiplex assay can be used to quickly quantify the presence of each pathogen.
© 2016 Her Majesty the Queen in Right of Canada © 2016 The Society for Applied
Microbiology. Reproduced with the permission of the Office of the Agriculture and
Agri-Food Canada.
DOI: 10.1111/lam.12566
301. Ying Yong Sheng Tai Xue Bao. 2015 Oct;26(10):3059-65.
[Soil dryness characteristics of alfalfa cropland and optimal growth years of

alfalfa on the Loess Plateau of central Gansu, China].
Luo ZZ, Li LL, Niu YN, Cai LQ, Zhang RZ, Xie JH.
This paper investigated soil moisture in alfalfa (Medicago sativa) cropland with
different growth years (1, 3, 8, 12 and 14 years) and discussed the optimum
growth years of alfalfa on the Loess Plateau of central Gansu. The results showed
that the soil moisture along 0-300 cm soil profile of alfalfa croplands with
different growth years was obviously lower than that of the local soil stable
moisture. The soil water contents in croplands with alfalfa that had grown for 12
and 14 years were only 9.2% and 7.1% of local soil stable moisture, respectively,
which were even lower than the lower limit of alfalfa growth. The average soil
dryness indexes along 0-300 cm soil profile in 1, 3, 8, 12 and 14 years alfalfa
croplands were 125.4%, 30.5%, 18.4%, -34.2% and -83.3% respectively. The results
indicated that soil dryness occurred to varying degrees with different growth
years except croplands with alfalfa grown for 1 year. With the increase of growth
years of alfalfa, the soil dryness intensity increased and the soil dryness rate
decreased. According to the soil moisture and alfalfa productivity results in
this study, it could be concluded that the optimum growth years of alfalfa are
8-10 years in semiarid areas of the Loess Plateau.

Mar
16.
Production, composition, and oxidative stability of milk highly enriched in

polyunsaturated fatty acids from dairy cows fed alfalfa protein concentrate or
supplemental vitamin E.
Fauteux MC(1), Gervais R(1), Rico DE(1), Lebeuf Y(1), Chouinard PY(2).
Author information:
(1)Département des Sciences Animales, Université Laval, Québec, QC, Canada G1V
0A6.
(2)Département des Sciences Animales, Université Laval, Québec, QC, Canada G1V
0A6. Electronic address: yvan.chouinard@fsaa.ulaval.ca.
Given its elevated content of carotenoids, alfalfa protein concentrates (APC)

have the potential to prevent oxidation of milk enriched in polyunsaturated fatty
acids. The effects of feeding APC or supplemental vitamin E on production,
composition, and oxidative stability of milk enriched in polyunsaturated fatty
acids were evaluated using 6 lactating Holstein cows (224±18d in milk) in a
replicated 3×3 Latin square (21-d periods, 14d for adaptation). Treatment diets
contained (dry matter basis) (1) 9% soybean meal (control, CTL); (2) 9% soybean
meal + 300 IU of vitamin E/kg (VitE treatment); or (3) 9% APC (APC treatment).
Cows received a continuous abomasal infusion of 450g/d of linseed oil. As a
result, milk fat content of cis-9,cis-12 18:2 increased from 1.08±0.13 to
3.9±0.40% (mean ± SD), whereas cis-9,cis-12,cis-15 18:3 increased from 0.40±0.04
to 14.27±1.81% during the experimental period compared with the pretrial period.
Milk yield tended to be higher for APC (14.7kg/d) compared with CTL (13.4kg/d),
and was greater than that for VitE (13.0kg/d). Protein yield was higher in cows
fed APC (518g/d) compared with VitE (445g/d) but was not different from that in
cows fed CTL (483g/d). These effects resulted in improved milk N efficiency in
cows fed APC (26.1% of N intake secreted in milk) compared with CTL (23.0%) and
VitE (22.9%). Feeding APC increased milk fat content of lutein (252μg/g) compared
with CTL (204μg/g) and VitE (190μg/g). Milk fat content of vitamin E was higher
for APC (34.5μg/g) compared with CTL (19.0μg/g) and tended to be lower than that
with VitE (44.9μg/g). Redox potential of fresh milk from cows fed APC (152mV) was
similar to that of VitE (144mV), but lower than that of CTL (189mV). Treatments
had no effect on fresh milk contents of dissolved oxygen (8.1±1.5mg/L), and
conjugated diene hydroperoxides (2.7±0.5mmol/L). The concentrations of volatile
lipid oxidation products (propanal, hexanal, hept-cis-4-enal, 1-octen-3-one)
tended to be decreased by APC relative to CTL, whereas similar values were
observed for VitE, except for hexanal, which was reduced by 40% in VitE. In
conclusion, feeding APC to lactating dairy cows could serve as a source of
dietary protein that improves dietary N utilization efficiency, and also as a
preharvest technology to increase natural antioxidant levels in milk to limit
oxidation.

DOI: 10.3168/jds.2015-10722

Mar
16.
Effect of exchanging Onobrychis viciifolia and Lotus corniculatus for Medicago

sativa on ruminal fermentation and nitrogen turnover in dairy cows.
Grosse Brinkhaus A(1), Bee G(2), Silacci P(2), Kreuzer M(3), Dohme-Meier F(4).
Author information:
(1)Agroscope, Institute for Livestock Sciences ILS, Tioleyre 4, 1725 Posieux,
Switzerland; ETH Zurich, Institute of Agricultural Science, Universitaetstrasse
2, 8092 Zurich, Switzerland.
Switzerland.
(3)ETH Zurich, Institute of Agricultural Science, Universitaetstrasse 2, 8092
Zurich, Switzerland.
Switzerland. Electronic address: frigga.dohme-meier@agroscope.admin.ch.
The objective of the study was to determine the effect of feeding sainfoin (SF;
Onobrychis viciifolia) and birdsfoot trefoil (BT; Lotus corniculatus), 2
temperate climate forage legumes that contain condensed tannins (CT), on ruminal
fermentation and N turnover in dairy cows. Six ruminally cannulated multiparous
dairy cows (milk yield=40kg/d; 36 d in milk) were used in a replicated 3×3 Latin
square design. All animals were fed basal diets containing 20% pelleted SF (223g
of CT/kg of dry matter), BT (30.3g of CT/kg of dry matter), or alfalfa (AL) and
concentrate to meet their predicted nutrient requirements. Each experimental
period consisted of a 21-d adaptation period in a tiestall, followed by a 7-d
collection period in metabolic crates, where feces and urine were collected
quantitatively. During the 7-d period, milk yield was recorded daily and milk
samples were taken at each milking. Blood, ruminal fluid, and papillae were
sampled on d 2 and 5. The relative abundance of selected bacterial strains in
ruminal fluid and the gene expression of transporter genes in the papillae were
determined with quantitative PCR. Total volatile fatty acids and the abundance of
the cellulolytic bacteria Prevotella spp. and Ruminococcus flavefaciens decreased
with SF compared with AL. The relative gene expression of the monocarboxylate
transporter 1 was increased with BT compared with AL and SF. Total yields of
milk, milk fat, and milk protein were similar among treatments. The proportion of
18:3n-3 in milk fat was greater and those of 22:5n-3 and 22:6n-3 were lower with
SF than with BT. The contents of urea N in blood (2.71, 3.45, and 3.90mmol/L for
SF, AL, and BT, respectively), milk (79.8, 100.1, and 110.9mg/kg for SF, AL, and
BT, respectively), and urine were lower with SF than with AL and BT, and a trend
toward a lower ruminal ammonia content occurred with SF compared with BT. Intake
and excretion of N with milk were similar among treatments, but urine N was lower
with SF than with AL. The N excretion to N intake relation showed a shift in a
part of urine N (17.5, 20.8, and 19.5% for SF, AL, and BT, respectively) to fecal
N (45.2, 41.3, and 38.5% for SF, AL, and BT respectively) with SF compared with
AL and BT. In conclusion, SF and BT differed in their effects on fermentation and
milk fatty acid profile and SF also showed potential to decrease metabolic and
environmental loads. The main reason for the different efficiency was likely a
higher CT content of SF compared with BT.

DOI: 10.3168/jds.2015-9911
304. Anim Sci J. 2016 Dec;87(12):1472-1479. doi: 10.1111/asj.12600. Epub 2016 Mar
17.
Effects of chemical additives on the fermentation quality and N distribution of

alfalfa silage in south of China.
Li P(1), Ji S(1), Hou C(1), Tang H(1), Wang Q(1), Shen Y(1).
Author information:
(1)College of Grassland Science, Nanjing Agricultural University, Nanjing, China.
In order to better utilize the last cut alfalfa harvested before killing frost in
a high moisture environment, the effects of chemical additives on the quality of
alfalfa silage were studied in south of China. The alfalfa was freshly harvested
at branching stage, and wilted by dry matter content of about 300 g/kg (fresh
matter basis). Silage was prepared by using a small-scale silage fermentation
system, where sucrose, potassium citrate, sodium carbonate, formic acid, acetic
acid and propionic acid were used as silage additives, and no additives served as
control. These silos were stored at ambient temperature (5-20°C), and the silage
qualities were analyzed after 120 days of fermentation. All additive treatments
affected the chemical composition and N distribution, increased the water-soluble
content and crude protein contents, decreased non-protein nitrogen (NPN) content,
and enhanced the in vitro ruminal dry matter digestibility (except for sodium
carbonate). Silages treated with organic acids were preserved with significantly
(P < 0.05) lower pH value, ethanol content and NPN content compared with control.
When the fermentation quality, chemical composition and N distribution were
considered, the treatment with sucrose or organic acids resulted in high quality
of alfalfa silage ensiled before killing frost, with formic acid having the best
effect.
DOI: 10.1111/asj.12600
305. Genet Mol Res. 2016 Feb 26;15(1):15018299. doi: 10.4238/gmr.15018299.
Salt stress alters DNA methylation levels in alfalfa (Medicago spp).
Al-Lawati A(1), Al-Bahry S(1), Victor R(1), Al-Lawati AH(2), Yaish MW(1).
Author information:
(1)Department of Biology, College of Science, Sultan Qaboos University, Alkhuod,
Muscat, Oman.
(2)Oman Animal and Plant Genetic Resources Center, The Research Council, Muscat,
Oman.
Modification of DNA methylation status is one of the mechanisms used by plants to

adjust gene expression at both the transcriptional and posttranscriptional levels
when plants are exposed to suboptimal conditions. Under abiotic stress, different
cultivars often show heritable phenotypic variation accompanied by epigenetic
polymorphisms at the DNA methylation level. This variation may provide the raw
materials for plant breeding programs that aim to enhance abiotic stress
tolerance, including salt tolerance. In this study, methylation-sensitive
amplified polymorphism (MSAP) analysis was used to assess cytosine methylation
levels in alfalfa (Medicago spp) roots exposed to increasing NaCl concentrations
(0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in
addition to a salt-tolerant cultivar that was used as a control. There was a
slight increase in DNA methylation upon exposure to high levels of soil salinity.
Phylogenetic analysis using MSAP showed epigenetic variation within and between
the alfalfa landraces when exposed to saline conditions. Based on MSAP and
enzyme-linked immunosorbent assay results, we found that salinity increased
global DNA methylation status, particularly in plants exposed to the highest
level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain
reaction indicated that this might be mediated by the overexpression of
methyltransferase homolog genes after exposure to saline conditions. DNA
demethylation using 5-azacytidine reduced seedling lengths and dry and fresh
weights, indicating a possible decrease in salinity tolerance. These results
suggest that salinity affects DNA methylation flexibility.
DOI: 10.4238/gmr.15018299
306. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2016;51(7):561-70. doi:
10.1080/10934529.2016.1141622. Epub 2016 Mar 16.
Metals in agricultural produce associated with acid-mine drainage in Mount Morgan

(Queensland, Australia).
Vicente-Beckett VA(1), McCauley GJ(1), Duivenvoorden LJ(1).
Author information:
(1)a Central Queensland University, School of Medical and Applied Sciences ,
Rockhampton , Queensland , Australia.
Acid-mine drainage (AMD) into the Dee River from the historic gold and copper
mine in Mount Morgan, Queensland (Australia) has been of concern to farmers in
the area since 1925. This study sought to determine the levels of AMD-related
metals and sulfur in agricultural produce grown near the mine-impacted Dee River,
compare these with similar produce grown in reference fields (which had no known
AMD influence), and assess any potential health risk using relevant Australian or
US guidelines. Analyses of lucerne (Medicago sativa; also known as alfalfa) from
five Dee fields showed the following average concentrations (mg/kg dry basis): Cd
< 1, Cu 11, Fe 106, Mn 52, Pb < 5, Zn 25 and S 3934; similar levels were found in
lucerne hay (used as cattle feed) from two Dee fields. All lucerne and lucerne
hay data were generally comparable with levels found in the lucerne reference
fields, suggesting no AMD influence; the levels were within the US National
Research Council (US NRC) guidelines for maximum tolerable cattle dietary intake.
Pasture grass (also cattle feed) from two fields in the Dee River floodplains
gave mean concentrations (mg/kg dry) of Cd 0.14, Cu 12, Fe 313, Mn 111, Pb 1.4,
Zn 86 and S 2450. All metal levels from the Dee and from reference sites were
below the US NRC guidelines for maximum tolerable cattle dietary intake; however,
the average Cd, Cu and Fe levels in Dee samples were significantly greater than
the corresponding levels in the pasture grass reference sites, suggesting AMD
influence in the Dee samples. The average levels in the edible portions of
mandarin oranges (Citrus reticulata) from Dee sites (mg/kg wet weight) were Cd
0.011, Cu 0.59, Fe 2.2, Mn 0.56, Pb 0.18, S 91 and Zn 0.96. Cd and Zn were less
than or close to, average Fe and Mn levels were at most twice, Cd 1.8 or
6.5 times, and Pb 8.5 or 72 times the maximum levels in raw oranges reported in
the US total diet study (TDS) or the Australian TDS, respectively. Average Cd,
Fe, Mn, Pb and Zn levels in the citrus reference samples were found to exceed the
maximum reported in one or both TDS surveys. Cu, Fe, Mn, Pb and Zn plant-soil
transfer factor (TF) values were < 1 for all agricultural samples from both Dee
and reference sites, suggesting relatively poor transfer of these metals from
soil to plant. In the case of Cd, TF values for Dee pasture grass and citrus
fruit samples were 0.14 and 0.73, respectively; lucerne and lucerne hay from both
Dee and reference sites gave TF = 10, suggesting some potential risk to cattle,
although this conclusion is tentative because Cd levels were close to or less
than the detection limit. TF values for S in lucerne, lucerne hay, pasture grass
and mandarin oranges from Dee sites were 18, 14, 3 and 3.6, respectively,
indicating that S in soil was readily available to plant or fruit. Sulfur in
pasture grass and citrus fruit (TF = 11 for both) was apparently more
bioavailable at the reference sites than at the Dee sites (TF = 3.0 for pasture
grass; TF = 3.6 for citrus fruit).
DOI: 10.1080/10934529.2016.1141622
307. Mol Plant Microbe Interact. 2016 Jun;29(6):458-67. doi:

10.1094/MPMI-07-15-0148-R. Epub 2016 Apr 25.
Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein

Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis.
Mao YH(1), Li F(1), Ma JC(1), Hu Z(1), Wang HH(1).
Author information:
(1)Guangdong Provincial Key Laboratory of Protein Function and Regulation in
Agricultural Organisms, College of Life Sciences, South China Agricultural
University, Guangzhou, Guangdong 510642, China.
In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the
symbiotic plasmid. Although strong sequence similarity (53% amino acid
identities) between S. meliloti NodG and Escherichia coli FabG was reported in
1992, it has not been determined whether S. meliloti NodG plays a role in fatty
acid synthesis. We report that expression of S. meliloti NodG restores the growth
of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive
conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze
the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic
reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause
any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti
fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted
without any effects on growth or fatty acid composition. This indicates that S.
meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when
expressed at sufficiently high levels. Thus, a bacterium can simultaneously
possess two or more OARs that can play a role in fatty acid synthesis. Our data
also showed that, although SmnodG increases alfalfa nodulation efficiency, it is
not essential for alfalfa nodulation.
DOI: 10.1094/MPMI-07-15-0148-R
308. Protein Pept Lett. 2016;23(5):495-502.
Genetic Engineering of Alfalfa (Medicago sativa L.).
Wang D, Khurshid M, Sun ZM, Tang YX, Zhou ML, Wu YM(1).

Author information:
(1)Biotechnology Research Institute, Chinese Academy of Agricultural Sciences
(CAAS), Beijing 100081, China. wuyanmin211@hotmail.com.
Alfalfa is excellent perennial legume forage for its extensive ecological

adaptability, high nutrition value, palatability and biological nitrogen
fixation. It plays a very important role in the agriculture, animal husbandry and
ecological construction. It is cultivated in all continents. With the development
of modern plant breeding and genetic engineering techniques, a large amount of
work has been carried out on alfalfa. Here we summarize the recent research
advances in genetic engineering of alfalfa breeding, including transformation,
quality improvement, stress resistance and as a bioreactor. The review article
can enables us to understand the research method, direction and achievements of
genetic engineering technology of Alfalfa.
309. Plant Sci. 2016 Apr;245:119-27. doi: 10.1016/j.plantsci.2016.01.012. Epub 2016

Feb 2.
Strigolactones in the Rhizobium-legume symbiosis: Stimulatory effect on bacterial

surface motility and down-regulation of their levels in nodulated plants.
Peláez-Vico MA(1), Bernabéu-Roda L(1), Kohlen W(2), Soto MJ(1), López-Ráez JA(3).
Author information:
(1)Department of Soil Microbiology and Symbiotic Systems, Estación Experimental
del Zaidín-Consejo Superior de Investigaciones Científicas (EEZ-CSIC), Profesor
Albareda 1, 18008 Granada, Spain.
(2)Department of Plant Sciences, Laboratory of Molecular Biology, Wageningen
University, Droevendaalsesteeg 1, 6708 PB, Wageningen, Netherlands.
(3)Department of Soil Microbiology and Symbiotic Systems, Estación Experimental
del Zaidín-Consejo Superior de Investigaciones Científicas (EEZ-CSIC), Profesor
Albareda 1, 18008 Granada, Spain. Electronic address: juan.lopezraez@eez.csic.es.
Strigolactones (SLs) are multifunctional molecules acting as modulators of plant

responses under nutrient deficient conditions. One of the roles of SLs is to
promote beneficial association with arbuscular mycorrhizal (AM) fungi belowground
under such stress conditions, mainly phosphorus shortage. Recently, a role of SLs
in the Rhizobium-legume symbiosis has been also described. While SLs' function in
AM symbiosis is well established, their role in the Rhizobium-legume interaction
is still emerging. Recently, SLs have been suggested to stimulate surface
motility of rhizobia, opening the possibility that they could also act as
molecular cues. The possible effect of SLs in the motility in the alfalfa
symbiont Sinorhizobium meliloti was investigated, showing that the synthetic SL
analogue GR24 stimulates swarming motility in S. meliloti in a dose-dependent
manner. On the other hand, it is known that SL production is regulated by
nutrient deficient conditions and by AM symbiosis. Using the model alfalfa-S.
meliloti, the impact of phosphorus and nitrogen deficiency, as well as of
nodulation on SL production was also assessed. The results showed that phosphorus
starvation promoted SL biosynthesis, which was abolished by nitrogen deficiency.
In addition, a negative effect of nodulation on SL levels was detected,
suggesting a conserved mechanism of SL regulation upon symbiosis establishment.
Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DOI: 10.1016/j.plantsci.2016.01.012
310. Mol Plant Pathol. 2017 Feb;18(2):187-194. doi: 10.1111/mpp.12389. Epub 2016
Jul
10.
Genotyping-by-sequencing-based genome-wide association studies on Verticillium

wilt resistance in autotetraploid alfalfa (Medicago sativa L.).
Yu LX(1), Zheng P(2), Zhang T(1), Rodringuez J(3), Main D(2).
Author information:
Germplasm Introduction and Testing Research, 24106 N Bunn Road, Prosser, WA,
99350 USA.
(2)Department of Horticulture, Washington State University, Pullman, WA, 99164
USA.
(3)Forage Genetics International, Inc., 7661 Becker Road, Davis, CA, 95618 USA.
Verticillium wilt (VW) is a fungal disease that causes severe yield losses in
alfalfa. The most effective method to control the disease is through the
development and use of resistant varieties. The identification of marker loci
linked to VW resistance can facilitate breeding for disease-resistant alfalfa. In
the present investigation, we applied an integrated framework of genome-wide
association with genotyping-by-sequencing (GBS) to identify VW resistance loci in
a panel of elite alfalfa breeding lines. Phenotyping was performed by manual
inoculation of the pathogen to healthy seedlings, and scoring for disease
resistance was carried out according to the standard test of the North America
Alfalfa Improvement Conference (NAAIC). Marker-trait association by linkage
disequilibrium identified 10 single nucleotide polymorphism (SNP) markers
significantly associated with VW resistance. Alignment of the SNP marker
sequences to the M. truncatula genome revealed multiple quantitative trait loci
(QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and
8, respectively. Resistance loci found on chromosomes 7 and 8 in the present
study co-localized with the QTLs reported previously. A pairwise alignment
(blastn) using the flanking sequences of the resistance loci against the M.
truncatula genome identified potential candidate genes with putative disease
resistance function. With further investigation, these markers may be implemented
into breeding programmes using marker-assisted selection, ultimately leading to
improved VW resistance in alfalfa.
PUBLISHED 2016. THIS ARTICLE IS A U.S. GOVERNMENT WORK AND IS IN THE PUBLIC
DOMAIN IN THE USA.
DOI: 10.1111/mpp.12389
311. J Dairy Sci. 2016 May;99(5):3497-3511. doi: 10.3168/jds.2015-10380. Epub 2016

Feb
28.
Storage characteristics, nutritive value, and fermentation characteristics of

alfalfa packaged in large-round bales and wrapped in stretch film after extended
time delays.
Coblentz WK(1), Coffey KP(2), Chow EA(3).

Author information:
(2)Department of Animal Science, University of Arkansas, Fayetteville 72701.
(3)Kuraray America Inc., Pasadena, TX 77507.
The production of baled silage is attractive to producers because it offers

advantages over dry hay, particularly by limiting risks associated with wet or
unstable weather conditions. Our objectives were to test the effects of delayed
wrapping on silage fermentation, storage characteristics, and the nutritive value
of baled alfalfa silages. To accomplish this, large-round bales of alfalfa were
wrapped in plastic film within 4h of baling (d 0), or after delays of 1, 2, or 3
d. A secondary objective was to evaluate a prototype bale wrap containing an
O2-limiting barrier (OB) against an identical polyethylene wrap without the O2
barrier (SUN). Sixty-four 1.19×1.25-m bales of alfalfa were made from 4 field
blocks at a mean moisture concentration of 59.1±4.3% with a mean initial wet bale
weight of 473±26.4kg. Two bales per field block were assigned to each combination
of bale wrap (SUN or OB) and wrapping time (0, 1, 2, or 3 d postbaling), and one
bale of each pair was fitted with a thermocouple placed in the geometric center
of each bale. All bales were sampled after a 97-d storage period. Internal bale
temperatures, recorded at the time bales were wrapped, were greater for all bales
with wrapping delays compared with bales wrapped on d 0 (54.9 vs. 34.9°C), and
increased to a maximum of 63.9°C after a 3-d delay exhibiting a linear effect of
time delay. Total silage fermentation acids (lactic, acetic, propionic, butyric,
and isobutyric) were greatest when bales were wrapped on d 0 compared with all
bales wrapped with time delays (4.64 vs. 2.26% of DM), and declined with linear
and quadratic effects of wrapping delay. Total fermentation acids also were
related quadratically to internal bale temperature by regression [y (% of
DM)=0.0042x(2) - 0.50x + 17.1; R(2)=0.725]. Similar responses were observed for
lactic acid, except that trends were linear, both for orthogonal contrasts
evaluating length of wrapping delay, and in regressions on internal bale
temperature [y (% of DM)=-0.046x + 3.5; R(2)=0.663]. Butyric acid also was
detected, regardless of treatment, but was greatest within bales wrapped on d 0
compared with those with wrapping delays (0.99 vs. 0.38% of DM), and a similar
response (0.68 vs. 0.52% of DM) was observed for NH3-N, suggesting that
clostridial activity occurred during silage fermentation. Based on these results,
silage fermentation characteristics and the nutritive value declined with time
delays before wrapping, but responses were exacerbated when delays exceeded 1 d.

DOI: 10.3168/jds.2015-10380

eCollection 2016.
Bioinformatic Identification and Analysis of Extensins in the Plant Kingdom.
Liu X(1)(2), Wolfe R(3), Welch LR(2)(3), Domozych DS(4), Popper ZA(5), Showalter
AM(1)(2).
Author information:
(1)Department of Environmental and Plant Biology, Ohio University, Athens, Ohio,
(2)Molecular and Cellular Biology Program, Ohio University, Athens, Ohio, United
States of America.
(3)Russ College of Engineering and Technology, Center for Intelligent,
Distributed and Dependable Systems, Ohio University, Athens, Ohio, United States
of America.
(4)Department of Biology, Skidmore College, Saratoga Springs, New York, United
States of America.
(5)Botany and Plant Science and Ryan Institute for Environmental, Marine, and
Energy Research, School of Natural Sciences, National University of Ireland
Galway, Galway, Ireland.
Extensins (EXTs) are a family of plant cell wall hydroxyproline-rich

glycoproteins (HRGPs) that are implicated to play important roles in plant
growth, development, and defense. Structurally, EXTs are characterized by the
repeated occurrence of serine (Ser) followed by three to five prolines (Pro)
residues, which are hydroxylated as hydroxyproline (Hyp) and glycosylated. Some
EXTs have Tyrosine (Tyr)-X-Tyr (where X can be any amino acid) motifs that are
responsible for intramolecular or intermolecular cross-linkings. EXTs can be
divided into several classes: classical EXTs, short EXTs, leucine-rich repeat
extensins (LRXs), proline-rich extensin-like receptor kinases (PERKs),
formin-homolog EXTs (FH EXTs), chimeric EXTs, and long chimeric EXTs. To guide
future research on the EXTs and understand evolutionary history of EXTs in the
plant kingdom, a bioinformatics study was conducted to identify and classify EXTs
from 16 fully sequenced plant genomes, including Ostreococcus lucimarinus,
Chlamydomonas reinhardtii, Volvox carteri, Klebsormidium flaccidum,
Physcomitrella patens, Selaginella moellendorffii, Pinus taeda, Picea abies,
Brachypodium distachyon, Zea mays, Oryza sativa, Glycine max, Medicago
truncatula, Brassica rapa, Solanum lycopersicum, and Solanum tuberosum, to
supplement data previously obtained from Arabidopsis thaliana and Populus
trichocarpa. A total of 758 EXTs were newly identified, including 87 classical
EXTs, 97 short EXTs, 61 LRXs, 75 PERKs, 54 FH EXTs, 38 long chimeric EXTs, and
346 other chimeric EXTs. Several notable findings were made: (1) classical EXTs
were likely derived after the terrestrialization of plants; (2) LRXs, PERKs, and
FHs were derived earlier than classical EXTs; (3) monocots have few classical
EXTs; (4) Eudicots have the greatest number of classical EXTs and Tyr-X-Tyr
cross-linking motifs are predominantly in classical EXTs; (5) green algae have no
classical EXTs but have a number of long chimeric EXTs that are absent in
embryophytes. Furthermore, phylogenetic analysis was conducted of LRXs, PERKs and
FH EXTs, which shed light on the evolution of three EXT classes.
PMCID: PMC4769139
313. Sensors (Basel). 2016 Feb 19;16(2):255. doi: 10.3390/s16020255.
UAV-Based Estimation of Carbon Exports from Heterogeneous Soil Landscapes--A Case

Study from the CarboZALF Experimental Area.
Wehrhan M(1), Rauneker P(2), Sommer M(3)(4).
Author information:
(1)Leibniz Centre for Agricultural Landscape Research (ZALF), Institute of Soil
Landscape Research, Eberswalder Straße 84, Müncheberg 15374, Germany.
wehrhan@zalf.de.
(2)Leibniz Centre for Agricultural Landscape Research (ZALF), Institute of
Landscape Hydrology, Eberswalder Straße 84, Müncheberg 15374, Germany.
philipp.rauneker@zalf.de.
(3)Leibniz Centre for Agricultural Landscape Research (ZALF), Institute of Soil
Landscape Research, Eberswalder Straße 84, Müncheberg 15374, Germany.
sommer@zalf.de.
(4)University of Potsdam, Institute of Earth and Environmental Sciences,
Karl-Liebknecht-Str. 24-25, Potsdam 14476, Germany. sommer@zalf.de.
The advantages of remote sensing using Unmanned Aerial Vehicles (UAVs) are a high
spatial resolution of images, temporal flexibility and narrow-band spectral data
from different wavelengths domains. This enables the detection of spatio-temporal
dynamics of environmental variables, like plant-related carbon dynamics in
agricultural landscapes. In this paper, we quantify spatial patterns of fresh
phytomass and related carbon (C) export using imagery captured by a 12-band
multispectral camera mounted on the fixed wing UAV Carolo P360. The study was
performed in 2014 at the experimental area CarboZALF-D in NE Germany. From
radiometrically corrected and calibrated images of lucerne (Medicago sativa), the
performance of four commonly used vegetation indices (VIs) was tested using band
combinations of six near-infrared bands. The highest correlation between
ground-based measurements of fresh phytomass of lucerne and VIs was obtained for
the Enhanced Vegetation Index (EVI) using near-infrared band b899. The resulting
map was transformed into dry phytomass and finally upscaled to total C export by
harvest. The observed spatial variability at field- and plot-scale could be
attributed to small-scale soil heterogeneity in part.
DOI: 10.3390/s16020255
PMCID: PMC4801631
314. Biochem Biophys Res Commun. 2016 Mar 25;472(1):75-82. doi:

10.1016/j.bbrc.2016.02.067. Epub 2016 Feb 21.
Co-transforming bar and CsLEA enhanced tolerance to drought and salt stress in
transgenic alfalfa (Medicago sativa L.).
Zhang J(1), Duan Z(1), Zhang D(1), Zhang J(1), Di H(1), Wu F(1), Wang Y(2).
Author information:
Agriculture Science and Technology, Lanzhou University, China.
Agriculture Science and Technology, Lanzhou University, China. Electronic
address: yrwang@lzu.edu.cn.
Drought and high salinity are two major abiotic factors that restrict alfalfa
productivity. A dehydrin protein, CsLEA, from the desert grass Cleistogenes
songorica was transformed into alfalfa (Medicago sativa L.) via
Agrobacterium-mediated transformation using the bar gene as a selectable marker,
and the drought and salt stress tolerances of the transgenic plants were
assessed. Thirty-nine of 119 transformants were positive, as screened by Basta,
and further molecularly authenticated using PCR and RT-PCR. Phenotype
observations revealed that the transgenic plants grew better than the wild-type
(WT) plants after 15d of drought stress and 10d of salt stress: the leaves of WT
alfalfa turned yellow, whereas the transgenic alfalfa leaves only wilted; after
rewatering, the transgenic plants returned to a normal state, though the WT
plants could not be restored. Evaluation of physiologic and biochemical indices
during drought and salt stresses showed a relatively lower Na(+) content in the
leaves of the transgenic plants, which would reduce toxic ion effects. In
addition, the transgenic plants were able to maintain a higher relative water
content (RWC), higher shoot biomass, fewer photosystem changes, decreased
membrane injury, and a lower level of osmotic stress injury. These results
demonstrate that overexpression of the CsLEA gene can enhance the drought and
salt tolerance of transgenic alfalfa; in addition, carrying the bar gene in the
genome may increase herbicide resistance.
DOI: 10.1016/j.bbrc.2016.02.067
315. J Bacteriol. 2016 Apr 14;198(9):1374-83. doi: 10.1128/JB.00013-16. Print 2016

May.
Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to

Bacterial Growth and Efficient Symbiosis with Medicago sativa.
Wippel K(1), Long SR(2).
Author information:
(1)Department of Biology, Stanford University, Stanford, California, USA.
(2)Department of Biology, Stanford University, Stanford, California, USA
srl@stanford.edu.
The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA
polymerase-binding protein DksA, is triggered by limiting nutrient conditions.
For some bacteria, it is involved in regulation of virulence. We investigated the
role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont
Sinorhizobium meliloti in free-living culture and in interaction with its host
plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and
SMc00049, differ in the constitution of two major domains required for function
in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc
finger domain. Using mutant analyses of single, double, and triple deletions for
SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but
not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced
nodulation on the host plant, and lower nitrogen fixation activity in early
nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe
pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA
complemented the metabolic defects of an Escherichia coli dksA mutant.
Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function.
Our results imply a role for transcriptional regulator DksA in the S. meliloti-M.
sativa symbiosis.IMPORTANCE: The stringent response is a bacterial transcription
regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a
soil bacterium establishing agriculturally important root nodule symbioses with
legume plants, undergoes constant molecular adjustment during host interaction.
Analyzing the components of the stringent response in this alphaproteobacterium
helps understand molecular control regarding the development of plant
interaction. Using mutant analyses, we describe how the lack of DksA influences
symbiosis with Medicago sativa and show that a second paralogous S. meliloti
protein cannot substitute for this missing function. This work contributes to the
field by showing the similarities and differences of S. meliloti DksA-like
proteins to orthologs from other species, adding information to the diversity of
the stringent response regulatory system.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
DOI: 10.1128/JB.00013-16
PMCID: PMC4836237
316. Plant Cell Rep. 2016 May;35(5):1205-8. doi: 10.1007/s00299-016-1947-5. Epub
2016
Feb 16.
mRNA biogenesis-related helicase eIF4AIII from Arabidopsis thaliana is an

important factor for abiotic stress adaptation.
Pascuan C(1)(2), Frare R(1), Alleva K(1), Ayub ND(1)(2), Soto G(3)(4).
Author information:
(1)CONICET, Avda. Rivadavia 1917, C1033AAJ CABA, Buenos Aires, Argentina.
(2)IGEAF- INTA, De los reseros S/N, Castelar C25 (1712), Buenos Aires, Argentina.
(3)CONICET, Avda. Rivadavia 1917, C1033AAJ CABA, Buenos Aires, Argentina.
soto.gabrielacinthia@inta.gob.ar.
(4)IGEAF- INTA, De los reseros S/N, Castelar C25 (1712), Buenos Aires, Argentina.
soto.gabrielacinthia@inta.gob.ar.
Similar to other plant species, Arabidopsis has a huge repertoire of predicted

helicases, including the eIF4AIII factor, a putative component of the exon
junction complex related to mRNA biogenesis. In this article, we integrated
evolutionary and functional approaches to have a better understanding of eIF4AIII
function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related
helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from
Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably
functional equivalences between mRNA biogenesis and stress-related plant
helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII
during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its
function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress
adaptation.
DOI: 10.1007/s00299-016-1947-5
317. Appl Microbiol Biotechnol. 2016 Jun;100(12):5581-93. doi:

10.1007/s00253-016-7362-6. Epub 2016 Feb 15.
Changes in the soil microbial community after reductive soil disinfestation and
cucumber seedling cultivation.
Huang X(1)(2)(3)(4), Liu L(1), Wen T(1), Zhang J(1)(2)(4), Wang F(1), Cai
Z(5)(6)(7)(8).
Author information:
(1)School of Geography Science, Nanjing Normal University, Nanjing, 210023,
China.
(2)Jiangsu Center for Collaborative Innovation in Geographical Information
Resource Development and Application, Nanjing, 210023, China.
(3)Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control,
Nanjing Normal University, Nanjing, 210023, China.
(4)Key Laboratory of Virtual Geographical Environment (VGE), Ministry of
Education, Nanjing Normal University, Nanjing, 210023, China.
(5)School of Geography Science, Nanjing Normal University, Nanjing, 210023,
China. zccai@njnu.edu.cn.
(6)Jiangsu Center for Collaborative Innovation in Geographical Information
Resource Development and Application, Nanjing, 210023, China. zccai@njnu.edu.cn.
(7)Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control,
Nanjing Normal University, Nanjing, 210023, China. zccai@njnu.edu.cn.
(8)Key Laboratory of Virtual Geographical Environment (VGE), Ministry of
Education, Nanjing Normal University, Nanjing, 210023, China. zccai@njnu.edu.cn.
Reductive soil disinfestation (RSD) has been proven to be an effective and

environmentally friendly way to control many soilborne pathogens and diseases. In
this study, the RSDs using ethanol (Et-RSD) and alfalfa (Al-RSD) as organic
carbons were performed in a Rhizoctonia solani-infected soil, and the
dissimilarities of microbial communities during the RSDs and after planting two
seasons of cucumber seedlings in the RSDs-treated soil were respectively
investigated by MiSeq pyrosequencing. The results showed that, as for bacteria,
Coprococcus, Flavisolibacter, Rhodanobacter, Symbiobacterium, and
UC-Ruminococcaceae became the dominant bacterial genera at the end of Al-RSD. In
contrast, Et-RSD soil involved more bacteria belonging to Firmicutes, such as
Sedimentibacter, UC-Gracilibacteraceae, and Desulfosporosinus. For fungi,
Chaetomium significantly increased at the end of RSDs, while Rhizoctonia and
Aspergillus significantly decreased. After planting two seasons of cucumber
seedlings, those bacteria belonging to Firmicutes significantly decreased, but
Lysobacter and Rhodanobacter belonging to the phylum Proteobacteria as well as
UC-Sordariales and Humicola belonging to Ascomycota alternatively increased in
Al- and Et-RSD-treated soils. Besides, some nitrification, denitrification, and
nitrogen fixation genes were apparently increased in the RSD-treated soils, but
the effect was more profound in Al-RSD than Et-RSD. Overall, Et-RSD could induced
more antagonists belonging to Firmicutes under anaerobic condition, whereas
Al-RSD could continuously stimulate some functional microorganisms (Lysobacter
and Rhodanobacter) and further improve nitrogen transformation activities in the
soil at the coming cropping season.
DOI: 10.1007/s00253-016-7362-6
318. J Anim Physiol Anim Nutr (Berl). 2016 Oct;100(5):820-7. doi:

10.1111/jpn.12442.
Epub 2016 Feb 12.
Effects of forage offering method on performance, rumen fermentation, nutrient

digestibility and nutritional behaviour in Holstein dairy calves.
EbnAli A(1), Khorvash M(1), Ghorbani GR(1), Mahdavi AH(1), Malekkhahi M(2),
Mirzaei M(3), Pezeshki A(4), Ghaffari MH(5).
Author information:
(1)Department of Animal Science, College of Agriculture, Isfahan University of
Technology, Isfahan, Iran.
(2)Department of Animal Science, Faculty of Agriculture, Ferdowsi University of
Mashhad, Mashhad, Iran.
(3)Department of Animal Science, Faculty of Agriculture and Natural Resources,
Arak University, Arak, Iran.
(4)Department of Production Animal Health, Faculty of Veterinary Medicine,
Gastrointestinal Research Group, Calvin, Phoebe and Joan Snyder Institute for
Chronic Diseases, University of Calgary, Calgary, AB, Canada.
Technology, Isfahan, Iran. morteza.h.g@gmail.com.
The potential effect of dietary forage supplementation on the performance and

rumen development in dairy calves is well established. However, limited research
has been directed to the comparative effects of forage offering methods on calf
performance. The objective of the present study was to determine the effects of
forage provision methods (total mixed ration or free choice) on the performance,
nutrient digestibility, rumen fermentation and nutritional behaviour in newborn
calves. Forty-five Holstein dairy calves (3 days of age and 41 ± 2 kg of body
weight) were assigned to the following three groups (n = 15): (i) starter without
forage provision (CON), (ii) starter supplemented with 10% alfalfa hay (AH) as a
total mixed ration (AH-TMR) and (iii) starter and AH as a free-choice provision
(AH-FC) for a period of 70 days. All the calves were offered 5 l of milk/day from
day 3 to 50, and 2.5 l/day from day 50 until weaning on day 56. Dry matter intake
(DMI) was greater (p < 0.01) in the AH-TMR and AH-FC treatments than in the CON
during the pre- and post-weaning periods. Calves fed the AH-FC diet showed the
highest post-weaning DMI among the treatments. The calves receiving ad libitum
forage tended (p = 0.08) to increase crude protein digestibility and overall
volatile fatty acids (VFA) concentrations in the rumen. No differences were
observed among the treatments at the time spent on standing, lying, eating and
performing non-nutritive oral behaviours. Compared to CON calves, animals in the
AH-TMR treatment spent more time (p < 0.05) ruminating. In conclusion, our data
suggest that forage supplementation in both forage offering methods increased
total DMI, ruminal pH and ruminating time in dairy calves. Hence, there is no
benefit in the free-choice provision of AH in dairy calves.
DOI: 10.1111/jpn.12442
319. Plant Cell Physiol. 2016 Feb;57(2):407-22. doi: 10.1093/pcp/pcw001. Epub 2016
Feb
9.
Combining -Omics to Unravel the Impact of Copper Nutrition on Alfalfa (Medicago

sativa) Stem Metabolism.
Printz B(1), Guerriero G(2), Sergeant K(3), Audinot JN(4), Guignard C(2), Renaut
J(2), Lutts S(5), Hausman JF(2).
Author information:
and Innovation (ERIN) Department, Esch/Alzette, Luxembourg Université Catholique
de Louvain, Earth and Life Institute Agronomy (ELI-A), Groupe de Recherche en
Physiologie Végétale (GRPV), Louvain-la-Neuve, Belgium.
and Innovation (ERIN) Department, Esch/Alzette, Luxembourg.
and Innovation (ERIN) Department, Esch/Alzette, Luxembourg
kjell.sergeant@list.lu.
(4)Luxembourg Institute of Science and Technology (LIST), Materials Research and
Technology (MRT) Department, Esch/Alzette, Luxembourg.
(5)Université Catholique de Louvain, Earth and Life Institute Agronomy (ELI-A),
Groupe de Recherche en Physiologie Végétale (GRPV), Louvain-la-Neuve, Belgium.
Copper can be found in the environment at concentrations ranging from a shortage

up to the threshold of toxicity for plants, with optimal growth conditions
situated in between. The plant stem plays a central role in transferring and
distributing minerals, water and other solutes throughout the plant. In this
study, alfalfa is exposed to different levels of copper availability, from
deficiency to slight excess, and the impact on the metabolism of the stem is
assessed by a non-targeted proteomics study and by the expression analysis of key
genes controlling plant stem development. Under copper deficiency, the plant stem
accumulates specific copper chaperones, the expression of genes involved in stem
development is decreased and the concentrations of zinc and molybdenum are
increased in comparison with the optimum copper level. At the optimal copper
level, the expression of cell wall-related genes increases and proteins playing a
role in cell wall deposition and in methionine metabolism accumulate, whereas
copper excess imposes a reduction in the concentration of iron in the stem and a
reduced abundance of ferritins. Secondary ion mass spectrometry (SIMS) analysis
suggests a role for the apoplasm as a copper storage site in the case of copper
toxicity.

Society of Plant Physiologists.
DOI: 10.1093/pcp/pcw001
PMCID: PMC4771972
320. Planta. 2016 May;243(5):1169-89. doi: 10.1007/s00425-016-2469-7. Epub 2016 Feb

10.
Medicago truncatula Gaertn. as a model for understanding the mechanism of growth

promotion by bacteria from rhizosphere and nodules of alfalfa.
Kisiel A(1), Kępczyńska E(2).
Author information:
(1)Department of Plant Biotechnology, Faculty of Biology, University of Szczecin,
Wąska 13, 71-415, Szczecin, Poland.
(2)Department of Plant Biotechnology, Faculty of Biology, University of Szczecin,
Wąska 13, 71-415, Szczecin, Poland. ekepcz@univ.szczecin.pl.
MAIN CONCLUSION: The present study showed all the 16 strains isolated and
identified from the alfalfa rhizosphere and nodules, and registered in GenBank,
to be good candidates for targeted use in studies addressing the rather weak
known mechanism of plant growth promotion, including that of Medicago truncatula,
a molecular crop model. Based on physiological, biochemical and molecular
analysis, the 16 isolates obtained were ascribed to the following five families:
Bacillaceae, Rhizobiaceae, Xantomonadaceae, Enterobacteriaceae and
Pseudomonadaceae, within which 9 genera and 16 species were identified. All these
bacteria were found to significantly enhance fresh and dry weight of root, shoots
and whole 5-week-old seedlings. The bacteria were capable of the in vitro use of
tryptophan to produce indolic compounds at various concentrations. The ability of
almost all the strains to enhance growth of seedlings and individual roots was
positively correlated with the production of the indolic compounds (r = 0.69; P =
0.0001), but not with the 1-aminocyclopropane-1-carboxylate deaminase (ACCD)
activity (no correlation). For some strains, it was difficult to conclude whether
the growth promotion was related to the production of indolic compounds or to the
ACCD activity. It is likely that promotion of M. truncatula root development
involves also root interaction with pseudomonads, known to produce
2,4-diacetylphloroglucinol (DAPG), a secondary metabolite reported to alter the
root architecture by interacting with an auxin-dependent signaling pathway.
Inoculation of seedlings with Pseudomonas brassicacearum KK 5, a bacterium known
for its lowest ability to produce indolic compounds, the highest ACCD activity
and the presence of the phlD gene responsible for DAPG precursor synthesis,
resulted in a substantial promotion of root development. Inoculation with the
strain increased the endogenous IAA level in M. truncatula leaves after
inoculation of 5-week-old seedlings. Three other strains examined in this study
also increased the IAA level in the leaves upon inoculation. Moreover, several
other factors such as mobilization of phosphorus and zinc to make them available
to plants, iron sequestration by siderophore production and the ability to
ammonia production also contributed substantially to the phytostimulatory
biofertilizing potential of isolated strains. There is, thus, evidence that
Medicago truncatula growth promotion by rhizobacteria involves more than one
mechanism.
DOI: 10.1007/s00425-016-2469-7
PMCID: PMC4837224
321. Saudi J Biol Sci. 2016 Jan;23(1):79-86. doi: 10.1016/j.sjbs.2015.06.008. Epub

2015 Jun 17.
Characterization of the plant growth promoting bacterium, Enterobacter cloacae

MSR1, isolated from roots of non-nodulating Medicago sativa.
Khalifa AY(1), Alsyeeh AM(2), Almalki MA(2), Saleh FA(3).
Author information:
(1)Biological Sciences Department, College of Science, King Faisal University,
Saudi Arabia; Botany and Microbiology Department, Faculty of Science, University
of Beni-Suef, Beni-Suef, Egypt.
(2)Biological Sciences Department, College of Science, King Faisal University,
Saudi Arabia.
(3)Department of Food Science and Nutrition, College of Agricultural and Food
Sciences, King Faisal University, Saudi Arabia; Department of Special Food and
Nutrition, Food Technology Research Institute, Agricultural Research Center,
Giza, Egypt.
The aim of the present study was to characterize the endophytic bacterial strain
designated MSR1 that was isolated from inside the non-nodulating roots of
Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter
cloacae using both 16S rDNA gene sequence analysis and API20E biochemical
identification system (Biomerieux, France). Furthermore, this bacterium was
characterized using API50CH kit (Biomerieux, France) and tested for antibacterial
activities against some food borne pathogens. The results showed that E. cloacae
consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin
melibiose as a sole carbon source and certain amino acids such as arginine,
tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple
plant-growth promoting characteristics; phosphate solubility, production of
phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with
MSR1 significantly improved the growth parameters (the length and dry weight) of
this economically important grain legume compared to the non-treated plants. To
our knowledge, this is the first report addressing E. cloacae which exist in
roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E.
cloacae exhibited traits for plant growth promoting and could be developed as an
eco-friendly biofertilizer for P. sativum and probably for other important plant
species in future.
DOI: 10.1016/j.sjbs.2015.06.008
PMCID: PMC4705252
PMID: 26858542
322. G3 (Bethesda). 2016 Apr 7;6(4):925-38. doi: 10.1534/g3.115.026021.
Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene

Transcription, and Genome Methylation.
Rosellini D(1), Ferradini N(2), Allegrucci S(2), Capomaccio S(2), Zago ED(3),
Leonetti P(4), Balech B(5), Aversano R(6), Carputo D(6), Reale L(2), Veronesi
F(2).
Author information:
(1)Department of Agricultural Food and Environmental Sciences, University of
Perugia, Borgo XX giugno 74, 06121, Perugia, Italy daniele.rosellini@unipg.it.
Perugia, Borgo XX giugno 74, 06121, Perugia, Italy.
(3)Department of Biotechnology, University of Verona, Ca' Vignal 1, Strada Le
Grazie 15, 37134 Verona, Italy.
(4)Institute for Sustainable Plant Protection, CNR, Via Amendola 122/D, 70126
Bari, Italy.
(5)Istituto di Biomembrane e Bioenergetica, CNR, Via Giovanni Amendola 165/A,
70126 Bari, Italy.
(6)Department of Agriculture, University of Naples Federico II, via Università
100, 80055 Portici, Naples, Italy.
Polyploidization as the consequence of 2n gamete formation is a prominent

mechanism in plant evolution. Studying its effects on the genome, and on genome
expression, has both basic and applied interest. We crossed two diploid
(2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a
coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen,
respectively. Such a cross produced full-sib diploid and tetraploid
(2n = 4x = 32) hybrids, the latter being the result of bilateral sexual
polyploidization (BSP). These unique materials allowed us to investigate the
effects of BSP, and to separate the effect of intraspecific hybridization from
those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple
sequence repeat marker segregation demonstrated tetrasomic inheritance for all
chromosomes but one, demonstrating that these neotetraploids are true
autotetraploids. BSP brought about increased biomass, earlier flowering, higher
seed set and weight, and larger leaves with larger cells. Microarray analyses
with M. truncatula gene chips showed that several hundred genes, related to
diverse metabolic functions, changed their expression level as a consequence of
polyploidization. In addition, cytosine methylation increased in 2x, but not in
4x, hybrids. Our results indicate that sexual polyploidization induces
significant transcriptional novelty, possibly mediated in part by DNA
methylation, and phenotypic novelty that could underpin improved adaptation and
reproductive success of tetraploid M. sativa with respect to its diploid
progenitor. These polyploidy-induced changes may have promoted the adoption of
tetraploid alfalfa in agriculture.
Copyright © 2016 Rosellini et al.
DOI: 10.1534/g3.115.026021
PMCID: PMC4825662
323. Microb Cell Fact. 2016 Feb 4;15:27. doi: 10.1186/s12934-016-0424-8.
Transcriptome-enabled discovery and functional characterization of enzymes

related to (2S)-pinocembrin biosynthesis from Ornithogalum caudatum and their
application for metabolic engineering.
Guo L(1), Chen X(2)(3), Li LN(4), Tang W(5), Pan YT(6), Kong JQ(7).
Author information:
(1)Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College (State Key Laboratory of Bioactive Substance and Function
of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of
Natural Products), Beijing, 100050, China. guoleiglly@163.com.
Natural Products), Beijing, 100050, China. cx-pharmacy@126.com.
(3)School of Medicine of Wuhan University, Wuhan, China. cx-pharmacy@126.com.
Natural Products), Beijing, 100050, China. lena@imm.ac.cn.
(5)School of Medicine of Wuhan University, Wuhan, China. tangweidoctor@163.com.
(6)School of Chemical Engineering, Beijing Institute of Petrochemical Technology,
Beijing, China. panyiting@bipt.edu.cn.
Natural Products), Beijing, 100050, China. jianqiangk@imm.ac.cn.
BACKGROUND: (2S)-Pinocembrin is a chiral flavanone with versatile pharmacological

and biological activities. Its health-promoting effects have spurred on research
effects on the microbial production of (2S)-pinocembrin. However, an
often-overlooked salient feature in the analysis of microbial (2S)-pinocembrin is
its chirality.
RESULTS: Here, we presented a full characterization of absolute configuration of
microbial (2S)-pinocembrin from engineered Escherichia coli. Specifically, a
transcriptome-wide search for genes related to (2S)-pinocembrin biosynthesis from
Ornithogalum caudatum, a plant rich in flavonoids, was first performed in the
present study. A total of 104,180 unigenes were finally generated with an average
length of 520 bp. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
mapping assigned 26 unigenes, representing three enzyme families of
4-coumarate:coenzyme A ligase (4CL), chalcone synthase (CHS) and chalcone
isomerase(CHI), onto (2S)-pinocembrin biosynthetic pathway. A total of seven,
three and one full-length candidates encoding 4CL, CHS and CHI were then verified
by reverse transcription polymerase chain reaction, respectively. These
candidates were screened by functional expression in E. coli individual or
coupled multienzyme reaction systems based on metabolic engineering processes.
Oc4CL1, OcCHS2 and OcCHI were identified to be bona fide genes encoding
respective pathway enzymes of (2S)-pinocembrin biosynthesis. Then Oc4CL1, OcCHS2
and MsCHI from Medicago sativa, assembled as artificial gene clusters in
different organizations, were used for fermentation production of
(2S)-pinocembrin in E. coli. The absolute configuration of the resulting
microbial pinocembrin at C-2 was assigned to be 2S-configured by combination of
retention time, UV spectrum, LC-MS, NMR, optical rotation and circular dichroism
spectroscopy. Improvement of (2S)-pinocembrin titres was then achieved by
optimization of gene organizations, using of codon-optimized pathway enzymes and
addition of cerulenin for increasing intracellular malonyl CoA pools. Overall,
the optimized strain can produce (2S)-pinocembrin of 36.92 ± 4.1 mg/L.
CONCLUSIONS: High titre of (2S)-pinocembrin can be obtained from engineered E.
coli by an efficient method. The fermentative production of microbial
(2S)-pinocembrin in E. coli paved the way for yield improvement and further
pharmacological testing.
DOI: 10.1186/s12934-016-0424-8
PMCID: PMC4743118
324. Environ Sci Pollut Res Int. 2016 May;23(9):9215-26. doi:
10.1007/s11356-015-5972-7. Epub 2016 Feb 2.
Citric acid- and Tween(®) 80-assisted phytoremediation of a co-contaminated soil:

alfalfa (Medicago sativa L.) performance and remediation potential.
Agnello AC(1)(2), Huguenot D(3), van Hullebusch ED(1), Esposito G(2).
Author information:
(1)Université Paris-Est, Laboratoire Géomatériaux et Environnement (EA 4508),
UPEM, 77454, Marne-la-Vallée, France.
(2)Dipartimento di Ingegneria Civile e Meccanica, Università degli Studi di
Cassino e del Lazio Meridionale, via Di Biasio 43, 03043, Cassino, FR, Italy.
UPEM, 77454, Marne-la-Vallée, France. david.huguenot@u-pem.fr.
A pot experiment was designed to assess the phytoremediation potential of alfalfa

(Medicago sativa L.) in a co-contaminated (i.e., heavy metals and petroleum
hydrocarbons) soil and the influence of citric acid and Tween(®) 80 (polyethylene
glycol sorbitan monooleate), applied individually and combined together, for
their possible use in chemically assisted phytoremediation. The results showed
that alfalfa plants could tolerate and grow in a co-contaminated soil. Over a
90-day experimental time, shoot and root biomass increased and negligible plant
mortality occurred. Heavy metals were uptaken by alfalfa to a limited extent,
mostly by plant roots, and their concentration in plant tissues were in the
following order: Zn > Cu > Pb. Microbial population (alkane-degrading
microorganisms) and activity (lipase enzyme) were enhanced in the presence of
alfalfa with rhizosphere effects of 9.1 and 1.5, respectively, after 90 days.
Soil amendments did not significantly enhance plant metal concentration or total
uptake. In contrast, the combination of citric acid and Tween(®) 80 significantly
improved alkane-degrading microorganisms (2.4-fold increase) and lipase activity
(5.3-fold increase) in the rhizosphere of amended plants, after 30 days of
experiment. This evidence supports a favorable response of alfalfa in terms of
tolerance to a co-contaminated soil and improvement of rhizosphere microbial
number and activity, additionally enhanced by the joint application of citric
acid and Tween(®) 80, which could be promising for future phytoremediation
applications.
DOI: 10.1007/s11356-015-5972-7

eCollection 2016.
The Many Dimensions of Diet Breadth: Phytochemical, Genetic, Behavioral, and

Physiological Perspectives on the Interaction between a Native Herbivore and an
Exotic Host.
Harrison JG(1)(2), Gompert Z(3), Fordyce JA(4), Buerkle CA(5), Grinstead R(2),
Jahner JP(2), Mikel S(2), Nice CC(6), Santamaria A(2), Forister ML(1)(2).
Author information:
(1)Program in Ecology, Evolution, and Conservation Biology, Department of
Biology, University of Nevada, Reno, NV, 89557, United States of America.
(2)Department of Biology, University of Nevada, Reno, NV, 89557, United States of
America.
(3)Department of Biology, Utah State University, Logan, UT, 84322, United States
of America.
(4)Department of Ecology and Evolutionary Biology, University of Tennessee,
Knoxville, TN, 37996, United States of America.
(5)Department of Botany, University of Wyoming, Laramie, WY, 82071, United States
of America.
(6)Department of Biology, Texas State University, San Marcos, TX, 78666, United
States of America.
From the perspective of an herbivorous insect, conspecific host plants are not
identical, and intraspecific variation in host nutritional quality or defensive
capacity might mediate spatially variable outcomes in plant-insect interactions.
Here we explore this possibility in the context of an ongoing host breadth
expansion of a native butterfly (the Melissa blue, Lycaeides melissa) onto an
exotic host plant (alfalfa, Medicago sativa). We examine variation among seven
alfalfa populations that differed in terms of colonization by L. melissa;
specifically, we examined variation in phytochemistry, foliar protein, and plant
population genetic structure, as well as responses of caterpillars and adult
butterflies to foliage from the same populations. Regional patterns of alfalfa
colonization by L. melissa were well predicted by phytochemical variation, and
colonized patches of alfalfa showed a similar level of inter-individual
phytochemical diversity. However, phytochemical variation was a poor predictor of
larval performance, despite the fact that survival and weight gain differed
dramatically among caterpillars reared on plants from different alfalfa
populations. Moreover, we observed a mismatch between alfalfa supporting the best
larval performance and alfalfa favored by ovipositing females. Thus, the axes of
plant variation that mediate interactions with L. melissa depend upon herbivore
life history stage, which raises important issues for our understanding of
adaptation to novel resources by an organism with a complex life history.
PMCID: PMC4737494
Transcriptome analysis of Glomus mosseae/Medicago sativa mycorrhiza on atrazine

stress.
Song F(1), Li J(1), Fan X(1), Zhang Q(2), Chang W(1), Yang F(1), Geng G(1).
Author information:
(1)Bio-ecological Restoration, School of Life Science, Heilongjiang University,
Harbin, 74 Xuefu Road Heilongjiang 150080, China.
(2)Department of Biology, Washington University at St Louis, Saint Louis, 1
Brookings Drive MO 63130, USA.
Arbuscular mycorrhizal fungi (AMF) protect host plants against diverse biotic and
abiotic stresses, and promote biodegradation of various contaminants. In this
study effect of Glomus mosseae/Medicago sativa mycorrhiza on atrazine degradation
was investigated. It was observed that the atrazine degradation rates with any
addition level in mycorrhizal treatments were all significantly higher than those
in non-mycorrhizal treatments. When atrazine was applied at 20 mg kg(-1), the
removal efficiency was up to 74.65%. Therefore, G. mosseae can be considered as
ideal inhabitants of technical installations to facilitate phytoremediation.
Furthermore, a total of 10.4 Gb was used for de novo transcriptome assembly,
resulting in a comprehensive data set for the identification of genes
corresponding to atrazine stress in the AM association. After comparative
analysis with edgeR, a total of 2,060 differential expressed genes were
identified, including 570 up-regulated genes and 1490 down-regulated genes. After
excluding 'function unknown' and 'general function predictions only' genes, 172
up-regulated genes were obtained. The differentially expressed genes in AM
association with and without atrazine stress were associated with molecular
processes/other proteins, zinc finger protein, intracellular/extracellular
enzymes, structural proteins, anti-stress/anti-disease protein, electron
transport-related protein, and plant growth associated protein. Our results not
only prove AMF has important ecological significance on atrazine degradation but
also provide evidence for the molecular mechanisms of atrazine degradation by
AMF.
DOI: 10.1038/srep20245
PMCID: PMC4735738
327. Neotrop Entomol. 2016 Apr;45(2):211-6. doi: 10.1007/s13744-015-0357-7. Epub

2016
Feb 1.
Characterization of Feeding Injuries Caused by Ceresa nigripectus Remes Lenicov

(Hemiptera: Membracidae) on Alfalfa Stems.
Grosso TP(1), Mercado MI(2), Ponessa GI(3), Conci LR(1), Virla EG(4).
Author information:
(1)Centro de Investigaciones Agropecuarias (CIAP), Instituto de Patología Vegetal
(IPAVE), INTA, Córdoba, Argentina.
(2)Área Botánica, Instituto de Morfología Vegetal, Fundación Miguel Lillo, Miguel
Lillo 251, San Miguel de Tucumán,, T4000JFE, Argentina. mainesmer@yahoo.com.ar.
(3)Área Botánica, Instituto de Morfología Vegetal, Fundación Miguel Lillo, Miguel
Lillo 251, San Miguel de Tucumán,, T4000JFE, Argentina.
(4)Instituto de Entomología, CONICET and Fundación Miguel Lillo, San Miguel de
Tucumán, Argentina.
Piercing-sucking insects cause mechanical and physiological injury to plants.

Ceresa nigripectus Remes Lenicov is a pest of alfalfa in subtropical regions of
South America and a carrier of the ArAWB phytoplasma. The aim of this study was
to determine the feeding habits of this treehopper and to describe the effects of
the feeding injuries on stem vascular tissues in alfalfa. Adults and nymphs of C.
nigripectus inserted their stylets repeatedly girdling the stem. One week after
feeding, alfalfa stems exhibited numerous feeding canals with salivary deposits,
most of which reached the phloem. Two weeks after feeding, cortex and phloem
cells next to the salivary sheath collapsed, mature tracheal elements became
sparse and appeared with an increased cross-section area, and phenolic compounds
increased in cells and cell walls compared to undamaged plants. Three weeks after
feeding, an annular callus, formed by abnormal cell division and hypertrophy of
preexisting cortex and vascular cambium cells, appeared immediately above the
stem girdle. Parenchyma cells from the outer layers of the callus differentiated
to form secondary anomalous amphicribal bundles in the wound. The aerial parts
above the stem girdle eventually withered and died.
DOI: 10.1007/s13744-015-0357-7

10.1007/s11356-016-6135-1. Epub 2016 Jan 29.
Phytoremediation with Geosiphon-like symbiosis?
Wojtczak G(1), Janik P(2).
Author information:
(1)Institute of Nature Conservation (Protection), Polish Academy of Sciences,
Mickiewicza 33, Kraków, 31-120, Poland. g.wojtczak@gmail.com.
(2)W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, Krakow,
31-512, Poland.
Comment on
Environ Sci Pollut Res Int. 2015 Dec;22(23):19142-56.
DOI: 10.1007/s11356-016-6135-1
PMCID: PMC4819920

eCollection 2016.
Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral

Blood Natural Killer Cells: A Randomized, Double-Blind Study.
Müller L(1)(2), Meyer M(3), Bauer RN(1)(4), Zhou H(5), Zhang H(5), Jones S(1),
Robinette C(1), Noah TL(1)(6), Jaspers I(1)(6).
Author information:
(1)Center for Environmental Medicine, Asthma and Lung Biology, University of
North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of
America.
(2)University Children's Hospital Basel, Basel, Switzerland.
(3)Department of Microbiology and Immunology, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina, United States of America.
(4)Department of Pediatric Allergy and Immunology, Stanford University, Stanford,
California, United States of America.
(5)Department of Biostatistics, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina, United States of America.
(6)Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina, United States of America.
Enhancing antiviral host defense responses through nutritional supplementation

would be an attractive strategy in the fight against influenza. Using inoculation
with live attenuated influenza virus (LAIV) as an infection model, we have
recently shown that ingestion of sulforaphane-containing broccoli sprout
homogenates (BSH) reduces markers of viral load in the nose. To investigate the
systemic effects of short-term BSH supplementation in the context of
LAIV-inoculation, we examined peripheral blood immune cell populations in
non-smoking subjects from this study, with a particular focus on NK cells. We
carried out a randomized, double-blinded, placebo-controlled study measuring the
effects of BSH (N = 13) or placebo (alfalfa sprout homogenate, ASH; N = 16) on
peripheral blood mononuclear cell responses to a standard nasal vaccine dose of
LAIV in healthy volunteers. Blood was drawn prior to (day-1) and post (day2,
day21) LAIV inoculation and analyzed for neutrophils, monocytes, macrophages, T
cells, NKT cells, and NK cells. In addition, NK cells were enriched, stimulated,
and assessed for surface markers, intracellular markers, and cytotoxic potential
by flow cytometry. Overall, LAIV significantly reduced NKT (day2 and day21) and T
cell (day2) populations. LAIV decreased NK cell CD56 and CD158b expression, while
significantly increasing CD16 expression and cytotoxic potential (on day2). BSH
supplementation further increased LAIV-induced granzyme B production (day2) in NK
cells compared to ASH and in the BSH group granzyme B levels appeared to be
negatively associated with influenza RNA levels in nasal lavage fluid cells. We
conclude that nasal influenza infection may induce complex changes in peripheral
blood NK cell activation, and that BSH increases virus-induced peripheral blood
NK cell granzyme B production, an effect that may be important for enhanced
antiviral defense responses.TRIAL REGISTRATION: ClinicalTrials.gov NCT01269723.
PMCID: PMC4731143
330. Plant Methods. 2016 Jan 26;12:5. doi: 10.1186/s13007-016-0110-1. eCollection

2016.
An improved microtiter plate assay to monitor the oxidative burst in monocot and
dicot plant cell suspension cultures.
Melcher RL(1), Moerschbacher BM(1).
Author information:
(1)Institute for Biology and Biotechnology of Plants, University of Münster,
Schlossplatz 8, 48143 Münster, Germany.
BACKGROUND: A screening method for elicitor and priming agents does not only
allow detecting new bioactive substances, it can also be used to understand
structure-function relationships of known agents by testing different derivatives
of them. This can not only provide new lead compounds for the development of
novel, more environment-benign, bio-based agro-chemicals, it may eventually also
lead to a better understanding of defense mechanisms in plants. Reactive oxygen
species (ROS) are sensitive indicators of these mechanisms but current assay
formats are not suitable for multiplex screening, in particularly not in the case
of monocot systems.
RESULTS: Here we describe continuous monitoring of ROS in 96-well microtiter
plates using the chemiluminescent probe L012, a luminol derivative producing
chemiluminescence when oxidised by ROS like hydrogen peroxide, superoxide, or
hydroxyl radical that can thus be used as an indicator for these ROS. We were
able to measure ROS in both monocot (Oryza sativa) and dicot (Medicago
truncatula) cell suspension cultures and record dose dependencies for the
carbohydrate elicitors and priming agents ulvan and chitosan at low substrate
concentrations (0.3-2.5 µg/ml). The method was optimized in terms of cell
density, L012 concentration, and pre-incubation time. In contrast to the single
peak observed using a cuvette luminometer, the improved method revealed a double
burst in both cell systems during the 90-min measuring period, probably due to
the detection of multiple ROS rather than only H2O2.
CONCLUSION: We provide a medium throughput screening method for monocot and dicot
suspension-cultured cells that enables direct comparison of monocot and dicot
plant systems regarding their reaction to different signaling molecules.
DOI: 10.1186/s13007-016-0110-1
PMCID: PMC4729151
PMID: 26819624
331. Microbiology. 2016 Mar;162(3):459-65. doi: 10.1099/mic.0.000245. Epub 2016 Jan

26.
Natural variation in methane emission of sheep fed on a lucerne pellet diet is

unrelated to rumen ciliate community type.
Kittelmann S(1), Pinares-Patiño CS(1), Seedorf H(1), Kirk MR(1), McEwan JC(2),
Janssen PH(1).
Author information:
(1)1AgResearch Ltd, Grasslands Research Centre, Palmerston, North 4442, New
Zealand.
(2)2AgResearch Ltd, Invermay Agricultural Centre, Mosgiel 9053, New Zealand.
Only limited information is available on the roles of different rumen ciliate

community types, first described by Eadie in 1962, in enteric methane (CH4)
formation by their ruminant hosts. If the different types were differentially
associated with CH4 formation, then ciliate community typing could be used to
identify naturally high and low CH4-emitting animals. Here we measured the CH4
yields [g CH4 (kg feed dry matter intake, DMI)(-1)] of 118 sheep fed a standard
pelleted lucerne diet at two different times, at least 2 weeks apart. There were
significant differences (P < 2.2 × 10(-16), Wilcoxon rank sum test) in the CH4
yields (± sd) from sheep selected as high [16.7 ± 1.5 g CH4 (kg DMI)(-1)] and low
emitters [13.3 ± 1.5 g CH4 (kg DMI)(-1)]. A rumen sample was collected after each
of the two measurements, and ciliate composition was analysed using barcoded 454
Titanium pyrosequencing of 18S rRNA genes. The genera found, in order of mean
relative abundance, were Epidinium, Entodinium, Dasytricha, Eudiplodinium,
Polyplastron, Isotricha and Anoplodinium-Diplodinium, none of which was
significantly correlated with the CH4 emissions ranking associated with the rumen
sample. Ciliate communities naturally assembled into four types (A, AB, B and O),
characterized by the presence and absence of key genera. There was no difference
in CH4 yield between sheep that harboured different ciliate community types,
suggesting that these did not underlie the natural variation in CH4 yields.
Further research is needed to unravel the nature of interactions between ciliate
protozoa and other rumen micro-organisms, which may ultimately lead to
contrasting CH4 emission phenotypes.
DOI: 10.1099/mic.0.000245
332. Microbiology. 2016 Mar;162(3):552-63. doi: 10.1099/mic.0.000239. Epub 2016 Jan

25.
Sinorhizobium meliloti low molecular mass phosphotyrosine phosphatase SMc02309

modifies activity of the UDP-glucose pyrophosphorylase ExoN involved in
succinoglycan biosynthesis.
Medeot DB(1), Romina Rivero M(2), Cendoya E(2), Contreras-Moreira B(3), Rossi
FA(2), Fischer SE(2), Becker A(4), Jofré E(2).
Author information:
(1)1Department of Natural Sciences, FCEFQyN, National University of Río Cuarto,
Ruta Nacional 36 Km 601, Córdoba, Argentina 2Department of Molecular Biology,
FCEFQyN, National University of Río Cuarto, Ruta Nacional 36 Km 601, Córdoba,
Argentina.
(2)1Department of Natural Sciences, FCEFQyN, National University of Río Cuarto,
Ruta Nacional 36 Km 601, Córdoba, Argentina.
(3)3Laboratory of Computational Biology, Department of Genetics and Plant
Production, Estación Experimental de Aula Dei/CSIC, Av. Montañana 1005, Zaragoza,
Spain.
(4)4LOEWE-Center for Synthetic Microbiology, Philipps-Universität Marburg,
D-35032, Marburg, Germany.
In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role
in the control of exopolysaccharide (EPS) production. This study demonstrated
that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a
protein with significant sequence similarity to low molecular mass
protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb.
Unlike other well-characterized EPS biosynthesis gene clusters, which contain
neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene
cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical
assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate
(p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore,
we show evidence that SMc02309 is not the LMW-PTP of the bacterial
tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose
pyrophosphorylase involved in the first stages of EPS I biosynthesis, is
phosphorylated at tyrosine residues and constitutes an endogenous substrate of
the SMc02309 protein. Additionally, we show that the UDP-glucose
pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine
dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I
production and delays nodulation on Medicago sativa roots.
DOI: 10.1099/mic.0.000239
333. J Anim Sci. 2016 Jan;94(1):349-58. doi: 10.2527/jas.2015-9437.
Effect of dietary replacement of alfalfa with urea-treated almond hulls on

intake, growth, digestibility, microbial nitrogen, nitrogen retention, ruminal
fermentation, and blood parameters in fattening lambs.
Rad MI, Rouzbehan Y, Rezaei J.
The objective of this study was to assess the effect of dietary replacement of
alfalfa with urea-treated almond hulls (UAH) on DM and nutrients intakes, growth
performance, diet digestibility, microbial N supply (MNS), N retention, rumen
fermentation parameters, and blood metabolites in fattening male Shall lambs
(29.9 ± 1.9 kg initial BW). Three diets, with equal ME and CP concentrations and
a forage-to-concentrate ratio of 40 to 60, were formulated in which alfalfa was
replaced by different levels (0, 200, or 400 g/kg of diet DM) of UAH.
Experimental diets were randomly assigned to the 3 groups ( = 8/group) in a
completely randomized design for a 74-d period (14 d for adaptation and 60 d for
data collection). Diets were offered as a total mixed ration to ensure 10% orts.
Dry matter and nutrients intakes, animal growth, diet digestibility, MNS, N
retention, rumen fermentation parameters, and plasma metabolites were determined.
The dietary substitution of UAH for alfalfa had no effects on DMI (linear, =
0.96; quadratic, = 0.86), ADG (linear, = 0.35; quadratic, = 0.19), and G:F
(linear, = 0.66; quadratic, = 0.13). In vivo digestibility coefficients of DM
(linear, = 0.82; quadratic, = 0.42), OM (linear, = 0.73; quadratic, = 0.95), CP
(linear, = 0.24; quadratic, = 0.66), and ash-free NDF (linear, = 0.69; quadratic,
= 0.74) were not affected by the dietary treatment. Feeding lambs on diets
containing UAH instead of alfalfa had no effects on MNS (linear, = 0.63;
quadratic, = 0.68) and N retention (linear, = 0.44; quadratic, = 0.17). Rumen pH
(linear, = 0.26; quadratic, = 0.071), ammonia N (linear, = 0.39; quadratic, =
0.13), and VFA (linear, = 0.091; quadratic, = 0.86) concentrations, acetic
acid-to-propionic acid ratio (linear, = 0.93; quadratic, = 0.62), and protozoa
population (linear, = 0.62; quadratic, = 0.22) were not influenced by the
experimental diets. Substituting alfalfa with UAH had no effects on the plasma
concentrations of glucose (linear, = 0.55; quadratic, = 0.91), triglycerides
(linear, = 0.97; quadratic, = 0.44), cholesterol (linear, = 0.71; quadratic, =
0.70), urea N (linear, = 0.084; quadratic, = 0.12), total protein (linear, =
0.53; quadratic, = 0.96), albumin (linear, = 0.43; quadratic, = 0.39), and
globulin (linear, = 0.39; quadratic, = 0.25). It is concluded that UAH can be fed
to fattening Shall lambs as a total replacement (400 g/kg of diet DM) for alfalfa
without negative effects on animal performance. This byproduct can be a safe
feedstuff to use in sheep diets and could help to reduce environmental pollution.
DOI: 10.2527/jas.2015-9437
334. J Anim Sci. 2016 Jan;94(1):248-54. doi: 10.2527/jas.2015-9649.
Estimation of the indigestible fiber in different forage types.
Palmonari A, Gallo A, Fustini M, Canestrari G, Masoero F, Sniffen CJ, Formigoni

A.
The role of indigestible NDF is essential in relation to OM digestibility

prediction, total tract digestibility, rumen fill, passage rate, and digestion
kinetics. Moreover, the truly indigestible NDF (iNDF) represents a core point in
dynamic models used for diet formulations. However, despite its wide possible
applications, few trials have been conducted to quantify iNDF and even fewer to
investigate whether or not it is consistent among different forage sources. The
objective of this study was to predict the iNDF by measuring the residual NDF
after 240-h in vitro fermentation to determine the unavailable NDF (uNDF) within
and among various forage types. Finally, a mathematical approach was investigated
for the estimation of the uNDF fraction. In all, 688 forages were analyzed in
this study. This pool included 122 alfalfa hays, 282 corn silages, and 284 grass
hays. Values of uNDF varied among different forages and within the same type
(22.7% ± 4.48%, 20.1% ± 4.23%, and 11.8% ± 3.5% DM for grass hay, alfalfa hay,
and corn silages, respectively). The relationship among uNDF and ADL was not
constant and, for grass hay and corn silage, was different ( 0.05) from the 2.4 ×
lignin value applied by the traditional Chandler equation. The observed uNDF:ADL
ratio was 3.22 for grass hay and 3.11 for corn silage. Relationships among
chemical and biological parameters and uNDF were investigated via simple and
multiple regression equations. The greatest correlation with a single variable
was obtained by ADL and ADF when applied to the whole data set ( = 0.63). Greater
coefficients of determination resulted from a multiple regression equation for
the whole data set ( = 0.80) and within each forage type ( = 0.65, 0.77, and 0.54
for grass hay, alfalfa hay, and corn silage, respectively). In conclusion, a
regression approach requires specific equations and different regression
coefficients for each forage type. The direct measurement of uNDF represented the
best approach to obtain an accurate prediction of the iNDF and to optimize its
specific purpose in dynamic nutrition models.
DOI: 10.2527/jas.2015-9649
335. Plant Biotechnol J. 2016 Jul;14(7):1604-18. doi: 10.1111/pbi.12524. Epub 2016

Jan
24.
Metabolic engineering of proanthocyanidin production by repressing the isoflavone

pathways and redirecting anthocyanidin precursor flux in legume.
Li P(1), Dong Q(1), Ge S(2)(3), He X(3)(4), Verdier J(5), Li D(1), Zhao J(1).
Author information:
(1)National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural
University, Wuhan, China.
(2)College of Agronomy, Agricultural University of Hebei, Baoding, China.
(3)Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK, USA.
(4)Department of Plant and Microbial Biology, North Carolina State University,
Raleigh, NC, USA.
(5)Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Science, Shanghai, China.
MtPAR is a proanthocyanidin (PA) biosynthesis regulator; the mechanism underlying

its promotion of PA biosynthesis is not fully understood. Here, we showed that
MtPAR promotes PA production by a direct repression of biosynthesis of
isoflavones, the major flavonoids in legume, and by redirecting immediate
precursors, such as anthocyanidins, flux into PA pathway. Ectopic expression of
MtPAR repressed isoflavonoid production by directly binding and suppressing
isoflavone biosynthetic genes such as isoflavone synthase (IFS). Meanwhile, MtPAR
up-regulated PA-specific genes and decreased the anthocyanin levels without
altering the expression of anthocyanin biosynthetic genes. MtPAR may shift the
anthocyanidin precursor flux from anthocyanin pathway to PA biosynthesis. MtPAR
complemented PA-deficient phenotype of Arabidopsis tt2 mutant seeds,
demonstrating their similar action on PA production. We showed the direct
interactions between MtPAR, MtTT8 and MtWD40-1 proteins from Medicago truncatula
and Glycine max, to form a ternary complex to trans-activate PA-specific ANR
gene. Finally, MtPAR expression in alfalfa (Medicago sativa) hairy roots and
whole plants only promoted the production of small amount of PAs, which was
significantly enhanced by co-expression of MtPAR and MtLAP1. Transcriptomic and
metabolite profiling showed an additive effect between MtPAR and MtLAP1 on the
production of PAs, supporting that efficient PA production requires more
anthocyanidin precursors. This study provides new insights into the role and
mechanism of MtPAR in partitioning precursors from isoflavone and anthocyanin
pathways into PA pathways for a specific promotion of PA production. Based on
this, a strategy by combining MtPAR and MtLAP1 co-expression to effectively
improve metabolic engineering performance of PA production in legume forage was
developed.
© 2016 The Authors. Plant Biotechnology Journal published by Society for

Experimental Biology and The Association of Applied Biologists and John Wiley &
Sons Ltd.
DOI: 10.1111/pbi.12524
PMCID: PMC5066740
336. G3 (Bethesda). 2016 Jan 22;6(3):755-65. doi: 10.1534/g3.115.025981.
Genome-Wide Investigation of MicroRNAs and Their Targets in Response to Freezing

Stress in Medicago sativa L., Based on High-Throughput Sequencing.
Shu Y(1), Liu Y(2), Li W(2), Song L(2), Zhang J(2), Guo C(1).
Author information:
150025, China syjun2003@126.com kaku_2008@163.com.
150025, China.
Winter damage, especially in northern climates, is a major limitation of the
utilization of perennial forages such as alfalfa. Therefore, improving freezing
tolerance is imperative in alfalfa genetic breeding. However, freezing tolerance
is a complex trait that is determined by many genes. To understand the complex
regulation mechanisms of freezing tolerance in alfalfa, we performed small RNA
sequencing analysis under cold (4°) and freezing (-8°) stress. The sequencing
results revealed that 173 known, and 24 novel miRNAs were expressed, and that the
expression of 35 miRNAs was affected by cold and/or freezing stress. Meanwhile,
105 target genes cleaved by these miRNAs were characterized by degradome
sequencing. These targets were associated with biological regulation, cellular
processes, metabolic processes, and response to stress. Interestingly, most of
them were characterized as transcription factors (TFs), including auxin response
factors, SBP, NAC, AP2/ERF, and GRF, which play important roles in plant abiotic
responses. In addition, important miRNAs and mRNAs involved in nodulation were
also identified, for example, the relationship between miR169 and the TF CCAAT
(also named as NF-YA/HAP2), which suggested that nodulation has an important
function in freezing tolerance in alfalfa. Our results provide valuable
information to help determine the molecular mechanisms of freezing tolerance in
alfalfa, which will aid the application of these miRNAs and their targets in the
improvement of freezing tolerance in alfalfa and related plants.
Copyright © 2016 Shu et al.
DOI: 10.1534/g3.115.025981
PMCID: PMC4777136
337. Front Plant Sci. 2016 Jan 12;6:1256. doi: 10.3389/fpls.2015.01256. eCollection
2015.
Comparative Physiological and Transcriptional Analyses of Two Contrasting Drought

Tolerant Alfalfa Varieties.
Quan W(1), Liu X(1), Wang H(2), Chan Z(3).
Author information:
(1)Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan
Botanical Garden/Sino-Africa Joint Research Center - Chinese Academy of
SciencesWuhan, China; University of Chinese Academy of SciencesBeijing, China.
Institute of Plateau Biology - Chinese Academy of Sciences Xining, China.
(3)Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan
Botanical Garden/Sino-Africa Joint Research Center - Chinese Academy of Sciences
Wuhan, China.
Drought is one of major environmental determinants of plant growth and

productivity. Alfalfa (Medicago sativa) is a legume perennial forage crop native
to the arid and semi-arid environment, which is an ideal candidate to study the
biochemical and molecular mechanisms conferring drought resistance in plants. In
this study, drought stress responses of two alfalfa varieties, Longdong and
Algonquin, were comparatively assayed at the physiological, morphological, and
transcriptional levels. Under control condition, the drought-tolerant Longdong
with smaller leaf size and lower stomata density showed less water loss than the
drought-sensitive Algonquin. After exposing to drought stress, Longdong showed
less severe cell membrane damage, more proline, and ascorbate (ASC) contents and
less accumulation of H2O2 than Algonquin. Moreover, significantly higher
antioxidant enzymes activities after drought treatment were found in Longdong
when compared with Algonquin. In addition, transcriptional expression analysis
showed that Longdong exhibited significantly higher transcripts of
drought-responsive genes in leaf and root under drought stress condition. Taken
together, these results indicated that Longdong variety was more drought-tolerant
than Algonquin variety as evidenced by less leaf firing, more lateral root
number, higher relative aboveground/underground biomass per plant and survival
rate.
DOI: 10.3389/fpls.2015.01256
PMCID: PMC4709457
PMID: 26793226
338. J Dairy Sci. 2016 Mar;99(3):1895-1902. doi: 10.3168/jds.2015-9792. Epub 2016

Jan
13.
Effects of lactic acid bacteria with bacteriocinogenic potential on the

fermentation profile and chemical composition of alfalfa silage in tropical
conditions.
Silva VP(1), Pereira OG(2), Leandro ES(1), Da Silva TC(1), Ribeiro KG(1),
Mantovani HC(3), Santos SA(4).
Author information:
(1)Departamento de Zootecnia, Universidade Federal de Vicosa, Vicosa, Minas
Gerais, Brazil 36570000.
(2)Departamento de Zootecnia, Universidade Federal de Vicosa, Vicosa, Minas
Gerais, Brazil 36570000. Electronic address: odilon@ufv.br.
(3)Departamento de Microbiologia Agrícola, Universidade Federal de Vicosa,
Vicosa, Minas Gerais, Brazil 36570000.
(4)Departamento de Zootecnia, Universidade Federal da Bahia, Bahia, Brazil
40170110.
The fermentation profile, chemical composition, and microbial populations of

alfalfa silages treated with microbial inoculants (MI) at different fermentation
periods (T) were evaluated in tropical conditions. A 4×6 factorial arrangement
was used in a randomized design with 3 replicates. Fresh alfalfa was treated with
(1) no treatment (CTRL), (2) commercial inoculant (CIN), (3) Pediococcus
acidilactici (strain 10.6, S1), and (4) Pediococcus pentosaceus (strain 6.16,
S2). An inoculant application rate of 10(6) cfu/g of fresh forage was used. The
fermentation periods were 1, 3, 7, 14, 28, and 56 d. Alfalfa was harvested 82 d
after sowing at the early flowering stage, chopped into 1.5-cm particle size, and
ensiled in 25 × 35 cm vacuum-sealed plastic bags. The numbers of lactic acid
bacteria, enterobacteria, mold, and yeast in alfalfa before ensiling were 5.42,
5.58, 4.82, and 4.8 log cfu/g, respectively. Silage chemical composition was
evaluated only at 56 d. All parameters were affected by the interaction MI × T,
except the concentrations of lactic and propionic acids. Alfalfa silage treated
with S1 or S2 had lower pH values than CTRL from the first day until 28 d.
However, the inoculants resulted in similar pH after 56 d, and these values were
lower than the CTRL. The highest concentration of lactic acid was observed in the
silage treated with S1 and S2 at 7 and 14 d of ensiling. The concentration of
acetic acid was lower in the silages treated with S1 and S2 than the CTRL and CIN
at 3 and 28 d of fermentation. There was no effect of MI or MI × T interaction on
the microbial populations. However, the number of enterobacteria decreased over
the fermentation period until 14 d and increased slightly after this time point.
The chemical composition of alfalfa silage was not affected by MI at 56 d of
ensiling. The strain P. pentosaceus 6.16 was the most efficient in dominating the
fermentation process by decreasing the pH more quickly and increasing the
concentration of lactic acid, suggesting its potential use as a silage inoculant.

DOI: 10.3168/jds.2015-9792
339. Environ Microbiol. 2016 Sep;18(8):2534-47. doi: 10.1111/1462-2920.13221. Epub

2016 Feb 16.
Genomic resources for identification of the minimal N2 -fixing symbiotic genome.
diCenzo GC(1), Zamani M(1), Milunovic B(1), Finan TM(1).
Author information:
Ontario, Canada, L8S 4K1.
The lack of an appropriate genomic platform has precluded the use of

gain-of-function approaches to study the rhizobium-legume symbiosis, preventing
the establishment of the genes necessary and sufficient for symbiotic nitrogen
fixation (SNF) and potentially hindering synthetic biology approaches aimed at
engineering this process. Here, we describe the development of an appropriate
system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo
cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability
and symbiosis, was transferred from Sinorhizobium fredii to the ancestral
location on the S. meliloti chromosome, rendering the ETR region on pSymB
redundant. A derivative of this strain lacking both the large symbiotic replicons
(pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this
strain restored symbiotic capabilities with alfalfa. To delineate the location of
the single-copy genes essential for SNF on these replicons, we screened a
S. meliloti deletion library, representing > 95% of the 2900 genes of the
symbiotic replicons, for their phenotypes with alfalfa. Only four loci,
accounting for < 12% of pSymA and pSymB, were essential for SNF. These regions
will serve as our preliminary target of the minimal set of horizontally acquired
genes necessary and sufficient for SNF.
DOI: 10.1111/1462-2920.13221
Transcriptome responses in alfalfa associated with tolerance to intensive animal

grazing.
Wang J(1), Zhao Y(1), Ray I(2), Song M(3).
Author information:
(1)College of Ecology and Environmental Science, Inner Mongolia Agricultural
University, Hohhot, China.
(2)Department of Plant and Environmental Sciences, New Mexico State University,
Las Cruces, NM, USA.
(3)Department of Computer Science, New Mexico State University, Las Cruces, NM,
USA.
Tolerance of alfalfa (Medicago sativa L.) to animal grazing varies widely within
the species. However, the molecular mechanisms influencing the grazing tolerant
phenotype remain uncharacterized. The objective of this study was to identify
genes and pathways that control grazing response in alfalfa. We analyzed
whole-plant de novo transcriptomes from grazing tolerant and intolerant
populations of M. sativa ssp. falcata subjected to grazing by sheep. Among the
Gene Ontology terms which were identified as grazing responsive in the tolerant
plants and differentially enriched between the tolerant and intolerant
populations (both grazed), most were associated with the ribosome and
translation-related activities, cell wall processes, and response to oxygen
levels. Twenty-one grazing responsive pathways were identified that also
exhibited differential expression between the tolerant and intolerant
populations. These pathways were associated with secondary metabolite production,
primary carbohydrate metabolic pathways, shikimate derivative dependent pathways,
ribosomal subunit composition, hormone signaling, wound response, cell wall
formation, and anti-oxidant defense. Sequence polymorphisms were detected among
several differentially expressed homologous transcripts between the tolerant and
intolerant populations. These differentially responsive genes and pathways
constitute potential response mechanisms for grazing tolerance in alfalfa. They
also provide potential targets for molecular breeding efforts to develop
grazing-tolerant cultivars of alfalfa.
DOI: 10.1038/srep19438
PMCID: PMC4725929
Performances of survival, feeding behavior, and gene expression in aphids reveal

their different fitness to host alteration.
Lu H(1)(2), Yang P(3), Xu Y(2), Luo L(1), Zhu J(1), Cui N(1), Kang L(1), Cui
F(1).
Author information:
(1)State Key Laboratory of Integrated Management of Pest Insects &Rodents,
Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
(2)Plant Protection College, Shandong Agricultural University, Tai'an, Shandong,
China.
(3)Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing,
China.
Insect populations feeding on different plant species are under selection

pressure to adapt to these differences. A study integrating elements of the
ecology, behavior, and gene expression of aphids on different host plants has not
yet been well-explored. The present study explores the relationship between host
fitness and survival, feeding behavior, and salivary gland gene expression of a
pea (Pisum sativum) host race of Acyrthosiphon pisum feeding on a common host
Vicia faba and on three genetically-related hosts (Vicia villosa, Medicago
truncatula, and Medicago sativa). Life table data indicated that aphids on
non-favored hosts exhibited small size, low reproduction rate, slow population
increase and individual development, and long lifespan. Electrical penetration
graph results showed that the aphids spent significantly less time in passive
ingestion of phloem sap on all non-preferred host plants before acclimation.
After a period of acclimation on M. truncatula and V. villosa, pea host race
individuals showed improved feeding behavior. No individuals of the pea host race
completed its life history on M. sativa. Interestingly, the number of
host-specific differentially-expressed salivary gland genes was negatively
correlated with the fitness of aphids on this host plant. This study provided
important cues in host plant specialization in aphids.
DOI: 10.1038/srep19344
PMCID: PMC4725932

eCollection 2016.
The Effect of Root Exudate 7,4'-Dihydroxyflavone and Naringenin on Soil Bacterial

Community Structure.
Szoboszlay M(1), White-Monsant A(2), Moe LA(1).
Author information:
(1)Department of Plant and Soil Sciences, University of Kentucky, Lexington,
Kentucky, United States of America.
(2)Department of Animal, Plant and Soil Science, Centre for AgriBioscience, La
Trobe University, Melbourne, Australia.
Our goal was to investigate how root exudate flavonoids influence the soil
bacterial community structure and to identify members of the community that
change their relative abundance in response to flavonoid exudation. Using a model
system that approximates flavonoid exudation of Medicago sativa roots, we treated
a soil with 7,4'-dihydroxyflavone and naringenin in two separate experiments
using three different rates: medium (equivalent to the exudation rate of
7,4'-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and
low (0.1× the medium rate). Controls received no flavonoid. Soil samples were
subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid
treatments caused no significant change in the soil ATP content. With the high
7,4'-dihydroxyflavone treatment rate, operational taxonomic units (OTUs)
classified as Acidobacteria subdivision 4 increased in relative abundance
compared with the control samples, whereas OTUs classified as Gaiellales,
Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The
naringenin treatments did not cause significant changes in the soil bacterial
community structure. Our results suggest that the root exudate flavonoid
7,4'-dihydroxyflavone can interact with a diverse range of soil bacteria and may
have other functions in the rhizosphere in addition to nod gene induction in
legume-rhizobia symbiosis.
PMCID: PMC4709137
343. Biol Lett. 2016 Jan;12(1):20150925. doi: 10.1098/rsbl.2015.0925.
Deviation from niche optima affects the nature of plant-plant interactions along
a soil acidity gradient.
He L(1), Cheng L(1), Hu L(1), Tang J(2), Chen X(3).
Author information:
(1)College of Life Sciences, Zhejiang University, Hangzhou 310058, People's
Republic of China.
Republic of China chandt@zju.edu.cn.
Republic of China chen-tang@zju.edu.cn.
There is increasing recognition of the importance of niche optima in the shift of

plant-plant interactions along environmental stress gradients. Here, we
investigate whether deviation from niche optima would affect the outcome of
plant-plant interactions along a soil acidity gradient (pH = 3.1, 4.1, 5.5 and
6.1) in a pot experiment. We used the acid-tolerant species Lespedeza formosa
Koehne as the neighbouring plant and the acid-tolerant species Indigofera
pseudotinctoria Mats. or acid-sensitive species Medicago sativa L. as the target
plants. Biomass was used to determine the optimal pH and to calculate the
relative interaction index (RII). We found that the relationships between RII and
the deviation of soil pH from the target's optimal pH were linear for both target
species. Both targets were increasingly promoted by the neighbour as pH values
deviated from their optima; neighbours benefitted target plants by promoting soil
symbiotic arbuscular mycorrhizal fungi, increasing soil organic matter or
reducing soil exchangeable aluminium. Our results suggest that the shape of the
curve describing the relationship between soil pH and facilitation/competition
depends on the soil pH optima of the particular species.
© 2016 The Author(s).
DOI: 10.1098/rsbl.2015.0925
PMCID: PMC4785930
344. J Food Prot. 2016 Jan;79(1):37-42. doi: 10.4315/0362-028X.JFP-15-184.
Assessing the Public Health Impact and Effectiveness of Interventions To Prevent

Salmonella Contamination of Sprouts.
Ding H(1), Fu TJ(2).
Author information:
(1)U.S. Food and Drug Administration, Division of Food Processing Science &
Technology, 6502 South Archer Road, Bedford Park, Illinois 60501, USA; Center for
Drug Evaluation and Research, Division of Epidemiology, 20903 New Hampshire
Avenue, Silver Spring, MD 20993, USA. Hongliu.Ding@fda.hhs.gov.
(2)U.S. Food and Drug Administration, Division of Food Processing Science &
Technology, 6502 South Archer Road, Bedford Park, Illinois 60501, USA.
Tongjen.Fu@fda.hhs.gov.
Sprouts have been a recurring public health challenge due to microbiological

contamination, and Salmonella has been the major cause of sprout-associated
outbreaks. Although seed treatment and microbiological testing have been applied
as risk reduction measures during sprout production, the extent to which their
effectiveness in reducing the public health risks associated with sprouts has not
been well investigated. We conducted a quantitative risk assessment to measure
the risk posed by Salmonella contamination in sprouts and to determine whether
and how mitigation strategies can achieve a satisfactory risk reduction based on
the assumption that the risk reduction achieved by a microbiological sampling and
testing program at a given sensitivity is equivalent to that achieved by direct
inactivation of pathogens. Our results indicated that if the sprouts were
produced without any risk interventions, the health impact caused by sprouts
contaminated with Salmonella would be very high, with a median annual estimated
loss of disability-adjusted life years (DALYs) of 691,412. Seed treatment (with
20,000 ppm of calcium hypochlorite) or microbiological sampling and testing of
spent irrigation water (SIW) alone could reduce the median annual impact to 734
or 4,856 DALYs, respectively. Combining seed treatment with testing of the SIW
would further decrease the risk to 58 DALYs. This number could be dramatically
lowered to 3.99 DALYs if sprouts were produced under conditions that included
treating seeds with 20,000 ppm of calcium hypochlorite plus microbiological
testing of seeds, SIW, and finished products. Our analysis shows that the public
health impact due to Salmonella contamination in sprouts could be controlled if
seeds are treated to reduce pathogens and microbiological sampling and testing is
implemented. Future advances in intervention strategies would be important to
improve sprout safety further.
DOI: 10.4315/0362-028X.JFP-15-184
2015.
Co-transforming bar and CsALDH Genes Enhanced Resistance to Herbicide and Drought
and Salt Stress in Transgenic Alfalfa (Medicago sativa L.).
Duan Z(1), Zhang D(1), Zhang J(1), Di H(1), Wu F(1), Hu X(1), Meng X(1), Luo
K(1), Zhang J(1), Wang Y(1).
Author information:
(1)State Key Laborotary of Grassland Agro-Ecosystems, College of Pastoral
Drought and high salinity are two major abiotic factors that restrict the
productivity of alfalfa. By application of the Agrobacterium-mediated
transformation method, an oxidative responsive gene, CsALDH12A1, from the desert
grass Cleistogenes songorica together with the bar gene associated with herbicide
resistance, were co-transformed into alfalfa (Medicago sativa L.). From the all
90 transformants, 16 were positive as screened by spraying 1 mL L(-1) 10% Basta
solution and molecularly diagnosis using PCR. Real-time PCR analysis indicated
that drought and salt stress induced high CsALDH expression in the leaves of the
transgenic plants. The CsALDH expression levels under drought (15 d) and salt
stress (200 mM NaCl) were 6.11 and 6.87 times higher than in the control plants,
respectively. In comparison to the WT plants, no abnormal phenotypes were
observed among the transgenic plants, which showed significant enhancement of
tolerance to 15 d of drought and 10 d of salinity treatment. Evaluation of the
physiological and biochemical indices during drought and salt stress of the
transgenic plants revealed relatively lower Na(+) content and higher K(+) content
in the leaves relative to the WT plants, a reduction of toxic on effects and
maintenance of osmotic adjustment. In addition, the transgenic plants could
maintain a higher relative water content level, higher shoot biomass, fewer
changes in the photosystem, decreased membrane injury, and a lower level of
osmotic stress. These results indicate that the co-expression of the introduced
bar and CsALDH genes enhanced the herbicide, drought and salt tolerance of
alfalfa and therefore can potentially be used as a novel genetic resource for the
future breeding programs to develop new cultivars.
DOI: 10.3389/fpls.2015.01115
PMCID: PMC4679862
PMID: 26734025
346. Environ Sci Pollut Res Int. 2016 Apr;23(7):7022-31. doi:

10.1007/s11356-015-6008-z. Epub 2016 Jan 5.
Changes in the chemical properties and swelling coefficient of alfalfa root cell
walls in the presence of toluene as a toxic agent.
Sharifi M(1), Khoshgoftarmanesh AH(2), Hadadzadeh H(3).
Author information:
(1)Department of Soil Science, College of Agriculture, Isfahan University of
Technology, 84156-83111, Isfahan, Iran.
(2)Department of Soil Science, College of Agriculture, Isfahan University of
Technology, 84156-83111, Isfahan, Iran. amirhkhosh@cc.iut.ac.ir.
(3)Department of Chemistry, Isfahan University of Technology, 84156-83111,
Isfahan, Iran.
The influence of toluene pollution on the chemical properties and swelling

coefficient of root cell walls in alfalfa (Medicago sativa L.) was investigated.
Two sets of alfalfa seedlings were selected and one set was treated with 450 mg
L(-1) toluene in the nutrient solution under hydroponic culture. Thirty days
after treatment with toluene, alfalfa plants were harvested and the root cell
walls were isolated. Fourier-transform infrared (FTIR) spectroscopy was carried
out for the characterization of the root cell walls composition. The cation
exchange capacity (CEC) and the swelling coefficient of the root cell walls (Kcw)
were estimated at various pH values. The toluene contamination significantly
reduced the mass of the cell wall material in the alfalfa roots. According to the
FTIR spectra, the toluene pollution can change the alfalfa root cell wall
properties by reducing the cell wall functional groups. These functional groups
are probably related to the proteins and polysaccharides in the cell wall. Also,
toluene pollution strongly reduced CEC and Kcw of the root cell walls. The
results show that the decrease in the active sites of adsorption on the root cell
walls as a response to toluene pollution can affect the water flow rate and the
mineral nutrients uptake by roots.
DOI: 10.1007/s11356-015-6008-z
347. Mol Plant Microbe Interact. 2016 Mar;29(3):231-9. doi: 10.1094/MPMI-12-15-

0264-R.
Epub 2016 Feb 22.
Contribution of Individual Chemoreceptors to Sinorhizobium meliloti Chemotaxis

Towards Amino Acids of Host and Nonhost Seed Exudates.
Webb BA(1), Helm RF(2), Scharf BE(1).
Author information:
(1)1 Virginia Tech Department of Biological Sciences and.
(2)2 Virginia Tech Department of Biochemistry, Life Sciences I, Blacksburg, VA
24061, U.S.A.
Plant seeds and roots exude a spectrum of molecules into the soil that attract
bacteria to the spermosphere and rhizosphere, respectively. The alfalfa symbiont
Sinorhizobium meliloti utilizes eight chemoreceptors (McpT to McpZ and IcpA) to
mediate chemotaxis. Using a modified hydrogel capillary chemotaxis assay that
allows data quantification and larger throughput screening, we defined the role
of S. meliloti chemoreceptors in sensing its host, Medicago sativa, and a closely
related nonhost, Medicago arabica. S. meliloti wild type and most single-deletion
strains displayed comparable chemotaxis responses to host or nonhost seed
exudate. However, while the mcpZ mutant responded like wild type to M. sativa
exudate, its reaction to M. arabica exudate was reduced by 80%. Even though the
amino acid (AA) amounts released by both plant species were similar, synthetic AA
mixtures that matched exudate profiles contributed differentially to the S.
meliloti wild-type response to M. sativa (23%) and M. arabica (37%) exudates,
with McpU identified as the most important chemoreceptor for AA. Our results show
that S. meliloti is equally attracted to host and nonhost legumes; however, AA
play a greater role in attraction to M. arabica than to M. sativa, with McpZ
being specifically important in sensing M. arabica.
DOI: 10.1094/MPMI-12-15-0264-R
348. J Dairy Sci. 2016 Feb;99(2):1237-1241. doi: 10.3168/jds.2015-10016. Epub 2015

Dec
17.
Short communication: Comparison of predicted dietary phosphorus balance using

bioavailabilities from the NRC (2001) and Virginia Tech model.
Feng X(1), Jarrett JP(2), Knowlton KF(2), James RE(2), Hanigan MD(2).
Author information:
(1)Department of Dairy Science, Virginia Polytechnic Institute and State
University, Blacksburg 24061. Electronic address: yangxin@vt.edu.
(2)Department of Dairy Science, Virginia Polytechnic Institute and State
University, Blacksburg 24061.
The objective of the current work was to use digestion coefficients from the Feng
et al. (2015) model (Virginia Tech model) to calculate P bioavailability of
common feeds used in dairy production. Compared with the bioavailability
calculated by the Virginia Tech model, using the NRC (2001) P absorption
coefficient of 0.64 for forages would underestimate the bioavailabilities of
alfalfa hay, alfalfa silage, corn silage, grass hay, and mixed mainly legume
silage. For concentrates, using the NRC (2001) P absorption coefficient of 0.70
would overestimate the bioavailabilities of corn grain but underestimate
bioavailability of high-moisture corn. Two dairy diets were formulated using
nutrient values from the NRC (2001): a standard diet that includes minimal
by-products, and a by-product diet that has reduced corn and soybean meal which
was replaced with corn gluten feed, distillers grains, hominy feed, and wet
brewers grains. For each diet, total bioavailable P was calculated using
availability values from the NRC (2001) and the Virginia Tech model. Comparison
of P balance (the difference between required and bioavailable P) for each diet
was made using the 2 sets of bioavailabilities for a reference cow weighing 682
kg, producing 38.6 kg of milk/d (3.5% fat and 3.0% true protein, 100 d in milk),
and consuming 23 kg of dry matter/d, yielding an absorbed P requirement of 59.4
g/d. The standard diet supplied 56.69 and 53.52 g of bioavailable P per day using
bioavailabilities from the NRC (2001) and Virginia Tech models, respectively,
resulting in a P balance of -2.71 and -5.88±0.26 g/d. The by-product diet
provided 75.75 and 78.47 g/d of bioavailable P, yielding P balances of 16.35 and
19.07±0.37 g per day, respectively, using the 2 sets of bioavailabilities. Using
P bioavailabilities for individual ingredients that were based on the Virginia
Tech model resulted in different bioavailable P levels thus resulted in differing
dietary P balances in a field setting.

DOI: 10.3168/jds.2015-10016
349. Spectrochim Acta A Mol Biomol Spectrosc. 2016 Mar 15;157:265-270. doi:
10.1016/j.saa.2015.11.017. Epub 2015 Dec 1.
Association of protein structure, protein and carbohydrate subfractions with

bioenergy profiles and biodegradation functions in modeled forage.
Ji C(1), Zhang X(2), Yu P(3).
Author information:
(1)College of Animal Science and Veterinary Medicine, Tianjin Agricultural
University, 22 Jinjing Road, Tianjin 300384, China; Department of Animal and
Poultry Science, College of Agriculture and Bioresources, University of
Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada.
University, 22 Jinjing Road, Tianjin 300384, China. Electronic address:
zhangxuewei63@163.com.
University, 22 Jinjing Road, Tianjin 300384, China; Department of Animal and
Poultry Science, College of Agriculture and Bioresources, University of
Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada. Electronic address:
The objectives of this study were to detect unique aspects and association of
forage protein inherent structure, biological compounds, protein and carbohydrate
subfractions, bioenergy profiles, and biodegradation features. In this study,
common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was
used as a modeled forage for inherent structure profile, bioenergy,
biodegradation and their association between their structure and bio-functions.
The molecular spectral profiles were determined using non-invasive molecular
spectroscopy. The parameters included: protein structure amide I group, amide II
group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC);
carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and
undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2,
RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4,
RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy
profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1×, DE3×, ME3×, NEL3×; NEm, NEg). The
results show differences in protein and carbohydrate (CHO) subfractions in the
moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P=0.09),
slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P=0.02),
moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P=0.06) and slowly
degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced
origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there
were differences in RD of PB1 (417 vs. 349 g/kg CP, P=0.09), RD of PB2 (29 vs. 62
g/kg CP, P=0.02), RD of CB2 (251 vs. 198 g/kg DM, P=0.06), RD of CB3 (236 vs. 261
g/kg CHO, P=0.08). As to bioenergy profile, there were differences in total
digestible nutrient (TDN: 551 vs. 537 g/kg DM, P=0.06), and metabolic bioenergy
(P=0.095). As to protein molecular structure, there were differences in protein
structure 1st and 2nd amide groups (P<0.10), but no difference in the 1st to 2nd
amide group intensity ratios (P>0.05). These results indicate that the
sourced-origins and the internal molecular structure profiles affected biological
functions, nutrient bioavailability and biodegradation.
DOI: 10.1016/j.saa.2015.11.017
eCollection 2015.
Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in

Alfalfa Seed Production Areas in the United States.
Greene SL(1), Kesoju SR(2), Martin RC(3), Kramer M(4).
Author information:
(1)Plant and Animal Genetic Resource Preservation Unit, USDA, Agricultural
Research Service, Fort Collins, Colorado, United States of America.
(2)Irrigated Agriculture Research and Extension Center, Washington State
University, Prosser, Washington, United States of America.
(3)Forage Seed and Cereal Research Unit, USDA, Agricultural Research Service,
Corvallis, Oregon, United States of America.
(4)Statistics Group, USDA Agricultural Research Service, Beltsville, Maryland,
The potential environmental risks of transgene exposure are not clear for alfalfa
(Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by
insects. We gathered data on feral alfalfa in major alfalfa seed-production areas
in the western United States to (1) evaluate evidence that feral transgenic
plants spread transgenes and (2) determine environmental and agricultural
production factors influencing the location of feral alfalfa, especially
transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla
Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples
were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of
4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven
percent of these sites had transgenic plants. The frequency of sites having
transgenic feral plants varied among our study areas. Transgenic plants were
found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla
Walla, respectively. Spatial analysis suggested that feral populations started
independently and tended to cluster in seed and hay production areas, places
where seed tended to drop. Significant but low spatial auto correlation suggested
that in some instances, plants colonized nearby locations. Neighboring feral
plants were frequently within pollinator foraging range; however, further
research is needed to confirm transgene flow. Locations of feral plant clusters
were not well predicted by environmental and production variables. However, the
likelihood of seed spillage during production and transport had predictive value
in explaining the occurrence of transgenic feral populations. Our study confirms
that genetically engineered alfalfa has dispersed into the environment, and
suggests that minimizing seed spillage and eradicating feral alfalfa along road
sides would be effective strategies to minimize transgene dispersal.
PMCID: PMC4689365
2015.
Stoichiometric Characteristics of Carbon, Nitrogen, and Phosphorus in Leaves of

Differently Aged Lucerne (Medicago sativa) Stands.
Wang Z(1), Lu J(1), Yang M(1), Yang H(1), Zhang Q(1).

Author information:
Element concentration within a plant which is vital to function maintenance and

adaptation to environment, may change with plant growth. However, how carbon (C),
nitrogen (N), and phosphorus (P) vary stoichiometrically with stand growth, i.e.,
ages or cuts, was still untouched in perennial species. This study tested the
hypothesis that lucerne (Medicago sativa) C:N, C:P, and N:P should change with
stand age and cut. Leaf C:N, C:P, and N:P changed with stand age, showing various
trends in different cuts of lucerne. Generally the greatest stoichiometric ratios
were measured in 8 year stand and in the second cut. They were affected
significantly and negatively by total N and P concentrations of leaf, but not by
organic C concentration. There were significantly positive correlations among
leaf C:N, C:P, and N:P. However, leaf C:N, C:P, and N:P were hardly affected by
soil features. Conclusively, lucerne C, N, and P stoichiometry are age- and
cut-specific, and regulated mainly by leaf N, P concentrations and stoichiometry.
There are few correlations with soil fertility. To our knowledge, it is the first
try to elucidate the stoichiometry in the viewpoint of age and cut with a
perennial herbaceous legume.
DOI: 10.3389/fpls.2015.01062
PMCID: PMC4673304
PMID: 26697029
352. Spectrochim Acta A Mol Biomol Spectrosc. 2016 Mar 5;156:151-4. doi:
10.1016/j.saa.2015.11.036. Epub 2015 Dec 2.
Using non-invasive molecular spectroscopic techniques to detect unique aspects of

protein Amide functional groups and chemical properties of modeled forage from
different sourced-origins.
Ji C(1), Zhang X(2), Yu P(3).
Author information:
(1)Tianjin Agricultural University, 22 Jinjing Road, Tianjin 300384, China;
Department of Animal and Poultry Science, College of Agriculture and
Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada,
S7N 5A8.
(2)Tianjin Agricultural University, 22 Jinjing Road, Tianjin 300384, China.
Electronic address: zhangxuewei63@163.com.
(3)Tianjin Agricultural University, 22 Jinjing Road, Tianjin 300384, China;
Department of Animal and Poultry Science, College of Agriculture and
Bioresources, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK, Canada,
S7N 5A8. Electronic address: peiqiang.yu@usask.ca.
The non-invasive molecular spectroscopic technique-FT/IR is capable to detect the

molecular structure spectral features that are associated with biological,
nutritional and biodegradation functions. However, to date, few researches have
been conducted to use these non-invasive molecular spectroscopic techniques to
study forage internal protein structures associated with biodegradation and
biological functions. The objectives of this study were to detect unique aspects
and association of protein Amide functional groups in terms of protein Amide I
and II spectral profiles and chemical properties in the alfalfa forage (Medicago
sativa L.) from different sourced-origins. In this study, alfalfa hay with two
different origins was used as modeled forage for molecular structure and chemical
property study. In each forage origin, five to seven sources were analyzed. The
molecular spectral profiles were determined using FT/IR non-invasive molecular
spectroscopy. The parameters of protein spectral profiles included functional
groups of Amide I, Amide II and Amide I to II ratio. The results show that the
modeled forage Amide I and Amide II were centered at 1653 cm(-1) and 1545 cm(-1),
respectively. The Amide I spectral height and area intensities were from 0.02 to
0.03 and 2.67 to 3.36 AI, respectively. The Amide II spectral height and area
intensities were from 0.01 to 0.02 and 0.71 to 0.93 AI, respectively. The Amide I
to II spectral peak height and area ratios were from 1.86 to 1.88 and 3.68 to
3.79, respectively. Our results show that the non-invasive molecular
spectroscopic techniques are capable to detect forage internal protein structure
features which are associated with forage chemical properties.
DOI: 10.1016/j.saa.2015.11.036
353. Ying Yong Sheng Tai Xue Bao. 2015 Aug;26(8):2561-70.
[Heat-responsive mechanisms in plants revealed by proteomic analysis: A review].
Liu JM, Zhao Q, Yin ZP, Xu CX, Wang QH, Dai SJ.
Heat stress is a major abiotic stress that limits plant growth and productivity.
In recent years, proteomic investigations provide more information for
understanding the sophisticated heat-responsive molecular mechanism in plants at
systematic biological level. The heat-responsive proteomic patterns in several
plants, i. e., model plants (Arabidopsis thaliana), staple food crops (soybean,
rice and wheat), heat-tolerant plants (Agrostis stolonifera, Portulaca oleracea,
and Carissa spinarum), grapevine, Populus euphratica, Medicago sativa, and
Pinellia ternate, were reported. A total of 838 heat-responsive proteins have
been identified in these studies. Among them, 534 proteins were induced and the
expression of 304 proteins was reduced in plants under heat stress. In this
paper, the diverse protein patterns in plants under various heat stress
conditions (30-45 °C for 0-10 d) were analyzed integratively. This provided new
evidences and clues for further interpreting the signaling and metabolic
pathways, e.g., signaling, stress and defense, carbohydrate and energy
metabolism, photosynthesis, transcription, protein synthesis and fate, membrane
and transport, in heat-responsive networks, and laid a foundation for a holistic
understanding of the molecular regulatory mechanism in plants in response to heat
stress.
354. J Appl Microbiol. 2016 Mar;120(3):543-51. doi: 10.1111/jam.13031. Epub 2016

Feb
9.
Identification and isolation of Lactobacillus fructivorans from wilted alfalfa

silage with and without molasses.
Wu B(1), Nishino N(1).
Author information:
(1)Department of Animal Science, Okayama University, Okayama, Japan.
AIM: To gain insights into the bacterial species associated with anaerobic
storage and aerobic stability of alfalfa silage.
METHODS AND RESULTS: Wilted alfalfa silage (498 g dry matter kg(-1) ) was
prepared with and without the addition of molasses. Aerobic spoilage tests were
conducted at 5, 10 and 60 days after ensiling. The composition of fermentation
products and the bacterial communities of silage were determined at 1, 3, 5 and 7
days after silo opening. Silage without molasses had small amounts of lactic and
acetic acids detectable at silo opening but resisted deterioration due to aerobic
spoilage for at least 5 days after opening. Resistance to aerobic deterioration
in silage increased with the addition of molasses. The predominant bacterial
species in molasses-added silage was Lactobacillus fructivorans, which was
detected by denaturing gradient gel electrophoresis analysis. Different bacterial
growth media were used for Lact. fructivorans isolation from alfalfa silage with
added molasses: isolation was successful using liver infusion sake medium, but
was unsuccessful when de Man, Rogosa and Sharpe medium was used.
CONCLUSION: A nonconventional lactic acid bacterium (LAB) species may be involved
in the high aerobic stability of alfalfa silage.
SIGNIFICANCE AND IMPACT OF THE STUDY: The findings demonstrate that
culture-independent microbiota analysis may be useful in the isolation and
identification of nonconventional LAB species involved in fermentation and the
aerobic stability of silage.
DOI: 10.1111/jam.13031
355. Genet Mol Res. 2015 Dec 17;14(4):17204-18. doi: 10.4238/2015.December.16.20.
Overexpression of EsMcsu1 from the halophytic plant Eutrema salsugineum promotes

abscisic acid biosynthesis and increases drought resistance in alfalfa (Medicago
sativa L.).
Zhou C(1)(2), Ma ZY(1), Zhu L(2), Guo JS(2), Zhu J(2), Wang JF(1).
Author information:
(1)School of Life Science, Anhui Science and Technology University, Bengbu,
China.
(2)School of Life Science and Technology, Tongji University, Shanghai, China.
The stress phytohormone abscisic acid (ABA) plays pivotal roles in plants'
adaptive responses to adverse environments. Molybdenum cofactor sulfurases
influence aldehyde oxidase activity and ABA biosynthesis. In this study, we
isolated a novel EsMcsu1 gene encoding a molybdenum cofactor sulfurase from
Eutrema salsugineum. EsMcus1 transcriptional patterns varied between organs, and
its expression was significantly upregulated by abiotic stress or ABA treatment.
Alfalfa plants that overexpressed EsMcsu1 had a higher ABA content than wild-type
(WT) plants under drought stress conditions. Furthermore, levels of reactive
oxygen species (ROS), ion leakage, and malondialdehyde were lower in the
transgenic plants than in the WT plants after drought treatment, suggesting that
the transgenic plants experienced less ROS-mediated damage. However, the
expression of several stress-responsive genes, antioxidant enzyme activity, and
osmolyte (proline and total soluble sugar) levels in the transgenic plants were
higher than those in the WT plants after drought treatment. Therefore, EsMcsu1
overexpression improved drought tolerance in alfalfa plants by activating a
series of ABA-mediated stress responses.
DOI: 10.4238/2015.December.16.20
356. Food Microbiol. 2016 Feb;53(Pt B):9-14. doi: 10.1016/j.fm.2015.08.002. Epub

2015
Aug 5.
Effect of sequential dry heat and hydrogen peroxide treatment on inactivation of

Salmonella Typhimurium on alfalfa seeds and seeds germination.
Hong EJ(1), Kang DH(2).
Author information:
(1)Department of Food and Animal Biotechnology, Department of Agricultural
Biotechnology, Center for Food and Bioconvergence, Research Institute for
Agricultural and Life Sciences, Seoul National University, Seoul 151-921,
Republic of Korea; Institutes of Green Bio Science & Technology, Seoul National
University, Pyeongchang-gun, Gangwon-do 232-916, Republic of Korea.
(2)Department of Food and Animal Biotechnology, Department of Agricultural
Biotechnology, Center for Food and Bioconvergence, Research Institute for
Agricultural and Life Sciences, Seoul National University, Seoul 151-921,
Republic of Korea; Institutes of Green Bio Science & Technology, Seoul National
University, Pyeongchang-gun, Gangwon-do 232-916, Republic of Korea. Electronic
address: kang7820@snu.ac.kr.
The purpose of this study was to inactivate Salmonella Typhimurium on alfalfa

seeds without having negative effect on seed germination. Inoculated alfalfa
seeds were treated with dry heat at 60, 70 or 80 °C for 0, 12, 18 or 24 h
followed by 2% hydrogen peroxide solution (10 min). Populations of Salmonella on
alfalfa seeds treated with dry heat alone (60, 70 or 80 °C) for up to 24 h were
reduced by 0.26-2.76 log CFU/g, and sequential treatment with dry heat and H2O2
reduced populations by 1.66-3.60 log CFU/g. The germination percentage of seeds
subjected to sequential treatments was significantly enhanced to up to 97%,
whereas that of untreated seeds was only 79.5%. This study suggests that
sequential treatment with dry heat and hydrogen peroxide is applicable for
reducing levels of Salmonella on seeds while simultaneously enhancing seeds
germinability.
DOI: 10.1016/j.fm.2015.08.002
357. J Environ Sci Health B. 2016;51(3):170-5. doi: 10.1080/03601234.2015.1108810.

Epub 2015 Dec 16.
Risks and benefits of compost-like materials prepared by the thermal treatment of

raw scallop hepatopancreas for supplying cadmium and the growth of alfalfa
Ushijima K(1), Fukushima M(1), Kanno S(2), Kanno I(2), Ohnishi M(3).
Author information:
(1)a Laboratory of Chemical Resources, Division of Sustainable Resources
Engineering, Faculty of Engineering, Hokkaido University , Sapporo , Hokkaido ,
Japan.
(2)b Daiso-KET Institute Corporation , Asahikawa , Hokkaido , Japan.
(3)c Plan Policy Section, Shibetsu-Town Government Office , Shibetsu , Hokkaido ,
Japan.
Scallop hepatopancreas, fishery waste, contains relatively high levels of Cd and

organic nitrogen compounds, the latter of which represent a fertilizer. In this
study, raw scallop hepatopancreas tissue was thermally treated with sawdust and
red loam in the presence of an iron catalyst to produce compost-like materials
(CLMs). Two CLM samples were prepared by varying the content of raw scallop
hepatopancreas tissue: 46 wt.% for CLM-1 and 18 wt.% for CLM-2. Mixtures of
control soil (CTL) and CLMs (CLM content: 10 and 25 wt.%) were examined for the
growth of alfalfa (Medicago sativa L.) to evaluate the risks and benefits of
using this material for fertilization. The Cd content in shoots and roots of
alfalfa, that were grown in the presence of CLMs, was significantly higher than
those for the plants grown in the CTL, indicating that Cd had accumulated in the
plants from CLMs. The accumulation of Cd in the alfalfa roots was quite high in
the case of the 25% CLM-1 sample. However, alfalfa growth was significantly
promoted in the presence of 10% CLM-1. This can be attributed to the higher
levels of nitrogen and humic substances, which serve as fertilizer components.
Although the fertilization effect in case of CLM-1showed a potential benefit, the
accumulation of Cd in alfalfa was clearly increased in the presence of both CLMs.
In conclusion, the use of CLMs produced from raw scallop hepatopancreas tissue
can be considered to have a desirable benefit from standpoint of its use as
fertilizer, but is accompanied by a risk of the accumulation of Cd in alfalfa
plants.
DOI: 10.1080/03601234.2015.1108810

eCollection 2015.
Legume-Cereal Intercropping Improves Forage Yield, Quality and Degradability.
Zhang J(1), Yin B(1), Xie Y(1), Li J(2), Yang Z(1), Zhang G(1)(2).
Author information:
(1)College of Animal Sciences and Technology, Shandong Agricultural University,
Tai-an, Shandong 271018, P. R. China.
(2)Department of Biochemistry and Molecular Biology, College of Life Sciences,
Peking University, Beijing 100871, P. R. China.
Intercropping legume with cereal is an extensively applied planting pattern in

crop cultivation. However, forage potential and the degradability of harvested
mixtures from intercropping system remain unclear. To investigate the feasibility
of applying an intercropping system as a forage supply source to ruminants, two
consecutive experiments (experiments 1 and 2) involving a field cultivation trial
and a subsequent in vivo degradable experiment were conducted to determine the
forage production performance and the ruminally degradable characteristics of a
harvested mixture from an alfalfa/corn-rye intercropping system. In experiment 1,
the intercropping system was established by alternating alfalfa and corn or rye
with a row ratio of 5:2. Dry matter (DM) and nutrient yields were determined. In
experiment 2, forages harvested from the different treatments were used as
feedstuff to identify nutrient degradation kinetics and distribution of
components between the rapidly degradable (a), potentially degradable (b) and the
degradation rate constant (c) of 'b' fraction by in sacco method in Small-Tail
Han wether Sheep. The intercropping system of alfalfa and corn-rye provided
higher forage production performance with net increases of 9.52% and 34.81% in DM
yield, 42.13% and 16.74% in crude protein (CP) yield, 25.94% and 69.99% in
degradable DM yield, and 16.96% and 5.50% in degradable CP yield than rotation
and alfalfa sole cropping systems, respectively. In addition, the harvest mixture
from intercropping system also had greater 'a' fraction, 'b' fraction, 'c'
values, and effective degradability (E value) of DM and CP than corn or rye hay
harvested from rotation system. After 48-h exposure to rumen microbes,
intercropping harvest materials were degraded to a higher extent than separately
degraded crop stems from the sole system as indicated by visual microscopic
examination with more tissues disappeared. Thus, the intercropping of alfalfa and
corn-rye exhibited a greater forage production potential, and could be applied as
forage supply source for ruminants. The improved effective degradability of
harvest mixture material could be attributed to greater degradable components
involving the rapidly degradable fractions (a), potentially degradable (b)
fractions, and degradable rate constant
(c), than that of corn and rye hay.
PMCID: PMC4687681
359. Planta. 2016 Mar;243(3):733-47. doi: 10.1007/s00425-015-2440-z. Epub 2015 Dec

15.
Eukaryotic Hsp70 chaperones in the intermembrane space of chloroplasts.
Bionda T(1)(2), Gross LE(1), Becker T(1)(3), Papasotiriou DG(4)(5), Leisegang

MS(1)(6), Karas M(4), Schleiff E(7)(8)(9).
Author information:
(1)Molecular Cell Biology of Plants, Goethe University, Max von Laue Str. 9,
60438, Frankfurt, Germany.
(2)Institute of Biochemistry II, Goethe University, Theodor-Stern-Kai 7, 60590,
Frankfurt, Germany.
(3)Biochemistry and Molecular Biology, ZBMZ, and BIOSS Centre for Biological
Signalling Studies, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 17,
79104, Freiburg, Germany.
(4)Pharmaceutical Chemistry, Goethe University, Max von Laue Str. 9, 60438,
Frankfurt, Germany.
(5)Syngenta Ltd., Jealott's Hill International Research Centre, Bracknell,
Berkshire, RG42 6EY, UK.
(6)Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai
7, 60590, Frankfurt, Germany.
(7)Molecular Cell Biology of Plants, Goethe University, Max von Laue Str. 9,
60438, Frankfurt, Germany. schleiff@bio.uni-frankfurt.de.
(8)Molecular Cell Biology of Plants, Cluster of Excellence Frankfurt, Goethe
University, Max von Laue Str. 9, 60438, Frankfurt, Germany.
schleiff@bio.uni-frankfurt.de.
(9)Buchmann Institut for Molecular Life Sciences, Max von Laue Str. 9, 60438,
Frankfurt, Germany. schleiff@bio.uni-frankfurt.de.
MAIN CONCLUSION: Multiple eukaryotic Hsp70 typically localized in the cytoplasm

are also distributed to the intermembrane space of chloroplasts and might thereby
represent the missing link in energizing protein translocation. Protein
translocation into organelles is a central cellular process that is tightly
regulated. It depends on signals within the preprotein and on molecular machines
catalyzing the process. Molecular chaperones participate in transport and
translocation of preproteins into organelles to control folding and to provide
energy for the individual steps. While most of the processes are explored and the
components are identified, the transfer of preproteins into and across the
intermembrane space of chloroplasts is not yet understood. The existence of an
energy source in this compartment is discussed, because the required transit
peptide length for successful translocation into chloroplasts is shorter than
that found for mitochondria where energy is provided exclusively by matrix
chaperones. Furthermore, a cytosolic-type Hsp70 homologue was proposed as
component of the chloroplast translocon in the intermembrane space energizing the
initial translocation. The molecular identity of such intermembrane space
localized Hsp70 remained unknown, which led to a controversy concerning its
existence. We identified multiple cytosolic Hsp70s by mass spectrometry on
isolated, thermolysin-treated Medicago sativa chloroplasts. The localization of
these Hsp70s of M. sativa or Arabidopsis thaliana in the intermembrane space was
confirmed by a self-assembly GFP-based in vivo system. The localization of
cytosolic Hsp70s in the stroma of chloroplasts or different mitochondrial
compartments could not be observed. Similarly, we could not identify any
cytosolic Hsp90 in the intermembrane space of chloroplast. With respect to our
results we discuss the possible targeting and function of the Hsp70 found in the
intermembrane space.
DOI: 10.1007/s00425-015-2440-z
360. Nutr Hosp. 2015 Dec 1;32(6):2741-8. doi: 10.3305/nh.2015.32.6.9849.
[MEDICAGO SATIVA L: IMPROVEMENT AND NEW APPROACHES OF ITS NUTRITIONAL AND

FUNCTIONAL VALUE BY BACTERIAL CO-INOCULATION].
[Article in Spanish; Abstract available in Spanish from the publisher]
Martínez R(1), Nebot E(2), Porres JM(3), Kapravelou G(1), Del Moral A(4), Talbi
C(5), Bedmar EJ(6), López-Jurado M(7).
Author information:
(1)Departamento de Fisiología, Instituto de Nutrición y Tecnología de los
Alimentos, Facultad de Farmacia, Universidad de Granada, Granada..
rosariomz@ugr.es.
Alimentos, Facultad de Farmacia, Universidad de Granada, Granada.. enebot@ugr.es.
jmporres@ugr.es.
(4)Departamento de Microbiología, Facultad de Farmacia, Universidad de Granada,
Granada.. admoral@ugr.es.
(5)Departamento de Microbiología de Suelos y Sistemas Simbióticos, Estación
Experimental del Zaidín (CSIC), Granada, España.. chouhra@hotmail.com.
(6)Departamento de Microbiología de Suelos y Sistemas Simbióticos, Estación
Experimental del Zaidín (CSIC), Granada, España.. eulogio.bedmar@eez.csic.es.
mlopezj@ugr.es.
OBJECTIVE: to study the effect of co-inoculation with Ensifer meliloti and

Halomonas maura of the leguminous Medicago sativa L., on growth, nutritional and
functional value, grown under salinity conditions.
METHODS: plants of M. sativa were grown in a solution with a mixture of salts
(CaSO4, MgCl, NaCl and NaHCO 3) and were co-inoculated with its specific
rhizobium and the halophilic moderated bacterium H. maura. Different physiologic
parameters were determined, as well as, nitrogen and minerals content.
Furthermore, an assay of in vitro digestibility was carried out.
RESULTS: salinity had a negative effect on the plants; however, co-inoculation
increased yield, nitrogen content, total minerals, Ca and Mg. Moreover,
physiologic parameters as water potential and leghemoglobin content in fresh
nodules were higher compared to those of plants inoculated only with E. meliloti.
Both, salinity and bacterial treatment with E. meliloti and H. maura increased
the antioxidant capacity of the legume, in dialyzates and retentates collected
after an in vitro digestibility assay.
CONCLUSION: co-inoculation of plants with E. meliloti and H. maura could improve
the alfalfa yield under specific salinity conditions, increasing the nutritional
and functional value of the plants. M. sativa could be considered in the
formulations of nutritional supplements for the human diet.
Publisher: Objetivo: estudiar el efecto de la inoculación con Ensifer meliloti y

Halomonas maura sobre el crecimiento y el valor nutricional y funcional de la
leguminosa Medicago sativa L., cultivada bajo condiciones de salinidad. Método:
las plantas de M. sativa se cultivaron con una solución de mezcla de sales CaSO4,
MgCl, NaCl and Na- HCO3 y se coinocularon con su rizobio específico y la bacteria
H. maura. Se determinaron los parámetros fisiológicos de las plantas, así como el
contenido en nitrógeno y minerales, y se llevó a cabo un proceso de
digestibilidad in vitro. Resultados: la salinidad ejerció un efecto negativo
sobre las plantas; sin embargo, la coinoculación de las mismas incrementó su
productividad, el contenido en nitrógeno, minerales totales, Ca y Mg. Además, los
parámetros fisiológicos de potencial hídrico y concentración de leghemoglobina se
incrementaron. Tanto la salinidad como la coinoculación de las plantas aumentaron
la capacidad antioxidante de la leguminosa en los dializados y retenidos
obtenidos tras someter a la planta a un proceso de digestibilidad in vitro.
Conclusión: la coinoculación con E. meliloti y H. maura podría mejorar el cultivo
de la alfalfa bajo condiciones específicas de salinidad, aumentando su
composición nutricional y funcional, pudiendo considerarse en la formulación de
suplementos nutricionales para el consumo humano.
Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights
reserved.
DOI: 10.3305/nh.2015.32.6.9849
361. Stand Genomic Sci. 2015 Dec 10;10:126. doi: 10.1186/s40793-015-0119-5.

eCollection 2015.
High-quality permanent draft genome sequence of Ensifer medicae strain WSM244, a

microsymbiont isolated from Medicago polymorpha growing in alkaline soil.
Ardley J(1), Tian R(1), O'Hara G(1), Seshadri R(2), Reddy TB(2), Pati A(2), Woyke
T(2), Markowitz V(3), Ivanova N(2), Kyrpides N(4), Howieson J(1), Reeve W(1).
Author information:
(1)Centre for Rhizobium Studies, Murdoch University, Murdoch, Australia.
(2)DOE Joint Genome Institute, Walnut Creek, CA USA.
(3)Biological Data Management and Technology Center, Lawrence Berkeley National
Laboratory, Berkeley, CA USA.
(4)DOE Joint Genome Institute, Walnut Creek, CA USA ; Department of Biological
Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
Ensifer medicae WSM244 is an aerobic, motile, Gram-negative, non-spore-forming

rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago
species. WSM244 was isolated in 1979 from a nodule recovered from the roots of
the annual Medicago polymorpha L. growing in alkaline soil (pH 8.0) in Tel Afer,
Iraq. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced
to date. It is effective at fixing nitrogen with M. polymorpha L., as well as
with more alkaline-adapted Medicago spp. such as M. littoralis Loisel., M.
scutellata (L.) Mill., M. tornata (L.) Mill. and M. truncatula Gaertn. This
strain is also effective with the perennial M. sativa L. Here we describe the
features of E. medicae WSM244, together with genome sequence information and its
annotation. The 6,650,282 bp high-quality permanent draft genome is arranged into
91 scaffolds of 91 contigs containing 6,427 protein-coding genes and 68 RNA-only
encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE
Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root
Nodule Bacteria (GEBA-RNB) project proposal.
DOI: 10.1186/s40793-015-0119-5
PMCID: PMC4674904
PMID: 26664655
362. Plant Cell Rep. 2016 Mar;35(3):561-71. doi: 10.1007/s00299-015-1903-9. Epub

2015
Dec 8.
Global transcriptome profiling analysis reveals insight into saliva-responsive

genes in alfalfa.
Liu W(1), Zhang Z(1), Chen S(2), Ma L(1), Wang H(1), Dong R(1), Wang Y(1), Liu
Z(3).
Author information:
Agriculture Science and Technology, Lanzhou University, Lanzhou, China.
(2)Key Laboratory of Plant Resources, Institute of Botany, The Chinese Academy of
Sciences, Beijing, China.
lzp@lzu.edu.cn.
KEY MESSAGE: We studied the genome-wide multiple time-course transcriptome

dynamics after saliva deposition in alfalfa and demonstrate that saliva
deposition functions as a stress that negatively affects the regrowth of alfalfa.
Saliva deposition is one of the key factors influencing plant-herbivore
interactions during grazing. Although many studies have focused on the effects of
saliva deposition on plant regrowth, no consistent conclusions have been reached.
Alfalfa is the most extensively cultivated forage legume, yet most alfalfa
cultivars, thus far, are not grazing-tolerant. To better understand the
underlying mechanism, we undertook a study to evaluate the global changes in the
transcriptome of alfalfa after cow saliva deposition treatment. In this study,
cDNA libraries from alfalfa seedlings at 0, 4, 8, and 24 h after cow saliva
deposition were constructed and sequenced, resulting in the identification of
53,195 annotated unigenes, from which 4,814 unigenes were significantly
differentially expressed. A metabolic pathway enrichment analysis demonstrated
that saliva deposition functions as a stress that negatively affects the regrowth
of alfalfa by modifying jasmonic acid synthesis, enhancing the susceptibility to
pathogens and reducing the expression levels of ribosomal protein genes. In the
present study, we demonstrate the potential effects of saliva deposition on
alfalfa regrowth at the transcriptome level. These fundamental and important
findings could facilitate further investigations into the molecular mechanisms
underlying the responses of alfalfa and other related species to herbivore
grazing.
DOI: 10.1007/s00299-015-1903-9
363. J Anim Sci. 2015 Dec;93(12):5774-80. doi: 10.2527/jas.2015-9579.
Technical Note: Collection and preparation techniques change nutrient composition

of masticate collected from esophageally fistulated cattle.
Musgrave JA, Judy JV, Jenkins KH, Klopfenstein TJ, Stalker LA.
Two experiments determined effects of collection and preparation techniques on

nutrient composition of masticate samples from esophageally fistulated cattle. In
Exp. 1, 12 esophageally fistulated cattle were maintained on 2 precollection
diets, high CP (24% CP; = 6) or low CP (7.7% CP; = 6), for 8 d. On d 9, the
esophageal plug was removed, screen bottom bags were attached, and each cow was
offered fresh grass. Immediately after fresh grass sample collection was
complete, dry grass (hay) was offered and a sample was collected. Blood samples
were collected and analyzed for serum urea nitrogen concentration. Masticate
samples of both fresh and dry grass were divided and each was either squeezed by
hand until no more saliva could be removed or remained unsqueezed. In Exp. 2, 10
esophageally fistulated cattle were fitted with either screen ( = 5) or solid ( =
5) bottom collection bags after removal of the esophageal plug and presented
grass hay, fresh grass, alfalfa hay, or fresh alfalfa. In Exp. 1, the
precollection diet did not affect ( = 0.49) CP content of masticate even though
serum urea nitrogen tended to be greater ( = 0.08) for high- vs. low-CP
precollection diets. Forage harvest type offered (fresh vs. hay) interacted ( =
0.01) with preparation technique (squeezed vs. unsqueezed) for CP, where CP
decreased in squeezed fresh samples ( < 0.001) but not in squeezed grass hay
samples ( = 0.98). In Exp. 2, ingestion greatly increased levels of ash ( <
0.001). Crude protein was greater ( < 0.004) before ingestion for all samples
except grass hay ( = 0.43). Levels of NDF were similar before and after ingestion
( > 0.15) for all samples except fresh alfalfa, which was greater after ingestion
( = 0.002). Ingestion status did not affect in vitro OM disappearance (IVOMD; >
0.34) except for grass hay, which was greater after ingestion ( < 0.001). Bag
type (screen vs. solid) did not affect ash and NDF ( > 0.31), except for fresh
alfalfa, which were greater ( < 0.03) for solid bottom bags. Bag type did not
affect alfalfa CP ( = 0.71) but did affect grass CP, which was lower ( = 0.02)
for solid bottom bags. Bag type did not affect IVOMD ( > 0.33). More ( = 0.01)
fresh forage than hay was recovered through the esophageal opening. Previous diet
did not impact masticate samples but squeezing impacted CP levels of high-quality
forage and therefore should not be performed. Nutrient values should be reported
on an OM basis.
DOI: 10.2527/jas.2015-9579
364. J Anim Sci. 2015 Dec;93(12):5735-42. doi: 10.2527/jas.2015-9139.
Phosphorus digestibility and phytate degradation by yearlings and mature horses.
Fowler AL, Hansen TL, Strasinger LA, Harlow BE, Lawrence LM.
Inorganic P is often added to growing horse diets because organic P, or

phytate-P, is believed to have lower digestibility. If horses can efficiently
digest organic P, then the need for inorganic P may be reduced. Much of the P in
grain-based concentrates fed to growing horses is in the form of phytate-P.
Little is known about the ability of growing horses to degrade phytate-P or
whether horse age affects mineral digestion in horses. The objective of this
study was to examine the effect of age on P, Ca, and Mg digestibility as well as
phytate-P degradation. Four yearling geldings and 4 mature geldings were fed a
diet of alfalfa cubes, timothy cubes, and a pelleted concentrate. The diet
contained 0.28% total P and 17.4% of that P was in the phytate form. There was a
14-d diet adaptation period followed by a 4-d fecal collection period. Apparent
total tract P digestibility was higher for yearlings than mature geldings ( =
0.036; 7.7 and -6.6% for yearlings and mature geldings, respectively). Phytate-P
disappearance was 94.8% and did not differ between ages ( = 0.190). Apparent Ca
digestibility was lower in mature geldings ( = 0.043), but apparent Mg
digestibility did not differ between ages ( = 0.414). Phytate is broken down in
the gastrointestinal tract, but the low P digestibilities suggest that either
degradation occurs after the site of P absorption or liberated P is recycled back
into the gastrointestinal tract. Yearlings can utilize organic P as well as
mature horses; therefore, diets without inorganic P are acceptable for growing
horses.
DOI: 10.2527/jas.2015-9139
365. Pak J Pharm Sci. 2015 Nov;28(6):2061-74.
Evaluation of Medicago sativa L. sprouts as antihyperlipidemic and

antihyperglycemic agent.
Seida A(1), El-Hefnawy H(2), Abou-Hussein D(3), Mokhtar FA(2), Abdel-Naim A(4).
Author information:
(1)Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El
Aini, Cairo, Egypt / Department of Pharmacognosy, Faculty of Pharmacy, October 6
University, Sixth of October, Egypt.
Aini, Cairo, Egypt.
Aini, Cairo, Egypt / Department of Natural Products and Alternative Medicine,
Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia.
(4)Department of Pharmacogology and Toxicology, Faculty of Pharmacy, Ain Shams
University, Abbasia, Cairo, Egypt / Department of Pharmacogology and Toxicology,
Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia.
Medicago sativa L. (Alfalfa) is traditionally used to treat diabetes. This study

was designed to investigate the potential antihyperlipidemic and
antihyperglycemic activity of M. sativa sprouts in streptozotocin (STZ) induced
diabetes via i.p. injection of 55 mg/kg of STZ. Experimental animals were divided
into the following groups: GP1 (normal), GP2 (STZ-hyperlipidemic), GP3
(rouvastatin), GP4 (metformin), GP 5-9 (diabetic treated with methanolic,
petroleum ether, chloroform, ethyl acetate and butanol extracts). The
administration of the total methanolic extract (500 mg/kg), the petroleum ether
(32.5mg) and butanol fractions (60 mg) for 4 weeks significantly decreased
(p<0.05) triglycerides (TG), total cholesterol (TC), low-density lipoproteins
(LDL) and very low density lipoproteins (VLDL) in comparison to rouvastatin.
Petroleum ether fraction proved to exhibit the best activity as
antihyperlipidemic agent (12.23%). On the other hand, ethyl acetate fraction
retained the best activity (vs. metformin) as antihyperglycemic agent.
Histopathological evidences on liver, pancreas and spleen were in agreement with
the above mentioned results. Purification, characterization, and identification
of isolated compounds from the active fractions afforded 9 compounds:
β-sitosterol and stigmasterol from the petroleum ether fraction;
10-hydroxy-coumestrol, apigenin, genistein, p-hydroxy-benzoic-acid, 7, 4'-
dihydroxyflavone, quercetin-3-glucoside and sissotrin from the ethyl acetate
fraction.
366. BMC Plant Biol. 2015 Dec 1;15:283. doi: 10.1186/s12870-015-0649-4.
Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem

vascular tissue suggests a role for invertase in cell wall cellulose synthesis.
Samac DA(1)(2), Bucciarelli B(3), Miller SS(4), Yang SS(5)(6), O'Rourke JA(7)(8),
Shin S(9)(10), Vance CP(11)(12).
Author information:
(1)USDA-ARS-Plant Science Research Unit, St. Paul, MN, 55108, USA.
debby.samac@ars.usda.gov.
(2)Department of Plant Pathology, University of Minnesota, St. Paul, MN, 55108,
USA. debby.samac@ars.usda.gov.
bruna.bucciarelli@ars.usda.gov.
(4)Department of Plant Pathology, University of Minnesota, St. Paul, MN, 55108,
USA. sue.miller@ars.usda.gov.
sam.yang@monsanto.com.
(6)Present address: Monsanto Company, Chesterfield, MO, 63017, USA.
sam.yang@monsanto.com.
jamie.orourke@ars.usda.gov.
(8)Present address: USDA-ARS-Corn Insects and Crop Genetics Research Unit, Ames,
IA, 50011, USA. jamie.orourke@ars.usda.gov.
(9)Department of Agronomy and Plant Genetics, University of Minnesota, St. Paul,
MN, 55108, USA. shinx101@korea.kr.
(10)Present address: National Institute of Crop Science, Iksan, 570-080, Korea.
shinx101@korea.kr.
vance004@umn.edu.
MN, 55108, USA. vance004@umn.edu.
BACKGROUND: Alfalfa (Medicago sativa L.) is a widely adapted perennial forage

crop that has high biomass production potential. Enhanced cellulose content in
alfalfa stems would increase the value of the crop as a bioenergy feedstock. We
examined if increased expression of sucrose synthase (SUS; EC 2.4.1.13) would
increase cellulose in stem cell walls.
RESULTS: Alfalfa plants were transformed with a truncated alfalfa
phosphoenolpyruvate carboxylase gene promoter (PEPC7-P4) fused to an alfalfa
nodule-enhanced SUS cDNA (MsSUS1) or the β-glucuronidase (GUS) gene. Strong GUS
expression was detected in xylem and phloem indicating that the PEPC7-P4 promoter
was active in stem vascular tissue. In contrast to expectations, MsSUS1
transcript accumulation was reduced 75-90 % in alfalfa plants containing the
PEPC7-P4::MsSUS1 transgene compared to controls. Enzyme assays indicated that SUS
activity in stems of selected down-regulated transformants was reduced by greater
than 95 % compared to the controls. Although SUS activity was detected in xylem
and phloem of control plants by in situ enzyme assays, plants with the
PEPC7-P4::MsSUS1 transgene lacked detectable SUS activity in post-elongation stem
(PES) internodes and had very low SUS activity in elongating stem (ES)
internodes. Loss of SUS protein in PES internodes of down-regulated lines was
confirmed by immunoblots. Down-regulation of SUS expression and activity in stem
tissue resulted in no obvious phenotype or significant change in cell wall sugar
composition. However, alkaline/neutral (A/N) invertase activity increased in SUS
down-regulated lines and high levels of acid invertase activity were observed. In
situ enzyme assays of stem tissue showed localization of neutral invertase in
vascular tissues of ES and PES internodes.
CONCLUSIONS: These results suggest that invertases play a primary role in
providing glucose for cellulose biosynthesis or compensate for the loss of SUS1
activity in stem vascular tissue.
DOI: 10.1186/s12870-015-0649-4
PMCID: PMC4666122
367. BMC Genomics. 2015 Dec 1;16:1020. doi: 10.1186/s12864-015-2212-y.
Accuracy of genomic selection for alfalfa biomass yield in different reference

populations.
Annicchiarico P(1), Nazzicari N(2), Li X(3), Wei Y(4), Pecetti L(5), Brummer
EC(6).
Author information:
(1)Council for Agricultural Research and Economics (CREA), Research Centre for
Fodder Crops and Dairy Productions, 29 viale Piacenza, 26900, Lodi, Italy.
paolo.annicchiarico@entecra.it.
nelson.nazzicari@entecra.it.
(3)Department of Plant Sciences, North Dakota State University, 1340
Administration Avenue, Fargo, ND, 58108, USA. xuehui.li@ndsu.edu.
(4)Plant Sciences Department, University of California, Davis, Plant Breeding
Center, One Shields Avenue, Davis, CA, 95616, USA. yweigalee@gmail.com.
luciano.pecetti@entecra.it.
(6)Plant Sciences Department, University of California, Davis, Plant Breeding
Center, One Shields Avenue, Davis, CA, 95616, USA. ecbrummer@ucdavis.edu.
BACKGROUND: Genomic selection based on genotyping-by-sequencing (GBS) data could

accelerate alfalfa yield gains, if it displayed moderate ability to predict
parent breeding values. Its interest would be enhanced by predicting ability also
for germplasm/reference populations other than those for which it was defined.
Predicting accuracy may be influenced by statistical models, SNP calling
procedures and missing data imputation strategies.
RESULTS: Landrace and variety material from two genetically-contrasting reference
populations, i.e., 124 elite genotypes adapted to the Po Valley (sub-continental
climate; PV population) and 154 genotypes adapted to Mediterranean-climate
environments (Me population), were genotyped by GBS and phenotyped in separate
environments for dry matter yield of their dense-planted half-sib progenies. Both
populations showed no sub-population genetic structure. Predictive accuracy was
higher by joint rather than separate SNP calling for the two data sets, and using
random forest imputation of missing data. Highest accuracy was obtained using
Support Vector Regression (SVR) for PV, and Ridge Regression BLUP and SVR for Me
germplasm. Bayesian methods (Bayes A, Bayes B and Bayesian Lasso) tended to be
less accurate. Random Forest Regression was the least accurate model. Accuracy
attained about 0.35 for Me in the range of 0.30-0.50 missing data, and 0.32 for
PV at 0.50 missing data, using at least 10,000 SNP markers. Cross-population
predictions based on a smaller subset of common SNPs implied a relative loss of
accuracy of about 25% for Me and 30% for PV. Genome-wide association analyses
based on large subsets of M. truncatula-aligned markers revealed many SNPs with
modest association with yield, and some genome areas hosting putative QTLs. A
comparison of genomic vs. conventional selection for parent breeding value
assuming 1-year vs. 5-year selection cycles, respectively, indicated over
three-fold greater predicted yield gain per unit time for genomic selection.
CONCLUSIONS: Genomic selection for alfalfa yield is promising, based on its
moderate prediction accuracy, moderate value of cross-population predictions, and
lack of sub-population structure. There is limited scope for searching individual
QTLs with overwhelming effect on yield. Some of our results can contribute to
better design of genomic selection experiments for alfalfa and other crops with
similar mating systems.
DOI: 10.1186/s12864-015-2212-y
PMCID: PMC4667460
368. Methods Mol Biol. 2016;1385:89-98. doi: 10.1007/978-1-4939-3289-4_6.
Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop

with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes.
Ferradini N(1), Giancaspro A(2), Nicolia A(1)(3), Gadaleta A(2), Veronesi F(1),
Rosellini D(4).
Author information:
(1)Department of Agricultural, Environmental and Food Science, University of
Perugia, Borgo XX giugno 74, 06121, Perugia, Italy.
(2)Department of Soil, Plant and Food Science, University of Bari "Aldo Moro",
Via G. Amendola 165/A, 70126, Bari, Italy.
(3)ENEA Casaccia Research Center, via Anguillarese 301, 00123, Roma, Italy.
(4)Department of Agricultural, Environmental and Food Science, University of
Perugia, Borgo XX giugno 74, 06121, Perugia, Italy. daniele.rosellini@unipg.it.
Antibiotic-free, efficient in vitro selection in plant genetic engineering can

improve risk perception and speed up pre-market scrutiny of genetically modified
crops. We provide a protocol for genetic transformation of two important crops,
durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker
gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde
aminotransferase (GSA) enzymes. These methods can potentially be applied, with
minor adaptations, to many other monocot and dicot crop plants.
DOI: 10.1007/978-1-4939-3289-4_6
369. J Dairy Sci. 2016 Jan;99(1):205-20. doi: 10.3168/jds.2015-9952. Epub 2015 Nov
18.
Ability of 3 tanniferous forage legumes to modify quality of milk and

Gruyère-type cheese.
Girard M(1), Dohme-Meier F(2), Wechsler D(3), Goy D(3), Kreuzer M(4), Bee G(5).
Author information:
(1)Institute for Livestock Sciences ILS, Agroscope, Tioleyre 4, 1725 Posieux,
Switzerland; Institute of Agricultural Science, ETH Zurich, Universitaetstrasse
2, 8092 Zurich, Switzerland.
Switzerland.
(3)Institute for Food Sciences IFS, Agroscope, Schwarzenburgstrasse 161, 3003
Bern, Switzerland.
(4)Institute of Agricultural Science, ETH Zurich, Universitaetstrasse 2, 8092
Switzerland. Electronic address: giuseppe.bee@agroscope.admin.ch.
Condensed tannins (CT) may affect ruminal biohydrogenation of dietary

polyunsaturated fatty acids. A feeding experiment was conducted with 24 Holstein
cows to evaluate whether diets containing CT from different forage legumes can
increase polyunsaturated fatty acids, especially n-3 fatty acid content in milk
and cheese, without affecting negatively their physicochemical and sensorial
properties. Cows were assigned to 4 treatment groups (n=6) for 52 d, divided into
2 periods: a control period (CoP) and an experimental period (ExP). During the
CoP, cows received a basal diet composed of hay, corn silage, ExtruLin (Trinova
Handel & Marketing AG, Wangen, Switzerland), concentrate, and alfalfa (AF) in a
ratio of 45:25:5:7:18. In the ExP, in 3 of the 4 groups AF was replaced by either
sainfoin (SF; 19% CT in dry matter) or 1 of 2 cultivars of birdsfoot trefoil
[Polom (BP), 3% CT; Bull (BB), 5% CT]. At the end of each period, milk was
collected on 3 consecutive days and analyzed for milk gross composition and fatty
acid profile and was processed to Gruyère-type cheese. A trained panel assessed
the sensory quality of raw milk and cheese using discriminative and descriptive
tests. This experimental design consisting of AF in both the CoP and ExP allowed
us to quantify effects due to lactation stage and experimental diets. In both the
CoP and ExP, dry matter intake and milk yield did not differ among treatment
groups. From the CoP to the ExP, milk urea content was reduced by 23% with SF,
remained unchanged with BP, and tended to increase with AF and BB. The odor of
the raw BB milk was judged to be different from AF milk. With SF, switching from
the CoP to the ExP resulted in a 17% increase of the 18:3n-3 proportion in milk
and cheese lipids. In BP cheese, the increase was 3%, whereas it tended to
decrease in BB cheese. Additionally, the 20:5n-3 and 22:5n-3 proportions tended
to increase in SF cheese from the CoP to the ExP. Compared with the AF cheeses,
cheeses from cows fed CT-containing legumes were judged harder and tended to be
less adhesive to the palate. In addition, SF and BP cheeses had less rind. In
conclusion, feeding SF compared with BB and BP increased the content of 18:3n-3
in the milk and the cheese without a negative effect on flavor of the cheese.
Despite a similar CT content, the 2 birdsfoot trefoil cultivars had opposite
effects on milk urea and 18:3n-3 deposition, suggesting that, besides the
content, the chemical structure may have had an important effect on the CT
efficacy.

DOI: 10.3168/jds.2015-9952
370. Genet Mol Res. 2015 Nov 23;14(4):14857-70. doi: 10.4238/2015.November.18.51.
Development of primer pairs from diverse chloroplast genomes for use in plant
phylogenetic research.
Yang YC(1), Kung TL(1), Hu CY(2), Lin SF(1).

Author information:
(1)Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic
of China.
(2)Wenshan Branch, Tea Research and Extension Station, Taipei, Taiwan, Republic
of China.
Variation in the chloroplast DNA sequence is useful for plant phylogenetic

studies. However, the number of variable sequences provided by chloroplast DNA
for suggested genes or genomic regions in plant phylogenetic analyses is often
inadequate. To identify conserved regions that can be used to design primers and
amplify variable sequences for use in plant phylogenetic studies, the complete
chloroplast genomic sequences of six plant species (including Oryza sativa,
Arabidopsis thaliana, Glycine max, Lotus japonicus, Medicago truncatula, and
Phaseolus vulgaris), searched from the taxonomy database of NCBI were
investigated. A total of 93 conserved regions, 32 in large single copy and 61 in
inverted repeat regions, were identified. A set of five primer pairs were
designed according to the conserved sequences located in the psbA~trnK,
psbB~psbH, rpl23~trnI, trnR~trnN, and trnY~trnD regions to amplify variable DNA
fragments. An additional 18 plant accessions from 14 species were used to
validate their utility. Each of the tested species could be distinguished by
length polymorphisms of fragments amplified with the five primer pairs. trnR~trnN
and rpl23~trnI amplified fragments specific to monocot and legume species,
respectively. Three primer pairs located in the psbA~trnK, psbB~psbH, and
trnR~trnN regions were applied to amplify variable DNA sequences for phylogenetic
analysis using the maximum parsimony method. The consistent result between
taxonomy and phylogenetic analysis on the variable sequences amplified with these
three primer pairs was revealed. The five newly developed primer pairs are
recommended as tools for use in the identification of plant species and in
phylogenetic studies.
DOI: 10.4238/2015.November.18.51
371. PLoS One. 2015 Nov 23;10(11):e0142851. doi: 10.1371/journal.pone.0142851.

eCollection 2015.
Mapping the Centimeter-Scale Spatial Variability of PAHs and Microbial

Populations in the Rhizosphere of Two Plants.
Bourceret A(1)(2), Leyval C(1)(2), de Fouquet C(3), Cébron A(1)(2).
Author information:
(1)CNRS, LIEC UMR7360, Faculté des Sciences et Technologies, Bd des Aiguillettes,
BP70239, 54506 Vandoeuvre-lès-Nancy, France.
(2)Université de Lorraine, LIEC UMR7360, Faculté des Sciences et Technologies, Bd
des Aiguillettes, BP 70239, 54506 Vandoeuvre-lès-Nancy, France.
(3)MINES ParisTech, Centre de Géosciences Géostatistique, Ecole Nationale
Supérieure des Mines de Paris, 35 Rue Saint-Honoré, 77305 Fontainebleau, France.
Rhizoremediation uses root development and exudation to favor microbial activity.

Thus it can enhance polycyclic aromatic hydrocarbon (PAH) biodegradation in
contaminated soils. Spatial heterogeneity of rhizosphere processes, mainly linked
to the root development stage and to the plant species, could explain the
contrasted rhizoremediation efficiency levels reported in the literature. Aim of
the present study was to test if spatial variability in the whole plant
rhizosphere, explored at the centimetre-scale, would influence the abundance of
microorganisms (bacteria and fungi), and the abundance and activity of
PAH-degrading bacteria, leading to spatial variability in PAH concentrations. Two
contrasted rhizospheres were compared after 37 days of alfalfa or ryegrass growth
in independent rhizotron devices. Almost all spiked PAHs were degraded, and the
density of the PAH-degrading bacterial populations increased in both rhizospheres
during the incubation period. Mapping of multiparametric data through
geostatistical estimation (kriging) revealed that although root biomass was
spatially structured, PAH distribution was not. However a greater variability of
the PAH content was observed in the rhizosphere of alfalfa. Yet, in the
ryegrass-planted rhizotron, the Gram-positive PAH-degraders followed a reverse
depth gradient to root biomass, but were positively correlated to the soil pH and
carbohydrate concentrations. The two rhizospheres structured the microbial
community differently: a fungus-to-bacterium depth gradient similar to the root
biomass gradient only formed in the alfalfa rhizotron.
PMCID: PMC4657893
372. Phytomedicine. 2015 Dec 1;22(13):1186-94. doi: 10.1016/j.phymed.2015.09.005.

Epub
2015 Oct 24.
Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce
apoptosis and overcome multidrug resistance in leukemia P388 cells.
Gatouillat G(1), Magid AA(2), Bertin E(3), El btaouri H(4), Morjani H(5), Lavaud
C(2), Madoulet C(6).
Author information:
(1)Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA,
Reims, France.
(2)Laboratoire de Pharmacognosie, Faculté de Pharmacie, SFR Cap Santé, ICMR-CNRS
UMR 7312, Reims, France.
(3)Service d'endocrinologie, de diabétologie et de nutrition, CHU Robert-Debré,
Reims, France.
(4)MEDyC UMR CNRS/URCA no. 7369, Faculté des Sciences, SFR Cap Santé, URCA,
Reims, France.
(5)MEDyC UMR CNRS/URCA no. 7369, Faculté de Pharmacie, SFR Cap Santé, URCA,
Reims, France.
(6)Laboratoire de Biochimie et Biologie Moléculaire, Faculté de Pharmacie, URCA,
Reims, France. Electronic address: claudie.madoulet@univ-reims.fr.
BACKGROUND: High consumption of flavonoids has been associated with a decrease

risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in
traditional medicine and is currently used as a dietary supplement because of
their high nutrient content. We previously reported the cytotoxic activity of
alfalfa leaf extracts against several sensitive and multidrug resistant tumor
cell lines.
HYPOTHESIS/PURPOSE: We aimed to determine whether medicarpin and millepurpan, two
isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects
against drug-sensitive (P388) and multidrug resistant P388 leukemia cells
(P388/DOX).
STUDY DESIGN/METHODS: Cells were incubated with medicarpin or millepurpan for the
appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation
was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by
flow cytometry technics. Caspases 3 and 9 activities were measured using Promega
caspACE assay kits. Proteins and genes expression were visualized respectively by
western-blot using specific antibodies and RT-PCR assay.
RESULTS: P-glycoprotein-expressing P388/DOX cells did not show resistance to
medicarpin (IC50 ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC50 = 54
µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with
medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug
resistant P388 cells. These effects were mediated through the mitochondrial
pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM
doxorubicin alone could not induce cell death in P388/DOX cells, concomitant
treatment with doxorubicin and subtoxic concentration of medicarpin or
millepurpan restored the pro-apoptotic cascade. Each compound increased
sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in
sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX
cells (IC50 = 210 nM to 23 and 25 nM with medicarpin and millepurpan,
respectively). This improved sensitivity was mediated by an increased uptake of
doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by
exposure to medicarpin and millepurpan.
CONCLUSION: These data indicate that medicarpin and millepurpan possess
pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in
multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of
drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to
enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.
DOI: 10.1016/j.phymed.2015.09.005
14.
Milk production responses to different strategies for feeding supplements to

grazing dairy cows.
Auldist MJ(1), Marett LC(2), Greenwood JS(2), Wright MM(2), Hannah M(2), Jacobs
JL(2), Wales WJ(2).
Author information:
(1)Agriculture Research Division, Department of Economic Development, Jobs,
Transport and Resources, Ellinbank VIC 3821, Australia. Electronic address:
Martin.Auldist@ecodev.vic.gov.au.
(2)Agriculture Research Division, Department of Economic Development, Jobs,
Transport and Resources, Ellinbank VIC 3821, Australia.
Milk production responses of grazing cows offered supplements in different ways

were measured. Holstein-Friesian cows, averaging 45 d in milk, were allocated
into 8 groups of 24, with 2 groups randomly assigned to each of 4 feeding
strategies. These were control: cows grazed a restricted allowance of perennial
ryegrass pasture supplemented with milled wheat grain fed in the milking parlor
and alfalfa hay offered in the paddock; FGM: same pasture and allowance as the
control supplemented with a formulated grain mix containing wheat grain, corn
grain, and canola meal fed in the parlor and alfalfa hay fed in the paddock;
PMRL: same pasture and allowance as the control, supplemented with a PMR
consisting of the same FGM but mixed with alfalfa hay and presented on a feed pad
after each milking; and PMRH: same PMR fed in the same way as PMRL but with a
higher pasture allowance. For all strategies, supplements provided the same
metabolizable energy and grain:forage ratio [75:25, dry matter (DM) basis]. Each
group of 24 cows was further allocated into 4 groups of 6, which were randomly
assigned to receive 8, 12, 14, or 16 kg of DM supplement/cow per d. Thus, 2
replicated groups per supplement amount per dietary strategy were used. The
experiment had a 14-d adaptation period and a 14-d measurement period. Pasture
allowance, measured to ground level, was approximately 14 kg of DM/d for control,
FGM, and PMRL cows, and 28 kg of DM/d for the PMRH cows, and was offered in
addition to the supplement. Positive linear responses to increasing amounts of
supplement were observed for yield of milk, energy-corrected milk, fat, and
protein for cows on all 4 supplement feeding strategies. Production of
energy-corrected milk was greatest for PMRH cows, intermediate for FGM and PMRL
cows, and lowest for control cows. Some of these differences in milk production
related to differences in intake of pasture and supplement. Milk fat
concentration decreased with increasing amount of supplement for all feeding
strategies, but the decline was most marked for the control cows. Milk protein
concentration increased for all groups as the amount of supplement increased, but
was greater for FGM, PMRL, and PMRH cows than control cows. It is concluded that
when supplements are fed to grazing dairy cows, inclusion of corn grain and
canola meal can increase milk production even at similar metabolizable energy
intakes, and that it does not matter whether these supplements are fed as a PMR
or in the parlor and paddock.

DOI: 10.3168/jds.2015-9834
374. Cryo Letters. 2015 Jul-Aug;36(4):252-63.
PHYSIOLOGICAL, CYTOLOGICAL AND BIOCHEMICAL STABILITY OF Medicago sativa L. CELL

CULTURE AFTER 27 YEARS OF CRYOGENIC STORAGE.
Volkova LA(1), Urmantseva VV(1), Popova EV(2), Nosov AM(3).
Author information:
(1)Department of Cell Biology and Biotechnology, Timiryazev Institute of Plant
Physiology of Russian Academy of Sciences, Moscow, Russia.
Physiology of Russian Academy of Sciences, Moscow, Russia. Gosling Research
Institute for Plant Preservation, Department of Plant Agriculture, University of
Guelph, Ontario, Canada. epopova@uoguelph.ca.
Physiology of Russian Academy of Sciences; Faculty of Biology, Lomonosov Moscow
State University, Moscow, Russia.
BACKGROUND: The efficiency of long-term cryogenic storage to prevent somaclonal

variations in plant cell cultures and retain their major cytogenetic and
biochemical traits remains under debate. In particular, it is not clear how
stress conditions associated with cryopreservation, such as low temperature,
dehydration and toxic action of some cryoprotectants (DMSO in particular), affect
post-storage regrowth and genetic integrity of cell samples.
OBJECTIVE: We assessed growth, cytogenetic and biochemical characteristics of the
peroxidase-producing strain of Medicago sativa L. cell culture recovered after 27
years of cryogenic storage as compared to the same culture before
cryopreservation.
MATERIALS AND METHODS: In 1984, M. sativa L. cell culture was cryopreserved using
programmed freezing and 7% DMSO as a cryoprotectant. In 2011, after rewarming in
a water bath at 40 degree C for 90 s, cell culture was recovered and
proliferated. Viability, growth profile, mitotic index, ploidy level, peroxidase
activity and cell response to hypothermia and osmotic stress were compared
between the recovered and the initial cell cultures using the records available
from 1984.
RESULTS: Viability of alfalfa cell culture after rewarming was below 20% but it
increased to 80% by the 27th subculture cycle. Recovered culture showed higher
mitotic activity and increased number of haploid and diploid cells compared to
the initial cell line. Both peroxidase activity and response to abiotic stress in
the recovered cell culture were similar to that of the initial culture.
CONCLUSION: Cryopreservation by programmed freezing was effective at retaining
the main characteristics of M. sativa undifferentiated cell culture after 27
years of storage. According to available data, this is longest period of
successful cryopreservation of plant cell cultures reported so far. After
storage, there was no evidence that DMSO had any detrimental effect on cell
viability, growth or cytogenetics.
375. Plant Cell Rep. 2016 Feb;35(2):439-53. doi: 10.1007/s00299-015-1895-5. Epub

2015
Nov 14.
MsZEP, a novel zeaxanthin epoxidase gene from alfalfa (Medicago sativa), confers
drought and salt tolerance in transgenic tobacco.
Zhang Z(1), Wang Y(1), Chang L(1), Zhang T(1), An J(1), Liu Y(1), Cao Y(1), Zhao
X(1), Sha X(1), Hu T(2), Yang P(3).
Author information:
(1)Department of Grassland Science, College of Animal Science and Technology,
Northwest A&F University, Yangling, 712100, Shaanxi, China.
Northwest A&F University, Yangling, 712100, Shaanxi, China. hutianming@126.com.
Northwest A&F University, Yangling, 712100, Shaanxi, China. yangpeizhi@126.com.
KEY MESSAGE: The zeaxanthin epoxidase gene ( MsZEP ) was cloned and characterized
from alfalfa and validated for its function of tolerance toward drought and salt
stresses by heterologous expression in Nicotiana tabacum. Zeaxanthin epoxidase
(ZEP) plays important roles in plant response to various environment stresses due
to its functions in ABA biosynthetic and the xanthophyll cycle. To understand the
expression characteristics and the biological functions of ZEP in alfalfa
(Medicago sativa), a novel gene, designated as MsZEP (KM044311), was cloned,
characterized and overexpressed in Nicotiana tabacum. The open reading frame of
MsZEP contains 1992 bp nucleotides and encodes a 663-amino acid polypeptide.
Amino acid sequence alignment indicated that deduced MsZEP protein was highly
homologous to other plant ZEP sequences. Phylogenetic analysis showed that MsZEP
was grouped into a branch with other legume plants. Real-time quantitative PCR
revealed that MsZEP gene expression was clearly tissue-specific, and the
expression levels were higher in green tissues (leaves and stems) than in roots.
MsZEP expression decreased in shoots under drought, cold, heat and ABA treatment,
while the expression levels in roots showed different trends. Besides, the
results showed that nodules could up-regulate the MsZEP expression under
non-stressful conditions and in the earlier stage of different abiotic stress.
Heterologous expression of the MsZEP gene in N. tabacum could confer tolerance to
drought and salt stress by affecting various physiological pathways, ABA levels
and stress-responsive genes expression. Taken together, these results suggested
that the MsZEP gene may be involved in alfalfa responses to different abiotic
stresses and nodules, and could enhance drought and salt tolerance of transgenic
tobacco by heterologous expression.
DOI: 10.1007/s00299-015-1895-5
376. Animal. 2016 Apr;10(4):607-15. doi: 10.1017/S1751731115002499. Epub 2015 Nov

16.
Determining optimum age of Holstein dairy calves when adding chopped alfalfa hay
to meal starter diets based on measures of growth and performance.
Hosseini SM(1), Ghorbani GR(1), Rezamand P(2), Khorvash M(1).
Author information:
(1)1Department of Animal Sciences,Isfahan University of Technology,Isfahan
84156,Iran.
(2)2Department of Animal and Veterinary Science,University of Idaho,Moscow,ID
83844,USA.
The present study was conducted to determine the optimum age of Holstein dairy
calves for an effective inclusion of alfalfa hay (AH) in starter feed on
performance, apparent digestibility and feeding behavior. A total of 40 Holstein
dairy calves (20 female and 20 male) were used in a completely randomized design
in which calves were randomly assigned to one of four different dietary
treatments including control (CON) calves fed starter feed without any forage and
three treatments consisting of the same starter feed plus 15% chopped AH fed when
calves were at the 2nd (AH2), 4th (AH4) or 6th (AH6) week of age. Calves were
individually housed and bedded with sand that was replaced every other day. Feed
and water were available ad libitum throughout the experiment. Calves were fed
milk at 10% of birth BW twice daily until d 57. The study concluded when calves
were 73 days old. Starter intake was recorded daily and BW was measured weekly.
Data were analyzed as a complete randomized design by MIXED procedures of SAS.
Results demonstrate that calves receiving AH treatments numerically consumed more
starter feed (0.62 v. 0.78, 0.71 and 0.65 kg/day for CON, AH2, AH4 and AH6,
respectively) and had greater average daily gain (ADG) compared with CON (0.48 v.
0.57, 0.49 and 0.49 kg/day for CON, AH2, AH4 and AH6), although the significant
difference was observed only between AH2 and CON. Among AH treatments, calves in
AH2 had better performance than AH6 in several cases including starter intake,
ADG. No detectable differences were observed, however, in apparent dry matter,
organic matter or CP digestibility among treatments. Ruminal pH and NH3
concentrations, measured on weeks 4, 6, 8 and 10, were lower for calves fed CON
compared with other treatments, with ammonia concentrations decreasing over time.
Calves in the AH treatments spent more time eating and ruminating compared with
CON. Calves fed CON, however, spent more time on laying down compared with other
treatments. Overall, results from the present study illustrated that inclusion of
alfalfa in starter feed for calves at 2 weeks of age may improve feed intake, ADG
and stimulate rumination in young Holstein dairy calves. Results, however, should
be viewed with caution as the number of calves per treatment was small and large
calf-to-calf variation may have affected the results reported.
DOI: 10.1017/S1751731115002499
377. J Sci Food Agric. 2016 Aug;96(10):3469-76. doi: 10.1002/jsfa.7530. Epub 2015
Dec
21.
The effects of Mg enrichment of vegetable sprouts on Mg concentration, yield and

ROS generation.
Przybysz A(1), Wrochna M(1), Małecka-Przybysz M(1), Gawrońska H(1), Gawroński
SW(1).
Author information:
(1)Laboratory of Basic Research in Horticulture, Faculty of Horticulture,
Biotechnology and Landscape Architecture, Warsaw University of Life
Sciences - SGGW, 02-776, Warsaw, Poland.
BACKGROUND: Two-thirds of the world's population do not consume the recommended

amount of Mg, hence the demand for the production of Mg-enriched plants. Sprouts
represent promising targets for enrichment. This study evaluated the effects of
enriching broccoli, radish, alfalfa and mung bean sprouts with Mg (50-300 mg
L(-1) ) on (i) the concentration of Mg and other ions, (ii) biomass accumulation,
(iii) levels of reactive oxygen species (ROS), and (iv) the activity/content of
enzymatic and non-enzymatic components of antioxidative systems.
RESULTS: Enrichment of sprouts with Mg led to a significant increase in Mg
concentration, especially in alfalfa (increase of 23-152 %), without depletion of
other ions. A higher Mg concentration had a minor effect on biomass accumulation,
but increased, often significantly, ROS generation and affected enzymatic and
non-enzymatic antioxidative systems. The level of O2 (•-) increased most in
broccoli, by 59-158%, while OH(•) increased most in radish, by 200-350%.
CONCLUSIONS: Enrichment of sprouts with Mg is possible, but attention must be
paid to elevated ROS levels in food. Mung bean sprouts are best suited to
enrichment as they make a considerable contribution to the daily supplementation
of Mg, at still low levels of ROS in enriched plants. © 2015 Society of Chemical
Industry.
DOI: 10.1002/jsfa.7530
378. Plant Mol Biol. 2016 Feb;90(3):207-15. doi: 10.1007/s11103-015-0404-0. Epub

2015
Nov 11.
NOD promoter-controlled AtIRT1 expression functions synergistically with NAS and

FERRITIN genes to increase iron in rice grains.
Boonyaves K(1), Gruissem W(1), Bhullar NK(2).
Author information:
(1)Plant Biotechnology, Department of Biology, ETH Zurich (Swiss Federal
Institute of Technology Zurich), Universitaetsstrasse 2, 8092, Zurich,
Switzerland.
(2)Plant Biotechnology, Department of Biology, ETH Zurich (Swiss Federal
Institute of Technology Zurich), Universitaetsstrasse 2, 8092, Zurich,
Switzerland. bhullarn@ethz.ch.
Rice is a staple food for over half of the world's population, but it contains
only low amounts of bioavailable micronutrients for human nutrition.
Consequently, micronutrient deficiency is a widespread health problem among
people who depend primarily on rice as their staple food. Iron deficiency anemia
is one of the most serious forms of malnutrition. Biofortification of rice grains
for increased iron content is an effective strategy to reduce iron deficiency.
Unlike other grass species, rice takes up iron as Fe(II) via the IRON REGULATED
TRANSPORTER (IRT) in addition to Fe(III)-phytosiderophore chelates. We expressed
Arabidopsis IRT1 (AtIRT1) under control of the Medicago sativa EARLY NODULIN 12B
promoter in our previously developed high-iron NFP rice lines expressing
NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN. Transgenic rice lines expressing
AtIRT1 alone had significant increases in iron and combined with NAS and FERRITIN
increased iron to 9.6 µg/g DW in the polished grains that is 2.2-fold higher as
compared to NFP lines. The grains of AtIRT1 lines also accumulated more copper
and zinc but not manganese. Our results demonstrate that the concerted expression
of AtIRT1, AtNAS1 and PvFERRITIN synergistically increases iron in both polished
and unpolished rice grains. AtIRT1 is therefore a valuable transporter for iron
biofortification programs when used in combination with other genes encoding iron
transporters and/or storage proteins.
DOI: 10.1007/s11103-015-0404-0
PMCID: PMC4717176
5.
Short communication: Regulation of hepatic gluconeogenic enzymes by dietary

glycerol in transition dairy cows.
White HM(1), Carvalho ER(1), Koser SL(1), Schmelz-Roberts NS(1), Pezzanite LM(1),
Slabaugh AC(1), Doane PH(2), Donkin SS(3).
Author information:
(1)Department of Animal Sciences, Purdue University, West Lafayette, IN 47907.
(2)Archer Daniels Midland Company, Decatur, IL 62521.
(3)Department of Animal Sciences, Purdue University, West Lafayette, IN 47907.
Electronic address: sdonkin@purdue.edu.
Nutritional status and glucose precursors are known regulators of gluconeogenic

gene expression. Glycerol can replace corn in diets fed to dairy cows and use of
glycerol is linked to increased rumen propionate production. The effect of
dietary glycerol on the regulation of gluconeogenic enzymes is unknown. The
objective of this study was to examine the effect of glycerol on expression of
pyruvate carboxylase (PC), cytosolic and mitochondrial phosphoenolpyruvate
carboxykinase (PEPCK-C and PEPCK-M), and glucose-6-phosphatase. Twenty-six
multiparous Holstein cows were fed either a control diet or a diet where
high-moisture corn was replaced by glycerol from -28 through +56 d relative to
calving (DRTC). Liver tissue was collected via percutaneous liver biopsy at -28,
-14, +1, +14, +28, and +56 DRTC for RNA analysis. Expression of PC mRNA increased
6-fold at +1 and 4-fold at +14 DRTC relative to precalving levels. Dietary
glycerol did not alter expression of PC mRNA expression. Expression of PEPCK-C
increased 2.5-fold at +14 and 3-fold at +28 DRTC compared with +1 DRTC. Overall,
dietary glycerol increased PEPCK-C expression compared with that of cows fed
control diets. The ratio of PC to PEPCK-C was increased 6.3-fold at +1 DRTC
compared with precalving and tended to be decreased in cows fed glycerol. We
detected no effect of diet or DRTC on PEPCK-M or glucose-6-phosphatase mRNA, and
there were no interactions of dietary treatment and DRTC for any transcript
measured. Substituting corn with glycerol increased the expression of PEPCK-C
mRNA during transition to lactation and suggests that dietary energy source
alters hepatic expression. The observed increase in PEPCK-C expression with
glycerol feeding may indicate regulation of hepatic gene expression by changes in
rumen propionate production.

DOI: 10.3168/jds.2015-9953
380. J Exp Biol. 2015 Nov;218(Pt 21):3425-34. doi: 10.1242/jeb.128165.
Decreasing methane yield with increasing food intake keeps daily methane
emissions constant in two foregut fermenting marsupials, the western grey
kangaroo and red kangaroo.
Vendl C(1), Clauss M(2), Stewart M(3), Leggett K(4), Hummel J(5), Kreuzer M(6),
Munn A(7).
Author information:
(1)Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty,
University of Zurich, 8057 Zurich, Switzerland Centre for Sustainable Ecosystems
Solutions, School of Biological Sciences, University of Wollongong, Wollongong,
NSW 2522, Australia.
(2)Clinic for Zoo Animals, Exotic Pets and Wildlife, Vetsuisse Faculty,
University of Zurich, 8057 Zurich, Switzerland.
(3)Centre for Sustainable Ecosystems Solutions, School of Biological Sciences,
University of Wollongong, Wollongong, NSW 2522, Australia.
(4)Fowlers Gap Arid Zone Research Station, School of Biological, Earth and
Environmental Sciences, University of New South Wales, Fowlers Gap, Broken Hill,
NSW 2880, Australia.
(5)Department of Animal Sciences, Ruminant Nutrition, University of Göttingen,
37077 Göttingen, Germany.
(6)ETH Zurich, Institute of Agricultural Sciences, 8092 Zurich, Switzerland.
(7)Centre for Sustainable Ecosystems Solutions, School of Biological Sciences,
University of Wollongong, Wollongong, NSW 2522, Australia Fowlers Gap Arid Zone
Research Station, School of Biological, Earth and Environmental Sciences,
University of New South Wales, Fowlers Gap, Broken Hill, NSW 2880, Australia
amunn@uow.edu.au.
Fundamental differences in methane (CH4) production between macropods (kangaroos)

and ruminants have been suggested and linked to differences in the composition of
the forestomach microbiome. Using six western grey kangaroos (Macropus
fuliginosus) and four red kangaroos (Macropus rufus), we measured daily absolute
CH4 production in vivo as well as CH4 yield (CH4 per unit of intake of dry
matter, gross energy or digestible fibre) by open-circuit respirometry. Two food
intake levels were tested using a chopped lucerne hay (alfalfa) diet. Body
mass-specific absolute CH4 production resembled values previously reported in
wallabies and non-ruminant herbivores such as horses, and did not differ with
food intake level, although there was no concomitant proportionate decrease in
fibre digestibility with higher food intake. In contrast, CH4 yield decreased
with increasing intake, and was intermediate between values reported for
ruminants and non-ruminant herbivores. These results correspond to those in
ruminants and other non-ruminant species where increased intake (and hence a
shorter digesta retention in the gut) leads to a lower CH4 yield. We hypothesize
that rather than harbouring a fundamentally different microbiome in their
foregut, the microbiome of macropods is in a particular metabolic state more
tuned towards growth (i.e. biomass production) rather than CH4 production. This
is due to the short digesta retention time in macropods and the known distinct
'digesta washing' in the gut of macropods, where fluids move faster than
particles and hence most likely wash out microbes from the forestomach. Although
our data suggest that kangaroos only produce about 27% of the body mass-specific
volume of CH4 of ruminants, it remains to be modelled with species-specific
growth rates and production conditions whether or not significantly lower CH4
amounts are emitted per kg of meat in kangaroo than in beef or mutton production.
© 2015. Published by The Company of Biologists Ltd.
DOI: 10.1242/jeb.128165
2015.
Nodulation by Sinorhizobium meliloti originated from a mining soil alleviates Cd

toxicity and increases Cd-phytoextraction in Medicago sativa L.
Ghnaya T(1), Mnassri M(1), Ghabriche R(1), Wali M(1), Poschenrieder C(2), Lutts
S(3), Abdelly C(1).
Author information:
(1)Laboratoire des Plantes Extremophiles, Centre de Biotechnologies de la
Technopole de Borj Cedria Hammam Lif, Tunisia.
(2)Departamento de Fisiologia Vegetal, Facultad de Ciencias, Universidad Autonoma
de Barcelona Barcelona, Spain.
(3)Groupe de Recherche en Physiologie Végétale, Earth and Life Institute -
Agronomy, Université Catholique de Louvain Louvain-la-Neuve, Belgium.
Besides their role in nitrogen supply to the host plants as a result of symbiotic
N fixation, the association between legumes and Rhizobium could be useful for the
rehabilitation of metal-contaminated soils by phytoextraction. A major limitation
presents the metal-sensitivity of the bacterial strains. The aim of this work was
to explore the usefulness of Sinorhizobium meliloti originated from a mining site
for Cd phytoextraction by Medicago sativa. Inoculated and non-inoculated plants
were cultivated for 60 d on soils containing 50 and/or 100 mg Cd kg(-1) soil. The
inoculation hindered the occurrence of Cd- induced toxicity symptoms that
appeared in the shoots of non-inoculated plants. This positive effect of S.
meliloti colonization was accompanied by an increase in biomass production and
improved nutrient acquisition comparatively to non-inoculated plants. Nodulation
enhanced Cd absorption by the roots and Cd translocation to the shoots. The
increase of plant biomass concomitantly with the increase of Cd shoot
concentration in inoculated plants led to higher potential of Cd-phytoextraction
in these plants. In the presence of 50 mg Cd kg(-1) in the soil, the amounts of
Cd extracted in the shoots were 58 and 178 μg plant(-1) in non-inoculated and
inoculated plants, respectively. This study demonstrates that this association M.
sativa-S. meliloti may be an efficient biological system to extract Cd from
contaminated soils.
DOI: 10.3389/fpls.2015.00863
PMCID: PMC4604267
PMID: 26528320
382. Sci Total Environ. 2016 Sep 1;563-564:693-703. doi:

Comparative bioremediation of heavy metals and petroleum hydrocarbons

co-contaminated soil by natural attenuation, phytoremediation, bioaugmentation
and bioaugmentation-assisted phytoremediation.
Agnello AC(1), Bagard M(2), van Hullebusch ED(3), Esposito G(4), Huguenot D(5).
Author information:
UPEM, 77454 Marne-la-Vallée, France; Università degli Studi di Cassino e del
Lazio Meridionale, Dipartimento di Ingegneria Civile e Meccanica, via Di Biasio
43, 03043 Cassino, FR, Italy.
(2)Université Paris-Est Créteil, Institut d'écologie et des sciences de
l'environnement de Paris UMR 1392, Équipe Interactions plantes-environnement,
Créteil Cedex, France.
UPEM, 77454 Marne-la-Vallée, France.
(4)Università degli Studi di Cassino e del Lazio Meridionale, Dipartimento di
Ingegneria Civile e Meccanica, via Di Biasio 43, 03043 Cassino, FR, Italy.
UPEM, 77454 Marne-la-Vallée, France. Electronic address: david.huguenot@u-pem.fr.
Biological remediation technologies are an environmentally friendly approach for

the treatment of polluted soils. This study evaluated through a pot experiment
four bioremediation strategies: a) natural attenuation, b) phytoremediation with
alfalfa (Medicago sativa L.), c) bioaugmentation with Pseudomonas aeruginosa and
d) bioaugmentation-assisted phytoremediation, for the treatment of a
co-contaminated soil presenting moderate levels of heavy metals (Cu, Pb and Zn at
87, 100 and 110mgkg(-1) DW, respectively) and petroleum hydrocarbons
(3800mgkg(-1) DW). As demonstrated by plant biomass and selected physiological
parameters alfalfa plants were able to tolerate and grow in the co-contaminated
soil, especially when soil was inoculated with P. aeruginosa, which promoted
plant growth (56% and 105% increase for shoots and roots, respectively) and
appeared to alleviate plant stress. The content of heavy metals in alfalfa plants
was limited and followed the order: Zn>Cu>Pb. Heavy metals were mainly
concentrated in plant roots and were poorly translocated, favouring their
stabilization in the root zone. Bioaugmentation of planted soil with P.
aeruginosa generally led to a decrease of plant metal concentration and
translocation. The highest degree of total petroleum hydrocarbon removal was
obtained for bioaugmentation-assisted phytoremediation treatment (68%), followed
by bioaugmentation (59%), phytoremediation (47%) and natural attenuation (37%).
The results of this study demonstrated that the combined use of plant and
bacteria was the most advantageous option for the treatment of the present
co-contaminated soil, as compared to natural attenuation, bioaugmentation or
phytoremediation applied alone.
DOI: 10.1016/j.scitotenv.2015.10.061
PMID: 26524994
Early supplementation of starter pellets with alfalfa improves the performance of

pre- and postweaning Hu lambs.
Yang B, He B, Wang SS, Liu JX, Wang JK.
This study aims to determine the effects of alfalfa supplementation on the pre-
and postweaning performance, rumen development, and feed transition in starter
diet-fed lambs. Six of 66 male Hu lambs were slaughtered at the age of 10 d to
serve as a control. The other 60 lambs were randomly allocated to 2 dietary
treatments: milk replacer and starter pellets without (STA) or with free-choice
chopped alfalfa (S-ALF). The animals were offered 300 g/d of the concentrate
mixture and had free access to alfalfa after weaning at the end of wk 4 (age 38
d). The alfalfa inclusion in the S-ALF group tended to increase the starter
intake before weaning, significantly increased the concentrate intake soon after
weaning ( < 0.05), and increased the BW ( < 0.01) and ADG ( < 0.10) in pre- and
postweaning lambs. The S-ALF group had heavier carcasses ( < 0.05), rumens ( <
0.05), reticula ( < 0.05), omasums ( < 0.10), abomasums ( < 0.05), and visceral
organs ( < 0.10) than the STA lambs after weaning. Alfalfa supplementation
increased ( < 0.05) the rumen papillae length and the ratio of the duodenal
villus height to the crypt depth; it also decreased ( < 0.05) the concentration
and molar proportion of propionate in wk 1 and 5. The STA lambs had higher ( <
0.01) blood concentrations of globulin and blood urea nitrogen and lower
β-hydroxybutyrate after weaning. The STA group also had a higher incidence of
feed plaque. From the above results, we infer that the free-choice addition of
chopped alfalfa to starter diets is beneficial to rumen development, relieves
weaning stress, and improves the performance of lambs.
DOI: 10.2527/jas.2015-9266
384. Plant Cell Environ. 2016 Apr;39(4):883-96. doi: 10.1111/pce.12672. Epub 2016
Jan
21.
The presence of nodules on legume root systems can alter phenotypic plasticity in
response to internal nitrogen independent of nitrogen fixation.
Goh CH(1), Nicotra AB(2), Mathesius U(1).
Author information:
(1)Division of Plant Science, Australian National University, Canberra, ACT,
0200, Australia.
(2)Division of Evolution, Ecology and Genetics, Research School of Biology,
Australian National University, Canberra, ACT, 0200, Australia.
All higher plants show developmental plasticity in response to the availability

of nitrogen (N) in the soil. In legumes, N starvation causes the formation of
root nodules, where symbiotic rhizobacteria fix atmospheric N2 for the host in
exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic
responses to internal [N] of legumes are altered by their symbionts. Glasshouse
experiments compared root phenotypes of three legumes, Medicago truncatula,
Medicago sativa and Trifolium subterraneum, inoculated with their compatible
symbiont partners and grown under four nitrate levels. In addition, six strains
of rhizobia, differing in their ability to fix N2 in M. truncatula, were compared
to test if plastic responses to internal [N] were dependent on the rhizobia or N2
-fixing capability of the nodules. We found that the presence of rhizobia
affected phenotypic plasticity of the legumes to internal [N], particularly in
root length and root mass ratio (RMR), in a plant species-dependent way. While
root length responses of M. truncatula to internal [N] were dependent on the
ability of rhizobial symbionts to fix N2 , RMR response to internal [N] was
dependent only on initiation of nodules, irrespective of N2 -fixing ability of
the rhizobia strains.
DOI: 10.1111/pce.12672
385. Oecologia. 2016 Jul;181(3):659-72. doi: 10.1007/s00442-015-3469-1. Epub 2015

Oct
31.
Enemy-free space promotes maintenance of host races in an aphid species.
Vosteen I(1), Gershenzon J(2), Kunert G(2).
Author information:
(1)Max-Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07749, Jena,
Germany. ivosteen@ice.mpg.de.
(2)Max-Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07749, Jena,
Germany.
The enormous biodiversity of herbivorous insects may arise from ecological

speciation via continuous host-plant switches. Whether such switches are
successful depends on the trade-off between different selection pressures that
act on herbivores. Decreased herbivore performance due to suboptimal nutrition
might be compensated for by a reduced natural enemy pressure. As a consequence,
an "enemy-free space" on a certain plant might facilitate host-plant switches and
maintain biotypes. To test this hypothesis, we used the pea aphid (Acyrthosiphon
pisum) complex, which consists of at least 11 genetically distinct host races
that are native to specific legume host plants but can all develop on the
universal host plant Vicia faba. Three A. pisum host races native to Trifolium
pratense, Pisum sativum, and Medicago sativa were investigated in experiments on
their respective host plants and on the universal host plant V. faba. We found
that hoverflies preferred to oviposit on P. sativum and the universal host V.
faba. Since feeding by hoverfly larvae suppressed aphid population growth on
these host plants, the native hosts M. sativa and T. pratense provided enemy-free
space for the respective A. pisum races. Mobile predators, such as ants and
ladybird beetles, preferred Pisum race aphids on V. faba over P. sativum. Thus,
all three of the native host plants studied supply enemy-free space for A. pisum
compared to the universal host V. faba. Reducing encounters between aphid races
on V. faba would reduce gene flow among them and could contribute to maintaining
the host races.
DOI: 10.1007/s00442-015-3469-1
386. Int J Syst Evol Microbiol. 2015 Nov;65(11):3853-3860. doi:

10.1099/ijsem.0.000505.
Paenibacillus medicaginis sp. nov. a chitinolytic endophyte isolated from a root

nodule of alfalfa (Medicago sativa L.).
Lai WA, Hameed A, Lin SY, Hung MH, Hsu YH, Liu YC, Shahina M, Shen FT, Young CC.
A Gram-stain-variable, short-rod-shaped, endospore-forming, strictly aerobic,

non-motile, chitinolytic and endophytic bacterium, designated strain
CC-Alfalfa-19T, exhibiting unusual bipolar appendages was isolated from a root
nodule of alfalfa (Medicago sativa L.) in Taiwan and subjected to a polyphasic
taxonomic study. Based on 16S rRNA gene sequence analysis, strain CC-Alfalfa-19T
was found to be most closely related to Paenibacillus puldeungensis CAU 9324T
(95.2 %), whereas other species of the genus Paenibacillus shared ≤ 95.0 %
sequence similarity. The phylogenetic analysis revealed a distinct phyletic
lineage established by strain CC-Alfalfa-19T with respect to other species of the
genus Paenibacillus. Fatty acids comprised predominantly anteiso-C15 : 0,
C16 : 0, anteiso-C17 : 0 and iso-C16 : 0. Menaquinone 7 (MK-7) was identified as
the sole respiratory quinone and the genomic DNA G+C content was 42.7 mol%. Polar
lipids included diphosphatidylglycerol, phosphatidylglycerol,
phosphatidylmonomethylethanolamine, phosphatidylethanolamine, an unidentified
glycolipid and an unidentified lipid. The diagnostic diamino acid found in the
cell-wall peptidoglycan was meso-diaminopimelic acid. Based on the polyphasic
taxonomic evidence that was in line with the genus Paenibacillus and additional
distinguishing characteristics, strain CC-Alfalfa-19T is considered to represent
a novel species, for which the name Paenibacillus medicaginis sp. nov. (type
strain CC-Alfalfa-19T = BCRC 80441T = JCM 18446T) is proposed.
DOI: 10.1099/ijsem.0.000505
PMID: 28875919
387. Genes Genet Syst. 2015;90(3):175-80. doi: 10.1266/ggs.90.175.
Selection of reliable reference genes for quantitative real-time RT-PCR in

alfalfa.
Wang X(1), Fu Y, Ban L, Wang Z, Feng G, Li J, Gao H.
Author information:
(1)Institute of Animal Science, Chinese Academy of Agricultural Sciences.
Real-time quantitative RT-PCR (qRT-PCR) is the most commonly used method for
accurately detecting gene expression patterns. As part of qRT-PCR analysis,
normalization of the data requires internal control gene(s) that display uniform
expression under different biological conditions. However, no invariable internal
control gene exists, and therefore more than one reference gene is needed to
normalize RT-PCR results. In this study, we assessed the expression of eight
candidate internal control genes, namely 18S ribosomal RNA (18S rRNA), elongation
factor-1alpha, β-Actin, E2 ubiquitin-conjugating enzyme, β-Tubulin (TUB), ACTIN2,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Msc27 of unknown function,
in a diverse set of 16 alfalfa (Medicago sativa) samples representing different
tissues and abiotic stress challenges, using geNorm and BestKeeper software. The
results revealed that the eight candidate genes are inconsistently expressed
under different experimental conditions. Msc27 and 18S rRNA are suitable
reference genes for comparing different tissue types. Under different abscisic
acid and NaCl conditions, three reference genes are necessary. Finally, GAPDH,
TUB and β-Actin are unsuitable for normalization of qRT-PCR data under these
given conditions in alfalfa. The relative expression level of MsWRKY33 was
analyzed using selected reference genes. These results provide an experimental
guideline for future research on gene expression in alfalfa using qRT-PCR.
DOI: 10.1266/ggs.90.175
388. Crit Rev Food Sci Nutr. 2016 Jul 29;56 Suppl 1:S149-61. doi:
10.1080/10408398.2015.1038378.
Therapeutic Potential of Temperate Forage Legumes: A Review.
Cornara L(1), Xiao J(2)(3), Burlando B(4)(5).
Author information:
(1)a Dipartimento di Scienze della Terra dell'Ambiente e della Vita , Università
degli Studi di Genova , Genova , Italy.
(2)b Institute of Chinese Medical Sciences, State Key Laboratory of Quality
Research in Chinese Medicine, University of Macau , Taipa , Macau.
(3)c College of Food Science, Fujian Agriculture and Forestry University , Fuzhou
, Fujian , China.
(4)d Dipartimento di Scienze e Innovazione Tecnologica , Università del Piemonte
Orientale , Alessandria , Italy.
(5)e Istituto di Biofisica, Consiglio Nazionale delle Ricerche , Genova , Italy.
The discovery of bioactive molecules from botanical sources is an expanding

field, preferentially oriented to plants having a tradition of use in medicine
and providing high yields and availability. Temperate forage legumes are Fabaceae
species that include worldwide-important crops. These plants possess therapeutic
virtues that have not only been used in veterinary and folk medicine, but have
also attracted the interest of official medicine. We have examined here Medicago
sativa (alfalfa), Trifolium pratense and T. repens (clovers), Melilotus albus and
M. officinalis (sweet clovers), Lotus corniculatus (birdsfoot trefoil),
Onobrychis viciifolia (sainfoin), Lespedeza capitata (roundhead lespedeza), and
Galega officinalis (goat's rue). The phytochemical complexes of these species
contain secondary metabolites whose pharmacological potentials deserve
investigation. Major classes of compounds include alkaloids and amines,
cyanogenic glycosides, flavonoids, coumarins, condensed tannins, and saponins.
Some of these phytochemicals have been related to antihypercholesterolemia,
antidiabetic, antimenopause, anti-inflammatory, antiedema, anthelmintic, and
kidney protective effects. Two widely prescribed drugs have been developed
starting from temperate forage legumes, namely, the antithrombotic warfarin,
inspired from sweet clover's coumarin, and the antidiabetic metformin, a
derivative of sainfoin's guanidine. Available evidence suggests that temperate
forage legumes are a potentially important resource for the extraction of active
principles to be used as nutraceuticals and pharmaceuticals.
DOI: 10.1080/10408398.2015.1038378
389. BMC Plant Biol. 2015 Oct 26;15:260. doi: 10.1186/s12870-015-0651-x.
The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis.
van Zeijl A(1), Liu W(2)(3), Xiao TT(4), Kohlen W(5), Yang WC(6), Bisseling T(7),
Geurts R(8).
Author information:
(1)Department of Plant Science, Laboratory of Molecular Biology, Wageningen
University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
arjan.vanzeijl@wur.nl.
wliu@genetics.ac.cn.
(3)State Key Laboratory of Molecular and Developmental Biology, Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101,
China. wliu@genetics.ac.cn.
tingting.xiao@wur.nl.
wouter.kohlen@wur.nl.
(6)State Key Laboratory of Molecular and Developmental Biology, Institute of
Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101,
China. wcyang@genetics.ac.cn.
ton.bisseling@wur.nl.
rene.geurts@wur.nl.
BACKGROUND: Strigolactones are a class of plant hormones whose biosynthesis is

activated in response to phosphate starvation. This involves several enzymes,
including the carotenoid cleavage dioxygenases 7 (CCD7) and CCD8 and the
carotenoid isomerase DWARF27 (D27). D27 expression is known to be responsive to
phosphate starvation. In Medicago truncatula and rice (Oryza sativa) this
transcriptional response requires the GRAS-type proteins NSP1 and NSP2; both
proteins are essential for rhizobium induced root nodule formation in legumes. In
line with this, we questioned whether MtNSP1-MtNSP2 dependent MtD27 regulation is
co-opted in rhizobium symbiosis.
RESULTS: We provide evidence that MtD27 is involved in strigolactone biosynthesis
in M. truncatula roots upon phosphate stress. Spatiotemporal expression studies
revealed that this gene is also highly expressed in nodule primordia and
subsequently becomes restricted to the meristem and distal infection zone of a
mature nodules. A similar expression pattern was found for MtCCD7 and MtCCD8.
Rhizobium lipo-chitooligosaccharide (LCO) application experiments revealed that
of these genes MtD27 is most responsive in an MtNSP1 and MtNSP2 dependent manner.
Symbiotic expression of MtD27 requires components of the symbiosis signaling
pathway; including MtDMI1, MtDMI2, MtDMI3/MtCCaMK and in part MtERN1. This in
contrast to MtD27 expression upon phosphate starvation, which only requires
MtNSP1 and MtNSP2.
CONCLUSION: Our data show that the phosphate-starvation responsive strigolactone
biosynthesis gene MtD27 is also rapidly induced by rhizobium LCO signals in an
MtNSP1 and MtNSP2-dependent manner. Additionally, we show that MtD27 is
co-expressed with MtCCD7 and MtCCD8 in nodule primordia and in the infection zone
of mature nodules.
DOI: 10.1186/s12870-015-0651-x
PMCID: PMC4624177
390. Food Funct. 2016 Jan;7(1):364-71. doi: 10.1039/c5fo01115a.
The isoflavone content of two new alfalfa-derived products for instant beverage
preparation.
Soto-Zarazúa MG(1), Rodrigues F(2), Pimentel FB(2), Bah MM(3), Oliveira MB(2).
Author information:
(1)REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of
Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
beatoliv@ff.up.pt and Facultad de Química, Universidad Autónoma de Querétaro,
Centro Universitario, Cerro de las Campanas, C.P. 76010, Querétaro, Qro., México.
(2)REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of
Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
beatoliv@ff.up.pt.
(3)Facultad de Química, Universidad Autónoma de Querétaro, Centro Universitario,
Cerro de las Campanas, C.P. 76010, Querétaro, Qro., México.
The frequent use of plant-based products to promote health leads to the search
for scientific information related to efficacy and safety of those products for
human consumption. Two alfalfa-derived products (ADP), freeze-dried juice (FDJ)
and dehydrated powder (DP), from alfalfa harvested in Mexico, are being developed
as new possible nutraceuticals. To the best of our knowledge, any study reports
the real composition of such products used to prepare instant beverages in what
concerns isoflavone contents. Seven isoflavones (glycitein, formononetin,
biochanin A, daidzein, genistein, daidzin and genistin) were assessed by HPLC-DAD
analysis as well as its variation in five different batches of these products.
Different solvents were tested in order to choose the best one to extract
isoflavones. The results showed the presence of daidzein, genistein, genistin and
daidzin in most samples while glycitein, formononetin and biochanin A were not
detected. Significant differences between isoflavone contents were found with
different solvent systems. Water was the best option to extract daidzein
(0.40-1.08 mg per unit and 1.30-4.90 mg per unit for DP and FDJ, respectively)
whereas the water-methanol-formic acid mixture was efficient to extract genistein
(0.19-0.43 mg per unit and 0.15-0.72 mg per unit for DP and FDJ, respectively).
In all cases, the total isoflavone content was higher in freeze-dried juices than
in dehydrated powders. Genistein and daidzein were the more abundant isoflavones
quantified. Further physiological and nutritional studies are needed to complete
the validation of effectiveness and safety of these products.
DOI: 10.1039/c5fo01115a
391. J Agric Food Chem. 2015 Nov 4;63(43):9590-600. doi: 10.1021/acs.jafc.5b03717.

Epub 2015 Oct 22.
Transformation with TT8 and HB12 RNAi Constructs in Model Forage (Medicago
sativa, Alfalfa) Affects Carbohydrate Structure and Metabolic Characteristics in
Ruminant Livestock Systems.
Li X(1)(2), Zhang Y(2), Hannoufa A(3), Yu P(1)(4).
Author information:
(1)College of Agriculture and Bioresources, University of Saskatchewan , 51
Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8.
(2)College of Animal Science and Technology, Northeast Agricultural University ,
Harbin 150030, China.
(3)Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario,
Canada N5V 4T3.
(4)Tianjin Agricultural University , Tianjin 300384, China.
Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative

impact on feed digestibility. TT8 and HB12 genes were shown to have low
expression levels in low-lignin tissues of alfalfa, but to date, there has been
no study on the effect of down-regulation of these two genes in alfalfa on
nutrient chemical profiles and availability in ruminant livestock systems. The
objectives of this study were to investigate the effect of transformation of
alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO
nutritive value in ruminant livestock systems. The results showed that
transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded
CHO fractions (RDCA4, P = 0.06; RDCB1, P < 0.01) and totally degraded CHO
fraction (TRDCHO, P = 0.08). Both HB12 and TT8 populations had significantly
higher in vitro digestibility of neutral detergent fiber (NDF) at 30 h of
incubation (ivNDF30) compared to the control (P < 0.01). The TT8 populations had
highest ivDM30 and ivNDF240. Transformation of alfalfa with TT8 and HB12 RNAi
constructs induced molecular structure changes. Different CHO functional groups
had different sensitivities and different responses to the transformation. The
CHO molecular structure changes induced by the transformation were associated
with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8
RNAi could improve forage quality by increasing the availability of both NDF and
DM. Further study is needed on the relationship between the
transformation-induced structure changes at a molecular level and nutrient
utilization in ruminant livestock systems when lignification is much higher.
DOI: 10.1021/acs.jafc.5b03717
392. Int J Food Microbiol. 2016 Jan 18;217:49-58. doi:

10.1016/j.ijfoodmicro.2015.10.011. Epub 2015 Oct 22.
Development of prototypes of bioactive packaging materials based on immobilized

bacteriophages for control of growth of bacterial pathogens in foods.
Lone A(1), Anany H(2), Hakeem M(1), Aguis L(1), Avdjian AC(1), Bouget M(1),
Atashi A(3), Brovko L(1), Rochefort D(3), Griffiths MW(1).
Author information:
(1)Canadian Research Institute for Food Safety, Department of Food Science,
University of Guelph, Guelph, ON N1G 2W1, Canada.
(2)Canadian Research Institute for Food Safety, Department of Food Science,
University of Guelph, Guelph, ON N1G 2W1, Canada; Department of Microbiology,
Faculty of Science, Ain Shams University, Egypt. Electronic address:
hanany@alumni.uoguelph.ca.
(3)Département de chimie, Université de Montréal, CP6128 Succ. Centre-Ville,
Montréal, Québec H3C 3J7, Canada.
Due to lack of adequate control methods to prevent contamination in fresh produce

and growing consumer demand for natural products, the use of bacteriophages has
emerged as a promising approach to enhance safety of these foods. This study
sought to control Listeria monocytogenes in cantaloupes and RTE meat and
Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage
conditions by using specific lytic bacteriophage cocktails applied either free or
immobilized. Bacteriophage cocktails were introduced into prototypes of packaging
materials using different techniques: i) immobilizing on positively charged
modified cellulose membranes, ii) impregnating paper with bacteriophage
suspension, and iii) encapsulating in alginate beads followed by application of
beads onto the paper. Phage-treated and non-treated samples were stored for
various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free
phage cocktail was added, L. monocytogenes counts dropped below the detection
limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C
and 12°C. However, at 25°C, counts below the detection limit were observed after
3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the
immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria
count was observed by the end of the storage period for all tested storage
temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage
application technique (spraying of free phage suspension, bringing in contact
with bacteriophage-based materials (paper coated with encapsulated bacteriophage
or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was
below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log
cycle reduction in E. coli count was observed on the germinated sprouts by day 5.
In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to
significantly reduce the growth of L. monocytogenes at both storage temperatures,
4°C and 10°C, for 25 days regardless of bacteriophage application format
(immobilized or non-immobilized (free)). In conclusion, the developed phage-based
materials demonstrated significant antimicrobial effect, when applied to the
artificially contaminated foods, and can be used as prototypes for developing
bioactive antimicrobial packaging materials capable of enhancing the safety of
fresh produce and RTE meat.
393. Genom Data. 2014 Sep 28;2:305-7. doi: 10.1016/j.gdata.2014.09.007. eCollection

2014 Dec.
Deep sequencing of the microRNA expression in fall dormant and non-dormant

alfalfa.
Fan W(1), Shi P(1), Wang C(1).
Author information:
MicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation

that down-regulates target genes by mRNA degradation or translational repression.
Evidence is increasing for their crucial roles during plant development.
Identification of miRNAs at the global genome-level by high-throughput sequencing
is essential to functionally characterize miRNAs in plants. Alfalfa (Medicago
sativa L.) is one of the most widely cultivated perennial forage legumes
worldwide. Fall dormancy is an adaptive character related to the biomass
production and winter survival in alfalfa. However, little is known about
miRNA-mediated developmental regulation of fall dormancy in alfalfa. Here, we
provide detailed experimental methods and analysis pipeline in our study to
identify miRNAs that were responsive to fall dormancy (Fan W et al., Genome-wide
identification of different dormant Medicago sativa L. microRNAs in response to
fall dormancy, submitted for publication) for reproducible research. The data
generated in our work provide meaningful information for understanding the roles
of miRNAs in response to seasonal change and growth regulation in alfalfa.
DOI: 10.1016/j.gdata.2014.09.007
PMCID: PMC4535959
PMID: 26484115
394. Genom Data. 2014 Sep 6;2:282-4. doi: 10.1016/j.gdata.2014.08.008. eCollection

2014 Dec.
Transcriptome profiling of gene expression in fall dormant and nondormant

alfalfa.
Zhang S(1), Wang C(1).
Author information:
Fall dormancy (FD) is an adaptive trait in alfalfa (Medicago sativa L.). It

appeared in the regrowth process in late summer or early autumn after alfalfa was
harvested. FD affects the biomass accumulation and winter survival in high
latitude area. However, the molecular mechanism under FD is still not clear at
present. Performing RNA-seq of fall dormant and nondormant alfalfa varieties at
different time points, we obtained differentially expressed genes between
different FD types or time points. These differentially expressed genes may
relate to FD in alfalfa. Here, we provide detailed experimental methods and
analysis pipeline in our study (Zhang S et al., De novo Characterization of Fall
Dormant and Nondormant Alfalfa (Medicago sativa L.) Transcriptome and
Identification of Candidate Genes Relate to Fall Dormancy, submitted for
publication) for reproducible research. Data generated in our work provide a
resource to help decipher the molecular mechanism of FD in alfalfa.
DOI: 10.1016/j.gdata.2014.08.008
PMCID: PMC4536029
PMID: 26484109
395. J Econ Entomol. 2015 Aug;108(4):1748-56. doi: 10.1093/jee/tov120. Epub 2015

May
25.
Revisiting the Economic Injury Level and Economic Threshold Model for Potato
Leafhopper (Hemiptera: Cicadellidae) in Alfalfa.
Chasen EM(1), Undersander DJ(2), Cullen EM(3).
Author information:
(1)Department of Entomology, University of Wisconsin, 1630 Linden Dr., Madison,
WI 53706. emchasen@gmail.com.
(2)Department of Agronomy, University of Wisconsin, 1575 Linden Dr., Madison, WI
53706.
(3)Department of Entomology, University of Wisconsin, 1630 Linden Dr., Madison,
WI 53706. Department of Plant Science, California State Polytechnic University,
3801 West Temple Avenue, Pomona, CA 91768.
The economic injury level for potato leafhopper, Empoasca fabae (Harris), in
alfalfa (Medicago sativa L.) was developed over 30 yr ago. In response to
increasing market value of alfalfa, farmers and consultants are interested in
reducing the economic threshold for potato leafhopper in alfalfa. To address this
question, caged field trials were established on two consecutive potato
leafhopper susceptible crops in 2013. Field cages were infested with a range of
potato leafhopper densities to create a linear regression of alfalfa yield
response. The slopes, or yield loss per insect, for the linear regressions of
both trials were used to calculate an economic injury level for a range of
current alfalfa market values and control costs. This yield-loss relationship is
the first quantification that could be used to help assess whether the economic
threshold should be lowered, given the increased market value of alfalfa.

DOI: 10.1093/jee/tov120
396. J Econ Entomol. 2014 Dec;107(6):2077-87. doi: 10.1603/EC14205.
Population Responses of Potato Leafhopper (Hemiptera: Cicadellidae) to

Insecticide in Glandular-Haired and Non-glandular-Haired Alfalfa Cultivars.
Sulc RM(1), McCormick JS(2), Hammond RB(3), Miller DJ(4).
Author information:
(1)Department of Horticulture and Crop Science, The Ohio State University, 2021
Coffey Rd., Columbus, OH 43210. sulc.2@osu.edu.
(2)Department of Horticulture and Crop Science, The Ohio State University, 2021
Coffey Rd., Columbus, OH 43210.
(3)Department of Entomology, Ohio Agricultural and Development Research Center,
The Ohio State University, 1680 Madison Ave., Wooster, OH 44691.
(4)D.J. Miller, Pioneer Hi-Bred Int'l., Inc., Johnston, IA 50131.
Conflicting results have been reported on the ability of glandular-haired alfalfa

(Medicago sativa L.) cultivars to reduce potato leafhopper, Empoasca fabae
Harris, population abundance in field environments. We measured potato leafhopper
adult and nymph abundance and yield responses in a cultivar selected for high
potato leafhopper resistance ('54H91') and in a non-glandular-haired susceptible
cultivar ('54V54') with and without insecticide treatment across 3 yr. Treatments
included no insecticide and insecticide applied either early or late in each
summer growth cycle. Date × cultivar × treatment interactions were found for
potato leafhopper population abundance. In the absence of insecticides, total
potato leafhopper abundance (adults + nymphs per sweep) was lower in 54H91 than
in 54V54 on 85% of sampling dates; cultivar differences were especially evident
as potato leafhopper abundance peaked. Insecticide treatment reduced potato
leafhopper populations in both cultivars, but populations recovered and often
exceeded the normal action threshold in both cultivars within 2-3 wk of
insecticide application. Yield gain from early insecticide treatment of 54V54 was
>400 kg/ha in 11 of 14 summer harvests, whereas in 54H91 the yield gain was <250
kg/ha in 10 of 14 summer harvests. We conclude that glandular-haired alfalfa
cultivars with high levels of potato leafhopper resistance significantly suppress
potato leafhopper adult and nymph abundance, reduce yield losses in the absence
of insecticides, and have potential within an integrated pest management strategy
to reduce insecticide use in alfalfa production systems.
© 2014 Entomological Society of America.
DOI: 10.1603/EC14205
397. J Environ Sci (China). 2015 Oct 1;36:112-20. doi: 10.1016/j.jes.2015.03.032.

Epub
2015 Jul 10.
Effect of liming on sulfate transformation and sulfur gas emissions in degraded

vegetable soil treated by reductive soil disinfestation.
Meng T(1), Zhu T(2), Zhang J(3), Cai Z(4).
Author information:
(1)School of Geography Sciences, Nanjing Normal University, Nanjing 210023,
China; Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution
Control, Nanjing 210023, China. Electronic address: mtianzhu@gmail.com.
Control, Nanjing 210023, China.
Control, Nanjing 210023, China; Jiangsu Center for Collaborative Innovation in
Geographical Information Resource Development and Application, Nanjing 210023,
China; Key Laboratory of Virtual Geographic Environment (VGE), Ministry of
Education, Nanjing Normal University, Nanjing 210023, China; Jiangsu Key
Laboratory of Environmental Change and Ecological Construction, Nanjing 210023,
China.
Control, Nanjing 210023, China; Jiangsu Center for Collaborative Innovation in
Geographical Information Resource Development and Application, Nanjing 210023,
China; Key Laboratory of Virtual Geographic Environment (VGE), Ministry of
Education, Nanjing Normal University, Nanjing 210023, China; Jiangsu Key
Laboratory of Environmental Change and Ecological Construction, Nanjing 210023,
China. Electronic address: zccai@njnu.edu.cn.
Reductive soil disinfestation (RSD), namely amending organic materials and

mulching or flooding to create strong reductive status, has been widely applied
to improve degraded soils. However, there is little information available about
sulfate (SO4(2-)) transformation and sulfur (S) gas emissions during RSD
treatment to degraded vegetable soils, in which S is generally accumulated. To
investigate the effects of liming on SO4(2-) transformation and S gas emissions,
two SO4(2-)-accumulated vegetable soils (denoted as S1 and S2) were treated by
RSD, and RSD plus lime, denoted as RSD0 and RSD1, respectively. The results
showed that RSD0 treatment reduced soil SO4(2-) by 51% and 61% in S1 and S2,
respectively. The disappeared SO4(2-) was mainly transformed into the undissolved
form. During RSD treatment, hydrogen sulfide (H2S), carbonyl sulfide (COS), and
dimethyl sulfide (DMS) were detected, but the total S gas emission accounted for
<0.006% of total S in both soils. Compared to RSD0, lime addition stimulated the
conversion of SO4(2-) into undissolved form, reduced soil SO4(2-) by 81% in S1
and 84% in S2 and reduced total S gas emissions by 32% in S1 and 57% in S2,
respectively. In addition to H2S, COS and DMS, the emissions of carbon disulfide,
methyl mercaptan, and dimethyl disulfide were also detected in RSD1 treatment.
The results indicated that RSD was an effective method to remove SO4(2-), liming
stimulates the conversion of dissolved SO4(2-) into undissolved form, probably
due to the precipitation with calcium.
DOI: 10.1016/j.jes.2015.03.032
398. Anim Reprod Sci. 2015 Nov;162:62-72. doi: 10.1016/j.anireprosci.2015.09.009.

Epub
2015 Sep 25.
Reproductive performance of ewes grazing lucerne during different periods around

mating.
Robertson SM(1), Clayton EH(2), Friend MA(3).
Author information:
(1)School of Animal and Veterinary Sciences, Charles Sturt University, Wagga
Wagga, NSW 2678, Australia; Graham Centre for Agricultural Innovation (NSW
Department of Primary Industries and Charles Sturt University), Locked Bag 588,
Wagga Wagga, NSW 2678, Australia. Electronic address: surobertson@csu.edu.au.
(2)NSW Department of Primary Industries, Wagga Wagga Agricultural Institute,
Wagga Wagga, NSW 2650, Australia; Graham Centre for Agricultural Innovation (NSW
Wagga Wagga, NSW 2678, Australia.
(3)School of Animal and Veterinary Sciences, Charles Sturt University, Wagga
Wagga, NSW 2678, Australia; Graham Centre for Agricultural Innovation (NSW
Wagga Wagga, NSW 2678, Australia. Electronic address: mfriend@csu.edu.au.
High intake of lucerne pastures or feeding of other high quality diets during
early pregnancy may increase embryo mortality, negating any benefit of improved
nutrition on ovulation rate in ewes. This study was conducted to determine
whether grazing ewes on lucerne (Medicago sativa) pastures for 7 days prior to
and throughout joining would result in greater foetal numbers than if ewes were
removed 7 days after the commencement of joining, or if ewes grazed senescent
pasture throughout the joining period. Merino ewes (300) were allocated to two
replicates of three treatments, grazing pastures between Days -7 and 36 of an
unsynchronised, natural autumn joining. Grazing lucerne to Day 7 of joining
resulted in 30% more (P<0.05) foetuses per ewe than grazing senescent pasture
(1.60±0.07 and 1.31±0.07, respectively), and 19% more lambs marked per ewe
joined. Extending grazing of lucerne past Day 7 of joining did not result in
additional foetuses per ewe (1.61±0.06) in comparison with only grazing lucerne
to Day 7 of joining. Greater than 80% of ewes mated during the first 14 days of
joining, and the proportions of ewes returning to oestrus and re-mating
(0.18±0.022) and of non-pregnant (0.09±0.017) ewes were similar (P>0.05) among
all treatment groups, suggesting no differences between treatments in embryo
mortality. Grazing naturally cycling ewes on lucerne prior to and during joinings
in autumn is recommended as a means to increase the number of lambs born,
although additional gains may not be obtained by grazing past day seven of
joining.
DOI: 10.1016/j.anireprosci.2015.09.009
399. Microb Ecol. 2016 Apr;71(3):711-24. doi: 10.1007/s00248-015-0682-8. Epub 2015

Oct
6.
The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated
Soil is Affected by Plant Cover and Edaphic Parameters.
Bourceret A(1)(2), Cébron A(3)(4), Tisserant E(5), Poupin P(6)(7), Bauda P(6)(7),
Beguiristain T(1)(2), Leyval C(1)(2).
Author information:
(1)CNRS, LIEC UMR7360, Faculté des Sciences et Technologies, BP70239, 54506,
Vandoeuvre-lès-Nancy, France.
(2)Université de Lorraine, LIEC UMR7360, Faculté des Sciences et Technologies, BP
70239, 54506, Vandoeuvre-lès-Nancy, France.
(3)CNRS, LIEC UMR7360, Faculté des Sciences et Technologies, BP70239, 54506,
Vandoeuvre-lès-Nancy, France. aurelie.cebron@univ-lorraine.fr.
(4)Université de Lorraine, LIEC UMR7360, Faculté des Sciences et Technologies, BP
70239, 54506, Vandoeuvre-lès-Nancy, France. aurelie.cebron@univ-lorraine.fr.
(5)INRA, IAM UMR1136, Centre INRA de Nancy, 54280, Champenoux, France.
(6)Université de Lorraine, LIEC UMR7360, Campus Bridoux-Rue du Général
Delestraint, 57070, Metz Borny, France.
(7)CNRS, LIEC UMR7360, Campus Bridoux-Rue du Général Delestraint, 57070, Metz
Borny, France.
Industrial wasteland soils with aged PAH and heavy metal contaminations are
environments where pollutant toxicity has been maintained for decades. Although
the communities may be well adapted to the presence of stressors, knowledge about
microbial diversity in such soils is scarce. Soil microbial community dynamics
can be driven by the presence of plants, but the impact of plant development on
selection or diversification of microorganisms in these soils has not been
established yet. To test these hypotheses, aged-contaminated soil samples from a
field trial were collected. Plots planted with alfalfa were compared to bare soil
plots, and bacterial and fungal diversity and abundance were assessed after 2 and
6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that
the bacterial community was dominated by Proteobacteria, Actinobacteria, and
Bacteroidetes and was characterized by low Acidobacteria abundance, while the
fungal community was mainly represented by members of the Ascomycota. The
short-term toxic impact of pollutants usually reduces the microbial diversity,
yet in our samples bacterial and fungal species richness and diversity was high
suggesting that the community structure and diversity adapted to the contaminated
soil over decades. The presence of plants induced higher bacterial and fungal
diversity than in bare soil. It also increased the relative abundance of
bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders
and of most fungal orders. Multivariate analysis showed correlations between
microbial community structure and heavy metal and PAH concentrations over time,
but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen
concentrations).
DOI: 10.1007/s00248-015-0682-8

10.1016/j.scitotenv.2015.09.108.
Epub 2015 Oct 3.
Effects of legume species introduction on vegetation and soil nutrient

development on abandoned croplands in a semi-arid environment on the Loess
Plateau, China.
Yuan ZQ(1), Yu KL(2), Epstein H(2), Fang C(3), Li JT(3), Liu QQ(3), Liu XW(3),
Gao WJ(3), Li FM(4).
Author information:
(1)State Key Laboratory of Grassland Agro-Ecosystem, Institute of Arid
Agroecology, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000,
China; State Key Laboratory of Frozen Soil Engineering, Cold and Arid Regions
Environmental and Engineering Research Institute, Chinese Academy of Science,
Lanzhou, Gansu 730000, China.
(2)Department of Environmental Sciences, University of Virginia, Charlottesville,
VA 22904, USA.
China.
China; The UWA Institute of Agriculture, The University of Western Australia,
M082, Perth WA 6009 Australia. Electronic address: fmli@lzu.edu.cn.
Revegetation facilitated by legume species introduction has been used for soil
erosion control on the Loess Plateau, China. However, it is still unclear how
vegetation and soil resources develop during this restoration process, especially
over the longer term. In this study, we investigated the changes of plant
aboveground biomass, vegetation cover, species richness and density of all
individuals, and soil total nitrogen, mineral nitrogen, total phosphorus and
available phosphorus over 11 years from 2003 to 2013 in three treatments (natural
revegetation, Medicago sativa L. introduction and Melilotus suaveolens L.
introduction) on the semi-arid Loess Plateau. Medicago significantly increased
aboveground biomass and vegetation cover, and soil total nitrogen and mineral
nitrogen contents. The Medicago treatment had lower species richness and density
of all individuals, lower soil moisture in the deep soil (i.e., 1.4-5m), and
lower soil available phosphorus. Melilotus introduction significantly increased
aboveground biomass in only the first two years, and it was not an effective
approach to improve vegetation biomass and cover, and soil nutrients, especially
in later stages of revegetation. Overall, our study suggests that M. sativa can
be the preferred plant species for revegetation of degraded ecosystems on the
Loess Plateau, although phosphorus fertilizer should be applied for the
sustainability of the revegetation.
DOI: 10.1016/j.scitotenv.2015.09.108
401. AoB Plants. 2015 Oct 3;7. pii: plv116. doi: 10.1093/aobpla/plv116.
An empirical model that uses light attenuation and plant nitrogen status to
predict within-canopy nitrogen distribution and upscale photosynthesis from leaf
to whole canopy.
Louarn G(1), Frak E(2), Zaka S(2), Prieto J(3), Lebon E(4).
Author information:
(1)INRA UR4 URP3F, BP6, F86600 Lusignan, France gaetan.louarn@lusignan.inra.fr.
(2)INRA UR4 URP3F, BP6, F86600 Lusignan, France.
(3)INTA EEA Mendoza, San Martín 3853, Luján de Cuyo (5507), Mendoza, Argentina.
(4)INRA, UMR 759, LEPSE, 2 place Viala, F34060 Montpellier, France.
Modelling the spatial and temporal distribution of leaf nitrogen (N) is central
to specify photosynthetic parameters and simulate canopy photosynthesis. Leaf
photosynthetic parameters depend on both local light availability and whole-plant
N status. The interaction between these two levels of integration has generally
been modelled by assuming optimal canopy functioning, which is not supported by
experiments. During this study, we examined how a set of empirical relationships
with measurable parameters could be used instead to predict photosynthesis at the
leaf and whole-canopy levels. The distribution of leaf N per unit area (Na)
within the canopy was related to leaf light irradiance and to the nitrogen
nutrition index (NNI), a whole-plant variable accounting for plant N status. Na
was then used to determine the photosynthetic parameters of a leaf gas exchange
model. The model was assessed on alfalfa canopies under contrasting N nutrition
and with N2-fixing and non-fixing plants. Three experiments were carried out to
parameterize the relationships between Na, leaf irradiance, NNI and
photosynthetic parameters. An additional independent data set was used for model
evaluation. The N distribution model showed that it was able to predict leaf N on
the set of leaves tested. The Na at the top of the canopy appeared to be related
linearly to the NNI, whereas the coefficient accounting for N allocation remained
constant. Photosynthetic parameters were related linearly to Na irrespective of N
nutrition and the N acquisition mode. Daily patterns of gas exchange were
simulated accurately at the leaf scale. When integrated at the whole-canopy
scale, the model predicted that raising N availability above an NNI of 1 did not
result in increased net photosynthesis. Overall, the model proposed offered a
solution for a dynamic coupling of leaf photosynthesis and canopy N distribution
without requiring any optimal functioning hypothesis.

DOI: 10.1093/aobpla/plv116
PMCID: PMC4635319
PMID: 26433705
402. Nat Prod Commun. 2015 Jul;10(7):1263-7.
Soluble Phenolic Compounds in Different Cultivars of Red Clover and Alfalfa, and
their Implication for Protection against Proteolysis and Ammonia Production in
Ruminants.
Kagan IA, Goff BM, Flythe MD.
Red clover (Trifolium pratense) contains soluble phenolic compounds with roles in
inhibiting proteolysis and ammonia production. Alfalfa (Medicago sativa) has been
found to have a low phenolic content, but few alfalfa and red clover cultivars
have been compared for phenolic content. Total soluble phenolics were quantified
by a Folin-Ciocalteu colorimetric assay in nine red clover and 27 alfalfa
cultivars. Mean total phenolic contents of red clover and alfalfa were 36.5 ± 4.3
mg/gdw and 15.8 ± 1.4 mg/gdw, respectively, with the greater standard deviation
of red clover possibly indicating more diversity in phenolic content. Because
different phenolic standards had different response factors in the colorimetric
assay, the red clover and 11 alfalfa cultivars were analyzed by HPLC to determine
if the differences in total soluble phenolics between genera reflected
differences in the amounts of phenolics or in the classes of phenolics responding
to the colorimetric assay. Two red clover cultivars differed in total phenolics
and phaselic acid. Alfalfa produced different phenolic compounds from red clover,
at lower concentrations. Extracts of two red clover cultivars were separated by
thin-layer chromatography (TLC), and the bands were assayed for activity against
Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium
(HAB). Only biochanin A had anti-HAB activity. Inhibitory amounts indicated that
five red clover cultivars could be suitable sources of anti-HAB activity.

Sep
26.
Herbage intake and milk production of late-lactation dairy cows offered a

second-year chicory crop during summer.
Muir SK(1), Ward GN(2), Jacobs JL(2).
Author information:
(1)Department of Economic Development, Jobs, Transport and Resources,
Warrnambool, Vic 3280, Australia. Electronic address:
stephanie.muir@ecodev.vic.gov.au.
(2)Department of Economic Development, Jobs, Transport and Resources,
Warrnambool, Vic 3280, Australia.
Chicory (Cichorum intybus L.) is a summer-active forage herb which has been
proposed as an option to increase summer feed supply, increase dry matter intake,
nutrient intake, and milk yield from nonirrigated dairy production systems in
southern Australia. Dry matter intake, nutrient intake, milk yield, and yield of
milk fat and protein of predominantly Holstein-Friesian dairy cows in late
lactation consuming 3 herbage-based diets (4 replicates per treatment) were
measured. The 3 grazed herbages were second-year chicory (CHIC) and perennial
ryegrass (Lolium perenne L.; PRG) monocultures and a mixed sward (~50:50) of
chicory and perennial ryegrass (MIX). All diets (CHIC, PRG, and MIX) were
supplemented with alfalfa (Medicago sativa L.) hay (5.5kg of DM/cow per day) and
an energy-based concentrate pellet (4.0kg of DM/cow per day). There were no
significant differences in milk yield (12.0 to 12.6kg/d across the treatments) or
the yield of milk fat (539 to 585g/d) and milk protein (433 to 447g/d) between
the 3 herbage-based diets. No differences in DMI (17.9 to 19.2kg/d) or estimated
metabolizable energy intake (173 to 185MJ/d) were noted between treatments.
Estimated metabolizable energy concentrations in the forages on offer were lower
in CHIC than PRG (7.6 vs. 8.2MJ/kg of dry matter), but the concentration in
consumed herbage was not different (9.1 vs. 9.2MJ/kg of dry matter); as such,
potential for increased milk yield in cows offered CHIC was limited. Increased
concentration of polyunsaturated fatty acids was observed in chicory herbage
compared with perennial ryegrass. This was associated with increased milk
conjugated linoleic acid and milk polyunsaturated fatty acids when chicory formed
part of the diet (CHIC compared to PRG and MIX). Chicory could be used as an
alternative to perennial ryegrass in summer; however, the developmental stage of
chicory will influence concentrations of metabolizable energy and neutral
detergent fiber and, therefore, intake and milk production responses compared
with perennial ryegrass.

DOI: 10.3168/jds.2014-9147
404. PLoS One. 2015 Sep 25;10(9):e0138931. doi: 10.1371/journal.pone.0138931.

eCollection 2015.
Identification of Loci Associated with Drought Resistance Traits in Heterozygous

Autotetraploid Alfalfa (Medicago sativa L.) Using Genome-Wide Association Studies
with Genotyping by Sequencing.
Zhang T(1), Yu LX(1), Zheng P(2), Li Y(1), Rivera M(1), Main D(2), Greene SL(3).
Author information:
(1)Plant and Germplasm Introduction and Testing Research, United States
Department of Agriculture-Agricultural Research Service, Prosser, Washington,
(2)Department of Horticulture, Washington State University, Pullman, Washington,
(3)National Center for Genetic Resource Preservation, United States Department of
Agriculture-Agricultural Research Service, Fort Collins, Colorado, United States
of America.
Drought resistance is an important breeding target for enhancing alfalfa

productivity in arid and semi-arid regions. Identification of genes involved in
drought tolerance will facilitate breeding for improving drought resistance and
water use efficiency in alfalfa. Our objective was to use a diversity panel of
alfalfa accessions comprised of 198 cultivars and landraces to identify genes
involved in drought tolerance. The panel was selected from the USDA-ARS National
Plant Germplasm System alfalfa collection and genotyped using genotyping by
sequencing. A greenhouse procedure was used for phenotyping two important traits
associated with drought tolerance: drought resistance index (DRI) and relative
leaf water content (RWC). Marker-trait association identified nineteen and
fifteen loci associated with DRI and RWC, respectively. Alignments of target
sequences flanking to the resistance loci against the reference genome of M.
truncatula revealed multiple chromosomal locations. Markers associated with DRI
are located on all chromosomes while markers associated with RWC are located on
chromosomes 1, 2, 3, 4, 5, 6 and 7. Co-localizations of significant markers
between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated
with DRI in this work overlap with the reported QTLs associated with biomass
under drought in alfalfa. Additional significant markers were targeted to several
contigs with unknown chromosomal locations. BLAST search using their flanking
sequences revealed homology to several annotated genes with functions in stress
tolerance. With further validation, these markers may be used for marker-assisted
breeding new alfalfa varieties with drought resistance and enhanced water use
efficiency.
PMCID: PMC4583413
405. ARYA Atheroscler. 2015 Mar;11(2):133-8.
Inhibitory potential of pure isoflavonoids, red clover, and alfalfa extracts on

hemoglobin glycosylation.
Hosseini M(1), Asgary S(1), Najafi S(1).
Author information:
(1)Isfahan Cardiovascular Research Center, Cardiovascular Research Institute,
Isfahan University of Medical Sciences, Isfahan, Iran.
BACKGROUND: Non-enzymatic glycosylation of hemoglobin is complications of

diabetes. Antioxidant system imbalance can result in the emergence of free
radicals' destructive effects in the long-term. Red clover (Trifolium pratense
L.) and alfalfa (Medicago sativa L.) contain isoflavonoids and have antioxidant
activity. This experimental study evaluated the inhibitory activity of pure
isoflavonoids (daidzein and genistein), red clover and alfalfa extracts on
hemoglobin glycosylation.
METHODS: This study was performed in Iran. Stock solution of hydroalcoholic
extracts of red clover and alfalfa in concentrations of 1 and 10 g/100 ml and
stock solution of daidzein and genistein in concentrations of 250 ng, 500 ng, 25
µg and 250 µg/100 ml were prepared as case groups. Control group was without
hydroalcoholic extracts of plants and pure isoflavonoids. All experiments were
performed in triplicate. Hemoglobin was prepared and antioxidant activities were
investigated to estimate degree of nonenzymatic hemoglobin glycosylation.
RESULTS: There was no significantly difference between used extracts (extract of
red clover and alfalfa) and control of the hemoglobin glycosylation but using
daidzein (P = 0.046, 0.029 and 0.021, respectively) and genistein (P = 0.034,
0.036 and 0.028) significantly inhibited (P < 0.050) this reaction in 25 µg/100
ml, 250 and 500 ng/100 ml concentrations when compared to control. in 25 µg/100
ml, 250 ng and 500 ng/100 ml concentrations percentage of inhibition were 32, 80
and 74.5% respectively with used of daidzein and were 21, 83 and 76% respectively
with consumption of genistein.
CONCLUSION: According to decrease of glycation of hemoglobin with isoflavonoids,
two used plant in this study containing isoflavonoid may be useful on diabetes.
PMCID: PMC4568198
PMID: 26405442
406. Animal. 2016 Feb;10(2):203-11. doi: 10.1017/S1751731115001949. Epub 2015 Sep
24.
Prediction of enteric methane emissions from Holstein dairy cows fed various
forage sources.
Rico DE(1), Chouinard PY(1), Hassanat F(2), Benchaar C(2), Gervais R(1).
Author information:
(1)1Département des sciences animales,Université Laval,2425 rue de
l'Agriculture,Québec,QC,Canada,G1V 0A6.
(2)2Agriculture and Agri-Food Canada,Dairy and Swine Research and Development
Centre,2000 College Street,Sherbrooke,QC,Canada,J1M 0C8.
Milk fatty acid (FA) profile has been previously used as a predictor of enteric
CH4 output in dairy cows fed diets supplemented with plant oils, which can
potentially impact ruminal fermentation. The objective of this study was to
investigate the relationships between milk FA and enteric CH4 emissions in
lactating dairy cows fed different types of forages in the context of commonly
fed diets. A total of 81 observations from three separate 3×3 Latin square design
(32-day periods) experiments including a total of 27 lactating cows (96±27 days
in milk; mean±SD) were used. Dietary forages were included at 60% of ration dry
matter and were as follows: (1) 100% corn silage, (2) 100% alfalfa silage, (3)
100% barley silage, (4) 100% timothy silage, (5) 50:50 mix of corn and alfalfa
silages, (6) 50:50 mix of barley and corn silages and (7) 50:50 mix of timothy
and alfalfa silages. Enteric CH4 output was measured using respiration chambers
during 3 consecutive days. Milk was sampled during the last 7 days of each period
and analyzed for components and FA profile. Test variables included dry matter
intake (DMI; kg/day), NDF (%), ether extract (%), milk yield (kg/day), milk
components (%) and individual milk FA (% of total FA). Candidate multivariate
models were obtained using the Least Absolute Shrinkage and Selection Operator
and Least-Angle Regression methods based on the Schwarz Bayesian Criterion. Data
were then fitted into a random regression using the MIXED procedure including the
random effects of cow, period and study. A positive correlation was observed
between CH4 and DMI (r=0.59, P0.19). Milk FA profile and DMI can be used to
predict CH4 emissions in dairy cows across a wide range of dietary forage
sources.
DOI: 10.1017/S1751731115001949
407. Plant Sci. 2015 Oct;239:230-7. doi: 10.1016/j.plantsci.2015.08.005. Epub 2015

Aug
11.
Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl

transferase and coumarate 3-hydroxylase significantly increases cellulose content
in transgenic alfalfa (Medicago sativa L.).
Tong Z(1), Li H(2), Zhang R(3), Ma L(4), Dong J(5), Wang T(6).
Author information:
China Agricultural University, Beijing 100193, China; Institute of Animal
Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Electronic address: dradon.tong@163.com.
China Agricultural University, Beijing 100193, China. Electronic address:
henry-0123@163.com.
zrx1230@163.com.
malei2002@163.com.
dongjl@cau.edu.cn.
China Agricultural University, Beijing 100193, China; National Energy R&D Center
for Non-food Biomass, China Agricultural University, Beijing 100193, China.
Electronic address: wangt@cau.edu.cn.
Lignin is a component of the cell wall that is essential for growth, development,
structure and pathogen resistance in plants, but high lignin is an obstacle to
the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin
and cellulose contents can be a good approach to overcoming that obstacle.
Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a
model plant for the genetic modification of lignin in this study. Two key enzymes
in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate
hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were
co-downregulated. Compared to wild-type plants, the lignin content in the
modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme
saccharification efficiency was increased by 10.9%, and cell wall digestibility
was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but
normal above ground biomass. This approach provides a new strategy for reducing
lignin and increasing cellulose contents and creates a new genetically modified
crop with enhanced value for biofuel.
DOI: 10.1016/j.plantsci.2015.08.005
408. J Plant Physiol. 2015 Sep 1;188:1-8. doi: 10.1016/j.jplph.2015.07.014. Epub

2015
Sep 10.
The coupling of the plant and microbial catabolisms of phenanthrene in the

rhizosphere of Medicago sativa.
Muratova A(1), Dubrovskaya E(2), Golubev S(2), Grinev V(2), Chernyshova M(2),
Turkovskaya O(2).
Author information:
(1)Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, 13
Prospekt Entuziastov, 410049 Saratov, Russia. Electronic address:
amuratova@yahoo.com.
Prospekt Entuziastov, 410049 Saratov, Russia.
We studied the catabolism of the polycyclic aromatic hydrocarbon phenanthrene by

four rhizobacterial strains and the possibility of enzymatic oxidation of this
compound and its microbial metabolites by the root exudates of alfalfa (Medicago
sativa L.) in order to detect the possible coupling of the plant and microbial
metabolisms under the rhizospheric degradation of the organic pollutant. A
comparative study of phenanthrene degradation pathways in the PAH-degrading
rhizobacteria Ensifer meliloti, Pseudomonas kunmingensis, Rhizobium petrolearium,
and Stenotrophomonas sp. allowed us to identify the key metabolites from the
microbial transformation of phenanthrene, including 9,10-phenanthrenequinone,
2-carboxybenzaldehyde, and 1-hydroxy-2-naphthoic, salicylic, and o-phthalic
acids. Sterile alfalfa plants were grown in the presence and absence of
phenanthrene (0.03 g kg(-1)) in quartz sand under controlled environmental
conditions to obtain plant root exudates. The root exudates were collected,
concentrated by ultrafiltration, and the activity of oxidoreductases was detected
spectrophotometrically by the oxidation rate for various substrates. The most
marked activity was that of peroxidase, whereas the presence of oxidase and
tyrosinase was detected on the verge of the assay sensitivity. Using alfalfa root
exudates as a crude enzyme preparation, we found that in the presence of the
synthetic mediator, the plant peroxidase could oxidize phenanthrene and its
microbial metabolites. The results indicate the possibility of active
participation of plants in the rhizospheric degradation of polycyclic aromatic
hydrocarbons and their microbial metabolites, which makes it possible to speak
about the coupling of the plant and microbial catabolisms of these contaminants
in the rhizosphere.
DOI: 10.1016/j.jplph.2015.07.014
409. Microbiology. 2015 Dec;161(12):2341-51. doi: 10.1099/mic.0.000182. Epub 2015

Sep
21.
Proline auxotrophy in Sinorhizobium meliloti results in a plant-specific

symbiotic phenotype.
diCenzo GC(1), Zamani M(1), Cowie A(1), Finan TM(1).
Author information:
(1)Department of Biology, McMaster University, 1280 Main Street West, Hamilton,
Ontario L8S 4K1, Canada.
In order to effectively manipulate rhizobium-legume symbioses for our benefit, it

is crucial to first gain a complete understanding of the underlying genetics and
metabolism. Studies with rhizobium auxotrophs have provided insight into the
requirement for amino acid biosynthesis during the symbiosis; however, a paucity
of available L-proline auxotrophs has limited our understanding of the role of
L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently
described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was
observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph
displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a
decrease in nodule mass formed and therefore a reduction in nitrogen fixed per
plant. However, the proline auxotroph formed nodules on white sweet clover
(Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover
nodulation by the auxotroph was slightly delayed, but the final number of nodules
per plant was not impacted. Examination of white sweet clover nodules by confocal
microscopy and transmission electron microscopy revealed the presence of the S.
meliloti proline auxotroph cells within the host legume cells, but few
differentiated bacteroids were identified compared with the bacteroid-filled
plant cells of WT nodules. Overall, these results indicated that L-proline
biosynthesis is a general requirement for a fully effective nitrogen-fixing
symbiosis, likely due to a transient requirement during bacteroid
differentiation.
DOI: 10.1099/mic.0.000182
410. Drug Dev Ind Pharm. 2016;42(6):880-9. doi: 10.3109/03639045.2015.1088865. Epub

2015 Sep 22.
Exploring the antioxidant potentiality of two food by-products into a topical

cream: stability, in vitro and in vivo evaluation.
Rodrigues F(1)(2), Sarmento B(3)(4), Amaral MH(5), Oliveira MB(1).
Author information:
(1)a Department of Chemical Sciences, Faculty of Pharmacy , REQUIMTE, University
of Porto , Porto , Portugal .
(2)b Fourmag, Lda., Parque Industrial Do Cruzeiro , Moreira De Cónegos , Portugal
.
(3)c INEB - Institute of Biomedical Engineering, University of Porto , Porto ,
Portugal .
(4)d Department of Pharmaceutical Sciences , CESPU, IINFACTS - Institute for
Research and Advanced Training in Health Sciences and Technologies, Instituto
Superior De Ciências Da Saúde-Norte , Gandra , PRD , Portugal , and.
(5)e Department of Drug Sciences, Laboratory of Pharmaceutical Technology,
Faculty of Pharmacy , University of Porto , Porto , Portugal.
CONTEXT: Coffee silverskin (CS), a food by-product of the coffee roasting

industry, has been studied as an active ingredient for skin care products due to
its high potential of antioxidant activity and low cytotoxicity. Another food
waste used as ingredient with promising characteristics is obtained from Medicago
sativa (MS), which antioxidants and isoflavones content is high.
OBJECTIVE: The aim of this study is to evaluate and characterize a new body
formulation containing two food by-products extracts.
MATERIALS AND METHODS: Different parameters (such as pH, rheological behavior,
color, antioxidant content and microbiological analysis) of a body cream
formulation containing by-products (CSMS) and a formulation without extracts (F)
were evaluated under a stability study during 180 days at different temperatures.
Moreover, the in vitro cell toxicity and the in vivo skin safety and protective
effects were also assessed.
RESULTS: Formulation showed stable physical properties and antioxidant activity
during 180 days of storage. In vitro toxicity was screened in two skin cell lines
(fibroblasts and keratinocytes) and any toxicity was reported. The in vivo test
carried out showed that, with respect to irritant effects, CSMS formulation can
be regarded as safe for topical application and the skin hydratation improved
after 30 days of its use. Also, considering the consumer acceptance, more than
90% of volunteers classified it as very pleasant.
CONCLUSIONS: CSMS formulation is stable and safe for topical use as no adverse
and/or side effects were observed during the application period of testing,
improving skin protective properties.
DOI: 10.3109/03639045.2015.1088865
411. Animal. 2016 Mar;10(3):410-7. doi: 10.1017/S1751731115001986. Epub 2015 Sep

14.
Effects of different sources of physically effective fiber on rumen microbial

populations.
Shaw CN(1), Kim M(1), Eastridge ML(1), Yu Z(1).
Author information:
(1)Department of Animal Sciences,The Ohio State University,2029 Fyffe
Court,Columbus,OH, 43210USA.
Physically effective fiber is needed by dairy cattle to prevent ruminal acidosis.

This study aimed to examine the effects of different sources of physically
effective fiber on the populations of fibrolytic bacteria and methanogens. Five
ruminally cannulated Holstein cows were each fed five diets differing in
physically effective fiber sources over 15 weeks (21 days/period) in a Latin
Square design: (1) 44.1% corn silage, (2) 34.0% corn silage plus 11.5% alfalfa
hay, (3) 34.0% corn silage plus 5.1% wheat straw, (4) 36.1% corn silage plus
10.1% wheat straw, and (5) 34.0% corn silage plus 5.5% corn stover. The impact of
the physically effective fiber sources on total bacteria and archaea were
examined using denaturing gradient gel electrophoresis. Specific real-time PCR
assays were used to quantify total bacteria, total archaea, the genus
Butyrivibrio, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus
flavefaciens and three uncultured rumen bacteria that were identified from
adhering ruminal fractions in a previous study. No significant differences were
observed among the different sources of physical effective fiber with respect to
the microbial populations quantified. Any of the physically effective fiber
sources may be fed to dairy cattle without negative impact on the ruminal
microbial community.
DOI: 10.1017/S1751731115001986
412. J Dairy Sci. 2015 Nov;98(11):7982-92. doi: 10.3168/jds.2015-9348. Epub 2015

Sep
9.
Effects of feeding birdsfoot trefoil hay on neutral detergent fiber digestion,

nitrogen utilization efficiency, and lactational performance by dairy cows.
Christensen RG(1), Yang SY(1), Eun JS(2), Young AJ(1), Hall JO(1), MacAdam JW(3).
Author information:
(1)Department of Animal, Dairy, and Veterinary Sciences, Utah State University,
Logan 84322.
Logan 84322. Electronic address: jseun@usu.edu.
(3)Department of Plants, Soils, and Climate, Utah State University, Logan 84322.
This experiment was conducted to determine effects of feeding birdsfoot trefoil

hay-based diets in comparison with an alfalfa hay-based diet on N utilization
efficiency, ruminal fermentation, and lactational performance by mid-lactation
dairy cows. Nine multiparous lactating Holstein cows (131 ± 22.6 d in milk), 3 of
which were rumen fistulated, were fed 3 experimental diets in a replicated 3 × 3
Latin square design with 3 periods of 14 d of adaptation and 7 d of data and
sample collection. Within squares, cows were randomly assigned to diets as
follows: alfalfa hay-based diet (AHT), alfalfa and birdsfoot trefoil hay-based
diet (ABT), and birdsfoot trefoil hay-based diet (BT). Intakes of dry matter and
crude protein were similar across treatments, whereas ABT and BT diets resulted
in decreased fiber intake compared with AHT. Feeding BT tended to increase
neutral detergent fiber digestibility compared with AHT and ABT. Milk yield
tended to increase for cows consuming ABT or BT diets. Milk true protein
concentration and yield were greater for cows consuming ABT relative to those fed
AHT. Concentration of total volatile fatty acids tended to increase by cows fed
BT compared with those fed AHT and ABT. Feeding birdsfoot trefoil hay in a total
mixed ration resulted in a tendency to decrease acetate proportion, but it tended
to increase propionate proportion, leading to a tendency to decrease
acetate-to-propionate ratio. Whereas concentration of ammonia-N was similar
across treatments, cows offered BT exhibited greater microbial protein yield
relative to those fed AHT and ABT. Cows offered birdsfoot trefoil hay diets
secreted more milk N than AHT, resulting in improved N utilization efficiency for
milk N. The positive effects due to feeding birdsfoot trefoil hay were attributed
to enhanced neutral detergent fiber digestion, and thus it could replace alfalfa
hay in high-forage dairy diets while improving N utilization efficiencies and
maintaining lactational performance compared with alfalfa hay.

DOI: 10.3168/jds.2015-9348
413. Int J Phytoremediation. 2016;18(6):598-604. doi:

10.1080/15226514.2015.1086296.
Differential uptake of silver, copper and zinc suggests complementary

species-specific phytoextraction potential.
Desjardins D(1), Pitre FE(1), Nissim WG(1)(2), Labrecque M(1).
Author information:
(1)a Institut de recherche en biologie végétale, Université de Montréal, Jardin
botanique de Montréal , Montréal , Canada.
(2)b Department of Agri-food and Environmental Science , University of Florence,
Viale delle Idee , Sesto Fiorentino ( FI ) Italy.
The aim of our study, conducted as a pot experiment, was to assess the potential
of willow (Salix miyabeana), alfalfa (Medicago sativa), tall fescue (Festuca
arundinacea), and Indian mustard (Brassica juncea) to remediate two brownfield
soils differentially contaminated with Ag, Cu and Zn (up to 113.60, 47.50, and
117.00 mg kg(-1) respectively). While aboveground Ag accumulation was highest in
B. juncea (4.60 ± 2.58 mg kg(-1)), lower levels were also measured in M. sativa
and F. arundinacea. Cu accumulation was observed in all species, but only in
underground parts, and was highest in F. arundinacea (269.20 ± 74.75 mg kg(-1)),
with a bioconcentration factor of 13.85. Salix miyabeana was found to have the
highest Zn aerial tissue concentration (119.96 ± 20.04 mg kg(-1)). Because of its
high Ag uptake, the remediation potential of B. juncea should be evaluated more
extensively on the site from which we excavated the soil for this study. Given
the multiple forms of contamination on the site and the differential
specie-related uptake evident in our findings, we hypothesize that an optimal
plantation allowing expression of complementary remediation functions would
include B. juncea for extraction of Ag, in combination with F. arundinacea for
stabilization of Cu and S. miyabeana for extraction of Zn.
DOI: 10.1080/15226514.2015.1086296

10.1016/j.scitotenv.2015.08.127.
Epub 2015 Sep 8.
Microbiote shift in the Medicago sativa rhizosphere in response to cyanotoxins

extract exposure.
El Khalloufi F(1), Oufdou K(2), Bertrand M(3), Lahrouni M(2), Oudra B(2), Ortet
P(3), Barakat M(3), Heulin T(3), Achouak W(4).
Author information:
(1)CEA, DSV, IBEB, Lab Ecol Microb Rhizosphere & Environ Extrem (LEMiRE), 13108,
Saint Paul-Lez-Durance, France; CNRS, BVME UMR 7265, ECCOREV FR 3098, 13108,
Saint Paul-Lez-Durance, France; Aix Marseille Université, 13284 Marseille Cedex
07, France; Laboratory of Biology and Biotechnology of Microorganisms, Faculty of
Sciences Semlalia, Cadi Ayyad University, PO Box 2390, Marrakech, Morocco;
University Hassan 1st. Polydisciplinary Faculty of Khouribga, BP. 145, 25000
Khouribga, Morocco.
(2)Laboratory of Biology and Biotechnology of Microorganisms, Faculty of Sciences
Semlalia, Cadi Ayyad University, PO Box 2390, Marrakech, Morocco.
07, France.
07, France. Electronic address: wafa.achouak@cea.fr.
The bloom-containing water bodies may have an impact due to cyanotoxins

production on other microorganisms and aquatic plants. Where such water is being
used for crops irrigation, the presence of cyanotoxins may also have a toxic
impact on terrestrial plants and their rhizosphere microbiota. For that purpose,
PCR-based 454 pyrosequencing was applied to phylogenetically characterize the
bacterial community of Medicago sativa rhizosphere in response to cyanotoxins
extract. This analysis revealed a wide diversity at species level, which
decreased from unplanted soil to root tissues indicating that only some
populations were able to compete for nutrients and niches in this selective
habitat. Gemmatimonas, Actinobacteria, Deltaproteobacteria and Opitutae mainly
inhabited the bulk soil, whereas, the root-adhering soil and the root tissues
were inhabited by Gammaproteobacteria and Alphaproteobacteria. The proportion of
these populations fluctuated in response to cyanotoxins extract exposure.
Betaproteobacteria proportion increased in the three studied compartments,
whereas Gammaproteobacteria proportion decreased except in the bulk soil. This
study revealed the potential toxicity of cyanotoxins extract towards
Actinobacteria, Gemmatimonas, Deltaproteobacteria, and Gammaproteobacteria,
however Clostridia, Opitutae and bacteria related with Betaproteobacteria, were
stimulated denoting their tolerance. Altogether, these data indicate that crop
irrigation using cyanotoxins containing water might alter the rhizosphere
functioning.
DOI: 10.1016/j.scitotenv.2015.08.127
415. Molecules. 2015 Aug 26;20(9):15550-71. doi: 10.3390/molecules200915550.
Optimization of Ultrasonic-Assisted Extraction of Flavonoid Compounds and

Antioxidants from Alfalfa Using Response Surface Method.
Jing CL(1), Dong XF(2), Tong JM(3).
Author information:
Beijing 100193, China. jingchangliang2008@163.com.
Beijing 100193, China. xiaofangdong@iascaas.net.cn.
Beijing 100193, China. tjm606@iascaas.net.cn.
Ultrasonic-assisted extraction (UAE) was used to extract flavonoid-enriched

antioxidants from alfalfa aerial part. Response surface methodology (RSM), based
on a four-factor, five-level central composite design (CCD), was employed to
obtain the optimal extraction parameters, in which the flavonoid content was
maximum and the antioxidant activity of the extracts was strongest. Radical
scavenging capacity of the extracts, which represents the amounts of antioxidants
in alfalfa, was determined by using 2,2'-azino-bis
(3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and
2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods. The results showed good fit with
the proposed models for the total flavonoid extraction (R² = 0.9849), for the
antioxidant extraction assayed by ABTS method (R² = 0.9764), and by DPPH method
(R² = 0.9806). Optimized extraction conditions for total flavonoids was a ratio
of liquid to solid of 57.16 mL/g, 62.33 °C, 57.08 min, and 52.14% ethanol. The
optimal extraction parameters of extracts for the highest antioxidant activity by
DPPH method was a ratio of liquid to solid 60.3 mL/g, 54.56 °C, 45.59 min, and
46.67% ethanol, and by ABTS assay was a ratio of liquid to solid 47.29 mL/g,
63.73 °C, 51.62 min, and 60% ethanol concentration. Our work offers optimal
extraction conditions for total flavonoids and antioxidants from alfalfa.
DOI: 10.3390/molecules200915550
416. Int J Mol Sci. 2015 Sep 1;16(9):20913-42. doi: 10.3390/ijms160920913.
Biological Networks Underlying Abiotic Stress Tolerance in Temperate Crops--A

Proteomic Perspective.
Kosová K(1), Vítámvás P(2), Urban MO(3), Klíma M(4), Roy A(5), Prášil IT(6).
Author information:
(1)Laboratory of Plant Stress Biology and Biotechnology, Division of Crop
Genetics and Breeding, Crop Research Institute, Drnovská 507/73, 16106 Prague,
Czech Republic. kosova@vurv.cz.
Czech Republic. vitamvas@vurv.cz.
Czech Republic. olinek.vcelar@seznam.cz.
Czech Republic. klima@vurv.cz.
(5)Research Institute of Agricultural Engineering, Drnovská 507, 16106 Prague,
Czech Republic. roy.amitava@vuzt.cz.
Czech Republic. prasil@vurv.cz.
Abiotic stress factors, especially low temperatures, drought, and salinity,

represent the major constraints limiting agricultural production in temperate
climate. Under the conditions of global climate change, the risk of damaging
effects of abiotic stresses on crop production increases. Plant stress response
represents an active process aimed at an establishment of novel homeostasis under
altered environmental conditions. Proteins play a crucial role in plant stress
response since they are directly involved in shaping the final phenotype. In the
review, results of proteomic studies focused on stress response of major crops
grown in temperate climate including cereals: common wheat (Triticum aestivum),
durum wheat (Triticum durum), barley (Hordeum vulgare), maize (Zea mays);
leguminous plants: alfalfa (Medicago sativa), soybean (Glycine max), common bean
(Phaseolus vulgaris), pea (Pisum sativum); oilseed rape (Brassica napus); potato
(Solanum tuberosum); tobacco (Nicotiana tabaccum); tomato (Lycopersicon
esculentum); and others, to a wide range of abiotic stresses (cold, drought,
salinity, heat, imbalances in mineral nutrition and heavy metals) are summarized.
The dynamics of changes in various protein functional groups including signaling
and regulatory proteins, transcription factors, proteins involved in protein
metabolism, amino acid metabolism, metabolism of several stress-related
compounds, proteins with chaperone and protective functions as well as structural
proteins (cell wall components, cytoskeleton) are briefly overviewed. Attention
is paid to the differences found between differentially tolerant genotypes. In
addition, proteomic studies aimed at proteomic investigation of multiple stress
factors are discussed. In conclusion, contribution of proteomic studies to
understanding the complexity of crop response to abiotic stresses as well as
possibilities to identify and utilize protein markers in crop breeding processes
are discussed.
DOI: 10.3390/ijms160920913
PMCID: PMC4613235
417. J Anim Physiol Anim Nutr (Berl). 2016 Aug;100(4):758-67. doi:

10.1111/jpn.12384.
Epub 2015 Sep 3.
Effect of treating alfalfa silage with pistachio by-products extract on Saanen

dairy goats performance and microbial nitrogen synthesis.
Mokhtarpour A(1)(2), Naserian AA(2), Pourmollae F(3), Ghaffari MH(2).
Author information:
(1)Research Center of Special Domestic Animals, University of Zabol, Zabol, Iran.
(3)Agricultural Jihad Organization of Khorasan-Razavi, Mashhad, Iran.
A lactation experiment was conducted to determine the influence of addition of

pistachio by-products extract (PBE) to alfalfa silage (AS) on performance, rumen
fermentation, milk yield and composition, and microbial nitrogen synthesis. Eight
multiparous dairy goats (1.8 ± 0.25 kg of milk yield) were used in a replicated
4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments to
compare two types of AS (supplemented with or without PBE) with two levels of
dietary crude protein (14% vs. 16% CP). Dietary treatments were (i) AS with 14%
CP of DM diet without PBE (14%CP-PBE), (ii) AS with 14% CP of DM diet with PBE
(14%CP + PBE), (iii) AS with 16% CP of DM diet without PBE (16%CP-PBE) and (iv)
AS with 16% CP of DM diet with PBE (16%CP + PBE). PBE was sprayed on fresh
alfalfa at a ratio of 500 ml/kg alfalfa DM to get the final concentration of 1%
tannin as tannic acid equivalent on DM basis. Intake of CP was greater (p < 0.01)
in goats fed 16% CP diets than those fed 14% CP diets, regardless of PBE
supplementation. Supplementation of PBE tended to decrease (p = 0.09) rumen NH3
-N concentration regardless of the level of CP in the diet. Supplementation of
PBE tended (p = 0.09) to decrease total purine derivatives regardless of the
level of CP in the diet with no significant change in microbial nitrogen supply.
Efficiency of microbial nitrogen synthesis (EMNS) had a tendency (p = 0.07) to
decrease in PBE supplemented diets. There was also a tendency (p = 0.10) for more
EMNS in 14% CP fed goats than those fed 16% CP diets. Therefore, AS supplemented
with PBE may lead to less concentration of ruminal NH3 -N because of decreased
degradation of CP by rumen micro-organisms in response to pistachio by-products
tannins.
DOI: 10.1111/jpn.12384
418. Front Plant Sci. 2015 Aug 11;6:574. doi: 10.3389/fpls.2015.00574. eCollection
2015.
Leaf δ(15)N as a physiological indicator of the responsiveness of N2-fixing

alfalfa plants to elevated [CO2], temperature and low water availability.
Ariz I(1), Cruz C(1), Neves T(1), Irigoyen JJ(2), Garcia-Olaverri C(3), Nogués
S(4), Aparicio-Tejo PM(5), Aranjuelo I(6).
Author information:
(1)Faculdade de Ciências, Centro Ecologia Evolução e Alterações Ambientais,
Universidade de Lisboa Lisboa, Portugal.
(2)Grupo de Fisiología del Estrés en Plantas, Departamento de Biología Ambiental,
Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV Logroño, Spain.
(3)Departamento de Estadística e Investigación Operativa, Universidad Pública de
Navarra Pamplona, Spain.
(4)Departamento de Biología Vegetal, Facultat de Biologia, Universidad de
Barcelona Barcelona, Spain.
(5)Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra
Pamplona, Spain.
(6)Plant Biology and Ecology Department, Science and Technology Faculty,
University of the Basque Country Leioa, Spain ; Instituto de Agrobiotecnología
(IdAB), Universidad Pública de Navarra-CSIC-Gobierno de Navarra Mutilva Baja,
Spain.
The natural (15)N/(14)N isotope composition (δ(15)N) of a tissue is a consequence

of its N source and N physiological mechanisms in response to the environment. It
could potentially be used as a tracer of N metabolism in plants under changing
environmental conditions, where primary N metabolism may be complex, and losses
and gains of N fluctuate over time. In order to test the utility of δ(15)N as an
indicator of plant N status in N2-fixing plants grown under various environmental
conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct
conditions of [CO2] (400 vs. 700 μmol mol(-1)), temperature (ambient vs. ambient
+4°C) and water availability (fully watered vs. water deficiency-WD). As
expected, increased [CO2] and temperature stimulated photosynthetic rates and
plant growth, whereas these parameters were negatively affected by WD. The
determination of δ(15)N in leaves, stems, roots, and nodules showed that leaves
were the most representative organs of the plant response to increased [CO2] and
WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD
conditions reflected decreased transpiration rates, but could also be related to
a higher N demand in leaves, as suggested by the decreased leaf N and total
soluble protein (TSP) contents detected at 700 μmol mol(-1) [CO2] and WD
conditions. In summary, leaf δ(15)N provides relevant information integrating
parameters which condition plant responsiveness (e.g., photosynthesis, TSP, N
demand, and water transpiration) to environmental conditions.
DOI: 10.3389/fpls.2015.00574
PMCID: PMC4531240
PMID: 26322051
419. J Insect Sci. 2015 Aug 28;15. pii: 121. doi: 10.1093/jisesa/iev097. Print
2015.
Population Dynamics of Empoasca fabae (Hemiptera: Cicadellidae) in Central Iowa

Alfalfa Fields.
Weiser Erlandson LA(1), Obrycki JJ(2).
Author information:
(1)Department of Entomology, Iowa State University, Ames, IA 50011 Texas A&M
University-Central Texas, Department of Science and Mathematics, Killeen, TX
76549 laura.erlandson@tamuct.edu.
(2)Department of Entomology, Iowa State University, Ames, IA 50011 University of
Kentucky, Department of Entomology, Lexington, KY 40546-0091.
Adults and nymphs of Empoasca fabae Harris (Hemiptera: Cicadellidae) and adults
of predatory species in the families Coccinellidae, Anthocoridae, Nabidae,
Chrysopidae, and Hemerobiidae were sampled in Iowa alfalfa fields from June to
September in 1999 and 2000. The relationship between each predatory taxa and E.
fabae was examined using regression analysis. In 2000, all predators were found
to be positively correlated with the presence of E. fabae during all periods
sampled and most likely contributed to mortality. Orius insidiosus (Say)
(Hemiptera: Anthoridae) was the most numerous insect predatory species;
population numbers ranged from 0 to 1 and 0.1 to 3.7 adults per 0.25 m(2) in 1999
and 2000, respectively. Partial life tables were constructed for E. fabae nymphs
for two alfalfa-growing periods. Nymphs were grouped into three age intervals:
first and second, third and fourth, and fifth instars. For the first alfalfa
growing period examined, E. fabae nymphal mortality was 70% in 1999 and 49% in
2000. During the last growing period of each season (August-September), total
nymphal mortality was relatively low (<25%). Adult E. fabae density ranged from
5.4 to 25.6 and 1.4-9.2 per 0.25 m(2) in 1999 and 2000, respectively. E. fabae
population peaks were similar for each age interval in all growing periods. This
study provides further information on the population dynamics of E. fabae and its
relationship with select predatory species in Iowa alfalfa fields.
© The Author 2015. Published by Oxford University Press on behalf of the

Entomological Society of America.
DOI: 10.1093/jisesa/iev097
PMCID: PMC4672220
420. Environ Entomol. 2015 Aug;44(4):1125-38. doi: 10.1093/ee/nvv088. Epub 2015 Jun
13.
Resource Effects on Solitary Bee Reproduction in a Managed Crop Pollination

System.
Pitts-Singer TL(1).
Author information:
(1)USDA ARS Pollinating Insects Research Unit, Utah State University, Logan, UT
84322. Theresa.Pitts-Singer@ars.usda.gov.
Population density may affect solitary bee maternal resource allocation. The
number of Megachile rotundata (F.), alfalfa leafcutting bee, females released for
seed production of Medicago sativa L., alfalfa, may limit flower availability for
nest provisioning. In turn, pollinator abundance also may affect crop yield. The
M. sativa pollination system presents an opportunity to test for effects of
density dependence and maternal manipulation on M. rotundata reproduction. A
multiyear study was performed on M. sativa fields upon which M. rotundata
densities were altered to induce low, medium, and high density situations.
Numbers of adult bees and open flowers were recorded weekly; bee reproduction
variables were collected once. Fields varied in plant performance for each site
and year, and the intended bee densities were not realized. Therefore, the
variable density index (DI) was derived to describe the number of female bees per
area of flowers over the study period. As DI increased, percentages of pollinated
flowers, established females, and healthy brood significantly increased, and the
number of pollinated flowers per female and of dead or diseased brood
significantly decreased. Sex ratio was significantly more female biased as DI
increased. Overwintered offspring weights were similar regardless of DI, but
significantly differed by year for both sexes, and for males also by field and
year × field interaction. Overall, resource limitation was not found in this
field study. Other density-dependent factors may have induced a bee dispersal
response soon after bees were released in the fields that circumvented the need
for, or impact of, maternal manipulation.
Published by Oxford University Press on behalf of Entomological Society of

America 2015. This work is written by a US Government employee and is in the
public domain in the US.
DOI: 10.1093/ee/nvv088
421. Environ Entomol. 2015 Aug;44(4):1052-64. doi: 10.1093/ee/nvv104. Epub 2015 Jul
9.
Impacts of Contrasting Alfalfa Production Systems on the Drivers of Carabid

Beetle (Coleoptera: Carabidae) Community Dynamics.
Goosey HB(1), McKenzie SC(2), Rolston MG(3), O'Neill KM(2), Menalled FD(2).
Author information:
(1)Department of Animal and Range Sciences, 103 Animal Bioscience Building, P.O.
Box 172900, Montana State University, Bozeman, MT 59717-3120.
hgoosey@montana.edu.
(2)Department of Land Resources and Environmental Sciences, 334 Leon Johnson
Hall, P.O. Box 3120, Montana State University, Bozeman, MT 59717-3120.
(3)Department of Animal and Range Sciences, 103 Animal Bioscience Building, P.O.
Box 172900, Montana State University, Bozeman, MT 59717-3120.
Growing concerns about the environmental consequences of chemically based pest

control strategies have precipitated a call for the development of integrated,
ecologically based pest management programs. Carabid or ground beetles
(Coleoptera:Carabidae) are an important group of natural enemies of common
agricultural pests such as aphids, slugs, and other beetles. Alfalfa (Medicago
sativa L.) is one of the most common forage crop species in the semi-arid western
United States. In 2011, Montana alone produced 4.0 × 10(6 )Mg of alfalfa on
8.1 × 10(5 )ha for gross revenue in excess of US$4.3 × 10(8), making it the third
largest crop by revenue. We conducted our study over the 2012 and 2013 growing
seasons. Each year, our study consisted of three sites each with adjacent systems
of monoculture alfalfa, alfalfa nurse cropped with hay barley, and an
uncultivated refuge consisting of a variety of forbs and grasses. Carabid
community structure differed and strong temporal shifts were detected during both
2012 and 2013. Multivariate fuzzy set ordination suggests that variation in
canopy height among the three vegetation systems was primarily responsible for
the differences observed in carabid community structure. Land managers may be
able to enhance carabid species richness and total abundance by creating a
heterogeneous vegetation structure, and nurse cropping in particular may be
effective strategy to achieve this goal.

DOI: 10.1093/ee/nvv104
422. Environ Entomol. 2015 Jun;44(3):529-45. doi: 10.1093/ee/nvv029. Epub 2015 Apr
8.
Effects of Season and Management of Irrigated Cotton Fields on Collembola

(Hexapoda) in New South Wales, Australia.
Lytton-Hitchins JA(1), Greenslade P(2), Wilson LJ(3).
Author information:
(1)Department of Agricultural Chemistry and Soil Science, University of Sydney,
NSW 2006, Australia. CSIRO Entomology, GPO Box 1700, ACT 2601, Australia.
(2)School of Applied and Biomedical Sciences, Faculty of Science and Technology,
University Drive, Mt Helen, Ballarat, 3350, Australia. South Australian Museum,
North Terrace, Adelaide, South Australia 5000. Department of Biology, Australian
National University, Australian Capital Territory, 0200, Australia.
Pgreenslade@federation.edu.au.
(3)CSIRO Plant Industry, Locked Bag 59, Narrabri, NSW 2390, Australia.
The effects of production practices on the relative abundance of springtails

(Collembola) in irrigated cotton fields of northern New South Wales (NSW) were
studied over 2 yr to examine effects of farm management on these decomposer
organisms. Pitfall trapping and soil core extraction was undertaken in both
pseudoreplicated plots within whole fields on cotton farms and on experimental
replicate plots of Envirofeast cotton and Lucerne. The relative abundance of
surface-active springtails in cotton rows and densities of soil species from the
rhizosphere were calculated. Twenty-three species of Collembola were collected
from 5 fields, 19 in pitfall traps, and 11 in soil cores. Five species,
Setogaster sp., Proisotoma minuta, Entomobrya unostrigata, Entomobrya
multifasciata grp, and Lepidobrya sp. were numerically dominant on the ground at
86-96% of individuals and Mesaphorura sp., Folsomides parvulus, and Hemisotoma
thermophila grp dominant in the soil. Native grassland samples contained 15
species of which a probable 10 were native and 8 were not found in cotton.
Nineteen species of the 24 species identified from cotton were predominantly
fungal feeders. Highest catches of Collembola occurred after flowering and soil
Collembola increased with depth and during cotton growth on unsprayed plots but
decreased on sprayed plots. Surface soil moistures affected daily catch rates
with decomposing residues, crop stage, predator abundance, and season as
secondary factors. Insecticide (endosulfan, pyrethroid, carbamate, and
organophosphate) and predator effects were either negligible or unclear depending
on the factor involved. Springtails appear to be predominately food limited
during times of adequate soil moisture in cotton fields.

DOI: 10.1093/ee/nvv029
423. Anal Bioanal Chem. 2015 Oct;407(26):8047-58. doi: 10.1007/s00216-015-8977-7.

Epub
2015 Aug 26.
Using non-targeted direct analysis in real time-mass spectrometry (DART-MS) to

discriminate seeds based on endogenous or exogenous chemicals.
Subbaraj AK(1), Barrett BA(1), Wakelin SA(2), Fraser K(3).
Author information:
(1)AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston
North, 4442, New Zealand.
(2)AgResearch Ltd., Lincoln Research Centre, Private Bag 4749, Christchurch,
8140, New Zealand.
(3)AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Palmerston
North, 4442, New Zealand. karl.fraser@agresearch.co.nz.
Forage seeds are a highly traded agricultural commodity, and therefore, quality
control and assurance is high priority. In this study, we have used direct
analysis in real time-mass spectrometry (DART-MS) as a tool to discriminate
forage seeds based on their non-targeted chemical profiles. In the first
experiment, two lots of perennial ryegrass (Lolium perenne L.) seed were
discriminated based on exogenous residues of N-(3,
4-dichlorophenyl)-N,N-dimethylurea (Diuron(TM)), a herbicide. In a separate
experiment, washed and unwashed seeds of the forage legumes white clover
(Trifolium repens L.) and alfalfa (Medicago sativa L.) were discriminated based
on the presence or absence of oxylipins, a class of endogenous antimicrobial
compounds. Unwashed seeds confer toxicity towards symbiotic, nitrogen-fixing
rhizobia which are routinely coated on legume seeds before planting, resulting in
reduced rhizobial count. This is the first report of automatic introduction of
intact seeds in the DART ion source and detecting oxylipins using DART-MS. Apart
from providing scope to investigate legume-rhizobia symbiosis further in the
context of oxylipins, the results presented here will enable future studies aimed
at classification of seeds based on chemicals bound to the seed coat, thereby
offering an efficient screening device for industry.
DOI: 10.1007/s00216-015-8977-7
424. Syst Appl Microbiol. 2015 Oct;38(7):506-14. doi: 10.1016/j.syapm.2015.07.003.

Epub 2015 Jul 26.
Rhizobial strains isolated from nodules of Medicago marina in southwest Spain are
abiotic-stress tolerant and symbiotically diverse.
Alías-Villegas C(1), Cubo MT(2), Lara-Dampier V(3), Bellogín RA(4), Camacho M(5),
Temprano F(6), Espuny MR(7).
Author information:
(1)Departamento de Microbiología, Facultad de Biología, Universidad de Sevilla,
Avda. Reina Mercedes 6, Sevilla 41012, Spain. Electronic address: calias@us.es.
Avda. Reina Mercedes 6, Sevilla 41012, Spain. Electronic address: cubo@us.es.
Avda. Reina Mercedes 6, Sevilla 41012, Spain. Electronic address:
victorialara@live.com.
Avda. Reina Mercedes 6, Sevilla 41012, Spain. Electronic address: bellogin@us.es.
(5)Centro Las Torres-Tomejil (IFAPA), Apartado Oficial, Alcalá del Río, Sevilla,
Spain. Electronic address: mariag.camachomartinez@juntadeandalucia.es.
(6)Centro Las Torres-Tomejil (IFAPA), Apartado Oficial, Alcalá del Río, Sevilla,
Spain. Electronic address: francisco.temprano@juntadeandalucia.es.
Avda. Reina Mercedes 6, Sevilla 41012, Spain. Electronic address: espuny@us.es.
The isolation and characterisation of nitrogen-fixing root nodule bacteria from

Medicago marina, a tolerant legume species, were studied in two areas from
southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR
fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S
rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S
rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS,
including the partial sequence of the housekeeping gene glnII and the symbiotic
gene nodA of some representatives), physiological (utilisation of sole carbon
sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and
symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis,
M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M.
marina nodules belonged to Ensifer meliloti, except for one strain that belonged
to E. medicae. To determine the nodulation range of M. marina, 10 different
Ensifer species were tested for their ability to nodulate on this plant. E.
kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only
Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most
of the M. marina-nodulating strains showed tolerance to stress factors and all of
them shared the presence of a gene similar to cadA, a gene that encodes for a
PIB-type ATPase, which is a transporter belonging to the large superfamily of
ATP-driven pumps involved in the transport of metals across cell membranes.
DOI: 10.1016/j.syapm.2015.07.003

Aug
20.
Effects of ruminal dosing of Holstein cows with Megasphaera elsdenii on milk fat
production, ruminal chemistry, and bacterial strain persistence.
Weimer PJ(1), Da Silva Cabral L(2), Cacite F(2).

Author information:
(1)USDA-Agricultural Research Service, US Dairy Forage Research Center, Madison,
WI 53706; Department of Bacteriology, University of Wisconsin, Madison 53706.
Electronic address: Paul.Weimer@ars.usda.gov.
(2)Department of Animal Science, Federal University of Mato Grosso, 78060-900
Cuiabá, Mato Grosso, Brazil.
Megasphaera elsdenii is a lactate-utilizing bacterium whose ruminal abundance has

been shown to be greatly elevated during milk fat depression (MFD). To further
examine this association, a total of 23 cannulated multiparous Holstein cows were
examined in 3 experiments in which strains of M. elsdenii were directly dosed
into the rumen (~2 × 10(12) cells/dose); control cows were dosed with sterile
lactate-free culture medium. Cows were fed a total mixed ration (292 g of
starch/kg of dry matter) that contained primarily corn silage, alfalfa silage,
finely ground high-moisture corn, supplemental protein, and corn oil (3 g/kg of
dry matter). Experiments differed in stage of lactation of the cows (early or
late), dosing events (single dose, or 4 doses over a 5-d period), timing of dose
(prefeed or 4 h postfeed), and M. elsdenii strain (laboratory strain YI9 or 3
strains isolated from cows in the same herd). Dry matter intake and milk yield
and composition were measured from 5 to 0 d before dosing and 1 to 7d after first
dosing, plus later time points that varied by experiment. Milk yield and
composition were not affected by dosing. Megasphaera elsdenii was quantified in
the liquid phase of ruminal contents by automated ribosomal intergenic spacer
analysis, or by PCR with relative quantification (M. elsdenii 16S rRNA gene copy
number as a percentage of total bacterial 16S rRNA gene copies). Neither the M.
elsdenii-dosed or control cows displayed MFD after dosing, and in almost all
cases M. elsdenii populations returned to low baseline levels (<0.02% of 16S rRNA
gene copy number) within 24 h of dosing. This rapid decline in M. elsdenii also
occurred in several cows that were dosed with a strain of M. elsdenii that had
been isolated from that particular cow during a previous bout of MFD. Ruminal pH
and total millimolar volatile fatty acids and lactate did not differ between
dosed and control cows, although acetate-to-propionate ratio declined in both
groups and butyrate increased after dosing with M. elsdenii. The results confirm
that establishing exogenously added bacterial strains in the rumen is difficult,
even for strains previously isolated from the recipient cow. The potential role
of M. elsdenii as an agent of MFD remains unclear in the absence of successful
establishment of the dosed strains.

DOI: 10.3168/jds.2015-9721

Aug
20.
Growth performance and sorting characteristics of corn silage-alfalfa haylage

diets with or without forage dilution offered to replacement Holstein dairy
heifers.
Coblentz WK(1), Esser NM(2), Hoffman PC(3), Akins MS(3).
Author information:
(2)University of Wisconsin Marshfield Agricultural Research Station, Marshfield
54449.
Gravid heifers consuming high-quality forage diets are susceptible to excessive

weight gains and overconditioning. One approach for controlling this problem is
to dilute diets with low-energy forages, such as straw, that reduce the caloric
density and dry matter intake (DMI) of that diet by heifers. These diluting
agents are often sortable by dairy heifers, but previous visual evidence has
suggested that eastern gamagrass haylage may be a nonsortable alternative. Our
objectives were (1) to compare the growth performance of dairy heifers offered a
high-quality forage diet (control) with diets containing 1 of 3 diluting agents
[eastern gamagrass haylage (EGH), chopped wheat straw (WS), or chopped corn
fodder (CF)]; and (2) evaluate sorting behaviors of heifers offered these forage
diets. Holstein heifers (n=128) were stratified (32 heifers/block) on the basis
of initial body weight (heavy, 560 ± 27.7 kg; medium-heavy, 481 ± 17.7 kg;
medium-light, 441 ± 22.0 kg; and light, 399 ± 14.4 kg), and then assigned to 1 of
16 identical research pens (4 pens/block; 8 heifers/pen), where each of the 4
research diets were assigned to 1 pen within each block. Diets were offered in a
118-d feeding trial with heifers crowded to 133% of capacity at the feed bunk.
Inclusion of low-energy forages was effective in reducing both diet energy
density and DMI. Concentrations of physically effective fiber (pef) particles did
not change during the 24-h period following feeding for either the control or EGH
diets; however, this response for pef particles masked the competing (and
cancelling) responses for individual large and medium particles, which heifers
sorted with discrimination and preference, respectively. Sorting against pef
particles was detected for WS, and much more severely for the CF diet. Sorting of
forage particles by heifers could not be related to heifer performance. Compared
with control (1.16 kg/d), average daily gains (ADG) were reduced by dilution in
all cases, but were virtually identical between EGH (0.98 kg/d) and CF (0.97
kg/d), which exhibited no sorting and extensive sorting of pef, respectively.
Furthermore, ADG for WS was approximately 0.2 kg/d less than EGH or CF, despite
exhibiting sorting characteristics intermediate between EGH and CF. Diets diluted
with low-energy forages were formulated to be isonitrogenous and isocaloric;
within that context, WS was most effective in reducing DMI and maintaining ADG
within typical recommendations for Holstein heifers.

DOI: 10.3168/jds.2015-9491
427. Ecotoxicol Environ Saf. 2015 Dec;122:268-74. doi:

10.1016/j.ecoenv.2015.08.003.
Epub 2015 Sep 20.
Phytotoxicity of three plant-based biodiesels, unmodified castor oil, and Diesel

fuel to alfalfa (Medicago sativa L.), lettuce (Lactuca sativa L.), radish
(Raphanus sativus), and wheatgrass (Triticum aestivum).
Bamgbose I(1), Anderson TA(2).
Author information:
(1)The Institute of Environmental and Human Health (TIEHH), Department of
Environmental Toxicology, Texas Tech University, 1207 Gilbert Drive Box 41163,
Lubbock, TX 79409-1163, USA. Electronic address: ait109@aol.com.
(2)The Institute of Environmental and Human Health (TIEHH), Department of
Environmental Toxicology, Texas Tech University, 1207 Gilbert Drive Box 41163,
Lubbock, TX 79409-1163, USA.
The wide use of plant-based oils and their derivatives, in particular biodiesel,
have increased extensively over the past decade to help alleviate demand for
petroleum products and improve the greenhouse gas emissions profile of the
transportation sector. Biodiesel is regarded as a clean burning alternative fuel
produced from livestock feeds and various vegetable oils. Although in theory
these animal and/or plant derived fuels should have less environmental impact in
soil based on their simplified composition relative to Diesel, they pose an
environmental risk like Diesel at high concentrations when disposed. The aim of
the present study was to ascertain the phytotoxicity of three different
plant-derived biodiesels relative to conventional Diesel. For phytotoxicological
analysis, we used seeds of four crop plants, Medicago sativa, Lactuca sativa,
Raphanus sativus, and Triticum aestivum to analyze the germination of seeds in
contaminated soil samples. The toxicological experiment was conducted with two
different soil textures: sandy loam soil and silt loam soil. The studied
plant-based biodiesels were safflower methyl-ester, castor methyl ester, and
castor ethyl-ester. Biodiesel toxicity was more evident at high concentrations,
affecting the germination and survival of small-seeded plants to a greater
extent. Tolerance of plants to the biodiesels varied between plant species and
soil textures. With the exception of R. sativus, all plant species were affected
and exhibited some sensitivity to the fuels, such as delayed seedling emergence
and slow germination (average=10 days) at high soil concentrations (0.85% for
Diesel and 1.76% for the biodiesels). Tolerance of plants to soil contamination
had a species-specific nature, and on average, decreased in the following order:
Raphanus sativus (0-20%)>Triticum aestivum (10-40%) ≥ Medicago sativa> Lactuca
sativa (80-100%). Thus, we conclude that there is some phytotoxicity associated
with plant-based biodiesels. Further, the findings of this study can be useful
for selecting the least fuel-tolerant species as a soil contamination
bio-indicator and for determining the risks of biodiesel contamination.
DOI: 10.1016/j.ecoenv.2015.08.003
428. PLoS One. 2015 Aug 17;10(8):e0135934. doi: 10.1371/journal.pone.0135934.

eCollection 2015.
Contrasting Strategies of Alfalfa Stem Elongation in Response to Fall Dormancy in

Early Growth Stage: The Tradeoff between Internode Length and Internode Number.
Liu Z(1), Li X(1), Wang Z(2), Sun Q(3).
Author information:
Hohhot, P.R. China; Graduate School of Chinese Academy of Agricultural Sciences,
Beijing, P.R. China.
Hohhot, P.R. China; Animal husbandry department of Ministry of Agriculture,
Beijing, P.R. China.
Hohhot, P.R. China.
Fall dormancy (FD) in alfalfa (Medicago sativa L.) can be described using 11 FD
ratings, is widely used as an important indicator of stress resistance,
productive performance and spring growth. However, the contrasting growth
strategies in internode length and internode number in alfalfa cultivars with
different FD rating are poorly understood. Here, a growth chamber study was
conducted to investigate the effect of FD on plant height, aboveground biomass,
internode length, and internode number in alfalfa individuals in the early growth
stages. In order to simulate the alfalfa growth environment in the early stage,
11 alfalfa cultivars with FD ratings from one to 11 were chosen and seeded at the
greenhouse, and then were transplanted into an artificial growth chamber. The
experimental design was a randomized complete block in a split-plot arrangement
with three replicates. Plant height, above-ground biomass, internode length, and
internode number were measured in early growth stage in all individuals. Our
findings showed that plant height and the aboveground biomass of alfalfa did not
significantly differ among 11 different FD rated cultivars. Also, internode
length and internode number positively affected plant height and the aboveground
biomass of alfalfa individuals and the average internode length significantly
increased with increasing FD rating. However, internode number tended to sharply
decline when the FD rating increased. Moreover, there were no correlations,
slightly negative correlations, and strongly negative correlations between
internode length and internode number in alfalfa individuals among the three
scales, including within-FD ratings, within-FD categories and inter-FD ratings,
respectively. Therefore, our results highlighted that contrasting growth
strategies in stem elongation were adopted by alfalfa with different FD ratings
in the early growth stage. Alfalfa cultivars with a high FD rating have longer
internodes, whereas more dormant alfalfa cultivars have a larger number of
internodes. There were tradeoffs between internode length and internode number in
response to FD in alfalfa, which reflected certain scale-dependence.
PMCID: PMC4539217
429. J Dairy Sci. 2015 Oct;98(10):7238-47. doi: 10.3168/jds.2015-9427. Epub 2015

Aug
12.
Feeding preferences and voluntary feed intake of dairy cows: Effect of

conservation and harvest time of birdsfoot trefoil and chicory.
Lombardi D(1), Vasseur E(1), Berthiaume R(2), DeVries TJ(3), Bergeron R(4).
Author information:
(1)Organic Dairy Research Centre, Université de Guelph, Campus d'Alfred, Alfred,
ON, K0B 1A0 Canada.
(2)Valacta, Ste-Anne-de-Bellevue, QC, H9X 3R4 Canada.
(3)Department of Animal and Poultry Science, University of Guelph, Kemptville
Campus, Kemptville, ON, K0G 1J0 Canada.
(4)Organic Dairy Research Centre, Université de Guelph, Campus d'Alfred, Alfred,
ON, K0B 1A0 Canada. Electronic address: rbergero@uoguelph.ca.
Bioactive forages contain compounds, such as tannins, that are active against
pathogens. They have been successfully used in ruminants to control parasite
infections. Because cattle may find bioactive forages unpalatable, it is of
interest to know if an afternoon harvest time, which has been shown to increase
the percentage of nonstructural carbohydrates (NSC), hence palatability, may
mitigate this. The objectives of this study were to quantify voluntary intake and
preference of dairy cows for 2 bioactive forages, harvested in the morning and
evening, in addition to determining their time spent grazing on each forage
species. The forage species evaluated were fresh chicory harvested at 0700 h
(FCAM) and 1800 h (FCPM), fresh birdsfoot trefoil harvested at 0700 h (FBAM) and
1800 h (FBPM), birdsfoot trefoil baleage harvested the previous summer at 0700 h
(BBAM) and at 1800 h (BBPM), and third-cut alfalfa baleage harvested the previous
summer and used as control (CON). Single forages were offered ad libitum in
30-min tests to 14 dairy cows to determine intake in a 7 × 7 Latin square
(experiment 1). Every possible pair of forages (21 pairs) was then presented for
a 30-min test to 8 different dairy cows, and feed intake was measured (experiment
2). Finally, time spent grazing on chicory and birdsfoot trefoil was measured on
12 dairy cows (experiment 3). The tests consisted of 2 d of restriction on 1 of
the 2 fields for 1h, and 2 d of free-choice sessions (1h) between the 2 fields
adjacent to each other. Grazing time and location of the animals on the field was
assessed through 2-min scan sampling. In experiment 1, the highest voluntary
intakes were for CON, BBPM, and BBAM. In experiment 2, BBPM was preferentially
consumed over all other forages followed by CON and BBAM. Multidimensional
scaling showed that preference for BBPM, CON, and BBAM in dimension 1 was
positively associated with dry matter and nitrogen content, and negatively
associated with hemicellulose and soluble N/total N. No relationships between
dimension coordinates and any of the measured chemical composition variables
could be found for the other 2 dimensions. In experiment 3, cows spent 71% of
their time grazing in the birdsfoot trefoil field and 23% in the chicory field
during the free-choice sessions. In conclusion, cows in the present experiments
showed an overall preference toward baled forages compared with fresh forages,
most notably toward birdsfoot trefoil baleage. Cow preference did not appear to
be linked to harvest time (a.m. vs. p.m.).

DOI: 10.3168/jds.2015-9427

Aug
12.
Rumen fermentation, blood metabolites, and growth performance of calves during

transition from liquid to solid feed: Effects of dietary level and particle size
of alfalfa hay.
Nemati M(1), Amanlou H(1), Khorvash M(2), Moshiri B(3), Mirzaei M(4), Khan MA(5),
Ghaffari MH(6).
Author information:
(1)Department of Animal Science, Faculty of Agricultural Sciences, University of
Zanjan, Zanjan 313-45195, Iran.
Technology, Isfahan 84156-83111, Iran.
(3)Ghiam Dairy Complex, Isfahan 833111-4761, Iran.
Arak University, 38156-8-8349 Arak, Iran.
(5)Animal Nutrition and Health Group, Grasslands Research Centre, AgResearch
Limited Palmerston North 4442, New Zealand.
Technology, Isfahan 84156-83111, Iran. Electronic address: morteza.h.g@gmail.com.
This study evaluated the effects of particle size (PS) and dietary level of
alfalfa hay (AH) on rumen fermentation parameters, blood metabolites, eating
behavior, and growth performance in dairy calves during transition from liquid to
solid feed. Sixty newborn dairy calves (41 ± 2.5,kg of body weight) were used in
a 2 × 2 factorial arrangement with the factors dietary AH level (medium, 12.5%,
or high, 25%, on DM basis) and PS (fine = 1mm or medium = 3mm, as geometric
means) of AH. Hence, the dietary treatments were (1) medium level of AH with fine
PS (M-FPS), (2) medium level of AH with medium PS (M-MPS), (3) high level of AH
with fine PS (H-FPS), and (4) high level of AH with medium PS (H-MPS). Particle
size of AH did not affect total DMI (TDMI) during the preweaning period, although
TDMI was greater for calves fed MPS than in those fed FPS during the postweaning
and overall periods. Calves fed MPS spent more time eating solid feed and
ruminating and less time on nonnutritive oral behaviors compared with FPS calves.
The dietary level of AH did not affect behavioral parameters. Average daily gain
of calves was not affected by dietary treatment before weaning. During the
postweaning and overall periods, average daily gain was greater in calves fed MPS
than in those fed FPS at the 25% AH level, but this effect was absent with 12.5%
AH. Furthermore, the rumen pH values on d 35 and 70 of the study were greater for
MPS than for FPS, regardless of the dietary level of AH. Effects of AH level, PS,
and their interaction did not affect blood glucose concentrations in developing
calves. These results indicate that feed intake, feeding behavior, rumen
fermentation parameters, and blood β-hydroxybutyrate concentration may be
affected by rations differing in forage PS; thus, providing calves with MPS can
improve calf performance and reduce their nonnutritive oral behaviors.

DOI: 10.3168/jds.2014-9144
431. Plant Biotechnol J. 2016 Mar;14(3):964-75. doi: 10.1111/pbi.12451. Epub 2015

Aug
13.
Co-expression of tonoplast Cation/H(+) antiporter and H(+)-pyrophosphatase from

xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity,
drought and field conditions.
Bao AK(1), Du BQ(1)(2), Touil L(1)(3), Kang P(1), Wang QL(1), Wang SM(1).
Author information:
(2)Lanzhou Animal Husbandry and Veterinary Institute, Lanzhou, China.
(3)Laboratory of arid and oasis cropping, Institute of Arid Area (IRA), Medenine,
Tunisia.
Salinity and drought are major environmental factors limiting the growth and
productivity of alfalfa worldwide as this economically important legume forage is
sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa
lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from
the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium
tumefaciens-mediated transformation. Compared with wild-type (WT) plants,
transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with
greater plant height and dry mass under normal or stress conditions (NaCl or
water-deficit) in the greenhouse. The growth performance of transgenic alfalfa
plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and
roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation
contributed to maintaining intracellular ions homoeostasis and osmoregulation of
plants and thus conferred higher leaf relative water content and greater
photosynthesis capacity in transgenic plants compared to WT when subjected to
NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing
ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and
most importantly, exhibited enhanced photosynthesis capacity by maintaining
higher net photosynthetic rate, stomatal conductance, and water-use efficiency
than WT plants. Our results indicate that co-expression of tonoplast NHX and
H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa,
and enhanced its tolerance to high salinity and drought. This study laid a solid
basis for reclaiming and restoring saline and arid marginal lands as well as
improving forage yield in northern China.
© 2015 Society for Experimental Biology, Association of Applied Biologists and

John Wiley & Sons Ltd.
DOI: 10.1111/pbi.12451
432. Plant Cell Rep. 2015 Dec;34(12):2127-36. doi: 10.1007/s00299-015-1856-z. Epub

2015 Aug 12.
A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase

confers gabaculine resistance when expressed in tobacco plastids.
Bellucci M(1), De Marchis F(1), Ferradini N(2), Pompa A(1), Veronesi F(2),
Rosellini D(3).
Author information:
(1)Institute of Biosciences and Bioresources, Research Division of Perugia,
National Research Council (CNR), via della Madonna Alta 130, 06128, Perugia,
Italy.
Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy.
Perugia, Borgo XX Giugno 74, 06121, Perugia, Italy. daniele.rosellini@unipg.it.
KEY MESSAGE: A mutant glutamate 1-semialdehyde aminotransferase gene from the

Synechococcus , inserted into tobacco plastid DNA by means of particle
bombardment and antibiotic selection, conferred gabaculine resistance allowing to
attain homoplasmy. Many plant species are recalcitrant to plastid genome
transformation. New selections systems may help to overcome this limitation and
to extend the application of this technology. A mutant hemL gene from the
photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive
glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable
marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since
GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco
plastid genome along with the conventional antibiotic resistance aadA gene, in
the attempt to develop a new selection system for plastome transformation.
Although we were unable to directly regenerate gabaculine resistant
transplastomic plants, we demonstrated the functionality of hemL in tobacco
plastids by using gabaculine selection in the second and third rounds of in vitro
selection that permitted to obtain the homoplasmic state in transgenic plants.
Thus, the mutant hemL gene functions as a secondary selection marker in tobacco
plastids. Our results encourage further attempts to test gabaculine resistant GSA
for plastome transformation of crop plants in which gabaculine has stronger
regeneration-inhibiting effects with respect to tobacco.
DOI: 10.1007/s00299-015-1856-z
433. Animal. 2015 Dec;9(12):2006-16. doi: 10.1017/S1751731115001585. Epub 2015 Aug
12.
Foraging behaviour, nutrient intake from pasture and performance of free-range

growing pigs in relation to feed CP level in two organic cropping systems.
Jakobsen M(1), Kongsted AG(1), Hermansen JE(1).
Author information:
(1)Department of Agroecology, Faculty of Science and Technology,Blichers Allé
20,Aarhus University,P.O. Box 50,DK-8830 Tjele,Denmark.
In organic pig production one of the major challenges is to be able to fulfil

amino acid requirements based on organic and locally grown protein feed crops.
The pig is an opportunistic omnivore with a unique capacity for foraging above
and below the soil surface. It is hypothesized that direct foraging in the range
area can pose an important contribution in terms of fulfilling nutrient
requirements of growing pigs. Foraging activity, lucerne nutrient intake and pig
performance were investigated in 36 growing pigs, foraging on lucerne or grass
and fed either a standard organic pelleted feed mixture (HP: high protein) or a
grain mixture containing 48% less CP (LP: low protein) compared with the high
protein feed mixture, from an average live weight of 58 kg to 90 kg in a complete
block design in three replicates. The pigs were fed 80% of energy recommendations
and had access to 4 m2 of pasture/pig per day during the 40 days experimental
period from September to October 2013. Behavioural observations were carried out
12 times over the entire experimental period. For both crops, LP pigs rooted
significantly more compared with HP pigs but the effect of CP level was more
pronounced in grass (44% v. 19% of all observations) compared with lucerne (28%
v. 16% of all observations). Feed protein level turned out not to have any
significant effect on grazing behaviour but pigs foraging on lucerne grazed
significantly more than pigs foraging on grass (10% v. 4% of all observations).
Daily weight gain and feed conversion ratio were significantly affected by feed
protein and forage crop interactions. Compared to HP pigs, LP treated pigs had
33% lower daily weight gain (589 v. 878 g) and 31% poorer feed conversion ratio
(3.75 v. 2.59 kg feed/kg weight gain) in grass paddocks, whereas in lucerne
paddocks LP pigs only had 18% lower daily weight gain (741 v. 900 g) and a 14%
poorer feed conversion ratio (2.95 v. 2.54 kg feed/kg weight gain) compared with
HP pigs. LP pigs foraging on lucerne used 169 g less concentrate CP/kg weight
gain, compared with HP pigs, indicating the nitrogen efficiency of the system.
The results indicate that direct foraging of lucerne may be a valuable strategy
in terms of accommodating CP and lysine requirements of organic growing pigs.
DOI: 10.1017/S1751731115001585
434. Mikrobiologiia. 2015 May-Jun;84(3):263-80.
[Root Nodule Bacteria Sinorhizobium meliloti: Tolerance to Salinity and Bacterial

Genetic Determinants].
Roumiantseva ML, Muntyan VS.
The theoretical and experimental data on salt tolerance of root nodule bacteria
Sinorhizobium meliloti (Ensifer meliloti), an alfalfa symbiont, and on genetic
determination of this feature are reviewed. Extensive data on the genes affecting
adaptation of proteobacteria are provided, as well as on the groups of genes with
activity depending on the osmolarity of the medium. Structural and functional
polymorphism of the bet genes involved in betaine synthesis and transport in S.
meliloti is discussed. The phenotypic and. genotypic polymorphism in 282
environmental rhizobial strains isolated from the centers of alfalfa diversity
affected by aridity and salinity is discussed. The isolates from the Aral Sea
area and northern Caucasus were shown to possess the betC gene represented by two
types of alleles: the dominant A-type allele found in Rm 1021 and the less common
divergent E-type allele, which was revealed in regions at the frequencies at the
frequencies of 0.35 and 0.48, respectively. In the isolates with the
salt-tolerant phenotype, which were isolated from root nodules and subsequently
formed less effective symbioses with alfalfa, the frequency of E-type alleles was
2.5 times higher. Analysis of the nucleotide and amino acid sequences of the
E-type allele of the betC gene revealed that establishment of this allele in the
population was a result of positive selection. It is concluded that
diversification of the functionally diverse bet genes occurring in S. meliloti
affects the salt tolerance and symbiotic effectivity of rhizobia.
435. Plant Sci. 2015 Sep;238:13-25. doi: 10.1016/j.plantsci.2015.05.014. Epub 2015

May
22.
Ups and downs in alfalfa: Proteomic and metabolic changes occurring in the
growing stem.
Printz B(1), Guerriero G(2), Sergeant K(3), Renaut J(2), Lutts S(4), Hausman
JF(2).
Author information:
(1)Environmental Research and Innovation (ERIN) Department, Luxembourg Institute
of Science and Technology (LIST), 5, Avenue des Hauts-Fourneaux, L-4362
Esch/Alzette, Luxembourg; Groupe de Recherche en Physiologie végétale (GRPV),
Earth and Life Institute Agronomy (ELI-A), Université catholique de Louvain, 5
(bte 7.07.13) Place Croix du Sud, B-1348 Louvain-la-Neuve, Belgium.
Esch/Alzette, Luxembourg.
Esch/Alzette, Luxembourg. Electronic address: kjell.sergeant@list.lu.
(4)Groupe de Recherche en Physiologie végétale (GRPV), Earth and Life Institute
Agronomy (ELI-A), Université catholique de Louvain, 5 (bte 7.07.13) Place Croix
du Sud, B-1348 Louvain-la-Neuve, Belgium.
The expanding interest for using lignocellulosic biomass in industry spurred the
study of the mechanisms underlying plant cell-wall synthesis. Efforts using
genetic approaches allowed the disentanglement of major steps governing stem
fibre synthesis. Nonetheless, little is known about the relations between the
stem maturation and the evolution of its proteome. During Medicago sativa L.
maturation, the different internodes grow asynchronously allowing the
discrimination of various developmental stages on a same stem. In this study, the
proteome of three selected regions of the stem of alfalfa (apical, intermediate
and basal) was analyzed and combined with a compositional analysis of the
different stem parts. Interestingly, the apical and the median regions share many
similarities: high abundance of chloroplast- and mitochondrial-related proteins
together with the accumulation of proteins acting in the early steps of fibre
production. In the mature basal region, forisomes and stress-related proteins
accumulate. The RT-qPCR assessment of the expression of genes coding for members
of the cellulose synthase family likewise indicates that fibres and the machinery
responsible for the deposition of secondary cell walls are predominantly formed
in the apical section. Altogether, this study reflects the metabolic change from
the fibre production in the upper stem regions to the acquisition of
defence-related functions in the fibrous basal part.
Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights
reserved.
DOI: 10.1016/j.plantsci.2015.05.014
436. J Environ Sci (China). 2015 Aug 1;34:86-92. doi: 10.1016/j.jes.2015.03.010.

Epub
2015 Jun 3.
Ractopamine up take by alfalfa (Medicago sativa) and wheat (Triticum aestivum)

from soil.
Shelver WL(1), DeSutter TM(2).
Author information:
(1)USDA-ARS Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, ND
58102, USA. Electronic address: Weilin.Shelver@ars.usda.gov.
(2)Department of Soil Science, North Dakota State University, Walster Hall,
Albrecht Boulevard, Fargo, ND 58108, USA.
Ractopamine is a beta adrenergic agonist used as a growth promoter in swine,

cattle and turkeys. To test whether ractopamine has the potential to accumulate
in plants grown in contaminated soil, a greenhouse study was conducted with
alfalfa (Medicago sativa) and wheat (Triticum aestivum) grown in two soils having
different concentrations of organic matter (1.3% and 2.1%), amended with 0, 0.5,
and 10 μg/g of ractopamine. Plant growth ranged from 2.7 to 8.8 g dry weight (dw)
for alfalfa, and 8.7 to 40 g dw for wheat and was generally greater in the higher
organic matter content soil. The uptake of ractopamine in plant tissues ranged
from non-detectable to 897 ng/g and was strongly dependent on soil ractopamine
concentration across soil and plant tissue. When adjusted to the total fortified
quantities, the amount of ractopamine taken up by the plant tissue was low,
<0.01% for either soil.
DOI: 10.1016/j.jes.2015.03.010
437. Environ Sci Pollut Res Int. 2015 Dec;22(23):19142-56. doi:

10.1007/s11356-015-5094-2. Epub 2015 Aug 7.
Effect of combined microbes on plant tolerance to Zn-Pb contaminations.
Ogar A(1), Sobczyk Ł(2), Turnau K(3)(4).
Author information:
(1)Plant-Microbial Interaction Research Group, Institute of Environmental
Sciences, Jagiellonian University, Krakow, Poland. anna.ogar@uj.edu.pl.
(2)Ecosystem Ecology Research Group, Institute of Environmental Sciences,
Jagiellonian University, Krakow, Poland. lukasz.sobczyk@uj.edu.pl.
(3)Plant-Microbial Interaction Research Group, Institute of Environmental
Sciences, Jagiellonian University, Krakow, Poland. katarzyna.turnau@uj.edu.pl.
(4)The Malopolska Center of Biotechnology, Jagiellonian University, Krakow,
Poland. katarzyna.turnau@uj.edu.pl.
Comment in
Environ Sci Pollut Res Int. 2016 Mar;23(6):5992-4.
The presence and composition of soil microbial communities has been shown to have
a large impact on plant-plant interactions and consequently plant diversity and
composition. The goal of the present study was to evaluate impact of arbuscular
mycorrhizal fungi (AMF) and nitrogen-fixing bacteria, which constitutes an
essential link between the soil and the plant's roots. A greenhouse pot
experiment was conducted to evaluate the feasibility of using selected microbes
to improve Hieracium pilosella and Medicago sativa growth on Zn-Pb-rich site.
Results of studies revealed that biomass, the dry mass of shoots and roots,
increased significantly when plants were inoculated with mycorrhizal fungi and
nitrogen-fixing bacteria. The addition of Azospirillum sp. and Nostoc edaphicum
without mycorrhiza suppressed plant growth. Single bacterial inoculation alone
does not have a positive effect on M. sativa growth, while co-inoculation with
AMF improved plant growth. Plant vitality (expressed by the performance index)
was improved by the addition of microbes. However, our results indicated that
even dry heat sterilization of the substratum created imbalanced relationships
between soil-plant and plants and associated microorganisms. The studies
indicated that AMF and N2-fixers can improve revegetation of heavy metal-rich
industrial sites, if the selection of interacting symbionts is properly
conducted.
DOI: 10.1007/s11356-015-5094-2
PMCID: PMC4669377
438. Int J Syst Evol Microbiol. 2015 Aug 4. doi: 10.1099/ijsem.0.000505. [Epub
ahead
of print]
Paenibacillus medicaginis sp. nov. a chitinolytic endophyte isolated from the

root nodule of alfalfa (Medicago sativa L.).
Lai WA, Hameed A, Lin SY, Hung MH, Hsu YH, Liu YC, Shahina M, Shen FT, Young CC.
A Gram-stain-variable, short-rod-shaped, endospore-forming, strictly aerobic,

non-motile, chitinolytic and endophytic bacterium, designated strain
CC-Alfalfa-19T, exhibiting unusual bipolar appendages was isolated from the root
nodule of alfalfa (Medicago sativa L.) in Taiwan and subjected to a polyphasic
taxonomy. Based on 16S rRNA gene sequence analysis, strain CC-Alfalfa-19T was
found to be most closely related to Paenibacillus puldeungensis CAU 9324T (95.2
%), whereas other Paenibacillus species shared ≤95.0 % sequence similarity. The
phylogenetic analysis revealed a distinct phyletic lineage established by strain
CC-Alfalfa-19T with respect to other Paenibacillus species. Fatty acids comprised
predominantly anteiso-C15:0, C16:0, anteiso-C17:0 and iso-C16. Menaquinone 7
(MK-7) was identified as the sole respiratory quinone and the genomic DNA G+C
content was 42.7 mol%. Polar lipids included diphosphatidylglycerol,
phosphatidylglycerol, phosphatidylmonomethylethanolamine,
phosphatidylethanolamine, an unidentified glycolipid and an unidentified lipid.
The diagnostic diamino acid found in the cell-wall peptidoglycan was
meso-diaminopimelic acid. Based on the polyphasic taxonomic evidences that were
in line with the genus Paenibacillus and additional distinguishing
characteristics, strain CC-Alfalfa-19T is considered to represent a novel
species, for which the name Paenibacillus medicaginis sp. nov. (type strain
CC-Alfalfa-19T = BCRC 80441T = JCM 18446T) is proposed.
DOI: 10.1099/ijsem.0.000505
PMID: 26242932
439. Animal. 2015 Dec;9(12):1970-8. doi: 10.1017/S1751731115001470. Epub 2015 Aug

5.
Replacement of grass and maize silages with lucerne silage: effects on

performance, milk fatty acid profile and digestibility in Holstein-Friesian dairy
cows.
Sinclair LA(1), Edwards R(1), Errington KA(1), Holdcroft AM(1), Wright M(1).
Author information:
(1)Department of Animal Production,Welfare and Veterinary Sciences,Harper Adams
University,Newport,Shropshire TF10 8NB,UK.
In total, 20 multiparous Holstein-Friesian dairy cows received one of four diets

in each of four periods of 28-day duration in a Latin square design to test the
hypothesis that the inclusion of lucerne in the ration of high-yielding dairy
cows would improve animal performance and milk fatty acid (FA) composition. All
dietary treatments contained 0.55 : 0.45 forage to concentrates (dry matter (DM)
basis), and within the forage component the proportion of lucerne (Medicago
sativa), grass (Lolium perenne) and maize silage (Zea mays) was varied (DM
basis): control (C)=0.4 : 0.6 grass : maize silage; L20=0.2 : 0.2 : 0.6 lucerne :
grass : maize silage; L40=0.4 : 0.6 lucerne : maize silage; and L60=0.6 : 0.4
lucerne : maize silage. Diets were formulated to contain a similar CP and
metabolisable protein content, with the reduction of soya bean meal and feed
grade urea with increasing content of lucerne. Intake averaged 24.3 kg DM/day and
was lowest in cows when fed L60 (P0.05) by dietary treatment. Digestibility of
DM, organic matter, CP and fibre decreased (P<0.01) with increasing content of
lucerne in the diet, although fibre digestibility was similar in L40 and L60. It
is concluded that first cut grass silage can be replaced with first cut lucerne
silage without any detrimental effect on performance and an improvement in the
milk FA profile, although intake and digestibility was lowest and plasma urea
concentrations highest in cows when fed the highest level of inclusion of
lucerne.
DOI: 10.1017/S1751731115001470
440. Sci Rep. 2015 Aug 3;5:12729. doi: 10.1038/srep12729.
Innate positive chemotaxis to pollen from crops and banker plants in predaceous
biological control agents: towards new field lures?
Li S(1), Tan X(2), Desneux N(3), Benelli G(4), Zhao J(1), Li X(2), Zhang F(5),
Gao X(6), Wang S(5).
Author information:
(1)1] Institute of Plant and Environment Protection, Beijing Academy of
Agriculture and Forestry Sciences, Beijing, 100097, China [2] Department of
Entomology, China Agricultural University, Beijing 100193, China.
(2)Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
(3)French National Institute for Agricultural Research (INRA), UMR1355-ISA, 06903
Sophia-Antipolis, France.
(4)Department of Agriculture, Food and Environment, University of Pisa, 56124
Pisa, Italy.
(5)Institute of Plant and Environment Protection, Beijing Academy of Agriculture
and Forestry Sciences, Beijing, 100097, China.
(6)Department of Entomology, China Agricultural University, Beijing 100193,
China.
Predator-prey interactions form the core of biological control of arthropod

pests. Which tools can be used to monitor and collect carnivorous arthropods in
natural habitats and targeted crops? Eco-friendly and effective field lures are
urgently needed. In this research, we carried out olfactometer experiments assess
innate positive chemotaxis to pollen of seven crop and banker plant by two
important predatory biological control agents: the coccinellid Propylea japonica
(Thunberg) and the anthocorid Orius sauteri (Poppius). We compared the
attractiveness of pollens from crops and banker plants to that of common prey
homogenates (aphids and thrips, respectively). Attractiveness of the tested odor
sources was checked via field trapping experiments conducted in organic apple
orchards and by release-recapture assays in organic greenhouse tomato crops.
Maize and canola pollen were attractive to both P. japonica and O. sauteri, in
laboratory and field assays. P. japonica was highly attracted by balm mint
pollen, whereas O. sauteri was attracted by alfalfa pollen. Our results encourage
the use of pollen from crops and banker plants as low-cost and eco-friendly
attractors to enhance the monitoring and attraction of arthropod predators in
biological control programs.
DOI: 10.1038/srep12729
PMCID: PMC4522688

Jul
29.
Effects of partly replacing dietary starch with fiber and fat on milk production
and energy partitioning.
Boerman JP(1), Potts SB(1), VandeHaar MJ(1), Lock AL(2).
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing 48824.
Electronic address: allock@msu.edu.
The effects of partly replacing dietary starch with fiber and fat to provide a
diet with similar net energy for lactation (NEL) density on yields of milk and
milk components and on energy partitioning were evaluated in a crossover design
experiment. Holstein cows (n = 32; 109 ± 22 d in milk, mean ± standard deviation)
were randomly assigned to treatment sequence. Treatments were a high-starch diet
containing 33% corn grain (mixture of dry ground and high-moisture corn; HS) or a
high-fiber, high-fat diet containing 2.5% palmitic acid-enriched fatty acid (FA)
supplement (HFF). Diets contained corn silage, alfalfa silage, and wheat straw as
forage sources; HS contained 32% starch, 3.2% FA, and 25% neutral detergent
fiber, whereas HFF contained 16% starch, 5.4% FA, and 33% neutral detergent
fiber. Compared with HS, the HFF treatment reduced milk yield, milk protein
concentration, and milk protein yield, but increased milk fat concentration, milk
fat yield, milk energy output, and milk to feed ratio (energy-corrected milk/dry
matter intake). The HFF treatment reduced the yield of de novo synthesized (<
16-carbon) milk FA and increased the yield of 16-carbon milk FA. Yield of
preformed (> 16-carbon) milk FA was not different. The HFF treatment increased
plasma concentrations of triglycerides and nonesterified fatty acids, but
decreased plasma concentration of insulin. Compared with HS, the HFF treatment
reduced body weight gain, change in body condition score, and fat thickness over
the rump and rib. Calculated body energy gain, as a fraction of NEL use, was less
for HFF than HS, whereas milk energy as a fraction of NEL use was increased for
HFF. We concluded that the 2 treatments resulted in similar apparent NEL
densities and intakes, but the HS treatment partitioned more energy toward body
gain whereas the HFF treatment partitioned more energy toward milk. A high-fiber,
high-fat diet might diminish the incidence of over conditioning in mid-lactation
cows while maintaining high milk production.

DOI: 10.3168/jds.2015-9467
442. Proc Natl Acad Sci U S A. 2015 Aug 25;112(34):10663-8. doi:

10.1073/pnas.1504124112. Epub 2015 Jul 30.
An inhibitor persistently decreased enteric methane emission from dairy cows with
no negative effect on milk production.
Hristov AN(1), Oh J(2), Giallongo F(2), Frederick TW(2), Harper MT(2), Weeks
HL(2), Branco AF(3), Moate PJ(4), Deighton MH(4), Williams SR(4), Kindermann
M(5), Duval S(6).
Author information:
(1)Department of Animal Science, The Pennsylvania State University, University
Park, PA 16802; anh13@psu.edu.
(2)Department of Animal Science, The Pennsylvania State University, University
Park, PA 16802;
(3)Departamento de Zootecnia, Universidade Estadual de Maringá, PR 87020-900,
Brazil;
(4)Agriculture Research Division, Department of Economic Development Jobs
Transport and Resources, Ellinbank Centre, Ellinbank 3821, Victoria, Australia;
(5)Animal Nutrition and Health, DSM Nutritional Products, Basel CH-4002,
Switzerland;
(6)Research Centre for Animal Nutrition and Health, DSM Nutritional Products
France, Saint Louis Cedex 68305, France.
Erratum in
Proc Natl Acad Sci U S A. 2015 Sep 15;112(37):E5218.
A quarter of all anthropogenic methane emissions in the United States are from
enteric fermentation, primarily from ruminant livestock. This study was
undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP),
on enteric methane emission in lactating Holstein cows. An experiment was
conducted using 48 cows in a randomized block design with a 2-wk covariate period
and a 12-wk data collection period. Feed intake, milk production, and fiber
digestibility were not affected by the inhibitor. Milk protein and lactose yields
were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP,
averaging about 30% lower than the control. Methane emission per unit of feed dry
matter intake or per unit of energy-corrected milk were also about 30% less for
the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was
80% greater than control cows during the 12-wk experiment. The experiment
demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry
matter, decreased methane emissions from high-producing dairy cows by 30% and
increased body weight gain without negatively affecting feed intake or milk
production and composition. The inhibitory effect persisted over 12 wk of
treatment, thus offering an effective methane mitigation practice for the
livestock industries.
DOI: 10.1073/pnas.1504124112
PMCID: PMC4553761
443. J Food Prot. 2015 Aug;78(8):1586-91. doi: 10.4315/0362-028X.JFP-15-029.
Inactivation of Human Norovirus and Its Surrogates on Alfalfa Seeds by Aqueous

Ozone.
Wang Q(1), Markland S(1), Kniel KE(2).
Author information:
(1)Department of Animal and Food Sciences, University of Delaware, Newark,
Delaware 19716, USA.
Delaware 19716, USA. kniel@udel.edu.
Alfalfa sprouts have been associated with numerous foodborne outbreaks. Previous
studies investigated the effectiveness of aqueous ozone on bacterially
contaminated seeds, yet little is known about the response of human norovirus
(huNoV). This study assessed aqueous ozone for the disinfection of alfalfa seeds
contaminated with huNoV and its surrogates. The inactivation of viruses without a
food matrix was also investigated. Alfalfa seeds were inoculated with huNoV
genogroup II, Tulane virus (TV), and murine norovirus (MNV); viruses alone or
inoculated on seeds were treated in deionized water containing 6.25 ppm of
aqueous ozone with agitation at 22°C for 0.5, 1, 5, 15, or 30 min. The data
showed that aqueous ozone resulted in reductions of MNV and TV infectivity from
1.66 ± 1.11 to 5.60 ± 1.11 log PFU/g seeds; for all treatment times,
significantly higher reductions were observed for MNV (P < 0.05). Viral genomes
were relatively resistant, with a reduction of 1.50 ± 0.14 to 3.00 ± 0.14 log
genomic copies/g seeds; the reduction of TV inoculated in water was similar to
that of huNoV, whereas MNV had significantly greater reductions in genomic copies
(P < 0.05). Similar trends were observed in ozone-treated viruses alone, with
significantly higher levels of inactivation (P < 0.05), especially with reduced
levels of infectivity for MNV and TV. The significant inactivation by aqueous
ozone indicates that ozone may be a plausible substitute for chlorine as an
alternative treatment for seeds. The behavior of TV was similar to that of huNoV,
which makes it a promising surrogate for these types of scenarios.
DOI: 10.4315/0362-028X.JFP-15-029

10.1007/s11356-015-5077-3. Epub 2015 Jul 25.
Metal uptake of tomato and alfalfa plants as affected by water source, salinity,
and Cd and Zn levels under greenhouse conditions.
Gharaibeh MA(1), Marschner B(2), Heinze S(2).
Author information:
(1)Department of Natural Resources and the Environment, Faculty of Agriculture,
Jordan University of Science and Technology, P.O. Box 3030, Irbid, 22110, Jordan.
mamoun@just.edu.jo.
(2)Department of Soil Science/Soil Ecology, Institute of Geography,
Ruhr-University Bochum, Bochum, Germany.
Irrigation with wastewater is a promising option to improve crop yields and to

reduce pressure on freshwater sources. However, heavy metal concentrations in
wastewater may cause health concerns. A greenhouse pot experiment was conducted
in order to determine cadmium (Cd) and zinc (Zn) concentrations in sandy soil and
plant tissues of tomato (Lycopersicon esculentum L.) and alfalfa (Medicago sativa
L.). A 2 × 2 × 4 × 2 factorial treatment arrangement was utilized. Two water
sources, fresh (FW) or treated wastewater (TWW), at two salinity levels (1 and 3
dS m(-1)) containing different levels of Cd and Zn were used. Samples were
collected after a 90-day growth period. It was observed that the growth of both
plants was depressed at the highest metal level (L3). Metal accumulation in plant
parts increased with the increase of metal concentration and salinity in
irrigation water. At low salinity, water source was the main factor which
controlled metal accumulation, whereas, at high salinity, chloride appeared to be
the principal factor controlling metal uptake regardless of water source. Metal
translocation from roots to shoots increased in TWW-irrigated plants, even in the
controls. Tomatoes accumulated Cd up to and above critical levels safe for human
consumption, even though Cd concentration in irrigation water did not exceed the
current recommended values. Therefore, food production in sandy soils may well
pose a health hazard when irrigated with TWW containing heavy metals.
Complexation with dissolved organic compounds (DOC) in TWW may be to be the
principal factor responsible for increased metal uptake and transfer at low
salinity, thereby increasing the risk of heavy metal contamination of food and
forage crops.
DOI: 10.1007/s11356-015-5077-3
445. Int J Mol Sci. 2015 Jul 16;16(7):16104-24. doi: 10.3390/ijms160716104.
Analysis of Cell Wall-Related Genes in Organs of Medicago sativa L. under

Different Abiotic Stresses.
Behr M(1)(2), Legay S(3), Hausman JF(4), Guerriero G(5).
Author information:
(1)Environmental Research and Innovation (ERIN), Luxembourg Institute of Science
and Technology (LIST), 5, Avenue des Hauts-Fourneaux, L-4362 Esch/Alzette,
Luxembourg. marc.behr@list.lu.
(2)Groupe de Recherche en Physiologie végétale, Earth and Life
Institute-Agronomy, Université catholique de Louvain, 5 (bte 7.07.13) Place Croix
du Sud, B-1348 Louvain-la-Neuve, Belgium. marc.behr@list.lu.
Luxembourg. sylvain.legay@list.lu.
Luxembourg. jean-francois.hausman@list.lu.
Luxembourg. gea.guerriero@list.lu.
Abiotic constraints are a source of concern in agriculture, because they can have
a strong impact on plant growth and development, thereby affecting crop yield.
The response of plants to abiotic constraints varies depending on the type of
stress, on the species and on the organs. Although many studies have addressed
different aspects of the plant response to abiotic stresses, only a handful has
focused on the role of the cell wall. A targeted approach has been used here to
study the expression of cell wall-related genes in different organs of alfalfa
plants subjected for four days to three different abiotic stress treatments,
namely salt, cold and heat stress. Genes involved in different steps of cell wall
formation (cellulose biosynthesis, monolignol biosynthesis and polymerization)
have been analyzed in different organs of Medicago sativa L. Prior to this
analysis, an in silico classification of dirigent/dirigent-like proteins and
class III peroxidases has been performed in Medicago truncatula and M. sativa.
The final goal of this study is to infer and compare the expression patterns of
cell wall-related genes in response to different abiotic stressors in the organs
of an important legume crop.
DOI: 10.3390/ijms160716104
PMCID: PMC4519941
446. Plant Biotechnol J. 2016 Mar;14(3):895-904. doi: 10.1111/pbi.12439. Epub 2015

Jul
20.
Combining enhanced biomass density with reduced lignin level for improved forage
quality.
Gallego-Giraldo L(1), Shadle G(1), Shen H(1)(2), Barros-Rios J(1), Fresquet

Corrales S(3), Wang H(4), Dixon RA(1).
Author information:
(1)Department of Biological Sciences, University of North Texas, Denton, TX, USA.
(2)Chromatin Inc, Chicago, IL, USA.
(3)Instituto de Biologia Molecular y Celular de Plantas, Universidad Politecnica
de Valencia-CSIC, Valencia, Spain.
(4)Department of Plant Science & Landscape Architecture, University of
Connecticut, Storrs, CT, USA.
To generate a forage crop with increased biomass density that retains forage
quality, we have genetically transformed lines of alfalfa (Medicago sativa L.)
expressing antisense constructs targeting two different lignin pathway
biosynthetic genes with a construct for down-regulation of a WRKY family
transcription factor that acts as a repressor of secondary cell wall formation in
pith tissues. Plants with low-level expression of the WRKY dominant repressor
construct produced lignified cell walls in pith tissues and exhibited enhanced
biomass and biomass density, with an increase in total sugars in the cell wall
fraction; however, lines with high expression of the WRKY dominant repressor
construct exhibited a very different phenotype, with loss of interfascicular
fibres associated with repression of the NST1 transcription factor. This latter
phenotype was not observed in transgenic lines in which the WRKY transcription
factor was down-regulated by RNA interference. Enhanced and/or ectopic deposition
of secondary cell walls was also seen in corn and switchgrass expressing WRKY
dominant repressor constructs, with enhanced biomass in corn but reduced biomass
in switchgrass. Neutral detergent fibre digestibility was not impacted by WRKY
expression in corn. Cell walls from WRKY-DR-expressing alfalfa plants with
enhanced secondary cell wall formation exhibited increased sugar release
efficiency, and WRKY dominant repressor expression further increased sugar
release in alfalfa down-regulated in the COMT, but not the HCT, genes of lignin
biosynthesis. These results suggest that significant enhancements in forage
biomass and quality can be achieved through engineering WRKY transcription
factors in both monocots and dicots.

DOI: 10.1111/pbi.12439
447. Food Chem. 2015 Dec 15;189:27-37. doi: 10.1016/j.foodchem.2015.02.050. Epub

2015
Feb 27.
Exposure assessment of cattle via roughages to plants producing compounds of

concern.
van Raamsdonk LW(1), Ozinga WA(2), Hoogenboom LA(3), Mulder PP(3), Mol JG(3),
Groot MJ(3), van der Fels-Klerx HJ(3), de Nijs M(3).
Author information:
(1)RIKILT, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands.
Electronic address: leo.vanraamsdonk@wur.nl.
(2)Alterra, Wageningen UR, P.O. Box 47, 6700 AA Wageningen, The Netherlands;
Radboud University, Nijmegen, The Netherlands.
(3)RIKILT, Wageningen UR, P.O. Box 230, 6700 AE Wageningen, The Netherlands.
Food producing animals are exposed to biologically active plant compounds through
feed and roughages, presenting a potential risk to the animal but also consumers
of food of animal origin. To evaluate to which plant compounds of concern dairy
cows in the Netherlands are exposed, a ranking filter model was developed,
combining information on abundance of plant species in vegetation plots in the
Netherlands (183,905 plots of three different vegetation types) with
plant-compound combinations (700), and with consumption data of fresh grass,
grass silage and corn silage by cattle. The most abundant plant genera are those
producing cyanogenic glycosides, coumarins and benzofuranocoumarins, being
predominantly fodder plants (alfalfa, clover and some grasses) considered to be
safe. Highest exposures were estimated for plant genera producing piperidine
alkaloids (horsetail), furanocoumarins (parsley and relatives), pyrrolizidine
alkaloids (Symphytum, Senecio, Leucanthemum, Eupatorium) and essential oils. The
current results allow to prioritise future scientific research on these
compounds.
DOI: 10.1016/j.foodchem.2015.02.050
448. Environ Toxicol Chem. 2016 Jan;35(1):144-51. doi: 10.1002/etc.3169. Epub 2015
Nov
24.
Effects of the herbicide dicamba on nontarget plants and pollinator visitation.

Bohnenblust EW(1), Vaudo AD(1), Egan JF(2), Mortensen DA(3), Tooker JF(1).
Author information:
(1)Department of Entomology, The Pennsylvania State University, University Park,
Pennsylvania, USA.
(2)Pasture Systems and Watershed Management Research Unit, USDA Agricultural
Research Service, University Park, Pennsylvania, USA.
(3)Department of Plant Science, The Pennsylvania State University, University
Park, Pennsylvania, USA.
Nearly 80% of all pesticides applied to row crops are herbicides, and these
applications pose potentially significant ecotoxicological risks to nontarget
plants and associated pollinators. In response to the widespread occurrence of
weed species resistant to glyphosate, biotechnology companies have developed
crops resistant to the synthetic-auxin herbicides dicamba and
2,4-dichlorophenoxyacetic acid (2,4-D); and once commercialized, adoption of
these crops is likely to change herbicide-use patterns. Despite current limited
use, dicamba and 2,4-D are often responsible for injury to nontarget plants; but
effects of these herbicides on insect communities are poorly understood. To
understand the influence of dicamba on pollinators, the authors applied several
sublethal, drift-level rates of dicamba to alfalfa (Medicago sativa L.) and
Eupatorium perfoliatum L. and evaluated plant flowering and floral visitation by
pollinators. The authors found that dicamba doses simulating particle drift (≈1%
of the field application rate) delayed onset of flowering and reduced the number
of flowers of each plant species; however, plants that did flower produced
similar-quality pollen in terms of protein concentrations. Further, plants
affected by particle drift rates were visited less often by pollinators. Because
plants exposed to sublethal levels of dicamba may produce fewer floral resources
and be less frequently visited by pollinators, use of dicamba or other
synthetic-auxin herbicides with widespread planting of herbicide-resistant crops
will need to be carefully stewarded to prevent potential disturbances of plant
and beneficial insect communities in agricultural landscapes.
© 2015 SETAC.
DOI: 10.1002/etc.3169
449. Environ Sci Pollut Res Int. 2016 Feb;23(4):3120-35. doi:

10.1007/s11356-015-4984-7. Epub 2015 Jul 16.
Plant responses to a phytomanaged urban technosol contaminated by trace elements

and polycyclic aromatic hydrocarbons.
Marchand L(1)(2)(3), Sabaris CQ(4), Desjardins D(5), Oustrière N(6)(7), Pesme

E(8), Butin D(8), Wicart G(8), Mench M(6)(7).
Author information:
(1)INRA, UMR 1202 BIOGECO, 69 route d'Arcachon, FR-33612, Cestas cedex, France.
lilian.marchand@hotmail.fr.
(2)University of Bordeaux, UMR 1202 BIOGECO, Bat B2, Allée Geoffroy St-Hilaire,
CS50023, FR-33615, Pessac cedex, France. lilian.marchand@hotmail.fr.
(3)Mairie de BORDEAUX, Service Aménagements Paysagers, Direction des Parcs, des
Jardins et des Rives, 77 Boulevard Alfred Daney, 33000, Bordeaux, France.
lilian.marchand@hotmail.fr.
(4)Departamento Biología Vegetal y Ecología, Facultad de Ciencia y Tecnologia,
Universidad del País Vasco/EHU, 48080, Bilbao, Spain.
(5)Institut de Recherche en Biologie Végétale (IRBV), Université de
Montréal-Jardin Botanique de Montréal, 4101 Rue Sherbrooke, Est Montréal, QC, H1X
2B2, Canada.
(6)INRA, UMR 1202 BIOGECO, 69 route d'Arcachon, FR-33612, Cestas cedex, France.
(7)University of Bordeaux, UMR 1202 BIOGECO, Bat B2, Allée Geoffroy St-Hilaire,
CS50023, FR-33615, Pessac cedex, France.
(8)Mairie de BORDEAUX, Service Aménagements Paysagers, Direction des Parcs, des
Jardins et des Rives, 77 Boulevard Alfred Daney, 33000, Bordeaux, France.
Medicago sativa was cultivated at a former harbor facility near Bordeaux (France)
to phytomanage a soil contaminated by trace elements (TE) and polycyclic aromatic
hydrocarbons (PAH). In parallel, a biotest with Phaseolus vulgaris was carried
out on potted soils from 18 sub-sites to assess their phytotoxicity. Total soil
TE and PAH concentrations, TE concentrations in the soil pore water, the foliar
ionome of M. sativa (at the end of the first growth season) and of Populus nigra
growing in situ, the root and shoot biomass and the foliar ionome of P. vulgaris
were determined. Despite high total soil TE, soluble TE concentrations were
generally low, mainly due to alkaline soil pH (7.8-8.6). Shoot dry weight (DW)
yield and foliar ionome of P. vulgaris did not reflect the soil contamination,
but its root DW yield decreased at highest soil TE and/or PAH concentrations.
Foliar ionomes of M. sativa and P. nigra growing in situ were generally similar
to the ones at uncontaminated sites. M. sativa contributed to bioavailable TE
stripping by shoot removal (in g ha(-1) harvest(-1)): As 0.9, Cd 0.3, Cr 0.4, Cu
16.1, Ni 2.6, Pb 4, and Zn 134. After 1 year, 72 plant species were identified in
the plant community across three subsets: (I) plant community developed on bare
soil sowed with M. sativa; (II) plant community developed in unharvested plots
dominated by grasses; and (III) plant community developed on unsowed bare soil.
The shoot DW yield (in mg ha(-1) harvest(-1)) varied from 1.1 (subset I) to 6.9
(subset II). For subset III, the specific richness was the lowest in plots with
the highest phytotoxicity for P. vulgaris.
DOI: 10.1007/s11356-015-4984-7
450. Sci Rep. 2015 Jul 13;5:12130. doi: 10.1038/srep12130.
Cutting improves the productivity of lucerne-rich stands used in the revegetation

of degraded arable land in a semi-arid environment.
Yuan ZQ(1), Yu KL(2), Wang BX(1), Zhang WY(1), Zhang XL(1), Siddique KH(3),
Stefanova K(3), Turner NC(4), Li FM(1).
Author information:
AgroEcology, School of Life Sciences, Lanzhou University, Lanzhou, Gansu 730000,
China.
(2)Department of Environmental Sciences, University of Virginia, Charlottesville,
Virginia 22904, USA.
(3)The UWA Institute of Agriculture, The University of Western Australia, 35
Stirling Highway, Crawley, WA 6009, Australia.
(4)1] The UWA Institute of Agriculture, The University of Western Australia, 35
Stirling Highway, Crawley, WA 6009, Australia [2] Centre for Plant Genetics and
Breeding, School of Plant Biology, The University of Western Australia, M080, 35
Stirling Highway, Crawley, WA 6009, Australia.
Understanding the relationships between vegetative and environmental variables is

important for revegetation and ecosystem management on the Loess Plateau, China.
Lucerne (Medicago sativa L.) has been widely used in the region to improve
revegetation, soil and water conservation, and to enhance livestock production.
However, there is little information on how environmental factors influence
long-term succession in lucerne-rich vegetation. Our objective was to identify
the main environmental variables controlling the succession process in
lucerne-rich vegetation such that native species are not suppressed after sowing
on the Loess Plateau. Vegetation and soil surveys were performed in 31 lucerne
fields (three lucerne fields without any management from 2003-2013 and 28 fields
containing 11-year-old lucerne with one cutting each year). Time after planting
was the most important factor affecting plant species succession. Cutting
significantly affected revegetation characteristics, such as aboveground biomass,
plant density and diversity. Soil moisture content, soil organic carbon, soil
available phosphorus and slope aspect were key environmental factors affecting
plant species composition and aboveground biomass, density and diversity.
Long-term cutting can cause self-thinning in lucerne, maintain the stability of
lucerne production and slow its degradation. For effective management of lucerne
fields, phosphate fertilizer should be applied and cutting performed.
DOI: 10.1038/srep12130
PMCID: PMC4499809
451. J Dairy Sci. 2015 Sep;98(9):6291-303. doi: 10.3168/jds.2014-8662. Epub 2015

Jul
8.
Effects of varying dietary ratios of corn silage to alfalfa silage on digestion

of neutral detergent fiber in lactating dairy cows.
Lopes F(1), Cook DE(1), Combs DK(2).
Author information:
Electronic address: dkcombs@wisc.edu.
An in vivo study was performed to test an in vitro procedure and model that
predicts total-tract neutral detergent fiber (NDF) digestibility for lactating
dairy cattle. Corn silage (CS) and alfalfa silage (AS) were used as forages for
this study. These forages had similar NDF composition, but fiber in the CS
contained less indigestible NDF compared with AS (35.5 and 47.8% of indigestible
NDF, respectively). The in vitro method estimated rate of digestion of alfalfa
potentially digestible NDF to be approximately 2 times faster than CS fiber (6.11
and 3.21%/h, respectively). Four diets were formulated containing different
proportions of CS to AS: 100CS:0AS, 67CS:33AS, 33CS:67AS, and 0CS:100AS, as
percentage of diet DM basis. The objective was to construct diets that contained
approximately similar levels of NDF but with different pool sizes and rates of
digestion of potentially digestible NDF. Diets were fed to 8 ruminally
cannulated, multiparous, lactating dairy cows in a replicated 4×4 Latin square
with 21-d periods. Total-tract fiber digestibility and fiber digestion kinetic
parameters observed in vivo were compared with the values predicted by the in
vitro assay and model. Total-tract NDF digestibility coefficients were similar
(41.8 and 40.6% of total NDF) for the in vitro and in vivo methods, respectively.
As the proportion of dietary alfalfa increased, the digestibility of NDF
increased. The rate of digestion of potentially digestible NDF predicted from the
in vitro assay was also similar to what was observed in vivo. Results suggest
that the in vitro total-tract NDF digestibility model could be used to predict
rate of fiber digestion and NDF digestibility for lactating dairy cattle.
DOI: 10.3168/jds.2014-8662

Epub 2015 Jul 10.
Partial replacements of Stylosanthes scabra forage for lucerne in total mixed

ration diet of Saanen goats.
Mpanza TD(1), Hassen A(2).
Author information:
(1)Faculty of Natural and Agricultural Sciences, Department of Animal and
Wildlife Sciences, University of Pretoria, Pretoria, 0002, South Africa.
thamidempanza@webmail.co.za.
(2)Faculty of Natural and Agricultural Sciences, Department of Animal and
Wildlife Sciences, University of Pretoria, Pretoria, 0002, South Africa.
Abubeker.Hassen@up.ac.za.
The inclusion of Stylosanthes scabra cv. Seca forage in the total mixed ration
(TMR) as partial replacement of lucerne (alfalfa) was evaluated for its effects
on voluntary feed intake, nutrient digestibility and nitrogen balance in Saanen
goats. Three experimental diets were formulated having 0 % Seca (T1), 15 % Seca
(T2) and 30 % Seca (T3) as partial replacement of lucerne forage in the TMR diet
for goats. Eighteen Saanen goats of about 7 months old were divided into three
groups of six animals per group. Each group was randomly assigned to one of the
three dietary treatments in a complete randomised design, and the study lasted
for a period of 21 days. There was an increase in fibre and mineral content of
the diets as Seca inclusion increased, but this resulted in the decrease of crude
protein contents and in vitro organic matter digestibility. Animals that were fed
15 % Seca recorded higher voluntary dry matter and nutrient (organic matter and
fibres) intake, but the difference was not statistically significant (P > 0.05)
as compared to the other treatments. Nutrient digestibility as well as nitrogen
balance was not significantly different across the three diets. The lack of
significant differences in feed intake, nutrient digestibility and nitrogen
utilisation following the inclusion of Seca in the TMR suggests that S. scabra
forage can partially replace lucerne in the TMR diet of goats.
DOI: 10.1007/s11250-015-0876-6
453. Glob J Health Sci. 2015 Apr 23;7(7 Spec No):137-42. doi:
10.5539/gjhs.v7n7p137.
The Effects of Medicago Sativa and Allium Porrum on Iron Overload in Rats.
Mirzaei A(1), Delaviz H, Mirzaei M, Tolooei M.
Author information:
(1). mirzaee3a2003@yahoo.com.
PURPOSE: Iron overload may occur due to regular blood transfusions and high
intestinal iron absorption. Currently, there is no effective drug without side
effects for the treatment of iron excess in thalassemia and other iron storage
diseases, except chelation therapy, which is the only safe method for iron
excretion. Thus, scientists are more focused on medicinal plants rich in
phytochemical compounds for the removal of iron in thalassemia. Therefore this
study was managed to discover the therapeutic potential of hydro-alcoholic
extract of Allium porrum and Medicago sativa for iron chelating potential.
METHODS: Aerial parts of Allium porrum and Medicago sativa were collected in
Yasuj Iran. Rats were divided into seven groups each containing six. Extracts
were administrated in four groups (two groups for each extract) by single doses
of each plant with 200 and 400 mg/kg body weight by (i.p.) route every other day
for 28 days. Group 1 as negative control received saline (0.5 ml/kg) by (i.p.)
route. Positive control received iron dextran 200 mg/kg body weight. Experimental
groups 1 and 2 for each plant extract were fed with 200 and 400 mg/kg,
hydro-alcoholic extract respectively via (i.p.) route, 1 h after the injection of
iron dextran. Standard group was treated with deferoxamine (DF) 50 mg/kg by
(i.p.) route 1 h after the injection of iron dextran. Serum iron (SI) and serum
total iron binding capacity (TIBC) were determined .The serum ferritin was then
measured using enzyme immunoassay ELISA kit for rat. For Analysis of data ANOVA
test was used.
RESULTS: Hydro-alcoholic extract of Medicago sativa and Allium porrum at 400
mg/kg showed significant (p<0.05) iron chelating activity compared to control.
The plant extracts with dose 200 mg/kg also reduced the iron and ferritin content
but the effect was lower level compared to higher doses. The plant extract
effects were similar to that of standard drug deferoxamine. Iron and ferritin
levels were significantly reduced in experimental groups when compared to
positive group especially in Medicago sativap<0.05. There was no difference
between standard drugs and last concentration of plant extracts.
CONCLUSION: Protective effect of M. sativa and A. Porrum against iron overload in
rat model was reported. Significant decrease in serum ferritin and iron
concentration was reported in iron overload rats which induced by iron dextran.
DOI: 10.5539/gjhs.v7n7p137
PMCID: PMC4803991
454. BMC Genomics. 2015 Jul 7;16:502. doi: 10.1186/s12864-015-1718-7.
The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for
investigating expression differences between Medicago sativa subspecies.
O'Rourke JA(1), Fu F(2), Bucciarelli B(3), Yang SS(4)(5), Samac DA(6), Lamb
JF(7), Monteros MJ(8), Graham MA(9), Gronwald JW(10), Krom N(11), Li J(12), Dai
X(13), Zhao PX(14), Vance CP(15)(16).
Author information:
(1)USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA, 50011, USA.
Jamie.ORourke@ars.usda.gov.
MN, 55108, USA. fuxxx026@umn.edu.
Bruna.Bucciarelli@ars.usda.gov.
yangsh38@hotmail.com.
(5)Present Address: Monsanto Company, Molecular Breeding Technology,
Chesterfield, MO, 63167, USA. yangsh38@hotmail.com.
Debby.Samac@ars.usda.gov.
JoAnn.Lamb@ars.usda.gov.
(8)Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA.
mjmonteros@noble.org.
(9)USDA-ARS, Corn Insects and Crop Genetics Research Unit, Ames, IA, 50011, USA.
michelle.graham@ars.usda.gov.
gronw001@umn.edu.
(11)Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA. ndkrom@noble.org.
(12)Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA. jli@noble.org.
(13)Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA. xdai@noble.org.
(14)Samuel Roberts Noble Foundation, Ardmore, OK, 73401, USA. pzhao@noble.org.
MN, 55108, USA. vance004@umn.edu.
vance004@umn.edu.
BACKGROUND: Alfalfa (Medicago sativa L.) is the primary forage legume crop
species in the United States and plays essential economic and ecological roles in
agricultural systems across the country. Modern alfalfa is the result of
hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp.
falcata. Due to its large and complex genome, there are few genomic resources
available for alfalfa improvement.
RESULTS: A de novo transcriptome assembly from two alfalfa subspecies, M. sativa
ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina
RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves,
flowers, elongating stem internodes, and post-elongation stem internodes were
assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626
unique transcript sequences. Nodule-specific and transcripts involved in cell
wall biosynthesis were identified. Statistical analyses identified 20,447
transcripts differentially expressed between the two subspecies. Pair-wise
comparisons of each tissue combination identified 58,932 sequences differentially
expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing
transcript abundance in floral tissues of B47 and F56 identified expression
differences in sequences involved in anthocyanin and carotenoid synthesis, which
determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to
each M. sativa subspecies (110,241) were identified.
CONCLUSIONS: The Medicago sativa Gene Index 1.2 increases the expressed sequence
data available for alfalfa by ninefold and can be expanded as additional
experiments are performed. The MSGI 1.2 transcriptome sequences, annotations,
expression profiles, and SNPs were assembled into the Alfalfa Gene Index and
Expression Database (AGED) at http://plantgrn.noble.org/AGED/ , a publicly
available genomic resource for alfalfa improvement and legume research.
DOI: 10.1186/s12864-015-1718-7
PMCID: PMC4492073
455. Bioresour Technol. 2015 Oct;193:288-96. doi: 10.1016/j.biortech.2015.06.096.

Epub
2015 Jun 25.
Mechanism of improved cellulosic bio-ethanol production from alfalfa stems via

ambient-temperature acid pretreatment.
Zhou S(1), Runge TM(2).
Author information:
(1)Department of Biological System Engineering, University of Wisconsin-Madison,
Agricultural Engineering Building, Madison, WI 53706, USA.
(2)Department of Biological System Engineering, University of Wisconsin-Madison,
Agricultural Engineering Building, Madison, WI 53706, USA. Electronic address:
trunge@wisc.edu.
Model compounds and recalcitrant biomass were studied to elucidate the mechanism
of ambient-temperature acid pretreatment of cellulosic biomass for bio-ethanol
production. Pure cellulose, a pure hemicellulose and alfalfa stems were
pretreated with sulfuric acid under ambient temperature with varied acid loading
and time. Changes in water-soluble carbohydrates (WSCs) and chemical components
of substrates were determined, and ethanol production via simultaneous
saccharification and fermentation (SSF) was studied. The results showed
significant amount of WSCs formed, and the WSCs increased with increasing acid
loading and pretreatment time. The ethanol yields from pure cellulose were
primarily affected by the added ash. Acid loading showed significant positive
effect on ethanol production from alfalfa stems, whereas pretreatment time showed
much weaker positive effect. However, non-significant amounts of WSCs were
removed by washing of dried substrates. It was hypothesized to be because the
WSCs adsorbed onto bulk substrates during the freeze-drying step, as supported by
experimental results.
DOI: 10.1016/j.biortech.2015.06.096
456. J Environ Sci (China). 2015 Jul 1;33:245-53. doi: 10.1016/j.jes.2014.12.013.

Epub
2015 Apr 2.
Effect of petroleum on decomposition of shrub-grass litters in soil in Northern

Shaanxi of China.
Zhang X(1), Liu Z(2), Yu Q(3), Luc NT(4), Bing Y(3), Zhu B(5), Wang W(5).
Author information:
(1)Institute of Soil and Water Conservation, Northwest A&F University, Yangling
712100, China. Electronic address: zhangxiaoxi712100@gmail.com.
(2)College of Natural Resources and Environment, Northwest A&F University,
Yangling 712100, China; Key Laboratory of Plant Nutrition and the
Agri-environment in Northwest China, Ministry of Agriculture, Yangling 712100,
China. Electronic address: zengwenliu2003@aliyun.com.
(3)College of Forestry, Northwest A&F University, Yangling 712100, China.
712100, China; Department of Agriculture and Rural Development of Lao Cai, Lao
Cai City 330100, Vietnam.
(5)College of Natural Resources and Environment, Northwest A&F University,
Yangling 712100, China.
The impacts of petroleum contamination on the litter decomposition of shrub-grass

land would directly influence nutrient cycling, and the stability and function of
ecosystem. Ten common shrub and grass species from Yujiaping oil deposits were
studied. Litters from these species were placed into litterbags and buried in
petroleum-contaminated soil with 3 levels of contamination (slight, moderate and
serious pollution with petroleum concentrations of 15, 30 and 45 g/kg,
respectively). A decomposition experiment was then conducted in the lab to
investigate the impacts of petroleum contamination on litter decomposition rates.
Slight pollution did not inhibit the decomposition of any litters and
significantly promoted the litter decomposition of Hippophae rhamnoides, Caragana
korshinskii, Amorpha fruticosa, Ziziphus jujuba var. spinosa, Periploca sepium,
Medicago sativa and Bothriochloa ischaemum. Moderate pollution significantly
inhibited litter decomposition of M. sativa, Coronilla varia, Artemisia vestita
and Trrifolium repens and significantly promoted the litter decomposition of C.
korshinskii, Z. jujuba var. spinosa and P. sepium. Serious pollution
significantly inhibited the litter decomposition of H. rhamnoides, A. fruticosa,
B. ischaemum and A. vestita and significantly promoted the litter decomposition
of Z. jujuba var. spinosa, P. sepium and M. sativa. In addition, the impacts of
petroleum contamination did not exhibit a uniform increase or decrease as
petroleum concentration increased. Inhibitory effects of petroleum on litter
decomposition may hinder the substance cycling and result in the degradation of
plant communities in contaminated areas.
DOI: 10.1016/j.jes.2014.12.013

eCollection 2015.
Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid

Alfalfa by 454 Sequencing.
Rocher S(1), Jean M(2), Castonguay Y(1), Belzile F(2).
Author information:
(1)Centre de Recherche et de Développement sur les Sols et les Grandes Cultures,
Agriculture et agroalimentaire Canada, Quebec City (QC), Canada.
(2)Département de Phytologie and Institut de Biologie Intégrative et des
Systèmes, Université Laval, Quebec City (QC), Canada.
Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput

genotyping technology based on next generation sequencing and is applicable to
orphan species with no reference genome. A combination of genome complexity
reduction and multiplexing with DNA barcoding provides a simple and affordable
way to resolve allelic variation between plant samples or populations. GBS was
performed on ApeKI libraries using DNA from 48 genotypes each of two
heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa):
the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained
after five cycles of recurrent selection for superior tolerance to freezing (TF).
Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000
sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK)
pipeline designed for species with no reference genome. Following the application
of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci
were obtained. About 60% had a significant match on the Medicago truncatula
syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS
data was directly assessed using 454 sequencing on a subset of SNP loci scored in
eight plant samples. Sequencing depth in this study was not sufficient for
accurate tetraploid allelic dosage, but reliable genotype calls based on diploid
allelic dosage were obtained when using additional quality filtering. Principal
Component Analysis of SNP loci in plant samples revealed that a small proportion
(<5%) of the genetic variability assessed by GBS is able to differentiate ATF0
and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable
approach for genome-wide discovery of SNP loci in outcrossed polyploids.
PMCID: PMC4482585
458. J Anim Sci. 2015 Jun;93(6):3044-54. doi: 10.2527/jas.2014-8758.
Effect of vitamin E supplementation or alfalfa grazing on fatty acid composition

and expression of genes related to lipid metabolism in lambs.
González-Calvo L, Joy M, Blanco M, Dervishi E, Molino F, Sarto P, Ripoll G,

Serrano M, Calvo JH.
The aim of this study was to investigate the effects of vitamin E (VE)
supplementation and alfalfa grazing during fattening on fatty acid composition
and mRNA expression of genes related to lipid metabolism in the LM of Rasa
Aragonesa light lambs. After weaning, 48 lambs were kept indoors and fed a
commercial concentrate and a VE supplemented concentrate (480 mg DL-α-tocopheryl
acetate/kg DM) for 0 (control [CON]), 10 (VE10d), 20 (VE20d), and 30 d (VE30d)
before slaughtering at 22 to 24 kg. Simultaneously, 8 unweaned lambs grazed in
alfalfa (154 mg α-tocopherol/kg DM) paddocks with their dams and supplemented
with the commercial concentrate (ALF). Immediately after slaughter, LM was
sampled to determine gene expression. After 24 h of cooling at 4°C, LM was
extracted to determine intramuscular fat (IMF) content and fatty acid
composition. The IMF content did not differ with the dietary treatment ( =
0.212). Unweaned grazing alfalfa lambs had greater concentration of rumenic acid
(C18:2 c9,t11; P < 0.001) and lower oleic acid (C18:1 c9; = 0.001) content and
PUFA n-6:n-3 ratio (P < 0.001) but similar expression of genes implicated in
lipid metabolism compared to the concentrate-fed lambs. Vitamin E supplementation
did not modify muscle fatty acid composition; however, it increased the
expression of FADS2 and ELOVL6, which are involved in desaturation of long-chain
fatty acid and the elongation of SFA and MUFA. The results showed that a short
period of VE supplementation, especially 10 (VE10d) and 20 d (VE20d), modified
gene expression. Overall, the results showed that VE may be acting as a
regulatory factor for transcriptional control of genes related to lipid
metabolism in the muscle of Rasa Aragonesa light lambs (22-24 kg live weight and
younger than 90 d old).
DOI: 10.2527/jas.2014-8758
459. Biomed Res Int. 2015;2015:202597. doi: 10.1155/2015/202597. Epub 2015 May 31.
Biochemical and Molecular Phylogenetic Study of Agriculturally Useful Association

of a Nitrogen-Fixing Cyanobacterium and Nodule Sinorhizobium with Medicago sativa
L.
Karaushu EV(1), Lazebnaya IV(2), Kravzova TR(3), Vorobey NA(4), Lazebny OE(5),
Kiriziy DA(4), Olkhovich OP(1), Taran NY(1), Kots SY(4), Popova AA(6), Omarova
E(7), Koksharova OA(8).
Author information:
(1)Educational and Scientific "Institute of Biology", Taras Shevchenko National
University of Kyiv, 64/13 Volodymyrska Street, Kyiv 01601, Ukraine.
(2)N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences,
Gubkin Street 3, Moscow 119333, Russia.
(3)Lomonosov Moscow State University, Biocenter, Leninskie Gory 1-12, Moscow
119991, Russia.
(4)Institute of Plant Physiology and Genetics, National Academy of Sciences of
Ukraine, 31/17 Vasylkivska Street, Kyiv 03022, Ukraine.
(5)N. K. Kol'tsov Institute of Developmental Biology, Russian Academy of
Sciences, Vavilova Street 26, Moscow 119334, Russia.
(6)Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182,
Russia.
(7)Lomonosov Moscow State University, Belozersky Institute of Physical-Chemical
Biology, Leninskie Gory 1-40, Moscow 119992, Russia.
(8)Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182,
Russia ; Lomonosov Moscow State University, Belozersky Institute of
Physical-Chemical Biology, Leninskie Gory 1-40, Moscow 119992, Russia.
Seed inoculation with bacterial consortium was found to increase legume yield,
providing a higher growth than the standard nitrogen treatment methods. Alfalfa
plants were inoculated by mono- and binary compositions of nitrogen-fixing
microorganisms. Their physiological and biochemical properties were estimated.
Inoculation by microbial consortium of Sinorhizobium meliloti T17 together with a
new cyanobacterial isolate Nostoc PTV was more efficient than the
single-rhizobium strain inoculation. This treatment provides an intensification
of the processes of biological nitrogen fixation by rhizobia bacteria in the root
nodules and an intensification of plant photosynthesis. Inoculation by bacterial
consortium stimulates growth of plant mass and rhizogenesis and leads to
increased productivity of alfalfa and to improving the amino acid composition of
plant leaves. The full nucleotide sequence of the rRNA gene cluster and partial
sequence of the dinitrogenase reductase (nifH) gene of Nostoc PTV were deposited
to GenBank (JQ259185.1, JQ259186.1). Comparison of these gene sequences of Nostoc
PTV with all sequences present at the GenBank shows that this cyanobacterial
strain does not have 100% identity with any organisms investigated previously.
Phylogenetic analysis showed that this cyanobacterium clustered with high
credibility values with Nostoc muscorum.
DOI: 10.1155/2015/202597
PMCID: PMC4465650
460. Environ Sci Technol. 2015 Jul 21;49(14):8451-60. doi: 10.1021/acs.est.5b01147.

Epub 2015 Jul 13.
Speciation Matters: Bioavailability of Silver and Silver Sulfide Nanoparticles to

Alfalfa (Medicago sativa).
Stegemeier JP, Schwab F, Colman BP(1), Webb SM(2), Newville M(3), Lanzirotti
A(3), Winkler C(4), Wiesner MR, Lowry GV.
Author information:
(1)∥Department of Biology, Duke University, Durham, North Carolina 27708, United
States.
(2)⊥Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park,
California 94025, United States.
(3)#Center for Advanced Radiation Sources, The University of Chicago, Chicago,
Illinois 60637, United States.
(4)⊗ICTAS Nanoscale Characterization and Fabrication Laboratory, Virginia Tech,
Blacksburg, Virginia 24061, United States.
Terrestrial crops are directly exposed to silver nanoparticles (Ag-NPs) and their
environmentally transformed analog silver sulfide nanoparticles (Ag2S-NPs) when
wastewater treatment biosolids are applied as fertilizer to agricultural soils.
This leads to a need to understand their bioavailability to plants. In the
present study, the mechanisms of uptake and distribution of silver in alfalfa
(Medicago sativa) were quantified and visualized upon hydroponic exposure to
Ag-NPs, Ag2S-NPs, and AgNO3 at 3 mg total Ag/L. Total silver uptake was measured
in dried roots and shoots, and the spatial distribution of elements was
investigated using transmission electron microscopy (TEM) and synchrotron-based
X-ray imaging techniques. Despite large differences in release of Ag(+) ions from
the particles, Ag-NPs, Ag2S-NPs, and Ag(+) became associated with plant roots to
a similar degree, and exhibited similarly limited (<1%) amounts of translocation
of silver into the shoot system. X-ray fluorescence (XRF) mapping revealed
differences in the distribution of Ag into roots for each treatment. Silver
nanoparticles mainly accumulated in the (columella) border cells and elongation
zone, whereas Ag(+) accumulated more uniformly throughout the root. In contrast,
Ag2S-NPs remained largely adhered to the root exterior, and the presence of
cytoplasmic nano-SixOy aggregates was observed. Exclusively in roots exposed to
particulate silver, NPs smaller than the originally dosed NPs were identified by
TEM in the cell walls. The apparent accumulation of Ag in the root apoplast
determined by XRF, and the presence of small NPs in root cell walls suggests
uptake of partially dissolved NPs and translocation along the apoplast.
DOI: 10.1021/acs.est.5b01147
461. Theor Appl Genet. 2015 Sep;128(9):1865-79. doi: 10.1007/s00122-015-2553-6.

Epub
2015 Jun 24.
A simple model for pollen-parent fecundity distributions in bee-pollinated forage

legume polycrosses.
Riday H(1), Smith MA, Peel MD.
Author information:
(1)U.S. Dairy Forage Research Center, USDA-ARS, Madison, WI, 53706, USA,
heathcliffe.riday@ars.usda.gov.
A simple Weibull distribution based empirical model that predicts pollen-parent

fecundity distributions based on polycross size alone has been developed in
outbred forage legume species for incorporation into quantitative genetic theory.
Random mating or panmixis is a fundamental assumption in quantitative genetic
theory. Random mating is sometimes thought to occur in actual fact, although a
large body of empirical work shows that this is often not the case in nature.
Models have been developed to explain many non-random mating phenomena. This
paper measured pollen-parent fecundity distributions among outbred perennial
forage legume species [autotetraploid alfalfa (Medicago sativa L.), autohexaploid
kura clover (Trifolium ambiguum M. Bieb.), and diploid red clover (Trifolium
pratense L.)] in ten polycrosses ranging in size (N) from 9 to 94 pollinated with
bee pollinators [Bumble Bees (Bombus impatiens Cr.) and leafcutter bees
(Megachile rotundata F.)]. A Weibull distribution best fit the observed
pollen-parent fecundity distributions. After standardizing data among the 10
polycrosses, a single Weibull distribution-based model was obtained with an R (2)
of 0.978. The model is able to predict pollen-parent fecundity distributions
based on polycross size alone. The model predicts that the effective polycross
size will be approximately 9 % smaller than under random mating (i.e., N e/N ~
0.91). The model is simple and can easily be incorporated into other models or
simulations requiring a pollen-parent fecundity distribution. Further work is
needed to determine how widely applicable the model is.
DOI: 10.1007/s00122-015-2553-6
462. J Dairy Sci. 2015 Aug;98(8):5672-87. doi: 10.3168/jds.2015-9563.
Replacing dietary soybean meal with canola meal improves production and
efficiency of lactating dairy cows.
Broderick GA(1), Faciola AP(2), Armentano LE(3).
Author information:
(1)Agricultural Research Service, USDA, US Dairy Forage Research Center, 1925
Linden Drive, Madison, WI 53706. Electronic address: gbroderi@wisc.edu.
(2)Department of Agriculture, Nutrition, and Veterinary Sciences, 1664 North
Virginia Street, University of Nevada, Reno 89557.
(3)Department of Dairy Science, 1675 Observatory Drive, University of Wisconsin,
Madison 53706.
Previous research suggested that crude protein (CP) from canola meal (CM) was
used more efficiently than CP from solvent soybean meal (SBM) by lactating dairy
cows. We tested whether dietary CP content influenced relative effectiveness of
equal supplemental CP from either CM or SBM. Fifty lactating Holstein cows were
blocked by parity and days in milk into 10 squares (2 squares with ruminal
cannulas) in a replicated 5×5 Latin square trial. Five squares were fed: (1) low
(14.5-14.8%) CP with SBM, (2) low CP with CM, (3) low CP with SBM plus CM, (4)
high (16.4-16.7%) CP with SBM, and (5) high CP with CM; the other 5 squares were
fed the same diets except with rumen-protected Met plus Lys (RPML) added as
Mepron (Degussa Corp., Kennesaw, GA) and AminoShure-L (Balchem Corp., New
Hampton, NY), which were assumed to provide 8g/d of absorbed dl-Met and 12g/d of
absorbed l-Lys. Diets contained [dry matter (DM) basis] 40% corn silage, 26%
alfalfa silage, 14 to 23% corn grain, 2.4% mineral-vitamin premixes, and 29 to
33% neutral detergent fiber. Periods were 3wk (total 15wk), and data from the
last week of each period were analyzed using the Mixed procedures of SAS (SAS
Institute Inc., Cary, NC). The only effects of RPML were increased DM intake and
milk urea N (MUN) and urinary N excretion and trends for decreased milk lactose
and solids-not-fat concentrations and milk-N:N intake; no significant RPML ×
protein source interactions were detected. Higher dietary CP increased milk fat
yield and tended to increase milk yield but also elevated MUN, urine volume,
urinary N excretion, ruminal concentrations of ammonia and branched-chain
volatile fatty acids (VFA), lowered milk lactose concentration and milk-N:N
intake, and had no effect on milk true protein yield. Feeding CM instead of SBM
increased feed intake, yields of milk, energy-corrected milk, and true protein,
and milk-N:N intake, tended to increase fat and lactose yields, and reduced MUN,
urine volume, and urinary N excretion. At low CP, MUN was lower and intake tended
to be greater on SBM plus CM versus SBM alone, but MUN and N excretion were not
reduced to the same degree as on CM alone. Interactions of parity × protein
source and parity × CP concentration indicated that primiparous cows were more
responsive than multiparous cows to improved supply of metabolizable protein.
Replacing SBM with CM reduced ruminal ammonia and branched-chain VFA
concentrations, indicating lower ruminal degradation of CM protein. Replacing SBM
with CM improved milk and protein yield and N-utilization in lactating cows fed
both low- and high-CP diets.

DOI: 10.3168/jds.2015-9563
463. J Zhejiang Univ Sci B. 2015 Jun;16(6):479-86. doi: 10.1631/jzus.B1400343.

Effects of alfalfa saponin extract on mRNA expression of Ldlr, LXRα, and FXR in
BRL cells.
Liang XP(1), Zhang DQ, Chen YY, Guo R, Wang J, Wang CZ, Shi YH.
Author information:
University, Zhengzhou 450002, China.
We studied the effects of alfalfa saponin extract (ASE) on low density

lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor
(FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and
hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells
treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells
treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and
FXR mRNA expression levels were measured by quantitative real-time polymerase
chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL
cells was significantly up-regulated by ASE treatment and mRNA expressions of
LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic
BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by
regulating genes involved in cholesterol metabolism, including up-regulation of
Ldlr as well as down-regulation of LXRα and FXR.
DOI: 10.1631/jzus.B1400343
PMCID: PMC4471599
464. Planta. 2015 Oct;242(4):1009-24. doi: 10.1007/s00425-015-2342-0. Epub 2015 Jun

9.
Transgenic alfalfa (Medicago sativa) with increased sucrose phosphate synthase

activity shows enhanced growth when grown under N2-fixing conditions.
Gebril S(1), Seger M, Villanueva FM, Ortega JL, Bagga S, Sengupta-Gopalan C.
Author information:
Las Cruces, NM, 88003, USA.
MAIN CONCLUSION: Overexpression of SPS in alfalfa is accompanied by early

flowering, increased plant growth and an increase in elemental N and protein
content when grown under N2-fixing conditions. Sucrose phosphate synthase (SPS;
EC 2.3.1.14) is the key enzyme in the synthesis of sucrose in plants. The outcome
of overexpression of SPS in different plants using transgenic approaches has been
quite varied, but the general consensus is that increased SPS activity is
associated with the production of new sinks and increased sink strength. In
legumes, the root nodule is a strong C sink and in this study our objective was
to see how increasing SPS activity in a legume would affect nodule number and
function. Here we have transformed alfalfa (Medicago sativa, cv. Regen SY), with
a maize SPS gene driven by the constitutive CaMV35S promoter. Our results showed
that overexpression of SPS in alfalfa, is accompanied by an increase in nodule
number and mass and an overall increase in nitrogenase activity at the whole
plant level. The nodules exhibited an increase in the level of key enzymes
contributing to N assimilation including glutamine synthetase and asparagine
synthetase. Moreover, the stems of the transformants showed higher level of the
transport amino acids, Asx, indicating increased export of N from the nodules.
The transformants exhibited a dramatic increase in growth both of the shoots and
roots, and earlier flowering time, leading to increased yields. Moreover, the
transformants showed an increase in elemental N and protein content. The overall
conclusion is that increased SPS activity improves the N status and plant
performance, suggesting that the availability of more C in the form of sucrose
enhances N acquisition and assimilation in the nodules.
DOI: 10.1007/s00425-015-2342-0
2015.
Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various

plants.
Bekešová S(1), Komis G(1), Křenek P(1), Vyplelová P(1), Ovečka M(1), Luptovčiak
I(1), Illés P(1), Kuchařová A(1), Šamaj J(1).
Author information:
(1)Department of Cell Biology, Centre of the Region Haná for Biotechnological and
Agricultural Research, Palacký University Olomouc Olomouc, Czech Republic.
The aim of the present study is to rationalize acrylamide pendant Phos-Tag™

in-gel discrimination of phosphorylated and non-phosphorylated plant protein
species with standard immunoblot analysis, and optimize sample preparation,
efficient electrophoretic separation and transfer. We tested variants of the
method including extraction buffers suitable for preservation of phosphorylated
protein species in crude extracts from plants and we addressed the importance of
the cation (Mn(2+) or Zn(2+)) used in the gel recipe for efficient transfer to
PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of
Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in
stress-treated wild type plants and transgenic SIMKK RNAi line. We further show
the hyperosmotically-induced phosphorylation of the previously uncharacterized
HvMPK4 of barley. The method is validated using inducible phosphorylation of
barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant
Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and
Arabidopsis circumventing radioactive labeling and the use of phosphorylation
specific antibodies.
DOI: 10.3389/fpls.2015.00336
PMCID: PMC4429547
PMID: 26029234
466. J Dairy Sci. 2015 Aug;98(8):5642-52. doi: 10.3168/jds.2015-9543. Epub 2015 May
28.
Brown midrib corn shredlage in diets for high-producing dairy cows.
Vanderwerff LM(1), Ferraretto LF(1), Shaver RD(2).
Author information:
Electronic address: rdshaver@wisc.edu.
A novel method of harvesting whole-plant corn silage, shredlage, may increase

kernel processing and physically effective fiber. Improved fiber effectiveness
may be especially advantageous when feeding brown midrib (BMR) corn hybrids,
which have reduced lignin content. The objective of this study was to determine
the effect of feeding TMR containing BMR corn shredlage (SHRD) compared with BMR
conventionally processed corn silage (KP) or KP plus chopped alfalfa hay (KPH) on
intake, lactation performance, and total-tract nutrient digestibility in dairy
cows. The KP was harvested using conventional rolls (2-mm gap) and the
self-propelled forage harvester set at 19mm of theoretical length of cut, whereas
SHRD was harvested using novel cross-grooved rolls (2-mm gap) and the
self-propelled forage harvester set at 26mm of theoretical length of cut.
Holstein cows (n=120; 81±8 d in milk at trial initiation), stratified by parity,
days in milk, and milk yield, were randomly assigned to 15 pens of 8 cows each.
Pens were randomly assigned to 1 of 3 treatment diets, SHRD, KP, or KPH, in a
completely randomized design using a 2-wk covariate period with cows fed a common
diet followed by a 14-wk treatment period with cows fed their assigned treatment
diet. The TMR contained (dry matter basis) KP or SHRD forages (45%), alfalfa
silage (10%), and a concentrate mixture (45%). Hay replaced 10% of KP silage in
the KPH treatment TMR (dry matter basis). Milk, protein, and lactose yields were
3.4, 0.08, and 0.16kg/d greater, respectively, for cows fed KP and SHRD than KPH.
A week by treatment interaction was detected for milk yield, such that cows fed
SHRD produced or tended to produce 1.5kg/d per cow more milk, on average, than
cows fed KP during 6 of the 14 treatment weeks. Component-corrected milk yields
were similar among treatments. Cows fed KPH had greater milk fat concentration
than cows fed KP and SHRD (3.67 vs. 3.30% on average). Consumption of dry matter,
rumination activity, and sorting behavior were similar among treatments. Ruminal
in situ starch digestibility was greater for SHRD than KP forages, and
total-tract dietary starch digestibility was greater for SHRD than KP. Milk yield
and starch digestibility were greater for SHRD than KP. Lack of improvement in
milk fat content and rumination activity for SHRD compared with KP and reduced
milk fat content for SHRD compared with KPH, however, suggest no improvement in
physically effective fiber from the longer theoretical length of cut used with
SHRD in a BMR hybrid.

DOI: 10.3168/jds.2015-9543
467. Int J Syst Evol Microbiol. 2015 Sep;65(9):2960-7. doi: 10.1099/ijs.0.000365.

Epub
2015 May 29.
Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and
Pisum sativum.
Zhang YJ(1), Zheng WT(1), Everall I(2), Young JP(2), Zhang XX(3), Tian CF(1), Sui
XH(1), Wang ET(4), Chen WX(1).
Author information:
(1)1 State Key Lab for Agro-Biotechnology, Ministry of Agriculture Key Lab of
Soil Microbiology, College of Biological Sciences, China Agricultural University,
Beijing 100193, PR China.
(2)2 Department of Biology, University of York, York YO10 5DD, UK.
(3)3 Institute of Agricultural Resources and Regional Planning, Chinese Academy
of Agricultural Sciences, Beijing 100081, PR China.
(4)1 State Key Lab for Agro-Biotechnology, Ministry of Agriculture Key Lab of
Soil Microbiology, College of Biological Sciences, China Agricultural University,
Beijing 100193, PR China 4 Escuela Nacional de Ciencias Biológicas, Instituto
Politécnico Nacional, 11340 México D. F., Mexico.
Four rhizobia-like strains, isolated from root nodules of Pisum sativum and Vicia
faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus
Rhizobium but were distinct from all recognized species of the genus Rhizobium by
phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequences
of the housekeeping genes atpD, recA and glnII for strain CCBAU 23252(T) showed
86.9 to 95% similarity to those of known species of the genus Rhizobium. All four
strains had nodC and nifH genes and could form effective nodules with Pisum
sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did
not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or
Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA
relatedness and a series of phenotypic tests also separated these strains from
members of closely related species. Based on all the evidence, we propose a novel
species, Rhizobium anhuiense sp. nov., and designate CCBAU 23252(T) ( = CGMCC
1.12621(T) = LMG 27729(T)) as the type strain. This strain was isolated from a
root nodule of Vicia faba and has a DNA G+C content of 61.1 mol% (Tm).
DOI: 10.1099/ijs.0.000365
468. Anim Reprod Sci. 2015 Jul;158:75-85. doi: 10.1016/j.anireprosci.2015.05.001.

Epub
2015 May 12.
Reproductive performance in ewes fed varying levels of cut lucerne pasture around
conception.
Robertson SM(1), Clayton EH(2), Morgan B(3), Friend MA(4).
Author information:
(1)School of Animal and Veterinary Sciences, Charles Sturt University, Locked Bag
588, Wagga Wagga, NSW 2678, Australia; Graham Centre for Agricultural Innovation
(NSW Department of Primary Industries and Charles Sturt University), Locked Bag
588, Wagga Wagga, NSW 2678, Australia. Electronic address:
surobertson@csu.edu.au.
(2)NSW Department of Primary Industries, Wagga Wagga Agricultural Institute,
Wagga Wagga, NSW 2650, Australia; Graham Centre for Agricultural Innovation (NSW
Wagga Wagga, NSW 2678, Australia.
588, Wagga Wagga, NSW 2678, Australia.
588, Wagga Wagga, NSW 2678, Australia. Electronic address: mfriend@csu.edu.au.
Elevated intakes of protein and energy may increase embryo mortality, but it is
not clear whether fresh lucerne (Medicago sativa) pasture poses a risk. A
two-year pen study using oestrous synchronised and artificially inseminated
Merino ewes (n=175 in 2013 and 215 in 2014) evaluated whether feeding freshly cut
lucerne pasture (mean crude protein 19.7%, metabolisable energy 9.4MJ/kg DM) at
maintenance or ad libitum during different periods around insemination altered
reproductive performance in comparison with ewes fed a Control diet (mean crude
protein 7.8%, metabolisable energy 9.0MJ/kg DM) of pelleted faba bean hulls and
oat grain hulls at maintenance. The proportion of pregnant ewes carrying multiple
fetuses was reduced (P=0.026) when ewes were fed lucerne ad libitum between days
0 and 17 after insemination compared with the Control diet (0.18 and 0.34,
respectively), but not when ewes were fed lucerne ad libitum between days 0 and 7
after insemination (0.22). Reproductive performance, including the proportion of
ewes pregnant and the proportion with multiple fetuses, was not different
(P>0.05) when ewes were fed lucerne at maintenance between days 0 and 7 compared
with the Control diet. While reproductive performance was similar when ewes were
fed lucerne at maintenance between 0 and 17 days after artificial insemination
compared with pellets at maintenance, fetal numbers per pregnant ewe were reduced
by feeding lucerne ad libitum after insemination.
469. J Anim Sci. 2015 Apr;93(4):1729-40. doi: 10.2527/jas.2014-8667.
Forage choice in pasturelands: Influence on cattle foraging behavior and

performance.
Villalba JJ, Cabassu R, Gunter SA.
We determined if combinations of adjacent pastures of 3 forage species led to

complementary relationships that influenced animal behavior and performance over
monocultures. Grazing bouts, behavioral levels of activity, blood urea N (BUN),
chemical composition of feces, BW, and herbage biomass before and after grazing
were monitored when beef calves strip-grazed 3 replications of 4 treatments from
June 14 through August 23, 2013 (9 animals/treatment). Animals grazed
monocultures of: 1) tall fescue (TF), 2) alfalfa (ALF), 3) sainfoin (SAN), or 4)
a choice of strips of forages TF, ALF, and SAN (CHOICE). The lowest and greatest
incidence of foraging bouts occurred for cattle in CHOICE and SAN, respectively
(P < 0.01). Animals in CHOICE grazed SAN > ALF > TF (P < 0.01). Animals on TF and
CHOICE took greater number of steps than animals grazing a monocultures of either
legume (P = 0.01). Calves in TF had lower BUN (P < 0.01) and fecal CP
concentration (P < 0.01) than calves grazing the remaining treatments, whereas
animals in SAN showed the greatest concentrations of fecal CP (P < 0.01). Fecal
NDF concentration was the greatest for animals grazing TF and the lowest for
animals grazing SAN (P < 0.01), whereas fecal ADF concentration was greater for
animals grazing TF and SAN than for animals grazing CHOICE and ALF (P = 0.02).
Calcium, Mg, and Zn concentrations were the lowest in feces from calves grazing
TF and the greatest for calves grazing a monoculture of either legume (P < 0.05).
When averaging both periods, animals grazing SAN, ALF, or CHOICE gained more BW
than animals grazing TF (P < 0.01). Thus, calves in CHOICE incorporated tall
fescue into their diets, were more active, and displayed a lower number of
grazing bouts than calves grazing monoculture of either legume. Herbage diversity
may lead to levels of ADG comparable to legume monocultures with the potential
benefit of maintaining plant species diversity in pasturelands.
DOI: 10.2527/jas.2014-8667
470. Protoplasma. 2016 Mar;253(2):531-42. doi: 10.1007/s00709-015-0831-4. Epub 2015

May 27.
Hexavalent chromium-induced differential disruption of cortical microtubules in

some Fabaceae species is correlated with acetylation of α-tubulin.
Eleftheriou EP(1), Adamakis ID(2), Michalopoulou VA(2).
Author information:
(1)Department of Botany, School of Biology, Aristotle University of Thessaloniki,
541 24, Thessaloniki, Greece. eelefth@bio.auth.gr.
(2)Department of Botany, School of Biology, Aristotle University of Thessaloniki,
541 24, Thessaloniki, Greece.
The effects of hexavalent chromium [Cr(VI)] on the cortical microtubules (MTs) of

five species of the Fabaceae family (Vicia faba, Pisum sativum, Vigna sinensis,
Vigna angularis, and Medicago sativa) were investigated by confocal laser
scanning microscopy after immunolocalization of total tubulin with conventional
immunofluorescence techniques and of acetylated α-tubulin with the specific
6-11B-1 monoclonal antibody. Moreover, total α-tubulin and acetylated α-tubulin
were quantified by Western immunoblotting and scanning densitometry. Results
showed the universality of Cr(VI) detrimental effects to cortical MTs, which
proved to be a sensitive and reliable subcellular marker for monitoring Cr(VI)
toxicity in plant cells. However, a species-specific response was recorded, and a
correlation of MT disturbance with the acetylation status of α-tubulin was
demonstrated. In V. faba, MTs were depolymerized at the gain of cytoplasmic
tubulin background and displayed low α-tubulin acetylation, while in P. sativum,
V. sinensis, V. angularis, and M. sativa, MTs became bundled and changed
orientation from perpendicular to oblique or longitudinal. Bundled MTs were
highly acetylated as determined by both immunofluorescence and Western
immunoblotting. Tubulin acetylation in P. sativum and M. sativa preceded MT
bundling; in V. sinensis it followed MT derangement, while in V. angularis the
two phenomena coincided. Total α-tubulin remained constant in all treatments.
Should acetylation be an indicator of MT stabilization, it is deduced that
bundled MTs became stabilized, lost their dynamic properties, and were rendered
inactive. Results of this report allow the conclusion that Cr(VI) toxicity
disrupts MTs and deranges the MT-mediated functions either by depolymerizing or
stabilizing them.
DOI: 10.1007/s00709-015-0831-4
471. Physiol Plant. 2015 May 26. doi: 10.1111/ppl.12350. [Epub ahead of print]
Overexpression of GsGSTU13 and SCMRP in Medicago sativa confers increased

salt-alkaline tolerance and methionine content.
Jia B(1), Sun M(1), Sun X(2), Li R(3), Wang Z(1), Wu J(1), Wei Z(1), DuanMu H(1),
Xiao J(1), Zhu Y(1).
Author information:
(1)Key Laboratory of Agricultural Biological Functional Gene, Northeast
Agricultural University, Harbin, 150030, P.R. China.
(2)Agricultural College, Heilongjiang Bayi Agricultural University, Daqing,
163319, P.R. China.
(3)Key Laboratory of Molecular Biology, College of Heilongjiang Province,
Heilongjiang University, Harbin, 150080, P.R. China.
Tau-class glutathione S-transferases (GSTUs) are ubiquitous proteins encoded by a

large gene family in plants, which play important roles in combating different
environmental stresses. In previous studies, we constructed a Glycine soja
transcriptional profile, and identified three GSTUs (GsGSTU13, GsGSTU14 and
GsGSTU19) as potential salt-alkaline stress-responsive genes. Two of them,
GsGSTU14 and GsGSTU19, have been shown to positively regulate plant salt-alkaline
tolerance. In this study, we further demonstrated the positive function of
GsGSTU13 in plant salt-alkaline stress responses by overexpressing it in Medicago
sativa. Stress tolerance tests suggested that GsGSTU13 transgenic lines showed
better growth and physiological indicators than wild alfalfa (cv. Zhaodong) under
alkaline stress. Considering the shortage of methionine in alfalfa, we then
co-transformed GsGSTU13 into two main alfalfa cultivars in Heilongjiang Province
(cv. Zhaodong and cv. Nongjing No. 1) together with SCMRP, a synthesized gene
that could improve the methionine content. We found that GsGSTU13/SCMRP
transgenic alfalfa displayed not only higher methionine content but also higher
tolerance to alkaline and salt stresses, respectively. Taken together, our
results demonstrate that GsGSTU13 acts as a positive regulator in plant responses
to salt and alkaline stresses, and can be used as a good candidate for generation
of salt-alkaline tolerant crops.
© 2015 Scandinavian Plant Physiology Society.
DOI: 10.1111/ppl.12350
PMID: 26010993
472. Virology. 2015 Sep;483:275-83. doi: 10.1016/j.virol.2015.05.001. Epub 2015 May

21.
Complete genome sequence and integrated protein localization and interaction map
for alfalfa dwarf virus, which combines properties of both cytoplasmic and
nuclear plant rhabdoviruses.
Bejerman N(1), Giolitti F(2), de Breuil S(2), Trucco V(2), Nome C(2), Lenardon
S(2), Dietzgen RG(3).
Author information:
(1)Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones
Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA),
Camino a 60 Cuadras k 5,5, Córdoba X5020ICA, Argentina; Queensland Alliance for
Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD
4072, Australia. Electronic address: n.bejerman@uq.edu.au.
Camino a 60 Cuadras k 5,5, Córdoba X5020ICA, Argentina.
(3)Queensland Alliance for Agriculture and Food Innovation, The University of
Queensland, St Lucia, QLD 4072, Australia.
We have determined the full-length 14,491-nucleotide genome sequence of a new

plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs)
were identified in the antigenomic orientation of the negative-sense,
single-stranded viral RNA, in the order 3'-N-P-P3-M-G-P6-L-5'. The ORFs are
separated by conserved intergenic regions and the genome coding region is flanked
by complementary 3' leader and 5' trailer sequences. Phylogenetic analysis of the
nucleoprotein amino acid sequence indicated that this alfalfa-infecting
rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently
expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins
accumulated in the cell periphery, but unexpectedly P protein was localized
exclusively in the nucleus. ADV P protein was shown to have a homotypic, and
heterotypic nuclear interactions with N, P3 and M proteins by bimolecular
fluorescence complementation. ADV appears unique in that it combines properties
of both cytoplasmic and nuclear plant rhabdoviruses.

DOI: 10.1016/j.virol.2015.05.001
473. Nature. 2015 May 21;521(7552):S66. doi: 10.1038/521S66a.
Q&A: Charles Michener. A life with bees.
Michener C, Gould J.
DOI: 10.1038/521S66a
474. Molecules. 2015 May 15;20(5):8759-71. doi: 10.3390/molecules20058759.
Development and Characterization of Transcription Factor Gene-Derived

Microsatellite (TFGM) Markers in Medicago truncatula and Their Transferability in
Leguminous and Non-Leguminous Species.
Liu W(1), Jia X(1), Liu Z(1), Zhang Z(1), Wang Y(2), Liu Z(1), Xie W(1).
Author information:
Agricultural Science and Technology, Lanzhou University, Lanzhou 730020, China.
Agricultural Science and Technology, Lanzhou University, Lanzhou 730020, China.
yrwang@lzu.edu.cn.
Transcription factors (TFs) are critical adaptor molecules that regulate many
plant processes by controlling gene expression. The recent increase in the
availability of TF data has made TFs a valuable resource for genic functional
microsatellite marker development. In the present study, we developed TF
gene-derived microsatellite (TFGM) markers for Medicago truncatula and assessed
their cross-species transferability. A total of 203 SSRs were identified from
1467 M. truncatula TF coding sequences, 87.68% of which were trinucleotide
repeats, followed by mono- (4.93%) and hexanucleotide repeats (1.48%). Further,
142 TFGM markers showed a high level of transferability to the leguminous
(55.63%-85.21%) and non-leguminous (28.17%-50.00%) species. Polymorphisms of 27
TFGM markers were evaluated in 44 alfalfa accessions. The allele number per
marker ranged from two to eight with an average of 4.41, and the PIC values
ranged from 0.08 to 0.84 with an average of 0.60. Considering the high
polymorphism, these TFGM markers developed in our study will be valuable for
genetic relationship assessments, marker-assisted selection and comparative
genomic studies in leguminous and non-leguminous species.
DOI: 10.3390/molecules20058759

May
14.
Milk production responses to a change in dietary starch concentration vary by

production level in dairy cattle.
Boerman JP(1), Potts SB(1), VandeHaar MJ(1), Allen MS(1), Lock AL(2).
Author information:
Electronic address: allock@msu.edu.
The effects of dietary starch concentration on yield of milk and milk components
were evaluated in a crossover design experiment. Holstein cows (n=32; 115±22 d in
milk) with a wide range in milk yield (28 to 62kg/d) were assigned randomly
within level of milk yield to a treatment sequence. Treatments were diets
containing 30% dry ground corn (CG) or 30% soyhulls (SH) on a DM basis. Diets
containing corn silage and alfalfa silage were formulated to contain 16% crude
protein, 24% forage neutral detergent fiber, and either 27 or 44% neutral
detergent fiber and 30 or 12% starch for CG and SH, respectively. Cows were fed a
diet intermediate to the treatments during a preliminary 14-d period. Treatment
periods were 28 d with measurements taken throughout the period for energy
calculations and the final 5 d used for data and sample collection for production
variables. Compared with SH, CG increased dry matter intake, and yields of milk,
milk protein, milk fat, and energy-corrected milk, as well as milk protein
concentration. Treatment did not affect milk fat concentration. Yield of de novo
synthesized and preformed milk fatty acids increased with CG. Treatment
interacted with level of preliminary milk production for several response
variables (yields of milk, milk protein, milk fat, energy-corrected milk, and
3.5% fat-corrected milk). Compared with SH, the CG treatment increased
energy-corrected milk in higher-producing cows with a lesser response to CG as
milk yield decreased. The CG treatment increased milk:feed compared with the SH
treatment, but not body weight or body condition score. In conclusion,
higher-producing cows benefited from the high-starch diet, and lower-producing
cows were able to maintain production when most of the starch was replaced with
nonforage fiber.

DOI: 10.3168/jds.2014-8999
476. Int J Phytoremediation. 2015;17(7):669-77. doi: 10.1080/15226514.2014.964837.
Phytotoxicity of citric acid and Tween® 80 for potential use as soil amendments
in enhanced phytoremediation.
Agnello AC(1), Huguenot D, van Hullebusch ED, Esposito G.
Author information:
(1)a Università degli Studi di Cassino e del Lazio Meridionale, Dipartimento di
Ingegneria Civile e Meccanica , Cassino , (FR) , Italia.
Enhanced phytoremediation adding biodegradable amendments like low molecular

weight organic acids and surfactants is an interesting area of current research
to overcome the limitation that represents low bioavailability of pollutants in
soils. However, prior to their use in assisted phytoremediation, it is necessary
to test if amendments per se exert any toxic effect to plants and to optimize
their application mode. In this context, the present study assessed the effects
of citric acid and Tween® 80 (polyethylene glycol sorbitan monooleate) on the
development of alfalfa (Medicago sativa) plants, as influenced by their
concentration and frequency of application, in order to evaluate the feasibility
for their future use in enhanced phytoremediation of multi-contaminated soils.
The results showed that citric acid negatively affected plant germination, while
it did not have any significant effect on biomass or chlorophyll content. In
turn, Tween® 80 did not affect plant germination and showed a trend to increase
biomass, as well as it did not have any significant effect on chlorophyll levels.
M. sativa appeared to tolerate citric acid and Tween® 80 at the tested
concentrations, applied weekly. Consequently, citric acid and Tween® 80 could
potentially be utilized to assist phytoremediation of contaminated soils
vegetated with M. sativa.
DOI: 10.1080/15226514.2014.964837
477. Genet Mol Res. 2015 May 4;14(2):4438-47. doi: 10.4238/2015.May.4.1.
Analysis of genetic diversity of salt-tolerant alfalfa germplasms.
Jiang J(1), Yang BL(2), Xia T(3), Yu SM(3), Wu YN(2), Jin H(2), Li JR(4).
Author information:
(1)Biological Science Department, College of Environment and Resources, Dalian
Nationalities University, Dalian, Liaoning, China jjx@dlnu.edu.cn.
(2)Biological Science Department, College of Environment and Resources, Dalian
Nationalities University, Dalian, Liaoning, China.
(3)Grassland Institute, Branch of Animal Science, Jilin Academy of Agricultural
Sciences, Gongzhuling, Jilin, China.
(4)Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
Random amplified polymorphic DNA technology was used to analyze the genetic
diversity of 25 salt-tolerant alfalfa varieties using 30 different primers.
Results showed that the percentage of polymorphic loci between single-plant DNA
was 81.52%, and that between mixed DNA of various varieties was 61.65%. Compared
to the mixed DNA samples, single-plant DNA samples can better reveal the level of
genetic variation among and between alfalfa varieties. The gene differentiation
coefficients of 18 Chinese salt-tolerant alfalfa varieties and 7 American
salt-tolerant alfalfa varieties were 0.271 and 0.152, respectively, showing that
the exchange of genes between Chinese salt-tolerant alfalfa germplasms was more
frequent than that of American germplasms. As a topical cross-pollinated plant,
the genetic structure of biological populations of alfalfa was directly linked to
its breeding system. According to the analysis of genetic distance (GD), 25
varieties can be divided into 9 groups, among which, the GD of Tumu No. 1 and
Tumu No. 2 was the shortest (0.148), and the GD of Jieda No. 1 and Tumu was the
longest (0.786). The analysis of genetic diversity of salt-tolerant alfalfa
germplasms provided a theoretical basis for the creation of an alfalfa
salt-tolerant core germplasm repository and for the selection and breeding of new
salt-tolerant varieties.
DOI: 10.4238/2015.May.4.1

eCollection 2015.
Assessment of heat shock protein 70 induction by heat in alfalfa varieties and

constitutive overexpression in transgenic plants.
Ferradini N(1), Iannacone R(2), Capomaccio S(1), Metelli A(1), Armentano N(2),
Semeraro L(2), Cellini F(2), Veronesi F(1), Rosellini D(1).
Author information:
(1)Department of Agricultural, Food and Environmental Sciences, University of
Perugia, Perugia, Italy.
(2)ALSIA- Research Center Metapontum Agrobios, S.S. Jonica 106 Km 448,2-75012
Metaponto, Italy.
Heat shock proteins (HSPs) are molecular chaperones involved in many cellular
functions. It has been shown that mammalian cytosolic HSP70 binds antigenic
peptides mediating the activation of the immune system, and that it plays a
determining role in tumour immunogenicity. This suggests that HSP70 may be used
for the production of conjugated vaccines. Human and plant HSPs share high
sequence similarity and some important biological functions in vitro. In
addition, plant HSPs have no endotoxic side effects. Extraction of HSP70 from
plants for use as vaccine adjuvant requires enhancing its concentration in plant
tissues. In this work, we explored the possibility to produce HSP70 in both
transgenic and non-transgenic plants, using alfalfa as a model species. First, a
transcriptional analysis of a constitutive and an inducible HSP70 genes was
conducted in Arabidopsis thaliana. Then the coding sequence of the inducible form
was cloned and introduced into alfalfa by Agrobacterium-mediated transformation,
and the accumulation of the protein in leaf tissue of transgenic plants was
demonstrated. We also tested diverse alfalfa varieties for heat-inducible
expression of endogenous HSP70, revealing variety-specific responses to heat
shock.
PMCID: PMC4423914
479. Int J Phytoremediation. 2015;17(12):1145-52. doi:

10.1080/15226514.2015.1045123.
Fluorene and Phenanthrene Uptake and Accumulation by Wheat, Alfalfa and Sunflower
from the Contaminated Soil.
Salehi-Lisar SY(1), Deljoo S(1), Harzandi AM(2).
Author information:
(1)a Department of Plant Sciences , Faculty of Natural Sciences, University of
Tabriz , Tabriz , East Azerbaijan , Iran.
(2)b Laboratory of Water Analysis, Water and Wastewater Organization , Tabriz ,
East Azerbaijan , Iran.
Polycyclic Aromatic Hydrocarbons (PAHs) are diverse organic contaminants released

into the environment by both natural and anthropogenic activities. These
compounds have negative impacts on plants growth and development. Although there
are many reports on their existence in different parts of plant, their uptake and
translocation pathways and mechanisms are not well understood yet. This paper
highlights the uptake, translocation and accumulation of PAHs by wheat, sunflower
and alfalfa through an experimental study under controlled conditions. Seeds were
cultivated in a soil containing 50 mg/kg of phenanthrene and fluorene and their
concentrations in plants roots and shoots were determined using a gas
chromatograph after 7 and 14 days. The results showed that phenanthrene and
fluorene concentrations in the treated plants were increased over the time. PAHs
bioavailability was time and species dependent and generally, phenanthrene uptake
and translocation was faster than that of fluorene, probably due to their higher
Kow. Fluorene tended to accumulate in roots, but phenanthrene was transported to
aerial parts of plants.
DOI: 10.1080/15226514.2015.1045123

eCollection 2015.
Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced
abiotic stress tolerance.
Wang Z(1), Ke Q(2), Kim MD(3), Kim SH(3), Ji CY(2), Jeong JC(2), Lee HS(2), Park
WS(4), Ahn MJ(4), Li H(5), Xu B(5), Deng X(5), Lee SH(6), Lim YP(7), Kwak SS(2).
Author information:
Bioscience and Biotechnology (KRIBB), Daejeon, 305-4432, Korea; Department of
Horticulture, Chungnam National University, Daejeon, Korea; State Key Laboratory
of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and
Water Conservation, Chinese Academy of Science and Ministry of Water Resources,
Northwest A & F University, Yangling, Shaanxi, P.R. China.
Bioscience and Biotechnology (KRIBB), Daejeon, 305-4432, Korea; Department of
Green Chemistry and Environmental Biotechnology, Korea University of Science &
Technology, Daejeon, Korea.
Bioscience and Biotechnology (KRIBB), Daejeon, 305-4432, Korea.
(4)College of Pharmacy and Research Institute of Pharmaceutical Sciences,
Gyeongsang National University, Jinju, Korea.
Institute of Soil and Water Conservation, Chinese Academy of Science and Ministry
of Water Resources, Northwest A & F University, Yangling, Shaanxi, P.R. China.
(6)Grassland and Forages Division, National Institute of Animal Science, Rural
Development Administration, Cheonan, Korea.
(7)Department of Horticulture, Chungnam National University, Daejeon, Korea.
Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional
content, is widely distributed in various environments worldwide. We recently
demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing
carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In
this study, in an effort to improve the nutritional quality and environmental
stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv.
Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase
(SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among
the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2,
SOR3, and SOR8) selected based on their IbOr transcript levels were examined for
their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc
assay. The SOR plants exhibited less damage in response to MV-mediated oxidative
stress and salt stress than non-transgenic plants. The SOR plants also exhibited
enhanced tolerance to drought stress, along with higher total carotenoid levels.
The results suggest that SOR alfalfa plants would be useful as forage crops with
improved nutritional value and increased tolerance to multiple abiotic stresses,
which would enhance the development of sustainable agriculture on marginal lands.
PMCID: PMC4422619
481. J Sep Sci. 2015 Jul;38(14):2545-50. doi: 10.1002/jssc.201500138. Epub 2015 Jul
7.
Elucidation of the absolute configuration of rhizopine by chiral supercritical

fluid chromatography and vibrational circular dichroism.
Krief A(1), Dunkle M(2), Bahar M(3), Bultinck P(4)(5), Herrebout W(5)(6), Sandra
P(2).
Author information:
(1)Department of Chemistry, University of Namur, Namur, Belgium.
(2)Research Institute for Chromatography (R.I.C.), Kortrijk, Belgium.
(3)Department of Plant Protection & Agricultural Biotechnology, College of
Agriculture, Isfahan University of Technology, Isfahan, Iran.
(4)Department of Inorganic and Physical Chemistry, Ghent University, Krijgslaan
281 (S3), Ghent, Belgium.
(5)Scaldis Spectroscopy, www.scaldis-spectroscopy.com.
(6)Department of Chemistry, University of Antwerp, Antwerp, Belgium.
The absolute configuration of rhizopine, an opine-like natural product present in

nitrogen-fixing nodules of alfalfa infected by rhizobia, is elucidated using a
combination of state-of-the-art analytical and semi-preparative supercritical
fluid chromatography and vibrational circular dichroism spectroscopy. A synthetic
peracetylated racemate was fractionated into its enantiomers and subjected to
absolute configuration analysis revealing that natural rhizopine exists as a
single enantiomer. The stereochemistry of non-derivatized natural rhizopine
corresponds to (1R,2S,3R,4R,5S,6R)-4-amino-6-methoxycyclohexane-1,2,3,5-tetraol.
DOI: 10.1002/jssc.201500138
482. Pak J Biol Sci. 2014 Apr;17(4):535-9.
Nodulation study of natural forage legume in semiarid region, Turkey.
Küçük C, Cevheri C.
In this study, we investigated the natural nodulation of legume forage crops were
widely grown in the natural pastures in Sanliurfa, Turkey. This legume forage
crops are Vicia sativa L. subsp. sativa L., Vicia narbonensis L. var. narbonensis
L., Vicia palaestina Boiss., Vicia hybrida L., Vicia lutea L. var. lutea Boiss.
ET Ball., Pisum sativum L. subsp. sativum L. var. sativum L, Cicer echinospermum
P.H. Davis, Trifolium tomentosum L., Trifolium retusum L., Trifolium campestre
SCHREB., Medicago truncatula GAERTN. var. truncatula Schultz Bip., Trigonella
mesopotamica Hub.-Mor., Lens culinaris Medik., Onobrychis crista-galli, Lathyrus
cassius Boiss., Melilotus officinalis (L.) DESR., Coronilla scorpioides (L.)
W.D.J. Koch. Nodulation, nodule colors and shapes were examined at the blooming
period of forage legumes. In this study, the colour of the interior of nodules
are pink-red colour and may be related to high rates of nitrogen fixation in
legume crops.

eCollection 2015.
Molecular Diversity and Population Structure of a Worldwide Collection of

Cultivated Tetraploid Alfalfa (Medicago sativa subsp. sativa L.) Germplasm as
Revealed by Microsatellite Markers.
Qiang H(1), Chen Z(2), Zhang Z(1), Wang X(1), Gao H(1), Wang Z(1).
Author information:
(1)Institute of Animal Sciences, Chinese Academy of Agriculture Sciences (CAAS),
(2)National Animal Husbandry Service, Ministry of Agriculture, Beijing 100125,
China.
Information on genetic diversity and population structure of a tetraploid alfalfa

collection might be valuable in effective use of the genetic resources. A set of
336 worldwide genotypes of tetraploid alfalfa (Medicago sativa subsp. sativa L.)
was genotyped using 85 genome-wide distributed SSR markers to reveal the genetic
diversity and population structure in the alfalfa. Genetic diversity analysis
identified a total of 1056 alleles across 85 marker loci. The average expected
heterozygosity and polymorphism information content values were 0.677 and 0.638,
respectively, showing high levels of genetic diversity in the cultivated
tetraploid alfalfa germplasm. Comparison of genetic characteristics across
chromosomes indicated regions of chromosomes 2 and 3 had the highest genetic
diversity. A higher genetic diversity was detected in alfalfa landraces than that
of wild materials and cultivars. Two populations were identified by the
model-based population structure, principal coordinate and neighbor-joining
analyses, corresponding to China and other parts of the world. However, lack of
strictly correlation between clustering and geographic origins suggested
extensive germplasm exchanges of alfalfa germplasm across diverse geographic
regions. The quantitative analysis of the genetic diversity and population
structure in this study could be useful for genetic and genomic analysis and
utilization of the genetic variation in alfalfa breeding.
PMCID: PMC4406709
484. Ying Yong Sheng Tai Xue Bao. 2014 Nov;25(11):3191-6.
[Effects of continuous cropping of wheat and alfalfa on soil enzyme activities

and nutrients].
Zhang LQ, Hao MD, Zang YF, Li LX.
Based on a long-term rotation and fertilization experiment in Changwu, Shaanxi,

China, we determined the enzymatic activities and nutrients in soils after 27
years continuous cropping of alfalfa and wheat, respectively. The activities of
invertase, urease and phosphatase were not affected by fertilization treatment
within each cropping system, but they were significantly higher in the alfalfa
continuous cropping system than in the wheat continuous cropping system under
each fertilization treatment. The activity of hydrogen peroxidase was not
affected by the type of cropping system or fertilization treatment. Across the
cropping systems, the activities of soil urease, phosphatase and hydrogen
peroxidase were higher while soil invertase activity was lower in N, P and manure
(NPM) combined treatment compared with the other fertilization treatments. The
accumulations of soil organic matter, total nitrogen and available nitrogen were
greater in the alfalfa cropping system than in the wheat continuous cropping
system, and the NPM treatment could improve the soil fertility.
[Effects of shading on the aboveground biomass and stiochiometry characteristics

of Medicago sativa].
Ma ZL, Yang WQ, Wu FZ, Gao S.
In order to provide scientific basis for inter-planting alfalfa in abandoned

farmland, a shading experiment was conducted to simulate the effects of different
light intensities on the aboveground biomass, the contents of carbon, nitrogen,
phosphorus and potassium, and the stoichiometric characteristics of alfalfa under
the plantation. The results showed that the aboveground biomass of alfalfa
correlated significantly with the light intensity, and shading treatment reduced
the aboveground biomass of alfalfa significantly. The aboveground alfalfa tissues
under the 62% shading treatment had the highest contents of carbon, nitrogen and
phosphorus, which was 373.73, 34.38 and 5.47 g · kg(-1), respectively, and
significantly higher than those of the control. However, shading treatments had
no significant effect on the potassium content of aboveground part. The C/N ratio
in aboveground tissues under the 72% shading treatment was significantly higher
than that of the control, but no significant differences among other treatments
were found. The ratios of N/P and C/P in aboveground tissues showed a tendency
that decreased firstly and then increased with the increase of light intensity.
486. BMC Microbiol. 2015 Mar 3;15:58. doi: 10.1186/s12866-015-0390-z.
Biofilm formation assessment in Sinorhizobium meliloti reveals interlinked

control with surface motility.
Amaya-Gómez CV(1), Hirsch AM(2), Soto MJ(3).
Author information:
(1)Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC),
Profesor Albareda 1, 18008, Granada, Spain. carolamaya@gmail.com.
(2)Department of Molecular, Cell, and Developmental Biology and Molecular Biology
Institute, University of California-Los Angeles, Los Angeles, CA, 90095-1606,
USA. ahirsch@ucla.edu.
Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC),
Profesor Albareda 1, 18008, Granada, Spain. mariajose.soto@eez.csic.es.
BACKGROUND: Swarming motility and biofilm formation are opposite, but related
surface-associated behaviors that allow various pathogenic bacteria to colonize
and invade their hosts. In Sinorhizobium meliloti, the alfalfa endosymbiont,
these bacterial processes and their relevance for host plant colonization are
largely unexplored. Our previous work demonstrated distinct swarming abilities in
two S. meliloti strains (Rm1021 and GR4) and revealed that both environmental
cues (iron concentration) and bacterial genes (fadD, rhb, rirA) play crucial
roles in the control of surface motility in this rhizobial species. In the
current study, we investigate whether these factors have an impact on the ability
of S. meliloti to establish biofilms and to colonize host roots.
RESULTS: We found that strain GR4, which is less prone to translocate on solid
surfaces than strain Rm1021, is more efficient in developing biofilms on glass
and plant root surfaces. High iron conditions, known to prevent surface motility
in a wild-type strain of S. meliloti, promote biofilm development in Rm1021 and
GR4 strains by inducing the formation of more structured and thicker biofilms
than those formed under low iron levels. Moreover, three different S. meliloti
mutants (fadD, rhb, and rirA) that exhibit an altered surface translocation
behavior compared with the wild-type strain, establish reduced biofilms on both
glass and alfalfa root surfaces. Iron-rich conditions neither rescue the defect
in biofilm formation shown by the rhb mutant, which is unable to produce the
siderophore rhizobactin 1021 (Rhb1021), nor have any impact on biofilms formed by
the iron-response regulator rirA mutant. On the other hand, S. meliloti FadD
loss-of-function mutants do not establish normal biofilms irrespective of iron
levels.
CONCLUSIONS: Our studies show that siderophore Rhb1021 is not only required for
surface translocation, but also for biofilm formation on glass and root surfaces
by strain Rm1021. In addition, we present evidence for the existence of control
mechanisms that inversely regulate swarming and biofilm formation in S. meliloti,
and that contribute to efficient plant root colonization. One of these mechanisms
involves iron levels and the iron global regulator RirA. The other mechanism
involves the participation of the fatty acid metabolism-related enzyme FadD.
DOI: 10.1186/s12866-015-0390-z
PMCID: PMC4381460
487. Meat Sci. 2015 Aug;106:31-7. doi: 10.1016/j.meatsci.2015.03.021. Epub 2015 Apr
3.
The effect of dietary alfalfa and flax sprouts on rabbit meat antioxidant
content, lipid oxidation and fatty acid composition.
Dal Bosco A(1), Castellini C(2), Martino M(2), Mattioli S(2), Marconi O(2),
Sileoni V(2), Ruggeri S(2), Tei F(2), Benincasa P(2).
Author information:
Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy. Electronic address:
alessandro.dalbosco@unipg.it.
Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
The aim of this study was to determine the effect of dietary supplementation with
flax and alfalfa sprouts on fatty acid, tocopherol and phytochemical contents of
rabbit meat. Ninety weaned New Zealand White rabbits were assigned to three
dietary groups: standard diet (S); standard diet+20g/d of alfalfa sprouts (A);
and standard diet+20g/d of flax sprouts (F). In the F rabbits the Longissimus
dorsi muscle showed a higher thio-barbituric acid-reactive value and at the same
time significantly higher values of alpha-linolenic acid, total polyunsaturated
and n-3 fatty acids. Additionally n-3/n-6 ratio and thrombogenic indices were
improved. The meat of A rabbits showed intermediate values of the previously
reported examined parameters. Dietary supplementation with sprouts produced meat
with a higher total phytoestrogen content. The addition of fresh alfalfa and flax
sprouts to commercial feed modified the fat content, fatty acid and phytochemical
profile of the meat, but the flax ones worsened the oxidative status of meat.
DOI: 10.1016/j.meatsci.2015.03.021
488. J Dairy Sci. 2015 Jun;98(6):3980-7. doi: 10.3168/jds.2014-8836. Epub 2015 Apr
8.
Effect of replacing alfalfa with panicled-tick clover or sericea lespedeza in

corn-alfalfa-based substrates on in vitro ruminal methane production.
Naumann HD(1), Lambert BD(2), Armstrong SA(3), Fonseca MA(4), Tedeschi LO(4),
Muir JP(2), Ellersieck MR(5).
Author information:
(1)Division of Plant Sciences, University of Missouri, Columbia 65211. Electronic
address: naumannhd@missouri.edu.
(2)Department of Wildlife, Sustainability, and Ecosystem Sciences, Tarleton State
University, Stephenville, TX 76402; Texas A&M AgriLife Research, Stephenville
76401.
(3)Oregon State University, Corvallis 97331.
(4)Department of Animal Science, Texas A&M University, College Station 77843.
(5)Department of Statistics, University of Missouri, Columbia 65211.
Methane emissions from ruminant livestock contribute to total anthropogenic

greenhouse gas emissions and reduce metabolizable energy intake by the animal.
Condensed tannins (CT) are polyphenolic plant secondary compounds commonly
produced by some perennial forage legumes that characteristically bind to
protein, carbohydrates, and minerals. The degree to which CT may affect ruminant
nutrition depends upon the concentration, structural composition, and biological
activity of the CT. The objective of our experiment was to determine the effect
of replacing alfalfa in a corn-alfalfa-based substrate with a legume containing
CT on in vitro CH4 production and the dynamics of fermentation using an in vitro
gas production technique. All fermented substrates contained 50% ground corn as
the energy concentrate portion, whereas the forage portion (50%) of each diet was
comprised of alfalfa (control) or some combination of alfalfa and sericea
lespedeza (SL) or panicled-tick clover (PTC). Our treatments consisted of PTC or
SL 15, 30, and 45, which corresponded with 15, 30, or 45% replacement of the diet
(alfalfa component) with either PTC or SL. Substrates containing 45% PTC or SL
reduced in vitro CH4 production. Treatments did not affect total gas production
as compared with that of the control. Replacement of alfalfa with SL or PTC
increased fermentable organic matter (FOM). The PTC treatment increased FOM by as
much as 1.8% at the 45% replacement level, whereas FOM of SL 45 was increased by
less than 1%. The replacement of alfalfa with PTC increased substrate nutritive
value greater than replacement with SL. There were no correlations between any
physicochemical constituent of the substrates and CH4 production. A combination
of factors associated with the inclusion of PTC and SL contributed to the in
vitro CH4 production, and CT in these forages was likely a major contributing
factor. Further confirmation of these results on in situ or in vivo animal
systems is required. If proven effective in an in vivo production scenario,
replacement of commonly fed non-CT-containing legumes, such as alfalfa, with
legumes containing CT might be a viable method to decrease the effect of animal
agriculture on greenhouse gas production.
DOI: 10.3168/jds.2014-8836
489. Environ Sci Pollut Res Int. 2015 Aug;22(15):11690-9. doi:

10.1007/s11356-015-4410-1. Epub 2015 Apr 9.
Relationships among bulk soil physicochemical, biochemical, and microbiological

parameters in an organic alfalfa-rice rotation system.
Lopes AR(1), Bello D, Prieto-Fernández Á, Trasar-Cepeda C, Manaia CM, Nunes OC.
Author information:
(1)LEPABE, Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e
Energia, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias,
4200-465, Porto, Portugal.
The microbial communities of bulk soil of rice paddy fields under an ancient
organic agriculture regimen, consisting on an alfalfa-rice rotation system, were
characterized. The drained soil of two adjacent paddies at different stages of
the rotation was compared before rice seeding and after harvesting. The
relationships among the soil microbial, physicochemical, and biochemical
parameters were investigated using multivariate analyses. In the first year of
rice cropping, aerobic cultivable heterotrophic populations correlated with
lineages of presumably aerobic bacteria (e.g., Sphingobacteriales,
Sphingomonadales). In the second year of rice cropping, the total C content
correlated with presumable anaerobic bacteria (e.g., Anaerolineae). Independently
of the year of rice cropping, before rice seeding, proteolytic activity
correlated positively with the cultivable aerobic heterotrophic and ammonifier
populations, the soil catabolic profile and with presumable aerobes (e.g.,
Sphingobacteriales, Rhizobiales) and anaerobes (e.g., Bacteroidales,
Anaerolineae). After harvesting, strongest correlations were observed between
cultivable diazotrophic populations and bacterial groups described as comprising
N2 fixing members (e.g., Chloroflexi-Ellin6529, Betaproteobacteria,
Alphaproteobacteria). It was demonstrated that chemical parameters and microbial
functions were correlated with variations on the total bacterial community
composition and structure occurring during rice cropping. A better understanding
of these correlations and of their implications on soil productivity may be valid
contributors for sustainable agriculture practices, based on ancient processes.
DOI: 10.1007/s11356-015-4410-1
490. Acta Crystallogr D Biol Crystallogr. 2015 Apr;71(Pt 4):907-17. doi:

10.1107/S1399004715001935. Epub 2015 Mar 27.
Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone

isomerase.
Thomsen M(1), Tuukkanen A(2), Dickerhoff J(1), Palm GJ(1), Kratzat H(1), Svergun
DI(2), Weisz K(1), Bornscheuer UT(1), Hinrichs W(1).
Author information:
(1)Institut für Biochemie, Universität Greifswald, Felix-Hausdorff-Strasse 4,
17489 Greifswald, Germany.
(2)EMBL, c/o DESY, Notkestrasse 85, Gebäude 25A, 22603 Hamburg, Germany.
Flavonoids represent a large class of secondary metabolites produced by plants.

These polyphenolic compounds are well known for their antioxidative abilities,
are antimicrobial phytoalexins responsible for flower pigmentation to attract
pollinators and, in addition to other properties, are also specific bacterial
regulators governing the expression of Rhizobium genes involved in root
nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from
Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway
by ring opening of (2S)-naringenin to form naringenin chalcone. The structural
biology and enzymology of plant CHIs have been well documented, whereas the
existence of bacterial CHIs has only recently been elucidated. This first
determination of the structure of a bacterial CHI provides detailed structural
insights into the key step of the flavonoid-degradation pathway. The active site
could be confirmed by co-crystallization with the substrate (2S)-naringenin. The
stereochemistry of the proposed mechanism of the isomerase reaction was verified
by specific (1)H/(2)H isotope exchange observed by (1)H NMR experiments and was
further supported by mutagenesis studies. The active site is shielded by a
flexible lid, the varying structure of which could be modelled in different
states of the catalytic cycle using small-angle X-ray scattering data together
with the crystallographic structures. Comparison of bacterial CHI with the plant
enzyme from Medicago sativa reveals that they have unrelated folds, suggesting
that the enzyme activity evolved convergently from different ancestor proteins.
Despite the lack of any functional relationship, the tertiary structure of the
bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite
dismutase and the stress-related protein SP1.
DOI: 10.1107/S1399004715001935
1.
Effects of alfalfa hay and its physical form (chopped versus pelleted) on
performance of Holstein calves.
Jahani-Moghadam M(1), Mahjoubi E(2), Hossein Yazdi M(3), Cardoso FC(4), Drackley
JK(4).
Author information:
(1)Department of Animal Science, Sari Agricultural Sciences and Natural Resources
University, Sari, Iran.
(2)Department of Animal Science, University of Zanjan, Zanjan, Iran 45195.
Electronic address: e_mahjoubi@znu.ac.ir.
(3)Department of Animal Science, University of Zanjan, Zanjan, Iran 45195.
(4)Department of Animal Sciences, University of Illinois, Urbana 61801.
Inclusion of forage and its physical form in starter may affect rumen
development, average daily gain (ADG), and dry matter intake (DMI) of dairy
calves. To evaluate the effects of forage and its physical form (chopped vs.
pelleted) on growth of calves under a high milk feeding regimen, 32 Holstein
calves (38.8±1.1kg) were assigned at birth to 1 of 3 treatments in a completely
randomized block design. Dietary treatments (% of dry matter) were (1) 100%
semi-texturized starter (CON); (2) 90% semi-texturized starter + 10% chopped
alfalfa hay (mean particle size=5.4mm) as a total mixed ration (TMR; CH); and (3)
90% semi-texturized starter + 10% pelleted alfalfa (mean=5.8mm) hay as a TMR
(PH). Data were subjected to mixed model analysis with contrasts used to evaluate
effect of forage inclusion. Calves were weaned at 76 d of age and the experiment
finished 2 wk after weaning. Individual milk and solid feed consumption were
recorded daily. Solid feed consumption and ADG increased as age increased (effect
of week), but neither forage inclusion nor physical form of forage affected these
variables pre- or postweaning. Plasma urea N was affected by treatments such that
the CON group had a lower concentration than forage-fed groups. Forage inclusion,
but not physical form, resulted in increased total protein in plasma. Although
days with elevated rectal temperature, fecal score, and general appearance were
not affected by dietary treatments, calves fed alfalfa hay during the first month
of life had fewer days with respiratory issues, regardless of physical form of
hay. We concluded that provision of forage does have some beneficial effects in
calves fed large amounts of milk replacer, but pelleted alfalfa hay did not
result in any improvement in calf performance or health.

DOI: 10.3168/jds.2014-9126
1.
Feed conversion efficiency in dairy cows: Repeatability, variation in digestion

and metabolism of energy and nitrogen, and ruminal methanogens.
Arndt C(1), Powell JM(2), Aguerre MJ(1), Crump PM(3), Wattiaux MA(4).
Author information:
(2)US Dairy Forage Research Center, USDA-Agricultural Research Service, Madison,
WI 53706.
(3)Department of Computing and Biometry, University of Wisconsin, Madison 53706.
Electronic address: wattiaux@wisc.edu.
The objective was to study repeatability and sources of variation in feed

conversion efficiency [FCE, milk kg/kg dry matter intake (DMI)] of lactating cows
in mid to late lactation. Trials 1 and 2 used 16 cows (106 to 368 d in milk)
grouped in 8 pairs of 1 high- and 1 low-FCE cow less than 16 d in milk apart.
Trial 1 determined the repeatability of FCE during a 12-wk period. Trial 2
quantified the digestive and metabolic partitioning of energy and N with a 3-d
total fecal and urine collection and measurement of CH4 and CO2 emission. Trial 3
studied selected ruminal methanogens in 2 pairs of cows fitted with rumen
cannulas. Cows received a single diet including 28% corn silage, 27% alfalfa
silage, 17% crude protein, and 28% neutral detergent fiber (dry matter basis). In
trial 1, mean FCE remained repeatedly different and averaged 1.83 and 1.03 for
high- and low-FCE cows, respectively. In trial 2, high-FCE cows consumed 21% more
DMI, produced 98% more fat- and protein-corrected milk, excreted 42% less manure
per kilogram of fat- and protein-corrected milk, but emitted the same daily
amount of CH4 and CO2 compared with low-FCE cows. Percentage of gross energy
intake lost in feces was higher (28.6 vs. 25.9%), but urinary (2.76 vs. 3.40%)
and CH4 (5.23 vs. 6.99%) losses were lower in high- than low-FCE cows.
Furthermore, high-FCE cows partitioned 15% more of gross energy intake toward net
energy for maintenance, body gain, and lactation (37.5 vs. 32.6%) than low-FCE
cows. Lower metabolic efficiency and greater heat loss in low-FCE cows might have
been associated in part with greater energy demand for immune function related to
subclinical mastitis, as somatic cell count was 3.8 fold greater in low- than
high-FCE cows. As a percentage of N intake, high-FCE cows tended to have greater
fecal N (32.4 vs. 30.3%) and had lower urinary N (32.2 vs. 41.7%) and greater
milk N (30.3 vs. 19.1%) than low-FCE cows. In trial 3, Methanobrevibacter spp.
strain AbM4 was less prevalent in ruminal content of high-FCE cows, which emitted
less CH4 per unit of DMI and per unit of neutral detergent fiber digested than
low-FCE cows. Thus lower digestive efficiency was more than compensated by
greater metabolic efficiencies in high- compared with low-FCE cows. There was not
a single factor, but rather a series of mechanisms involved in the observed
differences in efficiency of energy utilization of the lactating cows in this
study.

DOI: 10.3168/jds.2014-8449

eCollection 2015.
Transcriptome analysis of resistant and susceptible alfalfa cultivars infected

with root-knot nematode Meloidogyne incognita.
Postnikova OA, Hult M, Shao J, Skantar A, Nemchinov LG.
Nematodes are one of the major limiting factors in alfalfa production. Root-knot
nematodes (RKN, Meloidogyne spp.) are widely distributed and economically
important sedentary endoparasites of agricultural crops and they may inflict
significant damage to alfalfa fields. As of today, no studies have been published
on global gene expression profiling in alfalfa infected with RKN or any other
plant parasitic nematode. Very little information is available about molecular
mechanisms that contribute to pathogenesis and defense responses in alfalfa
against these pests and specifically against RKN. In this work, we performed root
transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan)
alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot
nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end
reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both
cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and
Lahontan, respectively. Bioinformatic analysis revealed a number of common and
unique genes that were differentially expressed in susceptible and resistant
lines as a result of nematode infection. Although the susceptible cultivar showed
a more pronounced defense response to the infection, feeding sites were
successfully established in its roots. Characteristically, basal gene expression
levels under normal conditions differed between the two cultivars as well, which
may confer advantage to one of the genotypes toward resistance to nematodes.
Differentially expressed genes were subsequently assigned to known Gene Ontology
categories to predict their functional roles and associated biological processes.
Real-time PCR validated expression changes in genes arbitrarily selected for
experimental confirmation. Candidate genes that contribute to protection against
M. incognita in alfalfa were proposed and alfalfa-nematode interactions with
respect to resistance are discussed.
PMCID: PMC4379156

eCollection 2015.
Effect of rhizosphere enzymes on phytoremediation in PAH-contaminated soil using
five plant species.
Liu R(1), Dai Y(2), Sun L(3).
Author information:
(1)Key Laboratory of Pollution Ecology and Environmental Engineering, Institute
of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China.
(2)Key Laboratory of Pollution Ecology and Environmental Engineering, Institute
of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, China;
University of Chinese Academy of Sciences, Beijing, China.
(3)Applied Engineering Department, Hangzhou Wanxiang Polytechnic, Hangzhou,
Zhejiang, China.
A pot experiment was performed to study the effectiveness of remediation using

different plant species and the enzyme response involved in remediating
PAH-contaminated soil. The study indicated that species Echinacea purpurea,
Festuca arundinacea Schred, Fire Phoenix (a combined F. arundinacea), and
Medicago sativa L. possess the potential for remediation in PAH-contaminated
soils. The study also determined that enzymatic reactions of polyphenol oxidase
(except Fire Phoenix), dehydrogenase (except Fire Phoenix), and urease (except
Medicago sativa L.) were more prominent over cultivation periods of 60d and 120d
than 150d. Urease activity of the tested species exhibited prominently linear
negative correlations with alkali-hydrolyzable nitrogen content after the tested
plants were cultivated for 150d (R2 = 0.9592). The experiment also indicated that
alkaline phosphatase activity in four of the five tested species (Echinacea
purpurea, Callistephus chinensis, Festuca arundinacea Schred and Fire Phoenix)
was inhibited during the cultivation process (at 60d and 120d). At the same time,
the study determined that the linear relationship between alkaline phosphatase
activity and effective phosphorus content in plant rhizosphere soil exhibited a
negative correlation after a growing period of 120d (R2 = 0.665).
Phytoremediation of organic contaminants in the soil was closely related to
specific characteristics of particular plant species, and the catalyzed reactions
were the result of the action of multiple enzymes in the plant rhizosphere soil.
PMCID: PMC4378954
495. Plant Sci. 2015 May;234:110-8. doi: 10.1016/j.plantsci.2014.11.016. Epub 2014

Dec
4.
Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.
Zhang WJ(1), Wang T(2).
Author information:
(1)Department of Grassland Science, China Agricultural University, No. 2
Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China. Electronic
address: wjzhang@cau.edu.cn.
(2)State Key Laboratory of Agro-biotechnology, China Agricultural University, 2
Yuanmingyuan West Road, Haidian District, Beijing 100193, PR China. Electronic
address: wangt@cau.edu.cn.
Generating salt tolerance forage plant is essential for use of the land affected
by high salinity. A salt tolerance gene rstB was used as a selectable marker gene
in Agrobacterium-mediated transformation of tobacco under a selective regime of
170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To
improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced
into alfalfa genome by Agrobacterium-mediated transformation. No abnormal
phenotype was observed among the transgenic plants when compared with wild type
(wt) plants. Significant enhancement of resistance to salt-shock treatment was
noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds
showed improved germination rate and seedling growth under salt-stress condition.
Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the
rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in
transgenic plants was also verified by cytohistochemical localization of calcium.
Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their
life-cycle but the wt plants had no flower formation. The results demonstrated
that the expression of rstB gene improved salt tolerance in transgenic alfalfa.
DOI: 10.1016/j.plantsci.2014.11.016
496. J Plant Res. 2015 Jul;128(4):697-707. doi: 10.1007/s10265-015-0715-x. Epub

2015
Mar 24.
Isolation and functional characterization of salt-stress induced RCI2-like genes

from Medicago sativa and Medicago truncatula.
Long R(1), Zhang F, Li Z, Li M, Cong L, Kang J, Zhang T, Zhao Z, Sun Y, Yang Q.
Author information:
Salt stress is one of the most significant adverse abiotic factors, causing crop
failure worldwide. So far, a number of salt stress-induced genes, and genes
improving salt tolerance have been characterized in a range of plants. Here, we
report the isolation and characterization of a salt stress-induced Medicago
sativa (alfalfa) gene (MsRCI2A), which showed a high similarity to the yeast
plasma membrane protein 3 gene (PMP3) and Arabidopsis RCI2A. The sequence
comparisons revealed that five genes of MtRCI2(A-E) showed a high similarity to
MsRCI2A in the Medicago truncatula genome. MsRCI2A and MtRCI2(A-E) encode small,
highly hydrophobic proteins containing two putative transmembrane domains,
predominantly localized in the plasma membrane. The transcript analysis results
suggest that MsRCI2A and MtRCI2(A-D) genes are highly induced by salt stress. The
expression of MsRCI2A and MtRCI2(A-C) in yeast mutants lacking the PMP3 gene can
functionally complement the salt sensitivity phenotype resulting from PMP3
deletion. Overexpression of MsRCI2A in Arabidopsis plants showed improved salt
tolerance suggesting the important role of MsRCI2A in salt stress tolerance in
alfalfa.
DOI: 10.1007/s10265-015-0715-x
497. Ecotoxicol Environ Saf. 2015 Jun;116:143-9. doi: 10.1016/j.ecoenv.2015.03.005.

Epub 2015 Mar 21.
The influence of multiwalled carbon nanotubes on polycyclic aromatic hydrocarbon

(PAH) bioavailability and toxicity to soil microbial communities in alfalfa
rhizosphere.
Shrestha B(1), Anderson TA(2), Acosta-Martinez V(3), Payton P(3), Cañas-Carrell

JE(2).
Author information:
(1)Department of Environmental Toxicology, The Institute of Environmental and
Human Health (TIEHH), Texas Tech University, Lubbock, TX, United States; Pegasus
Technical Services Inc., Cincinnati, OH, United States. Electronic address:
shrestha.babina@epa.gov.
(2)Department of Environmental Toxicology, The Institute of Environmental and
Human Health (TIEHH), Texas Tech University, Lubbock, TX, United States.
(3)USDA-ARS, Cropping Systems Research Laboratory, Lubbock, TX, United States.
Carbon nanotubes (CNTs) may affect bioavailability and toxicity of organic

contaminants due to their adsorption properties. Recent studies have observed the
influence of multiwalled carbon nanotubes (MWNTs) on the fate of polycyclic
aromatic hydrocarbons (PAHs) and other organic contaminants. Greenhouse studies
(49 d) were conducted with alfalfa plants in two different soil types. Four
treatment conditions (0, 25, 50, or 100 mg/kg MWNTs+100 mg/kg PAHs mixture-pyrene
and phenanthrene) were tested in order to determine their effects on soil
microbial community composition and PAH residues. Microbial community structure
in the two highest treatments (50 mg/kg and 100 mg/kg MWNTs) showed a dramatic
shift in the presence of MWNTs in sandy loam soil (1% organic matter) in
comparison to the control (0 mg/kg MWNTs). Many microbial fatty acid methyl ester
(FAMEs) markers (i15:0, 16:1ω5c, 10Me17:0, 10Me16:0) were missing in the control
soil. However, there was a lower abundance of these FAMEs in the 25 mg/kg MWNT
treatment (except 10Me17:0) and a higher presence of these FAMEs in the 50 mg/kg
and 100 mg/kg MWNT treatments compared to control. In contrast, microbial
community composition was not influenced by the MWNT treatments in sandy clay
loam soil (5.9% organic matter). However, pyrene degradation in sandy clay loam
soil significantly increased by 21% in the highest MWNT treatment group (100
mg/kg) and 9.34% in 50 mg/kg MWNT treatment. Under the conditions tested in this
study, MWNTs significantly impacted the soil microbial community distribution and
PAH degradation and effects were dependent on soil types, specifically organic
matter content.
DOI: 10.1016/j.ecoenv.2015.03.005

eCollection 2015.
De novo characterization of fall dormant and nondormant alfalfa (Medicago sativa

L.) leaf transcriptome and identification of candidate genes related to fall
dormancy.
Zhang S(1), Shi Y(1), Cheng N(1), Du H(1), Fan W(1), Wang C(1).
Author information:
Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial
forage legumes worldwide. Fall dormancy is an adaptive character related to the
biomass production and winter survival in alfalfa. The physiological, biochemical
and molecular mechanisms causing fall dormancy and the related genes have not
been well studied. In this study, we sequenced two standard varieties of alfalfa
(dormant and non-dormant) at two time points and generated approximately 160
million high quality paired-end sequence reads using sequencing by synthesis
(SBS) technology. The de novo transcriptome assembly generated a set of 192,875
transcripts with an average length of 856 bp representing about 165.1 Mb of the
alfalfa leaf transcriptome. After assembly, 111,062 (57.6%) transcripts were
annotated against the NCBI non-redundant database. A total of 30,165 (15.6%)
transcripts were mapped to 323 Kyoto Encyclopedia of Genes and Genomes pathways.
We also identified 41,973 simple sequence repeats, which can be used to generate
markers for alfalfa, and 1,541 transcription factors were identified across 1,350
transcripts. Gene expression between dormant and non-dormant alfalfa at different
time points were performed, and we identified several differentially expressed
genes potentially related to fall dormancy. The Gene Ontology and pathways
information were also identified. We sequenced and assembled the leaf
transcriptome of alfalfa related to fall dormancy, and also identified some genes
of interest involved in the fall dormancy mechanism. Thus, our research focused
on studying fall dormancy in alfalfa through transcriptome sequencing. The
sequencing and gene expression data generated in this study may be used further
to elucidate the complete mechanisms governing fall dormancy in alfalfa.
PMCID: PMC4370819
499. J Dairy Sci. 2015 Jun;98(6):4030-40. doi: 10.3168/jds.2014-9015. Epub 2015 Mar
18.
Effect of technical cashew nut shell liquid on rumen methane emission and
lactation performance of dairy cows.
Branco AF(1), Giallongo F(2), Frederick T(2), Weeks H(2), Oh J(2), Hristov AN(3).
Author information:
(1)Departamento de Zootecnia, Universidade Estadual de Maringá, Paraná, Brazil
87020-900.
(2)Department of Animal Science, Pennsylvania State University, University Park
16802.
(3)Department of Animal Science, Pennsylvania State University, University Park
16802. Electronic address: anh13@psu.edu.
Erratum in
J Dairy Sci. 2015 Jul;98(7):5018.
Technical-grade cashew nut shell liquid (TCNSL) is a by-product of the cashew nut
industry in tropical countries, and is known to exhibit a wide range of
biological activities, including inhibitory effect against gram-positive
bacteria. This study was conducted to investigate the effects of TCNSL (73.3%
cardanol, 16.4% cardol, and 3.0% methylcardol) on rumen methane emission,
nutrient digestibility, dry matter intake, and milk yield and composition in
dairy cows. Eight multiparous Holstein cows were used in a crossover design trial
with two 21-d experimental periods. The diet was based on corn silage and alfalfa
haylage and was formulated to meet or exceed the energy and metabolizable protein
requirements of the cows. Treatments were control (no TCNSL supplementation) or
30 g/cow per day of TCNSL. Rumen carbon dioxide emission was not affected by
TCNSL. Treatment had no effect on methane emission (542 vs. 511±35.3 g/cow per
day, respectively) and methane emission intensity (15.0 vs. 13.9±0.58 g/kg of
energy-corrected milk, respectively) and tended to decrease methane emission per
kilogram of dry matter intake (20.2 vs. 18.6±1.04 g/kg, respectively). Dry matter
intake (average 26.9±1.00 kg/d), milk yield (40.0±1.73 kg/d), and milk
composition were not different between treatments. The TCNSL had no effect on N
losses in urine and feces and total-tract apparent digestibility of nutrients,
except digestibility of neutral detergent fiber tended to be increased compared
with the control. Plasma urea and glucose concentrations were not affected by
TCNSL. Concentration of milk C18:0 tended to be decreased (17%) by TCNSL compared
with the control. In this study, TCNSL did not alter absolute methane emission in
the rumen, but tended to decrease it by 8% per kilogram of dry matter intake. The
TCNSL had no effect on milk yield and composition in dairy cows.

DOI: 10.3168/jds.2014-9015
2015.
Selenium hyperaccumulators harbor a diverse endophytic bacterial community

characterized by high selenium resistance and plant growth promoting properties.
Sura-de Jong M(1), Reynolds RJ(2), Richterova K(3), Musilova L(3), Staicu LC(2),
Chocholata I(3), Cappa JJ(2), Taghavi S(4), van der Lelie D(4), Frantik T(5),
Dolinova I(6), Strejcek M(3), Cochran AT(2), Lovecka P(3), Pilon-Smits EA(2).
Author information:
(1)Department of Biochemistry and Microbiology, University of Chemistry and
Technology in Prague Prague, Czech Republic ; Life Sciences and Technology, Van
Hall Larenstein University of Applied Sciences Leeuwarden, Netherlands.
(2)Biology Department, Colorado State University Fort Collins, CO, USA.
(3)Department of Biochemistry and Microbiology, University of Chemistry and
Technology in Prague Prague, Czech Republic.
(4)FMC Corporation, Center of Excellence for Agricultural Biosolutions, Research
Triangle Park NC, USA.
(5)Institute of Botany, Academy of Sciences of the Czech Republic Pruhonice,
Czech Republic.
(6)The Institute for Nanomaterials, Advanced Technology and Innovation, Technical
University of Liberec Liberec, Czech Republic.
Selenium (Se)-rich plants may be used to provide dietary Se to humans and

livestock, and also to clean up Se-polluted soils or waters. This study focused
on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of
dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis
was used to compare the diversity of endophytic bacteria of hyperaccumulators
Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those
from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa
(Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and
non-accumulators showed equal T-RF diversity. Parsimony analysis showed that
T-RFs from individuals of the same species were more similar to each other than
to those from other species, regardless of plant Se content or spatial proximity.
Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were
further identified and characterized. The 66 bacterial morphotypes were shown by
MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of
Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella,
Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and
selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce
nitrite and produce siderophores. Seven isolates were selected for plant
inoculation and found to have plant growth promoting properties, both in pure
culture and when co-cultivated with crop species Brassica juncea (Brassicaceae)
or M. sativa. There were no effects on plant Se accumulation. We conclude that Se
hyperaccumulators harbor an endophytic bacterial community in their natural
seleniferous habitat that is equally diverse to that of comparable
non-accumulators. The hyperaccumulator endophytes are characterized by high Se
resistance, capacity to produce elemental Se and plant growth promoting
properties.
DOI: 10.3389/fpls.2015.00113
PMCID: PMC4345804
PMID: 25784919
501. Plant Methods. 2015 Feb 25;11:10. doi: 10.1186/s13007-015-0053-y. eCollection

2015.
An ontology approach to comparative phenomics in plants.
Oellrich A(#)(1), Walls RL(#)(2), Cannon EK(3), Cannon SB(4)(5), Cooper L(6),
Gardiner J(7), Gkoutos GV(8), Harper L(4), He M(7), Hoehndorf R(9), Jaiswal P(6),
Kalberer SR(4), Lloyd JP(10), Meinke D(11), Menda N(12), Moore L(6), Nelson
RT(4), Pujar A(12), Lawrence CJ(5)(7), Huala E(13).
Author information:
(1)Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CB10
1SA UK.
(2)iPlant Collaborative, University of Arizona, 1657 E. Helen St., Tucson,
Arizona 85721 USA.
(3)Department of Electrical and Computer Engineering Iowa State University, 1018
Crop Informatics Lab, Ames, Iowa 50011 USA.
(4)USDA-ARS Corn Insects and Crop Genetics Research Unit, Iowa State University,
Crop Genome Informatics Lab, Iowa State University, Ames, IA 50011 USA.
(5)Department of Agronomy, Agronomy Hall, Iowa State University, Ames, IA 50010
USA.
(6)Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State
University, Corvallis, OR 97331 USA.
(7)Department of Genetics, Development and Cell Biology, Roy J Carver
Co-Laboratory, Iowa State University, Ames, IA 50010 USA.
(8)Department of Computer Science, Aberystwyth University, Llandinam Building,
Aberystwyth, SY23 3DB UK.
(9)Computer, Electrical and Mathematical Sciences & Engineering Division and
Computational Bioscience Research Center, King Abdullah University of Science and
Technology, 4700 King Abdullah University of Science and Technology, P.O. Box
2882, Thuwal, 23955-6900 Kingdom of Saudi Arabia.
(10)Department of Plant Biology, Michigan State University, 220 Trowbridge Rd,
East Lansing, MI 48824 USA.
(11)Department of Botany, Oklahoma State University, 301 Physical Sciences,
Stillwater, OK 74078 USA.
(12)Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, NY 14853
USA.
(13)Phoenix Bioinformatics, 643 Bair Island Rd Suite 403, Redwood City, CA 94063
USA.
BACKGROUND: Plant phenotype datasets include many different types of data,

formats, and terms from specialized vocabularies. Because these datasets were
designed for different audiences, they frequently contain language and details
tailored to investigators with different research objectives and backgrounds.
Although phenotype comparisons across datasets have long been possible on a small
scale, comprehensive queries and analyses that span a broad set of reference
species, research disciplines, and knowledge domains continue to be severely
limited by the absence of a common semantic framework.
RESULTS: We developed a workflow to curate and standardize existing phenotype
datasets for six plant species, encompassing both model species and crop plants
with established genetic resources. Our effort focused on mutant phenotypes
associated with genes of known sequence in Arabidopsis thaliana (L.) Heynh.
(Arabidopsis), Zea mays L. subsp. mays (maize), Medicago truncatula Gaertn.
(barrel medic or Medicago), Oryza sativa L. (rice), Glycine max (L.) Merr.
(soybean), and Solanum lycopersicum L. (tomato). We applied the same ontologies,
annotation standards, formats, and best practices across all six species, thereby
ensuring that the shared dataset could be used for cross-species querying and
semantic similarity analyses. Curated phenotypes were first converted into a
common format using taxonomically broad ontologies such as the Plant Ontology,
Gene Ontology, and Phenotype and Trait Ontology. We then compared ontology-based
phenotypic descriptions with an existing classification system for plant
phenotypes and evaluated our semantic similarity dataset for its ability to
enhance predictions of gene families, protein functions, and shared metabolic
pathways that underlie informative plant phenotypes.
CONCLUSIONS: The use of ontologies, annotation standards, shared formats, and
best practices for cross-taxon phenotype data analyses represents a novel
approach to plant phenomics that enhances the utility of model genetic organisms
and can be readily applied to species with fewer genetic resources and less
well-characterized genomes. In addition, these tools should enhance future
efforts to explore the relationships among phenotypic similarity, gene function,
and sequence similarity in plants, and to make genotype-to-phenotype predictions
relevant to plant biology, crop improvement, and potentially even human health.
DOI: 10.1186/s13007-015-0053-y
PMCID: PMC4359497
PMID: 25774204
2015.
Proteomic analyses reveal differences in cold acclimation mechanisms in

freezing-tolerant and freezing-sensitive cultivars of alfalfa.
Chen J(1), Han G(2), Shang C(3), Li J(3), Zhang H(3), Liu F(3), Wang J(3), Liu
H(3), Zhang Y(3).
Author information:
(1)College of Life Sciences and Technology, Harbin Normal University Harbin,
China.
(2)College of Life Sciences and Technology, Harbin Normal University Harbin,
China ; Institute of Grass Research, Heilongjiang Academy of Agricultural
Sciences Harbin, China.
(3)Institute of Grass Research, Heilongjiang Academy of Agricultural Sciences
Harbin, China.
Cold acclimation in alfalfa (Medicago sativa L.) plays a crucial role in cold
tolerance to harsh winters. To examine the cold acclimation mechanisms in
freezing-tolerant alfalfa (ZD) and freezing-sensitive alfalfa (W5), holoproteins,
and low-abundance proteins (after the removal of RuBisCO) from leaves were
extracted to analyze differences at the protein level. A total of 84 spots were
selected, and 67 spots were identified. Of these, the abundance of 49 spots and
24 spots in ZD and W5, respectively, were altered during adaptation to chilling
stress. Proteomic results revealed that proteins involved in photosynthesis,
protein metabolism, energy metabolism, stress and redox and other proteins were
mobilized in adaptation to chilling stress. In ZD, a greater number of changes
were observed in proteins, and autologous metabolism and biosynthesis were slowed
in response to chilling stress, thereby reducing consumption, allowing for
homeostasis. The capability for protein folding and protein biosynthesis in W5
was enhanced, which allows protection against chilling stress. The ability to
perceive low temperatures was more sensitive in freezing-tolerant alfalfa
compared to freezing-sensitive alfalfa. This proteomics study provides new
insights into the cold acclimation mechanism in alfalfa.
DOI: 10.3389/fpls.2015.00105
PMCID: PMC4343008
PMID: 25774161
503. FEMS Microbiol Ecol. 2015 Jan;91(1):1-13. doi: 10.1093/femsec/fiu015. Epub

2014
Dec 5.
Plant assemblage composition and soil P concentration differentially affect

communities of AM and total fungi in a semi-arid grassland.
Klabi R(1), Bell TH(2), Hamel C(3), Iwaasa A(3), Schellenberg M(3), Raies A(4),
St-Arnaud M(5).
Author information:
(1)Biodiversity Centre, Institut de recherche en biologie végétale, Université de
Montréal and Jardin botanique de Montréal, 4101 rue Sherbrooke est, Montreal, QC
H1X 2B2, Canada Semiarid Prairie Agricultural Research Centre, Agriculture and
Agri-food Canada, PO Box 1030, Airport Road, Swift Current, SK S9H 3X2, Canada
Département de Sciences Biologique, Faculté des Sciences de Tunis, Université
Tunis El-Manar, Campus universitaire, Tunis 1060, Tunisia.
H1X 2B2, Canada.
(3)Semiarid Prairie Agricultural Research Centre, Agriculture and Agri-food
Canada, PO Box 1030, Airport Road, Swift Current, SK S9H 3X2, Canada.
(4)Département de Sciences Biologique, Faculté des Sciences de Tunis, Université
Tunis El-Manar, Campus universitaire, Tunis 1060, Tunisia.
H1X 2B2, Canada marc.st-arnaud@umontreal.ca.
Adding inorganic P- and N-fixing legumes to semi-arid grasslands can increase

forage yield, but soil nutrient concentrations and plant cover may also interact
to modify soil fungal populations, impacting short- and long-term forage
production. We tested the effect of plant assemblage (seven native grasses, seven
native grasses + the domesticated N-fixing legume Medicago sativa, seven native
grasses + the native N-fixing legume Dalea purpurea or the introduced grass
Bromus biebersteinii + M. sativa) and soil P concentration (addition of 0 or 200
P2O5 kg ha(-1) at sowing) on the diversity and community structure of arbuscular
mycorrhizal (AM) fungi and total fungi over two consecutive years, using
454-pyrosequencing of 18S rDNA and ITS amplicons. Treatment effects were stronger
in the wet year (2008) than the dry year (2009). The presence of an N-fixing
legume with native grasses generally increased AM fungal diversity, while the
interaction between soil P concentration and plant assemblage modified total
fungal community structure in 2008. Excluding interannual variations, which are
likely driven by moisture and plant productivity, AM fungal communities in
semi-arid grasslands appear to be primarily affected by plant assemblage
composition, while the composition of other fungi is more closely linked to soil
P.

DOI: 10.1093/femsec/fiu015
504. Ying Yong Sheng Tai Xue Bao. 2014 Sep;25(9):2677-82.
[Population dynamics of ground carabid beetles and spiders in a wheat field along
the wheat-alfalfa interface and their response to alfalfa mowing].
Liu WH, Hu YJ, Hu WC, Hong B, Guan XQ, Ma SY, He DH.
Taking the wheat-alfalfa and wheat-wheat interfaces as model systems, sampling

points were set by the method of pitfall trapping in the wheat field at the
distances of 3 m, 6 m, 9 m, 12 m, 15 m, 18 m, 21 m, 24 m, and 27 m from the
interface. The species composition and abundance of ground carabid beetles and
spiders captured in pitfalls were investigated. The results showed that, to some
extent there was an edge effect on species diversity and abundance of ground
carabid beetles and spiders along the two interfaces. A marked edge effect was
observed between 15 m and 18 m along the alfalfa-wheat interface, while no edge
effect was found at a distance over 20 m. The edge effect along the wheat-wheat
interface was weaker in comparison to the alfalfa-wheat interface. Alfalfa mowing
resulted in the migration of a large number of ground carabid beetles and spiders
to the adjacent wheat filed. During ten days since mowing, both species and
abundance of ground carabid beetles and spiders increased in wheat filed within
the distance of 20 m along the alfalfa-wheat interface. The spatial distribution
of species diversity of ground beetles and spiders, together with the population
abundance of the dominant Chlaenius pallipes and Pardosa astrigera, were
depicted, which could directly indicate the migrating process of natural enemy
from alfalfa to wheat field.
505. J Agric Food Chem. 2015 Apr 1;63(12):3219-28. doi: 10.1021/acs.jafc.5b00494.

Epub
2015 Mar 19.
Structural, thermal, and anti-inflammatory properties of a novel pectic

polysaccharide from alfalfa (Medicago sativa L.) stem.
Chen L, Liu J, Zhang Y, Dai B, An Y, Yu LL(1).
Author information:
(1)§Department of Nutrition and Food Science, University of Maryland, College
Park, Maryland 20742, United States.
A pectic polysaccharide (APPS) was purified from the cold alkali extract of
alfalfa stem and characterized to be a rhamnogalacturonan I (RG-I) type pectin
with the molecular weight of 2.38 × 10(3) kDa and a radius of 123 nm. The primary
structural analysis indicated that APPS composed of a
→2)-α-l-Rhap-(1→4)-α-d-GalpA-(1→ backbone with 12% branching point at C-4 of Rhap
forming side chains by l-arabinosyl and d-galactosyl oligosaccharide units.
Transmission electron microscopy (TEM) analysis revealed a primary linear-shaped
structure with a few branches in its assembly microstructures. The thermal
decomposition evaluation revealed the stability of APPS with an apparent
activation energy (Ea) of 226.5 kJ/mol and a pre-exponential factor (A) of 2.10 ×
10(25)/s, whereas its primary degradation occurred in the temperature range from
215.6 to 328.0 °C. In addition, APPS showed significant anti-inflammatory effect
against mRNA expressions of the pro-inflammatory cytokine genes, especially for
IL-1β, suggesting its potential utilization in functional foods and dietary
supplement products.
DOI: 10.1021/acs.jafc.5b00494
506. Plant Mol Biol. 2015 Apr;87(6):645-54. doi: 10.1007/s11103-015-0304-3. Epub

2015
Mar 6.
A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers

tolerance to cold and oxidative stress.
He X(1), Sambe MA, Zhuo C, Tu Q, Guo Z.
Author information:
(1)State Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Guangdong Grassland Science Engineering Research Center,
College of Life Sciences, South China Agricultural University, Guangzhou, 510642,
China.
Temperature-induced lipocalins (TIL) are plasmalemma-localized proteins and

responsive to environmental stresses. Physiological functions of MfTIL1 from
Medicago sativa subsp. falcata (L.) Arcang. (hereafter falcata), a forage legume
with cold and drought tolerance, were investigated in this study. MfTIL1
expression was greatly induced by 4-96 h of cold treatment, while transcript
levels of the orthologs in Medicago truncatula, a model legume plant with lower
cold tolerance than falcata, were reduced or not altered within 48-96 h. MfTIL1
expression was not responsive to dehydration and salinity. Compared to the wild
type, transgenic tobacco plants overexpressing MfTIL1 had lower temperature
(LT50) that resulted in 50 % lethal and elevated survival rate in response to
freezing, elevated F v/F m and decreased ion leakage after treatments with
chilling, high light and methyl viologen (MV). H2O2 and O2 (-) were less
accumulated in transgenic plants than in the wild type after treatments with
chilling, high light and MV, while antioxidant enzyme activities showed no
difference between the two types of plants prior to or following treatments.
Higher transcript levels of NtDREB3 and NtDREB4 genes were observed in transgenic
plants than in the wild type under non-stressed conditions, but higher transcript
levels of NtDREB1, NtDREB2, NtDREB4 and NtCOR15a genes under chilling conditions.
It is suggested that MfTIL1 plays an important role in plant tolerance to cold
and oxidative stress through promoted scavenging of reactive oxygen species and
up-regulating expression of multiple cold responsive genes.
DOI: 10.1007/s11103-015-0304-3
507. Plant Physiol Biochem. 2015 Apr;89:112-22. doi: 10.1016/j.plaphy.2015.02.015.
Epub 2015 Feb 24.
Screening for salt-responsive proteins in two contrasting alfalfa cultivars using

a comparative proteome approach.
Rahman MA(1), Alam I(2), Kim YG(1), Ahn NY(1), Heo SH(1), Lee DG(3), Liu G(4),
Lee BH(5).
Author information:
(1)Division of Applied Life Sciences (BK21Plus), IALS, PMBBRC, Gyeongsang
National University, Jinju 660-701, Republic of Korea.
National University, Jinju 660-701, Republic of Korea; National Institute of
Biotechnology, Ganakbari, Savar, Dhaka 1349, Bangladesh.
(3)Division of Life Science, Korea Basic Science Institute, Daejeon 305-806,
Republic of Korea.
(4)Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China.
National University, Jinju 660-701, Republic of Korea. Electronic address:
hyun@gnu.ac.kr.
A comparative proteomic approach was carried out between two contrasting alfalfa
cultivars, nonomu (NM-801; salt tolerant) and vernal (VN; salt intolerant) in
terms of salt tolerance. Seedlings were subjected to salt stress (50 and 100 mM
NaCl) for three days. Several physiological parameters (leaf water, chlorophyll,
root Na(+), K(+), and Ca(2+)) and root proteome profile were analyzed. Comparison
of physiological status revealed that NM-801 is more tolerant to salt than VN.
Eighty three differentially expressed proteins were found on 2-DE maps, of which
50 were identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry. These
proteins were involved in ion homeostasis, protein turnover and signaling,
protein folding, cell wall components, carbohydrate and energy metabolism,
reactive oxygen species regulation and detoxification, and purine and fatty acid
metabolism. The comparative proteome analysis showed that 33 salt-responsive
proteins were significantly changed in both cultivars, while 17 (14 in VN and 3
in NM-801) were cultivar-specific. Peroxidase, protein disulfide-isomerase, NAD
synthetase, and isoflavone reductase were up-regulated significantly only in
NM-801 in all salt concentrations. In addition, we identified novel proteins
including NAD synthetase and biotin carboxylase-3 that were not reported
previously as salt-responsive. Taken together, these results provide new insights
of salt stress tolerance in alfalfa.
Copyright © 2015 Elsevier Masson SAS. All rights reserved.
DOI: 10.1016/j.plaphy.2015.02.015
508. J Agric Food Chem. 2015 Mar 25;63(11):2970-8. doi: 10.1021/acs.jafc.5b00063.

Epub
2015 Mar 11.
A novel alkaline hemicellulosic heteroxylan isolated from alfalfa (Medicago

sativa L.) stem and its thermal and anti-inflammatory properties.
Chen L, Liu J, Zhang Y, Niu Y, Dai B, Yu LL(1).
Author information:
(1)§Department of Nutrition and Food Science, University of Maryland, College
Park, Maryland 20742, United States.
A novel hemicellulosic polysaccharide (ACAP) was purified from the cold alkali
extraction of alfalfa stems and characterized as a heteroxylan with a
weight-average molecular weight of 7.94 × 10(3) kDa and a radius of 58 nm.
Structural analysis indicated that ACAP consisted of a 1,4-linked β-D-Xylp
backbone with 4-O-MeGlcpA and T-L-Araf substitutions at O-2 and O-3 positions,
respectively. Transmission electron microscopy (TEM) examination revealed the
entangled chain morphology of ACAP molecules. The evaluation of thermal
degradation property revealed a primary decomposition temperature range of
238.8-314.0 °C with an apparent activation energy (Ea) and a pre-exponential
factor (A) of 220.0 kJ/mol and 2.81 × 10(24)/s, respectively. ACAP also showed
significant inhibitory activities on IL-1β, IL-6, and COX-2 gene expressions in
cultured RAW 264.7 mouse macrophage cells. These results suggested the potential
utilization of ACAP in functional foods and dietary supplement products.
DOI: 10.1021/acs.jafc.5b00063
509. J Dairy Sci. 2015 May;98(5):3309-22. doi: 10.3168/jds.2014-8798. Epub 2015 Feb
26.
Saturated fat supplementation interacts with dietary forage neutral detergent

fiber content during the immediate postpartum and carryover periods in Holstein
cows: Production responses and digestibility of nutrients.
Piantoni P(1), Lock AL(1), Allen MS(2).
Author information:
Electronic address: allenm@msu.edu.
Forty-eight multiparous cows were used in a randomized complete block design

experiment with a 2×2 factorial arrangement of treatments to determine the
interaction between a highly saturated free FA supplement (SFFA) and dietary
forage neutral detergent fiber (fNDF) content on production responses and
nutrient digestibility of dairy cows in the postpartum period. Treatment diets
were offered from 1 to 29d postpartum (postpartum period; PP) and contained 20 or
26% fNDF (50:50 corn silage:alfalfa silage and hay, dry matter basis) and 0 or 2%
SFFA [Energy Booster 100 (Milk Specialties Global, Eden Prairie, MN); 96.1% FA:
46.2% C18:0 and 37.0% C16:0]. From 30 to 71d postpartum (carryover period), a
common diet (~23% fNDF, 0% SFFA) was offered to all cows to evaluate carryover
effects of the treatment diets early in lactation. During the PP, higher fNDF
decreased dry matter intake (DMI) by 2.0 kg/d, whereas SFFA supplementation
increased it by 1.4kg/d. In addition, high fNDF with 0% SFFA decreased DMI
compared with the other diets and this difference increased throughout the PP.
Treatments did not affect 3.5% fat-corrected milk yield during the PP but did
during the carryover period when SFFA supplementation decreased 3.5%
fat-corrected milk yield for the low-fNDF diet (51.1 vs. 58.7kg/d), but not for
the high-fNDF diet (58.5 vs. 58.0kg/d). During the PP, lower fNDF and SFFA
supplementation decreased body condition score loss. A tendency for an
interaction between fNDF and SFFA indicated that low fNDF with 2% SFFA decreased
body condition score loss compared with the other diets (-0.49 vs. -0.89). During
the PP, lower fNDF and 2% SFFA supplementation decreased feed efficiency (3.5%
fat-corrected milk/DMI) by 0.30 and 0.23 units, respectively. The low-fNDF diet
with 2% SFFA decreased feed efficiency compared with other diets early in the PP,
but this difference decreased over time. Supplementation of SFFA in the PP
favored energy partitioning to body reserves and limited DMI depression for the
high-fNDF diet, which might allow higher-fNDF diets to be fed to cows in the PP.
However, SFFA supplemented in the low-fNDF diet during the PP affected production
negatively in the carryover period. Dietary fNDF and SFFA interacted, affecting
performance in the PP with carryover effects when cows were fed a common diet in
early lactation.

DOI: 10.3168/jds.2014-8798
510. Plant Cell. 2015 Mar;27(3):823-38. doi: 10.1105/tpc.114.131326. Epub 2015 Feb
27.
Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and

rice.
Sun J(1), Miller JB(1), Granqvist E(1), Wiley-Kalil A(2), Gobbato E(1), Maillet
F(3), Cottaz S(4), Samain E(4), Venkateshwaran M(2), Fort S(4), Morris RJ(1), Ané
JM(2), Dénarié J(3), Oldroyd GE(5).
Author information:
(1)John Innes Centre, Norwich NR4 7UH, United Kingdom.
(2)Department of Agronomy, University of Wisconsin-Madison, Madison, Wisconsin
53706.
(3)INRA, Laboratoire des Interactions Plantes-Microorganismes, UMR441, F-31326
Castanet-Tolosan, France CNRS, Laboratoire des Interactions
Plantes-Microorganismes, UMR2594, F-31326 Castanet-Tolosan, France.
(4)Centre de Recherche sur les Macromolécules Végétales, CNRS (affiliated to
Université de Grenoble), 38041 Grenoble Cedex 9, France.
(5)John Innes Centre, Norwich NR4 7UH, United Kingdom giles.oldroyd@jic.ac.uk.
Establishment of arbuscular mycorrhizal interactions involves plant recognition

of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs)
and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that
associate with leguminous plants also signal to their hosts via LCOs, the
so-called Nod factors. Here, we have assessed the induction of symbiotic
signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in
legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl
chitotetraose (CO4) activate the common symbiosis signaling pathway, with
resultant calcium oscillations in root epidermal cells of Medicago truncatula and
Lotus japonicus. The nature of the calcium oscillations is similar for LCOs
produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs
activate distinct gene expression. Calcium oscillations were activated in rice
atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs
activated calcium oscillations in rice trichoblasts. In contrast, stimulation of
lateral root emergence occurred following treatment with Myc-LCOs, but not CO4,
in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work
indicates that legumes and non-legumes differ in their perception of Myc-LCO and
CO signals, suggesting that different plant species respond to different
components in the mix of signals produced by arbuscular mycorrhizal fungi.
DOI: 10.1105/tpc.114.131326
PMCID: PMC4558648
511. J Food Prot. 2015 Mar;78(3):609-14. doi: 10.4315/0362-028X.JFP-14-229.
Presence and correlation of some enteric indicator bacteria, diarrheagenic

Escherichia coli pathotypes, and Salmonella serotypes in alfalfa sprouts from
local retail markets in Pachuca, Mexico.
Rangel-Vargas E(1), Gómez-Aldapa CA(1), Torres-Vitela Mdel R(2), Villarruel-López

A(2), Gordillo-Martínez AJ(1), Castro-Rosas J(3).
Author information:
(1)Centro de Investigaciones Químicas, Instituto de Ciencias Básicas e
Ingeniería, Universidad Autónoma del Estado de Hidalgo, Ciudad del Conocimiento,
Carretera Pachuca-Tulancingo Kilometer 4.5, 42183 Mineral de la Reforma, Hidalgo,
Mexico.
(2)Laboratorio de Microbiología Sanitaria, Centro Universitario de Ciencias
Exactas e Ingeniería, Universidad de Guadalajara, Marcelino García Barragán 1451,
44430 Guadalajara, Jalisco, Mexico.
Ingeniería, Universidad Autónoma del Estado de Hidalgo, Ciudad del Conocimiento,
Carretera Pachuca-Tulancingo Kilometer 4.5, 42183 Mineral de la Reforma, Hidalgo,
Mexico. jcastro@uaeh.edu.mx.
Data on the presence of diarrheagenic Escherichia coli pathotypes (DEPs) in

alfalfa sprouts and correlations between the presence of coliform bacteria (CB),
fecal coliforms (FC), E. coli, DEPs, and Salmonella in alfalfa sprouts are not
available. The presence of and correlations between CB, FC, E. coli, DEPs, and
Salmonella in alfalfa sprouts were determined. One hundred sprout samples were
collected from retail markets in Pachuca, Hidalgo State, Mexico. The presence of
indicator bacteria and Salmonella was determined using conventional culture
procedures. DEPs were identified using two multiplex PCR procedures. One hundred
percent of samples were positive for CB, 90% for FC, 84% for E. coli, 10% for
DEPs, and 4% for Salmonella. The populations of CB ranged from 6.2 up to 8.6 log
CFU/g. The FC and E. coli concentrations were between , 3 and 1,100 most probable
number (MPN)/g. The DEPs identified included enterotoxigenic E. coli (ETEC; 2%),
enteropathogenic E. coli (EPEC; 3%), and Shiga toxin-producing E. coli (STEC;
5%). No E. coli O157:H7 strains were detected in any STEC-positive samples. In
samples positive for DEPs, the concentrations ranged from 210 to 240 MPN/g for
ETEC, 28 to 1,100 MPN/g for EPEC, and 3.6 to 460 MPN/g for STEC. The Salmonella
isolates identified included Salmonella enterica serotype Typhimurium in three
samples and Salmonella enterica serotype Enteritidis in one. STEC and Salmonella
Typhimurium were identified together in one sample. Positive correlations were
observed between FC and E. coli, between FC and DEPs, and between E. coli and
DEPs. Negative correlations occurred between CB and DEPs and between CB and
Salmonella. Neither FC nor E. coli correlated with Salmonella in the sprout
samples. To our knowledge, this is the first report of ETEC, EPEC, and STEC
isolated from alfalfa sprouts and the first report of correlations between
different indicator groups versus DEPs and Salmonella.
DOI: 10.4315/0362-028X.JFP-14-229
512. Genetika. 2014 Apr;50(4):433-42.
[Natural nucleotide polymorphism of the Srlk gene that determines salt stress
tolerance in alfalfa (Medicago sativa L)].
Vishnevskaia MS, Pavlov AV, Dziubenko EA, Dziubenko NI, Potokina EK.
Based on legume genome syntheny, the nucleotide sequence of Srlk gene, key role
of which in response to salt stress was demonstrated for the model species
Medicago truncatula, was identified in the major forage and siderate crop alfalfa
(Medicago sativa). In twelve alfalfa samples originating from regions with
contrasting growing conditions, 19 SNPs were revealed in the Srlk gene. For two
nonsynonymous SNPs, molecular markers were designed that could be further used to
analyze the association between Srlk gene nucleotide polymorphism and the
variability in salt stress tolerance among alfalfa cultivars.
513. Genetika. 2014 Apr;50(4):400-12.
[ITS-polymorphism of salt-tolerant and salt-sensitive native isolates of

Sinorhizoblum meliloti--symbionts of alfalfa, clover and fenugreek plants].
Rumiantseva ML, Muntian VS, Mengoni A, Simarov BV.
Polymorphism of rrs-rrl sequence of ribosomal operons (intergenic sequence, ITS)

was studied among 81 isolates of Sinorhizobium meliloti (AK001-AK210) derived
from the collection of alfalfa nodulating bacteria of the Laboratory of genetics
of ARRIAM, by using species-specific primers FGPS 1490/FGPL132VM. Isolates were
obtained from nodules of different species of wild host plants from Medicago,
Melilotus and Trigonella genera grown in salinized North-Western region of
Kazakhstan. The typical structure of ITS, similar to that of test strain Rm1021,
was dominant in native rhizobia population, while in one third of the isolates
(33.3%) this sequence was divergent. Among the latter, the ITS type of strain
AK83 (RCAM00182) was dominant. Here, we show for the first time that isolates
with reduced level of salt-tolerance had more diverse intergenic sequences of
rrn-operons. No phylogenetic separation was observed between isolates grouped on
the basis of their tolerance or sensitivity towards 0.6 M NaCl. However, the
frequency of divergent ITS types within the two groups of rhizobia depended on
the host symbiotic preference observed in natural environment, allowing to
speculate about the existence of a chromosome types specific for S. meliloti
isolates with differential salt tolerance. In conclusion, we propose that in the
area subjected to secondary salinization, which are also the centre of
introgressive hybridization of alfalfa, micro-evolutionary processes, affecting
rrn-operons and associated with salt adaptation, are also occurring in symbiotic
root nodule bacteria populations.
514. Plant Sci. 2015 Apr;233:165-173. doi: 10.1016/j.plantsci.2015.01.013. Epub

2015
Jan 29.
Specific mechanisms of tolerance to copper and cadmium are compromised by a

limited concentration of glutathione in alfalfa plants.
Flores-Cáceres ML(1), Hattab S(2), Hattab S(3), Boussetta H(4), Banni M(4),
Hernández LE(5).
Author information:
(1)Laboratory of Plant Physiology, Department of Biology, Universidad Autónoma de
Madrid, Spain; Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo,
Mendoza, Argentina.
Madrid, Spain; Laboratory of Biochemistry and Environmental Toxicology, Institute
Supérieur Agronomique de Chott-Mariem, Sousse, Tunisia; Centre Regional de
Recherches en Horticulture et Agriculture Biologique, Chott-Mariem, Sousse,
Tunisia.
Madrid, Spain; Laboratory of Biochemistry and Environmental Toxicology, Institute
Supérieur Agronomique de Chott-Mariem, Sousse, Tunisia.
(4)Laboratory of Biochemistry and Environmental Toxicology, Institute Supérieur
Agronomique de Chott-Mariem, Sousse, Tunisia; Centre Regional de Recherches en
Horticulture et Agriculture Biologique, Chott-Mariem, Sousse, Tunisia.
Madrid, Spain. Electronic address: luise.hernandez@uam.es.
The induction of oxidative stress is a characteristic symptom of metal

phytotoxicity and is counteracted by antioxidants such as glutathione (GSH) or
homoglutathione (hGSH). The depletion of GSH│hGSH in fifteen-day-old alfalfa
(Medicago sativa) plants pre-incubated with 1mM buthionine sulfoximine (BSO)
affected antioxidant responses in a metal-specific manner under exposure to
copper (Cu; 0, 6, 30 and 100μM) or cadmium (Cd; 0, 6 and 30μM) for 7 days. The
phytotoxic symptoms observed with excess Cu were accompanied by an inhibition of
root glutathione reductase (GR) activity, a response that was augmented in
Cd-treated plants but reverted when combined with BSO. The synthesis of
phytochelatins (PCs) was induced by Cd, whereas the biothiol concentration
decreased in Cu-treated plants, which did not accumulate PCs. The depletion of
GSH│hGSH by BSO also produced a strong induction of oxidative stress under excess
Cu stress, primarily due to impaired GSH│hGSH-dependent redox homeostasis. In
addition, the synthesis of PCs was required for Cd detoxification, apparently
also determining the distribution of Cd in plants, as less metal was translocated
to the shoots in BSO-incubated plants. Therefore, specific GSH│hGSH-associated
mechanisms of tolerance were triggered by stress due to each metal.
DOI: 10.1016/j.plantsci.2015.01.013

eCollection 2015.
Transcriptome analysis of resistant and susceptible alfalfa cultivars infected

with root-knot nematode Meloidogyne incognita.
Postnikova OA(1), Hult M(2), Shao J(1), Skantar A(2), Nemchinov LG(1).
Author information:
(1)USDA-ARS, Beltsville Agricultural Research Center, Molecular Plant Pathology
Laboratory, Beltsville, Maryland, United States of America.
(2)USDA-ARS, Beltsville Agricultural Research Center, Nematology Laboratory,
Beltsville, Maryland, United States of America.
Nematodes are one of the major limiting factors in alfalfa production. Root-knot
nematodes (RKN, Meloidogyne spp.) are widely distributed and economically
important sedentary endoparasites of agricultural crops and they may inflict
significant damage to alfalfa fields. As of today, no studies have been published
on global gene expression profiling in alfalfa infected with RKN or any other
plant parasitic nematode. Very little information is available about molecular
mechanisms that contribute to pathogenesis and defense responses in alfalfa
against these pests and specifically against RKN. In this work, we performed root
transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan)
alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot
nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end
reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both
cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and
Lahontan, respectively. Bioinformatic analysis revealed a number of common and
unique genes that were differentially expressed in susceptible and resistant
lines as a result of nematode infection. Although the susceptible cultivar showed
a more pronounced defense response to the infection, feeding sites were
successfully established in its roots. Characteristically, basal gene expression
levels under normal conditions differed between the two cultivars as well, which
may confer advantage to one of the genotypes toward resistance to nematodes.
Differentially expressed genes were subsequently assigned to known Gene Ontology
categories to predict their functional roles and associated biological processes.
Real-time PCR validated expression changes in genes arbitrarily selected for
experimental confirmation. Candidate genes that contribute to protection against
M. incognita in alfalfa were proposed and alfalfa-nematode interactions with
respect to resistance are discussed.
PMCID: PMC4339843
516. Mycorrhiza. 2015 Oct;25(7):533-46. doi: 10.1007/s00572-015-0631-x. Epub 2015

Feb
24.
High functional diversity within species of arbuscular mycorrhizal fungi is

associated with differences in phosphate and nitrogen uptake and fungal phosphate
metabolism.
Mensah JA(1), Koch AM(2), Antunes PM(3), Kiers ET(4), Hart M(2), Bücking H(5).
Author information:
(1)Biology and Microbiology Department, South Dakota State University, Brookings,
SD, 57007, USA.
(2)Department of Biology, University of British Columbia Okanagan, Kelowna,
British Columbia, V1V 1V7, Canada.
(3)Department of Biology, Algoma University, Sault Ste. Marie, Ontario, P6A 2G4,
Canada.
(4)Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081
HV, Amsterdam, The Netherlands.
(5)Biology and Microbiology Department, South Dakota State University, Brookings,
SD, 57007, USA. Heike.Bucking@sdstate.edu.
Plant growth responses following colonization with different isolates of a single

species of an arbuscular mycorrhizal (AM) fungus can range from highly beneficial
to detrimental, but the reasons for this high within-species diversity are
currently unknown. To examine whether differences in growth and nutritional
benefits are related to the phosphate (P) metabolism of the fungal symbiont, the
effect of 31 different isolates from 10 AM fungal morphospecies on the P and
nitrogen (N) nutrition of Medicago sativa and the P allocation among different P
pools was examined. Based on differences in the mycorrhizal growth response,
high, medium, and low performance isolates were distinguished. Plant growth
benefit was positively correlated to the mycorrhizal effect on P and N nutrition.
High performance isolates increased plant biomass by more than 170 % and
contributed substantially to both P and N nutrition, whereas the effect of medium
performance isolates particularly on the N nutrition of the host was
significantly lower. Roots colonized by high performance isolates were
characterized by relatively low tissue concentrations of inorganic P and
short-chain polyphosphates and a high ratio between long- to short-chain
polyphosphates. The high performance isolates belonged to different morphospecies
and genera, indicating that the ability to contribute to P and N nutrition is
widespread within the Glomeromycota and that differences in symbiotic performance
and P metabolism are not specific for individual fungal morphospecies.
DOI: 10.1007/s00572-015-0631-x
517. Talanta. 2015 May;136:128-35. doi: 10.1016/j.talanta.2014.11.070. Epub 2014

Dec
22.
Data visualization of Salmonella Typhimurium contamination in packaged fresh

alfalfa sprouts using a Kohonen network.
Siripatrawan U(1), Harte BR(2).
Author information:
(1)Department of Food Technology, Faculty of Science, Chulalongkorn University,
Bangkok, Thailand. Electronic address: ubonratana.s@chula.ac.th.
(2)School of Packaging, Michigan State University, East Lansing, MI, USA.
Class visualization of multi-dimensional data from analysis of volatile metabolic

compounds monitored using an electronic nose based on metal oxide sensor array
was attained using a Kohonen network. An array of 12 metal oxide based chemical
sensors was used to monitor changes in the volatile compositions from the
headspace of packaged fresh sprouts with and without Salmonella Typhimurium
contamination. Kohonen‫׳‬s self-organizing map (SOM) was then created for learning
different patterns of volatile metabolites. The Kohonen network comprising 225
nodes arranged into a two-dimensional hexagonal map was used to locate the
samples on the map to facilitate sample classification. Graphical maps including
the unified matrix, component planes, and hit histograms were described to
characterize the relation between samples. The clustering of samples with
different levels of S. Typhimurium contamination could be visually
distinguishable on the SOM. The Kohonen network proved to be advantageous in
visualization of multi-dimensional nonlinear data and provided a clearer
separation of different sample groups than a conventional linear principal
component analysis (PCA) approach. The sensor array integrated with the Kohonen
network could be used as a rapid and nondestructive method to distinguish samples
with different levels of S. Typhimurium contamination. Although the analyses were
performed on samples with natural background microbiota of about 7 Log(CFU/g),
this microbiota did not affect the S. Typhimurium detection. The proposed method
has potential to rapidly detect a target foodborne pathogen in real-life food
samples instantaneously without subsequently culturing stages.
DOI: 10.1016/j.talanta.2014.11.070
518. Sci Total Environ. 2015 May 15;515-516:60-9. doi:

10.1016/j.scitotenv.2015.02.014. Epub 2015 Feb 17.
Comparative phytotoxicity of ZnO NPs, bulk ZnO, and ionic zinc onto the alfalfa
plants symbiotically associated with Sinorhizobium meliloti in soil.
Bandyopadhyay S(1), Plascencia-Villa G(2), Mukherjee A(1), Rico CM(3),

José-Yacamán M(2), Peralta-Videa JR(4), Gardea-Torresdey JL(5).
Author information:
(1)Environmental Science and Engineering PhD program, The University of Texas at
El Paso, 500 West University Ave., El Paso, TX 79968, United States; University
of California Center for Environmental Implications of Nanotechnology (UC CEIN),
The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968,
United States.
(2)Department of Physics and Astronomy, The University of Texas at San Antonio,
One UTSA Circle, San Antonio, TX 78249, United States.
(3)Department of Chemistry, The University of Texas at El Paso, 500 West
University Ave., El Paso, TX 79968, United States; University of California
Center for Environmental Implications of Nanotechnology (UC CEIN), The University
of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, United States.
El Paso, 500 West University Ave., El Paso, TX 79968, United States; Department
of Chemistry, The University of Texas at El Paso, 500 West University Ave., El
Paso, TX 79968, United States; University of California Center for Environmental
Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, 500
West University Ave., El Paso, TX 79968, United States.
El Paso, 500 West University Ave., El Paso, TX 79968, United States; Department
of Chemistry, The University of Texas at El Paso, 500 West University Ave., El
Paso, TX 79968, United States; University of California Center for Environmental
Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, 500
West University Ave., El Paso, TX 79968, United States. Electronic address:
jgardea@utep.edu.
ZnO nanoparticles (NPs) are reported as potentially phytotoxic in hydroponic and

soil media. However, studies on ZnO NPs toxicity in a plant inoculated with
bacterium in soil are limited. In this study, ZnO NPs, bulk ZnO, and ZnCl₂ were
exposed to the symbiotic alfalfa (Medicago sativa L.)-Sinorhizobium meliloti
association at concentrations ranging from 0 to 750 mg/kg soil. Plant growth, Zn
bioaccumulation, dry biomass, leaf area, total protein, and catalase (CAT)
activity were measured in 30 day-old plants. Results showed 50% germination
reduction by bulk ZnO at 500 and 750 mg/kg and all ZnCl₂ concentrations. ZnO NPs
and ionic Zn reduced root and shoot biomass by 80% and 25%, respectively.
Conversely, bulk ZnO at 750 mg/kg increased shoot and root biomass by 225% and
10%, respectively, compared to control. At 500 and 750 mg/kg, ZnCl₂ reduced CAT
activity in stems and leaves. Total leaf protein significantly decreased as
external ZnCl₂ concentration increased. STEM-EDX imaging revealed the presence of
ZnO particles in the root, stem, leaf, and nodule tissues. ZnO NPs showed less
toxicity compared to ZnCl₂ and bulk ZnO found to be growth enhancing on measured
traits. These findings are significant to reveal the toxicity effects of
different Zn species (NPs, bulk, and ionic Zn) into environmentally important
plant-bacterial system in soil.
DOI: 10.1016/j.scitotenv.2015.02.014
519. Plant Cell Rep. 2015 Apr;34(4):557-72. doi: 10.1007/s00299-015-1772-2. Epub

2015
Feb 19.
Advances in the understanding of cuticular waxes in Arabidopsis thaliana and crop

species.
Lee SB(1), Suh MC.
Author information:
(1)Department of Bioenergy Science and Technology, Chonnam National University,
Gwangju, Korea.
The aerial parts of plants are covered with a cuticle, a hydrophobic layer
consisting of cutin polyester and cuticular waxes that protects them from various
environmental stresses. Cuticular waxes mainly comprise very long chain fatty
acids and their derivatives such as aldehydes, alkanes, secondary alcohols,
ketones, primary alcohols, and wax esters that are also important raw materials
for the production of lubricants, adhesives, cosmetics, and biofuels. The major
function of cuticular waxes is to control non-stomatal water loss and gas
exchange. In recent years, the in planta roles of many genes involved in
cuticular wax biosynthesis have been characterized not only from model organisms
like Arabidopsis thaliana and saltwater cress (Eutrema salsugineum), but also
crop plants including maize, rice, wheat, tomato, petunia, Medicago sativa,
Medicago truncatula, rapeseed, and Camelina sativa through genetic, biochemical,
molecular, genomic, and cell biological approaches. In this review, we discuss
recent advances in the understanding of the biological functions of genes
involved in cuticular wax biosynthesis, transport, and regulation of wax
deposition from Arabidopsis and crop species, provide information on cuticular
wax amounts and composition in various organs of nine representative plant
species, and suggest the important issues that need to be investigated in this
field of study.
DOI: 10.1007/s00299-015-1772-2
520. Huan Jing Ke Xue. 2014 Oct;35(10):3842-50.
[Soil organic carbon storage changes with land reclamation under vegetation
reconstruction on opencast coal mine dump].
Li JC, Dang TH, Guo SL, Xue J, Tang J.
Vegetation reconstruction was an effective solution to reclaim the opencast coal

mine dump which was formed in the process of mining. To understand the impact of
the vegetation reconstruction patterns' on the mine soil organic carbon (SOC)
storage was essential for selecting the methods of vegetation restoration and
also important for accurately estimating the potential of the soil carbon
sequestration. The study area was on the Heidaigou opencast coal mine, which was
15 years reclaimed coal mine dump in Zhungeer, Inner Mongolia autonomous region,
we selected 5 vegetation reconstruction patterns (natural recovery land,
grassland, bush land, mixed woodland of arbor and bush, arbor land), and 16
vegetation types, 408 soil samples (0-100 m), to study the effect of the
vegetation reconstruction patterns on the SOC storage. The results were showed as
follows: (1) on the reclaimed coal mine dump, the vegetation reconstruction
patterns significantly affected the SOC content and its distribution in the soil
profile (P < 0.05). The surface 0-10 cm SOC content was grassland > shrub land >
arbor forest > mixed forest of arbor and shrub > natural recovery land, in which
the grassland, shrub land and arbor forest were about 2.2, 1.3, and 1.3 times of
natural recovery land (2.14 g · kg(-1)) respectively. The total nitrogen (TN)
showed the similar trends. (2) Among the vegetation types, Medicago sativa had
the highest surface SOC content (5.71 g · kg(-1)) and TN content (0.49 g ·
kg(-1)), that were 171.3% and 166.7% higher than the natural recovery land, and
two times of Hippophae rhamnoides, Amorpha fruticosa + Pinus tabulaeformis and
Robinia pseudoacacia. (3) The effect of vegetation types on SOC mainly
concentrated in the 0-20 cm depth, and the effect on TN accounted for 40 cm. (4)
For the SOC storage, the order was original landform area > reclaimed dump > new
dump and grassland > woodland (including arbor and shrub land). After 15 years
revegetation, the soil carbon storage of the grassland, shrub land and arbor land
were increased by 15.47 t · hm(-2), 6.93 t · hm(-2) and 6.95 t · hm(-2)
respectively in the 100 cm depth, which were equivalent to 2/3, 1/2 and 1/2 of
the original landform levels. The results showed a great ability of carbon
sequestration.
521. J Sci Food Agric. 2016 Jan 30;96(2):650-5. doi: 10.1002/jsfa.7136. Epub 2015
Mar
3.
Microbial release of ferulic and p-coumaric acids from forages and their
digestibility in lactating cows fed total mixed rations with different forage
combinations.
Cao BB(1), Jin X(1), Yang HJ(1), Li SL(1), Jiang LS(2).
Author information:
Technology, China Agricultural University, Beijing, 100193, China.
(2)Beijing Key Laboratory for Dairy Cow Nutrition, Beijing University of
Agriculture, Beijing, 102206, China.
BACKGROUND: Ferulic acid (FA) and p-coumaric acid (PCA) are widely distributed in
graminaceous plant cell walls. This study investigated the in vitro and in vivo
digestibility of ester-linked FA (FAest) and PCA (PCAest) in lactating dairy
cows.
RESULTS: Regarding corn stover, ensiled corn stover, whole corn silage, Chinese
wild ryegrass and alfalfa hay with different phenolic acid profiles, the in vitro
rumen digestibility of forage FAest and PCAest was negatively correlated with the
ether-linked FA content and original PCA/FA ratio in the forages. The
concentration of both phenolic acids in culture fluids was low after a 72 h
incubation, and the mixed rumen microorganisms metabolized nearly all phenolic
acids released into the culture fluids. FAest digestibility in the whole
digestive tract was negatively correlated with dietary PCA/FA ratio, but a
converse result occurred with dietary PCAest digestibility. The digestibility in
either the rumen or the whole digestive tract was greater for FAest than for
PCAest.
CONCLUSION: Forage PCAest in comparison with FAest is not easily digested in
either the rumen or the whole digestive tract, and they were negatively affected
by forage FAeth content and lignification extent indicated by the original
dietary PCA/FA ratio.
DOI: 10.1002/jsfa.7136
522. Nat Prod Res. 2015;29(24):2332-5. doi: 10.1080/14786419.2015.1008474. Epub

2015
Feb 12.
Chemical characterisation of bioactive compounds in Medicago sativa growing in

the desert of Oman.
Hanif MA(1)(2), Al-Maskari AY(2), Al-Sabahi JN(3), Al-Hdhrami I(2), Khan MM(2),
Al-Azkawi A(4), Hussain AI(5).
Author information:
(1)a Department of Chemistry , University of Agriculture , Faisalabad , Pakistan.
(2)b Department of Crop Sciences , College of Agricultural and Marine Sciences,
Sultan Qaboos University , Muscat , Oman.
(3)c Central Instrumental Laboratory, College of Agricultural and Marine
Sciences, Sultan Qaboos University , Muscat , Oman.
(4)d Central Analytical and Applied Research Facility, College of Science, Sultan
Qaboos University , Muscat , Oman.
(5)e Department of Applied Chemistry , Government College University , Faisalabad
, Pakistan.
Medicago sativa Linn growing in Omani desert were chemically characterised using
flame photometry, inductively coupled plasma, gas chromatography-mass
spectrometry and high performance liquid chromatographic (HPLC) analysis. HPLC
analyses were performed to determine the phenolics and flavonoids present in M.
sativa. The major compounds detected in M. sativa leaves were protchaechenic acid
(3.22%), hydroxyl benzoic acid (1.05%), β-Phenyl caffate (0.97%) and kaempherol
(0.89%). Pterostilbene, a cholesterol-lowering compound, was detected in M.
sativa.
DOI: 10.1080/14786419.2015.1008474
523. FEMS Microbiol Lett. 2015 Jan;362(2):1-6. doi: 10.1093/femsle/fnu006. Epub

2014
Dec 4.
Pseudomonas fluorescens Pf-5 genome-wide mutant screen for resistance to the

antimicrobial peptide alfalfa snakin-1.
Ayub ND(1), Fox AR(1), García AN(2), Mozzicafreddo M(3), Cuccioloni M(3),
Angeletti M(3), Pagano E(2), Soto G(4).
Author information:
(1)Instituto de Genética Ewald A. Favret (CICVyA-INTA), De los reseros S/N,
Castelar C25 (1712), Buenos Aires, Argentina Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Avda. Rivadavia 1917, C1033AAJ, Cuidad Autónoma
de Buenos Aires, Argentina.
Castelar C25 (1712), Buenos Aires, Argentina.
(3)School of Biosciences and Biotechnology, University of Camerino, 62032
Camerino (MC), Italy.
Castelar C25 (1712), Buenos Aires, Argentina Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Avda. Rivadavia 1917, C1033AAJ, Cuidad Autónoma
de Buenos Aires, Argentina gabysoto80@gmail.com.
Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic

activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the
mode of action against target organisms is poorly understood. As a first step to
elucidate the mechanism, we screened a mutation library of Pseudomonas
fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1
(MsSN1). We identified three biofilm formation-related Pseudomonas mutants that
showed increased resistance to MsSN1. Genetic, physiological and bioinformatics
analysis validated the results of the mutant screens, indicating that bacterial
adhesion protein lapA is probably the target of MsSN1. Collectively, these
findings suggest that snakin-1 acts on microbial adhesion properties.

DOI: 10.1093/femsle/fnu006
CORRIGENDUM: Micromonospora from nitrogen fixing nodules of alfalfa (Medicago

sativa L.). A new promising Plant Probiotic Bacteria.
Martínez-Hidalgo P(1), Galindo-Villardón P(2), Trujillo ME(1), Igual JM(3),

Martínez-Molina E(1).
Author information:
(1)1] Department of Microbiology and Genetics. University of Salamanca. Plaza
Doctores de la Reina s/n. 37007 Salamanca, Spain [2] Unidad Asociada USAL-CSIC
''Interacción Planta-Microorganismo''
(2)Department of Statistics. University of Salamanca. Plaza Doctores de la Reina
s/n. 37007 Salamanca, Spain.
(3)1] Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), Cordel
de Merinas 40-52, 37008 Salamanca, Spain [2] Unidad Asociada USAL-CSIC
''Interacción Planta-Microorganismo''
Erratum for
Sci Rep. 2014;4:6389. Trujillo, Martha E [added].
DOI: 10.1038/srep08271
PMCID: PMC4323650
PMID: 25670343
525. Phytochemistry. 2015 Mar;111:114-23. doi: 10.1016/j.phytochem.2015.01.008.

Epub
2015 Feb 3.
Cell wall integrity, genotoxic injury and PCD dynamics in alfalfa saponin-treated
white poplar cells highlight a complex link between molecule structure and
activity.
Paparella S(1), Tava A(2), Avato P(3), Biazzi E(4), Macovei A(5), Biggiogera
M(6), Carbonera D(7), Balestrazzi A(8).
Author information:
(1)Dipartimento di Biologia e Biotecnologie 'L. Spallanzani', via Ferrata 1,
27100 Pavia, Italy. Electronic address: stefania.paparella01@ateneopv.it.
(2)Consiglio per la Ricerca e la Sperimentazione in Agricoltura - Centro di
Ricerca per le Produzioni Foraggere e Lattiero-Casearie, viale Piacenza 29, 29600
Lodi, Italy. Electronic address: aldo.tava@entecra.it.
(3)Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari
Aldo Moro, via Orabona 4, 70125 Bari, Italy. Electronic address:
pinarosa.avato@uniba.it.
(4)Consiglio per la Ricerca e la Sperimentazione in Agricoltura - Centro di
Ricerca per le Produzioni Foraggere e Lattiero-Casearie, viale Piacenza 29, 29600
Lodi, Italy. Electronic address: elisa.biazzi@entecra.it.
27100 Pavia, Italy. Electronic address: a.macovei@irri.org.
27100 Pavia, Italy. Electronic address: marco.biggiogera@unipv.it.
27100 Pavia, Italy. Electronic address: daniela.carbonera@unipv.it.
27100 Pavia, Italy. Electronic address: alma.balestrazzi@unipv.it.
In the present work, eleven saponins and three sapogenins purified from Medicago
sativa were tested for their cytotoxicity against highly proliferating white
poplar (Populus alba L.) cell suspension cultures. After preliminary screening,
four saponins with different structural features in terms of aglycone moieties
and sugar chains (saponin 3, a bidesmoside of hederagenin; saponins 4 and 5,
monodesmoside and bidesmoside of medicagenic acid respectively, and saponin 10, a
bidesmoside of zanhic acid) and different cytotoxicity were selected and used for
further investigation on their structure-activity relationship. Transmission
Electron Microscopy (TEM) analyses provided for the first time evidence of the
effects exerted by saponins on plant cell wall integrity. Exposure to saponin 3
and saponin 10 resulted into disorganization of the outer wall layer and the
effect was even more pronounced in white poplar cells treated with the two
medicagenic acid derivatives, saponins 4 and 5. Oxidative burst and nitric oxide
accumulation were common hallmarks of the response of white poplar cells to
saponins. When DNA damage accumulation and DNA repair profiles were evaluated by
Single Cell Gel Electrophoresis, induction of single and double strand breaks
followed by effective repair was observed within 24h. The reported data are
discussed in view of the current issues dealing with saponin structure-activity
relationship.
DOI: 10.1016/j.phytochem.2015.01.008
526. Plant Biotechnol J. 2015 Sep;13(7):974-82. doi: 10.1111/pbi.12338. Epub 2015

Feb
6.
Protein accumulation and rumen stability of wheat γ-gliadin fusion proteins in

tobacco and alfalfa.
Sun X(1), Chi-Ham CL(1), Cohen-Davidyan T(2), DeBen C(2), Getachew G(3), DePeters
E(3), Putnam D(2), Bennett A(1).
Author information:
(1)Public Intellectual Property Resource for Agriculture, Department of Plant
Sciences, University of California, Davis, CA, USA.
(2)Department of Plant Sciences, University of California, Davis, CA, USA.
(3)Department of Animal Science, University of California, Davis, CA, USA.
Erratum in
Plant Biotechnol J. 2016 Sep;14 (9):1936.
The nutritional value of various crops can be improved by engineering plants to

produce high levels of proteins. For example, because methionine deficiency
limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa
plants that accumulate high levels of a methionine-rich protein could increase
the nutritional value of that crop. We used three strategies in designing
methionine-rich recombinant proteins that could accumulate to high levels in
plants and thereby serve as candidates for improving the protein quality of
alfalfa forage. In tobacco, two fusion proteins, γ-gliadin-δ-zein and γ-δ-zein,
as well as δ-zein co-expressed with β-zein, all formed protein bodies. However,
the γ-gliadin-δ-zein fusion protein accumulated to the highest level,
representing up to 1.5% of total soluble protein (TSP) in one transformant. In
alfalfa, γ-gliadin-δ-zein accumulated to 0.2% of TSP, and in an in vitro rumen
digestion assay, γ-gliadin-δ-zein was more resistant to microbial degradation
than Rubisco. Additionally, although it did not form protein bodies, a
γ-gliadin-GFP fusion protein accumulated to much higher levels, 7% of TSP, than a
recombinant protein comprised of an ER localization signal fused to GFP in
tobacco. Based on our results, we conclude that γ-gliadin-δ-zein is a potential
candidate protein to use for enhancing methionine levels in plants and for
improving rumen stability of forage protein. γ-gliadin fusion proteins may
provide a general platform for increasing the accumulation of recombinant
proteins in transgenic plants.

DOI: 10.1111/pbi.12338
527. Proc Natl Acad Sci U S A. 2015 Feb 17;112(7):E757-65. doi:

10.1073/pnas.1421748112. Epub 2015 Feb 3.
Novel mixed-linkage β-glucan activated by c-di-GMP in Sinorhizobium meliloti.
Pérez-Mendoza D(1), Rodríguez-Carvajal MÁ(2), Romero-Jiménez L(1), Farias Gde

A(1), Lloret J(3), Gallegos MT(1), Sanjuán J(4).
Author information:
(1)Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008
Granada, Spain;
(2)Departamento Química Orgánica, Facultad de Química, Universidad de Sevilla,
41001 Sevilla, Spain; and.
(3)Departamento Biología, Facultad de Ciencias, Universidad Autónoma de Madrid,
28049 Madrid, Spain.
(4)Departamento Microbiología del Suelo y Sistemas Simbióticos, Estación
Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, 18008
Granada, Spain; Juan.Sanjuan@eez.csic.es.
Erratum in
Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3632.
An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium

meliloti 8530, a bacterium that does not carry known cellulose synthesis genes,
leads to overproduction of a substance that binds the dyes Congo red and
calcofluor. Sugar composition and methylation analyses and NMR studies identified
this compound as a linear mixed-linkage (1 → 3)(1 → 4)-β-D-glucan (ML β-glucan),
not previously described in bacteria but resembling ML β-glucans found in plants
and lichens. This unique polymer is hydrolyzed by the specific endoglucanase
lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic →
4)-β-D-Glcp-(1 → 3)-β-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA
required for production of this ML β-glucan is conserved among several genera
within the order Rhizobiales, where bgsA encodes a glycosyl transferase with
domain resemblance and phylogenetic relationship to curdlan synthases and to
bacterial cellulose synthases. ML β-glucan synthesis is subjected to both
transcriptional and posttranslational regulation. bgsBA transcription is
dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system,
and posttranslational regulation seems to involve allosteric activation of the ML
β-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our
knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 →
4)-β-glucan produced by a bacterium. The S. meliloti ML β-glucan participates in
bacterial aggregation and biofilm formation and is required for efficient
attachment to the roots of a host plant, resembling the biological role of
cellulose in other bacteria.
DOI: 10.1073/pnas.1421748112
PMCID: PMC4343143
528. Biotechnol J. 2015 Apr;10(4):599-609. doi: 10.1002/biot.201400637. Epub 2015

Feb
18.
Production of curcuminoids from tyrosine by a metabolically engineered

Escherichia coli using caffeic acid as an intermediate.
Rodrigues JL(1), Araújo RG, Prather KL, Kluskens LD, Rodrigues LR.
Author information:
(1)Centre of Biological Engineering, University of Minho, Braga, Portugal;
MIT-Portugal Program, Cambridge, MA and Lisbon, Portugal.
Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we

report an engineered artificial pathway in Escherichia coli to produce natural
curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase
and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were
used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic
acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower
concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and
ferulic acid, respectively. Additionally, curcuminoids were produced from
tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine
ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from
Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyltransferase from
Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic
acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of
curcumin were produced, respectively. This is the first time DCS and CURS1 were
used in vivo to produce curcuminoids and that curcumin was produced by feeding
tyrosine. We have shown that curcumin can be produced using a pathway involvoing
caffeic acid. This alternative pathway represents a step forward in the
heterologous production of curcumin using E. coli.
Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/biot.201400637
529. Sensors (Basel). 2015 Jan 28;15(2):2920-43. doi: 10.3390/s150202920.
Estimation of biomass and canopy height in bermudagrass, alfalfa, and wheat using
ultrasonic, laser, and spectral sensors.
Pittman JJ(1), Arnall DB(2), Interrante SM(3), Moffet CA(4), Butler TJ(5).
Author information:
(1)The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA.
jjpittman@noble.org.
(2)Oklahoma State University Department of Plant and Soil Sciences, Stillwater,
OK 74078, USA. b.arnall@okstate.edu.
sminterrante@noble.org.
camoffet@noble.org.
tjbutler@noble.org.
Non-destructive biomass estimation of vegetation has been performed via remote

sensing as well as physical measurements. An effective method for estimating
biomass must have accuracy comparable to the accepted standard of destructive
removal. Estimation or measurement of height is commonly employed to create a
relationship between height and mass. This study examined several types of
ground-based mobile sensing strategies for forage biomass estimation. Forage
production experiments consisting of alfalfa (Medicago sativa L.), bermudagrass
[Cynodon dactylon (L.) Pers.], and wheat (Triticum aestivum L.) were employed to
examine sensor biomass estimation (laser, ultrasonic, and spectral) as compared
to physical measurements (plate meter and meter stick) and the traditional
harvest method (clipping). Predictive models were constructed via partial least
squares regression and modeled estimates were compared to the physically measured
biomass. Least significant difference separated mean estimates were examined to
evaluate differences in the physical measurements and sensor estimates for canopy
height and biomass. Differences between methods were minimal (average percent
error of 11.2% for difference between predicted values versus machine and quadrat
harvested biomass values (1.64 and 4.91 t·ha(-1), respectively), except at the
lowest measured biomass (average percent error of 89% for harvester and quad
harvested biomass < 0.79 t·ha(-1)) and greatest measured biomass (average percent
error of 18% for harvester and quad harvested biomass >6.4 t·ha(-1)). These data
suggest that using mobile sensor-based biomass estimation models could be an
effective alternative to the traditional clipping method for rapid, accurate
in-field biomass estimation.
DOI: 10.3390/s150202920
PMCID: PMC4367341
530. Plant Cell. 2015 Jan;27(1):71-85. doi: 10.1105/tpc.114.133991. Epub 2015 Jan
29.
Genome-wide analysis of alternative splicing landscapes modulated during

plant-virus interactions in Brachypodium distachyon.
Mandadi KK(1), Scholthof KB(2).
Author information:
(1)Department of Plant Pathology and Microbiology, Texas A&M University, College
Station, Texas 77843.
(2)Department of Plant Pathology and Microbiology, Texas A&M University, College
Station, Texas 77843 kbgs@tamu.edu.
Comment in
Plant Cell. 2015 Jan;27(1):7.
In eukaryotes, alternative splicing (AS) promotes transcriptome and proteome

diversity. The extent of genome-wide AS changes occurring during a plant-microbe
interaction is largely unknown. Here, using high-throughput, paired-end RNA
sequencing, we generated an isoform-level spliceome map of Brachypodium
distachyon infected with Panicum mosaic virus and its satellite virus. Overall,
we detected ∼44,443 transcripts in B. distachyon, ∼30% more than those annotated
in the reference genome. Expression of ∼28,900 transcripts was ≥2 fragments per
kilobase of transcript per million mapped fragments, and ∼42% of multi-exonic
genes were alternatively spliced. Comparative analysis of AS patterns in B.
distachyon, rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor),
Arabidopsis thaliana, potato (Solanum tuberosum), Medicago truncatula, and poplar
(Populus trichocarpa) revealed conserved ratios of the AS types between monocots
and dicots. Virus infection quantitatively altered AS events in Brachypodium with
little effect on the AS ratios. We discovered AS events for >100 immune-related
genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription
factors, RNA silencing, and splicing-associated proteins. Cloning and molecular
characterization of SCL33, a serine/arginine-rich splicing factor, identified
multiple novel intron-retaining splice variants that are developmentally
regulated and modulated during virus infection. B. distachyon SCL33 splicing
patterns are also strikingly conserved compared with a distant Arabidopsis SCL33
ortholog. This analysis provides new insights into AS landscapes conserved among
monocots and dicots and uncovered AS events in plant defense-related genes.
DOI: 10.1105/tpc.114.133991
PMCID: PMC4330581
531. Environ Microbiol. 2015 Aug;17(8):2791-804. doi: 10.1111/1462-2920.12778. Epub

2015 Mar 4.
Community composition and population genetics of insect pathogenic fungi in the

genus Metarhizium from soils of a long-term agricultural research system.
Kepler RM(1), Ugine TA(2), Maul JE(3), Cavigelli MA(3), Rehner SA(1).
Author information:
(1)Systematic Mycology and Microbiology Laboratory, 10300 Baltimore Ave., Bldg.
010A, Beltsville, MD, 20705, USA.
(2)Department of Entomology, Cornell University, Ithaca, NY, 14853, USA.
(3)Sustainable Agricultural Systems Laboratory, USDA-ARS, 10300 Baltimore Ave.,
Bldg. 001, BARC-W, Beltsville, MD, 20705, USA.
Fungi in the genus Metarhizium are insect pathogens able to function in other
niches, including soil and plant rhizosphere habitats. In agroecosystems,
cropping and tillage practices influence soil fungal communities with unknown
effects on the distribution of Metarhizium, whose presence can reduce populations
of crop pests. We report results from a selective media survey of Metarhizium in
soils sampled from a long-term experimental farming project in the mid-Atlantic
region. Field plots under soybean cultivation produced higher numbers of
Metarhizium colony-forming units (cfu) than corn or alfalfa. Plots managed
organically and via chisel-till harboured higher numbers of Metarhizium cfu than
no-till plots. Sequence typing of Metarhizium isolates revealed four species,
with M. robertsii and M. brunneum predominating. The M. brunneum population was
essentially fixed for a single clone as determined by multilocus microsatellite
genotyping. In contrast, M. robertsii was found to contain significant diversity,
with the majority of isolates distributed between two principal clades. Evidence
for recombination was observed only in the most abundant clade. These findings
illuminate multiple levels of Metarhizium diversity that can be used to inform
strategies by which soil Metarhizium populations may be manipulated to exert
downward pressure on pest insects and promote plant health.
DOI: 10.1111/1462-2920.12778
Chemosensillum immunolocalization and ligand specificity of chemosensory proteins

in the alfalfa plant bug Adelphocoris lineolatus (Goeze).
Sun L(1), Zhou JJ(2), Gu SH(3), Xiao HJ(4), Guo YY(3), Liu ZW(5), Zhang YJ(3).
Author information:
(1)1] State Key Laboratory for Biology of Plant Diseases and Insect Pests,
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing,
100193, China [2] Key Laboratory of Tea Plants Biology and Resources Utilization
of Agriculture Ministry, Tea Research Institute, Chinese Academy of Agricultural
Sciences, Hangzhou, 310008, China [3] Key Laboratory of Integrated Management of
Crop Diseases and Pests (Ministry of Education), College of Plant Protection,
Nanjing Agricultural University, Nanjing, 210095, China.
(2)Department of Biological Chemistry and Crop Protection, Rothamsted Research,
Harpenden, AL5 2JQ, UK.
China.
(4)1] State Key Laboratory for Biology of Plant Diseases and Insect Pests,
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing,
100193, China [2] Institute of Entomology, Jiangxi Agricultural University,
Nanchang, 330045, China.
(5)Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry
of Education), College of Plant Protection, Nanjing Agricultural University,
Nanjing, 210095, China.
Insect chemosensory proteins (CSPs) are a family of small soluble proteins. To
date, their physiological functions in insect olfaction remain largely
controversial in comparison to odorant binding proteins (OBPs). In present study,
we reported the antenna specific expression of three CSPs (AlinCSP4-6) from
Adelphocoris lineolatus, their distinct chemosensillum distribution as well as
ligand binding capability thus providing the evidence for the possible roles that
they could play in semiochemical detection of the plant bug A. lineolatus. The
results of qRT-PCR and western blot assay clearly showed that all of these three
CSPs are highly expressed in the adult antennae, the olfactory organ of insects.
Further cellular investigation of their immunolocalization revealed their dynamic
protein expression profiles among different types of antennal sensilla. In a
fluorescence competitive binding assay, the selective ligand binding was observed
for AlinCSP4-6. In ad`dition, a cooperative interaction was observed between two
co-expressed CSPs resulting in an increase of the binding affinities by a mixture
of AlinCSP5 and AlinCSP6 to terpenoids which do not bind to individual CSPs.
These findings in combination with our previous data for AlinCSP1-3 indicate a
possible functional differentiation of CSPs in the A. lineolatus olfactory
system.
DOI: 10.1038/srep08073
PMCID: PMC4308698
533. Appl Microbiol Biotechnol. 2015 May;99(9):4071-84. doi:

10.1007/s00253-014-6320-4. Epub 2015 Jan 16.
Enrichment of aliphatic, alicyclic and aromatic acids by oil-degrading bacteria

isolated from the rhizosphere of plants growing in oil-contaminated soil from
Kazakhstan.
Mikolasch A(1), Omirbekova A, Schumann P, Reinhard A, Sheikhany H, Berzhanova R,

Mukasheva T, Schauer F.
Author information:
(1)Department of Applied Microbiology, Institute of Microbiology, University
Greifswald, Friedrich-Ludwig-Jahn-Str. 15, 17487, Greifswald, Germany,
annett.mikolasch@uni-greifswald.de.
Three microbial strains were isolated from the rhizosphere of alfalfa (Medicago
sativa), grass mixture (Festuca rubra, 75 %; Lolium perenne, 20 %; Poa pratensis,
10 %), and rape (Brassica napus) on the basis of their high capacity to use crude
oil as the sole carbon and energy source. These isolates used an unusually wide
spectrum of hydrocarbons as substrates (more than 80), including n-alkanes with
chain lengths ranging from C12 to C32, monomethyl- and monoethyl-substituted
alkanes (C12-C23), n-alkylcyclo alkanes with alkyl chain lengths from 4 to 18
carbon atoms, as well as substituted monoaromatic and diaromatic hydrocarbons.
These three strains were identified as Gordonia rubripertincta and Rhodococcus
sp. SBUG 1968. During their transformation of this wide range of hydrocarbon
substrates, a very large number of aliphatic, alicyclic, and aromatic acids was
detected, 44 of them were identified by GC/MS analyses, and 4 of them are
described as metabolites for the first time. Inoculation of plant seeds with
these highly potent bacteria had a beneficial effect on shoot and root
development of plants which were grown on oil-contaminated sand.
DOI: 10.1007/s00253-014-6320-4
534. J Hazard Mater. 2015 Apr 9;286:269-75. doi: 10.1016/j.jhazmat.2014.12.049.
Epub
2014 Dec 27.
Enhanced tolerance and remediation to mixed contaminates of PCBs and 2,4-DCP by

transgenic alfalfa plants expressing the 2,3-dihydroxybiphenyl-1,2-dioxygenase.
Wang Y(1), Ren H(2), Pan H(3), Liu J(3), Zhang L(4).
Author information:
Road, Changchun, Jilin 130021, People's Republic of China; College of Plant
Sciences, Jilin University, 5333 Xi'an Road, Changchun, Jilin 130062, People's
Republic of China.
Road, Changchun, Jilin 130021, People's Republic of China. Electronic address:
renhejun@126.com.
(3)College of Plant Sciences, Jilin University, 5333 Xi'an Road, Changchun, Jilin
130062, People's Republic of China.
Road, Changchun, Jilin 130021, People's Republic of China.
Polychlorinated biphenyls (PCBs) and 2,4-dichlorophenol (2,4-DCP) generally led

to mixed contamination of soils as a result of commercial and agricultural
activities. Their accumulation in the environment poses great risks to human and
animal health. Therefore, the effective strategies for disposal of these
pollutants are urgently needed. In this study, genetic engineering to enhance
PCBs/2,4-DCP phytoremediation is a focus. We cloned the
2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC.B) from a soil metagenomic library,
which is the key enzyme of aerobic catabolism of a variety of aromatic compounds,
and then it was expressed in alfalfa driven by CaMV 35S promoter using
Agrobacterium-mediated transformation. Transgenic line BB11 was selected out
through PCR, Western blot analysis and enzyme activity assays. Its disposal and
tolerance to both PCBs and 2,4-DCP were examined. The tolerance capability of
transgenic line BB11 towards complex contaminants of PCBs/2,4-DCP significantly
increased compared with non-transgenic plants. Strong dissipation of PCBs and
high removal efficiency of 2,4-DCP were exhibited in a short time. It was
confirmed expressing BphC.B would be a feasible strategy to help achieving
phytoremediation in mixed contaminated soils with PCBs and 2,4-DCP.
DOI: 10.1016/j.jhazmat.2014.12.049
535. Appl Microbiol Biotechnol. 2015 Jun;99(11):4855-65. doi:

10.1007/s00253-014-6347-6. Epub 2015 Jan 15.
Alfalfa microsymbionts from different ITS and nodC lineages of Ensifer meliloti
and Ensifer medicae symbiovar meliloti establish efficient symbiosis with alfalfa
in Spanish acid soils.
Ramírez-Bahena MH(1), Vargas M, Martín M, Tejedor C, Velázquez E, Peix Á.
Author information:
(1)Instituto de Recursos Naturales y Agrobiología (IRNASA-CSIC), Cordel de
Merinas 40-52, 37008, Salamanca, Spain.
Alfalfa (Medicago sativa L.) is an important crop worldwide whose cropping in

acid soils is hampered by the poor nodulation and yield commonly attributed to
the sensitivity of its endosymbionts to acid pH. In this work, we isolated
several acid-tolerant strains from alfalfa nodules in three acid soils in
northwestern Spain. After grouping by RAPD fingerprinting, most strains were
identified as Ensifer meliloti and only two strains as Ensifer medicae according
to their 16S-23S intergenic spacer (ITS) sequences that allowed the
differentiation of two groups within each one of these species. The two ITS
groups of E. meliloti and the ITS group I of E. medicae have been previously
found in Medicago nodules; however, the group II of E. medicae has been only
found to date in Prosopis alba nodules. The analysis of the nodC gene showed that
all strains isolated in this study belong to the symbiovar meliloti, grouping
with the type strains of E. meliloti or E. medicae, but some harboured nodC gene
alleles different from those found to date in alfalfa nodules. The strains of E.
medicae belong to the symbiovar meliloti which should be also recognised in this
species, although they harboured a nodC allele phylogenetically divergent to
those from E. meliloti strains. Microcosm experiments showed that inoculation of
alfalfa with selected acid-tolerant strains significantly increased yields in
acid soils representing a suitable agricultural practice for alfalfa cropping in
these soils.
DOI: 10.1007/s00253-014-6347-6
536. J Agric Food Chem. 2015 Jan 21;63(2):406-14. doi: 10.1021/jf503658n. Epub 2015
Jan 9.
Interrelations between herbage yield, α-tocopherol, β-carotene, lutein, protein,

and fiber in non-leguminous forbs, forage legumes, and a grass-clover mixture as
affected by harvest date.
Elgersma A(1), Søegaard K, Jensen SK.
Author information:
(1)Independent Scientist , P.O. Box 323, 6700 AH Wageningen, The Netherlands.
Pastures with diverse botanical composition may enhance animal-derived product

quality. A recent study demonstrated high vitamin concentrations and yields in
some forb species. The objectives of the present study were to investigate
interrelations between herbage yields, vitamin concentrations, protein and fiber
contents and analyze the effect of harvest date. We hypothesized that
interrelations would be similar across investigated forage species. Four
nonleguminous forbs: salad burnet (Sanguisorba minor), caraway (Carum carvi),
chicory (Cichorium intybus), and ribwort plantain (Plantago lanceolata), three
legumes: yellow sweet clover (Melilotus officinalis), lucerne (Medicago sativa),
and birdsfoot trefoil (Lotus corniculatus) and a perennial ryegrass (Lolium
perenne)-white clover (Trifolium repens) mixture were sown in a field trial with
two replicated and randomized blocks. Forage in 1.5 m × 9 m plots was grown in
two consecutive years and cut four times per year (May-October). Analyses of
variance were performed. In most herbages, α-tocopherol and β-carotene were
positively correlated as were β-carotene and lutein; all vitamins were negatively
correlated with fiber content and herbage yield. β-Carotene was positively
correlated with protein content. α-Tocopherol and β-carotene contents were
generally highest in October and lowest in July. Our results showed similar
interrelationships in most investigated species, and we suggest that these
species may be mixed when designing novel biodiverse mixtures for particular
product quality characteristics.
DOI: 10.1021/jf503658n
537. Plant Mol Biol. 2015 Feb;87(3):317-27. doi: 10.1007/s11103-014-0278-6. Epub

2014
Dec 31.
The wheat NHX antiporter gene TaNHX2 confers salt tolerance in transgenic alfalfa
by increasing the retention capacity of intracellular potassium.
Zhang YM(1), Zhang HM, Liu ZH, Li HC, Guo XL, Li GL.
Author information:
(1)Institute of Genetics and Physiology, Hebei Academy of Agriculture and
Forestry Sciences, Plant Genetic Engineering Center of Hebei Province,
Shijiazhuang, 050051, China, zhym63@sohu.com.
Previous studies have shown that TaNHX2 transgenic alfalfa (Medicago sativa L.)
accumulated more K(+) and less Na(+) in leaves than did the wild-type plants. To
investigate whether the increased K(+) accumulation in transgenic plants is
attributed to TaNHX2 gene expression and whether the compartmentalization of
Na(+) into vacuoles or the intracellular compartmentalization of potassium is the
critical mechanism for TaNHX2-dependent salt tolerance in transgenic alfalfa,
aerated hydroponic culture was performed under three different stress conditions:
control condition (0.1 mM Na(+) and 6 mM K(+) inside culture solution),
K(+)-sufficient salt stress (100 mM NaCl and 6 mM K(+)) and K(+)-insufficient
salt stress (100 mM NaCl and 0.1 mM K(+)). The transgenic alfalfa plants had
lower K(+) efflux through specific K(+) channels and higher K(+) absorption
through high-affinity K(+) transporters than did the wild-type plants. Therefore,
the transgenic plants had greater K(+) contents and [K(+)]/[Na(+)] ratios in leaf
tissue and cell sap. The intracellular compartmentalization of potassium is
critical for TaNHX2-induced salt tolerance in transgenic alfalfa.
DOI: 10.1007/s11103-014-0278-6
538. Nat Plants. 2015 Mar 30;1(4):15033. doi: 10.1038/nplants.2015.33.
Complementary effects of species and genetic diversity on productivity and

stability of sown grasslands.
Prieto I(1), Violle C(1), Barre P(2), Durand JL(2), Ghesquiere M(2), Litrico
I(2).
Author information:
(1)CNRS, CEFE UMR 5175, Université de Montpellier - Université Paul Valéry -
EPHE, 1919 Route de Mende, Montpellier Cedex 5 34293, France.
(2)INRA, URP3F, RD 150, site du chêne, BP 86006, Lusignan 86600, France.
Plant species diversity regulates the productivity(1-3) and stability(2,4) of

natural ecosystems, along with their resilience to disturbance(5,6). The
influence of species diversity on the productivity of agronomic systems is less
clear(7-10). Plant genetic diversity is also suspected to influence ecosystem
function(3,11-14), although empirical evidence is scarce. Given the large range
of genotypes that can be generated per species through artificial selection,
genetic diversity is a potentially important leverage of productivity in
cultivated systems. Here we assess the effect of species and genetic diversity on
the production and sustainable supply of livestock fodder in sown grasslands,
comprising single and multispecies assemblages characterized by different levels
of genetic diversity, exposed to drought and non-drought conditions. Multispecies
assemblages proved more productive than monocultures when subject to drought,
regardless of the number of genotypes per species present. Conversely, the
temporal stability of production increased only with the number of genotypes
present under both drought and non-drought conditions, and was unaffected by the
number of species. We conclude that taxonomic and genetic diversity can play
complementary roles when it comes to optimizing livestock fodder production in
managed grasslands, and suggest that both levels of diversity should be
considered in plant breeding programmes designed to boost the productivity and
resilience of managed grasslands in the face of increasing environmental hazards.
DOI: 10.1038/nplants.2015.33
539. Plants (Basel). 2015 Aug 11;4(3):548-72. doi: 10.3390/plants4030548.
Abscisic Acid: Hidden Architect of Root System Structure.
Harris JM(1).
Author information:
(1)Department of Plant Biology, University of Vermont, Burlington, VT 05401, USA.
Jeanne.harris@uvm.edu.
Plants modulate root growth in response to changes in the local environment,

guided by intrinsic developmental genetic programs. The hormone Abscisic Acid
(ABA) mediates responses to different environmental factors, such as the presence
of nitrate in the soil, water stress and salt, shaping the structure of the root
system by regulating the production of lateral roots as well as controlling root
elongation by modulating cell division and elongation. Curiously, ABA controls
different aspects of root architecture in different plant species, perhaps
providing some insight into the great diversity of root architecture in different
plants, both from different taxa and from different environments. ABA is an
ancient signaling pathway, acquired well before the diversification of land
plants. Nonetheless, how this ancient signaling module is implemented or
interacts within a larger signaling network appears to vary in different species.
This review will examine the role of ABA in the control of root architecture,
focusing on the regulation of lateral root formation in three plant species,
Arabidopsis thaliana, Medicago truncatula and Oryza sativa. We will consider how
the implementation of the ABA signaling module might be a target of natural
selection, to help contribute to the diversity of root architecture in nature.
DOI: 10.3390/plants4030548
PMCID: PMC4844405
PMID: 27135341
540. BMC Bioinformatics. 2014 Dec 30;15:423. doi: 10.1186/s12859-014-0423-x.
Prediction of plant pre-microRNAs and their microRNAs in genome-scale sequences

using structure-sequence features and support vector machine.
Meng J(1), Liu D(2), Sun C(3), Luan Y(4).

Author information:
(1)School of Computer Science and Technology, Dalian University of Technology,
Dalian, Liaoning, 116023, China. mengjun@dlut.edu.cn.
Dalian, Liaoning, 116023, China. kobe_liudong@mail.dlut.edu.cn.
Dalian, Liaoning, 116023, China. 253376871@qq.com.
(4)School of Life Science and Biotechnology, Dalian University of Technology,
Dalian, Liaoning, 116023, China. luanyush@dlut.edu.cn.
BACKGROUND: MicroRNAs (miRNAs) are a family of non-coding RNAs approximately 21

nucleotides in length that play pivotal roles at the post-transcriptional level
in animals, plants and viruses. These molecules silence their target genes by
degrading transcription or suppressing translation. Studies have shown that
miRNAs are involved in biological responses to a variety of biotic and abiotic
stresses. Identification of these molecules and their targets can aid the
understanding of regulatory processes. Recently, prediction methods based on
machine learning have been widely used for miRNA prediction. However, most of
these methods were designed for mammalian miRNA prediction, and few are available
for predicting miRNAs in the pre-miRNAs of specific plant species. Although the
complete Solanum lycopersicum genome has been published, only 77 Solanum
lycopersicum miRNAs have been identified, far less than the estimated number.
Therefore, it is essential to develop a prediction method based on machine
learning to identify new plant miRNAs.
RESULTS: A novel classification model based on a support vector machine (SVM) was
trained to identify real and pseudo plant pre-miRNAs together with their miRNAs.
An initial set of 152 novel features related to sequential structures was used to
train the model. By applying feature selection, we obtained the best subset of 47
features for use with the Back Support Vector Machine-Recursive Feature
Elimination (B-SVM-RFE) method for the classification of plant pre-miRNAs. Using
this method, 63 features were obtained for plant miRNA classification. We then
developed an integrated classification model, miPlantPreMat, which comprises
MiPlantPre and MiPlantMat, to identify plant pre-miRNAs and their miRNAs. This
model achieved approximately 90% accuracy using plant datasets from nine plant
species, including Arabidopsis thaliana, Glycine max, Oryza sativa,
Physcomitrella patens, Medicago truncatula, Sorghum bicolor, Arabidopsis lyrata,
Zea mays and Solanum lycopersicum. Using miPlantPreMat, 522 Solanum lycopersicum
miRNAs were identified in the Solanum lycopersicum genome sequence.
CONCLUSIONS: We developed an integrated classification model, miPlantPreMat,
based on structure-sequence features and SVM. MiPlantPreMat was used to identify
both plant pre-miRNAs and the corresponding mature miRNAs. An improved feature
selection method was proposed, resulting in high classification accuracy,
sensitivity and specificity.
DOI: 10.1186/s12859-014-0423-x
PMCID: PMC4310204
541. Environ Toxicol Chem. 2015 May;34(5):968-76. doi: 10.1002/etc.2854. Epub 2015
Mar
18.
Fate of flame retardants and the antimicrobial agent triclosan in planted and
unplanted biosolid-amended soils.
Davis EF(1), Gunsch CK, Stapleton HM.

Author information:
(1)Nicholas School of the Environment, Duke University, Durham, North Carolina,
USA.
A comprehensive understanding of the fate of contaminant-laden biosolids is

needed to fully evaluate the environmental impacts of biosolid land application.
The present study examined the fate of several flame retardants and triclosan in
biosolid-amended soil in a 90-d greenhouse experiment. Objectives included
evaluating the persistence of these compounds in soil, their phytoaccumulation
potential by alfalfa (Medicago sativa), and potential degradation reactions.
Concentrations of the polybrominated diphenyl ether (PBDE) congeners BDE-47 and
BDE-209 and the antimicrobial triclosan declined significantly over time in
biosolid-amended soil planted with alfalfa and then reached a steady state by day
28. In contrast, no significant losses of those analytes were observed from soil
in nonvegetated pots. The amount of an analyte lost from vegetated soil ranged
from 43% for the flame retardant di(2-ethylhexyl)-2,3,4,5-tetrabromophthalate to
61% for triclosan and was significantly and negatively related to the log
octanol-water partition coefficient. Alfalfa roots and shoots were monitored for
the compounds, but no clear evidence of phytoaccumulation was observed. Methyl
triclosan formation was observed in the biosolid-amended soils during the study
period, indicating in situ biotransformation of triclosan. The present study
demonstrates that, although they are highly recalcitrant, PBDEs, selected
alternate brominated flame retardants, and triclosan are capable of undergoing
dissipation from biosolid-amended soils in the presence of plants.
© 2014 SETAC.
DOI: 10.1002/etc.2854

eCollection 2014.
Identification of molecular markers associated with Verticillium wilt resistance

in alfalfa (Medicago sativa L.) using high-resolution melting.
Zhang T(1), Yu LX(1), McCord P(1), Miller D(2), Bhamidimarri S(2), Johnson D(3),
Monteros MJ(4), Ho J(5), Reisen P(5), Samac DA(6).
Author information:
(1)United States Department of Agriculture, Agricultural Research Service, Plant
Germplasm Introduction and Testing Research, 24106 N Bunn Road, Prosser,
Washington, United States of America.
(2)DuPont Pioneer, W8131 State HWY 60, Arlington, Wisconsin, United States of
America.
(3)Alforex Seeds, N4505 CTH M, West Salem, Wisconsin, United States of America.
(4)Forage Improvement Division, Samuel Roberts Noble Foundation, 2510 Sam Noble
Parkway, Ardmore, Oklahoma, United States of America.
(5)Forage Genetics International, Inc. 7661 Becker Road, Davis, California,
(6)United States Department of Agriculture, Agricultural Research Service, 495
Borlaug Hall, 1991 Upper Buford Circle, Saint Paul, Minnesota, United States of
America.
Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one

of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To
identify loci associated with resistance to Verticillium wilt, a bulk segregant
analysis was conducted in susceptible or resistant pools constructed from 13
synthetic alfalfa populations, followed by association mapping in two F1
populations consisted of 352 individuals. Simple sequence repeat (SSR) and single
nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was
done by manual inoculation of the pathogen to replicated cloned plants of each
individual and disease severity was scored using a standard scale. Marker-trait
association was analyzed by TASSEL. Seventeen SNP markers significantly
associated with Verticillium wilt resistance were identified and they were
located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2,
4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in
M. truncatula. Additional markers identified on chromosomes 1 and 8 located the
regions where no Verticillium resistance locus has been reported. This study
highlights the value of SNP genotyping by high resolution melting to identify the
disease resistance loci in tetraploid alfalfa. With further validation, the
markers identified in this study could be used for improving resistance to
Verticillium wilt in alfalfa breeding programs.
PMCID: PMC4275272
543. Plant Biotechnol J. 2015 Aug;13(6):779-90. doi: 10.1111/pbi.12308. Epub 2014

Dec
23.
MicroRNA156 as a promising tool for alfalfa improvement.
Aung B(1)(2), Gruber MY(3), Amyot L(1), Omari K(1), Bertrand A(4), Hannoufa
A(1)(2).
Author information:
(2)Biology Department, Western University, London, ON, Canada.
(3)Agriculture and Agri-Food Canada, Saskatoon, SK, Canada.
(4)Agriculture and Agri-Food Canada, St. Foy, QC, Canada.
A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa

(Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five
predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13)
contain miR156 cleavage sites and their expression was down-regulated in
transgenic alfalfa plants overexpressing miR156. These transgenic plants had
reduced internode length and stem thickness, enhanced shoot branching, increased
trichome density, a delay in flowering time and elevated biomass production.
Minor effects on sugar, starch, lignin and cellulose contents were also observed.
Moreover, transgenic alfalfa plants had increased root length, while nodulation
was maintained. The multitude of traits affected by miR156 may be due to the
network of genes regulated by the three target SPLs. Our results show that the
miR156/SPL system has strong potential as a tool to substantially improve quality
and yield traits in alfalfa.

DOI: 10.1111/pbi.12308
544. J Dairy Sci. 2015 Mar;98(3):1938-49. doi: 10.3168/jds.2014-8634. Epub 2014 Dec
18.
Milk production responses to dietary stearic acid vary by production level in
dairy cattle.
Piantoni P(1), Lock AL(1), Allen MS(2).
Author information:
Electronic address: allenm@msu.edu.
Effects of stearic acid supplementation on feed intake and metabolic and

production responses of dairy cows with a wide range of milk production (32.2 to
64.4 kg/d) were evaluated in a crossover design experiment with a covariate
period. Thirty-two multiparous Holstein cows (142±55 d in milk) were assigned
randomly within level of milk yield to treatment sequence. Treatments were diets
supplemented (2% of diet dry matter) with stearic acid (SA; 98% C18:0) or control
(soyhulls). The diets were based on corn silage and alfalfa and contained 24.5%
forage neutral detergent fiber, 25.1% starch, and 17.3% crude protein. Treatment
periods were 21 d with the final 4 d used for data and sample collection.
Compared with the control, SA increased dry matter intake (DMI; 26.1 vs. 25.2
kg/d) and milk yield (40.2 vs. 38.5 kg/d). Stearic acid had no effect on the
concentration of milk components but increased yields of fat (1.42 vs. 1.35
kg/d), protein (1.19 vs. 1.14 kg/d), and lactose (1.96 vs. 1.87 kg/d). The SA
treatment increased 3.5% fat-corrected milk (3.5% FCM; 40.5 vs. 38.6 kg/d) but
did not affect feed efficiency (3.5% FCM/DMI, 1.55 vs. 1.53), body weight, or
body condition score compared with the control. Linear interactions between
treatment and level of milk yield during the covariate period were detected for
DMI and yields of milk, fat, protein, lactose, and 3.5% FCM; responses to SA were
positively related to milk yield of cows. The SA treatment increased crude
protein digestibility (67.4 vs. 65.5%), tended to increase neutral detergent
fiber digestibility (43.6 vs. 42.3%), decreased fatty acid (FA) digestibility
(56.6 vs. 76.1%), and did not affect organic matter digestibility. Fatty acid
yield response, calculated as the additional FA yield secreted in milk per unit
of additional FA intake, was only 13.3% for total FA and 8.2% for C18:0 plus
cis-9 C18:1. Low estimated digestibility of the SA supplement was at least partly
responsible for the low FA yield response. Treatment did not affect plasma
insulin, glucagon, glucose, and nonesterified FA concentrations. Results show
that stearic acid has the potential to increase DMI and yields of milk and milk
components, without affecting conversion of feed to milk, body condition score,
or body weight. Moreover, effects on DMI and yields of milk and milk components
were more pronounced for higher-yielding cows than for lower-yielding cows.

DOI: 10.3168/jds.2014-8634
545. Parasite. 2014;21:71. doi: 10.1051/parasite/2014074. Epub 2014 Dec 22.
Anthelmintic effect of carob pods and sainfoin hay when fed to lambs after
experimental trickle infections with Haemonchus contortus and Trichostrongylus
colubriformis.
Arroyo-Lopez C(1), Manolaraki F(2), Saratsis A(3), Saratsi K(3), Stefanakis A(3),
Skampardonis V(3), Voutzourakis N(3), Hoste H(2), Sotiraki S(3).
Author information:
(1)Veterinary Research Institute - Hellenic Agricultural Organization Demeter,
57001 Thermi, Thessaloniki, Greece - UMR 1225 IHAP INRA/ENVT, École Nationale
Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex, France.
(2)UMR 1225 IHAP INRA/ENVT, École Nationale Vétérinaire de Toulouse, 23 Chemin
des Capelles, 31076 Toulouse Cedex, France.
(3)Veterinary Research Institute - Hellenic Agricultural Organization Demeter,
57001 Thermi, Thessaloniki, Greece.
The aim of the study was to compare the in vivo anthelmintic activity of sainfoin
hay (Onobrychis viciifolia) and carob pod meal (Ceratonia siliqua) against
gastrointestinal nematodes. Seven days before infection, 64 naive lambs were
assigned to four different groups: Group S received sainfoin hay and group CAR
was fed with carob pods. The remaining lambs received lucerne hay (Medicago
sativa) and were assigned to positive (non-treated, NT) and negative (treated, T)
control groups (treatment with albendazole). On day 0, lambs were artificially
trickle infected for 6 weeks, with a mixture of infective larvae of Haemonchus
contortus and Trichostrongylus colubriformis. Parasitological and
pathophysiological parameters were measured repeatedly during the 2-month study.
Compared to the NT group, decreases in egg excretion were observed in the CAR and
S groups with significant differences only found for sainfoin (p < 0.05). At
necropsy, group S showed decreases in the total worm numbers of both nematode
species with significant differences for H. contortus. In contrast, no
differences were noticed for the CAR group. Compared to the NT group, lower
values for fecundity of female H. contortus were found in the S and CAR groups,
however differences were non-significant. No differences in body weight gains
were found between groups. Consistent results were found showing significantly
higher packed cell volume (PCV) values in the T and S groups compared to NT and
CAR groups. Overall, these results confirm a positive effect associated with the
feeding of lambs with tanniniferous resources on host resilience (PCV values) and
against gastrointestinal parasitic nematodes by affecting some biological traits
of worm populations (e.g. eggs per gram of faeces and worm numbers). However, the
anthelmintic effects differed between the two tannin-containing resources, which
might be associated with the quantity and/or quality of secondary metabolites
(condensed tannins and/or other polyphenols).
© C. Arroyo-Lopez et al., published by EDP Sciences, 2014.
DOI: 10.1051/parasite/2014074
PMCID: PMC4271655
546. Curr Microbiol. 2015 Apr;70(4):556-61. doi: 10.1007/s00284-014-0744-z. Epub

2014
Dec 18.
Characterization of a novel two-component system in Burkholderia cenocepacia.
Merry CR(1), Perkins M, Mu L, Peterson BK, Knackstedt RW, Weingart CL.
Author information:
(1)Case Western University, Cleveland, USA, merrycallie@gmail.com.
Two-component systems are important regulatory systems that allow bacteria to

adjust to environmental conditions, and in some bacteria are used in
pathogenesis. We identified a novel two-component system in Burkholderia
cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis
(CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a
response regulator. Our studies indicated that the bceR mutant showed a
statistically significant decrease in protease, swimming motility, and quorum
sensing when compared to the wild-type, but there was no significant difference
in phospholipase C activity, swarming, and biofilm formation. In addition, the
mutant showed a statistically significant reduction in virulence compared to the
wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia
complex (a group of organisms that are phenotypically similar, but genotypically
distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans,
B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been
associated with CF patients. The collective results indicate that BceSR
influences various activities important in Burkholderia physiology and possibly
pathogenesis. This information could be important in the design of novel
therapeutics for Burkholderia infections.
DOI: 10.1007/s00284-014-0744-z
547. Plant Cell Physiol. 2015 Jan;56(1):e9. doi: 10.1093/pcp/pcu188. Epub 2014 Dec
11.
Plant Omics Data Center: an integrated web repository for interspecies gene
expression networks with NLP-based curation.
Ohyanagi H(1), Takano T(2), Terashima S(2), Kobayashi M(3), Kanno M(4), Morimoto
K(4), Kanegae H(4), Sasaki Y(3), Saito M(4), Asano S(3), Ozaki S(3), Kudo T(3),
Yokoyama K(4), Aya K(5), Suwabe K(6), Suzuki G(7), Aoki K(8), Kubo Y(9), Watanabe
M(10), Matsuoka M(5), Yano K(11).
Author information:
(1)School of Agriculture, Meiji University, Kawasaki, 214-8571 Japan CREST, JST,
Saitama, 332-0012 Japan Tsukuba Division, Mitsubishi Space Software Co., Ltd.,
Tsukuba, 305-0032 Japan Plant Genetics Laboratory, National Institute of
Genetics, Mishima, 411-8540 Japan These authors contributed equally to this work.
Saitama, 332-0012 Japan These authors contributed equally to this work.
Saitama, 332-0012 Japan.
(4)School of Agriculture, Meiji University, Kawasaki, 214-8571 Japan.
(5)Bioscience and Biotechnology Center, Nagoya University, Nagoya, 464-8601
Japan.
(6)Graduate School of Bioresources, Mie University, Tsu, 514-8507 Japan.
(7)Division of Natural Science, Osaka Kyoiku University, Kashiwara, 582-8582
Japan.
(8)Graduate School of Life and Environmental Sciences, Osaka Prefecture
University, Sakai, 599-8531 Japan.
(9)Graduate School of Environmental and Life Science, Okayama University,
Okayama, 700-8530 Japan.
(10)Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan.
Saitama, 332-0012 Japan kyano@isc.meiji.ac.jp.
Comprehensive integration of large-scale omics resources such as genomes,

transcriptomes and metabolomes will provide deeper insights into broader aspects
of molecular biology. For better understanding of plant biology, we aim to
construct a next-generation sequencing (NGS)-derived gene expression network
(GEN) repository for a broad range of plant species. So far we have incorporated
information about 745 high-quality mRNA sequencing (mRNA-Seq) samples from eight
plant species (Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Sorghum
bicolor, Vitis vinifera, Solanum tuberosum, Medicago truncatula and Glycine max)
from the public short read archive, digitally profiled the entire set of gene
expression profiles, and drawn GENs by using correspondence analysis (CA) to take
advantage of gene expression similarities. In order to understand the
evolutionary significance of the GENs from multiple species, they were linked
according to the orthology of each node (gene) among species. In addition to
other gene expression information, functional annotation of the genes will
facilitate biological comprehension. Currently we are improving the given gene
annotations with natural language processing (NLP) techniques and manual
curation. Here we introduce the current status of our analyses and the web
database, PODC (Plant Omics Data Center; http://bioinf.mind.meiji.ac.jp/podc/),
now open to the public, providing GENs, functional annotations and additional
comprehensive omics resources.

Society of Plant Physiologists.
DOI: 10.1093/pcp/pcu188
PMCID: PMC4301748
548. Genet Mol Res. 2014 Dec 4;13(4):10184-93. doi: 10.4238/2014.December.4.13.
Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2

in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique.
Gui Z(1), Liu HQ(1), Wang Y(2), Yuan QH(2), Xin N(1), Zhang X(1), Li XL(2), Pi
YS(1), Gao JM(3).
Author information:
(1)Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin
Agricultural University, Tianjin, China.
(2)Institute of Animal Science, Chinese Academy of Agricultural Science, Beijing,
China.
(3)Key Laboratory of Crop Genetics and Breeding, Department of Agronomy, Tianjin
Agricultural University, Tianjin, China gao11799713@163.com.
In this study, 2 approaches were adopted to obtain good single-strand

conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were
added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP
conditions for a 331-bp fragment in the coding region of
polygalacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized,
and the results showed that the best SSCP gel pattern could be obtained when the
loading mixture was made by mixing 1 μL PCR products, 0.2 to 0.8 μL unlabeled
primers (50 μM) and 4 to 16 μL loading buffer. Furthermore, the use of the
fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2
complementary single DNA strands, thus making the determination of alleles and
idiotypes a relatively easy task. In addition, the results of sequencing prove
that the determination of alleles and idiotypes were accurate based on SSCP
analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for
MsPGIP2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed
great genetic variation, and the addition of primers to the PCR products in
combination with the fluorescent labeling of primers could significantly improve
the sensitivity and resolution of SSCP analysis. This technique could be used for
genetic diversity detection and marker-assisted breeding of useful genes in
autopolyploid species such as alfalfa.
DOI: 10.4238/2014.December.4.13
549. Food Microbiol. 2015 Apr;46:280-7. doi: 10.1016/j.fm.2014.08.020. Epub 2014

Aug
30.
Comparison of eight different agars for the recovery of clinically relevant

non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro,
alfalfa sprouts and raw milk.
Kase JA(1), Maounounen-Laasri A(2), Son I(3), Lin A(4), Hammack TS(5).
Author information:
(1)Division of Microbiology, Center for Food Safety and Applied Nutrition, United
States Food and Drug Administration, College Park, MD 20740, USA. Electronic
address: julie.kase@fda.hhs.gov.
(2)Oak Ridge Research Institute for Science and Education, Oak Ridge, TN 37830,
USA. Electronic address: Anna.Laasri@fda.hhs.gov.
address: Insook.Son@fda.hhs.gov.
(4)Office of Regulatory Affairs-San Francisco Laboratory, United States Food and
Drug Administration, Alameda, CA 94502, USA. Electronic address:
Andrew.Lin@fda.hhs.gov.
address: Thomas.Hammack@fda.hhs.gov.
The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several

agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not
all have been thoroughly tested for recovering STECs from food. Using E. coli
strains representing ten clinically relevant O serogroups (O26, O45, O91, O103,
O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh
produce--bagged baby spinach, alfalfa sprouts, cilantro, and raw milk--we
evaluated the performance of 8 different agars. Performance was highly dependent
upon strain used and the presence of inhibitors, but not necessarily dependent on
food matrix. Tellurite resistant-negative strains, O91:-, O103:H6, O104:H21,
O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a
modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to
CHROMagar STEC and mRB agars improved overall performance; however, this also
reversed the inhibition of non-target bacteria provided by original formulations.
Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB
agars difficult. Study results support a strategy using inclusive agars (e.g.
L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7,
CHROMagar STEC) to allow for recovery of the most STECs while increasing the
probability of recovering STEC in high bacterial count matrices.
DOI: 10.1016/j.fm.2014.08.020
550. Environ Sci Process Impacts. 2015 Jan;17(1):177-85. doi: 10.1039/c4em00551a.

Epub
2014 Dec 4.
Toxic effects of copper-based nanoparticles or compounds to lettuce (Lactuca

sativa) and alfalfa (Medicago sativa).
Hong J(1), Rico CM, Zhao L, Adeleye AS, Keller AA, Peralta-Videa JR,
Gardea-Torresdey JL.
Author information:
(1)Environmental Sience and Engineering PhD Program, The University of Texas at
El Paso, 500 W. Univ. Av., El Paso, Texas 79968, USA. jgardea@utep.edu.
The increased production and use of nanoparticles (NPs) has generated concerns
about their impact on living organisms. In this study, nCu, bulk Cu, nCuO, bulk
CuO, Cu(OH)2 (CuPRO 2005, Kocide 3000), and CuCl2 were exposed for 15 days to 10
days-old hydroponically grown lettuce (Lactuca sativa) and alfalfa (Medicago
sativa). Each compound was applied at 0, 5, 10, and 20 mg L(-1). At harvest, we
measured the size of the plants and determined the concentration of Cu, macro and
microelements by using ICP-OES. Catalase and ascorbate peroxidase activity was
also determined. Results showed that all Cu NPs/compounds reduced the root length
by 49% in both plant species. All Cu NPs/compounds increased Cu, P, and S (>100%,
>50%, and >20%, respectively) in alfalfa shoots and decreased P and Fe in lettuce
shoot (>50% and >50%, respectively, excluding Fe in CuCl2 treatment). Biochemical
assays showed reduced catalase activity in alfalfa (root and shoot) and increased
ascorbate peroxidase activity in roots of both plant species. Results suggest
that Cu NPs/compounds not only reduced the size of the plants but altered
nutrient content and enzyme activity in both plant species.
DOI: 10.1039/c4em00551a
PMCID: PMC4326043

eCollection 2014.
Genome-wide identification of different dormant Medicago sativa L. MicroRNAs in

response to fall dormancy.
Fan W(1), Zhang S(1), Du H(1), Sun X(1), Shi Y(1), Wang C(1).
Author information:
BACKGROUND: MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that
regulate gene post-transcriptional expression in plants and animals.
High-throughput sequencing technology is capable of identifying small RNAs in
plant species. Alfalfa (Medicago sativa L.) is one of the most widely cultivated
perennial forage legumes worldwide, and fall dormancy is an adaptive
characteristic related to the biomass production and winter survival in alfalfa.
Here, we applied high-throughput sRNA sequencing to identify some miRNAs that
were responsive to fall dormancy in standard variety (Maverick and CUF101) of
alfalfa.
RESULTS: Four sRNA libraries were generated and sequenced from alfalfa leaves in
two typical varieties at distinct seasons. Through integrative analysis, we
identified 51 novel miRNA candidates of 206 families. Additionally, we identified
28 miRNAs associated with fall dormancy in standard variety (Maverick and
CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput
sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated
and six known miRNA members were down-regulated in response to fall dormancy in
standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five
miRNAs were up-regulated and three miRNAs were down-regulated in response to fall
dormancy in standard variety (Maverick and CUF101), and five of them were
confirmed by Northern blot analysis.
CONCLUSION: We identified 20 known miRNAs and eight new miRNA candidates that
were responsive to fall dormancy in standard variety (Maverick and CUF101) by
high-throughput sequencing of small RNAs from Medicago sativa. Our data provide a
useful resource for investigating miRNA-mediated regulatory mechanisms of fall
dormancy in alfalfa, and these findings are important for our understanding of
the roles played by miRNAs in the response of plants to abiotic stress in general
and fall dormancy in alfalfa.
PMCID: PMC4256440
552. Mar Pollut Bull. 2015 Jan 15;90(1-2):150-9. doi:

10.1016/j.marpolbul.2014.11.002.
Epub 2014 Nov 17.
Scouting contaminated estuaries: heavy metal resistant and plant growth promoting
rhizobacteria in the native metal rhizoaccumulator Spartina maritima.
Mesa J(1), Mateos-Naranjo E(2), Caviedes MA(1), Redondo-Gómez S(2), Pajuelo E(1),
Rodríguez-Llorente ID(3).
Author information:
(1)Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla,
C/Profesor García González 2, 41012 Sevilla, Spain.
(2)Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad
de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla, Spain.
(3)Departamento de Microbiología, Facultad de Farmacia, Universidad de Sevilla,
C/Profesor García González 2, 41012 Sevilla, Spain. Electronic address:
irodri@us.es.
Spartina maritima is a native endangered heavy metal rhizoaccumulator cordgrass

naturally growing in southwest coasts of Spain, where is used as a biotool to
rehabilitate degraded salt marshes. Fifteen bacterial strains were isolated from
the rhizosphere of S. maritima growing in the estuary of the Tinto River, one of
the most polluted areas in the world. A high proportion of bacteria were
resistant towards several heavy metals. They also exhibited multiple plant growth
promoting (PGP) properties, in the absence and the presence of Cu. Bacillus
methylotrophicus SMT38, Bacillusaryabhattai SMT48, B. aryabhattai SMT50 and
Bacilluslicheniformis SMT51 were selected as the best performing strains. In a
gnobiotic assay, inoculation of Medicago sativa seeds with the selected isolates
induced higher root elongation. The inoculation of S. maritima with these
indigenous metal-resistant PGP rhizobacteria could be an efficient method to
increase plant adaptation and growth in contaminated estuaries during restoration
programs.
DOI: 10.1016/j.marpolbul.2014.11.002
14.
Effect of dietary starch concentration and fish oil supplementation on milk yield
and composition, diet digestibility, and methane emissions in lactating dairy
cows.
Pirondini M(1), Colombini S(1), Mele M(2), Malagutti L(1), Rapetti L(1), Galassi
G(1), Crovetto GM(3).
Author information:
(1)Università degli Studi di Milano, Dipartimento di Scienze Agrarie e
Ambientali, 20133 Milan, Italy.
(2)Università degli Studi di Pisa, Dipartimento di Scienze Agrarie, Alimentari e
Agroambientali, 56124 Pisa, Italy.
Ambientali, 20133 Milan, Italy. Electronic address: matteo.crovetto@unimi.it.
The aim of this study was to evaluate the effects of diets with different starch
concentrations and fish oil (FO) supplementation on lactation performance, in
vivo total-tract nutrient digestibility, N balance, and methane (CH4) emissions
in lactating dairy cows. The experiment was conducted as a 4×4 Latin square
design with a 2×2 factorial arrangement: 2 concentrations of dietary starch [low
vs. high: 23.7 and 27.7% on a dry matter (DM) basis; neutral detergent
fiber/starch ratios: 1.47 and 1.12], the presence or absence of FO supplement
(0.80% on a DM basis), and their interaction were evaluated. Four Italian
Friesian cows were fed 1 of the following 4 diets in 4 consecutive 26-d periods:
(1) low starch (LS), (2) low starch plus FO (LSO), (3) high starch (HS), and (4)
high starch plus FO (HSO). The diets contained the same amount of forages (corn
silage, alfalfa and meadow hays). The starch concentration was balanced using
different proportions of corn meal and soybean hulls. The cows were housed in
metabolic stalls inside open-circuit respiration chambers to allow measurement of
CH4 emission and the collection of separate urine and feces. No differences among
treatments were observed for DM intake. We observed a trend for FO to increase
milk yield: 29.2 and 27.5kg/d, on average, for diets with and without FO,
respectively. Milk fat was affected by the interaction between dietary starch and
FO: milk fat decreased only in the HSO diet. Energy-corrected milk (ECM) was
affected by the interaction between starch and FO, with a positive effect of FO
on the LS diet. Fish oil supplementation decreased the n-6:n-3 ratio of milk
polyunsaturated fatty acids. High-starch diets negatively influenced all
digestibility parameters measured except starch, whereas FO improved neutral
detergent fiber digestibility (41.9 vs. 46.1% for diets without and with FO,
respectively, and ether extract digestibility (53.7 vs. 67.1% for diets without
and with FO, respectively). We observed a trend for lower CH4 emission (g/d) and
intensity (g/kg of milk) with the high-starch diets compared with the low-starch
diets: 396 versus 415g/d on average, respectively, and 14.1 versus 14.9g/kg of
milk, respectively. Methane intensity per kilogram of ECM was affected by the
interaction between starch and FO, with a positive effect of FO for the LS diet:
14.5 versus 13.3g of CH4/kg of ECM for LS and LSO diets, respectively.

DOI: 10.3168/jds.2014-8092
14.
Performance, digestion, nitrogen balance, and emission of manure ammonia, enteric

methane, and carbon dioxide in lactating cows fed diets with varying alfalfa
silage-to-corn silage ratios.
Arndt C(1), Powell JM(2), Aguerre MJ(1), Wattiaux MA(3).
Author information:
WI 53706.
Electronic address: wattiaux@wisc.edu.
Two trials were conducted simultaneously to study the effects of varying alfalfa
silage (AS) to corn silage (CS) ratio in diets formulated to avoid excess protein
or starch on lactating dairy cow performance, digestibility, ruminal parameters,
N balance, manure production and composition, and gaseous emissions [carbon
dioxide (CO2), methane (CH4), and ammonia-N (NH3-N)]. In trial 1 all
measurements, except gas emissions, were conducted on 8 rumen-cannulated cows in
replicated 4×4 Latin squares. In trial 2, performance and emissions were measured
on 16 cows randomly assigned to 1 of 4 air-flow controlled chambers in a 4×4
Latin square. Dietary treatments were fed as total mixed rations with
forage-to-concentrate ratio of 55:45 [dietary dry matter (DM) basis] and AS:CS
ratios of 20:80, 40:60, 60:40, and 80:20 (forage DM basis). Measurements were
conducted the last 3d of each 21-d period. Treatments did not affect DM intake,
DM digestibility, and milk/DM intake. However, responses were quadratic for
fat-and-protein-corrected milk, fat, and protein production, which reached
predicted maxima for AS:CS ratio of 50:50, 49:51, and 34:66, respectively.
Nitrogen use efficiency (milk N/N intake) decreased from 31 to 24g/100g as AS:CS
ratio increased from 20:80 to 80:20. Treatments did not alter NH3-N/milk-N but
tended to have a quadratic effect on daily NH3-N emission. Treatments had a
quadratic effect on daily CH4 emission, which was high compared with current
literature; they influenced CH4 emission per unit of neutral detergent fiber
(NDF) intake and tended to influence CO2/NDF intake. Ruminal
acetate-to-propionate ratio and total-tract NDF digestibility increased linearly
with increasing AS:CS ratio. In addition, as AS:CS ratio increased from 20:80 to
80:20, NDF digested increased linearly from 2.16 to 3.24kg/d, but CH4/digested
NDF decreased linearly from 270 to 190g/kg. These 2 counterbalancing effects
likely contributed to the observed quadratic response in daily CH4 emission,
which may have been influenced also by increasing starch with increasing CS in
the diet as reflected by the increased ruminal propionate molar proportion.
Overall, production performances were greatest for the intermediate AS:CS ratios
(40:60 and 60:40), but daily excretion of urine, manure, fecal N, urinary urea N,
and urinary N decreased with increasing proportion of CS in the diet, whereas
daily CH4 emission was reduced for the 2 extreme AS:CS ratios (20:80 and 80:20).
However, the proportion of AS and CS in the diet did not affect
CH4/fat-and-protein corrected milk.

DOI: 10.3168/jds.2014-8298
555. J Dairy Sci. 2014 Dec;97(12):7777-87. Epub 2014 Oct 18.
Evaluation of the systemic innate immune response and metabolic alterations of

nonlactating cows with diet-induced subacute ruminal acidosis.
Rodríguez-Lecompte JC(1), Kroeker AD, Ceballos-Márquez A, Li S, Plaizier JC,

Gomez DE.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T
2N2. jrodriguez@upei.ca
Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the

rumen, which might translocate into the systemic circulation, triggering a
cascade of clinical and immunological alterations. The objective of this study
was to characterize the clinical immune and metabolic responses to
ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2
challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA
challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3 × 3 Latin
square arrangement of treatments with 4-wk experimental cycles. All cows received
the control diet containing 70% forage and 30% mixed concentrates (dry matter
basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley
pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed
concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins
and immunology-related proteins, acute phase proteins, blood cells, serum
chemistry, mRNA gene expression of peripheral blood cell surface markers, and
selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both
groups with induced SARA compared with a control group. Ruminal endotoxins were
higher in GBSC; however, plasma endotoxin was not detected in any study group. No
significant differences in feed intake, rectal temperature, white blood cell
counts, or differentials were found between control and SARA challenge groups;
changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma
proteins were lower in both SARA groups, and acute phase proteins were higher in
GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes
was not affected by SARA induction. The induction of SARA as a result of GBSC or
APSC challenge was successful; however, LPS was not detected in plasma. Changes
in clinical, metabolic, and inflammatory responses were not observed in the
SARA-challenged cows, suggesting that, in this study, SARA was not associated
with a systemic response to inflammation.
DOI: 10.3168/jds.2014-8319
556. Bioresour Technol. 2015 Jan;175:97-101. doi: 10.1016/j.biortech.2014.10.054.

Epub
2014 Oct 18.
Production of medium-chain volatile fatty acids by mixed ruminal microorganisms

is enhanced by ethanol in co-culture with Clostridium kluyveri.
Weimer PJ(1), Nerdahl M(2), Brandl DJ(2).
Author information:
(1)US Dairy Forage Research Center, Agricultural Research Service, United States
Department of Agriculture, Madison, WI 53706, USA; Department of Bacteriology,
University of Wisconsin-Madison, Madison, WI 53706, USA. Electronic address:
Paul.Weimer@ars.usda.gov.
(2)Department of Bacteriology, University of Wisconsin-Madison, Madison, WI
53706, USA.
Mixed bacterial communities from the rumen ferment cellulosic biomass primarily
to C2-C4 volatile fatty acids, and perform only limited chain extension to
produce C5 (valeric) and C6 (caproic) acids. The aim of this study was to
increase production of caproate and valerate in short-term in vitro incubations.
Co-culture of mixed ruminal microbes with a rumen-derived strain of the bacterium
Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass
herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as
the major fermentation products over a 48-72h run time. Concentrations of
caproate reached 6.1gL(-1), similar to or greater than those reported in most
conventional carboxylate fermentations that employ substantially longer run
times.
DOI: 10.1016/j.biortech.2014.10.054
557. Eur J Med Chem. 2015 Jan 27;90:351-9. doi: 10.1016/j.ejmech.2014.11.055. Epub
2014 Nov 27.
Catechol-based substrates of chalcone synthase as a scaffold for novel inhibitors

of PqsD.
Allegretta G(1), Weidel E(1), Empting M(1), Hartmann RW(2).
Author information:
(1)Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2(3),
66123 Saarbrücken, Germany.
(2)Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Campus C2(3),
66123 Saarbrücken, Germany; Pharmaceutical and Medicinal Chemistry, Saarland
University, Campus C2(3), 66123 Saarbrücken, Germany. Electronic address:
rolf.hartmann@helmholtz-hzi.de.
A new strategy for treating Pseudomonas aeruginosa infections could be disrupting

the Pseudomonas Quinolone Signal (PQS) quorum sensing (QS) system. The goal is to
impair communication among the cells and, hence, reduce the expression of
virulence factors and the formation of biofilms. PqsD is an essential enzyme for
the synthesis of PQS and shares some features with chalcone synthase (CHS2), an
enzyme expressed in Medicago sativa. Both proteins are quite similar concerning
the size of the active site, the catalytic residues and the electrostatic surface
potential at the entrance of the substrate tunnel. Hence, we evaluated selected
substrates of the vegetable enzyme as potential inhibitors of the bacterial
protein. This similarity-guided approach led to the identification of a new class
of PqsD inhibitors having a catechol structure as an essential feature for
activity, a saturated linker with two or more carbons and an ester moiety bearing
bulky substituents. The developed compounds showed PqsD inhibition with IC50
values in the single-digit micromolar range. The binding mode of these compounds
was investigated by Surface Plasmon Resonance (SPR) experiments revealing that
their interaction with the protein is not influenced by the presence of the
anthranilic acid bound to active site cysteine. Importantly, some compounds
reduced the signal molecule production in cellulo.
DOI: 10.1016/j.ejmech.2014.11.055
558. Plant Cell Physiol. 2015 Jan;56(1):e1. doi: 10.1093/pcp/pcu179. Epub 2014 Nov
28.
MTGD: The Medicago truncatula genome database.

Krishnakumar V(1), Kim M(2), Rosen BD(2), Karamycheva S(2), Bidwell SL(2), Tang
H(2), Town CD(2).
Author information:
(1)Plant Genomics Group, J. Craig Venter Institute, 9704 Medical Center Dr,
Rockville, MD 20850, USA vkrishna@jcvi.org.
(2)Plant Genomics Group, J. Craig Venter Institute, 9704 Medical Center Dr,
Rockville, MD 20850, USA.
Medicago truncatula, a close relative of alfalfa (Medicago sativa), is a model

legume used for studying symbiotic nitrogen fixation, mycorrhizal interactions
and legume genomics. J. Craig Venter Institute (JCVI; formerly TIGR) has been
involved in M. truncatula genome sequencing and annotation since 2002 and has
maintained a web-based resource providing data to the community for this entire
period. The website (http://www.MedicagoGenome.org) has seen major updates in the
past year, where it currently hosts the latest version of the genome (Mt4.0),
associated data and legacy project information, presented to users via a rich set
of open-source tools. A JBrowse-based genome browser interface exposes tracks for
visualization. Mutant gene symbols originally assembled and curated by the
Frugoli lab are now hosted at JCVI and tie into our community annotation
interface, Medicago EuCAP (to be integrated soon with our implementation of
WebApollo). Literature pertinent to M. truncatula is indexed and made searchable
via the Textpresso search engine. The site also implements MedicMine, an instance
of InterMine that offers interconnectivity with other plant 'mines' such as
ThaleMine and PhytoMine, and other model organism databases (MODs). In addition
to these new features, we continue to provide keyword- and locus identifier-based
searches served via a Chado-backed Tripal Instance, a BLAST search interface and
bulk downloads of data sets from the iPlant Data Store (iDS). Finally, we
maintain an E-mail helpdesk, facilitated by a JIRA issue tracking system, where
we receive and respond to questions about the website and requests for specific
data sets from the community.

Society of Plant Physiologists. All rights reserved. For permissions, please
DOI: 10.1093/pcp/pcu179
559. Transgenic Res. 2015 Jun;24(3):475-88. doi: 10.1007/s11248-014-9851-7. Epub

2014
Nov 29.
Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a

modified bacterial atrazine chlorohydrolase gene.
Vail AW(1), Wang P, Uefuji H, Samac DA, Vance CP, Wackett LP, Sadowsky MJ.
Author information:
(1)BioTechnology Institute, University of Minnesota, 1479 Gortner Ave., 140
Gortner Labs, St. Paul, MN, 55108, USA.
The widespread use of atrazine and other s-triazine herbicides to control weeds
in agricultural production fields has impacted surface and groundwater in the
United States and elsewhere. We previously reported the cloning, sequencing, and
expression of six genes involved in the atrazine biodegradation pathway of
Pseudomonas sp. strain ADP, which is initiated by atzA, encoding atrazine
chlorohydrolase. Here we explored the use of enhanced expression of a modified
bacterial atrazine chlorohydrolase, p-AtzA, in transgenic grasses (tall fescue,
perennial ryegrass, and switchgrass) and the legume alfalfa for the
biodegradation of atrazine. Enhanced expression of p-AtzA was obtained by using
combinations of the badnavirus promoter, the maize alcohol dehydrogenase first
intron, and the maize ubiquitin promoter. For alfalfa, we used the first intron
of the 5'-untranslated region tobacco alcohol dehydrogenase gene and the cassava
vein mosaic virus promoter. Resistance of plants to atrazine in agar-based and
hydroponic growth assays was correlated with in vivo levels of gene expression
and atrazine degradation. The in planta expression of p-atzA enabled transgenic
tall fescue to transform atrazine into hydroxyatrazine and other metabolites.
Results of our studies highlight the potential use of transgenic plants for
bioremediating atrazine in the environment.
DOI: 10.1007/s11248-014-9851-7
560. Environ Sci Pollut Res Int. 2015 May;22(9):6778-88. doi:

10.1007/s11356-014-3868-6. Epub 2014 Nov 29.
Evaluation of ferrihydrite as amendment to restore an arsenic-polluted mine soil.
Abad-Valle P(1), Álvarez-Ayuso E, Murciego A.
Author information:
(1)Department of Environmental Geochemistry, IRNASA (CSIC), C/ Cordel de Merinas
40-52, 37008, Salamanca, Spain.
The effectiveness of ferrihydrite as amendment to restore the soil habitat

functioning of a soil polluted with As by mining activities was evaluated. Its
influence on As mobility and phytoavailability was also assessed. Soil treated
with increasing amendment doses (0, 1, 2, and 5 %) were analyzed for soil
microbiological parameters such as basal soil respiration and dehydrogenase,
β-glucosidase, urease, acid and alkaline phosphatase, and arylsulfatase
activities. Batch leaching tests and plant growth experiments using ryegrass and
alfalfa plants were performed. The treatment with ferrihydrite was effective to
reduce As mobility and plant As uptake, translocation, and accumulation.
Likewise, the soil microbiological status was generally improved as derived from
basal soil respiration and dehydrogenase and acid and alkaline phosphatase
activities, which showed increases up to 85, 45, 11, and 47 %, respectively, at a
ferrihydrite addition rate of 5 %.
DOI: 10.1007/s11356-014-3868-6
561. Appl Environ Microbiol. 2015 Feb;81(3):861-73. doi: 10.1128/AEM.02985-14. Epub

2014 Nov 21.
De novo amino acid biosynthesis contributes to salmonella enterica growth in

Alfalfa seedling exudates.
Kwan G(1), Pisithkul T(2), Amador-Noguez D(2), Barak J(3).
Author information:
(1)Department of Plant Pathology, University of Wisconsin-Madison, Madison,
Wisconsin, USA.
(2)Department of Bacteriology, University of Wisconsin-Madison, Madison,
Wisconsin, USA.
Wisconsin, USA barak@plantpath.wisc.edu.
Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in

sprouting-seed exudates is rapid; however, the active metabolic networks
essential in this environment are unknown. To examine the metabolic requirements
of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa
seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from
planktonic cells. Over half the proteins had known metabolic functions, and they
are involved in over one-quarter of the known metabolic reactions. Ion and
metabolite transport accounted for the majority of detected reactions. Proteins
involved in amino acid transport and metabolism were highly represented,
suggesting that amino acid metabolic networks may be important for S. enterica
growth in association with roots. Amino acid auxotroph growth phenotypes agreed
with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were
detected in our screen developed growth defects by 48 h. When the perceived
sufficiency of each amino acid was expressed as a ratio of the calculated biomass
requirement to the available concentration and compared to growth of each amino
acid auxotroph, a correlation between nutrient availability and bacterial growth
was found. Furthermore, glutamate transport acted as a fitness factor during S.
enterica growth in association with roots. Collectively, these data suggest that
S. enterica metabolism is robust in the germinating-alfalfa environment; that
single-amino-acid metabolic pathways are important but not essential; and that
targeting central metabolic networks, rather than dedicated pathways, may be
necessary to achieve dramatic impacts on bacterial growth.
DOI: 10.1128/AEM.02985-14
PMCID: PMC4292483
562. Plant Cell Rep. 2015 Feb;34(2):211-21. doi: 10.1007/s00299-014-1700-x. Epub

2014
Nov 21.
Evaluation of somatic embryos of alfalfa for recombinant protein expression.
Fu G(1), Grbic V, Ma S, Tian L.
Author information:
KEY MESSAGE: Somatic embryos of alfalfa can accumulate higher levels of

recombinant proteins comparing to vegetative organs. Somatic embryos may be
explored as a new system for new protein production for plants. Plants have been
explored via genetic engineering as an inexpensive system for recombinant protein
production. However, protein expression levels in vegetative tissues have been
low, which limits the commercial utilization of plant expression systems. Somatic
embryos resemble zygotic embryos in many aspects and may accumulate higher levels
of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago
sativa L.) was investigated for the expression of recombinant proteins. Three
heterologous genes, including the standard scientific reporter uid that codes for
β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit
(CTB), and hIL-13 coding for human interleukin 13, were independently introduced
into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were
subsequently induced from transgenic plants carrying these genes. Somatic embryos
accumulated approximately twofold more recombinant proteins than vegetative
organs including roots, stems, and leaves. The recombinant proteins of CTB and
hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa
somatic embryos, respectively. The recombinant proteins expressed in somatic
embryos also exhibited biological activities. As somatic embryos can be induced
in many plant species and their production can be scaled up via different
avenues, somatic embryos may be developed as an efficient expression system for
recombinant protein production.
DOI: 10.1007/s00299-014-1700-x
563. New Phytol. 2015 Apr;206(1):303-14. doi: 10.1111/nph.13162. Epub 2014 Nov 18.
Sapogenin content variation in Medicago inter-specific hybrid derivatives

highlights some aspects of saponin synthesis and control.
Carelli M(1), Biazzi E, Tava A, Losini I, Abbruscato P, Depedro C, Scotti C.
Author information:
(1)Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di
Ricerca per le Produzioni Foraggere e Lattiero-Casearie, viale Piacenza 29,
26900, Lodi, Italy.
In the Medicago genus, saponins are a complex mixture of triterpene glycosides

showing a broad spectrum of biological properties. Here we analyzed the variation
in the sapogenin content and composition of inter-specific hybrid Medicago
sativa × Medicago arborea derivatives to highlight the pattern of this variation
in plant organs (leaves/roots) and the possible mechanisms underlying it. In
Sativa Arborea Cross (SAC) leaves and roots, saponins and sapogenins were
evaluated using chromatographic methods. Phenotypic correlations between
sapogenin content and bio-agronomic traits were examined. Expression studies on
β-amyrin synthase and four cytochromes P450 (CYPs) involved in sapogenin
biosynthesis and sequence analysis of the key gene of the hemolytic sapogenin
pathway (CYP716A12) were performed. Chromatographic analyses revealed a different
pattern of among-family variation for hemolytic and nonhemolytic sapogenins and
saponins and for the two organs/tissues. Different correlation patterns of gene
expression in roots and leaves were found. Diachronic analysis revealed a
relationship between sapogenin content and gene transcriptional levels in the
early stages of the productive cycle. The results suggest that there are
different control mechanisms acting on sapogenin biosynthesis for leaves and
roots, which are discussed. A key role for medicagenic acid in the control of
sapogenin content in both the tissues is proposed and discussed.
DOI: 10.1111/nph.13162
564. J Exp Bot. 2015 Feb;66(2):613-23. doi: 10.1093/jxb/eru439. Epub 2014 Nov 16.
Amino acid-mediated impacts of elevated carbon dioxide and simulated root

herbivory on aphids are neutralized by increased air temperatures.
Ryalls JM(1), Moore BD(2), Riegler M(2), Gherlenda AN(2), Johnson SN(2).
Author information:
(1)Hawkesbury Institute for the Environment, University of Western Sydney, Locked
Bag 1797, Penrith, NSW 2751, Australia J.Ryalls@uws.edu.au.
(2)Hawkesbury Institute for the Environment, University of Western Sydney, Locked
Bag 1797, Penrith, NSW 2751, Australia.
Changes in host plant quality, including foliar amino acid concentrations,

resulting from global climate change and attack from multiple herbivores, have
the potential to modify the pest status of insect herbivores. This study
investigated how mechanically simulated root herbivory of lucerne (Medicago
sativa) before and after aphid infestation affected the pea aphid (Acyrthosiphon
pisum) under elevated temperature (eT) and carbon dioxide concentrations (eCO2).
eT increased plant height and biomass, and eCO2 decreased root C:N. Foliar amino
acid concentrations and aphid numbers increased in response to eCO2, but only at
ambient temperatures, demonstrating the ability of eT to negate the effects of
eCO2. Root damage reduced aboveground biomass, height, and root %N, and increased
root %C and C:N, most probably via decreased biological nitrogen fixation. Total
foliar amino acid concentrations and aphid colonization success were higher in
plants with roots cut early (before aphid arrival) than those with roots cut late
(after aphid arrival); however, this effect was counteracted by eT. These results
demonstrate the importance of amino acid concentrations for aphids and identify
individual amino acids as being potential factors underpinning aphid responses to
eT, eCO2, and root damage in lucerne. Incorporating trophic complexity and
multiple climatic factors into plant-herbivore studies enables greater insight
into how plants and insects will interact in the future, with implications for
sustainable pest control and future crop security.
for Experimental Biology.
DOI: 10.1093/jxb/eru439
PMCID: PMC4286407
565. ScientificWorldJournal. 2014;2014:717562. doi: 10.1155/2014/717562. Epub 2014

Oct
21.
The response of durum wheat to the preceding crop in a Mediterranean environment.
Ercoli L(1), Masoni A(2), Pampana S(2), Mariotti M(3), Arduini I(2).
Author information:
(1)Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127 Pisa,
Italy.
Borghetto 80, 56124 Pisa, Italy.
(3)Department of Veterinary Science, University of Pisa, Via delle Piagge 2,
56124 Pisa, Italy.
Crop sequence is an important management practice that may affect durum wheat
(Triticum durum Desf.) production. Field research was conducted in 2007-2008 and
2008-2009 seasons in a rain-fed cold Mediterranean environment to examine the
impact of the preceding crops alfalfa (Medicago sativa L.), maize (Zea mays L.),
sunflower (Helianthus annuus L.), and bread wheat (Triticum aestivum L.) on yield
and N uptake of four durum wheat varieties. The response of grain yield of durum
wheat to the preceding crop was high in 2007-2008 and was absent in the 2008-2009
season, because of the heavy rainfall that negatively impacted establishment,
vegetative growth, and grain yield of durum wheat due to waterlogging. In the
first season, durum wheat grain yield was highest following alfalfa, and was 33%
lower following wheat. The yield increase of durum wheat following alfalfa was
mainly due to an increased number of spikes per unit area and number of kernels
per spike, while the yield decrease following wheat was mainly due to a reduction
of spike number per unit area. Variety growth habit and performance did not
affect the response to preceding crop and varieties ranked in the order Levante >
Saragolla = Svevo > Normanno.
DOI: 10.1155/2014/717562
PMCID: PMC4221887
566. Plant Physiol Biochem. 2014 Dec;85:31-40.
Overexpression of codA gene confers enhanced tolerance to abiotic stresses in

alfalfa.
Li H, Wang Z, Ke Q, Ji CY, Jeong JC, Lee HS, Lim YP, Xu B, Deng XP, Kwak SS.
We generated transgenic alfalfa plants (Medicago sativa L. cv. Xinjiang Daye)

expressing a bacterial codA gene in chloroplasts under the control of the SWPA2
promoter (referred to as SC plants) and evaluated the plants under various
abiotic stress conditions. Three transgenic plants (SC7, SC8, and SC9) were
selected for further characterization based on the strong expression levels of
codA in response to methylviologen (MV)-mediated oxidative stress. SC plants
showed enhanced tolerance to NaCl and drought stress on the whole plant level due
to induced expression of codA. When plants were subjected to 250 mM NaCl
treatment for 2 weeks, SC7 and SC8 plants maintained higher chlorophyll contents
and lower malondialdehyde levels than non-transgenic (NT) plants. Under drought
stress conditions, all SC plants showed enhanced tolerance to drought stress
through maintaining high relative water contents and increased levels of
glycinebetaine and proline compared to NT plants. Under normal conditions, SC
plants exhibited increased growth due to increased expression of auxin-related
IAA genes compared to NT plants. These results suggest that the SC plants
generated in this study will be useful for enhanced biomass production on global
marginal lands, such as high salinity and arid lands, yielding a sustainable
agricultural product.
DOI: 10.1016/j.plaphy.2014.10.010
567. Insect Sci. 2016 Feb;23(1):145-56. doi: 10.1111/1744-7917.12185. Epub 2015 Jan
21.
Intercrop movement of convergent lady beetle, Hippodamia convergens (Coleoptera:

Coccinellidae), between adjacent cotton and alfalfa.
Bastola A(1), Parajulee MN(1), Porter RP(1), Shrestha RB(2), Chen FJ(3), Carroll
SC(1).
Author information:
(1)Texas A&M University AgriLife Research and Extension Center, Lubbock, Texas,
USA.
(2)Department of Entomology, Iowa State University, Ames, Iowa, USA.
(3)Department of Entomology, Nanjing Agricultural University, Nanjing, China.
A 2-year study was conducted to characterize the intercrop movement of convergent

lady beetle, Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae)
between adjacent cotton and alfalfa. A dual protein-marking method was used to
assess the intercrop movement of the lady beetles in each crop. In turns field
collected lady beetles in each crop were assayed by protein specific ELISA to
quantify the movement of beetles between the crops. Results indicated that a high
percentage of convergent lady beetles caught in cotton (46% in 2008; 56% in 2009)
and alfalfa (46% in 2008; 71% in 2009) contained a protein mark, thus indicating
that convergent lady beetle movement was largely bidirectional between the
adjacent crops. Although at a much lower proportion, lady beetles also showed
unidirectional movement from cotton to alfalfa (5% in 2008 and 6% in 2009) and
from alfalfa to cotton (9% in 2008 and 14% in 2009). The season-long
bidirectional movement exhibited by the beetles was significantly higher in
alfalfa than cotton during both years of the study. The total influx of lady
beetles (bidirectional and unidirectional combined) was significantly higher in
alfalfa compared with that in cotton for both years. While convergent lady
beetles moved between adjacent cotton and alfalfa, they were more attracted to
alfalfa when cotton was not flowering and/or when alfalfa offered more
opportunities for prey. This study offers much needed information on intercrop
movement of the convergent lady beetle that should facilitate integrated pest
management decisions in cotton utilizing conservation biological control.
© 2014 Institute of Zoology, Chinese Academy of Sciences.
DOI: 10.1111/1744-7917.12185
568. Mol Plant Microbe Interact. 2014 Dec;27(12):1307-17. doi:

10.1094/MPMI-06-14-0168-R.
Exopolysaccharide production in response to medium acidification is correlated

with an increase in competition for nodule occupancy.
Geddes BA, González JE, Oresnik IJ.
Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon

source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously
shown to be more competitive for nodule occupancy. In this work, we show that
strains carrying this mutation have galactose-dependent exopolysaccharide (EPS)
phenotypes that were manifested as aberrant Calcofluor staining as well as
decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor
staining was correlated with changes in the pH of the growth medium. Strains
carrying dgoK mutations were subsequently demonstrated to show earlier
acidification of their growth medium that was correlated with an increase
expression of genes associated with succinoglycan biosynthesis as well as
increased accumulation of high and low molecular weight EPS in the medium. In
addition, it was shown that the acidification of the medium was dependent on the
inability of S. meliloti strains to initiate the catabolism of galactose. To more
fully understand why strains carrying the dgoK allele were more competitive for
nodule occupancy, early nodulation phenotypes were investigated. It was found
that strains carrying the dgoK allele had a faster rate of nodulation. In
addition, nodule competition experiments using genetic backgrounds unable to
synthesize either succinoglycan or EPSII were consistent with the hypothesis that
the increased competition phenotype was dependent upon the synthesis of
succinoglycan. Fluorescent microscopy experiments on infected root-hair cells,
using the acidotropic dye Lysotracker Red DND-99, provide evidence that the
colonized curled root hair is an acidic compartment.
DOI: 10.1094/MPMI-06-14-0168-R

eCollection 2014.
The positive regulatory roles of the TIFY10 proteins in plant responses to

alkaline stress.
Zhu D(1), Li R(2), Liu X(3), Sun M(4), Wu J(4), Zhang N(4), Zhu Y(4).
Author information:
(1)College of Life Science, Qingdao Agricultural University, Qingdao, P.R. China;
Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin, P.R.
China.
(2)Key Laboratory of Molecular Biology, College of Heilongjiang Province,
Heilongjiang University, Harbin, P.R. China.
(3)College of Life Science, Qingdao Agricultural University, Qingdao, P.R. China.
(4)Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin,
P.R. China.
The TIFY family is a novel plant-specific protein family, and is characterized by

a conserved TIFY motif (TIFF/YXG). Our previous studies indicated the potential
roles of TIFY10/11 proteins in plant responses to alkaline stress. In the current
study, we focused on the regulatory roles and possible physiological and
molecular basis of the TIFY10 proteins in plant responses to alkaline stress. We
demonstrated the positive function of TIFY10s in alkaline responses by using the
AtTIFY10a and AtTIFY10b knockout Arabidopsis, as evidenced by the relatively
lower germination rates of attify10a and attify10b mutant seeds under alkaline
stress. We also revealed that ectopic expression of GsTIFY10a in Medicago sativa
promoted plant growth, and increased the NADP-ME activity, citric acid content
and free proline content but decreased the MDA content of transgenic plants under
alkaline stress. Furthermore, expression levels of the stress responsive genes
including NADP-ME, CS, H+-ppase and P5CS were also up-regulated in GsTIFY10a
transgenic plants under alkaline stress. Interestingly, GsTIFY10a overexpression
increased the jasmonate content of the transgenic alfalfa. In addition, we showed
that neither GsTIFY10a nor GsTIFY10e exhibited transcriptional activity in yeast
cells. However, through Y2H and BiFc assays, we demonstrated that GsTIFY10a, not
GsTIFY10e, could form homodimers in yeast cells and in living plant cells. As
expected, we also demonstrated that GsTIFY10a and GsTIFY10e could heterodimerize
with each other in both yeast and plant cells. Taken together, our results
provided direct evidence supporting the positive regulatory roles of the TIFY10
proteins in plant responses to alkaline stress.
PMCID: PMC4222965
570. Bull Entomol Res. 2014 Dec;104(6):689-701. doi: 10.1017/S0007485314000443.
Pea aphid biotype performance on diverse Medicago host genotypes indicates highly
specific virulence and resistance functions.
Kanvil S(1), Powell G(1), Turnbull C(1).
Author information:
(1)Department of Life Sciences,Imperial College London,London SW7 2AZ,UK.
Aphid-plant interactions depend on genotypes of both organisms, which determine
the two-way molecular exchange that leads to compatible or incompatible outcomes.
The underlying genes are mostly unknown, making it difficult to predict
likelihood of aphid success or host resistance, and hampering crop genetic
improvement. Here we screened eight pea aphid clonal genotypes collected from
diverse legume hosts, on a species-wide panel of Medicago truncatula (Mt)
genotypes. Aphid virulence was measured by survival, fecundity and growth rate,
together with scores for chlorosis and necrosis as host response indicators.
Outcomes were highly dependent on the specific aphid-host genotype combinations.
Only one Mt line was fully resistant against all clones. Aphid-induced host
chlorosis and necrosis varied greatly, but correlated with resistance only in a
few combinations. Bi-clustering analysis indicated that all aphid clones could be
distinguished by their performance profiles across the host genotypes tested,
with each clone being genetically differentiated and potentially representing a
distinct biotype. Clones originating from Medicago sativa ranged from highly
virulent to almost completely avirulent on both Medicago species, indicating that
some were well adapted, whereas others were most likely migrants. Comparisons of
closely related pairs of Australian Mt genotypes differing in aphid resistance
revealed no enhanced resistance to European pea aphid clones. Based on the
extensive variation in pea aphid adaptation even on unfamiliar hosts, most likely
reflecting multiple biotype-specific gene-for-gene interactions, we conclude that
robust defences require an arsenal of appropriate resistance genes.
DOI: 10.1017/S0007485314000443
571. BMC Genomics. 2014 Nov 3;15:950. doi: 10.1186/1471-2164-15-950.
Comprehensive characterization and RNA-Seq profiling of the HD-Zip transcription

factor family in soybean (Glycine max) during dehydration and salt stress.
Belamkar V, Weeks NT, Bharti AK, Farmer AD, Graham MA, Cannon SB(1).
Author information:
(1)Department of Agronomy, Iowa State University, Ames, IA 50011, USA.
steven.cannon@ars.usda.gov.
BACKGROUND: The homeodomain leucine zipper (HD-Zip) transcription factor family

is one of the largest plant specific superfamilies, and includes genes with roles
in modulation of plant growth and response to environmental stresses. Many HD-Zip
genes are characterized in Arabidopsis (Arabidopsis thaliana), and members of the
family are being investigated for abiotic stress responses in rice (Oryza
sativa), maize (Zea mays), poplar (Populus trichocarpa) and cucumber (Cucmis
sativus). Findings in these species suggest HD-Zip genes as high priority
candidates for crop improvement.
RESULTS: In this study we have identified members of the HD-Zip gene family in
soybean cv. 'Williams 82', and characterized their expression under dehydration
and salt stress. Homology searches with BLASTP and Hidden Markov Model guided
sequence alignments identified 101 HD-Zip genes in the soybean genome. Phylogeny
reconstruction coupled with domain and gene structure analyses using soybean,
Arabidopsis, rice, grape (Vitis vinifera), and Medicago truncatula homologues
enabled placement of these sequences into four previously described subfamilies.
Of the 101 HD-Zip genes identified in soybean, 88 exist as whole-genome
duplication-derived gene pairs, indicating high retention of these genes
following polyploidy in Glycine ~13 Mya. The HD-Zip genes exhibit ubiquitous
expression patterns across 24 conditions that include 17 tissues of soybean. An
RNA-Seq experiment performed to study differential gene expression at 0, 1, 6 and
12 hr soybean roots under dehydration and salt stress identified 20
differentially expressed (DE) genes. Several of these DE genes are orthologs of
genes previously reported to play a role under abiotic stress, implying
conservation of HD-Zip gene functions across species. Screening of HD-Zip
promoters identified transcription factor binding sites that are overrepresented
in the DE genes under both dehydration and salt stress, providing further support
for the role of HD-Zip genes in abiotic stress responses.
CONCLUSIONS: We provide a thorough description of soybean HD-Zip genes, and
identify potential candidates with probable roles in dehydration and salt stress.
Expression profiles generated for all soybean genes, under dehydration and salt
stress, at four time points, will serve as an important resource for the soybean
research community, and will aid in understanding plant responses to abiotic
stress.
DOI: 10.1186/1471-2164-15-950
PMCID: PMC4226900
572. J Agric Food Chem. 2014 Nov 19;62(46):11030-6. doi: 10.1021/jf5049063. Epub
2014
Nov 10.
Triterpenoid glycosides from Medicago sativa as antifungal agents against

Pyricularia oryzae.
Abbruscato P(1), Tosi S, Crispino L, Biazzi E, Menin B, Picco AM, Pecetti L,

Avato P, Tava A.
Author information:
(1)Genomica del Riso, Parco Tecnologico Padano , Via Einstein Località Cascina
Codazza, 26900 Lodi, Italy.
The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.)
tops and roots, the corresponding mixtures of prosapogenins from tops, and
purified saponins and sapogenins against the causal agent of rice blast
Pyricularia oryzae isolates are presented. In vitro experiments highlighted a
range of activities, depending upon the assayed metabolite. The antifungal
effects of the most promising prosapogenin mixture from alfalfa tops were
confirmed by means of in planta tests using three different Italian cultivars of
rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low
blast resistance. The evidenced antifungal properties of the tested metabolites
allowed some considerations on their structure-activity relationship. Results
indicate that prosapogenins are active compounds to prevent the fungal attack of
P. oryzae on different rice cultivars. Therefore, if properly formulated, these
substances could represent a promising and environmentally friendly treatment to
control rice blast.
DOI: 10.1021/jf5049063
573. Mol Plant Microbe Interact. 2015 Feb;28(2):134-42. doi:

10.1094/MPMI-08-14-0229-R.
The Pattern of Secreted Molecules During the Co-Inoculation of Alfalfa Plants

With Sinorhizobium meliloti and Delftia sp. strain JD2: An Interaction That
Improves Plant Yield.
Morel MA, Cagide C, Minteguiaga MA, Dardanelli MS, Castro-Sowinski S.

Delftia sp. strain JD2 is a plant-growth-promoting bacterium that enhances legume
nodulation and growth, acting as nodule-assisting bacterium during the
co-inoculation of plants with rhizobial strains. In this work, we evaluate how
the co-inoculation of alfalfa with Sinorhizobium meliloti U143 and JD2 increases
plant yield under greenhouse conditions and we analyze the pattern of secreted
bioactive compounds which may be involved in the microbe-plant communication. The
chemical composition of extracellular cultures (EC) produced in hydroponic
conditions (collected 4, 7, and 14 days after bacterial treatment) were
characterized using different chromatographic and elucidation techniques. In
addition, we assessed the effect that plant irrigation with cell-free EC,
produced during co-inoculation experiments, would have on plant yield. Results
showed increased alfalfa shoot and root matter, suggesting that U143-JD2
co-inoculation might be a beneficial agricultural practice. The pattern of
secreted secondary metabolites among treatments showed important differences.
Qualitative and quantitative changes in phenolic compounds (including
flavonoids), organic acids, and volatile compounds were detected during the early
microbe-plant interaction, suggesting that the production of some molecules
positively affects the microbe-plant association. Finally, the irrigation of
co-inoculated plants with cell-free EC under greenhouse conditions increased
plant yield over agronomic expectations. This effect might be attributed to the
bioactive secondary metabolites incorporated during the irrigation.
DOI: 10.1094/MPMI-08-14-0229-R
2014.
A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal

increases nodule numbers in Medicago truncatula independent of autoregulation.
Veliz-Vallejos DF(1), van Noorden GE(1), Yuan M(1), Mathesius U(1).
Author information:
(1)Department of Plant Science, Research School of Biology, Australian National
University Canberra, ACT, Australia.
N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate
cell-density dependent behaviors in many gram-negative bacteria, in particular
those important for plant-microbe interactions. AHLs can also be recognized by
plants, and this may influence their interactions with bacteria. Here we tested
whether the exposure to AHLs affects the nodule-forming symbiosis between legume
hosts and rhizobia. We treated roots of the model legume, Medicago truncatula,
with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti,
or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis.
We found increased numbers of nodules formed on root systems treated with the S.
meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM,
while the other AHLs did not result in significant changes to nodule numbers. We
did not find any evidence for altered nodule invasion by the rhizobia.
Quantification of flavonoids that could act as nod gene inducers in S. meliloti
did not show any correlation with increased nodule numbers. The effects of AHLs
were specific for an increase in nodule numbers, but not lateral root numbers or
root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone
treatment were under control of autoregulation of nodulation and were still
observed in the autoregulation mutant, sunn4 (super numeric nodules4). However,
increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the
ethylene-insensitive sickle mutant. A comparison between M. truncatula with M.
sativa (alfalfa) and Trifolium repens (white clover) showed that the observed
effects of AHLs on nodule numbers were specific to M. truncatula, despite M.
sativa nodulating with the same symbiont. We conclude that plant perception of
the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers
in M. truncatula via an ethylene-dependent, but autoregulation-independent
mechanism.
DOI: 10.3389/fpls.2014.00551
PMCID: PMC4196514
PMID: 25352858
575. Microb Biotechnol. 2014 Nov;7(6):570-9. doi: 10.1111/1751-7915.12165.
A comparison of the retention of pathogenic Escherichia coli O157 by sprouts,

leaves and fruits.
Mathews SL(1), Smith RB, Matthysse AG.
Author information:
(1)Department of Biology, University of North Carolina, Chapel Hill, NC,
27599-3280, USA.
The retention (binding to or association with the plant) of Escherichia coli by

cut leaves and fruits after vigorous water washing was compared with that by
sprouts. Retention by fruits and leaves was similar but differed from retention
by sprouts in rate, effect of wounding and requirement for
poly-β,1-6-N-acetyl-D-glucosamine. Escherichia coli was retained by cut ends of
lettuce leaves within 5 min while more than 1 h was required for retention by the
intact epidermis of leaves and fruits, and more than 1 day for sprouts. Retention
after 5 min at the cut leaf edge was specific for E. coli and was not shown by
the plant-associated bacteria Agrobacterium tumefaciens and Sinorhizobium
meliloti. Escherichia coli was retained by lettuce, spinach, alfalfa, bean,
tomato, Arabidopsis thaliana, cucumber, and pepper leaves and fruits faster than
by sprouts. Wounding of leaves and fruits but not sprouts increased bacterial
retention. Mutations in the exopolysaccharide synthesis genes yhjN and wcaD
reduced the numbers of bacteria retained. PgaC mutants were retained by cut
leaves and fruits but not by sprouts. There was no significant difference in the
retention of an O157 and a K12 strain by fruits or leaves. However, retention by
sprouts of O157 strains was significantly greater than K12 strains. These
findings suggest that there are differences in the mechanisms of E coli
retention among sprouts, and leaves and fruits.
© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd
and Society for Applied Microbiology.
DOI: 10.1111/1751-7915.12165
PMCID: PMC4265075
576. J Sci Food Agric. 2015 Sep;95(12):2454-61. doi: 10.1002/jsfa.6972. Epub 2014
Nov
24.
Effect of white wheat bread and white wheat bread added with bioactive compounds
on hypercholesterolemic and steatotic mice fed a high-fat diet.
Pozzo L(1), Pucci L(1), Buonamici G(2), Giorgetti L(1), Maltinti M(3), Longo
V(1).
Author information:
(1)Istituto di Biotecnologia e Biotecnologia Agraria (IBBA), CNR, I-56124 Pisa,
Italy.
(2)Agriproject, Calci, Pisa, Italy.
(3)Fondazione G. Monasterio, CNR-Regione Toscana, Pisa, Italy.
BACKGROUND: The effects of white wheat bread and white wheat bread added with a
bioactive compound mixture (Cyclanthera pedata, Glycine max, Monascus-fermented
red mold rice, Cynara scolymus and Medicago sativa) were examined on
hypercholesterolemic and steatotic mice, divided into four groups: control diet
(CTR), high-fat diet (HFD), high-fat diet with white wheat bread added with 1.5 g
kg(-1) of mixture (HFD+AB) and high-fat diet with white wheat bread (HFD+B).
RESULTS: Total serum cholesterol in the HFD+AB and HFD+B groups and hepatic
triglycerides in the HFD+AB group decreased compared with the HFD group. Liver
histology confirmed lower lipid drop accumulation in the HFD+AB group than in the
HFD and HFD+B groups. HFD+AB caused a 7.0-fold increase and a 3.5-fold reduction
in CYP7A1 and SREBP-1c gene expression respectively compared with the HFD group.
Moreover, the HFD+B group showed a 2.2-, 8.4- and 1.5-fold increase in HMG CoA
reductase, CYP7A1 and LDLr gene expression respectively compared with the HFD
group.
CONCLUSION: Both the white wheat bread and the added white wheat bread induced
cholesterol reduction by increasing CYP7A1. Moreover, the added white wheat bread
improved steatosis by decreasing SREBP-1c gene expression.
DOI: 10.1002/jsfa.6972
577. PLoS Genet. 2014 Oct 23;10(10):e1004742. doi: 10.1371/journal.pgen.1004742.

eCollection 2014 Oct.
Examination of prokaryotic multipartite genome evolution through experimental

genome reduction.
diCenzo GC(1), MacLean AM(1), Milunovic B(1), Golding GB(1), Finan TM(1).
Author information:
(1)Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Many bacteria carry two or more chromosome-like replicons. This occurs in

pathogens such as Vibrio cholerea and Brucella abortis as well as in many
N2-fixing plant symbionts including all isolates of the alfalfa root-nodule
bacteria Sinorhizobium meliloti. Understanding the evolution and role of this
multipartite genome organization will provide significant insight into these
important organisms; yet this knowledge remains incomplete, in part, because
technical challenges of large-scale genome manipulations have limited
experimental analyses. The distinct evolutionary histories and characteristics of
the three replicons that constitute the S. meliloti genome (the chromosome (3.65
Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good
model to examine this topic. We transferred essential genes from pSymB into the
chromosome, and constructed strains that lack pSymB as well as both pSymA and
pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a
prokaryotic genome to date and the first removal of an essential chromid.
Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize
55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur,
yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This
suggests that the core chromosome is sufficient for growth in a bulk soil
environment and that the pSymA and pSymB replicons carry genes with more
specialized functions such as growth in the rhizosphere and interaction with the
plant. These experimental data support a generalized evolutionary model, in which
non-chromosomal replicons primarily carry genes with more specialized functions.
These large secondary replicons increase the organism's niche range, which
offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution
with the chromosome then leads to the formation of a chromid through the
acquisition of functions core to all niches (e.g. pSymB).
DOI: 10.1371/journal.pgen.1004742
PMCID: PMC4207669
578. Animal. 2015 Mar;9(3):516-25. doi: 10.1017/S1751731114002559. Epub 2014 Oct

22.
Preference by sheep for endophyte-infected tall fescue grown adjacent to or at a

distance from alfalfa.
Friend MA(1), Provenza FD(2), Villalba JJ(2).
Author information:
(1)1Graham Centre for Agricultural Innovation (NSW DPI and Charles Sturt
University),Locked Bag 588,Wagga Wagga,NSW 2678,Australia.
(2)3Department of Wildland Resources,Utah State University,Logan,UT
84322-5230,USA.
Two experiments were conducted to assess preference by sheep for

endophyte-infected tall fescue growing in monoculture at least 5 m away from
alfalfa (fescue-middle (FM)) over endophyte-infected tall fescue growing adjacent
(0.2 to 1 m; fescue-alfalfa (FA)) to alfalfa (FA), and the effect of legume scent
on preference for endophyte-infected tall fescue. In Experiment 1, 10
six-month-old lambs were offered for 12 days a choice of freshly harvested FA and
FM. On days 13 and 14, lambs were offered the same choice, except cages (to allow
access only to scent) containing freshly harvested alfalfa were put in the
feeders containing FA, whereas cages containing freshly harvested FM were
included with the feeders containing FM. Forage intake was measured 1 h after
feeding and at three consecutive 2-h intervals thereafter. FA contained greater
(P<0.002) concentrations of the alkaloid ergovaline (360 ± 27 ppm) and CP (8 ±
0.4%) than FM (219 ± 27 ppm and 6 ± 0.4%, respectively). Lambs preferred (P<0.05)
FA to FM during the 1st hour of feeding, but the differences became smaller and
disappeared in later feeding periods (P<0.005). Lambs offered FA with alfalfa
scent or FM with FM scent preferred (P<0.05) FA but only on the 2nd day. In
Experiment 2, 10 six-month-old lambs were offered a choice of FM with cages (to
allow access only to scent) containing freshly harvested alfalfa or FM for 8
days. During the following 4 days, FM in the cages was replaced with freshly
harvested sainfoin. Preference was greater (P<0.05) for FM offered with alfalfa
scent than for FM offered with FM scent only on days 4 and 8. When lambs were
offered FM with alfalfa or sainfoin in cages, they preferred (P<0.05) tall fescue
with sainfoin scent over fescue with alfalfa scent, but intake was variable
across hours and days (P<0.001). It is concluded that (1) lambs adjusted their
intake of and preference for FA and FM over successive feeding bouts within each
day, likely owing to an attempt to balance intakes of nutrients and alkaloids and
(2) olfactory cues influenced preference, but to a lesser extent than nutrients
and alkaloids in endophyte-infected tall fescue.
DOI: 10.1017/S1751731114002559

eCollection 2014.
Hyperspectral detection of a subsurface CO2 leak in the presence of water

stressed vegetation.
Bellante GJ(1), Powell SL(1), Lawrence RL(1), Repasky KS(2), Dougher T(3).
Author information:
(1)Department of Land Resources and Environmental Sciences, Montana State
University, Bozeman, Montana, United States of America.
(2)Department of Electrical and Computer Engineering, Montana State University,
Bozeman, Montana, United States of America.
(3)Department of Plant Sciences and Plant Pathology, Montana State University,
Bozeman, Montana, United States of America.
Remote sensing of vegetation stress has been posed as a possible large area
monitoring tool for surface CO2 leakage from geologic carbon sequestration (GCS)
sites since vegetation is adversely affected by elevated CO2 levels in soil.
However, the extent to which remote sensing could be used for CO2 leak detection
depends on the spectral separability of the plant stress signal caused by various
factors, including elevated soil CO2 and water stress. This distinction is
crucial to determining the seasonality and appropriateness of remote GCS site
monitoring. A greenhouse experiment tested the degree to which plants stressed by
elevated soil CO2 could be distinguished from plants that were water stressed. A
randomized block design assigned Alfalfa plants (Medicago sativa) to one of four
possible treatment groups: 1) a CO2 injection group; 2) a water stress group; 3)
an interaction group that was subjected to both water stress and CO2 injection;
or 4) a group that received adequate water and no CO2 injection. Single date
classification trees were developed to identify individual spectral bands that
were significant in distinguishing between CO2 and water stress agents, in
addition to a random forest classifier that was used to further understand and
validate predictive accuracies. Overall peak classification accuracy was 90%
(Kappa of 0.87) for the classification tree analysis and 83% (Kappa of 0.77) for
the random forest classifier, demonstrating that vegetation stressed from an
underground CO2 leak could be accurately discerned from healthy vegetation and
areas of co-occurring water stressed vegetation at certain times. Plants appear
to hit a stress threshold, however, that would render detection of a CO2 leak
unlikely during severe drought conditions. Our findings suggest that early
detection of a CO2 leak with an aerial or ground-based hyperspectral imaging
system is possible and could be an important GCS monitoring tool.
PMCID: PMC4203680

eCollection 2014.
Alfalfa (Medicago sativa L.)/maize (Zea mays L.) intercropping provides a

feasible way to improve yield and economic incomes in farming and pastoral areas
of northeast China.
Sun B(1), Peng Y(1), Yang H(1), Li Z(1), Gao Y(1), Wang C(1), Yan Y(1), Liu Y(1).
Author information:
(1)Key Laboratory of Vegetation Ecology, Northeast Normal University, Changchun,
China.
Given the growing challenges to food and eco-environmental security as well as

sustainable development of animal husbandry in the farming and pastoral areas of
northeast China, it is crucial to identify advantageous intercropping modes and
some constraints limiting its popularization. In order to assess the performance
of various intercropping modes of maize and alfalfa, a field experiment was
conducted in a completely randomized block design with five treatments: maize
monoculture in even rows, maize monoculture in alternating wide and narrow rows,
alfalfa monoculture, maize intercropped with one row of alfalfa in wide rows and
maize intercropped with two rows of alfalfa in wide rows. Results demonstrate
that maize monoculture in alternating wide and narrow rows performed best for
light transmission, grain yield and output value, compared to in even rows. When
intercropped, maize intercropped with one row of alfalfa in wide rows was
identified as the optimal strategy and the largely complementary ecological
niches of alfalfa and maize were shown to account for the intercropping
advantages, optimizing resource utilization and improving yield and economic
incomes. These findings suggest that alfalfa/maize intercropping has obvious
advantages over monoculture and is applicable to the farming and pastoral areas
of northeast China.
PMCID: PMC4199727
Alfalfa (Medicago sativa L.).
Fu C(1), Hernandez T, Zhou C, Wang ZY.
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510 Sam
Noble Parkway, Ardmore, OK, 73401, USA.
Alfalfa (Medicago sativa L.) is a high-quality forage crop widely grown

throughout the world. This chapter describes an efficient protocol that allows
for the generation of large number of transgenic alfalfa plants by
sonication-assisted Agrobacterium-mediated transformation. Binary vectors
carrying different selectable marker genes that confer resistance to
phosphinothricin (bar), kanamycin (npt II), or hygromycin (hph) were used to
generate transgenic alfalfa plants. Intact trifoliates collected from clonally
propagated plants in the greenhouse were sterilized with bleach and then
inoculated with Agrobacterium strain EHA105. More than 80 % of infected leaf
pieces could produce rooted transgenic plants in 4-5 months after
Agrobacterium-mediated transformation.
DOI: 10.1007/978-1-4939-1695-5_17
582. Physiol Plant. 2015 Aug;154(4):511-25. doi: 10.1111/ppl.12297. Epub 2014 Nov
8.
Physiological and morphological adaptations of herbaceous perennial legumes allow

differential access to sources of varyingly soluble phosphate.
Pang J(1)(2), Yang J(3), Lambers H(1)(2), Tibbett M(4)(5), Siddique KH(2), Ryan
MH(1)(2).
Author information:
(1)School of Plant Biology, The University of Western Australia, Perth,
Australia.
(2)The UWA Institute of Agriculture, The University of Western Australia, Perth,
Australia.
(3)The Southern Grassland Ecosystem Research Station, South China Botanical
Garden, Chinese Academy of Sciences, Guangzhou, 510650, China.
(4)School of Earth and Environment, The University of Western Australia, Perth,
Australia.
(5)Department of Environmental Science and Technology, School of Applied
Sciences, Cranfield University, Cranfield, UK.
The aim of this study was to investigate the capacity of three perennial legume
species to access sources of varyingly soluble phosphorus (P) and their
associated morphological and physiological adaptations. Two Australian native
legumes with pasture potential (Cullen australasicum and Kennedia prostrata) and
Medicago sativa cv. SARDI 10 were grown in sand under two P levels (6 and 40 µg P
g(-1) ) supplied as Ca(H2 PO4 )2 ·H2 O (Ca-P, highly soluble, used in many
fertilizers) or as one of three sparingly soluble forms: Ca10 (OH)2 (PO4 )6
(apatite-P, found in relatively young soils; major constituent of rock
phosphate), C6 H6 O24 P6 Na12 (inositol-P, the most common form of organic P in
soil) and FePO4 (Fe-P, a poorly-available inorganic source of P). All species
grew well with soluble P. When 6 µg P g(-1) was supplied as sparingly soluble P,
plant dry weight (DW) and P uptake were very low for C. australasicum and M.
sativa (0.1-0.4 g DW) with the exception of M. sativa supplied with apatite-P
(1.5 g). In contrast, K. prostrata grew well with inositol-P (1.0 g) and Fe-P
(0.7 g), and even better with apatite-P (1.7 g), similar to that with Ca-P
(1.9 g). Phosphorus uptake at 6 µg P g(-1) was highly correlated with total root
length, total rhizosphere carboxylate content and total rhizosphere acid
phosphatase (EC 3.1.3.2) activity. These findings provide strong indications that
there are opportunities to utilize local Australian legumes in low P pasture
systems to access sparingly soluble soil P and increase perennial legume
productivity, diversity and sustainability.
DOI: 10.1111/ppl.12297

Oct
3.
Influence of corn silage hybrid type on lactation performance by Holstein dairy

cows.
Akins MS(1), Shaver RD(2).
Author information:
(1)School of Agriculture, University of Wisconsin-Platteville, Platteville 53818.
Electronic address: msakins@wisc.edu.
The primary objective of this study was to determine lactation performance by
dairy cows fed nutridense (ND), dual-purpose (DP), or brown midrib (BM) corn
silage hybrids at the same concentration in the diets. A secondary objective was
to determine lactation performance by dairy cows fed NutriDense corn silage at a
higher concentration in the diet. One hundred twenty-eight Holstein and Holstein
× Jersey cows (105 ± 38 d in milk) were stratified by breed and parity and
randomly assigned to 16 pens of 8 cows each. Pens were then randomly assigned to
1 of 4 treatments. Three treatment total mixed rations (TMR; DP40, BM40, and
ND40) contained 40% of dry matter (DM) from the respective corn silage hybrid and
20% of DM from alfalfa silage. The fourth treatment TMR had ND corn silage as the
sole forage at 65% of DM (ND65). A 2-wk covariate adjustment period preceded the
treatment period, with all pens receiving a TMR with equal proportions of DP40,
BM40, and ND40. Following the covariate period, cows were fed their assigned
treatment diets for 11 wk. nutridense corn silage had greater starch and lower
neutral detergent fiber (NDF) content than DP or BM, resulting in ND40 having
greater energy content (73.2% of total digestible nutrients, TDN) than DP40 or
BM40 (71.9 and 71.4% TDN, respectively). Cows fed BM40 had greater milk yield
than DP40, whereas ND40 tended to have greater milk yield and had greater protein
and lactose yields compared with DP40. No differences in intake,
component-corrected milk yields, or feed efficiency were detected between DP40,
BM40, and ND40. Milk yield differences may be due to increased starch intake for
ND40 and increased digestible NDF intake for BM40 compared with DP40. Intake and
milk yield and composition were similar for ND40 compared with BM40, possibly due
to counteracting effects of higher starch intake for ND40 and higher digestible
NDF intake for BM40. Feeding ND65 reduced intake, and thus milk and component
yields, compared with ND40 due to either increased ruminal starch digestibility
or increased rumen fill for ND65. Nutridense corn silage was a viable alternative
to both DP and BM at 40% of diet DM; however, lactation performance was reduced
when nutridense corn silage was fed at 65% of DM.

DOI: 10.3168/jds.2014-8400

eCollection 2014.
Cadmium-induced hydrogen sulfide synthesis is involved in cadmium tolerance in

Medicago sativa by reestablishment of reduced (homo)glutathione and reactive
oxygen species homeostases.
Cui W(1), Chen H(2), Zhu K(1), Jin Q(1), Xie Y(1), Cui J(1), Xia Y(1), Zhang
J(1), Shen W(1).
Author information:
Agricultural University, Jiangsu Province, Nanjing, China.
(2)Key Laboratory of Protection and Development Utilization of Tropical Crop
Germplasm Resources, Hainan University, Haikou, China.
Until now, physiological mechanisms and downstream targets responsible for the
cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H2S) have been
elusive. To address this gap, a combination of pharmacological, histochemical,
biochemical and molecular approaches was applied. The perturbation of reduced
(homo)glutathione homeostasis and increased H2S production as well as the
activation of two H2S-synthetic enzymes activities, including L-cysteine
desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD), in alfalfa seedling roots
were early responses to the exposure of Cd. The application of H2S donor sodium
hydrosulfide (NaHS), not only mimicked intracellular H2S production triggered by
Cd, but also alleviated Cd toxicity in a H2S-dependent fashion. By contrast, the
inhibition of H2S production caused by the application of its synthetic inhibitor
blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and
reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses
were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS
responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed
by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP)
signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H2S
might be an important signaling molecule in the enhancement of Cd toxicity in
alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS
homeostases.
PMCID: PMC4183592

Sep
26.
Influence of maturity on alfalfa hay nutritional fractions and indigestible fiber

content.
Palmonari A(1), Fustini M(2), Canestrari G(2), Grilli E(2), Formigoni A(2).
Author information:
(1)Department Of Veterinary Medicine, Università di Bologna, 40084 Bologna,
Italy. Electronic address: alberto.palmonari2@unibo.it.
(2)Department Of Veterinary Medicine, Università di Bologna, 40084 Bologna,
Italy.
This study focused on changes in fibrous and protein fractions, changes in fiber
digestibility and amount of indigestible neutral detergent fiber (NDF) as a
consequence of increased maturity in alfalfa. A total area of 720 m(2) was
divided in 18 blocks randomly assigned to 3 treatments, differing in cutting
intervals. Treatment 1 was harvested with a 21-d cutting schedule, at a prebloom
stage; treatment 2 with a 28-d schedule, at about first-bloom stage; whereas a
full bloom was observed in treatment 3, harvested with a 35-d cutting schedule.
Treatments were replicated 4 times through the spring-summer period for 2
subsequent years, 2011 and 2012. Statistical differences were observed for crude
protein [treatment 1: 20.8%, treatment 2: 17.3%, and treatment 3: 17.0%; standard
error of the mean (SEM)=0.83], soluble protein, and nonprotein nitrogen among
treatments on a dry matter basis. Similar results were observed for acid
detergent lignin (6.3, 6.9, and 7.3%, respectively; SEM=0.39), lower in treatment
1 compared with others, and in vitro NDF digestibility at 24 or 240 h.
Indigestible NDF at 240 h resulted in lower values for treatment 1 compared with
treatments 2 and 3 (15.5, 17.2, and 18.3%, respectively; SEM=1.54). Moreover, the
indigestible NDF:acid detergent lignin ratio varied numerically but not
statistically among treatments, being as much as 9% greater than the 2.4 fixed
value applied for rate of digestion calculation and Cornell Net Carbohydrate
Protein System (Cornell University, Ithaca, NY)-based model equations. Assuming
the diet composition remained unchanged, treatment 3 (35-d cutting interval)
would be expected to yield 1.4 kg less milk per day based on energy supply, and
2.8 kg less milk daily based on protein supply than treatment 1.
DOI: 10.3168/jds.2014-8123

Sep
26.
Effects of alfalfa and cereal straw as a forage source on nutrient digestibility

and lactation performance in lactating dairy cows.
Wang B(1), Mao SY(2), Yang HJ(3), Wu YM(1), Wang JK(1), Li SL(3), Shen ZM(2), Liu
JX(4).
Author information:
(1)Institute of Dairy Science, MoE Key Laboratory of Molecular Animal Nutrition,
College of Animal Sciences, Zhejiang University, Hangzhou 310058, P. R. China.
(2)College of Animal Science and Technology, Nanjing Agricultural University,
Nanjing 210095, P. R. China.
Technology, China Agricultural University, Beijing 100094, P. R. China.
(4)Institute of Dairy Science, MoE Key Laboratory of Molecular Animal Nutrition,
College of Animal Sciences, Zhejiang University, Hangzhou 310058, P. R. China.
Electronic address: liujx@zju.edu.cn.
Erratum in
J Dairy Sci. 2015 Jan;98(1):719.
This study was conducted to investigate the nutrient digestibility and lactation
performance when alfalfa was replaced with rice straw or corn stover in the diet
of lactating cows. Forty-five multiparous Holstein dairy cows were blocked based
on days in milk (164 ± 24.8 d; mean ± standard deviation) and milk yield (29.7 ±
4.7 kg; mean ± standard deviation) and were randomly assigned to 1 of 3
treatments. Diets were isonitrogenous, with a forage-to-concentrate ratio of
45:55 [dry matter (DM) basis] and contained identical concentrate mixtures and
15% corn silage, with different forage sources (on a DM basis): 23% alfalfa hay
and 7% Chinese wild rye hay (AH), 30% corn stover (CS), and 30% rice straw (RS).
The experiment was conducted over a 14-wk period, with the first 2 wk for
adaptation. The DM intake of the cows was not affected by forage source. Yield of
milk, milk fat, protein, lactose, and total solids was higher in cows fed diets
of AH than diets of RS or CS, with no difference between RS and CS. Contents of
milk protein and total solids were higher in AH than in RS, with no difference
between CS and AH or RS. Feed efficiency (milk yield/DM intake) was highest for
cows fed AH, followed by RS and CS. Cows fed AH excreted more urinary purine
derivatives, indicating that the microbial crude protein yield may be higher for
the AH diet than for RS and CS, which may be attributed to the higher content of
fermentable carbohydrates in AH than in RS and CS. Total-tract apparent
digestibilities of all the nutrients were higher in cows fed the AH diet than
those fed CS and RS. The concentration of rumen volatile fatty acids was higher
in the AH diet than in CS or RS diets, with no difference between CS and RS
diets. When the cereal straw was used to replace alfalfa as a main forage source
for lactating cows, the shortage of fermented energy may have reduced the rumen
microbial protein synthesis, resulting in lower milk protein yield, and lower
nutrient digestibility may have restricted milk production.

DOI: 10.3168/jds.2014-7961

Sep
26.
Feeding protein supplements in alfalfa hay-based lactation diets improves

nutrient utilization, lactational performance, and feed efficiency of dairy cows.
Neal K(1), Eun JS(2), Young AJ(1), Mjoun K(3), Hall JO(1).
Author information:
Logan 84322.
(3)Alltech, Brookings, SD 57006.
Due to the increasing cost of soybean meal and concerns of excess N being
excreted into the environment, new protein supplements have been developed. Two
products that have shown potential in increasing N utilization efficiency are
slow-release urea (SRU; Optigen; Alltech Inc., Nicholasville, KY) and
ruminal-escape protein derived from yeast (YMP; DEMP; Alltech Inc.). The
objective of this study was to assess the effects of feeding these 2 supplements
in alfalfa hay-based [45.7% of forage dietary dry matter (DM)] dairy diets on
nutrient utilization, feed efficiency, and lactational performance of dairy cows.
Twelve multiparous dairy cows were used in a triple 4 × 4 Latin square design
with one square consisting of ruminally cannulated cows. Treatments included (1)
control, (2) SRU-supplemented total mixed ration (SRUT), (3) YMP-supplemented
total mixed ration (YMPT), and (4) SRU- and YMP-supplemented total mixed ration
(SYT). The control consisted only of a mixture of soybean meal and canola meal in
a 50:50 ratio. The SRU and the YMP were supplemented at 0.49 and 1.15% DM,
respectively. The experiment consisted of 4 periods lasting 28 d each (21 d of
adaptation and 7 d of sampling). Cows fed YMPT and SYT had decreased intake of
DM, and all supplemented treatments had lower crude protein intake compared with
those fed the control. Milk yield tended to have the greatest increase in YMPT
compared with the control (41.1 vs. 39.7 kg/d) as well as a tendency for
increased milk fat and protein yields. Feed efficiencies based on yields of milk,
3.5% fat-corrected milk, and energy-corrected milk increased at 10 to 16% due to
protein supplementation. Cows fed protein supplements partitioned less energy
toward body weight gain, but tended to partition more energy toward milk
production. Efficiency of use of feed N to milk N increased by feeding SRUT and
YMPT, and milk N-to-manure N ratio increased with YMPT. Overall results from this
experiment indicate that replacing the mixture of soybean meal and canola meal
with SRU and YMP in alfalfa hay-based dairy diets can be a good approach to
improve nutrient utilization efficiencies in lactating dairy cows.

DOI: 10.3168/jds.2014-8033

Sep
26.
Effects of altering alfalfa hay quality when feeding steam-flaked versus

high-moisture corn grain on ruminal fermentation and lactational performance of
dairy cows.
Eun JS(1), Kelley AW(2), Neal K(2), Young AJ(2), Hall JO(2).
Author information:
Logan 84322.
This experiment was performed to test a hypothesis that nutritive benefits of

feeding high-moisture corn (HMC) would be different when fed with different
qualities of alfalfa hay (AH) due to associative effects on ruminal fermentation
and nutrient utilization efficiency. Eight multiparous lactating Holstein cows
were used; 4 were surgically fitted with ruminal cannulas. Days in milk averaged
184 ± 10.7 at the start of the experiment. The experiment was performed in a
duplicate 4 × 4 Latin square design. Within each square, cows were randomly
assigned to a sequence of 4 diets during each of the four 21-d periods (14 d of
treatment adaptation and 7 d of data collection and sampling). A 2 × 2 factorial
arrangement was used; fair-quality AH [FAH; 39.6% neutral detergent fiber (NDF)
and 17.9% crude protein (CP)] or high-quality AH (HAH; 33.6% NDF and 21.9% CP)
was combined with steam-flaked corn (SFC) or HMC to form 4 treatments: FAH with
SFC, FAH with HMC, HAH with SFC, and HAH with HMC. The AH was fed at 32% dry
matter (DM) content, whereas SFC or HMC was included at 17% DM content. Quality
of AH did not affect DM intake, whereas feeding HMC decreased DM intake,
regardless of quality of AH. Digestibility of DM was greater for cows fed HAH
compared with those fed FAH (70.1 vs. 67.6%). Digestibility of NDF increased by
feeding HMC (67.6 vs. 58.4%), but not by quality of AH. Under FAH, starch
digestibility decreased by feeding HMC compared with SFC (85.7 vs. 95.0%), but it
was similar under HAH, resulting in an interaction between quality of AH and type
of corn grain (CG). Feeding different qualities of AH did not affect milk yield;
however, feeding HMC decreased milk yield in FAH diet, causing an AH × CG
interaction. Efficiency of milk yield/DM intake was improved due to feeding HMC,
regardless of the quality of the AH. In addition, dietary N utilization for milk
N tended to increase by feeding HMC, but it was not influenced by quality of AH.
Yield of microbial protein increased by feeding HAH diets compared with FAH
diets, whereas feeding the HMC diet increased microbial protein yield under the
HAH diet, leading to an interaction between and AH and CG. Overall results in
this experiment indicate that feeding HMC in AH-based diets improved feed
efficiency as well as N utilization efficiency, regardless of quality of AH.

DOI: 10.3168/jds.2014-8425
589. J Chem Ecol. 2014 Oct;40(10):1099-109. doi: 10.1007/s10886-014-0504-3. Epub

2014
Sep 28.
Stem nematode counteracts plant resistance of aphids in alfalfa, Medicago sativa.

Ramirez RA(1), Spears LR.
Author information:
(1)Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT,
84322, USA, ricardo.ramirez@usu.edu.
Plants are exploited by a diverse community of insect herbivores and

phytopathogens that interact indirectly through plant-mediated interactions.
Generally, plants are thought to respond to insects and pathogens through
different defensive signaling pathways. As plants are selected for resistance to
one phytophagous organism type (insect vs. pathogen) in managed systems, it is
not clear how this selection may affect community interactions. This study
examined the effect of nematode-resistant varieties on aphid (Acyrthosiphon
pisum) suppression, and then determined how infection by the stem nematode,
Ditylenchus dipsaci, mediated ecological effects on aphids and on plant defense
proteins. Four alfalfa (Medicago sativa) varieties were selected with resistance
to nematodes only (+,-), aphids only (-,+), nematodes and aphids (+,+), and
susceptibility to nematodes and aphids (-,-). Field and greenhouse experiments
were conducted to isolate the effect of nematode infection and aphid abundance on
each variety. We found that varieties resistant to nematode, regardless of aphid
resistance, had the lowest aphid counts, suggesting possible cross-resistance.
Aphid abundance, however, increased when plants were exposed to nematodes.
Resistant varieties were associated with elevated saponins but these compounds
were not affected by insect or pathogen feeding. Concentrations of peroxidases
and trypsin inhibitors, however, were increased in nematode resistant varieties
when exposed to nematodes and aphids, respectively. The patterns of plant defense
were variable, and a combination of resistance traits and changes in nutrient
availability may drive positive interactions between nematodes and aphids
aboveground.
DOI: 10.1007/s10886-014-0504-3
PMCID: PMC4244557
590. J Chem Ecol. 2014 Sep;40(9):1051-61. doi: 10.1007/s10886-014-0498-x. Epub 2014

Sep 27.
Ambient has become strained. Identification of Acacia dealbata Link volatiles

interfering with germination and early growth of native species.
Souza-Alonso P(1), González L, Cavaleiro C.
Author information:
(1)Plant Biology and Soil Science Department, University of Vigo, 36310, Vigo,
Spain, souza@uvigo.es.
Acacia dealbata Link is a widespread invader in Mediterranean type ecosystems,

and traits promoting its invasiveness are currently under investigation. Due to
the dense atmosphere below its canopy, we hypothesized that volatile organic
compounds (VOCs) released from flowers, leaves, litter, or a mixture of
treatments exert inhibitory effects on the natives Trifolium subterraneum, Lolium
multiflorum, Medicago sativa, and also on its own seeds. We reported that VOCs
from flowers significantly reduced germination in L. multiflorum and A. dealbata;
moreover, root length, stem length, aboveground and belowground biomass were also
reduced in all species studied. Volatile organic compounds from flowers and the
mixture also increased significantly malondialdehyde content in T. subterraneum
and L. multiflorum. The effects of VOCs on antioxidant enzymatic activities were
species dependent. Flowers enhanced peroxidase but decreased superoxide dismutase
activity in T. subterraneum. In contrast, VOCs released from leaves increased the
activity of superoxide dismutase in L. multiflorum. GC/MS analyses revealed 27
VOCs in the volatile fraction from flowers, 12 of which were exclusive to this
fraction. Within them, heptadecadiene, n-nonadecane, n-tricosane, and octadecene
represent 62% of the fraction. We present evidence that the VOCs released from A.
dealbata flowers strongly inhibited germination and seedling growth of selected
species, and mainly on its own seedlings. As far as we know, this is the first
evidence of phytotoxicity induced by VOCs in invasive species belonging to the
Acacia genus.
DOI: 10.1007/s10886-014-0498-x

Sep
18.
Effects of dairy slurry on silage fermentation characteristics and nutritive

value of alfalfa.
Coblentz WK(1), Muck RE(2), Borchardt MA(3), Spencer SK(3), Jokela WE(3), Bertram
MG(4), Coffey KP(5).
Author information:
(1)USDA-Agricultural Research Service, US Dairy Forage Research Center,
Marshfield, WI 54449. Electronic address: wayne.coblentz@ars.usda.gov.
(2)USDA-Agricultural Research Service, US Dairy Forage Research Center, Madison,
WI 53706.
(3)USDA-Agricultural Research Service, US Dairy Forage Research Center,
Marshfield, WI 54449.
(4)University of Wisconsin Arlington Agricultural Research Station, Arlington
53911.
Dairy producers frequently ask questions about the risks associated with applying
dairy slurry to growing alfalfa (Medicago sativa L.). Our objectives were to
determine the effects of applying dairy slurry on the subsequent nutritive value
and fermentation characteristics of alfalfa balage. Dairy slurry was applied to
0.17-ha plots of alfalfa; applications were made to the second (HARV1) and third
(HARV2) cuttings during June and July of 2012, respectively, at mean rates of
42,400 ± 5271 and 41,700 ± 2397 L/ha, respectively. Application strategies
included (1) no slurry, (2) slurry applied directly to stubble immediately after
the preceding harvest, (3) slurry applied after 1 wk of post-ensiled regrowth, or
(4) slurry applied after 2 wk of regrowth. All harvested forage was packaged in
large, rectangular bales that were ensiled as wrapped balage. Yields of DM
harvested from HARV1 (2,477 kg/ha) and HARV2 (781 kg/ha) were not affected by
slurry application treatment. By May 2013, all silages appeared to be well
preserved, with no indication of undesirable odors characteristic of clostridial
fermentations. Clostridium tyrobutyricum, which is known to negatively affect
cheese production, was not detected in any forage on either a pre- or
post-ensiled basis. On a pre-ensiled basis, counts for Clostridium cluster 1 were
greater for slurry-applied plots than for those receiving no slurry, and this
response was consistent for HARV1 (4.44 vs. 3.29 log10 genomic copies/g) and
HARV2 (4.99 vs. 3.88 log10 genomic copies/g). Similar responses were observed on
a post-ensiled basis; however, post-ensiled counts also were greater for HARV1
(5.51 vs. 5.17 log10 genomic copies/g) and HARV2 (5.84 vs. 5.28 log10 genomic
copies/g) when slurry was applied to regrowth compared with stubble. For HARV2,
counts also were greater following a 2-wk application delay compared with a 1-wk
delay (6.23 vs. 5.45 log10 genomic copies/g). These results suggest that the risk
of clostridial fermentations in alfalfa silages is greater following applications
of slurry. Based on pre- and post-ensiled clostridial counts, applications of
dairy slurry on stubble are preferred (and less risky) compared with delayed
applications on growing alfalfa.

DOI: 10.3168/jds.2014-8582
592. J Plant Physiol. 2014 Nov 15;171(18):1774-81. doi:

10.1016/j.jplph.2014.07.027.
Epub 2014 Sep 6.
Increased photosynthetic acclimation in alfalfa associated with arbuscular

mycorrhizal fungi (AMF) and cultivated in greenhouse under elevated CO2.
Goicoechea N(1), Baslam M(2), Erice G(3), Irigoyen JJ(2).
Author information:
(1)Grupo de Fisiología del Estrés en Plantas (Unidad Asociada al CSIC, EEAD,
Zaragoza e ICVV, Logroño), Departamento de Biología Ambiental, Facultades de
Ciencias y Farmacia, Universidad de Navarra, C/ Irunlarrea, 1, 31008 Pamplona,
Spain. Electronic address: niegoi@unav.es.
(2)Grupo de Fisiología del Estrés en Plantas (Unidad Asociada al CSIC, EEAD,
Zaragoza e ICVV, Logroño), Departamento de Biología Ambiental, Facultades de
Ciencias y Farmacia, Universidad de Navarra, C/ Irunlarrea, 1, 31008 Pamplona,
Spain.
(3)Institute for Genomic Biology, University of Illinois at Urbana-Champaign,
1206W. Gregory Drive, Urbana, IL 61801, USA.
Medicago sativa L. (alfalfa) can exhibit photosynthetic down-regulation when

grown in greenhouse conditions under elevated atmospheric CO2. This forage legume
can establish a double symbiosis with nitrogen fixing bacteria and arbuscular
mycorrhizal fungi (AMF), which may increase the carbon sink effect of roots. Our
aim was to assess whether the association of alfalfa with AMF can avoid, diminish
or delay the photosynthetic acclimation observed in previous studies performed
with nodulated plants. The results, however, showed that mycorrhizal (M) alfalfa
at the end of their vegetative period had lower carbon (C) discrimination than
non-mycorrhizal (NM) controls, indicating photosynthetic acclimation under ECO2
in plants associated with AMF. Decreased C discrimination was due to the
acclimation of conductance, since the amount of Rubisco and the expression of
genes codifying both large and small subunits of Rubisco were similar or slightly
higher in M than in NM plants. Moreover, M alfalfa accumulated a greater amount
of soluble sugars in leaves than NM plants, thus favoring a down-regulation
effect on photosynthetic rates. The enhanced contents of sugars in leaves
coincided with a reduced percentage of arbuscules in roots, suggesting decreased
sink of carbohydrates from shoots to roots in M plants. The shorter life cycle of
alfalfa associated with AMF in comparison with the NM controls may also be
related to the accelerated photosynthetic acclimation in M plants. Further
research is needed to clarify to what extent this behavior could be extrapolated
to alfalfa cultivated in the field and subjected to periodic cutting of shoots
under climatic change scenarios.

DOI: 10.1016/j.jplph.2014.07.027
593. Plant Physiol Biochem. 2014 Nov;84:67-77. doi: 10.1016/j.plaphy.2014.08.025.

Epub
2014 Sep 6.
Transgenic alfalfa plants expressing AtNDPK2 exhibit increased growth and

tolerance to abiotic stresses.
Wang Z(1), Li H(2), Ke Q(3), Jeong JC(3), Lee HS(3), Xu B(4), Deng XP(4), Lim
YP(5), Kwak SS(6).
Author information:
Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806,
Republic of Korea; Department of Horticulture, Chungnam National University,
Daejeon, Republic of Korea.
Republic of Korea; State Key Laboratory of Soil Erosion and Dryland Farming on
the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of
Science and Ministry of Water Resources, Northwest A & F University, Yangling,
Shaanxi 712100, PR China.
Republic of Korea; Department of Green Chemistry and Environmental Biotechnology,
Korea University of Science and Technology, Daejeon, Republic of Korea.
Institute of Soil and Water Conservation, Chinese Academy of Science and Ministry
of Water Resources, Northwest A & F University, Yangling, Shaanxi 712100, PR
China.
(5)Department of Horticulture, Chungnam National University, Daejeon, Republic of
Korea.
Republic of Korea; Department of Green Chemistry and Environmental Biotechnology,
Korea University of Science and Technology, Daejeon, Republic of Korea.
Electronic address: sskwak@kribb.re.kr.
In this study, we generated and evaluated transgenic alfalfa plants (Medicago

sativa L. cv. Xinjiang Daye) expressing the Arabidopsis nucleoside diphosphate
kinase 2 (AtNDPK2) gene under the control of the oxidative stress-inducible SWPA2
promoter (referred to as SN plants) to develop plants with enhanced tolerance to
various abiotic stresses. We selected two SN plants (SN4 and SN7) according to
the expression levels of AtNDPK2 and the enzyme activity of NDPK in response to
methyl viologen (MV)-mediated oxidative stress treatment using leaf discs for
further characterization. SN plants showed enhanced tolerance to high
temperature, NaCl, and drought stress on the whole-plant level. When the plants
were subjected to high temperature treatment (42 °C for 24 h), the non-transgenic
(NT) plants were severely wilted, whereas the SN plants were not affected because
they maintained high relative water and chlorophyll contents. The SN plants also
showed significantly higher tolerance to 250 mM NaCl and water stress treatment
than the NT plants. In addition, the SN plants exhibited better plant growth
through increased expression of auxin-related indole acetic acid (IAA) genes
(MsIAA3, MsIAA5, MsIAA6, MsIAA7, and MsIAA16) under normal growth conditions
compared to NT plants. The results suggest that induced overexpression of AtNDPK2
in alfalfa will be useful for increasing biomass production under various abiotic
stress conditions.
DOI: 10.1016/j.plaphy.2014.08.025
594. Interdiscip Sci. 2015 Mar;7(1):7-20. doi: 10.1007/s12539-013-0017-8. Epub 2014

Sep 19.
Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis

vinifera and Oryza brachyantha using comparative modelling.
Sagar M(1), Pandey N, Qamar N, Singh B, Shukla A.
Author information:
(1)Department of Bioinformatics, University Institute of Engineering and
Technology, Chhatrapati Shahuji Maharaj University, Kanpur, 208024, India,
mamta1060@yahoo.com.
The long chain fatty acids incorporated into plant lipids are derived from the
iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA
after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants.
This study provides functional characterization of three 3 ketoacyl CoA synthase
like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins).
Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity
to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in
Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type
III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and
3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very
long chain 3-keto acyl CoA domain is absent. All three proteins were found to
have Chalcone and stilbene synthases C terminal domain which is similar to domain
of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7
protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in
N-terminal domain is responsible for distinguish activity. The J3MF16 protein of
Oryza brachyantha contains N terminal domain and C terminal domain and
characterized using annotation of these domains. Domains Gcs (streptomyces
coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera
hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in
three proteins. This similarity points toward anthocyanin biosynthetic process.
Similarity to chalcone synthase 2 reveals its possible role in Naringenine and
Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza
brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16
protein that are common in these three protein at different positions. Structural
variations among dimer interface, product binding site, malonyl-CoA binding
sites, were predicted in localized combination of conserved residues.
DOI: 10.1007/s12539-013-0017-8
595. Ecotoxicol Environ Saf. 2014 Dec;110:129-35. doi:

10.1016/j.ecoenv.2014.08.031.
Epub 2014 Sep 18.
Pyrene removal and transformation by joint application of alfalfa and exogenous

microorganisms and their influence on soil microbial community.
Ye J(1), Yin H(2), Peng H(3), Bai J(1), Li Y(1).
Author information:
(1)Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of
Guangdong Higher Education Institutes, School of Environment, Jinan University,
Guangzhou 510632, China.
(2)Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters,
College of Environment and Energy, South China University of Technology,
Guangzhou 510006, China. Electronic address: huayin@scut.edu.cn.
(3)Department of Chemistry, Jinan University, Guangzhou 510632, China.
Phytoremediation is an attractive approach for the cleanup of polycyclic aromatic

hydrocarbons-contaminated soil. The joint effect of alfalfa and microorganisms,
including Arthrobacter oxydans, Staphylococcus auricularis and Stenotrophomonas
maltophilia, on pyrene removal was investigated. The results showed that the
joint effect primarily contributed to pyrene removal, and the concentration of
residual pyrene in rhizosphere soil was lower than that in non-rhizosphere soil.
After joint treatment for 45d, pyrene in rhizosphere soils decreased from 11.3,
52.5 and 106.0mg/kg to 2.0-3.0, 15.0-18.7, and 41.2-44.8mg/kg, respectively.
These bacteria significantly enhanced pyrene accumulation and microbial community
diversity, and increased soil dehydrogenase and polyphenol oxidase activities.
Pyrene was initially degraded through ring cleavage. One of the main metabolites
4-dihydroxy-phenanthrene was transformed into naphthol and
1,2-dihydroxynaphthalene, which were further degraded through salicylic acid
pathway and phthalic acid pathway, separately.
DOI: 10.1016/j.ecoenv.2014.08.031
596. BMC Plant Biol. 2014 Sep 17;14:248. doi: 10.1186/s12870-014-0248-9.
Alfalfa snakin-1 prevents fungal colonization and probably coevolved with

rhizobia.
García AN, Ayub ND, Fox AR, Gómez MC, Diéguez MJ, Pagano EM, Berini CA,
Muschietti JP, Soto G.
BACKGROUND: The production of antimicrobial peptides is a common defense strategy

of living cells against a wide range of pathogens. Plant snakin peptides inhibit
bacterial and fungal growth at extremely low concentrations. However, little is
known of their molecular and ecological characteristics, including origin,
evolutionary equivalence, specific functions and activity against beneficial
microbes. The aim of this study was to identify and characterize snakin-1 from
alfalfa (MsSN1).
RESULTS: Phylogenetic analysis showed complete congruence between snakin-1 and
plant trees. The antimicrobial activity of MsSN1 against bacterial and fungal
pathogens of alfalfa was demonstrated in vitro and in vivo. Transgenic alfalfa
overexpressing MsSN1 showed increased antimicrobial activity against virulent
fungal strains. However, MsSN1 did not affect nitrogen-fixing bacterial strains
only when these had an alfalfa origin.
CONCLUSIONS: The results reported here suggest that snakin peptides have
important and ancestral roles in land plant innate immunity. Our data indicate a
coevolutionary process, in which alfalfa exerts a selection pressure for
resistance to MsSN1 on rhizobial bacteria. The increased antimicrobial activity
against virulent fungal strains without altering the nitrogen-fixing symbiosis
observed in MsSN1-overexpressing alfalfa transgenic plants opens the way to the
production of effective legume transgenic cultivars for biotic stress resistance.
DOI: 10.1186/s12870-014-0248-9
PMCID: PMC4177055
597. Sci Rep. 2014 Sep 17;4:6389. doi: 10.1038/srep06389.
Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A

new promising Plant Probiotic Bacteria.
Martínez-Hidalgo P(1), Galindo-Villardón P(2), Trujillo ME, Igual JM(3),

Martínez-Molina E(1).
Author information:
(1)1] Department of Microbiology and Genetics. University of Salamanca. Plaza
Doctores de la Reina s/n. 37007 Salamanca, Spain [2] Unidad Asociada USAL-CSIC
"Interacción Planta-Microorganismo"
(2)Department of Statistics. University of Salamanca. Plaza Doctores de la Reina
s/n. 37007 Salamanca, Spain.
(3)1] Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), Cordel
de Merinas 40-52, 37008 Salamanca, Spain [2] Unidad Asociada USAL-CSIC
"Interacción Planta-Microorganismo"
Erratum in
Sci Rep. 2015;5:8271. Trujillo, Martha E [added].
Biotic interactions can improve agricultural productivity without costly and

environmentally challenging inputs. Micromonospora strains have recently been
reported as natural endophytes of legume nodules but their significance for plant
development and productivity has not yet been established. The aim of this study
was to determine the diversity and function of Micromonospora isolated from
Medicago sativa root nodules. Micromonospora-like strains from field alfalfa
nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene
sequencing. The ecological role of the interaction of the 15 selected
representative Micromonospora strains was tested in M. sativa. Nodulation, plant
growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain
abundant and highly diverse populations of Micromonospora, both at the intra- and
at interspecific level. Selected Micromonospora isolates significantly increase
the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the
plant for nitrogen nutrition. Moreover, they promote aerial growth, the
shoot-to-root ratio, and raise the level of essential nutrients. Our results
indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and
has probiotic effects, promoting plant growth and increasing nutrition
efficiency. Its ecological role, biotechnological potential and advantages as a
plant probiotic bacterium (PPB) are also discussed.
DOI: 10.1038/srep06389
PMCID: PMC4165979
598. J Agric Food Chem. 2014 Oct 8;62(40):9657-68. doi: 10.1021/jf503221c. Epub
2014
Sep 29.
Chemical modification and degradation of atrazine in Medicago sativa through

multiple pathways.
Zhang JJ(1), Lu YC, Yang H.
Author information:
Agricultural University , Nanjing 210095, China.
Atrazine is a member of the triazine herbicide family intensively used to control

weeds for crop production. In this study, atrazine residues and its degraded
products in alfalfa (Medicago sativa) were characterized using UPLC-TOF-MS/MS.
Most of atrazine absorbed in plants was found as chemically modified derivatives
like deisopropylated atrazine (DIA), dehydrogenated atrazine (DHA), or methylated
atrazine (MEA), and some atrazine derivatives were conjugated through different
functional groups such as sugar, glutathione, and amino acids. Interestingly, the
specific conjugates DHA+hGSH (homoglutathione) and MEA-HCl+hGSH in alfalfa were
detected. These results suggest that atrazine in alfalfa can be degraded through
different pathways. The increased activities of glycosyltransferase and
glutathione S-transferase were determined to support the atrazine degradation
models. The outcome of the work uncovered the detailed mechanism for the residual
atrazine accumulation and degradation in alfalfa and will help to evaluate
whether the crop is suitable to be cultivated in the atrazine-polluted soil.
DOI: 10.1021/jf503221c
599. Water Sci Technol. 2014;70(5):795-802. doi: 10.2166/wst.2014.297.
Development of a natural treatment system consisting of red ball earth and

alfalfa for the post-treatment of anaerobically digested livestock wastewater.
Chen X(1), Fukushi K(2).
Author information:
(1)Department of Urban Engineering, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan E-mail: chenxiaochen1984@gmail.com.
(2)Integrated Research System for Sustainability Science, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan.
With the objective of developing a post-treatment process for anaerobically

digested livestock wastewater, an innovative natural treatment system composed of
two units is proposed. The first trickling filter unit further reduced
biochemical oxygen demand and achieved a certain degree of nitrification. The
second soil-plant unit was targeted at the removal and recovery of nutrients N, P
and K. For the feasibility study, a bench-scale soil column test was carried out,
in which red ball earth and alfalfa were utilized for treating synthetic
nutrient-enriched wastewater. Through long-term operation, the nitrification
function was well established in the top layers, especially the top 20 cm,
although a supplementary denitrification process was still required before
discharge. P and K were retained by the soil through different mechanisms, and
their plant-available forms that remained in the soil were considered suitable
for indirect nutrient reuse. As for alfalfa, with wastewater application it fixed
more N from the atmosphere, and directly recovered 6% of P and 4% of K input from
wastewater. More importantly, alfalfa was verified to have an indispensable role
in stimulating the soil nitrifying microorganisms by sustaining their abundance
during substrate (NH3) and oxygen scarcity, and enhancing cell-specific
nitrification potential during substrate (NH3) and oxygen sufficiency. The
proposed system is expected to be further improved, and adopted as a sound
countermeasure for livestock wastewater pollution.
DOI: 10.2166/wst.2014.297
600. Ying Yong Sheng Tai Xue Bao. 2014 Jun;25(6):1688-92.
[Changes of phenols and lignin contents in alfalfa leaf damaged by Odontothrips

loti].
Wang XS, Yang CL, Wang SS, Hu GX.
In order to investigate the changes of secondary metabolites content of alfalfa

induced by thrips Odontothrips loti damaging, two alfalfa strains, one resistant
to thrips (R-1) and the other susceptible to thrips (I-1) , were chosen to
measure the phenols and lignin contents of alfalfa leaves under infestation with
thrips at different densities (0, 1, 3, 5, 7 thrips x branch(-1), and 0 thrip x
branch(-1) as control). After infestation 7 days, the polyphenols, tannin and
condensed tannin contents increased in both leaves of R-l and I-i with the
increasing thrips density, the simple phenols content had no significant
difference, while the lignin content increased significantly compared with the
control. After infestation 14 days, the polyphenols, tannin, condensed tannin and
lignin contents in both leaves of R-1 and I-1 increased obviously with the
increasing thrips density, while the simple phenols content had no significant
difference. The lignin content increased significantly, and was significantly
higher under 7 thrips x branch(-1) than under the control. After infestation 21
days, the polyphenols, tannin, and lignin contents in both leaves of R-1 and I-1
increased obviously with the increasing thrips density, and were the highest
under 7 thrips x branch(-1). Simple phenols content of I-1 strain was increased
significantly, but that of R-1 strain had no significant change. The condensed
tannin content in both leaves of R-1 and I-1 was not obvious compared with the
control. Phenols and lignin contents in R-1 and I-1 leaves increased obviously
after thrips infestation, and the polyphenols, tannin and lignin contents
increased faster in R-1 strain than in I-1 strain. Thrips infestation had
inductive effects on phenols and lignin contents of alfalfa, which could be used
to evaluate the resistance of alfalfa.
[Genetic diversity of rhizobia isolated from shrubby and herbaceous legumes in

Shenmu arid area, Shaanxi, China].
Nie G, Chen WM, Wei GH.
Legume, with a strong resistance to the adverse environmental conditions, is one

of pioneer plants in the desert region and plays an important role in the
protection of the ecological environment. In this study, the symbiosis of
rhizobia associating with shrubby and herbaceous legumes in Shenmu area, Shaanxi,
China was characterized by the 16S rRNA PCR-RFLP and sequence analysis of
involved genes. A total of 55 strains were isolated and purified, including 30
strains from the shrubby legume Amorpha fruticosa and Caragana microphylla, and
25 strains from herbaceous plants Astragalus adsurgens, Medicago sativa and
Astragalus melilotoides. Results showed that there were 11 16S rRNA genotypes.
The strains isolated from herbaceous legumes belonged to five genus including
Mesorhizobium, Ensifer, Rhizobium, Phyllobacterium and Agrobacterium, which were
very close related to M. huakuii, M. mediterraneum, M. robiniae, E. fredii, E.
meliloti, R. indigoferae, R. radiobacter, P. ifriqiyense and Ag. tumefaciens
through the phylogenetic analysis. The strains isolated from shrubby legumes
belonged to Mesorhizobium, and they were very close related to M. huakuii and M.
mediterraneum which were shared simultaneously by shrubby and herbaceous legumes.
All of these indicated the choice of rhizobia by the two types of legumes in the
arid area was different, and it might depend on the species of host plant and
environmental factors.
[Allelopathic effects of the humus soils from Betula platyphylla and Quercus
liaotungensis pure plantations on 9 kinds of common shrubs and herbs].
Huang LJ, Liu ZW, Zhu BC, Bing YH, Zhang XX, Lü C.
The humus soils were collected from Betula platyphylla and Quercus liaotungensis
pure plantations and woodless land separately where the site conditions were
basically the same, and taken as medium for potting culture test of 9 kinds of
shrubs or herbs in plastic greenhouse to assess the allelopathic effects of humus
soils of pure plantations on shrubs or herbs. Humus soils from B. platyphylla
plantation significantly inhibited the seed germinations of Medicago sativa and
Melilotus officinalis, decreased the catalase (CAT) activity of M. officinalis,
Coronilla varia, M. sativa and Lespedeza davurica, and improved malondialdehyde
(MDA) contents in seedlings of Caragana kor-shinskii, C. varia and Astragalus
adsurgens. The biomass growths of C. varia, Amorpha fruticosa, M. sativa, M.
officinalis and A. adsurgens in humus soils from B. platyphylla plantation were
significantly decreased by 48.2%, 45.1%, 44.3%, 37.3% and 36.0%, respectively. In
addition, humus soil of Q. liaotungensis plantation significantly decreased the
germination rates of M. sativa and A. adsurgens, the chlorophyll contents of
Vicia villosa, A. fruticosa and M. sativa, and improved malondialdehyde (MDA)
contents in seedlings of Lespedeza davurica, Caragana korshinskii, M. officinalis
and A. adsurgens. The biomass growths of A. adsurgens, M. sativa, M. officinalis
and A. fruticosa were significantly decreased by 52.6% , 43.8%, 35.5% and 34.6%,
respectively. B. platyphylla plantation humus soil had obvious inhibition
effects on M. sativa, M. officinalis and A. fruticosa, while Q. liaotungensis
plantation humus soil had obvious inhibition effects on M. sativa, A. adsurgens
and A. fruticosa.
603. Environ Entomol. 2014 Oct;43(5):1264-74. doi: 10.1603/EN14008. Epub 2014 Sep
8.
Conventional and organic soil fertility management practices affect corn plant
nutrition and Ostrinia nubilalis (Lepidoptera: Crambidae) larval performance.
Murrell EG(1), Cullen EM.
Author information:
(1)Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706,
USA.
Few studies compare how different soil fertilization practices affect plant
mineral content and insect performance in organic systems. This study examined:
1) The European corn borer, Ostrinia nubilalis (Hübner), larval response on corn
(Zea mays L.) grown in field soils with different soil management histories; and
2) resilience of these plants to O. nubilalis herbivory. Treatments included: 1)
standard organic--organically managed soil fertilized with dairy manure and 2 yr
of alfalfa (Medicago sativa L.) in the rotation; 2) basic cation saturation
ratio--organically managed soil fertilized with dairy manure and alfalfa nitrogen
credits, plus addition of gypsum (CaSO4·2H2O) according to the soil balance
hypothesis; and 3) conventional--conventionally managed soil fertilized with
synthetic fertilizers. Corn plants were reared to maturity in a greenhouse, and
then infested with 0-40 O. nubilalis larvae for 17 d. O. nubilalis exhibited
negative competitive response to increasing larval densities. Mean development
time was significantly faster for larvae consuming basic cation saturation ratio
plants than those on standard organic plants, with intermediate development time
on conventional plants. Neither total yield (number of kernels) nor proportion
kernels damaged differed among soil fertility treatments. Soil nutrients differed
significantly in S and in Ca:Mg and Ca:K ratios, but principal components
analysis of plant tissue samples taken before O. nubilalis infestation showed
that S, Fe, and Cu contributed most to differences in plant nutrient profiles
among soil fertility treatments. Results demonstrate that different fertilization
regimens can significantly affect insect performance within the context of
organic systems, but the effects in this study were relatively minor compared
with effects of intraspecific competition.
DOI: 10.1603/EN14008
604. J Food Prot. 2014 May;77(5):706-12. doi: 10.4315/0362-028X.JFP-13-387.
Interactive effects of temperature, pH, and water activity on the growth kinetics
of Shiga toxin-producing Escherichia coli O104:H4 3.
Juneja VK, Mukhopadhyay S, Ukuku D, Hwang CA, Wu VC, Thippareddi H.
The risk of non-O157 Shiga toxin-producing Escherichia coli strains has become a
growing public health concern. Several studies characterized the behavior of E.
coli O157:H7; however, no reports on the influence of multiple factors on E. coli
O104:H4 are available. This study examined the effects and interactions of
temperature (7 to 46°C), pH (4.5 to 8.5), and water activity (aw ; 0.95 to 0.99)
on the growth kinetics of E. coli O104:H4 and developed predictive models to
estimate its growth potential in foods. Growth kinetics studies for each of the
23 variable combinations from a central composite design were performed. Growth
data were used to obtain the lag phase duration (LPD), exponential growth rate,
generation time, and maximum population density (MPD). These growth parameters as
a function of temperature, pH, and aw as controlling factors were analyzed to
generate second-order response surface models. The results indicate that the
observed MPD was dependent on the pH, aw, and temperature of the growth medium.
Increasing temperature resulted in a concomitant decrease in LPD. Regression
analysis suggests that temperature, pH, and aw significantly affect the LPD,
exponential growth rate, generation time, and MPD of E. coli O104:H4. A
comparison between the observed values and those of E. coli O157:H7 predictions
obtained by using the U. S. Department of Agriculture Pathogen Modeling Program
indicated that E. coli O104:H4 grows faster than E. coli O157:H7. The developed
models were validated with alfalfa and broccoli sprouts. These models will
provide risk assessors and food safety managers a rapid means of estimating the
likelihood that the pathogen, if present, would grow in response to the
interaction of the three variables assessed.
DOI: 10.4315/0362-028X.JFP-13-387
605. Stand Genomic Sci. 2014 Jan 25;9(3):602-13. doi: 10.4056/sigs.4929626.

eCollection 2014 Jun 15.
Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant

strain RRI128.
Reeve W(1), Ballard R(2), Drew E(2), Tian R(1), Bräu L(3), Goodwin L(4),
Huntemann M(5), Han J(5), Tatiparthi R(5), Chen A(6), Mavrommatis K(6), Markowitz
V(6), Palaniappan K(6), Ivanova N(5), Pati A(5), Woyke T(5), Kyrpides N(5).
Author information:
(1)Centre for Rhizobium Studies, Murdoch University, Western Australia,
Australia.
(2)South Australian Research and Development Institute, Urrbrae, South Australia,
Australia.
(3)School of Life and Environmental Sciences, Deakin University, Victoria,
Australia.
(4)Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico,
USA.
(5)DOE Joint Genome Institute, Walnut Creek, California, USA.
Laboratory, Berkeley, California, USA.
Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative,

non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots
of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with
soil collected from Victoria, Australia. The strain is used in commercial
inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a
diverse range of lucerne cultivars (Medicago sativa) and several species of
annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms
an ineffective symbiosis with Medicago polymorpha. Here we describe the features
of E. meliloti strain RRI128, together with genome sequence information and
annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157
contigs, contains 6,683 protein-coding genes and 87 RNA-only encoding genes, and
is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome
Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria
(GEBA-RNB) project.
DOI: 10.4056/sigs.4929626
PMCID: PMC4149011
PMID: 25197447
606. Stand Genomic Sci. 2013 Dec 31;9(3):473-83. doi: 10.4056/sigs.4828625.

eCollection 2014 Jun 15.
Genome sequence of Ensifer arboris strain LMG 14919(T); a microsymbiont of the

legume Prosopis chilensis growing in Kosti, Sudan.
Reeve W(1), Tian R(1), Bräu L(2), Goodwin L(3), Munk C(3), Detter C(3), Tapia
R(3), Han C(3), Liolios K(4), Huntemann M(4), Pati A(4), Woyke T(4), Mavrommatis
K(5), Markowitz V(5), Ivanova N(4), Kyrpides N(4), Willems A(6).
Author information:
Australia.
Australia.
USA.
(6)Laboratory of Microbiology, Department of Biochemistry and Microbiology,
Faculty of Sciences, Ghent University, Belgium.
Ensifer arboris LMG 14919(T) is an aerobic, motile, Gram-negative,

non-spore-forming rod that can exist as a soil saprophyte or as a legume
microsymbiont of several species of legume trees. LMG 14919(T) was isolated in
1987 from a nodule recovered from the roots of the tree Prosopis chilensis
growing in Kosti, Sudan. LMG 14919(T) is highly effective at fixing nitrogen with
P. chilensis (Chilean mesquite) and Acacia senegal (gum Arabic tree or gum
acacia). LMG 14919(T) does not nodulate the tree Leucena leucocephala, nor the
herbaceous species Macroptilium atropurpureum, Trifolium pratense, Medicago
sativa, Lotus corniculatus and Galega orientalis. Here we describe the features
of E. arboris LMG 14919(T), together with genome sequence information and its
annotation. The 6,850,303 bp high-quality-draft genome is arranged into 7
scaffolds of 12 contigs containing 6,461 protein-coding genes and 84 RNA-only
encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE
Nodule Bacteria (GEBA-RNB) project.
DOI: 10.4056/sigs.4828625
PMCID: PMC4148966
PMID: 25197433
607. Animal. 2015 Feb;9(2):258-66. doi: 10.1017/S1751731114002249. Epub 2014 Sep 8.
Total mixed ration pellets for light fattening lambs: effects on animal health.
Blanco C(1), Giráldez FJ(1), Prieto N(1), Benavides J(1), Wattegedera S(2), Morán
L(1), Andrés S(1), Bodas R(1).
Author information:
(1)1Instituto de Ganadería de Montaña (CSIC-Universidad de León),E-24346
Grulleros,León,Spain.
(2)2Moredun Research Institute,Bush Loan,Penicuik,Midlothian,Scotland EH26
0PZ,UK.
Fifty male Merino lambs (6 to 8 weeks, 14.1 kg; n=10 per group) were used to
study the effect of feeding system: barley straw in long form and concentrate
pellets in separate troughs (Control), ad libitum alfalfa supplemented with
concentrate in separate troughs (Alfalfa) or including various levels of ground
barley straw in concentrate pellets (B05, B15 and B25 for 50, 150 and 250 g
barley straw/kg), on rumen characteristics, acid-base status, blood cell counts
and lymphocyte stimulation. Alfalfa lambs had the heaviest digestive tract
contents, highest rumen pH values, lowest volatile fatty acid concentration,
highest papillae counts and best mucosa colour and the greatest blood pCO2
values, lowest sodium and chloride and highest potassium concentrations (P<0.05).
Including ground barley straw in the concentrate pellet or providing straw in
long form separately from the concentrate reduces rumen pH and darkens ruminal
mucosa as compared with alfalfa-fed lambs, thus affecting acid-base status.
DOI: 10.1017/S1751731114002249
608. J Econ Entomol. 2014 Aug;107(4):1695-9.
Quadrat method for assessing the population abundance of a commercially managed

native soil-nesting bee, Nomia melanderi (Hymenoptera: Halictidae), in proximity
to alfalfa seed production in the western United States.
Vinchesi AC, Walsh DB.
Alkali bees, Nomia melanderi Cockerell, are solitary, gregarious, soil-nesting

bees native to the western United States that are commercially managed in the
Walla Walla Valley of Washington State to provide pollination service on alfalfa,
Medicago sativa L., produced as a seed crop. In 2010 and 2011, we compared two
techniques for estimating N. melanderi abundance in commercially managed bee
beds. The first technique involved quantifying the abundance of emergence holes
in 24 0.5-m2 quadrats on the surface of 13 bee beds during the peak period of N.
melanderi foraging activity in July of both years. When we counted emergence
holes, we marked a subset of eight quadrats per bee bed with plastic tabs.
Subsequently, in late October of the same year, 0.014-m3 soil cores were
collected in close proximity to the plastic tabs. The soil cores were teased
apart in the laboratory and the absolute abundance of overwintering prepupae was
quantified per core. Simple regression was highly significant between the means
of emergence holes within the 0.5-m2 soil surface quadrats and the means of the
counts from the 0.014-m3 soil cores. Using mean emergence hole counts, mean
prepupae counts from the soil cores, and the surface area of the bee beds, we
were able to calculate the estimated abundance of N. melanderi in each bee bed.
We conclude that the nondestructive quadrat method of sampling N. melanderi
abundance in commercially managed beds is robust compared with the destructive,
labor-intensive, absolute soil core method.
609. Development. 2014 Sep;141(18):3517-28. doi: 10.1242/dev.110775.
Fate map of Medicago truncatula root nodules.
Xiao TT(1), Schilderink S(1), Moling S(1), Deinum EE(2), Kondorosi E(3), Franssen
H(1), Kulikova O(1), Niebel A(4), Bisseling T(5).
Author information:
University, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands.
University, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands Department
of Systems Biophysics, FOM institute AMOLF, Science Park 104, Amsterdam 1098 XG,
The Netherlands.
(3)Institute of Biochemistry, Biological Research Centre, Hungarian Academy of
Sciences, Szeged 6726, Hungary.
(4)INRA, Laboratoire des Interactions Plantes-Microorganismes (LIPM), UMR441,
Castanet-Tolosan F-31326, France CNRS, Laboratoire des Interactions
Plantes-Microorganismes (LIPM), UMR2594, Castanet-Tolosan F-31326, France.
University, Droevendaalsesteeg 1, Wageningen 6708 PB, The Netherlands College of
Science, King Saud University, Post Office Box 2455, Riyadh 11451, Saudi Arabia
ton.bisseling@wur.nl.
Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in
an intracellular manner. These nodules are formed by reprogramming differentiated
root cells. The model legume Medicago truncatula forms indeterminate nodules with
a meristem at their apex. This organ grows by the activity of the meristem that
adds cells to the different nodule tissues. In Medicago sativa it has been shown
that the nodule meristem is derived from the root middle cortex. During nodule
initiation, inner cortical cells and pericycle cells are also mitotically
activated. However, whether and how these cells contribute to the mature nodule
has not been studied. Here, we produce a nodule fate map that precisely describes
the origin of the different nodule tissues based on sequential longitudinal
sections and on the use of marker genes that allow the distinction of cells
originating from different root tissues. We show that nodule meristem originates
from the third cortical layer, while several cell layers of the base of the
nodule are directly formed from cells of the inner cortical layers, root
endodermis and pericycle. The latter two differentiate into the uninfected
tissues that are located at the base of the mature nodule, whereas the cells
derived from the inner cortical cell layers form about eight cell layers of
infected cells. This nodule fate map has then been used to re-analyse several
mutant nodule phenotypes. This showed, among other things, that intracellular
release of rhizobia in primordium cells and meristem daughter cells are regulated
in a different manner.
© 2014. Published by The Company of Biologists Ltd.
DOI: 10.1242/dev.110775
610. Plant Physiol Biochem. 2014 Oct;83:217-24. doi: 10.1016/j.plaphy.2014.08.005.

Epub 2014 Aug 17.
Identification of active site residues of Fenugreek β-amylase: chemical

modification and in silico approach.
Srivastava G(1), Singh VK(2), Kayastha AM(3).
Author information:
(1)School of Biotechnology, Faculty of Science, Banaras Hindu University,
Varanasi, India. Electronic address: garimasri99@gmail.com.
Varanasi, India. Electronic address: officerbiotech.1@gmail.com.
Varanasi, India. Electronic address: kayasthabhu@gmail.com.
The amino acid sequence of Fenugreek β-amylase is not available in protein data
bank. Therefore, an attempt has been made to identify the catalytic amino acid
residues of enzyme by employing studies of pH dependence of enzyme catalysis,
chemical modification and bioinformatics. Treatment of purified Fenugreek
β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group
specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics
and resulted in effective inactivation of enzyme. The reaction with EDAC in
presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of
two carboxyl groups per molecule of enzyme and presence of one accessible
sulfhydryl group at the active site, per molecule of enzyme was ascertained by
titration with DTNB. The above results were supported by the prevention of
inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of
purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was
found to be the best match. To further confirm the amino acid involved in
catalysis, homology modelling of β-amylase of M. sativa was performed. The
sequence alignment, superimposition of template and target models, along with
study of interactions involved in docking of sucrose and maltose at the active
site, led to identification of Glu187, Glu381 and Cys344 as active site residues.
DOI: 10.1016/j.plaphy.2014.08.005
611. Int J Phytoremediation. 2015;17(1-6):49-55. doi: 10.1080/15226514.2013.828017.
Medicago sativa--Sinorhizobium meliloti Symbiosis Promotes the Bioaccumulation of

Zinc in Nodulated Roots.
Zribi K(1), Nouairi I, Slama I, Talbi-Zribi O, Mhadhbi H.
Author information:
(1)a Laboratory of Legumes , Centre of Biotechnology of Borj Cedria , Hammam ,
Tunisia.
In this study we investigated effects of Zn supply on germination, growth,

inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago
sativa This plant was cultivated with and without Zn (2 mM). Treatments were
plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain
(S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa
germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation,
Zn increased plant biomass production. When grown with symbionts, Zn supply had
no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot
and roots biomasses. However, in symbiosis with S532, an increase of roots
biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in
their roots than nitrogen fertilised plants. Zn supply results in an increase of
Ca concentration in roots of fertilised nitrogen plants. However, under Zn
supply, Fe concentration decreased in roots and increased in nodules of plants
with S112. Zn supply showed contrasting effects on Mg concentrations for plants
with nitrogen fertilisation (increase) and plants with S112 (decrease). The
capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its
use in phytostabilisation processes.
DOI: 10.1080/15226514.2013.828017

Aug
28.
Alfalfa baleage with increased concentration of nonstructural carbohydrates

supplemented with a corn-based concentrate did not improve production and
nitrogen utilization in early lactation dairy cows.
Brito AF(1), Tremblay GF(2), Bertrand A(2), Castonguay Y(2), Bélanger G(2),
Michaud R(2), Lafrenière C(3), Martineau R(4), Berthiaume R(5).
Author information:
(1)University of New Hampshire, Department of Biological Sciences, Durham 03824.
Canada, Québec City, QC, Canada G1V 2J3.
(3)Université du Québec en Abitibi-Témiscamingue, Rouy-Noranda, QC, Canada J9X
5E4.
(4)Dairy and Swine Research and Development Centre, Agriculture and Agri-Food
Canada, Sherbrooke, QC, Canada J1M 1Z3.
(5)Valacta, Dairy Production Centre of Expertise Québec-Atlantic,
Sainte-Anne-de-Bellevue, QC, Canada H9X 3R4. Electronic address:
rberthiaume@valacta.com.
The objective of this study was to investigate the effects of feeding alfalfa
baleage with different concentrations of nonstructural carbohydrates (NSC)
supplemented with a common corn-based concentrate on performance, ruminal
fermentation profile, N utilization, and omasal flow of nutrients in dairy cows
during early lactation. Ten multiparous (8 ruminally cannulated) and 8
primiparous Holstein cows were randomly assigned to treatments (high- or low-NSC
diet) in a crossover design. The difference in NSC concentration between the 2
alfalfa baleages fed from d14 to 21 averaged 14 g of NSC/kg of dry matter (DM).
Forages and concentrate were offered in separate meals with forages fed once and
concentrate offered 3 times daily. Except for the molar proportion of valerate,
which was lowest in cows fed the high-NSC diet, no other changes in ruminal
fermentation were observed. Omasal flows of most nitrogenous fractions, including
bacterial nonammonia N and AA, were not affected by treatments. Apparent ruminal
digestibilities of neutral and acid detergent fiber and N were lowest, whereas
that of total ethanol-soluble carbohydrates was highest when feeding the high-NSC
diet. Postruminal digestibilities of DM, organic matter, fiber, and N were
highest in cows fed the high-NSC diet, resulting in no difference in total-tract
digestibilities. Total-tract digestibility of total ethanol-soluble carbohydrates
was highest in cows fed the high-NSC diet, but that of starch did not differ
across treatments. Although milk yield and total DM intake did not differ between
treatments, yields of milk fat and 4% fat-corrected milk decreased significantly
in cows fed the high-NSC diet. Milk concentration of urea N was lowest, and that
of ruminal NH3-N highest, in cows fed the high-NSC diet. Plasma urea N
concentration tended to be decreased in cows fed the high-NSC diet, but
concentrations of AA were not affected by treatments, with the exception of Asp
and Cys, both of which were lowest in cows fed the low-NSC diet. Feeding diets
with contrasting NSC concentrations did not improve milk production, N
utilization, or bacterial protein synthesis, possibly because intakes of NSC and
DM were similar between treatments. Overall, results from the current study
should be interpreted cautiously because of the lack of difference in dietary NSC
intake between treatments and reduced N and fiber intakes when feeding the
high-NSC diet.

DOI: 10.3168/jds.2013-7305
613. Animal. 2015 Jan;9(1):58-66. doi: 10.1017/S1751731114002183. Epub 2014 Aug 28.
Effect of intake on fasting heat production, respiratory quotient and plasma

metabolites measured using the washed rumen technique.
Kim DH(1), McLeod KR(1), Koontz AF(1), Foote AP(1), Klotz JL(2), Harmon DL(1).
Author information:
(1)1Department of Animal and Food Sciences,University of Kentucky,Lexington,KY
40546,USA.
(2)2USDA-ARS,Forage-Animal Production Research Unit,Lexington,KY 40546,USA.
The objective was to investigate the effect of intake before fasting on

concentrations of metabolites and hormones, respiratory quotient (RQ) and fasting
heat production (HP) using the washed rumen technique and to compare these values
with those from the fed state. Six Holstein steers (360±22 kg) were maintained at
21°C and fed three different energy intakes within a replicated 3×3 Latin square
design with 21-day periods. Steers were fed alfalfa cubes to provide 1.0, 1.5 and
2.0×NEm during 19 days of each experimental period. Steers were placed in
individual metabolism stalls fitted with indirect calorimetry head-boxes on day
20 of each experimental period (FED steers) and fed their normal meal. On day 21
of each period the reticulorumen was emptied, washed and refilled with ruminal
buffer (NaCl=96; NaHCO3=24; KHCO3=30; K2HPO4=2; CaCl2=1.5; MgCl2=1.5 mmol/kg of
buffer) aerated with 75% N2 and 25% CO2 before introduction to the rumen (steers
were not fed; WASHED steers). Each gas exchange was measured over 24 h. HP for
1.0, 1.5 and 2.0×NEm were 479, 597 and 714 kJ/daykg0.75 (s.e.m. =16),
respectively. The plateau RQ was 0.756, 0.824 and 0.860 for the 1.0, 1.5 and
2.0×NEm intakes for the FED steers, respectively. After rumen washing, fasting HP
was 331, 359 and 400 kJ/daykg0.75 (s.e.m.=13) for 1.0, 1.5, and 2.0×NEm intakes
before fasting, respectively. The RQ for WASHED rumen steers was 0.717, 0.710 and
0.719, respectively. Cortisol and β-hydroxybutyrate concentrations in WASHED
rumen steers did not exceed threshold levels for severe energy deficit and stress
as can be induced from prolonged fasting. This study demonstrates that a fasting
state can be emulated using the washed rumen technique, minimizing the time
required as opposed to traditional fasting methodologies, without causing a
severe energy deficit and stress.
DOI: 10.1017/S1751731114002183
614. Environ Microbiol. 2014 Dec;16(12):3714-29. doi: 10.1111/1462-2920.12608.
A vapBC-type toxin-antitoxin module of Sinorhizobium meliloti influences

symbiotic efficiency and nodule senescence of Medicago sativa.
Lipuma J(1), Cinege G, Bodogai M, Oláh B, Kiers A, Endre G, Dupont L, Dusha I.
Author information:
(1)Institut Sophia Agrobiotech (ISA), INRA UMR 1355, CNRS UMR 7254, Université de
Nice Sophia Antipolis, 400 Route des Chappes - BP167, Sophia Antipolis Cedex,
F-06903, France.
The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a

large number of toxin-antitoxin (TA) modules both on the chromosome and
megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems
was characterized here. It encodes an active toxin vapC-5, and was shown to be
controlled negatively by the complex of its own proteins. Different mutants of
the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during
interaction with the host plant Medicago sativa. The absence of the entire
vapBC-5 region had no influence on nodule formation and nitrogen fixation
properties. The strain carrying an insertion in the antitoxin gene showed a
reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast,
when the toxin gene was mutated, the strain developed more efficient symbiosis
with the host plant. The nitrogen fixing root nodules had a delayed senescent
phenotype and contained elevated level of plant-derived molecules characteristic
of later steps of nodule development. The longer bacteroid viability and
abundance of active nitrogen fixing zone resulted in increased production of
plant material. These data indicate that modification of the toxin/antitoxin
production may influence bacteroid metabolism and may have an impact on the
adaptation to changing environmental conditions.
DOI: 10.1111/1462-2920.12608
615. Physiol Plant. 2015 May;154(1):13-27. doi: 10.1111/ppl.12266. Epub 2014 Oct 7.
Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs

from roots of Medicago sativa and Medicago truncatula.
Long RC(1), Li MN, Kang JM, Zhang TJ, Sun Y, Yang QC.
Author information:
Small 21- to 24-nucleotide (nt) ribonucleic acids (RNAs), notably the microRNA
(miRNA), are emerging as a posttranscriptional regulation mechanism. Salt stress
is one of the primary abiotic stresses that cause the crop losses worldwide. In
saline lands, root growth and function of plant are determined by the action of
environmental salt stress through specific genes that adapt root development to
the restrictive condition. To elucidate the role of miRNAs in salt stress
regulation in Medicago, we used a high-throughput sequencing approach to analyze
four small RNA libraries from roots of Zhongmu-1 (Medicago sativa) and Jemalong
A17 (Medicago truncatula), which were treated with 300 mM NaCl for 0 and 8 h.
Each library generated about 20 million short sequences and contained
predominantly small RNAs of 24-nt length, followed by 21-nt and 22-nt small RNAs.
Using sequence analysis, we identified 385 conserved miRNAs from 96 families,
along with 68 novel candidate miRNAs. Of all the 68 predicted novel miRNAs, 15
miRNAs were identified to have miRNA*. Statistical analysis on abundance of
sequencing read revealed specific miRNA showing contrasting expression patterns
between M. sativa and M. truncatula roots, as well as between roots treated for 0
and 8 h. The expression of 10 conserved and novel miRNAs was also quantified by
quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR).
The miRNA precursor and target genes were predicted by bioinformatics analysis.
We concluded that the salt stress related conserved and novel miRNAs may have a
large variety of target mRNAs, some of which might play key roles in salt stress
regulation of Medicago.
DOI: 10.1111/ppl.12266
616. G3 (Bethesda). 2014 Aug 25;4(10):2003-12. doi: 10.1534/g3.114.011874.
Genomic characterization of the LEED..PEEDs, a gene family unique to the medicago

lineage.
Trujillo DI(1), Silverstein KA(1), Young ND(2).
Author information:
(1)Department of Plant Biology, University of Minnesota, St. Paul, Minnesota
55108.
55108 neviny@umn.edu.
The LEED..PEED (LP) gene family in Medicago truncatula (A17) is composed of 13

genes coding small putatively secreted peptides with one to two conserved domains
of negatively charged residues. This family is not present in the genomes of
Glycine max, Lotus japonicus, or the IRLC species Cicer arietinum. LP genes were
also not detected in a Trifolium pratense draft genome or Pisum sativum nodule
transcriptome, which were sequenced de novo in this study, suggesting that the LP
gene family arose within the past 25 million years. M. truncatula accession HM056
has 13 LP genes with high similarity to those in A17, whereas M. truncatula ssp.
tricycla (R108) and M. sativa have 11 and 10 LP gene copies, respectively. In M.
truncatula A17, 12 LP genes are located on chromosome 7 within a 93-kb window,
whereas one LP gene copy is located on chromosome 4. A phylogenetic analysis of
the gene family is consistent with most gene duplications occurring prior to
Medicago speciation events, mainly through local tandem duplications and one
distant duplication across chromosomes. Synteny comparisons between R108 and A17
confirm that gene order is conserved between the two subspecies, although a
further duplication occurred solely in A17. In M. truncatula A17, all 13 LPs are
exclusively transcribed in nodules and absent from other plant tissues, including
roots, leaves, flowers, seeds, seed shells, and pods. The recent expansion of LP
genes in Medicago spp. and their timing and location of expression suggest a
novel function in nodulation, possibly as an aftermath of the evolution of
bacteroid terminal differentiation or potentially associated with rhizobial-host
specificity.
Copyright © 2014 Trujillo et al.
DOI: 10.1534/g3.114.011874
PMCID: PMC4199706
617. Bioresour Technol. 2014 Oct;170:286-292. doi: 10.1016/j.biortech.2014.08.002.

Epub 2014 Aug 9.
Improving ethanol production from alfalfa stems via ambient-temperature acid

pretreatment and washing.
Zhou S(1), Weimer PJ(2), Hatfield RD(3), Runge TM(4), Digman M(5).
Author information:
(1)Department of Biological System Engineering, University of Wisconsin,
szhou34@wisc.edu.
(2)USDA-ARS, U.S. Dairy Forage Research Center, Madison, WI 53706, USA.
Electronic address: Paul.Weimer@ars.usda.gov.
(3)USDA-ARS, U.S. Dairy Forage Research Center, Madison, WI 53706, USA.
Electronic address: Ronald.Hatfield@ars.usda.gov.
(4)Department of Biological System Engineering, University of Wisconsin,
trunge@wbi.wisc.edu.
(5)KUHN North America, 1501 West Seventh Avenue, Brodhead, WI 53520, USA.
Electronic address: matthew.digman@kuhn.com.
The concept of co-production of liquid fuel (ethanol) along with animal feed on
farm was proposed, and the strategy of using ambient-temperature acid
pretreatment, ensiling and washing to improve ethanol production from alfalfa
stems was investigated. Alfalfa stems were separated and pretreated with sulfuric
acid at ambient-temperature after harvest, and following ensiling, after which
the ensiled stems were subjected to simultaneous saccharification and
fermentation (SSF) for ethanol production. Ethanol yield was improved by
ambient-temperature sulfuric acid pretreatment before ensiling, and by washing
before SSF. It was theorized that the acid pretreatment at ambient temperature
partially degraded hemicellulose, and altered cell wall structure, resulted in
improved cellulose accessibility, whereas washing removed soluble ash in
substrates which could inhibit the SSF. The pH of stored alfalfa stems can be
used to predict the ethanol yield, with a correlation coefficient of +0.83 for
washed alfalfa stems.
DOI: 10.1016/j.biortech.2014.08.002
618. G3 (Bethesda). 2014 Aug 21;4(10):1971-9. doi: 10.1534/g3.114.012245.
A saturated genetic linkage map of autotetraploid alfalfa (Medicago sativa L.)

developed using genotyping-by-sequencing is highly syntenous with the Medicago
truncatula genome.
Li X(1), Wei Y(1), Acharya A(1), Jiang Q(1), Kang J(2), Brummer EC(3).
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore,
Oklahoma 73401.
(2)The Institute of Animal Science, Chinese Academy of Agricultural Science,
Beijing, China 100193.
(3)Plant Breeding Center and Department of Plant Sciences, The University of
California, Davis, California 95616 ecbrummer@ucdavis.edu.
A genetic linkage map is a valuable tool for quantitative trait locus mapping,
map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa,
one of the most important forage crops in the world, is autotetraploid,
allogamous, and highly heterozygous, characteristics that have impeded the
construction of a high-density linkage map using traditional genetic marker
systems. Using genotyping-by-sequencing (GBS), we constructed low-cost,
reasonably high-density linkage maps for both maternal and paternal parental
genomes of an autotetraploid alfalfa F1 population. The resulting maps contain
3591 single-nucleotide polymorphism markers on 64 linkage groups across both
parents, with an average density of one marker per 1.5 and 1.0 cM for the
maternal and paternal haplotype maps, respectively. Chromosome assignments were
made based on homology of markers to the M. truncatula genome. Four linkage
groups representing the four haplotypes of each alfalfa chromosome were assigned
to each of the eight Medicago chromosomes in both the maternal and paternal
parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and
clearly identified the known translocation between Chromosomes 4 and 8. In
addition, a small inversion on Chromosome 1 was identified between M. truncatula
and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that
greatly improved genome coverage relative to previous maps and that will
facilitate investigation of genome structure. GBS could be used in breeding
populations to accelerate molecular breeding in alfalfa.
Copyright © 2014 Li et al.
DOI: 10.1534/g3.114.012245
PMCID: PMC4199703
619. Physiol Plant. 2015 Mar;153(3):355-64. doi: 10.1111/ppl.12249. Epub 2014 Sep
13.
A cold-induced myo-inositol transporter-like gene confers tolerance to multiple

abiotic stresses in transgenic tobacco plants.
Sambe MA(1), He X, Tu Q, Guo Z.
Author information:
Agro-bioresources, Guangdong Key Laboratory for Innovative Development and
Utilization of Forest Plant Germplasm, South China Agricultural University,
Guangzhou, 510642, China.
A full length cDNA encoding a myo-inositol transporter-like protein, named as

MfINT-like, was cloned from Medicago sativa subsp. falcata (herein falcata), a
species with greater cold tolerance than alfalfa (M. sativa subsp. sativa).
MfINT-like is located on plasma membranes. MfINT-like transcript was induced 2-4
h after exogenous myo-inositol treatment, 24-96 h with cold, and 96 h by
salinity. Given that myo-inositol accumulates higher in falcata after 24 h of
cold treatment, myo-inositol is proposed to be involved in cold-induced
expression of MfINT-like. Higher levels of myo-inositol was observed in leaves of
transgenic tobacco plants overexpressing MfINT-like than the wild-type but not in
the roots of plants grown on myo-inositol containing medium, suggesting that
transgenic plants had higher myo-inositol transport activity than the wild-type.
Transgenic plants survived better to freezing temperature, and had lower ion
leakage and higher maximal photochemical efficiency of photosystem II (Fv /Fm )
after chilling treatment. In addition, greater plant fresh weight was observed in
transgenic plants as compared with the wild-type when plants were grown under
drought or salinity stress. The results suggest that MfINT-like mediated
transport of myo-inositol is associated with plant tolerance to abiotic stresses.
DOI: 10.1111/ppl.12249
620. Carbohydr Polym. 2014 Nov 4;112:608-14. doi: 10.1016/j.carbpol.2014.06.047.

Epub
2014 Jun 24.
Purification, characterisation and protective effects of polysaccharides from

alfalfa on hepatocytes.
Wang S(1), Dong X(2), Ma H(3), Cui Y(4), Tong J(5).
Author information:
Beijing 100193, China. Electronic address: shaopu.1988@163.com.
Beijing 100193, China. Electronic address: xiaofangd1124@sina.com.
(3)Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Electronic address: majordan@163.com.
Beijing 100193, China. Electronic address: cymnongkeyuan@163.com.
Beijing 100193, China. Electronic address: tjm606@263.net.
The objective of this study was to determine the preliminary characteristics and
protective effects of alfalfa polysaccharides (APS) on hepatocytes in vitro. The
crude APS was purified by DEAE-cellulose and Sephadex G-100 chromatography,
resulting in the four purified fractions: APS-1, APS-2, APS-3 and APS-4. The
results indicated that APS-3 had higher carbohydrate and uronic acid contents and
that APS-4 had a more complicated monosaccharide composition compared to the
other purified fractions. The average molecular weights of APS-1, APS-2, APS-3
and APS-4 were 48,536, 6,221, 66,559 and 13,076 Da, respectively. Furthermore,
APS (crude and its purified fractions) restored the activities of antioxidant
enzymes and increased the total antioxidant capacity of hepatocytes subjected to
H2O2-induced oxidative stress. Furthermore, APS treatment counteracted the
increases in lactic dehydrogenase and malonaldehyde in the culture supernatant.
These results clearly demonstrate that APS possesses a protective effect against
oxidative injury in hepatocytes.
DOI: 10.1016/j.carbpol.2014.06.047
621. Plant Sci. 2014 Sep;226:22-9. doi: 10.1016/j.plantsci.2014.05.008. Epub 2014

May
24.
Growth, photosynthetic acclimation and yield quality in legumes under climate

change simulations: an updated survey.
Irigoyen JJ(1), Goicoechea N(1), Antolín MC(1), Pascual I(1), Sánchez-Díaz M(1),
Aguirreolea J(1), Morales F(2).
Author information:
(1)Grupo de Fisiología del Estrés en Plantas (Dpto. de Biología Ambiental),
Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV, Logroño, Facultades de Ciencias y
Farmacia, Universidad de Navarra, Irunlarrea 1, 31008 Pamplona, Spain.
(2)Estación Experimental de Aula Dei (EEAD), CSIC, Dpto. Nutrición Vegetal, Apdo.
13034, 50080 Zaragoza, Spain; Grupo de Fisiología del Estrés en Plantas (Dpto. de
Biología Ambiental), Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV, Logroño,
Facultades de Ciencias y Farmacia, Universidad de Navarra, Irunlarrea 1, 31008
Pamplona, Spain. Electronic address: fmorales@eead.csic.es.
Continued emissions of CO2, derived from human activities, increase atmospheric

CO2 concentration. The CO2 rise stimulates plant growth and affects yield
quality. Effects of elevated CO2 on legume quality depend on interactions with
N2-fixing bacteria and mycorrhizal fungi. Growth at elevated CO2 increases
photosynthesis under short-term exposures in C3 species. Under long-term
exposures, however, plants generally acclimate to elevated CO2 decreasing their
photosynthetic capacity. An updated survey of the literature indicates that a key
factor, perhaps the most important, that characteristically influences this
phenomenon, its occurrence and extent, is the plant source-sink balance. In
legumes, the ability of exchanging C for N at nodule level with the N2-fixing
symbionts creates an extra C sink that avoids the occurrence of photosynthetic
acclimation. Arbuscular mycorrhizal fungi colonizing roots may also result in
increased C sink, preventing photosynthetic acclimation. Defoliation (Anthyllis
vulneraria, simulated grazing) or shoot cutting (alfalfa, usual management as
forage) largely increases root/shoot ratio. During re-growth at elevated CO2, new
shoots growth and nodule respiration function as strong C sinks that counteracts
photosynthetic acclimation. In the presence of some limiting factor, the legumes
response to elevated CO2 is weakened showing photosynthetic acclimation. This
survey has identified limiting factors that include an insufficient N supply from
bacterial strains, nutrient-poor soils, low P supply, excess temperature
affecting photosynthesis and/or nodule activity, a genetically determined low
nodulation capacity, an inability of species or varieties to increase growth (and
therefore C sink) at elevated CO2 and a plant phenological state or season when
plant growth is stopped.
DOI: 10.1016/j.plantsci.2014.05.008
622. Plant Physiol. 2014 Sep;166(1):281-92. doi: 10.1104/pp.114.246595. Epub 2014

Aug
5.
Auxin perception is required for arbuscule development in arbuscular mycorrhizal

symbiosis.
Etemadi M(1), Gutjahr C(1), Couzigou JM(1), Zouine M(1), Lauressergues D(1),
Timmers A(1), Audran C(1), Bouzayen M(1), Bécard G(1), Combier JP(2).
Author information:
(1)Université Paul Sabatier Toulouse, Unité Mixte de Recherche 5546, Laboratoire
de Recherche en Sciences Végétales, F-31326 Castanet-Tolosan cedex, France (M.E.,
J.-M.C., D.L., G.B., J.-P.C.);Centre National de la Recherche Scientifique, Unité
Mixte de Recherche 5546, F-31326 Castanet-Tolosan cedex, France (M.E., J.-M.C.,
D.L., G.B., J.-P.C.);Institut National Polytechnique-Ecole Nationale Supérieure
Agronomique Toulouse, Génomique et Biotechnologie des Fruits, F-31326
Castanet-Tolosan, France (M.E., M.Z., C.A., M.B.);Institut National de la
Recherche Agronomique, Génomique et Biotechnologie des Fruits, F-52627 Auzeville,
France (M.E., M.Z., C.A., M.B.);Faculty of Biology, Genetics, University of
Munich, 82152 Martinsried, Germany (C.G.); andLaboratoire des Interactions
Plantes-Microorganismes, Unité Mixte de Recherche 441/2594 Institut National de
la Recherche Agronomique-Centre National de la Recherche Scientifique, F-31326
Castanet-Tolosan cedex, France (A.T.).
(2)Université Paul Sabatier Toulouse, Unité Mixte de Recherche 5546, Laboratoire
de Recherche en Sciences Végétales, F-31326 Castanet-Tolosan cedex, France (M.E.,
J.-M.C., D.L., G.B., J.-P.C.);Centre National de la Recherche Scientifique, Unité
Mixte de Recherche 5546, F-31326 Castanet-Tolosan cedex, France (M.E., J.-M.C.,
D.L., G.B., J.-P.C.);Institut National Polytechnique-Ecole Nationale Supérieure
Agronomique Toulouse, Génomique et Biotechnologie des Fruits, F-31326
Castanet-Tolosan, France (M.E., M.Z., C.A., M.B.);Institut National de la
Recherche Agronomique, Génomique et Biotechnologie des Fruits, F-52627 Auzeville,
France (M.E., M.Z., C.A., M.B.);Faculty of Biology, Genetics, University of
Munich, 82152 Martinsried, Germany (C.G.); andLaboratoire des Interactions
Plantes-Microorganismes, Unité Mixte de Recherche 441/2594 Institut National de
la Recherche Agronomique-Centre National de la Recherche Scientifique, F-31326
Castanet-Tolosan cedex, France (A.T.) combier@lrsv.ups-tlse.fr.
Most land plant species live in symbiosis with arbuscular mycorrhizal fungi.
These fungi differentiate essential functional structures called arbuscules in
root cortical cells from which mineral nutrients are released to the plant. We
investigated the role of microRNA393 (miR393), an miRNA that targets several
auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the
precursors of the miR393 was down-regulated during mycorrhization in three
different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza
sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with
concentrations of synthetic auxin analogs that did not affect root development
stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter
for auxin response, was preferentially expressed in root cells containing
arbuscules. Finally, overexpression of miR393 in root tissues resulted in
down-regulation of auxin receptor genes (transport inhibitor response1 and
auxin-related F box) and underdeveloped arbuscules in all three plant species.
These results support the conclusion that miR393 is a negative regulator of
arbuscule formation by hampering auxin perception in arbuscule-containing cells.
DOI: 10.1104/pp.114.246595
PMCID: PMC4149713
623. J Anim Sci Biotechnol. 2014 Jun 4;5(1):31. doi: 10.1186/2049-1891-5-31.

eCollection 2014.
Evaluation of nutritive value and in vitro rumen fermentation gas accumulation of

de-oiled algal residues.
Han KJ(1), McCormick ME(2).
Author information:
(1)Louisiana State University Agricultural Center, School of Plant,
Environmental, and Soil Sciences, 104 M.B. Sturgis Hall, Baton Rouge, LA 70803,
USA.
(2)Louisiana State University Agricultural Center, Southeast Region Office, 21549
Old Covington, Hammond, LA 70403, USA.
BACKGROUND: Algae are widely recognized for their high oil content and for
exponentially accumulating biomass with particular potential to provide single
cell protein for human consumption or animal feed. It is believed that along with
biodiesel from algae, the high protein de-oiled algal residue may become an
alternative feed supplement option in the future. This study was conducted to
investigate de-oiled algal residue obtained from the common Chlorella species,
Thalassiosira weissflogii, Selenarstrum capricornutum, Scenedesmus sp., and
Scenedesmus dimorphus for assessment as potential feed supplements for ruminants
by comparing with soybean (Glycine max) meal and alfalfa (Medicago sativa) hay.
RESULTS: With the exception of T. weissflogii, algal residue had higher
concentrations of Cu, Zn, and Mn and lower concentration of Ca, Mg, and K than
soybean meal and alfalfa hay. The algal residue CP (crude protein) concentrations
ranged from 140 to 445 g/kg DM and varied among the de-oiled residues. In vitro
rumen fermentation gas accumulation curves indicated that algal biomass
degradation potential was less than that of soybean meal or alfalfa hay by up to
41.7%. The gas production curve, interpreted with a dual pool logistic model,
confirmed that the fraction sizes for fast fermenting and slow fermenting of
de-oiled algal residues were smaller than those in soybean meal and alfalfa hay,
and the fermenting rate of the fractions was also low.
CONCLUSIONS: Inferior in vitro rumen gas accumulation from the five de-oiled
algal residues suggests that these algal byproducts are less degradable in the
rumen.
DOI: 10.1186/2049-1891-5-31
PMCID: PMC4105884
PMID: 25093078
624. Meat Sci. 2014 Dec;98(4):629-36. doi: 10.1016/j.meatsci.2014.06.040. Epub 2014

Jul 2.
Meat goat kids finished on alfalfa, red clover or orchardgrass pastures: carcass
merit and meat quality.
Turner KE(1), Cassida KA(2), Zerby HN(3).
Author information:
(1)Animal Science, USDA, ARS, Grazinglands Research Laboratory, El Reno, OK
73036, USA. Electronic address: Ken.Turner@ars.usda.gov.
(2)Agronomy, Department of Plant, Soil, and Microbial Sciences, Michigan State
University, East Lansing, MI 48824, USA.
(3)Meat Science, Department of Animal Sciences, The Ohio State University,
Columbus, OH 43210, USA.
This experiment was conducted in 2005-2007 to evaluate carcass and chevon (goat
meat) quality parameters when meat-goat kids (n=72) were finished on alfalfa
(Medicago sativa L; ALF); red clover (Trifolium pratense L.; RCG); or
orchardgrass (Dactylis glomerata L.; OGR) pastures. Carcass conformation score
was greater (P=0.08) when meat-goat kids were finished on ALF compared to OGR
with RCG intermediate. Chevon meat samples from goats finished on the three
pasture treatments did not differ in ash, intramuscular fat, or crude protein
content or in concentrations of omega6 and omega3 fatty acids, or the omega6 to
omega3 ratio. Goats finished on OGR had higher (P<0.001) 18:1 trans-11 fatty
acids (FA) compared to ALF or RCG. Overall, meat-goat kids finished on ALF, RCG,
or ORG produced desirable carcass weights for most niche markets in the USA.
Chevon is a low-fat meat option with high desirable fatty acids for human diets.
DOI: 10.1016/j.meatsci.2014.06.040

Jun 16.
Influence of fresh alfalfa supplementation on fat skatole and indole

concentration and chop odour and flavour in lambs grazing a cocksfoot pasture.
Devincenzi T(1), Prunier A(2), Meteau K(3), Nabinger C(4), Prache S(5).
Author information:
(1)Institut National de la Recherche Agronomique (INRA), UMR1213 Herbivores,
F-63122 St-Genès-Champanelle, France; Universidade Federal do Rio Grande do Sul
(UFRGS), Av. Bento Gonçalves 7712, CEP 91501-970 Porto Alegre-RS, Brazil.
(2)Institut National de la Recherche Agronomique (INRA), UMR1348 Physiologie,
Environnement et Génétique pour l'Animal et les Systèmes d'Élevage, F-35590
St-Gilles, France.
(3)Institut National de la Recherche Agronomique (INRA), UE1206 Elevage
Alternatif et Santé des Monogastriques, Le Magneraud, F-17700 St-Pierre-d'Amilly,
France.
(4)Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves 7712,
CEP 91501-970 Porto Alegre-RS, Brazil.
(5)Institut National de la Recherche Agronomique (INRA), UMR1213 Herbivores,
F-63122 St-Genès-Champanelle, France. Electronic address:
sophie.prache@clermont.inra.fr.
We investigated the influence of the level of fresh alfalfa supplementation on

fat skatole and indole concentration and chop sensory attributes in grazing
lambs. Four groups of nine male Romane lambs grazing a cocksfoot pasture were
supplemented with various levels of alfalfa for at least 60days before slaughter.
Perirenal fat skatole concentration was higher for lambs that consumed alfalfa
than for those that consumed only cocksfoot. The intensity of 'animal' odour in
the lean part of the chop and of 'animal' flavour in both the lean and fat parts
of the chop, evaluated by a trained sensory panel, increased from the lowest
level of alfalfa supplementation onwards and did not increase further with
increasing levels of alfalfa supplementation. The outcome of this study therefore
suggests that these sensory attributes may reach a plateau when perirenal fat
skatole concentration is in the range 0.16-0.24μg/g of liquid fat.
DOI: 10.1016/j.meatsci.2014.06.008
626. PLoS One. 2014 Aug 1;9(8):e103808. doi: 10.1371/journal.pone.0103808.

eCollection
2014.
Alfalfa Cellulose synthase gene expression under abiotic stress: a Hitchhiker's

guide to RT-qPCR normalization.
Guerriero G(1), Legay S(1), Hausman JF(1).
Author information:
(1)Department Environment and Agro-biotechnologies (EVA), Centre de Recherche
Public, Gabriel Lippmann, Belvaux, Luxembourg.
Abiotic stress represents a serious threat affecting both plant fitness and
productivity. One of the promptest responses that plants trigger following
abiotic stress is the differential expression of key genes, which enable to face
the adverse conditions. It is accepted and shown that the cell wall senses and
broadcasts the stress signal to the interior of the cell, by triggering a cascade
of reactions leading to resistance. Therefore the study of wall-related genes is
particularly relevant to understand the metabolic remodeling triggered by plants
in response to exogenous stresses. Despite the agricultural and economical
relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has
addressed specifically the wall-related gene expression changes in response to
exogenous stresses in this important crop, by monitoring the dynamics of wall
biosynthetic gene expression. We here identify and analyze the expression
profiles of nine cellulose synthases, together with other wall-related genes, in
stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt
stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main
responses for specific groups of genes, i.e. a salt/heat-induced and a
cold/heat-repressed group of genes. Prior to this analysis we identified
appropriate reference genes for expression analyses in alfalfa, by evaluating the
stability of 10 candidates across different tissues (namely leaves, stems,
roots), under the different abiotic stresses and time points chosen. The results
obtained confirm an active role played by the cell wall in response to exogenous
stimuli and constitute a step forward in delineating the complex pathways
regulating the response of plants to abiotic stresses.
PMCID: PMC4118957
627. Genet Mol Res. 2014 Jul 24;13(3):5350-60. doi: 10.4238/2014.July.24.14.
Salicornia europaea L. Na⁺/H⁺ antiporter gene improves salt tolerance in

transgenic alfalfa (Medicago sativa L.).
Zhang LQ(1), Niu YD(1), Huridu H(1), Hao JF(1), Qi Z(1), Hasi A(2).
Author information:
(1)Inner Mongolia Key Laboratory of Herbage & Endemic Crop Biotechnology, College
of Life Sciences, Inner Mongolia University, Hohhot, China.
(2)Inner Mongolia Key Laboratory of Herbage & Endemic Crop Biotechnology, College
of Life Sciences, Inner Mongolia University, Hohhot, China hasind@sina.com.
In order to obtain a salt-tolerant perennial alfalfa (Medicago sativa L.), we

transferred the halophyte Salicornia europaea L. Na(+)/H(+) antiporter gene,
SeNHX1, to alfalfa by using the Agrobacterium-mediated transformation method. The
transformants were confirmed by both PCR and RT-PCR analyses. Of 197 plants that
were obtained after transformation, 36 were positive by PCR analysis using 2
primer pairs for the CaMV35S-SeNHX1 and SeNHX1-Nos fragments; 6 plants survived
in a greenhouse. RT-PCR analysis revealed that SeNHX1 was expressed in 5 plants.
The resultant transgenic alfalfa had better salt tolerance. After stress
treatment for 21 days with 0.6% NaCl, the chlorophyll and MDA contents in
transgenic plants were lower, but proline content and SOD, POD, and CAT
activities were higher than those in wild-type plants. These results suggest that
the salt tolerance of transgenic alfalfa was improved by the overexpression of
the SeNHX1 gene.
DOI: 10.4238/2014.July.24.14
628. Foodborne Pathog Dis. 2014 Aug;11(8):635-44. doi: 10.1089/fpd.2013.1705.
Outbreaks caused by sprouts, United States, 1998-2010: lessons learned and

solutions needed.
Dechet AM(1), Herman KM, Chen Parker C, Taormina P, Johanson J, Tauxe RV, Mahon
BE.
Author information:
(1)1 Medical Education, Portland Providence Medical Center , Portland, Oregon.
After a series of outbreaks associated with sprouts in the mid-1990s, the U.S.
Food and Drug Administration (FDA) published guidelines in 1999 for sprouts
producers to reduce the risk of contamination. The recommendations included
treating seeds with an antimicrobial agent such as calcium hypochlorite solution
and testing spent irrigation water for pathogens. From 1998 through 2010, 33
outbreaks from seed and bean sprouts were documented in the United States,
affecting 1330 reported persons. Twenty-eight outbreaks were caused by
Salmonella, four by Shiga toxin-producing Escherichia coli, and one by Listeria.
In 15 of the 18 outbreaks with information available, growers had not followed
key FDA guidelines. In three outbreaks, however, the implicated sprouts were
produced by firms that appeared to have implemented key FDA guidelines. Although
seed chlorination, if consistently applied, reduces pathogen burden on sprouts,
it does not eliminate the risk of human infection. Further seed and sprouts
disinfection technologies, some recently developed, will be needed to enhance
sprouts safety and reduce human disease. Improved seed production practices could
also decrease pathogen burden but, because seeds are a globally distributed
commodity, will require international cooperation.
DOI: 10.1089/fpd.2013.1705

Jul
23.
Methane production, nutrient digestion, ruminal fermentation, N balance, and milk

production of cows fed timothy silage- or alfalfa silage-based diets.
Hassanat F(1), Gervais R(2), Massé DI(1), Petit HV(1), Benchaar C(3).
Author information:
(1)Agriculture and Agri-Food Canada, Dairy and Swine Research and Development
Centre, 2000 College Street, Sherbrooke, Quebec, Canada J1M 0C8.
(2)Université Laval, Département des Sciences Animales, Québec, Québec, Canada
G1V 0A6.
Centre, 2000 College Street, Sherbrooke, Quebec, Canada J1M 0C8. Electronic
address: chaouki.benchaar@agr.gc.ca.
The objective of this study was to investigate the effects of changing forage
source in dairy cow diets from timothy silage (TS) to alfalfa silage (AS) on
enteric CH₄ emissions, ruminal fermentation characteristics, digestion, milk
production, and N balance. Nine ruminally cannulated lactating cows were used in
a replicated 3 × 3 Latin square design (32-d period) and fed (ad libitum) a total
mixed ration (TMR; forage:concentrate ratio of 60:40, dry matter basis), with the
forage portion consisting of either TS (0% AS; 0% AS and 54.4% TS in the TMR), a
50:50 mixture of both silages (50% AS; 27.2% AS and 27.2% TS in the TMR), or AS
(100% AS; 54.4% AS and 0% TS in the TMR). Compared with TS, AS contained less
(36.9 vs. 52.1%) neutral detergent fiber but more (20.5 vs. 13.6%) crude protein
(CP). In sacco 24-h ruminal degradability of organic matter (OM) was higher for
AS than for TS (73.5 vs. 66.9%). Replacement of TS with AS in the diet entailed
increasing proportions of corn grain and bypass protein supplement at the expense
of soybean meal. As the dietary proportion of AS increased, CP and starch
concentrations increased, whereas fiber content declined in the TMR. Dry matter
intake increased linearly with increasing AS proportions in the diet. Apparent
total-tract digestibility of OM and gross energy remained unaffected, whereas CP
digestibility increased linearly and that of fiber decreased linearly with
increasing inclusion of AS in the diet. The acetate-to-propionate ratio was not
affected, whereas ruminal concentration of ammonia (NH₃) and molar proportion of
branched-chain VFA increased as the proportion of AS in the diet increased. Daily
CH₄ emissions tended to increase (476, 483, and 491 g/d for cows fed 0% AS, 50%
AS, and 100% AS, respectively) linearly as cows were fed increasing proportions
of AS. Methane production adjusted for dry matter intake (average=19.8 g/kg) or
gross energy intake (average=5.83%) was not affected by increasing AS inclusion
in the diet. When expressed on a fat-corrected milk or energy-corrected milk
yield basis, CH₄ production increased linearly with increasing AS dietary
proportion. Urinary N excretion (g/d) increased linearly when cows were fed
increasing amounts of AS in the diet, suggesting a potential for higher nitrous
oxide (N₂O) and NH₃ emissions. Efficiency of dietary N use for milk protein
secretion (g of milk N/g of N intake) declined with the inclusion of AS in the
diet. Despite marked differences in chemical composition and ruminal
degradability, under the conditions of this study, replacing TS with AS in dairy
cow diets was not effective in reducing CH₄ energy losses.

DOI: 10.3168/jds.2014-8069

Jul
23.
Symplastic phloem loading in poplar.
Zhang C(1), Han L(2), Slewinski TL(2), Sun J(2), Zhang J(2), Wang ZY(2), Turgeon
R(1).
Author information:
(1)Department of Plant Biology, Cornell University, Ithaca, New York 14853 (C.Z.,
T.L.S., J.S., J.Z., R.T.); andForage Improvement Division, The Noble Foundation,
Ardmore, Oklahoma 73401 (L.H., Z-Y.W.) cz46@cornell.edu ert2@cornell.edu.
(2)Department of Plant Biology, Cornell University, Ithaca, New York 14853 (C.Z.,
T.L.S., J.S., J.Z., R.T.); andForage Improvement Division, The Noble Foundation,
Ardmore, Oklahoma 73401 (L.H., Z-Y.W.).
Sap is driven through phloem sieve tubes by an osmotically generated pressure

gradient between source and sink tissues. In many plants, source pressure results
from thermodynamically active loading in which energy is used to transfer sucrose
(Suc) from mesophyll cells to the phloem of leaf minor veins against a
concentration gradient. However, in some species, almost all trees, correlative
evidence suggests that sugar migrates passively through plasmodesmata from
mesophyll cells into the sieve elements. The possibility of alternate loading
mechanisms has important ramifications for the regulation of phloem transport and
source-sink interactions. Here, we provide experimental evidence that, in gray
poplar (Populus tremula × Populus alba), Suc enters the phloem through
plasmodesmata. Transgenic plants were generated with yeast invertase in the cell
walls to prevent Suc loading by this route. The constructs were driven either by
the constitutive 35S promoter or the minor vein-specific galactinol synthase
promoter. Transgenic plants grew at the same rate as the wild type without
symptoms of loading inhibition, such as accumulation of carbohydrates or leaf
chlorosis. Rates of photosynthesis were normal. In contrast, alfalfa (Medicago
sativa) plants, which have limited numbers of plasmodesmata between mesophyll and
phloem, displayed typical symptoms of loading inhibition when transformed with
the same DNA constructs. The results are consistent with passive loading of Suc
through plasmodesmata in poplar. We also noted defense-related symptoms in leaves
of transgenic poplar when the plants were abruptly exposed to excessively high
temperatures, adding to evidence that hexose is involved in triggering the
hypersensitive response.
DOI: 10.1104/pp.114.245845
PMCID: PMC4149716
631. Foodborne Pathog Dis. 2014 Oct;11(10):759-68. doi: 10.1089/fpd.2014.1766. Epub

2014 Jul 23.
Effectiveness of calcium hypochlorite on viral and bacterial contamination of

alfalfa seeds.
Wang Q(1), Kniel KE.
Author information:
(1)Department of Animal and Food Sciences, University of Delaware , Newark,
Delaware.
Alfalfa sprouts have been involved in numerous foodborne outbreaks, which has
increased the awareness for seed and sprout safety. This study compared the
effectiveness of calcium hypochlorite (Ca(OCl)2) on the inactivation of bacteria
and viruses on alfalfa seeds and in the presence of a simulated organic load.
Alfalfa seeds were inoculated with human norovirus (huNoV) genogroup II (GII),
murine norovirus (MNV), Tulane virus (TV), Escherichia coli O104:H4, and
Salmonella enterica serovar Agona. Seeds were treated with Ca(OCl)2 (2000 ppm or
20,000 ppm with the average of free chlorine 1388 ± 117 mg/L and 11,472 ± 1500
mg/L, respectively, pH adjusted to 7.00). The reduction of huNoV genomic copies
indicated that huNoV was relatively resistant to Ca(OCl)2 regardless of
concentrations. Significant reductions were observed in the order of TV <
Salmonella Agona < MNV < E. coli O104:H4 at 20,000 ppm Ca(OCl)2. A similar trend
was found at 2000 ppm Ca(OCl)2 in the order of TV, Salmonella Agona, MNV < E.
coli O104:H4. Ca(OCl)2 at 20,000 ppm was more effective than 2000 ppm for all the
organisms tested. This trend was also observed in samples containing an
artificial organic material load. Ca(OCl)2 activity on virus inactivation
decreased as the organic load increased. Reduction was greater in fetal bovine
serum-containing samples compared to alfalfa seeds, indicating a close
relationship between the organisms and alfalfa seeds. Ca(OCl)2 could not
completely inactivate bacteria or viruses inoculated on seeds, and high levels of
E. coli O104:H4 and Salmonella Agona were present on sprouts from sanitized seed
samples following a 7-day germination period.
DOI: 10.1089/fpd.2014.1766

10.1007/s11356-014-3335-4. Epub 2014 Jul 23.
Effect of lead treatment on medicarpin accumulation and on the gene expression of

key enzymes involved in medicarpin biosynthesis in Medicago sativa L.
Ghelich S(1), Zarinkamar F, Soltani BM, Niknam V.
Author information:
(1)Faculty of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad
Highway, P.O.Box: 14115-111, Tehran, Iran.
Lead (Pb) is the most common heavy metal contaminant in the environment. The
present study was undertaken to determine the effect of Pb treatment on
medicarpin production and accumulation in Medicago sativa L. To this aim, 7- and
30-day-old plants were treated with 0, 120, 240, 500, and 1,000 μM Pb during 10
days. The content of medicarpin was determined by HPLC, and the extent of
medicarpin production was deduced from the result of semiquantitative RT-PCR
performed on PAL, CHS, and VR genes. HPLC results indicated that medicarpin
concentration has been reduced in the roots, while its exudation to the culture
medium has been increased. RT-PCR results indicated that the transcript levels of
PAL, CHS, and VR genes have not been affected following Pb stress in seedlings.
At the vegetative stage, transcript levels of PAL and CHS genes have been reduced
in the roots. However, the transcript level of VR gene increased at 120 and 240
μM Pb, while it decreased at higher concentrations. In the shoot, the transcript
levels of PAL, CHS, and VR genes were increased following increased concentration
of lead in the medium. Overall, q-PCR results suggest that medicarpin
biosynthesis has been induced in the shoots and reduced in the roots of the
plants treated with a toxic concentration of Pb.
DOI: 10.1007/s11356-014-3335-4
633. Photochem Photobiol Sci. 2014 Oct;13(10):1410-9. doi: 10.1039/c4pp00128a.
Sunlight-promoted photocatalytic hydrogen gas evolution from water-suspended

cellulose: a systematic study.
Speltini A(1), Sturini M, Dondi D, Annovazzi E, Maraschi F, Caratto V, Profumo A,

Buttafava A.
Author information:
(1)Department of Chemistry, University of Pavia, via Taramelli 12, 27100 Pavia,
Italy. andrea.speltini@unipv.it michela.sturini@unipv.it.
This work presents a systematic study of cellulose (CLS) as a sacrificial biomass

for photocatalytic H2 evolution from water. The idea is indeed to couple a
largely available and not expensive biomass, and water, with a renewable energy
like solar radiation. An aqueous CLS suspension irradiated either at 366 nm
(UV-A) or under sunlight in the presence of Pt/TiO2 behaves as a H2 evolving
system. The effects of irradiation time, catalyst and CLS concentrations, pH and
water salinity are studied. Addition of CLS to the sample significantly improved
H2 evolution from water splitting, with yields up to ten fold higher than those
observed in neat water. The mechanism of the photocatalytic process relies on the
TiO2-mediated CLS hydrolysis, under irradiation. The polysaccharide
depolymerisation generates water-soluble species and intermediates, among them
5-hydroxymethylfurfural (HMF) was identified. These intermediates are readily
oxidized following the glucose photoreforming, thus enhancing water hydrogen ion
reduction to give gas-phase H2. The formation of "colored" by-products from HMF
self-polymerization involves a sort of "in situ dye sensitization" that allows an
effective photoreaction even under solar light. The procedure is evaluated and
successfully extended on cellulosic biomasses, i.e. rice husk and alfalfa
(Medicago sativa) stems, not previously investigated for this application.
DOI: 10.1039/c4pp00128a
634. Asian-Australas J Anim Sci. 2014 Jun;27(6):825-31. doi:

10.5713/ajas.2013.13718.
Determination of Nutrient Contents and In vitro Gas Production Values of Some
Legume Forages Grown in the Harran Plain Saline Soils.
Boga M(1), Yurtseven S(1), Kilic U(2), Aydemir S(3), Polat T(4).
Author information:
(1)Department of Animal Science, Faculty of Agriculture, Harran University,
TR-63100 Şanlıurfa, Turkey .
(2)Department of Animal Science, Faculty of Agriculture, Ondokuz Mayıs
University, TR 55139, Samsun, Turkey .
(3)Department of Soil Science, Faculty of Agriculture, Harran University,
TR-63100, Şanlıurfa, Turkey .
(4)Department of Field Crops, Faculty of Agriculture, Harran University,
TR-63100, Şanlıurfa, Turkey .
The aim of this study was to determine the nutritive value of some legume species
in salt-affected soils of South-East Anatolian region using chemical composition
and in vitro gas production kinetics. In this study, Lotus corniculatus,
Trifolium alexandrinum, Medicago sativa were sown and tested in four different
locations. A 3 by 4 factorial design with 3 legume species and 4 salt levels (non
salty electrical conductivity (EC)<4 dS/m; low salt: 4 dS/m>EC<8 dS/m, medium
saline: 8 dS/m>EC<16 dS/m and high salt: 16 dS/m>EC) was used in the study.
Results indicated that salinity and plants had no significant effect on ash and
ether extract. Dry matter (DM), acid detergent fiber, digestible dry matter, dry
matter intake (DMI) were affected by plant, salinity and plant×salinity
interaction. On the other hand neutral detergent fiber, relative feed value
(RFV), and DMI were affected by salinity and plant×salinity interaction. Mineral
contents were affected by plant species, salinity and salinity×plants
interactions. In vitro gas production, their kinetics and estimated parameters
such as were not affected by salinity whereas the gas production up to 48 h,
organic matter digestibility, metabolizable energy (ME), and net energy lactation
(NEL) were affected by plant and plant×salt interaction. Generally RFVs of all
species ranged from 120 to 210 and were quite satisfactory in salty conditions.
Current results show that the feed value of Medicago sativa is higher compared to
Lotus corniculatus and Trifolium alexandrinum.
DOI: 10.5713/ajas.2013.13718
PMCID: PMC4093174
PMID: 25050020
635. J Sci Food Agric. 2015 Apr;95(6):1294-302. doi: 10.1002/jsfa.6822. Epub 2014
Aug
19.
Management practices and forage quality affecting the contamination of milk with
anaerobic spore-forming bacteria.
Zucali M(1), Bava L, Colombini S, Brasca M, Decimo M, Morandi S, Tamburini A,

Crovetto GM.
Author information:
Ambientali, via Celoria 2, 20133, Milan, Italy.
BACKGROUND: Anaerobic spore-forming bacteria (ASFB) in milk derive from the farm
environment, and the use of silages and management practices are the main
responsible of milk ASFB contamination. The aim of this study was to evaluate the
relationships between feeding, milking routine and cow hygiene and milk and Grana
Padano cheese (produced with and without lysozyme) ASFB contamination.
RESULTS: The study involved 23 dairy farms. ASFB in corn silage were on average
2.34 ± 0.87 log10 MPN g(-1). For grass, Italian ryegrass and alfalfa, ASFB (log10
MPN g(-1)) were numerically higher for silages (3.22) than hays (2.85). The use
of corn silages of high quality (high lactic and acetic acids concentrations)
decreased the milk ASFB contamination, whilst the use of herbage silages did not
affect it. The presence (>40%) of cows with dirty udders increased the ASFB
contamination of milk, while forestripping had a positive effect (-9% ASFB).
Ripened Grana Padano had an ASFB count below the analytical limit; Clostridium
tyrobutyricum DNA was found only in wheels produced without lysozyme, which also
showed late blowing.
CONCLUSION: The factors increasing milk spore contamination were corn silage
quality, cow udder hygiene and inadequate milking routine. Late blowing was
present only in cheeses without lysozyme.
DOI: 10.1002/jsfa.6822
636. Anim Sci J. 2015 Feb;86(2):159-65. doi: 10.1111/asj.12256. Epub 2014 Jul 12.
Nitrogen digestion and urea recycling in Hokkaido native horses fed hay-based
diets.
Obitsu T(1), Hata H, Taniguchi K.
Author information:
(1)Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima,
Japan.
Nitrogen (N) digestion and urea-N metabolism in Hokkaido native horses fed
roughage-based diets containing different types and levels of protein sources
were studied. Horses (173 ± 4.8 kg) fitted with an ileum cannula were fed four
diets consisting of 100% timothy hay (TH), 88% TH and 12% soybean meal (SBM), 79%
TH and 21% SBM, and 51% TH and 49% alfalfa hay at 2.2% of body weight. Dietary
protein content varied from 5% to 15% of dry matter. Apparent N digestibilities
in the pre-cecum and total tract for the TH diet were lower than those for other
diets. However, the proportion of post-ileum N digestion to N intake was not
affected by the diets. Urea-N production was linearly related to N intake, but
gut urea-N entry was not affected by the diets. The proportion of gut urea-N
entry to urea-N production tended to be higher for the TH diet (57%) than the two
SBM diets (39%). Anabolic use of urea-N entering the gut was not affected by the
diets (20-36% of gut urea-N entry). These results indicate that urea-N recycling
provides additional N sources for microbial fermentation in the hindgut of
Hokkaido native horses fed low-quality roughages.
DOI: 10.1111/asj.12256

10.1111/jpn.12229.
Epub 2014 Jul 14.
Effects of supplementation level and particle size of alfalfa hay on growth

characteristics and rumen development in dairy calves.
Mirzaei M(1), Khorvash M, Ghorbani GR, Kazemi-Bonchenari M, Riasi A, Nabipour A,

van den Borne JJ.
Author information:
(1)Department of Animal Sciences, College of Agriculture, Isfahan University of
The aim of this study was to assess the effects of particle size (PS) of alfalfa
hay on growth characteristics and rumen development in dairy calves at two levels
of alfalfa supplementation. Fifty newborn dairy calves (42.7 ± 2.2 kg BW) were
used in a 2 × 2 factorial arrangement with the factors supplementation level
(low, 8%; or high, 16% on DM basis) and PS (medium, 2.92 mm; or long, 5.04 mm as
geometrical means) of alfalfa hay. In addition, a control group without alfalfa
hay was used. Hence, treatments were: control (C); low level with medium PS (LM);
low level with long PS (LL); high level with medium PS (HM) or high level with
long PS (HL). Growth performance of alfalfa-fed calves did not differ from
control calves, but alfalfa supplementation decreased corneum thickness of the
rumen wall. In alfalfa-fed calves, post-weaning starter intake was greater for LL
calves than for LM calves. During the entire rearing period, starter intake was
26-32% higher for LL and HM calves than for LM calves. Pre-weaning average daily
gain was higher for LL and HM calves than for HL calves, but this effect was not
persistent over the entire rearing period. Final body weight decreased from 86 to
79 kg when the level of long PS alfalfa hay increased from 8 to 16%, but
increased from 78 to 87 kg when the level of medium PS alfalfa increased from 8
to 16%. Regardless of PS and level, morphometric characteristics of rumen wall
were generally similar among alfalfa feeding groups, but corneum thickness
decreased from 8.7 to 6.1 μm with greater PS at the low level. These results
indicate that adequate, but not excessive, physical stimulation is required for
appropriate rumen development and growth performance of dairy calves.
DOI: 10.1111/jpn.12229
638. J Hazard Mater. 2014 Aug 30;279:67-74. doi: 10.1016/j.jhazmat.2014.06.063.

Epub
2014 Jul 5.
Effectiveness of recombinant protein AlnA in enhancing the extractability of

polychlorinated biphenyls from contaminated soils.
Wang Y(1), Wang C(2), Ren H(3), Jia B(4), Zhang L(2).
Author information:
(1)Key Laboratory of Groudwater Resources and Environment of the Ministry of
Education, College of Environment and Resources, Jilin University, Changchun,
Jilin 130021, China; College of Plant Sciences, Jilin University, Changchun,
Jilin 130062, China.
Jilin 130021, China.
Jilin 130021, China. Electronic address: renhejun@126.com.
(4)College of Plant Sciences, Jilin University, Changchun, Jilin 130062, China.
One of the major limitations in bioremediation of polychlorinated biphenyls
(PCBs) in contaminated soils is their low water solubility and high
adsorbability. In the present study, effectiveness of recombinant protein AlnA as
a biosurfactant in increasing extractability of PCBs was first investigated. The
AlnA surface tension measurements allowed for determination of the minimum value
of 37.89mN/m with critical micelle concentration of 1.07g/L. The solubilization
of PCBs and desorption experiments were performed. It was observed that AlnA at
20μg/mL could enhance solubility of selected crystal PCBs varying from 1.87 to
6.12 folds and it is easier to recover PCBs from contaminated soils compared with
Tween-80 and cyclodextrin. Pot experiments indicated AlnA elevated 1.4-2.1-fold
PCBs accumulation in roots by Alfalfa. The homology modeling and docking revealed
PCBs congeners had different interactions with AlnA and higher-chlorinated PCBs
possessed higher binding affinity. Results showed AlnA had considerable
application potential in remediation of PCBs pollution.
DOI: 10.1016/j.jhazmat.2014.06.063
639. Ecotoxicol Environ Saf. 2015 Apr;114:312-7. doi: 10.1016/j.ecoenv.2014.04.044.

Epub 2014 Jul 16.
The difference in antioxidant capacity of four alfalfa cultivars in response to

Zn.
Dai HP(1), Shan CJ(2), Zhao H(3), Li JC(4), Jia GL(5), Jiang H(3), Wu SQ(3), Wang
Q(3).
Author information:
(1)College of Biological Science & Engineering, Shaanxi University of Technology,
Hanzhong, Shaanxi 723001, PR China. Electronic address: daihp72@aliyun.com.
(2)Henan Institute of Science and Technology, Xinxiang 453003, PR China.
(3)College of Biological Science & Engineering, Shaanxi University of Technology,
Hanzhong, Shaanxi 723001, PR China.
(4)College of Life Science, Northwest Agriculture & Forestry University,
Yangling, Shaanxi 712100, PR China.
(5)College of Science, Northwest Agriculture & Forestry University, Yangling,
Shaanxi 712100, PR China.
The aim of this study was to evaluate antioxidative responses in roots, stem and
leaves of four alfalfa cultivars to different concentrations of zinc (Zn) (0,
300, 600 and 900 μM) for 23 days. Among the four cultivars, Aohan displayed the
highest Zn concentrations in tissues and the largest Zn amount in aerial parts.
Zn stress induced the production of H2O2 and increased the content of free
proline and activities of antioxidative enzymes in roots, stem and leaves of
Aohan. Based on the above results, we concluded that Aohan is superior to other
three cultivars for Zn phyto-remediation, which indicated that Aohan is a novel
Zn accumulator and able to tolerate Zn-induced toxicity by activating the
antioxidative defense system.
DOI: 10.1016/j.ecoenv.2014.04.044
640. Int J Food Microbiol. 2014 Sep 18;187:15-21. doi:
10.1016/j.ijfoodmicro.2014.06.030. Epub 2014 Jul 3.
Effectiveness of a novel spontaneous carvacrol nanoemulsion against Salmonella

enterica Enteritidis and Escherichia coli O157:H7 on contaminated mung bean and
alfalfa seeds.
Landry KS(1), Chang Y(2), McClements DJ(3), McLandsborough L(4).
Author information:
(1)Department of Food Science, Massachusetts Agricultural Experiment Station,
University of Massachusetts, Amherst, MA 01003, USA. Electronic address:
kslandry@foodsci.umass.edu.
yuhua@foodsci.umass.edu.
mcclements@foodsci.umass.edu.
lm@foodsci.umass.edu.
Outbreaks of foodborne illness from consumption of sprouts have been linked to

contaminated seeds prior to germination. Due to the long sprouting period at
ambient temperatures and high humidity, germinating seeds contaminated with low
pathogen levels (0.1logCFU/g) can result in sprouts with high numbers
(≥10(8)CFU/g) of pathogens. Currently, the recommended treatment method involves
soaking seeds in 20,000ppm (2%) calcium hypochlorite prior to germination. In
this study, an alternative treatment involving soaking seeds in a carvacrol
nanoemulsion was tested for its efficacy against Salmonella enterica subspecies
enterica serovar Enteritidis (ATCC BAA-1045) or EGFP expressing E. coli O157:H7
(ATCC 42895) contaminated mung bean and alfalfa seeds. The antimicrobial
treatment was performed by soaking inoculated seed batches in the spontaneous
nanoemulsion (4000 or 8000ppm) for 30 or 60min. The spontaneous nanoemulsion was
formed by titrating the oil phase (carvacrol and medium chain triglycerides) and
water-soluble surfactant (Tween 80®) into sodium citrate buffer. Following
treatment, the numbers of surviving cells were determined by suspending the seeds
in TSB and performing plate counts and/or Most Probable Number (MPN) enumeration.
Treated seeds were sprouted and tested for the presence of the appropriate
pathogen. This treatment successfully inactivated low levels (2 and 3logCFU/g) of
S. Enteritidis and E. coli on either seed types when soaked for either 30 or
60min at nanoemulsion concentrations corresponding to 4000 (0.4%) or 8000 (0.8%)
ppm carvacrol. Inoculated alfalfa seeds treated with 4000ppm nanoemulsion,
required a 60min treatment time to show a similar 2-3 log reduction. Complete
inactivation was confirmed by germinating treated seeds and performing
microbiological testing. Total sprout yield was not compromised by any of the
tested treatments. These results show that carvacrol nanoemulsions may be an
alternative antimicrobial treatment method for mung bean and alfalfa seeds.
641. ScientificWorldJournal. 2014;2014:676242. doi: 10.1155/2014/676242. Epub 2014

Jun
17.
Phytotoxic activity of Ocimum tenuiflorum extracts on germination and seedling
growth of different plant species.
Islam AK(1), Kato-Noguchi H(1).
Author information:
(1)Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki, Kita, Kagawa
761-0795, Japan.
Phytotoxic activity of Ocimum tenuiflorum (Lamiaceae) plant extracts was

investigated against the germination and seedling growth of cress (Lepidium
sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), Italian ryegrass
(Lolium multiflorum), barnyard grass (Echinochloa crus-galli), and timothy
(Phleum pratense) at four different concentrations. The plant extracts at
concentrations greater than 30 mg dry weight equivalent extract mL(-1) reduced
significantly the total germination percent (GP), germination index (GI),
germination energy (GE), speed of emergence (SE), seedling vigour index (SVI),
and coefficient of the rate of germination (CRG) of all test species except
barnyard grass and GP of lettuce. In contrast, time required for 50% germination
(T 50) and mean germination time (MGT) were increased at the same or higher than
this concentration. The increasing trend of T 50 and MGT and the decreasing trend
of other indices indicated a significant inhibition or delay of germination of
the test species by O. tenuiflorum plant extracts and vice versa. In addition,
the shoot and root growth of all test species were significantly inhibited by the
extracts at concentrations greater than 10 mg dry weight equivalent extract
mL(-1). The I 50 values for shoot and root growth were ranged from 26 to 104 mg
dry weight equivalent extract mL(-1). Seedling growth was more sensitive to the
extracts compared to seed germination. Results of this study suggest that O.
tenuiflorum plant extracts have phytotoxic properties and thus contain phytotoxic
substances. Isolation and characterization of those substances from this plant
may act as a tool for new natural, biodegradable herbicide development to control
weeds.
DOI: 10.1155/2014/676242
PMCID: PMC4086237

Jun 16.
Endogenous hydrogen sulfide enhances salt tolerance by coupling the

reestablishment of redox homeostasis and preventing salt-induced K⁺ loss in
seedlings of Medicago sativa.
Lai D(1), Mao Y(1), Zhou H(1), Li F(2), Wu M(2), Zhang J(1), He Z(1), Cui W(1),
Xie Y(3).
Author information:
(2)Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China.
yjxie@njau.edu.cn.
Despite the external application of hydrogen sulfide (H2S) conferring plant

tolerance against various environmental cues, the physiological significance of
l-cysteine desulfhydrase (L-DES)-associated endogenous H2S production involved in
salt-stress signaling was poorly understood. To address this gap, the
participation of in planta changes of H2S homeostasis involved in alfalfa salt
tolerance was investigated. The increasing concentration of NaCl (from 50 to 300
mM) progressively caused the induction of total l-DES activity and the increase
of endogenous H2S production. NaCl-triggered toxicity symptoms (175 mM),
including seedling growth inhibition and lipid peroxidation, were alleviated by
sodium hydrosulfide (NaHS; 100 μM), a H2S donor, whereas aggravated by an
inhibitor of l-DES or a H2S scavenger. A weaker or negative response was observed
in lower or higher dose of NaHS. Further results showed that endogenous
l-DES-related H2S modulated several genes/activities of antioxidant defence
enzymes, and also regulated the contents of antioxidant compounds, thus
counterbalancing the NaCl-induced lipid peroxidation. Moreover, H2S maintained
K(+)/Na(+) homeostasis by preventing the NaCl-triggered K(+) efflux, which might
be result form the impairment of SKOR expression. Together, our findings
indicated that endogenous H2S homeostasis enhance salt tolerance by coupling the
reestablishment of redox balance and restraining K(+) efflux in alfalfa
seedlings.
DOI: 10.1016/j.plantsci.2014.06.006
643. Bull Environ Contam Toxicol. 2014 Sep;93(3):263-7.
A simple control for sediment-toxicity exposures using the amphipod, Hyalella

azteca.
Lasier PJ, Urich ML.
Sediment-toxicity exposures comparing survival and growth of the freshwater

amphipod, Hyalella azteca, are often components of aquatic-habitat assessments.
Standardized exposure methods have been established and require evaluations for
quality assurance. Test acceptability using performance-based criteria can be
determined from exposures to control sediments, which are collected from the
environment or formulated from commercially available components. Amending sand
with leached alfalfa solids provided a simple formulated sediment that elicited
consistently acceptable survival and growth in 28-day exposures with and without
a daily feeding regime. A procedure is described for preparing the sediment along
with results from comparisons among sand, amended sand, and field-collected
sediments that incorporated three feeding regimes.
DOI: 10.1007/s00128-014-1329-z
644. Anticancer Agents Med Chem. 2014;14(10):1324-31.
White poplar (Populus alba L.) suspension cultures as a model system to study
apoptosis induced by alfalfa saponins.
Balestrazzi A, Carbonera D, Avato P, Tava A(1).
Author information:
(1)Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia,
via Ferrata 1, 27100- Pavia, Italy. alma.balestrazzi@unipv.it.
In animal cells, the anticancer function played by plant saponins involves a
complex network of molecular processes that still deserves investigation and
apoptosis seems to be the outstanding pathway. An intriguing aspect of the
biological activity of saponins is related to their effects on genome integrity.
As demonstrated by the studies carried out in white poplar (Populus alba L., cv
Villafranca) cell suspension cultures, plant cells can as well be used as a model
system to unravel the molecular mechanisms activated by plant saponins. These
recent studies have evidenced that animal and plant cells share common features
in their response to saponins, paving the way for novel opportunities for both
basic and applied research. Indeed, there is a certain interest in replacing the
animal models for pharmacological research, at least when preliminary large-scale
cytotoxicity tests are performed on wide collections of natural extracts and/or
purified compounds. The review provides an up-date of the molecular pathways
(signal transduction, antioxidant response, DNA repair) associated with plant
saponin bioactivity, with an emphasis on apoptosis induced by alfalfa (Medicago
sativa L.) saponins. The comparison between animal and plant cells as tools for
the study of saponin bioactivity is also discussed in view of the most recent
literature and innovative future applications.
645. J Dairy Sci. 2014 Sep;97(9):5645-56. doi: 10.3168/jds.2013-7724. Epub 2014 Jul
2.
A statistical filtering procedure to improve the accuracy of estimating

population parameters in feed composition databases.
Yoder PS(1), St-Pierre NR(2), Weiss WP(3).
Author information:
(1)Department of Animal Sciences, Ohio Agricultural Research and Development
Center, The Ohio State University, Wooster 44691.
(2)Department of Animal Sciences, The Ohio State University, Columbus 43210.
Center, The Ohio State University, Wooster 44691. Electronic address:
weiss.6@osu.edu.
Accurate estimates of mean nutrient composition of feeds, nutrient variance

(i.e., standard deviation), and covariance (i.e., correlation) are needed to
develop a more quantitative approach of formulating diets to reduce risk and
optimize safety factors. Commercial feed-testing laboratories have large
databases of composition values for many feeds, but because of potentially
misidentified feeds or poorly defined feed names, these databases are possibly
contaminated by incorrect results and could generate inaccurate statistics. The
objectives of this research were to (1) design a procedure (also known as a
mathematical filter) that generates accurate estimates of the first 2 moments
[i.e., the mean and (co)variance] of the nutrient distributions for the largest
subpopulation within a feed in the presence of outliers and multiple
subpopulations, and (2) use the procedure to generate feed composition tables
with accurate means, variances, and correlations. Feed composition data
(>1,300,000 samples) were collected from 2 major US commercial laboratories. A
combination of a univariate step and 2 multivariate steps (principal components
analysis and cluster analysis) were used to filter the data. On average, 13.5% of
the total samples of a particular feed population were removed, of which the
multivariate steps removed the majority (66% of removed samples). For some feeds,
inaccurate identification (e.g., corn gluten feed samples included in the corn
gluten meal population) was a primary reason for outliers, whereas for other
feeds, subpopulations of a broader population were identified (e.g., immature
alfalfa silage within a broad population of alfalfa silage). Application of the
procedure did not usually affect the mean concentration of nutrients but greatly
reduced the standard deviation and often changed the correlation estimates among
nutrients. More accurate estimates of the variation of feeds and how they tend to
vary will improve the economic evaluation of feeds and risk assessment of diets,
and provide the ability to implement stochastic programming.

DOI: 10.3168/jds.2013-7724
2.
Effect of reducing dietary forage in lower starch diets on performance, ruminal

characteristics, and nutrient digestibility in lactating Holstein cows.
Farmer ER(1), Tucker HA(1), Dann HM(2), Cotanch KW(1), Mooney CS(1), Lock AL(3),
Yagi K(4), Grant RJ(1).
Author information:
(1)William H. Miner Agricultural Research Institute, Chazy, NY 12921.
(2)William H. Miner Agricultural Research Institute, Chazy, NY 12921. Electronic
address: dann@whminer.com.
(3)Department of Animal Sciences, Michigan State University, East Lansing 48824.
(4)ZEN-NOH National Federation of Agricultural Co-operative Associations, Tokyo
100-6832, Japan.
This experiment evaluated the effect of feeding a lower starch diet (21% of dry
matter) with different amounts of forage (52, 47, 43, and 39% of dry matter) on
lactational performance, chewing activity, ruminal fermentation and turnover,
microbial N yield, and total-tract nutrient digestibility. Dietary forage
consisted of a mixture of corn and haycrop silages, and as dietary forage content
was reduced, chopped wheat straw (0-10% of dry matter) was added in an effort to
maintain chewing activity. Dietary concentrate was adjusted (corn meal, nonforage
fiber sources, and protein sources) to maintain similar amounts of starch and
other carbohydrate and protein fractions among the diets. Sixteen lactating
Holstein cows were used in replicated 4×4 Latin squares with 21-d periods. Dry
matter intake increased while physically effective neutral detergent fiber
(peNDF1.18) intake was reduced as forage content decreased from 52 to 39%.
However, reducing dietary forage did not influence milk yield or composition,
although we observed changes in dry matter intake. Time spent chewing, eating,
and ruminating (expressed as minutes per day or as minutes per kilogram of NDF
intake) were not affected by reducing dietary forage. However, addition of
chopped wheat straw to the diets resulted in greater time spent chewing and
eating per kilogram of peNDF1.18 consumed. Reducing dietary forage from 52 to 39%
did not affect ruminal pH, ruminal digesta volume and mass, ruminal pool size of
NDF or starch, ruminal digesta mat consistency, or microbial N yield. Ruminal
acetate-to-propionate ratio was reduced, ruminal turnover rates of NDF and starch
were greater, and total-tract digestibility of fiber diminished as dietary forage
content decreased. Reducing the dietary forage content from 52 to 39% of dry
matter, while increasing wheat straw inclusion to maintain chewing and rumen
function, resulted in similar milk yield and composition although feed intake
increased. With the lower starch diets in this short-term study, the minimal
forage content to maintain lactational performance was between 39 and 43%.
DOI: 10.3168/jds.2014-7963
2.
Chewing, rumen pool characteristics, and lactation performance of dairy cows fed
2 concentrations of a corn wet-milling coproduct with different forage sources.
Shepherd DM(1), Firkins JL(2), VonBehren P(3).
Author information:
Electronic address: firkins.1@osu.edu.
(3)Cargill, 1705 Kellie Drive, Blair, NE 68008.
We used a novel corn wet-milling coproduct [CMP; approximately 70% dry matter,
28% crude protein, 36% neutral detergent fiber (NDF), and 18% nonstructural
carbohydrates] in diets formulated to contain 18.4% forage NDF, 17.4% crude
protein, 20.2% starch, and 3.7% sugar. Six primiparous, rumen-cannulated Jersey
cows were assigned to a 6 × 6 Latin square design with a 2 × 3 factorial
arrangement of treatments. Diets were formulated to contain 20 and 30% CMP with 3
forage sources [corn silage (CS) and 40.5% NDF, CS replaced with 10% alfalfa hay
(AH) and 45.0% NDF, or CS replaced with 7% grass hay (GH) and 67.4% NDF], with
each providing 18.4% forage NDF in the diet. Total-tract digestibilities of NDF,
N, and organic matter were not affected by treatment. Similarly, no treatment
effects were detected for kinetics of NDF disappearance in situ from CMP or
respective forage source or for N disappearance in situ from CMP. Grass hay
increased total and liquid pool size of rumen contents compared with AH (by 3.2
and 3.0kg, respectively). Total time spent chewing increased in cows fed GH by
over 35min compared with those fed AH, partially due to a trend for increased
minutes spent ruminating. Mean particle size of rumen contents also tended to be
higher in the GH (0.55mm) than AH (0.69mm) diets. No effects on production of
milk or milk components were detected, but dry matter intake (DMI) tended to
decrease when CMP increased from 20 to 30%. Gross feed efficiency (fat-corrected
milk/DMI) tended to be greater when cows were fed AH and GH compared with CS and
was greater for AH than GH diets. In diets containing low starch, increasing CMP
from 20 to 30% potentially maintained similar fat-corrected milk production with
lower DMI. However, more consideration also should be given to interactions among
forages with respect to fill, digestion, and passage of fiber with increased
inclusion rates of CMP.

DOI: 10.3168/jds.2014-8169
648. Stand Genomic Sci. 2013 Dec 15;9(2):315-24. doi: 10.4056/sigs.4608286.

eCollection 2013 Dec 20.
Genome sequence of Ensifer meliloti strain WSM1022; a highly effective

microsymbiont of the model legume Medicago truncatula A17.
Terpolilli J(1), Hill Y(1), Tian R(1), Howieson J(1), Bräu L(2), Goodwin L(3),
Han J(4), Liolios K(4), Huntemann M(4), Pati A(5), Woyke T(4), Mavromatis K(5),
Markowitz V(5), Ivanova N(3), Kyrpides N(3), Reeve W(1).
Author information:
Australia.
Australia.
USA.
Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming

rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago.
WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual
Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022
is highly effective at fixing nitrogen with M. truncatula and other annual
species such as M. tornata and M. littoralis and is also highly effective with
the perennial M. sativa (alfalfa or lucerne). In common with other characterized
E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha
and M. sphaerocarpos and does not nodulate M. murex. Here we describe the
features of E. meliloti WSM1022, together with genome sequence information and
its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121
scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only
encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE
Nodule Bacteria (GEBA-RNB) project.
DOI: 10.4056/sigs.4608286
PMCID: PMC4062635
PMID: 24976888
649. Biotechnol Lett. 2014 Nov;36(11):2331-41. doi: 10.1007/s10529-014-1598-y. Epub

2014 Jun 30.
Overexpression of an alfalfa GDP-mannose 3, 5-epimerase gene enhances acid,

drought and salt tolerance in transgenic Arabidopsis by increasing ascorbate
accumulation.
Ma L(1), Wang Y, Liu W, Liu Z.
Author information:
(1)State Key Laboratory of Grassland Agro-ecosystems, School of Pastoral
Agricultural Science and Technology, Lanzhou University, Lanzhou, 730020,
People's Republic of China, ma-lichao@163.com.
GDP-mannose 3', 5'-epimerase (GME) catalyses the conversion of GDP-D-mannose to

GDP-L-galactose, an important step in the ascorbic acid (ascorbic acid)
biosynthetic pathway in higher plants. In this study, a novel cDNA fragment
(MsGME) encoding a GME protein was isolated and characterised from alfalfa
(Medicago sativa). An expression analysis confirmed that MsGME expression was
induced by salinity, PEG and acidity stresses. MsGME overexpression in
Arabidopsis enhanced tolerance of the transgenic plants to salt, drought and
acid. Real-time PCR analysis revealed that the transcript levels of GDP-D-mannose
pyrophosphorylase (GMP), L-galactose-phosphate 1-P phosphatase (GP) and
GDP-L-galactose phosphorylase (GGP) were increased in transgenic Arabidopsis (T3
generation). Moreover, the ascorbate content was increased in transgenic
Arabidopsis. Our results suggest that MsGME can effectively enhance tolerance of
transgenic Arabidopsis to acid, drought and salt by increasing ascorbate
accumulation.
DOI: 10.1007/s10529-014-1598-y
650. Res Microbiol. 2014 Sep;165(7):508-16. doi: 10.1016/j.resmic.2014.06.001. Epub

2014 Jun 21.
Biogeography of Sinorhizobium meliloti nodulating alfalfa in different Croatian

regions.
Donnarumma F(1), Bazzicalupo M(2), Blažinkov M(3), Mengoni A(4), Sikora S(3),
Babić KH(3).
Author information:
(1)Institute of Plant Genetics, National Research Council (IGV-CNR), Via Madonna
del Piano 10, 50019 Sesto Fiorentino, Italy.
(2)Department of Biology, University of Firenze, Via Madonna del Piano 6, 50019
Sesto Fiorentino, Italy. Electronic address: marco.bazzicalupo@unifi.it.
(3)Department of Microbiology (AGR), University of Zagreb, Grad Zagreb, Croatia.
(4)Department of Biology, University of Firenze, Via Madonna del Piano 6, 50019
Sesto Fiorentino, Italy.
Sinorhizobium meliloti is a nitrogen-fixing rhizobium symbiont of legumes,

widespread in many temperate environments the high genetic diversity of which
enables it to thrive as a symbiont of host legumes and free-living in soil. Soil
type, together with geographic differences and host plant genotype, seem to be
prominent factors in shaping rhizobial genetic diversity. While a large body of
research supports the idea that the genetic structure of free-living microbial
taxa exhibits a clear biogeographic pattern, few investigations have been
performed on the biogeographic pattern of S. meliloti genotypes in a restricted
geographic range. In the present study, a collection of 128 S. meliloti isolates
from three different regions in Croatia was investigated to analyze the
relationship between genetic diversity, geographic distribution, soil features
and isolate phenotypes by using amplified fragment length polymorphism (AFLP) as
a genome-wide scanning method. Results obtained led to the conclusion that the
genotypes of isolates cluster according to the region of origin and that the
differentiation of S. meliloti populations can be mainly ascribed to geographic
isolation following an isolation-by-distance model, with a strong distance-decay
relationship of genetic similarity with distance, in which local soil conditions
are not the major component influencing the isolate phenotypes or their genomic
differentiation.
Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights
reserved.
DOI: 10.1016/j.resmic.2014.06.001
651. Ticks Tick Borne Dis. 2014 Sep;5(5):528-36. doi: 10.1016/j.ttbdis.2014.03.009.

Epub 2014 Jun 19.
Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus
glutathione S-transferase.
Guneidy RA(1), Shahein YE(2), Abouelella AM(3), Zaki ER(2), Hamed RR(2).
Author information:
(1)Department of Molecular Biology, National Research Center, Cairo, Egypt.
Electronic address: Rasha_a_m_g@hotmail.com.
(2)Department of Molecular Biology, National Research Center, Cairo, Egypt.
(3)Department of Radiation Biology, NCRRT, Cairo, Egypt.
Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes

severe production losses in the cattle industry. This study aims to evaluate the
in vitro effects of tannic acid, hematin (GST inhibitors) and different plant
extracts (rich in tannic acid) on the activity of the recombinant glutathione
S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order
to confirm their ability to inhibit the parasitic essential detoxification enzyme
glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus
(kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata
(banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus
(seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory
capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol
extraction of red pomegranate peel contained the highest content of phenolic
compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant
extracts. The highest inhibition activities of rRaGST were obtained with kenaf
and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and
0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor
of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl
(IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and
caffeic acid had almost no effect on tick GST activity. The IC50 values qualify
ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron
blue and hematin showed a considerable inhibition effect on rRaGST activity, and
their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was
highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic
activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid
and hematin inhibited rRaGST activity non-competitively with respect to GSH and
competitively with respect to CDNB. While red pomegranate extracts inhibited
rRaGST activity competitively with respect to GSH, uncompetitive inhibition was
observed with respect to CDNB.
DOI: 10.1016/j.ttbdis.2014.03.009
652. J Dairy Sci. 2014 Sep;97(9):5718-28. doi: 10.3168/jds.2014-7911. Epub 2014 Jun
18.
Effect of flaxseed physical form on digestibility of lactation diets fed to

Holstein steers.
Schroeder JW(1), Bauer ML(2), Bork NR(3).
Author information:
(1)Department of Animal Sciences, North Dakota State University, Fargo 58108.
Electronic address: jw.schroeder@ndsu.edu.
(2)Department of Animal Sciences, North Dakota State University, Fargo 58108.
(3)Standard Nutrition Services, Luverne, MN 56156.
Four multicannulated (rumen, duodenum, and ileum) Holstein steers (459.7±46.4kg

of initial body weight) were used in a 4×4 Latin square design to determine the
effect of flaxseed processing method on ruminal fermentation and digestibility.
Treatments were based on inclusion of (1) 7.5% linseed meal (control), (2) 10%
whole flaxseed, (3) 10% rolled flaxseed, or (4) 10% ground flaxseed on a dry
matter (DM) basis, and were formulated to mimic typical high-producing dairy cow
lactation diets. The control diet contained linseed meal in a proportion to
provide crude protein (CP) equal to the amount of CP contributed by the flaxseed
in the other treatments. Diets were fed for ad libitum intake and contained 30%
corn silage, 17% chopped alfalfa hay, 6% sugar beet pulp, and 47% concentrate
(comprising ground corn, supplemental protein, trace minerals and vitamins, and
either flaxseed or linseed meal (DM basis). Diets were formulated to contain 17%
CP, 34% neutral detergent fiber, 21% acid detergent fiber, and 4% fatty acid (DM
basis). Periods were 14 d long and consisted of 7 d of adaptation and 7 d of
sample collection. Dry matter intake (as a % of body weight) was similar
(2.41±0.17) for all treatments. The inclusion of flaxseed, regardless of
processing method, tended to decrease total-tract organic matter digestibility
relative to the linseed control, but no differences in CP intake, duodenal CP
flow (bacterial, apparent feed, or total), ileal CP flow, fecal CP output,
microbial efficiency, or CP digestibility (apparent ruminal, true ruminal, small
intestine, large intestine, or total tract) were observed between treatments.
Method of processing did not alter ruminal pH, ammonia, or volatile fatty acids
production. The ground flaxseed treatment had the fastest rate of in situ DM
degradation (11.25%/h), followed by the control (7.46%/h), rolled flaxseed
(4.53%/h), and whole flaxseed (0.57%/h) treatments. Degradability of CP and fat
followed the same pattern as DM degradability for processed flaxseed. In situ
degradation rates of alfalfa hay neutral detergent fiber and acid detergent fiber
tended to be fastest for the ground flaxseed treatment. Taken together, the
digestibility, fermentation, and in situ data indicate that rolling and grinding
are both acceptable methods of processing flaxseed. The in situ data strongly
support the need for processing flaxseed before inclusion in lactation diets.

DOI: 10.3168/jds.2014-7911
653. Appl Environ Microbiol. 2014 Sep;80(17):5265-73. doi: 10.1128/AEM.01393-14.

Epub
2014 Jun 20.
Transcriptional regulator LsrB of Sinorhizobium meliloti positively regulates the

expression of genes involved in lipopolysaccharide biosynthesis.
Tang G(1), Wang Y(2), Luo L(3).
Author information:
(1)Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Shanghai
University, Shanghai, China State Key Laboratory of Plant Molecular Genetics,
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai, China University of the Chinese
Academy of Sciences, Beijing, China.
(2)State Key Laboratory of Plant Molecular Genetics, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China.
(3)Shanghai Key Laboratory of Bio-energy Crops, School of Life Sciences, Shanghai
University, Shanghai, China liluo@shu.edu.cn.
Rhizobia induce nitrogen-fixing nodules on host legumes, which is important in

agriculture and ecology. Lipopolysaccharide (LPS) produced by rhizobia is
required for infection or bacteroid survival in host cells. Genes required for
LPS biosynthesis have been identified in several Rhizobium species. However, the
regulation of their expression is not well understood. Here, Sinorhizobium
meliloti LsrB, a member of the LysR family of transcriptional regulators, was
found to be involved in LPS biosynthesis by positively regulating the expression
of the lrp3-lpsCDE operon. An lsrB in-frame deletion mutant displayed growth
deficiency, sensitivity to the detergent sodium dodecyl sulfate, and acidic pH
compared to the parent strain. This mutant produced slightly less LPS due to
lower expression of the lrp3 operon. Analysis of the transcriptional start sites
of the lrp3 and lpsCDE gene suggested that they constitute one operon. The
expression of lsrB was positively autoregulated. The promoter region of lrp3 was
specifically precipitated by anti-LsrB antibodies in vivo. The promoter DNA
fragment containing TN11A motifs was bound by the purified LsrB protein in vitro.
These new findings suggest that S. meliloti LsrB is associated with LPS
biosynthesis, which is required for symbiotic nitrogen fixation on some ecotypes
of alfalfa plants.
DOI: 10.1128/AEM.01393-14
PMCID: PMC4136089
654. Plant Physiol. 2014 Aug;165(4):1440-1456. Epub 2014 Jun 19.
Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic

Acid O-Methyltransferase and the Structural Impact of Three brown midrib12
Mutations.
Green AR(1), Lewis KM(1), Barr JT(1), Jones JP(1), Lu F(1), Ralph J(1), Vermerris
W(1), Sattler SE(1), Kang C(2).
Author information:
(1)School of Molecular Biosciences (A.R.G., C.K.) and Department of Chemistry
(K.M.L., J.T.B., J.P.J., C.K.), Washington State University, Pullman, Washington
99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy
Research Center, University of Wisconsin, Madison, Wisconsin 53726 (F.L.,
J.R.);Department of Microbiology and Cell Science and Genetics Institute,
University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States
Department of Agriculture-Agricultural Research Service, Grain Forage and
Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.).
(2)School of Molecular Biosciences (A.R.G., C.K.) and Department of Chemistry
(K.M.L., J.T.B., J.P.J., C.K.), Washington State University, Pullman, Washington
99164;Department of Biochemistry and Department of Energy Great Lakes Bioenergy
Research Center, University of Wisconsin, Madison, Wisconsin 53726 (F.L.,
J.R.);Department of Microbiology and Cell Science and Genetics Institute,
University of Florida, Gainesville, Florida 32610 (W.V.); andUnited States
Department of Agriculture-Agricultural Research Service, Grain Forage and
Bioenergy Research Unit, Lincoln, Nebraska 68583 (S.E.S.) chkang@wsu.edu.
Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase

from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its
preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the
mechanism of SbCOMT and understand the observed reduction in the lignin
syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene
missense mutations, we determined the apo-form and S-adenosyl-methionine binary
complex SbCOMT crystal structures and established the ternary complex structure
with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed
many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa
(Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data
suggest a random bi-bi mechanism. Based on our structural, kinetic, and
thermodynamic results, we propose that the observed reactivity hierarchy among
4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol,
and acid substrates arises from the ability of the aldehyde to stabilize the
anionic intermediate that results from deprotonation of the 5-hydroxyl group by
histidine-267. Additionally, despite the presence of other phenylpropanoid
substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by
its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates,
the aldehydes exhibit product inhibition, and we propose that this is due to
nonproductive binding of the S-cis-form of the aldehydes inhibiting productive
binding of the S-trans-form. The S-cis-aldehydes most likely act only as
inhibitors, because the high rotational energy barrier around the 2-propenyl bond
prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond
rotational energy barrier enables rapid S-cis/S-trans-interconversion.
DOI: 10.1104/pp.114.241729
PMCID: PMC4119030
PMID: 24948836
655. Int J Phytoremediation. 2014;16(7-12):1241-56.
Phytoremediation efficiency of a pcp-contaminated soil using four plant species

as mono- and mixed cultures.
Hechmi N, Aissa NB, Abdenaceur H, Jedidi N.
Bioremediation of soil polluted by pentachlorophenol (PCP) is of great importance

due to the persistence and carcinogenic properties of PCP. Phytoremediation has
long been recognized as a promising approach for removal of PCP from soil. The
present study was conducted to investigate the capability of four plant species;
white clover, ryegrass, alfalfa, and rapeseed grown alone and in combination to
remediate pentachlorophenol contaminated soil. After 60 days cultivation, white
clover, raygrass, alfalfa, and rapeseed all significantly enhanced the
degradation of PCP in soils. Alfalfa showed highest efficiency for the removal of
PCP in single cropping flowed by rapeseed and ryegrass. Mixed cropping
significantly enhanced the remediation efficiencies as compared to single
cropping; about 89.84% of PCP was removed by mixed cropping of rapeseed and
alfalfa, and 72.01% of PCP by mixed cropping of rape and white clover. Mixed
cropping of rapeseed with alfalfa was however far better for the remediation of
soil PCP than single cropping. An evaluation of soil biological activities as a
monitoring mechanism for the bioremediation process of a PCP-contaminated soil
was made using measurements of microbial counts and dehydrogenase activity.
DOI: 10.1080/15226514.2013.828009
656. Int J Phytoremediation. 2014;16(7-12):671-83.

Effects of rapeseed oil on the rhizodegradation of polyaromatic hydrocarbons in
contaminated soil.
Gartler J, Wimmer B, Soja G, Reichenauer TG.
Plants have the ability to promote degradation of polycyclic aromatic

hydrocarbons (PAHs) in contaminated soil by supporting PAH degrading
microorganisms in the rhizosphere (rhizodegradation). The aim of this study was
to evaluate if rapeseed oil increases rhizodegradation because various studies
have shown that vegetable oils are able to act as extractants for PAHs in
contaminated soils and therefore might increase bioavailability of PAHs for
microbial degradation. In this study different leguminous and grass species were
tested. The results suggested a significant impact of vegetable oil (1 and 3%
w/w) on plant growth (decrease of plant height and biomass). The results of the
pot experiment showed a decrease in the PAH content of the soil without amendment
of rapeseed oil after six months. In soil amended with 1% and 3% of oil, there
was no decrease in PAH content within this period. Although no enhancement of PAH
degradation by plants could be measured in the bulk soil of the pot experiments,
a rhizobox experiment showed a significant reduction of PAH content in the
rhizosphere of alfalfa (Medicago sativa cv. Europe). Our investigations also
showed significant differences in the degradation behaviour of the 16
individually analysed PAHs.
DOI: 10.1080/15226514.2013.856841
657. J Dairy Sci. 2014;97(8):5088-100. doi: 10.3168/jds.2013-7653. Epub 2014 Jun

13.
Effects of feeding lauric acid or coconut oil on ruminal protozoa numbers,

fermentation pattern, digestion, omasal nutrient flow, and milk production in
dairy cows.
Faciola AP(1), Broderick GA(2).
Author information:
(1)Department of Agriculture, Nutrition, and Veterinary Science, University of
Nevada, Reno 89557. Electronic address: afaciola@cabnr.unr.edu.
(2)United States Dairy Forage Research Center, Agricultural Research Service,
USDA, Madison, WI 53706.
The objectives of this study were to evaluate the feeding of coconut oil (CO), in
which lauric acid (La) comprises about 50% of the fatty acid composition, as a
practical rumen protozoa (RP) suppressing agent, to assess whether the source of
La affects ruminal fermentation and animal performance and to test whether
suppressing RP improves N utilization, nutrient digestion, nutrient flow at the
omasal canal, and milk production. Fifteen multiparous Holstein cows (3 fitted
with ruminal cannulas) and 15 primiparous Holstein cows (3 fitted with ruminal
cannulas) were used in a replicated 3×3 Latin square experiment with 14d of
adaptation and 14d of sample collection. Diets were fed as total mixed ration and
contained (dry matter basis) 10% corn silage, 50% alfalfa silage, and 40%
concentrate. The control diet contained 3% (dry matter basis) calcium soaps of
palm oil fatty acids (Megalac, Church & Dwight Co. Inc., Princeton, NJ) as a
ruminally inert fat source and had no added La or CO. Diets with La and CO were
formulated to contain equal amounts of La (1.3%, dry matter basis). Dry matter
intake was not affected by treatment. Both CO and La reduced RP numbers by about
40%. Lauric acid reduced yield of milk and milk components; however, CO did not
affect yield of milk and yields of milk components. Both La and CO caused small
reductions in total VFA concentration; CO increased molar proportion of ruminal
propionate, reduced ruminal ammonia and branched-chain volatile fatty acids,
suggesting reduced protein degradation, and reduced milk urea N and blood urea N
concentrations, suggesting improved protein efficiency. Lauric acid reduced
total-tract apparent digestibility of neutral detergent fiber and acid detergent
fiber as well as ruminal apparent digestibility of neutral detergent fiber and
acid detergent fiber as measured at the omasal canal; however, CO did not alter
fiber digestion. Microbial protein flow at the omasal canal, as well as the flow
of N fractions at the omasal canal, did not differ among treatments. Results from
this experiment have confirmed that dietary La is not a practical agent for
suppressing RP population in dairy cows, mainly because of its negative effects
on fiber digestion and ruminal fermentation. Intake of CO appeared to reduce
ruminal and improve protein efficiency, but did not improve milk production, milk
composition, or increase microbial outflow from the rumen. Based on the results
of this study, a 40% reduction of RP population is not sufficient to improve N
utilization in dairy cows.

DOI: 10.3168/jds.2013-7653
658. Int J Phytoremediation. 2014;16(6):609-20.
Plant uptake and enhanced dissipation of di(2-ethylhexyl) phthalate (DEHP) in

spiked soils by different plant species.
Li YW, Cai QY, Mo CH, Zeng QY, Lü H, Li QS, Xu GS.
This study was conducted to investigate the uptake, accumulation and the enhanced
dissipation of di(2-ethylhexyl) phthalate (DEHP) spiked in soil (with a
concentration of 117.4 +/- 5.2 mg kg(-1)) by eleven plants including eight maize
(Zea mays) cultivars and three forage species (alfalfa, ryegrass and teosinte).
The results showed that, after 40 days of treatment, the removal rates of DEHP
ranged from 66.8% (for the control) to 87.5% (for the maize cultivar of
Huanong-1). Higher removal rate was observed during the first 10 days than the
following days. Plants enhanced significantly the dissipation of DEHP in soil.
Enhanced dissipation amount in planted soil was 13.3-122 mg pot(-1) for DEHP, and
a net removal of 2.2%-20.7% of the initial DEHP was obtained compared with
non-plant soil. The contribution of plant uptake to the total enhanced
dissipation was < 0.3%, and the enhanced dissipation of soil DEHP might be
derived from plant-promoted biodegradation and sorption stronger to the soil.
Nevertheless, the capability in accumulation and enhanced dissipation of DEHP
from spiked soils varied within different species and cultivars.
DOI: 10.1080/15226514.2013.803021
Immunofluorescent localization of MAPKs in Steedman's wax sections.
Ovečka M(1), Samajová O, Baluška F, Samaj J.
Author information:
(1)Centre of the Region Haná for Biotechnological and Agricultural Research,
Faculty of Science, Palacký University Olomouc, Šlechtitelů 11, 783 71, Olomouc,
Czech Republic, miroslav.ovecka@upol.cz.
Signals of different nature are transduced in cells through signal transduction

pathways, where mitogen-activated protein kinases (MAPKs) play an important role
as signaling molecules. Views into intracellular localization of MAPKs are
critical for the understanding of their spatial and temporal functions, like
activation-based relocation, compartmentation, or interactions with local
substrates. Localization of MAPKs in cells is thus very useful cell biological
approach, extending complex mode of cell signaling characterization in plants.
Here, we present a method for subcellular immunofluorescence localization of
MAPKs using protein- or phospho-specific antibodies, performed on sectioned fixed
plant samples. It is based on embedding of samples in the Steedman's wax, a
low-melting point polyester wax embedding medium, which maintains high
antigenicity of studied proteins. In addition, exposure of dewaxed sections to
antibodies allows for their efficient penetration. Altogether, it makes this
simple method a good tool in the efficient subcellular localization of diverse
proteins, including plant MAPKs.
DOI: 10.1007/978-1-4939-0922-3_10
660. J Biotechnol. 2014 Aug 20;184:201-8. doi: 10.1016/j.jbiotec.2014.05.020. Epub

2014 Jun 4.
Expression of an evolved engineered variant of a bacterial glycine oxidase leads

to glyphosate resistance in alfalfa.
Nicolia A(1), Ferradini N(2), Molla G(3), Biagetti E(2), Pollegioni L(3),
Veronesi F(2), Rosellini D(2).
Author information:
(1)Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of
Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy. Electronic address:
alessandro.nicolia@gmail.com.
(2)Dipartimento di Scienze Agrarie, Alimentari e Ambientali, University of
Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy.
(3)Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi
dell'Insubria, via J.H. Dunant 3, 21100 Varese, Italy; Centro Interuniversitario
di Ricerca in Biotecnologie Proteiche The Protein Factory, Politecnico di Milano,
ICRM CNR Milano and Università degli studi dell'Insubria, via Mancinelli 7, 20131
Milano, Italy.
The main strategy for resistance to the herbicide glyphosate in plants is the
overexpression of an herbicide insensitive, bacterial
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). A glyphosate resistance
strategy based on the ability to degrade the herbicide can be useful to reduce
glyphosate phytotoxicity to the crops. Here we present the characterization of
glyphosate resistance in transgenic alfalfa (Medicago sativa L.) expressing a
plant-optimized variant of glycine oxidase (GO) from Bacillus subtilis, evolved
in vitro by a protein engineering approach to efficiently degrade glyphosate. Two
constructs were used, one with (GO(TP+)) and one without (GO(TP-)) the pea rbcS
plastid transit peptide. Molecular and biochemical analyses confirmed the stable
integration of the transgene and the correct localization of the plastid-imported
GO protein. Transgenic alfalfa plants were tested for glyphosate resistance both
in vitro and in vivo. Two GO(TP+) lines showed moderate resistance to the
herbicide in both conditions. Optimization of expression of this GO variant may
allow to attain sufficient field resistance to glyphosate herbicides, thus
providing a resistance strategy based on herbicide degradation.
DOI: 10.1016/j.jbiotec.2014.05.020

eCollection
2014.
Digital gene-expression profiling analysis of the cholesterol-lowering effects of

alfalfa saponin extract on laying hens.
Zhou L(1), Shi Y(1), Guo R(1), Liang M(1), Zhu X(1), Wang C(1).
Author information:
BACKGROUND: To prevent cardiovascular disease, people are advised to limit their

intake of dietary cholesterol to less than 300 mg/day. Egg consumption has been
seriously reduced because of the high levels of cholesterol. The purpose of the
present study was to evaluate the cholesterol-lowering effects of alfalfa saponin
extract (ASE) in yolk and the molecular mechanisms underlying these effects using
digital gene-expression profiling analysis. Liver and ovary tissues were isolated
from laying hens fed with ASE for RNA sequencing.
RESULTS: The cholesterol content of the yolks of eggs from hens fed 120 mg/kg ASE
declined considerably on day 60. Other groups (60, 240, 480 mg/kg ASE group) also
showed decreases, but they were not significant. Digital gene expression
generated over nine million reads per sample, producing expression data for least
12,384 genes. Among these genes, 110 genes showed greater than normal expression
in the liver and 107 genes showed greater than normal expression in the ovary.
Cholesterol 7 alpha-hydroxylase (Cyp7a1) and apolipoprotein H (Apoh), which act
in the synthesis of bile acid and cholesterol efflux, showed more expression in
the livers of hens given dietary ASE supplementation. In the ovary, levels of
very low density lipoprotein receptor (Vldlr), apolipoprotein B (Apob),
apovitellenin 1 (ApovldlII) and vitellogenin (VtgI, VtgII and VtgIII) in ovary
decreased with dietary ASE supplementation.
CONCLUSION: Transcriptome analysis revealed that the molecular mechanisms
underlying the cholesterol-lowering effects of ASE were partially mediated by
enhancement of cholesterol efflux in the liver and this reduced of cholesterol
deposition in the ovary.
PMCID: PMC4041749
662. Huan Jing Ke Xue. 2014 Mar;35(3):1125-30.
[Growth responses of six leguminous plants adaptable in Northern Shaanxi to

petroleum contaminated soil].
Shan BQ, Zhang YT, Cao QL, Kang ZY, Li SY.

To select appropriate native species in Northern Shaanxi for phytoremediation,
the growth index of six kinds of leguminous plants planted in petroleum
contaminated soils were investigated through pot culture. Petroleum
concentrations were set at 0, 5 000, 10 000, 20 000, 40 000 mg x kg(-1)
respectively with three replicates. Using different levels of seed germination
rate, germination time, individual height, wilting rate, dry weight and
chlorophyll content in leaves of tested plants as the ecological indicator. The
results showed that tested plants have significantly different responses to
petroleum pollution. Compared with those planted in clean soils, seed germination
rate and individual height were promoted when petroleum concentration was lower
than 5000 mg x kg(-1), but inhibition occurred when petroleum concentrations were
higher than 10000 mg x kg(-1). Strong endurance of Medicago sativa was observed
to petroleum polluted soil, especially at lower petroleum concentration. Leaf
wilting of Robinia pseudoacacia was unobserved even when petroleum concentration
was 40 000 mg x kg(-1), thus displaying the potential of remediating petroleum
contaminated soils. The petroleum concentration was significantly and negatively
correlated with seed germination rate, individual height and dry weight, but
positively correlated with chlorophyll content in leaves.
663. Poult Sci. 2014 Jul;93(7):1868-74. doi: 10.3382/ps.2013-03831. Epub 2014 May
26.
Low-fiber alfalfa (Medicago sativa L.) meal in the laying hen diet: effects on
productive traits and egg quality.
Laudadio V(1), Ceci E(2), Lastella NM(1), Introna M(1), Tufarelli V(3).
Author information:
(1)Department of Emergency and Organ Transplantation (DETO), Section of
Veterinary Science and Animal Production, and.
(2)Department of Veterinary Medicine, University of Bari "Aldo Moro," Valenzano
70010, Italy.
(3)Department of Emergency and Organ Transplantation (DETO), Section of
Veterinary Science and Animal Production, and vincenzo.tufarelli@uniba.it.
This study was designed to determine the effects on laying performance and egg
quality resulting from partial substitution of soybean meal (SBM) with low-fiber
alfalfa (LFA; Medicago sativa L.) meal in the diet of early-phase laying hens.
ISA Brown layers, 18 wk of age, were randomly allocated to 2 dietary treatments
and fed for 10 wk. The hens were fed 2 wheat middling-based diets: a control
diet, which contained SBM (15% of diet), and a test diet containing LFA (15% of
diet) as the main protein source. Low-fiber alfalfa meal was obtained by a
combination of sieving and air-classification processes. Feed intake was recorded
daily, and egg production was calculated on a hen-day basis; eggs from each group
were weekly collected to evaluate egg components and quality. The partial
substitution of SBM with LFA had no adverse effect on growth performance of
early-phase laying hens. Egg production and none of the egg-quality traits
examined were influenced by dietary treatment, except for yolk color (P < 0.001)
and yolk percentage (P < 0.05) as well as yolk cholesterol and β-carotene
contents (P < 0.001), which were improved in hens fed the LFA diet. Including LFA
increased serum β-carotene and reduced serum cholesterol concentrations (P <
0.001). Our results suggest that partially replacing conventional SBM as protein
source with low-fiber alfalfa meal in the laying-hen diet can positively
influence yolk quality without adversely affecting productive traits.
© 2014 Poultry Science Association Inc.
DOI: 10.3382/ps.2013-03831
664. J Med Microbiol. 2014 Aug;63(Pt 8):1076-86. doi: 10.1099/jmm.0.075291-0. Epub

2014 May 21.
New pharmacological properties of Medicago sativa and Saponaria officinalis

saponin-rich fractions addressed to Candida albicans.
Sadowska B(1), Budzyńska A(1), Więckowska-Szakiel M(1), Paszkiewicz M(1),

Stochmal A(2), Moniuszko-Szajwaj B(2), Kowalczyk M(2), Różalska B(3).
Author information:
(1)Department of Infectious Biology, Faculty of Biology and Environmental
Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland.
(2)Department of Biochemistry, Institute of Soil Science and Plant Cultivation,
State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland.
(3)Department of Infectious Biology, Faculty of Biology and Environmental
Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland
rozab@biol.uni.lodz.pl.
The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa
(aerial parts and roots) and Saponaria officinalis (used as a well-known source
of plant saponins) against Candida albicans reference and clinical strains, their
yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated.
Direct fungicidal/fungistatic properties of the tested phytochemicals used alone,
as well as their synergy with azoles (probably resulting from yeast cell wall
instability) were demonstrated. Here, to the best of our knowledge, we report for
the first time the ability of saponin-rich extracts of M. sativa and S.
officinalis to inhibit C. albicans germ tube formation, limit hyphal growth,
reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida
biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the
range of concentrations which were active modulators of Candida virulence
factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These
properties seem to be very promising in the context of using plant-derived SFs as
potential novel antifungal therapeutics supporting classic drugs or as
ingredients of disinfectants.
© 2014 The Authors.
DOI: 10.1099/jmm.0.075291-0
665. J Dairy Sci. 2014 Jul;97(7):4412-22. doi: 10.3168/jds.2013-7585. Epub 2014 May
16.
Milk production response to varying protein supply is independent of forage

digestibility in dairy cows.
Alstrup L(1), Weisbjerg MR(2), Hymøller L(2), Larsen MK(3), Lund P(2), Nielsen
MO(4).
Author information:
(1)Department of Animal Science, AU Foulum, Aarhus University, DK 8830 Tjele,
Denmark. Electronic address: Lene.Alstrup@agrsci.dk.
(2)Department of Animal Science, AU Foulum, Aarhus University, DK 8830 Tjele,
Denmark.
(3)Department of Food Science, AU Foulum, Aarhus University, DK 8830 Tjele,
Denmark.
(4)Department of Veterinary Clinical and Animal Sciences, Faculty of Health and
Medical Sciences, University of Copenhagen, DK 1870 Frederiksberg C, Denmark.
The aim of this experiment was to examine whether the positive response in milk
production to increased crude protein (CP) supply in dairy cows was dependent on
the digestibility of the forage. Forty-eight lactating Danish Holstein cows were
used in a 4 × 4 Latin square design experiment with 4 rations: (1) high
digestibility and high CP concentration (HdHp), (2) high digestibility and low CP
concentration (HdLp), (3) low digestibility and high CP concentration (LdHp), and
(4) low digestibility and low CP concentration (LdLp). All rations contained 30%
corn silage, 25% grass-clover silage, and 45% concentrate on a dry matter (DM)
basis. Different digestibilities were obtained by replacing a high-digestible
grass-clover silage combined with a high-digestible corn silage with a
low-digestible grass-clover silage combined with a low-digestible corn silage.
Organic matter digestibilities were 79.8 and 74.7% in the high- and
low-digestibility rations, respectively. Dietary CP concentration in the ration
was increased by substituting barley and sugar beet pulp with rapeseed meal and
soybean meal, whereby CP increased from 13.9 to 14.0% (Lp) to 15.7 to 16.0% (Hp).
All cows were offered 3 kg of the same concentrate per day in the automatic
milking system in addition to the mixed ration. Every feeding period lasted 3 wk,
and DM intake and milk yield were measured in the last week in each period, and
milk samples for determining milk composition, including fatty acid content, and
blood samples were taken during the last 3d of each period. Dry matter intake
increased by 2.2 kg/d on Hd compared with Ld and by 0.7 kg/d on Hp compared with
Lp. The positive effect on DM intake was reflected in the energy-corrected milk
(ECM) yield, as a higher ration digestibility increased the ECM yield by 1.7 kg/d
and a higher CP concentration increased it by 1.2 kg/d. We detected no
interaction between forage digestibility and CP concentration on milk production.
Reduced digestibility was accompanied by an increase in the plasma level of
glucose, suggesting that other nutrients were limiting to milk production. In
conclusion, milk production responses to dietary CP supply appeared independent
of forage digestibility.

DOI: 10.3168/jds.2013-7585

eCollection
2014.
The impact of using alternative forages on the nutrient value within slurry and
its implications for forage productivity in agricultural systems.
Crotty FV(1), Fychan R(1), Theobald VJ(1), Sanderson R(1), Chadwick DR(2), Marley
CL(1).
Author information:
University, Gogerddan, Aberystwyth, United Kingdom.
(2)Environment Centre Wales, School of Environment, Natural Resources and
Geography, Bangor University, Bangor, Gwynedd, United Kingdom.
Alternative forages can be used to provide valuable home-grown feed for ruminant
livestock. Utilising these different forages could affect the manure value and
the implications of incorporating these forages into farming systems, needs to be
better understood. An experiment tested the hypothesis that applying slurries
from ruminants, fed ensiled red clover (Trifolium pratense), lucerne (Medicago
sativa) or kale (Brassica oleracea) would improve the yield of hybrid ryegrass
(Lolium hybridicum), compared with applying slurries from ruminants fed ensiled
hybrid ryegrass, or applying inorganic N alone. Slurries from sheep offered one
of four silages were applied to ryegrass plots (at 35 t ha⁻¹) with 100 kg N ha⁻¹
inorganic fertiliser; dry matter (DM) yield was compared to plots only receiving
ammonium nitrate at rates of 0, 100 and 250 kg N ha⁻¹ year-1. The DM yield of
plots treated with 250 kg N, lucerne or red clover slurry was significantly
higher than other treatments (P<0.001). The estimated relative fertiliser N
equivalence (FNE) (fertiliser-N needed to produce same yield as slurry N), was
greatest for lucerne (114 kg) >red clover (81 kg) >kale (44 kg) >ryegrass (26 kg
ha⁻¹ yr⁻¹). These FNE values represent relative efficiencies of 22% (ryegrass),
52% (kale), 47% (red clover) and 60% for lucerne slurry, with the ryegrass slurry
efficiency being lowest (P = 0.005). Soil magnesium levels in plots treated with
legume slurry were higher than other treatments (P<0.001). Overall, slurries from
ruminants fed alternative ensiled forages increased soil nutrient status, forage
productivity and better N efficiency than slurries from ruminants fed ryegrass
silage. The efficiency of fertiliser use is one of the major factors influencing
the sustainability of farming systems, these findings highlight the cascade in
benefits from feeding ruminants alternative forages, and the need to ensure their
value is effectively captured to reduce environmental risks.
PMCID: PMC4022616
667. Appl Environ Microbiol. 2014 Jul;80(14):4286-93. doi: 10.1128/AEM.00597-14.

Epub
2014 May 9.
Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within

Medicago sativa nodules.
Bianco C(1), Senatore B(1), Arbucci S(2), Pieraccini G(3), Defez R(4).
Author information:
(1)Institute of Biosciences and BioResources, Naples, Italy.
(2)Institute of Genetics and Biophysics Adriano Buzzati Traverso, Naples, Italy.
(3)CISM-Centro di servizi di Spettrometria di Massa, Università degli Studi di
Firenze, Florence, Italy.
(4)Institute of Biosciences and BioResources, Naples, Italy
roberto.defez@ibbr.cnr.it.
To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on

Medicago plant growth and dry weight production, we increased the synthesis of
IAA in both free-living and symbiosis-stage rhizobial bacteroids during
Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was
applied to modify an 85-bp promoter sequence, driving the expression of iaaM and
tms2 genes for IAA biosynthesis. A positive correlation was found between the
higher expression of IAA biosynthetic genes in free-living bacteria and the
increased production of IAA under both free-living and symbiotic conditions.
Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA
concentration, showed a significant (up to 73%) increase in the shoot fresh
weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated
by the wild-type strain. When these plants were analyzed by confocal microscopy,
using an anti-IAA antibody, the strongest signal was observed in bacteroids of
Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the
senescent nodule zone. We show here a simple system to modulate endogenous IAA
biosynthesis in bacteria nodulating legumes suitable to investigate which is the
maximum level of IAA biosynthesis, resulting in the maximal increase of plant
growth.
DOI: 10.1128/AEM.00597-14
PMCID: PMC4068685
668. Physiol Plant. 2015 Jan;153(1):91-104. doi: 10.1111/ppl.12222. Epub 2014 Jun
25.
Effect of shoot removal on remobilization of carbon and nitrogen during regrowth

of nitrogen-fixing alfalfa.
Aranjuelo I(1), Molero G, Erice G, Aldasoro J, Arrese-Igor C, Nogués S.
Author information:
(1)Instituto de Agrobiotecnología (IdAB), Universidad Pública de
Navarra-CSIC-Gobierno de Navarra, ES-31192, Mutilva Baja, Spain; Unitat de
Fisologia Vegetal, Facultat de Biologia, Universitat de Barcelona, ES-08028,
Barcelona, Spain.
The contribution of carbon and nitrogen reserves to regrowth following shoot

removal has been studied in the past. However, important gaps remain in
understanding the effect of shoot cutting on nodule performance and its relevance
during regrowth. In this study, isotopic labelling was conducted at root and
canopy levels with both (15) N2 and (13) C-depleted CO2 on exclusively
nitrogen-fixing alfalfa plants. As expected, our results indicate that the roots
were the main sink organs before shoots were removed. Seven days after regrowth
the carbon and nitrogen stored in the roots was invested in shoot biomass
formation and partitioned to the nodules. The large depletion in nodule
carbohydrate availability suggests that root-derived carbon compounds were
delivered towards nodules in order to sustain respiratory activity. In addition
to the limited carbohydrate availability, the upregulation of nodule peroxidases
showed that oxidative stress was also involved during poor nodule performance.
Fourteen days after cutting, and as a consequence of the stimulated
photosynthetic and N2 -fixing machinery, availability of Cnew and Nnew strongly
diminished in the plants due to their replacement by C and N assimilated during
the post-labelling period. In summary, our study indicated that during the first
week of regrowth, root-derived C and N remobilization did not overcome C- and
N-limitation in nodules and leaves. However, 14 days after cutting, leaf and
nodule performance were re-established.
DOI: 10.1111/ppl.12222
669. Ecotoxicol Environ Saf. 2014 Jul;105:103-11. doi:

10.1016/j.ecoenv.2014.04.009.
Epub 2014 May 7.
Hydrogen-rich water confers plant tolerance to mercury toxicity in alfalfa

seedlings.
Cui W(1), Fang P(1), Zhu K(1), Mao Y(1), Gao C(1), Xie Y(1), Wang J(2), Shen
W(3).
Author information:
China.
210095, China.
In this report, the effect of hydrogen-rich water (HRW), which was used to
investigate the physiological roles of hydrogen gas (H2) in plants recently, on
the regulation of plant adaptation to mercury (Hg) toxicity was studied. Firstly,
we observed that the exposure of alfalfa seedlings to HgCl2 triggered production
of reactive oxygen species (ROS), growth stunt and increased lipid peroxidation.
However, such effects could be obviously blocked by HRW. Meanwhile, significant
decreases in the relative ion leakage and Hg accumulation were observed.
Hg-induced increases in total and isozymatic activities of superoxide dismutase
(SOD) were significantly reversed by HRW. Further results suggested that
HRW-induced the activities of guaiacol peroxidase (POD) and ascorbate peroxidase
(APX), two hydrogen peroxide-scavenging enzymes, was at transcriptional levels.
Meanwhile, obvious increases of the ratios of reduced/oxidized glutathione (GSH),
homoglutathione (hGSH), and ascorbic acid (AsA) and corresponding gene expression
were consistent with the decreased oxidative damage in seedling roots. In
summary, the results of this investigation indicated that HRW was able to
alleviate Hg toxicity in alfalfa seedlings by (i) alleviating growth stunt and
reducing Hg accumulation, and (ii) avoidance of oxidative stress and
reestablishment of redox homeostasis.
DOI: 10.1016/j.ecoenv.2014.04.009
670. AoB Plants. 2014 Apr 16;6(0). pii: plu012. doi: 10.1093/aobpla/plu012. Print
2014.
Giving drought the cold shoulder: a relationship between drought tolerance and
fall dormancy in an agriculturally important crop.
Pembleton KG(1), Sathish P.
Author information:
(1)Tasmanian Institute of Agriculture, University of Tasmania, PO Box 3523,
Burnie, TAS 7320, Australia.
The growth of fall dormant/freezing tolerant plants often surpasses the growth of
non-fall dormant/non-freezing tolerant types of the same species under
water-limited conditions, while under irrigated conditions non-fall dormant types
exhibit superior yield performance. To investigate the mechanism behind this
phenomenon, we exposed seven diverse alfalfa (Medicago sativa) cultivars to
water-limited and fully watered conditions and measured their shoot growth, shoot
water potential and gas exchange parameters and the relative abundance of taproot
RNA transcripts associated with chilling stress/freezing tolerance. Fall dormant
cultivars had greater shoot growth relative to the fully watered controls under a
mild water deficit (a cumulative water deficit of 625 mL pot(-1)) and did not
close their stomata until lower shoot water potentials compared with the more
non-fall dormant cultivars. Several gene transcripts previously associated with
freezing tolerance increased in abundance when plants were exposed to a mild
water deficit. Two transcripts, corF (encodes galactinol synthase) and cas18
(encodes a dehydrin-like protein), increased in abundance in fall dormant
cultivars only. Once water deficit stress became severe (a cumulative water
deficit of 2530 mL pot(-1)), the difference between fall dormancy groups
disappeared with the exception of the expression of a type 1 sucrose synthase
gene, which decreased in fall dormant cultivars. The specific adaptation of fall
dormant cultivars to mild water deficit conditions and the increase in abundance
of specific genes typically associated with freezing tolerance in these cultivars
is further evidence of a link between freezing tolerance/fall dormancy and
adaption to drought conditions in this species.
DOI: 10.1093/aobpla/plu012
PMCID: PMC4038438
PMID: 24790133
671. Animal. 2014 Jul;8(7):1191-200. doi: 10.1017/S1751731114000998. Epub 2014 Apr

30.
Grazing increases the concentration of CLA in dairy cow milka.
Lahlou MN(1), Kanneganti R(1), Massingill LJ(1), Broderick GA(1), Park Y(2),
Pariza MW(3), Ferguson JD(4), Wu Z(4).
Author information:
(1)1US Dairy Forage Research Center,USDA-ARS,Madison,WI 53706,USA.
(2)2Department of Food Science,University of Massachusetts,Amherst,MA 01003,USA.
(3)3Food Research Institute,University of Wisconsin,Madison,WI 53706,USA.
(4)4New Bolton Center,University of Pennsylvania,Kennett Square,PA 19348,USA.
An experiment was conducted to examine whether increased CLA in milk of dairy

cows fed fresh pasture compared with alfalfa and corn silages was because of
ruminal or endogenous synthesis. Eight Holsteins were fed a total mixed ration
using alfalfa and corn silages as the forage source in confinement or grazed in a
replicated crossover design. The proportion of total fatty acids as CLA
(primarily c9, t11-18:2) in g/100 g was 0.44 v. 0.28 in ruminal digesta, 0.89 v.
0.53 in omasal digesta and 0.71 v. 1.06 in milk during confinement feeding and
grazing, respectively. Blood plasma CLA was 0.54 v. 1.05 mg/l for the two
treatments, respectively. The increased concentration of CLA in milk with grazing
likely resulted from increased synthesis through desaturation of t11-18:1 in the
mammary gland.
DOI: 10.1017/S1751731114000998
672. Plant Cell. 2014 Apr;26(4):1818-1830. Epub 2014 Apr 29.
A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice

and Medicago truncatula.
Wang E(1), Yu N(2), Bano SA(3), Liu C(4), Miller AJ(4), Cousins D(4), Zhang X(5),
Ratet P(6), Tadege M(7), Mysore KS(7), Downie JA(4), Murray JD(4), Oldroyd GE(4),
Schultze M(3).
Author information:
(1)National Key Laboratory of Plant Molecular Genetics, Institute of Plant
Academy of Sciences, Shanghai 200032, China John Innes Centre, Norwich NR4 7UH,
United Kingdom etwang@sibs.ac.cn.
Academy of Sciences, Shanghai 200032, China John Innes Centre, Norwich NR4 7UH,
United Kingdom.
(3)Department of Biology, University of York, York YO10 5DD, United Kingdom.
(4)John Innes Centre, Norwich NR4 7UH, United Kingdom.
Academy of Sciences, Shanghai 200032, China.
(6)Institut des Sciences du Végétale, Centre National de la Recherche
Scientifique, 91198 Gif-sur-Yvette, France.
(7)Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401.
Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which
facilitate the uptake of mineral nutrients such as phosphate from the soil.
Several transporters, particularly proton-coupled phosphate transporters, have
been identified on both the plant and fungal membranes and contribute to
delivering phosphate from fungi to plants. The mechanism of nutrient exchange has
been studied in plants during mycorrhizal colonization, but the source of the
electrochemical proton gradient that drives nutrient exchange is not known. Here,
we show that plasma membrane H+-ATPases that are specifically induced in
arbuscule-containing cells are required for enhanced proton pumping activity in
membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago
truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired
nutrient uptake by the host plant through the mycorrhizal symbiosis.
Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the
plasma membrane potential, suggesting that this H+-ATPase plays a key role in
energizing the periarbuscular membrane, thereby facilitating nutrient exchange in
arbusculated plant cells.
DOI: 10.1105/tpc.113.120527
PMCID: PMC4036588
PMID: 24781115
673. Phytopathology. 2014 Nov;104(11):1241-50. doi: 10.1094/PHYTO-11-13-0309-R.
Genetic Diversity, Reassortment, and Recombination in Alfalfa mosaic virus

Population in Spain.
Bergua M, Luis-Arteaga M, Escriu F.
The variability and genetic structure of Alfalfa mosaic virus (AMV) in Spain was
evaluated through the molecular characterization of 60 isolates collected from
different hosts and different geographic areas. Analysis of nucleotide sequences
in four coding regions--P1, P2, movement protein (MP), and coat protein
(CP)--revealed a low genetic diversity and different restrictions to variation
operating on each coding region. Phylogenetic analysis of Spanish isolates along
with previously reported AMV sequences showed consistent clustering into types I
and II for P1 and types I, IIA, and IIB for MP and CP regions. No clustering was
observed for the P2 region. According to restriction fragment length polymorphism
analysis, the Spanish AMV population consisted of seven haplotypes, including two
haplotypes generated by reassortment and one involving recombination. The most
frequent haplotypes (types for P1, MP, and CP regions, respectively) were I-I-I
(37%), II-IIB-IIB (30%), and one of the reassortants, II-I-I (17%). Distribution
of haplotypes was not uniform, indicating that AMV population was structured
according to the geographic origin of isolates. Our results suggest that
agroecological factors are involved in the maintenance of AMV genetic types,
including the reassortant one, and in their geographic distribution.
DOI: 10.1094/PHYTO-11-13-0309-R
674. Ann Bot. 2014 Jun;113(7):1107-20. doi: 10.1093/aob/mcu072. Epub 2014 Apr 25.
Legume genomics: understanding biology through DNA and RNA sequencing.
O'Rourke JA(1), Bolon YT(2), Bucciarelli B(1), Vance CP(3).
Author information:
(1)United States Department of Agriculture, Agricultural Research Service,
University of Minnesota, St. Paul, MN 55108, USA Department of Agronomy and Plant
Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108,
USA.
(2)Department of Agronomy and Plant Genetics, University of Minnesota, 1991 Upper
Buford Circle, St. Paul, MN 55108, USA.
(3)United States Department of Agriculture, Agricultural Research Service,
University of Minnesota, St. Paul, MN 55108, USA Department of Agronomy and Plant
Genetics, University of Minnesota, 1991 Upper Buford Circle, St. Paul, MN 55108,
USA vance004@umn.edu.
BACKGROUND: The legume family (Leguminosae) consists of approx. 17 000 species. A

few of these species, including, but not limited to, Phaseolus vulgaris, Cicer
arietinum and Cajanus cajan, are important dietary components, providing protein
for approx. 300 million people worldwide. Additional species, including soybean
(Glycine max) and alfalfa (Medicago sativa), are important crops utilized mainly
in animal feed. In addition, legumes are important contributors to biological
nitrogen, forming symbiotic relationships with rhizobia to fix atmospheric N2 and
providing up to 30 % of available nitrogen for the next season of crops. The
application of high-throughput genomic technologies including genome sequencing
projects, genome re-sequencing (DNA-seq) and transcriptome sequencing (RNA-seq)
by the legume research community has provided major insights into genome
evolution, genomic architecture and domestication.
SCOPE AND CONCLUSIONS: This review presents an overview of the current state of
legume genomics and explores the role that next-generation sequencing
technologies play in advancing legume genomics. The adoption of next-generation
sequencing and implementation of associated bioinformatic tools has allowed
researchers to turn each species of interest into their own model organism. To
illustrate the power of next-generation sequencing, an in-depth overview of the
transcriptomes of both soybean and white lupin (Lupinus albus) is provided. The
soybean transcriptome focuses on analysing seed development in two near-isogenic
lines, examining the role of transporters, oil biosynthesis and nitrogen
utilization. The white lupin transcriptome analysis examines how phosphate
deficiency alters gene expression patterns, inducing the formation of cluster
roots. Such studies illustrate the power of next-generation sequencing and
bioinformatic analyses in elucidating the gene networks underlying biological
processes.
© The Author 2014. Published by Oxford University Press on behalf of the Annals
of Botany Company. All rights reserved. For Permissions, please email:
DOI: 10.1093/aob/mcu072
PMCID: PMC4030821
675. Plant Sci. 2014 Jun;223:16-24. doi: 10.1016/j.plantsci.2014.02.005. Epub 2014

Feb
25.
Involvement of abscisic acid in the response of Medicago sativa plants in

symbiosis with Sinorhizobium meliloti to salinity.
Palma F(1), López-Gómez M(2), Tejera NA(1), Lluch C(1).
Author information:
(1)Departamento de Fisiología Vegetal, Facultad de Ciencias, Universidad de
Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain.
Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain. Electronic address:
mlgomez@ugr.es.
Legumes are classified as salt-sensitive crops with their productivity

particularly affected by salinity. Abcisic acid (ABA) plays an important role in
the response to environmental stresses as signal molecule which led us to study
its role in the response of nitrogen fixation and antioxidant metabolism in root
nodules of Medicago sativa under salt stress conditions. Adult plants inoculated
with Sinorhizobium meliloti were treated with 1 μM and 10 μM ABA two days before
200 mM salt addition. Exogenous ABA together with the salt treatment provoked a
strong induction of the ABA content in the nodular tissue which alleviated the
inhibition induced by salinity in the plant growth and nitrogen fixation.
Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione
reductase (GR) were induced by ABA pre-treatments under salt stress conditions
which together with the reduction of the lipid peroxidation, suggest a role for
ABA as signal molecule in the activation of the nodular antioxidant metabolism.
Interaction between ABA and polyamines (PAs), described as anti-stress molecules,
was studied being detected an induction of the common polyamines spermidine (Spd)
and spermine (Spm) levels by ABA under salt stress conditions. In conclusion, ABA
pre-treatment improved the nitrogen fixation capacity under salt stress
conditions by the induction of the nodular antioxidant defenses which may be
mediated by the common PAs Spd and Spm that seems to be involved in the
anti-stress response induced by ABA.
DOI: 10.1016/j.plantsci.2014.02.005
676. BMC Biotechnol. 2014 Apr 26;14:31. doi: 10.1186/1472-6750-14-31.
Formulation of enzyme blends to maximize the hydrolysis of alkaline peroxide

pretreated alfalfa hay and barley straw by rumen enzymes and commercial
cellulases.
Badhan A, Wang Y, Gruninger R, Patton D, Powlowski J, Tsang A, McAllister T(1).
Author information:
(1)Agriculture and Agri food Canada, Lethbridge research Centre, Lethbridge,
Alberta, Canada. Tim.McAllister@AGR.GC.CA.
BACKGROUND: Efficient conversion of lignocellulosic biomass to fermentable sugars

requires the synergistic action of multiple enzymes; consequently enzyme mixtures
must be properly formulated for effective hydrolysis. The nature of an optimal
enzyme blends depends on the type of pretreatment employed as well the
characteristics of the substrate. In this study, statistical experimental design
was used to develop mixtures of recombinant glycosyl hydrolases from thermophilic
and anaerobic fungi that enhanced the digestion of alkaline peroxide treated
alfalfa hay and barley straw by mixed rumen enzymes as well as commercial
cellulases (Accelerase 1500, A1500; Accelerase XC, AXC).
RESULTS: Combinations of feruloyl and acetyl xylan esterases (FAE1a;
AXE16A_ASPNG), endoglucanase GH7 (EGL7A_THITE) and polygalacturonase
(PGA28A_ASPNG) with rumen enzymes improved straw digestion. Inclusion of
pectinase (PGA28A_ASPNG), endoxylanase (XYN11A_THITE), feruloyl esterase (FAE1a)
and β-glucosidase (E-BGLUC) with A1500 or endoglucanase GH7 (EGL7A_THITE) and
β-xylosidase (E-BXSRB) with AXC increased glucose release from alfalfa hay.
Glucose yield from straw was improved when FAE1a and endoglucanase GH7
(EGL7A_THITE) were added to A1500, while FAE1a and AXE16A_ASPNG enhanced the
activity of AXC on straw. Xylose release from alfalfa hay was augmented by
supplementing A1500 with E-BGLUC, or AXC with EGL7A_THITE and XYN11A_THITE.
Adding arabinofuranosidase (ABF54B_ASPNG) and esterases (AXE16A_ASPNG;
AXE16B_ASPNG) to A1500, or FAE1a and AXE16A_ASPNG to AXC enhanced xylose release
from barley straw, a response confirmed in a scaled up assay.
CONCLUSION: The efficacy of commercial enzyme mixtures as well as mixed enzymes
from the rumen was improved through formulation with synergetic recombinant
enzymes. This approach reliably identified supplemental enzymes that enhanced
sugar release from alkaline pretreated alfalfa hay and barley straw.
DOI: 10.1186/1472-6750-14-31
PMCID: PMC4022426
677. Environ Entomol. 2014 Apr;43(2):363-9. doi: 10.1603/EN13128.
Spatial density and movement of the Lygus spp. parasitoid Peristenus relictus
(Hymenoptera: Braconidae) in organic strawberries with alfalfa trap crops.
Swezey SL(1), Nieto DJ, Pickett CH, Hagler JR, Bryer JA, Machtley SA.
Author information:
(1)Center for Agroecology and Sustainable Food Systems, University of California,
1156 High St., Santa Cruz, CA 95064, USA.
Alfalfa trap crops are currently used to manage Lygus spp. in organic strawberry
fields on the California Central Coast. The retention of Lygus spp. in alfalfa
creates aggregated distributions that provide improved opportunities for
biological control by the introduced parasitoid Peristenus relictus (Ruthe). The
abundance and distribution of P. relictus between two trap crops separated by 50
strawberry rows were analyzed in 2008 and 2010. Parasitism of Lygus spp. nymphs
by P. relictus (measured by larval abundance and % parasitism) was greatest in
alfalfa trap crops compared with strawberry rows. A significantly positive
correlation between host nymphs and P. relictus larvae in and between trap crops
was found. Movement of P. relictus adults from a marked alfalfa trap crop into
adjacent strawberry rows or trap crops was also studied in 2008 and 2009 using a
chicken egg-albumin enzyme-linked immunosorbent assay mark-capture technique. In
2008 and 2009, 85 and 49% of protein-marked wasps were captured from central trap
crops, respectively, indicating that alfalfa trap crops act as a concentrated
"host-density anchor" in organic strawberry fields.
DOI: 10.1603/EN13128

eCollection
2014.
Assessment of the soil organic carbon sink in a project for the conversion of
farmland to forestland: a case study in Zichang county, Shaanxi, China.
Mu L(1), Liang Y(1), Han R(1).
Author information:
Shaanxi Province, China; Institute of Soil and Water Conservation, Chinese
Academy of Sciences and Ministry of Water Resources, Yangling, Shaanxi Province,
China.
The conversion of farmland to forestland not only changes the ecological

environment but also enriches the soil with organic matter and affects the global
carbon cycle. This paper reviews the influence of land use changes on the soil
organic carbon sink to determine whether the Chinese "Grain-for-Green"
(conversion of farmland to forestland) project increased the rate of SOC content
during its implementation between 1999 and 2010 in the hilly and gully areas of
the Loess Plateau in north-central China. The carbon sink was quantified, and the
effects of the main species were assessed. The carbon sink increased from
2.26×106 kg in 1999 to 8.32×106 kg in 2010 with the sustainable growth of the
converted areas. The black locust (Robinia pseudoacacia L.) and alfalfa (Medicago
sativa L.) soil increased SOC content in the top soil (0-100 cm) in the initial
7-yr period, while the sequestration occurred later (>7 yr) in the 100-120 cm
layer after the "Grain-for-Green" project was implemented. The carbon sink
function measured for the afforested land provides evidence that the
Grain-for-Green project has successfully excavated the carbon sink potential of
the Shaanxi province and served as an important milestone for establishing an
effective organic carbon management program.
PMCID: PMC3988087

eCollection
2014.
Investigating the toxicity, uptake, nanoparticle formation and genetic response

of plants to gold.
Taylor AF(1), Rylott EL(1), Anderson CW(2), Bruce NC(1).
Author information:
(1)Centre for Novel Agricultural Products, Department of Biology, University of
York, York, United Kingdom.
(2)Institute of Agriculture and Environment, Massey University, Palmerston North,
New Zealand.
We have studied the physiological and genetic responses of Arabidopsis thaliana

L. (Arabidopsis) to gold. The root lengths of Arabidopsis seedlings grown on
nutrient agar plates containing 100 mg/L gold were reduced by 75%. Oxidized gold
was subsequently found in roots and shoots of these plants, but gold
nanoparticles (reduced gold) were only observed in the root tissues. We used a
microarray-based study to monitor the expression of candidate genes involved in
metal uptake and transport in Arabidopsis upon gold exposure. There was
up-regulation of genes involved in plant stress response such as glutathione
transferases, cytochromes P450, glucosyl transferases and peroxidases. In
parallel, our data show the significant down-regulation of a discreet number of
genes encoding proteins involved in the transport of copper, cadmium, iron and
nickel ions, along with aquaporins, which bind to gold. We used Medicago sativa
L. (alfalfa) to study nanoparticle uptake from hydroponic culture using ionic
gold as a non-nanoparticle control and concluded that nanoparticles between 5 and
100 nm in diameter are not directly accumulated by plants. Gold nanoparticles
were only observed in plants exposed to ionic gold in solution. Together, we
believe our results imply that gold is taken up by the plant predominantly as an
ionic form, and that plants respond to gold exposure by up-regulating genes for
plant stress and down-regulating specific metal transporters to reduce gold
uptake.
PMCID: PMC3988041
680. J Dairy Sci. 2014;97(6):3790-9. doi: 10.3168/jds.2013-7398. Epub 2014 Apr 14.
Effects of ethyl-3-nitrooxy propionate and 3-nitrooxypropanol on ruminal

fermentation, microbial abundance, and methane emissions in sheep.
Martínez-Fernández G(1), Abecia L(1), Arco A(1), Cantalapiedra-Hijar G(2),

Martín-García AI(1), Molina-Alcaide E(1), Kindermann M(3), Duval S(4), Yáñez-Ruiz
DR(5).
Author information:
(1)Estación Experimental del Zaidín (Consejo Superior de Investigaciones
Científicas), Camino del Jueves s/n, 18100 Armilla, Granada, Spain.
Científicas), Camino del Jueves s/n, 18100 Armilla, Granada, Spain; Institut
National de la Recherche Agronomique, UMR 1213, Unité Mixte de Recherches sur les
Herbivores, Theix,63122 St Genès Champanelle, France.
(3)DSM Nutritional Products, PO Box 2676, Bldg. 241/858, CH-4002 Basel,
Switzerland.
(4)DSM Nutritional Products, Research Centre for Animal Nutrition and Health, BP
170, F-68305 Saint-Louis Cedex, France.
Científicas), Camino del Jueves s/n, 18100 Armilla, Granada, Spain. Electronic
address: david.yanez@eez.csic.es.
The aim of this work was to investigate the effect of feeding ethyl-3-nitrooxy
propionate (E3NP) and 3-nitrooxypropanol (3 NP), 2 recently developed compounds
with potential antimethanogenic activity, in vitro and in vivo in nonlactating
sheep on ruminal methane production, fermentation pattern, the abundance of major
microbial groups, and feed degradability. Three experiments were conducted, 1 in
vitro and 2 in vivo. The in vitro batch culture trial (experiment 1) tested 2
doses of E3NP and 3 NP (40 and 80 μL/L), which showed a substantial reduction of
methane production (up to 95%) without affecting concentration of volatile fatty
acids (VFA). The 2 in vivo trials were conducted over 16 d (experiment 2) and 30
d (experiment 3) to study their effects in sheep. In experiment 2, 6 adult
nonpregnant sheep, with permanent rumen cannula and fed alfalfa hay and oats
(60:40), were treated with E3NP at 2 doses (50 and 500 mg/animal per day). After
7, 14, and 15 d of treatment, methane emissions were recorded in respiration
chambers and rumen fluid samples were collected for VFA analysis and
quantification of bacterial, protozoal, and archaeal numbers by real-time PCR.
Methane production decreased by 29% compared with the control with the higher
dose of E3NP on d 14 to 15. A decrease in the acetate:propionate ratio was
observed without detrimental effects on dry matter intake. In experiment 3, 9
adult nonpregnant sheep, with permanent rumen cannula and fed with alfalfa hay
and oats (60:40), were treated with E3NP or 3 NP at one dose (100mg/animal per
day) over 30 d. On d 14 and d 29 to 30, methane emissions were recorded in
respiration chambers. Rumen fluid samples were collected on d 29 and 30 for VFA
analysis and quantification of bacterial, protozoal, and archaeal numbers by
real-time PCR. In addition, on d 22 and 23, samples of oats and alfalfa hay were
incubated in the rumen of sheep to determine dry matter ruminal degradation over
24 and 48 h, respectively; no effect was observed (78.6, 78.3, and 78.8% of
alfalfa and 74.2, 74.0, and 70.6% of oats in control, E3NP, and 3 NP groups,
respectively). A reduction in methane production was observed for both additives
at d 14 and d 29 to 30. In both treatments, the acetate:propionate ratio was
significantly decreased. Likewise, total concentrations of the analyzed microbial
groups in the rumen showed no difference among treatments and doses for both
experiments. Both tested compounds showed promise as methane inhibitors in the
rumen, with no detrimental effects on fermentation or intake, which would need to
be confirmed in lactating animals.

DOI: 10.3168/jds.2013-7398
681. Rev Argent Microbiol. 2014 Jan-Mar;46(1):49-52. doi:

10.1016/S0325-7541(14)70048-3.
Natural occurrence of entomophthoroid fungi of aphid pests on Medicago sativa L.

in Argentina.
Manfrino RG(1), Zumoffen L(2), Salto CE(3), Lastra CC(4).
Author information:
(1)Instituto Nacional de Tecnología Agropecuaria (INTA), Área Investigación
Agronomía, Protección Vegetal, Rafaela, Santa Fe, Argentina; CONICET, INTA,
Rafaela, Santa Fe, Argentina. Electronic address: manfrino.romina@inta.gob.ar.
Agronomía, Protección Vegetal, Rafaela, Santa Fe, Argentina; CONICET, INTA,
Rafaela, Santa Fe, Argentina.
Agronomía, Protección Vegetal, Rafaela, Santa Fe, Argentina.
(4)Centro de Estudios Parasitológicos y de Vectores (CEPAVE), UNLP-CONICET, La
Plata, Buenos Aires, Argentina.
Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales:

Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae),
Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites
fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora,
Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of
Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites
(Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province
of Santa Fe. In this study, Zoophthora radicans was the most important pathogen
and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully
isolated and maintained in pure cultures. This study is the first report of
entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina.
Copyright © 2014 Asociación Colombiana de Psiquiatría. Publicado por Elsevier

España. All rights reserved.
DOI: 10.1016/S0325-7541(14)70048-3
682. Toxicol Pathol. 2015 Apr;43(3):435-48. doi: 10.1177/0192623314526318. Epub

2014
Apr 8.
Genistein protects female nonobese diabetic mice from developing type 1 diabetes
when fed a soy- and alfalfa-free diet.
Guo TL(1), Germolec DR(2), Zheng JF(3), Kooistra L(4), Auttachoat W(3), Smith
MJ(3), White KL(3), Elmore SA(2).
Author information:
(1)Department of Pharmacology and Toxicology, Virginia Commonwealth University,
Richmond, Virginia, USA tlguo1@uga.edu.
(2)Division of the National Toxicology Program, NIEHS, Research Triangle Park,
North Carolina, USA.
(3)Department of Pharmacology and Toxicology, Virginia Commonwealth University,
Richmond, Virginia, USA.
(4)Charles River Laboratories, Durham, North Carolina, USA.
The objective of this study was to determine the effects of the phytoestrogen
genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes
(T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage
once every day for up to 180 days. Five groups of mice (approximately 24
animals/group; 6-7 weeks of age) were included: naive control, vehicle control
(25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20
mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the
study and were monitored for blood glucose changes every week. When compared to
the vehicle control, exposure to 2-mg/kg GEN produced significant decreases
ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250
mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the
study. However, during the later stages of the study (i.e., after week 23), the
2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg
and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were
observed starting at week 16, while the incidence of severe diabetes was
significantly decreased with the changes being observed initially at weeks 18 and
17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines
of evidence, including histopathological analysis, suggested that GEN protected
the pancreas from autoimmune destruction. However, this protective effect of GEN
was absent when female NOD mice were maintained on NTP-2000 rodent diet, which
contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of
phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN
reduced the incidence and increased the time to onset of T1D in female NOD mice
but only when fed a soy- and alfalfa-free diet.
© 2014 by The Author(s).
DOI: 10.1177/0192623314526318
PMCID: PMC4190109
683. Environ Technol. 2014 May-Jun;35(9-12):1077-90.
Methylene blue biosorption by pericarp of corn, alfalfa, and agave bagasse

wastes.
Rosas-Castor JM, Garza-González MT, García-Reyes RB, Soto-Regalado E,

Cerino-Córdova FJ, García-González A, Loredo-Medrano JA.
The presence of dyes in effluent is a matter of concern due to their toxicologic

and aesthetical effects. In this research, locally available agro-industrial
wastes (Zea mays pericarp, ZMP; Agave tequilana bagasse, ATB; and Medicago sativa
waste, MSW) were used as alternative low-cost adsorbents for the removal of
methylene blue (MB) from aqueous solutions. The adsorbents were characterized
physically and chemically by Fourier transform infrared, scanning electron
microscopy, potentiometric titrations, and N2 physisorption. MB adsorption
experiments were carried out in batch systems and experimental data were used to
calculate the adsorption isotherm model parameters (Langmuir, Freundlich, and
Temkin) and the adsorption kinetic model parameters (pseudo-first- and
pseudo-second-order models). MB-loaded biosorbents were desorbed with deionized
water, ethanol (10% and 50% v/v), hydrochloric acid (0.01 and 0.05 N), and sodium
hydroxide (0.1 N) at room temperature, and the best eluent was used in various
adsorption-desorption cycles. The selected agricultural wastes can be considered
as promising adsorbents for dye uptake from water since they exhibit considerable
MB adsorption capacity (MSW 202.6 mg g(-1), ATB 156.2mg g(-1), and ZMP 110.9mg
g(-1)), but it is lower than that reported for activated carbon; however, the
biosorbents show higher adsorption rate than powdered activated carbon.
Furthermore, the adsorbents can be economically regenerated with HCl solutions
and reused for seven adsorption-desorption cycles.
DOI: 10.1080/09593330.2013.861022
684. Ecology. 2014 Feb;95(2):486-94.
Species interactions and a chain of indirect effects driven by reduced

precipitation.
Barton BT, Ives AR.
Climate change can affect species directly and indirectly by altering

interactions between species within communities. These indirect effects can
ramify through a community and affect many species, including some that may not
have been directly affected by the perturbation. Identifying these chains of
indirect effects is difficult, and most studies only follow indirect effects
across two or three species. Here, we use a factorial field experiment to
demonstrate that precipitation affects spotted aphids through a complex chain of
indirect interactions that are mediated by other herbivores and a generalist
predator. We experimentally simulated drought, which reduced water content in
alfalfa plants. While water stress in alfalfa had no direct effect on spotted
aphids, it lowered the population growth rate of pea aphids, another common
alfalfa pest. Because ladybeetle predators were attracted to high pea aphid
densities, predator densities were lower in drought treatments. Consequently,
spotted aphid densities were released from top-down control (apparent
competition) in drought treatments and reached densities three times higher than
spotted aphids in ambient treatments with high pea aphid densities. Thus, drought
affected spotted aphids in the interaction chain: drought --> alfalfa --> pea
aphids --> predators --> spotted aphids. This result illustrates the lengthy path
that indirect effects of climate change may take through a community, as well as
the importance of community-level experiments in determining the net effect of
climate change.
685. Genet Mol Res. 2014 Mar 17;13(1):1893-904. doi: 10.4238/2014.March.17.17.
Characterization of the β-1,3-glucanase gene in peanut (Arachis hypogaea L.) by

cloning and genetic transformation.
Qiao LX(1), Ding X(1), Wang HC(1), Sui JM(1), Wang JS(2).
Author information:
(1)Key Lab of Plant Biotechnology in Universities of Shandong, College of Life
Science, Qingdao Agriculture University, Qingdao, China.
(2)Key Lab of Plant Biotechnology in Universities of Shandong, College of Life
Science, Qingdao Agriculture University, Qingdao, China jswang319@126.com.
Plant β-1,3-glucanases are commonly involved in disease resistance. This report

describes the cloning and genetic transformation of a β-1,3-glucanase gene from
peanut. The gene was isolated from both the genomic DNA and cDNA of peanut
variety Huayu20 by polymerase chain reaction (PCR) and reverse transcription PCR
(RT-PCR), respectively. The DNA sequence contained 1471 bp including two exons
and one intron, and the coding sequence contained 1047 bp that coded for a
348-amino acid protein with a calculated molecular weight of 38.8 kDa. The
sequence was registered in NCBI (GenBank accession No. JQ801335) and was
designated as Ah-Glu. As determined by BLAST analysis, the Ah-Glu protein has
42-90% homology with proteins from Oryza sativa (BAC83070.1), Zea mays
(NP_001149308), Arabidopsis thaliana (NP_200470.1), Medicago sativa (ABD91577.1),
and Glycine max (XP_003530515.1). The over-expression vector pCAMBIA1301-Glu
containing Ah-Glu was constructed, confirmed by PCR and restriction enzyme
digestion, and transformed into peanut variety Huayu22 by Agrobacterium
EHA105-mediated transformation. The putative transformed plants (T0) were
confirmed by PCR amplification. RT-PCR analysis and β-glucuronidase (GUS)
staining showed that the transferred Ah-Glu was expressed as mRNA and protein. In
a laboratory test, the transgenic plants were found to be more resistant to the
fungal pathogen Cercospora personata than the non-transgenic plants were.
DOI: 10.4238/2014.March.17.17
686. J Anim Sci. 2014 Feb;92(2):830-5. doi: 10.2527/jas.2013-6768. Epub 2014 Jan
14.
Effects of residual feed intake classification and method of alfalfa processing

on ewe intake and growth.
Redden RR(1), Surber LM, Grove AV, Kott RW.
Author information:
(1)Department of Animal Science, North Dakota State University, Fargo 58108.
The objective of this research was to evaluate the effects of residual feed
intake (RFI) determined under ad libitum feeding conditions on DMI and
performance of yearling ewes fed either chopped or pelleted alfalfa hay. In Exp.
1, 45 ewe-lambs had ad libitum access to a pelleted grower diet for 63 d and
individual DMI was determined using an electronic feed delivery system. Residual
feed intake values were assigned to each ewe-lamb as a measure of feed
efficiency. Sixteen ewe-lambs with the most positive RFI values were classified
as high RFI (inefficient) and 16 ewe-lambs with the most negative RFI values were
classified as low RFI (efficient). In Exp. 2, half of the ewes from each
efficiency group were placed into 1 of 2 pens and provided ad libitum access to
either pelleted or chopped alfalfa hay. Individual DMI was again determined using
an electronic feed delivery system. Body weight, LM area (LMA), and 12th-rib back
fat thickness (BF) were measured at the beginning and end of both experiments. In
Exp. 1, DMI by ewe-lambs in the low RFI group was 9% less (P = 0.01) than by
ewe-lambs in the high RFI group (2.21 vs. 2.43 kg/d); however, ADG and initial
and final BW, LMA, and BF did not differ (P > 0.27) among RFI groups. In Exp. 2,
there were no feed processing × RFI group interactions (P > 0.14) for any trait.
By design, RFI values were lower (P < 0.01) by yearling ewes in the low than high
RFI group (-0.27 vs. 0.27); however, RFI values did not differ (P = 1.0) between
yearling ewes fed chopped versus pelleted alfalfa. Dry matter intake was 22% less
(P < 0.01) by yearling ewes in the low than high RFI group (2.5 vs. 3.2 kg/d) and
59% less (P < 0.01) by yearling ewes fed chopped versus pelleted alfalfa (2.2 vs.
3.5 kg/d). Initial and final BW, ADG, and G:F did not differ (P > 0.45) between
RFI groups but were greater (P < 0.01) by yearling ewes fed pelleted alfalfa
compared to chopped alfalfa. Final LMA did not differ (P = 0.77) between RFI
groups, but final BF tended to be greater (P = 0.06) for high than low RFI
yearling ewes (0.63 vs. 0.57 cm). Final LMA and BF were greater (P < 0.01) by
yearling ewes fed pelleted than chopped alfalfa. Low RFI ewes had lower DMI and
BF compared to high RFI ewes. Ewe efficiency as determined by RFI was repeatable
on subsequent forage based diets; however, differences in intake and efficiency
were more apparent when alfalfa was pelleted.
DOI: 10.2527/jas.2013-6768
687. J Anim Sci. 2014 Apr;92(4):1621-9. doi: 10.2527/jas.2013-6740. Epub 2014 Mar
18.
Comparative digestibility by cattle versus sheep: effect of forage quality.
Soto-Navarro SA(1), Lopez R, Sankey C, Capitan BM, Holland BP, Balstad LA,
Krehbiel CR.
Author information:
(1)Department of Animal and Range Sciences, New Mexico State University, Las
Cruces 88003.
The objective was to determine the effect of forage quality on apparent total
tract digestibility and ruminal fermentation in cattle versus sheep. Five
yearling English crossbred (Hereford × Angus) steers (440.4 ± 35.6 kg of initial
BW) and 5 yearling whiteface (Rambouillet × Columbia × Debouillet) wethers (44.4
± 4.6 kg of initial BW), each fitted with a ruminal cannula, were randomly
assigned to 1 of 3 forage sources within ruminant specie, and the study was
conducted over 3 periods. For forage source, both animal and period served as the
blocking factor with all forage sources represented once within each animal and
all forage sources represented at least once within each period. The treatment
structure was arranged in a 2 × 3 factorial with ruminant species (2) and forage
source (3) as the factors. Forage sources were 1) alfalfa hay (Medicago sativa;
17.5% CP and 34.1% NDF, DM basis), 2) warm-season grass hay mix (Bothriochloa
ischaemum and Cynodon dactylon; 7.3% CP and 74.7% NDF, DM basis), and 3)
lovegrass hay (Eragrostis curvula; 2.5% CP and 81.9% NDF, DM basis). As a percent
of BW, steers and wethers consumed similar (P ≤ 0.06) amounts of forage, and
intake was more influenced by forage quality (P < 0.001) than ruminant species (P
= 0.35). When expressed per unit of metabolic BW, cattle consumed more (P <
0.001) DM, NDF, and N than sheep. Apparent total tract digestibility was similar
among steers and wethers when alfalfa or grass hay was fed, but decreased to a
greater extent in wethers when low-quality lovegrass hay was fed (ruminant
species × diet interaction, P ≤ 0.01). Rate (%/h) of ruminal NDF disappearance
was greater (P = 0.02) for alfalfa and grass hay than lovegrass, but was not
influenced (P = 0.12) by ruminant species. In addition, ruminal DM fill was
influenced more (P < 0.01) by forage than by ruminant species (P = 0.07). Steers
and wethers had greater (P < 0.01) DM fill from grass hay and lovegrass hay than
alfalfa before and 5 h after feeding. Ruminal VFA were generally not influenced
(P ≥ 0.06) by ruminant specie. Results suggest that apparent total tract
digestibilities are more similar among ruminant species when moderate- to
high-quality forages are evaluated. However, sheep are not an adequate model for
cattle when low-quality forages are compared because cattle digest low-quality
forages to a greater extent than sheep. Expressing digestibility as digestible
intake per unit of BW allows for a wider range of forage qualities to be compared
when substituting sheep for cattle.
DOI: 10.2527/jas.2013-6740
688. Microbiology. 2014 Jun;160(Pt 6):1237-51. doi: 10.1099/mic.0.079137-0. Epub

2014
Mar 24.
Functional diversity of five homologous Cu+-ATPases present in Sinorhizobium

meliloti.
Patel SJ(1), Padilla-Benavides T(1), Collins JM(1), Argüello JM(2).
Author information:
(1)Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100
Institute Road, Worcester, MA 01609, USA.
(2)Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100
Institute Road, Worcester, MA 01609, USA arguello@wpi.edu.
Copper is an important element in host-microbe interactions, acting both as a

catalyst in enzymes and as a potential toxin. Cu(+)-ATPases drive cytoplasmic
Cu(+) efflux and protect bacteria against metal overload. Many pathogenic and
symbiotic bacteria contain multiple Cu(+)-ATPase genes within particular genetic
environments, suggesting alternative roles for each resulting protein. This
hypothesis was tested by characterizing five homologous Cu(+)-ATPases present in
the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to
different phenotypes and abnormal nodule development in the alfalfa host.
Distinct responses were detected in free-living S. meliloti mutant strains
exposed to metal and redox stresses. Differential gene expression was detected
under Cu(+), oxygen or nitrosative stress. These observations suggest that CopA1a
maintains the cytoplasmic Cu(+) quota and its expression is controlled by Cu(+)
levels. CopA1b is also regulated by Cu(+) concentrations and is required during
symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are
necessary for the assembly of two different cytochrome c oxidases at different
stages of bacterial life. CopA3 is a phylogenetically distinct Cu(+)-ATPase that
does not contribute to Cu(+) tolerance. It is regulated by redox stress and
required during symbiosis. We postulated a model where non-redundant homologous
Cu(+)-ATPases, operating under distinct regulation, transport Cu(+) to different
target proteins.
© 2014 The Authors.
DOI: 10.1099/mic.0.079137-0
689. J Anim Physiol Anim Nutr (Berl). 2014 Dec;98(6):1110-6. doi:

10.1111/jpn.12185.
Epub 2014 Mar 24.
Effects of grain source and marginal change in lucerne hay particle size on feed
sorting, eating behaviour, chewing activity, and milk production in mid-lactation
Holstein dairy cows.
Nasrollahi SM(1), Ghorbani GR, Khorvash M, Yang WZ.
Author information:
(1)Department of Animal Sciences, Isfahan University of Technology, Isfahan,
Iran.
This study investigated the effects of grain source and lucerne hay (LH) particle
length on eating behaviour, chewing activity, and milk production of lactating
dairy cows. Eight Holstein dairy cows (175 ± 21 days in milk) were used in a
replicated 4 × 4 Latin square design with four 21-days periods. The experiment
was a 2 × 2 factorial arrangement of treatments with barley grain alone or equal
blend of barley and maize grains combined with short (15 mm) and long (30 mm) LH.
Diets were fed ad libitum as total mixed ration with a concentrate to forage
ratio of 60:40. Interactions between grain source and LH particle length on feed
particle distributions, sorting index, chewing activity, and milk production were
minimal. Partially replacing barley grain with maize in the diet overall did not
change diurnal distributions of particles retained on the sieves of Penn State
Particle Separator but reduced the proportion of particles on 1.18-sieve and
increased that of particles on pan (p < 0.05). Grain source did not affect
sorting index and chewing activity. However, feeding long LH increased (p < 0.01)
intakes of long particles retained on 19- and 8-mm of sieve, prolonged (p < 0.05)
eating time, and lowered eating rate (p < 0.05). Interestingly, cows fed with
long LH ate more coarse particle during critical-early time post feeding (i. e.
1.5 h), where eating time increased and eating rate decreased (p < 0.05).
Increasing particle length of dietary LH tended to increase milk fat-to-protein
ratio (p = 0.08). The results suggested that the increased eating time and
decreased eating rate as a result of marginally increasing LH particle length
would be beneficial to alleviate reduction of ruminal pH and milk fat percentage
following the ingestion of highly fermentable diets.
DOI: 10.1111/jpn.12185
690. Appl Environ Microbiol. 2014 Jun;80(11):3404-15. doi: 10.1128/AEM.00115-14.
Epub
2014 Mar 21.
Sinorhizobium meliloti chemoreceptor McpU mediates chemotaxis toward host plant

exudates through direct proline sensing.
Webb BA(1), Hildreth S, Helm RF, Scharf BE.
Author information:
(1)Virginia Tech, Department of Biological Sciences, Blacksburg, Virginia, USA.
Bacterial chemotaxis is an important attribute that aids in establishing

symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a
spectrum of molecules into the soil to attract their bacterial symbionts. The
alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense
its environment and mediate chemotaxis toward its host. The methyl accepting
chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors
and an important sensor for the potent attractant proline. We established a
dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass
spectrometry analysis determined that a single germinating seed exudes 3.72 nmol
of proline, producing a millimolar concentration near the seed surface which can
be detected by the chemosensory system of S. meliloti. Complementation analysis
of the mcpU deletion strain verified McpU as the key proline sensor. A
structure-based homology search identified tandem Cache (calcium channels and
chemotaxis receptors) domains in the periplasmic region of McpU. Conserved
residues Asp-155 and Asp-182 of the N-terminal Cache domain were determined to be
important for proline sensing by evaluating mutant strains in capillary and swim
plate assays. Differential scanning fluorimetry revealed interaction of the
isolated periplasmic region of McpU (McpU40-284) with proline and the importance
of Asp-182 in this interaction. Using isothermal titration calorimetry, we
determined that proline binds with a Kd (dissociation constant) of 104 μM to
McpU40-284, while binding was abolished when Asp-182 was substituted by Glu. Our
results show that McpU is mediating chemotaxis toward host plants by direct
proline sensing.
DOI: 10.1128/AEM.00115-14
PMCID: PMC4018860
691. Biotechnol Biofuels. 2014 Mar 20;7(1):39. doi: 10.1186/1754-6834-7-39.
Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall

digestibility.
Badhan A, Jin L, Wang Y, Han S, Kowalczys K, Brown DC, Ayala CJ, Latoszek-Green
M, Miki B, Tsang A, McAllister T(1).
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge Research Centre, 5403 1st Avenue
South, Lethbridge, AB T1J 4B1, Canada. tim.mcallister@agr.gc.ca.
BACKGROUND: Alfalfa (Medicago sativa) is an important forage crop in North

America owing to its high biomass production, perennial nature and ability to fix
nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell
walls and has the potential to further improve alfalfa as biomass for biofuel
production.
RESULTS: In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and
sub-cloned into a novel pEACH vector containing different signaling peptides to
target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast,
endoplasmic reticulum and vacuole. Four constructs harboring faeB were
transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels
in all target sites, except chloroplast. Stable transformed lines of alfalfa were
subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136
transgenic plants regenerated, 18 independent lines exhibited FAEB activity.
Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy
(FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell
wall morphology and composition with a reduction in ester linkages and elevated
lignin content. Consequently, they were more recalcitrant to digestion by mixed
ruminal microorganisms. Interestingly, delignification by alkaline peroxide
treatment followed by exposure to a commercial cellulase mixture resulted in
higher glucose release from transgenic lines as compared to the control line.
CONCLUSION: Modifying cell wall crosslinking has the potential to lower
recalcitrance of holocellulose, but also exhibited unintended consequences on
alfalfa cell wall digestibility due to elevated lignin content. The combination
of efficient delignification treatment (alkaline peroxide) and transgenic
esterase activity complement each other towards efficient and effective digestion
of transgenic lines.
DOI: 10.1186/1754-6834-7-39
PMCID: PMC3999942
PMID: 24650274
692. J Exp Bot. 2014 Jun;65(9):2335-50. doi: 10.1093/jxb/eru115. Epub 2014 Mar 19.
Salt-induced subcellular kinase relocation and seedling susceptibility caused by

overexpression of Medicago SIMKK in Arabidopsis.
Ovečka M(1), Takáč T(1), Komis G(1), Vadovič P(1), Bekešová S(1), Doskočilová
A(1), Šamajová V(1), Luptovčiak I(1), Samajová O(1), Schweighofer A(2), Meskiene
I(2), Jonak C(3), Křenek P(1), Lichtscheidl I(4), Škultéty L(5), Hirt H(6), Šamaj
J(7).
Author information:
Department of Cell Biology, Faculty of Science, Palacký University, Šlechtitelů
11, 783 71 Olomouc, Czech Republic.
(2)Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Dr
Bohr-Gasse 9, A-1030 Vienna, Austria.
(3)Gregor Mendel Institute of Molecular Plant Biology GmbH, Dr Bohr-Gasse 3,
A-1030 Vienna, Austria.
(4)Institution of Cell Imaging and Ultrastructure Research, University of Vienna,
Althanstrasse 14, A-1090 Vienna, Austria.
(5)Department of Rickettsiology, Institute of Virology, Slovak Academy of
Sciences, Dubravska cesta 9, Bratislava, 845 05, Slovakia.
(6)Unité de Recherche en Genomique Végétale, Université d'Evry-Val-d'essone, 2,
rue Gaston Crémieux, F-91057 Evry, France.
Department of Cell Biology, Faculty of Science, Palacký University, Šlechtitelů
11, 783 71 Olomouc, Czech Republic jozef.samaj@upol.cz.
Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the

immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY
motif within the activation loop of MAPKs, allowing them to interact with and
regulate multiple substrates. Often, the activation of MAPKs triggers their
nuclear translocation. However, the spatiotemporal dynamics and the physiological
consequences of the activation of MAPKs, particularly in plants, are still poorly
understood. Here, we studied the activation and localization of the Medicago
sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the
inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus.
Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK
and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic
shuttling of SIMK correlated temporally with the dual phosphorylation of the
pTEpY motif. SIMKK function was further studied in Arabidopsis plants
overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants
showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt
treatment and exhibited high sensitivity against salt stress at the seedling
stage, although they were salt insensitive during seed germination. Proteomic
analysis of SIMKK-YFP overexpressors indicated the differential regulation of
proteins directly or indirectly involved in salt stress responses. These proteins
included catalase, peroxiredoxin, glutathione S-transferase, nucleoside
diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and
finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings
overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their
proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt
response pathway.
for Experimental Biology.
DOI: 10.1093/jxb/eru115
PMCID: PMC4036504

eCollection
2014.
Development and characterization of simple sequence repeat (SSR) markers based on

RNA-sequencing of Medicago sativa and in silico mapping onto the M. truncatula
genome.
Wang Z(1), Yu G(2), Shi B(1), Wang X(1), Qiang H(1), Gao H(1).
Author information:
Beijing, China.
Beijing, China; Department of Grassland Science, College of Animal Science and
Technology, Northwest A&F University, Yangling, Shaanxi, China.
Erratum in
PLoS One. 2014;9(7):e103682.
Sufficient codominant genetic markers are needed for various genetic

investigations in alfalfa since the species is an outcrossing autotetraploid.
With the newly developed next generation sequencing technology, a large amount of
transcribed sequences of alfalfa have been generated and are available for
identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant
unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology.
Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide
repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT
(19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent
top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer
pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear
and scored PCR products and 372 (70.6%) exhibited polymorphisms. High
transferability was observed for ssp falcata at 99.2% (523) and 71.7% (378) in M.
truncatula. In addition, 313 of 527 SSR marker sequences were in silico mapped
onto the eight M. truncatula chromosomes. Thirty-six polymorphic SSR primer pairs
were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated
accessions generating a total of 199 scored alleles. The mean observed
heterozygosity and polymorphic information content were 0.767 and 0.635,
respectively. The codominant markers not only enriched the current resources of
molecular markers in alfalfa, but also would facilitate targeted investigations
in marker-trait association, QTL mapping, and genetic diversity analysis in
alfalfa.
PMCID: PMC3958398
694. Sci Total Environ. 2014 Jun 1;482-483:36-41. doi:

The sky is falling II: Impact of deposition produced during the static testing of
solid rocket motors on corn and alfalfa.
Doucette WJ(1), Mendenhall S(2), McNeill LS(3), Heavilin J(3).
Author information:
(1)Utah Water Research Laboratory, Utah State University, 8200 Old Main Hill,
Logan, UT 84322, USA. Electronic address: william.doucette@usu.edu.
(2)Molzen-Corbin, 2701 Miles Road SE, Albuquerque, NM 87106, USA.
(3)Utah Water Research Laboratory, Utah State University, 8200 Old Main Hill,
Logan, UT 84322, USA.
Tests of horizontally restrained rocket motors at the ATK facility in Promontory,

Utah, USA result in the deposition of an estimated 1.5million kg of entrained
soil and combustion products (mainly aluminum oxide, gaseous hydrogen chloride
and water) on the surrounding area. The deposition is referred to as test fire
soil (TFS). Farmers observing TFS deposited on their crops expressed concerns
regarding the impact of this material. To address these concerns, we exposed corn
and alfalfa to TFS collected during a September 2009 test. The impact was
evaluated by comparing the growth and tissue composition of controls relative to
the treatments. Exposure to TFS, containing elevated levels of chloride (1000
times) and aluminum (2 times) relative to native soils, affected the germination,
growth and tissue concentrations of various elements, depending on the type and
level of exposure. Germination was inhibited by high concentrations of TFS in
soil, but the impact was reduced if the TFS was pre-leached with water. Biomass
production was reduced in the TFS amended soils and corn grown in TFS amended
soils did not develop kernels. Chloride concentrations in corn and alfalfa grown
in TFS amended soils were two orders of magnitude greater than controls. TFS
exposed plants contained higher concentrations of several cations, although the
concentrations were well below livestock feed recommendations. Foliar
applications of TFS had no impact on biomass, but some differences in the
elemental composition of leaves relative to controls were observed. Washing the
TFS off the leaves lessened the impact. Results indicate that the TFS deposition
could have an effect, depending on the amount and growth stage of the crops, but
the impact could be mitigated with rainfall or the application of additional
irrigation water. The high level of chloride associated with the TFS is the main
cause of the observed impacts.
DOI: 10.1016/j.scitotenv.2014.02.089
695. J Dairy Sci. 2014 May;97(5):3061-72. doi: 10.3168/jds.2013-7604. Epub 2014 Mar
13.
Diurnal variations of progesterone, testosterone, and

androsta-1,4-diene-3,17-dione in the rumen and in vitro progesterone
transformation by mixed rumen microorganisms of lactating dairy cows.
Ren QC(1), Yang HJ(2), Li SL(1), Wang JQ(3).
Author information:
Technology, China Agricultural University, Beijing 100193, PR China.
Technology, China Agricultural University, Beijing 100193, PR China. Electronic
address: yang_hongjian@sina.com.
(3)State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese
Academy of Agricultural Sciences, Beijing 100193, PR China.
Five Holstein lactating dairy cows fed 5 total mixed rations (TMR) with different
forage combinations were used in a 5 × 5 Latin square design to investigate
diurnal variations of progesterone (P4), testosterone, and
androsta-1,4-diene-3,17-dione (ADD) concentrations in the rumen. Meanwhile,
different P4 inclusion levels [0 (control), 2, 20, 40, 80, and 100 ng/mL in
culture fluids] were incubated in vitro for 6, 12, 24, 36, 48, and 72 h together
with rumen mixed microorganisms grown on a maize-rich feed mixture (maize
meal:Chinese ryegrass hay = :1) with an aim to determine microbial P4
transformation into testosterone and ADD. Ruminal P4, testosterone, and ADD
concentrations of lactating dairy cows were greater in the TMR with forage
combination of corn silage plus alfalfa hay or Chinese wild ryegrass hay than the
TMR with the corn stover-based forage combination. The diurnal fluctuation
pattern showed that P4, testosterone, and ADD concentrations in the rumen were
greater at nighttime than daytime and peaked 6h after feeding in the morning or
afternoon. The in vitro batch cultures showed that the P4 elimination rate was
highest at the P4 addition of 20 ng/mL and declined with the further increased
addition of P4. The treatments after dosing P4 exhibited a shorter time than the
control group until half of the initial P4 inclusion was eliminated (i.e., half
time), and the lowest half time (1.46 h) occurred at the P4 addition of 20 ng/mL.
In summary, the ruminal steroids concentration was affected by forage type and
quality, and the rumen microorganisms exhibited great ability to transform P4
into testosterone and ADD, depending on incubation time and initial P4 addition
level, suggesting that the host might affect the metabolism of its rumen
microorganisms via the endogenous steroids.

DOI: 10.3168/jds.2013-7604
696. Nutr Cancer. 2014;66(3):483-91. doi: 10.1080/01635581.2014.884228. Epub 2014

Mar
14.
Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in

sensitive and multidrug-resistant tumor cells.
Gatouillat G(1), Magid AA, Bertin E, Okiemy-Akeli MG, Morjani H, Lavaud C,

Madoulet C.
Author information:
(1)a Laboratoire de Biochimie et Biologie Moléculaire , Faculté de Pharmacie,
URCA , Reims , France.
Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments.
However, only a few studies have reported its anticancer effects. In this study,
extracts were obtained from alfalfa leaves and their cytotoxic effects were
assessed on several sensitive and multidrug-resistant tumor cells lines. Using
the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart
(P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf
extracts was mediated through the induction of apoptosis, as evidenced by DNA
fragmentation analysis. The execution of programmed cell death was achieved via
the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene
extract (To-1), the most active extract obtained from crude extract, led to the
identification of 3 terpene derivatives and 5 flavonoids. Among them,
(-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed
cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate
that alfalfa leaf extract may have interesting potential in cancer
chemoprevention and therapy.
DOI: 10.1080/01635581.2014.884228
697. J Sci Food Agric. 2014 Nov;94(14):3030-7. doi: 10.1002/jsfa.6651. Epub 2014
Apr
16.
Composted organic wastes from the pharmaceutical and agro-food industries induce
soil bioactivity and nodulation in alfalfa.
Fornes F(1), Jaramillo CX, García-de-la-Fuente R, Belda RM, Lidón A.
Author information:
(1)Grupo RESIAGRI, Instituto Agroforestal Mediterráneo, Universitat Politècnica
de València, Camino de Vera s/n, E-46020, Valencia, Spain.
BACKGROUND: Environmentally friendly agriculture needs to reduce the use of

synthetic fertilizers and to reclaim nutrients from organic wastes. In this study
the effect of five doses (0, 12, 24, 48 and 96 t ha(-1) ) of two two-phase olive
mill waste (TPOMW)-based composts on the bioactivity and chemical characteristics
of an agricultural soil and their potential to fertilize alfalfa (Medicago
sativa) and stimulate nodulation were assessed during a two-year incubation
experiment. The two composts were prepared either with the olive mill waste alone
(compost A), which served as control, or mixed with a liquid fatty-proteinaceous
hydrolyzate waste (FPH) from the pharmaceutical industry (compost AH).
RESULTS: Compost AH resulted in greater N immobilization than compost A because
the former supplied the soil with easily degradable C and N, which increased
microbial biomass and activity. Both compost mineralizations during the first
year of incubation supplied the soil with more nutrients (mainly N), more so with
A than with AH. Nevertheless, plant growth was similar in soils amended with
either A or AH. Both composts induced nodulation similarly and the highest dose
(96 t ha(-1) ) increased the formation of nodules by a factor of 11 compared with
the four lower doses.
CONCLUSION: TPOMW serves as an effective ground material for co-composting with
liquid wastes such as FPH. TPOMW supplies key nutrients and stimulates nodulation
in alfalfa.
DOI: 10.1002/jsfa.6651
698. Lett Appl Microbiol. 2014 Jul;59(1):79-85. doi: 10.1111/lam.12247. Epub 2014
Apr
4.
Metabolomics analysis reveals large effect of roughage types on rumen microbial

metabolic profile in dairy cows.
Zhao S(1), Zhao J, Bu D, Sun P, Wang J, Dong Z.
Author information:
(1)State Key Laboratory of Microbial Resources, Institute of Microbiology,
Chinese Academy of Sciences, Beijing, China; State Key Laboratory of Animal
Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural
Sciences, Beijing, China.
The aim of our study was to determine the effect of diets with different types of
roughage on the ruminal microbial metabolite profile in dairy cows. Holstein
dairy cows were fed a diet containing either corn stover (CS group) or a mixture
of alfalfa hay, Leymus chinensis hay and corn silage (MF group) at 0700 and 1900
h daily. Rumen fluid was sampled from each cow through a ruminal cannula at 0630
and 1030 h, and the mixed ruminal fluid from 3 day in each cow was analysed using
nuclear magnetic resonance (NMR) spectroscopy. A multivariate analysis revealed a
significant difference between the ruminal metabolome of the CS and MF groups at
both time points. The MF group had higher levels of acetate, valerate,
hydrocinnamate and methylamine and lower levels of glucose, glycine, propionate
and isovalerate than those in the CS group. Our results showed that different
types of roughages can significantly influence the ruminal microbial metabolome,
especially with regard to organic acids, amines and amino acids.SIGNIFICANCE AND
IMPACT OF THE STUDY: The microbial metabolites in the rumen provide nutritional
precursors that are critical for general health and milk production in dairy
cows. However, studies of the effect of diet on ruminal microbial metabolism are
scant. In our current study, we analysed the ruminal microbial metabolite profile
of cows fed different types of roughage. We found that the ruminal microbial
metabolite profile of cows fed a mixed-roughage diet differed significantly from
that of cows fed a single type of roughage. Certain metabolites, such as acetate,
hydrocinnamate and methylamine, were closely correlated with specific types of
roughage. Our findings provide insight into the effects of different roughages on
ruminal microbial fermentation in dairy cows.
DOI: 10.1111/lam.12247
699. Bioinformation. 2014 Feb 19;10(2):101-4. doi: 10.6026/97320630010101.

eCollection
2014.
Study on Folate Binding Domain of Dihydrofolate Reductase in Different Plant

species and Human beings.
Samanta A(1), Datta AK(1), Datta S(2).
Author information:
(1)Department of Botany, Cytogenetics, Genetics and Plant Breeding Section,
Kalyani University, Kalyani-741235, West Bengal, India.
(2)Department of Biotechnology, Haldia Institute of Technology, Haldia-721657,
West Bengal, India.
Data base (NCBI and TIGR) searches are made to retrieve protein sequences of
different plant species namely Medicago truncatula, Pisum sativum, Ricinus
communis, Arabidopsis thaliana, Vitis vinifera, Glycine max, Daucus carota, Oryza
sativa Japonica Group, Arabidopsis lyrata subsp. lyrata, Brachypodium distachyon,
Oryza sativa Indica Group, Zea mays and careful alignment of derived sequences
shows 95% or higher identity. Similarly, DHFR sequence of human being is also
retrieved from NCBI. A phylogenetic tree is constructed from different plant and
human DHFR domain using the Neighbour - Joining method in MEGA 5.05. Conservation
score is performed by using PARALINE. Result suggests that folate binding domain
of dihydrofolare reductase is conserved (score 8.06) and excepting some minor
variations the basic structure of the domain in both plant species and human
being is rather similar. Human DHFR domain contains PEKN sequence near active
site, though proline is common for all the selected organisms but the other
sequences are different in plants. The plant domain is always associated with TS
(Thymidylate synthase). Plant based system is predicted to be an effective model
for assessment of MTX (Methotrexate) and other antifolate drugs.
DOI: 10.6026/97320630010101
PMCID: PMC3937584
PMID: 24616563
5.
Responses of late-lactation cows to forage substitutes in low-forage diets

supplemented with by-products.
Hall MB(1), Chase LE(2).
Author information:
(1)US Dairy Forage Research Center, USDA Agricultural Research Service (ARS),
Madison, WI 53706. Electronic address: marybeth.hall@ars.usda.gov.
(2)Department of Animal Science, Cornell University, Ithaca, NY 14853.
In response to drought-induced forage shortages along with increased corn and soy
prices, this study was conducted to evaluate lactation responses of dairy cows to
lower-forage diets supplemented with forage substitutes. By-product feeds were
used to completely replace corn grain and soybean feeds. Forty-eight
late-lactation cows were assigned to 1 of 4 diets using a randomized complete
block design with a 2-wk covariate period followed by a 4-wk experimental period.
The covariate diet contained corn grain, soybean meal, and 61% forage.
Experimental diets contained chopped wheat straw (WS)/sugar beet pulp at 0/12,
3/9, 6/6, or 9/3 percentages of diet dry matter (DM). Corn silage (20%), alfalfa
silage (20%), pelleted corn gluten feed (25.5%), distillers grains (8%), whole
cottonseed (5%), cane molasses/whey blend (7%), and vitamin and mineral mix with
monensin (2.5%) comprised the rest of diet DM. The WS/sugar beet pulp diets
averaged 16.5% crude protein, 35% neutral detergent fiber, and 11% starch (DM
basis). Cows consuming the experimental diets maintained a 3.5% fat- and
protein-corrected milk production (35.2 kg; standard deviation=5.6 kg) that was
numerically similar to that measured in the covariate period (35.3 kg; standard
deviation=5.0 kg). Intakes of DM and crude protein declined linearly as WS
increased, whereas neutral detergent fiber intake increased linearly. Linear
increases in time spent ruminating (from 409 to 502 min/d) and eating (from 156
to 223 min/d) were noted as WS inclusion increased. Yields of milk fat and 3.5%
fat-and protein-corrected milk did not change as WS increased, but those of
protein and lactose declined linearly. Phosphorous intakes were in excess of
recommended levels and decreased linearly with increasing WS inclusion.
Nutritional model predictions for multiparous cows were closest to actual
performance for the National Research Council 2001 model when a metabolizable
protein basis was used; primiparous cow performance was better predicted by
energy-based predictions made with the National Research Council or Cornell Net
Carbohydrate and Protein System models. Model predictions of performance showed a
quadratic diet effect with increasing WS. Lactating dairy cows maintained
production on low-forage diets that included forage substitutes, and in which
by-product feeds fully replaced corn grain and soybean. However, longer-term
studies are needed to evaluate animal performance and to improve model
predictions of performance on these nontraditional diets.

DOI: 10.3168/jds.2013-7539
Integration and Expression of gfp in the plastid of Medicago sativa L.
Xing S(1), Wei Z, Wang Y, Liu Y, Lin C.
Author information:
(1)Agro-Biotechnology Research Institute, Jilin Academy of Agricultural Sciences,
Changchun, P. R. China.
Here we describe a protocol of alfalfa (Medicago sativa L.) plastid

transformation by which gfp, a gene encoding the green fluorescent protein (GFP),
is inserted into plastid genome via particle bombardment and homoplastomic plant
is obtained. Plastid engineering is likely to make a significant contribution to
the genetic improvement of this crop and the production of vaccines and
therapeutic proteins.
DOI: 10.1007/978-1-62703-995-6_25
702. J Dairy Res. 2014 May;81(2):183-92. doi: 10.1017/S0022029914000065. Epub 2014

Mar
5.
Effects of dietary cottonseed oil and tannin supplements on protein and fatty
acid composition of bovine milk.
Aprianita A(1), Donkor ON(1), Moate PJ(2), Williams SR(2), Auldist MJ(2),
Greenwood JS(2), Hannah MC(2), Wales WJ(2), Vasiljevic T(1).
Author information:
(1)Advanced Food Systems Research Unit, College of Health and Biomedicine,
Victoria University, Werribee Campus, Victoria 3030, Australia.
(2)Department of Environment and Primary Industries, Future Farming Systems
Research Division, Ellinbank, Victoria 3821, Australia.
This experiment was conducted to determine the effects of diets supplemented with
cottonseed oil, Acacia mearnsii-condensed tannin extract, and a combination of
both on composition of bovine milk. Treatment diets included addition of
cottonseed oil (800 g/d; CSO), condensed tannin from Acacia mearnsii (400 g/d;
TAN) or a combination of cottonseed oil (800 g/d) and condensed tannin (400 g/d;
CPT) with a diet consisting of 6·0 kg dry matter (DM) of concentrates and alfalfa
hay ad libitum, which also served as the control diet (CON). Relative to the CON
diet, feeding CSO and CPT diets had a minor impact on feed intake and yield of
lactose in milk. These diets increased yields of milk and protein in milk. In
contrast to the TAN diet, the CSO and CPT diets significantly decreased milk fat
concentration and altered milk fatty acid composition by decreasing the
proportion of saturated fatty acids but increasing proportions of monounsaturated
and polyunsaturated fatty acids. The CPT diet had a similar effect to the CSO
diet in modifying fatty acid profile. Overall, reduction in milk fat
concentration and changes in milk fatty acid profile were probably due to
supplementation of linoleic acid-rich cottonseed oil. The TAN diet had no effect
on feed intake, milk yield and milk protein concentration. However, a reduction
in the yields of protein and lactose occurred when cows were fed this diet.
Supplemented tannin had no significant effect on fat concentration and changes in
fatty acid profile in milk. All supplemented diets did not affect protein
concentration or composition, nitrogen concentration, or casein to total protein
ratio of the resulting milk.
DOI: 10.1017/S0022029914000065

10.1094/MPMI-09-13-0284-R.
The Sinorhizobium meliloti EmrR regulator is required for efficient colonization

of Medicago sativa root nodules.
Santos MR, Marques AT, Becker JD, Moreira LM.
The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse

conditions encountered during its symbiosis with leguminous plants. We
characterized a new symbiotically relevant gene, emrR (SMc03169), whose product
belongs to the TetR family of repressors and is divergently transcribed from
emrAB genes encoding a putative major facilitator superfamily-type efflux pump.
An emrR deletion mutant produced more succinoglycan, displayed increased
cell-wall permeability, and exhibited higher tolerance to heat shock. It also
showed lower tolerance to acidic conditions, a reduced production of
siderophores, and lower motility and biofilm formation. The simultaneous deletion
of emrA and emrR genes restored the mentioned traits to the wild-type phenotype,
except for survival under heat shock, which was lower than that displayed by the
wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR
mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules
and competed poorly with the wild-type strain for nodule colonization. Expression
profiling of the ΔemrR mutant showed decreased expression of genes involved in
Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of
genes directing the biosynthesis of succinoglycan and other polysaccharides were
increased. EmrR may therefore be involved in a regulatory network targeting
membrane and cell wall modifications in preparation for colonization of root
hairs during symbiosis.
DOI: 10.1094/MPMI-09-13-0284-R

eCollection
2014.
Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant

alkaline stress tolerance and methionine content.
Sun M(1), Sun X(1), Zhao Y(1), Zhao C(1), Duanmu H(1), Yu Y(1), Ji W(1), Zhu
Y(1).
Author information:
(1)Key Laboratory of Agricultural Biological Functional Gene, Northeast
Agricultural University, Harbin, P.R. China.
So far, it has been suggested that phosphoenolpyruvate carboxylases (PEPCs) and

PEPC kinases (PPCKs) fulfill several important non-photosynthetic functions.
However, the biological functions of soybean PPCKs, especially in alkali stress
response, are not yet well known. In previous studies, we constructed a Glycine
soja transcriptional profile, and identified three PPCK genes (GsPPCK1, GsPPCK2
and GsPPCK3) as potential alkali stress responsive genes. In this study, we
confirmed the induced expression of GsPPCK3 under alkali stress and investigated
its tissue expression specificity by using quantitative real-time PCR analysis.
Then we ectopically expressed GsPPCK3 in Medicago sativa and found that GsPPCK3
overexpression improved plant alkali tolerance, as evidenced by lower levels of
relative ion leakage and MDA content and higher levels of chlorophyll content and
root activity. In this respect, we further co-transformed the GsPPCK3 and SCMRP
genes into alfalfa, and demonstrated the increased alkali tolerance of
GsPPCK3-SCMRP transgenic lines. Further investigation revealed that GsPPCK3-SCMRP
co-overexpression promoted the PEPC activity, net photosynthetic rate and citric
acid content of transgenic alfalfa under alkali stress. Moreover, we also
observed the up-regulated expression of PEPC, CS (citrate synthase), H(+)-ATPase
and NADP-ME genes in GsPPCK3-SCMRP transgenic alfalfa under alkali stress. As
expected, we demonstrated that GsPPCK3-SCMRP transgenic lines displayed higher
methionine content than wild type alfalfa. Taken together, results presented in
this study supported the positive role of GsPPCK3 in plant response to alkali
stress, and provided an effective way to simultaneously improve plant alkaline
tolerance and methionine content, at least in legume crops.
PMCID: PMC3934933
705. Nanoscale Res Lett. 2014 Mar 2;9(1):101. doi: 10.1186/1556-276X-9-101.
In vivo synthesis of nanomaterials in plants: location of silver nanoparticles

and plant metabolism.
Marchiol L(1), Mattiello A, Pošćić F, Giordano C, Musetti R.

Author information:
(1)Department of Agriculture and Environmental Sciences, University of Udine, via
delle Scienze 206, Udine 33100, Italy. marchiol@uniud.it.
Metallic nanoparticles (MeNPs) can be formed in living plants by reduction of the

metal ions absorbed as soluble salts. It is very likely that plant metabolism has
an important role in MeNP biosynthesis. The in vivo formation of silver
nanoparticles (AgNPs) was observed in Brassica juncea, Festuca rubra and Medicago
sativa. Plants were grown in Hoagland's solution for 30 days and then exposed for
24 h to a solution of 1,000 ppm AgNO3. In the leaf extracts of control plants,
the concentrations of glucose, fructose, ascorbic acid, citric acid and total
polyphenols were determined. Total Ag content in plant fractions was determined
by inductively coupled plasma atomic emission spectroscopy. Despite the short
exposure time, the Ag uptake and translocation to plant leaves was very high,
reaching 6,156 and 2,459 mg kg-1 in B. juncea and F. rubra, respectively.
Ultrastructural analysis was performed by transmission electron microscopy (TEM),
and AgNPs were detected by TEM X-ray microanalysis. TEM images of plant fractions
showed the in vivo formation of AgNPs in the roots, stems and leaves of the
plants. In the roots, AgNPs were present in the cortical parenchymal cells, on
the cell wall of the xylem vessels and in regions corresponding to the pits. In
leaf tissues, AgNPs of different sizes and shapes were located close to the cell
wall, as well as in the cytoplasm and within chloroplasts. AgNPs were not
observed in the phloem of the three plant species. This is the first report of
AgNP synthesis in living plants of F. rubra. The contents of reducing sugars and
antioxidant compounds, proposed as being involved in the biosynthesis of AgNPs,
were quite different between the species, thus suggesting that it is unlikely
that a single substance is responsible for this process. MSC 2010: 92 Biology and
other natural sciences; 92Cxx Physiological, cellular and medical topics; 92C80
Plant biology.
DOI: 10.1186/1556-276X-9-101
PMCID: PMC3973990
PMID: 24581279
706. J Dairy Res. 2014 May;81(2):156-63. doi: 10.1017/S002202991400003X. Epub 2014

Feb
24.
Influence of different systems for feeding supplements to grazing dairy cows on

milk fatty acid composition.
Akbaridoust G(1), Plozza T(2), Trenerry VC(2), Wales WJ(3), Auldist MJ(3),
Dunshea FR(1), Ajlouni S(1).
Author information:
(1)Department of Agriculture and Food Systems, Melbourne School of Land and
Environment, The University of Melbourne, Victoria 3010, Australia.
(2)Future Farming Systems Research Division, Department of Primary Industries,
621 Sneydes Road, Werribee, Victoria 3030, Australia.
1301 Hazeldean Road, Ellinbank, Victoria 3820, Australia.
This study investigated the effects of different strategies for feeding

supplements to grazing dairy cows on the proportions of fatty acids in milk. Two
hundred and sixteen cows were fed supplementary grain and forage according to one
of 3 different strategies; (1) CONTROL: cows grazed perennial ryegrass pasture
(14 kg dry matter/d) supplemented with milled barley grain fed in the milking
parlour and pasture silage offered in the paddock; (2) Partial mixed ration 1
(PMR1): same pasture allotment and supplement as CONTROL strategy, but the
supplements presented as a mixed ration after each milking in feedpad, and; (3)
Partial mixed ration 2 (PMR2): same pasture allotment, supplemented with a mixed
ration of milled barley grain, alfalfa hay, corn silage and crushed corn grain
fed in a feedpad. Within each strategy, cows were assigned to receive either 6,
8, 10 or 12 kg dry matter supplement/cow per d. Milk fatty acid proportions from
cows fed CONTROL and PMR1 strategies were similar and different from those fed
PMR2, particularly at 10 to 12 kg dry matter supplement/cow per d. The reduction
in milk fat yield and concentration in cows fed high amounts of supplement as
CONTROL and PMR1 was coincident with 4 × increase in 10t-18:1 proportion. The
composition of the partial mixed ration (PMR) and the amount offered affected
milk fatty acid proportions and milk fat content, however, the method of
supplementation did not.
DOI: 10.1017/S002202991400003X
707. Mol Genet Genomics. 2014 Jun;289(3):457-68. doi: 10.1007/s00438-014-0823-7.

Epub
2014 Feb 21.
In silico identification of transcription factors in Medicago sativa using

available transcriptomic resources.
Postnikova OA(1), Shao J, Nemchinov LG.
Author information:
(1)Molecular Plant Pathology Laboratory, Beltsville Agricultural Research Center,
United States Department of Agriculture, 10300 Baltimore Avenue, Beltsville, MD,
20705, USA.
Transcription factors (TFs) are proteins that govern organismal development and
response to the environment by regulating gene expression. Information on the
amount and diversity of TFs within individual plant species is critical for
understanding of their biological roles and evolutionary history across the plant
kingdom. Currently, only scattered information on separate TFs is available for
alfalfa, the most extensively cultivated forage legume in the world. In the
meantime, several large transcriptomic resources that can be used to identify and
characterize alfalfa TF genes are freely accessible online. In this study, we
have performed an in silico analysis of transcriptome data generated in our
laboratory and publicly acquirable from other sources to reveal and systematize
alfalfa transcription factors. Transcriptome-wide mining enabled prediction of
983 TFs along with their sequence features and putative phylogenies of the
largest families. All data were assembled into a simple open-access database
named AlfalfaTFDB ( http://plantpathology.ba.ars.usda.gov/alfalfatfdb.html ).
Transcriptomic analysis used in this work represents an effective approach for
the identification of TF genes in plants with incomplete genomes, such as
alfalfa. Integrated TF repertoires of Medicago sativa will provide an important
tool for studying regulation of gene expression in other complex non-model
species of agricultural significance.
DOI: 10.1007/s00438-014-0823-7
708. J Biotechnol. 2014 Apr 20;176:40-1. doi: 10.1016/j.jbiotec.2014.02.008. Epub

2014
Feb 17.
Genome sequence of the acid-tolerant strain Rhizobium sp. LPU83.
Wibberg D(1), Tejerizo GT(2), Del Papa MF(2), Martini C(2), Pühler A(1), Lagares
A(2), Schlüter A(3), Pistorio M(2).
Author information:
(1)Center for Biotechnology (CeBiTec), Bielefeld University, Institute for Genome
Research and Systems Biology, D-33615 Bielefeld, Germany.
Nacional de La Plata, Calles 47 y 115, 1900 La Plata, Argentina.
Research and Systems Biology, D-33615 Bielefeld, Germany. Electronic address:
aschluet@CeBiTec.Uni-Bielefeld.DE.
Rhizobia are important members of the soil microbiome since they enter into
nitrogen-fixing symbiosis with different legume host plants. Rhizobium sp. LPU83
is an acid-tolerant Rhizobium strain featuring a broad-host-range. However, it is
ineffective in nitrogen fixation. Here, the improved draft genome sequence of
this strain is reported. Genome sequence information provides the basis for
analysis of its acid tolerance, symbiotic properties and taxonomic
classification.
DOI: 10.1016/j.jbiotec.2014.02.008
709. Guang Pu Xue Yu Guang Pu Fen Xi. 2013 Nov;33(11):3010-3.
[Alfalfa quality evaluation in the field by near-infrared reflectance

spectroscopy].
Xu RX(1), Li DN(2), Yang DH(2), Lin JH(3), Xiang M(3), Zhang YJ(3).
Author information:
China. hsuruixuan520@foxmail.com
(2)Maosheng Grass Co., Ltd. of Ningxia Land Reclamation, Yinchuan 750023, China.
China.
To explore the feasibility of using near-infrared reflectance spectroscopy (NIRS)

to evaluate alfalfa quality rapidly in the field and try to find the appropriate
machine and sample preparation method, the representative population of 170 fresh
alfalfa samples collected from different regions with different stages and
different cuts were scanned by a portable NIRS spectrometer (1 100 - 1 800 nm).
This is the first time to build models of fresh alfalfa to rapidly estimate
quality in the field for harvesting in time. The calibrations of dry matter (DM),
crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF)
were developed through the partial least squares regression (PLS). The
determination coefficients of cross-validation (R2((CV)) were 0.831 4, 0.597 9,
0.803 6, 0.786 1 for DM, CP, NDF, ADF, respectively; the root mean standard error
of cross-validation (RMSECV) were 1.241 1, 0.261 4, 0.990 3, 0.830 6; The
determination coefficients of validation (R2(V)) were 0.815 0, 0.401 1, 0.784 9,
0.752 1 and the root mean standard errors of validation(RMSEP)were 1.06, 0.31,
0.95, 0.80 for DM, CP, NDF, ADF, respectively. For fresh alfalfa ,the calibration
of DM, NDF, ADF can do rough quantitative analysis but the CP's calibration is
failed. however, as CP in alfalfa hay is enough for animal and the DM, NDF and
ADF is the crucial indicator for evaluating havest time, the model of DM, NDF and
ADF can be used for evaluating the alfalfa quality rapidly in the field.

eCollection
2014.
Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes

substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago
sativa L.).
Tong Z(1), Xie C(1), Ma L(1), Liu L(1), Jin Y(1), Dong J(1), Wang T(1).
Author information:
China Agricultural University, Beijing, China.
Erratum in
PLoS One. 2014;9(8):e105182.
Alfalfa (Medicago sativa L.) is one of the most important forage crops used to
feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content
of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK)
catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in
the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR)
catalyzes the conversion of activated sulfate to sulfite, providing reduced
sulfur for the synthesis of cysteine, methionine, and other essential metabolites
and secondary compounds. To reduce the feedback inhibition of other metabolites,
we cloned bacterial AK and APR genes, modified AK, and introduced them into
alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by
30% and that of methionine increased substantially by 60%. In addition, a
substantial increase in the abundance of essential amino acids (EAAs), such as
aspartate and lysine, was found. The results also indicated a close connection
between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total
amino acid content and the forage biomass tested showed no significant changes in
the transgenic plants. This approach provides a new method for increasing SAAs
and allows for the development of new genetically modified crops with enhanced
nutritional value.
PMCID: PMC3919742
711. Plant Biotechnol J. 2014 Jun;12(5):601-12. doi: 10.1111/pbi.12166. Epub 2014

Feb
12.
Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced

S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers
cold tolerance through up-regulating polyamine oxidation.
Guo Z(1), Tan J, Zhuo C, Wang C, Xiang B, Wang Z.
Author information:
Agro-bioresources, Guangdong Key Laboratory for Innovative Development and
Utilization of Forest Plant Germplasm, South China Agricultural University,
Guangzhou, China.
S-adenosylmethionine synthetase (SAMS) is the key enzyme catalysing the formation

of S-adenosylmethionine (SAM), a precursor of polyamines and ethylene. To
investigate the potential role of SAMS in cold tolerance, we isolated MfSAMS1
from the cold-tolerant germplasm Medicago sativa subsp. falcata and analysed the
association of SAM-derived polyamines with cold tolerance. The expression of
MfSAMS1 in leaves was greatly induced by cold, abscisic acid (ABA), H2O2 and
nitric oxide (NO). Our data revealed that ABA, H2O2 and NO interactions mediated
the cold-induced MfSAMS1 expression and cold acclimation in falcata. SAM,
putrescine, spermidine and spermine levels, ethylene production and polyamine
oxidation were sequentially altered in response to cold, indicating that
SAMS-derived SAM is preferentially used in polyamine synthesis and homeostasis
during cold acclimation. Antioxidant enzyme activities were also induced in
response to cold and showed correlation with polyamine oxidation. Overexpression
of MfSAMS1 in tobacco resulted in elevated SAM levels, but polyamine levels and
ethylene production in the transgenic plants were not significantly changed.
Compared to the wild type, transgenic plants had increased levels of apoplastic
H2O2, higher transcript levels of genes involved in polyamine synthesis and
oxidation, and higher activities of polyamine oxidation and antioxidant enzymes.
The results showed that overexpression of MfSAMS1 promoted polyamine synthesis
and oxidation, which in turn improved H2 O2 -induced antioxidant protection, as a
result enhanced tolerance to freezing and chilling stress in transgenic plants.
This is the first report demonstrating that SAMS plays an important role in plant
tolerance to cold via up-regulating polyamine oxidation.

DOI: 10.1111/pbi.12166
712. Virus Genes. 2014 Jun;48(3):562-5. doi: 10.1007/s11262-014-1045-0. Epub 2014

Feb
8.
Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa

(Medicago sativa L.) in Argentina.
Trucco V(1), de Breuil S, Bejerman N, Lenardon S, Giolitti F.
Author information:
Camino 60 cuadras Km. 5.5, X5020ICA, Córdoba, Argentina.
The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate

infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The
virus genome has the typical organization described for AMV, and comprises 3,643,
2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome
sequence and each encoding region were compared with those of other four isolates
that have been completely sequenced from China, Italy, Spain and USA. The
nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and
from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and
coat protein (CP) encoding regions, whereas the amino acid identity percentages
of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to
P2. CP sequences of AMV-Arg were compared with those of other 25 available
isolates, and the phylogenetic analysis based on the CP gene was carried out. The
highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with
a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from
Italy, one from Brazil and the remaining one from China. The phylogenetic
analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To
our knowledge, this is the first report of a complete nucleotide sequence of AMV
from South America and the first worldwide report of complete nucleotide sequence
of AMV isolated from alfalfa as natural host.
DOI: 10.1007/s11262-014-1045-0
713. Appl Environ Microbiol. 2014 Apr;80(8):2429-39. doi: 10.1128/AEM.04088-13.

Epub
2014 Feb 7.
ExpR coordinates the expression of symbiotically important, bundle-forming Flp

pili with quorum sensing in Sinorhizobium meliloti.
Zatakia HM(1), Nelson CE, Syed UJ, Scharf BE.
Author information:
(1)Department of Biological Sciences, Virginia Polytechnic Institute and State
University, Blacksburg, Virginia, USA.
Type IVb pili in enteropathogenic bacteria function as a host colonization factor

by mediating tight adherence to host cells, but their role in bacterium-plant
symbiosis is currently unknown. The genome of the symbiotic soil bacterium
Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili
of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the
role of Flp pili in the symbiotic interaction of S. meliloti and its host,
Medicago sativa, we deleted pilA1, which encodes the putative pilin subunit in
the chromosomal flp-1 cluster and conducted competitive nodulation assays. The
pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission
electron microscopy revealed the presence of bundle-forming pili protruding from
the polar and lateral region of S. meliloti wild-type cells. The putative pilus
assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral
localization. Transcriptional reporter gene assays demonstrated that expression
of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing
regulator ExpR, which also controls production of exopolysaccharides and
motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter
was confirmed with electrophoretic mobility shift assays. A 17-bp consensus
sequence for ExpR binding was identified within the 28-bp protected region by
DNase I footprinting analyses. Our results show that Flp pili are important for
efficient symbiosis of S. meliloti with its plant host. The temporal inverse
regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve
a coordinated expression of cellular processes during early stages of host
interaction.
DOI: 10.1128/AEM.04088-13
PMCID: PMC3993167
714. J Dairy Sci. 2014;97(4):2270-80. doi: 10.3168/jds.2012-6332. Epub 2014 Feb 6.
Interactions of alfalfa hay and sodium propionate on dairy calf performance and
rumen development.
Beiranvand H(1), Ghorbani GR(2), Khorvash M(2), Nabipour A(3), Dehghan-Banadaky

M(4), Homayouni A(2), Kargar S(2).
Author information:
Technology, Isfahan 84156-83111, Iran. Electronic address:
hamedbeiran669@gmail.com.
Technology, Isfahan 84156-83111, Iran.
(3)Department of Basic Sciences, College of Veterinary Medicine, Ferdowsi
University of Mashhad, Mashhad 91779-48974, Iran.
(4)Department of Animal Sciences, University College of Agriculture and Natural
Resources, University of Tehran, Karaj 31587-77871, Iran.
The objective of this experiment was to investigate the effects of different

levels of alfalfa hay (AH) and sodium propionate (Pro) added to starter diets of
Holstein calves on growth performance, rumen fermentation characteristics, and
rumen development. Forty-two male Holstein calves (40±2kg of birth weight) were
used in a complete randomized design with a 3×2 factorial arrangement of
treatments. Dietary treatments were as follows: (1) control = concentrate only;
(2) Pro = concentrate with 5% sodium propionate [dry matter (DM) basis]; (3) 5%
AH = concentrate + 5% alfalfa hay (DM basis); (4) 5% AH + Pro = concentrate + 5%
alfalfa hay + 5% sodium propionate (DM basis); (5) 10% AH = concentrate + 10%
alfalfa hay (DM basis); and (6) 10% AH + Pro = concentrate + 10% alfalfa hay + 5%
sodium propionate (DM basis). All calves were housed in individual pens bedded
with sawdust until 10wk of age. They were given ad libitum access to water and
starter throughout the experiment and were fed 2L of milk twice daily. Dry matter
intake was recorded daily and body weight weekly. Calves from the control, 10%
AH, and 10% AH + Pro treatments were euthanized after wk 10, and rumen wall
samples were collected. Feeding of forage was found to increase overall dry
matter intake, average daily gain, and final weight; supplementing sodium
propionate had no effect on these parameters. Calves consuming forage had lower
feed efficiency than those on the Pro diet. Rumen fluid in calves consuming
forage had higher pH and greater concentrations of total volatile fatty acids and
molar acetate. Morphometric parameters of the rumen wall substantiated the effect
of AH supplementation, as plaque formation decreased macroscopically. Overall,
the interaction between forage and sodium propionate did not affect calf
performance parameters measured at the end of the experiment. Furthermore,
inclusion of AH in starter diets positively enhanced the growth performance of
male Holstein calves and influenced both the macroscopic and microscopic
appearances of the rumen wall. These benefits, however, were small when only
sodium propionate was offered.

DOI: 10.3168/jds.2012-6332

eCollection
2014.
The regulation of alfalfa saponin extract on key genes involved in hepatic

cholesterol metabolism in hyperlipidemic rats.
Shi Y(1), Guo R(1), Wang X(1), Yuan D(1), Zhang S(1), Wang J(1), Yan X(1), Wang
C(1).
Author information:
University, Zhengzhou, Henan, China.
To investigate the cholesterol-lowering effects of alfalfa saponin extract (ASE)

and its regulation mechanism on some key genes involved in cholesterol
metabolism, 40 healthy 7 weeks old male Sprague Dawley (SD) rats were randomly
divided into four groups with 10 rats in each group: control group,
hyperlipidemic group, ASE treatment group, ASE prevention group. The body weight
gain, relative liver weight and serum lipid 1evels of rats were determined. Total
cholesterol (TC) and total bile acids (TBA) levels in liver and feces were also
measured. Furthermore, the activity and mRNA expressions of Hmgcr, Acat2, Cyp7a1
and Ldlr were investigated. The results showed the following: (1) The abnormal
serum lipid levels in hyperlipidemic rats were ameliorated by ASE administration
(both ASE prevention group and treatment group) (P<0.05). (2) Both ASE
administration to hyperlipidemic rats significantly reduced liver TC and
increased liver TBA level (P<0.05). TC and TBA levels in feces of hyperlipidemic
rats were remarkably elevated by both ASE administration (P<0.05). (3) mRNA
expressions of Hmgcr and Acat2 in the liver of hyperlipidemic rats were
remarkably down-regulated (P<0.05), as well as mRNA expressions of Cyp7a1 and
Ldlr were dramatically up-regulated by both ASE administration (P<0.05). The
activities of these enzymes also paralleled the observed changes in mRNA levels.
(4) There was no significant difference between ASE treatment and ASE prevention
group for most parameters evaluated. Our present study indicated that ASE had
cholesterol-lowering effects. The possible mechanism could be attributed to (1)
the down-regulation of Hmgcr and Acat2, as well as up-regulation of Cyp7a1 and
Ldlr in the liver of hyperlipidemic rats, which was involved in cholesterol
biosynthesis, uptake, and efflux pathway; (2) the increase in excretion of
cholesterol. The findings in our study suggested ASE had great potential
usefulness as a natural agent for treating hyperlipidemia.
PMCID: PMC3914959
716. Zhongguo Zhong Yao Za Zhi. 2013 Nov;38(21):3782-5.
[Quality standard of uygur medicine Medicago sativa seeds].
Ding WH(1), Xu HY(1), Wang DD(1), Li J(1), Tian SG(1).
Author information:
(1)Xinjiang Key Laboratory of Famous Prescription and Science of Formulas, Urumqi
830011, China.
In this paper, microscopic identification method was adopted to observe the

microscopic characters of ten batches of Medicago sativa seeds. And M. sativa
seeds were identificated by TLC method in contrast to trigonelline and
stachydrine hydrochloride. The impurities, moisture, ash, sour insoluble ash were
detected based on Chinese Pharmacopoeia 2010 version (Vol I ). An HPLC method was
also established for determination of trigonelline in the M. sativa seeds. The
contents of impurities, moisture, ash, sour insoluble ash should not exceed 5%,
10%, 6%, and 2%, respectively. The content of trigonelline should be not less
than 0.795 6 mg x g(-1). The experimental methods were accurate and reliable, and
can be used as the quality control of the seeds of M. sativa.
717. J Anim Sci. 2014 Mar;92(3):1037-43. doi: 10.2527/jas.2013-6436. Epub 2014 Feb
3.
Standardized ileal digestibility of amino acids in alfalfa meal, sugar beet pulp,
and wheat bran compared to wheat and protein ingredients for growing pigs.
Eklund M(1), Rademacher M, Sauer WC, Blank R, Mosenthin R.
Author information:
(1)Institute of Animal Nutrition, University of Hohenheim, 70593 Stuttgart,
Germany.
A total of 11 (8 + 3 for replacement) barrows with an initial BW of 23 kg and

fitted with a simple T-cannula at the distal ileum were used in 2 consecutive
experiments (Exp. 1 and Exp. 2) to determine the standardized ileal digestibility
(SID) of AA in 7 assay feed ingredients according to 2 consecutive duplicated 4 ×
4 Latin square designs. In Exp. 1, 3 corn starch-based assay diets were
formulated to contain 170 g CP/kg (as-fed basis) from either soybean meal (SBM),
canola meal (CM), or meat-and-bone meal (MBM) and 1 assay diet that contained 136
g CP/kg (as-fed basis) from wheat as commonly used feed ingredients for pigs. In
Exp. 2, the pigs were fed 4 assay diets formulated to contain 170 g CP/kg (as-fed
basis) from either the same SBM as in Exp. 1 or a combination of this SBM and
alfalfa meal (AM), sugar beet pulp (SB), or wheat bran (WB) to compare the SID of
AA in these feed ingredients with those used in Exp. 1. The SID of AA in CM was
lower compared to SBM (P < 0.05) with intermediate values for MBM and wheat.
Among fiber rich feed ingredients, SID values were lower in SB compared to WB (P
< 0.05) with intermediate values for AM. In AM, SID values ranged between 29 and
45% for Lys, Cys, Thr, and Phe and between 51 and 71% for Arg, His, Ile, Leu,
Met, and Val. In SB, SID values ranged between -21 and 46% for Cys, Thr, Phe, and
Val and between 51 and 61% for Arg, His, Ile, Leu, Lys, and Met. In WB, SID
values were between 55 and 64% for Lys, Cys, Phe, Thr, and Val and between 68 and
80% for Arg, His, Ile, Leu, and Met. The SID values in WB, SB, and AM provided in
the present study may improve diet formulation when these feed ingredients are
used in diet formulation for pigs.
DOI: 10.2527/jas.2013-6436
[Effects of different water harvesting modes on alfalfa planting in semi-arid

areas of Northwest China].
Huo HL(1), Wang Q(2), Zhang EH(3), Shi SL(2), Ren X(2), Wang HL(4), Wang TT(3),
Liu QL(3).
Author information:
(1)College of Grassland Science, Gansu Agricultural University, Lanzhou 730070,
China. hhl8599118@163.com
(2)College of Grassland Science, Gansu Agricultural University, Lanzhou 730070,
China.
(3)College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China.
(4)Gansu Province Key Laboratory of Arid Climate Change and Reducing Disaster,
Institute of Arid Meteorology, China Meteorological Administration, Lanzhou
730020, China.
A field experiment with complete random design was conducted to investigate the
effects of different mulching materials [common plastic film (CMR), biodegradable
mulch film (BMR), and soil crust (SR)] and different ratios of furrow to ridge
(60 cm:30 cm, 60 cm:45 cm, and 60 cm:60 cm) on the runoff efficiency, soil water
storage, soil water content, and hay yield and water use efficiency of alfalfa in
semiarid areas of the Loess Plateau. The runoff efficiency in treatments SR, BMR,
and CMR was 32.0%, 90.7%, and 96.4%, respectively. In the early growth period of
alfalfa (from April to June) , the soil water storage between the treatments had
no significant difference, but in the late growth period (from July to
September), the soil water storage in CMR and BMR was significantly higher than
that in SR. The soil water storage in SR was significantly higher than that in
traditional planting (TP). At budding stage, the soil water storage in TP, SR,
BMR, and CMR was 223.27, 248.56, and 277. 81, and 284.16 mm, respectively. In the
whole growth period, the hay yield of alfalfa in TP, SR, BMR, and CMR was 4112.1,
3397.5, 4317.8, and 4523.8 kg x hm(-2), and the water use efficiency was 11.08,
10.48, 14.56, and 14.95 kg x mm(-1) x hm(-2), respectively. The ratio of furrow
to ridge had no significant effects on the water use efficiency in the same
treatments. When the ratio of furrow to ridge was 60 cm:44 cm, the hay yield in
CMR and BMR reached the maximum.
719. Pest Manag Sci. 2014 Jul;70(7):1017-22. doi: 10.1002/ps.3745. Epub 2014 Mar
17.
Banning of methyl bromide for seed treatment: could Ditylenchus dipsaci again
become a major threat to alfalfa production in Europe?
Mouttet R(1), Escobar-Gutiérrez A, Esquibet M, Gentzbittel L, Mugniéry D,

Reignault P, Sarniguet C, Castagnone-Sereno P.
Author information:
(1)ANSES, Laboratoire de la Santé des Végétaux, Montferrier-sur-Lez, France.
In Europe, the stem and bulb nematode Ditylenchus dipsaci has been listed as a
quarantine pest by EPPO: without any control, it may cause complete failure of
alfalfa crops. Movement of nematodes associated with seeds is considered to be
the highest-risk pathway for the spread of this pest. Since the 2010 official
withdrawal of methyl bromide in Europe, and in the absence of any alternative
chemical, fumigation of contaminated seed batches is no longer possible, which
makes the production of nematode-free alfalfa seeds difficult to achieve and
leads to unmarketable seed batches. Thermotherapy is being considered as a
realistic alternative strategy, but its efficiency still remains to be validated.
The combination of the currently available methods (i.e. use of resistant
cultivars, seed production according to a certification scheme, mechanical
sieving, seed batch inspection) could significantly reduce the likelihood of seed
contamination. However, it does not guarantee a total eradication of the
nematode. Although it is already widely distributed all over Europe,
reclassification of D. dipsaci as a regulated non-quarantine pest to reduce the
possibility of further introductions and the rate of spread of this pest appears
to be a risky strategy because of the lack of up-to-date documented data to
evaluate damage thresholds and determine acceptable tolerance levels.
DOI: 10.1002/ps.3745
720. Nat Prod Rep. 2014 Mar;31(3):356-80. doi: 10.1039/c3np70104b.
Natural product biosynthesis in Medicago species.
Gholami A(1), De Geyter N, Pollier J, Goormachtig S, Goossens A.
Author information:
(1)Department of Plant Systems Biology, VIB, Ghent University, Technologiepark
927, B-9052 Gent, Belgium. alain.goossens@psb.vib-ugent.be.
The genus Medicago, a member of the legume (Fabaceae) family, comprises 87

species of flowering plants, including the forage crop M. sativa (alfalfa) and
the model legume M. truncatula (barrel medic). Medicago species synthesize a
variety of bioactive natural products that are used to engage into symbiotic
interactions but also serve to deter pathogens and herbivores. For humans, these
bioactive natural products often possess promising pharmaceutical properties. In
this review, we focus on the two most interesting and well characterized
secondary metabolite classes found in Medicago species, the triterpene saponins
and the flavonoids, with a detailed overview of their biosynthesis, regulation,
and profiling methods. Furthermore, their biological role within the plant as
well as their potential utility for human health or other applications is
discussed. Finally, we give an overview of the advances made in metabolic
engineering in Medicago species and how the development of novel molecular and
omics toolkits can influence a better understanding of this genus in terms of
specialized metabolism and chemistry. Throughout, we critically analyze the
current bottlenecks and speculate on future directions and opportunities for
research and exploitation of Medicago metabolism.
DOI: 10.1039/c3np70104b
721. J Dairy Sci. 2014 Mar;97(3):1436-45. doi: 10.3168/jds.2013-7205. Epub 2014 Jan
25.
Residual feed intake of lactating Holstein-Friesian cows predicted from

high-density genotypes and phenotyping of growing heifers.
Davis SR(1), Macdonald KA(2), Waghorn GC(2), Spelman RJ(3).
Author information:
(1)LIC, Private Bag 3016, Hamilton 3240, New Zealand. Electronic address:
steve.davis@lic.co.nz.
(2)DairyNZ, Private Bag 3221, Hamilton 3240, New Zealand.
(3)LIC, Private Bag 3016, Hamilton 3240, New Zealand.
A genomic prediction for residual feed intake (RFI) developed in growing dairy
heifers (RFIgro) was used to predict and test breeding values for RFI in
lactating cows (RFIlac) from an independent, industry population. A selection of
3,359 cows, in their third or fourth lactation during the study, of above average
genetic merit for milk production, and identified as at least 15/16ths
Holstein-Friesian breed, were selected for genotyping from commercial dairy
herds. Genotyping was carried out using the bovine SNP50 BeadChip (Illumina Inc.,
San Diego, CA) on DNA extracted from ear-punch tissue. After quality control
criteria were applied, genotypes were imputed to the 624,930 single nucleotide
polymorphisms used in the growth study. Using these data, genomically estimated
breeding values (GEBV) for RFIgro were calculated in the selected cow population
based on a genomic prediction for RFIgro estimated in an independent group of
growing heifers. Cows were ranked by GEBV and the top and bottom 310 identified
for possible purchase. Purchased cows (n=214) were relocated to research
facilities and intake and body weight (BW) measurements were undertaken in 99
"high" and 98 "low" RFIgro animals in 4 consecutive groups [beginning at d 61 ±
1.0 standard error (SE), 91 ± 0.5 SE, 145 ± 1.3 SE, and 191 ± 1.5 SE d in milk,
respectively] to measure RFI during lactation (RFIlac). Each group of ~50 cows
(~25 high and ~25 low RFIgro) was in a feed intake facility for 35 d, fed
pasture-alfalfa cubes ad libitum, milked twice daily, and weighed every 2 to 3 d.
Milk composition was determined 3 times weekly. Body weight change and BW at
trial mid-point were estimated by regression of pre- and posttrial BW
measurements. Residual feed intake in lactating cows was estimated from a linear
model including BW, BW change, and milk component yield (as MJ/d); RFIlac
differed consistently between the high and low selection classes, with the
overall means for RFIlac being +0.32 and -0.31 kg of dry matter (DM) per day for
the high and low classes, respectively. Further, we found evidence of sire
differences for RFIlac, with one sire, in particular, being highly represented in
the low RFIgro class, having a mean RFIlac of -0.83 kg of DM per day in 47
daughters. In conclusion, genomic prediction of RFIgro based on RFI measured
during growth will discriminate for RFIlac in an independent group of lactating
cows.

DOI: 10.3168/jds.2013-7205

10.1111/jpn.12167.
Epub 2014 Jan 27.
Effects of alfalfa meal on the intestinal microbial diversity and immunity of

growing ducks.
Jiang JF(1), Song XM, Wu JL, Jiang YQ.
Author information:
(1)Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of
Agricultural Sciences, Hangzhou, China.
This study was conducted to investigate the effects of alfalfa meal diets on the
intestinal microbial diversity and immunity of growing egg-type ducks. A total of
128 healthy 7-week-old female egg-type Shaoxing ducks were selected and randomly
assigned into four dietary treatments: 0%, 3%, 6% and 9% alfalfa meal for 8
weeks. Each treatment consisted of four replicates of eight ducks each.
Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) was
used to characterize the microbiota. The results showed that the DGGE
fingerprints of the V6-V8 fragments of the 16S rRNA from the caeca and faeces of
ducks fed 3%, 6% and 9% alfalfa meal had significantly higher microbiota species
richness than those fed 0% alfalfa meal (p < 0.05). The Shannon-Weiner index of
the microbiota from the caeca and faeces of ducks fed 3%, 6% and 9% alfalfa meal
was significantly higher than those fed 0% alfalfa meal (p < 0.05). Molecular
analysis of the caecal and faecal DNA extracts showed that the alfalfa meal diet
promotes the intestinal microbial diversity, as indicated by their higher species
richness and Shannon-Weiner index. However, the groups did not significantly
differ in terms of average daily gain, feed intake and gain-to-feed ratio (p >
0.05), and the 3-9% alfalfa meal did not affect the growth performance of the
growing egg-type ducks. The proliferation of T and B lymphocytes was
significantly greater (p < 0.05) in the groups supplemented with 3%, 6% and 9% of
alfalfa meal than the unsupplemented control group, and alfalfa meal promoted the
lymphocytes proliferation of the growing egg-type ducks. Dietary alfalfa meal
supplementation increases intestinal microbial community diversity and improves
of the immune response growing egg-type ducks.
DOI: 10.1111/jpn.12167
17.
Technical note: Evaluation of markers for estimating duodenal digesta flow and
ruminal digestibility: Acid detergent fiber, sulfuric acid detergent lignin, and
n-alkanes.
Kozloski GV(1), Stefanello CM(2), Mesquita FR(2), Alves TP(2), Ribeiro Filho
HM(3), Almeida JG(3), Moraes Genro TC(4).
Author information:
(1)Departamento de Zootecnia (Animal Science Department), Universidade Federal de
Santa Maria, Santa Maria, RS, Brazil. Electronic address: kozloski@smail.ufsm.br.
(2)Departamento de Zootecnia (Animal Science Department), Universidade Federal de
Santa Maria, Santa Maria, RS, Brazil.
(3)Departamento de Zootecnia (Animal Science Department), Centro de Ciências
Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, SC, Brazil.
(4)Empresa Brasileira de Pesquisa Agropecuária, Centro de Pesquisa Pecuária Sul
(CPPSUL), Bagé, RS, Brazil.
The amount of digesta flowing to the duodenum is a relevant measurement for the
evaluation of nutrient supply to ruminants, which is usually estimated in animals
fitted with a duodenal T-type cannula using internal or external markers. This
study evaluated acid detergent fiber (ADF) compared with external (C32n-alkane)
and internal [sulfuric acid lignin (ADL) and n-alkanes C31 and C33] markers for
estimating duodenal flow and(or) ruminal digestibility of dry matter (DM) in
cattle and sheep. In the first assay, 4 duodenally cannulated Holstein steers
housed in metabolism cages, dosed with C32n-alkane, and fed Avena strigosa plus
concentrate and increasing levels of tannin extract to reduce ruminal
digestibility, were used in a Latin square design. The mobile-bag technique was
used to measure the intestinal disappearance of ADL and ADF from forage (Avena
strigosa, Pennisetum purpureum, Cynodon dactylon, and Medicago sativa) and
concentrate (corn grain, soybean meal, and sunflower meal) samples that were
previously incubated in the rumen of additional fistulated steer for 12, 24, 36,
or 48 h. The ADF concentration in residues recovered in the feces was strongly
related to the ADF concentration in residues at the duodenum (R(2)=0.93, standard
deviation=30.0, n=901). This relationship showed a lower precision for ADL
fraction (R(2)=0.88, standard deviation=12.6, n=590). In a second assay, duodenal
flow and ruminal DM digestibility were calculated from the duodenal and fecal
concentration of either marker. We observed a significant effect of marker type
on ruminal DM digestibility values, and the effect of tannin treatments was
observed only when ADF or ADL was used as the marker. The lowest residual error
was obtained for ADF. Ruminal DM digestibility was, on average, higher for C31
and C(33)n-alkanes, and the use of dosed C(32)n-alkane resulted in a negative
value. In the third assay, a data set of 235 individual observations was compiled
from digestibility trials to compare ADF and ADL as markers for estimating
duodenal digesta flow in wethers (n=204) and cattle (n=31). We observed a strong
relationship between markers (R(2)=0.84 in sheep and R(2)=0.88 in cattle), but
variance analysis within trials indicated that ADF was more precise than ADL. In
conclusion, in digestibility trials in which fecal output was measured and spot
samples of the duodenal digesta were obtained, duodenal flow and ruminal
digestibility of the DM may be estimated from the relationship between the ADF
concentration in feces and that in the duodenal digesta of ruminants.

DOI: 10.3168/jds.2013-7414
11.
Effect of feeding extruded flaxseed with different grains on the performance of

dairy cows and milk fatty acid profile.
Neveu C(1), Baurhoo B(2), Mustafa A(3).
Author information:
(1)Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC, H9X
3V9, Canada.
(2)Belisle Solution and Nutrition Inc., Saint-Mathias sur Richelieu, QC, J3L 6A7,
Canada. Electronic address: bbaurhoo@belisle.net.
3V9, Canada. Electronic address: arif.mustafa@mcgill.ca.
Sixteen Holsteins cows were used in a Latin square design experiment to determine
the effects of extruded flaxseed (EF) supplementation and grain source (i.e.,
corn vs. barley) on performance of dairy cows. Extruded flaxseed diets contained
10% [dry matter (DM) basis] of an EF product that consisted of 75% flaxseed and
25% ground alfalfa meal. Four lactating Holsteins cows fitted with rumen fistulas
were used to determine the effects of dietary treatments on ruminal fermentation.
Intakes of DM (23.2 vs. 22.2 kg/d), crude protein (4.2 vs. 4.0 kg/d), and neutral
detergent fiber (8.3 vs. 7.9 kg/d) were greater for cows fed EF diets than for
cows fed diets without EF. Milk yield and composition were not affected by
dietary treatments. However, 4% fat-corrected milk (30.5% vs. 29.6 kg/d) and
solids-corrected milk (30.7 vs. 29.9 kg/d) were increased by EF supplementation.
Ruminal pH and total volatile fatty acid concentration were not influenced by EF
supplementation. However, feeding barley relative to corn increased molar
proportions of acetate and butyrate and decreased that of propionate. Ruminal
NH3-N was lower for cows fed barley than for cows fed corn. Milk fatty acid
composition was altered by both grain source and EF supplementation. Cows fed EF
produced milk with higher polyunsaturated and lower saturated fatty acid
concentrations than cows fed diets without EF. Feeding EF or corn increased the
milk concentration of C18:0, whereas that of C16:0 was decreased by EF
supplementation only. Extruded flaxseed supplementation increased milk fat
α-linolenic acid content by 60% and conjugated linoleic acid content by 29%.
Feeding corn relative to barley increased milk conjugated linoleic acid by 29%
but had no effect on milk α-linolenic concentration. Differences in animal
performance and milk fatty acid composition were mainly due to EF
supplementation, whereas differences in ruminal fermentation were mostly due to
grain source.

DOI: 10.3168/jds.2013-6728

eCollection
2014.
Development of an alfalfa SNP array and its use to evaluate patterns of

population structure and linkage disequilibrium.
Li X(1), Han Y(1), Wei Y(1), Acharya A(1), Farmer AD(2), Ho J(3), Monteros MJ(1),
Brummer EC(1).
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore,
Oklahoma, United States of America.
(2)National Center for Genome Resources, Santa Fe, New Mexico, United States of
America.
(3)Forage Genetics International, Davis, California, United States of America.
A large set of genome-wide markers and a high-throughput genotyping platform can

facilitate the genetic dissection of complex traits and accelerate molecular
breeding applications. Previously, we identified about 0.9 million SNP markers by
sequencing transcriptomes of 27 diverse alfalfa genotypes. From this SNP set, we
developed an Illumina Infinium array containing 9,277 SNPs. Using this array, we
genotyped 280 diverse alfalfa genotypes and several genotypes from related
species. About 81% (7,476) of the SNPs met the criteria for quality control and
showed polymorphisms. The alfalfa SNP array also showed a high level of
transferability for several closely related Medicago species. Principal component
analysis and model-based clustering showed clear population structure
corresponding to subspecies and ploidy levels. Within cultivated tetraploid
alfalfa, genotypes from dormant and nondormant cultivars were largely assigned to
different clusters; genotypes from semidormant cultivars were split between the
groups. The extent of linkage disequilibrium (LD) across all genotypes rapidly
decayed to 26 Kbp at r(2) = 0.2, but the rate varied across ploidy levels and
subspecies. A high level of consistency in LD was found between and within the
two subpopulations of cultivated dormant and nondormant alfalfa suggesting that
genome-wide association studies (GWAS) and genomic selection (GS) could be
conducted using alfalfa genotypes from throughout the fall dormancy spectrum.
However, the relatively low LD levels would require a large number of markers to
fully saturate the genome.
PMCID: PMC3887001
726. Int J Food Microbiol. 2014 Mar 3;173:99-104. doi:

10.1016/j.ijfoodmicro.2013.12.020. Epub 2013 Dec 29.
Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104

from sprouts.
Baranzoni GM(1), Fratamico PM(2), Rubio F(3), Glaze T(3), Bagi LK(4), Albonetti
S(1).
Author information:
(1)Department of Veterinary Medical Science, University of Bologna, 50 via Tolara
di Sopra, Ozzano dell'Emilia (BO), Italy.
(2)USDA, Agricultural Research Service, Eastern Regional Research Center, 600 E.
Mermaid Lane, Wyndmoor, PA, United States. Electronic address:
pina.fratamico@ars.usda.gov.
(3)Abraxis, LLC, 54 Steamwhistle Drive, Warminster, PA, United States.
(4)USDA, Agricultural Research Service, Eastern Regional Research Center, 600 E.
Mermaid Lane, Wyndmoor, PA, United States.
Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O104

have been associated with sporadic cases of illness and have caused outbreaks
associated with milk and sprouts. An outbreak that occurred in Europe in 2011
linked to fenugreek sprouts was caused by E. coli O104:H4 that had
characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that
encoded for Shiga toxin 2. In this study, methods were developed for detection of
this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR
assays for enteroaggregative STEC O104:H4 targeted the stx2, aggR, and wzx104
genes, and for STEC O104 targeted the stx1-2, ehxA, and wzx104 genes. After
incubating artificially contaminated sprouts at 4 °C for 48 h and overnight
enrichment in modified buffered peptone water with pyruvate supplemented with
three antibiotics (mBPWp), the pathogens were detected in all samples inoculated
at a level of ca. 100CFU/25 g. Several samples inoculated at lower concentrations
of ca. 10CFU/25 g were negative by the PCR assays, and this could have been due
to cells not surviving or not being able to recover after the stress treatment at
4 °C for 48 h. For isolation of the pathogens, immunomagnetic separation (IMS)
using magnetic beads coated with antibodies against O104 were employed, and this
was followed by plating the beads onto mRBA and CHROMagar STEC O104 for isolation
of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7.
Presumptive colonies were confirmed by agglutination using latex particles
attached to antibodies against serogroup O104 and by the multiplex PCR assays.
The methodologies described in this study for detection of enteroaggregative STEC
O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup
O104, and they enhance the ability to detect and isolate these pathogens from
sprouts and potentially other foods, as well.
Published by Elsevier B.V.
727. J Hazard Mater. 2014 Feb 28;267:40-7. doi: 10.1016/j.jhazmat.2013.12.029. Epub

2013 Dec 27.
Hydrogen-rich water alleviates aluminum-induced inhibition of root elongation in

alfalfa via decreasing nitric oxide production.
Chen M(1), Cui W(1), Zhu K(1), Xie Y(1), Zhang C(2), Shen W(3).
Author information:
China.
210095, China.
One of the earliest and distinct symptoms of aluminum (Al) toxicity is the
inhibition of root elongation. Although hydrogen gas (H2) is recently described
as an important bio-regulator in plants, whether and how H2 regulates Al-induced
inhibition of root elongation is largely unknown. To address these gaps,
hydrogen-rich water (HRW) was used to investigate a physiological role of H2 and
its possible molecular mechanism. Individual or simultaneous (in particular)
exposure of alfalfa seedlings to Al, or a fresh but not old nitric oxide
(NO)-releasing compound sodium nitroprusside (SNP), not only increased NO
production, but also led to a significant inhibition of root elongation. Above
responses were differentially alleviated by pretreatment with 50% saturation of
HRW. The addition of HRW also alleviated the appearance of Al toxicity symptoms,
including the improvement of seedling growth and less accumulation of Al.
Subsequent results revealed that the removal of NO by the NO scavenger, similar
to HRW, could decrease NO production and alleviate Al- or SNP-induced inhibition
of root growth. Thus, we proposed that HRW alleviated Al-induced inhibition of
alfalfa root elongation by decreasing NO production. Such findings may be
applicable to enhance crop yield and improve stress tolerance.
DOI: 10.1016/j.jhazmat.2013.12.029
728. Plant Physiol. 2014 Mar;164(3):1139-50. doi: 10.1104/pp.113.232421. Epub 2014

Jan
9.
Lignin modification leads to increased nodule numbers in alfalfa.
Gallego-Giraldo L(1), Bhattarai K, Pislariu CI, Nakashima J, Jikumaru Y, Kamiya

Y, Udvardi MK, Monteros MJ, Dixon RA.
Author information:
(1)Plant Biology Division , Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401.
Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by

down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme
A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased
digestibility and processing ability of lignocellulose. However, these
modifications are often also associated with dwarfing and other changes in plant
growth. Given the importance of nitrogen fixation for legume growth, we evaluated
the impact of constitutively targeted lignin modification on the belowground
organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants
exhibit a striking reduction in root growth accompanied by an unexpected increase
in nodule numbers when grown in the greenhouse or in the field. This phenotype is
associated with increased levels of gibberellins and certain flavonoid compounds
in roots. Although HCT down-regulation reduced biomass yields in both the
greenhouse and field experiments, the impact on the allocation of nitrogen to
shoots or roots was minimal. It is unlikely, therefore, that the altered growth
phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation
or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no
measurable effect on carbon allocation to roots in either greenhouse or 3-year
field trials.
DOI: 10.1104/pp.113.232421
PMCID: PMC3938609
729. Mol Plant Microbe Interact. 2014 May;27(5):437-45. doi:

10.1094/MPMI-11-13-0338-R.
Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of

the bifunctional RibBA protein.
Yurgel SN, Rice J, Domreis E, Lynch J, Sa N, Qamar Z, Rajamani S, Gao M, Roje S,

Bauer WD.
Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago spp.

and other legumes, secretes a considerable amount of riboflavin. This precursor
of the cofactors flavin mononucleotide and flavin adenine dinucleotide is a
bioactive molecule that has a beneficial effect on plant growth. The ribBA gene
of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone
phosphate synthase and guanosine triphosphate (GTP) cyclohydrolase II activities,
catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here
that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect
the ability of S. meliloti to establish an effective symbiosis with the host
plant but does affect the ability of the bacteria to secrete flavins, colonize
host-plant roots, and compete for nodulation. A strain missing the RibBA protein
retains considerable GTP cyclohydrolase II activity. Based on these results, we
hypothesize that S. meliloti has two partly interchangeable modules for
biosynthesis of riboflavin, one fulfilling the internal need for flavins in
bacterial metabolism and the other producing riboflavin for secretion. Our data
also indicate that bacteria-derived flavins play a role in communication between
rhizobia and the legume host and that the RibBA protein is important in this
communication process even though it is not essential for riboflavin biosynthesis
and symbiosis.
DOI: 10.1094/MPMI-11-13-0338-R
730. Foodborne Pathog Dis. 2014 Apr;11(4):332-4. doi: 10.1089/fpd.2013.1637. Epub

2014
Jan 8.
Large-scale gaseous acetic acid treatment to disinfect alfalfa seeds inoculated

with Escherichia coli.
Nei D(1), Enomoto K, Yamamoto K.
Author information:
(1)1 National Food Research Institute , Tsukuba, Japan .
Most outbreaks of foodborne illness related to sprout consumption are ascribed to
bacterial contamination of its seeds, and they need disinfection before
sprouting. Recently, gaseous acetic acid (GAA) treatment received great attention
as a method for seed disinfection. In this study, the effect of GAA treatment on
alfalfa seed disinfection was evaluated in a large-scale device to simulate
practical applications. Alfalfa seeds (3 kg) inoculated with Escherichia coli
were treated with 8.7% (vol/vol) GAA at 55°C for 1-3 h. The population of E. coli
was significantly reduced (p<0.05), and the reduction was larger with longer
exposure times. After 3-h treatment, a maximum decrease by more than 5 log
colony-forming units/g was observed. The germination ratio of alfalfa seeds was
not affected by the treatments under all the conditions. The results indicated
that the GAA treatment has a potential for practical application to reduce the
risk of foodborne illness caused by consumption of sprouts.
DOI: 10.1089/fpd.2013.1637
731. Plant Sci. 2014 Feb;215-216:92-9. doi: 10.1016/j.plantsci.2013.10.012. Epub

2013
Nov 8.
Expression of the alfalfa CCCH-type zinc finger protein gene MsZFN delays
flowering time in transgenic Arabidopsis thaliana.
Chao Y(1), Zhang T(1), Yang Q(2), Kang J(1), Sun Y(3), Gruber MY(4), Qin Z(5).
Author information:
100193, People's Republic of China. Electronic address: qchyang66@163.com.
Beijing 100193, People's Republic of China.
(4)Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science
Place, Saskatoon, Saskatchewan S7N0X2, Canada.
100193, People's Republic of China; Bioengineering College of Chongqing
University, Chongqing 400030, People's Republic of China.
Zinc finger proteins comprise a large family and function in various

developmental processes. CCCH type zinc finger protein is one kind of zinc finger
protein, which function is little known. MsZFN gene encoding a CCCH type zinc
finger protein was first discovered by its elevated transcript level in a
salt-induced alfalfa SSH cDNA library. The previous experiment had showed that
MsZFN protein was localized to the nucleus and little is known about the function
of MsZFN protein and its homologous proteins in other plants including model
plant, Arabidopsis thaliana. In the current study, we found that MsZFN transcript
levels increased in alfalfa under continuous dark conditions and that expression
was strongest in leaves and weakest in unopened flowers under light/dark
conditions. Expression of MsZFN in transgenic Arabidopsis plants resulted in late
flowering phenotypes under long day conditions. Yeast two-hybrid and bimolecular
fluorescence complementation assays indicated that MsZFN protein can interact
with itself. Transcript analyses of floral regulatory genes in MsZFN(+)
transgenic Arabidopsis showed enhanced expression of the flowering repressor
FLOWERING LOCUS C and decreased expression of three key flowering time genes,
FLOWERING LOCUS T, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS and GIGANTEA. These
results suggest that MsZFN primarily controls flowering time by repressing
flowering genes expression under long day conditions.
DOI: 10.1016/j.plantsci.2013.10.012
732. J Dairy Sci. 2014 Mar;97(3):1427-35. doi: 10.3168/jds.2013-7227. Epub 2013 Dec
28.
Holstein-Friesian calves selected for divergence in residual feed intake during

growth exhibited significant but reduced residual feed intake divergence in their
first lactation.
Macdonald KA(1), Pryce JE(2), Spelman RJ(3), Davis SR(3), Wales WJ(4), Waghorn
GC(5), Williams YJ(6), Marett LC(4), Hayes BJ(2).
Author information:
(1)DairyNZ, Hamilton 3240, New Zealand. Electronic address:
kevin.macdonald@dairynz.co.nz.
(2)Biosciences Research Division, Department of Environment and Primary
Industries Victoria, Bundoora, VIC 3083, Australia; Dairy Futures Cooperative
Research Centre, Bundoora, VIC 3083, Australia.
(3)Livestock Improvement Corporation, Hamilton 3240, New Zealand.
(4)Future Farming Systems Research Division, Department of Environment and
Primary Industries, Victoria, Ellinbank, VIC 3820, Australia.
(5)DairyNZ, Hamilton 3240, New Zealand.
(6)Future Farming Systems Research Division, Department of Environment and
Primary Industries, Victoria, Tatura, VIC 3616, Australia.
Residual feed intake (RFI), as a measure of feed conversion during growth, was
estimated for around 2,000 growing Holstein-Friesian heifer calves aged 6 to 9 mo
in New Zealand and Australia, and individuals from the most and least efficient
deciles (low and high RFI phenotypes) were retained. These animals (78 New
Zealand cows, 105 Australian cows) were reevaluated during their first lactation
to determine if divergence for RFI observed during growth was maintained during
lactation. Mean daily body weight (BW) gain during assessment as calves had been
0.86 and 1.15 kg for the respective countries, and the divergence in RFI between
most and least efficient deciles for growth was 21% (1.39 and 1.42 kg of dry
matter, for New Zealand and Australia, respectively). At the commencement of
evaluation during lactation, the cows were aged 26 to 29 mo. All were fed alfalfa
and grass cubes; it was the sole diet in New Zealand, whereas 6 kg of crushed
wheat/d was also fed in Australia. Measurements of RFI during lactation occurred
for 34 to 37 d with measurements of milk production (daily), milk composition (2
to 3 times per week), BW and BW change (1 to 3 times per week), as well as body
condition score (BCS). Daily milk production averaged 13.8 kg for New Zealand
cows and 20.0 kg in Australia. No statistically significant differences were
observed between calf RFI decile groups for dry matter intake, milk production,
BW change, or BCS; however a significant difference was noted between groups for
lactating RFI. Residual feed intake was about 3% lower for lactating cows
identified as most efficient as growing calves, and no negative effects on
production were observed. These results support the hypothesis that calves
divergent for RFI during growth are also divergent for RFI when lactating. The
causes for this reduced divergence need to be investigated to ensure that genetic
selection programs based on low RFI (better efficiency) are robust.

DOI: 10.3168/jds.2013-7227

10.1111/jpn.12157.
Epub 2013 Dec 30.
Volatile fatty acid profile for grass hay or alfalfa hay fed to alpacas (Vicugna
pacos).
Oldham CL(1), Robinson TF, Hunter ZR, Taylor L, White J, Johnston NP.
Author information:
(1)Department of Plant and Wildlife Sciences, Brigham, Young University, Provo,
UT, USA.
The purpose of this study was to determine the diurnal composition and
concentration of volatile fatty acids (VFA) and to determine VFA composition and
concentration differences between stomach compartment 1 (C1) and caecum of
alpacas fed grass and alfalfa hay. The study was divided into two experiments. In
Experiment 1 (EXP 1), 10 male alpacas (3+ years old, 65 kg BW) were divided into
two groups, housed in drylot pens, provided ad libitum water and fed alfalfa (AH)
or grass hay (GH) for 30 days. The alpacas were slaughtered and the digestive
tract collected, divided into sub-tract sections, weighed and digesta sampled for
pH, dry matter (DM) and NDF. Volatile fatty acid composition and concentration
were determined on C1 and caecal material. Four adult male (3+ years old,
60 kg BW), C1 fistulated alpacas were housed in metabolism crates and divided
into two forage groups for Experiment 2 (EXP 2). Alpacas were fed the forages as
in EXP 1. Diurnal C1 VFA samples were drawn at 1, 3, 6, 9, 12, 18 and 24 h
post-feeding. There were no differences between forages for tract weight, C1 and
caecum digesta DM or NDF. Differences were noted (p < 0.05) for pH between
forages and sub-tract site. Volatile fatty acids concentrations were different
(p < 0.05) for forage and site, and total VFA was higher for AH than GH (110.6
and 79.1 mm) and C1 than caecum (40.7 and 27.6 mm). Proportion of VFA was
significant (p < 0.05) for forage and site, C1 acetate highest for GH (84.8 vs.
74.0 mm) and caecum acetate 83.7 and 76.2 mm for GH and AH respectively. These
data demonstrate the level of VFA produced in C1 and the caecum of alpacas and
the diurnal VFA patterns. Composition of VFA is similar to other ruminant
species.
DOI: 10.1111/jpn.12157
734. Plants (Basel). 2014 Aug 7;3(3):324-47. doi: 10.3390/plants3030324.
Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of

Contributing to Auxin Biosynthesis.
Sánchez-Parra B(1), Frerigmann H(2), Alonso MM(3), Loba VC(4), Jost R(5),
Hentrich M(6), Pollmann S(7).
Author information:
(1)Center for Plant Biotechnology and Genomics (U.P.M.-I.N.I.A.), Technical
University Madrid, Montegancedo Campus, Crta. M-40, km 38, 28223 Pozuelo de
Alarcón (Madrid), Spain. beatriz.sanchez@upm.es.
(2)Department of Plant Physiology, Faculty of Biology and Biotechnology,
Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany.
henning.frerigmann@uni-koeln.de.
Alarcón (Madrid), Spain. martamarina.perez@upm.es.
Alarcón (Madrid), Spain. victorcarrascoloba@gmail.com.
(5)School of Plant Biology, University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009, Australia. ricarda.jost@uwa.edu.au.
(6)Department of Plant Physiology, Faculty of Biology and Biotechnology,
Ruhr-University Bochum, Universitätsstraße 150, 44801 Bochum, Germany.
mathias.hentrich@rub.de.
Alarcón (Madrid), Spain. stephan.pollmann@upm.es.
Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the

major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved
in auxin de novo biosynthesis. With the emerging amount of genomics data, it was
possible to identify over forty proteins with substantial homology to the already
characterized amidases from Arabidopsis and tobacco. The observed high
conservation of amidase-like proteins throughout the plant kingdom may suggest an
important role of theses enzymes in plant development. Here, we report cloning
and functional analysis of four, thus far, uncharacterized plant amidases from
Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa.
Intriguingly, we were able to demonstrate that the examined amidases are also
capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA),
an auxin endogenous to several plant species including Arabidopsis. Furthermore,
we compared the subcellular localization of the enzymes to that of Arabidopsis
AMI1, providing further evidence for similar enzymatic functions. Our results
point to the presence of a presumably conserved pathway of auxin biosynthesis via
IAM, as amidases, both of monocot, and dicot origins, were analyzed.
DOI: 10.3390/plants3030324
PMCID: PMC4844348
PMID: 27135507
735. Pol J Microbiol. 2014;63(4):375-86.
Nodulation competitiveness of Ensifer meliloti alfalfa nodule isolates and their

potential for application as inoculants.
Marek-Kozaczuk M, Wielbo J, Pawlik A, Skorupska A.
Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into

nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an
autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown
in arable soil was used as the basis for selection of potential inoculants.
Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA,
recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed
different symbiotic performance and diversity in the number of plasmids and
molecular weight. Isolates that were the most efficient in symbiotic nitrogen
fixation were tagged with a constitutively expressed gusA gene carried by a
stable plasmid vector pJBA21Tc and used in competition experiments in soil under
greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively
competed with indigenous soil rhizobia, were selected. The metabolic profiles of
these selected strains showed differences in the use of carbon and energy
sources. In addition, the LU09 strain exhibited bacteriocin production and LU12
mineral phosphate solubilization, which are valuable traits for soil survival.
These strains may be considered as potential biofertilizers for alfalfa
cultivation.
736. Stand Genomic Sci. 2014 Dec 8;9:4. doi: 10.1186/1944-3277-9-4. eCollection
2014.
Genome sequence of Ensifer medicae Di28; an effective N2-fixing microsymbiont of

Medicago murex and M. polymorpha.
Garau G(1), Terpolilli J(2), Hill Y(2), Tian R(2), Howieson J(2), Bräu L(3),
Goodwin L(4), Han J(5), Reddy T(5), Huntemann M(5), Pati A(6), Woyke T(5),
Mavromatis K(6), Markowitz V(6), Ivanova N(4), Kyrpides N(7), Reeve W(2).
Author information:
(1)Dipartimento di Agraria, S.T.A.A., University of Sassari, Sassari, Italy.
(2)Centre for Rhizobium Studies, Murdoch University, Murdoch, WA, Australia.
(3)School of Life and Environmental Sciences, Deakin University, Deakin, VIC,
Australia.
(4)Los Alamos National Laboratory, Bioscience Division, Los Alamos, NM, USA.
(5)DOE Joint Genome Institute, Walnut Creek, CA, USA.
Laboratory, Berkeley, CA, USA.
(7)Los Alamos National Laboratory, Bioscience Division, Los Alamos, NM, USA ;
Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi
Arabia.
Ensifer medicae Di28 is an aerobic, motile, Gram-negative, non-spore-forming rod

that can exist as a soil saprophyte or as a legume microsymbiont of Medicago spp.
Di28 was isolated in 1998 from a nodule recovered from the roots of M. polymorpha
growing in the south east of Sardinia (Italy). Di28 is an effective microsymbiont
of the annual forage legumes M. polymorpha and M. murex and is capable of
establishing a partially effective symbiotic association with the perennial M.
sativa. Here we describe the features of E. medicae Di28, together with genome
sequence information and its annotation. The 6,553,624 bp standard draft genome
is arranged into 104 scaffolds of 104 contigs containing 6,394 protein-coding
genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100
sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for
Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.
DOI: 10.1186/1944-3277-9-4
PMCID: PMC4334989
PMID: 25780497
737. BMC Biotechnol. 2013 Dec 20;13:111. doi: 10.1186/1472-6750-13-111.
CdSe/ZnS quantum dots trigger DNA repair and antioxidant enzyme systems in
Medicago sativa cells in suspension culture.
Santos AR, Miguel AS, Macovei A, Maycock C, Balestrazzi A, Oliva A, Fevereiro

P(1).
Author information:
(1)Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
psalema@itqb.unl.pt.
BACKGROUND: Nanoparticles appear to be promising devices for application in the

agriculture and food industries, but information regarding the response of plants
to contact with nano-devices is scarce. Toxic effects may be imposed depending on
the type and concentration of nanoparticle as well as time of exposure. A number
of mechanisms may underlie the ability of nanoparticles to cause genotoxicity,
besides the activation of ROS scavenging mechanisms. In a previous study, we
showed that plant cells accumulate 3-Mercaptopropanoic acid-CdSe/ZnS quantum dots
(MPA-CdSe/ZnS QD) in their cytosol and nucleus and increased production of ROS in
a dose dependent manner when exposed to QD and that a concentration of 10 nM
should be cyto-compatible.
RESULTS: When Medicago sativa cells were exposed to 10, 50 and 100 nM
MPA-CdSe/ZnS QD a correspondent increase in the activity of Superoxide dismutase,
Catalase and Glutathione reductase was registered. Different versions of the
COMET assay were used to assess the genotoxicity of MPA-CdSe/ZnS QD. The number
of DNA single and double strand breaks increased with increasing concentrations
of MPA-CdSe/ZnS QD. At the highest concentrations, tested purine bases were more
oxidized than the pyrimidine ones. The transcription of the DNA repair enzymes
Formamidopyrimidine DNA glycosylase, Tyrosyl-DNA phosphodiesterase I and DNA
Topoisomerase I was up-regulated in the presence of increasing concentrations of
MPA-CdSe/ZnS QD.
CONCLUSIONS: Concentrations as low as 10 nM MPA-CdSe/ZnS Quantum Dots are
cytotoxic and genotoxic to plant cells, although not lethal. This sets a limit
for the concentrations to be used when practical applications using nanodevices
of this type on plants are being considered. This work describes for the first
time the genotoxic effect of Quantum Dots in plant cells and demonstrates that
both the DNA repair genes (Tdp1β, Top1β and Fpg) and the ROS scavenging
mechanisms are activated when MPA-CdSe/ZnS QD contact M. sativa cells.
DOI: 10.1186/1472-6750-13-111
PMCID: PMC3901376

eCollection 2013.
Global transcriptome sequencing using the Illumina platform and the development
of EST-SSR markers in autotetraploid alfalfa.
Liu Z(1), Chen T(1), Ma L(1), Zhao Z(2), Zhao PX(3), Nan Z(1), Wang Y(1).
Author information:
Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
(2)Key Laboratory of Cell Activities and Stress Adaptations, Ministry of
Education, School of Life Sciences, Lanzhou University, Lanzhou, China.
(3)Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore,
Oklahoma, United States of America.
BACKGROUND: Alfalfa is the most widely cultivated forage legume and one of the
most economically valuable crops in the world. The large size and complexity of
the alfalfa genome has delayed the development of genomic resources for alfalfa
research. Second-generation Illumina transcriptome sequencing is an efficient
method for generating a global transcriptome sequence dataset for gene discovery
and molecular marker development in alfalfa.
METHODOLOGY/PRINCIPAL FINDINGS: More than 28 million sequencing reads (5.64 Gb of
clean nucleotides) were generated by Illumina paired-end sequencing from 15
different alfalfa tissue samples. In total, 40,433 unigenes with an average
length of 803 bp were obtained by de novo assembly. Based on a sequence
similarity search of known proteins, a total of 36,684 (90.73%) unigenes were
annotated. In addition, 1,649 potential EST-SSRs were identified as potential
molecular markers from unigenes with lengths exceeding 1 kb. A total of 100 pairs
of PCR primers were randomly selected to validate the assembly quality and
develop EST-SSR markers from genomic DNA. Of these primer pairs, 82 were able to
amplify sequences in initial screening tests, and 27 primer pairs successfully
amplified DNA fragments and detected significant amounts of polymorphism among 10
alfalfa accessions.
CONCLUSIONS/SIGNIFICANCE: The present study provided global sequence data for
autotetraploid alfalfa and demonstrates the Illumina platform is a fast and
effective approach to EST-SSR markers development in alfalfa. The use of these
transcriptome datasets will serve as a valuable public information platform to
accelerate studies of the alfalfa genome.
PMCID: PMC3861513
739. J Dairy Sci. 2014 Feb;97(2):952-60. doi: 10.3168/jds.2013-7046. Epub 2013 Dec
15.
Effect of substituting brown rice for corn on lactation and digestion in dairy
cows fed diets with a high proportion of grain.
Miyaji M(1), Matsuyama H(2), Hosoda K(2).
Author information:
(1)NARO Institute of Livestock and Grassland Science, Nasushiobara, Tochigi,
329-2793, Japan. Electronic address: mmiyaji@affrc.go.jp.
(2)NARO Institute of Livestock and Grassland Science, Nasushiobara, Tochigi,
329-2793, Japan.
The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea
mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain
on feed intake, lactation performance, ruminal fermentation, digestion, and N
utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk)
were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a
diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn
(dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7%
alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked
grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and
then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo.
Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed
40% corn. The ruminal pH and total volatile fatty acid concentrations were not
affected by dietary treatment. The ruminal ammonia-N concentration decreased as
the percentage of BR in the diets was elevated. The proportion of acetate
decreased, and that of propionate and butyrate increased with the increasing
levels of BR. Plasma urea-N concentrations was lower and glucose and insulin
concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The
whole-tract apparent digestibility of dry matter, organic matter, and starch
increased, and the digestibility of neutral detergent fiber and acid detergent
fiber decreased with the increasing BR level in the diet, with no dietary effect
on crude protein digestion. As a proportion of N intake, the urinary N excretion
was lower and the retention of N was higher for cows fed 40% BR than for cows fed
40% corn, with no dietary effect observed on N secretion in milk and fecal N
excretion. These results show that substituting BR for corn decreases urinary N
losses and improves N utilization, but causes adverse effects on milk production
when cows are fed high-grain diets at 40% of dietary dry matter.

DOI: 10.3168/jds.2013-7046
740. Genome Announc. 2013 Dec 12;1(6). pii: e01035-13. doi: 10.1128/genomeA.01035-
13.
High-Quality Draft Genome Sequence of Xanthomonas alfalfae subsp. alfalfae Strain

CFBP 3836.
Jacques MA(1), Bolot S, Charbit E, Darrasse A, Briand M, Arlat M, Gagnevin L,

Koebnik R, Noël LD, Portier P, Carrère S, Boureau T.
Author information:
(1)INRA, Institut de Recherche en Horticulture et Semences (IRHS), UMR 1345 SFR
4207 QUASAV, Beaucouzé, France.
We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp.

alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in
lucerne (Medicago sativa). Comparative genomics will help to decipher the
mechanisms provoking disease and triggering the defense responses of this
pathogen of the model legume Medicago truncatula.
DOI: 10.1128/genomeA.01035-13
PMCID: PMC3861426
PMID: 24336373
741. Anaerobe. 2014 Oct;29:52-8. doi: 10.1016/j.anaerobe.2013.11.012. Epub 2013 Dec

11.
Molecular comparative assessment of the microbial ecosystem in rumen and faeces

of goats fed alfalfa hay alone or combined with oats.
Mohammadzadeh H(1), Yáñez-Ruiz DR(2), Martínez-Fernandez G(2), Abecia L(3).
Author information:
(1)Animal Nutrition Institute, Estación Experimental del Zaidín, CSIC, Profesor
Albareda 1, 18008 Granada, Spain; Department of Animal Science, Faculty of
Agriculture, University of Tabriz, Iran.
Albareda 1, 18008 Granada, Spain.
Albareda 1, 18008 Granada, Spain. Electronic address: leticia.abecia@eez.csic.es.
The objective of this work was to compare the biomass and community structure of
bacteria, protozoa and archaea communities in samples of rumen and faeces of
goats and to what extent the diet (alfalfa hay with or without supplemented oats)
offered to them exert an influence. Four cannulated adult goats fistulated in the
rumen were used in a cross over design experiment in two experimental periods of
26 days, consisting in 14 days of adaptation, 7 days of sampling rumen contents
and 5 days of digestibility measurement. Bacterial, protozoa and archaeal biomass
and the communities' structure was assessed by real time PCR (qPCR) and
denaturing gradient gel electrophoresis (DGGE), respectively. The numbers of
archaea and bacteria in both rumen and faeces were higher and lower,
respectively, in animals fed AH diet (P < 0.005). Contrary, protozoal numbers
were not affected by the diet but were lower (P < 0.001) in faeces than in rumen.
The analysis of the community structure revealed a consistently different
population in structure in rumen and faeces for the three studied microbial
groups and that supplementing alfalfa hay with oats led to a decrease in the
similarity between sites in the rumen and faeces: similarity indexes for bacteria
(57 and 27%), archaea (26 and 9%) and protozoa (62 and 22%) in animals fed AH and
AHO diets, respectively.
DOI: 10.1016/j.anaerobe.2013.11.012
742. Animal. 2014 Mar;8(3):420-30. doi: 10.1017/S1751731113002206. Epub 2013 Dec

13.
Effects of dehydrated lucerne and soya bean meal on milk production and
composition, nutrient digestion, and methane and nitrogen losses in dairy cows
receiving two different forages.
Doreau M(1), Ferlay A(1), Rochette Y(1), Martin C(1).
Author information:
(1)INRA, UMR1213 Herbivores, 63122 Saint-Genès-Champanelle, France; Clermont
Université, VetAgro Sup, UMR1213 Herbivores, BP 10448, 63000 Clermont-Ferrand,
France.
Dehydrated lucerne is used as a protein source in dairy cow rations, but little
is known about the effects of lucerne on greenhouse gas production by animals.
Eight Holstein dairy cows (average weight: 582 kg) were used in a replicated 4 ×
4 Latin square design. They received diets based on either maize silage (M) or
grass silage (G) (45% of diet on dry matter (DM) basis), with either soya bean
meal (15% of diet DM) completed with beet pulp (15% of diet DM) (SP) or
dehydrated lucerne (L) (30% of diet DM) as protein sources; MSP, ML, GSP and GL
diets were calculated to meet energy requirements for milk production by dairy
cows and degradable protein for rumen microbes. Dry matter intake (DMI) did not
differ among diets (18.0 kg/day DMI); milk production was higher with SP diets
than with L diets (26.0 v. 24.1 kg/day), but milk production did not vary with
forage type. Milk fatty-acid (FA) composition was modified by both forage and
protein sources: L and G diets resulted in less saturated FA, less linoleic acid,
more trans-monounsaturated FA, and more linolenic acid than SP and M diets,
respectively. Enteric methane (CH4) production, measured by the SF6 tracer
method, was higher for G diets than for M diets, but did not differ with protein
source. The same effects were observed when CH4 was expressed per kg milk. Minor
effects of diets on rumen fermentation pattern were observed. Manure CH4
emissions estimated from faecal organic matter were negatively related to diet
digestibility and were thus higher for L than SP diets, and higher for M than G
diets; the resulting difference in total CH4 production was small. Owing to diet
formulation constraints, N intake was higher for SP than for L diets; interaction
between forage type and protein source was significant for N intake. The same
statistical effects were found for N in milk. Faecal and urinary N losses were
determined from total faeces and urine collection. Faecal N output was lower for
M than for G diets but did not differ between protein sources. Urinary N output
did not differ between forage types, but was lower for cows fed L diets than for
cows fed SP diets, potentially resulting in lower ammonia emissions with L diets.
Replacing soya bean meal plus beet pulp with dehydrated lucerne did not change
CH4 production, but resulted in more N in faeces and less N in urine.
DOI: 10.1017/S1751731113002206
743. J Agric Food Chem. 2014 Jan 8;62(1):270-8. doi: 10.1021/jf402463w. Epub 2013
Dec
20.
Characterization and phylogenetic analysis of allergenic Tryp_alpha_amyl protein

family in plants.
Wang J(1), Yang L, Zhao X, Li J, Zhang D.
Author information:
(1)National Center for Molecular Characterization of Genetically Modified
Organisms, State Key Laboratory of Hybrid Rice, School of Life Science and
Biotechnology, Shanghai Jiao Tong University , 800 Dongchuan Road, Shanghai
Most known allergenic proteins in rice ( Oryza sativa ) seed belong to the
Tryp_alpha_amyl family (PF00234), but the sequence characterization and the
evolution of the allergenic Tryp_alpha_amyl family members in plants have not
been fully investigated. In this study, two specific motifs were found besides
the common alpha-amylase inhibitors (AAI) domain from the allergenic
Tryp_alpha_amyl family members in rice seeds (trRSAs). To understand the
evolution and functional importance of the Tryp_alpha_amy1 family and the
specific motifs for the allergenic one, a BLAST search identified 75 homologous
proteins of trRSAs (trHAs) from 22 plant species including main crops such as
rice, maize ( Zea mays ), wheat ( Triticum aestivum ), and sorghum ( Sorghum
bicolor ) from all available sequences in the public databases. Statistical
analysis showed that the allergenicity of trHAs is closely associated with these
two motifs with high number of cysteine residues (p value = 0.00026), and the
trHAs with and without the two motifs were clustered into separate clades,
respectively. Furthermore, significant difference was observed on the secondary
and tertiary structures of allergenic and nonallergenic trHAs. In addition,
expression analysis showed that trHA-encoding genes of purple false brome (
Brachypodium distachyon ), barrel medic ( Medicago truncatula ), rice, and
sorghum are dominantly expressed in seeds. This work provides insight into the
understanding of the properties of allergens in the Tryp_alpha_amyl family and is
helpful for allergy therapy.
DOI: 10.1021/jf402463w

eCollection
2013.
Proteomics analysis of alfalfa response to heat stress.
Li W(1), Wei Z, Qiao Z, Wu Z, Cheng L, Wang Y.

Author information:
(1)College of Animal Science and Technology, Yangzhou University, Yangzhou,
China.
The proteome responses to heat stress have not been well understood. In this
study, alfalfa (Medicago sativa L. cv. Huaiyin) seedlings were exposed to 25 °C
(control) and 40 °C (heat stress) in growth chambers, and leaves were collected
at 24, 48 and 72 h after treatment, respectively. The morphological,
physiological and proteomic processes were negatively affected under heat stress.
Proteins were extracted and separated by two-dimensional polyacrylamide gel
electrophoresis (2-DE), and differentially expressed protein spots were
identified by mass spectrometry (MS). Totally, 81 differentially expressed
proteins were identified successfully by MALDI-TOF/TOF. These proteins were
categorized into nine classes: including metabolism, energy, protein synthesis,
protein destination/storage, transporters, intracellular traffic, cell structure,
signal transduction and disease/defence. Five proteins were further analyzed for
mRNA levels. The results of the proteomics analyses provide a better
understanding of the molecular basis of heat-stress responses in alfalfa.
PMCID: PMC3855785
745. J Bacteriol. 2014 Feb;196(4):811-24. doi: 10.1128/JB.01104-13. Epub 2013 Dec

6.
Cell growth inhibition upon deletion of four toxin-antitoxin loci from the
megaplasmids of Sinorhizobium meliloti.
Milunovic B(1), diCenzo GC, Morton RA, Finan TM.
Author information:
(1)Center for Environmental Genomics, Department of Biology, McMaster University,
Hamilton, Ontario, Canada.
Toxin and antitoxin (TA) gene pairs are addiction systems that are present in
many microbial genomes. Sinorhizobium meliloti is an N2-fixing bacterial symbiont
of alfalfa and other leguminous plants, and its genome consists of three large
replicons, a circular chromosome (3.7 Mb) and the megaplasmids pSymA (1.4 Mb) and
pSymB (1.7 Mb). S. meliloti carries 211 predicted type II TA genes, each encoding
a toxin or an antitoxin. We constructed defined deletion strains that
collectively removed the entire pSymA and pSymB megaplasmids except for their
oriV regions. Of approximately 100 TA genes on pSymA and pSymB, we identified
four whose loss was associated with cell death or stasis unless copies of the
genes were supplied in trans. Orthologs of three of these loci have been
characterized in other organisms (relB/E [sma0471/sma0473], Fic [DOC] [sma2105],
and VapC [PIN] [orf2230/sma2231]), and this report contains the first
experimental proof that RES/Xre (smb21127/smb21128) loci can function as a TA
system. Transcriptome sequencing (RNA-seq) analysis did not reveal
transcriptional differences between the TA systems to account for why deletion of
the four "active" systems resulted in cell toxicity. These data suggest that
severe cell growth phenotypes result from the loss of a few TA systems and that
loss of most TA systems may result in more subtle phenotypes. These four TA
systems do not appear to play a direct role in the S. meliloti-alfalfa symbiosis,
as strains lacking these TA systems had a symbiotic N2 fixation phenotype that
was indistinguishable from the wild type.
DOI: 10.1128/JB.01104-13
PMCID: PMC3911179
746. J Dairy Sci. 2014 Feb;97(2):1072-6. doi: 10.3168/jds.2013-6989. Epub 2013 Dec
6.
Short communication: Effects of molasses supplementation on performance of

lactating cows fed high-alfalfa silage diets.
Baurhoo B(1), Mustafa A(2).
Author information:
(1)Bélisle Solution & Nutrition Inc., Saint-Mathias sur Richelieu, QC, J3L 6A7,
Canada. Electronic address: bbaurhoo@belisle.net.
3V9, Canada. Electronic address: arif.mustafa@mcgill.ca.
Twelve Holstein cows were used in a replicated Latin square experiment to

determine the effect of adding dried molasses to high-alfalfa silage diets on
dairy cow performance. Three isonitrogenous diets were formulated with a 68:32
forage:concentrate ratio, with alfalfa silage as the only forage source. Dietary
treatments were a control diet with no added molasses and 3 and 6% dried molasses
diets. Three lactating Holstein cows fitted with ruminal cannulas were used to
determine the effects of dietary treatments on ruminal fermentation. Dietary
treatments had no effect on dry matter (average 23.3 kg/d), crude protein
(average 4.4 kg/d), or neutral detergent fiber (average 7.4 kg/d) intake. Milk
yield, energy-corrected milk (average 35.4 kg/d), and 4% fat-corrected milk
(average 33.8 kg/d) were not influenced by dietary treatments. Cows fed the
control diet produced milk with less milk urea nitrogen concentration than those
fed molasses-supplemented diets. Ruminal pH, NH3-N concentration, and total
volatile fatty acids were not different among dietary treatments. The molar
proportion of acetate linearly increased, whereas the molar proportion of
propionate linearly decreased as the level of dried molasses increased. It was
concluded that addition of dried molasses to high-alfalfa silage diets at 6% of
the diet (dry matter basis) increased milk urea nitrogen but had no effect on
animal performance.

DOI: 10.3168/jds.2013-6989
747. Microb Biotechnol. 2014 Nov;7(6):528-44. doi: 10.1111/1751-7915.12104. Epub

2013
Dec 6.
Transcriptional profile of Salmonella enterica subsp. enterica serovar

Weltevreden during alfalfa sprout colonization.
Brankatschk K(1), Kamber T, Pothier JF, Duffy B, Smits TH.
Author information:
(1)Plant Protection Division, Agroscope Changins-Wädenswil ACW, Schloss 1,
Wädenswil, CH-8820, Switzerland.
Sprouted seeds represent a great risk for infection by human enteric pathogens
because of favourable growth conditions for pathogens during their germination.
The aim of this study was to identify mechanisms of interactions of Salmonella
enterica subsp. enterica Weltevreden with alfalfa sprouts. RNA-seq analysis of
S. Weltevreden grown with sprouts in comparison with M9-glucose medium showed
that among a total of 4158 annotated coding sequences, 177 genes (4.3%) and 345
genes (8.3%) were transcribed at higher levels with sprouts and in minimal medium
respectively. Genes that were higher transcribed with sprouts are coding for
proteins involved in mechanisms known to be important for attachment, motility
and biofilm formation. Besides gene expression required for phenotypic adaption,
genes involved in sulphate acquisition were higher transcribed, suggesting that
the surface on alfalfa sprouts may be poor in sulphate. Genes encoding structural
and effector proteins of Salmonella pathogenicity island 2, involved in survival
within macrophages during infection of animal tissue, were higher transcribed
with sprouts possibly as a response to environmental conditions. This study
provides insight on additional mechanisms that may be important for pathogen
interactions with sprouts.
© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd
and Society for Applied Microbiology.
DOI: 10.1111/1751-7915.12104
PMCID: PMC4265072
748. Chemosphere. 2014 Feb;97:47-53. doi: 10.1016/j.chemosphere.2013.10.089. Epub

2013
Nov 27.
Effects of plant growth-promoting bacteria isolated from copper tailings on

plants in sterilized and non-sterilized tailings.
Liu W(1), Yang C(2), Shi S(3), Shu W(4).
Author information:
(1)Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun
Yat-sen University, Guangzhou 510275, China. Electronic address:
lsslwq@mail.sysu.edu.cn.
330862941@qq.com.
(3)College of Life Science & Technology, Southwest University for Nationalities,
Chengdu 610041, China. Electronic address: 936079637@qq.com.
shuws@mail.sysu.edu.cn.
Ten strains of Cu-tolerant bacteria with potential plant growth-promoting ability

were isolated by selecting strains with the ability to use
1-aminocyclopropane-1-carboxylate as a sole nitrogen source (designated ACC-B) or
fix nitrogen (designated FLN-B) originating from the rhizosphere of plants
growing on copper tailings. All 10 strains proved to have intrinsic ability to
produce indole acetic acid and siderophores, and most of them could mobilize
insoluble phosphate. In addition, a greenhouse study showed that ACC-B, FLN-B and
a mixture of both had similar, potent ability to stimulate growth of Pennisetum
purpureum, Medicago sativa and Oenothera erythrosepala plants grown on sterilized
tailings. For instance, above-ground biomass of P. purpureum was 278-357% greater
after 60d growth on sterilized tailings in their presence. They could also
significantly promote the growth of the plants grown on non-sterilized tailings,
though the growth-promoting effects were much weaker. So, strategies for using of
the plant growth-promoting bacteria in the practice of phytoremediation deserve
further studies to get higher growth-promoting efficiency.
749. Huan Jing Ke Xue. 2013 Sep;34(9):3601-6.
[Comparison of soil fertility among open-pit mine reclaimed lands in Antaibao

regenerated with different vegetation types].
Wang X(1), Li JC, Yue JY, Zhou XM, Guo CY, Lu N, Wang YH, Yang SQ.
Author information:
(1)Biology Institute of Shanxi, Taiyuan 030006, China.
Re-vegetation is mainly applied into regeneration in opencast mine to improve the

soil quality. It is very important to choose feasible vegetation types for soil
restoration. In this study, three typical forest restoration types were studied
at Antaibao mine, namely, Medicago sativa, mixed forests Pinus
taebelaefolius-Robinia pseudoacacia-Caragana korshinskii and Elaeagnus
angustifolia-Robinia pseudoacacia-Caragana korshinskii-Hipophae rhamnoides, to
determine the nutrient contents and enzyme activities in different soil layers.
The results showed that re-vegetation markedly increased soil nutrient contents
and the enzyme activities during the restoration process. The nutrient content of
soil in the P. taebelaefolius-R. pseudoacacia-C. korshinskii mixed forest field
was significantly higher than those in other plots. It was found that the soil of
the P. taebelaefolius-R. pseudoacacia-C. korshinskii mixed forest had the highest
integrated fertility index values. In conclusion, the restoration effects of the
P. zaebelaefolius-R. pseudoacacia-C. Korshinskii mixed forest was better than
that of E. angustifolia-R. pseudoacacia-C. korshinskii-H. rhamnoides, while M.
sativa grassland had the least effect.
750. Bull Environ Contam Toxicol. 2014 Apr;92(4):490-6. doi:

10.1007/s00128-013-1166-5. Epub 2013 Nov 28.
Germination and root elongation bioassays in six different plant species for
testing Ni contamination in soil.
Visioli G(1), Conti FD, Gardi C, Menta C.
Author information:
(1)Department of Life Sciences, University of Parma, 43124, Parma, Italy,
giovanna.visioli@unipr.it.
In vitro short-term chronic phytotoxicity germination and root elongation test

were applied to test the effects of nickel (Ni) in seed germination and root
elongation in six plants species: Cucumis sativus (Cucurbitaceae), Lepidium
sativum and Brassica nigra (Brassicaceae), Trifolium alexandrinum and Medicago
sativa (Fabaceae), Phacelia tanacetifolia (Boraginaceae). A naturally Ni rich
soil was used to compare the results obtained. Unlike root elongation,
germination was not affected by Ni in any of the six species tested. EC50 values,
calculated on the root elongation, showed that Ni toxicity decreases in the
following order: P. tanacetifolia > B. nigra > C. sativus > L. sativum > M.
sativa > T. alexandrinum. The test conducted using soil elutriate revealed a
significantly lower effect in both seed germination and root elongation when
compared to the results obtained using untreated soil. Conversely, the test
performed on soil confirmed the high sensitivity of C. sativus, P. tanacetifolia
and L. sativum to Ni.
DOI: 10.1007/s00128-013-1166-5
751. Front Plant Sci. 2013 Nov 11;4:445. doi: 10.3389/fpls.2013.00445. eCollection
2013.
Effects of elevated temperature and CO2 on aboveground-belowground systems: a

case study with plants, their mutualistic bacteria and root/shoot herbivores.
Ryalls JM(1), Riegler M, Moore BD, Lopaticki G, Johnson SN.
Author information:
(1)Hawkesbury Institute for the Environment, University of Western Sydney
Richmond, NSW, Australia.
Interactions between above- and belowground herbivores have been prominent in the
field of aboveground-belowground ecology from the outset, although little is
known about how climate change affects these organisms when they share the same
plant. Additionally, the interactive effects of multiple factors associated with
climate change such as elevated temperature (eT) and elevated atmospheric carbon
dioxide (eCO2) are untested. We investigated how eT and eCO2 affected larval
development of the lucerne weevil (Sitona discoideus) and colonization by the pea
aphid (Acyrthosiphon pisum), on three cultivars of a common host plant, lucerne
(Medicago sativa). Sitona discoideus larvae feed on root nodules housing
N2-fixing rhizobial bacteria, allowing us to test the effects of eT and eCO2
across trophic levels. Moreover, we assessed the influence of these factors on
plant growth. eT increased plant growth rate initially (6, 8 and 10 weeks after
sowing), with cultivar "Sequel" achieving the greatest height. Inoculation with
aphids, however, reduced plant growth at week 14. eT severely reduced root
nodulation by 43%, whereas eCO2 promoted nodulation by 56%, but only at ambient
temperatures. Weevil presence increased net root biomass and nodulation, by 31
and 45%, respectively, showing an overcompensatory plant growth response. Effects
of eT and eCO2 on root nodulation were mirrored by weevil larval development; eT
and eCO2 reduced and increased larval development, respectively. Contrary to
expectations, aphid colonization was unaffected by eT or eCO2, but there was a
near-significant 10% reduction in colonization rates on plants with weevils
present belowground. The contrasting effects of eT and eCO2 on weevils
potentially occurred through changes in root nodulation patterns.
DOI: 10.3389/fpls.2013.00445
PMCID: PMC3822287
PMID: 24273544
752. J Agric Food Chem. 2013 Dec 11;61(49):11913-20. doi: 10.1021/jf403195v. Epub
2013
Dec 2.
Fatty acids, α-tocopherol, β-carotene, and lutein contents in forage legumes,
forbs, and a grass-clover mixture.
Elgersma A(1), Søegaard K, Jensen SK.
Author information:
(1)Independent Scientist, P.O. Box 323, 6700 AH Wageningen, The Netherlands.
Fresh forages are an important natural source of vitamins and fatty acids in
ruminant diets, and their concentrations in forage species are important for the
quality of animal-derived foods such as dairy and meat products. The aims of this
study were to obtain novel information on vitamins and fatty acids (FA) in a
variety of forage legumes and non-legume forb species compared to a grass-clover
mixture and to explore implications for animal-derived products. Seven
dicotyledons [four forbs (salad burnet ( Sanguisorba minor ), caraway ( Carum
carvi ), chicory ( Cichorium intybus ), and ribwort plantain ( Plantago
lanceolata )) and three legume species (yellow sweet clover ( Melilotus
officinalis ), lucerne ( Medicago sativa ), and birdsfoot trefoil ( Lotus
corniculatus ))] and a perennial ryegrass-white clover mixture were investigated
in a cutting trial with four harvests (May-October) during 2009 and 2010. The
experimental design was a randomized complete block, and analyses of variance
were performed. In addition, three other forbs were grown: borage ( Borago
officinalis ), viper's bugloss ( Echium vulgare ), and chervil ( Anthriscus
cerefolium ). Lucerne and yellow sweet clover had the lowest α-tocopherol
concentrations (21-23 mg kg(-1) DM) and salad burnet and ribwort plantain the
highest (77-85 mg kg(-1) DM); β-carotene concentrations were lowest in lucerne,
salad burnet, and yellow sweet clover (26-33 mg kg(-1) DM) and highest in
caraway, birdsfoot trefoil, and ribwort plantain (56-61 mg kg(-1) DM). Total FA
concentrations were lowest in lucerne, ribwort plantain, chicory, and yellow
sweet clover (15.9-19.3 g kg(-1) DM) and highest in caraway and birdsfoot trefoil
(24.5-27.0 g kg(-1) DM). Birdsfoot trefoil had the highest (53.6 g 100 g(-1) FA)
and caraway and lucerne the lowest (33.7-35.7 g 100 g(-1) FA) proportions of n-3
FA. This study demonstrated higher vitamin concentrations in some forbs compared
with major forages such as lucerne and grass-clover, more total FA in salad
burnet, caraway, and birdsfoot trefoil than in lucerne, and higher n-3 FA
concentrations in all forbs than in lucerne. Opportunities are discussed to
develop novel biodiverse pastures for particular product quality characteristics.
DOI: 10.1021/jf403195v
753. Anim Sci J. 2014 Apr;85(4):405-10. doi: 10.1111/asj.12152. Epub 2013 Nov 21.
Physical and nutritional properties of buffalo meat finished on hay or maize

silage-based diets.
Cifuni GF(1), Contò M, Amici A, Failla S.
Author information:
(1)Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di
Ricerca per la Produzione delle Carni e il Miglioramento Genetico, Monterotondo,
Italy.
The current study examines the effect of different finishing diets (hay- vs.
maize-silage on meal ration) on carcass quality, physical, chemical and sensory
properties, and fatty acid profiles of buffalo meat. Twenty male Italian
Mediterranean buffaloes (246 ± 9.00 kg live weight) were distributed at random
into two groups at the beginning of the finishing period (368 ± 20 days). The
buffaloes were offered two finishing diets: a maize silage (MS) or an alfalfa hay
(AH) diet. No significant differences were found between dietary treatments for
live and carcass weight. Meat chemical composition was influenced by dietary
treatment. A higher fat content was detected in meat from animals finished with
MS than AH (P < 0.05). Overall, the data indicated differences between the fatty
acid profiles of meat as a consequence of different feeding systems. The higher
fat deposition in the MS group resulted in meat with a less favorable fatty acid
profile (i.e. a lower polyunsaturated/saturated fatty acid ratio and α-linolenic
fatty acid content) in relation to human health compared with meat from animals
fed the AH diet.
DOI: 10.1111/asj.12152
754. Plant Physiol Biochem. 2014 Jan;74:16-23. doi: 10.1016/j.plaphy.2013.10.028.

Epub
2013 Nov 5.
The thiol compounds glutathione and homoglutathione differentially affect cell

development in alfalfa (Medicago sativa L.).
Pasternak T(1), Asard H(2), Potters G(3), Jansen MA(4).
Author information:
(1)Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020
Antwerp, Belgium; Institute of Biology II/Molecular Plant Physiology,
Albert-Ludwigs-Universität Freiburg, Schänzlestr. 1, 79104 Freiburg, Germany.
Electronic address: taras.pasternak@biologie.uni-freiburg.de.
Antwerp, Belgium. Electronic address: han.asard@ua.ac.be.
(3)Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan
171, B-2020 Antwerp, Belgium; Antwerp Maritime Academy, Noordkasteel Oost 6,
B-2030 Antwerp, Belgium. Electronic address: geert.potters@hzs.be.
Antwerp, Belgium; School of Biological, Earth and Environmental Sciences,
University College Cork, Distillery Field, North Mall, Cork, Ireland. Electronic
address: m.jansen@ucc.ie.
Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS),

precursor of metal chelating phytochelatins, xenobiotic defence compound and
regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that
is present in several taxa in the family of Fabaceae. It is thought that hGSH
performs many of the stress-defence roles typically ascribed to GSH, yet little
is known about the potential involvement of hGSH in controlling cell
proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and
that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell
cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in
impaired cytokinesis in isolated protoplasts, showing the critical importance of
these thiol-compounds for cell division. However, exposure of isolated
protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was
found to inhibit the same process. We conclude that GSH and hGSH have distinct
functional roles in cell cycle regulation in Medicago sativa L. GSH is associated
with meristemic cells, and promotes cell cycle activation and induction of
somatic embryogenesis, while hGSH is associated with differentiated cells and
embryo proliferation.
DOI: 10.1016/j.plaphy.2013.10.028
755. FEMS Microbiol Ecol. 2014 Mar;87(3):650-63. doi: 10.1111/1574-6941.12253. Epub

2013 Dec 3.
Bacterial community variations in an alfalfa-rice rotation system revealed by 16S

rRNA gene 454-pyrosequencing.
Lopes AR(1), Manaia CM, Nunes OC.
Author information:
(1)Laboratório de Processos Ambiente e Energia (LEPAE), Departamento de
Engenharia Química, Faculdade de Engenharia, Universidade do Porto, Porto,
Portugal; Centro de Biotecnologia e Química Fina (CBQF), Laboratório Associado,
Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Porto,
Portugal.
Crop rotation is a practice harmonized with the sustainable rice production.

Nevertheless, the implications of this empirical practice are not well
characterized, mainly in relation to the bacterial community composition and
structure. In this study, the bacterial communities of two adjacent paddy fields
in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot
were characterized before rice seeding and after harvesting, using
454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria,
Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all
the samples, there were variations in relative abundance of these groups. Samples
from the 3rd and 4th years of the crop rotation differed on the higher abundance
of groups of presumable aerobic bacteria and of presumable anaerobic and
acidobacterial groups, respectively. Members of the phylum Nitrospira were more
abundant after rice harvest than in the previously sampled period. Rice cropping
was positively correlated with the abundance of members of the orders
Acidobacteriales and 'Solibacterales' and negatively with lineages such as
Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations
occurring in the microbial communities in soils under sustainable rice
production, based on real-world data.
© 2013 Federation of European Microbiological Societies. Published by John Wiley

& Sons Ltd. All rights reserved.
DOI: 10.1111/1574-6941.12253
756. Microb Ecol. 2014 Jan;67(1):195-204.
Worldwide populations of the aphid Aphis craccivora are infected with diverse
facultative bacterial symbionts.
Brady CM, Asplen MK, Desneux N, Heimpel GE, Hopper KR, Linnen CR, Oliver KM,
Wulff JA, White JA.
Facultative bacterial endosymbionts can play an important role in the

evolutionary trajectory of their hosts. Aphids (Hemiptera: Aphididae) are
infected with a wide variety of facultative endosymbionts that can confer
ecologically relevant traits, which in turn may drive microevolutionary processes
in a dynamic selective environment. However, relatively little is known about how
symbiont diversity is structured in most aphid species. Here, we investigate
facultative symbiont species richness and prevalence among world-wide populations
of the cowpea aphid, Aphis craccivora Koch. We surveyed 44 populations of A.
craccivora, and detected 11 strains of facultative symbiotic bacteria,
representing six genera. There were two significant associations between
facultative symbiont and aphid food plant: the symbiont Arsenophonus was found at
high prevalence in A. craccivora populations collected from Robinia sp. (locust),
whereas the symbiont Hamiltonella was almost exclusively found in A. craccivora
populations from Medicago sativa (alfalfa). Aphids collected from these two food
plants also had divergent mitochondrial haplotypes, potentially indicating the
formation of specialized aphid lineages associated with food plant
(host-associated differentiation). The role of facultative symbionts in this
process remains to be determined. Overall, observed facultative symbiont
prevalence in A. craccivora was lower than that of some other well-studied aphids
(e.g., Aphis fabae and Acyrthosiphon pisum), possibly as a consequence of A.
craccivora's almost purely parthenogenetic life history. Finally, most (70 %) of
the surveyed populations were polymorphic for facultative symbiont infection,
indicating that even when symbiont prevalence is relatively low,
symbiont-associated phenotypic variation may allow population-level evolutionary
responses to local selection.
DOI: 10.1007/s00248-013-0314-0

10.1094/MPMI-09-13-0282-R.
The Sinorhizobium meliloti EmrAB efflux system is regulated by flavonoids through

a TetR-like regulator (EmrR).
Rossbach S, Kunze K, Albert S, Zehner S, Göttfert M.
The divergently oriented Sinorhizobium meliloti emrAB (SMc03168 and SMc03167) and
emrR (SMc03169) genes are predicted to encode an efflux system of the major
facilitator superfamily and a TetR-like transcriptional regulator, respectively.
The transcription of the emrA gene was found to be inducible by flavonoids,
including luteolin and apigenin, which are known inducers of the nodulation genes
in S. meliloti. Interestingly, quercetin, which does not induce nodulation genes,
was also a potent inducer of emrA, indicating that NodD is not directly involved
in regulation of emrA. The likely regulator of emrAB is EmrR, which binds to
palindrome-like sequences in the intergenic region. Several modifications of the
palindromes, including an increase of the spacing between the two half sites,
prevented binding of EmrR. Binding was also impaired by the presence of luteolin.
Mutations in emrA had no obvious effect on symbiosis. This was in contrast to the
emrR mutant, which exhibited a symbiotic deficiency with Medicago sativa.
Conserved binding sites for TetR-like regulators within the intergenic regions
between the emrAB and emrR genes were identified in many symbiotic and pathogenic
members of the order Rhizobiales.
DOI: 10.1094/MPMI-09-13-0282-R

eCollection
2013.
Water-limiting conditions alter the structure and biofilm-forming ability of
bacterial multispecies communities in the alfalfa rhizosphere.
Bogino P(1), Abod A, Nievas F, Giordano W.
Author information:
(1)Departamento de Biología Molecular, Facultad de Ciencias Exactas,
Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto,
Córdoba, Argentina.
Biofilms are microbial communities that adhere to biotic or abiotic surfaces and
are enclosed in a protective matrix of extracellular compounds. An important
advantage of the biofilm lifestyle for soil bacteria (rhizobacteria) is
protection against water deprivation (desiccation or osmotic effect). The
rhizosphere is a crucial microhabitat for ecological, interactive, and
agricultural production processes. The composition and functions of bacterial
biofilms in soil microniches are poorly understood. We studied multibacterial
communities established as biofilm-like structures in the rhizosphere of Medicago
sativa (alfalfa) exposed to 3 experimental conditions of water limitation. The
whole biofilm-forming ability (WBFA) for rhizospheric communities exposed to
desiccation was higher than that of communities exposed to saline or nonstressful
conditions. A culture-dependent ribotyping analysis indicated that communities
exposed to desiccation or saline conditions were more diverse than those under
the nonstressful condition. 16S rRNA gene sequencing of selected strains showed
that the rhizospheric communities consisted primarily of members of the
Actinobacteria and α- and γ-Proteobacteria, regardless of the water-limiting
condition. Our findings contribute to improved understanding of the effects of
environmental stress factors on plant-bacteria interaction processes and have
potential application to agricultural management practices.
PMCID: PMC3817132
759. Ann Bot. 2014 Jan;113(1):145-57. doi: 10.1093/aob/mct251. Epub 2013 Nov 7.
Plant development controls leaf area expansion in alfalfa plants competing for
light.
Baldissera TC(1), Frak E, Carvalho PC, Louarn G.
Author information:
(1)INRA UR4 URP3F, BP6, F86600 Lusignan, France.
BACKGROUND AND AIMS: The growth of crops in a mixture is more variable and
difficult to predict than that in pure stands. Light partitioning and crop leaf
area expansion play prominent roles in explaining this variability. However, in
many crops commonly grown in mixtures, including the forage species alfalfa, the
sensitivity and relative importance of the physiological responses involved in
the light modulation of leaf area expansion are still to be established. This
study was designed to assess the relative sensitivity of primary shoot
development, branching and individual leaf expansion in alfalfa in response to
light availability.
METHODS: Two experiments were carried out. The first studied isolated plants to
assess the potential development of different shoot types and growth periods. The
second consisted of manipulating the intensity of competition for light using a
range of canopies in pure and mixed stands at two densities so as to evaluate the
relative effects on shoot development, leaf growth, and plant and shoot
demography.
KEY RESULTS: Shoot development in the absence of light competition was
deterministic (constant phyllochrons of 32·5 °Cd and 48·2 °Cd for primary axes
and branches, branching probability of 1, constant delay of 1·75 phyllochron
before axillary bud burst) and identical irrespective of shoot type and
growth/regrowth periods. During light competition experiments, changes in plant
development explained most of the plant leaf area variations, with average leaf
size contributing to a lesser extent. Branch development and the number of shoots
per plant were the leaf area components most affected by light availability.
Primary axis development and plant demography were only affected in situations of
severe light competition.
CONCLUSIONS: Plant leaf area components differed with regard to their sensitivity
to light competition. The potential shoot development model presented in this
study could serve as a framework to integrate light responses in alfalfa crop
models.
DOI: 10.1093/aob/mct251
PMCID: PMC3864722
760. Mycorrhiza. 2014 May;24(4):239-45. doi: 10.1007/s00572-013-0536-5. Epub 2013

Nov
7.
Contrasting impacts of defoliation on root colonization by arbuscular mycorrhizal

and dark septate endophytic fungi of Medicago sativa.
Saravesi K(1), Ruotsalainen AL, Cahill JF.
Author information:
(1)Department of Biology, University of Oulu, PO Box 3000, 90014, Oulu, Finland,
karita.saravesi@oulu.fi.
Individual plants typically interact with multiple mutualists and enemies

simultaneously. Plant roots encounter both arbuscular mycorrhizal (AM) and dark
septate endophytic (DSE) fungi, while the leaves are exposed to herbivores. AMF
are usually beneficial symbionts, while the functional role of DSE is largely
unknown. Leaf herbivory may have a negative effect on root symbiotic fungi due to
decreased carbon availability. However, evidence for this is ambiguous and no
inoculation-based experiment on joint effects of herbivory on AM and DSE has been
done to date. We investigated how artificial defoliation impacts root
colonization by AM (Glomus intraradices) and DSE (Phialocephala fortinii) fungi
and growth of Medicago sativa host in a factorial laboratory experiment.
Defoliation affected fungi differentially, causing a decrease in arbuscular
colonization and a slight increase in DSE-type colonization. However, the
presence of one fungal species had no effect on colonization by the other or on
plant growth. Defoliation reduced plant biomass, with this effect independent of
the fungal treatments. Inoculation by either fungal species reduced root/shoot
ratios, with this effect independent of the defoliation treatments. These results
suggest AM colonization is limited by host carbon availability, while DSE may
benefit from root dieback or exudation associated with defoliation. Reductions in
root allocation associated with fungal inoculation combined with a lack of effect
of fungi on plant biomass suggest DSE and AMF may be functional equivalent to the
plant within this study. Combined, our results indicate different controls of
colonization, but no apparent functional consequences between AM and DSE
association in plant roots in this experimental setup.
DOI: 10.1007/s00572-013-0536-5
761. Eur J Soil Biol. 2013 Nov;59:8-14.
Oribatida (Acari) in grassy arable fallows are more affected by soil properties
than habitat age and plant species.
Wissuwa J(1), Salamon JA(1), Frank T(1).
Author information:
(1)University of Natural Resources and Life Sciences, Department of Integrative
Biology & Biodiversity Research, Institute of Zoology, Gregor Mendel Straße 33,
Oribatid mites are one of the numerically dominant arthropod groups in soils.
They play an important role in soil food webs via regulating the decomposition of
organic matter and propagating microorganisms within the soil. To our knowledge,
the influence of different plant functional groups on oribatid mites has not been
studied in abandoned farmland with undisturbed succession before. The density
and assemblage structure of oribatid mites in nine grassy arable fallows relative
to three habitat age classes (2-3, 6-8, 12-15 years) and three selected plant
species (legume: Medicago sativa, forb: Taraxacum officinale, grass: Bromus
sterilis) were investigated in soil associated with single plants. Mite density
declined marginally not significant with habitat age because of high abundances
of the ubiquitous species Tectocepheus velatus sarekensis and Punctoribates
punctum in young and mid-aged fallows and their subsequent decline in old
fallows. Oribatid mite density and species assemblage were not affected by plant
species. Only P. punctum had significantly higher densities in B. sterilis
samples than in T. officinale samples due to a higher amount of fine roots.
Distance-based linear models revealed that 65% of the variation in mite
assemblage was explained by soil properties, soil type, exposition and geographic
position, while habitat age was of minor importance. Canonical correspondence
analysis revealed that the mite assemblage was best explained by soil organic and
microbial carbon, water content and pH.
DOI: 10.1016/j.ejsobi.2013.08.002
PMCID: PMC4461176
PMID: 26109839
762. Microb Ecol. 2014 Feb;67(2):430-42. doi: 10.1007/s00248-013-0305-1. Epub 2013

Oct
26.
Trophic structure of amoeba communities near roots of Medicago sativa after

contamination with fuel oil no. 6.
Cortés-Pérez S(1), Rodríguez-Zaragoza S, Mendoza-López MR.
Author information:
(1)Laboratorio de Microbiología, UBIPRO, Fes-Iztacala UNAM, Av. De los Barrios #1
Col. Los Reyes Iztacala, Tlalnepantla Edo., México, C.P. 54090, Mexico,
p5_azul@rocketmail.com.
Root exudation increases microbial activity, selecting bacterial and fungal

communities that metabolize organic matter such as hydrocarbons. However, a
strong contamination pulse of hydrocarbons around plant roots may reorganize the
soil's microbial trophic structure toward amoebae feeding on bacteria. We
conducted a microcosm experiment to elucidate the effect of Medicago sativa on
the trophic structure of naked amoebae after a strong pulse of pollution (50,000
ppm of fuel oil no. 6, which is a mixture of long chains ranging from C10 to
C28). Plants were seeded 24 h after contamination and species of amoebae in the
microcosms were identified at 1, 30, and 60 days after pollution. Several species
from three trophic groups of naked amoeba were still alive 24 h after the
hydrocarbon pulse. Non-planted microcosms harbored three trophic groups after 60
days, while planted ones nourished four groups. The bacterivore group was the
most diverse in all microcosms, followed by protist-eaters and omnivores. The
quantity of amoebae was significantly higher (3.4×10(3) organisms/g soil) in the
planted pots than in the non-planted ones (1.3×10(3) organisms/g soil after 30
days of pollution (P ≤ 0.01). The shortest hydrocarbon chains (C10-C14)
disappeared or diminished in all microcosms, and the longest ones increased in
the planted ones. M. sativa thus exerted a positive effect on species richness,
quantity, and the composition of amoebae trophic groups in contaminated soil.
This indirect effect on bacterial predators is another key factor underlying
hydrocarbon assimilation by living organisms during phytoremediation.
DOI: 10.1007/s00248-013-0305-1
763. Can Vet J. 2013 May;54(5):456-62.
Cantharidin toxicosis in 2 alpacas.
Simpson KM(1), Streeter RN, De Souza P, Genova SG, Morgan SE.
Author information:
(1)Department of Veterinary Clinical Sciences (Simpson, Streeter, Genova),
Department of Veterinary Pathobiology (De Souza), Department of Veterinary
Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State
University, Stillwater, Oklahoma 74078, USA; Department of Veterinary
Physiological Sciences, Oklahoma Animal Disease Diagnostic Laboratory,
Stillwater, Oklahoma 74078, USA (Morgan).
Two adult alpacas were presented for recumbency and reluctance to rise.
Cantharidin toxicosis was suspected based on clinical and ancillary diagnostic
findings. The diagnosis was confirmed by gas chromatography-mass spectrometry of
gastric contents and urine. Despite medical treatment, neither alpaca survived.
Blister beetle toxicosis has not been previously described in camelids.
Challenges in treatment of affected ruminants or pseudoruminants are noted.
Publisher: Toxicose à la cantharidine chez 2 alpagas. Deux alpagas adultes ont

été présentés pour décubitus et une réticence à se lever. La toxicose à la
cantharidine a été soupçonnée en se fondant sur des résultats diagnostiques
cliniques et auxiliaires. Le diagnostic a été confirmé par chromatographie en
phase gazeuse et spectromètre de masse du contenu gastrique et de l’urine. Malgré
un traitement médical, les deux alpagas n’ont pas survécu. La toxicose aux
cantharides n’avait pas été décrite antérieurement chez les camélidés. Les
difficultés du traitement des ruminants ou des pseudoruminants sont
signalées.(Traduit par Isabelle Vallières).
PMCID: PMC3624911
764. J Anim Sci. 2013 Dec;91(12):5813-20. doi: 10.2527/jas.2013-6333. Epub 2013 Oct
21.
Effect of hay steaming on forage nutritive values and dry matter intake by
horses.
Earing JE(1), Hathaway MR, Sheaffer CC, Hetchler BP, Jacobson LD, Paulson JC,
Martinson KL.
Author information:
(1)Tennessee Farmers Cooperative, La Vergne, 37086.
Management strategies for horses with respiratory disease include soaking hay
before feeding. Hay steaming is an alternative to this practice; however, little
is known about its impact on forage nutritive values or intake. The objective was
to determine the effect of steaming on forage nutritive value and intake by
horses. Two alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.)
mixed hays were evaluated: a low moldy (NM) and moderately moldy (MM) hay. Six
mature horses were used in a 10 d crossover design. Three horses were assigned to
each hay type and treatments were switched on d 6. Each day, one bale of each hay
was sampled (pre- and poststeaming) and steamed for 90 min using a commercial hay
steamer. Two flakes of steamed or unsteamed NM or MM hay were weighed and offered
simultaneously to each horse in individual hay nets. Horses were allowed access
to hay for 2 h, orts were collected, and 2 h DMI was calculated. Six additional
bales of NM and MM were used to evaluate the effect of steaming on total
suspended particulate (TSP). Flakes of unsteamed or steamed hay were agitated in
an electric cement mixer, and TSP were recorded every min for 30 min using a
tapered element oscillating microbalance sampler. Paired t tests and PROC MIXED
of SAS (SAS Inst. Inc., Cary, NC) were used to compare steamed and unsteamed hay
nutritive values, mold concentration, TSP, and 2 h DMI. Steaming increased hay
moisture and therefore reduced DM to 77 and 81% for NM and MM, respectively (P <
0.001). In NM and MM hay, steaming reduced P content by 16 and 17%, respectively
(P ≤ 0.007). Steaming reduced water-soluble carbohydrates (WSC) and
ethanol-soluble carbohydrates (ESC) by 13% (P = 0.001) and 27% (P = 0.003),
respectively, for MM but had no effect on NM (P > 0.05). Steaming reduced mold
concentrations in both hays by ≥ 91% (P < 0.001). Total suspended particulate of
MM hay was reduced by 55% (P = 0.043), but TSP in NM hay was not affected by
steaming (P = 0.445). Dry matter intake of NM was increased by steaming; horses
ingested 0.64 kg of unsteamed and 2.02 kg of steamed hay (P < 0.001). Dry matter
intake of MM was not affected by steaming (P > 0.05). For NM hay, steaming
decreased P and mold concentrations and increased DMI of the hay but had no
effect on TSP. In MM hay, steaming reduced P, WSC, ESC, mold concentrations, and
TSP but did not affect DMI. Steaming represents a strategy for reducing TSP and
mold concentrations and increasing DMI in some hays but can result in leaching of
essential nutrients.
DOI: 10.2527/jas.2013-6333
765. Exp Parasitol. 2013 Dec;135(4):661-8. doi: 10.1016/j.exppara.2013.10.003. Epub

2013 Oct 17.
Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against

Meloidogyne exigua and in silico interaction of one chalcone with a putative
caffeic acid 3-O-methyltransferase from Meloidogyne incognita.
Nunes AS(1), Campos VP, Mascarello A, Stumpf TR, Chiaradia-Delatorre LD, Machado
AR, Santos Júnior HM, Yunes RA, Nunes RJ, Oliveira DF.
Author information:
(1)Universidade Federal de Lavras, Departamento de Química, 37200-000 Lavras, MG,
Brazil.
Meloidogyne exigua is a parasitic nematode of plants that causes great losses to

coffee farmers. In an effort to develop parasitic controls, 154 chalcones were
synthesized and screened for activity against this nematode. The best results
were obtained with
(2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50%
lethal concentration (LC50) of 171 μg/ml against M. exigua second-stage
juveniles, in comparison to the commercially-available nematicide carbofuran
which had an LC50 of 260 μg/ml under the same conditions. When coffee plants were
used, 6 reduced the nematode population to ~50% of that observed in control
plants. To investigate the mechanism of action of 6, an in silico study was
carried out, which indicated that 6 may act against M. exigua through inhibition
of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid
sequence of which was determined by examining the genome of Meloidogyne
incognita.
DOI: 10.1016/j.exppara.2013.10.003
766. Environ Entomol. 2013 Dec;42(6):1166-73. doi: 10.1603/EN13041. Epub 2013 Oct
14.
Health status of alfalfa leafcutting bee larvae (Hymenoptera: Megachilidae) in

United States alfalfa seed fields.
James RR(1), Pitts-Singer TL.
Author information:
(1)USDA-ARS Pollinating Insects Research Unit, Department of Biology, UMC 5310,
Utah State University, Logan, UT 84322-5310, USA.
We conducted a broad geographic survey in the northwestern United States to

quantify production losses in the alfalfa leafcutting bee (Megachile rotundata
(F.), Hymenoptera: Megachilidae), a solitary pollinator used extensively in
alfalfa seed production. Viable larvae were found in only 47.1% of the nest cells
collected at the end of the season. Most of the rest of the cells contained
pollen balls (typified by a provision but no larva; 16.7%), unknown causes of
mortality (15.5%), or larvae killed by chalkbrood (8.0%). Prevalence of pollen
balls was correlated positively with bee release rates and negatively with
alfalfa stand age. The unknown mortality was correlated with the U.S. Department
of Agriculture-Plant Hardiness Zone, and thus, some of the mortality may be
caused by high temperature extremes, although the nesting season degree-days were
not correlated with this mortality. Chalkbrood prevalence was correlated with
possible nesting-resource or crowding-related factors, such as the number of bees
released per hectare and the number of shelters used, but not with nesting board
disinfection practices. Vapona is used to control parasitoids when the parent
bees are incubated before release, and use of this fumigant was associated with
an increase in both chalkbrood and diapausing offspring, although any reason for
these correlations are unknown. This survey quantifies the variation in the
quality of alfalfa leafcutting bee cocoons produced across much of the U.S.
alfalfa seed production area.
DOI: 10.1603/EN13041
767. Toxicon. 2013 Dec 15;76:167-77. doi: 10.1016/j.toxicon.2013.10.003. Epub 2013

Oct
12.
Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to

cyanobacterial toxins (Microcystins) exposure.
El Khalloufi F(1), Oufdou K, Lahrouni M, Faghire M, Peix A, Ramírez-Bahena MH,

Vasconcelos V, Oudra B.
Author information:
(1)Laboratory of Biology and Biotechnology of Microorganisms, Environmental
Microbiology and Toxicology Unit, Faculty of Sciences Semlalia, Cadi Ayyad
University, PO Box 2390, Marrakech, Morocco.
Toxic cyanobacteria in freshwaters can induce potent harmful effects on growth

and development of plants irrigated with contaminated water. In this study, the
effect of cyanobacteria extract containing Microcystins (MC) on Medicago
sativa-rhizobia symbiosis was investigated in order to explore plants response
through biomass production, photosynthetic pigment and antioxidant enzymes
analysis: Peroxidase (POD), Polyphenoloxidase (PPO) and Catalase (CAT). Alfalfa
plants were inoculated with two endosymbiotic rhizobial strains: RhOL1 (MC less
sensitive strain) and RhOL3 (MC more sensitive strain), to evaluate the rhizobial
contribution on the plant response cultured under cyanobacterial toxins stress.
The two rhizobia strains were identified as Ensifer meliloti by sequence analysis
of their rrs and atpD genes. The chronic exposure to MC extract showed shoot,
root and nodules dry weight decrease, in both symbiosis cultures. The rate of
decline in plants inoculated with RhOL3 was higher than that in symbiosis with
RhOL1 mainly at 20 μg L(-1) of MC. Cyanotoxins also reduced photosynthetic
pigment content and generated an oxidative stress observed at cellular level.
POD, PPO and CAT activities were significantly increased in leaves, roots and
nodules of alfalfa plants exposed to MC. These enzyme activities were higher in
plants inoculated with RhOL3 especially when alfalfa plants were exposed to
20 μg L(-1) of MC. The present paper reports new scientific finding related to
the behavior of rhizobia-M. sativa associations to MC (Microcystins) for later
recommendation concerning the possible use of these symbiosis face to crops
exposure to MC contaminated water irrigation.
DOI: 10.1016/j.toxicon.2013.10.003
768. J Appl Microbiol. 2014 Jan;116(1):14-22. doi: 10.1111/jam.12356. Epub 2013 Oct
24.
Fluorescence in situ hybridization probing of protozoal Entodinium spp. and their

methanogenic colonizers in the rumen of cattle fed alfalfa hay or triticale
straw.
Xia Y(1), Kong YH, Seviour R, Forster RJ, Kisidayova S, McAllister TA.
Author information:
(1)Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB,
Canada; Department of Biological Science and Technology, Kunming University,
Kunming, China.
AIMS: To develop and test a fluorescence in situ hybridization (FISH) based

technique and to identify and quantify simultaneously those methanogenic
populations colonizing Entodinium spp. in the rumen of cows fed different
forages.
METHODS AND RESULTS: New FISH probes targeting protozoal Entodinium spp. were
designed and used together with FISH probes for methanogens in the cow rumen. The
composition and relative abundance of methanogenic populations colonizing
Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were
similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter
millerae and Methanobrevibacter smithii, and members of Methanomicrobium and
Methanosphaera were generally the predominant colonizers of protozoa, regardless
of the forage fed to cattle. Individual animals appeared to differ in which
ruminal methanogenic populations colonized each of the individual Entodinium spp.
CONCLUSIONS: Simultaneous FISH probing is shown here to be a reliable and
effective approach to investigate the dynamics of symbiotic relationships between
ruminal protozoa and methanogens at a single cell level. Phylogenetically closely
related Entodinium spp. were colonized by similar methanogenic populations
regardless of the forage fed.
SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the
methanogenic archaeal populations that specifically colonize Entodinium spp. as
identified using simultaneous FISH probing.
Her Majesty the Queen in Right of Canada © 2013 Society for Applied Microbiology
Reproduced with the permission of the Minister of Agriculture and Agri-Food
Canada.
DOI: 10.1111/jam.12356
769. Plant Cell Environ. 2014 Apr;37(4):911-21. doi: 10.1111/pce.12207. Epub 2013
Oct
25.
Moderating mycorrhizas: arbuscular mycorrhizas modify rhizosphere chemistry and

maintain plant phosphorus status within narrow boundaries.
Nazeri NK(1), Lambers H, Tibbett M, Ryan MH.
Author information:
(1)School of Plant Biology and Institute of Agriculture, The University of
Western Australia, 35 Stirling Highway, Perth, Western Australia, 6009,
Australia; Future Farm Industries Cooperative Research Centre, The University of
Western Australia, 35 Stirling Highway, Perth, Western Australia, 6009,
Australia.
Pastures often experience a pulse of phosphorus (P) when fertilized. We examined

the role of arbuscular mycorrhizal fungi (AMF) in the uptake of P from a pulse.
Five legumes (Kennedia prostrata, Cullen australasicum, Bituminaria bituminosa,
Medicago sativa and Trifolium subterraneum) were grown in a moderate P,
sterilized field soil, either with (+AMF) or without (-AMF) addition of
unsterilized field soil. After 9-10 weeks, half the pots received 15 mg P kg(-1)
of soil. One week later, we measured: shoot and root dry weights; percentage of
root length colonized by AMF; plant P, nitrogen and manganese (Mn)
concentrations; and rhizosphere carboxylates, pH and plant-available P. The P
pulse raised root P concentration by a similar amount in uncolonized and
colonized plants, but shoot P concentration increased by 143% in uncolonized
plants and 53% in colonized plants. Inoculation with AMF decreased the amount of
rhizosphere carboxylates by 52%, raised rhizosphere pH by ∼0.2-0.7 pH units and
lowered shoot Mn concentration by 38%. We conclude that AMF are not simply a
means for plants to enhance P uptake when P is limiting, but also act to maintain
shoot P within narrow boundaries and can affect nutrient uptake through their
influence on rhizosphere chemistry.
DOI: 10.1111/pce.12207
770. Int J Biol Macromol. 2013 Nov;62:387-96. doi: 10.1016/j.ijbiomac.2013.09.029.

Epub 2013 Oct 1.
Optimization of enzyme-assisted extraction of polysaccharides from alfalfa and

its antioxidant activity.
Wang S(1), Dong X, Tong J.
Author information:
Beijing 100193, China. Electronic address: shaopu.1988@163.com.
In this present study, an efficient complex enzyme-assisted extraction technology

was developed and optimized to extract polysaccharides from alfalfa using four
factors at five levels central composite rotatable response surface design
(CCRD). The experimental data was fitted to a second order polynomial equation
with high coefficient of determination values (R(2)>0.95). The results of
statistical analysis showed that the linear and quadratic terms of these four
variables had significant effects (P<0.05) on the yield of polysaccharides from
alfalfa. The optimum conditions were as follows: enzyme concentration of 2.5%,
2.0%, 3.0% (weight of alfalfa) of cellulase, papain and pectase, extraction
temperature 52.7 °C, extraction pH 3.87, ratio of water to raw material 78.92
mL/g and extraction time 2.73 h. Under the optimal conditions, the experimental
extraction yield of alfalfa polysaccharides was 5.05 ± 0.02%, which was well
matched with the value (5.09%) predicted by the CCRD model. Moreover, evaluation
of the antioxidant activity of polysaccharides from alfalfa in vitro suggested
that the polysaccharides had good antioxidant effect, especially scavenging
activity for hydroxyl radical and DPPH radical, which indicated that the
polysaccharides from alfalfa may be explored as a novel natural antioxidant.
DOI: 10.1016/j.ijbiomac.2013.09.029
771. Appl Microbiol Biotechnol. 2013 Dec;97(24):10489-98. doi:

10.1007/s00253-013-5276-0. Epub 2013 Oct 5.
Biosafety and colonization of Burkholderia multivorans WS-FJ9 and its

growth-promoting effects on poplars.
Li GX(1), Wu XQ, Ye JR.
Author information:
(1)College of Forest Resources and Environment, Nanjing Forestry University,
Nanjing, Jiangsu, 210037, China.
Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting

biological characteristics, which make them simultaneously beneficial and harmful
to humans. They have been exploited for biocontrol, bioremediation, and plant
growth promotion. However, their capacity as opportunistic bacteria that infect
humans restricts their biotechnological applications. Therefore, the risks of
using these bacteria should be assessed. In this study, Burkholderia multivorans
WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient
in solubilizing phosphate, was evaluated with respect to its biosafety,
colonization in poplar rhizosphere, and growth-promoting effects on poplar
seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined
experimentally using onion and tobacco as model plants. Onion bulb inoculated
with B. multivorans WS-FJ9 showed slight hypersensitive responses around the
inoculation points, but effects were not detectable based on the inner color and
odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited
slightly water-soaked spots around the inoculation points, which did not expand
or develop into lesions even with repeated incubation. Pathogenicity of the
strain in alfalfa, which has been suggested as an alternative Bcc model for mice,
was not detectable. Results from gene-specific polymerase chain reactions showed
that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA
virulence genes. Scanning electron microscopy revealed that the colonization of
the WS-FJ9 strain reached 1.4 × 10(4) colony forming units (cfu) g(-1)
rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain
could colonize the rhizosphere as well as the root tissues and cells of poplars.
Greenhouse evaluations in both sterilized and non-sterilized soils indicated that
B. multivorans WS-FJ9 significantly promoted growth in height, root collar
diameter, and plant biomass of inoculated poplar seedlings compared with
controls. Phosphorus contents of roots and stems of treated seedlings were 0.57
and 0.55 mg g(-1) higher than those of the controls, respectively. Phosphorus
content was lower in the rhizosphere soils by an average of 1.03 mg g(-1)
compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a
nonpathogenic strain that could colonize the roots and significantly promote the
growth of poplar seedlings.
DOI: 10.1007/s00253-013-5276-0
1.
Effect of selenium concentration on feed preferences by cattle and sheep.
Pfister JA(1), Davis TZ, Hall JO.
Author information:
(1)USDA-ARS Poisonous Plant Research Laboratory, Logan, UT 84341.
Selenium-accumulator plants are reputed to be unpalatable to livestock. The

objective of this study was to determine if sheep and cattle could discriminate
between forages and feeds with different concentrations of Se. In the first
study, cattle and sheep preferences for intermediate wheatgrass (Thinopyrum
intermedium), alfalfa (Medicago sativa), and western aster (Symphyotrichum
ascendens) of varying Se concentrations were assessed. The Se concentrations
ranged from 0.8 to 50 mg/kg (DM) in grass, 1.4 to 275 mg/kg in alfalfa, and 4 to
4,455 mg/kg in aster. Selenium concentration had no influence (P > 0.05) on the
initial or subsequent preferences of sheep or cattle for grass or alfalfa. Cattle
developed an aversion to aster after consuming 95% of the plant material during
the first brief exposure and subsequently refused to eat any aster. Sheep
consumption of aster was variable, but their preference was not driven by Se
concentration. In the next study, cattle and sheep were offered pellets at 1.5%
of BW (as fed) that contained increasing concentrations of Se from aster (control
and 5, 25, 45, and 110 mg/kg Se). In trial 1, all pellets were offered. In Trials
2 and 3, all pellets were offered with the exception of the 5 mg/kg Se pellet and
the 5 and 25 mg/kg Se pellets, respectively. In trial 1, consumption of the
control pellet by cattle was greater on all days compared with other Se pellets
(P < 0.001). Cattle ate more (P < 0.001) of the 5 mg/kg Se pellet than the higher
Se pellets on d 3, 4, and 5. Sheep ate greater amounts of the control and 5 and
110 mg/kg Se pellets compared with the 25 and 45 mg/kg Se pellets (P < 0.0001) on
d 1, and sheep consumed primarily the control and 5 mg/kg Se pellets thereafter.
In trial 2, cattle and sheep consumed more (P < 0.0001) of the control Se pellet
than the 25, 45, and 110 mg/kg Se pellets. In trial 3, cattle consumption of the
control and 45 and 110 mg/kg Se pellets differed on d 2 and 3 (P < 0.001), except
there was no difference (P > 0.95) in cattle consumption of the control and 45
mg/kg Se pellets on d 1. Sheep consumed primarily the control and 45 mg/kg Se
pellets. We conclude that high Se concentrations in fresh forages had no effect
on initial consumption by cattle or sheep. When given Se pellets, initial
responses were variable, but the results indicate that cattle and sheep adjusted
their intake over time to avoid excessive intake of Se.
DOI: 10.2527/jas.2013-6595
773. Exp Appl Acarol. 2014 Feb;62(2):195-214. doi: 10.1007/s10493-013-9729-z. Epub

2013 Sep 26.
A qualitative and quantitative study of mites in similar alfalfa fields in

Greece.
Badieritakis EG(1), Fantinou AA, Emmanouel NG.
Author information:
(1)Laboratory of Agricultural Zoology and Entomology, Agricultural University of
Athens, Iera Odos 75, Votanicos, 118 55, Athens, Greece, ebadieritakis@yahoo.gr.
The present study investigated the mite fauna and the relative abundance of mites
present in foliage and litter of two adjacent and similar alfalfa fields,
differing only in the number of cuttings, in Kopais Valley (Central Greece)
through 2008-2010. We also examined the relationship between assemblage patterns
of Mesostigmata, Oribatida and Prostigmata by comparing their population
fluctuation, population density, species richness and diversity. Spatial
distribution of common dominant and influent mite species was also estimated. Our
results showed that both fields supported a very rich and similar mite fauna with
eight new species records for alfalfa of Greece, although these species have been
previously reported from other habitats in Greece. The pattern of population
fluctuation in foliage was similar in both fields, unlike the fluctuation in
litter. Population density significantly differed between fields, being higher in
the less harvested field, except Prostigmata. Species richness in litter was
higher in the less harvested field, whereas it was higher in the foliage of the
more harvested field, apart from that of prostigmatic mites in litter, which was
higher in the more harvested field, and that of oribatid mites in foliage, which
was higher in the less harvested field. The diversity of mites was higher in the
more harvested field, with the exception of prostigmatic mites. The spatial
distribution of mites in foliage and litter was aggregated in both fields. Our
results indicate that despite the considerable similarity of the study fields,
the different harvesting frequency might have disturbed differently the mite
communities hosted in foliage and litter.
DOI: 10.1007/s10493-013-9729-z
774. J Exp Bot. 2013 Nov;64(16):5157-72. doi: 10.1093/jxb/ert305. Epub 2013 Sep 21.
Characterization and genetic dissection of resistance to spotted alfalfa aphid

(Therioaphis trifolii) in Medicago truncatula.
Kamphuis LG(1), Lichtenzveig J, Peng K, Guo SM, Klingler JP, Siddique KH, Gao LL,
Singh KB.
Author information:
(1)CSIRO Plant Industry, Private Bag 5, Wembley, WA 6913, Australia.
Aphids cause significant yield losses in agricultural crops worldwide. Medicago

truncatula, a model legume, cultivated pasture species in Australia and close
relative of alfalfa (Medicago sativa), was used to study the defence response
against Therioaphis trifolii f. maculate [spotted alfalfa aphid (SAA)]. Aphid
performance and plant damage were compared among three accessions. A20 is highly
susceptible, A17 has moderate resistance, and Jester is strongly resistant.
Subsequent analyses using A17 and A20, reciprocal F1s and an A17×A20 recombinant
inbred line (RIL) population revealed that this moderate resistance is phloem
mediated and involves antibiosis and tolerance but not antixenosis. Electrical
penetration graph analysis also identified a novel waveform termed extended
potential drop, which occurred following SAA infestation of M. truncatula.
Genetic dissection using the RIL population revealed three quantitative trait
loci on chromosomes 3, 6, and 7 involved in distinct modes of aphid defence
including antibiosis and tolerance. An antibiosis locus resides on linkage group
3 (LG3) and is derived from A17, whereas a plant tolerance and antibiosis locus
resides on LG6 and is derived from A20, which exhibits strong temporary
tolerance. The loci identified reside in regions harbouring classical resistance
genes, and introgression of these loci in current medic cultivars may help
provide durable resistance to SAA, while elucidation of their molecular
mechanisms may provide valuable insight into other aphid-plant interactions.
DOI: 10.1093/jxb/ert305
PMCID: PMC3830491
775. Mol Biol Rep. 2013 Nov;40(11):6451-8. doi: 10.1007/s11033-013-2760-7. Epub

2013
Sep 22.
Characterization of a novel Medicago sativa NAC transcription factor gene

involved in response to drought stress.
Wang YX(1).
Author information:
(1), Beijing, China, wang001224@163.com.
Relying on the regulation of transcription factors, plants resist to various

abiotic and biotic stresses. NAC (NAM, ATAF1/2, CUC2) are one of the largest
families of plant-specific transcription factors and known to play important
roles in plant development and response to environmental stresses. A new NAC gene
was cloned on the basis of 503 bp EST fragment from the SSH cDNA library of
Medicago sativa. It was 1,115 bp including an 816 bp ORF and encodes 271 amino
acids. A highly conserved region is located from the 7th amino acid to the 315th
amino acid in its N-terminal domain. The NAC protein is subcellularly localized
in the nucleus of onion epidemical cells and possible functions as a
transcription factor. The relative quantitative real-time RT-PCR was performed at
different stress time. The results revealed that the transcription expression of
NAC gene could be induced by drought, high salinity and ABA. The transgenic
Arabidopsis with NAC gene has the drought tolerance better than the wild-type.
DOI: 10.1007/s11033-013-2760-7
776. J Dairy Sci. 2013;96(11):7110-9. doi: 10.3168/jds.2012-6462. Epub 2013 Sep 18.
Effects of alfalfa hay particle size in high-concentrate diets supplemented with

unsaturated fat: chewing behavior, total-tract digestibility, and milk production
of dairy cows.
Kahyani A(1), Ghorbani GR, Khorvash M, Nasrollahi SM, Beauchemin KA.
Author information:
(1)Department of Animal Sciences, Isfahan University of Technology, Isfahan
84156-83111, Iran.
This study evaluated the effects of increasing the physically effective neutral
detergent fiber (peNDF) intake of lactating dairy cows fed high-concentrate diets
supplemented with unsaturated fat on intake, eating behavior, diet sorting,
chewing activity, total-tract digestibility, and milk production and composition.
Diets contained 24% alfalfa hay (AH), 16% corn silage, 58% concentrate, and 2%
yellow grease [dry matter (DM) basis], and dietary peNDF content was increased by
varying the particle size (PS) of the AH. Nine multiparous cows averaging 87.8 ±
14.8d in milk and weighing 653 ± 53 kg were randomly assigned to a triplicate 3 ×
3 Latin square. During each 21-d period, cows were offered 1 of 3 total mixed
rations that varied in PS of AH: fine, medium, and long, with a geometric mean
particle length of 3.00, 3.57 and 3.87 mm, respectively. Increasing PS
quadratically affected DM intake (DMI; 24.7, 25.4, and 23.7 kg/d, for fine,
medium, and long, respectively), but cumulative DMI at 2, 4, and 6h after feeding
was similar across treatments, averaging 23.4, 35.6 and 46.4% of total DMI for
the 3 time points, respectively. Increased peNDF intake did not affect feed
sorting, but increased daily eating time, and eating and total chewing time per
kilogram of DMI. Daily rumination time exhibited a quadratic response, with
highest rumination time for the medium diet. Dietary PS had no effects on
digestibility in the total tract, but we observed, for fine, medium, and long
diets, quadratic responses in milk production (41.5, 43.3, and 40.4 kg/d), 4%
fat-corrected milk production, and milk protein yield. Milk fat content decreased
linearly with increasing PS, but milk fat content and fat:protein ratio were low
for all treatments, likely due to adding unsaturated fat to a diet containing a
high level of nonfiber carbohydrates (42.2% of DM). The composition, degree of
saturation, and total conjugated linoleic acid content of fatty acids in milk fat
were not affected by the change in peNDF content of the diet. The study indicates
that a moderate increase in the PS of AH in diets containing unsaturated fat
elevates peNDF intake and increases chewing activity, DMI, milk yield and milk
fat production. However, the effects of dietary PS were quadratic, with maximum
DMI and milk production observed with diets supplying 24% dietary peNDF (measured
as the proportion of the ration retained on sieves >1.18 mm multiplied by dietary
neutral detergent fiber content; DM basis).
DOI: 10.3168/jds.2012-6462
777. Appl Environ Microbiol. 2013 Dec;79(23):7150-9. doi: 10.1128/AEM.02225-13.

Epub
2013 Sep 13.
The Sinorhizobium meliloti ntrX gene is involved in succinoglycan production,

motility, and symbiotic nodulation on alfalfa.
Wang D(1), Xue H, Wang Y, Yin R, Xie F, Luo L.
Author information:
(1)School of Life Science, Anhui University, Heifei, Anhui, China.
Rhizobia establish a symbiotic relationship with their host legumes to induce the
formation of nitrogen-fixing nodules. This process is regulated by many rhizobium
regulators, including some two-component regulatory systems (TCSs). NtrY/NtrX, a
TCS that was first identified in Azorhizobium caulinodans, is required for
free-living nitrogen metabolism and symbiotic nodulation on Sesbania rostrata.
However, its functions in a typical rhizobium such as Sinorhizobium meliloti
remain unclear. Here we found that the S. meliloti response regulator NtrX but
not the histidine kinase NtrY is involved in the regulation of exopolysaccharide
production, motility, and symbiosis with alfalfa. A plasmid insertion mutant of
ntrX formed mucous colonies, which overproduced succinoglycan, an
exopolysaccharide, by upregulating its biosynthesis genes. This mutant also
exhibited motility defects due to reduced flagella and decreased expression of
flagellins and regulatory genes. The regulation is independent of the known
regulatory systems of ExoR/ExoS/ChvI, EmmABC, and ExpR. Alfalfa plants inoculated
with the ntrX mutant were small and displayed symptoms of nitrogen starvation.
Interestingly, the deletion mutant of ntrY showed a phenotype similar to that of
the parent strain. These findings demonstrate that the S. meliloti NtrX is a new
regulator of succinoglycan production and motility that is not genetically
coupled with NtrY.
DOI: 10.1128/AEM.02225-13
PMCID: PMC3837732
778. Meat Sci. 2014 Jan;96(1):606-9. doi: 10.1016/j.meatsci.2013.08.027. Epub 2013

Aug
27.
Effect of dietary alfalfa on the fatty acid composition and indexes of lipid
metabolism of rabbit meat.
Dal Bosco A(1), Mugnai C, Roscini V, Mattioli S, Ruggeri S, Castellini C.
Author information:
(1)Department of Applied Biology, University of Perugia, Borgo XX Giugno 74,
06121 Perugia, Italy. Electronic address: dalbosco@unipg.it.
The objective of the study was to analyse and increase the quality of rabbit meat
by increasing the levels of natural bioactive compounds through providing fresh
alfalfa to rabbits as complementary feed. At 50 days of age, forty rabbits were
divided into two homogeneous groups and fed pelleted feed (control group) or
pelleted feed plus fresh alfalfa (alfalfa group). The lipid content of meat was
significantly higher in the control group likely due to the higher feed
consumption observed in this group. The same trend was observed in the contents
of myristic, palmitoleic, oleic and linoleic acid. On the contrary, the
supplementation of fresh alfalfa increased the stearic, linolenic,
eicosatrienoic, eicosapentaenoic, docosapentaenoic, docosahexaenoic and total
polyunsaturated fatty acid contents; also the total monounsaturated fatty acids
and the α- and γ-tocopherol content was significantly lower. Moreover, alfalfa
ingestion decreased the n-6 content, n-6/n-3 ratio and thrombogenicity index.
© 2013.
DOI: 10.1016/j.meatsci.2013.08.027
779. New Phytol. 2014 Jan;201(1):116-30. doi: 10.1111/nph.12486. Epub 2013 Sep 13.
Early transcriptional responses to mercury: a role for ethylene in

mercury-induced stress.
Montero-Palmero MB(1), Martín-Barranco A, Escobar C, Hernández LE.
Author information:
(1)Laboratorio de Fisiología Vegetal, Departamento de Biología, Universidad
Autónoma de Madrid, 28049, Madrid, Spain; Laboratorio de Fisiología Vegetal,
Departamento de Ciencias Ambientales, Universidad de Castilla-La Mancha, Campus
Fábrica de Armas, 45070, Toledo, Spain.
Understanding the cellular mechanisms of plant tolerance to mercury (Hg) is

important for developing phytoremediation strategies of Hg-contaminated soils.
The early responses of alfalfa (Medicago sativa) seedlings to Hg were studied
using transcriptomics analysis. A Medicago truncatula microarray was hybridized
with high-quality root RNA from M. sativa treated with 3 μM Hg for 3, 6 and 24 h.
The transcriptional pattern data were complementary to the measurements of root
growth inhibition, lipid peroxidation, hydrogen peroxide (H2 O2 ) accumulation
and NADPH-oxidase activity as stress indexes. Of 559 differentially expressed
genes (DEGs), 91% were up-regulated. The majority of DEGs were shared between the
3 and 6 h (60%) time points, including the 'stress', 'secondary metabolism' and
'hormone metabolism' functional categories. Genes from ethylene metabolism and
signalling were highly represented, suggesting that this phytohormone may be
relevant for metal perception and homeostasis. Ethylene-insensitive alfalfa
seedlings preincubated with the ethylene signalling inhibitor
1-methylcyclopronene and Arabidopsis thaliana ein2-5 mutants confirmed that
ethylene participates in the early perception of Hg stress. It modulates root
growth inhibition, NADPH-oxidase activity and Hg-induced apoplastic H2 O2
accumulation. Therefore, ethylene signalling attenuation could be useful in
future phytotechnological applications to ameliorate stress symptoms in
Hg-polluted plants.
DOI: 10.1111/nph.12486
780. Appl Environ Microbiol. 2013 Nov;79(22):7021-7. doi: 10.1128/AEM.01704-13.

Epub
2013 Sep 6.
Survival of murine norovirus, Tulane virus, and hepatitis A virus on alfalfa

seeds and sprouts during storage and germination.
Wang Q(1), Hirneisen KA, Markland SM, Kniel KE.
Author information:
Delaware, USA.
Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several
produce-associated outbreaks and identified as major food-borne viral etiologies.
In this study, the survival of huNoV surrogates (murine norovirus [MNV] and
Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and
postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers
of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue
culture infectious doses (TCID50)/g, respectively. Inoculated seeds were stored
for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5,
10, and 15. Following storage, virus presence was monitored over a 1-week
germination period. Viruses remained infectious after 50 days, with titers of
1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50/g for
MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and
TV, without a statistically significant reduction over 20 days (<1 log TCID50/g);
however, relatively high levels of genomic copies of all viruses persisted over
the testing time period. Low titers of viruses were found on sprouts and were
located in all tissues as well as in sprout-spent water sampled on days 1, 3, and
6 following seed planting. Results revealed the persistence of viruses in seeds
for a prolonged period of time, and perhaps of greater importance these data
suggest the ease of which virus may transfer from seeds to sprouts and spent
water during germination. These findings highlight the importance of sanitation
and prevention procedures before and during germination.
DOI: 10.1128/AEM.01704-13
PMCID: PMC3811553
781. Can J Microbiol. 2013 Sep;59(9):598-603. doi: 10.1139/cjm-2013-0289. Epub 2013

Jul 11.
Impact of D-amino acid dehydrogenase on virulence factor production by a

Pseudomonas aeruginosa.
Oliver KE(1), Silo-Suh L.
Author information:
(1)a Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA.
Chronic Pseudomonas aeruginosa infections remain the leading cause of lung

dysfunction and mortality for cystic fibrosis (CF) patients. Many other bacteria
inhabit the CF lung, but P. aeruginosa utilizes novel strategies that allow it to
colonize this environment as the predominant bacterial pathogen. D-Amino acid
dehydrogenase encoded by dadA is highly expressed by P. aeruginosa within the CF
lung, and it is required for optimal production of hydrogen cyanide by some
CF-adapted isolates. To better understand the increased significance of D-amino
acid dehydrogenase in P. aeruginosa physiology, we characterized the contribution
of the dad operon to virulence factor production. In this study, we determined
that DadA is required for optimal production of pyocyanin, pyoverdine, and
rhamnolipid by CF-adapted and non-CF-adapted isolates of P. aeruginosa. In
addition, DadA is required for optimal production of alginate, biofilm formation,
and virulence of a CF-adapted isolated of P. aeruginosa in an alfalfa seedling
model of infection. Taken together, the results indicate that DadA plays a
pleiotropic role in the production of important virulence factors by P.
aeruginosa.
DOI: 10.1139/cjm-2013-0289
782. Food Chem. 2013 Dec 15;141(4):3738-43. doi: 10.1016/j.foodchem.2013.06.090.

Epub
2013 Jun 28.
Study of selenocompounds from selenium-enriched culture of edible sprouts.
Funes-Collado V(1), Morell-Garcia A, Rubio R, López-Sánchez JF.
Author information:
(1)Departament de Química Analítica, Facultat de Química, Universitat de
Barcelona, C/Martí i Franquès 1-11, E-08028 Barcelona, Spain.
Selenium is recognised as an essential micronutrient for humans and animals. One

of the main sources of selenocompounds in the human diet is vegetables.
Therefore, this study deals with the Se species present in different edible
sprouts grown in Se-enriched media. We grew alfalfa, lentil and soy in a
hydroponic system amended with soluble salts, containing the same proportion of
Se, in the form of Se(VI) and Se(IV). Total Se in the sprouts was determined by
acidic digestion in a microwave system and by ICP/MS. Se speciation was carried
out by enzymatic extraction (Protease XIV) and measured by LC-ICP/MS. The study
shows that the Se content of plants depends on the content in the growth culture,
and that part of the inorganic Se was biotransformed mainly into SeMet. These
results contribute to our understanding of the uptake of inorganic Se and its
biotransformation by edible plants.
DOI: 10.1016/j.foodchem.2013.06.090
783. Meat Sci. 2014 Jan;96(1):394-9. doi: 10.1016/j.meatsci.2013.07.028. Epub 2013

Jul
29.
Meat quality, proximate composition and muscle fatty acid profile of young llamas
(Lama glama) supplemented with hay or concentrate during the dry season.
Mamani-Linares LW(1), Gallo CB.
Author information:
(1)Becario MECESUP2 AUS 0601, Programa de Doctorado en Ciencias Veterinarias,
Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile;
Instituto de Ciencia Animal, Facultad Ciencias Veterinarias, Universidad Austral
de Chile, Valdivia, Chile.
Thirty llamas were used to study the effect of a 90 day feed supplementation on
meat quality, chemical composition and muscle fatty acid profile. Treatments
were: GR=llama on native pasture until slaughter; GR+SH=like GR, but with
overnight free access to barley/alfalfa hay; and GR+SC=like GR, but with
overnight free access to a wheat bran/sorghum grain concentrate. The
supplementation had no effect on postmortem pH and temperature decline in the
Longissimus lumborum muscle (LLM), cooking losses nor Warner-Bratzler shear force
values (P>0.05). Meat from GR+SC llama had higher fat content in LLM (P<0.05)
compared to GR and GR+SH llama. Intramuscular fat from GR+SH llama showed higher
(P<0.01) proportions of polyunsaturated fatty acids, higher (P<0.05)
polyunsaturated fatty acids/saturated fatty acids and desirable fatty acids
ratio, lower (P<0.05) omega-6/omega-3 (n-6/n-3) ratio, and higher (P<0.01)
conjugated linoleic acid.
© 2013.
DOI: 10.1016/j.meatsci.2013.07.028
784. J Anim Sci. 2013 Oct;91(10):4757-64. doi: 10.2527/jas.2013-6307. Epub 2013 Aug
21.
Effect of graded levels of fiber from alfalfa meal on intestinal nutrient and
energy flow, and hindgut fermentation in growing pigs.
Chen L(1), Zhang HF, Gao LX, Zhao F, Lu QP, Sa RN.
Author information:
(1)State Key Laboratory of Animal Nutrition, Institute of Animal Sciences,
Chinese Academy of Agricultural Sciences, Beijing, China 100193.
The objective of this experiment was to determine the effects of fiber level from
alfalfa meal and collection period on intestinal nutrient and energy flow, and
hindgut fermentation in growing pigs. Twenty-four pigs (initial BW = 21.4 ± 1.5
kg) were prepared by T-cannula insertion into the distal ileum and allotted to 4
treatments. The pigs were provided a corn-soybean meal control diet or a diet in
which corn and soybean meal were partly replaced by 5%, 10%, or 20% alfalfa meal
to give the graded levels of dietary fiber during two 10-d collection periods.
The BW of pigs at the start of periods 1 and 2 were 32.2 ± 2.4 and 37.7 ± 4.2 kg,
respectively. The final BW at the end of period 2 was 46.8 ± 4.0 kg. The apparent
ileal digestibility (AID) and apparent total tract digestibility (ATTD) of
nutrients and energy were measured. The VFA concentration was also determined in
the ileal and fecal samples. Nutrient digestibility was not affected by inclusion
of 5% alfalfa meal in the diet. The AID, ATTD, and hindgut fermentation of DM,
carbohydrates (CHO), and GE decreased (linear, P < 0.05) as the level of alfalfa
meal in the diet increased. The intestinal flow of DM, CHO, ADF, and GE increased
(linear, P < 0.05) by increasing the level of dietary alfalfa. The pH in ileal
digesta and concentration of acetate, propionate, and total VFA in the feces
increased (linear, P < 0.01) as the level of alfalfa meal in the diet increased.
From experiment period 1 to 2, ATTD and hindgut fermentation of CHO decreased (P
< 0.01), whereas concentrations of propionate, valerate, and total VFA in ileal
samples increased (P < 0.05). A multiple linear regression analysis, taking into
account both soluble dietary fiber and insoluble dietary fiber intake, explained
the variation (P < 0.01) in total tract flow of NDF (93%), ADF (84%), GE (73%),
DM (71%), and CHO (62%). In conclusion, the diet containing 5% of alfalfa meal
did not affect nutrient and energy digestion. Intestinal flow of CHO decreased
during the 10-d collection period. The VFA concentration increased with fiber
level and 10-d exposure to the experimental diets. Soluble and insoluble fibers
from alfalfa meal have differential roles in nutrient digestion, which may help
explain the variation observed in the intestinal flow of nutrients.
DOI: 10.2527/jas.2013-6307
785. J Biol Chem. 2013 Oct 4;288(40):28845-58. doi: 10.1074/jbc.M113.493155. Epub

2013
Aug 20.
Cloning and structure-function analyses of quinolone- and acridone-producing

novel type III polyketide synthases from Citrus microcarpa.
Mori T(1), Shimokawa Y, Matsui T, Kinjo K, Kato R, Noguchi H, Sugio S, Morita H,

Abe I.
Author information:
(1)From the Graduate School of Pharmaceutical Sciences, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033.
Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone
synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino
acid sequence of C. microcarpa QNS is unique, and it shared only 56-60%
identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and
the previously reported Aegle marmelos QNS. In contrast to the quinolone- and
acridone-producing A. marmelos QNS, C. microcarpa QNS produces
4-hydroxy-N-methylquinolone as the "single product" by the one-step condensation
of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows
broad substrate specificities and produces not only acridone and quinolone but
also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA,
benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal
structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions,
respectively, revealed wide active site entrances in both enzymes. The wide
active site entrances thus provide sufficient space to facilitate the binding of
the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the
active site cavity volume of C. microcarpa ACS (760 Å(3)) is almost as large as
that of M. sativa CHS (750 Å(3)), and ACS produces acridone by employing an
active site cavity and catalytic machinery similar to those of CHS. In contrast,
the cavity of C. microcarpa QNS (290 Å(3)) is significantly smaller, which makes
this enzyme produce the diketide quinolone. These results as well as mutagenesis
analyses provided the first structural bases for the anthranilate-derived
production of the quinolone and acridone alkaloid by type III polyketide
synthases.
DOI: 10.1074/jbc.M113.493155
PMCID: PMC3789980

Aug
16.
Corn silage hybrid type and quality of alfalfa hay affect dietary nitrogen
utilization by early lactating dairy cows.
Holt MS(1), Neal K, Eun JS, Young AJ, Hall JO, Nestor KE Jr.
Author information:
Logan 84322.
This experiment was conducted to determine the effects of corn silage (CS)
hybrids and quality of alfalfa hay (AH) in high-forage dairy diets on N
utilization, ruminal fermentation, and lactational performance by early-lactating
dairy cows. Eight multiparous Holstein cows were used in a duplicated 4 × 4 Latin
square experiment with a 2 × 2 factorial arrangement of dietary treatments. The 8
cows (average days in milk = 23 ± 11.2) were surgically fitted with ruminal
cannula, and the 2 squares were conducted simultaneously. Within square, cows
were randomly assigned to a sequence of 4 diets: conventional CS (CCS) or brown
midrib CS (BMR) was combined with fair-quality AH [FAH: 46.7% neutral detergent
fiber (NDF) and 18.4% crude protein (CP)] or high-quality AH (HAH: 39.2% NDF and
20.7% CP) to form 4 treatments: CCS with FAH, CCS with HAH, BMR with FAH, and BMR
with HAH. Diets were isonitrogenous across treatments, averaging 15.9% CP. Each
period lasted a total of 21 d, with 14 d for treatment adaptation and 7d for data
collection and sampling. Intake of DM and milk yield did not differ in response
to CS hybrids or AH quality. Although feeding BMR-based diets decreased urinary N
output by 24%, it did not affect fecal N output. Feeding HAH decreased urinary N
output by 15% but increased fecal N output by 20%. Nitrogen efficiency [milk N
(g/d)/intake N (g/d)] tended to increase for BMR treatments. Ruminal ammonia-N
concentration was lower for cows fed BMR-based diets than for those fed CCS-based
diets but was not affected by quality of AH. Feeding BMR-based diets or HAH
decreased milk urea N concentration by 23 or 15%, respectively, compared with
CCS-based diets or FAH. Total volatile fatty acid concentration increased with
HAH but was not influenced by CS hybrids. Feeding BMR-based diets decreased
urinary N-to-fecal N ratio (UN:FN), and it was further reduced by feeding HAH.
Although cows fed the BMR-based diets tended to increase milk N-to-manure N
ratio, the quality of AH did not affect the ratio. The lower ratio of UN:FN with
a higher ratio of milk N-to-manure N ratio for the BMR-based diets indicates that
feeding BMR may reduce manure ammonia-N by reducing excretion of urinary N and
increasing secretion of milk N per unit of manure N excreted.

DOI: 10.3168/jds.2013-6689

Aug
16.
Effects of short-term variation in forage quality and forage to concentrate ratio

on lactating dairy cows.
Yoder PS(1), St-Pierre NR, Daniels KM, O'Diam KM, Weiss WP.
Author information:
Center, The Ohio State University, Wooster 44691.
Within-farm variation in forage composition can be substantial and potentially

costly, and it presents challenges for sampling the forage accurately. We
hypothesized that day-to-day variation in forage neutral detergent fiber (FNDF)
concentrations and diet variation caused by sampling error would have negative
effects on production measures in lactating dairy cows. Twenty-four Holstein cows
(73 d in milk) were used in 8 replicated 3 × 3 Latin squares with 21-d periods.
Treatments were (1) control (CON), (2) variable (VAR), and (3) overreacting
(ORR). On average, over the 21-d period, all 3 treatments were the same [24.7%
FNDF and 48.2% forage dry matter (DM) composed of 67% alfalfa silage and 33%
grass silage]. The CON treatment was essentially consistent day-to-day in total
forage and FNDF concentrations and proportion of alfalfa and grass silages. The
VAR treatment changed daily (in a random pattern) in proportion of alfalfa and
grass silages fed, which resulted in day-to-day changes in FNDF (range was 21.5
to 28%). The ORR treatment varied in a 5-d cyclic pattern in total forage and
FNDF concentrations (26, 24, 28, and 21.5% FNDF). Over the 21 d, ORR (25.1 kg/d)
had higher DM intake compared with CON (24.5 kg/d) and VAR (24.3 kg/d). Milk
production (42.8 kg/d), milk fat (3.5%), and milk protein (2.8%) were not
affected by treatment; however, a treatment × day interaction was observed for
milk production. Lower daily milk yields for VAR and ORR compared with CON were
rare; they only followed sustained 4- and 5-d periods of feeding higher FNDF
diets compared with CON. In contrast, increased daily milk yields for VAR and ORR
versus CON were more frequent and followed sustained diet changes of only 2 or
3d. Lipolytic and lipogenic-related enzyme mRNA abundances in subcutaneous
adipose tissue were not affected by treatment. Treatment × day interactions were
observed for milk fatty acid markers of cellulolytic bacteria (iso-14:0,
iso-15:0, iso-16:0) and lipolysis (18:0) and generally followed the expected
response to changes in daily rations. Overall, extreme daily fluctuations in FNDF
had no cumulative negative effect on production measures over a 21-d period, and
daily responses to transient increases in FNDF were less than expected.

DOI: 10.3168/jds.2013-6724

10.1111/jpn.12120.
Epub 2013 Aug 20.
Effects of pistachio by-products in replacement of alfalfa hay on populations of

rumen bacteria involved in biohydrogenation and fermentative parameters in the
rumen of sheep.
Ghaffari MH(1), Tahmasbi AM, Khorvash M, Naserian AA, Ghaffari AH, Valizadeh H.
Author information:
The objective of this study was to investigate the effect of sundried pistachio
by-products (PBP) as a replacement of alfalfa hay (AH) on blood metabolites,
rumen fermentation and populations of rumen bacteria involved in biohydrogenation
(BH) in Baluchi sheep. Four adult male Baluchi sheep (41 ± 1.3 kg, BW) fitted
with ruminal cannulae were randomly assigned to four experimental diets in a 4 ×
4 Latin square design. The dietary treatments were as follows: (i) control, (ii)
12% PBP (0.33 of AH in basal diet replaced by PBP), (iii) 24% PBP (0.66 of AH in
basal diet replaced by PBP) and (iv) 36% PBP (all of AH in basal diet replaced by
PBP). The basal diet was 360 g/kg dry matter (DM) alfalfa hay, 160 g/kg DM wheat
straw and 480 g/kg DM concentrate. The trial consisted of four periods, each
composed of 16 days adaptation and 4 days data collection including measurement
of blood metabolites, rumen fermentation and population of bacteria. No
differences were observed in rumen pH among the treatments, while rumen ammonia-N
concentrations were decreased (p< 0.05) with increasing PBP by up to 36% DM of
the diets. Using of 36% PBP in the diet reduced (p < 0.05) total volatile fatty
acids (VFA) concentrations and the molar proportion of acetate, while the
concentration of propionate, butyrate and acetate to propionate ratio were
similar to all other treatments. The concentration of blood urea nitrogen (BUN)
decreased (p < 0.01) with increasing PBP by up to 36% DM in the diets of sheep.
However, other blood metabolites were not affected by the experimental diets. It
was concluded that PBP in replacement of AH had no effects on the relative
abundance of Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus in
relation to the control diet.
DOI: 10.1111/jpn.12120
789. J Mol Evol. 2013 Aug;77(1-2):3-7. doi: 10.1007/s00239-013-9578-8. Epub 2013

Aug
11.
Exploring the intrinsic limits of nitrogenase transfer from bacteria to

eukaryotes.
Soto G(1), Fox AR, Ayub ND.
Author information:
(1)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Avda.
Rivadavia 1917, C1033AAJ, Cuidad Autónoma de Buenos Aires, Argentina.
Biological nitrogen fixation is widespread among the Eubacteria and Archae

domains but completely absent in eukaryotes. The lack of lateral transfer of
nitrogen-fixation genes from prokaryotes to eukaryotes has been partially
attributed to the physiological requirements necessary for the function of the
nitrogenase complex. However, symbiotic bacterial nitrogenase activity is
protected by the nodule, a plant structure whose organogenesis can be trigged in
the absence of bacteria. To explore the intrinsic potentiality of this plant
organ, we generated rhizobium-independent nodules in alfalfa by overexpressing
the MsDMI3 kinase lacking the autoinhibitory domain. These transgenic nodules
showed similar levels of leghemoglobin, free oxygen, ATP, and NADPH to those of
efficient Sinorhizobium meliloti B399-infected nodules, suggesting that the
rhizobium-independent nodules can provide an optimal microenvironment for
nitrogenase activity. Finally, we discuss the intrinsic evolutionary constraints
on transfer of nitrogen-fixation genes between bacteria and eukaryotes.
DOI: 10.1007/s00239-013-9578-8
790. J Hazard Mater. 2013 Sep 15;260:1100-7. doi: 10.1016/j.jhazmat.2013.06.065.

Epub
2013 Jul 3.
Enhanced phytoremediation of mixed heavy metal (mercury)-organic pollutants

(trichloroethylene) with transgenic alfalfa co-expressing glutathione
S-transferase and human P450 2E1.
Zhang Y(1), Liu J, Zhou Y, Gong T, Wang J, Ge Y.
Author information:
(1)Department of Pharmaceutics, Qingdao University of Science and Technology, 53
Zhengzhou Road, PO Box 70, Qingdao 266042, China.
Soil contamination is a global environmental problem and many efforts have been
made to find efficient remediation methods over the last decade. Moreover,
remediation of mixed contaminated soils are more difficult. In the present study,
transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and
human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury
(Hg)-trichloroethylene (TCE) contaminants. Simultaneous expression of GST and
CYP2E1 may produce a significant synergistic effect, and leads to improved
resistance and accumulation to heavy metal-organic complex contaminants. Based on
the tolerance and accumulation assays, pKHCG transgenic plants were more
resistant to Hg/TCE complex pollutants and many folds higher in
Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated
soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy
to help achieve mixed heavy metal-organic pollutants phytoremediation.
DOI: 10.1016/j.jhazmat.2013.06.065
791. PLoS One. 2013 Jul 29;8(7):e69890. doi: 10.1371/journal.pone.0069890. Print

2013.
Evaluation of genome sequencing quality in selected plant species using expressed

sequence tags.
Shangguan L(1), Han J, Kayesh E, Sun X, Zhang C, Pervaiz T, Wen X, Fang J.
Author information:
(1)College of Horticulture, Nanjing Agricultural University, Nanjing City,
Jiangsu Province, China.
BACKGROUND: With the completion of genome sequencing projects for more than 30
plant species, large volumes of genome sequences have been produced and stored in
online databases. Advancements in sequencing technologies have reduced the cost
and time of whole genome sequencing enabling more and more plants to be subjected
to genome sequencing. Despite this, genome sequence qualities of multiple plants
have not been evaluated.
METHODOLOGY/PRINCIPAL FINDING: Integrity and accuracy were calculated to evaluate
the genome sequence quality of 32 plants. The integrity of a genome sequence is
presented by the ratio of chromosome size and genome size (or between scaffold
size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of
genome sequence was presented by the ratio between matched EST and selected ESTs
where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs
could be mapped to chromosome and scaffold sequences, respectively. According to
the integrity, accuracy and other analysis of each plant species, thirteen plant
species were divided into four levels. Arabidopsis thaliana, Oryza sativa and Zea
mays had the highest quality, followed by Brachypodium distachyon, Populus
trichocarpa, Vitis vinifera and Glycine max, Sorghum bicolor, Solanum
lycopersicum and Fragaria vesca, and Lotus japonicus, Medicago truncatula and
Malus × domestica in that order. Assembling the scaffold sequences into
chromosome sequences should be the primary task for the remaining nineteen
species. Low GC content and repeat DNA influences genome sequence assembly.
CONCLUSION: The quality of plant genome sequences was found to be lower than
envisaged and thus the rapid development of genome sequencing projects as well as
research on bioinformatics tools and the algorithms of genome sequence assembly
should provide increased processing and correction of genome sequences that have
already been published.
PMCID: PMC3726750

Epub 2013 Jul 13.
Characterization of physiological responses of two alfalfa half-sib families with

improved salt tolerance.
Anower MR(1), Mott IW, Peel MD, Wu Y.
Author information:
(1)Department of Biology and Microbiology, South Dakota State University,
Brookings, SD 57007, USA.
Alfalfa (Medicago sativa L.) is a major forage crop worldwide that is relatively
sensitive to soil salinity. Improved cultivars with high production on saline
soil will benefit many producers and land managers. This study reports the
characterization of physiological responses of two unrelated experimental alfalfa
half-sib families, HS-A and HS-B, selected for their improved survival under
saline conditions (up to EC 18). Six-week-old plants were subjected to
NaCl-nutrient solution treatment for three weeks starting at an electrical
conductivity (EC) of 3 dS m(-1) with incremental increases of 3 dS m(-1) every
week, reaching 9 dS m(-1) in the third week. HS-B showed greater leaf number
(72%) and stem length (44%) while HS-A showed better leaf production (84%) under
salt treatment compared to the initial genetic backgrounds from which they were
developed. This improved growth is associated with 208% and 78% greater
accumulation of chlorophyll content in HS-B and HS-A, respectively. Both HS-A and
HS-B also showed improved capability to maintain water content (RWC) under salt
stress compared to the initial populations. Differing from its initial
populations (P-B), HS-B did not accumulate Na in shoots after salt treatment.
HS-B also maintained K(+)/Na(+) and Ca(2+)/Na(+) ratios, while P-B showed 59% and
69% decrease in these ion ratios, respectively. Na(+) content in HS-A was not
different from its initial populations (P-A) after salt treatment. However, HS-A
showed an enhanced accumulation of Ca(2+) and maintained the levels of Mg(2+) and
K(+) in shoots compared to the P-A populations. This study provides physiological
support of improved salt tolerance in HS-A and HS-B and suggests that these
plants maintain ion homeostasis but have different mechanisms of coping with high
salinity.
Published by Elsevier Masson SAS.
DOI: 10.1016/j.plaphy.2013.06.026
793. J Insect Sci. 2013;13:41. doi: 10.1673/031.013.4101.
Fixed-precision sequential sampling plans for estimating alfalfa caterpillar,

Colias lesbia, egg density in alfalfa, Medicago sativa, fields in Córdoba,
Argentina.
Serra GV(1), Porta NC, Avalos S, Mazzuferi V.

Author information:
(1)Catedra de Zoología Agrícola, Facultad de Ciencias Agropecuarias, Universidad
Nacional de Córdoba, Av. Valparaiso s/n, 5000 Córdoba, Argentina.
The alfalfa caterpillar, Colias lesbia (Fabricius) (Lepidoptera: Pieridae), is a

major pest of alfalfa, Medicago sativa L. (Fabales: Fabaceae), crops in
Argentina. Its management is based mainly on chemical control of larvae whenever
the larvae exceed the action threshold. To develop and validate fixed-precision
sequential sampling plans, an intensive sampling programme for C. lesbia eggs was
carried out in two alfalfa plots located in the Province of Córdoba, Argentina,
from 1999 to 2002. Using Resampling for Validation of Sampling Plans software, 12
additional independent data sets were used to validate the sequential sampling
plan with precision levels of 0.10 and 0.25 (SE/mean), respectively. For a range
of mean densities of 0.10 to 8.35 eggs/sample, an average sample size of only 27
and 26 sample units was required to achieve a desired precision level of 0.25 for
the sampling plans of Green and Kuno, respectively. As the precision level was
increased to 0.10, average sample size increased to 161 and 157 sample units for
the sampling plans of Green and Kuno, respectively. We recommend using Green's
sequential sampling plan because it is less sensitive to changes in egg density.
These sampling plans are a valuable tool for researchers to study population
dynamics and to evaluate integrated pest management strategies.
DOI: 10.1673/031.013.4101
PMCID: PMC3740930
794. Mol Microbiol. 2013 Oct;90(1):54-71. doi: 10.1111/mmi.12347. Epub 2013 Aug 19.
The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in
Sinorhizobium meliloti.
Pini F(1), Frage B, Ferri L, De Nisco NJ, Mohapatra SS, Taddei L, Fioravanti A,
Dewitte F, Galardini M, Brilli M, Villeret V, Bazzicalupo M, Mengoni A, Walker
GC, Becker A, Biondi EG.
Author information:
(1)Interdisciplinary Research Institute USR3078, CNRS-Université Lille Nord de
France, 50 avenue de Halley, Villeneuve d'Ascq Cedex, France.
Sinorhizobium meliloti is a soil bacterium that invades the root nodules it

induces on Medicago sativa, whereupon it undergoes an alteration of its cell
cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid
with higher membrane permeability. In Caulobacter crescentus, a related
alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by
the phosphorylated response regulator DivK. The phosphorylation of DivK depends
on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK.
Despite the importance of the DivJ in C. crescentus, the mechanistic role of this
kinase has never been elucidated in other Alphaproteobacteria. We show here that
the histidine kinases DivJ together with CbrA and PleC participate in a complex
phosphorylation system of the essential response regulator DivK in S. meliloti.
In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn
CtrA inactivation, thereby controlling correct cell cycle progression and the
integrity of the cell envelope. In contrast, the essential PleC presumably acts
as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to
elicit nodules and enter plant cells, but fails to establish an effective
symbiosis suggesting that proper envelope and/or low CtrA levels are required for
symbiosis.
DOI: 10.1111/mmi.12347
PMCID: PMC3793127
795. J Food Prot. 2013 Aug;76(8):1429-33. doi: 10.4315/0362-028X.JFP-13-060.
Behavior of non-O157 Shiga toxin-producing Escherichia coli, enteroinvasive E.

coli, enteropathogenic E. coli, and enterotoxigenic E. coli strains on alfalfa
sprouts.
Gómez-Aldapa CA(1), Rangel-Vargas E, Torres-Vitela Mdel R, Villarruel-López A,

Castro-Rosas J.
Author information:
Ingeniería, Universidad Autónoma del Estado de Hidalgo, Centro Universitario,
Carretera Pachuca-Tulancingo Km. 4.5, Mineral de la Reforma, Hidalgo, C.P. 42183,
Mexico.
Data about the behavior of non-O157 Shiga toxin-producing Escherichia coli

(non-O157 STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC),
and enteropathogenic E. coli (EPEC) on seeds and alfalfa sprouts are not
available. The behavior of STEC, EIEC, ETEC, and EPEC was determined during
germination and sprouting of alfalfa seeds at 20 ± 2°C and 30 ± 2°C and on
alfalfa sprouts at 3 ± 2°C. When alfalfa seeds were inoculated with STEC, EIEC,
ETEC, or EPEC strains, all these diarrheagenic E. coli pathotypes (DEPs) grew
during germination and sprouting of seeds, reaching counts of approximately 5 and
6 log CFU/g after 1 day at 20 ± 2°C and 30 ± 2°C, respectively. However, when the
sprouts were inoculated after 1 day of seed germination and stored at 20 ± 2°C or
30 ± 2°C, no growth was observed for any DEP during sprouting at 20 ± 2°C or 30 ±
2°C for 9 days. Refrigeration reduced significantly (P < 0.0.5) the number of
viable DEPs on sprouts after 20 days in storage; nevertheless, these decreases
have no practical significance for the safety of the sprouts.
DOI: 10.4315/0362-028X.JFP-13-060
796. Environ Entomol. 2013 Aug;42(4):770-8. doi: 10.1603/EN12353.
Dispersion, distribution, and movement of Lygus spp. (Hemiptera: Miridae) in

trap-cropped organic strawberries.
Swezey SL(1), Nieto DJ, Hagler JR, Pickett CH, Bryer JA, Machtley SA.
Author information:
Santa Cruz, CA 95064, USA. findit@ucsc.edu
Alfalfa (Medicago sativa L.) is a highly attractive plant host to Lygus spp. and
is used as a trap crop in California organic strawberries to influence the
dispersion and dispersal of these pests, particularly Lygus hesperus Knight. The
abundance and distribution of Lygus spp. nymphs between two trap crops separated
by 50 strawberry rows was analyzed in 2008 and 2010. Nymphs demonstrated a
bimodal distribution in strawberries between trap crops, where nymphs were most
abundant and aggregated in alfalfa, when compared with interior strawberry rows,
where nymphs were less abundant. The majority of nymphs were concentrated in trap
crops and nymphal densities in interior strawberry rows were well below economic
thresholds. The movement of Lygus spp. from a marked alfalfa trap crop into
adjacent strawberry rows or trap crops was also studied in 2008 and 2009 using a
chicken egg albumin enzyme-linked immunosorbent assay mark-capture technique. The
majority of marked-captured L. hesperus adults and Lygus spp. nymphs remained in
alfalfa trap crops, rather than dispersing out into strawberry rows at 24 h, 48
h, and 2 wk, postprotein application. The attenuation of Lygus spp. movement in
alfalfa associated with organic strawberries is a key component of successful
trap cropping. A small percentage of marked adults and nymphs were captured in
neighboring alfalfa trap crops, located 62 m from the point of protein
application, highlighting the dispersal capacity of this key pest.
DOI: 10.1603/EN12353
797. Environ Sci Pollut Res Int. 2014 Jan;21(2):1285-91. doi:

10.1007/s11356-013-2009-y. Epub 2013 Jul 31.
Germination of Medicago sativa is inhibited by soluble compounds in cement dust.
Lafragüeta C(1), García-Criado B, Arranz A, Vázquez-de-Aldana BR.
Author information:
(1)Instituto de Recursos Naturales y Agrobiología de Salamanca; Consejo Superior
de Investigaciones Cientificas (IRNASA-CSIC), Cordel de Merinas 40-52, 37008,
Salamanca, Spain.
Deposition of cement dust on soils and plant surfaces is known to affect plant
growth and the species composition of plant communities, but little is known
about its effects (and those of its pH and constituents) on germination.
Therefore, the aim of this study was to assess the toxicity of an aqueous cement
extract, constituents of the extract and pH on the germination of seeds of a
selected species, Medicago sativa. First, the effects of the extract were tested
in assays with concentrations and exposure durations ranging from 0 to 1.0 g/mL
and 4 to 96 h, respectively. At 0.8 g/mL, the extract strongly inhibited
germination; a 4-h exposure reduced the germination rate, from 77 ± 1.8 to
50 ± 2.6% (mean ± SE), while 8-h exposure completely inhibited it. Further,
treatment at this concentration killed the non-germinating seeds, thus the
inhibition was due to toxic effects. Neither the pH of the extract nor the
concentration of its main soluble elements separately (K, Ca, S, Na, or Cr)
caused the toxicity since germination rates were not significantly reduced when
these variables were tested individually. However, a mixture of the elements in
solution reduced germination rates, suggesting that they have adverse synergistic
effects.
DOI: 10.1007/s11356-013-2009-y
798. J Plant Physiol. 2013 Dec 15;170(18):1609-19. doi:

10.1016/j.jplph.2013.06.012.
Epub 2013 Jul 26.
Identification of duplicated and stress-inducible Aox2b gene co-expressed with

Aox1 in species of the Medicago genus reveals a regulation linked to gene
rearrangement in leguminous genomes.
Cavalcanti JH(1), Oliveira GM, Saraiva KD, Torquato JP, Maia IG, de Melo DF,
Costa JH.
Author information:
(1)Department of Biochemistry and Molecular Biology, Federal University of Ceara,
60455-760 Fortaleza, Ceará, Brazil.
In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes

distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is
reported as a stress-responsive gene, but in the leguminous Vigna unguiculata
Aox2b is also induced by stress. In this work we investigated the Aox genes from
two leguminous species of the Medicago genus (Medicago sativa and Medicago
truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named
here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M.
sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed
germination. Similar analyses in leaves and roots under different treatments (SA,
PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but
with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of
expression under control conditions and were induced by stress in leaves and
roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress
conditions in leaves, and showed basal expression levels in roots that were
induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and
during seed germination than in roots and appeared to be not responsive to
stress. The Aox expression profiles obtained from a M. truncatula microarray
dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2
in leaves and roots. These results reinforce the stress-inducible co-expression
of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates
that this regulation is linked to Aox1/Aox2b proximity in the genome as a result
of the gene rearrangement that occurred in some leguminous plants during
evolution. The differential expression of Aox2b1/2b2 suggests that a second gene
has been originated by recent gene duplication with neofunctionalization.
DOI: 10.1016/j.jplph.2013.06.012
799. Acta Biochim Biophys Sin (Shanghai). 2013 Oct;45(10):882-8. doi:

10.1093/abbs/gmt083. Epub 2013 Jul 24.
The Sinorhizobium meliloti LysR family transcriptional factor LsrB is involved in

regulation of glutathione biosynthesis.
Lu D(1), Tang G, Wang D, Luo L.
Author information:
(1)School of Life Science, Anhui University, Heifei 230039, China.
Glutathione, a key antioxidant in Sinorhizobium meliloti, is required for the

development of alfalfa (Medicago sativa) nitrogen-fixing nodules. This tripeptide
can be synthesized by both γ-glutamyl cysteine synthetase (GshA) and glutathione
synthetase (GshB) in Escherichia coli and S. meliloti. Genetic evidence has
indicated that the null mutant of S. meliloti gshA or gshB1 does not establish
efficient symbiosis on alfalfa. However, the transcriptional regulation of gshA
and gshB has not been well understood. Here, S. meliloti LsrB, a member of LysR
family transcriptional factors, was found to positively regulate glutathione
biosynthesis by activating the transcription of gshA and gshB1 under both
free-living and symbiotic conditions. The decrease in glutathione production in
the lsrB in-frame deletion mutant (lsrB1-2) was determined by using quadrupole
time-of-flight liquid chromatography-mass spectrometry. The expression of gshA
and gshB1 was correspondingly reduced in the mutant under free-living and
symbiotic conditions by analyses of real-time quantitative reverse
transcription-polymerase chain reaction and promoter-GUS fusions. Interestingly,
LsrB positively regulated the transcription of oxyR, which encodes another member
of LysR family regulators and responds to oxidative stresses in S. meliloti. The
oxyR null mutant produced less glutathione, in which the transcription of gshA
was consistently down-regulated. These findings demonstrate that glutathione
biosynthesis is positively regulated by both LsrB and OxyR in S. meliloti.
DOI: 10.1093/abbs/gmt083
800. Animal. 2013 Oct;7(10):1714-20. doi: 10.1017/S1751731113001262. Epub 2013 Jul

23.
Cattle and sheep develop preference for drinking water containing grape seed
tannin.
Kronberg SL(1), Schauer CS.
Author information:
(1)United States Department of Agriculture, Agriculture Research Service,
Northern Great Plains Research Laboratory, PO Box 459, Mandan, North Dakota
58554, USA. scott.kronberg@ars.usda.gov
Ingestion of small amounts of some types of condensed tannins (CTs) by ruminant

livestock can provide nutritional, environmental and economic benefits. However,
practical methods are needed to make these tannins more available to ruminant
livestock. Results from previous trials with crude quebracho and black wattle
tannin indicated that cattle and/or sheep would not preferentially drink water
containing these tannins. Therefore, we conducted preference trials to determine
if cattle and sheep would learn to prefer water containing purified grape seed
tannin (GST) that provided up to 2% of their daily dry matter (DM) intake. After
gradual exposure to increasing amounts of this tannin in water during a pre-trial
period, five adult ewes and five yearling heifers fed lucerne (Medicago sativa)
pellets (19% CP) were offered water and several concentrations of GST solutions
for either 15 (sheep trial) or 20 days (cattle trial). We measured intake of all
liquids daily. Concentrations of blood urea were also measured for heifers when
they drank only tannin solutions or water. Both sheep and cattle developed
preferences for water with GST in it over water alone (P < 0.01) although this
preference appeared earlier in the trial for sheep than for cattle. For the
sheep, mean daily intake of water alone and all tannin solutions (in total) was
0.6 and 6.1 l, respectively. For the cattle, mean daily intake of water and all
tannin solutions in total was 21.8 and 20.6 l, respectively, in the first half of
the trial and 10.8 and 26.1 l, respectively, in the second half of the trial.
Compared with the other tannin solutions, both sheep and cattle drank more of the
solution with the highest tannin concentration (2% of daily DM intake as GST)
than of water on more trial days (P < 0.05). Ingestion of water with the highest
concentration of GST reduced blood plasma urea concentration in the cattle by 9%
to 14% (P ≤ 0.10) compared with ingestion of water alone. Results from the trials
suggest that providing grape seed and perhaps other CTs via drinking water may be
a practical way to introduce CTs into sheep and cattle diets.
DOI: 10.1017/S1751731113001262
Epub
2013 Jul 3.
Overexpression of GsZFP1 enhances salt and drought tolerance in transgenic

alfalfa (Medicago sativa L.).
Tang L(1), Cai H, Ji W, Luo X, Wang Z, Wu J, Wang X, Cui L, Wang Y, Zhu Y, Bai X.
Author information:
(1)College of Life Science, Northeast Agricultural University, Harbin 150030,
China. Electronic address: tanglili19861126@126.com.
GsZFP1 encodes a Cys2/His2-type zinc-finger protein. In our previous study, when

GsZFP1 was heterologously expressed in Arabidopsis, the transgenic Arabidopsis
plants exhibited enhanced drought and cold tolerance. However, it is still
unknown whether GsZFP1 is also involved in salt stress. GsZFP1 is from the wild
legume Glycine soja. Therefore, the aims of this study were to further elucidate
the functions of the GsZFP1 gene under salt and drought stress in the forage
legume alfalfa and to investigate its biochemical and physiological functions
under these stress conditions. Our data showed that overexpression of GsZFP1 in
alfalfa resulted in enhanced salt tolerance. Under high salinity stress, greater
relative membrane permeability and malondialdehyde (MDA) content were observed
and more free proline and soluble sugars accumulated in transgenic alfalfa than
in the wild-type (WT) plants; in addition, the transgenic lines accumulated less
Na(+) and more K(+) in both the shoots and roots. Overexpression of GsZFP1 also
enhanced the drought tolerance of alfalfa. The fold-inductions of
stress-responsive marker gene expression, including MtCOR47, MtRAB18, MtP5CS, and
MtRD2, were greater in transgenic alfalfa than those of WT under drought stress
conditions. In conclusion, the transgenic alfalfa plants generated in this study
could be used for farming in salt-affected as well as arid and semi-arid areas.
DOI: 10.1016/j.plaphy.2013.06.024
802. Genetika. 2013 Apr;49(4):448-53.
[Factor analysis of interactions between alfalfa nodule bacteria (Sinorhizobium

meliloti) genes that regulate symbiotic nitrogen fixation].
Provorov NA, Chuklina E, Vorob'ev NI, Onishchuk OP, Simarov BV.
Factor analysis has been conducted for the data on the interaction between the
genes of the root nodule bacteria (rhizobia), which influence the efficiency of
symbiosis with leguminous plants, including dctA (encoding succinate permease),
dctBD (activating the dctA gene due to binding its enhancer in the presence of
succinate), rpoN (activating the promoters of dctA and nitrogenase genes nifHDK),
and nifA (activating the nitrogenase genes due to binding their enhancers). The
analysis of the alfalfa rhizobia (Sinorhizobium meliloti) recombinants that
contain additional copies ofthese genes suggested the antagonistic (epistatic)
interaction between nifA and rpoN. It may be associated either with the
competition for C compounds imported into the nodules between the energy
production and nitrogen assimilation processes or with the competition for redox
potentials between the oxidative phosphorylation and nitrogen fixation processes.
Since the phenotypic effects of the studied genes depend on the activity of
nitrogen export into the aerial parts of plants, we suppose that its accumulation
in bacteroids impairs the activation of the nifHDK genes by the NifA protein due
to its interaction with the GlnB protein (the nitrogen metabolism regulator) or
with the FixLJ and ActSR proteins (the redox potential regulators).
803. J Econ Entomol. 2013 Jun;106(3):1164-72.
Survival of Hessian fly (Diptera: Cecidomyiidae) puparia exposed to simulated hay

harvest conditions, location and windrow drying in Washington and California.
Yokoyama VY(1), Cambron SE.
Author information:
(1)USDA-ARS, San Joaquin Valley Agricultural Sciences Center, 9611 South
Riverbend Ave., Parlier, CA 93648, USA. victoria.yokoyama@ars.usda.gov
Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae) puparia are of

regulatory concern in exported hay, and drying after harvest was evaluated as a
cultural control technique for bales shipped from the western states. In total
16,836; 31,122; and 48,051 puparia were tested under drying conditions in
environmental chambers, open air on location, and hay windrows, respectively.
Regression lines for percentage of total adults emerging from puparia exposed to
simulated drying conditions for 1-7 d in environmental chambers was significant
for 1 September, Kittitas Valley, WA; 3 June, East Columbia Basin, WA; 15 May and
15 July, San Joaquin Valley, CA; and 15 May, 20 July, and 15 September, Imperial
Valley, CA. In open air drying on location for 1-7 d, total percentage of puparia
surviving to adults for all exposure days was 0.4% for 18 June, Kittitas Valley;
1.2% for 15 May, San Joaquin Valley; and 0% for 16 July, Imperial Valley; and
significantly different between controls and exposure durations. In hay windrow
drying for 1-6 d, total percentage of puparia surviving to adults for all
exposure days was 5.4% on 28 June and 24.2% on 7 September in timothy, Phleum
pretense, in the Kittitas Valley; 3.8% on 28 June in timothy in the East Columbia
Basin; 2.2% on 20 July in alfalfa, Medicago sativa, in the San Joaquin Valley;
and 6.3% on 21 July in Sudan grass, Sorghum bicolor sudanensis, in the Imperial
Valley. The number of puparia surviving to adults in open air drying and in
windrows was significantly different between controls and exposure durations for
all test dates and locations. Puparial survival in field tests was related to
mild temperatures and high humidities. Hay drying with subsequent field baling,
storage, and export bale compression is discussed in relation to a systems
approach for quarantine control of Hessian fly in exported hay.
804. Anal Chim Acta. 2013 Jul 30;789:100-6. doi: 10.1016/j.aca.2013.06.031. Epub
2013
Jun 27.
On-line capillary electrophoresis enrichment by combining chitosan trapping with

surfactant assisted sample stacking for the ultratrace determination of organic
acids in Plateau alfalfa roots.
Li X(1), Ju Y, Xu Y, Wang W, Dong Y, Ma Y, Chen X.
Author information:
(1)State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou
730000, China.
In this paper, four organic acids constituents of Plateau alfalfa roots have been
identified and detected by a novel capillary electrophoresis (CE) strategy which
combined chitosan (CS) trapping and cetyltrimethyl ammonium bromide (CTAB)
assisted sample stacking. Under the optimized condition, organic acids, i.e.,
aconitic acid, gallic acid, citric acid and l-malic acid were concentrated and
separated within 3 min. Validation parameters of this method (such as detection
limits, linearity and precision) were also investigated and the limit of
detection (LOD) was 2.41-53.9 ng mL(-1). Linearity was obtained over the
magnitude range of 5-4000 ng mL(-1) approximately for different organic acids and
3×10(2)-1.5×10(4) folds enrichment was achieved. The method has been applied to
the determination of organic acids in roots of normal grown Plateau alfalfa and
stressing affected Plateau alfalfa. Satisfactory results and recoveries were
obtained in the analysis without costly and complicated sample pretreatment.
DOI: 10.1016/j.aca.2013.06.031
805. J Hazard Mater. 2013 Sep 15;260:715-24. doi: 10.1016/j.jhazmat.2013.06.032.

Epub
2013 Jun 21.
Alleviation of cadmium toxicity in Medicago sativa by hydrogen-rich water.
Cui W(1), Gao C, Fang P, Lin G, Shen W.
Author information:
China.
Hydrogen gas (H₂) induces plant tolerance to several abiotic stresses, including
salinity and paraquat exposure. However, the role of H₂ in cadmium (Cd)-induced
stress amelioration is largely unknown. Here, pretreatment with hydrogen-rich
water (HRW) was used to characterize physiological roles and molecular mechanisms
of H₂ in the alleviation of Cd toxicity in alfalfa plants. Our results showed
that the addition of HRW at 10% saturation significantly decreased contents of
thiobarbituric acid reactive substances (TBARS) caused by Cd, and inhibited the
appearance of Cd toxicity symptoms, including the improvement of root elongation
and seedling growth. These responses were related to a significant increase in
the total or isozymatic activities of representative antioxidant enzymes, or
their corresponding transcripts. In vivo imaging of reactive oxygen species
(ROS), and the detection of lipid peroxidation and the loss of plasma membrane
integrity provided further evidence for the ability of HRW to improve Cd
tolerance significantly, which was consistent with a significant enhancement of
the ratio of reduced/oxidized (homo)glutathione ((h)GSH). Additionally, plants
pretreated with HRW accumulated less amounts of Cd. Together, this study
suggested that the usage of HRW could be an effective approach for Cd
detoxification and could be explored in agricultural production systems.

DOI: 10.1016/j.jhazmat.2013.06.032
806. J Environ Manage. 2013 Oct 15;128:586-93. doi: 10.1016/j.jenvman.2013.05.045.

Epub 2013 Jul 6.
Forage and tree seedling growth in a soil with an encased swine sludge layer.
Penn CJ(1), Will R, Fultz L, Hamilton D.
Author information:
(1)Oklahoma State University, Department of Plant and Soil Science, 367
Agricultural Hall, Stillwater, OK 47078-1020, USA. chad.penn@okstate.edu
The closure of swine farms requires decommissioning of lagoons that contain large
amounts of swine solids (sludge). Sludge is typically transported and land
applied to soils. However, in some cases this process could be economically
prohibitive and/or unpractical. An alternative idea is to encase sludge with
lagoon soil berms after removing overlying effluent, followed by establishment of
forages or short-rotation woody crops on the encased sludge. The objective of
this study was to investigate growth potential for several forages and tree
species into a pure layer of swine sludge. Alfalfa (Meticago sativa),
bermudagrass (Cynodon dactylon), switchgrass (Panicum virgatum), green ash
(Fraxinus pennsylvanica), black locust (Robinia pseudoacacia), and sycamore
(Platanus occidentalis) were established in 40 cm deep pots consisting of a
lagoon berm soil overlaying a sludge layer for 12 w followed by analysis of
aboveground and belowground biomass production. "New" and "old" sludge was
collected from an active 10 year old lagoon and decommissioned 50 year old
lagoon, respectively. A control (soil only) was used. Encased sludge treatments
increased forage biomass production. Sycamore and green ash were sensitive to new
sludge but not old sludge as these species had less biomass production in new
sludge than control and showed tissue trace nutrient deficiencies. While both
sludge materials contained adequate nutrients, the new sludge had a salt
concentration 1.8 times higher than old sludge as indicated by electrical
conductivity (12.4 mS). Thus, the forage crops and black locust were able to
thrive in new sludge due to their salt tolerance.
DOI: 10.1016/j.jenvman.2013.05.045
807. J Anim Sci. 2013 Sep;91(9):4451-61. doi: 10.2527/jas.2012-5405. Epub 2013 Jul
3.
Effect of summer forage species grazed during finishing on animal performance,

carcass quality, and meat quality.
Schmidt JR(1), Miller MC, Andrae JG, Ellis SE, Duckett SK.
Author information:
(1)School of Agriculture, Forestry and Environmental Science, Clemson University,
Clemson, SC 29634, USA.
Angus-cross steers (n = 60) were used to assess the effect of forage species
[alfalfa (AL; Medicago sativa L.), bermudagrass (BG; Cynodon dactylon), chicory
(CH; Cichorium intybus L.), cowpea (CO; Vigna unguiculata L.), and pearl millet
(PM; Pennisetum glaucum (L. R Br.)] in replicated 2-ha paddocks for finishing on
cattle performance, carcass quality, and meat quality in a 2-yr study. Steers
were blocked by BW and assigned randomly to finishing-forage treatments before
the start of the experiment. Steers grazing AL and CH had greater (P < 0.05) ADG
than those grazing for BG, CO, and PM, whereas AL produced more (P < 0.05)
gain/ha than CH, CO, and PM. Days steers spent grazing were longest (P < 0.05)
for PM and shortest (P < 0.05) for CO. Steers grazing BG and CO produced heavier
(P < 0.05) HCW than steers grazing BG and PM. Dressing percentage was greatest (P
< 0.05) in steers grazing CO, and grazing AL resulted in greater (P < 0.05)
dressing percentages than grazing BG, CH, and PM. Grazing AL and CH produced
carcasses with more (P < 0.05) fat at the 12th rib than steers grazing
warm-season grasses (BG and PM). Marbling scores tended to be greater (P = 0.06)
for CO, but carcasses from steers grazing CO received greater (P < 0.05) quality
grades than AL and CH. Trans-11 vaccenic (C18:1 trans-11; TVA) acid concentration
in the LM was greater (P < 0.05) for BG than CH, CO, and AL. Conjugated linoleic
acid, cis-9 trans-11 isomer, concentration was greatest (P < 0.05) for BG and PM
than AL, CH, and CO. Grazing CH and PM increased (P < 0.05) the ratio of omega-6
to omega-3 fatty acids in the LM compared with AL, BG, and CO. Grazing legumes
(AL and CO) resulted in lower (P < 0.05) Warner-Bratzler shear force values than
other forage species. Consumers rated steaks from steers finished on AL and CO
pastures greatest (P < 0.05) and steaks from steers finished on BG and CH least
(P < 0.05) for overall palatability. Consumer preference was greatest (P < 0.05)
for steaks from steers finished on AL and least (P < 0.05) for steaks from steers
finished on BG and CH. Finishing steers on AL and CH during summer increased
steer performance (> 1 kg/d). Finishing on legumes (AL and CO) increased dressing
percentage, reduced Warner-Bratzler shear force values, and increased consumers
preference, whereas finishing on grasses (BG and PM) enhanced anticarcinogenic
fatty acid concentrations.
DOI: 10.2527/jas.2012-5405
808. Animal. 2013 Oct;7(10):1622-30. doi: 10.1017/S1751731113001225. Epub 2013 Jul

3.
Energy metabolism by splanchnic tissues of mature sheep fed varying levels of

lucerne hay cubes.
EL-Sabagh M(1), Goto M, Sugino T, Obitsu T, Taniguchi K.
Author information:
(1)Graduate School of Biosphere Science, Hiroshima University,
Higashihiroshima-shi, 739-8528, Japan.
The objective of this study was to determine the pattern of energy metabolites
net flux across the portal-drained viscera (PDV) and total splanchnic tissues
(TSP) in mature sheep fed varying levels of lucerne hay cubes. Four Suffolk
mature sheep (61.4 ± 3.6 kg BW) surgically fitted with multi-catheters were fed
four levels of dry matter intake (DMI) of lucerne hay cubes ranging from 0.4- to
1.6-fold the metabolizable energy (ME) requirements for maintenance. Six sets of
blood samples were simultaneously collected from arterial and venous catheters at
30-min intervals. With increasing DMI, apparent total tract digestibility
increased linearly and quadratically for dry matter (P < 0.05), quadratically (P
< 0.05) with a linear tendency (P < 0.1) for organic matter and tended to
increase quadratically (P < 0.1) for NDF. PDV release of volatile fatty acids
(VFA) and β-hydroxybutyric acid was relatively low at 0.4 M and then linearly
increased (P < 0.05) with increasing DMI. Net PDV flux of non-esterified fatty
acids showed curvilinear decrease from 0.4 to 1.2 M and then increased at 1.6 M.
The respective proportions of each VFA appearing in the portal blood differed (P
< 0.05) with DMI and this difference was more obvious from 0.4 to 0.8 M than from
0.8 to 1.6 M. Heat production, as a percentage of ME intake (MEI), decreased
linearly (P < 0.05) with increasing DMI accounting for 37%, 21%, 16% and 13% for
PDV and 62%, 49%, 33% and 27% for TSP at 0.4, 0.8, 1.2 and 1.6 M, respectively.
As a proportion of MEI, total energy recovery including heat production,
decreased linearly with increasing DMI (P < 0.05) accounting for 113%, 83%, 62%
and 57% for PDV and 140%, 129%, 86% and 83% for TSP at 0.4, 0.8, 1.2 and 1.6 M,
respectively. Regression analysis revealed a linear response between MEI (MJ/day
per kg BW) and total energy release (MJ/day per kg BW) across the PDV and TSP,
respectively. However, respective contributions of energy metabolites to net
energy release across the PDV and TSP were highly variable among treatments and
did not follow the same pattern of changes in DMI.
DOI: 10.1017/S1751731113001225
809. J Plant Physiol. 2013 Oct 15;170(15):1328-36. doi:

10.1016/j.jplph.2013.05.014.
Epub 2013 Jun 28.
Up-regulation of heme oxygenase-1 contributes to the amelioration of

aluminum-induced oxidative stress in Medicago sativa.
Cui W(1), Zhang J, Xuan W, Xie Y.
Author information:
(1)College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu
Province 210095, China.
In this report, pharmacological, histochemical and molecular approaches were used

to investigate the effect of heme oxygenase-1 (HO-1) up-regulation on the
alleviation of aluminum (Al)-induced oxidative stress in Medicago sativa.
Exposure of alfalfa to AlCl3 (0-100 μM) resulted in a dose-dependent inhibition
of root elongation as well as the enhancement of thiobarbituric acid reactive
substances (TBARS) content. 1 and 10 μM (in particular) Al(3+) increased alfalfa
HO-1 transcript or its protein level, and HO activity in comparison with the
decreased changes in 100 μM Al-treated samples. After recuperation, however,
TBARS levels in 1 and 10 μM Al-treated alfalfa roots returned to control values,
which were accompanied with the higher levels of HO activity. Subsequently,
exogenous CO, a byproduct of HO-1, could substitute for the cytoprotective
effects of the up-regulation of HO-1 in alfalfa plants upon Al stress, which was
confirmed by the alleviation of TBARS and Al accumulation, as well as the
histochemical analysis of lipid peroxidation and loss of plasma membrane
integrity. Theses results indicated that endogenous CO generated via heme
degradation by HO-1 could contribute in a critical manner to its protective
effects. Additionally, the pretreatments of butylated hydroxytoluene (BHT) and
hemin, an inducer of HO-1, exhibited the similar cytoprotective roles in the
alleviation of oxidative stress, both of which were impaired by the potent
inhibitor of HO-1, zinc protoporphyrin IX (ZnPP). However, the Al-induced
inhibition of root elongation was not influenced by CO, BHT and hemin,
respectively. Together, the present results showed up-regulation of HO-1
expression could act as a mechanism of cell protection against oxidative stress
induced by Al treatment.
DOI: 10.1016/j.jplph.2013.05.014

10.1007/s11356-013-1917-1. Epub 2013 Jun 27.
Comparison of bioavailable vanadium in alfalfa rhizosphere soil extracted by an

improved BCR procedure and EDTA, HCl, and NaNO₃ single extractions in a pot
experiment with V-Cd treatments.
Yang J(1), Teng Y, Zuo R, Song L.
Author information:
(1)College of Water Sciences, Beijing Normal University, Beijing, 100875, China.
The BCR sequential extraction procedure was compared with EDTA, HCl, and NaNO3
single extractions for evaluating vanadium bioavailability in alfalfa rhizosphere
soil. The amounts of vanadium extracted by these methods were in the following
order: BCR (bioavailable V) > EDTA ≈ HCl > NaNO3. Both correlation analysis and
stepwise regression were adopted to illustrate the extractable vanadium between
different reagents. The correlation coefficients between extracted vanadium and
the vanadium contents in alfalfa roots were R NaNO3 = 0.948, R HCl = 0.902, R
EDTA = 0.816, and R bioavailable V = 0.819. The stepwise multiple regression
equation of the NaNO3 extraction was the most significant at a 95 % confidence
interval. The influence of pH, total organic carbon, and cadmium content of soil
to vanadium bioavailability were not definite. In summary, both the BCR
sequential extraction and the single extraction methods were valid approaches for
predicting vanadium bioavailability in alfalfa rhizosphere soil, especially the
single extractions.
DOI: 10.1007/s11356-013-1917-1
811. Zoo Biol. 2013 Sep-Oct;32(5):484-9. doi: 10.1002/zoo.21080. Epub 2013 Jun 25.
Short-term digestible energy intake in captive moose (Alces alces) on different

diets.
Clauss M(1), Kohlschein GM, Peemöller A, Hummel J, Hatt JM.
Author information:
(1)Vetsuisse Faculty, Clinic for Zoo Animals, Exotic Pets and Wildlife,
University of Zurich, Zurich, Switzerland. mclauss@vetclinics.uzh.ch.
Moose (Alces alces) are regularly described as problematic animals in captivity,

mainly because of their particular digestive physiology and resulting feeding
demands. According to the literature, moose regularly reject non-browse forages
offered in captivity, which may indirectly lead to an overproportional ingestion
of easily digestible feeds and thus chronic acidosis, which may in turn be the
cause of their low life expectancy in captivity. By feeding experiments in four
animals, this study aimed at testing whether maintaining moose on roughage-only
diets appears feasible. The diets used consisted of the typical zoo ration with
mixed feeds (including alfalfa hay), and exclusive diets of alfalfa hay,
combinations of alfalfa hay and grass hay, alfalfa hay and grass hay and dried
browse leaves, and dried browse leaves only. Whereas results confirmed that moose
do not ingest grass hay in relevant amounts, digestible energy (DE) intake on
alfalfa hay was, at 0.67 ± 0.15 DE MJ kg(-0.75) day(-1), above the estimated
maintenance requirement of 0.6, and higher on the browse diets. At least for
short-time periods, results contradict previous reports in the literature that
alfalfa hay only is not a suitable maintenance diet for moose. At the same time
the results promote feeding moose in captivity forage-based diets.
© 2013 Wiley Periodicals, Inc.
DOI: 10.1002/zoo.21080
812. Vet Rec. 2013 Jul 27;173(4):95. doi: 10.1136/vr.101635. Epub 2013 Jun 25.
Alfalfa dodder (Cuscuta campestris) toxicity in horses: clinical, haematological

and serum biochemical findings.
Abutarbush SM(1).
Author information:
(1)Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine,
Jordan University of Science and Technology, Irbid 22110, Jordan.
sameeh75@hotmail.com
The objective of this observational study is to describe clinical, haematological

and serum biochemical findings of horses affected with alfalfa dodder (Cuscuta
campestris) toxicity. Twenty horses naturally exposed to alfalfa dodder toxicity
were examined and information was collected on history and clinical signs.
Physical examination was done on horses in the premises (n=20), and venous blood
samples of 12 horses were submitted for haematology and serum biochemical
examination for each horse. Abnormal clinical signs started around 36 hours after
horses were fed the contaminated alfalfa. Abnormal signs were seen in 11 horses
and those included diarrhoea (n=8), decreased appetite (n=7), neurological signs
(n=4) and abdominal pain (n=1). Some horses had multiple clinical signs of the
above. The results of complete blood cell count revealed leukocytopenia,
neutropenia and thrombocytopenia. Serum biochemical analysis revealed decreased
ALP, AST and CPK levels and increased direct bilirubin level. The used alfalfa
was stopped immediately and a different alfalfa from a new container that did not
contain any weeds was fed. Horses on the premises were observed closely, and the
abnormal clinical signs resolved within three days. No treatment was implemented.
Knowledge about toxicity of horses by Cuscuta species is scarce in the English
veterinary literature and very limited.
DOI: 10.1136/vr.101635
813. J Anim Sci. 2013 Jul;91(7):3315-21. doi: 10.2527/jas.2012-5942.
Effects of roughage concentration in dry-rolled corn-based diets containing wet

distillers grains with solubles on performance and carcass characteristics of
finishing beef steers.
Hales KE(1), Freetly HC, Shackelford SD, King DA.
Author information:
(1)USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933, USA.
Kristin.Hales@ars.usda.gov
Distillers grains and distillers solubles are by-products of grain fermentation

used to produce ethanol and contain greater concentrations of NDF and ADF,
compared with other grains and concentrates they replace in feedlot diets.
Typical finishing diets in the United States contain 8.3% and 9.0% roughage.
Therefore, it is plausible that the dietary concentration of roughage can be
altered when distillers grains are included in feedlot diets. The effects of
roughage concentration in dry-rolled, corn-based diets containing wet distillers
grains with solubles (WDGS) were evaluated in steers (n = 128; initial BW = 339
kg), using Calan gates. Each diet was based on dry-rolled corn and contained 25%
WDGS with coarsely ground alfalfa hay (AH), replacing corn at 2% (AH-2), 6%
(AH-6), 10% (AH-10), and 14% (AH-14) of DM. Feed offered was recorded daily, orts
were measured weekly, and BW was measured on d 0, 1, 35, 70, 105, 140, 174, and
175. After commercial harvest and chilling, carcasses were evaluated on-line with
a beef carcass grading camera to assess marbling and yield grade traits. The data
were analyzed using the Mixed Procedure of SAS, in which contrast statements were
used to separate linear and quadratic effects of AH inclusion. Decreasing
concentrations of AH in the finishing diet resulted in a tendency for a quadratic
response (P = 0.07) in final BW, where BW increased from 2 to 6% AH inclusion but
then decreased from 6 to 14% inclusion. Similarly, ADG from d 0 to end responded
quadratically (P < 0.01), in which ADG increased from 2 to 6% yet subsequently
decreased from 6 to 14% AH inclusion. Dry matter intake from d 0 to end increased
linearly (P = 0.02) as AH inclusion increased in the diet, whereas G:F increased
from 2 to 6% AH inclusion and then decreased linearly (P < 0.01) from 6 to 14% AH
inclusion. Concentration of AH in the finishing diet did not affect HCW, marbling
score, or the proportion of cattle grading USDA choice (P ≥ 0.18). However,
dressing percent and LM area did respond in a quadratic manner (P < 0.02), in
which they decreased from 2 to 10% AH inclusion and increased from 10 to 14% AH
in the diet. Yield grade and adjusted 12th rib fat responded quadratically (P <
0.01), in which both increased from 2 to 6% AH inclusion and decreased from 6 to
14% inclusion. Analysis of responses of G:F and ADG on AH predict the apex at 3%
and 7% for G:F and ADG, respectively, when fed in diets containing 25% WDGS.
DOI: 10.2527/jas.2012-5942

10.1111/jpn.12089.
Epub 2013 Jun 24.
Forage and sugar in dairy calves' starter diet and their interaction on
performance, weaning age and rumen fermentation.
Beiranvand H(1), Ghorbani GR, Khorvash M, Kazemi-Bonchenari M.
Author information:
The effects of sugar and forage inclusion in calves' starter and their
interaction on animal performance and rumen fermentation parameters were
investigated. Twenty-eight neonatal Holstein male calves 3 days of age with
average body weights of 42 ± 4 kg were allocated to four different treatments.
All calves were fed a similar basal diet consisting of milk and concentrate. The
experimental treatments were: (i) basal diet with no supplementation (Control,
hereafter designated by C), (ii) basal diet plus 5% granular sugar cane (Sugar,
designated by S), (iii) basal diet plus 5% forage (Forage, designated by F) and
(iv) basal diet plus 5% forage with 5% granular sugar cane (F × S). Supplement
ingredients were used on a dry matter (DM) basis. Rumen fluid parameters were
measured twice on days 35 and 70 of the study period. The calves were weaned when
they could consume 1 kg of starter for three consecutive days. The results show
that starter intake was not affected by treatment; however, the lowest ADG was
observed with calves in the sugar treatment. Weaning age was affected by
treatments, and forage showed to reduce milk consumption period down to its
shortest. Forage-sugar interaction was found to have no effects on animal
performance. The structural body indices as well as the health status of the
calves were similar in different treatments. Rumen pH did not differ among the
treatment groups. Among the rumen parameters, total VFA concentration and molar
proportions of butyrate and propionate did not exhibit any significant
differences among the treatments. However, ruminal acetate concentration
decreased in calves that fed sugar cane during the early weeks of the study
period. Comparison of forage and sugar included in the starter diets revealed
that forage reduced weaning age, while sugar cane had a negative effect on
calves' performance.
DOI: 10.1111/jpn.12089
815. J Agric Food Chem. 2013 Aug 7;61(31):7421-30. doi: 10.1021/jf401122d. Epub
2013
Jul 24.
Gene expression patterns, localization, and substrates of polyphenol oxidase in

red clover ( Trifolium pratense L.).
Webb KJ(1), Cookson A, Allison G, Sullivan ML, Winters AL.
Author information:
University , Gogerddan, Aberystwyth, Ceredigion SY23 3EE, Wales, U.K.
Polyphenol oxidase (PPO) genes and their corresponding enzyme activities occur in
many plants; natural PPO substrates and enzyme/substrate localization are less
well characterized. Leaf and root PPO activities in Arabidopsis and five legumes
were compared with those of high-PPO red clover ( Trifolium pratense L.). Red
clover PPO enzyme activity decreased leaves > stem > nodules > peduncle = petiole
> embryo; PPO1 and PPO4 genes were expressed early in leaf emergence, whereas
PPO4 and PPO5 predominated in mature leaves. PPO1 was expressed in embryos and
nodules. PPO substrates, phaselic acid and clovamide, were detected in leaves,
and clovamide was detected in nodules. Phaselic acid and clovamide, along with
caffeic and chlorogenic acids, were suitable substrates for PPO1, PPO4, and PPO5
genes expressed in alfalfa ( Medicago sativa L.) leaves. PPO enzyme presence and
activity were colocalized in leaves and nodules by cytochemistry. Substrates and
PPO activity were localized in developing squashed cell layer of nodules,
suggesting PPO may have a developmental role in nodules.
DOI: 10.1021/jf401122d
816. J Econ Entomol. 2013 Apr;106(2):576-86.
Intended release and actual retention of alfalfa leafcutting bees (Hymenoptera:

Megachilidae) for pollination in commercial alfalfa seed fields.
Pitts-Singer TL(1).
Author information:
(1)USDA-ARS Pollinating Insect Research Unit, Utah State University, Logan, UT
84322, USA. theresa.pitts-singer@ars.usda.gov
Low, medium, and high stocking densities (15,000; 30,000; and 45,000-50,000 bees
per acre, respectively) of Megachile rotundata (F.) (Hymenoptera: Megachilidae),
the alfalfa leafcutting bee, were released over 4 yr in three research plots of
Utah alfalfa, Medicago sativa L. (Fabaceae), planted at seed-production rates. A
low percentage of bees (46-79% of released) survived the incubation and
field-emergence processes; of those bees, the number of females that established
at the nesting sites was 25-100%. Of the three field sites, one site consistently
retained more females and produced more completed nests than the other sites, all
of which usually had poor female establishment and progeny production. In
addition, floral resources were depleted over time, but many flowers remained
unvisited over the season. Nest production decreased over time, as numbers of
flowers and female bees declined. Significant positive relationships were found
between the intended stocking densities and 1) the number of females that
actually survived incubation and field emergence and 2) the number of females
that established nests. The number of females that established nests was
positively affected by the number of females that survived to emerge in the
field. The percentage of females that established was not significantly affected
by the intended number of released bees, countering a prediction that the release
of fewer bees would allow more females to establish nests and achieve high
reproductive success. For growers, it may be more frugal to use modest numbers of
M. rotundata for pollination, because many of the bees at medium and high
stocking densities do not nest in grower-provided bee boards.
Variation in alfalfa leafcutting bee (Hymenoptera: Megachilidae) reproductive

success according to location of nests in United States commercial domiciles.
Pitts-Singer TL(1).
Author information:
(1)U.S. Department of Agriculture-Agriculture Research Services, Pollinating
Insects Research Unit, 1410 N 800 E, Utah State University, Logan, UT 84322-5310,
USA. theresa.pitts-singer@ars.usda.gov
The alfalfa leafcutting bee, Megachile rotundata F., is used extensively to

pollinate alfalfa for seed production in western North America. However, it
usually is not possible to sustain bee populations in the United States. In the
managed pollination system, variable microenvironments are experienced by
developing M. rotundata during the nesting season that may influence bee
reproductive success. In this study, bee cells were produced in a small alfalfa
field and collected from domiciles according to nesting boards and different
portions of boards. Examination of cells showed that the production of live
overwintering prepupae, diseased larvae, other dead eggs and larvae, and cells
attacked by natural enemies varied according to board orientations and positions,
and portions of the boards. Northeast-facing board samples were significantly
heavier and had more pollen balls and chalkbrood compared with southwest-facing
boards. Lower boards in stacks had significantly more chalkbrood than upper
boards. Outer portions of boards had significantly more live prepupae and less
chalkbrood than middle and inner portions, and outer and middle portions had less
summer-emerging bees. These results suggest that reproductive success could be
altered by changing the orientation of boards within domiciles or changing the
designs of domiciles. To accurately assess the quality of bee populations managed
in the commercial loose cell system, a sample of bees should be taken from a
pooled sample from all boards in each domicile. Each domicile sample should then
be evaluated according to its proportional contribution to the whole field's
progeny production (e.g., by weight or volume).
818. Anim Sci J. 2013 Dec;84(12):774-81. doi: 10.1111/asj.12065. Epub 2013 Jun 17.
Characterization of peptides in ensiled alfalfa treated with different chemical

additives.
Ding W(1), Guo X, Ataku K.
Author information:
(1)State Key Laboratory of Pastoral Agricultural Ecosystem, College of Pastoral
Effects of different chemical additives on peptide composition in ensiled alfalfa

(Medicago sativa L.) were investigated by using gel filtration and determination
of N characteristics. The alfalfa silages were prepared untreated (control) or
with formic acid, formaldehyde or tannic acid as additives at ensiling. All
additives reduced non-protein N (NPN), ammonia nitrogen (NH3 -N) and amino acid N
(AA-N) in the ensiled forage, and the most effective reduction of NPN and AA-N
was observed in the formaldehyde-treated silages. Peptides in the control silage
were mainly dipeptides to peptides with five amino acid residues. Most peptides
in the formic acid-treated silage contained 4-12 amino acid residues. Although
most peptides in the formaldehyde-treated silages contained 4-6 amino acid
residues, there was a considerable proportion of peptides with 7-11 amino acid
residues. Tannic acid had little effect on peptide size of ensiled alfalfa
extract in which most peptides contained 5-6 amino acid residues. Peptide size in
formic acid-treated alfalfa silage was greater than that in the other treatments.
Addition of formic acid and formaldehyde not only increased the peptide
concentration in alfalfa silage, but enlarged the peptide size.
DOI: 10.1111/asj.12065
819. Plant Mol Biol. 2013 Nov;83(4-5):317-28. doi: 10.1007/s11103-013-0088-2. Epub

2013 Jun 15.
Improved recombinant cellulase expression in chloroplast of tobacco through

promoter engineering and 5' amplification promoting sequence.
Jung S(1), Lee DS, Kim YO, Joshi CP, Bae HJ.
Author information:
(1)Department of Forest Products and Technology, Chonnam National University,
Kwangju, 500-757, Republic of Korea.
Economical production of bioethanol from lignocellulosic biomass still faces many

technical limitations. Cost-effective production of fermentable sugars is still
not practical for large-scale production of bioethanol due to high costs of
lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are
used as bioreactors, was developed for the mass production of cell wall degrading
enzymes that will help reduce costs. Subcellular targeting is also potentially
more suitable for the accumulation of recombinant cellulases. Herein, we
generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated
Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A
promoter and contained a small subunit of the rubisco complex transit peptide.
The generated transformants possessed high specific BglB activity and did not
show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A
promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to
MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB
plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of
BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble
protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal
production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight,
respectively. These results suggests that suitable recombinant expression of
cellulases in subcellular compartments such as chloroplasts will contribute to
the cost-effective production of enzymes, and will serve as the solid foundation
for the future commercialization of bioethanol production via plant molecular
farming.
DOI: 10.1007/s11103-013-0088-2
820. Pest Manag Sci. 2014 Apr;70(4):610-4. doi: 10.1002/ps.3589. Epub 2013 Jul 4.
Development of a female attractant for the click beetle pest Agriotes brevis.
Vuts J(1), Furlan L, Csonka ÉB, Woodcock CM, Caulfield JC, Mayon P, Pickett JA,
Birkett MA, Tóth M.
Author information:
(1)Rothamsted Research, Harpenden, Herts, UK.
BACKGROUND: Traps suitable for catching female Agriotes click beetles may provide
better reconnaissance than pheromone-baited traps which catch only males, thereby
contributing to more efficient crop protection. The basis for this study came
from (i) observations of female Agriotes brevis (Candeze) aggregating beneath
foliage of Medicago sativa (L.) and Lolium italicum (A. Br.) placed on plastic
sheets on bare soil, and (ii) field tests demonstrating attraction of females to
traps baited with foliage from these plants. The aim was to identify and field
test volatile compounds from M. sativa and L. italicum leaves.
RESULTS: A number of electrophysiologically active chemicals were identified from
headspace extracts of M. sativa and L. italicum. Three different synthetic blends
of the identified compounds, comprising four, seven and nine components, were
field tested. The four- and nine-component blends caught more female A. brevis
than unbaited traps, with the proportion of females not differing between blends.
CONCLUSION: The plant-derived blends were shown to catch female A. brevis under
field conditions when applied in traps. Of these, the four-component blend, given
its relatively simple composition [(Z)-3-hexenyl acetate:methyl
benzoate:(Z)-3-hexen-1-ol:methyl salicylate 300:5:30:30 mg bait(-1)], may be a
suitable 'standard' blend for bait optimisation.
DOI: 10.1002/ps.3589
10.1007/s11356-013-1883-7. Epub 2013 Jun 9.
Effect of EDTA and citric acid on phytoremediation of Cr- B[a]P-co-contaminated

soil.
Chigbo C(1), Batty L.
Author information:
(1)School of Geography, Earth and Environmental Sciences, University of
Birmingham, Birmingham, UK, chuddychigbo@yahoo.com.
Polycyclic aromatic hydrocarbons and heavy metals in the environment are a

concern, and their removal to acceptable level is required. Phytoremediation, the
use of plants to treat contaminated soils, could be an interesting alternative to
conventional remediation processes. This work evaluates the role of single and
combined applications of chelates to single or mixed Cr + benzo[a]pyrene
(B[a]P)-contaminated soil. Medicago sativa was grown in contaminated soil and was
amended with 0.3 g citric acid, 0.146 g ethylenediaminetetraacetic acid (EDTA),
or their combination for 60 days. The result shows that in Cr-contaminated soil,
the application of EDTA + citric acid significantly (p<0.05) decreased the shoot
dry matter of M. sativa by 55 % and, as such, decreased the Cr removal potential
from the soil. The soluble Cr concentration in single Cr or Cr +
B[a]P-contaminated soil was enhanced with the amendment of all chelates; however,
only the application of citric acid in Cr-contaminated soil (44 %) or EDTA and
EDTA + citric acid in co-contaminated soil increased the removal of Cr from the
soil (34 and 54 %, respectively). The dissipation of B[a]P in single
B[a]P-contaminated soil was effective even without planting and amendment with
chelates, while in co-contaminated soil, it was related to the application of
either EDTA or EDTA + citric acid. This suggests that M. sativa with the help of
chelates in single or co-contaminated soil can be effective in phytoextraction of
Cr and promoting the biodegradation of B[a]P.
DOI: 10.1007/s11356-013-1883-7
822. Acta Biol Hung. 2013 Jun;64(2):207-17. doi: 10.1556/ABiol.64.2013.2.7.
Enzymatic and non-enzymatic antioxidant responses of alfalfa leaves and roots

under different salinity levels.
Dehghan G(1), Amjad L, Nosrati H.
Author information:
(1)Department of Plant Biology, University of Tabriz, Tabriz, Iran.
gdehghan@tabrizu.ac.ir
The effect of increasing NaCl concentrations on biomass, hydrogen peroxide

(H2O2), ascorbic acid (ASC), proline and total thiol, and the activity of some
antioxidant enzymes in alfalfa (Medicago sativa L. cv. Gara-Yonjeh) were
investigated. The dry weights of roots and shoots with increasing NaCl
concentrations decreased progressively, and the strongest toxicity was detected
at NaCl treatment of 200 mM. Superoxide dismutase (SOD) activity in the leaves
increased gradually up to NaCl concentrations of 100, while the higher
concentration of NaCl reduced SOD activity in both leaves and roots. The maximum
levels of ascorbate peroxidase (APX) activity were increased at 150 mM and 100 mM
NaCl in leaves and roots of Gara-Yonjeh, respectively. Peroxidase (POD) activity
in roots of Gara-Yonjeh increased (82% at 200 mM) by salinity, while it decreased
(43% at 200 mM) in leaves. In contrast, catalase (CAT) activity increased (84% at
200 mM) in leaves, and decreased (57% at 200 mM) in the roots of Gara-Yonjeh.
Electrophoresis analysis suggested that different patterns in SOD, CAT and POD
isoenzymes depend on NaCl concentrations, and the staining intensities of these
isoforms are supported the results obtained from the spectrophotometric
determinations. In POD and CAT, activity of isoform III was detected at all
concentrations, by a "low-high-low" pattern, with the maximum activity at 50 mM
of NaCl. Results imply that the function of antioxidant systems in higher NaCl
concentration is responsible for the salt tolerance observed in Gara-Yonjeh.
DOI: 10.1556/ABiol.64.2013.2.7
823. Bioresour Technol. 2013 Aug;142:88-94. doi: 10.1016/j.biortech.2013.05.016.

Epub
2013 May 14.
Fermentation of alfalfa wet-fractionation liquids to volatile fatty acids by

Streptococcus bovis and Megasphaera elsdenii.
Weimer PJ(1), Digman MF.
Author information:
(1)United States Department of Agriculture, Agricultural Research Service, US
Dairy Forage Research Center, Madison, WI, USA. Paul.Weimer@ars.usda.gov
"Green juice", obtained by squeezing fresh alfalfa leaves inoculated with lactic
acid bacteria, was fermented at room temperature for 7-21 d to obtain 12-47 g
lactic acid L(-1). Inoculation of green juice with Streptococcus bovis and
incubation at 39°C reduced fermentation time to 8-12h. The resulting "brown
juice" from either fermentation had a pH of ∼4.5 and a protein precipitate. Upon
adjustment to pH 5.2-6.8 and inoculation with Megasphaera elsdenii, brown juice
was fermented within 48 h to up to 18 g of mixed volatile fatty acids (VFA)
L(-1). Single-stage fermentation of green juice by both species in coculture
typically resulted in overgrowth of S. bovis and acid inhibition of M. elsdenii,
inhibiting VFA production. Because the juice fermentations are conducted without
sterilization or supplemental nutrients, they can potentially contribute to an
integrated process featuring protein recovery and fermentation of fractionated
solids to VFA and other products.
DOI: 10.1016/j.biortech.2013.05.016
824. Appl Environ Microbiol. 2013 Aug;79(15):4613-9. doi: 10.1128/AEM.00443-13.

Epub
2013 May 24.
Evaluation of aerated steam treatment of alfalfa and mung bean seeds to eliminate
high levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica, and
Listeria monocytogenes.
Studer P(1), Heller WE, Hummerjohann J, Drissner D.

Author information:
(1)Research Station Agroscope Changins-Wädenswil, Wädenswil, Switzerland.
Sprouts contaminated with human pathogens are able to cause food-borne diseases
due to the favorable growth conditions for bacteria during germination and
because of minimal processing steps prior to consumption. We have investigated
the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung
bean seeds which have been artificially contaminated with Escherichia coli
O157:H7, Salmonella enterica subsp. enterica serovar Weltevreden, and Listeria
monocytogenes Scott A. In addition, a recently collected E. coli O178:H12
isolate, characterized by a reduced heat sensitivity, was exposed to the
treatment described. Populations of E. coli O157:H7 and S. enterica on alfalfa
and mung bean seeds could be completely eliminated by a 300-s treatment with
steam at 70 ± 1°C as revealed by enrichment studies. L. monocytogenes and E. coli
O178:H12 could not be completely eliminated from artificially inoculated seeds.
However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa
and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung
beans was not affected by the 300-s treatment compared to the germination rate of
untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%.
This chemical-free method is an effective alternative to the 20,000-ppm
hypochlorite treatment presently recommended by the U.S. Food and Drug
Administration (FDA).
DOI: 10.1128/AEM.00443-13
PMCID: PMC3719507
825. Guang Pu Xue Yu Guang Pu Fen Xi. 2013 Mar;33(3):766-9.
[Study on relationship between alfalfa canopy spectral reflectance and leaf water
content].
Fu YB(1), Fan YM, Sheng JD, Li N, Wu HQ, Li MT, Li L, Zhao Y.
Author information:
(1)fyb6915028@163.com
In the present study, alfalfa canopy reflectance was researched at alfalfa

squaring period under different irrigation amount at the hutubi county grassland
ecological station. Determining the spectral diagnostic model of alfalfa leaf
moisture content was determined by spectrometry. The results showed that (1) The
spectral reflectance of alfalfa canopy gradually decreases with the increase in
the leaf water content in the near infrared. (2) The spectral inversion model of
alfalfa leaf moisture content established by normalized reflectance spectra is
superior to the original reflectance spectra, and the prediction model
established in the 1,344-1,660 nm band has the lowest average relative error
(7.8%). (3) In this study, the spectral diagnostic model of the leaf moisture
content is: Y=0.962 - 7.560X1451 + 5.295X1473. The spectral prediction model of
the alfalfa leaf moisture content can provide a basis for decision making for
scientific irrigation of alfalfa.
826. Bull Entomol Res. 2013 Oct;103(5):611-8. doi: 10.1017/S0007485313000291. Epub

2013 May 23.
Resolving multiple host use of an emergent pest of cotton with microsatellite

data and chloroplast markers (Creontiades dilutus Stål; Hemiptera, Miridae).
Hereward JP(1), DeBarro PJ, Walter GH.
Author information:
(1)School of Biological Sciences, The University of Queensland, Brisbane,
Queensland, Australia. j.hereward@uq.edu.au
Following the global uptake of transgenic cotton several Hemipteran pests have
emerged as primary targets for pesticide control. Previous research on one such
emergent pest: the green mirid, Creontiades dilutus, indicated differential use
of two crop hosts, cotton (Gossypium hirsutum, Malvaceae) and lucerne (alfalfa)
(Medicago sativa, Fabaceae). We tested the hypothesis that this apparent
demographic independence of lucerne and cotton inhabiting mirids is the result of
cryptic species being associated with these two crops. We assessed gene flow
using microsatellite markers across adjacent cotton and lucerne crops at three
geographically separated sites (up to 900 km apart). We also analysed the recent
feeding behaviour of these insects by amplifying chloroplast markers from their
gut contents. We find high gene flow between these two crops (mean pair wise F ST
between host plants=0.0141 within localities), and no evidence of cryptic
species. Furthermore, the gut analyses revealed evidence of substantial recent
movement between these two crops. We discuss the implications of these results
for interpreting multiple host use in this species and setting future research
priorities for this economically important pest.
DOI: 10.1017/S0007485313000291
827. J Anim Physiol Anim Nutr (Berl). 2014 Apr;98(2):290-9. doi: 10.1111/jpn.12079.
Epub 2013 May 21.
Interactive effects of molasses by homofermentative and heterofermentative

inoculants on fermentation quality, nitrogen fractionation, nutritive value and
aerobic stability of wilted alfalfa (Medicago sativa L) silage.
Hashemzadeh-Cigari F(1), Khorvash M, Ghorbani GR, Ghasemi E, Taghizadeh A, Kargar

S, Yang WZ.
Author information:
(1)Department of Animal Sciences, Isfahan University of Technology, Isfahan,
Iran.
The effect of adding molasses (0, UM or 50 g/kg on DM basis, M) and two types of
inoculant including homofermentative (HO) and a combination of homofermentative
and propionate-producing bacterial (HOPAB) inoculants on silage fermentation
quality, nitrogen fractionation and aerobic stability of pre-bloom, wilted
alfalfa (AS) was determined in laboratory silos. The HOPAB inoculant was more
effective than HO in reducing the alfalfa silage pH but increased propionate
content in the absence of M (p < 0.05). Inoculation of HOPAB reduced (p < 0.01)
acid detergent fibre (ADF) and increased (p < 0.01) lactate to acetate ratio
compared with uninoculated AS. Acetate concentration was lower (p < 0.01) in
HOPAB-inoculated than other AS. This difference was more pronounced in M-added AS
(inoculants × M interaction, p = 0.01). Both inoculants reduced (p < 0.01)
ammonia-N content in AS added with M, whereas only HOPAB decreased (p < 0.01)
ammonia-N concentration in silage without M. Inoculants increased (p < 0.01) B2
fraction in AS with M addition but had no effect on AS without M. Treating
silages with HO-UM increased (p < 0.05) C fraction (acid-detergent insoluble-N)
but HOPAB decreased C fraction at two levels of M. Treating alfalfa crop with M
and HOPAB improved aerobic stability by increasing the concentration of acetate
and propionate of AS respectively. Adding M tended (p < 0.10) to increase
short-chain fatty acids (SCFA) and cumulative gas production (CGP). HOPAB alone
increased DM disappearance at 24 h post-incubation and effective degradability
assuming outflow rate of 8%/h relative to untreated AS (p < 0.05). It was
concluded that adding M had no pronounced effects on AS fermentation quality, but
increased aerobic stability. HOPAB-inoculated AS with no addition of M improved
fermentation quality and increased DM degradability compared with HO.
DOI: 10.1111/jpn.12079
828. PLoS One. 2013 May 14;8(5):e56699. doi: 10.1371/journal.pone.0056699. Print

2013.
Preservation and faithful expression of transgene via artificial seeds in

alfalfa.
Liu W(1), Liang Z, Wang X, Sibbald S, Hunter D, Tian L.
Author information:
(1)College of Life Sciences, Northwest A & F University, Shaanxi, China.
Proper preservation of transgenes and transgenic materials is important for wider

use of transgenic technology in plants. Here, we report stable preservation and
faithful expression of a transgene via artificial seed technology in alfalfa. DNA
constructs containing the uid reporter gene coding for β-glucuronidase (GUS)
driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via
Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently
induced from transgenic alfalfa plants via in vitro technology. These embryos
were treated with abscisic acid to induce desiccation tolerance and were
subjected to a water loss process. After the desiccation procedure, the water
content in dried embryos, or called artificial seeds, was about 12-15% which was
equivalent to that in true seeds. Upon water rehydration, the dried somatic
embryos showed high degrees of viability and exhibited normal germination. Full
plants were subsequently developed and recovered in a greenhouse. The progeny
plants developed from artificial seeds showed GUS enzyme activity and the GUS
expression level was comparable to that of plants developed from somatic embryos
without the desiccation process. Polymerase chain reaction analysis indicated
that the transgene was well retained in the plants and Southern blot analysis
showed that the transgene was stably integrated in plant genome. The research
showed that the transgene and the new trait can be well preserved in artificial
seeds and the progeny developed. The research provides a new method for
transgenic germplasm preservation in different plant species.
PMCID: PMC3653898
16.
Replacing alfalfa silage with corn silage in dairy cow diets: Effects on enteric
methane production, ruminal fermentation, digestion, N balance, and milk
production.
Hassanat F(1), Gervais R, Julien C, Massé DI, Lettat A, Chouinard PY, Petit HV,
Benchaar C.
Author information:
Centre, 2000 College Street, Sherbrooke, Quebec, Canada J1M 0C8.
Erratum in
J Dairy Sci. 2014 Feb;97(2):1169.
The objective of this study was to determine the effects of replacing alfalfa
silage (AS) with corn silage (CS) in dairy cow total mixed rations (TMR) on
enteric CH4 emissions, ruminal fermentation characteristics, apparent total-tract
digestibility, N balance, and milk production. Nine ruminally cannulated
lactating cows were used in a replicated 3×3 Latin square design (32-d period)
and fed (ad libitum) a TMR [forage:concentrate ratio of 60:40; dry matter (DM)
basis], with the forage portion consisting of either alfalfa silage (0% CS; 56.4%
AS in the TMR), a 50:50 mixture of both silages (50% CS; 28.2% AS and 28.2% CS in
the TMR), or corn silage (100% CS; 56.4% CS in the TMR). Increasing the CS
proportion (i.e., at the expense of AS) in the diet was achieved by decreasing
the corn grain proportion and increasing that of soybean meal. Intake of DM and
milk yield increased quadratically, whereas DM digestibility increased linearly
as the proportion of CS increased in the diet. Increasing the dietary CS
proportion resulted in changes (i.e., lower ruminal pH and acetate:propionate
ratio, reduced fiber digestibility, decreased protozoa numbers, and lower milk
fat and higher milk protein contents) typical of those observed when cows are fed
high-starch diets. A quadratic response in daily CH4 emissions was observed in
response to increasing the proportion of CS in the diet (440, 483, and 434 g/d
for 0% CS, 50% CS, and 100% CS, respectively). Methane production adjusted for
intake of DM, and gross or digestible energy was unaffected in cows fed the 50%
CS diet, but decreased in cows fed the 100% CS diet (i.e., quadratic effect).
Increasing the CS proportion in the diet at the expense of AS improved N
utilization, as reflected by the decreases in ruminal NH3 concentration and
manure N excretion, suggesting low potential NH3 and N2O emissions. Results from
this study, suggest that total replacement of AS with CS in dairy cow diets
offers a means of decreasing CH4 output and N losses. However, the reduction in
fiber degradation and the resulting increase in volatile solids content of the
manure may lead to increased CH4 emissions from manure storage.

DOI: 10.3168/jds.2012-6480
16.
Particle size alterations of feedstuffs during in situ neutral detergent fiber

incubation.
Krämer M(1), Nørgaard P, Lund P, Weisbjerg MR.
Author information:
(1)Department of Animal Science, Aarhus University, AU Foulum, Blichers Allé 20,
PO BOX 50, DK-8830 Tjele, Denmark. Monika.Kramer@agrsci.dk
Particle size alterations during neutral detergent fiber (NDF) determination and
in situ rumen incubation were analyzed by dry sieving and image analysis to
evaluate the in situ procedure for estimation of NDF degradation parameters and
indigestible NDF concentration in terms of particle size. Early-cut and late-cut
grass silages, corn silage, alfalfa silage, rapeseed meal, and dried distillers
grains were examined. Treatments were (1) drying and grinding of forage samples
and grinding of concentrates; (2) neutral detergent-soluble (NDS) extraction; (3)
machine washing and NDS extraction; (4) 24-h rumen incubation, machine washing,
and NDS extraction; and (5) 288-h rumen incubation, machine washing, and NDS
extraction. Degradation profiles for potentially degradable NDF were determined
and image analysis was used to estimate particle size profiles and thereby the
risk for particle loss. Particle dimensions changed during NDF determination and
in situ rumen incubation and variations depended on feedstuff and treatment. Corn
silage and late-cut grass silage varied most in particle area among feedstuffs,
with an increase of 139% between 0 and 24h and a decrease of 77% between 24 and
288 h for corn silage and a decrease of 74% for late-cut grass silage between 24-
and 288-h in situ rumen incubation. Especially for late-cut grass silage residues
after 288 h in situ rumen incubation, a high mass proportion in the critical zone
for escape was found. Particle area decreased linearly with increasing incubation
time. Particle loss during in situ rumen incubation cannot be excluded and is
likely to vary among feedstuffs.

DOI: 10.3168/jds.2013-6651
16.
The amino acid composition of rumen-undegradable protein: a comparison between

forages.
Edmunds B(1), Südekum KH, Bennett R, Schröder A, Spiekers H, Schwarz FJ.
Author information:
(1)Institute of Animal Science, University of Bonn, 53115 Bonn, Germany.
The objective of this study was to improve knowledge regarding the amino acid
profile of the insoluble portion of ingested forage escaping rumen degradation.
Six forage categories were analyzed. Categories varied in botanical composition
and each contained 2 samples. Samples within categories were derived from the
same parent material but differed in harvest, maturity, or conservation type. The
rumen-undegradable protein of all forages was measured by incubation for 16h in
the rumen of 3 nonlactating cows. All residues were corrected for microbial
colonization. The AA profile of the residue was different to the original
profile. Degradation trends of individual AA, in terms of increase or decrease
relative to the original concentration, were similar between all forages. The AA
profiles of forage residues, both within and between categories, were more
similar to each other than to their respective original profile. This information
may aid in improving the accuracy of estimating postruminal AA supply from
forages while decreasing the number of samples required to be analyzed.

DOI: 10.3168/jds.2012-6536
832. Plant Sci. 2013 Jul;208:75-82. doi: 10.1016/j.plantsci.2013.03.015. Epub 2013

Apr
8.
Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis

with Sinorhizobium meliloti by preventing nitrogen fixation inhibition.
Palma F(1), López-Gómez M, Tejera NA, Lluch C.
Author information:
Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain.
In this work we have investigated the contribution of pretreatment with 0.1 and
0.5mM salicylic acid (SA) to the protection against salt stress in root nodules
of Medicago sativa in symbiosis with Sinorhizobium meliloti. SA alleviated the
inhibition induced by salinity in the plant growth and photosynthetic capacity of
M. sativa-S. meliloti symbiosis. In addition, SA prevented the inhibition of the
nitrogen fixation capacity under salt stress since nodule biomass was not
affected by salinity in SA pretreated plants. Antioxidant enzymes peroxidase
(POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), dehidroascorbate
reductase (DHAR) and glutathione reductase (GR), key in the main pathway that
scavenges H2O2 in plants, were induced by SA pretreatments which suggest that SA
may participate in the redox balance in root nodules under salt stress. Catalase
activity (CAT) was inhibited around 40% by SA which could be behind the increase
of H2O2 detected in nodules of plants pretreated with SA. The accumulation of
polyamines (PAs) synthesized in response to salinity was prevented by SA which
together with the induction of 1-aminocyclopropane-l-carboxylic acid (ACC)
content suggest the prevalence of the ethylene signaling pathway induced by SA in
detriment of the synthesis of PAs. In conclusion, SA alleviated the negative
effect of salt stress in the M. sativa-S. meliloti symbiosis through the
increased level of nodule biomass and the induction of the nodular antioxidant
metabolism under salt stress. The H2O2 accumulation and the PAs inhibition
induced by SA in nodules of M. sativa suggest that SA activates a hypersensitive
response dependent on ethylene.
DOI: 10.1016/j.plantsci.2013.03.015
833. J Nat Med. 2014 Jan;68(1):220-4. doi: 10.1007/s11418-013-0779-7. Epub 2013 May
16.
Botanical origin of dietary supplements labeled as "Kwao Keur", a folk medicine

from Thailand.
Maruyama T(1), Kawamura M, Kikura-Hanajiri R, Goda Y.
Author information:
(1)Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of
Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo, 158-8501, Japan,
t-maruya@nihs.go.jp.
In the course of our study on the quality of dietary supplements in Japan, both
the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron
sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a
result, the DNA sequence of Pueraria candollei var. mirifica, which is the source
plant of Kwao Keur, was observed in only about half of the products. Inferred
from the determined sequences, source plants in the other products included
Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas,
etc. These inferior products are estimated to lack the efficacy implied by their
labeling. In order to guarantee the quality of dietary supplements, it is
important to identify the source materials exactly; in addition, an
infrastructure that can exclude these inferior products from the market is needed
for the protection of consumers from potential damage to their health and
finances. The DNA analysis performed in this study is useful for this purpose.
DOI: 10.1007/s11418-013-0779-7
834. PLoS One. 2013 May 13;8(5):e63666. doi: 10.1371/journal.pone.0063666. Print

2013.
Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application
to improve plant growth under nitrogen-deficient conditions.
Setten L(1), Soto G, Mozzicafreddo M, Fox AR, Lisi C, Cuccioloni M, Angeletti M,

Pagano E, Díaz-Paleo A, Ayub ND.
Author information:
(1)Instituto de Genética Ewald A. Favret-CICVyA-INTA, Castelar, Buenos Aires,
Argentina.
Erratum in
PLoS One. 2013;8(10).
doi:10.1371/annotation/279fe0d7-d9b1-4d05-a45a-5ff00b4606b7.
Nitrogen is the second most critical factor for crop production after water. In
this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was
genetically modified to fix nitrogen using the genes encoding the nitrogenase of
Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L
medium without nitrogen, displayed high nitrogenase activity and released
significant quantities of ammonium to the medium. Pf-5 X940 also showed
constitutive expression and enzymatic activity of nitrogenase in ammonium medium
or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar
to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and
Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri
transformed with cosmid X940 showed constitutive nitrogenase activity and high
ammonium production, suggesting that this phenotype depends on the genome context
and that this technology to obtain nitrogen-fixing bacteria is not restricted to
Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize
with Pf-5 X940 increased the ammonium concentration in soil and plant
productivity under nitrogen-deficient conditions. In conclusion, these results
open the way to the production of effective recombinant inoculants for nitrogen
fixation on a wide range of crops.
PMCID: PMC3652814
2013.
Mitochondrial Genome Sequence of the Legume Vicia faba.
Negruk V(1).
Author information:
(1)Biotechnology Research Lab, Miami Dade College Miami, FL, USA.
The number of plant mitochondrial genomes sequenced exceeds two dozen. However,
for a detailed comparative study of different phylogenetic branches more plant
mitochondrial genomes should be sequenced. This article presents sequencing data
and comparative analysis of mitochondrial DNA (mtDNA) of the legume Vicia faba.
The size of the V. faba circular mitochondrial master chromosome of cultivar
Broad Windsor was estimated as 588,000 bp with a genome complexity of 387,745 bp
and 52 conservative mitochondrial genes; 32 of them encoding proteins, 3 rRNA,
and 17 tRNA genes. Six tRNA genes were highly homologous to chloroplast genome
sequences. In addition to the 52 conservative genes, 114 unique open reading
frames (ORFs) were found, 36 without significant homology to any known proteins
and 29 with homology to the Medicago truncatula nuclear genome and to other plant
mitochondrial ORFs, 49 ORFs were not homologous to M. truncatula but possessed
sequences with significant homology to other plant mitochondrial or nuclear ORFs.
In general, the unique ORFs revealed very low homology to known closely related
legumes, but several sequence homologies were found between V. faba, Beta
vulgaris, Nicotiana tabacum, Vitis vinifera, and even the monocots Oryza sativa
and Zea mays. Most likely these ORFs arose independently during angiosperm
evolution (Kubo and Mikami, 2007; Kubo and Newton, 2008). Computational analysis
revealed in total about 45% of V. faba mtDNA sequence being homologous to the
Medicago truncatula nuclear genome (more than to any sequenced plant
mitochondrial genome), and 35% of this homology ranging from a few dozen to
12,806 bp are located on chromosome 1. Apparently, mitochondrial rrn5, rrn18,
rps10, ATP synthase subunit alpha, cox2, and tRNA sequences are part of
transcribed nuclear mosaic ORFs.
DOI: 10.3389/fpls.2013.00128
PMCID: PMC3646248
PMID: 23675376
836. Int J Genomics. 2013;2013:678969. doi: 10.1155/2013/678969. Epub 2013 Mar 10.
Conservation, Divergence, and Genome-Wide Distribution of PAL and POX A Gene

Families in Plants.
Rawal HC(1), Singh NK, Sharma TR.
Author information:
(1)Genoinformatics Laboratory, National Research Centre on Plant Biotechnology,
Indian Agricultural Research Institute, Pusa Campus, New Delhi 110 012, India.
Genome-wide identification and phylogenetic and syntenic comparison were

performed for the genes responsible for phenylalanine ammonia lyase (PAL) and
peroxidase A (POX A) enzymes in nine plant species representing very diverse
groups like legumes (Glycine max and Medicago truncatula), fruits (Vitis
vinifera), cereals (Sorghum bicolor, Zea mays, and Oryza sativa), trees (Populus
trichocarpa), and model dicot (Arabidopsis thaliana) and monocot (Brachypodium
distachyon) species. A total of 87 and 1045 genes in PAL and POX A gene families,
respectively, have been identified in these species. The phylogenetic and
syntenic comparison along with motif distributions shows a high degree of
conservation of PAL genes, suggesting that these genes may predate
monocot/eudicot divergence. The POX A family genes, present in clusters at the
subtelomeric regions of chromosomes, might be evolving and expanding with higher
rate than the PAL gene family. Our analysis showed that during the expansion of
POX A gene family, many groups and subgroups have evolved, resulting in a high
level of functional divergence among monocots and dicots. These results will act
as a first step toward the understanding of monocot/eudicot evolution and
functional characterization of these gene families in the future.
DOI: 10.1155/2013/678969
PMCID: PMC3647544
PMID: 23671845
837. Mol Biol Rep. 2013 Jul;40(7):4597-603. doi: 10.1007/s11033-013-2552-0. Epub

2013
May 14.
Molecular cloning and characterization of a gene regulating flowering time from

Zhang T(1), Chao Y, Kang J, Ding W, Yang Q.
Author information:
Genes that regulate flowering time play crucial roles in plant development and
biomass formation. Based on the cDNA sequence of Medicago truncatula (accession
no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis
revealed high homology with FLO/LFY family genes of other plants. When fused to
the green fluorescent protein, MsLFY protein was localized in the nucleus of
onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression
patterns showed that the expression of MsLFY gene was at a low level in roots,
stems, leaves and pods, and the expression level in floral buds was the highest.
The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression
vector was constructed and transformed into Arabidopsis by the
agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis
genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome.
Overexpression of MsLFY specifically caused early flowering under long day
conditions compared with non-transgenic plants. These results indicated MsLFY
played roles in promoting flowering time.
DOI: 10.1007/s11033-013-2552-0
838. Bull Exp Biol Med. 2013 Apr;154(6):755-7.
Hemorheological effects of complex isoflavonoid preparation in ovariectomized

rats.
Anishchenko AM(1).
Author information:
(1)Institute of Pharmacology, Russian Academy of Medical Sciences, Tomsk, Russia.
nuska-80@mail.ru
We have shown that rats with simulated hormone deficiency develop the
hyperviscosity syndrome. Course intragastric administration (14 days) of the
composition of extracts from red clover (100 mg/kg) and alfalfa (100 mg/kg)
prevents the development of hyperviscosity syndrome in rats with hormone
deficiency.
839. Mol Plant Microbe Interact. 2013 Sep;26(9):1089-105. doi:

10.1094/MPMI-03-13-0087-R.
The succinoglycan endoglycanase encoded by exoK is required for efficient

symbiosis of Sinorhizobium meliloti 1021 with the host plants Medicago truncatula
and Medicago sativa (Alfalfa).
Mendis HC(1), Queiroux C, Brewer TE, Davis OM, Washburn BK, Jones KM.
Author information:
(1)Department of Biological Science, Florida State University, Tallahassee, FL
32306-4370, USA.
The acidic polysaccharide succinoglycan produced by the nitrogen-fixing rhizobial

symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the
host plant Medicago truncatula and to efficiently invade the host plant M. sativa
(alfalfa). The β-glucanase enzyme encoded by exoK has previously been
demonstrated to cleave succinoglycan and participate in producing the low
molecular weight form of this polysaccharide. Here, we show that exoK is required
for efficient S. meliloti invasion of both M. truncatula and alfalfa. Deletion
mutants of exoK have a substantial reduction in symbiotic productivity on both of
these plant hosts. Insertion mutants of exoK have an even less productive
symbiosis than the deletion mutants with the host M. truncatula that is caused by
a secondary effect of the insertion itself, and may be due to a polar effect on
the expression of the downstream exoLAMON genes.
DOI: 10.1094/MPMI-03-13-0087-R
840. J Environ Manage. 2013 Sep 30;127:1-9. doi: 10.1016/j.jenvman.2013.04.033.

Epub
2013 May 4.
From soil to leaves--aluminum fractionation by single step extraction procedures

in polluted and protected areas.
Frankowski M(1), Zioła-Frankowska A, Siepak J.
Author information:
(1)Department of Water and Soil Analysis, Faculty of Chemistry, Adam Mickiewicz
University in Poznań, Umultowska 89b, 61-614 Poznań, Poland.
marcin.frankowski@amu.edu.pl
The paper presents the fractionation of aluminum in the samples of soil and
plants of different species using a selective single-step extraction method. The
study was conducted in the area located near a chemical plant, which for many
years served as a post-crystallization leachate disposal site storing chemical
waste (sector I), and in the area around the site: in Wielkopolski National Park,
Rogalin Landscape Park and toward the infiltration ponds at the "Dębina"
groundwater well-field for the city of Poznań (Poland) (sector II). The results
of aluminum fractionation in samples of soil, leaves and plants showed heavy
pollution with aluminum, especially in the water soluble aluminum fraction - Alsw
(maximum concentration of aluminum in soil extract was 234.8 ± 4.8 mg kg(-1), in
the leaves of Betula pendula it was 107.4 ± 1.8 mg kg(-1) and in the plants of
Artemisia vulgaris (root) and Medicago sativa (leaves) it amounted to
464.7 ± 10.7 mg kg(-1)and 146.8 ± 1.2 mg kg(-1) respectively). In addition, the
paper presents the problem of organic aluminum fractionation in biological
samples and it shows the relationship between aluminum concentration in soil and
the analysed woody and herbaceous species.
DOI: 10.1016/j.jenvman.2013.04.033
841. PLoS One. 2013 Apr 25;8(4):e62528. doi: 10.1371/journal.pone.0062528. Print

2013.
Effects of CO2 and temperature on tritrophic interactions.
Dyer LA(1), Richards LA, Short SA, Dodson CD.
Author information:
(1)Department of Biology, University of Nevada Reno, Reno, Nevada, USA.
nolaclimber@gmail.com
There has been a significant increase in studies of how global change parameters
affect interacting species or entire communities, yet the combined or interactive
effects of increased atmospheric CO2 and associated increases in global mean
temperatures on chemically mediated trophic interactions are mostly unknown.
Thus, predictions of climate-induced changes on plant-insect interactions are
still based primarily on studies of individual species, individual global change
parameters, pairwise interactions, or parameters that summarize communities. A
clear understanding of community response to global change will only emerge from
studies that examine effects of multiple variables on biotic interactions. We
examined the effects of increased CO2 and temperature on simple laboratory
communities of interacting alfalfa, chemical defense, armyworm caterpillars, and
parasitoid wasps. Higher temperatures and CO2 caused decreased plant quality,
decreased caterpillar development times, developmental asynchrony between
caterpillars and wasps, and complete wasp mortality. The effects measured here,
along with other effects of global change on natural enemies suggest that
biological control and other top-down effects of insect predators will decline
over the coming decades.
PMCID: PMC3636099
842. Theor Appl Genet. 2013 Jul;126(7):1885-96. doi: 10.1007/s00122-013-2103-z.

Epub
2013 May 1.
Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa

L.).
Yu F(1), Lei Y, Li Y, Dou Q, Wang H, Chen Z.
Author information:
(1)Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Plateau
Institute of Biology, Chinese Academy of Sciences, Xining 810001, China.
Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH
and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences
(MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric,
of which three were satellite DNAs and two were minisatellite DNAs. Monomers of
144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively,
while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively.
Three repetitive sequences were characterized as subtelomeric; one repetitive
sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had
fragments of 204 and 327 bp. Sequence analysis revealed homology (70-80 %)
between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp.
caerulea. Three identified repetitive sequences produced hybridization signals at
multiple sites in a few of the chromosomes; one repetitive sequence was
identified as the E180 satellite DNA previously isolated from M. sativa, while
the other 163 and 227 bp fragments had distinct sequences. Physical mapping of
the repetitive sequences with double-target FISH revealed different patterns.
Thus, nine novel tandemly repetitive sequences that can be adopted as distinct
chromosome markers in alfalfa were identified in this study. Furthermore, the
chromosome distribution of each sequence was well described. Though significant
chromosome variations were detected within and between cultivars, a molecular
karyotype of alfalfa was suggested with the chromosome markers we identified.
Therefore, these novel chromosome markers will still be a powerful tool for
genome composition analysis, phylogenetic studies, and breeding applications.
DOI: 10.1007/s00122-013-2103-z
843. J Med Food. 2013 May;16(5):410-20. doi: 10.1089/jmf.2012.2679. Epub 2013 Apr
30.
Chloroform extract of alfalfa (Medicago sativa) inhibits

lipopolysaccharide-induced inflammation by downregulating ERK/NF-κB signaling and
cytokine production.
Choi KC(1), Hwang JM, Bang SJ, Kim BT, Kim DH, Chae M, Lee SA, Choi GJ, Kim DH,
Lee JC.
Author information:
(1)Grassland and Forages Research Center, National Institute of Animal Science,
Cheonan, Korea.
Alfalfa (Medicago sativa L.) is commonly used as a traditional medicine and

functional food. This study investigated the anti-inflammatory potential of
alfalfa and the mechanisms involved. The chloroform extract of alfalfa aerial
parts inhibited lipopolysaccharide (LPS)-stimulated immune responses more than
ether, butanol, or water soluble extracts. Treatment with 1 μg/mL LPS increased
nitrite concentrations to 44.3 μM in RAW267.4 macrophages, but it was reduced to
10.6 μM by adding 100 μg/mL chloroform extract. LPS treatment also increased the
concentrations of tumor necrosis factor-α, interleukin (IL)-6, and IL-1β to 41.3,
11.6, and 0.78 ng/mL in culture supernatants of the cells, but these cytokine
levels decreased to 12.5, 3.1, and 0.19 ng/mL, respectively, by pretreating with
100 μg/mL of the extract. ICR mice injected with LPS (30 mg/kg body weight) alone
showed a 0% survival rate after 48 h of the injection, but 48-h survival of the
mice increased to 60% after oral administration of the extract. Subfractions of
the chloroform extract markedly suppressed LPS-mediated activation of the
extracellular signal-regulated kinase and nuclear factor kappa-B. Cinnamic acid
derivatives and fatty acids were found to be active constituents of the extract.
This research demonstrated that alfalfa aerial parts exert anti-inflammatory
activity and may be useful as a functional food for the prevention of
inflammatory disorders.
DOI: 10.1089/jmf.2012.2679
844. J Dairy Sci. 2013 Jul;96(7):4100-11. doi: 10.3168/jds.2012-6109. Epub 2013 Apr
28.
Grain-based versus alfalfa-based subacute ruminal acidosis induction experiments:

Similarities and differences between changes in milk fatty acids.
Colman E(1), Khafipour E, Vlaeminck B, De Baets B, Plaizier JC, Fievez V.
Author information:
(1)Laboratory for Animal Nutrition and Animal Product Quality, Ghent University,
Proefhoevestraat 10, 9090 Melle, Belgium.
Subacute ruminal acidosis (SARA) is one of the most important metabolic

disorders, traditionally characterized by low rumen pH, which might be induced by
an increase in the dietary proportion of grains as well as by a reduction of
structural fiber. Both approaches were used in earlier published experiments in
which SARA was induced by replacing part of the ration by a grain mixture or
alfalfa hay by alfalfa pellets. The main differences between both experiments
were the presence of blood lipopolysaccharide and Escherichia coli and associated
effects on the rumen microbial population in the rumen of grain-based induced
SARA animals as well as a great amount of quickly fermentable carbohydrates in
the grain-based SARA induction experiment. Both induction approaches changed
rumen pH although the pH decrease was more substantial in the alfalfa-based SARA
induction protocol. The goal of the current analysis was to assess whether both
acidosis induction approaches provoked similar shifts in the milk fatty acid (FA)
profile. Similar changes of the odd- and branched-chain FA and the C18
biohydrogenation intermediates were observed in the alfalfa-based SARA induction
experiment and the grain-based SARA induction experiment, although they were more
pronounced in the former. The proportion of trans-10 C18:1 in the last week of
the alfalfa-based induction experiment was 6 times higher than the proportion
measured during the control week. The main difference between both induction
experiments under similar rumen pH changes was the decreasing sum of iso FA
during the grain-based SARA induction experiment whereas the sum of iso FA
remained stable during the alfalfa-based SARA induction experiment. The
cellulolytic bacterial community seemed to be negatively affected by either the
presence of E. coli and the associated lipopolysaccharide accumulation in the
rumen or by the amount of starch and quickly fermentable carbohydrates in the
diet. In general, changes in the milk FA profile were related to changes in rumen
pH. Nevertheless, feed characteristics (low in structural fiber vs. high in
starch) also affected the milk FA profile and, as such, both effects should be
taken into account when subacute acidosis occurs.

DOI: 10.3168/jds.2012-6109
845. Zhongguo Zhong Yao Za Zhi. 2012 Dec;37(24):3773-6.
[Molecular identification of astragali radix and its adulterants by ITS

sequences].
Cui ZH(1), Li Y, Yuan QJ, Zhou LS, Li MH.
Author information:
(1)Institute of Chinese Materia Medica, China Academy of Chinese Medical Science,
OBJECTIVE: To explore a new method for identification Astragali Radix from its
adulterants by using ITS sequence.
METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples
of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and
Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced
unidirectionally. The interspecific K-2-P distances of Astragali Radix and its
adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA
4.
RESULT: ITS sequences were obtained from 19 samples respectively, there were
Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea
rosea 728 bp, which were registered in the GenBank. Phylogeny trees
reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can
effectively distinguish Astragali Radix from adulterants.
CONCLUSION: ITS sequence can be used to identify Astragali Radix from its
adulterants successfully and is an efficient molecular marker for authentication
of Astragali Radix and its adulterants.
846. Biotechnol Appl Biochem. 2013 Mar-Apr;60(2):170-89. doi: 10.1002/bab.1075.
Structure-function analyses and molecular modeling of caffeic

acid-O-methyltransferase and caffeoyl-CoA-O-methyltransferase: revisiting the
basis of alternate methylation pathways during monolignol biosynthesis.
Naaz H(1), Pandey VP, Singh S, Dwivedi UN.
Author information:
(1)Department of Biochemistry, Bioinformatics Infrastructure Facility, Centre of
Excellence in Bioinformatics, University of Lucknow, Lucknow, U.P., India.
Ten protein sequences, each of caffeic acid-O-methyltransferase (COMT) and

caffeoyl-coenzyme A-O-methyltransferase (CCoAOMT), catalyzing methylation of
precursors of monolignol from selected dicots and monocots have been analyzed and
compared on the basis of their amino acid sequence, motifs/domains,
three-dimensional (3D) structure, and substrate binding. The isoelectric points
of all the COMT and CCoAOMT sequences analyzed were found to vary in the pH range
of 5 to 6. Molecular weight analyses suggested CCoAOMT to be smaller monomeric
proteins (27-29 kDa) as compared with those of COMTs (39-40 kDa), which were
dimeric. On the basis of phylogenetic analysis, COMT and CCoAOMT were clustered
into two major groups, each of which could be further divided into two subgroups
of monocots and dicots. Modeling and superimposition of COMT and CCoAOMT
sequences of alfalfa (Medicago sativa) revealed that both were quite different at
the 3D levels, although they had similarity in the core region. Molecular docking
of 16 putative substrates (intermediates of monolignol biosynthesis pathway)
revealed that both enzymes interact with all 16 substrates in a similar manner,
with thiol esters being the most potent and binding of these putative substrates
to CCoAOMT being more efficient.
© 2013 International Union of Biochemistry and Molecular Biology, Inc.
DOI: 10.1002/bab.1075
847. Plant Cell Physiol. 2013 Jul;54(7):1041-55. doi: 10.1093/pcp/pct056. Epub 2013
Apr 15.
Analysis of the alfalfa root transcriptome in response to salinity stress.
Postnikova OA(1), Shao J, Nemchinov LG.
Author information:
(1)USDA/ARS, Plant Sciences Institute, Molecular Plant Pathology Laboratory,
10300 Baltimore Avenue, Beltsville, MD 20705, USA.
Salinity is one of the major abiotic factors affecting alfalfa productivity.

Identifying genes that control this complex trait will provide critical insights
for alfalfa breeding programs. To date, no studies have been published on a deep
sequencing-based profiling of the alfalfa transcriptome in response to salinity
stress. Observations gathered through research on reference genomes may not
always be applicable to alfalfa. In this work, Illumina RNA-sequencing was
performed in two alfalfa genotypes contrasting in salt tolerance, in order to
estimate a broad spectrum of genes affected by salt stress. A total of
367,619,586 short reads were generated from cDNA libraries originated from roots
of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained
and, among them, 74.5% had a significant similarity to proteins in the NCBI
database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496
SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that
the expression of 1,165 genes, including 86 transcription factors (TFs), was
significantly altered under salt stress. About 40% of differentially expressed
genes were assigned to known gene ontology (GO) categories using Arabidopsis GO.
A random check of differentially expressed genes by quantitative real-time PCR
confirmed the bioinformatic analysis of the RNA-seq data. A number of
salt-responsive genes in both tested genotypes were identified and assigned to
functional classes, and gene candidates with roles in the adaptation to salinity
were proposed. Alfalfa-specific data on salt-responsive genes obtained in this
work will be useful in understanding the molecular mechanisms of salinity
tolerance in alfalfa.
DOI: 10.1093/pcp/pct056
848. Environ Sci Pollut Res Int. 2013 Sep;20(9):6337-43. doi:

10.1007/s11356-013-1691-0. Epub 2013 Apr 16.
The linear accumulation of atmospheric mercury by vegetable and grass leaves:

Potential biomonitors for atmospheric mercury pollution.
Niu Z(1), Zhang X, Wang S, Ci Z, Kong X, Wang Z.
Author information:
(1)Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
P.O. Box 2871, Beijing, 100085, China.
One question in the use of plants as biomonitors for atmospheric mercury (Hg) is
to confirm the linear relationships of Hg concentrations between air and leaves.
To explore the origin of Hg in the vegetable and grass leaves, open top chambers
(OTCs) experiment was conducted to study the relationships of Hg concentrations
between air and leaves of lettuce (Lactuca sativa L.), radish (Raphanus sativus
L.), alfalfa (Medicago sativa L.) and ryegrass (Lolium perenne L.). The influence
of Hg in soil on Hg accumulation in leaves was studied simultaneously by soil
Hg-enriched experiment. Hg concentrations in grass and vegetable leaves and roots
were measured in both experiments. Results from OTCs experiment showed that Hg
concentrations in leaves of the four species were significantly positively
correlated with those in air during the growth time (p < 0.05), while results
from soil Hg-enriched experiment indicated that soil-borne Hg had significant
influence on Hg accumulation in the roots of each plant (p < 0.05), and some
influence on vegetable leaves (p < 0.05), but no significant influence on Hg
accumulation in grass leaves (p > 0.05). Thus, Hg in grass leaves is mainly
originated from the atmosphere, and grass leaves are more suitable as potential
biomonitors for atmospheric Hg pollution. The effect detection limits (EDLs) for
the leaves of alfalfa and ryegrass were 15.1 and 22.2 ng g(-1), respectively, and
the biological detection limit (BDL) for alfalfa and ryegrass was 3.4 ng m(-3).
DOI: 10.1007/s11356-013-1691-0
849. Plant Cell Rep. 2013 Aug;32(8):1289-98. doi: 10.1007/s00299-013-1443-0. Epub

2013
Apr 13.
Overexpression of a novel salt stress-induced glycine-rich protein gene from

alfalfa causes salt and ABA sensitivity in Arabidopsis.
Long R(1), Yang Q, Kang J, Zhang T, Wang H, Li M, Zhang Z.
Author information:
(1)School of Life Science, Chongqing University, Chongqing, China.
KEY MESSAGE: We cloned a novel salt stress-induced glycine-rich protein gene (

MsGRP ) from alfalfa. Its overexpression retards seed germination and seedling
growth of transgenic Arabidopsis after salt and ABA treatments. Since soil
salinity is one of the most significant abiotic stresses, salt tolerance is
required to overcome salinity-induced reductions in crop productivity. Many
glycine-rich proteins (GRPs) have been implicated in plant responses to
environmental stresses, but the function and importance of some GRPs in stress
responses remain largely unknown. Here, we report on a novel salt stress-induced
GRP gene (MsGRP) that we isolated from alfalfa. Compared with some glycine-rich
RNA-binding proteins, MsGRP contains no RNA recognition motifs and localizes in
the cell membrane or cell wall according to the subcellular localization result.
MsGRP mRNA is induced by salt, abscisic acid (ABA), and drought stresses in
alfalfa seedlings, and its overexpression driven by a constitutive cauliflower
mosaic virus-35S promoter in Arabidopsis plants confers salinity and ABA
sensitivity compared with WT plants. MsGRP retards seed germination and seedling
growth of transgenic Arabidopsis plants after salt and ABA treatments, which
implies that MsGRP may affect germination and growth through an ABA-dependent
regulation pathway. These results provide indirect evidence that MsGRP plays
important roles in seed germination and seedling growth of alfalfa under some
abiotic stress conditions.
DOI: 10.1007/s00299-013-1443-0

Epub 2013 Apr 11.
The effect of feeding rations with different ratios of concentrate to alfalfa hay
on blood hematological and biochemical parameters of farmed ostriches (Struthio
camelus).
Ghasemi HA(1), Kazemi-Bonchenari M, Khaltabadi-Farahani AH, Khodaei Motlagh M.
Author information:
Arak University, Arak, 38156-8-8349, Iran, h-ghasemi@araku.ac.ir.
A two-month trial was conducted to investigate the effects of pelleting rations

with 70:30, 65:35, and 60:40 concentrate to high-quality roughage (alfalfa hay)
ratios on hematological and biochemical parameters of ostriches. A total of 18
seven-month-old male ostrich chicks, initially weighing an average of 60-70 kg,
were distributed into three different outdoor paddocks at a stocking density of
six birds per paddock. In the morning, blood collection was made from the wing
vein after about 12 h of fasting. All rations resulted in no significant change
in hematological parameters. However, with increasing alfalfa inclusion rate, the
heterophil/lymphocyte ratio tended to be lower (P = 0.089); whereas the values of
hemoglobin, packed cell volume (PCV), and mean corpuscular hemoglobin (MCH)
tended to be higher (P values 0.069, 0.072 and 0.094, respectively). In terms of
plasma biochemical parameters, increasing the ratio of concentrate to alfalfa hay
in ostrich diets resulted in significant (P < 0.05) depression in the values of
glucose (up to 20.2 %), total cholesterol (up to 19.2 %), creatine kinase (up to
27.9 %), and aspartate aminotransferase (up to 29.9 %). Additionally, the 65:35
or 60:40 rations caused a decrease in low-density lipoprotein cholesterol level
(27.9 and 42 %, respectively; P = 0.008) compared to the 70:40 ration. Without
exception, no rations affected the values of leukocyte subsets, total protein,
albumin, globulin, uric acid, alanine aminotransferase, gamma glutamyl
transferase, triglycerides, high-density lipoprotein cholesterol, and very low
density lipoprotein. Based on our results, the concentrate to alfalfa ratio of
60:40 could be suggested as optimum ratio for good health conditions of juvenile
ostriches.
DOI: 10.1007/s11250-013-0409-0
851. Mol Plant Pathol. 2013 Aug;14(6):602-9. doi: 10.1111/mpp.12028. Epub 2013 Apr
10.
Novel receptor-like kinases in cacao contain PR-1 extracellular domains.
Teixeira PJ(1), Costa GG, Fiorin GL, Pereira GA, Mondego JM.
Author information:
(1)Laboratório de Genômica e Expressão, Departamento de Genética, Evolução e
Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas-Uicamp, CP
6109, Campinas, SP 13083-970, Brazil.
Members of the pathogenesis-related protein 1 (PR-1) family are well-known

markers of plant defence responses, forming part of the arsenal of the secreted
proteins produced on pathogen recognition. Here, we report the identification of
two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and
serine/threonine kinase domains, in a manner characteristic of receptor-like
kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor
kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao
indicated that PR-1RKs originated from a fusion between sequences encoding PR-1
and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase).
Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was
involved in the origin of PR-1RKs. Genes with a similar domain architecture to
cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago
truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However,
their kinase domains differed from those found in LecRLKs, indicating the
occurrence of convergent evolution. TcPR-1g expression was up-regulated in the
biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during
cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal
by interacting with a PR-1 ligand.
© 2013 BSPP AND JOHN WILEY & SONS LTD.
DOI: 10.1111/mpp.12028
852. Environ Toxicol Chem. 2013 Aug;32(8):1746-51. doi: 10.1002/etc.2237. Epub 2013
Jun 6.
Ecotoxicological responses of three ornamental herb species to cadmium.
Liu Z(1), He X, Chen W, Zhao M.
Author information:
(1)State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology,
Chinese Academy of Sciences, Shenyang, China.
Cadmium is one of the most toxic elements. The ideal vegetal cover should be
ensured by the selection of appropriate plant species for successful
phytoremediation. In the present study, the ecotoxicological effects of Cd on the
following 3 ornamental herbs were investigated: Italian ryegrass (Lolium
multiflorum Lam.), white clover (Trifolium repens L.), and alfalfa (Medicago
sativa L.). Based on the inhibition rate of seed germination, root and shoot
elongation, early seedling growth, median inhibition concentration (IC50) values,
and index of tolerance (IT) values, ecotoxicological indicators were determined.
The results showed that 10 μM to 50 μM Cd had little effect on seed germination
or root and shoot elongation of the 3 ornamental herbs (p > 0.01). With an
increase in Cd concentrations, alfalfa (M. sativa) was the most sensitive to Cd
toxicity in terms of seed germination and root elongation. Based on the IC50 of
root elongation, Italian ryegrass (L. multiflorum) was the least sensitive to Cd.
Based on the IC50 of seed germination and shoot elongation, white clover had the
least sensitivity to Cd. Among the 3 ornamental herbs, based on the IC50 of seed
germination and root and shoot elongation, alfalfa (M. sativa) was all the most
sensitive plant. According to the index of tolerance, Italian ryegrass (L.
multiflorum) was the most tolerant plant.
Copyright © 2013 SETAC.

DOI: 10.1002/etc.2237
853. Biodegradation. 2013 Jul;24(4):569-81. doi: 10.1007/s10532-013-9628-3. Epub

2013
Mar 30.
Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans

in rhizospheric sand and soil microcosms: microbial interactions and PAH
dissipation.
Thion C(1), Cébron A, Beguiristain T, Leyval C.
Author information:
(1)Université de Lorraine, CNRS, LIMOS, UMR 7137, Vandoeuvre-lès-Nancy Cedex,
France.
Very little is known about the influence of bacterial-fungal ecological

interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils.
Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro.
We investigated their interactions and their effect on the dissipation of three
PAHs-phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)-in planted
microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms
planted with alfalfa, the two microbes survived and grew, without any significant
effect of co-inoculation. Co-inoculation led to the dissipation of 46 % of PHE
after 21 days. In soil microcosms, whether planted with alfalfa or not, both
strains persisted throughout the 46 days of the experiment, without any effect of
co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level
indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer
specific to the genus Fusarium. The microbial community was analyzed by temporal
temperature gradient electrophoresis and real-time PCR targeting bacterial and
fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were
modified by PAH pollution, which selected PAH-degrading bacteria, by the presence
of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR
concentrations significantly decreased (91 and 46 %, respectively) whatever the
treatment, but DBA concentration significantly decreased (30 %) in planted and
co-inoculated microcosms only.
DOI: 10.1007/s10532-013-9628-3
854. PLoS One. 2013;8(3):e58188. doi: 10.1371/journal.pone.0058188. Epub 2013 Mar

11.
Effect of feeding selenium-fertilized alfalfa hay on performance of weaned beef

calves.
Hall JA(1), Bobe G, Hunter JK, Vorachek WR, Stewart WC, Vanegas JA, Estill CT,
Mosher WD, Pirelli GJ.
Author information:
(1)Department of Biomedical Sciences, College of Veterinary Medicine, Oregon
State University, Corvallis, Oregon, United States of America.
Jean.Hall@oregonstate.edu
Selenium (Se) is an essential micronutrient in cattle, and Se-deficiency can

affect morbidity and mortality. Calves may have greater Se requirements during
periods of stress, such as during the transitional period between weaning and
movement to a feedlot. Previously, we showed that feeding Se-fertilized forage
increases whole-blood (WB) Se concentrations in mature beef cows. Our current
objective was to test whether feeding Se-fertilized forage increases WB-Se
concentrations and performance in weaned beef calves. Recently weaned beef calves
(n = 60) were blocked by body weight, randomly assigned to 4 groups, and fed an
alfalfa hay based diet for 7 wk, which was harvested from fields fertilized with
sodium-selenate at a rate of 0, 22.5, 45.0, or 89.9 g Se/ha. Blood samples were
collected weekly and analyzed for WB-Se concentrations. Body weight and health
status of calves were monitored during the 7-wk feeding trial. Increasing
application rates of Se fertilizer resulted in increased alfalfa hay Se content
for that cutting of alfalfa (0.07, 0.95, 1.55, 3.26 mg Se/kg dry matter for Se
application rates of 0, 22.5, 45.0, or 89.9 g Se/ha, respectively). Feeding
Se-fertilized alfalfa hay during the 7-wk preconditioning period increased WB-Se
concentrations (P Linear<0.001) and body weights (P Linear = 0.002) depending
upon the Se-application rate. Based upon our results we suggest that soil-Se
fertilization is a potential management tool to improve Se-status and performance
in weaned calves in areas with low soil-Se concentrations.
PMCID: PMC3594272
855. Acta Microbiol Immunol Hung. 2013 Mar;60(1):29-39. doi:

10.1556/AMicr.60.2013.1.4.
Isolation of Acanthamoeba from the rhizosphere of maize and lucerne plants.
Orosz E(1), Farkas A, Ködöböcz L, Becságh P, Danka J, Kucsera I, Füleky G.
Author information:
(1)Department of Parasitology, National Center for Epidemiology, Budapest,
Hungary. orosz.erika@oek.antsz.hu
Acanthamoeba species are free-living amoebae that can be found in almost every
range of environments. Within this genus, a number of species are recognized as
human pathogens, potentially causing Acanthamoeba keratitis, granulomatous
amoebic encephalitis, and chronic granulomatous lesions. Soil and water samples
were taken from experimental station at Julianna Major of Plant Protection
Institute of Centre for Agricultural Research, Hungarian Academy of Sciences (CAR
HAS). We detected living Acanthamoeba spp. based on culture-confirmed detection
combined with the molecular taxonomic identification method. Living Acanthamoeba
spp. were detected in thirteen (65%) samples. The presence of Acanthamoeba spp.
in the samples depends significantly on the rhizosphere plants. The most
frequently identified living Acanthamoeba genotype was T4 followed by T11, T2/T6
and T17. Genotypes T4 and T11 of Acanthamoeba, are responsible for Acanthamoeba
keratitis as well as granulomatous amoebic encephalitis, and should therefore be
considered as a potential health risk associated with human activities in the
environment.
DOI: 10.1556/AMicr.60.2013.1.4

19.
Early-season host switching in Adelphocoris spp. (Hemiptera: Miridae) of
differing host breadth.
Pan H(1), Lu Y, Wyckhuys KA.
Author information:
of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
The mirid bugs Adelphocoris suturalis (Jakovlev), Adelphocoris lineolatus (Goeze)

and Adelphocoris fasciaticollis (Reuter) (Hemiptera: Miridae) are common pests of
several agricultural crops. These three species have vastly different
geographical distributions, phenologies and abundances, all of which are linked
to their reliance on local plants. Previous work has shown notable differences in
Adelphocoris spp. host use for overwintering. In this study, we assessed the
extent to which each of the Adelphocoris spp. relies on some of its major
overwinter hosts for spring development. Over the course of four consecutive
years (2009-2012), we conducted population surveys on 77 different plant species
from 39 families. During the spring, A. fasciaticollis used the broadest range of
hosts, as it was found on 35 plant species, followed by A. suturalis (15 species)
and A. lineolatus (7 species). Abundances of the species greatly differed between
host plants, with A. fasciaticollis reaching the highest abundance on Chinese
date (Ziziphus jujuba Mill.), whereas both A. suturalis and A. lineolatus
preferred alfalfa (Medicago sativa L.). The host breadths of the three
Adelphocoris spp. differed greatly between subsequent spring and winter seasons.
The generalist species exhibited the least host fidelity, with A. suturalis and
A. lineolatus using 8 of 22 and 4 of 12 overwinter host species for spring
development, respectively. By contrast, the comparative specialist A.
fasciaticollis relied on 9 of its 11 overwinter plants as early-season hosts. We
highlight important seasonal changes in host breadth and interspecific
differences in the extent of host switching behavior between the winter and
spring seasons. These findings benefit our understanding of the evolutionary
interactions between mirid bugs and their host plants and can be used to guide
early-season population management.
PMCID: PMC3602548
857. Curr Microbiol. 2013 Aug;67(2):240-8. doi: 10.1007/s00284-013-0353-2. Epub

2013
Mar 23.
Ribosomal intergenic spacer analysis as a tool for monitoring methanogenic

Archaea changes in an anaerobic digester.
Ciesielski S(1), Bułkowska K, Dabrowska D, Kaczmarczyk D, Kowal P, Możejko J.
Author information:
(1)Department of Environmental Biotechnology, Faculty of Environmental Sciences,
University of Warmia and Mazury in Olsztyn, Olsztyn, Poland. slavcm@uwm.edu.pl
The applicability of a newly-designed PCR primer pair in examination of

methanogenic Archaea in a digester treating plant biomass was evaluated by
Ribosmal Intergenic Spacer Analysis (RISA). To find a suitable approach, three
variants of RISA were tested: (1) standard, polyacrylamide gel-based, (2)
automated, utilized capillary electrophoresis (GA-ARISA), and (3) automated
microfluidics-based (MF-ARISA). All three techniques yielded a consistent picture
of archaeal community structure changes during anaerobic digestion monitored for
more than 6 weeks. While automated variants were more practical for handling and
rapid analysis of methanogenic Archaea, the gel-based technique was advantageous
when micro-organism identification was required. A DNA-sequence analysis of
dominant bands extracted from the gel revealed that the main role in methane
synthesis was played by micro-organisms affiliated with Methanosarcina barkeri.
The obtained results revealed that RISA is a robust method allowing for detailed
analysis of archaeal community structure during organic biomass conversion into
biogas. In addition, our results showed that GA-ARISA has a higher resolution and
reproducibility than other variants of RISA and could be used as a technique for
tracking changes in methanogenic Archaea in an anaerobic digester.
DOI: 10.1007/s00284-013-0353-2
PMCID: PMC3695317

Mar
25.
Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa)

and subsequent exposure to air.
McGarvey JA(1), Franco RB, Palumbo JD, Hnasko R, Stanker L, Mitloehner FM.
Author information:
(1)US Department of Agriculture, Agricultural Research Service, Western Regional
Research Center, Albany, CA 94133, USA. jeffery.mcgarvey@ars.usda.gov
AIMS: To describe, at high resolution, the bacterial population dynamics and

chemical transformations during the ensiling of alfalfa and subsequent exposure
to air.
METHODS AND RESULTS: Samples of alfalfa, ensiled alfalfa and silage exposed to
air were collected and their bacterial population structures compared using 16S
rRNA gene libraries containing approximately 1900 sequences each. Cultural and
chemical analyses were also performed to complement the 16S gene sequence data.
Sequence analysis revealed significant differences (P < 0·05) in the bacterial
populations at each time point. The alfalfa-derived library contained mostly
sequences associated with the Gammaproteobacteria (including the genera:
Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly
sequences associated with the lactic acid bacteria (LAB) (including the genera:
Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even
greater percentages of LAB, especially among the genus Lactobacillus, and a
significant drop in bacterial diversity.
CONCLUSIONS: In-depth 16S rRNA gene sequence analysis revealed significant
bacterial population structure changes during ensiling and again during exposure
to air.
SIGNIFICANCE AND IMPACT OF THE STUDY: This in-depth description of the bacterial
population dynamics that occurred during ensiling and simulated feed out expands
our knowledge of these processes.
© 2013 The Society for Applied Microbiology No claim to US Government works.
DOI: 10.1111/jam.12179
859. J Sci Food Agric. 2013 Sep;93(12):3072-80. doi: 10.1002/jsfa.6141. Epub 2013
Apr
29.
Protein molecular structures in alfalfa hay cut at three stages of maturity and
in the afternoon and morning and relationship with nutrient availability in
ruminants.
Yari M(1), Valizadeh R, Naserian AA, Jonker A, Yu P.
Author information:
(1)Department of Animal and Poultry Science, University of Saskatchewan,
Saskatoon, SK, Canada. m.yari@malayeru.ac.ir
BACKGROUND: Molecular structures in feed protein influence its digestive

behavior, availability and utilization. From a nutritive point of view, stage of
maturity and cutting time are important factors affecting nutrient profiles and
availability of alfalfa (Medicago sativa L.) hay in ruminants. The objectives of
this study were to determine protein molecular structures by Fourier transform
infrared spectroscopy (FTIR), and their relationship with nutrient profiles and
availability in ruminants of alfalfa hay cut at early bud, late bud and early
flower stages and in afternoon and morning.
RESULTS: With advancing maturity, molecular structure ratios of α-helix:β-sheets
decreased, while amide I:amide II increased (P ≤ 0.05). Alfalfa cutting in
afternoon versus morning increased protein structure α-helix:β-sheets and
α-helix:others ratios (P < 0.05) and tended to decrease the proportion of
β-sheets (P = 0.09). Positive correlations were found for α-helix:β-sheet ratio
(R ≥ 0.60; P < 0.05) with intermediately degradable protein (PB2) and ruminal
degradability and intestinal protein supply, and all these parameters correlated
negatively with amide I:amide II ratio (R ≤ -0.62; P < 0.05).
CONCLUSION: Protein molecular structures in alfalfa hay changed with advancing
maturity and during the day and these protein structures affected predicted
nutrient availability of alfalfa hay in ruminants.
DOI: 10.1002/jsfa.6141
15.
Effects of feeding algal meal high in docosahexaenoic acid on feed intake, milk
production, and methane emissions in dairy cows.
Moate PJ(1), Williams SR, Hannah MC, Eckard RJ, Auldist MJ, Ribaux BE, Jacobs JL,
Wales WJ.
Author information:
Ellinbank, Victoria 3821, Australia. peter.moate@dpi.vic.gov.au
This study examined effects on milk yield and composition, milk fatty acid
concentrations and methane (CH4) emissions when dairy cows were offered diets
containing different amounts of algal meal. The algal meal contained 20%
docosahexaenoic acid (DHA) and cows were offered either 0, 125, 250, or 375 g/cow
per d of algal meal corresponding to 0, 25, 50, or 75 g of DHA/cow per d.
Thirty-two Holstein cows in mid lactation were allocated to 4 treatment groups,
and cows in all groups were individually offered 5.9k g of dry matter (DM) per
day of concentrates [683 g/kg of cracked wheat (Triticum aestivum), 250 g/kg of
cold-pressed canola, 46 g/kg of granulated dried molasses, and 21 g/kg of mineral
mix] and ad libitum alfalfa (Medicago sativa) hay. The algal meal supplement was
added to the concentrate allowance and was fed during the morning and afternoon
milking, whereas the alfalfa hay was fed individually in pens. Cows were
gradually introduced to their diets over 7d and then fed their treatment diets
for a further 16d. Dry matter intake and milk yield were measured daily, and milk
composition was measured on a sample representative of the daily milk yield on
Thursday of each week. For the last 2d of the experiment, cows were individually
housed in respiration chambers to allow measurement of CH4 emissions. Dry matter
intake, milk yield and milk composition were also measured while cows were in the
respiration chambers. Cows ate all their offered concentrates, but measured
intake of alfalfa decreased with increasing dose of DHA by 16.2, 16.4, 15.1, and
14.3 kg of DM/d, respectively. Milk yield (22.6, 23.5, 22.6, and 22.6 kg/cow per
d) was not affected by DHA dose, but milk fat concentrations (49.7, 37.8, 37.0,
and 38.3g/kg) and, consequently, milk fat yields (1.08, 0.90, 0.83, and 0.85
kg/d) decreased with addition of DHA. The feeding of algal meal high in DHA was
associated with substantial increases in the concentrations of DHA (0.04, 0.36,
0.60, and 0.91 g/100g of milk fatty acids) and conjugated linoleic acid C18:2
cis-9,trans-11 (0.36, 1.09, 1.79, and 1.87 g/100g of milk fatty acids). Addition
of DHA did not affect total emissions of CH4 (543, 563, 553, and 520 g/cow per
d), nor emissions in terms of milk production (24.9, 22.1, 24.3, and 23.4 g of
CH4/kg of milk), but emissions were increased with respect to total intake (22.6,
23.5, 24.5, and 24.4 g of CH4/kg of DM). These findings indicate that CH4
emissions were not reduced when dairy cows were fed a forage-based diet
supplemented with DHA from algal meal.

DOI: 10.3168/jds.2012-6168
Legume-grass intercropping phytoremediation of phthalic acid esters in soil near

an electronic waste recycling site: a field study.
Ma TT(1), Teng Y, Luo YM, Christie P.
Author information:
(1)Key Laboratory of Soil Environment and Pollution Remediation of the Chinese
Academy of Sciences, Institute of Soil Science, Nanjing, China.
A field experiment was conducted to study the phytoremediation of phthalic acid

esters (PAEs) by legume (alfalfa, Medicago sativa L.)-grass (perennial ryegrass,
Lolium perenne L. and tall fescue, Festuca arundinacea) intercropping in
contaminated agricultural soil at one of the largest e-waste recycling sites in
China. Two compounds, DEHP and DnBP, were present in the soil and in the shoots
of the test plants at much higher concentrations than the other target PAEs
studied. Over 80% of 'total' (i.e., all six) PAEs were removed from the soil
across all treatments by the end of the experiment. Alfalfa in monoculture
removed over 90% of PAEs and alfalfa in the intercrop of the three plant species
contained the highest shoot concentration of total PAEs of about 4.7 mg kg(-1) DW
(dry weight). Calculation of phytoextraction efficiency indicated that the most
effective plant combinations in eliminating soil PAEs were the three-species
intercrop (1.78%) and the alfalfa monocrop (1.41%). Phytoremediation with alfalfa
was effective in both monoculture and intercropping. High bioconcentration
factors (BCFs) indicated the occurrence of significant extraction of PAEs by
plants from soil, suggesting that phytoremediation may have potential for the
removal of PAEs from contaminated soils.
DOI: 10.1080/15226514.2012.687016

4.
Detailed dimethylacetal and fatty acid composition of rumen content from lambs
fed lucerne or concentrate supplemented with soybean oil.
Alves SP(1), Santos-Silva J, Cabrita AR, Fonseca AJ, Bessa RJ.
Author information:
(1)Unidade de Produção Animal, Instituto Nacional dos Recursos Biológicos (INRB),
Vale de Santarém, Portugal. susanaalves@fmv.utl.pt
Lipid metabolism in the rumen is responsible for the complex fatty acid profile
of rumen outflow compared with the dietary fatty acid composition, contributing
to the lipid profile of ruminant products. A method for the detailed
dimethylacetal and fatty acid analysis of rumen contents was developed and
applied to rumen content collected from lambs fed lucerne or concentrate based
diets supplemented with soybean oil. The methodological approach developed
consisted on a basic/acid direct transesterification followed by thin-layer
chromatography to isolate fatty acid methyl esters from dimethylacetal, oxo-
fatty acid and fatty acid dimethylesters. The dimethylacetal composition was
quite similar to the fatty acid composition, presenting even-, odd- and
branched-chain structures. Total and individual odd- and branched-chain
dimethylacetals were mostly affected by basal diet. The presence of 18:1
dimethylacetals indicates that biohydrogenation intermediates might be
incorporated in structural microbial lipids. Moreover, medium-chain fatty acid
dimethylesters were identified for the first time in the rumen content despite
their concentration being relatively low. The fatty acids containing 18
carbon-chain lengths comprise the majority of the fatty acids present in the
rumen content, most of them being biohydrogenation intermediates of 18:2n-6 and
18:3n-3. Additionally, three oxo- fatty acids were identified in rumen samples,
and 16-O-18:0 might be produced during biohydrogenation of the 18:3n-3.
PMCID: PMC3587585
863. J Nematol. 2012 Dec;44(4):387-90.
Relationship of Resistance to Meloidogyne chitwoodi (race 2) and M. hapla in

Alfalfa.
McCord PH(1).
Author information:
(1)USDA-ARS, Vegetable and Forage Crops Research Unit, Prosser, WA 99350.
In the Pacific Northwest, alfalfa (Medicago sativa) is host to two species of

root-knot nematodes, including race 2 of the Columbia root-knot nematode
(Meloidogyne chitwoodi) and the northern root-knot nematode (Meloidogyne hapla).
In addition to the damage caused to alfalfa itself by M. hapla, alfalfa's host
status to both species leaves large numbers of nematodes available to damage
rotation crops, of which potato is the most important. A nematode-resistant
alfalfa germplasm release, W12SR2W1, was challenged with both nematode species,
to determine the correlation, if any, of resistance to nematode reproduction.
Thirty genotypes were screened in replicated tests with M. chitwoodi race 2 or M.
hapla, and the reproductive factor (RF) was calculated. The distribution of
natural log-transformed RF values was skewed for both nematode species, but more
particularly for M. chitwoodi race 2, where more than half the genotypes screened
were non-hosts. Approximately 30 percent of genotypes were non-hosts or very poor
hosts of M. hapla, but RF values for M. hapla on susceptible genotypes were
generally much higher than RF values for genotypes susceptible to M. chitwoodi
race 2. The Spearman rank correlation was positive (0.52) and significant
(p-value = 0.003), indicating there is some relationship between resistance to
these two species of root-knot nematode in alfalfa. However the relationship is
not strong enough to suggest genetic loci for resistance are identical, or
closely linked. Breeding for resistance or immunity will require screening with
each species separately, or with different DNA markers if marker-assisted
breeding is pursued. A number of genotypes were identified which are non-hosts to
both species. These plants will be intercrossed to develop a non-host germplasm.
PMCID: PMC3592361
PMID: 23483826
864. J Integr Plant Biol. 2013 Aug;55(8):721-34. doi: 10.1111/jipb.12047. Epub 2013
Jun 24.
Photosynthetic and molecular markers of CO₂-mediated photosynthetic

downregulation in nodulated alfalfa.
Sanz-Sáez A(1), Erice G, Aranjuelo I, Aroca R, Ruíz-Lozano JM, Aguirreolea J,

Irigoyen JJ, Sanchez-Diaz M.
Author information:
(1)Department of Plant Biology, University of Navarra, Pamplona E-31008, Spain.
alvaro_ssj@hotmail.com
Elevated CO₂ leads to a decrease in potential net photosynthesis in long-term

experiments and thus to a reduction in potential growth. This process is known as
photosynthetic downregulation. There is no agreement on the definition of which
parameters are the most sensitive for detecting CO₂ acclimation. In order to
investigate the most sensitive photosynthetic and molecular markers of CO₂
acclimation, the effects of elevated CO₂, and associated elevated temperature
were analyzed in alfalfa plants inoculated with different Sinorhizobium meliloti
strains. Plants (Medicago sativa L. cv. Aragón) were grown in summer or autumn in
temperature gradient greenhouses (TGG). At the end of the experiment, all plants
showed acclimation in both seasons, especially under elevated summer
temperatures. This was probably due to the lower nitrogen (N) availability caused
by decreased N₂-fixation under higher temperatures. Photosynthesis measured at
growth CO₂ concentration, rubisco in vitro activity and maximum rate of
carboxylation were the most sensitive parameters for detecting downregulation.
Severe acclimation was also related with decreases in leaf nitrogen content
associated with declines in rubisco content (large and small subunits) and
activity that resulted in a drop in photosynthesis. Despite the sensitivity of
rubisco content as a marker of acclimation, it was not coordinated with gene
expression, possibly due to a lag between gene transcription and protein
translation.
© 2013 Institute of Botany, Chinese Academy of Sciences.
DOI: 10.1111/jipb.12047
865. Ying Yong Sheng Tai Xue Bao. 2012 Dec;23(12):3309-16.
[Decomposition of different plant litters in Loess Plateau of Northwest China].
Li Y(1), Zhou JB, Dong YJ, Xia ZM, Chen ZJ.
Author information:
(1)College of Resources and Environment, Northwest A & F University, Yangling
712100, Shaanxi, China. wya313@yahoo.com.cn
Taking the litters of species Hippophae rhamnoides, Medicago sativa, Populus

simonii, Robinia pseudoacaci, Salix psammophila, and Stipa bungeana in the Loess
Plateau of Northeast China as test objects, and by using mesh bags, this paper
studied the dynamic changes of the litters mass, carbon, and nitrogen during
decomposition after buried in the field in semiarid region. The litters buried
were from one, two, or three of the plant species, and mixed thoroughly with
equal proportion of masses. During decomposition, the mass loss rate, total
carbon and nitrogen release rates, and total soluble carbon and nitrogen contents
of different litters were higher at the early than at the later decomposition
stage. After 412 d decomposition, the average mass loss rate of the litters was
in the order of mixed litters of three plant species > mixed litters of two plant
species > one plant species litter. By the end of this experiment, the average
release rates of the litter total carbon and nitrogen ranked as one plant species
litter > mixed litters of two plant species > mixed litters of three plant
species, the litter soluble organic carbon content was mixed litters of two plant
species > mixed litters of three plant species > one plant species litter, while
the litter soluble total nitrogen content was mixed litters of three plant
species > mixed litters of two plant species > one plant species litter.
Correlation analysis showed that the litter mass loss rate had definite
correlation with the litter soluble organic matter, especially soluble organic
carbon. From the viewpoint of mass loss rate, the mixture of the litters of P.
simonii, H. rhamnoide, and M. sativa was the optimum. It was suggested that in
the process of returning farmland into forestland and grassland in the gully and
valley region of Loess Plateau, it would be required to rationally increase plant
species diversity to improve soil fertility.
[Characters of soil organic matter functional groups in the fields planted with
alfalfa (Medicago sativa) for different years in hilly regions of south Ningxia,
Northwest China].
Li T(1), Zhao SW, Li XX, Ma S.

Author information:
Institute of Soil and Water Conservation, Northwest A & F University, Yangling
712100, Shaanxi, China. liting20053383@163.com
Soil samples were collected from the fields planted with alfalfa for different
years in the hilly region of southern Ningxia, and the technique of synchrotron
radiation-fourier transform infrared spectroscopy (SR-FTIR) was adopted to study
the characters of soil organic carbon (SOC) functional groups in different sizes
of soil aggregates. Planting alfalfa changed the contents of SOC functional
groups and their distribution in soil aggregates. With the increasing year of
planting alfalfa, the SOC content, percentage of macro-aggregates (> 0.25 mm),
aggregates stability, and relative content of SOC saturated alkyl all increased,
and the saturated alkyl was mainly distributed in 0.25-1 mm aggregates. However,
the relative contents of SOC aliphatic-C and alcohol-C decreased with the
increase of soil aggregate size. The increased aliphatic-C and alcohol-C were
mainly distributed in macro-aggregates, whereas the aromatic-C was mainly
distributed in < 0.25 mm aggregates. The variation range of the relative contents
of the functional groups in macro-aggregates was larger than that in < 0.25 mm
aggregates, showing that the SOC stability in micro-aggregates was higher than
that in macro-aggregates. Continuous planting of alfalfa made the increment of
SOC easily oxidizable functional groups (aliphatic-C and alcohol-C) be larger
than that of aromatic-C and the chemical stability of SOC increased, with the
best effect observed in 8-year planting. The aliphatic-C, alcohol-C, and
saturated alkyl in SOC had higher percentages, their variation was most active,
and had great contribution to the increase of SOC content.
8.
In situ protein degradation of alfalfa and birdsfoot trefoil hays and silages as
influenced by condensed tannin concentration.
Coblentz WK(1), Grabber JH.
Author information:
(1)US Department of Agriculture-Agricultural Research Service (USDA-ARS), US
Dairy Forage Research Center, Marshfield, WI 54449, USA.
wayne.coblentz@ars.usda.gov
Dairy cattle often make poor use of protein when offered diets comprising high
proportions of alfalfa (Medicago sativa L.) hay or silage because nonprotein N
formed during forage conservation and ruminal fermentation exceeds requirements
for rumen microbial protein synthesis; however, condensed tannins (CT) may reduce
proteolysis in the silo and in the rumen, thereby potentially improving the
efficiency of crude protein (CP) use in ruminant diets. Two harvests, yielding 12
hays and 12 silages made from alfalfa and birdsfoot trefoil (Lotus corniculatus
L.) that varied in concentrations of CT, were evaluated for in situ disappearance
kinetics of CP in 6 ruminally cannulated lactating Holstein dairy cows (627 ±
56.3 kg). Prior to conservation, alfalfa contained no detectable CT, whereas CT
in fresh lyophilized birdsfoot trefoil ranged from 1.16 to 2.77% of dry matter,
as determined by a modified acetone-butanol-HCl assay. Percentages of CP
remaining at each incubation time were fitted to nonlinear regression models with
or without a discrete lag time. Effective ruminal disappearance of CP
(rumen-degradable protein, RDP) was calculated by 3 procedures that included (1)
no discrete lag (RDPNL), (2) discrete lag (RDPL), and (3) discrete lag with a lag
adjustment (RDPLADJ). Regardless of the calculation method, RDP declined linearly
with increasing CT concentrations (R(2)=0.62 to 0.97). Generally, tests of
homogeneity showed that conservation type (hay or silage) or harvest (silage
only) affected intercepts, but not slopes in regressions of RDP on CT. A positive
relationship between lag time and CT suggests that the RDPLADJ approach may be
most appropriate for calculating RDP for legumes containing tannins. With this
approach, regression intercepts were mainly affected by conservation method, and
RDPLADJ averaged 77.5 and 88.7% of CP for hay and silage, respectively, when no
CT was present. Greater estimates of RDP for silages were related to extensive
proteolysis in laboratory silos resulting in conversions of protein into
nonprotein N forms, which readily washed out of Dacron bags. When RDPLADJ and CT
were expressed on a CP basis, regression slopes indicated that each unit of CT
protected 0.61 units of CP from ruminal degradation in hays and silages. Applying
this relationship to a typical mid-maturity forage legume containing 21% CP
suggests that a CT concentration of 3.8% of DM would be required to reduce RDP
from 81% to a 70% target considered optimal for improving protein utilization and
milk yields by dairy cattle.

DOI: 10.3168/jds.2012-6098
868. J Environ Manage. 2013 Apr 15;119:143-50. doi: 10.1016/j.jenvman.2013.02.004.

Epub 2013 Mar 8.
Bioaccumulation and degradation of pentachloronitrobenzene in Medicago sativa.
Li YY(1), Yang H.
Author information:
Pentachloronitrobenzene (PCNB) is a fungicide belonging to the organochlorine

family and used extensively in agriculture for crop production. Many studies have
implied that PCNB has become an environmental concern due to its widespread
contamination in eco-systems. However, whether PCNB is bioaccumulated, degraded
and phytotoxic in plants is poorly understood. In this study, several alfalfa
(Medicago sativa) cultivars were grown in soil with PCNB to investigate their
absorption and catabolism, including PCNB residues in the soil and PCNB-induced
toxic responses in plants. Alfalfa plants varied widely in their ability to
accumulate and degrade PCNB. The degradation rate of PCNB was 66.26-77.68% after
alfalfa growth in the soils for 20 d, while the rates in the control (soil
without alfalfa) were only 48.42%. Moreover, concentrations of PCNB residues in
the rhizosphere soil were significantly higher than those in the non-rhizosphere
soils. Alfalfa exposed to 10 mg kg(-1) PCNB showed inhibited growth and oxidative
damage, but the effects of PCNB on the cultivars differed significantly,
indicating that the alfalfa cultivars have different tolerance to PCNB.
Activities of invertase (INV), urease (URE), polyphenol oxidase (PPO), alkaline
phosphatase (ALP) and acid phosphatase (ACP) were assayed in the treated soils
and showed that the enzyme activities were altered after PCNB exposure. The URE,
PPO, ALP and ACP activities were increased in soil following the planting of
alfalfa. The objective of the study was to analyze the potential of different
cultivars of alfalfa to accumulate and degrade PCNB from the contaminated soil.
DOI: 10.1016/j.jenvman.2013.02.004
869. Animal. 2013 Jul;7(7):1128-36. doi: 10.1017/S1751731113000293. Epub 2013 Mar

8.
Data pre-processing to improve the mining of large feed databases.
Maroto-Molina F(1), Gómez-Cabrera A, Guerrero-Ginel JE, Garrido-Varo A, Sauvant

D, Tran G, Heuzé V, Pérez-Marín DC.
Author information:
(1)Servicio de Información sobre Alimentos, Universidad de Córdoba, Ctra.
Nacional IV km. 396, 14014, Córdoba, Spain. g02mamof@uco.es
The information stored in animal feed databases is highly variable, in terms of

both provenance and quality; therefore, data pre-processing is essential to
ensure reliable results. Yet, pre-processing at best tends to be unsystematic; at
worst, it may even be wholly ignored. This paper sought to develop a systematic
approach to the various stages involved in pre-processing to improve feed
database outputs. The database used contained analytical and nutritional data on
roughly 20 000 alfalfa samples. A range of techniques were examined for
integrating data from different sources, for detecting duplicates and,
particularly, for detecting outliers. Special attention was paid to the
comparison of univariate and multivariate solutions. Major issues relating to the
heterogeneous nature of data contained in this database were explored, the
observed outliers were characterized and ad hoc routines were designed for error
control. Finally, a heuristic diagram was designed to systematize the various
aspects involved in the detection and management of outliers and errors.
DOI: 10.1017/S1751731113000293
870. Clin Med (Lond). 2013 Feb;13(1):7-12.
Adverse effects of herbal medicines: an overview of systematic reviews.
Posadzki P(1), Watson LK, Ernst E.
Author information:
(1)Peninsula Medical School, Veysey Building, Exeter, Devon.
Paul.Posadzki@pcmd.ac.uk
Comment in
Clin Med (Lond). 2013 Aug;13(4):417-8.
This overview of systematic reviews (SRs) aims to evaluate critically the

evidence regarding the adverse effects of herbal medicines (HMs). Five electronic
databases were searched to identify all relevant SRs, with 50 SRs of 50 different
HMs meeting our inclusion criteria. Most had only minor weaknesses in methods.
Serious adverse effects were noted only for four HMs: Herbae pulvis
standardisatus, Larrea tridentate, Piper methysticum and Cassia senna. The most
severe adverse effects were liver or kidney damage, colon perforation, carcinoma,
coma and death. Moderately severe adverse effects were noted for 15 HMs:
Pelargonium sidoides, Perna canaliculus, Aloe vera, Mentha piperita, Medicago
sativa, Cimicifuga racemosa, Caulophyllum thalictroides, Serenoa repens,
Taraxacum officinale, Camellia sinensis, Commifora mukul, Hoodia gordonii, Viscum
album, Trifolium pratense and Stevia rebaudiana. Minor adverse effects were noted
for 31 HMs: Thymus vulgaris, Lavandula angustifolia Miller, Boswellia serrata,
Calendula officinalis, Harpagophytum procumbens, Panax ginseng, Vitex
agnus-castus, Crataegus spp., Cinnamomum spp., Petasites hybridus, Agave
americana, Hypericum perforatum, Echinacea spp., Silybum marianum, Capsicum spp.,
Genus phyllanthus, Ginkgo biloba, Valeriana officinalis, Hippocastanaceae,
Melissa officinalis, Trigonella foenum-graecum, Lagerstroemia speciosa, Cnicus
benedictus, Salvia hispanica, Vaccinium myrtillus, Mentha spicata, Rosmarinus
officinalis, Crocus sativus, Gymnema sylvestre, Morinda citrifolia and Curcuma
longa. Most of the HMs evaluated in SRs were associated with only moderately
severe or minor adverse effects.

Epub 2013 Mar 5.
Milk production performance of Friesian-Holstein cows fed diets containing

Medicago sativa, Centrosema pubescens, or groundnut haulms (Arachis hypogaea).
Chingala G(1), Mtimuni JP, Msiska H, Gondwe T, Chigwa FC.
Author information:
(1)Faculty of Agriculture, Bunda College of Agriculture, Lilongwe University of
Agriculture and Natural Resources, P.O. Box 219, Lilongwe, Malawi,
chingalagregory@gmail.com.
To investigate the effect of feeding dairy cows diets containing lucerne hay,
centrosema hay, and groundnut haulms (crop residue) on dry matter intake (DMI),
milk yield, and milk composition, nine multiparous Friesian-Holstein cows in
their mid-lactation stage were used in a 3 × 3 crossover design replicated three
times. Dairy cows fed lucerne hay had significantly (p < 0.001) higher DMI than
dairy cows fed centrosema hay. DMI for cows fed groundnut haulms and lucerne hay
was not significantly different. Daily milk yield for dairy cows fed diet
containing lucerne hay was significantly (p < 0.01) higher than that for dairy
cows fed diets containing groundnut haulms or centrosema hay. Milk composition
and body condition scores of the cows were not significantly affected by either
lucerne hay, groundnut haulms, or centrosema hay. Overall, the results in this
study indicated that feeding dairy cows diets containing lucerne hay increased
milk yield.
DOI: 10.1007/s11250-013-0386-3
872. Int J Mol Sci. 2013 Mar 4;14(3):5198-213. doi: 10.3390/ijms14035198.
Comparative Analysis of the Symbiotic Efficiency of Medicago truncatula and

Medicago sativa under Phosphorus Deficiency.
Sulieman S(1), Schulze J, Tran LS.
Author information:
(1)Signaling Pathway Research Unit, Plant Science Center, RIKEN Yokohama
Institute, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
tran@psc.riken.jp.
Phosphorus (P)-deficiency is a major abiotic stress that limits legume growth in

many types of soils. The relationship between Medicago and Sinorhizobium, is
known to be affected by different environmental conditions. Recent reports have
shown that, in combination with S. meliloti 2011, Medicago truncatula had a lower
symbiotic efficiency than Medicago sativa. However, little is known about how
Medicago-Sinorhizobium is affected by P-deficiency at the whole-plant level. The
objective of the present study was to compare and characterize the symbiotic
efficiency of N2 fixation of M. truncatula and M. sativa grown in sand under
P-limitation. Under this condition, M. truncatula exhibited a significantly
higher rate of N2 fixation. The specific activity of the nodules was much higher
in M. truncatula in comparison to M. sativa, partially as a result of an increase
in electron allocation to N2 versus H+. Although the main organic acid,
succinate, exhibited a strong tendency to decrease under P-deficiency, the more
efficient symbiotic ability observed in M. truncatula coincided with an apparent
increase in the content of malate in its nodules. Our results indicate that the
higher efficiency of the M. truncatula symbiotic system is related to the ability
to increase malate content under limited P-conditions.
DOI: 10.3390/ijms14035198
PMCID: PMC3634504
PMID: 23459233
873. World J Microbiol Biotechnol. 2013 Jul;29(7):1301-5. doi:

10.1007/s11274-013-1293-5. Epub 2013 Mar 1.
Modeling the impact of the indigenous microbial population on the maximum

population density of Salmonella on alfalfa.
Rijgersberg H(1), Franz E, Nierop Groot M, Tromp SO.
Author information:
(1)Wageningen University and Research Centre, Food and Biobased Research, P.O.
Box 17, 6700 AA, Wageningen, The Netherlands. hajo.rijgersberg@wur.nl
Within a microbial risk assessment framework, modeling the maximum population

density (MPD) of a pathogenic microorganism is important but often not
considered. This paper describes a model predicting the MPD of Salmonella on
alfalfa as a function of the initial contamination level, the total count of the
indigenous microbial population, the maximum pathogen growth rate and the maximum
population density of the indigenous microbial population. The model is
parameterized by experimental data describing growth of Salmonella on sprouting
alfalfa seeds at inoculum size, native microbial load and Pseudomonas fluorescens
2-79. The obtained model fits well to the experimental data, with standard errors
less than ten percent of the fitted average values. The results show that the MPD
of Salmonella is not only dictated by performance characteristics of Salmonella
but depends on the characteristics of the indigenous microbial population like
total number of cells and its growth rate. The model can improve the predictions
of microbiological growth in quantitative microbial risk assessments. Using this
model, the effects of preventive measures to reduce pathogenic load and a
concurrent effect on the background population can be better evaluated. If
competing microorganisms are more sensitive to a particular decontamination
method, a pathogenic microorganism may grow faster and reach a higher level. More
knowledge regarding the effect of the indigenous microbial population (size,
diversity, composition) of food products on pathogen dynamics is needed in order
to make adequate predictions of pathogen dynamics on various food products.
DOI: 10.1007/s11274-013-1293-5
874. Gene. 2013 May 15;520(2):139-47. doi: 10.1016/j.gene.2013.02.030. Epub 2013

Feb
27.
The isolation and identification of a light-induced protein in alfalfa sprouts

and the cloning of its specific promoter.
Su X(1), Xu WZ, Liu X, Zhuo RF, Wang CY, Zhang X, Kakutani K, You S.
Author information:
(1)The School of life Science and Biopharmaceutical, Shenyang Pharmaceutical
University, Shenyang, China.
We used 2D-PAGE to isolate a light-induced protein (AL-A) that is expressed

abundantly in light-growth alfalfa sprouts. The seven amino acids of the
N-terminal region of the protein were identified, and we searched for the protein
in GenBank using the BLAST program. The results of the homology analysis showed
that the amino acid sequence of the isolated protein is most similar to one from
a pea plastocyanin. To identify the protein, we amplified and sequenced the DNA
fragment encoding AL-A from genomic alfalfa DNA. We found that the AL-A gene was
highly homologous (90%) to the sequences from the pea plastocyanin via multiple
alignments, and the deduced protein precursor was predicted to be
chloroplast-specific via the ChloroP computer program. The protein was named
alfalfa-plastocyanin (AL-P). It was characterized as being a light-inducible
protein, and RT-PCR analysis showed that AL-P mRNA transcription only occurred in
the leaves of the alfalfa plant and the alfalfa seedlings growth in lighted
conditions. PCR was also used to amplify the DNA fragment encoding the AL-P
promoter (AL-Pp) from genomic alfalfa DNA. PlantCARE analysis of the promoter
sequence indicated that both a typical TATA box and a CAAT box were located in
the promoter sequence, and some of the cis-elements that are responsible for
light responsiveness were also identified within this promoter region. The AL-P
gene promoter fused to the β-glucuronidase (GUS) reporter gene has been examined
for expression in transgenic alfalfa seedlings. Our findings have a potential
application in plant genetic engineering; the AL-Pp may be used to drive the
expression of heterologous genes in transgenic alfalfa plants.
DOI: 10.1016/j.gene.2013.02.030
875. J AOAC Int. 2012 Nov-Dec;95(6):1656-68.
Samsung Salmonella Detection Kit. AOAC Performance Tested Method(SM) 021203.
Li J(1), Cheung WD, Opdyke J, Harvey J, Chong S, Moon CG.
Author information:
(1)Samsung Techwin Co., Ltd, Biotechnology Division, 6636 Virginia Manor Rd,
Beltsville, MD 20705, USA. jun36.li@samsung.com
Salmonella, one of the most common causes of foodborne illness, is a significant

public health concern worldwide. There is a need in the food industry for methods
that are simple, rapid, and sensitive for the detection of foodborne pathogens.
In this study, the Samsung Salmonella Detection Kit, a real-time PCR assay for
the detection of Salmonella, was evaluated according to the current AOAC
guidelines. The validation consisted of lot-to-lot consistency, stability,
robustness, and inclusivity/exclusivity studies, as well as a method comparison
of 10 different food matrixes. In the validation, the Samsung Salmonella
Detection Kit was used in conjunction with the Applied Biosystems StepOnePlus PCR
system and the Samsung Food Testing Software for the detection of Salmonella
species. The performance of the assays was compared to the U.S. Department of
Agriculture/Food Safety and Inspection Service-Microbiology Laboratory Guidebook
(USDA/FSIS-MLG) 4.05: Isolation and Identification of Salmonella from Meat,
Poultry, Pasteurized Egg, and Catfish and the and U.S. Food and Drug
Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella
reference methods. The validation was conducted using an unpaired study design
for detection of Salmonella spp. in raw ground beef, raw pork, raw ground pork,
raw chicken wings, raw salmon, alfalfa sprouts, pasteurized orange juice, peanut
butter, pasteurized whole milk, and shell eggs. The Samsung Salmonella Detection
Kit demonstrated lot-to-lot consistency among three independent lots as well as
ruggedness with minor modifications to changes in enrichment incubation time,
enrichment incubation temperature, and DNA sample volume for PCR reaction.
Stability was observed for 13 months at -20 degrees C and 3 months at 5 degrees
C. For the inclusivity/exclusivity study, the Samsung Salmonella Detection Kit
correctly identified 147 Salmonella species isolates out of 147 isolates tested
from each of three different enrichment broths (a total of 441 isolates
detected), and correctly excluded all 31 nontarget strains analyzed. For the
method comparison, statistical analysis was conducted according to the
Mantel-Haenszel Chi-square formula for unpaired test portions, and there was no
significant difference in the number of positive samples detected between the
Samsung Salmonella Detection Kit and the USDA/FSIS-MLG and FDA/BAM reference
methods for all 10 food matrixes.
876. J Econ Entomol. 2013 Feb;106(1):426-36.
Phylogenetic analysis of the alfalfa weevil complex (Coleoptera: Curculionidae)

in North America.
Böttger JA(1), Bundy CS, Oesterle N, Hanson SF.
Author information:
(1)Department of Entomology, Plant Pathology and Weed Science, New Mexico State
University, 945 College Drive, Room N141, Las Cruces, NM 88003, USA.
The Eastern, Western, and Egyptian strains of alfalfa weevil are pests introduced
to North America on three separate occasions, now they share partially
overlapping geographic ranges, covering most of the continental United States.
Behavior, susceptibility to parasites, and subtle morphological differences
separate the strains. The difficulty in differentiating among these strains
morphologically has led to the application of molecular phylogeny approaches
including restriction fragment-length polymorphism characterization and
sequencing of mitochondrial genes. While valuable for strain identification, this
approach cannot identify interstrain hybrids because mitochondrial markers are
maternally inherited. The work reported here extends previous findings by
comparing over 7 Kb of sequence from two mitochondrial and four nuclear loci to
increase the resolution of molecular phylogeny for these weevils. The related
clover leaf weevil, also an occasional pest of alfalfa, was included in the
analysis because the molecular phylogeny of this weevil has not been examined to
date. Analysis of nuclear loci indicate that the clover weevil is a distinct
species. Furthermore, while the three alfalfa weevil strains are separable based
on mitochondrial sequence data they cannot be separated using nuclearloci
suggesting that they are all recently diverged members of the same species. These
data refine the relationships among these strains and may find application in
design of better control strategies.
877. J Exp Bot. 2013 Feb;64(4):885-97. doi: 10.1093/jxb/ers367.
Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa)

under water restriction.
Aranjuelo I(1), Tcherkez G, Molero G, Gilard F, Avice JC, Nogués S.
Author information:
(1)Instituto de Agrobiotecnología (IdAB), Universidad Pública de
Navarra-CSIC-Gobierno de Navarra, Campus de Arrosadía, Mutilva Baja, Spain.
iker.aranjuelo@unavarra.es
Although the mechanisms of nodule N(2) fixation in legumes are now well
documented, some uncertainty remains on the metabolic consequences of water
deficit. In most cases, little consideration is given to other organs and,
therefore, the coordinated changes in metabolism in leaves, roots, and nodules
are not well known. Here, the effect of water restriction on exclusively
N(2)-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic,
metabolomic, and physiological analyses were carried out. It is shown that the
inhibition of nitrogenase activity caused by water restriction was accompanied by
concerted alterations in metabolic pathways in nodules, leaves, and roots. The
data suggest that nodule metabolism and metabolic exchange between plant organs
nearly reached homeostasis in asparagine synthesis and partitioning, as well as
the N demand from leaves. Typically, there was (i) a stimulation of the
anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g.
glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to
alanine and serine/glycine; and (iii) subtle changes in redox metabolites
suggesting the implication of a slight oxidative stress. Furthermore, water
restriction caused little change in both photosynthetic efficiency and
respiratory cost of N(2) fixation by nodules. In other words, the results suggest
that under water stress, nodule metabolism follows a compromise between
physiological imperatives (N demand, oxidative stress) and the lower input to
sustain catabolism.
DOI: 10.1093/jxb/ers367
PMCID: PMC3580806
878. PLoS One. 2013;8(2):e57338. doi: 10.1371/journal.pone.0057338. Epub 2013 Feb

21.
Comparative transcriptional profiling provides insights into the evolution and

development of the zygomorphic flower of Vicia sativa (Papilionoideae).
Liu Z(1), Ma L, Nan Z, Wang Y.
Author information:
Agricultural Science and Technology, Lanzhou University, Lanzhou, China.
lzp@lzu.edu.cn
BACKGROUND: Vicia sativa (the common vetch) possesses a predominant zygomorphic

flower and belongs to the subfamily Papilionoideae, which is related to
Arabidopsis thaliana in the eurosid II clade of the core eudicots. Each vetch
flower consists of 21 concentrically arranged organs: the outermost five sepals,
then five petals and ten stamens, and a single carpel in the center.
METHODOLOGY/PRINCIPAL FINDINGS: We explored the floral transcriptome to examine a
genome-scale genetic model of the zygomorphic flower of vetch. mRNA was obtained
from an equal mixture of six floral organs, leaves and roots. De novo assembly of
the vetch transcriptome using Illumina paired-end technology produced 71,553
unigenes with an average length of 511 bp. We then compared the expression
changes in the 71,553 unigenes in the eight independent organs through RNA-Seq
Quantification analysis. We predominantly analyzed gene expression patterns
specific to each floral organ and combinations of floral organs that corresponded
to the traditional ABC model domains. Comparative analyses were performed in the
floral transcriptomes of vetch and Arabidopsis, and genomes of vetch and Medicago
truncatula.
CONCLUSIONS/SIGNIFICANCE: Our comparative analysis of vetch and Arabidopsis
showed that the vetch flowers conform to a strict ABC model. We analyzed the
evolution and expression of the TCP gene family in vetch at a whole-genome level,
and several unigenes specific to three different vetch petals, which might offer
some clues toward elucidating the molecular mechanisms underlying floral
zygomorphy. Our results provide the first insights into the genome-scale
molecular regulatory network that controls the evolution and development of the
zygomorphic flower in Papilionoideae.
PMCID: PMC3578871
879. J Food Prot. 2013 Feb;76(2):314-7. doi: 10.4315/0362-028X.JFP-12-155.
Evaluation of PCR detection of Salmonella in alfalfa sprouts and spent irrigation

water collected during sprouting of naturally contaminated seed.
Maks N(1), Fu TJ.
Author information:
(1)Illinois Institute of Technology, Institute for Food Safety and Health,
Bedford Park, IL 60501, USA. maks@iit.edu
This study evaluated the efficacy of a PCR-based system (DuPont Qualicon BAX) for
detection of Salmonella in sprouts and spent irrigation water collected during
sprouting of seeds naturally contaminated with Salmonella. Alfalfa seeds were
grown in Mason jars at 20 and 30°C for 3 days. Levels of Salmonella present in
the water and sprouts were determined by most-probable-number (MPN) analysis.
Background microflora levels were also determined. Samples of spent irrigation
water and sprouts were enriched overnight individually in tetrathionate broth and
in buffered peptone water with novobiocin at 42°C and then run in the BAX system.
Samples were also enriched according to the U.S. Food and Drug Administration's
Bacteriological Analytical Manual (FDA BAM) method for Salmonella as a
comparison. Salmonella levels were lower at 20°C compared with 30°C for some
trials, and background microflora levels ranged from 10(7) to 10(8) CFU/g or ml
at 20°C and 10(8) to 10(9) CFU/g or ml at 30°C. In trials with a Salmonella level
>1.1 MPN/g or ml, both the BAX and FDA BAM methods were able to detect Salmonella
in all samples. In trials with lower levels (0.21 MPN/g or ml or lower) of
Salmonella, BAX was able to detect more positive samples than FDA BAM. For one
trial with <0.003 MPN/g or ml of Salmonella, the presence of the pathogen was not
indicated by either the BAX or the FDA BAM method. The results suggest that PCR
detected low levels of Salmonella in sprouts or spent irrigation water collected
from sprouting of naturally contaminated seeds.
DOI: 10.4315/0362-028X.JFP-12-155
[Effects of exogenous nitric oxide on the growth and nitrogen metabolism of

alfalfa seedlings under salt stress].
Zhou WH(1), Shi SL, Kou JT.
Author information:
(1)College of Pratacultural Science, Gansu Agricultural University, Lanzhou
730070, China. zhouwanhai@gsau.edu.cn
In order to explore the regulation approaches for improving the salt-tolerance of

alfalfa, the seedlings of Medicago sativa L. cv. Gannong No. 4 were taken to
study their growth and nitrogen metabolism under salt stress as affected by
NO-donor SNP, NO-scavenger c-PTIO, and sodium ferrocyanide (a SNP analogue with
NO not released). Exogenous NO could obviously alleviate the inhibition effects
of salt stress on the seedling growth and photosynthesis via increasing plant dry
matter and leaf chlorophyll content, net photosynthesis rate, transpiration rate,
and soluble protein content. Exogenous NO enhanced the activities of leaf nitrate
reductase, glutamine synthetase, and glutamate-oxoglutarate aminotransferase,
restrained the activities of protease and glutamate dehydrogenase, decreased the
free amino acid content, and improved the nitrate content and ammonium
assimilation under salt stress. Applying sodium ferrocyanide did not show any
alleviation effect on the seedling growth and nitrogen metabolism under salt
stress. As a NO-scavenger, c-PTIO inhibited the growth and nitrogen metabolism
under salt stress, but the inhibition effect could be mitigated by supplementing
SNP. It was suggested that exogenous and endogenous NO were involved in the
regulation of alfalfa nitrogen metabolism under salt stress.
881. Plant Physiol. 2013 Apr;161(4):1885-902. doi: 10.1104/pp.112.211383. Epub 2013

Feb 19.
A role for more axillary growth1 (MAX1) in evolutionary diversity in

strigolactone signaling upstream of MAX2.
Challis RJ(1), Hepworth J, Mouchel C, Waites R, Leyser O.
Author information:
(1)Department of Biology, University of York, York YO10 5DD, United Kingdom.
Strigolactones (SLs) are carotenoid-derived phytohormones with diverse roles.

They are secreted from roots as attractants for arbuscular mycorrhizal fungi and
have a wide range of endogenous functions, such as regulation of root and shoot
system architecture. To date, six genes associated with SL synthesis and
signaling have been molecularly identified using the shoot-branching mutants more
axillary growth (max) of Arabidopsis (Arabidopsis thaliana) and dwarf (d) of rice
(Oryza sativa). Here, we present a phylogenetic analysis of the MAX/D genes to
clarify the relationships of each gene with its wider family and to allow the
correlation of events in the evolution of the genes with the evolution of SL
function. Our analysis suggests that the notion of a distinct SL pathway is
inappropriate. Instead, there may be a diversity of SL-like compounds, the
response to which requires a D14/D14-like protein. This ancestral system could
have been refined toward distinct ligand-specific pathways channeled through
MAX2, the most downstream known component of SL signaling. MAX2 is tightly
conserved among land plants and is more diverged from its nearest sister clade
than any other SL-related gene, suggesting a pivotal role in the evolution of SL
signaling. By contrast, the evidence suggests much greater flexibility upstream
of MAX2. The MAX1 gene is a particularly strong candidate for contributing to
diversification of inputs upstream of MAX2. Our functional analysis of the MAX1
family demonstrates the early origin of its catalytic function and both
redundancy and functional diversification associated with its duplication in
angiosperm lineages.
DOI: 10.1104/pp.112.211383
PMCID: PMC3613463

Feb
15.
Storage characteristics, nutritive value, energy content, and in vivo

digestibility of moist, large rectangular bales of alfalfa-orchardgrass hay
treated with a propionic acid-based preservative.
Coblentz WK(1), Coffey KP(2), Young AN(2), Bertram MG(3).
Author information:
Dairy Forage Research Center, Marshfield, WI 54449. Electronic address:
wayne.coblentz@ars.usda.gov.
(3)Superintendent, University of Wisconsin Arlington Agricultural Research
Station, Arlington 53911.
Unstable weather, poor drying conditions, and unpredictable rainfall events often
place valuable hay crops at risk. Recent research with large round bales composed
of alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.) has
shown that these large-bale packages are particularly sensitive to spontaneous
heating and dry matter (DM) losses, as well as other undesirable changes with
respect to forage fiber, protein, and energy density. Various formulations of
organic acids have been marketed as preservatives, normally for use on hays that
are not desiccated adequately in the field to facilitate safe bale storage. Our
objectives for this study were to (1) evaluate the efficacy of applying a
commercial (buffered) propionic acid-based preservative at 3 rates (0, 0.6, and
1.0% of wet-bale weight) to hays baled at 3 moisture concentrations (19.6, 23.8,
and 27.4%) on the subsequent storage characteristics and poststorage nutritive
value of alfalfa-orchardgrass forages packaged in large rectangular (285-kg)
bales, and then (2) evaluate the in vivo digestibility of these hays in growing
lambs. Over a 73-d storage period, the preservative was effective at limiting
spontaneous heating in these hays, and a clear effect of application rate was
observed for the wettest (27.4%) bales. For drier hays, both acid-application
rates (1.0 and 0.6%) yielded comparable reductions in heating degree days >30°C
relative to untreated controls. Reductions in spontaneous heating could not be
associated with improved recovery of forage DM after storage. In this study, most
changes in nutritive value during storage were related to measures of spontaneous
heating in simple linear regression relationships; this suggests that the modest
advantages in nutritive value resulting from acid treatment were largely
associated with perturbations of normal heating patterns during bale storage.
Although somewhat erratic, apparent digestibilities of both DM (Y=-0.0080x +
55.6; R(2)=0.45) and organic matter (Y=-0.0085x + 55.5; R(2)=0.53) evaluated in
growing lambs were also directly related to heating degree days in simple linear
relationships. Based on these data, applying propionic acid-based preservatives
to large rectangular bales is likely to provide good insurance against
spontaneous heating during storage, as well as modest benefits with respect to
nutritive value and digestibility.

DOI: 10.3168/jds.2012-6145

Feb
15.
Effects of plant enzyme inactivation or sterilization on lipolysis and

proteolysis in alfalfa silage.
Ding WR(1), Long RJ(1), Guo XS(2).
Author information:
(1)State Key Laboratory of Grassland and Agro-Ecosystems, College of Pastoral
Agriculture Science and Technology, Lanzhou University, Lanzhou 730000, China.
(2)State Key Laboratory of Grassland and Agro-Ecosystems, School of Life
Sciences, Lanzhou University, Lanzhou 730000, China. Electronic address:
guoxsh07@lzu.edu.cn.
This experiment studied the contribution of plant enzymes and microbial activity
on lipolysis and proteolysis in ensiled alfalfa. Before ensiling, the wilted
alfalfa was treated with plant enzyme inactivation by autoclaving or with
sterilization by γ-ray irradiation. The treated alfalfa was then inoculated with
commercial lactic acid bacteria inoculants and ensiled for 40 d. Alfalfa without
treatment was ensiled as the control. The content of total fatty acid (FA) after
ensiling decreased 43% in the control silage and 28% in the γ-ray-treated silage,
but did not change in the autoclave-treated silage. Among the major FA (C16:0,
C18:2n-6, C18:3n-3), a considerable increase was observed in proportion of C16:0
in the control silage as compared with fresh alfalfa; conversely, decreases in
proportions of C18:2n-6 and C18:3n-3 occurred during ensilage. Silage treated
with γ-ray radiation at ensiling had a smaller proportion of C16:0 and greater
proportions of C18:2n-6 and C18:3n-3 than control silage. Autoclave treatment
further decreased proportions of C16:0 and most of the other FA, and increased
C18:2n-6 and C18:3n-3 proportions in comparison with γ-ray treatment. Proportions
of C16:0, C18:2n-6, C18:3n-3 and other detected FA (except for the proportion of
C15:0) did not differ between fresh forage and autoclave-treated silage.
Remarkably, smaller nonprotein nitrogen content was observed in the
autoclave-treated silage compared with the γ ray-treated silage or the control
silage. These results indicated that an extensive lipolysis occurred during
ensiling of alfalfa, and plant enzymes played a major role in lipolysis and
proteolysis.

DOI: 10.3168/jds.2012-6438

Mar
15.
Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under
free-living micro-oxic and symbiotic conditions.
Torres MJ(1), Hidalgo-García A, Bedmar EJ, Delgado MJ.
Author information:
(1)Estación Experimental del Zaidin, CSIC, Granada, Spain.
AIM: In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under
free-living and symbiotic conditions have been carried out.
METHODS AND RESULTS: Ensifer meliloti fixN1 mutant showed a defect in growth as
well as in TMPD-dependent oxidase activity when cells were incubated under
micro-oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a
fixP1 mutant identified two membrane-bound c-type cytochromes of 27 and 32 kDa,
present in microaerobically grown cells and in bacteroids, as the FixO and FixP
components of the E. meliloti cbb3 oxidase. Under symbiotic conditions, fixN1
mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown
in an N-free nutrient solution during 3 weeks. However, in plants grown for a
longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen
fixation.
CONCLUSIONS: The copy 1 of the fixNOQP operon is involved in E. meliloti
respiration and growth under micro-oxic conditions as well as in the expression
of the FixO and FixP components of the cbb3 oxidase present in free-living
microaerobic cultures and in bacteroids. This copy is important for nitrogen
fixation during the early steps of the symbiosis.
SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first time that a functional
analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this
work, the cytochromes c that constitute the cbb3 oxidase operating in free-living
micro-oxic cultures and in bacteroids of E. meliloti have been identified.
DOI: 10.1111/jam.12168
885. Biometals. 2013 Apr;26(2):321-8. doi: 10.1007/s10534-013-9615-5. Epub 2013 Feb

15.
Ferrous iron chelating property of low-molecular weight succinoglycans isolated

from Sinorhizobium meliloti.
Cho E(1), Choi JM, Kim H, Tahir MN, Choi Y, Jung S.

Author information:
(1)Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center &
Center for Biotechnology Research in UBITA, Konkuk University, 1 Hwayang-dong,
Gwangjin-gu, Seoul, 143-701, South Korea.
Iron is an essential nutrient for nitrogen-fixing legume root nodules, and the
chelation of ferrous iron plays an important role in the mobility and
availability of iron to the legume. In the present study, we investigated the
iron-binding properties of low-molecular weight succinoglycans isolated from the
nitrogen-fixing bacterium, Sinorhizobium meliloti. The low-molecular weight
succinoglycans comprising three monomers (M1-M3), four dimers (D1-D4), and six
trimers (T1-T6) of the succinoglycan repeating unit were purified by various
chromatographic techniques. Interestingly, the colorimetric ferrozine method
showed that the succinoglycans T6, M3, and D3 demonstrated a ferrous iron
chelating ability of 83, 63, and 38 % per mg, respectively. The individual
binding constants were determined as 43703, 2313, and 760 M(-1) for
succinoglycans T6, M3, and D3 using ultraviolet-visible spectroscopy. The
complexation of succinoglycan and ferrous iron can cause structural changes,
which were analyzed by circular dichroism spectroscopy. Furthermore, the complex
could provide antioxidant activity through an anti-Fenton reaction. These results
demonstrate that the low-molecular weight succinoglycans can effectively modulate
iron biochemistry as a novel ferrous iron-acquisition system of S. meliloti.
DOI: 10.1007/s10534-013-9615-5

7.
Predation determines different selective pressure on pea aphid host races in a

complex agricultural mosaic.
Balog A(1), Schmitz OJ.
Author information:
(1)School of Forestry and Environmental Studies, Yale University, New Haven,
Connecticut, USA. adalbert.balog@fulbrightmail.org
Field assessments were conducted to examine the interplay between host plant and
predation in complex agricultural mosaic on pea aphid clover and alfalfa races.
In one experiment, we examined the relative fitness on clover race (CR) and
alfalfa race (AR) pea aphids on broad bean, red clover and alfalfa alone. But
because clover is typically grown in a more complex agricultural mosaic with
alfalfa and broad bean, a second experiment was conducted to assess the fitness
consequences under predation in a more complex agricultural field setting that
also included potential apparent competition with AR pea aphids. In a third
experiment we tested for the effect of differential host race density on the
fitness of the other host race mediated by a predator effect. CR pea aphids
always had fitness losses when on broad bean (had lower fitness on broad bean
relative to red clover) and fitness benefits when on red clover (higher fitness
on red clover relative to broad bean), whether or not in apparent competition
with alfalfa race aphids on bean and alfalfa. AR suffered fitness loss on both
alfalfa and bean in apparent competition with CR on clover. Therefore we can
conclude that the predation rate between host races was highly asymmetrical. The
complexity of the agricultural mosaic thus can influence prey selection by
predators on different host plants. These may have evolutionary consequences
through context dependent fitness benefits on particular host plants.
PMCID: PMC3567016
887. J Anim Sci. 2013 Apr;91(4):1719-27. doi: 10.2527/jas.2012-5081. Epub 2013 Feb
13.
Effects of phytase supplementation in mature horses fed alfalfa hay and pelleted
concentrate diets.
Lavin TE(1), Nielsen BD, Zingsheim JN, O'Connor-Robison CI, Link JE, Hill GM,
Shelton J.
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing 48824,
USA. tara.lavin@gmail.com
An experiment was conducted to study P digestibility in mature horses because of

the growing environmental concerns regarding P runoff and previous equine
research focused mostly on young and growing animals or used ponies as a model.
Phytase supplementation of swine and poultry diets can result in greater
phytate-P digestibility, leading to a decreased need for inorganic P
supplementation and a decrease in P excreted to the environment; this, however,
has not been demonstrated in the horse. Six mature Arabian geldings were fed 6
diets consisting of pelleted concentrate and alfalfa hay. The concentrates
consisted mainly of soybean hulls, ground corn, wheat midds, broken rice, and
beet pulp, and phytase was added to the concentrates accordingly before
pelleting. There were 3 diet types: control (concentrate and hay), high P
(greater P concentrate and hay), and forage only, and each diet type included 1
phytase-supplemented diet and 1 non-phytase-supplemented diet, resulting in 6
total diets. Phytase supplementation for the forage only diet was accomplished by
feeding a nominal amount of concentrate formulated solely as a vehicle for the
phytase. Horses had unrestricted access to water throughout the experiment. Using
a Latin square design, all horses received all diets over a period of 12 wk. In
each week, the new diet was fed for 11 d of diet acclimation, which was followed
by a 3-d total collection of feces and urine for each horse. There was no effect
(P < 0.05) of phytase supplementation on P output in the urine or feces,
resulting in no differences in P apparent digestibility. Analysis of the feed and
feces for phytate revealed a 93% average disappearance rate of phytate,
indicating that horses are highly capable of degrading phytate and that phytase
supplementation was not beneficial. Thus, the results indicate that mature horses
are able to maintain a near 0 P balance, with adequate P provided in the diet
even as phytate, and increased P intakes above requirement may result in
increased potentially detrimental outputs to the environment.
DOI: 10.2527/jas.2012-5081
888. Environ Sci Technol. 2013 Apr 2;47(7):3082-90. doi: 10.1021/es303310t. Epub
2013
Mar 13.
Mercury localization and speciation in plants grown hydroponically or in a

natural environment.
Carrasco-Gil S(1), Siebner H, Leduc DL, Webb SM, Millán R, Andrews JC, Hernández
LE.
Author information:
Madrid, 28049 Madrid, Spain. carrasco.gil@eead.csic.es
Better understanding of mercury (Hg) accumulation, distribution, and speciation

in plants is required to evaluate potential risks for the environment and to
optimize phytostabilization strategies for Hg-contaminated soils. The behavior of
Hg in alfalfa (Medicago sativa) plants grown under controlled conditions in a
hydroponic system (30 μM HgCl2) was compared with that of naturally occurring
Horehound (Marrubium vulgare) plants collected from a mining soil polluted with
Hg (Almadenejos, Spain) to characterize common mechanisms of tolerance.
Synchrotron X-ray Fluorescence microprobe (μ-SXRF) showed that Hg accumulated at
the root apex of alfalfa and was distributed through the vascular system to the
leaves. Transmission electron microscopy (TEM) implied association of Hg with
cell walls, accompanied by their structural changes, in alfalfa roots. Extended
X-ray absorption fine structure (EXAFS) determined that Hg was principally bound
to biothiols and/or proteins in M. sativa roots, stems, and leaves. However, the
major fraction of Hg detected in M. vulgare plants consisted of mineral species,
possibly associated with soil components. Interestingly, the fraction of Hg bound
to biothiols/proteins (i.e., metabolically processed Hg) in leaves of both plants
(alfalfa and M. vulgare) was similar, in spite of the big difference in Hg
accumulation in roots, suggesting that some tolerance mechanisms might be shared.
DOI: 10.1021/es303310t
889. J Sci Food Agric. 2013 Aug 15;93(10):2421-8. doi: 10.1002/jsfa.6050. Epub 2013
Feb 11.
Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and
PPO substrates that can reduce post-harvest proteolysis.
Sullivan ML(1), Foster JL.
Author information:
(1)US Dairy Forage Research Center, Agricultural Research Service, US Department
of Agriculture, Madison, WI 53706, USA. michael.sullivan@ars.usda.gov
BACKGROUND: Studies of perennial peanut (Arachis glabrata Benth.) suggest its hay
and haylage have greater levels of rumen undegraded protein (RUP) than other
legume forages such as alfalfa (Medicago sativa L.). Greater RUP can result in
more efficient nitrogen utilization by ruminant animals with positive economic
and environmental effects. We sought to determine whether, like red clover
(Trifolium pretense L.), perennial peanut contains polyphenol oxidase (PPO) and
PPO substrates that might be responsible for increased RUP.
RESULTS: Perennial peanut extracts contain immunologically detectible PPO protein
and high levels of PPO activity (>100 nkatal mg(-1) protein). Addition of caffeic
acid (PPO substrate) to perennial peanut extracts depleted of endogenous
substrates reduced proteolysis by 90%. Addition of phenolics prepared from
perennial peanut leaves to extracts of either transgenic PPO-expressing or
control (non-expressing) alfalfa showed peanut phenolics could reduce proteolysis
>70% in a PPO-dependent manner. Two abundant likely PPO substrates are present in
perennial peanut leaves including caftaric acid.
CONCLUSIONS: Perennial peanut contains PPO and PPO substrates that together are
capable of inhibiting post-harvest proteolysis, suggesting a possible mechanism
for increased RUP in this forage. Research related to optimizing the PPO system
in other forage crops will likely be applicable to perennial peanut.
Published 2013. This article is a U.S. Government work and is in the public
domain in the USA.
DOI: 10.1002/jsfa.6050
890. Animal. 2013 Jul;7(7):1099-105. doi: 10.1017/S1751731113000116. Epub 2013 Feb

8.
Effects of nitrate adaptation by rumen inocula donors and substrate fiber

proportion on in vitro nitrate disappearance, methanogenesis, and rumen
fermentation acid.
Lin M(1), Schaefer DM, Zhao GQ, Meng QX.
Author information:
(1)College of Animal Science and Technology, State Key Laboratory of Animal
Nutrition, China Agricultural University, Beijing 100193, China.
A study was conducted to evaluate the main effects of dietary nitrate adaptation
by cattle and alfalfa cell wall to starch ratio in in vitro substrates on nitrate
disappearance and nitrite and volatile fatty acid (VFA) concentrations, as well
as hydrogen (H2) and methane (CH4) accumulations. Rumen fluid from steers fed
diets containing urea or nitrate was added into in vitro incubations containing
sodium nitrate as the sole nitrogen source and 20 cell wall : 80 starch or 80
cell wall : 20 starch as the carbohydrate source. The results showed that during
24 h incubation, rumen fluid inoculums from steers adapted to dietary nitrate
resulted in more rapid nitrate disappearance by 6 h of incubation (P < 0.01), no
significant effect on nitrite concentration and diminished CH4 accumulation (P <
0.05). Cell wall to starch ratio did not affect nitrate disappearance, CH4
accumulation and total VFA concentration. The higher cell wall ratio had the
lower total gas production and H2 concentration (P < 0.05). Ammonia-N (NH3-N)
concentration increased because of adaptation of donors to nitrate feeding (P <
0.05). Nitrate adaptation did not alter total VFA concentration, but increased
acetate, and decreased propionate and butyrate molar proportions (P < 0.01).
DOI: 10.1017/S1751731113000116
2013.
Defining the core proteome of the chloroplast envelope membranes.
Simm S(1), Papasotiriou DG, Ibrahim M, Leisegang MS, Müller B, Schorge T, Karas
M, Mirus O, Sommer MS, Schleiff E.
Author information:
(1)Institute of Molecular Cell Biology of Plants, Goethe University Frankfurt,
Germany.
High-throughput protein localization studies require multiple strategies. Mass

spectrometric analysis of defined cellular fractions is one of the complementary
approaches to a diverse array of cell biological methods. In recent years, the
protein content of different cellular (sub-)compartments was approached. Despite
of all the efforts made, the analysis of membrane fractions remains difficult, in
that the dissection of the proteomes of the envelope membranes of chloroplasts or
mitochondria is often not reliable because sample purity is not always warranted.
Moreover, proteomic studies are often restricted to single (model) species, and
therefore limited in respect to differential individual evolution. In this study
we analyzed the chloroplast envelope proteomes of different plant species,
namely, the individual proteomes of inner and outer envelope (OE) membrane of
Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and
Medicago sativa. The analysis of all three species yielded 341 identified
proteins in total, 247 of them being unique. 39 proteins were genuine envelope
proteins found in at least two species. Based on this and previous envelope
studies we defined the core envelope proteome of chloroplasts. Comparing the
general overlap of the available six independent studies (including ours)
revealed only a number of 27 envelope proteins. Depending on the stringency of
applied selection criteria we found 231 envelope proteins, while less stringent
criteria increases this number to 649 putative envelope proteins. Based on the
latter we provide a map of the outer and inner envelope core proteome, which
includes many yet uncharacterized proteins predicted to be involved in transport,
signaling, and response. Furthermore, a foundation for the functional
characterization of yet unidentified functions of the inner and OE for further
analyses is provided.
DOI: 10.3389/fpls.2013.00011
PMCID: PMC3565376
PMID: 23390424
892. Bioprocess Biosyst Eng. 2013 Jun;36(6):687-94. doi: 10.1007/s00449-013-0892-8.

Epub 2013 Feb 2.
Front-end recovery of protein from lignocellulosic biomass and its effects on

chemical pretreatment and enzymatic saccharification.
Kim TH(1), Yoo CG, Lamsal BP.
Author information:
(1)Department of Environmental Engineering, Kongju National University, Cheonan,
Chungnam, 330-717, Korea. thkim@kongju.ac.kr
Front-end protein recovery from biomass at different maturities, and its effects
on chemical pretreatment and enzyme hydrolysis of partially deproteinized fiber
were investigated. The protein recovery from alfalfa and switchgrass biomass
using sodium dodecyl sulfate and potassium hydroxide treatments was ~50-65 % of
initial biomass protein. When hot water was used as extraction media, the protein
recovery was 52.9 and 43.7 % of total protein in switchgrass and alfalfa,
respectively. For any treatment, relative protein recovery was higher from
switchgrass than from alfalfa. Only approximately half the total protein was
recovered from relatively mature (early fall) biomass compared with midsummer
harvested biomass. When protein was recovered partially using sodium dodecyl
sulfate or potassium hydroxide, and leftover fiber pretreated, aqueous ammonia
pretreatment removed 58.5-60.1 % of lignin and retained more cellulose in the
fiber compared with acid pretreatment (nearly no lignin removal). Protein removal
was helpful in the enzyme digestibility of fibers. Delignification of ammonia
pretreated partially deproteinized alfalfa fiber was in the range of 34.4-45 %,
while dilute sulfuric acid did not remove lignin effectively. Overall, the higher
delignification and enzyme digestibilities were observed in aqueous ammonia
pretreated partially deproteinized alfalfa fibers regardless of biomass type.
DOI: 10.1007/s00449-013-0892-8
893. Vet Pathol. 2013 May;50(3):530-3. doi: 10.1177/0300985812469634. Epub 2013 Jan
25.
Fatal Wedelia glauca intoxication in calves following natural exposure.
Giannitti F(1), Margineda CA, Cid MS, Montobbio C, Soteras CI, Caffarena RD, Diab
SS.
Author information:
(1)California Animal Health and Food Safety (CAHFS) Laboratory System, University
of California, West Health Sciences Drive, Davis, CA 95616, USA.
fgiannitti@ucdavis.edu
A group of 342 beef calves, corralled in the Patagonia region of Argentina, were
fed alfalfa hay that had been inadvertently contaminated with Wedelia glauca. A
total of 147 (43%) calves died within 4 days. Pathologic findings in 2 calves
were diffuse centrilobular hepatic necrosis and hemorrhage with edema in the
gallbladder, common bile duct, and choledochoduodenal junction. Epidermal
fragments of W. glauca were identified in rumen contents by microscopy. Intact W.
glauca plants and leaf fragments were found in the hay. Patches of defoliated W.
glauca were also identified in the alfalfa pasture from which the hay had been
baled.
DOI: 10.1177/0300985812469634

Epub
2013 Jan 25.
Development of a rapid, sensitive, and field-deployable razor ex BioDetection

system and quantitative PCR assay for detection of Phymatotrichopsis omnivora
using multiple gene targets.
Arif M(1), Fletcher J, Marek SM, Melcher U, Ochoa-Corona FM.
Author information:
(1)National Institute for Microbial Forensics & Food and Agricultural
Biosecurity, Oklahoma State University, Stillwater, Oklahoma, USA.
A validated, multigene-based method using real-time quantitative PCR (qPCR) and

the Razor Ex BioDetection system was developed for detection of Phymatotrichopsis
omnivora. This soilborne fungus causes Phymatotrichopsis root rot of cotton,
alfalfa, and other dicot crops in the southwestern United States and northern
Mexico, leading to significant crop losses and limiting the range of crops that
can be grown in soils where the fungus is established. It is on multiple lists of
regulated organisms. Because P. omnivora is difficult to isolate, accurate and
sensitive culture-independent diagnostic tools are needed to confirm infections
by this fungus. Specific PCR primers and probes were designed based on P.
omnivora nucleotide sequences of the genes encoding rRNA internal transcribed
spacers, beta-tubulin, and the second-largest subunit of RNA polymerase II
(RPB2). PCR products were cloned and sequenced to confirm their identity. All
primer sets allowed early detection of P. omnivora in infected but asymptomatic
plants. A modified rapid DNA purification method, which facilitates a quick
(∼30-min) on-site assay capability for P. omnivora detection, was developed.
Combined use of three target genes increased the assay accuracy and broadened the
range of detection. To our knowledge, this is the first report of a
multigene-based, field-deployable, rapid, and reliable identification method for
a fungal plant pathogen and should serve as a model for the development of
field-deployable assays of other phytopathogens.
DOI: 10.1128/AEM.03239-12
PMCID: PMC3623245
895. Environ Entomol. 2013 Feb;42(1):116-22. doi: 10.1603/EN12266.
Host density drives spatial variation in parasitism of the alfalfa weevil, Hypera
postica, across dryland and irrigated alfalfa cropping systems.
Rand TA(1).
Author information:
(1)USDA-ARS Northern Plains Agricultural Research Laboratory, Sidney, MT 59270,
USA. tatyana.rand@ars.usda.gov
Classical biological control against the alfalfa weevil, Hypera postica

(Gyllenhal), a destructive pest of alfalfa (Medicago sativa L.), has resulted in
the establishment of nine parasitoid species in the United States. Despite
widespread redistribution of a number of species, there remains little
postrelease data on their establishment and potential effectiveness in many
regions. I surveyed parasitoids associated with alfalfa weevil larvae across 30
or more sites in eastern Montana and western North Dakota over 2 yr. Replicate
sites were sampled in two habitat types that differ in their physical
characteristics, flood-irrigated and dryland alfalfa fields. Irrigated systems
are more productive but also more intensively disturbed habitats because of
increased harvest frequency and repeated flooding. Given evidence that both
habitat disturbance and herbivore density, which often increases with
productivity, can influence parasitoid dynamics, I predicted that parasitism
levels, the relative importance of different species, or both, would differ
across these two system types. Of four larval parasitoid species released
previously or recovered in the region, two were found in this study, Bathyplectes
curculionis (Thomson) and Oomyzus incertus (Ratzenberg), with average levels of
parasitism across habitat types and years of 37.2 and 3.5%, respectively.
Parasitism levels differed between habitat types, but the effect was driven by
concomitant differences in host densities that were higher in irrigated than
dryland fields. Parasitoid responses to host density varied across years and
species. B. curculionis exhibited positive density dependence in parasitism
across sites in 2009 and negative density dependence in 2010 when host densities
were higher regionally. In contrast, O. incertus exhibited positive density
dependence in 2010. Our results suggest that these species may be differentially
effective at different host densities. Thus, variation in host density could
represent an important axis along which parasitoids exhibit spatial
complementarity in function.
DOI: 10.1603/EN12266
896. Appl Environ Microbiol. 2013 Mar;79(6):2081-7. doi: 10.1128/AEM.02974-12. Epub

2013 Jan 18.
Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion

mutant library.
Yurgel SN(1), Mortimer MW, Rice JT, Humann JL, Kahn ML.
Author information:
(1)Institute of Biological Chemistry, Washington State University, Pullman,
Washington, USA.
Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid
libraries were used in a medium-throughput method to construct a set of 50
overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except
the replicon region. Each resulting pSymA derivative carried a defined deletion
of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration
activity, the ability of the mutants to grow on various carbon and nitrogen
sources, and the symbiotic effectiveness of the mutants with alfalfa, were
analyzed. This approach allowed us to systematically evaluate the potential
impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.
DOI: 10.1128/AEM.02974-12
PMCID: PMC3592244
897. J S Afr Vet Assoc. 2012 Sep 11;83(1):119. doi: 10.4102/jsava.v83i1.119.
Preliminary evaluation of selected minerals in liver samples from springbok

(Antidorcas marsupialis) from the National Zoological Gardens of South Africa.
Mbatha KR(1), Lane EP, Lander M, Tordiffe AS, Corr S.
Author information:
(1)Department of Research and Scientific Services, National Zoological Gardens of
South Africa, South Africa. khanyi@nzg.ac.za
Limited information is available on the mineral nutrition of captive antelope in

South Africa. Zoo animals are usually offered a very limited array of feeds,
which may result in nutritional imbalances. As a pilot study to investigate the
presence of myopathy in antelope at the National Zoological Gardens of South
Africa (NZG), stored liver samples from six springbok (Antidorcas marsupialis)
and seven other antelopes from the NZG, as well as selected food items, were
submitted for analysis of selenium, copper, manganese and zinc content by
spectrophotometry. Springbok liver levels of copper were 23.07 mg/kg ± 0.72
mg/kg, whilst manganese, selenium and zinc levels were 6.73 mg/kg ± 0.22 mg/kg,
0.14 mg/kg ± 0.05 mg/kg and 135.02 mg/kg ± 1.26 mg/kg, respectively. Liver
mineral levels in the other species were very variable. Food item copper levels
ranged from 4.00 mg/kg (Eragrostis tef) to 17.38 mg/kg (antelope cubes), lucerne
(Medicago sativa) and E. tef contained no detectable selenium. The highest zinc
levels were in antelope cubes (147.00 mg/kg) and the lowest were in lucerne
(20.80 mg/kg). Interpretation of these results was hampered by the small number
of samples and a paucity of information on liver mineral levels in free-ranging
and captive antelope; however, results suggested that, in the springbok, whilst
copper and manganese intake are likely adequate, selenium nutrition is probably
insufficient and may account for the myopathy diagnosed. Zinc liver levels are
possibly within the toxic range, perhaps as a result of the high levels of zinc
in the antelope cubes. This pilot study highlighted the need to establish
baseline mineral nutrition data for captive and freeranging antelope under South
African conditions.

898. Int J Syst Evol Microbiol. 2013 Aug;63(Pt 8):2806-12. doi:
10.1099/ijs.0.045831-0. Epub 2013 Jan 11.
Cohnella formosensis sp. nov., a xylanolytic bacterium isolated from the

rhizosphere of Medicago sativa L.
Hameed A(1), Hung MH, Lin SY, Hsu YH, Liu YC, Shahina M, Lai WA, Huang HC, Young
LS, Young CC.
Author information:
(1)Department of Soil & Environmental Sciences, College of Agriculture and
Natural Resources, National Chung Hsing University, Taichung 402, Taiwan, ROC.
A Gram-positive, spore-forming, aerobic, rod-shaped, xylanolytic bacterium

designated strain CC-Alfalfa-35(T) was isolated from the rhizosphere of Medicago
sativa L. in Taiwan. Phylogenetic analysis based on 16S rRNA gene sequence showed
that strain CC-Alfalfa-35(T) was affiliated to the genus Cohnella. Strain
CC-Alfalfa-35(T) shared 95.3 % pairwise 16S rRNA gene sequence similarity to the
type strain of the type species of the genus Cohnella (Cohnella thermotolerans
DSM 17683(T)) besides showing a similarity of 97.4-93.6 % with other recognized
species of the genus Cohnella. The DNA-DNA hybridization value between
CC-Alfalfa-35(T) and Cohnella thailandensis KCTC 22296(T) was 37.7 % ± 1.7 %
(reciprocal value, 55.7 % ± 3.0 %). Predominant cellular fatty acids were iso-C16
: 0 and anteiso-C15 : 0. The polar lipid profile constituted
diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol,
lysyl-phosphatidylglycerol, three unidentified phospholipids and three
unidentified aminophospholipids. The major respiratory quinone was MK-7 and the
DNA G+C content was 58.3 mol%. Strain CC-Alfalfa-35(T) contained
meso-diaminopimelic acid as the major diamino acid in the cell-wall
peptidoglycan. Based on the polar lipid and fatty acid profiles, which were in
line with those of C. thermotolerans DSM 17683(T), coupled with additional
distinguishing genotypic, phenotypic and chemotaxonomic features, strain
CC-Alfalfa-35(T) is proposed to represent a novel species within the genus
Cohnella, for which the name Cohnella formosensis sp. nov. is proposed. The type
strain is CC-Alfalfa-35(T) ( = JCM 18405(T) = BCRC 80428(T)).
DOI: 10.1099/ijs.0.045831-0
899. J Anim Sci. 2013 Feb;91(2):764-71. doi: 10.2527/jas.2012-5854. Epub 2013 Jan
10.
Influence of pasture intake on meat quality, lipid oxidation, and fatty acid
composition of geese.
Liu HW(1), Zhou DW.
Author information:
(1)Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences,
Jilin 130102, PR China.
This study was conducted to investigate the influence of pasture intake on meat
quality, lipid oxidation, and fatty acid composition of geese. One hundred twenty
Dongbei White male geese (a local breed; BW = 878 ± 13 g; 28 d old) were randomly
and equally divided into 2 treatments with 6 pens of 10 geese per treatment. The
2 treatments consisted of birds fed ad libitum a corn-based feed. One-half of the
birds had no access to pasture (control) while the other half had access to an
alfalfa (Medicago sativa)-based pasture (pasture). The study lasted 42 d. Body
weight and feed intake were recorded weekly. At the end of the study, geese were
slaughtered to collect meat samples. Results showed that pasture intake reduced
subcutaneous fat thickness (P < 0.05) and abdominal fat yield (P < 0.05) of geese
compared with control. Geese with access to pasture had greater cooking loss (P <
0.05) and lightness (L*) value (P < 0.05) and lower pH at 24 h postmortem (pH24;
P < 0.05) and thiobarbituric acid reacting substance values (P < 0.05) at 0 and
30 min of forced oxidation. Moreover, pasture intake increased linolenic acid
(C18:3n-3; P < 0.05) and eicosapentaenoic acid (C20:5n-3; P < 0.05) and reduced
the n-6:n-3 ratio (P < 0.05) in the breast muscle of geese compare with the
control. In conclusion, pasture intake did not enhance growth performance but
improved carcass characteristics and meat quality and changed fatty acid
composition of geese.
DOI: 10.2527/jas.2012-5854
4.
Effects of dietary forage sources on rumen microbial protein synthesis and milk
performance in early lactating dairy cows.
Zhu W(1), Fu Y, Wang B, Wang C, Ye JA, Wu YM, Liu JX.
Author information:
(1)Institute of Dairy Science, Zhejiang University, Hangzhou 310058, P.R. China.
The objective of this study was to evaluate the effects of dietary forage sources
on milk performance, rumen microbial protein synthesis, and N utilization in
early lactation dairy cows. Twelve primiparous Chinese Holstein dairy cows (45 ±
6.0 DIM) were used in a 3 × 3 Latin square design. Diets were isonitrogenous and
isocaloric, with a forage-to-concentrate ratio of 45:55 [dry matter (DM) basis]
and contained similar concentrate mixtures. Different forage sources were then
added (on a DM basis): 21% corn silage, 19% corn stover, and 5% alfalfa hay (CS);
19% corn silage, 21% Chinese wild rye hay and 5% alfalfa hay (CWR); or 19% corn
silage, 9% Chinese wild rye hay, and 17% alfalfa hay (AH). Each period lasted for
21 d, with the first 14 d for an adaptation period. Dry matter intake was not
affected by the source of dietary forage. Milk yield was higher for cows fed AH
than those fed CS, with an intermediate value for CWR. Milk protein content was
higher in the cows fed AH compared with CWR (3.02 vs. 2.92%), with CS (2.95%) at
an intermediate position. The contents of milk fat and lactose were not different
among the treatments. However, milk efficiency (milk yield/DM intake) was higher
for cows fed AH than those fed CS, with those fed CWR intermediate. Cows fed AH
had higher microbial protein yield and metabolizable protein than those fed CS or
CWR. The concentrations of urea N in the urine, blood, and milk were decreased
for cows fed AH, indicating an increased N conversion. The results indicated that
corn stover could replace Chinese wild rye grass in the diets for lactating cows
and that a high proportion of alfalfa hay in the diet is beneficial for milk
protein production by increasing microbial protein yield. This can be attributed
to the improving the supply of rumen-available energy.

DOI: 10.3168/jds.2012-5756
901. Insect Mol Biol. 2013 Apr;22(2):172-82. doi: 10.1111/imb.12012. Epub 2013 Jan
7.
Specific involvement of two amino acid residues in cis-nerolidol binding to

odorant-binding protein 5 AlinOBP5 in the alfalfa plant bug, Adelphocoris
lineolatus (Goeze).
Wang SY(1), Gu SH, Han L, Guo YY, Zhou JJ, Zhang YJ.
Author information:
Olfaction plays an important role in insects' survival and reproduction.

Odorant-binding proteins (OBPs) are considered to be one of the crucial proteins
in the insect olfactory pathway. In this study, an antenna-specific OBP of the
alfalfa plant bug, Adelphocoris lineolatus AlinOBP5, was expressed and purified
in vitro. The binding affinities of AlinOBP5 with sex pheromone analogues of the
Miridae and cotton volatiles were investigated by fluorescence competitive
binding assays. The binding sites of AlinOBP5 were predicted by three-dimensional
structure modelling and molecular docking, and site-directed mutagenesis.
AlinOBP5 could not effectively bind with sex pheromone analogues of Miridae but
showed high binding abilities with specific cotton volatiles, such as
cis-nerolidol, ethyl laurate, β-ionone, β-caryophyllene, 2,3-dimethylbenzoic acid
and (E)-farnesol. The strongest binding affinity was to cis-nerolidol, suggesting
a role of AlinOBP5 in general odorant chemoreception. Based on the relatively
strong binding affinity and the reported physiological activity of cis-nerolidol
in other insects, we chose cis-nerolidol for further homology modelling and
ligand docking. The results of molecular simulation and site-directed mutagenesis
indicated that two amino acids, Lys74 and Pro121, in the protein binding pocket
are the key amino acids involved in the binding of cis-nerolidol. The Lys74
residue may participate in specific recognition of ligands, and the Pro121
residue plays a crucial role in ligand binding and release by changing the
binding pocket environment and stabilizing the conformation of the C-terminus of
AlinOBP5.
© 2013 Royal Entomological Society.
DOI: 10.1111/imb.12012
902. J Evol Biol. 2013 Mar;26(3):545-52. doi: 10.1111/jeb.12070. Epub 2013 Jan 8.
Predation drives stable coexistence ratios between red and green pea aphid
morphs.
Balog A(1), Schmitz OJ.
Author information:
(1)School of Forestry and Environmental Studies, Yale University, New Haven, CT,
USA. adalbert.balog@ms.sapientia.ro
We conducted field surveys and experiments to evaluate the hypothesis that

predation is an important driving factor determining the degree of coexistence
between red and green morphs of the pea aphid Acyrthosiphon pisum. Theory
suggests that the different colour morphs are differentially susceptible to
natural enemies and selection by predation which in turn leads to variable
relative abundances of red and green morphs among host plants across landscapes.
Our field surveys on pea and alfalfa revealed, however, that the colour morphs
tended to coexist closely in a ratio of one red to three green aphids across
fields with different host plant monocultures. Experimentation involving
manipulation of the relative abundances of the two colour morphs on host plants
pea and alfalfa with and without predator presence revealed that red morphs had
higher or same fitness (per capita reproduction) than green morphs on both pea
and alfalfa only when in the proportion of one red/three green proportion.
Moreover, experimentation evaluating predator efficiency revealed that red morphs
are safest from predation when in a 1 : 3 ratio with green morphs. These results
suggest that in addition to predation selection effects, red morphs may
behaviourally choose to associate with green morphs in a narrow 1 : 3 ratio to
maximize their fitness. This evidence, along with existing published data on red
and green morph anti-predator behaviour indicates that a 1 : 3 red and green
morph coexistence ratio is driven by a balance between predation pressure and
behavioural assorting by red morphs across landscapes. In this way predators may
have ecological-evolutionary consequences for traits that affect the colour
morphs' proportion and tolerances to selective pressure.
© 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For
Evolutionary Biology.
DOI: 10.1111/jeb.12070
903. Mikrobiol Z. 2012 Nov-Dec;74(6):29-35.
[Efficiency of the preparation "Ekolan-M" for purification of oil polluted soil].
Nogina TM, Dumanskaia TU, Khomenko LA, Podgorskiĭ VS.
The efficiency of purification of oil contaminated loamy chernozem by the

preparation "Ekolan-M" was investigated. During 12 months a complex soil
bioremediation using the preparation and alfalfa, as the land-improving plant, at
the final stage of purification resulted in the reduction of hydrocarbon content
by 97.0%, and without the preparation - by 65.5 %. In the version of experiment
with the preparation a 100% decrease of soil phytotoxicity was achieved and a
significant stimulation of plant growth and development was observed. The process
of soil purification was accompanied by intensive development of
hydrocarbon-oxidizing microorganisms, the amount of which during the process of
oil concentration gradually decreased, approaching the level in the control
uncontaminated soil.
904. Vet Immunol Immunopathol. 2013 Feb 15;151(3-4):315-24. doi:

10.1016/j.vetimm.2012.12.004. Epub 2012 Dec 17.
Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants.
Peréz Aguirreburualde MS(1), Gómez MC, Ostachuk A, Wolman F, Albanesi G, Pecora

A, Odeon A, Ardila F, Escribano JM, Dus Santos MJ, Wigdorovitz A.
Author information:
(1)Virology Institute, CICVyA, Buenos Aires, Argentina. msperez@cnia.inta.gov.ar
Bovine viral diarrhea virus (BVDV) is considered an important cause of economic

loss within bovine herds worldwide. In Argentina, only the use of inactivated
vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is
variable due to its low immunogenicity. The use of recombinant subunit vaccines
has been proposed as an alternative to overcome this difficulty. Different
studies on protection against BVDV infection have focused the E2 protein,
supporting its putative use in subunit vaccines. Utilization of transgenic plants
expressing recombinant antigens for the formulation of experimental vaccines
represents an innovative and cost effective alternative to the classical
fermentation systems. The aim of this work was to develop transgenic alfalfa
plants (Medicago sativa, L.) expressing a truncated version of the structural
protein E2 from BVDV fused to a molecule named APCH, that target to antigen
presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa
leaves was 1 μg/g at fresh weight and its expression remained stable after
vegetative propagation. A methodology based an aqueous two phases system was
standardized for concentration and partial purification of APCH-tE2 from alfalfa.
Guinea pigs parentally immunized with leaf extracts developed high titers of
neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to
induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated
with 3 μg of APCH-tE2 produced in alfalfa transgenic plants showed complete
virological protection.
DOI: 10.1016/j.vetimm.2012.12.004
905. J Plant Physiol. 2013 Apr 15;170(6):577-82. doi: 10.1016/j.jplph.2012.11.015.

Epub 2012 Dec 31.
Allelopathy is involved in the formation of pure colonies of the fern Gleichenia

japonica.
Kato-Noguchi H(1), Saito Y, Ohno O, Suenaga K.
Author information:
University, Miki, Kagawa 761-0795, Japan. hisashi@ag.kagawa-u.ac.jp
The fern Gleichenia japonica is one of the most widely distributed fern and
occurs throughout East to South Asia. The species often dominates plant
communities by forming large monospecific colonies. However, the potential
mechanism for this domination has not yet been described. The objective of this
study was to test the hypothesis that allelochemicals are involved in the
formation of G. japonica colonies. An aqueous methanol extract of G. japonica
inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce
(Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense).
Increasing extract concentration increased the inhibition. These results suggest
that G. japonica contain allelopathic substances. The extract was then purified
by several chromatographies with monitoring the inhibitory activity and two
growth inhibitory substances causing the allelopathic effect were isolated. The
chemical structures of the two substances were determined by spectral data to be
a novel compound 3-O-β-allopyranosyl-13-O-β-fucopyranosyl-3β-hydroxymanool (1)
and 18-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-13-epitorreferol (2). These
compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa
(Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli)
at concentrations greater than 0.1-1.0mM. The concentrations required for 50%
growth inhibition of root and shoot growth of these test plants ranged from 0.72
to 3.49mM and 0.79 to 3.51mM for compounds 1 and 2, respectively. Concentration
of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and
5.7mM, respectively, indicating concentrations over those required for 50% growth
inhibition are potentially available under monocultural stands of these ferns.
Therefore, these compounds may contribute to the allelopathic effects caused by
presence of G. japonica and may thus contribute to the establishment of
monocultural stands by this fern.
DOI: 10.1016/j.jplph.2012.11.015
906. Br Poult Sci. 2012;53(5):681-8. doi: 10.1080/00071668.2012.731493.
Effects of alfalfa meal on carcase quality and fat metabolism of Muscovy ducks.
Jiang JF(1), Song XM, Huang X, Wu JL, Zhou WD, Zheng HC, Jiang YQ.
Author information:
(1)Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of
Agricultural Sciences, Hangzhou, China.
1. The effects of alfalfa meal on carcase quality and fat metabolism of Muscovy
duck were evaluated. The objective of this research was to establish whether
alfalfa meal can reduce fat content and improve carcase quality of Muscovy duck.
Animal products with a high fat content present a risk factor for many diseases.
Reducing fat content in poultry products is an important goal for the poultry
industry. 2. A total of 240 14-d-old white Muscovy ducks were selected and
randomly allocated to 1 of 4 dietary treatments containing 0, 3, 6, and 9% of
alfalfa meal for 5 weeks. Growth performances were recorded and carcase
characteristics and lipid parameters were analysed. 3. Results showed that 3, 6,
and 9% alfalfa meal in diet had no significant effects on growth performance of
Muscovy ducks from 14 to 49 d of age. Ducks given 3, 6, and 9% alfalfa meal had
significantly higher dressing percentage and lower abdominal fat percentage
compared with those given no alfalfa meal. Ducks given 9% alfalfa meal had higher
breast meat percentage compared with those given no alfalfa meal. The
concentrations of triglyceride, total cholesterol, low density lipoprotein (LDL),
very low density lipoprotein (VLDL) and free fatty acid in serum of ducks fed on
alfalfa meal decreased. Alfalfa meal in the diet decreased abdominal fat
percentage and improved carcase traits of Muscovy duck. 4. The study showed that
dietary alfalfa meal decreased abdominal fat percentage and improved carcase
traits, without an adverse effect on performance.
DOI: 10.1080/00071668.2012.731493
907. FEMS Microbiol Ecol. 2013 May;84(2):302-15. doi: 10.1111/1574-6941.12062. Epub

2013 Jan 8.
Biodiversity and composition of methanogenic populations in the rumen of cows fed

alfalfa hay or triticale straw.
Kong Y(1), Xia Y, Seviour R, Forster R, McAllister TA.

Author information:
(1)Department of Biological Science and Technology, Kunming University, Kunming,
China.
It is clear that methanogens are responsible for ruminal methane emissions, but
quantitative information about the composition of the methanogenic community in
the bovine rumen is still limited. The diversity and composition of rumen
methanogens in cows fed either alfalfa hay or triticale straw were examined using
a full-cycle rRNA approach. Quantitative fluorescence in situ hybridization
undertaken applying oligonucleotide probes designed here identified five major
methanogenic populations or groups in these animals: the Methanobrevibacter TMS
group (consisting of Methanobrevibacter thaueri, Methanobrevibacter millerae and
Methanobrevibacter smithii), Methanbrevibacter ruminantium-, Methanosphaera
stadtmanae-, Methanomicrobium mobile-, and Methanimicrococcus-related
methanogens. The TMS- and M. ruminantium-related methanogens accounted for on
average 46% and 41% of the total methanogenic cells in liquid (Liq) and solid
(Sol) phases of the rumen contents, respectively. Other prominent methanogens in
the Liq and Sol phases included members of M. stadtmanae (15% and 33%), M. mobile
(17% and 12%), and Methanimicrococcus (23% and 9%). The relative abundances of
these methanogens in the community varied among individual animals and across
diets. No clear differences in community composition could be observed with
dietary change using cloning techniques. This study extends the known
biodiversity levels of the methanogenic communities in the rumen of cows.
© 2012 Federation of European Microbiological Societies. Published by Blackwell

Publishing Ltd. All rights reserved.
DOI: 10.1111/1574-6941.12062
908. Bioinformation. 2012;8(20):974-9. doi: 10.6026/97320630008974. Epub 2012 Oct

13.
Sequence analysis and homology modeling of peroxidase from Medicago sativa.
Hooda V(1), Gundala PB, Chinthala P.
Author information:
(1)Department of Botany, Maharshi Dayanand University, Rohtak 124001, India.
Plant peroxidases are one of the most extensively studied group of enzymes which
find applications in the environment, health, pharmaceutical, chemical and
biotechnological processes. Class III secretary peroxidase from alfalfa (Medicago
sativa) has been characterized using bioinformatics approach Physiochemical
properties and topology of alfalfa peroxidase were compared with that of soybean
and horseradish peroxidase, two most popular commercially available peroxidase
preparations. Lower value of instability index as predicted by ProtParam and
presence of extra disulphide linkages as predicted by Cys_REC suggested alfalfa
peroxidase to be more stable than either of the commercial preparations. Multiple
Sequence Alignment (MSA) with other functionally similar proteins revealed the
presence of highly conserved catalytic residues. Three dimensional model of
alfalfa peroxidase was constructed based on the crystal structure of soybean
peroxidase (PDB Id: 1FHF A) by homology modelling approach. The model was checked
for stereo chemical quality by PROCHECH, VERIFY 3D, WHAT IF, ERRAT, 3D MATCH AND
ProSA servers. The best model was selected, energy minimized and used to analyze
structure function relationship with substrate hydrogen peroxide by Autodock 4.0.
The enzyme substrate complex was viewed with Swiss PDB viewer and one residue
ASP43 was found to stabilize the interaction by hydrogen bonds. The results of
the study may be a guiding point for further investigations on alfalfa
peroxidase.
DOI: 10.6026/97320630008974
PMCID: PMC3524945
PMID: 23275690
909. Mol Biol Rep. 2013 Apr;40(4):3291-8. doi: 10.1007/s11033-012-2404-3. Epub 2012
Dec 29.
Development of simple sequence repeat markers and diversity analysis in alfalfa

Wang Z(1), Yan H, Fu X, Li X, Gao H.
Author information:
Efficient and robust molecular markers are essential for molecular breeding in
plant. Compared to dominant and bi-allelic markers, multiple alleles of simple
sequence repeat (SSR) markers are particularly informative and superior in
genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The
objective of this study was to enrich SSR markers directly from alfalfa expressed
sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the
National Center for Biotechnology Information. Total 774 SSR-containing ESTs were
identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST
sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type,
followed by di-(26.1 %), tetra-(11.5 %), penta-(9.7 %), and hexanucleotide (3.9
%). One hundred EST-SSR primer pairs were successfully designed and 29 exhibited
polymorphism among 28 alfalfa accessions. The allele number per marker ranged
from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896
with an average of 0.608, indicating a high level of polymorphism of the EST-SSR
markers. Based on the 29 EST-SSR markers, assessment of genetic diversity was
conducted and found that Medicago sativa ssp. sativa was clearly different from
the other subspecies. The high transferability of those EST-SSR markers was also
found for relative species.
DOI: 10.1007/s11033-012-2404-3
910. Plants (Basel). 2013 May 31;2(2):343-53. doi: 10.3390/plants2020343.
The Hybridization Barrier between Herbaceous Medicago sativa and Woody M. arborea
Is Weakened by Selection of Seed Parents.
Bingham E(1), Armour D(2), Irwin J(3).
Author information:
(1)Agronomy Department, University of Wisconsin, Madison, WI 53706, USA.
ebingham@wisc.edu.
(2)School of Agriculture and Food Science, University of Queensland, Brisbane,
Qld 4072, Australia. d.armour@uq.edu.au.
(3)School of Agriculture and Food Science, University of Queensland, Brisbane,
Qld 4072, Australia. j.irwin@uq.edu.au.
Medicago sativa, alfalfa or lucerne, and M. arborea were considered
reproductively isolated until recently. Then, in 2003, an alfalfa genotype was
identified that produced a few seeds and progeny with hybrid traits after a large
number of pollinations by M. arborea. A derivative of this alfalfa genotype also
produced a low frequency of progeny with hybrid traits. Thus, the hybridization
barrier was weakened by selection of seed parents. Hybrids from both events
expressed traits from M. arborea and M. arborea-specific DNA bands, although more
of the M. sativa genome was retained, based on the DNA results. Thus, there was
chromatin elimination during embryogenesis, resulting in partial hybrids
(hereafter hybrids). However, more than 30 hybrids with an array of M. arborea
traits have been obtained thus far, and research continues on the nature of the
hybrids. Traits have been genetically transmitted in crosses, and selected traits
are in use for alfalfa breeding. This paper reviews the first hybrids and then
focuses on further weakening of the hybridization barrier with the discovery of a
more efficient hybridizer derived from crossing Medicago sativa subspecies,
sativa, coerulea and falcata. This genotype was found to have reproductive
abnormalities associated with its complex subspecies origin that are best
described as hybrid breakdown. In effect, this subspecies derivative is a
bridge-cross parent that consistently produces hybrids. Reproductive
abnormalities in the bridge-cross parent are reported and discussed.
DOI: 10.3390/plants2020343
PMCID: PMC4844362
PMID: 27137379
911. Physiol Plant. 2013 Sep;149(1):67-78. doi: 10.1111/ppl.12019. Epub 2013 Jan
28.
A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is

induced by myo-inositol and confers multiple tolerances to abiotic stresses.
Zhuo C(1), Wang T, Lu S, Zhao Y, Li X, Guo Z.
Author information:
Agro-bioresources, South China Agricultural University, Guangzhou, 510642, China.
Galactinol synthase (GolS, EC 2.4.1.123) catalyzes formation of galactinol and

the subsequent synthesis of raffinose family oligosaccharides. The relationship
of GolS to drought and salt tolerance has been well documented, however, little
information is available about the role of GolS gene in cold tolerance. A coding
sequence of MfGolS1 cDNA was cloned from Medicago sativa spp falcata (i.e. M.
falcata), a species that exhibits greater cold tolerance than alfalfa (M.
sativa). MfGolS1 transcript was not detected in untreated vegetative tissues
using RNA blot hybridization; however, it was greatly induced in leaves, but not
in stem and petiole, after cold treatment. Higher levels of MfGolS1 transcript
were induced and maintained in M. falcata than in M. sativa during cold
acclimation. Accordingly, more sugars including sucrose, galactinol, raffinose
and stachyose were accumulated in M. falcata than in M. sativa. The data
indicated that MfGolS1 transcript and its resultant sugar accumulation were
associated with the differential cold tolerance between M. falcata and M. sativa.
MfGolS1 transcript was weakly induced by dehydration and salt stresses, but not
responsive to abscisic acid. MfGolS1 could be induced by myo-inositol, which is
proposed to participate in cold-induced MfGolS1 expression. Overexpression of
MfGolS1 in tobacco resulted in elevated tolerance to freezing and chilling in
transgenic plants as a result of enhanced levels of galactinol, raffinose and
stachyose. Tolerance to drought and salt stresses was also increased in the
transgenic tobacco plants. It is suggested that MfGolS1 plays an important role
in plant tolerance to abiotic stresses.
DOI: 10.1111/ppl.12019
912. Environ Geochem Health. 2013 Aug;35(4):465-76. doi: 10.1007/s10653-012-9508-5.

Epub 2012 Dec 18.
Phthalate esters contamination in soil and plants on agricultural land near an

electronic waste recycling site.
Ma TT(1), Christie P, Luo YM, Teng Y.
Author information:
The accumulation of phthalic acid esters (PAEs) in soil and plants in

agricultural land near an electronic waste recycling site in east China has
become a great threat to the neighboring environmental quality and human health.
Soil and plant samples collected from land under different utilization, including
fallow plots, vegetable plots, plots with alfalfa (Medicago sativa L.) as green
manure, fallow plots under long-term flooding and fallow plots under alternating
wet and dry periods, together with plant samples from relative plots were
analyzed for six PAE compounds nominated as prior pollutants by USEPA. In the
determined samples, the concentrations of six target PAE pollutants ranged from
0.31-2.39 mg/kg in soil to 1.81-5.77 mg/kg in various plants (dry weight/DW), and
their bioconcentration factors (BCFs) ranged from 5.8 to 17.9. Health risk
assessments were conducted on target PAEs, known as typical environmental
estrogen analogs, based on their accumulation in the edible parts of vegetables.
Preliminary risk assessment to human health from soil and daily vegetable intake
indicated that DEHP may present a high-exposure risk on all ages of the
population in the area by soil ingestion or vegetable consumption. The potential
damage that the target PAE compounds may pose to human health should be taken
into account in further comprehensive risk assessments in e-waste recycling sites
areas. Moreover, alfalfa removed substantial amounts of PAEs from the soil, and
its use can be considered a good strategy for in situ remediation of PAEs.
DOI: 10.1007/s10653-012-9508-5
913. Physiol Plant. 2013 Sep;149(1):79-90. doi: 10.1111/ppl.12020. Epub 2013 Jan
18.
Commensalism in an agroecosystem: hydraulic redistribution by deep-rooted legumes

improves survival of a droughted shallow-rooted legume companion.
Pang J(1), Wang Y, Lambers H, Tibbett M, Siddique KH, Ryan MH.
Author information:
(1)School of Plant Biology, The University of Western Australia, Crawley, WA,
6009, Australia. Jiayin.Pang@csiro.au
We investigated commensalism of water use among annual shallow-rooted and

perennial deep-rooted pasture legumes by examining the effect of hydraulic lift
by Cullen pallidum (N.T.Burb.) J.W.Grimes and Medicago sativa on growth, survival
and nutrient uptake of Trifolium subterraneum L. A vertically split-root design
allowed separate control of soil water in top and bottom soil. Thirty-five days
after watering ceased in the top tube, but soil remained at field capacity in the
bottom tube, an increase in shallow soil water content by hydraulic lift was 5.6
and 5.9 g kg(-1) soil overnight for C. pallidum and M. sativa, respectively.
Trifolium subterraneum in this treatment maintained higher leaf water potentials
(with M. sativa) or exhibited a slower decline (with C. pallidum) than without
companion perennial plants; and shoot biomass of T. subterraneum was 56% (with C.
pallidum) and 67% (with M. sativa) of that when both top and bottom tubes were at
field capacity. Uptake of rubidium (a potassium analog) and phosphorus by T.
subterraneum was not facilitated by hydraulic lift. Interestingly, phosphorus
content was threefold greater, and shoot biomass 1.5-3.3-fold greater when T.
subterraneum was interplanted with C. pallidum compared with M. sativa, although
dry weight of C. pallidum was much greater than that of M. sativa. This study
showed that interplanting with deep-rooted perennial legumes has benefited the
survival of T. subterraneum.
DOI: 10.1111/ppl.12020
914. Mol Biol Rep. 2013 Feb;40(2):2035-44. doi: 10.1007/s11033-012-2262-z. Epub

2012
Dec 14.
Molecular cloning and characterization of three isoprenyl diphosphate synthase

genes from alfalfa.
Sun Y(1), Long R, Kang J, Zhang T, Zhang Z, Zhou H, Yang Q.
Author information:
Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid,

gibberellins, chlorophylls and many other products in plants. Saponins are an
important group of bioactive plant natural products. The alfalfa (Medicago sativa
L.) saponins are glycosides of different triterpene aglycones and possess many
biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS)
encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based
PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS
expressed in Escherichia coli indicated that they all had IDS activity.
Expression analysis of the three genes in different alfalfa tissues using real
time PCR displayed that they were expressed in all tissues although they had a
different expression patterns. MsFPPS and MsGPS displayed a significant increase
in transcript level in response to methyl jasmonate, but the transcript level of
MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their
overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in
tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS
showed high levels of squalene content when compared with control. However, no
differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In
addition, the overexpression of MsFPPS induced senescence response in transgenic
plant leaves. This result may indicate that MsFPPS performs a role not only in
phytosterol and triterpene biosynthesis, but also in growth regulation.
DOI: 10.1007/s11033-012-2262-z
915. Carbohydr Polym. 2013 Jan 30;92(1):817-23. doi: 10.1016/j.carbpol.2012.09.094.

Epub 2012 Oct 6.
Optimization of chitosan treatments for managing microflora in lettuce seeds

without affecting germination.
Goñi MG(1), Moreira MR, Viacava GE, Roura SI.
Author information:
(1)Concejo Nacional de Ciencia y Tecnología, Argentina. ggoni@fi.mdp.edu.ar
Many studies have focused on seed decontamination but no one has been capable of
eliminating all pathogenic bacteria. Two objectives were followed. First, to
assess the in vitro antimicrobial activity of chitosan against: (a) Escherichia
coli O157:H7, (b) native microflora of lettuce and (c) native microflora of
lettuce seeds. Second, to evaluate the efficiency of chitosan on reducing
microflora on lettuce seeds. The overall goal was to find a combination of
contact time and chitosan concentration that reduces the microflora of lettuce
seeds, without affecting germination. After treatment lettuce seeds presented no
detectable microbial counts (<10(2)CFU/50 seeds) for all populations. Moreover,
chitosan eliminated E. coli. Regardless of the reduction in the microbial load, a
90% reduction on germination makes imbibition with chitosan, uneconomical.
Subsequent treatments identified the optimal treatment as 10 min contact with a
10 g/L chitosan solution, which maintained the highest germination percentage.
DOI: 10.1016/j.carbpol.2012.09.094
916. Animal. 2012 Aug;6(8):1237-45. doi: 10.1017/S1751731112000122.
Grain source and marginal changes in forage particle size modulate digestive
processes and nutrient intake of dairy cows.
Nasrollahi SM(1), Khorvash M, Ghorbani GR, Teimouri-Yansari A, Zali A, Zebeli Q.
Author information:
84156-83111, Iran. smnasrolahi@gmail.com
This study investigated the effects of, and interactions between, dietary grain
source and marginal changes in alfalfa hay (AH) particle size (PS) on digestive
processes of dairy cows. A total of eight Holstein dairy cows (175 days in milk)
were allocated in a replicated 4 × 4 Latin square design with four 21-day
periods. The experiment was a 2 × 2 factorial arrangement with two levels of
theoretical PS of AH (fine = 15 mm or long = 30 mm) each combined with two
different sources of cereal grains (barley grain alone or barley plus corn grain
in a 50 : 50 ratio). Results showed that cows consuming diets supplemented with
corn had greater dry matter and nutrient intakes (P < 0.01), independent of
forage PS. In addition, the apparent digestibility of fiber fractions was greater
for diets supplemented with corn (P = 0.01). The feeding of barley grain-based
diets was associated with greater apparent digestibility of non-fiber
carbohydrates, and this variable was even greater when long AH was fed (P =
0.04). Moreover, the feeding of long AH resulted in longer time spent eating (P =
0.03) and higher pH (P < 0.01), as well as a tendency for higher
acetate-to-propionate ratio in the rumen fluid (P = 0.06) at 3 h post feeding. In
conclusion, the results indicated that the marginal increase of PS of AH may
prolong eating time and improve rumen fermentation, particularly in diets based
on barley grain. Partial substitution of barley grain by corn can improve feed
intake and fiber digestibility in mid-lactation dairy cows.
DOI: 10.1017/S1751731112000122
917. Huan Jing Ke Xue. 2012 Aug;33(8):2825-31.
[Response of soil microbial community to the bioremediation of soil contaminated

with PAHs].
Zhang J(1), Lin XG, Liu WW, Yin R.
Author information:
(1)State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil
Science, Chinese Academy of Sciences, Nanjing 210008, China. zhangj79@issas.ac.cn
The diversity of bacterial community in soil contaminated with polycyclic

aromatic hydrocarbons (PAHs) was investigated during the plant-microbe
remediation enhanced by biosurfactant rhamnolips (RH), using the polymerase chain
reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method. The results
showed that Shannon-Weaver diversity index was only 3.17 before bioremediation,
and increased to 3.24-3.45 after bioremediation, in particular, highest value was
found in the treatment of alfalfa (AL) inoculated with arbuscular mycorrhizal
fungi (AM) and PAHs-degrading bacteria (DB) among all the treatments. The
clustering analysis showed that the similarities of soil bacterial community of
AL, AL + RH, AL + AM and AL + AM + RH were above 90%. At the same time, the
similarity of AL + DB was much closer to those of the four treatments mentioned
above. Additionally, when the bacterial communities of AL + DB + RH, AL + DB + AM
and AL + DB + AM + RH were grouped together, the similarities of these three
treatments were also higher than 80%. By sequence alignment, it was found that
the predominant and characteristic bands in DGGE patterns were closely related
with PAHs-degrading bacteria, such as Bacillus, Pseudomonas, Acidobacteria,
Sphingmonas, Rhodopseudomonas, Firmicutes, and Methylocytaceae. Application of
rhamnolipids in plant-microbe bioremediation not only improved the
bioavailability of PAHs, but also had a simultaneous influence on the diversity
of soil bacterial community, resulting in the efficient promotion of PAHs removal
from soils.
918. J Exp Bot. 2013 Jan;64(1):317-32. doi: 10.1093/jxb/ers337. Epub 2012 Dec 3.
Natural diversity in the model legume Medicago truncatula allows identifying

distinct genetic mechanisms conferring partial resistance to Verticillium wilt.
Ben C(1), Toueni M, Montanari S, Tardin MC, Fervel M, Negahi A, Saint-Pierre L,

Mathieu G, Gras MC, Noël D, Prospéri JM, Pilet-Nayel ML, Baranger A, Huguet T,
Julier B, Rickauer M, Gentzbittel L.
Author information:
(1)Université de Toulouse; INP, UPS; Laboratoire d'Écologie Fonctionnelle et
Environnement (EcoLab); ENSAT, 18 chemin de Borderouge, 31326 Castanet Tolosan,
France.
Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other
crops. The model legume Medicago truncatula was used as a host for studying
resistance and susceptibility to Verticillium albo-atrum. In addition to
presenting well-established genetic resources, this wild plant species enables to
investigate biodiversity of the response to the pathogen and putative crosstalk
between disease and symbiosis. Symptom scoring after root inoculation and
modelling of disease curves allowed assessing susceptibility levels in
recombinant lines of three crosses between susceptible and resistant lines, in a
core collection of 32 lines, and in mutants affected in symbiosis with rhizobia.
A GFP-expressing V. albo-atrum strain was used to study colonization of
susceptible plants. Symptoms and colonization pattern in infected M. truncatula
plants were typical of Verticillium wilt. Three distinct major quantitative trait
loci were identified using a multicross, multisite design, suggesting that simple
genetic mechanisms appear to control Verticillium wilt resistance in M.
truncatula lines A17 and DZA45.5. The disease functional parameters varied
largely in lines of the core collection. This biodiversity with regard to disease
response encourages the development of association genetics and ecological
approaches. Several mutants of the resistant line, impaired in different steps of
rhizobial symbiosis, were affected in their response to V. albo-atrum, which
suggests that mechanisms involved in the establishment of symbiosis or disease
might have some common regulatory control points.
DOI: 10.1093/jxb/ers337
PMCID: PMC3528038
919. Mol Biol Rep. 2013 Mar;40(3):2083-90. doi: 10.1007/s11033-012-2266-8. Epub

2012
Dec 5.
Expression of the alfalfa FRIGIDA-like gene, MsFRI-L delays flowering time in

transgenic Arabidopsis thaliana.
Chao Y(1), Yang Q, Kang J, Zhang T, Sun Y.
Author information:
In Arabidopsis, allelic variation at the FRIGIDA (FRI) locus is a major

determinant of natural variation in flowering time. We have identified a FRIGIDA
like protein gene, MsFRI-L, with unknown function from Medicago sativa L.
Expression of MsFRI-L was increased in wild alfalfa under continuous cold
conditions and the gene was expressed in all tissues including roots, stems,
leaves and flowers, and weakest in flowers under light/dark conditions.
Expression of MsFRI-L in transgenic Arabidopsis plants resulted in late flowering
phenotypes under long day conditions or treated with vernalization. Subcellular
analysis showed that MsFRI-L was localized in the nucleus. Transcript profiling
of floral regulatory genes in MsFRI-L transgenic Arabidopsis showed enhanced
expression of the flowering repressor FLOWERING LOCUS C and decreased expression
of one key flowering time genes FLOWERING LOCUS T. These results suggest that
MsFRI-L delays flowering time by regulating flowering genes expression under long
day conditions.
DOI: 10.1007/s11033-012-2266-8
920. PLoS One. 2012;7(11):e50544. doi: 10.1371/journal.pone.0050544. Epub 2012 Nov

28.
Alfalfa carbon and nitrogen sequestration patterns and effects of temperature and
precipitation in three agro-pastoral ecotones of northern China.
Chang S(1), Liu N, Wang X, Zhang Y, Xie Y.
Author information:
(1)Institute of Grassland Science, China Agricultural University, Beijing,
Alfalfa (Medicago sativa L.) is a primary and widely cultivated forage crop in
China. As a perennial leguminous grass, continuous planted alfalfa may influence
carbon and nitrogen sequestration in soils. We evaluated the effect of alfalfa,
planted for different lengths of time, and temperature and precipitation on soil
organic carbon (SOC) and total nitrogen (TN) contents, and estimated soil SOC and
TN inventories from 0-60 cm in three agro-pastoral ecotones of northern China.
Alfalfa SOC and TN storage patterns were significantly different with increasing
soil depths between the three regions of northern China. Continuous alfalfa
grassland planted had a positive effect on accumulation of both SOC and TN in the
Northwest region, whereas SOC storage peaked 6-7 years after planting in the
Northeast and North region. Moreover, relatively higher TN storage appeared 7
years after planting in the Northeast and North regions. This study controlled as
many factors as possible, but we caution that such temporal inferences could be
artifacts of site selection. The regression analysis indicated that SOC and TN
accumulation was mainly dependent on temperature (≥10°C of effective total
accumulated temperature) in the North region. Precipitation in the growing season
was the main limiting factor for SOC storage in the Northwest region and TN
accumulation in the North regions. Therefore, the different climate factors
affecting SOC and TN sequestration in alfalfa occurred at a regional scale.
PMCID: PMC3509074
921. Theor Appl Genet. 2013 Mar;126(3):823-35. doi: 10.1007/s00122-012-2020-6. Epub

2012 Nov 28.
Characterization of two novel cold-inducible K3 dehydrin genes from alfalfa

(Medicago sativa spp. sativa L.).
Dubé MP(1), Castonguay Y, Cloutier J, Michaud J, Bertrand A.
Author information:
(1)Crops and Soils Research and Development Center, Agriculture and Agri-Food
Canada, 2560 Hochelaga Blvd., Quebec, QC, G1V 2J3, Canada.
Dehydrin defines a complex family of intrinsically disordered proteins with

potential adaptive value with regard to freeze-induced cell dehydration. Search
within an expressed sequence tags library from cDNAs of cold-acclimated crowns of
alfalfa (Medicago sativa spp. sativa L.) identified transcripts putatively
encoding K(3)-type dehydrins. Analysis of full-length coding sequences unveiled
two highly homologous sequence variants, K(3)-A and K(3)-B. An increase in the
frequency of genotypes yielding positive genomic amplification of the
K(3)-dehydrin variants in response to selection for superior tolerance to
freezing and the induction of their expression at low temperature strongly
support a link with cold adaptation. The presence of multiple allelic forms
within single genotypes and independent segregation indicate that the two K(3)
dehydrin variants are encoded by distinct genes located at unlinked loci. The
co-inheritance of the K(3)-A dehydrin with a Y(2)K(4) dehydrin restriction
fragment length polymorphism with a demonstrated impact on freezing tolerance
suggests the presence of a genome domain where these functionally related genes
are located. These results provide additional evidence that dehydrin play
important roles with regard to tolerance to subfreezing temperatures. They also
underscore the value of recurrent selection to help identify variants within a
large multigene family in allopolyploid species like alfalfa.
DOI: 10.1007/s00122-012-2020-6
22.
Effects of feeding brown midrib corn silage with a high dietary concentration of
alfalfa hay on lactational performance of Holstein dairy cows for the first 180
days of lactation.
Holt MS(1), Eun JS, Thacker CR, Young AJ, Dai X, Nestor KE Jr.
Author information:
Logan 84322, USA.
This experiment was conducted to test a hypothesis that lactating dairy cows fed
35% brown midrib (BMR) corn silage and 25% alfalfa hay (dry matter (DM) basis)
would consume more DM around peak lactation compared with those fed conventional
corn silage (CS), resulting in longer peak milk production. Twenty-eight
multiparous Holstein cows were used starting at the onset of lactation through
180 d in milk (DIM). Treatments were formulated to maintain a
forage-to-concentrate ratio of 60:40, differing only in the CS hybrids used. Two
dietary treatments were assessed in a completely randomized design: total mixed
ration based on conventional CS (CCS) and total mixed ration based on BMR silage.
Through peak lactation (1-60 DIM), DM intake was not different between dietary
treatments, whereas DM intake post-peak lactation (61-180 DIM) tended to increase
by feeding the BMR diet compared with the CCS diet (25.8 vs. 24.7 kg/d). Cows fed
the BMR diet tended to lose less body weight through peak lactation compared with
those fed the CCS diet (-0.22 vs. -0.52 kg/d). Although milk yield was not
different between dietary treatments through peak lactation, milk yield post-peak
lactation increased by feeding the BMR diet compared with the CCS diet (41.0 vs.
38.8 kg/d). Yield of 3.5% fat-corrected milk was similar between dietary
treatments throughout the experiment (41.4 kg/d, on average), but milk fat
concentration decreased by feeding the BMR diet compared with the CCS diet
post-peak lactation (3.47 vs. 3.80%). Overall milk protein concentration was
similar between dietary treatments throughout the experiment (2.96%, on average),
whereas milk protein yield tended to be higher for the BMR diet post-peak
lactation compared with the CCS diet (1.19 vs.1.13 kg/d). Feeding BMR silage with
a high dietary concentration of alfalfa hay maintained more body weight, but did
not affect milk production through peak lactation; however, cows fed the BMR diet
post-peak lactation consumed more feed and maintained longer peak milk yield,
leading to greater overall milk production and milk protein yield.
DOI: 10.3168/jds.2012-5856
923. J Bacteriol. 2013 Jan;195(2):389-98. doi: 10.1128/JB.01445-12. Epub 2012 Nov

16.
Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the

Mycobacterium tuberculosis BacA protein.
Arnold MF(1), Haag AF, Capewell S, Boshoff HI, James EK, McDonald R, Mair I,
Mitchell AM, Kerscher B, Mitchell TJ, Mergaert P, Barry CE 3rd, Scocchi M, Zanda
M, Campopiano DJ, Ferguson GP.
Author information:
(1)School of Medicine & Dentistry, Institute of Medical Sciences, University of
Aberdeen, Foresterhill, Aberdeen, United Kingdom.
The Sinorhizobium meliloti BacA ABC transporter protein plays an important role
in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The
Mycobacterium tuberculosis BacA homolog was found to be important for the
maintenance of chronic murine infections, yet its in vivo function is unknown. In
the legume plant as well as in the mammalian host, bacteria encounter host
antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was
able to partially complement the symbiotic defect of an S. meliloti
BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from
the antimicrobial activity of a synthetic legume peptide, NCR247, and a
recombinant human β-defensin 2 (HBD2). This finding was also confirmed using an
M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated
protection of the legume symbiont S. meliloti against legume defensins as well as
HBD2 is dependent on its attached ATPase domain. In addition, we show that M.
tuberculosis BacA mediates peptide uptake of the truncated bovine AMP,
Bac7(1-16). This process required a functional ATPase domain. We therefore
suggest that M. tuberculosis BacA is important for the transport of peptides
across the cytoplasmic membrane and is part of a complete ABC transporter. Hence,
BacA-mediated protection against host AMPs might be important for the maintenance
of latent infections.
DOI: 10.1128/JB.01445-12
PMCID: PMC3553841
924. J Econ Entomol. 2012 Oct;105(5):1603-11.
Early season host plants of Apolygus lucorum (Heteroptera: Miridae) in northern

China.
Lu Y(1), Jiao Z, Wu K.
Author information:
of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193,
China.
Apolygus lucorum (Meyer-Dür) (Heteroptera: Miridae) has become a severe pest of
cotton and many other crops in northern China as a result of the widespread
adoption of Bacillus thuringiensis (Berliner) cotton, with a corresponding
reduction of broad-spectrum insecticide application in cotton fields. From the
middle of April to middle June, A. lucorum feeds and develops on other host
plants before dispersing to cotton fields. Effective suppression of A. lucorum
populations before they enter cotton fields may be an excellent strategy for
reducing the occurrence and damage of their subsequent generations in cotton
fields. For that, basic information about the host plant range of A. lucorum
during the early season is needed. Between 2006 and 2010, a total of 94 plant
species from 41 families covering 39,956 square meters of land in natural
conditions were sampled using the standard white pan beat method. Sixty-six plant
species, including 45 weeds, 10 fruit trees, 5 timber trees, 4 pasture crops, and
2 arable crops were found to be hosts of A. lucorum. Among these species,
Descurainia sophia (L.) Webb ex Prantl, Humulus scandens (Loureiro) Merrill,
Zizyphus jujuba Miller, Vitis vinifera L., Viciafaba L., and Medicago sativa L.
were identified as dominant host species because of their wide distribution and
high population densities of A. lucorum. The results of this study provide useful
information about the early season host range of A. lucorum, which can be used to
develop effective strategies to control the pest before its dispersal to cotton
fields.
925. Meat Sci. 2013 Apr;93(4):906-13. doi: 10.1016/j.meatsci.2012.09.017. Epub 2012

Oct 23.
Effects of finishing period length with vitamin E supplementation and alfalfa

grazing on carcass color and the evolution of meat color and the lipid oxidation
of light lambs.
Ripoll G(1), González-Calvo L, Molino F, Calvo JH, Joy M.
Author information:
(1)Centro de Investigación y Tecnología Agroalimentaria de Aragón, Avda.
Montañana, 930, 50059 Zaragoza, Spain. gripoll@aragon.es
Indoor-kept concentrate-fed light lambs (n=54) were supplemented with 500 mg of

dl-α-tocopheryl acetate/kg concentrate for 0, 10, 20 and 30 d before slaughtering
at 22-24 kg BW. Simultaneously, 8 lambs with their dams were alfalfa-grazed and
the lambs were slaughtered at the same weight. The age at slaughter and carcass
characteristics were more affected by grazing than by supplementation with
α-tocopherol. The grazing lambs had similar α-tocopherol levels to the lambs fed
concentrate with dl-α-tocopheryl acetate for 10 days before slaughter. The length
of the feeding period affected the evolution of the color, delaying the blooming
and discoloration of the meat. Feeding lambs α-tocopherol enriched concentrate
during the last 10 days of life or grazing them on alfalfa drastically diminished
the lipid oxidation of the meat. Alfalfa grazing is a feasible alternative to
increase light lamb meat shelf life without using additives.
DOI: 10.1016/j.meatsci.2012.09.017
8.
Replacing alfalfa or red clover silage with birdsfoot trefoil silage in total
mixed rations increases production of lactating dairy cows.
Hymes-Fecht UC(1), Broderick GA, Muck RE, Grabber JH.
Author information:
Linden Drive, Madison, WI 53706.
The objective of this study was to compare milk production and nutrient
utilization in dairy cattle fed silage made from alfalfa (AL) or red clover (RC)
versus birdsfoot trefoil (BFT) selected for low, normal, and high levels of
condensed tannins. Condensed tannin contents of the 3 BFT silages were 8, 12, and
16 g/kg of DM by butanol-HCl assay. Twenty-five multiparous Holstein cows (5
fitted with ruminal cannulas) were blocked by days in milk and randomly assigned
within blocks to incomplete 5×5 Latin squares. Diets contained [dry matter (DM)
basis] about 60% AL, 50% RC, or 60% of 1 of the 3 BFT; the balance of dietary DM
was largely from high-moisture corn plus supplemental crude protein from soybean
meal. Diets were balanced to approximately 17% crude protein and fed for four
3-wk periods; 2 wk were allowed for adaptation and production data were collected
during the last week of each period. No differences existed in DM intake or milk
composition due to silage source, except that milk protein content was lowest for
RC. Yields of milk, energy-corrected milk, fat, protein, lactose, and
solids-not-fat were greater for the 3 BFT diets than for diets containing AL or
RC. Feeding BFT with the highest condensed tannin content increased yield of
milk, protein, and solids-not-fat compared with BFT containing the lowest amount
of condensed tannin. Moreover, milk-N/N-intake was higher, and milk urea nitrogen
concentration and urinary urea-N excretion were lower for diets with normal
levels of BFT than for AL or RC diets. Feeding RC resulted in the highest
apparent digestibility of DM, organic matter, neutral detergent fiber, acid
detergent fiber, and hemicellulose and lowest ruminal concentrations of ammonia
and free amino acids. Ruminal branched-chain volatile fatty acid levels were
lowest for RC diets and diets with high levels of BFT and highest for the AL
diet. Overall, diets containing BFT silage supported greater production than
diets containing silage from AL or RC. The results indicated that feeding BFT or
other legume silages containing condensed tannins can enhance performance and N
utilization in lactating dairy cows.

DOI: 10.3168/jds.2012-5724
8.
Energy partitioning in herbage-fed dairy cows offered supplementary grain during

an extended lactation.
Williams SR(1), Clarke T, Hannah MC, Marett LC, Moate PJ, Auldist MJ, Wales WJ.
Author information:
Ellinbank, Victoria 3821, Australia. richard.williams@dpi.vic.gov.au
An experiment was conducted to quantify the changes in energy partitioning
resulting from grain supplementation in herbage-fed dairy cows at 4 stages during
a 670-d lactation. The experiment used 16 lactating Holstein-Friesian cows, with
a control and a grain treatment being randomly allocated to 8 cows each. During 4
measurement periods (each of 4d in a metabolism stall and 3d in an indirect
calorimeter) beginning at approximately 110, 270, 450, and 560 d in milk (DIM),
the energy balance of each cow was measured. Cows in both groups were
individually offered freshly cut ryegrass pasture (Lolium hybridum L.) in periods
1 and 3 and ryegrass pasture silage and alfalfa (Medicago sativa L.) hay in
periods 2 and 4. In all periods, cows in the grain group were offered an
additional 4.4 to 5.0 kg of dry matter of cereal grain/cow per day. Adding grain
to the diet increased yields of fat and protein and tended to increase yields of
milk and lactose, but did not affect milk composition. Gross energy intake (GEI)
declined as lactation progressed. Adding grain to the diet decreased the
percentage of GEI in feces and urine, but the extent of these reductions did not
change as lactation progressed. Adding grain to the diet similarly reduced the
percentage of GEI lost to heat, but again the extent of the reduction remained
similar as lactation progressed. The magnitude of the increase in milk energy
resulting from grain supplementation did not change with advancing lactation, but
tissue energy retention was greater in the first 300 DIM compared with after 300
DIM. For herbage-based diets, CH(4) emissions ranged from 6.2 to 7.6% of GEI,
which corresponds to 24.0 to 25.8 g of CH(4)/kg of dry matter intake. For diets
supplemented with cereal grains, CH(4) emissions ranged from 6.3 to 7.3% of GEI,
which corresponds to 21.6 to 25.2 g of CH(4)/kg of dry matter intake. It was
concluded that, for cows producing <24 kg of milk/d and consuming herbage-based
diets supplemented with grain, the efficiency of utilizing the additional energy
in the grain, as measured by the loss of energy in heat, and its partitioning to
milk, did not change as lactation progressed from 110 to 560 DIM.

DOI: 10.3168/jds.2012-5787
928. PLoS One. 2012;7(11):e48669. doi: 10.1371/journal.pone.0048669. Epub 2012 Nov

6.
Arbuscular mycorrhizal colonization alters subcellular distribution and chemical

forms of cadmium in Medicago sativa L. and resists cadmium toxicity.
Wang Y(1), Huang J, Gao Y.
Author information:
(1)Institute of Organic Contaminant Control and Soil Remediation, College of
Resource and Environmental Sciences, Nanjing Agricultural University, Nanjing,
Jiangsu Province, P.R. China.
Some plants can tolerate and even detoxify soils contaminated with heavy metals.
This detoxification ability may depend on what chemical forms of metals are taken
up by plants and how the plants distribute the toxins in their tissues. This, in
turn, may have an important impact on phytoremediation. We investigated the
impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the
subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago
sativa L.) that were grown in Cd-added soils. The fungus significantly colonized
alfalfa roots by day 25 after planting. Colonization of alfalfa by G.
intraradices in soils contaminated with Cd ranged from 17% to 69% after 25-60
days and then decreased to 43%. The biomass of plant shoots with AM fungi showed
significant 1.7-fold increases compared to no AM fungi addition under the
treatment of 20 mg kg(-1) Cd. Concentrations of Cd in the shoots of alfalfa under
0.5, 5, and 20 mgkg(-1) Cd without AM fungal inoculation are 1.87, 2.92, and 2.38
times higher, respectively, than those of fungi-inoculated plants. Fungal
inoculation increased Cd (37.2-80.5%) in the cell walls of roots and shoots and
decreased in membranes after 80 days of incubation compared to untreated plants.
The proportion of the inactive forms of Cd in roots was higher in fungi-treated
plants than in controls. Furthermore, although fungi-treated plants had less
overall Cd in subcellular fragments in shoots, they had more inactive Cd in
shoots than did control plants. These results provide a basis for further
research on plant-microbe symbioses in soils contaminated with heavy metals,
which may potentially help us develop management regimes for phytoremediation.
PMCID: PMC3490862
929. Cytometry A. 2012 Dec;81(12):1067-75. doi: 10.1002/cyto.a.22202. Epub 2012 Nov

7.
Endoreduplication intensity as a marker of seed developmental stage in the

Fabaceae.
Rewers M(1), Sliwinska E.
Author information:
(1)Laboratory of Molecular Biology and Cytometry, Department of Plant Genetics
and Biotechnology, University of Technology and Life Sciences in Bydgoszcz,
85-789 Bydgoszcz, Poland.
Flow cytometry (FCM) can be used to study cell cycle activity in developing,
mature and germinating seeds. It provides information about a seed's
physiological state and therefore can be used by seed growers for assessing
optimal harvest times and presowing treatments. Because an augmented proportion
of 4C nuclei usually is indicative of high mitotic activity, the 4C/2C ratio is
commonly used to follow the progress of seed development and germination.
However, its usefulness for polysomatic (i.e., containing cells with different
DNA content) seeds is questioned. Changes in cell cycle/endoreduplication
activity in developing seeds of five members of the Fabaceae were studied to
determine a more suitable marker of seed developmental stages for polysomatic
species based on FCM measurements. Seeds of Phaseolus vulgaris, Medicago sativa,
Pisum sativum, Vicia sativa, and Vicia faba var. minor were collected 20, 30, 40,
50, and 60 days after flowering (DAF), embryos were isolated and the proportion
of nuclei with different DNA contents in the embryo axis and cotyledon was
established. The ratios 4C/2C and (Σ>2C)/2C were calculated. Dried seeds were
subjected to laboratory germination tests following international seed testing
association (ISTA) rules. Additionally, the absolute nuclear DNA content was
estimated in the leaves of the studied species. During seed development nuclei
with DNA contents from 2C to 128C were detected; the endopolyploidy pattern
depended on the species, seed organ and developmental stage. The cell
cycle/endoreduplication parameters correlated negatively with genome size. The
(Σ>2C)/2C ratio in the cotyledons reflected the seed developmental stage and
corresponded with seed germinability. Therefore, this ratio is recommended as a
marker in polysomatic seed research and production instead of the 4C/2C ratio,
which does not consider the occurrence of endopolyploid cells.
Copyright © 2012 International Society for Advancement of Cytometry.

DOI: 10.1002/cyto.a.22202
930. J Sci Food Agric. 2013 Aug 30;93(11):2858-63. doi: 10.1002/jsfa.5933. Epub
2012
Nov 1.
Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting
divergent cell wall composition.
Gronwald JW(1), Bucciarelli B.
Author information:
(1)USDA-ARS, Plant Science Research Unit, St Paul, MN, 55108, USA; Department of
Agronomy and Plant Genetics, University of Minnesota, St Paul, MN, 55108, USA.
BACKGROUND: In previous research, two alfalfa clonal lines (252 and 1283) were
identified that exhibited environmentally stable differences in stem cell walls.
Compared with stems of 1283, stems of 252 have a higher cell wall concentration
and greater amounts of lignin and cellulose but reduced levels of pectic sugar
residues. These results suggest greater deposition of secondary xylem and a
reduction in pith in stems of 252 compared with 1283.
RESULTS: The stem morphology and anatomy of first-cut and second-cut harvests of
field-grown 1283 and 252 were examined. For both harvests, stems of 1283 were
thicker and had a higher leaf/stem ratio compared with stems of 252. Stem
cross-sections of both genotypes were stained for lignin, and the proportions of
stem area that were pith and secondary xylem were measured using ImageJ. Stems of
252 exhibited greater deposition of secondary xylem and a reduction in pith
proportion compared with stems of 1283 for the first-cut harvest, but this
difference was not statistically significant for the second-cut harvest.
CONCLUSION: The results indicate that the proportions of secondary xylem and pith
are not environmentally stable in these two genotypes and hence cannot be the
sole basis for the differences in cell wall concentration/composition.
DOI: 10.1002/jsfa.5933
931. BMC Genomics. 2012 Oct 29;13:568. doi: 10.1186/1471-2164-13-568.
Prevalence of single nucleotide polymorphism among 27 diverse alfalfa genotypes

as assessed by transcriptome sequencing.
Li X(1), Acharya A, Farmer AD, Crow JA, Bharti AK, Kramer RS, Wei Y, Han Y, Gou
J, May GD, Monteros MJ, Brummer EC.
Author information:
BACKGROUND: Alfalfa, a perennial, outcrossing species, is a widely planted forage

legume producing highly nutritious biomass. Currently, improvement of cultivated
alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding
strategies can enhance alfalfa improvement efforts, particularly if many
genome-wide markers are available. Transcriptome sequencing enables efficient
high-throughput discovery of single nucleotide polymorphism (SNP) markers for a
complex polyploid species.
RESULT: The transcriptomes of 27 alfalfa genotypes, including elite breeding
genotypes, parents of mapping populations, and unimproved wild genotypes, were
sequenced using an Illumina Genome Analyzer IIx. De novo assembly of
quality-filtered 72-bp reads generated 25,183 contigs with a total length of 26.8
Mbp and an average length of 1,065 bp, with an average read depth of 55.9-fold
for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented
14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a
broad diversity of genes was represented in the resulting sequences. The
realignment of individual reads to the contigs enabled the detection of 872,384
SNPs and 31,760 InDels. High resolution melting (HRM) analysis was used to
validate 91% of 192 putative SNPs identified by sequencing. Both allelic variants
at about 95% of SNP sites identified among five wild, unimproved genotypes are
still present in cultivated alfalfa, and all four US breeding programs also
contain a high proportion of these SNPs. Thus, little evidence exists among this
dataset for loss of significant DNA sequence diversity from either domestication
or breeding of alfalfa. Structure analysis indicated that individuals from the
subspecies falcata, the diploid subspecies caerulea, and the tetraploid
subspecies sativa (cultivated tetraploid alfalfa) were clearly separated.
CONCLUSION: We used transcriptome sequencing to discover large numbers of SNPs
segregating in elite breeding populations of alfalfa. Little loss of SNP
diversity was evident between unimproved and elite alfalfa germplasm. The EST and
SNP markers generated from this study are publicly available at the Legume
Information System ( http://medsa.comparative-legumes.org/) and can contribute to
future alfalfa research and breeding applications.
DOI: 10.1186/1471-2164-13-568
PMCID: PMC3533575
932. Chemosphere. 2013 Jan;90(3):1267-73. doi: 10.1016/j.chemosphere.2012.09.081.

Epub
2012 Oct 26.
Metal toxicity and biodiversity in serpentine soils: application of bioassay

tests and microarthropod index.
Visioli G(1), Menta C, Gardi C, Conti FD.
Author information:
(1)Department of Environmental Sciences, Division of Genetics and Environmental
Biotechnologies, University of Parma, via G.P. Usberti 33/a, 43124 Parma, Italy.
giovanna.visioli@unipr.it
Eco-toxicological or bioassay tests have been intensively discussed as tools for

the evaluation of soil quality. Tests using soil organisms, including
microarthropods and plants, allow direct estimates to be made of important soil
characteristics and functions. In this study we compared the results obtained by
two in vitro standard bioassays following ISO or OECD guidelines: (i) the short
term-chronic phytotoxicity germination and root elongation test using three
different plant species Cucumis sativus L. (Cucurbitaceae), Lepidium sativum L.
(Brassicaceae), and Medicago sativa L. (Fabaceae) and (ii) the inhibition of
reproduction of Folsomia candida (Collembola) by soil pollutants to investigate
the toxicity of a serpentine soil present in the Italian Apennines, rich in heavy
metals such as Ni, Cr, and Co. In addition, microarthropod communities were
characterised to evaluate the effects of metal contents on the soil fauna in
natural conditions. Abundances, Acari/Collembola ratio, biodiversity indices and
the QBS-ar index were calculated. Our results demonstrate that the two in vitro
tests distinguish differences correlated with metal and organic matter contents
in four sub-sites within the serpentinite. Soil fauna characterisation, not
previously performed on serpentine soils, revealed differences in the most
vulnerable and adapted groups of microarthropods to soil among the four
sub-sites: the microarthropod community was found to be rich in term of
biodiversity in the sub-site characterised by a lower metal content and a higher
organic matter content and vegetation.
16.
Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets

varying in type of forage as monitored by real-time PCR and automated ribosomal
intergenic spacer analysis.
Saro C(1), Ranilla MJ, Carro MD.
Author information:
(1)Departamento de Producción Animal, Universidad de León, 24071 León, Spain.
Four ruminally cannulated sheep were used in a crossover design to assess the
postprandial changes of fiber-degrading microbes in the solid phase of the rumen
of sheep fed 2 high-forage diets. The diets had forage:concentrate ratio of 70:30
(DM basis) and either alfalfa (Medicago sativa) hay (AL) or grass hay (GR) as
forage (FOR). Sheep were fed twice daily, and samples from solid rumen digesta
were taken at 0, 4, and 8 h after the morning feeding. Postprandial changes of
DNA concentrations of all determined microbial populations were similar for the 2
diets. Samples taken at 4 h after feeding had lesser (P < 0.05) concentrations of
total bacterial DNA determined with real-time PCR and bacterial diversity and
greater (P < 0.05) protozoal DNA concentrations, relative abundance of fungal,
Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus DNA
compared with those taken at 0 and 8 h. No effect (P = 0.41 to 0.76) of FOR was
detected either on concentrations of bacterial and protozoal DNA or the relative
abundance of the 2 Ruminococcus DNA, but GR diet promoted greater (P < 0.001)
relative abundance of F. succinogenes and fungal DNA compared with AL diet.
Fibrobacter succinogenes was the most abundant (P < 0.05) of the 3 cellulolytic
bacteria for both diets, with no differences (P < 0.05) between the 2
Ruminococcus species. Rumen pH and carboxymethylcellulase, Avicelase, and amylase
activities were not affected (P = 0.15 to 0.69) by FOR, but xylanase activity was
greater (P = 0.01) for GR diet. The influence of FOR on microbial communities in
ruminal solid digesta was more evident in the first hours after feeding than at
later times after feeding, which highlights the influence of sampling time when
investigating dietary effects on rumen function and microbial populations.
DOI: 10.2527/jas.2012-5265
934. J Environ Qual. 2012 Sep-Oct;41(5):1525-30. doi: 10.2134/jeq2012.0062.
Evaluation of chloropyromorphite stability in the rhizosphere of and in a sand

culture.
Abbaspour A(1), Arocena JM.

Author information:
(1)Department of Soil Science, Shahrood University of Technology, Shahrood, Iran.
abbaspour2008@gmail.com
Chloropyromorphite (Pb(PO)Cl), CP, is the most stable lead (Pb) mineral under
normal environmental conditions and precipitates in Pb-contaminated soils by
addition of phosphorous (P). A sand culture experiment was conducted to evaluate
the efficiency of and roots to dissolve CP in the presence and in the absence of
P source. The results showed that the rhizosphere of the plants had lower soluble
P and Pb compared with the bulk, which can be attributed to a higher pH in the
rhizosphere. Mineralogical transformations of CP in the root surface of the
plants including lanarkite (PbSO.PbO) has been confirmed by X-ray diffraction and
scanning electron microscopy techniques. Decrease in soluble P in the rhizosphere
as a consequence of P uptake by the plant roots may be a reason for CP
dissolution. This study indicates that the dissolution of CP can be promoted by
rhizosphere processes.
Copyright © by the American Society of Agronomy, Crop Science Society of America,

and Soil Science Society of America, Inc.
DOI: 10.2134/jeq2012.0062
935. Chem Cent J. 2012 Oct 26;6(1):123. doi: 10.1186/1752-153X-6-123.
Design of optimal solvent for extraction of bio-active ingredients from six

varieties of Medicago sativa.
Caunii A(1), Pribac G, Grozea I, Gaitin D, Samfira I.
Author information:
(1)Plant Protection Department, Grassland Department, Banat's University of
Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului no,
119, Timisoara, 300645, Romania. samfiraionel@yahoo.ro.
BACKGROUND: Extensive research has been performed worldwide and important

evidences were collected to show the immense potential of plants used in various
traditional therapeutic systems. The aim of this work is to investigate the
different extracting solvents in terms of the influence of their polarity on the
extracting ability of bioactive molecules (phenolic compounds) from the M. sativa
flowers.
RESULTS: The total phenolic content of samples was determined using the Folin
Ciocalteu (FC) procedure and their antioxidant activity was assayed through in
vitro radical decomposing activity using the radical DPPH° assay (IUPAC name for
DPPH is (phenyl)-(2,4,6-trinitrophenyl) iminoazanium). The results showed that
water was better than methanol and acetic acid for extracting bioactive
compounds, in particular for total phenolic compounds from the flowers of
alfalfa. The average content of bioactive molecules in methanol extract was
263.5±1.02 mg GAE/100g of dry weight lyophilized extract. The total phenolic
content of the tested plant extracts was highly correlated with the radical
decomposing activity. However, all extracts were free-radical inhibitors, but the
water extract was more potent than the acetic and the methanol ones. The order of
inhibitor effectiveness (expressed by IC50) proved to be: water extract
(0.924mg/mL) > acetic acid extract (0.154mg/mL) > methanol (0.079mg/mL). The
profiles of each extract (fingerprint) were characterized by FT-MIR spectroscopy.
CONCLUSIONS: The present study compares the fingerprint of different extracts of
the M. sativa flowers, collected from the wild flora of Romania. The total
phenolic content of the tested plant extracts was highly correlated with the
radical decomposing activity. The dependence of the extract composition on the
solvent polarity (acetic acid vs. methanol vs. water) was revealed by UV-VIS
spectrometry and Infrared fingerprint.
DOI: 10.1186/1752-153X-6-123
PMCID: PMC3495705
PMID: 23098128
936. Mol Biol Rep. 2013 Feb;40(2):1227-39. doi: 10.1007/s11033-012-2165-z. Epub

2012
Oct 25.
Isolation and characterization of a gene from Medicago sativa L., encoding a bZIP
transcription factor.
Li Y(1), Sun Y, Yang Q, Fang F, Kang J, Zhang T.
Author information:
(1)Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2
Yuanmingyuan West Road, Haidian, Beijing, 100193, People's Republic of China.
caasliyan@yahoo.com.cn
A full-length cDNA of 1,537 nucleotides was cloned from Medicago sativa L. cv.
"Zhongmu No. 1" by rapid amplification of cDNA ends. It was designated as MsZIP,
encoding a protein of 340 amino acids. The protein molecular weight was
36.43 kDa, and the theoretical isoelectric point was 5.72. The MsZIP
preferentially localized in nucleus and have signal peptide. Blast analysis
revealed that MsZIP shared the highest homology with some bZIP proteins of M.
truncatula. The transcript of MsZIP was strongly enriched in leaf compared with
root and stem of mature alfalfa plants. MsZIP was strongly induced by 15 %
PEG6000 (polyethylene glycol), 50 μM abscisic acid, 200 mM NaCl, 70 μM
gibberellic acid, 5 mM salicylic acid and 200 μM methyl jasmonate. Physiological
resistance parameters were measured in the transgenic tobacco. Malondialdehyde
content, relative water content, soluble sugar content, soluble protein content
and proline content in transgenic tobacco increased compared with non-transgenic
tobacco under salt stress or drought stress. The results showed that accumulation
of the MsZIP protein in the vegetative tissues of transgenic plants enhanced
their tolerance to osmotic pressure stress. These results demonstrate a role for
the MsZIP protein in stress protection and suggest the potential of the MsZIP
gene for genetic engineering of salt tolerance and drought tolerance.
DOI: 10.1007/s11033-012-2165-z
937. Plant Cell Environ. 2013 May;36(5):956-69. doi: 10.1111/pce.12029. Epub 2012
Nov
19.
Hydrogen gas acts as a novel bioactive molecule in enhancing plant tolerance to

paraquat-induced oxidative stress via the modulation of heme oxygenase-1
signalling system.
Jin Q(1), Zhu K, Cui W, Xie Y, Han B, Shen W.
Author information:
(1)College of Life Sciences, Nanjing Agricultural University, Nanjing, China.
Hydrogen gas (H2) was recently proposed as a novel antioxidant and signalling
molecule in animals. However, the physiological roles of H2 in plants are less
clear. Here, we showed that exposure of alfalfa seedlings to paraquat stress
increased endogenous H2 production. When supplied with exogenous H2 or the heme
oxygenase-1 (HO-1)-inducer hemin, alfalfa plants displayed enhanced tolerance to
oxidative stress induced by paraquat. This was evidenced by alleviation of the
inhibition of root growth, reduced lipid peroxidation and the decreased hydrogen
peroxide and superoxide anion radical levels. The activities and transcripts of
representative antioxidant enzymes were induced after exposure to either H2 or
hemin. Further results showed that H2 pretreatment could dramatically increase
levels of the MsHO-1 transcript, levels of the protein it encodes and HO-1
activity. The previously mentioned H2-mediated responses were specific for HO-1,
given that the potent HO-1-inhibitor counteracted the effects of H2. The effects
of H2 were reversed after the addition of an aqueous solution of 50% carbon
monoxide (CO). We also discovered enhanced tolerance of multiple environmental
stresses after plants were pretreated with H2 . Together, these results suggested
that H2 might function as an important gaseous molecule that alleviates oxidative
stress via HO-1 signalling.
© 2012 Blackwell Publishing Ltd.
DOI: 10.1111/pce.12029
938. J Hazard Mater. 2012 Nov 30;241-242:379-86. doi:

10.1016/j.jhazmat.2012.09.056.
Epub 2012 Oct 1.
Comparative toxicity assessment of CeO2 and ZnO nanoparticles towards

Sinorhizobium meliloti, a symbiotic alfalfa associated bacterium: use of advanced
microscopic and spectroscopic techniques.
Bandyopadhyay S(1), Peralta-Videa JR, Plascencia-Villa G, José-Yacamán M,

Author information:
(1)Environmental Science and Engineering PhD Program, The University of Texas at
El Paso, 500 West University Avenue, El Paso, TX 79968, USA.
Cerium oxide (CeO(2)) and zinc oxide (ZnO) nanoparticles (NPs) are extensively
used in a variety of instruments and consumer goods. These NPs are of great
concern because of potential toxicity towards human health and the environment.
The present work aimed to assess the toxic effects of 10nm CeO(2) and ZnO NPs
towards the nitrogen fixing bacterium Sinorhizobium meliloti. Toxicological
parameters evaluated included UV/Vis measurement of minimum inhibitory
concentration, disk diffusion tests, and dynamic growth. Ultra high-resolution
scanning transmission electron microscopy (STEM) and infrared spectroscopy (FTIR)
were utilized to determine the spatial distribution of NPs and macromolecule
changes in bacterial cells, respectively. Results indicate that ZnO NPs were more
toxic than CeO(2) NPs in terms of inhibition of dynamic growth and viable cells
counts. STEM images revealed that CeO(2) and ZnO NPs were found on bacterial cell
surfaces and ZnO NPs were internalized into the periplasmic space of the cells.
FTIR spectra showed changes in protein and polysaccharide structures of extra
cellular polymeric substances present in bacterial cell walls treated with both
NPs. The growth data showed that CeO(2) NPs have a bacteriostatic effect, whereas
ZnO NPs is bactericidal to S. meliloti. Overall, ZnO NPs were found to be more
toxic than CeO(2) NPs.
DOI: 10.1016/j.jhazmat.2012.09.056
939. PLoS One. 2012;7(10):e47149. doi: 10.1371/journal.pone.0047149. Epub 2012 Oct

10.
Increasing cropping system diversity balances productivity, profitability and

environmental health.
Davis AS(1), Hill JD, Chase CA, Johanns AM, Liebman M.
Author information:
(1)United States Department of Agriculture/Agricultural Research Service, Global
Change and Photosynthesis Research Unit, Urbana, IL, USA. adam.davis@ars.usda.gov
Balancing productivity, profitability, and environmental health is a key

challenge for agricultural sustainability. Most crop production systems in the
United States are characterized by low species and management diversity, high use
of fossil energy and agrichemicals, and large negative impacts on the
environment. We hypothesized that cropping system diversification would promote
ecosystem services that would supplement, and eventually displace, synthetic
external inputs used to maintain crop productivity. To test this, we conducted a
field study from 2003-2011 in Iowa that included three contrasting systems
varying in length of crop sequence and inputs. We compared a conventionally
managed 2-yr rotation (maize-soybean) that received fertilizers and herbicides at
rates comparable to those used on nearby farms with two more diverse cropping
systems: a 3-yr rotation (maize-soybean-small grain + red clover) and a 4-yr
rotation (maize-soybean-small grain + alfalfa-alfalfa) managed with lower
synthetic N fertilizer and herbicide inputs and periodic applications of cattle
manure. Grain yields, mass of harvested products, and profit in the more diverse
systems were similar to, or greater than, those in the conventional system,
despite reductions of agrichemical inputs. Weeds were suppressed effectively in
all systems, but freshwater toxicity of the more diverse systems was two orders
of magnitude lower than in the conventional system. Results of our study indicate
that more diverse cropping systems can use small amounts of synthetic
agrichemical inputs as powerful tools with which to tune, rather than drive,
agroecosystem performance, while meeting or exceeding the performance of less
diverse systems.
PMCID: PMC3468434
940. Nucleic Acids Res. 2013 Jan;41(Database issue):D273-9. doi:

10.1093/nar/gks935.
Epub 2012 Oct 12.
PlantRNA, a database for tRNAs of photosynthetic eukaryotes.
Cognat V(1), Pawlak G, Duchêne AM, Daujat M, Gigant A, Salinas T, Michaud M,

Gutmann B, Giegé P, Gobert A, Maréchal-Drouard L.
Author information:
(1)Institut de Biologie Moléculaire des Plantes, UPR 2357-CNRS, Université de
Strasbourg, 12 rue du Général Zimmer, F-67084 Strasbourg Cedex, France.
PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA)

gene sequences retrieved from fully annotated plant nuclear, plastidial and
mitochondrial genomes. The set of annotated tRNA gene sequences has been manually
curated for maximum quality and confidence. The novelty of this database resides
in the inclusion of biological information relevant to the function of all the
tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and
B box sequences, region of transcription initiation and poly(T) transcription
termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and
enzymes responsible for tRNA maturation and modification. Finally, data on
mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the
respective tRNAs and tRNA-binding proteins are also included. The current
annotation concerns complete genomes from 11 organisms: five flowering plants
(Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and
Brachypodium distachyon), a moss (Physcomitrella patens), two green algae
(Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora
paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom
(Phaeodactylum tricornutum). The database will be regularly updated and
implemented with new plant genome annotations so as to provide extensive
information on tRNA biology to the research community.
DOI: 10.1093/nar/gks935
PMCID: PMC3531208
941. Environ Manage. 2012 Dec;50(6):1193-203. doi: 10.1007/s00267-012-9958-7. Epub

2012 Oct 12.
Differential effects of legume species on the recovery of soil microbial

communities, and carbon and nitrogen contents, in abandoned fields of the Loess
Plateau, China.
Li JH(1), Jiao SM, Gao RQ, Bardgett RD.
Author information:
(1)Lanzhou University, Lanzhou, People's Republic of China. lijinhuap@sohu.com
Plant-soil interactions are known to influence a wide range of ecosystem-level

functions. Moreover, the recovery of these functions is of importance for the
successful restoration of soils that have been degraded through intensive and/or
inappropriate land use. Here, we assessed the effect of planting treatments
commonly used to accelerate rates of grassland restoration, namely introduction
of different legume species Medicago sativa, Astragalus adsurgens, Melilotus
suaveolens, on the recovery of soil microbial communities and carbon and nitrogen
contents in abandoned fields of the Loess Plateau, China. The results showed
effects were species-specific, and either positive, neutral or negative depending
on the measure and time-scale. All legumes increased basal respiration and
metabolic quotient and had a positive effect on activity and functional diversity
of the soil microbial community, measured using Biolog EcoPlate. However, soil
under Astragalus adsurgens had the highest activity and functional diversity
relative to the other treatments. Soil carbon and nitrogen content and microbial
biomass were effectively restored in 3-5 years by introducing Medicago sativa and
Astragalus adsurgens into early abandoned fields. Soil carbon and nitrogen
content were retarded in 3-5 years and microbial biomass was retarded in the
fifth year by introducing Melilotus suaveolens. Overall, the restoration
practices of planting legumes can significantly affect soil carbon and nitrogen
contents, and the biomass, activity, and functional diversity of soil microbial
community. Therefore, we propose certain legume species could be used to
accelerate ecological restoration of degraded soils, hence assist in the
protection and preservation of the environment.
DOI: 10.1007/s00267-012-9958-7
942. Mol Ecol. 2012 Nov;21(22):5546-60. doi: 10.1111/mec.12021. Epub 2012 Oct 12.
Localizing F(ST) outliers on a QTL map reveals evidence for large genomic regions
of reduced gene exchange during speciation-with-gene-flow.
Via S(1), Conte G, Mason-Foley C, Mills K.
Author information:
(1)Department of Biology, University of Maryland, College Park, MD 21042, USA.
svia@umd.edu
Populations that maintain phenotypic divergence in sympatry typically show a

mosaic pattern of genomic divergence, requiring a corresponding mosaic of genomic
isolation (reduced gene flow). However, mechanisms that could produce the genomic
isolation required for divergence-with-gene-flow have barely been explored, apart
from the traditional localized effects of selection and reduced recombination
near centromeres or inversions. By localizing F(ST) outliers from a genome scan
of wild pea aphid host races on a Quantitative Trait Locus (QTL) map of key
traits, we test the hypothesis that between-population recombination and gene
exchange are reduced over large 'divergence hitchhiking' (DH) regions. As
expected under divergence hitchhiking, our map confirms that QTL and divergent
markers cluster together in multiple large genomic regions. Under divergence
hitchhiking, the nonoutlier markers within these regions should show signs of
reduced gene exchange relative to nonoutlier markers in genomic regions where
ongoing gene flow is expected. We use this predicted difference among nonoutliers
to perform a critical test of divergence hitchhiking. Results show that
nonoutlier markers within clusters of F(ST) outliers and QTL resolve the genetic
population structure of the two host races nearly as well as the outliers
themselves, while nonoutliers outside DH regions reveal no population structure,
as expected if they experience more gene flow. These results provide clear
evidence for divergence hitchhiking, a mechanism that may dramatically facilitate
the process of speciation-with-gene-flow. They also show the power of integrating
genome scans with genetic analyses of the phenotypic traits involved in local
adaptation and population divergence.
DOI: 10.1111/mec.12021
943. Environ Sci Technol. 2012 Nov 6;46(21):12021-9. doi: 10.1021/es301829r. Epub
2012
Oct 19.
Development of a constructed wetland water treatment system for selenium removal:

use of mesocosms to evaluate design parameters.
Huang JC(1), Passeport E, Terry N.
Author information:
(1)Department of Plant and Microbial Biology, University of California, Berkeley,
California 94720-3102, USA.
The Salton Sea in California is an important habitat for fish and waterfowl. Its
ecosystem is threatened due to diminishing water supplies and increasing
salinity. An alternative source of water to support species conservation habitat
may be obtained from local rivers (e.g., Alamo or New Rivers), provided that a
wetland treatment system can be developed to remove selenium (Se), fertilizer
nutrients, and other contaminants. Here, we used mesocosms to evaluate a number
of potential design options (e.g., plant species selection, sediment composition
and arrangement, forced aeration, organic amendments, etc.) to improve the
efficiency of Se removal using treatment wetlands. Our results show that, of five
different substrate arrangements tested for Se removal, the most efficient was
obtained for cattails growing in a substrate of cattail litter overlying sand and
peat moss sediment (water column Se was reduced from 15 μg Se/L to <0.1 μg Se/L
in 72 h). The addition of organic amendments in the form of alfalfa hay or
alfalfa meal was also helpful in lowering Se levels. These results suggest that
it may be possible to design constructed wetland water treatment systems capable
of reducing Se concentrations in river water to values below 1 μg Se/L.
DOI: 10.1021/es301829r
944. Oecologia. 2013 May;172(1):177-88. doi: 10.1007/s00442-012-2476-8. Epub 2012

Oct
4.
Specificity, rank preference, and the colonization of a non-native host plant by

the Melissa blue butterfly.
Forister ML(1), Scholl CF, Jahner JP, Wilson JS, Fordyce JA, Gompert Z, Narala
DR, Alex Buerkle C, Nice CC.
Author information:
(1)Department of Biology, University of Nevada, Reno, NV 89557, USA.
forister@gmail.com
Animals often express behavioral preferences for different types of food or other
resources, and these preferences can evolve or shift following association with
novel food types. Shifts in preference can involve at least two phenomena: a
change in rank preference or a change in specificity. The former corresponds to a
change in the order in which hosts are preferred, while a shift in specificity
can be an increase in the tendency to utilize multiple hosts. These possibilities
have been examined in relatively few systems that include extensive
population-level replication. The Melissa blue butterfly, Lycaeides melissa, has
colonized exotic alfalfa, Medicago sativa, throughout western North America. We
assayed the host preferences of 229 females from ten populations associated with
novel and native hosts. In four out of five native-associated populations, a
native host was preferred over the exotic host, while preference for a native
host characterized only two out of five of the alfalfa-associated populations.
Across all individuals from alfalfa-associated populations, there appears to have
been a decrease in specificity: females from these populations lay fewer eggs on
the native host and more eggs on the exotic relative to females from native-host
populations. However, females from alfalfa-associated populations did not lay
more eggs on a third plant species, which suggests that preferences for specific
hosts in this system can potentially be gained and lost independently. Geographic
variation in oviposition preference in L. melissa highlights the value of
surveying a large number of populations when studying the evolution of a complex
behavioral trait.
DOI: 10.1007/s00442-012-2476-8
945. Lett Appl Microbiol. 2013 Jan;56(1):30-6. doi: 10.1111/lam.12009. Epub 2012
Nov
2.
Changes in microbial contamination levels and prevalence of foodborne pathogens

in alfalfa (Medicago sativa) and rapeseed (Brassica napus) during sprout
production in manufacturing plants.
Kim SA(1), Kim OM, Rhee MS.
Author information:
(1)Division of Food Bioscience and Technology, College of Life Sciences and
Biotechnology, Korea University, Seoul, South Korea.
Samples were taken from three sprout processing plants at five different stages
of production (a total of 20 investigations). Quantitative analyses comprised
aerobic plate counts (APCs) and the measurement of coliforms and Bacillus cereus
levels, whereas qualitative analyses involved assessing the levels of Escherichia
coli and major foodborne pathogens (E. coli O157:H7, Listeria monocytogenes,
Salmonella spp., and Staphylococcus aureus). The APC for alfalfa seeds (3·71-4·61
log CFU g(-1)) and rapeseed (4·25-5·11 log CFU g(-1)) increased by approximately
3 log CFU g(-1) during sprouting, reaching 7·17-7·61 and 7·33-8·28 log CFU g(-1),
respectively, by the final stage of production. Similarly, increasing trends were
noted in the level of coliforms (0·58-4·03 log CFU g(-1) at the seed stage,
increasing to 5·52-6·99 log CFU g(-1) by the sprout stage). Bacillus cereus was
detected in eight alfalfa (40%) and 14 rapeseed (70%) sprouts, and L.
monocytogenes was isolated from one pregermination soaked alfalfa seed. A slight
reduction in the level of bacterial contamination was noted after washing the
sprouts with water prior to storage, indicating that improvements to the current
washing protocol, or other efficient intervention methods, may be needed. Taken
together, these results suggest that improved hygiene control during production
and processing and a more sanitary environment are needed. The present study
provides comprehensive information regarding the microbiological safety of seeds
and sprouts during manufacturing.SIGNIFICANCE AND IMPACT OF STUDY: The present
study investigated the levels of microbial contamination present in alfalfa and
rapeseed sprouts by examining the samples taken at different stages of the
manufacturing process in three actual plants. The results provide detailed
information regarding the levels of seed and sprout contamination during
production. The results may be useful to those involved in the sprout industry
and/or academic research in terms of developing hygienic control measures,
efficient intervention methods and appropriate guidelines.
DOI: 10.1111/lam.12009
946. Anim Sci J. 2012 Oct;83(10):690-5. doi: 10.1111/j.1740-0929.2012.01019.x. Epub

2012 Apr 25.
Improvement and validation of the method to determine neutral detergent fiber in

feed.
Hiraoka H(1), Fukunaka R, Ishikuro E, Enishi O, Goto T.
Author information:
(1)Food and Agricultural Materials Inspection Center, Kobe Center, Chuo, Kobe,
Hyougo, Japan.
To improve the performance of the analytical method for neutral detergent fiber
in feed with heat-stable α-amylase treatment (aNDFom), the process of adding
heat-stable α-amylase, as well as other analytical conditions, were examined. In
this new process, the starch in the samples was removed by adding amylase to
neutral detergent (ND) solution twice, just after the start of heating and
immediately after refluxing. We also examined the effects of the use of sodium
sulfite, and drying and ashing conditions for aNDFom analysis by this modified
amylase addition method. A collaborative study to validate this new method was
carried out with 15 laboratories. These laboratories analyzed two samples,
alfalfa pellet and dairy mixed feed, with blind duplicates. Ten laboratories used
a conventional apparatus and five used a Fibertec(®) type apparatus. There were
no significant differences in aNDFom values between these two refluxing
apparatuses. The aNDFom values in alfalfa pellet and dairy mixed feed were 388
g/kg and 145 g/kg, the coefficients of variation for the repeatability and
reproducibility (CV(r) and CV(R) ) were 1.3% and 2.9%, and the HorRat values were
0.8 and 1.1, respectively. This new method was validated with 5.8% uncertainty (k
= 2) from the collaborative study.
© 2012 The Authors. Animal Science Journal © 2012 Japanese Society of Animal
Science.
DOI: 10.1111/j.1740-0929.2012.01019.x
947. Animal. 2013 Feb;7(2):232-44. doi: 10.1017/S1751731112001437. Epub 2012 Jul

19.
Effect of harvest time and physical form of alfalfa silage on chewing time and
particle size distribution in boli, rumen content and faeces.
Kornfelt LF(1), Weisbjerg MR, Nørgaard P.
Author information:
(1)Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences,
University of Copenhagen, Denmark. kornfelt@life.ku.dk
The study examined the effects of physical form and harvest time of alfalfa
silage on eating and ruminating activity and particle size distribution in feed
boli, rumen content and faeces in dry cows. The alfalfa crop was harvested at two
stages of growth (early: NDF 37%, late: NDF 44% in dry matter (DM)), and from
each harvest, a chopped (theoretical cutting length: 19 mm) and an unchopped crop
was ensiled in bales. The silages were fed restrictively to four rumen cannulated
non-lactating Jersey cows (391 ± 26 kg) in a 4 × 4 Latin square design. The cows
were fed restrictively 80% of their ad libitum intake twice daily. Chewing
activity was recorded for 96 h continuously. Swallowed boli, rumen content, rumen
fluid and faeces samples were collected, washed in nylon bags (0.01 mm pore size)
and freeze-dried before dry sieving through 4.750, 2.360, 1.000, 0.500 and 0.212
mm pore sizes into six fractions. The length (PL) and width (PW) of particles
within each fraction was measured by the use of image analysis. The eating
activity (min/kg dry matter intake (P < 0.01) and min/kg NDF (P < 0.05)) was
affected by harvest time. The mean ruminating time (min/kg DM) was affected by
harvest time (P < 0.01), physical form (P < 0.05) and NDF intake per kg BW (P <
0.01). The proportion of washed particle DM of total DM in boli, rumen content,
rumen fluid and faeces was affected by harvest time (P < 0.01) and highest by
feeding late-harvested alfalfa silage. Two peaks on the probability density
distribution function (PDF) of PW and PL values of boli, rumen content and faeces
were identified. Chopping of the silage decreased the mean PL and PW, the most
frequent PL (mode) and 95% percentile PL and PW values in boli. In the rumen
content, chopping increased the mean PW (P < 0.05). The dimension sizes of faeces
particles were not significantly affected by chopping. The mode PW value was
lower in rumen content and faeces than in boli (P < 0.001), and the mode PL value
was higher in boli and lower in faeces compared with rumen contents (P < 0.001).
In conclusion, the mean total chewing activity per kg NDF decreased due to
chopping and early harvest time. The mean PL and PW in boli decreased due to
chopping and late harvest. The two peak values on the PDF (PL) and PDF (PW) of
boli, rumen content and faeces particles are most likely related to the leaf and
the stem residues.
DOI: 10.1017/S1751731112001437
948. Animal. 2012 Oct;6(10):1642-7. doi: 10.1017/S175173111200050X.
Rumen digestion of rice straw structural polysaccharides: effect of ammonia

treatment and lucerne extract supplementation in vitro.
Agbagla-Dohnani A(1), Cornu A, Broudiscou LP.
Author information:
(1)INRA, UMR Herbivores, St Genès Champanelle, France.
The combined effects of lucerne (Medicago sativa L.) extract supplementation and
ammonia treatment of rice straw (Oryza sativa, variety Thaibonnet) on the ruminal
digestion of cell wall components were investigated in six continuous culture
systems using a randomised complete block design. Data were fitted to
second-order polynomial models. Untreated rice straw had higher contents of
ash-free cell wall residues (CWR; 763 v. 687 g/kg dry matter (DM)) and
non-cellulosic sugars (191 v. 166 g/kg DM) than treated rice straw. Ammoniation
preferentially removed xylose, which resulted in a lower xylose-to-arabinose
ratio (5.1 v. 5.8). In absence of lucerne supplementation and ammoniation,
degradability coefficients were 0.54, 0.46, 0.58, 0.54, 0.42 and 0.60 for
cellulose-glucose, xylose, arabinose, galactose, mannose and uronic acids,
respectively. Both factors had significant effects on the microbial degradation
of structural polysaccharides. With lucerne extract at an optimal level, ammonia
treatment increased ash-free cell wall degradation by more than 10%. The
degradability coefficients were increased by ammoniation without any significant
interaction with lucerne extract, except for glucose, whose degradability was
mostly influenced by lucerne extract in a curvilinear way. The comparison of
regression coefficients in cell wall and CWR models suggested that ammoniation
improved the degradabilities of xylose, galactose and mannose by partly
solubilising the corresponding hemicelluloses and by improving the susceptibility
of the remaining fraction to microbial attack, whereas it increased the
degradability of arabinose only by favouring microbial attack.
DOI: 10.1017/S175173111200050X
949. Animal. 2013 Jan;7(1):82-92. doi: 10.1017/S1751731112001097. Epub 2012 May 29.
Mixing sainfoin and lucerne to improve the feed value of legumes fed to sheep by
the effect of condensed tannins.
Aufrère J(1), Dudilieu M, Andueza D, Poncet C, Baumont R.
Author information:
(1)INRA, UMR Herbivores, Saint-Genès Champanelle, France.
jocelyne.aufrere@clermont.inra.fr
The aim of this study was to investigate whether the use of sainfoin-based
condensed tannins (CT) enhances feed value when given with tannin-free legumes
(lucerne) to sheep. The experiments were conducted with fresh sainfoin and
lucerne harvested at two stages (vegetative stage as compared with early
flowering) in the first growth cycle. Fresh sainfoin and lucerne forages were
combined in ratios of 100 : 0, 75 : 25, 25 : 75 and 0 : 100 (denoted S100, S75,
S25 and S0, respectively). Voluntary intake, organic matter digestibility (OMD)
and nitrogen (N) retention were measured in sheep fed the different sainfoin and
lucerne mixtures. Loss of dry matter (DM) and N from polyester bags suspended in
the rumen, abomasum and small intestine (SI) was also measured using
rumen-fistulated sheep and intestinally fistulated sheep. The CT content in
sainfoin (S100) decreased with increasing percentage of lucerne in the mixture
(mean value from 58 g/kg DM for S100 to 18 g/kg DM for S25) and with growth stage
(S100: 64 to 52 g/kg DM). OMD did not differ between different sainfoin/lucerne
mixture ratios. Sainfoin and lucerne had an associative effect (significant
quadratic contrast) on voluntary intake, N intake, total-tract N digestibility, N
in faeces and urine (g/g N intake) and N retained (g/g N intake). Compared with
lucerne mixtures (S0 and S25), high-sainfoin-content mixtures (S100 and S75)
increased the in situ estimates of forage N escaping from the rumen (from 0.162,
0.188 for S0 and S25 to 0.257, 0.287 for S75 and S100) but decreased forage N
intestinal digestibility (from 0.496, 0.446 for S0 and S25 to 0.469, 0.335 for
S75 and S100). The amount of forage N disappearing from the bags in the SI (per g
forage N) was the highest for high-sainfoin mixtures (from 0.082, 0.108 for S100
and S75 to 0.056, 0.058 for S25 and S0, P < 0.001). Rumen juice total N (tN) and
ammonia N (NH3-N) values were the lowest in the high-sainfoin diet (mean tN 0.166
mg/g in S100 as compared with 0.514 mg/g in S0; mean NH3-N 0.104 mg/g in S100 as
compared with 0.333 mg/g in S0, P < 0.001).
DOI: 10.1017/S1751731112001097
950. Electrophoresis. 2012 Sep;33(18):2840-51. doi: 10.1002/elps.201200144.
Technical improvement of the TBP (tubulin-based polymorphism) method for plant

species detection, based on capillary electrophoresis.
Gavazzi F(1), Casazza AP, Depedro C, Mastromauro F, Breviario D.
Author information:
(1)Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle
Ricerche, Milan, Italy.
Nowadays, feed and food safety and traceability are of primary importance. Hence,
a correct labeling of the different products is highly desirable in general, but
mandatory for those people who are suffering from eating disorders and food
allergies. Among the technologies that have been developed for feed and food
analysis, the patented tubulin-based polymorphism (TBP) method emerges as an
easy, versatile, and inexpensive diagnostic tool. Initially used to fingerprint
different plant species and varieties, TBP was then successfully applied to trace
species in mixtures of plant origin such as commercial feeds. TBP is a DNA-based
molecular marker, that makes use of PCR for the selective amplification of plant
β-tubulin introns. Amplified fragments are then separated by PAGE and visualized
by silver staining. We have now developed an improved version of TBP. Based on
capillary electrophoresis and fluorescence detection, it makes the method
automatic, more sensible, reproducible, and faster. Compared to the classic TBP,
this new version allows to obtain a better data resolution and an easier
interpretation of the results, clearing the way to large-scale feed/food
diagnostics.
DOI: 10.1002/elps.201200144
951. Ann Pharmacother. 2012 Oct;46(10):1392-404. doi: 10.1345/aph.1R167. Epub 2012

Sep
25.
The use of galactogogues in the breastfeeding mother.
Forinash AB(1), Yancey AM, Barnes KN, Myles TD.
Author information:
(1)Department of Pharmacy Practice, St. Louis College of Pharmacy, MO, USA.
aforinash@stlcop.edu
OBJECTIVE: To review data regarding the efficacy of galactogogues available in

the US to increase breast milk production in postpartum mothers.
DATA SOURCES: Literature was sought using PubMed (1966-June 2012) and EMBASE
(1973-June 2012). Search terms included breastfeeding, breast milk, lactation,
galactogogue, metoclopramide, oxytocin, fenugreek, milk thistle, silymarin,
growth hormone, thyroid releasing hormone, medroxyprogesterone, domperidone,
goat's rue, beer, Asparagus racemosus, shatavari, Medicago sativa, alfalfa,
Onicus benedictus, blessed thistle, Galega officinalis, brewer's yeast, and
herbals.
STUDY SELECTION AND DATA EXTRACTION: All studies including humans and published
in English with data assessing the efficacy of galactogogues for increasing
breast milk production were evaluated.
DATA SYNTHESIS: Breast milk is considered the optimal food source for newborns
through 1 year of age. Many factors influence overall maternal production,
including maternal pain, illness, balance of time when returning to work,
anxiety, or emotional stress. Although a variety of herbal and pharmaceutical
options have anecdotal evidence of their ability to improve breast milk
production, peer-reviewed studies proving their efficacy are lacking.
Metoclopramide, oxytocin, fenugreek, and milk thistle have shown mixed results in
improving milk production; however, the trials were small and had a variety of
limitations.
CONCLUSIONS: Nonpharmacologic recommendations should be exhausted before adding
therapy. Although anecdotal evidence encourages the use of metoclopramide,
fenugreek, asparagus, and milk thistle for their galactogogue properties,
efficacy and safety data in the literature are lacking. Oxytocin and domperidone
are potentially available for compounding purposes, but safety data are limited.
More studies are needed to evaluate the effects of available galactogogues on
breast milk production.
DOI: 10.1345/aph.1R167
952. Huan Jing Ke Xue. 2012 Jul;33(7):2510-5.
[In-situ remediation of polychlorinated biphenyls polluted soil by ecological

controlling measures: a field trial].
Pan C(1), Teng Y, Luo YM, Tu C, Li XF, Ma TT, Zhang MY, Li ZG, Song J.
Author information:
cpan@issas.ac.cn
A field experiment was conducted to study the remediation of agricultural soils

polluted with polychlorinated biphenyls (PCBs) by several ecological controlling
measures. After sequential stages of liming, plowing, planting alfalfa and paddy,
a significant part of PCBs was removed from the soil, with an average removal
rate of 86.9%. The treatment also decreased the toxic equivalency of dioxin-like
PCBs in soil. Analysis of PCB congener composition showed that at stages of
liming, plowing and alfalfa planting, the low-chlorinated biphenyls were removed;
whereas at the stage of paddy planting, the high-chlorinated biphenyls were
degraded. The ecological controlling measures had little influence on the
microbial communities in soil. The results suggest that ecological control
techniques could be widely applied in the remediation of agricultural soils with
PCBs contamination.
953. Int J Syst Evol Microbiol. 2013 May;63(Pt 5):1760-5. doi:

10.1099/ijs.0.041368-0.
Epub 2012 Sep 21.
Endobacter medicaginis gen. nov., sp. nov., isolated from alfalfa nodules in an
acidic soil.
Ramírez-Bahena MH(1), Tejedor C, Martín I, Velázquez E, Peix A.
Author information:
(1)Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior
de Investigaciones Científicas (IRNASA-CSIC), Salamanca, Spain.
A bacterial strain designated M1MS02(T) was isolated from a surface-sterilized

nodule of Medicago sativa in Zamora (Spain). The 16S rRNA gene sequence of this
strain showed 96.5 and 96.2 % similarity, respectively, with respect to
Gluconacetobacter liquefaciens IFO 12388(T) and Granulibacter bethesdensis
CGDNIH1(T) from the family Acetobacteraceae. The novel isolate was a
Gram-stain-negative, non-sporulating, aerobic coccoid to rod-shaped bacterium
that was motile by a subpolar flagellum. The major fatty acid was C18 : 1ω7c and
the major ubiquinone was Q-10. The lipid profile consisted of
diphosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids, three
aminolipids, four glycolipids, two phospholipids and one lipid. Strain M1MS02(T)
was catalase-positive and oxidase- and urease-negative. Acetate and lactate were
not oxidized. Acetic acid was produced from ethanol in culture media supplemented
with 2 % CaCO3. Ammonium sulphate was assimilated in glucose medium. The strain
produced dihydroxyacetone from glycerol. Phylogenetic and phenotypic analyses
commonly used to differentiate genera within the family Acetobacteraceae showed
that strain M1MS02(T) should be classified as representing a novel species of a
new genus within this family, for which the name Endobacter medicaginis gen.
nov., sp. nov. is proposed. The type strain of the type species is M1MS02(T)
( = LMG 26838(T) = CECT 8088(T)). To our knowledge, this is the first report of a
member of the Acetobacteraceae occurring as a legume nodule endophyte.
DOI: 10.1099/ijs.0.041368-0
954. Mol Plant Microbe Interact. 2013 Feb;26(2):160-7. doi:

10.1094/MPMI-07-12-0186-CR.
Insights into the noncoding RNome of nitrogen-fixing endosymbiotic

α-proteobacteria.
Jiménez-Zurdo JI(1), Valverde C, Becker A.
Author information:
Consejo Superior de Investigaciones Científicas (CSIC), 18008 Granada, Spain.
jijz@eez.csic.es
Symbiotic chronic infection of legumes by rhizobia involves transition of

invading bacteria from a free-living environment in soil to an intracellular
state as differentiated nitrogen-fixing bacteroids within the nodules elicited in
the host plant. The adaptive flexibility demanded by this complex lifestyle is
likely facilitated by the large set of regulatory proteins encoded by rhizobial
genomes. However, proteins are not the only relevant players in the regulation of
gene expression in bacteria. Large-scale high-throughput analysis of prokaryotic
genomes is evidencing the expression of an unexpected plethora of small
untranslated transcripts (sRNAs) with housekeeping or regulatory roles. sRNAs
mostly act in response to environmental cues as post-transcriptional regulators
of gene expression through protein-assisted base-pairing interactions with target
mRNAs. Riboregulation contributes to fine-tune a wide range of bacterial
processes which, in intracellular animal pathogens, largely compromise virulence
traits. Here, we summarize the incipient knowledge about the noncoding RNome
structure of nitrogen-fixing endosymbiotic bacteria as inferred from genome-wide
searches for sRNA genes in the alfalfa partner Sinorhizobium meliloti and further
comparative genomics analysis. The biology of relevant S. meliloti RNA chaperones
(e.g., Hfq) is also reviewed as a first global indicator of the impact of
riboregulation in the establishment of the symbiotic interaction.
DOI: 10.1094/MPMI-07-12-0186-CR
955. Retina. 2013 Jan;33(1):89-96. doi: 10.1097/IAE.0b013e3182618a6d.
Posterior hyaloid detachment and internal limiting membrane peeling assisted by

anthocyanins from acai fruit (Euterpe oleracea) and 10 other natural vital dyes:
experimental study in cadaveric eyes.
Chen J(1), Ferreira MA, Farah ME, de Carvalho AM, Alves Ferreira RE, de Moraes
Filho MN, Souza Lima-Filho AA, Lago JH, Sartorelli P, Rodrigues EB, Ferreira E,
Peris C, Maia M.
Author information:
(1)Department of Ophthalmology, Vision Institute (IPEPO), Paulista School of
Medicine, Federal University of Sao Paulo, Brazil.
PURPOSE: The purpose of this study was to determine whether natural dyes
facilitate posterior hyaloid detachment (posterior vitreous detachment [PVD]) and
retinal internal limiting membrane (ILM) peeling in human eyes.
METHODS: Open-sky vitrectomy with posterior hyaloid and ILM removal was performed
in 86 human cadaveric eyes. After core vitrectomy, 11 different dyes were
injected into the vitreous cavity to aid hyaloid detachment and ILM removal. The
dyes were allowed to settle on the macula for 5 minutes after PVD and were
removed by mechanical aspiration. Intraocular forceps were used for ILM peeling,
which was confirmed by light microscopy of the peeled tissue. Acai fruit (Euterpe
oleracea) extract and 10 additional dyes from plants or animal sources were
tested: pomegranate (Punica granatum), logwood (Haematoxylum campechianum),
chlorophyll extract from alfalfa (Medicago sativa), cochineal (Dactylopius
coccus), hibiscus (Hibiscus rosa-sinensis), indigo (Indigofera tinctoria),
paprika (Capiscum annuum), turmeric (Curcuma longa), old fustic (Maclura
tinctoria), and grape (Vitis vinifera).
RESULTS: The dyes facilitated PVD and ILM peeling. Acai fruit (E. oleracea)
extract, logwood (H. campechianum), cochineal (D. coccus), and old fustic (M.
tinctoria) facilitated PVD in all cases; dye-assisted PVD was compared with
triamcinolone-assisted PVD performed previously in a comparative model. Acai
fruit (E. oleracea) extract, cochineal (D. coccus), and chlorophyll extract from
alfalfa (M. sativa) showed the best capability for ILM staining; dye-assisted ILM
removal was compared with the ILM peeling guided by indocyanine green staining
performed previously in a comparative model. Light microscopy confirmed the ILM
removal in all cases.
CONCLUSION: Anthocyanin dye of the acai fruit (E. oleracea) and the dyes from
cochineal (D. coccus) and chlorophyll extract from alfalfa (M. sativa) resulted
in the best capability for posterior hyaloid and ILM staining in human cadaveric
eyes and may be a useful tool for vitreoretinal surgery.
DOI: 10.1097/IAE.0b013e3182618a6d
956. J Food Prot. 2012 Jul;75(7):1338-41. doi: 10.4315/0362-028X.JFP-12-022.
Bacteriological survey of ready-to-eat lettuce, fresh-cut fruit, and sprouts

collected from the Swiss market.
Althaus D(1), Hofer E, Corti S, Julmi A, Stephan R.
Author information:
(1)Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of
Zurich, Winterthurerstr. 272, 8057 Zurich, Switzerland.
The consumption of ready-to-eat fresh vegetables has increased significantly in

the recent decades. So far, no data are available on the bacteriological burden
and the prevalence of foodborne pathogens in ready-to-eat lettuce, fresh-cut
fruit, and sprouts on the Swiss market. This study was based on investigations
carried out during 2 months of the summer season in 2011. Samples of 142 salads,
64 fresh-cut fruit, and 27 sprouts were included in this study. Escherichia coli,
an indicator microorganism for fecal contamination, was only found in 5 lettuce
samples, with amounts ranging between 2 and 3 log CFU/g. No Salmonella spp. were
detected from any of the 233 samples analyzed in this study, and a low occurrence
was found for contamination with L. monocytogenes, Shiga toxin-producing E. coli,
enteropathogenic E. coli, and Cronobacter. From the results of the present study,
we conclude that even in a country where the use of chlorine solutions to
sanitize fruits and vegetables in the fresh-cut industry is not allowed, it is
possible to produce ready-to-eat lettuce, fresh-cut fruit, and sprouts with high
microbiological standards. Strict maintenance of good practices of hygiene at
preharvest, harvest, and postharvest levels is of central importance to ensure
both public health protection and product quality.
DOI: 10.4315/0362-028X.JFP-12-022
957. J Vet Diagn Invest. 2012 Nov;24(6):1068-72. doi: 10.1177/1040638712458784.

Epub
2012 Sep 13.
Mortality of a captive axis deer (Axis axis) and a llama (Lama glama) due to
ingestion of Wedelia glauca.
Giannitti F(1), Margineda CA, Cid MS, Diab SS, Weber N, Rodríguez A, Campero CM,
Odriozola ER.
Author information:
(1)California Animal Health and Food Safety Laboratory System, University of
California, Davis, CA, USA. fgiannitti@ucdavis.edu
Erratum in
J Vet Diagn Invest. 2013 Jan;25(1):176.
The current study describes a naturally occurring cluster of cases of Wedelia

glauca intoxication. Seven of 14 axis deer (Axis axis) and 1 of 8 llamas (Lama
glama) in a zoo of Buenos Aires province, Argentina, died suddenly after
ingestion of a new batch of alfalfa (Medicago sativa) hay bales contaminated with
the hepatotoxic plant W. glauca. Necropsies of 1 deer and 1 llama were performed.
Pathological findings in both animals included severe diffuse acute centrilobular
hepatocellular necrosis and hemorrhage, and clear yellowish translucent
gelatinous edema on the wall of the gall bladder and the serosa of the
choledochoduodenal junction. Fragments of W. glauca plants were identified in the
hay based on the botanical characteristics of the leaves. Samples of gastric
contents were examined by microhistological analysis, which identified epidermal
fragments of W. glauca based on the presence of characteristic uniseriate
glandular hairs (trichomes), confirming recent ingestion of W. glauca in both
cases. The fragments were quantified and represented 5% of all examined vegetal
fragments in the deer and 10% in the llama.
DOI: 10.1177/1040638712458784
958. J R Soc Interface. 2012 Dec 7;9(77):3514-27. doi: 10.1098/rsif.2012.0535. Epub

2012 Sep 12.
Multiwalled carbon nanotubes in alfalfa and wheat: toxicology and uptake.
Miralles P(1), Johnson E, Church TL, Harris AT.
Author information:
(1)Laboratory for Sustainable Technology, School of Chemical and Biomolecular
Engineering, University of Sydney, New South Wales 2006, Australia.
Data on the bioavailability and toxicity of carbon nanotubes (CNTs) in the
environment, and, in particular, on their interactions with vascular plants, are
limited. We investigated the effects of industrial-grade multiwalled CNTs (75 wt%
CNTs) and their impurities on alfalfa and wheat. Phytotoxicity assays were
performed during both seed germination and seedling growth. The germinations of
both species were tolerant of up to 2560 mg l(-1) CNTs, and root elongation was
enhanced in alfalfa and wheat seedlings exposed to CNTs. Remarkably, catalyst
impurities also enhanced root elongation in alfalfa seedlings as well as wheat
germination. Thus the impurities, not solely the CNTs, impacted the plants. CNT
internalization by plants was investigated using electron microscopy and
two-dimensional Raman mapping. The latter showed that CNTs were adsorbed onto the
root surfaces of alfalfa and wheat without significant uptake or translocation.
Electron microscopy investigations of internalization were inconclusive owing to
poor contrast, so Fe(3)O(4)-functionalized CNTs were prepared and studied using
energy-filter mapping of Fe(3)O(4). CNTs bearing Fe(3)O(4) nanoparticles were
detected in the epidermis of one wheat root tip only, suggesting that
internalization was possible but unusual. Thus, alfalfa and wheat tolerated high
concentrations of industrial-grade multiwalled CNTs, which adsorbed onto their
roots but were rarely taken up.
DOI: 10.1098/rsif.2012.0535
PMCID: PMC3481593
959. PLoS One. 2012;7(9):e44742. doi: 10.1371/journal.pone.0044742. Epub 2012 Sep

6.
Heart of endosymbioses: transcriptomics reveals a conserved genetic program among

arbuscular mycorrhizal, actinorhizal and legume-rhizobial symbioses.
Tromas A(1), Parizot B, Diagne N, Champion A, Hocher V, Cissoko M, Crabos A,

Prodjinoto H, Lahouze B, Bogusz D, Laplaze L, Svistoonoff S.
Author information:
(1)Laboratoire Commun de Microbiologie IRD/ISRA/UCAD, Centre de Recherche de Bel
Air, Dakar, Senegal.
To improve their nutrition, most plants associate with soil microorganisms,

particularly fungi, to form mycorrhizae. A few lineages, including actinorhizal
plants and legumes are also able to interact with nitrogen-fixing bacteria hosted
intracellularly inside root nodules. Fossil and molecular data suggest that the
molecular mechanisms involved in these root nodule symbioses (RNS) have been
partially recycled from more ancient and widespread arbuscular mycorrhizal (AM)
symbiosis. We used a comparative transcriptomics approach to identify genes
involved in establishing these 3 endosymbioses and their functioning. We analysed
global changes in gene expression in AM in the actinorhizal tree C. glauca. A
comparison with genes induced in AM in Medicago truncatula and Oryza sativa
revealed a common set of genes induced in AM. A comparison with genes induced in
nitrogen-fixing nodules of C. glauca and M. truncatula also made it possible to
define a common set of genes induced in these three endosymbioses. The existence
of this core set of genes is in accordance with the proposed recycling of ancient
AM genes for new functions related to nodulation in legumes and actinorhizal
plants.
PMCID: PMC3435296
960. Protein J. 2012 Dec;31(8):667-73. doi: 10.1007/s10930-012-9446-1.
A novel malate dehydrogenase from Ceratonia siliqua L. seeds with potential

biotechnological applications.
Muccio C(1), Guida V, Di Petrillo A, Severino V, Di Maro A.
Author information:
(1)Department of Environmental, Biological and Pharmaceutical Sciences and
Technologies, Second University of Naples, Caserta, Italy.
A novel malate dehydrogenase (MDH; EC 3.1.1.1.37), hereafter MDHCs, from

Ceratonia siliqua seeds, commonly known as Carob tree, was purified by using
ammonium sulphate precipitation, ion exchange chromatography on SteamLine SP and
gel-filtration. The molecular mass of the native protein, obtained by analytical
gel-filtration, was about 65 kDa, whereas, by using SDS-PAGE analysis, with and
without reducing agent, was 34 kDa. The specific activity of purified MDHCs
(0.25 mg/100 g seeds) was estimated to be 188 U/mg. The optimum activity of the
enzyme is at pH 8.5, showing a decrease in the presence of Ca(2+), Mg(2+) and
NaCl. The N-terminal sequence of the first 20 amino acids of MDHCs revealed 95 %
identity with malate dehydrogenase from Medicago sativa L. Finally, the enzymatic
activity of MDHCs was preserved even after absorption onto a PVDF membrane. To
our knowledge, this is the first contribution to the characterization of an
enzyme from Carob tree sources.
DOI: 10.1007/s10930-012-9446-1

Epub
2012 Sep 7.
Effects of engineered Sinorhizobium meliloti on cytokinin synthesis and tolerance

of alfalfa to extreme drought stress.
Xu J(1), Li XL, Luo L.
Author information:
(1)State Key Lab of Plant Molecular Genetics, Institute of Plant Physiology and
Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai, China.
Cytokinin is required for the initiation of leguminous nitrogen fixation nodules

elicited by rhizobia and the delay of the leaf senescence induced by drought
stress. A few free-living rhizobia have been found to produce cytokinin. However,
the effects of engineered rhizobia capable of synthesizing cytokinin on host
tolerance to abiotic stresses have not yet been described. In this study, two
engineered Sinorhizobium strains overproducing cytokinin were constructed. The
tolerance of inoculated alfalfa plants to severe drought stress was assessed. The
engineered strains, which expressed the Agrobacterium ipt gene under the control
of different promoters, synthesized more zeatins than the control strain under
free-living conditions, but their own growth was not affected. After a 4-week
inoculation period, the effects of engineered strains on alfalfa growth and
nitrogen fixation were similar to those of the control strain under nondrought
conditions. After being subjected to severe drought stress, most of the alfalfa
plants inoculated with engineered strains survived, and the nitrogenase activity
in their root nodules showed no apparent change. A small elevation in zeatin
concentration was observed in the leaves of these plants. The expression of
antioxidant enzymes increased, and the level of reactive oxygen species decreased
correspondingly. Although the ipt gene was transcribed in the bacteroids of
engineered strains, the level of cytokinin in alfalfa nodules was identical to
that of the control. These findings suggest that engineered Sinorhizobium strains
synthesizing more cytokinin could improve the tolerance of alfalfa to severe
drought stress without affecting alfalfa nodulation or nitrogen fixation.
DOI: 10.1128/AEM.01276-12
PMCID: PMC3485951

2012 Sep 6.
Effects of dietary alfalfa inclusion on Salmonella typhimurium populations in

growing layer chicks.
Escarcha JF(1), Callaway TR, Byrd JA, Miller DN, Edrington TS, Anderson RC,
Nisbet DJ.
Author information:
(1)Agris Mundus-Sustainable Development in Agriculture, Montpellier SupAgro,
Ministere de l'Agriculture, Montpellier, France.
Reducing Salmonella in poultry has been a paramount goal of the poultry industry
in order to improve food safety. Inclusion of high-fiber fermentable feedstuffs
in chicken diets has been shown to reduce the incidence of Salmonella
colonization in laying hens, but no work has been performed in growing birds.
Therefore, the present study was designed to quantify differences in artificially
inoculated cecal Salmonella Typhimurium populations in growing layer chicks (n =
60 in each of two replications) fed 0%, 25%, and 50% of their diet (w/w) replaced
with alfalfa meal from day (d) 7 to d14 after hatch. Alfalfa supplementation
reduced cecal populations of Salmonella by 0.95 and 1.25 log10 colony-forming
unit per gram in the 25% and 50% alfalfa groups compared to controls. Alfalfa
feeding reduced (p < 0.05) the number of cecal- and crop-positive birds compared
to controls. Increasing levels of alfalfa increased (p < 0.05) total volatile
fatty acids (VFA) and the proportion of acetate in the cecum. Surprisingly,
alfalfa inclusion did not negatively impact average daily gain (ADG) in birds
over the 7-d feeding period. Alfalfa inclusion at 50% of the diet increased (p <
0.05) the number of bacterial genera detected in the cecum compared to controls,
and also altered proportions of the microbial population by reducing Ruminococcus
and increasing Clostridia populations. Results support the idea that providing a
fermentable substrate can increase gastrointestinal VFA production and bacterial
diversity which in turn can reduce colonization by Salmonella via natural
competitive barriers. However, further studies are obviously needed to more fully
understand the impact of changes made in diet or management procedures on poultry
production.
DOI: 10.1089/fpd.2012.1251
963. J Anim Sci. 2012 Dec;90(12):4441-8. doi: 10.2527/jas.2011-4332. Epub 2012 Sep
5.
Influence of feeding increasing levels of dry corn distillers grains plus

solubles in whole corn grain-based finishing diets on total tract digestion,
nutrient balance, and excretion in beef steers.
Salim H(1), Wood KM, Abo-Ismail MK, McEwen PL, Mandell IB, Miller SP, Cant JP,
Swanson KC.
Author information:
(1)Department of Animal and Poultry Science, University of Guelph, Guelph, ON,
Canada N1G 2W0.
Four crossbred steers (average BW = 478 ± 33 kg) were used in a 4 × 4 Latin

square design to determine the effects of dietary concentration of dry corn
distillers grains plus solubles (DDGS) in whole corn-based finishing diets on
total tract digestion and nutrient balance and excretion. The DDGS were fed at 0%
(control), 16.7%, 33.3%, and 50% of dietary DM. All diets contained 10% (DM
basis) alfalfa/grass haylage and were formulated to meet or exceed the estimated
requirements for CP. Steers were fed the experimental diets ad libitum for a 14-d
adaptation period followed by a 5-d period for fecal and urine collection.
Increasing concentration of DDGS in diets from 0 to 50% of DM linearly decreased
(P < 0.05) total tract DM and starch digestibility (from 77.8 to 72.9%, and 89.2
to 81.5%, respectively). Daily N and P intakes linearly increased (P = 0.06 and P
= 0.01, respectively) with increasing DDGS concentration. Fecal and urinary N, P,
S, Mg, and K excretion linearly increased (P < 0.05) with increasing DDGS
concentration; however, Se and Na excretion did not differ (P > 0.38) among
treatments. Retention (g/d; intake minus urinary and fecal excretion) of N did
not differ (P > 0.16) among treatments. Retention of P tended (P = 0.07) to
linearly increase and retention of S (g/d) linearly increased (P = 0.004), with
increasing DDGS concentration. There were no effects (P > 0.16) of dietary
treatment on digestion and retention of Se, Mg, K, and Na. Plasma P and S
concentrations increased (P = 0.03 and 0.01, respectively) with increasing DDGS
concentration. These data indicate that feeding DDGS up to 50% of dietary DM in
whole corn grain-based finishing diets does not have a negative effect on
nutrient retention but decreases digestibility. Total excretion of N, P, Ca, Mg,
S, and K increased as DDGS concentration increased.
DOI: 10.2527/jas.2011-4332
964. Huan Jing Ke Xue. 2012 Jun;33(6):2028-37.
[Effect of Cr(VI) stress on growth of three herbaceous plants and their Cr

uptake].
Wang AY(1), Huang SS, Zhong GF, Xu GB, Liu ZX, Shen XB.
Author information:
(1)College of Life Science and Technology, Central South University of Forestry
and Technology, Changsha 410004, China. wangaiyun12@yahoo.com.cn
In order to elucidate the toxic mechanisms of Trifolium repens, Festuca

arundinacea and Medicago sativa under chromium [Cr (VI)] stress, provide a
theoretic foundation for phytoremediation of Cr-contaminated soil, pot experiment
was conducted to investigate the effects of Cr(VI) on plant growth, physiological
characteristics, Cr accumulation and distribution in three herbaceous plants.
Soil sample was treated by adding K2Cr2O7 with the Cr(VI) concentration of 0,
100, 200, 300 and 400 mg x kg(-1), respectively. The results indicated that the
average tolerance indices of T. repens, F. arundinacea and M. sativa were 62.5,
48.3 and 36.33, respectively. Compared with control group, contents of
chlorophyll, the activity of superoxide dismutase(SOD) and peroxidase (POD) were
57.14%, 51.51%, 35.76% and 63.27%, 52.96%, 41.36% in T. repens, and F.
arundinacea, respectively, but M. sativa died in 400 mg x kg(-1) Cr(VI)
treatment. The plant height, root length, dry mass of roots and shoots decreased
under Cr(VI) stress in three herbaceous plants, and M. sativa > F. arundinacea >
T. repens, however, the content of malonyldialdehyde (MDA) increased compared to
the control, and the variation range of M. sativa was the highest, while T.
repens was the smallest among them. The tolerance of Cr( VI) was T. repens > F.
arundinacea > M. sativa. Cr mainly distributed in cell wall and then in the
cytoplasm, and less distributed in the mitochondrion and chloroplast in leaves of
three herbaceous plants, whereas the content of chlorophyll, MDA, the activity of
SOD and POD correlated well with Cr accumulation in the mitochondrion and
chloroplast. Cr concentration in the subcellular of leaves increased with the
adding Cr(VI) concentration,and M. sativa > F. arundinacea > T. repens. In
comparison with T. repens, F. arundinacea, Cr concentration in the leaves of M.
sativa was the maximal, i.e. 51.44 mg x kg(-1), and the proportions in the
mitochondrion (18.04%) and chloroplast (19.09%) were also higher in 300 mg x
kg(-1) Cr(VI). The average accumulation factors of shoots/roots were 1.22/1.54,
1.16/1.44 and 1.26/1.62, while the average translocation factors were 0.78, 0.78
and 0.74 in T. repens, F. arundinacea and M. sativa, respectively. The results
suggest that T. repens and F. arundinacea are promising for the phytoremediation
of Cr-contaminated soil.
965. Huan Jing Ke Xue. 2012 Jun;33(6):2021-7.
[Pb, Zn accumulation and nutrient uptake of 15 plant species grown in abandoned

mine tailings].
Shi X(1), Chen YT, Wang SF, Li JC.
Author information:
(1)The Research Institute of Subtropical Forestry, Chinese Academy of Forestry,
Fuyang 311400, China. shixiang284@sina.com
Vegetation restoration field test was carried out in the abandoned lead-zinc
tailings for 3 years. The study showed that accumulation of lead (Pb), zinc (Zn)
and nutrient uptake differed with plant species and organs, heavy metals, and
planting time. Pb was mainly accumulated in tree roots, and its content
distribution in tree organs was generally in the order of roots > leaves > stems.
But Zn concentrations in leaves of several tree species were higher than those in
roots and stems. Within the tested 15 species, Cercis Canadensis had the highest
concentrations of Pb and Zn in roots (1 803 and 2120 mg x kg(-1), respectively).
Rhus chinensis had the highest Pb concentration in stems and leaves (280 and 546
mg x kg(-1), respectively) and Zn concentration (1 507 mg x kg(-1)) in leaves. Zn
concentration in stems and leaves of Salix matsudana (729 and 1 153 mg x kg(-1),
respectively) were the highest. Among the tested 15 species, TF values for Pb of
Liquidambar formosana, Medicago sativa, and for Zn of Salix matsudana, Rhus
chinensis, Medicago sativa were higher than 1. BCF values for Pb were all lower
than 0.17, while that for Zn were all lower than 0.44. The N contents in
nitrogen-fixing plants, P contents in Rhus typhina and Ailanthus altissima, and K
content in Nerium indicum were significantly higher than those in other plants.
With the increase of planting time, concentrations of heavy metal in plant body
increased significantly; however the inverse trend were observed in nutritional
element content. The species have higher metal accumulation capacity, such as
Rhus chinensis, Salix matsudana and those nitrogen-fixing plants have higher
tolerance to metal contamination and nutrient deficiency, such as Amorpha
fruticosa, Medicago sativa, Lespedeza cuneata, and Alnus cremastogyne, they were
suitable as the phytostabilizers in abandoned mine tailings.

Aug
29.
Effects of natural and simulated rainfall on indicators of ensilability and

nutritive value for wilting alfalfa forages sampled before preservation as
silage.
Coblentz WK(1), Muck RE.
Author information:
Dairy Forage Research Center, Marshfield, WI 54449, USA.
The frustrations of forage producers attempting to conserve high-quality alfalfa

(Medicago sativa L.) silage during periods of unstable or inclement weather are
widely known. Our objectives for this series of studies were to (1) assess
indicators of ensilability, such as pH, buffering capacity, water-soluble
carbohydrates (WSC), and starch for wilting alfalfa forages receiving no rainfall
or damaged by simulated or natural rainfall events; (2) use these data as inputs
to calculate the threshold moisture concentration that would prohibit a
clostridially dominated fermentation; and (3) further evaluate the effects of
rain damage or no rain damage on measures of forage nutritive value. Rainfall
events were applied to wilting forages by both simulated and natural methods over
multiple studies distributed across 4 independent forage harvests. Generally,
simulated rainfall was applied to alfalfa under controlled conditions in which
forages were relatively wet at the time of application, and subsequently were
dried to final moisture endpoints under near ideal conditions within a constant
temperature/humidity environmental chamber, thereby limiting postwetting wilting
time to ≤21 h. As a result, indicators of ensilability, as well as measures of
nutritive value, changed only marginally as a result of treatment. Consistently,
reductions in concentrations of WSC and starch occurred, but changes in WSC were
relatively modest, and postwetting concentrations of WSC may have been buoyed by
hydrolysis of starch. When forages were subjected to natural rainfall events
followed by prolonged exposure under field conditions, indicators of ensilability
were much less desirable. In one study in which alfalfa received 49.3mm of
natural rainfall over a prolonged (8-d) field-exposure period, fresh pH increased
from 6.48 to 7.43 within all forages exposed to these extended, moist wilting
conditions. Furthermore, sharp reductions were observed in buffering capacity
(410 vs. 337 meq/kg of DM), WSC (6.13 vs. 2.90%), starch (2.28 vs. 0.45%), and
clostridially dominated fermentation (62.7 vs. 59.4%). Based on these
experiments, the potential for good fermentation is affected only minimally by
single rainfall events applied to relatively wet forages, provided these events
are followed by rapid dehydration; however, attaining acceptable silage
fermentations with forages subjected to prolonged exposure under poor drying
conditions is likely to be far more problematic.
DOI: 10.3168/jds.2012-5672
967. J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Sep 15;905:23-30. doi:
10.1016/j.jchromb.2012.07.034. Epub 2012 Aug 3.
Concentration and selective fractionation of an antihypertensive peptide from an

alfalfa white proteins hydrolysate by mixed ion-exchange centrifugal partition
chromatography.
Boudesocque L(1), Kapel R, Paris C, Dhulster P, Marc I, Renault JH.
Author information:
(1)Institut de Chimie Moléculaire de Reims UMR CNRS 7312, SFR CAP-Santé,
Université de Reims Champagne-Ardenne, Reims, France.
This article reports a promising use of the mixed ion-exchange centrifugal

partition chromatography (MIXCPC) technique in the field of downstream processes.
A complex alfalfa white protein concentrate hydrolysate (AWPC hydrolysate)
showing anti-hypertensive properties was successfully fractionated by MIXCPC to
yield a L-valyl-L-tryptophan (VW) enriched fraction in one run. This dipeptide
shows an interesting anti-angiotensin converting enzyme (anti-ACE) activity. An
analytical method based on RP-LC/MS-MS was developed to quantify the target VW
peptide in both the starting material and the enriched fractions. The best
results for the MIXCPC fractionation were obtained by the combined use of the
quaternary biphasic solvent system,
methyl-tert-butylether/acetonitrile/n-butanol/water (2:1:2:5, v/v) in the
descending mode, of the lipophilic di(2-ethylhexyl)phosphoric acid (DEHPA)
cation-exchanger with an exchanger (DEHPA)/peptides ratio of 15, and of two
displacers: calcium chloride and hydrochloric acid. The complexity of the
starting material involved the selectivity optimization by splitting the
stationary phase into two sections that differed by their triethylamine
concentration. From 1g of AWPC hydrolysate containing 0.26% of VW, 30.7 mg of a
VW enriched fraction were recovered with a purity of 10.9%, corresponding to a
purification factor of 41 and a recovery of 97%.
DOI: 10.1016/j.jchromb.2012.07.034
968. BMC Plant Biol. 2012 Aug 29;12:153. doi: 10.1186/1471-2229-12-153.
Comparative mapping in the Fagaceae and beyond with EST-SSRs.
Bodénès C(1), Chancerel E, Gailing O, Vendramin GG, Bagnoli F, Durand J,

Goicoechea PG, Soliani C, Villani F, Mattioni C, Koelewijn HP, Murat F, Salse J,
Roussel G, Boury C, Alberto F, Kremer A, Plomion C.
Author information:
(1)INRA, UMR1202 BIOGECO, Cestas F-33610, France. bodenes@pierroton.inra.fr
BACKGROUND: Genetic markers and linkage mapping are basic prerequisites for
comparative genetic analyses, QTL detection and map-based cloning. A large number
of mapping populations have been developed for oak, but few gene-based markers
are available for constructing integrated genetic linkage maps and comparing gene
order and QTL location across related species.
RESULTS: We developed a set of 573 expressed sequence tag-derived simple sequence
repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12
oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in
5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and
Quercus petraea (sessile oak). Consensus maps for the two species were
constructed and aligned. They showed a high degree of macrosynteny between these
two sympatric European oaks. We assessed the transferability of EST-SSRs to other
Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the
European chestnut. Reasonably high levels of macrosynteny were observed between
oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs,
to support further population genetic analyses with gene-based markers. Finally,
based on the orthologous relationships between the oak, Arabidopsis, grape,
poplar, Medicago, and soybean genomes and the paralogous relationships between
the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak
chromosomes from the eudicot ancestral karyotype.
CONCLUSIONS: This study provides map locations for a large set of EST-SSRs in two
oak species of recognized biological importance in natural ecosystems. This first
step toward the construction of a gene-based linkage map will facilitate the
assignment of future genome scaffolds to pseudo-chromosomes. This study also
provides an indication of the potential utility of new gene-based markers for
population genetics and comparative mapping within and beyond the Fagaceae.
DOI: 10.1186/1471-2229-12-153
PMCID: PMC3493355
969. Arch Anim Nutr. 2012 Aug;66(4):319-34.
Lucerne silage in high yielding dairy cows: effect of parity and analysis of
reasons in mixed-parity group housing using random regression test-day models.
Thamm K(1), Bulang M, Mielenz N, Elwert C, Spilke J.
Author information:
(1)Institute of Agricultural and Nutritional Sciences, Martin-Luther-University
Halle-Wittenberg, Halle (Saale), Germany.
A study with high-yielding dairy cows was re-analysed in order to test the
suitability of lucerne silage separately for primi- and multiparous cows as an
alternative to grass silage in maize-based total mixed rations (TMR). Lactation
curves were fitted using random regression test-day models for energy corrected
milk (ECM) and dry matter intake (DMI) as well as for number and duration of
feeder visits (NFV and DFV, respectively). Existing models for ECM and DMI were
extended by animal-specific random effects, which were formulated in their
dependency on days in milk. For NFV and DFV random regression models were applied
for the very first time. The chosen approach of statistical analysis permitted
comparisons of the lactation curves as well as of least square means for
sub-periods to answer nutritional questions. Whilst primiparous cows had
generally lower DMI and ECM as compared to multiparous cows, only in primiparous
cows a negative effect of lucerne TMR on ECM was observed, especially in early
lactation. Nutritional factors should be rejected because of very similar ECM
between the various TMR in multiparous cows. Traits of feeding behaviour
indicated that particle size could contribute to the decreased ECM. Even more
impact on the lower ECM should be addressed to domination behaviour of
multiparous cows. The resulting restlessness of primiparous cows caused a reduced
intake per minute spent at the feeder. Further studies should focus on optimising
the proportion and chopping length of lucerne in the diet and to improve flock
management to maximise feed intake of primiparous cows. Generally, statistical
analysis of lactation data became a very complex issue. It seems inevitable that
nutritionists and statisticians team up to address this problem.

Aug
23.
Effects of glucogenic and ketogenic feeding strategies on splanchnic glucose and

amino acid metabolism in postpartum transition Holstein cows.
Larsen M(1), Kristensen NB.
Author information:
(1)Department of Animal Science, Aarhus University, Foulum, DK-8830 Tjele,
Denmark. Mogens.Larsen@agrsci.dk
Nine periparturient Holstein cows catheterized in major splanchnic vessels were

used in a complete randomized design with repeated measurements to investigate
effects of glucogenic and ketogenic feeding strategies on splanchnic metabolism
of glucose and amino acids. At parturition, cows were assigned to 1 of 3 feeding
strategies: a glucogenic diet (GLCG) based on sodium hydroxide treated wheat
grain (56.5% of diet dry matter); a ketogenic diet (KETO) based on fodder beets
(40.5% of diet dry matter); or an alfalfa-glucogenic strategy (ALF-GLCG)
supplying 100% alfalfa (Medicago sativa L.) haylage at the day of parturition,
followed by a 6-d linear shift to the GLCG diet. Samples were obtained 14 d
before expected parturition as well as at 4, 15, and 29 d in milk (DIM). The net
portal release of glucose was greatest with GLCG, reflecting the higher intake of
ruminal escape starch with GLCG, as compared with a lower starch intake with
KETO. Postpartum, the portal recovery of feed starch was greater (28 ± 3%, mean ±
SEM) with KETO as compared with GLCG (15 ± 4%). At 4 DIM, the net hepatic release
of glucose was greatest with KETO and least with ALF-GLCG, whereafter it
increased as lactation progressed with ALF-GLCG and GLCG, but not with KETO. The
high alfalfa haylage allowance at 4 DIM with the ALF-GLCG treatment induced the
lowest net release of nutrients from the splanchnic tissues at 4 DIM. The hepatic
removal of lactate as percent of total influx (mean ± SEM) increased from 27 ± 3%
prepartum to 56 ± 3% at 4 DIM. The hepatic removal of lactate as percent of net
portal release increased from 144 ± 10% prepartum to 329 ± 17% at 4 DIM with
ALF-GLCG and KETO as compared with 242 ± 20% in GLCG. No clear evidence for an
amino acid sparing effect in splanchnic tissues from increasing small intestinal
glucose absorption was observed. In conclusion, the glucogenic feeding strategy
induced the highest glucogenic status among the tested feeding strategies due to
greater release of glucose from splanchnic tissues. In contrast, the immediate
postpartum high allowance of alfalfa haylage provided the lowest amount of
nutrients from the splanchnic tissues, inducing low glucogenic status, pointing
to the importance of allocating highly digestible diets to postpartum transition
cows. Salvaging glucogenic carbon via interorgan transfer of lactate from
peripheral tissues supported the immediate postpartum incremental increase in
hepatic glucose release rather than hepatic catabolism of amino acids.

DOI: 10.3168/jds.2012-5458
971. J Exp Bot. 2012 Sep;63(15):5521-34. doi: 10.1093/jxb/ers201. Epub 2012 Aug 21.
Haem oxygenase-1 is involved in salicylic acid-induced alleviation of oxidative

stress due to cadmium stress in Medicago sativa.
Cui W(1), Li L, Gao Z, Wu H, Xie Y, Shen W.
Author information:
China.
This work examines the involvement of haem oxygenase-1 (HO-1) in salicylic acid
(SA)-induced alleviation of oxidative stress as a result of cadmium (Cd) stress
in alfalfa (Medicago sativa L.) seedling roots. CdCl(2) exposure caused severe
growth inhibition and Cd accumulation, which were potentiated by pre-treatment
with zinc protoporphyrin (ZnPPIX), a potent HO-1 inhibitor. Pre-treatment of
plants with the HO-1 inducer haemin or SA, both of which could induce MsHO1 gene
expression, significantly reduced the inhibition of growth and Cd accumulation.
The alleviation effects were also evidenced by a decreased content of
thiobarbituric acid-reactive substances (TBARS). The antioxidant behaviour was
confirmed by histochemical staining for the detection of lipid peroxidation and
the loss of plasma membrane integrity. Furthermore, haemin and SA pre-treatment
modulated the activities of ascorbate peroxidase (APX), superoxide dismutase
(SOD), and guaiacol peroxidase (POD), or their corresponding transcripts.
Significant enhancement of the ratios of reduced/oxidized homoglutathione (hGSH),
ascorbic acid (ASA)/dehydroascorbate (DHA), and NAD(P)H/NAD(P)(+), and expression
of their metabolism genes was observed, consistent with a decreased reactive
oxygen species (ROS) distribution in the root tips. These effects are specific
for HO-1, since ZnPPIX blocked the above actions, and the aggravated effects
triggered by SA plus ZnPPIX were differentially reversed when carbon monoxide
(CO) or bilirubin (BR), two catalytic by-products of HO-1, was added. Together,
the results suggest that HO-1 is involved in the SA-induced alleviation of
Cd-triggered oxidative stress by re-establishing redox homeostasis.
DOI: 10.1093/jxb/ers201
PMCID: PMC3444266
972. Int J Phytoremediation. 2012 Jul;14(6):531-42.
Uptake of phosphorus and lead by Brassica juncea and Medicago sativa from
chloropyromorphite.
Abbaspour A(1), Arocena JM, Kalbasi M.
Author information:
(1)Department of Soil Science, Faculty of Agriculture, Shahrood University of
Technology, Shahrood, Semnan Province, Iran. abbaspour2008@gmail.com
In situ remediation of lead (Pb)-contaminated soils via phosphate amendments has

been extensively used to immobilize Pb as pyromorphite. However, in phosphorus
(P) deficient soils, plants may develop extensive root systems to access P in any
P-containing minerals, thereby affecting the stability of Pb5 (PO4)3Cl
(Chloropyromorphite; CP). We grew Brassica juncea and Medicago sativa in sand
culture to evaluate the stability of CP in the presence or absence of
hydroxyapatite (HA) as P source. Treatments (per kilogram of sand) watered with
P-nutrient solution were control [PC0, (without CP)], 1, and 5 g Pb as CP [PC1,
and PC5] and 0.45 g P as HA (PA), and those of watered with P-free nutrient
solution were 1 and 5 g Pb as CP [NC1 and NC5], 5 g Pb as CP plus 0.45 g P as HA
[NAC5], and 0.45 g P as HA [NA]. Plants in NC1 and NC5 treatments showed stunted
growth and reductions in shoot elongation and leaf size. Among CP treated pots,
the highest shoot Pb uptake was observed in NAC5 treatment. The results suggested
that Pb accumulation and translocation in the plants was markedly higher in
P-sufficient conditions than in P-deficient conditions.
DOI: 10.1080/15226514.2010.499918
973. PLoS One. 2012;7(8):e42871. doi: 10.1371/journal.pone.0042871. Epub 2012 Aug

10.
Functional characterizations of chemosensory proteins of the alfalfa plant bug

Adelphocoris lineolatus indicate their involvement in host recognition.
Gu SH(1), Wang SY, Zhang XY, Ji P, Liu JT, Wang GR, Wu KM, Guo YY, Zhou JJ, Zhang
YJ.
Author information:
Insect chemosensory proteins (CSPs) have been proposed to capture and transport
hydrophobic chemicals from air to olfactory receptors in the lymph of antennal
chemosensilla. They may represent a new class of soluble carrier protein involved
in insect chemoreception. However, their specific functional roles in insect
chemoreception have not been fully elucidated. In this study, we report for the
first time three novel CSP genes (AlinCSP1-3) of the alfalfa plant bug
Adelphocoris lineolatus (Goeze) by screening the antennal cDNA library. The
qRT-PCR examinations of the transcript levels revealed that all three genes
(AlinCSP1-3) are mainly expressed in the antennae. Interestingly, these CSP genes
AlinCSP1-3 are also highly expressed in the 5(th) instar nymphs, suggesting a
proposed function of these CSP proteins (AlinCSP1-3) in the olfactory reception
and in maintaining particular life activities into the adult stage. Using
bacterial expression system, the three CSP proteins were expressed and purified.
For the first time we characterized the types of sensilla in the antennae of the
plant bug using scanning electron microscopy (SEM). Immunocytochemistry analysis
indicated that the CSP proteins were expressed in the pheromone-sensitive
sensilla trichodea and general odorant-sensitive sensilla basiconica, providing
further evidence of their involvement in chemoreception. The antennal activity of
55 host-related semiochemicals and sex pheromone compounds in the host location
and mate selection behavior of A. lineolatus was investigated using
electroantennogram (EAG), and the binding affinities of these chemicals to the
three CSPs (AlinCSP1-3) were measured using fluorescent binding assays. The
results showed several host-related semiochemicals, (Z)-3-hexen-1-ol,
(E)-2-hexen-1-al and valeraldehyde, have a high binding affinity with AlinCSP1-3
and can elicit significant high EAG responses of A. lineolatus antennae. Our
studies indicate the three antennae-biased CSPs may mediate host recognition in
the alfalfa plant bug A. lineolatus.
PMCID: PMC3416781
974. Food Chem Toxicol. 2012 Nov;50(11):3920-6. doi: 10.1016/j.fct.2012.07.064.

Epub
2012 Aug 4.
Subchronic toxicity study of GH transgenic carp.
Yong L(1), Liu YM, Jia XD, Li N, Zhang WZ.
Author information:
(1)National Institute for Nutrition and Food Safety, China CDC, Beijing 100021,
China.
A subchronic toxicity study of GH (growth hormone) transgenic carp was carried

out with 60 SD rats aged 4 weeks, weight 115∼125 g. Ten male and 10 female rats
were allotted into each group. Animals of the three groups (transgenic carp group
(GH-TC), parental carp group (PC) and control group) were fed soy- and
alfalfa-free diet (SAFD) with 10% GH transgenic carp powder, 10% parental carp
powder or 10% common carp powder for 90 consecutive days, respectively. In the
end of study, animals were killed by exsanguination via the carotid artery under
diethyl ether anesthesia, then weights of heart, liver, kidneys, spleen, thymus,
brain, ovaries and uterus/testis were measured. Pathological examination of
organs was determined. Endocrine hormones of triiodothyronine (T3), thyroid
hormone (T4), follicle-stimulating hormone (FSH), 17β-estradiol (E2),
progesterone (P) and testosterone (T) levels were detected by specific ELISA kit.
Parameters of blood routine and blood biochemical were measured. The weights of
the body and organs of the rats, food intake, blood routine, blood biochemical
test and serum hormones showed no significant differences among the GH transgenic
carp-treated, parental carp-treated and control groups (P>0.05). Thus, it was
concluded that at the dose level of this study, GH transgenic carp showed no
subchronic toxicity and endocrine disruption to SD rats.
DOI: 10.1016/j.fct.2012.07.064
975. Planta. 2012 Dec;236(6):1687-700. doi: 10.1007/s00425-012-1725-8. Epub 2012

Aug
4.
Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain

display nitrogen-fixing activity with enhanced tolerance to salinity stress.
Redondo FJ(1), Coba de la Peña T, Lucas MM, Pueyo JJ.
Author information:
(1)Instituto de Ciencias Agrarias, CSIC, Serrano 115-bis, 28006, Madrid, Spain.
Nitrogen fixation by legumes is very sensitive to salinity stress, which can

severely reduce the productivity of legume crops and their soil-enriching
capacity. Salinity is known to cause oxidative stress in the nodule by generating
reactive oxygen species (ROS). Flavodoxins are involved in the response to
oxidative stress in bacteria and cyanobacteria. Prevention of ROS production by
flavodoxin overexpression in bacteroids might lead to a protective effect on
nodule functioning under salinity stress. Tolerance to salinity stress was
evaluated in alfalfa nodules elicited by an Ensifer meliloti strain that
overexpressed a cyanobacterial flavodoxin compared with nodules produced by the
wild-type bacteria. Nitrogen fixation, antioxidant and carbon metabolism enzyme
activities were determined. The decline in nitrogenase activity associated to
salinity stress was significantly less in flavodoxin-expressing than in wild-type
nodules. We detected small but significant changes in nodule antioxidant
metabolism involving the ascorbate-glutathione cycle enzymes and metabolites, as
well as differences in activity of the carbon metabolism enzyme sucrose synthase,
and an atypical starch accumulation pattern in flavodoxin-containing nodules.
Salt-induced structural and ultrastructural alterations were examined in detail
in alfalfa wild-type nodules by light and electron microscopy and compared to
flavodoxin-containing nodules. Flavodoxin reduced salt-induced structural damage,
which primarily affected young infected tissues and not fully differentiated
bacteroids. The results indicate that overexpression of flavodoxin in bacteroids
has a protective effect on the function and structure of alfalfa nodules
subjected to salinity stress conditions. Putative protection mechanisms are
discussed.
DOI: 10.1007/s00425-012-1725-8
976. PLoS One. 2012;7(7):e41233. doi: 10.1371/journal.pone.0041233. Epub 2012 Jul

27.
Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa.
Guan Q(1), Takano T, Liu S.
Author information:
(1)Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field
(SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center
(ASNESC), Northeast Forestry University, Harbin, China.
The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression
plants, of which expression level would be increasing under stressful conditions.
The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants
increased with increasing exposure time to different sodium chloride (NaCl) and
hydrogen peroxide (H(2)O(2))concentrations, as indicated by protein gel blot
analysis. The increased enzyme yield improved the ability of the plants to resist
the stress treatments. The OsAPx2 gene was localized in the cytoplasm of
epidermal onion cells as indicated by the instantaneous expression of green
fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants
transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by
specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the
individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation
of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT)
and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7
days, the results as shown of following: the root length of transgenic plant was
longer than WT plants, the H(2)O(2) content in roots of WT was more than of
transgenic plants, the APX activity under stresses increased by 2.89 times
compared with the WT, the malondialdehyde (MDA) content of the WT was higher than
the transgenic plants, the leaves of the WT turned yellow, but those of the
transgenic plants remained green and remained healthy. The chlorophyll content in
the WT leaves was less than in the transgenic plants, after soaking in solutions
of H(2)O(2), sodium sulfite (Na(2)SO(3)), and sodium bicarbonate (NaHCO(3)).
Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the
removal of H(2)O(2) and the salt-resistance compared with WT plants. A novel
strain of M. sativa carrying a salt-resistance gene was obtained.
PMCID: PMC3407167
977. Appl Environ Microbiol. 2012 Oct;78(19):7141-4. doi: 10.1128/AEM.01458-12.

Epub
2012 Jul 27.
Characterization of the twin-arginine transport secretome in Sinorhizobium

meliloti and evidence for host-dependent phenotypes.
Pickering BS(1), Yudistira H, Oresnik IJ.
Author information:
Canada.
The twin-arginine transport (Tat) system is essential for cell viability in

Sinorhizobium meliloti and may play a role during the development of root
nodules. Utilizing an in vivo recombination strategy, we have constructed 28
strains that contain deletions in predicted Tat substrates. Testing of these
mutations for symbiotic proficiency on the plant hosts alfalfa and sweet clover
shows that some of these mutations affect associations with these hosts
differentially.
DOI: 10.1128/AEM.01458-12
PMCID: PMC3457499
978. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2012;29(11):1679-
88.
doi: 10.1080/19440049.2012.702320. Epub 2012 Jul 24.
Liquid chromatography-tandem mass spectrometry method for the determination of

anthelmintics in alfalfa plants.
Islam MD(1), Haberhauer G, Gerzabek M, Cannavan A.
Author information:
(1)Food and Environmental Protection Laboratory, FAO/IAEA Agriculture and
Biotechnology Laboratories, Joint FAO/IAEA Division of Nuclear Techniques in Food
and Agriculture, International Atomic Energy Agency-IAEA, Vienna, Austria.
A simple and inexpensive liquid chromatography-tandem mass spectrometric method

for the determination of anthelmintics in alfalfa plants (Medicago sativa L.) was
developed and validated. Anthelmintics in plant leaves and stems (green chops)
were extracted with methanol/acetonitrile (7:3, v/v) followed by a concentration
and clean-up step using solid-phase extraction (Strata-X, 500 mg, 6 ml
cartridge). After drying with nitrogen gas, the adsorbed analytes were eluted
with methanol/acetonitrile (50:50, v/v) mixture followed by 100% acetonitrile.
Chromatographic separation was achieved on an Atlantis T-3 (2.1 × 100 mm × 3 µm)
analytical column with a Phenomenex guard cartridge (C8, 4 × 3 mm) attached to a
Waters triple quadrupole mass spectrometer operated in positive electrospray
ionisation mode with selected reaction monitoring. Samples were analysed using
gradient elution at a flow rate of 0.35 ml min⁻¹. The mobile phase consisted of a
10 mM ammonium formate solution in (A) water/acetonitrile (90:10, v/v) and (B)
methanol/acetonitrile (50:50, v/v). The method was validated for levamisole,
fenbendazole, fenbendazole sulphoxide and fenbendazole sulphone at 10, 20 and
40 µg kg⁻¹ and for eprinomectin at 20, 40 and 80 µg kg⁻¹. Limits of
quantification (LOQ) were 10 µg kg⁻¹ for all analytes except eprinomectin, which
had an LOQ of 20 µg kg⁻¹. The overall mean recovery in green plants was between
74.2% and 81.4% with repeatabilities ranging from 2.2% to 19.1% and
reproducibilities in the range 3.8-8.7%. The validated method was applied to
plant samples in a study on the behaviour of anthelmintic drugs in a soil, plant
and water system.
DOI: 10.1080/19440049.2012.702320
Short communication: Characteristics of proteolytic activities of endo- and

exopeptidases in alfalfa herbage and their implications for proteolysis in
silage.
Tao L(1), Guo XS, Zhou H, Undersander DJ, Nandety A.
Author information:
China Agricultural University, Beijing 100193, PR China.
The pH optimum and thermostability of both exopeptidases and endopeptidases were

investigated in this study to elucidate the possible role of plant proteases in
proteolysis during ensiling of alfalfa herbage. Proteolytic activities of 4
classes of endopeptidases (i.e., serine, metallo, aspartic, and cysteine
peptidase) and 5 classes of exopeptidases (i.e., aminopeptidase,
carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase)
were examined within pH values of 3 to 9, and within temperatures from 20 to
90°C. Serine and metalloproteases, the principal endopeptidases that hydrolyzed
most of the protein to nonprotein nitrogen in alfalfa silage, had optimum
activities at pH 4. Among the major exopeptidases contributing protein
degradation in ensiled alfalfa, dipeptidase and tripeptidyl-peptidase had stable
activities between pH 4 and 6, and carboxypeptidase activity was optimal at pH 5.
The optimum temperature for most peptidase activities was 40°C. Proteolytic
activities of both endo- and exopeptidases increased with the elevation of
incubating temperature from 20 to 40°C. The pH value in well-preserved alfalfa
silage is often above 4.0, and the temperatures in the ensiled mass range from 25
to 40°C. Therefore, high proteolytic activities between pH 4 and 6 and the
temperature range of ensiled alfalfa suggest that plant peptidases play a role in
hydrolyzing protein during prolonged storage.

DOI: 10.3168/jds.2012-5383
980. Am J Chin Med. 2012;40(4):779-93. doi: 10.1142/S0192415X12500589.
In vitro and in vivo antioxidant activity of alfalfa (Medicago sativa L.) on

carbon tetrachloride intoxicated rats.
Al-Dosari MS(1).
Author information:
(1)Department of Pharmacognosy, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia. msdosari@yahoo.com
The present study was conducted to determine whether lyophilized aqueous extract
of alfalfa, or Medicago sativa L. could exert antioxidant activity against carbon
tetrachloride-induced oxidative stress and liver injury in rats. The
hepatoprotective activity of alfalfa extract was determined by assessing the
levels of serum transaminases, ALP, bilirubin and lipid profile. Further, the
effect of the test substance on malondialdehyde (MDA), an end product of lipid
peroxidation; antioxidant liver enzyme non-protein sulfhydryl (NP-SH); and total
protein (TP) were also studied. Serum transaminase, ALP, bilirubin level, lipid
profile and liver MDA were significantly elevated and the antioxidant status in
liver NP-SH and TP contents were declined in animals treated with CCl (4) alone.
Pretreatment with alfalfa and silymarin for three weeks prior to the
administration of CCl (4) significantly prevented the increase in the serum
levels of hepatic marker, LDL, VLDL levels enzymes and reduced oxidative stress
indicated by elevated NP-SH and TP concentration. The histopathological
examination of the livers also showed that the alfalfa extract reduced the
incidence of liver lesions induced by CCl (4). The in vitro antioxidant
assessment of alfalfa extract on DPPH and carotene-linoleic assays demonstrated a
moderate antioxidant potential. Results suggest that the alfalfa extract
possesses hepatoprotective and antioxidative stress properties possibly through
its antioxidant phytochemical constituents and substantiates its use in various
liver disorders as a hepatoprotector.
DOI: 10.1142/S0192415X12500589
981. J Bacteriol. 2012 Sep;194(18):5044-53. doi: 10.1128/JB.00982-12. Epub 2012 Jul

13.
Inability to catabolize galactose leads to increased ability to compete for

nodule occupancy in Sinorhizobium meliloti.
Geddes BA(1), Oresnik IJ.
Author information:
Canada.
A mutant unable to utilize galactose was isolated in Sinorhizobium meliloti

strain Rm1021. The mutation was found to be in a gene annotated dgoK1, a putative
2-keto-3-deoxygalactonokinase. The genetic region was isolated on a complementing
cosmid and subsequently characterized. Based on genetic and bioinformatic
evidence, the locus encodes all five enzymes (galD, dgoK, dgoA, SMc00883, and
ilvD1) involved in the De Ley-Doudoroff pathway for galactose catabolism.
Although all five genes are present, genetic analysis suggests that the
galactonase (SMc00883) and the dehydratase (ilvD1) are dispensable with respect
to the ability to catabolize galactose. In addition, we show that the transport
of galactose is partially facilitated by the arabinose transporter (AraABC) and
that both glucose and galactose compete with arabinose for transport.
Quantitative reverse transcription-PCR (qRT-PCR) data show that in a dgoK
background, the galactose locus is constitutively expressed, and the induction of
the ara locus seems to be enhanced. Assays of competition for nodule occupancy
show that the inability to catabolize galactose is correlated with an increased
ability to compete for nodule occupancy.
DOI: 10.1128/JB.00982-12
PMCID: PMC3430339

Epub
2012 Jun 28.
Effect of elevated CO₂ and temperature on the oxidative stress response to

drought in Lolium perenne L. and Medicago sativa L.
Farfan-Vignolo ER(1), Asard H.
Author information:
(1)Laboratory for Molecular Plant Physiology and Biotechnology, Department of
Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium.
Studies addressing the combined impact of multiple climate factors on plant

abiotic stress responses are still scarce. We investigated physiological and
molecular (antioxidant), responses to water deficit, in grassland-model species,
Lolium perenne L. and Medicago lupulina L., under future climate conditions, i.e.
elevated CO₂ (+CO₂, +375 ppm) and elevated temperature (+T, +3 °C). Elevated CO₂,
but not warming, significantly increased biomass (gDW) in L. perenne, but not in
M. lupulina. Photosynthesis (A(sat)) and stomatal conductance (g(s)), were
differently affected by climate in each species, L. perenne generally being more
sensitive. Elevated CO₂ increased lipid peroxidation levels in M. lupulina, but
not in L. perenne, and had no effect on protein oxidation and little effect on
antioxidant levels. Drought stress caused severe inhibition in biomass and
photosynthesis, most severely in L. perenne, and strongly increased oxidative
damage. Elevated CO₂ protected against the drought-induced damage. Decreased
activities of APX and POX may indicate lower levels of oxidative challenge
(relaxation) at the level of H₂O₂ production. Polyphenols, tocopherols and
antioxidant capacity, increased under drought stress, in all climate conditions.
Elevated CO₂, increased reduced ascorbate (ASC) and reduced glutathione (GSH),
and their redox status, in both species, although to different levels. Changes in
activities of key ASC/GSH cycle enzymes, under stress and climate treatments,
showed weak correlations with ASC and GSH levels, indicating the complexity of
this network. Together this work supports the idea that redox changes are
involved in responses to climate changes, in the absence and presence of
water-deficit stress.
DOI: 10.1016/j.plaphy.2012.06.014
Evaluation of cytotoxicity of 3-mercaptopropionic acid-modified quantum dots on

Medicago sativa cells and tissues.
Santos AR(1), Miguel AS, Fevereiro P, Oliva A.
Author information:
(1)Diagnostic, Instituto de Tecnologia Química e Biológica, Universidade Nova de
Lisboa, Oeiras, Portugal. anaraqssantos@gmail.com
Like most of the new technologies designed to interact with biological systems,
the applications of -nanomaterials needs a proper assessment for their potential
impacts. It is only through addressing the issues raised by toxicological studies
that nanotechnology will be able to acquire its full potential. Here, we describe
the detailed protocols to study the responses of plant cells to their exposure to
nanoparticles, including viability, oxidative stress detection, and reactive
oxygen species enzymatic detoxification, as well as particle uptake.
DOI: 10.1007/978-1-61779-953-2_36
984. Theor Appl Genet. 2012 Nov;125(7):1517-23. doi: 10.1007/s00122-012-1930-7.

Epub
2012 Jul 13.
Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in
Medicago sativa.
Dudas B(1), Jenes B, Kiss GB, Maliga P.
Author information:
(1)Agricultural Biotechnology Center, Szent-Györgyi Albert u. 4, 2100, Gödöllő,
Hungary. dudasb@abc.hu
We report here the isolation of spectinomycin-resistant mutants in cultured cells

of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured
alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S
plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were
identified by their ability to form green shoots on plant regeneration medium
containing selective spectinomycin concentrations in the range of 25-50 mg/l.
Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is
due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants
transferred to the greenhouse developed normally and produced
spectinomycin-resistant seed progeny. In light of their absence in soybean, a
related leguminous plant, the isolation of spectinomycin-resistant mutants in M.
sativa was unexpected. The new mutations are useful for the study of plastid
inheritance, as demonstrated by detection of predominantly paternal plastid
inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective
markers in plastid transformation vectors to obtain cisgenic plants.
DOI: 10.1007/s00122-012-1930-7
985. J Sci Food Agric. 2013 Jan;93(2):219-26. doi: 10.1002/jsfa.5781. Epub 2012 Jul
6.
Efficacy of various naturally occurring caffeic acid derivatives in preventing

post-harvest protein losses in forages.
Sullivan ML(1), Zeller WE.
Author information:
(1)US Dairy Forage Research Center, ARS, USDA, Madison, WI 53706, USA.
michael.sullivan@ars.usda.gov
BACKGROUND: In red clover, oxidation of endogenous o-diphenols by polyphenol

oxidase (PPO) inhibits post-harvest proteolyis. This system is transferable to
alfalfa by providing PPO (via a transgene) and o-diphenol PPO substrates (via
exogenous application). To exploit the PPO system for protein protection, it
would be advantageous to produce PPO substrates in alfalfa, which lacks them. We
assessed the extent of PPO-mediated proteolytic inhibition by phenolic compounds,
especially those whose biosynthesis could be engineered into alfalfa.
RESULTS: Tested compounds included o-diphenols (caffeic acid, phaselic acid,
chlorogenic acid, clovamide) and monophenols (p-coumaric acid, p-coumaroyl-malic
acid). In the presence of PPO, 2 mmol o-diphenol g⁻¹ protein reduced 24 h
proteolysis 68-87% (P < 0.001) and as little as 0.25 mmol g⁻¹ protein still
decreased 24 h proteolysis 43-60% (P < 0.001). At high concentrations, clovamide
inhibited 24 h proteolysis 50% (P < 0.001) in the absence of PPO, likely due to
non-PPO oxidation. Monophenol p-coumaric acid did not inhibit 24 h proteolyis,
although high levels of its malate ester did exhibit PPO- and oxygen-independent
inhibition (37%, P < 0.001).
CONCLUSIONS: For PPO-mediated proteolytic inhibition, pathways for both phaselic
acid and chlorogenic acid may be good targets for engineering into alfalfa.
Clovamide may be useful for inhibiting proteolysis without PPO.
Published 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/jsfa.5781
986. Plant Cell Environ. 2013 Feb;36(2):288-99. doi: 10.1111/j.1365-

3040.2012.02573.x.
Epub 2012 Aug 1.
Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced

myo-inositol phosphate synthase that confers multiple resistances to abiotic
stresses.
Tan J(1), Wang C, Xiang B, Han R, Guo Z.
Author information:
Agro-bioresources, South China Agricultural University, Guangzhou 510642, China.
myo-Inositol phosphate synthase (MIPS) is the key enzyme of myo-inositol

synthesis, which is a central molecule required for cell metabolism and plant
growth as a precursor to a large variety of compounds. A full-length fragment of
MfMIPS1 cDNA was cloned from Medicago falcata that is more cold-tolerant than
Medicago sativa. While MfMIPS1 transcript was induced in response to cold,
dehydration and salt stress, MIPS transcript and myo-inositol were maintained
longer and at a higher level in M. falcata than in M. sativa during cold
acclimation at 5 °C. MfMIPS1 transcript was induced by hydrogen peroxide (H(2)
O(2)) and nitric oxide (NO), but was not responsive to abscisic acid (ABA).
Pharmacological experiments revealed that H(2) O(2) and NO are involved in the
regulation of MfMIPS1 expression by cold and dehydration, but not by salt.
Overexpression of MfMIPS1 in tobacco increased the MIPS activity and levels of
myo-inositol, galactinol and raffinose, resulting in enhanced resistance to
chilling, drought and salt stresses in transgenic tobacco plants. It is suggested
that MfMIPS1 is induced by diverse environmental factors and confers resistance
to various abiotic stresses.
DOI: 10.1111/j.1365-3040.2012.02573.x
987. PLoS One. 2012;7(6):e39440. doi: 10.1371/journal.pone.0039440. Epub 2012 Jun

29.
Modelling the dynamics of feral alfalfa populations and its management

implications.
Bagavathiannan MV(1), Begg GS, Gulden RH, Van Acker RC.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, Manitoba
Canada. muthu@uark.edu
BACKGROUND: Feral populations of cultivated crops can pose challenges to novel

trait confinement within agricultural landscapes. Simulation models can be
helpful in investigating the underlying dynamics of feral populations and
determining suitable management options.
METHODOLOGY/PRINCIPAL FINDINGS: We developed a stage-structured matrix population
model for roadside feral alfalfa populations occurring in southern Manitoba,
Canada. The model accounted for the existence of density-dependence and
recruitment subsidy in feral populations. We used the model to investigate the
long-term dynamics of feral alfalfa populations, and to evaluate the
effectiveness of simulated management strategies such as herbicide application
and mowing in controlling feral alfalfa. Results suggest that alfalfa populations
occurring in roadside habitats can be persistent and less likely to go extinct
under current roadverge management scenarios. Management attempts focused on
controlling adult plants alone can be counterproductive due to the presence of
density-dependent effects. Targeted herbicide application, which can achieve
complete control of seedlings, rosettes and established plants, will be an
effective strategy, but the seedbank population may contribute to new recruits.
In regions where roadside mowing is regularly practiced, devising a timely mowing
strategy (early- to mid-August for southern Manitoba), one that can totally
prevent seed production, will be a feasible option for managing feral alfalfa
populations.
CONCLUSIONS/SIGNIFICANCE: Feral alfalfa populations can be persistent in roadside
habitats. Timely mowing or regular targeted herbicide application will be
effective in managing feral alfalfa populations and limit
feral-population-mediated gene flow in alfalfa. However, in the context of novel
trait confinement, the extent to which feral alfalfa populations need to be
managed will be dictated by the tolerance levels established by specific
production systems for specific traits. The modelling framework outlined in this
paper could be applied to other perennial herbaceous plants with similar
life-history characteristics.
PMCID: PMC3387144

Jun 17.
Fat spectro-colorimetric characteristics of lambs switched from a low to a high

dietary carotenoid level for various durations before slaughter.
de Oliveira L(1), Carvalho PC, Prache S.

Author information:
(1)Institut National de la Recherche Agronomique, INRA, UR1213 Herbivores, Site
de Theix, F-63122 Saint-Genès-Champanelle, France.
This study investigated the changes in fat reflectance spectrum characteristics

and color in lambs switched from a low to a high dietary carotenoid level for
various durations before slaughter. Six treatments, feeding a high dietary
carotenoid level for 0, 15, 30, 45, 60 or 75 days before slaughter, were compared
in individually indoor penned lambs. Each treatment used 10 Romane lambs; feeding
management ensured similar growth pattern and carcass weight for all the
treatment groups. There was a change in reflectance spectrum characteristics and
yellowness of subcutaneous fat as early as 15 days after the switch. Mean
concentration of carotenoid pigments and yellowness of subcutaneous fat increased
linearly with the duration of the high dietary carotenoid level. In perirenal
fat, the change in reflectance spectrum characteristics was observed as early as
15 days after the switch, but the response to the duration of the high dietary
carotenoid level was curvilinear.
DOI: 10.1016/j.meatsci.2012.06.012
989. Soil Biol Biochem. 2012 Jul;50(6):96-107.
Effects of habitat age and plant species on predatory mites (Acari, Mesostigmata)
in grassy arable fallows in Eastern Austria.
Wissuwa J(1), Salamon JA, Frank T.
Author information:
(1)University of Natural Resources and Life Sciences, Department of Integrative
Biology and Biodiversity Research, Institute of Zoology, Gregor Mendel Straße 33,
Density, diversity and assemblage structure of Mesostigmata (cohorts Gamasina and

Uropodina) were investigated in nine grassy arable fallows according to a
factorial design with age class (2-3, 6-8, 12-15 years) and plant species
(legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) as
factors. The response of Mesostigmata to habitat age and plant species was
explored because this group belongs to the dominant acarine predators playing a
crucial role in soil food webs and being important as biological control agents.
To our knowledge, this combination of factors has never been studied before for
Mesostigmata. A further rarely applied aspect of the present study is the
micro-scale approach investigating the Mesostigmata assemblage of the soil
associated with single plants. Four plots were randomly chosen at each fallow in
May 2008. At each plot plant roots and the adjacent soil of five randomly
selected plant individuals per plant species were dug out with steel cylinders
for heat extraction of soil fauna and measurement of environmental parameters. In
total, 83 mite taxa were identified, with 50 taxa being new to Austria. GLM
analysis revealed a significant effect of plant species on mite density, with
significantly more mites in B. sterilis than in T. officinale samples, and
M. sativa samples being intermediate. This was in contrast to the assumption that
the mite density is highest in M. sativa samples due to the propagation of plant
quality effects to higher trophic levels. These results were probably caused by a
higher amount of fine roots in grass samples leading to high densities of
Collembola, which are preferred prey of predatory mites. Mite density did not
significantly differ between the three age classes. A canonical analysis of
principal coordinates (CAP) showed that the mite assemblage exhibited a weak yet
significant separation between plant species, and a highly significant separation
between age classes. Accordingly, different mite assemblages were found for the
three age classes, while only few mite species were clearly associated with a
single plant species. Finally, canonical correspondence analysis (CCA) revealed
that the mite assemblage was best explained by soil organic carbon, total density
of Collembola and water content.
DOI: 10.1016/j.soilbio.2012.02.025
PMCID: PMC3365242
PMID: 22761538
990. Phytochem Anal. 2013 Jan-Feb;24(1):47-52. doi: 10.1002/pca.2379. Epub 2012 Jun
29.
Free radical scavenging activities of polyphenolic compounds isolated from

Medicago sativa and Medicago truncatula assessed by means of thin-layer
chromatography DPPH˙ rapid test.
Cieśla Ł(1), Kowalska I, Oleszek W, Stochmal A.
Author information:
(1)Department of Biochemistry and Crop Quality, Institute of Soil Science and
Plant Cultivation-State Research Institute, 8 Czartoryskich Street, 24-100
Puławy, Poland. lukecarpenter@poczta.onet.pl
INTRODUCTION: The structure of polyphenolic compounds influences their

anti-oxidant potential. Finding a simple, rapid and reliable analytical method to
study the structure-activity relationships for numerous samples is challenging.
OBJECTIVE: To develop a simple thin-layer
chromatography-2,2-diphenyl-1-picrylhdrazyl (TLC-DPPH˙) protocol with image
processing to study the influence of the structure of polyphenols on observed
direct anti-oxidant properties.
METHODOLOGY: First, compounds exhibiting free radical scavenging activities were
chosen from among the isolated compounds with the application of a rapid TLC
dot-blot test. The active ones were further chromatographed on silica gel plates
using the mobile phase: acetonitrile:water:chloroform:formic acid (60:15:10:5,
v/v/v/v). Subsequently the plates were stained with DPPH˙ methanolic solution. An
improved image processing protocol was used to quantitatively measure the
polyphenols' activity.
RESULTS: The application of a properly optimised chromatographic system enabled
separation of the investigated compounds from dimethyl sulphoxide (DMSO) that
influences the results of an anti-oxidant test. New solutions enabling better
data processing are proposed. It has been discovered that acylation of flavonoid
glycosides with hydroxycinnamic acids increases their direct anti-oxidant
properties. Some of the analysed glycosides acylated with ferulic acid molecule
were found to be the most potent free radical scavengers from among those
analysed. The amount of sugar moieties as well as their type also influenced the
observed activity.
CONCLUSION: A simple, rapid and reliable TLC-DPPH˙ test with image processing has
been developed enabling comparison of free radical scavenging activity of plant
polyphenols. The influence of different structural features on the observed
activity was measured successfully.
Copyright © 2012 John Wiley & Sons, Ltd.
DOI: 10.1002/pca.2379
991. Acta Sci Pol Technol Aliment. 2012 Jul-Sep;11(3):303-10.
Chemical composition of lucerne leaf extract (EFL) and its applications as a

phytobiotic in human nutrition.
Gaweł E(1).
Author information:
(1)Department of Forage Crop Production, Institute of Soil Science and Plant
Cultivation in Puławy - NRI, Czartoryskich 8, 24-100 Puławy, Poland.
Eliza.Gawel@iung.pulawy.pl
Lucerne, a valuable plant grown mainly for animal feed, is rich in protein,
minerals (Ca, Cu, Fe, Mg, Mn, P, Zn, Si), vitamins (A, B, C, D, E, K, U),
phytochemical substances (carotene, chlorophyll, coumarins, isoflavones,
alkaloids, saponins), contains secondary metabolites of plants (phytoestrogens:
isoflavones and coumestrol), and antinutritional components (phytates,
L-canavanine, saponins). It may be used as a dietary supplement in human
nutrition. The proteins found in lucerne are comprised of numerous exogenous and
semi-exogenous amino acids which are desirable for human body. Extract from the
leaves of alfalfa (EFL - l'Extrait Foliaire de Luzerne) practically does not
contain mycotoxins and pesticide pollutants. It is a completely natural product,
safe even in a long-term supplementation. Extract from the leaves of alfalfa has
a positive, multidirectional impact on the human body. It increases the level of
estrogen, prevents atherosclerosis, helps blood circulation and strenghtens
immunity, protects against the development of dangerous diseases of the digestive
tract, combats anemia and many other health ailments. The results of preclinical
studies indicate that alfalfa leaf extract enriched with vitamin C (EFL) can be a
dietary supplement supporting the human body in fighting malnutrition, ischemic
diseases, and various disorders of digestive tract. It also strengthens and
enhances immunity.
992. J Anim Physiol Anim Nutr (Berl). 2013 Aug;97(4):773-84. doi:

10.1111/j.1439-0396.2012.01318.x. Epub 2012 Jun 28.
Assessment of in vitro digestibility and fermentation parameters of alfalfa

hay-based diet following direct incorporation of fenugreek seed (Trigonella
foenum) and asparagus root (Asparagus officinalis).
Naseri V(1), Hozhabri F, Kafilzadeh F.
Author information:
(1)Animal Science Department, Razi University, Kermanshah, Iran.
This study was completed to evaluate the effect of fenugreek seed (Trigonella
foenum; FS) and asparagus root (Asparagus officinalis; AR) on in vitro nutrient
digestibility and fermentation patterns. Different levels [0%, 5%, 10%, 15% and
20% of dry matter (DM)] of the medicinal plants were included using alfalfa hay
(AH) as a basal substrate at different incubation times (12, 18, 24 and 48 h).
Total phenolic components of AH, FS and AR were 5.9, 10 and 8.3 g/kg DM, whereas
total tannins were 0.4, 3.8 and 1.5 g/kg DM, respectively. Corresponding values
for saponins were 10.4, 27.3 and 40.3 g/kg DM. Fenugreek seed increased (p<0.05)
in vitro organic matter (OM) digestibility at different incubation times and
decreased (p<0.05) crude protein (CP) digestibility at 18 and 24 h of incubation.
Asparagus root also increased (p<0.05) in vitro OM digestibility and decreased
(p<0.05) CP digestibility at different incubation times. Neutral detergent fibre
digestibility was increased (p<0.05) by the addition of AR or FS at low levels,
but decreased (p<0.05) noticeably by increasing level of two plants in the basal
substrate. Ammonia-N concentration was markedly reduced (p<0.05) by the addition
of AR at different incubation times, and this reduction was accompanied by the
decrease in CP digestibility. True DM degradability and partitioning factor
(ratio of substrate DM truly degraded to gas volume produced at different times
of incubation) were increased, and total volatile fatty acid concentration and
total gas production were decreased (p<0.05) with the addition of FS (at 10% and
15% DM levels) or AR (at 5%, 10% and 15% DM levels) at different incubation
times. Results suggest that FS and AR may have potential as feed additives to
increase the efficiency of nutrients' utilization, particularly of nitrogen in
ruminant diets.
© 2012 Blackwell Verlag GmbH.
DOI: 10.1111/j.1439-0396.2012.01318.x
993. Environ Entomol. 2012 Jun;41(3):706-13. doi: 10.1603/EN11325.
Comparisons of developmental and reproductive biology between parthenogenetic and

sexual Echinothrips americanus (Thysanoptera: Thripidae).
Li XW(1), Zhang XC, Jiang HX, Feng JN.
Author information:
(1)Key Laboratory of Plant Protection Resources and Pest Management, Ministry of
Education, Northwest A&F University, Yangling, Shaanxi, China.
Echinothrips americanus Morgan, an invasive pest on various ornamentals and

greenhouse crops, was introduced into mainland China recently, posing a potential
threat to ornamentals and greenhouse crops. It exhibits two different
reproductive modes: arrhenotokous parthenogenesis and sexual reproduction.
Laboratory studies were conducted to compare the developmental and reproductive
biology of E. americanus in these two reproductive modes. Results showed that the
oviposition period, and longevity of female adults using sexual reproduction were
longer than those using parthenogenesis. Furthermore, sexual female adults had
higher fecundity and survival rates. However, no significant differences were
found among immature stages in the durations of first and second instars,
prepupae, and pupae between the two reproductive modes, with the exception of the
duration of the egg stadium. The survival rates for eggs and first and second
instars were higher in sexual E. americanus whereas there were no survival
differences for prepupae and pupae. These results provide valuable insights into
the mechanisms of parthenogenesis and sex determination in Thysanoptera.
DOI: 10.1603/EN11325
994. Mol Biol Rep. 2012 Aug;39(8):8559-69. doi: 10.1007/s11033-012-1712-y. Epub

2012
Jun 14.
Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger
protein promoter MsZPP.
Li Y(1), Sun Y, Yang Q, Kang J, Zhang T, Gruber MY, Fang F.
Author information:
(1)Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2
West Yuanmingyuan Road, Haidian, Beijing 100193, People's Republic of China.
caasliyan@yahoo.com.cn
A 1272 bp upstream sequence of MsZFN gene was cloned from alfalfa, which was
designed as MsZPP (Genbank accession number: FJ 161979.2) using an
adaptor-mediated genome walking method. A sole transcription start site was
located 69 bp upstream of the translation start site. Its pattern of expression
included roots, stem vascular tissues, floral reproductive organs, and leaves,
but the promoter did not express in seeds, petals or sepals. Transcription levels
can be stimulated by dark, MeJA, and IAA. However, GUS fusion activities had no
change by treatments of GA, ABA, drought and high salt for 3 days. Deletion
analysis revealed that all sections of the promoter can drive gus gene expression
in the root, stem, leaves and floral reproductive organs; however, only fragments
longer than the -460 bp promoter can stimulate strong gus gene expression in
these organs. In addition, the -460 bp promoter fragment can drive gus expression
not only in the vascular tissue, but also in leaf guard cells. The results
suggest that the promoter MsZPP plays roles in the regulation of transgene
expression, particularly due to its darkness, MeJA, and IAA responsiveness.
DOI: 10.1007/s11033-012-1712-y
995. Environ Eng Sci. 2012 Jun;29(6):494-501.
Treatment and Remediation of Petroleum-Contaminated Soils Using Selective

Ornamental Plants.
Liu R, Jadeja RN, Zhou Q, Liu Z.
Pot-culture experiments were carried out to assess the phytoremediation potential

of 14 ornamental plants in weathered petroleum-contaminated soil, which was
collected in the Shengli Oil Field, one of the biggest oil fields in China, by
examining their impact on the degradation potential of total petroleum
hydrocarbons (TPHs) and its composition. Results showed Gaillardia aristata,
Echinacea purpurea, Fawn (Festuca arundinacea Schreb), Fire Phoenix (a combined
F. arundinacea), and Medicago sativa L. could effectively reduce TPHs and its
composition in 10,000 mg kg(-1) TPH-contaminated soil. After a 30-day pot-culture
experiment, the removal rates were 37.16%, 46.74%, 49.42%, 41.00%, and 37.93%,
respectively, significantly higher than that in the control (only 12.93%).
Removal rates of TPH composition including saturated hydrocarbon, aromatic
hydrocarbon, asphaltene, and polar compound reached 39.41%, 38.47%, 45.11%,
42.92%, and 37.52%, respectively, also higher than that in the control (only
6.90%). Further, the total biomass did not significantly decrease for all plants
tested in 10,000 mg kg(-1) TPH-contaminated soil. Fourier transform infrared
spectroscopy confirmed the presence of oil in the plant tissues. These results
suggested that the typical ornamental species including G. aristata, E. purpurea,
Fawn, Fire Phoenix, and M. sativa can be adopted in phytoremediation of
oil-contaminated soil.
DOI: 10.1089/ees.2010.0490
PMCID: PMC3363014
PMID: 22693416
996. Plant Sci. 2012 Aug;191-192:16-23. doi: 10.1016/j.plantsci.2012.04.007. Epub
2012
Apr 24.
Effects of nitrogen source and water availability on stem carbohydrates and

cellulosic bioethanol traits of alfalfa plants.
Fiasconaro ML(1), Gogorcena Y, Muñoz F, Andueza D, Sánchez-Díaz M, Antolín MC.
Author information:
(1)Dpto. Biología Vegetal, Sección Biología Vegetal, Unidad Asociada al
CSIC-EEAD, Zaragoza, ICVV, Logroño, Facultades de Ciencias y Farmacia,
Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain.
Symbiotic association of legumes with rhizobia frequently results in higher

photosynthesis and soluble carbohydrates in comparison with nitrate-fed plants,
which might improve its potential for biomass conversion into bioethanol. A
greenhouse experiment was conducted to examine the effects of nitrogen source and
water availability on stem characteristics and on relationships between
carbohydrates, phenolic metabolism activity and cell wall composition in alfalfa
(Medicago sativa L. cv. Aragón). The experiment included three treatments: (1)
plants fed with ammonium nitrate (AN); (2) plants inoculated with rhizobia (R);
and (3) plants inoculated with rhizobia and amended with sewage sludge (RS). Two
levels of irrigation were imposed: (1) well-watered and (2) drought stress. Under
well-watered conditions, nitrogen-fixing plants have increased photosynthesis and
stem fermentable carbohydrate concentrations, which result in higher potential
for biomass conversion to bioethanol than in AN plants. The latter had higher
lignin due to enhanced activities of phenolic metabolism-related enzymes. Under
drought conditions, the potential for bioethanol conversion decreased to a
similar level in all treatments. Drought-stressed nitrogen-fixing plants have
high concentrations of fermentable carbohydrates and cell wall cellulose, but
ammonium nitrate-fed plants produced higher plant and stem biomass, which might
compensate the decreasing stem carbohydrates and cellulose concentrations.
DOI: 10.1016/j.plantsci.2012.04.007
997. Planta. 2012 Sep;236(3):901-18. doi: 10.1007/s00425-012-1670-6. Epub 2012 Jun

8.
An integrated approach to demonstrating the ANR pathway of proanthocyanidin

biosynthesis in plants.
Peng QZ(1), Zhu Y, Liu Z, Du C, Li KG, Xie DY.
Author information:
(1)Hunan Provincial Key Laboratory of Plant Resources Conservation and
Utilization, College of Biology and Environmental Sciences, Jishou University,
No. 120 Ren Min Nan Lu, Jishou City, 416000, Hunan Province, People's Republic of
China.
Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are

important to plant adaptation in extreme environmental conditions. The
characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin
reductase (LAR) has demonstrated the different biogenesis of four
stereo-configurations of flavan-3-ols. It is important to understand whether ANR
and the ANR pathway widely occur in the plant kingdom. Here, we report an
integrated approach to demonstrate the ANR pathway in plants. This includes
different methods to extract native ANR from different tissues of eight
angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa,
Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and
Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general
enzymatic analysis approach to demonstrate the ANR activity, high-performance
liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and
other flavan-3-ol molecules, and phytochemical analysis of PAs. Results
demonstrate that in addition to leaves of M. sativa, tissues of other eight
plants contain an active ANR pathway. Particularly, the leaves, flowers and pods
of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are
demonstrated to express an active ANR pathway. This finding suggests that the ANR
pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST
analysis reveals that ANR homologs have been sequenced in plants from both
gymnosperms and angiosperms. These data show that the ANR pathway to PA
biosynthesis occurs in both seed and seedless vascular plants.
DOI: 10.1007/s00425-012-1670-6
998. Sheng Wu Gong Cheng Xue Bao. 2012 Feb;28(2):203-13.
[Agrobacterium-mediated genetic transformation of secondary somatic embryos in

alfalfa].
Liu W(1), Duan Q, Liu J, Sun Y.
Author information:
(1)Department of Life Science, Northwest A & F University, Yangling 712100,
Shaanxi, China. lwt1635@yahoo.com.cn
We describe a genetic transformation method of secondary somatic embryogenesis in

alfalfa through cotyledon-stage somatic embryos of alfalfa infected by
Agrobacterium strain GV3101. The Agrobacterium strain GV3101 contained binary
vector pCAMBIA2301 that had gus gene as reporter and npt II gene as selectable
marker. The infected primary embryos were induced through series of medium under
75 mg/L kanamycin selection. We obtained the transgenic alfalfa plants. Then, GUS
expression in different tissue of transgenic alfalfa was tested by GUS
histochemical analysis. Further, the stable integration and transformation
efficiency were tested by polymerase chain reaction and Southern blotting
hybridization. The result showed that GUS expression was different in different
organs of transgenic alfalfa; the copy number of integrated npt II gene was from
1 to 4; the transformation efficiency via primary somatic embryogenesis was
65.82%.
999. Yi Chuan. 2012 May;34(5):621-34.
[Expression analysis of the Medicago truncatula floral specific expression

genes].
Ma LC(1), Wang YR, Liu ZP.
Author information:
(1)College of Pastoral Agriculture Science and Technology, Lanzhou University,
State Key Laboratory of Grassland Agro-Ecosystems, Lanzhou 730020, China.
ma-lichao@163.com
The expression of genes specific to floral organ is important for the floral
organ formation and development in Medicago truncatula. Screening of the genes
specifically expressed in M. truncatula flowers and comparing the expression
patterns of their orthologous homologous genes among different model plants can
provide novel insights into the functions of these genes in controlling the
floral organ development in M. truncatula. According to the expression profile
data of PISTILLATA (PI), we screened 97 genes specifically expressed in M.
truncatula floral organs (ratio≥10 and Z≥7.9). Their homolog genes were also
identified in Arabidopsis thaliana, soybean (Glycine max L.), Lotus japonicus,
and rice (Oryza sativa L.). The results of comparing the gene expression levels,
the gene expression patterns, and the gene functions among these species
indicated that the expression variation of the orthologous homolog genes was
small in the kindred species and was great in distant species. Furthermore, we
compared the cis-acting regulatory elements of the genes, which had large
expression variation among different plants. These results suggest that the great
discrepancy of the orthologous homolog gene expression caused by the different
character of cis-element in the promoter region.
1000. J Sci Food Agric. 2013 Jan 15;93(1):93-9. doi: 10.1002/jsfa.5735. Epub 2012
Jun
1.
Chemical composition and sensory quality of bovine milk as affected by type of

forage and proportion of concentrate in the feed ration.
Larsen MK(1), Kidmose U, Kristensen T, Beaumont P, Mortensen G.
Author information:
(1)Aarhus University, Department of Food Science, Blichers Allé 20, DK-8830
Tjele, Denmark.
BACKGROUND: The objective of this study was to investigate how some small changes
in the forage content of maize and lucerne silage and in the ration between
forage and concentrate in the diet of dairy cows affect milk quality. Milk
quality was assessed by quantitative descriptive sensory analysis and by analysis
of tocopherols and carotenoids as well as fatty acid composition.
RESULTS: Changing the ratio between maize silage and lucerne silage from 5:1 to
2:1 increased milk fat content of carotenoids (23-27%) and C18:3 n3 (15%), and
reduced stale aroma and creamy flavour. Increasing the proportion of concentrates
in the feed ration from 0.2 to 0.4 increased energy corrected milk yield (26%),
reduced fat content (-10%), increased C18 fatty acids (8-62%) and reduced C16
(-20%) content in milk fat. In addition, this milk type was described by the
sensory panel as less oily, less saturated and less yellow. The changes in milk
composition were related to differences in feed composition.
CONCLUSION: The study revealed the potential to produce milk with a distinct
composition and sensory quality based on even small changes in the feed
composition that are straightforward to implement by farmers.
Copyright © 2012 Society of Chemical Industry.
DOI: 10.1002/jsfa.5735
1001. Plant Cell Rep. 2012 Sep;31(9):1737-46. doi: 10.1007/s00299-012-1287-z. Epub

2012
May 30.
An alfalfa (Medicago sativa L.) ethylene response factor gene, MsERF11, enhances
salt tolerance in transgenic Arabidopsis.
Chen T(1), Yang Q, Zhang X, Ding W, Gruber M.
Author information:
Beijing, 100193, People's Republic of China. chentingting8701@163.com
A novel orthologue of ethylene response factor gene, MsERF11, was isolated from
alfalfa in this study. It has an open reading frame of 807 bp, encoding a
predicted polypeptide of 268 amino acids. Sequence similarity analysis clearly
suggested that MsERF11 encoded an ethylene response factor protein. The results
of transient expression of MsERF11 in onion epidermal cells indicated that
MsERF11 is a nuclear protein. The expression pattern of MsERF11 gene was analyzed
by real-time quantitative PCR and a higher level of expression was observed in
leaves than was observed in roots, stems, flower buds and flowers. Furthermore,
the expression was induced by PEG6000, NaCl, Al2(SO4)3 and six different
hormones. Over-expressing MsERF11 resulted in enhanced tolerances to salt stress
in transgenic Arabidopsis plants. This research indicates that MsERF11 has the
potential to be used for improving crop's salt tolerance in areas where salinity
is a limiting factor for agricultural productivity.KEY MESSAGE: MsERF11 was
isolated from alfalfa. Its expression was induced by different abiotic stresses
and hormones. Over-expressing MsERF11 resulted in enhanced salt tolerance in
transgenic Arabidopsis plants.
DOI: 10.1007/s00299-012-1287-z
1002. Am Nat. 2012 Jun;179(6):805-10. doi: 10.1086/665647. Epub 2012 Apr 19.
Use of an exotic host plant affects mate choice in an insect herbivore.
Forister ML(1), Scholl CF.
Author information:
(1)Program in Ecology, Evolution, and Conservation Biology, Department of
Biology, University of Nevada, Reno, Nevada 89557, USA. mforister@unr.edu
The colonization of exotic plants by herbivorous insects has provided

opportunities for investigating causes and consequences of the evolution of niche
breadth. The butterfly Lycaeides melissa utilizes exotic alfalfa, Medicago
sativa, which is a relatively poor larval resource, and previous studies have
found that caterpillars that consume M. sativa develop into smaller and less
fecund adults. Here we investigate the effect of smaller female body size on male
mate preference, a previously unexplored consequence of novel host use. Smaller
females, which developed on the exotic host, were less likely to be visited by
males. This result was confirmed with a second set of choice tests involving
females reared on a single plant species, thus ruling out host-specific
confounding factors. We suggest that an effect on mate choice be considered part
of the complex suite of factors determining persistence of herbivorous insects
following colonization of new habitats or resources.
DOI: 10.1086/665647
1003. J Dairy Sci. 2012 Jun;95(6):3288-97. doi: 10.3168/jds.2011-5022.
Rates of particle size reduction and passage are faster for legume compared with
cool-season grass, resulting in lower rumen fill and less effective fiber.
Kammes KL(1), Allen MS.
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing, MI
48824-1225, USA.
Effects of forage family on rates of particle size reduction in, and passage
from, the rumen and the relationship of these effects with preliminary dry matter
intake (pDMI) were evaluated using 13 ruminally and duodenally cannulated
Holstein cows in a crossover design with a 14-d preliminary period and two 18-d
treatment periods. During the preliminary period, pDMI of individual cows ranged
from 19.6 to 29.5 kg/d (mean = 25.9 kg/d). Experimental treatments were diets
containing either a) alfalfa silage (AL) or b) orchardgrass silage (OG) as the
sole forage. Silages were chopped to 10-mm theoretical length of cut and
contained 42.3 and 58.2% neutral detergent fiber (NDF) for alfalfa and
orchardgrass, respectively. Both diets contained approximately 25% forage NDF and
30% total NDF. Feed, orts, rumen, and duodenal samples were wet sieved to
fractionate particles above (large) and below (small) 2.36 mm. Indigestible NDF
(iNDF) was used as a flow marker. Preliminary DMI, an index of nutrient demand,
was determined during the last 4 d of the preliminary period when cows were fed a
common diet and used as a covariate. Main effects of forage family and their
interaction with pDMI were tested by ANOVA. Approximately 75% of the NDF consumed
was large and 25% was small for both treatments, but cows fed AL consumed more
iNDF and less potentially digestible NDF (pdNDF) than cows fed OG. The AL diet
increased the reduction rate (large to small) compared with OG despite less
rumination per unit of forage NDF for AL than OG, suggesting alfalfa NDF was more
fragile than orchardgrass NDF. Over 55% of particles in the rumen were below 2.36
mm for AL and OG, indicating that particle size was not a limiting constraint to
passage. Passage rates (k(p)) of large iNDF and large pdNDF were similar for AL
and OG, but AL increased k(p) of large pdNDF and OG decreased it as pDMI
increased. The AL diet increased k(p) of small iNDF and small pdNDF compared with
OG, resulting in lower rumen fill for AL than OG. The k(p) of small iNDF and
small pdNDF were similar within forage family, suggesting buoyancy was not
limiting passage. The OG diet increased rumen pool size of large NDF compared
with AL, which likely retained small NDF, contributing to the slower k(p) of
small iNDF and small pdNDF observed for OG. Particle size reduction was a
prerequisite to ruminal passage but not a constraint. Selective retention of
small particles was less for alfalfa than orchardgrass, resulting in lower rumen
fill and less effective fiber.

DOI: 10.3168/jds.2011-5022
Nutrient demand interacts with forage family to affect digestion responses in

dairy cows.
Kammes KL(1), Allen MS.
Author information:
48824-1225, USA.
Effects of forage family on dry matter intake (DMI), milk production, ruminal
pool sizes, digestion and passage kinetics, and chewing activity and the
relationship of these effects with preliminary DMI (pDMI), an index of nutrient
demand, were evaluated using 13 ruminally and duodenally cannulated Holstein cows
in a crossover design with a 14-d preliminary period and two 18-d treatment
periods. During the preliminary period, pDMI of individual cows ranged from 19.6
to 29.5 kg/d (mean=25.9 kg/d) and 3.5% fat-corrected milk yield ranged from 24.3
to 60.3 kg/d (mean=42.1 kg/d). Experimental treatments were diets containing
either a) alfalfa silage (AL) or b) orchardgrass silage (OG) as the sole forage.
Alfalfa and orchardgrass contained 42.3 and 58.2% neutral detergent fiber (NDF)
and 22.5 and 11.4% crude protein, respectively. Forage:concentrate ratios were
60:40 and 43:57 for AL and OG, respectively; both diets contained approximately
25% forage NDF and 30% total NDF. Preliminary DMI was determined during the last
4 d of the preliminary period when cows were fed a common diet and used as a
covariate. Main effects of forage family and their interaction with pDMI were
tested by ANOVA. Forage family and its interaction with pDMI did not affect feed
intake, milk yield, or milk composition. The AL diet increased indigestible NDF
(iNDF) intake and decreased potentially digestible NDF (pdNDF) intake compared
with OG. The AL diet increased ruminal pH, digestion rates of pdNDF and starch,
and passage rates of pdNDF and iNDF compared with OG, which affected ruminal
digestibility. Passage rate of iNDF was related to pDMI; AL increased iNDF
passage rate and OG decreased it as pDMI increased. The AL diet decreased ruminal
pool sizes of pdNDF, starch, organic matter, dry matter, and rumen digesta wet
weight and volume compared with OG. The AL diet decreased ruminating time per
unit of forage NDF consumed compared with OG, indicating that alfalfa provided
less physically effective fiber than orchardgrass. The AL diet, but not OG,
increased ammonia N, nonammonia nonmicrobial N, and nonammonia N fluxes as pDMI
increased. Efficiency of microbial protein synthesis was positively related to
pdNDF passage rate for OG, but not AL. The faster rates of digestion and passage
for AL compared with OG decreased rumen pool size but did not increase feed
intake for cows consuming AL. Digestion responses to forage family were affected
by nutrient demand of cows.

DOI: 10.3168/jds.2011-5021
1005. BMC Microbiol. 2012 May 20;12:78. doi: 10.1186/1471-2180-12-78.
Exploring the plant-associated bacterial communities in Medicago sativa L.

Pini F(1), Frascella A, Santopolo L, Bazzicalupo M, Biondi EG, Scotti C, Mengoni
A.
Author information:
(1)Department of Evolutionary Biology, University of Florence, via Romana 17,
I-50125, Florence, Italy.
BACKGROUND: Plant-associated bacterial communities caught the attention of

several investigators which study the relationships between plants and soil and
the potential application of selected bacterial species in crop improvement and
protection. Medicago sativa L. is a legume crop of high economic importance as
forage in temperate areas and one of the most popular model plants for
investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging
to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its
importance, no studies have been carried out looking at the total bacterial
community associated with the plant. In this work we explored for the first time
the total bacterial community associated with M. sativa plants grown in mesocosms
conditions, looking at a wide taxonomic spectrum, from the class to the single
species (S. meliloti) level.
RESULTS: Results, obtained by using Terminal-Restriction Fragment Length
Polymorphism (T-RFLP) analysis, quantitative PCR and sequencing of 16 S rRNA gene
libraries, showed a high taxonomic diversity as well as a dominance by members of
the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the
families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant
tissues, while soil Alphaproteobacteria were represented by the families of
Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At
the single species level, we were able to detect the presence of S. meliloti
populations in aerial tissues, nodules and soil. An analysis of population
diversity on nodules and soil showed a relatively low sharing of haplotypes
(30-40%) between the two environments and between replicate mesocosms, suggesting
drift as main force shaping S. meliloti population at least in this system.
CONCLUSIONS: In this work we shed some light on the bacterial communities
associated with M. sativa plants, showing that Alphaproteobacteria may constitute
an important part of biodiversity in this system, which includes also the well
known symbiont S. meliloti. Interestingly, this last species was also found in
plant aerial part, by applying cultivation-independent protocols, and a genetic
diversity analysis suggested that population structure could be strongly
influenced by random drift.
DOI: 10.1186/1471-2180-12-78
PMCID: PMC3412730
Host association of Spodoptera frugiperda (Lepidoptera: Noctuidae) corn and rice

strains in Argentina, Brazil, and Paraguay.
Juárez ML(1), Murúa MG, García MG, Ontivero M, Vera MT, Vilardi JC, Groot AT,
Castagnaro AP, Gastaminza G, Willink E.
Author information:
(1)Estación Experimental Agroindustrial Obispo Colombres, Av. William Cross 3150,
Las Talitas (T4101XAC), Tucumán, Argentina. lau_zoo@yahoo.com.ar
Spodoptera frugiperda (J.E. Smith) is composed of two genetically distinct

strains, the so-called corn strain and the rice strain. Whether the two strains
differ in their host use is unclear, because laboratory experiments have not been
able to show consistent host performance or preference differences between them,
and field studies showed high rates of hybridization, as well as some degree
asymmetric host use. To determine the distribution of the two strains and their
association with host plants, we collected fall armyworm larvae from different
crops (corn, rice, alfalfa, and sorghum) and grasses in 15 different localities
over 4 yr in Argentina, Brazil, and Paraguay. The strain identity was analyzed
using two polymorphisms in the mitochondrial cytochrome oxidase subunit I gene.
We identified the corn and rice haplotypes and three types of populations were
characterized based on the frequencies of the individuals that belonged to any of
these haplotypes: in 44% of populations the corn haplotype predominated, in 44%
of populations the rice haplotype was the most frequent, and 11% of populations
showed both haplotypes at similar proportions. In total, eight populations (47%)
showed the expected pattern, two populations (12%) were polymorphic within the
same field, and seven populations (41%) showed the inverse pattern. Taken
together, there was no consistent pattern of host association between the two
sympatric genotypes and their respective host plants. This investigation supports
the need for additional studies to determine which other forces keep the
genotypes separate, and what is the degree of genetic differentiation between
these populations.
1007. J Food Sci. 2012 Jun;77(6):S240-6. doi: 10.1111/j.1750-3841.2012.02720.x.

Epub
2012 May 16.
Gas chromatographic-olfactometric aroma profile and quantitative analysis of

volatile carbonyls of grilled beef from different finishing feed systems.
Resconi VC(1), del Mar Campo M, Montossi F, Ferreira V, Sañudo C, Escudero A.
Author information:
(1)Faculty of Veterinary, Univ of Zaragoza, 50013 Zaragoza, Spain.
resconi@unizar.es
In this study, the important odor impact volatiles generated in the meat of
grilled beef loin muscle were characterized. Animals were finished in 4 different
diet systems: T₁, pasture (a mixture of Medicago sativa, Trifolium repens, and
Festuca arundinacea); T₂, pasture supplemented with cracked corn grain (offered
at 0.6% live weight, LW); T₃, pasture supplemented with cracked corn grain
(offered at 1.2% LW); and T₄, concentrate (pellets with 85% corn and 12.8%
sunflower, on a dry-matter basis) plus alfalfa hay (both ad libitum). Aroma
compounds were assessed by dynamic headspace-solid phase extraction (DHS-SPE) and
gas chromatographic-olfactometric (GC-O) analysis. Most odorants were carbonyl
compounds, some of them reaching high GC-O scores, especially 1-octen-3-one,
(E)-2-octenal, methional, and hexanal. A specific quantitative analysis of
ketones and aldehydes was conducted through their derivatization with
o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride directly on the
headspace trap and analyzed by GC-MS, with the purpose of studying the effect of
finishing diet systems. From the 23 carbonyl compounds quantified, 2 were
especially affected by the diet system; methional was higher in the treatment
based on concentrates, whereas (E,E)-2,4-heptadienal was higher in the treatment
based only in pastures. The results are discussed considering previous published
productive and quality traits.PRACTICAL APPLICATION: The knowledge of how
production factors, such as animal feeding, can affect the flavor of meat is of
significant interest toward in achieving a high-quality and differentiated
product. The development of more specific and efficient methodologies is
necessary to analyze meat aroma compounds, which would be used as routine
analysis, that is for product authentication. In the future, the use of this
analysis would allow producing and designing specific foods according to
different markets.
© 2012 Institute of Food Technologists®
DOI: 10.1111/j.1750-3841.2012.02720.x
1008. FEMS Microbiol Ecol. 2012 Oct;82(1):169-81. doi:

10.1111/j.1574-6941.2012.01414.x. Epub 2012 Jun 13.
Long-term in situ dynamics of the fungal communities in a multi-contaminated soil

are mainly driven by plants.
Thion C(1), Cébron A, Beguiristain T, Leyval C.
Author information:
(1)LIMOS, CNRS UMR 7137, Faculté des Sciences, Lorraine University,
Vandoeuvre-lès-Nancy, France.
The fungal communities of a multi-contaminated soil polluted by polycyclic

aromatic hydrocarbons and heavy metals (NM) were studied within a long-term in
situ experiment of natural attenuation assisted by plants. Three treatments were
monitored: bare soil (NM-BS), soil planted with alfalfa and inoculated with
mycorrhizal fungi (NM-Msm), and soil with spontaneous vegetation (NM-SV). The
same soil after thermal desorption (TD) was planted with alfalfa and inoculated
with mycorrhizal fungi (TD-Msm). Twice a year for 5 years, the fungal abundance
and the community structure were evaluated by real-time PCR and temporal
temperature gradient gel electrophoresis targeting 18S rRNA genes. The fungal
abundance increased over time and was higher in planted than in bare NM soil and
in TD than in NM soil. The Shannon diversity index (H') increased during the
first 2 years with the emergence of more than 30 ribotypes, but decreased after
3 years with the selection of a few competitive species, mostly Ascomycetes. H'
was higher under complex plant assemblage (NM-SV) than in the NM-BS plots but did
not differ between NM and TD soils planted with alfalfa. These results indicated
that even in a highly polluted soil, the plant cover was the main driver of the
fungal community structure.

DOI: 10.1111/j.1574-6941.2012.01414.x
A comparative genomics screen identifies a Sinorhizobium meliloti 1021 sodM-like

gene strongly expressed within host plant nodules.
Queiroux C(1), Washburn BK, Davis OM, Stewart J, Brewer TE, Lyons MR, Jones KM.
Author information:
(1)Department of Biological Science, Florida State University, Biology Unit I,
230A, Tallahassee, FL 32306-4370, USA.
BACKGROUND: We have used the genomic data in the Integrated Microbial Genomes
system of the Department of Energy's Joint Genome Institute to make predictions
about rhizobial open reading frames that play a role in nodulation of host
plants. The genomic data was screened by searching for ORFs conserved in
α-proteobacterial rhizobia, but not conserved in closely-related
non-nitrogen-fixing α-proteobacteria.
RESULTS: Using this approach, we identified many genes known to be involved in
nodulation or nitrogen fixation, as well as several new candidate genes. We
knocked out selected new genes and assayed for the presence of nodulation
phenotypes and/or nodule-specific expression. One of these genes, SMc00911, is
strongly expressed by bacterial cells within host plant nodules, but is expressed
minimally by free-living bacterial cells. A strain carrying an insertion mutation
in SMc00911 is not defective in the symbiosis with host plants, but in contrast
to expectations, this mutant strain is able to out-compete the S. meliloti 1021
wild type strain for nodule occupancy in co-inoculation experiments. The SMc00911
ORF is predicted to encode a "SodM-like" (superoxide dismutase-like) protein
containing a rhodanese sulfurtransferase domain at the N-terminus and a
chromate-resistance superfamily domain at the C-terminus. Several other ORFs
(SMb20360, SMc01562, SMc01266, SMc03964, and the SMc01424-22 operon) identified
in the screen are expressed at a moderate level by bacteria within nodules, but
not by free-living bacteria.
CONCLUSIONS: Based on the analysis of ORFs identified in this study, we conclude
that this comparative genomics approach can identify rhizobial genes involved in
the nitrogen-fixing symbiosis with host plants, although none of the newly
identified genes were found to be essential for this process.
DOI: 10.1186/1471-2180-12-74
PMCID: PMC3462710
1010. J Anim Sci. 2012 Sep;90(9):3162-73. doi: 10.2527/jas.2011-4347. Epub 2012 May
14.
Comparison of in vitro and in situ methods in evaluation of forage digestibility

in ruminants.
Krizsan SJ(1), Nyholm L, Nousiainen J, Südekum KH, Huhtanen P.
Author information:
(1)Swedish University of Agricultural Sciences, Department of Agricultural
Research for Northern Sweden, S-901 83 Umeå, Sweden. sophie.krizsan@slu.se
The objective of this study was to compare the application of different in vitro
and in situ methods in empirical and mechanistic predictions of in vivo OM
digestibility (OMD) and their associations to near-infrared reflectance
spectroscopy spectra for a variety of forages. Apparent in vivo OMD of silages
made from alfalfa (n = 2), corn (n = 9), corn stover (n = 2), grass (n = 11),
whole crops of wheat and barley (n = 8) and red clover (n = 7), and fresh alfalfa
(n = 1), grass hays (n = 5), and wheat straws (n = 5) had previously been
determined in sheep. Concentrations of indigestible NDF (iNDF) in all forage
samples were determined by a 288-h ruminal in situ incubation. Gas production of
isolated forage NDF was measured by in vitro incubations for 72 h. In vitro
pepsin-cellulase OM solubility (OMS) of the forages was determined by a 2-step
gravimetric digestion method. Samples were also subjected to a 2-step
determination of in vitro OMD based on buffered rumen fluid and pepsin. Further,
rumen fluid digestible OM was determined from a single 96-h incubation at 38°C.
Digestibility of OM from the in situ and the in vitro incubations was calculated
according to published empirical equations, which were either forage specific or
general (1 equation for all forages) within method. Indigestible NDF was also
used in a mechanistic model to predict OMD. Predictions of OMD were evaluated by
residual analysis using the GLM procedure in SAS. In vitro OMS in a general
prediction equation of OMD did not display a significant forage-type effect on
the residuals (observed - predicted OMD; P = 0.10). Predictions of OMD within
forage types were consistent between iNDF and the 2-step in vitro method based on
rumen fluid. Root mean square error of OMD was least (0.032) when the prediction
was based on a general forage equation of OMS. However, regenerating a simple
regression for iNDF by omitting alfalfa and wheat straw reduced the root mean
square error of OMD to 0.025. Indigestible NDF in a general forage equation
predicted OMD without any bias (P ≥ 0.16), and root mean square error of
prediction was smallest among all methods when alfalfa and wheat straw samples
were excluded. Our study suggests that compared with the in vitro laboratory
methods, iNDF used in forage-specific equations will improve overall predictions
of forage in vivo OMD. The in vitro and in situ methods performed equally well in
calibrations of iNDF or OMD by near-infrared reflectance spectroscopy.
DOI: 10.2527/jas.2011-4347
1011. Brain Res. 2012 Jun 29;1463:21-9. doi: 10.1016/j.brainres.2012.04.057. Epub

2012
May 7.
Involvement of phosphatidylinositol 3-kinase/Akt on basic fibroblast growth

factor-induced glial cell line-derived neurotrophic factor release from rat
glioma cells.
Tanabe K(1), Matsushima-Nishiwaki R, Iida M, Kozawa O, Iida H.
Author information:
(1)Department of Anesthesiology and Pain Medicine, Gifu University Graduate
School of Medicine, Gifu 501-1194, Japan. kumiko-t@m2.gyao.ne.jp
Basic fibroblast growth factor (FGF-2) has a neuroprotective effect. Astrocytes

support neurons by releasing neurotrophic factors including glial cell
line-derived neurotrophic factor (GDNF). FGF-2 stimulates GDNF synthesis in
astrocytes and the release. It has been reported that FGF-2 induces the
activation of p44/p42 mitogen-activated protein (MAP) kinase, stress-activated
protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 MAP kinase in C6 glioma
cells, and that FGF-2 stimulates GDNF release through p44/p42 MAP kinase or
SAPK/JNK, but not p38 MAP kinase. In the present study, we investigated the exact
mechanism of FGF-2-induced GDNF release from C6 cells. FGF-2 induced the
phosphorylation of Akt and its substrate, glycogen synthase kinase 3β (GSK3β) in
addition to three MAP kinases in these cells. FGF-2-stimulated release of GDNF
was suppressed by wortmannin (a phosphatidylinositol 3 (PI3)-kinase inhibitor) or
LY294002 (another PI3-kinase inhibitor). The FGF-2-induced GDNF release from
PI3-kinase-downregulated C6 cells was decreased compared with that in control
siRNA-transfected cells. PD98059 (an inhibitor of MEK 1/2) or SP600125 (an
inhibitor of SAPK/JNK), which suppressed FGF-2-induced phosphorylation of p44/p42
MAP kinase or SAPK/JNK respectively, did not affect FGF-2-induced Akt
phosphorylation. Wortmannin or LY294002, which attenuated FGF-2-induced
phosphorylation of Akt and GSK3β, had no effect on FGF-2-induced phosphorylation
of p44/p42 MAP kinase or SAPK/JNK. These results strongly suggest that the
PI3-kinase/Akt pathway plays a positive role in FGF-2-stimulated GDNF release
independently of p44/p42 MAP kinase or SAPK/JNK in C6 glioma cells.

DOI: 10.1016/j.brainres.2012.04.057
1012. Plant Physiol. 2012 Jul;159(3):1204-20. doi: 10.1104/pp.112.195420. Epub 2012

May
7.
Expression of the R2R3-MYB transcription factor TaMYB14 from Trifolium arvense

activates proanthocyanidin biosynthesis in the legumes Trifolium repens and
Medicago sativa.
Hancock KR(1), Collette V, Fraser K, Greig M, Xue H, Richardson K, Jones C,

Rasmussen S.
Author information:
(1)AgResearch, Ltd., Palmerston North 4442, New Zealand.
Proanthocyanidins (PAs) are oligomeric flavonoids and one group of end products
of the phenylpropanoid pathway. PAs have been reported to be beneficial for human
and animal health and are particularly important in pastoral agricultural systems
for improved animal production and reduced greenhouse gas emissions. However, the
main forage legumes grown in these systems, such as Trifolium repens and Medicago
sativa, do not contain any substantial amounts of PAs in leaves. We have
identified from the foliar PA-accumulating legume Trifolium arvense an R2R3-MYB
transcription factor, TaMYB14, and provide evidence that this transcription
factor is involved in the regulation of PA biosynthesis in legumes. TaMYB14
expression is necessary and sufficient to up-regulate late steps of the
phenylpropanoid pathway and to induce PA biosynthesis. RNA interference silencing
of TaMYB14 resulted in almost complete cessation of PA biosynthesis in T.
arvense, whereas Nicotiana tabacum, M. sativa, and T. repens plants
constitutively expressing TaMYB14 synthesized and accumulated PAs in leaves up to
1.8% dry matter. Targeted liquid chromatography-multistage tandem mass
spectrometry analysis identified foliar PAs up to degree of polymerization 6 in
leaf extracts. Hence, genetically modified M. sativa and T. repens plants
expressing TaMYB14 provide a viable option for improving animal health and
mitigating the negative environmental impacts of pastoral animal production
systems.
DOI: 10.1104/pp.112.195420
PMCID: PMC3387705
1013. J Dairy Sci. 2012 May;95(5):2632-47. doi: 10.3168/jds.2011-4996.
Nutrient demand interacts with legume maturity to affect rumen pool sizes in
dairy cows.
Kammes KL(1), Ying Y, Allen MS.
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing,
Michigan 48824-1225, USA.
Effects of legume maturity on dry matter intake (DMI), milk production, ruminal
fermentation and pool sizes, and digestion and passage kinetics, and the
relationship of these effects with preliminary DMI (pDMI) were evaluated using 16
ruminally and duodenally cannulated Holstein cows in a crossover design with a
14-d preliminary period and two 17-d treatment periods. During the preliminary
period, the pDMI of individual cows ranged from 22.9 to 30.0 kg/d (mean=25.9
kg/d) and the 3.5% fat-corrected milk yield ranged from 34.1 to 68.2 kg/d
(mean=43.7 kg/d). Experimental treatments were diets containing alfalfa silage
harvested either a) early-cut, less mature (EC) or b) late-cut, more mature (LC)
as the sole forage. Early- and late-cut alfalfa contained 40.8 and 53.1% neutral
detergent fiber (NDF) and 23.7 and 18.1% crude protein, respectively.
Forage:concentrate ratios were 53:47 and 42:58 for EC and LC, respectively; both
diets contained approximately 22% forage NDF and 27% total NDF. Preliminary DMI,
an index of nutrient demand, was determined during the last 4d of the preliminary
period when cows were fed a common diet and used as a covariate. Main effects of
alfalfa maturity and their interaction with pDMI were tested by ANOVA. Alfalfa
maturity and its interaction with pDMI did not affect milk yield but EC increased
DMI compared with LC; thus, EC had lower efficiency of milk production than LC.
The EC diet decreased milk fat concentration more per kilogram of pDMI increase
than the LC diet, but milk fat yield was not affected. The lower concentration
and faster passage rate of indigestible NDF for EC resulted in lower rumen pools
of indigestible NDF, total NDF, and dry matter than did LC, which EC increased at
a slower rate than did LC as pDMI increased. The EC diet decreased starch intake
and increased ruminal pH compared with the LC diet. The rate of ruminal starch
digestion was related to level of intake, but this did not affect ruminal or
postruminal starch digestion. Total-tract digestibility of NDF, organic matter,
and dry matter was higher for EC than LC. Microbial efficiency tended to be
related to pDMI and the response differed by treatment. When alfalfa silage was
the only source of forage in the diet, cows supplemented with additional
concentrate to account for decreased protein and increased fiber concentrations
associated with LC produced similar fat-corrected milk yields with greater
efficiency than cows fed EC.

DOI: 10.3168/jds.2011-4996
Nutrient demand interacts with legume particle length to affect digestion

responses and rumen pool sizes in dairy cows.
Kammes KL(1), Ying Y, Allen MS.
Author information:
Michigan 48824-1225, USA.
Effects of legume particle length on dry matter intake (DMI), milk production,
ruminal fermentation and pool sizes, and digestion and passage kinetics, and the
relationship of these effects with preliminary DMI (pDMI) were evaluated using 13
ruminally and duodenally cannulated Holstein cows in a crossover design with a
14-d preliminary period and two 19-d treatment periods. During the preliminary
period, pDMI of individual cows ranged from 22.8 to 32.4 kg/d (mean=26.5 kg/d)
and 3.5% fat-corrected milk yield ranged from 22.9 to 62.4 kg/d (mean=35.1 kg/d).
Experimental treatments were diets containing alfalfa silage chopped to (1) 19 mm
(long cut, LC) or (2) 10 mm (short cut, SC) theoretical length of cut as the sole
forage. Alfalfa silages contained approximately 43% neutral detergent fiber
(NDF); diets contained approximately 47% forage and 20% forage NDF. Preliminary
DMI, an index of nutrient demand, was determined during the last 4 d of the
preliminary period, when cows were fed a common diet, and used as a covariate.
Main effects of legume particle length and their interaction with pDMI were
tested by ANOVA. Alfalfa particle length and its interaction with pDMI did not
affect milk yield or rumen pH. The LC diet decreased milk fat concentration more
per kilogram of pDMI increase than the SC diet and increased yields of milk fat
and fat-corrected milk less per kilogram of pDMI increase than the SC diet,
resulting in a greater benefit for LC at low pDMI and for SC at high pDMI. The LC
diet tended to decrease DMI compared with the SC diet. Ruminal digestion and
passage rates of feed fractions did not differ between LC and SC and were not
related to level of intake. The LC diet tended to decrease the rate of ruminal
turnover for NDF but increased NDF rumen pools at a slower rate than the SC diet
as pDMI increased. This indicated that the faster NDF turnover rate did not
counterbalance the higher DMI for SC, resulting in larger NDF rumen pools for SC
than LC. As pDMI increased, LC increased ruminal digestibility of potentially
digestible NDF and total NDF, and SC decreased them, but total-tract
digestibilities of potentially digestible NDF, total NDF, organic matter, and dry
matter were lower for LC than for SC. Ruminal digestibilities of starch and
organic matter interacted quadratically with level of intake. When legume silage
was the only source of forage in the diet, increasing chop length from 10 to 19
mm tended to decrease DMI but did not negatively affect productivity of cows.

DOI: 10.3168/jds.2011-4906
1015. Biometals. 2012 Jun;25(3):617-31. doi: 10.1007/s10534-012-9551-9. Epub 2012

Apr
27.
Roles of hydrogen sulfide and nitric oxide in the alleviation of cadmium-induced

oxidative damage in alfalfa seedling roots.
Li L(1), Wang Y, Shen W.
Author information:
(1)College of Life Sciences, Cooperative Demonstration Laboratory of Centrifuge
Technique of Nanjing Agricultural University and Beckman Coulter Ltd. Co.,
Nanjing 210095, People's Republic of China.
Despite hydrogen sulfide (H(2)S) and nitric oxide (NO) are important endogenous
signals or bioregulators involved in many vital aspects of plant growth and
responses against abiotic stresses, little information was known about their
interaction. In the present study, we evaluated the effects of H(2)S and NO on
alfalfa (Medicago sativa L.) plants exposed to cadmium (Cd) stress. Pretreatment
with an H(2)S donor sodium hydrosulfide (NaHS) and well-known NO donor sodium
nitroprusside (SNP) decreased the Cd toxicity. This conclusion was supported by
the decreases of lipid peroxidation as well as the amelioration of seedling
growth inhibition and Cd accumulation, in comparison with the Cd-stressed alone
plants. Total activities and corresponding transcripts of antioxidant enzymes,
including superoxide dismutase, peroxidase and ascorbate peroxidase were
modulated differentially, thus leading to the alleviation of oxidative damage.
Effects of H(2)S above were reversed by
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt
(cPTIO), the specific scavenger of NO. By using laser confocal scanning
microscope combined with Greiss reagent method, further results showed that NO
production increased significantly after the NaHS pretreatment regardless of
whether Cd was applied or not, all of which were obviously inhibited by cPTIO.
These decreases of NO production were consistent with the exaggerated syndromes
associated with Cd toxicity. Together, above results suggested that NO was
involved in the NaHS-induced alleviation of Cd toxicity in alfalfa seedlings, and
also indicated that there exists a cross-talk between H(2)S and NO responsible
for the increased abiotic stress tolerance.
DOI: 10.1007/s10534-012-9551-9
1016. Genet Mol Res. 2012 Mar 14;11(1):606-16. doi: 10.4238/2012.March.14.4.
Association of AFLP and SCAR markers with common leafspot resistance in

autotetraploid alfalfa (Medicago sativa).
Wang Y(1), Bi B, Yuan QH, Li XL, Gao JM.
Author information:
Beijing, China.
To identify amplified fragment length polymorphism (AFLP) markers associated with

resistance or susceptibility of alfalfa to common leafspot (CLS) caused by the
fungus Pseudopeziza medicaginis (Dermateaceae), bulked segregant analysis was
conducted based on an F(1(M × M)) population of 93 plants and a BC(1)S population
of 91 plants. Three AFLP markers, ACTCAA(R206), TAGCAC(R185), and GGACTA(S264),
were found to be associated with CLS resistance or susceptibility. All three
markers were found at significantly different frequencies (71.9, 80.3 and 91.8%)
compared to resistant or susceptible plants in the original population.
Subsequently, these three AFLP markers were converted into three SCAR markers,
ACTCAA(R136), TAGCAC(R128) and GGACTA(S254), which are easier to employ in
breeding programs. The three SCAR markers were used in a randomly selected
population with 50% resistance; the probability of finding one resistant plant
was increased to 67.3, 66.7 and 90.0% with markers ACTCAA(R136), TAGCAC(R128) and
GGACTA(S254), independently. If two of the SCAR markers were used simultaneously,
the probability would be higher than 89%. The three SCAR markers identified in
this study would be applicable for selection for CLS resistance in alfalfa
breeding programs. Moreover, the genetic analysis indicated that CLS resistance
in alfalfa is conferred by a single dominant gene.
DOI: 10.4238/2012.March.14.4
1017. Pest Manag Sci. 2012 Jul;68(7):959-62. doi: 10.1002/ps.3303. Epub 2012 Apr
20.
Evaluation of dazomet as fumigant for the control of brown root rot disease.
Fu CH(1), Hu BY, Chang TT, Hsueh KL, Hsu WT.
Author information:
(1)Taiwan Forestry Research Institute, Division of Forest Protection, Taipei,
Taiwan, People's Republic of China.
BACKGROUND: Brown root rot disease caused by Phellinus noxius is widespread in

approximately 216 tree and plant species in tropical and subtropical countries.
No direct means of protection against P. noxius infection have thus far been
developed. Therefore, in this field survey, a method was developed for preventing
and controlling brown root rot disease using fumigation with dazomet.
RESULTS: Tracers were used to monitor the effect of dazomet fumigation. The
results from field surveys and phytotoxicity tests showed that dazomet is able to
kill P. noxius without any side effects on plants.
CONCLUSION: The use of an environmentally friendly agent to control brown root
rot disease is needed, and prevention of the disease is more important than
treatment. Hence, chemical fumigation with 60 g m(-2) of 98% dazomet may be a
possible means of preventing P. noxius incursion in agriculture, gardening and
agroforestry.
DOI: 10.1002/ps.3303
1018. Plant Signal Behav. 2012 May;7(5):539-43. doi: 10.4161/psb.19780. Epub 2012
Apr
20.
Global regulation of reactive oxygen species scavenging genes in alfalfa root and
shoot under gradual drought stress and recovery.
Kang Y(1), Udvardi M.
Author information:
(1)The Samuel Roberts Noble Foundation, Ardmore, OK, USA.
Reactive oxygen species (ROS) production and scavenging in plants under drought
stress have been studied intensively in recent years. Here we report a global
analysis of gene expression for the major ROS generating and scavenging proteins
in alfalfa root and shoot under gradual drought stress followed by one-day
recovery. Data from two alfalfa varieties, one drought tolerant and one drought
sensitive, were compared and no qualitative differences in ROS gene regulation
between the two were found. Conserved, tissue-specific patterns of gene
expression in response to drought were observed for several ROS-scavenging gene
families, including ascorbate peroxidase, monodehydroascorbate reductase, and
peroxiredoxin. In addition, differential gene expression within families was
observed. Genes for the ROS-generating enzyme, NADPH oxidase were generally
induced under drought, while those for glycolate oxidase were repressed. Among
the ROS-scavenging protein genes, Ferritin, Cu/Zn superoxide dismutase (SOD), and
the majority of the glutathione peroxidase family members were induced under
drought in both roots and shoots of both alfalfa varieties. In contrast, Fe-SOD,
CC-type glutaredoxins, and thoiredoxins were downregulated.
DOI: 10.4161/psb.19780
PMCID: PMC3419014
1019. N Z Vet J. 2012 Jul;60(4):247-53. doi: 10.1080/00480169.2012.665343. Epub

2012
Apr 16.
Acute coenurosis of dairy sheep from 11 flocks in Greece.
Giadinis ND(1), Psychas V, Polizopoulou Z, Papadopoulos E, Papaioannou N,

Komnenou AT, Thomas AL, Petridou EJ, Kritsepi-Konstantinou M, Lafi SQ, Brellou
GD.
Author information:
(1)Department of Clinical Sciences, School of Veterinary Medicine, Aristotle
University, Thessaloniki , Greece. ngiadini@vet.auth.gr
CASE HISTORY: A syndrome of acute neurological dysfunction with increased

mortality was observed in lambs of 10 dairy sheep flocks and adult animals in one
flock in Central and Northern Greece. Each farmer completed a questionnaire
regarding the management and feeding of their flocks. In seven of the 11 flocks
the affected animals were grazing pasture, while in the remaining four flocks (5,
8, 9, 10) the animals were fed alfalfa hay (Medicago sativa) and concentrates
indoors. A follow-up study of the affected flocks was conducted during the next
12 months.
CLINICAL FINDINGS: Of 42 sheep with acute coenurosis that were examined, the most
prominent neurological abnormalities were ataxia, depression, blindness,
scoliosis, coma and dysmetria. Except for the four sheep that were comatose, all
other animals had normal body temperatures and their appetites remained normal or
were slightly decreased. Haematological findings of 15 examined sheep were within
normal limits. The affected sheep were subject to euthanasia. A histopathological
examination was performed in 13 cases. Faecal samples from dogs associated with
these flocks were negative for taeniid infections. During the following 12 months
cases of chronic coenurosis in these flocks were observed.
PATHOLOGICAL FINDINGS: In the 42 animals that were necropsied, the main gross
findings were cystic formations between 0.5-1 cm in diameter with translucent
walls that were seen lying free on the leptomeninges or partly penetrating the
brain tissue, sterile microabscecess and brain necrosis. Histopathological
evaluation of tissue sections of 13 brains showed multifocal purulent or
pyogranulomatous meningoencephalitis, accompanied by eosinophilic infiltrations.
No bacteria were isolated following bacterial culture of brain tissue
Parasitological examination of the cysts from five cases revealed whitish specks
on the transparent cyst wall and germination membrane representing the scolices.
DIAGNOSIS: Acute coenurosis was diagnosed in all cases studied.
CLINICAL RELEVANCE: Acute coenurosis can be one of the causes of acute
encephalopathy mainly in lambs, but also in adult sheep. This condition is
incurable, but can be controlled by changing the feeding regime. Cases of chronic
coenurosis may be seen a few months later in the same flock.
DOI: 10.1080/00480169.2012.665343
1020. Zhong Yao Cai. 2011 Nov;34(11):1709-11.
[Study on the chemical constituents from the aerial parts of Medicago sativa and
their hypolipidemic activity].
Yu CH(1), Liang DL, Guo JN, Mei QX, Yang DP.
Author information:
(1)School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006
China. ych1203@163.com
OBJECTIVE: To research the chemical constituents from the aerial parts of

Medicago sativa.
METHODS: Various column chromatographies were emoployed to isolate and purify the
constituents. Their structures were elucidated by spectral analysis (IR, UV, MS,
1H-NMR, 13C-NMR) and chemical evidence.
RESULTS: Two constituents were obtained and identified as soysaponin I (I),
azukisaponin V (II).
CONCLUSION: These two compounds are isolated from the arial parts of Mesicago
sativa for the first time. Moreover,the compounds are found to have hypolipidemic
activity.

Epub
2012 Apr 6.
A positive correlation between bacterial autoaggregation and biofilm formation in

native Sinorhizobium meliloti isolates from Argentina.
Sorroche FG(1), Spesia MB, Zorreguieta A, Giordano W.
Author information:
(1)Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Río
Cuarto, Córdoba, Argentina.
Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits

nodule formation on roots of alfalfa plants. S. meliloti produces two
exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote
symbiosis. EPS I and EPS II are secreted in two major fractions that reflect
differing degrees of subunit polymerization, designated high- and
low-molecular-weight fractions. We reported previously that EPSs are crucial for
autoaggregation and biofilm formation in S. meliloti reference strains and
isogenic mutants. However, the previous observations were obtained by use of
"domesticated" laboratory strains, with mutations resulting from successive
passages under unnatural conditions, as has been documented for reference strain
Rm1021. In the present study, we analyzed the autoaggregation and biofilm
formation abilities of native S. meliloti strains isolated from root nodules of
alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all
the native isolates revealed a high degree of identity with reference S. meliloti
strains. PCR analysis of the expR gene of all the isolates showed that, as in the
case of reference strain Rm8530, this gene is not interrupted by an insertion
sequence (IS) element. A positive correlation was found between autoaggregation
and biofilm formation abilities in these rhizobia, indicating that both processes
depend on the same physical adhesive forces. Extracellular complementation
experiments using mutants of the native strains showed that autoaggregation was
dependent on EPS II production. Our results indicate that a functional EPS II
synthetic pathway and its proper regulation are essential for cell-cell
interactions and surface attachment of S. meliloti.
DOI: 10.1128/AEM.07826-11
PMCID: PMC3370541
1022. Folia Microbiol (Praha). 2012 Jul;57(4):359-62. doi: 10.1007/s12223-012-0142-

5.
Epub 2012 Apr 11.
Growth and survival of lactic acid bacteria in lucerne silage.

Vlková E(1), Rada V, Bunešová V, Ročková S.
Author information:
(1)Department of Microbiology, Nutrition and Dietetics, Faculty of Agrobiology,
Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká
129, Prague 6, 165 21, Czech Republic. vlkova@af.czu.cz
A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one

strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were
used for the experimental production of lucerne silage. Laboratory silage without
inoculants served as a control. Counts of total anaerobes, total lactic acid
bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days
14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage
microflora, reaching a percentage between 59 and 95 % of total anaerobes.
Lactobacilli were found as a predominant group of LAB during the whole study.
Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g
in the control at the first observation. Their counts decreased to 4.23 and 4.92
log CFU/g in treated silage and the control, respectively, on day 63 of
fermentation. Similar decreases were observed in all bacterial groups. The
treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and
contained more lactic acid compared to control silage. The identity of
re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne
was evaluated by fingerprinting techniques. The fingerprint profiles of
re-isolated bacteria corresponded to the profiles of strains used for the
treatment. It could be concluded that supplemented LAB dominated in laboratory
silage and overgrew naturally occurring LAB.
DOI: 10.1007/s12223-012-0142-5
1023. Pharm Biol. 2012 Jul;50(7):878-82. doi: 10.3109/13880209.2011.641227. Epub

2012
Apr 10.
Evaluation of anxiolytic effect of Medicago sativa in mice.
Singh Bora K(1), Sharma A.
Author information:
(1)LR Institute of Pharmacy, Solan, Himachal Pradesh, India.
CONTEXT: Medicago sativa Linn. (Leguminosae) has a long tradition of use as an

Ayurvedic and Homoeopathic medicine in a variety of central nervous system (CNS)
disorders. Traditionally, M. sativa is used to improve the memory, as a
rejuvenator, antidiabetic, antioxidant, anti-inflammatory, and in CNS disorders.
Despite a long tradition of use, no systematic phytochemical and pharmacological
work has been carried out on this potential plant. M. sativa was subjected to
preliminary anti-anxiety screening studies, with a view to ascertain the verity
of its traditional use as an anxiolytic.
OBJECTIVE: Various extracts, viz., petroleum ether, chloroform, methanol and
aqueous extract from the aerial parts of M. sativa was subjected to preliminary
anti-anxiety screening studies, with a view to ascertain the truth on evidence of
its traditional use as an anxiolytic.
MATERIALS AND METHODS: The aerial parts of the plant were extracted using
solvents in order of increasing polarity, viz., petroleum ether (60-80 °C),
chloroform, methanol and distilled water. All the crude extracts were evaluated
for anti-anxiety activity in mice using elevated plus-maze apparatus. Diazepam
was used as the standard drug.
RESULTS: Among all extracts, only the methanol extract exhibited significant (p <
0.05) anti-anxiety activity by increasing the average time spent, and number of
entries in open arms at a dose of 100 mg/kg in mice with respect to the vehicle
treated control as well as the standard (2 mg/kg).
CONCLUSION: These results suggest that administration of M. sativa exerts
anxiolytic effect on mice, and it could serve as a new approach for the treatment
of anxiety.
DOI: 10.3109/13880209.2011.641227
1024. Vet Parasitol. 2012 Aug 13;188(1-2):1-9. doi: 10.1016/j.vetpar.2012.03.014.

Epub
2012 Mar 16.
In vivo and in vitro efficacy of sainfoin (Onobrychis viciifolia) against Eimeria

spp in lambs.
Saratsis A(1), Regos I, Tzanidakis N, Voutzourakis N, Stefanakis A, Treuter D,

Joachim A, Sotiraki S.
Author information:
(1)Veterinary Research Institute, National Agricultural Research Foundation,
NAGREF Campus, PO Box 60272, 57001 Thermi, Greece. saratsis@vri.gr
The effect of sainfoin (Onobrychis viciifolia) against ovine coccidia was

evaluated in vivo and in vitro. In 3 in vivo trials weaned lambs were allocated
into two treatment groups receiving diets with either lucerne (Medicago sativa)
or sainfoin. During the trials, which lasted for 7 (trial 1) or 8 weeks (trials 2
and 3), oocysts per gram of faeces (OPGs), faecal scores and weight gain were
recorded. In two of the experiments (trials 1 and 3) a reduction in the mean
oocyst excretion rates was observed, starting three to four weeks after sainfoin
hay feeding. This reduction ranged between 21.3% (trial 1) and 61.7% (trial 3)
compared to the control values. As a result, a decrease in the total number of
oocysts excreted (expressed as the mean area under the curve of the OPG) was
observed from week 4 to the end of the two trials, respectively (trial 1: 42.6%
reduction, p=0.05; trial 3: 52.4% reduction, p=0.06). The results did not show
any significant diet effect on lamb growth rates and faecal scores. In the in
vitro experiments the effect of 39 sainfoin extracts were tested in an oocyst
sporulation inhibition assay. The Eimeria oocysts sporulation inhibition
throughout the experiments did not exceed 10.7%, showing that extracts of this
forages do not have a significant inhibitory effect on Eimeria oocyst
sporulation. This was an initial attempt to investigate a possible anticoccidial
effect of sainfoin and further studies are needed in order to better understand
its mode of action against Eimeria.
DOI: 10.1016/j.vetpar.2012.03.014

30.
Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of

genetic structure among distant and habitat diverse populations.
Zhou C(1), Kandemir I, Walsh DB, Zalom FG, Lavine LC.
Author information:
(1)Department of Entomology, Washington State University, Pullman, Washington,
BACKGROUND: The western tarnished plant bug Lygus hesperus is an economically

important pest that belongs to a complex of morphologically similar species that
makes identification problematic. The present study provides evidence for the use
of DNA barcodes from populations of L. hesperus from the western United States of
America for accurate identification.
METHODOLOGY/PRINCIPAL FINDINGS: This study reports DNA barcodes for 134
individuals of the western tarnished plant bug from alfalfa and strawberry
agricultural fields in the western United States of America. Sequence divergence
estimates of <3% reveal that morphologically variable individuals presumed to be
L. hesperus were accurately identified. Paired estimates of F(st) and subsequent
estimates of gene flow show that geographically distinct populations of L.
hesperus are genetically similar. Therefore, our results support and reinforce
the relatively recent (<100 years) migration of the western tarnished plant bug
into agricultural habitats across the western United States.
CONCLUSIONS/SIGNIFICANCE: This study reveals that despite wide host plant usage
and phenotypically plastic morphological traits, the commonly recognized western
tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no
significant genetic structure was found for the geographically diverse
populations of western tarnished plant bug used in this study.
PMCID: PMC3316671
1026. Theor Appl Genet. 2012 Aug;125(3):577-90. doi: 10.1007/s00122-012-1854-2.

Epub
2012 Apr 5.
Patterns of linkage disequilibrium and association mapping in diploid alfalfa (M.

sativa L.).
Sakiroglu M(1), Sherman-Broyles S, Story A, Moore KJ, Doyle JJ, Charles Brummer
E.
Author information:
(1)Department of Bioengineering, Kafkas University, Kars, 36100, Turkey.
Association mapping enables the detection of marker-trait associations in

unstructured populations by taking advantage of historical linkage disequilibrium
(LD) that exists between a marker and the true causative polymorphism of the
trait phenotype. Our first objective was to understand the pattern of LD decay in
the diploid alfalfa genome. We used 89 highly polymorphic SSR loci in 374
unimproved diploid alfalfa (Medicago sativa L.) genotypes from 120 accessions to
infer chromosome-wide patterns of LD. We also sequenced four lignin biosynthesis
candidate genes (caffeoyl-CoA 3-O-methyltransferase (CCoAoMT),
ferulate-5-hydroxylase (F5H), caffeic acid-O-methyltransferase (COMT), and
phenylalanine amonialyase (PAL 1)) to identify single nucleotide polymorphisms
(SNPs) and infer within gene estimates of LD. As the second objective of this
study, we conducted association mapping for cell wall components and agronomic
traits using the SSR markers and SNPs from the four candidate genes. We found
very little LD among SSR markers implying limited value for genomewide
association studies. In contrast, within gene LD decayed within 300 bp below an r
(2) of 0.2 in three of four candidate genes. We identified one SSR and two highly
significant SNPs associated with biomass yield. Based on our results, focusing
association mapping on candidate gene sequences will be necessary until a dense
set of genome-wide markers is available for alfalfa.
DOI: 10.1007/s00122-012-1854-2
PMCID: PMC3397135
1027. J Dairy Sci. 2012 Apr;95(4):1992-2003. doi: 10.3168/jds.2011-4802.
A ring test of in vitro neutral detergent fiber digestibility: analytical

variability and sample ranking.
Hall MB(1), Mertens DR.
Author information:
WI 53706, USA. marybeth.hall@ars.usda.gov
In vitro neutral detergent fiber (NDF) digestibility (NDFD) is an empirical

measurement of fiber fermentability by rumen microbes. Variation is inherent in
all assays and may be increased as multiple steps or differing procedures are
used to assess an empirical measure. The main objective of this study was to
evaluate variability within and among laboratories of 30-h NDFD values analyzed
in repeated runs. Subsamples of alfalfa (n=4), corn forage (n=5), and grass (n=5)
ground to pass a 6-mm screen passed a test for homogeneity. The 14 samples were
sent to 10 laboratories on 3 occasions over 12 mo. Laboratories ground the
samples and ran 1 to 3 replicates of each sample within fermentation run and
analyzed 2 or 3 sets of samples. Laboratories used 1 of 2 NDFD procedures: 8 labs
used procedures related to the 1970 Goering and Van Soest (GVS) procedure using
fermentation vessels or filter bags, and 2 used a procedure with preincubated
inoculum (PInc). Means and standard deviations (SD) of sample replicates within
run within laboratory (lab) were evaluated with a statistical model that included
lab, run within lab, sample, and lab × sample interaction as factors. All factors
affected mean values for 30-h NDFD. The lab × sample effect suggests against a
simple lab bias in mean values. The SD ranged from 0.49 to 3.37% NDFD and were
influenced by lab and run within lab. The GVS procedure gave greater NDFD values
than PInc, with an average difference across all samples of 17% NDFD. Because of
the differences between GVS and PInc, we recommend using results in contexts
appropriate to each procedure. The 95% probability limits for within-lab
repeatability and among-lab reproducibility for GVS mean values were 10.2 and
13.4%, respectively. These percentages describe the span of the range around the
mean into which 95% of analytical results for a sample fall for values generated
within a lab and among labs. This degree of precision was supported in that the
average maximum difference between samples that were not declared different by
means separation was 4.4% NDFD. Although the values did not have great precision,
GVS labs were able to reliably rank sample data in order of 30-h NDFD (Spearman
correlation coefficient = 0.93) with 80% of the rankings correct or off by only 1
ranking. A relative ranking system for NDFD could reduce the effect of within-
and among-lab variation in numeric values. Such a system could give a more
accurate portrayal of the comparative values of samples than current numeric
values imply.

DOI: 10.3168/jds.2011-4802
1028. J Microsc. 2012 Jul;247(1):60-7. doi: 10.1111/j.1365-2818.2012.03611.x. Epub

2012
Mar 28.
Texture of cellulose microfibrils of root hair cell walls of Arabidopsis

thaliana, Medicago truncatula, and Vicia sativa.
Akkerman M(1), Franssen-Verheijen MA, Immerzeel P, Hollander LD, Schel JH, Emons
AM.
Author information:
(1)Laboratory of Cell Biology, Wageningen University, Droevendaalsesteeg 1, 6708
PB Wageningen, the Netherlands.
Cellulose is the most abundant biopolymer on earth, and has qualities that make
it suitable for biofuel. There are new tools for the visualisation of the
cellulose synthase complexes in living cells, but those do not show their
product, the cellulose microfibrils (CMFs). In this study we report the
characteristics of cell wall textures, i.e. the architectures of the CMFs in the
wall, of root hairs of Arabidopsis thaliana, Medicago truncatula and Vicia sativa
and compare the different techniques we used to study them. Root hairs of these
species have a random primary cell wall deposited at the root hair tip, which
covers the outside of the growing and fully grown hair. The secondary wall starts
between 10 (Arabidopsis) and 40 (Vicia) μm from the hair tip and the CMFs make a
small angle, Z as well as S direction, with the long axis of the root hair. CMFs
are 3-4 nm wide in thin sections, indicating that single cellulose synthase
complexes make them. Thin sections after extraction of cell wall matrix, leaving
only the CMFs, reveal the type of wall texture and the orientation and width of
CMFs, but CMF density within a lamella cannot be quantified, and CMF length is
always underestimated by this technique. Field emission scanning electron
microscopy and surface preparations for transmission electron microscopy reveal
the type of wall texture and the orientation of individual CMFs. Only when the
orientation of CMFs in subsequent deposited lamellae is different, their density
per lamella can be determined. It is impossible to measure CMF length with any of
the EM techniques.
© 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.
DOI: 10.1111/j.1365-2818.2012.03611.x
1029. Physiol Plant. 2013 Jan;147(1):64-74. doi: 10.1111/j.1399-3054.2012.01624.x.

Epub
2012 Apr 28.
Molecular physiology and breeding at the crossroads of cold hardiness

improvement.
Castonguay Y(1), Dubé MP, Cloutier J, Bertrand A, Michaud R, Laberge S.
Author information:
(1)Soils and Crops Research Development Center, Agriculture and Agri-Food Canada,
2560 Hochelaga Boulevard, Sainte-Foy, Québec G1V 2J3, Canada.
yves.castonguay@agr.gc.ca
Alfalfa (Medicago sativa L.) is a major forage legume grown extensively worldwide
with important agronomic and environmental attributes. Insufficient cold
hardiness is a major impediment to its reliable production in northern climates.
Improvement of freezing tolerance using conventional breeding approaches is
slowed by the quantitative nature of inheritance and strong interactions with the
environment. The development of gene-based markers would facilitate the
identification of genotypes with superior stress tolerance. Successive cycles of
recurrent selection were applied using an indoor screening method to develop
populations with significantly higher tolerance to freezing (TF). Bulk segregant
analysis of heterogeneous TF populations identified DNA variations that are
progressively enriched in frequency in response to selection. Polymorphisms
resulting from intragenic variations within a dehydrin gene were identified and
could potentially lead to the development of robust selection tools. Our results
illustrate the benefits of feedback interactions between germplasm development
programs and molecular physiology for a deeper understanding of the molecular and
genetic bases of cold hardiness.
Copyright © Physiologia Plantarum 2012.
DOI: 10.1111/j.1399-3054.2012.01624.x

16.
Modeling effective dosages in hormetic dose-response studies.
Belz RG(1), Piepho HP.
Author information:
(1)Agroecology Unit, University of Hohenheim, Institute of Plant Production and
Agroecology in the Tropics and Subtropics, Stuttgart, Germany.
regina.belz@uni-hohenheim.de
BACKGROUND: Two hormetic modifications of a monotonically decreasing log-logistic

dose-response function are most often used to model stimulatory effects of low
dosages of a toxicant in plant biology. As just one of these empirical models is
yet properly parameterized to allow inference about quantities of interest, this
study contributes the parameterized functions for the second hormetic model and
compares the estimates of effective dosages between both models based on 23
hormetic data sets. Based on this, the impact on effective dosage estimations was
evaluated, especially in case of a substantially inferior fit by one of the two
models.
METHODOLOGY/PRINCIPAL FINDINGS: The data sets evaluated described the hormetic
responses of four different test plant species exposed to 15 different chemical
stressors in two different experimental dose-response test designs. Out of the 23
data sets, one could not be described by any of the two models, 14 could be
better described by one of the two models, and eight could be equally described
by both models. In cases of misspecification by any of the two models, the
differences between effective dosages estimates (0-1768%) greatly exceeded the
differences observed when both models provided a satisfactory fit (0-26%). This
suggests that the conclusions drawn depending on the model used may diverge
considerably when using an improper hormetic model especially regarding effective
dosages quantifying hormesis.
CONCLUSIONS/SIGNIFICANCE: The study showed that hormetic dose responses can take
on many shapes and that this diversity can not be captured by a single model
without risking considerable misinterpretation. However, the two empirical models
considered in this paper together provide a powerful means to model, prove, and
now also to quantify a wide range of hormetic responses by reparameterization.
Despite this, they should not be applied uncritically, but after statistical and
graphical assessment of their adequacy.
PMCID: PMC3306408
1031. Animal. 2012 Apr;6(4):676-82. doi: 10.1017/S1751731111001996.
Phytochemical complementarities among endophyte-infected tall fescue, reed

canarygrass, birdsfoot trefoil and alfalfa affect cattle foraging.
Lyman TD(1), Provenza FD, Villalba JJ, Wiedmeier RD.
Author information:
(1)Department of Wildland Resources, Utah State University, Logan 84322-5230,
USA. tiffannyl@cc.usu.edu
We determined whether plant diversity and sequence of plant ingestion affected

foraging when cattle chose from plants that varied in concentrations of
alkaloids, tannins and saponins. We hypothesized cattle that ate high-alkaloid
grasses (endophyte-infected tall fescue (TF) or reed canarygrass (RCG)) would
prefer forages high in tannins (birdsfoot trefoil, BFT+) or saponins (alfalfa,
ALF+), because tannins and saponins can bind to alkaloids, presumably reducing
their absorption. We further hypothesized that forages with tannins or saponins
consumed before, rather than after, foraging on high-alkaloid grasses would
promote greater use of those grasses presumably by binding to alkaloids, thereby
reducing their absorption. In Phase 1, cattle (n = 32) grazed on either high (+)
or low (-) alkaloid grass (TF or RCG) pastures for 30 min each morning at 0600 h
and were then offered a choice of BFT+, BFT-, ALF+ and ALF- for 60 min each day
for 12 days. In Phase 2, cattle (n = 32) were first offered a choice of BFT+ or
ALF+ for 30 min at 0600 h and then placed on grass (TF+ or -, or RCG+ or -)
pastures for 60 min for 12 days. In both phases, we had four spatial replications
of four treatments with 2 per calves assigned to each of the 16 replications per
treatment combinations. Scan samples of individuals at 2-min intervals were used
to determine incidence of foraging on each plant species (%). Cattle grazed more
on RCG than on TF in Phases 1 (62% v. 27%; P = 0.0015) and 2 (71% v. 32%; P =
0.0005). In Phase 1, cattle that first foraged on RCG+ or TF- subsequently
preferred ALF over BFT, whereas cattle offered RCG- or TF+ foraged on ALF and BFT
equally. Foraging by cattle on RCG was cyclic during Phase 1, whereas cattle
foraging on TF markedly decreased incidence of use of TF from 41% to only 16% by
the end of the 12-day trial (P = 0.0029). Contrary to the cyclic (RCG) or
steadily declining (TF) use of grasses in Phase 1, cattle steadily and
dramatically increased foraging on both RCG and TF throughout Phase 2, when they
first grazed BFT+ or ALF+ followed by high-alkaloid grasses (P = 0.0159). Our
findings suggest that in plant species the sequence of ingestion influenced
foraging behavior of cattle and that secondary compounds influenced those
responses.
DOI: 10.1017/S1751731111001996
1032. Animal. 2012 Apr;6(4):624-32. doi: 10.1017/S1751731111002114.
Effects of alfalfa silage storage structure and roasting corn on ruminal

digestion and microbial CP synthesis in lactating dairy cows.
Krizsan SJ(1), Broderick GA, Promkot C, Colombini S.
Author information:
(1)Department of Agricultural Research for Northern Sweden, Swedish University of
Agricultural Sciences, Umeå, Sweden. sophie.krizsan@slu.se
The objective of this experiment was to quantify the effects of unroasted or

roasted ground-shelled corn (GSC), when fed with alfalfa ensiled in bag, bunker,
or O2-limiting tower silos on ruminal digestion and microbial CP synthesis in
lactating dairy cows. The roasted corn was heat-treated in a propane-fired
roasting system. Alfalfa was harvested as second cutting from fields with
regrowth of the same maturity. A portion of each field was allotted to each silo.
The diets with 3 × 2 factorial arrangement of treatments were fed to six
multiparous rumen-cannulated Holstein cows in a cyclic change-over design with
five 21-day periods. Experimental diets were comparable and averaged (on dry
matter (DM) basis): 410 g/kg alfalfa silage (AS), 150 g/kg corn silage, 350 g/kg
GSC, 50 g/kg soybean meal, 40 g/kg roasted soybeans, 177 g/kg CP, 264 g/kg NDF
and 250 g/kg starch. Nutrient flow was quantified by the omasal sampling
technique with use of three markers (Co, Yb and indigestible NDF). Continuous
infusion of 10% atom excess (15NH4)2SO4 was used to label microbial CP. None of
the interactions between storage structure of dietary AS and corn type were
significant. DM intake was not different among dietary treatments, averaging 24.5
kg/day across diets. Means of ADF digested in the rumen for cows fed diets with
AS from bag, bunker and O2-limiting tower silo were 2.1, 1.7 and 2.1 kg/day,
respectively, and was lower in cows fed AS from the bunker silo. This response
may partly be a reflection of the higher intake of ADF by cows fed AS ensiled in
the O2-limiting tower silo compared with the bunker. There was a slightly greater
supply of fermentable substrates for cows fed diets with roasted compared with
unroasted GSC. The small increases in yield of milk protein and lactose observed
in the previous production trial in cows fed diets containing roasted corn may
have occurred because of greater supply of fermentable substrates.
DOI: 10.1017/S1751731111002114
1033. Animal. 2012 Mar;6(3):449-58. doi: 10.1017/S1751731111001765.
The potential use of n-alkanes, long-chain alcohols and long-chain fatty acids as
diet composition markers: indoor validation with sheep and herbage species from
the rangeland of Inner Mongolia of China.
Lin LJ(1), Zhu XY, Jiang C, Luo HL, Wang H, Zhang YJ, Hong FZ.
Author information:
China Agricultural University, Beijing 100193, PR China.
To investigate the potential use of n-alkanes (alkanes), long-chain alcohols

(alcohols) and long-chain fatty acids (acids) for estimating the diet composition
of sheep, in a feeding trial. A total of 18 sheep were assigned randomly to three
different diets (diet A, diet B and diet C) containing up to eight herbage
species (Leymus chinensis, Leymus dasystachys, Elymus sibiricum, Chenopodium
album, Puccinellia chinampoensis, Medicago sativa, Saussurea sinuata and Bromus
inermis). Faecal recoveries of alkanes, alcohols and acids were determined, and
diet compositions were estimated using different combinations of alkanes,
alcohols and acids. The faecal concentrations of individual alkanes, alcohols and
acids were corrected using the mean recovery of the dietary treatment that the
respective animal belonged to (diet recovery), or the mean recovery across all
dietary treatments (general recovery). In general, diets did not affect the
faecal recovery values for alkanes, alcohols and acids, and no difference in
accuracy was found between diet composition estimates based on dietary recovery
and general recovery. The accuracy of diet composition estimates declined as the
number of dietary components increased from four to eight herbage species (P <
0.001). Better (P < 0.05) estimates of diet composition were obtained with the
combinations of two or three marker types instead of alkanes alone. Moreover,
results showed that excluding minor diet components from the calculations
decreased (P < 0.05) the accuracy of diet composition estimates, whereas
including extra non-grazed herbage species did not reduce (P > 0.05) the quality
of diet composition estimates. These results confirmed the usefulness of alkanes,
alcohols and acids as markers for determining complex diet composition of sheep.
However, a negative impact on the accuracy of diet composition estimates, caused
by missing minor diet components from the calculation of diet composition, could
happen when plant wax markers are used to estimate the diet composition of
free-ranging animals.
DOI: 10.1017/S1751731111001765
A degree-day model of sheep grazing influence on alfalfa weevil and crop

characteristics.
Goosey HB(1).
Author information:
(1)Department of Animal and Range Sciences, Montana State University, 103 Animal
Bioscience Building, P.O. Box 172900, Bozeman, MT 59717, USA. hgoosey@montana.edu
Domestic sheep (Ovis spp.) grazing is emerging as an integrated pest management

tactic for alfalfa weevil, Hypera postica (Gyllenhal), management and a
degree-day model is needed as a decision and support tool. In response to this
need, grazing exclosures with unique degree-days and stocking rates were
established at weekly intervals in a central Montana alfalfa field during 2008
and 2009. Analyses indicate that increased stocking rates and grazing degree-days
were associated with decreased crop levels of weevil larvae. Larval data
collected from grazing treatments were regressed against on-site and near-site
temperatures that produced the same accuracy. The near-site model was chosen to
encourage producer acceptance. The regression slope differed from zero, had an r2
of 0.83, and a root mean square error of 0.2. Crop data were collected to achieve
optimal weevil management with forage quality and yield. Differences were
recorded in crude protein, acid and neutral detergent fibers, total digestible
nutrients, and mean stage by weight. Stem heights differed with higher stocking
rates and degree-days recording the shortest alfalfa canopy height at harvest.
The degree-day model was validated at four sites during 2010 with a mean square
prediction error of 0.74. The recommendation from this research is to stock
alfalfa fields in the spring before 63 DD with rates between 251 and 583 sheep
days per hectare (d/ha). Sheep should be allowed to graze to a minimum of 106 and
maximum of 150 DD before removal. This model gives field entomologists a new
method for implementing grazing in an integrated pest management program.

Duration and frequency of a high temperature pulse affect survival of

emergence-ready Megachile rotundata (Hymenoptera: Megachilidae) during
low-temperature incubation.
Yocum GD(1), Rinehart JP, Kemp WP.
Author information:
(1)USDA-ARS Red River Valley Agricultural Research Center, Biosciences Research
Laboratory, 1605 Albrecht Blvd, Fargo, ND 58102-2765, USA.
george.yocum@ars.usda.gov
Synchronizing Megachile rotundata (F.) nesting activity with alfalfa bloom is

essential for ensuring optimal pollination for alfalfa seed production. This is
achieved by timing the initiation of spring bee incubation so that adults will
emerge -2 wk before peak bloom. If weather conditions change so as to delay the
bloom, bee managers will commonly expose the developing bees to a period of
low-temperature incubation to slow their development. We have previously
demonstrated survival during low-temperature incubation can be significantly
increased by using a fluctuating thermal regime (FTR) where the bees receive a
daily pulse at 20 degrees C. A FTR incubation protocol is composed of a number of
different components, such as the base and pulse temperatures, and the duration
and frequency of the pulse. In this investigation, the effect of the duration of
the pulse (5-120 min) and the frequency of a pulse (twice daily to weekly) on the
survival of developing M. rotundata was examined. A pulse as short as 5 min at 20
degrees C increased survival of the developing bees as compared with the constant
6 degrees C controls. Increasing the pulse duration induced a further increase in
tolerance to 6 degrees C. As with the pulse duration, increasing the pulse
frequency from once weekly to twice daily had a significant effect on improving
the bees tolerance to low-temperature incubation. This investigation further
strengthens the argument that a FTR protocol is superior to using a constant
low-temperature exposure for interrupting the spring incubation of M. rotundata.
1036. J Anim Sci. 2012 Aug;90(8):2754-62. doi: 10.2527/jas.2011-4879. Epub 2012 Mar
9.
Effects of different fresh-cut forages and their hays on feed intake,

digestibility, heat production, and ruminal methane emission by Boer x Spanish
goats.
Puchala R(1), Animut G, Patra AK, Detweiler GD, Wells JE, Varel VH, Sahlu T,
Goetsch AL.
Author information:
(1)American Institute for Goat Research, Langston University, Langston, OK 73050,
USA.
Twenty-four yearling Boer × Spanish wethers were used to assess effects of

different forages, either fresh (Exp. 1) or as hay (Exp. 2), on feed intake,
digestibilities, heat production, and ruminal methane emission. Treatments were:
1) Sericea lespedeza (SER; Lespedeza cuneata), a legume high in condensed tannins
(CT; 20% and 15% in fresh forage and hay, respectively), 2) SER supplemented with
polyethylene glycol (SER-PEG; 25 g/d), 3) alfalfa (Medicago sativa), a legume low
in CT (ALF), and 4) sorghum-sudangrass (Sorghum bicolor), a grass low in CT
(GRASS). Experiments were 22 d, which included 16 d for acclimatization followed
by a 6-d period for fecal and urine collection, and gas exchange measurement
(last 2 d). Intake of OM was 867, 823, 694, and 691 g/d (SEM = 20.1) with fresh
forage, and 806, 887, 681, and 607 g/d with hay for SER, SER-PEG, ALF, and GRASS,
respectively (SEM = 46.6). Apparent total tract N digestion was greater for
SER-PEG vs. SER (P < 0.001) with fresh forage (46.3%, 66.5%, 81.7%, and 73.2%;
SEM = 1.71) and hay (49.7%, 71.4%, 65.4%, and 54.8% for SER, SER-PEG, ALF, and
GRASS, respectively; SEM = 1.57). Intake of ME was similar among treatments with
fresh forage (8.24, 8.06, 7.42, and 7.70 MJ/d; SEM = 0.434) and with hay was
greater for SER-PEG than ALF (P < 0.03) and GRASS (P < 0.001) (8.63, 10.40, 8.15,
and 6.74 MJ/d for SER, SER-PEG, ALF, and GRASS, respectively; SEM = 0.655). The
number of ciliate protozoa in ruminal fluid was least for SER with fresh forage
(P < 0.01) (9.8, 20.1, 21.0, and 33.6 × 10(5)/ml; SEM = 2.76) and hay (P < 0.02)
(6.3, 11.4, 13.6, and 12.5 × 10(5)/ml for SER, SER-PEG, ALF, and GRASS,
respectively; SEM = 1.43). Methane emission as a percentage of DE intake was
lower (P < 0.01) for SER vs. ALF and GRASS with fresh forage (6.6, 8.3, 9.4, and
9.2%; SEM = 0.64) and hay (4.3, 4.9, 6.4, and 6.7% for SER, SER-PEG, ALF, and
GRASS, respectively; SEM = 0.38). In summary, methane emission in this short-term
experiment was similar between a legume and grass low in CT as fresh forage and
hay. The CT in SER markedly decreased N digestibility and elicited a moderate
decline in ruminal methane emission. Supplementation with PEG alleviated the
effect of CT on N digestibility but not ruminal methane emission, presumably
because of different modes of action. In conclusion, potential of using
CT-containing forage as a means of decreasing ruminal methane emission requires
further study, such as with longer feeding periods.
DOI: 10.2527/jas.2011-4879
1037. Appl Environ Microbiol. 2012 May;78(10):3630-7. doi: 10.1128/AEM.00035-12.

Epub
2012 Mar 9.
Intraradical dynamics of two coexisting isolates of the arbuscular mycorrhizal

fungus Glomus intraradices sensu lato as estimated by real-time PCR of
mitochondrial DNA.
Krak K(1), Janoušková M, Caklová P, Vosátka M, Štorchová H.
Author information:
(1)Institute of Botany, Academy of Sciences of the Czech Republic, Prùhonice,
Czech Republic.
Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established

tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this
genomic region does not allow the specific amplification of closely related
genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution
power, but mtDNA-based quantification has not been previously explored in AM
fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to
monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato
coexisting in the roots of medic (Medicago sativa). The mtDNA-based
quantification was compared to quantification in nrDNA. The ratio of copy numbers
determined by the nrDNA- and mtDNA-based assays consistently differed between the
two isolates. Within an isolate, copy numbers of the nuclear and the
mitochondrial genes were closely correlated. The two quantification approaches
revealed similar trends in the dynamics of both isolates, depending on whether
they were inoculated alone or together. After 12 weeks of cultivation,
competition between the two isolates was observed as a decrease in the mtDNA copy
numbers of one of them. The coexistence of two closely related isolates, which
cannot be discriminated by nrDNA-based assays, was thus identified as a factor
influencing the dynamics of AM fungal DNA in roots. Taken together, the results
of this study show that real-time PCR assays targeted to the large subunit of
mtDNA may become useful tools for the study of coexisting AM fungi.
DOI: 10.1128/AEM.00035-12
PMCID: PMC3346362
1038. Plant Physiol. 2012 May;159(1):501-16. doi: 10.1104/pp.112.193706. Epub 2012

Mar
7.
Silencing of the Rac1 GTPase MtROP9 in Medicago truncatula stimulates early

mycorrhizal and oomycete root colonizations but negatively affects rhizobial
infection.
Kiirika LM(1), Bergmann HF, Schikowsky C, Wimmer D, Korte J, Schmitz U, Niehaus

K, Colditz F.
Author information:
(1)Leibniz University of Hannover, Institute for Plant Genetics, Department III,
Plant Molecular Biology, D-30419 Hannover, Germany.
RAC/ROP proteins (ρ-related GTPases of plants) are plant-specific small G

proteins that function as molecular switches within elementary signal
transduction pathways, including the regulation of reactive oxygen species (ROS)
generation during early microbial infection via the activation of NADPH oxidase
homologs of plants termed RBOH (for respiratory burst oxidase homolog). We
investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9,
orthologous to Medicago sativa Rac1, via an RNA interference silencing approach.
Composite M. truncatula plants (MtROP9i) whose roots have been transformed by
Agrobacterium rhizogenes carrying the RNA interference vector were generated and
infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and
the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic
oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear
growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and
MtRBOH gene expression after microbial infection. Coincidently, antioxidative
compounds were not induced in infected MtROP9i roots, as documented by
differential proteomics (two-dimensional differential gel electrophoresis).
Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early
hyphal root colonization, while rhizobial infection was clearly impaired.
Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs
and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments
of MtROP9i led to deformed root hairs showing progressed swelling of its upper
regions or even of the entire root hair and spontaneous constrictions but reduced
branching effects occurring only at swollen root hairs. These results suggest a
key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.
DOI: 10.1104/pp.112.193706
PMCID: PMC3375982
1039. Ecol Lett. 2012 May;15(5):468-74. doi: 10.1111/j.1461-0248.2012.01759.x. Epub

2012 Mar 7.
Increasing antagonistic interactions cause bacterial communities to collapse at
high diversity.
Becker J(1), Eisenhauer N, Scheu S, Jousset A.
Author information:
(1)J.F. Blumenbach Institute for Zoology and Anthropology, Georg August
University Göttingen, Berliner Str. 28, 37073 Göttingen, Germany.
Biodiversity is a major determinant of ecosystem functioning. Species-rich

communities often use resources more efficiently thereby improving community
performance. However, high competition within diverse communities may also reduce
community functioning. We manipulated the genotypic diversity of Pseudomonas
fluorescens communities, a plant mutualistic species inhibiting pathogens. We
measured antagonistic interactions in vitro, and related these interactions to
bacterial community productivity (root colonisation) and ecosystem service (host
plant protection). Antagonistic interactions increased disproportionally with
species richness. Mutual poisoning between competitors lead to a 'negative
complementarity effect', causing a decrease in bacterial density by up to 98% in
diverse communities and a complete loss of plant protection. The results
emphasize that antagonistic interactions may determine community functioning and
cause negative biodiversity-ecosystem functioning relationships. Interference
competition may thus be an additional key for predicting the dynamics and
performance of natural assemblages and needs to be implemented in future
biodiversity models.
© 2012 Blackwell Publishing Ltd/CNRS.
DOI: 10.1111/j.1461-0248.2012.01759.x
[Effects of plant hedgerow on population dynamics of wheat aphid and its natural
enemies].
Shi GQ(1), Lin CW, Liu ZY, Li CR, Chen YB, Ma T, Wang QF, Liu ZL, Cai QN.
Author information:
(1)College of Agronomy & Biotechnology, China Agricultural University, Beijing
100193, China. shiguoqing19821100@126.com
This paper studied the effects of planting different kinds of plant hedgerow
(Amorpha fruticosa, Vetiveria zizanioides, Eulaliopsis binata, and Medicago
sativa) on the population dynamics of wheat aphid and its natural enemies in the
hillside wheat fields in Ziyang City of Sichuan Province, Southwest China. On the
20 degrees hillside, A. fruticosa hedgerow inhibited the occurrence of wheat
aphid in the wheat field significantly, and the parasitoid densities were equal
to or significantly lower than those in the wheat field with V. zizanioides
hedgerow. On the 12 degrees hillside, M. sativa and E. binata hedgerows delayed
the peak time of wheat aphid occurrence, and E. binata hedgerow suppressed the
wheat aphid population density significantly. In the wheat field with M. sativa
hedgerow, the parasitoid densities were significantly higher than those with no
hedgerow. An equal or significantly higher ladybird density was observed in the
field with M. sativa hedgerow. The olfactory responses showed that E. binata
volatiles had repellent effect on both Sitobion avenae and Rhopalosiphum padi,
but attracted a spider species of oxyopidae. M. sativa volatiles attracted S.
avenae, whereas neither M. sativa nor E. binata volatiles caused obvious behavior
response of ladybird adults. Therefore, planting A. fruticosa and E. binata as
the hedgerows in hillside wheat fields could not only prevent the soil erosion
from seasonal rainfall, but also benefit the control of pest insects.

22.
Models analyses for allelopathic effects of chicory at equivalent coupling of

nitrogen supply and pH level on F. arundinacea, T. repens and M. sativa.
Wang Q(1), Xie B, Wu C, Chen G, Wang Z, Cui J, Hu T, Wiatrak P.
Author information:
(1)Department of Grassland Science, College of Animal Sciences and Technology,
Northwest Agriculture and Forestry University, Yangling, Shaanxi Province,
People's Republic of China. wangquanzhen191@163.com
Alllelopathic potential of chicory was investigated by evaluating its effect on

seed germination, soluble sugar, malondialdehyde (MDA) and the chlorophyll
content of three target plants species (Festuca arundinacea, Trifolium repens and
Medicago sativa). The secretion of allelochemicals was regulated by keeping the
donor plant (chicory) separate from the three target plant species and using
different pH and nitrogen levels. Leachates from donor pots with different pH
levels and nitrogen concentrations continuously irrigated the target pots
containing the seedlings. The allelopathic effects of the chicory at equivalent
coupling of nitrogen supply and pH level on the three target plants species were
explored via models analyses. The results suggested a positive effect of nitrogen
supply and pH level on allelochemical secretion from chicory plants. The nitrogen
supply and pH level were located at a rectangular area defined by 149 to 168 mg/l
nitrogen supply combining 4.95 to 7.0 pH value and point located at nitrogen
supply 177 mg/l, pH 6.33 when they were in equivalent coupling effects; whereas
the inhibitory effects of equivalent coupling nitrogen supply and pH level were
located at rectangular area defined by 125 to 131 mg/l nitrogen supply combining
6.71 to 6.88 pH value and two points respectively located at nitrogen supply 180
mg/l with pH 6.38 and nitrogen supply 166 mg/l with pH 7.59. Aqueous extracts of
chicory fleshy roots and leaves accompanied by treatment at different sand pH
values and nitrogen concentrations influenced germination, seedling growth,
soluble sugar, MDA and chlorophyll of F. arundinacea, T. repens and M. sativa.
Additionally, we determined the phenolics contents of root and leaf aqueous
extracts, which were 0.104% and 0.044% on average, respectively.
PMCID: PMC3285180
1042. New Phytol. 2012 May;194(3):810-22. doi: 10.1111/j.1469-8137.2012.04090.x.

Epub
2012 Mar 1.
Establishment, persistence and effectiveness of arbuscular mycorrhizal fungal

inoculants in the field revealed using molecular genetic tracing and measurement
of yield components.
Pellegrino E(1), Turrini A, Gamper HA, Cafà G, Bonari E, Young JP, Giovannetti M.
Author information:
(1)Institute of Life Sciences, Scuola Superiore Sant'Anna, Pisa, Italy.
• Inoculation of crop plants by non-native strains of arbuscular mycorrhizal (AM)

fungi as bio-enhancers is promoted without clear evidence for symbiotic
effectiveness and fungal persistence. To address such gaps, the forage legume
Medicago sativa was inoculated in an agronomic field trial with two isolates of
Funneliformis mosseae differing in their nuclear rDNA sequences from native
strains. • The inoculants were traced by PCR with a novel combination of the
universal fungal NS31 and Glomeromycota-specific LSUGlom1 primers which target
the nuclear rDNA cistron. The amplicons were classified by restriction fragment
length polymorphism and sequencing. • The two applied fungal inoculants were
successfully traced and discriminated from native strains in roots sampled from
the field up to 2 yr post inoculation. Moreover, field inoculation with inocula
of non-native isolates of F. mosseae appeared to have stimulated root
colonization and yield of M. sativa. • Proof of inoculation success and sustained
positive effects on biomass production and quality of M. sativa crop plants hold
promise for the role that AM fungal inoculants could play in agriculture.
DOI: 10.1111/j.1469-8137.2012.04090.x
1043. J Plant Physiol. 2012 May 15;169(8):782-8. doi: 10.1016/j.jplph.2012.01.010.

Epub
2012 Feb 25.
Alfalfa forage digestibility, quality and yield under future climate change
scenarios vary with Sinorhizobium meliloti strain.
Sanz-Sáez Á(1), Erice G, Aguirreolea J, Muñoz F, Sánchez-Díaz M, Irigoyen JJ.
Author information:
(1)Departamento de Biología Vegetal, Sección Biología Vegetal, Universidad de
Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain. alvaro_ssj@hotmail.com
Elevated CO(2) may decrease alfalfa forage quality and in vitro digestibility
through a drop in crude protein and an enhancement of fibre content. The aim of
the present study was to analyse the effect of elevated CO(2), elevated
temperature and Sinorhizobium meliloti strains (102F78, 102F34 and 1032 GMI) on
alfalfa yield, forage quality and in vitro dry matter digestibility. This
objective is in line with the selection of S. meliloti strains in order to
maintain high forage yield and quality under future climate conditions. Plants
inoculated with the 102F34 strain showed more DM production than those inoculated
with 1032GMI; however, these strains did not show significant differences with
102F78 plants. Neutral or acid detergent fibres were not enhanced in plants
inoculated with the 102F34 strain under elevated CO(2) or temperature and hence,
in vitro dry matter digestibility was unaffected. Crude protein content, an
indicator of forage quality, was negatively related to shoot yield. Plants
inoculated with 102F78 showed a similar shoot yield to those inoculated with
102F34, but had higher crude protein content at elevated CO(2) and temperature.
Under these climate change conditions, 102F78 inoculated plants produced higher
quality forage. However, the higher digestibility of plants inoculated with the
102F34 strain under any CO(2) or temperature conditions makes them more suitable
for growing under climate change conditions. In general, elevated CO(2) in
combination with high temperature (Climate Change scenario) reduced IVDMD and CP
content and enhanced fibre content, which means that animal production will be
negatively affected.
DOI: 10.1016/j.jplph.2012.01.010
1044. J Dairy Sci. 2012 Mar;95(3):1462-71. doi: 10.3168/jds.2011-4670.
Measuring residual feed intake in dairy heifers fed an alfalfa (Medicago sativa)
cube diet.
Waghorn GC(1), Macdonald KA, Williams Y, Davis SR, Spelman RJ.
Author information:
(1)DairyNZ Ltd., Private Bag 3221, Hamilton, Waikato 3240, New Zealand.
Garry.waghorn@dairyNZ.co.nz
Selection for divergence between individuals for efficiency of feed utilization

(residual feed intake, RFI) has widespread application in the beef industry and
is usually undertaken when animals are fed diets based on silages with grain. The
objective of this research was to develop a feeding system (using Gallagher,
Hamilton, New Zealand, electronics) to measure RFI for growth in
Holstein-Friesian heifers (aged 5-9 mo), and identify divergent individuals to be
tested for RFI during lactation. A dry forage diet (alfalfa cubes) was fed
because intakes could be measured accurately, and the New Zealand dairy industry
(4.4 million milking cows in lactation) relies heavily on forage feeding. The
evaluation was undertaken over 3 yr with 1,052 animals fed in a facility for 7
wk, and weighed 3 times weekly. The mean age at the start of measurements was 215
d, body weight (BW) 189 kg, and mean daily dry matter intakes averaged 6.7 kg.
Body weight gain (all animals) averaged 0.88 kg/d. The RFI was determined as the
residuals from the regression of mean intake on mean BW(0.75) and daily BW gain
of individuals. Actual and fitted intakes were strongly related (R(2) = 0.82). In
terms of gross efficiency (feed intake/BW gain), RFI+year explained 43% of the
variation, BW gain+year explained 66%, and RFI+BW gain+year explained 79% of the
variation (all P<0.001). Daily BW gains (kg) of the most and least efficient 10%
averaged (± standard deviation) 0.88 ± 0.15 and 0.88 ± 0.12 (P = 0.568),
respectively, and the divergence between mean intakes was 1.46 kg of dry
matter/d. The most and least efficient animals will be tested for RFI during
lactation and genetic markers will be identified for the trait.

DOI: 10.3168/jds.2011-4670
1045. Mol Breed. 2012 Feb;29(2):489-501. Epub 2011 Mar 29.
High-resolution melting analysis for SNP genotyping and mapping in tetraploid

Han Y(1), Khu DM, Monteros MJ.

Author information:
Noble Parkway, Ardmore, OK 73401 USA.
Single nucleotide polymorphisms (SNPs) represent the most abundant type of

genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic
analysis of complex traits of agronomic importance, linkage and association
mapping, genome-wide selection, map-based cloning, and marker-assisted selection.
Current challenges for SNP genotyping in polyploid outcrossing species include
multiple alleles per loci and lack of high-throughput methods suitable for
variant detection. In this study, we report on a high-resolution melting (HRM)
analysis system for SNP genotyping and mapping in outcrossing tetraploid
genotypes. The sensitivity and utility of this technology is demonstrated by
identification of the parental genotypes and segregating progeny in six alfalfa
populations based on unique melting curve profiles due to differences in allelic
composition at one or multiple loci. HRM using a 384-well format is a fast,
consistent, and efficient approach for SNP discovery and genotyping, useful in
polyploid species with uncharacterized genomes. Possible applications of this
method include variation discovery, analysis of candidate genes, genotyping for
comparative and association mapping, and integration of genome-wide selection in
breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this
article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is
available to authorized users.
DOI: 10.1007/s11032-011-9566-x
PMCID: PMC3275744
PMID: 22363202
1046. Chem Biodivers. 2012 Feb;9(2):403-8. doi: 10.1002/cbdv.201100080.
A novel phytotoxic nonenolide from Phomopsis sp. HCCB03520.
Yang Z(1), Ge M, Yin Y, Chen Y, Luo M, Chen D.
Author information:
(1)Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, PR China.
A novel phytotoxic nonenolide,

(6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone (1), was isolated from solid
cultures of the endophytic fungus Phomopsis sp. HCCB03520, together with three
known compounds, cytochalasin H (2), cytochalasin N (3), and epoxycytochalasin H
(4). The structures of these compounds were elucidated through spectroscopic
analysis, and the absolute configurations were determined by CD spectroscopy.
Phytotoxic activities of compounds 1-4 were also investigated. Compound 1 showed
phytotoxic activity on germination and radicle growth of Medicago sativa,
Trifolium hybridum, and Buchloe dactyloides.
Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.
DOI: 10.1002/cbdv.201100080
1047. Int J Food Microbiol. 2012 Apr 16;155(3):247-56. doi:

10.1016/j.ijfoodmicro.2012.01.024. Epub 2012 Feb 3.
Comparative genomic analysis of Salmonella enterica subsp. enterica serovar

Weltevreden foodborne strains with other serovars.
Brankatschk K(1), Blom J, Goesmann A, Smits TH, Duffy B.
Author information:
(1)Agroscope Changins-Wädenswil ACW, Plant Protection Division, Wädenswil,
Switzerland.
Salmonella enterica subsp. enterica serovar Weltevreden is a dominant serovar

associated with foodborne gastroenteritis in South-East Asia and emerging in
Europe associated with fresh vegetables. Here we compared the genome of strain
2007-60-3289-1 linked to an alfalfa sprout outbreak in Scandinavia with a S.
Weltevreden strain isolated from scallops in the USA and with other S. enterica
serovars. A unique plasmid pSW82 was identified for S. Weltevreden carrying a
two-component type II non-ribosomal peptide synthase/polyketide synthase.
Analysis of all available complete S. enterica genomes identified differences for
presence of type VI secretion systems and carbohydrate metabolic pathways.
Differential transcription thereof was observed when S. Weltevreden strains were
grown in vitro or on sprouts.
1048. J Sci Food Agric. 2012 Nov;92(14):2891-8. doi: 10.1002/jsfa.5620. Epub 2012
Feb
15.
Fatty acid, tocopherol and carotenoid content in herbage and milk affected by
sward composition and season of grazing.
Larsen MK(1), Fretté XC, Kristensen T, Eriksen J, Søegaard K, Nielsen JH.
Author information:
(1)Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark.
mette.larsen@agrsci.dk
BACKGROUND: The aim of the present work was to study to what extent grazing large
amounts of white clover (WCL), red clover (RCL), lucerne (LUC) or chicory (CHI)
was suitable for production of bovine milk with a high milk fat content of
tocopherols, carotenoids, α-linolenic acid and conjugated linoleic acid.
RESULTS: WCL, RCL, LUC and CHI swards were established to investigate the
influence of herbage composition on the milk quality of grazing dairy cows at
three periods. Average milk fat composition showed high concentrations of
linolenic acid (12 mg g⁻¹), conjugated linoleic acid (13 mg g⁻¹), carotenoids (6
µg g⁻¹) and α-tocopherol (21 µg g⁻¹ milk fat). There were minor differences
between herbage types and periods, but multivariate analysis of these data showed
no clear grouping. Chemical composition of herbage varied with species as well as
period, but it was not possible to relate milk and feed contents of specific
fatty acids, carotenoids or tocopherols.
CONCLUSION: All four herbages tested were suitable for production of milk with a
high content of beneficial compounds. Thus any of these herbages could be used in
production of such differentiated milk based on a large proportion of grazing in
the ration.

DOI: 10.1002/jsfa.5620
1049. J Sci Food Agric. 2012 Aug 30;92(11):2265-73. doi: 10.1002/jsfa.5619. Epub
2012
Feb 15.
Fermentation, degradation and microbial nitrogen partitioning for three forage

colour phenotypes within anthocyanidin-accumulating Lc-alfalfa progeny.
Jonker A(1), Gruber MY, Wang Y, Narvaez N, Coulman B, McKinnon JJ, Christensen
DA, Azarfar A, Yu P.
Author information:
Saskatoon, SK, Canada. arjan.jonker@agresearch.co.nz
BACKGROUND: Alfalfa has the disadvantage of having a rapid initial rate of

protein degradation, which results in pasture bloat, low efficiency of protein
utilisation and excessive nitrogen (N) pollution into the environment for cattle.
Introducing a gene that stimulates the accumulation of monomeric/polymeric
anthocyanidins might reduce the ruminal protein degradation rate (by fixing
protein and/or direct interaction with microbes) and additionally reduce methane
emission. The objectives of this study were to evaluate in vitro fermentation,
degradation and microbial N partitioning of three forage colour phenotypes
(green, light purple-green (LPG) and purple-green (PG)) within newly developed
Lc-progeny and to compare them with those of parental green non-transgenic (NT)
alfalfa.
RESULTS: PG-Lc accumulated more anthocyanidin compared with Green-Lc (P < 0.05),
with LPG-Lc intermediate. Volatile fatty acids and potentially degradable dry
matter (DM) and N were similar among the four phenotypes. Gas, methane and
ammonia accumulation rates were slower for the two purple-Lc phenotypes compared
with NT-alfalfa (P < 0.05), while Green-Lc was intermediate. Effective degradable
DM and N were lower in the three Lc-phenotypes (P < 0.05) compared with
NT-alfalfa. Anthocyanidin concentration was negatively correlated (P < 0.05) with
gas and methane production rates and effective degradability of DM and N.
CONCLUSION: The Lc-alfalfa phenotypes accumulated anthocyanidin. Fermentation and
degradation parameters indicated a reduced rate of fermentation and effective
degradability for both purple anthocyanidin-accumulating Lc-alfalfa phenotypes
compared with NT-alfalfa.
DOI: 10.1002/jsfa.5619
1050. Plant Sci. 2012 Apr;185-186:65-77. doi: 10.1016/j.plantsci.2011.10.014. Epub

2011
Oct 25.
Modifying crops to increase cell wall digestibility.
Jung HJ(1), Samac DA, Sarath G.
Author information:
(1)USDA-Agricultural Research Service, Plant Science Research Unit, St. Paul, MN
55108, USA. Hans.Jung@ars.usda.gov
Improving digestibility of roughage cell walls will improve ruminant animal
performance and reduce loss of nutrients to the environment. The main
digestibility impediment for dicotyledonous plants is highly lignified secondary
cell walls, notably in stem secondary xylem, which become almost non-digestible.
Digestibility of grasses is slowed severely by lignification of most tissues, but
these cell walls remain largely digestible. Cell wall lignification creates an
access barrier to potentially digestible wall material by rumen bacteria if cells
have not been physically ruptured. Traditional breeding has focused on increasing
total dry matter digestibility rather than cell wall digestibility, which has
resulted in minimal reductions in cell wall lignification. Brown midrib mutants
in some annual grasses exhibit small reductions in lignin concentration and
improved cell wall digestibility. Similarly, transgenic approaches
down-regulating genes in monolignol synthesis have produced plants with reduced
lignin content and improved cell wall digestibility. While major reductions in
lignin concentration have been associated with poor plant fitness, smaller
reductions in lignin provided measurable improvements in digestibility without
significantly impacting agronomic fitness. Additional targets for genetic
modification to enhance digestibility and improve roughages for use as biofuel
feedstocks are discussed; including manipulating cell wall polysaccharide
composition, novel lignin structures, reduced lignin/polysaccharide
cross-linking, smaller lignin polymers, enhanced development of non-lignified
tissues, and targeting specific cell types. Greater tissue specificity of
transgene expression will be needed to maximize benefits while avoiding negative
impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter.
Published by Elsevier Ireland Ltd.
DOI: 10.1016/j.plantsci.2011.10.014
1051. J Environ Biol. 2011 Sep;32(5):559-65.
Assessment of genetic variation in lucerne (Medicago sativa L.) using protease

inhibitor activities and RAPD markers.
Chandra A(1), Pandey KC.
Author information:
(1)Crop Improvement Division, Indian Grassland and Fodder Research Institute,
Jhansi-284003, India. amaresh62@yahoo.com
The aim of the present study is to identify and characterize lucerne lines
resistance to weevil infestation. After three years of field screening for
resistance to weevil infestation, 13 lines of lucerne were selected to assess the
genotypic variations for lucerne weevil (Hypera postica Gyll.) at biochemical and
molecular levels. Total phenols varied from 0.15 to 0.91 mg g (DM) in these
genotypes. The highest trypsin (11.11 unit mg(-1) protein) and chymotrypsin (93.0
unit mg(-1) protein) inhibitors activities were recorded in G-1-02 and B-4-03
lines respectively, whereas highest alpha-amylases inhibitor activity (14.2 unit
mg(-1) protein) in C-6-01. Zymogram patterns for trypsin inhibitor activity
showed quantitative variations among the lines. In total 262 DNA fragments were
generated when 45 deca-mer random primers were employed. Genetic variation in
terms of genetic distance ranged from 0.65 to 0.85. Sequential Agglomerative
Hierarchical and Nested (SAHN) clustering using the Un-weighted Pair Group Method
with Arithmetic mean (UPGMA) algorithm yielded two clusters (cluster I and II)
which converged at 72% similarity level. Cluster I contained most of the lines
having low level of weevil infestation. High bootstrap values (>40) indicated the
significance of nodes embodied in these two clusters. However, SDS-PAGE analysis
of the leaf proteins of these 13 lines showed no major variations except minor
difference in the protein bands of molecular weights between 14 to 20 kD.
1052. J Food Sci. 2012 Feb;77(2):M121-6. doi: 10.1111/j.1750-3841.2011.02553.x.

Epub
2012 Feb 6.
E. coli o157:H7 population reduction from alfalfa seeds with malic acid and
thiamine dilauryl sulfate and quality evaluation of the resulting sprouts.
Fransisca L(1), Park HK, Feng H.
Author information:
(1)Dept of Food Science and Human Nutrition, Univ of Illinois at
Urbana-Champaign, 1304 W Pennsylvania Ave, Urbana, IL 61801, USA.
It has been reported that washing seeds with a 20000 ppm Ca(OCl)(2) solution as
recommended by the U.S. Food and Drug Administration is unable to eliminate E.
coli cells attached to seed surfaces, and the bacterial cells that have survived
a sanitation wash can proliferate during sprouting to a high population. The
objectives of this research were to examine the efficacy of malic acid (MA) and
thiamine dilauryl sulfate (TDS) combined treatments on the inactivation of E.
coli O157:H7 on alfalfa seeds, to study the growth of the remaining E. coli cells
during sprouting, and to evaluate the sprout quality. When 10 g of inoculated
alfalfa seeds were washed in a 10% MA-1% TDS solution, a complete elimination of
E. coli was achieved. The same result was observed by washing the seeds in a
20000 ppm Ca(OCl)(2) solution. However, when the seed size was increased to 50 g
while maintaining the same seed-to-sanitizer ratio, both the MA + TDS and the
20000 ppm chlorine washes failed to completely inactivate the E. coli cells on
the seeds. Nevertheless, the 10% MA-1% TDS solution was significantly more
effective in E. coli count reduction compared to the 20000 ppm chlorine wash. The
E. coli O157:H7 cells remaining on the seeds after treatments with both
sanitizers grew up to 7 to 8 log CFU/g sprout after 96 h of sprouting. Under the
treatment conditions used in this study, none of the treatments resulted in
significant differences in germination rate, yield, or quality of the
sprouts.PRACTICAL APPLICATION: The malic acid (MA) and thiamine dilauryl sulfate
(TDS) combined treatment may provide a new solution to secure the microbial
safety of seeds and sprouts. An important finding of this study is that seed
sample size has a significant impact on the inactivation of E. coli O157:H7 on
alfalfa seeds. The microbial inactivation results obtained in a laboratory set-up
cannot be directly applied to a large scale operation. A validation test on the
large scale has to be performed to evaluate the efficacy of the sanitizer.
© 2012 Institute of Food Technologists®
DOI: 10.1111/j.1750-3841.2011.02553.x
1053. Proc Natl Acad Sci U S A. 2012 Jan 31;109(5):1766-71. doi:

10.1073/pnas.1120916109. Epub 2012 Jan 17.
MtPAR MYB transcription factor acts as an on switch for proanthocyanidin

biosynthesis in Medicago truncatula.
Verdier J(1), Zhao J, Torres-Jerez I, Ge S, Liu C, He X, Mysore KS, Dixon RA,
Udvardi MK.
Author information:
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401,
USA.
MtPAR (Medicago truncatula proanthocyanidin regulator) is an MYB family

transcription factor that functions as a key regulator of proanthocyanidin (PA)
biosynthesis in the model legume Medicago truncatula. MtPAR expression is
confined to the seed coat, the site of PA accumulation. Loss-of-function par
mutants contained substantially less PA in the seed coat than the wild type,
whereas levels of anthocyanin and other specialized metabolites were normal in
the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was
expressed ectopically in transformed hairy roots of Medicago. Transcriptome
analysis of par mutants and MtPAR-expressing hairy roots, coupled with yeast
one-hybrid analysis, revealed that MtPAR positively regulates genes encoding
enzymes of the flavonoid-PA pathway via a probable activation of WD40-1.
Expression of MtPAR in the forage legume alfalfa (Medicago sativa) resulted in
detectable levels of PA in shoots, highlighting the potential of this gene for
biotechnological strategies to increase PAs in forage legumes for reduction of
pasture bloat in ruminant animals.
DOI: 10.1073/pnas.1120916109
PMCID: PMC3277187
1054. Huan Jing Ke Xue. 2011 Nov;32(11):3341-7.
[Cation exchanges during the process of Cd(2+) absorption by Alfalfa in aqueous

solutions].
Li YP(1), Yin H, Ye JS, Peng H, Qin HM, Long Y, He BY, Zhang N, Tong Y, Peng SF.
Author information:
(1)Department of Environmental Engineering, Jinan University, Guangzhou 510632,
China. lyp282008@163.com
A hydroponic experiment was conducted to investigate the cation exchanges during

the process of Cd2+ absorption by Alfalfa in aqueous solution. The absorption
efficiency of Alfalfa plants with 0-10 mg x L(-1) Cd2+ treatments, changes of
Na+, K+, Mg2+, Ca2+ and NH4(+) concentration, and the variation of pH values at
different absorption time (0, 1, 2, 4, 8, 12, 24 and 72 h) were studied
separately. The multiple linear regressions between Cd2+ absorption and cation
variation were analyzed. The results indicated that when Cd2+ concentrations were
0.1, 1, 5, 10 mg x L(-1), the absorption efficiencies of Cd2+ by Alfalfa after 72
h were 85.86%, 52.14%, 15.97% and 7.81%. Cation exchange was involved in the
removal of Cd2+ by Alfalfa in aqueous solution. Except for NH4(+), the
concentrations of cationic metals Na+, K+, Mg2+ and Ca2+ in aqueous solution
increased over time, which increased 11.30% - 61.72%, 21.44% - 98.73%, 24.09% -
8.90% and 37.04% - 191.96%, respectively. Kinetic studies illuminated that the
release of Na+, K+, Mg2+ and Ca2+ by Alfalfa in Cd2+ solution with initial
concentrations of 0, 0. 1, 1, 5, 10 mg x L(-1) best fitted pseudo-second-order
equation,while the NH4(+) release fitted this model when Cd2+ concentrations were
1, 5, 10 mg x L(-1). The gradual decrease of pH during adsorption of Cd2+ by
Alfalfa was observed. As the competition ion of Cd2+, H+ might affect the
capacity of Alfalfa root system to absorb Cd2+. The ternary linear equation
results demonstrated that the content of Cd2+ absorption by Alfalfa strongly
related with the release of Ca2+, Mg2+, Na+. And this exchange mainly occurred
among Cd2+ and divalent cations.
1055. J Food Prot. 2012 Feb;75(2):261-9. doi: 10.4315/0362-028X.JFP-11-279.
Effect of surface roughness on inactivation of Escherichia coli O157:H7 87-23 by

new organic acid-surfactant combinations on alfalfa, broccoli, and radish seeds.
Fransisca L(1), Feng H.
Author information:
(1)Department of Food Science and Human Nutrition, University of Illinois at
Urbana-Champaign, Urbana, IL 61801, USA.
Surface roughness has been reported as one of the factors affecting microbial
attachment and removal. Seed surfaces are complex, and different seed varieties
have different surface topographies. As a result, a sanitizer effective in
eliminating pathogenic bacteria on one seed may not be as effective when applied
to another seed. The objectives of this research were (i) to investigate the
efficacy of malic acid and thiamine dilaurylsulfate (TDS) combined treatments for
inactivation of E. coli O157:H7 strain 87-23 on alfalfa, broccoli, and radish
seeds, (ii) to quantify surface roughness of the seeds, and (iii) to determine
the correlation between microbial removal and surface roughness. The surface
roughness of each seed type was measured by confocal laser scanning microscopy
(CLSM) and surface profilometry. Surface roughness (Ra) values of the seeds were
then calculated from CLSM data. Seeds inoculated with E. coli O157:H7 87-23 were
washed for 20 min in malic acid and TDS solutions and rinsed for 10 min in tap
water. Radish seeds had the highest Ra values, followed by broccoli and alfalfa
seeds. A combination of 10% malic acid and 1% TDS was more effective than 20,000
ppm of Ca(OCl)(2) for inactivation of E. coli O157:H7 87-23 on broccoli seeds,
while the inactivation on radish and alfalfa seeds was not significantly
different compared with the 20,000-ppm Ca(OCl)(2) wash. Overall, a negative
correlation existed between the seeds' Ra values and microbial removal. Different
seeds had different surface roughness, contributing to discrepancies in the
ability of the sanitizers to eliminate E. coli O157:H7 87-23 on the seeds.
Therefore, the effectiveness of one sanitizer on one seed type should not be
translated to all seed varieties.
DOI: 10.4315/0362-028X.JFP-11-279
1056. Plant Physiol Biochem. 2012 Mar;52:162-8. doi: 10.1016/j.plaphy.2011.12.010.

Epub
2012 Jan 10.
The histone phosphatase inhibitory property of plant nucleosome assembly

protein-related proteins (NRPs).
Bíró J(1), Farkas I, Domoki M, Otvös K, Bottka S, Dombrádi V, Fehér A.
Author information:
Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary.
SET/I(2)(PP2A), a member of the family of nucleosome assembly proteins (NAPs),

has been previously described as a multifunctional protein inhibiting protein
phosphatase 2A (PP2A)-mediated histone H3((pSer10)) dephosphorylation during the
heat shock response in animal cells. In the present work we demonstrate that its
plant orthologs, designated as NAP-related proteins (NRPs), have a similar
in vitro biochemical activity and interact with PP2A and histone
H3((pSer10))in vivo. Although heat shock gene promoters were found to be
associated with histone H3((pSer10))-marked chromatin following a high
temperature treatment, heat shock gene expression was not affected in
NRP-deficient mutant Arabidopsis thaliana (L.) plantlets. These observations
indicate that NRPs are potential regulators of histone dephosphorylation in
plants, but they are dispensable for gene expression reorganization in response
to heat shock.
DOI: 10.1016/j.plaphy.2011.12.010
1057. Vet Parasitol. 2012 Jun 8;187(1-2):237-43. doi: 10.1016/j.vetpar.2011.12.040.

Epub 2012 Jan 5.
Anthelmintic efficacy of five tropical native Australian plants against

Haemonchus contortus and Trichostrongylus colubriformis in experimentally
infected goats (Capra hircus).
Moreno FC(1), Gordon IJ, Knox MR, Summer PM, Skerrat LF, Benvenutti MA, Saumell
CA.
Author information:
(1)INTA EEA Balcarce, Buenos Aires, Argentina. fmoreno@balcarce.inta.gov.ar
The study of the anthelmintic properties of plants rich in plant secondary

metabolites can provide ecologically sound methods for the treatment of parasites
on grazing animals. The purpose of the present study was to evaluate the
anthelmintic effect of five tropical native Australian plant species rich in
plant secondary metabolites on adult Haemonchus contortus and Trichostrongylus
colubriformis in experimentally infected goats. Thirty young, nematode-free goats
were infected with 2500 H. contortus and 5000 T. colubriformis infective larvae
thrice weekly for a week (day 1-7 of the experiment). On day 27 after first
infection, the goats were allocated into six groups of five animals per group.
From day 28 to day 35, fresh leaves from Acacia salicina, Acacia nilotica,
Eucalyptus corymbia, Casuarina cunninghamiana and Eucalyptus drepanophylla were
included in the goats diet. Five groups were offered leaves from one of these
plant species and one group, the untreated control, received only the basal diet
formulated with 20% Medicago sativa and 80% Avena sativa. Following plant
material administration, the goats were monitored daily until day 40 and then
slaughtered on day 41. Total faecal worm egg output, total production of larvae
recovered from faecal cultures, total post-mortem worm burdens and the per capita
fecundity of female worms were estimated. The toxicity of the plant species for
the goats was measured by histopathological analyses of liver and kidney samples.
Results showed that goats feeding on the plant material rich in plant secondary
metabolites had significantly lower egg output compared to the control goats
(P<0.05). A similar response was found for larval production in both H. contortus
and T. colubriformis supporting that egg output was affected in both species.
Although the total worm burdens were not affected by the plant material (P>0.05),
the per capita fecundity was significantly reduced by E. corymbia, A. nilotica
and A. salicina (P<0.05). No signs of toxicity were detected in the liver or
kidney samples. It is concluded that goats can benefit from the short-term
ingestion of plant secondary metabolites, which reduce the total faecal egg
output and thus decrease the potential for re-infection from the pasture.
DOI: 10.1016/j.vetpar.2011.12.040
1058. Food Microbiol. 2012 May;30(1):83-90. doi: 10.1016/j.fm.2011.12.003. Epub

2011
Dec 16.
Efficacy of a post enrichment acid treatment for isolation of Escherichia coli

O157:H7 from alfalfa sprouts.
Fedio WM(1), Jinneman KC, Yoshitomi KJ, Zapata R, Weagant SD.
Author information:
(1)Food Safety Laboratory, Center for Animal Health, Food Safety and Biosecurity,
New Mexico State University, 2990 Knox Street, Las Cruces, NM 88003, USA.
wfedio@nmsu.edu
The enrichment, detection and isolation procedure in the current US FDA BAM have
been shown effective for Escherichia coli O157:H7 in a wide variety of foods.
Recently reported modifications to the enrichment protocol, including
post-enrichment immunomagnetic separation (IMS) procedures have improved
sensitivity of the method for alfalfa sprouts. However, cultural isolation on
selective agar plates still presents a challenge in this food matrix. The focus
of this study was to reduce levels of competing microflora and enhance isolation
of E. coli O157:H7 on selective agars. We optimized the use of a short acid
treatment after enrichment and with post-enrichment IMS beads. The optimized acid
treatments were then evaluated in alfalfa sprouts artificially contaminated with
E. coli O157:H7. After 5h enrichment of alfalfa sprouts contaminated at 0.2cfu/g,
there was significant improvement in isolation on the selective plates following
acid treatment of two types of IMS beads. Following 24h enrichment of alfalfa
sprouts contaminated at low levels, E. coli O157:H7 was only recovered from 8/25
samples when no IMS or acid treatments were used. The use of only the acid
treatment improved recovery to 19/25 samples. Following IMS of the enrichment
broths, acid treatment increased isolation to 23/25 for Pathatrix™ and 24/25 for
BeadRetriever™ IMS. Acid treatment (pH 2) of the enrichment broth for 30min or
IMS beads for 7min is a simple and rapid way to greatly improve isolation of
E. coli O157 from alfalfa sprout enrichments by reducing the interfering
microflora on the selective media.
DOI: 10.1016/j.fm.2011.12.003

10.1111/j.1439-0396.2011.01269.x. Epub 2012 Jan 20.
Fermentation of six different forages in the semi-continuous fermentation

technique Caesitec.
Vosmer J(1), Liesegang A, Wanner M, Zeyner A, Suter D, Hoelzle L, Wichert B.
Author information:
(1)Institute of Animal Nutrition, Vetsuisse-Faculty University of Zurich, Zurich,
Switzerland. judith-vosmer@gmx.de
The aim of the present study was to compare carbohydrate degradation of forages
which store carbohydrates either predominantly as fructan or starch, in horses'
hindgut. The effects of an abrupt change from hay-based feeding to green
fodder-based feeding on the caecal flora were tested with the in vitro hindgut
simulation technique 'Caesitec'. Six trials with different forages (English
ryegrass, tall fescue, grass mixture-horses, grass mixture-cows, lucerne, white
clover) were conducted. During a 4-day stabilisation period, samples were taken
once a day before loading the fermenters with hay. After diet-change to
forage-based feeding, samples were taken four times a day. Ammonia and pH-value
were measured before and 1, 2 and 6 h after loading the 'Caesitec'. Gas formation
was measured daily. Bacterial numbers, lactate and short chain fatty acids were
detected at four time-points of each trial. The grass mixtures contained the
highest amounts of fructan. The pH-values were in the physiological range from pH
6 up to 7 (6.58-6.83) by feeding all forages. Gas formation, anaerobic and
aerobic bacterial numbers increased after diet change from hay to any forage. The
maximum amount of fructan (3.75 g/kg) in swiss pasture did not cause a permanent
pathological change in the hindgut-flora.
DOI: 10.1111/j.1439-0396.2011.01269.x
1060. AMB Express. 2012 Jan 20;2(1):6. doi: 10.1186/2191-0855-2-6.
Desiccation induces viable but Non-Culturable cells in Sinorhizobium meliloti

1021.
Vriezen JA(1), de Bruijn FJ, Nüsslein KR.
Author information:
(1)Department of Microbiology, University of Massachusetts, Amherst,
Massachusetts, USA, MA 01003. nusslein@microbio.umass.edu.
Sinorhizobium meliloti is a microorganism commercially used in the production of

e.g. Medicago sativa seed inocula. Many inocula are powder-based and production
includes a drying step. Although S. meliloti survives drying well, the quality of
the inocula is reduced during this process. In this study we determined survival
during desiccation of the commercial strains 102F84 and 102F85 as well as the
model strain USDA1021.The survival of S. meliloti 1021 was estimated during nine
weeks at 22% relative humidity. We found that after an initial rapid decline of
colony forming units, the decline slowed to a steady 10-fold reduction in colony
forming units every 22 days. In spite of the reduction in colony forming units,
the fraction of the population identified as viable (42-54%) based on the
Baclight live/dead stain did not change significantly over time. This change in
the ability of viable cells to form colonies shows (i) an underestimation of the
survival of rhizobial cells using plating methods, and that (ii) in a part of the
population desiccation induces a Viable But Non Culturable (VBNC)-like state,
which has not been reported before. Resuscitation attempts did not lead to a
higher recovery of colony forming units indicating the VBNC state is stable under
the conditions tested. This observation has important consequences for the use of
rhizobia. Finding methods to resuscitate this fraction may increase the quality
of powder-based seed inocula.
DOI: 10.1186/2191-0855-2-6
PMCID: PMC3293009
PMID: 22260437
1061. Environ Entomol. 2011 Aug;40(4):917-30. doi: 10.1603/EN10320.
Effect of temperature on post-wintering development and total lipid content of

alfalfa leafcutting bees.
O'Neill KM(1), O'Neill RP, Kemp WP, Delphia CM.
Author information:
(1)Department of Land Resources and Environmental Sciences, Montana State
University, Bozeman, MT 59717, USA. koneill@montana.edu
Temperature plays an important role in effective management of the alfalfa

leafcutting bee [Megachile rotundata (F.); Megachilidae], the major commercial
pollinator of seed alfalfa [Medicago sativa (L.); Fabaceae] in North America. To
improve our understanding of threshold and optimum rearing temperatures of M.
rotundata, we examined the effect of temperature on postwintering development by
using a greater number of temperature treatments than applied in previous studies
(19 versus eight or fewer) and analytical tools formulated to model nonlinear
relationships between temperature and insect development rates. We also tested
the hypothesis that rearing temperature influences adult body lipid content at
emergence, which could affect adult survival, establishment and performance as a
pollinator, and reproductive success. We found that the Lactin-2 and Briere-2
models provided the best fits to data and gave reasonable estimates of lower
(16-18°C) and upper (36-39°C) developmental thresholds and optimum (33-34°C)
rearing temperatures for maximizing development rate. Bees successfully emerged
over a broad range of temperatures (22-35°C), but variation in development rate
among individuals reared at the same temperature was lowest at 31-33°C. The
optimum rearing temperature to maximize the proportion of body lipids in adults
was 27-29°C. Our results are discussed in relation to previous findings and speak
to the difficulties in designing practical rearing guidelines that simultaneously
maximize development rate, survival, and adult condition, while synchronizing
adult emergence with alfalfa bloom.
DOI: 10.1603/EN10320
Field and vegetable crops as hosts of larval western spotted cucumber beetle
(Coleoptera: Chrysomelidae).
Pedersen AB(1), Godfrey LD.
Author information:
(1)Department of Entomology, University of California, Davis, Davis, CA 95616,
USA. abpedersen@ucdavis.edu
The western spotted cucumber beetle, Diabrotica undecimpunctata undecimpunctata

Mannerheim, is an important pest of melons (Cucurmis melo L.) in northern
California. Recent observations indicate that adults are using alfalfa (Medicago
sativa L.) as a feeding host and larvae may be developing on the roots.
Greenhouse studies were conducted during the winters of 2009 and 2010 in which
larval development was compared on the roots of six field and vegetable crops
commonly grown in the southern Sacramento Valley. The growth parameters used to
evaluate the hosts were larval and pupal head capsule width, body width, and body
length as well as total survival percentage and survival percentage to the third
instar. According to larval growth and survivorship in 2009, maize (Zea mays L.)
was the best host, followed by alfalfa and tomato (Solanum lycopersicum L.) that
were roughly equivalent to one another. Melon was a slightly weaker host than
alfalfa and tomato; sunflower (Helianthus annuus L.) was a completely
incompatible host and thus dropped from the 2010 study. In 2010, melon was the
weakest host for larval development. Maize was the superior host again followed
closely by alfalfa that performed slightly better than tomato and lima bean
(Phaseolus lunatus L.). Data suggest that larval western spotted cucumber beetles
may primarily develop outside of melon fields and the adults emigrate to melons.
DOI: 10.1603/EN10143
1063. Chemosphere. 2012 Apr;87(3):217-25. doi: 10.1016/j.chemosphere.2011.12.063.

Epub
2012 Jan 14.
Phytoremediation of pyrene contaminated soils amended with compost and planted

with ryegrass and alfalfa.
Wang MC(1), Chen YT, Chen SH, Chang Chien SW, Sunkara SV.
Author information:
(1)Department of Environmental Engineering and Management, Chaoyang University of
Technology, Wufong District 41349, Taichung City, Taiwan, ROC.
Ryegrass (Lolium perenne) and alfalfa (Medicago sativa) were planted in pots to
remediate pyrene contaminated quartz sand (as a control group), alluvial and red
soils amended with and without compost. The pyrene degradation percentages in
quartz sand, alluvial soil, and red soil amended with compost (5%, w/w) and
planted with ryegrass and alfalfa for 90 d growth were 98-99% and 97-99%,
respectively, while those of pyrene in the corresponding treatments amended
without compost but planted with ryegrass and alfalfa were 91-96% and 58-89%,
respectively. Further, those of pyrene in the respective treatments amended with
and without compost but unplanted were 54-77% and 51-63%, respectively. Pyrene
contents in both roots and aboveground parts of ryegrass and alfalfa after 90 d
growth in quartz sand and the two soils amended with or without compost were
trace amounts. Statistical analyses for the parameters of ryegrass planted in red
and alluvial soils including the concentrations of total water-soluble volatile
low molecular weight organic acids, microbial population, pyrene degradation
percentage, and spiked pyrene concentration show significant correlations at 5%
and mostly 1% probability levels, by the analysis of variance. It was thus
suggested that the interactions among the consortia of plant root exudates,
microorganisms, and amended compost in rhizosphere soils could facilitate
bioavailability of pyrene and subsequently enhance its dissipation.
Copyright Â© 2012 Elsevier Ltd. All rights reserved.
1064. FEMS Microbiol Ecol. 2012 Apr;80(1):159-67. doi:
10.1111/j.1574-6941.2011.01295.x. Epub 2012 Jan 18.
In situ identification of carboxymethyl cellulose-digesting bacteria in the rumen

of cattle fed alfalfa or triticale.
Kong Y(1), Xia Y, Seviour R, He M, McAllister T, Forster R.
Author information:
(1)Department of Biological Science and Technology, Kunming University, Kunming,
China.
A method was developed and used to arrest and stain reducing sugars (glucose)
produced by bacteria with cell-surface-associated carboxymethyl cellulase
(CMCase) and endoglucanase activities (CMC bacteria) in the rumen of cows fed
alfalfa or triticale. Precipitation of silver oxide on the surface of individual
cells was observed using cellulolytic bacterial pure cultures with known CMCase
activity and rumen mixed cultures. The CMC bacteria in the liquid and solid
fractions of the rumen digesta were identified using fluorescence in situ
hybridization (FISH) with currently available and newly designed oligonucleotide
probes. The CMC bacteria contributed between 8.2% and 10.1% to the total
bacterial cell numbers. Most of the CMC bacteria (75.2-78.5%) could be identified
by FISH probing. The known cellulolytic populations Ruminococcus flavefaciens,
R. albus, and Fibrobacter succinogenes constituted 44.5-53.1% of the total. Other
CMC bacteria identified hybridized with the probe Clo549 (11.2-23.0%) targeting
members of an uncharacterized genus in Clostridia, the probe Inc852 (8.9-10.7%)
targeting members of the family Incertae Sedis III and unclassified
Clostridiales, and the probe But1243 (< 1%) designed against members of genus
Butyrivibrio. Different forage feeds had no marked effects on the percentage
abundances of these identified CMC bacteria. All appeared to be involved in
cellulose degradation in the rumen of cows fed either alfalfa or triticale.

DOI: 10.1111/j.1574-6941.2011.01295.x
1065. J Insect Sci. 2011;11:144. doi: 10.1673/031.011.14401.
Foraging range of honey bees, Apis mellifera, in alfalfa seed production fields.
Hagler JR(1), Mueller S, Teuber LR, Machtley SA, Van Deynze A.
Author information:
(1)Arid Land Agricultural Research Center, USDA-ARS, 21881 North Cardon Lane,
Maricopa, AZ 85138, USA. james.hagler@ars.usda.gov
A study was conducted in 2006 and 2007 designed to examine the foraging range of
honey bees, Apis mellifera (Hymenoptera: Apidae), in a 15.2 km(2) area dominated
by a 128.9 ha glyphosate-resistant Roundup Ready® alfalfa seed production field
and several non-Roundup Ready alfalfa seed production fields (totaling 120.2 ha).
Each year, honey bee self-marking devices were placed on 112 selected honey bee
colonies originating from nine different apiary locations. The foraging bees
exiting each apiary location were uniquely marked so that the apiary of origin
and the distance traveled by the marked (field-collected) bees into each of the
alfalfa fields could be pinpointed. Honey bee self-marking devices were installed
on 14.4 and 11.2% of the total hives located within the research area in 2006 and
2007, respectively. The frequency of field-collected bees possessing a distinct
mark was similar, averaging 14.0% in 2006 and 12.6% in 2007. A grand total of
12,266 bees were collected from the various alfalfa fields on seven sampling
dates over the course of the study. The distances traveled by marked bees ranged
from a minimum of 45 m to a maximum of 5983 m. On average, marked bees were
recovered ~ 800 m from their apiary of origin and the recovery rate of marked
bees decreased exponentially as the distance from the apiary of origin increased.
Ultimately, these data will be used to identify the extent of pollen-mediated
gene flow from Roundup Ready to conventional alfalfa.
DOI: 10.1673/031.011.14401
PMCID: PMC3281370
1066. Mol Biol Rep. 2012 May;39(5):6067-75. doi: 10.1007/s11033-011-1421-y. Epub

2011
Dec 31.
Expression of an alfalfa (Medicago sativa L.) ethylene response factor gene

MsERF8 in tobacco plants enhances resistance to salinity.
Chen T(1), Yang Q, Gruber M, Kang J, Sun Y, Ding W, Zhang T, Zhang X.
Author information:
China Agricultural University, Beijing, 100193, People's Republic of China.
Ethylene response factors (ERF) play crucial roles in plant development and
response to stresses. Here, a novel cDNA fragment (MsERF8) encoding an ERF
protein with an AP2 domain was isolated and characterized from alfalfa. The
MsERF8 cDNA has an open reading frame of 603 bp and encodes a nuclear protein of
201 amino acids. Q-RT-PCR analysis revealed that MsERF8 was strongly enriched in
roots and leaves compared with stems, flower buds and flowers of mature alfalfa
plants. Bioinformatic analysis of the MsERF8 promoter indicated a number of
elements associated with stress-related responses, and MsERF8 transcripts in
alfalfa seedlings were induced by NaCl, PEG6000, Al(2)(SO(4))(3) and five
different hormones. Expression of MsERF8 in transgenic tobacco plants resulted in
higher tolerance to salinity than with non-transgenic plants. This data shows
that MsERF8 is a gene which prevents or alleviates salinity damage and has strong
potential to impart salt tolerance to other crop plants.
DOI: 10.1007/s11033-011-1421-y
1067. J Agric Food Chem. 2012 Jan 11;60(1):315-21. doi: 10.1021/jf204286r. Epub
2011
Dec 29.
Occurrence of pipecolic acid and pipecolic acid betaine (homostachydrine) in

Citrus genus plants.
Servillo L(1), Giovane A, Balestrieri ML, Ferrari G, Cautela D, Castaldo D.
Author information:
(1)Dipartimento di Biochimica e Biofisica, II Università degli Studi di Napoli,
via L De Crecchio 7, 80138 Naples, Italy. luigi.servillo@unina2.it
The presence of pipecolic acid and pipecolic acid betaine, also known as
homostachydrine, is herein reported for the first time in Citrus genus plants.
Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and
bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially
available, as a comparative source, extracts of alfalfa leaves ( Medicago sativa
L.) were used, in which homostachydrine is present at high concentration. Then,
the results where confirmed by comparison with an authentic standard synthesized
and purified starting from pipecolic acid. The synthesized standard was
characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When
subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike
its lower homologue proline betaine (also known as stachydrine), showed a pattern
of numerous ionic fragments that allowed unambiguous identification of the
compound. For the quantitation in the plant sources, high sensitivity and
specificity were achieved by monitoring the transition (158 → 72), which is
absent in the fragmentation patterns of other major osmolytes commonly used as
markers for studies of abiotic stress. As for the metabolic origin of
homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the
hypothesis that it could act as a homostachydrine precursor through direct
methylation.
DOI: 10.1021/jf204286r
1068. Rev Biol Trop. 2011 Dec;59(4):1651-62.
[Effect of spiders (Arachnida: Araneae) as predators of insect pest in alfalfa

crops (Medicago sativa) (Fabaceae) in Argentina].
[Article in Spanish]
Armendano A(1), González A.
Author information:
(1)Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CONICET), 2 No 584
(1900) La Plata, Argentina. aarmendano@hotmail.com
Spiders are predators that reduce insect pest populations in agroecosystems.

Trials were conducted to measure the selectivity against different insect preys,
the daily consumption, effect of predators alone and together with a known number
of preys, and the indirect effect of predators on vegetation. For this,
experimental units (1 x 1m) were used covered with a fine plastic mesh.
Misumenops pallidus, Oxyopes salticus and Araneus sp. were used as generalist
predators, and aphids, weevils, locusts, chrysomelids and Lepidoptera larvae as
their potential preys. Among the preys offered, the spiders preferred Lepidoptera
larvae compared to the other two pests groups (weevils and aphids). The maximum
consumption rate was of 93.33% for Lepidoptera larvae, 25.33% for aphids and
11.67% for weevils. The Q Index values for the three species of spiders showed a
positive selectivity only for defoliating larvae. O. salticus showed the highest
values of consumption rates while Rachiplusia nu was the most consumed. The
maximum value of consumption in 24 hours was showed by O. salticus on R. nu (C) =
2.8. The association of several species of predatory spiders increased the total
number of insects captured, and also showed that the addition of spiders caused a
decrease in the number of leaves damaged by the effect of lepidopterous larvae.

1069. J Microw Power Electromagn Energy. 2012;46(2):57-67.
The effect of microwave treatment on animal fodder.
Brodie G(1), Rath C(1), Devanny M(1), Reeve J(1), Lancaster C(1), Doherty T(1),
Harris G(1), Chaplin S(1), Laird C(1).
Author information:
(1)Melbourne School of Land and Environment, University of Melbourne, 3010
Victoria, Australia.
Preliminary research has suggested that in vitro dry matter disappearance (DMD)
of some poor quality animal fodder materials can be improved by microwave
treatment. Laboratory scale experiments revealed that dry matter percentage of
Lucerne hay increased by 1.7% as microwave treatment time increased from 0 to 80
seconds. The in vitro DMD of lucerne hay increased by 14.9% during the same
microwave treatment. In addition it was also demonstrated that microwave
treatment significantly increased starch digestion of oats compared to the
control samples. These experiments were followed up with a larger sample
experiment in which 25 kg bags of Lucerne fodder were treated for 7.5, 15, 22.5
or 30 minutes in an experimental 6 kW microwave chamber. Dry matter percentage
increased by 7.2% as microwave treatment time increased from 0 to 30 minutes.
Microwave treatment significantly increased DMD during an in vitro digestion
study; however there were no significant differences between the various
microwave treatment times. The 15 minute treatment resulted in the greatest
increase in dry matter disappearance (5.9%). The crude protein retained in the
digestion residues increased by 19.2% as microwave treatment increased from 0 to
30 minutes. These laboratory studies were followed up with an animal response
study in which a Merino sheep group being fed the microwave treated lucerne
gained 8.1% of their initial body weight by the end of the trial compared to a
0.4% increase in body weight for the control group.
1070. J Environ Sci (China). 2012;24(8):1476-82.
Effect of organic wastes on the plant-microbe remediation for removal of aged

PAHs in soils.
Zhang J(1), Lin X, Liu W, Wang Y, Zeng J, Chen H.
Author information:
The effectiveness of in-situ bioremediation of polycyclic aromatic hydrocarbons

(PAHs) may be inhibited by low nutrients and organic carbon. To evaluate the
effect of organic wastes on the PAHs removal efficiency of a plant-microbe
remediation system, contaminated agricultural soils were amended with different
dosages of sewage sludge (SS) and cattle manure (CM) in the presence of alfalfa
(Medicago sativa L.) and PAHs-degraders (Bacillus sp. and Flavobacterium sp.).
The results indicated that the alfalfa mean biomasses varied from 0.56 to 2.23
g/pot in root dry weight and from 1.80 to 4.88 g/pot in shoot dry weight. Low
dose amendments, with rates of SS at 0.1% and CM at 1%, had prominent effects on
plant growth and soil PAHs degradation. After 60-day incubation, compared with
about 5.6% in the control, 25.8% PAHs removal was observed for treatments in the
presence of alfalfa and PAHs-degraders; furthermore, when amended with different
dosages of SS and CM, the removed PAHs from soils increased by 35.5%-44.9% and
25.5%-42.3%, respectively. In particular, the degradation of
high-molecular-weight PAHs was up to 42.4%. Dehydrogenase activities (DH) ranged
between 0.41 and 1.83 microg triphenylformazan/(g dry soil x hr) and the numbers
of PAHs-degrading microbes (PDM) ranged from 1.14 x 10(6) to 16.6 x 10(6)
most-probable-number/g dry soil. Further investigation of the underlying
microbial mechanism revealed that both DH and PDM were stimulated by the addition
of organic wastes and significantly correlated with the removal ratio of PAHs. In
conclusion, the effect of organic waste application on soil PAHs removal to a
great extent is dependent on the interactional effect of nutrients and dissolved
organic matter in organic waste and soil microorganisms.
1071. J Anim Sci. 2012 Jun;90(6):1885-91. doi: 10.2527/jas.2011-4356. Epub 2011 Dec
28.
Free ferulic acid uptake in ram lambs.
Soberon MA(1), Cherney DJ, Cherney JH.
Author information:
(1)Cornell University, Ithaca, NY 14850, USA. mas329@cornell.edu
The objective of this research was to investigate the fate of free ferulic acid
(FA) in sheep. Ferulic acid is normally present in plants, bound to the
indigestible cell wall. If the FA present in a ruminant diet is released from the
cell wall with feed pretreatment methods, FA may be released into the rumen for
digestion or absorption into the bloodstream or both. Eight male Dorset × Finn
lambs were randomly assigned to 1 of 4 treatment (trt) concentrations, 0
(control), 3, 6, or 9 g/d free FA as part of a replicated 4 × 4 Latin square
design. Lambs were housed individually and consumed chopped alfalfa hay (Medicago
sativa; 22.8% CP, 39.3% NDF, 0.73 Mcal/kg NE(g)) ad libitum and 350 g corn grain
(Zea mays L.; 9.1% CP, 11.2% NDF, 1.52 Mcal/kg NE(g)) once daily at 0800 h. Basal
concentrations of FA in hay, grain, blood, feces, and urine were established
following a 14-d adjustment to diet and housing. An oral dose of free FA was
administered for 5 d via bolus after each morning feeding, after which hay,
grain, blood, feces, and urine were sampled. Body weights were recorded at the
beginning and end of each trt, and DMI was measured daily during trt periods. In
addition to trt, each lamb ingested a daily average of 3.78 g FA in its bound
form via the offered hay (2.67 mg/g FA; 1.0 kg/d DMI) and corn (3.17 mg/g FA;
0.35 kg/d DMI). The FA administered had a quadratic effect on average hay DMI
(1.25, 1.41, 1.41, and 1.29 kg/d for 0, 3, 6, or 9 g/d FA trt; P < 0.01; SE =
58.9 g), but lamb BW did not change as a result of FA trt (P = 0.28). The NDF
content and amount of FA in refusals were not affected by trt, (P = 0.30; P =
0.82, respectively). Fecal FA did not differ among trt or when compared with
basal FA (P = 0.53), while urine FA increased as FA dose increased (P < 0.01),
indicating that free FA was absorbed and transferred into urine. No free FA was
found in the plasma analyzed, suggesting that disappearance from the blood of
absorbed free FA occurred within the 5 h that passed between bolus dosage and
blood collection. An in vitro analysis was conducted to assess the degree of
inhibition of microbial NDF digestion caused by FA supplementation. In vitro, NDF
disappearance was not inhibited as a result of FA treatment (P = 0.80). These
data in combination with the results of the lamb study indicate that free FA as
0.24, 0.43, and 0.70% of DMI in lambs is absorbed and excreted in the urine as
opposed to the feces with no apparent effects on rumen microbial NDF digestion.
DOI: 10.2527/jas.2011-4356
1072. Oecologia. 2012 Jul;169(3):793-802. doi: 10.1007/s00442-011-2230-7. Epub 2011

Dec
24.
Grassland plant species diversity decreases invasion by increasing resource use.
Frankow-Lindberg BE(1).
Author information:
(1)Department of Crop Production Ecology, Swedish University of Agricultural
Sciences, Box 7043, SE-750 07, Uppsala, Sweden. bodil.frankow-lindberg@slu.se
Species richness of plant communities has been demonstrated to provide resistance

to invasion by unsown species, though the relationship with resource availability
varies between studies. The present work involved five grassland species grown in
monocultures and in four-species mixtures sown in accordance with a simplex
design. The species used represented different functional groups (i.e. grasses,
legumes and non-N(2)-fixing species), each of which differed internally in terms
of competitiveness. I hypothesized that sown diversity would negatively affect
invader performance by decreasing the availability of light and soil nitrogen (N)
for invading species, and that functional composition of the sown diversity would
affect the functional composition of the invading flora. The experimental plots
were harvested for two years, and were fertilized with 100 kg N ha(-1) each year.
The number of unsown species (classified into four functional groups) invading
each plot and their proportion of the biomass harvested were recorded. The
penetration of incoming light through the canopy, the apparent N uptake by the
sown species from the soil, and the mineral N content in the soil were measured.
I found that diverse communities captured more resources both above- and
belowground, and the number of invading species and their biomass production were
smaller in mixed than in monoculture plots. However, the sampling effect of one
grass was also strong. These results suggest that increased resource use in
diverse communities can reduce invasion.
DOI: 10.1007/s00442-011-2230-7
1073. J Dairy Sci. 2012 Jan;95(1):340-52. doi: 10.3168/jds.2011-4496.
Effects of a propionic acid-based preservative on storage characteristics,

nutritive value, and energy content for alfalfa hays packaged in large round
bales.
Coblentz WK(1), Bertram MG.
Author information:
(1)USDA-ARS, US Dairy Forage Research Center, Marshfield, WI 54449, USA.
During 2009 and 2010, alfalfa (Medicago sativa L.) hays from 2 cuttings harvested
from the same field site were used to evaluate the effects of a propionic
acid-based preservative on the storage characteristics and nutritive value of
hays stored as large round bales. A total of 87 large round bales (diameter =
1.5m) were included in the study; of these, 45 bales served as controls, whereas
42 were treated with a commercial propionic acid-based preservative at mean
application rates of 0.5±0.14 and 0.7±0.19% of bale weight, expressed on a wet
(as is) or dry matter basis, respectively. Initial bale moisture concentrations
ranged from 10.2 to 40.4%. Internal bale temperatures were monitored daily during
an outdoor storage period, and heating characteristics were summarized for each
bale as heating degree days (HDD) >30°C. For acid-treated bales, the regression
relationship between HDD and initial bale moisture was best fitted to a quadratic
model in which the linear term was dropped to improve fit (Y=2.02x(2) - 401;
R(2)=0.77); control hays were best fitted to a nonlinear model in which the
independent variable was squared [Y=4,112 - (4,549×e(-0.000559x*x)); R(2)=0.77].
Based on these regressions, acid-treated bales accumulated more HDD than control
hays when the initial bale moisture was >27.7%; this occurred largely because
acid treatment tended to prolong active heating relative to control hays. Linear
regressions of recoveries of dry matter on HDD did not differ on the basis of
treatment, yielding a common linear relationship of Y=-0.0066x+96.3 (R(2)=0.75).
Regressions relating changes (post-storage - pre-storage) in concentrations of
several nutritional components (neutral detergent fiber, lignin, ash, crude
protein, and total digestible nutrients) with HDD for acid-treated hays typically
exhibited more inflection points or were higher-ordered polynomial regressions
than those of control hays. These more complex responses probably reflected the
perturbation of normal heating patterns following acid treatment; however,
overall effects on post-storage nutritive value were relatively limited in scope.
The potential to improve nutritive value relative to cost for these large round
bales was not especially favorable, and hay producers may find that diligence to
achieve adequate field desiccation before baling, or use of oxygen-exclusion
methods, such as wrapping in plastic, may be better alternatives for preserving
moist hays.

DOI: 10.3168/jds.2011-4496
Changes in ruminal bacterial community composition following feeding of alfalfa

ensiled with a lactic acid bacterial inoculant.
Mohammed R(1), Stevenson DM, Beauchemin KA, Muck RE, Weimer PJ.
Author information:
(1)USDA-ARS, US Dairy Forage Research Center, Madison, WI 53706, USA.
Some silage inoculants help to improve silage quality and promote an increase in
milk production, possibly through altering the rumen microflora. We hypothesized
that rumen bacterial community composition (BCC) would be different in cows fed
alfalfa ensiled with the inoculant Lactobacillus plantarum MTD/1 (LP) compared
with those fed alfalfa ensiled without the inoculant (Ctrl). Eight ruminally
cannulated Holstein cows were allotted to 2 diets (Ctrl or LP) in a double
crossover design with four 28-d periods. Diets were formulated to contain (% dry
matter basis) 28.0% neutral detergent fiber and 16.2% crude protein, and
contained alfalfa silage, 50.9; corn silage, 20.6; high-moisture shelled corn,
21.4; soy hulls, 4.7; plus minerals and vitamins, 2.4. Ruminal digesta were
collected just before feeding on 3 consecutive days near the end of each period,
and were separated into solid and liquid phases. Microbial DNA was extracted from
each phase, amplified by PCR using domain-level bacterial primers, and subjected
to automated ribosomal intergenic spacer analysis. The pH was 4.56 and 4.86 and
the lactate-to-acetate ratio 9.8 and 4.4, respectively, for the treated and
untreated alfalfa silages. Dry matter intakes and milk production data were not
influenced by diets but showed a cow effect. Total volatile fatty acids (mM)
tended to be greater for LP compared with Ctrl. Individual volatile fatty acids
were not influenced by diets but showed a significant cow effect. Ruminal acetate
(mol/100 mol) and acetate-to-propionate ratio were lower and propionate (mol/100
mol) greater for the 2 milk fat-depressed (MFD; <3.2% fat content) cows compared
with the other 6 cows. Correspondence analysis of the 265 peaks in the automated
ribosomal intergenic spacer analysis profile across the 188 samples revealed that
the first 2 components contributed 7.1 and 3.8% to the total variation in the
profile. The ordination points representing the liquid and solid phases clustered
separately, indicating that these phases differed in BCC. The analysis of
similarity data showed differences between Ctrl and LP. The lactic acid bacterial
counts (log(10) cfu/g of wet silage) were 3.94 and 4.53 for the untreated and
treated silage, respectively, at ensiling. The relative population size (RPS) of
L. plantarum, determined by real-time PCR of 16S rRNA gene copies, was greater in
LP compared with Ctrl. The ordination points corresponding to certain individual
cows clustered separately, and the most distinctive bacterial communities were
those associated with MFD cows. The RPS of Megasphaera elsdenii was greater in 1
of the 2 MFD cows, although mean RPS of M. elsdenii did not differ between the
treatments. In addition to the differences in rumen BCC between LP and Ctrl, MFD
cows also displayed differences in BCC compared with cows with normal milk fat
yield.

DOI: 10.3168/jds.2011-4492
1075. Ann Bot. 2012 Mar;109(4):773-82. doi: 10.1093/aob/mcr309. Epub 2011 Dec 19.
Amplification, contraction and genomic spread of a satellite DNA family (E180) in

Medicago (Fabaceae) and allied genera.
Rosato M(1), Galián JA, Rosselló JA.
Author information:
(1)Jardín Botánico, Universidad de Valencia, Spain.
BACKGROUND AND AIMS: Satellite DNA is a genomic component present in virtually

all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an
important element in genome organization and evolution in plants. Here we assess
the presence and physical distribution of the repetitive DNA E180 family in
Medicago and allied genera. Our goals were to gain insight into the karyotype
evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic
and phylogenetic signal of a satellite DNA family in a genus hypothesized to have
a complex evolutionary history.
METHODS: Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium
were analysed by PCR to assess the presence of the repetitive E180 family, and
fluorescence in situ hybridization (FISH) was used for physical mapping in
somatic chromosomes.
KEY RESULTS: The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago,
eight of 12 species of Trigonella, six of seven species of Melilotus and in two
of 11 Trifolium species. Examination of the mitotic chromosomes revealed that
only 13 Medicago and two Trigonella species showed FISH signals using the E180
probe. Stronger hybridization signals were observed in subtelomeric and
interstitial loci than in the pericentromeric loci, suggesting this satellite
family has a preferential genomic location. Not all 13 Medicago species that
showed FISH localization of the E180 repeat were phylogenetically related.
However, nine of these species belong to the phylogenetically derived clade
including the M. sativa and M. arborea complexes.
CONCLUSIONS: The use of the E180 family as a phylogenetic marker in Medicago
should be viewed with caution. Its amplification appears to have been produced
through recurrent and independent evolutionary episodes in both annual and
perennial Medicago species as well as in basal and derived clades.
DOI: 10.1093/aob/mcr309
PMCID: PMC3286279
1076. Biol Trace Elem Res. 2012 Jun;147(1-3):315-9. doi: 10.1007/s12011-011-9290-8.

Epub 2011 Dec 13.
Using the pollen viability and morphology for fluoride pollution biomonitoring.
Malayeri BE(1), Noori M, Jafari M.
Author information:
(1)Bu-Ali Sina University, Hamedan, Islamic Republic of Iran. bmalayeri@gmail.com
The methods using plants for biomonitoring of air and soil quality are simple,
cheap, and fast and can supplement the classical physicochemical methods. In this
study, biological pollen characterization of some collected legume species from
an aluminum smelter area in Iran (IRALCO) was carried out to determine the actual
value of pollen as a bioindicator of the effects of soil and atmospheric
pollution. Young buds and flowers of six legumes (Cercis siliquastrum L.,
Medicago sativa L., Robinia pseudoacacia L., Melilotus officinalis (L.) lam,
Trifolium repens L., and Sophora alopecuroides L.) in polluted and control plants
were removed and compared. Studies of light and electron microscopic preparation
showed some abnormalities during pollen development in affect of fluoride
pollution. The viability of pollen grains estimated by staining with acetocarmine
shows sharp differences in smearing advanced pollen grains from abnormal ones.
Except M. officinalis, the pollen grains of C. siliquastrum, M. sativa, R.
pseudoacacia, T. repens, and S. alopecuroides in polluted areas showed light,
partial, or no staining with acetocarmine, whereas almost all of the control ones
clearly stained. Observation of the pollen grains by light microscopy and
scanning electron microscopy showed the significant effect of fluoride on shapes
and sizes of pollen grains. The stimulation and inhibition of these pollen
characteristics depend on the pollen species as well as on the pollutant and its
concentration. Therefore, pollen grains provide essential information on
biological impact of pollutants and they are good candidates for biomonitoring
the atmospheric and edaphic pollutions.
DOI: 10.1007/s12011-011-9290-8
1077. Appl Microbiol Biotechnol. 2012 Apr;94(2):461-6. doi: 10.1007/s00253-011-

3751-z.
Epub 2011 Dec 9.
Isolation, characterization, and quantification of Clostridium kluyveri from the

bovine rumen.
Weimer PJ(1), Stevenson DM.

Author information:
(1)United States Department of Agriculture, Madison, WI 53706, USA.
Paul.Weimer@ars.usda.gov
A strain of Clostridium kluyveri was isolated from the bovine rumen in a medium
containing ethanol as an electron donor and acetate and succinate (common
products of rumen fermentation) as electron acceptors. The isolate displayed a
narrow substrate range but wide temperature and pH ranges atypical of ruminal
bacteria and a maximum specific growth rate near the typical liquid dilution rate
of the rumen. Quantitative real-time PCR revealed that C. kluyveri was widespread
among bovine ruminal samples but was present at only very low levels (0.00002% to
0.0002% of bacterial 16S rRNA gene copy number). However, the species was present
in much higher levels (0.26% of bacterial 16S rRNA gene copy number) in lucerne
silage (but not maize silage) that comprised much of the cows' diet. While C.
kluyveri may account for several observations regarding ethanol utilization and
volatile fatty acid production in the rumen, its population size and growth
characteristics suggest that it is not a significant contributor to ruminal
metabolism in typical dairy cattle, although it may be a significant contributor
to silage fermentation. The ability of unadapted cultures to produce substantial
levels (12.8 g L(-1)) of caproic (hexanoic) acid in vitro suggests that this
strain may have potential for industrial production of caproic acid.
DOI: 10.1007/s00253-011-3751-z
1078. Zookeys. 2011;(143):23-45. doi: 10.3897/zookeys.143.2162. Epub 2011 Nov 1.
Nine eriophyoid mite species from Iran (Acari, Eriophyidae).
Xue XF(1), Sadeghi H, Hong XY, Sinaie S.
Author information:
(1)Department of Entomology, Nanjing Agricultural University, Nanjing, Jiangsu
210095, China.
Nine eriophyoid mites, including two new species and five new records, from Iran
are described and illustrated. They are Aceria acroptiloni Shevchenko & Kacalev,
1974, rec. n. on Rhaponticum repens (L.) Hidalgo (Asteraceae); Aceria anthocoptes
(Nalepa, 1892), rec. n. on Cirsium arvense (L.) Scop. (Asteraceae); Aceria
lactucae (Canestrini, 1893), rec. n. on Lactuca virosa L. (Asteraceae); Aceria
pulicarissp. n. on Pulicaria gnaphalodes (Vent.) Boiss. (Asteraceae); Aceria
tosichella Keifer, 1969 on Setaria viridis (L.) Beauv. (Poaceae); Eriophyes
rotundae Mohanasundaram, 1983 on Cyperus rotundus L. (Cyperaceae); Aculops
maroccensis Keifer, 1972, rec. n. on Mentha piperita L. (Lamiaceae); Aculus
medicagersp. n. on Medicago sativa L. (Leguminosae); Tetra lycopersici Xue &
Hong, 2005, rec. n. on Solanum nigrum L. (Solanaceae).
DOI: 10.3897/zookeys.143.2162
PMCID: PMC3208532
PMID: 22144865

10.1007/s11356-011-0672-4. Epub 2011 Dec 2.
Effects of alfalfa and organic fertilizer on benzo[a]pyrene dissipation in an

aged contaminated soil.
Fu D(1), Teng Y, Luo Y, Tu C, Li S, Li Z, Christie P.
Author information:
Soil Science, Chinese Academy of Sciences, Nanjing, China.
BACKGROUND: A climate-controlled pot experiment was conducted to investigate the

effects of planting alfalfa and applying organic fertilizer on the dissipation of
benzo[a]pyrene from an aged contaminated agricultural soil.
RESULTS: Short-term planting of alfalfa inhibited the dissipation of
benzo[a]pyrene from the soil by 8.9%, and organic fertilizer enhanced
benzo[a]pyrene removal from the soil by 11.6% compared with the unplanted and
unfertilized treatments, respectively. No significant interaction was observed
between alfalfa and organic fertilizer on benzo[a]pyrene dissipation.
Sterilization completely inhibited the removal of benzo[a]pyrene from the soil
indicating that its degradation by indigenous microorganisms may have been the
main mechanism of dissipation. Furthermore, significant positive relationships
were observed between benzo[a]pyrene removal and the contents of soil ammonium
nitrogen, nitrate nitrogen, and total mineral nitrogen at the end of the
experiment, suggesting that competition between plants and microorganisms for
nitrogen may have inhibited benzo[a]pyrene dissipation in the rhizosphere of
alfalfa and the addition of organic fertilizer may facilitate microbial
degradation of benzo[a]pyrene in the soil.
DOI: 10.1007/s11356-011-0672-4
PMCID: PMC3386484
1080. Biotechnol Lett. 2012 Jan;34(1):167-74. doi: 10.1007/s10529-011-0750-1. Epub

2011
Nov 30.
Overexpression of alfalfa mitochondrial HSP23 in prokaryotic and eukaryotic model

systems confers enhanced tolerance to salinity and arsenic stress.
Lee KW(1), Cha JY, Kim KH, Kim YG, Lee BH, Lee SH.
Author information:
(1)Grassland and Forages Division, National Institute of Animal Science, Rural
Development Administration, Cheonan, 330-801, Korea.
The cloning and characterization of a gene (MsHSP23) coding for a heat shock
protein in alfalfa in a prokaryotic and model plant system is described. MsHSP23
contains a 633 bp ORF encoding a polypeptide of 213 amino acids and exhibits
greater sequence similarity to mitochondrial sHSPs from dicotyledons than to
those from monocotyledons. When expressed in bacteria, recombinant MsHSP23
conferred tolerance to salinity and arsenic stress. Furthermore, MsHSP23 was
cloned in a plant expressing vector and transformed into tobacco, a eukaryotic
model organism. The transgenic plants exhibited enhanced tolerance to salinity
and arsenic stress under ex vitro conditions. In comparison to wild type plants,
the transgenic plants exhibited significantly lower electrolyte leakage.
Moreover, the transgenic plants had superior germination rates when placed on
medium containing arsenic. Taken together, these overexpression results imply
that MsHSP23 plays an important role in salinity and arsenic stress tolerance in
transgenic tobacco. This approach could be useful to develop stress tolerant
crops including forage crops.
DOI: 10.1007/s10529-011-0750-1
PMCID: PMC3235403
1081. Ying Yong Sheng Tai Xue Bao. 2011 Sep;22(9):2285-90.
[Effects of copper stress on Medicago sativa seedlings leaf antioxidative

system].
Wang SH(1), Zhang H, He QY.
Author information:
(1)College of Life Science, Anhui Science and Technology University, Bengbu
233100, Anhui, China. shwang70@yahoo.com.cn
This paper studied the effects of different concentration (0, 10, 30, 50, and 100
micromol x L(-1)) CuSO4 on the leaf physiological and biochemical characteristics
of Medicago sativa seedlings cultured with 1/4-strength Hoagland nutrient
solution. In treatments 30, 50, and 100 micromol x L(-1) of CuSO4, the leaf H2O2,
OH., and MDA contents and Fe-SOD and EST activities increased, and GSH and AsA
contents increased significantly. With increasing concentration Cu, the POD, GR,
and APX activities increased gradually, and the CAT and G6PDH activities
decreased after an initial increase. In treatments >10 micromol x L(-1) of Cu,
the capacity of leaf antioxidative system in reactive oxygen species scavenging
increased to prevent the injury from copper-induced oxidative stress.
1082. Bioinformation. 2011;7(5):264-70. Epub 2011 Oct 31.
Mining functional microsatellites in legume unigenes.
Roorkiwal M, Sharma PC.
Highly polymorphic and transferable microsatellites (SSRs) are important for

comparative genomics, genome analysis and phylogenetic studies. Development of
novel species-specific microsatellite markers remains a costly and
labor-intensive project. Therefore, interest has been shifted from genomic to
genic markers owing to their high inter-species transferability as they are
developed from conserved coding regions of the genome. This study concentrates on
comparative analysis of genic microsatellites in nine important legume (Arachis
hypogaea, Cajanus cajan, Cicer arietinum, Glycine max, Lotus japonicus, Medicago
truncatula, Phaseolus vulgaris, Pisum sativum and Vigna unguiculata) and two
model plant species (Oryza sativa and Arabidopsis thaliana). Screening of a total
of 228090 putative unique sequences spanning 219610522 bp using a microsatellite
search tool, MISA, identified 12.18% of the unigenes containing 36248
microsatellite motifs excluding mononucleotide repeats. Frequency of legume
unigene-derived SSRs was one SSR in every 6.0 kb of analyzed sequences. The
trinucleotide repeats were predominant in all the unigenes with the exception of
C. cajan, which showed prevalence of dinucleotide repeats over trinucleotide
repeats. Dinucleotide repeats along with trinucleotides counted for more than 90%
of the total microsatellites. Among dinucleotide and trinucleotide repeats, AG
and AAG motifs, respectively, were the most frequent. Microsatellite positive
chickpea unigenes were assigned Gene Ontology (GO) terms to identify the possible
role of unigenes in various molecular and biological functions. These unigene
based microsatellite markers will prove valuable for recording allelic variance
across germplasm collections, gene tagging and searching for putative candidate
genes.
PMCID: PMC3218422
PMID: 22125396
1083. Proc Natl Acad Sci U S A. 2011 Dec 20;108(51):20814-9. doi:

10.1073/pnas.1117873108. Epub 2011 Nov 28.
Salicylic acid mediates the reduced growth of lignin down-regulated plants.
Gallego-Giraldo L(1), Escamilla-Trevino L, Jackson LA, Dixon RA.
Author information:
USA.
Down-regulation of the enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl

transferase (HCT) in thale cress (Arabidopsis thaliana) and alfalfa (Medicago
sativa) leads to strongly reduced lignin levels, reduced recalcitrance of cell
walls to sugar release, but severe stunting of the plants. Levels of the stress
hormone salicylic acid (SA) are inversely proportional to lignin levels and
growth in a series of transgenic alfalfa plants in which lignin biosynthesis has
been perturbed at different biosynthetic steps. Reduction of SA levels by
genetically blocking its formation or causing its removal restores growth in
HCT-down-regulated Arabidopsis, although the plants maintain reduced lignin
levels. SA-mediated growth inhibition may occur via interference with gibberellic
acid signaling or responsiveness. Our data place SA as a central component in
growth signaling pathways that either sense flux into the monolignol pathway or
respond to secondary cell-wall integrity, and indicate that it is possible to
engineer plants with highly reduced cell-wall recalcitrance without negatively
impacting growth.
DOI: 10.1073/pnas.1117873108
PMCID: PMC3251142
1084. J Plant Physiol. 2012 Jan 15;169(2):176-82. doi: 10.1016/j.jplph.2011.08.016.

Epub 2011 Nov 25.
Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced

by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa).
Feng BH(1), Wu B, Zhang CR, Huang X, Chen YF, Huang XL.
Author information:
(1)The Key Laboratory of Gene Engineering of the Ministry of Education, School of
Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, China.
Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv.
Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin
(KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The
application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate
oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be
involved in the modulation of TDZ-induced morphogenesis responses. However, very
little is known about the genes involved in ethylene formation during somatic
embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved
in the effect of TDZ on inhibition of embryogenic competence of the alfalfa
callus. In this study we cloned full-length ACO cDNA from the alfalfa callus,
named MsACO, and observed changes in this gene expression during callus formation
and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis
showed that during the EC subcultural period, the expression level of MsACO in EC
was significantly increased on the 2nd day, rose to the highest level on the 8th
day and remained at this high level until the 21st day. However, the ACO
expression in the TDZ (0.93 μM)-treated callus was higher than in the EC
especially on the 8th day. Moreover the ACO expression level increased with
increasing TDZ concentration during the subcultural/maintenance period of the
callus. It is worth noting that comparing the treatment with TDZ alone, the
treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene
expressions and ACO activity in the treated callus. These results indicate that
the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene
expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic
competence of alfalfa callus could be mediated by ACO gene expression.
Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.
DOI: 10.1016/j.jplph.2011.08.016
1085. Appl Environ Microbiol. 2012 Feb;78(3):684-94. doi: 10.1128/AEM.06274-11.

Epub
2011 Nov 18.
Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in

Escherichia coli.
Malla S(1), Koffas MA, Kazlauskas RJ, Kim BG.
Author information:
(1)Laboratory of Molecular Biotechnology and Biomaterials, School of Chemical and
Biological Engineering, Seoul National University, Seoul, South Korea.
7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important

flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus
maculata, with various medicinal applications. To produce such valuable natural
flavonoids in large quantity, an Escherichia coli cell factory has been developed
to employ various plant biosynthetic pathways. Here, we report the generation of
7-OMA from its precursor, p-coumaric acid, in E. coli for the first time.
Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding
p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing
the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from
Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone
isomerase from Medicago sativa. In order to increase the availability of
malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α
and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA
synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced
NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis
thaliana, which was further methylated at position 7 to produce 7-OMA in the
presence of 7-O-methyltransferase from Streptomyces avermitilis.
Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as
intermediate products. Overexpression of the genes for flavanone biosynthesis and
modification pathways, along with malonyl-CoA overproduction in E. coli, produced
2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in
24 h, whereas the strain expressing only the flavanone modification enzymes
yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.
DOI: 10.1128/AEM.06274-11
PMCID: PMC3264098
1086. Nature. 2011 Nov 16;480(7378):520-4. doi: 10.1038/nature10625.
The Medicago genome provides insight into the evolution of rhizobial symbioses.
Young ND(1), Debellé F, Oldroyd GE, Geurts R, Cannon SB, Udvardi MK, Benedito VA,
Mayer KF, Gouzy J, Schoof H, Van de Peer Y, Proost S, Cook DR, Meyers BC,
Spannagl M, Cheung F, De Mita S, Krishnakumar V, Gundlach H, Zhou S, Mudge J,
Bharti AK, Murray JD, Naoumkina MA, Rosen B, Silverstein KA, Tang H, Rombauts S,
Zhao PX, Zhou P, Barbe V, Bardou P, Bechner M, Bellec A, Berger A, Bergès H,
Bidwell S, Bisseling T, Choisne N, Couloux A, Denny R, Deshpande S, Dai X, Doyle
JJ, Dudez AM, Farmer AD, Fouteau S, Franken C, Gibelin C, Gish J, Goldstein S,
González AJ, Green PJ, Hallab A, Hartog M, Hua A, Humphray SJ, Jeong DH, Jing Y,
Jöcker A, Kenton SM, Kim DJ, Klee K, Lai H, Lang C, Lin S, Macmil SL, Magdelenat
G, Matthews L, McCorrison J, Monaghan EL, Mun JH, Najar FZ, Nicholson C, Noirot
C, O'Bleness M, Paule CR, Poulain J, Prion F, Qin B, Qu C, Retzel EF, Riddle C,
Sallet E, Samain S, Samson N, Sanders I, Saurat O, Scarpelli C, Schiex T,
Segurens B, Severin AJ, Sherrier DJ, Shi R, Sims S, Singer SR, Sinharoy S, Sterck
L, Viollet A, Wang BB, Wang K, Wang M, Wang X, Warfsmann J, Weissenbach J, White
DD, White JD, Wiley GB, Wincker P, Xing Y, Yang L, Yao Z, Ying F, Zhai J, Zhou L,
Zuber A, Dénarié J, Dixon RA, May GD, Schwartz DC, Rogers J, Quétier F, Town CD,
Roe BA.
Author information:
(1)Department of Plant Pathology, University of Minnesota, St Paul, Minnesota
55108, USA. neviny@umn.edu
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their
ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a
process that takes place in a specialized structure known as the nodule. Legumes
belong to one of the two main groups of eurosids, the Fabidae, which includes
most species capable of endosymbiotic nitrogen fixation. Legumes comprise several
evolutionary lineages derived from a common ancestor 60 million years ago (Myr
ago). Papilionoids are the largest clade, dating nearly to the origin of legumes
and containing most cultivated species. Medicago truncatula is a long-established
model for the study of legume biology. Here we describe the draft sequence of the
M. truncatula euchromatin based on a recently completed BAC assembly supplemented
with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula
genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role
in shaping the M. truncatula genome and thereby contributed to the evolution of
endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome
experienced higher levels of rearrangement than two other sequenced legumes,
Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa
(Medicago sativa), a widely cultivated crop with limited genomics tools and
complex autotetraploid genetics. As such, the M. truncatula genome sequence
provides significant opportunities to expand alfalfa's genomic toolbox.
DOI: 10.1038/nature10625
PMCID: PMC3272368
1087. Theor Appl Genet. 2012 Mar;124(5):809-19. doi: 10.1007/s00122-011-1735-0.
Epub
2011 Nov 9.
Intron-length polymorphism identifies a Y2K4 dehydrin variant linked to superior

freezing tolerance in alfalfa.
Castonguay Y(1), Dubé MP, Cloutier J, Michaud R, Bertrand A, Laberge S.
Author information:
Canada, 2560 Hochelaga Blvd., Quebec, QC, Canada. yves.castonguay@agr.gc.ca
Erratum in
Theor Appl Genet. 2012 Mar;124(5):821-4.
Breeding alfalfa (Medicago sativa L.) with superior freezing tolerance could be
accelerated by the identification of molecular markers associated to that trait.
Dehydrins are a group of highly hydrophilic proteins that have been related to
low temperature tolerance. We previously identified a dehydrin restriction
fragment length polymorphism (RFLP) among populations recurrently selected for
superior tolerance to freezing (TF). Analysis of crosses between genotypes with
(D+) or without (D-) that RFLP revealed a significant impact on freezing
tolerance. In this study, we sought to develop a PCR marker for freezing
tolerance based on prior evidence of a relationship between size variation in
Y(2)K(4) dehydrins and the RFLP. Results confirm the enrichment of Y(2)K(4)
sequences of intermediate size (G2 group) in response to recurrent selection and
in the D+ progeny. Analysis of genomic sequences revealed significant
intron-length polymorphism (ILP) within the G2 group. G2 sequences with a
characteristic short intron were more frequently found in D+ genotypes.
Amplification using sequence-characterized amplified region (SCAR) primers
bordering the intron confirmed an increase in the number of fragments with small
introns in the D+ progeny and in the ATF5 population obtained after five cycles
of recurrent selection for superior TF within the cultivar Apica (ATF0).
Conversely, there was a reduction in the number of fragments with long introns in
the D+ progeny and in ATF5 as compared to ATF0. Recurrent selection for superior
tolerance to freezing in combination with ILP identified a sequence variant of
Y(2)K(4) dehydrins associated to the phenotypic response to selection.
DOI: 10.1007/s00122-011-1735-0
1088. Plant J. 2012 Mar;69(5):906-20. doi: 10.1111/j.1365-313X.2011.04842.x. Epub

2011
Dec 15.
The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal
arbuscule formation in rice.
Gutjahr C(1), Radovanovic D, Geoffroy J, Zhang Q, Siegler H, Chiapello M, Casieri

L, An K, An G, Guiderdoni E, Kumar CS, Sundaresan V, Harrison MJ, Paszkowski U.
Author information:
(1)Department of Plant Molecular Biology, University of Lausanne, Switzerland.
The central structure of the symbiotic association between plants and arbuscular
mycorrhizal (AM) fungi is the fungal arbuscule that delivers minerals to the
plant. Our earlier transcriptome analyses identified two half-size ABCG
transporters that displayed enhanced mRNA levels in mycorrhizal roots. We now
show specific transcript accumulation in arbusculated cells of both genes during
symbiosis. Presently, arbuscule-relevant factors from monocotyledons have not
been reported. Mutation of either of the Oryza sativa (rice) ABCG transporters
blocked arbuscule growth of different AM fungi at a small and stunted stage,
recapitulating the phenotype of Medicago truncatula stunted arbuscule 1 and 2
(str1 and str2) mutants that are deficient in homologous ABCG genes. This
phenotypic resemblance and phylogenetic analysis suggest functional conservation
of STR1 and STR2 across the angiosperms. Malnutrition of the fungus underlying
limited arbuscular growth was excluded by the absence of complementation of the
str1 phenotype by wild-type nurse plants. Furthermore, plant AM signaling was
found to be intact, as arbuscule-induced marker transcript accumulation was not
affected in str1 mutants. Strigolactones have previously been hypothesized to
operate as intracellular hyphal branching signals and possible substrates of STR1
and STR2. However, full arbuscule development in the strigolactone biosynthesis
mutants d10 and d17 suggested strigolactones to be unlikely substrates of
STR1/STR2. Interestingly, rice STR1 is associated with a cis-natural antisense
transcript (antiSTR1). Analogous to STR1 and STR2, at the root cortex level, the
antiSTR1 transcript is specifically detected in arbusculated cells, suggesting
unexpected modes of STR1 regulation in rice.
© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.
DOI: 10.1111/j.1365-313X.2011.04842.x
1089. Mol Plant Microbe Interact. 2012 Mar;25(3):355-62. doi: 10.1094/MPMI-09-11-

0249.
Nitrogen metabolism in Sinorhizobium meliloti-alfalfa symbiosis: dissecting the

role of GlnD and PII proteins.
Yurgel SN(1), Rice J, Kahn ML.
Author information:
(1)Institute of Biological Chemistry, Washington State University, Pullman
99164-6340, USA. syurgel@wsu.edu
To contribute nitrogen for plant growth and establish an effective symbiosis with
alfalfa, Sinorhizobium meliloti Rm1021 needs normal operation of the GlnD
protein, a bifunctional uridylyltransferase/uridylyl-cleavage enzyme that
measures cellular nitrogen status and initiates a nitrogen stress response (NSR).
However, the only two known targets of GlnD modification in Rm1021, the PII
proteins GlnB and GlnK, are not necessary for effectiveness. We introduced a
Tyr→Phe variant of GlnB, which cannot be uridylylated, into a glnBglnK background
to approximate the expected state in a glnD-sm2 mutant, and this strain was
effective. These results suggested that unmodified PII does not inhibit
effectiveness. We also generated a glnBglnK-glnD triple mutant and used this and
other mutants to dissect the role of these proteins in regulating the free-living
NSR and nitrogen metabolism in symbiosis. The glnD-sm2 mutation was dominant to
the glnBglnK mutations in symbiosis but recessive in some free-living phenotypes.
The data show that the GlnD protein has a role in free-living growth and in
symbiotic nitrogen exchange that does not depend on the PII proteins, suggesting
that S. meliloti GlnD can communicate with the cell by alternate mechanisms.
DOI: 10.1094/MPMI-09-11-0249
Impact of Lygus spp. (Hemiptera: Miridae) on damage, yield and quality of

lesquerella (Physaria fendleri), a potential new oil-seed crop.
Naranjo SE(1), Ellsworth PC, Dierig DA.
Author information:
(1)USDA-ARS, Arid-Land Agricultural Research Center, 21881 N. Cardon Ln.,
Maricopa, AZ 85138, USA. steve.naranjo@ars.usda.gov
Lesquerella, Physaria fendleri (A. Gray) S. Watson, is a mustard native to the

western United States and is currently being developed as a commercial source of
valuable hydroxy fatty acids that can be used in a number of industrial
applications, including biolubricants, biofuel additives, motor oils, resins,
waxes, nylons, plastics, corrosion inhibitors, cosmetics, and coatings. The plant
is cultivated as a winter-spring annual and in the desert southwest it harbors
large populations of arthropods, several of which could be significant pests once
production expands. Lygus spp. (Hemiptera: Miridae) are common in lesquerella and
are known pests of a number of agronomic and horticultural crops where they feed
primarily on reproductive tissues. A 4-yr replicated plot study was undertaken to
evaluate the probable impact of Lygus spp. on production of this potential new
crop. Plant damage and subsequent seed yield and quality were examined relative
to variable and representative densities of Lygus spp. (0.3-4.9 insects per sweep
net) resulting from variable frequency and timing of insecticide applications.
Increasing damage to various fruiting structures (flowers [0.9-13.9%], buds
[1.2-7.1%], and seed pods [19.4-42.5%]) was significantly associated with
increasing pest abundance, particularly the abundance of nymphs, in all years.
This damage, however, did not consistently translate into reductions in seed
yield (481-1,336 kg/ha), individual seed weight (0.5-0.7 g per 1,000 seed), or
seed oil content (21.8-30.4%), and pest abundance generally explained relatively
little of the variation in crop yield and quality. Negative effects on yield were
not sensitive to the timing of pest damage (early versus late season) but were
more pronounced during years when potential yields were lower due to weed
competition and other agronomic factors. Results suggest that if the crop is
established and managed in a more optimal fashion, Lygus spp. may not
significantly limit yield. Nonetheless, additional work will be needed once more
uniform cultivars become available and yield effects can be more precisely
measured. Densities of Lygus spp. in unsprayed lesquerella are on par with those
in other known agroecosystem level sources of this pest (e.g., forage and seed
alfalfa, Medicago sativa L.). Thus, lesquerella production may introduce new
challenges to pest management in crops such as cotton.
1091. Int J Phytoremediation. 2011;13 Suppl 1:245-63.
In situ assessment of phytotechnologies for multicontaminated soil management.
Ouvrard S(1), Barnier C, Bauda P, Beguiristain T, Biache C, Bonnard M, Caupert C,

Cébron A, Cortet J, Cotelle S, Dazy M, Faure P, Masfaraud JF, Nahmani J, Palais
F, Poupin P, Raoult N, Vasseur P, Morel JL, Leyval C.
Author information:
(1)Laboratoire Sols et Environnement, Nancy-Université, INRA,
Vandoeuvre-lès-Nancy, France. Stephanie.Ouvrard@ensaia.inpl-nancy.fr
Due to human activities, large volumes of soils are contaminated with organic
pollutants such as polycyclic aromatic hydrocarbons, and very often by metallic
pollutants as well. Multipolluted soils are therefore a key concern for
remediation. This work presents a long-term evaluation of the fate and
environmental impact of the organic and metallic contaminants of an industrially
polluted soil under natural and plant-assisted conditions. A field trial was
followed for four years according to six treatments in four replicates:
unplanted, planted with alfalfa with or without mycorrhizal inoculation, planted
with Noccaea caerulescens, naturally colonized by indigenous plants, and
thermally treated soil planted with alfalfa. Leaching water volumes and
composition, PAH concentrations in soil and solutions, soil fauna and microbial
diversity, soil and solution toxicity using standardized bioassays, plant
biomass, mycorrhizal colonization, were monitored. Results showed that plant
cover alone did not affect total contaminant concentrations in soil. However, it
was most efficient in improving the contamination impact on the environment and
in increasing the biological diversity. Leaching water quality remained an issue
because of its high toxicity shown by micro-algae testing. In this matter, prior
treatment of the soil by thermal desorption proved to be the only effective
treatment.
DOI: 10.1080/15226514.2011.568546
1092. Plant Cell. 2011 Oct;23(10):3853-65. doi: 10.1105/tpc.111.089771. Epub 2011

Oct
28.
Strigolactone biosynthesis in Medicago truncatula and rice requires the symbiotic

GRAS-type transcription factors NSP1 and NSP2.
Liu W(1), Kohlen W, Lillo A, Op den Camp R, Ivanov S, Hartog M, Limpens E, Jamil
M, Smaczniak C, Kaufmann K, Yang WC, Hooiveld GJ, Charnikhova T, Bouwmeester HJ,
Bisseling T, Geurts R.
Author information:
University, 6708 PB Wageningen, The Netherlands.
Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING

PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced
nodulation. Both proteins are considered to be Nod factor response factors
regulating gene expression after symbiotic signaling. However, legume NSP1 and
NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza
sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved
in higher plants. Here, we show that NSP1 and NSP2 are indispensable for
strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice.
Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is
essential for conversion of orobanchol into didehydro-orobanchol, which is the
main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2
mutant backgrounds correlates with reduced expression of DWARF27, a gene
essential for SL biosynthesis. Rice and M. truncatula represent distinct
phylogenetic lineages that split approximately 150 million years ago. Therefore,
we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral
function conserved in higher plants. NSP1 and NSP2 are single-copy genes in
legumes, which implies that both proteins fulfill dual regulatory functions to
control downstream targets after rhizobium-induced signaling as well as SL
biosynthesis in nonsymbiotic conditions.
DOI: 10.1105/tpc.111.089771
PMCID: PMC3229154
1093. N Engl J Med. 2011 Nov 10;365(19):1763-70. doi: 10.1056/NEJMoa1106482. Epub

2011
Oct 26.
German outbreak of Escherichia coli O104:H4 associated with sprouts.
Buchholz U(1), Bernard H, Werber D, Böhmer MM, Remschmidt C, Wilking H, Deleré Y,

an der Heiden M, Adlhoch C, Dreesman J, Ehlers J, Ethelberg S, Faber M, Frank C,
Fricke G, Greiner M, Höhle M, Ivarsson S, Jark U, Kirchner M, Koch J, Krause G,
Luber P, Rosner B, Stark K, Kühne M.
Author information:
(1)Department of Infectious Disease Epidemiology, Robert Koch Institute, Berlin,
Germany. buchholzu@rki.de
Comment in
N Engl J Med. 2011 Nov 10;365(19):1835-6.
BACKGROUND: A large outbreak of the hemolytic-uremic syndrome caused by

Shiga-toxin-producing Escherichia coli O104:H4 occurred in Germany in May 2011.
The source of infection was undetermined.
METHODS: We conducted a matched case-control study and a recipe-based restaurant
cohort study, along with environmental, trace-back, and trace-forward
investigations, to determine the source of infection.
RESULTS: The case-control study included 26 case subjects with the
hemolytic-uremic syndrome and 81 control subjects. The outbreak of illness was
associated with sprout consumption in univariable analysis (matched odds ratio,
5.8; 95% confidence interval [CI], 1.2 to 29) and with sprout and cucumber
consumption in multivariable analysis. Among case subjects, 25% reported having
eaten sprouts, and 88% reported having eaten cucumbers. The recipe-based study
among 10 groups of visitors to restaurant K included 152 persons, among whom
bloody diarrhea or diarrhea confirmed to be associated with Shiga-toxin-producing
E. coli developed in 31 (20%). Visitors who were served sprouts were
significantly more likely to become ill (relative risk, 14.2; 95% CI, 2.6 to ∞).
Sprout consumption explained 100% of cases. Trace-back investigation of sprouts
from the distributor that supplied restaurant K led to producer A. All 41 case
clusters with known trading connections could be explained by producer A. The
outbreak strain could not be identified on seeds from the implicated lot.
CONCLUSIONS: Our investigations identified sprouts as the most likely outbreak
vehicle, underlining the need to take into account food items that may be
overlooked during subjects' recall of consumption.
DOI: 10.1056/NEJMoa1106482
1094. Genome Biol. 2011 Oct 21;12(10):R106. doi: 10.1186/gb-2011-12-10-r106.
Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres

investigated by comparative transcriptomics.
Ramachandran VK(1), East AK, Karunakaran R, Downie JA, Poole PS.

Author information:
(1)School of Biological Sciences, University of Reading, Reading, RG6 6AJ, UK.
BACKGROUND: The rhizosphere is the microbe-rich zone around plant roots and is a
key determinant of the biosphere's productivity. Comparative transcriptomics was
used to investigate general and plant-specific adaptations during rhizosphere
colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres
of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet
(non-legume). Gene expression data were compared to metabolic and transportome
maps to understand adaptation to the rhizosphere.
RESULTS: Carbon metabolism was dominated by organic acids, with a strong bias
towards aromatic amino acids, C1 and C2 compounds. This was confirmed by
induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis
in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars,
suggesting that although numerous sugar and putative complex carbohydrate
transport systems are induced in the rhizosphere, they are less important carbon
sources than organic acids. A common core of rhizosphere-induced genes was
identified, of which 66% are of unknown function. Many genes were induced in the
rhizosphere of the legumes, but not sugar beet, and several were plant specific.
The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation
of R. leguminosarum to its host. Mutation of many of the up-regulated genes
reduced competitiveness for pea rhizosphere colonization, while two genes
specifically up-regulated in the pea rhizosphere reduced colonization of the pea
but not alfalfa rhizosphere.
CONCLUSIONS: Comparative transcriptome analysis has enabled differentiation
between factors conserved across plants for rhizosphere colonization as well as
identification of exquisite specific adaptation to host plants.
DOI: 10.1186/gb-2011-12-10-r106
PMCID: PMC3333776
1095. J Sci Food Agric. 2012 Mar 15;92(4):987-92. doi: 10.1002/jsfa.4681. Epub 2011
Oct
14.
Supplementing endophyte-infected tall fescue or reed canarygrass with alfalfa or

birdsfoot trefoil increases forage intake and digestibility by sheep.
Owens J(1), Provenza FD, Wiedmeier RD, Villalba JJ.
Author information:
(1)Department of Wildland Resources, Utah State University, Logan, UT 84322-5230,
USA.
BACKGROUND: We hypothesized that alfalfa (ALF) or birdsfoot trefoil (BFT) eaten

prior to a meal of endophyte-infected tall fescue (TF) or reed canarygrass (RCG)
would provide benefits not possible when TF or RCG are eaten alone. In trial 1,
four groups of five lambs were first offered ALF for 30 min followed by either TF
or RCG for 3.5 h in a 2 × 2 factorial arrangement of a completely randomized
design that included grass (TF or RCG) with or without ALF. Trial 2 was similar
to trial 1 except we used a new group of lambs fed BFT. Forage, fecal and urine
samples were collected and analyzed for dry matter intake and apparent
digestibility of dry matter, energy (in Kcal), nitrogen and neutral detergent
fiber.
RESULTS: A meal of either ALF or BFT prior to eating TF or RCG caused lambs to
eat more total dry matter and nitrogen (both in g d⁻¹) and energy (in MJ d⁻¹)
than lambs fed only TF or RCG (P < 0.05). Lambs fed ALF slightly reduced intake
of TF and RCG relative to unsupplemented animals. Lambs fed BFT ate slightly less
RCG, but much more TF than unsupplemented animals (P < 0.10). Lambs ate much less
BFT than ALF, but they were stimulated to eat TF to a greater degree by BFT than
by ALF.
CONCLUSIONS: The enhanced intake of TF by lambs provided BFT, as well as the
greater nutrient intake by lambs fed legumes and grasses was likely due in part
to complementary profiles of alkaloids, saponins, and tannins.
DOI: 10.1002/jsfa.4681
1096. Int J Syst Evol Microbiol. 2012 Aug;62(Pt 8):1871-6. doi:

10.1099/ijs.0.026880-0.
Epub 2011 Oct 7.
Rhizobium petrolearium sp. nov., isolated from oil-contaminated soil.
Zhang X(1), Li B, Wang H, Sui X, Ma X, Hong Q, Jiang R.
Author information:
(1)Agricultural Culture Collection of China, Institute of Agricultural Resources
and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081,
PR China.
Two Gram-negative, aerobic, rod-shaped bacteria, designated strains SL-1(T) and

F11, which had the ability to decompose polycyclic aromatic hydrocarbons (PAHs),
were isolated from soil samples contaminated by oil. The cells were motile by
polar or lateral flagella. According to comparison of 16S rRNA gene sequences,
strains SL-1(T) and F11 were identical and showed the greatest degree of
similarity (96.8%) to both Rhizobium oryzae Alt505(T) and Rhizobium mesosinicum
CCBAU 25010(T); however, only Rhizobium oryzae with SL-1(T) and F11 formed a
separate clade. There were low similarities (<90%) between the atpD and recA
sequences of the two strains and those of the genus of Rhizobium. The bacteria
grew at temperatures of 10-40 °C with an optimum of 30 °C. The pH range for
growth was 6.0-10.0 and optimum pH was 7.0-8.0. Growth occurred at NaCl
concentrations up to 3.0% (w/v). They were catalase- and oxidase-positive. The
main cellular fatty acids were summed feature 8 (18:1ω7c and/or 18:1ω6c) and
16:0. The DNA G+C content was 62.2 mol%. Strain SL-1(T) showed 29 and 0% DNA-DNA
relatedness, respectively, with the most related strains R. oryzae Alt505(T) and
R. mesosinicum CCBAU 25010(T) according to phylogenic analysis of the 16S rRNA
gene. According to physiological and biochemical characteristics and genotypic
data obtained in this work, the bacteria represent a novel species of the genus
Rhizobium, and the name Rhizobium petrolearium is proposed. The type strain is
SL-1(T) ( = ACCC 11238(T) = KCTC 23288(T)) and it could nodulate Medicago sativa
in nodulation tests.
DOI: 10.1099/ijs.0.026880-0
1097. Meat Sci. 2012 Feb;90(2):457-64. doi: 10.1016/j.meatsci.2011.09.007. Epub

2011
Sep 19.
Influence of alfalfa grazing-based feeding systems on carcass fat colour and meat
quality of light lambs.
Ripoll G(1), Albertí P, Joy M.
Author information:
(1)Centro de Investigación y Tecnología Agroalimentaria de Aragón, Avda Montañana
930, 50059 Zaragoza, Spain. gripoll@aragon.es
One hundred and twenty-seven lambs were fed as follows: lambs and dams grazing
alfalfa (Gr); the same as Gr but lambs had access to concentrate (Gr + S); ewes
grazed and lambs received milk and concentrate until weaning and thereafter
concentrate and straw (Rat-Gr); ewes and lambs were stall-fed (Ind). Lambs were
slaughtered at 22-24 kg live weight and fat and M. rectus abdominis colour was
measured instrumentally and subjectively. The evolution of the instrumental
colour and texture of M. longissimus lumborum was also recorded. There were
significant differences in the instrumental colour of subcutaneous fat amongst
feeding systems, but no differences were observed upon visual appraisal. On
average, M. rectus abdominis colour in the four feeding systems was classed as
pink. Differences in longissimus thoracis colour at 0 h disappeared at 24h of air
exposure and there were no differences in pH or cooking losses. At no time were
any differences in instrumental texture found.
DOI: 10.1016/j.meatsci.2011.09.007
1098. Sci Total Environ. 2011 Nov 15;409(24):5284-91. doi:

Phytotoxicity and uptake of nitroglycerin in a natural sandy loam soil.
Rocheleau S(1), Kuperman RG, Dodard SG, Sarrazin M, Savard K, Paquet L, Hawari J,
Checkai RT, Thiboutot S, Ampleman G, Sunahara GI.
Author information:
(1)Biotechnology Research Institute, National Research Council of Canada, 6100
Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
Nitroglycerin (NG) is widely used for the production of explosives and solid
propellants, and is a soil contaminant of concern at some military training
ranges. NG phytotoxicity data reported in the literature cannot be applied
directly to development of ecotoxicological benchmarks for plant exposures in
soil because they were determined in studies using hydroponic media, cell
cultures, and transgenic plants. Toxicities of NG in the present studies were
evaluated for alfalfa (Medicago sativa), barnyard grass (Echinochloa crusgalli),
and ryegrass (Lolium perenne) exposed to NG in Sassafras sandy loam soil. Uptake
and degradation of NG were also evaluated in ryegrass. The median effective
concentration values for shoot growth ranged from 40 to 231 mg kg(-1) in studies
with NG freshly amended in soil, and from 23 to 185 mg kg(-1) in studies with NG
weathered-and-aged in soil. Weathering-and-aging NG in soil did not significantly
affect the toxicity based on 95% confidence intervals for either seedling
emergence or plant growth endpoints. Uptake studies revealed that NG was not
accumulated in ryegrass but was transformed into dinitroglycerin in the soil and
roots, and was subsequently translocated into the ryegrass shoots. The highest
bioconcentration factors for dinitroglycerin of 685 and 40 were determined for
roots and shoots, respectively. Results of these studies will improve our
understanding of toxicity and bioconcentration of NG in terrestrial plants and
will contribute to ecological risk assessment of NG-contaminated sites.
DOI: 10.1016/j.scitotenv.2011.08.067
1099. Am J Bot. 2011 Oct;98(10):1633-46. doi: 10.3732/ajb.1000318. Epub 2011 Sep

29.
Complex patterns of autopolyploid evolution in alfalfa and allies (Medicago

sativa; Leguminosae).
Havananda T(1), Brummer EC, Doyle JJ.
Author information:
(1)Cornell University, Ithaca, New York 14853, USA. th263@cornell.edu
PREMISE OF THE STUDY: Although there is growing evidence that autopolyploidy is a

widespread and important evolutionary phenomenon, it has received less attention
than allopolyploidy. Medicago sativa comprises several diploid and autopolyploid
taxa, including autotetraploid cultivated alfalfa, and affords an opportunity to
elucidate the evolutionary history of a morphologically and genetically complex
autopolyploid system.
METHODS: Phylogenies and haplotype networks were constructed from two chloroplast
noncoding regions (rpl20-rps12 and trnS-trnG spacers) across seven diploid and
polyploid infraspecific taxa of M. sativa and five additional closely related
Medicago species, and genetic differentiation was estimated.
KEY RESULTS: The two most prominent M. sativa autopolyploids have contrasting
evolutionary histories. Chloroplast data support a simple autopolyploid origin of
subsp. sativa (alfalfa) from diploid subsp. caerulea, from which it is
distinguishable in several quantitative characters. In contrast, morphologically
identical diploid and autopolyploid cytotypes of subsp. falcata were found to
possess very different chloroplast haplotypes, suggesting past introgression from
M. prostrata into the polyploid. Despite the presence of hybrids between
tetraploid subspecies falcata and sativa, there was little evidence of
introgression of chloroplast genomes from either subspecies into the other.
CONCLUSIONS: Autopolyploid evolution in M. sativa is complicated and has followed
very different paths in different subspecific taxa. The potential exists for gene
flow in virtually all combinations of subspecies both within and between
ploidies, yet despite the existence of hybrids, morphologically and genetically
distinctive subspecies persist.
DOI: 10.3732/ajb.1000318
1100. Nat Biotechnol. 2011 Oct 2;29(10):922-7. doi: 10.1038/nbt.1976.
Comparative genomic analysis of the thermophilic biomass-degrading fungi

Myceliophthora thermophila and Thielavia terrestris.
Berka RM(1), Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N,

John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO,
Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D,
Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell
AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ,
Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A.
Author information:
(1)Novozymes, Inc., Davis, CA, USA.
Thermostable enzymes and thermophilic cell factories may afford economic

advantages in the production of many chemicals and biomass-based fuels. Here we
describe and compare the genomes of two thermophilic fungi, Myceliophthora
thermophila and Thielavia terrestris. To our knowledge, these genomes are the
first described for thermophilic eukaryotes and the first complete
telomere-to-telomere genomes for filamentous fungi. Genome analyses and
experimental data suggest that both thermophiles are capable of hydrolyzing all
major polysaccharides found in biomass. Examination of transcriptome data and
secreted proteins suggests that the two fungi use shared approaches in the
hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation.
Characterization of the biomass-hydrolyzing activity of recombinant enzymes
suggests that these organisms are highly efficient in biomass decomposition at
both moderate and high temperatures. Furthermore, we present evidence suggesting
that aside from representing a potential reservoir of thermostable enzymes,
thermophilic fungi are amenable to manipulation using classical and molecular
genetics.
DOI: 10.1038/nbt.1976
1101. Plant Physiol. 2011 Nov;157(3):1483-96. doi: 10.1104/pp.111.185140. Epub 2011

Sep
28.
From model to crop: functional analysis of a STAY-GREEN gene in the model legume
Medicago truncatula and effective use of the gene for alfalfa improvement.
Zhou C(1), Han L, Pislariu C, Nakashima J, Fu C, Jiang Q, Quan L, Blancaflor EB,

Tang Y, Bouton JH, Udvardi M, Xia G, Wang ZY.
Author information:
(1)Forage Improvement Division, Samuel Roberts Noble Foundation, Ardmore,
Oklahoma 73401, USA.
Medicago truncatula has been developed into a model legume. Its close relative
alfalfa (Medicago sativa) is the most widely grown forage legume crop in the
United States. By screening a large population of M. truncatula mutants tagged
with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1),
we identified a mutant line (NF2089) that maintained green leaves and showed
green anthers, central carpels, mature pods, and seeds during senescence. Genetic
and molecular analyses revealed that the mutation was caused by Tnt1 insertion in
a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed
that loss of the MtSGR function affected the expression of a large number of
genes involved in different biological processes. Further analyses revealed that
SGR is implicated in nodule development and senescence. MtSGR expression was
detected across all nodule developmental zones and was higher in the senescence
zone. The number of young nodules on the mutant roots was higher than in the wild
type. Expression levels of several nodule senescence markers were reduced in the
sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned,
and transgenic alfalfa lines were produced by RNA interference. Silencing of
MsSGR led to the production of stay-green transgenic alfalfa. This beneficial
trait offers the opportunity to produce premium alfalfa hay with a more greenish
appearance. In addition, most of the transgenic alfalfa lines retained more than
50% of chlorophylls during senescence and had increased crude protein content.
This study illustrates the effective use of knowledge gained from a model system
for the genetic improvement of an important commercial crop.
DOI: 10.1104/pp.111.185140
PMCID: PMC3252161
1102. Saudi J Biol Sci. 2011 Oct;18(4):317-21. doi: 10.1016/j.sjbs.2011.05.002.

Epub
2011 Jun 7.
Effect of sewage water on mineral nutritive potential of six fodder species grown
under semiarid conditions.
Ahmad K(1), Ibrahim M, Khan ZI, Rizwan Y, Ejaz A, Fardsous A, Gondal S, Lee DJ,
Al-Yemeni M.
Author information:
(1)Department of Biological Sciences, University of Sargodha, Sargodha 40100,
Pakistan.
Investigation was performed to assess the effect of different sewage water

treatments on the metal status of different fodder species consumed by ruminants
under semi-arid conditions. Five samples each of six fodder species viz.,
Trifolium alexandrinum, Cichorium intybus, Avena sativa, Medicago polymorpha,
Brassica campestris and Medicago sativa were collected from three fields
irrigated with canal water, mix water (canal water and sewage water) and sewage
water, respectively. Fodder samples were analyzed to determine the Mg, Co and Zn
concentrations in shoots. Higher values of these metals were found in fodder
species irrigated with sewage water relatively. Mg and Zn concentrations in all
the fodder species were found to be below the critical level among all
treatments. Whereas, concentrations of Co in the different fodder species were
significantly different (p < 0.05) and above the critical level. Consequently,
ruminants feeding on these fodder species need continued mineral supplementation
of Zn and Mg elements to prevent diseases caused by the deficiency of these
elements, and to support optimum animal productivity.
DOI: 10.1016/j.sjbs.2011.05.002
PMCID: PMC3730963
PMID: 23961142
1103. Int J Mol Sci. 2011;12(8):5449-60. doi: 10.3390/ijms12085449. Epub 2011 Aug
24.
Estimation of the genetic diversity in tetraploid alfalfa populations based on

RAPD markers for breeding purposes.
Nagl N(1), Taski-Ajdukovic K, Barac G, Baburski A, Seccareccia I, Milic D, Katic

S.
Author information:
(1)Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad 21000,
Serbia; E-Mails: nevena.nagl@ifvcns.ns.ac.rs (N.N.); dragan.milic@ifvcns.ns.ac.rs
(D.M.); slobodan.katic@ifvcns.ns.ac.rs (S.K.).
Alfalfa is an autotetraploid, allogamous and heterozygous forage legume, whose

varieties are synthetic populations. Due to the complex nature of the species,
information about genetic diversity of germplasm used in any alfalfa breeding
program is most beneficial. The genetic diversity of five alfalfa varieties,
involved in progeny tests at Institute of Field and Vegetable Crops, was
characterized based on RAPD markers. A total of 60 primers were screened, out of
which 17 were selected for the analysis of genetic diversity. A total of 156
polymorphic bands were generated, with 10.6 bands per primer. Number and
percentage of polymorphic loci, effective number of alleles, expected
heterozygosity and Shannon's information index were used to estimate genetic
variation. Variety Zuzana had the highest values for all tested parameters,
exhibiting the highest level of variation, whereas variety RSI 20 exhibited the
lowest. Analysis of molecular variance (AMOVA) showed that 88.39% of the total
genetic variation was attributed to intra-varietal variance. The cluster analysis
for individual samples and varieties revealed differences in their population
structures: variety Zuzana showed a very high level of genetic variation, Banat
and Ghareh were divided in subpopulations, while Pecy and RSI 20 were relatively
uniform. Ways of exploiting the investigated germplasm in the breeding programs
are suggested in this paper, depending on their population structure and
diversity. The RAPD analysis shows potential to be applied in analysis of
parental populations in semi-hybrid alfalfa breeding program in both, development
of new homogenous germplasm, and identification of promising, complementary
germplasm.
DOI: 10.3390/ijms12085449
PMCID: PMC3179177
1104. J Basic Microbiol. 2012 Feb;52(1):66-78. doi: 10.1002/jobm.201100058. Epub

2011
Sep 23.
Characterization of rhizobia from legumes of agronomic interest grown in

semi-arid areas of Central Spain relates genetic differences to soil properties.
Ruiz-Díez B(1), Fajardo S, Felipe Mdel R, Fernández-Pascual M.
Author information:
(1)Institute of Agricultural Sciences, CSIC, Madrid, Spain.
beatriz.ruiz@ccma.csic.es
A study of symbiotic bacteria from traditional agricultural legumes from Central

Spain was performed to create a collection of rhizobia from soils differing in
physicochemical, analytical and/or agroecological properties which could be
well-adapted to the environmental conditions of this region, and be used for
sustainable agricultural practices. Thirty-six isolates were obtained from
root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus
sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum
sativum, and Vicia spp.) from three agriculture areas with soils of different pHs
and from a forest area with undisturbed soils. Phenotypical characterization
revealed uniformity across the thirty-six isolates, with important exceptions in
terms of environmental tolerance (three isolates survived at high temperatures,
three at high salinity and three at acid pH). The molecular analysis of 16S rRNA
gene showed a close relationship of twenty-nine isolates to Rhizobium
leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to
Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The
sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with
the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv.
viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA
intergenic spacer (IGS) region allowed for intraspecific differentiation, so that
strains with equal 16S rRNA were grouped by means of their soil origin. These
results indicated that phenotypical and genetically related strains may be widely
distributed in this region and that soil abiotic characteristics could have a
substantial bearing on the selection of the strains living in each environment.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/jobm.201100058
1105. J Dairy Sci. 2011 Oct;94(10):5105-10. doi: 10.3168/jds.2011-4340.
Corn silage versus corn silage:alfalfa hay mixtures for dairy cows: effects of
dietary potassium, calcium, and cation-anion difference.
Erdman RA(1), Piperova LS, Kohn RA.
Author information:
(1)Animal and Avian Sciences Department, University of Maryland, College Park
20742, USA. erdman@umd.edu
Corn silage (CS) has replaced alfalfa hay (AH) and haylage as the major forage
fed to lactating dairy cows, yet many dairy producers believe that inclusion of
small amounts of alfalfa hay or haylage improves feed intake and milk production.
Alfalfa contains greater concentrations of K and Ca than corn silage and has an
inherently higher dietary cation-anion difference (DCAD). Supplemental dietary
buffers such as NaHCO(3) and K(2)CO(3) increase DCAD and summaries of studies
with these buffers showed improved performance in CS-based diets but not in
AH-based diets. We speculated that improvements in performance with AH addition
to CS-based diets could be due to differences in mineral and DCAD concentrations
between the 2 forages. The objective of this experiment was to test the effects
of forage (CS vs. AH) and mineral supplementation on production responses using
45 lactating Holstein cows during the first 20 wk postpartum. Dietary treatments
included (1) 50:50 mixture of AH and CS as the forage (AHCS); (2) CS as the sole
forage; and (3) CS fortified with mineral supplements (CaCO(3) and K(2)CO(3)) to
match the Ca and K content of the AHCS diet (CS-DCAD). Feed intake and milk
production were equivalent or greater for cows fed the CS and CS-DCAD diets
compared with those fed the AHCS diet. Fat percentage was greater in cows fed the
CS compared with the AHCS diet. Fat-corrected milk (FCM; 3.5%) tended to be
greater in cows fed the CS and CS-DCAD diets compared with the AHCS diet. Feed
efficiencies measured as FCM/dry matter intake were 1.76, 1.80, and 1.94 for the
AHCS, CS, and CS-DCAD diets, respectively. The combined effects of reduced feed
intake and increased FCM contributed to increased feed efficiency with the
CS-DCAD diet, which contained 1.41% K compared with 1.18% K in the CS diet, and
we speculate that this might be the result of added dietary K and DCAD effects on
digestive efficiency. These results indicate no advantage to including AH in
CS-based diets, but suggest that improving mineral supplementation in CS-based
diets may increase feed efficiency.

DOI: 10.3168/jds.2011-4340
1106. J Exp Bot. 2011 Nov;62(15):5699-711. doi: 10.1093/jxb/err287. Epub 2011 Sep
9.
Acetoacetyl-CoA thiolase regulates the mevalonate pathway during abiotic stress
adaptation.
Soto G(1), Stritzler M, Lisi C, Alleva K, Pagano ME, Ardila F, Mozzicafreddo M,

Cuccioloni M, Angeletti M, Ayub ND.
Author information:
(1)Instituto de Genética Ewald A. Favret (CICVyA-INTA), De reseros S/N, Castelar
C25 (1712), Provincia de Buenos Aires, Argentina.
Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two
molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step
in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase
II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was
experimentally demonstrated in planta and in heterologous systems. The
condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a
negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis
indicated that MsAACT1 expression is highly increased in roots and leaves under
cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the
MVA pathway, resulted in a decrease in squalene production, antioxidant activity,
and the survival of stressed plants. As expected, the presence of mevastatin did
not change chlorophyll and carotenoid levels, isoprenoids synthesized via the
plastidial MVA-independent pathway. The addition of vitamin C suppressed the
sensitive phenotype of plants challenged with mevastatin, suggesting a critical
function of the MVA pathway in abiotic stress-inducible antioxidant defence.
MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable
with empty vector transformed plants and enhanced production of squalene without
altering the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in
salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in
isoprenoid biosynthesis involved in abiotic stress adaptation.
DOI: 10.1093/jxb/err287
Host suitability and gas exchange response of grapevines to potato leafhopper

(Hemiptera: Cicadellidae).
Lamp WO(1), Miranda D, Culler LE, Alexander LC.
Author information:
(1)Department of Entomology, University of Maryland, College Park, MD 20742, USA.
lamp@umd.edu
Although potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), is

highly polyphagous, classic host studies do not recognize grapevines (Vitis
spp.), as suitable hosts. Recently, injury has been reported and reproduction
documented within grape vineyards, suggesting a host expansion for the
leafhopper. To document this apparent expansion in host use, we determined
whether grape plants were suitable hosts for potato leafhopper reproduction,
measured the consequence of feeding injury on gas exchange rates of grape leaves,
and compared the susceptibility to feeding injury among cultivars. We found that
potato leafhopper adults survived equally well on grape (Vitis vinifera L.),
alfalfa (Medicago sativa L.), and fava bean (Vicia faba L.). The total number of
offspring was greater on fava bean but did not differ between alfalfa and grape.
Injury to grapevines was assessed by measuring gas exchange responses of leaves
in field cages and in greenhouse tests. We found marginally significant declines
in photosynthesis and transpiration rates in the field (9.6 and 13.2%,
respectively), and much stronger effects in greenhouse tests (ranging between 22
and 52%). Our results verify that Vitis is a suitable host, and that potato
leafhopper is capable of injuring its gas exchange physiology. We discuss
possible explanations for the host expansion, and its potential to damage
commercial grapevines.
1108. J Plant Physiol. 2011 Nov 15;168(17):2007-14. doi:

10.1016/j.jplph.2011.07.007.
Epub 2011 Aug 30.
Photosynthesis, N(2) fixation and taproot reserves during the cutting regrowth
cycle of alfalfa under elevated CO(2) and temperature.
Erice G(1), Sanz-Sáez A, Aranjuelo I, Irigoyen JJ, Aguirreolea J, Avice JC,

Sánchez-Díaz M.
Author information:
(1)Departamento de Biología Vegetal, Sección Biología Vegetal, Universidad de
Navarra, Pamplona, Spain. erice@euskalnet.net
Future climatic conditions, including rising atmospheric CO(2) and temperature

may increase photosynthesis and, consequently, plant production. A larger
knowledge of legume performance under the predicted growth conditions will be
crucial for safeguarding crop management and extending the area under cultivation
with these plants in the near future. N(2) fixation is a key process conditioning
plant responsiveness to varying growth conditions. Moreover, it is likely to
increase under future environments, due to the higher photosynthate availability,
as a consequence of the higher growth rate under elevated CO(2). However, as
described in the literature, photosynthesis performance is frequently
down-regulated (acclimated) under long-term exposure to CO(2), especially when
affected by stressful temperature and water availability conditions. As growth
responses to elevated CO(2) are dependent on sink-source status, it is generally
accepted that down-regulation occurs in situations with insufficient plant C sink
capacity. Alfalfa management involves the cutting of shoots, which alters the
source-sink relationship and thus the photosynthetic behaviour. As the growth
rate decreases at the end of the pre-cut vegetative growth period, nodulated
alfalfa plants show photosynthetic down-regulation, but during regrowth following
defoliation, acclimation to elevated CO(2) disappears. The shoot harvest also
leads to a drop in mineral N uptake and C translocation to the roots, resulting
in a reduction in N(2) fixation due to the dependence on photosynthate supply to
support nodule function. Therefore, the production of new shoots during the first
days following cutting requires the utilization of reduced C and N compounds that
have been stored previously in reserve organs. The stored reserves are mediated
by phytohormones such as methyl jasmonate and abscisic acid and in situations
where water stress reduces shoot production this potentially enables the
enhancement of taproot protein levels in nodulated alfalfa, which may lead to
these plants being in better condition in the following cut/regrowth cycle.
Furthering our knowledge of legume performance under predicted climate change
conditions will be crucial for the development of varieties with better
adaptation that will achieve greater and more efficient production values.
Furthermore, for this purpose it will be necessary to improve existing
methodologies and create new ones for phenotype characterization. Such knowledge
will provide key information for future plant breeding programs.
DOI: 10.1016/j.jplph.2011.07.007
1109. J Biotechnol. 2011 Nov 10;156(2):147-52. doi: 10.1016/j.jbiotec.2011.08.015.

Epub
2011 Aug 22.
A point mutation in the Medicago sativa GSA gene provides a novel, efficient,
selectable marker for plant genetic engineering.
Ferradini N(1), Nicolia A, Capomaccio S, Veronesi F, Rosellini D.
Author information:
(1)Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo
XX Giugno 74, 06121 Perugia, Italy. nicoletta.ferradini@gmail.com
Bacterial selectable marker genes (SMG) conferring antibiotic resistance are

valuable tools in plant genetic engineering, but public concern and regulatory
requirements have stimulated the development of alternative selection systems. We
have previously demonstrated that a mutated Synechococcus elongatus HemL gene
encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in
alfalfa. In fact, GSA is irreversibly inhibited by gabaculine
(3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine
insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced
the Medicago sativa GSA cDNA and reproduced one of the two mutations associated
with gabaculine resistance in Synechococcus, a transversion resulting in a
methionine to isoleucine (M→I) substitution. This mutated gene was assessed as a
SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the
wild type gene. In tobacco, about 43% of the leaf explants produced green shoots,
whereas in alfalfa 47% of the explants produced green embryos in the presence of
30 μM gabaculine when the M→I GSA was introduced. Escapes were absent in tobacco
and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose
this new SMG as a widely acceptable alternative to those currently used.
DOI: 10.1016/j.jbiotec.2011.08.015
1110. Appl Biochem Biotechnol. 2011 Nov;165(5-6):1253-63. doi:

10.1007/s12010-011-9343-7. Epub 2011 Aug 26.
Cloning and characterization of a heme oxygenase-2 gene from alfalfa (Medicago

sativa L.).
Fu GQ(1), Jin QJ, Lin YT, Feng JF, Nie L, Shen WB, Zheng TQ.
Author information:
Technique, Nanjing Agricultural University, Nanjing 210095, People's Republic of
China.
Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs
vital roles in plant development and stress responses. Two HO isozymes exist in
plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2
usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has
been investigated previously, HO2 is still poorly understood. In this study, we
report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica
sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes
for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar
to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for
chloroplast import. Many conserved residues in plant HO were also conserved in
MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron
binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further
biochemical activity analysis of purified mature MsHO2 showed no HO activity,
suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR
confirmed its maximum expression in the germinating seeds. Importantly, the
expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and
H(2)O(2) (especially) treatment, respectively.
DOI: 10.1007/s12010-011-9343-7
1111. Environ Pollut. 2011 Dec;159(12):3627-33. doi: 10.1016/j.envpol.2011.08.001.

Epub
2011 Aug 24.
Screening of Cd tolerant genotypes and isolation of metallothionein genes in

Wang X(1), Song Y, Ma Y, Zhuo R, Jin L.
Author information:
(1)School of Pastoral Agriculture Science and Technology, Lanzhou University, PO
Box 61, Lanzhou 730020, China. xiaojuanwang@lzu.edu.cn
In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago

sativa) and to identify Cd tolerant genotypes which may potentially be useful for
restoring Cd-contaminated environments, thirty-six accessions of alfalfa were
screened under hydroponic culture. Our results showed that the relative root
growth rate varied from 0.48 to 1.0, which indicated that different alfalfa
accessions had various responses to Cd stress. The candidate fragments derived
from differentially expressed metallothionein (MT) genes were cloned from leaves
of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein
sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous
plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants
under control and Cd stress treatment, but MsMT2b only expressed under Cd stress
treatment. This suggested that MsMT2a was universally expressed in leaves of
alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.
DOI: 10.1016/j.envpol.2011.08.001
1112. J Appl Microbiol. 2011 Nov;111(5):1086-96. doi: 10.1111/j.1365-

2672.2011.05127.x.
Epub 2011 Sep 8.
Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by

rumen fungi growing in methanogenic co-culture.
Bootten TJ(1), Joblin KN, McArdle BH, Harris PJ.
Author information:
(1)School of Biological Sciences, The University of Auckland, Auckland, New
Zealand.
AIMS: To compare the abilities of the monocentric rumen fungi Neocallimastix

frontalis, Piromyces communis and Caecomyces communis, growing in coculture with
Methanobrevibacter smithii, to colonize and degrade lignified secondary cell
walls of lucerne (alfalfa) hay.
METHODS AND RESULTS: The cell walls of xylem cylinders isolated from stems of
lucerne contained mostly xylans, cellulose and lignin together with a small
proportion of pectic polysaccharides. All of these major components were removed
during incubation with the three fungi, and differing cell wall polysaccharides
were degraded to different extents. The greatest dry weight loss was found with
N. frontalis and least with C. communis, and scanning electron microscopy
revealed that these extensively colonized different cell types. C. communis
specifically colonized secondary xylem fibres and showed much less degradation
than N. frontalis and P. communis.
CONCLUSIONS: Neocallimastix frontalis and P. communis were efficient degraders of
the cell walls of lucerne xylem cylinders. Degradation occurred of pectic
polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders
probably resulted from the cleavage of xylan releasing xylan-lignin complexes.
SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike rumen bacteria, the rumen fungi N.
frontalis, P. communis and C. communis are able to degrade lignified secondary
walls in lucerne stems. These fungi could improve forage utilization by ruminants
and may have potential in the degradation of lignocellulosic biomass in the
production of biofuels.
© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for
Applied Microbiology.
DOI: 10.1111/j.1365-2672.2011.05127.x
1113. Guang Pu Xue Yu Guang Pu Fen Xi. 2011 Jun;31(6):1510-3.
[Study on estimation of fall dormancy in alfalfa by near infrared reflectance

spectroscopy and support vector machine model].
Wang HL(1), Yue ZW, Lu XS.
Author information:
(1)Grassland Resource and Ecology Laboratory, Beijing Forestry University,
Beijing 100083, China. liuliugirl@qq.com
The present study proposes a new approach to producing accurate estimates of fall
dormancy (FD) in alfalfa in a rapid manner. Using near infrared spectroscopy, the
approach produces results fast without causing damage to samples. Near infrared
reflectance spectroscopy was applied to measuring the spectra of samples. Then
principal component analysis (PCA) was conducted on the measurements. The top ten
principal components were selected based on their cumulative contribution rates
to build a support vector machine (SVM) model. Detailed analysis and discussions
were conducted over their parameter and kernel classifications. The experiment
found that when c = 0.339 2 and g = 32, the accuracy of the predictions of the
test set can reach 98.182%. Therefore the approach can estimate the FD in alfalfa
in a rapid and accurate manner. Moreover, it was compared with other approaches
such as principal component regression, partial least squares regression, BP
neural networks, and LVQ neural networks. The comparisons have shown that the
PCA-SVM model can effectively address the small-sample-size problem and avoid
local minimum.
1114. GM Crops. 2011 Jan-Mar;2(1):1-3. doi: 10.4161/gmcr.2.1.15458.
GM in the media.
Moses V(1).
Author information:
(1)King's College, London, UK. V.Moses@qmul.ac.uk
DOI: 10.4161/gmcr.2.1.15458
1115. GM Crops. 2010 Nov-Dec;1(5):344-50. doi: 10.4161/gmcr.1.5.15091.
Stable integration and expression of a plant defensin in tomato confers

resistance to fusarium wilt.
Abdallah NA(1), Shah D, Abbas D, Madkour M.
Author information:
(1)Faculty of Agriculture, Cairo University, Egypt.
Plant defensins are small cysteine-rich peptides which belong to a group of

pathogenasis related defense mechanism proteins. The proteins inhibit the growth
of a broad range of microbes and are highly stable under extreme environmental
stresses. Tomato cultivation is affected by fungal disease such as Fusarium wilt.
In order to overcome fungal damages, transgenic tomato plants expressing the
Medicago sativa defensin gene MsDef1 under the control of the CaMV 35S promoter
were developed. The Fusarium-susceptible tomato (Lycobersicum esculentum Mill)
cultivar CastleRock was used for transformation to acquire fungal resistance.
Hypocotyl with a part of cotyledon (hypocotyledonary) for young tomato seedlings
were used as an explant material and transformation was performed using the
biolistic delivery system. Bombarded shoots were selected on regeneration medium
supplemented with hygromycin and suitable concentrations of BA, zeatin ripozide
and AgNO(3). Putative transgenic plantlets of T(0) were confirmed by PCR analysis
using primers specific for the transgene and the transformation frequency
obtained was 52.3%. Transformation and transcription of transgenes were confirmed
in T(1) by PCR, Southern hybridizations, and reverse-transcription PCR (RT-PCR).
The copy numbers of integrated transgene into tomato genome ranged between 1-3
copies. Greenhouse bioassay was performed on the transgenic T(1) and T(2) young
seedlings and non-transgenic controls by challenging with a vigorous isolate of
the fungal pathogen Fusarium oxysporum f. sp. Lycopersici. The level of fungal
infectivity was determined using RT-PCR with tomatinase specific primers.
Transgenic lines were more resistant to infection by fusarium than the control
plants. These results indicated that overexpressing defensins in transgenic
plants confer resistance to fungal pathogens.
DOI: 10.4161/gmcr.1.5.15091
1116. Plant Physiol. 2011 Oct;157(2):552-62. doi: 10.1104/pp.111.181206. Epub 2011

Aug
15.
The rice miniature inverted repeat transposable element mPing is an effective

insertional mutagen in soybean.
Hancock CN(1), Zhang F, Floyd K, Richardson AO, Lafayette P, Tucker D, Wessler

SR, Parrott WA.
Author information:
(1)Institute for Plant Breeding, Genetics and Genomics/Center for Applied Genetic
Technologies, University of Georgia, Athens, Georgia 30602, USA. cnh@uga.edu
Insertional mutagenesis of legume genomes such as soybean (Glycine max) should

aid in identifying genes responsible for key traits such as nitrogen fixation and
seed quality. The relatively low throughput of soybean transformation
necessitates the use of a transposon-tagging strategy where a single
transformation event will produce many mutations over a number of generations.
However, existing transposon-tagging tools being used in legumes are of limited
utility because of restricted transposition (Ac/Ds: soybean) or the requirement
for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered
transposable element from rice (Oryza sativa), mPing, and the genes required for
its mobilization, were transferred to soybean to determine if it will be an
improvement over the other available transposon-tagging tools. Stable
transformation events in soybean were tested for mPing transposition. Analysis of
mPing excision at early and late embryo developmental stages revealed increased
excision during late development in most transgenic lines, suggesting that
transposition is developmentally regulated. Transgenic lines that produced
heritable mPing insertions were identified, with the plants from the highest
activity line producing at least one new insertion per generation. Analysis of
the mPing insertion sites in the soybean genome revealed that features displayed
in rice were retained including transposition to unlinked sites and a preference
for insertion within 2.5 kb of a gene. Taken together these findings indicate
that mPing has the characteristics necessary for an effective transposon-tagging
resource.
DOI: 10.1104/pp.111.181206
PMCID: PMC3192579
1117. Plant J. 2011 Dec;68(5):871-89. doi: 10.1111/j.1365-313X.2011.04738.x. Epub

2011
Oct 4.
System responses to long-term drought and re-watering of two contrasting alfalfa

varieties.
Kang Y(1), Han Y, Torres-Jerez I, Wang M, Tang Y, Monteros M, Udvardi M.
Author information:
(1)The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK
73401, USA.
Systems analysis of two alfalfa varieties, Wisfal (Medicago sativa ssp. falcata
var. Wisfal) and Chilean (M. sativa ssp. sativa var. Chilean), with contrasting
tolerance/sensitivity to drought revealed common and divergent responses to
drought stress. At a qualitative level, molecular, biochemical, and physiological
responses to drought stress were similar in the two varieties, indicating that
they employ the same strategies to cope with drought. However, quantitative
differences in responses at all levels were revealed that may contribute to
greater drought tolerance in Wisfal. These included lower stomatal density and
conductance in Wisfal; delayed leaf senescence compared with Chilean; greater
root growth following a drought episode, and greater accumulation of osmolytes,
including raffinose and galactinol, and flavonoid antioxidants in roots and/or
shoots of Wisfal. Genes encoding transcription factors and other regulatory
proteins, and genes involved in the biosynthesis of osmolytes and (iso)flavonoids
were differentially regulated between the two varieties and represent potential
targets for improving drought tolerance in alfalfa in the future.
DOI: 10.1111/j.1365-313X.2011.04738.x
Substrate analysis of Arabidopsis PP2C-type protein phosphatases.
Umbrasaite J(1), Schweighofer A, Meskiene I.
Author information:
(1)Max F. Perutz Laboratories, University of Vienna, Wien, Austria.
Protein phosphorylation by protein kinases can be reversed by the action of

protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among
the protein phosphatase families with the type 2C protein phosphatases (PP2Cs)
being the most abundant among them. PP2Cs are monomeric enzymes that require
metal cations for their activity and are insensitive to known phosphatase
inhibitors. PP2Cs were shown to counteract the mitogen-activated protein kinase
(MAP kinase/MAPK) activities in plants and to regulate developmental and stress
signaling pathways. Studies of PP2C activities can be performed in vitro using
recombinant proteins. The potential substrates of PP2Cs can be tested for
dephosphorylation by the phosphatase in vitro. We have found that the
stress-induced PP2Cs from alfalfa and Arabidopsis interact with stress-activated
MAPKs in yeast two-hybrid (Y2H) screens. Consequently, recombinant MAPKs were
employed as substrates for dephosphorylation by selected PP2Cs from different
family clusters. The members of the PP2C phosphatase family demonstrated
specificity toward the substrate already in vitro, supporting the notion that
protein phosphatases are specific enzymes. The PP2C from Arabidopsis thaliana
cluster B, Arabidopsis PP2C-type phosphatase (AP2C1), and its homolog from
Medicago sativa, Medicago PP2C-type phosphatase (MP2C), were able to
dephosphorylate and inactivate MAPKs, whereas the ABSCISIC ACID (ABA)-INSENSITIVE
2 (ABI2) and HOMOLOGY TO ABI1 (HAB1) PP2Cs from the distinct Arabidopsis cluster
A were not able to do so. The method described here can be used for the
determination of PP2C protein activity and for studying the effect of mutations
introduced into their catalytic domains.
DOI: 10.1007/978-1-61779-264-9_8
1119. Zoonoses Public Health. 2011 Dec;58(8):589-96. doi:
10.1111/j.1863-2378.2011.01408.x. Epub 2011 May 12.
A nationwide outbreak of Salmonella bovismorbificans associated with sprouted

alfalfa seeds in Finland, 2009.
Rimhanen-Finne R(1), Niskanen T, Lienemann T, Johansson T, Sjöman M, Korhonen T,

Guedes S, Kuronen H, Virtanen MJ, Mäkinen J, Jokinen J, Siitonen A, Kuusi M.
Author information:
(1)National Institute for Health and Welfare, Helsinki, Finland. fasihi@kmu.ac.ir
Salmonella enterica serotype Bovismorbificans is a rare serotype in Finland. In

June 2009, a nationwide outbreak of S. Bovismorbificans infections occurred, and
42 clinical isolates were identified. We conducted a case-control study enrolling
28 cases and 48 matched controls, and found ready-to-eat alfalfa sprouts
associated with the infection (odds ratio = 35.2, 95% confidence interval
2.8-435). The sprouts were traced back to a domestic producer, with the seeds
originating in Italy. Although finding microbiological evidence for sprouts as a
source of this outbreak was very challenging, S. Bovismorbificans was finally
found in sprouts germinated in the laboratory, and in soaking and rinse waters
during the germination process. The pulsed field gel electrophoresis showed that
these isolates were identical to the human outbreak isolates. Before sampling, it
is important to mix the seeds well and to take several large-size samples from
different seed lots. Instead of analysing seeds, the analysis should be targeted
to soaking or rinse water samples and to the sprouts germinated in a laboratory.
Accordingly, the sprout producers should only buy seeds that have been analysed
for Salmonella. The producers have to include Salmonella testing in their
internal quality control to ensure that Salmonella is absent from sprouts placed
on the market during their shelf-life. In order to reduce the health hazard
caused by sprouts, an effective and safe decontamination procedure should also be
developed for the seeds.
DOI: 10.1111/j.1863-2378.2011.01408.x
1120. Bioinformation. 2011;6(9):360-3. Epub 2011 Jul 19.
Survey and characterization of NBS-LRR (R) genes in Curcuma longa transcriptome.
Joshi RK(1), Kar B, Nayak S.
Author information:
(1)Centre of Biotechnology, School of Pharmaceutical Sciences, Siksha O
Anusandhan University, Bhubaneswar-751003, India.
Resistance genes are among the most important gene classes for plant breeding
purposes being responsible for activation of plant defense mechanisms. Among
them, the nucleotide binding site-leucine rich repeat (NBS-LRR) class R-genes are
the most abundant and actively found in all types of plants. Insilico
characterization of EST database resulted in the detection of 28 NBS types R-gene
sequences in Curcuma longa. All the 28 sequences represented the NB-ARC domain,
21 of which were found to have highly conserved motif characteristics and
categorized as regular NBS genes. The Open Reading Frames varied from 361
(CL.CON.3566) to 112 (CL.CON.1267) with an average of 279 amino acids. Most
alignment occurred with monocots (67.8%) with emphasis on Oryza sativa and
Zingiber sequences. All best alignments with dicots occurred with Arabidopsis
thaliana, Populus trichocarpa and Medicago sativa. These detected NBS type Rgenes
from Curcuma longa can be used as a valuable resource for molecular marker
development, molecular mapping of R-genes, and identification of resistance gene
analogs and functional and evolutionary characterization of NBS-LRR-encoding
resistance genes in asexually reproducing plants.
PMCID: PMC3143401
PMID: 21814396
1121. J Hazard Mater. 2011 Sep 15;192(3):1659-66. doi:

10.1016/j.jhazmat.2011.06.084.
Epub 2011 Jul 5.
Fluoride accumulation by plants grown in acid soils amended with flue gas
desulphurisation gypsum.
Álvarez-Ayuso E(1), Giménez A, Ballesteros JC.
Author information:
(1)Department of Environmental Geochemistry, IRNASA, CSIC, Apdo 257, 37071
Salamanca, Spain. esther.alvarez@irnasa.csic.es
The application of flue gas desulphurisation (FGD) gypsum as an acid soil

ameliorant was studied in order to establish the possible detrimental effects on
plants and animals feeding on them caused by the high fluoride content in this
by-product. A greenhouse experiment was conducted under controlled conditions to
determine the F accumulation by two plant species (alfalfa (Medicago sativa L.)
and ryegrass (Lolium perenne L.)) grown in acid soils amended with different FGD
gypsum doses (0-10%). The F concentrations in plant aerial parts were comprised
in the range 22-65 mg kg(-1), and those in plant roots varied from 49 to 135 mg
kg(-1). The F contents in the above-ground plant tissues showed to decrease with
the FGD gypsum application rate, whereas an inverse trend was manifested by plant
roots. The increase in the soil content of soluble Ca as a result of the FGD
gypsum addition seemed to play an important role in limiting the translocation of
F to plant aerial parts.
DOI: 10.1016/j.jhazmat.2011.06.084
1122. J Anim Sci. 2011 Dec;89(12):4081-92. doi: 10.2527/jas.2010-3825. Epub 2011

Jul
25.
Protein quality and utilization of timothy, oat-supplemented timothy, and alfalfa

at differing harvest maturities in exercised Arabian horses.
Woodward AD(1), Nielsen BD, Liesman J, Lavin T, Trottier NL.
Author information:
Michigan 48824, USA.
To evaluate the protein quality and postgut N utilization of full-bloom timothy

hay, oat-supplemented timothy-hay diets, and alfalfa hay harvested at different
maturities, apparent whole tract N digestibility, urinary N excretion, and serum
AA profiles were determined in light to moderately exercised Arabian horses. Six
Arabian geldings (16.0 ± 0.3 yr; 467 ± 11 kg of BW) were randomly allocated to a
6 × 6 Latin square design. Diets included full-bloom timothy grass hay (G), G +
0.2% BW oat (G1), G + 0.4% BW oat (G2), mid-bloom alfalfa (A1), early-bloom
alfalfa (A2), and early-bud alfalfa hay (A3). Forages were fed at 1.6% of the BW
of the horse (as-fed). Each period consisted of an 11-d adaptation period
followed by total collection of feces and urine for 3 d. Blood samples were taken
on d 11 for analysis of serum AA concentrations. During the 3-d collection
period, urine and feces were collected every 8 h and measured and weighed,
respectively. Approximately 10% of the total urine volume and fecal weight per
period was retained for N analyses. Fecal DM output was less (P < 0.05) in A1,
A2, or A3 compared with G, G1, or G2. Apparent whole tract N digestibility was
greater (P < 0.01) in A1, A2, and A3 compared with G, G1, or G2, and was greater
(P < 0.05) in G1 and G2 compared with G. Nitrogen retention was not different
from zero, and there were no differences (P > 0.05) in N retention among diets.
Urinary N excretion and total N excretion were greater (P < 0.05) in A1, A2, and
A3 compared with G, G1, or G2. Plasma concentrations for the majority of AA
increased curvilinearly in response to feeding G, A1, A2, and A3 (quadratic, P <
0.05), with values appearing to maximize 2-h postfeeding. Although alfalfa N
digestibility increased with decreasing harvest maturity, N retention did not
differ and urinary volume and N excretion increased, indicating that
postabsorptive N utilization decreased. In contrast, inclusion of oats at either
0.2 or 0.4% of the BW of the horse to timothy hay markedly enhanced N
digestibility without increasing N excretion, indicating improvement in postgut N
utilization. These findings indicate that feeding oat-supplemented timothy hay is
more environmentally sustainable than feeding alfalfa to the horse at maintenance
or under light to moderate exercise.
DOI: 10.2527/jas.2010-3825
Effectiveness of extruded rapeseed associated with an alfalfa protein concentrate

in enhancing the bovine milk fatty acid composition.
Dang Van QC(1), Bejarano L, Mignolet E, Coulmier D, Froidmont E, Larondelle Y,

Focant M.
Author information:
(1)Institut des Sciences de la Vie, Université catholique de Louvain, 2/8 Croix
du Sud, B-1348 Louvain-la-Neuve, Belgium. quynh.dangvan@uclouvain.be
Linseed and rapeseed, good sources of 18:3 n-3 and cis9-18:1, respectively, have
been shown to improve the bovine milk fatty acid (FA) profile. However, rapeseed,
unlike linseed, has little effect on the concentration of 18:3 n-3 in milk fat.
Alfalfa protein concentrate (APC), besides being a valuable protein source for
milk production, contains lipids rich in 18:3 n-3. Therefore, this experiment
aimed at (1) evaluating the transfer efficiency of unsaturated FA (UFA),
especially 18:3 n-3, of APC to bovine milk fat, and (2) evaluating whether
extruded rapeseed (ER) associated with APC is as effective as extruded linseed
(EL) in enhancing the bovine milk fat composition. Six lactating Holstein cows
were used in a replicated 2 × 2 Latin square design with 2 iso-energy,
iso-nitrogen and iso-FA corn silage-based diets (EL and ER-APC) and two 21-d
periods. Extruded linseed, as main UFA source, was included in the first diet,
whereas ER, as main UFA source, and APC, as supplemental 18:3 n-3, were included
in the second diet. Diets were distributed as a restricted total mixed ration.
Compared with the EL diet, the ER-APC diet, where ER was associated with APC,
increased milk concentration of 18:3 n-3 (1.18 vs. 1.31% of FA) and cis9-18:1
(18.35 vs. 20.01% of FA). The apparent transfer efficiency of 18:3 n-3 from diet
to milk was almost twice as much for the ER-APC diet than for the EL diet (7.4
vs. 3.8% of intake). Extruded linseed accounted for 84% of 18:3 n-3 provided in
the EL diet, whereas ER and APC accounted for 33 and 38% of 18:3 n-3 provided in
the ER-APC diet, respectively. Because both EL and ER underwent extrusion in
similar conditions, these results suggest that 18:3 n-3 of EL in the EL diet and
ER in the ER-APC diet were subjected to more extensive ruminal biohydrogenation
than 18:3 n-3 of APC in the ER-APC diet. This experiment shows that corn
silage-based diets supplemented with ER as the main UFA source, associated with
APC as supplemental 18:3 n-3, are as effective as corn silage-based diets
supplemented with EL as the main UFA source, in increasing bovine milk UFA and
18:3 n-3 contents. Furthermore, at similar levels of dietary incorporation, this
experiment shows that the ruminal bypass of 18:3 n-3 is higher for APC compared
with EL.

DOI: 10.3168/jds.2011-4204
Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling

of alfalfa.
Tao L(1), Zhou H, Guo XS, Long RJ, Zhu Y, Cheng W.
Author information:
(1)MOE Key Laboratory of Arid and Grassland Ecology, School of Life Science,
Lanzhou University, Lanzhou 730000, P. R. China.
The experiment was conducted to investigate the exopeptidase classes in alfalfa

(Medicago sativa L.) leaves, and to determine their contribution to the formation
of nonprotein nitrogen (NPN) components during ensiling. Six classes of
inhibitors that included bestatin (aminopeptidase inhibitor), potato
carboxypeptidase inhibitor (PCI, carboxypeptidase inhibitor), 1,10-phenanthroline
(dipeptidase inhibitor), diprotin A (dipeptidyl-peptidase inhibitor), butabindide
(tripeptidyl-peptidase inhibitor), and dipeptide Phe-Arg (peptidyl-dipeptidase
inhibitor) were used. To determine the contribution of each exopeptidase to the
formation of NPN products, aqueous extracts of fresh alfalfa were fermented to
imitate the proteolytic process of ensiled alfalfa and to ensure that each class
of exopeptidase inhibitor would have immediate contact with the proteases in the
alfalfa extract. Five classes of exopeptidases; namely, aminopeptidase,
carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase,
were shown to be present in alfalfa leaves, each playing a different role in
alfalfa protein degradation. Aminopeptidase, carboxypeptidase, and dipeptidase
were the main exopeptidases contributing to the formation of NH(3)-N. Among the 5
exopeptidases, tripeptidyl-peptidase appeared to be the principal exopeptidase in
hydrolyzing forage protein into peptides, whereas carboxypeptidase and
dipeptidase appeared to be more important in contributing to the formation of
amino acid-N. Dipeptidyl-peptidase and tripeptidyl-peptidase did not play a role
in the formation of NH(3)-N or amino acid-N. Dipeptidase, carboxypeptidase, and
tripeptidyl-peptidase were the principal exopeptidases for hydrolyzing forage
protein into NPN during ensilage, and treatment with a mixture of the 5
inhibitors reduced the total NPN concentration in the fermented alfalfa extract
to about 45% of that in the control after 21 d of fermentation.

DOI: 10.3168/jds.2010-3752
1125. Biotechnol Lett. 2011 Dec;33(12):2487-94. doi: 10.1007/s10529-011-0709-2.

Epub
2011 Jul 23.
Transformation of alfalfa chloroplasts and expression of green fluorescent

protein in a forage crop.
Wei Z(1), Liu Y, Lin C, Wang Y, Cai Q, Dong Y, Xing S.
Author information:
(1)Biotechnology Research Centre, Jilin Academy of Agricultural Sciences, 1363,
Caiyu Street, Changchun 130033, People's Republic of China, weizy932@yahoo.com.cn
The ability to transform chloroplasts in multiple species is important for

improving agricultural traits. Chloroplast transformation of alfalfa (Medicago
sativa L.), a useful forage plant with high market value, was achieved using a
vector carrying aadA and gfp genes being introduced into the chloroplasts of
alfalfa via particle bombardment using leaves and calli as explants. Resistant
somatic embryos were generated and developed into plantlets from explants. The
transformation efficiency was 1.3% for callus explants and 2.7% for leaf
explants. PCR and Southern blotting analyses revealed that the foreign genes were
integrated into the transformed chloroplast genome. The occurrence of GFP was
further confirmed by fluorescence microscopy. Expression of foreign genes in
alfalfa chloroplasts is therefore possible, and provides a novel means for
genetic improvement of agronomically important traits and production of
value-added proteins.
© Springer Science+Business Media B.V. 2011
DOI: 10.1007/s10529-011-0709-2
1126. Evid Based Complement Alternat Med. 2011;2011:792167. doi:

10.1093/ecam/neq019.
Epub 2011 Mar 13.
Evaluation of Antioxidant and Cerebroprotective Effect of Medicago sativa Linn.

against Ischemia and Reperfusion Insult.
Bora KS(1), Sharma A.
Author information:
(1)L.R. Institute of Pharmacy, Solan 173 223, India.
Antioxidants have been the focus of studies for developing neuroprotective agents
to be used in the therapy for stroke, which is an acute and progressive
neurodegenerative disorder. Medicago sativa (MS) has a long tradition of use as
ayurvedic and homoeopathic medicine in central nervous system disorders. The
plant has been reported to have antioxidant, anti-inflammatory and antidiabetic
effects. Therefore, the present study was designed to investigate the
neuroprotective effect of methanol extract of MS on ischemia and
reperfusion-induced cerebral injury in mice. Bilateral carotid artery occlusion
(BCAO) for 15 min followed by 24-h reperfusion, resulted in significant elevation
in infarct size, xanthine oxidase (XO) activity, superoxide anion (O(•-) (2))
production and thiobarbituric acid-reactive substance (TBARS) levels, and
significant depletion in endogenous antioxidant [reduced glutathione (GSH),
superoxide dismutase (SOD) and total tissue sulfhydryl (T-SH) groups] systems in
mice brain. Further, BCAO led to impairment in short-term memory and motor
coordination. Pre-treatment with MS (100 or 200 mg kg(-1), p.o.) markedly reduced
cerebral infarct size, XO, O(•-) (2) and TBARS levels, significantly restored
GSH, SOD and T-SH levels and attenuated impairment in short-term memory and motor
coordination. In addition, MS directly scavenged free radicals generated against
a stable radical 1,1-diphenyl-2-picrylhydrazyl and O(•-) (2) generated in
phenazine methosulphate-nicotinamide adenine dinucleotide systems, and also
inhibited XD/XO conversion and resultant O(•-) (2) production. The data from this
study suggest that treatment with MS enhances the antioxidant defense against
BCAO-induced global cerebral ischemia and exhibits neuroprotective activity.
DOI: 10.1093/ecam/neq019
PMCID: PMC3137587
PMID: 21785631
1127. Int J Food Microbiol. 2011 Oct 3;149(3):209-17. doi:

10.1016/j.ijfoodmicro.2011.06.008. Epub 2011 Jul 5.
Optimization and evaluation of a modified enrichment procedure combined with

immunomagnetic separation for detection of E. coli O157:H7 from artificially
contaminated alfalfa sprouts.
Weagant SD(1), Jinneman KC, Yoshitomi KJ, Zapata R, Fedio WM.
Author information:
(1)Food and Drug Administration, Retired, Poulsbo, WA, USA.
Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with
alfalfa sprouts. Detection of the organism in sprouts by standard cultural
methods can be difficult due to the high background microflora. The objective of
this study was to develop and optimize an enrichment protocol with and without
post-enrichment immunomagnetic separation (IMS) for the rapid detection by
real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially
contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure,
enriching samples in modified buffered peptone water with pyruvate and at 37°C
for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin
(mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR
detection and isolation for alfalfa sprouts artificially contaminated at
0.2cfu/g. The addition of post-enrichment IMS improved detection but not
isolation. This procedure was modified and optimized by changing to mBPWp with
cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior
to PCR detection and cultural isolation. Using the resulting protocol we were
able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated
at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation
was 84% without added post-enrichment IMS and 100% with IMS. The optimized
procedure was effective for detection and isolation of E. coli O157:H7 from this
difficult food matrix.

1128. Huan Jing Ke Xue. 2011 May;32(5):1462-70.
[Dynamic changes of physicochemical properties in phenanthrene-contaminated soil

under wheat and clover intercropping].
Zhang XB(1), Zhan XH, Zhou LX, Liang X.
Author information:
(1)College of Resources and Environment Science, Nanjing Agricultural University,
Nanjing 210095, China. 2008103079@njau.edu.cn
Soil physicochemical properties play an important role in the efficiency of

phytoremediation and soil arability after phytoremediation. Soil pot experiments
were conducted to investigate the dynamic changes of physicochemical properties
in phenanthrene (a representative of polycyclic aromatic
hydrocarbons)-contaminated soil under wheat and clover intercropping. The results
showed that plants improved the pH values of phenanthrene-polluted soil with a
maximum variation pH of 0.61. The difference in pH between wheat/clover
intercropping and wheat/clover single cropping was not significant. Soil organic
matter, total nitrogen, available nitrogen, total phosphorus, available
phosphorus, cation exchange capacity and available potassium decreased over the
experiment period due to biodecomposition and plant root absorption. The
intercropping of wheat and alfalfa accelerated the consumption of the
above-mentioned nutrients. And the decrease percentages ranged from 5.24% to
57.85%, more than those of wheat or alfalfa only planted with decrease
percentages between 6.29% and 39.09%. In particular, soil available nitrogen and
availiable phosphorus decreased more than the other nutrients with a maximum
reduction percentage of 57.85%. Therefore, the application of nitrogen and
phosphorus must be paid more attention during phytoremediation of polycyclic
aromatic hydrocarbon-contaminated soil in the wheat and alfalfa intercropping
system.
1129. J Proteome Res. 2011 Sep 2;10(9):3891-903. doi: 10.1021/pr101274f. Epub 2011
Aug
4.
Toward characterizing seed vigor in alfalfa through proteomic analysis of

germination and priming.
Yacoubi R(1), Job C, Belghazi M, Chaibi W, Job D.
Author information:
(1)Laboratoire de Biologie et Physiologie Cellulaire Végétales, Département de
Biologie, Université de Tunis, Tunisie. yacoubirafika@yahoo.fr
Alfalfa, the most widely grown leguminous crop in the world, is generally exposed
to severe salinity stress in Tunisia, notably affecting its germination
performance. Toward a better understanding of alfalfa seed vigor, we have used
proteomics to characterize protein changes occurring during germination and
osmopriming, a pretreatment that accelerates germination and improves seedling
uniformity particularly under stress conditions. The data revealed that
germination was accompanied by dynamic changes of 79 proteins, which are mainly
involved in protein metabolism, cell structure, metabolism, and defense.
Comparative proteomic analysis also revealed 63 proteins specific to osmopriming,
65 proteins preferentially varying during germination, and 14 proteins common to
both conditions. Thus, the present study unveiled the unexpected finding that
osmopriming cannot simply be considered as an advance of germination-related
processes but involves other mechanisms improving germination such as the
mounting of defense mechanisms enabling osmoprimed seeds to surmount
environmental stresses potentially occurring during germination. The present
results therefore provide novel avenues toward understanding the mechanisms of
invigoration of low vigor seeds by priming treatments that are widely used both
in commercial applications and in developing countries (on farm seed priming) to
better control crop yields.
DOI: 10.1021/pr101274f
Synchronization of Medicago sativa cell suspension culture.
Ayaydin F(1), Kotogány E, Abrahám E, Horváth GV.
Author information:
(1)Cellular Imaging Laboratory, Biological Research Centre, Hungarian Academy of
Sciences, H-6726, Szeged, Temesvárt krt. 62, Hungary. ferhan@brc.hu
Deepening our knowledge on the regulation of the plant cell division cycle
depends on techniques that allow for the enrichment of cell populations in
defined cell cycle phases. Synchronization of cell division can be achieved using
different plant tissues; however, well-established cell suspension cultures
provide the largest amount of biological sample for further analysis. Here we
describe the methodology of the establishment, propagation, and analysis of a
Medicago sativa suspension culture that can be used for efficient synchronization
of the cell division and also the application and removal of hydroxyurea blocking
agent. A novel method is used for the estimation of cell portion that enters S
phase during the assay. The protocol can be used in the case of other species as
well.
DOI: 10.1007/978-1-61779-182-6_15
1131. BMC Genomics. 2011 Jul 6;12:1-11. doi: 10.1186/1471-2164-12-350.
Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high
resolution melting analysis.
Han Y(1), Kang Y, Torres-Jerez I, Cheung F, Town CD, Zhao PX, Udvardi MK,
Monteros MJ.
Author information:
Noble Parkway, Ardmore, OK 73401, USA.
BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common type of
sequence variation among plants and are often functionally important. We describe
the use of 454 technology and high resolution melting analysis (HRM) for high
throughput SNP discovery in tetraploid alfalfa (Medicago sativa L.), a species
with high economic value but limited genomic resources.
RESULTS: The alfalfa genotypes selected from M. sativa subsp. sativa var.
'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water
stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated
plants grown under either well-watered or water-stressed conditions, and then
pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216
unique sequences were obtained including 24,144 tentative consensus (TCs)
sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with
an average length of 541 bp. We identified 40,661 candidate SNPs distributed
throughout the genome. A sample of candidate SNPs were evaluated and validated
using high resolution melting (HRM) analysis. A total of 3,491 TCs harboring
20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1) chromosomes.
Gene Ontology assignments indicate that sequences obtained cover a broad range of
GO categories.
CONCLUSIONS: We describe an efficient method to identify thousands of SNPs
distributed throughout the alfalfa genome covering a broad range of GO
categories. Validated SNPs represent valuable molecular marker resources that can
be used to enhance marker density in linkage maps, identify potential factors
involved in heterosis and genetic variation, and as tools for association mapping
and genomic selection in alfalfa.
DOI: 10.1186/1471-2164-12-350
PMCID: PMC3154875
1132. Br Poult Sci. 2011 Jun 1;52(3):302-9. doi: 10.1080/00071668.2011.569008.
Effects of dietary polysavone (Alfalfa extract) and chlortetracycline

supplementation on antioxidation and meat quality in broiler chickens.
Dong XF(1), Gao WW, Su JL, Tong JM, Zhang Q.
Author information:
Academy of Agricultural Sciences, Beijing, China.
1. A total of 360 1-d-old male commercial Arbor Acre broiler chickens were
randomly assigned to 5 groups (6 replicates of 12 birds each) to evaluate the
dietary effects of polysavone (0·5, 1·0 and 1·5 g/kg), a natural extract from
alfalfa, and 0·15 g/kg chlortetracycline (CTC) on growth performance,
antioxidation and meat quality of broiler chickens. 2. Over the 6-week study,
feed intake increased significantly with CTC supplementation and final body
weight (BW) was significantly higher for 1·0 g/kg polysavone and 0·15 g/kg CTC
treatments. Feed:gain ratio was not significantly affected by the dietary
treatments. 3. At 3 weeks of age, serum total superoxide dismutase (T-SOD)
activity for all polysavone treatments was significantly higher than controls,
liver T-SOD activity in 1·5 g/kg polysavone group was significantly higher than
the control and CTC groups, and serum glutathione peroxidase (GSHPx) activity for
1·5 g/kg polysavone and liver GSHPx activity in all polysavone groups were
significantly higher than CTC. 4. At 6 weeks of age, serum and liver T-SOD
activity in 1·5 g/kg polysavone group and liver GSHPx activity for all polysavone
treatments were higher significantly than the control and CTC groups, and serum
malondialdehyde (MDA) content for all polysavone treatments was significantly
lower than CTC. 5. Breast muscle T-SOD activity and pH value at 6 weeks of age
were significantly higher and MDA content was significantly lower in 1·0 and 1·5
g/kg polysavone groups than in the control and CTC groups. Breast muscle shear
force was significantly lower in l·5 g/kg polysavone group compared with the
control, and drip loss for all polysavone treatments was significantly lower than
CTC. 6. It was indicated that polysavone modulates antioxidation and modifies
meat quality, but with no adverse effect on performance of broiler chickens, and
that CTC can be beneficial to performance but has no beneficial effect on
antioxidant function or meat quality.
DOI: 10.1080/00071668.2011.569008
1133. Acta Biochim Pol. 2011;58(3):427-32. Epub 2011 Jul 4.
Effect of Medicago sativa Mhb1gene expression on defense response of Arabidopsis

thaliana plants.
Maassen A(1), Hennig J.
Author information:
(1)Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Warszawa, Poland.
Besides the previously described nitric oxide-detoxification activity we

identified new features of class-1 non-symbiotic hemoglobin from Medicago sativa
(Mhb1). Under in vitro conditions, using peroxidase in-gel activity assay, the
Mhb1 protein was shown to possess also peroxidase-like activity. Due to this
activity, in the presence of nitrite and hydrogen peroxide, the protein can
mediate autonitration and nitration of other proteins at tyrosine residues, as
revealed by tandem mass spectrometry and immune assay approaches. Mhb1 through
its multifunctional activities can affect different components of signal
transduction cascades operating during plant response to infections. This
influence is manifested by Mhb1-mediated selective up-regulation of expression of
certain pathogen inducible genes in Pseudomonas syringae infected Arabidopsis
thaliana plants which overproduce Mhb1, as revealed by reverse
transcription-quantitative real-time PCR analysis. Changes in expression level of
these genes can influence such processes as synthesis of secondary metabolites,
protein degradation and biosynthesis of ethylene. They can also result in
alteration of pathogen-induced defense response of Mhb1 transgenic plants.
1134. J Bacteriol. 2011 Sep;193(17):4405-16. doi: 10.1128/JB.05045-11. Epub 2011

Jul 1.
An ABC-type cobalt transport system is essential for growth of Sinorhizobium

meliloti at trace metal concentrations.
Cheng J(1), Poduska B, Morton RA, Finan TM.
Author information:
We report expression and mutant phenotypes for a gene cluster in Sinorhizobium

meliloti, designated cbtJKL, that has been shown to encode an ABC-type cobalt
transport system. Transcription of cbtJKL initiated 384 nucleotides upstream from
the cbtJ translation start codon, and the resulting 5' region contained a
putative B(12)riboswitch. Expression of the cbtJKL genes appeared to be
controlled by (cobalt-loaded) cobalamin interacting at the B(12)riboswitch, since
(i) a putative B(12)riboswitch was located within this large upstream region,
(ii) cbtJ transcription was repressed upon addition of cobalt or vitamin B(12),
and (iii) deletions in the B(12)riboswitch resulted in constitutive cbtJKL
transcription. Insertion mutants in cbtJKL failed to grow in LB medium, and
growth was restored through the addition of cobalt but not other metals. This
growth phenotype appeared to be due to the chelation of cobalt present in LB, and
cbtJKL mutants also failed to grow in minimal medium containing the chelating
agent EDTA unless the medium was supplemented with additional or excess cobalt.
In uptake experiments, (57)Co(2+)accumulation was high in wild-type cells
expressing the cbtJKL genes, whereas wild-type cells in which cbtJKL expression
was repressed showed reduced accumulation. In cbtJKL mutant cells,
(57)Co(2+)accumulation was reduced relative to that of the wild type, and
presumably, this residual cobalt transport occurred via an alternate ion uptake
system(s) that is not specific to cobalt. In symbiosis, the alternate system(s)
appeared to mediate cobalt transport into bacteroid cells, as low cbtJKL
expression was detected in bacteroids and cbtJKL mutants formed N(2)-fixing
nodules on alfalfa.
DOI: 10.1128/JB.05045-11
PMCID: PMC3165532
1135. J Dairy Sci. 2011 Jul;94(7):3605-8. doi: 10.3168/jds.2010-3536.
Short communication: effects of replacing part of corn silage and alfalfa hay
with Leymus chinensis hay on milk production and composition.
Yan R(1), Chen S, Zhang X, Han J, Zhang Y, Undersander D.
Author information:
China Agricultural University, Beijing 100193, P. R. China.
The objective of this research was to investigate the effects of replacing part
of corn silage (CS) and alfalfa hay (AH) with Leymus chinensis hay on milk
production and composition. Twenty multiparous Holstein dairy cows were used in a
randomized block design for a 14-week period and 2 treatments. Treatments were
(dry matter basis): (1) Non-Leymus chinensis hay diet (NLC; 35% CS, 15% AH) and
(2) added Leymus chinensis hay diet (ALC; 30% CS, 10% AH, 10% Leymus chinensis
hay). Adding Leymus chinensis hay increased neutral detergent fiber content and
in vitro digestibility of the diet. Cows receiving the ALC diet had higher dry
matter intake, milk yield, milk protein yield, lactose yield, solids-not-fat
yield, and milk fat content compared with those fed the NLC diet. Somatic cell
counts of cows decreased in the ALC compared with the NLC treatment. Cis-11 18:1
and 18:2 contents in milk increased, whereas trans-9 and cis-9 18:1 fatty acid
contents decreased. Trans-9, cis-11 conjugated linoleic acid content was not
influenced by adding Leymus chinensis hay to the diet. Leymus chinensis hay can
be used to replace part of CS and AH in diets of dairy cows to get higher milk
yield and good milk quality.

DOI: 10.3168/jds.2010-3536
Effect of varying total mixed ration particle size on rumen digesta and fecal
particle size and digestibility in lactating dairy cows.
Maulfair DD(1), Fustini M, Heinrichs AJ.
Author information:
(1)Department of Dairy and Animal Science, The Pennsylvania State University,
University Park 16802, USA.
The objective of this experiment was to evaluate the effects of feeding rations
of different particle sizes on rumen digesta and fecal matter particle size. Four
rumen-cannulated, multiparous, Holstein cows (104±15 d in milk) were randomly
assigned to treatments in a 4×4 Latin square design. The diets consisted of 29.4%
corn silage, 22.9% ground corn, 17.6% alfalfa haylage, and 11.8% dry grass hay
[20% of forage dry matter (DM)] on a DM basis. Dry grass hay was chopped to 4
different lengths to vary the total mixed ration (TMR) particle size. Geometric
mean particle sizes of the rations were 4.46, 5.10, 5.32, and 5.84 mm for short,
medium, long, and extra long diets, respectively. The ration affected rumen
digesta particle size for particles ≥3.35 mm, and had no effect on distribution
of particles <3.35 mm. All rumen digesta particle size fractions varied by time
after feeding, with soluble particle fractions increasing immediately after
feeding and 0.15, 0.6, and 1.18-mm particle size fractions decreasing slightly
after feeding. Particle fractions >1.18 mm had ration by time interactions. Fecal
neutral detergent fiber (NDF) and indigestible NDF concentrations decreased with
increasing TMR particle size. Fecal particle size expressed as total geometric
mean particle length followed this same tendency. Fecal particle size, expressed
as retained geometric mean particle length, averaged 1.13 mm with more than 36%
of particles being larger than 1.18 mm. All fecal nutrient concentrations
measured were significantly affected by time after feeding, with NDF and
indigestible NDF increasing after feeding and peaking at about 12h later and then
decreasing to preprandial levels. Starch concentrations were determined to have
the opposite effect. Additionally, apparent digestibility of diet nutrients was
analyzed and DM digestibility tended to decrease with increasing TMR particle
size, whereas other nutrient digestibilities were not different among rations.
These results show that the critical size for increased resistance to rumen
escape is larger than 1.18 mm and this critical size is constant throughout the
day. This study also concludes that, when using average quality grass hay to
provide the range of particle sizes fed, DM digestibility tends to decrease with
increasing ration particle size.

DOI: 10.3168/jds.2010-3718
1137. N Engl J Med. 2011 Nov 10;365(19):1771-80. doi: 10.1056/NEJMoa1106483. Epub

2011
Jun 22.
Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in

Germany.
Frank C(1), Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, Bernard H,
Fruth A, Prager R, Spode A, Wadl M, Zoufaly A, Jordan S, Kemper MJ, Follin P,
Müller L, King LA, Rosner B, Buchholz U, Stark K, Krause G; HUS Investigation
Team.
Collaborators: Abu Sin M, Adlhoch C, Alpers K, Altmann D, Altmann M, Arends K,

Atzpodien K, Behnke S, Benzler J, Bergholz A, Bielecke J, Böhmer M, Brodhun B,
Burger R, Cai W, Claus H, Christner M, Cyberski C, Dehnert M, Deleré Y, Dudareva
S, Eckmanns T, Espelage W, Falkenhorst G, Fiebig L, Fraedrich K, Gilsdorf A,
Greutélaers B, Gunsenheimer-Bartmeyer B, Haar K, Haas W, Hamouda O, Hauer B,
Hellenbrand W, Hermes J, Höhle M, Koch J, Köpke K, Krügermann K, Laude G, Lee MH,
Liss I, Lohse AW, Luchtenberg M, Marx M, Meyer D, Mielke M, Milde-Busch A, Mücke
I, Nachtnebel M, Neifer J, Nielsen S, Noll I, Offergeld R, Oh J, Pfeiffer Y,
Pohland R, Prahm K, Preuss U, Reiter S, Remschmidt C, Reuss A, Rexroth U, Richter
M, Rieck T, Rohde H, Sailer A, Santos-Hövener C, Schaade L, Schink S, Schmiedel
S, Schmidt D, Schoene C, Schöneberg I, Schuster M, Schwarz F, Schweickert B,
Stöcker P, Sü T, Takla A, Tietze E, Ultsch B, Ung M, Kang Z, Velasco E, Walter D,
Weiss B, Wilking H, Zimmermann R, Zhang W, Zunk J.
Author information:
(1)Department of Infectious Disease Epidemiology, Robert Koch Institute, Berlin,
Germany.
Comment in
N Engl J Med. 2011 Nov 10;365(19):1835-6.
N Engl J Med. 2012 Feb 23;366(8):766; author reply 766-7.
BACKGROUND: We describe an outbreak of gastroenteritis and the hemolytic-uremic

syndrome caused by Shiga-toxin-producing Escherichia coli in Germany in May,
June, and July, 2011. The consumption of sprouts was identified as the most
likely vehicle of infection.
METHODS: We analyzed data from reports in Germany of Shiga-toxin-producing E.
coli gastroenteritis and the hemolytic-uremic syndrome and clinical information
on patients presenting to Hamburg University Medical Center (HUMC). An outbreak
case was defined as a reported case of the hemolytic-uremic syndrome or of
gastroenteritis in a patient infected by Shiga-toxin-producing E. coli, serogroup
O104 or serogroup unknown, with an onset of disease during the period from May 1
through July 4, 2011, in Germany.
RESULTS: A total of 3816 cases (including 54 deaths) were reported in Germany,
845 of which (22%) involved the hemolytic-uremic syndrome. The outbreak was
centered in northern Germany and peaked around May 21 to 22. Most of the patients
in whom the hemolytic-uremic syndrome developed were adults (88%; median age, 42
years), and women were overrepresented (68%). The estimated median incubation
period was 8 days, with a median of 5 days from the onset of diarrhea to the
development of the hemolytic-uremic syndrome. Among 59 patients prospectively
followed at HUMC, the hemolytic-uremic syndrome developed in 12 (20%), with no
significant differences according to sex or reported initial symptoms and signs.
The outbreak strain was typed as an enteroaggregative Shiga-toxin-producing E.
coli O104:H4, producing extended-spectrum beta-lactamase.
CONCLUSIONS: In this outbreak, caused by an unusual E. coli strain, cases of the
hemolytic-uremic syndrome occurred predominantly in adults, with a preponderance
of cases occurring in women. The hemolytic-uremic syndrome developed in more than
20% of the identified cases.
DOI: 10.1056/NEJMoa1106483
1138. Plant Cell Rep. 2011 Nov;30(11):1991-2000. doi: 10.1007/s00299-011-1107-x.

Epub
2011 Jun 21.
Assessment of simple marker-free genetic transformation techniques in alfalfa.
Ferradini N(1), Nicolia A, Capomaccio S, Veronesi F, Rosellini D.
Author information:
(1)Dipartimento di Biologia Applicata, Università degli Studi di Perugia, Borgo
XX giugno 74, 06121, Perugia, Italy.
Methods to avoid the presence of selectable marker genes (SMG) in transgenic

plants are available but not implemented in many crop species. We assessed the
efficiency of simple marker-free Agrobacterium-mediated transformation techniques
in alfalfa: regeneration without selection, or marker-less, and co-transformation
with two vectors, one containing the SMG and one containing a non-selected gene.
To easily estimate the efficiency of marker-less transformation, the nptII and
the GUS markers were used as non-selected genes. After Agrobacterium treatment,
somatic embryos were regenerated without selection. The percentage of transgenic
embryos was determined by a second cycle of regeneration using the embryos as
starting material, in the presence of kanamycin, by PCR screening of T1
progenies, and by the GUS test. In two experiments, from 0 to 1.7% of the somatic
embryos were transgenic. Co-transformation was performed with two vectors, one
with the hemL SMG and one with the unselected nptII gene, each carried by a
different culture of Agrobacterium. Only 15 putative co-transformed plants were
regenerated from two experiments, with an average co-transformation percentage of
3.7. Southern blot hybridizations and/or T(1) progeny segregation were used to
confirm transgene integration, and qPCR was also used to estimate the T-DNA copy
number. In the T(1) progenies obtained by crossing with a non-transgenic
pollinator, marker-free segregants were obtained. Both marker-free approaches
showed very low efficiency.
DOI: 10.1007/s00299-011-1107-x
1139. Infect Immun. 2011 Aug;79(8):2984-97. doi: 10.1128/IAI.00170-11. Epub 2011

Jun
20.
Burkholderia cenocepacia ShvR-regulated genes that influence colony morphology,

biofilm formation, and virulence.
Subramoni S(1), Nguyen DT, Sokol PA.
Author information:
(1)Department of Microbiology and Infectious Diseases, Faculty of Medicine,
University of Calgary, Calgary, Alberta, Canada.
Burkholderia cenocepacia is an opportunistic pathogen that primarily infects

cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR
regulator, influences colony morphology, virulence, and biofilm formation and
regulates the expression of an adjacent 24-kb genomic region encoding 24 genes.
In this study, we report the functional characterization of selected genes in
this region. A Tn5 mutant with shiny colony morphology was identified with a
polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase.
Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is
required for rough colony morphology, biofilm formation, and virulence on alfalfa
seedlings. It was not possible to complement with BCAS0208 containing a mutation
in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide
(FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate
synthase, do not influence colony morphology but are required for optimum levels
of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to
pellicle formation, although individual mutations in each of these genes had no
appreciable effect on pellicle formation. A mutant with a polar insertion in
BCAS0208 was significantly less virulent in a rat model of chronic lung infection
as well as in the alfalfa model. Genes in this region were shown to influence
utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates,
l-arabinose, and branched-chain amino acids. Together, our data show that the
ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony
morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia.
DOI: 10.1128/IAI.00170-11
PMCID: PMC3147549
1140. Syst Appl Microbiol. 2011 Sep;34(6):453-61. doi: 10.1016/j.syapm.2011.04.006.
Phenotypic and genetic characterization of rhizobia associated with alfalfa in

the Hokkaido and Ishigaki regions of Japan.
Djedidi S(1), Yokoyama T, Tomooka N, Ohkama-Ohtsu N, Risal CP, Abdelly C,

Sekimoto H.
Author information:
(1)United Graduate School of Agricultural Science, Tokyo University of
Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa
inoculated with soil samples collected from the Sapporo region and Ishigaki
Island in Japan. To study their diversity and characterize them in relation to
the climatic conditions of their soils of origin, a polyphasic approach analyzing
stress tolerance, symbiotic and genetic properties was used. Stress tolerance
assays revealed marked variations in salinity, pH and temperature tolerance.
Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki
Island) tolerated high temperature, salinity and pH levels. Moreover, isolates
recovered from a temperate climate in acidic soil (Sapporo) were sensitive to
high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of
conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25
isolates to be closely related to Ensifer meliloti. Furthermore, the branch
patterns of phylogenetic trees constructed from different genes revealed the
existence of at least two E. meliloti types in the soils studied. These results
may be relevant to programs directed towards improving crop productivity through
biofertilization with locally adapted and genetically defined strains.
DOI: 10.1016/j.syapm.2011.04.006
1141. Plant Sci. 2011 Aug;181(2):140-50. doi: 10.1016/j.plantsci.2011.04.013. Epub

2011
Apr 30.
Elevation of free proline and proline-rich protein levels by simultaneous

manipulations of proline biosynthesis and degradation in plants.
Stein H(1), Honig A, Miller G, Erster O, Eilenberg H, Csonka LN, Szabados L,
Koncz C, Zilberstein A.
Author information:
(1)Department of Plant Sciences, Faculty of Life Sciences, Tel Aviv University,
Tel Aviv 69978, Israel. hstein@cbd-tech.com
Proline-rich proteins (PRP) are cell wall and plasma membrane-anchored factors
involved in cell wall maintenance and its stress-induced fortification. Here we
compare the synthesis of P5C as the proline (Pro) precursor in the cytosol and
chloroplast by an introduced alien system and evaluate correlation between PRP
synthesis and free Pro accumulation in plants. We developed a Pro over-producing
system by generating transgenic tobacco plants overexpressing E. coli P5C
biosynthetic enzymes; Pro-indifferent gamma-glutamyl kinase 74 (GK74) and
gamma-glutamylphosphate reductase (GPR), as well as antisensing proline
dehydrogenase (ProDH) transcription. GK74 and GPR enzymes were targeted either to
the cytosol or plastids. Molecular analyses indicated that the two bacterial
enzymes are efficiently expressed in plant cells, correctly targeted to the
cytosol or chloroplasts, and processed to active enzymatic complexes in the two
compartments. Maximal Pro increase is obtained when GK74 and GPR are active in
chloroplasts, and ProDH mRNA level is reduced by anti-sense silencing, resulting
in more than 50-fold higher Pro content compared to that of wild type tobacco
plants. The Pro over-producing system efficiently works in tobacco and
Arabidopsis. The elevation of Pro levels promotes accumulation of ectopically
expressed Cell Wall Linker Protein (AtCWLP), a membrane protein with an external
Pro-rich domain. These results suggest that the Pro-generating system can support
endogenous or alien PRP production in plants.
DOI: 10.1016/j.plantsci.2011.04.013
1142. Mol Biol Rep. 2012 Mar;39(3):2883-92. doi: 10.1007/s11033-011-1048-z. Epub

2011
Jun 18.
Isolation and functional characterization of a Medicago sativa L. gene, MsLEA3-1.
Bai Y(1), Yang Q, Kang J, Sun Y, Gruber M, Chao Y.
Author information:
Beijing 100193, People's Republic of China.
A full-length cDNA of 1,728 nt, called MsLEA3-1, was cloned from alfalfa by rapid
amplification of cDNA ends from an expressed sequence tag homologous to soybean
pGmPM10 (accession No. AAA91965.1). MsLEA3-1, encodes a deduced protein of 436
amino acids, a calculated molecular weight of 47.0 kDa, a theoretical isoelectric
point of 5.18, and closest homology with late embryogenesis abundant proteins in
soybean. Sequence homology suggested a signal peptide in the N terminus, and
subcellular localization with GFP revealed that MsLEA3-1 was localized
preferentially to the nucleolus. The transcript titre of MsLEA3-1 was strongly
enriched in leaves compared with roots and stems of mature alfalfa plants. Gene
expression of MsLEA3-1 was strongly induced when seedlings were treated with NaCl
and ABA. Expression of the MsLEA3-1 transgenic was detected in transgenic
tobacco. Malondialdehyde content and, electrical conductivity content were
reduced and electrical conductivity and proline content were increased in
transgenic tobacco compared with non-transgenic tobacco under salt stress. The
results showed that accumulation of the MsLEA3-1 protein in the vegetative
tissues of transgenic plants enhanced their tolerance to salt stress. These
results demonstrate a role for the MsLEA3-1 protein in stress protection and
suggest the potential of the MsLEA3-1 gene for genetic engineering of salt
tolerance.
DOI: 10.1007/s11033-011-1048-z
PMCID: PMC3271230
1143. Mol Biol Rep. 2012 Mar;39(3):2489-95. doi: 10.1007/s11033-011-1000-2. Epub

2011
Jun 11.
Harvest-inducibility of the promoter of alfalfa S-adenosyl-L-methionine:

trans-caffeoyl-CoA3-O-methyltransferase gene.
Zhang J(1), Erickson LR.
Author information:
(1)Plant Agriculture Department, University of Guelph, Guelph, ON N1G 2W1l,
Canada. jian.zhang@albertainnovates.ca
A major limitation on the expression of some foreign proteins in transgenic

plants is the toxic effect of such proteins on the host plant resulting in
inhibition of normal growth and development. A solution to this problem is to
control the expression of genes for such proteins by means of inducible
promoters, as is frequently done in microbial systems. A cDNA clone was obtained
from subtractive hybridization of non-harvested and harvested alfalfa leaf
tissue, named hi12. The hi12 cDNA was identified as part of the
S-adenosyl-L-methionine: trans-caffeoyl-CoA3-O-methyltransferase gene of alfalfa,
a gene encoding an essential key enzyme in lignin synthesis. The hi12 gene was
strongly induced by harvesting and wounding but not by heat shock. The promoter
of the hi12 gene, isolated by genomic walking, contained several stress response
cis-elements. Transgenic plants of tobacco and Medicago truncatula containing the
GUS gene driven by the promoter showed GUS expression following harvesting,
demonstrating the activity of these regulatory regions in other plant species.
DOI: 10.1007/s11033-011-1000-2
1144. Foodborne Pathog Dis. 2011 Oct;8(10):1089-94. doi: 10.1089/fpd.2011.0901.

Epub
2011 Jun 8.
Disinfection of radish and alfalfa seeds inoculated with Escherichia coli O157:H7
and Salmonella by a gaseous acetic acid treatment.
Nei D(1), Latiful BM, Enomoto K, Inatsu Y, Kawamoto S.
Author information:
(1)Food Hygiene Laboratory, National Food Research Institute, Kannondai-2-1-12,
Tsukuba, Japan. nei@affrc.go.jp
Abstract The majority of seed sprout-related outbreaks have been associated with
Escherichia coli O157:H7 and Salmonella. Therefore, we aimed to find an effective
method to inactivate these organisms on seeds before sprouting. Treatment with
8.7% (v/v) acetic acid at 55°C for 2-3 h reduced the population of E. coli
O157:H7 and Salmonella inoculated on alfalfa (Medicago sativa L.) and radish
seeds (Raphanus sativus L.) by more than 5.0 log CFU/g, and a longer treatment
time completely eliminated the E. coli O157:H7 population. The E. coli O157:H7
populations were reduced to an undetectable level with a gaseous acetic acid
treatment for 48 h. After enrichment, no E. coli O157:H7 were found in the
alfalfa and radish seeds (25 g). However, these treatments were unable to
eliminate Salmonella in both seed types. No significant difference between the
germination rates of treated alfalfa seeds and control seeds was found, and
germination rates greater than 95% were obtained for the radish seeds. Although
chlorine washing is commonly used for seed decontamination, chlorine washing at
200 and 20,000 ppm resulted in a reduction of pathogens by less than or equal to
3 log CFU/g. Therefore, these results suggested that gaseous acetic acid is more
effective than chlorine washing in controlling pathogenic bacteria on sprout
seeds.
DOI: 10.1089/fpd.2011.0901
1145. FEMS Microbiol Ecol. 2011 Sep;77(3):590-9. doi: 10.1111/j.1574-

6941.2011.01139.x.
Epub 2011 Jun 23.
Soil factors exhibit greater influence than bacterial inoculation on alfalfa

growth and nitrogen fixation.
Neumann U(1), Kosier B, Jahnke J, Priefer UB, Al-Halbouni D.
Author information:
(1)Department of Soil Ecology, Institute of Biology I, RWTH Aachen University,
Aachen, Germany.
In order to study the effects of soil factors and bacterial inoculation on

alfalfa (Medicago sativa), plants were inoculated with Ensifer meliloti L33 and
Azospirillum brasilense Sp7 in pot experiments using two different soils
separately as well as in a mixture. One soil was contaminated with chemical waste
products; the other was an arable soil. Soil factors, including the availability
of macro- and micronutrients as well as carbon and nitrogen contents, were found
to exhibit a much greater influence on the growth of alfalfa than any of the
inoculations. In contaminated soil, the shoot and root growth of alfalfa was
decreased and nodules were diminished and ineffective. Bacterial inoculations did
not significantly improve this hostile growth environment. However, in a mixture
(44% arable, 22% contaminated soil, 34% vermiculite), growth conditions for
alfalfa were improved so that shoot dry weight and nodule numbers increased up to
100- and 20-fold, respectively, compared with the contaminated soil. For the
strain L33, its persistence in the rhizosphere was correlated to the presence of
its host plant, but its dynamics were influenced by competition with indigenous
rhizobia. The strain Sp7, once provided with a suitable soil, was not dependent
on the plant's rhizosphere, but it enhanced the performance of L33 and native
rhizobia.

DOI: 10.1111/j.1574-6941.2011.01139.x
Epub
2011 May 31.
Prevalence of segregation distortion in diploid alfalfa and its implications for

genetics and breeding applications.
Li X(1), Wang X, Wei Y, Brummer EC.
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK
73401, USA.
Segregation distortion (SD) is often observed in plant populations; its presence

can affect mapping and breeding applications. To investigate the prevalence of SD
in diploid alfalfa (Medicago sativa L.), we developed two unrelated segregating
F(1) populations and one F(2) population. We genotyped all populations with SSR
markers and assessed SD at each locus in each population. The three maps were
syntenic and largely colinear with the Medicago truncatula genome sequence. We
found genotypic SD for 24 and 34% of markers in the F(1) populations and 68% of
markers in the F(2) population; distorted markers were identified on every
linkage group. The smaller percentage of genotypic SD in the F(1) populations
could be because they were non-inbred and/or due to non-fully informative
markers. For the F(2) population, 60 of 90 mapped markers were distorted, and
they clustered into eight segregation distortion regions (SDR). Most SDR
identified in the F(1) populations were also identified in the F(2) population.
Genotypic SD was primarily due to zygotic rather than allelic distortion,
suggesting zygotic not gametic selection is the main cause of SD. On the F(2)
linkage map, distorted markers in all SDR except two showed heterozygote excess.
The severe SD in the F(2) population likely biased genetic distances among
markers and possibly also marker ordering and could affect QTL mapping of
agronomic traits. To reduce the effects of SD and non-fully informative markers,
we suggest constructing linkage maps and conducting QTL mapping in advanced
generation populations.
DOI: 10.1007/s00122-011-1617-5
1147. PLoS Comput Biol. 2011 May;7(5):e1002047. doi: 10.1371/journal.pcbi.1002047.

Epub
2011 May 19.
Integrative analysis of transgenic alfalfa (Medicago sativa L.) suggests new

metabolic control mechanisms for monolignol biosynthesis.
Lee Y(1), Chen F, Gallego-Giraldo L, Dixon RA, Voit EO.
Author information:
(1)Integrative BioSystems Institute and The Wallace H. Coulter Department of
Biomedical Engineering, Georgia Institute of Technology and Emory University,
Atlanta, Georgia, United States of America.
The entanglement of lignin polymers with cellulose and hemicellulose in plant

cell walls is a major biological barrier to the economically viable production of
biofuels from woody biomass. Recent efforts of reducing this recalcitrance with
transgenic techniques have been showing promise for ameliorating or even
obviating the need for costly pretreatments that are otherwise required to remove
lignin from cellulose and hemicelluloses. At the same time, genetic manipulations
of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on
lignin composition, thus raising the question of whether the current
understanding of the pathway is indeed correct. To address this question
systemically, we developed and applied a novel modeling approach that, instead of
analyzing the pathway within a single target context, permits a comprehensive,
simultaneous investigation of different datasets in wild type and transgenic
plants. Specifically, the proposed approach combines static flux-based analysis
with a Monte Carlo simulation in which very many randomly chosen sets of
parameter values are evaluated against kinetic models of lignin biosynthesis in
different stem internodes of wild type and lignin-modified alfalfa plants. In
addition to four new postulates that address the reversibility of some key
reactions, the modeling effort led to two novel postulates regarding the control
of the lignin biosynthetic pathway. The first posits functionally independent
pathways toward the synthesis of different lignin monomers, while the second
postulate proposes a novel feedforward regulatory mechanism. Subsequent
laboratory experiments have identified the signaling molecule salicylic acid as a
potential mediator of the postulated control mechanism. Overall, the results
demonstrate that mathematical modeling can be a valuable complement to
conventional transgenic approaches and that it can provide biological insights
that are otherwise difficult to obtain.
DOI: 10.1371/journal.pcbi.1002047
PMCID: PMC3098223
1148. J Anim Sci. 2011 Nov;89(11):3501-13. doi: 10.2527/jas.2010-3145. Epub 2011

May
27.
Valine partitioning and kinetics between the gastrointestinal tract and hind
limbs in lambs with an adult Trichostrongylus colubriformis burden.
Bermingham EN(1), McNabb WC, Sinclair BR, Tavendale MH, Roy NC.
Author information:
(1)Food, Nutrition Genomics, Food and Bio-Based Products, AgResearch Grasslands,
Palmerston North, New Zealand. emma.bermingham@agresearch.co.nz
Intestinal parasitic infection increases the demand for AA because of increased

protein synthesis in the intestine and increased luminal losses of AA, and these
increased demands may be supported by increased mobilization of AA from the
skeletal muscles. Two experiments were conducted to determine the effects of
parasitic infection on valine kinetics within the gastrointestinal tract and hind
limbs of lambs fed fresh forages. On d 1, lambs were given 6,000 stage-3
Trichostrongylus colubriformis larvae per day for 6 d (n = 6) or kept as
parasite-free controls (n = 6) and fed fresh lucerne (Medicago sativa; Exp. 1) or
fresh sulla (Hedysarum coronarium; Exp. 2). On d 48, valine kinetics within the
mesenteric- (MDV) and portal-drained viscera (PDV) and hind limbs were obtained
by carrying out concurrent infusions of para-amminohippuric acid into the
mesenteric vein and indocyanin green into the abdominal aorta (for blood flow),
and [3,4-(3)H]valine into the jugular vein and [1-(13)C]valine into the abomasum
for 8 h (for kinetics). During the infusions, blood was collected from the
mesenteric and portal veins and from the mesenteric artery and vena cava, and
plasma was harvested. After the 8-h infusion, lambs were euthanized, ileal
digesta were collected, and tissues were sampled from the intestine and muscle
(biceps femoris). Tissues, digesta, and plasma were analyzed for valine
concentration, specific radioactivity, and isotopic enrichment. In both
experiments, intestinal worm burdens on d 48 were greater in parasitized lambs (P
= 0.0001 and 0.003). In Exp. 1, parasitic infection increased (P = 0.03) the
total valine irreversible loss rate (ILR) in the MDV and PDV. In Exp. 2, luminal
ILR of valine in the MDV was reduced (P = 0.01); however, ILR of valine in the
PDV was unaffected. Despite these changes within the MDV and PDV, parasitic
infection did not affect the ILR of valine within the hind limbs, and valine
transport rates were largely unchanged. We suggest that the increased
mobilization of AA from the hind limbs that might have occurred in the early
phase of inflammation was no longer required when the parasitic infection was
established. The MDV and PDV data may indicate that the non-MDV parts of the PDV
play an important role in this adaptation, which warrants further study.
DOI: 10.2527/jas.2010-3145
1149. Am J Bot. 2010 Jul;97(7):1142-55. doi: 10.3732/ajb.1000009.
Phylogeny and character evolution in Medicago (Leguminosae): Evidence from

analyses of plastid trnK/matK and nuclear GA3ox1 sequences.
Steele KP(1), Ickert-Bond SM, Zarre S, Wojciechowski MF.
Author information:
(1)Arizona State University, Department of Applied Sciences and Mathematics,
Polytechnic Campus, 6098 Backus Mall, Mesa, Arizona 85212 USA.
PREMISE OF THE STUDY: The genus Medicago, with about 87 species, includes the
model legume species M. truncatula, and a number of important forage species such
as M. sativa (alfalfa), M. scutellata (snail medic), and M. lupulina (black
medic). Relationships within the genus are not yet sufficiently resolved,
contributing to difficulty in understanding the evolution of a number of
distinguishing characteristics such as aneuploidy and polyploidy, life history,
structure of cotyledons, and number of seeds per fruit. •
METHODS: Phylogenetic relationships of 70-73 species of Medicago and its sister
genus Trigonella (including Melilotus) were reconstructed from nucleotide
sequences of the plastid trnK/matK region and the nuclear-encoded GA3ox1 gene
(gibberellin 3-β-hydroxylase) using maximum parsimony and Bayesian inference
methods. •
KEY RESULTS: Our results support certain currently recognized taxonomic groups,
e.g., sect. Medicago (with M. sativa) and sect. Buceras. However, other strongly
supported clades-the "reduced subsection Leptospireae clade" that includes M.
lupulina, the "polymorpha clade" that includes M. murex and M. polymorpha and the
"subsection Pachyspireae clade" that includes M. truncatula-each of which
includes species presently in different subsections of sect. Spirocarpos,
contradict the current classification. •
CONCLUSIONS: These results support the hypothesis that some characters considered
important in existing taxonomies, for example, single-seeded fruits that have
arisen more than once in both Medicago and Trigonella, are indeed homoplastic.
Others, such as the 2n = 14 chromosome number, have also arisen independently
within the genus. In addition, we demonstrate support for the utility of GA3ox1
sequences for phylogenetic analysis among and within closely related genera of
legumes.
DOI: 10.3732/ajb.1000009
PMID: 21616866
2011 May 25.
Development and evaluation of a real-time polymerase chain reaction assay

targeting iap for the detection of Listeria monocytogenes in select food
matrices.
Chen Y(1), Kumar N, Siddique N.
Author information:
(1)Center for Food Safety and Applied Nutrition, U.S. Food and Drug
Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA.
yi.chen@fda.hhs.gov
Listeria monocytogenes is an intracellular foodborne pathogen that has been

associated with severe human illnesses. Various rapid detection methods have been
developed for the specific detection of this pathogen. In the present study, a
real-time quantitative polymerase chain reaction (PCR) assay targeting iap, a
gene encoding extracellular protein p60, was developed for L. monocytogenes. The
PCR efficiency is above 85% and the limit of detection (LOD) is 30 copies of
genome per reaction for all strains tested. The assay exhibited 100% inclusivity
and exclusivity rates. The detection of L. monocytogenes in five food matrices,
whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts,
was evaluated with and without enrichment. Without enrichment, the LOD for all
food matrices were 4×10(3) CFU/mL food enrichment mix for whole milk and
4×10(4) CFU/mL for all other foods. With 24 h incubation in Buffered Listeria
Enrichment Broth, the LOD was 3 CFU/25 g food for whole milk, turkey deli meat,
and smoked salmon and 9 CFU/25 g food for soft cheese and alfalfa sprouts. With
48 h incubation, the LOD was 3 CFU/25 g food for all matrices. This quantitative
PCR appears to be a promising alternative for rapid detection of L. monocytogenes
in select foods.
DOI: 10.1089/fpd.2010.0820
1151. Equine Vet J Suppl. 2010 Nov;(38):382-6. doi: 10.1111/j.2042-

3306.2010.00208.x.
Sweat composition in Arabian horses performing endurance exercise on

forage-based, low Na rations.
Spooner HS(1), Nielsen BD, Schott HC 2nd, Harris PA.
Author information:
(1)West Virginia University, USA; and ‡Mars HorseCare UK, Leics, UK.
holly.spooner@mail.wvu.edu
REASONS FOR PERFORMING STUDY: Excessive sweat loss during endurance exercise may
lead to electrolyte disturbances and previous research suggests dietary factors
may affect hydration status. While investigating the effect of dietary fibre type
on hydration status, sweat samples were collected which allowed for the
evaluation of sweat composition in horses consuming forage-based, low sodium (Na)
rations.
OBJECTIVE: To investigate sweat composition in Arabian horses performing
endurance type exercise while fed forage-based, rations low in Na.
METHODS: Six 2-year-old Arabian horses were fed, according to a replicated 3 × 3
Latin square, either grass hay (G), 50:50 grass hay:alfalfa hay (GA), or 50:50
grass hay: chopped fibres (GM) without any additional electrolyte
supplementation. After 14 days on each diet, horses performed a 60 km treadmill
exercise test. Sweat was collected from sealed pouches on the dorsal thorax after
each of four 15 km exercise bouts.
RESULTS: Intake (g/day) of Na (2.5 ± 0.4), Cl (72 ± 16), and Mg (18 ± 3) were not
different between diets but K and Ca intakes (g/day) were greater (P < 0.05) on
GA (246 ± 35; 101 ± 14) than G (176 ± 38; 59 ± 14) or GM (168 ± 33; 62 ± 15).
There was no effect of diet on sweat pH (7.65 ± 0.04) or concentrations (mmol/l)
of K (46 ± 3), Cl (133 ± 7), Ca (8.5 ± 1.1), or Mg (2.3 ± 0.3); yet diet did
influence sweat Na concentration (P < 0.05, G 88 ± 5 mmol/l, GA 104 ± 5, GM 96 ±
6). Na and Cl concentrations were lower than those previously reported.
CONCLUSIONS: Differences in sweat constituents due to diet were observed, but
more importantly both Na and Cl concentration are lower than those previously
reported perhaps due to low dietary Na intake or breed of animal.
© 2010 EVJ Ltd.
DOI: 10.1111/j.2042-3306.2010.00208.x
Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium

meliloti.
Fontenelle C(1), Blanco C, Arrieta M, Dufour V, Trautwetter A.
Author information:
(1)UMR CNRS 6026, DUALS, Université de Rennes I, Campus de Beaulieu, Av. du
Général Leclerc, 35042 Rennes, France.
BACKGROUND: Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that

elicits nodules on roots of host plants Medicago sativa. During nodule formation
bacteria have to withstand oxygen radicals produced by the plant. Resistance to
H2O2 and superoxides has been extensively studied in S. meliloti. In contrast
resistance to organic peroxides has not been investigated while S. meliloti
genome encodes putative organic peroxidases. Organic peroxides are produced by
plants and are highly toxic. The resistance to these oxygen radicals has been
studied in various bacteria but never in plant nodulating bacteria.
RESULTS: In this study we report the characterisation of organic hydroperoxide
resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of
ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen
peroxide or menadione in vitro. The expression of ohr and ohrR genes is
specifically induced by organic peroxides. OhrR binds to the intergenic region
between the divergent genes ohr and ohrR. Two binding sites were characterised.
Binding to the operator is prevented by OhrR oxidation that promotes OhrR
dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen
fixation, suggesting that redundant enzymatic activity exists in this strain.
Both ohr and ohrR are expressed in nodules suggesting that they play a role
during nitrogen fixation.
CONCLUSIONS: This report demonstrates the significant role Ohr and OhrR proteins
play in bacterial stress resistance against organic peroxides in S. meliloti. The
ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a
role during nodulation.
© 2011 Fontenelle et al; licensee BioMed Central Ltd.
DOI: 10.1186/1471-2180-11-100
PMCID: PMC3107159
1153. BMC Genomics. 2011 May 12;12:235. doi: 10.1186/1471-2164-12-235.
Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium

meliloti.
Galardini M(1), Mengoni A, Brilli M, Pini F, Fioravanti A, Lucas S, Lapidus A,

Cheng JF, Goodwin L, Pitluck S, Land M, Hauser L, Woyke T, Mikhailova N, Ivanova
N, Daligault H, Bruce D, Detter C, Tapia R, Han C, Teshima H, Mocali S,
Bazzicalupo M, Biondi EG.
Author information:
(1)Department of Evolutionary Biology, University of Firenze, via Romana 17,
I-50125 Firenze, Italy.
BACKGROUND: Sinorhizobium meliloti is a model system for the studies of symbiotic

nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level
is present in natural populations of this species, especially in relation with
symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated
strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting
growth of the Medicago sativa plants than strain AK83. In order to investigate
the genetic determinants of the phenotypic diversification of S. meliloti strains
AK83 and BL225C, we sequenced the complete genomes for these two strains.
RESULTS: With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83
and BL225C are larger than the laboratory strain Rm1021. The core genome of
Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the
accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C
have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two
plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of
genes that were present only in the accessory genome, consequently responsible
for phenotypic diversity and putatively involved in plant-bacterium interaction.
Notably, the symbiosis inefficient AK83 lacked several genes required for
microaerophilic growth inside nodules, while several genes for accessory
functions related to competition, plant invasion and bacteroid tropism were
identified only in AK83 and BL225C strains. Presence and extent of polymorphism
in regulons of transcription factors involved in symbiotic interaction were also
analyzed. Our results indicate that regulons are flexible, with a large number of
accessory genes, suggesting that regulons polymorphism could also be a key
determinant in the variability of symbiotic performances among the analyzed
strains.
CONCLUSIONS: In conclusions, the extended comparative genomics approach revealed
a variable subset of genes and regulons that may contribute to the symbiotic
diversity.
DOI: 10.1186/1471-2164-12-235
PMCID: PMC3164228
1154. Biol Trace Elem Res. 2011 Dec;144(1-3):570-9. doi: 10.1007/s12011-011-9065-2.

Epub 2011 May 10.
Effects of different copper sources and levels on plasma superoxide dismutase,

lipid peroxidation, and copper status of lambs.
Cheng J(1), Ma H, Fan C, Zhang Z, Jia Z, Zhu X, Wang L.

Author information:
(1)College of Animal Science and Technology, Anhui Agricultural University,
Hefei, 230036, People's Republic of China.
This study was performed to determine the effects of different copper (Cu)
sources and levels on plasma superoxide dismutase (SOD), lipid peroxidation, and
Cu status of lambs. Fifty Dorper × Mongolia wether lambs (approximately 3 month
of age; average BW = 23.8 ± 0.6 kg) were divided into five equal groups each with
ten animals according to their weight. Treatments consisted of (1) control (no
supplemental Cu), (2) 10 mg Cu/kg DM from Cu-lysine, (3) 20 mg Cu/kg DM from
Cu-lysine, (4) 10 mg Cu/kg DM from tribasic copper chloride (Cu(2)(OH)(3)Cl;
TBCC), and (5) 20 mg Cu/kg DM from TBCC. The Cu concentration was 6.74 mg/kg DM
in the basal diet. Plasma copper concentrations and ceruloplasmin activities were
not affected on day 30 by Cu supplementation. Copper supplementation increased
plasma and liver copper concentrations and ceruloplasmin activities on day 60.
Muscle Cu concentrations were not affected by Cu supplementation. There were no
differences in plasma, liver, and muscle Cu concentrations and ceruloplasmin
activities between Cu-lysine and TBCC. Liver copper concentrations and plasma
ceruloplasmin activities were increased in lambs supplemented with 20 mg Cu/kg DM
than in those supplemented with 10 mg Cu/kg DM on day 60. However, copper levels
had no effects on Cu concentrations in plasma and muscle. Malondialdehyde (MDA)
concentrations were decreased in plasma and liver tissues, but not affected in
muscle by Cu supplementation. Plasma SOD activities were increased by Cu
supplementation. There were no differences in plasma, liver, and muscle MDA
concentrations and plasma SOD activities between Cu sources and levels. These
results indicated that Cu supplementation increased plasma SOD activity, lipid
oxidative stability, and copper status of lambs, but did not influence lipid
oxidative stability in sheep muscle. Cu-lysine and TBCC were of similar
availability when offered to finishing sheep.
DOI: 10.1007/s12011-011-9065-2
1155. Ying Yong Sheng Tai Xue Bao. 2011 Jan;22(1):105-13.
[Simulation on the restoration effect of soil moisture in alfalfa (Medicago

sativa)-grain rotation system in semi-arid and drought-prone regions of Loess
Plateau].
Wang XC(1), Li J, Fang XY, Sun J, Tahir MN.
Author information:
(1)College of Agronomy, Northwest A & F University, Yangling 712100, Shaanxi,
China.
With the combination of field survey and EPIC modeling, this paper simulated the
restoration effect of soil moisture in different alfalfa (Medicago sativa)-grain
rotation systems in semi-arid and drought-prone regions of Loess Plateau. In
perennial alfalfa field and in grain crop field after alfalfa, the correlation
coefficients between the simulated and observed values of soil moisture content
in 0-10 m layer were larger than 0.9 (P < 0.01), and their relative root mean
square errors were between 0.05 and 0.16, with the relative errors less than 10%.
The dynamic changes of the simulated soil moisture contents in different soil
layers were consistent with those of the observed values. In the study regions,
it was difficult for the restoration of soil moisture in the deep soil layers of
alfalfa field. During the cultivation of alfalfa, the soil moisture content in
the layers at 8-10 m depth should not be less than 5.7%. Considering the
sustainable development of agricultural production, the appropriate cultivation
duration of alfalfa should be 4-6 years and no more than 8 years. For the
restoration of soil moisture after alfalfa cultivation in the study regions, the
rotation system potato (Solanum tuberosum) --> potato --> spring wheat (Triticum
aestivum) could be adopted, and alfalfa could be cultivated again after 32-33
years.
1156. Meat Sci. 2011 Sep;89(1):91-7. doi: 10.1016/j.meatsci.2011.04.003. Epub 2011

Apr
14.
The effect of feeding system in the expression of genes related with fat
metabolism in semitendinous muscle in sheep.
Dervishi E(1), Serrano C, Joy M, Serrano M, Rodellar C, Calvo JH.
Author information:
(1)Unidad de Tecnología en Producción Animal, CITA, Zaragoza, Spain.
edervishi@aragon.es
The effect of feeding system on the expression of LPL, ACACA, FASN, FABP4, DGAT1,
SCD, CPT1B, PRKAA2, LEP, SREBP1, PPARG, PPARA and CEBPB genes in semitendinous
muscle was studied. Forty-four single born male lambs of the Rasa Aragonesa
breed, allocated to four different dietary treatments, were used: grazing
alfalfa, grazing alfalfa with supplement for lambs, indoor lambs with grazing
ewes and drylot. Significant differences were found in the expression of genes
LPL, ACACA, FASN, FABP4, CPT1B and SCD. Genes related to adipogenesis (LPL,
ACACA, FASN, FABP4, and SCD) are up-regulated in the intensive groups. In grazing
groups CPT1B gene expression, related to β-oxidation process, is up-regulated.
The relative expression of CPT1B was 1.54 fold higher in ALF+S, and 0.43 and 0.37
fold lower in IND- GRE and IND, respectively. The results support the hypothesis
that changes in fatty acid profile due to feeding system implicate changes in the
mRNA expression level of genes related with fat metabolism. Feeding strategy is
an important tool to manipulate intramuscular fatty acid profile in meat through
altering gene expression of enzymes related with fat metabolism.
DOI: 10.1016/j.meatsci.2011.04.003
1157. J Insect Sci. 2011;11:43. doi: 10.1673/031.011.0143.
Effects of the insect growth regulator, novaluron on immature alfalfa leafcutting

bees, Megachile rotundata.
Hodgson EW(1), Pitts-Singer TL, Barbour JD.
Author information:
(1)Department of Entomology, Iowa State University, Ames, Iowa 50011-3140, USA.
aewh@iastate.edu
Alfalfa leafcutting bees, Megachile rotundata F. (Hymenoptera: Megachilidae), are

the most common pollinators of alfalfa in the Pacific Northwest. Reports from
users of M. rotundata in Idaho, Utah and Colorado have indicated exceptionally
poor bee return from fields treated with novaluron to control Lygus spp. Our goal
was to evaluate novaluron toxicity to immature M. rotundata using two different
possible mechanisms of exposure. One goal was to assess immature mortality via
treating nectar-pollen provisions and adults with novaluron. Immature M.
rotundata mortality in all novaluron provision dosing treatments was
significantly higher than the water or blank controls, providing evidence that
novaluron is toxic to progeny in nest cells. The mean cumulative frequency showed
that more eggs and 1st-2nd instars died compared to older instars. Female M.
rotundata nested similarly in field cages during the field cage experiment;
however, there was greater immature mortality in cages where females were fed
sugar-water + novaluron compared to sugar-water only. Although females provided
adequate provisions, there was a low percentage of egg hatch and larval
development when females ingested novaluron before mating and nesting. Novaluron
was also present in egg provision of bees collecting resources from
novaluron-sprayed plants. At least 84% of progeny died when the females were
allowed to mate and nest 24 hours after a novaluron application. Novaluron could
be contributing to poor bee return in alfalfa grown for seed. Timely insecticide
applications to suppress Lygus spp. is an important consideration to improve
ongoing bee health.
DOI: 10.1673/031.011.0143
PMCID: PMC3281451
1158. Planta. 2011 Sep;234(3):445-57. doi: 10.1007/s00425-011-1416-x. Epub 2011 Apr

27.
Comparative studies on tolerance of Medicago truncatula and Medicago falcata to

freezing.
Zhang LL(1), Zhao MG, Tian QY, Zhang WH.
Author information:
(1)State Key Laboratory of Vegetation and Environmental Change, Institute of
Botany, the Chinese Academy of Sciences, Beijing 100093, People's Republic of
China.
Medicago falcata is a legume species that exhibits great capacity of tolerance to

abiotic stresses. To elucidate the mechanism underlying tolerance of M. falcata
to freezing, we compared the characteristics of M. falcata in response to cold
acclimation and freezing with those of the legume model plant Medicago
truncatula. M. falcata seedlings were more tolerant to freezing than M.
truncatula, as evidenced by a lower value of EL(50) (temperature at which 50%
electrolyte leakage after freezing) and greater survival rate for M. falcata than
M. truncatula. Cold acclimation led to greater reduction in EL(50) for M. falcata
than M. truncatula. Sucrose was the most abundant sugar in both M. falcta and M.
truncatula, and a greater accumulation of sucrose and Pro in M. falcata than in
M. truncatula during cold acclimation was observed. Cold acclimation induced
small amounts of raffinose and stachyose in M. falcata, but not in M. truncatula.
The activities of sucrose phosphate synthase and sucrose synthase were greater in
M. falcata than in M. truncatula. In contrast, the activity of acid invertase was
higher in M. truncatula than in M. falcata. There was an increase in transcript
of CRT binding factor (CBF) upon exposure to low temperature in the two species.
The low temperature-induced increase in transcript of CBF2 was much higher in M.
truncatula than in M. falcata, while transcript of CBF3 in M. falcata was greater
than that in M. truncatula. There were sustained increases in transcripts of cold
acclimation specific (CAS), a downstream target of CBF, during cold acclimation
and the increases were greater in M. falcata than in M. truncatula. These results
demonstrate that accumulation of greater amounts of soluble sugars coupled with
higher CBF3 and CAS transcript levels in M. falcata may play a role in conferring
greater tolerance of M. falcata to freezing than that of M. truncatula.
DOI: 10.1007/s00425-011-1416-x
1159. J Environ Qual. 2011 Mar-Apr;40(2):438-48.
Environmental and economic comparisons of manure application methods in farming

systems.
Rotz CA(1), Kleinman PJ, Dell CJ, Veith TL, Beegle DB.
Author information:
(1)USDA-ARS, Bldg. 3702, Curtin Rd., University Park, PA 16802, USA.
al.rotz@ars.usda.gov
Alternative methods for applying livestock manure to no-till soils involve

environmental and economic trade-offs. A process-level farm simulation model
(Integrated Farm System Model) was used to evaluate methods for applying liquid
dairy (Bos taurus L.) and swine (Sus scrofa L.) manure, including no application,
broadcast spreading with and without incorporation by tillage, band application
with soil aeration, and shallow disk injection. The model predicted ammonia
emissions, nitrate leaching, and phosphorus (P) runoff losses similar to those
measured over 4 yr of field trials. Each application method was simulated over 25
yr of weather on three Pennsylvania farms. On a swine and cow-calf beef operation
under grass production, shallow disk injection increased profit by $340 yr(-1)
while reducing ammonia nitrogen and soluble P losses by 48 and 70%, respectively.
On a corn (Zea mays L.)-and-grass-based grazing dairy farm, shallow disk
injection reduced ammonia loss by 21% and soluble P loss by 76% with little
impact on farm profit. Incorporation by tillage and band application with
aeration provided less environmental benefit with a net decrease in farm profit.
On a large corn-and-alfalfa (Medicago sativa L.)-based dairy farm where manure
nutrients were available in excess of crop needs, incorporation methods were not
economically beneficial, but they provided environmental benefits with relatively
low annual net costs ($13 to $18 cow). In all farming systems, shallow disk
injection provided the greatest environmental benefit at the least cost or
greatest profit for the producer. With these results, producers are better
informed when selecting manure application equipment.
1160. BMC Genomics. 2011 Apr 19;12:199. doi: 10.1186/1471-2164-12-199.
Using RNA-Seq for gene identification, polymorphism detection and transcript

profiling in two alfalfa genotypes with divergent cell wall composition in stems.
Yang SS(1), Tu ZJ, Cheung F, Xu WW, Lamb JF, Jung HJ, Vance CP, Gronwald JW.
Author information:
(1)USDA-Agricultural Research Service, Plant Science Research Unit, St, Paul, MN
55108, USA. sam.yang@ars.usda.gov
BACKGROUND: Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial
forage has potential for development as a cellulosic ethanol feedstock. However,
the genomics of alfalfa, a non-model species, is still in its infancy. The recent
advent of RNA-Seq, a massively parallel sequencing method for transcriptome
analysis, provides an opportunity to expand the identification of alfalfa genes
and polymorphisms, and conduct in-depth transcript profiling.
RESULTS: Cell walls in stems of alfalfa genotype 708 have higher cellulose and
lower lignin concentrations compared to cell walls in stems of genotype 773.
Using the Illumina GA-II platform, a total of 198,861,304 expression sequence
tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from
elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In
addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773
using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene
index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the
GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI
1.0 contains 124,025 unique sequences including 22,729 tentative consensus
sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a
total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In
addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted
between the two genotypes. Out of 55 SNPs randomly selected for experimental
validation, 47 (85%) were polymorphic between the two genotypes. We also
identified numerous allelic variations within each genotype. Digital gene
expression analysis identified numerous candidate genes that may play a role in
stem development as well as candidate genes that may contribute to the
differences in cell wall composition in stems of the two genotypes.
CONCLUSIONS: Our results demonstrate that RNA-Seq can be successfully used for
gene identification, polymorphism detection and transcript profiling in alfalfa,
a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled
in this study, and the SNPs, SSRs and candidate genes identified can be used to
improve alfalfa as a forage crop and cellulosic feedstock.
DOI: 10.1186/1471-2164-12-199
PMCID: PMC3112146
1161. Biol Lett. 2011 Oct 23;7(5):743-6. doi: 10.1098/rsbl.2011.0249. Epub 2011 Apr
13.
Symbiont infection affects aphid defensive behaviours.
Dion E(1), Polin SE, Simon JC, Outreman Y.
Author information:
(1)UMR 1099 INRA-Agrocampus Ouest-Université Rennes 1 Biologie des Organismes et
des Populations appliquée à la Protection des Plantes, 65 rue de Saint-Brieuc CS
84215, 35042, Rennes Cedex, France.
Aphids harbour both an obligate bacterial symbiont, Buchnera aphidicola, and a

wide range of facultative ones. Facultative symbionts can modify morphological,
developmental and physiological host traits that favour their spread within aphid
populations. We experimentally investigated the idea that symbionts may also
modify aphid behavioural traits to enhance their transmission. Aphids exhibit
many behavioural defences against enemies. Despite their benefits, these
behaviours have some associated costs leading to reduction in aphid reproduction.
Some aphid individuals harbour a facultative symbiont Hamiltonella defensa that
provides protection against parasitoids. By analysing aphid behaviours in the
presence of parasitoids, we showed that aphids infected with H. defensa exhibited
reduced aggressiveness and escape reactions compared with uninfected aphids. The
aphid and the symbiont have both benefited from these behavioural changes: both
partners reduced the fitness decrements associated with the behavioural defences.
Such symbiont-induced changes of behavioural defences may have consequences for
coevolutionary processes between host organisms and their enemies.
DOI: 10.1098/rsbl.2011.0249
PMCID: PMC3169066
1162. J Environ Qual. 2011 Jan-Feb;40(1):28-36.
Identification and quantitation of volatile organic compounds emitted from dairy

silages and other feedstuffs.
Malkina IL(1), Kumar A, Green PG, Mitloehner FM.
Author information:
(1)Dep. of Animal Science, Univ. of California, One Shields Ave., Davis, CA
95616, USA.
High ground-level ozone continues to be an important human, animal, and plant

health impediment in the United States and especially in California's San Joaquin
Valley (SJV). According to California state and regional air quality agencies,
dairies are one of the major sources of volatile organic compounds (VOCs) in the
SJV. A number of recently conducted studies reported emissions data from
different dairy sources. However, limited data are currently available for silage
and otherfeed storages on dairies, which could potentially contribute to ozone
formation. Because the impact of different VOCs on ozone formation varies
significantly from one molecular species to another, detailed characterization of
VOC emissions is essential to include all the important contributors to
atmospheric chemistry and especially atmospheric reactivity. The present research
study identifies and quantifies the VOCs emitted from various silages and other
feedstuffs. Experiments were conducted in an environmental chamber under
controlled conditions. Almost 80 VOCs were identified and quantified from corn
(Zea mays L.), alfalfa (Medicago sativa L.),and cereal (wheat [Triticum aestivum
L.] and oat [Avena sativava L.] grains) silages, total mixed ration (TMR), almond
(Amygdalus communis L.) shells and hulls using gas chromatography-mass
spectrometry and high performance liquid chromatography. The results revealed
high concentrations of emitted alcohols and other oxygenated species. Lower
concentrations of highly reactive alkenes and aldehydes were also detected.
Additional quantitation and monitoring of these emissions are essential for
assessment of and response to the specific needs of the regional air quality in
the SJV.
1163. Microbes Environ. 2011;26(1):36-45.
Stress tolerance and symbiotic and phylogenic features of root nodule bacteria
associated with Medicago species in different bioclimatic regions of Tunisia.
Djedidi S(1), Yokoyama T, Ohkama-Ohtsu N, Risal CP, Abdelly C, Sekimoto H.
Author information:
(1)United Graduate School of Agricultural Science, Tokyo University of
Agriculture and Technology, 3–5–8 Saiwai-cho, Fuchu, Tokyo 183–8509, Japan.
Thirty two rhizobial isolates were obtained from different bioclimatic regions of
Tunisia using as trap plants, Medicago sativa, Medicago ciliaris, Medicago
polymorpha and Medicago minima. To study their diversity and characterize them in
relation to Mediterranean conditions, abiotic stress resistance, symbiotic
properties and genetic diversity in terms of 16S rRNA and nodA sequences were
assessed. Five isolates from M. sativa, three from M. ciliaris and three from M.
minima could grow at 45°C. Only two isolates from M. sativa grew at 4% NaCl. The
most stress tolerant isolates were obtained from arid soils. A phylogenetic
analysis of 16S rRNA genes revealed 29 isolates to be closely related to Ensifer
including one (Pl.3-9) that showed a 16S rRNA sequence similar to that of Ensifer
meliloti and nodA sequence similar to that of Ensifer medicae. However, three
isolates were categorized into Agrobacterium containing the nodA of Ensifer.
Furthermore, these isolates developed nodules on original hosts. The results for
the four isolates suggest horizontal gene transfer between the species.
1164. J Anim Sci. 2011 Aug;89(8):2472-84. doi: 10.2527/jas.2010-3226. Epub 2011 Apr
8.
Effect of type of grinding of barley and dehydrated alfalfa on performance,

digestion, and crude mucin ileal concentration in growing rabbits.
Romero C(1), Nicodemus N, Rodríguez JD, García AI, de Blas C.
Author information:
(1)Departamento de Producción Animal, Universidad Politécnica de Madrid, Escuela
Técnica Superior de Ingenieros Agrónomos, 28040, Madrid, Spain.
carlos.romerom@upm.es
The effects of type of grinding of barley and dehydrated alfalfa (DA) were tested
in rabbits weaned at 35 d of age with an average BW of 846 ± 93 g. Four
nonmedicated diets were arranged in a 2 × 2 factorial structure, with type of
grinding (coarse grinding with a 4.5-mm screen or fine grinding with a 1.5-mm
screen) of barley (TGB) and DA (TGDA) as the main factors. A total of 1,056
mixed-sex rabbits (264 per diet) were fattened until d 63. Most of these rabbits
(216 per diet) were housed in pairs and were used only to record mortality rate.
Mortality was also recorded for the remaining 192 rabbits, which were housed
individually and used to determine growth performance. From this group, 100
rabbits were used to determine digestive traits. Apart from those rabbits, a
different group of 88 rabbits (44 housed individually and the remaining 44 housed
in pairs) was used in the digestibility trial. All rabbits in this group were
used to determine ileal digestibility (13 pools of ileal digesta per diet) and
ileal mucin concentration (6 pools of ileal digesta per diet), whereas only the
44 individually housed rabbits were used to assess the fecal digestibility
coefficients (11 rabbits per diet). Last, a jejunal sample was excised from 32 of
the 44 individually housed rabbits to determine mucosal histology. Treatments did
not affect ADG, ADFI, or G:F in the entire fattening period, but in the 49- to
63-d period, the diet containing both finely ground barley and DA reduced ADFI
(P=0.08) compared with the other treatments (130 vs. 137 g). Moreover, this diet
increased total digestive tract (4.76%, P=0.08) and cecal content (11.3%, P=0.08)
weights compared with the other 3 treatments. Pylorus (P=0.09) and mixed digesta
(P=0.06) pH, respectively, were reduced from 1.53 and 1.59 to 1.37 and 1.44 when
both barley and DA were finely instead of coarsely ground. Grinding both barley
and DA coarsely reduced the ileal digestibility of starch (0.899 vs. 0.936,
P=0.06), increased (P < 0.01) its ileal flow and content in the feces to 1.66 g/d
and 7.42 g/kg of DM, respectively, and led to decreased fecal digestibility
(0.932 vs. 0.951, P < 0.01) compared with fine grinding. Coarse DA shortened
villi (612 vs. 704 μm, P=0.02), increased crypt depth (121 vs. 92.1 μm, P=0.01),
and reduced the villus:crypt ratio (5.08 vs. 7.66, P < 0.01) compared with finely
ground DA. Furthermore, the greatest ileal crude mucin (148 vs. 107 g/kg of DMI,
P=0.02) and sialic acid (71.7 vs. 61.7 mg/kg of DMI, P=0.04) concentrations were
reported in rabbits receiving the diet with both coarsely ground barley and DA.
Finally, mortality rate was not influenced by treatments, with an average of
9.64%. In conclusion, the diet containing finely ground barley and coarsely
ground DA did not increase the weight of cecal contents, resulting in increased
feed intake and leading to increased ileal digestibility and reduced ileal flow
of starch.
© 2011 American Society of Animal Science. All rights reserved.
DOI: 10.2527/jas.2010-3226
1165. Bioresour Technol. 2011 Jun;102(11):6479-86. doi:

10.1016/j.biortech.2011.03.022.
Epub 2011 Mar 15.
Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol

production by altering lignin composition.
Dien BS(1), Miller DJ, Hector RE, Dixon RA, Chen F, McCaslin M, Reisen P, Sarath
G, Cotta MA.
Author information:
(1)Bioenergy Research Unit, United States Department of Agriculture, Agricultural
Research Service, National Center for Agricultural Utilization Research, Peoria,
IL 61604, USA. Bruce.Dien@ars.usda.gov
Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion
into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines
compared were a reduced S-lignin transgenic cultivar generated through down
regulation of the caffeic acid O-methyltransferase gene and a wild-type control.
Both were harvested at two maturities. All the samples had similar carbohydrate
contents including a mean composition of 316 g glucan and 497 g total neutral
carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield
of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were
significantly impacted by harvest maturity and lignin composition, whereas when
pretreated with dilute-ammonia, yield was solely affected by lignin composition.
Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the
ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol
yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly
enhanced for the reduced S lignin cultivars.
DOI: 10.1016/j.biortech.2011.03.022
1166. Trop Anim Health Prod. 2011 Aug;43(6):1169-74. doi: 10.1007/s11250-011-9819-

z.
Epub 2011 Apr 5.
Rumen development and growth of Balouchi lambs offered alfalfa hay pre- and
post-weaning.
Norouzian MA(1), Valizadeh R, Vahmani P.
Author information:
(1)Department of Animal Science, College of Aboureihan, University of Tehran,
Tehran, P.O. Box 11365-4117, Iran. manorouzian@ut.ac.ir
The consumption of solid feed is essential for successful transition from a

pre-ruminant to a functional digestive tract. Lambs fed starter rations
containing highly fermentable carbohydrates often experience dramatic changes in
concentrations of rumen and blood metabolites. The optimal amount of roughage
required in the diet of pre-ruminant animals is still unclear. The objective of
this study was to determine the effect of feeding alfalfa hay on performance and
rumen development in young Balouchi lambs. In a completely randomized design, 30
lambs were fed one of three experimental diets consisting of a control, without
alfalfa hay (C), a diet containing 7.5% alfalfa hay (A1), and a diet containing
15% alfalfa hay (A2). Lambs fed A1 and A2 diets had lower dry matter intake
during the pre-weaning period (P < 0.01) and overall (P = 0.02), but feed
conversion ratio and average daily gain were not affected by feeding alfalfa hay.
Concentration of beta-hydroxybutyric acid was higher in C compared with the A1
and A2 groups (P < 0.01). Concentrations of glucose and non-esterified fatty
acids did not differ among the groups of animals. Feeding alfalfa hay reduced
thickness of the rumen epithelial keratinized layer (P = 0.04) and increased
thickness of muscular layer (P = 0.05). We concluded that including 15% alfalfa
hay in the starter diet could reduce thickness of the keratinization layer and
increase muscularity of rumen wall without adverse effects on growth and
performance of newborn lambs.
DOI: 10.1007/s11250-011-9819-z
1167. Water Environ Res. 2011 Feb;83(2):116-27.
Factors affecting dissolved phosphorus and nitrate concentrations in ground and

surface water for a valley dairy farm in the northeastern United States.
Flores-López F(1), Easton ZM, Geohring LD, Steenhuis TS.
Author information:
(1)Stockholm Environment Institute, U.S. Center, Davis, California, USA.
Agriculture often is considered to be a contributor of soluble reactive

phosphorus (SRP) and nitrate-N (NO3- -N) to surface waters. This research
analyzed SRP and NO3- -N concentrations in groundwater and in a creek fed by
groundwater on a valley dairy farm in the Cannonsville basin of the New York City
(NYC) watershed. A total of 37 groundwater piezometers were installed to depths
of 0.3 to 1.5 m. Water-table depth and concentrations of SRP, NO3- -N, dissolved
organic carbon (DOC), and dissolved oxygen were measured at regular intervals
over a three-year period. A multivariate mixed model analysis of variance
indicated that the SRP and NO3- -N concentrations were controlled primarily by
three classes of variables: environmental variables, including precipitation and
water table depth; source variables, including manure applied and crop type; and
chemical variables, including DOC and dissolved oxygen concentrations in
groundwater. The highest groundwater concentrations of N03- -N and SRP were found
at the shallowest water-table depths, which has implications for agricultural
nutrient management in areas with shallow groundwater.
1168. Mol Genet Genomics. 2011 May;285(5):393-402. doi: 10.1007/s00438-011-0615-2.

Epub
2011 Mar 26.
Evolution of the number of LRRs in plant disease resistance genes.
Tamura M(1), Tachida H.
Author information:
(1)Graduate School of Systems Life Sciences, Kyushu University, 6-10-1 Hakozaki,
Fukuoka 812-8581, Japan. mtamura@kyudai.jp
The largest group of plant resistance (R) genes contain the regions that encode
the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains (NBS-LRR
genes). To gain new resistance, amino acid substitutions and changes in number of
the LRRs that recognize the presence of pathogens are considered important. In
this study, we focus on the evolution of the number of LRRs and analyze the
genome data of five plant species, Arabidopsis thaliana, Oryza sativa, Medicago
truncatula, Lotus japonicus and Populus trichocarpa. We first categorized the
NBS-LRR genes in each species into groups and subgroups based on the phylogenetic
relationships of their NBS domain sequences. Then we estimated the evolutionary
rate of the number of LRRs relative to the synonymous divergence in the NBS
domain sequences by a maximum likelihood method assuming the single stepwise
mutation model. The estimates ranged from 4.5 to 600 and differed between groups
in the same species or between species. This indicated different roles played by
different groups of the NBS-LRR genes within a species or the effects of various
life history characteristics, such as generation time, of the species. We also
tested the fit of the model to the data using the variance of number of LRRs in
each subgroup. In some subgroups in some plants (16 out of 174 subgroups), the
results of simulation using the estimated rates significantly deviated from the
observed data. Those subgroups may have undergone different modes of selection
from the other subgroups.
DOI: 10.1007/s00438-011-0615-2
1169. Bioinformatics. 2011 May 15;27(10):1368-76. doi:

10.1093/bioinformatics/btr153.
Epub 2011 Mar 26.
PlantMiRNAPred: efficient classification of real and pseudo plant pre-miRNAs.
Xuan P(1), Guo M, Liu X, Huang Y, Li W, Huang Y.
Author information:
(1)Department of Computer Science and Engineering, Harbin Institute of
Technology, Harbin, PR China.
MOTIVATION: MicroRNAs (miRNAs) are a set of short (21-24 nt) non-coding RNAs that
play significant roles as post-transcriptional regulators in animals and plants.
While some existing methods use comparative genomic approaches to identify plant
precursor miRNAs (pre-miRNAs), others are based on the complementarity
characteristics between miRNAs and their target mRNAs sequences. However, they
can only identify the homologous miRNAs or the limited complementary miRNAs.
Furthermore, since the plant pre-miRNAs are quite different from the animal
pre-miRNAs, all the ab initio methods for animals cannot be applied to plants.
Therefore, it is essential to develop a method based on machine learning to
classify real plant pre-miRNAs and pseudo genome hairpins.
RESULTS: A novel classification method based on support vector machine (SVM) is
proposed specifically for predicting plant pre-miRNAs. To make efficient
prediction, we extract the pseudo hairpin sequences from the protein coding
sequences of Arabidopsis thaliana and Glycine max, respectively. These pseudo
pre-miRNAs are extracted in this study for the first time. A set of informative
features are selected to improve the classification accuracy. The training
samples are selected according to their distributions in the high-dimensional
sample space. Our classifier PlantMiRNAPred achieves >90% accuracy on the plant
datasets from eight plant species, including A.thaliana, Oryza sativa, Populus
trichocarpa, Physcomitrella patens, Medicago truncatula, Sorghum bicolor, Zea
mays and G.max. The superior performance of the proposed classifier can be
attributed to the extracted plant pseudo pre-miRNAs, the selected training
dataset and the carefully selected features. The ability of PlantMiRNAPred to
discern real and pseudo pre-miRNAs provides a viable method for discovering new
non-homologous plant pre-miRNAs.
DOI: 10.1093/bioinformatics/btr153
1170. J Hazard Mater. 2011 Jun 15;190(1-3):81-6. doi:

10.1016/j.jhazmat.2011.02.086.
Epub 2011 Mar 6.
Accumulation and residue of napropamide in alfalfa (Medicago sativa) and soil

involved in toxic response.
Cui LE(1), Yang H.
Author information:
Agricultural University, Nanjing, China.
Napropamide belongs to the amide herbicide family and widely used to control
weeds in farmland. Intensive use of the herbicide has resulted in widespread
contamination to ecosystems. The present study demonstrated an analysis on
accumulation of the toxic pesticide napropamide in six genotypes of alfalfa
(Medicago sativa), along with biological parameters and its residues in soils.
Soil was treated with napropamide at 3 mg kg(-1) dry soil and alfalfa plants were
cultured for 10 or 30 d, respectively. The maximum value for napropamide
accumulation is 0.426 mg kg(-1) in shoots and 2.444 mg kg(-1) in roots. The
napropamide-contaminated soil with alfalfa cultivation had much lower napropamide
concentrations than the control (soil without alfalfa cultivation). Also, the
content of napropamide residue in the rhizosphere was significantly lower than
that in the non-rhizosphere soil. M. sativa exposed to 3 mg kg(-1) napropamide
showed inhibited growth. Further analysis revealed that plants treated with
napropamide accumulated more reactive oxygen species (O(2)(-) and H(2)O(2)) and
less amounts of chlorophyll. However, not all cultivars showed oxidative injury,
suggesting that the alfalfa cultivars display different tolerance to napropamide.
DOI: 10.1016/j.jhazmat.2011.02.086
1171. Plant J. 2011 Jul;67(1):181-94. doi: 10.1111/j.1365-313X.2011.04577.x. Epub
2011
Apr 28.
A high-definition native polyacrylamide gel electrophoresis system for the

analysis of membrane complexes.
Ladig R(1), Sommer MS, Hahn A, Leisegang MS, Papasotiriou DG, Ibrahim M, Elkehal
R, Karas M, Zickermann V, Gutensohn M, Brandt U, Klösgen RB, Schleiff E.
Author information:
(1)Institute of Biology - Plant Physiology, Martin Luther University
Halle-Wittenberg, Weinbergweg 10, D-06120 Halle/Saale, Germany.
Native polyacrylamide gel electrophoresis (PAGE) is an important technique for

the analysis of membrane protein complexes. A major breakthrough was the
development of blue native (BN-) and high resolution clear native (hrCN-) PAGE
techniques. Although these techniques are very powerful, they could not be
applied to all systems with the same resolution. We have developed an alternative
protocol for the analysis of membrane protein complexes of plant chloroplasts and
cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-)
PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the
well-studied respiratory chain complexes in mitochondria of bovine heart muscle
and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago
sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the
assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope
membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by
HDN-PAGE permitted us to resolve complexes such as the translocon of the outer
envelope migrating at approximately 700 kDa or of the inner envelope of about 230
and 400 kDa with high resolution. By immunodecoration and mass spectrometry of
these complexes we present new insights into the assembly/composition of these
translocation machineries. The HDN-PAGE technique thus provides an important tool
for future analyses of membrane complexes such as protein translocons.
DOI: 10.1111/j.1365-313X.2011.04577.x
1172. J Hazard Mater. 2011 May 15;189(1-2):357-62. doi:

10.1016/j.jhazmat.2011.02.042.
Epub 2011 Feb 22.
Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human

CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic
pollutants.
Zhang Y(1), Liu J.
Author information:
(1)Department of Pharmaceutics, Qingdao University of Science and Technology, 53
Zhengzhou Road, PO Box 70, Qingdao 266042, China.
Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione

S-transferase (GST) were generated from hypocotyl segments by the use of an
Agrobacterium transformation system for the phytoremediation of the mixed
contaminated soil with heavy metals and organic pollutants. The transgenic
alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST
and CYP2E1 activity and Western blot analysis. The capabilities of mixed
contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic
alfalfa plants became markedly increased compared with the transgenic alfalfa
plants expressing single gene (GST or CYP2E1) and the non-transgenic control
plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures
of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the
introduced GST and CYP2E1 in combination. Our results show that the pKHCG
transgenic alfalfa plants have good potential for phytoremediation because they
have cross-tolerance towards the complex contaminants of heavy metals and organic
pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and
human P450 CDNAs may have a great potential for phytoremediation of mixed
environmental contaminants.
DOI: 10.1016/j.jhazmat.2011.02.042
1173. Arch Oral Biol. 2011 Oct;56(10):960-71. doi:

10.1016/j.archoralbio.2011.02.014.
Epub 2011 Mar 15.
Mandibular corpus bone strains during mastication in goats (Capra hircus): a

comparison of ingestive and rumination chewing.
Williams SH(1), Stover KK, Davis JS, Montuelle SJ.
Author information:
(1)Department of Biomedical Sciences, Ohio University College of Osteopathic
Medicine, Athens, OH 45701, United States. Williams@oucom.ohiou.edu
OBJECTIVE: To compare the mechanical loading environment of the jaw in goats

during ingestive and rumination chewing.
DESIGN: Rosette strain gauges were attached to the external surface of the
mandibular corpus in five goats to record bone strains during the mastication of
hay and rumination.
RESULTS: Strain magnitudes and maximum physiological strain rates during the
mastication of hay are significantly higher than during rumination chewing on the
working and balancing sides. Principal strain ratios and orientations are similar
between the two chewing behaviours. Loading and chewing cycle duration are all
longer during rumination chewing, whereas chew duty factor and variances in load
and chewing cycle durations are higher during ingestive chewing. For most of the
variables, differences in strain magnitudes or durations are similar at all three
gauge sites, suggesting that rumination and ingestive chewing do not
differentially influence bone at the three gauge sites.
CONCLUSIONS: Despite lower strain magnitudes, the repetitive nature of rumination
chewing makes it an important component of the mechanical loading environment of
the selenodont artiodactyl jaw. However, similarities in principal strain
orientations and ratios indicate that rumination chewing need not be considered
as a unique loading behaviour influencing the biomechanics of the selenodont
artiodactyl jaw. Differences in loading and chewing cycle durations during
rumination and ingestion demonstrate flexibility in adult chewing frequencies.
Finally, although the low within-sequence variability in chewing cycle durations
supports the hypothesis that mammalian mastication is energetically efficient,
chewing during rumination may not be more efficient than during ingestion.
DOI: 10.1016/j.archoralbio.2011.02.014
1174. J Biotechnol. 2011 Aug 20;155(1):20-33. doi: 10.1016/j.jbiotec.2010.12.018.

Epub
2011 Mar 17.
The complete genome sequence of the dominant Sinorhizobium meliloti field isolate
SM11 extends the S. meliloti pan-genome.
Schneiker-Bekel S(1), Wibberg D, Bekel T, Blom J, Linke B, Neuweger H, Stiens M,

Vorhölter FJ, Weidner S, Goesmann A, Pühler A, Schlüter A.
Author information:
Research and Systems Biology, Universitätsstr. 27, D-33615 Bielefeld, Germany.
Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually

contain a chromosome and two large megaplasmids encoding functions that are
absolutely required for the specific interaction of the microsymbiont with
corresponding host plants leading to an effective symbiosis. The complete genome
sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d
(related to pSymB), was established for the dominant, indigenous S. meliloti
strain SM11 that had been isolated during a long-term field release experiment
with genetically modified S. meliloti strains. The chromosome, the largest
replicon of S. meliloti SM11, is 3,908,022bp in size and codes for 3785 predicted
protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319bp and it
contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is
1,632,395bp in size and comprises 1548 predicted coding sequences. The gene
content of the SM11 chromosome is quite similar to that of the reference strain
S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain
revealed that many gene regions of these replicons are variable, supporting the
assessment that pSymA is a major hot-spot for intra-specific differentiation.
Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of
pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae
WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g.
additional plasmid survival genes (partition, mobilisation and conjugative
transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase
involved in modulation of the phytohormone ethylene level and genes having
predicted functions in degradative capabilities, stress response, amino acid
metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c,
megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing
extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b
contains a new gene cluster predicted to be involved in polysaccharide
biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to
an extension of the S. meliloti pan-genome.
DOI: 10.1016/j.jbiotec.2010.12.018
1175. J Appl Microbiol. 2011 May;110(5):1351-8. doi: 10.1111/j.1365-

2672.2011.04993.x.
Epub 2011 Apr 4.
Alginate beads as a storage, delivery and containment system for genetically
modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens
F113.
Power B(1), Liu X, Germaine KJ, Ryan D, Brazil D, Dowling DN.
Author information:
(1)Department of Science and Health, Institute of Technology Carlow, Carlow,
Ireland.
AIMS: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere

bacterium with the potential to degrade polychlorinated biphenyls (PCBs).
F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB
bioavailability and biodegradation. The aim of this paper is to evaluate the use
of alginate beads as a storage, delivery and containment system for use of these
strains in PCB contaminated soils.
METHODS AND RESULTS: The survival and release of Ps. fluorescens F113Rifpcb from
alginate beads were evaluated. Two Ps. fluorescens F113-based biosensor strains
were encapsulated, and their ability to detect 3-chlorobenzoate (3-CBA) and
3-chlorobiphenyl (3-CBP) degradation in soil was assessed. After 250 days of
storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the
alginate formulation allowed for the timed release of the inoculant.
Encapsulation of the F113Rifpcb cells provided a more targeted approach for the
inoculation of plants and resulted in lower inoculum populations in the bulk
soil, which may reduce the risk of unintentional spread of these genetically
modified micro-organisms in the environment. Encapsulation of the biosensor
strains in alginate beads did not interfere with their ability to detect either
3-CBA or 3-CBP degradation. In fact, detection of 3-CBP degradation was enhanced
in encapsulated biosensors.
CONCLUSIONS: Alginate beads are an effective storage and delivery system for
PCB degrading inocula and biosensors.
SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens F113Rifpcb and the
F113 derivative PCB biosensor strains have excellent potential for detecting and
bioremediation of PCB contaminated soils. The alginate bead delivery system could
facilitate the application of these strains as biosensors.
© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for
Applied Microbiology.
DOI: 10.1111/j.1365-2672.2011.04993.x
1176. Res Microbiol. 2011 Nov;162(9):896-907. doi: 10.1016/j.resmic.2011.02.011.

Epub
2011 Mar 21.
Biological functioning of PAH-polluted and thermal desorption-treated soils

assessed by fauna and microbial bioindicators.
Cébron A(1), Cortet J, Criquet S, Biaz A, Calvert V, Caupert C, Pernin C, Leyval

C.
Author information:
(1)LIMOS, UMR 7137 CNRS - Nancy Université, Faculté des Sciences, BP70239, 54506
Vandoeuvre-les-Nancy cedex, France. aurelie.cebron@limos.uhp-nancy.fr
A large number of soil bioindicators were used to assess biological diversity and
activity in soil polluted with polycyclic aromatic hydrocarbons (PAHs) and the
same soil after thermal desorption (TD) treatment. Abundance and biodiversity of
bacteria, fungi, protozoa, nematodes and microarthropods, as well as functional
parameters such as enzymatic activities and soil respiration, were assessed
during a two year period of in situ monitoring. We investigated the influence of
vegetation (spontaneous vegetation and Medicago sativa) and TD treatment on
biological functioning. Multivariate analysis was performed to analyze the whole
data set. A principal response curve (PRC) technique was used to evaluate the
different treatments (various vegetation and contaminated vs. TD soil) contrasted
with control (bare) soil over time. Our results indicated the value of using a
number of complementary bioindicators, describing both diversity and functions,
to assess the influence of vegetation on soil and discriminate polluted from
thermal desorption (TD)-treated soil. Plants had an influence on the abundance
and activity of all organisms examined in our study, favoring the whole trophic
chain development. However, although TD-treated soil had a high abundance and
diversity of microorganisms and fauna, enzymatic activities were weak because of
the strong physical and chemical modifications of this soil.
Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights
reserved.
DOI: 10.1016/j.resmic.2011.02.011
1177. Nat Biotechnol. 2011 Mar;29(3):179-81. doi: 10.1038/nbt0311-179a.
Industry exhales as USDA okays glyphosate-resistant alfalfa.
Waltz E.
DOI: 10.1038/nbt0311-179a
1178. Mol Plant Microbe Interact. 2011 Apr;24(4):478-86. doi: 10.1094/MPMI-07-10-

0155.
Controlling the expression of rhizobial genes during nodule development with

elements and an inducer of the lac operon.
Box J(1), Noel KD.
Author information:
(1)Department of Biological Sciences, Marquette University, PO Box 1881,
Milwaukee, WI 53201, USA.
A simple strategy was tested for imposing artificial regulation of rhizobial

genes during nodule development. Isopropyl-β-d-1-thiogalactoside (IPTG) was added
to liquid root media to sustain expression of rhizobial genes controlled by
Escherichia coli lac promoter/operators and repressor gene lacI. Conversely, a
rinsing protocol was devised to remove IPTG sufficiently that genes could be
repressed after having been induced. gusA under this control exhibited clearly
delineated expression and repression in both the determinate Rhizobium
etli-Phaseolus vulgaris and the indeterminate Sinorhizobium meliloti-Medicago
sativa symbioses. Apparently, IPTG was taken up in sufficiently undegraded
concentrations that gene expression was derepressed even in interior portions of
the nodule. Moreover, the rinsing protocol led to obvious repression of gusA.
Importantly, no deleterious effects of IPTG on nodule development, infection, or
nitrogen fixation were observed. An R. etli CE3 gene required for
lipopolysaccharide O antigen and infection on bean was put under this control by
means of a two-plasmid construct. When this construct was added to a strain with
a null mutation in this gene, infection, nodule development, and nitrogenase
activity all depended on the length of time before IPTG was rinsed from the roots
after inoculation.
DOI: 10.1094/MPMI-07-10-0155
1179. Biochim Biophys Acta. 2011 Aug;1809(8):353-9. doi:

10.1016/j.bbagrm.2011.02.005.
Plant histone acetylation: in the beginning ...
Waterborg JH(1).
Author information:
(1)School of Biological Sciences, University of Missouri, Kansas City, MO 64110,
USA. WaterborgJ@umkc.edu
The study of histone acetylation in plants started with protein purification and
sequencing, with gel analysis and the use of radioactive tracers. In alfalfa,
acid urea Triton gel electrophoresis and in vivo labeling with tritated acetate
and lysine quantified dynamic acetylation of core histones and identified the
replication-coupled and -independent expression patterns of the histone H3.1 and
H3.2 variants. Pulse-chase analyses demonstrated protein turnover of newly
synthesized histone H3.2 and thereby identified the replacement H3 histones of
plants which maintain the nucleosome density of transcribed chromatin. Sequence
analysis of histone H4 revealed acetylation of lysine 20, a site typically
methylated in animals and yeasts. Histone deacetylase inhibitors butyrate and
trichostatin A are metabolized in alfalfa, but loss of TSA is slow, allowing its
use to induce transient hyperacetylation of histones H2B, H4 and H3. This article
is part of a Special Issue entitled: Epigenetic Control of cellular and
developmental processes in plants.
DOI: 10.1016/j.bbagrm.2011.02.005
1180. New Phytol. 2011 Jul;191(1):120-31. doi: 10.1111/j.1469-8137.2011.03670.x.

Epub
2011 Mar 4.
Effects of selenium hyperaccumulation on plant-plant interactions: evidence for

elemental allelopathy?
El Mehdawi AF(1), Quinn CF, Pilon-Smits EA.
Author information:
(1)Biology Department, Colorado State University, Fort Collins, CO 80523, USA.
• Few studies have investigated plant-plant interactions involving

hyperaccumulator plants. Here, we investigated the effect of selenium (Se)
hyperaccumulation on neighboring plants. • Soil and litter Se concentrations were
determined around the hyperaccumulators Astragalus bisulcatus and Stanleya
pinnata and around the nonhyperaccumulators Medicago sativa and Helianthus
pumilus. We also compared surrounding vegetative cover, species composition and
Se concentration in two plant species (Artemisia ludoviciana and Symphyotrichum
ericoides) growing either close to or far from Se hyperaccumulators. Then,
Arabidopsis thaliana germination and growth were compared on soils collected next
to the hyperaccumulators and the nonhyperaccumulators. • Soil collected around
hyperaccumulators contained more Se (up to 266 mg Se kg(-1) ) than soil collected
around nonhyperaccumulators. Vegetative ground cover was 10% lower around Se
hyperaccumulators compared with nonhyperaccumulators. The Se concentration was
higher in neighboring species A. ludoviciana and S. ericoides when growing close
to, compared with far from, Se hyperaccumulators. A. thaliana showed reduced
germination and growth, and higher Se accumulation, when grown on soil collected
around Se hyperaccumulators compared with soil collected around nonaccumulators.
• In conclusion, Se hyperaccumulators may increase the surrounding soil Se
concentration (phytoenrichment). The enhanced soil Se contents around
hyperaccumulators can impair the growth of Se-sensitive plant species, pointing
to a possible role of Se hyperaccumulation in elemental allelopathy.
DOI: 10.1111/j.1469-8137.2011.03670.x
1181. Huan Jing Ke Xue. 2010 Dec;31(12):3062-6.
[Phytoremediation of polychlorinated biphenyls contaminated soil by

leguminosae-gramineae intercropping: a field trial].
Tu C(1), Teng Y, Luo YM, Pan C, Sun XH, Li ZG.
Author information:
Soil Sciecne, Chinese Academy of Sciences, Nanjing 210008, China.
tchen@issas.ac.cn
Phytoremediation of polychlorinated biphenyls (PCBs) contaminated soil by

leguminosae (alfalfa) and gramineae (ryegrass and tall fescue) was studied in a
field trial. All planted treatments had significantly higher PCBs removal
efficiency compared with those of unplanted control after 270 days of in-situ
phytoremediation. Alfalfa monoculture received the highest removal efficiency of
soil PCBs by 59.6%. Analysis of PCBs congeners composition showed that the
percentage of di-chlorinated biphenyl decreased in all planted treatments.
Alfalfa produced the maximum biomass among the 3 plants. Total PCBs concentration
in alfalfa roots reached 355.1 microg/kg, which was significantly higher than
those in ryegrass and tall fescue. The phytoextraction efficiency of different
treatment was in order of alfalfa > alfalfa-ryegrass-tall fescue >
alfalfa-ryegrass > ryegrass > alfalfa-tall fescue > tall fescue. The results
suggest that alfalfa may be an ideal candidate for the phytoremediation of PCBs
contaminated soil.
1182. Huan Jing Ke Xue. 2010 Dec;31(12):2995-3003.
[Seasonal characteristics of soil respiration and affecting factors under typical

vegetations in the water-wind erosion crisscross region of the Loess Plateau].
Xie HH(1), Fan J, Qi LB, Hao MD.
Author information:
(1)College of Resources and Environment, Northwest A & F University, Yangling
712100, China. xiehui3108hh@gmail.com
By the soil respiration system, the characteristics of soil respiration were

investigated to explore the correlations between soil respiration and soil
temperature, soil water and soil nutrient under different land use patterns in
the water-wind erosion crisscross region of the Loess Plateau. The results
indicated that the seasonal changing characteristics of soil respiration were
distinguished significantly among different vegetations, and soil water content
and temperature were the main influencing factors. Soil respiration seasonal
changing ranges, such as bare land, crop land, Medicago sativa land, Caragana
korshinskii land, abandoned wild grass land, Stipa bungeana land, wild grass
land, degraded Medicago sativa land, sloping Medicago sativa land, sloping
abandoned wild grass land, sloping crop land and terraced crop land, were
0.32-0.82, 0.41-2.83, 0.74-2.81, 0.76-3.07, 0.67-2.79, 0.51-2.12, 0.56-2.05,
0.59-1.66, 0.42-2.09, 0.31-1.86, 0.32-1.93 and 0.41-3.17 micromol x (m2 x s)(-1).
Comparing seasonal changing magnitudes of soil respiration, crop land was the
biggest (167% - 203%), abandoned wild grass land (117% -154%), Caragana
korshinskii land (134%), Stipa bungeana land (129%), Medicago sativa land
(119%-120%) and bare land (94%) followed crop land. The smallest was degraded
Medicago sativa land (92%). Bare land and degraded Medicago sativa land had small
seasonal variation during the study period. Monthly average values of soil carbon
flux (soil respiration) of Medicago sativa land and Caragana korshinskii land
were maximal, but the maximum values under crop land were observed in July and
August. Besides Q10 of crop land was also maximal, which reached 1.86. There were
significant correlations between soil respiration and soil organic matter and
available K. Moreover, soil respiration was affected by soil organic matter and
total nitrogen in July and August when the water and heat condition were
plentiful, but soil ammonium nitrogen had negative impact on soil respiration in
the seasons when water and heat were not optimal.
1183. Can J Microbiol. 2011 Mar;57(3):186-95. doi: 10.1139/W10-118.
Malate synthase expression is deregulated in the Pseudomonas aeruginosa cystic

fibrosis isolate FRD1.
Hagins JM(1), Scoffield J, Suh SJ, Silo-Suh L.
Author information:
(1)Department of Biological Sciences, Auburn University, AL 36849, USA.
Pseudomonas aeruginosa causes chronic pulmonary infections, which can persist for
decades, in patients with cystic fibrosis (CF). Current evidence suggests that
the glyoxylate pathway is an important metabolic pathway for P. aeruginosa
growing within the CF lung. In this study, we identified glcB, which encodes for
the second key enzyme of the glyoxylate pathway, malate synthase, as a
requirement for virulence of P. aeruginosa on alfalfa seedlings. While expression
of glcB in PAO1, an acute isolate of P. aeruginosa, responds to some carbon
sources that use the glyoxylate pathway, expression of glcB in FRD1, a CF
isolate, is constitutively upregulated. Malate synthase activity is moderately
affected by glcB expression and is nearly constitutive in both backgrounds, with
slightly higher activity in FRD1 than in PAO1. In addition, RpoN negatively
regulates glcB in PAO1 but not in FRD1. In summary, the genes encoding for the
glyoxylate-specific enzymes appear to be coordinately regulated, even though they
are not located within the same operon on the P. aeruginosa genome. Furthermore,
both genes encoding for the glyoxylate enzymes can become deregulated during
adaptation of the bacterium to the CF lung.
DOI: 10.1139/W10-118

Epub
2011 Feb 25.
Effects of methanogenic inhibitors on methane production and abundances of

methanogens and cellulolytic bacteria in in vitro ruminal cultures.
Zhou Z(1), Meng Q, Yu Z.
Author information:
(1)College of Animal Science and Technology and State Key Laboratory of Animal
Nutrition, China Agricultural University, Beijing 100193, China.
The objective of this study was to systematically evaluate and compare the
effects of select antimethanogen compounds on methane production, feed digestion
and fermentation, and populations of ruminal bacteria and methanogens using in
vitro cultures. Seven compounds, including 2-bromoethanesulphonate (BES),
propynoic acid (PA), nitroethane (NE), ethyl trans-2-butenoate (ETB),
2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were
tested at a final concentration of 12 mM. Ground alfalfa hay was included as the
only substrate to simulate daily forage intake. Compared to no-inhibitor
controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to
99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate
(79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23%
without a significant effect on total VFAs, acetate, or propionate. BES
significantly reduced the propionate concentration but not the production of
methane, total VFAs, or acetate. ETB or NE had no significant effect on any of
the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed
that none of the inhibitors significantly affected total bacterial populations
but that they did reduce the Fibrobacter succinogenes population. SN reduced the
Ruminococcus albus population, while PA and 2NEOH increased the populations of
both R. albus and Ruminococcus flavefaciens. Archaeon-specific PCR-denaturing
gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the
methanogen population structure, while archaeon-specific qPCR revealed a
significant decrease in methanogen population in all treatments. These results
showed that EB, ETB, NE, and BES can effectively reduce the total population of
methanogens but that they reduce methane production to a lesser extent. The
results may guide future in vivo studies to develop effective mitigation of
methane emission from ruminants.
DOI: 10.1128/AEM.02779-10
PMCID: PMC3126374
1185. Phytochemistry. 2011 Jul;72(10):1020-7. doi: 10.1016/j.phytochem.2011.01.026.

Epub 2011 Feb 23.
A legume specific protein database (LegProt) improves the number of identified

peptides, confidence scores and overall protein identification success rates for
legume proteomics.
Lei Z(1), Dai X, Watson BS, Zhao PX, Sumner LW.
Author information:
(1)Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble
Parkway, Ardmore, OK 73401, USA.
A legume specific protein database (LegProt) has been created containing

sequences from seven legume species, i.e., Glycine max, Lotus japonicus, Medicago
sativa, Medicago truncatula, Lupinusalbus, Phaseolus vulgaris, and Pisum sativum.
The database consists of amino acid sequences translated from predicted gene
models and 6-frame translations of tentative consensus (TC) sequences assembled
from expressed sequence tags (ESTs) and singleton ESTs. This database was queried
using mass spectral data for protein identification and identification success
rates were compared to the NCBI nr database. Specifically, Mascot MS/MS ion
searches of tandem nano-LC Q-TOFMS/MS mass spectral data showed that relative to
the NCBI nr protein database, the LegProt database yielded a 54% increase in the
average protein score (i.e., from NCBI nr 480 to LegProt 739) and a 50% increase
in the average number of matched peptides (i.e., from NCBI nr 8 to LegProt 12).
The overall identification success rate also increased from 88% (NCBI nr) to 93%
(LegProt). Mascot peptide mass fingerprinting (PMF) searches of the LegProt
database using MALDI-TOFMS data yielded a significant increase in the
identification success rate from 19% (NCBI nr) to 34% (LegProt) while the average
scores and average number of matched peptides showed insignificant changes. The
results demonstrate that the LegProt database significantly increases legume
protein identification success rates and the confidence levels compared to the
commonly used NCBI nr. These improvements are primarily due to the presence of a
large number of legume specific TC sequences in the LegProt database that were
not found in NCBI nr. The LegProt database is freely available for download
(http://bioinfo.noble.org/manuscript-support/legumedb) and will serve as a
valuable resource for legume proteomics.
DOI: 10.1016/j.phytochem.2011.01.026
1186. J Plant Physiol. 2011 Jul 1;168(10):1076-83. doi:

10.1016/j.jplph.2010.12.017.
Epub 2011 Feb 23.
Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase

gene from Lactuca sativa.
Ren W(1), Zhao L, Zhang L, Wang Y, Cui L, Tang Y, Sun X, Tang K.
Author information:
(1)Plant Biotechnology Research Center, School of Agriculture and Biology, Key
Laboratory of Urban Agriculture South, Ministry of Agriculture,
Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Shanghai Jiao Tong
University, Shanghai 200240, PR China.
Vitamin E has been found to be associated with an important antioxidant property

in mammals and plants. In photosynthetic organisms, the enzyme
4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important
role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD
was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE).
The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open
reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence
analysis indicated that LsHPPD shared high identity with HPPD from Medicago
truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed
that LsHPPD was preferentially expressed in mature leaves compared with other
tissues and that the LsHPPD expression was sensitive to high light and drought
stress treatments. Transient expression of LsHPPD via agroinfiltration resulted
in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in
α-tocopherol content compared with the negative control. A decrease in
chlorophyll content and inhibition of photosystem II were observed during stress
treatments and agroinfiltration.
DOI: 10.1016/j.jplph.2010.12.017
Modeling degradation ratios and nutrient availability of

anthocyanidin-accumulating Lc-alfalfa populations in dairy cows.
Jonker A(1), Gruber MY, Wang Y, Coulman B, Azarfar A, McKinnon JJ, Christensen
DA, Yu P.
Author information:
(1)Department of Animal and Poultry Science, University of Saskatchewan, 51
Campus Drive, Saskatoon, SK, Canada. arj051@mail.usask.ca
Dairy cattle eating fresh or ensiled alfalfa as the main portion of their diet
often have low protein efficiency because of the rapid initial rate of ruminal
protein degradation of fresh and ensiled alfalfa. Ruminal protein degradation of
alfalfa might be reduced by introducing a gene that stimulates the accumulation
of mono- or polymeric anthocyanidins in alfalfa. The objectives of this study
were to fractionate protein and carbohydrates by in situ and chemical approaches,
to evaluate in situ ruminal degradation characteristics and synchronization
ratios, to determine protein availability to dairy cattle using the 2007
digestible intestinal protein/rumen-degraded protein balance (DVE/OEB) protein
system, and to determine net energy for lactation using the Dutch net energy for
lactation (VEM) system for 3 newly developed transgenic winter hardy
anthocyanidin-accumulating T(1)Lc-alfalfa populations. These T(1)Lc-alfalfa
populations, called (T1)BeavLc1, (T1)RambLc3, and (T1)RangLc4, had an average
anthocyanidin accumulation of 163.4 μg/g of DM, whereas AC Grazeland (selected
for a low initial rate of degradation) did not accumulate anthocyanidin. The
basic chemical composition of the original samples, soluble and potentially
degradable fractions, and degradation characteristics of crude protein and
carbohydrates were similar in T(1)Lc-alfalfa and AC Grazeland. The undegradable
in situ crude protein and neutral detergent fiber fraction had 1.3% lower CP and
4.8% lower CHO, respectively, in T(1)Lc-alfalfa compared with the amounts in AC
Grazeland. The T(1)Lc-alfalfa had a 0.34 MJ/kg of DM higher calculated net energy
for lactation and 1.9% of CP higher buffer soluble protein compared with that in
AC Grazeland. By the protein evaluation model, it was predicted that
T(1)Lc-alfalfa tended to have 11.9, 6.9, and 8.4 g/kg of DM higher rumen
degradable protein, OEB, and intestinal available protein, respectively, compared
with the amounts in AC Grazeland. The hourly OEB included an initial and
substantial peak (oversupply) of protein relative to energy, which was highest in
(T1)RangLc4 and lowest in (T1)RambLc3. The hourly OEB between 4 and 24h was
similar and more balanced for all 4 alfalfa populations. In conclusion,
T(1)Lc-alfalfa accumulated anthocyanidin, tended to have higher predicted
intestinal protein availability, and had higher predicted net energy of lactation
availability for dairy cattle than did AC Grazeland.

DOI: 10.3168/jds.2010-3604

Epub
2011 Feb 15.
First genomic analysis of the broad-host-range Rhizobium sp. LPU83 strain, a

member of the low-genetic diversity Oregon-like Rhizobium sp. group.
Tejerizo GT(1), Del Papa MF, Draghi W, Lozano M, Giusti Mde L, Martini C, Salas
ME, Salto I, Wibberg D, Szczepanowski R, Weidner S, Schlüter A, Lagares A,
Pistorio M.
Author information:
Nacional de La Plata, Calles 47 y 115, (1900) La Plata, Argentina.
Alfalfa (Medicago sativa) is the most cultivated forage legume for cattle and
animal feeding, occupying about 32 million hectares over the world. Management of
the N₂-fixing symbiosis of this plant to maximize crop production is therefore an
important objective. A fundamental constraint to this aim emerges when a
moderately low soil pH hampers the establishment of an effective symbiosis with
indigenous and/or inoculated rhizobia. Besides the association of alfalfa with
Ensifer (Sinorhizobium) meliloti, this legume is able to establish a symbiosis
with Ensifer (Sinorhizobium) medicae and with less characterized types of
rhizobia, such as the Oregon-like strains, Rhizobium sp. Or191 initially isolated
in the USA, and the Rhizobium sp. LPU83 strain, from Argentina. These strains are
acid-tolerant, highly competitive for acidic-soil-alfalfa nodulation, but
inefficient for biological nitrogen fixation with alfalfa. These features
position the Oregon-like rhizobia as strains of potential risk in agricultural
soils compared with the efficient symbiont E. meliloti. Moreover, the collected
genetic information has revealed that the genomic structure of these rhizobial
isolates is complex in terms of sequence similarities shared with other rhizobia.
Such a "patched" genetic composition has obviously imposed severe restrictions to
the classical taxonomy of these rhizobia. In this work we summarize the
accumulated knowledge about the Oregon-like rhizobia and present a phylogenetic
analysis based on genome sequence data of Rhizobium sp. LPU83 obtained by a
high-throughput sequencing on the Genome Sequencer FLX Titanium platform. The
accessibility of the complete genomic sequence will release up more experimental
possibilities since this information will then enable biochemical studies as well
as proteomics and transcriptomics approaches.
DOI: 10.1016/j.jbiotec.2011.01.011
1189. Plant Physiol Biochem. 2011 Jul;49(7):792-9. doi:

10.1016/j.plaphy.2011.01.018.
Epub 2011 Jan 21.
Molecular cloning, characterization, and expression of an alfalfa (Medicago

sativa L.) heme oxygenase-1 gene, MsHO1, which is pro-oxidants-regulated.
Fu GQ(1), Xu S, Xie YJ, Han B, Nie L, Shen WB, Wang R.
Author information:
Technique, Nanjing Agricultural University, Nanjing 210095, PR China.
It has been documented that plant heme oxygenase-1 (HO-1; EC 1.14.99.3) is both
development- and stress-regulated, thus it plays a vital role in light signalling
and stress responses. In this study, an alfalfa (Medica sativa L.) HO-1 gene
MsHO1 was isolated and sequenced. It contains four exons and three introns within
genomic DNA sequence and encodes a polypeptide with 283 amino acids. MsHO1 had a
conserved HO signature sequence and showed high similarity to other HOs in
plants, especially HO-1 isoform. The MsHO1:GFP fusion protein was localized in
the chloroplast. Further biochemical activity analysis of mature MsHO1, which was
expressed in Escherichia coli, showed that the Vmax was 48.78 nmol biliverdin-IXα
(BV) h⁻¹ nmol⁻¹ protein with an apparent Km value for hemin of 2.33 μM, and the
optimum Tm and pH were 37 °C and 7.2, respectively. Results of semi-quantitative
RT-PCR and western blot showed that the expressions of MsHO1 were higher in
alfalfa stems and leaves than those in germinating seeds and roots. Importantly,
MsHO1 gene expression and protein level were induced significantly by some
pro-oxidant compounds, including hemin and nitric oxide (NO) donor sodium
nitroprusside (SNP). In conclusion, MsHO1 may play an important role in oxidative
responses.
DOI: 10.1016/j.plaphy.2011.01.018
1190. J Econ Entomol. 2010 Dec;103(6):2009-18.
Development and population growth of Lygus hesperus on selected weed hosts,

artificial diet and cotton in the laboratory.
Chen C(1), Parajulee MN.
Author information:
(1)Texas A&M System AgriLife Research and Extension Center, 1102 E FM 1294,
Lubbock, TX 79403, USA.
Laboratory studies were conducted to obtain basic biological information of Lygus

hesperus Knight (Hemiptera: Miridae) on alfalfa (Medicago sativa L.), pigweed
(Amaranthus palmeri S. Wats.), Russian thistle (Salsola iberica Sennen and Pau),
green bean (Phaseolus vulgaris L.), artificial diet, and cotton (Gossypium
hirsutum L.) square and cotton boll at 27 degrees C. The nymphal developmental
duration was 28.8 d on cotton bolls, nearly twice longer, compared with
approximately 15 d in other hosts. The nymphal survivorship was significantly
lowest on the cotton boll (18%), whereas the survivorship in other hosts ranged
from 33% (cotton square) to 93% (green bean). The average total nymphal
survivorship was approximately 60% and the most nymphal mortalities occurred on
the first three instars. The life-table parameters were obtained only in cotton
square, artificial diet, green bean, and alfalfa. The average adult longevity in
artificial diet was the shortest (18.7 d),with the longest longevity observed
(36.1 d) on green beans. The intrinsic rates of population increase (r(m)) were
much higher in artificial diet (0.0771) and green beans (0.0671) than in alfalfa
(0.0327) and cotton square (0.0317). Although statistically significant, the
difference in r(m) values between alfalfa and cotton square was very small,
indicating the similarity in reproductive suitability of cotton square and
alfalfain a no-choice test. Thus, even though cotton is not a preferred host,
when blooming alfalfa and roadside weeds are mowed in the Texas High Plains
during June-July, it is the most likely time that Lygus bugs may move from
alfalfa and other host plants into adjacent cotton fields and become effective
pest of actively fruiting cotton.
1191. Environ Toxicol Chem. 2011 May;30(5):1044-9. doi: 10.1002/etc.474. Epub 2011
Feb
25.
Use of vegetated agricultural drainage ditches to decrease pesticide transport

from tomato and alfalfa fields in California, USA.
Moore MT(1), Denton DL, Cooper CM, Wrysinski J, Miller JL, Werner I, Horner G,
Crane D, Holcomb DB, Huddleston GM 3rd.
Author information:
(1)U.S. Department of Agriculture-Agricultural Research Service, National
Sedimentation Laboratory, Oxford, Mississippi, USA. matt.moore@ars.usda.gov
Irrigation and storm water runoff from agricultural fields has the potential to
cause impairment to downstream aquatic receiving systems. Over the last several
years, scientists have discovered the benefit of using edge-of-field practices,
such as vegetated agricultural drainage ditches, in the mitigation of pesticides
and sediment. After demonstrating this practice's feasibility in California,
field trials were initiated to document irrigation runoff pesticide mitigation in
California alfalfa and tomato fields. In the alfalfa field, chlorpyrifos
concentration was decreased by 20% from the inflow to the ditch outflow.
Thirty-two percent of the measured chlorpyrifos mass was associated with ditch
plant material. In the tomato field, permethrin concentration was decreased by
67% and there was a 35% reduction in suspended sediment concentration from inflow
to the ditch outflow. When surface water was not present in the ditch systems,
the sediment was a significant repository for pesticides. Based on the field
trials, vegetated agricultural drainage ditches can be successfully used as part
of a suite of management practices to reduce pesticide and sediment runoff into
aquatic receiving systems.
DOI: 10.1002/etc.474
1192. Plant Physiol Biochem. 2011 Mar;49(3):303-10. doi:

10.1016/j.plaphy.2011.01.004.
Epub 2011 Jan 13.
A stress-associated protein containing A20/AN1 zing-finger domains expressed in

Medicago truncatula seeds.
Gimeno-Gilles C(1), Gervais ML, Planchet E, Satour P, Limami AM, Lelievre E.
Author information:
(1)University of Angers, UMR-1191 Physiologie Moléculaire des Semences, IFR 149
Quasav, 2 Boulevard Lavoisier, Angers Cedex 01, France.
MtSAP1 (Medicago truncatula stress-associated protein 1) was revealed as a

down-regulated gene by suppressive subtractive hybridization between two mRNA
populations of embryo axes harvested before and after radicle emergence. MtSAP1
is the first gene encoding a SAP with A20 and AN1 zinc-finger domains
characterized in M. truncatula. MtSAP1 protein shares 54% and 62% homology with
AtSAP7 (Arabidopsis thaliana) and OsiSAP8 (Oryza sativa) respectively, with in
particular a strong homology in the A20 and AN1 conserved domains. MtSAP1 gene
expression increased in the embryos during the acquisition of tolerance to
desiccation, reached its maximum in dry seed and decreased dramatically during
the first hours of imbibition. Abiotic stresses (cold and hypoxia), abscisic acid
and desiccation treatments induced MtSAP1 gene expression and protein
accumulation in embryo axis, while mild drought stress did not affect
significantly its expression. This profile of expression along with the presence
of anaerobic response elements and ABRE sequences in the upstream region of the
gene is consistent with a role of MtSAP1 in the tolerance of low oxygen
availability and desiccation during late stages of seed maturation. Silencing of
MtSAP1 by RNA interference (RNAi) showed that the function of the encoded protein
is required for adequate accumulation of storage globulin proteins, vicilin and
legumin, and for the development of embryos able to achieve successful
germination.
DOI: 10.1016/j.plaphy.2011.01.004
1193. Science. 2011 Feb 4;331(6017):523. doi: 10.1126/science.331.6017.523.
Agbiotech. USDA decides against new regulation of GM crops.
Stokstad E.
DOI: 10.1126/science.331.6017.523
1194. Rapid Commun Mass Spectrom. 2011 Mar 15;25(5):599-607. doi: 10.1002/rcm.4895.
Measurement of 13C and 15N isotope labeling by gas

chromatography/combustion/isotope ratio mass spectrometry to study amino acid
fluxes in a plant-microbe symbiotic association.
Molero G(1), Aranjuelo I, Teixidor P, Araus JL, Nogués S.
Author information:
(1)Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona,
Spain. gemma.molero@ub.edu
We have developed a method based on a double labeling with stable isotopes and
gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)
analyses to study amino acid exchange in a symbiotic plant-microbe association.
Isotopic precision was studied for 21 standards including 15 amino acid
derivatives, three N-protected amino acid methyl esters, three amines and one
international standard. High correlations were observed between the δ(13)C and
δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer
(EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992,
respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for
δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship
between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were
simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which
was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules.
Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free
amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed
the pattern of C and N exchange between leaves and nodules, highlighting that
γ-aminobutanoic acid and glycine may represent an important form of C transport
from leaves to the nodules. The results confirmed the validity, reliability and
accuracy of the method for assessing C and N fluxes between plants and symbiotic
bacteria and support the use of this technique in a broad range of metabolic and
fluxomic studies.
Copyright © 2011 John Wiley & Sons, Ltd.
DOI: 10.1002/rcm.4895
1195. Syst Appl Microbiol. 2011 May;34(3):235-41. doi: 10.1016/j.syapm.2010.10.003.

Epub 2011 Feb 1.
Natural populations of lactic acid bacteria associated with silage fermentation

as determined by phenotype, 16S ribosomal RNA and recA gene analysis.
Pang H(1), Qin G, Tan Z, Li Z, Wang Y, Cai Y.
Author information:
(1)National Institute of Livestock and Grassland Science, Nasushiobara 329-2793,
Japan.
One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy
rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa
L.) silages prepared on dairy farms were screened, of which 110 isolates were
considered to be lactic acid bacteria (LAB) according to their Gram-positive and
catalase-negative characteristics and, mainly, the lactic acid metabolic
products. These isolates were divided into eight groups (A-H) based on the
following properties: morphological and biochemical characteristics,
γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were
identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc
citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides
(8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum
(4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly
present in corn silages, and L. plantarum was dominant on sorghum and forage
paddy rice silages, while L. pseudomesenteroides, L. plantarum and L.
paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and
forage paddy rice silages were well preserved with lower pH values and ammonia-N
concentrations, but had higher lactic acid content, while the alfalfa silage had
relatively poor quality with higher pH values and ammonia-N concentrations, and
lower lactic acid content. The present study confirmed the diversity of LAB
species inhabiting silages. It showed that the differing natural populations of
LAB on these silages might influence fermentation quality. These results will
enable future research on the relationship between LAB species and silage
fermentation quality, and will enhance the screening of appropriate inoculants
aimed at improving such quality.
DOI: 10.1016/j.syapm.2010.10.003
1196. J Appl Genet. 2011 May;52(2):111-24. doi: 10.1007/s13353-011-0028-2. Epub

2011
Jan 29.
Biotechnological advancements in alfalfa improvement.
Kumar S(1).
Author information:
(1)Division of Crop Improvement, Indian Grassland and Fodder Research Institute,
Jhansi, 284003, Uttar Pradesh, India. sureshkumar3_in@yahoo.co.uk
Review of biotechnology research in alfalfa shows that molecular techniques are

extensively being used for basic and applied research toward alfalfa improvement.
Biotechnological approaches have been used in two major areas, genomics and
transgenics. In genomics, molecular markers, structural and functional genomics
allowed identification of genes of interest and their regulatory components.
Alfalfa being obstinate to genetic and genomic analysis, comparative genomics is
used for molecular and genetic dissection of various plant processes in alfalfa.
Alternatively, transgenic approach involves incorporation of specific and useful
genes into alfalfa to improve the traits of interest. Input traits to improve
agronomic performance and output traits to improve forage quality, or to produce
novel industrial/pharmaceutical proteins, are the focus of current transgenic
research in alfalfa. However, transgenic approach is controversial requiring
cautious experimental design to combat bioisafety concerns. Ideally, forage
alfalfa needs to possess more fermentable carbohydrates, proteins with balanced
amino acid profile that degrade slower in rumen, improved winter hardiness,
better water use efficiency, pest resistance and no anti-quality factors.
Concerted efforts are required to bring together maximum of these characteristic
features into the alfalfa plant.
DOI: 10.1007/s13353-011-0028-2
1197. J Anim Physiol Anim Nutr (Berl). 2012 Feb;96(1):85-94. doi:

10.1111/j.1439-0396.2010.01125.x. Epub 2011 Jan 31.
Effects of an aqueous alfalfa extract on production performance, egg quality and

lipid metabolism of laying hens.
Deng W(1), Dong XF, Tong JM, Xie TH, Zhang Q.
Author information:
(1)State Key Lab of Animal Nutrition, Institute of Animal Science, Chinese
Academy of Agricultural Sciences, Beijing, China.
The present study was conducted to evaluate the effect of an aqueous alfalfa
extract (AAE) on production performance, egg quality and lipid metabolism of
laying hens between 28 and 36 weeks of age. Four groups of commercial hens
(Hy-Line Brown) were fed with diets containing 0, 0.05%, 0.1% and 0.15% AAE.
Dietary AAE had no effect on egg production parameters (p > 0.05). Shell strength
was increased (linear and quadratic effects; p < 0.05) with gradient addition of
AAE. Furthermore, shell strength was notably higher (p < 0.05) in 0.1% and 0.15%
AAE groups than the control group. With an exception of serum low and high
density lipoprotein cholesterol, serum total triglycerides and liver cholesterol
concentration (day 42), lipid indices in yolk, serum and liver were lowered
(linear and quadratic effects; p < 0.05) at day 42 and day 56 as AAE in diets
increased, as well as the enzyme activities of acetyl-CoA carboxylase (ACC) and
β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase. In relation to the control
group, cholesterol and triglycerides in yolk, liver and serum and serum very low
density lipoprotein cholesterol were lower in 0.15% AAE-treated birds at day 42
and day 56. It also significantly decreased activities of ACC, HMG-CoA reductase
and fatty acid synthetase (FAS) at day 56. The 0.1% AAE decreased triglycerides
in yolk, liver and serum, as well as the activities of ACC and FAS at day 56.
These data suggest that dietary AAE could be applied to improve egg shell quality
and modify the lipid status of laying hens and eggs.
DOI: 10.1111/j.1439-0396.2010.01125.x
1198. Plant Cell Environ. 2011 May;34(5):778-91. doi: 10.1111/j.1365-

3040.2011.02281.x.
Epub 2011 Mar 15.
Complexation of Hg with phytochelatins is important for plant Hg tolerance.
Carrasco-Gil S(1), Alvarez-Fernández A, Sobrino-Plata J, Millán R, Carpena-Ruiz

RO, Leduc DL, Andrews JC, Abadía J, Hernández LE.
Author information:
Madrid, 28049 Madrid.
Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea
mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg
speciation in root, with no symptoms of being poisoned. The largest pool (99%)
was associated with the particulate fraction, whereas the soluble fraction (SF)
accounted for a minor proportion (<1%). Liquid chromatography coupled with
electro-spray/time of flight mass spectrometry showed that Hg was bound to an
array of phytochelatins (PCs) in root SF, which was particularly varied in
alfalfa (eight ligands and five stoichiometries), a species that also accumulated
homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe
synchrotron X-ray fluorescence spectroscopy showed that most of the Hg
co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption
Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to
organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor
proportion of total Hg, Hg-PCs complexes in the SF might be important for
tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low
glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with
wild type plants. Interestingly, high-performance liquid
chromatography-electrospray ionization-time of flight analysis showed that none
of these mutants accumulated Hg-biothiol complexes.
DOI: 10.1111/j.1365-3040.2011.02281.x
1199. Proc Biol Sci. 2011 Sep 7;278(1718):2698-703. doi: 10.1098/rspb.2010.2193.

Epub
2011 Jan 26.
Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host

sanctions that reduce fitness of their reproductive clonemates.
Oono R(1), Anderson CG, Denison RF.
Author information:
(1)Department of Plant Biological Sciences, University of Minnesota, 250
Biological Sciences, 1445 Gortner Avenue, Saint Paul, MN 55108, USA.
oonox001@umn.edu
The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and

nitrogen are exchanged between the species. Nonetheless, the plant carbon that
fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by
'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit
of fixation by other rhizobia. Host sanctions can decrease the relative fitness
of less-beneficial reproductive bacteroids and prevent cheaters from breaking
down the mutualism. However, in certain legume species, only undifferentiated
rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix
nitrogen. Sanctions were, therefore, tested in two legume species that host
non-reproductive bacteroids. We demonstrate that even legume species that host
non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction
undifferentiated rhizobia when bacteroids within the same nodule fail to fix
N(2). Hence, host sanctions by a diverse set of legumes play a role in
maintaining N(2) fixation.
DOI: 10.1098/rspb.2010.2193
PMCID: PMC3136820
1200. J Dairy Sci. 2011 Feb;94(2):899-907. doi: 10.3168/jds.2010-3573.
Improved milk production efficiency in early lactation dairy cattle with dietary
addition of a developmental fibrolytic enzyme additive.
Holtshausen L(1), Chung YH, Gerardo-Cuervo H, Oba M, Beauchemin KA.
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge, AB, T1J 4B1 Canada.
A 3-part study was conducted to evaluate the effect of a developmental fibrolytic

enzyme additive on the digestibility of selected forages and the production
performance of early-lactation dairy cows. In part 1, 4 replicate 24-h batch
culture in vitro incubations were conducted with alfalfa hay, alfalfa silage, and
barley silage as substrates and ruminal fluid as the inoculum. A developmental
fibrolytic enzyme additive (AB Vista, Marlborough, UK) was added at 5 doses: 0,
0.5, 1.0, 1.5, and 2.0 μL/g of forage dry matter (DM). After the 24-h incubation,
DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) disappearance
were determined. For alfalfa hay, DM, NDF, and ADF disappearance was greater at
the highest dosage compared with no enzyme addition. Barley silage NDF and ADF
and alfalfa silage NDF disappearance tended to be greater for the highest enzyme
dosage compared with no enzyme addition. In part 2, 6 ruminally cannulated,
lactating Holstein dairy cows were used to determine in situ degradation of
alfalfa and barley silage, with (1.0 mL/kg of silage DM) and without added
enzyme. Three cows received a control diet (no enzyme added) and the other 3
received an enzyme-supplemented (1.0 mL/kg of diet DM) diet. Enzyme addition
after the 24h in situ incubation did not affect the disappearance of barley
silage or alfalfa silage. In part 3, 60 early-lactation Holstein dairy cows were
fed 1 of 3 diets for a 10-wk period: (1) control (CTL; no enzyme), (2) low enzyme
(CTL treated with 0.5 mL of enzyme/kg of diet DM), and (3) high enzyme (CTL
treated with 1.0 mL of enzyme/kg of diet DM). Adding enzyme to the diet had no
effect on milk yield, but dry matter intake was lower for the high enzyme
treatment and tended to be lower for the low enzyme treatment compared with CTL.
Consequently, milk production efficiency (kg of 3.5% fat-corrected milk/kg of DM
intake) linearly increased with increasing enzyme addition. Cows fed the low and
high enzyme diets were 5.3 (not statistically significant) and 11.3% more
efficient, respectively, compared with CTL cows. This developmental fibrolytic
enzyme additive has the potential to increase fiber digestibility of forages,
which could lead to greater milk production efficiency for dairy cows in early
lactation.
Copyright Â© 2011 American Dairy Science Association. Published by Elsevier Inc.

DOI: 10.3168/jds.2010-3573
1201. Genetika. 2010 Nov;46(11):1493-9.
Efficiency of phenotypic and DNA markers for a genetic diversity study of

alfalfa.
Tucak M(1), Popović S, Cupić T, Grljusić S, Meglic V, Jurković Z.
Author information:
(1)Agricultural Institute Osijek, 31000 Osijek, Croatia.
marijana.tucak@poljinos.hr
Information on genetic diversity and germplasm characterization is essential for

successful crop improvement. Diverse data sets (pedigree, morphological,
biochemical, DNA based-markers) are employed in various aspects of plant
analysis. The objective of this study was to determine the efficiency of
phenotypic and RAPD markers in diversity assessment often alfalfa (Medicago spp.)
accessions from Europe, North America and Australia. Field experiment was
designed as a randomised complete block with three replications over two
consecutive years (2004, 2005) at one location. Twelve morpho-agronomic traits
were recorded on 50 plants per each accession. Genomic DNA's from 16-20 randomly
selected individual plants per accession were used for RAPD analysis. Six primers
selected in this study generated a total of 93 polymorphic RAPD bands. The number
of polymorphic bands detected per primer ranged from 11 to 20. Genetic distances
(GD) among investigated accessions and two-dimensional principal coordinate
analysis (2D PCoA) based on phenotypic and molecular data were obtained. The
average GD between (0.283-0.416) and within (0.247-0.332) accessions based on
RAPD data was higher than GD values obtained by morpho-agronomic traits
(0.171-0.354 and 0.157-0.261, respectively). 2D PCoA based on GD from RAPD data
grouped most of the studied individual plants to four clusters according to their
geographical or taxonomy origin. 2D PCoA based only on morpho-agronomic data did
not group plants congruently to their origin, probably due to a strong
environmental influence on studied traits. Our results indicated that the RAPD
markers were effective in assessing genetic diversity within and between studied
alfalfa accessions. In addition, the obtained results suggested that the RAPD
markers might be useful for grouping of germplasm with similar genetic background
and for prescreening of potential heterotic groups in our breeding programme.
1202. Ecotoxicology. 2011 Mar;20(2):438-46. doi: 10.1007/s10646-011-0595-3. Epub

2011
Jan 21.
Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic

condition: characteristics, degradation products, enzyme analysis and
phytotoxicity.
Du LN(1), Wang S, Li G, Wang B, Jia XM, Zhao YH, Chen YL.
Author information:
(1)College of Life Science, Zhejiang University, Hangzhou, Zhejiang, People's
Republic of China.
Malachite green (MG), a widely-used and recalcitrant dye, has been confirmed to
be carcinogenic and mutagenic against many organisms. The main objective of this
study is to investigate the capability of Pseudomonas sp. strain DY1 to
decolorize MG, and to explore the possible mechanism. The results showed that
this strain demonstrated high decolorizing capability (90.3-97.2%) at high
concentrations of MG (100-1,000 mg/l) under shaking condition within 24 h. In
static conditions, lower but still effective decolorization (78.9-84.3%) was
achieved. The optimal pH and temperature for the decolorization was pH 6.6 and
28-30°C, respectively. Mg(2+) and Mn(2+) (1 mM) were observed to significantly
enhance the decolorization. The intermediates of the MG degradation under aerobic
condition identified by UV-visible, GC-MS and LC-MS analysis included malachite
green carbinol, (dimethyl amino-phenyl)-phenyl-methanone, N,N-dimethylaniline,
(methyl amino-phenyl)-phenyl-methanone, (amino phenyl)-phenyl methanone and
di-benzyl methane. The enzyme analysis indicated that Mn-peroxidase, NADH-DCIP
and MG reductase were involved in the biodegradation of MG. Moreover,
phytotoxicity of MG and detoxification for MG by the strain were observed.
Therefore, this strain could be potentially used for bioremediation of MG.
DOI: 10.1007/s10646-011-0595-3
1203. New Phytol. 2011 May;190(3):627-39. doi: 10.1111/j.1469-8137.2010.03621.x.

Epub
2011 Jan 20.
Selective lignin downregulation leads to constitutive defense response expression

in alfalfa (Medicago sativa L.).
Gallego-Giraldo L(1), Jikumaru Y, Kamiya Y, Tang Y, Dixon RA.
Author information:
USA.
• Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase

(HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage
quality and saccharification efficiency for bioethanol production. However, the
plants have reduced stature. It was previously reported that HCT-down-regulated
Arabidopsis have impaired auxin transport, but this has recently been disproved.
• To address the basis for the phenotypes of lignin-modified alfalfa, we measured
auxin transport, profiled a range of metabolites including flavonoids and
hormones, and performed in depth transcriptome analyses. • Auxin transport is
unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The
plants show increased cytokinin and reduced auxin levels, and gibberellin levels
and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic
acids are elevated, associated with massive upregulation of pathogenesis and
abiotic stress-related genes and enhanced tolerance to fungal infection and
drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive
activation of defense responses, triggered by release of bioactive cell wall
fragments and production of hydrogen peroxide as a result of impaired secondary
cell wall integrity.
DOI: 10.1111/j.1469-8137.2010.03621.x
1204. Plant Cell Rep. 2011 Jun;30(6):997-1006. doi: 10.1007/s00299-011-1003-4. Epub

2011 Jan 20.
Characterization of the mannan synthase promoter from guar (Cyamopsis

tetragonoloba).
Naoumkina M(1), Dixon RA.
Author information:
(1)Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble
Guar seed gum, consisting primarily of a high molecular weight galactomannan, is

the most cost effective natural thickener, having broad applications in the food,
cosmetics, paper, pharmaceutical and petroleum industries. The properties of the
polymer can potentially be enhanced by genetic modification. Development of
suitable endosperm-specific promoters for use in guar is desirable for metabolic
engineering of the seed gum. A ~1.6 kb guar mannan synthase (MS) promoter region
has been isolated. The MS promoter sequence was fused with the GUS reporter gene
and overexpressed in the heterologous species alfalfa (Medicago sativa). The
potential strength and specificity of the MS promoter was compared with those of
the constitutive 35S promoter and the seed specific β-phaseolin promoter.
Quantitative GUS assays revealed that the MS promoter directs GUS expression
specifically in endosperm in transgenic alfalfa. Thus, the guar MS promoter could
prove generally useful for directing endosperm-specific expression of transgenes
in legume species.
DOI: 10.1007/s00299-011-1003-4
1205. Insect Biochem Mol Biol. 2011 Apr;41(4):254-63. doi:

10.1016/j.ibmb.2011.01.002.
Epub 2011 Jan 11.
Identification and tissue distribution of odorant binding protein genes in the

lucerne plant bug Adelphocoris lineolatus (Goeze).
Gu SH(1), Wang SP, Zhang XY, Wu KM, Guo YY, Zhou JJ, Zhang YJ.
Author information:
of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193,
China.
Insect odorant binding proteins (OBPs) are required for insect olfaction
perception and play a key role in transporting hydrophobic semiochemicals across
the sensillum lymph to the olfactory receptors (Ors). We constructed two
high-quality cDNA libraries from the male and female antennae of the lucerne
plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae), a hemipteran
species in a large and economically important phylogenetic group of phytophagous
insects. A total of 1538 male ESTs and 1576 female ESTs were sequenced and
analyzed, which produced 2915 high-quality ESTs for further analysis. The 2915
ESTs were assembled to 1423 unigenes. Of the 1423 unigenes, 895 (63%) showed no
significant similarity with any known GenBank entry. The most prevalent
transcripts in the cDNA libraries are OBPs, chemosensory proteins (CSPs), protein
takeout precursors, antennae-specific proteins. We identified 14 genes encoding
12 "classical OBPs" with only six conserved cysteines and 2 "Plus-C OBPs" with
two additional conserved cysteines and a conserved proline immediately after the
sixth cysteine. AlinOBP4 has a very high amino acid identity of 89% to LAP, a
well studied OBP of the tarnished plant bug Lygus lineolaris. The expression
profiles of the 14 OBPs in different tissues (antennae, heads, thoraxes,
abdomens, legs and wings) were measured by real-time qPCR. The results revealed
some OBP genes are highly and differentially expressed in male and female
antennae, and four OBP genes have an extremely high transcript level in the legs.
Of two Plus-C OBP genes, one (AlinOBP14) is highly expressed only in the heads
and another (AlinOBP7) is expressed in all olfactory tissues with much lower
transcript levels comparing to other OBPs. The possible physiological functions
of these OBPs are discussed.
DOI: 10.1016/j.ibmb.2011.01.002
[Interactive effect of spent mushroom compost and rhamnolipids to enhance the

efficiency of alfalfa remediation of aged PAHs contaminated soil].
Zhang J(1), Lin XG, Li XZ, Yin R.
Author information:
Polycyclic aromatic hydrocarbons (PAHs) are a group of the most widespread

organic pollutants, which distributed widely in soil and sediment. Pot experiment
was conducted to improve efficiency of phytoremediation using alfalfa (Medicago
sativa L.) in aged PAHs contaminated soil by introducing spent mushroom compost
and rhamnolipids. Plant biomass, PAHs concentrations, number of soil
microorganism, soil enzyme activity and soil microbial functional diversity were
determined after 60 days of alfalfa growth. The results showed that within 60
days, removal ratio of PAHs in treatment of alfalfa alone (AL) reached to 14.43%,
while removal ratio of PAHs in treatments of "GZ + RH0.5, + AL" and "GZ + RH1.0 +
AL" reached to 32.64% and 36.95%, which were 115.45% and 156.06% higher than that
of phytoremediation. Contrasted to the control, the treatment of "GZ + RH1.0 +
AL" had more plant biomass than others, shoot and root dry weight were 1.05 g/pot
and 0.20 g/pot, respectively. During the process of phytoremediation, the number
of soil bacteria and fungi were greatly increased by "GZ + RH1.0 + AL" and
reached to 31.37 x 10(6) CFU x g(-1) and 5.86 x 10(6) CFU x g(-1), especially the
number of PAHs-degrading bacteria reached to 39.57 x 10(5) MPN x g(-1), which
were 29 times more than control treatment and 4 times more than treatment of
alfalfa alone (AL). Moreover, soil dehydrogenase activity and the functional
diversity of soil microbial community were increased significantly by the
treatment of "GZ + RH1.0 + AL", respectively. Therefore, interaction of spent
mushroom compost and rhamnolipids to enhance the phytoremediation efficiency had
satisfied results in removal aged PAHs from an agricultural soil, the feasibility
of this method needed to be further proved by large-area scale field experiment.
1207. J Food Prot. 2010 Nov;73(11):2010-7.
Inactivation of Escherichia coli O157:H7 and Salmonella typhimurium DT 104 on

alfalfa seeds by levulinic acid and sodium dodecyl sulfate.
Zhao T(1), Zhao P, Doyle MP.
Author information:
(1)Center for Food Safety, University of Georgia, 1109 Experiment Street,
Griffin, Georgia 30223, USA.
Studies were conducted to determine the best concentration and exposure time for
treatment of alfalfa seeds with levulinic acid plus sodium dodecyl sulfate (SDS)
to inactivate Escherichia coli O157:H7 and Salmonella without adversely affecting
seed germination. Alfalfa seeds inoculated with a five-strain mixture of E. coli
O157:H7 or Salmonella Typhimurium were dried in a laminar flow hood at 21°C for
up to 72 h. Inoculated alfalfa seeds dried for 4 h then treated for 5 min at 21°C
with 0.5% levulinic acid and 0.05% SDS reduced the population of E. coli O157:H7
and Salmonella Typhimurium by 5.6 and 6.4 log CFU/g, respectively. On seeds dried
for 72 h, treatment with 0.5% levulinic acid and 0.05% SDS for 20 min at 21°C
reduced E. coli O157:H7 and Salmonella Typhimurium populations by 4 log CFU/g.
Germination rates of alfalfa seeds treated with 0.5% levulinic acid plus 0.05%
SDS for up to 1 h at 21°C were compared with a treatment of 20,000 ppm of calcium
hypochlorite or tap water only. Treatment of alfalfa seeds with 0.5% levulinic
acid plus 0.05% SDS for 5 min at 21°C resulted in a >3.0-log inactivation of E.
coli O157:H7 and Salmonella.
1208. Anim Cogn. 2011 May;14(3):369-76. doi: 10.1007/s10071-010-0371-4. Epub 2011

Jan
4.
Do sheep (Ovis aries) categorize plant species according to botanical family?
Ginane C(1), Dumont B.
Author information:
(1)INRA, UR1213 Herbivores, 63122, Saint-Genès-Champanelle, France.
cecile.ginane@clermont.inra.fr
The ability of grazing herbivores to assign food types to categories by relying

on certain relevant criteria could considerably reduce cognitive demand and
increase their foraging efficiency when selecting among many different plant
items. Grasses and legumes differ functionally in vegetation communities as well
as in nutritive value. We aimed to determine whether sheep can generalize an
aversion they learnt for a grass or a legume species to another species of the
same functional type and consequently whether botanical family is a potential
level of categorization. Over four successive weeks, 12 lambs were conditioned
against either a freshly cut grass (tall fescue-Festuca arundinacea, N = 6) or
legume species (sainfoin-Onobrychis viciifolia, N = 6) using a negative
post-ingestive stimulus (lithium chloride) on day 1. Preference of all lambs
between another grass (cocksfoot-Dactylis glomerata) and another legume
(alfalfa-Medicago sativa) was assessed on day 3 by measuring their relative
consumptions. Preference for alfalfa progressively became lower for lambs that
were conditioned against sainfoin than against tall fescue, indicating that lambs
generalized the aversion between species along some perceptual gradient and
classed the considered grasses and legumes in distinct categories. Beyond this
original result, the question now is to identify which specific plant
characteristics or functional traits the animals rely on in order to form
categories.
DOI: 10.1007/s10071-010-0371-4
1209. J Exp Bot. 2011 Mar;62(6):2155-68. doi: 10.1093/jxb/erq413. Epub 2010 Dec 31.
Immunodetection of retinoblastoma-related protein and its phosphorylated form in

interphase and mitotic alfalfa cells.
Abrahám E(1), Miskolczi P, Ayaydin F, Yu P, Kotogány E, Bakó L, Otvös K, Horváth

GV, Dudits D.
Author information:
Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary.
Plant retinoblastoma-related (RBR) proteins are primarily considered as key

regulators of G(1)/S phase transition, with functional roles in a variety of
cellular events during plant growth and organ development. Polyclonal antibody
against the C-terminal region of the Arabidopsis RBR1 protein also specifically
recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition
assay. The MsRBR protein was detected in all cell cycle phases, with a moderate
increase in samples representing G(2)/M cells. Antibody against the human
phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR
protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment.
Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into
the S phase and remained high during the G(2)/M phases. Roscovitine treatment
abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR
protein level was significantly decreased in the treated cells. Colchicine block
increased the detected levels of both forms of MsRBR protein. Reduced levels of
the MsRBR protein in cells at stationary phase or grown in hormone-free medium
can be a sign of the division-dependent presence of plant RBR proteins.
Immunolocalization of the phospho-MsRBR protein indicated spots of variable
number and size in the labelled interphase nuclei and high signal intensity of
nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot
be recognized in later mitotic phases. Based on the presented western blot and
immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase
regulation in plant cells is discussed.
DOI: 10.1093/jxb/erq413
PMCID: PMC3060694
1210. FEBS Lett. 2011 Jan 21;585(2):300-6. doi: 10.1016/j.febslet.2010.12.039. Epub

2010 Dec 28.
Ultra-high resolution crystal structure of a dimeric defensin SPE10.
Song X(1), Zhang M, Zhou Z, Gong W.
Author information:
(1)National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese
Academy of Sciences, Beijing, China.
Defensins are key players of the innate immune system known to act against
bacteria, fungi and viruses. Here we report the 0.98-Å crystal structure of
SPE10, a dimeric plant defensin. SPE10 associates as a dimer through a unique
amino acid triplet involving residues R36-W42-R40. The helix from one subunit
interacts with arginines R36 and R40 from the other subunit, forming a sheet-like
dimer with a highly extended molecular surface. A conserved hydrophobic patch on
the molecular head largely overlaps with the putative receptor-binding site
previously reported for another defensin. Structural analysis and mutational
studies indicate that the dimeric association of SPE10 is relevant to its
function, and that the hydrophobic patch on the molecular head is required for
its antifungal activity.
Copyright © 2010 Federation of European Biochemical Societies. Published by

Elsevier B.V. All rights reserved.
DOI: 10.1016/j.febslet.2010.12.039
1211. Onderstepoort J Vet Res. 2011 Jul 15;78(1):307. doi: 10.4102/ojvr.v78i1.307.
Rumen protozoa in South African sheep with a summary of the worldwide

distribution of sheep protozoa.
Booyse D(1), Dehority BA.
Author information:
(1)Department of Anatomy and Physiology, University of Pretoria.
dbooyse@op.up.ac.za.
Protozoa species were identified in rumen contents of four domestic sheep (Ovis
aries) from South Africa. All animals were fed a forage diet which consisted of
50% lucerne and 50% teff hay. Ten new host records were identified, bringing the
total number of species and forms observed in sheep in South Africa to 30. The
occurrence and geographic distribution of ciliate protozoa in both domestic and
wild sheep from around the world are summarised. It was found that 15 genera and
131 species occur in domestic sheep globally.
Interactions of monensin with dietary fat and carbohydrate components on ruminal

fermentation and production responses by dairy cows.
Mathew B(1), Eastridge ML, Oelker ER, Firkins JL, Karnati SK.
Author information:
(1)Department of Animal Sciences, The Ohio State University, Columbus, OH 43210,
USA.
Variation in milk fat percentage resulting from monensin supplementation to

lactating dairy cows could be due to altered ruminal fermentation with
interactions of monensin with ruminal biohydrogenation of fat and ruminal
carbohydrate availability. The objective of the study was to determine the
effects of feeding monensin as Rumensin (R) in diets differing in starch
availability (ground or steam-flaked corn), effective fiber (long or short
alfalfa hay, LAH or SAH), and 4% fat (F) from distillers grains, roasted
soybeans, and an animal-vegetable blend on ruminal fermentation characteristics
and milk production in lactating dairy cows. Six ruminally cannulated lactating
Holstein cows were used in a balanced 6×6 Latin square design with 21-d periods.
The cows were fed 6 diets: (1) C=control diet with ground corn and LAH, (2) CR=C
plus R, (3) CRFL=CR plus F, (4) CRFS=ground corn, R, F, and SAH, (5)
SRFL=steam-flaked corn, R, F, and LAH, and (6) SRFS=steam-flaked corn, R, F, and
SAH. Mean particle size of LAH was 5.00 mm and 1.36 mm for SAH. All diets were
formulated to have 21% forage NDF and 40% NFC. The R tended to decrease DMI,
decreased milk fat yield, and numerically lowered milk fat percentage (3.41 vs.
2.98%). Addition of F to R diets did not affect milk fat percentage. By feeding
diets containing R and F, SAH tended to increase milk fat percentage for the
ground-corn diet, but SAH tended to decrease milk fat percentage with
steam-flaked corn (CRFL+SRFS vs. CRFS+SRFL). The steam-flaked corn increased
total-tract NDF digestibility (CRFL + CRFS vs. SRFL+SRFS; 51.1 vs. 56%). Addition
of F with R decreased total VFA concentration and increased rumen pH. Fat
addition with R decreased rumen NH3N and MUN (12.8 vs. 13.9 mg/dL), and SFC
decreased NH3N concentration compared with ground corn. Although R caused milk
fat depression, addition of F did not further exacerbate milk fat depression.
Fatty acid analysis did not implicate any particular biohydrogenation
intermediate as the causative factor for the milk fat depression.

DOI: 10.3168/jds.2010-3580
Population structure of rumen Escherichia coli associated with subacute ruminal

acidosis (SARA) in dairy cattle.
Khafipour E(1), Plaizier JC, Aikman PC, Krause DO.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2
Canada.
Previous studies indicated that only subacute ruminal acidosis (SARA), induced by
feeding a high-grain diet, is associated with an inflammatory response and
increased abundance of Escherichia coli in the rumen. We hypothesized that
ruminal E. coli in grain pellet-induced SARA carried virulence factors that
potentially contribute to the immune activation during SARA. One hundred
twenty-nine E. coli isolates were cultured from the rumens of 8 cows (4 animals
per treatment) in which SARA had been nutritionally induced by feeding a
high-grain diet (GPI-SARA) or a diet containing alfalfa pellets (API-SARA). The
population structure of the E. coli was evaluated with the ABD genotyping system
and repetitive sequence-based (rep)-PCR fingerprinting. Twenty-five virulence
factors were evaluated with PCR. Escherichia coli numbers were higher in the
GPI-SARA treatment than in the API-SARA treatment. The genetic structure of the
E. coli was significantly different between SARA challenge models. Isolates from
GPI-control (46%), API-control (70%), and API-SARA (53%) were closely related and
fell into one cluster, whereas isolates from GPI-SARA (54%) grouped separately.
The ABD typing indicated a shift from an A-type E. coli population to a B1-type
population only due to GPI-SARA. Of the 25 virulence factors tested, curli fiber
genes were highly associated with GPI. Curli fibers were first identified in E.
coli mastitis isolates and are potent virulence factors that induce a range of
immune responses. Results suggest that under low rumen pH conditions induced by a
grain diet, there is a burst in the number of E. coli with virulence genes that
can take advantage of these rumen conditions to trigger an inflammatory response.

DOI: 10.3168/jds.2010-3435
1214. J Hazard Mater. 2011 Feb 28;186(2-3):1271-6. doi:

10.1016/j.jhazmat.2010.11.126.
Epub 2010 Dec 4.
Influence of Rhizobium meliloti on phytoremediation of polycyclic aromatic

hydrocarbons by alfalfa in an aged contaminated soil.
Teng Y(1), Shen Y, Luo Y, Sun X, Sun M, Fu D, Li Z, Christie P.
Author information:
Microbe-assisted phytoremediation is emerging as one of the most effective means

by which plants and their associated rhizosphere microbes degrade organic
contaminants in soils. A pot study was conducted to examine the effects of
inoculation with Rhizobium meliloti on phytoremediation by alfalfa grown for 90
days in an agricultural soil contaminated with weathered polycyclic aromatic
hydrocarbons (PAHs). Planting with uninoculated alfalfa (P) and alfalfa
inoculated with R. meliloti (PR) significantly lowered the initial soil PAH
concentrations by 37.2 and 51.4% respectively compared with unplanted control
soil. Inoculation with R. meliloti significantly increased the counts of
culturable PAH-degrading bacteria, soil microbial activity and the carbon
utilization ability of the soil microbial community. The results suggest that the
symbiotic association between alfalfa and Rhizobium can stimulate the rhizosphere
microflora to degrade PAHs and its application may be a promising bioremediation
strategy for aged PAH-contaminated soils.
DOI: 10.1016/j.jhazmat.2010.11.126
1215. Ann Bot. 2011 Mar;107(3):407-13. doi: 10.1093/aob/mcq249. Epub 2010 Dec 17.
Arbuscular mycorrhizal fungi alter plant allometry and biomass-density

relationships.
Zhang Q(1), Zhang L, Weiner J, Tang J, Chen X.
Author information:
(1)College of Life Sciences, Zhejiang University, Hangzhou, China.
BACKGROUND AND AIMS: Plant biomass-density relationships during self-thinning are

determined mainly by allometry. Both allometry and biomass-density relationship
have been shown to vary with abiotic conditions, but the effects of biotic
interactions have not been investigated. Arbuscular mycorrhizal fungi (AMF) can
promote plant growth and affect plant form. Here experiments were carried out to
test whether AMF affect plant allometry and the self-thinning trajectory.
METHODS: Two experiments were conducted on Medicago sativa L., a leguminous
species known to be highly dependent on mycorrhiza. Two mycorrhizal levels were
obtained by applying benomyl (low AMF) or not (high AMF). Experiment 1
investigated the effects of AMF on plant growth in the absence of competition.
Experiment 2 was a factorial design with two mycorrhizal levels and two plant
densities (6000 and 17 500 seeds m(-2)). Shoot biomass, root biomass and canopy
radius were measured 30, 60, 90 and 120 d after sowing. The allometric
relationships among these aspects of size were estimated by standardized major
axis regression on log-transformed data.
KEY RESULTS: Shoot biomass in the absence of competition was lower under low AMF
treatment. In self-thinning populations, the slope of the log (mean shoot
biomass) vs. log density relationship was significantly steeper for the high AMF
treatment (slope = -1·480) than for the low AMF treatment (-1·133). The canopy
radius-biomass allometric exponents were not significantly affected by AMF level,
but the root-shoot allometric exponent was higher in the low AMF treatment. With
a high level of AMF, the biomass-density exponent can be predicted from the
above-ground allometric model of self-thinning, while this was not the case when
AMF were reduced by fungicide.
CONCLUSIONS: AMF affected the importance of below-ground relative to above-ground
interactions and changed root vs. shoot allocation. This changed allometric
allocation of biomass and altered the self-thinning trajectory.
DOI: 10.1093/aob/mcq249
PMCID: PMC3043928
1216. Int J Phytoremediation. 2010 Jul;12(5):516-33. doi:

10.1080/15226510903353120.
Influence of arbuscular mycorrhiza and Rhizobium on phytoremediation by alfalfa
of an agricultural soil contaminated with weathered PCBs: a field study.
Teng Y(1), Luo Y, Sun X, Tu C, Xu L, Liu W, Li Z, Christie P.
Author information:
Soil Science, Chinese Academy of Sciences, Nanjing, China.
A field experiment was conducted to study the effects of inoculation with the
arbuscular mycorrhizal fungus Glomus caledonium and/or Rhizobium meliloti on
phytoremediation of an agricultural soil contaminated with weathered PCBs by
alfalfa grown for 180 days. Planting alfalfa (P), alfalfa inoculated with G.
caledonium (P + AM), alfalfa inoculated with R. meliloti (P + R), and alfalfa
co-inoculated with R. meliloti and G. caledonium (P+AM+R) decreased significantly
initial soil PCB concentrations by 8.1, 12.0, 33.8, and 43.5%, respectively.
Inoculation with R. meliloti and/or G. caledonium (P+AM+R) increased the yield of
alfalfa, and the accumulation of PCBs in the shoots. Soil microbial counts and
the carbon utilization ability of the soil microbial community increased when
alfalfa was inoculated with R. meliloti and/or G. caledonium. Results of this
field study suggest that synergistic interactions between AMF and Rhizobium may
have great potential to enhance phytoremediation by alfalfa of an agricultural
soil contaminated with weathered PCBs.
DOI: 10.1080/15226510903353120
1217. J Agric Food Chem. 2011 Jan 12;59(1):131-7. doi: 10.1021/jf102997p. Epub 2010
Dec
15.
Phytoestrogenic compounds in alfalfa sprout (Medicago sativa) beyond coumestrol.
Hong YH(1), Wang SC, Hsu C, Lin BF, Kuo YH, Huang CJ.
Author information:
(1)Department of Biochemical Science and Technology, National Taiwan University,
Taipei, Taiwan.
Coumestrol has long been known as the phytoestrogenic compound in alfalfa.

However, it has been demonstrated that the ethyl acetate extract of alfalfa
sprout (AEA) attenuated the disease severity and increased survival and life span
of autoimmune-prone MRL-lpr/lpr mice. Coumestrol, on the contrary, decreased the
survival. This study thus aimed to isolate and identify phytoestrogenic compounds
other than coumestrol in AEA. AEA was fractionated and separated by successive
silica gel chromatography and preparative HPLC. The activity of collected
fractions was tracked by a transactivation assay for ERα and ERβ, respectively.
In addition to coumestrol, liquiritigenin, isoliquiritigenin, loliolide, and
(4S,6S)- and (4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one were isolated and
chemically identified. Except for loliolide, these compounds showed higher
transactivation via ERβ than via ERα. The maximal activation via ERα of
coumestrol reached 80% that of 1 nM 17β-estradiol (E(2)), whereas the activations
of the remaining five compounds as well as AEA ranged from 8 to 49%. In addition,
isoliquiritigenin, loliolide, and (4S,6S)- and
(4R,6S)-4-hydroxy-6-pentadecyltetrahydropyr-2-one, but not coumestrol,
preferentially inhibited 1 nM E(2) induced ERα activation, compared to that ERβ
activation. The selectivity of these phytoestrogens might account for the
difference between the effects of AEA and coumestrol in autoimmune-prone
MRL-lpr/lpr mice observed previously.
DOI: 10.1021/jf102997p
1218. Biol Lett. 2011 Jun 23;7(3):472-4. doi: 10.1098/rsbl.2010.1040. Epub 2010 Dec
8.
Plant mortality varies with arbuscular mycorrhizal fungal species identities in a

self-thinning population.
Zhang Q(1), Tang J, Chen X.
Author information:
Republic of China.
Because arbuscular mycorrhizal fungal (AMF) species differ in stimulating the

growth of particular host plant species, AMF species may vary in their effects on
plant intra-specific competition and the self-thinning process. We tested this
hypothesis using a microcosm experiment with Medicago sativa L. as a model plant
population and four AMF species. Our results showed that the AMF species Glomus
diaphanum stimulated host plant growth more than the other three AMF species did
when the plants were grown individually. Glomus diaphanum also induced the
highest rate of mortality in the self-thinning plant populations. We also found a
positive correlation between mortality and growth response to colonization. Our
results demonstrate that AMF species can affect plant mortality and the
self-thinning process by affecting plant growth differently.
DOI: 10.1098/rsbl.2010.1040
PMCID: PMC3097865
1219. Plant Biol (Stuttg). 2011 Jan;13(1):69-77. doi: 10.1111/j.1438-

8677.2009.00311.x.
Molecular characterisation of Ltchi7, a gene encoding a Class III endochitinase

induced by drought stress in Lotus spp.
Tapia G(1), Morales-Quintana L, Inostroza L, Acuña H.
Author information:
(1)Unidad de Recursos Genéticos, Instituto de Investigaciones Agropecuarias,
INIA-Quilamapu, Chillán, Chile. gtapia@inia.cl
Chitinases are enzymes that digest chitin molecules, present principally in

insects and fungi. In plants, these enzymes play an important role in defence
against pathogen attack, although they have also been described as induced by
mechanical damage, ozone, heavy metals, cold, salinity, etc. Using an annealing
control primer, we isolated a gene fragment whose translated sequence has high
homology with a class III endochitinase. The gene, named Ltchi7, consisted of one
ORF of 1005 bp, which codes for a peptide of 334 amino acids, including a deduced
signal peptide of 27 amino acid that directs protein to the extracellular space.
Phylogenetic analysis suggests that Ltchi7 is within a cluster that includes
Sesbania rostrata, Medicago sativa and Glycine max class III endochitinases. This
group is differentiated from other species of endochitinases by the presence of
an additional extension in carboxy-terminal region. Moreover, in comparison with
the majority of chitinases, Ltchi7 has two additional cysteine residues, which,
according to 3D modelling studies, are very close. Gene expression analysis
showed enhanced transcript abundance of this gene during drought stress in Lotus
tenuis and Lotus japonicus, compared with growth under normal conditions.
Furthermore, its expression is restricted to nodules and roots. Expression of
this gene was also induced by salt stress, hydrogen peroxide and weakly with
abscisic acid.
© 2010 German Botanical Society and The Royal Botanical Society of the
Netherlands.
DOI: 10.1111/j.1438-8677.2009.00311.x
1220. Guang Pu Xue Yu Guang Pu Fen Xi. 2010 Oct;30(10):2789-92.
[The influences of different vegetation ecosystems on heavy metals in soil in

semi-arid region].
Zhou MM(1), Pan ZH, Chen DD, Zhang LY, An PL, Pan XB, Zhao PY.
Author information:
(1)College of Resources and Environmental Sciences, China Agricultural
University, Beijing 100193, China.
The ecological environment of semi-arid regions in China is fragile, and the

situation of protecting environment is grim. So it is urgent to strengthen
environment protection and ecological construction in semi-arid region. Four
different vegetation ecosystems were selected in semi-arid region with Wuchuan
County in Inner Mongolia as a case study: the bushes, the trees, the alfalfa
land, and artificially mixed sowing grassland. The soil was sampled and carried
to laboratory for analysis of the content of lead, cadmium and chromium in the
soil in May and September (the start of the growing season and the end of the
growing season). It was showed that among the four different ecosystems, the lead
and cadmium contents in the soil were significantly different in variability,
while the chromium is not significant. And the changing rate trend of the content
of lead and cadmium is consistent: the contents of the both elements in May are
higher than in September obviously. The degree of the order of the content change
is also very close. The biggest of the change scope is from the bushes and
artificially mixed sowing grassland, followed by the alfalfa land, and finally
the trees. Therefore, it is more favorable to plant shrubs and grass for
absorbing heavy metals in the soil in the common local vegetation eco-system.
1221. Physiol Plant. 2011 Mar;141(3):227-38. doi: 10.1111/j.1399-3054.2010.01436.x.

Epub 2010 Dec 30.
Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba)
suspension cultures exposed to alfalfa saponins.
Balestrazzi A(1), Agoni V, Tava A, Avato P, Biazzi E, Raimondi E, Macovei A,

Carbonera D.
Author information:
(1)Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Ferrata 1,
Pavia, Italy.
The present work reports on the biological activity of alfalfa (Medicago sativa)
saponins on white poplar (Populus alba, cultivar 'Villafranca') cell suspension
cultures. The extracts from alfalfa roots, aerial parts and seeds were
characterized for their saponin content by means of thin layer chromatography
(TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative
saponin composition from the different plant extracts was determined considering
the aglycone moieties and determined by gas chromatography (GC) and gas
chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was
detected in the seed extract while several other saponins were found in the root
and leaf extracts. Actively proliferating white poplar cell cultures were
challenged with the different saponin extracts. Only alfalfa root saponins, at 50
µg ml⁻¹, induced significant cell death rates (75.00 ± 4.90%). Different cell
subpopulations with peculiar cell death morphologies were observed and the
programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin
concentrations. Enhancement of nitric oxide (NO) production was observed in white
poplar cells treated with root saponins (RSs) at 50 µg ml⁻¹ and release of
reactive oxygen species (ROS) in the culture medium was also demonstrated.
Saponin-induced NO production was sensitive to sodium azide and
N(G)-monomethyl-L-arginine, two specific inhibitors of distinct pathways for NO
biosynthesis in plant cells.
Copyright © Physiologia Plantarum 2010.
DOI: 10.1111/j.1399-3054.2010.01436.x
1222. Arch Environ Contam Toxicol. 2011 Feb;60(2):220-32. doi:

10.1007/s00244-010-9624-0. Epub 2010 Nov 24.
Effects of ten antibiotics on seed germination and root elongation in three plant
species.
Hillis DG(1), Fletcher J, Solomon KR, Sibley PK.
Author information:
(1)University of Guelph, ON, Canada.
We applied a screening-level phytotoxicity assay to evaluate the effects of 10

antibiotics (at concentrations ranging from 1 to 10,000 μg/L) on germination and
early plant growth using three plant species: lettuce (Lactuca sativa), alfalfa
(Medicago sativa), and carrot (Daucus carota). The range of phytotoxicity of the
antibiotics was large, with EC₂₅s ranging from 3.9 μg/L to >10,000 μg/L.
Chlortetracycline, levofloxacin, and sulfamethoxazole were the most phytotoxic
antibiotics. D. carota was the most sensitive plant species, often by an order of
magnitude or more, followed by L. sativa and then M. sativa. Plant germination
was insensitive to the antibiotics, with no significant decreases up to the
highest treatment concentration of 10,000 μg/L. Compared with shoot and total
length measurements, root elongation was consistently the most sensitive end
point. Overall, there were few instances where measured soil concentrations, if
available in the publicly accessible literature, would be expected to exceed the
effect concentrations of the antibiotics evaluated in this study. The use of
screening assays as part of a tiered approach for evaluating environmental
impacts of antibiotics can provide insight into relative species sensitivity and
serve as a basis by which to screen the potential for toxic effects of novel
compounds to plants.
DOI: 10.1007/s00244-010-9624-0
1223. J Chromatogr A. 2011 Sep 9;1218(36):6191-9. doi:

10.1016/j.chroma.2010.10.092.
Epub 2010 Oct 30.
Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go

counter-current chromatography and following on-line liquid chromatography
desalination.
Liang J(1), Yang Z, Cao X, Wu B, Wu S.
Author information:
(1)Research Center of Siyuan Natural Pharmacy and Biotoxicology, College of Life
Sciences, Zhejiang University, Hangzhou, Zhejiang Province 310058, China.
In this work, we have established a new stop-and-go two-dimensional

chromatography coupling of counter-current chromatography and liquid
chromatography (2D CCC × LC) for the preparative separation of two novel
antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC
column has been used as the first dimension to purify the target flavonoids using
a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1,
v/v) with the stop-and-go flow technique, and the LC column packed with
macroporous resin has been employed as the second dimension for on-line
absorption, desalination and desorption of the targeting effluents purified from
the first CCC dimension. As a result, two novel flavonoids,
6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and
6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated
from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their
structures have been identified by electrospray ionization mass spectrometry
(ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS)
and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR).
Further antioxidant assays showed that the first component possess a strong
antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC ×
LC method is very efficient for the separation of flavonoids of alfalfa and it
can also be applied to isolate other comprehensive multi-component natural
products.
DOI: 10.1016/j.chroma.2010.10.092

Epub
2010 Nov 16.
RhizoRegNet--a database of rhizobial transcription factors and regulatory

networks.
Krol E(1), Blom J, Winnebald J, Berhörster A, Barnett MJ, Goesmann A, Baumbach J,

Becker A.
Author information:
(1)Faculty of Biology, Albert-Ludwigs-University Freiburg, Schänzlestr. 1, 79104
Freiburg, Germany.
Sinorhizobium meliloti is a symbiotic soil bacterium that forms nitrogen-fixing

nodules on roots of leguminous plants, including Medicago truncatula (barrel
medic), and M. sativa (alfalfa). The Sinorhizobium-Medicago symbiosis is an
important symbiosis model system. Knowledge gained from this system can be
extended to other agriculturally important "rhizobial" symbioses. Since the
publication of the S. meliloti genome in 2001, many new genetic, biochemical and
physiological data have been generated. Effective methods to organize, store, and
mine this postgenome data are crucial for continued success of the S. meliloti
model system. In 2009, we introduced a portal for rhizobial genomes, RhizoGATE
(Becker et al., J. Biotechnol. 140, 45-50). The RhizoGATE portal combines
continuously updated S. meliloti genome annotation with postgenome data
resources. Here we report integration of a new component, RhizoRegNet, to
RhizoGATE. RhizoRegNet combines transcriptome data and operon predictions with
published data on regulatory interactions. By allowing searching and
visualisation of complex transcriptional regulatory networks, RhizoRegNet
advances our understanding of transcriptional regulation in S. meliloti. The
current version of RhizoRegNet is divided into 13 functional modules containing
information for 114 regulators, 475 regulated genes, and 178 transcription factor
binding motifs. In this report, we provide an example of how RhizoRegNet
facilitates visualisation and analysis of the regulatory network for
exopolysaccharide biosynthesis and motility. Presently, RhizoRegNet contains
regulatory network information for S. meliloti and the closely related bacterium,
S. medicae, but can be expanded to include other rhizobial species.
DOI: 10.1016/j.jbiotec.2010.11.004
Role of symbiotic auxotrophy in the Rhizobium-legume symbioses.
Prell J(1), Bourdès A, Kumar S, Lodwig E, Hosie A, Kinghorn S, White J, Poole P.
Author information:
(1)John Innes Centre, Norwich Research Park, Norwich, United Kingdom.
BACKGROUND: Rhizobium leguminosarum bv. viciae mutants unable to transport

branched-chain amino acids via the two main amino acid ABC transport complexes
AapJQMP and BraDEFGC produce a nitrogen starvation phenotype when inoculated on
pea (Pisum sativum) plants [1], [2]. Bacteroids in indeterminate pea nodules have
reduced abundance and a lower chromosome number. They reduce transcription of
pathways for branched-chain amino acid biosynthesis and become dependent on their
provision by the host. This has been called "symbiotic auxotrophy".
METHODOLOGY/PRINCIPAL FINDINGS: A region important in solute specificity was
identified in AapQ and changing P144D in this region reduced branched-chain amino
acid transport to a very low rate. Strains carrying P144D were still fully
effective for N(2) fixation on peas demonstrating that a low rate of branched
amino acid transport in R. leguminosarum bv. viciae supports wild-type rates of
nitrogen fixation. The importance of branched-chain amino acid transport was then
examined in other legume-Rhizobium symbioses. An aap bra mutant of R.
leguminosarum bv. phaseoli also showed nitrogen starvation symptoms when
inoculated on French bean (Phaseolus vulgaris), a plant producing determinate
nodules. The phenotype is different from that observed on pea and is accompanied
by reduced nodule numbers and nitrogen fixation per nodule. However, an aap bra
double mutant of Sinorhizobium meliloti 2011 showed no phenotype on alfalfa
(Medicago sativa).
CONCLUSIONS/SIGNIFICANCE: Symbiotic auxotrophy occurs in both determinate pea and
indeterminate bean nodules demonstrating its importance for bacteroid formation
and nodule function in legumes with different developmental programmes. However,
only small quantities of branched chain amino acids are needed and symbiotic
auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under
the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants
inoculated on different legumes probably reflects altered timing of amino acid
availability, development of symbiotic auxotrophy and nodule developmental
programmes.
PMCID: PMC2978685
1226. Appl Environ Microbiol. 2011 Jan;77(1):258-68. doi: 10.1128/AEM.01289-09.

Epub
2010 Nov 12.
Effect of environmental factors and influence of rumen and hindgut biogeography

on bacterial communities in steers.
Romero-Pérez GA(1), Ominski KH, McAllister TA, Krause DO.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada.
Feces from cattle production are considered important sources of bacterial

contamination of food and the environment. Little is known about the combined
effects of arctic temperatures and fodder tannins on rumen and hindgut bacterial
populations. Individual rumen liquor and rectal fecal samples from donor steers
fed either alfalfa silage or sainfoin (Onobrychis viciifolia Scop.) silage and
water ad libitum were collected weekly on the first three sampling days and
fortnightly afterwards. The daily ambient temperatures were registered and
averaged to weekly mean temperatures. Steers fed sainfoin silage had lower (P <
0.05) concentrations of branched-chain volatile fatty acids (VFA) than those fed
alfalfa silage. All VFA concentrations were higher (P < 0.001) in rumen liquor
samples than in fecal samples. The interaction of sample type and diet showed a
significant effect (P < 0.05) on the proportions of the bacterial community that
were from the phyla Proteobacteria and Verrucomicrobia. Ambient temperature had
an indirect effect (P < 0.05) on the phylum Firmicutes, as it affected its
proportional balance. The bacterial population diversity in samples appeared to
decrease concurrently with the ambient temperature. The phylum Firmicutes
explained the first principal component at 64.83 and 42.58% of the total variance
in rumen liquor and fecal samples, respectively. The sample type had a larger
effect on bacterial communities than diet and temperature. Certain bacterial
populations seemed to be better adapted than others to environmentally adverse
conditions, such as less access time to nutrients due to higher motility and rate
of passage of digesta caused by extreme temperatures, or antimicrobials such as
tannins, possibly due to an influence of their biogeographical location within
the gut.
DOI: 10.1128/AEM.01289-09
PMCID: PMC3019729
1227. J Plant Physiol. 2011 Apr 15;168(6):566-75. doi: 10.1016/j.jplph.2010.09.012.
Epub 2010 Nov 12.
Effect of oligogalacturonides on root length, extracellular alkalinization and

O₂⁻-accumulation in alfalfa.
Camejo D(1), Martí MC, Jiménez A, Cabrera JC, Olmos E, Sevilla F.
Author information:
(1)Department of Stress Biology and Plant Pathology, Centro de Edafología y
Biología Aplicada del Segura (CEBAS-CSIC), P.O. Box 164, E-30100 Murcia, Spain.
The effects of an oligogalacturonic acid (OGA) pool on root length of intact

alfalfa seedlings (Medicago sativa L.), on extracellular pH and on both
extracellular and intracellular O₂⁻ dynamics were examined in this study. Lower
OGA concentrations (25, 50 and 75 μg mL⁻¹)promoted root length, but 50 μg mL⁻¹
had a stronger effect in promoting growth, while the higher OGA concentration
(100 μg mL⁻¹)had no significant effect. Extracellular alkalinization was tested
only at concentrations higher than 50 μg mL⁻¹ OGA, showing that the response is
determined not only by the specific size of OGA, but also by the concentration of
OGA. The promoting effect of OGA on root growth at 25, 50 and 75 μg mL⁻¹ OGA
concentrations in alfalfa root appeared to be unrelated to extracellular
alkalinization. A possible explanation could be the induction of an O₂⁻ burst at
non-toxic levels, which could drive directly or indirectly several processes
associated with root elongation in 25, 50 and 75 μg mL⁻¹ OGA-treated seedlings.
Analyses using confocal microscopy showed that the increase in the O₂⁻
generation, mainly in the epidermal cells, induced by 50 μg mL⁻¹ OGA could be
related to the promoting effect on root growth. The combination of OGA with DPI
allowed us to demonstrate that there are different O₂⁻-generating sources in the
epidermal cells of the meristematic zone, likely NADPH oxidase and oxidases or
oxido-reductase enzymes, insensitive to DPI, that maintain detectable O₂⁻
accumulation at 60 and 120 min of treatment. These results suggest that OGA
induce an oxidative burst by several O₂⁻-generating sources in the active growth
zones.
DOI: 10.1016/j.jplph.2010.09.012
1228. Appl Microbiol Biotechnol. 2011 Mar;89(6):1773-82. doi:

10.1007/s00253-010-2989-1. Epub 2010 Nov 10.
Absolute configuration-dependent epoxide formation from isoflavan-4-ol

stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain
KF707.
Seo J(1), Kang SI, Won D, Kim M, Ryu JY, Kang SW, Um BH, Pan CH, Ahn JH, Chong Y,
Kanaly RA, Han J, Hur HG.
Author information:
(1)School of Environmental Science and Engineering, Gwangju Institute of Science
and Technology, Gwangju, South Korea.
Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in

Escherichia coli was found to exhibit monooxygenase activity toward four
stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites
revealed that the corresponding epoxides formed between C2' and C3' on the B-ring
of each isoflavan-4-ol substrate were the sole products. The relative reactivity
of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol >
(3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol >
(3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute
configuration of the 4-OH group on the isoflavanols, as explained by an
enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P.
pseudoalcaligenes strain KF707 were further abiotically transformed into
pterocarpan, the molecular structure of which is commonly found as part of
plant-protective phytoalexins, such as maackiain from Cicer arietinum and
medicarpin from Medicago sativa.
DOI: 10.1007/s00253-010-2989-1
Field trials of aggregation pheromones for the stink bugs Chlorochroa uhleri and
Chlorochroa sayi (Hemiptera: Pentatomidae).
Millar JG(1), McBrien HM, McElfresh JS.
Author information:
(1)Department of Entomology, University of California, Riverside, CA 92521, USA.
jocelyn.millar@ucr.edu
In field trials, adult Chlorochroa uhleri (Stål) (Heteroptera: Pentatomidae) of

both sexes were caught in significant numbers in cylindrical screen traps baited
with gray rubber septum lures loaded with the main component of the male-produced
pheromone, methyl (E)-6-2,3-dihydrofarnesoate. Addition of the two possible minor
components of the pheromone, methyl (E)-5-2,6,10-trimethyl-5,9-undecadienoate and
methyl (2E,6E)-farnesoate, did not affect attraction. Combining the pheromone
with different concentrations of volatiles mimicking the odors of a known host
plant, alfalfa (Medicago sativa L.), had no significant effect on attraction of
adult bugs, whereas combining the pheromone with the pheromones of two sympatric
stink bug species, Chlorochroa sayi (Stål) and Euschistus conspersus Uhler,
decreased trap captures, suggesting interference between the pheromones. Small
numbers of Chlorochroa ligata (Say) adults also were attracted, but numbers
caught were too low to allow statistical comparisons between lure blends. In
field trials with C. sayi, all three of the male-specific pheromone compounds
[methyl geranate, methyl citronellate, and methyl (E) -6-2,3-dihydrofarnesoate]
were required for optimal attraction. As with C. uhleri, adults of both sexes
were attracted to pheromone lures in approximately equal numbers. Because of the
decreased volatility (=release rate) of methyl (E)-6-2,3-dihydrofarnesoate in
comparison with the other two, lower molecular weight pheromone components, lures
needed to be loaded with a disproportionately high amount of methyl
(E)-6-2,3-dihydrofarnesoate to obtain the best trap catch. There was no
indication that the pheromone components of C. uhleri or E. conspersus interfered
with the attractiveness of the C. sayi pheromone in lures containing a blend of
all three pheromones.
1230. J Bacteriol. 2011 Jan;193(2):485-96. doi: 10.1128/JB.01129-10. Epub 2010 Nov

5.
Complex regulation of symbiotic functions is coordinated by MucR and quorum
sensing in Sinorhizobium meliloti.
Mueller K(1), González JE.
Author information:
(1)Department of Molecular and Cell Biology, University of Texas at Dallas,
Richardson, TX 75080-0688, USA.
In Sinorhizobium meliloti, the production of exopolysaccharides such as

succinoglycan and exopolysaccharide II (EPS II) enables the bacterium to invade
root nodules on Medicago sativa and establish a nitrogen-fixing symbiosis. While
extensive research has focused on succinoglycan, less is known concerning the
regulation of EPS II or the mechanism by which it mediates entrance into the host
plant. Previously, we reported that the ExpR/Sin quorum-sensing system is
required to produce the symbiotically active low-molecular-weight fraction of
this exopolysaccharide. Here, we show that this system induces EPS II production
by increasing expression of the expG-expC operon, encoding both a transcriptional
regulator (ExpG) and a glycosyl transferase (ExpC). ExpG derepresses EPS II
production at the transcriptional level from MucR, a RosR homolog, while
concurrently elevating expression of expC, resulting in the synthesis of the
low-molecular-weight form. While the ExpR/Sin system abolishes the role of MucR
on EPS II production, it preserves a multitude of other
quorum-sensing-independent regulatory functions which promote the establishment
of symbiosis. In planktonic S. meliloti, MucR properly coordinates a diverse set
of bacterial behaviors by repressing a variety of genes intended for expression
during symbiosis and enhancing the bacterial ability to induce root nodule
formation. Quorum sensing precisely modulates the functions of MucR to take
advantage of both the production of symbiotically active EPS II as well as the
proper coordination of bacterial behavior required to promote symbiosis.
DOI: 10.1128/JB.01129-10
PMCID: PMC3019836
1231. Food Microbiol. 2011 Feb;28(1):119-27. doi: 10.1016/j.fm.2010.09.004. Epub

2010
Sep 16.
Individual and combined application of dry heat with high hydrostatic pressure to
inactivate Salmonella and Escherichia coli O157:H7 on alfalfa seeds.
Neetoo H(1), Chen H.
Author information:
(1)Department of Animal and Food Sciences, University of Delaware, Newark, DE
19716-2150, USA.
Alfalfa sprouts are recurrently implicated in outbreaks of food-borne illnesses

as a result of contamination with Salmonella or Escherichia coli O157:H7. In the
majority of these outbreaks, the seeds themselves have been shown to be the most
likely source of contamination. The aims of this study were to comparatively
assess the efficacy of dry heat treatments alone or in conjunction with high
hydrostatic pressure (HHP) to eliminate a ∼5 log CFU/g load of Salmonella and E.
coli O157:H7 on alfalfa seeds. Dry heat treatments at mild temperatures of 55 and
60 °C achieved ≤1.6 and 2.2 log CFU/g reduction in the population of Salmonella
spp. after a 10-d treatment, respectively. However, subjecting alfalfa seeds to
more aggressive temperatures of 65 °C for 10 days or 70 °C for 24 h eliminated a
∼5 log population of Salmonella and E. coli O157:H7. We subsequently showed that
the sequential application of dry heating followed by HHP could substantially
reduce the dry heating exposure time while achieving equivalent decontamination
results. Dry heating at 55, 60, 65 and 70 °C for 96, 24, 12 and 6 h, respectively
followed by a pressure treatment of 600 MPa for 2 min at 35 °C were able to
eliminate a ∼5 log CFU/g initial population of both pathogens. Finally, we
evaluated the impact of selected treatments on the seed germination percentages
and yield ratios and showed that dry heating at 65 °C for 10 days did not bring
about any considerable decrease in the germination percentage. However, the
sprout yield of treated alfalfa seeds was reduced by 21%. Dry heating at 60 and
65 °C for 24 and 12 h respectively followed by the pressure treatment of 600 MPa
for 2 min at 35 °C did not significantly (P > 0.05) affect the germination
percentage of alfalfa seeds although a reduction in the sprouting yield was
observed.
DOI: 10.1016/j.fm.2010.09.004
1232. Proc Biol Sci. 2011 May 22;278(1711):1539-47. doi: 10.1098/rspb.2010.1959.

Epub
2010 Nov 3.
Ant association facilitates the evolution of diet breadth in a lycaenid

butterfly.
Forister ML(1), Gompert Z, Nice CC, Forister GW, Fordyce JA.
Author information:
mforister@unr.edu
The role of mutualistic interactions in adaptive diversification has not been

thoroughly examined. Lycaenid butterflies provide excellent systems for exploring
mutualistic interactions, as more than half of this family is known to use ants
as a resource in interactions that range from parasitism to mutualism. We
investigate the hypothesis that protection from predators offered to caterpillars
by ants might facilitate host-range evolution. Specifically, experiments with the
butterfly Lycaeides melissa investigated the role of ant association in the use
of a novel host, alfalfa, Medicago sativa, which is a sub-optimal host for larval
development. Survival on alfalfa is increased by the presence of ants, thus
supporting the hypothesis that interaction with ants might be important for
host-range evolution. Using a demographic model to explore ecological conditions
associated with host-range expansion in L. melissa, we conclude that the presence
of ants might be an essential component for populations persisting on the novel,
sub-optimal host.
DOI: 10.1098/rspb.2010.1959
PMCID: PMC3081752
1233. FEMS Microbiol Ecol. 2010 Dec;74(3):612-22. doi:

10.1111/j.1574-6941.2010.00977.x. Epub 2010 Nov 2.
Composition, spatial distribution, and diversity of the bacterial communities in

the rumen of cows fed different forages.
Kong Y(1), Teather R, Forster R.
Author information:
(1)Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB,
Canada.
The species composition, distribution, and biodiversity of the bacterial

communities in the rumen of cows fed alfalfa or triticale were investigated using
16S rRNA gene clone library analyses. The rumen bacterial community was
fractionated and analyzed as three separate fractions: populations in the
planktonic, loosely attached to rumen digesta particles, and tightly attached to
rumen digesta particles. Six hundred and thirteen operational taxonomic units
(OTUs) belonging to 32 genera, 19 families, and nine phyla of the domain Bacteria
were identified from 1014 sequenced clones. Four hundred and fifty one of the 613
OTUs were identified as new species. These bacterial sequences were distributed
differently among the three fractions in the rumen digesta of cows fed alfalfa or
triticale. Chao 1 estimation revealed that, in both communities, the populations
tightly attached to particulates were more diverse than the planktonic and those
loosely attached to particulates. S-Libshuff detected significant differences in
the composition between any two fractions in the rumen of cows with the same diet
and between the communities fed alfalfa and triticale diets. The species richness
estimated for the communities fed alfalfa and triticale is 1027 and 662,
respectively. The diversity of the rumen bacterial community examined in this
study is greater than previous studies have demonstrated and the differences in
the community composition between two high-fiber diets have implications for
sample selection for downstream metagenomics applications.
© 2010 Federation of European Microbiological Societies and Her Majesty the Queen
in Right of Canada, as represented by the Minister of Agriculture and Agri-Food
Canada.
DOI: 10.1111/j.1574-6941.2010.00977.x
1234. Ecotoxicol Environ Saf. 2011 Mar;74(3):431-8. doi:

10.1016/j.ecoenv.2010.10.006.
Epub 2010 Oct 27.
Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago

sativa-Rhizobia symbiosis.
El Khalloufi F(1), Oufdou K, Lahrouni M, El Ghazali I, Saqrane S, Vasconcelos V,

Oudra B.
Author information:
(1)Laboratory of Biology and Biotechnology of Microorganisms, Environmental
Microbiology and Toxicology Unit, Cadi Ayyad University, Faculty of Sciences
Semlalia, P.O. Box 2390, Marrakech, Morocco.
The eutrophication of water leads to massive blooms of cyanobacteria potentially

producers of highly toxic substances: cyanotoxins, especially microcystins (MC).
The contamination of water used for irrigation by these toxins, can cause several
adverse effects on plants and microorganisms. In this work, we report the
phytotoxic effects of microcystins on the development of symbiosis between the
leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of
rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC
ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh
L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19%
and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC
concentrations affects the whole stages of plant development. The germination
process has also been disrupted with an inhibition, which reaches 68.34% for a
22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were
also disrupted. The toxins reduced significantly the roots length and nodule
formation and leads to an oxidative stress. Thus, the MCs contained in lake water
and used for irrigation affect the development of symbiosis between M. sativa and
Rhizobia.
DOI: 10.1016/j.ecoenv.2010.10.006
1235. J Colloid Interface Sci. 2011 Jan 15;353(2):433-44. doi:

10.1016/j.jcis.2010.09.088. Epub 2010 Oct 25.
Facile synthesis, stabilization, and anti-bacterial performance of discrete Ag

nanoparticles using Medicago sativa seed exudates.
Lukman AI(1), Gong B, Marjo CE, Roessner U, Harris AT.
Author information:
(1)Laboratory for Sustainable Technology, School of Chemical and Biomolecular
Engineering, University of Sydney, NSW 2006, Australia.
The biogenic synthesis of metal nanomaterials offers an environmentally benign

alternative to the traditional chemical synthesis routes. Colloidal silver (Ag)
nanoparticles were synthesized by reacting aqueous AgNO(3) with Medicago sativa
seed exudates under non-photomediated conditions. Upon contact, rapid reduction
of Ag(+) ions was observed in <1 min with Ag nanoparticle formation reaching 90%
completion in <50 min. Effect of Ag concentration, quantity of exudate and pH on
the particle size and shape were investigated. At [Ag(+)]=0.01 M and 30°C,
largely spherical nanoparticles with diameters in the range of 5-51 nm were
generated, while flower-like particle clusters (mean size=104 nm) were observed
on treatment at higher Ag concentrations. Pre-dilution of the exudate induced the
formation of single-crystalline Ag nanoplates, forming hexagonal particles and
nanotriangles with edge lengths of 86-108 nm, while pH adjustment to 11 resulted
in monodisperse Ag nanoparticles with an average size of 12 nm. Repeated
centrifugation and redispersion enhanced the percentage of nanoplates from 10% to
75% in solution. The kinetics of nanoparticle formation were monitored using
ultraviolet-visible spectroscopy and the Ag products were characterized using
transmission electron microscopy, selected-area electron diffraction, scanning
electron microscopy, X-ray powder diffraction, and atomic force microscopy. X-ray
photoelectron spectroscopy was used to investigate the elements and chemical
environment in the top layers of the as-synthesized Ag nanoparticles, while the
metabolites in the exudate were analyzed using gas chromatography-mass
spectroscopy. To our knowledge, this is the first account of M. sativa seed
exudate assisted synthesis and stabilization of biogenic Ag nanoparticles; the
nanoplates are notably smaller and better faceted compared with those synthesized
by vascular plant extracts previously reported. Stabilized films of exudate
synthesized Ag nanoparticles were effective anti-bacterial agents.
DOI: 10.1016/j.jcis.2010.09.088
1236. J Bacteriol. 2011 Jan;193(1):30-9. doi: 10.1128/JB.01009-10. Epub 2010 Oct
22.
The nodulation of alfalfa by the acid-tolerant Rhizobium sp. strain LPU83 does
not require sulfated forms of lipochitooligosaccharide nodulation signals.
Torres Tejerizo G(1), Del Papa MF, Soria-Diaz ME, Draghi W, Lozano M, Giusti Mde
L, Manyani H, Megías M, Gil Serrano A, Pühler A, Niehaus K, Lagares A, Pistorio
M.
Author information:
(1)IBBM-Instituto de Biotecnología y Biología Molecular, CCT-CONICET-La Plata,
Nacional de La Plata, Calles 47 y 115 (1900), La Plata, Argentina.
pistorio@biol.unlp.edu.ar
The induction of root nodules by the majority of rhizobia has a strict

requirement for the secretion of symbiosis-specific lipochitooligosaccharides
(nodulation factors [NFs]). The nature of the chemical substitution on the NFs
depends on the particular rhizobium and contributes to the host specificity
imparted by the NFs. We present here a description of the genetic organization of
the nod gene cluster and the characterization of the chemical structure of the
NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium
capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod
gene cluster was located on the plasmid pLPU83b. The organization of the cluster
showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium
meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence
similarity observed was between the partial nod sequences of Rhizobium mongolense
USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic
analysis of the intergenic region nodEG positions strain LPU83 and the type
strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp.
strain LPU83 might represent an early alfalfa-nodulating genotype. The NF
chemical structures obtained for the wild-type strain consist of a trimeric,
tetrameric, and pentameric chitin backbone that shares some substitutions with
both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83
most of the NFs were sulfated in their reducing terminal residue, none of the NFs
isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained
supports the notion that the sulfate decoration of NFs in LPU83 is not necessary
for alfalfa nodulation.
DOI: 10.1128/JB.01009-10
PMCID: PMC3019937
1237. Appl Environ Microbiol. 2010 Dec;76(24):7972-80. doi: 10.1128/AEM.01972-10.

Epub
2010 Oct 22.
Inositol catabolism, a key pathway in sinorhizobium meliloti for competitive host

nodulation.
Kohler PR(1), Zheng JY, Schoffers E, Rossbach S.
Author information:
(1)Department of Biological Sciences, 3923 Wood Hall, Western Michigan
University, Kalamazoo, MI 49008-5410, USA.
The nitrogen-fixing symbiont of alfalfa, Sinorhizobium meliloti, is able to use
myo-inositol as the sole carbon source. Putative inositol catabolism genes (iolA
and iolRCDEB) have been identified in the S. meliloti genome based on their
similarities with the Bacillus subtilis iol genes. In this study, functional
mutational analysis revealed that the iolA and iolCDEB genes are required for
growth not only with the myo-isomer but also for growth with scyllo- and
d-chiro-inositol as the sole carbon source. An additional, hypothetical
dehydrogenase of the IdhA/MocA/GFO family encoded by the smc01163 gene was found
to be essential for growth with scyllo-inositol, whereas the idhA-encoded
myo-inositol dehydrogenase was responsible for the oxidation of d-chiro-inositol.
The putative regulatory iolR gene, located upstream of iolCDEB, encodes a
repressor of the iol genes, negatively regulating the activity of the myo- and
the scyllo-inositol dehydrogenases. Mutants with insertions in the iolA,
smc01163, and individual iolRCDE genes could not compete against the wild type in
a nodule occupancy assay on alfalfa plants. Thus, a functional inositol catabolic
pathway and its proper regulation are important nutritional or signaling factors
in the S. meliloti-alfalfa symbiosis.
DOI: 10.1128/AEM.01972-10
PMCID: PMC3008233
1238. Pharm Biol. 2011 Feb;49(2):211-20. doi: 10.3109/13880209.2010.504732. Epub

2010
Oct 25.
Phytochemical and pharmacological potential of Medicago sativa: a review.
Bora KS(1), Sharma A.
Author information:
(1)L.R. Institute of Pharmacy, Solan, Himachal Pradesh, India.
kundanresearch1381@gmail.com
CONTEXT: Many herbal remedies have so far been employed for the treatment and
management of various ailments since the beginning of human civilization.
Medicago is an extensive genus of the family Leguminosae, comprising about 83
different species. Medicago sativa (Linn.) has long been used as traditional
herbal medicine in China, Iraq, Turkey, India and America for the treatment of a
variety of ailments.
OBJECTIVES: The aim of this review was to collect all available scientific
literature published and combine it into this review. The present review
comprises the ethnopharmacological, phytochemical and therapeutic potential of M.
sativa.
METHODS: The present review includes 117 references compiled from major databases
as Chemical Abstracts, Science Direct, SciFinder, PubMed, Dr. Dukes Phytochemical
and Ethnobotany, CIMER, and InteliHealth.
RESULTS: An exhaustive survey of literature revealed that saponins, flavonoids,
phytoestrogens, coumarins, alkaloids, amino acids, phytosterols, vitamins,
digestive enzymes and terpenes constitute major classes of phytoconstituents of
this plant. Pharmacological reports revealed that it is used as neuroprotective,
hypocholesterolemic, antioxidant, antiulcer, antimicrobial, hypolipidemic,
estrogenic, and in the treatment of atherosclerosis, heart disease, stroke,
cancer, diabetes and menopausal symptoms in women.
CONCLUSION: M. sativa seems to hold great potential for in-depth investigation
for various biological activities, especially their effects on central nervous
and cardiovascular system. Through this review, the authors hope to attract the
attention of natural product researchers throughout the world to focus on the
unexplored potential of M. sativa, and it may be useful in developing new
formulations with more therapeutic value.
DOI: 10.3109/13880209.2010.504732
1239. J Dairy Sci. 2010 Nov;93(11):5397-407. doi: 10.3168/jds.2010-3411.
Effects of corn silage hybrids and dietary nonforage fiber sources on feed
intake, digestibility, ruminal fermentation, and productive performance of
lactating Holstein dairy cows.
Holt MS(1), Williams CM, Dschaak CM, Eun JS, Young AJ.
Author information:
Logan, Utah 84322-4815, USA.
This experiment was conducted to determine the effects of corn silage hybrids and
nonforage fiber sources (NFFS) in high forage diets formulated with high dietary
proportions of alfalfa hay (AH) and corn silage (CS) on ruminal fermentation and
productive performance by early lactating dairy cows. Eight multiparous Holstein
cows (4 ruminally fistulated) averaging 36±6.2 d in milk were used in a
duplicated 4 × 4 Latin square design experiment with a 2 × 2 factorial
arrangement of treatments. Cows were fed 1 of 4 dietary treatments during each of
the four 21-d replicates. Treatments were (1) conventional CS (CCS)-based diet
without NFFS, (2) CCS-based diet with NFFS, (3) brown midrib CS (BMRCS)-based
diet without NFFS, and (4) BMRCS-based diet with NFFS. Diets were isonitrogenous
and isocaloric. Sources of NFFS consisted of ground soyhulls and pelleted beet
pulp to replace a portion of AH and CS in the diets. In vitro 30-h neutral
detergent fiber (NDF) degradability was greater for BMRCS than for CCS (42.3 vs.
31.2%). Neither CS hybrids nor NFFS affected intake of dry matter (DM) and
nutrients. Digestibility of N, NDF, and acid detergent fiber tended to be greater
for cows consuming CCS-based diets. Milk yield was not influenced by CS hybrids
and NFFS. However, a tendency for an interaction between CS hybrids and NFFS
occurred, with increased milk yield due to feeding NFFS with the BMRCS-based
diet. Yields of milk fat and 3.5% fat-corrected milk decreased when feeding the
BMRCS-based diet, and a tendency existed for an interaction between CS hybrids
and NFFS because milk fat concentration further decreased by feeding NFFS with
BMRCS-based diet. Although feed efficiency (milk/DM intake) was not affected by
CS hybrids and NFFS, an interaction was found between CS hybrids and NFFS because
feed efficiency increased when NFFS was fed only with BMRCS-based diet. Total
volatile fatty acid production and individual molar proportions were not affected
by diets. Dietary treatments did not influence ruminal pH profiles, except that
duration (h/d) of pH <5.8 decreased when NFFS was fed in a CCS-based diet but not
in a BMRCS-based diet, causing a tendency for an interaction between CS hybrids
and NFFS. Overall measurements in our study reveal that high forage NDF
concentration (20% DM on average) may eliminate potentially positive effects of
BMRCS. In the high forage diets, NFFS exerted limited effects on productive
performance when they replaced AH and CS. Although the high quality AH provided
adequate NDF (38.3% DM) for optimal rumen fermentative function, the low NDF
concentration of the AH and the overall forage particle size reduced physically
effective fiber and milk fat concentration.

DOI: 10.3168/jds.2010-3411
Effect of feeding alfalfa hay or Tifton 85 bermudagrass haylage with or without a

cellulase enzyme on performance of Holstein cows.
Bernard JK(1), Castro JJ, Mullis NA, Adesogan AT, West JW, Morantes G.
Author information:
(1)Animal and Dairy Science Department, University of Georgia, Tifton, Georgia
31793-0748, USA. jbernard@uga.edu
Forty-four lactating Holstein cows (173±30 DIM, 42.5±6.8 kg of milk, 4.03±0.69%

fat, 674±78 kg of body weight) were used in an 8-wk, completely randomized trial
with a 2 × 2 factorial arrangement of treatments to determine the effect of
forage source and supplemental cellulase enzyme on performance. Treatments
included 2 forage combinations (corn silage plus 12.2% dry matter, DM, from
either alfalfa hay or Tifton 85 bermudagrass haylage) with or without a
commercial cellulase enzyme applied to the total mixed ration at the rate of 4
g/head per day (Promote N.E.T.-L, Cargill Animal Nutrition, Minneapolis, MN).
Experimental diets were formulated to provide similar concentrations of protein
(16.5% of DM), energy (1.63 Mcal of net energy for lactation/kg of DM), and
neutral detergent fiber (41.7% of DM) and were fed once daily as a total mixed
ration behind Calan doors for ad libitum intake. The cellulase enzyme provided
1,200 cellulase units of activity/g of product and was applied to the total mixed
ration and allowed to mix for 5min before feeding. Before beginning the trial,
all cows were trained to use Calan (American Calan, Northwood, NH) doors and then
fed the alfalfa hay-based diet for 2 wk. Data collected during wk 2 were used as
a covariate in the statistical analysis. At the beginning of the 6-wk
experimental period, cows were assigned randomly to 1 of the 4 experimental
diets. No interactions were observed between forage and enzyme for any measures.
Daily DM intake; milk yield; concentrations of milk fat, true protein, lactose,
and solids not fat; energy-corrected milk yield; and dairy efficiency were not
different among alfalfa or Tifton 85 bermudagrass rations with or without
cellulase enzyme supplementation. The results of this trial indicate that Tifton
85 bermudagrass haylage can replace alfalfa hay in diets fed to high-producing,
lactating dairy cows without depressing DM intake or milk yield when rations are
balanced for NDF. Although supplemental cellulase enzymes have been shown to
improve ration digestibility and animal performance in previous trials, no
advantages were observed in the current trial.

DOI: 10.3168/jds.2010-3111
1241. J Hazard Mater. 2011 Jan 30;185(2-3):703-9. doi:

10.1016/j.jhazmat.2010.09.076.
Epub 2010 Oct 1.
Arbuscular mycorrhizal phytoremediation of soils contaminated with phenanthrene

and pyrene.
Gao Y(1), Li Q, Ling W, Zhu X.

Author information:
(1)Institute of Organic Contaminant Control and Soil Remediation, College of
Resource and Environmental Sciences, Nanjing Agricultural University, Weigang
Road 1, Nanjing 210095, PR China. gaoyanzheng@njau.edu.cn
An available remediation technique--arbuscular mycorrhizal phytoremediation

(AMPR)--is further proposed for soils contaminated with phenanthrene and pyrene
as representative polycyclic aromatic hydrocarbons (PAHs) utilizing a greenhouse
pot experiment. The initial concentrations of phenanthrene and/or pyrene in soils
were 103 mg kg(-1) and 74 mg kg(-1), respectively. The host plant was alfalfa
(Medicago sativa L.), and the experimental arbuscular mycorrhizal fungi (AMF)
were Glomus mosseae and G. etunicatum. More than 98.6% and 88.1% of phenanthrene
and pyrene were degraded after 70 days in soils with AMPR. Use of multiple
mycorrhizal species significantly promoted degradation of PAHs in soils. The
co-contaminant (pyrene) present clearly inhibited the degradation of a single PAH
(phenanthrene) in soil. Mycorrhizal colonization caused increased accumulation of
PAHs in plant roots but a decrease in shoot. However, plant uptake contributed
negligibly to PAH dissipation in AMPR, and plant accumulated PAHs amounted to
less than 3.24% of total PAH degradation in mycorrhizal soils. In contrast, the
optimized microbiota in mycorrhizal association was responsible for PAH
degradation in AMPR. The high rate of PAH dissipation in mycorrhizal soils, the
evident promotion of PAH degradation by AM colonization, and the healthy plant
growth suggest encouraging opportunities for AMPR of PAH-contaminated soils.
DOI: 10.1016/j.jhazmat.2010.09.076
1242. Phytopathology. 2011 May;101(5):594-610. doi: 10.1094/PHYTO-04-10-0107.
Genetic and morphological evidence that Phoma sclerotioides, causal agent of

brown root rot of alfalfa, is composed of a species complex.
Wunsch MJ(1), Bergstrom GC.
Author information:
(1)Department of Plant Pathology and Plant-Microbe Biology, Cornell University,
Ithaca, NY, USA.
Phoma sclerotioides, causal agent of brown root rot of alfalfa, causes severe
root and crown lesions on alfalfa and other perennial forage legumes in regions
with harsh winters. Isolates of P. sclerotioides exhibit diverse cultural
morphologies on potato dextrose agar (PDA), suggesting that they may exhibit a
high degree of genetic diversity. To investigate the genetic relatedness of P.
sclerotioides isolates, 154 isolates from North America were sequenced at 10
loci. Maximum parsimony and maximum likelihood analyses of the complete 10-locus
data set placed isolates into multiple strongly supported clades, and analyses of
gene-jackknife and single-gene partitions of the data set indicated robust
support for six major clades and three subclades. Genetic differences
corresponded closely to differences in conidial size and septation, pycnidial
neck length, mycelial pigmentation, and growth rate in axenic culture at 18 and
25°C. Isolates exhibited morphologies broadly consistent with the species
description of P. sclerotioides, and new species were not designated. On the
basis of genetic and morphological differences, we propose establishing seven
infraspecific varieties within P. sclerotioides: P. sclerotioides var.
sclerotioides, champlainii, viridis, obscurus, steubenii, macrospora, and
saskatchewanii. All varieties of P. sclerotioides caused brown root rot of
alfalfa and grew well at low temperatures.
DOI: 10.1094/PHYTO-04-10-0107
1243. J Nanobiotechnology. 2010 Oct 7;8:24. doi: 10.1186/1477-3155-8-24.
The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension

culture.
Santos AR(1), Miguel AS, Tomaz L, Malhó R, Maycock C, Vaz Patto MC, Fevereiro P,
Oliva A.
Author information:
(1)Biomolecular Diagnostics Laboratory, Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
raquelsantos@itqb.unl.pt.
BACKGROUND: Nanotechnology has the potential to provide agriculture with new

tools that may be used in the rapid detection and molecular treatment of diseases
and enhancement of plant ability to absorb nutrients, among others. Data on
nanoparticle toxicity in plants is largely heterogeneous with a diversity of
physicochemical parameters reported, which difficult generalizations. Here a cell
biology approach was used to evaluate the impact of Quantum Dots (QDs)
nanocrystals on plant cells, including their effect on cell growth, cell
viability, oxidative stress and ROS accumulation, besides their cytomobility.
RESULTS: A plant cell suspension culture of Medicago sativa was settled for the
assessment of the impact of the addition of mercaptopropanoic acid coated
CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of
mercaptopropanoic acid -QDs was added during the exponential growth phase, with
less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs
addition. They were up taken by Medicago sativa cells and accumulated in the
cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of
the cellular response to internalization, Medicago sativa cells were found to
increase the production of Reactive Oxygen Species (ROS) in a dose and time
dependent manner. Using the fluorescent dye H2DCFDA it was observable that
mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive
and linear increase of ROS accumulation.
CONCLUSIONS: Our results showed that the extent of mercaptopropanoic acid coated
CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors
including QDs properties, dose and the environmental conditions of administration
and that, for Medicago sativa cells, a safe range of 1-5 nM should not be
exceeded for biological applications.
DOI: 10.1186/1477-3155-8-24
PMCID: PMC2958997
PMID: 20929583
1244. Int J Food Microbiol. 2010 Nov 15;144(1):141-6. doi:

10.1016/j.ijfoodmicro.2010.09.011.
Inactivation of Escherichia coli O157:H7 and Salmonella spp. on alfalfa seeds by

caprylic acid and monocaprylin.
Chang SS(1), Redondo-Solano M, Thippareddi H.

Author information:
(1)Department of Food Science and Technology, University of Nebraska-Lincoln,
Lincoln, NE 68583, USA.
Alfalfa and other seed sprouts have been implicated in several Escherichia coli
O157:H7 and Salmonella spp. human illness outbreaks in the U.S. Continuing food
safety issues with alfalfa seeds necessitate the need for discovery and use of
novel and effective antimicrobials. The potential use of caprylic acid (CA) and
monocaprylin (MC) for reducing E. coli O157:H7 and Salmonella spp. populations on
alfalfa seeds was evaluated. The effectiveness of three concentrations of CA and
MC (25, 50, and 75 mM) to reduce E. coli O157:H7 and Salmonella spp. populations
in 0.1% peptone water and on alfalfa seeds was evaluated. Surviving populations
of E. coli O157:H7 and Salmonella spp. were enumerated by direct plating on
tryptic soy agar (TSA). Non-inoculated alfalfa seeds were soaked for up to 120
min to evaluate the effect of CA and MC solutions on seed germination rate. For
planktonic cells, the efficacy of the treatments was: 75 MC > 50 MC > 25 MC > 75
CA > 50 CA > 25 CA. Both E. coli O157:H7 and Salmonella spp. were reduced to
below the detection limit (0.6 log CFU/ml) within 10 min of exposure to 75 MC
from initial populations of 7.65 ± 0.10 log CFU/ml and 7.71 ± 0.11 log CFU/ml,
respectively. Maximum reductions of 1.56 ± 0.25 and 2.56 ± 0.17 log CFU/g for E.
coli O157:H7 and Salmonella spp., respectively, were achieved on inoculated
alfalfa seeds (from initial populations of 4.74 ± 0.62 logCFU/g and
5.27±0.20logCFU/g, respectively) when treated with 75 MC for 90 min. Germination
rates of CA or MC treated seeds ranged from 84% to 99%. The germination rates of
CA or MC soaked seeds and water soaked seeds (control) were similar (P > 0.05)
for soaking times of ≤ 90 min. Monocaprylin (75 mM) can be used to reduce E. coli
O157:H7 and Salmonella spp. on alfalfa seeds without compromising seed viability.
1245. J Biol Chem. 2010 Dec 10;285(50):38961-8. doi: 10.1074/jbc.M110.137315. Epub

2010
Oct 4.
Increase in 4-coumaryl alcohol units during lignification in alfalfa (Medicago

sativa) alters the extractability and molecular weight of lignin.
Ziebell A(1), Gracom K, Katahira R, Chen F, Pu Y, Ragauskas A, Dixon RA, Davis M.
Author information:
(1)National Bioenergy Center, National Renewable Energy Laboratory, Golden,
Colorado 80401-3393, USA.
The lignin content of biomass can impact the ease and cost of biomass processing.
Lignin reduction through breeding and genetic modification therefore has
potential to reduce costs in biomass-processing industries (e.g. pulp and paper,
forage, and lignocellulosic ethanol). We investigated compositional changes in
two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of
p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate
hydroxycinnamoyltransferase (HCT). We investigated whether the difference in
reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the
naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters
the lignin structure. Sequential base extraction readily reduced the H monomer
content of the transgenic lines, leaving a residual lignin greatly enriched in H
subunits; the extraction profile highlighted the difference between the control
and transgenic lines. Gel permeation chromatography of isolated ball-milled
lignin indicated significant changes in the weight average molecular weight
distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500;
C3H9a, 4000; and HCT30a, 4000).
DOI: 10.1074/jbc.M110.137315
PMCID: PMC2998124
1246. J Anim Physiol Anim Nutr (Berl). 2011 Apr;95(2):267-72. doi:

10.1111/j.1439-0396.2010.01051.x. Epub 2010 Sep 29.
Maturity of coastal bermudagrass and alfalfa affects ruminal in situ and total
tract dry matter and phosphorus disappearance in cannulated steers.
Riojas-McCollister AV(1), Lambert BD, Muir JP.
Author information:
(1)Department of Animal Sciences, Tarleton State University, Stephenville, TX
76402, USA.
Variability of phosphorus (P) availability among forage species and plant

maturity is largely ignored when formulating ruminant diets. To determine if
variability in P availability changes with forage species and/or maturity,
ruminal in situ and total (ruminal+post-ruminal) dry matter (DM) and phosphorus
disappearance (PD) from alfalfa (ALF; Medicago sativa) and coastal bermudagrass
(CB; Cynodon dactylon) harvested at four stages of maturity was measured in
cattle. Forages were hand clipped at 14, 21, 28 and 35 days after first cutting.
Ruminal in situ DM disappearance (DMD) and PD were measured after 24 h ruminal
incubation in Dacron bags. Total tract DMD and PD were measured using the mobile
nylon bag technique. Disappearance of DM and P were greater (p≤0.05) in the rumen
than post-rumen for both species regardless of maturity; however, 80 g PD/kg DM
in 35-day ALF (9% of total PD) and 224 g PD/kg of 35-day CB (38% of total PD)
occurred post ruminally. Alfalfa DM disappeared to a greater (p ≤ 0.05) extent
than CB and showed 5% greater total tract PD at 14 days and 13% more at 35 days
compared to the grass. Alfalfa total tract PD decreased (p ≤ 0.05) 5.4% from 14-
to 35-day maturity while the decrease was far greater for CB, 12.4%. Results from
this study indicate that ruminant nutritionists should take into account forage
species and maturity when calculating PD in diets; these details can be used to
aid in formulating more precise rations that reduce fecal-phosphorus excretion
into the environment.
DOI: 10.1111/j.1439-0396.2010.01051.x
1247. Br J Nutr. 2011 Jan;105(1):62-70. doi: 10.1017/S0007114510003120. Epub 2010

Sep
28.
Alfalfa (Medicago sativa L.) shoot saponins: identification and bio-activity by

the assessment of aphid feeding.
Mazahery-Laghab H(1), Yazdi-Samadi B, Bagheri M, Bagheri AR.
Author information:
(1)Agronomy and Plant Breeding Department, Agriculture Faculty of Bu-Ali Sina
University, 6517833131, Hamedan, Iran. hojat.mazahery@yahoo.co.uk
Biochemical components in alfalfa (Medicago sativa L.), such as saponins, can act
as protecting factors against bio-stresses. Saponins are also antifeedants and
show oral toxicity towards higher and lower animals. Changes in saponins, such as
variation in the carbon skeleton, or hydrolysis of saponin glycosides and other
conjugates, may change their biological effects. The aims of this research were
to study saponin variation in different growth stages of alfalfa and to
investigate the biological role of saponins in the spotted alfalfa aphid,
Therioaphis maculata. Saponins from alfalfa shoots in different growth stages
were extracted, chemically purified and analysed by TLC. Specific saponins such
as soyasaponin1 from root and shoot and two bisdesmosides of medicagenic acid,
one from shoot and another from root tissues, were identified using reference
compounds allowing changes in saponin composition during plant development in
different shoot tissues of alfalfa to be assessed. The response of the alfalfa
aphid to feeding on alfalfa in different growth stages was studied. No
significant difference in the survival of aphids, from neonate to adult, was
observed, but due to the antibiotic effects of saponins, two differences were
found in the onset of nymph production and cumulative nymph production. The
results show that the saponin composition in alfalfa changes with plant
development and this, in turn, can often negatively affect the development of
specific insect pests such as the spotted alfalfa aphid, suggesting a possible
biological role of alfalfa saponins.
DOI: 10.1017/S0007114510003120
Distribution of glassy-winged sharpshooter and threecornered alfalfa hopper on

plant hosts in the San Joaquin Valley, California.
Wistrom C(1), Sisterson MS, Pryor MP, Hashim-Buckey JM, Daane KM.
Author information:
(1)Department of Environmental Science, Policy and Management, University of
California, Berkeley, CA 94720-3114, USA.
Homalodisca vitripennis (Germar) and Spissistilus festinus (Say) populations were

surveyed bimonthly for 14 mo in Kern County, CA, at five agricultural sites made
up of a variety of potential host plants. In addition, S. festinus populations
were surveyed in four alfalfa, Medicago sativa L., fields in Kern and Tulare
counties. Insects were collected by beats-sweeps and sticky traps. Data on host
plant condition and phenology, and ground cover presence and composition were
collected at the five agricultural sites, whereas data on mowing and insecticide
use were collected at the four alfalfa sites. Populations of both insects
persisted at the five agricultural locations despite insecticide applications
applied as part of a H. vitripennis areawide management program and standard
commercial operations. Plants colonized by H. vitripennis included eucalyptus
(Eucalyptus L'Hér.), jojoba [Simmondsia chinensis (Link) C. K. Schneid.], and
citrus (Citrus spp.). Populations of S. festinus were much greater in collections
from alfalfa fields than from the five agricultural sites. Insects collected from
the five mixed agricultural sites were negative for presence of X. fastidiosa. In
laboratory tests, S. festinus did not acquire or transmit X. fastidiosa in tests
with infected grape (Vitis spp.) as an acquisition source and grape, almond
[Prunus dulcis (Mill.) D.A.Webb], and alfalfa as inoculation hosts.
Recommendations for vector control, vegetation management, and targeted
monitoring to reduce insect populations and inoculum potential are discussed.
1249. Arch Microbiol. 2011 Jan;193(1):63-8. doi: 10.1007/s00203-010-0632-2. Epub

2010
Sep 21.
Delftia sp. JD2: a potential Cr(VI)-reducing agent with plant growth-promoting

activity.
Morel MA(1), Ubalde MC, Braña V, Castro-Sowinski S.
Author information:
(1)Unidad de Microbiología Molecular, Instituto de Investigaciones Biológicas
Clemente Estable, Avda Italia 3318, 11600 Montevideo, PC, Uruguay.
A chromium (Cr)-resistant bacterium isolated from soil containing 6,000 mg/kg of

Cr was identified based on 16S rRNA gene sequence analysis as Delftia, and
designated as JD2. Growth of JD2 was accompanied with reduction of Cr(VI) to
Cr(III) in liquid medium initially containing 100 mg/L Cr(VI), the maximum
concentration allowing growth. JD2 showed NADH/NADPH-dependent reductase activity
associated with the soluble fraction of cells. The results suggest that JD2 might
be a good candidate for the treatment of highly Cr(VI)-contaminated water and/or
industrial effluents. The isolate produced indole-3-acetic acid in the presence
and absence of Cr(VI) and showed free-living nitrogen-fixing activity possibly
attributable to a V-nitrogenase. JD2 did not counteract the harmful effect of
Cr(VI) during leguminous plant growth and nodulation by rhizobial strains but
functioned as a "helper" bacterium to enhance the performance of rhizobial
inoculant strains during inoculation of alfalfa and clover (used as model plants
to study plant growth-promoting activity) in the absence of Cr(VI).
DOI: 10.1007/s00203-010-0632-2
1250. J Environ Manage. 2012 Mar;95 Suppl:S188-92. doi:

10.1016/j.jenvman.2010.08.017.
Epub 2010 Sep 19.
Chemical properties and biological activity in soils of Mallorca following twenty

years of treated wastewater irrigation.
Adrover M(1), Farrús E, Moyà G, Vadell J.
Author information:
(1)Department of Biology, University of the Balearic Islands, 07122 Palma de
Mallorca, Spain. maria.adrover@uib.es
On the Mediterranean island of Mallorca, the use of secondary-treated municipal

wastewater in irrigation was introduced with the construction of the first
wastewater treatment plants in the 1970s. In this study, the chemical properties
and biological activity of 21 arable soils, irrigated for more than 20 years with
secondary-treated wastewater, were tested in order to assess their quality. Soil
quality was evaluated by measuring cation exchange capacity, pH, calcium
carbonate equivalent, soil organic matter, total nitrogen, available phosphorus,
water-soluble organic carbon, soil microbial biomass, soil basal respiration, and
the activities of the enzymes dehydrogenase, β-glucosidase and alkaline
phosphatase. No negative effects of the irrigation treatment were observed on the
measured soil parameters. Indeed, soil water-soluble organic carbon, soil
microbial biomass and β-glucosidase and alkaline phosphatase activities increased
under treated wastewater irrigation. Biological activity of soils irrigated with
treated wastewater was affected mainly by soil organic matter content. Although
the typical crop management of alfalfa, and other forage crops associated with
treated wastewater irrigation, may have contributed to the increase of these
parameters, the results suggest that irrigation with treated wastewater is a
strategy with many benefits to agricultural land management.
DOI: 10.1016/j.jenvman.2010.08.017
1251. Biometals. 2011 Feb;24(1):93-103. doi: 10.1007/s10534-010-9377-2. Epub 2010

Sep
16.
Cadmium-induced heme oxygenase-1 gene expression is associated with the depletion

of glutathione in the roots of Medicago sativa.
Cui W(1), Fu G, Wu H, Shen W.
Author information:
Technique, Nanjing Agricultural University, Nanjing 210095, People's Republic of
China.
Following previous findings that cadmium (Cd) induces heme oxygenase-1 (HO1) gene
expression in alfalfa seedling roots, we now show that the decreased glutathione
(GSH) and ascorbic acid (AsA) contents, induction of HO-1 gene expression and its
protein level by Cd was mimicked by a GSH depletor diethylmaleate (DEM).
Meanwhile, above Cd- or DEM-induced decreased GSH content followed by HO-1
up-regulation could be strengthened or reversed differentially by the application
of a selective inhibitor of GSH biosynthesis L: -buthionine-sulfoximine (BSO), or
exogenous GSH and AsA, respectively. The antioxidative behavior of HO-1 induction
was further confirmed by histochemical staining for the detection of loss of
membrane integrity in a short period of treatment time. Additionally, the
induction of HO-1 transcript was inhibited by the transcriptional inhibitor
actinomycin D (ActD) or protein synthesis inhibitor cycloheximide (CX,
especially). In contrast, the level of HO-2 transcript did not change upon
various treatments. Together, above results suggested that Cd-induced
up-regulation of HO-1 gene expression is associated with GSH depletion, which is
at least existing transcriptional regulation level, thus leading to enhanced
antioxidative capability transiently.
DOI: 10.1007/s10534-010-9377-2
1252. Lett Appl Microbiol. 2010 Oct;51(4):462-8. doi: 10.1111/j.1472-

765X.2010.02929.x.
Reduction of Salmonella on alfalfa seeds using peroxyacetic acid and a commercial

seed washer is as effective as treatment with 20 000 ppm of Ca(OCl)2.
Buchholz A(1), Matthews KR.
Author information:
(1)Department of Food Science, Rutgers, The State University of New Jersey, New
Brunswick, NJ 08901-8520, USA.
AIMS: The efficacy of a commercial seed washer and 1 and 3% peroxyacetic acid or
20 000 ppm calcium hypochlorite for reducing Salmonella on alfalfa seeds was
investigated.
METHODS AND RESULTS: Alfalfa seeds were inoculated with Salmonella Stanley to
achieve c. 5 log CFU g(-1). Seeds were then treated with 1 or 3% peroxyacetic
acid or 20 000 ppm calcium hypochlorite for 15 min in a commercial seed washer
that uses air to enhance contact of the sanitizer with the seed. Experiments were
also conducted using industry and laboratory methods. An c. 1-log reduction in
number of Salm. Stanley was demonstrated regardless of the chemical treatment or
method of treatment. Although this 1-log reduction was significant (P < 0.05),
differences among the treatments were not significant. Treating the seed with 1
and 3% peroxyacetic acid resulted in similar Salm. Stanley reductions of 1.77 and
1.34 log, respectively, not being statistically significant (P > 0.05).
CONCLUSIONS: These results suggest that under conditions tested, 1 or 3%
peroxyacetic acid solutions are equally effective as 20 000 ppm of Ca(OCl)2 in
the reduction of Salm. Stanley on alfalfa seed when used in conjunction with a
commercial seed washer.
SIGNIFICANCE AND IMPACT OF THE STUDY: A 1% peroxyacetic acid solution could
potentially be used in place of 20 000 ppm of Ca(OCl)2 for treatment of seeds
used for sprouting. The commercial seed washer did not enhance removal of Salm.
Stanley from alfalfa seeds, but did facilitate removal of excess soil from seeds.
© 2010 The Authors. Journal compilation © 2010 The Society for Applied
Microbiology.
DOI: 10.1111/j.1472-765X.2010.02929.x
1253. Environ Toxicol Chem. 2010 Dec;29(12):2859-68. doi: 10.1002/etc.356. Epub

2010
Oct 22.
Use of vegetated agricultural drainage ditches to decrease toxicity of irrigation

runoff from tomato and alfalfa fields in California, USA.
Werner I(1), Deanovic LA, Miller J, Denton DL, Crane D, Mekebri A, Moore MT,
Wrysinski J.
Author information:
(1)Aquatic Toxicology Laboratory, School of Veterinary Medicine, University of
California, Davis, California 95616, USA. iwerner@ucdavis.edu
The current study investigated the potential of vegetated drainage ditches for
mitigating the impact of agricultural irrigation runoff on downstream aquatic
ecosystems. Water column toxicity to larval fathead minnow (Pimephales
promelas),and the amphipod Hyalella azteca was measured for 12 h or less at the
ditch inflow and outflow, using custom-built in situ exposure systems. In
addition, water and sediment samples were subject to standard toxicity tests with
Ceriodaphnia dubia and H. azteca, respectively. No acute toxicity to larval
fathead minnow was observed; however, runoff was highly toxic to invertebrates.
Passage through a 389- to 402-m section of vegetated ditch had a mitigating
effect and reduced toxicity to some degree. However, runoff from an alfalfa field
treated with chlorpyrifos remained highly toxic to both invertebrate species, and
runoff from a tomato field treated with permethrin remained highly toxic to H.
azteca after passage through the ditch. Predicted toxic units calculated from
insecticide concentrations in runoff and 96-h median lethal concentration (LC50)
values generally agreed with C. dubia toxicity measured in the laboratory but
significantly underestimated in situ toxicity to H. azteca. Sediments collected
near the ditch outflow were toxic to H. azteca. Results from the current study
demonstrate that experimental vegetated ditches were unable to eliminate the risk
of irrigation runoff to aquatic ecosystems. In addition, protective measures
based on chemical concentrations or laboratory toxicity tests with C. dubia do
not ensure adequate protection of aquatic ecosystems from pyrethroid-associated
toxicity.
DOI: 10.1002/etc.356
1254. Curr Microbiol. 2011 Feb;62(2):597-605. doi: 10.1007/s00284-010-9749-4. Epub

2010
Sep 11.
Bacillus thuringiensis protein Cry6B (BGSC ID 4D8) is toxic to larvae of Hypera

postica.
Sharma A(1), Kumar S, Bhatnagar RK.
Author information:
(1)Insect Resistance Group, International Center for Genetic Engineering and
Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Insecticidal proteins produced by strains of Bacillus thuringenesis are specific

toward target pests. One of the Bt proteins, Cry 1Ac has been used successfully
for controlling crop predation by polyphagous pests Helicoverpa armigera.
Structurally, Bt proteins consist of three domains; domain I and III are fairly
homologous in various Bt proteins while domain II is hypervariable. The
hypervariable domain II is believed to be responsible for specificity toward
target pest. Successful deployment of Bt proteins requires knowledge of its
specificity toward the insect. Various Bt proteins have been characterized for
activity against coleopteran pests. Some Bt proteins of class Cry6 have been
found to be active against potato weevil. We have evaluated the activity of Cry6B
protein (BGSC-4D8) against lucerne weevil, Hypera postica, which is a major pest
of forage crop Medicago sativa. Results revealed that the purified Cry6B protein
is significantly active against the coleopteran pest with LC₅₀ value 280 ng/μl.
The leaves coated with the purified Cry6 toxin were three times less damaged as
compared with the negative control.
DOI: 10.1007/s00284-010-9749-4
1255. J Bacteriol. 2010 Nov;192(21):5725-35. doi: 10.1128/JB.00629-10. Epub 2010

Sep 3.
Characterization of a two-component regulatory system that regulates

succinate-mediated catabolite repression in Sinorhizobium meliloti.
Garcia PP(1), Bringhurst RM, Arango Pinedo C, Gage DJ.

Author information:
(1)University of Connecticut, Department of Molecular and Cell Biology, 91 N.
Eagleville Rd., U-3125, Storrs, CT 06269-3125, USA.
When they are available, Sinorhizobium meliloti utilizes C(4)-dicarboxylic acids

as preferred carbon sources for growth while suppressing the utilization of some
secondary carbon sources such as α- and β-galactosides. The phenomenon of using
succinate as the sole carbon source in the presence of secondary carbon sources
is termed succinate-mediated catabolite repression (SMCR). Genetic screening
identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown
in succinate plus lactose, maltose, or raffinose. sma0113 and the gene
immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine
kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking
a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of
catabolite repression compared to the wild type. sma0114 in-frame deletion
mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires
sma0113. Sma0113 may use its five PAS domains for redox level or energy state
monitoring and use that information to regulate catabolite repression and related
responses.
DOI: 10.1128/JB.00629-10
PMCID: PMC2953702
1256. Annu Rev Entomol. 2011;56:221-37. doi: 10.1146/annurev-ento-120709-144836.
The alfalfa leafcutting bee, Megachile rotundata: the world's most intensively
managed solitary bee.
Pitts-Singer TL(1), Cane JH.
Author information:
(1)USDA ARS Bee Biology & Systematics Laboratory, Utah State University, Logan,
Utah 84322, USA. Theresa.Pitts-Singer@ars.usda.gov
The alfalfa leafcutting bee (ALCB), Megachile rotundata F. (Megachildae), was

accidentally introduced into the United States by the 1940s. Nest management of
this Eurasian nonsocial pollinator transformed the alfalfa seed industry in North
America, tripling seed production. The most common ALCB management practice is
the loose cell system, in which cocooned bees are removed from nesting cavities
for cleaning and storage. Traits of ALCBs that favored their commercialization
include gregarious nesting; use of leaves for lining nests; ready acceptance of
affordable, mass-produced nesting materials; alfalfa pollination efficacy; and
emergence synchrony with alfalfa bloom. The ALCB became a commercial success
because much of its natural history was understood, targeted research was
pursued, and producer ingenuity was encouraged. The ALCB presents a model system
for commercializing other solitary bees and for advancing new testable hypotheses
in diverse biological disciplines.
DOI: 10.1146/annurev-ento-120709-144836
1257. Eur J Protistol. 2010 Nov;46(4):271-9. doi: 10.1016/j.ejop.2010.05.002.
Physiological characteristics of several rumen protozoa grown in vitro with

observations on within and among species variation.
Dehority BA(1).
Author information:
Center, The Ohio State University, Wooster, OH 44691-4096, USA.
dehority.1@osu.edu
When fed equal amounts of substrate, two Epidinium caudatum clone cultures of
markedly different size produced similar volumes of microbial protoplasm.
Addition of up to 50% volume of 72h culture medium had no inhibitory effects on
growth of Epidinium. Two clone cultures of Epidinium caudatum from Australia had
longer generation times and showed less substrate attachment when compared to
Ohio clones of this same species. Substitution of alfalfa for orchardgrass in the
normal substrate increased Epidinium concentrations, while feeding only ground
orchardgrass or alfalfa resulted in a marked decrease or disappearance of the
protozoa. Eudiplodinium impalae, isolated from rumen contents of a steer in
Australia, was successfully cultured, with generation times for this species
averaging 11.3h. Reducing particle size of the substrates by ball-milling was
detrimental for growth of Entodinium and Epidinium; however, Eudiplodinium
increased in concentration. Significant concentration differences were observed
among six clone cultures of Epidinium obtained from Europe. A generation time of
18.7h was measured for Enoploplastron triloricatum when the culture was
transferred every 12h. Lowering the incubation temperature to 34°C completely
inhibited protozoal growth of Epidinium and Entodinium exiguum after 12 days, but
not for Entodinium caudatum.
DOI: 10.1016/j.ejop.2010.05.002
1258. J Exp Bot. 2011 Jan;62(1):111-23. doi: 10.1093/jxb/erq249. Epub 2010 Aug 25.
Plant physiology and proteomics reveals the leaf response to drought in alfalfa
Aranjuelo I(1), Molero G, Erice G, Avice JC, Nogués S.
Author information:
(1)Unitat de Fisiologia Vegetal, Facultat de Biologia, Universitat de Barcelona,
Diagonal 645, E-08028 Barcelona, Spain. iker.aranjuelo@gmail.com
Despite its relevance, protein regulation, metabolic adjustment, and the

physiological status of plants under drought is not well understood in relation
to the role of nitrogen fixation in nodules. In this study, nodulated alfalfa
plants were exposed to drought conditions. The study determined the
physiological, metabolic, and proteomic processes involved in photosynthetic
inhibition in relation to the decrease in nitrogenase (N(ase)) activity. The
deleterious effect of drought on alfalfa performance was targeted towards
photosynthesis and N(ase) activity. At the leaf level, photosynthetic inhibition
was mainly caused by the inhibition of Rubisco. The proteomic profile and
physiological measurements revealed that the reduced carboxylation capacity of
droughted plants was related to limitations in Rubisco protein content,
activation state, and RuBP regeneration. Drought also decreased amino acid
content such as asparagine, and glutamic acid, and Rubisco protein content
indicating that N availability limitations were caused by N(ase) activity
inhibition. In this context, drought induced the decrease in Rubisco binding
protein content at the leaf level and proteases were up-regulated so as to
degrade Rubisco protein. This degradation enabled the reallocation of the
Rubisco-derived N to the synthesis of amino acids with osmoregulant capacity.
Rubisco degradation under drought conditions was induced so as to remobilize
Rubisco-derived N to compensate for the decrease in N associated with N(ase)
inhibition. Metabolic analyses showed that droughted plants increased amino acid
(proline, a major compound involved in osmotic regulation) and soluble sugar
(D-pinitol) levels to contribute towards the decrease in osmotic potential
(Ψ(s)). At the nodule level, drought had an inhibitory effect on N(ase) activity.
This decrease in N(ase) activity was not induced by substrate shortage, as
reflected by an increase in total soluble sugars (TSS) in the nodules. Proline
accumulation in the nodule could also be associated with an osmoregulatory
response to drought and might function as a protective agent against ROS. In
droughted nodules, the decrease in N(2) fixation was caused by an increase in
oxygen resistance that was induced in the nodule. This was a mechanism to avoid
oxidative damage associated with reduced respiration activity and the consequent
increase in oxygen content. This study highlighted that even though drought had a
direct effect on leaves, the deleterious effects of drought on nodules also
conditioned leaf responsiveness.
DOI: 10.1093/jxb/erq249
PMCID: PMC2993905
1259. J Plant Physiol. 2010 Dec 15;167(18):1558-65. doi:

10.1016/j.jplph.2010.06.015.
Epub 2010 Aug 13.
Photosynthetic down-regulation under elevated CO₂ exposure can be prevented by

nitrogen supply in nodulated alfalfa.
Sanz-Sáez A(1), Erice G, Aranjuelo I, Nogués S, Irigoyen JJ, Sánchez-Díaz M.
Author information:
(1)Departamento de Biología Vegetal, Sección Biología Vegetal (Unidad Asociada al
CSIC, EEAD, Zaragoza, ICVV, Logroño), Facultades de Ciencias y Farmacia,
Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Navarra, Spain.
Increasing atmospheric CO₂ concentrations are expected to enhance plant

photosynthesis and yield. Nevertheless, after long-term exposure, plants
acclimate and show a reduction in photosynthetic activity (called
down-regulation), which may cause a reduction in potential yield. Some authors
suggest that down-regulation is related to nutrient availability, and more
specifically, to an insufficient plant C sink strength caused by limited N
supply. In this paper, we tested whether or not N availability prevents
down-regulation of photosynthesis in nodulated alfalfa plants (Medicago sativa
L.). To do so, we examined the effect of the addition of different levels of
NH₄NO₃ (0, 10, and 15 mM) to 30-day-old nodulated alfalfa plants exposed to
ambient (approximately 400 μmol mol⁻¹) or elevated CO₂ (700 μmol mol⁻¹) during a
period of 1 month in growth chambers. After 2 weeks of exposure to elevated CO₂,
no significant differences were observed in plant growth or photosynthesis rates.
After 4 weeks of treatment, exclusively N₂ fixing alfalfa plants (0 mM NH₄NO₃)
showed significant decreases in photosynthesis and Vc(max). Photosynthetic
down-regulation of these plants was caused by the C/N imbalance as reflected by
the carbohydrate and N data. On the other hand, plants supplied with 15 mM NH₄NO₃
grown under elevated CO₂ maintained high photosynthetic rates owing to their
superior C/N adjustment. The intermediate N treatment, 10 mM NH₄NO₃, also showed
photosynthetic down-regulation, but to a lesser degree than with 0 mM treatment.
The present study clearly shows that external N supply can reduce or even avoid
acclimation of photosynthesis to elevated CO₂ as a consequence of the increase in
C sink strength associated with N availability.
DOI: 10.1016/j.jplph.2010.06.015
1260. Planta. 2010 Oct;232(5):1151-62. doi: 10.1007/s00425-010-1247-1. Epub 2010

Aug
13.
The 3' untranslated region of the two cytosolic glutamine synthetase (GS(1))
genes in alfalfa (Medicago sativa) regulates transcript stability in response to
glutamine.
Simon B(1), Sengupta-Gopalan C.
Author information:
(1)New Mexico State University, Las Cruces, NM 88003, USA.
Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of ammonia

with glutamate to produce glutamine. The GS enzyme is located either in the
chloroplast (GS(2)) or in the cytoplasm (GS(1)). GS(1) is encoded by a small gene
family and the members exhibit differential expression pattern mostly attributed
to transcriptional regulation. Based on our recent finding that a soybean GS(1)
gene, Gmglnβ ( 1 ) is subject to its 3'UTR-mediated post-transcriptional
regulation as a transgene in alfalfa (Medicago sativa) we have raised the
question of whether the 3'UTR-mediated transcript destabilization is a more
universal phenomenon. Gene constructs consisting of the CaMV35S promoter driving
the reporter gene, GUS, followed by the 3'UTRs of the two alfalfa GS(1) genes,
MsGSa and MsGSb, were introduced into alfalfa and tobacco. The analysis of these
transformants suggests that while both the 3'UTRs promote transcript turnover,
the MsGSb 3'UTR is more effective than the MsGSa 3'UTR. However, both the 3'UTRs
along with Gmglnβ ( 1 ) 3'UTR respond to nitrate as a trigger in transcript
turnover. More detailed analysis points to glutamine rather than nitrate as the
mediator of transcript turnover. Our data suggests that the 3'UTR-mediated
regulation of GS(1) genes at the level of transcript turnover is probably
universal and is used for fine-tuning the expression in keeping with the
availability of the substrates.
DOI: 10.1007/s00425-010-1247-1
1261. Plant Cell Rep. 2010 Nov;29(11):1235-50. doi: 10.1007/s00299-010-0909-6. Epub

2010 Aug 12.
A comparison of two class 10 pathogenesis-related genes from alfalfa and their

activation by multiple stresses and stress-related signaling molecules.
Bahramnejad B(1), Goodwin PH, Zhang J, Atnaseo C, Erickson LR.
Author information:
(1)Department of Plant Breeding, Faculty of Agriculture, University of Kurdistan,
Sanandaj, Iran.
A collection of 29 pathogenesis-related 10 (PR10) genes of Medicago sativa and
Medicago truncatula showed that they were almost all obtained from cDNA libraries
of tissues undergoing abiotic or biotic stresses. The predicted proteins could be
divided into two subclasses, PR10.1 and PR10.2, but in silico predicted models of
their three-dimensional structures revealed that they could be further divided
based on size of the hydrophobic internal cavity and number of β-bulges. A
comparison of the expression of two highly similar M. sativa subclass PR10.1
genes, MsPR10.1A and MsPR10.1B, predicted to have similar sized hydrophobic
internal cavities, but a different number of β-bulges revealed differences in
their expression patterns. MsPR10.1A was induced faster than MsPR10.1B by ABA,
ethylene, and X. campestris pv. alfalfae, but slower than MsPR10.1B by harvesting
and wounding. Unlike MsPR10.1A, MsPR10.1B expression was induced in non-harvested
tissues following harvesting, but was not induced by heat treatment.
Histochemical observations of Nicotiana benthamiana transformed with 657 bp of
the MsPR10.1A promoter fused to the β-glucuronidase (GUS) gene showed that GUS
expression was wound-inducible in leaves, which was consistent with MsPR10.1A
expression in alfalfa leaves. GUS expression in stems and leaves was mostly in
vascular tissue. The MsPR10.1A promoter may be valuable in controlling the
expression in vascular tissues and disease resistance.
DOI: 10.1007/s00299-010-0909-6

10.1094/MPMI-07-10-0151.
Sinorhizobium meliloti requires a cobalamin-dependent ribonucleotide reductase

for symbiosis with its plant host.
Taga ME(1), Walker GC.
Author information:
(1)Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, MA 02139, USA.
Vitamin B(12) (cobalamin) is a critical cofactor for animals and protists, yet
its biosynthesis is limited to prokaryotes. We previously showed that the
symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires
cobalamin to establish a symbiotic relationship with its plant host, Medicago
sativa (alfalfa). Here, the specific requirement for cobalamin in the S.
meliloti-alfalfa symbiosis was investigated. Of the three known
cobalamin-dependent enzymes in S. meliloti, the methylmalonyl CoA mutase (BhbA)
does not affect symbiosis, whereas disruption of the metH gene encoding the
cobalamin-dependent methionine synthase causes a significant defect in symbiosis.
Expression of the cobalamin-independent methionine synthase MetE alleviates this
symbiotic defect, indicating that the requirement for methionine synthesis does
not reflect a need for the cobalamin-dependent enzyme. To investigate the
function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by
nrdJ, S. meliloti was engineered to express an Escherichia coli
cobalamin-independent (class Ia) RNR instead of nrdJ. This strain is severely
defective in symbiosis. Electron micrographs show that these cells can penetrate
alfalfa nodules but are unable to differentiate into nitrogen-fixing bacteroids
and, instead, are lysed in the plant cytoplasm. Flow cytometry analysis indicates
that these bacteria are largely unable to undergo endoreduplication. These
phenotypes may be due either to the inactivation of the class Ia RNR by reactive
oxygen species, inadequate oxygen availability in the nodule, or both. These
results show that the critical role of the cobalamin-dependent RNR for survival
of S. meliloti in its plant host can account for the considerable resources that
S. meliloti dedicates to cobalamin biosynthesis.
DOI: 10.1094/MPMI-07-10-0151
PMCID: PMC2979309
1263. Genome Biol. 2010;11(8):R81. doi: 10.1186/gb-2010-11-8-r81. Epub 2010 Aug 9.
Multiple distinct small RNAs originate from the same microRNA precursors.
Zhang W(1), Gao S, Zhou X, Xia J, Chellappan P, Zhou X, Zhang X, Jin H.
Author information:
(1)Department of Computer Science and Engineering, Washington University in Saint
Louis, Campus Box 1045, Saint Louis, MO 63130, USA. weixiong.zhang@wustl.edu
BACKGROUND: MicroRNAs (miRNAs), which originate from precursor transcripts with

stem-loop structures, are essential gene expression regulators in eukaryotes.
RESULTS: We report 19 miRNA precursors in Arabidopsis that can yield multiple
distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA
precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes
with an approximately two-nucleotide 3'-end overhang. Their production depends on
the same biogenesis pathway as their sibling miRNAs and does not require
RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are
methylated, and many of them are associated with Argonaute proteins. Some of the
miRNA-like RNAs are differentially expressed in response to bacterial challenges,
and some are more abundant than the cognate miRNAs. Computational and expression
analyses demonstrate that some of these miRNA-like RNAs are potentially
functional and they target protein-coding genes for silencing. The function of
some of these miRNA-like RNAs was further supported by their target cleavage
products from the published small RNA degradome data. Our systematic examination
of public small-RNA deep sequencing data from four additional plant species
(Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus
trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans
and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.
CONCLUSIONS: We demonstrate that multiple miRNAs could derive from miRNA
precursors by sequential processing of Dicer or Dicer-like proteins. Our results
suggest that the pool of miRNAs is larger than was previously recognized, and
miRNA-mediated gene regulation may be broader and more complex than previously
thought.
DOI: 10.1186/gb-2010-11-8-r81
PMCID: PMC2945783
1264. Talanta. 2010 Jun 30;82(1):164-70. doi: 10.1016/j.talanta.2010.04.015. Epub

2010
May 4.
Determination of thiodicarb using a biosensor based on alfalfa sprout peroxidase

immobilized in self-assembled monolayers.
Moccelini SK(1), Vieira IC, de Lima F, Lucca BG, Barbosa AM, Ferreira VS.
Author information:
(1)Departamento de Química, Laboratório de Biossensores, Universidade Federal de
Santa Catarina, 88040-970 Florianópolis, SC, Brazil. sallykm@gmail.com
A biosensor based on alfalfa sprout (Medicago sativa) homogenate as a source of
peroxidase is proposed for the determination of thiodicarb by square-wave
voltammetry. This enzyme was immobilized in self-assembled monolayers of
l-cysteine on a gold electrode. Several parameters were investigated to evaluate
the optimum conditions for operation of the biosensor. The analytical curve was
linear for thiodicarb concentrations of 2.27 x 10(-6) to 4.40 x 10(-5) mol L(-1)
with a detection limit of 5.75 x 10(-7) mol L(-1). The lifetime of the Au-alfalfa
sprout-SAMs was 20 days (at least 220 determinations). The average recovery of
thiodicarb from samples of vegetable extracts ranged from 99.02 to 101.04%. The
results obtained for thiodicarb in vegetable extracts using the proposed method
are in close agreement with those using a high performance liquid chromatography
procedure at the 95% confidence level.
Copyright 2010 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.talanta.2010.04.015
1265. Anim Reprod Sci. 2010 Sep;121(3-4):242-8. doi:

10.1016/j.anireprosci.2010.06.007.
Epub 2010 Aug 3.
Short-term grazing of lucerne and chicory increases ovulation rate in

synchronised Merino ewes.
King BJ(1), Robertson SM, Wilkins JF, Friend MA.
Author information:
(1)EH Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga
Wagga, NSW, Australia. bking@csu.edu.au
This study evaluated the ability of short-term grazing of live=green pasture to

increase ovulation rate during late summer when annual pasture is generally dead
and of low quality. Ovulation rates, measured by the number of corpora lutea,
were compared between 4 nutritional treatments: senesced phalaris (Phalaris
aquatica), phalaris plus 500g lupin grain per day, lucerne (Medicago sativa) or
chicory (Chicorum intybus) pastures. The study used 100 Merino ewes per
treatment, divided between 2 replicates. The experiment was repeated in 3 years;
February 2006, and January 2007 and 2008. Oestrus was synchronised and the ewes
grazed the pastures for 9 days prior to ovulation at times corresponding to days
8-17 of the cycle in 2006, and days 6-14 in 2007 and 2008. The proportion of ewes
producing multiple ovulations was higher (P<0.05) in the lucerne and chicory
(0.36, 0.38) than the phalaris (0.27), and intermediate in the lupin (0.33)
treatment. Regression analysis showed that the proportion of ewes with multiple
ovulations increased with the quantity of live herbage (P<0.04). Responses were
achieved even at low levels of live herbage with 90% of the maximum proportion of
multiples occurring at 350kg DM/ha. It is concluded that providing short-term
grazing of live chicory or lucerne to ewes can increase ovulation rates relative
to ewes grazing senesced phalaris and to levels similar to those achieved by
lupin grain supplementation.
2010. Published by Elsevier B.V. All rights reserved.
1266. Chemosphere. 2010 Oct;81(6):755-66. doi: 10.1016/j.chemosphere.2010.07.012.
Epub
2010 Aug 3.
Comparison of biological and chemical measures of metal bioavailability in field

soils: test of a novel simulated earthworm gut extraction.
Smith BA(1), Greenberg B, Stephenson GL.
Author information:
(1)Department of Biology, University of Waterloo, 200 University Avenue West,
Waterloo, Ontario, Canada. ben.smith@phac-aspc.gc.ca
Bioavailability of metals in soil is a major factor influencing estimates of risk

associated with exposure of ecological receptors. Metal concentrations in soil
are often compared to ecological screening benchmarks, which are based on total
concentrations in soil. Often, the total concentration is not correlated with
toxicity. No standardised method exists for determining the bioavailability of
metals in soil to ecological receptors. Several surrogate measures of
bioavailability were compared to the results of a battery of toxicity tests using
copper (Cu), lead (Pb), and zinc (Zn)-contaminated soils collected from a former
industrial area. A calcium chloride (CaCl(2)) extraction, cyclodextrin (HPCD)
extraction, simulated earthworm gut (SEG) test, and earthworm bioaccumulation
test were performed using the soils. Extractable metals using the CaCl(2)
solution were not correlated with any biological responses of earthworms (Eisenia
andrei), collembola (Folsomia candida), northern wheatgrass (Elymus lanceolatus),
or alfalfa (Medicago sativa L.). Concentrations of metals in the HPCD extracts
were highly variable and were not adequate for revealing adverse effects. E.
andrei tissue concentrations were variable but were predictive of adverse effects
to invertebrates. The results of the SEG test correlated with most of the
biological endpoints. Bioavailable Cu was correlated with adverse effects to
invertebrates and plants using the SEG test. Overall, coefficients of
determination associated with the relationships between the biological responses
and each measure of bioavailability indicated that those for the SEG test were
greater than those for the other surrogate measures of bioavailability. Further
validation is required before this test is routinely used to estimate metal
bioavailability and toxicity.
1267. J Bacteriol. 2010 Oct;192(19):4944-53. doi: 10.1128/JB.00294-10. Epub 2010

Jul
30.
Pyruvate is synthesized by two pathways in pea bacteroids with different

efficiencies for nitrogen fixation.
Mulley G(1), Lopez-Gomez M, Zhang Y, Terpolilli J, Prell J, Finan T, Poole P.
Author information:
(1)Department of Molecular Microbiology, John Innes Centre, Norwich Research
Park, Colney Lane, Norwich NR4 7UH, United Kingdom.
Nitrogen fixation in legume bacteroids is energized by the metabolism of

dicarboxylic acids, which requires their oxidation to both oxaloacetate and
pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD+ malic
enzyme (Dme) but not NADP+ malic enzyme (Tme). However, we show that Rhizobium
leguminosarum has two pathways for pyruvate formation from dicarboxylates
catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP)
carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N2
fixation, but the PckA/PykA pathway supports N2 fixation at only 60% of that for
Dme. Double mutants of dme and pckA/pykA did not fix N2. Furthermore, dme pykA
double mutants did not grow on dicarboxylates, showing that they are the only
pathways for the production of pyruvate from dicarboxylates normally expressed.
PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement
for Dme for pyruvate formation and N2 fixation. When PckA was expressed from a
constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at
30% of the wild-type rate, although this level was insufficient to prevent
nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal
phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak
acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type
rate, and this was accompanied by increased nodule mass. Plants infected with Pta
deletion mutants did not have increased dry weight, demonstrating that there is
not a sustained change in nitrogen fixation throughout growth. This indicates a
complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation,
and plant growth.
DOI: 10.1128/JB.00294-10
PMCID: PMC2944551
1268. BMC Plant Biol. 2010 Jul 29;10:156. doi: 10.1186/1471-2229-10-156.
Structure-function relationships of wheat flavone O-methyltransferase: Homology

modeling and site-directed mutagenesis.
Zhou JM(1), Lee E, Kanapathy-Sinnaiaha F, Park Y, Kornblatt JA, Lim Y, Ibrahim

RK.
Author information:
(1)Plant Biochemistry Laboratory and Centre for Structural-Functional Genomics,
Concordia University, Montreal, QC, H4B 1R6, Canada.
BACKGROUND: Wheat (Triticum aestivum L.) O-methyltransferase (TaOMT2) catalyzes

the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin),
3',5'-dimethyl- (tricin) and 3',4',5'-trimethyl ether derivatives. Tricin, a
potential multifunctional nutraceutical, is the major enzyme reaction product.
These successive methylations raised the question as to whether they take place
in one, or different active sites. We constructed a 3-D model of this protein
using the crystal structure of the highly homologous Medicago sativa caffeic
acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT) as a template with the
aim of proposing a mechanism for multiple methyl transfer reactions in wheat.
RESULTS: This model revealed unique structural features of TaOMT2 which permit
the stepwise methylation of tricetin. Substrate binding is mediated by an
extensive network of H-bonds and van der Waals interactions. Mutational analysis
of structurally guided active site residues identified those involved in binding
and catalysis. The partly buried tricetin active site, as well as proximity and
orientation effects ensured sequential methylation of the substrate within the
same pocket. Stepwise methylation of tricetin involves deprotonation of its
hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of
SAM-methyl groups. We also demonstrate that Val309, which is conserved in a
number of graminaceous flavone OMTs, defines the preference of TaOMT2 for
tricetin as the substrate.
CONCLUSIONS: We propose a mechanism for the sequential methylation of tricetin,
and discuss the potential application of TaOMT2 to increase the production of
tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309,
determines its preference for tricetin as the substrate, and may define the
evolutionary differences between the two closely related proteins, COMT and
flavone OMT.
DOI: 10.1186/1471-2229-10-156
PMCID: PMC3017781
1269. Bioorg Med Chem Lett. 2010 Sep 1;20(17):5099-103. doi:

10.1016/j.bmcl.2010.07.022. Epub 2010 Jul 11.
Structure-based engineering of benzalacetone synthase.
Shimokawa Y(1), Morita H, Abe I.
Author information:
(1)Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo,
Japan.
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific type
III polyketide synthases (PKSs), sharing 70% amino acid sequence identity and
highly homologous overall protein structures. BAS catalyzes the decarboxylative
coupling of 4-coumaroyl-CoA with malonyl-CoA to produce the diketide
benzalacetone, whereas CHS produces the tetraketide chalcone by iterative
condensations with three molecules of malonyl-CoA, and folding the resulting
intermediate into a new aromatic ring system. Recent crystallographic analyses of
Rheum palmatum BAS revealed that the characteristic substitution of Thr132
(numbering of Medicago sativa CHS2), a conserved CHS residue lining the
active-site cavity, with Leu causes steric contraction of the BAS active-site to
produce the diketide, instead of the tetraketide. To test this hypothesis, we
constructed a set of R. palmatum BAS site-directed mutants (L132G, L132A, L132S,
L132C, L132T, L132F, L132Y, L132W and L132P), and investigated the mechanistic
consequences of the point mutations. As a result, the single amino acid
substitution L132T restored the chalcone-forming activity in BAS, whereas the
Ala, Ser, and Cys substitutions expanded the product chain length to produce
4-coumaroyltriacetic acid lactone (CTAL) after three condensations with
malonyl-CoA, but without the formation of the aromatic ring system. Homology
modeling suggested that this is probably caused by the restoration of the
'coumaroyl binding pocket' in the active-site cavity. These findings provide
further insights into the structural details of the catalytic mechanism of the
type III PKS enzymes.
Copyright 2010 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.bmcl.2010.07.022
1270. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Apr;28(2):94-
7.
[Changes of cytokine secretion in mice by immunization with leaf protein

extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa].
Ye YJ(1), Li WG.
Author information:
(1)Institute of Infectious and Parasitic Diseases, the First Affiliated Hospital,
Chongqing Medical University, Chongqing 400016, China.
OBJECTIVE: To investigate the weight reduction of hydatid cyst and the changes of
cytokine secretion in mice immunized by leaf protein extracted from Echinococcus
granulosus (Eg) Eg95-EgA31 transgenic alfalfa and challenged by Eg protoscoleces.
METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic
alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from
transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32
female BALB/c mice were randomly divided into 4 groups. Groups A and B were
immunized intragastrically (100 p1) and intranasally (10 microl) respectively by
leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated
intranasally by 10 microl. leaf protein with pBI121, group D was given
intragastrically 100 microl normal leaf protein. All mice were immunized once per
3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces
on the 8th week after vaccination and sacrificed on the 24th week after
infection. The weight of hydatid cysts was measured and weight-reduction rate was
calculated. Spleens were collected to prepare splenocytes which were cultured
under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS).
The supernatant was collected to measure Objective To investigate the weight
reduction of hydatid cyst and the changes of cytokine secretion in mice immunized
by leaf protein extracted from Echinococcus granulosus (Eg) Eg95-EgA31 transgenic
alfalfa and challenged by Eg protoscoleces.
METHODS: Leaf protein was extracted from E. granulosus Eg95-EgA31 transgenic
alfalfa by heat coagulation method. Meanwhile, leaf protein extracted from
transgenic alfalfa containing pBI121 and normal alfalfa served as control. 32
female BALB/c mice were randomly divided into 4 groups. Groups A and B were
immunized intragastrically (100 microl) and intranasally (10 microl) respectively
by leaf protein containing Eg95-EgA31 fusion antigen, group C was vaccinated
intranasally by 10 microl. leaf protein with pBI121, group D was given
intragastrically 100 microl normal leaf protein. All mice were immunized once per
3 days for 2 months. Mice in all groups were challenged with 50 Eg protoscoleces
on the 8th week after vaccination and sacrificed on the 24th week after
infection. The weight of hydatid cysts was measured and weight-reduction rate was
calculated. Spleens were collected to prepare splenocytes which were cultured
under stimulation with EgAg, concanavalin A (ConA) or lipopolysaccharide (LPS).
The supernatant was collected to measure the level of IL-12, IL-10, IFN-gamma and
TNF-alpha by ELISA.
RESULTS: The average weight of hydatid cysts in groups A, B, C, and D was (28.0
+/- 36.0), (41.0 +/- 33.0), (72.0 +/- 36.0) and (78.0 +/- 57.0) mg, respectively,
the cyst weight of group A was lower than that of group D (P < 0.05), decreased
by 64.1%. The levels of IFN-gamma, IL-12 and TNF-alpha in group A [(925.0 +/-
88.6), (22.5 +/- 2.7) and (82.5 +/- 11.7) pg/ml] were higher than those of group
D (P < 0.01), while the IL-10 level in group A [(125.0 +/- 26.7) pg/ml] was
significantly lower than that of group D (P < 0.01). The level of IFN-gamma
[(750.0 +/- 100.0) pg/ml] and TNF-alpha [(80.0 +/- 13.1) pg/ml] in group B was
significantly higher than those of group D (P < 0.01); but there was no
significant difference in the level of IL-12 and IL-10 between the two groups (P
> 0.05). No considerable difference in the cytokines was found between group C
and group D (P > 0.05). The levels of the 4 cytokines in groups stimulated by
EgAg, ConA or LPS were higher than those without stimulation (P < 0.05 or <
0.01), and those in groups stimulated by ConA or LPS were higher than groups
stimulated by EgAg (P < 0.05 or < 0.01).
CONCLUSION: Th1 response may be induced in mice by immunization with the leaf
protein extracted from Echinococcus granulosus Eg95-EgA31 transgenic alfalfa to
resist the challenge of Eg protoscoleces.
1271. Biol Trace Elem Res. 2011 Sep;142(3):787-95. doi: 10.1007/s12011-010-8777-z.

Epub
2010 Jul 23.
Vanadium uptake by alfalfa grown in V-Cd-contaminated soil by pot experiment.
Yang J(1), Teng Y, Wang J, Li J.
Author information:
(1)College of Water Sciences, Beijing Normal University, Beijing 100875, China.
In order to characterize uptake of vanadium in alfalfa grown in vanadium-cadmium

(Cd)-contaminated soil, 104 soil samples and 94 plant samples were collected from
pot experiment. The results showed alfalfa had strong metal adaptability (up to
400 mg kg(-1)) and high accumulation (up to 3,440.14 mg kg(-1)) of vanadium. Root
had higher contents and better absorption to vanadium than overground part.
Moreover, both root and overground part had direct correlation with vanadium in
soil, especially with the sum of first three fractions and reducible fraction.
With the increasing of vanadium, higher concentration of Cd may inhibit the
absorption of vanadium in alfalfa.
DOI: 10.1007/s12011-010-8777-z

Jul
21.
Arbuscular mycorrhizal mediation of biomass-density relationship of Medicago

sativa L. under two water conditions in a field experiment.
Zhang Q(1), Xu L, Tang J, Bai M, Chen X.
Author information:
(1)Agroecology Institute, College of Life Sciences, Zhejiang University,
Zijingang campus, Hangzhou, China.
The biomass-density relationship (whereby the biomass of individual plants

decreases as plant density increases) has generally been explained by competition
for resources. Arbuscular mycorrhizal fungi (AMF) are able to affect plant
interactions by mediating resource utilization, but whether this AMF-mediated
interaction will change the biomass-density relationship is unclear. We conducted
an experiment to test the hypothesis that AMF will shift the biomass-density
relationship by affecting intraspecific competition. Four population densities
(10, 100, 1,000, or 10,000 seedlings per square meter) of Medicago sativa L. were
planted in field plots. Water application (1,435 or 327.7 mm/year) simulated
precipitation in wet areas (sufficient water) and arid areas (insufficient
water). The fungicide benomyl was applied to suppress AMF in some plots
("low-AMF" treatment) and not in others ("high-AMF" treatment). The effect of the
AMF treatment on the biomass-density relationship depended on water conditions.
High AMF enhanced the decrease of individual biomass with increasing density (the
biomass-density line had a steeper slope) when water was sufficient but not when
water was insufficient. AMF treatment did not affect plant survival rate or
population size but did affect absolute competition intensity (ACI). When water
was sufficient, ACI was significantly higher in the high-AMF treatment than in
the low-AMF treatment, but ACI was unaffected by AMF treatment when water was
insufficient. Our results suggest that AMF status did not impact survival rate
and population size but did shift the biomass-density relationship via effects on
intraspecific competition. This effect of AMF on the biomass-density relationship
depended on the availability of water.
DOI: 10.1007/s00572-010-0331-5
1273. Transgenic Res. 2011 Apr;20(2):397-407. doi: 10.1007/s11248-010-9425-2. Epub

2010
Jul 16.
Occurrence of alfalfa (Medicago sativa L.) populations along roadsides in

southern Manitoba, Canada and their potential role in intraspecific gene flow.
Bagavathiannan MV(1), Gulden RH, Van Acker RC.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2,
Canada. umbagava@cc.umanitoba.ca
Alfalfa is a highly outcrossing perennial species that can be noticed in

roadsides as feral populations. There remains little information available on the
extent of feral alfalfa populations in western Canadian prairies and their role
in gene flow. The main objectives of this study were (a) to document the
occurrence of feral alfalfa populations, and (b) to estimate the levels of
outcrossing facilitated by feral populations. A roadside survey confirmed
widespread occurrence of feral alfalfa populations, particularly in alfalfa
growing regions. The feral populations were dynamic and their frequency ranged
from 0.2 to 1.7 populations km(-1). In many cases, the nearest feral alfalfa
population from alfalfa production field was located within a distance sufficient
for outcrossing in alfalfa. The gene flow study confirmed that genes can move
back and forth between feral and cultivated alfalfa populations. In this study,
the estimated outcrossing levels were 62% (seed fields to feral), 78% (feral to
seed fields), 82% (hay fields to feral) and 85% (feral to feral). Overall, the
results show that feral alfalfa plants are prevalent in alfalfa producing regions
in western Canada and they can serve as bridges for gene flow at landscape level.
Management of feral populations should be considered, if gene flow is a concern.
Emphasis on preventing seed spill/escapes and intentional roadside planting of
alfalfa cultivars will be particularly helpful. Further, realistic and pragmatic
threshold levels should be established for markets sensitive to the presence of
GE traits.
DOI: 10.1007/s11248-010-9425-2
Effects of spontaneous heating on estimates of total digestible nutrients for

alfalfa-orchardgrass hays packaged in large round bales.
Coblentz WK(1), Hoffman PC.

Author information:
Large round or large square hay packages are more likely to heat spontaneously
during storage than hay packaged in conventional (45 kg) bales, and the effects
of this phenomenon on the associated energy estimates for these hays can be
severe. Our objectives for this project were to assess the relationship between
estimates of total digestible nutrients (TDN) and spontaneous heating and to
describe any important differences in energy estimates that may result
specifically from 2 methods of estimating truly digestible fiber (TD-Fiber).
Using the summative approach to estimate TDN, TD-Fiber can be estimated from
inputs of protein-corrected neutral detergent fiber (NDFn) and acid detergent
lignin (TD-FiberLIG) or from NDFn and 48-h neutral detergent fiber digestibility
(TD-FiberNDFD). Throughout 2006 and 2007, mixed alfalfa (Medicago sativa
L.)-orchardgrass (Dactylis glomerata L.) hays from 3 individual harvests were
obtained from the same 8.2-ha research site near Stratford, Wisconsin. Both
options for estimating TD-Fiber (TD-FiberLIG or TD-FiberNDFD) were then used
independently via the summative approach to estimate the total TDN concentrations
(TDN-LIG or TDN-NDFD, respectively) within these hays. Estimates of both TDN-LIG
and TDN-NDFD then were related to heating degree days >30 degrees C accumulated
during storage by various regression techniques. Changes (poststorage -
prestorage) in TDN-LIG that occurred during storage (DeltaTDN-LIG) were best
fitted with a nonlinear decay model in which the independent variable was squared
[Y=(11.7 x e(-0.0000033xxxx)) - 11.6; R(2)=0.928]. For changes in TDN-NDFD
(DeltaTDN-NDFD), a quadratic regression model provided the best fit
(Y=0.0000027x(2) - 0.010x+0.4; R(2)=0.861). Generally, DeltaTDN-LIG estimates
were 2.0 to 4.0 percentage units lower than DeltaTDN-NDFD estimates when heating
exceeded 500 HDD. For regressions on maximum internal bale temperature, both
DeltaTDN-LIG (Y=-0.38x+16.3; R(2)=0.954) and DeltaTDN-NDFD (Y=-0.25x+10.2;
R(2)=0.848) were best fitted by linear models with heterogeneous (P<0.001) slopes
and intercepts. In both cases, coefficients of determination were high,
suggesting that simple measures of spontaneous heating are excellent predictors
of energy losses in heated forages. Regardless of method, reductions in TDN were
associated primarily with losses of nonfiber carbohydrate, which is known to
occur via oxidation of sugars during spontaneous heating. For heated forages,
some discrepancy between TDN-LIG and TDN-NDFD existed because the relationship
between NDFD and spontaneous heating was shown previously to be very poor,
resulting in minimal changes for estimates of TD-FiberNDFD as a consequence of
heating. In contrast, TD-FiberLIG declined in close association with heating,
largely because TD-FiberLIG was sensitive to changes in concentrations of both
NDFn and acid detergent lignin. Discrepancies between TDN-LIG and TDN-NDFD were
exacerbated further when neutral detergent fiber rather than NDFn was used to
estimate TD-FiberNDFD. Estimates of TDN declined by as much as 13.0 percentage
units within severely heated hays, and this is a serious consequence of
spontaneous heating.
Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc.
DOI: 10.3168/jds.2010-3133
Effects of feeding alfalfa hay on chewing, rumen pH, and milk fat concentration
of dairy cows fed wheat dried distillers grains with solubles as a partial
substitute for barley silage.
Zhang SZ(1), Penner GB, Abdelqader M, Oba M.
Author information:
(1)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, T6G 2P5, Canada.
The objective of this study was to determine the effects of feeding alfalfa hay
on chewing activity, rumen fermentation, and milk fat concentration of dairy cows
fed wheat-based dried distillers grains with solubles (DDGS) as a partial
replacement of barley silage. Thirty lactating Holstein cows (220+/-51 DIM), 6 of
which were ruminally cannulated, were used in a 3 x 3 Latin square design with
21-d periods. Cows were fed a control diet [CON; 50% barley silage and 50%
concentrate mix on a dry matter (DM) basis], a diet in which barley silage was
replaced with DDGS at 20% of dietary DM (DG), or a diet in which barley silage
was replaced with DDGS and alfalfa hay at 20 and 10% of dietary DM, respectively
(DG+AH). All diets contained approximately 20% crude protein. Compared with the
CON diet, cows fed DG and DG+AH diets respectively had greater DM intake (20.1
vs. 23.1 and 22.7 kg/d); yields of milk (24.5 vs. 27.3 and 28.1 kg/d), milk
protein (0.88 vs. 0.99 and 1.01 kg/d), and milk lactose (1.11 vs. 1.24 and 1.29
kg/d); and body weight gain (0.25 vs. 1.17 and 1.23 kg/d). However, compared with
cows fed the CON diet, cows fed the DG and DG+AH diets respectively had lower
chewing time (38.3 vs. 30.7 and 31.5 min/kg of DM intake), mean rumen pH (6.11
vs. 5.88 and 5.84), and minimum rumen pH (5.28 vs. 5.09 and 5.07) and a greater
duration that rumen pH was below 5.8 (7.3 vs. 11.2 and 12.0 h/d). However, these
response variables did not differ between cows fed the DG and DG+AH diets. Milk
fat concentration differed among the 3 diets (3.92, 3.60, and 3.38% for CON, DG,
and DG+AH, respectively), but milk fat yield was not affected by treatment. These
results indicate that partially replacing barley silage with DDGS can improve
productivity of lactating dairy cows but may decrease chewing time, rumen pH, and
milk fat concentration, and that dietary inclusion of alfalfa hay may not
alleviate such responses.
DOI: 10.3168/jds.2009-3011
Utilization of nitrogen in cows consuming wet distillers grains with solubles in

alfalfa and corn silage-based dairy rations.
Gehman AM(1), Kononoff PJ.
Author information:
(1)Department of Animal Science, University of Nebraska, Lincoln, USA.
The objectives of this experiment were to determine the effects of forage type on
nutrient digestibility, purine derivative excretion, nitrogen utilization, and
milk production in dairy cattle consuming rations containing high levels of wet
distillers grains with solubles (WDGS). Primiparous (n=8) and multiparous (n=20)
Holstein cows were used in a replicated 4 x 4 Latin square. Animals were fed 1 of
4 treatments during each 21-d period: 1) CONT-CS, 0% WDGS and high corn silage;
2) CONT-AS, 0% WDGS and high alfalfa silage; 3) WDGS-CS, 25% WDGS and high corn
silage; and 4) WDGS-AS, 25% WDGS and high alfalfa silage (dry matter basis).
Intake and milk data were collected daily and averaged for d 15 to 21 of each
period. Dry matter intake was lower for CONT-CS than for CONT-AS, WDGS-CS, and
WDGS-AS (22.5, 24.6, 24.6, and 24.8 kg/d, respectively). Digestibility of dry
matter, organic matter, neutral detergent fiber, and N were not affected by
treatment, averaging 59.6, 62.3, 40.1, and 58.6%, respectively. Excretion of
urinary purine derivatives was greatest for WDGS-AS, followed by WDGS-CS, and
then CONT-CS and CONT-AS. Thus, by calculation, estimated microbial protein flow
was highest for WDGS-AS (2,189.9 g/d) followed by WDGS-CS (1,996.2 g/d), CONT-AS
(1,640.0 g/d), and CONT-CS (1,627.0 g/d). Mass of fecal N was not different among
treatments (averaging 287.1+/-14.8 g/d), but urinary and manure N were reduced
for rations with WDGS compared with those not including WDGS. Observed 4%
fat-corrected milk was greatest for WDGS-AS, followed by WDGS-CS, and then
CONT-CS and CONT-AS (30.7, 29.7, 28.3, and 27.2 kg/d, respectively). Milk protein
yield was greatest for WDGS-AS (1.00 kg/d), followed by WDGS-CS, and then CONT-AS
and CONT-CS (0.96, 0.91, 0.86 kg/d, respectively). This research demonstrated
that rations can be balanced for dairy cattle to include up to 25% WDGS and
result in increased microbial protein synthesis, milk production, and milk
protein yield.
DOI: 10.3168/jds.2009-2962
1277. Environ Microbiol. 2010 Dec;12(12):3185-95. doi:

10.1111/j.1462-2920.2010.02291.x.
Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable

to form biofilms on abiotic surfaces.
Barahona E(1), Navazo A, Yousef-Coronado F, Aguirre de Cárcer D, Martínez-Granero

F, Espinosa-Urgel M, Martín M, Rivilla R.
Author information:
(1)Departamento de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens.

Mutants with reduced motility are poor competitors, and hypermotile, more
competitive phenotypic variants are selected in the rhizosphere. Flagellar
motility is a feature associated to planktonic, free-living single cells, and
although it is necessary for the initial steps of biofilm formation, bacteria in
biofilm lack flagella. To test the correlation between biofilm formation and
rhizosphere colonization, we have used P. fluorescens F113 hypermotile
derivatives and mutants affected in regulatory genes which in other bacteria
modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants
affected in these three genes and a hypermotile variant (V35) isolated from the
rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized
the alfalfa root apex as efficiently as the wild-type strain, indicating that
biofilm formation on abiotic surfaces and rhizosphere colonization follow
different regulatory pathways in P. fluorescens. Furthermore, a triple mutant
gacSsadBwspR (GSW) and V35 were more competitive than the wild-type strain for
root-tip colonization, suggesting that motility is more relevant in this
environment than the ability to form biofilms on abiotic surfaces. Microscopy
showed the same root colonization pattern for P. fluorescens F113 and all the
derivatives: extensive microcolonies, apparently held to the rhizoplane by a
mucigel that seems to be plant produced. Therefore, the ability to form biofilms
on abiotic surfaces does not necessarily correlates with efficient rhizosphere
colonization or competitive colonization.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.
DOI: 10.1111/j.1462-2920.2010.02291.x
1278. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2010 Aug;26(8):750-3.
[The study on immune responses in BALB/c mice by immunization with the transgenic
Alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus
granulosus].
Ye YJ(1), Li WG.
Author information:
(1)Institute of Infectious and Parasitic Diseases, the First Affiliated Hospital,
Chongqing Medical University, Chongqing 400016, China. YeYanju.happy@163.com
AIM: To investigate the immune responses and the protection induced by the
transgenic Alfalfa (Medicago sativa) containing Eg95-EgA31 fusion gene of
Echinococcus granulosus against Eg protoscoleces.
METHODS: The leaf protein was extracted from the transgenic alfalfa by
heat-coagulation method, its concentration was prepared for 20 g/L. BALB/c mice
were immunized intranasally or orally with the leaf protein once per 3 days for 2
months.At the same time, the leaf protein transfected with pBI-121 blank vector
and the normal leaf protein without foreign antigen was served as control. The
mice were then challenged intraperitoneally with Eg protoscoleces (50
protoscoleces per mouce)on week 8 after the last vaccination and sacrified on
week 24 postinfection to count the rate of reduced hydatid cyst. The specific
antibody (IgE, IgG and its subclasses)in the sera collected from the eyeballs was
evaluated by ELISA. Splenocytes were separated and cultured in vitro with EgAg,
ConA or LPS stimulus.The substes of CD4(+); and CD8(+); T cells were measured by
FCM. The splenocytes' proliferation was determined by MTT method.Then the cells
were collected, stained by PI and Annexin V-FITC, and analyzed by FCM to get the
splenocytes' apoptotic rate.The supernatant was collected to measure the level of
IL-12, IL-10, IFN-gamma and TNF-alpha by ELISA.
RESULTS: Compared with the control group, The hydatid cyst weight in the oral
immunization group decreased by 64.1%; the splenocytes' apoptotic rate got
obviously lower than that in the control group; the splenocytes' proliferation
increased significantly, the CD4(+); subsets and the ratio of the CD4(+);/CD8(+);
did so, and the similar trend about the specific antibody titer and the level of
cytokines could be seen in this group.
CONCLUSION: Apoptosis of splenocytes may be inhibited in mice by immunization
with the transgenic alfalfa, splenocytes' proliferation and Th1 response can be
induced in the mice against the challenge of Eg protoscoleces. CD4(+); T cell and
the specific antibody(IgG, IgG2b and IgE) may play important roles in the
protection induced by the transgenic alfalfa vaccine.
1279. Res Microbiol. 2010 Sep;161(7):556-64. doi: 10.1016/j.resmic.2010.05.004.

Epub
2010 May 31.
Identification of a TRAP transporter for malonate transport and its expression
regulated by GtrA from Sinorhizobium meliloti.
Chen AM(1), Wang YB, Jie S, Yu AY, Luo L, Yu GQ, Zhu JB, Wang YZ.
Author information:
Sinorhizobium meliloti can live as a saprophyte in soil or as a nitrogen-fixing

symbiont inside the root nodule cells of alfalfa and related legumes by utilizing
different organic compounds as its carbon source. Here we have identified the
matPQMAB operon in S. meliloti 1021. Within this operon, matP, matQ and the M
region of the fused gene matMA encode an extracytoplasmic solute receptor, a
small transmembrane protein and a large transmembrane protein, consisting of
three components of the tripartite ATP-independent periplasmic (TRAP) transporter
for malonate transport. The A region of the fused gene matMA and matB encode
malonate-metabolizing enzymes, malonyl-CoA decarboxylase and malonyl-CoA
synthetase. The null mutant of each matPQMAB gene is unable to grow on M9 minimal
medium containing malonate as the sole carbon source. However, these mutants can
induce the formation of efficient nitrogen-fixing root nodules on alfalfa. The
matPQMAB operon is expressed in free-living bacterial cells and symbiotic
bacterial cells from infection threads and root nodules. The GntR family
transcriptional regulator, GtrA, specifically binds the promoter of the matPQMAB
operon, positively regulating its expression. Moreover, the matPQMAB can be
transcriptionally induced by malonate. These results suggested that a
C(3)-dicarboxylic acid TRAP transporter is responsible for malonate transport in
S. meliloti.
Copyright 2010 Elsevier Masson SAS. All rights reserved.
DOI: 10.1016/j.resmic.2010.05.004
1280. Microb Ecol. 2010 Nov;60(4):816-28. doi: 10.1007/s00248-010-9705-7. Epub 2010

Jul
1.
In situ dynamics of microbial communities during decomposition of wheat, rape,

and alfalfa residues.
Pascault N(1), Cécillon L, Mathieu O, Hénault C, Sarr A, Lévêque J, Farcy P,

Ranjard L, Maron PA.
Author information:
(1)UMR Microbiologie du Sol et de l'Environnement, INRA/Université de Bourgogne,
CMSE, BP 86510, Dijon, France.
Microbial communities are of major importance in the decomposition of soil

organic matter. However, the identities and dynamics of the populations involved
are still poorly documented. We investigated, in an 11-month field experiment,
how the initial biochemical quality of crop residues could lead to specific
decomposition patterns, linking biochemical changes undergone by the crop
residues to the respiration, biomass, and genetic structure of the soil microbial
communities. Wheat, alfalfa, and rape residues were incorporated into the 0-15 cm
layer of the soil of field plots by tilling. Biochemical changes in the residues
occurring during degradation were assessed by near-infrared spectroscopy.
Qualitative modifications in the genetic structure of the bacterial communities
were determined by bacterial-automated ribosomal intergenic spacer analysis.
Bacterial diversity in the three crop residues at early and late stages of
decomposition process was further analyzed from a molecular inventory of the 16S
rDNA. The decomposition of plant residues in croplands was shown to involve
specific biochemical characteristics and microbial community dynamics which were
clearly related to the quality of the organic inputs. Decay stage and seasonal
shifts occurred by replacement of copiotrophic bacterial groups such as
proteobacteria successful on younger residues with those successful on more
extensively decayed material such as Actinobacteria. However, relative abundance
of proteobacteria depended greatly on the composition of the residues, with a
gradient observed from alfalfa to wheat, suggesting that this bacterial group may
represent a good indicator of crop residues degradability and modifications
during the decomposition process.
DOI: 10.1007/s00248-010-9705-7
1281. J Hazard Mater. 2010 Oct 15;182(1-3):210-6. doi:

10.1016/j.jhazmat.2010.06.017.
Epub 2010 Jun 9.
Relationship between photosynthetic capacity, nitrogen assimilation and nodule

metabolism in alfalfa (Medicago sativa) grown with sewage sludge.
Antolín MC(1), Fiasconaro ML, Sánchez-Díaz M.
Author information:
(1)Dpto. Biología Vegetal, Sección Biología Vegetal Unidad Asociada al CSIC
(EEAD, Zaragoza; ICVV, Logroño), Facultade de Ciencias, Universidad de Navarra,
C/ Irunlarrea 1, 31008 Pamplona, Spain. cantolin@unav.es
Sewage sludge has been used as N fertilizer because it contains some of inorganic
N, principally as nitrate and ammonium ions. However, sewage sludge addition to
legumes could result in impaired nodule metabolism due to the presence of
inorganic N from sludge. A greenhouse experiment was conducted to examine the
effects of sewage sludge on growth, photosynthesis, nitrogen assimilation and
nodule metabolism in alfalfa (Medicago sativa L. cv. Aragón). Plants were grown
in pots with a mixture of perlite and vermiculite (2:1, v/v). The experiment
included three treatments: (1) plants inoculated with rhizobia and amended with
sewage sludge at rate of 10% (w/w) (RS); (2) plants inoculated with rhizobia
without any amendment (R); and (3) non-inoculated plants fed with ammonium
nitrate (N). N(2)-fixing plants had lower growth and sucrose phosphate synthase
activity but higher photosynthesis than nitrate-fed plants because they
compensated the carbon cost of the rhizobia. However, sewage sludge-treated
plants evidenced a loss of carbon sink strength due to N(2) fixation by means of
decreased photosynthetic capacity, leaf chlorophylls and N concentration in
comparison to untreated plants. Sewage sludge did no affect nodulation but
decreased nodule enzyme activities involved in carbon and N metabolisms that may
lead to accumulation of toxic N-compounds.
2010 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.jhazmat.2010.06.017
1282. Bioresour Technol. 2010 Nov;101(21):8469-72. doi:

10.1016/j.biortech.2010.06.010.
Epub 2010 Jun 26.
Evaluation of feeding glycerol on free-fatty acid production and fermentation

kinetics of mixed ruminal microbes in vitro.
Krueger NA(1), Anderson RC, Tedeschi LO, Callaway TR, Edrington TS, Nisbet DJ.
Author information:
(1)United States Department of Agriculture, Agriculture Research Service,
Southern Plains Agriculture Research Center, Food and Feed Safety research Unit,
2881 F&B Road, College Station, TX 77845, USA.
Ruminant-derived foods contain high proportions of saturated fats as a result of

ruminal biohydrogenation that rapidly saturates and thus limits the availability
of free unsaturated fatty acids for assimilation. The objective of this study was
to evaluate the effects of glycerol on ruminal free-fatty acid (FFA) production
rates and in vitro fermentation kinetics of alfalfa hay. In vitro incubations
demonstrated 48% and 77% reductions in rates of FFA accumulation in incubations
supplemented with 2% and 20% glycerol as compared to controls. In vitro
incubations with alfalfa hay demonstrated that increasing levels of glycerol did
not affect NDF digestibility of the hay. Additionally, increasing amounts of
glycerol decreased the acetate to propionate ratio in the rumen. These results
suggest that inhibiting bacterial fat degradation may promote ruminal passage of
total lipid, thereby providing greater proportions of beneficial unsaturated fat
for incorporation into beef products.
DOI: 10.1016/j.biortech.2010.06.010
1283. Meat Sci. 2010 Oct;86(2):486-90. doi: 10.1016/j.meatsci.2010.05.040. Epub

2010
Jun 2.
Effect of protein-xanthophylls (PX) concentrate of alfalfa supplementation on

physico-chemical properties of turkey breast and thigh muscles during ageing.
Karwowska M(1), Stadnik J, Dolatowski ZJ, Grela ER.
Author information:
(1)Department of Meat Technology and Food Quality, University of Life Sciences in
Lublin, Skromna 8, 20-704 Lublin, Poland. malgorzata.karwowska@up.lublin.pl
The objective was to evaluate the effects of a dietary protein-xanthophylls (PX)

concentrate of alfalfa to turkey diets (at 15 and 30 g kg(-1) feed) on the
physico-chemical properties of breast and thigh muscles during ageing. The
experiment involved 120 turkeys (Big-6 type) allotted to 3 groups (group
I-control group; group II--with 1.5% supplementation of the protein-xanthophylls
(PX) concentrate; group III--with 3% supplementation of the protein-xanthophylls
(PX) concentrate). Measurements of pH, water holding capacity, color,
oxymyoglobin content, TBARS and oxidation-reduction potential showed that the
addition of protein-xanthophylls (PX) concentrate of alfalfa to a turkey diet did
not cause deterioration of breast and thigh meat quality. In addition, changes in
color, oxymyoglobin content, TBARS and oxidation-reduction potential values
suggested that the inclusion of the concentrate to turkey diets acts as an
antioxidant in the raw meat.
Copyright (c) 2010 The American Meat Science Association. Published by Elsevier
Ltd. All rights reserved.
DOI: 10.1016/j.meatsci.2010.05.040
1284. BMC Microbiol. 2010 Jun 23;10:180. doi: 10.1186/1471-2180-10-180.
Absence of functional TolC protein causes increased stress response gene

expression in Sinorhizobium meliloti.
Santos MR(1), Cosme AM, Becker JD, Medeiros JM, Mata MF, Moreira LM.
Author information:
(1)Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e
Química, Instituto Superior Técnico, Lisboa, Portugal.
BACKGROUND: The TolC protein from Sinorhizobium meliloti has previously been
demonstrated to be required for establishing successful biological nitrogen
fixation symbiosis with Medicago sativa. It is also needed in protein and
exopolysaccharide secretion and for protection against osmotic and oxidative
stresses. Here, the transcriptional profile of free-living S. meliloti 1021 tolC
mutant is described as a step toward understanding its role in the physiology of
the cell.
RESULTS: Comparison of tolC mutant and wild-type strains transcriptomes showed
1177 genes with significantly increased expression while 325 had significantly
decreased expression levels. The genes with an increased expression suggest the
activation of a cytoplasmic and extracytoplasmic stress responses possibly
mediated by the sigma factor RpoH1 and protein homologues of the CpxRA
two-component regulatory system of Enterobacteria, respectively. Stress
conditions are probably caused by perturbation of the cell envelope. Consistent
with gene expression data, biochemical analysis indicates that the tolC mutant
suffers from oxidative stress. This is illustrated by the elevated enzyme
activity levels detected for catalase, superoxide dismutase and glutathione
reductase. The observed increase in the expression of genes encoding products
involved in central metabolism and transporters for nutrient uptake suggests a
higher metabolic rate of the tolC mutant. We also demonstrated increased swarming
motility in the tolC mutant strain. Absence of functional TolC caused decreased
expression mainly of genes encoding products involved in nitrogen metabolism and
transport.
CONCLUSION: This work shows how a mutation in the outer membrane protein TolC,
common to many bacterial transport systems, affects expression of a large number
of genes that act in concert to restore cell homeostasis. This finding further
underlines the fundamental role of this protein in Sinorhizobium meliloti
biology.
DOI: 10.1186/1471-2180-10-180
PMCID: PMC2912261
Habitat affinity of resident natural enemies of the invasive Aphis glycines

(Hemiptera: Aphididae), on soybean, with comments on biological control.
Brewer MJ(1), Noma T.

Author information:
(1)Texas AgriLife Research and Extension Center, 10345 State Hgwy. 44, Corpus
Christi, TX 78406, USA. mjbrewer@ag.tamu.edu
We integrated a natural enemy survey of the broader landscape into a more

traditional survey for Aphis glycines Matsumura (Hemiptera: Aphididae),
parasitoids and predatory flies on soybean using A. glycines-infested soybean,
Glycine max (L.) Merr., placed in cropped and noncropped plant systems to
complement visual field observations. Across three sites and 5 yr, 18 parasitoids
and predatory flies in total (Hymenoptera: Aphelinidae [two species] and
Bracondae [seven species], Diptera: Cecidomyiidae [one species], Syrphidae [seven
species], Chamaemyiidae [one species]) were detected, with significant
variability in recoveries detected across plant system treatments and strong
contrasts in habitat affinity detected among species. Lysiphlebus testaceipes
Cresson was the most frequently detected parasitoid, and no differences in
abundance were detected in cropped (soybean, wheat [Triticum aestivum L.], corn
[Zea mays L.], and alfalfa [Medicago sativa L.]) and noncropped (poplar [Populus
euramericana (Dode) Guinier] and early successional vegetation) areas. In
contrast, Binodoxys kelloggensis Pike, Starý & Brewer had strong habitat affinity
for poplar and early successional vegetation. The low recoveries seasonally and
across habitats of Aphelinus asychis Walker, Aphelinus sp., and Aphidius colemoni
Viereck make their suitability to A. glycines on soybean highly suspect. The
widespread occurrence of many of the flies reflects their broad habitat affinity
and host aphid ranges. The consistent low field observations of parasitism and
predation suggest that resident parasitoids and predatory flies are unlikely to
contribute substantially to A. glycines suppression, at least during the
conventional time period early in the pest invasion when classical biological
control activities are considered. For selected species that were relatively well
represented across plant systems (i.e., L. testaceipes and Aphidoletes aphidimyza
Rondani), conservation biological control efforts may be fruitful. The additional
information gained from expanding the natural enemy survey into the broader
landscape was essential in making these distinctions relevant to conservation
biological control, while adding agroecosystem-specific information valuable to
classical biological control.
1286. Vet Parasitol. 2010 Aug 27;172(1-2):89-94. doi: 10.1016/j.vetpar.2010.04.018.

Epub 2010 Apr 28.
Effect of feeding bioactive forages on infection and subsequent development of

Haemonchus contortus in lamb faeces.
Valderrábano J(1), Calvete C, Uriarte J.
Author information:
(1)Centro de Investigación y Tecnología Agroalimentaria de Aragón, Avda. de
Montañana, 930, 50059 Zaragoza, Spain. jvalderrabano@aragon.es
Contrasting dried herbage diets were offered to lambs to evaluate their effects
on Haemonchus contortus infection and on subsequent development of larvae in
faeces. Artificially infected lambs (n=24, 4 months old) were allocated to one of
four treatment groups (n=6) and fed on hay of lucerne (Medicago sativa; Lu),
sulla (Hedysarum coronarium; Su), sainfoin (Onobrychis viciifolia; Sa) or
wormwood (Artemisia absinthium; Wo) included as 20% in ground lucerne pellets.
While voluntary intake was similar, lamb growth rates were notably affected by
diet. Additionally, necropsies carried out 30 days post-infection showed that
worm burden was reduced by 8 (Su), 13 (Sa) and 49% (Wo) with respect to that
found in lambs fed on lucerne, though only the difference with wormwood was
significant. Faecal egg excretion expressed on a dry matter basis was also
reduced by 22 (Su), 54 (Sa) and 73% (Wo), but differences were significant only
for wormwood and sainfoin, which was associated with a decrease in female
fecundity. Sulla and sainfoin diets significantly reduced egg hatching rates but
increased the percentage of L(3) in the faeces of the host animal, particularly
in the case of sulla hay compared to other forages. Consequently, the potential
risk of infection derived from faecal excretion could be notably altered. Further
work is needed to validate these findings and their implications in an applied
farming system.
(c) 2010 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.vetpar.2010.04.018
Effects of relay-intercropping sorghum with winter wheat, alfalfa, and cotton on

lady beetle (Coleoptera: Coccinellidae) abundance and species composition.
Phoofolo MW(1), Giles KL, Elliott NC.
Author information:
(1)Department of Biology, National University of Lesotho, PO Roma 180, Lesotho.
Creating conditions that enhance the abundance of resident populations of natural

enemies in agroecosystems is considered critical to the efficiency of biological
control of insect pests. We conducted a study to determine the potential of
relay-intercropping for enhancing the abundance of aphidophagous lady beetles in
sorghum. A relay-intercropping system consisting of alfalfa, winter wheat, and
cotton as intercrops and sorghum as a main crop was compared with sorghum
monoculture plots at two study sites in OK from 2003 to 2006. Lady beetles and
aphids were sampled throughout the season using sticky traps and field counts on
individual sorghum plants. Results from sticky traps and field counts show that
differences in abundance and species composition of lady beetles between
intercropped and monoculture sorghum were not statistically different during each
year of study. Also, the lady beetle-greenbug ratios in relay-intercropped and
monoculture plots were not significantly different. Lack of significant effects
of relay-intercropping in our study may have been attributable to the confounding
effects of spatial and temporal scale and the low number of aphids and other
alternative prey in the intercrops compared with high incidence of corn leaf
aphids in sorghum early in the season.
DOI: 10.1603/EN09122
Phenology and abundance of bean leaf beetle (Coleoptera: Chrysomelidae) in

eastern South Dakota on alfalfa and soybean relative to tillage, fertilization,
and yield.
Hammack L(1), Pikul JL Jr, West MS.

Author information:
(1)USDA-ARS North Central Agricultural Research Laboratory, Brookings, SD 57006,
USA. hammackl@mt-rushmore.net
Phenology and abundance of bean leaf beetles, Cerotoma trifurcata (Förster), were
examined throughout two eastern South Dakota growing seasons in relation to grain
yields in chisel- and ridge-tilled soybeans [Glycine max (L.) Merrill] grown in
2-yr rotation with corn (Zea mays L.) with and without added nitrogen (N).
Populations were also sampled early and late season in alfalfa (Medicago sativa
L.). Beetles were present in alfalfa by late May and most were reproductively
active within a week, but their presence in alfalfa did not always precede
soybean emergence. Most beetles taken from alfalfa in late fall were teneral; all
were previtellogenic and unmated. Reproductively active beetles were detected in
soybeans as soon as seedlings emerged. A partial second generation apparently
occurred each year. First-generation beetles started to emerge in soybean fields
during the first or third week of July but, whether emergence started early or
late, most beetles emerging after July seemingly failed to mature their eggs and
started leaving soybeans within several weeks of eclosion. This pattern suggested
that any second generation arose from only the earliest emerging beetles of the
first generation, with later emerging individuals having to overwinter before
reproducing. Thus, any factors delaying emergence of the first generation, such
as delayed soybean planting, could potentially limit its reproductive capacity
through winter mortality. Cumulative seasonal beetle counts were lower in
N-treated subplots and in ridge-tilled compared with chisel-tilled plots. Soybean
grain yield increased with decreases in peak abundance of first-generation
beetles and with N fertilization.
DOI: 10.1603/EN09153
1289. Guang Pu Xue Yu Guang Pu Fen Xi. 2010 Apr;30(4):988-90.
[Comparative analysis of alfalfa seeds between space flight mutation and its
control by Raman spectroscopy].
Ren WB(1), Zhang YW, Deng B, Xu Z, Chen LB.
Author information:
(1)Grassland Research Institute of Chinese Academy of Agricultural Sciences,
Huhhot 010010, China. rppcaucau@163.com
To realize the effect of space flight factors on chemical component of alfalfa

seeds and its possible mechanism, seeds were loaded onto satellite "Jianbing No.
1" in 2006 for 14 days' space flight and then analyzed by Raman spectroscopy.
Results showed that the intensity of two peaks (358 and 553 cm(-1)) of space
flight seeds had been increased and the intensity of four peaks (814, 1 122, 1
531 and 1 743 cm(-1)) of space flight seeds had been decreased compared with its
ground control. Based on the classification of Raman spectra, the increased peaks
of 358 and 553 cm(-1) are related to DNA and Ca2+ respectively, which mean that
the content of DNA and Ca2+ of alfalfa seeds had increased after space flight.
The decreased peaks of 814, 1 122 and 1 743 cm(-1) are related to saccharide and
fatty acid respectively, which mean that the content of reserve energy of alfalfa
seeds had decreased after space flight. These findings can be explained as
follows: (1) The increase in the content of DNA may be explained by the DNA
damage induced by space flight factors and DNA syntheses and duplication before
the cell division. (2) The increase in the content of Ca2+ may be stimulated by
the complexity of gravity during the space flight, especially the hypergravity.
Recent researches in Abrabidopsis thaliana have provided additional proof. (3)
The decrease of the energy materials such as saccharide and fatty acid may be
explained by the consumption both during the repair process of DNA damage induced
by cosmic radiation and during the germination of seeds because the dormancy of
alfalfa seeds had been broken up by space flight factors (cosmic radiation,
microgravity, vibration or others) which subsequently resulted in that nutritious
materials of alfalfa seeds were used earlier than its ground control.
1290. Acta Biochim Biophys Sin (Shanghai). 2010 Jun 15;42(6):410-7.
GGDEF and EAL proteins play different roles in the control of Sinorhizobium
meliloti growth, motility, exopolysaccharide production, and competitive
nodulation on host alfalfa.
Wang Y(1), Xu J, Chen A, Wang Y, Zhu J, Yu G, Xu L, Luo L.
Author information:
(1)East China Normal University, Shanghai, China.
A new bacterial secondary messenger, bis-(3',5')-cyclic dimeric guanosine

monophosphate (c-di-GMP), is usually synthesized or decomposed by proteins
containing GGDEF or glutamate-alanine-leucine (EAL) domain. They often act as
cyclase or phosphodiesterase of c-di-GMP and their genes are distributed among
almost all bacteria according to known genomic DNA sequences. However, the
systematic identification of GGDEF and EAL genes remains unclear in rhizobia,
soil bacteria that interact with compatible legumes to form nitrogen-fixing
nodules. In this study, 19 putative GGDEF and EAL genes were identified in a
model rhizobium, Sinorhizobium meliloti, by bioinformatic analysis (encoding 5
GGDEF proteins, 4 EAL proteins, and 10 GGDEF and EAL double-domain proteins).
Null mutants of 14 genes were constructed through systematic plasmid insertion.
All 14 gene mutants showed deficient growth in minimal medium and defective
motility, and 11 gene mutants produced a lot more exopolysaccharide and displayed
less competitive nodulation on the host plant, alfalfa. Our results suggested
that GGDEF and EAL proteins may play different roles in the control of S.
meliloti physiology, although they contain conserved catalytic (GGDEF or EAL)
domains. Our finding also implied that c-di-GMP may play an important role in the
interactions between this rhizobium and its host plants to establish efficient
symbiosis.
1291. Rev Biol Trop. 2010 Jun;58(2):757-67.
[Spider community (Arachnida, Araneae) of alfalfa crops (Medicago sativa) in

Buenos Aires, Argentina].
Armendano A(1), González A.
Author information:
(1)Centro de Estudios Parasitológicos y de Vectores (CEPAVE - CONICET), 2 No 584
(1900) La Plata, Argentina. aarmendano@hotmail.com
Over the last decades there has been an increasing interest in the use of natural
enemies to control pest insects, including spiders. We studied a spider community
in Argentina by sampling every two weeks during 2004-2006 in one-hectare lots.
Soil stratum spiders were collected using nets and pitfall traps. A total of 6229
specimens were collected (15 families and 50 species). Seven families were found
in the herbal stratum, the most abundant were Thomisidae (n=2012, 32.30%),
Araneidae (n=1516, 24.33%) and Oxyopidae (n=604, 9.70%). The soil had 14
families, mainly: Lycosidae (n=629, 10.10%) and Linyphiidae (n=427, 6.85%).
Hunting spiders predominated: ambushers (32.99%); stalkers (11.77%) and
ground-runners (10.84%) were less common. The most abundant web building spiders
were the orb weavers (27.56%). The diversity indexes were: H'=2.97, Dsp=0.11 and
J=0.79, evidencing a moderately diverse spider community with predominance of
Misumenops pallidus, Oxyopes salticus, Lycosa poliostoma and L. erythrognatha.
The spiders were present throughout the phenological development of the crop with
abundance peaks in spring and summer.
1292. Huan Jing Ke Xue. 2010 Apr;31(4):1079-84.
[Interaction of biosurfactant-microorganism to enhance phytoremediation of aged

polycyclic aromatic hydrocarbons (PAHS) contaminated soils with alfalfa (Medicago
sativa L.)].
Liu WW(1), Yin R, Lin XG, Zhang J, Chen XM, Li XZ, Yang T.
Author information:
Science, Chinese Academy of Sciences, Nanjing 210008, China. blww1221@163.com
A pot experiment in greenhouse was carried out to investigate the interactive

effect of rhamnolipids (RH) addition and PAHs-specific degrading bacteria (DB)
inoculation on the phytoremediation efficiency for removal polycyclic aromatic
hydrocarbons (PAHs) from agricultural soils. Results indicated that RH addition
and DB inoculation promoted alfalfa (Medicago sativa L. ) growth and PAHs
degradation in the soil. After 90 days, residual PAHs concentration in soil
reduced 30.0% and 49.6% for the treatment of RH and DB, respectively, but only
21.7% for control. For the treatment of RH + DB, residual PAHs concentration in
soil reduced 53.9%, showing synergy effect of RH addition and DB inoculation. In
addition, the average PAHs degradation gradually reduced with the increase of the
PAHs rings, but DB inoculation promoted the degradation of four-ring PAHs and
five-ring PAHs. The number of PAHs degrading bacteria and dehydrogenase activity
increased with the removal ratio of PAHs. Therefore RH addition and PAHs-specific
degrading bacteria inoculation were effective in enhancing the phytoremediation
efficiency of the long-term PAHs contaminated soils.
1293. J Environ Monit. 2010 Jul 8;12(7):1482-9. doi: 10.1039/c0em00029a. Epub 2010
Jun
4.
Beta-cyclodextrin enhanced phytoremediation of aged PCBs-contaminated soil from
e-waste recycling area.
Chen Y(1), Tang X, Cheema SA, Liu W, Shen C.
Author information:
(1)Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control,
Institute of Environmental Science and Technology, Zhejiang University, Hangzhou,
310029, P.R. China. yxchen@zju.edu.cn
The objective in the first phase of this study was to screen four plant species
(alfalfa, ryegrass, tall fescue and rice) for phytoremediation of aged
polychlorinated biphenyl (PCB)-contaminated soil from an electronic and electric
waste (e-waste) recycling site. Glucose, biphenyl and three surfactants
(TritonX-100, randomly methylated-beta-cyclodextrins and beta-cyclodextrin) were
used to enhance the phytoremediation process. During the second phase, the focus
was rhizosphere characteristics and plant uptake to investigate the mechanism of
PCB removal from soil. In the first phase, all the tested plant species showed a
significantly greater PCB removal percentage compared to the unplanted controls,
while different amendments showed no significant difference. The most effective
plant (ryegrass) combined with beta-cyclodextrin was selected for further
studies. During the rhizosphere characteristics and plant uptake study, the
highest PCB removal percentage (38.1%) was observed in the ryegrass planted soil
when beta-cyclodextrin was amended at 1.0% (w/w). The presence of plants
significantly increased the biological activity (microbial counts and enzyme
activity) of both beta-cyclodextrin amended and non-amended soils. Higher levels
of PCB removal were closely related to greater microbial counts and soil enzyme
activities by correlation analysis. After 120 days of plant growth, ryegrass
accumulated 708.7-820.1 ng PCBs/g in the root and 71.7-110.8 ng PCBs/g in the
shoot, resulting in about 0.08% PCBs removal from soil. It was concluded that
high PCB degradation was due to the increased PCB bioavailability as well as
biostimulation of microbial communities after plantation and beta-cyclodextrin
addition. Furthermore, results suggested that PCB removal was mainly contributed
by microbial degradation rather than plant uptake or abiotic dissipation.
DOI: 10.1039/c0em00029a
1294. Bioresour Technol. 2010 Oct;101(19):7545-50. doi:

10.1016/j.biortech.2010.04.097.
Epub 2010 May 26.
Fermentation of bioenergy crops into ethanol using biological abatement for

removal of inhibitors.
Nichols NN(1), Dien BS, Cotta MA.
Author information:
(1)Fermentation Biotechnology Research Unit, NCAUR, USDA-ARS, Peoria, IL 61604,
USA. nancy.nichols@ars.usda.gov
Biological abatement was used to condition dilute acid-pretreated hydrolysates of

three perennial herbaceous crops that are potential bioenergy feedstocks:
switchgrass, reed canarygrass, and alfalfa stems. Fungal isolate Coniochaeta
ligniaria was inoculated into the hydrolysates to metabolize and remove
inhibitory compounds prior to yeast fermentation of glucose. Switchgrass, reed
canarygrass, and alfalfa stem samples were pretreated with dilute acid at 10% w/w
biomass loading and subjected to bioabatement with strain NRRL30616, to prepare
the material for simultaneous saccharification of cellulose and fermentation by
Saccharomyces cerevisiae. Bioabatement eliminated the extended fermentation lag
times associated with inhibitory compounds and observed for the unconditioned
biomass hydrolysates controls. Bioabatement was as effective as lime conditioning
at reducing fermentation lag times. Prolonged incubations with the bioabatement
microbe resulted in consumption of some glucose and reduced production of
ethanol.
DOI: 10.1016/j.biortech.2010.04.097
1295. BMC Genomics. 2010 May 24;11:323. doi: 10.1186/1471-2164-11-323.
Transcript profiling of two alfalfa genotypes with contrasting cell wall

composition in stems using a cross-species platform: optimizing analysis by
masking biased probes.
Yang SS(1), Xu WW, Tesfaye M, Lamb JF, Jung HJ, VandenBosch KA, Vance CP,
Gronwald JW.
Author information:
(1)USDA-Agricultural Research Service, Plant Science Research Unit, St, Paul, MN
55108, USA. sam.yang@ars.usda.gov
BACKGROUND: The GeneChip(R) Medicago Genome Array, developed for Medicago

truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa
[Medicago sativa (L.) subsp. sativa]. However, previous research involving
cross-species hybridization (CSH) has shown that sequence variation between two
species can bias transcript profiling by decreasing sensitivity (number of
expressed genes detected) and the accuracy of measuring fold-differences in gene
expression.
RESULTS: Transcript profiling using the Medicago GeneChip(R) was conducted with
elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa
genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose
and lignin. A protocol was developed that masked probes targeting inter-species
variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold
was selected that optimized both sensitivity and accuracy. After masking for both
ISV regions and previously identified single-feature polymorphisms (SFPs), the
number of differentially expressed genes between the two genotypes in both ES and
PES internodes was approximately 2-fold greater than the number detected prior to
masking. Regulatory genes, including transcription factor and receptor kinase
genes that may play a role in development of secondary xylem, were significantly
over-represented among genes up-regulated in 252 PES internodes compared to 1283
PES internodes. Several cell wall-related genes were also up-regulated in
genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially
expressed regulatory and cell wall-related genes demonstrated increased
sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000
genes that were differentially expressed in ES and PES internodes of genotypes
252 and 1283 were mapped onto putative orthologous loci on M. truncatula
chromosomes. Clustering simulation analysis of the differentially expressed genes
suggested co-expression of some neighbouring genes on Medicago chromosomes.
CONCLUSIONS: The problems associated with transcript profiling in alfalfa stems
using the Medicago GeneChip as a CSH platform were mitigated by masking probes
targeting ISV regions and SFPs. Using this masking protocol resulted in the
identification of numerous candidate genes that may contribute to differences in
cell wall concentration and composition of stems of two alfalfa genotypes.
DOI: 10.1186/1471-2164-11-323
PMCID: PMC2893600
1296. J Plant Physiol. 2010 Sep 1;167(13):1112-5. doi: 10.1016/j.jplph.2010.03.014.

Epub 2010 May 21.
The argon-induced decline in nitrogenase activity commences before the beginning

of a decline in nodule oxygen uptake.
Fischinger SA(1), Schulze J.
Author information:
(1)Department of Crop Science, Plant Nutrition, Georg-August-University of
Goettingen, Carl-Sprengel Street 1, 37075 Goettingen, Germany.
Replacement of N(2) by argon in the air around nodules directs nitrogenase

electron flow in its total onto H(+) resulting in increased nodule H(2) evolution
(total nitrogenase activity (TNA)). However, argon application induces a
so-called argon-induced decline in nitrogenase activity (Ar-ID) connected with
decreased nodule oxygen permeability. Consequently, TNA measurements tend to
underestimate total nitrogenase activity. It is unclear whether the decline in
oxygen diffusion into nodules induces the Ar-ID, or whether a decline in
nitrogenase activity is followed by lower nodule O(2) uptake. The objective of
the present work was to examine the time sequence of the decline in nodule H(2)
evolution and O(2) uptake after argon application. In addition, the reliability
of TNA values, taken as quickly as possible after the switch to Ar/O(2), was
tested through comparative measurement of (15)N(2) uptake of the same plants.
Short-term TNA measurements in an optimized gas exchange measurement system
yielded reliable results, verified by parallel determination of (15)N(2) uptake.
A five min application of Ar/O(2) was without effect on the subsequent H(2)
evolution in ambient air. A parallel experiment on control plants revealed that a
decrease in nodule oxygen uptake began several minutes after the onset of the
decline in H(2) evolution. We conclude that the primary effect of the replacement
of N(2) by argon differs from oxygen diffusion control. A gas exchange system
allowing an immediate taking of TNA yields reliable results and does not disturb
nodule activity. Gas exchange measurements provide a powerful tool for studying
nodule physiology and should be combined with material from molecular studies.
Copyright 2010 Elsevier GmbH. All rights reserved.
DOI: 10.1016/j.jplph.2010.03.014
1297. Theor Appl Genet. 2010 Sep;121(5):865-76. doi: 10.1007/s00122-010-1356-z.

Epub
2010 May 16.
Association of a CONSTANS-LIKE gene to flowering and height in autotetraploid

alfalfa.
Herrmann D(1), Barre P, Santoni S, Julier B.
Author information:
(1)INRA, UR 4, Unité de Recherche Pluridisciplinaire Prairies et Plantes
Fourragères, BP 6, 86600, Lusignan, France.
In alfalfa (Medicago sativa), an autotetraploid forage legume, stem length is a

major component of forage yield, quality and competing ability. In this species,
flowering date is not a breeding criterion. Association mapping based on a
candidate gene approach has given good results in plants, including
autotetraploid species for which genetic analyses are complex. The role of a
CONSTANS-LIKE gene, identified as a candidate for stem elongation and flowering
date in the model legume M. truncatula, was tested for association with the same
traits in alfalfa. Four hundred genotypes from ten cultivars were evaluated for
stem height and flowering date in two locations during 4 years. They were
genotyped with simple sequence repeat markers and a low structuration was
noticed. Primers were designed to amplify and sequence two regions of the alfalfa
gene homologous to CONSTANS-LIKE. Single nucleotide polymorphisms (SNPs) were
detected and their allelic dose in each genotype was scored. Linkage
disequilibrium within CONSTANS-LIKE rapidly decreased as expected. Eight SNPs
with a frequency above 10% were detected over 1,010 bp (one SNP every 126 bp on
average) in the 400 genotypes. This number was lower than observed in a neutral
gene (a SNP every 31 bp on average). Highly significant associations of three
SNPs to flowering date and stem height were identified. Each SNP explained up to
4.2% of the genetic variance. Thus, as in the model species, the CONSTANS-LIKE
gene was shown to be involved in flowering date and stem height in alfalfa.
DOI: 10.1007/s00122-010-1356-z
1298. Sci Total Environ. 2010 Jul 15;408(16):3193-9. doi:

10.1016/j.scitotenv.2010.04.010. Epub 2010 May 14.
Toxicity of 2,4-dinitrotoluene to terrestrial plants in natural soils.
Rocheleau S(1), Kuperman RG, Simini M, Hawari J, Checkai RT, Thiboutot S,

Ampleman G, Sunahara GI.
Author information:
Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
The presence of energetic materials (used as explosives and propellants) at

contaminated sites is a growing international issue, particularly with respect to
military base closures and demilitarization policies. Improved understanding of
the ecotoxicological effects of these materials is needed in order to accurately
assess the potential exposure risks and impacts on the environment and its
ecosystems. We studied the toxicity of the nitroaromatic energetic material
2,4-dinitrotoluene (2,4-DNT) on alfalfa (Medicago sativa L.), barnyard grass
(Echinochloa crusgalli L. Beauv.), and perennial ryegrass (Lolium perenne L.)
using four natural soils varying in properties (organic matter, clay content, and
pH) that were hypothesized to affect chemical bioavailability and toxicity.
Amended soils were subjected to natural light conditions, and wetting and drying
cycles in a greenhouse for 13 weeks prior to toxicity testing to approximate
field exposure conditions in terms of bioavailability, transformation, and
degradation of 2,4-DNT. Definitive toxicity tests were performed according to
standard protocols. The median effective concentration (EC(50)) values for shoot
dry mass ranged from 8 to 229 mg kg(-1), depending on the plant species and soil
type. Data indicated that 2,4-DNT was most toxic in the Sassafras (SSL) and
Teller (TSL) sandy loam soils, with EC(50) values for shoot dry mass ranging
between 8 to 44 mg kg(-1), and least toxic in the Webster clay loam soil, with
EC(50) values for shoot dry mass ranging between 40 to 229 mg kg(-1). The
toxicity of 2,4-DNT for each of the plant species was significantly (p < or =
0.05) and inversely correlated with the soil organic matter content. Toxicity
benchmark values determined in the present studies for 2,4-DNT weathered-and-aged
in SSL or TSL soils will contribute to development of an Ecological Soil
Screening Level for terrestrial plants that can be used for ecological risk
assessment at contaminated sites.
Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.
DOI: 10.1016/j.scitotenv.2010.04.010
1299. Plant Physiol. 2010 Jul;153(3):1161-74. doi: 10.1104/pp.110.157347. Epub 2010

May
13.
Large-scale comparative phosphoproteomics identifies conserved phosphorylation

sites in plants.
Nakagami H(1), Sugiyama N, Mochida K, Daudi A, Yoshida Y, Toyoda T, Tomita M,

Ishihama Y, Shirasu K.
Author information:
(1)RIKEN Plant Science Center, Tsurumi-ku, Yokohama 230-0045, Japan.
Knowledge of phosphorylation events and their regulation is crucial to understand

the functional biology of plants. Here, we report a large-scale phosphoproteome
analysis in the model monocot rice (Oryza sativa japonica 'Nipponbare'), an
economically important crop. Using unfractionated whole-cell lysates of rice
cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate
the conservation of phosphoproteomes between plant species, we developed a novel
phosphorylation-site evaluation method and performed a comparative analysis of
rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine
phosphorylation in the phosphoresidues of rice was equivalent to those in
Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use
of different cell types, more than 50% of the phosphoproteins identified in rice
and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein
in the other species. Moreover, nearly half of the phosphorylated orthologous
pairs were phosphorylated at equivalent sites. Further comparative analyses
against the Medicago phosphoproteome also showed similar results. These data
provide direct evidence for conserved regulatory mechanisms based on
phosphorylation in plants. We also assessed the phosphorylation sites on
nucleotide-binding leucine-rich repeat proteins and identified novel conserved
phosphorylation sites that may regulate this class of proteins.
DOI: 10.1104/pp.110.157347
PMCID: PMC2899915
1300. J Pharm Pharm Sci. 2010;13(1):78-92.
Carboxymethyl-starch excipients for gastrointestinal stable oral protein

formulations containing protease inhibitors.
De Koninck P(1), Archambault D, Hamel F, Sarhan F, Mateescu MA.
Author information:
(1)Department of Chemistry, Université du Québec à Montréal, Montréal (Québec)
Canada.
PURPOSE: The aim of this study was to develop a formulation for bioactive
compounds using Carboxymethyl Starch (CMS) as excipient containing protease
inhibitors. This formulation provided gastro protection and enhanced stability
against pancreatic enzymes. Such stability is needed for formulation of oral
vaccines with specific antigens.
METHODS: CMS was synthesized by treatment of starch with monochloroacetic acid in
conditions leading to a substitution degree of about 1 meq/g and used as
excipient for monolithic devices (300 mg tablets). Pefabloc SC and Aprotinin
inhibitors were tested in dissolution media and in formulation to prevent the
degradation of released bioactive materials. To evaluate the structural integrity
and biological stability of plant proteins in the CMS formulation, albumin and
lipase were added to the plant protein extract as protein and respectively as
enzyme markers. The amounts of released and recovered proteins were evaluated by
SDS-PAGE and densitometric analysis.
RESULTS: It was found that 1.6 % (w/w) of Pefabloc SC provides 98 % protection of
the released plant proteins for formulations of 30 % alfalfa protein extract
(APE) with CMS. In addition, when bovine serum albumin (BSA) added to the plant
protein extract as a marker, 90 % protection of the released BSA was observed.
Furthermore, a much higher lipase activity was found in the releasing media when
the formulations contained Pefabloc SC.
CONCLUSION: Formulations with CM-Starch excipients and containing protease
inhibitors prevent protein degradation and protect lipase activity, showing a
marked potential to use for orally administered bioactive peptides and
therapeutic enzymes.
1301. J Hazard Mater. 2010 Aug 15;180(1-3):63-70. doi:

10.1016/j.jhazmat.2010.02.069.
Epub 2010 Mar 1.
Winery wastewater inhibits seed germination and vegetative growth of common crop
species.
Mosse KP(1), Patti AF, Christen EW, Cavagnaro TR.
Author information:
(1)School of Applied Sciences and Engineering, Monash University, Churchill, Vic.
3842, Australia. kim.mosse@sci.monash.edu.au
The ability to reuse winery wastewater would be of significant benefit to the

wine industry, as it could potentially be a cost-effective method of wastewater
management, whilst at the same time providing a valuable water resource. This
study investigated the effects of different dilutions of a semi-synthetic winery
wastewater on the growth and germination of four common crop species in a
glasshouse study; barley (Hordeum vulgare), millet (Pennisetum glaucum), lucerne
(Medicago sativa) and phalaris (Phalaris aquatica). The wastewater caused a
significant delay in the germination of lucerne, millet and phalaris, although
overall germination percentage of all species was not affected. Vegetative growth
was significantly reduced in all species, with millet being the most severely
affected. The germination index of barley correlated very highly (r(2)=0.99) with
barley biomass, indicating that barley seed germination bioassays are highly
relevant to plant growth, and therefore may be of use as a bioassay for winery
wastewater toxicity.
DOI: 10.1016/j.jhazmat.2010.02.069
1302. J Dairy Res. 2010 Aug;77(3):337-42. doi: 10.1017/S002202991000021X. Epub 2010

May
10.
Effect of grazing fresh legumes or feeding silage on fatty acids and enzymes
involved in the synthesis of milk fat in dairy cows.
Wiking L(1), Theil PK, Nielsen JH, Sørensen MT.
Author information:
(1)Department of Food Science, Faculty of Agricultural Sciences, Aarhus
University, Research Centre Foulum, PO Box 50, DK-8830 Tjele, Denmark.
lars.wiking@agrsci.dk
The impact of fresh legume types or silage on the composition of milk fatty acids
and transcription of enzymes involved in the synthesis of milk fat in cows was
studied. Three groups of cows grazed high proportions of white clover, red clover
and lucerne, respectively. A fourth group of cows was fed maize/grass silage. The
cows grazing high proportions of legumes produced significantly more 18:1
trans-11, 18:2 cis9-trans11, 18:2 trans10-cis12 and 18:3 fatty acids than cows
fed silage. White clover and lucerne grazing resulted in significantly lower
output of 18:1 trans9 in milk than red clover grazing and maize/grass silages.
Transcription of stearoyl-CoA desaturase (SCD) in mammary tissue was
significantly increased by grazing high proportions of legume whereas fatty acid
synthase and acetyl-CoA carboxylase were not affected by type of feeding.
Furthermore, average milk fat globule diameter was correlated to daily milk fat
yield but was not affected by feeding. Although the fresh forage affected the
transcription of SCD in mammary tissue, the largest effects were on the
trans11-based fatty acids. It is concluded that type of forage, i.e. fresh or
silage, had a greater impact on rumen fermentation pattern than on transcription
of enzymes involved in the synthesis of milk fat.
DOI: 10.1017/S002202991000021X
Modeling demographic response to constant temperature in Hypera postica

(Coleoptera: Curculionidae).
Zahiri B(1), Fathipour Y, Khanjani M, Moharramipour S, Zalucki MP.
Author information:
(1)Department of Entomology, Faculty of Agriculture, Tarbiat Modares University,
P.O. Box 14115-336, Tehran, Iran.
Alfalfa weevil, Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), is among

the most destructive pests of alfalfa, Medicago sativa L., in the world.
Survivorship and fecundity schedules of H. postica were investigated to
characterize the population growth potential of the weevil at six constant
temperatures: 11.5, 14.0, 19.0, 24.0, 29.0, and 31.5 degrees C. Preoviposition
period, oviposition period and female longevity significantly decreased with
rising temperature within the temperature range tested. At the respective
temperatures adult female lived an average of 294.2, 230.2, 163.6, 141.0, 84.10,
and 32.9 d, with average lifetime progeny production of 470, 814, 2209, 3619,
2656, and 338 eggs per female. The net reproductive rates (R0) were 86.9, 288.0,
869.7, 1,479.7, 989.8, and 107.8 females per female, respectively. Mean daily
fecundity (Mx) was modeled as a function of time by using both Enkegaard and
Analytis models. Survivorship data (l(x)) of adult females were summarized and
compared using the shape and scale parameters of the Weibull frequency
distribution model across the temperature range tested. Life table entropy values
within the range 14.0-31.5 degrees C (H < 0.5) indicates Slobodkin's type I
survivorship curve; however, the value of 0.806 at 11.5 degrees C (H > 0.5)
corresponds to type III. As temperature increased, the r(m) exhibited an
asymmetrical dome-shaped pattern, with a maximum value of 0.114 females per
female per d at 29.0 degrees C. The r(m)-temperature relation of weevils was
modeled and critical temperatures (T(Min), T(Opt), and T(Max)) for intrinsic rate
of increase of the weevil were computed as 8.83, 30.61, and 32.14 degrees C and
5.72, 29.94, and 32.12 degrees C by using Analytis/Allahyari and
Analytis/Briere-2 models, respectively.
Interrupted incubation and short-term storage of the alfalfa pollinator Megachile

rotundata (Hymenoptera: Megachilidae): a potential tool for synchronizing bees
with bloom.
Yocum GD(1), Rinehart JP, West M, Kemp WP.
Author information:
(1)USDA-ARS Red River Valley Agricultural Research Center, Biosciences Research
Laboratory, 1605 Albrecht Blvd., Fargo, ND 58105, USA. george.yocum@ars.usda.gov
A useful technique for synchronizing pollinators with the alfalfa, Medicago

savita L. (Fabaceae), bloom is to interrupt the late spring incubation of
developing bee pupae and pharate adults of Megachile rotundata (F.) (Hymenoptera:
Megachilidae) with short-term low-temperature storage. However, low-temperature
exposure can be stressful depending on the temperature, duration of exposure, and
the developmental stage exposed. To evaluate the effect of low-temperature
storage after development had already been initiated by exposure to 29 degrees C,
three developmental stages (eye pigment pupae, body pigment pupae, and pharate
adults ready to emerge) were exposed to 6, 12, or 18 degrees C for durations up
to 28 d. The effect of delaying the termination of overwintering storage (6
degrees C) from April to July ("April bees" and "July bees," respectively) also
was examined. The following observations were made: (1) All developmental stages
of the April bees and July bees examined can be stored without harmful effects,
as measured by percentage of survival, for 14 d at 12 degrees C and above. (2)
Postponing the termination of overwintering storage at 6 degrees C from April
until July significantly decreased the mean number of days to 95% adult emergence
in the July ready-to-emerge bees across the three temperatures tested. (3) The
increase of overwintering duration also caused a decrease in the postemergence
longevity of the ready-to-emerge adults stored at 6 degrees C for 14 d. (4) Of
the three storage temperatures examined, 18 degrees C seems to be the optimal
storage temperature for short-term storage of developing bees because of their
slow but continuing development without increased mortality either during storage
or after emergence.
1305. Ann Bot. 2010 May;105(5):755-67. doi: 10.1093/aob/mcq040.
Multiple adaptive responses of Australian native perennial legumes with pasture

potential to grow in phosphorus- and moisture-limited environments.
Suriyagoda LD(1), Ryan MH, Renton M, Lambers H.
Author information:
(1)School of Plant Biology, The University of Western Australia, 35 Stirling
Highway, Crawley, WA 6009, Australia. suriyl01@student.uwa.edu.au
BACKGROUND AND AIMS: Many Australian legumes have evolved in low-phosphorus (P)
soils and low-rainfall areas. Therefore a study was made of the interaction of
soil [P] and water availability on growth, photosynthesis, water-use efficiency
(WUE) and P nutrition of two Australian native legumes with pasture potential,
Cullen australasicum and C. pallidum, and the widely grown exotic pasture legume,
lucerne (Medicago sativa).
METHODS: Plants were grown in a glasshouse at 3, 10 and 30 mg P kg(-1) dry soil
for 5 months. At week 10, two drought treatments were imposed, total pot dried
(all-dry) and only top soil dried (top-dry), while control pots were maintained
at field capacity.
KEY RESULTS: Shoot dry weight produced by lucerne was never higher than that of
C. australasicum. For C. pallidum only, shoot dry weight was reduced at 30 mg P
kg(-1) dry soil. The small root system of the Cullen species was quite plastic,
allowing plants to access P and moisture efficiently. Lucerne always had a higher
proportion of its large root system in the top soil layer compared with Cullen
species. All species showed decreased photosynthesis, leaf water potential and
stomatal conductance when exposed to drought, but the reductions were less for
Cullen species, due to tighter stomatal control, and consequently they achieved a
higher WUE. All species showed highest rhizosphere carboxylate concentrations in
the all-dry treatment. For lucerne only, carboxylates decreased as P supply
increased. Citrate was the main carboxylate in the control and top-dry
treatments, and malate in the all-dry treatment.
CONCLUSIONS: Multiple adaptive responses of Cullen species and lucerne favoured
exploitation of low-P soils under drought. The performance of undomesticated
Cullen species, relative to that of lucerne, shows their promise as pasture
species for environments such as in south-western Australia where water and P are
limiting, especially in view of a predicted drying and warming climate.
DOI: 10.1093/aob/mcq040
PMCID: PMC2859915
1306. Nat Prod Commun. 2010 Mar;5(3):485-504.
Toxicity of non-protein amino acids to humans and domestic animals.
Nunn PB(1), Bell EA, Watson AA, Nash RJ.
Author information:
(1)School of Pharmacy and Biomedical Sciences, St Michael's Building, University
of Portsmouth, Portsmouth, PO1 2DT, UK.
Non-protein amino acids are common in plants and are present in widely consumed
animal feeds and human foods such as alfalfa (Medicago sativa), which contains
canavanine, and lentil (Lens culinaris), which contains homoarginine. Some occur
in wild species that are inadvertently harvested with crop species. Some
non-protein amino acids and metabolites can be toxic to humans, e.g. Lathyrus
species contain a neurotoxic oxalyl-amino acid. Some potential toxins may be
passed along a food chain via animal intermediates. The increased interest in
herbal medicines in the Western countries will increase exposure to such
compounds.

Epub
2010 Apr 23.
Application of multilocus sequence typing to study the genetic structure of

megaplasmids in medicago-nodulating rhizobia.
van Berkum P(1), Elia P, Eardly BD.
Author information:
(1)Soybean Genomics and Improvement Laboratory, Agricultural Research Service,
U.S. Department of Agriculture, Beltsville, Maryland 20705, USA.
peter.vanberkum@ars.usda.gov
A multilocus sequence typing (MLST) analysis was used to examine the genetic
structure and diversity within the two large extrachromosomal replicons in
Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae).
The allelic diversity within these replicons was high compared to the reported
diversity within the corresponding chromosomes of the same strains (P. van Berkum
et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized
linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD.
Although both of these observations could be explained by positive (or
diversifying) selection by plant hosts, results of tests for positive selection
did not provide consistent support for this hypothesis. The strong LD observed
between the nodC and nifD genes could also be explained by their close proximity
on the pSymA replicon. Evidence was obtained that some nodC alleles had a history
of intragenic recombination, while other alleles of this locus had a history of
intergenic recombination. Both types of recombination were associated with a
decline in symbiotic competence with Medicago sativa as the host plant. The
combined observations of LD between the nodC and nifD genes and intragenic
recombination within one of these loci indicate that the symbiotic gene region on
the pSymA plasmid has evolved as a clonal segment, which has been laterally
transferred within the natural populations.
DOI: 10.1128/AEM.00251-10
PMCID: PMC2893487
1308. Carbohydr Res. 2010 Jun 16;345(9):1163-73. doi: 10.1016/j.carres.2010.03.015.

Epub 2010 Mar 20.
Structural investigation of an exopolysaccharide substituted with a lactyl ether

group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl
exclusion zone.
Pillon M(1), Pau-Roblot C, Lequart V, Pilard S, Courtois B, Courtois J,
Pawlicki-Jullian N.
Author information:
(1)Laboratoire des Polysaccharides Microbiens et Végétaux, Université de Picardie
Jules Verne, IUT d'Amiens, Avenue des Facultés, Amiens, France.
Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago

sativa harvested in the Chernobyl exclusion zone, produces a non-referenced
high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was
determined using a combination approach including monosaccharide composition
(GLC-FID, HPAEC-PAD), determination of glycosylation sites (GLC-EIMS) and 1D/2D
NMR ((1)H, (13)C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from
mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a
repeating unit composed of D-galactose, D-glucose, D-mannose and D-glucuronic
acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose
and glucose residues, respectively, as presented in the following structure:
Copyright (c) 2010 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.carres.2010.03.015
1309. Meat Sci. 2010 Aug;85(4):597-605. doi: 10.1016/j.meatsci.2010.03.010. Epub

2010
Mar 23.
Effect of forage species on fatty acid content and performance of

pasture-finished steers.
Dierking RM(1), Kallenbach RL, Grün IU.
Author information:
(1)Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA.
rmdq44@mizzou.edu
Consumers are increasingly concerned with the form and quantity of fat present in
the foods they consume. This is leading to a shift in the way food is produced.
In particular the animal industry is increasing the number of organic and
naturally finished meat animals rather than finishing them on grains. The
objective of this study was to determine if different pasture compositions (i.e.
grass only or grass legume mixtures) would impact the fatty acid (FA) meat
composition of beef steers (Bos taurus L.). The pasture treatments were tall
fescue [Lollium arundinaceum (Schreb.) S.J. Darbysh] only, or tall fescue
combined with either red clover (Trifolium pretense L.) or alfalfa (Medicago
sativa L. ssp. sativa L.). Beef steers (n=9-10 per treatment) rotationally grazed
each pasture treatment. Forage from treatments were different for crude protein
(CP), in vitro true digestibility (IVTD) and the FA myristric (C14:0), palmitic
(C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), linoleic (C18:2),
and total FA. Steers grazing mixtures with either red clover (RC) or alfalfa
(ALF) had greater average daily gains (ADG) than tall fescue only. Additionally,
RC treatment steers had larger ribeye areas (REA) and greater finishing weights
than those in the ALF treatment. However, the differences found between pasture
treatments in FA concentration did not translate to any differences in the FA
concentration of meat harvested from steers. Thus, it is concluded that although
the pastures contained different FA concentrations, with the levels of legumes
present, this does not influence the FA composition of beef.
Copyright (c) 2010. Published by Elsevier Ltd.
DOI: 10.1016/j.meatsci.2010.03.010
1310. Microbiology. 2010 Jul;156(Pt 7):2092-101. doi: 10.1099/mic.0.038547-0. Epub

2010
Apr 22.
Autoregulation of Sinorhizobium meliloti exoR gene expression.
Lu HY(1), Cheng HP.
Author information:
(1)Biological Sciences Department, Lehman College, The City University of New
York, Bronx, NY 10468, USA.
The successful nitrogen-fixing symbiosis between the gram-negative soil bacterium

Sinorhizobium meliloti and its leguminous plant host alfalfa (Medicago sativa)
requires the bacterial exopolysaccharide succinoglycan. Succinoglycan and
flagellum production, along with the ability to metabolize more than 20 different
carbon sources and control the expression of a large number of S. meliloti genes,
is regulated by the ExoR-ExoS/ChvI signalling pathway. The ExoR protein interacts
with and suppresses the sensing activities of ExoS, the membrane-bound sensor of
the ExoS/ChvI two-component regulatory system. Here we show that exoR expression
is clearly upregulated in the absence of any functional ExoR protein. This
upregulation was suppressed by the presence of the wild-type ExoR protein but not
by a mutated ExoR protein lacking signal peptide. The levels of exoR expression
could be directly modified in real time by changing the levels of total ExoR
protein. The expression of exoR was also upregulated by the constitutively active
sensor mutation exoS96, and blocked by two single mutations, exoS* and
exoS(supA), in the ExoS sensing domain. Presence of the wild-type ExoS protein
further elevated the levels of exoR expression in the absence of functional ExoR
protein, and reversed the effects of exoS96, exoS* and exoS(supA) mutations.
Altogether, these data suggest that ExoR protein autoregulates exoR expression
through the ExoS/ChvI system, allowing S. meliloti cells to maintain the levels
of exoR expression based on the amount of total ExoR protein.
DOI: 10.1099/mic.0.038547-0
PMCID: PMC3068678
1311. Plant J. 2010 Jul 1;63(1):100-14. doi: 10.1111/j.1365-313X.2010.04223.x. Epub

2010 Apr 7.
An NAC transcription factor orchestrates multiple features of cell wall

development in Medicago truncatula.
Zhao Q(1), Gallego-Giraldo L, Wang H, Zeng Y, Ding SY, Chen F, Dixon RA.
Author information:
To identify genes controlling secondary cell wall biosynthesis in the model

legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant
population for plants with altered patterns of lignin autofluorescence. From more
than 9000 R1 plants screened, four independent lines were identified with a total
lack of lignin in the interfascicular region. The mutants also showed loss of
lignin in phloem fibers, reduced lignin in vascular elements, failure in anther
dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls.
Microarray and PCR analyses confirmed that the mutations were caused by the
insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like
protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING
PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but
has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in
different combinations to control cell wall composition in stems and anthers.
Loss of MtNST1 function resulted in reduced lignin content, associated with
reduced expression of most lignin biosynthetic genes, and a smaller reduction in
cell wall polysaccharide content, associated with reduced expression of putative
cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase
digestion released significantly more sugars from cell walls of nst1 mutants
compared with the wild type. We discuss the implications of these findings for
the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.
DOI: 10.1111/j.1365-313X.2010.04223.x
1312. Lett Appl Microbiol. 2010 Jun 1;50(6):578-84. doi:

10.1111/j.1472-765X.2010.02836.x. Epub 2010 Mar 11.
Characterization of Kluyveromyces marxianus as a potential feed additive for

ruminants.
Nooraee SE(1), Alimon AR, Ho YW, Abdullah N.
Author information:
(1)Institute of Tropical Agriculture, Universiti Putra Malaysia, Serdang,
Selangor, Malaysia.
AIM: The aim of this study was to find suitable yeast isolates as potential
microbial feed additives for ruminants.
METHODS AND RESULTS: Yeast isolates from traditional fermented food (tapai) and
home-made wine were selected based on their tolerance to volatile fatty acids
(VFA) mixture of acetic, propionic and butyric acids and to pH and temperature
according to the rumen condition. The ability to grow in and produce ethanol was
determined in yeast extract peptone glucose broth supplemented with a VFA mixture
(VFA-YEPG medium). Fifty-five isolates showed OD(660 nm) values between 0.35-0.6,
and 27 isolates showed ethanol production in the range of 0.17-0.30% (v/v). All
selected isolates were identified as Kluyveromyces marxianus base on biochemical
tests (BioLog kit; Biolog Inc., Hayward, CA) and molecular techniques. The best
isolate in terms of ethanol production (K. marxianus WJ1) significantly (P <
0.01) improved in vitro apparent dry matter (DM) digestibility of alfalfa
(Medicago sativa), guinea grass (Panicum maximum) and timothy (Phleum pretense)
hay by rumen microbes.
CONCLUSION: Yeast isolates from tapai and wine were able to grow in VFA-YEPG
medium, and K. marxianus WJ1 improved in vitro DM digestibility of plant
substrates.
SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated the possibility of
using K. marxianus as a microbial feed additive.
DOI: 10.1111/j.1472-765X.2010.02836.x
1313. Environ Sci Technol. 2010 May 15;44(10):4006-11. doi: 10.1021/es903385g.
Energy efficiency of conventional, organic, and alternative cropping systems for

food and fuel at a site in the U.S. Midwest.
Gelfand I(1), Snapp SS, Robertson GP.
Author information:
(1)W. K. Kellogg Biological Station, Michigan State University, Hickory Corners,
Michigan 49060, USA. igelfand@msu.edu
The prospect of biofuel production on a large scale has focused attention on

energy efficiencies associated with different agricultural systems and production
goals. We used 17 years of detailed data on agricultural practices and yields to
calculate an energy balance for different cropping systems under both food and
fuel scenarios. We compared four grain and one forage systems in the U.S.
Midwest: corn (Zea mays) - soybean (Glycine max) - wheat (Triticum aestivum)
rotations managed with (1) conventional tillage, (2) no till, (3) low chemical
input, and (4) biologically based (organic) practices, and (5) continuous alfalfa
(Medicago sativa). We compared energy balances under two scenarios: all
harvestable biomass used for food versus all harvestable biomass used for biofuel
production. Among the annual grain crops, average energy costs of farming for the
different systems ranged from 4.8 GJ ha(-1) y(-1) for the organic system to 7.1
GJ ha(-1) y(-1) for the conventional; the no-till system was also low at 4.9 GJ
ha(-1) y(-1) and the low-chemical input system intermediate (5.2 GJ ha(-1)
y(-1)). For each system, the average energy output for food was always greater
than that for fuel. Overall energy efficiencies ranged from output:input ratios
of 10 to 16 for conventional and no-till food production and from 7 to 11 for
conventional and no-till fuel production, respectively. Alfalfa for fuel
production had an efficiency similar to that of no-till grain production for
fuel. Our analysis points to a more energetically efficient use of cropland for
food than for fuel production and large differences in efficiencies attributable
to management, which suggests multiple opportunities for improvement.
DOI: 10.1021/es903385g
1314. Toxicon. 2010 Sep 1;56(3):411-7. doi: 10.1016/j.toxicon.2010.04.004. Epub

2010
Apr 14.
Influence of the cyanotoxin microcystin-LR on tocopherol in Alfalfa seedlings

(Medicago sativa).
Peuthert A(1), Pflugmacher S.
Author information:
(1)Leibniz Institute of Freshwater Ecology and Inland Fisheries, Department of
Aquaculture and Ecophysiology, Müggelseedamm 301, 12587 Berlin, Germany.
Spray irrigation of crop plants can represent a risk if the water is contaminated
with cyanotoxins. These secondary metabolites produced by many cyanobacteria can
have adverse effects on organisms, among others the induction of excess oxygen
radicals, so-called oxidative stress. Thereby tocopherol as a lipid antioxidant
is essential to maintain membrane integrity. In this study the effects of the
cyanotoxin microcystin-LR (MC-LR), and a Microcystis crude extract (Mic-CE)
containing MC-LR on tocopherol content and gene expression of Homogentisate
phytyltransferase (HPT), involved in tocopherol synthesis, were investigated in
seedlings of the crop plant Medicago sativa. Exposures to environmental relevant
concentrations showed that Mic-CE affects the tocopherols at a lower
concentration than the pure toxin. Changes during a 3-day exposure using 0.5mug
L(-1) MC-LR and Mic-CE revealed strong increases of alpha- and beta-tocopherol
content, whereas the influence on beta-tocopherol was far stronger than on
alpha-tocopherol. The HPT mRNA was elevated after 24 and 72h exposure to MC-LR.
These results show that also low concentrations of cyanobacterial toxins in the
water have strong influence on tocopherol in M. sativa seedlings and therefore
represent a stress for the plants.
DOI: 10.1016/j.toxicon.2010.04.004
1315. Environ Sci Pollut Res Int. 2010 Sep;17(8):1448-59. doi:

10.1007/s11356-010-0330-2.
The demography of feral alfalfa (Medicago sativa L.) populations occurring in

roadside habitats in Southern Manitoba, Canada: implications for novel trait
confinement.
Bagavathiannan MV(1), Gulden RH, Begg GS, Van Acker RC.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, Canada.
umbagava@cc.umanitoba.ca
PURPOSE: Feral populations of cultivated crops can act as reservoirs for novel
genetically engineered (GE) traits and aid in trait movement at the landscape
level. However, little information is available on the potential of cultivated
crops to become feral. In this study, we investigated the ferality of alfalfa
populations (non-GE version) occurring in roadside habitats. Knowledge on the
nature of roadside alfalfa populations would be useful for designing efficient
trait confinement protocols and coexistence strategies in alfalfa.
METHODS: We investigated roadside alfalfa populations from 2006 to 2009 in three
rural municipalities (Hanover, MacDonald, and Springfield) in Southern Manitoba,
Canada. We studied the demography of these populations including seedbank,
seedling recruitment, and fecundity and examined the impact of road verge mowing
on key life stages of these populations. We also compared the growth and
reproductive attributes of roadside and cultivated alfalfa populations.
RESULTS: Alfalfa is reproductively successful in roadside habitats and capable of
establishing self-perpetuating populations. A substantial portion of the alfalfa
seeds we extracted from seedbank samples were viable but not germinable,
suggesting some degree of seedbank persistence in roadside habitats. In the
roadside habitat, alfalfa seedlings recruited successfully, however, seedling
mortality was high when seedlings were in close proximity to well-established
alfalfa plants. Mowing dramatically reduced the reproductive success of roadside
alfalfa. Generally, the growth and reproduction of roadside alfalfa was
comparable to cultivated alfalfa except for total fecundity.
CONCLUSIONS: Considering the long lifespan (>10 years) of alfalfa and the levels
of fecundity, seedbank, and seedling survival we observed, long-term persistence
of roadside alfalfa populations seems reasonable. In the context of novel trait
confinement, our results suggest that feral alfalfa populations required to be
managed if there is a desire/need to confine novel traits in alfalfa.
DOI: 10.1007/s11356-010-0330-2
1316. Environ Entomol. 2010 Apr;39(2):576-82. doi: 10.1603/EN08308.
Frass analysis of diets of aphidophagous lady beetles (Coleoptera: Coccinellidae)

in Utah alfalfa fields.
Davidson LN(1), Evans EW.
Author information:
(1)Department of Biology, Utah State University, Logan, UT 84322-5305, USA;
Northern Plains Agricultural Research Laboratory, USDA-ARS, 1500 North Central
Avenue, Sidney MT 59270, USA.
Aphidophagous lady beetles enhance their foraging success in natural settings by

consuming other types of food in addition to aphids. Frass analysis was used to
examine natural diets of female lady beetles in fields of alfalfa (Medicago
sativa L.) in northern Utah. The first (spring) alfalfa crop was censused in 2004
and 2005 to determine the diet of female adults of the introduced Coccinella
septempunctata L., and two native species, C. transversoguttata richardsoni
Brown, and Hippodamia convergens Guerin. The proportion of females of the three
lady beetle species that fed on pea aphids [Acyrthosiphon pisum (Harris)] and
alfalfa weevil larvae [Hypera postica (Gyllenhal), an abundant alternative prey]
increased from early to late season during the first crop. A corresponding
seasonal decrease occurred in the proportion of females consuming other types of
arthropods (e.g., thrips and collembolans) and nonarthropod food (pollen and
fungal spores). Overall, frass analysis indicated that the diets of C.
septempunctata and the two native species in alfalfa were similar in their
inclusion of a broad variety of foods. The study shows that frass analysis can
provide a good overview of the diets of lady beetles in natural settings.
DOI: 10.1603/EN08308
1317. Ying Yong Sheng Tai Xue Bao. 2010 Jan;21(1):165-73.
[Soil microbial properties under different vegetation types in Loess hilly

region].
Zhang YY(1), Qu LY, Chen LD, Wei W.
Author information:
(1)State Key Laboratory of Urban and Regional Ecology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
zhangyanyans@gmail.com
By using fumigation-extract (FE) method and Biolog Ecoplate, this paper

investigated the microbial biomass and diversity in 0-20 cm soil layer under five
vegetation types, including artificial woodland, shrubland, cropland, abandoned
farmland, and natural grassland, in Dingxi of Gansu Province. In the meanwhile,
the relationships between soil microbes and soil nutrients were studied by path
analysis, and the five typical vegetation types were evaluated from the aspect of
soil microbes. Relative to cropland, "grain for green" project played a key role
in improving soil microbial resources. Microbial biomass carbon was the highest
in ridge grassland, abandoned farmland, and pine woodland, followed by in
Caragana korshinskii land, Medicago sativa land, restored land, and roadside
land, and in wheat field and potato field. Microbial biomass nitrogen was the
highest in ridge land, abandoned farmland, Pinus tabulaeformis woodland, Caragana
korshinskii land, and Medicago sativa land, followed by in restored land and
roadside land, and in wheat field and potato field. Caragana korshinskii land and
Medicago sativa land, due to the existence of N-fixing rhizobium, had the highest
ratio of soil microbial biomass nitrogen to soil total nitrogen. Owing to the
continual biomass loss and rare feedback, cropland had the lowest quantity and
activity of soil microbes. Through planting trees, shrubs and grasses or through
fallowing, soil microbial biomass and activity were recovered, and the effect was
increased with time. In 20-year old Caragana korshinskii land, the quantity and
activity of soil microbes were similar to those in 50-year old Pinus
tabulaeformis woodland, and the microbial community catabolic activity and soil
nutrient use efficiency were higher. Considering the features of soil microbes
under test vegetation types, Caragana korshinskii would be a good choice for
local vegetation restoration.
1318. Ecotoxicol Environ Saf. 2010 Jul;73(5):970-81. doi:

10.1016/j.ecoenv.2010.01.013.
Epub 2010 Apr 10.
Plant treatment, pollutant load, and soil type effects in rhizosphere ecology of
trace element polluted soils.
Belén Hinojosa M(1), Carreira JA, García-Ruíz R, Rodríguez-Maroto JM, Daniell TJ,
Griffiths BS.
Author information:
(1)Dpto Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, 23071
Jaén, Spain. MariaBelen.Hinojosa@uclm.es
Re-vegetation of trace element contaminated soils can alter the pH and chelating
capacity in the rhizosphere, increasing the mobility of pollutants, which, in
turn, may impact on rhizosphere ecology. In this study a short-term pot
experiment was carried out in order to investigate the multi-factorial effects
of: buffering capacity (sandy-loam and loam soils); pollutant load (0%, 1.3%, and
4% of pyrite sludge), and the presence/absence of plant (Lolium perenne L. and
Medicago sativa L.) on the mobility of trace elements, soil biochemical
functionality (hydrolase activities), and biological diversity (bacterial and
nematode communities). The experiment was carried out with representative soils
from the Guadiamar basin (SW Spain), an area where the Aznalcóllar mining spill
affected over 4000ha. Results indicated that the development of rhizospheres in
polluted soils (coarse-textured) increases the mobilization of trace elements. In
general the presence of roots has stimulatory effects on soil quality indicators
such as hydrolase activities and both bacterial and nematode communities.
However, the presence of high amount of metals interferes with these beneficial
effects. This study provided evidence about the complexity of the impact of
growing plants on trace element polluted soils. Trace element mobilization,
hydrolase activities and bacterial and nematode communities in the rhizosphere
are dependent on plant species, soil type, and pollution dose.
Copyright (c) 2010 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ecoenv.2010.01.013
1319. Int J Syst Evol Microbiol. 2011 Mar;61(Pt 3):512-7. doi:
10.1099/ijs.0.020156-0.
Epub 2010 Apr 9.
Rhizobium tubonense sp. nov., isolated from root nodules of Oxytropis glabra.
Zhang RJ(1), Hou BC, Wang ET, Li Y Jr, Zhang XX, Chen WX.
Author information:
(1)State Key Laboratories for Agro-Biotechnology, Ministry of Agriculture/College
of Biological Sciences, China Agricultural University, Beijing 100193, PR China.
Four rhizobial strains, designated CCBAU 85046(T), CCBAU 85051, CCBAU 85048 and
CCBAU 85049, isolated from root nodules of Oxytropis glabra grown in Tibet,
China, were previously defined, using amplified 16S rRNA gene restriction
analysis, as a novel group within the genus Rhizobium. To clarify their taxonomic
position, these strains were further analysed and compared with reference strains
of related bacteria using a polyphasic approach. The 16S rRNA gene analysis
showed that the four isolates formed a distinct phylogenetic lineage in the genus
Rhizobium. The isolates showed highest sequence similarity (97.8 %) to Rhizobium
indigoferae CCBAU 71042(T). Phenotypic and physiological tests, DNA-DNA
hybridization, phylogenetic analyses of housekeeping genes recA, atpD and glnII
and fatty acid profiles also indicated that these four strains constitute a novel
group distinct from recognized species of the genus Rhizobium. Based on this
evidence, strains CCBAU 85046(T), CCBAU 85051, CCBAU 85048 and CCBAU 85049
represent a novel species in the genus Rhizobium, for which the name Rhizobium
tubonense sp. nov. is proposed. The type strain is CCBAU 85046(T) (=LMG 25225(T)
=HAMBI 3066(T)) and its DNA G+C content is 59.52 % (T(m)). Strain CCBAU 85046(T)
could form effective nodules on plant species Vigna unguiculata and Medicago
sativa but not on its host of origin Oxytropis glabra.
DOI: 10.1099/ijs.0.020156-0
1320. Meat Sci. 2010 Mar;84(3):545-52. doi: 10.1016/j.meatsci.2009.10.010. Epub

2009
Oct 14.
Lucerne grazing compared with concentrate-feeding slightly modifies carcase and

meat quality of young bulls.
Blanco M(1), Casasús I, Ripoll G, Panea B, Albertí P, Joy M.
Author information:
(1)Unidad de Tecnología en Producción Animal, CITA-Gobierno de Aragón, Avda.
Montañana 930, 50059 Zaragoza, Spain. mblanco@pctad.com
Carcase and meat quality of young bulls raised on one of three fattening
strategies from 224 to 450 kg were compared. One group was fed concentrates
(CON), another group grazed on lucerne plus 1.8 kg DM barley/day (LUC) and the
last group had the same management as LUC young bulls for 3 months and was then
finished on concentrates for 2 months. Among carcase traits, only tissue
composition differed, with LUC young bulls having more muscle and less
subcutaneous and intermuscular fat than their counterparts. Concerning meat
quality, most attributes did not differ among fattening strategies but LUC young
bulls had the lowest intramuscular fat, which presented greatest n-3 PUFA
content. It can be concluded that lucerne grazing can be a good alternative to
concentrates for young bulls, with similar carcase and meat quality but with
lower fat content and healthier fatty acid composition than young bulls fed
concentrates during the finishing period.
DOI: 10.1016/j.meatsci.2009.10.010
1321. Nutr Cancer. 2010;62(3):351-61. doi: 10.1080/01635580903407213.
Resveratrol from transgenic alfalfa for prevention of aberrant crypt foci in

mice.
Kineman BD(1), Brummer EC, Paiva NL, Birt DF.
Author information:
(1)Iowa State University, Ames, Iowa 50011, USA.
Transgenic alfalfa (Medicago sativa L.), which accumulated resveratrol-glucoside

(RG), was incorporated into diets and fed to female, 6-wk-old CF-1 mice for 5 wk.
Mice fed diets containing transgenic alfalfa with supplemented alpha
-galactosidase had significantly fewer azoxymethane (AOM)-induced aberrant crypt
foci (ACF) in their colon relative to mice fed the transgenic alfalfa diets
without added alpha -galactosidase (P = 0.02). Resveratrol-aglycone (Rag) was
detected in the colon of 100% of mice fed transgenic alfalfa diets with
supplemented alpha -galactosidase and in 60% of mice fed transgenic alfalfa
without alpha -galactosidase (P < 0.05). Colonic concentrations of Rag (< 0.5
nmol/g tissue) in mice fed transgenic alfalfa with alpha -galactosidase (0.22 +/-
0.18 nmol/g tissue) tended to be higher than in animals fed diets without alpha
-galactosidase (0.1 +/- 0.08 nmol/g tissue; P = 0.09). The use of
N-(Bn-butyl)-deoxygalactonojirimycin, an inhibitor of lactase-phlorizin hydrolase
(LPH), in transport studies with everted jejunal sacs from CF-1 mice (N = 8)
suggested that LPH is involved in the intestinal deglycosylation of RG. Our
collective findings suggest that RG from transgenic alfalfa is metabolized and
absorbed in the upper intestine and does not reach the colon in sufficient
amounts to inhibit ACF.
DOI: 10.1080/01635580903407213
1322. J Sci Food Agric. 2010 Jan 15;90(1):78-84. doi: 10.1002/jsfa.3783.
Effect of inoculated corn silage enriched with sunflower oil on rumen

fermentation and lipid metabolism in an artificial rumen (RUSITEC).
Jalc D(1), Váradyová Z, Lauková A.
Author information:
(1)Institute of Animal Physiology, Slovak Academy of Sciences, Soltésovej 4-6,
040 01 Kosice, Slovak Republic. jalcd@saske.sk
BACKGROUND: Some rumen isolates are able to produce conjugated linoleic acid
(CLA) from linoleic acid (LA) in vitro. Effects of providing diets containing
corn silage (CS) and lucerne hay to an artificial rumen (RUSITEC) in which the
corn was not inoculated (CS), or inoculated with Lactobacillus plantarum CCM 4000
(CS + LP), Lactobacillus fermentum LF2 (CS + LF) or Enterococcus faecium CCM 4231
(CS + EF) and supplied with sunflower oil (SO; 30 g kg(-1); w/w) on rumen
metabolism were examined.
RESULTS: The SO affected the outputs of all fatty acids. TVA output of
uninoculated CS with SO was lower as compared to inoculated CS. The interaction
of the CS x SO in the daily output of TVA was detected (P < 0.001). The
biohydrogenation of oleic, linoleic, alpha-linolenic and total fatty acids was
influenced by SO (P < 0.05 and P < 0.001).
CONCLUSION: Inoculated silage induces changes in the rumen metabolism which might
be related to differences observed in the extent of rumen BH of PUFA in RUSITEC.
SO supplementation might positively enhances the production of some rumen
intermediates; however, relationships between inoculated silages and oil
supplementation can be presumed in the daily production of trans-vaccenic acid.
Copyright (c) 2009 Society of Chemical Industry.
DOI: 10.1002/jsfa.3783
1323. Genet Mol Biol. 2010 Apr;33(2):285-91. doi: 10.1590/S1415-47572010005000031.

Epub
2010 Jun 1.
Molecular cloning and expression profiling of a chalcone synthase gene from hairy
root cultures of Scutellaria viscidula Bunge.
Lei W(1), Tang SH, Luo KM, Sun M.
Author information:
(1)Key Laboratory of Eco-environments in Three Gorges Reservoir Region of
Ministry of Education, School of Life Science, Southwest University, Chongqing
China.
A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid

biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula
Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S.
viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp
with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of
390 amino acid residues, a calculated molecular mass of 42.56 kDa and a
theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed
that SvCHS shared high homology with CHS from other plants. Functional analysis
in silico indicated that SvCHS was a hydrophilic protein most likely associated
with intermediate metabolism. The active sites of the malonyl-CoA binding motif,
coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found
in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS
contained mainly α-helixes and random coils. Phylogenetic analysis showed that
SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement
with its function as an elicitor-responsive gene, the expression of Svchs was
induced and coordinated by methyl jasmonate. To our knowledge, this is the first
report to describe the isolation and expression of a gene from S. viscidula.
DOI: 10.1590/S1415-47572010005000031
PMCID: PMC3036846
PMID: 21637484

Epub
2010 Mar 30.
Inferring population structure and genetic diversity of broad range of wild

diploid alfalfa (Medicago sativa L.) accessions using SSR markers.
Sakiroğlu M(1), Doyle JJ, Charles Brummer E.
Author information:
(1)Department of Biology, Kafkas University, 36100, Kars, Turkey.
msakiroglu@kafkas.edu.tr
Diversity analyses in alfalfa have mainly evaluated genetic relationships of

cultivated germplasm, with little known about variation in diploid germplasm in
the M. sativa-falcata complex. A collection of 374 individual genotypes derived
from 120 unimproved diploid accessions from the National Plant Germplasm System,
including M. sativa subsp. caerulea, falcata, and hemicycla, were evaluated with
89 polymorphic SSR loci in order to estimate genetic diversity, infer the genetic
bases of current morphology-based taxonomy, and determine population structure.
Diploid alfalfa is highly variable. A model-based clustering analysis of the
genomic data identified two clearly discrete subpopulations, corresponding to the
morphologically defined subspecies falcata and caerulea, with evidence of the
hybrid nature of the subspecies hemicycla based on genome composition. Two
distinct subpopulations exist within each subsp. caerulea and subsp. falcata. The
distinction of caerulea was based on geographical distribution. The two falcata
groups were separated based on ecogeography. The results show that taxonomic
relationships based on morphology are reflected in the genetic marker data with
some exceptions, and that clear distinctions among subspecies are evident at the
diploid level. This research provides a baseline from which to systematically
evaluate variability in tetraploid alfalfa and serves as a starting point for
exploring diploid alfalfa for genetic and breeding experiments.
DOI: 10.1007/s00122-010-1319-4
1325. BMC Microbiol. 2010 Mar 27;10:92. doi: 10.1186/1471-2180-10-92.
Identification and characterization of the intracellular poly-3-hydroxybutyrate

depolymerase enzyme PhaZ of Sinorhizobium meliloti.
Trainer MA(1), Capstick D, Zachertowska A, Lam KN, Clark SR, Charles TC.
Author information:
(1)Department of Biology, University of Waterloo, 200 University Ave W, Waterloo,
ON N2L 3G1, Canada.
BACKGROUND: S. meliloti forms indeterminate nodules on the roots of its host

plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are
terminally differentiated and, unlike their non-terminally differentiated
counterparts in determinate nodules, do not accumulate large quantities of
Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB
depolymerase; it represents the first enzyme in the degradative arm of the PHB
cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that
remains uncharacterized.
RESULTS: The S. meliloti phaZ gene was identified by in silico analysis, the ORF
was cloned, and a S. meliloti phaZ mutant was constructed. This mutant exhibited
increased PHB accumulation during free-living growth, even when grown under
non-PHB-inducing conditions. The phaZ mutant demonstrated no reduction in
symbiotic capacity; interestingly, analysis of the bacteroids showed that this
mutant also accumulated PHB during symbiosis. This mutant also exhibited a
decreased capacity to tolerate long-term carbon starvation, comparable to that of
other PHB cycle mutants. In contrast to other PHB cycle mutants, the S. meliloti
phaZ mutant did not exhibit any decrease in rhizosphere competitiveness; however,
this mutant did exhibit a significant increase in succinoglycan biosynthesis.
CONCLUSIONS: S. meliloti bacteroids retain the capacity to synthesize PHB during
symbiosis; interestingly, accumulation does not occur at the expense of symbiotic
performance. phaZ mutants are not compromised in their capacity to compete for
nodulation in the rhizosphere, perhaps due to increased succinoglycan production
resulting from upregulation of the succinoglycan biosynthetic pathway. The
reduced survival capacity of free-living cells unable to access their accumulated
stores of PHB suggests that PHB is a crucial metabolite under adverse conditions.
DOI: 10.1186/1471-2180-10-92
PMCID: PMC2867953
The effect of wide swathing on wilting times and nutritive value of alfalfa
haylage.
Kung L Jr(1), Stough EC, McDonell EE, Schmidt RJ, Hofherr MW, Reich LJ,
Klingerman CM.
Author information:
(1)Department of Animal & Food Sciences, University of Delaware, Newark 19716,
USA. lksilage@udel.edu
On 3 consecutive cuttings, alfalfa from a single field was mowed with a John
Deere 946 mower-conditioner (4-m cut width; Moline, IL) to leave narrow swaths
(NS) ranging from 1.2 to 1.52 m wide (30-37% of cutter bar width) and wide swaths
(WS) ranging from 2.44 to 2.74 m wide (62-67% of cutter bar width). Samples were
collected from windrows and dry matter (DM) was monitored during wilting until a
target of 43 to 45% DM was obtained. Forage from random windrows (n=4-6) was
harvested by hand, chopped through a forage harvester before being packed in
replicated vacuum-sealed bags, and allowed to ensile for 65 d. There was no swath
width x cutting interaction for any parameter tested. Over all cuttings, the
resulting silage DM was not different between the NS silage (43.8%) and the WS
silage (44.9%). However, wide swathing greatly reduced the time of wilting before
making silage. The hours of wilting time needed to reach the targeted DM for the
NS silage compared with the WS silage at cuttings 1, 2, and 3 were 50 versus 29,
54 versus 28, and 25 versus 6, respectively. At the time of ensiling, the WS
silage had more water-soluble carbohydrates (5.1%) than did the NS silage (3.7%).
The WS silage had a lower pH (4.58) than did the NS silage (4.66), but swath
width did not affect fermentation end products (lactic acid, acetic acid, and
ethanol). The NS silage had more NH(3)-N (0.26%) than did the WS silage (0.21%).
Wide swathing did not affect the concentration of ash or the digestibility of
NDF, but it lowered the N content (NS=3.45%; WS=3.23%) and increased the ADF
content (NS=39.7%; WS=40.9%) of the resulting silage. Wide swathing can markedly
reduce the time that alfalfa must wilt before it can be chopped for silage, but
under good conditions, as in this study, the resulting silage quality was
generally not improved.
DOI: 10.3168/jds.2009-2451
1327. Mol Biol Rep. 2011 Jan;38(1):23-9. doi: 10.1007/s11033-010-0073-7. Epub 2010
Mar
18.
Isolation and characterization of a harvest-inducible gene hi11 and its promoter

from alfalfa.
Zhang J(1), Xiong AS, Erickson LR.
Author information:
(1)Plant Agriculture Department, University of Guelph, Guelph, ON, N1G 2W1l,
Canada. zhangj@arc.ab.ca
The harvesting and storing of alfalfa is a routine practice in the agricultural

industry worldwide. To investigate gene expression in harvested alfalfa, cDNA
from non-harvested and harvested plants in the field was subjected to subtractive
hybridization to identify, in particular, those genes that are induced by the
harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The
full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11
gene, which has high homology to a putative protein of unknown function in
Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a
wounding treatment. Northern blot analysis confirmed that the expression patterns
of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In
addition, genomic walking was performed to isolate the 5' flanking region of the
hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and
transferred to Medicago truncatula and tobacco. In all transgenic plants of M.
truncatula and tobacco, GUS gene expression was observed in harvested tissue,
especially in the transgenic tobacco plants, but not in the non-harvested control
tissue.
DOI: 10.1007/s11033-010-0073-7
1328. Protoplasma. 2010 Jun;242(1-4):69-80. doi: 10.1007/s00709-010-0125-9. Epub

2010
Mar 17.
NaCl effect on the distribution of wall ingrowth polymers and arabinogalactan

proteins in type A transfer cells of Medicago sativa Gabès leaves.
Boughanmi N(1), Thibault F, Decou R, Fleurat-Lessard P, Béré E, Costa G,

Lhernould S.
Author information:
(1)Département des Sciences de la Vie, Faculté des Sciences de Bizerte, 7021,
Zarzouna, Bizerte, Tunisia. nezighanem@yahoo.fr
We studied the distribution of wall ingrowth (WI) polymers by probing thin

sections of companion cells specialized as transfer cells in minor veins of
Medicago sativa cv Gabès blade with affinity probes and antibodies specific to
polysaccharides and glycoproteins. The wall polymers in the controls were similar
in WIs and in the primary wall but differently distributed. The extent of
labeling in these papillate WIs differed for JIM5 and JIM7 homogalacturonans but
was in the same range for LM5 and LM6 rhamnogalacturonans and xyloglucans. These
data show that WI enhancement probably requires arabinogalactan proteins (JIM8)
mainly localized on the outer part of the primary wall and WIs. By comparison,
NaCl-treated plants exhibited cell wall polysaccharide modifications indicating
(1) an increase in unesterified homogalacturonans (JIM5), probably implicated in
Na(+) binding and/or polysaccharide network interaction for limiting turgor
variations in mesophyll cells; (2) enhancement of the xyloglucan network with an
accumulation of fucosylated xyloglucans (CCRC-M1) known to increase the capacity
of cellulose binding; and (3) specific recognition of JIM8 arabinogalactan
proteins that could participate in both wall enlargement and cohesion by
increasing the number of molecular interactions with the other polymers. In
conclusion, the cell wall polysaccharide distribution in enlarged WIs might (1)
participate in wall resistance to sequestration of Na(+), allowing a better
control of hydric homeostasis in mesophyll cells to maintain metabolic activity
in source leaves, and (2) maintain tolerance of M. sativa to NaCl.
DOI: 10.1007/s00709-010-0125-9
1329. Food Microbiol. 2010 May;27(3):332-8. doi: 10.1016/j.fm.2009.11.003. Epub

2009
Nov 13.
Inactivation of Salmonella and Escherichia coli O157:H7 on artificially

contaminated alfalfa seeds using high hydrostatic pressure.
Neetoo H(1), Chen H.
Author information:
19716-2150, USA.
Alfalfa sprouts contaminated with Salmonella and Escherichia coli O157:H7 have
been implicated in several outbreaks of foodborne illnesses in recent years. The
seed used for sprouting appears to be the primary source of pathogens. Seed
decontamination prior to sprouting presents a unique challenge for the sprouting
industry since cells of the pathogenic survivors although undetectable after
sanitizing treatments, can potentially multiply back to hazardous levels. The
focus of this study was to therefore test the efficacy of high hydrostatic
pressure to eliminate a approximately 5 log CFU/g load of Salmonella and E. coli
O157:H7 on alfalfa seeds. Pressure treatment of 600 MPa for up to 25 min at 20
degrees C could not result in complete inactivation of Salmonella. High-pressure
treatment was then carried out either at sub-ambient (4 degrees C) or elevated
(40, 45 and 50 degrees C) temperatures to test the ability of high pressure to
eliminate Salmonella. Pressure treatment at 4 and 20 degrees C did not deliver
any satisfactory inactivation of Salmonella while high pressure at elevated
temperatures achieved complete kill. Pre-soaking seeds prior to high-pressure
treatment also enhanced pressure inactivation of Salmonella but at the expense of
seed viability. High-pressure treatment of 500 MPa for 2 min at 45 degrees C was
able to eliminate wild-type Salmonella and E. coli O157:H7 strains without
bringing about any appreciable decrease in the seed viability.
Copyright (c) 2009 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.fm.2009.11.003
1330. Guang Pu Xue Yu Guang Pu Fen Xi. 2009 Dec;29(12):3250-3.

[Near-infrared reflectance spectroscopy analytic models established for the crude
protein and crude fiber of alfalfa property].
Shang C(1), Zhang YX, Li Y, Hao RC, Tang FL, Han WB, Liu JL.
Author information:
(1)Life Science and Technology Institute, Harbin Normal University, Harbin
150025, China.
Near-infrared reflectance spectroscopy (NIRS) calibrations of crude protein and

crude fiber in 152 alfalfa samples were developed by means of partial
least-squares (PLS) regression. Different interval wavelengths of 1, 2 and 5 nm
and two kinds of samples (crude and fine) were set to collect spectral data and
establish calibration respectively. The accuracy and repeatability of
calibrations were verified so as to compare superiority and inferiority. Results
showed that the best validation result is the calibration by 2 nm interval
wavelength and fine sample, and the correlation coefficients of calibration (R2)
were 0.97 and 0.94 for crude protein and crude fiber, and the SECV of these
parameters were 0.42 and 0.78 respectively. The coefficients of determination for
validation (R2(val)) were 0.96 and 0.92, and the SEP of these parameters were
0.43 and 0.79. The experiment showed that it is feasible to evaluate the major
quality traits of alfalfa by NIRS. All together, it provided a new model to
verify the crude protein and fiber compositions of alfalfa.
1331. J Plant Physiol. 2010 Jun 15;167(9):683-92. doi: 10.1016/j.jplph.2009.12.016.

Epub 2010 Mar 6.
The efficiency of nitrogen fixation of the model legume Medicago truncatula

(Jemalong A17) is low compared to Medicago sativa.
Sulieman S(1), Schulze J.
Author information:
(1)Department of Crop Sciences, Plant Nutrition, Georg-August-University of
Goettingen, Goettingen, Germany.
Medicago truncatula (Gaertn.) (barrel medic) serves as a model legume in plant

biology. Numerous studies have addressed molecular aspects of the biology of M.
truncatula, while comparatively little is known about the efficiency of N(2)
fixation at the whole plant level. The objective of the present study was to
compare the efficiency of N(2) fixation of M. truncatula to the genetically
closely related Medicago sativa (L.) (alfalfa). The relative growth of both
species relying exclusively on N(2) fixation versus nitrate nutrition, H(2)
evolution, nitrogen assimilation, the concentration of amino acids and organic
acids in nodules, and (15)N(2) uptake and distribution were studied. M.
truncatula showed much lower efficiency of N(2) fixation. Nodule-specific
activity was several-fold lower when compared to M. sativa, partially as a result
of a lower electron allocation to N(2) versus H(+). M. truncatula or M. sativa
plants grown solely on N(2) fixation as a nitrogen source reached about 30% or
80% of growth, respectively, when compared to plants supplied with sufficient
nitrate. Moreover, M. truncatula had low %N in shoots and a lower allocation of
(15)N to shoots during 1h (15)N(2) labeling period. Amino acid concentration was
about 20% higher in M. sativa nodules, largely as a result of more asparagine,
while the organic acid concentration was about double in M. sativa, coinciding
with a six-fold higher concentration of malate. Total soluble protein in nodules
was about three times lower in M. truncatula and the pattern of enzyme activity
in that fraction was strongly different. Sucrose cleaving enzymes displayed
higher activity in M. truncatula nodules, while the activity of
phosphoenolpyruvate carboxylase (PEPC) was much lower. It is concluded that the
low efficiency of the M. truncatula symbiotic system is related to a low capacity
of organic acid formation and limited nitrogen export from nodules.
DOI: 10.1016/j.jplph.2009.12.016
1332. BMC Microbiol. 2010 Mar 6;10:71. doi: 10.1186/1471-2180-10-71.
The Sinorhizobium meliloti RNA chaperone Hfq influences central carbon metabolism
and the symbiotic interaction with alfalfa.
Torres-Quesada O(1), Oruezabal RI, Peregrina A, Jofré E, Lloret J, Rivilla R,

Toro N, Jiménez-Zurdo JI.
Author information:
CSIC, Profesor Albareda, 1, 18008 Granada, Spain.
BACKGROUND: The bacterial Hfq protein is able to interact with diverse RNA
molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is
recognized as a global post-transcriptional regulator of gene expression. Loss of
Hfq has an extensive impact in bacterial physiology which in several animal
pathogens influences virulence. Sinorhizobium meliloti is a model soil bacterium
known for its ability to establish a beneficial nitrogen-fixing intracellular
symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the
establishment of successful bacteria-eukaryote interactions, its function in S.
meliloti has remained unexplored.
RESULTS: Two independent S. meliloti mutants, 2011-3.4 and 1021Deltahfq, were
obtained by disruption and deletion of the hfq gene in the wild-type strains 2011
and 1021, respectively, both exhibiting similar growth defects as free-living
bacteria. Transcriptomic profiling of 1021Deltahfq revealed a general
down-regulation of genes of sugar transporters and some enzymes of the central
carbon metabolism, whereas transcripts specifying the uptake and metabolism of
nitrogen sources (mainly amino acids) were more abundant than in the wild-type
strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these
observations. Symbiotic tests showed that lack of Hfq led to a delayed
nodulation, severely compromised bacterial competitiveness on alfalfa roots and
impaired normal plant growth. Furthermore, a large proportion of nodules
(55%-64%) elicited by the 1021Deltahfq mutant were non-fixing, with scarce
content in bacteroids and signs of premature senescence of endosymbiotic
bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and
microoxic conditions revealed that Hfq contributes to regulation of nifA and
fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated
regulation of fixK is only aerobiosis dependent. Finally, we found that some of
the recently identified S. meliloti sRNAs co-inmunoprecipitate with a
FLAG-epitope tagged Hfq protein.
CONCLUSIONS: Our results support that the S. meliloti RNA chaperone Hfq
contributes to the control of central metabolic pathways in free-living bacteria
and influences rhizospheric competence, survival of the microsymbiont within the
nodule cells and nitrogen fixation during the symbiotic interaction with its
legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted
to participate in the Hfq regulatory network.
DOI: 10.1186/1471-2180-10-71
PMCID: PMC2848018
1333. Environ Manage. 2010 Oct;46(4):590-8. doi: 10.1007/s00267-010-9454-x. Epub

2010
Feb 27.
Allelopathic potential of switchgrass (Panicum virgatum L.) on perennial ryegrass

(Lolium perenne L.) and alfalfa (Medicago sativa L.).
Shui J(1), An Y, Ma Y, Ichizen N.
Author information:
(1)Institute of Soil and Water Conservation, Ministry of Water Resources and
Chinese Academy of Sciences, Northwest A&F University, 712100, Yangling, Shaanxi
Province, People's Republic of China.
This study investigated allelopathy and its chemical basis in nine switchgrass
(Panicum virgatum L.) accessions. Perennial ryegrass (Lolium perenne L.) and
alfalfa (Medicago sativa L.) were used as test species. Undiluted aqueous
extracts (5 g plant tissue in 50 ml water) from the shoots and roots of most of
the switchgrass accessions inhibited the germination and growth of the test
species. However, the allelopathic effect of switchgrass declined when extracts
were diluted 5- or 50-fold. Seedling growth was more sensitive than seed
germination as an indicator of allelopathic effect. Allelopathic effect was
related to switchgrass ecotype but not related to ploidy level. Upland accessions
displayed stronger allelopathic potential than lowland accessions. The aqueous
extract from one switchgrass accession was separated into phenols, organic acids,
neutral chemicals, and alkaloids, and then these fractions were bioassayed to
test for allelopathic potential. Alkaloids had the strongest allelopathic effect
among the four chemical fractions. In summary, the results indicated that
switchgrass has allelopathic potential; however, there is not enough evidence to
conclude that allelopathic advantage is the main factor that has contributed to
the successful establishment of switchgrass on China's Loess Plateau.
DOI: 10.1007/s00267-010-9454-x
1334. Bioresour Technol. 2010 Jul;101(14):5345-9. doi:

10.1016/j.biortech.2009.11.108.
Epub 2010 Mar 2.
Effect of nitroethane, dimethyl-2-nitroglutarate and 2-nitro-methyl-propionate on

ruminal methane production and hydrogen balance in vitro.
Anderson RC(1), Huwe JK, Smith DJ, Stanton TB, Krueger NA, Callaway TR, Edrington
TS, Harvey RB, Nisbet DJ.
Author information:
(1)United States Department of Agriculture/Agricultural Research Service,
Southern Plains Agricultural Research Center, Food & Feed Safety Research Unit,
2881 F&B Road, College Station, Texas 77845 USA. Robin.Anderson@ars.usda.gov
Ruminal methanogenesis is considered a digestive inefficiency that results in the
loss of 2-12% of the host's gross energy intake and contributes nearly 20% to the
United States annual CH(4) emissions. Presently, the effects of the known CH(4)
inhibitor, nitroethane, and two synthetic nitrocompounds,
dimethyl-2-nitroglutarate and 2-nitro-methyl-propionate, on ruminal CH(4)
production and fermentation were evaluated in vitro. After 24 h incubation at 39
degrees C under 100% CO(2), ruminal fluid cultures treated with 2.97 or 11.88
mumol ml(-1) of the respective nitrocompounds produced > 92% less CH(4) (P <
0.05) than non-treated controls. Quantification of fermentation end-products
produced and H(2) balance estimates indicate that fermentation efficiencies were
not compromised by the nitro-treatments.
DOI: 10.1016/j.biortech.2009.11.108
1335. J Anim Sci. 2010 Jun;88(6):2189-98. doi: 10.2527/jas.2009-2272. Epub 2010 Feb
26.
Selection of tannins by sheep in response to gastrointestinal nematode infection.
Villalba JJ(1), Provenza FD, Hall JO, Lisonbee LD.
Author information:
(1)Department of Wildland Resources, Utah State University, Logan 84322-5230,
USA. juan.villalba@usu.edu
Herbivores learn to select compounds that attenuate the aversive effects of plant
secondary metabolites (PSM), but can they increase intake of PSM they typically
avoid when these PSM provide medicinal effects? We hypothesized that herbivores
learn to increase intake of PSM-containing feeds when experiencing a
gastrointestinal parasitic infection. Ten lambs with natural gastrointestinal
parasitic burdens (PB) and 10 nonparasitized lambs (NP) were offered a choice of
alfalfa (Medicago sativa) and alfalfa mixed with 10% quebracho tannin (Schinopsis
quebracho-colorado; alfalfa:tannins) before and after they were conditioned with
the postingestive effects of tannins. Preference for alfalfa:tannins did not
differ between groups before experiencing the postingestive effects of tannins (P
= 0.85) or when parasite loads were terminated due to the administration of
ivermectin (P = 0.18). In contrast, when tested with a parasite burden, lambs in
PB consumed more alfalfa:tannins (P = 0.08), showed greater preference for
alfalfa:tannins (P = 0.07), and consumed less alfalfa than lambs in NP (P =
0.06). Ingestion of tannins by lambs in PB was followed by reduced fecal egg
counts (FEC; P = 0.006), and there was a direct proportional relationship between
preference for alfalfa:tannins and FEC (P = 0.07). In summary, parasitized lambs
increased their intake of alfalfa:tannins when they experienced a parasite
burden, which suggests they self-medicated with tannins against parasites.
Self-selection of PSM has implications for the quest for alternatives to
chemoprophylaxis in the treatment and well-being of parasitized wild and domestic
animals grazing in pasturelands and in confinement.
DOI: 10.2527/jas.2009-2272
1336. J Agric Food Chem. 2010 Mar 24;58(6):3689-93. doi: 10.1021/jf904472e.
X-ray absorption spectroscopy (XAS) corroboration of the uptake and storage of

CeO(2) nanoparticles and assessment of their differential toxicity in four edible
plant species.
López-Moreno ML(1), de la Rosa G, Hernández-Viezcas JA, Peralta-Videa JR,

Author information:
(1)Department of Chemistry, University of Texas at El Paso, El Paso, Texas 79968,
USA.
Fate, transport, and possible toxicity of cerium oxide nanoparticles (nanoceria,

CeO(2)) are still unknown. In this study, seeds of alfalfa (Medicago sativa),
corn (Zea mays), cucumber (Cucumis sativus), and tomato (Lycopersicon esculentum)
were treated with nanoceria at 0-4000 mg L(-1). The cerium uptake and oxidation
state within tissues were determined using inductively coupled plasma-optical
emission spectroscopy (ICP-OES) and X-ray absorption spectroscopy (XAS),
respectively. The germination rate and root elongation were also determined.
Results showed that nanoceria significantly reduced corn germination (about 30%
at 2000 mg L(-1); p < 0.05), and at 2000 mg L(-1), the germination of tomato and
cucumber was reduced by 30 and 20%, respectively (p < 0.05). The root growth was
significantly promoted (p < 0.05) by nanoceria in cucumber and corn but reduced
(p < 0.05) in alfalfa and tomato. At almost all concentrations, nanoceria
promoted shoot elongation in the four plant species. XAS data clearly showed the
nanoceria within tissues of the four plant species. To the authors' knowledge,
this is the first report on the presence nanoceria within plants.
DOI: 10.1021/jf904472e
PMCID: PMC2852460
1337. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2009 Nov;44(13):1476-83.
doi: 10.1080/10934520903217922.
Investigations on phosphorus recovery and reuse as soil amendment from electric

arc furnace slag filters.
Bird SC(1), Drizo A.
Author information:
(1)Department of Plant and Soil Science, University of Vermont, Burlington,
Vermont, USA. sbird@agrefresh.org
Electric arc furnace (EAF) steel slag has been identified as an effective filter
material for the removal of phosphorus (P) from both point and non-point sources.
To determine the feasibility of land-applying P saturated EAF steel slag this
study was undertaken to investigate (i) saturated EAF steel slag material's
potential as a P fertilizer or soil amendment and (ii) P desorption and metals
leachate from saturated EAF steel slag material to surface runoff. Medicago
sativa (alfalfa) was planted in a nutrient depleted washed sand media. Phosphorus
was added either as saturated EAF steel slag or as a standard commercial
phosphate fertilizer in order to assess the plant availability of the P from
saturated EAF steel slag. Four different P application levels were tested: a low
(20 lbs acre furrow slice(-1) (5.5 g P m(-3))) two medium (40 and 60 lbs. acre
f.s.(-1) (11 and 16.5 g P m(-3))) and a high (120 lbs. acre f.s.(-1) (33 g P
m(-3))). The above-ground biomass of half of the plants was harvested after 5
weeks and the second half at 10 weeks. All treatments regardless of the P source
used showed high rates of germination. At the first harvest period (5 weeks)
significantly higher above-ground biomass (p < 0.01) was seen at the 3 highest P
amendment rates in treatments with triple super phosphate fertilizer (TSP) than
with EAF steel slag. However, by the second harvest (10 weeks) only the highest
amendment rate of TSP showed a significantly higher amount of biomass (p < 0.01),
suggesting that EAF steel slag might be an effective slow release P source. In a
second experiment, a rain simulator was used to assess desorption of DRP, TP and
metals from a saturated and semi-saturated EAF steel slag. The results revealed
that the total amounts of DRP and TP released to surface runoff from EAF steel
slag were negligible when compared to the total quantities of P retained by this
material. Overall the results from this study demonstrated that once the EAF
steel slag filter reaches its saturation point, the material could be re-used as
soil amendment for the slow release of bioavailable P with minimal risk for loss
of P to surface runoff, bringing further benefits to the environment.
DOI: 10.1080/10934520903217922
Effects of spontaneous heating on forage protein fractions and in situ

disappearance kinetics of crude protein for alfalfa-orchardgrass hays packaged in
large round bales.
Coblentz WK(1), Hoffman PC, Martin NP.
Author information:
During 2006 and 2007, forages from 3 individual hay harvests were used to assess
the effects of spontaneous heating on concentrations of crude protein (CP),
neutral detergent insoluble CP (NDICP), acid detergent insoluble CP (ADICP), and
in situ disappearance kinetics of CP and NDICP for large round bales of mixed
alfalfa (Medicago sativa L.) and orchardgrass (Dactylis glomerata L.). Over the 3
harvests, 96 large round bales were made at preset bale diameters of 0.9, 1.2, or
1.5m and at moisture concentrations ranging from 9.3 to 46.6%. Internal bale
temperatures were monitored daily during an outdoor storage period. The change in
concentrations of NDICP (poststorage - prestorage) increased with heating degree
days (HDD) >30 degrees C in a relationship best explained with a nonlinear model
{Y=24.9 - [22.7 x (e(-0.000010 x x x x))]; R(2)=0.892} that became asymptotic at
+24.9 percentage units of CP, thereby indicating that NDICP increases rapidly
within bales that heat spontaneously. When maximum internal bale temperature
(MAX) was used as the independent variable, the best regression model was
quadratic and the coefficient of determination was still relatively high
(R(2)=0.716). The change in concentrations of ADICP (poststorage - prestorage;
DeltaADICP) also increased with HDD and was best fitted to a nonlinear model
{Y=14.9 - [15.7 x (e(-0.0000019 x x x x))]} with a very high coefficient of
determination (R(2)=0.934). A similar quartic response was observed for the
regression of DeltaADICP on MAX (R(2)=0.975). Increases in DeltaADICP as a result
of heating (HDD or MAX) were paralleled by concurrent increases in hemicellulose
at relatively low increments of heating, but the inverse relationship was
observed as hemicelluloses likely became reactive and concentrations decreased in
more severely heated hays. Changes in ruminal disappearance rate of CP were best
fitted to cubic models for regressions on both HDD (R(2)=0.939) and MAX
(R(2)=0.876); these changes represented an approximate 50% rate reduction in
severely heated hays relative to prestorage controls. Within ranges of heating
most commonly encountered under field conditions, changes in rumen-degradable
protein decreased in a primarily linear relationship with HDD or MAX. However,
the mean change in rumen-degradable protein for the 4 most severely heated hays
was only -2.6 percentage units of CP, which represents a minimal reduction from
prestorage controls and is far less than the maximum of -7.9 percentage units of
CP observed with less-severe heating. Interpretation of these results was
complicated by poor recovery of NDICP from our most severely heated hays
following machine rinsing of 0-h Dacron bags; theoretically, and by definition,
this unrecovered pool of NDICP is assumed to be entirely degradable in the rumen.
It remains unclear whether these responses could be corroborated in vivo or by
other analytical techniques, or whether the magnitude of HDD or MAX for our most
severely heated hays exceeds the reliable limits for estimating RDP via in situ
methodology.
DOI: 10.3168/jds.2009-2701
1339. Anim Sci J. 2009 Oct;80(5):546-55. doi: 10.1111/j.1740-0929.2009.00661.x.
The effect of dietary ratios of corn silage and alfalfa hay on carbohydrate
digestion and retention time of feed particles in the gastrointestinal tract of
steers.
Obitsu T(1), Goto M, Sugino T, Taniguchi K, Yukizane K, Imoto S, Yanagawa M,

El-Sabagh M.
Author information:
(1)Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima,
Japan. tobitsu@hiroshima-u.ac.jp
Four Holstein steers fitted with duodenal cannula were used in a 4 x 4 Latin
square design to investigate the effect of the combination of corn silage (CS)
and alfalfa hay (AH) in different ratios on the ruminal and intestinal digestion
of carbohydrates and feed particle passage rate. Steers were fed mixed diets
containing both CS and AH in ratios of 80:20, 60:40, 40:60 and 20:80 on a dry
matter basis at 95% of ad libitum feed intake. The increase of AH proportion in
diets increased dry matter intake and ruminal digestion of dry matter, non-fiber
carbohydrate, neutral and acid detergent fiber linearly. Ruminal digestibility of
neutral detergent fiber showed a quadratic response, and total digestibility
increased linearly with increasing AH proportion. Digestibility of acid detergent
fiber in the rumen was not affected by the dietary treatments, but the total
tract digestibility increased as the AH proportion increased. Mean retention time
of feed particles in total compartment increased when the AH proportion increased
from 20% to 60%, but decreased with further increase of the AH proportion. These
results indicate that moderate combinations of CS and AH have an associative
effect on ruminal fiber digestion, modifying particle movement in the rumen.
DOI: 10.1111/j.1740-0929.2009.00661.x
1340. Anim Sci J. 2009 Oct;80(5):532-41. doi: 10.1111/j.1740-0929.2009.00662.x.
Effect of different levels of Quebracho tannin on nitrogen utilization and growth

performance of Najdi sheep fed alfalfa (Medicago sativa) hay as a sole diet.
Al-Dobaib SN(1).
Author information:
(1)Department of Animal Production and Breeding, Faculty of Agriculture and
Veterinary Medicine, Qassim University, Buriedah, Saudi Arabia.
saldabeeb@yahoo.com
A commercial tannin source (Quebracho tannin, QT), containing 75% condensed

tannins (CT) in dry matter (DM) was used to evaluate the effects of addition of
different levels of QT to alfalfa hay on the in vitro degradation kinetics of
organic matter (OM) and nitrogen (N) in experiment 1 (Exp. 1), N utilization and
microbial N synthesis (MNS) in experiment 2 (Exp. 2) and growth performance of
growing Najdi lambs in experiment 3 (Exp. 3). Alfalfa hay was treated with QT at
the levels of 0, 1, 2 and 3% of DM to form four treatments of QT0, QT1, QT2 and
QT3 to have actual levels of CT being 0, 0.75, 1.5 and 2.25% of DM, respectively.
Degradation rate and the effective degradability of N were significantly
decreased (P < 0.05) for QT2 and QT3 as compared with the QT0. In Exp. 2,
digestibility coefficients for OM, neutral detergent fiber and acid detergent
fiber were significantly decreased (P < 0.05) at QT3, whereas QT1 and QT2 showed
no difference to QT0. In the metabolism trial (Exp. 2), digested N (DN) and
urinary N (UN) excretion for QT2 and QT3 were significantly decreased (P < 0.05)
compared to QT0 and QT1. The DN and UN as percentage of N intake were 79.2, 76.9,
75.5 and 69.8%; and 24.4, 22.6, 19.9 and 19.6% for QT0, QT1, QT2 and QT3,
respectively. QT2 had the highest MNS and the lowest value was in QT3, the MNS of
the treatments were 18.1, 18.7, 19.2 and 15.8 g/day for QT0, QT1, QT2 and QT3,
respectively. In the growth trial of 56 days duration (Exp. 3, n = 24 lambs), the
addition of QT at the level of QT2 enhanced (P < 0.05) the average daily gain and
feed conversion rate compared with the QT0, while, QT1 and QT3 had intermediate
values. It is concluded that alfalfa hay treated with QT at the level of 2% of DM
could be used to protect alfalfa N from ruminal degradation that would lead to
improve growth performance of lambs.
DOI: 10.1111/j.1740-0929.2009.00662.x
1341. Anim Sci J. 2009 Aug;80(4):403-10. doi: 10.1111/j.1740-0929.2009.00653.x.
Effects of diets containing alfalfa hay or barley flour mixed alfalfa silage on
feeding behavior, productivity, rumen fermentation and blood metabolites in
lactating cows.
Khadem AA(1), Sharifi M, Afzalzadeh A, Rezaeian M.
Author information:
(1)Department of Animal Science, Agricultural Faculty of Abouraihan, University
of Tehran, Tehran, Iran. akhadem@ut.ac.ir
The effects of barley flour on the fermentation parameters of alfalfa silage and
on the productivity of dairy cows were investigated. Alfalfa forage was ensiled
either with or without barley flour. Barley flour was soaked in water for 24 h
before being mixed with alfalfa (12 kg: 100 kg dry matter bases) at ensiling.
Eighteen multi-parous cows were assigned to three equal treatment groups using a
completely randomized design. Three isocaloric and isonitrogenous total mixed
rations containing alfalfa hay, ordinary alfalfa silage or barley flour mixed
alfalfa silage were then prepared. The concentrations of ammonia nitrogen, acetic
acid and butyric acid were lower in barley flour mixed alfalfa silage compared to
that in ordinary alfalfa silage but the concentration of lactic acid was lower in
the ordinary alfalfa silage. Feeding behavior, milk yield and composition,
ruminal fermentation and blood metabolites were measured. Although dry matter
intake and milk production were not affected, the effect of preparation of
alfalfa influenced feeding behavior and rumen fermentation parameters. Cows on
alfalfa silage diets spent longer ruminating compared to those fed alfalfa hay.
The ruminal ammonia nitrogen and blood urea were affected by ensiling (alfalfa
hay versus alfalfa silages) while both parameters were lower in cows fed on
barley flour mixed alfalfa silage than those fed on ordinary silage. Although
similar blood glucose was recorded for cows fed on alfalfa silages, it was higher
in cows fed on alfalfa hay. It is concluded that the addition of barely flour
when making alfalfa silage may improve both the fermentation process during
ensilage and the ruminal ammonia nitrogen utilization with no significant effects
on productivity.
DOI: 10.1111/j.1740-0929.2009.00653.x
12.
Using alfalfa leaf meal as a supplement in late-gestation beef heifer and nursing
beef calf diets.
Zehnder CM(1), Maddock TD, DiCostanzo A, Miller LR, Hall JM, Lamb GC.
Author information:
(1)Department of Animal Science, University of Minnesota, St Paul 55108, USA.
Two experiments were conducted to investigate using alfalfa leaf meal (ALM; 22%
CP, DM basis) in beef cattle diets. In Exp. 1, a total of 24 late-gestation Angus
heifers (initial BW 470 +/- 9 kg) were blocked by BW, calving date, and BCS to 1
of 4 dietary treatments in a randomized complete block design. All heifers were
offered a basal hay diet (7.4% CP and 67.6% NDF, DM basis). Treatments were
arranged as a 2 x 2 factorial consisting of CP supplied at 100 or 112.5% of the
recommended daily intake using either soybean meal (SBM) or ALM as the
supplemental protein source. Treatments were fed for an average of 100 d before
calving. Total DMI was unaffected by supplemental protein source, although
heifers consumed more (P < 0.001) ALM supplement than SBM supplement at the
expense of hay and corn. Feeding 112.5% of recommended CP to heifers increased
precalving rate of BW gain (P = 0.004) and DM digestibility (P = 0.003). Protein
source did not affect DM digestibility (P = 0.17). Neither supplemental protein
source nor protein amount affected changes in BCS or calving traits. In Exp. 2,
replicates of treatments were conducted over 2 consecutive years at 2 locations
in northern Minnesota to determine the effects of including ALM in creep-fed
supplements on nursing calf performance, supplement BW gain efficiency (GF; BW
gain over control/supplement intake), and cow performance. Treatments were
control (no supplement), ALM supplement (58% ALM, as-fed basis), or a wheat
middling- and soybean hull-based supplement (MIDD). Milk intake (estimated by the
weigh-suckle-weigh technique) was similar among treatments. Creep-fed calves had
greater (P < 0.001) ADG than control calves, whereas calves offered MIDD tended
to have greater ADG (P = 0.05) than those offered ALM (1.38 vs. 1.30 kg/d,
respectively). Calves offered MIDD had greater (P < 0.001) creep feed DMI than
those offered ALM (2.6 vs. 1.3 kg/d, respectively). A year x treatment
interaction was noted for GF (P = 0.02). In yr 1, GF for calves offered ALM was
greater (P = 0.006) than GF for calves offered MIDD, but in yr 2, there were no
differences. Alfalfa leaf meal may substitute for SBM in beef heifer wintering
diets and conventional creep feed ingredients. When included in creep feed diets,
ALM can result in slightly less ADG and less DMI, but supplement conversion
efficiency may be increased.
DOI: 10.2527/jas.2009-2592
1343. Water Sci Technol. 2010;61(3):625-32. doi: 10.2166/wst.2010.904.
Sustainable domestic effluent reuse via Subsurface Drip Irrigation (SDI): alfalfa
as a perennial model crop.
Kazumba S(1), Gillerman L, DeMalach Y, Oron G.
Author information:
(1)Department of Civil Engineering, Dar es Salaam Institute of Technology, Dar es
Salaam, Tanzania. shija.kazumba@dit.ac.tz
Scarcity of fresh high-quality water has heightened the importance of wastewater

reuse primarily in dry regions together with improving its efficient use by
implementing the Subsurface Drip Irrigation (SDI) method. Sustainable effluent
reuse combines soil and plant aspects, along with the maintainability of the
application system. In this study, field experiments were conducted for two years
on the commercial farm of Revivim and Mashabay-Sade farm (RMF) southeast of the
City of Beer-Sheva, Israel. The purpose was to examine the response of alfalfa
(Medicago sativa) as a perennial model crop to secondary domestic effluent
application by means of a SDI system as compared with conventional overhead
sprinkler irrigation. Emitters were installed at different depths and spacing.
Similar amounts of effluent were applied to all plots during the experimental
period. The results indicated that in all SDI treatments, the alfalfa yields were
11% to 25% higher than the ones obtained under sprinkler irrigated plots, besides
the one in which the drip laterals were 200 cm apart. The average Water Use
Efficiency (WUE) was better in all SDI treatments in comparison with the
sprinkler irrigated plots. An economic assessment reveals the dependence of the
net profit on the emitters' installation geometry, combined with the return for
alfalfa in the market.
DOI: 10.2166/wst.2010.904
Degree-day requirements for alfalfa weevil (Coleoptera: Curculionidae)

development in eastern Nebraska.
Stilwell AR(1), Wright RJ, Hunt TE, Blankenship EE.
Author information:
(1)Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE
68583-0816, USA. abbyrstilwell@gmail.com
The alfalfa weevil, Hypera postica (Gyllenhal), is a serious, yet sporadic

defoliator of alfalfa, Medicago sativa L., in Nebraska. A 2-yr study was
conducted in 2005 and 2006 to test for variation in degree-day requirements by
location in eastern Nebraska. Sampling took place along a latitudinal gradient in
three regions of eastern Nebraska. Three fields were sampled in each region
during the 2 yr of the study. Alfalfa weevil larval degree-day requirements were
found to vary by latitude in eastern Nebraska. Alfalfa weevil larvae were
discovered in southern regions after fewer developmental degree-days had
accumulated than in fields in the northern regions. Alfalfa weevils may be more
damaging to alfalfa in southern regions than in northern regions of eastern
Nebraska because they emerge earlier relative to alfalfa growth. Management
implications of this shift in alfalfa weevil phenology are discussed.
DOI: 10.1603/EN09048
Preimaginal development response to constant temperatures in Hypera postica

(Coleoptera: Curculionidae): picking the best model.
Zahiri B(1), Fathipour Y, Khanjani M, Moharramipour S, Zalucki MP.
Author information:
(1)Department of Entomology, Faculty of Agriculture, Tarbiat Modares University,
Tehran, Iran.
Immature survival and development of alfalfa weevil, Hypera postica (Gyllenhal)

(Coleoptera: Curculionidae), were examined at 15 constant temperatures ranging
from 9 to 37 degrees C. At 9 and 37 degrees C, eggs did not develop. Embryos
developed to adulthood between 11.5 and 36 degrees C, although survival was low
at both ends of the temperature range. Distribution of development times for all
stages of alfalfa weevil were skewed toward longer times mainly at moderate
temperatures. Constant proportion of time spent in egg, larva, and pupa indicated
rate isomorphy within the range 14-31.5 degrees C. No significant difference was
shown between development time of males and females at any of the temperatures
tested. Two linear and 23 nonlinear models were fitted to describe development
rate of immature stages of H. postica as a function of temperature, as well as
estimating the thermal constant and critical temperatures (i.e., T(min), T(opt),
and T(max)). There was no statistical difference between the estimated
parameters, using Ikemoto and ordinary linear methods. Of the nonlinear models
fitted, the Logan-6/Lactin-2, Analytis-3/Briere-2, and Analytis-3/Briere-2 models
were found to be the best for modeling development rate of egg, larva, and entire
immature stages of H. postica, respectively. Our findings will be incorporated in
more efficient phenological models of this pest and its population dynamics.
DOI: 10.1603/EN08239
Nest establishment, pollination efficiency, and reproductive success of Megachile

rotundata (Hymenoptera: Megachilidae) in relation to resource availability in
field enclosures.
Pitts-Singer TL(1), Bosch J.
Author information:
(1)USDA-ARS Bee Biology & Systematics Laboratory, Logan, UT 84326, USA.
Theresa.Pitts-Singer@ars.usda.gov
The alfalfa leafcutting bee, Megachile rotundata (Fabricius), is used to

pollinate alfalfa, Medicago sativa L., for seed production in the United States
and Canada. It is difficult to reliably sustain commercial M. rotundata
populations in the United States because of problems with disease, parasites,
predators, and unexplained mortality. One possible explanation for early immature
mortality is that, relative to floral availability, superfluous numbers of bees
are released in alfalfa fields where resources quickly become limited. Our
objective was to determine how M. rotundata density affects bee nesting,
pollination efficiency, and reproductive success. Various numbers of bees were
released into enclosures on an alfalfa field, but only 10-90% of released female
bees established nests. Therefore, a "bee density index" was derived for each
enclosure from the number of established females and number of open flowers over
time. As the density index increased, significant reductions occurred in the
number of pollinated flowers, number of nests, and number of cells produced per
bee, as well as the percentage of cells that produced viable prepupae by summer's
end and the percentage that produced adult bees. The percentage of cells
resulting in early brood mortality (i.e., pollen balls) significantly increased
as the density index increased. We conclude that bee nest establishment,
pollination efficiency, and reproductive success are compromised when bee
densities are high relative to floral resource availability. Open field studies
are needed to determine commercial bee densities that result in sustainable bee
populations and adequate pollination for profitable alfalfa seed production.
DOI: 10.1603/EN09077
1347. Theor Appl Genet. 2010 May;120(8):1611-9. doi: 10.1007/s00122-010-1280-2.

Epub
2010 Feb 9.
SRAP polymorphisms associated with superior freezing tolerance in alfalfa

(Medicago sativa spp. sativa).
Castonguay Y(1), Cloutier J, Bertrand A, Michaud R, Laberge S.
Author information:
Canada, 2560 Hochelaga Blvd, Quebec, QC, G1V 2J3, Canada.
yves.castonguay@agr.gc.ca
Sequence-related amplified polymorphism (SRAP) analysis was used to uncover

genetic polymorphisms among alfalfa populations recurrently selected for superior
tolerance to freezing (TF populations). Bulk DNA samples (45 plants/bulk) from
each of the cultivar Apica (ATF0), and populations ATF2, ATF4, ATF5, and ATF6
were evaluated with 42 different SRAP primer pairs. Several polymorphisms that
progressively intensified or decreased with the number of recurrent cycles were
identified. Four positive polymorphisms (F10-R14, Me4-R8, F10-R8 and F11-R9)
that, respectively, yielded 540-, 359-, 213-, and 180-bp fragments were selected
for further analysis. SRAP amplifications with genotypes within ATF populations
confirmed that the polymorphisms identified with bulk DNA samples were reflecting
changes in the frequency of their occurrence in response to selection. In
addition, the number of genotypes cumulating multiple polymorphisms markedly
increased in response to recurrent selection. Independent segregation of the four
SRAP polymorphisms suggests location at unlinked loci. Homology search gave
matches with BAC clones from syntenic Medicago truncatula for the four SRAP
fragments. Analysis of the relationship with low temperature tolerance showed
that multiple SRAP polymorphisms are more frequent in genotypes that maintain
superior regrowth after freezing. These results show that SRAP analysis of bulk
DNA samples from recurrent selections is an effective approach for the
identification of genetic polymorphisms associated with quantitative traits in
allogamous species. These polymorphisms could be useful tools for indirect
selection of freezing tolerance in alfalfa.
DOI: 10.1007/s00122-010-1280-2

[Effects of irrigation with regenerated water on the growth and quality of
alfalfa].
Zhang ZH(1), Chen WF, Shi YF, Tian SJ, Liu LJ.
Author information:
(1)Environment, Shandong Agricultural University, Tai'an 271018, Shandong, China.
zhihua1221@163.com
A pot experiment was conducted to study the effects of irrigation with

regenerated water on the growth, nutrient uptake, and quality of alfalfa. Four
treatments were installed, i.e., irrigation with fresh water (Q), irrigation with
regenerated water (Z), mixed irrigation with regenerated water and fresh water (Z
+ Q), and rotational irrigation with regenerated water and fresh water (Z/Q).
Comparing with treatment Q, treatment Z increased the stem length, lateral branch
number, and plant biomass significantly, but decreased leaf area. Treatments Z, Z
+ Q, and Z/Q increased plant soluble protein content by 78.43%, 83.68% and
72.53%, respectively, but treatment Z decreased plant soluble sugar content.
Treatment Z increased plant Ca and Mg contents by 27.78% and 26.61%, and
treatments Z and Z + Q decreased plant Fe content by 28.71% and 10.09%,
respectively. Treatments Z, Z + Q, and Z/Q increased the cadmium content in
above-ground part of alfalfa by 98.6%, 89.5%, and 59.0%, respectively, but the
cadmium and lead contents were still lower than the thresholds of National
Sanitation Standard (GB 13078-2001). It was suggested that regenerated water
could be used as a source of irrigation water in alfalfa growth, but its
long-term effect should be concerned.
Effects of nonstructural carbohydrate concentration in alfalfa on fermentation

and microbial protein synthesis in continuous culture.
Berthiaume R(1), Benchaar C, Chaves AV, Tremblay GF, Castonguay Y, Bertrand A,

Bélanger G, Michaud R, Lafrenière C, McAllister TA, Brito AF.
Author information:
Centre, Sherbrooke, Québec, Canada J1M 1Z3. Robert.Berthiaume@agr.gc.ca
Insufficient readily fermentable energy combined with extensive degradation of

proteins in alfalfa (Medicago sativa L.) may result in poor forage N utilization
by ruminants. Using the inherent genetic variability and differences between
harvests, our objective was to compare the effect of contrasting concentrations
of nonstructural carbohydrates (NSC) in alfalfa on rumen fermentation and
microbial protein synthesis. Individual genotypes of the alfalfa cultivar AC
Caribou grown near Québec City, Québec, Canada, were harvested at the vegetative
and early flowering stages, dried at 55 degrees C, ground, and analyzed for
soluble carbohydrates (fructose + sucrose + glucose + pinitol) and starch.
Approximately 20 genotypes having, respectively, the highest and lowest NSC
concentrations were pooled to constitute 2 contrasted 1-kg forage samples.
Samples of high- (17.9% DM) and low- (7.4% DM) NSC alfalfa were respectively
allocated to separate dual-flow fermenters in a completely randomized design with
3 replications. Rumen inoculum was obtained from 4 ruminally fistulated cows in
early lactation that were fed a TMR with a 50:50 forage to concentrate ratio. A
10-d incubation period was used, with the first 6 d serving as an adaptation
period followed by 4 d of sampling with solid and liquid dilution rates in the
fermenters set at approximately 2.0 and 4.3%/h, respectively. High versus low NSC
concentration in alfalfa significantly enhanced the apparent digestibility of OM
(59.1% for high-NSC alfalfa vs. 54.4% for low-NSC alfalfa) and DM (60.0 vs.
54.3%) and the true digestibility of DM (74.1 vs. 64.7%). Increasing NSC
concentration in alfalfa (high vs. low) significantly decreased ruminal pH (6.85
vs. 7.08) and NH(3)-N concentration (26.0 vs. 33.6 mg/dL) and increased total VFA
concentration (94.9 vs. 83.0mM). Molar proportions of acetate, isobutyrate, and
isovalerate significantly decreased, whereas molar proportions of propionate and
butyrate significantly increased with high-NSC alfalfa, resulting in a more
glucogenic fermentation. More importantly, microbial N flow (263 vs. 230 mg/d)
and bacterial N efficiency (41.1 vs. 29.6% of available N), measured using (15)N
as a microbial marker, both significantly increased with the high-NSC alfalfa.
These results indicate that increasing the concentration of NSC in alfalfa
promotes a glucogenic fermentation and enhances microbial N synthesis in the
rumen.
Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All
rights reserved.
DOI: 10.3168/jds.2009-2399
1350. BMC Microbiol. 2010 Jan 20;10:15. doi: 10.1186/1471-2180-10-15.
Phenotypic and genetic diversity in Sinorhizobium meliloti and S. medicae from

drought and salt affected regions of Morocco.
Elboutahiri N(1), Thami-Alami I, Udupa SM.
Author information:
(1)Institut National de la Recherche Agronomique (INRA), Centre Régional de la
Recherche Agronomique de Rabat, B,P, 415, Rabat, Morocco.
BACKGROUND: Sinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing

bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In
Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions
frequently affected by drought, extremes of temperature and soil pH, soil
salinity and heavy metals, which affect biological nitrogen fixing ability of
rhizobia and productivity of the host. This study examines phenotypic diversity
for tolerance to the above stresses and genotypic diversity at Repetitive
Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled
from marginal soils of arid and semi-arid regions of Morocco.
RESULTS: RsaI digestion of PCR amplified 16S rDNA of the 157 sampled isolates,
assigned 136 isolates as S. meliloti and the rest as S. medicae. Further
phenotyping of these alfalfa rhizobia for tolerance to the environmental stresses
revealed a large degree of variation: 55.41%, 82.16%, 57.96% and 3.18% of the
total isolates were tolerant to NaCl (>513 mM), water stress (-1.5 MPa), high
temperature (40 degrees C) and low pH (3.5), respectively. Sixty-seven isolates
of S. meliloti and thirteen isolates of S. medicae that were tolerant to salinity
were also tolerant to water stress. Most of the isolates of the two species
showed tolerance to heavy metals (Cd, Mn and Zn) and antibiotics
(chloramphenicol, spectinomycin, streptomycin and tetracycline). The phenotypic
clusters observed by the cluster analysis clearly showed adaptations of the S.
meliloti and S. medicae strains to the multiple stresses. Genotyping with rep-PCR
revealed higher genetic diversity within these phenotypic clusters and classified
all the 157 isolates into 148 genotypes. No relationship between genotypic
profiles and the phenotypes was observed. The Analysis of Molecular Variance
revealed that largest proportion of significant (P < 0.01) genetic variation was
distributed within regions (89%) than among regions (11%).
CONCLUSION: High degree of phenotypic and genotypic diversity is present in S.
meliloti and S. medicae populations from marginal soils affected by salt and
drought, in arid and semi-arid regions of Morocco. Some of the tolerant strains
have a potential for exploitation in salt and drought affected areas for
biological nitrogen fixation in alfalfa.
DOI: 10.1186/1471-2180-10-15
PMCID: PMC2823721
1351. Mol Biol (Mosk). 2009 Nov-Dec;43(6):1082-7.
[Compute simulation to characterize structure and function of chalcone synthase

from Scutellaria baicalensis Georgi].
Lei W, Sun M, Luo KM, Shui XR, Sun YM, Tang H.
Prediction and analysis of molecular structure and biochemical function are of

theoretical guiding significance for gene discovery and application, and
considered as one of the central problem of computational biology. Here, some
characteristic features of chalcone synthase (CHS) family from Scutellaria
baicalensis were described via bioinformatic analysis, and showed as following:
the nucleic acid sequences and amino acid sequences of three chs member
genesshared high similarity inthe molecular structures and physicochemical
properties; SbCHS proteins were localized to the cytosol, and possessed a good
hydrophobic nature, with lacking any transmembrane topological structure. The
phylogram analysis suggested that they were a group genes with significant
functional association and genetic conservation. The secondary structures of the
SbCHSs were mainly composed of alpha-helixes and random coils, and the tertiary
structures contained malonyl CoA linkers, besides, each of CHS-A and CHS-B with
N-glycosylation motif included. Taken together, these results demonstrate that
CHS family from S. baicalensis has the typical molecular structure and function
of chalcone synthase, compared with the experimental data for Medicago sativa CHS
protein.
1352. J Bacteriol. 2010 Mar;192(6):1710-8. doi: 10.1128/JB.01427-09. Epub 2010 Jan

15.
The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti

and alfalfa.
Barra-Bily L(1), Pandey SP, Trautwetter A, Blanco C, Walker GC.
Author information:
(1)Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139-4307, USA.
There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic
Brucella bacteria in terms of extensive gene synteny and the requirements for
intracellular survival in their respective hosts. The RNA chaperone Hfq is
essential for virulence for several bacterial groups, including Brucella;
however, its role in S. meliloti has not been investigated. Our studies of an S.
meliloti loss-of-function hfq mutant have revealed that Hfq plays a key role in
the establishment of the symbiosis between S. meliloti and its host Medicago
sativa. S. meliloti Hfq is involved in controlling the population density under a
free-living state and affects the growth parameters and nodulation. An hfq mutant
poorly colonizes the infection threads that are necessary for the bacteria to
invade the developing nodule. An hfq mutant is severely impaired in its ability
to invade plant cells within the nodule, which leads to the formation of small,
ineffective nodules unable to fix nitrogen. In culture, the hfq mutant did not
accumulate transcripts of nifA, which encodes a key regulator necessary for
nitrogen fixation. Hfq may be involved in regulation of several proteins relevant
to hfq mutant phenotypes. The crucial role of Hfq in symbiosis suggests that
small regulatory RNAs are important for its interactions with its plant host.
DOI: 10.1128/JB.01427-09
PMCID: PMC2832522
1353. J Bacteriol. 2010 Mar;192(6):1719-29. doi: 10.1128/JB.01429-09. Epub 2010 Jan

15.
Proteomic alterations explain phenotypic changes in Sinorhizobium meliloti

lacking the RNA chaperone Hfq.
Barra-Bily L(1), Fontenelle C, Jan G, Flechard M, Trautwetter A, Pandey SP,

Walker GC, Blanco C.
Author information:
(1)Interactions Cellulaires et Moleculaires, DUALS, CNRS UMR 6026, Université de
Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France.
The ubiquitous bacterial RNA-binding protein Hfq is involved in stress resistance

and pathogenicity. In Sinorhizobium meliloti, Hfq is essential for the
establishment of symbiosis with Medicago sativa and for nitrogen fixation. A
proteomic analysis identifies 55 proteins with significantly affected expression
in the hfq mutant; most of them are involved in cell metabolism or stress
resistance. Important determinants of oxidative stress resistance, such as CysK,
Gsh, Bfr, SodC, KatB, KatC, and a putative peroxiredoxine (SMc00072), are
downregulated in the hfq mutant. The hfq mutant is affected for H(2)O(2),
menadione, and heat stress resistance. Part of these defects could result from
the reductions of rpoE1, rpoE2, rpoE3, and rpoE4 expression levels in the hfq
mutant. Some proteins required for efficient symbiosis are reduced in the hfq
mutant, contributing to the drastic defect in nodulation observed in this mutant.
DOI: 10.1128/JB.01429-09
PMCID: PMC2832530
1354. J Hazard Mater. 2010 May 15;177(1-3):384-9. doi:

10.1016/j.jhazmat.2009.12.044.
Epub 2009 Dec 16.
Degradation of phenanthrene and pyrene in spiked soils by single and combined

plants cultivation.
Cheema SA(1), Imran Khan M, Shen C, Tang X, Farooq M, Chen L, Zhang C, Chen Y.
Author information:
(1)Institute of Environmental Science and Technology, Zhejiang University, 268
Kaixuan Road, Hangzhou 310029, Zhejiang, PR China.
The present study was conducted to investigate the capability of four plant
species (tall fescue, ryegrass, alfalfa, and rape seed) grown alone and in
combination to the degradation of phenanthrene and pyrene (polycyclic aromatic
hydrocarbons, PAHs) in spiked soil. After 65 days of plant growth, plant biomass,
dehydrogenase activity, water-soluble phenolic (WSP) compounds, plant uptake and
accumulation and residual concentrations of phenanthrene and pyrene were
determined. Our results showed that presence of vegetation significantly enhanced
the dissipation of phenanthrene and pyrene from contaminated soils. Higher
degradation rates of PAHs were observed in the combined plant cultivation
(98.3-99.2% phenanthrene and 88.1-95.7% pyrene) compared to the single plant
cultivation (97.0-98.0% phenanthrene and 79.8-86.0% pyrene). Contribution of
direct plant uptake and accumulation of phenanthrene and pyrene was very low
compared to the plant enhanced dissipation. By contrast, plant-promoted
biodegradation was the predominant contribution to the remediation enhancement.
The correlation analysis indicates a negative relation between biological
activities (dehydrogenase activity and WSP compounds) and residual concentrations
of phenanthrene and pyrene in planted soils. Our results suggest that
phytoremediation could be a feasible choice for PAHs contaminated soil. Moreover,
the combined plant cultivation has potential to enhance the process.
Copyright (c) 2010 Elsevier B.V. All rights reserved.
DOI: 10.1016/j.jhazmat.2009.12.044
1355. Mol Plant Microbe Interact. 2010 Feb;23(2):153-60. doi: 10.1094/MPMI-23-2-

0153.
Sinorhizobium meliloti 1021 loss-of-function deletion mutation in chvI and its

phenotypic characteristics.
Wang C(1), Kemp J, Da Fonseca IO, Equi RC, Sheng X, Charles TC, Sobral BW.
Author information:
(1)Virginia Bioinformatics Instutue, Virginia Polytechnic Institute and STate
University, Blacksburg 24061, USA. cwang@vbi.vt.edu
Bacterial two-component regulatory systems (TCS) are common components of complex

regulatory networks and cascades. In Sinorhizobium meliloti, the TCS ExoS/ChvI
controls exopolysaccharide succinoglycan production and flagellum biosynthesis.
Although this system plays a crucial role in establishing the symbiosis between
S. meliloti and its host plant, it is not well characterized. Attempts to
generate complete loss-of-function mutations in either exoS or chvI in S.
meliloti have been unsuccessful; thus, it was previously suggested that exoS or
chvI are essential genes for bacterial cell growth. We constructed a chvI mutant
by completely deleting the open reading frame encoding this gene. The mutant
strain failed to grow on complex medium, exhibited lower tolerance to acidic
condition, produced significantly less poly-3-hydroxybutyrate than the wild type,
was hypermotile, and exhibited an altered lipopolysaccharide profile. In
addition, this mutant was defective in symbiosis with Medicago truncatula and M.
sativa (alfalfa), although it induced root hair deformation as efficiently as the
wild type. Together, our results demonstrate that ChvI is intimately involved in
regulatory networks involving the cell envelope and metabolism; however, its
precise role within the regulatory network remains to be determined.
DOI: 10.1094/MPMI-23-2-0153
1356. Ecotoxicol Environ Saf. 2010 Jul;73(5):932-8. doi:

10.1016/j.ecoenv.2009.12.021.
Epub 2010 Jan 12.
Bioassay and use in irrigation of untreated and treated wastewaters from

phosphate fertilizer industry.
Gouider M(1), Feki M, Sayadi S.
Author information:
(1)Unité de Recherche Chimie Industrielle et Matériaux, Ecole Nationale
d'Ingénieurs de Sfax, BPW 1173-3038 Sfax, Tunisie. mbarkagouider@yahoo.fr
Wastewater from phosphate fertilizer industry that contains essentially a

significant amount of both fluoride and phosphate was treated by separative
precipitation of fluoride ions with hydrated lime. Thus, a phosphate-rich
effluent with low content of fluoride was obtained. The microtoxicity of the
treated wastewater was then monitored by LUMIStox and its phytotoxicity was
investigated on tomato (Lycopersicon esculentum), wheat (Triticum aestivum),
maize (Zea mays), ryegrass (Lolium perenne), and alfalfa (Medicago sativa) seed
germination and plant growth. The cress (Lepidium sativum) was used as a standard
species for the germination index and phytotoxicity evaluation. Seedlings of four
species (namely wheat, maize, ryegrass, and alfalfa) were grown in pots, which
were irrigated with untreated wastewater, treated wastewater, aqueous solution of
triple superphosphate fertilizer (TSP) or with tap water as control. LUMIStox
tests showed that lime treatment allowed a significant toxicity removal. The
treated water displayed beneficial fertilizing effect on plants. An increase in
the germination index from 100% to 119% was observed. However, the untreated
wastewater inhibited the species germination even when diluted 10 times. Neither
plants mortality nor growth inhibition was observed after 90 days of treated
wastewater application. Moreover, an improvement in plant growth, leaf number and
a root development were noticed in these plants when compared with those
irrigated with tap water or with fertilizer. In contrast, leaf necrosis and
growth inhibition were observed in plants amended with raw wastewater. The
irrigation with treated wastewater also improved soil labile P content. Indeed,
soils amended with treated wastewater had more a double labile P concentration
(38.15 mg kg(-1)) in comparison with control soil (15.53 mg kg(-1)).
Copyright (c) 2009 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ecoenv.2009.12.021
1357. New Phytol. 2010 Apr;186(2):514-25. doi: 10.1111/j.1469-8137.2009.03137.x.

Epub
2009 Jan 6.
Presence of three mycorrhizal genes in the common ancestor of land plants

suggests a key role of mycorrhizas in the colonization of land by plants.
Wang B(1), Yeun LH, Xue JY, Liu Y, Ané JM, Qiu YL.
Author information:
(1)Department of Ecology and Evolutionary Biology, University of Michigan, Ann
Arbor, MI 48109, USA.
*The colonization of land by plants fundamentally altered environmental

conditions on earth. Plant-mycorrhizal fungus symbiosis likely played a key role
in this process by assisting plants to absorb water and nutrients from soil.
*Here, in a diverse set of land plants, we investigated the evolutionary
histories and functional conservation of three genes required for mycorrhiza
formation in legumes and rice (Oryza sativa), DMI1, DMI3 and IPD3. *The genes
were isolated from nearly all major plant lineages. Phylogenetic analyses showed
that they had been vertically inherited since the origin of land plants. Further,
cross-species mutant rescue experiments demonstrated that DMI3 genes from
liverworts and hornworts could rescue Medicago truncatula dmi3 mutants for
mycorrhiza formation. Yeast two-hybrid assays also showed that bryophyte DMI3
proteins could bind to downstream-acting M. trunculata IPD3 protein. Finally,
molecular evolutionary analyses revealed that these genes were under purifying
selection for maintenance of their ancestral functions in all mycorrhizal plant
lineages. *These results indicate that the mycorrhizal genes were present in the
common ancestor of land plants, and that their functions were largely conserved
during land plant evolution. The evidence presented here strongly suggests that
plant-mycorrhizal fungus symbiosis was one of the key processes that contributed
to the origin of land flora.
DOI: 10.1111/j.1469-8137.2009.03137.x
1358. Chembiochem. 2010 Mar 1;11(4):573-80. doi: 10.1002/cbic.200900674.
Unnatural polyketide analogues selectively target the HER signaling pathway in

human breast cancer cells.
Kwon SJ(1), Kim MI, Ku B, Coulombel L, Kim JH, Shawky JH, Linhardt RJ, Dordick
JS.
Author information:
(1)Department of Chemical and Biological Engineering, Center for Biotechnology
and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180,
USA.
Receptor tyrosine kinases are critical targets for the regulation of cell
survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to
have more aggressive disease with poor clinical outcomes. As a result, human
epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3,
represent important therapeutic targets. Several plant polyphenols including the
type III polyketide synthase products (genistein, curcumin, resveratrol, and
epigallocatechin-3-galate) possess chemopreventive activity, primarily as a
result of RTK inhibition. However, only a small fraction of the polyphenolic
structural universe has been evaluated. Along these lines, we have developed an
in vitro route to the synthesis and subsequent screening of unnatural polyketide
analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme
A (CoA) and methylmalonyl-CoA as extender substrates. The resulting polyketide
analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone)
with variable side chains. Screening chalcone synthase (CHS) reaction products
against BT-474 cells resulted in identification of several
trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the
HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of
nontransformed MCF-10A breast cells (IC(50)>100 microM). Specifically,
4-trifluoromethylcinnamoyl pyrone (compound 2 e) was highly potent (IC(50)<200
nM) among the test compounds toward proliferation of several breast cancer cell
lines. This breadth of activity likely stems from the ability of compound 2 e to
inhibit the phosphorylation of HER1, HER2, and HER3. Therefore, these polyketide
analogues might prove to be useful drug candidates for potential breast cancer
therapy.
DOI: 10.1002/cbic.200900674
PMCID: PMC3094853
1359. J Food Prot. 2010 Jan;73(1):9-17.
Control of Salmonella on sprouting mung bean and alfalfa seeds by using a

biocontrol preparation based on antagonistic bacteria and lytic bacteriophages.
Ye J(1), Kostrzynska M, Dunfield K, Warriner K.
Author information:
(1)Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G
2W1.
The following reports on the application of a combination of antagonistic

bacteria and lytic bacteriophages to control the growth of Salmonella on
sprouting mung beans and alfalfa seeds. Antagonistic bacteria were isolated from
mung bean sprouts and tomatoes by using the deferred plate assay to assess
anti-Salmonella activity. From the isolates screened, an Enterobacter asburiae
strain (labeled "JX1") exhibited stable antagonistic activity against a broad
range of Salmonella serovars (Agona, Berta, Enteritidis, Hadar, Heidelberg,
Javiana, Montevideo, Muenchen, Newport, Saint Paul, and Typhimurium). Lytic
bacteriophages against Salmonella were isolated from pig or cattle manure
effluent. A bacteriophage cocktail prepared from six isolates was coinoculated
with E. asburiae JX1 along with Salmonella in broth culture. The combination of
E. asburiae JX1 and bacteriophage cocktail reduced the levels of Salmonella by
5.7 to 6.4 log CFU/ml. Mung beans inoculated with Salmonella and sprouted over a
4-day period attained levels of 6.72 + or - 0.78 log CFU/g. In contrast, levels
of Salmonella were reduced to 3.31 + or - 2.48 or 1.16 + or - 2.14 log CFU/g when
the pathogen was coinoculated with bacteriophages or E. asburiae JX1,
respectively. However, by using a combination of E. asburiae JX1 and
bacteriophages, the levels of Salmonella associated with mung bean sprouts were
only detected by enrichment. The biocontrol preparation was effective at
controlling the growth of Salmonella under a range of sprouting temperatures (20
to 30 degrees Celsius) and was equally effective at suppressing the growth of
Salmonella on sprouting alfalfa seeds. The combination of E. asburiae JX1 and
bacteriophages represents a promising, chemical-free approach for controlling the
growth of Salmonella on sprouting seeds.
1360. Commun Agric Appl Biol Sci. 2010;75(2):229-33.
Preliminary note on potential use of forage crops for soil phytoremediation of

dieldrin.
Donnarumma L(1), Annesi T, Pompi V, Rosati S, Conte E.
Author information:
(1)CRA- Plant Pathology Research Centre, Via C.G. Bertero, 22 -Rome, Italy.
lucia.donnarumma@entecra.it
The aim of this trial was to evaluate the feasibility of using tall fescue
(Festuca arundinacea) and alfalfa (Medicago sativa) for phytoremediation of
dieldrin. Experimental trial was carried out in greenhouse with temperature and
light control. Each tested crop were seeded in individual pots (10 plants/pot)
filled with contaminated soil (47 microg/kg dieldrin) and uncontaminated soil
collected in sites located in the province of Latina (Italy). Samples of soil,
root, and aerial part of plants were analysed at 3 and 6 months after seeding.
The analytical determinations in soil and plant samples were carried out by
GLC-ECD and confirmed by GLC-MS. After 6 months in the greenhouse, recoveries of
dieldrin from soil planted with tall fescue and alfalfa were significantly lower
than recoveries in unplanted control soil. Dieldrin residue values in root did
not differ between the two different sampling times for each forage crop tested,
but they were always higher in fescue than in alfalfa. Residue levels in aerial
part were low (< 10 microg/kg) in the two forage crops. Preliminary results seem
to confirm the ability of tested plants to enhance dissipation of dieldrin in
soil at low level of contamination.
Levels of hexachlorocyclohexanes in agricultural environment of Sacco river

valley.
Pompi V(1), Donnarumma L, Rosati S, Conte E.
Author information:
(1)CRA- Research Center on Plant Pathology, Via C.G. Bertero 22, Rome, Italy.
Aim of this trial was to verify the occurrence and the distribution of
hexachlorocyclohexanes (HCHs) in soil, sediment, straw, alfalfa, other animal
feed grown in farms with contaminated soil. In the present study two years of
monitoring activity in the province of Roma and Frosinone was reported.
Experimental trial in two contaminated sites was carried out on uptake and
translocation of HCHs in maize and alfalfa. In 19 sites soil, forage and weed has
been collected for two years, soil samples consisted in cores of 40 cm to test
the presence of HCHs at different deep. The analytical determinations in soil and
plant samples were carried out by gas liquid chromatography with electron capture
detector and confirmed by mass detector. In the first year (2005- 2006) 68% of
soil samples were contaminated (HCHs > LOQ) and 3% of vegetable samples. In the
second year (2006- 2007) 42% of soil samples resulted positive and 26% of
vegetable matrix. In particular B hexacyclohexane was detected in wheat stem
(0.037 mg/kg) with a soil contamination of 0.039 mg/kg and in alfalfa (0.012
mg/kg) with presence in soil of 0.004 mg/kg. Experimental trials on maize
evidenced a translocation factor for this isomer stem/soil of 0.006 mg/kg ? and
for grain of 0.005 mg/kg. On alfalfa translocation factor root/soil was 0.01 and
shot/soil 0.009. A propose to calculate the threshold value of soil contamination
to admit crop grown destined to animal feed, would be based on HCHs LOD values
weighted with translocation factor.
1362. Top Curr Chem. 2010;297:45-66.
Engineered biosynthesis of plant polyketides: structure-based and

precursor-directed approach.
Abe I(1).
Author information:
(1)Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. abei@mol.f.u-tokyo.ac.jp
Pentaketide chromone synthase (PCS) and octaketide synthase (OKS) are novel
plant-specific type III polyketide synthases (PKSs) obtained from Aloe
arborescens. Recombinant PCS expressed in Escherichia coli catalyzes iterative
condensations of five molecules of malonyl-CoA to produce a pentaketide
5,7-dihydroxy-2-methylchromone, while recombinant OKS carries out sequential
condensations of eight molecules of malonyl-CoA to yield octaketides SEK4 and
SEK4b, the longest polyketides produced by the structurally simple type III PKS.
The amino acid sequences of PCS and OKS are 91% identical, sharing 50-60%
identity with those of other chalcone synthase (CHS) superfamily type III PKSs of
plant origin. One of the most characteristic features is that the conserved
active-site Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely replaced
with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed
mutagenesis and X-ray crystallographic analyses demonstrated that the chemically
inert single residue lining the active-site cavity controls the polyketide chain
length and the product specificity depending on the steric bulk of the side
chain. On the basis of the crystal structures, an F80A/Y82A/M207G triple mutant
of the pentaketide-producing PCS was constructed and shown to catalyze
condensations of nine molecules of malonyl-CoA to produce an unnatural novel
nonaketide naphthopyrone, whereas an N222G mutant of the octaketides-producing
OKS yielded a decaketide benzophenone SEK15 from ten molecules of malonyl-CoA. On
the other hand, the type III PKSs exhibited broad substrate specificities and
catalytic potential. OKS accepted p-coumaroyl-CoA as a starter substrate to
produce an unnatural novel C19 hexaketide stilbene and a C21 heptaketide
chalcone. Remarkably, the C21 chalcone-forming activity was dramatically
increased in the structure-guided OKS N222G mutant. In addition, OKS N222G mutant
also yielded unnatural novel polyketides from phenylacetyl-CoA and benzoyl-CoA as
a starter substrate. These results suggested that the engineered biosynthesis of
plant polyketides by combination of the structure-based and the
precursor-directed approach would lead to further production of chemically and
structurally divergent unnatural novel polyketides.
1363. Theor Appl Genet. 2010 Apr;120(6):1163-74. doi: 10.1007/s00122-009-1243-7.

Epub
2009 Dec 29.
Dehydrin variants associated with superior freezing tolerance in alfalfa

Rémus-Borel W(1), Castonguay Y, Cloutier J, Michaud R, Bertrand A, Desgagnés R,

Laberge S.
Author information:
Canada, 2560 Hochelaga Blvd., Quebec, QC, G1V-2J3, Canada.
wilfried.remusborel@gmail.com
A cDNA (msaCIG) encoding a cold-inducible Y(2)K(4) dehydrin in alfalfa (Medicago

sativa spp. sativa) was shown to share extensive homology with sequences from
other species and subspecies of Medicago. Differences were mainly the result of
the occurrence of large indels, amino acids substitutions/deletions and sequence
duplications. Using a combination of a bulk segregant analysis and RFLP
hybridization, we uncovered an msaCIG polymorphism that increases in frequency in
response to recurrent selection for superior freezing tolerance. Progenies from
crosses between genotypes with (D+) or without (D-) the polymorphic dehydrin
significantly differed in their tolerance to subfreezing temperatures. Based on
the msaCIG sequence, we looked for intragenic variations that could be associated
to the polymorphism detected on Southern blots. Amplifications with primers
targeting the 3' half side of msaCIG revealed fragment size variations between
pools of genotypes with (+) or without (-) the polymorphism. Three major groups
of amplicons of approximately 370 nt (G1), 330 nt (G2), and 290 nt (G3) were
distinguished. The G2 group was more intensively amplified in pools of genotypes
with the polymorphic dehydrin and was associated to a superior freezing tolerance
phenotype. Sequences analysis revealed that size variation in the 3' half was
attributable to the variable occurrence of large indels. Single amino acid
substitutions and/or deletions caused major differences in the prediction of the
secondary structure of the polypeptides. The identification of dehydrin variants
associated to superior freezing tolerance paves the way to the development of
functional markers and the fixation of favorable alleles in various genetic
backgrounds.
DOI: 10.1007/s00122-009-1243-7
1364. Sex Plant Reprod. 2009 Jun;22(2):97-107. doi: 10.1007/s00497-009-0095-2. Epub

2009 Feb 26.
Isolation of genes from female sterile flowers in Medicago sativa.
Capomaccio S(1), Barone P, Reale L, Veronesi F, Rosellini D.
Author information:
(1)Department of Applied Biology, University of Perugia, Perugia, Italy.
capemaster@gmail.com
A better knowledge of female sporogenesis and gametogenesis could have several

practical applications, from commercial hybrid seed production to gene
containment in GM crops. With the purpose of isolating genes involved in the
megasporogenesis process, the cDNA-AFLP technique was employed to isolate
transcript-derived fragments (TDF) differentially expressed between
female-fertile and female-sterile full-sib alfalfa plants. This female sterility
trait involves female-specific arrest of sporogenesis at early prophase
associated with ectopic, massive callose deposition within the nucellus.
Ninety-six TDFs were generated and BLAST analyses revealed similarities with
genes involved in different Gene Ontology categories. Three TDFs were selected
based on their putative functions: showing high similarity to a soybean
flower-expressed beta 1,3-glucanase, to an Arabidopsis thaliana MAPKKK, and to an
A. thaliana eukaryotic initiation translation factor eIF4G III, respectively. The
full length mRNA sequences were obtained. RT-PCR and in situ hybridizations were
performed to confirm differential expression during flower development. The
genomic organization of the three genes was assessed through sequencing and
Southern experiments. Sequence polymorphisms were found between sterile and
fertile plants. Our approach based on differential display and bulked segregant
analysis was successful in isolating genes that were differentially expressed
between fertile and sterile alfalfa plants.
DOI: 10.1007/s00497-009-0095-2
1365. Environ Biosafety Res. 2009 Jul-Sep;8(3):149-51. doi: 10.1051/ebr/2009010.

Epub
2009 Sep 24.
Response to Bagavathiannan and Van Acker's "Transgenes and national boundaries -

The need for international regulations": Biotechnology developers and regulators
already consider transgene movement across national boundaries and the
environmental risks posed by adventitious presence of unapproved events are
overstated.
Nickson TE(1), Raybould AF.
Author information:
(1)Monsanto Company, St. Louis, MO 63167, USA. thomas.nickson@monsanto.com
Comment on
Environ Biosafety Res. 2009 Jul-Sep;8(3):141-8.
Bagavathiannan and Van Acker propose greater international cooperation and

information sharing in risk assessment for biotechnology-derived crops because
pollen- and seed-mediated gene flow across political boundaries may lead to the
adventitious presence of unapproved transgenes at sites along the borders of
neighboring countries. However, they fail to convince us that something is wrong
with the current situation and provide no details of how it could be improved.
DOI: 10.1051/ebr/2009010
1366. Environ Biosafety Res. 2009 Jul-Sep;8(3):141-8. doi: 10.1051/ebr/2009011.

Epub
2009 Sep 24.
Transgenes and national boundaries - The need for international regulation.
Bagavathiannan M(1), Van Acker R.
Author information:
(1)Department of Plant Science, The University of Manitoba, Winnipeg, R3T 2N2,
Canada. umbagava@cc.umanitoba.ca
Comment in
Environ Biosafety Res. 2009 Jul-Sep;8(3):149-51.
What happens when one nation cultivates a transgenic crop variety but neighboring
nations do not? Using alfalfa as a case study, we argue that the potential for
international transgene flow is substantial, and therefore, the need for
international cooperation in regulatory decisions concerning transgenic crops is
imperative. Alfalfa (Medicago sativa, L.) is the major forage crop in North
America. Recently, genetically modified (GM) alfalfa received a moratorium on
further cultivation in the US on the grounds that the approvals were based on
inadequate environmental impact assessments. With their deep root system,
symbiotic nitrogen fixation, prolific seed production and prolonged dormancy,
alfalfa plants are capable of establishing self-perpetuating (feral) populations
in unmanaged environments. Given what is known about alfalfa pollen dispersal,
such feral populations could facilitate gene flow between GM and non-GM fields.
The border between the US and Canada, particularly in farming areas, is very
narrow (< 10 m wide). We surveyed along the US-Canada border and found both
alfalfa fields and potentially feral alfalfa plants in the ditches along the
border. Our survey results provide evidence of the possibility of cross-border
transgene flow, suggesting a need for international co-operative risk assessment
initiatives between the US and Canada. Such situations could occur for other
crops, in other international border regions as well.
DOI: 10.1051/ebr/2009011
1367. Environ Entomol. 2009 Dec;38(6):1765-71.
Development, reproduction, and within-plant infestation patterns of Aphis

craccivora (Homoptera: Aphididae) on alfalfa.
Berberet RC(1), Giles KL, Zarrabi AA, Payton ME.
Author information:
(1)Department of Entomology and Plant Pathology, Oklahoma State University,
Stillwater, OK 74076, USA.
The goal of this research was to describe developmental rates, reproductive

rates, and infestation patterns of Aphis craccivora Koch on alfalfa (Medicago
sativa L.). All studies were conducted on the susceptible cultivar OK08 using
aphids reared from collections made in Oklahoma. To determine thermal
requirements for growth of A. craccivora, development from birth to adult was
recorded at 7.2, 12.8, 18.3, 23.9, and 29.4 degrees C. The same constant
temperature treatments (except for 7.2 degrees C being raised to 8.3 degrees C)
were used to assess the influence of temperature on reproductive rates.
Within-plant distribution patterns were determined by infesting three stems on
each of 24 plants and recording numbers of A. craccivora on leaf blades,
petioles, and internodal stems sections at 2-d intervals through 10 d after
infestation. Aphid counts were analyzed to determine significant differences
among node parts (leaf blades, petioles, and stem sections). The developmental
threshold temperature for A. craccivora was calculated to be 7.1 degrees C, and
the thermal constant for development from the first instar to reproducing adult
was 100 DD ( degrees C). The optimal temperature range for reproduction on
alfalfa was 18-24 degrees C, with a mean of 82 nymphs produced per female. From
the initial infestation of three apterae per stem, numbers increased to a mean of
510 per stem after 10 d. Plant profiles showed that the greatest numbers of
aphids were located in middle and lower portions of the plant canopy. On all
sampling dates, the proportion of aphids on internodal stem sections was
significantly greater (P < 0.05) than on petioles and leaf blades.
1368. Sci Total Environ. 2010 Feb 1;408(5):1007-13. doi:

10.1016/j.scitotenv.2009.11.031. Epub 2009 Dec 8.
Enhanced removal of polychlorinated biphenyls from alfalfa rhizosphere soil in a

field study: the impact of a rhizobial inoculum.
Xu L(1), Teng Y, Li ZG, Norton JM, Luo YM.
Author information:
Polychlorinated biphenyls (PCB) are persistent pollutants in soil environments

where they continue to present considerable human health risks. Successful
strategies to remediate contaminated soils are needed that are effective and of
low cost. Bioremediation approaches that include the use of plants and microbial
communities to promote degradation of PCB have significant potential but need
further assessment under field conditions. The effects of growth of alfalfa
(Medicago sativa L.) and inoculation with a symbiotic nitrogen fixing bacterium
(Rhizobium meliloti) on the removal of polychlorinated biphenyls (PCB) from
rhizosphere soil were evaluated in a field experiment. The initial PCB content of
the soil ranged from 414 to 498 microg kg(-)(1). PCB removal for the rhizosphere
soil was enhanced in the planted treatments, an average of 36% decrease in PCB
levels compared to a 5.4% decrease in the unplanted soil, and further enhanced
when plants were inoculated with the symbiotic Rhizobium (an average of 43%
decrease) when evaluated at 90 days after planting. Plant biomass production was
higher in the inoculated treatment. The total PCB content was increased from 3.30
microg kg(-)(1) to 26.72 microg kg(-)(1) in plant shoots, and from 115.07 microg
kg(-)(1) to 142.23 microg kg(-)(1) in roots in the inoculated treatment compared
to the planted treatment. Increased colony forming units (cfu) of total
heterotrophic bacteria, biphenyl-degrading bacteria and fungi were observed in
the rhizosphere of inoculated plants. PCB removal from the rhizosphere soil was
not significantly correlated with the direct PCB uptake by the plants in any of
the treatments but was significantly correlated with the stimulation of
rhizosphere microflora. Changes in the soil microbial community structure in the
planted and inoculated treatment were observed by profiling of bacterial
ribosomal sequences. Some bacteria, such as Flavobacterium sp., may have
contributed to the effective degradation of PCB and deserve further
investigation.
DOI: 10.1016/j.scitotenv.2009.11.031
1369. Parasitology. 2010 Apr;137(4):685-96. doi: 10.1017/S0031182009991399. Epub

2009
Dec 7.
Anthelmintic activity of some Mediterranean browse plants against parasitic

nematodes.
Manolaraki F(1), Sotiraki S, Stefanakis A, Skampardonis V, Volanis M, Hoste H.
Author information:
(1)NAGREF-VRI NAGREF Campus, Thermi 57001 PO Box 60272 Thessaloniki, Greece.
The anthelmintic properties of tannin-rich plants are being explored as an

alternative to chemical drugs. Most data have been acquired on legume forages,
but only few on browse plants. The present study aimed to (i) screen the in vitro
effects of extracts from 7 Mediterranean plants on Haemonchus contortus, (ii)
verify the role of tannins using an inhibitor, polyvinyl polypyrrolidone (PVPP)
and (iii) verify the in vivo effects of extracts from 4 plants. Significant
inhibition was shown in vitro using a larval migration inhibition (LMI) assay for
all extracts except that from Olea europaea var. koroneiki. After adding PVPP,
the LMI values were restored to control levels for all plants except Pistacia
lentiscus and Ceratonia siliqua, confirming a role for tannins in the activity.
In the in vivo experiment, 48 lambs composed 6 groups, depending on diet. On Day
0, groups G1-G5 received H. contortus and Trichostrongylus colubriformis larvae
and G6 remained uninfected. The various diets were distributed from Days 14 to
45: P. lentiscus (G1), Quercus coccifera (G2), C. siliqua (G3), Onobrychis
viciifolia (G4), or Medicago sativa for the 2 control groups (G5, G6). Egg
excretion, packed cell volumes (PCVs) and inorganic phosphate were measured
weekly throughout the entire experimental period. At slaughter, the worms were
enumerated and their fecundity assessed. Consumption of the 4 browser plants did
not provoke differences in pathophysiological measurements but there were
significant decreases in egg excretion, mainly explained by significant decreases
in worm fecundity for both species, without any statistical difference in worm
numbers.
DOI: 10.1017/S0031182009991399
1370. Ecotoxicol Environ Saf. 2010 Mar;73(3):436-42. doi:

10.1016/j.ecoenv.2009.08.022.
Epub 2009 Dec 2.
Sewage sludge application can induce changes in antioxidant status of nodulated

alfalfa plants.
Antolín MC(1), Muro I, Sánchez-Díaz M.
Author information:
(1)Dpto. Biología Vegetal, Sección Biología Vegetal (Unidad Asociada al CSIC,
EEAD, Zaragoza), Facultades de Ciencias y Farmacia, Universidad de Navarra,
C/Irunlarrea 1, 31008 Pamplona, Spain. cantolin@unav.es
A greenhouse experiment was conducted to investigate the oxidative stress

produced by sewage sludge addition on nodulated alfalfa (Medicago sativa L. cv.
Aragón) plants. Two types of sludge were incorporated into substrate: anaerobic
mesophilic digested (AM) and autothermal thermophilic aerobic digested (ATAD)
sludge. Pots without sludge but with inoculated plants were used as control
treatment for comparison. Results showed that sludge amended plants had increased
tissue accumulation of heavy metals that induced oxidative stress. This is
characterized by induction of the antioxidant enzymatic activities and
alterations in the redox state of ascorbate. ATAD sludge application produced a
reduction in nodulation, increased nodule antioxidant enzyme activities and
decreased ascorbate/dehydroascorbate ratio. As a consequence, nodules of ATAD
treatment suffered from oxidative damages as evidenced by high malondialdehyde
levels. By contrast, AM application enhanced plant growth and no deleterious
effects on nodulation were found. Nodules developed in AM sludge had increased
antioxidant enzyme activities, ascorbate/dehydroascorbate ratio and improved
capacity for thiol synthesis. Results clearly showed that nodulated alfalfa
performed better in AM than in ATAD sludge and suggest that differential response
appears to be mediated by plant ability to thiol synthesis and to maintenance of
a more equilibrated antioxidant status.
(c) 2009 Elsevier Inc. All rights reserved.
DOI: 10.1016/j.ecoenv.2009.08.022
1371. FEMS Microbiol Lett. 2010 Jan;302(2):123-30. doi:

10.1111/j.1574-6968.2009.01846.x. Epub 2009 Nov 6.
Cultural conditions required for the induction of an adaptive acid-tolerance

response (ATR) in Sinorhizobium meliloti and the question as to whether or not
the ATR helps rhizobia improve their symbiosis with alfalfa at low pH.
Draghi WO(1), Del Papa MF, Pistorio M, Lozano M, de Los Angeles Giusti M, Torres
Tejerizo GA, Jofré E, Boiardi JL, Lagares A.
Author information:
(1)IBBM - Instituto de Biotecnología y Biología Molecular, Departamento de
Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La
Plata, CCT-CONICET-La Plata, Buenos Aires, Argentina.
Sinorhizobium meliloti associates with Medicago and Melilotus species to develop

nitrogen-fixing symbioses. The agricultural relevance of these associations, the
worldwide distribution of acid soils, and the remarkable acid sensitivity of the
microsymbiont have all stimulated research on the responses of the symbionts to
acid environments. We show here that an adaptive acid-tolerance response (ATR)
can be induced in S. meliloti, as shown previously for Sinorhizobium medicae,
when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In
marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia
are grown in a chemostat under continuous cultivation at the same pH. The
adaptive ATR appears as a complex process triggered by an increased hydrogen-ion
concentration, but operative only if other--as yet unknown--concomitant factors
that depend on the culture conditions are present (although not provided under
continuous cultivation). Although the stability of the ATR and its influence on
acid tolerance has been characterized in rhizobia, no data have been available on
the effect of the adapted state on symbiosis. Coinoculation experiments showed
that acid-adapted indicator rhizobia (ATR+) were present in >90% of the nodules
when nodulation was performed at pH 5.6, representing a >30% increase in
occupancy compared with a control test. We show that the ATR represents a clear
advantage in competing for nodulation at low pH. It is not yet clear whether such
an effect results from an improved performance in the acid environment during
preinfection, an enhanced ability to initiate infections, or both conditions. The
practical use of ATR+ rhizobia will depend on validation experiments with soil
microcosms and on field testing, as well as on the possibility of preserving the
physiology of ATR+ bacteria in inoculant formulations.
DOI: 10.1111/j.1574-6968.2009.01846.x
[Suitable habitat of Therioaphis trifolii population on arable lands of southern

Ningxia based on GIS].
Zhang R(1), Zhu MM, Ma JH, Wang F.

Author information:
(1)Institute of Plant Protection, Ningxia Academy of Agriculture and Forestry
Sciences, Yinchuan, China. zhangrong_nx@yahoo.com.cn
Based on GIS techniques, the interpolation maps of Therioaphis trifolii

population on the arable lands in Yuanzhou District of Guyuan City in different
periods were overlapped with the ecological regionalization map and digital
altitude map, aimed to analyze the relations of T. trifolii population density
with its ecological environment and geographical distribution. In the study area,
the occurrence and distribution of T. trifolii had close relations with climate,
geomorphology, and altitude. The suitable habitat of T. trifolii in the District
was located in the semi-arid plain area and semi-arid holly area at the altitude
1440-2000 m, while the most possible occurrence area was the semi-arid plain at
the altitude 1600-1800 m. By combining the suitable habitat with the
interpolation maps of T. trifolii population in different periods, the spatial
dynamics of T. trifolii population in variety of ecological areas could be
analyzed quantitatively.
[Effects of grassland restoration from cropland on soil macrofaunal community in

Wuchuan County, Inner Mongolia].
Liu XM(1), Men LN.
Author information:
(1)College of Life Science and Technology, Inner Mongolia Normal University,
Huhhot, China. linxm6596671@163.com
By the method of hand-sorting, and using the indices individual number, group
number, and biodiversity, the changes in the soil macrofaunal community under the
grassland restoration from cropland in Wuchuan County, Inner Mongolia were
investigated. A total of 1479 soil macrofauna belonging to 3 classes, 7 orders
and 41 families were captured. After the grassland restoration from cropland, the
group number, individual number, and biomass of soil macrofauna all had an
increasing trend, and the biodiversity of soil macrofaunal community reflected by
D(Ma) and DG increased significantly. However, due to the short term of cropland
rehabilitation, the Shannon-Wiener index H' and the Pielou evenness index E of
the soil microfaunal community in grassland were not higher than those in
cropland. Different soil macrofaunal groups had different responses to the
grassland restoration from cropland. The preferable approaches for the grassland
restoration from cropland were planting Medicago sativa or planting Medicago
sativa + Artemisia sp.
1374. Planta. 2010 Jan;231(2):319-28. doi: 10.1007/s00425-009-1054-8. Epub 2009 Nov

17.
Red clover coumarate 3'-hydroxylase (CYP98A44) is capable of hydroxylating

p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the
biosynthesis of phaselic acid.
Sullivan ML(1), Zarnowski R.
Author information:
of Agriculture, 1925 Linden Drive, Madison, WI 53706, USA.
Red clover (Trifolium pratense) leaves accumulate several mumol of phaselic acid
[2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such
o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents
breakdown of forage protein during storage. Forages like alfalfa (Medicago
sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of
their protein upon harvest and storage results in economic losses and release of
excess nitrogen into the environment. Understanding how red clover synthesizes
o-diphenols such as phaselic acid will help in the development of forages
utilizing this natural system of protein protection. We have proposed
biosynthetic pathways in red clover for phaselic acid that involve a specific
hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether
the transfer reaction to malate to form phaselic acid involves caffeic acid or
p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate.
The latter would require a coumarate 3'-hydroxylase (C3'H) capable of
hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a
cytochrome P450 C3'H (CYP98A44) was identified and its gene cloned from red
clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula
and Arabidopsis thaliana C3'H proteins that are capable of hydroxylating
p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis.
CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves.
Immune serum raised against CYP98A44 recognizes a membrane-associated protein in
red clover stems and leaves and cross-reacts with C3'H proteins from other
species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of
hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding
indicates that in red clover, phaselic acid is likely formed by transfer of a
caffeoyl moiety to malic acid, although the existence of a second C3'H capable of
hydroxylating p-coumaroyl-malate cannot be definitively ruled out.
DOI: 10.1007/s00425-009-1054-8
1375. Ying Yong Sheng Tai Xue Bao. 2009 Jul;20(7):1711-5.
[Ecological responses of Brassica juncea-alfalfa intercropping to cadmium

stress].
Li XB(1), Xie JZ, Li BW, Wang W.
Author information:
(1)College of Resources and Environmental Sciences, Hebei Agricultural
University, Baoding 071001, Hebei, China. lixinbo2004@163.com
A pot experiment was conducted to study the effects of cadmium (Cd) stress on the
soil-plant system under Brassica juncea-alfalfa intercropping, and to evaluate
the Cd feed safety of alfalfa. Comparing with monoculture, when the soil Cd
content was in the range of 0.37-20.37 mg x kg(-1), intercropping decreased the
B. juncea biomass by 0.4%-11.8% while increased the alfalfa biomass by
55.3%-70.0%. Soil available Cd was mainly determined by soil total Cd and plant
species, and less affected by planting pattern. Comparing with monoculture, when
the soil Cd content was in the range of 0.37-5.37 mg x kg(-1), intercropping
increased the Cd content in above-ground part of B. juncea by 14.5%, but
decreased the Cd content in above-ground part of alfalfa by 57.1%. The Cd
contents in the above-ground parts of alfalfa under monoculture and intercropping
were 0.21 and 0.09 mg x kg(-1), respectively, neither of them being over the feed
safety standard (0.5 mg x kg(-1)). When the soil Cd content was in the range of
10.37-20.37 mg x kg(-1), though the Cd contents of mono-cultured and intercropped
alfalfa were both over the standard, the Cd contents in the above-ground parts of
alfalfa and B. juncea under intercropping were decreased by 2.8%-48.3% and
1.1%-48.6%, respectively. Under both monoculture and intercropping, the Cd
transport coefficient of B. juncea was far greater than that of alfalfa.
1376. Planta. 2010 Jan;231(2):233-44. doi: 10.1007/s00425-009-1043-y. Epub 2009 Nov

8.
Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA)

has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago
sativa L.).
Aleman L(1), Ortega JL, Martinez-Grimes M, Seger M, Holguin FO, Uribe DJ,
Garcia-Ibilcieta D, Sengupta-Gopalan C.
Author information:
(1)Graduate Program in Molecular Biology, New Mexico State University, Las
Cruces, NM 88003, USA.
Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of
sucrose in photosynthetic tissues. We characterized the expression of three
different isoforms of SPS belonging to two different SPS gene families in alfalfa
(Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms
belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced
expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf
and nodule SPS enzymes showed differences in chromatographic and electrophoretic
migration and differences in V (max) and allosteric regulation. The root nodules
in legume plants are a strong sink for photosynthates with its need for ATP,
reducing power and carbon skeletons for dinitrogen fixation and ammonia
assimilation. The expression of genes encoding SPS and other key enzymes in
sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was
analyzed in the leaves and nodules of plants inoculated with Sinorhizobium
meliloti. Based on the expression pattern of these genes, the properties of the
SPS isoforms and the concentration of starch and soluble sugars in nodules
induced by a wild type and a nitrogen fixation deficient strain, we propose that
SPS has an important role in the control of carbon flux into different metabolic
pathways in the symbiotic nodules.
DOI: 10.1007/s00425-009-1043-y
PMCID: PMC3881968
1377. Nat Biotechnol. 2009 Nov;27(11):970. doi: 10.1038/nbt1109-970c.
Courts upbraid USDA for 'lax review' of GM crops.

Waltz E.
DOI: 10.1038/nbt1109-970c

10.1094/MPMI-22-12-1656.
Production of succinoglycan polymer in Sinorhizobium meliloti is affected by

SMb21506 and requires the N-terminal domain of ExoP.
Jofré E(1), Becker A.
Author information:
(1)Departamento de Ciencias Naturales, Facultad de Ciencias Exactas,
Fisico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto,
Córdoba, Argentina. ejofre@exa.unrc.edu.ar
The protein tyrosine kinase ExoP, consisting of an N-terminal periplasmic and a

C-terminal cytoplasmic domain, is important for polymerization of the
exopolysaccharide succinoglycan (EPS I) in Sinorhizobium meliloti. We analyzed
the contribution of the ExoP paralogs ExoP2 and SMb21506 to the production of the
high molecular weight (HMW) form of EPS I. ExoP2, though not contributing to EPS
I or lipopolysaccharide biosynthesis, showed increased expression at high
osmolarity and was expressed in Medicago sativa nodules, suggesting an
involvement in the synthesis of an as-yet-unidentified polysaccharide.
Furthermore, a mutation in SMb21506 affected the production of HMW EPS I,
particularly in the absence of the C-terminal ExoP domain. High salinity induced
the production of HMW EPS I by the wild type and mutants whereas high osmolarity
had the opposite effect. It was shown that ExoP localizes at the inner membrane
of S. meliloti cells. Tyrosine phosphorylation of the C-terminal domain was
strongly increased by amino acid substitutions in the polysaccharide
co-polymerase motif (formerly proline-rich motif) located in the N-terminal
domain, suggesting that this phosphorylation could be modulated by conformational
changes of the N-terminal domain. Moreover, deletion of a coiled-coil motif
present in the N-terminal domain abolished phosphorylation and EPS I production
and, consequently, the ability to nodulate M. sativa.
DOI: 10.1094/MPMI-22-12-1656
1379. J Plant Physiol. 2010 Mar 1;167(4):286-91. doi: 10.1016/j.jplph.2009.09.014.

Epub
2009 Oct 30.
Reduced nodulation in alfalfa induced by arsenic correlates with altered

expression of early nodulins.
Lafuente A(1), Pajuelo E, Caviedes MA, Rodríguez-Llorente ID.
Author information:
(1)Departamento de Microbiología y Parasitología, Facultad de Farmacia,
Universidad de Sevilla, c/ Profesor García González, 2, 41012 Sevilla, Spain.
Arsenic (As) reduces legume nodulation by affecting the first stages of the
symbiotic interaction, which causes a 90% decrease in rhizobial infections. In
this paper, we examine molecular mechanisms underlying this toxic effect, using
the model system Medicago sativa-Sinorhizobium. In the presence and absence of
As, the expression patterns of seven nodulin genes, markers for the different
events leading to nodule formation, were analyzed by RT-PCR and by real-time
RT-PCR. A significant decrease was observed, especially from days 1-5 after the
inoculation, in the expression of four early nodulins: the genes coding the Nod
factor receptor (nork), the transcription factor NIN and the markers for
infection progression (N6) and nodule organogenesis (Enod2). On the contrary, the
expression of markers for primordium initiation (Enod40) and differentiation
(ccs52) was not significantly altered. Finally, the expression of a marker for
nitrogen fixation (Legbrc) was also reduced, probably due to the reduction in
nodule number induced by As. These results suggest that As affects the expression
of nodulation genes that have been associated with processes that take place in
the epidermis and the outer cortical cells, and that the expression of genes
associated with events that take place in the inner cortical cells is less
affected. This is the first report showing changes in the expression of nodulin
genes induced by the presence of any toxic metal(loid).
DOI: 10.1016/j.jplph.2009.09.014
1380. Microbiology. 2010 Feb;156(Pt 2):505-20. doi: 10.1099/mic.0.034058-0. Epub

2009
Oct 29.
Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa

(alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a
history of cultivation.
Bromfield ES(1), Tambong JT, Cloutier S, Prévost D, Laguerre G, van Berkum P, Thi
TV, Assabgui R, Barran LR.
Author information:
(1)Agriculture and Agri-Food Canada, Ottawa, Ontario K1A OC6, Canada.
eden.bromfield@agr.gc.ca
Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet

clover, grown at a site without a known history of cultivation, were identified
as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on
sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and
recA) genes, Southern hybridization/RFLP and a range of phenotypic
characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp.
predominated on alfalfa (frequency approximately 68 %) but was recovered
infrequently ( approximately 1 %) from sweet clover. A second genotype was
isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in
association with sweet clover and Phaseolus vulgaris, but were moderately
effective with alfalfa. They produced a near-neutral reaction on mineral salts
agar containing mannitol, which is atypical of the genus Rhizobium. A single
isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from
sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic
analysis indicated that Ensifer sp. strain T173 is closely related to, but
separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173
is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective
nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium
atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably
represent bacterial opportunists. Three genotypes of E. meliloti that were
symbiotically effective with alfalfa and sweet clover were encountered
infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were
encountered infrequently and were highly effective with alfalfa, sweet clover and
Medicago polymorpha. The ecological and practical implications of the findings
are discussed.
DOI: 10.1099/mic.0.034058-0
1381. Mol Microbiol. 2009 Dec;74(5):1223-37. doi: 10.1111/j.1365-2958.2009.06931.x.

Epub 2009 Oct 19.
Null mutations in Sinorhizobium meliloti exoS and chvI demonstrate the importance
of this two-component regulatory system for symbiosis.
Bélanger L(1), Dimmick KA, Fleming JS, Charles TC.
Author information:
(1)Department of Biology, University of Waterloo, Waterloo, ON, Canada.
Exopolysaccharides, either succinoglycan or galactoglucan, are essential for the

establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa
(alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator
of succinoglycan production but the genes that are directly regulated by ChvI
have not been determined. Difficulty isolating exoS and chvI null mutants has
prompted the suggestion that these genes are essential for S. meliloti viability.
We have successfully isolated exoS and chvI null mutants using a
merodiploid-facilitated strategy. We present evidence that the S. meliloti
ExoS/ChvI two-component regulatory system is essential for symbiosis with
alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate
that ExoS/ChvI controls both succinoglycan and galactoglucan production and is
required for growth on over 21 different carbon sources. These new findings
suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are
specific for succinoglycan biosynthesis but rather genes that have common
influence on both succinoglycan and galactoglucan production. Other studied
alpha-proteobacteria ExoS/ChvI orthologues are required for the bacteria to
invade or persist in host cells and thus we present more evidence that this
two-component regulatory system is essential for alpha-proteobacterial host
interaction.
DOI: 10.1111/j.1365-2958.2009.06931.x
From milk to diet: feed recognition for milk authenticity.
Ponzoni E(1), Gianì S, Mastromauro F, Breviario D.
Author information:
(1)Istituto Sperimentale Italiano "Lazzaro Spallanzani," Rivolta d'Adda (CR)
26027, Italy.
The presence of plastidial DNA fragments of plant origin in animal milk samples
has been confirmed. An experimental plan was arranged with 4 groups of goats,
each provided with a different monophytic diet: 3 fresh forages (oats, ryegrass,
and X-triticosecale) and one 2-wk-old silage (X-triticosecale). Feed-derived
rubisco (ribulose bisphosphate carboxylase, rbcL) DNA fragments were detected in
100% of the analyzed goat milk samples, and the nucleotide sequence of the
PCR-amplified fragments was found to be 100% identical to the corresponding
fragments amplified from the plant species consumed in the diet. Two additional
chloroplast-based molecular markers were used to set up an assay for
distinctiveness, conveniently based on a simple PCR. In one case, differences in
single nucleotides occurring within the gene encoding for plant maturase K (matK)
were exploited. In the other, plant species recognition was based on the
difference in the length of the intron present within the transfer RNA leucine
(trnL) gene. The presence of plastidial plant DNA, ascertained by the PCR-based
amplification of the rbcL fragment, was also assessed in raw cow milk samples
collected directly from stock farms or taken from milk sold on the commercial
market. In this case, the nucleotide sequence of the amplified DNA fragments
reflected the multiple forages present in the diet fed to the animals.
DOI: 10.3168/jds.2009-2239
1383. J Bacteriol. 2009 Dec;191(24):7490-9. doi: 10.1128/JB.00725-09. Epub 2009 Oct

16.
Importance of trehalose biosynthesis for Sinorhizobium meliloti Osmotolerance and

nodulation of Alfalfa roots.
Domínguez-Ferreras A(1), Soto MJ, Pérez-Arnedo R, Olivares J, Sanjuán J.
Author information:
Experimental del Zaidín, CSIC, Granada, Spain.
The disaccharide trehalose is a well-known osmoprotectant, and trehalose

accumulation through de novo biosynthesis is a common response of bacteria to
abiotic stress. In this study, we have investigated the role of endogenous
trehalose synthesis in the osmotolerance of Sinorhizobium meliloti. Genes coding
for three possible trehalose synthesis pathways are present in the genome of S.
meliloti 1021: OtsA, TreYZ, and TreS. Among these, OtsA has a major role in
trehalose accumulation under all of the conditions tested and is the main system
involved in osmoadaptation. Nevertheless, the other two systems are also
important for growth in hyperosmotic medium. Genes for the three pathways are
transcriptionally responsive to osmotic stress. The presence of at least one
functional trehalose biosynthesis pathway is required for optimal competitiveness
of S. meliloti to nodulate alfalfa roots.
DOI: 10.1128/JB.00725-09
PMCID: PMC2786593
1384. Food Microbiol. 2009 Dec;26(8):847-52. doi: 10.1016/j.fm.2009.05.007. Epub

2009
May 27.
Assessment of the microbiological safety of edible dried seeds from retail

premises in the United Kingdom with a focus on Salmonella spp.
Willis C(1), Little CL, Sagoo S, de Pinna E, Threlfall J.
Author information:
(1)Health Protection Agency, Food Water and Environmental Microbiology Network
(Southampton Laboratory), Level B South Block, Southampton General Hospital, SO16
6YD, UK. caroline.willis@hpa.org.uk
Sesame seed products have recently been associated with a number of Salmonella
outbreaks in the UK and elsewhere. Aside from sesame seeds, there is little
published information on the prevalence of Salmonella spp. in edible seeds. A
study of 3735 samples of retail edible dried seeds in the UK was therefore
carried out between October 2007 and March 2008 to assess their microbiological
safety in relation to Salmonella contamination and levels of Escherichia coli, an
indicator of faecal contamination. Overall, Salmonella was detected in 23 samples
(0.6%), of which over half (57%) were sesame seeds. Other seeds contaminated with
Salmonella were linseed (1 sample), sunflower (1 sample), alfalfa (1 sample),
melon (4 samples) and mixed seeds (3 samples). E. coli was detected in 9% of
samples, with 1.5% containing unsatisfactory levels (> or = 10(2)/g). These
included melon, pumpkin, sesame, hemp, poppy, linseed, sunflower and mixed seeds.
The UK retailers affected by the detection of Salmonella in their products
recalled the contaminated batches, and Food Standards Agency food alerts were
issued to advise against the consumption of affected seed products. This study
highlights the importance of good hygiene practices and effective decontamination
procedures during the production of these products.
DOI: 10.1016/j.fm.2009.05.007
1385. Mol Plant. 2008 Sep;1(5):858-77. doi: 10.1093/mp/ssn052.
Construction of a rice glycosyltransferase phylogenomic database and

identification of rice-diverged glycosyltransferases.
Cao PJ(1), Bartley LE, Jung KH, Ronald PC.
Author information:
(1)Department of Plant Pathology, University of California, Davis, CA 95616, USA.
Glycosyltransferases (GTs; EC 2.4.x.y) constitute a large group of enzymes that

form glycosidic bonds through transfer of sugars from activated donor molecules
to acceptor molecules. GTs are critical to the biosynthesis of plant cell walls,
among other diverse functions. Based on the Carbohydrate-Active enZymes (CAZy)
database and sequence similarity searches, we have identified 609 potential GT
genes (loci) corresponding to 769 transcripts (gene models) in rice (Oryza
sativa), the reference monocotyledonous species. Using domain composition and
sequence similarity, these rice GTs were classified into 40 CAZy families plus an
additional unknown class. We found that two Pfam domains of unknown function,
PF04577 and PF04646, are associated with GT families GT61 and GT31, respectively.
To facilitate functional analysis of this important and large gene family, we
created a phylogenomic Rice GT Database
(http://ricephylogenomics.ucdavis.edu/cellwalls/gt/). Through the database,
several classes of functional genomic data, including mutant lines and gene
expression data, can be displayed for each rice GT in the context of a
phylogenetic tree, allowing for comparative analysis both within and between GT
families. Comprehensive digital expression analysis of public gene expression
data revealed that most ( approximately 80%) rice GTs are expressed. Based on
analysis with Inparanoid, we identified 282 'rice-diverged' GTs that lack
orthologs in sequenced dicots (Arabidopsis thaliana, Populus tricocarpa, Medicago
truncatula, and Ricinus communis). Combining these analyses, we identified 33
rice-diverged GT genes (45 gene models) that are highly expressed in
above-ground, vegetative tissues. From the literature and this analysis, 21 of
these loci are excellent targets for functional examination toward understanding
and manipulating grass cell wall qualities. Study of the remainder may reveal
aspects of hormone and protein metabolism that are critical for rice biology.
This list of 33 genes and the Rice GT Database will facilitate the study of GTs
and cell wall synthesis in rice and other plants.
DOI: 10.1093/mp/ssn052
1386. Environ Entomol. 2009 Oct;38(5):1480-4.
Correlations between environmental factors and wild bee behavior on alfalfa

(Medicago sativa) in northwestern China.
Wang X(1), Liu H, Li X, Song Y, Chen L, Jin L.
Author information:
(1)School of Pastoral Agriculture Science and Technology, Lanzhou University, PO
Box 61, Lanzhou 730020, China.
To discover the effect of environmental factors on pollinator visitation to

flowering Medicago sativa, several field experiments were designed to examine the
diurnal movement patterns of wild bee species in the Hexi Corridor of
northwestern China. Our study results showed that Megachile abluta, M. spissula,
and Xylocopa valga showed unimodal diurnal foraging behavior, whereas Andrena
parvula and Anthophora melanognatha showed bimodal diurnal foraging behavior.
Correlation analysis indicated that diurnal foraging activities of pollinators
were significantly correlated with environmental factors. Correlations of
foraging activities versus environmental factors for M. abluta, M. spissula, and
X. valga best fit a linear model, whereas those of A. parvula and A. melanognatha
best fit a parallel quadratic model. Results of this study indicated that
solitary wild bees such as M. abluta, M. spissula, X. valga, A. parvula, and A.
melanognatha are potential alfalfa pollinators in the Hexi Corridor. An
understanding of the environmental factors that affect the behaviors of different
wild bees foraging in alfalfa are basic to the utilization of solitary wild bees
in a practical way for increased, or more consistent, pollination of alfalfa for
seed production.
1387. J Anim Sci. 2010 Jan;88(1):258-74. doi: 10.2527/jas.2008-1342. Epub 2009 Oct
9.
Evaluation of dried distillers grains and roughage source in steam-flaked corn

finishing diets.
Uwituze S(1), Parsons GL, Shelor MK, Depenbusch BE, Karges KK, Gibson ML,
Reinhardt CD, Higgins JJ, Drouillard JS.
Author information:
(1)Department of Animal Sciences and Industry, Kansas State University, Manhattan
66506-1600, USA.
Two studies were conducted to evaluate effects of dried distillers grains with
solubles (DDGS) and alfalfa hay (AH) or corn silage (CS) on feedlot performance,
carcass characteristics, ruminal fermentation, and diet digestibility in cattle
fed steam-flaked corn (SFC) diets. In trial 1, crossbred heifers (n = 358; BW =
353 +/- 13 kg) were used in a finishing trial to evaluate interactions between
corn-DDGS and roughage source (AH or CS) in terms of impact on feedlot
performance and carcass characteristics. Experimental diets (DM basis) consisted
of SFC and 11% CS without DDGS (SFC-CS), SFC and 11% CS with 25% DDGS (DDGS-CS),
SFC and 6% AH without DDGS (SFC-AH), and SFC with 25% DDGS and 6% AH (DDGS-AH).
Heifers were fed for ad libitum intake once daily for 97 d. Results indicated no
interaction between DDGS and roughage source with respect to animal performance.
Feeding DDGS did not affect ADG (P = 0.19), DMI (P = 0.14), or feed conversion (P
= 0.67). Heifers fed CS had greater DMI than those fed AH (P = 0.05), but ADG (P
= 0.56) and G:F (P = 0.63) were not different. There were no differences among
treatments with respect to HCW, dressing percentage, subcutaneous fat thickness,
quality grades, or yield grades (P > 0.20). Cattle fed CS tended (P = 0.10) to
have greater marbling scores than those fed AH. There was an interaction (P =
0.02) between roughage and DDGS with respect to incidence of liver abscess. The
greatest incidence was observed in cattle fed diets without DDGS when CS was fed,
and the least was observed in cattle fed diets without DDGS when AH was used. In
the second trial, ruminal fermentation characteristics and diet digestibility
were examined in 12 cannulated Holstein steers fed similar diets to those fed in
the finishing trial. Ruminal pH for all treatments was below 5.8 for 14 h after
feeding. Acetate:propionate ratios were less (P = 0.02) in steers fed 25% DDGS
but had greater (P = 0.02) ruminal lactate concentrations compared with cattle
fed 0% DDGS. Feeding 25% DDGS decreased (P < 0.01) ruminal ammonia
concentrations, and digestion of DM and OM was less (P < 0.01) compared with
diets without DDGS. The decrease in digestibility was largely attributable to
decreases in digestion of CP (P = 0.03) and NDF (P < 0.01). Feeding strategies
aimed at increasing ruminal pH and ruminally available protein may improve
digestion of DDGS in steam-flaked corn-based finishing diets.
DOI: 10.2527/jas.2008-1342
1388. J Exp Bot. 2010;61(1):121-30. doi: 10.1093/jxb/erp287.
Elevated CO2 concentration around alfalfa nodules increases N2 fixation.
Fischinger SA(1), Hristozkova M, Mainassara ZA, Schulze J.
Author information:
(1)Department of Crop Sciences, Plant Nutrition, Georg-August-University of
Goettingen, Carl-Sprengel-Street 1, D-37075 Goettingen, Germany.
Nodule CO2 fixation via PEPC provides malate for bacteroids and oxaloacetate for
N assimilation. The process is therefore of central importance for efficient
nitrogen fixation. Nodule CO2 fixation is known to depend on external CO2
concentration. The hypothesis of the present paper was that nitrogen fixation in
alfalfa plants is enhanced when the nodules are exposed to elevated CO2
concentrations. Therefore nodulated plants of alfalfa were grown in a hydroponic
system that allowed separate aeration of the root/nodule compartment that avoided
any gas leakage to the shoots. The root/nodule compartments were aerated either
with a 2500 microl l(-1) (+CO2) or zero microl l(-1) (-CO2) CO2-containing N2/O2
gas flow (80/20, v/v). Nodule CO2 fixation, nitrogen fixation, and growth were
strongly increased in the +CO2 treatment in a 3-week experimental period. More
intensive CO2 and nitrogen fixation coincided with higher per plant amounts of
amino acids and organic acids in the nodules. Moreover, the concentration of
asparagine was increased in both the nodules and the xylem sap. Plants in the
+CO2 treatment tended to develop nodules with higher %N concentration and
individual activity. In a parallel experiment on plants with inefficient nodules
(fix-) the +CO2 treatment remained without effect. Our data support the thesis
that nodule CO2 fixation is pivotal for efficient nitrogen fixation. It is
concluded that strategies which enhance nodule CO2 fixation will improve nitrogen
fixation and nodule formation. Moreover, sufficient CO2 application to roots and
nodules is necessary for growth and efficient nitrogen fixation in hydroponic and
aeroponic growth systems.
DOI: 10.1093/jxb/erp287
PMCID: PMC2791116
1389. Nucleic Acids Res. 2010 Jan;38(Database issue):D806-13. doi:

10.1093/nar/gkp818.
Epub 2009 Oct 6.
PMRD: plant microRNA database.
Zhang Z(1), Yu J, Li D, Zhang Z, Liu F, Zhou X, Wang T, Ling Y, Su Z.
Author information:
(1)State Key Laboratory of Plant Physiology and Biochemistry and State Key
Laboratory for Agricultural Biotechnology, College of Biological Sciences, China
Agricultural University, Beijing 100193, China.
MicroRNAs (miRNA) are approximately 21 nucleotide-long non-coding small RNAs,

which function as post-transcriptional regulators in eukaryotes. miRNAs play
essential roles in regulating plant growth and development. In recent years,
research into the mechanism and consequences of miRNA action has made great
progress. With whole genome sequence available in such plants as Arabidopsis
thaliana, Oryza sativa, Populus trichocarpa, Glycine max, etc., it is desirable
to develop a plant miRNA database through the integration of large amounts of
information about publicly deposited miRNA data. The plant miRNA database (PMRD)
integrates available plant miRNA data deposited in public databases, gleaned from
the recent literature, and data generated in-house. This database contains
sequence information, secondary structure, target genes, expression profiles and
a genome browser. In total, there are 8433 miRNAs collected from 121 plant
species in PMRD, including model plants and major crops such as Arabidopsis,
rice, wheat, soybean, maize, sorghum, barley, etc. For Arabidopsis, rice, poplar,
soybean, cotton, medicago and maize, we included the possible target genes for
each miRNA with a predicted interaction site in the database. Furthermore, we
provided miRNA expression profiles in the PMRD, including our local rice
oxidative stress related microarray data (LC Sciences miRPlants_10.1) and the
recently published microarray data for poplar, Arabidopsis, tomato, maize and
rice. The PMRD database was constructed by open source technology utilizing a
user-friendly web interface, and multiple search tools. The PMRD is freely
available at http://bioinformatics.cau.edu.cn/PMRD. We expect PMRD to be a useful
tool for scientists in the miRNA field in order to study the function of miRNAs
and their target genes, especially in model plants and major crops.
DOI: 10.1093/nar/gkp818
PMCID: PMC2808885
1390. Proc Natl Acad Sci U S A. 2009 Oct 6;106(40):16936-41. doi:

10.1073/pnas.0909417106. Epub 2009 Sep 28.
Experimental evidence for the conditions necessary to sustain meandering in

coarse-bedded rivers.
Braudrick CA(1), Dietrich WE, Leverich GT, Sklar LS.
Author information:
(1)Department of Earth and Planetary Science, University of California Berkeley,
Berkeley, CA 94720, USA.
Meandering rivers are common on Earth and other planetary surfaces, yet the
conditions necessary to maintain meandering channels are unclear. As a
consequence, self-maintaining meandering channels with cutoffs have not been
reproduced in the laboratory. Such experimental channels are needed to explore
mechanisms controlling migration rate, sinuosity, floodplain formation, and
planform morphodynamics and to test theories for wavelength and bend propagation.
Here we report an experiment in which meandering with near-constant width was
maintained during repeated cutoff and regeneration of meander bends. We found
that elevated bank strength (provided by alfalfa sprouts) relative to the
cohesionless bed material and the blocking of troughs (chutes) in the lee of
point bars via suspended sediment deposition were the necessary ingredients to
successful meandering. Varying flood discharge was not necessary. Scaling
analysis shows that the experimental meander migration was fast compared to most
natural channels. This high migration rate caused nearly all of the bedload
sediment to exchange laterally, such that bar growth was primarily dependent on
bank sediment supplied from upstream lateral migration. The high migration rate
may have contributed to the relatively low sinuosity of 1.19, and this suggests
that to obtain much higher sinuosity experiments at this scale may have to be
conducted for several years. Although patience is required to evolve them, these
experimental channels offer the opportunity to explore several fundamental issues
about river morphodynamics. Our results also suggest that sand supply may be an
essential control in restoring self-maintaining, actively shifting gravel-bedded
meanders.
DOI: 10.1073/pnas.0909417106
PMCID: PMC2761352
1391. Plant Mol Biol. 2010 Jan;72(1-2):75-89. doi: 10.1007/s11103-009-9552-4. Epub

2009
Sep 30.
Higher accumulation of F1-V fusion recombinant protein in plants after induction

of protein body formation.
Alvarez ML(1), Topal E, Martin F, Cardineau GA.
Author information:
(1)Center for Infectious Diseases and Vaccinology, The Biodesign Institute at
Arizona State University, 1001 South McAllister Avenue, Tempe, AZ 85287-5401,
USA. lucrecia.alvarez@asu.edu
Improving foreign protein accumulation is crucial for enhancing the commercial

success of plant-based production systems since product yields have a major
influence on process economics. Cereal grain evolved to store large amounts of
proteins in tightly organized aggregates. In maize, gamma-Zein is the major
storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in
specialized organelles called protein bodies (PB). Zera (gamma-Zein
ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that
is sufficient to induce the assembly of PB formation. Fusion of the Zera domain
to proteins of interest results in assembly of dense PB-like, ER-derived
organelles, containing high concentration of recombinant protein. Our main goal
was to increase recombinant protein accumulation in plants in order to enhance
the efficiency of orally-delivered plant-made vaccines. It is well known that
oral vaccination requires substantially higher doses than parental formulations.
As a part of a project to develop a plant-made plague vaccine, we expressed our
model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without
a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at
least 3x higher than F1-V alone when expressed in three different host plant
systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum
NT1 cells. We confirmed the feasibility of using Zera technology to induce
protein body formation in non-seed tissues. Zera expression and accumulation did
not affect plant development and growth. These results confirmed the potential
exploitation of Zera technology to substantially increase the accumulation of
value-added proteins in plants.
DOI: 10.1007/s11103-009-9552-4

Epub
2009 Sep 25.
Rumen microbiome composition determined using two nutritional models of subacute

ruminal acidosis.
Khafipour E(1), Li S, Plaizier JC, Krause DO.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, MB R3T 2N2,
Canada.
Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that

occurs during early and mid-lactation and has traditionally been characterized by
low rumen pH, but lactic acid does not accumulate as in acute lactic acid
acidosis. It is hypothesized that factors such as increased gut permeability,
bacterial lipopolysaccharides, and inflammatory responses may have a role in the
etiology of SARA. However, little is known about the nature of the rumen
microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen
samples taken from eight lactating Holstein dairy cattle using terminal
restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time
PCR. We used rumen samples from two published experiments in which SARA had been
induced with either grain or alfalfa pellets. The results of TRFLP analysis
indicated that the most predominant shift during SARA was a decline in
gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes
organisms was greater in alfalfa pellet-induced SARA than in mild or severe
grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was
also evident from the real-time PCR data for Prevotella albensis, Prevotella
brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The
real-time PCR data also indicated that severe grain-induced SARA was dominated by
Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was
dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated
by P. albensis. Using discriminant analysis, the severity of SARA and degree of
inflammation were highly correlated with the abundance of E. coli and not with
lipopolysaccharide in the rumen. We thus suspect that E. coli may be a
contributing factor in disease onset.
DOI: 10.1128/AEM.00739-09
PMCID: PMC2786511
1393. J Bacteriol. 2009 Dec;191(23):7216-24. doi: 10.1128/JB.01063-09. Epub 2009

Sep
25.
The low-molecular-weight fraction of exopolysaccharide II from Sinorhizobium

meliloti is a crucial determinant of biofilm formation.
Rinaudi LV(1), González JE.
Author information:
(1)Departamento de Biología Molecular, Universidad Nacional de Río Cuarto,
5800-Río Cuarto, Córdoba, Argentina.
Sinorhizobium meliloti is a soil bacterium that elicits the formation of root

organs called nodules on its host plant, Medicago sativa. Inside these
structures, the bacteria are able to convert atmospheric nitrogen into ammonia,
which is then used by the plant as a nitrogen source. The synthesis by S.
meliloti of at least one exopolysaccharide, succinoglycan or EPS II, is essential
for a successful symbiosis. While exopolysaccharide-deficient mutants induce the
formation of nodules, they fail to invade them, and as a result, no nitrogen
fixation occurs. Interestingly, the low-molecular-weight fractions of these
exopolysaccharides are the symbiotically active forms, and it has been suggested
that they act as signals to the host plant to initiate infection thread
formation. In this work, we explored the role of these rhizobial
exopolysaccharides in biofilm formation and their importance in the symbiotic
relationship with the host. We showed that the ExpR/Sin quorum-sensing system
controls biofilm formation in S. meliloti through the production of EPS II, which
provides the matrix for the development of structured and highly organized
biofilms. Moreover, the presence of the low-molecular-weight fraction of EPS II
is vital for biofilm formation, both in vitro and in vivo. This is the first
report where the symbiotically active fraction of EPS II is shown to be a
critical factor for biofilm formation and root colonization. Thus, the ability of
S. meliloti to properly attach to root surfaces and form biofilms conferred by
the synthesis of exopolysaccharides may embody the main function of these
symbiotically essential molecules.
DOI: 10.1128/JB.01063-09
PMCID: PMC2786570
1394. Chemosphere. 2009 Oct;77(6):709-13. doi: 10.1016/j.chemosphere.2009.08.050.

Epub
2009 Sep 22.
Water and phosphorus content affect PAH dissipation in spiked soil planted with
mycorrhizal alfalfa and tall fescue.
Zhou XB(1), Cébron A, Béguiristain T, Leyval C.
Author information:
(1)LIMOS, Nancy University, CNRS, BP 70239, 54506 Vandoeuvre-les-Nancy Cedex,
France.
Polycyclic aromatic hydrocarbon (PAH) dissipation efficiency can be increased in

the plant rhizosphere, but may be affected by various environmental factors. We
investigated the effects of the watering regime and phosphorus concentration on
PAH dissipation in the rhizosphere of mycorrhizal plants in a pot experiment. Two
plant species, alfalfa (Medicago sativa) and tall fescue (Festuca arundinacea),
were co-cultured and inoculated with an arbuscular mycorrhizal (AM) fungus
(Glomus intraradices) in PAH (phenanthrene (PHE)=500 mg kg(-1), pyrene (PYR)=500
mg kg(-1), dibenzo(a,h)anthracene (DBA)=65 mg kg(-1)) spiked agricultural soil
for 6 weeks. Treatments with different phosphorus concentrations and watering
regimes were compared. The PHE dissipation reached 90% in all treatments and was
not affected by the treatments. The major finding was the significant positive
impact of mycorrhizal plants on the dissipation of high molecular weight PAH
(DBA) in high-water low-phosphorus treatment. Such an effect was not observed in
high-water high-phosphorus and low-water low-phosphorus treatments, where AM
colonization was very low. A positive linear relationship was detected between
PYR dissipation and the percentage of Gram-positive PAH-ring hydroxylating
dioxygenase genes in high-water high-phosphorus treatments, but not in the other
two treatments with lower phosphorus concentrations and water contents. Such
results indicated that the phosphorus and water regime were important parameters
for the dissipation of HMW-PAH.
The development of lactic acid bacteria and Lactobacillus buchneri and their
effects on the fermentation of alfalfa silage.
Schmidt RJ(1), Hu W, Mills JA, Kung L Jr.
Author information:
(1)Department of Animal and Food Sciences, University of Delaware, Newark
19716-2150, USA.
This study was conducted to document the development of populations of lactic

acid bacteria (LAB) and Lactobacillus buchneri in alfalfa silage treated with
various inoculants. Wilted and chopped alfalfa (45% dry matter) was treated with
1) distilled water (untreated, U), 2) Lactobacillus buchneri 40788 (4 x 10(5)
cfu/g; LB), or 3) L. buchneri 40788 (4 x 10(5) cfu/g) and Pediococcus pentosaceus
(1 x 10(5) cfu/g; LBPP). Forages were packed into triplicate vacuum-sealed,
nylon-polyethylene bags per treatment, and ensiled for 2, 5, 45, 90, and 180 d.
Viable (cfu) LAB in forage and silage were quantified by traditional plating on
selective agar, and numbers of L. buchneri (cfu-equivalent, cfu-E) were
quantified by real-time quantitative PCR. Fresh, untreated forage had 5.52 log
cfu of LAB/g and 3.79 log cfu-E of L. buchneri/g. After 2 d of ensiling, numbers
of LAB increased to >8 log cfu/g in all silages. In contrast, numbers of L.
buchneri in U remained below 4 log cfu-E/g but reached approximately 7 log
cfu-E/g in LB and LBPP. From d 5 onward, numbers of L. buchneri in U remained
below 6 log cfu-E/g but approached 9 log cfu-E/g in LB and LBPP. The pH was lower
in LBPP compared with U and LB after 2 and 5 d of ensiling, but pH was lower for
U compared with LB and LBPP thereafter. Treatments LB and LBPP had more acetic
acid than U at 45 d of ensiling, which coincided with detectable amounts of 1,2
propanediol. Inoculation with LBPP resulted in silage with the highest
concentration of 1,2 propanediol after 180 d of ensiling. From d 45 onward, LB
and LBPP silages had lower concentrations of residual water-soluble carbohydrates
but had higher concentrations of ammonia-N than U. In conclusion, epiphytic L.
buchneri can be detected in alfalfa but this population is unable to lead the
silage fermentation. In contrast, when L. buchneri was added to silage as an
inoculant, the numbers of L. buchneri (cfu-E) increased markedly but did not
dictate fermentation until 45 d of ensiling. These findings help to explain why
the response (in increased acetic acid) from the addition of L. buchneri in
silages is not immediate.
DOI: 10.3168/jds.2008-1701
1396. Microb Ecol. 2010 Feb;59(2):379-89. doi: 10.1007/s00248-009-9583-z. Epub 2009

Sep
16.
Long-term phosphorus fertilization impacts soil fungal and bacterial diversity

but not AM fungal community in alfalfa.
Beauregard MS(1), Hamel C, Atul-Nayyar, St-Arnaud M.
Author information:
(1)Institut de recherche en biologie végétale, Université de Montréal and Jardin
botanique de Montréal, Montreal, QC, Canada H1X 2B2.
Soil function may be affected by cropping practices impacting the soil microbial
community. The effect of different phosphorus (P) fertilization rates (0, 20, or
40 kg P(2)O(5) ha(-1)) on soil microbial diversity was studied in 8-year-old
alfalfa monocultures. The hypothesis that P fertilization modifies soil microbial
community was tested using denaturing gradient gel electrophoresis and
phospholipids fatty acid (PLFA) profiling to describe soil bacteria, fungi, and
arbuscular mycorrhizal (AM) fungi diversity. Soil parameters related to fertility
(soil phosphate flux, soluble P, moisture, phosphatase and dehydrogenase assays,
and carbon and nitrogen content of the light fraction of soil organic matter)
were also monitored and related to soil microbial ribotype profiles. Change in
soil P fertility with the application of fertilizer had no effect on crop yield
in 8 years, but on the year of this study was associated with shifts in the
composition of fungal and bacterial communities without affecting their richness,
as evidenced by the absence of effect on the average number of ribotypes
detected. However, variation in soil P level created by a history of differential
fertilization did not significantly influence AM fungi ribotype assemblages nor
AM fungi biomass measured with the PLFA 16:1omega5. Fertilization increased P
flux and soil soluble P level but reduced soil moisture and soil microbial
activity, as revealed by dehydrogenase assay. Results suggest that soil P
fertility management could influence soil processes involving soil
microorganisms. Seasonal variations were also recorded in microbial activity,
soil soluble P level as well as in the abundance of specific bacterial and fungal
PLFA indicators of soil microbial biomass.
DOI: 10.1007/s00248-009-9583-z
1397. J Hazard Mater. 2009 Dec 30;172(2-3):1671-6. doi:

10.1016/j.jhazmat.2009.08.064.
Epub 2009 Aug 22.
Enhanced phytoremediation potential of polychlorinated biphenyl contaminated soil

from e-waste recycling area in the presence of randomly
methylated-beta-cyclodextrins.
Shen C(1), Tang X, Cheema SA, Zhang C, Khan MI, Liang F, Chen X, Zhu Y, Lin Q,
Chen Y.
Author information:
(1)Institute of Environmental Science and Technology, Zhejiang University,
Hangzhou 310029, PR China.
The crude recycling of electronic and electric waste (e-waste) is now creating
soil pollution problems with organic compounds such as polychlorinated biphenyls
(PCBs). The present study aimed to compare the phytoremediation potential of four
plant species (rice, alfalfa, ryegrass and tall fescue) for PCBs contaminated
soil from Taizhou city, one of the largest e-waste recycling centers in China. In
addition, the enhanced effects of randomly methylated-beta-cyclodextrins (RAMEB)
on PCBs phytoremediation potential were evaluated. Higher PCBs removal
percentages of 25.6-28.5% in rhizosphere soil were observed after 120 days,
compared with those of the non-rhizosphere (10.4-16.9%) and unplanted controls
(7.3%). The average PCBs removal percentages of four plant species increased from
26.9% to 37.1% in the rhizosphere soil with addition of RAMEB. Meanwhile,
relatively high microbial counts and dehydrogenase activity were detected in
planted soils and a stimulatory effect by RAMEB addition was found. The present
study indicated that all the plant candidates were feasible for phytoremediation
of PCBs contaminated soil from the e-waste recycling area, and tall fescue with
RAMEB amendment seemed as a promising remediation strategy. High PCBs removal
percentage was due to the increased PCBs bioavailability as well as
biostimulation of microbial communities after plantation and RAMEB addition.
DOI: 10.1016/j.jhazmat.2009.08.064
1398. J Sep Sci. 2009 Sep;32(18):3223-31. doi: 10.1002/jssc.200900184.
Characterization of green-tissue protein extract from alfalfa (Medicago sativa)

exploiting a 3-D technique.
Aguilar O(1), Glatz CE, Rito-Palomares M.
Author information:
(1)Departamento de Biotecnología e Ingeniería de Alimentos, Centro de
Biotecnología, Tecnológico de Monterrey, Campus Monterrey, Monterrey, NL, México.
There is a growing interest of pharmaceutical companies for plant-based

production systems. To facilitate the general acceptance of plants as
bioreactors, the establishment of efficient downstream operations is critical. It
has been proposed that a better understanding of the properties of the
contaminant proteins can benefit downstream processing design and operation. The
coupled application of 2-DE with aqueous two-phase partitioning has been
suggested as a practical 3-D method to characterize potential contaminant
proteins from plant extracts. The application of this novel 3-D approach to a
complex protein extract from alfalfa (Medicago sativa) containing a model
recombinant protein (human granulocyte colony stimulating factor (hG-CSF))
resulted in the quantification of 55 protein spots. The 3-D properties (M(r), pI,
and K(p)) obtained for 17 proteins comprising 69% of the alfalfa proteins,
allowed the proposal of a prefractionation step as well as the identification of
the target molecule (rG-CSF) from bulk of alfalfa proteins. The information
obtained from this experimental approach was useful for the identification of the
potential contaminant proteins that will occur in alfalfa when this plant is used
as a host for recombinant proteins. Additionally, this method will assist in the
design of adequate purification strategies for recombinant proteins expressed in
alfalfa green tissue.
DOI: 10.1002/jssc.200900184
1399. J Plant Physiol. 2010 Jan 15;167(2):114-20. doi: 10.1016/j.jplph.2009.07.016.
Biomass partitioning, morphology and water status of four alfalfa genotypes

submitted to progressive drought and subsequent recovery.
Erice G(1), Louahlia S, Irigoyen JJ, Sanchez-Diaz M, Avice JC.
Author information:
(1)INRA, UMR INRA-UCBN 950 Ecophysiologie Végétale, Agronomie & nutritions
N.C.S., Institut de Biologie Fondamentale et Appliquée, IFR 146 ICORE, Esplanade
de la Paix, F-14000 Caen, France.
The predicted worldwide increase of arid areas and water stress episodes will
strongly affect crop production. Numerous plants have developed specific
morphological and physiological mechanisms as a means to increase their tolerance
to drought. Water stress modifies dry matter partitioning and morphological
components such as leaf area ratio (LAR), specific leaf area (SLA) and leaf
weight ratio (LWR). Alfalfa has a wide-ranging distribution and is thus expected
to show differing levels of drought tolerance. The aim of our study was to
determine the effect of progressive drought and subsequent recovery in four
alfalfa genotypes differing in drought sensitivity: three cultivars adapted to a
Mediterranean climate, Tafilalet (TA), Tierra de Campos (TC) and Moapa (MO), and
another representative of an oceanic climate, Europe (EU). Mild drought did not
affect biomass production or water status in the studied varieties. Under
moderate drought conditions, TA and MO showed decreased leaf production, which
may help them to maintain relative water content (RWC). Despite observations that
water stress did not affect root growth, after the recovery period, TA increased
its root biomass, making higher water soil prospecting possible. Mediterranean
cultivars modified LAR and SLA depending on water availability, whereas EU alters
LWR. At the end of the experiment, TC was the most productive cultivar, but
severe drought did not predict differences among cultivars. Severe water stress
increased the root/shoot ratio in order to diminish water consumption and
increase absorption of water. In spite of all cultivars showing a decreased LWR,
TA also decreased SLA, which may suggest higher drought resistance. Morphological
traits from Mediterranean cultivars, including the ability to alter SLA or LAR
may be used for drought-tolerant cultivar improvement.
DOI: 10.1016/j.jplph.2009.07.016
Prewinter management affects Megachile rotundata (Hymenoptera: Megachilidae)

prepupal physiology and adult emergence and survival.
Pitts-Singer TL(1), James RR.
Author information:
(1)USDA-ARS Bee Biology & Systematics Laboratory, Logan, UT 84326, USA.
theresa.pitts-singer@ars.usda.gov
The alfalfa leafcutting bee, Megachile rotundata F. (Hymenoptera: Megachilidae),

is widely used as a pollinator for production of alfalfa, Medicago sativa L.,
seed, and populations of these bees can be maintained by alfalfa seed growers or
can be purchased from mostly Canadian bee providers. M. rotundata raised in
Canada have higher survival rates during the incubation that occurs after winter
storage than do bees produced in the northwestern United States, but no reason
has been found for this difference. We investigated whether storing immature M.
rotundata for various time periods at a warm temperature (16 degrees C) before
winter or allowing them to remain unmanaged at ambient temperatures affects
physiological aspects of prepupae during the winter as well as the survival and
longevity of adult bees after spring or summer incubation. Our results show that
the timing of the onset of winter storage and incubation does affect prepupal
weights, prepupal lipid and water contents, adult emergence, and adult female
longevity. Winter storage of prepupae in November or December with a late June
incubation resulted in heavier adults that emerged more readily than bees
incubated in late May. However, adult females incubated in May thrived longer
than June-incubated bees if fed a honey-water diet. Thus, some prewinter
management regimes for M. rotundata commercial stocks may be more effective than
others for achieving optimal adult emergence synchrony, as well as adult survival
and longevity for pollination of a summer crop.
DOI: 10.1603/029.102.0402
1401. J Bacteriol. 2009 Nov;191(22):6988-7000. doi: 10.1128/JB.00466-09. Epub 2009

Sep
4.
The rkp-1 cluster is required for secretion of Kdo homopolymeric capsular

polysaccharide in Sinorhizobium meliloti strain Rm1021.
Müller MG(1), Forsberg LS, Keating DH.
Author information:
(1)Department of Microbiology and Immunology, Loyola University Chicago, Maywood,
Illinois 60153, USA.
Under conditions of nitrogen stress, leguminous plants form symbioses with soil
bacteria called rhizobia. This partnership results in the development of
structures called root nodules, in which differentiated endosymbiotic bacteria
reduce molecular dinitrogen for the host. The establishment of rhizobium-legume
symbioses requires the bacterial synthesis of oligosaccharides,
exopolysaccharides, and capsular polysaccharides. Previous studies suggested that
the 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) homopolymeric capsular
polysaccharide produced by strain Sinorhizobium meliloti Rm1021 contributes to
symbiosis with Medicago sativa under some conditions. However, a conclusive
symbiotic role for this polysaccharide could not be determined due to a lack of
mutants affecting its synthesis. In this study, we have further characterized the
synthesis, secretion, and symbiotic function of the Kdo homopolymeric capsule. We
showed that mutants lacking the enigmatic rkp-1 gene cluster fail to display the
Kdo capsule on the cell surface but accumulate an intracellular polysaccharide of
unusually high M(r). In addition, we have demonstrated that mutations in kdsB2,
smb20804, and smb20805 affect the polymerization of the Kdo homopolymeric
capsule. Our studies also suggest a role for the capsular polysaccharide in
symbiosis. Previous reports have shown that the overexpression of rkpZ from
strain Rm41 allows for the symbiosis of exoY mutants of Rm1021 that are unable to
produce the exopolysaccharide succinoglycan. Our results demonstrate that
mutations in the rkp-1 cluster prevent this phenotypic suppression of exoY
mutants, although mutations in kdsB2, smb20804, and smb20805 have no effect.
DOI: 10.1128/JB.00466-09
PMCID: PMC2772494
1402. Int J Food Microbiol. 2009 Nov 30;136(1):66-70. doi:

10.1016/j.ijfoodmicro.2009.08.014. Epub 2009 Aug 19.
Potential use of supercritical carbon dioxide to decontaminate Escherichia coli

O157:H7, Listeria monocytogenes, and Salmonella typhimurium in alfalfa sprouted
seeds.
Jung WY(1), Choi YM, Rhee MS.
Author information:
(1)Division of Food Bioscience and Technology, College of Life Sciences and
Biotechnology, Korea University, Seoul, South Korea.
We sought to develop a method of decontaminating alfalfa sprouts of Escherichia

coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium without altering
the seed germination capability using supercritical carbon dioxide (SC-CO(2)).
Samples were treated with SC-CO(2) at 10, 15, or 20 MPa and temperatures of 35,
40, or 45 degrees C for 5, 10, or 15 min. The germination percentage was measured
after three days of germination. Generally, treating seeds with SC-CO(2) at
higher pressures, temperatures, or for longer treatment times resulted in greater
microbial reductions than treatments at lower pressures, temperatures, or for
shorter treatment times. SC-CO(2) treatment clearly reduced the microorganism
levels in alfalfa seeds; in particular, treatment at 20 MPa and 45 degrees C for
15 min reduced levels of the three pathogens by >7.0 log colony forming units
(CFU)/g. However, SC-CO(2) treatment at a high pressure and high temperature,
especially treatment at 20 MPa and 40 or 45 degrees C, impaired the seed
germination capability in some cases. Without impairing the germination
capability, the maximum reduction level of E. coli O157:H7 was 3.51 CFU/g with
SC-CO(2) treatment at 15 MPa and 35 degrees C for 10 min. Maximum reductions of
L. monocytogenes and S. typhimurium were 2.65 and 2.48 log CFU/g, respectively,
with treatment at 10 MPa and 45 degrees C for 5 min. Therefore, our results
indicate that SC-CO(2) treatment can be used to effectively improve alfalfa seed
safety.
1403. Chemosphere. 2009 Nov;77(7):946-54. doi: 10.1016/j.chemosphere.2009.08.007.

Epub
2009 Sep 3.
Differential alterations of antioxidant defenses as bioindicators of mercury and

cadmium toxicity in alfalfa.
Sobrino-Plata J(1), Ortega-Villasante C, Flores-Cáceres ML, Escobar C, Del Campo

FF, Hernández LE.
Author information:
Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain.
Several physiological parameters related to oxidative stress, which is a

characteristic of plants exposed to toxic metals, were studied in 3-week-old
alfalfa plants treated with cadmium (Cd) or mercury (Hg) at doses of 0, 3, 10 and
30 microM for 7d. The concentrations of biothiols, glutathione (GSH),
homoglutathione (hGSH) and phytochelatins (PCs) increased dramatically in
metals-treated plants, in particular in the presence of Cd. This was accompanied
by a remarkable up-regulation of gamma-glutamyl cysteine synthetase gene,
probably in response to the higher demand for GSH|hGSH needed for PC synthesis.
The presence of metals enhanced lipid peroxidation in shoots, while chlorophyll
content declined in a concentration dependent manner. Ascorbate peroxidase (APX)
activity increased moderately in roots of Cd-exposed plants, and a new basic root
peroxidase isoform was found in both Cd- and Hg-treated plants. Glutathione
reductase (GR) activity was enhanced in shoots of plants exposed to Cd and Hg.
However, this enzymatic activity showed a metal dependent response in roots, and
was enhanced in Cd-treated plants but was severely inhibited in roots of plants
treated with Hg. Inhibition of GR by Hg was confirmed in vitro by incubating a
commercially available GR and control shoot extracts with several doses of Hg and
Cd. Ascorbate concentrations were elevated with treatments of 3 microM Hg, 10
microM Cd and 30 microM Cd, indicating that this compound is necessary for redox
cellular homeostasis. The different responses observed with Cd and Hg treatments
might be the basis for specific stress bioindicators.
1404. Pest Manag Sci. 2009 Dec;65(12):1298-304. doi: 10.1002/ps.1840.
The integrated control concept and its relevance to current integrated pest
management in California fresh market grapes.
Bentley WJ(1).
Author information:
(1)University of California, Kearney Agricultural Center, Parlier, CA
559-646-6527, USA. walt@uckac.edu
The foundation of an integrated pest management program involves valid treatment

thresholds, accurate and simple monitoring methods, effective natural controls,
selective pesticides and trained individuals who can implement the concept. The
Integrated Control Concept written by Stern, Smith, van den Bosch and Hagen
elucidated each of these points in an alfalfa ecosystem. Alfalfa hay (Medicago
sativa L.) has a low per acre value, requires little hand labor and is primarily
marketed in the USA. In contrast, fresh market table grape (Vitis vinifera L.)
has a high per acre value, requires frequent hand labor operations, suffers
unacceptable cosmetic damage and is marketed throughout both the USA and the
world. Each of the components of a working IPM program is present in table grape
production. Marketing grapes to foreign countries presents special problems with
pests considered invasive and where residue tolerances for some selective
insecticides are lacking. However, fresh market grape farmers are still able to
deal with these special problems and utilize an IPM program that has resulted in
a 42% reduction in broad-spectrum insecticide use from 1995 to 2007.
(c) 2009 Society of Chemical Industry.
DOI: 10.1002/ps.1840
1405. Bioresour Technol. 2010 Jan;101(1):215-21. doi:

10.1016/j.biortech.2009.07.090.
Epub 2009 Aug 31.
Fiber length and pulping characteristics of switchgrass, alfalfa stems, hybrid

poplar and willow biomasses.
Ai J(1), Tschirner U.
Author information:
(1)Department of Bioproducts and Biosystems Engineering, University of Minnesota,
2004 Folwell Avenue, St. Paul, MN 55108, USA.
Switchgrass (Panicum virgatum), alfalfa stems (Medicago sativa), second year

growth hybrid poplar (Populus) and willow (Salix spp.) were examined to determine
fiber characteristics, pulping behavior and paper properties. Alfalfa stems and
switchgrass both showed length weighted average fiber length (LWW) of 0.78 mm, a
very narrow fiber length distribution and high fines content. Willow and hybrid
poplar have lower fines content but a very low average fiber length (0.42 and
0.48 mm LWW). In addition, the four biomass species showed distinctly different
chemical compositions. Switchgrass was defibered successfully using Soda and Soda
Anthraquinone (AQ) pulping and demonstrated good paper properties. Both
fast-growing wood species pulped well using the Kraft process, and showed
acceptable tensile strength, but low tear strength. Alfalfa stems reacted very
poorly to Soda and Soda AQ pulping but responded well to Kraft and Kraft AQ.
Pulps with tensile and tear strength considerably higher than those found for
commercial aspen pulps were observed for alfalfa. All four biomass species
examined demonstrated low pulp yield. The highest yields were obtained with
poplar and switchgrass (around 43%). Considering the short fibers and low yields,
all four biomass types will likely only be used in paper manufacturing if they
offer considerable economic advantage over traditional pulp wood.
DOI: 10.1016/j.biortech.2009.07.090
1406. Microbiology. 2009 Nov;155(Pt 11):3701-9. doi: 10.1099/mic.0.032029-0. Epub

2009
Aug 27.
Previously uncharacterized Salmonella enterica genes required for swarming play a

role in seedling colonization.
Barak JD(1), Gorski L, Liang AS, Narm KE.
Author information:
(1)Department of Plant Pathology, Rm 790 Russell Laboratories, 1630 Linden Drive,
University of Wisconsin-Madison, Madison, WI 53706, USA. barak@plantpath.wisc.edu
Incidences of bacterial foodborne illness caused by ingestion of fresh produce

are rising. Instead of this being due to incidental contamination, the animal
pathogen Salmonella enterica utilizes specific molecular mechanisms to attach to
and colonize plants. This work characterizes two S. enterica genes of unknown
function: a putative periplasmic protein, STM0278, and a putative protein with a
hydrolase in the C-terminus, STM0650. STM0278 and STM0650 are important for
seedling colonization but appear to have different roles during the process of
colonization. Mutants of either STM0278 or STM0650 showed reduced colonization of
alfalfa seedlings at 24 h, and the STM0278 mutant also showed reduced
colonization at 48 h. Both genes were expressed in planta at 4 h following
inoculation of 3-day-old seedlings and at 72 h after seed inoculation. This
suggests that the role of STM0650 in seedling colonization is less important
later in the process or is duplicated by other mechanisms. Mutants of STM0278 and
STM0650 were defective in swarming. The STM0278 mutant failed to swarm in 24 h,
while swarming of the STM0650 mutant was delayed. Addition of surfactant restored
swarming of the STM0278 mutant, suggesting that STM0278 is involved in surfactant
or osmotic agent production or deployment. Alfalfa seed exudates as the sole
nutrient source were capable of perpetuating S. enterica swarming. Sequence
analysis revealed sequences homologous to STM0278 and STM0650 in plant-associated
bacteria, but none in Escherichia coli. Phylogenetic analysis of STM0650 showed
similar sequences from diverse classes of plant-associated bacteria. Bacteria
that preferentially colonize roots, including S. enterica, may use a similar
hydrolase for swarming or biofilm production on plants. Multicellular behaviours
by S. enterica appear central to plant colonization. S. enterica genes involved
in plant colonization and survival outside of a host are most likely among the
'function unknown' genes of this bacterium.
DOI: 10.1099/mic.0.032029-0
1407. Plant Physiol. 2009 Nov;151(3):1114-29. doi: 10.1104/pp.109.144022. Epub 2009

Aug
26.
A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific

anthocyanin and proanthocyanidin biosynthesis but not for trichome development.
Pang Y(1), Wenger JP, Saathoff K, Peel GJ, Wen J, Huhman D, Allen SN, Tang Y,
Cheng X, Tadege M, Ratet P, Mysore KS, Sumner LW, Marks MD, Dixon RA.
Author information:
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401, USA.
WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins

(PAs), and mucilage in the seed and the development of trichomes and root hairs.
We have cloned and characterized a WD40 repeat protein gene from Medicago
truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of
MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic
compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in
the seed, reduces epicatechin levels without corresponding effects on other
flavonoids in flowers, reduces isoflavone levels in roots, but does not impair
trichome or root hair development. MtWD40-1 is expressed constitutively, with
highest expression in the seed coat, where its transcript profile temporally
parallels those of PA biosynthetic genes. Transcript profile analysis revealed
that many genes of flavonoid biosynthesis were down-regulated in a
tissue-specific manner in M. truncatula lines harboring retrotransposon
insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and
trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa
glabrous1 mutant. We discuss the function of MtWD40-1 in natural product
formation in M. truncatula and the potential use of the gene for engineering PAs
in the forage legume alfalfa (Medicago sativa).
DOI: 10.1104/pp.109.144022
PMCID: PMC2773055
1408. Public Health Nutr. 2010 Mar;13(3):418-23. doi: 10.1017/S1368980009991224.

Epub
2009 Aug 26.
Leaf concentrate as an alternative to iron and folic acid supplements for anaemic
adolescent girls: a randomised controlled trial in India.
Vyas S(1), Collin SM, Bertin E, Davys GJ, Mathur B.
Author information:
(1)Department of Foods and Nutrition, International College for Girls, Jaipur,
India.
OBJECTIVE: Despite public health campaigns based on Fe and folic acid

supplements, Fe-deficiency anaemia remains highly prevalent among women in India.
We investigated leaf concentrate as an alternative to Fe and folic acid
supplements for treating anaemia in adolescent girls.
DESIGN: Randomised controlled two-arm trial over 3 months: one group received
daily Fe and folic acid (IFA; 60 mg Fe, 500 microg folic acid); the other daily
leaf concentrate (LC; 5 mg Fe, 13 microg folic acid). Hb concentration, mean cell
volume, serum Fe, serum ferritin and total Fe-binding capacity were measured pre-
and post-intervention.
SETTING: Jaipur, India.
SUBJECTS: One hundred and two adolescent girls aged 14-18 years.
RESULTS: Of the 102 girls randomized to the two arms of the trial, four (3.9 %)
were severely anaemic (Hb < 7 g/dl), twenty-eight (27.5 %) were moderately
anaemic (Hb > or = 7 g/dl, <10 g/dl) and seventy (68.6 %) were mildly anaemic (Hb
> or = 10 g/dl, <12 g/dl). In the IFA group, eleven girls (20.4 %) withdrew due
to side-effects, compared with one girl (2.1 %) in the LC group (P = 0.005).
Total losses to follow-up were 14/54 in the IFA group and 2/48 in the LC group.
At the end of the trial, none of the eighty-six remaining girls were severely
anaemic, nine (10.5 %) were moderately anaemic and twenty-six (30.2 %) were
mildly anaemic; fifty-one (59.3 %) had normal Hb levels (> or = 12 g/dl). After
adjustment for baseline values, LC was as effective as IFA in improving serum Fe
parameters and treating anaemia.
CONCLUSIONS: Leaf concentrate is an effective, and more palatable, alternative to
Fe and folic acid supplements for treating anaemia in adolescent girls.
DOI: 10.1017/S1368980009991224
1409. Plant Signal Behav. 2008 Oct;3(10):791-4.
Multiple roles for cytokinin receptors and cross-talk of signaling pathways.
Coba de la Peña T(1), Cárcamo CB, Lucas MM, Pueyo JJ.
Author information:
(1)Department of Plant Physiology and Ecology; Instituto de Recursos Naturales;
Centro de Ciencias Medioambientales; CSIC; Madrid Spain.
Cytokinin receptors (CRs) are hybrid-type histidine kinases, membrane proteins

with a cytokinin-binding extracellular domain. CRs initiate and propagate
cytokinin signaling by means of phosphorylation and phosphotransfer to downstream
proteins. In legumes, some members of the CR multigenic family are essential for
nodulation. In two recent works, we investigated the involvement of two new CRs,
MsHK1 from Medicago sativa, and LaHK1 from Lupinus albus, in nodule
morphogenesis, senescence and stress response. LaHK1 expression increased during
the first stages of lupin nodule development, while MsHK1 expression was
localized in the meristem and the invasion zone of alfalfa nodules pointing to a
role for CRs in nodule cell proliferation and differentiation. Both CRs were also
induced during nodule senescence. MsHK1 expression increased under osmotic stress
and both genes were induced following dark stress, indicating that CRs are also
likely to play a significant role in the response to stress. We propose multiple
roles for CRs which, when analyzed jointly with recent results from other
authors, suggest coordinated cross-talk of different signaling systems.
PMCID: PMC2634374
PMID: 19704559
1410. Plant Signal Behav. 2008 Oct;3(10):823-6.
The involvement of reactive oxygen species (ROS) in the cell cycle activation
(G(0)-to-G(1) transition) of plant cells.
Fehér A(1), Otvös K, Pasternak TP, Szandtner AP.
Author information:
(1)Institute of Plant Biology; Biological Research Center; Hungarian Academy of
Sciences; Szeged Hungary.
Reactive oxygen species (ROS) are involved in various cellular processes in

plants. Among those, resistance to abiotic stress, defence mechanisms and cell
expansion have been intensively studied during the last years. We recently
demonstrated that ROS, in concert with auxin, have a role in cell cycle
activation of differentiated leaf cells.1 In this addendum we provide further
evidence to show that oxidative stress/ROS accelerate auxin-mediated cell cycle
entry (G(0)-to-G(1)) and may have a positive effect on the plant cell cycle
machinery. A generalized model for concentration-dependent synergistic effect of
auxin and ROS on differentiated plant cells is also shown.
PMCID: PMC2634385
PMID: 19704510
1411. FEMS Microbiol Ecol. 2009 Nov;70(2):62-70. doi: 10.1111/j.1574-

6941.2009.00752.x.
Epub 2009 Jul 29.
Influence of intercropping and intercropping plus rhizobial inoculation on

microbial activity and community composition in rhizosphere of alfalfa (Medicago
sativa L.) and Siberian wild rye (Elymus sibiricus L.).
Sun YM(1), Zhang NN, Wang ET, Yuan HL, Yang JS, Chen WX.
Author information:
(1)State Key Laboratory for Agrobiotechnology, College of Biological Sciences and
Center for Biomass Engineering, China Agricultural University, Beijing, China.
Alfalfa-Siberian wild rye intercropping is the predominant cropping system used

to produce forage in China. In this study, the effects of intercropping and
intercropping-rhizobial inoculation on soil enzyme activities, microbial biomass
and bacterial community composition in the rhizosphere were examined. In both
treatments, the yield of alfalfa, microbial biomass and activities of soil
urease, invertase and alkaline phosphatase in the alfalfa rhizosphere were
markedly increased, whereas there was a slight increase in the yield of Siberian
wild rye, few impacts on soil microbial biomass, and decreased enzyme activities
(except for urease) in the Siberian wild rye rhizosphere. Terminal restriction
fragment length polymorphism (T-RFLP) of 16S rRNA genes indicated that
Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,
Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the major
bacterial groups in the rhizosphere of both plants. However, intercropping and
rhizobial inoculation induced some shifts in the relative abundance of them.
Nitrosomonas and Nitrosospira groups were detected in all treatments by the
T-RFLP patterns of ammonia monooxygenase subunit A (amoA) gene, but the relative
abundance of Nitrosomonas increased and that of Nitrosospira decreased in the
intercropping-rhizobial inoculation treatment. Both treatments tended to increase
the diversity of amoA. Conclusively, the two treatments clearly affected soil
microbial composition and soil enzyme activities, which might be reflected in
changes in yield.
DOI: 10.1111/j.1574-6941.2009.00752.x
1412. Environ Entomol. 2009 Aug;38(4):1028-37.
Anthropogenic islands in the arid West: comparing the richness and diversity of
insect communities in cultivated fields and neighboring wildlands.
Forister ML(1).
Author information:
mforister@unr.edu
The interface between cultivated fields and wildlands has become a central focus
for conservation biology, particularly as previously uncultivated lands worldwide
are converted to agriculture at an escalating pace. Although research in some
parts of the world has highlighted the potential value of agricultural lands for
managing and preserving native animals, we know comparatively little about native
animals spanning the cultivated/wildlands interface in North America. The study
reported here investigated insect communities at three sets of paired sites
(cultivated alfalfa fields and native, sagebrush areas) on the western edge of
the Great Basin. Two hundred ninety-nine morphospecies were sorted from a
collection of >9,000 insects: 221 morphospecies were found in cultivated fields,
and 143 were found in the native areas. Insect communities in alfalfa fields were
higher in species richness and abundance than communities in adjacent, native
fields. However, communities in the cultivated habitat were relatively more
homogenous: species composition was more similar among cultivated fields than
among native fields. Considering the number of individual insects and
morphospecies found in the cultivated habitat, and the relatively small number of
species that overlap the two habitat types, the potential ecological consequences
of the widespread, anthropogenic habitat are discussed.
1413. Ecol Appl. 2009 Jul;19(5):1102-13.
Nitrogen fertilizer effects on soil carbon balances in midwestern U.S.

agricultural systems.
Russell AE(1), Cambardella CA, Laird DA, Jaynes DB, Meek DW.
Author information:
(1)Department of Natural Resource Ecology and Management, Iowa State University,
Ames, Iowa 50011, USA. arussell@iastate.edu
A single ecosystem dominates the Midwestern United States, occupying 26 million

hectares in five states alone: the corn-soybean agroecosystem [Zea mays
L.-Glycine max (L.) Merr.]. Nitrogen (N) fertilization could influence the soil
carbon (C) balance in this system because the corn phase is fertilized in 97-100%
of farms, at an average rate of 135 kg N x ha(-1) x yr(-1). We evaluated the
impacts on two major processes that determine the soil C balance, the rates of
organic-carbon (OC) inputs and decay, at four levels of N fertilization, 0, 90,
180, and 270 kg/ha, in two long-term experimental sites in Mollisols in Iowa,
USA. We compared the corn-soybean system with other experimental cropping systems
fertilized with N in the corn phases only: continuous corn for grain;
corn-corn-oats (Avena sativa L.)-alfalfa (Medicago sativa L.;
corn-oats-alfalfa-alfalfa; and continuous soybean. In all systems, we estimated
long-term OC inputs and decay rates over all phases of the rotations, based on
long-term yield data, harvest indices (HI), and root:shoot data. For corn, we
measured these two ratios in the four N treatments in a single year in each site;
for other crops we used published ratios. Total OC inputs were calculated as
aboveground plus belowground net primary production (NPP) minus harvested yield.
For corn, measured total OC inputs increased with N fertilization (P < 0.05, both
sites). Belowground NPP, comprising only 6-22% of total corn NPP, was not
significantly influenced by N fertilization. When all phases of the crop
rotations were evaluated over the long term, OC decay rates increased
concomitantly with OC input rates in several systems. Increases in decay rates
with N fertilization apparently offset gains in carbon inputs to the soil in such
a way that soil C sequestration was virtually nil in 78% of the systems studied,
despite up to 48 years of N additions. The quantity of belowground OC inputs was
the best predictor of long-term soil C storage. This indicates that, in these
systems, in comparison with increased N-fertilizer additions, selection of crops
with high belowground NPP is a more effective management practice for increasing
soil C sequestration.
1414. BMC Plant Biol. 2009 Aug 13;9:107. doi: 10.1186/1471-2229-9-107.
Comparative gene expression profiles between heterotic and non-heterotic hybrids

of tetraploid Medicago sativa.
Li X(1), Wei Y, Nettleton D, Brummer EC.
Author information:
(1)Center for Applied Genetic Technologies, University of Georgia, Athens,
Georgia 30602, USA. xligalee@uga.edu
BACKGROUND: Heterosis, the superior performance of hybrids relative to parents,

has clear agricultural value, but its genetic control is unknown. Our objective
was to test the hypotheses that hybrids expressing heterosis for biomass yield
would show more gene expression levels that were different from midparental
values and outside the range of parental values than hybrids that do not exhibit
heterosis.
RESULTS: We tested these hypotheses in three Medicago sativa (alfalfa) genotypes
and their three hybrids, two of which expressed heterosis for biomass yield and a
third that did not, using Affymetrix M. truncatula GeneChip arrays. Alfalfa
hybridized to approximately 47% of the M. truncatula probe sets. Probe set signal
intensities were analyzed using MicroArray Suite v.5.0 (MAS) and robust
multi-array average (RMA) algorithms. Based on MAS analysis, the two heterotic
hybrids performed similarly, with about 27% of genes showing differential
expression among the parents and their hybrid compared to 12.5% for the
non-heterotic hybrid. At a false discovery rate of 0.15, 4.7% of differentially
expressed genes in hybrids (approximately 300 genes) showed nonadditive
expression compared to only 0.5% (16 genes) in the non-heterotic hybrid. Of the
nonadditively expressed genes, approximately 50% showed expression levels that
fell outside the parental range in heterotic hybrids, but only one of 16 showed a
similar profile in the non-heterotic hybrid. Genes whose expression differed in
the parents were three times more likely to show nonadditive expression than
genes whose parental transcript levels were equal.
CONCLUSION: The higher proportions of probe sets with expression level that
differed from the parental midparent value and that were more extreme than either
parental value in the heterotic hybrids compared to a non-heterotic hybrid were
also found using RMA. We conclude that nonadditive expression of transcript
levels may contribute to heterosis for biomass yield in alfalfa.
DOI: 10.1186/1471-2229-9-107
PMCID: PMC2736959
1415. Syst Appl Microbiol. 2009 Dec;32(8):583-92. doi: 10.1016/j.syapm.2009.07.007.

Epub 2009 Aug 8.
Symbiotic diversity of Ensifer meliloti strains recovered from various legume

species in Tunisia.
Mnasri B(1), Badri Y, Saïdi S, de Lajudie P, Mhamdi R.
Author information:
(1)Laboratoire des Interactions Légumineuses-Microorganismes, Centre de
Biotechnologie de Borj-Cédria, BP 901, 2050 Hammam-Lif, Tunisia.
Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a

specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of
E. meliloti were recovered from root nodules of various legume species and their
symbiotic status still remains unclear. Here, we further investigate the
specificity of these strains. A collection of 47 E. meliloti strains isolated in
Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago
ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus
vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus
roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis,
Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR
fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K
intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation
range was also assessed by cross-inoculation experiments. No clear correlation
was found between chromosomal backgrounds and host plants of origin. The
nodulation polyvalence of the species E. meliloti was associated with a high
symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and
nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes
harboured distinct genes and possessed wider host ranges. Some strains did not
nodulate Medicago species. On the basis of nodA phylogeny, the majority of the
Tunisian strains, including strains from Medicago, harboured distinct nodA
alleles more related to those found in E. medicae than those found in E.
meliloti. However, more work is still needed to characterize this group further.
The diversity observed among M. laciniata isolates, which was supported by nodA
phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that
what was thought to be bv. medicaginis is certainly heterogeneous.
DOI: 10.1016/j.syapm.2009.07.007
1416. J Anim Physiol Anim Nutr (Berl). 2010 Aug 1;94(4):495-504. doi:
10.1111/j.1439-0396.2009.00934.x. Epub 2009 Aug 3.
Changes in the viscoelastic behaviour of the rumenal digesta mat of sheep fed on
pasture or chaffed lucerne hay.
Lentle RG(1), Stafford KJ, Bekkour K, Aserevujanon P, Sylvester S, Hemar Y.
Author information:
(1)Institute of Food Nutrition and Human Health, Massey University, Palmerston
North, New Zealand. R.G.Lentle@massey.ac.nz
We examined the changes in the physical properties of the digesta mat over a
period of 24 h after cessation of feeding, in sheep that had been maintained on
pasture or fed chaffed lucerne hay. The dry matter content of the digesta mat
declined at similar rates in both dietary groups, although it was generally
higher in sheep fed lucerne. Median particle size declined in the digesta mats of
both dietary groups at similar rates in samples taken after 8 h, but median
particle size was significantly greater in sheep fed chopped lucerne hay than in
those fed grass. Thus, particles were not reduced to a common size suggesting
that factors in addition to particle size governed the rate of breakdown of the
rumen mat. The relationship between the elastic and loss moduli was of a
consistent pattern in all samples taken from the rumen mat indicating that it
behaved as a weak gel. The elastic and loss moduli of the digesta mats of sheep
that had been fed pasture or chopped lucerne hay converged to similar values
after 12 h and declined broadly at similar rates after this. The relationship
between these two moduli and the dry matter content of the rumen mat were of
similar curvilinear form for sheep on both diets. These findings suggest that the
rate of breakdown of the rumen mat is more likely to be governed by its composite
behaviour than by the size of the constituent particles.
DOI: 10.1111/j.1439-0396.2009.00934.x
1417. Int J Syst Evol Microbiol. 2010 Mar;60(Pt 3):664-74. doi:

10.1099/ijs.0.012088-0.
Epub 2009 Aug 5.
Multilocus sequence analysis of root nodule isolates from Lotus arabicus

(Senegal), Lotus creticus, Argyrolobium uniflorum and Medicago sativa (Tunisia)
and description of Ensifer numidicus sp. nov. and Ensifer garamanticus sp. nov.
Merabet C(1), Martens M, Mahdhi M, Zakhia F, Sy A, Le Roux C, Domergue O, Coopman

R, Bekki A, Mars M, Willems A, de Lajudie P.
Author information:
(1)Laboratoire de Rhizobiologie, Université d'Oran, Es-Senia, Algeria.
Nine isolates from Argyrolobium uniflorum, Lotus creticus , Medicago sativa

(Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence
analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the
16S rRNA gene and the nodulation gene nodA. Analysis of the individual and
concatenated gene sequences demonstrated that the nine new strains constituted
three stable, well-supported (bootstrap and gene sequence similarity values)
monophyletic clusters, A, B and C, all belonging to the branch of the genus
Ensifer, regardless of the phylogenetic reconstruction method used (maximum
likelihood, maximum-parsimony, neighbour-joining). The three groups were further
characterized by API 100 auxanographic tests, host specificity and nodA gene
sequence analysis. On the basis of these data, clusters A and C are suggested as
representing two novel species within the genus Ensifer, for which the names
Ensifer numidicus sp. nov. (type strain ORS 1407(T)=LMG 24690(T)=CIP 109850(T))
and Ensifer garamanticus sp. nov. (type strain ORS 1400(T)=LMG 24692(T)=CIP
109916(T)) are proposed. The cluster B strains were assigned to Ensifer adhaerens
genomovar A.
DOI: 10.1099/ijs.0.012088-0
1418. Mol Plant Microbe Interact. 2009 Sep;22(9):1116-27. doi: 10.1094/MPMI-22-9-

1116.
Identification of a hydroxyproline transport system in the legume endosymbiont

Maclean AM(1), White CE, Fowler JE, Finan TM.
Author information:
Hydroxyproline-rich proteins in plants offer a source of carbon and nitrogen to

soil-dwelling microorganisms in the form of root exudates and decaying organic
matter. This report describes an ABC-type transport system dedicated to the
uptake of hydroxyproline in the legume endosymbiont Sinorhizobium meliloti. We
have designated genes involved in hydroxyproline metabolism as hyp genes and show
that an S. meliloti strain lacking putative transport genes (DeltahypMNPQ) is
unable to grow with or transport trans-4-hydroxy-l-proline when this compound is
available as a sole source of carbon. Expression of hypM is upregulated in the
presence of trans-4-hydroxy-l-proline and cis-4-hydroxy-d-proline, as modulated
by a repressor (HypR) of the GntR/FadR subfamily. Although alfalfa root nodules
contain hydroxyproline-rich proteins, we demonstrate that the transport system is
not highly expressed in nodules, suggesting that bacteroids are not exposed to
high levels of free hydroxyproline in planta. In addition to hypMNPQ, we report
that S. meliloti encodes a second independent mechanism that enables transport of
trans-4-hydroxy-l-proline. This secondary transport mechanism is induced in
proline-grown cells and likely entails a system involved in l-proline uptake.
This study represents the first genetic description of a prokaryotic
hydroxyproline transport system, and the ability to metabolize hydroxyproline may
contribute significantly toward the ecological success of plant-associated
bacteria such as the rhizobia.
DOI: 10.1094/MPMI-22-9-1116
1419. Int J Syst Evol Microbiol. 2009 Dec;59(Pt 12):3051-7. doi:

10.1099/ijs.0.009647-0. Epub 2009 Jul 30.
Rhizobium tibeticum sp. nov., a symbiotic bacterium isolated from Trigonella

archiducis-nicolai (Sirj.) Vassilcz.
Hou BC(1), Wang ET, Li Y Jr, Jia RZ, Chen WF, Gao Y, Dong RJ, Chen WX.
Author information:
(1)State Key Laboratories for Agro biotechnology/College of Biological Sciences,
China Agricultural University, 100193 Beijing, PR China.
Isolated from root nodules of Trigonella archiducis-nicolai (Sirj.) Vassilcz.

grown in Tibet, China, cells of the bacterial strains CCBAU 85039(T) and CCBAU
85027 were Gram-negative, aerobic, motile, non-spore-forming rods that formed
colonies that were semi-translucent and opalescent on yeast extract-mannitol
agar. In numerical taxonomy, SDS-PAGE analysis of whole-cell proteins and DNA-DNA
hybridization, the two strains were very similar and were different from
reference strains of defined Rhizobium species. In the phylogeny based on 16S
rRNA gene sequences, they were most similar to Rhizobium etli CFN 42(T) (98.2 %
similarity) and R. leguminosarum USDA 2370(T) (97.6 %). Sequence analyses of the
housekeeping genes recA, atpD and glnII and the 16S-23S rRNA intergenic spacer,
phenotypic characteristics and cellular fatty acid profiles strongly suggested
that these two strains represented a novel species within Rhizobium.
Cross-nodulation tests and sequencing of nifH and nodA genes showed that these
two strains were symbiotic bacteria that nodulated Trigonella archiducis-nicolai,
Medicago lupulina, Medicago sativa, Melilotus officinalis, Phaseolus vulgaris and
Trigonella foenum-graecum. Based on the results, the novel species Rhizobium
tibeticum sp. nov. is described to accommodate the two strains. The type strain
is CCBAU 85039(T) (=LMG 24453(T) =CGMCC 1.7071(T)). The DNA G+C content of this
strain is 59.7 mol% (T(m)).
DOI: 10.1099/ijs.0.009647-0
1420. J Biol Chem. 2009 Sep 25;284(39):26482-92. doi: 10.1074/jbc.M109.009340. Epub

2009 Jul 27.
Unraveling delta1-pyrroline-5-carboxylate-proline cycle in plants by uncoupled

expression of proline oxidation enzymes.
Miller G(1), Honig A, Stein H, Suzuki N, Mittler R, Zilberstein A.
Author information:
(1)Department of Plant Science, Tel Aviv University, Tel-Aviv 69978, Israel.
The two-step oxidation of proline in all eukaryotes is performed at the inner

mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH)
that produces Delta(1)-pyrroline-5-carboxylate (P5C) and P5C dehydrogenase
(P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in
plants during osmotic stress, allowing free Pro accumulation. We show here that
overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C
oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C
ratio under ambient and osmotic stress conditions, indicating that P5C excess was
reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH
and P5C reductase, exists in animal cells and now demonstrated in plants. As a
part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to
the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of
exogenous l-Pro is provided, generates mitochondrial reactive oxygen species
(ROS) by delivering electrons to O(2), as demonstrated by the
mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity
led to higher ROS production under dark and light conditions in the presence of
Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing
mitochondrial ROS production during increased Pro oxidation is therefore critical
for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by
P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from
electron run-off.
DOI: 10.1074/jbc.M109.009340
PMCID: PMC2785336
1421. Appl Environ Microbiol. 2009 Oct;75(19):6322-30. doi: 10.1128/AEM.02862-08.

Epub
2009 Jul 24.
Influence of vegetation on the in situ bacterial community and polycyclic

aromatic hydrocarbon (PAH) degraders in aged PAH-contaminated or
thermal-desorption-treated soil.
Cébron A(1), Beguiristain T, Faure P, Norini MP, Masfaraud JF, Leyval C.
Author information:
(1)Faculté des Sciences, LIMOS, Nancy Université, CNRS UMR 7137, 54506
Vandoeuvre-lès-Nancy Cedex, France. aurelie.cebron@limos.uhp-nancy.fr
The polycyclic aromatic hydrocarbon (PAH) contamination, bacterial community, and

PAH-degrading bacteria were monitored in aged PAH-contaminated soil
(Neuves-Maisons [NM] soil; with a mean of 1,915 mg of 16 PAHs.kg(-1) of soil dry
weight) and in the same soil previously treated by thermal desorption (TD soil;
with a mean of 106 mg of 16 PAHs.kg(-1) of soil dry weight). This study was
conducted in situ for 2 years using experimental plots of the two soils. NM soil
was colonized by spontaneous vegetation (NM-SV), planted with Medicago sativa
(NM-Ms), or left as bare soil (NM-BS), and the TD soil was planted with Medicago
sativa (TD-Ms). The bacterial community density, structure, and diversity were
estimated by real-time PCR quantification of the 16S rRNA gene copy number,
temporal thermal gradient gel electrophoresis fingerprinting, and band
sequencing, respectively. The density of the bacterial community increased the
first year during stabilization of the system and stayed constant in the NM soil,
while it continued to increase in the TD soil during the second year. The
bacterial community structure diverged among all the plot types after 2 years on
site. In the NM-BS plots, the bacterial community was represented mainly by
Betaproteobacteria and Gammaproteobacteria. The presence of vegetation (NM-SV and
NM-Ms) in the NM soil favored the development of a wider range of bacterial phyla
(Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Verrucomicrobia,
Actinobacteria, Firmicutes, and Chloroflexi) that, for the most part, were not
closely related to known bacterial representatives. Moreover, under the influence
of the same plant, the bacterial community that developed in the TD-Ms was
represented by different bacterial species (Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, and Actinobacteria) than that in the
NM-Ms. During the 2 years of monitoring, the PAH concentration did not evolve
significantly. The abundance of gram-negative (GN) and gram-positive (GP)
PAH-degrading bacteria was estimated by real-time PCR quantification of specific
functional genes encoding the alpha subunit of PAH-ring hydroxylating dioxygenase
(PAH-RHD(alpha)). The percentage of the PAH-RHD(alpha) GN bacterial genes
relative to 16S rRNA gene density decreased with time in all the plots. The GP
PAH-RHD(alpha) bacterial gene proportion decreased in the NM-BS plots but stayed
constant or increased under vegetation influence (NM-SV, NM-Ms, and TD-Ms).
DOI: 10.1128/AEM.02862-08
PMCID: PMC2753067

Jul
24.
The novel genes emmABC are associated with exopolysaccharide production,

motility, stress adaptation, and symbiosis in Sinorhizobium meliloti.
Morris J(1), González JE.
Author information:
The nitrogen-fixing symbiont Sinorhizobium meliloti senses and responds to

constantly changing environmental conditions as it makes its way through the soil
in search of its leguminous plant host, Medicago sativa (alfalfa). As a result,
this bacterium regulates various aspects of its physiology in order to respond
appropriately to stress, starvation, and competition. For example,
exopolysaccharide production, which has been shown to play an important role in
the ability of S. meliloti to successfully invade its host, also helps the
bacterium withstand osmotic changes and other environmental stresses. In an
effort to further elucidate the intricate regulation of this important cell
component, we set out to identify genetic factors that may affect its production.
Here we characterize novel genes that encode a small protein (EmmA) and a
putative two-component system (EmmB-EmmC). A mutation in any of these genes leads
to increased production of the symbiotically important exopolysaccharide
succinoglycan. In addition, emm mutants display membrane-associated defects, are
nonmotile, and are unable to form an optimal symbiosis with alfalfa, suggesting
that these novel genes may play a greater role in the overall fitness of S.
meliloti both during the free-living stage and in its association with its host.
DOI: 10.1128/JB.00760-09
PMCID: PMC2747899
1423. Foodborne Pathog Dis. 2009 Nov;6(9):1041-6. doi: 10.1089/fpd.2009.0267.
Use of 1% peroxyacetic acid sanitizer in an air-mixing wash basin to remove

bacterial pathogens from seeds.
Rajkowski KT(1), Ashurst K.
Author information:
(1)Food Safety Intervention Technologies Research Unit, Eastern Regional Research
Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor,
Pennsylvania, USA. kathleen.rajkowski@ars.usda.gov
To achieve the production of pathogen-free sprouts, there must be appropriate

mixing of liquid sanitizer with the seeds to assure contact. Commercial
treatments by irradiation or ozone gas of Salmonella spp. artificially inoculated
seeds were compared, and these resulted in a 1 log reduction after all
treatments. Use of peroxyacetic acid (1%) sanitizer on Salmonella spp. or
Escherichia coli O157:H7 inoculated alfalfa seeds consistently resulted in a
greater than 1 log reduction. In addition, during these studies debris was noted
after the seeds were removed. Based on this observation, an air-mixing wash basin
was developed for commercial use. Validation was done by commercial growers using
1% peroxyacetic acid sanitizer to wash seeds in the air-mixing basin, followed by
sprouting the seeds. No positive or false-positive pathogen results were reported
after the required testing of the sprout water (run-off during sprouting). Use of
1% peroxyacetic acid sanitizer in the air-mixing wash basin does provide the
sprout grower an effective means of sanitizing sprout seeds.
DOI: 10.1089/fpd.2009.0267
1424. Wei Sheng Wu Xue Bao. 2009 Mar;49(3):395-9.
[Isolation and identification of hydrogen-oxidizing bacteria producing

1-aminocyclopropane-1-carboxylate deaminase and the determination of enzymatic
activity].
Fu B(1), Wang W, Tang M, Chen X.
Author information:
(1)Key Laboratory of Resource Biology and Biotechnology in Western China,
Ministry of Education, Northwest University, Xi'an 710069, China.
lisa_265@163.com
OBJECTIVE: We used Medicago sativa rhizosphere in Shaanxi province of China to

isolate and identify hydrogen-oxidizing bacteria that produced ACC
(1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why
they can promote the growth of plants.
METHODS: Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation
system. We studied the morphological character, physiological characteristics,
16S rDNA sequence analysis and built the phylogenic tree. Thin layer
chromatography was used to isolate the strain that produced ACC deaminase.
Ninhydrin reaction was used to test the enzyme activity.
RESULTS: In total 37 strains were isolated, 8 of which could oxidize H2 strongly
and grow chemolithoautotrophically. We initially identified them as
hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these
8 strains. Morphological and physiological characteristics analysis showed that
strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA
sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7
was clustered together with Pseudomonas putida in phylogenetic tree, with the
sequence identity of 99%. Based on all these results, strain WMQ-7 was identified
as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/microg.
CONCLUSION: A strain producing ACC deaminase was identified and tested.
1425. J Anim Sci. 2009 Oct;87(10):3259-77. doi: 10.2527/jas.2008-1277. Epub 2009

Jul
17.
Fatty acid metabolism and deposition in subcutaneous adipose tissue of pasture-

and feedlot-finished cattle.
Fincham JR(1), Fontenot JP, Swecker WS, Herbein JH, Neel JP, Scaglia G, Clapham
WM, Notter DR.
Author information:
(1)Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and
State University, Blacksburg, VA 24061, USA.
An experiment was conducted to evaluate the effects of pasture finishing versus

feedlot finishing, over time, on fatty acid metabolism in Angus crossbred steers
(n = 24). Ruminal fluid, serum, and adipose tissue biopsies were obtained on d 0,
28, 84, and 140. Pasture forages and diet ingredient samples were obtained at
14-d intervals to determine nutritive value and fatty acid composition. The
feedlot diet consisted of corn silage, cracked corn grain, soybean meal, and a
vitamin and mineral supplement. The pasture-finished steers grazed sequentially
on triticale (x Triticosecale rimpaui)/annual ryegrass (Lolium multiflorum),
alfalfa (Medicago sativa)/orchardgrass (Dactylis glomerata), and a cool-season
grass/legume mixture. The feedlot diet contained an average of 57% of total fatty
acids as linoleic acid and 2% as linolenic acid. The pasture forages contained 9%
of total fatty acids as linoleic acid and 66% as linolenic acid. Concentrations
(% of total fatty acids) of linolenic acid were greater (P < 0.05) in ruminal
fluid, serum, and adipose tissue of the pasture-finished steers, compared with
the feedlot-finished steers. Concentrations (% of total fatty acids) of cis-9,
trans-11 CLA were greater (P < 0.05) in adipose tissue of the pasture-finished
steers than feedlot-finished steers. Concentrations of cis-9, trans-11 CLA in
adipose tissue declined (P < 0.05) in the feedlot-finished steers from d 0 to 28
to 84. In the pasture-finished steers, concentrations of cis-9, trans-11 CLA in
adipose tissue (mg/g of tissue) peaked (P < 0.05) on d 28 and remained elevated
(ranged from 9.91 to 12.80 mg/g of tissue) throughout the duration of the study.
In the pasture-finished steers, linolenic acid concentrations tended to peak (P =
0.07) on d 28 and remained elevated (ranged from 0.64 to 0.80% of total fatty
acids) throughout the study. It appears that only a short time is needed to alter
the n-3 and CLA composition of adipose tissue in cattle finished on pasture.
DOI: 10.2527/jas.2008-1277
1426. J Hazard Mater. 2009 Nov 15;171(1-3):879-85. doi:

10.1016/j.jhazmat.2009.06.083.
Epub 2009 Jun 23.
Effect of oil refinery sludges on the growth and antioxidant system of alfalfa
plants.
Martí MC(1), Camejo D, Fernández-García N, Rellán-Alvarez R, Marques S, Sevilla

F, Jiménez A.
Author information:
(1)Department of Stress Biology and Plant Pathology, Centro de Edafología y
Biología Aplicada del Segura (CEBAS-CSIC), E-30100 Murcia, Spain.
mamen.marti@gmail.com
The refining process in the petrochemical industry generates oil refinery

sludges, a potentially contaminating waste product, with a high content of
hydrocarbons and heavy metals. Faster degradation of hydrocarbons has been
reported in vegetated soils than in non-vegetated soils, but the impact of these
contaminants on the plants physiology and on their antioxidant system is not well
known. In this study, the effect of the addition of petroleum sludge to soil on
the physiological parameters, nutrient contents, and oxidative and antioxidant
status in alfalfa was investigated. An inhibition of alfalfa growth and an
induction of oxidative stress, as indicated by an increase in protein oxidation,
were found. Also, the superoxide dismutase isoenzymes, peroxidase, and those
enzymes involved in the ascorbate-glutathione cycle showed significant activity
increases, parallel to an enhancement of total homoglutathione, allowing plants
being tolerant to this situation. This information is necessary to establish
successful and sustainable plant-based remediation strategies.
DOI: 10.1016/j.jhazmat.2009.06.083
1427. J Biomed Sci. 2009 Jul 14;16:64. doi: 10.1186/1423-0127-16-64.
Ethyl acetate extracts of alfalfa (Medicago sativa L.) sprouts inhibit

lipopolysaccharide-induced inflammation in vitro and in vivo.
Hong YH(1), Chao WW, Chen ML, Lin BF.
Author information:
(1)Department of Biochemical Science and Technology, Institute of Microbiology
and Biochemistry, College of Life Science, National Taiwan University, Taipei,
Taiwan, Republic of China.
This study aimed to investigate if food components that exert anti-inflammatory

effects may be used for inflammatory disorders by examining alfalfa sprout ethyl
acetate extract (ASEA). The cytokine profile and life span of BALB/c mice with
acute inflammation after intra-peritoneal (ip) injection of 15 mg/kg BW
lipopolysaccharide (LPS) were determined. The results showed that the life span
of LPS-induced inflammatory mice were negatively correlated with serum levels of
TNF-alpha, IL-6, and IL-1beta at 9 hr after LPS-injection, which indicated that
suppressing these cytokines in the late phase of inflammation may be beneficial
for survival. The in vitro experiment then showed that ASEA significantly reduced
IL-6 and IL-1beta production and the NF-kappaB trans-activation activity of
mitogen-stimulated RAW264.7 cells. To further evaluate the anti-inflammatory
effects of ASEA in vivo, BALB/c mice were tube-fed with 25 mg ASEA/kg BW/day in
50 microl sunflower oil, while the control and PDTC (pyrrolidine dithiocarbamate,
an anti-inflammatory agent) groups were tube-fed with 50 microl sunflower oil/day
only. After one week of tube-feeding, the PDTC group was injected with 50 mg/kg
BW PDTC and one hour later, all of the mice were injected with 15 mg/kg BW LPS.
The results showed that the ASEA and PDTC groups had significantly lower serum
TNF-alpha, IL-6, and IL-1beta levels at 9 hr after LPS challenge, and
significantly higher survival rates than the control group. This study suggests
that ASEA supplementation can suppress the production of pro-inflammatory
cytokines and alleviate acute inflammatory hazards.
DOI: 10.1186/1423-0127-16-64
PMCID: PMC2720939
1428. Mol Plant Microbe Interact. 2009 Aug;22(8):921-31. doi: 10.1094/MPMI-22-8-

0921.
Full-size ABC transporters from the ABCG subfamily in medicago truncatula.
Jasinski M(1), Banasiak J, Radom M, Kalitkiewicz A, Figlerowicz M.
Author information:
(1)Institute of Bioorganic Chemistry PAS, Noskowskiego 12/14, 61-704 Poznań,
Poland.
Full-size ATP-binding cassette (ABC) transporters belonging to the ABCG subfamily

are unique for plants and fungi. There is growing evidence that certain of these
proteins play a role in plant defense or signaling systems. As yet, a complete
set of full-size ABCG protein genes has been inventoried and classified in only
two plants: Arabidopsis thaliana and Oryza sativa. Recently, a domain-based
clustering analysis has predicted the presence of at least 12 genes encoding such
proteins in the Lotus japonicus genome. Here, we identify and classify 19 genes
coding full-size ABCG proteins in Medicago truncatula, a model legume plant. We
have found that the majority of these genes are expressed in roots and flowers
whereas only a few are expressed in leaves. Expression of several has been
induced upon pathogenic infection in both roots and leaves. ABCG messenger RNAs
have been detected in root nodules forming during symbiosis of legume plants and
nitrogen-fixing bacteria. The data presented provide a scaffold for further
studies of the physiological function of Medicago ABCG transporters and their
possible role in modulating plant-microorganism interactions.
DOI: 10.1094/MPMI-22-8-0921
1429. Acta Biol Hung. 2009 Jun;60(2):211-9. doi: 10.1556/ABiol.60.2009.2.8.
Effect of foliar feeding on nitrogen assimilation in alfalfa plants at

insufficient molybdenum supply.
Hristozkova M(1), Geneva M, Stancheva I.
Author information:
(1)Acad. M. Popov, Institute of Plant Physiology, Bulgarian Academy of Sciences,
Acad. G. Bonchev Street, Building 21, Sofia 1113, Bulgaria.
The influence of foliar feeding on the nitrogen assimilation in alfalfa plants

under conditions of Mo shortage was studied. It was established that foliar
fertilization with 0.3% solution of Agroleaf® resulted in increase of nitrogen
fixation and nitrogen assimilation in the absence of Mo. Insufficient molybdenum
supply leads to significant reduction of plant Mo content and nitrogen-fixing
activity, while stress induced amino acids as alanine, GABA, threonine, proline
and serine increased repeatedly. The negative effect of Mo deficiency on the
enzyme activities related to the primary nitrogen assimilation (NR, GS, GOGAT)
and plant growth diminished due to the foliar absorbed nutrients.
DOI: 10.1556/ABiol.60.2009.2.8
1430. Appl Environ Microbiol. 2009 Sep;75(17):5708-13. doi: 10.1128/AEM.00752-09.

Epub
2009 Jul 6.
Characterization of mundticin L, a class IIa anti-Listeria bacteriocin from

Enterococcus mundtii CUGF08.
Feng G(1), Guron GK, Churey JJ, Worobo RW.
Author information:
(1)Department of Food Science and Technology, Cornell University, Geneva, NY
14456, USA.
Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa
sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a
high level of inhibitory activity against the genus Listeria. The
plasmid-associated operons containing genes for the mundticin L precursor, the
ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced.
The fifth residue of the conservative consensus sequence YGNGX in the mature
bacteriocin is leucine instead of valine in the sequences of the homologous
molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the
ABC transporter and the immunity protein are homologous but unique.
DOI: 10.1128/AEM.00752-09
PMCID: PMC2737909
1431. Pak J Biol Sci. 2009 Jan 15;12(2):192-4.
Investigation on effect of Populus alba stands distance on density of pests and

their natural enemies population under poplar/alfalfa agroforestry system.
Khabir ZH(1), Sadeghi SE, Hanifeh S, Eivazi A.
Author information:
(1)Agricultural and Natural Resources Research Center, West Azarbaijan, Orumieh,
Iran.
This study was carried out in order to distinguish the effect of agroforestry
system (combination of agriculture and forestry) on pests and natural enemy's
population in poplar research station. Wood is one of the first substances that
naturally was used for a long period of time. Forage is an important production
of natural resources too. Some factors such as proper lands deficit, lack of
economy, pest and disease attacks and faced production of these materials with
serious challenges. Agroforestry is a method for decrease of the mentioned
problems. The stands of poplar had have planted by complete randomized design
with 4 treatments (stand distance) of poplar/alfalfa include 3x4, 3x6.7, 3x8,
3x10 m and 2 control treatments, alfalfa and poplar. The results showed that
Chaitophorus populeti had the highest density in poplar and 3x10 m treatments.
Monosteira unicostata is another insect pest that had most density in 3x10 m
treatment. And alfalfa had high density of Chrysoperla carnea. The density of
Coccinella septempunctata, were almost equal in all treatments.

Jul 2.
Predicting milk and forage intake of nursing calves.
Tedeschi LO(1), Fox DG.
Author information:
(1)Department of Animal Science, Texas A & M University, College Station, TX
77843-2471, USA. luis.tedeschi@tamu.edu
A theoretical model was developed to predict forage intake of nursing calves

based on peak milk level (PML) and BW using data from 39 Holstein steer calves
individually fed for 200 d with milk replacer reconstituted to equal the fat and
protein contents of beef cow milk. Treatment levels were amounts of reconstituted
milk allowed per day based on lactation curves, which were based on PML of 2.72,
5.44, 8.16, 10.88, and 13.6 kg/d, respectively. Chopped alfalfa hay was offered
for ad libitum intake to allow maximum voluntary forage consumption in addition
to the reconstituted milk. We observed that calves receiving increased amounts of
milk (10.88 to 13.66 kg of milk/d at peak) consumed little forage during the
first 60 d of age. Their consumption of forage was also less than those calves
receiving reduced quantities of milk (2.72 to 5.44 kg of peak milk/d) at the same
BW because milk intake was prioritized. The forage DMI of the calf depended on
calf BW and quality of the forage. Furthermore, calf BW and forage DMI was
correlated with calf milk intake. A significant (P < 0.05) relationship between
total DE intake (DEI) and BW was identified. A theoretical model was developed to
predict forage DMI of nursing calves based on total DEI. The total DEI was
estimated using PML and BW. Equations were developed to adjust forage DMI for DE
content of the forage. A sensitivity analysis using Monte Carlo simulation
indicated that forage DMI of grazing, nursing calves is likely to be less than
4.26 kg/d 95% of the time and that variation in BW and PML have the greatest
impact on forage DMI. We concluded that equations developed in this study can be
used to evaluate different cow-calf production scenarios, including matching
forage quality and availability with dam milk production potential.
DOI: 10.2527/jas.2009-2014
1433. Mol Biol Rep. 2010 Feb;37(2):745-53. doi: 10.1007/s11033-009-9590-7. Epub

2009
Jul 2.
Screening of genes induced by salt stress from Alfalfa.
Jin H(1), Sun Y, Yang Q, Chao Y, Kang J, Jin H, Li Y, Margaret G.
Author information:
100193, Beijing, People's Republic of China.
An alfalfa cDNA library induced by salt stress was constructed by suppression

subtraction hybridization (SSH) technology. Total RNA from 10-day-old seedlings
was used as a "driver," and total RNA from seedlings induced by salt was used as
a "tester". One hundred and nineteen clones identified as positive clones by
reverse Northern dot-blotting resulted in 82 uni-ESTs comprised of 16 contigs and
66 singletons. Blast analysis of deduced protein sequences revealed that 51 ESTs
had identity similar to proteins with known function, while 24 could not be
annotated at all. Most of the annotated sequences were homologous to genes
involved in abiotic or biotic stress in plants. Among these proteins,
beta-amylase, fructose-1,6-bisphosphate, aldolase, and sucrose synthase are
related to osmolyte synthesis; a CCCH-type zinc finger protein, DNA binding
protein, His-Asp phosphotransfer protein, and the RelA/SpoT protein partake in
transcription regulation and signal transduction; and ribulose-l,5-bisphosphate
carboxylase/oxygenase, chlorophyll a/b binding proteins, and an early
light-inducible proteins are related to photosynthesis. In addition, several
ESTs, similar to genes from other plant species, closely involved in salt stress
were isolated from alfalfa, such as an aquaporin protein, a late
embryogenesis-abundant protein, and glutathione peroxidase.
DOI: 10.1007/s11033-009-9590-7
1434. BMC Syst Biol. 2009 Jun 16;3:63. doi: 10.1186/1752-0509-3-63.
The Symbiosis Interactome: a computational approach reveals novel components,

functional interactions and modules in Sinorhizobium meliloti.
Rodriguez-Llorente I(1), Caviedes MA, Dary M, Palomares AJ, Cánovas FM,

Peregrín-Alvarez JM.
Author information:
(1)Dpt, of Molecular Biology and Biochemistry, University of Malaga, Malaga,
Spain. irodri@us.es
BACKGROUND: Rhizobium-Legume symbiosis is an attractive biological process that

has been studied for decades because of its importance in agriculture. However,
this system has undergone extensive study and although many of the major factors
underpinning the process have been discovered using traditional methods, much
remains to be discovered.
RESULTS: Here we present an analysis of the 'Symbiosis Interactome' using novel
computational methods in order to address the complex dynamic interactions
between proteins involved in the symbiosis of the model bacteria Sinorhizobium
meliloti with its plant hosts. Our study constitutes the first large-scale
analysis attempting to reconstruct this complex biological process, and to
identify novel proteins involved in establishing symbiosis. We identified 263
novel proteins potentially associated with the Symbiosis Interactome. The
topology of the Symbiosis Interactome was used to guide experimental techniques
attempting to validate novel proteins involved in different stages of symbiosis.
The contribution of a set of novel proteins was tested analyzing the symbiotic
properties of several S. meliloti mutants. We found mutants with altered
symbiotic phenotypes suggesting novel proteins that provide key complementary
roles for symbiosis.
CONCLUSION: Our 'systems-based model' represents a novel framework for studying
host-microbe interactions, provides a theoretical basis for further experimental
validations, and can also be applied to the study of other complex processes such
as diseases.
DOI: 10.1186/1752-0509-3-63
PMCID: PMC2701930
Effects of alfalfa hay inclusion rate on productivity of lactating dairy cattle

fed wet corn gluten feed-based diets.
Mullins CR(1), Grigsby KN, Bradford BJ.
Author information:
66506, USA.
An experiment was conducted to evaluate the effects of varying the alfalfa

inclusion rate in diets containing 31% (dry matter basis) wet corn gluten feed
(Sweet Bran, Cargill Inc.). Eighty primiparous and multiparous Holstein cows
averaging 178 +/- 90 d in milk (mean +/- SD) were randomly assigned to 1 of 4
sequences in a 4 x 4 Latin square design with 28-d periods. Treatments were diets
containing 0, 7, 14, or 21% alfalfa on a dry matter basis, with corn silage, corn
grain, soybean meal, expeller soybean meal, and mineral supplements varying
across diets to maintain uniform nutrient densities. Diets were formulated for
similar crude protein, neutral detergent fiber, and nonfiber carbohydrate
concentrations. Feed intake, milk production, body weight, and body condition
score were monitored, and linear and quadratic effects of increasing the alfalfa
inclusion rate were assessed using mixed model analysis. As the alfalfa inclusion
rate increased, dry matter intake tended to increase linearly (26.7, 27.3, 27.4,
and 27.5 kg/d for 0, 7, 14, and 21% alfalfa, respectively), and solids-corrected
milk (29.9, 30.2, 30.8, and 30.5 kg/d) and energy-corrected milk production
(32.9, 33.3, 33.8, and 33.6 kg/d) tended to increase linearly. Body weight gain
decreased linearly (22.9, 18.0, 11.2, and 9.5 kg/28 d) with increasing alfalfa
inclusion rate. Although increasing the inclusion rate of alfalfa increased the
proportion of large particles in the diets, treatments had no effect on milk fat
yield or concentration. Feeding more alfalfa (up to 21% of dry matter) tended to
increase milk yield while decreasing body weight gain, suggesting that
metabolizable energy utilization shifted from body weight gain to milk production
in these treatments. However, adding alfalfa to the diet had only minor effects
on productivity.
DOI: 10.3168/jds.2008-1873
1436. Infect Immun. 2009 Sep;77(9):4102-10. doi: 10.1128/IAI.00398-09. Epub 2009

Jun
15.
Identification of specific and universal virulence factors in Burkholderia

cenocepacia strains by using multiple infection hosts.
Uehlinger S(1), Schwager S, Bernier SP, Riedel K, Nguyen DT, Sokol PA, Eberl L.
Author information:
(1)Department of Microbiology, Institute of Plant Biology, University of Zurich,
Over the past few decades, strains of the Burkholderia cepacia complex have
emerged as important pathogens for patients suffering from cystic fibrosis.
Identification of virulence factors and assessment of the pathogenic potential of
Burkholderia strains have increased the need for appropriate infection models. In
previous studies, different infection hosts, including mammals, nematodes,
insects, and plants, have been used. At present, however, the extent to which the
virulence factors required to infect different hosts overlap is not known. The
aim of this study was to analyze the roles of various virulence factors of two
closely related Burkholderia cenocepacia strains, H111 and the epidemic strain
K56-2, in a multihost pathogenesis system using four different model organisms,
namely, Caenorhabditis elegans, Galleria mellonella, the alfalfa plant, and mice
or rats. We demonstrate that most of the identified virulence factors are
specific for one of the infection models, and only three factors were found to be
essential for full pathogenicity in several hosts: mutants defective in (i)
quorum sensing, (ii) siderophore production, and (iii) lipopolysaccharide
biosynthesis were attenuated in at least three of the infection models and thus
may represent promising targets for the development of novel anti-infectives.
DOI: 10.1128/IAI.00398-09
PMCID: PMC2738042
1437. Plant Physiol. 2009 Aug;150(4):1866-79. doi: 10.1104/pp.109.136689. Epub 2009
Jun
12.
A novel red clover hydroxycinnamoyl transferase has enzymatic activities

consistent with a role in phaselic acid biosynthesis.
Sullivan M(1).
Author information:
(1)Dairy Forage Research Center, Agricultural Research Service, United States
Department of Agriculture, Madison, Wisconsin 53706, USA.
Red clover (Trifolium pratense) leaves accumulate several mumol g(-1) fresh
weight of phaselic acid [2-O-(caffeoyl)-l-malate]. Postharvest oxidation of such
o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of
forage protein during storage. Forage crops like alfalfa (Medicago sativa) lack
both polyphenol oxidase and o-diphenols, and breakdown of their protein upon
harvest and storage results in economic losses and release of excess nitrogen
into the environment. Understanding how red clover synthesizes o-diphenols such
as phaselic acid will help in the development of forage crops utilizing this
natural system of protein protection. A possible pathway for phaselic acid
biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming
caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs
were identified in red clover. HCT1 shares more than 75% amino acid identity with
a number of well-characterized shikimate O-HCTs implicated in monolignol
biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has
limited sequence identity to any previously identified HCT. Expression analyses
indicate that HCT1 mRNA accumulates to 4-fold higher levels in stems than in
leaves, whereas HCT2 mRNA accumulates to 10-fold higher levels in leaves than in
stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli
indicate that HCT1 transfers caffeoyl or p-coumaroyl moieties from a coenzyme
A-thiolester to shikimate but not malate, whereas HCT2 transfers caffeoyl or
p-coumaroyl moieties from a coenzyme A-thiolester to malate but not shikimate.
Together, these results indicate that HCT1 is involved in monolignol biosynthesis
and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.
DOI: 10.1104/pp.109.136689
PMCID: PMC2719126
1438. FEMS Microbiol Lett. 2009 Apr;293(2):177-87. doi:

10.1111/j.1574-6968.2009.01527.x.
Genetic characterization of oligopeptide uptake systems in Sinorhizobium

meliloti.
Nogales J(1), Muñoz S, Olivares J, Sanjuán J.
Author information:
(1)Dpto. Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental
del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
The genetic characterization of three ABC transport systems involved in

oligopeptide uptake by Sinorhizobium meliloti is reported. Oligopeptide permease
(Opp) encoded by the pSymB oppABCD operon, is required for uptake of
tetrapeptides and certain tripeptides like 3Ala and bialaphos. The chromosomally
encoded dipeptide permease (Dpp1), also able to import the toxic tripeptide
bialaphos, is required for utilization of dipeptides and tripeptides like 3Gly
and GlyGlyAla, with minor importance for utilization of 3Ala and tetrapeptides.
The ttp (tri and tetrapeptide uptake) operon, encodes a third ABC system (Ttp)
unable of transporting bialaphos and with minor role in the utilization of
tetrapeptides and tripeptides like 3-Ala. Despite the overlapping substrate
specificities of these ABC transporters, the corresponding gene operons displayed
distinct expression profiles: dpp1 showed high constitutive expression levels
under all conditions tested, in contrast to the low expression levels of ttp,
whereas opp was maximally expressed upon entry into stationary phase.
Nevertheless, complex interactions among the three systems at the transcriptional
level were observed: opp was negatively autoregulated via OppA and positively
regulated via DppA1, whereas dpp1 seems negatively autoregulated via DppA1. The
expression of both opp and dppl was reduced in Ttp mutants. The ABC transport
systems characterized in this work are not essential for the establishment of
nitrogen-fixing symbiosis with alfalfa.
DOI: 10.1111/j.1574-6968.2009.01527.x
1439. Biotechnol J. 2009 Jun;4(6):880-7. doi: 10.1002/biot.200800360.
Flow-through imaging cytometry for characterization of Salmonella subpopulations

in alfalfa sprouts, a complex food system.
Bisha B(1), Brehm-Stecher BF.
Author information:
(1)Rapid Microbial Detection and Control Laboratory, Department of Food Science
and Human Nutrition, Iowa State University, Ames, IA 50011, USA.
We recently developed an approach combining fluorescence in situ hybridization

(FISH) and flow cytometry for detecting low levels of Salmonella spp.
(approximately 10(3) cells/mL sprout wash) against high levels of naturally
occurring sprout flora (approximately 10(7)-10(8) CFU/g sprouts). Although this
"FISH and flow" approach provided rapid presence/absence testing for Salmonella
in this complex food system, it was not capable of more nuanced tasks, such as
probing the phenotypic complexity of the microbes present in sprouts or
determining the physical interactions of Salmonella with these microbes, or with
sprout debris. In the present study, we have combined rapid FISH-based labeling
of Salmonella spp. in sprout washes with flow-through imaging cytometry (FT-IC),
using the ImageStream 100, a commercial FT-IC instrument. This approach enables
image-based characterization of various subpopulations of interest occurring
within these samples. Here, we demonstrate the ability of FT-IC to unambiguously
identify cells, cell aggregates and other events within these subpopulations
based on both cell morphology and hybridization status after reaction with a
Salmonella-targeted probe cocktail. Our ability to directly explore the nature of
these events expands the layers of information possible from cytometric analyses
of these complex samples and clearly demonstrates that "a picture is worth a
thousand dots".
DOI: 10.1002/biot.200800360
1440. J Food Sci. 2009 May-Jul;74(4):M159-64. doi: 10.1111/j.1750-

3841.2009.01125.x.
Acidified sodium chlorite as an alternative to chlorine for elimination of
salmonella on alfalfa seeds.
Liao CH(1).
Author information:
(1)Eastern Regional Research Center, Agricultural Research Service, U.S. Dept. of
Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA.
ChingHsing.liao@ars.usda.gov
The health and environmental hazard associated with the use of chlorine for food
processing has been documented previously. This study was conducted to determine
if acidified sodium chlorite (ASC) could be used to replace calcium hypochlorite
(Ca[OCl](2)) for disinfection of alfalfa seeds. Contaminated seeds containing
approximately 1.5 x 10(7) CFU/g of Salmonella were treated with ASC or Ca(OCl)(2)
at different concentrations and for different periods of time. Results showed
that the efficacy of ASC and Ca(OCl)(2) for elimination of Salmonella on
contaminated seeds could be improved greatly by extending the treatment time from
the traditional 15 to 45 min. Treatment of seeds with 800 ppm of ASC for 45 min
reduced the number of Salmonella by 3.9 log units, approximately 1.2 log units
higher than that treated with 20000 ppm of Ca(OCl)(2). Treatment of seeds with a
lower concentration (100 to 400 ppm) of ASC for 45 min reduced the number of
Salmonella by 1.3 to 2.2 log units. Soaking alfalfa seeds in 800 ppm of ASC for
45 min did not affect seed germination. However, soaking seeds in 20000 ppm of
Ca(OCl)(2) for 45 min reduced seed germination by 20%. Unlike Ca(OCl)(2),
antimicrobial efficiency of ASC was not affected by pre-exposure to alfalfa
seeds. Data presented also showed that Salmonella on newly inoculated seeds that
had been stored at 4 degrees C for less than 7 d were more sensitive to sanitizer
treatment than those on seeds that had been stored for 4 wk or longer.
DOI: 10.1111/j.1750-3841.2009.01125.x
1441. Syst Appl Microbiol. 2009 Sep;32(6):413-20. doi: 10.1016/j.syapm.2009.04.003.

Epub 2009 May 23.
Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with

Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and
host range.
León-Barrios M(1), Lorite MJ, Donate-Correa J, Sanjuán J.
Author information:
(1)Departamento de Microbiología y Biología Celular, Facultad de Farmacia,
Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain. mileonba@ull.es
Eleven strains were isolated from root nodules of Lotus endemic to the Canary
Islands and they belonged to the genus Ensifer, a genus never previously
described as a symbiont of Lotus. According to their 16S rRNA and atpD gene
sequences, two isolates represented minority genotypes that could belong to
previously undescribed Ensifer species, but most of the isolates were classified
within the species Ensifer meliloti. These isolates nodulated Lotus
lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and
Lotus uliginosus were more restrictive hosts. However, effective nitrogen
fixation only occurred with the endemic L. lancerottensis. The E. meliloti
strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or
Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC
and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of
Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts
of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and
bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and
nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic
genotypes. According to their symbiotic gene sequences and host range, the Lotus
symbionts would represent a new biovar of E. meliloti for which bv. lancerottense
is proposed.
DOI: 10.1016/j.syapm.2009.04.003
1442. J Environ Qual. 2009 May 20;38(4):1466-72. doi: 10.2134/jeq2008.0285. Print

2009
Jul-Aug.
Photosynthetic pigment concentrations, gas exchange and vegetative growth for

selected monocots and dicots treated with two contrasting coal fly ashes.
Yunusa IA(1), Burchett MD, Manoharan V, Desilva DL, Eamus D, Skilbeck CG.
Author information:
(1)Plant Functional Biology & Climate Change Cluster, Dep. of Environmental
Sciences, Univ. of Technology, Sydney, PO Box 123, Broadway 2007, Australia.
isa.yunusa@uts.edu.au
There is uncertainty as to the rates of coal fly ash needed for optimum
physiological processes and growth. In the current study we tested the hypothesis
that photosynthetic pigments concentrations and CO(2) assimilation (A) are more
sensitive than dry weights in plants grown on media amended with coal fly ash. We
applied the Terrestrial Plant Growth Test (Guideline 208) protocols of the
Organization for Economic Cooperation and Development (OECD) to monocots [barley
(Hordeum vulgare) and ryegrass (Secale cereale)] and dicots [canola (Brasica
napus), radish (Raphanus sativus), field peas (Pisum sativum), and lucerne
(Medicago sativa)] on media amended with fly ashes derived from semi-bituminous
(gray ash) or lignite (red ash) coals at rates of 0, 2.5, 5.0, 10, or 20 Mg
ha(-1). The red ash had higher elemental concentrations and salinity than the
gray ash. Fly ash addition had no significant effect on germination by any of the
six species. At moderate rates (</= 10 Mg ha(-1)) both ashes increased (p < 0.05)
growth rates and concentrations of chlorophylls a and b, but reduced carotenoid
concentrations. Addition of either ash increased A in radish and transpiration in
barley. Growth rates and final dry weights were reduced for all of the six test
species when addition rates exceeded 10 Mg ha(-1) for gray ash and 5 Mg ha(-1)
for red ash. We concluded that plant dry weights, rather than pigment
concentrations and/or instantaneous rates of photosynthesis, are more consistent
for assessing subsequent growth in plants supplied with fly ash.
DOI: 10.2134/jeq2008.0285
1443. Spectrochim Acta A Mol Biomol Spectrosc. 2009 Sep 1;73(5):846-53. doi:
10.1016/j.saa.2009.04.006. Epub 2009 Apr 22.
Molecular basis of protein structure in proanthocyanidin and anthocyanin-enhanced

Lc-transgenic alfalfa in relation to nutritive value using synchrotron-radiation
FTIR microspectroscopy: a novel approach.
Yu P(1), Jonker A, Gruber M.

Author information:
(1)College of Agriculture and Bioresources, The University of Saskatchewan, 51
Campus Drive, Saskatoon, SK, S7N 5A8, Canada. peiqiang.yu@usask.ca
To date there has been very little application of synchrotron radiation-based

Fourier transform infrared microspectroscopy (SRFTIRM) to the study of molecular
structures in plant forage in relation to livestock digestive behavior and
nutrient availability. Protein inherent structure, among other factors such as
protein matrix, affects nutritive quality, fermentation and degradation behavior
in both humans and animals. The relative percentage of protein secondary
structure influences protein value. A high percentage of beta-sheets usually
reduce the access of gastrointestinal digestive enzymes to the protein. Reduced
accessibility results in poor digestibility and as a result, low protein value.
The objective of this study was to use SRFTIRM to compare protein molecular
structure of alfalfa plant tissues transformed with the maize Lc regulatory gene
with non-transgenic alfalfa protein within cellular and subcellular dimensions
and to quantify protein inherent structure profiles using Gaussian and Lorentzian
methods of multi-component peak modeling. Protein molecular structure revealed by
this method included alpha-helices, beta-sheets and other structures such as
beta-turns and random coils. Hierarchical cluster analysis and principal
component analysis of the synchrotron data, as well as accurate spectral analysis
based on curve fitting, showed that transgenic alfalfa contained a relatively
lower (P<0.05) percentage of the model-fitted alpha-helices (29 vs. 34) and
model-fitted beta-sheets (22 vs. 27) and a higher (P<0.05) percentage of other
model-fitted structures (49 vs. 39). Transgenic alfalfa protein displayed no
difference (P>0.05) in the ratio of alpha-helices to beta-sheets (average: 1.4)
and higher (P<0.05) ratios of alpha-helices to others (0.7 vs. 0.9) and
beta-sheets to others (0.5 vs. 0.8) than the non-transgenic alfalfa protein. The
transgenic protein structures also exhibited no difference (P>0.05) in the
vibrational intensity of protein amide I (average of 24) and amide II areas
(average of 10) and their ratio (average of 2.4) compared with non-transgenic
alfalfa. Cluster analysis and principal component analysis showed no significant
differences between the two genotypes in the broad molecular fingerprint region,
amides I and II regions, and the carbohydrate molecular region, indicating they
are highly related to each other. The results suggest that transgenic Lc-alfalfa
leaves contain similar proteins to non-transgenic alfalfa (because amide I and II
intensities were identical), but a subtle difference in protein molecular
structure after freeze drying. Further study is needed to understand the
relationship between these structural profiles and biological features such as
protein nutrient availability, protein bypass and digestive behavior of livestock
fed with this type of forage.
DOI: 10.1016/j.saa.2009.04.006
Effects of spontaneous heating on fiber composition, fiber digestibility, and in

situ disappearance kinetics of neutral detergent fiber for alfalfa-orchardgrass
hays.
Author information:
During 2006 and 2007, forages from 3 individual hay harvests were utilized to
assess the effects of spontaneous heating on concentrations of fiber components,
48-h neutral detergent fiber (NDF) digestibility (NDFD), and in situ
disappearance kinetics of NDF for large-round bales of mixed alfalfa (Medicago
sativa L.) and orchardgrass (Dactylis glomerata L.). Over the 3 harvests, 96
large-round bales were made at preset bale diameters of 0.9, 1.2, or 1.5 m, and
at moisture concentrations ranging from 9.3 to 46.6%. Internal bale temperatures
were monitored daily during an outdoor storage period, reaching maxima (MAX) of
77.2 degrees C and 1,997 heating degree days >30 degrees C (HDD) for one specific
combination of bale moisture, bale diameter, and harvest. Concentrations of all
fiber components (NDF, acid detergent fiber, hemicellulose, cellulose, and
lignin) increased in response to spontaneous heating during storage. Changes in
concentrations of NDF during storage (poststorage - prestorage; DeltaNDF) were
regressed on HDD using a nonlinear regression model (R(2) = 0.848) that became
asymptotic after DeltaNDF increased by 8.6 percentage units. Although the
specific regression model varied, changes (poststorage - prestorage) in
concentrations of acid detergent fiber, cellulose, and lignin also increased in
nonlinear relationships with HDD that exhibited relatively high coefficients of
determination (R(2) = 0.710 to 0.885). Fiber digestibility, as determined by
NDFD, was largely unaffected by heating characteristics except within bales
incurring the most extreme levels of HDD or MAX. In situ assessment of ruminal
NDF disappearance kinetics indicated that disappearance rate (K(d)) declined by
about 40% within the range of heating incurred over these hay harvests. The
change in K(d) during storage (DeltaK(d)) was related closely to both HDD and MAX
by nonlinear models exhibiting high R(2) statistics (0.907 and 0.883,
respectively). However, there was no regression relationship between changes
(poststorage - prestorage) in effective ruminal disappearance of NDF and
spontaneous heating, regardless of which heating measure was used as the
independent variable. The close regression relationship between DeltaK(d) and
measures of spontaneous heating indicates clearly that ruminal NDF disappearance
was altered negatively by some direct or indirect aspect of spontaneous heating.
However, it was equally apparent that these effects were offset by an expanding
pool of dry matter recovered as potentially degradable NDF.
DOI: 10.3168/jds.2008-1921
Effects of bale moisture and bale diameter on spontaneous heating, dry matter
recovery, in vitro true digestibility, and in situ disappearance kinetics of
alfalfa-orchardgrass hays.
Author information:
Alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.) hay was made in
96 large-round bales over 3 harvests during 2006 and 2007 to assess the effects
of spontaneous heating on dry matter (DM) recovery, in vitro true digestibility
(IVTD), and in situ disappearance kinetics of DM. Throughout these harvests,
bales were made at preset diameters of 0.9, 1.2, or 1.5 m and at moisture
concentrations ranging from 9.3 to 46.6%. Internal bale temperatures were
monitored daily during an outdoor storage period, reaching maxima of 77.2 degrees
C (MAX) and 1,997 heating degree days >30 degrees C (HDD) for one specific
combination of bale moisture, bale diameter, and harvest. Following storage,
regressions of DM recovery on HDD and MAX indicated that DM recovery declined
linearly in close association with measures of spontaneous heating. For HDD,
slopes and intercepts differed across bale diameters, probably because the
greater surface area per kilogram of DM for 0.9-m bales facilitated more rapid
dissipation of heat than occurred from 1.2- or 1.5-m-diameter bales. Regardless
of bale diameter, coefficients of determination were high (r(2) > or = 0.872)
when HDD was used as the independent variable. Regressions of DM recovery on MAX
also exhibited high r(2) statistics (> or = 0.833) and a common slope across bale
diameters (-0.32 percentage units of DM/ degrees C). Changes in concentrations of
IVTD during storage (poststorage - prestorage; DeltaIVTD) also were regressed on
HDD and MAX. For HDD, the data were best fit with a nonlinear model in which
DeltaIVTD became rapidly negative at <1,000 HDD, but was asymptotic thereafter.
When MAX was used as the independent variable, a simple linear model (y = -0.23x
+ 9.5) provided the best fit. In both cases, coefficients of determination were
comparable to those for DM recovery (R(2) or r(2) > or =0.820). Changes
(poststorage - prestorage) in ruminal DM degradation rate (DeltaK(d)) and
effective ruminal degradability of DM (DeltaDEG) were assessed similarly.
Although the most appropriate statistical model varied, DeltaK(d) and DeltaDEG
both became increasingly negative at low to moderate levels of heating, but
generally stabilized thereafter. Both HDD and MAX were excellent predictor
variables for both DeltaK(d) and DeltaDEG; r(2) or R(2) statistics ranged from
0.788 to 0.921. Measures of spontaneous heating are consistently effective
indicators of DM recovery following storage, as well as good indicators of
concurrent changes in IVTD or in situ disappearance of DM for heated
alfalfa-orchardgrass hays.
DOI: 10.3168/jds.2008-1920
1446. Guang Pu Xue Yu Guang Pu Fen Xi. 2009 Feb;29(2):386-8.
[Application of near-infrared reflectance spectroscopy to the discrimination of

salt tolerance of alfalfa cultivars].
Ren WB(1), Han JG, Zhang YW, Guo HQ.
Author information:
China.
Near infrared reflectance spectroscopy is a new developed method able to analyse

the content of specific compound in the detected sample quickly and efficiently,
and has been wildly used in many fields such as evaluation of nutrition value of
forage, cultivar discrimination and so on. In the present paper a new method was
developed for the discrimination of salt tolerance of alfalfa cultivars by
Fourier transform near infrared reflectance spectroscopy. Firstly absorbance
spectrum generated by Fourier transform near infrared spectrometry was used to
classify twenty alfalfa cultivars. These cultivars were obviously clustered into
two groups: salt tolerant and salt sensitive, similar to the result, screened by
the traditional methods. Based on these results, a discrimination model was built
for identification of salt tolerant cultivars. And then, six other cultivars were
used to verify the function of the discriminating model. Result showed that the
distinguishing rate of 100% was achieved with the performance index of 85.7%. It
is concluded that Fourier transform near infrared spectrum is useful for
classification and discrimination of salt tolerance of alfalfa cultivars. The
method can be applied to estimate salt tolerance of alfalfa cultivars, and works
more quickly and efficiently than the traditional screening methods. Application
of Fourier transform near infrared spectrum to discriminating salt tolerance of
alfalfa cultivars is significant in both academic and technical areas.
1447. MMWR Morb Mortal Wkly Rep. 2009 May 15;58(18):500-3.
Outbreak of Salmonella serotype Saintpaul infections associated with eating

alfalfa sprouts - United States, 2009.
Centers for Disease Control and Prevention (CDC).
On February 24, 2009, the Nebraska Department of Health and Human Services
identified six isolates of Salmonella serotype Saintpaul with collection dates
from February 7--14. Salmonella Saintpaul is not a commonly detected serotype;
during 2008, only three Salmonella Saintpaul isolates were identified in
Nebraska. This report summarizes the preliminary results of the investigation of
this outbreak, which has identified 228 cases in 13 states and implicated the
source as alfalfa sprouts produced at multiple facilities using seeds that likely
originated from a common grower. On April 26, the Food and Drug Administration
(FDA) and CDC recommended that consumers not eat raw alfalfa sprouts, including
sprout blends containing alfalfa sprouts, until further notice. On May 1, FDA
alerted sprout growers and retailers that a seed supplier was withdrawing
voluntarily from the market all lots of alfalfa seeds with a specific three-digit
prefix.
1448. J Food Prot. 2009 Apr;72(4):856-9.
Microbiological examination of vegetable seed sprouts in Korea.
Kim H(1), Lee Y, Beuchat LR, Yoon BJ, Ryu JH.
Author information:
(1)Division of Human Environmental Science and Institute of Biotechnology,
Wonkwang University, 344-2 Shinyong-dong, Iksan, Jeonbuk 570-749, Republic of
Korea.
Sprouted vegetable seeds used as food have been implicated as sources of

outbreaks of Salmonella and Escherichia coli O157:H7 infections. We profiled the
microbiological quality of sprouts and seeds sold at retail shops in Seoul,
Korea. Ninety samples of radish sprouts and mixed sprouts purchased at department
stores, supermarkets, and traditional markets and 96 samples of radish, alfalfa,
and turnip seeds purchased from online stores were analyzed to determine the
number of total aerobic bacteria (TAB) and molds or yeasts (MY) and the incidence
of Salmonella, E. coli O157:H7, and Enterobacter sakazakii. Significantly higher
numbers of TAB (7.52 log CFU/g) and MY (7.36 log CFU/g) were present on mixed
sprouts than on radish sprouts (6.97 and 6.50 CFU/g, respectively). Populations
of TAB and MY on the sprouts were not significantly affected by location of
purchase. Radish seeds contained TAB and MY populations of 4.08 and 2.42 log
CFU/g, respectively, whereas populations of TAB were only 2.54 to 2.84 log CFU/g
and populations of MY were 0.82 to 1.69 log CFU/g on alfalfa and turnip seeds,
respectively. Salmonella and E. coli O157:H7 were not detected on any of the
sprout and seed samples tested. E. sakazakii was not found on seeds, but 13.3% of
the mixed sprout samples contained this potentially pathogenic bacterium.
1449. J Nanosci Nanotechnol. 2009 Mar;9(3):1785-91.
Analysis of Ag nanoparticles synthesized by bioreduction.
Tavera-Davila L(1), Liu HB, Herrera-Becerra R, Canizal G, Balcazar M, Ascencio

JA.
Author information:
(1)Facultad de Química, Universidad Autónoma del Estado de México, Paseo Colón
Esq, Con Paseo Tollocan, Toluca, Estado de México, C.P. 50160, México.
Based on a green chemistry mechanism, small silver clusters were obtained by

using biosynthesis with alfalfa (medicago sativa), controlling the size of the
nanoparticles base don different pH conditions. The analysis of the samples was
made with help of transmission electron microscopy methods, mainly with high
angle annular dark field and high resolution transmission electron microscopy.
The optimal conditions were identified when the sample was obtained at pH10,
which allowed obtaining an average size of 4.09 nm and a standard deviation of
1.59, mainly based on cubic like structures.
1450. J Biotechnol. 2009 May 20;141(3-4):181-8. doi: 10.1016/j.jbiotec.2009.03.013.

Epub 2009 Mar 31.
Engineering of plant-specific phenylpropanoids biosynthesis in Streptomyces

venezuelae.
Park SR(1), Yoon JA, Paik JH, Park JW, Jung WS, Ban YH, Kim EJ, Yoo YJ, Han AR,
Yoon YJ.
Author information:
(1)Department of Chemistry and Nano Science, Ewha Womans University, Seoul,
Republic of Korea.
Phenylpropanoids, including flavonoids and stilbenes, are plant secondary

metabolites with potential pharmacological and nutraceutical properties. To
expand the applicability of Streptomyces venezuelae as a heterologous host to
plant polyketide production, flavonoid and stilbene biosynthetic genes were
expressed in an engineered strain of S. venezuelae DHS2001 bearing a deletion of
native pikromycin polyketide synthase gene. A plasmid expressing the
4-coumarate/cinnamate:coenzyme A ligase from Streptomyces coelicolor (ScCCL) and
the chalcone synthase from Arabidopsis thaliana (atCHS) under the control of a
single ermE* promoter was constructed and introduced into S. venezuelae DHS2001.
The resulting strain produced racemic naringenin and pinocembrin from 4-coumaric
acid and cinnamic acid, respectively. Placement of an additional ermE* promoter
upstream of the codon-optimized atCHS (atCHS(op)) gene significantly increased
the yield of both flavanones. Expression of codon-optimized chalcone isomerase
gene from Medicago sativa, together with ScCCL and atCHS(op) genes led to
production of (2S)-flavanones, but the yield was reduced. On the other hand, a
recombinant strain harboring the ScCCL and codon-optimized stilbene synthase gene
from Arachis hypogaea generated stilbenes such as resveratrol and pinosylvin.
This is the first report on the heterologous expression of plant phenylpropanoid
biosynthetic pathways in Streptomyces genus.
DOI: 10.1016/j.jbiotec.2009.03.013
1451. FEMS Microbiol Lett. 2009 May;294(2):183-190.
Fibrolytic capabilities of ruminal bacterium Fibrobacter succinogenes in relation

to its phylogenetic grouping.
Shinkai T, Ohji R, Matsumoto N, Kobayashi Y.
To characterize the fibrolytic function of Fibrobacter succinogenes strains in

relation to their phylogenetic grouping, 32 strains were newly isolated from the
rumen of sheep. All new strains were classified into phylogenetic groups 1 or 2
including a novel subgroup of group 2. Importantly, the majority of the strains
belonging to group 1 were isolated from ruminally incubated hay. Although almost
complete degradation of Avicel was observed among all strains, significantly
lower digestibility of three different forages was recorded for strain HM2 of
group 3 than for the strains of groups 1 and 2. In a comparison of all strains,
two group 1 strains showed significantly higher digestibility of alfalfa and
orchard grass hays, while two strains of the novel subgroup of group 2 had lower
digestibility of orchard grass hay. Adhesion ability of each strain did not
necessarily associate with the extent of digestibility. Maximum growth on Avicel
was higher in group 1 than in group 2 strains, and two group 1 strains showed a
shorter lag time. The results suggest that the ecological prominence of group 1
is due to a mixture of strains that are diverse in their fibrolytic capability
making this group highly adaptable to any forage.
1452. J Bacteriol. 2009 Jul;191(14):4681-6. doi: 10.1128/JB.00318-09. Epub 2009 May

8.
The Sinorhizobium meliloti LpxXL and AcpXL proteins play important roles in
bacteroid development within alfalfa.
Haag AF(1), Wehmeier S, Beck S, Marlow VL, Fletcher V, James EK, Ferguson GP.
Author information:
(1)School of Medicine & Dentistry, Institute of Medical Sciences, University of
Aberdeen, Foresterhill, Aberdeen, United Kingdom.
Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A

very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa.
We demonstrate that LpxXL and AcpXL play important but distinct roles in
bacteroid development and that LpxXL is essential for the modification of S.
meliloti bacteroid lipid A with VLCFAs.
DOI: 10.1128/JB.00318-09
PMCID: PMC2704725
1453. Rev Biol Trop. 2008 Sep;56(3):1053-61.
[Experimental tolerance to boron of the plant species Nicotiana glauca, Jacaranda

mimosifolia, Tecoma stans, Medicago sativa y Spinacea oleracea in Argentina].
de Viana ML(1), Albarracín Franco S.
Author information:
(1)Instituto de Ecología y Ambiente Humano, CIUNSa, Universidad Nacional de
Salta, Buenos Aires No 177, 4400, Salta, Argentina. mldeviana@arnet.com.ar
The activity of boron industries is a punctual and diffuse source of air, soil
and water pollution. Therefore, it is a priority to study possible ways of
reducing this impact. A relatively new technology for reducing soil pollution is
phytoremediation, which uses plants and associate microorganisms. The first step
in phytoremediation is to detect tolerant plant species, which is the objective
of this work. A laboratory experiment to assess the germination, survival and
growth of different species at different boron concentrations was carried out
following a factorial design with two factors: plant species and boron
concentration. Boron concentrations were determined at the beginning and the end
of the experiment, taking into account substrates with and without vegetation. We
found significant differences for treatment, species and the interaction
species*treatment. N. glauca, M. sativa and J. mimosifolia were the most tolerant
species. The other species had a decrease in the response variables, with the
concentration of the pollutant. All the species had a low survival at the highest
boron concentration. The reduction in boron concentration at the end of the
experiment was higher in the 30 ppm treatment with M. sativa and the lower was
registered in the 20 ppm treatment with J. mimosifolia and in 30 ppm with T.
stans and S. oleraceae. We conclude that N. glauca, M. sativa and J. mimosifolia
can be considered in remediation plans.
1454. J Bacteriol. 2009 Jul;191(13):4372-82. doi: 10.1128/JB.00376-09. Epub 2009

Apr
24.
Role of quorum sensing in Sinorhizobium meliloti-Alfalfa symbiosis.
Gurich N(1), González JE.
Author information:
Richardson, Texas 75080-0688, USA.
The ExpR/Sin quorum-sensing system of the gram-negative soil bacterium

Sinorhizobium meliloti plays an important role in the establishment of symbiosis
with its host plant Medicago sativa. A mutant unable to produce autoinducer
signal molecules (sinI) is deficient in its ability to invade the host, but
paradoxically, a strain lacking the quorum-sensing transcriptional regulator ExpR
is as efficient as the wild type. We compared the whole-genome expression profile
of the wild-type strain with strains missing one of the quorum-sensing regulatory
components to identify genes controlled by the ExpR/Sin system throughout the
different phases of the bacterial growth cycle, as well as in planta. Our
analyses revealed that ExpR is a highly versatile regulator with a unique ability
to show different regulatory capabilities in the presence or absence of an
autoinducer. In addition, this study provided us with insight into the plant
invasion defect displayed by the autoinducer mutant. We also discovered that the
ExpR/Sin quorum-sensing system is repressed after plant invasion. Therefore,
quorum sensing plays a crucial role in the regulation of many cell functions that
ensures the successful invasion of the host and is inactivated once symbiosis is
established.
DOI: 10.1128/JB.00376-09
PMCID: PMC2698488
1455. BMC Ecol. 2009 Apr 24;9:9. doi: 10.1186/1472-6785-9-9.
Resource heterogeneity and foraging behaviour of cattle across spatial scales.
Utsumi SA(1), Cangiano CA, Galli JR, McEachern MB, Demment MW, Laca EA.
Author information:
(1)Department of Animal and Rangeland Sciences, New Mexico State University, Las
Cruces, NM 88003, USA. sau@nmsu.edu
BACKGROUND: Understanding the mechanisms that influence grazing selectivity in

patchy environments is vital to promote sustainable production and conservation
of cultivated and natural grasslands. To better understand how patch size and
spatial dynamics influence selectivity in cattle, we examined grazing selectivity
under 9 different treatments by offering alfalfa and fescue in patches of 3 sizes
spaced with 1, 4, and 8 m between patches along an alley. We hypothesized that
(1) selectivity is driven by preference for the forage species that maximizes
forage intake over feeding scales ranging from single bites to patches along
grazing paths, (2) that increasing patch size enhances selectivity for the
preferred species, and that (3) increasing distances between patches restricts
selectivity because of the aggregation of scale-specific behaviours across
foraging scales.
RESULTS: Cows preferred and selected alfalfa, the species that yielded greater
short-term intake rates (P < 0.0001) and greater daily intake potential.
Selectivity was not affected by patch arrangement, but it was scale dependent.
Selectivity tended to emerge at the scale of feeding stations and became strongly
significant at the bite scale, because of differences in bite mass between plant
species. Greater distance between patches resulted in longer patch residence time
and faster speed of travel but lower overall intake rate, consistent with
maximization of intake rate. Larger patches resulted in greater residence time
and higher intake rate.
CONCLUSION: We conclude that patch size and spacing affect components of intake
rate and, to a lesser extent, the selectivity of livestock at lower hierarchies
of the grazing process, particularly by enticing livestock to make more even use
of the available species as patches are spaced further apart. Thus, modifications
in the spatial pattern of plant patches along with reductions in the temporal and
spatial allocation of grazing may offer opportunities to improve uniformity of
grazing by livestock and help sustain biodiversity and stability of plant
communities.
DOI: 10.1186/1472-6785-9-9
PMCID: PMC2684096
1456. Environ Entomol. 2009 Apr;38(2):333-44.
Population abundance of Frankliniella occidentalis (Thysanoptera: Thripidae) and

natural enemies on plant hosts in central Chile.
Ripa R(1), Funderburk J, Rodriguez F, Espinoza F, Mound L.
Author information:
(1)Instituto Nacionales de Investigaciones Agropecurios, Chorillos 86, La Cruz,
Chile.
Populations of the invasive Frankliniella occidentalis (Pergande) are serious

pests of agricultural crops in the Aconcagua Valley of central Chile. An
extensive survey was conducted of 55 plant species in 24 families to identify
plant hosts of F. occidentalis and to determine its relative abundance on each
host during each season. A more intensive study was conducted on selected plant
species serving as reproductive hosts to determine the population dynamics of F.
occidentalis and to evaluate the potential importance of Orius species and other
natural enemies for controlling F. occidentalis. Adults of F. occidentalis were
active during each season of the year inhabiting the flowers of 91% of the
sampled plant species in 22 families, and 86% of these plant species in 19
families served as reproductive hosts. The number of host plant species used was
greatest in the spring and least in the winter. All of the hosts except Medicago
sativa L. were used only when flowering. Populations of F. occidentalis were
significantly aggregated in M. sativa in the terminal buds over the leaves when
the host was not flowering, and in the flowers, followed by the terminal buds,
followed by the leaves when the host was flowering. Larvae were 1.3-2.3 times
more abundant on dates when M. sativa was flowering. There were no identifiable
patterns in plant hosts based on endemicity or plant family. Most of the plant
species used by F. occidentalis were inferior quality hosts where populations
either declined or were stable. Populations of F. occidentalis on low-quality
hosts generally escaped predation by Orius species and competition by other
species of thrips. Only 25% of the food hosts and 28% of the reproductive hosts
for F. occidentalis in the extensive survey, respectively, were host plants for
Orius. Parasitoids and other predators were not found to be important in
suppressing thrips on any of the plant hosts. Populations of F. occidentalis
increased on only a few hosts, including M. sativa and Sisymbrium officinale L.
Scop. These apparently are major sources of F. occidentalis adults invading
crops. We conclude that F. occidentalis is established in central Chile and that
it has replaced and possibly displaced the native Frankliniella australis
(Morgan) as the most common thrips species.
1457. New Phytol. 2009 Jun;182(4):829-37. doi: 10.1111/j.1469-8137.2009.02839.x.
Glomus intraradices induces changes in root system architecture of rice

independently of common symbiosis signaling.
Gutjahr C(1), Casieri L, Paszkowski U.
Author information:
(1)Department of Plant Molecular Biology, University of Lausanne, Biophore
Building, 1015 Lausanne, Switzerland.
Arbuscular mycorrhizal fungi colonize the roots of most monocotyledons and

dicotyledons despite their different root architecture and cell patterning. Among
the cereal hosts of arbuscular mycorrhizal fungi, Oryza sativa (rice) possesses a
peculiar root system composed of three different types of roots: crown roots;
large lateral roots; and fine lateral roots. Characteristic is the constitutive
formation of aerenchyma in crown roots and large lateral roots and the absence of
cortex from fine lateral roots. Here, we assessed the distribution of
colonization by Glomus intraradices within this root system and determined its
effect on root system architecture. Large lateral roots are preferentially
colonized, and fine lateral roots are immune to arbuscular mycorrhizal
colonization. Fungal preference for large lateral roots also occurred in sym
mutants that block colonization of the root beyond rhizodermal penetration.
Initiation of large lateral roots is significantly induced by G. intraradices
colonization and does not require a functional common symbiosis signaling pathway
from which some components are known to be needed for symbiosis-mediated lateral
root induction in Medicago truncatula. Our results suggest variation of symbiotic
properties among the different rice root-types and induction of the preferred
tissue by arbuscular mycorrhizal fungi. Furthermore, signaling for arbuscular
mycorrhizal-elicited alterations of the root system differs between rice and M.
truncatula.
DOI: 10.1111/j.1469-8137.2009.02839.x
1458. Plant J. 2009 Jul;59(1):136-49. doi: 10.1111/j.1365-313X.2009.03885.x. Epub

2009
Apr 2.
The LAP1 MYB transcription factor orchestrates anthocyanidin biosynthesis and

glycosylation in Medicago.
Peel GJ(1), Pang Y, Modolo LV, Dixon RA.
Author information:
MYB transcription factors help to control anthocyanin biosynthesis in plants, and

ectopic expression of the Arabidopsis Production of Anthocyanin Pigment 1 (PAP1)
transcription factor activates the anthocyanin pathway in tobacco, suggesting the
general utility of such factors for metabolic engineering of anthocyanins and
anthocyanin-derived compounds such as proanthocyanidins (condensed tannins).
However, PAP1 does not activate anthocyanin biosynthesis in the model legume
Medicago truncatula or in alfalfa (Medicago sativa). A related Legume Anthocyanin
Production 1 (LAP1) gene was identified from the genome of M. truncatula. When
constitutively expressed in transgenic alfalfa, M. truncatula or white clover,
LAP1 induced massive accumulation of anthocyanin pigments comprising multiple
glycosidic conjugates of cyanidin. Oligomeric/polymeric compounds with some
diagnostic characteristics of proanthocyanidins also accumulated in
LAP1-expressing plants, but these compounds were not composed of (epi)catechin
units. Over 260 and 70 genes were up-regulated in leaves of alfalfa or M.
truncatula, respectively, in response to constitutive expression of LAP1, many of
which are involved in anthocyanin biosynthesis. In particular, the
glucosyltransferase UGT78G1, previously identified as showing preference for
isoflavonoid substrates in vitro, was strongly up-regulated by LAP1, and appears
to function as an anthocyanin glycosyltransferase in vivo. Over-expression of
UGT78G1 in transgenic alfalfa resulted in increased anthocyanin accumulation when
plants were exposed to abiotic stress.
DOI: 10.1111/j.1365-313X.2009.03885.x
1459. PLoS One. 2009;4(4):e5142. doi: 10.1371/journal.pone.0005142. Epub 2009 Apr

9.
The use of flagella and motility for plant colonization and fitness by different
strains of the foodborne pathogen Listeria monocytogenes.
Gorski L(1), Duhé JM, Flaherty D.
Author information:
(1)US Department of Agriculture, Produce Safety and Microbiology Research Unit,
Agricultural Research Service, Albany, California, United States of America.
lisa.gorski@ars.usda.gov
The role of flagella and motility in the attachment of the foodborne pathogen
Listeria monocytogenes to various surfaces is mixed with some systems requiring
flagella for an interaction and others needing only motility for cells to get to
the surface. In nature this bacterium is a saprophyte and contaminated produce is
an avenue for infection. Previous studies have documented the ability of this
organism to attach to and colonize plant tissue. Motility mutants were generated
in three wild type strains of L. monocytogenes by deleting either flaA, the gene
encoding flagellin, or motAB, genes encoding part of the flagellar motor, and
tested for both the ability to colonize sprouts and for the fitness of that
colonization. The motAB mutants were not affected in the colonization of alfalfa,
radish, and broccoli sprouts; however, some of the flaA mutants showed reduced
colonization ability. The best colonizing wild type strain was reduced in
colonization on all three sprout types as a result of a flaA deletion. A mutant
in another background was only affected on alfalfa. The third, a poor alfalfa
colonizer was not affected in colonization ability by any of the deletions.
Fitness of colonization was measured in experiments of competition between
mixtures of mutant and parent strains on sprouts. Here the flaA and motAB mutants
of the three strain backgrounds were impaired in fitness of colonization of
alfalfa and radish sprouts, and one strain background showed reduced fitness of
both mutant types on broccoli sprouts. Together these data indicate a role for
flagella for some strains to physically colonize some plants, while the fitness
of that colonization is positively affected by motility in almost all cases.
PMCID: PMC2664462
1460. Cancer Prev Res (Phila). 2009 Apr;2(4):353-60. doi:

10.1158/1940-6207.CAPR-08-0192.
Dietary sulforaphane-rich broccoli sprouts reduce colonization and attenuate

gastritis in Helicobacter pylori-infected mice and humans.
Yanaka A(1), Fahey JW, Fukumoto A, Nakayama M, Inoue S, Zhang S, Tauchi M, Suzuki
H, Hyodo I, Yamamoto M.
Author information:
(1)Division of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Tokyo
University of Science, Chiba-Ken, Tokyo, Japan. ayanaka@rs.noda.tus.ac.jp
The isothiocyanate sulforaphane [SF; 1-isothiocyanato-4(R)-methylsulfinylbutane]

is abundant in broccoli sprouts in the form of its glucosinolate precursor
(glucoraphanin). SF is powerfully bactericidal against Helicobacter pylori
infections, which are strongly associated with the worldwide pandemic of gastric
cancer. Oral treatment with SF-rich broccoli sprouts of C57BL/6 female mice
infected with H. pylori Sydney strain 1 and maintained on a high-salt (7.5% NaCl)
diet reduced gastric bacterial colonization, attenuated mucosal expression of
tumor necrosis factor-alpha and interleukin-1beta, mitigated corpus inflammation,
and prevented expression of high salt-induced gastric corpus atrophy. This
therapeutic effect was not observed in mice in which the nrf2 gene was deleted,
strongly implicating the important role of Nrf2-dependent antioxidant and
anti-inflammatory proteins in SF-dependent protection. Forty-eight H.
pylori-infected patients were randomly assigned to feeding of broccoli sprouts
(70 g/d; containing 420 micromol of SF precursor) for 8 weeks or to consumption
of an equal weight of alfalfa sprouts (not containing SF) as placebo.
Intervention with broccoli sprouts, but not with placebo, decreased the levels of
urease measured by the urea breath test and H. pylori stool antigen (both
biomarkers of H. pylori colonization) and serum pepsinogens I and II (biomarkers
of gastric inflammation). Values recovered to their original levels 2 months
after treatment was discontinued. Daily intake of sulforaphane-rich broccoli
sprouts for 2 months reduces H. pylori colonization in mice and improves the
sequelae of infection in infected mice and in humans. This treatment seems to
enhance chemoprotection of the gastric mucosa against H. pylori-induced oxidative
stress.
DOI: 10.1158/1940-6207.CAPR-08-0192
1461. J Food Prot. 2009 Mar;72(3):631-6.
Combination treatments for killing Escherichia coli O157:H7 on alfalfa, radish,

broccoli, and mung bean seeds.
Bari ML(1), Nei D, Enomoto K, Todoriki S, Kawamoto S.
Author information:
(1)National Food Research Institute, Kannondai-2-1-12, Tsukuba 305-8642, Japan.
latiful@affrc.go.jp
In this study, the effectiveness of prolonged dry-heat treatment (50 degrees C)

alone or in combination with chemical treatments (1% oxalic acid, 0.03% phytic
acid, 50% ethanol, electrolyzed acidic water, and electrolyzed alkaline water) in
eliminating Escherichia coli O157:H7 on laboratory-inoculated alfalfa, radish,
broccoli, and mung bean seeds was compared with that of dry-heat treatment in
combination with irradiation treatment. Dry-heat treatment for 17 or 24 h alone
could reduce E. coli O157:H7 numbers to below detectable levels in radish,
broccoli, and alfalfa seeds, but was unable to reduce the pathogen numbers to
below the detectable level in mung bean seeds. In addition, dry-heat treatment
for 17 h plus sanitizer treatments were effective in greatly reducing pathogen
populations on radish, broccoli, and alfalfa seeds, without compromising the
quality of the sprouts, but these treatments did not eliminate the pathogen from
radish and alfalfa seeds. Seventeen hours of dry heat followed by a 1.0-kGy dose
of irradiation completely eliminated E. coli O157:H7 from radish and mung bean
seeds, whereas only a minimum radiation dose of 0.25 kGy was required to
completely eliminate the pathogen from broccoli and alfalfa seeds. Dry heat in
combination with radiation doses of up to 1.0 kGy did not negatively impact the
seed germination rate or length of alfalfa, broccoli, and radish seeds or the
length of alfalfa, broccoli, and radish sprouts, but did decrease the length of
mung bean sprouts.
1462. Int J Food Microbiol. 2009 May 31;131(2-3):218-23. doi:

10.1016/j.ijfoodmicro.2009.02.028. Epub 2009 Mar 12.
Factors affecting the efficacy of pressure inactivation of Escherichia coli

O157:H7 on alfalfa seeds and seed viability.
Neetoo H(1), Ye M, Chen H.
Author information:
19716-2150, USA.
The application of high hydrostatic pressure technology as a seed decontamination

technology was evaluated. Alfalfa seeds inoculated with approximately 10(5) CFU/g
of Escherichia coli O157:H7 were subjected to oscillatory pressure treatments at
600 MPa and 20 degrees C for up to five cycles with a holding time of 2
min/cycle. However, oscillatory pressurization was not able to eliminate E. coli
O157:H7. The application of pressure treatment at 600 MPa for 2 min at 20 degrees
C in the presence of chemicals such as calcium hypochlorite, calcium hydroxide,
lactic acid or sodium acid sulfate was subsequently investigated and it was
demonstrated that this "multiple hurdle" approach was unable to decontaminate
alfalfa seeds. Soaking seeds prior to pressure treatment was found to play a
critical role on enhancing the pressure inactivation of E. coli O157:H7; seeds
soaked in water for 60 min followed by treatment at 600 MPa for 2 min at 20
degrees C were decontaminated and had a germination rate of 91% which was 4%
lower than that of the untreated seeds (not statistically significant, P>0.05).
It was further demonstrated that a process involving soaking seeds in water for >
or = 10 min followed by treatment at 600 MPa for 15 min at 20 degrees C was
equally effective with respect to E. coli O157:H7 elimination and viability
retention of the seeds.
1463. Animal. 2009 Apr;3(4):548-56. doi: 10.1017/S1751731108003765.
Recovery of intravenously infused chromium EDTA and lithium sulphate in the urine
of cattle and their use as markers to measure urine volume.
Bowen MK(1), Poppi DP, McLennan SR.
Author information:
(1)1Schools of Animal Studies and Veterinary Science, University of Queensland,
St Lucia, QLD 4072, Australia.
A series of metabolism experiments investigated the recovery of continuous-,

intravenously infused chromium complexed with ethylenediamine tetra-acetic acid
(CrEDTA) and lithium sulphate in the urine of cattle with a view to using the
markers to estimate urine and metabolite output in grazing cattle. The recovery
of Cr in urine from these infusions was similar (90%) in metabolism trials when
cattle consumed three very contrasting diets: high-grain formulated pellet,
lucerne hay (Medicago sativa) or low-quality native grass hay (predominantly
Heteropogon contortus). By contrast, Li recovery in urine averaged 46.3 ± 0.40%
and 72.6 ± 0.43% for native pasture and lucerne hays, respectively, but was not
constant across days. There was negligible transfer of Cr from CrEDTA in blood
serum to the rumen or faeces, whereas appreciable quantities of infused Li were
found in both. The ratio of urine volume estimated by spot samples and marker
dilution of Cr, to urine volume measured gravimetrically, was 1.05. In grazing
studies using rumen-fistulated (RF) steers grazing seven different tropical and
temperate grass and legume pastures, the ratio of concentrations of purine
derivatives (PD) to Cr in spot samples of urine was shown to vary diurnally in
the range of 49% to 157% of the average 24 h value. This finding indicated the
need for regular sampling of urine to achieve an accurate average value for the
PD : Cr ratio in urine for use in estimating urinary PD excretion and hence
microbial protein production in the rumen. It was concluded that continuous,
intravenous infusion of CrEDTA resulted in a constant recovery of Cr in the urine
of cattle across diets and, provided an intensive sampling regime was followed to
account for diurnal variation, it would be suitable as a marker to estimate urine
volume and urinary output of PD in grazing cattle.
DOI: 10.1017/S1751731108003765
PMID: 22444379
1464. Int J Mol Sci. 2008 May;9(5):768-88. doi: 10.3390/ijms9050768. Epub 2008 May
20.
Perennial forages as second generation bioenergy crops.
Sanderson MA(1), Adler PR.
Author information:
(1)USDA-ARS, University Park, PA 16802-3702, USA. matt.sanderson@ars.usda.gov
The lignocellulose in forage crops represents a second generation of biomass

feedstock for conversion into energy-related end products. Some of the most
extensively studied species for cellulosic feedstock production include forages
such as switchgrass (Panicum virgatum L.), reed canarygrass (Phalaris arundinacea
L.), and alfalfa (Medicago sativa L.). An advantage of using forages as bioenergy
crops is that farmers are familiar with their management and already have the
capacity to grow, harvest, store, and transport them. Forage crops offer
additional flexibility in management because they can be used for biomass or
forage and the land can be returned to other uses or put into crop rotation.
Estimates indicate about 22.3 million ha of cropland, idle cropland, and cropland
pasture will be needed for biomass production in 2030. Converting these lands to
large scale cellulosic energy farming could push the traditional forage-livestock
industry to ever more marginal lands. Furthermore, encouraging bioenergy
production from marginal lands could directly compete with forage-livestock
production.
DOI: 10.3390/ijms9050768
PMCID: PMC2635706
PMID: 19325783
1465. Bull Entomol Res. 2010 Feb;100(1):67-73. doi: 10.1017/S0007485309006774. Epub

2009 Mar 27.
A comparison of alternative plant mixes for conservation bio-control by native

beneficial arthropods in vegetable cropping systems in Queensland Australia.
Qureshi SA(1), Midmore DJ, Syeda SS, Reid DJ.

Author information:
(1)Centre for Plant and Water Science, Central Queensland University, North
Rockhampton, Queensland 4702, Australia. s.qureshi@cqu.edu.au
Cucurbit crops host a range of serious sap-sucking insect pests, including

silverleaf whitefly (SLW) and aphids, which potentially represent considerable
risk to the Australian horticulture industry. These pests are extremely
polyphagous with a wide host range. Chemical control is made difficult due to
resistance and pollution, and other side-effects are associated with insecticide
use. Consequently, there is much interest in maximising the role of biological
control in the management of these sap-sucking insect pests. This study aimed to
evaluate companion cropping alongside cucurbit crops in a tropical setting as a
means to increase the populations of beneficial insects and spiders so as to
control the major sap-sucking insect pests. The population of beneficial and
harmful insects, with a focus on SLW and aphids, and other invertebrates were
sampled weekly on four different crops which could be used for habitat
manipulation: Goodbug Mix (GBM; a proprietary seed mixture including self-sowing
annual and perennial herbaceous flower species); lablab (Lablab purpureus L.
Sweet); lucerne (Medicago sativa L.); and niger (Guizotia abyssinica (L.f.)
Cass.). Lablab hosted the highest numbers of beneficial insects (larvae and
adults of lacewing (Mallada signata (Schneider)), ladybird beetles (Coccinella
transversalis Fabricius) and spiders) while GBM hosted the highest numbers of
European bees (Apis mellifera Linnaeus) and spiders. Lucerne and niger showed
little promise in hosting beneficial insects, but lucerne hosted significantly
more spiders (double the numbers) than niger. Lucerne hosted sig-nificantly more
of the harmful insect species of aphids (Aphis gossypii (Glover)) and Myzus
persicae (Sulzer)) and heliothis (Heliothis armigera Hübner). Niger hosted
significantly more vegetable weevils (Listroderes difficillis (Germar)) than the
other three species. Therefore, lablab and GBM appear to be viable options to
grow within cucurbits or as field boundary crops to attract and increase
beneficial insects and spiders for the control of sap-sucking insect pests. Use
of these bio-control strategies affords the opportunity to minimise pesticide
usage and the risks associated with pollution.
DOI: 10.1017/S0007485309006774
1466. Plant Physiol Biochem. 2009 Jul;47(7):570-7. doi:

10.1016/j.plaphy.2009.02.009.
Epub 2009 Feb 28.
Analysis of antioxidant enzyme activity during germination of alfalfa under salt

and drought stresses.
Wang WB(1), Kim YH, Lee HS, Kim KY, Deng XP, Kwak SS.
Author information:
(1)State Key Laboratory of Soil Erosion and Dryland Farming on Loess Plateau,
Northwest A&F University, Xinong Road No. 26, Yangling, Shaanxi 712100, P R
China.
To understand the adaptability of alfalfa (Medicago sativa L.) to environmental

stresses, we analyzed the activity of several antioxidant enzymes, including
superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and
catalase (CAT), in alfalfa shoots and roots subjected to salt and drought
stresses during germination. The germination rate of six alfalfa cultivars was
comparatively studied under 200 mM NaCl or 35% PEG treatment. Alfalfa Xinmu No. 1
and Northstar varieties were selected as stress-tolerant and -sensitive
cultivars, respectively, and were used for further characterization. After NaCl
or PEG treatment, Xinmu No. 1 showed enhanced seedling growth, compared with
Northstar. Xinmu No. 1 also exhibited low levels of hydrogen peroxide (H(2)O(2))
production and lipid peroxidation, compared with Northstar. In addition, Xinmu
No. 1 showed higher enzymatic activity of SOD, APX, CAT, and POD in its shoots
and roots than Northstar. These results seem to indicate that Xinmu No. 1
cultivar's tolerance to salt or drought stresses during germination is associated
with enhanced activity of antioxidant enzymes. This study highlights the
importance of antioxidant enzymes in the establishment of alfalfa seedlings under
drought and salinity conditions typical of desertification.
DOI: 10.1016/j.plaphy.2009.02.009
Technical note: effects of forage protein-binding polyphenols on chemistry of

dairy excreta.
Powell JM(1), Broderick GA, Grabber JH, Hymes-Fecht UC.
Author information:
(1)USDA-Agricultural Research Service, US Dairy Forage Research Center, 1925
Linden Drive West, Madison, WI 53706, USA. mark.powell@ars.usda.gov
Forage chemistry can affect intake, digestion, milk production, and manure
excretion. Although information is available on the effects of forage
protein-binding polyphenols on small ruminant production and manure excretion,
little information is available for dairy cattle. The objective of this study was
to compare fecal and urinary N excretion of diets formulated with alfalfa
(Medicago sativa L.) silage versus condensed tannin-containing birdsfoot trefoil
(Lotus corniculatus) or o-quinone-containing red clover (Trifolium pratense L.)
silages. Significantly higher concentrations of N were excreted in urine by
lactating Holstein dairy cows fed red clover and low-tannin birdsfoot trefoil
(8.2 g/L) than by cows fed high-tannin birdsfoot trefoil or alfalfa (7.1 g/L).
Fecal N concentrations were similar (33.6 g/kg) among all diets. Dairy cows fed
red clover had lower rates of urinary N excretion (5.0 g/h) compared with other
forages (6.6 g/h). Fecal N excretion rates were lowest for red clover (4.1 g/h),
intermediate for alfalfa (5.8 g/h), and greatest for cows fed high- and
low-tannin birdsfoot trefoil (6.4 g/h). The ratio of fecal N to urinary N was
highest for high-tannin trefoil, lowest for alfalfa and red clover, and higher in
excreta collected in morning than evening. Concentrations of neutral detergent
fiber (NDF) in feces, of N in NDF (NDIN) and acid detergent fiber (ADIN), and
relative amounts of NDIN and ADIN excreted in feces were significantly higher
from cows fed high-tannin birdsfoot trefoil than the other silage types. Study
results imply that collection of excreta for environmental studies needs to
consider forage polyphenol and diurnal effects on chemistry of dairy excreta.
DOI: 10.3168/jds.2008-1738
Alfalfa pellet-induced subacute ruminal acidosis in dairy cows increases

bacterial endotoxin in the rumen without causing inflammation.
Khafipour E(1), Krause DO, Plaizier JC.

Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, Manitoba,
Canada R3T 2N2.
A study was conducted to determine if subacute ruminal acidosis (SARA) induced by

feeding alfalfa pellets results in increases in free bacterial lipopolysaccharide
(LPS) in rumen fluid and peripheral blood, and acute phase proteins in plasma,
and to determine the effect of alfalfa pellet-induced SARA on feed intake, rumen
fermentation characteristics, milk production and composition, and blood
metabolites. Eight lactating Holstein cows, 4 of which were ruminally cannulated,
were used in a 6-wk experiment and were fed once daily at 0900 h. During wk 1,
cows received a diet containing 50% of DM as concentrate and 50% of DM chopped
alfalfa hay. Between wk 2 and wk 6, alfalfa hay was gradually replaced with
alfalfa pellets at the rate of 8% per week to reduce rumen pH. Rumen pH was
monitored continuously in the ruminally cannulated cows using indwelling pH
probes. Rumen fluid and peripheral blood were sampled 15 min before feed delivery
and at 6 h after feed delivery. Based on adopted threshold of SARA of at least
180 min/d below pH 5.6, SARA was induced from wk 3 onwards. Replacing 40% of
alfalfa hay with alfalfa pellets quadratically increased the DMI from 18.1 kg/d
in wk 1 to 23.4 kg/d in wk 6. This replacement linearly decreased milk yield
(32.7 vs. 35.9 kg/d) and milk fat percentage and yield (2.32 vs. 3.22%, and 0.77
vs. 1.14 kg/d, respectively), but increased milk protein percentage and yield
(3.80 vs. 3.04%, and 1.23 vs. 1.07 kg/d, respectively). This gradual replacement
also linearly increased the daily averages of total volatile fatty acids (90 to
121.9 mM), acetate (53.9 to 66.8 mM), propionate (21.5 to 39.6 mM), and
osmolality (277.7 to 293.8 mmol/kg) in the rumen and decreased the acetate to
propionate ratio from 2.62 to 1.73. Replacing alfalfa hay with alfalfa pellets
linearly increased blood lactate from 1.00 mM in wk 1 to a peak of 3.46 mM in wk
5. Induction of SARA in this study increased free rumen LPS concentration from
42,122 endotoxin unit (EU)/mL in wk 1 to 145,593 EU/mL in wk 6. However, this
increase was not accompanied by an increase in LPS (<0.05 EU/mL) and in acute
phase proteins serum amyloid-A, haptoglobin, and LPS-binding protein in
peripheral circulation. Results suggest that SARA induced by alfalfa pellets
increased LPS in the rumen without causing translocation of LPS and an immune
response.
DOI: 10.3168/jds.2008-1656
Near-infrared reflectance spectroscopy prediction of neutral detergent-soluble

carbohydrates in timothy and alfalfa.
Nie Z(1), Tremblay GF, Bélanger G, Berthiaume R, Castonguay Y, Bertrand A,

Michaud R, Allard G, Han J.
Author information:
China Agricultural University, Beijing, P R China, 100094.
Carbohydrates in forage crops can be divided into neutral detergent-insoluble

fiber and neutral detergent-soluble carbohydrates (NDSC); the latter includes
organic acids (OA), total ethanol:water-soluble carbohydrates (TESC), starch, and
neutral detergent-soluble fiber (NDSF). The accurate and efficient estimation of
NDSC in forage crops is essential for improving the performance of dairy cattle.
In the present study, visible and near-infrared reflectance spectroscopy (NIRS)
were applied to evaluate the feasibility of predicting OA, TESC, starch, NDSF,
NDSC, and all related constituents used to calculate these 5 carbohydrate
fractions in timothy and alfalfa. Forage samples (n = 1,008) of timothy and
alfalfa were taken at the first and second harvests at 2 sites in 2007; samples
were dried, ground, and then scanned (400 to 2,500 nm) using an NIRSystems 6500
monochromator. A calibration (n = 60) and a validation (n = 15) set of samples
were selected for each species and then chemically analyzed. Concentrations of
TESC and NDSC in timothy, as well as starch in alfalfa, were successfully
predicted, but many other carbohydrate fractions were not predicted accurately
when calibrations were performed using single-species sample sets. Both sets of
samples were combined to form new calibration (n = 120) and validation (n = 30)
sets of alfalfa and timothy samples. Calibration and validation statistics for
the combined sets of alfalfa and timothy samples indicated that TESC, starch, and
NDSC were predicted successfully, with coefficients of determination of
prediction of 0.92, 0.89, and 0.93, and a ratio of prediction to deviation (RPD)
of 3.3, 3.1, and 3.6, respectively. The NDSF prediction was classified as
moderately successful The NIRS prediction of OA was unsuccessful All related
constituents were predicted successfully by NIRS except ethanol-insoluble
residual OM, with Our results confirm the feasibility of using NIRS to predict
NDSC, its fractions, and other related constituents, except for OA and
ethanol-insoluble residual OM, in timothy and alfalfa forage samples.
DOI: 10.3168/jds.2008-1599
1470. Public Health Nutr. 2009 Mar;12(3):440.
Nourishing inmates in Malagasy prisons.
Andrianasolo F.
Comment on
Public Health Nutr. 2009 Jan;12(1):142.
Public Health Nutr. 2008 Jun;11(6):651.
1471. Public Health Nutr. 2009 Apr;12(4):587. doi: 10.1017/S1368980009005412.
Leaf concentrate. Undernutrition: testimony from a clinic in Senegal.
Coly A.
DOI: 10.1017/S1368980009005412
1472. J Agric Food Chem. 2009 Apr 8;57(7):2849-55. doi: 10.1021/jf803632t.
Design, synthesis, and biological activities of novel 2-cyanoacrylates containing

oxazole, oxadiazole, or quinoline moieties.
Zhao Q(1), Liu S, Li Y, Wang Q.
Author information:
(1)State Key Laboratory of Elemento-Organic Chemistry, Research Institute of
Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People's Republic
of China.
A series of novel 2-cyanoacrylates containing an oxazole, oxadiazole, or

quinoline moiety were designed and synthesized, and their structures were
characterized by (1)H NMR and elemental analysis (or high-resolution mass
spectrometry). Their herbicidal activities against four weeds were evaluated, and
the result indicated that some of the title compounds showed excellent herbicidal
activities against rape and amaranth pigweed in postemergence treatment at a dose
of 375 g/ha. Furthermore, most of these cyanoacrylates exhibited interesting
plant growth regulatory activities.
DOI: 10.1021/jf803632t
1473. Oecologia. 2009 Jun;160(3):551-61. doi: 10.1007/s00442-009-1310-4. Epub 2009

Mar
7.
Host range evolution is not driven by the optimization of larval performance: the
case of Lycaeides melissa (Lepidoptera: Lycaenidae) and the colonization of
alfalfa.
Forister ML(1), Nice CC, Fordyce JA, Gompert Z.
Author information:
(1)Department of Biology/MS 314, University of Nevada, Reno, NV 89557, USA.
mforister@unr.edu
Herbivorous insects that have recently incorporated novel hosts into their diet
provide unique opportunities for understanding factors that promote or constrain
the evolution of niche breadth. Lycaeides melissa has colonized both cultivated
and feral alfalfa (Medicago sativa) throughout much of North America within the
past 200 years. We investigated the quality of the novel host as a resource for
juvenile development, and asked if the novel host is a preferred host for
oviposition relative to a native host (Astragalus canadensis). Larval-performance
and oviposition-preference were examined using L. melissa individuals from a
population associated with both M. sativa and A. canadensis, and
oviposition-preference was also examined in another population associated
exclusively with M. sativa. In addition, we investigated the effects of M. sativa
and A. canadensis flowers on both preference and performance. Only one of the
hosts, M. sativa, has flowers that are accessible to nectaring butterflies, and
we hypothesized that the presence of flowers could affect female behavior. We
find that the novel host is a relatively poor larval resource: adults that were
reared as larvae on M. sativa were roughly one-third the size of adults that were
reared on the native host, A. canadensis. The native host, Astragalus canadensis,
is the preferred host in choice experiments involving only foliage. However, when
flowers were included in preference assays, the native and novel hosts received
similar numbers of eggs. Thus, the presence of flowers on hosts in the field
might influence the utilization of a novel and inferior larval resource. These
results are consistent with a model in which host shifts are driven by adult
behavior that does not directly optimize larval performance.
DOI: 10.1007/s00442-009-1310-4
1474. Int J Phytoremediation. 2008 Nov-Dec;10(6):486-502. doi:

10.1080/15226510802114920.
Phytoremediation of oil-sludge-contaminated soil.
Muratova AY(1), Dmitrieva TV, Panchenko LV, Turkovskaya OV.
Author information:
(1)Institute ofBiochemistry and Physiology of Plants and Microorganisms, Saratov,
Russia. amuratova@yahoo.com
The aim of this research was to select plant species that could be effective in
the phytoremediation ofa former oil-sludge pit. Seven crop plants (Triticum
aestivum L., Secale cereale L., Avena sativa L., Hordeum vulgare, Sorghum bicolor
L Moench, Panicum miliaceum L, and Zea mays L.),five wild grasses (Lolium perenne
L., Bromopsis inermis, Agropyron cristatum L., Agropyrum tenerum L., and Festuca
pratensis Huds.), and three legumes (Medicago sativa L., Trifolium pratense L.,
and Onobrychis antasiatica Khin.) were screened for phytotoxicity, including the
assessment of germination, shoot biomass, and root biomass, in a pot experiment.
The estimation of oil-sludge degradation in the root zone of the tested plants
showed that rye accelerated cleanup most effectively, degrading all of the main
contaminant fractions in the oil sludge by a total of 52%. Although alfalfa had a
lower phytoremediation potential than did rye, it maintained large numbers of
soil microorganisms, including polycyclic aromatic hydrocarbon degraders, in its
rhizosphere. Rye and alfalfa were chosen for a large-scale study to remediate an
oil-sludge pit on the grounds of a petroleum refinery. Remediation monitoring
confirmed the effectiveness of rye: the oil-sludge content decreased consistently
for 3 years and remained low in comparison with the results from other plant
species.
DOI: 10.1080/15226510802114920
[Research on predicting the nutrition value of pelletized alfalfa by near

infrared reflectance spectroscopy].
Hua R(1), Han JG, Qi X, Nie ZD, Li B.
Author information:
(1)Institute of Grassland Science, China Agricultural University, Beijing Major
Laboratory, Beijing, China. huarong9999@yahoo.com.cn
The present research aimed to predict the qualities of pelletized alfalfa by near
infrared reflectance spectroscopy. Sixty pelletized alfalfa samples were
collected, including 22 whole plant alfalfa samples, 19 stem samples and 19 leaf
samples. They were divided into a calibration sample set (45 samples) and a
validation sample set (15 samples). The Fourier transform-near infrared
reflectance spectroscopy (FT-NIRS) and the partial least square (PLS) were used
to calibrate models of the pelletized alfalfa nutrition value, involving crude
protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF)
contents. All models had great calibration performances. The correlation
coefficients of cross-validation (R(CV)) were between 0.96410 and 0.96887, and
the root mean square errors of cross-validation (RMSECV) were between 0.80% and
2.59%. Fifteen validation samples were used to predict the performances of these
models, all the correlation coefficients of NIRS value and chemical value (r)
were between 0.9669 and 0.9743, and the root mean square errors of prediction
(RMSEP) were between 0.85% and 2.07%. The RPD values of cross-validation and
prediction were all above 3. The results showed that pelletized alfalfa's CP,
NDF, ADF contents were exactly predicted by near infrared reflectance
spectroscopy.
[Evaluation of fermentation character of alfalfa silage through near infrared

reflectance spectroscopy (NIRS)].
Chen PF(1), Rong YP, Han JG.
Author information:
Beijing. d.p.f-001@163.com
It is very important to evaluate the fermentation character of alfalfa silage

using near infrared reflectance spectroscopy technology (NIRS) for animal
production, including the content of NH3-N, lactic acid, acetic acid and butyric
acid in silage. In order to evaluate the feasibility of using NIRS to analyze the
formation character of alfalfa silage, the near infrared reflectance spectroscopy
models were built for NH3-N, lactic acid, zcetic acid and butyric acid in this
experiment. Partial least square regression (PLS), Fourier transform technology
and sample preparation with liquid nitrogen technology were used to optimize the
model. The analyzed samples were obtained with different cultivars, maturity,
cuttings and ensiling method. The determination of cross validation was between
0.6024 and 0.9497. The standard errors of cross validation were between 5.59 x
10(-1) and 3.78 g x kg(-1) fresh weight. The validation samples were used to test
the performance of the models. The correlation coefficients between the chemical
value and the NIRS value were between 0.8826 and 0.9853, and the root mean square
errors of prediction were between 5.71 x 10(-1) and 3.15 g x kg(-1) fresh weight.
The results showed the NIRS could evaluate the fermentation of the fresh forage.
[Soil hydraulic properties and their influences on soil water content under
different land uses in Liudaogou watershed of Loess Plateau].
Liu CL(1), Shao MA.
Author information:
(1)Key Lab. of Soil Erosion and Dry Land Farming on the Loess Plateau, Inst of
Soil and Water Conservation, Chinese Academy of Sciences and Northwest Science
and Technology Univ. of Agri. and Forestry, Yangling 712100, Shaanxi, China
chunliliu@sdu.edu.cn
By the method of cross correlation coefficient in geo-statistics, this paper
studied the soil hydraulic properties and their influences on soil water
distribution under four land uses, i.e., farmland, waste-grassland, forestland,
and alfalfa land in Liudaogou watershed of Loess Plateau. The results showed that
farmland soil and waste-grassland soil had the similar physical characteristics,
as did forestland soil and alfalfa land soil. For any given soil suction,
farmland soil had the highest water content, while forestland soil had the lowest
one. The opposite was observed for the saturated hydraulic conductivity of soil
water. In forestland and alfalfa land, soil water content increased with
increasing depth during water consumption period, but decreased with soil depth
at any other time. The influence range of saturated hydraulic conductivity on
soil water content depended on soil water condition. During the compensation
period of soil water, the influenced lag distance of saturated hydraulic
conductivity to water content was 0-40 cm, and that of water content to saturated
hydraulic conductivity was 0-50 cm; while during the stable period of soil water,
the influenced lag distances of saturated hydraulic conductivity and water
content were both 0-60 cm. There were positive correlations between the two
properties under all land uses during these two periods. During water consumption
period, saturated hydraulic conductivity was positively correlated with soil
water content in farmland and waste-grassland. The influence range of saturated
hydraulic conductivity to water content was about 0-80 cm, while that of water
content to saturated hydraulic conductivity was 0-60 cm. However, negative
correlation was observed in forestland and alfalfa land, and the influence ranges
were also about 0-60 cm.
1478. Environ Manage. 2009 Jun;43(6):1061-70. doi: 10.1007/s00267-009-9284-x. Epub

2009
Feb 26.
Changes in soil aggregate, carbon, and nitrogen storages following the conversion
of cropland to alfalfa forage land in the marginal oasis of northwest China.
Su YZ(1), Liu WJ, Yang R, Chang XX.
Author information:
(1)Laboratory of Watershed Hydrology and Applied Ecology, Cold and Arid Regions
Environmental and Engineering Research Institute, Chinese Academy of Sciences,
Lanzhou, China. suyzh@lzb.ac.cn
Maintenance of soil organic carbon (SOC) is important for sustainable use of soil
resources due to the multiple effects of SOC on soil nutrient status and soil
structural stability. The objective of this study was to identify the changes in
soil aggregate distribution and stability, SOC, and nitrogen (N) concentrations
after cropland was converted to perennial alfalfa (Medicago sativa L. Algonguin)
grassland for 6 years in the marginal oasis of the middle of Hexi Corridor
region, northwest China. Significant changes in the size distribution of
dry-sieving aggregates and water-stable aggregates, SOC, and N concentrations
occurred after the conversion from crop to alfalfa. SOC and N stocks increased by
20.2% and 18.5%, respectively, and the estimated C and N sequestration rates were
0.4 Mg C ha(-1) year(-1) and 0.04 Mg N ha(-1) year(-1) following the conversion.
The large aggregate (>5 mm) was the most abundant dry aggregate size fraction in
both crop and alfalfa soils, and significant difference in the distribution of
dry aggregates between the two land use types occurred only in the >5 mm
aggregate fraction. The percentage of water-stable macroaggregates (>2, 2-0.25
mm) and aggregate stability (mean weight diameter of water-stable aggregates,
WMWD) were significantly higher in alfalfa soils than in crop soils. There was a
significant linear relationship between total SOC concentration and aggregate
parameters (mean weight diameter) for alfalfa soils, indicating that aggregate
stability was closely associated with increased SOC concentration following the
conversion of crops to alfalfa. The SOC and N concentrations and the C/N ratio
were greatest in the >2 mm water-stable aggregates and the smallest in the
0.25-0.05 mm aggregates in crop and alfalfa soils. For the same aggregate, SOC
and N concentrations in aggregate fractions increased with increasing total SOC
and N concentrations. The result showed that the conversion of annual crops to
alfalfa in the marginal land with coarse-texture soils can significantly increase
SOC and N stocks, and improve soil structure.
DOI: 10.1007/s00267-009-9284-x
1479. Prikl Biokhim Mikrobiol. 2009 Jan-Feb;45(1):84-91.
[Effects of natural and hybrid lectins on the legume-rhizobium interactions].
Baĭmiev AKh, Gubaĭdullin II, Baĭmiev AKh, Cheremis AV.
The effects of hybrid lectins--full-sized pea Pisum sativum lectin (PSL) with the
carbohydrate-binding region of white melilot Melilotus albus lectin or wild
licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL
region (PSL/MAL and PSL/AGL, correspondingly)--on the legume-rhizobium symbiosis
were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the
alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected
pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus
cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules
on alfalfa roots. This ability is associated with expanded and unusual
carbohydrate-binding properties (combined specificity for Gal and Glc) of this
hybrid protein as compared with the natural legume lectins.
Effect of alfalfa silage storage structure and rumen-protected methionine on

production in lactating dairy cows.
Broderick GA(1), Muck RE.
Author information:
(1)Agricultural Research Service, U.S. Department of Agriculture, U.S. Dairy
Forage Research Center, 1925 Linden Drive West, Madison, WI 53706, USA.
glen.broderick@ars.usda.gov
The objective of this study was to determine whether production and nutrient
utilization differed when lactating cows were fed diets based on 1 of 3 sources
of alfalfa silage (AS) and whether performance was altered by feeding
rumen-protected Met (RPM; fed as Mepron). Thirty-six lactating Holstein cows were
blocked by parity and days in milk, then assigned to a randomized complete block
design and fed a 3 x 2 arrangement of diets formulated from alfalfa ensiled in
bag, bunker, or oxygen-limited silos, and supplemented with either 0 or 8 g of
RPM/d. After feeding a covariate diet for 3 wk, treatment diets were fed for the
remaining 12 wk of the trial. Experimental diets averaged [dry matter (DM) basis]
41% AS, 24% corn silage, 24% high-moisture corn, 3.7% soybean meal, 4% roasted
soybeans, 2% ground shelled corn, 1.0% minerals and vitamins, 16.7% CP, and 31%
NDF. Alfalfa from the oxygen-limited silo was lower in ash, higher in lactate,
nonfiber carbohydrate, and in vitro NDF digestibility, had lower pH and ammonia
content, and gave rise to greater DM intake and ADF digestibility than silage
from the other 2 silos, indicating a more effective fermentation that, in turn,
resulted in greater nutrient preservation. However, the more favorable
composition, intake, and digestibility of alfalfa from the oxygen-limited silo
were not reflected in improved milk production, which was not different among
alfalfa sources. There was increased apparent N efficiency and trends for
improved feed efficiency and protein yield with RPM supplementation across all 3
silages. The National Research Council (2001) model predicted that feeding RPM
reduced Lys:Met ratio from 3.5 to 2.9, indicating that the diets were limiting in
Met.
DOI: 10.3168/jds.2008-1303
Alfalfa cut at sundown and harvested as baleage increases bacterial protein

synthesis in late-lactation dairy cows.
Brito AF(1), Tremblay GF, Lapierre H, Bertrand A, Castonguay Y, Bélanger G,

Michaud R, Benchaar C, Ouellet DR, Berthiaume R.
Author information:
Canada, Sherbrooke, Québec, Canada.
Alfalfa (Medicago sativa L.) cut at sundown (p.m.) has been shown to have a
greater concentration of total nonstructural carbohydrates (TNC) than when cut at
sunup (a.m.). Eight ruminally cannulated Holstein cows that were part of a larger
lactation trial were used in a crossover design (24-d periods) to investigate the
effects of alfalfa cutting time on digestibility and omasal flow of nutrients.
Alfalfa was cut at sundown or sunup, field-wilted, and harvested as baleage (530
+/- 15.0 g of dry matter/kg of fresh matter). The difference in TNC concentration
between p.m. and a.m. alfalfa within each pair of bales fed daily during the 10 d
of data and sample collection varied from -10 to 50 g/ kg of dry matter. Each
pair of bales was fed for ad libitum intake to cows once daily with no
concentrate. During the 3 d of omasal sampling, intake (+0.8 kg/d) and omasal
flow of organic matter (OM; +0.42 kg/d) tended to be greater when cows were fed
p.m. vs. a.m. alfalfa, but no differences were found for ruminal and postruminal
digestion of this nutrient. Similarly, N apparently digested ruminally and
postruminally did not differ when feeding p.m. vs. a.m. alfalfa. However, N truly
digested in the rumen, as a proportion of N intake, was significantly greater in
cows fed p.m. (79%) vs. a.m. alfalfa (74%), thus suggesting that longer wilting
time of alfalfa cut at sundown increased forage proteolysis. Supply of
rumen-degradable protein did not change (2,716 g/d) when averaged across
treatments, whereas omasal flow of non-NH(3) nonbacterial N was significantly
decreased (-29 g/d) when feeding p.m. vs. a.m. alfalfa. Omasal flow of total
bacterial non-NH(3)-N (NAN) increased (+21 g/d) significantly when cows were fed
p.m. vs. a.m. alfalfa possibly because bacteria from cows fed p.m. alfalfa
captured significantly more NH(3) than those from cows fed a.m. alfalfa.
Therefore, greater availability of fermentable energy as TNC appears to increase
the capacity of microbes to uptake NH(3)-N and convert it to microbial protein.
Enhanced OM intake can also explain the observed increase in bacterial protein
synthesis with p.m. alfalfa. Efficiency of bacterial protein synthesis, expressed
on a fermented OM basis or as grams of bacterial NAN per gram of rumen-degradable
N, did not differ between p.m. and a.m. alfalfa. Conversely, bacterial
efficiency, as grams of bacterial NAN per gram of N intake, was significantly
increased when cows were fed p.m. baleage. No significant difference between
forage treatments was found for the omasal flow of total AA from omasal true
digesta, suggesting no benefit of daytime cutting management on the passage of
total AA to the lower gastrointestinal tract. Enhancing energy intake and TNC
concentration of alfalfa by shifting forage cutting from sunup to sundown
increased protein synthesis and NH(3) uptake by ruminal bacteria indicating an
improvement in N utilization.
DOI: 10.3168/jds.2008-1469

Epub
2009 Feb 13.
Elimination of Escherichia coli O157:H7 from Alfalfa seeds through a combination

of high hydrostatic pressure and mild heat.
Neetoo H(1), Pizzolato T, Chen H.
Author information:
19716-2150, USA.
Escherichia coli O157:H7 has been associated with contaminated seed sprout
outbreaks. The majority of these outbreaks have been traced to sprout seeds
contaminated with low levels of pathogens. Sanitizing sprout seeds presents a
unique challenge in the arena of produce safety in that even a low residual
pathogen population remaining on contaminated seed after treatments appears
capable of growing to very high levels during sprouting. In this study, the
effectiveness of high-pressure treatment in combination with low and elevated
temperatures was assessed for its ability to eliminate E. coli O157:H7 on
artificially contaminated alfalfa seeds. Inoculated seed samples were treated at
600 MPa for 2 min at 4, 20, 25, 30, 35, 40, 45, and 50 degrees C. The pressure
sensitivity of the pathogenic bacteria was strongly dependent on the treatment
temperature. At 40 degrees C, the process was adequate in eliminating a
5-log-unit population on the seeds with no adverse effect on seed viability.
Three treatments carried out at reduced pressure levels and/or extended treatment
time, 550 MPa for 2 min at 40 degrees C, 300 MPa for 2 min at 50 degrees C, and
400 MPa for 5 min at 45 degrees C, were equally lethal to the pathogen. When all
three treatments were compared in terms of their impact on seed viability, the
process of 550 MPa for 2 min at 40 degrees C was the most desirable, achieving
final germination percentages and sprout sizes statistically similar to those of
control untreated seeds (P > 0.05).
DOI: 10.1128/AEM.02531-08
PMCID: PMC2663220

Epub
2009 Feb 13.
Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris
have characteristics in common with Sinorhizobium meliloti isolates from mainland
Spain.
Zurdo-Piñeiro JL(1), García-Fraile P, Rivas R, Peix A, León-Barrios M, Willems A,

Mateos PF, Martínez-Molina E, Velázquez E, van Berkum P.
Author information:
(1)Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca,
Spain.
The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial

isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the
Canary Islands, were identical to a less-common pattern found within
Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa
and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and
the group II LMW RNA S. meliloti isolates also were distinguishable in that both
had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed
spacer region that were not present in other strains of S. meliloti. The isolates
from P. vulgaris nodulated bean but not Medicago sativa, while those recovered
from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The
bean isolates also were distinguished from those of Medicago, Melilotus, and
Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean
isolates were most similar to those reported for S. meliloti bv. mediterranense
and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891
and AF217267, respectively). None of the evidence placed the bean isolates from
Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne
transmission of Rhizobium etli from the Americas to the Canaries as an
explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.
DOI: 10.1128/AEM.02811-08
PMCID: PMC2675199
1484. Vet Parasitol. 2009 May 12;161(3-4):248-54. doi:

10.1016/j.vetpar.2009.01.008.
Epub 2009 Jan 23.
Associations between nematode larval challenge and gastrointestinal tract size

that affect carcass productivity in sheep.
Jacobson C(1), Pluske J, Besier RB, Bell K, Pethick D.
Author information:
(1)School of Veterinary and Biomedical Sciences, Murdoch University, Western
Australia 6150, Australia. C.Bath@murdoch.edu.au
Effects of gastrointestinal parasitism on sheep productivity are usually

described using live weight change, however carcass productivity is more
accurately described using dressing percentage (carcass weight as a proportion of
live weight). This experiment had a 2x2x2 factorial design whereby 10-month-old
Merino wethers were fed lucerne (Medicago sativa) diets (fresh lucerne or lucerne
chaff) with 2 levels of carboxymethycellulose (CMC) inclusion (0% or 8% CMC) and
nematode larval challenge (no larval challenge or 10,000 Teladorsagia
circumcincta and 10,000 Trichostrongylus colubriformis per week). Sheep were
weighed and euthanased 50 or 51 days after larval challenge and CMC
supplementation commenced. Weight of the carcass (hot standard carcass weight)
and gastrointestinal organs (full and empty) were recorded and expressed as a
proportion of live weight. Larval challenged sheep had a worm egg count
(mean+/-standard error) of 173+/-38 eggs per gram of faeces and total worm count
of 30,237+/-2013 at slaughter. Larval challenged sheep had 1.3% lower dressing
percentage (p=0.048), and 2% heavier full (p=0.007) and 1.2% heavier empty
gastrointestinal tracts (p=0.012) compared to unchallenged sheep. There was no
effect of CMC inclusion or lucerne type (fresh or chaff) on gastrointestinal
tract weight or dressing percentage. Larval challenged sheep had 1.1% heavier
full (p<0.001) and 0.6% heavier empty (p<0.001) small intestines, and 0.6%
heavier full (p=0.005) and 0.3% heavier empty (p=0.026) large intestines compared
to unchallenged sheep. Use of live weight change or other measures based on live
weight (e.g. feed conversion efficiency) to assess the impact of nematode
challenge in sheep may underestimate carcass productivity losses associated with
larval challenge in sheep even at moderate levels of larval intake and without
overt clinical signs of parasitism. Measurement of carcass weight and/or lean
meat yield may better reflect the true economic effects of parasitism in sheep.
DOI: 10.1016/j.vetpar.2009.01.008
1485. Lupus. 2009 Mar;18(3):206-15. doi: 10.1177/0961203308095450.
The ethyl acetate extract of alfalfa sprout ameliorates disease severity of

autoimmune-prone MRL-lpr/lpr mice.
Hong YH(1), Huang CJ, Wang SC, Lin BF.
Author information:
(1)Department of Biochemical Science and Technology, Institute of Microbiology
and Biochemistry, College of Life Science, National Taiwan University, Taipei,
Taiwan.
Previous study showed that soy isoflavone supplement alleviates disease severity
in autoimmune-prone mice. As the ethyl acetate extract of alfalfa sprout (AS) has
selective oestrogenic and anti-inflammatory activity, this study evaluated the
effects of alfalfa sprout ethyl acetate extract (ASEA) on disease severity of
systemic lupus erythematosus, using autoimmune-prone female MRL-lpr/lpr mice. In
Experiment 1, five groups of 12-week-old female mice were per oral treated with
vehicle (control), lyophilized AS (550 mg wt/kg BW), ASEA (ASEA, 25 mg/kg BW),
coumestrol (CUM, 0.075 mg/kg BW) and tamoxifen (TAM, 0.375 mg/kg BW) as the
positive control. The onset of proteinuria was delayed, and the life span was
significantly longer in the ASEA and TAM groups but neither in the AS nor in the
CUM groups, compared to the control. To examine the changes in the immunological
parameters related to disease process, three more groups of MRL-lpr/lpr female
mice (control, ASEA and TAM) were fed in a similar manner for 6 weeks in the
Experiment 2. Flow cytometric analysis of splenocytes showed a significantly
lower percentage of activated T cells in the ASEA and TAM groups. The ex-vivo
interferon-gamma and interleukin (IL)-4 production from splenocytes and tumour
necrosis factor-alpha and IL-1beta production from peritoneal exudate cells were
also significantly lower in the ASEA group compared with the control. The ASEA
group also had less severe glomerulonephritis. Thus, ASEA attenuated cytokine and
inflammatory responses of self-reactive lymphocytes, decreased the disease
severity, increased survival and life span of the autoimmune-prone MRL-lpr/lpr
mice, suggesting a potential of ASEA in the treatment of autoimmune diseases.
DOI: 10.1177/0961203308095450
1486. Public Health Nutr. 2009 Mar;12(3):439-40. doi: 10.1017/S1368980008004746.
Nourishing child and adult patients in Congolese hospitals.
Bonnet MJ.
Comment on
Public Health Nutr. 2009 Jan;12(1):142.
DOI: 10.1017/S1368980008004746
1487. Lett Appl Microbiol. 2009 Mar;48(3):355-61. doi:

10.1111/j.1472-765X.2008.02532.x. Epub 2009 Jan 16.
Development of real-time PCR assay for detection and quantification of

Sinorhizobium meliloti in soil and plant tissue.
Trabelsi D(1), Pini F, Aouani ME, Bazzicalupo M, Mengoni A.
Author information:
(1)Laboratoire des Interactions Légumineuses-Microorganismes, Centre de
Biotechnologie de Borj-Cedria, Hammam-Lif, Tunisia.
AIMS: Sinorhizobium meliloti is a nitrogen-fixing alpha-proteobacterium present

in soil and symbiotically associated with root nodules of leguminous plants. To
date, estimation of bacterial titres in soil is achieved by most-probable-number
assays based on the number of nodules on the roots of test plants. Here, we
report the development of two real-time PCR (qPCR) assays to detect the presence
of S. meliloti in soil and plant tissues by targeting, in a species-specific
fashion, the chromosomal gene rpoE1 and the pSymA gene nodC.
METHODS AND RESULTS: rpoE1 and nodC primer pairs were tested on DNA extracted
from soil samples unspiked and spiked with known titres of S. meliloti and from
plant root samples nodulated with S. meliloti. Results obtained were well in
agreement with viable titres of S. meliloti cells estimated in the same samples.
CONCLUSIONS: The developed qPCR assays appear to be enough sensitive, precise and
species-specific to be used as a complementary tool for S. meliloti titre
estimation.
SIGNIFICANCE AND IMPACT OF THE STUDY: These two novel markers offer the
possibility of quick and reliable estimation of S. meliloti titres in soil and
plant roots contributing new tools to explore S. meliloti biology and ecology
including viable but nonculturable fraction.
DOI: 10.1111/j.1472-765X.2008.02532.x
1488. J Bacteriol. 2009 Apr;191(7):2133-43. doi: 10.1128/JB.01567-08. Epub 2009 Jan

30.
Role of potassium uptake systems in Sinorhizobium meliloti osmoadaptation and

symbiotic performance.
Domínguez-Ferreras A(1), Muñoz S, Olivares J, Soto MJ, Sanjuán J.
Author information:
Experimental del Zaidín, CSIC, Granada, Spain.
Stimulation of potassium uptake is the most rapid response to an osmotic upshock

in bacteria. This cation accumulates by a number of different transport systems
whose importance has not been previously addressed for rhizobia. In silico
analyses reveal the presence of genes encoding four possible potassium uptake
systems in the genome of Sinorhizobium meliloti 1021: Kup1, Kup2, Trk, and Kdp.
The study of the relevance of these systems under a number of different growth
conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential
for growth under laboratory conditions even in osmotically balanced media and the
absence of both systems leads to a reduced infectivity and competitiveness of the
bacteria in alfalfa roots. Trk is the main system involved in the accumulation of
potassium after an osmotic upshift and the most important system for growth of S.
meliloti under hyperosmotic conditions. The other three systems, especially Kup1,
are also relevant during the osmotic adaptation of the cell, and the relative
importance of the Kdp system increases at low potassium concentrations.
DOI: 10.1128/JB.01567-08
PMCID: PMC2655490
1489. Int J Phytoremediation. 2009 Feb;11(2):131-149. doi:

10.1080/15226510802378434.
EFFECT OF INDOLE-3-ACETIC ACID, KINETIN, AND ETHYLENEDIAMINETETRAACETIC ACID ON

PLANT GROWTH AND UPTAKE AND TRANSLOCATION OF LEAD, MICRONUTRIENTS, AND
MACRONUTRIENTS IN ALFALFA PLANTS.
López ML(1), Peralta-Videa JR(1), Parsons JG(1), Gardea-Torresdey JL(2),

Duarte-Gardea M(3).
Author information:
(1)a Chemistry Department , The University of Texas at El Paso , El Paso , Texas
, USA.
(2)b Chemistry Department and Environmental Sciences and Engineering Ph.D.
Program , The University of Texas at El Paso , El Paso , Texas , USA.
(3)c Department of Health Promotion, College of Health Sciences , The University
of Texas at El Paso , El Paso , Texas , USA.
Alfalfa plants germinated and grown for 15 d in soil containing 80 mg Pb kg-1

were treated with ethylenediaminetetraacetic acid (EDTA) at 0.8 mM and
indole-3-acetic acid-kinetin (IAA-KN) at 100 μM. Fifteen days after the treatment
application, the concentration of lead (Pb), macronutrients, and micronutrients
was determined using inductively coupled plasma/optical emission spectroscopy.
The chlorophyll content and plant growth were also measured. Roots of plants
exposed to Pb alone, Pb-EDTA, and Pb-EDTA-IAA-KN had 160, 140, and 150 mg Pb kg-1
DW, respectively. Pb was not detected in the stems of plants exposed to Pb alone;
however, stems of plants treated with EDTA and EDTA-IAA-KN had 78 and 142 mg Pb
kg-1 DW, respectively. While the Pb concentration in leaves of plants treated
with EDTA and EDTA-IAA-KN was 92 and 127 mg kg-1 DW, respectively. In addition,
EDTA and EDTA-IAA-KN significantly increased the translocation of zinc and
manganese to leaves. The x-ray absorption spectroscopic studies demonstrated that
Pb(II) was transported from roots to leaves without a change in the oxidation
state.
DOI: 10.1080/15226510802378434
1490. BMC Microbiol. 2009 Jan 27;9:17. doi: 10.1186/1471-2180-9-17.
The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane

efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation
of alfalfa plants.
van Dillewijn P(1), Sanjuán J, Olivares J, Soto MJ.
Author information:
(1)Departamento de Protección Ambiental, Estación Experimental del Zaidín, CSIC,
Granada, Spain. pieter.vandillewijn@eez.csic.es
BACKGROUND: Soil bacteria collectively known as Rhizobium, characterized by their

ability to establish beneficial symbiosis with legumes, share several common
characteristics with pathogenic bacteria when infecting the host plant. Recently,
it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in
the control of swarming, a type of co-ordinated movement previously associated
with pathogenicity, and is also impaired in nodulation efficiency on alfalfa
roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms
part of a cluster of genes involved in the regulation of pathogenicity factors.
In this work, we have investigated the role in swarming and symbiosis of
SMc02161, a S. meliloti fadD-linked gene.
RESULTS: The SMc02161 locus in S. meliloti shows similarities with members of the
Major Facilitator Superfamily (MFS) of transporters. A S. meliloti null-mutant
shows increased sensitivity to chloramphenicol. This indication led us to rename
the locus tep1 for transmembrane efflux protein. The lack of tep1 does not affect
the appearance of swarming motility. Interestingly, nodule formation efficiency
on alfalfa plants is improved in the tep1 mutant during the first days of the
interaction though nod gene expression is lower than in the wild type strain.
Curiously, a nodC mutation or the addition of N-acetyl glucosamine to the wild
type strain lead to similar reductions in nod gene expression as in the tep1
mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation.
CONCLUSION: tep1 putatively encodes a transmembrane protein which can confer
chloramphenicol resistance in S. meliloti by expelling the antibiotic outside the
bacteria. The improved nodulation of alfalfa but reduced nod gene expression
observed in the tep1 mutant suggests that Tep1 transports compounds which
influence nodulation. In contrast to Bradyrhizobium japonicum, we show that in S.
meliloti there is no feedback regulation of nodulation genes. Moreover, the Nod
factor precursor, N-acetyl glucosamine reduces nod gene expression and nodulation
efficiency when present at millimolar concentrations. A role for Tep1 in the
efflux of Nod factor precursors could explain the phenotypes associated with tep1
inactivation.
DOI: 10.1186/1471-2180-9-17
PMCID: PMC2637885
1491. Environ Pollut. 2009 May;157(5):1613-8. doi: 10.1016/j.envpol.2008.12.022.

Epub
2009 Jan 25.
Phenanthrene uptake by Medicago sativa L. under the influence of an arbuscular

mycorrhizal fungus.
Wu N(1), Huang H, Zhang S, Zhu YG, Christie P, Zhang Y.

Author information:
(1)State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
Centre for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871,
Phenanthrene uptake by Medicago sativa L. was investigated under the influence of

an arbuscular mycorrhizal fungus. Inoculation of lucerne with the arbuscular
mycorrhizal fungus Glomus etunicatum L. resulted in higher phenanthrene
accumulation in the roots and lower accumulation in the shoots compared to
non-mycorrhizal controls. Studies on sorption and desorption of phenanthrene by
roots and characterization of heterogeneity of mycorrhizal and non-mycorrhizal
roots using solid-state (13)C nuclear magnetic resonance spectroscopy ((13)C NMR)
demonstrated that increased aromatic components due to mycorrhizal inoculation
resulted in enhanced phenanthrene uptake by the roots but lower translocation to
the shoots. Direct visualization using two-photon excitation microscopy (TPEM)
revealed higher phenanthrene accumulation in epidermal cells of roots and lower
transport into the root interior and stem in mycorrhizal plants than in
non-mycorrhizal controls. These results provide some insight into the mechanisms
by which arbuscular mycorrhizal inoculation may influence the uptake of organic
contaminants by plants.
DOI: 10.1016/j.envpol.2008.12.022
1492. Mol Biol Rep. 2009 Nov;36(8):2315-21. doi: 10.1007/s11033-009-9450-5. Epub

2009
Jan 22.
Molecular cloning and characterization of a novel gene encoding zinc finger

protein from Medicago sativa L.
Chao Y(1), Kang J, Sun Y, Yang Q, Wang P, Wu M, Li Y, Long R, Qin Z.
Author information:
Beijing 100193, People's Republic of China. chaoyuehui@163.com
A suppression subtraction hybridization (SSH) cDNA library had been constructed

to identify differentially expressed genes. Based on the sequence of an expressed
sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA
(Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was
cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated
as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared
by blast revealed high homology with zinc finger protein of other plants.
Sequence comparison showed that there were five conserved typical zinc finger
motifs, and one sugar transfer protein signature. The calculated molecular weight
of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13.
The MsZFN localized in nucleus. Under normal growth conditions, differential
expression of MsZFN exhibited that the expression was the highest in leaf and the
lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and
reached its maximum at 30 min.
DOI: 10.1007/s11033-009-9450-5
1493. Phytochemistry. 2009 Jan;70(2):211-5. doi: 10.1016/j.phytochem.2008.12.013.

Epub
2009 Jan 18.
Fabacyl acetate, a germination stimulant for root parasitic plants from Pisum
sativum.
Xie X(1), Yoneyama K, Harada Y, Fusegi N, Yamada Y, Ito S, Yokota T, Takeuchi Y,

Yoneyama K.
Author information:
(1)Weed Science Center, Utsunomiya University, 350 Mine-machi, Utsunomiya
321-8505, Japan.
A germination stimulant, fabacyl acetate, was purified from root exudates of pea
(Pisum sativum L.) and its structure was determined as
ent-2'-epi-4a,8a-epoxyorobanchyl acetate
[(3aR,4R,4aR,8bS,E)-4a,8a-epoxy-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofu
ran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-decahydro-2H-indeno[1,2-b]furan-4-
yl acetate], by 1D and 2D NMR spectroscopic, ESI- and EI-MS spectrometric, X-ray
crystallographic analyses, and by comparing the (1)H NMR spectroscopic data and
relative retention times (RR(t)) in LC-MS and GC-MS with those of synthetic
standards prepared from (+)-orobanchol and (+)-2'-epiorobanchol. The (1)H NMR
spectroscopic data and RR(t) of fabacyl acetate were identical with those of an
isomer prepared from (+)-2'-epiorobanchol except for the opposite sign in CD
spectra. This is the first natural ent-strigolactone containing an epoxide group.
Fabacyl acetate was previously detected in root exudates of other Fabaceae plants
including faba bean (Vicia faba L.) and alfalfa (Medicago sativa L.).
DOI: 10.1016/j.phytochem.2008.12.013
1494. Wei Sheng Wu Xue Bao. 2008 Nov;48(11):1445-50.
[Identification of the Burkholderia cepacia complex genomovars and their

virulence in an alfalfa infection model].
Zhang L(1), Song J, Xie G.
Author information:
(1)College of Plant Protection, Anhui Agricultural University, Hefei 230036,
China. zhanglixinjoyce@163.com
OBJECTIVE: To confirm Burkholderia cepacia complex (Bcc) genomovars from

agricultural niches and clinical samples, and to evaluate their possible
virulence to human body on alfalfa infection model in China.
METHODS: A total of 57 Bcc strains were isolated and collected from the
rhizosphere, soil and clinical samples in China. The genomovars composition of
the Bcc strains was analyzed by species-specific PCR tests, and the virulence of
the Bce strains was tested on alfalfa seedlings.
RESULTS: Four genomovars of the ten genomovars were detected among the Bcc
strains, including B. cepacia (genomovar I), B. cenocepacia (genomovar III), B.
vietnamiensis (genomovarV) and B. pyrrocinia (genomovar IX). Bcc genomovars I and
III A from clinic, and genomovar III B from rhizosphere were the most virulent in
the alfalfa infection model, and caused symptoms in 69%, 68% and 55% of
seedlings, respectively. There were significant variances in the mean percentage
of seedlings with symptoms for genomovars I , III A and III B compared to those
for genomovar V and IX.
CONCLUSION: There was difference in the ability to cause disease in alfalfa for
different genomovar strains from agricultural inches. The strains of Bcc
genomovar III B from rhizosphere were more virulent similar to those of Bcc
genomovar III A from clinic.
[Combined remediation effects of arbuscular mycorrhizal fungi-legumes-rhizobium

symbiosis on PCBs contaminated soils].
Teng Y(1), Luo YM, Gao J, Li ZG.
Author information:
Soil Science, Chinese Academy of Sciences,Nanjing 210008, China.
yteng@issas.ac.cn
The combined remediation effects of dual inoculation with an arbuscular

mycorrhizal fungus (AM) and rhizabium (Rhizobium meliloti) with a host plant
(alfalfa) on PCBs contaminated soils was studied using pot experiments. The
results showed that alfalfa had a clear role in PCBs removal in soils compared
with treatments without alfalfa and inoculated microorganisms, PCBs concentration
in lightly and heavily polluted soils decreased 15.8% and 23.5%, respectively.
After planting alfalfa with single incubation of Glomus caledonium, PCBs
concentration decreased 14.8% and 24.1% from lightly and heavily polluted soils,
decreased 20.6% and 25.5% for single incubation of Rhizobium meliloti,
respectively. After dual incubation with Glomus caledonium and Rhizobium meliloi,
PCBs concentration in lightly and heavily polluted soils decreased 23.2% and
26.9%. We also observed that soil microbial communities in alfalfa rhizosphere
soils had a higher carbon utilization rate, improving the functional diversity of
the soil microbial community. The results suggest that dual incubation of Glomus
caledonium and Rhizobium meliloti has great potential in remediation of PCBs
contaminated soils.

10.1111/j.1439-0396.2008.00872.x.
Performance and dietary preferences of white-tailed deer grazing chicory,

birdsfoot trefoil or alfalfa in north central Alberta.
Chapman GA(1), Bork EW, Donkor NT, Hudson RJ.
Author information:
(1)Department of Agricultural, Food and Nutritional Science, University of
Alberta, Edmonton, AB, Canada.
Little information exists on the performance of deer on alternative forage

species in northern temperate environments during summer and fall, the period of
inherent maximum growth in deer. In performance and choice experiments, we
compared live weight gain (g/kg(0.75)/day), absolute [kg/ha dry matter (DM)] and
relative (% DM) herbage utilization, relative preference index (RPI) as well as
plant community visitation of white-tailed deer grazing alfalfa (Medicago
sativa), birdsfoot trefoil (Lotus corniculatus) or chicory (Cichorium intybus) in
north central Alberta, Canada. Herbage phytomass and quality was also measured on
the grazed pastures. Alfalfa had higher dry matter yields and crude protein
concentrations than chicory and trefoil. Chicory had lower neutral detergent
fiber concentrations than the other forages. Tannin concentrations were greatest
in birds foot trefoil (nearly 55 g/kg DM), well above those in the other forages
(<5 g/kg DM). Live weight gain was similar among deer feeding within the paddocks
seeded to birds foot trefoil and chicory, and more than two times higher (p <
0.05) than deer feeding in paddocks seeded to alfalfa. Deer spent more grazing
time (about 40%) on chicory pastures than on alfalfa and birds foot trefoil
pastures. RPI values were greatest for birds foot trefoil at 2.11, intermediate
for chicory at 1.40, and lowest for alfalfa at <0.60. Absolute herbage
utilization remained similar (p > 0.05) among the three forage species. In
contrast, relative herbage utilization was greater from birds foot trefoil (52%
DM) than chicory (40% DM) or alfalfa (25% DM). These results suggest that the use
of alfalfa with other alternative forages may prove beneficial to deer
production, rather than using alfalfa pasture alone.
DOI: 10.1111/j.1439-0396.2008.00872.x
[Diurnal dynamics of photosynthetic characteristics of alfalfa on Kerqin sandy

land].
Xu LJ(1), Wang B, Sun QZ.
Author information:
(1)Graduate School of the Chinese Academy of Agriculture Sciences, Beijing
100081, China. xulijun_nmg@163.com
By using a Li-6400 portable photosynthesis system, the diurnal dynamics of the

photosynthetic characteristics of three alfalfa varieties Medicago sativa L. cv.
Aohan, M. sativa L. cv. Rangelander, and M. sativa L. cv. Algonquin were studied.
The results showed that for the test alfalfa varieties, the diurnal variation
curves of their P(n), T(r), and WUE were not all presented double-peak. For Aohan
and Algonquin, the maximum P(n) and T(r) occurred at 9:00-11:00; while for
Rangelander, they appeared at 11:00. The minimum P(n) and T(r) of the three
varieties all appeared at 7:00 and 19:00. The maximum WUE of Rangelander and
Algonquin appeared at 7:00 and that of Aohan was at 9:00, and the minimum WUE of
the three varieties all appeared at 15:00-17:00. Comprehensive analysis on the
photosynthetic performance indices P(n), T(r), and WUE indicated that 2 years old
alfalfa was in the best state, followed by 1 year old, and 4 years old.
Correlation analysis showed that air temperature (T(a)) was the main
environmental factor affecting P(n) and T(r), followed by water vapor pressure
deficit (VPD) and relative humidity (RH).
1498. Microbiology. 2009 Jan;155(Pt 1):257-67. doi: 10.1099/mic.0.022079-0.

SMb20651 is another acyl carrier protein from Sinorhizobium meliloti.
Ramos-Vega AL(1), Dávila-Martínez Y, Sohlenkamp C, Contreras-Martínez S,

Encarnación S, Geiger O, López-Lara IM.
Author information:
(1)Programa de Ecología Genómica, Centro de Ciencias Genómicas, Universidad
Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos C. P. 62251,
Mexico.
Acyl carrier proteins (ACPs) are small acidic proteins that carry growing acyl
chains during fatty acid or polyketide synthesis. In rhizobia, there are four
different and well-characterized ACPs: AcpP, NodF, AcpXL and RkpF. The genome
sequence of Sinorhizobium meliloti 1021 reveals two additional ORFs that possibly
encode additional ACPs. One of these, smb20651, is located on the plasmid pSymB
as part of an operon. The genes of the operon encode a putative asparagine
synthetase (AsnB), the predicted ACP (SMb20651), a putative long-chain fatty
acyl-CoA ligase (SMb20650) and a putative ammonium-dependent NAD+ synthetase
(NadE1). When SMb20651 was overexpressed in Escherichia coli, [3H]beta-alanine, a
biosynthetic building block of 4'-phosphopantetheine, was incorporated into the
protein in vivo. The purified SMb20651 was modified with 4'-phosphopantetheine in
the presence of S. meliloti holo-ACP synthase (AcpS). Also, holo-SMb20651 was
modified in vitro with a malonyl group by malonyl CoA-ACP transacylase. In E.
coli, coexpression of SMb20651 together with other proteins such as AcpS and
SMb20650 led to the formation of additional forms of SMb20651. In this bacterium,
acylation of SMb20651 with C12 : 0 or C18 : 0 fatty acids was detected,
demonstrating that this protein is involved in fatty acid biosynthesis or
transfer. Expression of SMb20651 was detected in S. meliloti as holo-SMb20651 and
acyl-SMb20651.
DOI: 10.1099/mic.0.022079-0
1499. Plant Sci. 2009 Jan;176(1):119-129.
Repercussion of mesophyll-specific overexpression of a soybean cytosolic

glutamine synthetase gene in alfalfa (Medicago sativa L.) and tobacco (Nicotiana
tabaccum L.).
Seger M(1), Ortega JL(2), Bagga S(2), Gopalan CS(3).
Author information:
(1)Graduate Program in Molecular Biology, New Mexico State University, 945
College Ave, MSC-3Q, Las Cruces, NM 88003 USA.
945 College Ave, MSC-3Q, Las Cruces, NM 88003 USA.
(3)Graduate Program in Molecular Biology, New Mexico State University, 945
College Ave, MSC-3Q, Las Cruces, NM 88003 USA ; Department of Plant and
Environmental Sciences, New Mexico State University, 945 College Ave, MSC-3Q, Las
Cruces, NM 88003 USA.
Glutamine synthetase (GS) plays a central role in plant nitrogen metabolism.

Plant GS occurs as a number of isoenzymes present in either the cytosol (GS1) or
chloroplast/plastid (GS2). There are several reports of improved performance in
transgenic plants overexpressing GS1 transgenes driven by the constitutive
CaMV35S promoter. Improvement has been attributed to the GS1 transgene product
functioning to enhance re-assimilation of NH4+ released by photorespiration or
protein degradation. In this paper, alfalfa and tobacco transformants expressing
a soybean gene driven by a photosynthetic cell-specific promoter have been
compared to transformants with the same transgene driven by the stronger CaMV35S
promoter. The two classes of alfalfa and tobacco transformants showed differences
in the level of GS1 transcript and GS1 protein accumulation, but the difference
in the total GS activity was small. The discrepancy in the transgene expression
level and GS activity has been attributed to posttranslational regulation at the
level of holoprotein stability. Both classes of transformants exhibited similar
level of improvement in soluble protein and in the rates of photosynthesis and
photorespiration. The data supports the hypothesis that GS1 made in the mesophyll
cells is involved in the re-assimilation of NH4+ released via photorespiration
and/or protein degradation.
DOI: 10.1016/j.plantsci.2008.10.006
PMCID: PMC4108266
PMID: 25067868
Interaction of molasses and monensin in alfalfa hay- or corn silage-based diets

on rumen fermentation, total tract digestibility, and milk production by Holstein
cows.
Oelker ER(1), Reveneau C, Firkins JL.
Author information:
(1)Department of Animal Sciences, The Ohio State University, Columbus 43210, USA.
Sugar supplementation can stimulate rumen microbial growth and possibly fiber
digestibility; however, excess ruminal carbohydrate availability relative to
rumen-degradable protein (RDP) can promote energy spilling by microbes, decrease
rumen pH, or depress fiber digestibility. Both RDP supply and rumen pH might be
altered by forage source and monensin. Therefore, the objective of this study was
to evaluate interactions of a sugar source (molasses) with monensin and 2 forage
sources on rumen fermentation, total tract digestibility, and production and
fatty acid composition of milk. Seven ruminally cannulated lactating Holstein
cows were used in a 5 x 7 incomplete Latin square design with five 28-d periods.
Four corn silage diets consisted of 1) control (C), 2) 2.6% molasses (M), 3) 2.6%
molasses plus 0.45% urea (MU), or 4) 2.6% molasses plus 0.45% urea plus monensin
sodium (Rumensin, at the intermediate dosage from the label, 16 g/909 kg of dry
matter; MUR). Three chopped alfalfa hay diets consisted of 1) control (C), 2)
2.6% molasses (M), or 3) 2.6% molasses plus Rumensin (MR). Urea was added to corn
silage diets to provide RDP comparable to alfalfa hay diets with no urea. Corn
silage C and M diets were balanced to have 16.2% crude protein; and the remaining
diets, 17.2% crude protein. Dry matter intake was not affected by treatment, but
there was a trend for lower milk production in alfalfa hay diets compared with
corn silage diets. Despite increased total volatile fatty acid and acetate
concentrations in the rumen, total tract organic matter digestibility was lower
for alfalfa hay-fed cows. Rumensin did not affect volatile fatty acid
concentrations but decreased milk fat from 3.22 to 2.72% in corn silage diets but
less in alfalfa hay diets. Medium-chain milk fatty acids (% of total fat) were
lower for alfalfa hay compared with corn silage diets, and short-chain milk fatty
acids tended to decrease when Rumensin was added. In whole rumen contents,
concentrations of trans-10, cis-12 C(18:2) were increased when cows were fed corn
silage diets. Rumensin had no effect on conjugated linoleic acid isomers in
either milk or rumen contents but tended to increase the concentration of
trans-10 C(18:1) in rumen samples. Molasses with urea increased ruminal NH(3)-N
and milk urea N when cows were fed corn silage diets (6.8 vs. 11.3 and 7.6 vs.
12.0 mg/dL for M vs. MU, respectively). Based on ruminal fermentation
characteristics and fatty acid isomers in milk, molasses did not appear to
promote ruminal acidosis or milk fat depression. However, combinations of
Rumensin with corn silage-based diets already containing molasses and with a
relatively high nonfiber carbohydrate:forage neutral detergent fiber ratio
influenced biohydrogenation characteristics that are indicators of increased risk
for milk fat depression.
DOI: 10.3168/jds.2008-1432
1501. Mycorrhiza. 2009 Apr;19(4):255-266. doi: 10.1007/s00572-008-0217-y. Epub 2008

Dec
23.
Influence of soil organic matter decomposition on arbuscular mycorrhizal fungi in

terms of asymbiotic hyphal growth and root colonization.
Gryndler M(1), Hršelová H(2), Cajthaml T(2), Havránková M(3)(4), Řezáčová V(2),
Gryndlerová H(2), Larsen J(5).
Author information:
(1)Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20,
Prague, Czech Republic. gryndler@biomed.cas.cz.
(2)Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20,
Prague, Czech Republic.
(3)Department of Botany, Faculty of Science, Charles University, 128 01, Prague,
Czech Republic.
(4)Institute of Botany, Academy of Sciences of the Czech Republic, 252 43,
Průhonice, Czech Republic.
(5)Department of Integrated Pest Management, Faculty of Agricultural Science,
University of Aarhus, DK-4200, Slagelse, Denmark.
Soil organic matter is known to influence arbuscular mycorrhizal (AM) fungi, but
limited information is available on the chemical components in the organic matter
causing these effects. We studied the influence of decomposing organic matter
(pure cellulose and alfalfa shoot and root material) on AM fungi after 30, 100,
and 300 days of decomposition in nonsterile soil with and without addition of
mineral N and P. Decomposing organic matter affected maize root length colonized
by the AM fungus Glomus claroideum in a similar manner as other plant growth
parameters. Colonized root length was slightly increased by both nitrogen and
phosphorus application and plant materials, but not by application of cellulose.
In vitro hyphal growth of Glomus intraradices was increased by soil extracts from
the treatments with all types of organic materials independently of mineral N and
P application. Pyrolysis of soil samples from the different decomposition
treatments revealed in total 266 recognizable organic compounds and in vitro
hyphal growth of G. intraradices in soil extract positively correlated with 33 of
these compounds. The strongest correlation was found with 3,4,5-trimethoxybenzoic
acid methyl ester. This compound is a typical product of pyrolysis of phenolic
compounds produced by angiosperm woody plants, but in our experiment, it was
produced mainly from cellulose by some components of the soil microflora. In
conclusion, our results indicate that mycelia of AM fungi are influenced by
organic matter decomposition both via compounds released during the decomposition
process and also by secondary metabolites produced by microorganisms involved in
organic matter decomposition.
DOI: 10.1007/s00572-008-0217-y
Epub
2008 Dec 19.
Potential to reduce Escherichia coli shedding in cattle feces by using sainfoin

(Onobrychis viciifolia) forage, tested in vitro and in vivo.
Berard NC(1), Holley RA, McAllister TA, Ominski KH, Wittenberg KM, Bouchard KS,
Bouchard JJ, Krause DO.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, Manitoba,
Canada.
There is a growing concern about the presence of pathogens in cattle manure and
its implications on human and environmental health. The phytochemical-rich forage
sainfoin (Onobrychis viciifolia) and purified phenolics (trans-cinnamic acid,
p-coumaric acid, and ferulic acid) were evaluated for their ability to reduce the
viability of pathogenic Escherichia coli strains, including E. coli O157:H7. MICs
were determined using purified phenolics and acetone extracts of sainfoin and
alfalfa (Medicago sativa), a non-tannin-containing legume. Ground sainfoin or
pure phenolics were mixed with fresh cattle feces and inoculated with a
ciprofloxacin-resistant strain of E. coli, O157:H7, to assess its viability at
-20 degrees C, 5 degrees C, or 37 degrees C over 14 days. Forty steers were fed
either a sainfoin (hay or silage) or alfalfa (hay or silage) diet over a 9-week
period. In the in vitro study, the MICs for coumaric (1.2 mg/ml) and cinnamic
(1.4 mg/ml) acids were 10- to 20-fold lower than the MICs for sainfoin and
alfalfa extracts. In the inoculated feces, the -20 degrees C treatment had death
rates which were at least twice as high as those of the 5 degrees C treatment,
irrespective of the additive used. Sainfoin was less effective than coumaric acid
in reducing E. coli O157:H7 Cip(r) in the inoculated feces. During the animal
trial, fecal E. coli numbers declined marginally in the presence of sainfoin
(silage and hay) and alfalfa silage but not in the presence of hay, indicating
the presence of other phenolics in alfalfa. In conclusion, phenolic-containing
forages can be used as a means of minimally reducing E. coli shedding in cattle
without affecting animal production.
DOI: 10.1128/AEM.00983-08
PMCID: PMC2643585
1503. Plant Physiol. 2009 Feb;149(2):1166-78. doi: 10.1104/pp.108.129601. Epub 2008

Dec
19.
Overexpression of flavodoxin in bacteroids induces changes in antioxidant

metabolism leading to delayed senescence and starch accumulation in alfalfa root
nodules.
Redondo FJ(1), de la Peña TC, Morcillo CN, Lucas MM, Pueyo JJ.
Author information:
(1)Department of Plant Physiology and Ecology, Instituto de Recursos Naturales,
Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones
Científicas, E-28006 Madrid, Spain.
Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis
flavodoxin, a protein that is involved in the response to oxidative stress.
Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants
nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control
bacteria. The decline of nitrogenase activity and the nodule structural and
ultrastructural alterations that are associated with nodule senescence were
significantly delayed in flavodoxin-expressing nodules. Substantial changes in
nodule antioxidant metabolism, involving antioxidant enzymes and
ascorbate-glutathione cycle enzymes and metabolites, were detected in
flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in
flavodoxin-expressing nodules than in control nodules. The observed amelioration
of the oxidative balance suggests that the delay in nodule senescence was most
likely due to a role of the protein in reactive oxygen species detoxification.
Flavodoxin overexpression also led to high starch accumulation in nodules,
without reduction of the nitrogen-fixing activity.
DOI: 10.1104/pp.108.129601
PMCID: PMC2633860
1504. Guang Pu Xue Yu Guang Pu Fen Xi. 2008 Sep;28(9):2062-6.
[Research on predicting the qualities of stem of alfalfa hay by near infrared

reflectance spectroscopy].
Qi X(1), Han JG, Nie ZD, Liu FY, Zhang ZH, Li ML.
Author information:
(1)Institute of Grassland Science, China Agricultural University, Beijing Major
Laboratory, Beijing 100094, China. tq07mms@sina.com
The present research was attempted to predict the qualities of stem of alfalfa
(Medicago sativa L. ) without separation from the whole plant by near infrared
reflectance spectroscopy and discussed the feasibility of using the near infrared
reflectance spectra information of the whole object to predict the qualities of a
certain part. Sixty six whole alfalfa hay samples of separated stems from leaves
were collected and they were distinguishing by years, cultivars, cuts and growing
periods. There were 138 calibration samples and 60 validation samplers. Fourier
transform-near infrared reflectance spectroscopy (FT-NIRS) and partial least
square (PLS) were used to set up the calibration models of stem's crude protein
(CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude ash (CA)
and in vitro digestible dry matter (IVDDM) contents. All models showed great
calibration and prediction performances except the one of stem's NDF content. The
correlation coefficients of cross-validation (rCV) were between 0.8523 and
0.9007, the root mean square errors of cross-validation (RMSECV) were between
0.72% and 3.96% and the correlation coefficients of NIRS values and chemical
values (r) were between 0.9255 and 0.9512. However, rCV, RMSECV and r of the
model of stem's NDF content were 0.8214, 3.70% and 0.9020, respectively. It
wasn't exact enough and would be used for rough predicting only. All of the
results showed that near infrared reflectance spectra information of whole
alfalfa hay could be used to predict some components of its stem exactly. It was
the maiden attempt of using near infrared reflectance spectra information of the
whole objects to evaluated the qualities of a certain part.
PMID: 19093561
1505. Pak J Biol Sci. 2007 Dec 15;10(24):4426-31.
The effect of yeast (Saccharomyces cerevisiae) on nutrient intake, digestibility

and finishing performance of lambs fed a diet based on dried molasses sugar
beet-pulp.
Payandeh S(1), Kafilzadeh F.
Author information:
(1)Department of Animal Science, Faculty of Agriculture, Razi University,
Kermanshah, Iran.
This experiment was conducted to determine the effect of yeast (Saccharomyces

cerevisiae, SC47) on finishing performance, digestibility, some blood metabolites
and carcass characteristics of male lambs fed a diet based on dried Molasses
Sugar Beet-Pulp (MSBP). Eighteen Sanjabi male lambs (20.95 +/- 2.7 kg initial
body weight and 3 month of age) were used in a completely randomized design.
Animals were assigned to one of the two dietary treatments (with or without
yeast). Digestibility and nitrogen balance experiment was carried out using six
mature rams on finishing diet with and without yeast. Serum metabolites were
determined in samples taken from lambs at the end of finishing period. Dry matter
digestibility of finishing diet was significantly increased by yeast addition.
However, yeast did not have any significant effect on apparent digestibility of
OM, NDF, CP and energy. Nitrogen retention was also not affected by yeast
addition. Yeast resulted in a significant increase in the average daily gain, dry
matter and organic matter intake. However, feed conversion ratio was not
significantly affected by addition of yeast. The concentration of the serum
metabolites including glucose, urea, cholesterol, sodium, potassium, calcium,
phosphorous and cratinine were not affected significantly by yeast
supplementation, but triglyceride concentrations increased significantly when
yeast was fed. Addition of yeast to the diet did not have any significant effect
on the carcass characteristics. Results of this study suggest that feeding
saccharomyces cerevisiae with a diet based on MSBP can improve the performance of
fattening lambs without any change in carcass characteristics or cuts.
1506. Pak J Biol Sci. 2007 Dec 15;10(24):4406-12.
Soil decontamination of 2,4,6- trinitrotoluene by alfalfa (Medicago sativa).
Fallahi S(1), Habibi-Rezaei M, Khayami M, Heydari R.
Author information:
(1)Department of Biology, University of Urmia, Urmia, Iran.
Present study investigate the toxicity effect of 2,4,6-trinitrotoluene (TNT) on a

terrestrial plant, alfalfa (Medicago sativa) in artificial soils. In this study,
TNT toxicity assessment was performed on spiked silica with this nitroaromatic
compound by determination of the percent of emergence and shoots and roots
biomasses at the concentration range of 3.2-10000 mg kg(-1) Dry Weight (DW). The
emergence was reduced by 22-32% after 5 days of exposure at TNT concentrations up
to 100 mg kg(-1) DW; shoot and root biomasses were reduced by 48-50 and 63-74%,
respectively after 30 days exposure at TNT concentrations < or = 32 mg kg(-1) DW.
Concentrations higher than 100 mg kg(-1) DW can not be tolerated at all.
Concentrations of TNT and its metabolites in silica, root and shoot were measured
by High-Performance Liquid Chromatography (HPLC). Analyses of TNT spiked soil
extracts reveal hat during alfalfa cultivation for 30 days, TNT was partially
transformed at the extent of 15-27%. This transformation decreased at higher TNT
soil concentrations. TNT is taken up and metabolized by plants to its downstream
derivatives.

Epub
2008 Dec 16.
An orphan LuxR homolog of Sinorhizobium meliloti affects stress adaptation and

competition for nodulation.
Patankar AV(1), González JE.
Author information:
(1)Department of Molecular Biology and Immunology, University of North Texas
Health Science Center at Fort Worth, Fort Worth, Texas 76107, USA.
The Sin/ExpR quorum-sensing system of Sinorhizobium meliloti plays an important

role in the symbiotic association with its host plant, Medicago sativa. The
LuxR-type response regulators of the Sin system include the synthase
(SinI)-associated SinR and the orphan regulator ExpR. Interestingly, the S.
meliloti Rm1021 genome codes for four additional putative orphan LuxR homologs
whose regulatory roles remain to be identified. These response regulators contain
the characteristic domains of the LuxR family of proteins, which include an
N-terminal autoinducer/response regulatory domain and a C-terminal
helix-turn-helix domain. This study elucidates the regulatory role of one of the
orphan LuxR-type response regulators, NesR. Through expression and phenotypic
analyses, nesR was determined to affect the active methyl cycle of S. meliloti.
Moreover, nesR was shown to influence nutritional and stress response activities
in S. meliloti. Finally, the nesR mutant was deficient in competing with the
wild-type strain for plant nodulation. Taken together, these results suggest that
NesR potentially contributes to the adaptability of S. meliloti when it
encounters challenges such as high osmolarity, nutrient starvation, and/or
competition for nodulation, thus increasing its chances for survival in the
stressful rhizosphere.
DOI: 10.1128/AEM.01692-08
PMCID: PMC2643561
1508. Public Health Nutr. 2009 Jan;12(1):142. doi: 10.1017/S136898000800414X.
The greatest untapped food resource on earth?
Davys G.
Comment in
Public Health Nutr. 2009 Mar;12(3):440.
Public Health Nutr. 2009 Mar;12(3):439-40.
Comment on
DOI: 10.1017/S136898000800414X
1509. New Phytol. 2008 Jul;179(2):495-504. doi: 10.1111/j.1469-8137.2008.02460.x.
Lack of trehalose catabolism in Sinorhizobium species increases their nodulation

competitiveness on certain host genotypes.
Ampomah OY(1), Jensen JB, Bhuvaneswari TV.
Author information:
(1)Department of Biology, Faculty of Science, University of Tromsø, N-9037
Tromsø, Norway.
The role of host and bacterial genotypes in determining the competitiveness of

trehalose utilization mutants of Sinorhizobium meliloti and Sinorhizobium medicae
was investigated here. Trehalose utilization mutants of S. meliloti and S.
medicae were obtained by mutagenesis of their trehalose utilization gene thuB.
The mutant strains and the wild type were coinoculated on three cultivars of
alfalfa (Medicago sativa) and two cultivars of Medicago truncatula and assessed
for competitiveness in root colonization, and nodule occupancy. The thuB mutants
formed more nodules than their parent strains on two of the three alfalfa lines
tested and on one of the two M. truncatula lines tested. They were not more
competitive on the other alfalfa and M. truncatula lines. Their competitiveness
for nodule occupancy did not correlate positively with their ability to colonize
these roots but correlated with the extent of thuB induction in the infection
threads. Induction of thuB was shown to be dependent on the concentration of
trehalose in the environment. These results suggest a direct role for host
trehalose metabolism in early plant-symbiont interactions and show that the
ability to manage host-induced stresses during infection, rather than the ability
to colonize the root, is critical for competitive nodulation.
DOI: 10.1111/j.1469-8137.2008.02460.x
1510. Mol Plant Microbe Interact. 2009 Jan;22(1):7-17. doi: 10.1094/MPMI-22-1-0007.
Global gene expression profiling during Medicago truncatula-Phymatotrichopsis

omnivora interaction reveals a role for jasmonic acid, ethylene, and the
flavonoid pathway in disease development.
Uppalapati SR(1), Marek SM, Lee HK, Nakashima J, Tang Y, Sledge MK, Dixon RA,
Mysore KS.
Author information:
(1)Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK
73401, USA.
Phymatotrichopsis omnivora (Duggar) Hennebert causes a destructive root rot in

cotton, alfalfa (Medicago sativa), and many other dicot species. No consistently
effective control measures or resistant host germplasm for Phymatotrichum root
rot (PRR) are known. The relative genetic intractability of cotton and alfalfa
precludes their use as model pathosystem hosts for P. omnivora. Therefore, we
used the model legume M. truncatula and its available genetic and genomic
resources to investigate PRR. Confocal imaging of P. omnivora interactions with
M. truncatula roots revealed that the mycelia do not form any specialized
structures for penetration and mainly colonize cortical cells and, eventually,
form a mycelial mantle covering the root's surfaces. Expression profiling of M.
truncatula roots infected by P. omnivora identified several upregulated genes,
including the pathogenesis-related class I and class IV chitinases and genes
involved in reactive oxygen species generation and phytohormone (jasmonic acid
and ethylene) signaling. Genes involved in flavonoid biosynthesis were induced
(2.5- to 10-fold over mock-inoculated controls) at 3 days postinoculation (dpi)
in response to fungal penetration. However, the expression levels of flavonoid
biosynthesis genes returned to the basal levels with the progress of the disease
at 5 dpi. These transcriptome results, confirmed by real-time quantitative
polymerase chain reaction analyses, showed that P. omnivora apparently evades
induced host defenses and may downregulate phytochemical defenses at later stages
of infection to favor pathogenesis.
DOI: 10.1094/MPMI-22-1-0007
1511. J Agric Food Chem. 2008 Dec 24;56(24):11800-8. doi: 10.1021/jf802362g.
Effects of an alfalfa protein hydrolysate on the gene expression and activity of

enzymes of the tricarboxylic acid (TCA) cycle and nitrogen metabolism in Zea mays
L.
Schiavon M(1), Ertani A, Nardi S.
Author information:
(1)Dipartimento di Biotecnologie Agrarie, Universita degli Studi di Padova,
Agripolis, Viale dell'Universita 16, 35020 Legnaro, Padova, Italy.
The effects through which an alfalfa protein hydrolysate (EM) possessing

gibberellin- and auxin-like activity may promote plant nitrogen (N) nutrition
have been investigated in Zea mays L. Treatment with 0.01 or 0.1 mg L(-1) EM for
48 h resulted in enhanced plant growth and leaf sugar accumulation.
Concomitantly, the level of nitrates decreased, whereas total N percentage was
unchanged. The activity of a number of enzymes involved in carbon (C) metabolism
(malate dehydrogenase, MDH; isocitrate dehydrogenase, IDH; citrate synthase, CS)
and N reduction and assimilation (nitrate reductase, NR; nitrite reductase, NiR;
glutamine synthetase, GS; glutamate synthase, GOGAT; aspartate aminotransferase,
AspAT) was significantly induced by EM supply to plants, and the transcription
pattern of MDH, IDH, CS, and NR strongly correlated with data of enzyme activity.
The transcript accumulation of asparagine synthetase (AS) was also induced by EM
in the roots. The results suggest that EM might promote nitrogen assimilation in
plants through a coordinate regulation of C and N metabolic pathways and open the
way for further research on protein hydrolysates as a valid tool to improve N use
efficiency and, as a consequence, to reduce the intensive use of inorganic N
fertilizers in agriculture.
DOI: 10.1021/jf802362g
1512. J Dairy Sci. 2008 Dec;91(12):4755-64. doi: 10.3168/jds.2008-1302.
Corn silage partially replacing short alfalfa hay to optimize forage use in total
mixed rations for lactating cows.
Kowsar R(1), Ghorbani GR, Alikhani M, Khorvash M, Nikkhah A.

Author information:
84156, Iran.
We theorized that adding corn silage to a total mixed ration with alfalfa hay as
the sole dietary forage would improve nutrient intake and chewing activity and
thereby improve rumen fermentation and milk production. The objective of this
research was to determine the effects of partial replacement of short alfalfa
[physically effective (pe) neutral detergent fiber (NDF) >1.18 mm (peNDF(>1.18))
= 33.2%] with corn silage (CS, peNDF(>1.18) = 51.9%) in yellow
grease-supplemented total mixed rations on feed intake, chewing behavior, rumen
fermentation, and lactation performance by dairy cows. Four multiparous (138 +/-
3 d in milk) and 4 primiparous (115 +/- 10 d in milk) Holstein cows were used in
a 4 x 4 Latin square design experiment with four 21-d periods. Each period had 14
d of adaptation and 7 d of sampling, and parity was the square. Treatments were
diets [dry matter (DM) basis] with 1) 40% alfalfa hay (ALF), 2) 24% alfalfa hay +
16% CS (CS40), 3) 20% alfalfa hay + 20% CS (CS50), and 4) 16% alfalfa hay + 24%
CS (CS60). Diets had a forage-to-concentrate ratio of 40:60 on a DM basis. Cows
had greater intake of DM and thus greater intakes of net energy for lactation,
NDF, and peNDF when CS partially replaced alfalfa hay. Replacing alfalfa hay with
CS increased daily eating and chewing times in all cows, and increased rumen pH
at 4 h postfeeding in multiparous cows. Apparent total-tract digestibility
coefficients for crude protein (CP) and NDF were not different among cows fed
ALF, CS40, and CS50, but were lower for CS60 than for ALF. Energy-corrected milk
yield was greater for CS40 and CS60 than for ALF. Milk protein yield was
increased when CS replaced 40, 50, and 60% of alfalfa hay. Milk lactose was
greater only for CS60, but milk lactose yield was greater for CS50 and CS60 than
for ALF. Milk percentage and yield of fat did not differ among treatments.
Therefore, CS partially replacing short alfalfa hay increased DM intake,
consequently increased net energy for lactation and physically effective fiber
intakes, and thus, improved milk and milk protein and lactose yields.
DOI: 10.3168/jds.2008-1302
1513. Appl Environ Microbiol. 2009 Jan;75(2):446-53. doi: 10.1128/AEM.02207-08.

Epub
2008 Nov 21.
Regulatory and DNA repair genes contribute to the desiccation resistance of

Sinorhizobium meliloti Rm1021.
Humann JL(1), Ziemkiewicz HT, Yurgel SN, Kahn ML.
Author information:
(1)Institute of Biological Chemistry, Washington State University, Pullman,
Washington 99164-6340, USA.
Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with

alfalfa after bacteria in the soil infect emerging root hairs of the growing
plant. To be successful at this, the bacteria must be able to survive in the soil
between periods of active plant growth, including when conditions are dry. The
ability of S. meliloti to withstand desiccation has been known for years, but
genes that contribute to this phenotype have not been identified. Transposon
mutagenesis was used in combination with novel screening techniques to identify
four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the
transposon insertion sites identified three genes with regulatory functions
(relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the
mutants were determined, including their behavior on several indicator media and
in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To
test the hypothesis that UvrC-related excision repair was important in
desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also
constructed. These strains were sensitive to DNA damage induced by UV light and
4-NQO and were also desiccation sensitive. These data indicate that uvr
gene-mediated DNA repair and the regulation of stress-induced pathways are
important for desiccation resistance.
DOI: 10.1128/AEM.02207-08
PMCID: PMC2620701
1514. J Anim Sci. 2009 Mar;87(3):1097-105. doi: 10.2527/jas.2008-1262. Epub 2008

Nov
21.
A protease additive increases fermentation of alfalfa diets by mixed ruminal

microorganisms in vitro.
Colombatto D(1), Beauchemin KA.
Author information:
(1)Facultad de Agronomía, Universidad de Buenos Aires, Buenos Aires, Argentina.
In vitro experiments were conducted to examine the characteristics and mode of

action of a protease that increased the ruminal fiber digestibility of alfalfa
hay. A commercial source of protease (Protex 6L, Genencor Int., Rochester, NY),
already characterized for its main activities, was further analyzed to determine
protease activity in response to pH, molecular size by SDS-PAGE, specificity to
degrade model or feed substrates, response to autoclaving, and action of specific
protease inhibitors in the absence or presence of ruminal fluid. In addition,
batch culture in vitro incubations in buffered ruminal fluid were conducted to
compare the enzyme product with purified protease sources, and dose-response
studies (0 to 10 microL/g of forage DM) were carried out using alfalfa hay as a
substrate. The enzyme product was shown to be an alkaline protease (optimum pH
>8.5) of approximately 30 kDa. Specificity in the absence of ruminal fluid showed
that the enzyme was active against gelatin and casein to the same extent, whereas
it had limited (21% of the total) activity on BSA. In the presence of ruminal
fluid and with the use of feed substrates, the protease increased (P < 0.05) 22-h
IVDMD (%) of alfalfa hay, fresh corn silage, dry-rolled corn, and a total mixed
ration composed of the 3 ingredients (39.5 vs. 44.7; 50.3 vs. 54.5; 63.8 vs.
68.4; and 55.4 vs. 56.4 for control vs. protease for each feed, respectively).
Inhibitor studies in the absence of ruminal fluid indicated that the enzyme was
inhibited most by a serine protease inhibitor but not by cysteine- or
metalloprotease inhibitors (10 vs. 1.9 and 0.1%, respectively). In the presence
of ruminal fluid, the serine protease inhibitor reversed (P < 0.05) the increase
in alfalfa IVDMD achieved by the enzyme product, such that IVDMD was similar to
that of the control treatment. Comparisons among different proteases revealed
that only pure subtilisin achieved increases in IVDMD that were similar to those
with protease, suggesting the serine protease was subtilisin-like (EC 3.4.1.62).
Dose-response studies using alfalfa hay as substrate showed quadratic responses
in IVDMD, NDF digestion, and hemicellulose and protein disappearance. It is
postulated that this enzyme acts by removing structural proteins in the cell
wall, allowing ruminal microbes to gain faster access to digestible substrates.
DOI: 10.2527/jas.2008-1262
1515. J Basic Microbiol. 2009 Apr;49(2):178-86. doi: 10.1002/jobm.200800139.
Effect of poly-3-hydroxybutyrate synthase mutation on the metabolism of Ensifer

(formerly Sinorhizobium) meliloti.
Povolo S(1), Casella S.
Author information:
(1)Dipartimento di Biotecnologie Agrarie, Università degli Studi di Padova,
Padua, Italy.
In order to investigate the effect of poly-3-hydroxybutyrate synthase mutation

(phbC) on the synthesis of exopolysaccharides (EPS) and glycogen, on the
symbiotic properties and on the survival under specific conditions of Ensifer
meliloti (formerly Sinorhizobium), a new stable phbC mutant of Ensifer meliloti
41 was isolated and characterized.Under poly-3-hydroxybutyrate accumulation
conditions, the phbC -minus mutant (strain 41003) accumulates more glycogen and
less exopolysaccharides as compared to the wild-type strain, and grows poorly in
pyruvate as carbon source. The inactivation of aniA, encoding for a global carbon
flux regulator, restores in E. meliloti 41003 the ability to grow on pyruvate,
indicating a new role for this gene. Survival studies of E. meliloti 41 and 41003
under carbon free medium in both liquid and soil microcosms showed prolonged
survival of E. meliloti 41 under these adverse conditions as compared to the
mutant strain unable to accumulate the polyester. On the other hand, the
accumulation of P(3HB) gave no significant advantage in survival under
oxygen-limiting conditions. In both strains, E. meliloti 41 and 41003,
nodule-inducing ability on alfalfa plants and acetylene reduction activity did
not significantly differ from each other, although the mutant strain was less
competitive in terms of root colonization.
((c) 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).
DOI: 10.1002/jobm.200800139
1516. Proc Natl Acad Sci U S A. 2008 Dec 2;105(48):18958-63. doi:

10.1073/pnas.0808048105. Epub 2008 Nov 19.
A mutant GlnD nitrogen sensor protein leads to a nitrogen-fixing but ineffective

Sinorhizobium meliloti symbiosis with alfalfa.
Yurgel SN(1), Kahn ML.
Author information:
(1)Institute of Biological Chemistry and School of Molecular Biosciences,
Washington State University, Pullman, WA 99164-6340, USA.
The nitrogen-fixing symbiosis between rhizobia and legume plants is a model of

coevolved nutritional complementation. The plants reduce atmospheric CO(2) by
photosynthesis and provide carbon compounds to symbiotically associated bacteria;
the rhizobia use these compounds to reduce (fix) atmospheric N(2) to ammonia, a
form of nitrogen the plants can use. A key feature of symbiotic N(2) fixation is
that N(2) fixation is uncoupled from bacterial nitrogen stress metabolism so that
the rhizobia generate "excess" ammonia and release this ammonia to the plant. In
the symbiosis between Sinorhizobium meliloti and alfalfa, mutations in GlnD, the
major bacterial nitrogen stress response sensor protein, led to a symbiosis in
which nitrogen was fixed (Fix(+)) but was not effective (Eff(-)) in substantially
increasing plant growth. Fixed (15)N(2) was transported to the shoots, but most
fixed (15)N was not present in the plant after 24 h. Analysis of free-living S.
meliloti strains with mutations in genes related to nitrogen stress response
regulation (glnD, glnB, ntrC, and ntrA) showed that catabolism of various
nitrogen-containing compounds depended on the NtrC and GlnD components of the
nitrogen stress response cascade. However, only mutants of GlnD with an amino
terminal deletion had the unusual Fix(+)Eff(-) symbiotic phenotype, and the data
suggest that these glnD mutants export fixed nitrogen in a form that the plants
cannot use. These results indicate that bacterial nitrogen stress regulation is
important to symbiotic productivity and suggest that GlnD may act in a novel way
to influence symbiotic behavior.
DOI: 10.1073/pnas.0808048105
PMCID: PMC2596199
1517. J Hazard Mater. 2009 Jun 15;165(1-3):366-71. doi:

10.1016/j.jhazmat.2008.10.024.
Epub 2008 Oct 15.
Study of the heavy metal phytoextraction capacity of two forage species growing
in an hydroponic environment.
Bonfranceschi BA(1), Flocco CG, Donati ER.
Author information:
(1)Facultad de Ciencias Exactas (UNLP), Centro de Investigación y Desarrollo en
Fermentaciones Industriales, UNLP-CCT La Plata, CONICET, La Plata, Argentina.
Sorghum and alfalfa are two important forage crops. We studied their capacity for
accumulating heavy metals in hydroponic experiments. Cadmium, nickel (as divalent
cations) and chromium (trivalent and hexavalent) were added individually to the
nutrient solution in a range of concentrations from 1 to 80 mg/l. Cr(III) was
complexed with EDTA to increase its bioavailability. In alfalfa the increases in
the concentration of Cr(III) and Cr(VI) favoured translocation of the metals to
the upper parts of the plants, while with Ni(II) the level of translocated metal
remained almost unchanged. In sorghum, both Cr(VI) and Ni(II) produced similar
results to those in alfalfa, but increases in the concentrations of Cd(II) and
Cr(III) in the solution lead to a higher accumulation of the metal at the root
level. The concentrations referred to the dry biomass of alfalfa were 500 mg/kg
(aerial parts) and 1500 mg/kg (roots) of Cr(III), simultaneously enhancing plant
growth. Sorghum captured 500 and 1100 mg/kg (in aerial parts) and 300 and 2000
mg/kg (in roots) for Ni(II) and Cd(II) respectively, without significant damage
to its biomass. The results show that alfalfa and sorghum can not only grow in
the presence of high heavy metal concentration but also capture and translocate
them to the aerial parts; because of these results special attention should be
given to these crop plants for their possible use in phytoremediation of large
contaminated areas but especially to avoid the possible introduction of the
metals accumulated in aerial parts into the food chain when those plants grow in
contaminated areas.
DOI: 10.1016/j.jhazmat.2008.10.024
1518. Int J Food Microbiol. 2009 Jan 15;128(3):453-9. doi:

10.1016/j.ijfoodmicro.2008.10.014. Epub 2008 Oct 22.
Use of bacteriophages as biocontrol agents to control Salmonella associated with

seed sprouts.
Kocharunchitt C(1), Ross T, McNeil DL.
Author information:
(1)Tasmanian Institute of Agricultural Research, University of Tasmania, Hobart
TAS 7001, Australia. ck5@utas.edu.au
Two Salmonella bacteriophages (SSP5 and SSP6) were isolated and characterized
based on their morphology and host range, and evaluated for their potential to
control Salmonella Oranienburg in vitro and on experimentally contaminated
alfalfa seeds. Phages SSP5 and SSP6 were classified as members of the Myoviridae
and Siphoviridae families, respectively. Both phages had a broad host range of
over 65% of the 41 Salmonella strains tested. During in vitro trials, the phages
resulted in incomplete lysis of Salmonella cultures, in spite of high levels of
phage remaining in the system. Phage SSP5 was more effective in reducing
Salmonella populations. Addition of phage SSP6 to alfalfa seeds previously
contaminated with S. Oranienburg caused an approximately 1 log(10) CFU g(-1)
reduction of viable Salmonella, which was achieved 3 h after phage application.
Thereafter the phage had no inhibitory effect on Salmonella population growth. A
second addition of the same (SSP6) or different (SSP5) phage to a Salmonella
culture treated with phage SSP6, did not affect Salmonella populations. It was
further shown that development of Salmonella permanently resistant to phage was
not evident in either seed or in vitro challenge trials, suggesting the existence
of a temporary, acquired, non-specific phage resistance phenomenon. These factors
may complicate the use of phages for biocontrol.
1519. Mol Microbiol. 2008 Dec;70(5):1261-73. doi: 10.1111/j.1365-2958.2008.06479.x.
The hmuUV genes of Sinorhizobium meliloti 2011 encode the permease and ATPase
components of an ABC transport system for the utilization of both haem and the
hydroxamate siderophores, ferrichrome and ferrioxamine B.
Cuív PO(1), Keogh D, Clarke P, O'Connell M.
Author information:
(1)School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Sinorhizobium meliloti, the endosymbiont of Medicago sativa, can use haem

compounds, including haemoglobin and leghaemoglobin, when growing in the
free-living state. The components of the system involved in haem acquisition were
confirmed to be ShmR, an outer membrane receptor, and HmuTUV, predicted to be an
ABC transport system comprising a periplasmic protein, a permease and an ATPase
respectively. The roles of HmuTUV in haem transport were confirmed in a
heterologous expression system in Escherichia coli in conjunction with HasR, the
outer membrane haem receptor of Serratia marcescens. hmuTUV mutants of S.
meliloti showed a reduced capacity to acquire haem, suggesting the presence of a
second haem acquisition system in the organism. S. meliloti can also acquire iron
from xenosiderophores and the genes encoding the outer membrane receptors for
ferrichrome and ferrioxamine B, fhuA1 and fhuA2, respectively, were identified.
In light of this it is proposed that fhuA2 should be renamed foxA in the S.
meliloti 1021 genome sequence. A siderophore reductase, FhuF, with the capacity
to complement an E. coli ferrioxamine B reductase mutant, was identified encoded
by a gene next to fhuA2. In the same transcriptional unit as fhuF the gene fhuP
was identified and shown to encode a protein necessary for transport of
ferrichrome and ferrioxamine B and predicted to be periplasmic. Interestingly,
the remaining components of the transport system for the siderophores are HmuU
and HmuV. Ferrichrome, ferrioxamine B and haem compounds therefore share
components of the same transport system in S. meliloti.
DOI: 10.1111/j.1365-2958.2008.06479.x
1520. Mol Biol Rep. 2009 Sep;36(7):1991-4. doi: 10.1007/s11033-008-9409-y. Epub

2008
Nov 7.
Isolation, sequence identification and tissue expression profile of two novel

soybean (glycine max) genes-vestitone reductase and chalcone reductase.
Liu GY(1).
Author information:
(1)Key Laboratory of Animal Nutrition and Feed of Yunnan Province, Yunnan
Agricultural University, Kunming, China. liuyg4567@163.com
The complete mRNA sequences of two soybean (glycine max) genes-vestitone

reductase and chalcone reductase, were amplified using the rapid amplification of
cDNA ends methods. The sequence analysis of these two genes revealed that soybean
vestitone reductase gene encodes a protein of 327 amino acids which has high
homology with the vestitone reductase of Medicago sativa (77%). The soybean
chalcone reductase gene encodes a protein of 314 amino acids that has high
homology with the chalcone reductase of kudzu vine (88%) and medicago sativa
(83%). The expression profiles of the soybean vestitone reductase and chalcone
reductase genes were studied and the results indicated that these two soybean
genes were differentially expressed in detected soybean tissues including leaves,
stems, roots, inflorescences, embryos and endosperm. Our experiment established
the foundation for further research on these two soybean genes.
DOI: 10.1007/s11033-008-9409-y
1521. Protoplasma. 1999;209(1-2):46-57. doi: 10.1007/BF01415700.
Immunolocalization of two lignin O-methyltransferases in stems of alfalfa

Kersey R(1), Inoue K, Schubert KR, Dixon RA.
Author information:
(1)Department of Botany and Microbiology, University of Oklahoma, Norman, USA.
Caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase

(CCOMT) catalyze parallel reactions that are believed to be involved in the
biosynthesis of lignin monomers. Antisera specific for alfalfa (Medicago sativa
L.) COMT or CCOMT were raised against the enzymes expressed inEscherichia coli,
and were used for immunolocalization studies in lignifying alfalfa stem tissue.
Both COMT and CCOMT were localized to xylem parenchyma cells, as assessed by
light microscopy and immunocytochemistry. Electron microscopy revealed that both
enzymes were located in the cytoplasm of xylem parenchyma cells, and to a lesser
extent, in the cytoplasm of phloem cells. There was no significant difference in
the localization pattern of COMT and CCOMT, suggesting that the two enzymes may
be part of a metabolic grid leading to production of lignin monomers in
lignifying tissue of mature alfalfa stem internodes.
DOI: 10.1007/BF01415700
PMID: 18987794
1522. J Nat Med. 2009 Apr;63(2):189-91. doi: 10.1007/s11418-008-0297-1. Epub 2008

Nov
7.
A new arylbenzofuran from the aerial parts of alfalfa.
Wang S(1), Zhang G, Guan J, Zhu L, Chen L, Pan C, Li P, Li L.
Author information:
(1)School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shenyang, China.
A new arylbenzofuran,
4-hydroxy-2-(4'-hydroxy-2'-methoxyphenyl)-6-methoxybenzofu-ran-3-carbaldehyde
(1), was isolated from the aerial parts of alfalfa (Medicago sativa L.), together
with three known compounds. The structure of the new compound (1) was determined
by spectroscopic methods.
DOI: 10.1007/s11418-008-0297-1
1523. Int J Syst Evol Microbiol. 2008 Nov;58(Pt 11):2610-8. doi:

10.1099/ijs.0.2008/000125-0.
Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov., isolated
from wild legumes growing in desert soils of Xinjiang, China.
Han TX(1), Han LL, Wu LJ, Chen WF, Sui XH, Gu JG, Wang ET, Chen WX.
Author information:
(1)Key Laboratory of Agro-Microbial Resource and Application, Ministry of
Agriculture/College of Biological Sciences, China Agricultural University,
Twenty-four Mesorhizobium strains were isolated from desert soils in the Xinjiang
region of China and were characterized by a polyphasic approach. These strains
grouped into three clusters in IGS-RFLP, SDS-PAGE analysis of whole-cell proteins
and BOX-PCR analysis, corresponding to genomic species V, VI and VII as found in
a previous study. The results were supported by sequencing analyses of rrs, IGS,
atpD and recA genes. Genospecies VII was most related to Mesorhizobium
septentrionale, while genospecies V and VI were both most closely related to
Mesorhizobium tianshanense, but were distinct from each other and from M.
tianshanense. The DNA-DNA hybridization value between the representative strain
CCBAU 83284 (genospecies VII) and the type strain of M. septentrionale was 90.1
%. Genospecies VII was thus defined as M. septentrionale. The DNA-DNA relatedness
value for representative strains of genospecies V or VI with the related
reference strains of recognized species were always lower than 60 %. Low values
of DNA-DNA hybridization (32.79 %) between representative strains of genospecies
V (CCBAU 83330(T)) and of VI (CCBAU 83306(T)) were also observed. Based upon
these results, two novel species are proposed: Mesorhizobium gobiense sp. nov.
represented by genospecies V (type strain, CCBAU 83330(T)=LMG 23949(T)=HAMBI
2974(T)) and Mesorhizobium tarimense sp. nov. represented by genospecies VI (type
strain, CCBAU 83306(T)=LMG 24338(T)=HAMBI 2973(T)). Strain CCBAU 83278 grouped as
the most peripheral member with genospecies VI in SDS-PAGE of whole-cell proteins
and BOX-PCR analysis and in the phylogenetic tree of 16S-23S rRNA intergenic
spacer (IGS) sequences. The results of analyses of rrs, atpD and recA gene
sequences, as well as those of DNA-DNA hybridization studies, strongly supported
the suggestion that this strain belonged to a species quite different from
genospecies V and VI and from any other recognized species of the genus
Mesorhizobium. As only one strain has been isolated to date, strain CCBAU 83278
was not proposed as a novel species in this study. Mesorhizobium gobiense sp.
nov. and Mesorhizobium tarimense sp. nov. could be differentiated from each other
as well as from recognized species of the genus Mesorhizobium on the basis of
phenotypic characteristics. The symbiotic loci (nodC and nifH) of the two novel
species formed two phylogenetic branches related to Mesorhizobium loti and M.
tianshanense. The type strains of the two novel species were able to nodulate
Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra and Robinia
pseudoacacia but not Astragalus membranaceus, Leucaena leucocephala, Phaseolus
vulgaris, Pisum sativum or Medicago sativa.
DOI: 10.1099/ijs.0.2008/000125-0
1524. Pak J Biol Sci. 2008 Aug 1;11(15):1923-8.
Genetic diversity of some Mediterranean populations of the cultivated alfalfa

(Medicago sativa L.) using SSR markers.
Touil L(1), Guesmi F, Fares K, Zagrouba C, Ferchichi A.
Author information:
(1)Institute of Arids Areas, Rue Eljorf Km 22.5, Medenine 4119, Tunisia.
This species study was to investigate the differentiation level among 26

populations in which 12 are locals originating from the Tunisian South and 14
introduced from Italy, Austerely, France and Morocco with two SSR markers. These
highly polymorphic and co dominant markers, together with recent population
genetic statistic extended to autotetraploids, offer tools to analyse genetic
diversity in alfalfa. The number of alleles per locus varied between 8 and 9. The
genetic similarity between these various populations is estimated by the index of
Rogers and Tanimoto. Genetic diversity is analysed by two statistical procedures:
Hierarchical classification and Correspondence Factorial Analysis (CFA). Four
large groups were obtained.
1525. Chem Pharm Bull (Tokyo). 2008 Nov;56(11):1505-14.
Engineering of plant polyketide biosynthesis.
Abe I(1).
Author information:
(1)School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
abei@u-shizuoka-ken.ac.jp
A growing number of functionally divergent the chalcone synthase (CHS)

superfamily type III polyketide synthases (PKSs) have been cloned and
characterized, which include recently obtained pentaketide chromone synthase
(PCS) and octaketide synthase (OKS) from aloe (Aloe arborescens). Recombinant PCS
expressed in Escherichia coli catalyzes iterative condensations of five molecules
of malonyl-CoA to produce a pentaketide, 5,7-dihydroxy-2-methylchromone, while
OKS carries out sequential condensations of eight molecules of malonyl-CoA to
yield aromatic octaketides, SEK4 and SEK4b, the longest polyketides generated by
the structurally simple type III PKS. The two enzymes share 91% amino acid
sequence identity, maintaining most of the active-site residues of CHS including
the Cys-His-Asn catalytic triad. One of the most characteristic features is that
the conserved Thr197 of CHS (numbering in Medicago sativa CHS) is uniquely
replaced with Met207 in PCS and with Gly207 in OKS, respectively. Site-directed
mutagenesis and X-ray crystallographic studies clearly demonstrated that the
chemically inert single residue lining the active-site cavity controls the
polyketide chain length and the product specificity depending on the steric bulk
of the side chain. Finally, on the basis of the crystal structures of both
wild-type and M207G-mutant PCS, a triple mutant PCS F80A/Y82A/M207G was
constructed and shown to catalyze condensations of nine molecules of malonyl-CoA
to produce a novel nonaketide naphthopyrone with a fused tricyclic ring system.
Structure-based engineering of the type III PKS superfamily enzymes would thus
lead to further production of chemically and structurally divergent unnatural
novel polyketides.
1526. Environ Toxicol Chem. 2009 Mar;28(3):619-28. doi: 10.1897/08-186.1. Epub 2008
Nov
3.
Application of ridge regression to quantify marginal effects of collinear soil

properties on phytoaccumulation of arsenic, cadmium, lead, and zinc.
Anderson RH(1), Basta NT.
Author information:
(1)School of Environment and Natural Resources, Ohio State University, Columbus,
Ohio 43210, USA.
Soil properties that mitigate hazardous effects of environmental contaminants

through soil chemical sequestration should be considered when evaluating
ecological risk from terrestrial contamination. The objective of this research
was to identify predominant soil chemical/physical properties that modify
phytoaccumulation of As, Cd, Pb, and Zn to the non-hyperaccumulating higher
plants: Alfalfa (Medicago sativa L.), perennial ryegrass (Lolium perenne L.), and
Japanese millet (Echinochloa crusgalli L.). Transmission coefficients were
estimated from a dose-response experiment with the use of aboveground tissue
contaminant concentrations and correlated with selected soil property
measurements to develop statistical prediction models for soil-specific
adjustments to ecological risk assessments. Significant correlations between soil
properties and transmission coefficients were observed for all four contaminants.
Intercorrelation was also observed among soil properties, including cation
exchange capacity (CEC) and soil pH (p = 0.035), CEC and total clay (p = 0.030),
organic carbon (OC) and total clay content (p = 0.085), reactive iron oxides
(FeOX) and OC (p = 0.078), and reactive Mn oxide (MnOX) and total clay content (p
< 0.001). Ridge regression, a technique that suppresses the effects of
multicollinearity and enables prediction, was used to assess the marginal
contributions of soil properties found to mitigate phytoaccumulation. Prediction
models were developed for all four contaminants. Significant variables were FeOX
for As or pH, OC, CEC, clay content, or a combination of factors for cationic
metal models. Ridge regression provides a powerful alternative to conventional
multiple regression techniques for ecotoxicological studies when intercorrelated
predictors are experimentally unavoidable, as with soil properties.
DOI: 10.1897/08-186.1
1527. J AOAC Int. 2008 Sep-Oct;91(5):1083-9.
Evaluation of sample preparation methods for the isolation of Salmonella from

alfalfa and mung bean seeds with the Bacteriological Analytical Manual's
Salmonella culture method.
Jacobson AP(1), Hammack TS, Andrews WH.
Author information:
(1)U.S. Food and Drug Administration, Center for Food Safety and Applied
Nutrition, College Park, MD 20740, USA. andrew.jacobson@fda.hhs.gov
Five pre-enrichment methods were evaluated for effectiveness with the U.S. Food
and Drug Administration's Bacteriological Analytical Manual Salmonella culture
method in recovering S. Stanley, S. Poona, and S. Muenchen from artificially
contaminated alfalfa seeds, and S. Saintpaul, S. Anatum, and S. Infantis from
artificially contaminated mung bean seeds. The methods included: (1) Soak.--Test
portions were inoculated into pre-enrichment media; (2) Rinse.--Test portions
were rinsed with pre-enrichment media, and the media was decanted from the test
portions; (3) Rinsed seed.--Pre-enrichment media was added to the test portions
that were rinsed in the rinse method; (4) Wet blend.--Test portions were blended
with the pre-enrichment media; and (5) Dry blend.--Test portions were blended
prior to pre-enrichment. The methods of pre-enrichment were also evaluated for
effectiveness in recovering Pantoea agglomerans from alfalfa and mung bean seeds
with a modified culture method for the recovery of Enterobacteriaceae from foods.
The purpose of these studies was to provide a model for the recovery of
Salmonella that may occur in seeds as a natural contaminant. The relative
effectiveness of the soak method was consistently superior to the rinse method in
isolating the selected Salmonella serovars from both seed types. Statistically,
the rinsed seed method was as effective as the soak method in all trials, except
1 of 3, with S. Muenchen and alfalfa seeds (P > 0.05). The relative effectiveness
of the methods in isolating P. agglomerans from alfalfa and mung bean seeds was
similar to that observed with the artificially contaminated test portions. The
soak method was consistently the most effective method and the rinse method was
consistently the most ineffective method. The rinsed seed, wet blend, and dry
blend methods were also as effective as the soak method in all 3 trials with each
seed type (P > 0.05).
1528. Plant Physiol. 2009 Jan;149(1):306-17. doi: 10.1104/pp.108.131540. Epub 2008

Oct
31.
Antiquity and function of CASTOR and POLLUX, the twin ion channel-encoding genes
key to the evolution of root symbioses in plants.
Chen C(1), Fan C, Gao M, Zhu H.
Author information:
Kentucky 40546, USA.
Root symbioses with arbuscular mycorrhizal fungi and rhizobial bacteria share a
common signaling pathway in legumes. Among the common symbiosis genes are CASTOR
and POLLUX, the twin homologous genes in Lotus japonicus that encode putative ion
channel proteins. Here, we show that the orthologs of CASTOR and POLLUX are
ubiquitously present and highly conserved in both legumes and nonlegumes. Using
rice (Oryza sativa) as a study system, we employ reverse genetic tools (knockout
mutants and RNA interference) to demonstrate that Os-CASTOR and Os-POLLUX are
indispensable for mycorrhizal symbiosis in rice. Furthermore, a cross-species
complementation test indicates that Os-POLLUX can restore nodulation, but not
rhizobial infection, to a Medicago truncatula dmi1 mutant.
DOI: 10.1104/pp.108.131540
PMCID: PMC2613720
1529. Environ Pollut. 2009 May;157(5):1445-52. doi: 10.1016/j.envpol.2008.09.013.

Epub
2008 Nov 1.
Responses to ozone pollution of alfalfa exposed to increasing salinity levels.
Maggio A(1), Chiarandà FQ, Cefariello R, Fagnano M.
Author information:
(1)DIAAT, Naples University Federico II, via Università 100, 80055 Portici (NA),
Italy.
Stomatal closure and biosynthesis of antioxidant molecules are two fundamental

components of the physiological machinery that lead to stress adaptation during
plant's exposure to salinity. Since high stomatal resistance may also contribute
in counteracting O(3) damages, we hypothesized that soil salinization may
increase O(3) tolerance of crops. An experiment was performed with alfalfa grown
in filtered (AOT40=0 in both years) and non-filtered (AOT40=9.7 in 2005 and 6.9
ppm h in 2006) open-top chambers. Alfalfa yield was reduced by O(3) (-33%) only
in plants irrigated with salt-free water, while the increasing levels of soil
salinity until 1.06 dS m(-1) reduced both stomatal conductance and plant O(3)
uptake, thus linearly reducing O(3) effects on yield. Therefore a reliable
flux-based model for assessing the effects of O(3) on crop yield should take into
account soil salinity.
DOI: 10.1016/j.envpol.2008.09.013
1530. Environ Microbiol. 2008 Nov;10(11):2922-30. doi:

10.1111/j.1462-2920.2008.01762.x.
Influence of different Sinorhizobium meliloti inocula on abundance of genes

involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago
sativa L.).
Babić KH(1), Schauss K, Hai B, Sikora S, Redzepović S, Radl V, Schloter M.
Author information:
(1)Department of Terrestrial Ecogenetics, Helmholtz Zentrum München, German
Research Center for Environmental Health, Institute of Soil Ecology,
Ingolstädter, Neuherberg, Germany. khuic@agr.hr
Inoculation of leguminous seeds with selected rhizobial strains is practised in

agriculture to ameliorate the plant yield by enhanced root nodulation and
nitrogen uptake of the plant. However, effective symbiosis between legumes and
rhizobia does not only depend on the capacity of nitrogen fixation but also on
the entire nitrogen turnover in the rhizosphere. We investigated the influence of
seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting
different efficiency concerning plant growth promotion on nitrogen turnover
processes in the rhizosphere during the growth of alfalfa. Quantification of six
target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within
the nitrogen cycle was performed in rhizosphere samples before nodule formation,
at bud development and at the late flowering stage. The results clearly
demonstrated that effectiveness of rhizobial inocula is related to abundance of
nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved
in nitrogen turnover had been affected by the inocula, e.g. higher numbers of
amoA copies were observed during flowering when the more effective strain had
been inoculated. However, the respective gene abundances differed overall to a
greater extent between the three plant development stages than between the
inoculation variants.
DOI: 10.1111/j.1462-2920.2008.01762.x
1531. Int J Food Microbiol. 2008 Dec 10;128(2):348-53. doi:

10.1016/j.ijfoodmicro.2008.09.011. Epub 2008 Sep 30.
Potential application of high hydrostatic pressure to eliminate Escherichia coli

O157:H7 on alfalfa sprouted seeds.
Neetoo H(1), Ye M, Chen H.
Author information:
19716-2150, USA.
Sprouts eaten raw are increasingly being perceived as hazardous foods as they
have been implicated in Escherichia coli O157:H7 outbreaks where the seeds were
found to be the likely source of contamination. The objective of our study was to
evaluate the potential of using high hydrostatic pressure (HHP) technology for
alfalfa seed decontamination. Alfalfa seeds inoculated with a cocktail of five
strains of E. coli O157:H7 were subjected to pressures of 500 and 600 MPa for 2
min at 20 degrees C in a dry or wet (immersed in water) state. Immersing seeds in
water during pressurization considerably enhanced inactivation of E. coli O157:H7
achieving reductions of 3.5 log and 5.7 log at 500 and 600 MPa, respectively.
When dry seeds were pressurized, both pressure levels reduced the counts by <0.7
log. To test the efficacy of HHP to completely decontaminate seeds whilst meeting
the FDA requirement of 5 log reductions, seeds inoculated with a ~5 log CFU/g of
E. coli O157:H7 were pressure-treated at 600 and 650 MPa at 20 degrees C for
holding times of 2 to 20 min. A >5 log reduction in the population was achieved
when 600 MPa was applied for durations of > or =6 min although survivors were
still detected by enrichment. When the pressure was stepped up to 650 MPa, the
threshold time required to achieve complete elimination was 15 min. Un-inoculated
seeds pressure-treated at 650 MPa for 15 min at 20 degrees C successfully
sprouted achieving a germination rate identical to untreated seeds after eight
days of sprouting. These results therefore demonstrate the promising application
of HHP on alfalfa seeds to eliminate the risk of E. coli O157:H7 infections
associated with consumption of raw alfalfa sprouts.
1532. Phytopathology. 2000 Jun;90(6):657-65. doi: 10.1094/PHYTO.2000.90.6.657.
Regression Analyses for Evaluating the Influence of Bacillus cereus on Alfalfa

Yield Under Variable Disease Intensity.
Kazmar ER, Goodman RM, Grau CR, Johnson DW, Nordheim EV, Undersander DJ,
Handelsman J.
ABSTRACT We developed and tested regression methods to exploit the variability in

disease inherent in field experiments, and applied the methods to evaluate
strains of Bacillus cereus for biocontrol efficacy. Four B. cereus strains were
tested for their effect on alfalfa (Medicago sativa) performance in 16 field
trials planted during 1993 to 1996 at multiple sites in Wisconsin. To evaluate
performance of the strains, we used the ratio of (metalaxyl response)/(untreated
control response) as a measure of disease intensity within the experiments. The
ratio of (Bacillus response)/(untreated control response) was then regressed as a
function of disease intensity. The slope of the resulting line provides a
statistical test to compare performance of the Bacillus strain with that of the
untreated seed (H(o): slope = 0) and metalaxyl controls (H(o): slope = 1). Under
conditions in which disease occurred, forage yield of plots planted with seed
treated with B. cereus strain AS4-12 exceeded yield from the untreated control
plots (P = 0.002) and was similar to yield of plots planted with
metalaxyl-treated seed (P = 0.14). Yield gain associated with AS4-12 and
metalaxyl seed treatment averaged 6.1 +/- 2.8% (+/-standard error) and 3.0 +/-
2.8%, respectively. In contrast to the regression approach, means analysis by
analysis of variance did not detect differences among treatments. Three other B.
cereus strains either did not increase alfalfa yield or increased yield less than
did AS4-12. Metalaxyl and three of the Bacillus strains increased seedling
emergence, but the improved stands were not predictive of increased forage yield.
In six additional studies conducted for one season in 1997, AS4-12 enhanced yield
of two cultivars at diverse locations in Wisconsin, but there was an apparent
cultivar-location interaction. A strong correlation between response to AS4-12
and metalaxyl treatment suggests that these treatments controlled similar
pathogens, most likely the oomycete pathogens Phytophthora medicaginis and
Pythium spp.
DOI: 10.1094/PHYTO.2000.90.6.657
PMID: 18944546
1533. Phytopathology. 2002 Oct;92(10):1038-47. doi: 10.1094/PHYTO.2002.92.10.1038.
Molecular identification of a new phytoplasma associated with alfalfa

witches'-broom in oman.
Khan AJ, Botti S, Al-Subhi AM, Gundersen-Rindal DE, Bertaccini AF.

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical
of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and
interior regions of the Sultanate of Oman. Phytoplasmas were detected from all
symptomatic samples by the specific amplification of their 16S-23S rRNA gene.
Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer
pairs, consistently amplified a product of expected lengths when DNA extract from
symptomatic samples was used as template. Asymptomatic plant samples and the
negative control yielded no amplification. Restriction fragment length
polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7
primer pair identified phytoplasmas belonging to peanut witches'-broom group
(16SrII or faba bean phyllody). Restriction enzyme profiles showed that the
phytoplasmas detected in all 300 samples belonged to the same ribosomal group.
Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I,
Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is
different from all the other phytoplasmas employed belonging to subgroup 16SrII,
except tomato big bud phytoplasma from Australia, and could be therefore
classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma
P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp
product. The homology search showed 99% similarity (1,667 of 1,690 base identity)
with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A
phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas,
mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma
species, with closest relationships to PapayaYC phytoplasmas from New Zealand and
Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable
from them considering the different associated plant hosts and the extreme
geographical isolation.
DOI: 10.1094/PHYTO.2002.92.10.1038
PMID: 18944213
1534. Phytopathology. 2006 Dec;96(12):1388-96. doi: 10.1094/PHYTO-96-1388.
Interactions Among a Soil Organic Amendment, Nematodes, and the Nematode-Trapping

Fungus Dactylellina candidum.
Jaffee BA.
ABSTRACT When alfalfa leaves (Medicago sativa) are added to soil, both the
nematode-trapping fungus Dactylellina candidum and microbivorous nematodes
increase. To determine whether the response of the fungus to alfalfa depends on
consumption of bacterivorous and fungivorous nematodes, soil microcosm
experiments were performed. D. candidum did not increase if alfalfa leaves were
added to soil lacking nematodes, but did increase if nematodes were added to soil
lacking alfalfa leaves. Although these results indicate that the response of D.
candidum to alfalfa depends on nematodes, D. candidum and microbivorous nematodes
did not exhibit classical predator-prey dynamics (i.e., D. candidum seldom
reduced numbers of resident nematodes and, after initially increasing in alfalfa
amended soil, numbers of D. candidum then decreased, whereas numbers of nematodes
continued to increase). Fungivorous nematodes were abun dant in alfalfa-amended
soil, and their potential to suppress trapping fungi requires more research.
DOI: 10.1094/PHYTO-96-1388
PMID: 18943672
1535. Phytopathology. 2006 Dec;96(12):1330-6. doi: 10.1094/PHYTO-96-1330.
Identification of Sources of Resistance to Phoma medicaginis Isolates in Medicago

truncatula SARDI Core Collection Accessions, and Multigene Differentiation of
Isolates.
Ellwood SR, Kamphuis LG, Oliver RP.
ABSTRACT Phoma medicaginis is a necrotrophic fungal pathogen, commonly found

infecting the annual medic Medicago truncatula. To differentiate eight P.
medicaginis isolates, five gene regions were examined: actin, beta-tubulin,
calmodulin, translation elongation factor 1-alpha (EF-1alpha), and the internal
transcribed spacer ribosomal DNA. Sequence comparisons showed that specimens
isolated from M. truncatula in Western Australia formed a group that was
consistently different from, but allied to, a P. medicaginis var. medicaginis
type specimen. EF-1alpha contained a hyper-variable 55-bp repeat unit, which
forms the basis of a rapid polymerase chain reaction-based method of reliably
distinguishing isolates. Characterization of three isolates showed that all
exhibited a narrow host range, causing disease only in M. sativa and M.
truncatula among eight commonly cultivated legume species sampled. Infection of
86 M. truncatula single-seeded accessions showed a continuous distribution in
disease phenotypes, with the majority of accessions susceptible. On a 1-to-5
disease reaction scale increasing in severity, individual fungal isolates showed
means of 2.6 to 3.2, and scores ranged from 1 to 4.8 among accessions. The
results presented here suggest that M. truncatula harbors specific and diverse
sources of resistance to individual P. medicaginis genotypes.
DOI: 10.1094/PHYTO-96-1330
PMID: 18943665
1536. Phytopathology. 2008 Oct;98(10):1075-83. doi: 10.1094/PHYTO-98-10-1075.
Occurrence and characterization of a Phytophthora sp. pathogenic to asparagus

(Asparagus officinalis) in Michigan.
Saude C(1), Hurtado-Gonzales OP, Lamour KH, Hausbeck MK.
Author information:
(1)Department of Plant Pathology, Michigan State University, East Lansing
48824-1311, USA.
A homothallic Phytophthora sp. was recovered from asparagus (Asparagus

officinalis) spears, storage roots, crowns, and stems in northwest and central
Michigan in 2004 and 2005. Isolates (n = 131) produced ovoid, nonpapillate,
noncaducous sporangia 45 microm long x 26 microm wide and amphigynous oospores of
25 to 30 microm diameter. Mycelial growth was optimum at 25 degrees C with no
growth at 5 and 30 degrees C. All isolates were sensitive to 100 ppm mefenoxam.
Pathogenicity studies confirmed the ability of the isolates to infect asparagus
as well as cucurbits. Amplified fragment length polymorphism analysis of 99
isolates revealed identical fingerprints, with 12 clearly resolved fragments
present and no clearly resolved polymorphic fragments, suggesting a single clonal
lineage. The internal transcribed spacer regions of representative isolates were
homologous with a Phytophthora sp. isolated from diseased asparagus in France and
a Phytophthora sp. from agave in Australia. Phylogenetic analysis supports the
conclusion that the Phytophthora sp. isolated from asparagus in Michigan is a
distinct species, and has been named Phytophthora asparagi.
DOI: 10.1094/PHYTO-98-10-1075
1537. Phytopathology. 2003 Jul;93(7):799-804. doi: 10.1094/PHYTO.2003.93.7.799.
Expression of Oryzacystatin I and II in Alfalfa Increases Resistance to the

Root-Lesion Nematode.
Samac DA, Smigocki AC.
ABSTRACT Digestive cysteine proteinases have been isolated from plant-parasitic

nematodes as well as coleopteran and hemipteran insects. Phytocystatins,
inhibitors of cysteine proteinases, are found in a number of plants where they
may play a role in defense against pathogens and pests. The cDNAs of the
phytocystatins from rice, oryzacystatin I (OC-I) and oryzacystatin II (OC-II),
were expressed in alfalfa (Medicago sativa) plants under the control of the
potato protease inhibitor II (PinII) promoter and the plants were evaluated for
resistance to the root-lesion nematode (Pratylenchus penetrans). A
PinII-beta-glucuronidase (GUS) gene was introduced into alfalfa to determine the
pattern of gene expression from this promoter. Constitutive GUS expression was
observed in leaf and root vascular tissue, and in some plants, expression was
observed in leaf mesophyll cells. Mechanical wounding of leaves increased GUS
expression approximately twofold over 24 h. Inoculation with root-lesion
nematodes resulted in localized GUS expression. Populations of root-lesion
nematodes in alfalfa roots from one line containing the PinII::OC-I transgene and
one line containing the PinII::OC-II transgene were reduced 29 and 32%,
respectively, compared with a transgenic control line. These results suggest that
oryzacystatins have the potential to confer increased resistance to the
root-lesion nematode in alfalfa.
DOI: 10.1094/PHYTO.2003.93.7.799
PMID: 18943160
1538. Poult Sci. 2008 Nov;87(11):2178-85. doi: 10.3382/ps.2008-00034.
The effects of alfalfa-based molt diets on skeletal integrity of white leghorns.
Kim WK(1), Herfel TM, Dunkley CS, Hester PY, Crenshaw TD, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station
77843-2472, USA.
Sixty White Leghorn hens were assigned to 1 of 6 treatments: pretrial control

(PC), full fed (FF), 9-d feed withdrawal (FW), 90% alfalfa-10% layer ration
(A90), 80% alfalfa-20% layer ration (A80), and 70% alfalfa/30% layer ration
(A70). For the PC, hens were killed and bones collected immediately before light
restriction. The FF hens were also subjected to light restriction. After hens
were fasted or fed different molting treatment diets for 9 d, they were fed a
maintenance diet for 14 d. At 23 d postmolt, hens were killed and bones
collected. Feed intakes per hen of the FF, A90, A80, and A70 treatments during a
9-d molting period were 97, 17, 44, and 46 g, respectively. Tibia dry weight of
the PC was greater than the FF group. The PC group had significantly greater
tibia ash weight than the FF, FW, A90, and A80 groups. Tibia ash concentration of
the PC group was significantly greater than the FF, A90, and A80 groups. Humerus
dry weights of the PC group were greater than the other groups except for hens on
FW. Humerus ash weight was lower for FF hens as compared with PC hens, but other
molting treatments had humerus ash weights similar to PC hens. Tibia mineral
content and density of the PC were significantly greater than the other
treatments. The FF group exhibited significantly lower humerus mineral content
and density compared with those of the PC group. The PC and A70 hens exhibited
significantly greater ultimate bending moment and ultimate stress compared with
the FF and A90 hens. The modulus of elasticity of the PC hens was greater than
that of the FF and A90. There were no significant differences in modulus of
elasticity among the PC, FW, A80, and A70 hens. In conclusion, hens at 23 d
postmolt experienced deterioration in skeletal integrity. This decrease in bone
quality occurred in all molted hens. However, A70 and A80 alfalfa-fed hens
retained mechanical properties of bones compared with the pretrial control.
DOI: 10.3382/ps.2008-00034
1539. Pak J Pharm Sci. 2008 Oct;21(4):460-4.
Effects of alfalfa on lipoproteins and fatty streak formation in

hypercholesterolemic rabbits.
Asgary S(1), Moshtaghian J, Hosseini M, Siadat H.
Author information:
(1)Isfahan Cardiovascular Research Center, Applied Physiology Research Center,
Isfahan University of Medical Science, Isfahan, Iran. sasgary2yahoo.com
Alfalfa (Medicago sativa) is a leguminous plant with high contents of

phytoestrogen and saponin which are both useful in preventing cardiovascular
disorders. This study was designed to evaluate the preventive effect of dietary
alfalfa on the development and the progression of atherosclerosis in
hyperlipidemic rabbits. Twenty male rabbits were obtained and kept under standard
conditions. After 2 weeks of accommodation to the new place, the animals were
semi-randomly distributed into four groups of five each. Animals in Group 1
received basic diet and the ones in Group 2 received the same diet to which 1%
cholesterol was added. Groups 3 and 4 received similar diets as Groups 1 and 2
respectively, but supplemented with alfalfa. Each of the four groups was fed with
its respected diet for a period of 12 weeks. Fasting blood samples were collected
from all animals at the beginning and at the end of the study and blood factors
were measured. At the end of the study samples from the right and the left
coronary arteries as well as the aorta were collected from all animals for
pathological evaluations. Though dietary alfalfa decreased total cholesterol, LDL
and triglyceride and this effect was not significant, but dietary alfalfa
significantly increased HDL. The formation of fatty streaks in the aorta, the
right and the left coronary arteries were significantly reduced under the
influence of dietary alfalfa. The indication that dietary alfalfa may have
preventive effects on the progression of fatty streak formation calls for more
studies to clarify the mechanisms of the effect.

Epub
2008 Oct 16.
Expression of a putative alfalfa helicase increases tolerance to abiotic stress

in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic
adjustment.
Luo Y(1), Liu YB, Dong YX, Gao XQ, Zhang XS.
Author information:
(1)State Key Laboratory of Crop Biology, College of Life Sciences, Shandong
Agricultural University, Taian, Shandong, PR China.
Plant helicases are known to be involved in salinity and low-temperature

tolerance. However, a functional involvement of helicases in the antioxidative
response of plants has not been described. We have isolated a DEAD-box-containing
cDNA sequence from Medicago sativa (alfalfa) that is a homolog of the pea DNA
helicase 45 (PDH45) and named it M. sativa helicase 1 (MH1). Transient
transfection of 35S::MH1-GFP to onion epidermis revealed that MH1 was localized
in the nucleus. Expression of MH1 was detected in roots, stems and leaves of
alfalfa. Furthermore, real-time PCR analysis revealed that mannitol, NaCl, methyl
viologen and abscisic acid induced the expression of MH1. The ectopic expression
of MH1 in Arabidopsis improved seed germination and plant growth under drought,
salt and oxidative stress. The capacity for osmotic adjustment, superoxide
dismutase (SOD) and ascorbate peroxidase (APX) activities and proline content
were also elevated in the transgenic Arabidopsis plants. Our results suggest that
MH1 responds to reactive oxygen species (ROS) and functions in drought and salt
stress tolerance by enhancing the capacities for ROS scavenging and osmotic
adjustment.
DOI: 10.1016/j.jplph.2008.06.018
1541. Roum Arch Microbiol Immunol. 2007 Jul-Dec;66(3-4):90-6.
Fungal control of pathogenic fungi isolated from wild plants in Taif Governorate,
Saudia Arabia.
Abou-Zeid AM(1), Altalhi AD, Abd El-Fattah RI.
Author information:
(1)Biology department, Faculty of Science, Taif University, Saudi Arabia. P.B.
abouzeid_alaa@hotmail.com
Twenty two plants were collected from Taif Governorate and identified as:
Euphorbia glomerifera, Juniperus procera, Launaea mucronata, Capparis dcidua,
Punica granatum, Opuntia ficus, Prunus persica, Eucalyptus globulus, Medicago
sativa, Artemisia monosperma, Trichodesma calathiforme, Artemisia judaica,
Foeniculum vulgare, Phagnalon sinaicum, Rumex dentatus, Asphodelus aestives,
Pulicaria crispa, Launae sonchoides, Forsskaolea tenacissima, Arnebia
hispidissima, Avena spp and Aerva lanata. Pathogenic fungi were isolated from
some of these plants and identified as Alternaria alternate, Ulocladium botrytis,
Cladosporium spp, Cephalosporium spp, Penicillium chrysogenum, Fusarium oxysporum
and Humicola grisea. Four antagonistic isolates were tested, 2 from Gliocladium
fungus and 2 from Trichoderma fungus. We found that all the four antagonistic
isolates (G. deliquescens, G. virens, T. viride and T. hamatum) significantly
inhibited the radial growth of the pathogenic fungi tested, with different
ratios. The results indicated that the antibiotics produced by the antagonists
were more effective than the fungus itself and differ with different fungi.
Coating plant stems with antagonists or with antagonist extracts reduce the
severity of the disease but not prevent it in all tested pathogens.
1542. Microb Pathog. 2008 Nov-Dec;45(5-6):394-402. doi:

10.1016/j.micpath.2008.09.002.
Epub 2008 Sep 27.
Characterization of Brucella abortus sigma factor sigma54 (rpoN): genetic

complementation of Sinorhizobium meliloti ntrA mutant.
Iannino F(1), Ugalde RA, Iñón de Iannino N.
Author information:
(1)Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de
Chascomús (IIB-INTECH), Consejo de Investigaciones Científicas y Técnicas,
Universidad Nacional de General San Martín (CONICET-UNSAM), Av. Gral Paz 5445,
Buenos Aires, Argentina.
The intracellular pathogen Brucella abortus has an alternative sigma factor

sigma54 (RpoN) highly similar to Sinorhizobium meliloti NtrA. RpoN was described
to be required for the transcription of a wide range of genes involved in diverse
physiological functions including the regulation of virulence-related factors in
both plants and animal pathogens. B. abortus rpoN gene restored the normal growth
of an S. meliloti ntrA mutant in minimal media with succinic acid as a sole
carbon source as well as the formation of functional nodules in alfalfa, thus
revealing that the gene is functional. B. abortus rpoN mutant and B. abortus
wild-type strain harboring a multicopy plasmid coding for a wild-type rpoN gene
displayed reduced survival under stationary-phase conditions suggesting that
expression of RpoN must be tightly regulated. Real-time PCR analysis revealed
that B. abortus rpoN expression is downregulated during the stationary phase of
growth. This regulation is absent in the rpoN mutant background, indicating that
RpoN regulates its own expression. Intracellular multiplication in HeLa or J774
cells, and survival in BALB/c mice of the rpoN mutant, are not affected. However
2weeks postinfection survival of rpoN mutant complemented with a multicopy
plasmid containing a wild-type rpoN gene is reduced, thus suggesting that
overexpression of rpoN may misregulate the expression of genes involved in this
stage of infection.
DOI: 10.1016/j.micpath.2008.09.002
1543. Genome. 2008 Oct;51(10):856-69. doi: 10.1139/G08-069.
Structural and expressional divergence of genes encoding O-methyltransferase in

wheat.
Jung JH(1), Hong MJ, Kim DY, Kim JY, Heo HY, Kim TH, Jang CS, Seo YW.
Author information:
(1)Bio Resources Research Center, KT&G Central Research Institute, 434
Dangsu-Dong, Gwonsun-Gu, Suwon, Gyunggi 441-480, Republic of Korea.
Enzymatic methylation, which is catalyzed by the large number of

O-methyltransferases (OMTs), is one of the important reactions in the flow of
primary and (or) secondary metabolism. In a previous study, the gene TaOMT1 was
induced by Hessian fly infestation of a wheat-rye translocation line. In this
study we considered other wheat OMT genes - TaOMT3, TaOMT4, and TaOMT5 - using a
bioinformatics approach and examined the TaOMT genes for their genomic
organization, tissue-specific expression, responses to abiotic stresses and
hormones, and cis-elements. There appeared to be a homoeologous relationship
between TaOMT4 (6DS) and TaOMT5 (6BS), whereas TaOMT1 and TaOMT3 were placed on
chromosome arms 7BL and 5DL, respectively. Differences in the tissue-specific,
constitutive, and stress-inducible expression patterns among the TaOMT genes were
found in both healthy and stressed plants. A number of cis-elements, which are
potentially correlated with the responses of the TaOMT genes, were detected in
the analysis of the TaOMT promoter sequences. In addition, evolutionary
perspectives of the TaOMT genes are discussed. The nucleotide sequences have been
deposited in the GenBank database under accession Nos. AAP23942 (TaCOMT1),
EF423610 (TaOMT5), EF423611 (TaOMT4), EF423612 (TaOMT3), EU831287 (5' upstream of
TaOMT1), EU831288 (5' upstream of TaOMT3), EU831289 (5' upstream of TaOMT4), and
EU831290 (5' upstream of TaOMT5).
DOI: 10.1139/G08-069
1544. Environ Sci Technol. 2008 Sep 15;42(18):6917-21.
No long-term persistence of bacterial pollution-induced community tolerance in

tylosin-polluted soil.
Demoling LA(1), Bååth E.
Author information:
(1)Department of Microbial Ecology, Ecology Building, Lund University, SE-223 62
Lund, Sweden. Louise.Alden@mbioekol.lu.se
Pollution-induced community tolerance (PICT) of soil bacteria to the antibiotic

tylosin was studied over 95 days. Tylosin was added at increasing concentrations,
together with different amounts of alfalfa to study the effects of substrate
addition on PICT and bacterial growth in soil. The leucine incorporation
technique was used to estimate bacterial growth and as a detection method in the
PICT concept. Direct inhibition of the bacterial growth rates, resulting in a
dose-response curve, was found above 50 mg of tylosin kg(-1) of soil two days
after tylosin addition (IC50 value of 960 mg tylosin kg(-1)). After 10 days of
exposure to at least 50 mg of tylosin kg(-1), the PICT was observed and
correlated to inhibition of bacterial growth by tylosin. A return of the PICT to
control levels was found over time, and after 95 days at 1500 mg of tylosin
kg(-1), essentially no PICT was found, as compared to the unpolluted control
soil. The return of PICT to pre-exposure levels was not totally reflected in the
recovery of bacterial growth. Alfalfa addition did not affect the inhibitory
effect of tylosin on bacterial growth rates; neither did it alter the PICT. Since
tylosin is relatively rapidly degraded in soil, our results indicate that the
PICT will return to prepollution levels when the selective pressure of the
toxicant is removed and will thus be a useful technique for monitoring
remediation measures.
1545. J Agric Food Chem. 2008 Nov 12;56(21):9797-802. doi: 10.1021/jf800888r. Epub
2008
Oct 8.
Interference of condensed tannin in lignin analyses of dry bean and forage crops.
Marles MA(1), Coulman BE, Bett KE.
Author information:
(1)Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive,
Saskatoon, SK, Canada S7N 5A8. susan.marles@usask.ca
Legumes with high concentrations of condensed tannin (pinto bean [Phaseolus

vulgaris L.], sainfoin [Onobrychis viciifolia Scop.], and big trefoil [Lotus
uliginosus Hoff.]), were compared to a selection of forages, with low or zero
condensed tannin (smooth bromegrass [ Bromus inermis Leyss], Lotus japonicus
[Regel] K. Larsen, and alfalfa [Medicago sativa L.]), using four methods to
estimate fiber or lignin. Protocols were validated by using semipurified
condensed tannin polymers in adulteration assays that tested low-lignin tissue
with polyphenolic-enriched samples. The effect on lignin assay methods by
condensed tannin concentration was interpreted using a multivariate analysis.
There was an overestimation of fiber or lignin in the presence of condensed
tannin in the acid detergent fiber (ADF) and Klason lignin (KL) assays compared
to that in the thioglycolic acid (TGA) and acid detergent lignin (ADL) methods.
Sulfite reagents (present in TGA lignin method) or sequential acidic digests at
high temperatures (ADF followed by ADL) were required to eliminate condensed
tannin. The ADF (alone) and KL protocols are not recommended to screen nonwoody
plants, such as forages, where condensed tannin has accumulated in the tissue.
DOI: 10.1021/jf800888r
Alfalfa cut at sundown and harvested as baleage improves milk yield of

late-lactation dairy cows.
Brito AF(1), Tremblay GF, Bertrand A, Castonguay Y, Bélanger G, Michaud R,

Lapierre H, Benchaar C, Petit HV, Ouellet DR, Berthiaume R.
Author information:
Canada, Sherbrooke, Québec, Canada.
Alfalfa (Medicago sativa L.) cut at sundown has been shown to contain greater
concentration of total nonstructural carbohydrates (TNC) than that cut at sunup.
Fourteen multiparous (8 ruminally cannulated) and 2 primiparous lactating dairy
cows were randomly assigned to 2 treatments in a crossover design (2 periods of
24 d) to investigate the effects of alfalfa daytime cutting management on ruminal
metabolism, nutrient digestibility, N balance, and milk yield. Half of each
alfalfa field (total of 3 fields) was cut at sundown (PM) after a sunny day,
whereas the second half was cut at sunup (AM) on the following day. Both PM and
AM cuts were field-wilted and harvested as baleage (531 +/- 15.0 g of dry
matter/kg of fresh matter). Bales (PM and AM) were ranked according to their
concentrations of TNC, paired, and each pair of PM and AM baleages was then
assigned to each experimental day (total of 48 d). The difference in TNC
concentration between PM and AM baleages fed during the 10 d of data and sample
collection varied from -10 to 50 g/kg of dry matter. Each pair of baleage was fed
ad libitum to cows once daily with no concentrate. Ruminal molar proportion of
acetate and total volatile fatty acid concentration were greater in animals fed
the AM baleage, whereas the proportion of valerate was greater with PM baleage;
no other significant changes in ruminal molar proportions of volatile fatty acids
were observed between forage treatments. Digestible organic matter intake,
organic matter digestibility, and plasma Lys concentration were significantly
greater in cows fed PM alfalfa, suggesting that more nutrients were available for
milk synthesis. Significantly lower body weight gain and retained N as a
proportion of N intake were observed in cows fed PM alfalfa, thus suggesting that
nutrients were channeled to milk synthesis rather than to body reserves. Intake
of dry matter (+1.0 kg/d), and yields of milk (+1.0 kg/d), milk fat (+70 g/d),
and milk protein (+40 g/d) were significantly greater in cows fed PM vs. AM
alfalfa. Concentration of milk urea N and excretion of urea N as a proportion of
total urinary N were significantly reduced, and milk N efficiency was increased
when feeding PM vs. AM alfalfa, indicating an improvement in N utilization.
Increasing the TNC concentration of alfalfa by shifting forage cutting from sunup
to sundown improved N utilization and milk production in late-lactation dairy
cows.
DOI: 10.3168/jds.2008-1282
1547. Plant J. 2009 Feb;57(3):542-54. doi: 10.1111/j.1365-313X.2008.03699.x. Epub

2008
Oct 30.
Effective, homogeneous and transient interference with cytosine methylation in

plant genomic DNA by zebularine.
Baubec T(1), Pecinka A, Rozhon W, Mittelsten Scheid O.
Author information:
(1)Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of
Sciences, Dr Bohr-Gasse 3, 1030 Vienna, Austria.
Covalent modification by methylation of cytosine residues represents an important

epigenetic hallmark. While sequence analysis after bisulphite conversion allows
correlative analyses with single-base resolution, functional analysis by
interference with DNA methylation is less precise, due to the complexity of
methylation enzymes and their targets. A cytidine analogue, 5-azacytidine, is
frequently used as an inhibitor of DNA methyltransferases, but its rapid
degradation in aqueous solution is problematic for culture periods of longer than
a few hours. Application of zebularine, a more stable cytidine analogue with a
similar mode of action that is successfully used as a methylation inhibitor in
Neurospora and mammalian tumour cell lines, can significantly reduce DNA
methylation in plants in a dose-dependent and transient manner independent of
sequence context. Demethylation is connected with transcriptional reactivation
and partial decondensation of heterochromatin. Zebularine represents a promising
new and versatile tool for investigating the role of DNA methylation in plants
with regard to transcriptional control, maintenance and formation of (hetero-)
chromatin.
DOI: 10.1111/j.1365-313X.2008.03699.x
PMCID: PMC2667684
1548. Pak J Biol Sci. 2008 Apr 15;11(8):1165-8.
Effects of gamma irradiation on ruminal DM and NDF degradation kinetics of

alfalfa hay.
Shahbazi HR(1), Sadeghi AA, Shawrang P, Raisali G.
Author information:
(1)Department of Veterinary Science, Islamic Azad University, Kerman-Shah Branch,
P.O. Box 67155-1774, Kerman-Shah, Iran.
The effects of gamma irradiation on ruminal dry matter, Neutral Detergent Fiber
(NDF) degradation of alfalfa hay were investigated. Alfalfa hay samples were
irradiated by gamma irradiator at doses of 50, 100 and 150 kGy under identical
conditions of temperature and humidity. Nylon bags of untreated or irradiated
samples were suspended in the rumen of three Taleshi bulls for up to 96 h and
resulting data were fitted to non-linear degradation model to calculate
degradation parameters. Results indicated that the washout fractions of dry
matter and NDF increased linearly (p < 0.001) with increasing irradiation dose.
The b fraction and the degradation rate of the b fraction (c) of DM and NDF were
the highest at 50 kGy dose. Effective degradability of DM and NDF increased
linearly with increasing irradiation dose. Gamma irradiation at doses of 50, 100
and 150 kGy increased the effective NDF degradability of alfalfa hay at rumen
outflow rate of 0.05 h(-1) by about 8, 11 and 12%, respectively. Gamma
irradiation affects on the hydrogenic bonds and with theirs breakdowning causes
the wander-valls power weaken, that results in the degradation of cellulose and
increasing of DM and NDF degradability.
1549. Biochem Biophys Res Commun. 2008 Nov 28;376(4):700-5. doi:

10.1016/j.bbrc.2008.09.064. Epub 2008 Sep 24.
Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7

MADS domain associated protein.
Páez-Valencia J(1), Valencia-Mayoral P, Sánchez-Gómez C, Contreras-Ramos A,

Hernández-Lucas I, Martínez-Barajas E, Gamboa-DeBuen A.
Author information:
(1)Departamento de Ecología Funcional, Instituto de Ecología, Universidad
Nacional Autónoma de México, Ciudad Universitaria 04510, México DF, Mexico.
We are interested in identifying proteins that interact with the MADS domain
protein NMH7 of Medicago sativa. We use an affinity column with a synthetic
peptide derived from the MADS domain of NMH7 which has been reported to mediate
protein-protein interaction with non-MADS domain interacting proteins. We
identified approximately 40 and approximately 80kDa specifically bound proteins
as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase
cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions
of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic
activity is not required for the interaction with NMH7. NMH7 and aldolase were
coimmunoprecipitated from non-inoculated seed and seedlings extracts.
Colocalization studies using confocal microscopy showed that aldolase and NMH7
are localized in the cytoplasm and the nucleus of the cortical cells. These data
together show that M. sativa aldolase is a novel MADS domain binding protein, and
suggest a broader functional repertory for this enzyme, as has been proposed for
other glycolytic enzymes.
DOI: 10.1016/j.bbrc.2008.09.064
1550. J Environ Qual. 2008 Sep-Oct;37(5 Suppl):S169-79. doi: 10.2134/jeq2007.0468.
Infiltration into cropped soils: effect of rain and sodium adsorption

ratio-impacted irrigation water.
Suarez DL(1), Wood JD, Lesch SM.
Author information:
(1)USDA-ARS, Salinity Lab., 450 W. Big Springs Road, Riverside, CA 92507, USA.
donald.suarez@ars.usda.gov
The sodium adsorption ratio (SAR) and salinity criteria for water suitability for
irrigation have been developed for conditions where irrigation water is the only
water source. It is not clear that these criteria are applicable to environments
where there is a combination of rain and irrigation during the growing season.
The interaction of rainfall with irrigation water is expected to result in
increased sodicity hazard because of the low electrical conductivity of rain. In
this study we examined the effects of irrigation waters of SAR 2, 4, 6, 8, and 10
mmol(1/2) L(-1/2) and electrical conductivities of 1 and 2 dS m(-1) on the
infiltration rate of two soils with alternating cycles of rain (simulated with a
rainfall sprinkler) and irrigation water, separated by drying cycles. The
infiltration rate of surface samples from two soils, Kobase silty clay (fine,
smectitic, frigid, Torrertic Haplustept) and Glendive very fine sandy loam
(coarse-loamy, mixed superactive, calcareous, frigid Aridic Ustifluvent) were
evaluated under alfalfa (Medicago sativa) cropped conditions for over 140 d and
under full canopy cover. Reductions in infiltration were observed for both soils
for SAR above 2, and the reductions became more severe with increasing SAR.
Saturated hydraulic conductivity measurements taken from undisturbed cores at the
end of the experiment were highly variable, suggesting that in situ infiltration
measurements may be preferred when evaluating SAR effects.
DOI: 10.2134/jeq2007.0468
Diversity cascades in alfalfa fields: from plant quality to agroecosystem

diversity.
Pearson CV(1), Massad TJ, Dyer LA.
Author information:
(1)Tulane University, 400 Lindy Boggs Bldg., New Orleans, LA 70118, USA.
To examine top-down and bottom-up influences on managed terrestrial communities,

we manipulated plant resources and arthropod abundance in alfalfa (Medicago
sativa L.) fields. We modified arthropod communities using three nonfactorial
manipulations: pitfall traps to remove selected arthropods, wooden crates to
create habitat heterogeneity, and an arthropod removal treatment using a
reversible leaf blower. These manipulations were crossed with fertilizer
additions, which were applied to half of the plots. We found strong effects of
fertilizer on plant quality and biomass, and these effects cascaded up to
increase herbivore abundance and diversity. The predator community also exhibited
a consistent positive effect on the maintenance of herbivore species richness and
abundance. These top-down changes in arthropods did not cascade down to affect
plant biomass; however, plant quality (saponin content) increased with higher
herbivore densities. These results corroborate previous studies in alfalfa that
show complex indirect effects, such as trophic cascades, can operate in
agricultural systems, but the specifics of the interactions depend on the
assemblages of arthropods involved.

1552. FEMS Microbiol Lett. 2008 Nov;288(1):40-6. doi: 10.1111/j.1574-
6968.2008.01329.x.
Diversity of Sinorhizobium strains nodulating Medicago sativa from different

Iranian regions.
Talebi MB, Bahar M, Saeidi G, Mengoni A, Bazzicalupo M.
Alfalfa is believed to have originated in north-western Iran and has a long

history of coexistence with its bacterial symbiont Sinorhizobium in soils of
Iran. However, little is known about the diversity of Sinorhizobium strains
nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations
were sampled from eight different Iranian sites using three cultivars of Medicago
sativa as trap host plants. A total of 982 rhizobial strains were isolated and
species were identified showing a large prevalence of Sinorhizobium meliloti over
Sinorhizobium medicae. Analysis of salt tolerance demonstrated a great phenotypic
diversity. The genetic diversity of the Sinorhizobium isolates was analysed using
BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Patterns
ofBOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the
role of plant variety and site of origin in the genetic variance observed.
Results indicated that most of the total molecular variance was attributable to
divergence among strains isolated from different sites and cultivars
(intrapopulation, strain-by-strain variance). Moreover, the analysis showed the
presence of two geographic populations (west and northwest), indicating that the
effect of the site of origin could be more relevant in shaping population genetic
diversity than the effect of cultivar or individual plant.
DOI: 10.1111/j.1574-6968.2008.01329.x
1553. J Proteome Res. 2008 Oct;7(10):4508-20. doi: 10.1021/pr800291z. Epub 2008 Sep
10.
Characterization of the secretome of suspension cultures of Medicago species

reveals proteins important for defense and development.
Kusumawati L(1), Imin N, Djordjevic MA.
Author information:
(1)Australian Research Council Centre of Excellence for Integrative Legume
Research, Genomic Interactions Group, Research School of Biological Sciences,
Australian National University, Canberra ACT 2601, Australia.
Molecular events occurring in the plant apoplast contribute to important

developmental and defense responses. To define the secretome of Medicago, we used
suspension cultures to isolate and identify secreted proteins as a first step to
determining their functions. Proteins in the extracellular medium of the
suspension cultures were examined using SDS-PAGE, tandem mass spectrometry
(MALDI-TOF/TOF) and bioinformatics tools. There were 39 proteins identified in
the cultures derived from M. sativa, M. truncatula 2HA (an embryogenic line), and
M. truncatula sickle (an ethylene-insensitive mutant). N-Terminal secretion
signals were detected in 34 proteins and five other proteins were predicted to be
secreted via a nonclassical (ER-independent) route. All samples possessed defense
related proteins including pathogenesis related (PR) proteins. The glycoprotein,
SIEP1L, was found only in M. sativa. Three secreted proteinases were identified
in M. truncatula, including a serine carboxypeptidase detected only in 2HA. Some
proteins were unique to a cell culture line. Quantitative real time RT-PCR was
used to determine mRNA expression of selected genes corresponding to proteins
found only in 2HA or sickle or in both. The results correlate well with the
proteomic data. For instance, a GDSL-lipase gene known to be regulated by
ethylene was found only in 2HA but not in the ethylene insensitive mutant.
Similarly, the PR1a protein, expressed from a well recognized ethylene-regulated
gene, was found in 2HA but not sickle. These experiments indicate that the
suspension culture systems established here are useful to avoid contamination
from cytoplasmic proteins and to identify secreted proteins in Medicago, and
should have application in other plant systems.
DOI: 10.1021/pr800291z
1554. New Phytol. 2008;180(2):311-5. doi: 10.1111/j.1469-8137.2008.02612.x. Epub

2008
Aug 29.
OsIPD3, an ortholog of the Medicago truncatula DMI3 interacting protein IPD3, is

required for mycorrhizal symbiosis in rice.
Chen C(1), Ané JM, Zhu H.
Author information:
(1)Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
40546, USA.
Medicago truncatula IPD3 (MtIPD3) is an interacting protein of DMI3 (does not

make infections 3), a Ca(2+)/calmodulin-dependent protein kinase (CCaMK)
essential for both arbuscular mycorrhizal (AM) and rhizobial symbioses. However,
the function of MtIPD3 in root symbioses has not been demonstrated in M.
truncatula, because of a lack of knockout mutants for functional analysis. In
this study, the availability of IPD3 knockout mutants in rice (Oryza sativa) was
exploited to test the function of OsIPD3 in AM symbiosis. Three independent
retrotransposon Tos17 insertion lines of OsIPD3 were selected and the phenotypes
characterized upon inoculation with the AM fungus Glomus intraradices. Phenotypic
and genetic analyses revealed that the Osipd3 mutants were unable to establish a
symbiotic association with G. intraradices. In conclusion, IPD3 represents a
novel gene required for root symbiosis with AM fungi in plants.
DOI: 10.1111/j.1469-8137.2008.02612.x
1555. Talanta. 2008 Sep 15;76(5):1130-5. doi: 10.1016/j.talanta.2008.05.013. Epub

2008
May 21.
Detection and quantification of additives (urea, biuret and poultry litter) in

alfalfas by NIR spectroscopy with fibre-optic probe.
González-Martín I(1), Hernández-Hierro JM.
Author information:
(1)Departamento de Química Analítica, Nutrición y Bromatología, Facultad de CC,
Químicas, Universidad de Salamanca, C/Plaza de la Merced s/n, 37008 Salamanca,
Spain.
The additives (urea, biuret and poultry litter) present in alfalfa, which
contribute non-proteic nitrogen, were analysed using near infrared spectroscopy
(NIRS) technology together with a remote reflectance fibre-optic probe. We used
75 samples of known alfalfa without additives and 75 samples with each of the
additives, urea (0.01-10%), biuret (0.01-10%) and poultry litter (1-25%). Using
the discriminant partial least squares (DPLS) algorithm, the presence or absence
of the additives urea, biuret and poultry litter is classified and predicted with
a high prediction rate of 96.9%, 100% and 100%, obtaining the equations of
discrimination for each additive. The regression method employed for the
quantification was modified partial least squares (MPLS). The equations were
developed using the fibre-optic probe to determine the content of urea, biuret
and poultry litter with multiple correlation coefficients (RSQ) and prediction
corrected standard errors (SEP (C)) of 0.990, 0.28% for urea, 0.991, 0.29% for
biuret and 0.925, 2.08% for poultry litter. The work permits the instantaneous
and simultaneous prediction and determination of urea, biuret and poultry litter
in alfalfas, applying the fibre-optic directly on the ground samples of alfalfa.
DOI: 10.1016/j.talanta.2008.05.013
1556. Trop Anim Health Prod. 2009 Apr;41(4):579-86. doi: 10.1007/s11250-008-9227-1.

Epub 2008 Aug 31.
Effect of supplementing urea-treated barley straw with lucerne or vetch hays on

feed intake, digestibility and growth of Arsi Bale sheep.
Abate D(1), Melaku S.
Author information:
(1)Sinana Agricultural Research Center, P.O. Box 208, Bale Robe, Ethiopia.
The study was conducted at Sinana Agricultural Research Center, Ethiopia to

assess the supplementation of graded levels of vetch (Vicia dasycarpa 'lana') and
lucerne (Medicago sativa,' Hunter river') hay on feed intake, digestibility and
body weight (BW) change of Arsi-Bale sheep fed urea treated barley straw (UTBS).
A 7 day- digestibility and a 90 day- feed intake trials were conducted using 28
and 35 sheep, respectively. The experimental design was a randomized complete
block design with seven dietary treatments that consisted of feeding UTBS (T1) as
the control treatment, UTBS plus 150, 250 and 350 g dry matter (DM) per day of
vetch for T2, T3, T4, respectively and UTBS plus 150, 250 and 350 g DM per day of
lucerne for T5, T6 and T7, respectively. Intake of UTBS was not affected (P >
0.05) by inclusion of lucerne hay at 25-35% of daily DM intake. The supplements
increased daily intake of total DM, organic matter (OM), neutral detergent fiber
(NDF), acid detergent fiber (ADF) and metabolizable energy (ME) (P < 0.001) as
well as apparent digestibility of DM, OM (P < 0.001), NDF (P < 0.01), ADF, crude
protein (CP) (P < 0.05) and daily BW gain (P < 0.001). Supplementation with
lucerne than vetch hay promoted higher (P < 0.001) CP and ME intakes and daily BW
gain. Feeding with the UTBS without supplementation was enough to meet the
maintenance requirements of the sheep and allow small BW gain. The results of the
study showed that urea treatment of barley straw in conjunction with
supplementation of lucerne or vetch hay could serve as a useful strategy in
improving smallholder sheep production in the tropics.
DOI: 10.1007/s11250-008-9227-1
1557. Animal. 2008 Sep;2(9):1331-9. doi: 10.1017/S1751731108002450.

In vivo and in situ measurements of the digestive characteristics of sainfoin in
comparison with lucerne fed to sheep as fresh forages at two growth stages and as
hay.
Aufrère J(1), Dudilieu M, Poncet C.
Author information:
(1)1Herbivore Research Unit, INRA-Theix, 63122 St - Genes Champanelle, France.
In vivo and in situ digestive characteristics of sainfoin (Onobrychis viciifolia

L., a tannin-rich forage) and lucerne (Medicago sativa L., a tannin-free forage)
were compared to evaluate the effects of condensed tannins (CT) and growth stage
(vegetative v. early flowering) in experiment 1. In experiment 2, the hays of the
two forages, harvested at early flowering, were compared. Ingestibility, organic
matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed
sainfoin and lucerne fresh forages and hays. The loss of dry matter (DM) and N
from polyester bags suspended in the rumen, abomasum and small intestine was also
measured using rumen fistulated sheep and other intestine fistulated sheep.
Nitrogen content was lower in sainfoin than in lucerne. Content of CT in sainfoin
decreased with growth stage (3.5 to 2.5 g CT/kg DM) and was lower for sainfoin
hay (0.6 g CT/kg DM). Ingestibility and OMD did not differ between fresh-fed
forage species. Total N tract digestibility in vivo was much lower for sainfoin
than for lucerne fresh forages (mean value 0.540 v. 0.721, P < 0.001) and for
sainfoin hay than lucerne hay (0.464 v. 0.683, P < 0.001). In both species, N
digestibility was not altered by growth stage. The rumen degradation of N was
lower in sainfoin than in lucerne, resulting in a lower proportion of N intake
excreted in urine. The intestinal digestibility of sainfoin was also lower than
that of lucerne, resulting in a higher N excretion in faeces. Hence the
efficiency of N utilisation by sheep (ENr) was similar (mean value 0.205 and
0.199 g N retained/g N intake for fresh sainfoin and lucerne, respectively). The
coefficient of N retention by the animal was higher for sainfoin at the
vegetative stage than for all the other forages. Nitrogen degradability in the
rumen determined by the nylon bag technique (DegN) was lower for sainfoin than
for lucerne when forages were studied both fresh (mean value 0.608 and 0.818,
respectively) and as hays (0.631 and 0.767). The efficiency of forage N digestion
(ENd) was higher for sainfoin at the vegetative stage. Compared with lucerne,
sainfoin greatly increased the in situ estimate of forage N escaping the rumen
but decreased its intestinal digestibility.
DOI: 10.1017/S1751731108002450
PMID: 22443822

Epub
2008 Aug 27.
An allelopathic substance in red pine needles (Pinus densiflora).
Kato-Noguchi H(1), Fushimi Y, Shigemori H.
Author information:
University, Miki, Kagawa, Japan. hisashi@ag.kagawa-u.ac.jp
Aqueous methanol extracts of red pine (Pinus densiflora) needles inhibited the
growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa),
alfalfa (Medicago sativa), ryegrass (Lolium multiflorum), timothy (Pheleum
pratense), Digitaria sanguinalis and Echinochloa crus-galli. Increasing the
extract concentration increased inhibition, suggesting that the pine needles may
have growth inhibitory substances and possess allelopathic potential. The aqueous
methanol extract of the pine needles was purified, and a main inhibitory
substance was isolated and determined by spectral data as
9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid. This substance inhibited root
and shoot growth of cress and Echinochloa crus-galli seedlings at concentrations
greater than 0.1 mM. The endogenous concentration of the substance was 0.13
mmol/kg pine needle. These results suggest that
9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth
inhibitory effect of the pine needles and may play an important role in the
allelopathy of red pine.
DOI: 10.1016/j.jplph.2008.06.012
1559. Sci China C Life Sci. 2000 Aug;43(4):387-94. doi: 10.1007/BF02879303.
Regeneration of foreign genes co-transformed plants of Medicago sativa L by

Agrobacterium rhizogenes.
Lü D(1), Cao X, Tang S, Tian X.
Author information:
(1)Institute of Genetics, Chinese Academy of Sciences, Beijing, China.
Gene encoding sulphur amino acid-rich protein (HNP) and rol genes were
transferred into Medicago sativa L (alfalfa) mediated by Agrobacterium
tumafeciens. Regeneration of transgenic plants was induced successfully from
hairy root tissue of cotyledon in alfalfa. Cotyledon tissues were an ideally
transformed recipient. There was a negative correlation between age of hairy
roots and embryogenesis frequency in alfalfa. Production of co-transformed plants
with greater yield and super quality was important for development of new alfalfa
varieties.
DOI: 10.1007/BF02879303
PMID: 18726342
1560. J Food Prot. 2008 Aug;71(8):1556-62.
Comparison of the stress response of Listeria monocytogenes strains with sprout

colonization.
Gorski L(1), Flaherty D, Duhé JM.
Author information:
(1)U.S. Department of Agriculture, Agricultural Research Service, Produce Safety
and Microbiology Research Unit, 800 Buchanan Street, Albany, California 94710,
USA. lgorski@pw.usda.gov
Twenty-nine strains of the foodborne pathogen Listeria monocytogenes were tested

for their ability to colonize alfalfa, radish, and broccoli sprouts and their
capacity to withstand acid and oxidative stress, two stresses common to the
sprouting environment. Wide variation in the ability of different strains to
colonize alfalfa sprouts were confirmed, but the variations among radish and
broccoli sprouts were not as large. With a few exceptions, strains that were poor
colonizers of alfalfa tended to be among the poorer colonizers of radish and
broccoli and vice versa. The strains also were variable in their resistance to
both acid and oxidative stress. Statistical analysis revealed no correlation
between acid stress and sprout colonization, but there was a positive correlation
between resistance to oxidative stress and colonization of all three sprout
types. Although the response to oxidative stress is important for L.
monocytogenes virulence, it also may be important for life outside of a host.
1561. Proc Natl Acad Sci U S A. 2008 Aug 26;105(34):12164-9. doi:

10.1073/pnas.0802518105. Epub 2008 Aug 21.
Alfalfa benefits from Medicago truncatula: the RCT1 gene from M. truncatula
confers broad-spectrum resistance to anthracnose in alfalfa.
Yang S(1), Gao M, Xu C, Gao J, Deshpande S, Lin S, Roe BA, Zhu H.
Author information:
(1)Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY
40546, USA.
Alfalfa is economically the most important forage legume worldwide. A recurrent

challenge to alfalfa production is the significant yield loss caused by disease.
Although knowledge of molecular mechanisms underlying host resistance should
facilitate the genetic improvement of alfalfa, the acquisition of such knowledge
is hampered by alfalfa's tetrasomic inheritance and outcrossing nature. However,
alfalfa is congeneric with the reference legume Medicago truncatula, providing an
opportunity to use M. truncatula as a surrogate to clone the counterparts of many
agronomically important genes in alfalfa. In particular, the high degree of
sequence identity and remarkably conserved genome structure and function between
the two species enables M. truncatula genes to be used directly in alfalfa
improvement. Here we report the map-based cloning of RCT1, a host resistance (R)
gene in M. truncatula that confers resistance to multiple races of Colletotrichum
trifolii, a hemibiotrophic fungal pathogen that causes anthracnose disease of
alfalfa. RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding
site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers
broad-spectrum anthracnose resistance when transferred into susceptible alfalfa
plants. Thus, RCT1 provides a novel resource to develop anthracnose-resistant
alfalfa cultivars and contributes to our understanding of host resistance against
the fungal genus Colletotrichum. This work demonstrates the potential of using M.
truncatula genes for genetic improvement of alfalfa.
DOI: 10.1073/pnas.0802518105
PMCID: PMC2527883

Aug
15.
Sinorhizobium meliloti mutants deficient in phosphatidylserine decarboxylase

accumulate phosphatidylserine and are strongly affected during symbiosis with
alfalfa.
Vences-Guzmán MA(1), Geiger O, Sohlenkamp C.

Author information:
(1)Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av.
Universidad s/n, Apdo. Postal 565-A, Cuernavaca, Morelos CP62210, México.
Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin,

phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids.
PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss)
condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the
second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to
form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A
sinorhizobial mutant deficient in psd is unable to form PE but accumulates the
anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or
Psd were compared with those of the S. meliloti wild type. Whereas both
PE-deficient mutants grew in a wild-type-like manner on many complex media, they
were unable to grow on minimal medium containing high phosphate concentrations.
Surprisingly, the psd-deficient mutant could grow on minimal medium containing
low concentrations of inorganic phosphate, while the pss-deficient mutant could
not. Addition of choline to the minimal medium rescued growth of the
pss-deficient mutant, CS111, to some extent but inhibited growth of the
psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were
analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with
their alfalfa host plant, they behaved strikingly differently. The Pss-deficient
mutant, CS111, initiated nodule formation at about the same time point as the
wild type but did form about 30% fewer nodules than the wild type. In contrast,
the PS-accumulating mutant, MAV01, initiated nodule formation much later than the
wild type and formed 90% fewer nodules than the wild type. The few nodules formed
by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen.
Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish,
indicating that the plants were starved for nitrogen. Therefore, changes in lipid
composition, including the accumulation of bacterial PS, prevent the
establishment of a nitrogen-fixing root nodule symbiosis.
DOI: 10.1128/JB.00610-08
PMCID: PMC2566212

10.1111/j.1439-0396.2008.00848.x. Epub 2008 Aug 12.
The effect of mixing and changing the order of feeding oats and chopped alfalfa
to horses on: glycaemic and insulinaemic responses, and breath hydrogen and
methane production.
Vervuert I(1), Voigt K, Hollands T, Cuddeford D, Coenen M.
Author information:
Veterinary Medicine, University of Leipzig, Leipzig, Germany.
Ingrid.Vervuert@vetmed.uni-leipzig.de
The aim of this study was to investigate the effects of feeding oats alone before
or after feeding chopped alfalfa or, in admixture with the alfalfa on the
glycaemic and insulinaemic responses of horses as well as post-prandial breath
hydrogen and methane excretion. Horses were fed in a randomized order, chopped
alfalfa as a source of dietary fibre and unprocessed oats as a source of starch.
Chopped alfalfa intake was adjusted to a crude fibre intake of 0.5 g/kg
bodyweight (BW) per meal and the oats intake was adjusted to a starch intake of 2
g/kg BW per meal. The feeds were offered in three different ways: (i) alfalfa
followed by oats (A/O), (ii) oats followed by alfalfa (O/A) or (iii) a mixture of
alfalfa and oats (A + O). Oats alone were used as a control. Blood and breath
were collected after the test meal was fed at the end of a 11.5-h overnight fast
following a 10-day acclimatization period. The highest glycaemic and insulinaemic
responses were measured when the A/O and O/A diets orders were fed, whereas most
hydrogen was produced after feeding oats alone. It was concluded that adding
alfalfa chaff to a meal of oats prolonged the pre-caecal digestion of starch, but
there was no evidence for any effect on pre-caecal starch digestibility.
DOI: 10.1111/j.1439-0396.2008.00848.x
1564. Mol Plant Microbe Interact. 2008 Sep;21(9):1232-41. doi: 10.1094/MPMI-21-9-

1232.
Auxotrophy accounts for nodulation defect of most Sinorhizobium meliloti mutants

in the branched-chain amino acid biosynthesis pathway.
de las Nieves Peltzer M(1), Roques N, Poinsot V, Aguilar OM, Batut J, Capela D.
Author information:
(1)Instituto de Biotecnologia y Biologia Molecular, CCT-La Plata, Universidad
Nacional de La Plata, Facultad de Ciencias Exactas, La Plata, Argentina.
Some Sinorhizobium meliloti mutants in genes involved in isoleucine, valine, and

leucine biosynthesis were previously described as being unable to induce nodule
formation on host plants. Here, we present a reappraisal of the interconnection
between the branched-chain amino acid biosynthesis pathway and the nodulation
process in S. meliloti. We characterized the symbiotic phenotype of seven mutants
that are auxotrophic for isoleucine, valine, or leucine in two closely related S.
meliloti strains, 1021 and 2011. We showed that all mutants were similarly
impaired for nodulation and infection of the Medicago sativa host plant. In most
cases, the nodulation phenotype was fully restored by the addition of the missing
amino acids to the plant growth medium. This strongly suggests that auxotrophy is
the cause of the nodulation defect of these mutants. However, we confirmed
previous findings that ilvC and ilvD2 mutants in the S. meliloti 1021 genetic
background could not be restored to nodulation by supplementation with exogenous
amino acids even though their Nod factor production appeared to be normal.
DOI: 10.1094/MPMI-21-9-1232
1565. J Environ Qual. 2008 Aug 8;37(5):1701-9. doi: 10.2134/jeq2007.0521. Print

2008
Sep-Oct.
Partitioning species variability from soil property effects on phytotoxicity: ECx

normalization using a plant contaminant sensitivity index.
Anderson RH(1), Basta NT, Lanno RP.
Author information:
(1)School of Environment and Natural Resources, Ohio State Univ., 2021 Coffey
Rd., Columbus, OH 43210, USA.
Soil properties mitigate hazardous effects of contaminants through soil chemical

sequestration and should be considered when evaluating ecological risk from
terrestrial contamination. Empirical models that quantify relationships between
soil properties and toxicity to ecological receptors are necessary for
site-specific adjustments to ecological risk assessments. However, differential
sensitivities of test organisms in dose-response studies may limit the utility of
such models. We present a novel approach to toxicity estimation that partitions
the effect of differential sensitivities of test organisms from that of soil
chemical/physical properties. Five soils that ranged in selected properties were
spiked with five concentrations of sodium arsenate. Bioassays were conducted
where above ground dry matter growth and the corresponding tissue arsenic
concentrations were evaluated for three terrestrial plants (Alfalfa, Medicago
sativa L.; Perennial ryegrass, Lolium perrene L.; and Japanese millet,
Echinochloa crusgalli L.). Estimates were combined into a plant contaminant
sensitivity index (PCSI) and used to normalize phytotoxicity parameters to the
most sensitive species (i.e., alfalfa) where necessary. Simple linear regression
and ANCOVA indicated a 36.5% increase in the explanatory power of the modifying
effects of soil properties on phytotoxicity when differential arsenate
sensitivities were accounted for by PCSI (r(2) = 0.477-0.833). Normalization of
ecotoxicity parameters by PCSI is a seemingly effective approach to quantify the
modifying effects of soil properties on phytotoxicity endpoints when it is of
interest to consider multiple plant species (or varieties within a species) with
differential sensitivities to experimental contaminants.
DOI: 10.2134/jeq2007.0521
1566. J Integr Plant Biol. 2008 Jan;50(1):29-39. doi: 10.1111/j.1744-

7909.2007.00607.x.
Eco-physiological characteristics of alfalfa seedlings in response to various

mixed salt-alkaline stresses.
Peng YL(1), Gao ZW, Gao Y, Liu GF, Sheng LX, Wang DL.
Author information:
(1)Institute of Grassland Science, Northeast Normal University, Key Laboratory of
Vegetation Ecology, Ministry of Education, Changchun 130024, China.
Soil salinization and alkalization frequently co-occur in nature, but little is

known about the mixed effects of salt-alkaline stresses on plants. An experiment
with mixed salts (NaCl, Na(2)SO(4), NaHCO(3) and Na(2)CO(3)) and 30 salt-alkaline
combinations (salinity 24-120 mmol/L and pH 7.03-10.32) treating Medicago sativa
seedlings was conducted. The results demonstrated that salinity and alkalinity
significantly affected total biomass and biomass components of seedlings. There
were interactive effects of salt composition and concentration on biomass (P<or=
0.001). The interactions between salinity and alkalinity stresses led to changes
in the root activity along the salinity gradient (P<or= 0.001). The effects of
alkalinity on seedling survival rate were more significant than those of
salinity, and the seedlings demonstrated some physiological responses (leaf
electrolyte leakage rate and proline content) in order to adapt to mixed
salt-alkaline stresses. It was concluded that the mixed salt-alkaline stresses,
which differ from either salt or alkali stress, emphasize the significant
interaction between salt concentration (salinity) and salt component
(alkalinity). Further, the effects of the interaction between high alkalinity and
salinity are more severe than those of either salt or alkali stress, and such a
cooperative interaction results in more sensitive responses of ecological and
physiological characteristics in plants.
DOI: 10.1111/j.1744-7909.2007.00607.x
Utilization of kura clover-reed canarygrass silage versus alfalfa silage by

Kammes KL(1), Heemink GB, Albrecht KA, Combs DK.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison, Wisconsin
53706, USA.
The mixture of kura clover (Trifolium ambiguum M. Bieb.) and reed canarygrass
(Phalaris arundinacea L.) has proven to be extremely persistent in the northern
United States, but information about dairy cow performance on this mixture is
lacking. Twenty lactating Holstein cows were used in a crossover design to
compare dry matter (DM) intake and milk production from diets containing kura
clover-reed canarygrass silage (KRS) or alfalfa (Medicago sativa L.) silage (AS).
Forages were cut, wilted, ensiled in horizontal plastic bags, and allowed to
ferment for at least 50 d before beginning the feeding experiment. The KRS was
approximately 40% kura clover and 60% reed canarygrass. Treatments were total
mixed rations formulated with either 57% of total DM from 1) AS or 2) KRS.
Experimental periods were 28 d, with the first 14 d for diet adaptation and the
last 14 d for measurement of intake and milk production. The neutral detergent
fiber (NDF) concentrations of AS and KRS were 37.3 and 47.3%, respectively. The
fermentation analyses indicated that both silages underwent a restricted
fermentation, producing primarily lactic acid and some acetic acid. Dry matter
intake (24.2 vs. 22.8 kg) and 4% fat-corrected milk (32.8 vs. 30.9 kg) were
significantly higher for cows fed AS than for cows fed KRS. Cows consumed less
NDF (6.7 vs. 8.0 kg) and less digestible NDF (3.0 vs. 4.4 kg) when fed AS diets
compared with KRS diets, but the pool of ruminally undegraded NDF was similar
(3.7 kg) between diets. Cows produced 1.5 kg of milk/kg of DM consumed regardless
of the diet, indicating that digestible NDF of KRS was utilized with similar
efficiency as the cell wall constituents of AS, but the intake of cows fed KRS
may have been limited by rumen fill. Milk fat concentration tended to be higher
for cows fed AS, but the milk true protein concentration and yields of fat and
protein did not differ by treatment. Milk urea nitrogen content was higher when
cows consumed AS (16.4 mg/ dL) compared with KRS (13.4 mg/dL). The cows fed KRS
consumed more NDF but less total DMI, based on the results from this trial with
diets formulated to contain approximately 60% of DM as forage, resulting in
slightly lower milk yields than cows fed excellent-quality AS. This grass-legume
mixture has the potential to be a source of quality forage for dairy cows in
regions where alfalfa persistence is a problem.
DOI: 10.3168/jds.2007-0865
1568. Evolution. 2008 Oct;62(10):2508-24. doi: 10.1111/j.1558-5646.2008.00468.x.

Epub
2008 Aug 28.
Population differentiation and genetic variation in performance on eight hosts in

the pea aphid complex.
Ferrari J(1), Via S, Godfray HC.

Author information:
(1)Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS,
United Kingdom. julia.ferrari@zoo.ox.ac.uk
Phytophagous insects frequently use multiple host-plant species leading to the

evolution of specialized host-adapted populations and sometimes eventually to
speciation. Some insects are confronted with a large number of host-plant
species, which may provide complex routes of gene flow between host-adapted
populations. The pea aphid (Acyrthosiphon pisum) attacks a broad range of plants
in the Fabaceae and it is known that populations on Trifolium pratense and
Medicago sativa can be highly specialized at exploiting these species. To find
out whether adaptation to a broad range of co-occurring hosts has occurred, we
tested the performance of pea aphid clones collected from eight host-plant genera
on all of these plants in a reciprocal transfer experiment. We provide evidence
for pervasive host-plant specialization. The high performance of all aphid clones
on Vicia faba suggests that this host plant could be a site of gene flow between
different populations that could limit further host-associated divergence. The
genetic variance in host-plant usage was partitioned into within- and
among-population components, which represent different levels of host adaptation.
Little evidence of within-population trade-offs in performance on different plant
species was found.
DOI: 10.1111/j.1558-5646.2008.00468.x
1569. J Biol Chem. 2008 Sep 12;283(37):25247-55. doi: 10.1074/jbc.M801967200. Epub

2008
Jul 17.
Root-microbe communication through protein secretion.
De-la-Peña C(1), Lei Z, Watson BS, Sumner LW, Vivanco JM.
Author information:
(1)Department of Horticulture and Landscape Architecture and Center for
Rhizosphere Biology, Colorado State University, Fort Collins, CO 80523, USA.
Biotic interactions in the rhizosphere are biologically important, and although

many of those interactions have been well studied, the role of secreted proteins
in the cross-talk between microbes and roots has not been investigated. Here,
protein secretion was studied during the communication between the roots of two
plants (Medicago sativa and Arabidopsis thaliana) and the bacterial symbiont of
one of these species (Sinorhizobium meliloti strain Rm1021) and an opportunistic
bacterial pathogen of A. thaliana (Pseudomonas syringae pv. tomato DC3000) using
a proteomic approach. It was found that protein exudation in the M. sativa-S.
meliloti interaction caused an increase in the secretion of seven plant proteins,
such as hydrolases, peptidases, and peroxidases among others in two or more time
points compared with the plant control. In addition, four proteins, all of
bacterial origin, were increased 1.5-fold more in this interaction compared with
S. meliloti alone. However, these proteins were not induced when M. sativa was
inoculated with P. syringae DC3000. The interaction between A. thaliana and P.
syringae DC3000 highly induced the secretion of several plant proteins related to
defense soon after initial contact with P. syringae, but these proteins were not
secreted in the incompatible interaction with S. meliloti. The results of this
study reveal a specific, protein level cross-talk between roots and microbes.
These results suggest that secreted proteins may be a critical component in the
process of signaling and recognition that occurs between compatible and
incompatible interactions.
DOI: 10.1074/jbc.M801967200
1570. Int J Food Microbiol. 2008 Sep 30;127(1-2):37-42. doi:

10.1016/j.ijfoodmicro.2008.06.008. Epub 2008 Jun 12.
Presence and potential for horizontal transfer of antibiotic resistance in

oxidase-positive bacteria populating raw salad vegetables.
Bezanson GS(1), MacInnis R, Potter G, Hughes T.
Author information:
(1)Food Safety and Quality Program, Atlantic Food and Horticulture Research
Centre, Agriculture and AgriFood Canada, Kentville, Nova Scotia, Canada B4N
1J5(2). bezansong@agr.gc.ca
To assess whether domestically grown fresh salad vegetables constitute a possible

reservoir of antibiotic resistance for Canadian consumers, aerobic bacteria
capable of forming colonies at 30 degrees C on nutrient-limited media were
recovered from a single sampling of Romaine lettuce, Savoy spinach and alfalfa
sprouts, then examined for their susceptibility to ten antibiotics and the
carriage of potentially mobile R-plasmids and integrons. Of the 140 isolates
resistant to one or more antibiotic, 93.5 and 90.0% were resistant to ampicillin
and cephalothin; 35.7% to chloramphenicol, 10.0% to streptomycin, 4.2% to
nalidixic acid, 4.2% to kanamycin, and 2.8% to gentamicin. Gram-positive isolates
accounted for less than 4% of the antibiotic resistant strains. A small portion
(23.1%) of the predominant oxidase-positive, gram-negative isolates was resistant
to two or more antimicrobials. Members of the Pseudomonas fluorescens/putida
complex were most prevalent among the 34 resistant strains identified.
Sphingobacterium spp. and Acinetobacter baumanni also were detected. Ten of 52
resistant strains carried plasmids, 3 of which were self-transmissible and bore
resistance to ampicillin and kanamycin. Eighteen of 48 gave PCR evidence for
integron DNA. Class 2 type integrons were the most prevalent, followed by class
1. We conclude that the foods examined here carry antibiotic resistant bacteria
at the retail level. Further, our determination that resistant strains contain
integron-specific DNA sequences and self-transmissible R-plasmids indicates their
potential to influence the pool of antibiotic resistance in humans via lateral
gene transfer subsequent to ingestion.
1571. New Phytol. 2008;179(4):1133-41. doi: 10.1111/j.1469-8137.2008.02540.x.
Long-distance transport of mRNA via parenchyma cells and phloem across the
host-parasite junction in Cuscuta.
David-Schwartz R(1), Runo S, Townsley B, Machuka J, Sinha N.
Author information:
(1)Section of Plant Biology, University of California, Davis, CA 95616, USA.
It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes
a continuous vascular system through which water and nutrients are drawn. Along
with solutes, viruses and proteins, mRNA transcripts are transported from the
host to the parasite. The path of the transcripts and their stability in the
parasite have yet to be revealed. To discover the route of mRNA transportation,
the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique
was used to locally amplify host transcript within parasitic tissue. The
stability of host mRNA molecules was also checked by monitoring specific
transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits
of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit
of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC
ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum
lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells
and to the phloem. LePFP transcripts were found in the growing dodder stem up to
30 cm from the tomato-dodder connection. These results suggest that mRNA
molecules are transferred from host to parasite via symplastic connections
between parenchyma cells, move towards the phloem, and are stable for a long
distance in the parasite. This may allow developmental coordination between the
parasite and its host.
DOI: 10.1111/j.1469-8137.2008.02540.x
1572. FEMS Microbiol Ecol. 2008 Sep;65(3):391-9. doi: 10.1111/j.1574-

6941.2008.00544.x.
Epub 2008 Jul 8.
Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving

rhizobia.
Ratcliff WC(1), Kadam SV, Denison RF.
Author information:
(1)Ecology, Evolution and Behavior, University of Minnesota, Minneapolis, MN
55108, USA. ratcl009@umn.edu
The carbon that rhizobia in root nodules receive from their host powers both N(2)
fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia
also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance
rhizobium survival when they are carbon limited, either in nodules or in the soil
between hosts. There can be a conflict of interest between rhizobia and legumes
over the rate of PHB accumulation, due to a metabolic tradeoff between N(2)
fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited
rhizobia, populations of genetically uniform rhizobia with high vs. low PHB
(confirmed by flow cytometry) were generated by fractionating Sinorhizobium
meliloti via density gradient centrifugation, and also by harvesting cells at
early vs. late stationary phase. These rhizobia were starved for 165 days. PHB
use during starvation was highly predictive of both initial reproduction and
long-term population maintenance. Cultured S. meliloti accumulated enough PHB to
triple their initial population size when starved, and to persist for c. 150 days
before the population fell below its initial value. During the first 21 days of
nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated
enough PHB to support significant increases in reproduction and survival during
starvation.
DOI: 10.1111/j.1574-6941.2008.00544.x
1573. Mol Microbiol. 2008 Jul;69(2):479-90.
A Sinorhizobium meliloti osmosensory two-component system required for cyclic

glucan export and symbiosis.
Griffitts JS(1), Carlyon RE, Erickson JH, Moulton JL, Barnett MJ, Toman CJ, Long
SR.
Author information:
(1)Department of Microbiology and Molecular Biology, Brigham Young University,
Provo, UT 84602, USA. joelg@byu.edu
screen for novel symbiotic mutants of the nitrogen-fixing legume symbiont

Sinorhizobium meliloti uncovered a crucial role for the putative response
regulator FeuP in the symbiotic infection process. Transcriptome analysis shows
that FeuP controls the transcription of at least 16 genes, including ndvA, which
encodes an ATP-dependent exporter of cyclic beta glucans. Loss of feuP function
gives rise to traits associated with cyclic beta glucan biosynthetic defects,
including poor growth and motility under hypoosmotic conditions, and the
inability to invade plant tissue during the early stages of symbiotic infection.
Analysis of cyclic glucans indicates that the feuP mutant is able to synthesize
intracellular cyclic beta glucans, but is unable to export them. Cyclic beta
glucan export can be restored to feuP mutant cells by constitutive expression of
ndvA; likewise, the symbiotic phenotype of a feuP mutant is rescued by ectopic
ndvA expression. We further show that the linked sensor kinase gene, feuQ, is
also important for modulating ndvA transcription, and that signalling through the
FeuP/FeuQ pathway is responsive to extracellular osmotic conditions, with low
osmolarity stimulating ndvA expression.
1574. Lipids. 2008 Sep;43(9):853-65. doi: 10.1007/s11745-008-3210-5. Epub 2008 Jul

15.
Significance of coprophagy for the fatty acid profile in body tissues of rabbits
fed different diets.
Leiber F(1), Meier JS, Burger B, Wettstein HR, Kreuzer M, Hatt JM, Clauss M.
Author information:
(1)Department of Agricultural and Food Science, ETH Zurich, Universitätstrasse 2,
8092, Zurich, Switzerland. fleiber@ethz.ch
Four groups of eight New Zealand hybrid rabbits were fattened with ad libitum
access to the following pelleted experimental diets: ryegrass meal or alfalfa
meal fed either alone or with oats meal in a ratio of 1:1. After 25 weeks they
were slaughtered and dissected. Fatty acid (FA) profiles of caecotrophs
(re-ingested fermentation products of the caecum), perirenal adipose tissue and
intramuscular fat in the Musculus quadriceps were determined. With high
proportions of branched-chain FA (BFA) and trans FA, and increased proportions of
saturated FA relative to the diets, the caecotroph FA profile showed a clear
fingerprint of anaerobe microbial lipid metabolism including biohydrogenation. By
contrast, the FA profiles of adipose and lean tissue comprised high proportions
of polyunsaturated FA (PUFA), whilst BFA and trans FA occurred in much lower
proportions compared to the caecotrophs. Thus, coprophagy did not substantially
modify the FA composition of the tissues investigated. Use of forage-only diets,
compared to the oats supplemented diets, led to extraordinary high proportions of
n-3 PUFA (including 18:3 and long-chain n-3) in the fat of adipose (21.3 vs.
6.7%) and lean tissue (15.4 vs. 5.7%). The forage type diet (grass vs. alfalfa)
had smaller effects on the FA profiles. Indications of diet effects on endogenous
desaturation, chain elongation and differential distribution of functional FA
between the two tissues investigated were found.
DOI: 10.1007/s11745-008-3210-5
1575. Food Microbiol. 2008 Sep;25(6):762-70. doi: 10.1016/j.fm.2008.05.001. Epub

2008
May 13.
Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables

by application of washing solutions containing enterocin AS-48 alone and in
combination with other antimicrobials.
Cobo Molinos A(1), Abriouel H, Lucas López R, Ben Omar N, Valdivia E, Gálvez A.
Author information:
(1)Area de Microbiología, Departamento de Ciencias de la Salud, Facultad de
Ciencias Experimentales, Universidad de Jaén, 23071 Jaén, Spain.
Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by

Enterococcus faecalis. In the present study, the bacteriocin was tested alone and
in combination with other antimicrobials for decontamination of Bacillus
inoculated on alfalfa, soybean sprouts and green asparagus. Washing with
enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and
Bacillus weihenstephanensis by 1.0-1.5 and by 1.5-2.38 log units right after
application of treatment, respectively. In both cases, the bacteriocin was
effective in reducing the remaining viable population below detection levels
during further storage of the samples at 6 degrees C, but failed to prevent
regrowth in samples stored at 15 or 22 degrees C. Application of washing
treatments containing enterocin AS-48 in combination with several other
antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol,
polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium
hypochlorite) greatly enhanced the bactericidal effects. The combinations of
AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride
provided the best results. After application of the combined treatments on
alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable
bacilli were not detected or remained at very low concentrations in the treated
samples during a 1-week storage period at 15 degrees C. Inhibition of B. cereus
by in situ produced bacteriocin was tested by cocultivation with the AS-48
producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32
was able to grow and produce bacteriocin on sprouts both at 15 and 22 degrees C.
At 15 degrees C, growth of B. cereus was completely inhibited in the cocultures,
while a much more limited effect was observed at 22 degrees C. The results
obtained for washing treatments are very encouraging for the application of
enterocin AS-48 in the decontamination of sprouts. Application of washing
treatments containing AS-48 alone can serve to reduce viable cell counts of
bacilli in samples stored under refrigeration, while application of combined
treatments should be recommended to avoid proliferation of the surviving bacilli
under temperature-abuse conditions.
DOI: 10.1016/j.fm.2008.05.001
1576. Toxicon. 2008 Jul;52(1):84-90. doi: 10.1016/j.toxicon.2008.04.172. Epub 2008

May
29.
In vivo influence of cyanobacterial toxins on enzyme activity and gene expression
of protein phosphatases in Alfalfa (Medicago sativa).
Peuthert A(1), Lawton L, Pflugmacher S.
Author information:
(1)Leibniz Institute of Freshwater Ecology and Inland Fisheries, RG Biochemical
Regulation, Müggelseedamm 301, 12587 Berlin, Germany.
Irrigation of crop plants with surface water can be a threat if cyanobacterial

toxins are present in the water. Cyanotoxins are known to cause adverse effects
in plants. Microcystin (MC), a cyclic heptapeptide, with more than 70 structural
variants, is a frequently occurring toxin. MC is a specific inhibitor of
serine/threonine protein phosphatases 1 and 2A (PP1 and 2A), important regulatory
enzymes in eukaryotic cells. Protein phosphatases consist of a catalytic subunit
and one or more regulatory subunits. In Alfalfa several isoforms of the catalytic
subunit of PP1 (MsPP1alpha, MsPP1beta, MsPP1gamma, MsPP1delta, MsPP1varepsilon)
and PP2A (MsPP2A Calpha/beta/gamma) are known along with isoforms of the
regulatory subunits of PP2A (MsPP2A Aalpha/beta, MsPP2A Balpha/beta). The in vivo
effect of environmentally relevant concentrations of cyanobacterial components on
the mRNA transcript level of the subunits of protein phosphatases 1 and 2A in
Alfalfa (Medicago sativa) was examined using semi-quantitative RT-PCR. Plants
were exposed for one week to 5 microg L(-1) microcystin-LR, microcystin-LW,
okadaic acid and to cell-free cyanobacterial crude extracts from Microcystis
aeruginosa containing 5 microg L(-1) microcystin-LR and a toxin-free crude
extract from Synechocystis spp. The protein phosphatase activity in vivo was
inhibited when exposed to toxins and crude extract containing microcystin-LR, no
change was induced by Synechocystis crude extract. The gene expression of the
MsPP1gamma subunit and the MsPP1varepsilon subunit was induced in plants exposed
to MC-LW.
DOI: 10.1016/j.toxicon.2008.04.172

Jul
9.
A novel type of thioredoxin dedicated to symbiosis in legumes.
Alkhalfioui F(1), Renard M, Frendo P, Keichinger C, Meyer Y, Gelhaye E, Hirasawa

M, Knaff DB, Ritzenthaler C, Montrichard F.
Author information:
(1)Physiologie Moléculaire des Semences, UMR 1191 Université d'Angers-Institut
National d'Horticulture-INRA, IFR 149 QUASAV, ARES, 49045 Angers cedex 01,
France.
Thioredoxins (Trxs) constitute a family of small proteins in plants. This family

has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which
contains six different Trx types: f, m, x, and y in chloroplasts, o in
mitochondria, and h mainly in cytosol. A detailed study of this family in the
model legume Medicago truncatula, realized here, has established the existence of
two isoforms that do not belong to any of the types previously described. As no
possible orthologs were further found in either rice (Oryza sativa) or poplar
(Populus spp.), these novel isoforms may be specific for legumes. Nevertheless,
on the basis of protein sequence and gene structure, they are both related to
Trxs m and probably have evolved from Trxs m after the divergence of the higher
plant families. They have redox potential values similar to those of the
classical Trxs, and one of them can act as a substrate for the M. truncatula
NADP-Trx reductase A. However, they differ from classical Trxs in that they
possess an atypical putative catalytic site and lack disulfide reductase activity
with insulin. Another important feature is the presence in both proteins of an
N-terminal extension containing a putative signal peptide that targets them to
the endoplasmic reticulum, as demonstrated by their transient expression in
fusion with the green fluorescent protein in M. truncatula or Nicotiana
benthamiana leaves. According to their pattern of expression, these novel
isoforms function specifically in symbiotic interactions in legumes. They were
therefore given the name of Trxs s, s for symbiosis.
DOI: 10.1104/pp.108.123778
PMCID: PMC2528116
Do weather conditions correlate with findings in failed, provision-filled nest

cells of Megachile rotundata (Hymenoptera: Megachilidae) in western North
America?
Pitts-Singer TL(1), James RR.
Author information:
(1)USDA-ARS Bee Biology & Systematics Laboratory; Utah State University; Logan,
UT 84322-5310, USA. theresa.pitts-singer@ars.usda.gov
Cavity-nesting alfalfa leafcutting bees, Megachile rotundata (F.) (Hymenoptera:

Megachilidae), are excellent pollinators of alfalfa, Medicago savita L., for seed
production. In commercial settings, artificial cavities are placed in field
domiciles for nesting and, thereby, bee populations are sustained for future use.
For this study, cells from leafcutting bee nests were collected in late summer
from commercial seed fields. Over 3 yr (2003-2005), 39 samples in total of
approximately equal to 1,000 cells each were taken from several northwestern U.S.
states and from Manitoba, Canada. X-radiography of 500 cells from each sample was
used to identify "pollen balls" (i.e., cells in which the pollen-nectar provision
remained, but the egg or larva, if present, was not detectable on an
x-radiograph). Most U.S. samples seemed to have higher proportions of pollen ball
cells than Manitoba samples. Pollen ball cells were dissected to determine the
moisture condition of the mass provision and true contents of each cell. Most
pollen ball cells from Manitoba samples contained fungus, the frequency of which
was positively correlated with cool, wet weather. In the United States, most
pollen ball cells had moist provisions, and many of them lacked young brood.
Correlation analysis revealed that pollen ball cells occurred in greater
proportions in fields with more hot days (above 38 degrees C). Broodless pollen
ball cells occurred in greater proportions under cool conditions, but dead small
larvae (second-third instars) seemed to occur in greater proportions under hot
conditions. Pollen ball cells with unhatched eggs and first instars (in the
chorion) occurred in lesser proportions under hot conditions.

Laboratory bioassays to evaluate fungicides for chalkbrood control in larvae of
the alfalfa leafcutting bee (Hymenoptera: Megachilidae).
Huntzinger CI(1), James RR, Bosch J, Kemp WP.
Author information:
(1)USDA-ARS Pollinating Insects Research Unit, Department Biology, Utah State
University Logan, UT 84322-5310, USA.
Chalkbrood, a fungal disease in bees, is caused by several species of

Ascosphaera. A. aggregata is a major mortality factor in populations of the
alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae)
used in commercial alfalfa seed production. Four formulated fungicides, Benlate
50 WP, Captan, Orbit, and Rovral 50 WP were tested in the laboratory for efficacy
against hyphal growth of A. aggregata cultures. The same fungicides, with the
addition of Rovral 4 F, were tested for their effects on incidence of chalkbrood
disease, and toxicity to M. rotundata larvae. Benlate, Rovral 50 WP, and Rovral 4
F reduced incidence of chalkbrood with minimal mortality on larval bees. Benlate
and Rovral 50 WP also reduced hyphal growth. Orbit was effective in reducing
hyphal growth, but it did not reduce incidence of chalkbrood and was toxic to bee
larvae. Captan was not effective in reducing hyphal growth or chalkbrood
incidence, and it was toxic to bee larvae. Fungicides that reduce incidence of
chalkbrood and larval mortality in this laboratory study are candidates for
further study for chalkbrood control.
1580. J Plant Physiol. 2008 Oct 9;165(15):1647-51. doi:

10.1016/j.jplph.2008.05.005.
Epub 2008 Jul 7.
Accumulation of overproduced ferritin in the chloroplast provides protection

against photoinhibition induced by low temperature in tobacco plants.
Hegedus A(1), Janda T, Horváth GV, Dudits D.
Author information:
(1)Department of Applied Chemistry, Faculty of Food Science, Corvinus University
of Budapest, Budapest, Budapest, Hungary. hegedus.attila@uni-corvinus.hu
Wild-type tobacco plants (Nicotiana tabacum L. cv. Petit Havanna line SR1) and
plants transformed with full-length alfalfa ferritin cDNA with the chloroplast
transit peptide under the control of a Rubisco small subunit gene promoter (C3
and C8) were cold-treated at 0 degrees C with continuous light (250 micromol
m(-2)s(-1)). These transgenic plants had higher chlorophyll content and higher
F(v)/F(m) chlorophyll-a fluorescence induction parameters than wild-type plants
after 2 or 3d of cold treatment in C3 and C8 transgenic plants, respectively.
Thermoluminescence studies on the high-temperature bands suggest that these
plants suffered less oxidative damage in comparison to the wild-type genotype.
The present experiments provide evidence that transgenic tobacco lines
overexpressing alfalfa ferritin, which is accumulated in the chloroplasts, may
show higher tolerance to various stress factors, generating ROS including low
temperature-induced photoinhibition.
DOI: 10.1016/j.jplph.2008.05.005
1581. Comp Biochem Physiol A Mol Integr Physiol. 2008 Sep;151(1):114-25. doi:
10.1016/j.cbpa.2008.06.007. Epub 2008 Jun 14.
Digestive parameters and water turnover of the leopard tortoise.
McMaster MK(1), Downs CT.
Author information:
(1)School of Biological and Conservation Sciences, University of KwaZulu-Natal,
Private Bag X01, Scottsville, Pietermaritzburg 3209, South Africa.
Leopard tortoises (Stigmochelys pardalis) experience wide fluctuations in

environmental conditions and unpredictable availability of food and water within
the Nama-Karoo biome. It was hypothesised that tortoises fed two diets differing
in preformed water and fibre content would have differing food intake, gut
transit rate, assimilation efficiency, faecal and urinary water loss, and urine
concentrations. It was predicted that tortoises fed these contrasting diets would
attempt to maintain energy and water balance by altering their digestive
parameters. Leopard tortoises fed lucerne (Medicago sativa) had a low food intake
coupled with long gut transit times, which resulted in the lowest amount of
faecal energy and faecal water lost. Tortoises fed tomatoes (Solanum
lycopersicum) had higher food intake and faster gut transit times, but more
energy and water was lost in the faeces. However, daily energy assimilated and
assimilation efficiency were comparable between tortoises fed the two diets.
Urine osmolality was significantly different between tortoises on the two diets.
Results indicate that leopard tortoises can adjust parameters such as transit
rate, food intake, water loss and urine osmolality to maintain body mass, water
and energy balance in response to a high fibre, low water content and a low
fibre, high water content diet. This study suggests that this digestive
flexibility allows leopard tortoises in the wild to take advantage of
unpredictable food and water resources.
DOI: 10.1016/j.cbpa.2008.06.007
1582. Int J Syst Evol Microbiol. 2008 Jul;58(Pt 7):1693-9. doi:

10.1099/ijs.0.65568-0.
Rhizobium multihospitium sp. nov., isolated from multiple legume species native
of Xinjiang, China.
Han TX(1), Wang ET, Wu LJ, Chen WF, Gu JG, Gu CT, Tian CF, Chen WX.
Author information:
(1)Key Laboratory of Agro-Microbial Resource and Application, Ministry of
Agriculture/College of Biological Sciences, China Agricultural University,
Thirty-one rhizobial strains isolated from nodules of legumes native of Xinjiang,

China, were characterized. These strains were classified as belonging to the
genus Rhizobium based on amplified 16S rDNA restriction analysis (ARDRA). The
strains were distinguished from recognized Rhizobium species using analysis of
16S-23S rDNA intergenic spacers (IGS-RFLP), SDS-PAGE analysis of whole proteins
and BOX-PCR; the test strains always formed a distinct cluster with patterns that
were quite different from those of the reference rhizobial strains used.
According to the phylogenetic analysis based on the 16S rRNA gene, the test
strains belonged to the genus Rhizobium, with Rhizobium tropici, Rhizobium
rhizogenes and Rhizobium lusitanum as the closest related species, with 99.6,
99.2 and 99.4 % sequence similarities, respectively, between the type strains of
the three Rhizobium species and strain CCBAU 83401(T). Phylogenetic analyses of
the representative strains using IGS and atpD, recA and glnII genes all confirmed
the phylogenetic arrangements obtained using the 16S rRNA gene. The DNA-DNA
relatedness values between strain CCBAU 83401(T) and strains CCBAU 83364, CCBAU
83345 and CCBAU 83523 ranged from 80.8 to 100 %, showing that they belong to the
same species. The DNA-DNA relatedness between strain CCBAU 83401(T) and R.
tropici IIB CIAT 899(T), R. tropici IIA CFN 299, R. rhizogenes LMG 150(T) and R.
lusitanum P1-7(T) were 26.9, 27.7, 38.2 and 22.6 %, respectively, clearly
indicating that strain CCBAU 83401(T) represents a novel species. Phenotypic
characterization of four representative strains, CCBAU 83401(T), CCBAU 83364,
CCBAU 83345 and CCBAU 83523, showed several distinctive features that
differentiated them from closely related species. The 31 strains had identical
nodD and nifH genes, which were very similar to those of the bean-nodulating R.
lusitanum, Devosia neptuniae and R. tropici IIB. Based upon these results, the
strains from this study are considered to represent a novel species, for which
the name Rhizobium multihospitium sp. nov. is proposed. The DNA G+C content
ranged from 65.3 to 66.0 mol% (T(m)). The type strain is CCBAU 83401(T) (=LMG
23946(T)=HAMBI 2975(T)), which nodulates Robinia pseudoacacia, but not Leucaena
leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.
DOI: 10.1099/ijs.0.65568-0

Jul
3.
Seven in absentia proteins affect plant growth and nodulation in Medicago

truncatula.
Den Herder G(1), De Keyser A, De Rycke R, Rombauts S, Van de Velde W, Clemente

MR, Verplancke C, Mergaert P, Kondorosi E, Holsters M, Goormachtig S.
Author information:
(1)Department of Plant Systems Biology, Flanders Institute for Biotechnology and
Department of Molecular Genetics, Ghent University, B-9052 Gent, Belgium.
Protein ubiquitination is a posttranslational regulatory process essential for

plant growth and interaction with the environment. E3 ligases, to which the seven
in absentia (SINA) proteins belong, determine the specificity by selecting the
target proteins for ubiquitination. SINA proteins are found in animals as well as
in plants, and a small gene family with highly related members has been
identified in the genome of rice (Oryza sativa), Arabidopsis (Arabidopsis
thaliana), Medicago truncatula, and poplar (Populus trichocarpa). To acquire
insight into the function of SINA proteins in nodulation, a dominant negative
form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M.
truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants,
fewer nodules were formed than in control plants, and most nodules remained small
and white, a sign of impaired symbiosis. Defects in rhizobial infection and
symbiosome formation were observed by extensive microscopic analysis. Besides the
nodulation phenotype, transgenic plants were affected in shoot growth, leaf size,
and lateral root number. This work illustrates a function for SINA E3 ligases in
a broad spectrum of plant developmental processes, including nodulation.
DOI: 10.1104/pp.108.119453
PMCID: PMC2528092
1584. Animal. 2008 Jul;2(7):1037-44. doi: 10.1017/S1751731108002176.
Intestinal amino acid absorption in lambs fed fresh Lucerne ( Medicago sativa)
during an established Trichostrongylus colubriformis infection.
Bermingham EN(1), Roy NC, Sutherland IA, Waghorn GC, Sinclair BR, Peters JS,
McNabb WC.
Author information:
(1)1Food, Metabolism & Microbiology, AgResearch Grasslands, Palmerston North
4442, New Zealand.
The effects of an established Trichostrongylus colubriformis infection on amino

acid (AA) absorption from the small intestine and their availability to other
tissues were determined in lambs 48 days post infection. The lambs were fed fresh
Lucerne (Medicago sativa; 800 g dry matter (DM)/day) and dosed with 6000 L3 T.
colubriformis larvae for 6 days (n = 5) or kept as parasite free controls (n =
6). Faecal egg production was monitored every second day from day 22 to day 48. A
nitrogen (N) balance was conducted on days 35 to 43 after infection, and digesta
flow and AA concentration measurements were made on day 44. On day 48 after
infection, blood was continuously collected from the mesenteric artery and vein,
plasma harvested and AA concentrations measured. Faecal egg production peaked on
the 26th day after infection (P < 0.001) and intestinal worm burdens on day 48
were greater (P < 0.001) in the infected lambs. Feed intake and liveweight gain
were similar (P > 0.10) between control and infected lambs. Digestibility and
flow of DM and N through the digestive tract were also unaffected (P > 0.10) by
parasite infection. Despite a trend towards higher abomasal AA flux in the
parasitised lambs (P < 0.10), apparent AA absorption from the small intestine and
AA availability to other tissues were unaffected (P > 0.10) by infection. These
results suggest that an established parasite infection had little effect on the
intestinal absorption and availability of AA to other tissues in lambs fed fresh
Lucerne.
DOI: 10.1017/S1751731108002176
PMID: 22443704
1585. Poult Sci. 2008 Jul;87(7):1263-75. doi: 10.3382/ps.2007-00179.
In vitro fermentation response of laying hen cecal bacteria to combinations of

fructooligosaccharide prebiotics with alfalfa or a layer ration.
Donalson LM(1), Kim WK, Chalova VI, Herrera P, McReynolds JL, Gotcheva VG,
Vidanovic D, Woodward CL, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station, TX
77843-2472, USA.
The objective of this in vitro study was to evaluate the effects of combining a
prebiotic with alfalfa on fermentation by laying hen cecal bacteria. Cecal
contents from laying hens were diluted to a 1:3,000 concentration with an
anaerobic dilution solution and added to serum tubes filled with ground alfalfa
or a layer ration with or without fructooligosaccharide (FOS) prebiotic. Samples
were processed in an anaerobic hood, pressurized by using a pressure manifold,
and incubated at 37 degrees C. Volatile fatty acid (VFA) and lactic acid
concentrations were quantified at 6 and 24 h of substrate fermentation. In this
study, fermentation of alfalfa resulted in greater production of acetate, VFA,
and lactic acid compared with the layer ration. Although with a relative
inconsistency in data between trials, the amendment of FOS to both alfalfa and
the layer ration appeared to further increase fermentation as demonstrated by
overall higher propionate, butyrate, VFA, and lactic acid concentrations. The
effect was more pronounced after 24 h of fermentation, implying time constraints
for the optimal production of fermentation products in the chicken
gastrointestinal tract. These data indicate that in vitro cecal fermentation can
be enhanced by the addition of FOS.
DOI: 10.3382/ps.2007-00179
1586. Poult Sci. 2008 Jul;87(7):1253-62. doi: 10.3382/ps.2007-00166.
The influence of a fructooligosaccharide prebiotic combined with alfalfa molt

diets on the gastrointestinal tract fermentation, Salmonella enteritidis
infection, and intestinal shedding in laying hens.
Donalson LM(1), McReynolds JL, Kim WK, Chalova VI, Woodward CL, Kubena LF, Nisbet
DJ, Ricke SC.
Author information:
77843-2472, USA.
Molting is a natural process, which birds undergo to rejuvenate their

reproductive organs. The US poultry egg production industry has used feed
withdrawal to effectively induce molt; however, susceptibility of Salmonella
Enteritidis has encouraged the development of alternative methods. Previous
research conducted in our laboratory showed that alfalfa is effective at molt
induction and provides equivalent postmolt production numbers and quality when
compared with feed withdrawal. In the attempt to further increase the efficacy of
alfalfa molt diet and decrease the chicken susceptibility to Salmonella
Enteritidis during molt, fructooligosaccharide (FOS) was added to a combination
of 90% alfalfa and 10% layer ration in 2 levels (0.750 and 0.375%). Ovary and
liver colonization by Salmonella Enteritidis in 3 and 2 of the 4 trials,
respectively, were reduced (P <or= 0.05) in hens fed FOS-containing diets
compared with hens subjected to feed withdrawal. Significant decreases in ce-cal
Salmonella Enteritidis counts were also observed in 2 of the 4 trials. In 3 of
the 4 trials, the same diets did not affect (P > 0.05) the production of cecal
total volatile fatty acids when compared with hens undergoing feed withdrawal.
However, in all 3 alfalfa molt diets, the concentrations of lactic acid were
greater (P <or= 0.05) than hens with feed withdrawal, but no differences (P >
0.05) were observed among hens fed alfalfa combined with FOS and hens fed
alfalfa/layer ration without FOS. Overall, given the similarities between hens
fed 0.750% FOS (H) and 0.375% FOS (L), molt diets combined with the lower level
of FOS should be sufficient.
DOI: 10.3382/ps.2007-00166
1587. Trop Anim Health Prod. 2008 Aug;40(6):413-7. doi: 10.1007/s11250-007-9102-5.

Epub
2007 Nov 23.
Effects of non-feed removal molting methods on egg quality traits in commercial
brown egg laying hens in Turkey.
Petek M(1), Gezen SS, Alpay F, Cibik R.
Author information:
(1)Department of Zootechnics, Faculty of Veterinary Medicine, University of
Uludag, Bursa 16059, Turkey. petek@uludag.edu.tr
Non-feed removal molting programme in commercial brown laying hens and its
influence on pre-molting, post-molting and end of cycle egg quality traits were
investigated. Overall 54 birds were randomly divided into three treatment groups
and each group was fed with one of the following diets during 10 days of molting
period: (i) grain barley, (ii) alfalfa meal, or (iii) commercial layer ration
(non-molted control group). Eggs obtained from groups in pre-molting,
post-molting and end of cycle periods were examined for several quality
performance traits such as egg weight, specific gravity, shape index, shell
strength, shell thickness, eggshell weight, haugh unit, albumen index, yolk index
and yolk color. Results indicated that non-feed removal molting programme based
particularly on grain barley had positive effect on egg quality traits in laying
hens. Notably, yolk color and haugh unit, which are considered as the most
important quality parameters from the consumer point of view, were relatively
improved in barley molted group.
DOI: 10.1007/s11250-007-9102-5
1588. J Anim Sci. 2008 Nov;86(11):2998-3013. doi: 10.2527/jas.2007-0070. Epub 2008

Jun
20.
Effects of calcium magnesium carbonate and roughage level on feedlot performance,

ruminal metabolism, and site and extent of digestion in steers fed high-grain
diets.
Crawford GI(1), Keeler CD, Wagner JJ, Krehbiel CR, Erickson GE, Crombie MB,
Nunnery GA.
Author information:
(1)Department of Animal Science, University of Nebraska, Lincoln 68583, USA.
A feedlot growth performance experiment and 2 metabolism experiments were

conducted to evaluate dietary roughage concentration and calcium magnesium
carbonate in steers fed a high-grain diet. In Exp. 1, one hundred ninety-two
crossbred yearling steers (320 +/- 10 kg of initial BW) were fed diets based on
steam-flaked corn with 0, 0.75, or 1.5% CaMg(CO(3))(2). There were no effects (P
> or = 0.13) on ADG, DMI, G:F, or total water intake due to CaMg(CO(3))(2). In
Exp. 2, five ruminally and duodenally fistulated steers (263 +/- 9 kg of initial
BW) were used in a 5 x 5 Latin square design, with 5 dietary treatments arranged
in a 2 x 2 + 1 factorial: 1) 3.8% dietary roughage and no CaMg(CO(3))(2); 2) 7.6%
dietary roughage and no CaMg(CO(3))(2); 3) 11.4% dietary roughage and no
CaMg(CO(3))(2); 4) 3.8% dietary roughage and 1.5% CaMg(CO(3))(2); and 5) 7.6%
dietary roughage and 1.5% CaMg(CO(3))(2). Water consumption was less (quadratic,
P = 0.003) when 7.6% dietary roughage was fed compared with 3.8 or 11.4% dietary
roughage. Intake of DM was not affected (P > or = 0.16) by dietary roughage or by
CaMg(CO(3))(2). Poststomach and total tract starch digestion decreased (linear, P
< 0.01) as dietary roughage increased. Ruminal pH tended (P = 0.08) to increase
as dietary roughage increased but was not affected (P = 0.60) by CaMg(CO(3))(2).
In Exp. 3, DMI and ruminal pH were continuously monitored in a 6 x 6 Latin square
design using 6 ruminally and duodenally fistulated Holstein steers (229 +/- 10 kg
of initial BW). A 3 x 2 factorial treatment structure was utilized, with factors
consisting of dietary roughage concentration (4.5, 9.0, or 13.5%) and
CaMg(CO(3))(2) inclusion (0 or 1.0%) to replace MgO and partially replace
lime-stone. A dietary roughage x CaMg(CO(3))(2) interaction (P = 0.01) occurred
as steers consuming 13.5% roughage, 1.0% CaMg(CO(3))(2) had greater DMI per meal
than those consuming 4.5% dietary roughage, no CaMg(CO(3))(2) and 9.0% dietary
roughage, 1.0% CaMg(CO(3))(2). Steers consuming 13.5% dietary roughage, 1.0%
CaMg(CO(3))(2) and 9.0% dietary roughage, no CaMg(CO(3))(2) had greater meal
length (min/meal; P = 0.01) than steers consuming 4.5% dietary roughage, no
CaMg(CO(3))(2). Total tract OM digestibility decreased linearly (P = 0.01), and
ruminal pH increased linearly (P = 0.01) with increasing dietary roughage
concentration. Inclusion of CaMg(CO(3))(2) can replace limestone and MgO but did
not produce ruminal pH responses similar to those observed by increasing dietary
roughage in high-concentrate diets.
DOI: 10.2527/jas.2007-0070
Nutrient demand interacts with forage family to affect intake and digestion
responses in dairy cows.
Voelker Linton JA(1), Allen MS.
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing 48824,
USA.
The effect of feed intake in the preliminary period on responses to diets

containing alfalfa silage or orchardgrass silage was evaluated using 8 ruminally
and duodenally cannulated Holstein cows in a crossover design experiment with a
14-d preliminary period and two 15-d treatment periods. Responses measured were
DMI, rates of fiber digestion and passage, and milk production. Cows were 139 +/-
83 (mean +/- SD) d in milk at the beginning of the preliminary period. During the
preliminary period, 3.5% fat-corrected milk yield ranged from 23.9 to 47.6 kg/d
(mean = 36.9 kg/d) and preliminary voluntary DMI (pVDMI) ranged from 14.2 to 21.3
kg/d (mean = 18.6 kg/d). The 2 treatments were a diet containing alfalfa silage
as the sole forage (AL) and a diet containing orchardgrass silage as the sole
forage (OG). Alfalfa silage contained 43% neutral detergent fiber (NDF;
dry-matter basis) and orchardgrass silage contained 48% NDF; diets contained
approximately 23% forage NDF and 27% total NDF, so forage-to-concentrate ratio
was 53:47 for AL and 48:52 for OG. Digestibility of NDF was lower for AL in the
rumen and whole tract compared with OG, and milk fat concentration tended to be
greater for OG than for AL. Mean 3.5% fat-corrected milk yield and DMI were not
different between AL and OG. Response of DMI to forage family depended on pVDMI,
as indicated by a significant interaction between treatment and pVDMI in
predicting DMI. As pVDMI increased, DMI increased when cows were fed AL but not
when they were fed OG. That is, as appetite increased, intake was more restricted
for the more physically filling OG than for the less physically filling AL. This
more positive DMI response to AL over OG among high-pVDMI cows is corroborated by
interactions between treatments and pVDMI for both ruminal NDF turnover rate and
indigestible NDF passage rate response. Therefore, the effects of alfalfa and
orchardgrass forages on intake and fiber digestion depended on the extent to
which fill limited feed intake of an individual cow.
DOI: 10.3168/jds.2007-0897
1590. J Agric Food Chem. 2008 Jul 9;56(13):5443-50. doi: 10.1021/jf0733923. Epub
2008
Jun 13.
Perchlorate in the feed-dairy continuum of the southwestern United States.
Sanchez CA(1), Blount BC, Valentin-Blasini L, Lesch SM, Krieger RI.
Author information:
(1)Department of Soil, Water, and Environmental Sciences, Yuma Agricultural
Center, The University of Arizona, Yuma, Arizona 85364, USA.
Perchlorate has the potential to cause thyroid dysfunction by inhibiting iodide

uptake by the sodium iodide symporter. Perchlorate-contaminated waters may lead
to human exposure through drinking water and food chain transfer in crops by way
of irrigation water. Perchlorate has been found in dairy milk collected
nationally and internationally. This study was conducted to evaluate perchlorate
in the feed-dairy continuum in the southwestern United States. All feed products
collected at dairies in this study had detectable levels of perchlorate as
analyzed by ion chromatography-tandem mass spectrometry. The calculated total
perchlorate intake across dairies ranged from 1.9 to 12.7 mg/cow per day. The
variation in total perchlorate intake across dairies was largely associated with
variation in forage and silage products. Alfalfa products were the single most
important source of perchlorate intake variability among dairies. The estimated
perchlorate intake from drinking water ranged from 0.01 mg per cow per day and
was generally less than 2% of the total perchlorate intake. The perchlorate
content of milk ranged from 0.9 to 10.3 microg/L and was similar to levels
reported by the Food and Drug Administration's Total Diet Study. The perchlorate
content of milk was significantly related to the presence of perchlorate in feed
but the variation of perchlorate in milk could not be explained by feed intake
alone.
DOI: 10.1021/jf0733923
1591. Biotechnol Bioeng. 2008 Aug 15;100(6):1066-77. doi: 10.1002/bit.21856.
Regulation and characterization of Thermobifida fusca carbohydrate-binding module

proteins E7 and E8.
Moser F(1), Irwin D, Chen S, Wilson DB.
Author information:
(1)Department of Molecular Biology and Genetics, Cornell University, 458
Biotechnology Building, Ithaca, New York 14853, USA.
E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a
non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII
domain, followed by a Family 2 CBM, were cloned, expressed, purified, and
characterized. Western blots showed that E7 and E8 were induced and secreted when
Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa
as well as on beta-1,3 linked glucose molecules such as laminaribiose or
pachyman. E8 bound well to alpha- and beta-chitin and bacterial microcrystalline
cellulose (BMCC) at all pHs tested. E7 bound strongly to beta-chitin, less well
to alpha-chitin and more weakly to BMCC than E8. Filter paper binding assays
showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain
from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A
C-terminal 6xHis tag influenced binding of both E7 and E8 to these substrates.
Filter paper activity assays showed enhanced activity of T. fusca cellulases when
E7 or E8 was present. This effect was observed at very low concentrations of
cellulases or at very long times into the reaction and was mainly independent of
the type of cellulase and the number of cellulases in the mixture. E8, and to a
lesser extent E7, significantly enhanced the activity of Serratia marscescens
Chitinase C on beta-chitin.
2008 Wiley Periodicals, Inc.
DOI: 10.1002/bit.21856
1592. Genomics. 2008 Aug;92(2):115-21. doi: 10.1016/j.ygeno.2008.04.007. Epub 2008

Jun
12.
Maturation of the nodule-specific transcript MsHSF1c in Medicago sativa may

involve interallelic trans-splicing.
He ZS(1), Zou HS, Wang YZ, Zhu JB, Yu GQ.
Author information:
(1)National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology
and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China.
In nonplant species, many heat-shock transcription factors (HSFs) undergo

spatiotemporal-specific alternative splicing. However, little is known about the
spatiotemporal-specific splicing of HSFs in plants. Previously, we reported that
the alfalfa HSF gene MsHSF1 undergoes multiple alternative splicing events in
various tissues. Here, we identified another spliced transcript isoform, MsHSF1c,
containing a 177-base tandem repeat, and showed that the low-abundance MsHSF1c is
a nodule-specific transcript of MsHSF1. We also found that MsHSF1 presents
multiple alleles with single-base variations and the expression of MsHSF1 alleles
has allele-specific differences in alfalfa nodules. Because single-base
variations at position 1006 change the AT of MsHSF1b to GT in MsHSF1b-3, creating
a pair of donor/acceptor sites with the AG of MsHSF1b/1b-1 at position 827-828
for pre-mRNA splicing, we suggest that MsHSF1c may be generated by trans-splicing
between alleles MsHSF1b-3 and MsHSF1b or MsHSF1b-1. These results provide new
insight into the role of tissue-specific contribution in the transcription of
plant HSF genes.
DOI: 10.1016/j.ygeno.2008.04.007

2008
Jun 28.
Multi-site genetic modification of monolignol biosynthesis in alfalfa (Medicago

sativa): effects on lignin composition in specific cell types.
Nakashima J(1), Chen F, Jackson L, Shadle G, Dixon RA.

Author information:
* Independent antisense down-regulation of 10 individual enzymes in the

monolignol pathway has generated a series of otherwise isogenic alfalfa (Medicago
sativa) lines with varying lignin content and composition. These plants show
various visible growth phenotypes, and possess significant differences in
vascular cell size and number. * To better understand the phenotypic consequences
of lignin modification, the distributions of lignin content and composition in
stems of the various alfalfa lines at the cellular level were studied by confocal
microscopy after staining for specific lignin components, and by chemical
analysis of laser capture dissected tissue types. * Although all antisense
transgenes were driven by the same promoter with specificity for vascular, fiber
and parenchyma tissues, the impact of down-regulating a specific transgene varied
in the different tissue types. For example, reducing expression of ferulate
5-hydroxylase reduced accumulation of syringyl lignin in fiber and parenchyma
cells, but not in vascular elements. * The results support a model for cell
type-specific regulation of lignin content and composition at the level of the
monolignol pathway, and illustrate the use of laser capture microdissection as a
new approach to spatially resolved lignin compositional analysis.
DOI: 10.1111/j.1469-8137.2008.02502.x
1594. J Microbiol. 2008 Apr;46(2):137-45. doi: 10.1007/s12275-007-0145-0. Epub 2008

Jun
11.
The GntR-type regulators gtrA and gtrB affect cell growth and nodulation of
Wang Y(1), Chen AM, Yu AY, Luo L, Yu GQ, Zhu JB, Wang YZ.
Author information:
Academy of Sciences, Shanghai, 200032, P R China.
GntR-type transcriptional regulators are involved in the regulation of various

biological processes in bacteria, but little is known about their functions in
Sinorhizobium meliloti. Here, we identified two GntR-type transcriptional
regulator genes, gtrA and gtrB, from S. meliloti strain 1021. Both the gtrA1
mutant and the gtrB1 mutant had lower growth rates and maximal cell yields on
rich and minimal media, as well as lower cell motility on swimming plates, than
did the wild-type strain. Both mutants were also symbiotically deficient. Alfalfa
plants inoculated with wild-type strain 1021 formed pink elongated nodules on
primary roots. In contrast, the plants inoculated with the gtrA1 and gtrB1
mutants formed relatively smaller, round, light pink nodules mainly on lateral
roots. During the first 3 approximately 4 weeks post-inoculation, the plants
inoculated with the gtrA1 and gtrB1 mutants were apparently stunted, with lower
levels of nitrogenase activity, but there was a remarkable increase in the number
of nodules compared to those inoculated with the wild-type strain. Moreover, the
gtrA1 and gtrB1 mutants not only showed delayed nodulation, but also showed
markedly reduced nodulation competition. These results demonstrated that both
GtrA and GtrB affect cell growth and effective symbiosis of S. meliloti. Our work
provides new insight into the functions of GntR-like transcriptional regulators.
DOI: 10.1007/s12275-007-0145-0
1595. J Agric Food Chem. 2008 Jul 9;56(13):5437-42. doi: 10.1021/jf800489c. Epub
2008
Jun 11.
Chelation of molybdenum in Medicago sativa (alfalfa) grown on reclaimed mine

tailings.
Steinke DR(1), Majak W, Sorensen TS, Parvez M.
Author information:
(1)Agriculture and Agri-Food Canada, 3015 Ord Road, Kamloops, B.C., Canada.
steinked@agr.gc.ca
Extraction and analytical procedures were developed from 1999 to 2005 for the
chemical investigation of molybdenum (Mo) in aerial portions of alfalfa (
Medicago sativa L.) grown on a reclaimed mine tailings site at the Highland
Valley Copper Mine in British Columbia, Canada. The purification procedures were
guided by colorimetric analyses specific for Mo. The Mo levels in freeze-dried
plant samples exceeded 100 ppm, which is more than 20 times the maximum level
recommended for livestock feed. In contrast to previous work, which detected the
inorganic molybdate anion (MoO4(2-)) in alfalfa from the site, the present study
identified the major pool of Mo as a chelate bound to malic acid in five sources
of plant material. However, the inorganic form of Mo was characterized in aqueous
tailings samples, but once imbibed by vegetation, the anion was chelated to the
alpha-hydroxy organic acid. Synthetic chelates were synthesized to differentiate
the Mo-malate complex from the Mo-citrate by 95Mo NMR. Crystal structure of the
synthetic Mo-malate determined that the Mo was bound to two malato ligands as Na
2[MoO2(malate) 2] x 5H2O, which confirmed the structure of the isolates deduced
by 95Mo NMR. The chelation of Mo at the site may well explain the apparent lack
of long-term clinical effects in cattle grazing the site.
DOI: 10.1021/jf800489c
1596. Pol J Vet Sci. 2008;11(1):35-9.
Effect of a preparation containing lactic fermentation bacteria on the hygienic

status and aerobic stability of silages.
Selwet M(1).
Author information:
(1)Department of Agricultural Microbiology, August Cieszkowski Agricultural
University in Poznań, 60-656 Poznań, Poland. miksel70@jay.au.poznan.pl
The objective of this study was to determine the influence of biological silage
additive (Bonsilage) on the hygiene quality and nutritive value of maize and
grass-legume silages. The experiments were conducted on FAO 240 maize (Zea mays
L.) and a mixture of italian ryegrass (Lolium multiflorum L.), 50% with alfalfa
(Medicago media Pers.), 50%. Group 1 was a control and comprised silage without
any additives, group 2 was ensiled with the addition of 4 cm3 kg(-1) biological
silage additive. After 60 days of silage process individual silages were
subjected to microbiological composition, and chemical analyses of silages were
also determined. Similar analyses were repeated at day 7 following exposure to
oxygen. The applied biological silage additive was found to reduce (P<0.05)
numbers of Clostridium, Enterobacteriaceae, yeasts and mold fungi cells, and
increase (P<0.05) the number of LAB (lactic acid bacteria) in comparison with the
control in both silages. Chemical analysis of the maize silage showed that the
biological additive caused an increase (P<0.05) in DM (dry matter), CP (crude
protein), WSC (water soluble carbohydrates), LA (lactic acid), AA (acetic acid),
ethanol, and a decrease (P<0.05) in the concentration of BA (butyric acid), N-NH3
and pH value in comparison with the control. Chemical analysis of silage samples
from the grass-legume mixture showed that the additive caused an increase
(P<0.05) in the content of DM, CP, WSC, LA and AA in comparison with the control.
Samples of silage with the addition of an inoculant were characterized by a lower
(P<0.05) content of BA, N-NH3, ethanol and pH value. The biological additive
impoved the aerobic stability of silages in the aerobic phase.

Jun 6.
Influence of slice baling on feeding value of alfalfa hay in receiving and

finishing diets for feedlot cattle.
Loya-Olguin F(1), Avendaño-Reyes L, Encinias AM, Walker DA, Elam NA, Soto-Navarro
SA.
Author information:
(1)Clayton Livestock Research Center, New Mexico State University, Clayton 88415,
USA.
Three studies were conducted to evaluate the feeding value of slice alfalfa hay
in feedlot diets. In Exp. 1, 108 steer calves (183.1 +/- 1.2 kg initial BW; 6
pens/treatment) were used in a completely randomized design to evaluate the
effect of baling method on performance and morbidity of newly received calves.
The study lasted 28 d. Treatments consisted of a 65% concentrate receiving diet
containing 1) ground or 2) slice alfalfa hay. Steer calves were fed daily at 0800
h. Animals also received long-stem sudangrass hay the first 7 d. Steers were
weighed on d 0, 16, and 28. Feed, sudangrass hay, or feed plus sudangrass hay
intakes were not affected (P > 0.25) by treatment. Conversely, ADG from d 0 to 16
was greater (P < 0.001) for slice than ground (1.27 vs. 0.81 +/- 0.067 kg/d,
respectively) and from d 0 to 28 (1.23 vs. 0.91 +/- 0.042 kg/d, respectively). In
addition, G:F was greater (P < 0.001) for slice than ground hay from d 0 to 16
(0.39 vs. 0.25 +/- 0.021), and from d 0 to 28 (0.31 vs. 0.24 +/- 0.013 for slice
and ground, respectively). Moreover, morbidity (40.5 +/- 3.9%; P = 0.20) and
retreatment rates (30.7 +/- 7.5%; P = 0.14) were similar for slice and ground. In
Exp. 2, 176 crossbred steers (393.9 +/- 10.8 kg initial BW) were used in an 84-d
feeding experiment (4 pens/treatment) in a randomized complete block experimental
design with a 2 x 2 factorial arrangement of treatments to evaluate effects of
alfalfa baling method (ground or slice) and forage level (8 or 14%) on growth
performance. Experimental diets were based on steam-flaked corn. Daily BW gain
was greater (P = 0.10) for steers consuming ground compared with the slice hay
diet. A baling method x forage level interaction (P = 0.07) was observed for DMI.
Baling method did not (P = 0.98) influence DMI with 8% roughage level. But with
14% roughage, DMI was greater (P = 0.02) for steers consuming ground hay than the
slice diet. The G:F ratio was affected (P = 0.03) only by forage level (0.194 vs.
0.182 +/- 0.003 for 8 and 14% roughage, respectively). In Exp. 3, 4 ruminally
cannulated mixed-breed steers were used in a 4 x 4 Latin square design to
evaluate effects on digestive function. No baling method effects (P >or= 0.16)
were detected for DM, OM, CP, or NDF intakes or DM, OM, and NDF total tract
digestibility. Digestibility of NDF and OM were greater (P <or= 0.08) for diets
which contained 14% forage compared with diets that contained 8% forage. Slice
baling improved alfalfa hay feeding value for feedlot receiving cattle. However,
no major effects of slice baling alfalfa on finishing performance and digestion
were observed.
DOI: 10.2527/jas.2007-0637
1598. Mol Plant Microbe Interact. 2008 Jul;21(7):979-87. doi: 10.1094/MPMI-21-7-

0979.
Mutational analysis of the Sinorhizobium meliloti short-chain

dehydrogenase/reductase family reveals substantial contribution to symbiosis and
catabolic diversity.
Jacob AI(1), Adham SA, Capstick DS, Clark SR, Spence T, Charles TC.
Author information:
(1)Department of Biology, University of Waterloo, 200 University Avenue West,
Waterloo, ON N2L 3G1, Canada.
The short-chain dehydrogenase/reductase (SDR) family is one of the largest and

most ubiquitous protein families in bacterial genomes. Despite there being a few
well-characterized examples, the substrate specificities or functions of most
members of the family are unknown. In this study, we carried out a large-scale
mutagenesis of the SDR gene family in the alfalfa root nodule symbiont
Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for
mutants of 21 of the SDR-encoding genes. This brings the total number of S.
meliloti SDR-encoding genes with known function or associated phenotype to 25.
Several of the mutants were deficient in the utilization of specific carbon
sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago
sativa), ranging from partial ineffectiveness to complete inability to form root
nodules. Five of the mutants had both symbiotic and carbon utilization
phenotypes. These results clearly demonstrate the importance of the SDR family in
both symbiosis and saprotrophy, and reinforce the complex nature of the
interaction of S. meliloti with its plant hosts. Further analysis of the genes
identified in this study will contribute to the overall understanding of the
biology and metabolism of S. meliloti in relation to its interaction with
alfalfa.
DOI: 10.1094/MPMI-21-7-0979
1599. Mol Plant Microbe Interact. 2008 Jul;21(7):947-57. doi: 10.1094/MPMI-21-7-

0947.
The outer membrane protein TolC from Sinorhizobium meliloti affects protein
secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis.
Cosme AM, Becker A, Santos MR, Sharypova LA, Santos PM, Moreira LM.
Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation

relationship with Medicago sativa. During this process, it must cope with diverse
environments and has evolved different types of transport systems that help its
propagation in the plant roots. TolC protein family members are the
outer-membrane components of several transport systems involved in the export of
diverse molecules, playing an important role in bacterial survival. In this work,
we have characterized the protein TolC from S. meliloti 2011. An insertional
mutation in the tolC gene strongly affected the resistance phenotype to
antimicrobial agents and induced higher susceptibility to osmotic and oxidative
stresses. Immunodetection experiments and comparison of the extracellular
proteins present in the supernatant of the wild-type versus tolC mutant strains
showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the
product of open reading frame SMc04171, a putative hemolysin-type calcium-binding
protein, are secreted by a TolC-dependent secretion system. In the absence of
TolC, neither succinoglycan nor galactoglucan were detected in the culture
supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of
nonfixing nitrogen nodules in M. sativa roots. Taken together, our results
confirm the importance of TolC in protein secretion, exopolysaccharide
biosynthesis, antimicrobials resistance, and symbiosis.
DOI: 10.1094/MPMI-21-7-0947
1600. Ying Yong Sheng Tai Xue Bao. 2008 Mar;19(3):691-4.
[Effects of cutting on the population dynamics of main insect pests on alfalfa].
Liu CZ(1), Yan L, Wei LX, Zhang F, Qian XJ.
Author information:
(1)Department of Entomology, College of Grassland Science, Gansu Agricultural
University, Lanzhou 730070, China. liuchzh@gsau.edu.cn
A systematic investigation was made on the effects of cutting on the population

dynamics of main insect pests on alfalfa in Dingxi of Gansu Province. The results
showed that the effects of cutting varied significantly with the kinds of insect
pests. The seasonal average population densities of Therioaphis trifolii,
Acyrthosiphon pisum and thrips decreased significantly and maintained at a low
level under the first cutting in early June, but less variation was observed
under the second cutting in mid-July. Plant bug presented somewhat different
picture. In the early period of first cutting, its seasonal average population
density decreased significantly but increased then, and was significantly higher
in early August, compared with that in uncut field. Opposite with T. trifolii, A.
pisum and thrips, the seasonal average population density of plant bug was more
affected by second cutting.
1601. Environ Toxicol. 2008 Oct;23(5):607-16. doi: 10.1002/tox.20405.
Plant response to heavy metal toxicity: comparative study between the

hyperaccumulator Thlaspi caerulescens (ecotype Ganges) and nonaccumulator plants:
lettuce, radish, and alfalfa.
Benzarti S(1), Mohri S, Ono Y.
Author information:
(1)The Graduate School of Environmental Science, Okayama University. 3-1-1,
Tsushimanaka, Okayama 700-8530, Japan. dev18310@cc.okayama-u.ac.jp
Thlaspi caerulescens (alpine pennycress) is one of the best-known heavy metal

(HM) hyperaccumulating plant species. It exhibits the ability to extract and
accumulate various HM at extremely high concentrations. In this hydroponic study,
the performance of T. caerulescens (ecotype Ganges) to accumulate Cd, Zn, and Cu
was compared with that of three nonaccumulator plants: alfalfa (Medicago sativa),
radish (Raphanus sativus), and lettuce (Lactuca sativa). Plants were exposed to
the separately dissolved HM salts for 7 days at a wide range of increasing
concentrations: 0 (control: 1/5 Hoagland nutrient solution), 0.1, 1, 10, 100, and
1000 microM. The comparative study combined chemical, physiological, and
ecotoxicological assessments. Excessive concentrations of HM (100 and 1000
microM) affected plant growth, photosynthesis, and phytoaccumulation efficiency.
Root exudation for all plant species was highly and significantly correlated to
HM concentration in exposure solutions and proved its importance to counter
effect toxicity. T. caerulescens resisted better the phytotoxic effects of Cd and
Zn (at 1000 microM each), and translocated them significantly within tissues (366
and 1290 microg g(-1), respectively). At the same HM level, T. caerulescens
exhibited lower performances in accumulating Cu when compared with the rest of
plant species, mainly alfalfa (298 microg g(-1)). Root elongation inhibition test
confirmed the selective aptitude of T. caerulescens to better cope with Cd and Zn
toxicities. MetPLATE bioassay showed greater sensitivity to HM toxicity with much
lower EC(50) values for beta-galactosidase activity in E. coli. Nevertheless,
exaggerated HM concentrations coupled with relatively short exposure time did not
allow for an efficient metal phytoextraction thus a significant reduction of
ecotoxicity.
DOI: 10.1002/tox.20405
1602. J Food Prot. 2008 May;71(5):888-96.
Factors influencing the growth of Salmonella during sprouting of naturally

contaminated alfalfa seeds.
Fu TJ(1), Reineke KF, Chirtel S, VanPelt OM.
Author information:
(1)U.S. Food and Drug Administration, Summit-Argo, Illinois 60501, USA.
tong.fu@fda.hhs.gov
In this study, the factors that affect Salmonella growth during sprouting of
naturally contaminated alfalfa seeds associated with two previous outbreaks of
salmonellosis were examined. A minidrum sprouter equipped with automatic
irrigation and rotation systems was built to allow sprouting to be conducted
under conditions similar to those used commercially. The growth of Salmonella
during sprouting in the minidrum was compared with that observed in sprouts grown
in glass jars under conditions commonly used at home. The level of Salmonella
increased by as much as 4 log units after 48 h of sprouting in jars but remained
constant during the entire sprouting period in the minidrum. The effect of
temperature and irrigation frequency on Salmonella growth was examined.
Increasing the sprouting temperature from 20 to 30 degrees C increased the
Salmonella counts by as much as 2 log units on sprouts grown both in the minidrum
and in the glass jars. Decreasing the irrigation frequency from every 20 min to
every 2 h during sprouting in the minidrum or from every 4 h to every 24 h during
sprouting in the glass jars resulted in an approximately 2-log increase in
Salmonella counts. The levels of total aerobic mesophilic bacteria, coliforms,
and Salmonella in spent irrigation water closely reflected those found in
sprouts, confirming that monitoring of spent irrigation water is a good way to
monitor pathogen levels during sprouting.
1603. Seikagaku. 2008 Apr;80(4):300-7.
[Occurrence of D-amino acids in higher plants and foods and the enzymes related
to their metabolism].
[Article in Japanese]
Oikawa T(1).
Author information:
(1)Department of Life Science and Biotechnology, Faculty of Chemistry, Materials
and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita-Shi, Osaka-Fu
564-8680, Japan.
1604. Int J Cosmet Sci. 1998 Aug;20(4):223-33. doi: 10.1046/j.1467-

2494.1998.176608.x.
Study of the refirming effect of a plant complex.
Benaiges A(1), Marcet P, Armengol R, Betes C, Gironés E.
Author information:
(1)Provital S.A. Pol. Ind. Santiga, Talleres 6, Barberádel Vallés, Barcelona,
Spain.
Loss of skin elasticity is one of the main problems of ageing. This is a

mechanical property influenced by elastin, a protein in the dermis which,
together with collagen and glycosaminoglycans, makes up the connective tissue.
This tissue is affected by a large number of events (such as cutaneous ageing,
pregnancy, slimming processes and cellulitis) which eventually cause it to
change. At the same time, the metabolism of the proteins of the connective tissue
decreases and there is an ever greater presence of enzymes, principally elastases
and collagenases, which are responsible for breaking down the elastin and the
collagen. One way to prevent such a loss of elasticity is to use active
ingredients that are able to inhibit elastase enzymes. A plant complex was
prepared using the following plants: lady's thistle (Silybum marianum GAERTN),
alchemilla or yarrow (Alchemilla vulgaris L.), horsetail (Equisetum arvense L.)
as well as germinated seeds (Glycine soja Siebold and Zucc., Triticum vulgare
Vilars, Medicago sativa L., Raphanus sativus L.). The complex was standardized to
give the corresponding active principles, silybin, tannins, silicon and peptides,
respectively, and in vitro enzymatic tests were carried out to establish its
ability to inhibit elastase. The study of enzymatic inhibition was carried out
using two enzymes: (1) porcine pancreatic elastase (PPE), and (2) human leukocyte
elastase (HLE). The results showed that the plant complex presents
non-competitive inhibition in the order of 41.0% against PPE and 50.0% against
HLE. An in vivo test was made alongside the in vitro test using an SEM 474
Cutometer (Courage & Khazaka) to study the elasticity of the skin, and positive
effects were obtained when applying a cosmetic formulation containing 5% of the
plant complex. Image analysis of duplicates of the cutaneous surface, before and
after treatment began with a product containing 5% of plant complex and showed
that wrinkles were decreased by 36.7%.
DOI: 10.1046/j.1467-2494.1998.176608.x
PMID: 18505506
1605. Plant Cell Rep. 2008 Aug;27(8):1271-9. doi: 10.1007/s00299-008-0546-5. Epub

2008
May 27.
Dwarf plants of diploid Medicago sativa carry a mutation in the gibberellin

3-beta-hydroxylase gene.
Dalmadi A(1), Kaló P, Jakab J, Saskoi A, Petrovics T, Deák G, Kiss GB.
Author information:
(1)Institute of Genetics Agricultural Biotechnology Center, Szent-Györgyi A. St.
4, 2100, Gödöllo, Hungary.
In this paper we describe the identification of a gene, MsDWF1 coding for a

putative gibberellin 3-beta-hydroxylase (GA3ox), whose natural mutation is
conditioning a dwarf growth phenotype in Medicago sativa. The dwarf phenotype
could not be complemented with grafting, which indicates that the bioactive
gibberellin compound necessary for shoot elongation is immobile. On the contrary,
exogenously added gibberellic acid restored normal growth. The genetic position
of the Msdwf1 gene was mapped to linkage group 2 (LG2) and the physical location
was delimited by map-based cloning using Medicago truncatula genomic resources.
Based on the similar appearance and behavior of the dwarf Medicago sativa plants
to the pea stem length mutant (le) as well as the synthenic map position of the
two genes it was postulated that MsDWF1 and pea Le are orthologs. The comparison
of wild type and mutant allele sequences of MsGA3ox revealed an amino acid change
in a conserved position in the mutant allele, which most probably impaired the
function of the enzyme. Our results indicate that the dwarf phenotype was the
consequence of this mutation.
DOI: 10.1007/s00299-008-0546-5
1606. J Anim Sci. 2008 Sep;86(9):2277-87. doi: 10.2527/jas.2007-0082. Epub 2008 May
23.
Nutrient net absorption across the portal-drained viscera of forage-fed beef

steers: Quantitative assessment and application to a nutritional prediction
model.
El-Kadi SW(1), McLeod KR, Elam NA, Kitts SE, Taylor CC, Harmon DL, Bequette BJ,
Vanzant ES.
Author information:
(1)Department of Animal and Food Sciences, University of Kentucky, Lexington
40546, USA.
This study aimed to establish the relationship between ME intake and energy and
nutrient absorption across the portal-drained viscera (PDV) of forage-fed beef
steers. Eight Angus (328 +/- 40 kg of BW) steers were surgically fitted with
portal, mesenteric arterial, and mesenteric venous catheters, and were fed
alfalfa cubes in a replicated 4 x 4 Latin square design with 4 levels of energy
intake between 1 and 2 times maintenance energy requirements. On d 28 of each
experimental period, p-aminohippuric acid was infused to measure blood and plasma
flow across the PDV, and blood samples (1 every hour, for 6 h) were collected
simultaneously from arterial and venous catheters for net absorption
measurements. Oxygen utilization, and therefore energy utilization, increased (P
< 0.05) linearly in relation to ME intake. Glucose net uptake was unaffected, but
lactate net release increased linearly in response to ME intake (P < 0.05). Net
absorption of all AA except tryptophan, glutamate, and glutamine increased
linearly with ME intake (P < 0.05). The constant net absorption of glutamate and
glutamine indicated increased net utilization of these AA when dietary supply was
increased. These data provide quantitative measures of the PDV effects on energy
and AA availability for productive tissues, and suggest that the greater net
utilization of some AA when ME intake is increased could relate to their
catabolism for energy production. Prediction estimates of small intestinal AA
absorption, based on the Cornell Net Carbohydrate and Protein System (CNCPS),
exceeded observed net AA PDV absorption. Mean bias represented the greatest
proportion (87 to 96%) of the deviation between individual AA absorption and
observed net AA PDV absorption, suggesting that the CNCPS model may be used to
predict AA net absorption when factors describing AA utilization by the PDV are
applied to model predictions.
DOI: 10.2527/jas.2007-0082
1607. Plant Biotechnol J. 2008 Sep;6(7):702-21. doi: 10.1111/j.1467-

7652.2008.00353.x.
Epub 2008 May 19.
Down-regulated expression of plant-specific glycoepitopes in alfalfa.
Sourrouille C(1), Marquet-Blouin E, D'Aoust MA, Kiefer-Meyer MC, Seveno M,

Pagny-Salehabadi S, Bardor M, Durambur G, Lerouge P, Vezina L, Gomord V.
Author information:
(1)CNRS UMR 6037, IFRMP 23, GDR 2590, Université de Rouen, 76821 Mont Saint
Aignan, France.
SUMMARY: Compared with other plant expression systems used for pharmaceutical
protein production, alfalfa offers the advantage of very homogeneous
N-glycosylation. Therefore, this plant was selected for further attempts at
glycoengineering. Two main approaches were developed in order to humanize
N-glycosylation in alfalfa. The first was a knock-down of two plant-specific
N-glycan maturation enzymes, beta1,2-xylosyltransferase and
alpha1,3-fucosyltransferases, using sense, antisense and RNA interference
strategies. In a second approach, with the ultimate goal of rebuilding the whole
human sialylation pathway, human beta1,4-galactosyltransferase was expressed in
alfalfa in a native form or in fusion with a targeting domain from
N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early
Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies
strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and
beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However,
recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa
completely prevented the accumulation of the Lewis a glycoepitope on complex
N-glycans.
DOI: 10.1111/j.1467-7652.2008.00353.x
1608. Rev Biol Trop. 2006 Jun;54(2):505-13.
[Feeding preferences of the spider Misumenops pallidus (Araneae: Thomisidae) on

potential prey insects from alfalfa crops].
Cheli G(1), Armendano A, González A.
Author information:
(1)CEPAVE, CONICET, Facultad de Ciencias Naturales y Museo, UNLP, Argentina,
Calle 2 No 584 (1900) La Plata, Argentina. chelicera@arnet.com.ar
The spider Misumenops pallidus (Thomisidae) is commonly found in alfalfa crops.

We studied its predatory preferences on potential insect prey, particularly
regarding agroecosystems pests. Two kinds of tests were done under normal
laboratory conditions: simultaneous presentation of prey (n = 215) and
alternative prey test (n = 45). The spiders preferred insects that were mobile,
small, without defensive glands and with thin exoskeletons. According to the
amount of prey consumed, we established four predation levels: high (> 55%, on
adult Drosophila melanogaster flies); intermediate (30%-55% on the defoliator
larvae of Rachiplusia nu and adult heteropterans: Horciasinus argentinus and
Halticus spegazzinii); and low (10%-30% on the chrysomelids Colapsis sp. and
Diabrotica speciosa). The pentatomid Piezodorus guildinii, the curculionid
Naupactus sp. and the aphid Acyrthosiphom pisum were not accepted as food. Once
the spider captured a prey item it did not accept another, independently of prey
item species (82% of trials).
1609. Poult Sci. 2008 Jun;87(6):1005-11. doi: 10.3382/ps.2006-00386.
Behavioral responses of laying hens to different alfalfa-layer ration

combinations fed during molting.
Dunkley CS(1), Friend TH, McReynolds JL, Woodward CL, Kim WK, Dunkley KD, Kubena
LF, Nisbet DJ, Ricke SC.
Author information:
(1)Texas A&M University, Department of Poultry Science, College Station
77843-2472, USA.
Induced molting by feed withdrawal has been a common practice in the commercial
layer industry and usually involves the removal of feed for a period of up to 14
d. However, this is a practice that is believed to adversely influence the
welfare of the hens and there is a need to examine behavioral responses to
alternative molt regimens. The behavioral patterns of hens on 90% alfalfa:10%
layer ration, 80% alfalfa:20% layer ration, and 70% alfalfa:30% layer ration molt
diets were compared with feed withdrawal (FW) hens, and fully fed (FF) hens. The
White Leghorn laying hens were approximately 54 wk old and were placed in 3
identical climate-controlled rooms. The hens were individually housed in 2-tier
wire battery cages and provided treatment rations and water ad libitum.
Nonnutritive pecking, walking, drinking, feeder activity, preening, aggression,
and head movement were quantified during two 10-min periods each day for 6 hens
from each treatment. Over the 9-d treatment period, hens in the FW, 70%
alfalfa:30% layer ration, and 80% alfalfa:20% layer ration groups spent
significantly more time walking than hens in the 90% alfalfa:10% layer ration
group. The FF and 70% alfalfa:30% layer ration hens spent half as much time
preening, whereas the FW hens displayed nearly twice as much nonnutritive pecking
when compared with other treatments. Most differences in head movements occurred
at the beginning of the molt period, whereas during the last half of molt,
alfalfa-fed hens exhibited feeder activity similar to FF hens, and all were
significantly higher than that of FW hens. After some initial adjustment by the
hens, consumption of alfalfa molt diets appeared to reduce nonnutritive pecking
behavior, which is characteristically associated with FW hens.
DOI: 10.3382/ps.2006-00386
Hot topic: application of support vector machine method in prediction of alfalfa

protein fractions by near infrared reflectance spectroscopy.
Nie Z(1), Han J, Liu T, Liu X.
Author information:
China Agricultural University, Beijing 10094, China.
The object of this study was to explore the potential for support vector machine
(SVM) to improve the precision of predicting protein fractions by near infrared
reflectance spectroscopy (NIRS). Generally, most protein fractions determined in
Cornell Net Carbohydrate and Protein System (CNCPS), especially the neutral
detergent insoluble protein (NDFCP) and acid detergent insoluble protein (ADFCP),
could not be accurately predicted by the commonly used partial least squares
(PLS) method. A recently developed chemometric method, SVM, was applied in NIRS
prediction of alfalfa protein fractions in this study. Two hundred thirty alfalfa
samples were scanned on a near infrared reflectance spectrophotometer, and
analyzed for crude protein (CP), true protein precipitated in tungstic acid
(TCP), borate-phosphate buffer-insoluble protein (BICP), NDFCP, and ADFCP. These
5 laboratory proteins and the CNCPS protein fractions A, B1, B2, B3, and C were
predicted by NIRS using the PLS and SVM methods. According to PLS-NIRS
regression, CP, TCP, BICP, A, and B2 obtained the determination coefficient of
prediction (R(p)(2)) of 0.96, 0.91, 0.94, 0.94, and 0.93, and the ratios of
standard deviation of prediction samples: standard error of prediction samples
(RPD) values were 5.07, 3.31, 3.98, 3.96, and 3.91. Neutral detergent insoluble
protein, ADFCP (fraction C), B1, and B3 were predicted with R(p)(2) of 0.75,
0.83, 0.30, and 0.62, and RPD values of 1.98, 2.42, 1.20, and 1.62; Calibrated by
the SVM-NIRS method, R(p)(2) values of CP, TCP, BICP, NDFCP, ADFCP(C), A, and B2
achieved 0.99, 0.97, 0.97, 0.90, 0.93, 0.97, and 0.97, respectively. The RPD
values of those fractions were 8.68, 8.26, 6.11, 3.08, 3.69, 5.97, and 5.81,
respectively. The R(p)(2) and RPD values of fractions B1 and B3 were 2.67 and
0.87 (B1) and 2.51 and 0.75 (B3) directly predicted by SVM-NIRS model. In this
study, the chemical analysis results of B1 and B3 were also correlated with
calculated results from TCP-BICP and NDFCP-ADFCP, which were predicted by
SVM-NIRS models. The B1 protein fraction achieved R(p)(2) and RPD values of 0.87
and 3.61, whereas values for B2 were 0.75 and 2.00. Data suggested that use of
SVM methods in NIRS technology could improve the accuracy of predicting protein
fractions. This study showed the potential of increasing the NIRS prediction
accuracy to a level of practical use for all protein fractions, except B3.
DOI: 10.3168/jds.2008-0985
1611. Int Immunopharmacol. 2008 Jul;8(7):1023-32. doi:

10.1016/j.intimp.2008.03.007.
Epub 2008 Apr 7.
The majority of in vitro macrophage activation exhibited by extracts of some

immune enhancing botanicals is due to bacterial lipoproteins and
lipopolysaccharides.
Pugh ND(1), Tamta H, Balachandran P, Wu X, Howell J, Dayan FE, Pasco DS.
Author information:
(1)National Center for Natural Products Research, Research Institute of
Pharmaceutical Sciences, School of Pharmacy, University of Mississippi,
University, MS 38677, USA.
We have identified potent monocyte/macrophage activating bacterial lipoproteins

within commonly used immune enhancing botanicals such as Echinacea, American
ginseng and alfalfa sprouts. These bacterial lipoproteins, along with
lipopolysaccharides, were substantially more potent than other bacterially
derived components when tested in in vitro monocyte/macrophage activation
systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the
majority (85-98%) of the activity within extracts from eight immune enhancing
botanicals was eradicated by treatment with agents (lipoprotein lipase and
polymyxin B) known to target these two bacterial components. Alfalfa sprouts
exhibited the highest activity of those botanicals tested but the appearance of
this activity during the germination of surface sterilized seeds was abolished by
the presence of antibiotics. These studies indicate that the majority of the in
vitro macrophage activating properties in extracts from these botanicals can be
attributed to the presence of lipoproteins and lipopolysaccharides derived from
bacteria and that bacterial endophytes may be a significant source of these
components.
DOI: 10.1016/j.intimp.2008.03.007
PMCID: PMC2467439
1612. Anal Biochem. 2008 Aug 1;379(1):66-72. doi: 10.1016/j.ab.2008.04.034. Epub

2008
Apr 25.
Plant N-glycan profiling of minute amounts of material.
Séveno M(1), Cabrera G, Triguero A, Burel C, Leprince J, Rihouey C, Vézina LP,

D'Aoust MA, Rudd PM, Royle L, Dwek RA, Harvey DJ, Lerouge P, Cremata JA, Bardor
M.
Author information:
(1)CNRS-FRE 3090, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan Cédex,
France; Medicago Inc., Québec, QC G1V 3V9, Canada.
Development of convenient strategies for identification of plant N-glycan

profiles has been driven by the emergence of plants as an expression system for
therapeutic proteins. In this article, we reinvestigated qualitative and
quantitative aspects of plant N-glycan profiling. The extraction of plant
proteins through a phenol/ammonium acetate procedure followed by deglycosylation
with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides
an oligosaccharide preparation containing reduced amounts of contaminants from
plant cell wall polysaccharides. Such a preparation was also suitable for
accurate qualitative and quantitative evaluation of the N-glycan content by mass
spectrometry. Combining these approaches allows the profiling to be carried out
from as low as 500 mg of fresh leaf material. We also demonstrated that
collision-induced dissociation (CID) mass spectrometry in negative mode of
N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal
GlcNAc leads to specific fragmentation patterns, thereby allowing the
discrimination of plant N-glycans from those arising from mammalian
contamination.
DOI: 10.1016/j.ab.2008.04.034
1613. Guang Pu Xue Yu Guang Pu Fen Xi. 2008 Feb;28(2):317-20.
[Prediction of leaf concentration in alfalfa using near infrared reflectance

spectroscopy].
Nie ZD(1), Han JG, Yu Z, Zhang LD.
Author information:
China.
Leaf concentration in alfalfa is an important factor affecting the nutritive

value, forage intake and digestibility. Estimates of leaf concentrations commonly
used currently involve a labor intensive process of hand separating leaf and stem
fractions. In the present study, a total of 41 artificial alfalfa samples were
mixed with different leaf concentrations ranging from 15% to 55%. The object was
to develop 3 calibrations for predicting alfalfa leaf concentrations using 15, 25
and 35 calibrated samples by near infrared reflectance spectroscopy. The root
mean square error of prediction(RMSEP)was 1.02, 1.97 and 0.51, respectively.
External validation had a coefficient of determination (r2) ranging from 0.79 8
to 0.998 9. The ratio of performance to standard deviation (RPD) varied from 2.85
to 25.93. The results showed that 15 samples could develop accurate NIRS model of
alfalfa leaf concentrations; the calibration equations got better accuracy with
the increase in calibrated samples numbers from 15 to 35.
1614. Genome. 1997 Dec;40(6):887-95.
Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago
species.
Diwan N, Bhagwat AA, Bauchan GB, Cregan PB.
Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to
function well in plant and mammalian species for genetic map construction and
genotype identification. The objectives of the work reported here were to search
GenBank for the presence of SSR-containing sequences from the genus Medicago, to
assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.)
L. &L.) genome, and to examine the function of selected markers in a spectrum of
perennial and annual Medicago species. The screening of an alfalfa genomic DNA
library and sequencing of clones putatively containing SSRs indicated
approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid
genome. Inheritance was consistent with Mendelian expectations at four selected
SSR loci with different core motifs. Additionally, genotypes of a range of
Medicago species, including 10 perennial subspecies of the M. sativa complex and
other perennial and annual Medicago species, were analyzed at each of the loci to
ascertain the presence, number, and size of SSR alleles at each locus in each
genotype. These studies indicate that SSR markers can function in alfalfa for the
construction of genetic maps and will also be useful in a range of Medicago
species for purposes of assessing genetic relatedness and taxonomic
relationships, and for genotype identification.
PMID: 18464874
1615. Mol Biol Evol. 2008 Aug;25(8):1566-75. doi: 10.1093/molbev/msn102. Epub 2008
May
2.
Substitution of the gene for chloroplast RPS16 was assisted by generation of a

dual targeting signal.
Ueda M(1), Nishikawa T, Fujimoto M, Takanashi H, Arimura S, Tsutsumi N, Kadowaki

K.
Author information:
(1)Genetic Diversity Department, National Institute of Agrobiological Sciences,
Kannondai, Tsukuba, Ibaraki, Japan.
Organelle (mitochondria and chloroplasts in plants) genomes lost a large number

of genes after endosymbiosis occurred. Even after this major gene loss, organelle
genomes still lose their own genes, even those that are essential, via gene
transfer to the nucleus and gene substitution of either different organelle
origin or de novo genes. Gene transfer and substitution events are important
processes in the evolution of the eukaryotic cell. Gene loss is an ongoing
process in the mitochondria and chloroplasts of higher plants. The gene for
ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher
plants but not in Medicago truncatula and Populus alba. Here, we show that these
2 species have compensated for loss of the rps16 from the chloroplast genome by
having a mitochondrial rps16 that can target the chloroplasts as well as
mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and
Oryza sativa, whose chloroplast genomes encode the rps16, we show that the
product of the mitochondrial rps16 has dual targeting ability. These results
suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts
emerged before the divergence of monocots and dicots (140-150 MYA). The gene
substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher
plants is the first report about ongoing gene substitution by dual targeting and
provides evidence for an intermediate stage in the formation of this
heterogeneous organelle.
DOI: 10.1093/molbev/msn102
1616. J Environ Qual. 2008 May 2;37(3):772-9. doi: 10.2134/jeq2007.0157. Print 2008
May-Jun.
Soil sulfur amendments suppress selenium uptake by alfalfa and Western

wheatgrass.
Mackowiak CL(1), Amacher MC.
Author information:
(1)University of Florida, North Florida Research and Education Center, 155
Research Drive, Quincy, FL 32351-5677, USA.
Selenium (Se) is a potential soil contaminant in many parts of the world where it
can pose a health risk to livestock and wildlife. Phosphate ore mining in
Southeast Idaho has resulted in numerous waste rock dumps revegetated with
forages to stabilize the dumps and support grazing. Alfalfa (Medicago sativa L.),
smooth brome (Bromus inermis Leyss.), and western wheat grass [Pascopyrum smithii
(Rydb.) A. Löve] are the dominant forage species on these lands. To demonstrate
the feasibility of using sulfur (S) as a soil amendment to restrict plant Se
uptake, 3 kg pots containing 50:50 w/w soil and waste shale were uniformly mixed
with 0, 0.5, 1.0, or 2.0 Mg ha(-1) S as either elemental S or gypsum. Pots were
seeded with alfalfa or western wheat grass. Dry mass and tissue Se were monitored
over several clippings. Soils were sampled at the conclusion of the study and
analyzed for water-soluble, oxalate-extractable, and total Se. Sulfur amendments
as either elemental S or gypsum at 1.0 Mg ha(-1) or greater equally suppressed Se
uptake over 60% in both forage species. Alfalfa accumulated more Se than western
wheat grass. Plant removal via successive clippings resulted in lower tissue Se
accumulation over time than the use of S soil amendments alone. Alfalfa-planted
soils contained lower water-soluble and oxalate-extractable Se than did the
non-planted controls while western wheat grass-planted soils contained lower
water-soluble Se. Applying S to these shale-based soils may be an economically
viable option for treating Se-impacted, revegetated lands.
DOI: 10.2134/jeq2007.0157
1617. J Anim Sci. 2008 Sep;86(9):2344-56. doi: 10.2527/jas.2007-0545. Epub 2008 Apr
25.
Comparing relative feed value with degradation parameters of grass and legume
forages.
Hackmann TJ(1), Sampson JD, Spain JN.
Author information:
(1)Division of Animal Sciences, University of Missouri, Columbia 65211, USA.
Relative feed value (RFV) was evaluated relative to in situ degradation

parameters of grass and legume forages. Early-cut alfalfa (n = 20), late-cut
alfalfa (n = 26), cool-season grass (n = 11), warm-season grass (n = 4), and
grass-legume (n = 20) samples were collected from duplicate hay bales submitted
to the 2002 and 2003 Missouri State Fair Hay Contests. Subsamples were incubated
in the rumen of 2 lactating Holstein cows for 0, 6 or 8, 12, 24, and 48 h to
determine in situ degradation of DM, ADF, NDF, CP, and hemicellulose over time.
Degradation data were fit to a variety of candidate models to estimate
degradation parameters. Correlation coefficients between degradation parameter
estimates [sorted according to forage (early-cut alfalfa, late-cut alfalfa,
grass, or grass-legume)] and RFV were determined. For further comparison,
correlations between NDF degradation parameter estimates and digestible DMI were
determined with data from a previous study. Degradation data were best fit to a
single, gamma 2-distributed pool model without a lag phase. Relative feed value
was significantly correlated (P < 0.05) with potentially digestible DM and CP for
early-cut alfalfa, potentially digestible DM for late-cut alfalfa, and
potentially digestible DM, NDF, and hemicellulose for grass-legume. The
percentage of significant correlations (10.7%) across the entire data set was low
and no correlations were significant for grass. Relative feed value did not
account for the variation in degradation parameters, especially for grasses. A
further correlation analysis, which compared digestible DMI with degradation
parameter estimates reported from another data set, revealed that digestible DMI
and degradation parameter estimates were related for grass but not for alfalfa
forages. These results suggest that RFV is limited by its failure to include
degradation parameters.
DOI: 10.2527/jas.2007-0545
1618. J Biotechnol. 2008 Jun 1;135(2):224-31. doi: 10.1016/j.jbiotec.2008.03.006.

Epub
2008 Mar 22.
Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein

in transgenic alfalfa for the development of an edible vaccine against bovine
pneumonic pasteurellosis.
Lee RW(1), Cornelisse M, Ziauddin A, Slack PJ, Hodgins DC, Strommer JN, Shewen
PE, Lo RY.
Author information:
(1)Department of Molecular and Cellular Biology, University of Guelph, Guelph,
ON, Canada N1G 2W1.
The GS60 antigen is one of the protective antigens of Mannheimia haemolytica A1.
GS60 contains conserved domains belonging to the LppC family of bacterial outer
membrane lipoproteins. A high antibody titer to GS60 has been shown to be
significantly correlated with resistance to pneumonic pasteurellosis. Calves
vaccinated with a commercial vaccine (Presponse) and demonstrating protection
against M. haemolytica A1 produced antibodies directed against GS60. Alfalfa was
chosen as the platform for an edible vaccine. Agrobacterium tumefaciens was used
to mediate the transformation of alfalfa with sequences encoding a slightly
shortened derivative of the GS60 antigen (GS60(54)). Stable transgenic alfalfa
lines were recovered and production of GS60(54) was examined by Western
immunoblot analysis. The antigen is stable in dried transgenic plant material
stored at ambient temperature for more than a year. The plant-produced GS60(54)
protein was shown to be immunogenic when injected into rabbits. Feeding of the
dried transgenic alfalfa expressing the GS60(54) to rabbits is capable of
inducing seroconversion, suggesting that GS60(54) could be an effective oral
antigen for stimulating mucosal immune responses.
DOI: 10.1016/j.jbiotec.2008.03.006
1619. Bull Entomol Res. 2008 Jun;98(3):217-21. doi: 10.1017/S000748530800597X. Epub

2008 Apr 28.
Does host plant influence parasitism and parasitoid species composition in Lygus
rugulipennis? A molecular approach.
Gariepy TD(1), Kuhlmann U, Gillott C, Erlandson M.
Author information:
(1)Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan,
Canada. taradawn@hawaii.edu
Lygus Hahn plant bugs (Hemiptera: Miridae) are serious pests of a wide variety of
economically important crops in North America. European Peristenus digoneutis
Loan and P. relictus Ruthe (Hymenoptera: Braconidae) are being considered for
release in Canada as part of a classical biological control program for Lygus.
The attractiveness of different host plants to European Peristenus has not been
addressed, but may be an important consideration prior to parasitoid release.
Lygus rugulipennis Poppius nymphs were collected in the Northern Temperate
Atlantic (NTA) ecoregion on red clover (Trifolium pratense L.; Fabaceae) and
chamomile (Matricaria recutita L.; Asteraceae), and in the Western European
Broadleaf Forest (WEBF) ecoregion on red clover and alfalfa (Medicago sativa L.;
Fabaceae). Parasitism levels and parasitoid species were determined using a
multiplex PCR assay for P. digoneutis, P. relictus, and P. pallipes Curtis. Mean
parasitism levels in L. rugulipennis were 45-49% in the NTA ecoregion and 25-32%
in the WEBF ecoregion. However, in neither ecoregion were parasitism levels and
parasitoid species compositions significantly different in nymphs from different
host plant species. Furthermore, multiparasitism was low despite the fact that P.
digoneutis and P. relictus share the same host species.
DOI: 10.1017/S000748530800597X
1620. Plant Mol Biol. 2008 Jul;67(5):499-510. doi: 10.1007/s11103-008-9334-4. Epub

2008
Apr 27.
Characterization and comparison of intron structure and alternative splicing

between Medicago truncatula, Populus trichocarpa, Arabidopsis and rice.
Baek JM(1), Han P, Iandolino A, Cook DR.
Author information:
(1)College of Agricultural and Environmental Sciences Genomics Facility,
University of California, 117 Robbins hall, Davis, CA 95616, USA.
jbaek@ucdavis.edu
Alignment of transcripts and genome sequences yielded a set of alternatively

spliced transcripts in four angiosperm genomes: three dicotyledon species
Medicago truncatula (Medicago), Populus trichocarpa (poplar) and Arabidopsis
thaliana (Arabidopsis), and the monocotyledon Oryzae sativa (rice). Intron
retention was the predominant mode of alternative splicing (AS) in each species,
consistent with previous reports for Arabidopsis and rice. We analyzed the
structure of 5'-splice junctions and observed commonalities between species.
There was dependency of base composition between sites flanking the 5'-splice
junction, with the potential to create a subset of splice sites that interact
more weakly or strongly than average with U1 snRNA. Such altered nucleotide
composition was correlated with splicing fidelity in all four species. For
Medicago, poplar and Arabidopsis, but not in rice, alternative splicing was most
prevalent for introns with decreased UA content, consistent with lower UA content
for monocot introns and potentially reflecting evolved differences in splicing
mechanisms. Similarly, the occurrence of AS between transcript Gene Ontology
categories was positively correlated between Arabidopsis and Medicago, with no
correlation between dicots and rice. Analysis of within-species paralogs and
between-species reciprocal best-hit homologs yielded rare cases of potentially
conserved AS events. Reverse transcriptase PCR and amplicon sequencing were used
to confirm a subset of the in silico-predicted AS events within Medicago, as well
as to characterize conserved AS events between Medicago and Arabidopsis.
DOI: 10.1007/s11103-008-9334-4

2008
Apr 14.
The model legume Medicago truncatula A17 is poorly matched for N2 fixation with
the sequenced microsymbiont Sinorhizobium meliloti 1021.
Terpolilli JJ(1), O'Hara GW, Tiwari RP, Dilworth MJ, Howieson JG.
Author information:
(1)Centre for Rhizobium Studies, School of Biological Sciences and Biotechnology,
Murdoch University, South St, Murdoch, WA 6150, Australia.
j.terpolilli@murdoch.edu.au
Comment in
New Phytol. 2008;179(1):3-5.
Medicago truncatula (barrel medic) A17 is currently being sequenced as a model

legume, complementing the sequenced root nodule bacterial strain Sinorhizobium
meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M.
truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness
was examined with eight Medicago species and three accessions of M. truncatula
with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant
nitrogen content and nodule distribution, morphology and number were analysed.
Compared with nitrogen-fed controls, Sm1021 was ineffective or partially
effective on all hosts tested (excluding M. sativa), as measured by reduced dry
weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula
accessions produced more nodules, which were small, pale, more widely distributed
on the root system and with fewer infected cells. The Sm1021-M. truncatula
symbiosis is poorly matched for N(2) fixation and the strain could possess
broader N(2) fixation deficiencies. A possible origin for this reduction in
effectiveness is discussed. An alternative sequenced strain, effective at N(2)
fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.
DOI: 10.1111/j.1469-8137.2008.02464.x
1622. Poult Sci. 2008 May;87(5):815-22. doi: 10.3382/ps.2006-00385.
Behavior of laying hens on alfalfa crumble molt diets.
Dunkley CS(1), Friend TH, McReynolds JL, Kim WK, Dunkley KD, Kubena LF, Nisbet
DJ, Ricke SC.
Author information:
(1)Texas A&M University, Department of Poultry Science, College Station, TX
77843-2472, USA.
Several dietary alternatives to feed withdrawal have been proposed to induce a
molt in laying hens. This study compared the behavior of laying hens on an
alfalfa crumble diet (ALC) to hens that were either on a conventional layer diet
(FF) or hens that had feed withdrawn (FW) during a 9-d trial. Each treatment
consisted of 24 hens (3 hens per battery cage), and treatment began after a
2-week acclimation period. Video cameras connected to a digital multiplexer
recorded the behavior of the hens. The percentages of observations performing
nonnutritive pecking, feeder activity, drinking, walking, preening, head
movement, and aggression were quantified for two 10-min periods at daily
intervals. The FF hens spent significantly more (P < or = 0.05) time drinking
than the other treatments, whereas FW hens displayed the most head movements.
From d 1 through 7, FW hens walked less than ALC hens except on d 8 when FW hens
walked more than ALC and FF hens. On d 4 and 6, the FW hens spent an increased
amount of time preening compared with FF hens until the last few days of the molt
period. For the most part, FW hens generally displayed more nonnutritive pecking
than ALC and FF hens throughout the molt period. However, FW hen visits to the
feeders declined as the trial proceeded, whereas ALC and FF hens generally spent
more time at the feeder. In summary, the ALC diet showed potential as an
alternative to FW for inducing a molt in laying hens based on reduced
nonnutritive pecking behavior, head movements, and greater feeding activity.
DOI: 10.3382/ps.2006-00385
Short communication: salivary secretion during meals in lactating dairy cattle.
Beauchemin KA(1), Eriksen L, Nørgaard P, Rode LM.
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge,
Alberta, Canada T1J 4B1. beauchemink@agr.gc.ca
Four multiparous Holstein cows in midlactation were used in a 4 x 4 Latin square

to evaluate whether source of forage influenced salivary secretion during eating
in lactating dairy cows. The forages were allocated separately from the pelleted
concentrates. Cows were offered 1 of 4 forages each period: barley silage,
alfalfa silage, long-stemmed alfalfa hay, or chopped barley straw. Saliva
secretion was measured during the morning meal by collecting masticates through
the rumen cannula at the cardia of each cow. Rate of salivation (213 g/min) was
not affected by forage source. However, the forage sources differed in eating
rate (g of DM/min), which led to differences in ensalivation of forages (g of
saliva/g of DM and g of saliva/g of NDF). On the basis of DM, ensalivation (g of
saliva/g of DM) was greatest for straw (7.23) and similar for barley silage,
alfalfa silage, and alfalfa hay (4.15, 3.40, and 4.34 g/g of DM, respectively).
Higher ensalivation of straw could be accounted for by its higher neutral
detergent fiber (NDF) content; ensalivation of NDF (g of saliva/g of NDF) was
actually greatest for long-stemmed alfalfa hay (12.4) and similar for the other
chopped forages (8.9). Cows consumed concentrate about 3 to 12 times faster than
the various forages (DM basis), and ensalivation of concentrate was much lower
(1.12 g of saliva/g of DM) than for forages. Feed characteristics such as
particle size, DM, and NDF content affect salivary output during eating by
affecting the eating rate. Slower eating rate and greater time spent eating may
help prevent ruminal acidosis by increasing the total daily salivary secretion in
dairy cows.
DOI: 10.3168/jds.2007-0726
Effects of the chop lengths of alfalfa silage and oat silage on feed intake, milk
production, feeding behavior, and rumen fermentation of dairy cows.
Bhandari SK(1), Li S, Ominski KH, Wittenberg KM, Plaizier JC.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, R3T
2N2 Canada.
Effects of chop length (shorter: 6 mm, or longer: 19 mm) of alfalfa silage and
oat silage were determined in 16 mid-lactation Holstein cows, 4 of which were
rumen cannulated, using a replicated 4 x 4 Latin square design with a 2 x 2
arrangement of treatments. Experimental periods were 21 d long and consisted of
14 d of adaptation and 7 d of sampling. Cows received a total mixed ration
containing [dry matter (DM) basis] 42.0% barley grain-based energy supplement,
10% protein supplement, and 24% of DM longer chop or shorter chop alfalfa silage
and 24% of DM longer chop or shorter chop oat silage. Rumen pH was measured
continuously, and rumen liquid flow rates were determined in rumen-cannulated
cows. Feeding behavior was determined by videotaping, and meal patterns were
determined by continuously weighing the feed in the bunk of 8 cows. Reducing the
chop length of alfalfa silage and oat silage reduced the average geometric
particle length from 14.2 to 10.9 mm and from 13.4 to 10.4 mm, respectively.
Reducing the alfalfa chop length did not affect feed intake, whereas reducing the
oat silage chop length increased DM intake from to 19.4 to 21.2 kg/d. Reducing
the chop lengths of alfalfa silage and oat silage chop length did not affect milk
production, rumen fermentation, feeding behavior, meal patterns, and blood
metabolites. Daily milk yield, milk fat percentage, and milk protein percentage
averaged 36.1 kg/d, 3.00%, and 3.16%, respectively, across diets. The low milk
fat percentages suggest that the diets induced subacute ruminal acidosis. This
was also substantiated by the rumen pH, which was below 5.6 for more than 122
min/d for all diets. The onset of subacute ruminal acidosis despite apparently
adequate dietary neutral detergent fiber content and particle size distribution
as well as the long duration of chewing might be attributed to sorting against
long feed particles.
DOI: 10.3168/jds.2007-0358
Effect of feeding corn, hull-less or hulled barley on fermentation by mixed

cultures of ruminal microorganisms.
Fellner V(1), Burns JC, Marshall DS.
Author information:
(1)Department of Animal Science, North Carolina State University, Raleigh, NC
27695, USA. Vivek_Fellner@ncsu.edu
Increased demands for corn grain warrant the evaluation of alternative grain
types for ruminant production systems. This study was conducted to determine the
effects of hulled and hull-less barley (Hordeum vulgare L.) cultivars compared
with corn (Zea mays L.) as an alternative grain type on fermentation in cultures
of mixed ruminal microorganisms. Three continuous fermentors were fed 14 g of dry
feed per day (divided equally between 2 feedings) consisting of alfalfa (Medicago
sativa L.) hay pellets (40% of dry matter) and 1) ground corn, 2) hulled barley,
or 3) hull-less barley concentrate (60% of dry matter) in each fermentor.
Following an adaptation period of 5 d, culture samples were taken at 2 h after
the morning feeding on d 6, 7, and 8 of each period for analysis. A second run of
the fermentors followed the same treatment sequence to provide replication.
Culture pH was reduced with corn (5.55) and did not differ between barley
cultivars (average pH 5.89). Total volatile fatty acid concentration and acetate
to propionate ratio were not different across grain type or barley cultivar with
the exception of greater total volatile fatty acid concentrations with hull-less
barley. Corn produced less methane (14.6 mmol/d) and ammonia-N (7.3 mg/100 mL)
compared with barley (33.1 mmol/d and 22 mg/100 mL, respectively); methane was
greater with hull-less barley but ammonia-N concentration was similar between the
2 barley cultivars. Hull-less barley had greater digestibility compared with
hulled barley, and corn had reduced digestibility compared with barley.
Concentrations of C18:0 were greater and those of C18:1 and C18:2 lesser in
cultures fed hulled and hull-less barley compared with corn. Our data indicate
that grain type and barley cultivar have an impact on ruminal fermentation. The
lesser starch concentration of barley minimized the drop in culture pH and
improved digestibility.
DOI: 10.3168/jds.2007-0078
1626. J Anim Sci. 2008 Aug;86(8):2032-42. doi: 10.2527/jas.2007-0407. Epub 2008 Apr
11.
Forage systems for cow-calf production in the Appalachian region.
Scaglia G(1), Swecker WS Jr, Fontenot JP, Fiske D, Fike JH, Abaye AO, Peterson
PR, Clapham W, Hall JB.
Author information:
(1)Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and
State University, Blacksburg 24061-0306, USA. billgs@vt.edu
Erratum in
J Anim Sci. 2008 Dec;86(12):3666.. Peterson, P R [added].
Small cow-calf operations are common in the Appalachian region. Tall fescue
[Lolium arundinaceum (Schreb.) S. J. Darbyshire] is the dominant forage in these
systems for direct grazing as well as for stockpiling. The present study was
conducted from 2001 to 2005. A total of 108 Angus and Angus crossbred cows were
allotted randomly to 6 forage systems and then to 3 replicates within each
system. In brief, system 1 had a stocking rate of 0.91 ha/cow in a Middleburg
3-paddock (A, B, and C) system. System 2 was similar to system 1 except for a
stocking rate of 0.71 ha/cow. A stocking rate of 0.71 ha/cow also was used in
systems 3 through 6. All A paddocks had tall fescue, whereas B paddocks had tall
fescue/white clover (Trifolium repens L.) except in system 6, which had tall
fescue/lespedeza [Lespedeza cuneata (Dum. Cours.) G. Don]. System 3 evaluated a
2-paddock (A and B) rotational grazing system, and system 4 evaluated a 3-paddock
(A, B, and C) rotational grazing system, with paddock C containing orchardgrass
(Dactylis glomerata L.) and alfalfa (Medicago sativa L.). Systems 5 and 6
differed from system 2 in the areas of paddocks B and C as well as in the forage
mixtures used. In paddock C, system 5 had switchgrass (Panicum virgatum L.) and
system 6 had tall fescue and birdsfoot trefoil (Lotus corniculatus L.). System 1
had the greatest average herbage availability from weaning until breeding (P <
0.05) with the least amount of hay fed (P = 0.03) when compared with the
remainder of the systems. Differences (P > 0.05) in percentage of ground cover
were not detected among systems. There was no year x system interaction effect on
the cow or calf performance variables evaluated and no treatment effect on cow
performance variables. There was a treatment effect on calf performance
variables. System 2 produced the greatest adjusted weaning weight, kilograms of
calf weaned per hectare, and kilograms of calf per kilograms of cow at weaning (P
< 0.05). Numerical ranking for total calf production per hectare from the
greatest to least was system 2, 6, 3, 5, 4, and 1. Systems evaluated did not
affect cow performance although differences in calf performance and overall
productivity of the systems were observed.
DOI: 10.2527/jas.2007-0407
1627. FEMS Microbiol Lett. 2008 May;282(2):273-81. doi:

10.1111/j.1574-6968.2008.01139.x. Epub 2008 Apr 9.
Stable low molecular weight RNA profiling showed variations within Sinorhizobium
meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa
cross-inoculation group.
Del Villar M(1), Rivas R, Peix A, Mateos PF, Martínez-Molina E, van Berkum P,
Willems A, Velázquez E.
Author information:
(1)Departamento de Microbiología y Genética, Universidad de Salamanca, Spain.
Four different low molecular weight (LMW) RNA profiles, designated I-IV, among
179 isolates from Medicago, Melilotus and Trigonella species growing in a field
site in Northern Spain were identified. From sequence analysis of the 16S rRNA,
atpD and recA genes as well as DNA-DNA hybridization analysis with
representatives of each LMW RNA profile it was evident that isolates with LMW RNA
profiles I and II belonged to Sinorhizobium meliloti and those displaying
profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA
electrophoretic mobility profiles were found within each of these two species.
Collectively, LMW RNA profiles I and II (identified as S. meliloti) were
predominant in Melilotus alba, Melilotus officinalis and Medicago sativa.
Profiles III and IV (identified as S. medicae) were predominant in Melilotus
parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella
foenum-graecum. All the four LMW RNA profiles were identified among isolates from
Trigonella monspelliaca nodules. These results revealed a different specificity
by the hosts of the alfalfa cross-inoculation group towards the two bacterial
species found in this study.
DOI: 10.1111/j.1574-6968.2008.01139.x
1628. Theor Appl Genet. 2008 Jul;117(2):149-56. doi: 10.1007/s00122-008-0761-z.

Epub
2008 Apr 8.
Transfer of anthracnose resistance and pod coiling traits from Medicago arborea
to M. sativa by sexual reproduction.
Armour DJ(1), Mackie JM, Musial JM, Irwin JA.

Author information:
(1)School of Integrative Biology, The University of Queensland, Brisbane, QLD,
Australia. d.armour@uq.edu.au
Five asymmetric hybrid plants were obtained between Medicago sativa (2n = 4x =
32) and Medicago arborea (2n = 4x = 32) through sexual reproduction and the use
of a cytoplasmically male sterile M. sativa genotype. Over 2,000 pollinations
were made to obtain these hybrids. Amplified fragment length polymorphism (AFLP)
analysis showed that in the most studied hybrid (WA2273), 4% of the bands unique
to the M. arborea parent were present, versus 72% for the unique M. sativa bands.
This suggests that only a single M. arborea chromosome or chromosome parts has
been transferred. WA2273 had 7% of AFLP bands which were not present in either
parent, which is suggestive of chromosome rearrangements as would be expected if
only chromosome parts or a single part had been transferred from M. arborea.
Phenotypic evidence for hybridity was obtained for pod coiling (1.4 coils in
WA2273 versus three coils in the M. sativa parent and its self and testcross
populations, and one coil in M. arborea), and Colletotrichum trifolii race 2
resistance (transferred from the resistant M. arborea parent, as the M. sativa
parent and the self populations were highly susceptible). The hybrids were self
sterile, but were female fertile to a high level when crossed with 4x, but not
2x, M. sativa, indicating they were at or near 4x. Both the pod coiling trait and
anthracnose resistance segregated in the progeny of testcrosses between WA2273
and M. sativa. The work demonstrates that agronomically useful traits can be
introgressed into M. sativa from M. arborea by use of male sterile M. sativa and
sexual reproduction.
DOI: 10.1007/s00122-008-0761-z
1629. J Environ Qual. 2008 Mar-Apr;37(2):542-50.
Nitrous oxide emissions from a Northern Great Plains soil as influenced by

nitrogen management and cropping systems.
Dusenbury MP(1), Engel RE, Miller PR, Lemke RL, Wallander R.
Author information:
(1)Dep. of Land Resources and Environmental Sciences, Montana State Univ.,
Bozeman, MT 59717-3120, USA.
Field measurements of N2O emissions from soils are limited for cropping systems
in the semiarid northern Great Plains (NGP). The objectives were to develop N2O
emission-time profiles for cropping systems in the semiarid NGP, define important
periods of loss, determine the impact of best management practices on N2O losses,
and estimate direct N fertilizer-induced emissions (FIE). No-till (NT) wheat
(Triticum Aestivum L.)-fallow, wheat-wheat, and wheat-pea (Pisum sativum), and
conventional till (CT) wheat-fallow, all with three N regimes (200 and 100 kg N
ha(-1) available N, unfertilized control); plus a perennial grass-alfalfa
(Medicago sativa L.) system were sampled over 2 yr using vented chambers.
Cumulative 2-yr N2O emissions were modest in contrast to reports from more humid
regions. Greatest N2O flux activity occurred following urea-N fertilization
(10-wk) and during freeze-thaw cycles. Together these periods comprised up to 84%
of the 2-yr total. Nitrification was probably the dominant process responsible
for N2O emissions during the post-N fertilization period, while denitrification
was more important during freeze-thaw cycles. Cumulative 2-yr N2O-N losses from
fertilized regimes were greater for wheat-wheat (1.31 kg N ha(-1)) than
wheat-fallow (CT and NT) (0.48 kg N ha(-1)), and wheat-pea (0.71 kg N ha(-1)) due
to an additional N fertilization event. Cumulative losses from unfertilized
cropping systems were not different from perennial grass-alfalfa (0.28 kg N
ha(-1)). Tillage did not affect N2O losses for the wheat-fallow systems. Mean FIE
level was equivalent to 0.26% of applied N, and considerably below the
Intergovernmental Panel on Climate Change mean default value (1.25%).
1630. Microbiology. 2008 Apr;154(Pt 4):1258-70. doi: 10.1099/mic.0.2007/014894-0.
The Sinorhizobium meliloti MsbA2 protein is essential for the legume symbiosis.
Beck S(1), Marlow VL, Woodall K, Doerrler WT, James EK, Ferguson GP.
Author information:
(1)Institute of Cell Biology and Centre for Science at Extreme Conditions, School
of Biological Sciences, University of Edinburgh, Edinburgh, UK.
Sinorhizobium meliloti is a beneficial legume symbiont, closely related to

Brucella species, which are chronic mammalian pathogens. We discovered that the
S. meliloti MsbA2 protein is essential to ensure the symbiotic interaction with
the host plant, alfalfa. S. meliloti invades plant cells via plant-derived
structures known as infection threads. However, in the absence of MsbA2, S.
meliloti remains trapped within abnormally thickened infection threads and
induces a heightened plant defence response, characterized by a substantial
thickening of the nodule endodermis layer and the accumulation of polyphenolic
compounds. The S. meliloti MsbA2 protein is homologous to the Escherichia coli
lipopolysaccharide/phospholipid trafficking protein MsbA. However, MsbA2 was not
essential for the membrane transport of either lipopolysaccharide or
phospholipids in S. meliloti. We determined that the msbA2 gene is transcribed in
free-living S. meliloti and that in the absence of MsbA2 the polysaccharide
content of S. meliloti is altered. Consequently, we propose a model whereby the
altered polysaccharide content of the S. meliloti msbA2 mutant could be
responsible for its symbiotic defect by inducing an inappropriate host response.
DOI: 10.1099/mic.0.2007/014894-0

2008
Mar 26.
Characterization of root mucilage from Melastoma malabathricum, with emphasis on

its roles in aluminum accumulation.
Watanabe T(1), Misawa S, Hiradate S, Osaki M.
Author information:
(1)Research Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kitaku,
Sapporo 060-8589, Japan.
Plant roots exude viscous polysaccharides, called mucilage. One of the suggested
roles of mucilage is immobilization of toxic metal cations, including aluminum
(Al), in the rhizosphere. Mucilage exuded from roots of Melastoma malabathricum
(Al accumulator) was characterized in comparison with that of Zea mays (maize; Al
nonaccumulator). Removal of mucilage significantly reduced Al accumulation in M.
malabathricum. The cation adsorption affinity of M. malabathricum mucilage was
higher for Al and lanthanum (La) than for barium (Ba), whereas that of maize
mucilage was in the order Ba > La > Al. A (27)Al nuclear magnetic resonance (NMR)
spectrum of the Al-adsorbed mucilage and bioassay with alfalfa seedlings
indicated that the concentrated Al in the mucilage of M. malabathricum, unlike
that of maize, bound very weakly to cation exchange sites of mucilage. The higher
charge density in M. malabathricum mucilage, derived from unmethylated uronic
acid, is inferred to be related to preferential adsorption of trivalent cation.
Not only a higher degree of methylation in the uronic acid (glucuronic acid) but
also H(+) release from roots to the mucilage appears to be responsible for the
loose binding of Al in M. malabathricum mucilage. These characteristics of
mucilage may help Al hyperaccumulation in M. malabathricum.
DOI: 10.1111/j.1469-8137.2008.02397.x
1632. Plant Cell Rep. 2008 Jul;27(7):1147-56. doi: 10.1007/s00299-008-0538-5. Epub

2008
Mar 28.
Excretion of polyamines in alfalfa and tobacco suspension-cultured cells and its

possible role in maintenance of intracellular polyamine contents.
Cvikrová M(1), Gemperlová L, Eder J, Zazímalová E.
Author information:
(1)Institute of Experimental Botany v.v.i, Academy of Sciences of the Czech
Republic, Rozvojová 236, 16502 Prague 6, Czech Republic. cvikrova@ueb.cas.cz
Changes in polyamines (PAs) in cells and cultivation media of alfalfa (Medicago

sativa L.) and tobacco bright yellow 2 (BY-2) (Nicotiana tabacum L.) cell
suspension cultures were studied over their growth cycles. The total content of
PAs (both free and conjugated forms) was nearly 10 times higher in alfalfa, with
high level of free putrescine (Put) (in exponential growth phase it represented
about 65-73% of the intracellular Put pool). In contrast, the high content of
soluble Put conjugates was found in tobacco cells (in exponential phase about 70%
of the intracellular Put). Marked differences occurred in the amount of PAs
excreted into the cultivation medium: alfalfa cells excreted at the first day
after inoculation 2117.0, 230.5, 29.0 and 88.0 nmol g(-1) of cell fresh weight
(FW) of Put, spermidine (Spd), spermine (Spm) and cadaverine (Cad), respectively,
while at the same time tobacco cells excreted only small amount of Put and Spd
(12.7 and 2.4 nmol g(-1) FW, respectively). On day 1 the amounts of Put, Spd, Spm
and Cad excreted by alfalfa cells represented 21, 38, 12 and 15% of the total
pool (intra- plus extra-cellular contents) of Put, Spd, Spm and Cad,
respectively. In the course of lag-phase and the beginning of exponential phase
the relative contents of extracellular PAs continually decreased (with the
exception of Cad). On day 10, the extracellular Put, Spd, Spm and Cad still
represented 11.3, 10.9, 2.1 and 27% of their total pools. The extracellular PAs
in tobacco cells represented from day 3 only 0.1% from their total pools. The
possible role of PA excretion into the cultivation medium in maintenance of
intracellular PA contents in the cells of the two cell culture systems, differing
markedly in growth rate and PA metabolism is discussed.
DOI: 10.1007/s00299-008-0538-5

The effect of silage cutting height on the nutritive value of a normal corn
silage hybrid compared with brown midrib corn silage fed to lactating cows.
Kung L Jr(1), Moulder BM, Mulrooney CM, Teller RS, Schmidt RJ.
Author information:
19716-2150, USA. lksilage@udel.edu
A brown midrib (BMR) hybrid and a silage-specific non-BMR (7511FQ) hybrid were
harvested at a normal cut height leaving 10 to 15 cm of stalk in the field. The
non-BMR hybrid was also cut at a greater height leaving 45 to 50 cm of stalk.
Cutting high increased the concentrations of dry matter (+4%), crude protein
(+5%), net energy for lactation (+3%), and starch (+7%), but decreased the
concentrations of acid detergent fiber (-9%), neutral detergent fiber (-8%), and
acid detergent lignin (-13%) for 7511FQ. As expected, the BMR corn silage was 30%
lower in lignin concentration than 7511FQ. After 30 h of in vitro ruminal
fermentation, the digestibility of neutral detergent fiber for normal cut 7511FQ,
the same hybrid cut high, and the normal cut BMR hybrid were 51.7, 51.4, and
63.5%, respectively. Twenty-seven multiparous lactating cows were fed a total
mixed ration composed of the respective silages (45% of dry matter) with alfalfa
haylage (5%), alfalfa hay (5%), and concentrate (45%) (to make the TMR isocaloric
and isonitrogenous) in a study with a 3 x 3 Latin square design with 21-d
periods. Milk production was greater for cows fed the BMR hybrid (48.8 kg/d)
compared with those fed the normal cut 7511FQ (46.8 kg/d) or cut high (47.7
kg/d). Dry matter intake was not affected by treatment. Feed efficiency for cows
fed the BMR silage (1.83) was greater than for those fed high-cut 7511FQ (1.75),
but was not different from cows fed the normal cut 7511FQ (1.77). Cows fed the
BMR silage had milk with greater concentrations of lactose but lower milk urea
nitrogen than cows on other treatments. Harvesting a silage-specific, non-BMR
corn hybrid at a high harvest height improved its nutritive content, but the
improvement in feeding value was not equivalent to that found when cows were fed
BMR corn silage.
DOI: 10.3168/jds.2007-0236
1634. Appl Spectrosc. 2008 Mar;62(3):327-32. doi: 10.1366/000370208783759722.
A multi-source portable light emitting diode spectrofluorometer.
Obeidat S(1), Bai B, Rayson GD, Anderson DM, Puscheck AD, Landau SY, Glasser T.
Author information:
(1)Box 30001 MSC 3C, Department of Chemistry and Biochemistry, New Mexico State
University, Las Cruces, New Mexico 88003-0001, USA.
A portable luminescence spectrofluorometer weighing only 1.5 kg that uses

multiple light emitting diodes (LEDs) as excitation sources was developed and
evaluated. Excitation using a sequence of seven individual broad-band LED
emission sources enabled the generation of excitation-emission spectra using a
light weight (<1.5 kg) spectrometer. Limits of detection for rhodamine 6G,
rhodamine B, and fluorescein were 2.9, 3.2, and 11.0 nM, respectively. Generation
of excitation-emission matrices (EEMs) enabled the analysis of samples containing
mixtures of rhodamine B and fluorescein. Buffered saline plant and animal feed
extracts were also analyzed using this instrument. These samples included the
woody plants Pistacia lentiscus (Evergreen pistache or Mastic) and Philyria
latifolia, and the herbaceous species Medicago sativa (alfalfa), Trifolium spp.
(clover), and a feed concentrate. Application of multi-way principal component
analysis (MPCA) to the resulting three-dimensional data sets enabled discernment
among these various diet constituents.
DOI: 10.1366/000370208783759722
1635. J Anim Physiol Anim Nutr (Berl). 2008 Apr;92(2):157-67. doi:

10.1111/j.1439-0396.2007.00721.x.
Effects of maize grain and lucerne particle size on ruminal fermentation,

digestibility and performance of cows in midlactation.
Cao ZJ(1), Li SL, Xing JJ, Ma M, Wang LL.
Author information:
Technology, China Agricultural University, Beijing, China.
This study evaluated the effects of, and interactions between, maize grain
particle size (MPS) and lucerne particle size (LPS) on dry matter intake, milk
production, milk composition, ruminal fermentation, microbial yield, chewing
activity and nutrient digestibility in midlactation cows. Four multiparous
Holstein cows with ruminal cannulas were assigned randomly to a 4 x 4 Latin
square design, averaged 595 kg (SD = 52) of body weight and 121 days in milk (SD
= 21) at the start of the experiment. Experimental periods were 21 days in length
(14 days of treatment adaptation and 7 days of data collection). All diets were
fed as total mixed ration and were formulated to meet or exceed the requirements
of a 600 kg multiparous cow producing 20 kg milk/day with 4.0% fat. The ratio of
concentrate to forage was 39:61 (dry matter basis). Treatments were arranged in a
2 x 2 factorial design; two levels of LPS (2.54 and 6.22 mm) were combined with
concentrates based on either ground maize grain (711 mum) or cracked maize grain
(1755 mum). Maize grain and LPS did not affect milk production and milk fat
percentage. Milk protein percentage increased when MPS was decreased (p = 0.04).
Milk urea nitrogen was lower for cows fed ground maize grain compared with
cracked maize grain (118-134 mg/l, p = 0.05). Estimated microbial N supply
increased 41.9 g/day for ground maize grain compared with cracked maize grain.
Cows fed long lucerne (LL) hay spent more time ruminating compared with cows fed
short lucerne (SL) hay ranging from 293 to 336 min/day (p < 0.001). Total time
spent chewing by cows increased from 505 to 574 min/day (p = 0.002) for SL and LL
respectively. Based on the results from this study, midlactation cows can be fed
diets that contain ground maize grain and SL hay without leading to negative
effects on ruminal pH and nutrient digestibility.
DOI: 10.1111/j.1439-0396.2007.00721.x
1636. Acta Biochim Biophys Sin (Shanghai). 2008 Mar;40(3):209-16.
Cloning and characterization of a heat shock protein 70 gene, MsHSP70-1, in

Medicago sativa.
He Z(1), Xie R, Wang Y, Zou H, Zhu J, Yu G.
Author information:
and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200032, China.
Some members of the heat shock protein 70 (HSP70) family have important functions
in organism development. Here, we identified an alfalfa (Medicago sativa L. cv.
Algonquin) HSP70 gene, MsHSP70-1, using cDNA array and reverse
transcription-polymerase chain reaction. This gene contains a 1947 bp open
reading frame encoding a protein of 649 amino acids. This protein contains all
conserved domains, motifs, and characteristic sequences of plant HSP70s. The
expression of MsHSP70-1 is enhanced in nodule compared with root, stem, leaf, and
flower, and throughout the process of nodule development. Northern hybridization
analysis indicated that the expression of MsHSP70-1 in nodule requires the active
bacA gene of rhizobia. These results suggested that MsHSP70-1 might play an
important role in alfalfa nodule development.
1637. Phytochemistry. 2008 Apr;69(6):1339-47. doi: 10.1016/j.phytochem.2008.01.011.

Epub 2008 Mar 6.
Phytotoxic activity and conformational analysis of thymol analogs from

Hofmeisteria schaffneri.
Pérez-Vásquez A(1), Linares E, Bye R, Cerda-García-Rojas CM, Mata R.
Author information:
(1)Facultad de Química, Universidad Nacional Autónoma de México, México DF 04510,
Mexico.
Bioassay-guided fractionation of two phytotoxic extracts (a CH(2)Cl(2)-MeOH (1:1)

and an aqueous) prepared from the aerial parts of Hofmeisteria schaffneri led to
isolation of thymol analogs 3-5, along with seven known compounds, 1, 2 and 6-10.
Compounds 3-5 were identified by spectroscopic methods as
1,4-bis(2'-hydroxy-4'-methylphenyl)butane-1,4-dione (3),
2-isopropyl-5-methylphenyl (2Z)-2-methylbut-2-enoate (4) and
2-hydroxy-2-(2-hydroxy-4-methylphenyl)propane-1,3-diyl
(2Z,2'Z)bis(2-methylbut-2-enoate) (5) and designated trivial names of
hofmeisterins II-IV, respectively. Their conformational behavior was also studied
by molecular modeling using density functional theory calculations at the
B3LYP/DGDZVP level. Compounds 1-4 and 6-10 significantly inhibited radicle growth
of seedlings of Amaranthus hypochondriacus and Echinochloa crus-galli in the
Petri dish bioassay with IC(50)'s10(-4)M. Furthermore, the northymol analog 3
provoked significant bleaching of seedlings of A. hypochondriacus. However, none
of the isolates affected either seedling growth or germination of Medicago
sativa.
DOI: 10.1016/j.phytochem.2008.01.011
1638. Appl Environ Microbiol. 2008 May;74(9):2921-3. doi: 10.1128/AEM.00075-08.

Epub
2008 Mar 7.
Functional nodFE genes are present in Sinorhizobium sp. strain MUS10, a symbiont
of the tropical legume Sesbania rostrata.
Krishnan HB(1), Chronis D.

Author information:
(1)Plant Genetics Research Unit, USDA-ARS, 108 Curtis Hall, University of
Missouri, Columbia, MO 65211, USA. KrishnanH@missouri.edu
We have cloned the nodFE operon from Sinorhizobium sp. strain MUS10. MUS10 NodF
shows sequence homology to acyl carrier protein and enables an S. meliloti nodF
mutant to effectively nodulate alfalfa. Our results demonstrate the occurrence of
nodFE in a symbiont that nodulates a legume host not belonging to the galegoid
group.
DOI: 10.1128/AEM.00075-08
PMCID: PMC2394878
1639. Mol Plant Microbe Interact. 2008 Apr;21(4):404-10. doi: 10.1094/MPMI-21-4-

0404.
Transcription of ENOD8 in Medicago truncatula nodules directs ENOD8 esterase to

developing and mature symbiosomes.
Coque L(1), Neogi P, Pislariu C, Wilson KA, Catalano C, Avadhani M, Sherrier DJ,
Dickstein R.
Author information:
(1)University of North Texas, Department of Biological Sciences, Chestnut and
Avenue C, Denton 76203-5220, USA.
In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces

atmospheric dinitrogen into nitrogenous compounds that the legume uses for its
own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes,
which include the rhizobial cell, the symbiosome membrane surrounding it, and the
matrix between the bacterium and the symbiosome membrane, termed the symbiosome
space. Here, we describe the localization of ENOD8, a nodule-specific esterase.
The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the
genes that support the nitrogen fixation capabilities of the nodule. Expression
of an ENOD8 promoter-gusA fusion in nodulated hairy roots of composite
transformed M. truncatula plants indicated that ENOD8 is expressed from the
proximal end of interzone II to III to the proximal end of the nodules. Confocal
immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein
was detected in the same zones. ENOD8 protein was localized in the symbiosome
membrane or symbiosome space around the bacteroids in the infected nodule cells.
Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8
protein was found in the symbiosome membrane and symbiosome space, but not in the
bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a
first step in understanding its role in symbiosome membrane and space during
nodule formation and function.
DOI: 10.1094/MPMI-21-4-0404
1640. Sci Total Environ. 2008 May 15;394(2-3):230-6. doi:

Enhanced dissipation of phenanthrene in spiked soil by arbuscular mycorrhizal

alfalfa combined with a non-ionic surfactant amendment.
Wu N(1), Zhang S, Huang H, Christie P.
Author information:
Center for Eco-Environmental Sciences, Chinese Academy of Sciences, PO Box 2871,
Experiments were conducted to assess the role of colonization of alfalfa roots by

an arbuscular mycorrhizal (AM) fungus (Glomus etunicatum) in conjunction with a
non-ionic surfactant (Triton X-100) in dissipation of phenanthrene in a soil
spiked with phenanthrene at 0, 2.5, 5.0 and 10.0 mg kg(-1). After plant harvest
the residual phenanthrene concentration in the soil decreased markedly.
Mycorrhizal treatment enhanced phenanthrene dissipation in the rhizosphere and
bulk soils irrespective of phenanthrene application rate. Addition of Triton
X-100 resulted in the highest phenanthrene concentration in the rhizosphere soil
among the treatments, while the lowest phenanthrene concentration in the bulk
soil was obtained by AM inoculation and amendment with Triton X-100. AM
inoculation and addition of the surfactant consistently promoted phenanthrene
dissipation in the soil and decreased the microbial biomass based on phospholipid
fatty acid (PLFA) analysis. PLFA profiles demonstrated that AM inoculation
together with addition of Triton X-100 altered the microbial community structure
in the rhizosphere soil. The results of this study provide a reference value for
phytoremediation of soil contaminated by organic pollutants.
DOI: 10.1016/j.scitotenv.2008.02.003

Epub
2008 Feb 29.
Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid

are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa
sprouts and K-12 strains to plastic but not for binding to epithelial cells.
Matthysse AG(1), Deora R, Mishra M, Torres AG.
Author information:
(1)Department of Biology, Coker Hall, University of North Carolina, Chapel Hill,
NC 27599-3280, USA. ann_matthysse@unc.edu
When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in
water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12
strains of E. coli do not bind to sprouts under similar conditions. The roles of
E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and
exopolysaccharides in binding to sprouts were examined. An LPS mutant had no
effect on the binding of the pathogenic strain. Cellulose synthase mutants showed
a significant reduction in binding; colanic acid mutants were more severely
reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was
barely detectable. The addition of a plasmid carrying a cellulose synthase gene
to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps
biosynthesis genes had only a marginal effect on the binding of K-12 bacteria.
However, the introduction of the same plasmid allowed Sinorhizobium meliloti and
a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments.
These results suggest that although multiple redundant protein adhesins are
involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides
required for binding are not redundant and each polysaccharide may play a
distinct role. PGA, colanic acid, and cellulose were also required for biofilm
formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7
to mammalian cells.
DOI: 10.1128/AEM.01854-07
PMCID: PMC2293172
1642. Environ Toxicol Pharmacol. 2008 Mar;25(2):144-7. doi:

10.1016/j.etap.2007.10.009.
Epub 2007 Oct 13.
Dechlorination of PCB in the presence of plant nitrate reductase.
Magee KD(1), Michael A, Ullah H, Dutta SK.
Author information:
(1)Molecular Genetics Laboratory, Department of Biology, Howard University, 415
College Street, NW, Washington, DC 20059, USA.
The dechlorination of PCB, specifically the noncoplanar congener PCB 153, has
been observed in the presence of a crude nitrate reductase extract from Medicago
sativa leaves. These observations were further confirmed using a commercially
available and pure nitrate reductase from Zea mays. The presence of nitrate
reductase increased PCB 153 dechlorination. Then, the addition of molybdenum, the
enzyme's cofactor, enhanced dechlorination of the environmental contaminant. The
ability of plant nitrate reductase to dechlorinate PCB is a new observation.
DOI: 10.1016/j.etap.2007.10.009
PMID: 21783850
1643. Plant Cell Rep. 2008 May;27(5):893-901. doi: 10.1007/s00299-008-0517-x. Epub

2008
Feb 28.
Bacterial citrate synthase expression and soil aluminum tolerance in transgenic

alfalfa.
Barone P(1), Rosellini D, Lafayette P, Bouton J, Veronesi F, Parrott W.
Author information:
(1)Department of Crop and Soil Sciences, University of Georgia, 30602, Athens,
GA, USA. pib8@uiuc.edu
Alfalfa is very sensitive to soil acidity and its yield and stand duration are
compromised due to inhibited root growth and reduced nitrogen fixation caused by
Al toxicity. Soil improvement by liming is expensive and only partially
effective, and conventional plant breeding for Al tolerance has had limited
success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa
citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al,
alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis
Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen
transgenic plants were assayed for exclusion of Al from the root tip, for
internal citrate content, for growth in in vitro assays, or for shoot and root
growth in either hydroponics or in soil assays. Overall, only the soil assays
yielded consistent results. Based on the soil assays, two transgenic events were
identified that were more aluminum-tolerant than the non-transgenic control,
confirming that citrate synthase overexpression can be a useful tool to help
achieve aluminum tolerance.
DOI: 10.1007/s00299-008-0517-x
1644. Bull Entomol Res. 2008 Aug;98(4):371-7. doi: 10.1017/S0007485308005671. Epub

2008
Feb 25.
Olfactory conditioning in mate searching by the parasitoid Aphidius ervi

(Hymenoptera: Braconidae).
Villagra CA(1), Vásquez RA, Niemeyer HM.
Author information:
(1)Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de
Chile. cavillagra@gmail.com
Despite the fact that insect learning capacity has been broadly demonstrated, the
role that this process plays during mate searching has been scarcely explored. We
studied whether the sexual behaviour of a male parasitic wasp can be conditioned
to the odours from two alternative host plant complexes (HPCs) present during its
first copulation. The experimental subjects were newly emerged males of the aphid
parasitoid, Aphidius ervi, and two alternative HPCs (alfalfa or wheat). In the
training protocol, copulation experience corresponded to an unconditioning
stimulus and HPC odours to the conditioning stimuli. The initial (just after
eclosion) and trained responses were assessed in a glass Y-olfactometer. The
results showed that neither alfalfa HPC nor wheat HPC stimuli elicited
sexual-related behaviours in initial male responses. Conversely, both HPCs
triggered strong attraction and wing fanning courtship behaviour in trained
responses when the male was exposed to a female plus HPC during training. In
males trained with females plus a given HPC but tested with the alternative HPC
in the olfactometer, trained response showed a similar trend to the
non-associative treatments. Hence, through learning, the olfactory stimulus
context present during copulation could become a predictive cue for further mate
searching. These results are discussed in terms of parasitic wasp ecology and
host fidelity.
DOI: 10.1017/S0007485308005671
Effect of supplementing rumen-protected methionine on production and nitrogen

excretion in lactating dairy cows.
Broderick GA(1), Stevenson MJ, Patton RA, Lobos NE, Olmos Colmenero JJ.
Author information:
Linden Drive West, Madison, WI 53706, USA. glen.broderick@ars.usda.gov
Two 4 x 4 Latin square trials (4-wk periods; 16 wk total) were conducted to see
whether supplementing rumen-protected Met (RPM; fed as Mepron) would allow
feeding less crude protein (CP), thereby reducing urinary N excretion, but
without losing production. In trial 1, 24 Holsteins were fed 4 diets as total
mixed rations containing [dry matter (DM) basis]: 18.6% CP and 0 g of RPM/d;
17.3% CP and 5 g of RPM/d; 16.1% CP and 10 g of RPM/d; or 14.8% CP and 15 g of
RPM/d. Dietary CP was reduced by replacing soybean meal with high-moisture
shelled corn. All diets contained 21% alfalfa silage, 28% corn silage, 4.5%
roasted soybeans, 5.8% soyhulls, 0.6% sodium bicarbonate, 0.5% vitamins and
minerals, and 27% neutral detergent fiber. There was no effect of diet on intake,
weight gain, or yields of protein, lactose, and solids-not-fat. However,
production was greater at 17.3% CP plus RPM and 16.1% CP plus RPM than on the
other 2 diets. Apparent N efficiency (milk N:N intake) was greatest on the lowest
CP diet containing the most RPM. Linear reductions in milk urea N and urinary N
excretion were observed with lower dietary CP. In trial 2, 32 Holsteins were fed
4 diets as total mixed rations, formulated from ingredients used in trial 1 and
containing 16.1 or 17.3% CP with 0 or 10 g of RPM/d. On average, cows were
calculated to be in negative N balance on all diets because of lower than
expected DM intake. There was no effect of RPM supplementation on any production
trait. However, higher CP gave small increases in yields of milk, protein, and
solids-not-fat and tended to increase DM intake and lactose yield. Apparent N
efficiency was greater, and milk urea nitrogen was lower, on 16.1% CP. In trial
1, feeding lower CP diets supplemented with RPM resulted in improved N efficiency
and reduced urinary N excretion. However, in trial 2, reducing dietary CP from
17.3 to 16.1% reduced milk secretion, an effect that was not reversed by RPM
supplementation at low DM intakes when cows were apparently mobilizing body
protein.
DOI: 10.3168/jds.2007-0769
1646. Environ Entomol. 2007 Dec;36(6):1457-65.
Control of western tarnished plant bug Lygus hesperus Knight (Hemiptera: Miridae)
in California organic strawberries using alfalfa trap crops and tractor-mounted
vacuums.
Swezey SL(1), Nieto DJ, Bryer JA.
Author information:
Santa Cruz, CA 95064, USA. findit@ucsc.edu
A key economic pest of strawberries in California is the western tarnished plant

bug, Lygus hesperus Knight (Hemiptera:Miridae). Alfalfa (Medicago sativa L.) is a
highly attractive plant host to western tarnished plant bug, and we hypothesized
that it can be successfully managed as a trap crop for pest suppression in
strawberries. Completely randomized design trap cropping experiments were
established on an organic strawberry farm from 2002 to 2004. Western tarnished
plant bug adults and nymphs were significantly more abundant in alfalfa trap
crops than in comparable edge strawberry rows. Over 3 experimental yr,
twice-weekly summer vacuuming of alfalfa trap crops with a tractor-mounted
vacuuming device reduced adult and nymph abundance by 72 and 90%, respectively,
in trap crops. This summer vacuuming of alfalfa trap crops also significantly
reduced damage caused by western tarnished plant bug in associated unvacuumed
organic strawberries (June and July 2002, June 2003, and June and July 2004)
compared with either an untreated control (2003) or the organic strawberry
grower's standard whole field vacuuming treatment. Vacuuming of alfalfa trap
crops reduces an organic grower's costs (tractor, tractor fuel, and driver time)
by 78% compared with current whole field vacuuming practices. An economic
analysis of a whole hectare model indicates that a positive return from the use
of vacuumed trap crops could be realized in 2004. The overall potential positive
net return for the 3 mo of vacuumed alfalfa trap crop treatments in 2004 was
calculated at +$1,829/ha.
1647. Mol Microbiol. 2008 Mar;67(6):1292-306. doi: 10.1111/j.1365-

2958.2008.06123.x.
Epub 2008 Feb 13.
A symbiotic mutant of Sinorhizobium meliloti reveals a novel genetic pathway

involving succinoglycan biosynthetic functions.
Griffitts JS(1), Long SR.
Author information:
(1)Department of Microbiology and Molecular Biology, Brigham Young University,
Provo, UT 84602, USA. joelg@byu.edu
A large-scale screen for symbiotic mutants was carried out using the model root
nodulating bacterium Sinorhizobium meliloti. Several mutations in the previously
uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the
ATP-binding cassette exporter family. This protein family is known to export a
wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for
the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule
primordium morphogenesis, but failing to invade plant tissue beyond the epidermal
cell layer. msbA2 mutants do not exhibit any of the free-living traits often
found to correlate with symbiotic defects, suggesting that MsbA2 may take part in
a specifically symbiotic function. In strains that overproduce the symbiotic
signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely
deleterious. This synthetic lethal phenotype can be suppressed by disrupting the
succinoglycan biosynthetic genes exoY or exoA. It can also be suppressed by
disrupting putative glycosyltransferase-encoding genes found upstream of msbA2.
Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by
secondary mutations in these upstream transferase genes, indicating that the
msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel
polysaccharide that is synthesized from succinoglycan precursors.
DOI: 10.1111/j.1365-2958.2008.06123.x
1648. Bioresour Technol. 2008 Oct;99(15):6745-50. doi:

10.1016/j.biortech.2008.01.010.
Epub 2008 Feb 20.
Contrasting effects of municipal compost on alfalfa growth in clay and in sandy

soils: N, P, K, content and heavy metal toxicity.
Mbarki S(1), Labidi N, Mahmoudi H, Jedidi N, Abdelly C.
Author information:
(1)The Laboratory of Plant Adaptation to Abiotic Stress (LAPSA), Biotechnology
Center at the Technopark of Borj-Cedria (CBBC), BP 901, Hammam-Lif 2050, Tunisia.
The limits for loading soils with Tunisian urban compost for cultivating Medicago
sativa were determined in a 6-month experiment in a greenhouse. Mature municipal
solid waste compost (MSWC) from Tunis city was applied to clay and sandy soils
from cultivated fields at rates equivalent to 40, 80, 120tha(-1). In the absence
of MSWC, the shoot biomass (dry weight) cumulated over four cuts was 2-2.5 lower
in sandy soil than in clay soil. It was 20-25% augmented upon MSWC addition in
clay soil, independently of MSWC dose. The opposite trend was observed in sandy
soil, the shoot yields being diminished by MSWC in a dose dependent manner. In
MSWC-amended clay soil but not in sandy soil, Cd, Cu, Zn, and Pb concentrations
in shoots remained below or close to the tolerated values according to EEC norms.
The MSWC might be used as conditioner for clay soil, but not for sandy soil.
DOI: 10.1016/j.biortech.2008.01.010
1649. J Bacteriol. 2008 Apr;190(8):2947-56. doi: 10.1128/JB.01917-07. Epub 2008 Feb

15.
Sinorhizobium meliloti mutants lacking phosphotransferase system enzyme HPr or

EIIA are altered in diverse processes, including carbon metabolism, cobalt
requirements, and succinoglycan production.
Pinedo CA(1), Bringhurst RM, Gage DJ.
Author information:
(1)Department of Molecular and Cell Biology, University of Connecticut, 91 N.
Eagleville Rd., U-3125, Storrs, CT 06269-3125, USA.
Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen

when it is in a symbiotic relationship. Genes for an incomplete
phosphotransferase system (PTS) have been found in the genome of S. meliloti. The
genes present code for Hpr and ManX (an EIIA(Man)-type enzyme). HPr and EIIA
regulate carbon utilization in other bacteria. hpr and manX in-frame deletion
mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr
resulted in partial relief of succinate-mediated catabolite repression, extreme
sensitivity to cobalt limitation, rapid die-off during stationary phase, and
altered succinoglycan production. Loss of ManX decreased expression of melA-agp
and lac, the operons needed for utilization of alpha- and beta-galactosides,
slowed growth on diverse carbon sources, and enhanced accumulation of
high-molecular-weight succinoglycan. A strain with both hpr and manX deletions
exhibited phenotypes similar to those of the strain with a single hpr deletion.
Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation
and nitrogen fixation when they were inoculated onto Medicago sativa. The results
show that HPr and ManX (EIIA(Man)) are involved in more than carbon regulation in
S. meliloti and suggest that the phenotypes observed occur due to activity of HPr
or one of its phosphorylated forms.
DOI: 10.1128/JB.01917-07
PMCID: PMC2293241
1650. Oecologia. 2008 May;156(1):125-35. doi: 10.1007/s00442-008-0978-1. Epub 2008

Feb
15.
UV-B effects on the nutritional chemistry of plants and the responses of a

mammalian herbivore.
Thines NJ(1), Shipley LA, Bassman JH, Slusser JR, Gao W.

Author information:
(1)Department of Natural Resource Sciences, Washington State University, Pullman,
WA 99164-6410, USA. nikkithines@yahoo.com
Stratospheric ozone depletion has caused ground-level ultraviolet-B (UV-B)

radiation to rise in temperate latitudes of both hemispheres. Because the effects
of enhanced UV-B radiation on the nutrition of food consumed by mammalian
herbivores are unknown, we measured nutritional and chemical constituents of 18
forages and related changes to in vitro dry matter digestibility. We also
measured intake and in vivo digestibility of Pacific willow (Salix lasiandra) and
alfalfa (Medicago sativa L.) by blue duikers (Cephalophus monticola). Forages
were irradiated for 3 months with ambient (1x) or supplemental (1.6 x) UV-B
radiation representing a 15% ozone depletion for Pullman, Washington, USA.
Enhanced UV-B radiation had minimal and inconsistent effects on the nutritional
content, in vitro dry matter digestibility, and protein-binding capacity of
forages. However, flavonoid compounds increased in seven of the 13 forbs and
woody dicots that were evaluated. Flavonoids were found to decrease only in
yarrow (Achillea millefolium). When offered simultaneously, blue duikers
preferred 1x and 1.6 x UV-B irradiated plants of alfalfa equally, but ate 26%
less willow grown under 1.6 x UV-B radiation. However, when fed to duikers in
separate feeding experiments, total dry matter intake and in vivo digestibility
of dry matter, fiber, protein, and apparent energy did not differ between alfalfa
and willow grown under 1x and 1.6 x UV-B radiation. We conclude that expected
increases in UV-B radiation from ozone depletion would have minimal effects on
intake and digestion of ruminant herbivores.
DOI: 10.1007/s00442-008-0978-1
13.
Effects of cis-beta-ocimene, cis-sabinene hydrate, and monoterpene and

sesquiterpene mixtures on alfalfa pellet intake by lambs.
Estell RE(1), Fredrickson EL, Anderson DM, Remmenga MD.
Author information:
(1)USDA/ARS Jornada Experimental Range, Las Cruces, New Mexico 88003, USA.
restell@nmsu.edu
The transition of grasslands to shrub-dominated scrubland reduces livestock

productivity and contributes to impoverished conditions for humans in arid and
semiarid regions worldwide. Many shrubs that are increasing in dominance contain
secondary compounds that deter browsing by herbivores. Knowledge concerning the
effects of specific compounds in herbivore diets is limited but may provide
useful insights into desertification. Flourensia cernua is a dominant shrub in
the northern Chihuahuan Desert that contains an abundance of terpenes. Four
experiments were conducted to determine the effects of terpenes on intake of
alfalfa pellets by lambs. Two individual monoterpenes (cis-beta-ocimene and
cis-sabinene hydrate) were examined in Exp. 1 and 2, and mixtures of monoterpenes
(borneol, camphene, camphor, 1,8-cineole, limonene, myrcene, and alpha-pinene)
and sesquiterpenes (beta-caryophyllene, caryophyllene oxide, alpha-copaene, and
alpha-humulene) were examined in Exp. 3 and 4, respectively. Forty-five lambs (9
lambs/treatment) were individually fed treated alfalfa pellets for 20 min each
morning for 5 d. Five treatments (0x, 0.5x, 1x, 2x, and 10x; multiples of the
concentrations of the same terpenes in F. cernua) were applied to alfalfa pellets
(637 g, DM basis) in an ethanol carrier. The experiments were preceded by a 10-d
adaptation period of the lambs to untreated pellets. Except during the 20-min
test, the lambs were maintained outdoors and fed untreated alfalfa pellets (total
mean intake = 4.7% of BW, DM basis). Day x treatment interactions were detected
(P < 0.04) in Exp. 1 and 4 because of a greater intake for 0x than for the other
treatments on d 1 (Exp. 1) and a lower intake for the 10x treatment on d 1 and 2
(Exp. 4). A trend for decreased intake (g/kg of BW) as the concentration of the
sesquiterpene mixture increased was observed in Exp. 3 (P = 0.093 for the linear
contrast). Although there was a tendency for the sesquiterpene mixture to
decrease intake, cis-beta-ocimene, cis-sabinene hydrate, and the monoterpene
mixture did not appear to affect intake by lambs. Thus, sesquiterpenes may exert
antiherbivory properties under certain conditions that may contribute to shrub
dominance with extended periods of livestock foraging.
DOI: 10.2527/jas.2007-0699
[Water potential productivity and soil desiccation effect of alfalfa grasslands

in different type arid areas of Loess Plateau: a simulation study].
Li J(1), Chen B, Li XF, Shao MA, Cheng JM.
Author information:
(1)College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi,
China. junli@nwsuaf.edu.cn
Based on model validation and database establishment, the dynamic changes of

water potential productivity, available water amount in 0-10 m soil layer, and
moisture distribution in soil profiles on the alfalfa grasslands at Changwu of
semi-humid area, Guyuan of semi-arid area, and Haiyuan of semi-arid prone drought
area on Loess Plateau were simulated with WinEPIC model in a 20-30 years period.
The results showed that the simulated water potential productivity of alfalfa
grasslands at Changwu, Guyuan and Haiyuan fluctuated but declined with
precipitation, and the mean value was 8.81, 3.83 and 2.48 t x hm(-2),
respectively. The simulated monthly available water amount in 0-10 m soil layer
also declined obviously but with fluctuation. On the 4-8 years old alfalfa
grasslands, soil desiccation was quite serious, and soil available water amount
fluctuated at low level with precipitation for a long period. With the increasing
year of alfalfa growth, the thickness and distribution depth of desiccated soil
layer increased gradually, and the duration for the desiccated soil depth over 10
m was 6 years at Changwu, 6 years at Guyuan, and 4 years at Haiyuan. After then,
the soil layer beneath rainfall infiltration depth lasted permanently in a
desiccated status. The reasonable duration for water sustainable use on alfalfa
grasslands was 8-10 years on semi-humid area, 6-8 years on semi-arid years, and
4-6 years on semi-arid prone drought area.
1653. Physiol Plant. 2007 Oct;131(2):191-200. doi: 10.1111/j.1399-

3054.2007.00959.x.
Metabolic fate of nicotinamide in higher plants.

Matsui A(1), Yin Y, Yamanaka K, Iwasaki M, Ashihara H.
Author information:
(1)Department of Biological Sciences, Graduate School of Humanities and Sciences,
Ochanomizu University, Bunkyo-ku, Tokyo 112-8610, Japan.
Metabolism of [carbonyl-14C]nicotinamide was surveyed in various plant materials

including the model plants, Arabidopsis thaliana, Oryza sativa and Lotus
japonicus. In all plants studied, nicotinamide was used for the pyridine
(nicotinamide adenine) nucleotide synthesis, probably after conversion to
nicotinic acid. Radioactivity from [carbonyl-14C]nicotinamide was incorporated
into trigonelline (1-N-methylnicotinic acid) and/or into nicotinic acid
1N-glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures
of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum, Medicago
sativa and Raphanus sativus. Trigonelline synthesis from nicotinamide is
generally greater in leaves than in roots. Na-Glc was formed as the major
nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In
seedlings of Chrysanthemum coronarium and Theobroma cacao, both trigonelline and
Na-Glc were synthesized from [carbonyl-14C]nicotinamide. Trigonelline is
accumulated in some seeds, mainly Leguminosae species. The pattern of formation
of the nicotinic acid conjugates differs between species and organs.
DOI: 10.1111/j.1399-3054.2007.00959.x
1654. Anal Biochem. 2008 Apr 15;375(2):354-60. doi: 10.1016/j.ab.2008.01.001. Epub

2008
Jan 9.
Rapid quantification of global DNA methylation by isocratic cation exchange

high-performance liquid chromatography.
Rozhon W(1), Baubec T, Mayerhofer J, Mittelsten Scheid O, Jonak C.
Author information:
Sciences, 1030 Vienna, Austria. wilfried.rozhon@univie.ac.at
The DNA of many eukaryotes is methylated at specific cytosine residues in

connection with gene regulation. Here we report a method for the quantification
of global cytosine methylation based on enzymatic hydrolysis of DNA,
dephosphorylation, and subsequent high-performance cation exchange
chromatography. Nucleosides are separated in less than 3 min under isocratic
conditions on a benzenesulfonic acid-modified silica phase and detected by UV
absorption. As little as 1 microg of DNA is sufficient to measure
5-methyldeoxycytosine levels with a typical relative standard deviation of less
than 3%. As a proof of concept, the method was applied for analysis of DNA from
several Arabidopsis thaliana mutants affected in DNA methylation and from
Medicago sativa seedlings treated with the environmental pollutant chromium(VI).
DOI: 10.1016/j.ab.2008.01.001
1655. BMC Dev Biol. 2008 Feb 4;8:12. doi: 10.1186/1471-213X-8-12.
Peanut gene expression profiling in developing seeds at different reproduction

stages during Aspergillus parasiticus infection.
Guo B(1), Chen X, Dang P, Scully BT, Liang X, Holbrook CC, Yu J, Culbreath AK.
Author information:
(1)USDA-ARS, Crop Protection and Management Research Unit, Tifton, Georgia 31793,
USA. baozhu.guo@ars.usda.gov
BACKGROUND: Peanut (Arachis hypogaea L.) is an important crop economically and

nutritionally, and is one of the most susceptible host crops to colonization of
Aspergillus parasiticus and subsequent aflatoxin contamination. Knowledge from
molecular genetic studies could help to devise strategies in alleviating this
problem; however, few peanut DNA sequences are available in the public database.
In order to understand the molecular basis of host resistance to aflatoxin
contamination, a large-scale project was conducted to generate expressed sequence
tags (ESTs) from developing seeds to identify resistance-related genes involved
in defense response against Aspergillus infection and subsequent aflatoxin
contamination.
RESULTS: We constructed six different cDNA libraries derived from developing
peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a
susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to Aspergillus
infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20'
(resistant to Aspergillus with reduced aflatoxin contamination and susceptible to
TSWV). The developing peanut seed tissues were challenged by A. parasiticus and
drought stress in the field. A total of 24,192 randomly selected cDNA clones from
six libraries were sequenced. After removing vector sequences and quality
trimming, 21,777 high-quality EST sequences were generated. Sequence clustering
and assembling resulted in 8,689 unique EST sequences with 1,741 tentative
consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification
was performed according to MIPS functional catalogue criteria. The unique EST
sequences were divided into twenty-two categories. A similarity search against
the non-redundant protein database available from NCBI indicated that 84.78% of
total ESTs showed significant similarity to known proteins, of which 165 genes
had been previously reported in peanuts. There were differences in overall
expression patterns in different libraries and genotypes. A number of sequences
were expressed throughout all of the libraries, representing constitutive
expressed sequences. In order to identify resistance-related genes with
significantly differential expression, a statistical analysis to estimate the
relative abundance (R) was used to compare the relative abundance of each gene
transcripts in each cDNA library. Thirty six and forty seven unique EST sequences
with threshold of R > 4 from libraries of 'GT-C20' and 'Tifrunner', respectively,
were selected for examination of temporal gene expression patterns according to
EST frequencies. Nine and eight resistance-related genes with significant
up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively.
Among them, three genes were common in both genotypes. Furthermore, a comparison
of our EST sequences with other plant sequences in the TIGR Gene Indices
libraries showed that the percentage of peanut EST matched to Arabidopsis
thaliana, maize (Zea mays), Medicago truncatula, rapeseed (Brassica napus), rice
(Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum) ESTs ranged
from 33.84% to 79.46% with the sequence identity >/= 80%. These results revealed
that peanut ESTs are more closely related to legume species than to cereal crops,
and more homologous to dicot than to monocot plant species.
CONCLUSION: The developed ESTs can be used to discover novel sequences or genes,
to identify resistance-related genes and to detect the differences among alleles
or markers between these resistant and susceptible peanut genotypes.
Additionally, this large collection of cultivated peanut EST sequences will make
it possible to construct microarrays for gene expression studies and for further
characterization of host resistance mechanisms. It will be a valuable genomic
resource for the peanut community. The 21,777 ESTs have been deposited to the
NCBI GenBank database with accession numbers ES702769 to ES724546.
DOI: 10.1186/1471-213X-8-12
PMCID: PMC2257936
1656. Int J Food Microbiol. 2008 Mar 31;123(1-2):121-9. doi:

10.1016/j.ijfoodmicro.2007.12.013. Epub 2008 Jan 30.
Microbiological quality of fresh, minimally-processed fruit and vegetables, and

sprouts from retail establishments.
Abadias M(1), Usall J, Anguera M, Solsona C, Viñas I.
Author information:
(1)IRTA, Centre UdL-IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida,
Catalonia, Spain. isabel.abadias@irta.cat
A survey of fresh and minimally-processed fruit and vegetables, and sprouts was
conducted in several retail establishments in the Lleida area (Catalonia, Spain)
during 2005-2006 to determine whether microbial contamination, and in particular
potentially pathogenic bacteria, was present under these commodities. A total of
300 samples--including 21 ready-to-eat fruits, 28 whole fresh vegetables, 15
sprout samples and 237 ready-to-eat salads containing from one to six
vegetables--were purchased from 4 supermarkets. They were tested for mesophilic
and psychrotrophic aerobic counts, yeasts and moulds, lactic acid bacteria,
Enterobacteriaceae, presumptive E. coli and Listeria monocytogenes counts as well
as for the presence of Salmonella, E. coli O157:H7, Yersinia enterocolitica and
thermotolerant Campylobacter. Results for the fresh-cut vegetables that we
analyzed showed that, in general, the highest microorganism counts were
associated with grated carrot, arugula and spinach (7.8, 7.5 and 7.4 log cfu
g(-1) of aerobic mesophilic microorganisms; 6.1, 5.8 and 5.2 log cfu g(-1) of
yeast and moulds; 5.9, 4.0 and 5.1 log cfu g(-1) lactic acid bacteria and 6.2,
5.3 and 6.0 log cfu g(-1) of Enterobacteriaceae). The lowest counts were
generally associated with fresh-cut endive and lettuce (6.2 and 6.3 log cfu g(-1)
of aerobic mesophilic microorganisms; 4.4 and 4.6 log cfu g(-1) of yeast and
moulds; 2.7 and 3.8 log cfu g(-1) lactic acid bacteria and 4.8 and 4.4 log cfu
g(-1) of Enterobacteriaceae). Counts of psychrotrophic microorganisms were as
high as those of mesophilic microorganisms. Microbiological counts for fresh-cut
fruit were very low. Sprouts were highly contaminated with mesophilic (7.9 log
cfu g(-1)), psychrotrophic microorganisms (7.3 log cfu g(-1)) and
Enterobacteriaceae (7.2 log cfu g(-1)) and showed a high incidence of E. coli
(40% of samples). Of the samples analyzed, four (1.3%) were Salmonella positive
and two (0.7%) harboured L. monocytogenes. None of the samples was positive for
E. coli O157:H7, pathogenic Y. enterocolitica or thermotolerant Campylobacter.
1657. J Food Prot. 2008 Jan;71(1):200-4.
Bacterial communities associated with retail alfalfa sprouts.
Loui C(1), Grigoryan G, Huang H, Riley LW, Lu S.
Author information:
(1)Program in Infectious Diseases and Immunity, School of Public Health, 140
Warren Hall, University of California, Berkeley, California 94720, USA.
Fresh produce, including salad, is increasingly implicated in foodborne

outbreaks. Although studies have been carried out to detect specific human
pathogens from fresh produce, the total bacterial community associated with fresh
produce is poorly understood. In this study, we characterized the bacterial
community associated with alfalfa sprouts, using a culture-independent method.
Four retail-purchased alfalfa sprout samples were obtained from different
producers, and the bacterial community associated with each sample was determined
by 16S rDNA profiling. Our results indicate that alfalfa sprouts sampled in our
study shared significant similarities in their bacterial communities.
Proteobacteria was the dominant phylum detected from all alfalfa sprout samples,
with Enterobacteriaceae, Oxalobacteraceae, Moraxellaceae, and Sphingomonadaceae
as the most frequently detected families. These results indicate that growth
conditions of alfalfa sprouts should be taken into consideration to prevent the
proliferation of pathogenic proteobacteria such as Escherichia coli O157 and
Salmonella.
1658. Plant Physiol. 2008 Mar;146(3):1242-54. doi: 10.1104/pp.107.108779. Epub 2008

Jan
24.
Comparative genomic sequence and expression analyses of Medicago truncatula and

alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes.
Pennycooke JC(1), Cheng H, Stockinger EJ.
Author information:
(1)Department of Horticulture and Crop Science, The Ohio State University/Ohio
Agricultural Research and Development Center, Wooster, OH 44691, USA.
In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes

encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key
step in cold acclimation. CBFs in turn activate a battery of downstream genes
known as the CBF regulon, which collectively act to increase tolerance to low
temperatures. Fundamental questions are: What determines the size and scope of
the CBF regulon, and is this is a major determinant of the low-temperature
tolerance capacity of individual plant species? Here we have begun to address
these questions through comparative analyses of Medicago truncatula and Medicago
sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold
acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C.
Both species possessed low-temperature-induced CBFs but differed in the
expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF
targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31
homolog was not. M. falcata also possessed additional CAS30 homologs in
comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple
pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs,
the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one
CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a
promoter structure comprising modules proximal and distal to the coding sequence.
CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs,
but only in the distal module. However, fusion of the MtCAS15 promoter, including
the distal module, to a reporter gene did not result in low-temperature
responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and
MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31
fusion was less robust than the MfCAS30 fusion. From these studies we conclude
that CAS genes harbor CRT/DRE motifs, their proximity to one another is likely
key to regulatory output in Medicago, and they may be located kilobases distal to
the transcriptional start site. We hypothesize that these differences in CRT/DRE
copy numbers in CAS30/CAS31 upstream regions combined with differences in gene
copy numbers may be a factor in determining differences in low-temperature
tolerance between M. truncatula and M. falcata.
DOI: 10.1104/pp.107.108779
PMCID: PMC2259090
Seasonal diet affects ammonia emissions from tie-stall dairy barns.
Powell JM(1), Broderick GA, Misselbrook TH.
Author information:
(1)USDA-ARS US Dairy Forage Research Center, Madison, WI 53706, USA.
mark.powell@ars.usda.gov
Federal and state regulations are being promulgated under the Clean Air Act to
reduce hazardous air emissions from livestock operations. Although much is known
about air emissions from livestock operations in Europe, few data are available
on emissions from livestock facilities in the United States and the management
practices that may minimize these emissions. The objective of this study was to
measure seasonal and diet effects on ammonia emissions from experimental
tie-stall dairy barns located in central Wisconsin. Four experimental chambers
each housed 4 lactating Holstein dairy cows for three 28-d trial periods
corresponding to spring, early fall, and winter. A 4 x 4 Latin square statistical
design was used to evaluate 4 diets [corn silage (CS)- or alfalfa silage
(AS)-based diets at low or high crude protein] in each chamber for a 4-d ammonia
monitoring period. Partially due to higher crude protein levels, average
ammonia-N emissions during spring (18.8 g/cow per d) were approximately twice the
emissions recorded during early fall (8.4 g/cow per d) and 3 times greater than
emissions during winter (6.7 g/cow per d). Ammonia-N emissions accounted for
approximately 1 to 3% of consumed feed N, 2 to 5% of excreted manure N, and 4 to
11% of manure ammonical N. Nighttime ammonia emissions were on average 30% lower
than daytime emissions. Forage type did not affect ammonia emissions during
winter or early fall. Only during early spring were ammonia emissions lower from
chambers containing cows fed low-CP diets than from cows fed high-CP diets. Of
the total chamber N inputs (feed and bedding), 93, 91, and 95% were recovered in
N outputs (milk, manure, body weight change, and ammonia N) during spring, early
fall, and winter trials, respectively. Confidence in the accuracy of ammonia
emission results was gained by the relatively high chamber N balances and
favorable comparisons of study data with published relationships among the
variables of feed N intake, milk urea N, manure N, and urine N excretion, and
ammonia emissions.
DOI: 10.3168/jds.2007-0588
Milk fatty acid composition of grazing dairy cows when supplemented with linseed
oil.
Flowers G(1), Ibrahim SA, AbuGhazaleh AA.
Author information:
(1)Department of Animal Science, Food and Nutrition, Southern Illinois
University, Carbondale, IL 62901, USA.
The effects of varying amounts of linseed oil (LSO) in grazing dairy cows' diet
on milk conjugated linoleic acid (cis-9, trans-11 CLA) were investigated in this
study. Twelve Holstein cows in midlactation (150 +/- 19 DIM) were placed on
alfalfa-based pasture and assigned to 4 treatments using a 4 x 4 Latin square
design with 3-wk periods. Treatments were: 1) control grain supplement; 2)
control grain supplement containing 170 g of LSO (LSO1); 3) control grain
supplement containing 340 g of LSO (LSO2); and 4) control grain supplement
containing 510 g of LSO (LSO3). Grain supplements were offered at 7 kg/d.
Additional 100 g/d of algae, divided evenly between the 2 feeding times, were
added to every treatment diet. Milk samples were collected during the last 3 d of
each period and analyzed for chemical and fatty acid composition. Treatments had
no effect on milk production (18.9, 18.5, 19.6, and 19.1 kg/d for treatments 1 to
4, respectively). Linseed oil supplementation caused a quadratic increase in milk
fat (3.23, 3.44, 3.35, and 3.27% for treatments 1 to 4, respectively) and protein
(3.03, 3.19, 3.12, and 3.08%) contents. Concentrations (g/100 g of fatty acids)
of milk cis-9, trans-11 CLA (1.12, 1.18, 1.39, and 1.65 for treatments 1 to 4,
respectively) and VA (3.39, 3.62, 4.25, and 4.89) linearly increased with LSO
supplementations. Results from this trial suggest that the increase in milk
cis-9, trans-11 CLA was proportional to the amounts of LSO fed. In conclusion,
adding LSO to grazing dairy cow diets can improve the nutritional value of milk
without compromising milk composition or cow performance.
DOI: 10.3168/jds.2007-0410
Alfalfa containing the glyphosate-tolerant trait has no effect on feed intake,

milk composition, or milk production of dairy cattle.
Combs DK(1), Hartnell GF.
Author information:
53706, USA. dkcombs@wisc.edu
The objective of this experiment was to assess if feeding glyphosate-tolerant

alfalfa affects feed intake, milk composition, or milk production of dairy cows.
One alfalfa (Medicago sativa), variety expressing the CP4 EPSPS protein and grown
in southeastern Washington State was harvested at the late vegetative stage as
hay. Three commercial conventional varieties of alfalfa hay of similar nutrient
composition and harvested in the same geographic region were fed to cows as
controls. The commercial hays were selected to be similar in crude protein [18%
of dry matter (DM)] and neutral detergent fiber (40% of DM) to the
glyphosate-tolerant hay. Sixteen multiparous Holstein cows were fed diets
containing alfalfa hay (39.7% of diet DM) from either the glyphosate-tolerant
alfalfa, or 1 of the 3 conventional varieties. Diets contained at least 15.7%
crude protein and 29% neutral detergent fiber. Experimental design was a
replicated 4 x 4 Latin square. Periods were 28 d and feed intake, milk yield, and
milk composition were summarized over the last 14 d of each period. Daily milk
yield (38.0 kg) and 4% fat-corrected milk (34.7 kg) were not affected by
treatment. Milk fat (3.44%) and milk true protein (2.98%) were also not affected
by source of hay. Milk lactose (4.72%) and soldis-not-fat (8.5%) did not differ
due to treatment. Dry matter intake was similar across treatments (24.4 kg/d).
These results are consistent with data from feeding trials with other
glyphosate-tolerant crops and previously reported compositional comparisons of
glyphosate-tolerant alfalfa with controls. Milk production, milk composition,
feed intake, and feed efficiency were not affected by feeding diets that
contained nearly 40% glyphosate-tolerant alfalfa hay to lactating dairy cows.
DOI: 10.3168/jds.2007-0611
1662. FEMS Microbiol Ecol. 2008 Mar;63(3):350-8. doi: 10.1111/j.1574-

6941.2008.00440.x.
Epub 2008 Jan 16.
Examining the fungal and bacterial niche overlap using selective inhibitors in
soil.
Rousk J(1), Demoling LA, Bahr A, Bååth E.
Author information:
(1)Department of Microbial Ecology, Lund University, Lund, Sweden.
johannes.rousk@mbioekol.lu.se
It is important to know the contributions of bacteria and fungi to decomposition

in connection with both the structure of the food web and the functioning of the
ecosystem. However, the extent of the competition between these groups of
organisms is largely unknown. The bacterial influence on fungal growth in a soil
system was studied by applying three different bacterial inhibitors - bronopol,
tylosin and oxytetracycline - in a series of increasing concentrations, and
comparing the resulting bacterial and fungal growth rates measured using leucine
and acetate-in-ergosterol incorporation, respectively. Direct measurements of
growth showed that fungi increased after adding inhibitors; the level of increase
in fungal growth corresponded to that of the decrease in bacterial growth,
irrespective of the bacterial inhibitor used. Similar antagonistic effects of the
bacteria on fungal growth were also found after adding the bacterial inhibitors
together with additional substrate (alfalfa or straw plant material). The
resulting responses in bacterial and fungal growth indirectly indicated that the
negative interaction between fungi and bacteria was mostly attributable to
exploitation competition. The results of this study also emphasize the increased
sensitivity of using growth-related, instead of biomass-based, measurements when
studying bacterial and fungal interactions in soil.
DOI: 10.1111/j.1574-6941.2008.00440.x
1663. J Anim Sci. 2008 Apr;86(4):936-48. doi: 10.2527/jas.2007-0410. Epub 2008 Jan
11.
Effects of supplemental fat on growth performance and quality of beef from steers
fed corn finishing diets.
Nelson ML(1), Busboom JR, Ross CF, O'Fallon JV.
Author information:
(1)Department of Animal Sciences, Washington State University, Pullman
99164-6351, USA. nelsonm@wsu.edu
To measure the effects of dietary fat on feedlot performance and carcass

characteristics, and on beef appearance, moisture binding, shelf life,
palatability, and fatty acid content, 126 crossbred beef steers (321.1 +/- 0.57
kg of BW) were allotted to a randomized complete block (3) design with a 3 x 2 +
1 factorial arrangement of dietary treatments. The main effects were level of
yellow grease (0, 3, or 6%) and alfalfa hay (3.5 or 7%) in corn-based diets
containing 15% potato by-product (PB). The added treatment was 6% tallow and 7%
alfalfa in a barley-based diet containing 15% PB. Dry matter intake and ADG were
not affected by diet; however, G:F and diet NE content increased linearly (P <
0.10) with yellow grease. Kidney, pelvic, and heart fat (2.0 to 2.3 +/- 0.07) and
yield grade (2.8 to 3.1 +/- 0.09) increased linearly (P < or = 0.05) with yellow
grease. Steers fed corn plus 6% yellow grease had lower (P < 0.05) beef firmness
and beef texture scores but greater (P < 0.01) fat color score than those fed
barley plus 6% tallow. Moisture retention of beef was not affected by dietary
treatment, except purge score during retail storage, which was decreased linearly
(P < 0.01) from 2.1 to 1.6 +/- 0.06 by level of yellow grease. Steaks from steers
fed barley plus 6% tallow had greater (P < 0.05) shear force than those from
steers fed corn plus 6% yellow grease, and beef flavor increased linearly (P <
0.05) from 6.2 to 6.7 +/- 0.11 as the level of yellow grease increased. Level of
yellow grease linearly increased (P < 0.01) transvaccenic acid (TVA) by 61% and
CLA content of beef by 48%. Beef from steers fed corn plus yellow grease had
lower (P < 0.05) palmitoleic and oleic acids and greater (P < 0.05) linoleic,
TVA, and CLA than beef from steers fed the barley-tallow diet. Feeding yellow
grease increased diet energy content, which increased carcass fatness, and
altered beef fatty acid content, which increased beef flavor without affecting
moisture retention, shelf life, or cooking properties of the beef. Additionally,
beef from steers fed corn plus 6% yellow grease was more tender and had more
polyunsaturated fatty acid content and CLA than beef from steers fed barley plus
6% tallow.
DOI: 10.2527/jas.2007-0410
1664. Environ Sci Technol. 2007 Dec 1;41(23):8165-70.
Gibberellic acid, kinetin, and the mixture indole-3-acetic acid-kinetin assisted

with EDTA-induced lead hyperaccumnulation in alfalfa plants.
López ML(1), Peralta-Videa JR, Parsons JG, Benitez T, Gardea-Torresdey JL.
Author information:
(1)Environmental Science and Engineering Ph.D. Program, University of Texas, El
Paso 79968, USA.
There are a few plant species considered potential hyperaccumulators for heavy
metals, particularly lead (Pb). In this study, alfalfa plants grown in
hydroponics were exposed to Pb at 40 mg/L, ethylenediaminetetraacetic acid (EDTA)
equimolar to Pb, and 1, 10, and 100 microM concentrations of the phytohormones
indole-3-acetic acid (IAA), gibberellic acid (GA), and kinetin (KN) and a mixture
of IAA and KN at 100 microM each. Metal quantification by inductively coupled
plasma/optical emission spectroscopy demonstrated that plants treated with
Pb/EDTA plus KN at 1, 10, and 100 microM increased the Pb concentration in
alfalfa leaves (compared to Pb alone) by factors of 17, 43, and 67, respectively,
and by factors of 2, 5, and 8, respectively, compared to the Pb/EDTA treatment.
The correlation coefficient between the Pb concentration in leaves and the
concentrations of KN in the medium was 0.9993. In addition, the leaves of plants
exposed to a Pb/EDTA/100 microM IAA-KN mixture had approximately 9500 mg of Pb/kg
of dry weight, demonstrating that non-Pb hyperaccumulating plants could
hyperaccumulate Pb when treated with EDTA and a mixture of IAA-KN. The X-ray
absorption spectroscopic studies demonstrated that the absorption and
translocation of Pb was in the same oxidation state as the supplied Pb(II).
1665. J Anim Physiol Anim Nutr (Berl). 2008 Feb;92(1):9-17. doi:

10.1111/j.1439-0396.2007.00685.x.
Fermentation characteristics and microbial growth promoted by diets including

two-phase olive cake in continuous fermenters.
Moumen A(1), Yáñez-Ruiz DR, Martín-García I, Molina-Alcaide E.
Author information:
(1)Unidad de Nutrición Animal, Estación Experimental del Zaidín (CSIC), Camino
del Jueves s/n, Armilla, Granada, Spain.
Two-phase olive cake (2POC) is the by-product obtained from the so called
'two-phase' procedure to extract olive oil by mechanical methods. After the
mechanical extraction the 2POC is dried and most of the remaining oil extracted
by chemical means. The production of the crude by-product may reach more than 4
millions t/year in Spain (MAPA, 2003), most of it in areas (Southeast) with
shortage of pastures and conventional feeds for ruminants. Six continuous
fermenters, inoculated with ruminal liquor from wethers or goats, were fed
lucerne hay (LH); LH plus a concentrate including dried two-phase olive cake
(LHCO) and; diet LHCO added with polyethylene glycol (LHCOP). The highest pH
values and ammonia N (NH(3)-N) output were found in fermenters fed diet LH (6.19
and 6.35 for pH, and 53.7 and 68.9 mg NH(3)N/day, respectively, in fermentes
inoculated with rumen liquor from sheep and goats) without differences due to the
inoculum origin. The digestibility of carbohydrates (CHO) was affected (p <
0.001) by inoculum (67.0 and 58.8%, respectively, for goats and wethers) the
lowest values being for diet LHCO (53.2 and 57.0% with inoculum from wethers and
goats, respectively). The main volatile fatty acid (VFA) was acetic acid with
higher (p < 0.01) values in fermenters with inoculum from goats than from wethers
(80.2 and 63.0 mmol/day respectively). The efficiency of bacterial protein
synthesis (EBS) was not different (p > 0.05) with inoculum from wethers and goats
[26.4 and 28.1 and 35.2 and 33.5 g bacterial N/kg digested CHO, respectively,
obtained by using diamino pimelic acid (DAPA) and purine bases (PB) as microbial
markers]. The lowest (p < 0.05) values were found in fermenters fed diets LHCOP
and LH, estimated, respectively, from DAPA and PB (21.9 and 29.0 g bacterial N/kg
digested CHO). The substitution of a part of lucerne hay by a concentrate
including dried 2POC does not seem to cause important differences in efficiency
of VFA production. Results concerning bacterial protein synthesis are not so
clear since values estimated from DAPA and PB did not show similar trends.
Neither inoculum origin nor PEG had important effects on fermentation
characteristics. The DAPA could be an adequate marker in continuous fermenters,
with comparative purposes, as protozoa are not present and, estimated protein
synthesis values are similar to those obtained in vivo with similar diets using
urinary excretion of PD (Yáñez Ruiz et al., 2004b). Further research is needed to
state the optimal proportion of 2POC in practical diets for ruminants at both
maintenance and production stages.
DOI: 10.1111/j.1439-0396.2007.00685.x
1666. Mol Plant Microbe Interact. 2008 Feb;21(2):219-31. doi: 10.1094/MPMI-21-2-
0219.
Identification of genes relevant to symbiosis and competitiveness in

Sinorhizobium meliloti using signature-tagged mutants.
Pobigaylo N(1), Szymczak S, Nattkemper TW, Becker A.
Author information:
(1)Institute for Genome Research and Systems Biology, Center for Biotechnology,
Bielefeld University, 33594 Bielefeld, Germany.
Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the

formation of nitrogen-fixing nodules. In order to identify S. meliloti genes
required for symbiosis and competitiveness, a method of signature-tagged
mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants
with a known transposon insertion site, were used in an experiment in planta. As
a result, 67 mutants showing attenuated symbiotic phenotypes were identified,
including most of the exo, fix, and nif mutants in the sets. For 38 mutants in
genes previously not described to be involved in competitiveness or symbiosis in
S. meliloti, attenuated competitiveness phenotypes were tested individually. A
large part of these phenotypes was confirmed. Moreover, additional symbiotic
defects were observed for mutants in several novel genes such as infection
deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA,
thiC, thiO, and thiD mutants).
DOI: 10.1094/MPMI-21-2-0219
1667. Lett Appl Microbiol. 2008 Feb;46(2):232-6. doi: 10.1111/j.1472-

765X.2007.02302.x.
Epub 2007 Dec 20.
Growth of Salmonella on sprouting alfalfa seeds as affected by the inoculum size,

native microbial load and Pseudomonas fluorescens 2-79.
Liao CH(1).
Author information:
(1)Eastern Regional Research Center, Agricultural Research Service, U.S.
Department of Agriculture, Wyndmoor, PA 19038, USA. chinghsing.liao@ars.usda.gov
AIMS: To investigate the growth of salmonellae on sprouting alfalfa seeds as

affected by the inoculum size, microbial load and Pseudomonas fluorescens 2-79.
METHODS AND RESULTS: Alfalfa seeds pre-inoculated with < or =10(1)-10(3) CFU
g(-1) of salmonellae and with or without Ps. fluorescens 2-79 were sprouted in
glass jars and the population of salmonellae were determined daily for up to 6
days. The population of salmonellae on germinating seeds reached the maximum 2-3
days after sprouting when total bacterial count reached the maximum (10(9) CFU
g(-1)). The population of salmonellae on sprouting seeds not treated with Ps.
fluorescens 2-79 showed a net increase of 3-4 log units. However, the population
of salmonellae on alfalfa seeds treated with Ps. fluorescens 2-79 showed a net
increase of only 1-2 log units. Disinfection of seeds with calcium hypochlorite
enhanced the growth of salmonellae.
CONCLUSIONS: Treatment of seeds with Ps. fluorescens 2-79 reduced the growth of
salmonellae by 2-3 log units.
SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of Ps. fluorescens 2-79 as a
biological agent for use in control of salmonellae on sprouting seeds was
demonstrated and warrants further investigation.
DOI: 10.1111/j.1472-765X.2007.02302.x
1668. Photochem Photobiol. 2008 Jan-Feb;84(1):236-42. doi:

10.1111/j.1751-1097.2007.00240.x.
Effect of X-radiation on lipid peroxidation and antioxidant systems in rats

treated with saponin-containing compounds.
Yalinkilic O(1), Enginar H.
Author information:
(1)Kumluca Anatolian Vocation High School, Kumluca, Antalya, Turkey.
The aim of this study was to investigate the effect of three saponin-containing
plant species extracts (Aesculuc hippocastanum L. seed extract [AHE], Medicago
sativa L. extract [MSE] and Spinacia oleracea L. extract [SOE]) on lipid
peroxidation and on antioxidant systems in rats exposed to X-rays (XR). The rats
were divided into three categories. The first category served as controls and
received only a standard diet. The second category served as the radiation group
and received 5 and 10 Gy XR dose. The third category (XR+extract-treated)
received plant extracts (25.0 or 50.0 mg kg(-1) live weight) and 5 or 10 Gy XR
dose. Blood samples were analyzed for their content of malondialdehyde (MDA),
reduced glutathione (GSH), plasma vitamin C, beta-carotene and retinol. In
animals receiving XR, the plasma MDA (P < 0.001) value significantly increased
but the level of GSH (P < 0.01), vitamin C (P < 0.001), retinol and beta-carotene
(P < 0.001) decreased significantly with increasing XR doses. In the
XR+extract-treated groups, the concentrations of MDA increased significantly with
increasing radiation but their concentrations decreased significantly with
increasing extract concentrations. Plasma concentrations of GSH, beta-carotene,
retinol and vitamin C in XR+extract-treated groups decreased significantly with
increasing XR dose but their concentrations increased with increasing extract
doses. Further, comparison of blood samples of XR+extract-treated groups with
those from the control group showed that GSH, beta-carotene, retinol and vitamin
C values increased significantly but that MDA values decreased significantly. The
results showed that all extracts have enhanced the antioxidant status and
decreased the incidence of free radical-induced lipid peroxidation in blood
samples of rats exposed to XR. However, the antioxidant effect of
AHE-administered animals was more effective than that of MSE- and
SOE-administered whole-body XR rats. We conclude that the supplementation with
saponin-containing extracts may serve to reinforce the antioxidant systems, thus
having protective effect against cell damage by XR.
DOI: 10.1111/j.1751-1097.2007.00240.x
1669. Bioresour Technol. 2008 Sep;99(14):6692-6. doi:

10.1016/j.biortech.2007.11.045.
Epub 2007 Dec 27.
Organ weight and serum triglyceride responses of older (80 week) commercial
laying hens fed an alfalfa meal molt diet.
Landers KL(1), Moore RW, Herrera P, Landers DA, Howard ZR, McReynolds JL, Bryd
JA, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
(1)Poultry Science Department, Texas A&M University, College Station, TX 77843,
USA.
Since one of the costs in the commercial egg industry is that of replacement
pullets, commercial egg layer managers have opted to induce molt older hens in
order to extend their productive life for additional egg laying cycles.
Conventional molt induction involves the complete removal of feed for several
days. However, this management practice can lead to deleterious physiological
responses by the hen and subsequent susceptibility to infection by pathogens.
Consequently less stressful molting regimens involving the feeding of low energy
diets such as alfalfa have been developed. In this study, 80 week old laying hens
that were deprived of feed or fed alfalfa meal during a nine day induced molt.
Full fed hens were used as the control. On day 8 serum triglycerides were
quantified and on day 9 hens were euthanized and the liver, spleen, heart,
intestine, pancreas, ovary, and kidney were collected and weighed. Intestinal
weight were highest in the non-molted hens, lower in the hens fed alfalfa, and
lower still in the hens deprived of feed. Molted hens exhibited reduced weights
of liver, heart, ovary, and pancreas compared to the non-molted hens. Serum
triglycerides were highest in the non-molted hens, less in feed deprived hens,
and the lowest in alfalfa fed hens. These results suggest that a comparable molt
could be achieved with feeding alfalfa meal to 80 week hens compared to feed
deprivation.
DOI: 10.1016/j.biortech.2007.11.045
[Effects of imidacloprid on arthropod community structure and its dynamics in

alfalfa field].
Liu CZ(1), Wang G, Yan L.
Author information:
(1)Department of Entomology, College of Grassland Science, Gansu Agricultural
University, Lanzhou 730070, China. liuchzh@gsau.edu.cn
With species richness (S), individual number (N), Shannon's index (H') and
evenness (E) as the indices of biodiversity, this paper studied the arthropod
community structure and its seasonal dynamics in alfalfa field under effects of
imidacloprid. The results showed that imidacloprid more affected the species
number of natural enemies rather than that of insect pests, resulting in a
significant increase of the dominance concentration of both total arthropod
community and pest sub-community, but spraying the insecticide by the end of May
had little effects on the dominance concentration of pollination bee
sub-community. The numbers of dominant pests such as aphid and thrip reduced
significantly at the initial stage of spraying imidacloprid, increased rapidly
after 7 days, and exceeded the control after 40 days. As for the natural enemy
sub-community, its species number decreased greatly and its species diversity and
evenness were lower at the initial stage of spraying imidacloprid, its species
number increased gradually after 7 days and approached to the level of the
control thereafter, while its individual number was lower than the control all
the time.
1671. Bioinformation. 2008;3(5):198-204. Epub 2008 Dec 31.
Pharmacovigilance: effects of herbal components on human drugs interactions

involving cytochrome P450.
Saxena A(1), Tripathi KP, Roy S, Khan F, Sharma A.
Author information:
(1)Bioinformatics and In Silico Biology Division, Central Institute of Medicinal
and Aromatic Plants (Council of Scientific and Industrial Research), Lucknow
226015 (UP), India.
Cytochrome P450 (CYP P450) enzymes are a superfamily of mono-oxygenases that are
found in all kingdoms of life. The CYP P450 enzymes constitute a large
superfamily of haem-thiolate proteins involved in the metabolism of a wide
variety of both exogenous and endogenous compounds. The CYP activities have been
shown to be involved in numerous interactions especially between drugs and herbal
constituents. The majority of serious cases of drug interactions are as a result
of the interference of the metabolic clearance of one drug by yet another
co-administered drug, food or natural product. Gaining mechanistic knowledge
towards such interactions has been accepted as an approach to avoid adverse
reactions. The inductions and inhibition of CYP enzymes by natural products in
the presence of a prescribed drug has led to adverse effects. Herbal medicines
such as St. John's wort (Hypericum perforatum), garlic (Allium sativa), piperine
(from Piper sp.), ginseng (Ginseng sp.), gingko (Gingko biloba), soya beans
(Glycine max), alfalfa (Medicago sativa) and grape fruit juice show clinical
interactions when co-administered with medicines. This review documents the
involvement of CYP enzymes in the metabolism of known available drugs and herbal
products. We also document the interactions between herbal constituents & CYP
enzymes showing potential drug-herb interactions. Data on CYP450 enzymes in
activation (i.e. induction or inhibition) with natural constituents is also
reviewed.
PMCID: PMC2646189
PMID: 19255634
1672. Cell Biol Int. 2008 May;32(5):549-53. Epub 2007 Nov 21.
Non-invasive microscopy of tip-growing root hairs as a tool for study of dynamic

and cytoskeleton-based vesicle trafficking.
Ovecka M(1), Baluska F, Lichtscheidl I.
Author information:
(1)Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, SK-84523
Bratislava, Slovakia. miroslav.ovecka@savba.sk
The techniques of live cell imaging by electronic light microscopy and confocal
microscopy were used to analyse the cytoarchitecture, organelle dynamics and
membrane trafficking in living root hairs of Arabidopsis thaliana and Medicago
sativa. We focused on the motility of vesicles in the tip of growing root hairs,
the internalisation of plasma membrane by endocytosis and the fate of endocytic
compartments. Vesicles as well as their trafficking to and contact with the
plasma membrane were visualised in the tip of root hairs. We showed rapid
endocytosis using a pulse treatment with lipophillic FM dyes in growing root
hairs. Endocytosis was active at the very tip and labelled endocytic membranes
progressed further down the endocytic network through dynamic compartments
merging with the vacuole by their fusion with highly dynamic tonoplast.
DOI: 10.1016/j.cellbi.2007.11.007
1673. Int J Food Microbiol. 2008 Feb 29;122(1-2):156-61. Epub 2007 Dec 5.
Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating

methods and LAMP method: a collaborative study.
Hara-Kudo Y(1), Konishi N, Ohtsuka K, Hiramatsu R, Tanaka H, Konuma H, Takatori

K.
Author information:
(1)Division of Microbiology, National Institute of Health Sciences, Setagaya-ku,
1-18-1 Kamiyoga, Tokyo 158-8501, Japan. ykudo@nihs.go.jp
In order to establish a rapid and sensitive method for the detection of

Verotoxigenic Escherichia coli O157 and O26, a collaborative study was conducted
focusing on a comparison of the efficiency of loop-mediated amplification (LAMP)
assay targeting the Verocytotoxin (also called Shiga toxin) gene, utilizing a
direct plating method and a plating method with immunomagnetic separation
(IMS-plating method) using various agar media. In combination with enrichment
with the modified EC supplemented with novobiocin, E. coli O157 was detected in
most samples of ground beef and alfalfa sprouts by LAMP assay, the direct plating
method and the IMS-plating method. E. coli O26 was detected in approximately 100%
of the food samples by LAMP assay. However, the IMS-plating and direct plating
methods recovered 80 and 50% in ground beef samples, respectively. As a result,
it was demonstrated the LAMP assay is superior to the IMS-plating method. Based
on these results, it appears LAMP assay is effective as a screening assay to
detect E. coli O157 and O26 from positive samples.
1674. J Dairy Sci. 2008 Jan;91(1):229-35.
Effects of feeding forage soybean silage on milk production, nutrient digestion,

and ruminal fermentation of lactating dairy cows.
Vargas-Bello-Pérez E(1), Mustafa AF, Seguin P.
Author information:
(1)Department of Animal Science, Macdonald Campus, McGill University,
Sainte-Anne-de-Bellevue, Québec, Canada.
The objective of this study was to determine the feeding value of forage soybean
silage (SS) for dairy cows relative to a fourth-cut alfalfa silage (AS). Forage
soybean was harvested at full pod stage. Two isonitrogenous diets were formulated
with a 48:52 forage:concentrate ratio. Soybean silage and AS constituted 72% of
the forage in each diet, with corn silage constituting the remaining 28%. Twenty
Holsteins cows in early lactation were used in a switchback design. Four
lactating Holsteins cows fitted with ruminal cannulas were used to determine the
effects of dietary treatments on ruminal fermentation parameters and in vivo
total tract nutrient utilization. Relative to AS, SS contained 15, 28, and 25%
more neutral detergent fiber, acid detergent fiber, and crude protein,
respectively. Dry matter intake (23.5 vs. 25.1 kg/d) and milk yield (35.5 vs.
37.2 kg/d) were lower for cows fed SS than for those fed AS. However,
energy-corrected milk and milk efficiency were similar for both dietary
treatments. Milk protein, lactose, and total solids concentrations were not
influenced by dietary treatments (average 3.0, 4.7, and 12.6%, respectively).
However, cows fed SS produced milk with greater milk fat (3.8 vs. 3.6%) and milk
urea nitrogen concentrations (15.6 vs. 14.3 mg/dL) compared with cows fed AS.
Ruminal pH was lower, whereas ruminal NH3-N concentration was greater in cows fed
SS than in cows fed AS. Total tract digestibilities of dry matter, crude protein,
and neutral detergent fiber were not influenced by silage type. We concluded that
forage SS, when compared with AS, had a negative impact on feed intake and milk
yield, whereas energy-corrected milk, milk efficiency, and total tract nutrient
digestion were similar.
DOI: 10.3168/jds.2007-0484
1675. Environ Health Perspect. 2007 Dec;115(12):A579.
Agriculture: pesticides disrupt nitrogen fixation.
Potera C.
PMCID: PMC2137115
1676. Chemosphere. 2008 Apr;71(8):1593-8. Epub 2007 Dec 20.
Promotion of pyrene degradation in rhizosphere of alfalfa (Medicago sativa L.).
Fan S(1), Li P, Gong Z, Ren W, He N.
Author information:
(1)Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016,
China.
Pot experiment was conducted to evaluate the phytoremediation of

pyrene-contaminated soil using alfalfa (Medicago sativa L.). Alfalfa biomasses,
microbial viable counts, dehydrogenase activity, residual pyrene concentration
and pyrene removal percentage were determined after 60 days of alfalfa growth.
The results indicated that pyrene had an inhibitive effect on alfalfa growth, and
higher pyrene concentration seriously affected alfalfa growth. In addition, the
inhibitive effect on the root was more severe than that on the shoot. When pyrene
concentration reached 492 mg kg(-1) in soil, the shoot and root biomasses were
only 34% and 22% of those of alfalfa growing in non-spiked soil, respectively.
The rhizospheric bacterial and fungi counts were 5.0-7.5 and 1.8-2.3 times higher
than those in non-rhizosphere soil, respectively. The residual concentrations of
pyrene in the rhizosphere soil were lower than those in the non-rhizosphere soil.
After 60 days, 69-85% and 59-80% of spiked pyrene disappeared from the
rhizosphere and non-rhizosphere soils, respectively. The removal percentage
decreased with increasing pyrene concentration. However, the average removal of
pyrene in the rhizosphere soil was 6% higher than that in the non-rhizosphere
soil. Therefore, the presence of alfalfa roots was effective in promoting the
phytoremediation of freshly added pyrene into the soil.
1677. Environ Sci Technol. 2007 Nov 15;41(22):7818-23.
Improved approaches for modeling the sorption of phenanthrene by a range of plant

species.
Zhu Y(1), Zhang S, Zhu YG, Christie P, Shan X.
Author information:
State Key Laboratory of Environmental Chemistry and Ecotoxicology, P.O. Box 2871,
Equilibrium sorption of phenanthrene and its relationship with plant lipid

contents were investigated using roots and shoots of alfalfa, ryegrass, tomato,
potato, carrot, cucumber, zucchini, and pumpkin. Lipid extractions using
chloroform and hexane were compared, and the influence of dechlorophyllization on
lipid determinations was evaluated. The sorption isotherms were close to linear
(R2 > 0.923, P < 0.05) and the plant-water partition coefficients (K(pl)) of
phenanthrene obtained from the isotherms exhibited significant and positive
correlations with plantlipid contents (R2 > 0.664, P < 0.05). The correlations
were more significant (R2 > 0.906, P < 0.001) when dechlorophyllization was
included in the lipid extraction. The measured sorption was higher than that
estimated using the octanol-water partition coefficient (K(ow)) but was very
close to the estimate using the triolein-water partition coefficient (K(tw)).
This study leads us to conclude that dechlorophyllization is necessary for plant
lipid determination and that K(tw) is more accurate as a substitute for the
lipid-water partition coefficient (K(lip)) than K(ow). These novel approaches may
provide substantial improvements in the application of partition-limited models
for the estimation of plant uptake of organic contaminants.
1678. J Agric Saf Health. 2007 Nov;13(4):367-74.
Noise exposures during potato processing and manufacture of animal feed.
Achutan C(1), Tubbs RL.
Author information:
(1)National Institute for Occupational Safety and Health, Cincinnati, Ohio 45226,
USA. cma4@cdc.gov
Erratum in
J Agric Saf Health. 2008 Jan;14(1):table of contents.
A noise exposure survey was conducted at an agricultural facility to evaluate

noise exposures during potato processing and during the manufacture of alfalfa
pellets. Of the 19 employees monitored, five reached or exceeded the National
Institute for Occupational Safety and Health recommended exposure limit for
occupational noise. Four of these employees were from the potato processing area,
and one was from the alfalfa pellet-mill operation. Two of the five (bagger and
pellet-mill operator) also exceeded the Occupational Safety and Health
Administration action level. The facility has a well-managed hearing conservation
program for employees in the potato processing area, but not in the alfalfa
manufacturing area.
1679. Nucleic Acids Res. 2008 Jan;36(Database issue):D970-6. Epub 2007 Dec 10.
PlantTribes: a gene and gene family resource for comparative genomics in plants.
Wall PK(1), Leebens-Mack J, Müller KF, Field D, Altman NS, dePamphilis CW.
Author information:
(1)Department of Biology, Institute of Molecular Evolutionary Genetics, and The
Huck Institutes of the Life Sciences, The Pennsylvania State University,
University Park, PA 16802, USA.
The PlantTribes database (http://fgp.huck.psu.edu/tribe.html) is a plant gene

family database based on the inferred proteomes of five sequenced plant species:
Arabidopsis thaliana, Carica papaya, Medicago truncatula, Oryza sativa and
Populus trichocarpa. We used the graph-based clustering algorithm MCL [Van Dongen
(Technical Report INS-R0010 2000) and Enright et al. (Nucleic Acids Res. 2002;
30: 1575-1584)] to classify all of these species' protein-coding genes into
putative gene families, called tribes, using three clustering stringencies (low,
medium and high). For all tribes, we have generated protein and DNA alignments
and maximum-likelihood phylogenetic trees. A parallel database of microarray
experimental results is linked to the genes, which lets researchers identify
groups of related genes and their expression patterns. Unified nomenclatures were
developed, and tribes can be related to traditional gene families and conserved
domain identifiers. SuperTribes, constructed through a second iteration of MCL
clustering, connect distant, but potentially related gene clusters. The global
classification of nearly 200 000 plant proteins was used as a scaffold for
sorting approximately 4 million additional cDNA sequences from over 200 plant
species. All data and analyses are accessible through a flexible interface
allowing users to explore the classification, to place query sequences within the
classification, and to download results for further study.
DOI: 10.1093/nar/gkm972
PMCID: PMC2238917
1680. J Agric Food Chem. 2008 Jan 9;56(1):272-80. Epub 2007 Dec 11.
Three polyphenol oxidases from red clover (Trifolium pratense) differ in

enzymatic activities and activation properties.
Schmitz GE(1), Sullivan ML, Hatfield RD.
Author information:
of Agriculture, Madison, WI 53706, USA.
Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause

browning reactions in many wounded fruits, vegetables, and plants including the
forage crop red clover (Trifolium pratense L.). Production of o-quinones in red
clover inhibits postharvest proteolysis during the ensiling process. The cDNAs
encoding three red clover PPOs were expressed individually in alfalfa (Medicago
sativa L.), which lacks detectable endogenous foliar PPO activity and
o-diphenols. Several physical and biochemical characteristics of the red clover
PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red
clover PPOs exist in a latent state and are activated (10-40-fold increase in
activity) by long incubations (>2 days) at ambient temperature or short
incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at
high temperatures than PPO2 or PPO3. During incubation at ambient temperature,
the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The
apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1,
respectively, and latent PPO1 is slightly activated (~5-fold) by low pH.
Activation of the PPOs shifts the pH optima to approximately 7, and the activated
PPOs retain substantial levels of activity as the pH increases above their
optima. The latent and activated PPOs were surveyed for ability to oxidize
various o-diphenols, and activation of the PPOs had little effect on substrate
specificity. Activation increases the V max but not the affinity of the PPO
enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and
kinetic changes during activation and provide new insights to their effects in
postharvest physiology.
DOI: 10.1021/jf072488u
1681. Mol Microbiol. 2008 Jan;67(2):350-63. Epub 2007 Dec 7.
Multiple Ku orthologues mediate DNA non-homologous end-joining in the free-living

form and during chronic infection of Sinorhizobium meliloti.
Kobayashi H(1), Simmons LA, Yuan DS, Broughton WJ, Walker GC.
Author information:
(1)Department of Biology, Massachusetts Institute of Technology, Cambridge, MA
02139, USA.
The bacterial non-homologous end-joining (NHEJ) apparatus is a two-component

system that uses Ku and LigD to repair DNA double-strand breaks. Although the
reaction mechanism has been extensively studied, much less is known about the
physiological role of bacterial NHEJ. Recent studies suggest that NHEJ acts under
conditions where DNA replication is reduced or absent (such as in a spore or
stationary phase). Interestingly, genes encoding Ku and LigD have been identified
in a wide range of bacteria that can chronically infect eukaryotic hosts.
Strikingly, Sinohizobium meliloti, an intracellular symbiont of legume plants,
carries four genes encoding Ku homologues (sku1 to sku4). Deletion analysis of
the sku genes indicated that all Ku homologues are functional. One of these
genes, sku2, is strongly expressed in free-living cells, as well as in bacteroid
cells residing inside of the host plant. To visualize the NHEJ apparatus in vivo,
SKu2 protein was fused to yellow fluorescent protein (YFP). Ionizing radiation
(IR) induced focus formation of SKu2-YFP in free-living cells in a
dosage-dependent manner. Moreover, SKu2-YFP foci formed in response to IR in
non-dividing bacteroids, indicating that NHEJ system is functional even during
the chronic infection phase of symbiosis.
DOI: 10.1111/j.1365-2958.2007.06036.x
PMCID: PMC2770583
1682. J Bacteriol. 2008 Feb;190(3):1118-23. Epub 2007 Nov 30.
A highly conserved protein of unknown function is required by Sinorhizobium

meliloti for symbiosis and environmental stress protection.
Davies BW(1), Walker GC.
Author information:
We report here the first characterization of the Sinorhizobium meliloti open

reading frame SMc01113. The SMc01113 protein is a member of a highly conserved
protein family, universal among bacteria. We demonstrate that the SMc01113 gene
is absolutely required for S. meliloti symbiosis with alfalfa and also for the
protection of the bacterium from a wide range of environmental stresses.
DOI: 10.1128/JB.01521-07
PMCID: PMC2223554
1683. Euro Surveill. 2007 Nov 29;12(11):E071129.4.
Outbreak of Salmonella Weltevreden infections in Norway, Denmark and Finland

associated with alfalfa sprouts, July-October 2007.
Emberland KE(1), Ethelberg S, Kuusi M, Vold L, Jensvoll L, Lindstedt BA, Nygard

K, Kjelsø C, Torpdahl M, Sørensen G, Jensen T, Lukinmaa S, Niskanen T, Kapperud
G.
Author information:
(1)Division of Infectious Disease Control, Folkehelseinstituttet (FHI, Norwegian
Institute of Public Health), Oslo, Norway. Knut.Erik.Emberland@fhi.no
1684. J Food Prot. 2007 Nov;70(11):2602-5.
Mathematical modeling and assessment of microbial migration during the sprouting

of alfalfa in trays in a nonuniformly contaminated seed batch using Enterobacter
aerogenes as a surrogate for Salmonella Stanley.
Liu B(1), Schaffner DW.
Author information:
(1)Food Risk Analysis Initiative, Rutgers University, 65 Dudley Road, New
Brunswick, New Jersey 08901-8520, USA.
Raw seed sprouts have been implicated in several food poisoning outbreaks in the
past 10 years. The U.S. Food and Drug Administration recommends that sprout
growers use interventions (such as testing of spent irrigation water) to control
the presence of pathogens in the finished product. During the sprouting process,
initially low concentrations of pathogen may increase, and contamination may
spread within a batch of sprouting seeds. A model of pathogen growth as a
function of time and distance from the contamination spot during the sprouting of
alfalfa in trays has been developed with Enterobacter aerogenes. The probability
of detecting contamination was assessed by logistic regression at various time
points and distances by sampling from sprouts or irrigation water. Our results
demonstrate that microbial populations and possibility of detection were greatly
reduced at distances of > or = 20 cm from the point of contamination in a seed
batch during tray sprouting; however, the probability of detecting microbial
contamination at distances less than 10 cm from the point of inoculation was
almost 100% at the end of the sprouting process. Our results also show that
sampling irrigation water, especially large volumes of water, is highly effective
at detecting contamination: by collecting 100 ml of irrigation water for membrane
filtration, the probability of detection was increased by three to four times
during the first 6 h of seed germination. Our findings have quantified the degree
to which a small level of contamination will spread throughout a tray of
sprouting alfalfa seeds and subsequently be detected by either sprout or
irrigation water sampling.
1685. Environ Pollut. 2008 Jul;154(2):203-11. Epub 2007 Nov 26.
Toxic effects of arsenic on Sinorhizobium-Medicago sativa symbiotic interaction.
Pajuelo E(1), Rodríguez-Llorente ID, Dary M, Palomares AJ.
Author information:
(1)Department of Microbiology and Parasitology, Faculty of Pharmacy, University
of Seville, 41012 Seville, Spain.
Recently, the Rhizobium-legume symbiotic interaction has been proposed as an

interesting tool in bioremediation. However, little is known about the effect of
most common contaminants on this process. The phytotoxic effects of arsenic on
nodulation of Medicago sativa have been examined in vitro using the highly
arsenic resistant and symbiotically effective Sinorhizobium sp. strain MA11. The
bacteria were able to grow on plates containing As concentrations as high as 10
mM. Nevertheless, as little as 25-35 microM arsenite produced a 75% decrease in
the total number of nodules, due to a 90% reduction in the number of rhizobial
infections, as could be determined using the strain MA11 carrying a lacZ reporter
gene. This effect was associated to root hair damage and a shorter infective root
zone. However, once nodulation was established nodule development seemed to
continue normally, although earlier senescence could be observed in nodules of
arsenic-grown plants.
DOI: 10.1016/j.envpol.2007.10.015
1686. FEMS Microbiol Ecol. 2008 Jan;63(1):118-31. Epub 2007 Nov 20.
Prevalence of pSmeSM11a-like plasmids in indigenous Sinorhizobium meliloti

strains isolated in the course of a field release experiment with genetically
modified S. meliloti strains.
Kuhn S(1), Stiens M, Pühler A, Schlüter A.
Author information:
(1)Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld,
Bielefeld, Germany.
Plasmid pSmeSM11a, residing in the indigenous Sinorhizobium meliloti strain SM11

originating from a field in Strassmoos (Bavaria, Germany), was analysed
previously at the genomic level. Thirty-seven indigenous S. meliloti strains,
originating from two different locations in Germany, were screened for genes
identified previously on pSmeSM11a. Seven of these strains harbour accessory
plasmids that are very similar to pSmeSM11a. The identified pSmeSM11a-like
plasmids are c. 130-150 kb in size and possess nearly identical restriction
profiles. Up to 30 genes identified previously on pSmeSM11a could be detected on
these plasmids by hybridisation experiments, e.g., the nodulation genes nodP and
nodQ, the ethylene level modulation gene acdS and the taurine metabolism gene
tauD. A few pSmeSM11a genes were also detected on other plasmids. The reference
plasmid pSmeSM11a contains a region that is similar to a segment of S. meliloti
strain Rm1021 pSymA. Regions with similarity to pSymA were also detected on the
aforementioned seven pSmeSM11a-like plasmids. The specifications of these regions
are nearly identical to the one on pSmeSM11a and differ from Rm1021 pSymA as
determined by nucleotide sequence analysis. Two further plasmids similar to
pSmeSM11a completely lack the pSymA-region. Those strains carrying accessory
plasmids that contain the acdS gene encoding 1-aminocyclopropane-1-carboxylate
deaminase are able to grow on 1-aminocyclopropane-1-carboxylate as the sole
source of nitrogen, demonstrating functionality of the acdS gene product. About
36% of the analysed plasmids, including three pSmeSM11a-like plasmids, could be
transferred to another S. meliloti recipient strain, allowing for their
dissemination in S. meliloti populations.
DOI: 10.1111/j.1574-6941.2007.00399.x
1687. FEMS Microbiol Lett. 2007 Dec;277(2):210-6.
Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba

in North Spain.
Iglesias O, Rivas R, García-Fraile P, Abril A, Mateos PF, Martinez-Molina E,

Velázquez E.
Prosopis is a Mimosaceae legume tree indigenous to South America and not

naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba
roots were isolated from a soil in North Spain that belong to eight different
randomly amplified polymorphic DNA groups phylogenetically related to
Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according
to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of
isolates close to S. medicae and S. meliloti were identical to those of S.
medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these
strains were able to nodulate both alfalfa and Prosopis. These nodC genes were
phylogenetically divergent from those of the isolates close to R. giardinii that
were identical to that of R. giardinii H152(T) and therefore all these strains
formed nodules in common beans and Prosopis. The nodC genes of the strains
isolated in Spain were phylogenetically divergent from that carried by
Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T)
nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a
promiscuous host which can establish symbiosis with strains carrying very
divergent nodC genes and this promiscuity may be an important advantage for this
legume tree to be used in reforestation.
DOI: 10.1111/j.1574-6968.2007.00968.x
1688. Poult Sci. 2007 Dec;86(12):2502-8.
Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles.

IV. Immune and stress protein response.
Dunkley CS(1), McReynolds JL, Dunkley KD, Njongmeta LN, Berghman LR, Kubena LF,
Nisbet DJ, Ricke SC.
Author information:
77843-2472, USA.
Immunological responses of molting hens either infected or not infected with

Salmonella enterica serovar Enteritidis were compared in 2 trials with Single
Comb White Leghorn hens >50 wk old. The hens were placed into 6 treatment groups
with 12 hens per group: nonmolted Salmonella Enteritidis positive (FF+),
non-molted Salmonella Enteritidis negative (FF-), feed withdrawal Salmonella
Enteritidis positive (FW+), FW Salmonella Enteritidis negative (FW-), alfalfa
Salmonella Enteritidis positive (ALC+), and ALC Salmonella Enteritidis negative
(ALC-). Each hen in the Salmonella Enteritidis-positive groups was challenged on
d 4 of the study with 1 mL of 10(6)-cfu Salmonella Enteritidis, and diets were
administered for 12 d. Blood samples were collected on d 2, 5, 9, and 12, and
blood smears were enumerated for heterophil to lymphocyte (H:L) ratios. Serum
samples were also analyzed for alpha(1)-acid glycoprotein (AGP) levels and
antibody level. On d 12, hens were euthanized and bile samples from the gall
bladder and sections of the ileum and the ceca were collected, and an ELISA was
used to determine the intestinal, serum, and bile antibody responses. The FW+
hens produced more (P <or= 0.05) cecal IgA than hens in all the other treatments
except the FW-hens in trial 2. Bile IgA production in all Salmonella
Enteritidis-positive groups was higher (P <or= 0.05) than in the Salmonella
Enteritidis-negative groups, with the exception of the ALC- hens. In trial 1 on d
5, FW+ hens produced less (P <or= 0.05) serum IgG than ALC-, FF-, and FW- hens
but not FF+ and ALC+ hens, whereas in trial 2, only FW- hens had lower IgG levels
than FW+ hens. On d 9, FW groups exhibited increases in the H:L ratios, but on d
12, these declined to levels more similar to the other treatments. The FW hen
groups yielded generally higher AGP levels compared with the other treatments on
d 9 and 12 in both trials. The ALC molt diets appeared to reduce stress and
inflammation, based on the H:L ratios and AGP levels in the serum, when compared
with FW-molted hens.
DOI: 10.3382/ps.2006-00401
1689. Poult Sci. 2007 Dec;86(12):2492-501.
Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles.

III. Blood plasma metabolite response.
Dunkley CS(1), McReynolds JL, Dunkley KD, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
77843-2472, USA.
The objective of this study was to examine an alfalfa crumble diet as an

alternative molt diet and compare the physiological response of hens to the
responses of feed-deprived molted hens. Hens >50 wk old were placed into 6
treatment groups (12 hens per group in trial 1 and 10 hens per group in trial 2):
nonmolted Salmonella enterica serovar Enteritidis positive (FF+), nonmolted
Salmonella Enteritidis negative (FF-), feed withdrawal Salmonella Enteritidis
positive (FW+), FW Salmonella Enteritidis negative (FW), alfalfa Salmonella
Enteritidis positive (ALC+), and ALC Salmonella Enteritidis negative (ALC-). Each
hen in the Salmonella Enteritidis-positive groups was challenged on the fourth
day of the study with 1 mL of 10(6)-cfu Salmonella Enteritidis. Blood was
collected on d 2, 5, 9, and 12 of the trial. Blood plasma was collected and
metabolite concentrations were analyzed for glucose, calcium, cholesterol, uric
acid, total protein, and triglycerides. The feed intakes of the FF hens were 4-
to 6-fold greater (P <or= 0.05) than those of the ALC birds in both trials. Over
the 12 d of molt, the FW+ hens lost more (P <or= 0.05) BW than all other groups
except the FW- hens, whereas the FW-, ALC-, and ALC+ hens lost more BW than the
FF hen groups. Uric acid concentrations were generally lower in molted hens
compared with the FF hen groups during the initial stages of molt. On d 9 of both
trials, concentrations of calcium and total protein were higher (P <or= 0.05) in
the FF hens than in the other groups. The FF hen groups exhibited higher (P <or=
0.05) concentrations of triglycerides than the FW and ALC groups in both trials
on d 5, 9, and 12. Based on the results from these studies, ALC diets can limit
some of the potential physiological stress indicators that accompany feed
deprivation during an induced molt.
DOI: 10.3382/ps.2006-00400
1690. New Phytol. 2008;177(1):155-66. Epub 2007 Nov 20.
Carbon monoxide alleviates cadmium-induced oxidative damage by modulating

glutathione metabolism in the roots of Medicago sativa.
Han Y(1), Zhang J, Chen X, Gao Z, Xuan W, Xu S, Ding X, Shen W.
Author information:
(1)College of Life Sciences, Co. Laboratory of Nanjing Agricultural University
and Carl Zeiss Far East, Nanjing Agricultural University, Nanjing 210095, PR
China.
Using pharmacological and biochemical approaches, the role of cadmium

(Cd)-induced carbon monoxide (CO) release and the relationship between CO and
oxidative stress conferred by Cd exposure in the root tissues of alfalfa
(Medicago sativa) plants were investigated. Cd treatments showed a dose-dependent
enhancement in lipid peroxidation. Both 100 and 200 microm CdCl(2 )treatments
caused the increase of CO release, which is consistent with the changes in the
activity of the CO synthetic enzyme heme oxygenase (HO) and its HO-1 transcript.
A 100 microm CdCl(2) exposure enhanced the formation of nonprotein thiols (NPT),
and reduced glutathione (GSH) to oxidized glutathione (GSSG), which was
potentiated by the pretreatment of CO scavenger hemoglobin (Hb). Plants
pretreated for 6 h with 50% CO-saturated aqueous solution, which induced the
rapid endogenous CO release followed by a gradual decrease when subsequently
exposed to 100 microm CdCl(2) for 72 h, effectively decreased oxidative damage.
Meanwhile, CO pretreatment modulated several enzymes responsible for GSH
metabolism, thus resulting in the partial restoration of GSH : GSSG ratio, which
was significantly blocked by Hb. These results are suggestive of a role for CO
release as a signal element for the alleviation of Cd-induced oxidative damage by
modulating glutathione metabolism.
DOI: 10.1111/j.1469-8137.2007.02251.x
1691. New Phytol. 2008;177(3):715-24. Epub 2007 Nov 17.
Selenium hyperaccumulation reduces plant arthropod loads in the field.
Galeas ML(1), Klamper EM, Bennett LE, Freeman JL, Kondratieff BC, Quinn CF,
Pilon-Smits EA.
Author information:
(1)Department of Biology, Colorado State University, Fort Collins, CO 80523, USA.
Comment in
New Phytol. 2008;177(3):569-72.
The elemental defense hypothesis proposes that some plants hyperaccumulate toxic
elements as a defense mechanism. In this study the effectiveness of selenium (Se)
as an arthropod deterrent was investigated under field conditions. Arthropod
loads were measured over two growing seasons in Se hyperaccumulator habitats in
Colorado, USA, comparing Se hyperaccumulator species (Astragalus bisulcatus and
Stanleya pinnata) with nonhyperaccumulators (Camelina microcarpa, Astragalus
americanus, Descurainia pinnata, Medicago sativa, and Helianthus pumilus). The Se
hyperaccumulating plant species, which contained 1000-14 000 microg Se g(-1) DW,
harbored significantly fewer arthropods (c. twofold) and fewer arthropod species
(c. 1.5-fold) compared with nonhyperaccumulator species that contained < 30
microg Se g(-1) DW. Arthropods collected on Se-hyperaccumulating plants contained
three- to 10-fold higher Se concentrations than those found on
nonhyperaccumulating species, but > 10-fold lower Se concentrations than their
hyperaccumulator hosts. Several arthropod species contained > 100 microg Se g(-1)
DW, indicating Se tolerance and perhaps feeding specialization. These results
support the elemental defense hypothesis and suggest that invertebrate herbivory
may have contributed to the evolution of Se hyperaccumulation.
DOI: 10.1111/j.1469-8137.2007.02285.x
1692. J Dairy Sci. 2007 Dec;90(12):5698-705.
Effects of a mixture of lactic acid bacteria applied as a freeze-dried or fresh

culture on the fermentation of alfalfa silage.
Kizilsimsek M(1), Schmidt RJ, Kung L Jr.
Author information:
(1)University of Kahramanmaras, Sutcu Imam Field Crops Department, Kahramanmaras,
Turkey.
Alfalfa (approximately 31% DM) was untreated or treated with a silage inoculant
containing the lactic acid bacteria Lactobacillus lactis, Lactobacillus plantarum
L-54, and L. plantarum Aber F1. The inoculant was added at a normal and a high
dose as a freeze-dried powder that had been mixed with water just prior to
application, or it was grown with nutrients the day before and added as a fresh
culture. The actual application rate of lactic acid bacteria was 1.19 x 10(5) for
the normal dose, 4.30 x 10(5) for the high dose, and 5.10 x 10(5) for the fresh
culture. All inoculated silages showed a faster increase in the rate of lactic
acid production and a decrease in the drop in pH over the first 24 h of ensiling
compared with untreated silage. The effect was greatest for silage treated with
the fresh culture and was supported by the fact that this treatment had numbers
of lactic acid bacteria that increased faster than in other treatments.
Inoculation also generally resulted in a fermentation profile that was more
homolactic (more lactic acid and less acetic acid, ethanol, and NH(3)-N) than for
untreated silage, but the effect was greatest for the fresh culture. Inoculation
did not affect in vitro neutral detergent fiber digestion or the concentrations
of neutral detergent fiber or total N in silages. The recovery of dry matter was
greater in silage that was treated with a high level of the freeze-dried culture
or with the fresh culture when compared with the untreated control. This study
showed that application of a silage inoculant as a freeze-dried culture or as a
fresh culture resulted in alfalfa silage with a more homolactic fermentation
profile. The effect was greatest from addition of the fresh culture.
DOI: 10.3168/jds.2007-0448
1693. J Dairy Sci. 2007 Dec;90(12):5587-99.
Dried distillers grains plus solubles with corn silage or alfalfa hay as the
primary forage source in dairy cow diets.
Kleinschmit DH(1), Schingoethe DJ, Hippen AR, Kalscheur KF.
Author information:
(1)Dairy Science Department, South Dakota State University, Brookings 57007-0647,
USA. david.schingoethe@sdstate.edu
Nine multiparous (250 +/- 6 d in milk) and 3 primiparous (204 +/- 6 d in milk)
Holstein cows were utilized in a 3 x 3 Latin square design to evaluate the
lactation performance of cows fed a diet containing dried distillers grains plus
solubles (DDGS) with either corn silage or alfalfa hay as forage. Cows were fed
total mixed diets containing corn silage (CS), 50% corn silage and 50% alfalfa
hay (CSAH), or alfalfa hay (AH) as the forage source. All diets had a 50:50
forage-to-concentrate ratio, contained 15% DDGS, and were formulated to be equal
in metabolizable protein. Dry matter intake increased when cows were fed CSAH
(24.9 kg/d) compared with CS (21.9 kg/d) and AH (20.9 kg/d). Yields of milk
(26.5, 28.4, 29.0 kg/d for CS, CSAH, and AH, respectively) increased linearly as
proportions of alfalfa fed increased but 4% fat-corrected milk and
energy-corrected milk were not affected by treatment. Feed efficiency (1.28,
1.23, and 1.45 kg of energy-corrected milk/kg of intake) improved when AH was fed
compared with CS or CSAH. Milk fat concentration (3.67, 3.55, and 3.49%)
decreased linearly when alfalfa replaced corn silage, but was observed only in
primiparous cows, not multiparous cows. Milk protein concentration (3.32, 3.29,
and 3.29%) was not affected by diet although yield (0.90, 0.96, and 0.98 kg/d)
tended to increase linearly when alfalfa was added to the diet. This may have
been due to an increase in essential amino acid (AA) availability and uptake by
the mammary gland or to greater crude protein intake in cows fed AH. In addition,
replacing corn silage with alfalfa increased the uptake of Lys by the mammary
gland. Methionine was the first-limiting AA based on the transfer efficiency of
AA in arterial plasma to milk protein. However, Lys was the first-limiting AA in
CS and CSAH and Met was first limiting in AH for mammary gland extraction
efficiency of AA from plasma. In conclusion, replacing corn silage with alfalfa
hay in diets containing 15% DDGS increased milk yield and tended to increase milk
protein yield linearly in cows during late lactation. Feeding alfalfa hay as the
sole forage source improved feed efficiency compared with diets containing corn
silage.
DOI: 10.3168/jds.2006-753
1694. J Bacteriol. 2008 Feb;190(3):861-71. Epub 2007 Nov 16.
Regulation of motility by the ExpR/Sin quorum-sensing system in Sinorhizobium

meliloti.
Hoang HH(1), Gurich N, González JE.
Author information:
A successful symbiotic relationship between Sinorhizobium meliloti and its host

Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the
biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our
laboratory has shown that a quorum-sensing mechanism controls the production of
the symbiotically active EPS II. Recent microarray analysis of the whole-genome
expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system
regulates additional physiological processes that include low-molecular-weight
succinoglycan production, nitrogen utilization, metal transport, motility, and
chemotaxis. Nearly half of the flagellar genes and their dependence on quorum
sensing are prominently displayed in our microarray analyses. We extend those
observations in this work and confirm the findings by real-time PCR expression
analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB
genes, involved in motility and chemotaxis. These genes code for regulators of
flagellum synthesis, the chemotactic response, or parts of the flagellar
apparatus. Gene expression analyses and visualization of flagella by electron
microscopy performed at different points in the growth phase support our proposed
model in which quorum sensing downregulates motility in S. meliloti. We
demonstrate that the ExpR/Sin quorum-sensing system controls motility gene
expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent
two-component system suppresses motility gene expression through VisN and Rem in
parallel to quorum sensing. This study contributes to our understanding of the
mechanisms that govern motility in S. meliloti.
DOI: 10.1128/JB.01310-07
PMCID: PMC2223547
1695. Plant Physiol Biochem. 2008 Jan;46(1):93-9. Epub 2007 Oct 7.
Characterization of a pathogenesis-related class 10 protein (PR-10) from

Astragalus mongholicus with ribonuclease activity.
Yan Q(1), Qi X, Jiang Z, Yang S, Han L.
Author information:
(1)Bioresource Utilization Laboratory, College of Engineering, China Agricultural
University, Beijing, China. yanqj@cau.edu.cn
A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first

isolated from the Chinese medicinal material Astragalus mongholicus using a
combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange
chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The
purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in
SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by
gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical
subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS)
staining and its neutral carbohydrate content was 13.7%. The carbohydrate was
mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w)
fructose on the basis of high-performance anion exchange chromatography (HPAEC)
analysis. Its N-terminal sequence of 15 amino acid residues was determined as
GVISFNEETISTVAP, and showed significant sequence homology to some
pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with
the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3%
identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting
it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase)
activity as do some other PR-10 proteins. The optimal pH and temperature for
RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was
stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The
purification and characterization of AmPR-10 in this investigation furnish
additional data to the relatively scanty literature pertaining to Astragali radix
proteins.
DOI: 10.1016/j.plaphy.2007.10.002
1696. Environ Toxicol Chem. 2007 Dec;26(12):2717-23.
Lead toxicity in alfalfa plants exposed to phytohormones and

ethylenediaminetetraacetic acid monitored by peroxidase, catalase, and amylase
activities.
López ML(1), Peralta-Videa JR, Castillo-Michel H, Martinez-Martinez A,

Duarte-Gardea M, Gardea-Torresdey JL.
Author information:
(1)Environmental Science and Engineering Program, University of Texas, El Paso
79968, USA.
This manuscript describes the toxicity of lead in alfalfa plants treated with
ethylenediaminetetraacetic acid (EDTA) and the phytohormones indole-3-acetic-acid
(IAA), gibberellic acid (GA), and kinetin (KN), on catalase (CAT), ascorbate
peroxidase (APOX), and total amylase activity (TAA). In all cases Pb was used at
40 mg/L; EDTA at 0.2 mM (equimolar to Pb); and IAA, GA, and KN at 1, 10, and 100
microM, respectively. An experiment containing Pb at 40 mg/L, 0.2 mM EDTA, and
IAA and KN at 100 microM each was performed to determine changes in TAA. A
control (plain nutrient solution) also was used for comparison. In all cases the
treatments were performed in triplicate. Standard procedures were followed to
determine the activity of the respective enzymes. After 10 d of exposure to the
treatments, the leaves were harvested, homogenized, and centrifuged, and the
supernatants were analyzed for CAT, APOX, and TAA. All determinations were
performed in triplicate. The results demonstrated that CAT was reduced
significantly (p < 0.05) by all treatments containing Pb, IAA, and GA at 10 and
100 microM. However, only the treatments Pb/EDTA/KN at 1, 10, and 100 microM
reduced the APOX. The TAA in leaves of alfalfa plants was increased significantly
(p < 0.05) by all treatments. Overall, the results suggest that the CAT tests
showed no lead toxicity to the alfalfa seedlings. However IAA at 10 and 100 muM
revealed toxicity to the CAT enzyme. In addition, the APOX tests exhibited no
toxicity to the peroxidase enzyme with the exception of Pb/EDTA/KN treatments.
Finally, the TAA tests showed high Pb/EDTA/phytohormone toxicity to the amylase
enzyme in alfalfa seedlings.
DOI: 10.1897/07-302.1
1697. Appl Microbiol Biotechnol. 2008 Jan;77(6):1371-8. Epub 2007 Nov 15.
Degradation of 2,4-DB in Argentinean agricultural soils with high humic matter

content.
Cuadrado V(1), Merini LJ, Flocco CG, Giulietti AM.
Author information:
(1)Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires,
Argentina.
The dissipation of 4-(2,4-dichlorophenoxy) butyric acid (2,4-DB) in

high-humic-matter-containing soils from agricultural fields of the Argentinean
Humid Pampa region was studied, employing soil microcosms under different
experimental conditions. The added herbicide was dissipated almost completely by
soils with and without history of herbicide use by day 28. At 500 ppm, both soils
showed the same degradation rates; but at 5-ppm concentration, the chronically
exposed soil demonstrated a faster degradation of the herbicide. 2,4-DB addition
produced increases in herbicide-degrading bacteria of three and 1.5 orders of
magnitude in soils with and without history of herbicide use, respectively, in
microcosms with 5 ppm. At 500-ppm concentration, the increase in 2,4-DB degraders
was five orders of magnitude after 14 days, independent of the history of
herbicide use. No differences were observed in either 2,4-DB degradation rates or
in degrader bacteria numbers in the presence and absence of alfalfa plants, in
spite of some differential characteristics in patterns of 2,4-DB metabolite
accumulation. The main factor affecting 2,4-DB degradation rate would be the
history of herbicide use, as a consequence of the adaptation of the indigenous
microflora to the presence of herbicides in the field.
DOI: 10.1007/s00253-007-1260-x
1698. Euro Surveill. 2007 Oct 18;12(10):E071018.2.
Outbreak of Salmonella Stanley in Sweden associated with alfalfa sprouts,

July-August 2007.
Werner S(1), Boman K, Einemo I, Erntell M, de Jong B, Lindqvist A, Löfdahl M,

Lofdahl S, Meeuwisse A, Ohlen G, Olsson M, Stamer U, Sellstrom E, Andersson Y.
Author information:
(1)Swedish Institute for Infectious Disease Control (SMI), Stockholm, Sweden.
simon.werner@skane.se
1699. J Plant Physiol. 2008 May 26;165(8):895-900. Epub 2007 Nov 7.
Induction of peroxidases and superoxide dismutases in transformed embryogenic

calli of alfalfa (Medicago sativa L.).
Platisa J(1), Veljović-Jovanović S, Kukavica B, Vinterhalter B, Smigocki A,

Ninković S.
Author information:
(1)Center for Multidisciplinary Studies, University of Belgrade, Kneza Viseslava
1a, 11030 Belgrade, Serbia.
Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed
in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.)
carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I
antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the
transformed lines analyzed had elevated levels of all POD isoforms. Three POD
isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional
pair of isoforms with a pI value of approximately 8.8 were separated from tissue
extracts of all transgenic lines. Isoelectrofocusing patterns revealed the
induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines
compared with non-transformed embryogenic tissue. These results indicate that the
process of transformation may disrupt redox homeostasis in alfalfa tissues.
DOI: 10.1016/j.jplph.2007.07.021
1700. Plant Cell. 2007 Nov;19(11):3369-78. Epub 2007 Nov 9.
Compound leaf development and evolution in the legumes.
Champagne CE(1), Goliber TE, Wojciechowski MF, Mei RW, Townsley BT, Wang K, Paz
MM, Geeta R, Sinha NR.
Author information:
(1)Section of Plant Biology, University of California, Davis, California 95616,
USA.
Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a

critical role in the development of compound leaves. An exception to this trend
is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog
of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1
genes to regulate compound leaf development. To assess the phylogenetic
distribution of KNOX1-independent compound leaf development, a survey of KNOX1
protein expression across the Fabaceae was undertaken. The majority of
compound-leafed Fabaceae have expression of KNOX1 proteins associated with
developing compound leaves. However, in a large subclade of the Fabaceae, the
inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression
is not associated with compound leaves. These data suggest that the FLO/LFY gene
may function in place of KNOX1 genes in generating compound leaves throughout the
IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae
outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in
transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY
expression showed only slight reductions in leaflet number. Overexpression of a
KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an
increase in leaflet number. This implies that KNOX1 targets, which promote
compound leaf development, are present in alfalfa and are still sensitive to
KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may
have played partially overlapping roles in compound leaf development in ancestral
Fabaceae but that the FLO/LFY gene took over this role in the IRLC.
DOI: 10.1105/tpc.107.052886
PMCID: PMC2174894
1701. FEMS Microbiol Ecol. 2007 Dec;62(3):258-67.
Fungal and bacterial growth in soil with plant materials of different C/N ratios.
Rousk J(1), Bååth E.
Author information:
(1)Department of Microbial Ecology, Lund University, Lund, Sweden.
johannes.rousk@mbioekol.lu.se
Fungal (acetate-in-ergosterol incorporation) and bacterial (leucine/thymidine

incorporation) growth resulting from alfalfa (C/N=15) and barley straw (C/N=75)
addition was studied in soil microcosms for 64 days. Nitrogen amendments were
used to compensate for the C/N difference between the substrates. Fungal growth
increased to a maximum after 3-7 days, at five to eight times the controls,
following the addition of straw, and three to four times the controls following
the addition of alfalfa. After 20-30 days, the fungal growth rate converged with
the controls, resulting in a cumulative fungal growth two to three times the
controls following straw addition and about 20% higher than the controls
following alfalfa addition. The bacterial growth rate reached rates five times
the controls following alfalfa addition and twice that of the controls following
straw addition after 3-7 days. It remained elevated after 64 days. The cumulative
bacterial growth was two and four times the controls following straw and alfalfa
addition, respectively. A negative correlation was found between N addition and
bacterial growth, while N stimulated fungal growth. Thus, the C/N ratio of the
additions (substrate and extra N) could not entirely explain the different
results regarding fungal and bacterial growths. Respiration was not always
related to the combined growth of the microorganisms, emphasizing the requirement
for a better understanding of growth efficiencies of fungi and bacteria.
DOI: 10.1111/j.1574-6941.2007.00398.x
1702. Huan Jing Ke Xue. 2007 Sep;28(9):2080-4.
[Study on phytoremediation of phenanthrene-contaminated soil with alfalfa

(Medicago sativa L.)].
Fan SX(1), Li PJ, Gong ZQ, He N, Zhang LH, Ren WX, Verkhozina VA.
Author information:
China.
Pot experiment was used to investigate phytoremediation of

phenanthrene-contaminated soil with alfalfa (Medicago sativa L.). Results
indicated that phenanthrene had inhibitive effect on alfalfa growth, and higher
phenanthrene concentration seriously prevent alfalfa growth. When the
concentration was 445.22 mg/kg, the shoot and root biomasses were only 57.31% and
31.20% of control respectively. Alfalfa significantly promoted phenanthrene
degradation in the soil. After 60 days, 85.68%-91.40% and 75.25%-86.61% of spiked
phenanthrene disappeared from the rhizosphere and non-rhizosphere soils
respectively. And the average removal ratio of phenanthrene in rhizosphere soils
was 6.33% higher than that in non-rhizoshpere soils. The residual concentration
of phenanthrene in the rhizosphere was lower than that in the non-rhizosphere but
the dehydrogenase activity was on the contrary. With phenanthrene concentration
increase the removal ratio and dehydrogenase activity decreased. A positive
correlation was observed between the soil dehydrogenase activity and the removal
ratio of phenanthrene in both the rhizosphere and non-rhizosphere soils.
Therefore the presence of alfalfa roots was effective in promoting the
phytoremediation of phenanthrene.
1703. Anal Chem. 2007 Dec 15;79(24):9385-90. Epub 2007 Nov 7.
Method for determining molar concentrations of metabolites in complex solutions

from two-dimensional 1H-13C NMR spectra.
Lewis IA(1), Schommer SC, Hodis B, Robb KA, Tonelli M, Westler WM, Sussman MR,
Markley JL.
Author information:
(1)Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock
Drive, Madison, Wisconsin 53706, USA.
One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectroscopy is used

extensively for high-throughput analysis of metabolites in biological fluids and
tissue extracts. Typically, such spectra are treated as multivariate statistical
objects rather than as collections of quantifiable metabolites. We report here a
two-dimensional (2D) (1)H-(13)C NMR strategy (fast metabolite quantification,
FMQ, by NMR) for identifying and quantifying the approximately 40 most abundant
metabolites in biological samples. To validate this technique, we prepared
mixtures of synthetic compounds and extracts from Arabidopsis thaliana,
Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical
error 2.7%) molar concentrations can be determined in 12 min using our
quantitative 2D (1)H-(13)C NMR strategy. In contrast, traditional 1D (1)H NMR
analysis resulted in 16.2% technical error under nearly ideal conditions. We
propose FMQ by NMR as a practical alternative to 1D (1)H NMR for metabolomics
studies in which 50-mg (extract dry weight) samples can be obtained.
DOI: 10.1021/ac071583z
PMCID: PMC2533272
1704. Planta. 2008 Mar;227(4):769-79. Epub 2007 Nov 6.
A salt stress-responsive cytokinin receptor homologue isolated from Medicago

sativa nodules.
Coba de la Peña T(1), Cárcamo CB, Almonacid L, Zaballos A, Lucas MM, Balomenos D,
Pueyo JJ.
Author information:
(1)Department of Plant Physiology and Ecology, Instituto de Recursos Naturales,
Centro de Ciencias Medioambientales, CSIC, Serrano 115-bis, 28006 Madrid, Spain.
A new cytokinin receptor homologue, MsHK1, was isolated from Medicago sativa root
nodules. MsHK1 expression was induced in alfalfa seedlings by exogenous
application of the cytokinin trans-zeatin. Transcript accumulation was detected
in different plant organs. MsHK1 expression was induced by salt stress in alfalfa
roots, leaves and nodules, and transcript accumulation in the vascular bundles
pointed to a putative role in osmosensing for MsHK1 and/or other close cytokinin
receptor homologues. Expression in the meristem and the invasion zone of the
nodule suggest a role for cytokinin receptors in cytokinin sensing during nodule
cell division and differentiation.
DOI: 10.1007/s00425-007-0655-3
1705. Chemosphere. 2008 Feb;70(8):1383-9. Epub 2007 Nov 5.
Saprobe fungi decreased the sensitivity to the toxic effect of dry olive mill
residue on arbuscular mycorrhizal plants.
Sampedro I(1), Aranda E, Díaz R, García-Sanchez M, Ocampo JA, García-Romera I.
Author information:
Experimental del Zaidín, CSIC, Prof. Albareda 1, Apdo. 419, 18008 Granada, Spain.
We studied the influence of olive mill dry residue (DOR) treated with saprobe
fungi on growth of tomato and alfalfa colonized by Glomus deserticola. The
application of 25g kg(-1) of dry DOR to soil decreased the shoot and root dry
weight of tomato and alfalfa. Plants were more sensitive to the toxicity of DOR
when colonized with the arbuscular mycorrhizal (AM) fungi. The sensitivity of
both plants to the toxicity of DOR differed according to whether they were
colonized by G. deserticola or by indigenous AM fungi. The phytotoxicity of DOR
towards tomato and alfalfa was decreased by incubation the residue before
planting with saprobe fungi for 20wk. The beneficial effects of AM fungi on plant
growth added with DOR incubated with saprobe fungi depend of the type of the
plant and AM fungi. The contribution of AM fungi to the beneficial effect of DOR
incubated with saprobe fungi varied according to the type of the plant and AM
fungi. G. deserticola increased the shoot and root dry weight of plants when they
were grown in the presence of DOR incubated with saprobe fungi for 20wk. The
beneficial effect of saprobe fungi on the dry weight and the level of AM
mycorrhization of plants seem to be related to the decrease caused by these fungi
in the phenol concentration in DOR. However, the toxicity of DOR due to
substances other than phenols can not be ignored. The use of certain saprobe and
AM fungi allows the possibility of using DOR as an organic fertilizer.
1706. Biochem Biophys Res Commun. 2007 Dec 28;364(4):1056-61. Epub 2007 Oct 30.
Structure and alternative splicing of a heat shock transcription factor gene,

MsHSF1, in Medicago sativa.
He ZS(1), Xie R, Zou HS, Wang YZ, Zhu JB, Yu GQ.
Author information:
& Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, 300 Fenglin Road, Shanghai 200032, PR China.
Plant heat shock transcription factors (HSF) are highly complex. In this study,
we identified an alfalfa HSF gene MsHSF1 that is composed of four exons and three
introns in the encoding region. The intron1-exon2-intron2-exon3-intron3 as an
intervening sequence was inserted at the conserved position that separates the
coding region for the DNA-binding domain by single intron in other known plant
HSF genes. Alternative splicing of MsHSF1 has generated five transcript isoforms.
Spliced transcript MsHSF1b consisted of exon1 and exon4, encodes a class A1 HSF
protein that can specifically bind to the heat shock elements in vitro. Other
four spliced transcripts (MsHSF1a-1 to 4) consist of exon1, part of the
intervening sequence and exon4. These transcripts carry the premature termination
codon and are low-abundant. Apparently these transcripts are the targets of
nonsense-mediated mRNA decay (NMD). These results provide new insight into roles
in the expression regulation of plant HSF genes.
DOI: 10.1016/j.bbrc.2007.10.131
1707. Infect Immun. 2008 Jan;76(1):38-47. Epub 2007 Oct 29.
A LysR-type transcriptional regulator in Burkholderia cenocepacia influences

colony morphology and virulence.
Bernier SP(1), Nguyen DT, Sokol PA.
Author information:
(1)Department of Microbiology and Infectious Diseases, University of Calgary
Health Sciences Centre, 3330 Hospital Dr., N.W., Calgary, Alberta T2N 4N1,
Canada.
Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on

agar medium; however, shiny colony variants (shv) can appear spontaneously. These
shv all had a minimum of 50% reduction in biomass formation and were generally
avirulent in an alfalfa seedling infection model. Three shv-K56-2 S15, K56-2 S76,
and K56-2 S86-were analyzed for virulence in a chronic agar bead model of
respiratory infection and, although all shv were able to establish chronic
infection, they produced significantly less lung histopathology than the rough
K56-2. Transmission electron microscopy revealed that an extracellular matrix
surrounding bacterial cells was absent or reduced in the shv compared to the
rough wild type. Transposon mutagenesis was performed on the rough wild-type
strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a
putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm
production, increased N-acyl homoserine lactone production, and avirulence in
alfalfa. The rough parental colony morphotype, biofilm formation, and virulence
in alfalfa were restored by providing BCAS0225 in trans in the
BCAS0225::pGSVTp-luxCDABF mutant. Introduction of BCAS0225 restored the rough
morphotype in several shv which were determined to have spontaneous mutations in
this gene. In the present study, we show that the conversion from rough wild type
to shv in B. cenocepacia correlates with reduced biofilm formation and virulence,
and we determined that BCAS0225 is one gene involved in the regulation of these
phenotypes.
DOI: 10.1128/IAI.00874-07
PMCID: PMC2223663
1708. J Environ Sci (China). 2007;19(8):961-7.
Evaluation of phytoextracting cadmium and lead by sunflower, ricinus, alfalfa and

mustard in hydroponic culture.
Zhi-xin N(1), Sun LN, Sun TH, Li YS, Wang H.
Author information:
China. jesonniu@hotmail.com
Soil contaminated with heavy metals cadmium (Cd) and lead (Pb) is hard to be
remediated. Phytoremediation may be a feasible method to remove toxic metals from
soil, but there are few suitable plants which can hyperaccumulate metals. In this
study, Cd and Pb accumulation by four plants including sunflower (Helianthus
annuus L.), mustard (Brassica juncea L.), alfalfa (Medicago sativa L.), ricinus
(Ricinus communis L.) in hydroponic cultures was compared. Results showed that
these plants could phytoextract heavy metals, the ability of accumulation
differed with species, concentrations and categories of heavy metals. Values of
BCF (bioconcentration factor) and TF (translocation factor) indicated that four
species had dissimilar abilities of phytoextraction and transportation of heavy
metals. Changes on the biomass of plants, pH and Eh at different treatments
revealed that these four plants had distinct responses to Cd and Pb in cultures.
Measurements should be taken to improve the phytoremediation of sites
contaminated with heavy metals, such as pH and Eh regulations, and so forth.
1709. Plant Physiol. 2007 Dec;145(4):1619-28. Epub 2007 Oct 26.
Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene

encoding a Ca2+/calmodulin-dependent protein kinase.
Chen C(1), Gao M, Liu J, Zhu H.
Author information:
Kentucky 40546, USA.
In natural ecosystems, many plants are able to establish mutually beneficial

symbioses with microorganisms. Of critical importance to sustainable agriculture
are the symbioses formed between more than 80% of terrestrial plants and
arbuscular mycorrhizal (AM) fungi and between legumes and nitrogen-fixing
rhizobial bacteria. Interestingly, the two symbioses share overlapping signaling
pathways in legumes, suggesting that the evolutionarily recent root nodule
symbiosis may have acquired functions from the ancient AM symbiosis. The Medicago
truncatula DMI3 (DOESN'T MAKE INFECTIONS3) gene (MtDMI3) and its orthologs in
legumes are required for both bacterial and fungal symbioses. MtDMI3 encodes a
Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for the transduction
of the Ca(2+) signal induced by the perception of Nod factors. Putative orthologs
of MtDMI3 are also present in non-legumes, but their function in AM symbiosis has
not been demonstrated in any non-legume species. Here, we combine reverse genetic
approaches and a cross-species complementation test to characterize the function
of the rice (Oryza sativa) ortholog of MtDMI3, namely, OsDMI3, in AM symbiosis.
We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also
is able to complement a M. truncatula dmi3 mutant, indicating an equivalent role
of MtDMI3 orthologs in non-legumes.
DOI: 10.1104/pp.107.109876
PMCID: PMC2151686
1710. J Dairy Sci. 2007 Nov;90(11):5226-36.
Fibrolytic enzymes to increase the nutritive value of dairy feedstuffs.
Elwakeel EA(1), Titgemeyer EC, Johnson BJ, Armendariz CK, Shirley JE.
Author information:
66506-1600, USA.
In vitro studies and a lactation trial were conducted to investigate the effects
of fibrolytic enzyme mixtures at different inclusion amounts. Seven enzymes in
amounts designed to mimic addition of 1, 5, 15, or 30 g/d to dairy diets were
incubated in vitro with either soybean hulls or alfalfa for 24 or 48 h. Enzyme
treatments generally increased in vitro dry matter disappearance (IVDMD), but not
volatile fatty acid production. For some enzyme mixtures, lesser amounts of
enzymes led to greater increases in IVDMD, whereas for others there were no
differences among the amounts tested. The enzyme mixture with the most cellulase
activity was the most effective enzyme in improving IVDMD. In additional in vitro
experiments, the same enzymes were used at an amount of 5 g/d, as well as at
other amounts that showed promising responses in the first trial. Preincubation
of substrates with enzymes before fermentation also was tested. Alfalfa, soybean
hulls, corn silage, and corn gluten feed were used as substrates. Preincubation
of the substrate with enzymes for 18 h before in vitro fermentation improved
IVDMD. The effect on substrate solubilization of incubating substrates with the
enzymes but without rumen fluid was also studied. Addition of enzymes to
substrates without subsequent fermentation did not solubilize significant amounts
of dry matter, indicating that the positive effect of preincubation cannot be
attributed directly to hydrolysis of substrates before the in vitro fermentation
with ruminal microbes. The fibrolytic enzyme that appeared most promising in
vitro did not affect lactational performance when fed to dairy cows.
DOI: 10.3168/jds.2007-0305
1711. J Dairy Sci. 2007 Nov;90(11):5159-64.
Effect of application of ammonium chloride and calcium chloride on alfalfa

cation-anion content and yield.
Goff JP(1), Brummer EC, Henning SJ, Doorenbos RK, Horst RL.
Author information:
(1)Periparturient Diseases of Cattle Research Unit, National Animal Disease
Center, USDA-Agricultural Research Service, Ames, IA 50010-0070, USA.
jesseg@westcentral.net
A major factor predisposing the cow to periparturient hypocalcemia, or milk

fever, is being fed a prepartum ration with a high dietary cation-anion
difference (DCAD). The DCAD can be favorably altered to prevent milk fever by
decreasing K and Na or increasing Cl and S in forages for cows in late gestation.
The objective of this study was to test the hypothesis that application of Cl to
alfalfa could increase Cl in forage, thereby lowering DCAD. We conducted a field
experiment at 2 Iowa locations in which established plots of alfalfa were treated
in April 2001 with 0, 56, 112, or 168 kg of Cl/ha using ammonium chloride,
calcium chloride, or a mix of the 2 sources with equal amounts of chloride coming
from each source. Plots were harvested 4 times in 2001 and once in 2002 and plant
tissue analyzed for mineral composition. Applying chloride from either source
once in the spring resulted in increased plant chloride content over all 4
cuttings for that year. Averaged across both locations, chloride levels were
elevated from 0.52% in control plots to 0.77, 0.87, and 0.89% Cl in plots treated
with 56, 112, and 168 kg of Cl/ha, respectively. Chloride application had no
effect on plant potassium, sodium, calcium, magnesium, or phosphorus. These
results suggest chloride application can elevate chloride content and lower DCAD
values of alfalfa, and also maintain crop yield.
DOI: 10.3168/jds.2007-0070
1712. J Dairy Sci. 2007 Nov;90(11):5115-25.
Inoculant effects on alfalfa silage: in vitro gas and volatile fatty acid
production.
Muck RE(1), Filya I, Contreras-Govea FE.
Author information:
(1)USDA, Agricultural Research Service, US Dairy Forage Research Center, Madison,
WI 53706, USA. richard.muck@ars.usda.gov
Alfalfa silages from 2 similar trials were analyzed for in vitro ruminal gas
production. In both trials, there were 15 treatments: alfalfa treated at ensiling
with 1 of 14 lactic acid bacterial inoculants or untreated alfalfa. First-cut
(477 g of dry matter/kg) and second-cut (393 g of dry matter/kg) alfalfa were
ensiled in glass jars for a minimum of 35 d at room temperature (approximately 22
degrees C). At opening, a portion of each silage was wet-ground with a mixer.
Each silage was then assessed for in vitro ruminal gas production in 3 replicate
runs with the wet-ground silage, 1 on the fresh silage and 2 on frozen and thawed
silage. In vitro gas production was measured in 160-mL sealed serum vials
incubated at 39 degrees C. One gram of silage was incubated with 17.1 mL of
nutrient solution, 0.9 mL of reducing solution, and 12 mL of ruminal inoculum
(1:2 vol/vol mixture of rumen fluid and buffer). Gas production was measured
manually by using a pressure gauge at 3, 6, 9, 24, 48, and 96 h. At 96 h, the
rumen fluid was analyzed for pH and volatile fatty acids. In the 2 trials, the
untreated control silage produced either numerically the highest or one of the
highest levels of gas production per unit of dry matter incubated. In first-cut
silage, 9 of the inoculant treatments at 9 h and 4 treatments at 96 h had reduced
gas production compared with the control. In second-cut silage, 10 inoculant
treatments at both 9 and 96 h had reduced gas production compared with the
control. Furthermore, in first-cut silage, the fraction of total gas production
at 3, 6, and 9 h was numerically the highest for the control, and only 4
treatments were not significantly lower than the control at 9 h. In second-cut
silage, 2 of 14 inoculated treatments produced faster fractional rates of gas
production than the control, but most inoculated treatments had numerically
slower fractional rates (4 significant) in the first 9 h. The in vitro fermented
wet-ground control silages had one of the highest acetate:propionate ratios in
both trials, significantly higher than 12 and 8 of the inoculated treatments in
first- and second-cut silage, respectively. The response in acetate:propionate
ratio in both cuts was similar, even though the control silage was highest in
lactic acid in one trial and lowest in the other. Overall, inoculation of crops
at ensiling appears to affect in vitro ruminal fermentation of wet-ground
silages, even in the absence of large effects during silage fermentation.
DOI: 10.3168/jds.2006-878
1713. J Dairy Sci. 2007 Nov;90(11):5108-14.
Inoculant effects on alfalfa silage: fermentation products and nutritive value.
Filya I(1), Muck RE, Contreras-Govea FE.
Author information:
(1)Department of Animal Science, Faculty of Agriculture, Uludag University, 16059
Bursa, Turkey.
The effect of 14 microbial inoculants on the fermentation and nutritive value of

alfalfa silages was studied under laboratory conditions. The first cut (477 g of
dry matter/kg) and second cut (393 g of dry matter/kg) of a second-year alfalfa
stand were ensiled in 2 trials. In both trials alfalfa was harvested with
standard field equipment. All inoculants were applied at 1.0 x 10(6) cfu/g of
crop. Uninoculated silages served as controls. After inoculants were added, the
chopped forages were ensiled in 1.0- and 0.5-L anaerobic glass jars,
respectively, at a density of 500 g/L. Each trial had 15 treatments (uninoculated
control and 14 inoculants), with 4 silos per treatment. Silos were stored for a
minimum of 30 d at room temperature (approximately 22 degrees C). In first-cut
silage, all inoculants but one reduced pH relative to the uninoculated control,
and all but 2 of the homofermentative strains shifted fermentation toward lactic
acid. In second-cut silage, the epiphytic lactic acid bacterial population was
2.7 x 10(7) cfu/g, and only commercial inoculants produced significant shifts in
fermentation. Overall, microbial inoculants generally had a positive effect on
alfalfa silage characteristics in terms of lower pH and shifting fermentation
toward lactic acid with homofermentative lactic acid bacteria or toward acetic
acid with heterofermentative lactic acid bacteria, Lactobacillus buchneri. These
effects were stronger in the commercial products tested. In spite of the positive
effects on silage fermentation, 48-h in vitro true DM digestibility was not
improved by inoculation with lactic acid bacteria.
DOI: 10.3168/jds.2006-877
1714. J Appl Microbiol. 2007 Nov;103(5):1707-15.
Quality of silages from Italian farms as attested by number and identity of

microbial indicators.
Rossi F(1), Dellaglio F.
Author information:
(1)Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona,
Verona, Italy. rossi@sci.univr.it
AIMS: This study evaluated the quality and possible hygiene risks related to
farm-made silages by analysing the presence and number of micro-organisms that
influence the preservation and safety in samples from four Italian regions.
METHODS AND RESULTS: Lactic acid bacteria, clostridia, lactate-fermenting yeasts
and propionibacteria (PAB) were isolated and identified by random amplified
polymorphic DNA PCR, sequencing of the V2-V3 16S rRNA gene region, 5.8S-ITS rDNA
RFLP and species-specific PCR. The Lactobacillus plantarum cluster was the most
numerous and comprised strains mostly isolated from alfalfa silage. The
Lactobacillus buchneri cluster, second in number, comprised isolates from both
alfalfa and maize silage. Anaerobic spore formers were assigned to the species
Clostridium baratii, Clostridium beijerinkii, Clostridium butyricum, Clostridium
perfringens, Clostridium saccharolyticum, Clostridium tyrobutyricum and
Paenibacillus macerans. Yeast isolates were identified as Candida apicola,
Candida mesenterica and Pichia fermentans. PAB strains, detected only in unifeed,
were all identified as Propionibacterium acidipropionici.
CONCLUSIONS: The occurrence of spoiling micro-organisms was frequent and the
possibility of contamination by potentially pathogenic clostridia was
demonstrated.
SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest the need for improved
ensiling practices and appropriate control measures to safeguard the hygienic and
nutritional quality of silages produced in farms.
DOI: 10.1111/j.1365-2672.2007.03416.x
1715. Biochem Biophys Res Commun. 2007 Dec 14;364(2):325-31. Epub 2007 Oct 15.
Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase

receptors Toc159 and thylakoidal SRP.
Hernández Torres J(1), Maldonado MA, Chomilier J.
Author information:
(1)Laboratorio de Biología Molecular, CINBIN, Universidad Industrial de
Santander, Bucaramanga, Apartado Aéreo 678, Colombia. hernanj@uis.edu.co
The evolutionary origin of some nuclear encoded proteins that translocate

proteins across the chloroplast envelope remains unknown. Therefore, sequences of
GTPase proteins constituting the Arabidopsis thaliana translocon at the outer
membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In
particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do
not have proven homologues of prokaryotic or eukaryotic ancestry. We established
that the three domains commonly referred to as A, G, and M originate from the
GTPase G domain, tandemly repeated, and probably evolving toward an unstructured
conformation in the case of the A domain. It resulted from this study a putative
common ancestor for these proteins and a new domain definition, in particular the
splitting of A into three domains (A1, A2, and A3), has been proposed. The family
of Toc159, previously containing A. thaliana and Pisum sativum, has been extended
to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza
sativa. They have also been compared to GTPase subunits involved in the cpSRP
system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing
proteins of A. thaliana, and repetitions of a GTPase domain were also found in
cpSRP protein receptors, by means of HCA analysis.
DOI: 10.1016/j.bbrc.2007.10.006
1716. Environ Pollut. 2008 Jun;153(3):677-86. Epub 2007 Oct 18.
Environmental risks of farmed and barren alkaline coal ash landfills in Tuzla,
Bosnia and Herzegovina.
Dellantonio A(1), Fitz WJ, Custovic H, Repmann F, Schneider BU, Grünewald H,

Gruber V, Zgorelec Z, Zerem N, Carter C, Markovic M, Puschenreiter M, Wenzel WW.
Author information:
(1)University of Natural Resources and Applied Life Sciences, Vienna - BOKU,
Department of Forest and Soil Sciences, Peter Jordan Strasse 82, A-1190 Vienna,
Austria.
The disposal of coal combustion residues (CCR) has led to a significant

consumption of land in the West Balkan region. In Tuzla (Bosnia and Herzegovina)
we studied previously soil-covered (farmed) and barren CCR landfills including
management practises, field ageing of CCR and the transfer of trace elements into
crops, wild plants and wastewaters. Soil tillage resulted in mixing of cover soil
with CCR. Medicago sativa showed very low Cu:Mo ratios (1.25) which may cause
hypocuprosis in ruminants. Total loads of inorganic pollutants in the CCR
transport water, but not pH ( approximately 12), were below regulatory limits of
most EU countries. Arsenic concentrations in CCR transport water were
<2microgl(-1) whereas reductive conditions in an abandoned landfill significantly
enhanced concentrations in leachates (44microgl(-1)). The opposite pattern was
found for Cr likely due to large initial leaching of CrVI. Public use of
landfills, including farming, should be based on a prior risk assessment due to
the heterogeneity of CCR.
DOI: 10.1016/j.envpol.2007.08.032
1717. Guang Pu Xue Yu Guang Pu Fen Xi. 2007 Jul;27(7):1308-11.
[Quality prediction of alfalfa hay using Fourier transform near infrared

reflectance spectroscopy].
Nie ZD(1), Han JG, Yu Z, Zhang LD, Li JH, Zhong Y, Liu FY.
Author information:
(1)Institute of Grassland Science China Agricultural University, Beijing 100094,
China.
Alfalfa hay has high nutritive value, and it is one of the most important protein
feed for domestic animals. The quality parameters of alfalfa hay, including CP,
Ash, NDF, ADF, ADL and IVDMD, were predicted using Fourier transform near
infrared reflectance spectroscopy with PLS regression in this test. Then the 6
models were validated by cross-validation and external-validation. The results
indicated that FT-NIR models of alfalfa hay quality have considerable accuracy
and precision: the correlation coefficient of cross-validation is 0.953 88 to 0.
990 19, and the RMSECV is 1.980-0.345; The correlation coefficient of
external-validation is 0.963-0. 990. By using FT-NIR, analysis can rapidly and
accurately determine the quality of alfalfa without any chemical reagent. This
method is of great significance for analysing the trait of alfalfa production,
the quality determination, the estimation of germ plasm resource, and the
identifying and selecting of hybridized generations in alfalfa research of China.
1718. Guang Pu Xue Yu Guang Pu Fen Xi. 2007 Jul;27(7):1304-7.
[Evaluation of fresh sample of alfalfa silage through near infrared reflectance

spectroscopy (NIRS)].
Chen PF(1), Rong YP, Han JG, Wang JH, Zhang LD, Xu XJ.
Author information:
It is very important to evaluate the fresh sample of alfalfa silage using near
infrared reflectance spectroscopy technology (NIRS) for animal production. The
nutrient content of forage means the contents of dry matter (DM), crude protein
(CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) in the
forage. Because of the high moisture content, it is difficult to make uniform
samples for fresh forage and to get useful information from the spectrum.
Therefore, it is hard to use NIRS analysis. In order to evaluate the feasibility
of using NIRS to analyse the fresh alfalfa silage, the DM, CP, NDF and ADF
contents of fresh alfalfa silage were evaluated by the near infrared reflectance
spectroscopy model in this experiment using partial least square regression
(PLS), Fourier transform technology and sample preparation with liquid nitrogen
technology. The analysis samples were obtained through different cultivars,
maturity, cuttings and ensiling method. The cross validation was determined
between 0.884 6-0. 989 8. The standard error of cross validation was between 3.9
and 9.7 g x kg(-1) fresh weight. Fifty samples were used to test the performance
of the models. The coefficients of correlation between the chemical value and the
NIRS value are between 0.939 7 and 0.994 9, and the root mean square errors of
prediction are between 1.9 and 8.3 g x kg(-1) fresh weight. The results showed
that NIRS could be used to evaluate the nutrition of fresh forage.
1719. J Anim Sci. 2008 Jan;86(1):134-45. Epub 2007 Oct 16.
Effect of intake level and alfalfa substitution for grass hay on ruminal kinetics
of fiber digestion and particle passage in beef cattle.
Bhatti SA(1), Bowman JG, Firkins JL, Grove AV, Hunt CW.
Author information:
(1)Department of Animal & Range Sciences, Montana State University, Bozeman
59717, USA.
Two experiments were conducted to evaluate digestion kinetics of alfalfa

(Medicago sativa L.) substitution for grass hay in beef cattle. In Exp. 1, forage
combinations evaluated in situ consisted of 0% alfalfa-100% big bluestem
(Andropogon gerardi Vitman), 25% alfalfa-75% big bluestem, 50% alfalfa-50% big
bluestem, and 100% alfalfa-0% big bluestem. Nonlinear regression was used to
determine the immediately soluble fraction A, the potentially degradable fraction
B, the undegraded fraction C, and the disappearance rate of DM and NDF. Dry
matter fraction A increased linearly (P = 0.03), and DM and NDF fraction B
decreased linearly (P = 0.01) with increasing alfalfa substitution. Rate of DM
and NDF disappearance increased linearly (P </= 0.02) with increasing alfalfa
substitution. In Exp. 2, treatments were arranged as a 2 x 2 factorial testing
alfalfa substitution [none or 25% (as-fed basis)] to orchardgrass hay (Dactylis
glomerata L.) and intake level [restricted to 1% of BW daily (DM basis) or ad
libitum]. Nutrient intakes were lowest (P </= 0.05) by steers fed restricted
diets, intermediate by steers fed orchardgrass ad libitum, and greatest by steers
fed orchardgrass plus alfalfa ad libitum. Intake level and forage source had no
effect (P >/= 0.23) on total tract apparent digestibility of all nutrients except
CP. Steers fed orchardgrass plus alfalfa had 33% greater (P = 0.01) total tract
apparent digestibility for CP than those fed orchardgrass alone. Lag time of DM
and NDF disappearance was not affected (P >/= 0.20) by alfalfa supplementation or
intake level. Rate of DM and NDF disappearance of orchardgrass was faster (P </=
0.01) in steers fed orchardgrass plus alfalfa, at both restricted and ad libitum
levels of feeding, than in animals fed orchardgrass alone. Mean retention times
of large and small particles of orchardgrass tended to be shorter (P </= 0.06)
when steers consumed ad libitum vs. restricted diets. Small orchardgrass
particles tended to have a faster (P = 0.09) rate of passage under ad libitum
feeding conditions and with alfalfa addition. Ad libitum intake was associated
with a shorter mean retention time of orchardgrass and faster rate of passage of
small orchardgrass particles, whereas alfalfa addition increased the rate of
passage of small orchardgrass particles and the rate of DM and NDF disappearance.
DOI: 10.2527/jas.2006-693
1720. J Bacteriol. 2007 Dec;189(23):8741-5. Epub 2007 Oct 5.
H2O2 is required for optimal establishment of the Medicago sativa/Sinorhizobium

meliloti symbiosis.
Jamet A(1), Mandon K, Puppo A, Hérouart D.
Author information:
(1)Interactions Plantes-Microorganismes et Santé Végétale, UMR IPMSV INRA 1064 -
CNRS 6192, Université de Nice-Sophia Antipolis, Centre Sophia Agrobiotech, 400
route des chappes, BP 167, F-06903 Sophia Antipolis, France.
The symbiotic interaction between Medicago sativa and Sinorhizobium meliloti

RmkatB(++) overexpressing the housekeeping catalase katB is delayed, and this
delay is combined with an enlargement of infection threads. This result provides
evidence that H(2)O(2) is required for optimal progression of infection threads
through the root hairs and plant cell layers.
DOI: 10.1128/JB.01130-07
PMCID: PMC2168964
1721. Mol Microbiol. 2007 Nov;66(3):771-86. Epub 2007 Oct 1.
Glucosylceramide synthase is essential for alfalfa defensin-mediated growth

inhibition but not for pathogenicity of Fusarium graminearum.
Ramamoorthy V(1), Cahoon EB, Li J, Thokala M, Minto RE, Shah DM.
Author information:
(1)Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO
63132, USA.
Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth
of a fungal pathogen, Fusarium graminearum, at micromolar concentrations.
However, molecular mechanisms by which they inhibit the growth of this fungus are
not known. We have characterized a functional role of the fungal sphingolipid
glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A
null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a
mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to
MsDef1, but not to MtDef4. It shows a significant change in the conidial
morphology and displays dramatic polar growth defect, and its mycelia are
resistant to cell wall degrading enzymes. Contrary to its essential role in the
pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not
required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant
successfully colonizes wheat heads and corn silk, but its ability to spread in
these tissues is significantly reduced as compared with the wild-type PH-1
strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis
thaliana floral and foliar tissues. Based on our findings, we conclude that
glucosylceramide is essential for MsDef1-mediated growth inhibition of F.
graminearum, but its role in fungal pathogenesis is host-dependent.
DOI: 10.1111/j.1365-2958.2007.05955.x
1722. Chemosphere. 2008 Feb;70(8):1500-9. Epub 2007 Oct 1.
Biological detection and analysis of mercury toxicity to alfalfa (Medicago

sativa) plants.
Zhou ZS(1), Wang SJ, Yang ZM.
Author information:
(1)Department of Biochemistry and Molecular Biology, College of Life Science,
Nanjing Agricultural University, Nanjing 210095, China.
Mercury has become one of the major causes of toxic metal pollution in
agricultural lands. Accumulation of mercury by plants may disrupt many cellular
functions and block growth and development. To assess mercury toxicity, we
performed an experiment focusing on the responses of alfalfa (Medicago sativa) to
Hg(2+)-induced oxidative stress. Alfalfa plants were treated with 0-40microM
HgCl(2) for 7d. The concentrations of Hg(2+) were positively correlated with the
generation of O2- and H(2)O(2) in leaves. Treatment with Hg(2+) increased the
activities of NADH oxidase and lipoxygenase (LOX) and damaged the biomembrane
lipids. To understand biochemical responses under Hg stress, activities of
several antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD),
catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were
assayed. Analysis of SOD activity by non-denaturing polyacrylamide gel
electrophoresis revealed five isoforms in leaves, but they showed different
patterns. Also, eight isoenzymes of APX and seven of POD in leaves were detected.
However, only one isoform of CAT was visualized. The total activities of APX, POD
and CAT were generally enhanced. We also measured several antioxidative
metabolites such as ascorbate and glutathione (GSH), and found that both
differentially accumulated in leaves. These results indicate that the increased
levels of O2- and H(2)O(2) under Hg stress were closely linked to the improved
capacity of antioxidant enzymes. The data not only provide the important
information for better understanding of the toxic and tolerance mechanisms, but
as well can be used as a bio-indicator for soil contamination by Hg.
1723. Pak J Biol Sci. 2007 Oct 1;10(19):3314-21.
Identification of two types of Iranian alfalfa gene pool-based on

agro-morphological traits.
Abbasi MR(1), Vaezi S, Hemati F.
Author information:
(1)Department of Plant Genetics and Genetic Resources, Seed and Plant Improvement
Institute (SPII), Karaj, Iran.
There are two types of alfalfa landraces in Iran. The first is temperate
landraces such as Hamedani, Gharah Younjeh and the second is sub-tropical
landraces such as Bami, Nikshahri and Yazdi. Do they have different gene pools
and could they be identified using agro-morphological traits. A total of 332
landraces which had been collected from different parts of Iran were planted in
the experimental field. During 1998-2002, agro-morphological traits were
evaluated according to IPGRI descriptors. One way ANOVA analysis showed that the
variance among accessions within provinces is not significant for mostly of
traits, whereas it was significant among provinces. Cluster analysis revealed
that there are five clusters which are grouped in two main clusters. The first
main cluster consisted of landraces of central to Northern provinces. The other
main cluster consisted of landraces of central to Southern provinces. The results
of t-test among quantitative traits in two main clusters and distribution of
collected accessions of each province in the produced plot based on two first
factor of factor analysis, confirmed the cluster analysis results. Therefore
these results revealed that those accessions which were collected from Northern
parts of the country were genetically different from Southern accessions. As a
result, there are two types of gene pools for alfalfa landraces in Iran. Also
this study showed that using agro-morphological traits could be used for
distinguishing between two types of gene pools of alfalfa landraces in Iran.
1724. Fortune. 2007 Sep 3;156(5):108-12, 114, 116.
Flight of the honey bee.
Stipp D.
1725. DNA Seq. 2008 Jun;19(3):274-81.
Isolation and characterization of a cDNA encoding a papain-like cysteine protease

from alfalfa.
Yan L(1), Han J, Yang Q, Sun Y, Kang J, Liu Z, Wu M.
Author information:
(1)Institute of Grassland Sciences, China Agricultural University, Beijing,
Protein hydrolyzation is activated and involved in response to various stress

signals. In the present study, a full-length cDNA, named MsCP1, encoding a
papain-like cysteine protease was obtained by degenerated primers and 3'- and
5'-RACE from salt-tolerant alfalfa. The cDNA contained an open reading frame
encoding a deduced protein of 350 amino acids with a putative N-terminal signal
peptide, NPIR vacuole-sorting signal sequence and potential N-linked
glycosylation sites. The deduced sequence showed a high similarity to deduced
proteins from pea, tobacco, tomato and ryegrass. Fusion expression analysis in
Escherichia coli showed that the putative eukaryotic signal peptide prevented its
expression in prokaryotic system. The integration and transcript of the
expression elements in transgenic tobacco plants were detected with Southern blot
and RT-PCR analysis.
DOI: 10.1080/10253890701575166
1726. Environ Monit Assess. 2008 Sep;144(1-3):261-7. Epub 2007 Sep 22.
Vegetated ditches as a management practice in irrigated alfalfa.
Gill SL(1), Spurlock FC, Goh KS, Ganapathy C.
Author information:
(1)Environmental Monitoring Branch, California Department of Pesticide
Regulation, 1001 I Street, Sacramento, CA 95814, USA. sgill@cdpr.ca.gov
The organophosphate (OP) insecticides diazinon and chlorpyrifos have been

frequently detected in the San Joaquin River, California, USA. Irrigation tail
waters are a significant source of OP pesticides in the watershed. This study
tested several management practices for reducing offsite movement of chlorpyrifos
to surface water from flood irrigated alfalfa. Management practices evaluated
include (1) a constructed, vegetated irrigation tailwater return ditch and (2)
increased lag time between chlorpyrifos application and start of flood
irrigation. Chlorpyrifos concentrations in whole-water samples of irrigation
runoff were variable and ranged from 0.22 microg/l to a maximum of 1.67 microg/l.
The median concentration reduction at the end of a 200 m vegetated ditch was 38%
compared to 1% in an adjacent conventional tail water ditch. Runoff data
collected represented first flush runoff from sets that were irrigated between 48
and 144 h after chlorpyrifos application. There was no consistent effect of
irrigation lag time on chlorpyrifos concentration in tailwater for lag times of
up to 144 h. Consequently these data indicate that delayed irrigation is not an
effective management practice for reducing chlorpyrifos off-site movement to
surface water in California flood irrigated alfalfa.
DOI: 10.1007/s10661-007-9988-4
1727. J Dairy Sci. 2007 Oct;90(10):4793-804.
Effect of alfalfa silage storage structure and roasting corn on production and
ruminal metabolism of lactating dairy cows.
Krizsan SJ(1), Broderick GA, Muck RE, Promkot C, Colombini S, Randby AT.
Author information:
(1)Department of Animal and Aquacultural Sciences, Norwegian University of Life
Sciences, NO-1432 As, Norway. sophie.krizsan@umb.no
The objective of this study was to determine if feeding roasted corn would
improve production and nutrient utilization when supplemented to lactating cows
fed 1 of 3 different alfalfa silages (AS). Forty-two lactating Holstein cows (6
fitted with ruminal cannulas) averaging 77 d in milk and 43 kg of milk/d pretrial
were assigned to 2 cyclic changeover designs. Treatments were AS ensiled in bag,
bunker, or O2-limiting tower silos and supplemented with ground shelled corn
(GSC) or roasted GSC (RGSC). Silages were prepared from second-cutting alfalfa,
field-wilted an average of 24 h, and ensiled over 2 d. Production and N
utilization were evaluated in 36 cows during four 28-d periods, and ruminal
fermentation was evaluated with 6 cows during five 21-d periods. Experimental
diets contained 40% AS, 15% corn silage, and 35% of either GSC or RGSC on a dry
matter basis. No significant interactions between AS and corn sources were
detected for any production trait. Although the chemical composition of the 3 AS
was similar, feeding AS from the O2-limited tower silo elicited positive
production responses. Yields of 3.5% fat-corrected milk and fat were increased
1.7 kg/ d and 150 g/d, and milk fat content was increased 0.3% when cows were fed
diets based on AS from the O2-limiting silo compared with the other 2 silages.
The responses in milk fat were paralleled by an average increase in acid
detergent fiber digestibility of 270 g/d for cows fed AS from the O2-limiting
tower silo. However, ruminal concentrations of lipogenic volatile fatty acids
were unchanged with AS source. Cows fed RGSC consumed 0.6 kg/d more dry matter
and yielded 30 g/ d more protein and 50 g/d more lactose than cows fed GSC diets.
There was no evidence of increased total tract digestibility of organic matter or
starch, or reduced ruminal NH(3) concentration, when feeding RGSC. Free amino
acids increased, and isovalerate decreased in rumen fluid from cows fed RGSC
diets. However, responses in production with roasted corn were mainly due to
increased dry matter intake, which increased the supply of energy and nutrients
available for synthesis of milk and milk components.
DOI: 10.3168/jds.2006-816
1728. Mycorrhiza. 2007 Dec;18(1):1-14. doi: 10.1007/s00572-007-0147-0. Epub 2007

Sep
19.
The cultivation bias: different communities of arbuscular mycorrhizal fungi

detected in roots from the field, from bait plants transplanted to the field, and
from a greenhouse trap experiment.
Sýkorová Z(1), Ineichen K(1), Wiemken A(1), Redecker D(2).
Author information:
(1)Institute of Botany, University of Basel, Hebelstrasse 1, 4056, Basel,
Switzerland.
(2)Institute of Botany, University of Basel, Hebelstrasse 1, 4056, Basel,
Switzerland. dirk.redecker@unibas.ch.
The community composition of arbuscular mycorrhizal fungi (AMF) was investigated

in roots of four different plant species (Inula salicina, Medicago sativa,
Origanum vulgare, and Bromus erectus) sampled in (1) a plant species-rich
calcareous grassland, (2) a bait plant bioassay conducted directly in that
grassland, and (3) a greenhouse trap experiment using soil and a transplanted
whole plant from that grassland as inoculum. Roots were analyzed by AMF-specific
nested polymerase chain reaction, restriction fragment length polymorphism
screening, and sequence analyses of rDNA small subunit and internal transcribed
spacer regions. The AMF sequences were analyzed phylogenetically and used to
define monophyletic phylotypes. Overall, 16 phylotypes from several lineages of
AMF were detected. The community composition was strongly influenced by the
experimental approach, with additional influence of cultivation duration,
substrate, and host plant species in some experiments. Some fungal phylotypes,
e.g., GLOM-A3 (Glomus mosseae) and several members of Glomus group B, appeared
predominantly in the greenhouse experiment or in bait plants. Thus, these
phylotypes can be considered r strategists, rapidly colonizing uncolonized
ruderal habitats in early successional stages of the fungal community. In the
greenhouse experiment, for instance, G. mosseae was abundant after 3 months, but
could not be detected anymore after 10 months. In contrast, other phylotypes as
GLOM-A17 (G. badium) and GLOM-A16 were detected almost exclusively in roots
sampled from plants naturally growing in the grassland or from bait plants
exposed in the field, indicating that they preferentially occur in late
successional stages of fungal communities and thus represent the K strategy. The
only phylotype found with high frequency in all three experimental approaches was
GLOM A-1 (G. intraradices), which is known to be a generalist. These results
indicate that, in greenhouse trap experiments, it is difficult to establish a
root-colonizing AMF community reflecting the diversity of these fungi in the
field roots because fungal succession in such artificial systems may bias the
results. However, the field bait plant approach might be a convenient way to
study the influence of different environmental factors on AMF community
composition directly under the field conditions. For a better understanding of
the dynamics of AMF communities, it will be necessary to classify AMF phylotypes
and species according to their life history strategies.
DOI: 10.1007/s00572-007-0147-0
1729. Poult Sci. 2007 Oct;86(10):2101-9.
Molting in Salmonella Enteritidis-challenged laying hens fed alfalfa crumbles.

II. Fermentation and microbial ecology response.
Dunkley KD(1), McReynolds JL, Hume ME, Dunkley CS, Callaway TR, Kubena LF, Nisbet
DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station 77843,
USA.
The objective of this study was to examine microbial population shifts and
short-chain fatty acid (SCFA) responses in the gastrointestinal tract of
Salmonella Enteritidis-challenged molted and nonmolted hens fed different dietary
regimens. Fifteen Salmonella-free Single Comb Leghorn hens (>50 wk old) were
assigned to 3 treatment groups of 5 birds each based on diet in 2 trials: 100%
alfalfa crumbles (ALC), full-fed (FF, nonmolted) 100% commercial layer ration,
and feed withdrawal (FW). A forced molt was induced by either a 12-d alfalfa diet
or FW. In all treatment groups, each hen was challenged by crop gavage orally 4 d
after molt induction with a 1-mL inoculum containing 10(6) cfu of Salmonella
Enteritidis. Fecal and cecal samples (d 4, 6, 8, 11, and necropsy on d 12) were
collected postchallenge. Microbial population shifts were evaluated by PCR-based
16S ribosomal RNA gene amplification and denaturing gradient gel electrophoresis,
and SCFA concentrations were measured. Total SCFA in fecal and cecal contents for
FW molted hens were generally lower (P < or = 0.05) in the later stages of the
molt period when compared to ALC and FF treatment groups. The overall trend of
SCFA in cecal and fecal samples exhibited similar patterns. In trials 1 and 2,
hens molted with ALC diet generally yielded more similar amplicon band patterns
with the FF hens in both fecal and cecal samples by the end of the molting period
than with FW hens. The results of these studies suggest that ALC molted hens
supported microflora and fermentation activities, which were more comparable to
FF hens than FW hens by the end of the molting period.
DOI: 10.1093/ps/86.10.2101
1730. Environ Geochem Health. 2007 Dec;29(6):473-81. Epub 2007 Sep 12.
Increase of multi-metal tolerance of three leguminous plants by arbuscular

mycorrhizal fungi colonization.
Lin AJ(1), Zhang XH, Wong MH, Ye ZH, Lou LQ, Wang YS, Zhu YG.
Author information:
(1)Department of Environmental Science and Engineering, Beijing University of
Chemical Technology, Beijing, 100029, PR China.
A greenhouse pot experiment was conducted to investigate the effects of the

colonization of arbuscular mycorrhizal fungus (AMF) Glomus mosseae on the growth
and metal uptake of three leguminous plants (Sesbania rostrata, Sesbania
cannabina, Medicago sativa) grown in multi-metal contaminated soil. AMF
colonization increased the growth of the legumes, indicating that AMF
colonization increased the plant's resistance to heavy metals. It also
significantly stimulated the formation of root nodules and increased the N and P
uptake of all of the tested leguminous plants, which might be one of the
tolerance mechanisms conferred by AMF. Compared with the control, colonization by
G. mosseae decreased the concentration of metals, such as Cu, in the shoots of
the three legumes, indicating that the decreased heavy metals uptake and growth
dilution were induced by AMF treatment, thereby reducing the heavy metal toxicity
to the plants. The root/shoot ratios of Cu in the three legumes and Zn in M.
sativa were significantly increased (P<0.05) with AMF colonization, indicating
that heavy metals were immobilized by the mycorrhiza and the heavy metal
translocations to the shoot were decreased.
DOI: 10.1007/s10653-007-9116-y
1731. Transgenic Res. 2008 Aug;17(4):587-97. Epub 2007 Sep 13.
Development of an in planta method for transformation of alfalfa (Medicago

sativa).
Weeks JT(1), Ye J, Rommens CM.
Author information:
(1)J. R. Simplot Company, Simplot Plant Sciences, Boise, ID 83706, USA.
Conventional methods in transforming alfalfa (Medicago sativa) require multiple

tissue culture manipulations that are time-consuming and expensive, while
applicable only to a few highly regenerable genotypes. Here, we describe a simple
in planta method that makes it possible to transform a commercial variety without
employing selectable marker genes. Basically, young seedlings are cut at the
apical node, cold-treated, and vigorously vortexed in an Agrobacterium suspension
also containing sand. About 7% of treated seedlings produced progenies
segregating for the T-DNA. The vortex-mediated seedling transformation method was
applied to transform alfalfa with an all-native transfer DNA comprising a
silencing construct for the caffeic acid o-methyltransferase (Comt) gene.
Resulting intragenic plants accumulated reduced levels of the indigestible fiber
component lignin that lowers forage quality. The absence of both selectable
marker genes and other foreign genetic elements may expedite the governmental
approval process for quality-enhanced alfalfa.
DOI: 10.1007/s11248-007-9132-9
1732. Mol Plant Microbe Interact. 2007 Sep;20(9):1083-91.
The role of cellulose and O-antigen capsule in the colonization of plants by

Salmonella enterica.
Barak JD(1), Jahn CE, Gibson DL, Charkowski AO.
Author information:
(1)Produce Safety and Microbiology Research Unit, United States Department of
Agriculture-Agricultural Research Service, Albany, CA 94710, USA.
jbarak@pw.usda.gov
Numerous salmonellosis outbreaks have been associated with vegetables, in

particular sprouted seed. Thin aggregative fimbriae (Tafi), a component of the
extracellular matrix responsible for multicellular behavior, are important for
Salmonella enterica attachment and colonization of plants. Here, we demonstrate
that the other surface polymers composing the extracellular matrix, cellulose,
and O-antigen capsule also play a role in colonization of plants. Mutations in
bacterial cellulose synthesis (bcsA) and O-antigen capsule assembly and
translocation (yihO) reduced the ability to attach to and colonize alfalfa
sprouts. A colanic acid mutant was unaffected in plant attachment or
colonization. Tafi, cellulose synthesis, and O-antigen capsule, all of which
contribute to attachment and colonization of plants, are regulated by AgfD,
suggesting that AgfD is a key regulator for survival outside of hosts of
Salmonella spp. The cellulose biosynthesis regulator adrA mutant was not affected
in the ability to attach to or colonize plants; however, promoter probe assays
revealed expression by cells attached to alfalfa sprouts. Furthermore,
quantitative reverse-transcriptase polymerase chain reaction revealed
differential expression of agfD and adrA between planktonic and plant-attached
cells. In addition, there was no correlation among mutants between biofilm
formation in culture and attachment to plants. Outside of animal hosts, S.
enterica appears to rely on an arsenal of adhesins to persist on plants, which
can act as vectors and perpetuate public health concerns.
DOI: 10.1094/MPMI-20-9-1083
1733. Planta. 2008 Jan;227(2):331-9. Epub 2007 Sep 12.
Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth.
Allen A(1), Snyder AK, Preuss M, Nielsen EE, Shah DM, Smith TJ.
Author information:
(1)The Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO
63132, USA.
Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins

that are thought to constitute an important component of plant defense against
fungal pathogens. There are a number of such defensins expressed in various plant
tissues with differing antifungal activity and spectrum. Relatively little is
known about the modes of action and biological roles of these proteins. Our
previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and
subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated
that some of these proteins specifically blocked calcium channels in both fungi
and animals. The results presented here demonstrate that KP4 and three plant
defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating
Arabidopsis seeds at low micromolar concentrations. We have previously
demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced
yellow fluorescent protein (EYFP) is dependent upon calcium gradients for
localization to the tips of the growing root hairs in Arabidopsis thaliana. Using
this tip-localized fusion protein, we demonstrate that all four proteins rapidly
depolarize the growing root hair and block growth in a reversible manner. This
inhibitory activity on root and root hair is not directly correlated with the
antifungal activity of these proteins and suggests that plants apparently express
targets for these antifungal proteins. The data presented here suggest that plant
defensins may have roles in regulating plant growth and development.
DOI: 10.1007/s00425-007-0620-1
1734. Trop Anim Health Prod. 2007 May;39(4):265-70.
Effect of diet on the metabolic profile of ostriches (Struthio camelus var.

domesticus).
Bovera F(1), Moniello G, De Riu N, Di Meo C, Pinna W, Nizza A.
Author information:
(1)Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, Faculty of
Veterinary Medicine, University of Naples Federico II, via F. Delpino 1, 80137
Napoli, Italy. bovera@unina.it
In order to study the metabolic profile of ostriches in relation to diet, 40

animals of both sexes were divided equally into two groups and fed two diets ad
libitum consisting, on a dry matter basis, of the same commercial concentrate
(60%) for the two groups and of corn silage (group A) or alfalfa hay (group B).
In the morning, after about 12 h of fasting, blood was collected from the wing
vein. The following haematological parameters were determined with an automatic
system (Ektachem 250 analyser, Kodak): glucose, cholesterol, triglycerides,
lactate (LAC), total protein (TP), uric acid, total bilirubin (Tbil), creatinine
(CREA), calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), potassium (K),
chloride (Cl-), iron (Fe), aspartate aminotransferase (AST), alanine
aminotransferase (ALT), alkaline phosphatase (AP), cholinesterase (ChE),
alpha-amylase (Amyl), lipase (LIP) and gamma-glutamyltrasferase (GGT). Diet
significantly affected some parameters of the metabolic profile. Indeed, owing to
the presence of alfalfa hay in the diet, group B showed, in comparison to group
A, significantly higher values of uric acid (222.5 vs 387.5 mmol/L, p < 0.01),
GGT (8.50 vs 11.3 U/L, p < 0.05), Tbil (8.50 vs 10.7 mmol/L, p < 0.05), Ca (2.41
vs 2.83 micromol/L, p < 0.01), Mg (1.01 vs 1.18 micromol/L, p < 0.05) and K (2.71
vs 3.16 micromol/L, p < 0.01). The levels of creatinine (27.3 vs 32.6 mmol/L, p <
0.05) and AST (344.9 vs 461.4 U/l, p < 0.01) were also higher for group B.
1735. Biotechnol J. 2007 Oct;2(10):1250-62.
Biosynthesis of 5-deoxyflavanones in microorganisms.
Yan Y(1), Huang L, Koffas MA.

Author information:
(1)Department of Chemical and Biological Engineering, University at Buffalo,
State University of New York, Buffalo, NY 14260, USA.
Flavanones are the common precursors of plant polyphenolic compounds collectively

known as flavonoids. Leguminous plants have evolved a distinct class of flavanone
molecules, known as 5-deoxyflavanones that play important roles in their
symbiotic interactions. A four-step metabolic circuit was constructed in
Escherichia coli with plant genes from heterologous origins: 4-coumarate:coenzyme
A ligase from Petroselinum crispum, chalcone synthases (CHS) from Medicago sativa
and Petunia x hybrida and chalcone reductase and chalcone isomerase from M.
sativa. Evaluation of the different recombinant strains in shake flask
experiments demonstrated that P. hybrida rather than M. sativa CHS resulted in
the highest liquiritigenin production levels in glucose minimal medium, starting
from precursor p-coumaric acid. Expression of the same recombinant pathway in
Saccharomyces cerevisiae resulted in the accumulation of both 5-hydroxyflavanone
and 5-deoxyflavanone, with the yields of the later lower than that achieved in E.
coli. Other phenylpropanoid acid precursors, such as cinnamic acid and caffeic
acid could also be metabolized through the recombinant pathway, yielding
corresponding 5-deoxyflavanone compounds. The construction of such recombinant
strains for 5-deoxyflavanone biosynthesis offers an alternative way to
biochemically characterize flavonoid biosynthetic enzymes and promising
production platforms for the biosynthesis of such high-value natural products.
DOI: 10.1002/biot.200700119
1736. New Phytol. 2007;176(1):96-107.
Rapid alteration of cellular redox homeostasis upon exposure to cadmium and

mercury in alfalfa seedlings.
Ortega-Villasante C(1), Hernández LE, Rellán-Alvarez R, Del Campo FF,

Carpena-Ruiz RO.
Author information:
(1)Departamento de Biología, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa)

seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure,
expanding from a few minutes to 24 h, were studied. Intracellular oxidative
stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular
hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth
inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione
(hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes
related to GSH metabolism were also determined. Both Cd and Hg increased cellular
reactive oxygen species (ROS) production and extracellular H(2)O(2) formation,
but in different ways. The increase was mild and slow with Cd, but more rapid and
transient with Hg. Hg treatments also caused a higher cell death rate,
significant oxidation of hGSH, as well as increased APX activity and transient
overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and
homoglutathione synthetase genes. However, Cd caused minor alterations. Hg
accumulation was one order of magnitude higher than Cd accumulation. The
different kinetics of early physiological responses in vivo to Cd and Hg might be
relevant to the characterization of their mechanisms of toxicity. Thus, high
accumulation of Hg might explain the metabolism poisoning observed in Hg-treated
seedlings.
DOI: 10.1111/j.1469-8137.2007.02162.x
1737. Vet Rec. 2007 Sep 1;161(9):312-3.
Photosensitisation in sheep grazing alfalfa infested with aphids and ladybirds.
Ferrer LM(1), Ortín A, Loste A, Fernández A, Verde MT, Ramos JJ.
Author information:
(1)Department of Animal Pathology, Veterinary Faculty, University of Zaragoza,
Miguel Servet 177, 50013 Zaragoza, Spain.
[Effects of Ca2+ on acid tolerance of Medicago sativa and Rhizobium meliloti].
Zhang Q(1), Zhang L, Wei SQ, Li YB, Zhang C.
Author information:
(1)College of Resource and Environment, Southwest University, Chongqing 400716,
China. jhtabszq@sina.com
Soil acidity is an important environmental factor hampering the effective

symbiotic nitrogen fixation of Rhizobium meliloti and its host plant Medicag
sativa. The study on the effects of Ca2 + on the acid tolerance of M. sativa and
R. meliloti showed that applying 5 and 10 mmol . L(-1) of Ca2+ could promote the
growth of R. meliloti and advance its log growth phase. Under neutral pH
condition, applying Ca2+ didn't have any significant effects on root hair
deformation, while under low pH condition, Ca2+ demonstrated positive functions.
The higher the Ca2+ concentration, the more significantly it affected, indicating
that Ca2+ might play an important role in the recognition between R. meliloti and
its host plant. Applying Ca2+ under low pH made the nodulation ahead of time and
the nodulation rate enhanced. Definite concentration of Ca2+ could increase the
number of nodules in the same period, which was more obvious at anaphase stage or
under lower pH condition.
1739. Mol Plant Microbe Interact. 2007 Aug;20(8):986-93.
A thioredoxin of Sinorhizobium meliloti CE52G is required for melanin production

and symbiotic nitrogen fixation.
Castro-Sowinski S(1), Matan O, Bonafede P, Okon Y.
Author information:
(1)Department of Plant Pathology and Microbiology, The Otto Warburg Minerva
Center for Agricultural Biotechnology, The Hebrew University of Jerusalem,
Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot,
Israel. scs@iibce.edu.uy
A miniTn5-induced mutant of a melanin-producing strain of Sinorhizobium meliloti

(CE52G) that does not produce melanin was mapped to a gene identified as a
probable thioredoxin gene. It was proved that the thiol-reducing activity of the
mutant was affected. Addition to the growth medium of substrates that induce the
production of melanin (L-tyrosine, guaiacol, orcinol) increased the
thioredoxin-like (trxL) mRNA level in the wild-type strain. The mutant strain was
affected in the response to paraquat-induced oxidative stress, symbiotic nitrogen
fixation, and both laccase and tyrosinase activities. The importance of
thioredoxin in melanin production in bacteria, through the regulation of laccase
or tyrosinase activities, or both, by the redox state of structural or catalytic
SH groups, is discussed.
DOI: 10.1094/MPMI-20-8-0986
Small-scale spatial pattern of web-building spiders (Araneae) in alfalfa:

relationship to disturbance from cutting, prey availability, and intraguild
interactions.
Birkhofer K(1), Scheu S, Wise DH.
Author information:
(1)University of Technology Darmstadt, Department of Animal Ecology,
Schnittspahnstrasse 3, 64287 Darmstadt, Germany. Birkhofer@bio.uni-giessen.de
Understanding the development of spatial patterns in generalist predators will

improve our ability to incorporate them into biological control programs. We
studied the small-scale spatial patterns of spider webs in alfalfa by analyzing
the relationship between web locations over distances ranging from 4 to 66 cm.
Using a coordinate-based spatial statistic (O-ring) and assuming a heterogeneous
distribution of suitable web sites, we analyzed the impact of cutting and changes
in spider abundance on web distribution. We analyzed the influence of small-scale
variation in prey availability by comparing web distributions to the pattern of
sticky-trap captures of Aphididae and Diptera described by a count-based spatial
statistic (SADIE). Cutting of alfalfa reduced the overall density of web-building
spiders but had no immediate impact on the spatial distribution of their webs.
Availability of aphids was highest before the alfalfa was cut and was clumped at
a scale of 66 cm. Spider webs, however, were not clumped at any scale or date. In
contrast, webs were regularly distributed at smaller distances (<20 cm)
immediately before and after cutting. Because cursorial and web-building spiders
were most active during this period, we hypothesize that the development of
small-scale regularity in web locations was driven by intraguild interactions.
Our results suggest that intraguild interactions contribute to the development of
small-scale spatial patterns of spider webs in alfalfa. Variation in prey
availability may have more of an influence on web distribution in crops with a
different vegetation structure or if patterns are studied at larger spatial
scales.

Host plants of the tarnished plant bug (Heteroptera: Miridae) in Central Texas.
Esquivel JF(1), Mowery SV.
Author information:
(1)USDA-ARS, Areawide Pest Management Research Unit, 2771 F & B Road, College
Station, TX 77845, USA. zeus@usda-apmru.tamu.edu
The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), has taken on
added importance as a pest of cotton in the Cotton Belt after successful
eradication efforts for the boll weevil (Anthonomus grandis grandis Boheman).
Because the Southern Blacklands region of Central Texas is in advanced stages of
boll weevil eradication, blooming weeds and selected row crops were sampled
during a 3-yr study to determine lygus species composition and associated
temporal host plants. L. lineolaris was the sole lygus species in the region.
Thirteen previously unreported host plants were identified for L. lineolaris, of
which 69% supported reproduction. Rapistrum rugosum L. Allioni and Ratibida
columnifera (Nuttall) Wooton and Standley were primary weed hosts during the
early season (17 March to 31 May). Conyza canadensis L. Cronquist variety
canadensis and Ambrosia trifida L. were primary weed hosts during the midseason
(1 June to 14 August) and late-season (15 August to 30 November), respectively.
Sisymbrium irio L. and Lamium amplexicaule L. sustained L. lineolaris populations
during the overwintering period (1 December to 16 March). The proportion of
females and numbers of nymphs found in R. rugosum, C. canadensis, A. trifida, and
S. irio suggests these weeds supported reproductive adults during the early,
mid-, and late season and overwintering period, respectively. Medicago sativa L.
was the leading crop host for L. lineolaris; Glycine max L. Merrill did not yield
L. lineolaris. Few L. lineolaris were collected in Gossypium hirsutum L. These
results provide a more comprehensive assessment of host plants contributing to L.
lineolaris populations in central Texas.
1742. Poult Sci. 2007 Sep;86(9):1955-9.
Effect of polysavone (alfalfa extract) on abdominal fat deposition and immunity

in broiler chickens.
Dong XF(1), Gao WW, Tong JM, Jia HQ, Sa RN, Zhang Q.
Author information:
Academy of Agricultural Sciences, Beijing, China 100094.
Two hundred 1-day-old male commercial Arbor Acres broiler birds were randomly
distributed to a control group and a polysavone group (5 replicates of 20 birds
each) to investigate the influence of polysavone, a natural extract from alfalfa,
on abdominal fat deposition and immunity in broiler chickens. Birds in the
control group were supplied with a basal diet, and 0.06% polysavone was added to
the basal diet of birds in the polysavone group. Body weight and feed consumption
for each replicate were recorded weekly. At 3, 4, 5, and 6 wk of age, 4 birds
from each replicate were randomly selected for blood and organ sampling.
Polysavone had no significant effect on feed intake, BW, or feed:gain ratio in
the experimental period, and it decreased the abdominal fat weights at 5 and 6 wk
of age. Polysavone improved (P <0.05) the relative thymus and spleen weights at 6
wk of age and the bursa weights at 4 and 5 wk of age compared with the control
group. At 4 and 6 wk of age, the proliferation of T and B lymphocytes in the
polysavone group was significantly greater (P <0.05) than that in the control
group. When birds were 4 and 5 wk of age, polysavone resulted in a significant
increase (P <0.05) in serum anti-Newcastle disease virus hemagglutination
inhibition antibody titer. These results showed that polysavone may decrease
abdominal fat deposition and enhance immunity without an adverse effect on the
performance of broiler chickens.
DOI: 10.1093/ps/86.9.1955
1743. Poult Sci. 2007 Sep;86(9):1821-30.
Molt performance and bone density of cortical, medullary, and cancellous bone in
laying hens during feed restriction or alfalfa-based feed molt.
Kim WK(1), Donalson LM, Bloomfield SA, Hogan HA, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
77843, USA.
A study was conducted to evaluate the effects of alfalfa-based molt diets on

molting performance and bone qualities. A total of 36 Single Comb White Leghorn
hens were used for the study. There were 6 treatments: pretrial control (PC),
fully fed (FF), feed withdrawal (FW), 90% alfalfa:10% layer ration (A90), 80%
alfalfa:20% layer ration (A80), and 70% alfalfa:30% layer ration (A70). For the
PC treatment, hens were euthanized by CO(2) gas, and bones were collected before
molt was initiated. At the end of the 9-d molt period, hens were euthanized, and
femurs and tibias were collected to evaluate bone qualities by peripheral
quantitative computed tomography, mechanical testing, and conventional ash
weights. The hens fed alfalfa-based molt diets and FW stopped laying eggs within
5 d after molt started, and all hens in these groups had reduced ovary weights
compared with those of the FF hens. In the FW and A90 groups, total femur
volumetric bone mineral densities (vBMD) at the midshaft were significantly
lower, but those of the A80 and A70 groups were not significantly different from
the values for the PC and FF hens. In cortical bone density, the midshaft tibial
vBMD were significantly higher for FF and A70 hens than for PC hens. The
medullary bone densities at the midshaft femur or tibia of the FW, A90, A80, and
A70 hens were reduced compared with those of the PC hens. Femur cancellous
densities at the distal femur for the FW and A90 hens were significantly reduced
compared with those of the PC and FF hens. The FW, A80, and A70 hens yielded
significantly higher elastic moduli, and the A80 hens had higher ultimate stress
compared with the PC hens, suggesting that the mechanical integrity of the
midshaft bone was maintained even though the medullary vBMD was reduced. These
results suggest that alfalfa-based molt diets exhibit molt performance similar to
FW, that medullary and cancellous bones are labile bone compartments during
molting, and that alfalfa-based molt diets may be beneficial to maintain the
mechanical properties of bones during molt.
DOI: 10.1093/ps/86.9.1821
1744. J Dairy Sci. 2007 Sep;90(9):4356-60.
Manure nutrient excretion by lactating cows fed exogenous phytase and cellulase.
Knowlton KF(1), Taylor MS, Hill SR, Cobb C, Wilson KF.
Author information:
(1)Virginia Polytechnic Institute and State University, Blacksburg 24061, USA.
Knowlton@vt.edu
The effect of an exogenous phytase and cellulase-containing enzyme formulation on

nutrient digestibility and excretion was evaluated in 24 Holstein cows. Cows were
fed corn silage- and alfalfa silage-based diets with or without a
cellulase-phytase blend for 31 d in a continuous random design. Treatment groups
were balanced for parity, days in milk, and mature-equivalent projected milk
yield. Diets contained 37% forage, 18.3% crude protein, 35.4% neutral detergent
fiber, 18% acid detergent fiber, and 0.42% P (no supplemental P). Cows were fed
once daily in Calan doors and milked 2 times daily. Body weight and milk yield
were recorded at each milking. Milk samples were collected on d 28 to 31 at 8
consecutive milkings. On d 28 to 31, fecal grab samples were collected every 8 h,
with sampling times advanced by 2 h each day. Feces samples were pooled by cow.
Feed and feces samples were analyzed for acid detergent lignin (used as an
internal marker) and for N, P, neutral detergent fiber, and acid detergent fiber.
Days in milk were similar between treatments, and body weight and milk yield were
unaffected by treatment. Cows fed the enzyme formulation had reduced fecal dry
matter, neutral detergent fiber, and acid detergent fiber excretion and reduced
fecal excretion of N and P. Apparent digestibility of dry matter, acid detergent
fiber, neutral detergent fiber, and N tended to increase with the enzyme
formulation. Addition of an exogenous phytase and cellulase enzyme formulation to
diets for lactating cows reduced fecal nutrient excretion.
DOI: 10.3168/jds.2006-879
1745. J Food Prot. 2007 Jul;70(7):1698-703.
Thermal inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seeds.
Feng G(1), Churey JJ, Worobo RW.
Author information:
(1)Department of Food Science and Technology, Cornell University, Geneva, New
York 14456, USA.
Alfalfa seeds inoculated with five strains of Salmonella or Escherichia coli

O157:H7 were subjected to dry heat at 55 degrees C for up to 8 days. Five-log
reductions in Salmonella or E. coli O157:H7 on seeds were observed. No pathogens
were detected on the sprouted seeds, which were initially inoculated with ca. 2
log CFU/g of Salmonella or more than 8 log CFU/g of E. coli O157:H7. The
percentages of germination of the alfalfa seeds did not significantly decrease
after 6 days of heating at 55 degrees C. These results showed that heat treatment
of alfalfa seeds at 55 degrees C for up to 6 days was effective in enhancing the
safety of alfalfa sprouts without affecting germination significantly.
1746. Int J Syst Evol Microbiol. 2007 Aug;57(Pt 8):1704-10.
'Candidatus Phytoplasma lycopersici', a phytoplasma associated with 'hoja de

perejil' disease in Bolivia.
Arocha Y(1), Antesana O, Montellano E, Franco P, Plata G, Jones P.
Author information:
(1)National Centre for Animal and Plant Health (CENSA), Apdo 10, San José de Las
Lajas, Havana, Cuba. yaimaarocha@yahoo.es
New diseases known locally as 'hoja de perejil' of tomato (Lycopersicon

esculentum Mill) and 'brotes grandes' of potato (Solanum tuberosum L.) were first
recognized in surveys of production fields in Bolivia during 2000-2003. Alfalfa
(Medicago sativa) witches' broom and little leaf diseases of native weeds
Morrenia variegata and mora-mora (Serjania perulacea) were also identified near
to production fields. Phytoplasma aetiology was attributed to each of these
diseases following detection and initial identification of aster yellows group
(16SrI) phytoplasmas in all five diseased plant species. While potato, alfalfa
and mora-mora plants contained indistinguishable 16SrI-B strains, 'hoja de
perejil' (THP) and morrenia little leaf (MVLL)-associated phytoplasma strains
shared 97.5 % 16S rRNA gene sequence similarity with 'Candidatus Phytoplasma
asteris' and related strains and <95 % similarity with all other 'Candidatus
Phytoplasma' species. Phylogenetic analysis of 16S rRNA gene sequences indicated
that the THP and MVLL phytoplasmas represent a novel lineage within the aster
yellows (16SrI) group and, on the basis of unique 16S rRNA gene sequences, we
propose that THP and MVLL phytoplasmas represent 'Candidatus Phytoplasma
lycopersici', with THP as the reference strain.
DOI: 10.1099/ijs.0.64851-0
1747. Theor Appl Genet. 2007 Oct;115(6):859-67. Epub 2007 Aug 4.
Analyses of a multi-parent population derived from two diverse alfalfa

germplasms: testcross evaluations and phenotype-DNA associations.
Maureira-Butler IJ(1), Udall JA, Osborn TC.
Author information:
(1)Department of Agronomy, University of Wisconsin, Madison, WI 53706, USA.
imaureira@inia.cl
In a previous study, we showed that the genetic variation present in the Medicago
sativa subsp. sativa Peruvian and M. sativa subsp. falcata WISFAL germplasms
could be used to improve forage yields when favorable alleles were recombined and
used in hybrid combination with cultivated alfalfa. In this paper, we present
testcross forage yield and fall growth data for two seasons of a C0 population
generated after intermating the Peruvian x WISFAL population for several
generations. In addition, we conducted marker-trait association analysis as an
attempt to identify Peruvian and WISFAL genomics regions affecting the targeted
traits. Five and seven genomic regions were found significantly associated with
forage yield and fall growth, respectively. In the case of fall growth, alleles
from both accessions were positively associated with plant height. However, more
alleles from WISFAL were positively associated with forage yield than from
Peruvian. WISFAL is known for its winter hardiness and genomic regions with large
effects on winter survival may have masked the effect of forage yield from
Peruvian. The fact that most of the genomic regions discovered in this study have
been previously associated with traits involved in winter hardiness validates our
findings and suggests that associations between DNA fragments and agronomic
traits can be detected without the necessity of developing bi-parental mapping
populations.
DOI: 10.1007/s00122-007-0614-1
1748. Sci Total Environ. 2007 Oct 15;385(1-3):297-309. Epub 2007 Jul 27.
Groundwater nitrate following installation of a vegetated riparian buffer.
Yamada T(1), Logsdon SD, Tomer MD, Burkart MR.
Author information:
(1)National Institute of Public Health, 2-3-6, Minami, Wako, Saitama 351-0197
Japan. t-yamada@niph.go.jp
Substantial questions remain about the time required for groundwater nitrate to
be reduced below 10 mg L(-1) following establishment of vegetated riparian
buffers. The objective of this study was to document changes in groundwater
nitrate-nitrogen (NO3-N) concentrations that occurred within a few years of
planting a riparian buffer. In 2000 and 2001 a buffer was planted adjacent to a
first-order stream in the deep loess region of western Iowa with strips of walnut
and cottonwood trees, alfalfa and brome grass, and switch grass. Non-parametric
statistics showed significant declines in NO3-N concentrations in shallow
groundwater following buffer establishment, especially mid 2003 and later. The
dissolved oxygen generally was >5 mg L(-1) beneath the buffer, and neither NO3-N
nor DO changed significantly under a non-buffered control area. These short-term
changes in groundwater NO3-N provide evidence that vegetated riparian buffers may
yield local water-quality benefits within a few years of planting.
DOI: 10.1016/j.scitotenv.2007.06.035
1749. Rev Sci Instrum. 2007 Jul;78(7):073706.
Application of a charge-coupled device photon-counting technique to

three-dimensional element analysis of a plant seed (alfalfa) using a full-field
x-ray fluorescence imaging microscope.
Hoshino M(1), Ishino T, Namiki T, Yamada N, Watanabe N, Aoki S.
Author information:
(1)Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1
Tennoudai, Tsukuba, Ibaraki 305-8571, Japan. hoshino@aokilab.bk.tsukuba.ac.jp
A full-field x-ray fluorescence imaging microscope using a Wolter mirror was

constructed at Photon Factory BL3C2. White x rays from a bending magnet were used
to excite x-ray fluorescence and to enhance the x-ray fluorescence intensity. A
photon-counting method using a charge-coupled device was applied to obtain an
x-ray fluorescence spectrum at the image plane. The spatial distributions of some
specific atoms such as Fe and Zn were obtained from photon-counting calculations.
An energy resolution of 220 eV at the Fe Kalpha line was obtained from the x-ray
fluorescence spectrum by the photon-counting method. The newly developed
three-dimensional element mappings of the specific atoms were accomplished by the
photon-counting method and a reconstruction technique using computed tomography.
DOI: 10.1063/1.2756632
1750. J Genet. 2007 Apr;86(1):59-63.
Population genetic structure based on SSR markers in alfalfa (Medicago sativa L.)
from various regions contiguous to the centres of origin of the species.
Falahati-Anbaran M(1), Habashi AA, Esfahany M, Mohammadi SA, Ghareyazie B.
Author information:
(1)Agricultural Biotechnology Research Institute of North Region of Iran, Rasht
Ghazvin Road, Rasht 41635-4115, Iran. falahati_m@abrii.ac.ir
1751. Ying Yong Sheng Tai Xue Bao. 2007 May;18(5):990-6.
[Comparison of ecophysiological characteristics of seven plant species in

semiarid loess hilly-gully region].
Xu BC(1), Shan L, Li FM.
Author information:
Institute of Soil and Water Conservation, Chinese Academy of Sciences & Ministry
of Water Resource, Northwest A & F University, Yangling 712100, Shaanxi, China.
Bcxu@163.com
The diurnal course of photosynthetic rate, transpiration rate, and leaf water
potential (psi L) of five plant species in North Shaanxi loess hilly-gully region
were measured in dry seasons. Based on the daily maximum photosynthetic and
transpiration rates, daily total assimilation and transpiration, and diurnal
change characteristics of psi L, the test plants were classified into different
eco-adaptation types. Panicum virgatum L. had high photosynthetic rate, low
transpiration rate and high water use efficiency (WUE), and its drought
adaptation strategy was to delay dehydration by developing high psi L. Medicago
sativa had high photosynthetic and transpiration rates but low WUE, while
Lespedeza dahurica had low photosynthetic and transpiration rates and low WUE.
Their drought adaptation strategies were the same, namely, by increasing psi L
delay dehydration. Bothriochloa ischaemum had high photosynthetic rate, relative
high transpiration rate and medium WUE, and its drought-resistant strategy was to
decrease psi L to endure dehydration. Astragalus adsurgens had similar
characteristics in diurnal courses of photosynthesis with B. ischaemum, and its
drought adaption strategy was to delay dehydration by developing low psi psi L.
1752. J Bacteriol. 2007 Oct;189(19):7077-88. Epub 2007 Jul 20.
The ExpR/Sin quorum-sensing system controls succinoglycan production in

Glenn SA(1), Gurich N, Feeney MA, González JE.

Author information:
Sinorhizobium meliloti is a gram-negative soil bacterium capable of forming a

symbiotic nitrogen-fixing relationship with its plant host, Medicago sativa.
Various bacterially produced factors are essential for successful nodulation. For
example, at least one of two exopolysaccharides produced by S. meliloti
(succinoglycan or EPS II) is required for nodule invasion. Both of these polymers
are produced in high- and low-molecular-weight (HMW and LMW, respectively)
fractions; however, only the LMW forms of either succinoglycan or EPS II are
active in nodule invasion. The production of LMW succinoglycan can be generated
by direct synthesis or through the depolymerization of HMW products by the action
of two specific endoglycanases, ExsH and ExoK. Here, we show that the ExpR/Sin
quorum-sensing system in S. meliloti is involved in the regulation of genes
responsible for succinoglycan biosynthesis as well as in the production of LMW
succinoglycan. Therefore, quorum sensing, which has been shown to regulate the
production of EPS II, also plays an important role in succinoglycan biosynthesis.
DOI: 10.1128/JB.00906-07
PMCID: PMC2045190
1753. Nat Rev Microbiol. 2007 Aug;5(8):619-33.
How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model.
Jones KM(1), Kobayashi H, Davies BW, Taga ME, Walker GC.
Author information:
(1)Department of Biology, Massachusetts Institute of Technology, Building 68,
Room 633, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Nitrogen-fixing rhizobial bacteria and leguminous plants have evolved complex

signal exchange mechanisms that allow a specific bacterial species to induce its
host plant to form invasion structures through which the bacteria can enter the
plant root. Once the bacteria have been endocytosed within a host-membrane-bound
compartment by root cells, the bacteria differentiate into a new form that can
convert atmospheric nitrogen into ammonia. Bacterial differentiation and nitrogen
fixation are dependent on the microaerobic environment and other support factors
provided by the plant. In return, the plant receives nitrogen from the bacteria,
which allows it to grow in the absence of an external nitrogen source. Here, we
review recent discoveries about the mutual recognition process that allows the
model rhizobial symbiont Sinorhizobium meliloti to invade and differentiate
inside its host plant alfalfa (Medicago sativa) and the model host plant barrel
medic (Medicago truncatula).
DOI: 10.1038/nrmicro1705
PMCID: PMC2766523
1754. Bioresour Technol. 2008 May;99(8):3084-93. Epub 2007 Jul 16.
Profiling of a microbial community under confined conditions in a fed-batch

garbage decomposer by denaturing gradient gel electrophoresis.
Horisawa S(1), Sakuma Y, Nakamura Y, Doi S.

Author information:
(1)Department of Environmental Systems Engineering, Faculty of Engineering, Kochi
University of Technology, 185 Miyanokuchi, Tosayamada, Kami-gun, Kochi 782-8502,
Japan. horisawa.sakae@kochi-tech.ac.jp <horisawa.sakae@kochi-tech.ac.jp>
In order to determine the conditions for the maximum performance of a fed-batch

composting (FBC) reactor, polymerase chain reaction-denaturing gradient gel
electrophoresis (PCR-DGGE) was used to analyze the microbial communities
established under the confined conditions of moisture content and environmental
temperature. To evaluate the effects of microbial community structures on the
performance of FBC reactors, degradation experiments using small-scale reactors
and model waste were conducted under confined environmental conditions. A high
degradation rate was observed under a wide range of MC conditions (30-60%) and at
higher than usual temperatures (30-50 degrees C). The microbial communities that
formed in the experimental FBC reactors were analyzed by DGGE of PCR-amplified
16S rRNA genes. The DGGE banding patterns at the same level as the degradation
rates were similar even if the environmental conditions were different. Sequence
analysis of the DGGE bands revealed the primary microbes which act in the
reactor.
DOI: 10.1016/j.biortech.2007.05.054
1755. Pak J Biol Sci. 2007 Jul 15;10(14):2379-88.
De-inking sludge and phosphorus effects on growth and symbiotic dinitrogen

fixation in forage legumes.
Allahdadi I(1), Beauchamp CJ, Califour FP, Khalaj H, Labafi MR.
Author information:
(1)Department of Agronomy and Plant Breeding, Aburaihan Campus, University of
Tehran, Tehran, Iran.
The de-inking process produces a waste by-product, called de-inking paper sludge
(DS), that contains paper fibers, clay particles and inks and high carbon (C)
concentrations combined with low nitrogen (N) and phosphorus (P) concentrations.
The use of high rates of DS to increase the soil organic matter thus requires
provision of high rates of N and P for adequate plant growth. Using dinitrogen
(N2)-fixing forage legumes is an alternative to N fertilization under such
circumstances. In a greenhouse study, DS rates of 0, 50 or 100 Mg ha(-1) and five
rates of P (40, 80, 120, 160, or 200 kg P2O5 ha(-1)) were applied on two soil
types, a clay loam (Pintendre) and a silty clay loam (St-Augustin). Nitrogen
uptake and symbiotic N2 fixation (SNF) were estimated in alfalfa (Medicago sativa
L.), sweetclover (Melilotus officinalis L.) and red clover (Trifolium pratense
L.); Bromegrass (Bromus inermis L.) and alfalfa ineffective for N2 fixation were
used as the reference (non-N2 fixing) crops. Atmospheric N2 fixation was
estimated by natural abundance of 15N (815N). Under controlled conditions, high
rates of DS substantially reduced delta15N values, particularly with high rates
of P. In addition, N uptake of legumes generally increased with increased P
concentrations and it peaked with 120 or 160 kg P2O5 ha(-1). Correlated with the
trends observed with delta15N values and it peaked with 120 or 160 kg P2O5
ha(-1). Present results showed that under high rates of application of DS and
adequate P supply, forage legumes fixed more atmospheric N2. delta15N can be a
good indicator of SNF under the above-mentioned conditions.
1756. Poult Sci. 2007 Aug;86(8):1633-9.
Molting in Salmonella enteritidis-challenged laying hens fed alfalfa crumbles. I.

Salmonella enteritidis colonization and virulence gene hilA response.
Dunkley KD(1), McReynolds JL, Hume ME, Dunkley CS, Callaway TR, Kubena LF, Nisbet
DJ, Ricke SC.
Author information:
77843, USA.
The objectives of this study were to enumerate Salmonella enterica serovar

Enteritidis colonization in fecal, cecal, and internal organs, and to compare the
level of virulence gene expression (hilA) of experimentally challenged laying
hens fed different dietary molt-induction regimens. Twelve Salmonella-free Single
Comb Leghorn hens (>50 wk old) hens were randomly assigned to each of 6 treatment
groups designated based on diet in 2 trials: 1) feed withdrawal Salmonella
Enteritidis-positive (FW+), 2) fully fed Salmonella Enteritidis-positive (FF+),
3) 100% alfalfa crumble Salmonella Enteritidis-positive (ALC+), 4) feed
withdrawal Salmonella Enteritidis-negative, 5) fully fed Salmonella
Enteritidis-negative, and 6) 100% alfalfa crumble Salmonella
Enteritidis-negative. A forced molt was induced by a 12-d alfalfa diet and a
feed-withdrawal regimen. On d 4 of the molt, all hens in groups 1, 2, and 3 were
challenged by crop gavage with 1 mL of inocula containing approximately 10(6) cfu
of nalidixic acid- and novobiocin-resistant Salmonella Enteritidis (phage type
13A). At the conclusion of both trials, all hens were euthanized and Salmonella
Enteritidis colonization was enumerated in the cecal contents, liver, spleen, and
ovaries. In addition, fecal (d 4 and 8) and cecal samples (necropsy at d 12) were
collected postchallenge from treatment groups 1, 2, and 3 (Salmonella
Enteritidis-positive) to quantify hilA expression by PCR. In both trials, all
nonchallenged birds were Salmonella Enteritidis-negative; therefore, no further
analysis was done. In trial 1, a 2-fold reduction in Salmonella Enteritidis
colonization was observed in the ALC+ hens (log10 Salmonella Enteritidis of 1.99)
compared with the FW+ hens (log(10) Salmonella Enteritidis of 3.89). In trial 2,
a 4-fold reduction in Salmonella Enteritidis colonization was observed in the
ALC+ hens (log(10) Salmonella Enteritidis of 1.27) compared with the FW+ hens
(log(10) Salmonella Enteritidis of 5.12). In trial 2, Salmonella Enteritidis
colonization in spleens was higher (P <or= 0.05) in FW+ hens compared with ALC+
and FF+ hens. Relative expression of hilA was higher (P <or= 0.05) in FW+
compared with FF+ hens, whereas the FF+ and ALC+ groups were not different (P >
0.05). In trial 2, hilA expression in FW+ hens was higher (P <or= 0.05) for d 6,
11, and 12, respectively, when compared with ALC+ and FF+ hens. The results of
these studies support the concept that changes in the gastrointestinal tract
microenvironment, such as those created during feed deprivation, encourage
Salmonella Enteritidis virulence and susceptibility in molted hens.
DOI: 10.1093/ps/86.8.1633
1757. Waste Manag. 2008;28(8):1423-31. Epub 2007 Jul 10.
Impact of DOM from composted "alperujo" on soil structure, AM fungi, microbial

activity and growth of Medicago sativa.
Kohler J(1), Tortosa G, Cegarra J, Caravaca F, Roldán A.
Author information:
(1)CSIC-Centro de Edafología y Biología Aplicada del Segura., Department of Soil
and Water Conservation. P.O. Box 164, Campus de Espinardo, 30100 Murcia, Spain.
Water-soluble extracts from compost may represent an alternative nutrient and

organic matter source for crop production under drip irrigation. Dissolved
organic matter (DOM), extracted from composted "alperujo", the main by-product
from the Spanish olive oil industry, was applied to soil alone or in combination
with either Glomus intraradices Schenck and Smith or a mixture of G.
intraradices, Glomus deserticola (Trappe, Bloss. and Menge) and Glomus mosseae
(Nicol and Gerd.) Gerd. and Trappe. Response measurements included mycorrhizal
colonisation, nutrient uptake and growth of Medicago sativa and microbiological
and physical properties in the rhizosphere. Dissolved organic matter was added to
soil at concentrations of 0, 50, 100 or 300mgCkg(-1) substrate. During the four
months of the experiment, the plants were harvested three times. Both mycorrhizal
inoculation treatments significantly increased soil aggregate stability. Only the
mycorrhizal inoculations increased microbial biomass C and protease and
phosphatase activities and decreased water-soluble C, particularly the mixture of
arbuscular mycorrhizal fungi. At the third harvest, the greatest increase in
growth of M. sativa was observed in the inoculated plants with shoot biomass
being 38% greater than for plants grown in the soil amended with the highest dose
of DOM and 57% greater than for control plants. The addition of DOM was not
sufficient to restore soil structure and microbial activity and did not affect
the mycorrhizal development of introduced populations of arbuscular mycorrhizal
fungi, but, depending on the dose, its fertiliser efficiency for improving plant
growth was apparent.
DOI: 10.1016/j.wasman.2007.05.008
1758. Nat Biotechnol. 2007 Jul;25(7):746-8.
Loosening lignin's grip on biofuel production.
Chapple C(1), Ladisch M, Meilan R.
Author information:
(1)Department of Biochemistry, 175 S. University Street, West Lafayette, IN
47902-2063, USA. chapple@purdue.edu
Comment on
Nat Biotechnol. 2007 Jul;25(7):759-61.
DOI: 10.1038/nbt0707-746
1759. Prikl Biokhim Mikrobiol. 2007 May-Jun;43(3):318-24.
[The effect of combined and separate inoculation of alfalfa plants with

Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and
nitrogen-fixing activities].
Furina EK, Bonartseva GA.

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and
root nodule bacteria Sinorhizobium meliloti after combined and separate
inoculation of alfalfa seedlings on the background of mineral nitrogen applied at
various times were studied. It was demonstrated that exudates of the alfalfa
seedlings with the first pair of cotyledonary leaves already provide a high
activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate
was transformed into N2O when the binary preparation of these bacteria was used.
In an extended experiment (30 days), an active reduction of nitrates to N2O (11
micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all
of the experimental variants during the formation of legume-rhizobial and
associative symbioses and simultaneous introduction of nitrates and bacteria. The
most active enzyme fixation was observed in the case of a late (after 14 days)
application of nitrates in the variants with both separate inoculations and
inoculation with the binary preparation of A. lipoferum and S. meliloti.
Separation in time of the application of bacterial preparations and mineral
nitrogen assisted its preservation in all of the experimental variants. The
variant of alfalfa inoculation with the binary preparation of A. lipoferum and S.
meliloti and application of nitrates 2 weeks after inoculation was optimal for
active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification
activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied
developments aimed at the use of bacterial and mineral fertilizers for leguminous
plants.
1760. J Exp Bot. 2007;58(11):2799-810. Epub 2007 Jul 5.
Molecular cloning of a bifunctional beta-xylosidase/alpha-L-arabinosidase from

alfalfa roots: heterologous expression in Medicago truncatula and substrate
specificity of the purified enzyme.
Xiong JS(1), Balland-Vanney M, Xie ZP, Schultze M, Kondorosi A, Kondorosi E,

Staehelin C.
Author information:
(1)State Key Laboratory of Biocontrol, 135, Xingangxi Road, School of Life
Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China.
Glycoside hydrolases are often members of a multigene family, suggesting

individual roles for each isoenzyme. Various extracellular glycoside hydrolases
have an important but poorly understood function in remodelling the cell wall
during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa
(Medicago sativa L.) belonging to the glycoside hydrolase family 3
(beta-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected
in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the
control of the CaMV 35S promoter was expressed in the model legume Medicago
truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants
exhibited 5-8-fold increased activities towards three p-nitrophenyl (PNP)
glycosides, namely PNP-beta-D-xyloside, PNP-alpha-L-arabinofuranoside, and
PNP-alpha-L-arabinopyranoside. An antiserum raised against a synthetic peptide
recognized MsXyl1, which was processed to a 65 kDa form. To characterize the
substrate specificity of MsXyl1, the recombinant protein was purified from
transgenic M. truncatula leaves by concanavalin A and anion chromatography.
MsXyl1cleaved beta-1,4-linked D-xylo-oligosaccharides and alpha-1,5-linked
L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar
beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant
to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall
polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a
multifunctional
beta-xylosidase/alpha-L-arabinofuranosidase/alpha-L-arabinopyranosidase
implicated in cell wall turnover of arabinose and xylose, particularly in rapidly
growing root tips. Moreover, the findings of this study demonstrate that stable
transgenic M. truncatula plants serve as an excellent expression system for
purification and characterization of proteins.
DOI: 10.1093/jxb/erm133
Development of lucerne (Medicago sativa L.) treated with mineral fertilizer and
manure at optimal and water deficit conditions.
Vasileva V(1), Kostov O, Vasilev E.
Author information:
(1)Institute of Forage Crops, 89 General Vladimir Vazov, 5800 Pleven, Bulgaria.
A study on the effect of different rates of mineral fertilizer and manure on

yield parameters of lucerne under optimal and water deficit conditions was
carried out. Leached chernozem soil and lucerne cultivar Victoria were used. The
soil was treated with ammonium nitrate and fully matured cattle manure. The
plants were grown under optimum moisture content of 80% and 40% of field
capacity. The water deficit stress decreased top and root biomass by 11-75% and
3-29% at mineral and organic fertilization, respectively. The applied mineral and
organic N strongly depressed nodules development. Both mineral fertilizer and
organic manure at dose of 210 mg N kg(-1) soil completely inhibited the
appearance of nodules. Next to nitrogen, water deficit stress further inhibited
the development of nodules. Nitrogen fertilization increased seed productivity in
the two experimental moisture conditions. The water deficit stress decreased seed
productivity by 18 to 33% as compared to optimum conditions. The plant treatments
with manure were much more resistant to water deficit and recovering ability of
plants was faster as compared to treatments with mineral fertilizer. The
application of manure stimulates development of drought-stress tolerance in
lucerne. However, the results obtained can be considered for the soil type and
experimental conditions used.
1762. J Chem Ecol. 2007 Aug;33(8):1598-606. Epub 2007 Jul 4.
Deterrence and toxicity of plant saponins for the pea aphid acyrthosiphon pisum
harris.
Goławska S(1).
Author information:
(1)Department of Biochemistry and Molecular Biology, University of Podlasie, ul.
Prusa 12, 08-110 Siedlce, Poland. sylwia@ap.siedlce.pl
Plant saponins are widely distributed among plants and have a wide range of
biological properties. Three alfalfa saponins - zanhic acid tridesmoside, 3GlcA,
28AraRhaXyl medicagenic acid glycoside, and 3GlcA, 28AraRha medicagenic acid
glycoside - were tested for their settling inhibition effects on feeding behavior
of the aphid Acyrthosiphon pisum using the electrical penetration graph method.
Application of saponins to artificial diets affected the insects' probing
behavior. In general, saponins incorporated into sucrose-agarose gels
significantly reduced the number of aphid probes and extended their duration.
Lower saponin concentrations (50 ppm) extended aphid activity and corresponded to
phloem sap ingestion. In contrast, higher concentrations (100 ppm) strongly
reduced aphid ability to ingest phloem and xylem sap.
DOI: 10.1007/s10886-007-9333-y
1763. J Anim Sci. 2007 Nov;85(11):3054-61. Epub 2007 Jul 3.
A dose-response study relating the concentration of carotenoid pigments in blood

and reflectance spectrum characteristics of fat to carotenoid intake level in
sheep.
Dian PH(1), Chauveau-Duriot B, Prado IN, Prache S.
Author information:
(1)INRA, UR1213 Herbivores, Site de Theix, 63122 St-Genès-Champanelle, France.
Erratum in
J Anim Sci. 2008 Jan;86(1):235.
This study was conducted to describe the dose-response curve relating the
concentration of carotenoid pigments in plasma and reflectance spectrum
characteristics of fat to the carotenoid intake level in sheep, and to
investigate the extent to which incorporation of dehydrated alfalfa in the diet
affects the reliability of the discrimination between concentrate-fed and
pasture-fed lambs based on these measurements. In Exp. 1, 6 treatments were
compared in individually penned lambs: feeding 0, 250, 500, 750, 1,000, or 1,250
g/d of dehydrated alfalfa for 60 d before slaughter. Each treatment (T0 to
T1,250) consisted of 8 male Romanov x Berrichon lambs with an initial average BW
of 24.8 kg (SD 2.6). All lambs received straw for ad libitum intake and T0 to
T1,000 lambs received a concentrate free of green vegetative matter in amounts to
produce similar ADG in all treatments. In Exp. 2, 33 male Romanov x Berrichon
lambs grazed a natural pasture maintained in a leafy green vegetative stage for
at least 59 d before slaughter. Initial BW when turning out to pasture was 14.2
kg (SD 2.3). Plasma carotenoid concentration was measured at slaughter by
spectrophotometry. Reflectance spectrum, lightness, redness, and yellowness were
measured after 24 h of shrinkage in subcutaneous caudal and perirenal fat. The
spectra were translated to 0 reflectance at 510 nm, and the integral of the
translated spectrum was calculated between 450 and 510 nm (i.e., the range of
light absorption by carotenoids). Reflectance measurement was replicated 5 times,
from which we calculated the absolute value of the mean integral (AVMI). In Exp.
1, plasma carotenoid concentration at slaughter (PCCS) increased linearly with
mean daily carotenoid intake (P < 0.01). Both subcutaneous caudal and perirenal
fat AVMI increased linearly (P < 0.01) with mean daily carotenoid intake and
PCCS, the slopes of the regressions being greater for perirenal than for
subcutaneous caudal fat. The mean PCCS was greater for lambs of Exp. 2 than for
lambs on any treatment of Exp. 1 (P < 0.01). We established the dose-response
curves relating PCCS and AVMI of subcutaneous and perirenal fat to carotenoid
intake level. The combined use of PCCS and of perirenal fat AVMI enabled
discrimination of pasture-fed lambs of Exp. 2 from the lambs of Exp. 1 that
received up to 500 g/d of dehydrated alfalfa.
DOI: 10.2527/jas.2006-477
1764. Plant Physiol Biochem. 2007 Aug;45(8):535-41. Epub 2007 May 5.
Inhibition of the catalase activity from Phaseolus vulgaris and Medicago sativa
by sodium chloride.
Tejera García NA(1), Iribarne C, Palma F, Lluch C.
Author information:
Granada, Campus de Fuentenueva s/n, 18071 Granada, Spain. natejera@ugr.es
Changes in catalase activity during the development of the Rhizobium-legume

symbiosis as well as its response in salinized plants of Phaseolus vulgaris and
Medicago sativa, was studied. Besides, it was examined the behavior of the
enzyme, isolated from leaves and root nodules, during in vitro incubation with
NaCl doses. Nodule catalase activities of both legumes were assayed with several
enzyme inhibitors and also purified. Leaf catalase activity of Phaseolus vulgaris
and Medicago sativa decreased and increased respectively throughout the ontogeny,
but root nodule catalase kept a high and stable value. This last result suggests
that both legumes require the maintenance of high nodule catalase in
nitrogen-fixing nodules. Under salt stress conditions leaf and nodule catalase
activity decreased in both, grain and pasture legumes. Because catalase from leaf
of Medicago sativa and nodules of Phaseolus vulgaris were relatively sensitive to
NaCl during in vitro experiments, the detoxifying role of this enzyme for
H(2)O(2) should be limited in such conditions. Both catalases, from determinate
and indeterminate nodules, were affected neither by oxygen nor superoxide
radicals but showed a strong (Phaseolus vulgaris) or partial (Medicago sativa)
inhibition with dithiothreitol, dithionite and beta-mercaptoethanol. Besides,
cyanide was the most potent inhibitor of nodule catalases. Finally, catalases
partially purified by immobilized metal ion affinity chromatography migrated at
42 (Phaseolus vulgaris) and 46kDa (Medicago sativa) on SDS-PAGE, whereas native
forms on sephacryl S-300 columns exhibited a molecular mass of 59 and 48kDa
(Phaseolus vulgaris) and 88 and 53kDa (Medicago sativa).
DOI: 10.1016/j.plaphy.2007.04.008
1765. J Dairy Sci. 2007 Jul;90(7):3410-21.
Altering physically effective fiber intake through forage proportion and particle
length: digestion and milk production.
Yang WZ(1), Beauchemin KA.
Author information:
(1)Agriculture and Agri-Food Canada, Research Centre, Lethbridge, Alberta, T1J
4B1, Canada.
Intake of physically effective neutral detergent fiber (peNDF) of dairy cows was
altered by adjusting the proportion of forage in the diet and forage particle
length, and effects on nutrient intake, site and extent of digestion, microbial N
synthesis, and milk production were measured. The experiment was designed as a
triplicated 4 x 4 Latin square using 12 lactating dairy cows, with 4 that were
ruminally and duodenally cannulated, 4 that were ruminally cannulated, and 4 that
were intact. Thus, the site and extent of digestion, and microbial N synthesis
were measured in a single 4 x 4 Latin square. Treatments were arranged in a 2 x 2
factorial design; 2 forage particle lengths (FPL) of alfalfa silage (short and
long) were combined with low (35:65) and high (60:40) forage:concentrate (F:C)
ratios (dry matter basis). Dietary peNDF content was determined from the sum of
the proportion (dry matter basis) of dietary dry matter retained either on the 2
screens (8- and 19-mm) or on the 3 screens (1.18-, 8-, and 19-mm) of the Penn
State Particle Separator multiplied by the neutral detergent fiber content of the
diet. An increased F:C ratio reduced intakes of dry matter and starch by 9 and
46%, respectively, but increased intake of fiber from forage sources by 53%.
Digestibility of dry matter in the total tract was not affected, whereas total
digestion of fiber and N was improved by increasing the F:C ratio. Improved total
fiber digestion resulted from higher ruminal digestion, which was partially due
to a shift in starch digestion from the rumen to the intestine with the increased
F:C ratio. Actual milk yield was decreased but production of 4% fat-corrected
milk was similar between the low and high F:C diets because of increased milk fat
content. Increased FPL increased intake of peNDF, especially when the high F:C
diet was fed. However, nutrient intakes, N metabolism in the digestive tract, and
milk production were not affected. Digestibility of neutral detergent fiber in
the total tract was increased because of improved fiber digestion in the rumen
with increased FPL. These results indicate that feeding dairy cows a low F:C diet
is beneficial in terms of increasing feed intake, microbial N synthesis, and milk
production. However, low F:C diets do not maximize feed digestion and production
efficiency because of the effects of subacute ruminal acidosis. Increased FPL
improves fiber utilization with minimal effects on the digestion of other
nutrients and milk production. Increasing dietary peNDF, through an increased
proportion of forage or increased FPL, improves fiber digestion because of
improved rumen function.
DOI: 10.3168/jds.2006-818
1766. Nat Biotechnol. 2007 Jul;25(7):759-61. Epub 2007 Jun 17.
Lignin modification improves fermentable sugar yields for biofuel production.
Chen F(1), Dixon RA.
Author information:
Parkway, Ardmore, Oklahoma 73401, USA. fchen@noble.org
Comment in
Nat Biotechnol. 2007 Jul;25(7):746-8.
Recalcitrance to saccharification is a major limitation for conversion of

lignocellulosic biomass to ethanol. In stems of transgenic alfalfa lines
independently downregulated in each of six lignin biosynthetic enzymes,
recalcitrance to both acid pretreatment and enzymatic digestion is directly
proportional to lignin content. Some transgenics yield nearly twice as much sugar
from cell walls as wild-type plants. Lignin modification could bypass the need
for acid pretreatment and thereby facilitate bioprocess consolidation.
DOI: 10.1038/nbt1316
1767. Nutr Cancer. 2007;58(1):66-74.
Transgenic alfalfa that accumulates piceid

(trans-resveratrol-3-O-beta-D-glucopyranoside) requires the presence of
beta-glucosidase to inhibit the formation of aberrant crypt foci in the colon of
CF-1 mice.
Kineman BD(1), Au A, Paiva NL, Kaiser MS, Brummer EC, Birt DF.
Author information:
(1)Department of Food Science and Human Nutrition, Iowa State University, Ames,
IA 50011, USA.
Plants have been genetically enhanced to produce a number of products for

agricultural, industrial and pharmaceutical purposes. This technology could
potentially be applied to providing chemoprevention strategies to the general
population. Resveratrol (3,5,4'-trihydroxystilbene) is a compound that has been
shown to have protective activity against a number of cancers and could be an
ideal candidate for such an application. Alfalfa that was genetically modified to
express resveratrol-synthase was used as a model in applying biotechnological
approaches to cancer prevention. The transgenic alfalfa, which accumulates
resveratrol as a glucoside (piceid =
trans-resveratrol-3-O-Beta-D-glucopyranoside) (152 +/- 17.5 microg piceid/g dry
weight), was incorporated into a standard mouse diet at 20% of the diet by weight
and fed for 5 wk to 6-wk-old, female CF-1 mice (N = 17-30) that were injected
with a single dose of azoxymethane (5 mg/kg body weight). While the addition of
resveratrol-aglycone (20 mg/kg diet) to the basal diet reduced the number of
aberrant crypt foci/mouse, the transgenic alfalfa did not inhibit the number,
size, or multiplicity of aberrant crypt foci in the colon of the CF-1 mice
relative to control alfalfa which does not accumulate resveratrol-glucoside.
However, diets containing transgenic alfalfa with an exogenous Beta-glucosidase
(860 U/kg diet) did significantly inhibit the number of aberrant crypt foci in
the distal 2 cm of the colon of the mice relative to mice fed diets containing
the transgenic alfalfa without the enzyme (P < 0.05; Fisher's Combination of
p-values). The Beta-glucosidase alone appeared to have no effect on the
inhibition of aberrant crypt foci. These results suggest that piceid in
transgenic piceid-accumulating alfalfa was not bioavailable.
DOI: 10.1080/01635580701308208
1768. Environ Pollut. 2008 Feb;151(3):524-31. Epub 2007 Jun 11.
Influence of papermill sludge on growth of Medicago sativa, Festuca rubra and

Agropyron trachycaulum in gold mine tailings: a greenhouse study.
Green S(1), Renault S.
Author information:
(1)Department of Botany, University of Manitoba, Winnipeg, Manitoba, Canada.
A greenhouse study was undertaken to determine the suitability of adding

papermill sludge to neutral/alkaline gold mine tailings to improve the
establishment of Festuca rubra, Agropyron trachycaulum and Medicago sativa.
Festuca rubra root and shoot biomass and A. Trachycaulum shoot biomass were
increased with papermill sludge amendment. The addition of papermill sludge and
fertilizer drastically increased the shoot and root biomass of M. sativa (20-30
times) while A. trachycaulum and F. rubra showed a more moderate increase in
growth. Photosynthetic pigment content of the leaves was higher in papermill
sludge treatments than in the treatments without papermill sludge. The organic
carbon content, macro-aggregate content and field capacity of the gold mine
tailings were increased while the bulk density was decreased by the addition of
papermill sludge. This study suggests that addition of papermill sludge and
adequate fertilization can alleviate some of the adverse conditions of
neutral/alkaline gold mine tailings.
DOI: 10.1016/j.envpol.2007.04.016
1769. Environ Pollut. 2008 Feb;151(3):569-75. Epub 2007 Jun 8.
DDT uptake by arbuscular mycorrhizal alfalfa and depletion in soil as influenced

by soil application of a non-ionic surfactant.
Wu N(1), Zhang S, Huang H, Shan X, Christie P, Wang Y.
Author information:
Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing,
China.
A greenhouse pot experiment was conducted to investigate the colonization of

alfalfa roots by the arbuscular mycorrhizal (AM) fungus Glomus etunicatum and
application of the non-ionic surfactant Triton X-100 on DDT uptake by alfalfa and
depletion in soil. Mycorrhizal colonization led to an increase in the
accumulation of DDT in roots but a decrease in shoots. The combination of AM
inoculation and Triton X-100 application enhanced DDT uptake by both the roots
and shoots. Application of Triton X-100 gave much lower residual concentrations
of DDT in the bulk soil than in the rhizosphere soil or in the bulk soil without
Triton X-100. AM colonization significantly increased bacterial and fungal counts
and dehydrogenase activity in the rhizosphere soil. The combined AM inoculation
of plants and soil application of surfactant may have potential as a
biotechnological approach for the decontamination of soil polluted with DDT.
DOI: 10.1016/j.envpol.2007.04.005
1770. Proc Natl Acad Sci U S A. 2007 Jun 12;104(24):10282-7. Epub 2007 Jun 4.
Pesticides reduce symbiotic efficiency of nitrogen-fixing rhizobia and host

plants.
Fox JE(1), Gulledge J, Engelhaupt E, Burow ME, McLachlan JA.
Author information:
(1)Center for Ecology and Evolutionary Biology, University of Oregon, 335 Pacific
Hall, Eugene, OR 97403, USA.
Unprecedented agricultural intensification and increased crop yield will be

necessary to feed the burgeoning world population, whose global food demand is
projected to double in the next 50 years. Although grain production has doubled
in the past four decades, largely because of the widespread use of synthetic
nitrogenous fertilizers, pesticides, and irrigation promoted by the "Green
Revolution," this rate of increased agricultural output is unsustainable because
of declining crop yields and environmental impacts of modern agricultural
practices. The last 20 years have seen diminishing returns in crop yield in
response to increased application of fertilizers, which cannot be completely
explained by current ecological models. A common strategy to reduce dependence on
nitrogenous fertilizers is the production of leguminous crops, which fix
atmospheric nitrogen via symbiosis with nitrogen-fixing rhizobia bacteria, in
rotation with nonleguminous crops. Here we show previously undescribed in vivo
evidence that a subset of organochlorine pesticides, agrichemicals, and
environmental contaminants induces a symbiotic phenotype of inhibited or delayed
recruitment of rhizobia bacteria to host plant roots, fewer root nodules
produced, lower rates of nitrogenase activity, and a reduction in overall plant
yield at time of harvest. The environmental consequences of synthetic chemicals
compromising symbiotic nitrogen fixation are increased dependence on synthetic
nitrogenous fertilizer, reduced soil fertility, and unsustainable long-term crop
yields.
DOI: 10.1073/pnas.0611710104
PMCID: PMC1885820
1771. Am J Bot. 2007 Jun;94(6):912-25. doi: 10.3732/ajb.94.6.912.
Reaction tissue formation and stem tensile modulus properties in wild-type and
p-coumarate-3-hydroxylase downregulated lines of alfalfa, Medicago sativa
(Fabaceae).
Patten AM(1), Jourdes M, Brown EE, Laborie MP, Davin LB, Lewis NG.
Author information:
(1)The Institute of Biological Chemistry, Washington State University, Pullman,
Washington 99164-6340 USA;
To our knowledge, xylary reaction tissue has never been reported in a forage crop
species. Here we report the discovery of reaction tissue in a transgenic line of
Medicago sativa (pC3H, for the gene for p-coumarate-3-hydroxylase) with reduced
lignin content and in the wild-type (WT) line. Based on microscopy and
biomechanical testing of internodal alfalfa branch sections, the transgenic
(pC3H-I) line, relative to the WT (1) apparently formed more reaction tissue
containing gelatinous fibers with adjacent thick-walled fibers (presumed to be
"intermediate" tissue) more rapidly, (2) had more xylem tissue, and (3) had
comparable tensile dynamic modulus properties. These findings thus establish the
(limited) ability of this perennial angiosperm to form (inducible) reaction
tissue in a manner somewhat analogous to that of woody arborescent angiosperms.
The potential of effectuating reductions in lignin amounts in (woody) angiosperms
with increased formation of reaction (tension wood) tissue is discussed because
reaction tissues are often viewed as a deleterious trait in processing for many
agronomic/industrial applications, especially with the current interest in
biofuels.
DOI: 10.3732/ajb.94.6.912
PMID: 21636460
1772. Cell Mol Biol (Noisy-le-grand). 2007 May 15;53(3):64-73.
In vitro reconstitution of legumin (11S) mRNA and binding proteins as related to

post-transcriptional regulation of protein synthesis in developing alfalfa
embryos.
Pramanik SK(1), Williams AL, Bewley JD.
Author information:
(1)Department of Biology, Morgan State University, Baltimore, MD 21251, USA.
There is undetectable transcription of 11S storage protein (medicagin) mRNA by

nuclei isolated from pre-cotyledonary-stage somatic embryos of alfalfa (Medicago
sativa L). However, this message exists at steady-state levels in the embryos at
this stage of development without concomitant synthesis of the storage protein.
At the pre-cotyledonary stage, therefore, the transcriptional rate for 11S mRNA
is low; what message is transcribed is sequestered in the form of mRNP complexes
and is not recruited into polysomes in vivo (33). Both transcription (in vivo and
in isolated nuclei) and translation of the 11S mRNA are evident at the onset of
cotyledon development in somatic and zygotic embryos, reaching a maximum during
expansion of the cotyledons and then declining as the embryos mature.
Pre-cotyledonary-stage somatic embryos which do not utilize the 11S-mRNA in
polysomes lack certain mRNA-binding proteins (32, 36 and 38 kD) which are present
at later stages of development. These mRNA-binding proteins may be responsible
for the initiation of large polysome formation since they were exclusively
present in the translational extracts of cotyledonary somatic and zygotic embryos
in which there was no repression of storage protein synthesis. In contrast, the
pre-cotyledonary somatic embryos contained a different set of 11S-mRNA-binding
proteins (28, 50, 55, and 62 kD) whose presence in the cotyledonary stage embryos
was very rare or non-existent; these could be responsible for preventing
translation.
1773. In Silico Biol. 2006;6(6):607-20.
A database of simple sequence repeats from cereal and legume expressed sequence
tags mined in silico: survey and evaluation.
Jayashree B(1), Punna R, Prasad P, Bantte K, Hash CT, Chandra S, Hoisington DA,
Varshney RK.
Author information:
(1)Bioinformatics and Biometrics Unit, International Crops Research Institute for
Semi-Arid Tropics, Patancheru, Andhra Pradesh, India. b.jayashree@cgiar.org
Simple sequence repeats (SSRs) or microsatellites are an important class of

molecular markers for genome analysis and plant breeding applications. In this
paper, the SSR distributions within ESTs from the legumes soybean (Glycine max,
representing 135.86 Mb), medicago (Medicago truncatula, 121.1 Mb) and lotus
(Lotus japonicus, 45.4 Mb) have been studied relative to the distributions in
cereals such as sorghum (Sorghum bicolor, 98.9 Mb), rice (Oryza sativa, 143.9 Mb)
and maize (Zea mays, 183.7 Mb). The relative abundance, density, composition and
putative annotations of di-, tri-, tetra- and penta-nucleotide repeats have been
compared and SSR containing ESTs (SSR-ESTs) have been clustered to give a
non-redundant set of EST-SSRs, available in a database. Further, a subset of such
candidate EST-SSRs from sorghum have been tested for their ability to detect
polymorphism between Striga-susceptible, stay-green drought tolerant mapping
population parent 'E 36-1' and its Striga-resistant, non-stay-green counterpart
'N13'. Primer sets for 64% of the EST-SSRs tested produced a clear and specific
PCR product band and 34% of these detected scorable polymorphism between the N13
and E 36-1 parental lines. Over half of these markers have been genotyped on 94
RILs from the (N13 x E 36-1)-based mapping population, with 42 markers mapping
onto the ten sorghum linkage groups. This establishes the value of this database
as a resource of molecular markers for practical applications in cereal and
legume genetics and breeding. The primer pairs for non-redundant EST-SSRs have
been designed and are freely available through the database
(http://intranet.icrisat.org/gt1/ssr/ssrdatabase.html).
1774. J Dairy Sci. 2007 Jun;90(6):2839-51.
Enhancing in vitro degradation of alfalfa hay and corn silage using feed enzymes.
Eun JS(1), Beauchemin KA.
Author information:
4B1, Canada.
A series of in vitro fermentation experiments was performed to assess the effects

of 4 feed enzyme products (FE) that varied in enzymatic activities on the
degradation of alfalfa hay and corn silage. The FE contained a range of
endoglucanase, exoglucanase, xylanase, and protease activities, and a range of
dose rates (DR) was used. The objective of the study was to identify effective
formulations and optimum DR, and to establish if combining FE would further
improve fiber degradation. For alfalfa hay, quadratic increases in gas production
and degradation of dry matter (DM) and fiber were observed for all FE, with
maximum responses at low to medium DR. For corn silage, none of the FE increased
gas production or DM degradation, but all FE increased NDF degradation, with
optimum DR in the low to medium range. The proteolytic enzyme papain improved
fiber degradation of alfalfa hay and corn silage in a manner similar to that
observed for polysaccharidase FE. Among the polysaccharidase FE, added activities
of endoglucanase and exoglucanase were positively correlated with improvement in
neutral detergent fiber (NDF) degradability of corn silage, whereas only added
endoglucanase activity tended to be correlated with improvement in NDF
degradability of alfalfa hay. Combining effective polysaccharidase FE further
improved fiber degradation of both forages, with greater improvements for corn
silage. Combining polysaccharidase and proteolytic FE further improved NDF
degradation of corn silage, but not alfalfa hay. Combination treatments generally
resulted in additive effects with increases in fiber degradation equal to the sum
of the improvements for the individual enzyme components. Improved fiber
degradation of corn silage was associated with decreased acetate to propionate
ratios. Enzyme products that improve in vitro degradation of forages may have the
potential to improve lactational performance of dairy cows.
DOI: 10.3168/jds.2006-820
1775. J Dairy Sci. 2007 Jun;90(6):2826-38.
Altering physically effective fiber intake through forage proportion and particle
length: chewing and ruminal pH.
Yang WZ(1), Beauchemin KA.
Author information:
4B1, Canada.
Alfalfa silages varying in theoretical chop length and diets high and low in
forage proportion were used to evaluate whether increasing the physically
effective (pe) neutral detergent fiber (NDF) content of dairy cow diets reduces
the risk of acidosis. The experiment was designed as a replicated 4 x 4 Latin
square using 8 ruminally cannulated lactating dairy cows. Treatments were
arranged in a 2 x 2 factorial design; 2 forage particle lengths (FPL) of alfalfa
silage (short and long) were combined with low (35:65) and high (60:40)
forage:concentrate (F:C) ratios [dry matter (DM) basis]. Dietary peNDF content
(DM basis) was determined from the sum of the proportion of dietary DM retained
on either the 2 sieves (8 and 19 mm) or the 3 sieves (1.18, 8, and 19 mm) of the
Penn State Particle Separator multiplied by the NDF content of the diet. The
dietary peNDF contents ranged from 9.6 to 19.8% using 2 sieves, or from 28.6 to
34.0% using 3 sieves. Intake of peNDF was increased by increasing both the F:C
ratio and the FPL of the diets. However, F:C ratio and FPL affected chewing
activity differently; increasing F:C ratio increased chewing time but increasing
FPL only increased chewing when a high-forage diet was fed. Mean ruminal pH was
increased by 0.5 and 0.2 units with increasing F:C ratio and FPL, respectively.
Cows fed the low F:C diet had > 10 or 7 h daily in which ruminal pH was below 5.8
or 5.5, respectively, compared with 1.2 and 0.1 h for cows fed the high F:C ratio
diet. Increased F:C ratio reduced ruminal VFA concentration from 135 to 121 mM
but increased the acetate:propionate ratio from 1.82 to 3.13. Dietary peNDF
content when measured using 2 sieves was positively correlated to chewing time (r
= 0.61) and mean ruminal pH (r = 0.73), and negatively correlated to the time
that pH was below 5.8 or 5.5 (r = -0.46). This study shows that the risk of
ruminal acidosis is high for cows fed a low F:C diet, particularly when finely
chopped silage is used. Intake of dietary peNDF is a good indicator of ruminal pH
status of dairy cows. Increasing the proportion of forage in the diet helps
prevent ruminal acidosis through increased chewing time, a change in meal
patterns, and decreased ruminal acid production. Increasing FPL elevates ruminal
pH, but in low-forage diets increased FPL does not completely alleviate subacute
acidosis because the fermentability of the diet is high and changes in chewing
activity are marginal.
DOI: 10.3168/jds.2007-0032
1776. Plant Physiol Biochem. 2007 Jun-Jul;45(6-7):508-14. Epub 2007 Mar 28.
MsCYS1, a developmentally-regulated cystatin from alfalfa.
Rivard D(1), Girard C, Anguenot R, Vézina LP, Trépanier S, Michaud D.
Author information:
(1)CRH/INAF, Pavillon des Services (INAF), Université Laval, Québec, Québec G1K
7P4, Canada.
Several roles have been attributed to cystatins in plants, ranging from the
regulation of host [endogenous] cysteine proteases to the inhibition of
herbivorous pest [exogenous] proteases. We report here the cloning, expression
and functional characterization of a novel cystatin from alfalfa, Medicago sativa
L. The new sequence, isolated from a cDNA expression library prepared from young
leaves, encodes a protein, MsCYS1, with the typical inhibitory motifs of
cystatins, namely the central signature motif QxVxG, a GG doublet in the
N-terminal trunk, and a W residue in the C-terminal region, about 30 amino acids
distant from the central inhibitory motif. As shown by a protein-based
phylogenetic reconstruction, MsCYS1 is a close relative of other cystatins from
Fabaceae presumably involved in the regulation of endogenous proteases. This
cystatin is developmentally regulated in stems and leaves, and not induced by
stress signals including methyl jasmonate, known to activate cystatins involved
in plant defense. A recombinant form of MsCYS1 expressed in Escherichia coli was
shown to strongly inhibit alfalfa leaf cysteine proteases while showing weak
affinity for the digestive cysteine proteases of different herbivorous pests.
Overall, these observations suggest an endogenous protease regulatory role for
MsCYS1, possibly associated with the early development of stems and leaves.
DOI: 10.1016/j.plaphy.2007.03.028
1777. Can J Microbiol. 2007 Jan;53(1):106-15.
The effect of Penicillium fungi on plant growth and phosphorus mobilization in

neutral to alkaline soils from southern Australia.
Wakelin SA(1), Gupta VV, Harvey PR, Ryder MH.
Author information:
(1)Commmonwealth Scientific and Industrial Research Organisation Land and Water,
Glen Osmond, SA, Australia. Steven.Wakelin@csiro.au
The phosphate solubilizing fungi Penicillium radicum, Penicillium bilaiae (strain

RS7B-SD1), and an unidentified Penicillium sp. designated strain KC6-W2 were
tested for their ability to increase the growth and phosphorus (P) nutrition of
wheat, medic, and lentil in three soils of neutral to alkaline pH reaction. The
strongest plant growth promoting (PGP) strain was Penicillium sp. KC6-W2, which
stimulated significant increases in shoot growth and dry mass in seven of the
nine experiments conducted. Levels of PGP by Penicillium sp. KC6-W2 ranged from
6.6% to 19% and were associated with increased uptake of P to the shoot. The PGP
properties of Penicillium sp. KC6-W2 were evident on each of the three different
plant species and soil types, a level of reliability not observed in other
strains tested. Inoculation of seed with P. radicum increased lentil growth by
5.5% (P < 0.05) in soil from Tarlee but did not affect plant growth in the eight
other experiments. Inoculation of plant seed with P. bilaiae RS7B-SD1 resulted in
significant PGP in two of the nine experiments conducted. However, when
significant, stimulation of PGP by P. bilaiae RS7B-SD1 was strong and resulted in
increases in medic dry matter (19%) and lentil shoot dry matter (15%). A soil
microcosm experiment investigated the effect of Penicillium fungi on cycling of
soil P. Penicillium bilaiae RS7B-SD1 was the only fungus to significantly
increase HCO3-extractable P (23% increase; P < 0.05). Production of phosphatase
enzymes was not associated with increased HCO3-extractable P. Addition of carbon
in the form of ryegrass seed significantly increased microbial respiration and
movement of P to the microbial biomass (P < 0.05), but these parameters were
irrespective of Penicillium treatment. This work has established the potential
for use of Penicillium inoculants to increase plant growth on alkaline soils in
Australia. The role of Penicillium fungi in plant P uptake and soil P cycling
requires further exploration.
DOI: 10.1139/w06-109
1778. Ecol Appl. 2007 Apr;17(3):675-91.
Life-cycle assessment of net greenhouse-gas flux for bioenergy cropping systems.

Adler PR(1), Del Grosso SJ, Parton WJ.
Author information:
(1)Pasture Systems and Watershed Management Research Unit, United States
Department of Agriculture-Agricultural Research Service, University Park,
Pennsylvania 16802, USA. paul.adler@ars.usda.gov
Bioenergy cropping systems could help offset greenhouse gas emissions, but
quantifying that offset is complex. Bioenergy crops offset carbon dioxide
emissions by converting atmospheric CO2 to organic C in crop biomass and soil,
but they also emit nitrous oxide and vary in their effects on soil oxidation of
methane. Growing the crops requires energy (e.g., to operate farm machinery,
produce inputs such as fertilizer) and so does converting the harvested product
to usable fuels (feedstock conversion efficiency). The objective of this study
was to quantify all these factors to determine the net effect of several
bioenergy cropping systems on greenhouse-gas (GHG) emissions. We used the DAYCENT
biogeochemistry model to assess soil GHG fluxes and biomass yields for corn,
soybean, alfalfa, hybrid poplar, reed canarygrass, and switchgrass as bioenergy
crops in Pennsylvania, USA. DAYCENT results were combined with estimates of
fossil fuels used to provide farm inputs and operate agricultural machinery and
fossil-fuel offsets from biomass yields to calculate net GHG fluxes for each
cropping system considered. Displaced fossil fuel was the largest GHG sink,
followed by soil carbon sequestration. N20 emissions were the largest GHG source.
All cropping systems considered provided net GHG sinks, even when soil C was
assumed to reach a new steady state and C sequestration in soil was not counted.
Hybrid poplar and switchgrass provided the largest net GHG sinks, >200 g CO2e-C x
m(-2) x yr(-1) for biomass conversion to ethanol, and >400 g CO2e-C x m(-2) x
yr(-1) for biomass gasification for electricity generation. Compared with the
life cycle of gasoline and diesel, ethanol and biodiesel from corn rotations
reduced GHG emissions by approximately 40%, reed canarygrass by approximately
85%, and switchgrass and hybrid poplar by approximately 115%.
1779. Pak J Biol Sci. 2007 May 15;10(10):1713-7.
Acacia nilotica and Medicago sativa, suitable plants for agro-forestry in

southern coasts of Iran.
Emtehani MH(1), Tabari M.
Author information:
(1)Department of Rehabilitation of Desert and Arid Regions, Faculty of Natural
Resources, University of Yazd, Ira.
Habitats of the multipurpose tree, Acacia nilotica, were identified along the
coastline of the Persian Gulf and Oman Sea, south of Iran. Four sites were
randomly chosen and in each one, vegetation as well as climatic and soil
characteristics were studied. Likewise, biometry of Acacia trees was conducted
and compared in the sites. The fodder values in leaf and fruit of Acacia were
determined and compared with those of in foliage of Medicago sativa being managed
under an agro-forestry system. By analyzing data it was revealed that the biggest
trees were found in Dashteyari region and the smallest in Bamani region (both in
the Oman Sea coast). Values in most of nutritional elements were higher in
foliage of Medicago than in leaf and fruit of Acacia, respectively. From this
investigation it is concluded that in south of Iran where the site is favorable
for Acacia plantation, cultivation of Medicago or other adaptable crops together
with Acacia can be developed as agroforestry systems (such as undercropping and
intercropping) if water is available.
1780. Pak J Biol Sci. 2007 May 15;10(10):1639-42.
Allelopathic effects of Medicago sativa L. and Vicia cracca L. leaf and root
extracts on weeds.
Koloren O(1).
Author information:
(1)Department of Plant Protection, Agricultural Faculty, Cukurova University,
01330 Adana, Turkey.
In this study, the allelopathic potential of different concentration (5, 25 and

50%) of M. sativa and V. cracca leaf and root extracts were evaluated on
germination and radicle length of four weed species (Amaranthus retroflexus L.,
Lolium perenne L., Ipomoea hederacea L. and Portulaca oleracea L.) in laboratory
condition. As a result, germination and radicle length of all species were
reduced by the extract from M. sativa and V. cracca leaf and root at different
percentage. Increasing the water extract concentrations from 5 to 50% of test
plants parts significantly increased the inhibition of all weed species
germination and radicle length.
1781. Genetika. 2007 Mar;43(3):323-32.
[TolC mutant of Sinorhizobium meliloti strain SKHM1-188 fails to establish

effective symbiosis with alfalfa].
Zatovskaia TV, Sharypova LA, Seliverstova EV, Simarov BV.
The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5
mutagenesis in the effective strain SKhM1-188. The mutant did not produce
fluorescent halos in UV light on the LB medium containing calcofluor white, which
suggests that modification occurred in the production of exopolysaccharide EPS1.
Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS
media, and this was most likely connected with the absence of the second
exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis
with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula
white round Fix- nodules or nodules of irregular shape. These nodules possessed
the structure usually described for nodules of EPS1 mutants. According to the
data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63
contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome.
This gene encodes the protein sharing homology with the TolC protein, a component
of a type I secretion system responsible for the export of protein toxins and
proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase
of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to
be exported by the type I secretion system, was tested in cultural supernatants
of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was
ascertained that secretory proteins ExsH and ExpE1 are absent in the culture
medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved
in the excretion of proteins ExsH and ExpE1.
1782. Plant Physiol. 2007 Jun;144(2):1104-14. Epub 2007 Apr 27.
The response of carbon metabolism and antioxidant defenses of alfalfa nodules to

drought stress and to the subsequent recovery of plants.
Naya L(1), Ladrera R, Ramos J, González EM, Arrese-Igor C, Minchin FR, Becana M.
Author information:
(1)Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo
Superior de Investigaciones Científicas, 50080 Zaragoza, Spain.
Alfalfa (Medicago sativa) plants were exposed to drought to examine the

involvement of carbon metabolism and oxidative stress in the decline of
nitrogenase (N(2)ase) activity. Exposure of plants to a moderate drought (leaf
water potential of -1.3 MPa) had no effect on sucrose (Suc) synthase (SS)
activity, but caused inhibition of N(2)ase activity (-43%), accumulation of
succinate (+36%) and Suc (+58%), and up-regulation of genes encoding cytosolic
CuZn-superoxide dismutase (SOD), plastid FeSOD, cytosolic glutathione reductase,
and bacterial MnSOD and catalases B and C. Intensification of stress (-2.1 MPa)
decreased N(2)ase (-82%) and SS (-30%) activities and increased malate (+40%),
succinate (+68%), and Suc (+435%). There was also up-regulation (mRNA) of
cytosolic ascorbate peroxidase and down-regulation (mRNA) of SS, homoglutathione
synthetase, and bacterial catalase A. Drought stress did not affect nifH mRNA
level or leghemoglobin expression, but decreased MoFe- and Fe-proteins.
Rewatering of plants led to a partial recovery of the activity (75%) and proteins
(>64%) of N(2)ase, a complete recovery of Suc, and a decrease of malate (-48%)
relative to control. The increase in O(2) diffusion resistance, the decrease in
N(2)ase-linked respiration and N(2)ase proteins, the accumulation of respiratory
substrates and oxidized lipids and proteins, and the up-regulation of antioxidant
genes reveal that bacteroids have their respiratory activity impaired and that
oxidative stress occurs in nodules under drought conditions prior to any
detectable effect on SS or leghemoglobin. We conclude that a limitation in
metabolic capacity of bacteroids and oxidative damage of cellular components are
contributing factors to the inhibition of N(2)ase activity in alfalfa nodules.
DOI: 10.1104/pp.107.099648
PMCID: PMC1914205
1783. Phytochemistry. 2007 Jun;68(11):1521-9. Epub 2007 Apr 26.
Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase

in transgenic alfalfa affects lignification, development and forage quality.
Shadle G(1), Chen F, Srinivasa Reddy MS, Jackson L, Nakashima J, Dixon RA.
Author information:
Erratum in
Phytochemistry. 2007 Jul;68(14):2023.
The recently discovered enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl

transferase (HCT) catalyzes the reactions both immediately preceding and
following the insertion of the 3-hydroxyl group into monolignol precursors. A
number of independent transgenic lines of alfalfa (Medicago sativa L.) were
generated in which the levels of HCT were reduced through antisense HCT
expression under control of the bean PAL2 promoter which is preferentially
expressed in vascular tissue. Reduction of enzyme activity in these lines was
from at least 15-50%. The most severely down-regulated lines exhibited
significant stunting, reduction of biomass and delayed flowering. HCT
down-regulation resulted in strongly reduced lignin content and striking changes
in lignin monomer composition, with predominant deposition of 4-hydroxyphenyl
units in the lignin. Vascular structure was impaired in the most strongly
down-regulated lines. Analysis of forage quality parameters showed strong
reductions of neutral- and acid-detergent fiber in the down-regulated lines, in
parallel with large increases (up to 20%) in dry matter forage digestibility.
Although manipulation of lignin biosynthesis can greatly improve forage
digestibility, accompanying effects on plant development need to be better
understood.
DOI: 10.1016/j.phytochem.2007.03.022
1784. FEMS Microbiol Lett. 2007 Jun;271(2):297-309. Epub 2007 Apr 27.
Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a

dominant Sinorhizobium meliloti strain identified during a long-term field
release experiment.
Stiens M(1), Schneiker S, Pühler A, Schlüter A.
Author information:
(1)Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach
100131, D-33501 Bielefeld, Germany.
The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11,
belonging to a dominant indigenous S. meliloti subpopulation identified during a
long-term field release experiment, was sequenced. This plasmid has 166 coding
sequences (CDSs), 42% of which encode proteins with homology to proteins of known
function. Plasmid pSmeSM11b is a member of the repABC replicon family and
contains a large gene region coding for a conjugation system similar to that of
other self-transmissible plasmids in Rhizobium and Agrobacterium. Another
pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide
catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the
genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b
encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia
CcI3, and which are likely to be involved in the synthesis of a secondary
metabolite. Several ORFs of pSmeSM11b were predicted to play a role in
nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic
elements, which contribute to the mosaic composition of the plasmid.
DOI: 10.1111/j.1574-6968.2007.00731.x
1785. Pak J Biol Sci. 2007 May 1;10(9):1513-8.

Alterations in nitrogen metabolites after putrescine treatment in alfalfa under
drought stress.
Zeid IM(1), Shedeed ZA.
Author information:
(1)Department of Botany, Faculty of Science, Helwan University, Cairo 11792,
Egypt.
Alfalfa (Medicago sativa, Siwa 1) seeds were subjected to drought stress during
germination by using polyethylene glycol (PEG 4000) for studying the changes in
some enzyme activities involved in nitrogen metabolism and the content of
nitrogenous compounds during the first four days of growth after putrescine (Put)
treatment. Decreasing the external water potential reduced activities of
glutamate-pyruvate transferase (GPT), glutamate-oxaloacetate transferase (GOT)
and RNase. Some free amino acids such as proline and glycine increased, while
alanine and aspartic acid decreased. Nucleic acids content also decreased.
Polyamines e.g., spermidine (Spd) and spermine (Spm) increased at the water
potential -0.4 MPa. Put treatment increased activities of GOT, GPT and RNase.
Furthermore, Put treatment increased nucleic acids content and the endogenous
polyamines under drought stress. Drought stress was imposed during seedling stage
by decreasing soil moisture content. GOT, GPT and RNase activities increased in
leaves of alfalfa seedlings under drought stress. Soluble nitrogenous compounds
accumulated under drought stress, while nucleic acids content decreased. Except
glutamic acid, all free amino acids detected increased under drought stress. Put
treatment decreased activities of GOT, GPT and RNase, as well as reduced the
accumulation of the total soluble nitrogenous compounds, but increased DNA, RNA
and protein contents.
1786. Talanta. 2007 Apr 30;72(2):682-90. doi: 10.1016/j.talanta.2006.11.045. Epub

2007
Jan 16.
Application of multi-way data analysis on excitation-emission spectra for plant

identification.
Obeidat SM(1), Glasser T, Landau SY, Anderson DM, Rayson GD.
Author information:
(1)Department of Chemistry and Biochemistry, New Mexico State University, Las
Cruces, Box 30001, NM 88003, USA.
The ability to distinguish among diets fed to Damascus goats using

excitation-emission luminescence spectra was investigated. These diets consisted
of Medicago sativa L. (alfalfa), Trifolium spp. (clover), Pistacia lentiscus,
Phyllirea latifolia and Pinus brutia. The three-dimensional luminescence response
surface from phosphate buffered saline (PBS) extracts of each material was
analyzed using muti-way analysis chemometric tools (MPCA) and parallel factor
analysis (PARAFAC). Using three principal components, the spectra from each diet
material were distinguished. Additionally, fecal samples from goats fed diets of
either alfalfa or clover hays were investigated. The application of MPCA and
PARAFAC to these samples using models derived from the pre-digested diet
materials was strongly suggestive of the utility of similarly derive training
samples for the elucidation of botanical diet composition for animals.
DOI: 10.1016/j.talanta.2006.11.045
PMID: 19071673
Parasitic hymenoptera fauna on agromyzidae (diptera) colonizing weeds in

ecological compensation areas in northern Italian agroecosystems.
Burgio G(1), Lanzoni A, Navone P, Van Achterberg K, Masetti A.
Author information:
(1)Dipartimento di Scienze e Teenologie Agroambientali-Entomologia, Alma Mater
Studiorum-Università di Bologna, viale G. Fanin 42, 40127 Bologna, Italy.
gburgio@entom.agrsci.unibo.it
Parasitoids (Hymenoptera) associated with agromyzid leafminers (Diptera:

Agromyzidae) were studied in three rural farms located in northern Italy. The
parasitoids were reared from mined foliage of weeds growing in field margins. We
reared 998 Hymenoptera specimens, representing five families, 23 genera, and 53
species, from leafminers infesting weeds. Eulophidae was the most abundant family
(67.64%), followed by Braconidae (28.86%), Eucoilinae (1.40%), Tetracampidae
(1.40%), and Pteromalidae (0.7%). Braconids was the most species rich family,
accounting for 28 species; eulophids were represented by 19 species, pteromalids
by four species, and eucoilins and tetracampids by one species each. The dominant
parasitoid was the eulophid Pediobius metallicus (Nees), representing 18.17% of
the total, followed by Diglyphus isaea (Walker) (12.73%), and Neochrysocharis
formosa (Westwood) (10.82%). The most abundant braconid parasitoid was Dacnusa
maculipes Thomson (9.62%). More than 80% of parasitoids were recovered from 10
plant species: Cirsium arvense (L.) Scopoli, Plantago lanceolata L., Sonchus
asper (L.) Hill, Papaver rhoeas L., Picris echioides L., Lactuca serriola L.,
Myagrum perfoliatum L., Ranunculus velutinus Tenore, Arctium lappa L., and
Medicago sativa L. The retention and the management of wild plants within field
margins can be crucial tools to enhance the populations of biological control
agents of agromyzids and to conserve rare parasitic wasp species.
1788. J Environ Sci Health B. 2007 Mar-Apr;42(3):265-9.
Influence of crop residues on trifluralin mineralization in a silty clay loam

soil.
Farenhorst A(1).
Author information:
(1)Department of Soil Science, University of Manitoba, Winnipeg, Manitoba,
Canada. farenhor@ms.umanitoba.ca
Trifluralin is typically applied onto crop residues (trash, stubble) at the soil
surface, or onto the bare soil surface after the incorporation of crop residues
into the soil. The objective of this study was to quantify the effect of the type
and amount of crop residues in soil on trifluralin mineralization in a Wellwood
silty clay loam soil. Leaves and stubble of Potato (Solanum tuberosum) (P);
Canola (Brassica napus) (C), Wheat (Triticum aestivum) (W), Oats (Avena sativa),
(O), and Alfalfa (Medicago sativa) (A) were added to soil microcosms at rates of
2%, 4%, 8% and 16% of the total soil weight (25 g). The type and amount of crop
residues in soil had little influence on the trifluralin first-order
mineralization rate constant, which ranged from 3.57E-03 day(-1) in soil with 16%
A to 2.89E-02 day(-1) in soil with 8% W. The cumulative trifluralin
mineralization at 113 days ranged from 1.15% in soil with 16% P to 3.21% in soil
with 4% C, again demonstrating that the observed differences across the
treatments are not of agronomic or environmental importance.
DOI: 10.1080/10934520601134122
1789. Environ Entomol. 2007 Apr;36(2):416-24.
Alfalfa living mulch advances biological control of soybean aphid.
Schmidt NP(1), O'neal ME, Singer JW.
Author information:
(1)Department of Entomology, Iowa State University, Ames, IA 50011-3140, USA.
Despite evidence for biological control in North America, outbreaks of the

invasive soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), continue
to occur on soybean (Glycine max L. Merr.). Our objectives were to determine
whether natural enemies delay aphid establishment and limit subsequent population
growth and whether biological control can be improved by altering the
within-field habitat. We hypothesized that a living mulch would increase the
abundance of the aphidophagous community in soybean and suppress A. glycines
establishment and population growth. We measured natural enemy and A. glycines
abundance in soybean grown with and without an alfalfa (Medicago sativa L.)
living mulch. Soybean grown with an alfalfa living mulch had 45% more natural
enemies and experienced a delay in A. glycines establishment that resulted in
lower peak populations. From our experiments, we concluded that the current
natural enemy community in Iowa can delay A. glycines establishment, and an
increase in aphidophagous predator abundance lowered the rate of A. glycines
population growth preventing economic populations (i.e., below the current
economic threshold) from occurring. Incorporation of a living mulch had an
unexpected impact on A. glycines population growth, lowering the aphids'
intrinsic rate of growth, thus providing a bottom-up suppression of A. glycines.
We suggest future studies of living mulches or cover crops for A. glycines
management should address both potential sources of suppression. Furthermore, our
experience suggests that more consistent biological control of A. glycines may be
possible with even partial resistance that slows but does not prevent
reproduction.
1790. BMC Bioinformatics. 2007 Apr 19;8:129.
CGKB: an annotation knowledge base for cowpea (Vigna unguiculata L.) methylation
filtered genomic genespace sequences.
Chen X(1), Laudeman TW, Rushton PJ, Spraggins TA, Timko MP.
Author information:
(1)Department of Microbiology, University of Virginia Health System,
Charlottesville, VA 29908, USA. xc3m@virginia.edu <xc3m@virginia.edu>
BACKGROUND: Cowpea [Vigna unguiculata (L.) Walp.] is one of the most important
food and forage legumes in the semi-arid tropics because of its ability to
tolerate drought and grow on poor soils. It is cultivated mostly by poor farmers
in developing countries, with 80% of production taking place in the dry savannah
of tropical West and Central Africa. Cowpea is largely an underexploited crop
with relatively little genomic information available for use in applied plant
breeding. The goal of the Cowpea Genomics Initiative (CGI), funded by the
Kirkhouse Trust, a UK-based charitable organization, is to leverage modern
molecular genetic tools for gene discovery and cowpea improvement. One aspect of
the initiative is the sequencing of the gene-rich region of the cowpea genome
(termed the genespace) recovered using methylation filtration technology and
providing annotation and analysis of the sequence data.
DESCRIPTION: CGKB, Cowpea Genespace/Genomics Knowledge Base, is an annotation
knowledge base developed under the CGI. The database is based on information
derived from 298,848 cowpea genespace sequences (GSS) isolated by methylation
filtering of genomic DNA. The CGKB consists of three knowledge bases: GSS
annotation and comparative genomics knowledge base, GSS enzyme and metabolic
pathway knowledge base, and GSS simple sequence repeats (SSRs) knowledge base for
molecular marker discovery. A homology-based approach was applied for annotations
of the GSS, mainly using BLASTX against four public FASTA formatted protein
databases (NCBI GenBank Proteins, UniProtKB-Swiss-Prot, UniprotKB-PIR (Protein
Information Resource), and UniProtKB-TrEMBL). Comparative genome analysis was
done by BLASTX searches of the cowpea GSS against four plant proteomes from
Arabidopsis thaliana, Oryza sativa, Medicago truncatula, and Populus trichocarpa.
The possible exons and introns on each cowpea GSS were predicted using the
HMM-based Genscan gene predication program and the potential domains on annotated
GSS were analyzed using the HMMER package against the Pfam database. The
annotated GSS were also assigned with Gene Ontology annotation terms and
integrated with 228 curated plant metabolic pathways from the Arabidopsis
Information Resource (TAIR) knowledge base. The UniProtKB-Swiss-Prot ENZYME
database was used to assign putative enzymatic function to each GSS. Each GSS was
also analyzed with the Tandem Repeat Finder (TRF) program in order to identify
potential SSRs for molecular marker discovery. The raw sequence data, processed
annotation, and SSR results were stored in relational tables designed in
key-value pair fashion using a PostgreSQL relational database management system.
The biological knowledge derived from the sequence data and processed results are
represented as views or materialized views in the relational database management
system. All materialized views are indexed for quick data access and retrieval.
Data processing and analysis pipelines were implemented using the Perl
programming language. The web interface was implemented in JavaScript and Perl
CGI running on an Apache web server. The CPU intensive data processing and
analysis pipelines were run on a computer cluster of more than 30 dual-processor
Apple XServes. A job management system called Vela was created as a robust way to
submit large numbers of jobs to the Portable Batch System (PBS).
CONCLUSION: CGKB is an integrated and annotated resource for cowpea GSS with
features of homology-based and HMM-based annotations, enzyme and pathway
annotations, GO term annotation, toolkits, and a large number of other facilities
to perform complex queries. The cowpea GSS, chloroplast sequences, mitochondrial
sequences, retroelements, and SSR sequences are available as FASTA formatted
files and downloadable at CGKB. This database and web interface are publicly
accessible at http://cowpeagenomics.med.virginia.edu/CGKB/.
DOI: 10.1186/1471-2105-8-129
PMCID: PMC1868039
1791. J Sep Sci. 2007 Mar;30(4):491-501.

Composition and content of seed flavonoids in forage and grain legume crops.
Prati S(1), Baravelli V, Fabbri D, Schwarzinger C, Brandolini V, Maietti A,

Tedeschi P, Benvenuti S, Macchia M, Marotti I, Bonetti A, Catizone P, Dinelli G.
Author information:
(1)Inter-departmental Centre for Research in Environmental Sciences (C.I.R.S.A.),
University of Bologna, Ravenna, Italy.
Flavonoid content and composition in 124 accessions of forage and grain legumes
was assessed by means of HPLC-DAD and HPLC-ESI-MS analyses of seed extracts.
Excluding soybean, only 7 out of 77 grain legume accessions exhibited a total
flavonoid content higher than 0.1 mg/g fresh seed weight (mean content 0.33 +/-
0.31 mg/ g fresh seed weight); while in 38 out of 47 forage legume, the mean
flavonoid content was 1.49 +/- 0.89 mg/g fresh seed weight. On the basis of their
higher seed flavonoid content and agronomic relevance, two grain and five forage
legume accessions were selected for further HPLC-ESI-MS analyses aimed to
identify bioactive compounds. Chemical analysis of the selected accessions
permitted the identification of several flavonoids with pharmaceutical properties
such as vitexin in Vigna angularis and Trigonella foenum-graecum, quercetin in
all Trifolium spp and Medicago sativa, taxifolin in Trifolium pratense and
Trifolium alexandrinum, myricetin in Trifolium repens, kaempferol in Phaseolus
vulgaris and T. repens and luteolin in M. sativa and T. alexandrinum. Since much
of literature information is regarding flavonoid content and composition of the
green part of legume species, the obtained results contributed to fill the gap in
knowledge about flavonoid content at the seed level.
1792. J Mol Biol. 2007 May 25;369(1):265-76. Epub 2007 Mar 21.
Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.).
Shao H(1), Dixon RA, Wang X.
Author information:
Isoflavonoids are commonly found in leguminous plants, where they play important
roles in plant defense and have significant health benefits for animals and
humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction
of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid
phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.)
vestitone reductase has been determined at 1.4 A resolution. The structure
contains a classic Rossmann fold domain in the N terminus and a small C-terminal
domain. Sequence and structural analysis showed that vestitone reductase is a
member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the
low levels of sequence identity, and the prominent structural differences from
other SDR enzymes with known structures. The putative binding sites for the
co-factor NADPH and the substrate (3R)-vestitone were defined and located in a
large cleft formed between the N and C-terminal domains of enzyme. Potential key
residues for enzyme activity were also identified, including the catalytic triad
Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but
not the (3S) isomer, forms favored interactions with the co-factor and catalytic
triad, thus providing an explanation for the enzyme's strict substrate
stereo-specificity.
DOI: 10.1016/j.jmb.2007.03.040
1793. J Dairy Sci. 2007 May;90(5):2355-66.
Effects of chop length of alfalfa and corn silage on milk production and rumen
fermentation of dairy cows.
Bhandari SK(1), Ominski KH, Wittenberg KM, Plaizier JC.
Author information:
(1)Department of Animal Science University of Manitoba, Winnipeg, Manitoba,
Canada R3T 2N2.
Effects of chop length (shorter = 10 mm or longer = 19 mm) of alfalfa silage and

corn silage were determined in 16 midlactation Holstein cows using a 4 x 4 Latin
square design with a 2 x 2 arrangement of treatments. Experimental periods were
21 d long and consisted of 14 d of adaptation and 7 d of sampling. Cows received
total mixed ration containing (dry matter basis) 44.0% barley grain-based energy
supplement, 12.6% protein supplement, and 21.7% longer chop or shorter chop
alfalfa silage and 21.7% longer chop or shorter chop corn silage. Reducing the
chop length of alfalfa silage and corn silage reduced the average geometric
particle length from 14.4 to 11.0 mm and from 14.2 to 10.4 mm, respectively.
Reducing the chop length of both silages reduced the proportion of the diets
retained by the 8- and 19-mm screen of the Penn State Particle Separator from
55.0 to 46.0% of dry matter. Reducing the alfalfa chop length increased total
rumen volatile fatty acids at 4 to 5 h after feeding but did not affect rumen pH
at 4 to 5 h after feeding, feed intake, and milk production. Reducing the corn
silage chop length increased dry matter intake from 22.3 to 23.2 kg/d, increased
rumen pH at 4 to 5 h after feeding from 6.12 to 6.20, but did not alter rumen
volatile fatty acids at 4 to 5 h after feeding or milk production. Daily milk
yield, milk fat percentage, and milk protein percentage averaged 38.2 kg/d,
2.62%, and 3.29%, respectively, across all diets. The low milk fat percentages
suggest that all diets induced subacute ruminal acidosis (SARA), whereas the
rumen pH did not indicate SARA. This discrepancy could be due to a difference in
the time of rumen pH measurement and the time of the lowest rumen pH. Hence, the
pH data need to be interpreted with caution. Diets could have induced SARA,
because for all experimental diets the content of forage neutral detergent fiber
was lower than recommended for barley grain-based diets.
DOI: 10.3168/jds.2006-609
1794. Nat Biotechnol. 2007 Apr;25(4):367-8. Epub 2007 Apr 3.
US courts thwart GM alfalfa and turf grass.
Fox JL.
DOI: 10.1038/nbt0407-367
1795. Environ Pollut. 2008 Jan;151(1):200-4. Epub 2007 Apr 6.

Amelioration of alkali soil using flue gas desulfurization byproducts:
productivity and environmental quality.
Wang SJ(1), Chen CH, Xu XC, Li YJ.
Author information:
(1)Key Laboratory for Thermal Science and Power Engineering of Ministry of
Education, Department of Thermal Engineering, Tsinghua University, Beijing
100084, PR China. wangshuj@tsinghua.edu.cn
In this study, flue gas desulfurization (FGD) byproducts are used to ameliorate
alkali soil. The average application rates for soils with low exchangeable sodium
percentage (ESP), mid ESP, and high ESP are 20.9, 30.6, and 59.3 Mg ha(-1),
respectively. The experimental results obtained for 3 consecutive years reveal
that the emergence ratios and yields of the crops were 1.1-7.6 times and 1.1-13.9
times those of the untreated control, respectively. The concentrations of Cr, Pb,
Cd, As, and Hg in the treated soils are far below the background values
stipulated by the Environmental Quality Standard for Soils (GB15618-1995). Their
concentrations in the seeds of corn and alfalfa grown in the treated soils are
far below the tolerance limits regulated by National Food Standards of China. The
results of this research demonstrate that the amelioration of alkali soils using
FGD byproducts is promising.
DOI: 10.1016/j.envpol.2007.02.014
1796. J Bacteriol. 2007 Jun;189(11):4204-16. Epub 2007 Mar 30.
An extracytoplasmic function sigma factor acts as a general stress response

regulator in Sinorhizobium meliloti.
Sauviac L(1), Philippe H, Phok K, Bruand C.
Author information:
(1)Laboratoire des Interactions Plantes-Microorganismes, UMR 2594-441 CNRS-INRA,
BP52627, 31326 Castanet-Tolosan Cedex, France.
Sinorhizobium meliloti genes transcriptionally up-regulated after heat stress, as

well as upon entry into stationary phase, were identified by microarray analyses.
Sixty stress response genes were thus found to be up-regulated under both
conditions. One of them, rpoE2 (smc01506), encodes a putative extracytoplasmic
function (ECF) sigma factor. We showed that this sigma factor controls its own
transcription and is activated by various stress conditions, including heat and
salt, as well as entry into stationary phase after either carbon or nitrogen
starvation. We also present evidence that the product of the gene cotranscribed
with rpoE2 negatively regulates RpoE2 activity, and we therefore propose that it
plays the function of anti-sigma factor. By combining transcriptomic,
bioinformatic, and quantitative reverse transcription-PCR analyses, we identified
44 RpoE2-controlled genes and predicted the number of RpoE2 targets to be higher.
Strikingly, more than one-third of the 60 stress response genes identified in
this study are RpoE2 targets. Interestingly, two genes encoding proteins with
known functions in stress responses, namely, katC and rpoH2, as well as a second
ECF-encoding gene, rpoE5, were found to be RpoE2 regulated. Altogether, these
data suggest that RpoE2 is a major global regulator of the general stress
response in S. meliloti. Despite these observations, and although this sigma
factor is well conserved among alphaproteobacteria, no in vitro nor in planta
phenotypic difference from the wild-type strain could be detected for rpoE2
mutants. This therefore suggests that other important actors in the general
stress response have still to be identified in S. meliloti.
DOI: 10.1128/JB.00175-07
PMCID: PMC1913381
1797. Int J Syst Evol Microbiol. 2007 Apr;57(Pt 4):844-8.
Rhizobium cellulosilyticum sp. nov., isolated from sawdust of Populus alba.
García-Fraile P(1), Rivas R, Willems A, Peix A, Martens M, Martínez-Molina E,

Mateos PF, Velázquez E.
Author information:
(1)Departamento de Microbiología y Genética, Universidad de Salamanca, Spain.
During a study of polysaccharide-hydrolysing bacteria present in different plant

sources, two strains were isolated from pulverized decaying wood of Populus alba
and classified in the genus Rhizobium on basis of their almost complete 16S rRNA
gene sequences. Their closest phylogenetic relatives were Rhizobium galegae USDA
4128(T) and Rhizobium huautlense S02(T), with 98.2 and 98.1 % 16S rRNA gene
sequence similarity, respectively. recA and atpD sequence analysis showed that
these species have less than 88 and 92 % similarity, respectively, to the novel
strains. In contrast to their closest phylogenetic relatives, the two strains
showed strong cellulase activity on plates containing CM-cellulose as a carbon
source. They were also distinguishable from these species on the basis of other
phenotypic characteristics. The strains were able to induce ineffective nodules
on Medicago sativa and the sequence of their nodD gene was phylogenetically close
to that of Ensifer meliloti 1021 (99.6 % similarity). DNA-DNA hybridization
values ranged from 10 to 22 % with respect to R. galegae USDA 4128(T) and 14 to
25 % with respect to R. huautlense S02(T), showing that the strains from this
study belong to a novel species, for which the name Rhizobium cellulosilyticum
sp. nov. is proposed. The type strain is ALA10B2(T) (=LMG 23642(T)=DSM
18291(T)=CECT 7176(T)).
DOI: 10.1099/ijs.0.64680-0
1798. Commun Agric Appl Biol Sci. 2006;71(3 Pt A):715-23.
Tillage and residue burning affects weed populations and seed banks.
Narwal S(1), Sindel BM, Jessop RS.
Author information:
(1)School of Rural Science and Agriculture, The University of New England,
Cooperative Research Centre for Australian Weed Management, Armidale, New South
Wales 2351, Australia. snarwal@une.edu.au
An integrated weed management approach requires alternative management practices

to herbicide use such as tillage, crop rotations and cultural controls to reduce
soil weed seed banks. The objective of this study was to examine the value of
different tillage practices and stubble burning to exhaust the seed bank of
common weeds from the northern grain region of Australia. Five tillage and
burning treatments were incorporated in a field experiment, at Armidale (30
degrees 30'S, 151 degrees 40'E), New South Wales, Australia in July 2004 in a
randomized block design replicated four times. The trial was continued and
treatments repeated in July 2005 with all the mature plants from the first year
being allowed to shed seed in their respective treatment plots. The treatments
were (i) no tillage (NT), (ii) chisel ploughing (CP), (iii) mould board ploughing
(MBP), (iv) wheat straw burning with no tillage (SBNT) and (v) wheat straw
burning with chisel ploughing (SBC). Soil samples were collected before applying
treatments and before the weeds flowered to establish the seed bank status of the
various weeds in the soil. Wheat was sown after the tillage treatments. Burning
treatments were only initiated in the second year, one month prior to tillage
treatments. The major weeds present in the seed bank before initiating the trial
were Polygonum aviculare, Sonchus oleraceus and Avena fatua. Tillage promoted the
germination of other weeds like Hibiscus trionum, Medicago sativa, Vicia sp. and
Phalaris paradoxa later in the season in 2004 and Convolvulus erubescens emerged
as a new weed in 2005. The MBP treatment in 2004 reduced the weed biomass to a
significantly lower level of 55 g/m2 than the other treatments of CP (118 g/m2)
and NT plots (196 g/m2) (P < 0.05). However, in 2005 SBC and MBP treatments were
similar in reducing the weed biomass. In 2004, the grain yield trend of wheat was
significantly different between CP and NT, and MBP and NT (P < 0.05) with maximum
yield of 5898 kg/ha in CP and 5731 kg/ha in MBP. Rainfall before the start of the
second trial season promoted the germination of a large numbers of weeds. SBC and
MBP treatments reduced the numbers of most of the individual weed species
compared with CP, SBNT and NT. SBC was able to destroy a large proportion of
seeds most likely through burning and burying some in the soil and was found to
be the best treatment in exhausting the seed bank followed closely by MBP which
probably buried large number of seeds deep in the soil and promoted others to
germinate. CP might have buried some of the seeds in the top 5-10 cm but also
promoted parts of the seed bank to germinate. SBNT and NT provided an ideal
medium for weeds to germinate and resulted in heavy infestations of weeds.
1799. J Exp Bot. 2007;58(7):1663-75. Epub 2007 Mar 26.
Dicot and monocot plants differ in retinoblastoma-related protein subfamilies.
Lendvai A(1), Pettkó-Szandtner A, Csordás-Tóth E, Miskolczi P, Horváth GV,

Györgyey J, Dudits D.
Author information:
Sciences, Szeged, H-6726, Temesvári krt. 62, Hungary.
The present study supports the view that the retinoblastoma functions are shared
by two distinct retinoblastoma-related (RBR) protein subfamilies in the monocot
cereal species, whereas dicot plants have only a single RBR protein. Genes
encoding RBR proteins were identified and characterized in alfalfa (Medicago
sativa), rice (Oryza sativa), and wheat (Triticum aestivum). The alfalfa MsRBR
gene encodes a new member of the dicot RBR proteins (subfamily A). A comparison
was made of two rice genes, OsRBR1 (subfamily B) and OsRBR2 (subfamily C), which
exhibit differences in exon-intron organization and share only 52% amino acid
sequence identity. The plant RBR proteins can be categorized into three distinct
subfamilies, in which the similarity between members is greater than the
similarity to other RBR proteins from the same species. Comparison of the
transcript levels in various tissues revealed that the expression of the OsRBR1
gene was high in embryos or cultured cells and gradually decreased from the basal
region to the tip of the leaves. The OsRBR2 gene displayed more transcripts in
differentiated tissues, such as leaves and roots. In contrast, the mRNA level of
the MsRBR gene did not differ significantly in either mature leaves or cultured
cells. The results of yeast two-hybrid pairwise interaction assays demonstrated
differences between the rice RBR variants in the interactions with the
phosphatase 2A B'' regulatory subunit and an unknown protein. The in silico and
functional data presented in this work highlight considerable differences between
dicot and monocot species in the retinoblastoma regulatory pathways and permit an
improved classification of RBR proteins in higher plants.
DOI: 10.1093/jxb/erm022
1800. Anal Chim Acta. 2007 Jan 2;581(1):63-70. Epub 2006 Aug 12.
Solid phase microextraction/gas chromatography/mass spectrometry integrated with

chemometrics for detection of Salmonella typhimurium contamination in a packaged
fresh vegetable.
Siripatrawan U(1), Harte BR.
Author information:
Bangkok, Thailand. ubonratana.s@chula.ac.th <ubonratana.s@chula.ac.th>
A rapid method for detection of Salmonella typhimurium contamination in packaged

alfalfa sprouts using solid phase microextraction/gas chromatography/mass
spectrometry (SPME/GC/MS) integrated with chemometrics was investigated. Alfalfa
sprouts were inoculated with S. typhimurium, packed into commercial LDPE bags and
stored at 10+2 degrees C for 0, 1, 2 and 3 days. Uninoculated sprouts were used
as control samples. A SPME device was used to collect the volatiles from the
headspace above the samples and the volatiles were identified using GC/MS.
Chemometric techniques including linear discriminant analysis (LDA) and
artificial neural network (ANN) were used as data processing tools. Numbers of
Salmonella were followed using a colony counting method. From LDA, it was able to
differentiate control samples from sprouts contaminated with S. typhimurium. The
potential to predict the number of contaminated S. typhimurium from the
SPME/GC/MS data was investigated using multilayer perceptron (MLP) neural network
with back propagation training. The MLP comprised an input layer, one hidden
layer, and an output layer, with a hyperbolic tangent sigmoidal transfer function
in the hidden layer and a linear transfer function in the output layer. The MLP
neural network with a back propagation algorithm could predict number of S.
typhimurium in unknown samples using the volatile fingerprints. Good prediction
was found as measured by a regression coefficient (R(2)=0.99) between actual and
predicted data.
DOI: 10.1016/j.aca.2006.08.007
1801. FEMS Microbiol Ecol. 2007 Jun;60(3):477-89. Epub 2007 Mar 26.
Population genetic structure of Sinorhizobium meliloti and S. medicae isolated

from nodules of Medicago spp. in Mexico.
Silva C(1), Kan FL, Martínez-Romero E.
Author information:
(1)Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México,
Cuernavaca, Morelos, Mexico. csilva@ccg.unam.mx
We studied the genetic structure of 176 bacterial isolates from nodules of
Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in
three localities in Mexico. The strains were characterized by multilocus enzyme
electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism
of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes,
and partial sequences of glnII, recA and nodB. Most of the strains were
classified as Sinorhizobium meliloti, and a high genetic diversity was recorded.
Six strains were classified as Sinorhizobium medicae, with no genetic variation.
Phylogenetic and population genetic analyses revealed evidence of frequent
recombination and migration within species.
DOI: 10.1111/j.1574-6941.2007.00301.x
1802. Bioorg Med Chem Lett. 2007 Jun 1;17(11):3161-6. Epub 2007 Mar 15.
Structure function analysis of benzalacetone synthase from Rheum palmatum.
Abe T(1), Morita H, Noma H, Kohno T, Noguchi H, Abe I.
Author information:
(1)School of Pharmaceutical Sciences, The COE21 Program, University of Shizuoka,
52-1 Yada, Shizuoka 422-8526, Japan.
Benzalacetone synthase (BAS) is a plant-specific chalcone synthase (CHS)

superfamily type III polyketide synthase (PKS) that catalyzes a one-step
decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA. The diketide
forming activity of Rheum palmatum BAS is attributed to the characteristic
substitution of the conserved active-site Phe215 with Leu (numbering in Medicago
sativa CHS). To further understand the structure and function of R. palmatum BAS,
four site-directed mutants (C197T, C197G, G256L, and S338V) were newly
constructed. All the mutants did not change the product pattern, however, the
activity was 2-fold increased in S338V, while reduced to half in G256L mutant. On
the other hand, the C197 mutants were functionally almost identical to wild-type
BAS, excluding the possibility that the second active-site Cys is involved in the
enzyme reaction. Instead, homology modeling suggested a possibility that, unlike
the case of CHS, BAS utilizes an alternative pocket to lock the coumaroyl moiety
for the diketide formation reaction.
DOI: 10.1016/j.bmcl.2007.03.029
1803. J Agric Food Chem. 2007 Apr 18;55(8):2918-22. Epub 2007 Mar 20.
Qualitative and quantitative polymerase chain reaction assays for an alfalfa

(Medicago sativa)-specific reference gene to use in monitoring transgenic
cultivars.
Alexander TW(1), Reuter T, McAllister TA.
Author information:
(1)Agriculture and Agri-Food Canada Research Centre, P.O. Box 3000, Lethbridge,
Alberta, Canada.
Genetically modified (GM) alfalfa (Medicago sativa) was marketed for the first
time in 2005. For countries with established thresholds for GM plants, methods to
detect and quantify their adventitious presence are required. We selected acetyl
CoA carboxylase as a reference gene for the detection and quantification of GM
alfalfa. Two qualitative polymerase chain reaction (PCR) assays (Acc1 and Acc2)
were designed to detect alfalfa. Both were specific to alfalfa, amplifying DNA
from 12 separate cultivars and showing negative results for PCR of 15 nonalfalfa
plants. The limits of detection for Acc1 and Acc2 were 0.2 and 0.01%,
respectively. A quantitative real-time PCR assay was also designed, having high
linearity (r > 0.99) over alfalfa standard concentrations ranging from 100 to 2.0
x 10(5) pg of alfalfa DNA per PCR. The real-time PCR assay was effective in
quantifying alfalfa DNA from forage- and concentrate-based mixed diets containing
different amounts of alfalfa meal.
DOI: 10.1021/jf0630116
1804. Theor Appl Genet. 2007 May;114(8):1367-78. Epub 2007 Mar 14.
Genome mapping of white clover (Trifolium repens L.) and comparative analysis
within the Trifolieae using cross-species SSR markers.
Zhang Y(1), Sledge MK, Bouton JH.
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, 2510, Sam
Noble Parkway, Ardmore, OK 73401, USA. yzhang@noble.org
Allotetraploid white clover (Trifolium repens L.), a cool-season perennial legume

used extensively as forage for livestock, is an important target for
marker-assisted breeding. A genetic linkage map of white clover was constructed
using simple sequence repeat (SSR) markers based on sequences from several
Trifolieae species, including white clover, red clover (T. pratense L.), Medicago
truncatula (Gaertn.) and soybean (Glycine max L.). An F(1) population consisting
of 179 individuals, from a cross between two highly heterozygous genotypes, GA43
and Southern Regional Virus Resistant, was used for genetic mapping. A total of
1,571 SSR markers were screened for amplification and polymorphism using DNA from
two parents and 14 F(1)s of the mapping population. The map consists of 415 loci
amplified from 343 SSR primer pairs, including 83 from white clover, 181 from red
clover, 77 from M. truncatula, and two from soybean. Linkage groups for all eight
homoeologous chromosome pairs of allotetraploid white clover were detected. Map
length was estimated at 1,877 cM with 87% genome coverage. Map density was
approximately 5 cM per locus. Segregation distortion was detected in six segments
of the genome (homoeologous groups A1, A2, B1, B2, C1, and D1). A comparison of
map locations of markers originating from white clover, red clover, and alfalfa
(M. sativa L.) revealed putative macro-colinearity between the three Trifolieae
species. This map can be used to link quantitative trait loci with SSR markers,
and accelerate the improvement of white clover by marker-assisted selection and
breeding.
DOI: 10.1007/s00122-007-0523-3
PMCID: PMC1950584
Identification of QTL for reaction to three races of Colletotrichum trifolii and

further analysis of inheritance of resistance in autotetraploid lucerne.
Mackie JM(1), Musial JM, Armour DJ, Phan HT, Ellwood SE, Aitken KS, Irwin JA.
Author information:
4072, Australia.
Anthracnose, caused by Colletotrichum trifolii, is one of the most serious

diseases of lucerne worldwide. The disease is managed through deployment of
resistant cultivars, but new pathotypes present a challenge to the successful
implementation of this strategy. This paper reports the genetic map locations of
quantitative trait loci (QTL) for reaction to races 1, 2 and 4 of C. trifolii in
a single autotetraploid lucerne clone, designated W126 from the Australian cv.
Trifecta. Resistance was mapped in a backcross population of 145 individuals, and
reaction was assessed both by spray and injection inoculation of stems.
Resistance to injection inoculation with races 1 and 4 was incompletely dominant
and closely linked (phenotypic markers 2.2 cM apart); these resistances mapped to
a linkage group homologous to Medicago truncatula linkage group 8. When the spray
inoculation data were subjected to QTL analysis, the strongest QTL for resistance
was located on linkage group 8; six QTL were identified for race 1 and four for
race 4. Resistance to race 2 was incompletely recessive; four QTL were identified
and these include one QTL on linkage group 4 that was also identified for race 1.
Modelling of the interactions between individual QTL and marker effects allowed a
total of 52-63% of the phenotypic variation to be described for each of the
different races. These markers will have value in breeding lucerne, carrying
multiple sources of resistance to the three known races of C. trifolii.
DOI: 10.1007/s00122-007-0527-z
Identification of QTL for resistance and susceptibility to Stagonospora meliloti

in autotetraploid lucerne.
Musial JM(1), Mackie JM, Armour DJ, Phan HT, Ellwood SE, Aitken KS, Irwin JA.
Author information:
4072, Australia. j.musial@uq.edu.au
In eastern Australia and California, USA, one of the major lethal fungal diseases
of lucerne (Medicago sativa) is Stagonospora root and crown rot, caused by
Stagonospora meliloti. Quantitative trait loci (QTL) involved in resistance and
susceptibility to S. meliloti were identified in an autotetraploid lucerne
backcross population of 145 individuals. Using regression analysis and interval
mapping, we detected one region each on linkage groups 2, 6 and 7 that were
consistently associated with disease reaction to S. meliloti in two separate
experiments. The largest QTL on linkage group 7, which is associated with
resistance to S. meliloti, contributed up to 17% of the phenotypic variation. The
QTL located on linkage group 2, which is potentially a resistance allele in
repulsion to the markers for susceptibility to S. meliloti, contributed up to 8%
of the phenotypic variation. The QTL located on linkage group 6, which is
associated with susceptibility to S. meliloti, contributed up to 16% of the
phenotypic variation. A further two unlinked markers contributed 5 and 8% of the
phenotypic variation, and were detected in only one experiment. A total of 517
simple sequence repeat (SSR) markers from Medicago truncatula were screened on
the parents of the mapping population. Only 27 (6%) SSR markers were polymorphic
and could be incorporated into the autotetraploid map of M. sativa. This allowed
alignment of our M. sativa linkage map with published M. truncatula maps. The
markers linked to the QTL we have reported will be useful for marker assisted
selection for partial resistance to S. meliloti in lucerne.
DOI: 10.1007/s00122-007-0528-y
1807. Environ Entomol. 2007 Feb;36(1):195-203.
Physiological response of glandular-haired alfalfa to potato leafhopper

(Hemiptera: Cicadellidae) injury.
Lamp WO(1), Alexander LC, Nguyen M.
Author information:
(1)Department of Entomology, University of Maryland, College Park, MD 20742, USA.
lamp@umd.edu
Plant tolerance to herbivory is a key approach for managing pests. In alfalfa,

Medicago sativa, the potato leafhopper, Empoasca fabae, is a major pest as a
result of the cascade of plant responses to piercing-sucking injury. To identify
tolerance to its injury based on alfalfa physiology, experiments were conducted
in the field and greenhouse. In our comparison of the response of field-grown
alfalfa cultivars to standardized leafhopper densities, net photosynthesis and
transpiration rates of 'Geneva' leaves were reduced by 18 and 21%, respectively,
by leafhopper presence compared with a rate change of <1% of resistant
'EverGreen' leaves. Under greenhouse conditions, alfalfa clones varied in their
level of gas exchange (net photosynthesis and transpiration) and stem elongation
responses to leafhopper injury. For example, in the comparison of seven clones,
net photosynthesis declined an average of 40.7% with leafhopper injury, although
individual clones varied from 26.6 to 74.3% reduction. Internode elongation after
2 d was 60.3% less on injured stems compared with healthy stems, but again, the
individual clones varied from 17.3 to 91.9%. In a time-course study of selected
clones, clones varied in their level of injury just after and 3 d after insect
removal. Gas exchange responses of all clones recovered by 7 d after cessation of
injury. In a choice test, leafhoppers spent similar amounts of time on the
susceptible clone and the most tolerant clone; however, their precise feeding
behaviors were not measured. Thus, the variable response of clones to injury may
be either true physiological tolerance or antixenosis from a change in feeding
behavior. This study showed putative tolerance to leafhopper injury among alfalfa
genotypes, suggesting that tolerance could be the basis for crop protection in
alfalfa from potato leafhopper injury.
1808. BMC Bioinformatics. 2007 Mar 12;8:87.
GeneBins: a database for classifying gene expression data, with application to

plant genome arrays.
Goffard N(1), Weiller G.
Author information:
(1)ARC Centre of Excellence for Integrative Legume Research and Bioinformatics
Laboratory, Genomic Interactions Group, Research School of Biological Sciences,
Australian National University, Canberra, Australia. nicolas.goffard@anu.edu.au
BACKGROUND: To interpret microarray experiments, several ontological analysis
tools have been developed. However, current tools are limited to specific
organisms.
RESULTS: We developed a bioinformatics system to assign the probe set sequences
of any organism to a hierarchical functional classification modelled on KEGG
ontology. The GeneBins database currently supports the functional classification
of expression data from four Affymetrix arrays; Arabidopsis thaliana, Oryza
sativa, Glycine max and Medicago truncatula. An online analysis tool to identify
relevant functions is also provided.
CONCLUSION: GeneBins provides resources to interpret gene expression results from
microarray experiments. It is available at
http://bioinfoserver.rsbs.anu.edu.au/utils/GeneBins/
DOI: 10.1186/1471-2105-8-87
PMCID: PMC1828068
1809. J Agric Food Chem. 2007 Apr 4;55(7):2645-52. Epub 2007 Mar 10.
Flavonoids from barrel medic (Medicago truncatula) aerial parts.
Kowalska I(1), Stochmal A, Kapusta I, Janda B, Pizza C, Piacente S, Oleszek W.
Author information:
ul. Czartoryskich 8, 24-100 Pulawy, Poland.
Twenty-three flavonoids have been identified in the aerial parts of barrel medic,
and their structures were established by spectrometric and spectroscopic
(ESI-MS/MS and NMR) techniques. Eight of the identified compounds, including
apigenin
7-O-beta-D-glucuronopyranosyl-(1-->3)-O-beta-D-glucuronopyranosyl-(1-->2)-O-beta-
D-glucuronopyranoside, apigenin
7-O-[2'-O-sinapoyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside
], apigenin
7-O-{2-O-feruloyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl
-(1-->2)-O-beta-D-glucopyranoside}, chrysoeriol
7-O-[beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside, chrysoeriol
7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyran
osyl(1-->2)-O-beta-D-glucuronopyranoside}, tricin
7-O-beta-D-glucuronopyranosyl-4'-O-glucopyranoside, tricin
7-O-[2'-O-feruloyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucopyranoside],
and tricin
7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyran
osyl(1-->2)-O-beta-D-glucuronopyranoside}, have not been reported before in the
plant kingdom. Additionally, the presence of two luteolin, three apigenin, one
chrysoeriol, and six tricin glycosides, previously identified in alfalfa
(Medicago sativa), was confirmed in M. truncatula. Moreover, besides the above
flavones, the aerial parts of this species contained three flavonols including
rutin, laricitrin 3,7,5'-triglucoside, and laricitrin 3,5'-diglucoside.
DOI: 10.1021/jf063635b
1810. J Food Prot. 2007 Feb;70(2):316-22.
Quantitative analysis of the growth of Salmonella stanley during alfalfa

sprouting and evaluation of Enterobacter aerogenes as its surrogate.
Liu B(1), Schaffner DW.
Author information:
(1)Food Risk Analysis Initiative, Rutgers University, New Brunswick, New Jersey
08901-8520, USA.
Raw seed sprouts have been implicated in several food poisoning outbreaks in the
last 10 years. Few studies have included investigations of factors influencing
the effectiveness of testing spent irrigation water, and in no studies to date
has a nonpathogenic surrogate been identified as suitable for large-scale
irrigation water testing trials. Alfalfa seeds were inoculated with Salmonella
Stanley or its presumptive surrogate (nalidixic acid-resistant Enterobacter
aerogenes) at three concentrations (-3, -30, and -300 CFU/g) and were then
transferred into either flasks or a bench top-scale sprouting chamber. Microbial
concentrations were determined in seeds, sprouts, and irrigation water at various
times during a 4-day sprouting process. Data were fit to logistic regression
models, and growth rates and maximum concentrations were compared using the
generalized linear model procedure of SAS. No significant differences in growth
rates were observed among samples taken from flasks or the chamber. Microbial
concentrations in irrigation water were not significantly different from
concentrations in sprout samples obtaihed at the same time. E. aerogenes
concentrations were similar to those of Salmonella Stanley at corresponding time
points for all three inoculum concentrations. Growth rates were also constant
regardless of inoculum concentration or strain, except that lower inoculum
concentrations resulted in lower final concentrations proportional to their
initial concentrations. This research demonstrated that a nonpathogenic
easy-to-isolate surrogate (nalidixic acid-resistant E. aerogenes) provides
results similar to those obtained with Salmonella Stanley, supporting the use of
this surrogate in future large-scale experiments.
1811. Bioresour Technol. 2008 Feb;99(3):604-8. Epub 2007 Mar 1.
Immunological cell and serum metabolite response of 60-week-old commercial laying

hens to an alfalfa meal molt diet.
Landers KL(1), Moore RW, Dunkley CS, Herrera P, Kim WK, Landers DA, Howard ZR,
McReynolds JL, Bryd JA, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
(1)Poultry Science Department, Texas A&M University, College Station, TX, USA.
The practice of induced molting involves the restriction of light, feed removal
and optionally water for 5-14 days. However, there is growing concern regarding
feed removal and animal welfare issues. With this in mind, alternative diets have
been developed to produce similar molting effects as that of feed deprivation.
Alfalfa, which largely consists of insoluble fiber, can be used as a molting
diet. In this study, heterophil and lymphocyte counts, serum chemistry, and organ
weight parameters were evaluated in hens that were deprived of feed or fed
alfalfa during a nine day induced molt. Full-fed hens were used as the control.
Blood serum parameters assessed included calcium, magnesium, glucose, total
protein, ketone bodies, uric acid, and cholesterol. White blood cells were
counted and categorized by cell type. On the ninth day of the trial, the hens
were euthanized and the liver, spleen, heart, intestine, pancreas, ovary,
oviduct, and kidney were collected and weighed. On day 8 birds molted with
alfalfa or by feed deprivation had significantly higher (P<0.05) levels of ketone
bodies and cholesterol and lower levels of calcium, and magnesium compared to the
full-fed hens while birds molted by feed deprivation exhibited significantly
lower levels of uric acid. Birds molted by both methods exhibited significant
reductions in ovary, oviduct, liver and pancreas weights and increased spleen
weights when compared to the non-molted hens. On days 0, 2, and 6 there were no
significant differences (P>0.05) in either heterophil or lymphocyte percentages.
However, heterophil percentages were higher in feed withdrawal birds than
full-fed birds on day 4 but lymphocyte percentages were higher in full-fed birds
compared to feed withdrawal birds. On day 8 of the induced molt lymphocyte
percentages were higher from full-fed birds when compared to feed withdrawal
birds but no significant differences were detectable for heterophil percentages.
Based on reproductive organ weight loss and changes in serum and immunological
responses of birds during molt, it appears that alfalfa meal can be an effective
molt induction alternative.
DOI: 10.1016/j.biortech.2006.12.036
1812. Plant Cell Rep. 2007 Jul;26(7):1035-44. Epub 2007 Feb 27.
Non-antibiotic, efficient selection for alfalfa genetic engineering.
Rosellini D(1), Capomaccio S, Ferradini N, Savo Sardaro ML, Nicolia A, Veronesi

F.
Author information:
(1)Dipartimento di Biologia Vegetale Biotecnologie Agroambientali e Zootecniche,
Università degli Studi di Perugia, Borgo XX giugno 74, 06121 Perugia, Italy.
roselli@unipg.it
A selectable marker gene (SMG), usually conferring resistance to an antibiotic or

herbicide, is generally introduced into the plant cells with the gene(s) for the
trait of interest to allow only the cells that have integrated and express the
foreign sequences to regenerate into a plant. The availability of several SMGs
for each plant species is useful for both basic and applied research to combine
several genes of interest in the same plant. A selection system based on
gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the
bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde
aminotransferase (GSA-AT)] as the SMG was previously used for genetic
transformation of tobacco. The hemL gene is a good candidate for a safe SMG,
because GSA-AT is present in all plants and is likely involved in one metabolic
step only, so that unintended effects of its overexpression in plants are not
probable. In this work, we have compared this new selection system with the
conventional, kanamycin-based system for alfalfa Agrobacterium-mediated
transformation. The hemL and NptII genes were placed together into a T-DNA under
the control of identical promoters and terminators. We show that the
gabaculine-based system is more efficient than the conventional, kanamycin-based
system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect
of its expression was observed.
DOI: 10.1007/s00299-007-0321-z
1813. J Environ Qual. 2007 Mar 1;36(2):532-9. Print 2007 Mar-Apr.

Freezing and drying effects on potential plant contributions to phosphorus in
runoff.
Roberson T(1), Bundy LG, Andraski TW.
Author information:
(1)Department of Soil Science, University of Wisconsin, Madison, WI 53706-1299,
USA.
Phosphorus (P) in runoff from landscapes can promote eutrophication of natural

waters. Soluble P released from plant material can contribute significant amounts
of P to runoff particularly after plant freezing or drying. This study was
conducted to evaluate P losses from alfalfa or grass after freezing or drying as
potential contributors to runoff P. Alfalfa (Medicago sativa L.) and grass
(principally, Agropyron repens L.) plant samples were subjected to freezing and
drying treatments to determine P release. Simulated rainfall runoff and natural
runoff from established alfalfa fields and a grass waterway were collected to
study P contributions from plant tissue to runoff. The effects of freezing and
drying on P released from plant tissue were simulated by a herbicide treatment in
selected experiments. Soluble reactive P (SP) extracted from alfalfa and grass
samples was markedly increased by freezing or drying. In general, SP extracted
from plant samples increased in the order fresh < frozen < frozen/thawed < dried,
and averaged 1, 8, 14, and 26% of total P in alfalfa, respectively. Soluble
reactive P extracted from alfalfa after freezing or drying increased with
increasing soil test P (r(2) = 0.64 to 0.68), suggesting that excessive soil P
levels increased the risk of plant P contributions to runoff losses. In simulated
rainfall studies, paraquat (1,1'-dimethyl-4, 4''-bipyridinium ion) treatment of
alfalfa increased P losses in runoff, and results suggested that this treatment
simulated the effects of drying on plant P loss. In contrast to the simulated
rainfall results, natural runoff studies over 2 yr did not show higher runoff P
losses that could be attributed to P from alfalfa. Actual P losses likely depend
on the timing and extent of plant freezing and drying and of precipitation events
after freezing.
DOI: 10.2134/jeq2006.0169
1814. Plant Methods. 2007 Feb 27;3:4.
A database of PCR primers for the chloroplast genomes of higher plants.
Heinze B(1).
Author information:
(1)Department of Genetics, Federal Research Centre for Forests, Hauptstrasse 7,
Vienna, Austria. berthold.heinze@bfw.gv.at
BACKGROUND: Chloroplast genomes evolve slowly and many primers for PCR
amplification and analysis of chloroplast sequences can be used across a wide
array of genera. In some cases 'universal' primers have been designed for the
purpose of working across species boundaries. However, the essential information
on these primer sequences is scattered throughout the literature.
RESULTS: A database is presented here which assembles published primer
information for chloroplast DNA. Additional primers were designed to fill gaps
where little or no primer information could be found. Amplicons are either the
genes themselves (typically useful in studies of sequence variation in
higher-order phylogeny) or they are spacers, introns, and intergenic regions (for
studies of phylogeographic patterns within and among species). The current list
of 'generic' primers consists of more than 700 sequences. Wherever possible, we
give the locations of the primers in the thirteen fully sequenced chloroplast
genomes (Nicotiana tabacum, Atropa belladonna, Spinacia oleracea, Arabidopsis
thaliana, Populus trichocarpa, Oryza sativa, Pinus thunbergii, Marchantia
polymorpha, Zea mays, Oenothera elata, Acorus calamus, Eucalyptus globulus,
Medicago trunculata).
CONCLUSION: The database described here is designed to serve as a resource for
researchers who are venturing into the study of poorly described chloroplast
genomes, whether for large- or small-scale DNA sequencing projects, to study
molecular variation or to investigate chloroplast evolution.
DOI: 10.1186/1746-4811-3-4
PMCID: PMC1821018
PMID: 17326828
1815. Microbiology. 2007 Mar;153(Pt 3):727-36.
Sinorhizobium meliloti pSymB carries genes necessary for arabinose transport and
catabolism.
Poysti NJ(1), Loewen ED, Wang Z, Oresnik IJ.
Author information:
(1)Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2,
Canada.
Arabinose is a known component of plant cell walls and is found in the

rhizosphere. In this work, a previously undeleted region of the megaplasmid pSymB
was identified as encoding genes necessary for arabinose catabolism, by Tn5-B20
random mutagenesis and subsequent complementation. Transcription of this region
was measured by beta-galactosidase assays of Tn5-B20 fusions, and shown to be
strongly inducible by arabinose, and moderately so by galactose and seed exudate.
Accumulation of [(3)H]arabinose in mutants and wild-type was measured, and the
results suggested that this operon is necessary for arabinose transport. Although
catabolite repression of the arabinose genes by succinate or glucose was not
detected at the level of transcription, both glucose and galactose were found to
inhibit accumulation of arabinose when present in excess. To determine if glucose
was also taken up by the arabinose transport proteins, [(14)C]glucose uptake
rates were measured in wild-type and arabinose mutant strains. No differences in
glucose uptake rates were detected between wild-type and arabinose catabolism
mutant strains, indicating that excess glucose did not compete with arabinose for
transport by the same system. Arabinose mutants were tested for the ability to
form nitrogen-fixing nodules on alfalfa, and to compete with the wild-type for
nodule occupancy. Strains unable to utilize arabinose did not display any
symbiotic defects, and were not found to be less competitive than wild-type for
nodule occupancy in co-inoculation experiments. Moreover, the results suggest
that other loci are required for arabinose catabolism, including a gene encoding
arabinose dehydrogenase.
DOI: 10.1099/mic.0.29148-0
1816. Science. 2007 Feb 23;315(5815):1069.
Environmental regulation. U.S. courts say transgenic crops need tighter scrutiny.
Charles D.
DOI: 10.1126/science.315.5815.1069
1817. Plant J. 2007 Mar;49(6):1076-90. Epub 2007 Feb 22.
A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and

carbohydrate metabolism.
Kempa S(1), Rozhon W, Samaj J, Erban A, Baluska F, Becker T, Haselmayer J,

Schleiff E, Kopka J, Hirt H, Jonak C.
Author information:
Sciences, Vienna Biocenter, A-1030 Vienna, Austria.
Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of

glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of
glucose, and serves as an energy and carbon store. Starch is the main
carbohydrate store in plants. Regulation of starch metabolism, in particular in
response to environmental cues, is of primary importance for carbon and energy
flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel
Medicago sativa GSK-3-like kinase, connects stress signalling with carbon
metabolism. MsK4 was found to be a plastid-localized protein kinase that is
associated with starch granules. High-salt stress rapidly induced the in vivo
kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines
revealed changes in sugar metabolism, including increased amounts of maltose, the
main degradation product of starch in leaves. Plants over-expressing MsK4 showed
improved tolerance to salt stress. Moreover, under high-salinity conditions,
MsK4-over-expressing plants accumulated significantly more starch and showed
modified carbohydrate content compared with wild-type plants. Overall, these data
indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism
to environmental stress.
DOI: 10.1111/j.1365-313X.2006.03025.x
PMCID: PMC1865003
1818. Chemosphere. 2007 Jun;68(2):259-65. Epub 2007 Feb 21.
Dissipation of 2,4-D in soils of the Humid Pampa region, Argentina: a microcosm

study.
Merini LJ(1), Cuadrado V, Flocco CG, Giulietti AM.
Author information:
(1)Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires Junín 956 (1113), Ciudad Autónoma de Buenos Aires,
Argentina.
Phenoxy herbicides like 2,4-dichlorophenoxyacetic acid (2,4-D) are widely used in

agricultural practices. Although its half life in soil is 7-14d, the herbicide
itself and its first metabolite 2,4-dichlorophenol (2,4-DCP) could remain in the
soil for longer periods, as a consequence of its intensive use. Microcosms assays
were conducted to study the influence of indigenous microflora and plants
(alfalfa) on the dissipation of 2,4-D from soils of the Humid Pampa region,
Argentina, with previous history of phenoxy herbicides application. Results
showed that 2,4-D was rapidly degraded, and the permanence of 2,4-DCP in soil
depended on the presence of plants and soil microorganisms. Regarding soil
microbial community, the presence of 2,4-D degrading bacteria was detected even
in basal conditions in this soil, possibly due to the adaptation of the
microflora to the herbicide. There was an increment of two orders of magnitude in
herbicide degraders after 15d from 2,4-D addition, both in planted and unplanted
microcosms. Total heterotrophic bacteria numbers were about 1x10(8) CFUg(-1) dry
soil and no significant differences were found between different treatments.
Overall, the information provided by this work indicates that the soil under
study has an important intrinsic degradation capacity, given by a microbial
community adapted to the presence of phenoxy herbicides.
1819. Mol Plant Microbe Interact. 2007 Feb;20(2):138-45.
Identification and characterization of a nodH ortholog from the

alfalfa-nodulating Or191-like rhizobia.
Del Papa MF(1), Pistorio M, Draghi WO, Lozano MJ, Giusti MA, Medina C, van
Dillewijn P, Martínez-Abarca F, Moron Flores B, Ruiz-Sainz JE, Megías M, Pühler
A, Niehaus K, Toro N, Lagares A.
Author information:
(1)Instituto de Bioquímica y Biología Molecular, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Calles 47 y 115 (1900) La Plata, Argentina.
Nodulation of Medicago sativa (alfalfa) is known to be restricted to

Sinorhizobium meliloti and a few other rhizobia that include the poorly
characterized isolates related to Rhizobium sp. strain Or191. Distinctive
features of the symbiosis between alfalfa and S. meliloti are the marked
specificity from the plant to the bacteria and the strict requirement for the
presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its
reducing end. Here, we present evidence of the presence of a functional
nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene,
present in single copy, maps to a high molecular weight megaplasmid. As in S.
meliloti, a nodF homolog was identified immediately upstream of nodH that was
transcribed in the opposite direction (local synteny). This novel nodH ortholog
was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes
when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH
disruption in the Or191-like bacteria did not abolish their ability to nodulate
alfalfa, resulting instead in a severely delayed nodulation. In agreement with
evidence from other authors, the nodH sequence analysis strongly supports the
idea that the Or191-like rhizobia most likely represent a genetic mosaic
resulting from the horizontal transfer of symbiotic genes from a sinorhizobial
megaplasmid to a not yet clearly identified ancestor.
DOI: 10.1094/MPMI-20-2-0138
1820. Sci China C Life Sci. 2006 Dec;49(6):603-12.
Plant species, atmospheric CO2 and soil N interactively or additively control C

allocation within plant-soil systems.
F U S(1), Ferris H.
Author information:
(1)Institute of Ecology, South China Botanical Garden, Chinese Academy of
Sciences, Guangzhou 510650, China. sfu@scbg.ac.cn
Two plant species, Medicago truncatula (legume) and Avena sativa (non-legume),
were grown in low- or high-N soils under two CO2 concentrations to test the
hypothesis whether C allocation within plant-soil system is interactively or
additively controlled by soil N and atmospheric CO2 is dependent upon plant
species. The results showed the interaction between plant species and soil N had
a significant impact on microbial activity and plant growth. The interaction
between CO2 and soil N had a significant impact on soil soluble C and soil
microbial biomass C under Madicago but not under Avena. Although both CO2 and
soil N affected plant growth significantly, there was no interaction between CO2
and soil N on plant growth. In other words, the effects of CO2 and soil N on
plant growth were additive. We considered that the interaction between N2
fixation trait of legume plant and elevated CO2 might have obscured the
interaction between soil N and elevated CO2 on the growth of legume plant. In
low-N soil, the shoot-to-root ratio of Avena dropped from 2.63 +/- 0.20 in the
early growth stage to 1.47 +/- 0.03 in the late growth stage, indicating that
Avena plant allocated more energy to roots to optimize nutrient uptake (i.e. N)
when soil N was limiting. In high-N soil, the shoot-to-root ratio of Medicago
increased significantly over time (from 2.45 +/- 0.30 to 5.43 +/- 0.10),
suggesting that Medicago plants allocated more energy to shoots to optimize
photosynthesis when N was not limiting. The shoot-to-root ratios were not
significantly different between two CO2 levels.
1821. Environ Pollut. 2007 Sep;149(1):99-103. Epub 2007 Feb 16.
Alfalfa nutritive quality for ruminant livestock as influenced by ambient air

quality in west-central Alberta.
Lin JC(1), Nosal M, Muntifering RB, Krupa SV.
Author information:
(1)Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA.
Alfalfa (Medicago sativa) nutritive quality response to ambient ozone (O(3)),

sulfur dioxide (SO(2)) and oxides of nitrogen (NO(x)) were assessed at three
locations in west-central Alberta, Canada (1998-2002). Yield data were segregated
into high and low relative to overall median yield. Ozone concentrations (hourly
median and 95th-percentile) and precipitation (P) contributed 69 and 29%,
respectively, to the variability in crude protein (CP) concentration in
low-yielding alfalfa, whereas mean temperature (T) and relative humidity (RH)
collectively influenced 98% of the variation in CP in high-yielding alfalfa.
Three-fourths of the accounted variation in relative feed value (RFV) of
low-yielding alfalfa was attributable to P, T and RH, whereas median and
95th-percentile hourly O(3) concentrations and SO(2) and NO(x) exposure integrals
contributed 25%. In contrast, air quality, (mainly O(3)) influenced 86% of the
accounted variation in RFV of high-yielding alfalfa, and T and P collectively
contributed 14%.
DOI: 10.1016/j.envpol.2006.12.009
1822. J Biosci. 2006 Dec;31(5):599-605.
Potential application of urea-derived herbicides as cytokinins in plant tissue

culture.
Srinivasan M(1), Nachiappan V, Rajasekharan R.
Author information:
(1)Department of Biochemistry, Indian Institute of Science, Bangalore 560 012,
India.
Various urea-derived herbicides and different cytokinin analogues were used to

determine their effects on callusing response and shoot regenerating capacity of
alfalfa (Medicago sativa L.) and Coleus (Coleus forskohlii Briq.). The herbicides
monuron and diuron evoked profuse callusing response from Coleus leaf segments
and alfalfa petiole explants on Murashige and Skoog medium. Shoot regeneration by
monuron (2.0 mg/l) showed a maximum of 3 multiple shoots both in alfalfa and
Coleus with a frequency of 92% and 75%, respectively. Whereas diuron (0.5 mg/l)
showed a high frequency of shoot regeneration (89%)with a mean number of 5 shoots
in alfalfa, in C.forskohlii, the frequency of regeneration was 90%with a mean
number of 6 shoots. Diuron with two chloride groups in the phenyl ring showed
significantly higher cytokinin-like activity than single chloride substitution
monuron. This study demonstrates the potential use of monuron and diuron as
cytokinins in plant tissue culture.
1823. Pak J Biol Sci. 2007 Feb 15;10(4):590-7.
Effects of live yeast Saccharomyces cerevisiae on fermentation parameters and

microbial populations of rumen, total tract digestibility of diet nutrients and
on the in situ degradability of alfalfa hay in Iranian Chall sheep.
Khadem AA(1), Pahlavan M, Afzalzadeh A, Rezaeian M.
Author information:
(1)Department of Animal Science, Abourayhan Institute, University of Tehran, P.O.
Box 11365-4117, Tehran, Iran.
The effects of live yeast Saccharomyces cerevisiae (LYSC, strain Sc 47) on rumen
fermentation and microbial populations, digestibility of nutrients, degradability
of alfalfa hay and on the performances of sheep were investigated in two
experiments. In both experiments, animals in treatment groups were received 0,
2.5, 5 and 7.5 g of LYSC per sheep per day and were defined as control, 2.5, 5.0
and 7.5 g LYSC treatment groups, respectively. In the first experiment, four
fistulated Chall sheep (49 +/- 0.5 kg BW) were kept in individual metabolic
crates under a 4x4 Latin square design and fed a Total Mixed Ration (TMR)
containing of barely (48%), wheat bran (16%), shelled corn (5%), mineral-vitamin
mix (1%) and of alfalfa hay (30%). In the second experiment, 28 sheep (48 +/- 0.5
kg BW) were assigned into four treatment groups under a complete randomized
design and fed a TMR containing of barely (60%), wheat bran (7%), cottonseed meal
(2%), mineral-vitamin mix (1%) and of alfalfa hay (15%) and wheat straw (15%).
The highest and the lowest ruminal pH values (p < 0.01) were recorded for sheep
in 2.5 g LYSC and control groups, respectively. At 3 h post-feeding, the total
VFA of rumen fluid was increased (p < 0.01) from 91.26 to 103.34 mmol L(-1) in
control vs. 2.5 g LYSC groups. The ruminal NH3-N of sheep was decreased (p <
0.01) from 159.63 to 128.90 mg L(-1) in control vs. 2.5 g LYSC groups. Bacterial
populations of rumen fluids were differed from 14 to 43% in treatment groups
although the differences were not significant. Compared to the other groups,
voluntary feed intake was higher (p < 0.01) in 5.0 g LYSC group and this was
ended to an inappropriate feed to gain ratio in this group. It can be concluded
that the use of LYSC at a level of 2.5 g per sheep per day could improve the
ruminal fermentation and resulted in a relatively better performances in Chall
sheep.
1824. J Dairy Sci. 2007 Mar;90(3):1440-51.
Use of exogenous fibrolytic enzymes to enhance in vitro fermentation of alfalfa

hay and corn silage.
Eun JS(1), Beauchemin KA, Schulze H.
Author information:
4B1, Canada.
Two in vitro experiments were performed to identify promising exogenous

fibrolytic enzyme products (EFE) and optimum dose rates (DR) for improving the
degradation of alfalfa hay and corn silage. The relationship between enzymatic
activity and fermentation responses was examined to identify optimum
formulations. In experiment 1, 5 EFE containing mainly endoglucanase and xylanase
activities, with different ratios between the 2 activities, were assessed at a DR
of 0.7, 1.4, and 2.1 mg/g of DM forage. Milled alfalfa hay or corn silage was
incubated in an in vitro batch culture with buffer, ruminal fluid, and EFE. Gas
production (GP) was measured during 24 h of incubation, and degradabilities of DM
and fiber were measured after terminating the incubation at 24 h. Two (E1 and E3)
EFE substantially improved GP and degradation of alfalfa hay and corn silage
fiber. The optimum DR of these EFE was 1.4 mg/g of DM for both forages with
improvements in NDF degradability up to 20.6% for alfalfa hay and up to 60.3% for
corn silage. Whereas added activities of endoglucanase and exoglucanase were
positively correlated with improvement in NDF degradability for alfalfa hay and
corn silage, there was no relationship between added xylanase activity and NDF
degradability. The 2 most promising EFE from experiment 1 were reevaluated in
experiment 2, alone and in combination with a high xylanase EFE, to determine
whether their effectiveness could be enhanced by decreasing the endoglucanase to
xylanase ratio. The 2 EFE improved GP and fiber degradation in a manner similar
to that observed in experiment 1, but the combination treatments resulted in no
further beneficial effects. Exogenous fibrolytic enzyme products can greatly
improve forage utilization, but DR and the activities supplied are critical for
achieving this response. Products used with alfalfa hay and corn silage should
contain high endoglucanase activity, with an ideal ratio of endoglucanse to
xylanase.
DOI: 10.3168/jds.S0022-0302(07)71629-6
1825. J Dairy Sci. 2007 Mar;90(3):1405-16.
Kinetics of fatty acid biohydrogenation in vitro.

Ribeiro CV(1), Eastridge ML, Firkins JL, St-Pierre NR, Palmquist DL.
Author information:
Biohydrogenation (BH) of fatty acids (FA) from fresh alfalfa and alfalfa hay with
varying levels of supplemental sucrose and media pH was evaluated in vitro. A
multicompartmental model was then developed to estimate pool size and flux of
vaccenic acid (VA) during BH of FA in fresh alfalfa. To vary incubation pH,
alfalfa samples were inoculated with rumen fluid in 2 media differing in molarity
of the bicarbonate buffer. Samples were incubated for 0, 1, 2, 3, 4, 6, 9, and 12
h; pH was measured and tubes were put in ice and stored until analysis. The BH
rates of linoleic acid (18:2) and linolenic acid (18:3) were estimated by PROC
NLIN of SAS (single pool, first-order kinetic model) and SAAM II (multiple pools,
first-order kinetic model). Both methods gave similar estimates for the BH rates
of 18:2 and 18:3 as well as the temporal pool size of VA. The BH rates (%/h) in
the strong (SB) and weak buffers (WB) were 27.4 (+/-0.7) and 23.5 (+/-0.9) for
18:2, and 43.8 (+/-0.2) and 30.3 (+/-0.6) for 18:3, respectively. The WB
decreased the BH rates of 18:2 and 18:3 for both forage sources. However, BH
rates of 18:3 were higher from fresh alfalfa than alfalfa hay. There was no
effect of sucrose addition on the BH rates of 18:2 and 18:3. Moreover, there was
no effect of buffer on the BH of VA estimated by the multiple pools model between
the SB and WB (12.5 +/- 2.1 and 14.1 +/- 3.7%/h, respectively). The BH rates of
the conjugated linoleic acid isomers were not different between the SB and WB
treatments (36.7 +/- 19.8 and 25.9 +/- 27.2, respectively). Because we could
estimate fluxes as well as mass of the VA pools, more information was generated
from the data when a multiple pools model was used compared with a single pool,
first-order kinetic model.
DOI: 10.3168/jds.S0022-0302(07)71626-0
1826. J Dairy Sci. 2007 Mar;90(3):1392-404.
Effects of feeding formate-treated alfalfa silage or red clover silage on omasal

nutrient flow and microbial protein synthesis in lactating dairy cows.
Brito AF(1), Broderick GA, Olmos Colmenero JJ, Reynal SM.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison 53706, USA.
Eight ruminally cannulated Holstein cows that were part of a larger lactation
trial were blocked by days in milk and randomly assigned to replicated 4 x 4
Latin squares to quantify effects of nonprotein N (NPN) content of alfalfa silage
(AS) and red clover silage (RCS) on omasal nutrient flows. Diets, fed as total
mixed rations, contained 50% dry matter from control AS (CAS), ammonium
tetraformate-treated AS (TAS), late maturity RCS (RCS1), or early maturity RCS
(RCS2). Silages differed in NPN and acid detergent insoluble N (% of total N): 50
and 4% (CAS); 45 and 3% (TAS); 27 and 8% (RCS1); 29 and 4% (RCS2). The CAS, TAS,
and RCS2 diets had 36% high-moisture shelled corn and 3% soybean meal, and the
RCS1 diet had 31% high-moisture shelled corn and 9% soybean meal. All diets
contained 10% corn silage, 27% neutral detergent fiber, and 17 to 18% crude
protein. Compared with RCS, feeding AS increased the supply of rumen-degraded
protein and omasal flows of nonammonia N and microbial protein, which may explain
the improved milk yield observed in the companion lactation trial. However,
omasal flow of rumen-undegraded protein was 34% greater on RCS. Except for Arg,
omasal flows of individual AA, branched-chain AA, nonessential AA, essential AA,
and total AA did not differ between cows fed AS vs. RCS. Within AS diets, no
differences in omasal AA flows were observed. However, omasal flows of Asp, Ser,
Glu, Cys, Val, Ile, Tyr, Lys, total nonessential AA, and total AA all were higher
in cows fed RCS1 vs. cows fed RCS2. In this trial, there was no advantage to
reducing NPN content of hay-crop silage.
1827. J Dairy Sci. 2007 Mar;90(3):1378-91.
Effects of feeding formate-treated alfalfa silage or red clover silage on the

production of lactating dairy cows.
Broderick GA(1), Brito AF, Olmos Colmenero JJ.
Author information:
(1)Agricultural Research Service, USDA US Dairy Forage Research Center, 1925
Linden Drive West, Madison 53706, USA. gbroderi@wisc.edu
In trial 1, 15 Holsteins were fed 3 total mixed rations (TMR) with 33% neutral
detergent fiber in 3 x 3 Latin squares (28-d periods). Two TMR contained (dry
matter basis): 40% control alfalfa silage (CAS) or 40% ammonium
tetraformate-treated alfalfa silage (TAS), 20% corn silage (CS), 33%
high-moisture shelled corn (HMSC), 6% solvent soybean meal (SSBM), and 18% crude
protein (CP); the third TMR contained 54% red clover silage (RCS), 6% dried
molasses, 33% HMSC, 6% SSBM, and 16.3% CP. Silages differed in nonprotein N (NPN)
and acid detergent insoluble N (ADIN; % of total N): 50 and 4% (CAS); 45 and 3%
(TAS); 27 and 8% (RCS). Replacing CAS with TAS increased intake, yields of milk,
fat-corrected milk, protein, and solids-not-fat, and apparent dry matter and N
efficiency. Replacing CAS with RCS increased intake and N efficiency but not milk
yield. Replacing CAS or TAS with RCS lowered milk urea N, increased apparent
nutrient digestibility, and diverted N excretion from urine to feces. In trial 2,
24 Holsteins (8 ruminally cannulated) were fed 4 TMR in 4 x 4 Latin squares (28-d
periods). Diets included the CAS, TAS, and RCS (RCS1) fed in trial 1 plus an
immature RCS (RCS2; 29% NPN, 4% ADIN). The CAS, TAS, and RCS2 diets contained 36%
HMSC and 3% SSBM and the RCS1 diet contained 31% HMSC and 9% SSBM. All TMR had
50% legume silage, 10% CS, 27% neutral detergent fiber, and 17 to 18% CP. Little
difference was observed between cows fed CAS and TAS. Intakes of DM and yields of
milk, fat-corrected milk, fat, protein, lactose, and solids-not-fat, and milk fat
and protein content were greater on alfalfa silage vs. RCS. Blood urea N, milk
urea N, ruminal ammonia, and total urinary N excretion were reduced on RCS,
suggesting better N utilization on the lower NPN silage. Apparent N efficiency
tended to be higher for cows fed RCS but there was no difference when N
efficiency was expressed as kilograms of milk yield per kilogram of total N
excreted.
[Community structure and its dynamics of predatory arthropod in jujube orchards

intercropped with different herbage species].
Shi G(1), Wang Y, Miao Z, Li D, Zhang T, Yu T, Ji Q, Dong H.
Author information:
(1)Key Laboratory of New Technology of Agricultural Application, Beijing 102206,
China. glshi@126.com
By using community structural characteristic indices and principal component

analysis, this paper studied the community structure and its dynamics of
predatory arthropod in the jujube orchards intercropped with Astrugalus
complanatus, Trifolium repen, Lotus comiculotus, and Medicago sativa. The results
showed that in all test jujube orchards, spider and predatory insects were the
predominant components of the predatory arthropod community, and their relative
abundances were 48.3% - 52.7% and 38.8% - 44.4% , respectively. There were
significant differences (P < 0.05) in the mean density, diversity, and evenness
of the most common predatory arthropod groups in the jujube orchards intercropped
with different herbage species, with the sequence of intercropped with Lotus
comiculotus > Medicago sativa > Astrugalus complanatu > Trifolium repens, but for
dominant concentration index, the sequence was intercropped with Trifolium repens
> Astrugalus complanatu > Medicago sativa > Lotus comiculotus. The average
density of predatory spiders was significant higher (P < 0.05) than that of
predatory insects in all test jujube orchards. The individuals of Coccinellidae,
Pentatomidae, inoccllidae, Chrysopidae, Thomisidae, Araneidae and Phytoseiidae
played the dominant role in the community.
1829. Pak J Biol Sci. 2007 Feb 1;10(3):481-5.
Comparison of somatic embryogenesis in Medicago sativa and Medicago truncatula.
Hoori F(1), Ehsanpour AA, Mostajeran A.
Author information:
(1)Department of Biology, Faculty of Science, Isfahan University, Isfahan, Iran.
In this study, the regeneration through embryogenesis of two species of Medicago

were studied. Seeds of Medicago sativa cv. Rehnani and M. truncatula line A17
were grown on MS medium. After 4-6 weeks, segments of leaf and stem from two
species were transferred to MS medium containing 2 mg L(-1) NAA, 2,4-D and
Kinetin. The results indicated that callus formation from leaf explants of M.
sativa was higher than M. trancatula. In the next stage, media with different
combinations of auxin, cytokinin or ethinyl estradiol were provided for
regeneration. Then in two stages, explants of leaf and stem of two species were
transferred on these media. Results after 3-6 weeks showed that in medium
containing NAA and TDZ, stem pieces ofM. sativa produced shoots while leaf pieces
on NAA and ethinyl estradiol formed roots. Leaf explants of M. truncatula in the
medium containing NAA and BAP, produced somatic embryos. Also in media with auxin
and ethinyl estradiol, somatic embryos were formed on calli of two species.
Ethinyl estradiol and auxin together can induce somatic embryogenesis and root
production on calli and stem or leaf explants.
1830. Microbiology. 2007 Feb;153(Pt 2):388-98.

Roles of poly-3-hydroxybutyrate (PHB) and glycogen in symbiosis of Sinorhizobium
meliloti with Medicago sp.
Wang C(1), Saldanha M, Sheng X, Shelswell KJ, Walsh KT, Sobral BW, Charles TC.
Author information:
(1)Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State
University, Blacksburg, VA 24061, USA. cwang@vbi.vt.edu
Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in

Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume
symbiosis are not fully understood. Glycogen synthase mutations were constructed
by in-frame deletion (glgA1) or insertion (glgA2). These mutations were combined
with a phbC mutation to make all combinations of double and triple mutants. PHB
was not detectable in any of the mutants containing the phbC mutation; glycogen
was not detectable in any of the mutants containing the glgA1 mutation. PHB
levels were significantly lower in the glgA1 mutant, while glycogen levels were
increased in the phbC mutant. Exopolysaccharide (EPS) was not detected in any of
the phbC mutants, while the glgA1 and glgA2 mutants produced levels of EPS
similar to the wild-type. Symbiotic properties of these strains were investigated
on Medicago truncatula and Medicago sativa. The results indicated that the
strains unable to synthesize PHB, or glycogen, were still able to form nodules
and fix nitrogen. However, phbC mutations caused greater nodule formation delay
on M. truncatula than on M. sativa. Time-course studies showed that (1) the
ability to synthesize PHB is important for N(2) fixation in M. truncatula nodules
and younger M. sativa nodules, and (2) the blocking of glycogen synthesis
resulted in lower levels of N(2) fixation on M. truncatula and older nodules on
M. sativa. These data have important implications for understanding how PHB and
glycogen function in the interactions of S. meliloti with Medicago spp.
DOI: 10.1099/mic.0.29214-0
1831. Microbiology. 2007 Feb;153(Pt 2):375-87.
A Sinorhizobium meliloti minE mutant has an altered morphology and exhibits

defects in legume symbiosis.
Cheng J(1), Sibley CD, Zaheer R, Finan TM.
Author information:
Hamilton, Ontario L8S 4K1, Canada.
Sinorhizobium meliloti differentiates from rod-shaped, free-living cells into

pleomorphic, non-dividing, N(2)-fixing bacteroids within alfalfa root nodules.
Here, the role of the minCDE genes in bacteroid differentiation and in
free-living cell division is examined. Disruption of the minE gene resulted in
large, swollen and branched free-living cells, and in symbiosis a minE mutation
resulted in a defect in nitrogen fixation with activity reduced by approximately
70 % compared to the wild-type. It has been demonstrated that the minCDE genes
form an operon driven by a promoter located 173 bp upstream of minC. The minCDE
genes were expressed in free-living cells and in both the infection zone and the
symbiotic zone of alfalfa nodules; however, no changes in the free-living cell
morphology, growth or symbiotic N(2) fixation were detected as a result of
deletion of these genes. Induced production of individual or combinations of Min
proteins in S. meliloti altered its rod-shaped cell morphology. Moreover, cell
morphologies resulting from the overexpression of the S. meliloti Min proteins in
Escherichia coli suggested similar functions for the E. coli and S. meliloti min
genes. These data suggest that there is greater redundancy in the roles of cell
division genes in S. meliloti compared with E. coli.
DOI: 10.1099/mic.0.2006/001362-0
1832. Cell Microbiol. 2007 Jun;9(6):1491-506. Epub 2007 Jan 23.
Two mitogen-activated protein kinase signalling cascades mediate basal resistance

to antifungal plant defensins in Fusarium graminearum.
Ramamoorthy V(1), Zhao X, Snyder AK, Xu JR, Shah DM.
Author information:
(1)Donald Danforth Plant Science Center, St Louis, MO 63132, USA.
Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth
of Fusarium graminearum, which causes head blight disease in cereals. In order to
determine the signalling cascades that are modulated by these defensins, we have
isolated several insertional mutants of F. graminearum that exhibit
hypersensitivity to MsDef1, but not to MtDef4. The molecular characterization of
two of these mutants, designated enhanced sensitivity to defensin (esd), has
revealed that the Mgv1 and Gpmk1 MAP kinase signalling cascades play a major role
in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4. The
Hog1 MAP kinase signalling cascade, which is responsible for adaptation of this
fungus to hyperosmotic stress, does not participate in the fungal response to
these defensins. Significantly, the esd mutants also exhibit hypersensitivity to
other tested defensins and are highly compromised in their pathogenesis on wheat
heads and tomato fruits. The studies reported here for the first time implicate
two MAP kinase signalling cascades in a plant defensin-mediated alteration of
fungal growth. Based on our findings, we propose that specific MAP kinase
signalling cascades are essential for protection of a fungal pathogen from the
antimicrobial proteins of its host plant.
DOI: 10.1111/j.1462-5822.2006.00887.x
1833. Curr Microbiol. 2007 Mar;54(3):167-74.
Resuscitation of viable but not culturable Sinorhizobium meliloti 41 pRP4-luc:

effects of oxygen and host plant.
Basaglia M(1), Povolo S, Casella S.
Author information:
(1)Dipartimento di Biotecnologie Agrarie, Agripolis-Università di Padova, Viale
dell'Università 16, 35020 Legnaro, Padova, Italy.
A plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and stably

inherited in Sinorhizobium meliloti 41 as a reporter gene. The strain obtained,
S. meliloti 41/pRP4-luc, and its parental strain served as a model system for
viable but not culturable (VBNC) resuscitation experiments in both in vitro and
soil samples. Incubation under oxygen (02) concentrations varying from 1% to
atmospheric levels did not result in resuscitation. A demonstration of recovery
was attained through exposure to the appropriate concentrations of antibiotics,
bacteriostatic chloramphenicol, and bactericidal ampicillin. The resuscitation
ratio was 1 recovered VBNC cell in every 10(5) 5-cyano-2,3-di-4-tolyl-tetrazolium
chloride (CTC+) bacteria. Although isolated VBNC rhizobia were unable to nodulate
Medicago sativa, which apparently did not enhance VBNC reversion, resuscitated
bacteria maintained their symbiotic properties. Soil experiments showed that the
lack of O2 leads to onset of VBNC status as in liquid microcosm, but the number
of recoverable and culturable cells decreased more drastically in soil.
DOI: 10.1007/s00284-005-0482-3
1834. New Phytol. 2007;173(3):630-40.
Independent action and contrasting phenotypes of resistance genes against spotted

alfalfa aphid and bluegreen aphid in Medicago truncatula.
Klingler JP(1), Edwards OR, Singh KB.
Author information:
(1)Commonwealth Scientific and Industrial Research Organization Entomology,
Private Bag 5, Wembley, WA 6913, Australia. kli026@csiro.au
Host resistance to aphids is poorly understood. Medicago truncatula, a model

legume and cultivated pasture species, was used to elucidate defense against two
aphid species, Therioaphis trifolii f. maculata (spotted alfalfa aphid, SAA) and
Acyrthosiphon kondoi (bluegreen aphid, BGA). Aphid performance and plant damage
were compared between near-isogenic cultivars, Mogul and Borung, that differ in
resistance to both aphids. Analyses of aphid resistance in Mogul x Borung F2
plants and their progeny revealed modes of action and chromosome locations of
resistance genes. Separate genes were identified for SAA resistance (TTR) and BGA
resistance (AKR); both mapped to chromosome 3 but were found to act independently
to reduce survival and growth of their target aphid species. The TTR locus
controls distinct, and contrasting, local and systemic plant responses between
the near-isogenic cultivars. TTR-mediated plant responses imply interaction
between a resistance factor(s) in vascular tissue and a bioactive component(s) of
SAA saliva. Features of both resistance traits suggest homology to aphid
resistance in other legumes; elucidation of their molecular mechanisms will
likely apply to other aphid-plant interactions.
DOI: 10.1111/j.1469-8137.2006.01939.x
1835. J Bacteriol. 2007 May;189(9):3591-602. Epub 2007 Jan 19.
The symbiosis regulator CbrA modulates a complex regulatory network affecting the
flagellar apparatus and cell envelope proteins.
Gibson KE(1), Barnett MJ, Toman CJ, Long SR, Walker GC.
Author information:
02139, USA.
Sinorhizobium meliloti participates in a nitrogen-fixing symbiosis with legume

plant host species of the genera Medicago, Melilotus, and Trigonella. We recently
identified an S. meliloti two-component sensory histidine kinase, CbrA, which is
absolutely required to establish a successful symbiosis with Medicago sativa (K.
E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol.
188:4508-4521, 2006). In addition to having a symbiotic defect, the cbrA::Tn5
mutant also has free-living phenotypes that suggest a cell envelope perturbation.
Because the bases for these phenotypes are not well understood, we undertook an
identification of CbrA-regulated genes. We performed a microarray analysis and
compared the transcriptome of the cbrA::Tn5 mutant to that of the wild type. Our
global analysis of gene expression identified 162 genes that are differentially
expressed in the cbrA::Tn5 mutant, including those encoding proteins involved in
motility and chemotaxis, metabolism, and cell envelope function. With regard to
those genes with a known role in symbiosis, we observed increased expression of
nine genes with overlapping functions in bacterial invasion of its host, which
suggests that the mutant could be competent for invasion. Since these
CbrA-repressed genes are vital to the invasion process, it appears that
down-regulation of CbrA activity is important at this stage of nodule
development. In contrast, our previous work showed that CbrA is required for
bacteria to establish themselves within the host as nitrogen-fixing symbionts.
Therefore, we propose a model in which CbrA functions as a developmental switch
during symbiosis.
DOI: 10.1128/JB.01834-06
PMCID: PMC1855900
1836. J Dairy Sci. 2007 Feb;90(2):886-97.
Effects of essential oils on digestion, ruminal fermentation, rumen microbial

populations, milk production, and milk composition in dairy cows fed alfalfa
silage or corn silage.
Benchaar C(1), Petit HV, Berthiaume R, Ouellet DR, Chiquette J, Chouinard PY.
Author information:
Canada, Sherbrooke, Quebec, Canada J1M 1Z3. benchaarc@agr.gc.ca
Four Holstein cows fitted with ruminal cannulas were used in a 4 x 4 Latin square
design (28-d periods) with a 2 x 2 factorial arrangement of treatments to
investigate the effects of addition of a specific mixture of essential oil
compounds (MEO; 0 vs. 750 mg/d) and silage source [alfalfa silage (AS) vs. corn
silage (CS)] on digestion, ruminal fermentation, rumen microbial populations,
milk production, and milk composition. Total mixed rations containing either AS
or CS as the sole forage source were balanced to be isocaloric and
isonitrogenous. In general, no interactions between MEO addition and silage
source were observed. Except for ruminal pH and milk lactose content, which were
increased by MEO supplementation, no changes attributable to the administration
of MEO were observed for feed intake, nutrient digestibility, end-products of
ruminal fermentation, microbial counts, and milk performance. Dry matter intake
and milk production were not affected by replacing AS with CS in the diet.
However, cows fed CS-based diets produced milk with lower fat and higher protein
and urea N concentrations than cows fed AS-based diets. Replacing AS with CS
increased the concentration of NH(3)-N and reduced the acetate-to-propionate
ratio in ruminal fluid. Total viable bacteria, cellulolytic bacteria, and
protozoa were not influenced by MEO supplementation, but the total viable
bacteria count was higher with CS- than with AS-based diets. The apparent
digestibility of crude protein did not differ between the AS and CS treatments,
but digestibilities of neutral detergent fiber and acid detergent fiber were
lower when cows were fed CS-based diets than when they were fed AS-based diets.
Duodenal bacterial N flow, estimated using urinary purine derivatives and the
amount of N retained, increased in cows fed CS-based diets compared with those
fed AS-based diets. Feeding cows AS increased the milk fat contents of cis-9,
trans-11 18:2 (conjugated linoleic acid) and 18:3 (n-3 fatty acid) compared with
feeding cows CS. Results from this study showed limited effects of MEO
supplementation on nutrient utilization, ruminal fermentation, and milk
performance when cows were fed diets containing either AS or CS as the sole
forage source.
DOI: 10.3168/jds.S0022-0302(07)71572-2
1837. J Dairy Sci. 2007 Feb;90(2):823-32.
Milk from forage as affected by rumen degradable protein and corn grinding when
feeding corn- and alfalfa silage-based diets.
Charbonneau E(1), Chouinard PY, Allard G, Lapierre H, Pellerin D.
Author information:
(1)Département des sciences animales, Université Laval, Quebec, G1K 7P4 Canada.
To increase the production of milk from forage (MF), a previous experiment with
alfalfa silage showed the importance of a complementary combination of
concentrates and forages offered. When corn silage is fed with alfalfa,
increasing the rumen degradable protein (RDP) content in the diet should allow a
better utilization of forage energy. To evaluate this hypothesis, 8 multiparous
Holstein cows in early lactation were used in a replicated 4 x 4 Latin square
design with 3-wk periods. Diets were fed as total mixed rations and were
formulated to provide similar levels of net energy for lactation and crude
protein but differing in RDP. Corn and alfalfa silages were used. Treatments
were: 1) cracked corn-based concentrate providing low RDP [level recommended by
the NRC (2001); RDP = 11.1% of dry matter (DM)]; 2) cracked corn-based
concentrate providing medium RDP (RDP = 12.8% of DM); 3) cracked corn-based
concentrate providing high RDP (RDP = 14.5% of DM); and 4) ground corn-based
concentrate providing high RDP (RDP = 13.6% of DM). The first 3 treatments, using
cracked corn, were compared on the basis of their RDP level. For these
treatments, MF, calculated on a protein basis, decreased and the average of MF
calculated on an energy basis and MF calculated on a protein basis tended to
decrease as RDP increased. There was no difference for MF calculated on an energy
basis between treatments. Increasing dietary RDP levels decreased the milk yield
(from 32.8 to 30.7 kg/d) and milk protein yield (from 1,094 to 1,005 g/d) but not
the milk fat yield. The milk urea N concentration increased as RDP increased.
This suggests that there is no advantage of feeding RDP above the NRC
recommendations when diets are based on corn and alfalfa silage. At high RDP
levels (treatments 3 and 4), ground corn supported higher DM intake and yields of
milk and protein than did cracked corn. Milk from forage, calculated on a protein
basis, was higher and milk urea N decreased with ground corn. Even with corn
silage in the diet, grinding corn grain proved to be beneficial to milk yield and
MF production.
DOI: 10.3168/jds.S0022-0302(07)71566-7
1838. Environ Microbiol. 2007 Feb;9(2):465-73.
Plant-microbe association for rhizoremediation of chloronitroaromatic pollutants

with Comamonas sp. strain CNB-1.
Liu L(1), Jiang CY, Liu XY, Wu JF, Han JG, Liu SJ.
Author information:
(1)State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese
Academy of Sciences, Beijing 100080, China.
Comamonas sp. strain CNB-1, isolated from activated sludge and having a strong
ability to degrade 4-chloronitrobenzene (4CNB), was applied for rhizoremediation
of 4CNB-polluted soil through association with alfalfa. Confocal laser scanning
microscopy revealed that strain CNB-1 successfully colonized alfalfa roots.
Determination of strain CNB-1 populations by cultivation method and by
quantitative competitive PCR technique targeting the chloronitrobenzene
nitroreductase gene showed that the population of strain CNB-1 in the rhizosphere
was about 10-100 times higher than that in the bulk soil. Gnotobiotic and outdoor
experiments showed that pollutant 4CNB was completely removed within 1 or 2 days
after 4CNB application into soil, and that its phytotoxicity to alfalfa was
eliminated by inoculation of strain CNB-1. Results from PCR-denaturing gradient
gel electrophoresis and analysis of 16S rRNA gene libraries revealed that the
indigenous soil microbial community mainly consisted of alphaproteobacteria,
betaproteobacteria, gammaproteobacteria, the CFB bacteria
(Cytophaga-Flavabacterium-Bacteriodes), and Acidobacteria. This microbial
community was not significantly influenced by inoculation of strain CNB-1. Thus,
this study has developed a Comamonas-alfalfa system for rhizoremediation of 4CNB.
DOI: 10.1111/j.1462-2920.2006.01163.x
1839. Theor Appl Genet. 2007 Mar;114(5):901-13. Epub 2007 Jan 12.
Quantitative trait loci and candidate gene mapping of aluminum tolerance in

diploid alfalfa.
Narasimhamoorthy B(1), Bouton JH, Olsen KM, Sledge MK.
Author information:
(1)The Samuel Roberts Noble Foundation, 2510, Sam Noble Pkway, Ardmore, OK,
73402, USA. bnarasimhamoorthy@noble.org
Aluminum (Al) toxicity in acid soils is a major limitation to the production of

alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant
alfalfa cultivars is one approach to overcome this constraint. Accessions of wild
diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of
useful genes for Al tolerance. Previously, two genomic regions associated with Al
tolerance were identified in this diploid species using restriction fragment
length polymorphism (RFLP) markers and single marker analysis. This study was
conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to
identify simple sequence repeat (SSR) markers that flank the previously
identified QTLs; to map candidate genes associated with Al tolerance from other
plant species; and to test for co-localization with mapped QTLs. A genetic
linkage map was constructed using EST-SSR markers in a population of 130
BC(1)F(1) plants derived from the cross between Al-sensitive and Al-tolerant
genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III,
explaining 38, 16 and 27% of the phenotypic variation, respectively, were
identified. Six candidate gene markers designed from Medicago truncatula ESTs
that showed homology to known Al-tolerance genes identified in other plant
species were placed on the QTL map. A marker designed from a candidate gene
involved in malic acid release mapped near a marginally significant QTL (LOD
2.83) on LG I. The SSR markers flanking these QTLs will be useful for
transferring them to cultivated alfalfa via marker-assisted selection and for
pyramiding Al tolerance QTLs.
DOI: 10.1007/s00122-006-0488-7
PMCID: PMC1805042
1840. Ann Bot. 2007 Feb;99(2):275-84. Epub 2007 Jan 11.
Elevated atmospheric CO2 and strain of rhizobium alter freezing tolerance and
cold-induced molecular changes in alfalfa (Medicago sativa).
Bertrand A(1), Prévost D, Bigras FJ, Castonguay Y.
Author information:
(1)Agriculture and Agri-Food Canada, 2560, Soils and Crops Research and
Development Centre, 2560 Hochelaga Boulevard, Quebec, Quebec, G1V 2J3, Canada.
bertranda@agr.gc.ca
BACKGROUND AND AIMS: The objective of the study was to assess the impact of
elevated CO2 in interaction with rhizobial strains on freezing tolerance and
cold-induced molecular changes in alfalfa.
METHODS: Alfalfa inoculated with two different strains of rhizobium (A2 and
NRG34) was grown and cold acclimated (2 weeks at 2 degrees C) under either 400
(ambient) or 800 micromol mol(-1) (elevated) CO2.
KEY RESULTS: Plants acclimated under 400 micromol mol(-1) CO2 were more freezing
tolerant than those maintained under 800 micromol mol(-1). Cryoprotective sugars
typically linked with the acquisition of freezing tolerance such as sucrose,
stachyose and raffinose increased in roots in response to low temperature but did
not differ between CO2 treatments. Similarly high CO2 did not alter the
expression of many cold-regulated (COR) genes although it significantly increased
the level of transcripts encoding a COR gene homologous to
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). A significant effect of
rhizobial strain was observed on both freezing tolerance and gene expression.
Plants of alfalfa inoculated with strain A2 were more freezing tolerant than
those inoculated with strain NRG34. Transcripts of COR genes homologous to a
pathogenesis-related protein (PR-10) and to a nuclear-targeted protein were
markedly enhanced in roots of alfalfa inoculated with strain A2 as compared with
strain NRG34. Transcripts encoding the vegetative storage proteins (VSPs)
beta-amylase and chitinase were more abundant in roots of non-acclimated plants
inoculated with strain NRG34 than with strain A2.
CONCLUSIONS: Taken together, the results suggest that elevated CO2 stimulates
plant growth and reduces freezing tolerance. The acquisition of cold tolerance is
also influenced by the rhizobial strain, as indicated by lower levels of
expression of COR genes and sustained accumulation of VSP-encoding transcripts in
alfalfa inoculated with strain NRG34 as compared with strain A2.
DOI: 10.1093/aob/mcl254
PMCID: PMC2802994
1841. Mol Genet Genomics. 2007 Apr;277(4):413-25. Epub 2007 Jan 11.
Validation of in silico-predicted genic SNPs in white clover (Trifolium repens

L.), an outbreeding allopolyploid species.
Cogan NO(1), Drayton MC, Ponting RC, Vecchies AC, Bannan NR, Sawbridge TI, Smith
KF, Spangenberg GC, Forster JW.
Author information:
(1)Primary Industries Research Victoria, Victorian AgriBiosciences Centre, La
Trobe Research and Development Park, Bundoora, VIC, 3083, Australia.
White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid

forage legume. Gene-associated SNPs provide the optimum genetic system for
improvement of such crop species. An EST resource obtained from multiple cDNA
libraries constructed from numerous genotypes of a single cultivar has been used
for in silico SNP discovery and validation. A total of 58 from 236 selected
sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by
genotypic analysis across the parents and progeny of several two-way
pseudo-testcross mapping families. The clusters include genes belonging to a
broad range of predicted functional categories. Polymorphic SNP-containing ESTs
have also been used for comparative genomic analysis by comparison with whole
genome data from model legume species, as well as Arabidopsis thaliana. A total
of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal
locations in Medicago truncatula, which is closely related to white clover within
the Trifolieae tribe of the Fabaceae. This analysis provides access to
translational data from model species. The efficiency of in silico SNP discovery
in white clover is limited by paralogous and homoeologous gene duplication
effects, which are resolved unambiguously by the transmission test. This approach
will also be applicable to other agronomically important cross-pollinating
allopolyploid plant species.
DOI: 10.1007/s00438-006-0198-5
[Diurnal dynamics of photosynthetic and transpiration rates of alfalfa under dry

farming and their relationships with environmental factors].
Liu Y(1), Shi J, Jia Z, Han Q.
Author information:
(1)College of Agronomy, Northwest Sci-Tech University of Agriculture and
Forestry, Yangling 712100, China. liuyuhua00@sina.com.cn
In this paper, the diurnal dynamics of photosynthetic rate (Pn) and transpiration
rate ( Tr) of four years planted alfalfa under dry farming, as well as those of
photosynthetic active radiation (PAR) , CO2 concentration in field ( Ca ) ,
relative humidity (RH) and air temperature ( Ta) were measured, and the
relationships between the diurnal dynamics of Pn, Tr and climatic factors were
analyzed by correlation coefficient, path coefficient, and decision coefficient
analyses. The results showed that T. had the greatest effect on the diurnal
dynamics of Pn , while RH, PAR and C, affected P,, mainly through their acting on
Ta PAR had the greatest effect on the diurnal changes of Tr, and RH, Ta and Ca
affected Tr indirectly by acting on PAR. For the characteristics of
photosynthesis and transpiration, PAR was the primary determining factor, and Ta
was the main limiting factor.

1843. J Bacteriol. 2007 Mar;189(6):2510-20. Epub 2007 Jan 5.
Sinorhizobium meliloti SyrA mediates the transcriptional regulation of genes

involved in lipopolysaccharide sulfation and exopolysaccharide biosynthesis.
Keating DH(1).
Author information:
(1)Department of Microbiology and Immunology, Loyola University Chicago, Building
105, 2160 S. First Avenue, Maywood, IL 60153, USA. dkeati1@lumc.edu
Sinorhizobium meliloti is a gram-negative soil bacterium found either in

free-living form or as a nitrogen-fixing endosymbiont of leguminous plants such
as Medicago sativa (alfalfa). S. meliloti synthesizes an unusual sulfate-modified
form of lipopolysaccharide (LPS). A recent study reported the identification of a
gene, lpsS, which encodes an LPS sulfotransferase activity in S. meliloti.
Mutants bearing a disrupted version of lpsS exhibit an altered symbiosis, in that
they elicit more nodules than wild type. However, under free-living conditions,
the lpsS mutant displayed no change in LPS sulfation. These data suggest that the
expression of lpsS is differentially regulated, such that it is transcriptionally
repressed during free-living conditions but upregulated during symbiosis. Here, I
show that the expression of lpsS is upregulated in strains that constitutively
express the symbiotic regulator SyrA. SyrA is a small protein that lacks an
apparent DNA binding domain and is predicted to be located in the cytoplasmic
membrane yet is sufficient to upregulate lpsS transcription. Furthermore, SyrA
can mediate the transcriptional upregulation of exo genes involved in the
biosynthesis of the symbiotic exopolysaccharide succinoglycan. The SyrA-mediated
transcriptional upregulation of lpsS and exo transcription is blocked in mutants
harboring a mutation in chvI, which encodes the response regulator of a conserved
two-component system. Thus, SyrA likely acts indirectly to promote
transcriptional upregulation of lpsS and exo genes through a mechanism that
requires the ExoS/ChvI two-component system.
DOI: 10.1128/JB.01803-06
PMCID: PMC1899389
1844. Bioresour Technol. 2007 Dec;98(18):3547-54. Epub 2007 Jan 4.
Solid-state cultures of Fusarium oxysporum transform aromatic components of

olive-mill dry residue and reduce its phytotoxicity.
Sampedro I(1), D'Annibale A, Ocampo JA, Stazi SR, García-Romera I.
Author information:
(1)Departamento Microbiología, Estación Experimental del Zaidín, CSIC, Prof.
Albareda 1, E-18008 Granada, Spain.
The present study mainly investigated the ability of solid-state cultures of the
non-pathogenic Fusarium oxysporum strain BAFC 738 to transform aromatic
components to reduce the phytotoxicity in olive-mill dry residue (DOR), the waste
from the two-phase manufacturing process. Lignin, hemicellulose, fats and
water-soluble extractives contents of DOR colonized by the fungus for 20 weeks
were reduced by 16%, 25%, 71% and 13%, respectively, while the cellulose content
increased by 25%. In addition, the ethyl acetate-extractable phenolic fraction of
the waste was reduced by 65%. However, mass-balance ultra-filtration and
size-exclusion chromatography experiments suggested that the apparent removal of
that fraction, mainly including 2-(3,4-dihydroxyphenyl)ethyl alcohol and
2-(4-hydroxyphenyl)ethyl alcohol, was due to polymerization. Mn-peroxidase and
Mn-independent peroxidase activities were found in F. oxysporum solid-state
cultures, while laccase and aryl alcohol oxidase activities were not detected.
Tests performed with seedlings of tomato (Lycopersicum esculentum L.), soybean
(Glycine maximum Merr.), and alfalfa (Medicago sativa L.) grown on soils
containing 6% (w/w) of bioconverted DOR (kg soil)(-1) showed that the waste's
phytotoxicity was removed by 20 weeks-old fungal cultures. By contrast, the same
material exhibited a high residual toxicity towards lettuce (Lactuca sativa L.).
DOI: 10.1016/j.biortech.2006.11.015
1845. Plant Biotechnol J. 2007 Jan;5(1):16-25.
Engineering of a sialic acid synthesis pathway in transgenic plants by expression

of bacterial Neu5Ac-synthesizing enzymes.
Paccalet T(1), Bardor M, Rihouey C, Delmas F, Chevalier C, D'Aoust MA, Faye L,

Vézina L, Gomord V, Lerouge P.
Author information:
(1)CNRS-UMR 6037, GDR2590, IFRMP 23, Université de Rouen, 76821 Mont Saint Aignan
Cédex, France.
Plants are a low-cost and contamination-free factory for the production of

recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ
from their mammalian homologues by the structure of their N-linked glycans. For
instance, most mammalian glycoproteins harbour terminal sialic acids that control
their half-life in the bloodstream. The absence of the whole sialylation
machinery in plants is of major concern as non-sialylated plant-made
pharmaceuticals may not perform at their full potential in humans, because of
their removal from the circulation through the involvement of hepatic cell
receptors. In this context, we have investigated the synthesis of
N-acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the
re-routing of the endogenous 3-deoxy-d-manno-2-octulosonic acid (Kdo)
biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes.
In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use
N-acetyl d-mannosamine as a substrate, and thus re-routing of the Kdo pathway for
the synthesis of Neu5Ac is not possible. Consequently, we expressed genes
encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase (neuB2) from
Campylobacter jejuni in plants. These resulted in the production of functional
enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in
vitro. Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco
cells and Medicago sativa (alfalfa), the perennial legume crop.
DOI: 10.1111/j.1467-7652.2006.00211.x
1846. Anal Bioanal Chem. 2007 Mar;387(6):2199-205. Epub 2007 Jan 5.
Use of NIRS technology with a remote reflectance fibre-optic probe for predicting
mineral composition (Ca, K, P, Fe, Mn, Na, Zn), protein and moisture in alfalfa.
González-Martín I(1), Hernández-Hierro JM, González-Cabrera JM.
Author information:
(1)Departamento de Química Analítica, Nutrición y Bromatología, Facultad de
Ciencias Químicas, C/ Plaza de la Merced s/n, 37008, Salamanca, Spain.
inmaglez@usal.es
In the present work we study the use of near-infrared spectroscopy (NIRS)

technology together with a remote reflectance fibre-optic probe for the analysis
of major (Ca, K, P) and minor (Fe, Mn, Na, Zn) elements, protein and moisture in
alfalfa. The method allows immediate analysis of the alfalfa without prior sample
treatment or destruction through direct application of the fibre-optic probe on
ground samples in the case of the mineral composition and on-ground and compacted
(baled) samples in the case of protein and humidity. The regression method
employed was modified partial least-squares (MPLS). The calibration results
obtained using samples of alfalfa allowed the determination of Ca, K, P, Fe, Mn,
Na and Zn, with a standard error of prediction (SEP(C)) and a correlation
coefficient (RSQ) expressed in mg/kg of alfalfa of 1.37x10(3) and 0.878 for Ca,
1.10x10(3) and 0.899 for K, 227 and 0.909 for P, 103 and 0.948 for Fe, 5.1 and
0.843 for Mn, 86.2 and 0.979 for Na, and of 1.9 and 0.853 for Zn, respectively.
The SEP(C) and RSQ values (in %) for protein and moisture in ground samples were
0.548 and 0.871 and 0.150 and 0.981, respectively; while in the compacted samples
they were 0.564 and 0.826 and 0.262 and 0.935, respectively. The prediction
capacity of the model and the robustness of the method were checked in the
external validation in alfalfa samples of unknown composition, and the results
confirmed the suitability of the method.
DOI: 10.1007/s00216-006-1039-4
1847. J Agric Food Chem. 2007 Jan 10;55(1):32-7.
Evaluation of microwave irradiation for analysis of carbonyl sulfide, carbon

disulfide, cyanogen, ethyl formate, methyl bromide, sulfuryl fluoride, propylene
oxide, and phosphine in hay.
Ren Y(1), Mahon D.
Author information:
(1)CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia.
Yonglin.Ren@csiro.au
Fumigant residues in hay were "extracted" by microwave irradiation. Hay, in

gastight glass flasks, was placed in a domestic microwave oven, and fumigants
were released into the headspace by microwave irradiation. Power settings for
maximum release of fumigants were determined for carbonyl sulfide (COS), carbon
disulfide (CS(2)), cyanogen (C(2)N(2)), ethyl formate (EF), methyl bromide
(CH(3)Br), sulfuryl fluoride (SF), propylene oxide (PPO), and phosphine (PH(3)).
Recoveries of fortified samples were >91% for COS, CS(2), CH(3)Br, SF, PPO, and
PH(3) and >76% for C(2)N(2) and EF. Completeness of extraction was assessed from
the amount of fumigant retained by the microwaved hay. This amount was determined
from further microwave irradiation and was always small (<5% of the amount
obtained from the initial procedure). Limits of quantification were <0.1 mg/kg
for COS, CS(2), C(2)N(2), EF, and PH(3) and <0.5 mg/kg for CH(3)Br, SF, and PPO.
These low limits were essentially due to the absence of interference from
solvents and no necessity to inject large-volume gas samples. The microwave
method is rapid and solvent-free. However, care is required in selecting the
appropriate power setting. The safety implications of heating sealed flasks in
microwave ovens should be noted.
DOI: 10.1021/jf061952c
1848. Theor Appl Genet. 2007 Feb;114(4):647-57. Epub 2006 Dec 6.
Wide- versus specific-adaptation strategy for lucerne breeding in northern Italy.
Annicchiarico P(1).
Author information:
(1)CRA - Istituto Sperimentale per le Colture Foraggere, 29 viale Piacenza, 26900
Lodi, Italy. bred@iscf.it
This study is aimed at comparing wide- versus specific-adaptation strategies for

lucerne in northern Italy on the basis of actual dry matter yield gains over 12
harvests from phenotypic selection, assessing the value of specific genetic bases
and selecting environments for the contrasting subregion A (no drought
stress/sandy-loam soil) and subregion C (summer drought stress/silty-clay soil).
A second aim is to investigate the adaptive responses of five sets of 18 half-sib
progenies. The following selected populations were evaluated along with five
cultivars: GW-SW, GA-SA, GA-SC, GC-SC and GC-SA (where GW, GA and GC are the
genetic bases for wide adaptation, subregions A and C; SW, SA and SC are the
selection environments for wide adaptation, subregions A and C). The selection
and test environments were four artificial environments created by the factorial
combination of two drought stress levels by two soil types. Two environments
represented the subregions A and C whereas the combination of the other two
environments represented the intermediate subregion B. Genotype x environment
interaction (P < or = 0.001) due to both environmental factors and implying
cross-over interaction between the contrasting subregions occurred for the
populations and the five selections. Specific genetic bases (GA and GC) implied
gains in their target subregions of 5.2% for subregion A and 2.9% for subregion C
compared with the widely adapted one (GW). The gain of SA ('no stress/sandy-loam
soil') over SC ('stress/silty-clay soil') decreased from subregion A (10.6%)
through subregion C (1.7%) but exhibited an advantage per se across environments
of 5.4%. The best specific selections (GA-SA for subregions A and B; GC-SA for
subregion C) implied higher yields of 9.8% in subregion A and 6.5% in subregion
C, and over twofold greater selection efficiency across the region, relative to
GW-SW. Half-sib progeny x artificial environment interaction (P < or = 0.05)
occurred in three sets of progenies whose parents belonged to cultivars with
different or similar adaptation.
DOI: 10.1007/s00122-006-0465-1
1849. J Exp Bot. 2007;58(5):969-78. Epub 2006 Dec 21.
Unique properties of NADP-thioredoxin reductase C in legumes.
Alkhalfioui F(1), Renard M, Montrichard F.
Author information:
(1)Physiologie Moléculaire des Semences, UMR 1191 Université d'Angers-Institut
National d'Horticulture-INRA, ARES, 16 boulevard Lavoisier, 49045 Angers cedex
01, France.
NADP-thioredoxin reductases (NTRs) reduce thioredoxins (Trxs), using NADPH as a

reductant, together constituting complete redox systems (NTS). Beside NTRA and
NTRB targeted to both cytosol and mitochondria of plant cells, there is in
chloroplasts an unusual NTR (NTRC) harbouring a Trx domain in a C-terminal
extension, as recently reported in Oryza sativa. Although NTRC may constitute a
complete NTS, it was described as a bifunctional enzyme. Because the gene is only
present in photosynthetic organisms and the protein in green tissues, NTRC was
thought to have a role restricted to photosynthetic cells. To determine whether
NTRC from dicot plants is a bifunctional enzyme or a complete NTS, as well as to
identify its putative target, NTRC from Medicago truncatula was cloned and NTRA
was cloned for comparison. Here evidence is presented that MtNTRC (i) acts as an
NTS and reduces dithiobisnitrobenzoate (DTNB) with a turnover (0.62 s(-1))
similar to that measured with MtNTRA in the presence of a Trxh (0.81 s(-1)); (ii)
is able to use both NADPH (k(M)=2.4 microM) and NADH (k(M)=11 microM) as
cofactors; (iii) efficiently reduces BAS1, a plastidial peroxiredoxin; and (iv)
is expressed in both leaves and stems but unexpectedly is even more abundant in
cotyledons from dry and germinating seeds. Because BAS1 is also present in both
green tissues and seeds, NTRC/BAS1 may be involved in the scavenging of peroxides
produced in green tissues during the day or the night and in seeds during
germination. These results suggest different roles for NTRC in monocot and dicot
plants.
DOI: 10.1093/jxb/erl248
1850. Biochim Biophys Acta. 2006 Nov-Dec;1759(11-12):543-51. Epub 2006 Nov 18.
Identification and characterization of genes associated with the induction of

embryogenic competence in leaf-protoplast-derived alfalfa cells.
Domoki M(1), Györgyey J, Bíró J, Pasternak TP, Zvara A, Bottka S, Puskás LG,
Dudits D, Fehér A.
Author information:
(1)Laboratory of Functional Cell Biology, Institute of Plant Biology, Biological
Research Center, Hungarian Academy of Sciences, H-6701, P. O. Box 521, Szeged,
Hungary.
Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending
on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial
culture medium. In order to reveal gene expression changes during the
establishment of embryogenic competence, we compared the cell types developed in
the presence of 1 and 10 microM 2,4-D, respectively, at the time of their first
cell divisions (fourth day of culture) using a PCR-based cDNA subtraction
approach. Although the subtraction efficiency was relatively low, applying an
additional differential screening step allowed the identification of 38 10 microM
2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated
and representatives of various functional groups were selected for more detailed
gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used
to determine relative expression of the selected genes in 2,4-D-treated leaves as
well as during the whole process of somatic embryogenesis. Gene expression
patterns confirmed 2,4-D inducibility for all but one of the 11 investigated
genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The
characterized genes exhibited differential expression patterns during the early
induction phase and the late embryo differentiation phase of somatic
embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related
protein, a cell division-related ribosomal (S3a) protein, an ARF-type small
GTPase and the nucleosome assembly factor family SET protein exhibited higher
relative expression not only during the induction of somatic embryogenesis but at
the time of somatic embryo differentiation as well. This may indicate that the
expression of these genes is associated with developmental transitions
(differentiation as well as de-differentiation) during the process of somatic
embryogenesis.
DOI: 10.1016/j.bbaexp.2006.11.005
1851. J Biol Chem. 2007 Feb 23;282(8):5249-55. Epub 2006 Dec 19.
A Proteasome-regulated glycogen synthase kinase-3 modulates disease response in

plants.
Wrzaczek M(1), Rozhon W, Jonak C.
Author information:
(1)Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr.
Bohrgasse 9, A-1030 Vienna, Austria.
Glycogen synthase kinase-3 (GSK-3) is a key player in various important signaling

pathways in animals. The activity of GSK-3 is known to be modulated by protein
phosphorylation and differential complex formation. However, little information
is available regarding the function and regulation of plant GSK-3/shaggy-like
kinases (GSKs). Analysis of the in vivo kinase activity of MsK1, a GSK from
Medicago sativa, revealed that MsK1 is active in healthy plants and that MsK1
activity is down-regulated by the elicitor cellulase in a time- and
dose-dependent manner. Surprisingly, cellulase treatment triggered the
degradation of the MsK1 protein in a proteasome-dependent manner suggesting a
novel mechanism of GSK-3 regulation. Inhibition of MsK1 kinase activity and
degradation of the protein were two successive processes that could be uncoupled.
In a transgenic approach, stimulus-induced inhibition of MsK1 was impeded by
constant replenishment of MsK1 by a strong constitutive promoter. MsK1
overexpressing plants exhibited enhanced disease susceptibility to the virulent
bacterial pathogen Pseudomonas syringae. MAP kinase activation in response to
pathogen infection was compromised in plants with elevated MsK1 levels. These
data strongly suggest that tight regulation of the plant GSK-3, MsK1, may be
important for innate immunity to limit the severity of virulent bacterial
infection.
DOI: 10.1074/jbc.M610135200
1852. Plant Cell. 2006 Dec;18(12):3656-69. Epub 2006 Dec 15.
Structural basis for dual functionality of isoflavonoid O-methyltransferases in

the evolution of plant defense responses.
Liu CJ(1), Deavours BE, Richard SB, Ferrer JL, Blount JW, Huhman D, Dixon RA,
Noel JP.
Author information:
(1)Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology
and Proteomics, Salk Institute for Biological Studies, La Jolla, California,
92037, USA.
In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa),

barrel medic (Medicago truncatula), and chickpea (Cicer arietinum),
4'-O-methylation of isoflavonoid natural products occurs early in the
biosynthesis of defense chemicals known as phytoalexins. However, among these
four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid
isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for
chemical resistance to the pathogenic fungus Nectria hematococca. While barrel
medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures
are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro,
hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits
nearly identical steady state kinetic parameters for the 4'-O-methylation of the
isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the
3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid
intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate
complexes revealed identically bound conformations for the 2S,3R-stereoisomer of
2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of
6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to
seemingly distinct chemical substrates allowed leguminous plants to use
homologous enzymes for two different biosynthetic reactions. The
three-dimensional similarity of natural small molecules represents one
explanation for how plants may rapidly recruit enzymes for new biosynthetic
reactions in response to changing physiological and ecological pressures.
DOI: 10.1105/tpc.106.041376
PMCID: PMC1785397
1853. J Bacteriol. 2007 Mar;189(5):2101-9. Epub 2006 Dec 15.
Disruption of sitA compromises Sinorhizobium meliloti for manganese uptake

required for protection against oxidative stress.
Author information:
02139, USA.
During the initial stages of symbiosis with the host plant Medicago sativa,
Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in
order for proper symbiotic development to continue. While identifying mutants
defective in symbiosis and oxidative stress defense, we isolated a mutant with a
transposon insertion mutation of sitA, which encodes the periplasmic binding
protein of the putative iron/manganese ABC transporter SitABCD. Disruption of
sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide
and superoxide. Disruption of sitA leads to elevated catalase activity and a
severe decrease in superoxide dismutase B (SodB) activity and protein level. The
decrease in SodB level strongly correlates with the superoxide sensitivity of the
sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant
can be rescued by the addition of exogenous manganese but not iron, a result that
strongly implies that SitABCD plays an important role in manganese uptake in S.
meliloti.
DOI: 10.1128/JB.01377-06
PMCID: PMC1855753
1854. J Bacteriol. 2007 Mar;189(5):2110-3. Epub 2006 Dec 15.
Identification of novel Sinorhizobium meliloti mutants compromised for oxidative

stress protection and symbiosis.
Author information:
02139, USA.
Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants

that were both sensitive to hydrogen peroxide and symbiotically defective on the
host plant Medicago sativa. The mutations affect a wide variety of cellular
processes and represent both novel and previously identified genes important in
symbiosis.
DOI: 10.1128/JB.01802-06
PMCID: PMC1855713
1855. Wei Sheng Wu Xue Bao. 2006 Oct;46(5):767-72.
[Composition diversity of lactic acid bacteria (LAB) community Al2 used for
alfalfa silage].
Wang XF(1), Gao LJ, Yang HY, Wang WD, Cui ZJ.
Author information:
(1)College of Agronomy and Biotechnology, China Agricultural University, Beijing
100094, China. wxiaofen_16@yahoo.com.cn
Alfalfa is the most important forage grass that is difficult to ensile for good
quality. Using silage inoculants are the important way for preservation of
alfalfa silage. Through continuous restricted subcultivation, a lactic acid
bacteria (LAB) community Al2 was selected from well-fermented alfalfa silage.
Plate isolation and Denaturing Gradient Gel Electrophoresis (DGGE), construction
of 16S rDNA clone library were used to identify the composition diversity of Al2
community, with 7 strains detected, and they were all belonged to Lactobacillus.
The composition ratios of the 7 strains were 55.21%, 19.79%, 14.58%, 3.13%,
3.13%, 3.13%, 1.03% according to 16S rDNA clone library. Al2-1i, Al2-2i, Al2-3i,
corresponding to L. plantarum (99.9%), L. kimchii (99.4%), L. farciminis (100%)
were detected by plate isolation. Among 3 isolates, Al2-1i had the highest
ability of dropping pH and producing lactic acid, and the amount of lactic acid
was reach to 18g/L at 24h cultivated in MRS media. The ability of dropping pH and
producing lactic acid of Al2-3i was the lowest. From DGGE profiles, the dominant
strains in Al2 community were L. plantarum and L. kimchii. L. plantarum was
detected during the whole process, and L. kimchii was detected in the later
phase.
1856. Plant Biotechnol J. 2005 Jan;3(1):71-9.
Enhanced levels of methionine and cysteine in transgenic alfalfa (Medicago sativa

L.) plants over-expressing the Arabidopsis cystathionine gamma-synthase gene.
Avraham T(1), Badani H, Galili S, Amir R.

Author information:
(1)Plant Science Laboratory, Migal-Galilee Technology Center, PO Box 831, Kiryat
Shmona 11016, Israel. Rachel@migal.org.il
With the aim of increasing the methionine level in alfalfa (Medicago sativa L.)
and thus improving its nutritional quality, we produced transgenic alfalfa plants
that expressed the Arabidopsis cystathionine gamma-synthase (AtCGS), the enzyme
that controls the synthesis of the first intermediate metabolite in the
methionine pathway. The AtCGS cDNA was driven by the Arabidopsis rubisco small
subunit promoter to obtain expression in leaves. Thirty transgenic plants were
examined for the transgene protein expression, and four lines with a high
expression level were selected for further work. In these lines, the contents of
methionine, S-methylmethionine (SMM), and methionine incorporated into the
water-soluble protein fraction increased up to 32-fold, 19-fold, and 2.2-fold,
respectively, compared with that in wild-type plants. Notably, in these four
transgenic lines, the levels of free cysteine (the sulphur donor for methionine
synthesis), glutathione (the cysteine storage and transport form), and
protein-bound cysteine increased up to 2.6-fold, 5.5-fold, and 2.3-fold,
respectively, relative to that in wild-type plants. As the transgenic alfalfa
plants over-expressing AtCGS had significantly higher levels of both soluble and
protein-bound methionine and cysteine, they may represent a model and target
system for improving the nutritional quality of forage crops.
DOI: 10.1111/j.1467-7652.2004.00102.x
PMID: 17168900
1857. Sci Total Environ. 2007 Jul 1;379(2-3):226-34. Epub 2006 Dec 6.
The arbuscular mycorrhizal fungus Glomus mosseae gives contradictory effects on

phosphorus and arsenic acquisition by Medicago sativa Linn.
Chen B(1), Xiao X, Zhu YG, Smith FA, Xie ZM, Smith SE.
Author information:
(1)Department of Soil Environmental Sciences/State Key Laboratory of
Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences, Beijing 100085, P.R. China.
Mycorrhizal fungi may play an important role in protecting plants against arsenic
(As) contamination. However, little is known about the direct and indirect
involvement of arbuscular mycorrhizal fungi (AMF) in detoxification mechanisms. A
compartmented pot cultivation system ('cross-pots') is used here to investigate
the roles of AMF Glomus mosseae in plant phosphorus (P) and As acquisition by
Medicago sativa, and P-As interactions. The results indicate that fungal
colonization dramatically increased plant dry weight by a factor of around 6, and
also substantially increased both plant P and As contents (i.e. total uptake).
Irrespective of P and As addition levels, AM plants had shoot and root P
concentrations 2 fold higher, but As concentrations significantly lower, than
corresponding uninoculated controls. The decreased shoot As concentrations were
largely due to "dilution effects" that resulted from stimulated growth of AM
plants and reduced As partitioning to shoots. The study provides further evidence
for the protective effects of AMF on host plants against As contamination, and
have uncovered key aspects of underlying mechanisms. The possible application of
AMF in remediation practices is discussed.
DOI: 10.1016/j.scitotenv.2006.07.038
1858. Plant Cell Rep. 2007 May;26(5):611-5. Epub 2006 Dec 6.
Delay of leaf senescence in Medicago sativa transformed with the ipt gene
controlled by the senescence-specific promoter SAG12.
Calderini O(1), Bovone T, Scotti C, Pupilli F, Piano E, Arcioni S.
Author information:
(1)CNR Istituto di Genetica Vegetale, via Madonna Alta 130, Perugia, Italy.
We report the successfull delay of leaf senescence in Medicago sativa. A highly

regenerable clone of alfalfa was transformed with the construct SAG12-IPT, an
approach that has already proved efficient in other crops. Several independent
transformants were obtained as determined by Southern analysis and all the
transformants expressed the transgene as measured by RT-PCR. In vitro and in vivo
analyses showed that SAG12-IPT plants exhibited a stay-green phenotype that has
the potential to greatly improve the quantity and quality of alfalfa forage.
DOI: 10.1007/s00299-006-0262-y
1859. Plant J. 2006 Dec;48(6):843-56. Epub 2006 Nov 21.
A CDPK isoform participates in the regulation of nodule number in Medicago

truncatula.
Gargantini PR(1), Gonzalez-Rizzo S, Chinchilla D, Raices M, Giammaria V, Ulloa

RM, Frugier F, Crespi MD.
Author information:
(1)Instituto de Investigaciones en Ingeniería Genética y Biología Molecular,
Vuelta de Obligado 2490, 2 piso, 1428 Buenos Aires, Argentina.
Medicago spp. are able to develop root nodules via symbiotic interaction with
Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in
various signalling pathways in plants, and we found that expression of MtCPK3, a
CDPK isoform present in roots of the model legume Medicago truncatula, is
regulated during the nodulation process. Early inductions were detected 15 min
and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers
was also present in inoculated M. truncatula dmi mutants and in wild-type roots
subjected to salt stress, indicating that this rapid response is probably
stress-related. In contrast, the later response was concomitant with cortical
cell division and the formation of nodule primordia, and was not observed in
wild-type roots inoculated with nod (-) strains. This late induction correlated
with a change in the subcellular distribution of CDPK activities. Accordingly, an
anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the
particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in
epidermal onion cells, a localization that depends on myristoylation and
palmitoylation sites of the protein, suggesting a dual subcellular localization.
MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone
linked to the regulation of cortical cell division and other nodulation-related
responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium
rhizogenes-transformed roots. Although no major phenotype was detected in these
roots, when infected with rhizobia, the total number of nodules was, on average,
twofold higher than in controls. This correlates with the lack of MtCPK3
induction in the inoculated super-nodulator sunn mutant. Our results suggest that
CPK3 participates in the regulation of the symbiotic interaction.
DOI: 10.1111/j.1365-313X.2006.02910.x
Potential for phytoremediation of polychlorinated biphenyl-(PCB-)contaminated

soil.
Zeeb BA(1), Amphlett JS, Rutter A, Reimer KJ.
Author information:
(1)Environmental Sciences Group, Department of Chemistry and Chemical
Engineering, Royal Military College of Canada, Kingston, Ontario, Canada.
zeeb-b@rmc.ca
Weathered soils contaminated with commercial-grade Aroclor 1260 from three sites
in Canada were used to investigate the polychlorinated biphenyl (PCB)
phytoextraction potential of nine plant species (Festuca arundinacea, Glycine
max, Medicago sativa, Phalaris arundinacea, Lolium multiflorum, Carex normalis,
and three varieties of Cucurbita pepo ssp. pepo) under controlled greenhouse
conditions. The soils used varied in PCB concentration (90-4200 microg/g) and
total organic content (0.06-2.02%). Greenhouse experiments controlled for PCB
volatilization through the use of a vented enclosure and by isolating the
contaminated soils with parafilm. After 8 wks, PCB concentrations of 47-6700
microg/g were observed in root tissues. Although PCB concentrations in shoot
tissues were lower (< 1-470 microg/g), the absolute amounts of PCBs observed in
shoot tissue were significant (1.7-290 microg) once shoot biomass was accounted
for. Congener signatures indicated that tetra- to hexa-chlorobiphenyls
contributed the largest proportions to shoot tissues, but hepta-to
nona-chorobiphenyls were also present in measurable amounts. Overall, the results
indicate that varieties of C. pepo were more effective at extracting PCBs from
soil than other plants screened The evidence suggests that this was mainly due to
root uptake of PCBs and tranlocation to the shoots, rather than volatilization of
PCBs from soil. All plants screened showed signs of stress in the most highly
contaminated soil (4200 microg/g), but not in the two lower PCB contaminated
soils (250 and 90 microg/g, respectively). No detectable decreases in soil PCB
concentrations were observed in these short-term greenhouse experiments, but the
results suggest that this may be achievable through multiple plantings.
DOI: 10.1080/15226510600846749
1861. J Insect Sci. 2005 Dec 22;5:44.
Parasitism of Lygus spp. nymphs by the parasitoid wasp, Peristenus howardi, in

the alfalfa seed-growing region of the Pacific Northwest.
Seymour LM(1), Mowry TM, Day WH, Barbour JD.
Author information:
(1)Department of Plant, Soil and Entomological Sciences, University of Idaho,
Parma Research and Extension Center, Parma, ID 83660-6699, USA.
The parasitoid, Peristenus howardi Shaw (Hymenoptera: Braconidae) has been found
to parasitize a large proportion of Lygus species in some Washington and Idaho
alfalfa seed fields. During 2002-2003 a survey was conducted to estimate the
proportion of Lygus spp. (Hemiptera: Miridae) parasitized and the amountof that
parasitism attributable to P. howardi in crop and non-crop plants attacked by
Lygus in the alfalfa seed growing region of southwestern Idaho and eastern
Oregon. Percentage parasitism was estimated from dissection of field-collected
Lygus nymphs. Polymerase chain reaction (PCR) was used to amplify DNA extracted
from parasitoid larvae followed by restriction endonuclease digestion of PCR
products to distinguish P. howardi from other potentially co-occurring Peristenus
species. Peak parasitism of Lygus nymphs occurred between the first and third
weeks of July for both years for all host plants sampled. Of the parasitoid
larvae recovered from Lygus nymphs in our study, 75% to 80% tested positive as
Peristenus spp. and 76% of these matched the endonuclease digestion banding
pattern for P. howardi. The identity of the remaining 20% to 25% of the
parasitoids is not known.
PMCID: PMC1615251
1862. Appl Microbiol Biotechnol. 2007 Mar;74(3):540-6. Epub 2006 Nov 16.
Batch fermentations on synthetic mixed sugar and starch medium with amylolytic
lactic acid bacteria.
Thomsen MH(1), Guyot JP, Kiel P.
Author information:
(1)Plant Research Department, Risoe National Laboratory, BIO-NRG-Building 301,
P.O. Box 49, 4000, Roskilde, Denmark. mette.hedegaard.thomsen@risoe.dk
The green crop drying industry in Denmark uses Italian rye grass, clover, and
alfalfa as raw materials for the production of green pellets. The green crop
drying industry solves its energy economical problems by heating and pressing of
the green crop before drying. The produced sidestream is called brown juice.
Brown juice was shown to be an excellent medium for lactic acid fermentation. The
aim of this study was to investigate the utilisation of brown juice in the
production of polylactic acid, where wheat starch would be added to increase the
lactic acid yield and, thus, the feasibility of the process. A number of
amylolytic lactic acid bacteria have been identified, and in this work, six
different strains were tested for their ability to produce alpha-amylase and to
utilise all sugars with high lactic acid yield in a medium with a complex
composition of free sugars (brown juice) and starch. Lactobacillus plantarum A6
was the only strain that showed both a good lactic acid production and
utilisation of starch in this medium. The growth rate of this strain was
approximately 0.4 h(-1) and the lactic acid yield was 0.7.
DOI: 10.1007/s00253-006-0694-x
1863. J Dairy Sci. 2006 Dec;89(12):4784-94.
Evaluation of various sources of corn dried distillers grains plus solubles for
lactating dairy cattle.
Kleinschmit DH(1), Schingoethe DJ, Kalscheur KF, Hippen AR.
Author information:
(1)Dairy Science Department, South Dakota State University, Brookings, SD 57007,
USA.
Erratum in
J Dairy Sci. 2007 Jan;90(1):522.
The objectives of this study were to evaluate the effects of feeding dried
distillers grains plus solubles (DDGS) from different sources on milk production
and composition in dairy cows. Eight multiparous and 4 primiparous Holstein cows
were used in a replicated 4 x 4 Latin square design with 28-d periods. Treatments
consisted of total mixed diets containing no DDGS (CON), or DDGS from source 1
(DDGS-1), source 2 (DDGS-2), or source 3 (DDGS-3) at 20% of diet dry matter. The
processing of DDGS-2 and DDGS-3 was intended to decrease heat damage and improve
nutritional quality. The DDGS in the diets replaced a portion of the ground corn
and soybean meal, allowing them to be isonitrogenous at 16% crude protein. All
diets had a forage-to-concentrate ratio of 55:45. Dry matter intake (21.4 kg/d)
did not differ among diets, but cows fed diets containing DDGS had greater yields
of milk (34.6 vs. 31.2 kg/d), 4% fat-corrected milk (32.7 vs. 29.6 kg/d), and
energy-corrected milk (35.4 vs. 32.3) compared with cows fed the CON diet. Feed
efficiency was greater in cows fed DDGS compared with CON (1.78 vs. 1.63). Milk
fat yield was greater in cows fed DDGS compared with those fed CON (1.26 vs. 1.14
kg/d). Milk protein percentages (3.28, 3.13, 3.19, and 3.17% for CON, DDGS-1,
DDGS-2, and DDGS-3, respectively) were greater for CON vs. DDGS and tended to be
lower for DDGS-1 than for DDGS-2 and DDGS-3. Milk protein yields tended to be
greater for cows fed DDGS than for those fed CON (1.09 vs. 1.02 kg/d).
Concentrations of milk urea nitrogen were lower in cows fed DDGS compared with
CON (9.36 vs. 10.6 mg/dL). Feeding DDGS decreased arterial plasma concentrations
of Arg, Ile, Lys, and Thr and increased His and Leu compared with CON. Arterial
plasma from cows fed DDGS-2 and DDGS-3 had greater concentrations of Ile, Trp,
and Val compared with DDGS-1. In all diets, Lys, Met, and Phe were the first 3
limiting amino acids for protein synthesis with Lys being first limiting in
DDGS-1 and DDGS-3 and Met being first limiting for CON and DDGS-2. Inclusion of
DDGS did not affect the molar proportions of ruminal acetate or propionate
compared with CON. Ruminal proportions of butyrate were lower in CON compared
with DDGS. Total concentrations of VFA were greater in CON compared with DDGS.
The concentrations of rumen ammonia were greater in CON (7.2 mg/dL) compared with
DDGS (4.5 mg/dL). Overall, the source of DDGS used in this study did not affect
lactation performance.
DOI: 10.3168/jds.S0022-0302(06)72528-0
1864. J Dairy Sci. 2006 Dec;89(12):4736-46.
Effect of chestnut tannin on fermentation quality, proteolysis, and protein rumen

degradability of alfalfa silage.
Tabacco E(1), Borreani G, Crovetto GM, Galassi G, Colombo D, Cavallarin L.
Author information:
(1)Dipartimento di Agronomia, Selvicoltura e Gestione del Territorio, Università
degli Studi di Torino, Grugliasco, Torino, Italy.
Two experiments were conducted on alfalfa to investigate the effects of the

addition of commercial chestnut hydrolyzable tannin at ensiling on 1) silage
fermentation quality in lab-scale silos and protein degradation in the rumen, and
2) silage fermentation quality and proteolysis in bale silages. Wilted alfalfa
was prepared with 4 tannin levels (0, 2, 4, and 6% on a dry matter (DM) basis;
T0, T1, T2, T3, respectively) and ensiled in lab-scale silos. Silages (33% DM)
were analyzed for fermentation quality, protein rumen degradability in situ, and
organic matter digestibility in vitro through gas production after 120 d of
conservation. Wilted alfalfa containing 0 and 4% tannin (T0 and T2) was harvested
at 40% DM (wilting level I) and 53% DM (wilting level II) for bale (600 mm
diameter) silage. Silages were analyzed for fermentation quality after 78 d of
conservation. All the silages were well fermented with no butyric acid. Lab-scale
silages showed reductions in ammonia, nonprotein nitrogen (NPN) and DM losses in
T2 and T3 treatments, while the fermentation acid profiles were unaffected. In
experiment 1, the untreated silage (T0) had the highest protein degradability
after being incubated in the rumen. The addition of tannin reduced crude protein
ruminal disappearance in a dose-dependent manner. However, the tannin reduced the
organic matter digestibility by 5.1% for all of the tannin addition levels. The
tannin positively affected the silage quality in the round bale silages, in
particular reducing ammonia and NPN in the lowest wilting level. In both
experiments, T2 treatment reduced proteolysis without any influence of DM on the
binding reaction and reduced the NPN by 15% in comparison to the control.
DOI: 10.3168/jds.S0022-0302(06)72523-1
1865. Methods Mol Biol. 2007;355:79-92.
Isolation of cell wall proteins from Medicago sativa stems.
Watson BS(1), Sumner LW.
Author information:
(1)Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.
Plant cell walls are highly dynamic and chemically active components of plant
cells. Cell walls consist primarily of polysaccharides, with proteins comprising
approx 10% of the cell wall mass. These proteins are difficult to isolate with a
high degree of purity from the complex carbohydrate matrix. This matrix traps
proteins and is a source of contamination for subsequent 2-DE analysis. Mature
plant tissues provide a further challenge owing to the formation of secondary
walls that contain phenolic compounds. This chapter discusses protein extraction
from cell walls and presents a specific method for the isolation of proteins from
Medicago sativa stem cell walls. The method includes cell disruption by grinding,
copious washes with both aqueous and organic solutions to remove cytosolic
proteins and small molecule contaminants, and two different salt extractions that
provide a highly enriched cell wall protein fraction from alfalfa stem cell
walls. Following treatment with a commercial clean-up kit, the protein extracts
yield high-quality and high-resolution 2-DE separations from which proteins can
be readily identified by mass spectrometry.
DOI: 10.1385/1-59745-227-0:79
1866. J Inorg Biochem. 2007 Jan;101(1):1-9. Epub 2006 Jun 7.
Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago

sativa L.
Zhou ZS(1), Huang SQ, Guo K, Mehta SK, Zhang PC, Yang ZM.
Author information:
(1)Department of Biochemistry and Molecular Biology, College of Life Science, No.
1 Weigang, Outside the Zhoungshan Men, Building of Life Science, Nanjing
Alfalfa (Medicago sativa) roots were treated with mercuric ions in a

concentration- and time-dependent manner, and lipid peroxidation was studied
biochemically as well as histochemically along with other physiological
responses. Histochemical staining with Schiff's reagent and Evans blue revealed
that the peroxidation of membrane lipids and loss of plasma membrane integrity in
Hg-treated roots occurred in the meristem and the elongation zone. The
histochemical observations were supported by the quantitative determinations of
thiobarbituric acid reactive substances (TBARS). However, under the mercuric ions
stress, the alfalfa plants showed no significant alteration of hydrogen peroxide
in roots. Analysis of lipoxygenase activity by non-denaturing polyacrylamide gel
electrophoresis (PAGE) showed that there were two isoforms in the root of alfalfa
plants, but they showed quite different patterns under the Hg exposure. Also,
using non-denaturing PAGE, activities of superoxide dismutase (SOD) and
peroxidase (POD) were determined in roots after treatment with Hg ions. The total
activities of SOD and POD increased in roots after Hg treatment of roots.
Activity of ascorbate peroxides (APX) was stimulated at relatively high
concentration of Hg (40microM), and after prolonged Hg exposure (20microM, 24h).
In contrast, glutathione reductase activity was depressed at higher
concentrations of Hg (10-20microM). Treatments of seedlings with 10-40microM Hg
decreased the ascorbate and glutathione amounts but increased total non-protein
thiols. The above results indicated that Hg exerted its toxic effect on the root
growth of alfalfa by induction of oxidative stress.
DOI: 10.1016/j.jinorgbio.2006.05.011
1867. Int J Syst Evol Microbiol. 2006 Nov;56(Pt 11):2631-7.
Rhizobium lusitanum sp. nov. a bacterium that nodulates Phaseolus vulgaris.
Valverde A(1), Igual JM, Peix A, Cervantes E, Velázquez E.
Author information:
(1)Departamento de Producción Vegetal, IRNASA-CSIC, Salamanca, Spain.
The species Phaseolus vulgaris is a promiscuous legume nodulated by several

species of the family Rhizobiaceae. During a study of rhizobia nodulating this
legume in Portugal, we isolated several strains that nodulate P. vulgaris
effectively and also Macroptilium atropurpureum and Leucaena leucocephala, but
they form ineffective nodules in Medicago sativa. According to phylogenetic
analysis of the 16S rRNA gene sequence, the strains from this study belong to the
genus Rhizobium, with Rhizobium rhizogenes and Rhizobium tropici as the closest
related species, with 99.9 and 99.2% similarity, respectively, between the type
strains of these species and strain P1-7T. The nodD and nifH genes carried by
strain P1-7T are phylogenetically related to those of other species nodulating
Phaseolus. This strain does not carry virulence genes present in the type strain
of R. rhizogenes, ATCC 11325T. Analysis of the recA and atpD genes confirms this
phylogenetic arrangement, showing low similarity with respect to those of R.
rhizogenes ATCC 11325T (91.9 and 94.1% similarity, respectively) and R. tropici
IIB CIAT 899T (90.6% and 91.8% similarity, respectively). The intergenic spacer
(ITS) of the strains from this study is phylogenetically divergent from those of
R. rhizogenes ATCC 11235T and R. tropici CIAT 899T, with 85.9 and 82.8%
similarity, respectively, with respect to strain P1-7T. The tRNA profile and
two-primer random amplified polymorphic DNA pattern of strain P1-7T are also
different from those of R. rhizogenes ATCC 11235T and R. tropici CIAT 899T. The
strains isolated in this study can be also differentiated from R. rhizogenes and
R. tropici by several phenotypic characteristics. The results of DNA-DNA
hybridization showed means of 28 and 25% similarity between strain P1-7T and R.
rhizogenes ATCC 11235T and R. tropici CIAT 899T, respectively. All these data
showed that the strains isolated in this study belong to a novel species of the
genus Rhizobium, for which we propose the name Rhizobium lusitanum sp. nov.; the
type strain is P1-7T (=LMG 22705T=CECT 7016T).
DOI: 10.1099/ijs.0.64402-0
1868. J Environ Qual. 2006 Oct 27;35(6):2302-12. Print 2006 Nov-Dec.
Nitrogen- vs. phosphorus-based dairy manure applications to field crops: nitrate

and phosphorus leaching and soil phosphorus accumulation.
Toth JD(1), Dou Z, Ferguson JD, Galligan DT, Ramberg CF Jr.
Author information:
(1)Section for Animal Production Systems, School of Veterinary Medicine, Univ. of
Pennsylvania, 382 West Street Rd., Kennett Square, PA 19348, USA.
jdtoth@vet.upenn.edu
Management of animal manures to provide nutrients for crop growth has generally
been based on crop N needs. However, because manures have a lower N/P ratio than
most harvested crops, N-based manure management often oversupplies the crop-soil
system with P, which can be lost into the environment and contribute to
eutrophication of water bodies. We examined the effects of N- vs. P-based manure
applications on N and P uptake by alfalfa (Medicago sativa L.), corn (Zea mays
L.) for silage, and orchardgrass (Dactylis glomerata L.), leaching below the root
zone, and accumulation of P in soil. Treatments included N- and P-based manure
rates, with no nutrient input controls and inorganically fertilized plots for
comparison. Nitrate concentrations in leachate from inorganic fertilizer or
manure treatments averaged 14 mg NO(3)-N L(-1), and did not differ by nutrient
treatment. Average annual total P losses in leachate did not exceed 1 kg ha(-1).
In the top 5 cm of soil in plots receiving the N-based manure treatment, soil
test P increased by 47%, from 85 to 125 mg kg(-1). Nitrogen- and P-based manure
applications did not differ in ability to supply nutrients for crop growth, or in
losses of nitrate and total P in leachate. However, the N-based manure led to
significantly greater accumulation of soil test P in the surface 5 cm of soil.
Surface soil P accumulation has implications for increased risk of off-field P
movement.
DOI: 10.2134/jeq2005.0479
1869. J Bacteriol. 2007 Jan;189(1):160-8. Epub 2006 Oct 27.
Malic enzyme cofactor and domain requirements for symbiotic N2 fixation by

Mitsch MJ(1), Cowie A, Finan TM.
Author information:
1280 Main Street West, Hamilton, Ontario, Canada.
The NAD(+)-dependent malic enzyme (DME) and the NADP(+)-dependent malic enzyme
(TME) of Sinorhizobium meliloti are representatives of a distinct class of malic
enzymes that contain a 440-amino-acid N-terminal region homologous to other malic
enzymes and a 330-amino-acid C-terminal region with similarity to
phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of
S. meliloti fail to fix N(2) (Fix(-)) in alfalfa root nodules, whereas tme
mutants are unimpaired in their N(2)-fixing ability (Fix(+)). Here we report that
the amount of DME protein in bacteroids is 10 times greater than that of TME. We
therefore investigated whether increased TME activity in nodules would allow TME
to function in place of DME. The tme gene was placed under the control of the dme
promoter, and despite elevated levels of TME within bacteroids, no symbiotic
nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme
from the tme promoter resulted in a large reduction in DME activity and symbiotic
N(2) fixation. Hence, TME cannot replace the symbiotic requirement for DME. In
further experiments we investigated the DME PTA-like domain and showed that it is
not required for N(2) fixation. Thus, expression of a DME C-terminal deletion
derivative or the Escherichia coli NAD(+)-dependent malic enzyme (sfcA), both of
which lack the PTA-like region, restored wild-type N(2) fixation to a dme mutant.
Our results have defined the symbiotic requirements for malic enzyme and raise
the possibility that a constant high ratio of NADPH + H(+) to NADP in
nitrogen-fixing bacteroids prevents TME from functioning in N(2)-fixing
bacteroids.
DOI: 10.1128/JB.01425-06
PMCID: PMC1797227
1870. Br Poult Sci. 2006 Oct;47(5):561-6.
Effects of carotenoids from lucerne, marigold and tomato on egg yolk pigmentation
and carotenoid composition.
Karadas F(1), Grammenidis E, Surai PF, Acamovic T, Sparks NH.
Author information:
(1)Faculty of Agriculture, Department of Animal Science, University of Yüzüncü
Yil, Van, Turkey. fkaradas@yyu.edu.tr
1. The effects of various sources of natural carotenoids (Px alfalfa concentrate,

tomato powder and marigold extract) as feed additives in quail diets on egg yolk
pigmentation and carotenoid composition were investigated. 2. Adult Japanese
quail were fed one of 5 different diets for 23 d: three diets each contained Px
alfalfa concentrate (PX) or tomato powder (TP) or marigold extract (MG), one diet
contained marigold extract and tomato powder (MG + TP) and a control diet
(wheat/barley based) was low in carotenoid. All products were added at a rate of
2%, apart from marigold extract which was added at a rate of 0.2%. 3. Visual
assessment of yolk colour (Roche colour fan) showed a stabilised yolk colour of
1.6, 7.7, 8.5, 8.8 and 10.6 for the control, PX, TP, MG and MG + TP treatments,
respectively. 4. The total carotenoid concentration of the egg yolks were 2.2,
22.4, 4.1, 39.0 and 37.7 microg/g for the experimental groups fed the following
diets: control, PX, TP, MG and MG + TP, respectively. Deposition of retinol,
alpha-tocopherol and gamma-tocopherol in the egg yolk was unaffected by
treatments. 5. Lutein was shown to be the major carotenoid in the egg yolk,
comprising 1.65, 17.97, 2.03, 31.14 and 28.57 microg/g in control, PX, TP, MG and
MG + TP, respectively. Inclusion of TP in the quail diet resulted in lycopene
transfer to the egg. 6. It was concluded that, in comparison with the control
group, there was an increase in the yolk concentrations of lutein, zeaxanthin,
lycopene and beta-carotene in eggs produced by female quail fed diets
supplemented with natural carotenoids.
DOI: 10.1080/00071660600962976
1871. Biochim Biophys Acta. 2006 Dec;1761(12):1419-28. Epub 2006 Sep 14.
Hydroperoxide lyases (CYP74C and CYP74B) catalyze the homolytic isomerization of

fatty acid hydroperoxides into hemiacetals.
Grechkin AN(1), Brühlmann F, Mukhtarova LS, Gogolev YV, Hamberg M.
Author information:
(1)Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences,
P.O. Box 30, Kazan, 420111 Russia. grechkin@mail.knc.ru
The conversion of linoleic acid 9-hydroperoxide (9-HPOD) by recombinant melon

(Cucumis melo L.) hydroperoxide lyase (HPL, CYP74C subfamily) was studied. Short
(5 s-1 min) incubations at 0 degrees C followed by rapid extraction and
trimethylsilylation made it possible to trap a new unstable (t(1/2) <30 s)
product, i.e. the hemiacetal (1'E,3'Z)-9-hydroxy-9-(1',3'-nonadienyloxy)-nonanoic
acid. Identification was performed by GC-MS analysis and substantiated by the
formation of trimethylsilyl 9-trimethylsilyloxy-9-nonyloxy-nonanoate upon
catalytic hydrogenation and by (2)H-labelling experiments. Both (18)O atoms of
[(18)O(2)-hydroperoxy]9-HPOD were incorporated into the hemiacetal. Along with
the hemiacetal, three chain-cleavage products, i.e. the enol (1E,3Z)-nonadienol
and the hydrates of 3(Z)-nonenal and 9-oxononanoic acid, were trapped as their
trimethylsilyl derivatives. The kinetics of (18)O incorporation from
[(18)O(2)]9-HPOD provided strong evidence that the cleavage products originated
in the hemiacetal. Linolenic and linoleic acid 13-hydroperoxides served as
substrates for recombinant HPLs of melon, alfalfa (Medicago sativa) and guava
(Psidium guajava), and in each case hemiacetals and enols were detectable by the
trapping technique. The data obtained demonstrated that CYP74C and CYP74B HPLs
act as isomerases performing a homolytic rearrangement of fatty acid
hydroperoxides into short-lived hemiacetals which upon decomposition produce
3(Z)-nonenal, 3(Z)-hexenal and other short chain aldehydes.
DOI: 10.1016/j.bbalip.2006.09.002
1872. Plant Physiol. 2006 Dec;142(4):1642-55. Epub 2006 Oct 13.
Geminivirus infection up-regulates the expression of two Arabidopsis protein

kinases related to yeast SNF1- and mammalian AMPK-activating kinases.
Shen W(1), Hanley-Bowdoin L.
Author information:
(1)Department of Molecular and Structural Biochemistry, North Carolina State
University, Raleigh, North Carolina 27695-7622, USA.
Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein

kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that
a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We
show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid
studies. Using specific antibodies, we showed that both Arabidopsis kinases are
elevated in infected leaves. Immunoblot analysis of healthy plants revealed that
GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or
undetectable in mature tissues. Immunohistochemical staining showed that the
kinases accumulate in the shoot apical meristem, leaf primordium, and emerging
petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show
a small increase during infection and do not change significantly during
development. Treating healthy seedlings and infected leaves with the proteasome
inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas
treatment with the translation inhibitor cycloheximide reduced both kinases,
demonstrating that their accumulation is modulated by posttranscriptional
processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related
kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the
yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate
the yeast kinase SNF1 and its mammalian homolog AMPK, respectively.
Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1
and GRIK2 can functionally replace the yeast kinases, suggesting that the
Arabidopsis kinases mediate one or more processes during early plant development
and geminivirus infection by activating SNF1-related kinases.
DOI: 10.1104/pp.106.088476
PMCID: PMC1676070
1873. Environ Pollut. 2007 May;147(1):187-93. Epub 2006 Oct 6.
Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.).
Kong WD(1), Zhu YG, Liang YC, Zhang J, Smith FA, Yang M.
Author information:
A series of experiments were conducted in a hydroponic system to investigate the

uptake of oxytetracycline (OTC) and its toxicity to alfalfa (Medicago sativa L.).
OTC inhibited alfalfa shoot and root growth by up to 61% and 85%, respectively.
The kinetics of OTC uptake could be well described by Michaelis-Menten equation
with Vmax of 2.25 micromol g-1 fresh weight h-1, and Km of 0.036 mM. The uptake
of OTC by alfalfa was strongly inhibited by the metabolic inhibitor, 2,4-DNP
(2,4-dinitrophenol), at pH 3.5 and 6.0, but not by the aquaporin competitors,
glycerol and Ag+. OTC uptake, however, was significantly inhibited by Hg2+,
suggesting that the inhibition of influx was due to general cellular stress
rather than the specific action of Hg2+ on aquaporins. Results from the present
study suggested that OTC uptake into alfalfa is an energy-dependent process.
DOI: 10.1016/j.envpol.2006.08.016
1874. Arch Microbiol. 2007 Jan;187(1):79-85. Epub 2006 Sep 22.
Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective
for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv.
mediterranense) of Sinorhizobium meliloti.
Mnasri B(1), Mrabet M, Laguerre G, Aouani ME, Mhamdi R.
Author information:
(1)Laboratoire Interactions Légumineuses-Microorganismes, Centre de
Biotechnologie à la Technopole de Borj-Cédria, BP 901, Hammam-lif, 2050, Tunisia.
Nodulation of common bean was explored in six oases in the south of Tunisia.
Nineteen isolates were characterized by PCR-RFLP of 16S rDNA. Three species of
rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium
meliloti. The diversity of the symbiotic genes was then assessed by PCR-RFLP of
nodC and nifH genes. The majority of the symbiotic genotypes were conserved
between oases and other soils of the north of the country. Sinorhizobia isolated
from bean were then compared with isolates from Medicago truncatula plants grown
in the oases soils. All the nodC types except for nodC type p that was specific
to common bean isolates were shared by both hosts. The four isolates with nodC
type p induced N(2)-fixing effective nodules on common bean but did not nodulate
M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC
genes showed that these isolates carry symbiotic genes different from those
previously characterized among Medicago and bean symbionts, but closely related
to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with
common bean but unable to nodulate soybean. The creation of a novel biovar shared
by S. meliloti and S. fredii, bv. mediterranense, was proposed.
DOI: 10.1007/s00203-006-0173-x
1875. Anal Bioanal Chem. 2006 Nov;386(5):1553-8. Epub 2006 Sep 22.
Near-infrared spectroscopy (NIRS) reflectance technology for the determination of

tocopherols in alfalfa.
González-Martín I(1), Hernández-Hierro JM, Bustamante-Rangel M, Barros-Ferreiro

N.
Author information:
(1)Departamento de Química Analítica, Nutrición y Bromatología, Facultad de
Ciencias Químicas, Universidad de Salamanca, Plaza de la Merced s/n, 37008
Salamanca, Spain. inmaglez@usal.es
The vitamin E (alpha- and (beta+gamma)-tocopherol) contents present in alfalfa

(fresh or dehydrated) were analysed using near-infrared spectroscopy (NIRS)
technology together with a remote reflectance fibre-optic probe. The range of
vitamin E was 0.55-5.16 mg/100 g for alpha-tocopherol and 0.07-0.48 for
(beta+gamma)-tocopherol. The regression method employed was modified partial
least squares (MPLS). The equations developed using the fibre-optic probe for 69
samples of alfalfa (dehydrated and fresh) to determine the content of vitamin E
in feeds had multiple correlation coefficients (RSQs) and prediction corrected
standard errors (SEP (C)) of 0.946 and 0.321 mg/100 g for alpha-tocopherol and
0.956 and 0.022 mg/100 g for (beta+gamma)-tocopherol. The predicted values of
vitamin E in feeds using NIRS technology applying the fibre-optic probe directly
on the sample with neither previous treatment nor manipulation are comparable to
those obtained using the chemical method, which included alkaline hydrolysis and
hexane extraction of the vitamin from the unsaponifiable fraction before
chromatographic determination.
DOI: 10.1007/s00216-006-0666-0
1876. Phytother Res. 2006 Dec;20(12):1061-6.

Antidermatophyte activity of medicago extracts and contained saponins and their
structure-activity relationships.
Houghton P(1), Patel N, Jurzysta M, Biely Z, Cheung C.
Author information:
(1)Department of Pharmacy, King's College London. UK. peter.houghton@kcl.ac.uk
Saponin-rich extracts of Medicago sativa L. have been shown to display antifungal

activity against a range of common fungibut only a few individual saponins have
been tested against dermatophytes. Total saponins were separately obtained from
roots and aerial parts of Medicago sativa, M. murex, M. arabica and M. hybrida.
Nineteen saponins and three parent triterpenoids, were tested against three
dermatophytic fungi Microsporum gypseum, Trichophyton interdigitale and T.
tonsurans using mircowell plate serial dilution assay to determine the Minimum
Inhibitory Concentration (MIC). Miconazole was used as a positive control. T.
tonsurans appeared to be the most sensitive of the dermatophytes to the active
compounds. Aglycones showed less antifungal effect than the glycosides, which
displayed a range of activities. Monodesmosidic glycosides of medicagenic acid
were the most active compounds, especially the 3-O-beta-D-glucopyranoside, which
displayed MIC less than 0.09mm against all three fungi, although those of
hederagenin and zanhic acid also showed weak activity (MIC 3.3mm against two
fungal species). Bidesmosidic saponins had weaker activity than monodesmosidic
ones. This corresponds to activity of these and similar compounds against other
fungal species but this is the first comprehensive report of activity of Medicago
saponins against dermatophytes.
DOI: 10.1002/ptr.1995
1877. Cell Res. 2006 Oct;16(10):818-29. Epub 2006 Sep 26.
Sinorhizobium meliloti nifA mutant induces different gene expression profile from
wild type in Alfalfa nodules.
Gong ZY(1), He ZS, Zhu JB, Yu GQ, Zou HS.
Author information:
(1)National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant
Several studies have demonstrated that the Rhizobium nifA gene is an activator of
nitrogen fixation acting in nodule bacteria. To understand the effects of the
Sinorhizobium meliloti nifA gene on Alfalfa, the cDNA-AFLP technique was employed
to study the changes in gene expression in nifA mutant nodules. Among the
approximately 3,000 transcript-derived fragments, 37 had differential expression
levels. These expression levels were subsequently confirmed by reverse Northern
blot and RT-polymerase chain reaction. Sequence analyses revealed that 21 cDNA
fragments corresponded to genes involved in signal communication, protein
degradation, nutrient metabolism, cell growth and development.
DOI: 10.1038/sj.cr.7310096
1878. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2006;41(11):2503-16.
Rhizosphere microbial activity during phytoremediation of diesel-contaminated
soil.
Kim J(1), Kang SH, Min KA, Cho KS, Lee IS.
Author information:
(1)Department of Environmental Science and Engineering, Ewha Womans University,
Seoul, Korea. jkimtamu@ewha.ac.kr
To know microbial activity and diesel-removal efficiency influencing through

plant roots, we examined the effect of the rhizosphere on phytoremediation of
diesel-contaminated soils by alfalfa (Medicago sativa L.). Pots were treated with
and without diesel and allowed to stabilize for 7 weeks, at which time four
experimental/control groups were prepared: (1) planted diesel-contaminated soil,
(2) unplanted diesel-contaminated soil, (3) planted uncontaminated soil, and (4)
unplanted uncontaminated soil. Samples of rhizosphere and bulk soils were
separately taken from all planted pots. After 7 weeks of alfalfa growth from
seeds, the removal efficiencies in rhizosphere and bulk soil samples were 82.5%
and 36.5 approximately 59.4%, respectively. The total microbial activity was
highest in diesel-contaminated rhizosphere soils. Significantly more culturable
soil bacteria and hydrocarbon-degraders were found in diesel-contaminated
rhizosphere soil versus unplanted and uncontaminated bulk soil, with a greater
increase seen in hydrocarbon-degraders (172-fold) versus general soil bacteria
(14-fold). DGGE (Denaturing Gel Gradient Electrophoresis) analysis revealed that
the bacterial community structure was most highly influenced by the combined
presence of diesel contamination and plant roots (39.13% similarity compared to
the control), but that diesel contamination alone had a higher influence (42.31%
similarity compared to the control) than the rhizosphere (50.00% similarity
compared to the control). Sequence analysis and BLAST searches revealed that all
samples were dominated by members of alpha -, gamma -, delta - and epsilon
-proteobacteria, and Chloroflexi. The rhizosphere samples additionally contained
novel dominant members of alpha -proteobacteria and
Cytophaga-Flexibacter-Bacteroides, while the diesel samples contained additional
dominant alpha -proteobacteria and the rhizosphere plus diesel samples contained
other epsilon -proteobacteria. Collectively, these findings indicate that the
presence of plant roots (i.e., a rhizosphere) had a greater effect on bacterial
activity in diesel contamination than did the absence of diesel contamination,
whereas diesel contamination had a greater effect on bacterial community
structure. These novel findings provide new insight into the mechanisms of
phytoremediation.
DOI: 10.1080/10934520600927658
1879. New Phytol. 2006;172(2):347-57.
Functional diversity of arbuscular mycorrhizal fungal isolates in relation to

extraradical mycelial networks.
Avio L(1), Pellegrino E, Bonari E, Giovannetti M.
Author information:
(1)Institute of Biology and Agricultural Biotechnology, UO Pisa, CNR, Via del
Borghetto 80, 56124 Pisa, Italy.
We investigated the functional significance of extraradical mycorrhizal networks

produced by geographically different isolates of the arbuscular mycorrhizal
fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional
experimental system was used to visualize and quantify intact extraradical
mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and
nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The
AMF isolates were characterized by differences in extent and interconnectedness
of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density,
hyphal length per mm of total or colonized root length, were positively
correlated with M. sativa growth response variables, such as total shoot biomass
and plant P content. The utilization of an experimental system in which size,
growth rate, viability and interconnectedness of ERM extending from mycorrhizal
roots are easily quantified under realistic conditions allows the simultaneous
evaluation of different isolates and provides data with a predictive value for
selection of efficient AMF.
DOI: 10.1111/j.1469-8137.2006.01839.x
1880. Methods Mol Biol. 2006;343:301-11.
Samac DA(1), Austin-Phillips S.
Author information:
(1)USDA-ARS-Plant Science Research Unit and Department of Plant Pathology,
University of Minnesota, St. Paul, MN, USA.
A protocol for rapid, highly efficient transformation of alfalfa is described.

Leaf explants from growth chamber-grown plants of a highly regenerable genotype
are surface-sterilized, the margins are removed, and explants are inoculated with
Agrobacterium tumefaciens strain LBA4404 carrying the T-DNA vector of interest.
The explants and bacteria are cocultured for 7 to 8 d. Bacteria are removed by
rinsing explants in sterile distilled water and by culture on regeneration medium
containing the antibiotics carbenicillin or ticarcillin. Transformed callus is
selected using kanamycin. Somatic embryos are induced by culture of callus on
medium lacking plant growth regulators. As mature cotyledonary stage embryos
arise, they are transferred to a fresh medium for shoot development and finally
to a medium lacking kanamycin for continued shoot and root development.
Transgenic plants can be produced in 9 wk with this protocol. Typically 60 to 80%
of inoculated explants produce transgenic plants, and escapes are rare.
DOI: 10.1385/1-59745-130-4:301
1881. Environ Toxicol Chem. 2006 Sep;25(9):2381-7.
Effects of cyanobacterial toxins and cyanobacterial cell-free crude extract on

germination of alfalfa (Medicago sativa) and induction of oxidative stress.
Pflugmacher S(1), Jung K, Lundvall L, Neumann S, Peuthert A.
Author information:
(1)Leibniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm
301, 12587 Berlin, Germany. pflugmacher@igb-berlin.de
Cyanobacterial toxins have adverse effects on both terrestrial and aquatic

plants. Microcystins are cyclic heptapeptides and an important group of
cyanotoxins. When lake water contaminated with cyanobacterial blooms is used for
spray irrigation, these toxins can come in contact with agricultural plants.
During the exposure to these toxins, reactive oxygen species can form. These
reactive oxygen species have a strong reactivity and are able to interact with
other cellular compounds (lipids, protein, and DNA). Plants have antioxidative
systems that will limit the negative effects caused by reactive oxygen species.
These systems consist of enzymes, such as superoxide dismutase, catalase, and
ascorbate peroxidase, and nonenzymatic substances, such as reduced glutathione or
vitamins. The aim of the present study was to investigate the effects of
cyanobacterial toxins (microcystins and anatoxin-a) and cyanobacterial cell-free
crude extract on alfalfa (Medicago sativa) seedlings. Inhibition of germination
and root growth was observed with toxin concentrations of 5.0 microg/L. Also,
oxidative damage, such as lipid peroxidation, was detected after the exposure of
alfalfa seedlings to the toxin. Reactive oxygen detoxifying enzymes were
elevated, showing a marked response in alfalfa to oxidative stress caused by the
exposure to cyanobacterial metabolites that might influence the growth and
development of these plants negatively.
1882. Plant J. 2006 Oct;48(1):113-24.
Multi-site genetic modulation of monolignol biosynthesis suggests new routes for

formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago
sativa L.).
Chen F(1), Srinivasa Reddy MS, Temple S, Jackson L, Shadle G, Dixon RA.
Author information:
Parkway, Ardmore, Oklahoma 73401, USA.
Genes encoding seven enzymes of the monolignol pathway were independently

downregulated in alfalfa (Medicago sativa) using antisense and/or RNA
interference. In each case, total flux into lignin was reduced, with the largest
effects arising from the downregulation of earlier enzymes in the pathway. The
downregulation of l-phenylalanine ammonia-lyase, 4-coumarate 3-hydroxylase,
hydroxycinnamoyl CoA quinate/shikimate hydroxycinnamoyl transferase, ferulate
5-hydroxylase or caffeic acid 3-O-methyltransferase resulted in compositional
changes in lignin and wall-bound hydroxycinnamic acids consistent with the
current models of the monolignol pathway. However, downregulating caffeoyl CoA
3-O-methyltransferase neither reduced syringyl (S) lignin units nor wall-bound
ferulate, inconsistent with a role for this enzyme in 3-O-methylation ofS
monolignol precursors and hydroxycinnamic acids. Paradoxically, lignin
composition differed in plants downregulated in either cinnamate 4-hydroxylase or
phenylalanine ammonia-lyase. No changes in the levels of acylated flavonoids were
observed in the various transgenic lines. The current model for monolignol and
ferulate biosynthesis appears to be an over-simplification, at least in alfalfa,
and additional enzymes may be needed for the 3-O-methylation reactions of S
lignin and ferulate biosynthesis.
DOI: 10.1111/j.1365-313X.2006.02857.x
1883. J Zhejiang Univ Sci B. 2006 Oct;7(10):788-93.
A kinetic approach to evaluate salinity effects on carbon mineralization in a

plant residue-amended soil.
Nourbakhsh F(1), Sheikh-Hosseini AR.
Author information:
(1)Department of Soil Science, School of Agriculture, Isfahan University of
Technology, Isfahan 84154, Iran. farshid@cc.iut.ac.ir
The interaction of salinity stress and plant residue quality on C mineralization

kinetics in soil is not well understood. A laboratory experiment was conducted to
study the effects of salinity stress on C mineralization kinetics in a soil
amended with alfalfa, wheat and corn residues. A factorial combination of two
salinity levels (0.97 and 18.2 dS/m) and four levels of plant residues (control,
alfalfa, wheat and corn) with three replications was performed. A first order
kinetic model was used to describe the C mineralization and to calculate the
potentially mineralizable C. The CO(2)-C evolved under non-saline condition,
ranged from 814.6 to 4842.4 mg CO(2)-C/kg in control and alfalfa residue-amended
soils, respectively. Salinization reduced the rates of CO(2) evolution by 18.7%,
6.2% and 5.2% in alfalfa, wheat and corn residue-amended soils, respectively.
Potentially mineralizable C (C(0)) was reduced significantly in salinized alfalfa
residue-treated soils whereas, no significant difference was observed for control
treatments as well as wheat and corn residue-treated soils. We concluded that the
response pattern of C mineralization to salinity stress depended on the plant
residue quality and duration of incubation.
DOI: 10.1631/jzus.2006.B0788
PMCID: PMC1599805
1884. J Anim Sci. 2006 Oct;84(10):2801-8.
Effects of alfalfa extract, anise, capsicum, and a mixture of cinnamaldehyde and

eugenol on ruminal fermentation and protein degradation in beef heifers fed a
high-concentrate diet.
Cardozo PW(1), Calsamiglia S, Ferret A, Kamel C.
Author information:
(1)Animal Nutrition, Management, and Welfare Research Group, Departament de
Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193,
Bellaterra, Spain.
Four Holstein heifers (360 +/- 22 and 450 +/- 28 kg of BW in Exp. 1 and 2,
respectively) fitted with ruminal trocars were used in 4 x 4 Latin square designs
to evaluate the effects on ruminal microbial fermentation of the following: Exp.
1, no additive, alfalfa extract (30 g/d, AEX), a mixture of cinnamaldehyde (0.18
g/d) and eugenol (0.09 g/d; CIE1), and AEX and CIE1 in combination; and Exp. 2,
no additive, anise oil (2 g/d), capsicum oil (1 g/d), and a mixture of
cinnamaldehyde (0.6 g/d) and eugenol (0.3 g/d). Heifers were fed a 90:10
concentrate:barley straw diet (16% CP; 25% NDF) for ad libitum intake. Each
period consisted of 15 d for adaptation and 6 d for sampling. On d 16 to 18, DM
and water intakes were measured. On d 19 to 21 ruminal contents were sampled at
0, 3, 6, 9, and 12 h after feeding to determine ruminal pH and the concentrations
of VFA, L-lactate, large peptides, small peptides plus AA (SPep+AA), and ammonia
N. On d 20 and 21, samples of ruminal fluid were collected at 0 and 3 h after
feeding to determine protozoal counts. In Exp. 1, CIE1 and AEX decreased (P <
0.05) total DMI, concentrate DMI, and water intake. The increase (P < 0.05) in
SPep+AA and the decrease (P < 0.05) in ammonia N when supplementing CIE1 suggest
that deamination was inhibited. Treatment AEX increased (P < 0.05) the acetate to
propionate ratio, which is less efficient for beef production. Treatment CIE1
increased (P < 0.05) counts of holotrichs. Effects of AEX and CIE1 were not
additive for many of the measured metabolites. In Exp. 2, treatments had no
effect on ruminal pH, total VFA concentration, and butyrate proportion. The
capsicum oil treatment increased (P < 0.05) DMI, water intake, and SPep+AA N
concentration and decreased (P < 0.05) acetate proportion, branched-chain VFA
concentration, and large peptide N concentration. The cinnamaldehyde (0.6 g/d)
and eugenol (0.3 g/d) treatment decreased (P < 0.05) water intake, acetate
proportion, branched-chain VFA, L-lactate, and ammonia N concentrations and
increased (P < 0.05) propionate proportion and SPep+AA N concentration. The anise
oil treatment decreased (P < 0.05) acetate to propionate ratio, branched-chain
VFA and ammonia N concentrations, and protozoal counts. The results indicate that
at the doses used a mixture of cinnamaldehyde and eugenol, anise oil, and
capsicum oil may be useful as modifiers of rumen fermentation in beef production
systems.
DOI: 10.2527/jas.2005-593
1885. J Agric Food Chem. 2006 Sep 20;54(19):7187-92.
Glyphosate-tolerant alfalfa is compositionally equivalent to conventional alfalfa

McCann MC(1), Rogan GJ, Fitzpatrick S, Trujillo WA, Sorbet R, Hartnell GF, Riodan
SG, Nemeth MA.
Author information:
(1)Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167,
USA. melinda.c.mccann@monsanto.com
Glyphosate-tolerant alfalfa (GTA) was developed to withstand over-the-top

applications of glyphosate, the active ingredient in Roundup agricultural
herbicides. As a part of the safety assessment, GTA (designated J101 x J163) was
grown under controlled field conditions at geographically diverse locations
within the United States during the 2001 and 2003 field seasons along with
control and other conventional alfalfa varieties for compositional assessment.
Field trials were conducted using a randomized complete block design with four
replication blocks at each site. Alfalfa forage was harvested at the late bud to
early bloom stage from each plot at five field sites in 2001 (establishment year)
and from four field sites in 2003 (third year of stand). The concentration of
proximate constituents, fibers, amino acids, coumestrol, and minerals in the
forage was measured. The results showed that the forage from GTA J101 x J163 is
compositionally equivalent to forage from the control and conventional alfalfa
varieties.
DOI: 10.1021/jf061482m
1886. Appl Environ Microbiol. 2006 Nov;72(11):7156-67. Epub 2006 Sep 8.
An integrated approach to functional genomics: construction of a novel reporter

gene fusion library for Sinorhizobium meliloti.
Cowie A(1), Cheng J, Sibley CD, Fong Y, Zaheer R, Patten CL, Morton RM, Golding
GB, Finan TM.
Author information:
Hamilton, Ontario L8S 4K1, Canada.
As a means of investigating gene function, we developed a robust transcription

fusion reporter vector to measure gene expression in bacteria. The vector,
pTH1522, was used to construct a random insert library for the Sinorhizobium
meliloti genome. pTH1522 replicates in Escherichia coli and can be transferred
to, but cannot replicate in, S. meliloti. Homologous recombination of the DNA
fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional
fusions to either the reporter genes gfp(+) and lacZ or gusA and rfp, depending
on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298
clones were identified by DNA sequence analysis, and the plasmid clones were
recombined into S. meliloti. Reporter enzyme activities following growth of these
recombinants in complex medium (LBmc) and in minimal medium with glucose or
succinate as the sole carbon source allowed the identification of genes highly
expressed under one or more growth condition and those expressed at very low to
background levels. In addition to generating reporter gene fusions, the vector
allows Flp recombinase-directed deletion formation and gene disruption, depending
on the nature of the cloned fragment. We report the identification of genes
essential for growth on complex medium as deduced from an inability to recover
recombinants from pTH1522 clones that carried fragments internal to gene or
operon transcripts. A database containing all the gene expression activities
together with a web interface showing the precise locations of reporter fusion
junctions has been constructed (www.sinorhizobium.org).
DOI: 10.1128/AEM.01397-06
PMCID: PMC1636157
1887. J Dairy Sci. 2006 Oct;89(10):3939-53.
Effect of varying dietary ratios of alfalfa silage to corn silage on omasal flow
and microbial protein synthesis in dairy cows.
Brito AF(1), Broderick GA, Reynal SM.
Author information:
53706, USA.
Eight ruminally cannulated multiparous Holstein cows that were part of a larger
production trial were used to study the effects of varying dietary ratios of
alfalfa silage (AS) to corn silage (CS) on omasal flow of nutrients and microbial
protein. Cows were blocked by DIM and randomly assigned to 2 replicated 4 x 4
Latin squares (28-d periods). Diets fed contained (dry matter basis): A) 51% AS,
43% rolled high-moisture shelled corn (HMSC), and 3% solvent soybean meal (SSBM);
B) 37% AS, 13% CS, 39% HMSC, and 7% SSBM; C) 24% AS, 27% CS, 35% HMSC, and 12%
SSBM; or D) 10% AS, 40% CS, 31% HMSC, and 16% SSBM. Crude protein (CP) contents
were 17.2, 16.9, 16.6, and 16.2% for diets A, B, C, and D. All 4 diets were high
in energy, averaging 49% nonfiber carbohydrates and 24% neutral detergent fiber.
Total microbial nonammonia nitrogen flow was lower on diet D (423 g/d) compared
with diets A (465 g/d), B (479 g/d), and C (460 g/d). A significant quadratic
effect indicated that microbial protein synthesis was maximal at 38% AS. Supply
of rumen-degraded protein decreased linearly from 3,068 g/d (diet A) to 2,469 g/d
(diet D). Omasal flow of rumen-undegraded protein did not differ among diets and
averaged 1,528 g/d. However, when expressed as a percentage of dry matter intake,
rumen-undegraded protein increased linearly from 5.59% (diet A) to 6.13% (diet
D), probably because CP from SSBM was more resistant to degradation than CP from
AS. Essential AA flow was lowest on diet D, and Lys flow tended to be lower on
diet D, which may explain the lower milk and protein yields observed on that
diet.
DOI: 10.3168/jds.S0022-0302(06)72436-5
1888. J Dairy Sci. 2006 Oct;89(10):3924-38.
Effect of varying dietary ratios of alfalfa silage to corn silage on production

and nitrogen utilization in lactating dairy cows.
Brito AF(1), Broderick GA.
Author information:
53706, USA.
Twenty-eight (8 ruminally cannulated) lactating, multiparous Holstein cows were

blocked by DIM and randomly assigned to 7 replicated 4 x 4 Latin squares (28-d
periods) to investigate the effects of different dietary ratios of alfalfa silage
(AS) to corn silage (CS) on production, N utilization, apparent digestibility,
and ruminal metabolism. The 4 diets contained (dry matter basis): A) 51% AS, 43%
rolled high-moisture shelled corn (HMSC), and 3% solvent soybean meal (SSBM); B)
37% AS, 13% CS, 39% HMSC, and 7% SSBM; C) 24% AS, 27% CS, 35% HMSC, and 12% SSBM;
and D) 10% AS, 40% CS, 31% HMSC, and 16% SSBM. Dietary crude protein contents
were 17.2, 16.9, 16.6, and 16.2% for diets A, B, C, and D. All 4 diets were high
in energy, averaging 49% nonfiber carbohydrates and 24% neutral detergent fiber.
Intake of dry matter, yield of milk, 3.5% fat-corrected milk and fat, milk fat
content, and apparent digestibility of neutral detergent fiber and acid detergent
fiber all decreased linearly when CS replaced AS. Effects on fiber digestion and
milk fat may have been due to increasing fluctuation in ruminal pH and time the
pH remained < 6.0 when CS replaced AS. Milk protein content increased linearly
with increasing CS, but there were no differences in protein yield. There were
linear increases in apparent N efficiency and decreases in N excreted in urine
and feces when CS replaced AS. Production was depressed on the diet highest in
CS. Quadratic analysis indicated that milk and protein yields were maximal at
dietary AS:CS ratios of, respectively, 37:13 and 31:19. No diet minimized N
excretion without negatively affecting production. Diet C, with an AS:CS ratio of
24:27, was the best compromise between improved N efficiency and sustained
production. Because CS is complementary with AS, it is recommended that CS be fed
in AS-based diets to maintain milk yield while improving N utilization.
DOI: 10.3168/jds.S0022-0302(06)72435-3
Metabolite profiles of nodulated alfalfa plants indicate that distinct stages of

nodule organogenesis are accompanied by global physiological adaptations.
Barsch A(1), Tellström V, Patschkowski T, Küster H, Niehaus K.
Author information:
(1)Proteom und Metabolomforschung, Fakultät für Biologie, Universität Bielefeld,
Postfach 100131, D-33501 Bielefeld, Germany. aiko.barsch@Genetik.Uni-Bielefeld.DE
An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago
sativa is dependent on a balanced physiological interaction enabling the
microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic
interaction is regulated by still poorly understood control mechanisms. A first
step toward a better understanding of nodule metabolism was the determination of
characteristic metabolites for alfalfa root nodules. Furthermore, nodules
arrested at different developmental stages were analyzed in order to address
metabolic changes induced during the progression of nodule formation. Metabolite
profiles of bacteroid-free pseudonodule extracts indicated that early nodule
developmental processes are accompanied by photosynthate translocation but no
massive organic acid formation. To determine metabolic adaptations induced by the
presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains
lacking the nitrogenase protein were analyzed. The bacteroids are unable to
provide ammonium to the host plant, which is metabolically reflected by reduced
levels of characteristic amino acids involved in ammonium fixation. Elevated
levels of starch and sugars in Fix(-) nodules provide strong evidence that plant
sanctions preventing a transformation from a symbiotic to a potentially parasitic
interaction are not strictly realized via photosynthate supply. Instead,
metabolic and gene expression data indicate that alfalfa plants react to
nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis
and an early induction of senescence. Noneffective symbiotic interactions
resulting from plants nodulated by mutant rhizobia also are reflected in
characteristic metabolic changes in leaves. These are typical for nitrogen
deficiency, but also highlight metabolites potentially involved in sensing the N
status.
DOI: 10.1094/MPMI-19-0998
1890. Plant Sci. 2006 Sep;171(3):332-44. doi: 10.1016/j.plantsci.2006.04.007. Epub

2006
May 17.
Isolation and characterization of class A4 heat shock transcription factor from

alfalfa.
Friedberg JN(1), Bowley SR, McKersie BD, Gurley WB, Czarnecka-Verner E.
Author information:
(1)Department of Plant Agriculture, Crop Science Building, University of Guelph,
Guelph, Ont. N1G 2W1, Canada.
Plant heat shock transcription factors (HSFs) regulate transcription of heat

shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been classified into
groups A-C. Members of class A act as typical transcriptional activators, whereas
B HSFs function as coactivators or repressors depending on promoter context. The
function of class C HSFs is still unclear. Here, we present the isolation and
characterization of the first HSF from alfalfa (Medicago sativa L.) and designate
it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined
to be single copy and was detected at two separate genetic loci in the tetraploid
Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll protoplasts
resulted in weak transcriptional activity, similar to that exhibited by
Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains three putative HSE
elements, and the gene itself is activated both by heat and cold stress.

DOI: 10.1016/j.plantsci.2006.04.007
PMID: 22980202
1891. J Nematol. 2006 Sep;38(3):391-8.
Effect of Rotation Crops on Heterodera glycines Population Density in a

Greenhouse Screening Study.
Warnke SA(1), Chen SY, Wyse DL, Johnson GA, Porter PM.
Author information:
(1)Department of Plant Pathology and Agronomy and Plant Genetics, University of
Minnesota, Saint Paul, MN 55108. Southern Research and Outreach Center,
University of Minnesota, Waseca, MN 56093.
Crop rotation is a common means of reducing pathogen populations in soil. Several

rotation crops have been shown to reduce soybean cyst nematode (Heterodera
glycines) populations, but a comprehensive study of the optimal crops is needed.
A greenhouse study was conducted to determine the effect of growth and
decomposition of 46 crops on population density of H. glycines. Crops were sown
in soil infested with H. glycines. Plants were maintained until 75 days after
planting, when the soil was mixed, a sample of the soil removed to determine egg
density, and shoots and roots chopped and mixed into the soil. After 56 days,
soil samples were again taken for egg counts, and a susceptible soybean
('Sturdy') was planted in the soil as a bioassay to determine egg viability. Sunn
hemp (Crotalaria juncea), forage pea (Pisum sativum), lab-lab bean (Lablab
purpureus), Illinois bundleflower (Desman-thus illinoensis), and alfalfa
(Medicago sativa) generally resulted in smaller egg population density in soil or
number of cysts formed on soybean in the bioassay than the fallow control. Sunn
hemp most consistently showed the lowest numbers of eggs and cysts. As a group,
legumes resulted in lower egg population densities than monocots, Brassica
species, and other dicots.
PMCID: PMC2586703
PMID: 19259545
Yield and forage quality of glandular-haired alfalfa under alfalfa weevil

(Coleoptera: Curculionidae) and potato leafhopper (Hemiptera: Cicadellidae) pest
pressure in Virginia.
Dellinger TA(1), Youngman RR, Laub CA, Brewster CC, Kuhar TP.
Author information:
(1)Department of Entomology, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061, USA.
Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are

currently available and marketed as being resistant to potato leafhopper,
Empoasca fabae (Harris) (Hemiptera: Cicadellidae). 54H69 and a standard,
nonglandular-haired alfalfa 'Choice' were evaluated at two locations in Virginia
over a 3-yr period. Dry matter yields and concentrations of crude protein and
acid detergent fiber were compared at the first, second, and third harvests.
Overall, the two cultivars produced similar dry matter yields of comparable
forage quality in the absence of insecticides at both locations in each year.
Untreated 54H69 did not produce greater dry matter yields than untreated Choice
under either light or heavier potato leafhopper pest pressure. Concentrations of
crude protein did not vary between the two cultivars at any harvest. Some
differences in concentrations of acid detergent fiber were detected between
cultivars, but these differences were not consistent among years, harvests, or
between locations. Further comparisons between untreated 54H69 and treated Choice
were made, but few significant differences were detected in dry matter yields or
forage quality. An economic analysis for the study indicated that a grower
planting 54H69 would realize less net revenue than a grower planting Choice,
largely because of the seed premium for the glandular-haired cultivar and the
evident need to treat 54H69 with insecticide for control of alfalfa weevil,
Hypera postica (Gyllenhal) (Coleoptera: Curculionidae), and potato leafhopper.
Cropping systems for phytoremediation of phosphorus-enriched soils.
Kratochvil RJ(1), Coale FJ, Momen B, Harrison MR Jr, Pearce JT, Schlosnagle S.
Author information:
(1)Department of Natural Resource Sciences and Landscape Architecture, University
of Maryland, College Park, Maryland 20742, USA. rkratoch@umd.edu
Eutrophication of freshwater bodies is frequently attributed to elevated

phosphorus (P) concentrations in surface runoff from P-enriched agricultural
soils. Forage and grain-cropping systems were compared for their effectiveness at
remediating P-enriched soils. At each of four locations, one of three forage
systems (Forage I = cereal rye silage and corn silage annually; Forage II =
alfalfa; Forage III = annual ryegrass and corn silage annually) and the grain
system (corn, small grain, and soybean rotation) were maintained for 3 yr on
soils with five distinct initial soil P concentrations that were established by
using four annual applications (1994-1997) of five different rates (0, 100, 200,
300, and 400 kg total P ha(-1) y(-1)) of poultry manure, dairy manure, or
commercial fertilizer. Across all manure P treatments at all locations, the
forage systems had greater removal of P than the grain system. Soil P
concentration changes (2001-2004) did not reflect differences in crop P removal.
Few significant reductions in soil P concentration were observed for either crop
system. When reductions did occur, they were for the more highly enriched soil P
treatments. No significant reductions in soil P concentration have occurred for
the lowest manure P treatments. Considerable variability in crop P concentrations
was observed among species at locations and among years produced. However, crop P
concentrations did increase uniformly as soil P concentration increased,
indicating that luxury consumption of P does occur in agronomic species produced
on P-enriched soils.
DOI: 10.1080/15226510600678456
1894. J Food Prot. 2006 Aug;69(8):1844-50.
Detection of Escherichia coli in packaged alfalfa sprouts with an electronic nose

and an artificial neural network.
Siripatrawan U(1), Linz JE, Harte BR.
Author information:
Bangkok, Thailand. ubonratana.s@chula.ac.th
A rapid method for the detection of Escherichia coli (ATCC 25922) in packaged
alfalfa sprouts was developed. Volatile compounds from the headspace of packaged
alfalfa sprouts, inoculated with E. coli and incubated at 10 degrees C for 1, 2,
and 3 days, were collected and analyzed. Uninoculated sprouts were used as
control samples. An electronic nose with 12 metal oxide electronic sensors was
used to monitor changes in the composition of the gas phase of the package
headspace with respect to volatile metabolites produced by E. coli. The
electronic nose was able to differentiate between samples with and without E.
coli. To predict the number of E. coli in packaged alfalfa sprouts, an artificial
neural network was used, which included an input layer, a hidden layer, and an
output layer, with a hyperbolic tangent sigmoidal transfer function in the hidden
layer and a linear transfer function in the output layer. The network was shown
to be capable of correlating voltametric responses with the number of E. coli. A
good prediction was possible, as measured by a regression coefficient (R2 =
0.903) between the actual and predicted data. In conjunction with the artificial
neural network, the electronic nose proved to have the ability to detect E. coli
in packaged alfalfa sprouts.
1895. Microb Ecol. 2006 Oct;52(3):583-95. Epub 2006 Aug 19.
Long-term field release of bioluminescent Sinorhizobium meliloti strains to

assess the influence of a recA mutation on the strains' survival.
Selbitschka W(1), Keller M, Miethling-Graff R, Dresing U, Schwieger F, Krahn I,

Homann I, Dammann-Kalinowski T, Pühler A, Tebbe CC.
Author information:
(1)Lehrstuhl für Genetik, Universität Bielefeld, Postfach 100131, D-33501
Bielefeld, Germany. Werner.Selbitschka@Genetik.Uni-Bielefeld.DE
A field release experiment was carried out to study the fate of the isogenic,
firefly luciferase (luc) gene-tagged Sinorhizobium meliloti strains L1 (RecA-)
and L33 (RecA+) in the environment. Both strains were released at concentrations
of approximately 10(6) cfu g(-1) soil in replicate and randomized field plots,
which had been sown with alfalfa (Medicago sativa). The survival of both strains
during the following 7 years could be subdivided into three phases: a sharp
decline for more than two orders of magnitude within the first 4 months (phase
I), followed by fluctuations around an average number of 10(4) cfu g(-1) soil for
nearly 4 years (phase II), and a further decline to approximately 60 cfu g(-1)
(phase III). At most sampling dates, no significant differences in the survival
of both strains were detected, indicating that the recA gene function was
dispensable under these environmental conditions. During the field inoculation,
both strains were dispersed accidentally by wind in small numbers to
noninoculated field plots. Strain L33 established at a concentration of more than
10(3) cfu g(-1) soil with subsequent seasonal fluctuations. Although strain L1
must have been disseminated to a similar extent, it could never be recovered from
noninoculated field plots, indicating that the recA mutation interfered with the
strain's capability to establish there. At the beginning of the field experiment,
an indigenous alfalfa-nodulating population was below the limit of detection. In
the following years, however, an indigenous population arose, which finally
outcompeted both strains for saprophytic growth and alfalfa nodulation. RecA-
strain L1 was outcompeted for alfalfa nodulation slightly faster than its RecA+
counterpart L33. The diversity of the indigenous population was characterized by
employing the Enterobacterial Repetitive Intergenic Consensus polymerase chain
reaction fingerprint method. Typing of 2731 root nodule isolates revealed a total
of 38 fingerprint groups. More than 80% of the isolates could be grouped into six
dominant fingerprint groups, indicating that a few dominant bacterial strain
types had outcompeted the released strains.
DOI: 10.1007/s00248-006-9056-6
1896. Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):13068-73. Epub 2006 Aug 21.
Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis.
Maussang D(1), Verzijl D, van Walsum M, Leurs R, Holl J, Pleskoff O, Michel D,

van Dongen GA, Smit MJ.
Author information:
(1)Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry,
Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV, Amsterdam,
The Netherlands.
Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a

role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a
broad spectrum of chemokines and constitutively activates various pathways linked
to proliferation. Our studies reveal that expression of US28 induces a
proangiogenic and transformed phenotype by up-regulating the expression of
vascular endothelial growth factor and enhancing cell growth and cell cycle
progression. US28-expressing cells promote tumorigenesis when injected into nude
mice. The G protein-uncoupled constitutively inactive mutant of US28, induces
delayed and attenuated tumor formation, indicating the importance of constitutive
receptor activity in the early onset of tumor development. Importantly, also in
glioblastoma cells infected with the newly isolated clinical HCMV strain Titan,
US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence,
the constitutively activated chemokine receptor US28 might act as a viral
oncogene and enhance and/or promote HCMV-associated tumor progression.
DOI: 10.1073/pnas.0604433103
PMCID: PMC1559754
1897. Bull Entomol Res. 2006 Aug;96(4):327-35.
Adult movements of newly introduced alien Diabrotica virgifera virgifera

(Coleoptera: Chrysomelidae) from non-host habitats.
Toepfer S(1), Levay N, Kiss J.
Author information:
(1)Department of Plant Protection, St Istvan University, Pater K. Street 1,
H-2100 Godollo, Hungary. stoepfer@gmx.net
Mark-release-recapture experiments were undertaken in order to investigate the

movement of adult Diabrotica virgifera virgifera LeConte from accidental
introduction points towards suitable habitats, such as its host plant, Zea mays
L. In Hungary in 2003 and 2004, nine mark-release-recapture experiments were
carried out in a grass steppe area and lucerne field, in which two small maize
fields (10x10 m) had been planted 300 m distant from the central release point.
After each release of 5500 to 6000 marked D. v. virgifera, beetle recaptures were
recorded three times using non-baited yellow sticky traps placed on 30, 105, 205
and 305 m radii around the release point. In seven out of 15 recapture periods
(47%), beetle populations showed no directional movements, and their movements
towards any particular habitat cannot be predicted. During five recapture periods
(33%), beetle populations showed a uni-directional movement, and in three cases
(20%) a bi-directional movement was observed. In 10 out of 15 recapture periods
(67%), the released populations moved in a direction that was comparable with the
mean wind direction during these periods; thus, beetle movements were slightly
correlated with wind direction. On average over sites and years, beetles were not
preferentially moving towards the two small maize fields (located 300 m from the
release point) compared to other directions. However, beetles moved significantly
more frequently in the direction of naturally-occurring maize fields within a
radius of 1500 m than towards other habitats. Beetles stayed more frequently
within flowering lucerne fields out to a radius of 300 and 600 m than in
non-flowering lucerne or other habitats. On average, 2.8% (SD 3.2) of all
recaptured beetles arrived in one of the two small maize fields located 300 m
from their release point indicating that there is a high risk of a founder
population establishing. Habitat management cannot be suggested as a means of
preventing the beetle's initial dispersal because movement was usually
non-directional, and alternative food plants were used prior to reaching maize.
1898. Ecology. 2006 Jul;87(7):1861-6.
The allometry of plant spacing that regulates food intake rate in mammalian
herbivores.
Fortin D(1).
Author information:
(1)Département de biologie, Université Laval, Sainte-Foy, Québec GLK 7P4, Canada.
Daniel.Fortin@bio.laval.ca
The distance that mammalian herbivores can travel without interrupting food
processing corresponds to a distance threshold (d*) in plant spacing where change
occurs in the mechanisms regulating the functional response. The instantaneous
rate of food consumption is controlled by food encounter rate when plant spacing
exceeds d*. Below this threshold, food processing in the mouth controls
instantaneous intake rate. The distance threshold provides a mechanistic
definition of the scale of heterogeneity of the food resource. Recent work
indicates that d* should scale positively with the body mass of mammalian
herbivores. Here I evaluated the empirical evidence for this positive scaling by
investigating (1) herbivores consuming only alfalfa (Medicago sativa), (2)
grazers, and (3) herbivores consuming any kind of vegetation. Overall, I found
greater evidence for a negative than for a positive scaling of d*. Out of the
three groups, only herbivores consuming alfalfa could yield a positive
covariation between d* and body mass. However, even this positive scaling became
negative when herbivores consumed bites of alfalfa that were only a fraction of
their maximum size. Finally, d* also scaled negatively among herbivores foraging
in similar food patches. Overall, differences in foraging decisions among
mammalian herbivores seem more likely to have been shaped by a negative than a
positive scaling of d*.
1899. Plant Cell Environ. 2006 Sep;29(9):1841-52.
Isolation of a novel nodulin: a molecular marker of osmotic stress in Glycine

max/Bradyrhizobium japonicum nodule.
Clement M(1), Boncompagni E, de Almeida-Engler J, Herouart D.
Author information:
(1)Unité Mixte de Recherche (UMR) 'Interactions Plantes Microorganimes et Santé
Végétale' INRA1064-CNRS 6192-Université de Nice-Sophia Antipolis, Institut Sophia
Agrobiotech, 400 Routes des Chappes, 06903 Sophia-Antipolis Cedex, France.
Symbiotic N(2) fixation of legume crops is highly sensitive to drought, which

results in a dramatic drop of N accumulation and yield. The symbiosis between
soybean (Glycine max) and Bradyrhizobium japonicum, because of its extreme
sensitivity to drought, was chosen as a model to analyse the response to drought
stress at a molecular level. The mRNA differential display technique was
performed to isolate cDNA markers differentially expressed in well-watered [100%
of N(2) fixation capacity (NFC)] and drought-stressed nodules (40% NFC). One gene
noted, G93, appeared strongly down-regulated by drought and fully recovered after
rehydration. In situ hybridization showed that G93 transcripts were localized in
N(2)-fixing cells of mature nodules, indicating that G93 could be considered as a
late nodulin. However, G93 expression was not directly correlated to N(2)
fixation but mainly responded to osmotic stress. Other stresses that lead to
decrease of N(2) fixation did not affect G93 expression. Sequence analyses showed
that G93 presented a strong homology with two soybean expressed sequence tags
(ESTs) and with the ZR1 protein of Medicago sativa. Putative roles of this
nodulin in adaptation of soybean nodule to osmotic stress are proposed.
DOI: 10.1111/j.1365-3040.2006.01558.x
Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine

transporter, in bacteroids from Medicago sativa nodules sustains nitrogen
fixation during early salt stress adaptation.
Boscari A(1), Van de Sype G, Le Rudulier D, Mandon K.
Author information:
(1)Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR 6192 CNRS-
1064 INRA-Université de Nice-Sophia Antipolis, Centre de Recherche INRA
Agrobiotech, 400 Route des Chappes, BP 167, 06903 Sophia Antipolis, France.
Sinorhizobium meliloti possesses several betaine transporters to cope with salt

stress, and BetS represents a crucial high-affinity glycine and proline betaine
uptake system involved in the rapid acquisition of betaines by cells subjected to
osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we
showed that betS is expressed during the establishment of the symbiosis and in
mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were
impaired in a betS mutant. BetS is functional in isolated bacteroids, and its
activity is strongly activated by high osmolarity. In bacteroids from a betS
mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%,
indicating that BetS is a major component of the overall betaine uptake activity
in bacteroids in response to osmotic stress. Upon betS overexpression (strain
UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than
in the wild-type strain. Interestingly, the accumulation of proline betaine, the
endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349
bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl)
than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen
fixation activity was observed in 7-day-salinized plants nodulated with the
overexpressing strain than in wild-type nodulated plants. Taken altogether, these
results are consistent with the major role of BetS as an emergency system
involved in the rapid uptake of betaines in isolated and in planta osmotically
stressed bacteroids of S. meliloti.
DOI: 10.1094/MPMI-19-0896
1901. J Dairy Sci. 2006 Sep;89(9):3505-7.
Short communication: effect of number of extractions on percentage of long-chain

fatty acids from fresh alfalfa.
Ribeiro CV(1), Eastridge ML.
Author information:
(1)Department of Animal Sciences, The Ohio State University, Columbus, Ohio
43210, USA.
Accurate determination of fatty acids in fresh forage is very important when

studying biohydrogenation. Fatty acids from fresh alfalfa were extracted by
hexane:isopropanol (H:IP, 3:2 vol/vol) in 3 sequential extractions. The
percentage and profile of fatty acids from each of the 3 extractions were
evaluated by a randomized complete block design with repeated measures in space.
Samples of fresh alfalfa were randomly harvested and immediately submerged in
liquid nitrogen. For the first extraction, approximately 5 g of the frozen
alfalfa was mixed with 18 mL of H:IP per gram of material. Samples were then
centrifuged and the supernatant was collected. The second and third extractions
were done by adding H:IP to the pellet (3 mL/g of the original sample weight),
mixing for 2 min, and then centrifuging. Samples were submerged in H:IP and
stored in the dark at 8 degrees C at all times. The solvent from each extraction
was partially evaporated and the fatty acids methylated by methanolic HCl.
Repeated extractions increased the percentage of total fatty acids recovered from
the samples. The concentration of fatty acids in the alfalfa after 3 extractions
was 4.0%. The first, second, and third extractions resulted in 92.7, 4.8, and
2.6% of the total fatty acids extracted, respectively. There was no effect of
extraction on the proportion of 16:0, 18:0, 18:1, and 18:2 fatty acids. However,
the proportion of 18:3 in the extract decreased from the first to the second
extraction and the ratio of saturated to unsaturated fatty acid increased from
the first to the second extraction. The results of this experiment revealed that
the profile of fatty acids can vary with the number of extractions performed. The
higher amount of 18:3 in the first extraction may reflect the higher proportion
of linolenic acid in the more easily extracted plant fractions.
DOI: 10.3168/jds.S0022-0302(06)72390-6
1902. Mycol Res. 2006 Aug;110(Pt 8):887-97. Epub 2006 Aug 7.
Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm

roots: an underestimated experimental method.
Dreyer B(1), Morte A, Pérez-Gilabert M, Honrubia M.
Author information:
(1)Departamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia,
Campus de Espinardo, E-30100 Murcia, Spain.
The aim of this study was to reassess the use of autofluorescence for evaluating
AM colonization in mycorrhizal roots in the light of criticisms of this method
that affirmed that only metabolically inactive arbuscules autofluoresce. It was
also investigated whether other mycorrhizal structures, such as hyphae, vesicles
and spores, could be detected by autofluorescence, and whether the
autofluorescence pattern of AM fungal structures could be exploited
methodologically, for example, in the detection and sorting of spores by flow
cytometry. Mycorrhizal roots of the palm species Brahea armata, Chamaerops
humilis, Phoenix canariensis and Phoenix dactylifera were sectioned and observed
by means of fluorescence microscopy. In addition, fungal structures isolated from
mycorrhizal roots of P. dactylifera were examined. The same root sections and
isolated fungal structures were subjected to vital staining with nitro blue
tetrazolium to determine their metabolic state (active or inactive). Moreover,
spores of Glomus intraradices, and Glomus clarum were studied by epifluorescence
and flow cytometry. Mycorrhizal whole roots of Medicago sativa were also assessed
by autofluorescence detection. In contrast to previous reports, the results
presented in this paper clearly demonstrate that all fungal structures, both
intra- and extraradical, autofluoresced under blue light excitation, regardless
of their state (dead or alive). Some arbuscules isolated from roots and mature
spores showed further autofluorescence under green light excitation. The source
of the autofluorescence was localized in the fungal cell wall. It was shown that
AM spores can be detected by flow cytometry. The results support the use of
autofluorescence for the evaluation of AM colonization, at least in palm species,
and refute previous criticisms of the method.
DOI: 10.1016/j.mycres.2006.05.011
1903. Autoimmun Rev. 2006 Jul;5(6):429-35. Epub 2005 Dec 29.
Role of non-protein amino acid L-canavanine in autoimmunity.
Akaogi J(1), Barker T, Kuroda Y, Nacionales DC, Yamasaki Y, Stevens BR, Reeves
WH, Satoh M.
Author information:
(1)Division of Rheumatology and Clinical Immunology, Department of Medicine,
University of Florida, Gainesville, FL 32610-0221, USA.
Association of SLE and alfalfa was first reported in a volunteer who developed
lupus-like autoimmunity while ingesting alfalfa seed for a hypercholesterolemia
study. This was corroborated with studies in monkeys fed with alfalfa sprout that
developed SLE. Re-challenge with L-canavanine relapsed the disease. Arginine
homologue L-canavanine, present in alfalfa, was suspected as a cause.
L-canavanine can be charged by arginyl tRNA synthetase to replace L-arginine
during protein synthesis. Aberrant canavanyl proteins have disrupted structure
and functions. Induction or exacerbation of SLE by alfalfa tablets reported in a
few cases remains controversial. Epidemiological studies on the relationship
between alfalfa and SLE are sparse. In mice, NZB/W F1, NZB, and DBA/2 mice fed
with L-canavanine show exacerbation/triggering of the SLE, however, BALB/c
studies were negative. L-canavanine incorporation may be more efficient in the
presence of inflammation or other conditions that can cause arginine deficiency.
The L-canavanine induced apoptotic cells can be phagocytosed and a source of
autoantigens processed by endosomal proteases. Endogenous canavanyl proteins are
ubiquitinated and processed via proteasome. Incorporation of L-canavanine into
proteasome or endosome can also cause disruption of antigen processing.
Alfalfa/L-canavanine-induced lupus will be an interesting model of autoimmunity
induced by the modification of self-proteins at the translational level.
DOI: 10.1016/j.autrev.2005.12.004
1904. Wiad Parazytol. 2001;47(4):839-44.
[The effect of Medicago spp. on growth of Trichophyton mentagrophytes in

microculture].
[Article in Polish]
Spiewak R(1), Szostak W, Jurzysta M, Biały Z, Maleszka R, Rzepecka B, Mazurek M.
Author information:
(1)Instytut Medycyny Wsi, ul. Jaczewskiego 2, 20-090 Lublin.
The study aimed at assessing effect of dried root and aerial parts of Medicago
spp. on growth of Trichophryton mentagrophytes. Fungus strains were inoculated
onto microcultures with Sabouraud agar supplemented each with 1 g of dried and
pulverised roots or aerial parts of 3 species: Medicago arabica, M. sativa, and
M. murex. The strongest inhibitory effect on T. mentagrophytes growth was that of
aerial parts of M. arabica (median diameter 6 mm compared to 13 mm of control),
followed by root of M. arabica (10 mm) and root of M. murex (10.5 mm)--in all
cases p < 0.001. Slight inhibitory effect was also found in the case of aerial
parts of M. murex (median diameter 12 mm, p = 0.03). In contrast, M. sativa has
shown stimulating effect on growth of T. mentagrophytes (15 mm for root and 16.5
mm for aerial part, p<0.001).
1905. Appl Environ Microbiol. 2006 Aug;72(8):5421-7.
Peptidoglycan from Bacillus cereus mediates commensalism with rhizosphere

bacteria from the Cytophaga-Flavobacterium group.
Peterson SB(1), Dunn AK, Klimowicz AK, Handelsman J.
Author information:
(1)Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI
53706, USA.
Previous research in our laboratory revealed that the introduction of Bacillus

cereus UW85 can increase the populations of bacteria from the
Cytophaga-Flavobacterium (CF) group of the Bacteroidetes phylum in the soybean
rhizosphere, suggesting that these rhizosphere microorganisms have a beneficial
relationship (G. S. Gilbert, J. L. Parke, M. K. Clayton, and J. Handelsman,
Ecology 74:840-854, 1993). In the present study, we determined the frequency at
which CF bacteria coisolated with B. cereus strains from the soybean rhizosphere
and the mechanism by which B. cereus stimulates the growth of CF rhizosphere
strains in root exudate media. In three consecutive years of sampling, CF strains
predominated among coisolates obtained with B. cereus isolates from field-grown
soybean roots. In root exudate media, the presence of B. cereus was required for
CF coisolate strains to reach high population density. However, rhizosphere
isolates from the phylum Proteobacteria grew equally well in the presence and
absence of B. cereus, and the presence of CF coisolates did not affect the growth
of B. cereus. Peptidoglycan isolated from B. cereus cultures stimulated growth of
the CF rhizosphere bacterium Flavobacterium johnsoniae, although culture
supernatant from B. cereus grown in root exudate media did not. These results
suggest B. cereus and CF rhizosphere bacteria have a commensal relationship in
which peptidoglycan produced by B. cereus stimulates the growth of CF bacteria.
DOI: 10.1128/AEM.02928-05
PMCID: PMC1538759
1906. J Food Prot. 2006 Jul;69(7):1571-8.
Inactivation of escherichia coli 0157:H7 and Salmonella on mung beans, alfalfa,

and other seed types destined for sprout production by using an oxychloro-based
sanitizer.
Kumar M(1), Hora R, Kostrzynska M, Waites WM, Warriner K.
Author information:
(1)Department of Food Science, University of Guelph, Guelph, Ontario, Canada.
The efficacy of a stabilized oxychloro-based food grade sanitizer to

decontaminate seeds destined for sprout production has been evaluated. By using
mung bean seeds as a model system, it was demonstrated that the sanitizer could
be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or
Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more
tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels
of >150 and >50 ppm, respectively, being required to ensure pathogen-free
sprouts. The decontamination efficacy was also found to be dependent on treatment
time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized
oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout
yields were not significantly (P > 0.05) different as compared with untreated
controls. Although human pathogens could be effectively eliminated from mung
beans, the aerobic plate count of native microflora on sprouts grown from treated
seed was not significantly (P > 0.05) different from the controls. The diversity
of microbial populations (determined through 16S rRNA denaturing gradient gel
electrophoresis analysis) associated with bean sprouts was not significantly
affected by the sanitizer treatment. However, it was noted that Klebsiella and
Herbasprillum (both common plant endophytes) were absent in sprouts derived from
decontaminated seed but were present in control sprouts. When a further range of
seed types was evaluated, it was found that alfalfa, cress, flax, and soybean
could be decontaminated with the stabilized oxychloro sanitizer. However, the
decontamination efficacy with other seed types was less consistent. It appears
that the rate of seed germination and putative activity of sanitizer sequestering
system(s), in addition to other factors, may limit the efficacy of the
decontamination method.
1907. Environ Sci Technol. 2006 Jul 1;40(13):4181-8.
Sorption of uranyl cations onto inactivated cells of alfalfa biomass investigated

using chemical modification, ICP-OES, and XAS.
Parsons JG(1), Tiemann KJ, Peralta-Videa JR, Gardea-Torresdey JL.
Author information:
(1)University of Texas at El Paso, Department of Chemistry, 500 W University
Avenue, El Paso, Texas 79968-0513, USA.
Studies have shown that biomaterials have the capacity to adsorb heavy metals and
metal oxo-cations from aqueous solution. In addition, previous studies have shown
that biomaterials have the ability to bind uranyl cations from solution with
capacities that are comparable to or greater than some commercially available
synthetic ion-exchange resins. By using chemical modification, inductively
coupled plasma optical emission spectroscopy (ICP-OES), and X-ray absorption
spectroscopy (XAS), we have found that the primary functional group on alfalfa
biomass responsible for the binding of uranyl cations from aqueous solution is
the carboxyl functionality. Batch pH dependency experiments show a direct
relationship between the increase in binding and the increase in pH (up to pH
4.5). XAS experiments showed that the major ligand involved in the binding of
uranyl cations from aqueous solution was either a nitrogen or oxygen ligand with
coordination numbers ranging from 6 to 10 +/- 1.
1908. J Bacteriol. 2006 Aug;188(15):5417-27.
Diverse flavonoids stimulate NodD1 binding to nod gene promoters in Sinorhizobium

meliloti.
Peck MC(1), Fisher RF, Long SR.
Author information:
(1)Department of Biological Sciences, Gilbert Lab, 371 Serra Mall, Stanford
University, Stanford, CA 94305, USA.
NodD1 is a member of the NodD family of LysR-type transcriptional regulators that

mediates the expression of nodulation (nod) genes in the soil bacterium
Sinorhizobium meliloti. Each species of rhizobia establishes a symbiosis with a
limited set of leguminous plants. This host specificity results in part from a
NodD-dependent upregulation of nod genes in response to a cocktail of flavonoids
in the host plant's root exudates. To demonstrate that NodD is a key determinant
of host specificity, we expressed nodD genes from different species of rhizobia
in a strain of S. meliloti lacking endogenous NodD activity. We observed that nod
gene expression was initiated in response to distinct sets of flavonoid inducers
depending on the source of NodD. To better understand the effects of flavonoids
on NodD, we assayed the DNA binding activity of S. meliloti NodD1 treated with
the flavonoid inducer luteolin. In the presence of luteolin, NodD1 exhibited
increased binding to nod gene promoters compared to binding in the absence of
luteolin. Surprisingly, although they do not stimulate nod gene expression in S.
meliloti, the flavonoids naringenin, eriodictyol, and daidzein also stimulated an
increase in the DNA binding affinity of NodD1 to nod gene promoters. In vivo
competition assays demonstrate that noninducing flavonoids act as competitive
inhibitors of luteolin, suggesting that both inducing and noninducing flavonoids
are able to directly bind to NodD1 and mediate conformational changes at nod gene
promoters but that only luteolin is capable of promoting the downstream changes
necessary for nod gene induction.
DOI: 10.1128/JB.00376-06
PMCID: PMC1540014
1909. Plant Physiol. 2006 Sep;142(1):265-79. Epub 2006 Jul 14.
The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family

includes NFP and new nodule-expressed genes.
Arrighi JF(1), Barre A, Ben Amor B, Bersoult A, Soriano LC, Mirabella R, de

Carvalho-Niebel F, Journet EP, Ghérardi M, Huguet T, Geurts R, Dénarié J, Rougé
P, Gough C.
Author information:
(1)Laboratoire des Interactions Plantes-Microorganismes, Institut National de la
Recherche Agronomique-Centre National de la Recherche Scientifique, Unité Mixte
de Recherche 441/2594, 31326 Castanet-Tolosan, France.
Erratum in
Plant Physiol. 2007 Feb;143(2):1078.
Rhizobial Nod factors are key symbiotic signals responsible for starting the
nodulation process in host legume plants. Of the six Medicago truncatula genes
controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was
reported as a candidate Nod factor receptor gene. Here, we provide further
evidence for this by showing that NFP is a lysin [corrected] motif
(LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in
association with infection thread development and to be involved in the infection
process. Consistent with deviations from conserved kinase domain sequences, NFP
did not show autophosphorylation activity, suggesting that NFP needs to associate
with an active kinase or has unusual functional characteristics different from
classical kinases. Identification of nine new M. truncatula LysM-RLK genes
revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis
thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into
three subfamilies. Three LysM domains could be structurally predicted for all M.
truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was
characterized by deviations from conserved kinase sequences. Most of the newly
identified genes were found to be expressed in roots and nodules, suggesting this
class of receptors may be more extensively involved in nodulation than was
previously known.
DOI: 10.1104/pp.106.084657
PMCID: PMC1557615
1910. Poult Sci. 2006 Jul;85(7):1123-8.
Effect of various combinations of alfalfa and standard layer diet on

susceptibility of laying hens to Salmonella enteritidis during forced molt.
McReynolds JL(1), Moore RW, Kubena LF, Byrd JA, Woodward CL, Nisbet DJ, Ricke SC.
Author information:
(1)US Department of Agriculture, Agricultural Research Service, Food and Feed
Safety Research Unit, Southern Plains Agricultural Research Center, College
Station, TX 77845, USA. mcreynolds@ffsru.tamu.edu
Feed deprivation is commonly used by the poultry industry to induce molting and
stimulate multiple egg-laying cycles. However, feed deprivation has been observed
experimentally to increase susceptibility of poultry to Salmonella infections.
Previous studies indicated that alfalfa was efficacious in reducing Salmonella;
the present investigation was designed to evaluate the efficacy of combined
alfalfa and layer diets on Salmonella colonization. Leghorn hens over 50 wk of
age were divided into 12 groups of hens and placed in individual laying cages.
One week prior to dietary changes, hens were put on an 8L:16D photoperiod that
continued for the 9-d experiment. Hens were challenged orally with 104 cfu of
Salmonella Enteritidis (SE) on d 4 of treatment and cultured for SE at the
termination of the 9-d study. Two independent experiments were conducted
consisting of the following treatment groups: nonfed hens, full-fed standard
commercial layer diet, 100% alfalfa meal diet, a 90% alfalfa meal/10% standard
commercial layer diet, and a 70% alfalfa meal/30% standard commercial layer diet.
When evaluating SE colonization in the ceca (Exp. 1), a reduction (P < 0.05) was
seen in the 100% alfalfa meal diet and the 70% alfalfa meal/30% standard
commercial layer diet treatment groups when compared with the controls with Log10
values of 0.54, 0.44, and 2.82, respectively. Evaluation of physiological
parameters showed the alfalfa treatment groups had reductions (P < 0.05) in
weight loss, ovary weight, and feed consumption when compared with the full-fed
standard commercial layer diet hens, and these results were comparable with the
nonfed hens. In Exp. 2, all of the treatment groups had a reduction (P < 0.05) in
SE colonization of the ceca when compared with the controls. There were also
similar physiological reductions in weight loss, ovary weight, and feed
consumption when birds were fed the alfalfa diets in Exp. 2. These data suggest
that alfalfa can potentially be combined with layer ration to limit SE infection
and still induce a molt comparable with feed withdrawal.
DOI: 10.1093/ps/85.7.1123
1911. J Environ Qual. 2006 Jul 6;35(4):1248-59. Print 2006 Jul-Aug.
Role of rainfall intensity and hydrology in nutrient transport via surface

runoff.
Kleinman PJ(1), Srinivasan MS, Dell CJ, Schmidt JP, Sharpley AN, Bryant RB.
Author information:
(1)USDA-ARS, Pasture Systems and Watershed Management Research Unit, 3702 Curtin
Road, University Park, PA 16802, USA. Peter.Kleinman@ars.usda.gov
Loss of soil nutrients in runoff accelerates eutrophication of surface waters.

This study evaluated P and N in surface runoff in relation to rainfall intensity
and hydrology for two soils along a single hillslope. Experiments were initiated
on 1- by 2-m plots at foot-slope (6%) and mid-slope (30%) positions within an
alfalfa (Medicago sativa L.)-orchardgrass (Dactylis glomerata L.) field. Rain
simulations (2.9 and 7.0 cm h(-1)) were conducted under wet (spring) and dry
(late-summer) conditions. Elevated, antecedent soil moisture at the foot-slope
during the spring resulted in less rain required to generate runoff and greater
runoff volumes, compared with runoff from the well-drained mid-slope in spring
and at both landscape positions in late summer. Phosphorus in runoff was
primarily in dissolved reactive form (DRP averaged 71% of total P), with DRP
concentrations from the two soils corresponding with soil test P levels. Nitrogen
in runoff was mainly nitrate (NO3-N averaged 77% of total N). Site hydrology, not
chemistry, was primarily responsible for variations in mass N and P losses with
landscape position. Larger runoff volumes from the foot-slope produced higher
losses of total P (0.08 kg ha(-1)) and N (1.35 kg ha(-1)) than did runoff from
the mid-slope (0.05 total P kg ha(-1); 0.48 kg N ha(-1)), particularly under wet,
spring-time conditions. Nutrient losses were significantly greater under the high
intensity rainfall due to larger runoff volumes. Results affirm the critical
source area concept for both N and P: both nutrient availability and hydrology in
combination control nutrient loss.
DOI: 10.2134/jeq2006.0015
1912. FEMS Microbiol Ecol. 2006 Jul;57(1):106-15.
Diversity of a stable enrichment culture which is useful for silage inoculant and
its succession in alfalfa silage.
Wang X(1), Haruta S, Wang P, Ishii M, Igarashi Y, Cui Z.
Author information:
(1)College of Agronomy and Biotechnology, China Agricultural University, Haidian
District, Beijing, China.
Alfalfa is a kind of forage that is difficult to ensile for good quality.

Therefore, inoculants are always used to enhance the preservation of alfalfa
silage. Through continuous restricted subcultivation, a lactic acid bacteria
community (Al2) was selected from well-fermented alfalfa silage, which sharply
decreased the pH level and produced a large amount of lactic acid. The adding of
Al2 to alfalfa at ensiling resulted in a more rapid drop in pH and higher levels
of lactic acid, and it also reduced the ammonia-nitrogen content significantly (P
< 0.01). Plate isolation, denaturing gradient gel electrophoresis (DGGE) and the
construction of a 16S rRNA gene clone library were used to identify the
composition diversity of the Al2 community; seven strains were detected in the
community, the predominant strain belonging to Lactobacillus plantarum. Samples
of alfalfa silages of duration 0, 2, 5, 10, 20 and 30 days were studied with DGGE
analysis. The DGGE band patterns of Al2-treated and non inoculated were rather
different, and the components of Al2 were the dominant bacteria in Al2-treated
silages, especially L. plantarum, while Pediococcus pentosaceaus was predominant
in naturally fermented alfalfa silage.
DOI: 10.1111/j.1574-6941.2006.00099.x
Effects of three-dimensional and color patterns on nest location and progeny

mortality in alfalfa leafcutting bee (Hymenoptera: Megachilidae).
Guédot C(1), Bosch J, James RR, Kemp WP.
Author information:
(1)Biology Department, Utah State University, Logan, UT 84322-5310, USA.
ABSTRACT In alfalfa, Medicago sativa L., seed production where high bee densities
are released, alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera:
Megachilidae), females may enter several nesting holes before locating their
nests. Such levels of "wrong hole" visits lead to an increase in the time spent
by females locating their own nests, thereby decreasing alfalfa pollination
efficiency and possibly healthy brood production. The objectives of this study
were to determine the effect of different nesting board configurations in
commercial alfalfa leafcutting bee shelters (separating nesting boards, applying
a three-dimensional pattern to the boards, applying a color contrast pattern, or
applying a combination of three-dimensional and color contrast patterns) on nest
location performance, on the incidence of chalkbrood disease, and on the
incidence of broodless provisions. Separating the nesting boards inside shelters
improved the ability of females to locate their nests. An increase in nest
location performance also occurred in boards with the three-dimensional pattern
and the combined three-dimensional and color contrast pattern, compared with the
uniform board (a standard configuration currently used commercially). The
percentage of provisioned cells that were broodless was not statistically
different between treatments, but the percentage of larvae infected with
chalkbrood decreased by half in the three-dimensional board design, compared with
the uniform board.
1914. J Exp Bot. 2006;57(10):2325-37. Epub 2006 Jun 23.
VSP accumulation and cold-inducible gene expression during autumn hardening and
overwintering of alfalfa.
Dhont C(1), Castonguay Y, Avice JC, Chalifour FP.
Author information:
(1)Département de Phytologie, Université Laval, Québec, QC, Canada G1K 7P4.
This study describes the time-course of the accumulation of total soluble

proteins (TSPs) and vegetative storage proteins (VSPs) and of the transcripts of
cold-inducible (CI) and VSP-encoding genes in taproots of two alfalfa cultivars
(AC Caribou and Europe) during their acclimation to natural autumn hardening and
overwintering conditions in eastern Canada. The impact of a defoliation in
September on these winter hardening-related changes was also assessed. Both
concentrations and pools of VSPs increased significantly between early September
and mid-October and remained unchanged thereafter, concomitantly with the
disappearance of VSP-encoding transcripts. Other soluble protein constituents
continued to increase later in the autumn and early winter and accounted for
nearly 60% of taproot TSP pools in winter. As a result, VSP abundance relative to
TSPs decreased markedly during the winter. The increase in the levels of CI
transcripts was induced by lowering temperatures, and distinct patterns suggest
differences in the regulation of their accumulation. RNA analyses revealed that
the accumulation of VSP transcripts during the autumn precedes the accumulation
of CI transcripts. Autumn defoliation interrupted the accumulation of both TSPs
and VSPs during autumn hardening and repressed the transcript levels of two CI
genes differentially between cultivars. The well-documented impact of autumn
defoliation on the vigour of spring regrowth and long-term persistence of alfalfa
could be related to its negative impact on the accumulation of VSPs and TSPs and
on the expression of genes encoding CI proteins potentially involved in cold
tolerance and pathogen resistance.
DOI: 10.1093/jxb/erj204
1915. J Bacteriol. 2006 Jul;188(13):4890-902.
FixJ: a major regulator of the oxygen limitation response and late symbiotic
functions of Sinorhizobium meliloti.
Bobik C(1), Meilhoc E, Batut J.
Author information:
(1)Laboratoire des Interactions Plantes Microorganismes (LIPM), UMR441-2594
CNRS/INRA BP52627, Chemin de Borde Rouge, Auzeville, 31326 Castanet-Tolosan
Cedex, France.
Sinorhizobium meliloti exists either in a free-living state in the soil or in

symbiosis within legume nodules, where the bacteria differentiate into
nitrogen-fixing bacteroids. Expression of genes involved in nitrogen fixation and
associated respiration is governed by two intermediate regulators, NifA and FixK,
respectively, which are controlled by a two-component regulatory system FixLJ in
response to low-oxygen conditions. In order to identify the FixLJ regulon, gene
expression profiles were determined in microaerobic free-living cells as well as
during the symbiotic life of the bacterium for the wild type and a fixJ
null-mutant strain. We identified 122 genes activated by FixJ in either state,
including 87 novel targets. FixJ controls 74% of the genes induced in
microaerobiosis (2% oxygen) and the majority of genes expressed in mature
bacteroids. Ninety-seven percent of FixJ-activated genes are located on the
symbiotic plasmid pSymA. Transcriptome profiles of a nifA and a fixK mutant
showed that NifA activates a limited number of genes, all specific to the
symbiotic state, whereas FixK controls more than 90 genes, involved in
free-living and/or symbiotic life. This study also revealed that FixJ has no
other direct targets besides those already known. FixJ is involved in the
regulation of functions such as denitrification or amino acid/polyamine
metabolism and transport. Mutations in selected novel FixJ targets did not affect
the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa
roots. From these results, we propose an updated model of the FixJ regulon.
DOI: 10.1128/JB.00251-06
PMCID: PMC1482993
1916. J Agric Food Chem. 2006 Jun 28;54(13):4758-65.
Circulating fluidized bed combustion product addition to acid soil: alfalfa

(Medicago sativa L.) composition and environmental quality.
Chen L(1), Dick WA, Kost D.
Author information:
Wooster, Ohio 44691, USA.
To reduce S emissions, petroleum coke with a high concentration of S was

combusted with limestone in a circulating fluidized bed (CFB) boiler. The
combustion process creates a bed product that has potential for agricultural
uses. This CFB product is often alkaline and enriched in S and other essential
plant nutrients, but also contains high concentrations of Ni and V. Agricultural
land application of CFB product is encouraged, but little information is
available related to plant responses and environmental impacts. CFB product and
agricultural lime (ag-lime) were applied at rates of 0, 0.5, 1.0, and 2.0 times
the soil's lime requirement (LR) to an acidic soil (Wooster silt loam). The 2.0x
LR application rate of CFB product was equivalent to 67.2 Mg ha(-1). Alfalfa
yield was increased 4.6 times by CFB product and 3.8 times by ag-lime compared to
untreated control. Application of CFB product increased the concentration of V in
soil and alfalfa tissue, but not in soil water, and increased the concentration
of Ni in soil and soil water, but not in alfalfa tissue. However, these
concentrations did not reach levels that might cause environmental problems.
DOI: 10.1021/jf0603275
1917. J Agric Food Chem. 2006 Jun 28;54(13):4524-30.
Selenium species in aqueous extracts of alfalfa sprouts by two-dimensional liquid

chromatography coupled to inductively coupled plasma mass spectrometry and
electrospray mass spectrometry detection.
Gergely V(1), Montes-Bayón M, Fodor P, Sanz-Medel A.
Author information:
(1)Department of Physical and Analytical Chemistry, Faculty of Chemistry,
University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain.
The complementary use of two different liquid chromatographic mechanisms coupled

to inductively coupled plasma mass spectrometry (ICP-MS) for selenium (Se)
specific detection has permitted the screening of the most abundant Se-containing
fractions in selenized alfalfa sprouts (Medicago sativa). Aqueous extracts of the
sprouts were fractionated first by size exclusion chromatography (SEC) using a
Superdex Peptide column and a mobile phase containing an ammonium acetate buffer
(pH 7). Further purification of the individual SEC Se-containing fractions was
carried out using two different chromatographic systems: a Shodex Ashaipack
column, with a mixed mechanism of size exclusion and ion exchange, and a
conventional reversed phase C8 using ion-pairing reagents. In both cases, the
columns were coupled to an inductively coupled plasma mass spectrometer equipped
with an octapole reaction system for Se specific detection. This system allowed
the on-line monitoring of the most abundant Se isotopes (78Se, 80Se) by reducing
the possible polytomic interferences affecting these ions by adding hydrogen (2
mL min(-1)) to the octapole reaction cell. The results obtained by both
separation mechanisms were highly comparable, revealing the presence of
Se-methionine and Se-methyl selenocysteine. Both compounds were then confirmed by
analyzing the corresponding fractions by electrospray quadrupole-time-of-flight
(ESI-Q-TOF) mass spectrometry. Finally, an additional Se-containing species
showing Se isotope distribution was detected at a molecular ion m/z 239 in the
ESI-Q-TOF. The collision-induced dissociation of the m/z 239 and 237 ions
(corresponding to 80Se and 78Se isotopes, respectively) revealed the possible
presence as well of a derivative of the Se-2-propenyl selenocysteine.
DOI: 10.1021/jf060320n
1918. J Environ Sci Health B. 2006;41(5):595-603.
Dissipation of phosalone and diazinon in fresh and dried alfalfa.
Talebi K(1).
Author information:
(1)Department of Plant Protection, College of Agriculture, University of Tehran,
Karaj, Iran. khtalebi@ut.ac.ir
The objectives of this study were to determine the persistence of phosalone

(S-6-chloro-2,3-dihydro-2-oxobenzoxazol-3-ylmethyl O, O-diethyl
phosphorodithioate) and diazinon (O,O-diethyl
O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate) residues in fresh and
baled alfalfa under field conditions. Plots of alfalfa were sprayed with each
insecticide. Fresh alfalfa was sampled up to 20 days after treatment, and dried
alfalfa was sampled up to 25 weeks after baling. Samples were analyzed for
residues using high performance liquid chromatography (HPLC) equipped with a UV
detector. The half-lives of diazinon and phosalone in fresh alfalfa were 1.8 and
3.3 days, respectively. In baled alfalfa the half-life of diazinon and phosalone
were 2.8 and 16.7 weeks, respectively. No diazinon residues were detected in
baled alfalfa, sampled after week 9, although the concentration of phosalone
found at week 25 was 5.51 mg/kg.
DOI: 10.1080/03601230600701759
1919. Toxicology. 2006 Jul 25;224(3):229-37. Epub 2006 Jun 14.
Alteration of an essential NK cell signaling pathway by low doses of tributyltin

in human natural killer cells.
Aluoch AO(1), Odman-Ghazi SO, Whalen MM.
Author information:
(1)Department of Biological Sciences, Tennessee State University, Nashville, TN
37209, USA.
Tributyltin (TBT), a toxic and widespread environmental contaminant, has been

shown to inhibit natural killer (NK) cell cytotoxic function significantly.
Inhibition of NK cell cytotoxic function has the potential to increase viral
infections and tumor growth. Upon NK cell binding to lysis-sensitive tumor cells,
an intracellular pathway is activated, which generally begins with activation of
non-receptor protein tyrosine kinases (PTKs) and ends with mitogen-activated
protein kinase (MAPK)-mediated release of lytic granules toward the contacted
target cell. In the current studies, we used a cytotoxicity assay to examine how
low doses (200nM or lower) of TBT affect cytotoxic function. Additionally, we
investigated how low doses of TBT modulate the signaling pathway that dictates
lytic granule exocytosis. A 1h exposure to 200, 100, 50 and 25nM TBT
significantly decreased cytotoxic function 6d later. We also saw significant
activation of p38 and p44/42 by as low as 50nM TBT within ten minutes of
exposure. The observed activation of MAPKs, p38 and p44/42, implicated their
upstream activators MAPK kinases (MAP2Ks). On examining MAP2Ks, MKK3/6 and
MEK1/2, activation was seen within ten minutes. However, when the most upstream
signaling molecules in this pathway, non-receptor protein tyrosine kinases (PTKs)
such as Syk, ZAP-70, Pyk2 and Src were examined, no significant activation was
seen. These data imply that upstream activators of MAP2Ks, MAP2K kinases
(MAP3Ks), are activated by TBT exposures and/or that MAP2K phosphatases are being
inhibited by TBT. Taken together, these data suggest that TBT-induced activation
of MAPKs, p38 and p44/42, is caused by their upstream activators MAP2Ks, MKK3/6
and MEK1/2, respectively.
DOI: 10.1016/j.tox.2006.05.002
1920. J Dairy Sci. 2006 Jul;89(7):2603-7.
Brisket boards reduce freestall use.
Tucker CB(1), Zdanowicz G, Weary DM.
Author information:
(1)Animal Welfare Program, University of British Columbia, Vancouver, Canada V6T
1Z4. cassandra.tucker@agresearch.co.nz
We examined how the presence of a brisket board influenced cow preference, stall
use, and position within the stall. When given a choice between stalls with or
without a brisket board, 15 nonlactating cows spent 68% of their time lying in
the stalls without a brisket board, indicating that they preferred this option.
When 13 cows had access to either stalls with a brisket board or ones without,
they spent, on average, 1.2 h/d more time lying down in stalls without a brisket
board. Resting cows positioned themselves relatively forward in the stalls in 98
+/- 5% (mean +/- SE) of lying bouts when the brisket board was absent, compared
with 67 +/- 5% of bouts when the board was present. Longer cows were more likely
than shorter cows to move forward in the stalls without a brisket board. Cows
also had longer lying bouts in stalls without the brisket board (absent: 1.7 +/-
0.08; present: 1.5 +/- 0.08 h/bout). Although it seems likely that the brisket
board helps keep stalls clean by positioning cows closer to the curb, our results
indicate that brisket boards also make stalls less comfortable for cows. Stall
features designed to reduce stall maintenance may compromise cow comfort. We
suggest that new approaches to cow housing are now required.
DOI: 10.3168/jds.S0022-0302(06)72337-2
1921. J Bacteriol. 2006 Jun;188(12):4508-21.
CbrA is a stationary-phase regulator of cell surface physiology and legume

symbiosis in Sinorhizobium meliloti.
Gibson KE(1), Campbell GR, Lloret J, Walker GC.
Author information:
02139, USA.
Sinorhizobium meliloti produces an exopolysaccharide called succinoglycan that

plays a critical role in promoting symbiosis with its host legume, alfalfa
(Medicago sativa). We performed a transposon mutagenesis and screened for mutants
with altered succinoglycan production and a defect in symbiosis. In this way, we
identified a putative two-component histidine kinase associated with a PAS
sensory domain, now designated CbrA (calcofluor-bright regulator A). The
cbrA::Tn5 mutation causes overproduction of succinoglycan and results in
increased accumulation of low-molecular-weight forms of this exopolysaccharide.
Our results suggest the cbrA::Tn5 allele leads to this succinoglycan phenotype
through increased expression of exo genes required for succinoglycan biosynthesis
and modification. Interestingly, CbrA-dependent regulation of exo and exs genes
is observed almost exclusively during stationary-phase growth. The cbrA::Tn5
mutant also has an apparent cell envelope defect, based on increased sensitivity
to a number of toxic compounds, including the bile salt deoxycholate and the
hydrophobic dye crystal violet. Growth of the cbrA mutant is also slowed under
oxidative-stress conditions. The CbrA-regulated genes exsA and exsE encode
putative inner membrane ABC transporters with a high degree of similarity to
lipid exporters. ExsA is homologous to the Escherichia coli MsbA protein, which
is required for lipopolysaccharide transport, while ExsE is a member of the
eukaryotic family of ABCD/hALD peroxisomal membrane proteins involved in
transport of very long-chain fatty acids, which are a unique component of the
lipopolysaccharides of alphaproteobacteria. Thus, CbrA could play a role in
regulating the lipopolysaccharide or lipoprotein components of the cell envelope.
DOI: 10.1128/JB.01923-05
PMCID: PMC1482976
1922. J Insect Physiol. 2006 Jul;52(7):737-43. Epub 2006 Apr 25.
Effect of low and high-saponin lines of alfalfa on pea aphid.
Sylwia G(1), Leszczynski B, Wieslaw O.
Author information:
(1)Department of Biochemistry, University of Podlasie, ul. Prusa 12, 08-110
Siedlce, Poland.
Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction

and survival, and disturbance in development of its population. This line
negatively influenced aphid probing behaviour, particularly prolonging the
non-probing period and probing of the peripheral tissues (epidermis and
mesophyll) and shortening the period of phloem sap ingestion. The high-saponin
line of alfalfa differed from the low-saponin one by the presence of zanhic acid
tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid
glycoside. The saponins incorporated into sucrose-agarose gels significantly
reduced number of the aphid probes into the gels and extended their duration in
comparison to the control gels (without tested compounds). Role of zanhic acid
tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential
factors for partial resistance of alfalfa towards the pea aphid is discussed.
DOI: 10.1016/j.jinsphys.2006.04.001
1923. J Environ Qual. 2006 May 31;35(4):1060-71. Print 2006 Jul-Aug.
Estimating yields of salt- and water-stressed forages with remote sensing in the
visible and near infrared.
Poss JA(1), Russell WB, Grieve CM.
Author information:
(1)USDA-ARS George E. Brown, Jr. Salinity Laboratory, 450 West Big Springs Road,
Riverside, CA 92507, USA. Jposs@ussl.ars.usda.gov
In arid irrigated regions, the proportion of crop production under deficit

irrigation with poorer quality water is increasing as demand for fresh water
soars and efforts to prevent saline water table development occur. Remote sensing
technology to quantify salinity and water stress effects on forage yield can be
an important tool to address yield loss potential when deficit irrigating with
poor water quality. Two important forages, alfalfa (Medicago sativa L.) and tall
wheatgrass (Agropyron elongatum L.), were grown in a volumetric lysimeter
facility where rootzone salinity and water content were varied and monitored.
Ground-based hyperspectral canopy reflectance in the visible and near infrared
(NIR) were related to forage yields from a broad range of salinity and water
stress conditions. Canopy reflectance spectra were obtained in the 350- to
1000-nm region from two viewing angles (nadir view, 45 degrees from nadir). Nadir
view vegetation indices (VI) were not as strongly correlated with leaf area index
changes attributed to water and salinity stress treatments for both alfalfa and
wheatgrass. From a list of 71 VIs, two were selected for a multiple
linear-regression model that estimated yield under varying salinity and water
stress conditions. With data obtained during the second harvest of a
three-harvest 100-d growing period, regression coefficients for each crop were
developed and then used with the model to estimate fresh weights for preceding
and succeeding harvests during the same 100-d interval. The model accounted for
72% of the variation in yields in wheatgrass and 94% in yields of alfalfa within
the same salinity and water stress treatment period. The model successfully
predicted yield in three out of four cases when applied to the first and third
harvest yields. Correlations between indices and yield increased as canopy
development progressed. Growth reductions attributed to simultaneous salinity and
water stress were well characterized, but the corrections for effects of varying
tissue nitrogen (N) and very low leaf area index (LAI) are necessary.
DOI: 10.2134/jeq2005.0204
1924. J Nanosci Nanotechnol. 2006 Apr;6(4):1044-9.
Neodymium nanoparticles: biosynthesis and structural analysis.
Ascencio JA(1), Canizal G, Medina-Flores A, Bejar L, Tavera L, Matamoros H, Liu

HB.
Author information:
(1)Instituto Mexicano Del Petroleo, Atepehuacan, Mexico DF.
Small metallic nanoparticles of neodymium are obtained by a facile route based on

the biosynthesis and the pH conditions that demonstrate the possibility of
obtaining particles of 1-8 nm. The size is controlled by synthesis conditions.
Smaller clusters were obtained with pH = 5, while for pH = 10 evidences of
nanorods productions are found and this opens the perspective to use this
rare-earth element for zero and one dimensional based applications. Using
transmission electron microscopy techniques, the size distribution and structure
are studied. Density functional theory-based calculations allow the determination
of the lowest energy configuration, which is based on the hexagonal bulk
symmetry. Theoretical models are used to simulate the high resolution
transmission electron microscopy to identify the experimental image, determining
that the synthesized nanoparticles reach the lowest energy hexagonal
configurations.
1925. J Chem Ecol. 2006 Apr;32(4):895-906. Epub 2006 May 5.
Identification of phytotoxic substances from early growth of barnyard grass

(Echinochloa crusgalli) root exudates.
Xuan TD(1), Chung IM, Khanh TD, Tawata S.

Author information:
(1)Department of Bioscience and Biotechnology, Faculty of Agriculture, University
of the Ryukyus, Okinawa, 903-0213, Japan.
Barnyard grass is a problematic weed worldwide. It competes with crops and causes
reduction in crop yields. In this study, barnyard grass suppressed rice
emergence, and the degree of rice inhibition was proportional to the density of
barnyard grass. Root exudates of barnyard grass reduced germination and growth of
lettuce, rice, and monochoria. Fifteen compounds potentially involved in the
phytotoxic activities of barnyard grass were isolated and identified, including
phenolics, long-chain fatty acids, lactones, diethyl phthalate, acenaphthene, and
derivatives of phthalic acids, benzoic acid, and decane. Quantities of diethyl
phthalate, decanoic acid, myristic acid, stearic acid,
7,8-dihydro-5,6-dehydrokavain, and 7,8-dihydrokavain were 2.7, 11.1, 19.6, 35.5,
10.3, and 15.5 microg/ml of barnyard grass root exudates, respectively. The two
lactones exhibited the greatest inhibition, followed by the phenolics and the
derivatives of phthalic acids. Fatty acids had stronger suppression than diethyl
phthalate and ethyl ester-4-ethoxy-benzoic acid. The acenaphthene and decane
derivatives were the least phytotoxic. The phytotoxins released by barnyard grass
roots showed strong inhibition on growth of broadleaf indicator plants and paddy
weeds, but were less effective on barnyard grass itself and rice. Our study
revealed that in addition to competition, barnyard grass also interferes with
rice and other plants in its surroundings by chemical means.
DOI: 10.1007/s10886-006-9035-x
1926. Anaerobe. 2006 Apr;12(2):85-92. Epub 2006 Jan 24.
Effects of nitro compounds and feedstuffs on in vitro methane production in

chicken cecal contents and rumen fluid.
Saengkerdsub S(1), Kim WK, Anderson RC, Nisbet DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station, Room 101
Kleberg Center, TX 77843-2472, USA.
Short-chain volatile fatty acids (VFA) and methane are the products from a wide
variety of microorganisms living in the gastrointestinal tract. The objective of
this study was to examine effects of feedstuff and select nitro compounds on VFA
and methane production during in vitro incubation of laying hen cecal contents
and rumen fluid from cattle and sheep. In the first experiment, one of the three
nitro compound was added to incubations containing cecal contents from laying
hens supplemented with either alfalfa (AF) or layer feed (LF). Both feed material
influenced VFA production and acetic acid was the primary component. Incubations
with nitro ethanol and 2-nitropropanol (NP) had significantly (P<0.05) higher
propionate concentrations than incubations with added nitroethane (NE). The
results further indicated that incubations containing LF produced significantly
(P<0.05) more butyrate than incubations with added AF. Addition of NP and LF to
incubations of avian cecal flora may promote Gram-positive, saccharolytic,
VFA-producing bacteria, especially Clostridium spp. which is the predominant
group in ceca. Similar to VFA production, both feed materials fostered methane
production in the incubations although methane was lower (P<0.05) in incubations
with added nitro compound, particularly NE. In experiments 3-8, NE was examined
in incubations of bovine or ovine rumen fluid or cecal contents containing either
AF or LF. Unlike cecal contents, LF significantly (P<0.05) supported in vitro
methane production in incubations of both rumen fluids. The results show that NE
impedes methane production, especially in incubations of chicken cecal contents.
DOI: 10.1016/j.anaerobe.2005.11.006
1927. J Anim Sci. 2006 Jun;84(6):1429-38.
Changes in heat production by mature cows after changes in feeding level.
Freetly HC(1), Nienaber JA, Brown-Brandl T.
Author information:
(1)USDA, ARS, US Meat Animal Research Center, Clay Center, NE 68933, USA.
freetly@email.marc.usda.gov
We hypothesized that adaptation of heat production in the realimented cow would

occur over an extended period, and the length of time would be influenced by the
level of feed. Our objectives were to quantify the changes in heat production of
cows after feed restriction and to quantify the effect of level of realimentation
on the dynamics of heat production in lightweight cows. Forty 4-yr-old
nonpregnant, nonlacting cows (4-breed composite: 1/4 Hereford, 1/4 Angus, 1/4 Red
Poll, and 1/4 Pinzgauer) were randomly assigned to receive 1 of 4 levels of a
common alfalfa hay source. All cows were feed-restricted [50.0 g of DM/metabolic
body size (MBS, kg of BW(0.75)); period 1], and individual fed heat production
measurements were taken 0, 7, 13, 28, 56, and 91 d after feed restriction (period
1). In period 2, cows were fed their assigned feed level for their treatment
after d 91 of restriction: 50.0 (T50.0), 58.5 (T58.5), 67.0 (T67.0), and 75.5
(T75.5) g of DM/MBS. Measures were taken at 7, 13, 28, 42, 56, 91, 119, and 175
d. In period 3, all cows were fed 75.5 g of DM/MBS after their 175-d measurement,
and measures were taken at 7, 14, 28, 56, and 112 d later. In period 1, heat
production decreased rapidly during the first 7 d of feed restriction, and heat
production continued to decrease during the 91-d restriction. Heat production
increased rapidly within the first 7 d, but chronic adaptation continued for
T75.5 and T67.0 cows. In period 3, heat production increased rapidly during the
first 7 d. Heat production scaled for metabolic body size tended to differ among
treatments (P = 0.11). Daily heat production increased by 2.5 kcal/d. These data
suggest that there is not a lag in heat production during realimentation and that
increased recovered energy is associated with a rapid increase in heat
production.
Influence of plant ontogeny and abiotic factors on resistance of glandular-haired

alfalfa to potato leafhopper (Hemiptera: Cicadellidae).
Casteel CL(1), Ranger CM, Backus EA, Ellersieck MR, Johnson DW.
Author information:
(1)Department of Entomology, University of Missouri, Columbia 65211, USA.
ccastee2@igb.uiuc.edu
Laboratory experiments were conducted to characterize the trichome-based defense

of glandular-haired alfalfa, Medicago sativa L., against the potato leafhopper,
Empoasca fabae (Harris). Within-plant variability in leafhopper resistance was
examined by caging adult leafhoppers to either basal or apical stem internodes of
the leafhopper-resistant, glandular-haired M. sativa genotype G98A or the
susceptible, nonglandular-haired M. sativa 'Ranger'. Young, actively secreting
glandular trichomes are located on apical internodes of G98A, whereas senesced
gland heads are found on older, basal internodes of G98A. After 96 h, the highest
cumulative leafhopper mortality and lowest number of excretory droplets were
associated with apical internodes of G98A. No difference was detected in
mortality and feeding levels among insects caged to basal internodes of G98A and
basal and apical internodes of Ranger. The influence of abiotic factors on
leafhopper resistance was evaluated by caging adult leafhoppers to either G98A or
Ranger under four combinations of low and high light (250 and 1,000 micromol
s(-1) m(-2)) and temperature regimes (17 and 30 degrees C). After 96 h, the
highest cumulative mortality was associated with leafhoppers confined to G98A
under high light and high temperature conditions. Temperature level and plant
type also had an effect on the production of excretory droplets, resulting in the
highest number of excretory droplets being associated with Ranger under the high
temperature regime. These results indicate that certain regions of M. sativa G98A
are better protected against the potato leafhopper than others and that
temperature influences resistance levels of glandular-haired alfalfa.
1929. Folia Microbiol (Praha). 2005;50(6):509-14.
Evaluation of abundance of aerobic bacteria in the rhizosphere of transgenic and

non-transgenic alfalfa lines.
Faragová N(1), Faragó J, Drábeková J.
Author information:
(1)Department of Breeding Methods, Section of Genetic and Breeding Research,
Research Institute of Plant Production, Piest'any, Slovakia. faragova@vurv.sk
Fourteen genetically modified lines of alfalfa (Medicago sativa) containing the

gene Ov from Japanese quail, coding for a methionine-rich protein ovalbumin, were
evaluated for nodulation ability and concentration of aerobic bacteria in the
rhizosphere. The transgenic lines were derived from a highly regenerable genotype
Rg9/I-14-22, selected from cv. Lucia. On selective media, a higher concentration
of ammonifying bacteria, bacterial spores, denitrifying and nitrifying bacteria
were observed in the rhizosphere of transgenic clonesand, on the other hand,
lower concentration of cellulolytic bacteria and Azotobacter spp. compared with
the rhizosphere of non-transgenic clone SE/22-GT2. A statistically significant
difference in the concentration of all the bacterial types was found between
samples taken from two types of substrates (i.e. sterile vs. nonsterile). Higher
bacterial concentration (measured as colony forming units per g soil dry mass)
were observed for all tested groups of culturable bacteria in the non-sterile
substrate. The presence of Azotobacter spp. was found only in the rhizosphere of
plants grown in non-sterile soil in which the highest number of fertile soil
particles (97 %) was observed in transgenic clones SE/22-9-1-12 and
SE/22-11-1-1S.1. Concentration of bacteria involved in the N cycle in the soil
was increased in the rhizosphere of transgenic clones and decreased in the
rhizosphere of non-transgenic plants compared with the average value. In spite of
some differences in colony numbers in samples isolated from the root rhizosphere
of transgenic and nontransgenic alfalfa plants, we could not detect any
statistically significant difference between individual lines.
1930. Appl Environ Microbiol. 2006 May;72(5):3662-72.
Sequence analysis of the 144-kilobase accessory plasmid pSmeSM11a, isolated from

a dominant Sinorhizobium meliloti strain identified during a long-term field
release experiment.
Stiens M(1), Schneiker S, Keller M, Kuhn S, Pühler A, Schlüter A.
Author information:
(1)Fakultät für Biologie, Lehrstuhl für Genetik, Universität Bielefeld, Postfach
100131, D-33501 Bielefeld, Germany.
The genome of Sinorhizobium meliloti type strain Rm1021 consists of three

replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally,
many indigenous S. meliloti strains possess one or more smaller plasmids, which
represent the accessory genome of this species. Here we describe the complete
nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was
isolated from a dominant indigenous S. meliloti subpopulation in the context of a
long-term field release experiment with genetically modified S. meliloti strains.
Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and
has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a
total of 160 coding sequences. Functional predictions could be made for 43% of
the genes, whereas 57% of the genes encode hypothetical or unknown gene products.
Two plasmid replication modules, one belonging to the repABC replicon family and
the other belonging to the plasmid type A replicator region family, were
identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of
the type IV secretion system-related genes traG and traA and a putative mobC
gene. A large continuous region that is about 42 kb long is very similar to a
corresponding region located on S. meliloti Rm1021 megaplasmid pSymA.
Single-base-pair deletions in the homologous regions are responsible for
frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a
carries additional copies of the nodulation genes nodP and nodQ that are
responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a
putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on
pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS
gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is
proposed to be involved in reducing the phytohormone ethylene, thus influencing
nodulation events. The presence of numerous insertion sequences suggests that
these elements mediated acquisition of accessory plasmid modules.
DOI: 10.1128/AEM.72.5.3662-3672.2006
PMCID: PMC1472397
Rhizosphere selection of highly motile phenotypic variants of Pseudomonas

fluorescens with enhanced competitive colonization ability.
Martínez-Granero F(1), Rivilla R, Martín M.
Author information:
(1)Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de
Madrid, c/Darwin 2, 28049 Madrid, Spain.
Phenotypic variants of Pseudomonas fluorescens F113 showing a translucent and
diffuse colony morphology show enhanced colonization of the alfalfa rhizosphere.
We have previously shown that in the biocontrol agent P. fluorescens F113,
phenotypic variation is mediated by the activity of two site-specific
recombinases, Sss and XerD. By overexpressing the genes encoding either of the
recombinases, we have now generated a large number of variants (mutants) after
selection either by prolonged laboratory cultivation or by rhizosphere passage.
All the isolated variants were more motile than the wild-type strain and appear
to contain mutations in the gacA and/or gacS gene. By disrupting these genes and
complementation analysis, we have observed that the Gac system regulates swimming
motility by a repression pathway. Variants isolated after selection by prolonged
cultivation formed a single population with a swimming motility that was equal to
the motility of gac mutants, being 150% more motile than the wild type. The
motility phenotype of these variants was complemented by the cloned gac genes.
Variants isolated after rhizosphere selection belonged to two different
populations: one identical to the population isolated after prolonged cultivation
and the other comprising variants that besides a gac mutation harbored additional
mutations conferring higher motility. Our results show that gac mutations are
selected both in the stationary phase and during rhizosphere colonization. The
enhanced motility phenotype is in turn selected during rhizosphere colonization.
Several of these highly motile variants were more competitive than the wild-type
strain, displacing it from the root tip within 2 weeks.
DOI: 10.1128/AEM.72.5.3429-3434.2006
PMCID: PMC1472367
1932. Mol Ecol. 2006 May;15(6):1589-602.
Domestication history in the Medicago sativa species complex: inferences from

nuclear sequence polymorphism.
Muller MH(1), Poncet C, Prosperi JM, Santoni S, Ronfort J.
Author information:
(1)UMR Diversité et Génomes des Plantes Cultivées, INRA, Domaine de Melgueil,
Mauguio, France. marie-helene.muller@ensam.inra.fr
DNA sequence polymorphism carries genealogical information and allows for testing
hypotheses on selection and population history, especially through
coalescent-based analysis. Understanding the evolutionary forces at work in plant
domestication and subsequent selection is of critical importance for the
management of genetic resources. In this study, we surveyed DNA sequence
diversity at two assumed neutral nuclear loci in the wild-domesticated species
complex of alfalfa (Medicago sativa L.). A high level of polymorphism was
detected. The domesticated pool contains on average 31% less diversity than the
wild pool, but with a high heterogeneity among loci. Coalescent simulations of
the domestication process showed that this result cannot be explained by assuming
a constant population size but is rather consistent with a demographic bottleneck
during domestication. A very low level of divergence was detected between the
wild and the domesticated forms as well as between the related subspecies of the
M. sativa species complex. However, the originality of the Spanish wild
populations, already observed based on mitochondrial DNA polymorphism, was
confirmed. These results, together with patterns of intrapopulation polymorphism,
suggest that nuclear sequence polymorphism could be a promising tool,
complementary to mitochondrial DNA and phenotypic evaluations, to investigate
historical demographic and evolutionary processes.
DOI: 10.1111/j.1365-294X.2006.02851.x
1933. J Food Prot. 2006 Apr;69(4):719-28.
Inhibition of Salmonella enterica by plant-associated pseudomonads in vitro and

on sprouting alfalfa seed.
Fett WF(1).
Author information:
(1)Food Safety Intervention Technologies Research Unit, Eastern Regional Research
Center, Agricultural Research Service, US Department of Agriculture, Wyndmoor,
Pennsylvania 19038, USA. wfett@arserrc.gov
Foodborne illness due to the consumption of contaminated raw or lightly cooked

sprouts is a continuing food safety concern. In this study, we tested several
plant-associated pseudomonads for their ability to inhibit the growth of
Salmonella enterica both in vitro and in situ. An agar spot bioassay method was
used with three different media. Only Pseudomonas fluorescens 2-79 produced clear
zones of inhibition when tested against five serovars of S. enterica, and
activity was dependent on media type and serovar. The antibiosis by derivative
strains of P. fluorescens 2-79 defective in the production of
phenazine-1-carboxylic acid and fluorescent siderophore was not reduced,
indicating that these known antimicrobial metabolites were not responsible for
the inhibition observed in our studies. However, mutants defective in the
regulatory gene gacS (global antibiotic and cyanide control) were severely
reduced in inhibitory activity. In tryptic soy broth, the control cultures of a
cocktail of S. enterica strains reached approximately 10 log CFU/ml by 24 h but,
when coinoculated with P. fluorescens 2-79, reached only approximately 5 log
CFU/ml. The addition of P. fluorescens 2-79 to the seed soak water prior to the
germination of alfalfa seed previously inoculated with a cocktail of S. enterica
strains led to an average reduction of 5 log CFU/g at 6 days of sprouting without
an adverse effect on sprout yield or appearance. Time course studies indicated
that S. enterica outgrowth was controlled on days 1 through 6 of sprouting.
Competitive exclusion as a potential food safety intervention for seed sprouts
merits further study.
The effect of predators and hymenopterous parasites on population fluctuations of

alfalfa aphids.
Rassoulian GR(1).
Author information:
(1)Dept. of plant protection, Agricultural college of Karaj, University of
Tehran, Karaj, Iran.
Among alfalfa pests in Iran three aphid species, green alfalfa aphid
Acyrthosiphon pisum Harris spotted alfalfa aphid Therioaphis trifolii forma
maculata Buckton and blue alfalfa aphid, Acyrthosiphon kondoi Shinji are
important pests. The green alfalfa aphid can be observed all along the growing
season particularly from late May to mid June at Karaj climate conditions. During
this period, the mean monthly maximum temperature and relative humidity were
about 28 degrees C and 60-65% respectively. This aphid overwinters as nymph and
viviparous female. Sexual forms and eggs could not be seen under field
conditions. Spotted alfalfa aphid, Therioaphis trifolii fonna maculata is the
most prevalent aphid in summer time, when the mean monthly maximum temperature
and relative humidity are about 33-34 degrees C and 44-58% respectively. Sexual
individuals have been observed in the laboratory but not in the field. Among
predators (Coccinella septempunctata, Adonia variegata, Syrphus cinctus, S.
corolae, S. grassulariae, Chrysoperla carnea and Nabis capsiformis) one
coccinellid species, C. septempunctata, had greatest impact on fluctuations of
population. Among hymenopterous parasitoids two species have been collected from
alfalfa field they were Aphidius ervi and Praon palitans. These parasitoids
destroyed a good percent of aphids and statistically proved to lower aphid
populations significantly.
1935. Biochim Biophys Acta. 2006 Mar;1761(3):324-34. Epub 2006 Mar 31.
Plant fatty acid (ethanol) amide hydrolases.
Shrestha R(1), Kim SC, Dyer JM, Dixon RA, Chapman KD.
Author information:
(1)University of North Texas, Center for Plant Lipid Research, Department of
Biological Sciences, Denton, P.O. Box 305220, TX 76203, USA.
Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous
N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an
"endocannabinoid" signaling pathway that regulates diverse physiological and
behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was
identified which catalyzed the hydrolysis of NAEs in vitro suggesting a
FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs.
Here, we provide evidence to support this concept by identifying candidate FAAH
genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have
similar, but not identical, exon-intron organizations. Corresponding M.
truncatula and rice cDNAs were isolated and cloned into prokaryotic expression
vectors and expressed as recombinant proteins in Escherichia coli. NAE
amidohydrolase assays confirmed that these proteins indeed catalyzed the
hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition
properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH,
but not identical. Sequence alignments and motif analysis of plant FAAH enzymes
revealed a conserved domain organization for these members of the amidase
superfamily. Five amino-acid residues determined to be important for catalysis by
rat FAAH were absolutely conserved within the FAAH sequences of six plant
species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal
structure as a template revealed a conserved protein core that formed the active
site of each enzyme. Collectively, these results indicate that plant and
mammalian FAAH proteins have similar structure/activity relationships despite
limited overall sequence identity. Defining the molecular properties of NAE
amidohydrolase enzymes in plants will help to better understand the metabolic
regulation of NAE lipid mediators.
DOI: 10.1016/j.bbalip.2006.03.004
1936. Phytother Res. 2006 Jun;20(6):454-7.
Antimicrobial activity of saponins from Medicago sp.: structure-activity

relationship.
Avato P(1), Bucci R, Tava A, Vitali C, Rosato A, Bialy Z, Jurzysta M.
Author information:
(1)Dipartimento Farmaco-Chimico, Via Orabona 4, Università, I-70125 Bari, Italy.
avato@farmchim.uniba.it
The antimicrobial activity of saponins from Medicago sativa, M. arborea and M.

arabica against a selection of medically important yeasts, Gram-positive and
-negative bacteria was investigated. Structure-activity growth inhibitory effects
of related prosapogenins and sapogenins are also described. Increasing antibiotic
activity was observed going from the saponin extracts to the sapogenin samples,
suggesting that the sugar moiety is not important for the antimicrobial efficacy.
Activity was especially high against Gram-positive bacteria (Bacillus cereus, B.
subtilis, Staphylococcus aureus and Enterococcus faecalis) with M. arabica being
the species showing a broader spectrum of action. Discrete antifungal activity
was also observed, mainly against Saccharomyces cerevisiae. The observed
antimicrobial properties of M. sativa and M. arborea were related to the content
of medicagenic acid, while hederagenin seems to contribute to the bioactivity of
M. arabica total sapogenins.
DOI: 10.1002/ptr.1876
1937. Phytochemistry. 2006 May;67(9):856-60. Epub 2006 Apr 17.
Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for
the presence of an amino acid racemase in plants.
Ono K(1), Yanagida K, Oikawa T, Ogawa T, Soda K.
Author information:
(1)Department of Biotechnology, Faculty of Engineering, Kansai University,
Suita-Shi, Osaka-Fu, Japan.
Erratum in
Phytochemistry. 2006 Jun;67(12):1298.
We demonstrated several kinds of D-amino acids in plant seedlings, and moreover

alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is
the first evidence for the presence of amino acid racemase in plant. The enzyme
was effectively induced by the addition of L- or D-alanine, and we highly
purified the enzyme to show enzymological properties. The enzyme exclusively
catalyzed racemization of L- and D-alanine. The K(m) and V(max) values of enzyme
for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are
12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K(eq) value was estimated to
be about 1 and indicated that the enzyme catalyzes a typical racemization of both
enantiomers of alanine. The enzyme was inactivated by hydroxylamine,
phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors.
Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and
enzymologically resembled bacterial alanine racemases studied so far.
DOI: 10.1016/j.phytochem.2006.02.017
1938. J Endocrinol. 2006 Apr;189(1):67-75.
Ghrelin enhances glucose-induced insulin secretion in scheduled meal-fed sheep.
Takahashi H(1), Kurose Y, Kobayashi S, Sugino T, Kojima M, Kangawa K, Hasegawa Y,

Terashima Y.
Author information:
(1)School of Veterinary Medicine and Animal Science, Kitasato University,
Towada-shi, Aomori 034-8628, Japan.
The purpose of this study was to investigate the effects of physiologic levels of
ghrelin on insulin secretion and insulin sensitivity (glucose disposal) in
scheduled fed-sheep, using the hyperglycemic clamp and hyperinsulinemic
euglycemic clamp respectively. Twelve castrated Suffolk rams (69.8 +/- 0.6 kg)
were conditioned to be fed alfalfa hay cubes (2% of body weight) once a day.
Three hours after the feeding, synthetic ovine ghrelin was intravenously
administered to the animals at a rate of 0.025 and 0.05 mug/kg body weight (BW)
per min for 3 h. Concomitantly, the hyperglycemic clamp or the hyperinsulinemic
euglycemic clamp was carried out. In the hyperglycemic clamp, a target glucose
concentration was clamped at 100 mg/100 ml above the initial level. In the
hyperinsulinemic euglycemic clamp, insulin was intravenously administered to the
animals for 3 h at a rate of 2 mU/kg BW per min. Basal glucose concentrations
(44+/- 1 mg/dl) were maintained by variably infusing 100 mg/dl glucose solution.
In both clamps, plasma ghrelin concentrations were dose-dependently elevated and
maintained at a constant level within the physiologic range. Ghrelin infusions
induced a significant (ANOVA; P < 0.01) increase in plasma GH concentrations. In
the hyperglycemic clamp, plasma insulin levels were increased by glucose infusion
and were significantly (P < 0.05) greater in ghrelin-infused animals. In the
hyperinsulinemic euglycemic clamp, glucose infusion rate, an index of insulin
sensitivity, was not affected by ghrelin infusion. In conclusion, the present
study has demonstrated for the first time that ghrelin enhances glucose-induced
insulin secretion in the ruminant animal.
DOI: 10.1677/joe.1.06310
1939. J Anim Sci. 2006 May;84(5):1205-14.
Wet corn gluten feed and alfalfa hay levels in dry-rolled corn finishing diets:
effects on finishing performance and feedlot nitrogen mass balance.
Farran TB(1), Erickson GE, Klopfenstein TJ, Macken CN, Lindquist RU.
Author information:
(1)Department of Animal Science, University of Nebraska, Lincoln, 68583-0908,
USA.
One-hundred ninety-two crossbred steers (initial BW = 351 +/- 11 kg) were used to
determine the effects of removing alfalfa hay (AH) from dry-rolled corn-based
diets containing wet corn gluten feed (WCGF) on animal performance and nutrient
(N and OM) mass balance in open feedlot pens. Steers were stratified by weight
and assigned randomly to 24 pens (2 x 3 factorial) and fed for 132 d from June to
October 2002. Experimental diets contained either 0 or 35% WCGF and 0, 3.75, or
7.5% AH, and were formulated to be isonitrogenous. For efficiency of gain, an
interaction occurred (P = 0.09) between AH and WCGF. Feed efficiencies of cattle
fed 35% WCGF were improved 4.4% (P = 0.10) compared with efficiencies of cattle
fed no WCGF at 0% AH; there was a marked improvement in ADG for cattle fed WCGF
compared with no WCGF in diets with 0% AH. Within 35% WCGF diets, efficiency
decreased as AH inclusion increased (P = 0.06). Efficiency was equal across AH
levels when 0% WCGF was fed; however, ADG was decreased when AH was removed.
Interactions between AH and WCGF were not detected for other performance or
carcass criteria; therefore, main effects of AH and WCGF are discussed. Daily
intake, ADG, and HCW increased linearly (P < 0.05) as dietary AH level increased.
Feeding 35% WCGF also resulted in greater DMI (P < 0.01) and a tendency for
greater ADG and HCW (P < or = 0.10) compared with steers fed no WCGF.
Interactions between AH and WCGF were not observed for feedlot N mass balance. As
level of AH increased across diets, N intake, N retention, and N excretion
increased (P < 0.05). Steers fed 35% WCGF consumed and excreted more N (P < 0.01)
than those fed no WCGF. More manure DM (P = 0.11), OM, and N (P < 0.01) were
removed from pens housing steers fed 35% WCGF as well as greater OM and N
recovery in finished compost. More N (kilogram/steer) was also lost to
volatilization as a result of greater N excretion when WCGF was fed. Expressed as
a percentage of N excretion, loss of N from pens housing steers fed 0 and 35%
WCGF was not different, averaging nearly 80%. These data suggest that AH has less
value when dry-rolled corn-based diets contain 35% WCGF and can be decreased from
conventional levels. Furthermore, loss of N from open feedlot pens is high during
the summer months, and feeding WCGF may not reduce N losses during these times of
year.
1940. Mol Plant Microbe Interact. 2006 Apr;19(4):363-72.
Sinorhizobium meliloti differentiation during symbiosis with alfalfa: a

transcriptomic dissection.
Capela D(1), Filipe C, Bobik C, Batut J, Bruand C.
Author information:
(1)Laboratoire des Interactions Plantes-Microorganismes, UMR INRA-CNRS 441-2594,
BP52627, 31326 Castanet-Tolosan, France.
Sinorhizobium meliloti is a soil bacterium able to induce the formation of

nodules on the root of specific legumes, including alfalfa (Medicago sativa).
Bacteria colonize nodules through infection threads, invade the plant
intracellularly, and ultimately differentiate into bacteroids capable of reducing
atmospheric nitrogen to ammonia, which is directly assimilated by the plant. As a
first step to describe global changes in gene expression of S. meliloti during
the symbiotic process, we used whole genome microarrays to establish the
transcriptome profile of bacteria from nodules induced by a bacterial mutant
blocked at the infection stage and from wild-type nodules harvested at various
timepoints after inoculation. Comparison of these profiles to those of cultured
bacteria grown either to log or stationary phase as well as examination of a
number of genes with known symbiotic transcription patterns allowed us to
correlate global gene-expression patterns to three known steps of symbiotic
bacteria bacteroid differentiation, i.e., invading bacteria inside infection
threads, young differentiating bacteroids, and fully differentiated,
nitrogen-fixing bacteroids. Finally, analysis of individual gene transcription
profiles revealed a number of new potential symbiotic genes.
DOI: 10.1094/MPMI-19-0363
1941. J Dairy Sci. 2006 May;89(5):1704-12.
Effect of dietary crude protein concentration on milk production and nitrogen

utilization in lactating dairy cows.
Colmenero JJ(1), Broderick GA.
Author information:
Forty lactating Holstein cows, including 10 with ruminal cannulas, were blocked
by days in milk into 8 groups and then randomly assigned to 1 of 8 incomplete 5 x
5 Latin squares to assess the effects of 5 levels of dietary crude protein (CP)
on milk production and N use. Diets contained 25% alfalfa silage, 25% corn
silage, and 50% concentrate, on a dry matter (DM) basis. Rolled high-moisture
shelled corn was replaced with solvent-extracted soybean meal to increase CP from
13.5 to 15.0, 16.5, 17.9, and 19.4% of DM. Each of the 4 experimental periods
lasted 28 d, with 14 d for adaptation and 14 d for data collection. Spot sampling
of ruminal digesta, blood, urine, and feces was conducted on d 21 of each period.
Intake of DM was not affected by diet but milk fat content as well as ruminal
acetate, NH3, and branched-chain volatile fatty acids, urinary allantoin, and
blood and milk urea all increased linearly with increasing CP. Milk and protein
yield showed trends for quadratic responses to dietary CP and were, respectively,
38.3 and 1.18 kg/d at 16.5% CP. As a proportion of N intake, urinary N excretion
increased from 23.8 to 36.2%, whereas N secreted in milk decreased from 36.5 to
25.4%, as dietary protein increased from 13.5 to 19.4%. Under the conditions of
this study, yield of milk and protein were not increased by feeding more than
16.5% CP. The linear increase in urinary N excretion resulted from a sharp
decline in N efficiency as dietary CP content increased.
DOI: 10.3168/jds.S0022-0302(06)72238-X
1942. J Dairy Sci. 2006 May;89(5):1694-703.
Effect of dietary crude protein concentration on ruminal nitrogen metabolism in

Author information:
Ten lactating Holstein cows fitted with ruminal cannulas that were part of a
larger feeding trial were blocked by days in milk into 2 groups and then randomly
assigned to 1 of 2 incomplete 5 x 5 Latin squares. Diets contained [dry matter
(DM) basis] 25% alfalfa silage, 25% corn silage, and 50% concentrate. Rolled
high-moisture shelled corn was replaced with solvent-extracted soybean meal to
increase crude protein (CP) from 13.5% to 15.0, 16.5, 17.9, and 19.4% of DM. Each
of the 4 experimental periods lasted 28 d with data and sample collection
performed during the last 8 d. Digesta samples were collected from the omasum to
quantify the ruminal outflow of different N fractions. Intake of DM was not
affected but showed a quadratic trend with maxima of 23.9 kg/d at 16.5% CP.
Ruminal outflow of total bacterial nonammonia N (NAN) was not different among
diets but a significant linear effect of dietary CP was detected for this
variable. Bacterial efficiency (g of total bacterial NAN flow/kg of organic
matter truly digested in the rumen) and omasal flows of dietary NAN and total NAN
also showed positive linear responses to dietary CP. Total NAN flow increased
from 574 g/d at 13.5% CP to 688 g/d at 16.5% CP but did not increase further with
the feeding of more CP. Under the conditions of this study, 16.5% of dietary CP
appeared to be sufficient for maximal ruminal outflow of total bacterial NAN and
total NAN.
DOI: 10.3168/jds.S0022-0302(06)72237-8
1943. J Dairy Sci. 2006 May;89(5):1644-53.
Effect of corn silage hybrid and metabolizable protein supply on nitrogen

metabolism of lactating dairy cows.
Weiss WP(1), Wyatt DJ.
Author information:
Center, The Ohio State University, Wooster 44691, USA. weiss.6@osu.edu
This experiment was conducted to determine the effects of corn silage hybrid and
supply of metabolizable protein (MP) on manure excretion and N metabolism by
lactating dairy cows. Eight Holstein cows in midlactation (replicated 4 x 4 Latin
square with 21-d periods) were fed 1 of 4 treatments, arranged factorially. Diets
contained 55% corn silage made from a dual-purpose hybrid or a brown midrib (BMR)
hybrid, and 45% concentrate that contained either a low or high concentration of
rumen undegradable protein (altered by the addition of fish meal and treated
soybean meal). Crude protein averaged 14.0 and 17.5% and supply of MP averaged
2,360 and 2,990 g/d for the low and high MP treatments (not affected by hybrid).
Increasing supply of MP greatly increased urine output and tended to increase
total manure output, whereas diets with BMR silage tended to reduce manure
output. Increased MP supply increased daily excretion of manure N by 25% (465 vs.
374 g/d), fecal N by 27 g, and urinary N by 64 g. When the effect of N intake was
removed, cows fed BMR silage excreted about 15 g/d less N via manure than cows
fed the other silage. Rumen ammonia, volatile fatty acid concentrations, and pH
were not affected by treatment. Dry matter intake (overall mean = 24.9 kg/d)
tended to be increased with increased MP but was not affected by hybrid. Milk
production for cows fed BMR was higher than for cows fed the dual-purpose hybrid
(36.9 vs. 35.3 kg/d), but because of changes in fat concentration, yield of
energy-corrected milk was not affected by treatment. The only interaction
observed was increased yield of milk protein when BMR silage was combined with
increased supply of MP.
DOI: 10.3168/jds.S0022-0302(06)72231-7
1944. J Dairy Sci. 2006 May;89(5):1635-43.
Effect of amount and ruminal degradability of soybean meal protein on performance

of lactating dairy cows.
Author information:
Twenty-eight Holstein cows (4 with ruminal cannulas) were blocked by days in milk
into 7 groups and then randomly assigned to 1 of 7 balanced 4 x 4 Latin square
diet sequences. The diets contained [dry matter (DM) basis] 20% alfalfa silage,
35% corn silage, and 45% concentrate mainly from high-moisture corn and soybean
meal. Diets differed in crude protein (CP) content and source of protein
supplement: diet A) 15.6% CP, 3.7% solvent-extracted soybean meal (SSBM), 4.5%
expeller soybean meal (ESBM); diet B) 16.6% CP, 9.6% SSBM, 0% ESBM; diet C) 16.6%
CP, 4.6% SSBM, 5.9% ESBM; and diet D) 17.6% CP, 11.7% SSBM, 0% ESBM. Each
experimental period consisted of 14 d for adaptation plus 14 d for collection of
production data. Sampling of ruminal digesta and spot sampling of blood, feces,
and urine was done on d 26 and 27 of each period. Planned contrasts compared
included diet A vs. diet B, diet B vs. diet C, and diet B vs. diet D. There were
no effects of diet on most of the production traits measured. However, milk yield
tended to be higher for diet B vs. A. Trends were also detected for higher DM
intake and weight gain and lower milk yield/DM intake in cows fed diet D vs. diet
B. Milk lactose content was higher on diets A and C than on B. Ruminal NH3 was
higher on diet D vs. B, but other ruminal metabolites, apparent nutrient
digestibility, and estimated bacterial CP synthesis did not differ across diets.
Blood and milk urea-N were higher on diets C and D than on B; milk urea-N was
higher on diet B than on A. Increasing dietary CP from 16.6% (diet B) to 17.6%
(diet D) increased urinary N excretion by 54 g/d and reduced apparent N
efficiency (milk N/N intake) by 2.5 percentage units, without altering yield.
Under the conditions of this trial, milk production was not improved by feeding
rumen-undegraded protein from ESBM or greater amounts of rumen-degraded protein
from SSBM. Feeding more than 16.6% CP depressed N efficiency.
DOI: 10.3168/jds.S0022-0302(06)72230-5
1945. J Dairy Sci. 2006 May;89(5):1580-91.
Effect of total mixed ration composition on fermentation and efficiency of

ruminal microbial crude protein synthesis in vitro.
Boguhn J(1), Kluth H, Rodehutscord M.
Author information:
(1)Institut für Ernährungswissenschaften, Martin-Luther-Universität
Halle-Wittenberg, 06099 Halle, Saale, Germany.
The goal of this study was to identify dietary factors that affect fermentation
and efficiency of microbial crude protein (CP(M)) synthesis in the rumen in
vitro. We used 16 total mixed, dairy cow rations with known digestibilities that
varied in ingredient composition and nutrient content. Each ration was incubated
in a Rusitec (n = 3) for 15 d, and fermentation of different fractions was
assessed. Observed extents of fermentation in 24 h were 35 to 47% for organic
matter, 25 to 60% for crude protein, 3 to 28% for neutral detergent fiber, and 31
to 45% for gross energy. Organic matter fermentation depended on the content of
crude protein and neutral detergent fiber in the ration. We studied net synthesis
of CP(M) using an 15N dilution technique and found that 7 d of continuous 15N
application are needed to achieve an 15N enrichment plateau in the N of isolated
microbes in this type of study. The efficiency of CP(M) synthesis was 141 to 286
g/kg of fermented organic matter or 4.9 to 11.1 g/MJ of metabolizable energy, and
these ranges agree with those found in the literature. Multiple regressions to
predict the efficiency of CP(M) synthesis by diet data showed that crude protein
was the only dietary chemical fraction that had a significant effect. Fat content
and the inclusion rate of corn silage in the ration also tended to improve
efficiency. We suggest that microbial need for preformed amino acids may explain
the crude protein effect. A large part of the variation in efficiency of
microbial activity still remains unexplained.
DOI: 10.3168/jds.S0022-0302(06)72225-1
1946. J Dairy Sci. 2006 May;89(5):1571-9.
NutriDense corn grain and corn silage for dairy cows.
Benefield BC(1), Liñeiro M, Ipharraguerre IR, Clark JH.
Author information:
(1)Department of Animal Sciences, University of Illinois, Urbana 61801, USA.
Twenty multiparous Holstein cows, 4 of them surgically fitted with ruminal

cannulas, were used in a replicated 4 x 4 Latin square to compare the effects of
whole-plant silage and grain produced from NutriDense (ND), leafy NutriDense
(LND), or a conventional yellow dent (YD) hybrid on ruminal fermentation, total
tract nutrient digestibility, and performance of lactating dairy cows. On a DM
basis, diets contained 30.6% corn silage and 27.7% corn grain provided from the 3
hybrids according to the following combinations: 1) YD grain and YD silage, 2) YD
grain and LND silage, 3) ND grain and YD silage, and 4) ND grain and LND silage.
The average concentrations of crude protein, neutral and acid detergent fiber,
and ether extract of LND silage and ND grain were higher, but the contents of
nonfibrous carbohydrates and starch were lower than those of their YD
counterparts. Although DM intake was similar among treatments, feeding ND grain,
LND silage, or both reduced the intakes of nonfibrous carbohydrates and starch
but increased the intake of ether extract. Apparent digestibility of starch in
the total tract was highest for the diet that contained LND silage and YD grain,
whereas the amount and percentage of ether extract that were apparently digested
in the total tract was increased and tended to be increased, respectively, by the
addition of ND grain, LND silage, or both to the diets. Ruminal fermentation
parameters were unaffected by treatments except for the concentration of ammonia
nitrogen in the ruminal fluid, which tended to be increased by the feeding of ND
grain, LND silage, or both. Production of milk, crude and true protein, fat,
lactose, and total solids did not differ among diets. Concentration of milk urea
nitrogen increased when the ND grain, LND silage, or both were fed to the cows.
Results indicate that ND grain and LND silage were similar to the conventional
grain and silage for the feeding of lactating dairy cows.
DOI: 10.3168/jds.S0022-0302(06)72224-X
1947. Biochim Biophys Acta. 2006 Jan-Feb;1759(1-2):108-15. Epub 2006 Mar 29.
Characterization of three Rop GTPase genes of alfalfa (Medicago sativa L.).
Szucs A(1), Dorjgotov D, Otvös K, Fodor C, Domoki M, Györgyey J, Kaló P, Kiss GB,
Dudits D, Fehér A.
Author information:
(1)Institute of Plant Biology, Biological Research Center of the Hungarian
Academy of Sciences, Temesvári krt. 62., 6726 Szeged, Hungary.
Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The
represented genes could be assigned to various linkage groups by genetic mapping.
They were expressed in all investigated plant organs, although at different
level. Relative gene expression patterns in response to Sinorhizobium infection
of roots as well as during somatic embryogenesis indicated their differential
participation in these processes. DNA sequences coding for altogether six
different Medicago sp. Rop GTPases could be identified in sequence databases.
Based on their homology to each other and to their Arabidopsis counterparts, a
unified nomenclature is suggested for Medicago Rop GTPases.
DOI: 10.1016/j.bbaexp.2006.03.001
1948. J Mol Biol. 2006 May 19;358(5):1341-52. Epub 2006 Mar 29.
Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).
Wang X(1), He X, Lin J, Shao H, Chang Z, Dixon RA.
Author information:
Parkway, Ardmore, OK 73401, USA. xwang@noble.org
Isoflavonoids play important roles in plant defense and exhibit a range of

mammalian health-promoting activities. Isoflavone reductase (IFR) specifically
recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction
to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion
of residues 39-47 has been determined at 1.6A resolution. Structural analysis,
molecular modeling and docking, and comparison with the structures of other
NADPH-dependent enzymes, defined the putative binding sites for co-factor and
substrate and potential key residues for enzyme activity and substrate
specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic
residue. This study provides a structural basis for understanding the enzymatic
mechanism and substrate specificity of IFRs as well as the functions of IFR-like
proteins.
DOI: 10.1016/j.jmb.2006.03.022
1949. J Evol Biol. 2006 Mar;19(2):392-401.
Ecological specialization correlates with genotypic differentiation in sympatric

host-populations of the pea aphid.
Frantz A(1), Plantegenest M, Mieuzet L, Simon JC.
Author information:
(1)UMR INRA/Agrocampus Rennes Biologie des Organismes et des Populations
appliquée à la Protection des Plantes, BP 35327--Domaine de la Motte-au-Vicomte,
35653--Le Rheu, Cedex, France. adrien.frantz@rennes.inra.fr
The pea aphid, Acyrthosiphon pisum, encompasses distinct host races specialized
on various Fabaceae species, but the extent of genetic divergence associated with
ecological specialization varies greatly depending on plant and geographic
origins of aphid populations. Here, we studied the genetic structure of French
sympatric pea aphid populations collected on perennial (pea and faba bean) and
annual (alfalfa and red clover) hosts using 14 microsatellite loci. Classical and
Bayesian population genetics analyses consistently identified genetic clusters
mostly related to plant origin: the pea/faba bean cluster was highly divergent
from the red clover and the alfalfa ones, indicating they represent different
stages along the continuum of genetic differentiation. Some genotypes were
assigned to a cluster differing from the one expected from their plant origin
while others exhibited intermediate genetic characteristics. These results
suggest incomplete barriers to gene flow. However, this limited gene flow seems
insufficient to prevent ecological specialization and genetic differentiation in
sympatry.
DOI: 10.1111/j.1420-9101.2005.01025.x
1950. Appl Environ Microbiol. 2006 Apr;72(4):2738-48.
Identification of Sinorhizobium meliloti early symbiotic genes by use of a

positive functional screen.
Zhang XS(1), Cheng HP.
Author information:
York, 250 Bedford Park Boulevard, West Bronx, NY 10468.
The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis

with its leguminous host plant, alfalfa, following a series of continuous signal
exchanges. The complexity of the changes of alfalfa root structures during
symbiosis and the amount of S. meliloti genes with unknown functions raised the
possibility that more S. meliloti genes may be required for early stages of the
symbiosis. A positive functional screen of the entire S. meliloti genome for
symbiotic genes was carried out using a modified in vivo expression technology. A
group of genes and putative genes were found to be expressed in early stages of
the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes
were further separated into two groups based on their responses to apigenin, a
known nodulation (nod) gene inducer. The group of six genes not inducible by
apigenin included the lsrA gene, which is essential for the symbiosis, and the
dgkA gene, which is involved in the synthesis of cyclic beta-1,2-glucan required
for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible
genes, most have not been previously characterized in S. meliloti, and none of
them belongs to the nod gene family. The identification of this large group of
alfalfa root exudate-inducible S. meliloti genes suggests that the interactions
in the early stages of the S. meliloti and alfalfa symbiosis could be complex and
that further characterization of these genes will lead to a better understanding
of the symbiosis.
DOI: 10.1128/AEM.72.4.2738-2748.2006
PMCID: PMC1449070
1951. J Environ Qual. 2006 Apr 3;35(3):776-85. Print 2006 May-Jun.
Environmental impact of irrigation in La Violada District (Spain): II. Nitrogen

fertilization and nitrate export patterns in drainage water.
Isidoro D(1), Quílez D, Aragüés R.
Author information:
(1)Unidad de Suelos y Riegos, Centro de Investigación y Tecnología
Agroalimentaria (CITA-DGA) and Laboratorio de Agronomía y Medio Ambiente
(DGA-CSIC), Apdo. 727, 50080-Zaragoza, Spain. disidoro@aragon.es
Fertilizer leaching affects farm profitability and contributes to nonpoint-source

pollution of receiving waters. This work aimed to establish nitrate nitrogen
export from La Violada Gully in relation to nitrogen fertilization practices in
its basin (La Violada Gully watershed, VGW, 19,637 ha) and especially in La
Violada Irrigation District (VID, 5282 ha). Nitrogen (N) fertilization in VID
(and VGW) was determined through interviews with local farmers for the hydrologic
years 1995 and 1996 and NO3-N load in the gully was monitored from 1995 to 1998.
The N fertilizer applied in VGW was 2175 Mg in 1995 and 2795 Mg in 1996. About
43% was applied in VID (945 Mg in 1995 and 1161 Mg in 1996). The most fertilized
crop was corn: 398 kg N ha-1 (665 Mg) in 1995 and 453 kg N ha-1 (911 Mg) in 1996.
Nitrogen fertilization was higher than N uptake for irrigated crops, especially
for corn and rice. Nitrate N load in La Violada Gully averaged 427.4 Mg yr-1.
Seventy-five percent of the exports took place during the irrigation season
(321.8 Mg). During the non-irrigation season maximum NO3-N loads (3.1 Mg NO3-N
d-1) were found after heavy rains following the N side-dressing of wheat in the
rain-fed area of VGW (February). During the irrigation season NO3-N load was
determined by outflow from the district (caused by irrigation) and to a lesser
extent by changes in NO3 concentration (caused by fertilization), showing peaks
in April (pre-sowing corn N fertilization and first irrigations) and June to
August (highest irrigation months and corn side-dress N applications, maximum 6.3
Mg NO3-N d-1 in July). Adjusting N fertilization to crops' needs, improving
irrigation efficiencies, and better scheduling N fertilization and irrigation in
corn could reduce N export from VID.
DOI: 10.2134/jeq2005.0065
1952. Mikrobiologiia. 2006 Jan-Feb;75(1):94-100.
[Symbiosis between the nodule bacterium Sinorhizobium meliloti and alfalfa

(Medicago sativa) under salinization conditions].
Ibragimova MV, Rumiantseva ML, Onishchuk OP, Belova VS, Kurchak ON, Andronov EE,
Dziubenko NI, Simarov BV.
Two hundred forty-three isolates of alfalfa nodule bacteria (Sinorhizobium

meliloti) were obtained from legume nodules and soils sampled in the northern
Aral region, experiencing secondary salinization. Isolates obtained from nodules
(N isolates) were significantly more salt-tolerant than those from soils (S
isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild
species of alfalfa, melilot, and trigonella preferably formed symbioses with
salt-tolerant nodule bacteria in both salinized and nonsalinized soils. Only two
alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses
in soils contrasting in salinity. The formation of efficient symbiosis with
alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and S)
differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed
efficient symbioses in the presence of salt. The increase in the dry weight of
the plants was 25-68% higher than in the control group. The efficiency of
symbiotic interaction under salinization conditions depended on the efficiency of
the isolates under standard conditions but did not correlate with the source of
nodule bacteria (soil or nodule) or their salt tolerance. The results indicate
that nodule bacterium strains forming efficient symbioses under salinization
conditions can be found.
1953. J Exp Biol. 2006 Apr;209(Pt 8):1535-47.
Forage fibre digestion, rates of feed passage and gut fill in juvenile and adult
red kangaroos Macropus rufus Desmarest: why body size matters.
Munn AJ(1), Dawson TJ.
Author information:
(1)School of Biological, Earth and Environmental Sciences, University of New
South Wales, Sydney, Australia. a.munn@unswalumni.com
Using red kangaroos Macropus rufus Desmarest, a large (>20 kg) marsupial
herbivore, we compared the digestive capabilities of juveniles with those of
mature, non-lactating females on high-quality forage (chopped lucerne Medicago
sativa hay) of 43+/-1% neutral-detergent fibre (NDF) and poorer quality,
high-fibre forage (chopped oaten Avena sativa hay) of 64+/-1% NDF. On chopped
lucerne apparent dry matter (DM) digestibilities by young-at-foot (YAF) red
kangaroos (an age that would normally be taking some milk from their mother),
weaned juveniles and mature females were similar (55-59%). On chopped oaten hay
apparent DM digestibility was lower in the YAF (35.9+/-2.3%) followed by weaned
(43.4+/-2.8%) and mature females (44.6+/-1%). The digestion of NDF and its
components (mainly cellulose and hemicellulose) was lowest among the YAF followed
by weaned and then mature females. The YAF and weaned kangaroos could not sustain
growth on the poor-quality diet, and appeared to be at or near maximal gut fill
on both forages; the values being 114-122 g DM for YAF and 151-159 g DM for
weaned kangaroos. Mean retention times (MRT) of particle and solute markers were
significantly longer for the YAF and weaned kangaroos on oaten hay than on
lucerne hay, and DM intake (g d(-1)) was approximately 50% lower on the oaten
hay. In contrast, solute and particle MRTs in the mature females were not
significantly affected by diet; they maintained DM intakes by increasing DM gut
fill from 264+/-24 g on chopped lucerne to 427+/-26 g DM on chopped oaten hay.
Clearly, the mature female kangaroos did not maximise gut fill on the
high-quality forage, presumably as a consequence of their proportionally lower
energy requirements compared with still-growing juveniles. Overall, we have
provided the first mechanistic link between the physiological constraints faced
by juvenile red kangaroos in relation to their drought-related mortalities,
rainfall and forage quality.
DOI: 10.1242/jeb.02137
1954. Physiol Biochem Zool. 2006 Mar-Apr;79(2):424-36. Epub 2005 Jan 30.
Endogenous nitrogen excretion by red kangaroos (Macropus rufus): effects of

animal age and forage quality.
Munn AJ(1), Dawson TJ, Hume ID.
Author information:
(1)School of Biological, Earth, and Environmental Sciences, University of New
South Wales, Sydney, New South Wales 2052, Australia.
Red kangaroos (Macropus rufus) are large (>20 kg) herbivorous marsupials common
to arid and semiarid Australia. The population dynamics of red kangaroos are
linked with environmental factors, operating largely through juvenile survival. A
crucial period is the young-at-foot (YAF) stage, when juveniles have permanently
left the mother's pouch but still take milk from a teat in the pouch. Forage
quantity and quality have been implicated in drought-related mortalities of
juvenile kangaroos. Here we compared how forage quality affected nitrogen (N)
intake and excretion by YAF, weaned, and mature, nonlactating female red
kangaroos. On high-quality forage (chopped lucerne hay, Medicago sativa) low in
neutral-detergent fiber (43%+/-1%) and high in N (2.9%+/-0.1%), YAF and weaned
kangaroos had ideal growth rates and retained 460-570 mg dietary N kg(-0.75)
d(-1). But on poor-quality forage (chopped oaten hay, Avena sativa) high in
neutral-detergent fiber (64%+/-1%) and low in N (0.9%+/-0.1%), YAF and weaned
kangaroos could not sustain growth and were in negative N balance at -103+/-26 mg
and -57+/-31 mg N kg(-0.75) d(-1), respectively. Notably, the YAF kangaroos
excreted 64% of their truly digestible N intake from forage as nondietary fecal N
(NDFN). By weaning age, the situation had improved, but the juveniles still lost
40% of their truly digestible N intake as NDFN compared with only 30% by the
mature females. Our findings support field observations that forage quality, and
not just quantity, is a major factor affecting the mortality of juvenile red
kangaroos during drought.
DOI: 10.1086/499980
1955. Plant J. 2006 Apr;46(1):111-23.
Activation of an alfalfa cyclin-dependent kinase inhibitor by calmodulin-like

domain protein kinase.
Pettkó-Szandtner A(1), Mészáros T, Horváth GV, Bakó L, Csordás-Tóth E, Blastyák

A, Zhiponova M, Miskolczi P, Dudits D.
Author information:
(1)Institute of Plant Biology, Biological Research Centre, Hungarian Academy of
Sciences, Szeged, H-6726, Temesvári krt. 62, Hungary.
Kip-related proteins (KRPs) play a central role in the regulation of the cell
cycle and differentiation through modulation of cyclin-dependent kinase (CDK)
functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt)
by a yeast two-hybrid screen. The KRPMt gene was expressed in all plant organs
and cultured cells, and its transcripts accumulated after abscisic acid and NaCl
treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST
motif that is also detected in various Arabidopsis KRPs. In the yeast two-hybrid
test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D-type cyclins.
However, in the pull-down assays, B-type CDK complexes were also detectable.
Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in
phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was
strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most
sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by
the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded
weakly to this inhibitor protein. Kinase complexes from G2/M cells showed
increased sensitivity towards the inhibitor compared with those isolated from
G1/S-phase cells. In vitro phosphorylation of Medicago retinoblastoma-related
protein was also reduced in the presence of KRPMt. Phosphorylation of this
inhibitor protein by the recombinant calmodulin-like domain protein kinase
(MsCPK3) resulted in enhanced inhibition of CDK function. The data presented
emphasize the selective sensitivity of various cyclin-dependent kinase complexes
to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in
cross-talk between Ca2+ signalling and regulation of cell-cycle progression in
plants.
DOI: 10.1111/j.1365-313X.2006.02677.x
1956. J Anim Sci. 2006 Apr;84(4):886-93.
Determination of undegradable intake protein digestibility of forages using the

mobile nylon bag technique.
Haugen HL(1), Ivan SK, MacDonald JC, Klopfenstein TJ.
Author information:
(1)Department of Animal Science, University of Nebraska, Lincoln, 68583-0908,
USA.
Two experiments were conducted using 2 ruminally and duodenally fistulated steers
to determine the digestibility of undegradable intake protein (UIP) of smooth
bromegrass (Bromis inermis), birdsfoot trefoil (Lotus coniculatus L.), and
heat-treated alfalfa (Medicago sativa) using the mobile nylon bag technique.
Undegradable intake protein was determined using neutral detergent insoluble CP
at a single in situ incubation time point based on 75% of the total mean
retention time estimated from IVDMD plus a 10-h passage lag. In Exp. 1, UIP (%
DM) of smooth bromegrass in June and July were 1.82 and 1.71, respectively (P =
0.11). Undegradable intake protein (% DM) of birdsfoot trefoil increased from
1.30 in June to 1.94 in July (P < 0.01). Total tract indigestible protein of
smooth brome-grass and birdsfoot trefoil increased in July (P < 0.05).
Digestibility of UIP decreased in July for smooth brome-grass (P < 0.01) but
tended to increase for birdsfoot trefoil (P = 0.07). In Exp. 2, alfalfa from
plots fertilized with low (66 kg of N/ha) or high (200 kg of N/ha) amounts of N
were dried to simulate 3 preservation methods: dehydrated (100 degrees C, 10 h),
sun-cured (50 degrees C, 15 h), and lyophilized (-50 degrees C, 72 h) alfalfa.
Undegradable intake protein (% DM) was estimated as in Exp. 1 and was 3.13, 2.10,
and 1.84 for dehydrated, sun-cured, and lyophilized alfalfa, respectively. Total
tract indigestible protein (% DM) was increased (P < 0.05) for dehydrated alfalfa
(1.66) compared with sun-cured (1.54) or lyophilized (1.57) alfalfa. As a result
of greater UIP flow to the lower tract, digestibility (%) of UIP was greater (P <
0.01) for dehydrated (46.4) than for sun-cured (25.6) or lyophilized (14.7)
alfalfa. Heat-treated alfalfa samples increased net UIP absorption in the lower
tract because 1.47, 0.56, and 0.27 percentage units of UIP (% DM) of dehydrated,
sun-cured, and lyophilized alfalfa, respectively, disappeared. Overall, the
digestibility of the UIP of these forages was low in the lower tract.
1957. J Food Prot. 2006 Mar;69(3):582-90.
Inactivation of Escherichia coli O157:H7, Salmonella typhimurium DT104, and

Listeria monocytogenes on inoculated alfalfa seeds with a fatty acid-based
sanitizer.
Pierre PM(1), Ryser ET.
Author information:
(1)Department of Food Science and Human Nutrition, 2108 South Anthony Hall,
Michigan State University, East Lansing, Michigan 48824, USA.
Alfalfa seeds were inoculated with a three-strain cocktail of Escherichia coli

O157:H7, Salmonella enterica subsp. enterica serovar Typhimurium DT104, or
Listeria monocytogenes by immersion to contain approximately 6 to 8 log CFU/g and
then treated with a fatty acid-based sanitizer containing 250 ppm of peroxyacid,
1,000 ppm of caprylic and capric acids (Emery 658), 1,000 ppm of lactic acid, and
500 ppm of glycerol monolaurate at a reference concentration of 1X. Inoculated
seeds were immersed at sanitizer concentrations of 5X, 10X, and 15X for 1, 3, 5,
and 10 min and then assessed for pathogen survivors by direct plating. The lowest
concentration that decreased all three pathogens by >5 log was 15. After a 3-min
exposure to the 15X concentration, populations of E. coli O157:H7, Salmonella
Typhimurium DT104, and L. monocytogenes decreased by >5.45, >5.62, and >6.92 log,
respectively, with no sublethal injury and no significant loss in seed
germination rate or final sprout yield. The components of this 15x concentration
(treatment A) were assessed independently and in various combinations to optimize
antimicrobial activity. With inoculated seeds, treatment C (15,000 ppm of Emery
658, 15,000 ppm of lactic acid, and 7,500 ppm of glycerol monolaurate) decreased
Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes by 6.23 and 5.57
log, 4.77 and 6.29 log, and 3.86 and 4.21 log after 3 and 5 min of exposure,
respectively. Treatment D (15,000 ppm of Emery 658 and 15,000 ppm of lactic acid)
reduced Salmonella Typhimurium by >6.90 log regardless of exposure time and E.
coli )157:H7 and L. monocytogenes by 4.60 and >5.18 log and 3.55 and 3.14 log
after 3 and 5 min, respectively. No significant differences (P > 0.05) were found
between treatments A, C, and D. Overall, treatment D, which contained Emery 658
and lactic acid as active ingredients, reduced E. coli O157:H7, Salmonella
Typhimurium, and L. monocytogenes populations by 3.55 to >6.90 log and may
provide a viable alternative to the recommended 20,000 ppm of chlorine for
sanitizing alfalfa seeds.
Strain distribution of alfalfa weevil (Coleoptera: Curculionidae) in an

intergrade zone.
Bundy CS(1), Smith PF, English LM, Sutton D, Hanson S.
Author information:
(1)Department of Entomology, Plant Pathology, and Weed Science, New Mexico State
University, Las Cruces, NM 88003, USA.
Strain distribution for the alfalfa weevil, Hypera postica (Gyllenhal), was
determined for the five major alfalfa-growing regions of New Mexico.
Mitochondrial DNA from field-collected weevils at each location was digested and
sequenced to differentiate among strains. Eastern, Egyptian, and western strains
were recovered, along with a population not matching any known strain. The
majority of sites examined had populations of two strains intermingled within a
field. This is the first confirmation of the presence of all three strains within
the same geographic region. Possible implications for alfalfa weevil management
are discussed.

1959. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4634-9. Epub 2006 Mar 1.
Sinorhizobium meliloti bluB is necessary for production of

5,6-dimethylbenzimidazole, the lower ligand of B12.
Campbell GR(1), Taga ME, Mistry K, Lloret J, Anderson PJ, Roth JR, Walker GC.
Author information:
(1)Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, MA 02139, USA.
Comment in
Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):4799-800.
An insight into a previously unknown step in B(12) biosynthesis was unexpectedly

obtained through our analysis of a mutant of the symbiotic nitrogen fixing
bacterium Sinorhizobium meliloti. This mutant was identified based on its
unusually bright fluorescence on plates containing the succinoglycan binding dye
calcofluor. The mutant contains a Tn5 insertion in a gene that has not been
characterized previously in S. meliloti. The closest known homolog is the bluB
gene of Rhodobacter capsulatus, which is implicated in the biosynthesis of B(12)
(cobalamin). The S. meliloti bluB mutant is unable to grow in minimal media and
fails to establish a symbiosis with alfalfa, and these defects can be rescued by
the addition of vitamin B(12) (cyanocobalamin) or the lower ligand of cobalamin,
5,6-dimethylbenzimidazole (DMB). Biochemical analysis demonstrated that the bluB
mutant does not produce cobalamin unless DMB is supplied. Sequence comparison
suggests that BluB is a member of the NADH/flavin mononucleotide (FMN)-dependent
nitroreductase family, and we propose that it is involved in the conversion of
FMN to DMB.
DOI: 10.1073/pnas.0509384103
PMCID: PMC1450223
1960. Appl Environ Microbiol. 1997 Dec;63(12):4978-81.
Regulation of the Phosphate Stress Response in Rhizobium meliloti by PhoB.
Al-Niemi TS, Summers ML, Elkins JG, Kahn ML, McDermott TR.
Alkaline phosphatase activity and phosphate transport rates in Rhizobium meliloti

increased significantly when medium phosphate levels decreased to approximately
10 (mu)M. Both responses were abolished in a Tn5:: phoB mutant, but the mutant
could be complemented by a plasmid that contained cloned R. meliloti phoB. The
PhoB(sup-) mutant had a normal symbiosis phenotype under growth conditions that
supplied either limiting or nonlimiting levels of phosphate to the host plant
Medicago sativa, suggesting that induction of genes by PhoB was not required for
normal symbiotic function.
PMCID: PMC1389314
PMID: 16535758
1961. Appl Environ Microbiol. 1997 Nov;63(11):4573-7.
Occurrence of flavonoids and nucleosides in agricultural soils.

Phillips DA, Joseph CM, Hirsch PR.
AN ECOLOGICALLY RELEVANT SOIL EXTRACTION PROCEDURE SEPARATED TWO TYPES OF

MOLECULES IMPORTANT FOR BACTERIA: flavonoids and small hydrophilic organic
compounds. Two flavonoids, identified previously as inducers of nodulation genes
in Rhizobium meliloti, were detected in rhizosphere soil from alfalfa (Medicago
sativa L.). In addition, biologically significant quantities (micromoles per
kilogram) of ribonucleosides and deoxyribonucleosides were found in all soils
tested. Long-term wheat (Triticum aestivum L.) plots that had received manure
contained elevated amounts of nucleosides, and in a separate experiment, the
presence of legumes in a wheat-cropping sequence increased soil nucleosides.
Intact bacterial cells accounted for less than 1% of the free nucleosides
detected. These results suggest new testable hypotheses for molecular ecologists
and differ from those obtained with older, harsher techniques.
PMCID: PMC1389295
PMID: 16535739
1962. Plant Cell Rep. 2006 Aug;25(8):799-806. Epub 2006 Mar 14.
Strategies to obtain stable transgenic plants from non-embryogenic lines:

complementation of the nn1 mutation of the NORK gene in Medicago sativa MN1008.
Perhald A(1), Endre G, Kevei Z, Kiss GB, Kereszt A.
Author information:
(1)Institute of Genetics, Biological Research Center of the Hungarian Academy of
Sciences, Temesvari korut 62, 6726, Szeged, Hungary.
Strategies to introduce genes into non-embryogenic plants for complementation of

a mutation are described and tested on tetraploid alfalfa (Medicago sativa).
Genes conditioning embryogenic potential, a mutant phenotype, and a gene to
complement the mutation can be combined using several different crossing and
selection steps. In the successful strategy used here, the M. sativa genotype
MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn ( 1 ) was
crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid
individuals were identified from the F1 progeny. After transformation of these
hybrids with the wild-type gene (NORK), an F2 generation segregating for the
mutation and transgene were produced. Plants homozygous for the mutant allele and
carrying the wild-type NORK transgene could form root nodules after inoculation
with Sinorhizobium meliloti demonstrating successful complementation of the nn (
1 ) mutation.
DOI: 10.1007/s00299-006-0117-6
Nuclear DNA endoreduplication and expression of the mitotic inhibitor Ccs52

associated to determinate and lupinoid nodule organogenesis.
González-Sama A(1), de la Peña TC, Kevei Z, Mergaert P, Lucas MM, de Felipe MR,
Kondorosi E, Pueyo JJ.
Author information:
(1)Institut des Sciences du Vegetal, Centre National de la Recherche
Scientifique, Unité Propre de Recherche 2355, Avenue de la Terrasse, 91198
Gif-sur-Yvette, France.
Lotus japonicus determinate nodules differ greatly from indeterminate nodules in

their organogenesis and morphological characteristics, whereas Lupinus albus
lupinoid nodules share features of determinate and indeterminate nodules. The
mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent
cell enlargement during symbiotic cell differentiation in Medicago truncatula
indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus
nodules; the deduced Ccs52A proteins showed high sequence similarity with other
Cdh-1-type activators of the anaphase-promoting complex and were grouped with
A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was
restricted to the earlier stages of nodule development, whereas ccs52A
transcripts accumulated in lotus nodule primordia and, to a lesser extent, in
mature nodules. Nodule development in Lupinus albus involved a progressive
increase in nuclear and cellular size and ploidy level; similarly, Lotus
japonicus nodules contained polyploid nuclei and enlarged cells in the infected
zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A
expression in the inner cortex cells of the lupin nodule primordium, probably
associated to the increased size of these cells in mature nodules. In view of our
results, Ccs52A-mediated endoreduplication appears to be a universal mechanism
required for nodule cell differentiation during the establishment of
nitrogen-fixing symbioses.
DOI: 10.1094/MPMI-19-0173
1964. N Z Vet J. 2006 Feb;54(1):21-7.
In vivo anthelmintic activity of Dorycnium rectum and grape seed extract against
Ostertagia (Teladorsagia) circumcincta and Trichostrongylus colubriformis in
sheep.
Waghorn TS(1), Molan AL, Deighton M, Alexander RA, Leathwick DM, McNabb WC,
Meagher LP.
Author information:
(1)AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston
North, New Zealand. tania.waghorn@agresearch.co.nz
AIM: To assess the in vivo anthelmintic activity of condensed tannins (CT) in the
forage species Dorycnium rectum and Medicago sativa, and in an extract from grape
(Vitus vinifera) seeds (GSE), against two species of parasite, Teladorsagia
(Ostertagia) circumcincta and Trichostrongylus colubriformis, at different stages
of their life cycle, in sheep that were parasite-naïve or previously exposed to
nematodes.
METHODS: In Trial 1, a factorial treatment structure was used to compare faecal
nematode egg counts (FEC) and worm burdens in 40 weaned Romney lambs fed either
the CT-containing forage D. rectum (12% dry matter; DM) or M. sativa (lucerne;
0.2% DM). Twenty naïve and 20 previously-exposed lambs were drenched free of
parasites then reinfected with known species and numbers of parasites, and housed
in pens indoors on a diet of lucerne pellets and chaffed hay. Groups of lambs
(n=5 lambs per group) were fed one of the forages over one of two time periods
within the parasite's life cycle. Six to nine days after the last feeding of
fresh forages, faecal samples were collected for FEC, and all lambs were
slaughtered and worm counts conducted. In Trial 2, 12 Suffolk x Romney lambs were
surgically implanted with an abomasal cannula and then housed indoors in
metabolism crates. After infection with parasites, six lambs were infused
continuously over a 14-day period with a commercially available CT GSE (96% DM,
made up to 34 g/L in water); the remaining lambs were infused with water. During
infusion, samples were collected for egg hatch and larval development assays.
After infusion, samples were collected for FEC, and all lambs were slaughtered
and worm counts conducted.
RESULTS: In Trial 1, there was a significant (p<0.001) difference in burdens of
O. circumcincta between naïve lambs and those previously exposed to parasites,
but no other differences were recorded. In Trial 2, lambs infused with GSE had
significantly (p<0.05) fewer T. colubriformis at slaughter and significantly
(p<0.001) fewer eggs hatched in the egg hatch assay (EHA) than for lambs infused
with water. Overall, the differences attributable to GSE were small in magnitude,
being an 11% drop in egg hatch, and an 18% drop in numbers of adult T.
colubriformis after 14 days of continuous infusion. No other differences were
recorded.
CONCLUSION: The results indicate that the in vivo anthelmintic activity of these
CT sources is, at best, modest and is unlikely to be of any practical value.
Further, these data emphasise that in vitro activity is an unreliable indicator
of in vivo efficacy for CT-containing forages and extracts.
DOI: 10.1080/00480169.2006.36599
1965. Yi Chuan. 2006 Feb;28(2):184-8.
[Physical localization of ribosomal genes and chromosome DAPI banding by in situ

hybridization in Medicago sativa L].
Chen JM(1), Hong YH, Wang YP, Bowley S, Wan JM.
Author information:
(1)College of Agriculture, Nanjing Agricultural University, Nanjing 210095,
China. jmchen@yzu.edu.cn
Physical localization of ribosomal genes in diploid and tetraploid alfalfa

(Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It
was revealed that 45s gene was only located at nucleolus organizer region (NOR)
with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3
loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as
probe to hybridize with tetraploid species in alfalfa, both diploid species were
successfully hybridized with tetraploid chromosomes, only showing the difference
in hybridization signals in different numbers of chromosomes. Chromosomes of
alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of
distribution of DAPI banding were consistent with C-banding for M. coerulea. The
possible origination of tetraploid alfalfa was discussed based on DAPI banding
patterns and FISH analysis for ribosomal genes .
1966. J Anim Physiol Anim Nutr (Berl). 2006 Apr;90(3-4):89-102.
[Studies on the use of lucerne silage source for high-milk-producing dairy cows].
[Article in German]
Bulang M(1), Kluth H, Engelhard T, Spilke J, Rodehutscord M.
Author information:
(1)Institut für Ernährungswissenschaften, Martin-Luther-Universität
Halle-Wittenberg, Halle, Saale, Germany.
Studies on the use of lucerne silage as a forage source for high-yielding dairy
cows. It was the main objective of this study to investigate effects of lucerne
silage inclusion in maize silage-based rations on dry matter intake, milk yield
and metabolic parameters of high yielding dairy cows. Three total mixed rations
(TMR) were used. They contained as the main forage source on a dry matter basis
41.5% maize silage and 12% grass silage (ration maize) or 18% maize silage and
29% of either grass silage (ration grass) or lucerne silage (ration lucerne).
Organic matter digestibility, determined with wether sheep, was 77 and 65% for
the grass and the lucerne silage, respectively. Each TMR was fed to 4 wether
sheep to determine crude nutrient digestibilities. The content of net energy for
lactation (NEL), as calculated from digestible crude nutrients, was (MJ/kg dry
matter (DM)): 7.1 (maize), 7.2 (grass) and 6.8 (lucerne). Based on the results of
a chemical fractionation of crude protein it was calculated that the content of
utilisable crude protein (nXP) was not below 167 g/kg DM and that the N balance
in the rumen was not negative for all 3 TMR. The TMR were fed for 169 days to at
least 28 dairy cows per treatment which were in their first half of lactation.
Feed intake, milk performance data and physiological indicators were measured for
each individual cow. The DM intake was significantly higher in treatment lucerne
(23.2 kg/d) than in treatments grass (21.2 kg/d) and maize (22.6 kg/d). Daily
intakes of NEL and nXP were significantly lower in treatment grass than in the
other two treatments. Milk yield was significantly higher in treatment maize
(41.7 kg/d) than in treatments grass (37.3 kg/d) and lucerne (38.5 kg/d). This
was associated with a significantly lower milk fat content in treatment maize
(3.5%) in comparison with the other two treatments (3.9% each). Milk protein
content was unaffected by treatment (3.4% on average). Similarly, the acetone
content of milk was not significantly different between treatments. The
beta-hydroxybutyrate content of the deproteinized blood was significantly higher
in treatment lucerne at start of lactation, but this difference disappeared in
subsequent parts of the experimental period. Measurements of back fat thickness
indicated mobilisation and retention of energy by cows during the experimental
period to be similar in all treatments. It is concluded that lucerne silage is a
suitable forage source for high yielding dairy cows in spite of its low
digestibility.
DOI: 10.1111/j.1439-0396.2005.00568.x
1967. Vet Parasitol. 2006 Jun 15;138(3-4):308-17. Epub 2006 Mar 3.
Effects of legume forages on ovine gastrointestinal parasite development,

migration and survival.
Marley CL(1), Fraser MD, Roberts JE, Fychan R, Jones R.
Author information:
(1)Institute of Grassland and Environmental Research, Aberystwyth, SY23 3EB, UK.
christina.marley@bbsrc.ac.uk
Lambs grazing certain legumes have reduced parasite intensities compared to lambs
grazing ryegrass swards. Eighteen replicates of white clover (cv. AberHerald),
lucerne (cv. Luzelle), red clover (cv. Merviot) and perennial ryegrass (cv.
Abersilo) were sown at equivalent field rates in 25 cm diameter PVC pots and
maintained outside for 6 months. On day 0, forage in each pot was cut to 50 mm
from soil level and the pots were placed in a glasshouse (at 19-25 degrees C and
70% humidity) in a randomised block design. Ten grams sheep faeces containing
2,133 Haemonchus contortus eggs per gram were placed on the soil in each pot. Six
replicates of each forage were destructively sampled on days 14, 21 and 29.
Forage samples were cut at 50 mm from the soil surface and at the soil surface to
give two samples per pot. The number of nematodes was determined by a
modification of the Whitehead tray method. The ratio of free-living to
infective-stage larvae was determined from at least 10% of the larvae. The number
of H. contortus larvae kgdrymatter(-1) (DM) forage was calculated and the data
rank transformed prior to analysis by ANOVA. There were fewer larvae on legumes
compared with ryegrass on samples from forage above 50 mm (P<0.001) but there was
no forage effect on larvae below this height. The sum of larvae present on all
forage per kilogram DM showed fewer larvae on red clover compared with ryegrass
on day 21 (P<0.05). There was an effect of day on the total number of larvae on
forage (P<0.001) but there were no foragexday interactions. Analysis of the data
according to the leaf area above 50 mm from the soil surface confirmed these
results, that there were fewer larvae on legume forages than ryegrass above this
height (P<0.01). Overall, red clover affected the development of H. contortus and
all legumes affected larval migration above 50 mm compared with ryegrass but
survival of larvae was similar on all forages. Further work is needed to
determine if these effects of legume forages would reduce the number of parasitic
larvae ingested by livestock under field conditions.
DOI: 10.1016/j.vetpar.2006.02.001
[Responses of Medicago sativa and Astragalus adsurgens seedlings growth and water
use to soil moisture regime].
Xu B(1), Shan L, Li F.
Author information:
Institute of Soil and Water Conservation, CAS and MWR, Northwest A & F
University, Yangling 712100, China. Bcxu@163.com
In the semi-arid area of Loess Plateau, seasonal drought often occurs during the
vegetative stage of grass plants, leading to the subsequent serious reduction of
their yield. Aimed to study the responses of the seedlings growth and water use
of two perennial leguminous grasses Medicago sativa and Astragalus adsurgens to
different soil moisture regimes, a pot experiment was installed with five
treatments, i.e., adequate water supply (HW), moderate water stress (LW), soil
drying gradually from HW (DHW) and LW (DLW), and refilling water to LW after soil
drying from LW (RWL). The results showed that under HW, the seedlings of both M.
sativa and A. adsurgens had the highest biomass and transpiration water use
efficiency (TWUE), and M. sativa had a significantly higher biomass than A.
adsurgens (P < 0.05). When the soil moisture content declined, M. sativa had a
higher reduction rate in biomass and TWUE than A. adsurgens, and after the soil
moisture regime changed from LW and DLW to RWL, the biomass of M. sativa and A.
adsurgens was reduced by 47.8% and 27.9%, respectively, as compared to LW (P <
0.05). At the same time, the root/shoot ratio (R/S) of M. sativa and its water
consumption per unit root increased significantly, while the TWUE decreased
significantly (P < 0.05); but for A. adsurgens, its R/S decreased significantly
(P < 0.05), while the TWUE and the water consumption per unit root had no
significant change.
[Degradation process and plant diversity of alfalfa grassland in North Loess

Plateau of China].
Li Y(1), Shao M.
Author information:
(1)Key Laboratory of Water Cycle and Related Land Surface Processes, Institute of
Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences,
Beijing 100101, China. liyy@igsnrr.ac.cn
Vegetation recovery and reconstruction is the key of ecosystem restoration in the

North Loess Plateau. With the Liudaogou catchment of Shenmu County, Shaanxi
Province as test area, this paper studied the dynamics of the plant diversity of
artificial alfalfa (Medicago sativa) grassland during its degradation process.
The results showed that the degradation process of the grassland could be divided
into three stages, i.e., artificial alfalfa grassland stage (1-6 yrs),
transitional stage from artificial to natural grassland (6-10 yrs), and secondary
natural grassland stage dominated by Stipa bungeana ( >10 yrs). In the whole
degradation process from 1-to 30-yrs, 32 species belonging to 13 families and 28
genera were found, of which, 90% appeared in the initial 6-yrs. The dynamics of
accumulatively appeared family, genus, and species in the vegetation succession
process were well described by logarithmic function. During the process of
community succession, the species richness (Gleason index and Margalef
index),plant diversity (Shannon-Wiener index), and Pielou evenness index were
changed with a similar tendency, i.e., increased more rapidly at the first stage,
and the climax phase appeared in the second stage. The plant diversity and
evenness decreased slightly, and gradually became stable later. Planting alfalfa
could markedly accelerate the natural vegetation succession process in the
forest-steppe ecotone of Loess Plateau, mainly due to the intensive soil water
consumption of artificial grassland, which accelerated the process of soil
aridification. To develop artificial grasslands in the Loess Plateau is an
optimal joint between accelerating natural vegetation recovery and increasing
farmers' income by stockbreeding, and is also a favorable paradigm both for the
ecological and for the economic benefits in the eco-environmental construction in
West China.
1970. J Dairy Sci. 2006 Mar;89(3):1052-6.
Short communication: Further validation of the fat sub-model in the

Cornell-Penn-Miner Dairy model.
Moate PJ(1), Boston RC, Lean IJ, Chalupa W.
Author information:
(1)University of Pennsylvania, Kennett Square, 19348, USA. moate@vet.upenn.edu
Recently, a fat sub-model was introduced into the Cornell-Penn-Miner Dairy model
(CPM-Dairy; Moate et al., 2004). The principal aim of the work reported here was
to validate this fat sub-model in terms of its accuracy in predicting the
apparent absorption (intake - feces) of total long-chain fatty acids (LCFA) in
lactating dairy cows. The fat sub-model in CPM-Dairy was used to predict the
amounts (g/d) of total LCFA apparently absorbed from 63 diets described in 14
published experiments. These predicted amounts (PLCFA) were regressed against the
amounts reported to be apparently absorbed (RLCFA). The regression equation was:
PLCFA = - 24.8 +/- 25.2 + 1.011 x 0.029 x RLCFA; R2 = 0.95, RMSE = 55.2 g/d. The
results show that for a diverse range of diets, the fat model in CPM-Dairy can
accurately predict apparent absorption of dietary total LCFA.
DOI: 10.3168/jds.S0022-0302(06)72172-5
1971. J Food Prot. 2006 Feb;69(2):441-3.
Incidence of enterohemorrhagic Escherichia coli, Escherichia coli O157,

Salmonella, and Listeria monocytogenes in retail fresh ground beef, sprouts, and
mushrooms.
Samadpour M(1), Barbour MW, Nguyen T, Cao TM, Buck F, Depavia GA, Mazengia E,
Yang P, Alfi D, Lopes M, Stopforth JD.
Author information:
(1)Institute for Environmental Health Inc, Seattle, Washington 98155, USA.
mansour@u.washington.edu
The objective of this study was to determine the prevalence of enterohemorrhagic

Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in
retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef
(1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were
collected over a 12-month period and analyzed for the presence of these
pathogens. PCR assays, followed by culture confirmation were used to determine
the presence or absence of each organism. Of the 1,750 ground beef samples
analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive
for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef
samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L.
monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3
(1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%)
were positive for Salmonella and none were positive for L. monocytogenes. Among
the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4
samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of
the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.
1972. Ann Bot. 2006 May;97(5):785-91. Epub 2006 Feb 22.
Temperature sensitivity of the low-moisture-content limit to negative seed

longevity--moisture content relationships in hermetic storage.
Ellis RH(1), Hong TD.

Author information:
(1)Department of Agriculture, The University of Reading, Earley Gate, PO Box 237,
Reading RG6 6AR, UK. r.h.ellis@reading.ac.uk
BACKGROUND AND AIMS The negative logarithmic relationship between orthodox seed
longevity and moisture content in hermetic storage is subject to a
low-moisture-content limit (m(c)), but is m(c) affected by temperature?METHODS:
Red clover (Trifolium pratense) and alfalfa (Medicago sativa) seeds were stored
hermetically at 12 moisture contents (2-15 %) and five temperatures (-20, 30, 40,
50 and 65 degrees C) for up to 14.5 years, and loss in viability was estimated.
KEY RESULTS: Viability did not change during 14.5 years hermetic storage at -20
degrees C with moisture contents from 2.2 to 14.9 % for red clover, or 2.0 to
12.0 % for alfalfa. Negative logarithmic relationships between longevity and
moisture contents >m(c) were detected at 30-65 degrees C, with discontinuities at
low moisture contents; m(c) varied between 4.0 and 5.4 % (red clover) or 4.2 and
5.5 % (alfalfa), depending upon storage temperature. Within the ranges
investigated, a reduction in moisture content below m(c) at any one temperature
had no effect on longevity. Estimates of m(c) were greater the cooler the
temperature, the relationship (P < 0.01) being curvilinear. Above m(c), the
estimates of C(H) and C(Q) (i.e. the temperature term of the seed viability
equation) did not differ (P > 0.10) between species, whereas those of K(E) and
C(W) did (P < 0.001).
CONCLUSIONS: The low-moisture-content limit to negative logarithmic relationships
between seed longevity and moisture content in hermetic storage increased the
cooler the storage temperature, by approx. 1.5 % over 35 degrees C (4.0-4.2 % at
65 degrees C to 5.4-5.5 % at 30-40 degrees C) in these species. Further reduction
in moisture content was not damaging. The variation in m(c) implies greater
sensitivity of longevity to temperature above, compared with below, m(c). This
was confirmed (P < 0.005).
DOI: 10.1093/aob/mcl035
PMCID: PMC2803418
1973. Poult Sci. 2006 Jan;85(1):15-20.
Effects of alfalfa and fructooligosaccharide on molting parameters and bone

qualities using dual energy X-ray absorptiometry and conventional bone assays.
Kim WK(1), Donalson LM, Mitchell AD, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station
77843-2472, USA.
The aim of this study was to evaluate effects of alfalfa and

fructooligosaccharides (FOS) on molting performance and bone parameters compared
with the conventional feed withdrawal molting procedure. A total of 36 Single
Comb White Leghorn hens (84 wk of age) were used for this experiment. The hens
were divided into 6 treatment groups with 6 birds per treatment: pre-trial
control (PC), full fed (FF), feed withdrawal (FW), 100% alfalfa (A100), A100 +
0.375% FOS (A100L), and A100 + 0.75% FOS (A100H). At the end of the 9-d molt
period, hens were euthanized, and tibia and femurs were collected to evaluate
bone qualities using dual-energy x-ray absorptiometry (DXA), Instron (Model 1011
Instron Universal Testing Machine, Instron Corp., Canton. MA), and conventional
bone assays. Egg production was recorded during the molting period to evaluate
first day out of production, and ovary was also collected to measure ovary
weight. Alfalfa molting diets had comparable molting parameters, such as
percentage of BW loss, ovary weight, and first day out of egg production, to the
conventional feed withdrawal molting procedure, and FOS supplementation did not
have any detrimental effects on molting performance. Conventional bone assay and
DXA results suggest that hens lose a considerable amount of bone minerals during
a molting period. The tibia and femur bone strengths of the FF, FW, A100, and
A100L hens were significantly lower than the PC hens, whereas hens fed A100H had
similar tibia bone breaking strength to that of the PC hens. The bone parameters
measured by conventional assays, bone breaking strength measured by Instron, and
bone density and mineral content measured by DXA were highly correlated to each
other.
DOI: 10.1093/ps/85.1.15
1974. J Virol Methods. 2006 Jun;134(1-2):217-22. Epub 2006 Feb 20.
Expression of avian reovirus sigmaC protein in transgenic plants.
Huang LK(1), Liao SC, Chang CC, Liu HJ.
Author information:
(1)Institute of Biotechnology, National Cheng Kung University, Tainan 701,
Taiwan.
Avian reovirus (ARV) structural protein, sigmaC encoded by S1 genome segment, is

the prime candidate to become a vaccine against ARV infection. Two plant nuclear
expression vectors with expression of sigmaC-encoding gene driven by CaMV 35S
promoter and rice actin promoter were constructed, respectively. Agrobacterium
containing the S1 expression constructs were used to transform alfalfa, and
transformants were selected using hygromysin. The integration of S1 transgene in
alfalfa chromosome was confirmed by PCR and histochemical GUS staining. Western
blot analysis using antiserum against sigmaC was carried out to determine the
expression of sigmaC protein in transgenic alfalfa cells. The highest expression
levels of sigmaC protein in the cellular extracts of selected p35S-S1 and
pAct1-S1 transgenic alfalfa lines were 0.008% and 0.007% of the total soluble
protein, respectively. The transgenic alfalfa cells with expression of sigmaC
protein pave the way for the development of edible vaccine.
DOI: 10.1016/j.jviromet.2006.01.013
1975. Bioresour Technol. 2007 Jan;98(1):104-11. Epub 2006 Feb 9.
Anaerobic digestion of alfalfa silage with recirculation of process liquid.
Nordberg A(1), Jarvis A, Stenberg B, Mathisen B, Svensson BH.
Author information:
(1)Swedish Institute of Agricultural and Environmental Engineering, PO Box 7033,
S-750 07 Uppsala, Sweden. ake.nordberg@jti.slu.se
Process liquid recirculation initially stimulated one-phase anaerobic digestion

of alfalfa silage in two semi-continuously fed and stirred tank reactors. Thus,
with increased pH, alkalinity and stability it was possible to increase the
organic loading rate to 3 g VS L(-1) d(-1), as compared to 2.25 g VS L(-1) d(-1)
in a control reactor without recirculation. However, the recirculation of liquid
eventually caused an accumulation of organic and inorganic substances, leading to
an inhibition of hydrolysis and methanogenesis. This inhibition of microbial
activity was prevented in one of the processes by replacing 50% of the
recirculated process liquid with water during the second half of the operation
period. A multiple linear regression model of principal components using seven
input variables explained the variance in output variables nearly as well as the
original model using all 23 measured input variables. The results show that it is
necessary to adjust the degree of liquid recirculation to reach an optimal
process.
DOI: 10.1016/j.biortech.2005.11.027
1976. J Anim Sci. 2006 Mar;84(3):669-77.
Ruminal in situ disappearance kinetics of nitrogen and neutral detergent

insoluble nitrogen from common crabgrass forages sampled on seven dates in
northern Arkansas.
Ogden RK(1), Coblentz WK, Coffey KP, Turner JE, Scarbrough DA, Jennings JA,
Richardson MD.
Author information:
(1)Division of Agriculture, Department of Animal Science, University of Arkansas,
Fayetteville, 72701, USA.
Southern crabgrass (Digitaria ciliaris [Retz.] Koel.) is often an undesirable

species in field and forage crops, but visual observations suggest that livestock
prefer it to many other summer forages. The objectives of this study were to
assess the nutritive value of crabgrass sampled weekly between July 11 and August
22, 2001 and then to determine ruminal in situ disappearance kinetics of N and
neutral detergent insoluble N (NDIN) for these forages. A secondary objective was
to compare these kinetic estimates for crabgrass with those of alfalfa (Medicago
sativa L.), bermudagrass (Cynodon dactylon [L.] Pers.), and or-chardgrass
(Dactylis glomerata L.) as control hays. All kinetic evaluations were conducted
with 5 ruminally cannulated Gelbvieh x Angus x Brangus steers (383 +/- 22.7 kg).
Concentrations of N for crabgrass decreased linearly (P < or = 0.002) across
sampling dates for leaf, stem, and whole-plant tissues. Conversely, percentages
of the total N pool within NDIN and ADIN fractions generally increased over
sampling dates in mostly linear patterns. For crabgrass, the immediately soluble
portion of the total N pool (fraction A; overall mean = 54.6% of N) was greater
(P < 0.001) than for all control hays. Crabgrass exhibited a more rapid N
disappearance rate (overall mean = 0.093/h; expressed as a proportion
disappearing/h) than that of bermudagrass (0.046/h; P < 0.001), but the
disappearance rate for alfalfa N (0.223/h) was considerably faster (P < 0.001)
than for crabgrass. The effective ruminal disappearance of N was greater (P <
0.001) for crabgrass (overall mean = 85.4%) than for the alfalfa (83.3%),
bermudagrass (72.3%), or orchardgrass (76.0%) control hays. For alfalfa, the
ruminal disappearance rate of NDIN (0.150/h) was more rapid (P < 0.001) than for
crabgrass (overall mean = 0.110/h); however, the disappearance rate for crabgrass
was faster than that for bermudagrass (0.072/h; P < 0.001) or for orchardgrass
(0.098/h; P = 0.010). Effective ruminal disappearance of NDIN was greater (P <
0.001) for crabgrass (overall mean = 72.0%) than for the bermudagrass (69.0%) or
alfalfa hays (50.5%), but there was no difference (P = 0.865) between crabgrass
and orchardgrass (72.1%). Although crabgrass forages exhibited concentrations of
total N that were comparable with those of alfalfa and rates of ruminal N
disappearance that were < 50% of those for the alfalfa hay control, improvements
in N use efficiency relative to alfalfa are questionable because of the
excessively large Fraction A for crabgrass.
Rapid plant-cover establishment on gold mine tailings in southern New Zealand:

glasshouse screening trials.
Schroeder K(1), Rufaut CG, Smith C, Mains D, Craw D.
Author information:
(1)School of Environmental Sciences, University of Otago, Dunedin, New Zealand.
The use of a short-term vegetation cover to temporarily control the negative

environmental effects of inactive tailings ponds is notfrequently practiced
during operational mining, but could have some merit This article reports on a
glasshouse trial designed to examine some of the issues associated with
short-term vegetation: fast germination of a high proportion of seed, the ability
of seedlings to survive in unamended substrates, and potentially toxic substrate.
Five nonindigenous plant species were tested--barley (Hordeum vulgare), rye corn
(Secale cereale), Italian ryegrass (Lolium multifiorum), red clover (Trifolium
pratense), and lucerne (Medicago sativa)--in five different types of substrate:
unamended tailings, tailings and fertilizer, tailings and greenwaste,
biosolid-blend compost, and local topsoil. The nutrient and heavy metal status
(As, Cu, Cd, Ni, Pb) of each substrate type was determined Plant species
performance was monitored over 14 wk Substrate metal concentrations were low
except for As, which was elevated in all substrate types. Plants in unamended
tailings grew less vigorously than plants in tailings and compost or in topsoil.
Plant performance in tailings and fertiliser was greatly suppressed following a
high fertilization rate. Metal uptake in plants was highest for As (0.4-77 mg
kg(-1) DW) and Cu (5.3-50.3 mg kg(-1) DW). Future field trials are necessary to
authenticate findings, but barley and rye corn are promising species for a
short-term tailings cover.
DOI: 10.1080/16226510500327178
1978. Exp Cell Res. 2006 Apr 15;312(7):1050-64. Epub 2006 Feb 3.
Alfalfa Mob1-like proteins are involved in cell proliferation and are localized
in the cell division plane during cytokinesis.
Citterio S(1), Piatti S, Albertini E, Aina R, Varotto S, Barcaccia G.
Author information:
(1)Dipartimento di Scienze dell'Ambiente e del Territorio, University of
Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy.
Mps-one-binder (Mob) proteins play a crucial role in yeast cytokinesis. After

cloning two Mob1-like genes, MsMob1-A and MsMob1-B from alfalfa (Medicago sativa
L.) we show that, although they are constitutively expressed in roots, stems,
leaves, flowers and pods, their transcripts and proteins are mostly produced in
actively proliferating tissues. A polyclonal antibody specifically raised against
MsMob1 proteins was used for immunolocalization studies in synchronized root tip
cells. The subcellular localization of MsMob1-like proteins is demonstrated to be
cell cycle-regulated. Cytoplasmic localization is faint and diffused during G1
and S. It becomes concentrated in punctuate and fibrillar structures in G2 as
well as M phase. At the stage of cytokinesis, the protein is found at the
emerging cell plate marking the progressive formation of the septum. Mob1
proteins partially co-localize with microtubules structures functionally related
to the spindles and important for cytokinesis in eukaryotic cells. The MsMob1
expression cannot rescue the lethality of the yeast mob1 mutant, suggesting that
interaction of Mob1 proteins with their effectors may be species-specific.
Localization of Mob1 proteins in the inner layer of the root cap indicates an
additional function for this class of proteins in plants, which is likely related
to the onset of programmed cell death.
DOI: 10.1016/j.yexcr.2005.12.032
1979. Plant J. 2006 Mar;45(5):832-46.
The 3' untranslated region of a soybean cytosolic glutamine synthetase (GS1)

affects transcript stability and protein accumulation in transgenic alfalfa.
Ortega JL(1), Moguel-Esponda S, Potenza C, Conklin CF, Quintana A,

Sengupta-Gopalan C.
Author information:
(1)Department of Agronomy and Horticulture, New Mexico State University, Las
Higher plants assimilate nitrogen in the form of ammonia through the concerted
activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS
enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2).
Glutamine synthetase 1 is regulated in different plants at the transcriptional
level and there are some reports of regulation at the level of protein stability.
Here we present data that clearly establish that GS1 in plants is also regulated
at the level of transcript turnover and at the translational level. Using a
Glycine max (soybean) GS1 transgene, with and without its 3' untranslated region
(UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa)
and Nicotiana tabacum (tobacco), we show that the 3' UTR plays a major role in
both transcript turnover and translation repression in both the leaves and the
nodules. Our data suggest that the 3' UTR mediated turnover of the transcript is
regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that
the 3' UTR of the gene for the soybean GS1 confers post-transcriptional
regulation on a reporter gene. Our dissection of post-transcriptional and
translational levels of regulation of GS in plants shows that the situation in
plants strongly resembles that in other organisms where GS is regulated at almost
all levels. Multistep regulation of GS shows the high priority given by organisms
to regulating and ensuring optimal control of nitrogen substrates and preventing
overproduction of glutamine and drainage of the glutamate pool.
DOI: 10.1111/j.1365-313X.2005.02644.x
PMCID: PMC3881554
1980. Bioresour Technol. 2007 Jan;98(1):38-45. Epub 2006 Jan 25.
Compression of fractionated sun-cured and dehydrated alfalfa chops into

cubes--specific energy models.
Adapa P(1), Schoenau G, Tabil L, Sokhansanj S, Singh A.

Author information:
(1)Department of Mechanical Engineering, University of Saskatchewan, 57 Campus
Drive, Saskatoon SK, Canada S7N 5A9.
The objective of this study was to determine the specific energy requirements for
the compression of fractionated sun-cured and dehydrated alfalfa chops, when
subjected to different pressures and holding times. The compression behavior of
fractionated sun-cured and dehydrated alfalfa chops was studied using a single
cubing unit capable of making one cube in a single stroke of the plunger. The
cube die dimensions were 30 mm x 30 mm in cross-section and an effective depth of
compression of 0.38 m. The initial moisture content of dehydrated and sun-cured
chops were 6% and 7% (wb), respectively. A stack of two sieves (instead of five)
was used along with a pan to achieve leaf and stem separation. The nominal
opening sizes of two sieves with square holes were 3.96 and 1.17 mm,
respectively. Leaf and stem fractions were combined later to obtain five
different samples each for sun-cured and dehydrated alfalfa with leaf content
ranging from 0% to 100% by mass in increments of 25%. The chop moisture content
and preheat temperature before compaction was 10% (wb) and 75 degrees C,
respectively. The cube die temperature was maintained at 90+/-5 degrees C. The
mass of chops used for making each cube was 23+/-02 g. A hydraulic press was used
to apply 9.0, 12.0 and 14.0 MPa of pressures through a plunger. After
compression, the plunger was held in place for 10 and 30s, before the compacted
forage was extracted. Empirical equations were fitted to the data relating
specific energy for cube making to pressure, residence time, and leaf content.
DOI: 10.1016/j.biortech.2005.11.032
1981. Br J Nutr. 2006 Jan;95(1):88-98.
A comparison of the effect of forage type and level of feeding on the

digestibility and gastrointestinal mean retention time of dry forages given to
cattle, sheep, ponies and donkeys.
Pearson RA(1), Archibald RF, Muirhead RH.
Author information:
(1)Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush
Veterinary Centre, Roslin, Midlothian EH25 9RG, UK. anne.pearson@ed.ac.uk
Four cattle, sheep, ponies and donkeys were fed dehydrated lucerne, early-cut
hay, later-cut hay or barley straw in a Latin square-based design for four
periods of 35 d. In the first sub-period animals were fed the diets ad libitum
(1-21 d) and in the second sub-period they were fed the same diet restricted to
0.75 of ad libitum intake (days 22-35). Measurements of forage intake, apparent
digestibilities and gastrointestinal mean retention times (MRT) were made in the
last 7 d of each sub-period. Differences between species in voluntary DM intake
(VDMI; g/kg live weight (LW)(0.75) and g/LW) were greatest on the lucerne and
least on barley straw. Cattle VDMI (g/kg LW(0.75)) compared with intake of the
other species was > ponies > sheep > donkeys on lucerne. On barley straw VDMI
(g/kg LW(0.75)) of cattle compared with intake of the other species was = donkey
= ponies > sheep. VDMI of hays were intermediate between the lucerne and straw
forages. Apparent digestibilities of DM, organic matter (OM), neutral-detergent
fibre (NDF) and acid-detergent fibre (ADF) of the lucerne and hays were higher in
the ruminants than in the equids. Effect of feeding level was not significant.
Gastrointestinal MRT was shorter in the equids than in the ruminants. On straw
diets donkeys showed similar apparent digestibilities of feed components to those
of the cattle, whilst apparent digestibility of the straw diet by the ponies was
lowest. Results are discussed in relation to evolutionary differences in feeding
and digestion strategy associated with fore- or hind-gut fermentation in
ruminants and equids.
1982. Vet Parasitol. 2006 Mar 31;136(3-4):363-6. Epub 2006 Jan 19.
Efficacy of short-term feeding of sulla (Hedysarum coronarium) to young goats

against a mixed burden of gastrointestinal nematodes.
Pomroy WE(1), Adlington BA.
Author information:
(1)Institute of Veterinary, Animal and Biomedical Science, Massey University,
Palmerston North, New Zealand. w.pomroy@massey.ac.nz
The aim was to investigate the efficacy of feeding sulla to young goats (<6
months of age) against a naturally acquired infection of gastrointestinal
nematodes. Eleven goats were removed from the field, housed and were randomly
divided into two treatment groups. Group 1 (n = 5) was fed a diet comprising
lucerne-based pellets and aged hay. Group 2 (n = 6) were fed a diet of only sulla
offered ad lib. The sulla contained 2.6% free condensed tannins (CT), 1.8%
protein-bound CT and 0.1% fibre-bound CT. Goats in Gp 2 ate the sulla very
readily from the first day. The goats were fed these diets for 10 days, then
killed and total worm counts obtained. The arithmetic mean worm counts (in
parentheses) for Gp 1 and Gp 2, respectively, were Teladorsagia circumcincta
(5792, 5690), Trichostrongylus colubriformis (272, 262), T. vitrinus (1060, 1804)
and Trichuris spp. (32, 30). There were no significant differences (p > 0.05)
between any of these treatments. Short-term feeding of sulla had no effect on
mixed burdens of gastrointestinal nematodes.
DOI: 10.1016/j.vetpar.2005.11.018
1983. J Biol Chem. 2006 Mar 31;281(13):8843-53. Epub 2006 Jan 17.
Effects of coumarate 3-hydroxylase down-regulation on lignin structure.
Ralph J(1), Akiyama T, Kim H, Lu F, Schatz PF, Marita JM, Ralph SA, Reddy MS,
Chen F, Dixon RA.
Author information:
(1)United States Dairy Forage Research Center, United States Department of
Agriculture-Agricultural Research Service, Madison, Wisconsin 53706, USA.
jralph@wisc.edu
Down-regulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa

massively but predictably increased the proportion of p-hydroxyphenyl (P) units
relative to the normally dominant guaiacyl (G) and syringyl (S) units. Stem
levels of up to approximately 65% P (from wild-type levels of approximately 1%)
resulting from down-regulation of C3H were measured by traditional degradative
analyses as well as two-dimensional 13C-1H correlative NMR methods. Such levels
put these transgenics well beyond the P:G:S compositional bounds of normal
plants; p-hydroxyphenyl levels are reported to reach a maximum of 30% in
gymnosperm severe compression wood zones but are limited to a few percent in
dicots. NMR also revealed structural differences in the interunit linkage
distribution that characterizes a lignin polymer. Lower levels of key beta-aryl
ether units were relatively augmented by higher levels of phenylcoumarans and
resinols. The C3H-deficient alfalfa lignins were devoid of beta-1 coupling
products, highlighting the significant differences in the reaction course for
p-coumaryl alcohol versus the two normally dominant monolignols, coniferyl and
sinapyl alcohols. A larger range of dibenzodioxocin structures was evident in
conjunction with an approximate doubling of their proportion. The nature of each
of the structural units was revealed by long range 13C-1H correlation
experiments. For example, although beta-ethers resulted from the coupling of all
three monolignols with the growing polymer, phenylcoumarans were formed almost
solely from coupling reactions involving p-coumaryl alcohol; they resulted from
both coniferyl and sinapyl alcohol in the wild-type plants. Such structural
differences form a basis for explaining differences in digestibility and pulping
performance of C3H-deficient plants.
DOI: 10.1074/jbc.M511598200
1984. Syst Appl Microbiol. 2006 Nov;29(7):526-38. Epub 2006 Jan 18.
Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar

(bv. medicaginis) of S. meliloti.
Villegas Mdel C(1), Rome S, Mauré L, Domergue O, Gardan L, Bailly X, Cleyet-Marel

JC, Brunel B.
Author information:
(1)Laboratoire des Symbioses Tropicales et Méditerranéennes Cirad Ensa.M Ird
UmII, TA10/J, Campus International de Baillarguet, 34 398 Montpellier Cedex 5,
France.
Sixty-eight new rhizobial isolates were obtained from root-nodules of Medicago

laciniata and from Mediterranean soils in Tunisia and France. All of them were
identified as Sinorhizobium meliloti on the basis of PCR-RFLP analyses of 16S
rDNA and the intergenic spacer sequence between 16S and 23S rDNAs. DNA/DNA
hybridization, phenotypic characterization and 16S rRNA gene sequencing led to
the conclusion that they belong the same taxon. All new isolates shared the
ability to nodulate and fix nitrogen with M. laciniata except 11 of them not
capable of fixing nitrogen with this plant and originating from French soils
containing no efficiently adapted symbionts with M. laciniata. The
nitrogen-fixing rhizobia on M. laciniata differed markedly from the other S.
meliloti or Sinorhizobium medicae isolates and references in their symbiotic
traits such as nifDK RFLP diversity, nodA sequences and nitrogen effectiveness
with tree other different annual Medicago species (M. truncatula, M. polymorpha
and M. sauvagei). Two infrasubspecific (biovar) divisions are therefore proposed
within S. meliloti: bv. medicaginis for Sinorhizobium efficient on M. laciniata
and bv. meliloti for the classically known S. meliloti group represented by the
strains ATCC9930(T) and RCR 2011 efficient on M. sativa.
DOI: 10.1016/j.syapm.2005.12.008
1985. Plant Physiol. 2006 Feb;140(2):693-703. Epub 2006 Jan 11.
Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene

(Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell
division-dependent manner.
Zhiponova MK(1), Pettkó-Szandtner A, Stelkovics E, Neer Z, Bottka S, Krenács T,

Dudits D, Fehér A, Szilák L.
Author information:
(1)Institute of Plant Biology, Biological Research Center, Szeged H-6701,
Hungary.
Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating

the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs)
with preferential protein accumulation at G2/M-phases; however, their exact
functions are still enigmatic. Here we describe the functional characterization
of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in
transgenic plants and cell lines. It is shown that the activity of the analyzed
promoter was characteristic for proliferating meristematic regions in planta and
specific for cells in the G2/M-phases in synchronized cell cultures.
Immunohistochemical analysis of transgenic root sections further confirmed the
correlation of the expression of the CDKB2;1 promoter-linked reporter genes with
the accumulation of the correspondent kinase. It was found that, in addition to
auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both
the reporter and endogenous genes in transgenic leaf explants. Furthermore,
ethylene, known as a wound-response mediator, had a similar effect. The gene
activation in response to wounding or ethephon was faster and occurred without
the induction of cell cycle progression in contrast to the control auxin
treatment. In silico analysis of this promoter indeed revealed the presence of a
set of cis-elements, indicating not only cell cycle- but wound- and
ethylene-dependent regulation of this CDK gene. Based on the presented data, we
discuss the functional significance of the complex regulation of mitosis-specific
CDK genes in plants.
DOI: 10.1104/pp.105.072173
PMCID: PMC1361335
1986. Genetika. 2005 Dec;41(12):1617-23.
[Identification of Sinorhizobium meliloti genes influencing synthesis of surface

polysaccharides and competitiveness].
Onishchuk OP, Sharypova LA, Kurchak ON, Becker A, Simarov BV.
A new approach to isolating mutants with altered composition of capsular

polysaccharides (CPS) and lipopolysaccharides (LPS) in nodule bacteria of alfalfa
Sinorhizobium meliloti based on analysis of their respiratory activity was
proposed. Random Tn5-mob mutants of symbiotically effective strain SKhM1-105 were
tested for slime-production ability and coloration on diagnostic media containing
the indicator of reducing equivalents, triphenyltetrasolium chloride (TTC), the
inhibitor of respiratory activity, 2-methyl-4-chlorphenoxybutyric acid and Congo
Red, the stain for LPS and exopolysaccharides (EPS). Electrophoretic analysis
(SDS-PAGE) of polysaccharides in seven mutants, markedly differing from the
parental strain with respect to their growth on diagnostic media demonstrated
that (1) the production of CPS was either decreased (in mutants T64 and T835) or
blocked (in T71, T94, T124, T134, and T170); (2) the R form of LPS had changed
mobility (in T134); (3) the S form of LPS contained only one component instead of
two (T71). In symbiosis with alfalfa Medicago sativa, all mutants exhibited a
decreased competitive ability for adsorption on roots of host alfalfa plant,
compared to the parental strain. At early stages of symbiosis, mutants had a
tendency toward a decrease in the number of nodules, in comparison with the
parental strain (a statistically significant decrease was observed in mutants T71
and T64). In mutants T64, T71, and T134, nodulation competitiveness was lower
than in the parental strain. Analysis of Tn5-mob tagged sequences of the mutants
revealed homologies with the genes encoding methionine synthase, luciferase-like
monooxygenase, UDP-glucuronic acid epimerase, sensor hystidine kinase,
acetoacetyl-CoA-reductase, oligopeptide uptake ABC transporter, and a
transcription activator.
1987. Plant Biol (Stuttg). 2005 Nov;7(6):601-10.
Contribution of current photosynthates to root respiration of non-nodulated

Medicago sativa: effects of light and nitrogen supply.
Lötscher M(1), Gayler S.
Author information:
(1)Plant Science Department, Technische Universität München, Am Hochanger 1,
85350 Freising-Weihenstephan, Germany. loetscher@wzw.tum.de
The effects of light (PFD) and nitrogen (N) supply on root respiration of new C
(currently assimilated carbon, R(new)) and old C ( R(old)) were analysed in
non-nodulated Medicago sativa. Plants were pre-treated with high/low PFD and
high/low N supply with a regular 16/8 h light/dark cycle. Five to eight weeks
after planting current photosynthates were labelled with (13)C and their
contribution to root respiration was continuously measured during a 24 h
day/night cycle. PFD conditions during labelling were either those of the
pre-treatments (control, 25 or 6 mol m(-2) d(-1)) or, for high PFD plants, 6 mol
m(-2) d(-1) by shortening the photoperiod or reducing irradiance. The fraction of
new C in the respiratory CO2 increased during the light period, but remained
constant in the dark period. In control plants, R(new) contributed 40 % to the
daily root respiration in high PFD/high N conditions. Continuously low PFD
increased (50 %) and low N decreased (26 %) the contribution of R(new). Exposing
plants from high PFD pre-treatments to a short photoperiod or to low PFD
stimulated R(old), indicating mobilisation of reserve C. This stimulation was
more pronounced in plants with high N supply than in those with low N supply.
Comparison with other legumes suggested that R(new) in root respiration was
mainly defined by the ratio between the assimilatory capacity of the shoots and
the maintenance costs of roots with a short-term capacity of buffering
respiratory demand by mobilisation of reserves in situations of fluctuating PFD.
DOI: 10.1055/s-2005-872881
1988. Plant Physiol Biochem. 2005 Dec;43(12):1039-43. Epub 2005 Dec 13.
The de novo designed nutritive protein MB-1Trp does not resist proteolytic
degradation in alfalfa leaves.
Khoudi H(1), Beauregard M.

Author information:
(1)Plant Biology Research Group, Département de chimie-biologie, Université du
Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Que., Canada G9A5H7.
We previously reported on a de novo designed protein "milk bundle-1Trp" (MB-1Trp)

as a source of selected essential amino acids (EAA) for ruminant feeding. Here,
we attempt to express this de novo designed protein in alfalfa. The microbial
version of the gene encoding the protein was modified in order to achieve two
expression strategies in transgenic alfalfa plants. Chimeric MB-1Trp genes alone
or fused to a signal peptide and an endoplasmic reticulum retention sequence were
introduced into alfalfa via Agrobacterium-mediated transformation. Polymerase
chain reaction and reverse transcriptase polymerase chain reaction analysis
performed on individual transgenic lines demonstrated that the MB-1Trp gene was
correctly integrated and transcribed into mRNA. However, under our conditions, it
was impossible to detect MB-1Trp protein expression in any of the transgenic
plants analyzed. In order to assess MB-1Trp stability in alfalfa, Escherichia
coli-derived MB-1Trp was incubated with proteins extracted from leaves of a
non-transgenic plant. This study revealed a high susceptibility of mature MB-1Trp
to alfalfa proteases, which may have contributed to its lack of accumulation.
DOI: 10.1016/j.plaphy.2005.10.007
1989. Microbiology. 2006 Jan;152(Pt 1):223-32.
A cysteine/methionine auxotroph of the opportunistic fungus Aspergillus flavus is

associated with host-range restriction: a model for emerging diseases.
Scully LR(1), Bidochka MJ.
Author information:
(1)Department of Biological Sciences, 500 Glenridge Avenue, Brock University, St
Catharines, Ontario, Canada L2S 3A1.
The evolution of host specialization in pathogens is a topic of considerable

interest, particularly since it can represent a decisive step in the emergence of
infectious diseases. Aspergillus flavus is an opportunistic fungus capable of
infecting a wide variety of hosts, including plants, insects and mammals,
although with low virulence. Here the derivation of an A. flavus strain that
exhibits severe host restriction is reported. This strain exhibited a severe
diminution or a complete lack of conidial production on a variety of standard
agar media and on various plant species. However, it retained its ability to
infect insects from various orders and to re-emerge from and adequately conidiate
on the insect cadavers as a culmination of the pathogenic life cycle. This
strain, demonstrating insect-dependent conidiation, was discovered to be a
cysteine/methionine auxotroph due to an inability to reduce sulfate to sulfite.
However, other A. flavus auxotrophs tested for plant and insect host range failed
to show insect-dependent conidiation. An association between this specific
auxotroph and a decreased host range is shown, emphasizing the role of nutrition
in the host-pathogen relationship with respect to host restriction and evolution
towards obligate pathogenesis.
DOI: 10.1099/mic.0.28452-0
1990. Vaccine. 2006 Mar 20;24(13):2387-94. Epub 2005 Dec 9.

F4 (K88) fimbrial adhesin FaeG expressed in alfalfa reduces F4+ enterotoxigenic
Escherichia coli excretion in weaned piglets.
Joensuu JJ(1), Verdonck F, Ehrström A, Peltola M, Siljander-Rasi H, Nuutila AM,

Oksman-Caldentey KM, Teeri TH, Cox E, Goddeeris BM, Niklander-Teeri V.
Author information:
(1)Department of Applied Biology, P.O. Box 27, FIN-00014 University of Helsinki,
Finland. jussi.joensuu@helsinki.fi
Transgenic plants are attractive bioreactors to large-scale production of

recombinant proteins because of their relatively low cost. This study reports for
the first time the use of transgenic plants to reduce enterotoxigenic Escherichia
coli (ETEC) excretion in its natural host species. The DNA sequence encoding the
major subunit and adhesin FaeG of F4+ ETEC was transformed into edible alfalfa
plants. Targeting of FaeG production to chloroplasts led to FaeG levels of up to
1% of the total soluble protein fraction of the transgenic alfalfa. Recombinant
plant-produced FaeG (pFaeG) remained stable for 2 years when the plant material
was dried and stored at room temperature. Intragastric immunization of piglets
with pFaeG induced a weak F4-specific humoral response. Co-administration of
pFaeG and the mucosal adjuvant cholera toxin (CT) enhanced the immune response
against FaeG, reflected a better induction of an F4-specific immune response. In
addition, the intragastric co-administration of CT with pFaeG significantly
reduced F4+ E. coli excretion following F4+ ETEC challenge as compared with pigs
that had received nontransgenic plant material. In conclusion, transgenic plants
producing the FaeG subunit protein could be used for production and delivery of
oral vaccines against F4+ ETEC infections.
DOI: 10.1016/j.vaccine.2005.11.056
1991. J Dairy Sci. 2006 Jan;89(1):283-93.
Milk from forage as affected by carbohydrate source and degradability with

alfalfa silage-based diets.
Charbonneau E(1), Chouinard PY, Allard G, Lapierre H, Pellerin D.
Author information:
(1)Département des Sciences Animales, Université Laval, QC, G1K 7P4, Canada.
Milk from forage (MF) is an estimation of the milk produced solely from forage
intake. It is calculated by subtracting milk production theoretically allowed by
concentrates from total milk production, assuming that maintenance requirements
are covered by the forage portion of the diet. Eight multiparous Holstein cows in
early lactation were used in a replicated 4 x 4 Latin square design to evaluate
the impact on MF of different sources of carbohydrate with forage that was high
in RDP. Diets were alfalfa-based total mixed rations that were formulated to
provide similar concentrations of NEL and CP while differing in rumen
degradability of concentrate carbohydrates. Treatments were 1) cracked corn
(control), 2) ground corn (GC), 3) GC plus wheat starch (GC+S), and 4) GC plus
dried whey permeate (GC+W). The GC and the GC+S treatments increased MF as
calculated on a protein basis (14.8 vs. 10.5 kg) and increased average MF
production (8.6 vs. 5.5 kg) compared with the control. Protein of forage was used
more efficiently with GC and with GC+S, as shown by the lower differences between
allowable MF, which estimates the potential for milk production from forage, and
MF on a protein basis for these 2 treatments when compared with the control.
Compared with the control, DMI increased with GC and GC+S; GC+W yielded the
highest DMI. Milk production with GC+W (35.8 kg/d) was lower than with GC and
GC+S (37.5 kg/d) but was higher than the control (34.0 kg/d). Milk fat
concentration was higher with GC+W and lower with GC+S; GC and the control had
intermediate values. Milk urea was higher with the control diet compared with the
other 3 treatments. Results emphasize the advantage of using concentrates of
higher degradability in the rumen to improve MF and milk production when feeding
silage with high rumen-degradable protein.
DOI: 10.3168/jds.S0022-0302(06)72093-8
1992. J Dairy Sci. 2006 Jan;89(1):174-87.
Dietary forage and nonfiber carbohydrate contents influence B-vitamin intake,

duodenal flow, and apparent ruminal synthesis in lactating dairy cows.
Schwab EC(1), Schwab CG, Shaver RD, Girard CL, Putnam DE, Whitehouse NL.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison, 53706, USA.
The objective of this experiment was to quantify intakes, duodenal flows, and
ruminal apparent synthesis (AS) of B-vitamins in lactating dairy cows fed diets
varying in forage and nonfiber carbohydrate (NFC) contents. Eight (4 primiparous
and 4 multiparous) ruminally and duodenally cannulated Holstein cows were
assigned to 4 dietary treatments in a replicated 21-d period, 4 x 4 Latin square
design with a 2 x 2 factorial treatment arrangement. Diets, fed as TMR, contained
(DM basis) 2 levels of forage (35 and 60%) and 2 levels of NFC (30 and 40%). The
forage portion of the diets contained 50% corn silage, 33% alfalfa hay, and 17%
grass hay. Soybean hulls and beet pulp (2:1) and corn meal and ground barley
(2:1) were included to achieve desired NFC concentrations. No supplemental
B-vitamins were fed. B-vitamin AS was calculated as the amount of a specific
B-vitamin flowing to the duodenum minus its daily orts-corrected intake. Dry
matter and organic matter intakes were higher for cows fed the 35% forage diets
and the 40% NFC diets. Increasing dietary forage content decreased ruminal AS of
pyridoxine, folic acid, and B12. Increasing dietary NFC content increased ruminal
AS of nicotinic acid, nicotinamide, niacin, pyridoxal, B6, and folic acid but
decreased AS of B12. Across diets, amounts of B-vitamins synthesized were highest
for niacin, followed by riboflavin, B12, thiamin, B6, and folic acid. Biotin AS
values were negative for all diets, suggesting either no ruminal synthesis or
that destruction by ruminal microflora was greater than synthesis. B-vitamin
intake, duodenal flow, and ruminal synthesis are influenced by dietary forage and
NFC contents.
DOI: 10.3168/jds.S0022-0302(06)72082-3
1993. Mol Plant Microbe Interact. 2005 Nov;18(11):1235-42.
Characterization of the binding of diarrheagenic strains of E. coli to plant

surfaces and the role of curli in the interaction of the bacteria with alfalfa
sprouts.
Jeter C(1), Matthysse AG.
Author information:
(1)Department of Biology, University of North Carolina, Chapel Hill 27599-3280,
USA.
Diarrheagenic Escherichia coli were able to bind to plant surfaces, including

alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana
seedlings incubated in water. The characteristics of the binding differed with
the bacterial strain examined. Laboratory K12 strains of E. coli failed to show
significant binding to any of the plant surfaces examined, suggesting that some
of the genes present and expressed in pathogenic strains and absent or
unexpressed in K12 strains may be required for binding to plants. When a plasmid
carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid
carrying the operons that encode the synthesis of curli (csgA-G) was introduced
into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA
mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction
in their ability to bind to sprouts. Thus, the production of curli appears to be
sufficient to allow K12 strains to bind, but curli are not necessary for the
binding of pathogenic strains, suggesting that pathogenic strains may have more
than one mechanism for binding to plant surfaces.
DOI: 10.1094/MPMI-18-1235

Dec
9.
Organic and mineral fertilization, respectively, increase and decrease the

development of external mycelium of arbuscular mycorrhizal fungi in a long-term
field experiment.
Gryndler M(1), Larsen J(2), Hršelová H(3), Řezáčová V(3), Gryndlerová H(3), Kubát
J(4).
Author information:
(1)Institute of Microbiology CAS, Division of Ecology, Vídeňská 1083, CZ 142 20,
Prague 4, Czech Republic. gryndler@biomed.cas.cz.
(2)Danish Institute of Agricultural Sciences, Department of Integrated Pest
Management, Research Centre Flakkebjerg, Flakkebjerg, 4200, Slagelse, Denmark.
(3)Institute of Microbiology CAS, Division of Ecology, Vídeňská 1083, CZ 142 20,
Prague 4, Czech Republic.
(4)Research Institute for Crop Production, Drnovská 507, CZ 161 06, Prague 6,
Czech Republic.
Effects of long-term mineral fertilization and manuring on the biomass of

arbuscular mycorrhizal fungi (AMF) were studied in a field experiment. Mineral
fertilization reduced the growth of AMF, as estimated using both measurements of
hyphal length and the signature fatty acid 16:1omega5, whereas manuring alone
increased the growth of AMF. The results of AMF root colonization followed the
same pattern as AMF hyphal length in soil samples, but not AMF spore densities,
which increased with increasing mineral and organic fertilization. AMF spore
counts and concentration of 16:1omega5 in soil did not correlate positively,
suggesting that a significant portion of spores found in soil samples was dead.
AMF hyphal length was not correlated with whole cell fatty acid (WCFA)
18:2omega6,9 levels, a biomarker of saprotrophic fungi, indicating that visual
measurements of the AMF mycelium were not distorted by erroneous involvement of
hyphae of saprotrophs. Our observations indicate that the measurement of WCFAs in
soil is a useful research tool for providing information in the characterization
of soil microflora.
DOI: 10.1007/s00572-005-0027-4
1995. Microbiol Res. 2006;161(1):1-8. Epub 2005 Jun 2.
Zoospore encystment and pathogenicity of Phytophthora and Pythium species on

plant roots.
Raftoyannis Y(1), Dick MW.
Author information:
(1)TEI Lamias, Department of Forestry, Karpenisi 36100, Greece. rafto@teilam.gr
Seven plant species (lucerne, maize, oat, sugarbeet, sorghum, tomato, wheat) and
12 Pythium and Phytophthora species were used in a comparative study designed to
investigate the effects of plant and oomycete inter-specific variation on
zoospore encystment density and pathogenicity. Zoospores showed differential
encystment behaviour and they encysted more on dicotyledonous than on
monocotyledonous plants. Pythium aphanidermatum, P. deliense, and Phytophthora
nicotianae were the most aggressive species. Sugarbeet was the most severely
attacked plant species followed by tomato while oat plants were relatively
unaffected. The relationship between zoospore encystment on roots and disease
severity depended on the oomycete-plant combination. Correlation analysis between
zoospore encystment density and disease severity indicated low and no significant
levels (p.05) of association for most plant-oomycete combinations.
DOI: 10.1016/j.micres.2005.04.003
Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial

cells, and plastic is mediated by a variety of surface structures.
Torres AG(1), Jeter C, Langley W, Matthysse AG.
Author information:
(1)Department of Microbiology and Immunology, Department of Pathology, and Sealy
Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX
77555-1070, USA. altorres@utmb.edu
Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts,

has been implicated in food poisoning and outbreaks of disease in the United
States. Adhesion to cell surfaces is a key component for bacterial establishment
and colonization on many types of surfaces. Several E. coli O157:H7 surface
proteins are thought to be important for adhesion and/or biofilm formation.
Therefore, we examined whether mutations in several genes encoding potential
adhesins and regulators of adherence have an effect on bacterial binding to
plants and also examined the role of these genes during adhesion to Caco-2 cells
and during biofilm formation on plastic in vitro. The genes tested included those
encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA,
yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD,
and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci)
into an E. coli K-12 strain markedly increased its ability to bind to alfalfa
sprouts and seed coats. The addition of more than one of these genes did not show
an additive effect. In contrast, deletion of one or more of these genes in a
strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the
absence of the ompA gene had a significant effect on binding, and the
plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In
contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only
slightly affected in adhesion to Caco-2 cells and during biofilm formation. These
findings suggest that some adhesins alone are sufficient to promote binding to
alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing
it to compensate for the loss of one or more of these systems. Binding to the
three types of surfaces appeared to be mediated by overlapping but distinct sets
of genes. The only gene which appeared to be irreplaceable for binding to plant
surfaces was ompA.
DOI: 10.1128/AEM.71.12.8008-8015.2005
PMCID: PMC1317338
Effect of immersion solutions containing enterocin AS-48 on Listeria

monocytogenes in vegetable foods.
Molinos AC(1), Abriouel H, Ben Omar N, Valdivia E, López RL, Maqueda M, Cañamero
MM, Gálvez A.
Author information:
(1)Area de Microbiología, Departamento de Ciencias de la Salud, Facultad de
Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas s/n,
23071-Jaén, Spain. agalvez@ujaen.es.
The effect of immersion solutions containing enterocin AS-48 alone or in

combination with chemical preservatives on survival and proliferation of Listeria
monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and
green asparagus was tested. Immersion treatments (5 min at room temperature) with
AS-48 solutions (25 microg/ml) reduced listeria counts of artificially
contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g
compared to a control immersion treatment in distilled water. The same
bacteriocin immersion treatment applied on green asparagus had a very limited
effect. During storage of vegetable samples treated with immersion solutions of
12.5 and 25 microg of AS-48/ml, viable listeria counts were reduced below
detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15
degrees C, as well as green asparagus at 15 degrees C. Only a limited inhibition
of listeria proliferation was detected during storage of bacteriocin-treated
alfalfa sprouts and green asparagus at 22 degrees C. Treatment with solutions
containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium
nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate,
n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester,
hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced
viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log
CFU/g) upon application of the immersion treatment and/or further storage for 24
h, depending of the chemical preservative concentration. Significant increases of
antimicrobial activity were also detected for AS-48 plus potassium permanganate
and in some combinations with acetic acid, citric acid, sodium propionate, and
potassium sorbate.
DOI: 10.1128/AEM.71.12.7781-7787.2005
PMCID: PMC1317399
1998. PLoS Biol. 2006 Jan;4(1):e1.
Functional diversity of plant-pollinator interaction webs enhances the

persistence of plant communities.
Fontaine C(1), Dajoz I, Meriguet J, Loreau M.
Author information:
(1)UMR 7618, Biogéochimie et Ecologie des Milieux Continentaux (BIOEMCO), Ecole
Normale Supérieure, Paris, France. colin.fontaine@biologie.ens.fr
Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm

species. Thus, pollinators provide an essential ecosystem service to humankind.
However, the impact of human-induced biodiversity loss on the functioning of
plant-pollinator interactions has not been tested experimentally. To understand
how plant communities respond to diversity changes in their pollinating fauna, we
manipulated the functional diversity of both plants and pollinators under natural
conditions. Increasing the functional diversity of both plants and pollinators
led to the recruitment of more diverse plant communities. After two years the
plant communities pollinated by the most functionally diverse pollinator
assemblage contained about 50% more plant species than did plant communities
pollinated by less-diverse pollinator assemblages. Moreover, the positive effect
of functional diversity was explained by a complementarity between functional
groups of pollinators and plants. Thus, the functional diversity of pollination
networks may be critical to ecosystem sustainability.
DOI: 10.1371/journal.pbio.0040001
PMCID: PMC1310649
1999. J Bacteriol. 2005 Dec;187(24):8427-36.
L-Canavanine made by Medicago sativa interferes with quorum sensing in

Keshavan ND(1), Chowdhary PK, Haines DC, González JE.
Author information:
(1)Department of Molecular and Cell Biology, University of Texas at Dallas, FO
3.1, Richardson, TX 75083-0688, USA.
Sinorhizobium meliloti is a gram-negative soil bacterium, capable of establishing

a nitrogen-fixing symbiosis with its legume host, alfalfa (Medicago sativa).
Quorum sensing plays a crucial role in this symbiosis, where it influences the
nodulation process and the synthesis of the symbiotically important
exopolysaccharide II (EPS II). S. meliloti has three quorum-sensing systems (Sin,
Tra, and Mel) that use N-acyl homoserine lactones as their quorum-sensing signal
molecule. Increasing evidence indicates that certain eukaryotic hosts involved in
symbiotic or pathogenic relationships with gram-negative bacteria produce
quorum-sensing-interfering (QSI) compounds that can cross-communicate with the
bacterial quorum-sensing system. Our studies of alfalfa seed exudates suggested
the presence of multiple signal molecules capable of interfering with
quorum-sensing-regulated gene expression in different bacterial strains. In this
work, we choose one of these QSI molecules (SWI) for further characterization.
SWI inhibited violacein production, a phenotype that is regulated by quorum
sensing in Chromobacterium violaceum. In addition, this signal molecule also
inhibits the expression of the S. meliloti exp genes, responsible for the
production of EPS II, a quorum-sensing-regulated phenotype. We identified this
molecule as l-canavanine, an arginine analog, produced in large quantities by
alfalfa and other legumes.
DOI: 10.1128/JB.187.24.8427-8436.2005
PMCID: PMC1317012
2000. J Food Prot. 2005 Nov;68(11):2256-63.
A simple method for the direct detection of Salmonella and Escherichia coli
O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR.
Johnston LM(1), Elhanafi D, Drake M, Jaykus LA.
Author information:
(1)Department of Food Science, College of Life Science and Agriculture, North
Carolina State University, Raleigh, North Carolina 27695-7624, USA.
The U.S. Food and Drug Administration recognizes that raw seed sprouts are an
important cause of foodborne disease and is now recommending that either spent
irrigation water or final product be screened for Salmonella and Escherichia coli
O157:H7 as a means of assuring the safety of product intended for consumption. In
an effort to streamline such testing efforts, a simple method to preconcentrate
pathogens from sprouts and spent irrigation water was investigated to facilitate
the direct (without prior cultural enrichment) detection of pathogens using the
PCR technique. Alfalfa sprouts and spent irrigation water were seeded with
Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106
CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then
centrifuged at high speed to sediment the total bacterial population. The
precipitate was processed for DNA isolation, PCR amplification, and amplicon
confirmation by Southern hybridization. Mean pathogen recoveries after
centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using
primers targeting the invA gene for Salmonella Typhimurium and the stx genes of
E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at
seeding concentrations as low as 10 CFU/g. PCR detection limits for both
pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100
CFU/liter of water. Because spent irrigation water is constitutionally simple, it
is particularly well suited for bacterial concentration by simple centrifugation
steps. In this study, progress was made toward development of a rapid,
inexpensive, and sensitive method for the detection of sprout-associated
pathogens that is relevant to current industrial practices and needs.
2001. J Dairy Sci. 2005 Dec;88(12):4342-55.
Processing whole cottonseed moderates fatty acid metabolism and improves

performance by dairy cows.
Reveneau C(1), Ribeiro CV, Eastridge ML, St-Pierre NR, Firkins JL.
Author information:
(1)Department of Animal Sciences, The Ohio State University, Columbus, 43210,
USA.
Pelleting cottonseed (CS) improves handling characteristics. Our objectives were

to determine whether increasing the particle size of the CS pellet or dilution of
a smaller pellet with delinted CS would limit the rate of CS oil release to
optimize digestibility of fatty acids (FA) and fiber while maintaining milk fat
production. In a 5 x 5 Latin square design with 3-wk periods, 5 rumen-cannulated
cows were fed 1) control with CS hulls (CSH) and CS meal plus tallow and Ca soaps
of FA, 2) whole CS (WCS), 3) small CS pellets (SP; 0.44-cm die diameter), 4)
larger CS pellets (LP; 0.52-cm die diameter), or 5) a blend of 1/2 SP plus 1/2
partially delinted CS (SPD). Diets contained 39.6% concentrate, 14.4% CS, and 46%
forage (40:60, alfalfa hay:corn silage) on a DM basis and were balanced to have
similar concentrations of CS protein, CS fiber, and total fat. In a production
trial, dietary treatments were 1) WCS control, 2) LP, 3) SPD, and 4) SPD fed at
90%. Sixty cows averaging 105 d in milk were fed the WCS diet for 2 wk and then
assigned to one of the 4 diets for 12 wk. Total tract digestibility of NDF was
unaffected, but N digestibility was lower for SPD than for other treatments.
Fatty acid digestibility was higher for SP and LP (82.6 and 82.3%) than for CSH
or SPD treatments (78.8 and 75.3%), and WCS was intermediate (81.1%). The
trans-11 C18:1 from cows fed SP and LP (6.58 and 6.24% of total milk FA) was
greater than that from cows fed CSH, WCS, and SPD (3.23, 3.79, and 3.97%). The
trans-10 C18:1 in milk fat from SP and LP (0.508 and 0.511%) was higher than that
in WCS and SPD diets (0.316 and 0.295%); CSH was intermediate (0.429%). Using
passage rates estimated from the NRC, disappearance of total FA in situ was
estimated to be 17.7, 44.2, 46.6, and 35.0% for WCS, SP, LP, and SPD,
respectively. In the production trial, a diet x week interaction was explained by
a trend for progressively greater milk production for SPD and SPD90 than for WCS
or LP. Milk fat was lower for LP (2.74%) and SPD90 (2.85%) than for WCS or SPD
(3.07 and 3.08%). The fat yield was lower for LP than for SPD (1.09 and 1.30
kg/d); WCS and SPD90 were intermediate (1.23 and 1.21 kg/d). Although having a
lower FA digestibility, SPD appeared to minimize negative effects of free oil
from SP in the rumen, explaining higher DMI and milk production compared with WCS
or LP.
DOI: 10.3168/jds.S0022-0302(05)73121-0
2002. Planta. 2006 Apr;223(5):1041-6. Epub 2005 Nov 12.
Class-1 hemoglobin and antioxidant metabolism in alfalfa roots.
Igamberdiev AU(1), Stoimenova M, Seregélyes C, Hill RD.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2,
Canada.
In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked
to antioxidant metabolism and affects the cellular redox level. The influence of
Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H(2)O(2)
and ascorbate was investigated in alfalfa root cultures transformed to
over-express (Hb+) or down-regulate (Hb-) class-1 Hb. Hb+ lines had substantially
increased ascorbate levels as well as elevated monodehydroascorbate reductase and
ascorbate peroxidase activities. Hb- lines showed significant increases in
dehydroascorbate reductase and glutathione reductase activities. The observed
changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both
at high (40 kPa) and low (3 kPa) O(2) pressures. Hb- lines had significantly
reduced levels of the NO- and H(2)O(2)-sensitive enzyme, aconitase, as compared
to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb-
lines, as treatment of plant extracts with the NO-donor DEANO also affected
aconitase activity. The H(2)O(2) levels were not significantly different amongst
the lines and showed no variation with change in oxygen partial pressure. In
conclusion, the expression of class-1 Hb improves the antioxidant status through
increased ascorbate levels and increased activity of enzymes involved in H(2)O(2)
removal.
DOI: 10.1007/s00425-005-0145-4
2003. BMC Genomics. 2005 Nov 10;6:158.
Large-scale genetic variation of the symbiosis-required megaplasmid pSymA

revealed by comparative genomic analysis of Sinorhizobium meliloti natural
strains.
Giuntini E(1), Mengoni A, De Filippo C, Cavalieri D, Aubin-Horth N, Landry CR,

Becker A, Bazzicalupo M.
Author information:
(1)Dipartimento di Biologia Animale e Genetica, Università di Firenze, via Romana
17, I-50125 Firenze, Italy.
BACKGROUND: Sinorhizobium meliloti is a soil bacterium that forms nitrogen-fixing

nodules on the roots of leguminous plants such as alfalfa (Medicago sativa). This
species occupies different ecological niches, being present as a free-living soil
bacterium and as a symbiont of plant root nodules. The genome of the type strain
Rm 1021 contains one chromosome and two megaplasmids for a total genome size of 6
Mb. We applied comparative genomic hybridisation (CGH) on an oligonucleotide
microarrays to estimate genetic variation at the genomic level in four natural
strains, two isolated from Italian agricultural soil and two from desert soil in
the Aral Sea region.
RESULTS: From 4.6 to 5.7 percent of the genes showed a pattern of hybridisation
concordant with deletion, nucleotide divergence or ORF duplication when compared
to the type strain Rm 1021. A large number of these polymorphisms were confirmed
by sequencing and Southern blot. A statistically significant fraction of these
variable genes was found on the pSymA megaplasmid and grouped in clusters. These
variable genes were found to be mainly transposases or genes with unknown
function.
CONCLUSION: The obtained results allow to conclude that the symbiosis-required
megaplasmid pSymA can be considered the major hot-spot for intra-specific
differentiation in S. meliloti.
DOI: 10.1186/1471-2164-6-158
PMCID: PMC1298293
Rhizobium daejeonense sp. nov. isolated from a cyanide treatment bioreactor.
Quan ZX(1), Bae HS, Baek JH, Chen WF, Im WT, Lee ST.
Author information:
(1)Department of Biological Sciences, Korea Advanced Institute of Science and
Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Korea.
A polyphasic study was carried out to determine the taxonomic position of two
aerobic, cyanide-degrading bacterial strains, designated L61T and L22, which had
been isolated from a bioreactor for the treatment of nickel-complexed cyanide.
The two isolates exhibited almost identical taxonomic characteristics.
Phylogenetic analysis inferred from comparative 16S rRNA gene sequences indicated
that the isolates fall in a sublineage of the genus Rhizobium comprising the type
strains of Rhizobium giardinii, Rhizobium radiobacter, Rhizobium rubi, Rhizobium
larrymoorei, Rhizobium vitis, Rhizobium undicola, Rhizobium loessense, Rhizobium
galegae and Rhizobium huautlense. Cells of the two isolates are Gram-negative,
aerobic, motile and non-spore-forming rods (0.6-0.7x1.1-1.3 microm), with
peritrichous flagella. The DNA G+C content is 60.1-60.9 mol%. Cellular fatty
acids are C(16 : 0) (2.2-3.3 %), C(18 : 0) (2.1-3.2 %), C(19 : 0) cyclo omega8c
(9.9-16.8 %), C(20 : 3)omega6,9,12c (2.7-3.3 %), summed feature 3 (7.2-7.7 %) and
summed feature 7 (67.8-73.7 %). The strains formed nodules on a legume plant,
Medicago sativa. A nifH gene encoding denitrogenase reductase, the key component
of the nitrogenase enzyme complex, was detected in L61T by PCR amplification by
using a nifH-specific primer system. Strains L61T and L22 were distinguished from
the type strains of recognized Rhizobium species in the same sublineage based on
low DNA-DNA hybridization values (2-4 %) and/or a 16S rRNA gene sequence
similarity value of less than 96 %. Moreover, some phenotypic properties with
respect to substrate utilization as a carbon or nitrogen source, antibiotic
resistance and growth conditions could be used to discriminate L61T and L22 from
Rhizobium species in the same sublineage. Based on the results obtained in this
study, L61T and L22 are considered to be representatives of a novel species of
Rhizobium, for which the name Rhizobium daejeonense sp. nov. is proposed. The
type strain is L61T (=KCTC 12121T=IAM 15042T=CCBAU 10050T).
DOI: 10.1099/ijs.0.63667-0
2005. Br J Nutr. 2005 Nov;94(5):771-82.
The effect of enzyme treatment on the in vitro fermentation of lucerne incubated

with equine faecal inocula.
Murray JA(1), Longland AC, Moore-Colyer MJ, Dunnett C.
Author information:
(1)Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth
SY23 3EB, UK. Jo-Anne.Murray@ed.ac.uk
A series of experiments was conducted to determine the effects of a fibrolytic

enzyme preparation (enzyme 1; E1) on the in vitro fermentation of lucerne
incubated with equine faecal inocula. In experiment 1, high-temperature-dried
(HT) lucerne was treated with five levels of E1 (0 to 2.4 ml/g DM) and incubated
at 50 degrees C for 20 h. Samples then received a simulated foregut digestion
(SFD) treatment before DM and NSP analysis. In experiment 2, HT lucerne was
treated with the same enzyme levels used in experiment 1. Samples were then split
into two groups; plus or minus an SFD treatment before in vitro fermentation
using an equine faecal inoculum. In experiment 3, fresh and wilted lucerne were
treated with the same levels of E1 as experiments 1 and 2, incubated at 50
degrees C for 20 h, then fermented in vitro. For experiment 4, fresh and wilted
lucerne were treated with low levels (0 to 0.008 ml/g DM) of E1 before
fermentation. E1 significantly (P<0.05) enhanced DM and NSP losses from HT
lucerne following SFD treatment compared with the control. High levels of E1
significantly (P<0.05) enhanced the rate, but not extent, of fermentation of HT,
wilted and fresh lucerne; however, low levels of E1 were ineffective. At higher
application levels, E1 appears to have considerable potential to enhance the
nutritive value of lucerne for horses. Information on the fermentation kinetics
of the substrates was valuable; all end-point measurements showed no effect of
enzyme treatment.
2006. Mol Genet Genomics. 2005 Dec;274(6):644-57. Epub 2005 Nov 5.
Significant microsynteny with new evolutionary highlights is detected between

Arabidopsis and legume model plants despite the lack of macrosynteny.
Kevei Z(1), Seres A, Kereszt A, Kaló P, Kiss P, Tóth G, Endre G, Kiss GB.
Author information:
Sciences, P. O. Box 521, 6701, Szeged, Hungary.
The increased amount of data produced by large genome sequencing projects allows
scientists to carry out important syntenic studies to a great extent. Detailed
genetic maps and entirely or partially sequenced genomes are compared, and macro-
and microsyntenic relations can be determined for different species. In our
study, the syntenic relationships between key legume plants and two model plants,
Arabidopsis thaliana and Populus trichocarpa were investigated. The comparison of
the map position of 172 gene-based Medicago sativa markers to the organization of
homologous A. thaliana genes could not identify any sign of macrosynteny between
the two genomes. A 276 kb long section of chromosome 5 of the model legume
Medicago truncatula was used to investigate potential microsynteny with the other
legume Lotus japonicus, as well as with Arabidopsis and Populus. Besides the
overall correlation found between the legume plants, the comparison revealed
several microsyntenic regions in the two more distant plants with significant
resemblance. Despite the large phylogenetic distance, clear microsyntenic regions
between Medicago and Arabidopsis or Populus were detected unraveling new
intragenomic evolutionary relations in Arabidopsis.
DOI: 10.1007/s00438-005-0057-9
2007. Yakugaku Zasshi. 2005 Nov;125(11):895-8.
Introduction of grapes and alfalfa into China: a reflection on the role of Zhang
Qian.
Uchibayashi M(1).
Author information:
(1)Takeda Science Foundation, Osaka, Japan.
There is a prevailing legend that grapes and alfalfa were introduced to China
from West Asia by Zhang Qian in 126 BC. This paper intends to show the legend to
be a misconception. The three crucial documents were thoroughly examined, and the
author believes he can demonstrate that it was a later traveler who actually
brought these two plants to China following the trade route pioneered by Zhang
Qian.

2008. Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16573-8. Epub 2005 Nov 1.
Targeted down-regulation of cytochrome P450 enzymes for forage quality

improvement in alfalfa (Medicago sativa L.).
Reddy MS(1), Chen F, Shadle G, Jackson L, Aljoe H, Dixon RA.
Author information:
(1)Plant Biology Division and Agriculture Division, Samuel Roberts Noble
Foundation, Ardmore, OK 73401, USA.
Improving the digestibility of forages provides a means to enhance animal

performance and protect the environment against excessive animal waste. Increased
lignin content during maturity, and corresponding changes in lignin composition,
correlate with decreased digestibility of forages. These relationships have yet
to be investigated in isogenic systems. By targeting three specific cytochrome
P450 enzymes of the lignin pathway for antisense down-regulation, we generated
transgenic alfalfa lines with a range of differences in lignin content and
composition. There was a strong negative relationship between lignin content and
rumen digestibility, but no relationship between lignin composition and
digestibility, in these transgenic lines. Models for genetic manipulation of
forage digestibility based on the changes in lignin composition that increase
paper-pulping efficiency in trees are therefore invalid. Down-regulation of
4-coumarate 3-hydroxylase provided the largest improvements in digestibility yet
seen in a forage crop.
DOI: 10.1073/pnas.0505749102
PMCID: PMC1283808
2009. Ann Bot. 2006 Jan;97(1):109-20. Epub 2005 Oct 31.
Nitrogen reserves, spring regrowth and winter survival of field-grown alfalfa

(Medicago sativa) defoliated in the autumn.
Dhont C(1), Castonguay Y, Nadeau P, Bélanger G, Drapeau R, Laberge S, Avice JC,

Chalifour FP.
Author information:
(1)Département de Phytologie, Université Laval, Québec, QC, Canada G1K 7P4.
AIMS: The objective of the study was to characterize variations in proline,

arginine, histidine, vegetative storage proteins, and cold-inducible gene
expression in overwintering roots of field-grown alfalfa, in response to autumn
defoliation, and in relation to spring regrowth and winter survival.
METHODS: Field trials, established in 1996 in eastern Canada, consisted of two
alfalfa cultivars ('AC Caribou' and 'WL 225') defoliated in 1997 and 1998 either
only twice during the summer or three times with the third defoliation taken 400,
500 or 600 growing degree days (basis 5 degrees C) after the second summer
defoliation.
KEY RESULTS: The root accumulation of proline, arginine, histidine and soluble
proteins of 32, 19 and 15 kDa, characterized as alfalfa vegetative storage
proteins, was reduced the following spring by an early autumn defoliation at 400
or 500 growing degree days in both cultivars; the 600-growing-degree-days
defoliation treatment had less or no effect. Transcript levels of the
cold-inducible gene msaCIA, encoding a glycine-rich protein, were markedly
reduced by autumn defoliation in 'WL 225', but remained unaffected in the more
winter-hardy cultivar 'AC Caribou'. The expression of another cold-inducible
gene, the dehydrin homologue msaCIG, was not consistently affected by autumn
defoliation. Principal component analyses, including components of root organic
reserves at the onset of winter, along with yield and plant density in the
following spring, revealed that (a) amino acids and soluble proteins are
positively related to the vigour of spring regrowth but poorly related to winter
survival and (b) winter survival, as indicated by plant density in the spring, is
associated with higher concentrations of cryoprotective sugars in alfalfa roots
the previous autumn.
CONCLUSIONS: An untimely autumn defoliation of alfalfa reduces root accumulation
of specific N reserves such as proline, arginine, histidine and vegetative
storage proteins that are positively related to the vigour of spring regrowth but
poorly related to winter survival.
DOI: 10.1093/aob/mcj006
PMCID: PMC2000764
2010. Vet Res Commun. 2005 Aug;29 Suppl 2:363-5.
Effect of plant maturity and germplasm on in situ rumen degradability and rate of
passage of alfalfa hay.
Dorigo M(1), Gottardo F, Berzaghi P, Cozzi G.
Author information:
(1)Department of Animal Science, University of Padua, 35020, Legnaro, PD, Italy.
martina.dorigo@unipd.it
DOI: 10.1007/s11259-005-0082-5
2011. Plant Physiol. 2005 Nov;139(3):1338-49. Epub 2005 Oct 21.
Plant-derived transfer DNAs.
Rommens CM(1), Bougri O, Yan H, Humara JM, Owen J, Swords K, Ye J.
Author information:
(1)J.R. Simplot Company, Simplot Plant Sciences, Boise, IA 83706, USA.
crommens@simplot.com
Erratum in
Plant Physiol. 2013 Apr;161(4):2182.
The transfer of DNA from Agrobacterium to plant cell nuclei is initiated by a

cleavage reaction within the 25-bp right border of Ti plasmids. In an effort to
develop all-native DNA transformation vectors, 50 putative right border
alternatives were identified in both plant expressed sequence tags and genomic
DNA. Efficacy tests in a tobacco (Nicotiana tabacum) model system demonstrated
that 14 of these elements displayed at least 50% of the activity of conventional
Agrobacterium transfer DNA borders. Four of the most effective plant-derived
right border alternatives were found to be associated with intron-exon junctions.
Additional elements were embedded within introns, exons, untranslated trailers,
and intergenic DNA. Based on the identification of a single right border
alternative in Arabidopsis and three in rice (Oryza sativa), the occurrence of
this motif was estimated at a frequency of at least 0.8x10(-8). Modification of
plasmid DNA sequences flanking the alternative borders demonstrated that both
upstream and downstream sequences play an important role in initiating DNA
transfer. Optimal DNA transfer required the elements to be preceded by pyrimidine
residues interspaced by AC-rich trinucleotides. Alteration of this organization
lowered transformation frequencies by 46% to 93%. Despite their weaker
resemblance with left borders, right border alternatives also functioned
effectively in terminating DNA transfer, if both associated with an upstream
A[C/T]T[C/G]A[A/T]T[G/T][C/T][G/T][C/G]A[C/T][C/T][A/T] domain and tightly linked
cytosine clusters at their junctions with downstream DNA. New insights in border
region requirements were used to construct an all-native alfalfa (Medicago
sativa) transfer DNA vector that can be used for the production of intragenic
plants.
DOI: 10.1104/pp.105.068692
PMCID: PMC1283770
2012. DNA Seq. 2005 Oct;16(5):352-7.
Cloning and expression analysis of a vacuolar Na+/H+ antiporter gene from

alfalfa.
Yang Q(1), Wu M, Wang P, Kang J, Zhou X.
Author information:
(1)Institute of Animal Sciences, Chinese Academy of Agricultural Science (CAAS),
Turf & Forage Science, Beijing, 100094, China.
A full-length cDNA, named MsNHX1, encoding a vacuolar Na+/H+ antiporter was

cloned from alfalfa (Medicago sativa), using degenerate primers, followed by 3'
and 5' RACE. The cDNA sequence was 2225 bp long and included an open reading
frame encoding a deduced protein of 541-amino-acid polypeptide. The deduced amino
acid sequence showed high similarity (more than 73%) to those of the previously
cloned Na+/H+ antiporters form Arabidopsis thaliana, Qryza sativa, Atriplex
gemlinin, Beta vulgaris and Hordeum vulgare. Southern hybridization showed that
MsNHX1 has only one copy in alfalfa genome. Semiquantitative RT-PCR analysis
indicated that the mRNA level of MsNHX1 was moderate without stress and markedly
up-regulated after treatment by NaCl and ABA (abscisic acid). Those results
suggest that the MsNHX1 product play an important role in salt tolerance of the
alfalfa, and its transcript expression is possibly partially regulated through
ABA-dependent signaling pathway.
DOI: 10.1080/10425170500272742
2013. Plant Mol Biol. 2005 Aug;58(6):789-807.
Alfalfa Mob 1-like genes are expressed in reproductive organs during meiosis and
gametogenesis.
Citterio S(1), Albertini E, Varotto S, Feltrin E, Soattin M, Marconi G, Sgorbati

S, Lucchin M, Barcaccia G.
Author information:
(1)Dipartimento di Scienze dell'Ambiente e del Territorio, University of Milano -
Bicocca, Piazza della Scienza 1, 20126, Milano, Italy.
Mps-one-binder (Mob) proteins play an important role in chromosome separation and

cell plate formation in yeast. We cloned two Mob 1-like genes from alfalfa
(Medicago sativa L.) and show that one gene is constitutively expressed while the
other is expressed only in flower buds during sporogenesis and gametogenesis. For
the analysis of gene expression during reproduction in alfalfa wild-types and
apomeiotic mutants, a specific antisense riboprobe was designed for MsMob 1
transcripts and a polyclonal antibody was raised against MsMob 1 proteins. In
situ mRNA localization as well as protein immunolocalization proved that
MsMob1-like genes are specifically expressed in degenerating megaspores of normal
ovules and in enlarged megaspore mother cells and embryo sacs of apomeiotic
ovules. Gene products were also found in microspore tetrads at the beginning of
pollen development as well as in tapetum cells of anthers undergoing programmed
cell death to allow pollen dispersal at maturity. Overall results suggest that
MsMob 1-like genes can play a key role during the reproductive pathway in plants.
DOI: 10.1007/s11103-005-8104-9
2014. J Biosci Bioeng. 2004;98(3):187-92.
Effects of the fermentation product of herbs by lactic acid bacteria against

phytopathogenic filamentous fungi and on the growth of host plants.
Kuwaki S(1), Ohhira I, Takahata M, Hirota A, Murata Y, Tada M.
Author information:
(1)Department of Biological Resources Chemistry, Faculty of Agriculture, Okayama
University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
The fermentation product of herbs by lactic acid bacteria (FHL) was assayed for
antifungal activities against Rosellinia necatrix, Helicobasidium mompa, Fusarium
oxysporum, Pythium graminicola and Pyricularia oryzae. FHL completely inhibited
the growth of R. necatrix, H. mompa, P. graminicola and P. oryzae, and reduced
the growth of F. oxysporum by 35%. When the seeds of Medicago sativa L.
(alfalfa), Asparagus officinalis L. (asparagus), Brassica campestris L.
(komatsuna), Oryza sativa L. (rice), Spinacia oleracea L. (spinach), Festuca
arundinacea Schreb. (tall fescue), and Lycopersicum esculentum Mill. (tomato)
were put on plates containing 0.69 mg/ml FHL, their germination rates did not
decrease. The root elongation of A. officinalis, B. campestris, O. sativa, and L.
esculentum seedlings was suppressed on plates containing 6.92 mg/ml FHL, but the
root elongation of M. sativa was not suppressed on the 6.92 mg/ml FHL plate. When
FHL was diluted to less than 1.73 mg/ml, the diluted FHL solution did not
suppress the germination of B. campestris seeds, but it suppressed the root
elongation of B. campestris seedlings. An FHL concentration higher than 0.35
mg/ml hastened the growth of seedlings of B. campestris in the presence of a
chemical fertilizer but delayed the growth of these seedlings in the absence of
the chemical fertilizer, suggesting that inorganic elements could affect the
efficiency of FHL.
DOI: 10.1016/S1389-1723(04)00264-6
PMID: 16233688
2015. J Dairy Sci. 2005 Nov;88(11):4045-64.
Effect of dietary level of rumen-degraded protein on production and nitrogen

metabolism in lactating dairy cows.
Reynal SM(1), Broderick GA.

Author information:
sreynal@wisc.edu .
Twenty-eight (8 with ruminal cannulas) lactating Holstein cows were assigned to 4

x 4 Latin squares and fed diets with different levels of rumen-degraded protein
(RDP) to study the effect of RDP on production and N metabolism. Diets contained
[dry matter (DM) basis] 37% corn silage, 13% alfalfa silage, and 50% concentrate.
The concentrate contained solvent and lignosulfonate-treated soybean meal and
urea, and was adjusted to provide RDP at: 13.2, 12.3, 11.7, and 10.6% of DM in
diets A to D, respectively. Intake of DM and yield of milk, fat-corrected milk,
and fat were not affected by treatments. Dietary RDP had positive linear effects
on milk true protein content and microbial non-ammonia N (NAN) flow at the omasal
canal, and a quadratic effect on true protein yield, with maximal protein
production at 12.3% RDP. However, dietary RDP had a positive linear effect on
total N excretion, with urinary N accounting for most of the increase, and a
negative linear effect on environmental N efficiency (kg of milk produced per kg
of N excreted). Therefore, a compromise between profitability and environmental
quality was achieved at a dietary RDP level of 11.7% of DM. Observed microbial
NAN flow and RDP supply were higher and RUP flow was lower than those predicted
by the NRC (2001) model. The NRC (2001) model overpredicted production responses
to RUP compared with the results in this study. Replacing default NRC degradation
rates for protein supplements with rates measured in vivo resulted in similar
observed and predicted values, suggesting that in situ degradation rates used by
the NRC are slower than apparent rates in this study.
DOI: 10.3168/jds.S0022-0302(05)73090-3
2016. J Dairy Sci. 2005 Nov;88(11):4007-17.
Biohydrogenation of fatty acids and digestibility of fresh alfalfa or alfalfa hay

plus sucrose in continuous culture.
Ribeiro CV(1), Karnati SK, Eastridge ML.
Author information:
The pattern of biohydrogenation of fatty acids from fresh alfalfa or alfalfa hay
supplemented with 3 concentrations (0, 4, and 8%) of sucrose was studied at a
constant pH of 6.2. Four continuous culture fermenters were used in a 4 x 4 Latin
square design to test the hypothesis that fresh forage would increase flow of
vaccenic acid (VA) from the fermenters compared with the same forage in hay form
and that this difference would be diminished by adding sucrose to the hay diet by
changing the bacterial community profile. Effluent was collected from each of the
4 fermenters during the last 3 d of each 10-d period. Nutrient digestibility,
volatile fatty acids (VFA), and fatty acids in the effluent were measured. Flow
of bacterial organic matter (OM) and neutral and acid detergent fiber and acid
detergent fiber digestibilities were higher for fresh alfalfa than alfalfa hay.
True OM digestibility of alfalfa hay tended to linearly decrease with sucrose
supplementation. However, microbial efficiency and flow of bacterial OM (g/d)
linearly increased with sucrose addition. There was no change in total VFA
concentration; however, proportion of acetate linearly decreased and proportion
of butyrate linearly increased with sucrose addition. Fresh alfalfa increased
total biohydrogenation of fatty acids compared with than hay. Vaccenic acid flow
(mg/d) was much higher for fresh alfalfa compared with alfalfa hay (216 vs. 41)
and VA was the predominant 18:1 isomer, followed by trans-13 18:1; however,
sucrose had no effect on VA flow. The percentage of VA (of total trans-18:1) was
not different between fresh alfalfa and hay, whereas percentage of trans-10 18:1
was much lower for fresh alfalfa. Therefore, the ratio of VA to trans-10 18:1 was
higher for fresh alfalfa. Flow of trans-12 18:1 linearly increased, whereas flows
of cis-12 and total cis-18:1 had quadratic responses to sucrose supplementation.
Total biohydrogenation and biohydrogenation of linoleic and linolenic acids
linearly decreased with sucrose; however, there was no effect of sucrose on total
trans fatty acid flow. Sucrose may be more detrimental to the last step of
biohydrogenation of VA. The effects of sucrose on biohydrogenation and
concentration of VFA may have been caused by a shift in microbial population by
mechanisms that are independent of pH.
DOI: 10.3168/jds.S0022-0302(05)73087-3
2017. J Dairy Sci. 2005 Nov;88(11):4000-6.
Lactational response of cows fed condensed corn distillers solubles.
Da Cruz CR(1), Brouk MJ, Schingoethe DJ.
Author information:
USA.
Fifteen Holstein cows (6 multiparous and 9 primiparous) in early lactation were

used in a replicated 3 x 3 Latin square design with 5-wk periods to evaluate the
use of condensed corn distillers solubles (CCDS) in the total mixed ration. Diets
were control (0%), low (5%), or high (10% of dry matter) CCDS. All diets
contained alfalfa hay, corn silage, rolled corn, and a concentrate supplement
formulated to yield isonitrogenous diets. Condensed corn distillers solubles
replaced a portion of the rolled corn and soybean meal in the control diet. Data
were collected from wk 3 to 5 of each 5-wk period. Dry matter intakes (23.4,
24.4, and 22.5 kg/d for control, low, and high CCDS, respectively) were similar
for CCDS and control and tended to be greater for low than for high CCDS. Milk
yields (34.1, 35.5, and 35.8 kg/d) were greater for added CCDS vs. control, with
no additional benefit found by feeding a high over a low amount of CCDS. Milk fat
percentage decreased slightly, whereas protein and lactose production increased
when cows were fed CCDS. Medium-chain fatty acids in milk fat were decreased and
long-chain fatty acids increased by feeding CCDS, and similar changes were found
comparing high with low level of CCDS. Unsaturated fatty acids in milk fat tended
to be higher for diets containing CCDS compared with control and were higher for
high compared with low inclusion. Ruminal ammonia and serum urea nitrogen
concentrations were lower when cows were fed CCDS. This study indicated that it
is advantageous to feed CCDS at 5% of dry matter; however, some beneficial
changes in milk fatty acid composition were achieved by doubling that amount.
DOI: 10.3168/jds.S0022-0302(05)73086-1
2018. J Dairy Sci. 2005 Nov;88(11):3982-5.
Short communication: Prediction of mean particle size and proportion of very long
fiber particles from simplified sieving results.
Armentano LE(1), Taysom D.

Author information:
learment@wisc.edu
Mean particle size of alfalfa silage and corn silage can be predicted based on
material retained above a screen with square-hole diagonal of 9 mm. The
regression equation is mean particle length (mm) = 1.16 + 13.00 x cumulative
fraction of as-fed mass trapped on or above the 9-mm screen; r2 = 0.89. For mixed
rations, the intercept was 0.54 and the slope 11.84, with r2 = 0.78. Using data
from the screen with a 5.6-mm diagonal also provided reasonable estimates of mean
particle size.
DOI: 10.3168/jds.S0022-0302(05)73084-8
2019. J Environ Qual. 2005 Oct 12;34(6):2005-17. Print 2005 Nov-Dec.
Movement of diuron and hexazinone in clay soil and infiltrated pond water.
Prichard T(1), Troiano J, Marade J, Guo F, Canevari M.
Author information:
(1)University of California, Stockton, 95215, USA. tlprichard@ucdavis.edu
Pre-emergence herbicide residues were detected in domestic wells sampled near

Tracy, CA. This study sought to determine the source of contamination by
comparing soil distribution of diuron [N'-(3,4-dichlorophenyl)-N,N-dimethylurea]
and hexazinone
[3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4(1H,3H)-dione] in an
agricultural field where the soil was a cracking clay to infiltration of residues
in water captured by an adjacent holding pond. Diuron and hexazinone were applied
in December to a 3-yr-old alfalfa (Medicago sativa L.) crop. Water content of
soil taken after major rainfall but before irrigation at 106 d after application
was elevated at the lowest depth sampled centered at 953 mm, indicating water was
available for percolation. Herbicide residues (reporting limit 8 microg kg(-1))
were confined above the 152 mm soil depth, even after subsequent application of
two border-check surface irrigations. The pattern of distribution and
concentration of residues in the soil were similar to results obtained from the
LEACHM model, suggesting that macropore flow was limited to a shallow depth of
soil. Herbicide residues were measured in runoff water at the first irrigation at
20 microg L(-1) for diuron and 1 microg L(-1) for hexazinone. Runoff water
captured in the pond rapidly infiltrated into the subsurface soil, causing a
concomitant rise in ground water elevation near the pond. Herbicide residues were
also detected in the sampled ground water. We concluded that the pond was the
predominant source for movement to ground water. Since addition of a surfactant
to the spray mixture did not reduce concentrations in runoff water, mitigation
methods will focus on minimizing infiltration of water from the pond.
DOI: 10.2134/jeq2004.0253
2020. New Phytol. 2005 Nov;168(2):313-22.
The dark side of green fluorescent protein.
Zhou X(1), Carranco R, Vitha S, Hall TC.

Author information:
(1)Institute of Developmental and Molecular Biology and Department of Biology,
Texas A & M University, College Station, 77843-3155, USA.
Here, severe interference of chlorophyll with green fluorescent protein (GFP)

fluorescence is described for medicago (Medicago truncatula), rice (Oryza sativa)
and arabidopsis (Arabidopsis thaliana). This interference disrupts the
proportional relationship between GFP content and fluorescence that is intrinsic
to its use as a quantitative reporter. The involvement of chlorophyll in the loss
of GFP fluorescence with leaf age was shown in vivo, by the removal of
chlorophyll through etiolation or by ethanol extraction, and in vitro, by
titration of a GFP solution with chlorophyll solutions of various concentrations.
A substantial decrease in fluorescence in early development of medicago and rice
leaves correlated with chlorophyll accumulation. In all three species tested,
removal of chlorophyll yielded up to a 10-fold increase in fluorescence. Loss of
GFP fluorescence in vitro was 4-fold greater for chlorophyll b than for
chlorophyll a. Differences exist between plant species for the discrepancy
between apparent GFP fluorescence and its actual level in green tissues.
Substantial errors in estimating promoter activity from GFP fluorescence can
occur if pigment interference is not considered.
DOI: 10.1111/j.1469-8137.2005.01489.x
2021. Pol J Microbiol. 2005;54(1):43-8.
Pesticide side effect on the symbiotic efficiency and nitrogenase activity of

Rhizobiaceae bacteria family.
Niewiadomska A(1), Klama J.
Author information:
(1)Department of Agricultural Microbiology, August Cieszkowski Agricultural
University of Poznań.
The laboratory experiments tested the influence of selected pesticides on the

symbiotic efficiency and nitrogenase activity of Rhizobium leguminosarumin bv.
trifolii KGL, Sinorhizobiuni melilotii Bp and Badyrhizobium sp. Ornithopus B
bacteria entering into symbiosis with clover, lucerne and serradella,
respectively. The results obtained indicate that the pesticides used in the
experiments (Funaben T seed dressing and Pivot 100SL herbicide) caused reduced
nitrogenase activity in active strains tested. In addition, a toxic effect of the
applied pesticides on the nodulation and root growth of the tested plants was
observed.
2022. J Ind Microbiol Biotechnol. 2005 Dec;32(11-12):561-6. Epub 2005 Oct 6.
Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced

PCB bioremediation.
Chen Y(1), Adam A, Toure O, Dutta SK.
Author information:
(1)Molecular Genetics Laboratory, Department of Biology, Howard University, 415
College Street, Washington DC 20059, USA.
The genome of the nitrogen-fixing soil bacterium Sinorhizobium meliloti does not
possess genes for bioremediation of aromatic pollutants. It has the well-known
ability to interact specifically with the leguminous alfalfa plant, Medicago
sativa. Our previous work has shown enhanced degradation of the nitroaromatic
compound 2,4-dinitrotoluene (DNT) when a plasmid containing degradative genes was
introduced in it. In this study we report molecular evidence of the transfer of a
polychlorinated biphenyl (PCB)-biodegradative plasmid pE43 to S. meliloti strain
USDA 1936. Several standard analytical tests and plant growth chamber studies
were conducted to test the ability of S. meliloti to degrade 2',3,4-PCB congener.
Alfalfa plant alone was able to degrade 30% of PCBs compared with control. No
enhanced dechlorination was noted when alfalfa plant was grown with wild-type S.
meliloti, and when alfalfa plant was grown with the S. meliloti
electrotransformants (genetically modified) dechlorination of PCBs was more than
twice that when alfalfa plant was grown with wild-type S. meliloti. When alfalfa
plant was grown with uncharacterized mixed culture (containing nodule formers),
almost equally significant PCB degradation was observed. The significance of this
work is that the naturally occurring nitrogen-fixing soil bacterium S. meliloti
(genetically modified) has the ability to enhance fertility of soil in
association with the leguminous alfalfa plant while simultaneously enhancing
bioremediation of PCB-contaminated soils. Enhanced bioremediation of PCB and
robust alfalfa plant growth was also noted when uncharacterized mixed cultures
containing alfalfa plant nodule formers were used.
DOI: 10.1007/s10295-005-0039-2
2023. Planta Med. 2005 Sep;71(9):841-6.
Identification of botanicals and potential contaminants through RFLP and

sequencing.
Lum MR(1), Potter E, Dang T, Heber D, Hardy M, Hirsch AM.
Author information:
(1)Department of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, CA 90095-1606, USA.
Botanical supplements for health enhancement are being increasingly used in the
United States, but no safeguards are formally in place to ensure that they are
not contaminated with non-efficacious or potentially harmful plant material. A
molecular approach, which allows the authentication of botanical ingredients and
detection of contaminating plant material by analyzing the ITS-1 region by
PCR-RFLP and subsequent sequencing, is described. When using starting material
from which DNA can be obtained, this method has the potential for identifying
both primary and contaminating plant material in botanical dietary supplements.
DOI: 10.1055/s-2005-871230
2024. Appl Microbiol Biotechnol. 2006 Jul;71(3):329-38. Epub 2005 Oct 5.
Use of real time PCR to determine population profiles of individual species of

lactic acid bacteria in alfalfa silage and stored corn stover.
Stevenson DM(1), Muck RE, Shinners KJ, Weimer PJ.
Author information:
(1)Department of Biological Systems Engineering, University of Wisconsin-Madison,
Madison, 53706, USA.
Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species
of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or
absence of four commercial inoculants and in uninoculated corn stover harvested
and stored under a variety of field conditions. Species-specific PCR primers were
designed based on recA gene sequences. Commercial inoculants improved the quality
of alfalfa silage, but species corresponding to those in the inoculants displayed
variations in persistence over the next 96 h. Lactobacillus brevis was the most
abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96
h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed
in inoculated silages. Pediococcus pentosaceus populations increased over time
but did not exceed 2% of the total. Small populations (0.1 to 1%) of
Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while
Lactobacillus pentosus and Enterococcus faecium were near or below detection
limits. Corn stover generally displayed higher populations of L. plantarum and L.
brevis and lower populations of other LAB species. The data illustrate the
utility of RT-PCR for quantifying individual species of LAB in conserved forages
prepared under a wide variety of conditions.
DOI: 10.1007/s00253-005-0170-z
2025. Appl Environ Microbiol. 2005 Oct;71(10):5685-91.
Salmonella enterica virulence genes are required for bacterial attachment to

plant tissue.
Barak JD(1), Gorski L, Naraghi-Arani P, Charkowski AO.
Author information:
(1)USDA, ARS, WRRC, Produce Safety and Microbiology, 800 Buchanan St., Albany, CA
94710, USA. jbarak@pw.usda.gov
Numerous Salmonella enterica food-borne illness outbreaks have been associated

with contaminated vegetables, in particular sprouted seeds, and the incidence of
reported contamination has steadily risen. In order to understand the physiology
of S. enterica serovar Newport on plants, a screen was developed to identify
transposon mutants that were defective in attachment to alfalfa sprouts. Twenty
independent mutants from a pool of 6,000 were selected for reduced adherence to
alfalfa sprouts. Sixty-five percentage of these mutants had insertions in
uncharacterized genes. Among the characterized genes were strains with insertions
in the intergenic region between agfB, the surface-exposed aggregative fimbria
(curli) nucleator, and agfD, a transcriptional regulator of the LuxR superfamily,
and rpoS, the stationary-phase sigma factor. Both AgfD and RpoS have been
reported to regulate curli and cellulose production and RpoS regulates other
adhesins such as pili. The intergenic and rpoS mutants were reduced in initial
attachment to alfalfa sprouts by 1 log unit compared to the wild type. Mutations
of agfA, curli subunit, and agfB in S. enterica serovar Enteritidis
differentially affected attachment to plant tissue. The agfA mutation was not
reduced in ability to attach to or colonize alfalfa sprouts, whereas the agfB
mutation was reduced. Thus, agfB alone can play a role in attachment of S.
enterica to plant tissue. These results reveal that S. enterica genes important
for virulence in animal systems are also required for colonization of plants, a
secondary host that can serve as a vector of S. enterica from animal to animal.
DOI: 10.1128/AEM.71.10.5685-5691.2005
PMCID: PMC1265987
2026. Chemosphere. 2005 Oct;61(4):595-8. Epub 2005 Apr 7.
Enhancement of lead uptake by alfalfa (Medicago sativa) using EDTA and a plant
growth promoter.
López ML(1), Peralta-Videa JR, Benitez T, Gardea-Torresdey JL.
Author information:
(1)Environmental Sciences and Engineering Ph.D. Program, University of Texas-El
Paso, 500 W. University Ave. El Paso, TX 79968, USA.
Phytoremediation is a novel cleanup technology for the removal of contaminants

from polluted waters and soils. In phytoremediation, the plant uptake capability
and the availability of the pollutant in the media are important. Here we show
the results of a study on the combined effects of ethylenediaminetetraacetic acid
(EDTA) and the phytohormone indole-3-acetic acid (IAA) on Pb uptake by Medicago
sativa (alfalfa). Plants were grown in hydroponics media containing a nutrient
solution amended with Pb at 0.2mM and different combinations of EDTA, and IAA.
After 10d of treatment, the Pb content in plant tissues was quantified using an
Inductively Coupled Plasma Optical Emission Spectrometer (ICP/OES). The results
showed that the combination of 100 microM IAA/0.2 mM EDTA increased the Pb
accumulation in leaves by about 2800% and by about 600%, as compared to Pb
content in leaves of plants exposed to Pb alone and those cultivated with
Pb/EDTA, respectively. These results indicate that non-metal hyperaccumulator
plants could increase their hyperaccumulating potential without genetic
manipulation.
2027. Pest Manag Sci. 2005 Dec;61(12):1202-8.
Capsicum oleoresin: development of an in-soil repellent for pocket gophers.
Sterner RT(1), Shumake SA, Gaddis SE, Bourassa JB.
Author information:
(1)USDA, APHIS, Wildlife Services, National Wildlife Research Center, 4101
LaPorte Avenue, Fort Collins, CO 80521-2154, USA. ray.t.sterner@aphis.usda.gov
A pre- and post-monitoring study was conducted of the potential use of capsicum
oleoresin as an in-soil repellent for northern pocket gophers (Thomomys
talpoides). Pocket gophers were captured in irrigated alfalfa (Medicago sativa
L), affixed with radio transmitters, and monitored daily for location. Six plots
(4.87 x 4.87 m) each were randomly assigned to capsicum oleoresin and soybean oil
treatments; these were set up based upon the centers of initial core areas of
gophers. Mean (+/-SD) volumes of capsicum oleoresin and water and soybean oil and
water mixtures (10 + 90 by volume) dispensed onto plots equaled 178.5 (+/-4.7)
and 175.7 (+/-14.0) liters, respectively. Movements (m) of the radio-transmitted
gophers from plot centers were computed for four daily readings (i.e., 0801-1000,
1101-1300, 1501-1700 and 1801-2000 h). Spectrophotometric analysis of soil
samples from capsicum oleoresin plots validated the presence of capsicum on plots
and the absence of capsicum on placebo- and off-plot locations. Analysis of
variance for movement distances of gophers yielded a Date main effect [F(11, 103)
= 2.08, P < or = 0.03] and a Date x Reading (time) interaction [F(32, 299) =
3.21, P < or = 0.01]. Results showed that gophers were located farther from plot
centers for the 0801-1000 and 1501-1700 h telemetry readings for < or =3 days
post-chemical application-a probable 'disturbance' effect rather than a
chemical-induced avoidance. In a prior laboratory study, capsicum oleoresin and
soil treatments of 1.5% w/w capsicum caused nearly a 50% decrease in soil contact
time by gophers relative to placebo-exposed control animals. This implies that
procedural variables warrant revision before abandoning this approach. The
potential for soil insertion of repellents as a technique for expelling pocket
gophers from territories and some methodological changes for future research of
the technique are discussed.
DOI: 10.1002/ps.1118
2028. Bull Entomol Res. 2005 Oct;95(5):449-55.
Application of DL-beta-aminobutyric acid (BABA) as a root drench to legumes

inhibits the growth and reproduction of the pea aphid Acyrthosiphon pisum
(Hemiptera: Aphididae).
Hodge S(1), Thompson GA, Powell G.
Author information:
(1)Division of Biology, Imperial College London, Wye Campus, Ashford, Kent, UK.
s.hodge@imperial.ac.uk
DL-beta-aminobutyric acid (BABA) is a non-protein amino acid that is an effective

inducer of resistance against a variety of plant pathogens. However, examples of
BABA-induced resistance against insect herbivores have not been reported. We
applied BABA as a soil drench to legumes and monitored its effects on the pea
aphid Acyrthosiphon pisum (Harris). On tic bean (Vicia faba var. minor), BABA
increased aphid mortality, caused a reduction in the mean relative growth rate of
individual insects and lessened the intrinsic rate of population increase (rm).
BABA also caused significant reductions in the growth rate of A. pisum on pea
(Pisum sativa), broad bean (Vicia faba var. major), runner bean (Phaseolus
coccineus), red clover (Trifolium pratense) and alfalfa (Medicago sativa). No
direct toxic effects of BABA against A. pisum were found, and no phytotoxic
effects that may have caused a reduction in aphid performance were detected.
Possible mechanisms behind this BABA-induced inhibition of aphid performance are
discussed.
2029. J Agric Food Chem. 2005 Oct 5;53(20):7654-60.
Determination of saponins in aerial parts of barrel medic (Medicago truncatula)

by liquid chromatography-electrospray ionization/mass spectrometry.
Kapusta I(1), Janda B, Stochmal A, Oleszek W.
Author information:
State Research Institute, ul. Czartoryskich 8, 24-100 Pulawy, Poland.
Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17,
M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var.
truncatula were profiled and quantified using reverse-phase liquid chromatography
with on-line photodiode array detection and electrospray ionization mass
spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves
obtained for the 18 available saponin standards, previously isolated from
Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins
previously identified and also a substantial amount of astragaloside VIII
(3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The
compositions of saponin mixtures were very similar in the three subspecies with
three dominant groups, recognized as zanhic acid, medicagenic acid, and
soyasapogenol glycosides. Relative proportions of these three groups were also
similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var.
truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of
zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively.
Jemalong A-17 had 30% lower total content of saponins as compared to M.
truncatula var. longispina and M. truncatula var. truncatula; in relation to the
dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong
A-17, 0.15% dry matter of saponins. If one takes into consideration that this
determination was performed on spring-collected samples, it can be concluded that
the concentration of saponins in M. truncatula is similar to the concentration in
alfalfa (Medicago sativa); the proportions of the three groups of saponins in
these species are slightly different from those found in alfalfa, having a higher
content of zanhic acid glycosides.
DOI: 10.1021/jf051256x
2030. Environ Pollut. 2006 Mar;140(1):1-3. Epub 2005 Sep 26.
Short-term exposure to ozone altered the relative feed value of an alfalfa

cultivar.
Muntifering RB(1), Manning WJ, Lin JC, Robinson GB.
Author information:
(1)Department of Animal Sciences, Auburn University, Auburn, AL 36849-5415, USA.
muntirb@auburn.edu
DOI: 10.1016/j.envpol.2005.07.002
2031. Trans R Soc Trop Med Hyg. 2006 Feb;100(2):158-66. Epub 2005 Sep 21.
Risk factors for Fasciola hepatica infection in children: a case-control study.
Marcos L(1), Maco V, Samalvides F, Terashima A, Espinoza JR, Gotuzzo E.
Author information:
(1)Institute of Tropical Medicine Alexander von Humboldt (IMTAvH), Universidad
Peruana Cayetano Heredia (UPCH), Av. Honorio Delgado 430, Urb. Ingeniería, San
Martín de Porres AP 4314, Lima 100, Peru. egh@upch.edu.pe
We conducted a case-control study to clarify the risk factors for human

fascioliasis in the Northern Peruvian Altiplano. Cases (n=61) were individuals
who were diagnosed as having chronic fascioliasis by faecal and/or serologic
(Fas2-ELISA) studies. Controls (n=61) had negative serologic and coprological
results for Fasciola hepatica. We identified an association between fascioliasis
and four variables (40 analysed): the habit of drinking alfalfa juice (OR=4.5;
95% CI 1.8-11.1; P<0.001); familiarity with aquatic plants (OR=4.3; 95% CI
1.8-10.6; P<0.001); dog ownership (OR=5; 95% CI 1.7-15.1; P=0.002); and raising
more than five sheep (OR=0.3; 95% CI 0.1-0.8; P=0.01). According to clinical
presentation and laboratory studies, dizzy spells (P=0.01), history of jaundice
(P=0.01), peripheral eosinophilia (P=0.005) and Ascaris lumbricoides in stools
(P=0.001) were associated with fascioliasis. The principal exposure factor for F.
hepatica infection was drinking alfalfa juice. In conclusion, we suggest that
human fascioliasis in Peru should be suspected in patients from livestock-rearing
areas, who present with recurrent episodes of jaundice and who have a history of
consumption of alfalfa juice or aquatic plants, or who have eosinophilia.
DOI: 10.1016/j.trstmh.2005.05.016
2032. Can J Microbiol. 2005 Jul;51(7):599-604.
Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti

PHB synthesis and degradation pathway mutants.
Aneja P(1), Zachertowska A, Charles TC.
Author information:
(1)Department of Natural Resource Sciences, Mcgill University, Canada.
The competitive abilities of Sinorhizobium meliloti mutant strains containing

lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were
compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa
host plants when inoculated alone, in mixed inoculation experiments it was found
to be less competitive than the wild type for nodule occupancy. Long-term
survival of the bdhA mutant on a carbon-limiting medium was not affected.
However, when subjected to competition with the wild-type strain in periodic
subculturing through alternating carbon-limiting and carbon-excess conditions,
the bdhA mutant performed poorly. A more severe defect in competition for growth
and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC).
These results indicate that the ability to efficiently deposit cellular PHB
stores is a key factor influencing competitive survival under conditions of
fluctuating nutrient carbon availability, whereas the ability to use these stores
is less important.
DOI: 10.1139/w05-042
Phosphorus-free membrane lipids of Sinorhizobium meliloti are not required for

the symbiosis with alfalfa but contribute to increased cell yields under
phosphorus-limiting conditions of growth.
López-Lara IM(1), Gao JL, Soto MJ, Solares-Pérez A, Weissenmayer B, Sohlenkamp C,

Verroios GP, Thomas-Oates J, Geiger O.
Author information:
(1)Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo.
Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico.
The microsymbiont of alfalfa, Sinorhizobium meliloti, possesses

phosphatidylglycerol, cardiolipin, phosphatidylethanolamine, and
phosphatidylcholine as major membrane phospholipids, when grown in the presence
of sufficient accessible phosphorus sources. Under phosphate-limiting conditions
of growth, S. meliloti replaces its phospholipids by membrane lipids that do not
contain any phosphorus in their molecular structure and, in S. meliloti, these
phosphorus-free membrane lipids are sulphoquinovosyl diacylglycerols (SL),
ornithine-containing lipids (OL), and diacylglyceryl-N,N,N-trimethylhomoserines
(DGTS). In earlier work, we demonstrated that neither SL nor OL are required for
establishing a nitrogen-fixing root nodule symbiosis with alfalfa. We now report
the identification of the two structural genes btaA and btaB from S. meliloti
required for DGTS biosynthesis. When the sinorhizobial btaA and btaB genes are
expressed in Escherichia coli, they cause the formation of DGTS in this latter
organism. A btaA-deficient mutant of S. meliloti is unable to form DGTS but can
form nitrogen-fixing root nodules on alfalfa, demonstrating that sinorhizobial
DGTS is not required for establishing a successful symbiosis with the host plant.
Even a triple mutant of S. meliloti, unable to form any of the phosphorus-free
membrane lipids SL, OL, or DGTS is equally competitive for nodule occupancy as
the wild type. Only under growth-limiting concentrations of phosphate in culture
media did mutants that could form neither OL nor DGTS grow to lesser cell
densities.
DOI: 10.1094/MPMI-18-0973
2034. J Dairy Sci. 2005 Oct;88(10):3619-32.
Milk production and nitrogen excretion of dairy cows fed different amounts of
protein and varying proportions of alfalfa and corn silage.
Groff EB(1), Wu Z.
Author information:
(1)Department of Dairy and Animal Science, Pennsylvania State University,
University Park 16802, USA.
Four trials were conducted to determine the effect of dietary protein amount on
lactation performance and N utilization. Each trial used one of the following
alfalfa-to-corn-silage ratios for the forage part of the diet: 100:0, 75:25,
50:50, and 25:75. All trials utilized 16 mid-lactation Holstein cows (days in
milk averages ranging from 80 to 140 among trials) in a replicated 4 x 4 Latin
square design with 3-wk periods, including 2 wk for adaptation and 1 wk for data
collection. Diets consisted of 50% forage and 50% concentrate (dry matter basis)
and were formulated to contain 15.00, 16.25, 17.50, or 18.75% protein in each
trial. The analyzed protein content of the diets was 15.7, 16.9, 18.0, and 19.2%
when averaged across trials. Milk yield was similar among dietary protein levels
in each trial, ranging from 35.2 to 36.1 kg/d when data were combined across
trials. Changes in milk fat and protein due to the protein content of the diet
were small and inconsistent. Both milk urea nitrogen and blood urea nitrogen
concentrations increased linearly as the protein content of the diet was
increased, ranging from 9.9 to 13.1 and from 9.9 to 13.8 mg/dL, respectively,
across trials. As dietary protein was increased from the lowest to the highest
concentrations when data were combined and analyzed, mean fecal N concentration
increased from 2.8 to 3.0%, and urinary N from 5.8 to 7.3 g/L. At the same time,
mean total N excretion increased from 484 to 571 g/d, and conversion of intake N
to milk N decreased from 0.27 to 0.22, resulting in an average change of 18%. Of
the N excreted, urinary N accounted for an increasing proportion, ranging from 41
to 48%, as dietary protein was increased. Overall, based on N utilization as well
as milk production, 17% protein in diets utilizing various proportions of alfalfa
and corn silage as the forage source appeared sufficient for cows producing 38
kg/d of milk in this study.
DOI: 10.3168/jds.S0022-0302(05)73047-2
Alfalfa leafcutting bee population dynamics, flower availability, and pollination

rates in two Oregon alfalfa fields.
Bosch J(1), Kemp WP.
Author information:
(1)Biology Department, Utah State University, Logan, UT 84322-5310, USA.
Since the 1970s, it has become increasingly difficult for U.S. alfalfa seed
producers to maintain Megachile rotundata (F.) populations used for alfalfa,
Medicago sativa L., pollination. In 1998, we monitored M. rotundata population
dynamics and foraging behavior, as well as alfalfa bloom and pollination rates in
two fields in eastern Oregon. Despite marked differences in bee management,
establishment was very similar in the two fields (approximately 0.5 females per
nesting cavity) and lagged peak bloom by approximately 2 wk. Pollination rates
increased from 0-10% in the first 3 wk to 80-90% in week 4-5. By then, M.
rotundata females had difficulty finding untripped (nonpollinated) flowers and
visited large numbers of already tripped or not fully matured flowers. M.
rotundata progeny mortality was very high (54-78%). Estimated seed yields were
similar in both fields. We contend similar seed yields, and improved bee
production, could be accomplished with smaller bee populations, better timed with
alfalfa bloom.
DOI: 10.1603/0022-0493-98.4.1077
2036. Poult Sci. 2005 Aug;84(8):1186-90.
Evaluation of Salmonella enteritidis in molting hens after administration of an

experimental chlorate product (for nine days) in the drinking water and feeding
an alfalfa molt diet.
McReynolds J(1), Kubena L, Byrd J, Anderson R, Ricke S, Nisbet D.
Author information:
(1)USDA-ARS-SPARC, 2881 F and B Road, College Station, Texas 77845, USA.
mcreynolds@ffsru.tamu.edu
The method most commonly used to induce molting and stimulate multiple egg-laying
cycles in laying hens for commercial egg production is to fast the hens.
Unfortunately, increased risk of Salmonella enteritidis (SE) infection may result
from the use of this method. Methods to stimulate multiple egg-laying cycles
without increasing the risk of SE infection are needed. Hens over 50 wk of age
were divided into 12 groups of 11 hens each and placed in individual laying
cages. One week prior to dietary changes, hens were placed on an 8-h light and
16-h dark photoperiod that continued for the 9-d molt. All hens were challenged
orally with 10(6) cfu of SE on the fourth day of the molt. Treatments were nonfed
hens with distilled water (NFD), nonfed hens with the experimental chlorate
product (ECP, which provided 15 mM chlorate ion) water (NFECP), alfalfa diets
with distilled water (ALD), and alfalfa diets with ECP water (ALECP). In the NFD
hens, 67% (log10 2.74) of the crops and 94% (log10 5.62) of the ceca were
colonized, whereas for the NFECP hens significant reductions to 22% (log10 1.05)
of the crops and 61% (log10 2.44) of the ceca were observed. In the ALD hens, 61%
(log10 2.52) of the crops and 94% (log10 4.06) of the ceca were colonized. In the
ALECP hens, highly significant reductions to 11% (log10 1.26) of the crops and
39% (log10 1.12) of the ceca were observed. When compared with the NFD hens,
significant reductions in SE invasion of the ovary, liver, and spleen occurred in
all other treatments, except the ovary in the ALD hens. The low alfalfa intake is
probably a factor in our lowered protection against SE when compared with
previous results. For several parameters, these results suggest that ECP or the
combination of ECP and alfalfa may be a useful tool to reduce the risk of SE
during an induced molt.
DOI: 10.1093/ps/84.8.1186
2037. J AOAC Int. 2005 Jul-Aug;88(4):995-7.
Evaluation of two procedures to determine acid and neutral detergent fibers in

ruminant feeds of the temperate region of Argentina.
Fay JP(1), Guaita MS, Danelón JL, Chifflet S, Wawrzkiewicz M, Fernández HM, Ross
DA.
Author information:
(1)National Institute of Agricultural Technology, Balcarce Agricultural
Experiment Station, CC 276, Balcarce, 7620 Argentina. pfay@balcarce.inta.gov.ar
The objective of this work was to compare the traditional Van Soest's procedure
to analyze neutral detergent fibers (NDF) and acid detergent fibers (ADF) using
filtering crucibles (VS) with a semiautomatic method which uses filter bags in an
ANKOM Technology Corp. instrument (ANK). Nine ruminant feeds widely used in the
temperate region of Argentina were analyzed: soybean meal expeller, alfalfa,
pasture silage, ryegrass, corn silage, weeping lovegrass, tall wheatgrass, guinea
grass, and barley grass. Four runs were done for each technique and feed. The
comparison of ADF and NDF means by the means test for paired samples showed no
significant differences between techniques (alpha = 0.01). The variability among
runs was greater with the VS method than the ANK, both for NDF, standard
deviation (SD) = 0.71 vs 0.39, and for ADF, SD = 0.83 vs 0.56, but the
differences were not significant. The linear regressions were VS = 1.43 + 0.95
ANK; R2 = 0.99, and VS = 0.53 + 0.98 ANK; R2 = 0.99 for NDF and ADF,
respectively, which indicated a strong linear relationship among the results of
both procedures. It was concluded that the ANK procedure gave results comparable
to those of the VS method when ruminant feeds from the temperate region of
Argentina were analyzed.
2038. Theor Appl Genet. 2005 Nov;111(7):1420-9. Epub 2005 Sep 6.
Genetic diversity among alfalfa (Medicago sativa) cultivars coming from a

breeding program, using SSR markers.
Flajoulot S(1), Ronfort J, Baudouin P, Barre P, Huguet T, Huyghe C, Julier B.
Author information:
(1)Jouffray-Drillaud, 86600 Lusignan, France.
Alfalfa (Medicago sativa) is an autotetraploid, allogamous and heterozygous

species whose cultivars are synthetic populations. The breeders apply selection
pressure for some agronomic traits within a breeding pool to increase the
frequency of favorable individuals. The objective of this study was to
investigate the differentiation level among seven cultivars originating from one
breeding program, and between these cultivars and the breeding pool, with eight
SSR markers. These highly polymorphic and codominant markers, together with
recent population genetic statistics extended to autotetraploids, offer tools to
analyse genetic diversity in alfalfa. The number of alleles per locus varied
between 3 and 24. All loci were at a panmictic equilibrium in the cultivars,
except one, probably because of null alleles. With seven SSR loci, each cultivar
was at panmictic equilibrium. The mean gene diversity was high, ranging from
0.665 to 0.717 in the cultivars. The parameter F(ST) indicated a low but
significant diversity among cultivars. Among 21 pairs of cultivars, 15 were
significantly different. The breeding pool also had a high diversity, and was
significantly different from each cultivar except the most recent one.
Considering the characteristics of the breeding program and the mode of cultivar
elaboration, we found that they were unable to generate a large variety
differentiation. Estimation of population genetics parameters at SSR loci can be
applied for assessing the differences between cultivars or populations, either
for variety distinction or the management of genetic resources.
DOI: 10.1007/s00122-005-0074-4
2039. J Environ Qual. 2005 Sep 8;34(5):1811-9. Print 2005 Sep-Oct.
Herbaceous vegetation productivity, persistence, and metals uptake on a

biosolids-amended mine soil.
Evanylo GK(1), Abaye AO, Dundas C, Zipper CE, Lemus R, Sukkariyah B, Rockett J.
Author information:
(1)Department of Crop and Soil Environmental Sciences, Virginia Polytechnic
Institute and State University, Blacksburg, VA 24060, USA. gevanylo@vt.edu
The selection of plant species is critical for the successful establishment and
long-term maintenance of vegetation on reclaimed surface mined soils. A study was
conducted to assess the capability of 16 forage grass and legume species in
monocultures and mixes to establish and thrive on a reclaimed Appalachian surface
mine amended with biosolids. The 0.15-ha coarse-textured, rocky, non-acid forming
mined site was prepared for planting by grading to a 2% slope and amending
sandstone overburden materials with a mixture of composted and dewatered,
anaerobically digested biosolids at a rate of 368 Mg ha(-1) (dry weight). Tall
fescue (Festuca arundinacea Schreb.), orchardgrass (Dactylis glomerata L.),
switchgrass (Panicum virgatum L.), caucasian bluestem (Bothriochloa caucasia L.),
reed canarygrass (Phalaris arundinacea L.), ladino clover (Trifolium repens L.),
birdsfoot trefoil (Lotus corniculatus L.), crownvetch (Coronilla varia L.),
alfalfa (Medicago sativa L.), common sericea lespedeza and AULotan sericea
lespedeza (Lespedeza cuneata L.), tall fescue-ladino clover, tall fescue-alfalfa,
orchardgrass-birdsfoot trefoil, switchgrass-AULotan, and an herbaceous species
mix intended for planting on reforested sites consisting of foxtail millet
[Setaria italica (L.) Beauv.], perennial ryegrass (Lolium perenne L.), redtop
(Agrostis alba L.), kobe lespedeza (Kummerowia striata L.), appalow lespedeza
(Lespedeza cuneata L.), and birdsfoot trefoil were established between spring
1990 and 1991. Vegetative biomass and/or persistence were assessed in 1996, 1997,
1998, 2000, 2001, and 2002. The high rate of biosolids applied provided favorable
soil chemical properties but could not overcome physical property limitations due
to shallow undeveloped soil perched atop a compacted soil layer at 25 cm depth.
The plant species whose persistence and biomass production were the greatest
after a decade or more of establishment (i.e., switchgrass, sericea lespedeza,
reed canarygrass, tall fescue, and crownvetch) shared the physiological and
reproductive characteristics of low fertility requirements, drought and moisture
tolerance, and propagation by rhizome and/or stolons. Of these five species, two
(tall fescue and sericea lespedeza) are or have been seeded commonly on
Appalachian coal surface mines, and often dominate abandoned pasture sites.
Despite the high rates of heavy metal-bearing biosolids applied to the soil,
plant uptake of Cd, Cu, Ni, and Zn were well within critical concentrations more
than a decade after establishment of the vegetation.
DOI: 10.2134/jeq2004.0329
2040. Plant J. 2005 Sep;43(6):849-60.
Nitric oxide is required for, and promotes auxin-mediated activation of, cell
division and embryogenic cell formation but does not influence cell cycle
progression in alfalfa cell cultures.
Otvös K(1), Pasternak TP, Miskolczi P, Domoki M, Dorjgotov D, Szucs A, Bottka S,

Dudits D, Fehér A.
Author information:
(1)Laboratory of Functional Cell Biology, Institute of Plant Biology, Biological
Research Centre, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726,
Szeged, Hungary.
It is now well established that nitric oxide (NO) serves as a signaling molecule
in plant cells. In this paper experimental data are presented which indicate that
NO can stimulate the activation of cell division and embryogenic cell formation
in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was
found that various NO-releasing compounds promoted auxin-dependent division (as
shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa
cells. In contrast, application of NO scavenger or NO synthesis inhibitor
inhibited the same process. Both the promotion and the inhibition of cell cycle
activation correlated with the amount and activity of the cognate alfalfa p34cdc2
protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was
transient, and protoplast-derived cells spending more than 3 days in culture
become insensitive to the inhibitor as far as cell cycle progression was
concerned. L-NMMA had no effect on the cell cycle parameters of cycling
suspension-cultured cells, but had a moderate transient inhibitory effect on
cells re-entering the cell cycle following phosphate starvation. Cycling cultured
cells, however, could respond to NO, as indicated by the sodium nitroprusside
(SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
(PTIO)-dependent accumulation of the ferritin protein. Based on these
observations, it is hypothesized that L-NMMA-sensitive generation of NO is
involved in the activation, but not the progression of the plant cell division
cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin
[2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of
embryogenic cell clusters expressing the MsSERK1 gene; this further supports a
link between auxin- and NO-dependent signaling pathways in plant cells.
DOI: 10.1111/j.1365-313X.2005.02494.x
2041. J Chem Ecol. 2005 Sep;31(9):2049-68. Epub 2005 Aug 17.
Characterization of condensed tannins purified from legume forages: chromophore

production, protein precipitation, and inhibitory effects on cellulose digestion.
McAllister TA(1), Martinez T, Bae HD, Muir AD, Yanke LJ, Jones GA.
Author information:
(1)Agriculture and Agri-Food Canada Research Centre, Lethbridge, Alberta, Canada,
T1J 4B1. mcallister@agr.gc.ca
To identify simple screening tools for selecting condensed tannin (CT)-containing

forages as candidate sources for further study, CT were isolated from nine
legumes, and their molecular weights (MW), chromophore production, capacity to
precipitate bovine serum albumin (BSA) and Fraction 1 protein (Rubisco) isolated
from alfalfa, and inhibition of filter paper digestion were compared. Sources
were as follows: leaves of sericea lespedeza (Lespedeza cuneata Dum.-Cours.),
crown vetch (Coronilla varia L.), and sainfoin (Onobrychis viciifolia Scop.);
stems of hedysarum (Hedysarum alpinum L.); seeds of alfalfa (Medicago sativa L.);
and whole plants of birdsfoot trefoil (Lotus corniculatus var. corniculatus L.)
and three varieties of big trefoil (Lotus pedunculatus Cav.), viz., Lotus
uliginosus Schkuhr, L. uliginosus var. glabriusculus, and L. uliginosus var.
villosus. Molecular weights and sizes (degrees of polymerization) of the CT
varied considerably within and among plant species. Average MW ranged from 3036
Da (crown vetch) to 7143 Da (lespedeza). All CT exhibited greater capacity (w/w
basis) to bind alfalfa Rubisco than BSA. Relative astringencies (microg CT
required to precipitate 1 mg protein) against BSA ranged from 262.5 for CT from
lespedeza to 435.5 for CT from L. corniculatus, and against Rubisco, from 49.6
(sainfoin) to 108.2 (alfalfa seed). Including CT at 300 microg/ml in cultures of
Fibrobacter succinogenes reduced digestion of cellulose filter paper by 19.8%
(sainfoin) to 92.4% (crown vetch) and increased the specific activity of
cell-associated endoglucanase. There were no correlations between inhibitory
effects of CT on filter paper digestion and (1) chromophore formation during CT
assay by butanol-HCl, vanillin-HCl, or H2SO4; (2) precipitation of BSA or alfalfa
Rubisco; and (3) MW of CT. The most inhibitory CT for cellulose digestion
included those with broad and with narrow MW distributions. Sainfoin was the most
desirable source of CT, as it had the highest capacity to bind alfalfa protein
and was least inhibitory to cellulose digestion by F. succinogenes. This study
suggests that these properties are not easily defined via chemical means, and
that biological assays using rumen bacteria may help identify those CT with
properties of nutritional interest.
DOI: 10.1007/s10886-005-6077-4
2042. Genome. 2005 Jun;48(3):556-61.
Variation in rDNA locus number and position among legume species and detection of
2 linked rDNA loci in the model Medicago truncatula by FISH.
Abirached-Darmency M(1), Prado-Vivant E, Chelysheva L, Pouthier T.

Author information:
(1)Institut national de la recherche agronomique (INRA), Unité de recherche sur
les legumineuses (URLEG), Dijon, France. mona@epoisses.inra.fr
Within Fabaceae, legume species have a variable genome size, chromosome number,
and ploidy level. The genome distribution of ribosomal genes, easily detectable
by fluorescent in situ hybridization (FISH), is a good tool for anchoring
physical and genetic comparative maps. The organisation of 45S rDNA and 5S loci
was analysed by FISH in the 4 closely related species: Pisum sativum, Medicago
truncatula, Medicago sativa (2 diploid taxa), and Lathyrus sativus. The 2 types
of rDNA arrays displayed interspecific variation in locus number and location,
but little intraspecific variation was detected. In the model legume, M.
truncatula, the presence of 2 adjacent 45S rDNA loci was demonstrated, and the
location of the rDNA loci was independent of the general evolution of the genome
DNA. The different parameters relative to clustering of the rDNA loci in specific
chromosome regions and the possible basis of rDNA instability are discussed.
DOI: 10.1139/g05-015
2043. Chemosphere. 2006 Jan;62(4):545-58. Epub 2005 Aug 19.
Phytotoxicity of nitroaromatic energetic compounds freshly amended or weathered

and aged in sandy loam soil.
Rocheleau S(1), Kuperman RG, Martel M, Paquet L, Bardai G, Wong S, Sarrazin M,

Dodard S, Gong P, Hawari J, Checkai RT, Sunahara GI.
Author information:
Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
The toxicities of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB),

2,4-dinitrotoluene (2,4-DNT), and 2,6-dinitrotoluene (2,6-DNT) to terrestrial
plants alfalfa (Medicago sativa L.), Japanese millet (Echinochloa crusgalli L.),
and perennial ryegrass (Lolium perenne L.) were determined in Sassafras sandy
loam soil using seedling emergence, fresh shoot, and dry mass measurement
endpoints. A 13-week weathering and aging of energetic materials in soils, which
included wetting and drying cycles, and exposure to sunlight of individual soil
treatments, was incorporated into the study design to better reflect the soil
exposure conditions in the field than toxicity determinations in freshly amended
soils. Definitive toxicity tests showed that dinitrotoluenes were more phytotoxic
for all plant species in freshly amended treatments based on EC20 values for dry
shoot ranging from 3 to 24mgkg(-1) compared with values for TNB or TNT ranging
from 43 to 62mgkg(-1). Weathering and aging of energetic materials (EMs) in soil
significantly decreased the toxicity of TNT, TNB or 2,6-DNT to Japanese millet or
ryegrass based on seedling emergence, but significantly increased the toxicity of
all four EMs to all three plant species based on shoot growth. Exposure of the
three plant species to relatively low concentrations of the four compounds
initially stimulated plant growth before the onset of inhibition at greater
concentrations (hormesis).
2044. Wei Sheng Wu Xue Bao. 2004 Dec;44(6):729-32.

[A Sinorhizoboium meliloti strain that can nodulate soybean plants].
Lin RS(1), Du BH, Li XH, Wang L, Yang SS.
Author information:
(1)College of Biological Science, China Agricultural University, Key Laboratory
for Microbial Research and Application of Agriculture Ministry, Beijing 100094,
China.
Sinorhizobium meliloti XJ96077 was isolated from root nodules of alfalfa

(Medicago sativa) in Xinjiang Region of China. Nodulation experiments showed that
both soybean and alfalfa were effectively nodulated by XJ96077. The DNA (G+ C)
mol% of strain XJ96077 was 61.9%. The DNA homologies of strain XJ96077 were 93%
and 80% with S. meliloti USDA1002T and 042BM, respectively. These results showed
that XJ96077 belongs to Sinorhizobium meliloti. To prove the capability of
XJ96077 to nodulate both soybean and alfalfa, constitutively expressed green
fluorescence protein gene gfp was introduced to XJ96077, and the recombinant
strain XJ96077(G) was obtained. Root nodules of the soybean and alfalfa
inoculated with XJ96077(G) and the expression of gfp were observed using the
confocal laser scanning microscope. XJ96077 showed various nodulation capacities
with different soybean cultivars.
2045. J Anim Sci. 2005 Sep;83(9):2225-34.
Behavior, preference for, and use of alfalfa, tall fescue, white clover, and
buffalograss by pregnant gilts in an outdoor production system.
Rachuonyo HA(1), Allen VG, McGlone JJ.
Author information:
(1)Pork Industry Institute, Department of Animal and Food Sciences, Texas Tech
University, Lubbock, 79409, USA.
Sustainable outdoor pig production requires vegetation that can maintain ground
cover, assimilate manure nutrients, and prevent soil erosion. Two experiments
were conducted to evaluate the suitability of four forages: alfalfa (Medicago
sativa), tall fescue (Festuca arundinacea), white clover (Trifolium repens), and
buffalograss (Buchloe dactyloides) for grazing or ground cover in pastures for
pigs. Each forage plot covered 7.5 m2, with nine replicates in a randomized block
design. In Exp. 1, eight pregnant gilts had free access to all forages during a
2-d adjustment period. Immediately thereafter, pairs of gilts were assigned
randomly to one of each of four blocks of the four forages during a 2-d
measurement period. The percentage of ground cover for each forage was visually
estimated at 0, 24, and 48 h of study. Behavioral data, including walking,
eating, grazing, rooting, drinking, standing, lying, and time spent in hut were
video-monitored continuously for 48 h. Initial percentage of ground cover was
100% for all species. By 48 h, percentage of ground cover decreased (P < 0.001)
for white clover (11.3 +/- 0.88%) and alfalfa (36.3 +/- 0.88%), but not for tall
fescue (98.0 +/- 0.88%) or buffalograss (98.0 +/- 0.88%). Gilts spent more (P <
0.01) time grazing white clover (16.3 +/- 1.97 min/d) and alfalfa (11.2 +/- 1.97)
than tall fescue (0.8 +/- 1.97) or buffalograss (0.3 +/- 1.97), and rooted more
(P < 0.04) white clover than other forages. In Exp. 2, six gilts from the initial
group were put on six blocks of the four forages. Each gilt was assigned randomly
to three replicates of each forage, including alfalfa, tall fescue, or
buffalograss (white clover was excluded because of damage by gilts during Exp.
1), and gilts grazed single forages for 2 d. After this grazing period, the
percentage of ground cover was less (P < 0.01) for alfalfa than for buffalograss
or tall fescue (37.5 +/- 0.38, 96.7 +/- 0.39, 96.3 +/- 0.39%, respectively). With
access to a single forage, pregnant gilts spent more (P < 0.01) time grazing
alfalfa (15.8 +/- 2.36 min/d) than buffalograss (1.5 +/- 2.36) or tall fescue
(0.7 +/- 2.37). These gilts clearly preferred grazing white clover and alfalfa,
and rooting and eating white clover compared with buffalograss or tall fescue.
Rates of ground cover loss were less (P < 0.01) for tall fescue and buffalograss
than for the more preferred forages. Less preferred forages could have potential
as pasture for swine when the primary objective is ground cover maintenance
rather than nutrient supply.
DOI: 10.2527/2005.8392225x
2046. Theor Appl Genet. 2005 Sep;111(5):980-92. Epub 2005 Aug 2.
An expressed sequence tag SSR map of tetraploid alfalfa (Medicago sativa L.).
Sledge MK(1), Ray IM, Jiang G.
Author information:
(1)The Samuel Roberts Noble Foundation, Ardmore, 73402 OK, USA.
mksledge@noble.org
A genetic map constructed from a population segregating for a trait of interest

is required for QTL identification. The goal of this study was to construct a
molecular map of tetraploid alfalfa (Medicago sativa.) using simple sequence
repeat (SSR) markers derived primarily from expressed sequence tags (ESTs) and
bacterial artificial chromosome (BAC) inserts of M. truncatula. This map will be
used for the identification of drought tolerance QTL in alfalfa. Two first
generation backcross populations were constructed from a cross between a
water-use efficient, M. sativa subsp. falcata genotype and a low water-use
efficient M. sativa subsp. sativa genotype. The two parents and their F(1) were
screened with 1,680 primer pairs designed to amplify SSRs, and 605 single dose
alleles (SDAs) were amplified. In the F(1), 351 SDAs from 256 loci were mapped to
41 linkage groups. SDAs not inherited by the F(1), but transmitted through the
recurrent parents and segregating in the backcross populations, were mapped to 43
linkage groups, and 44 of these loci were incorporated into the composite maps.
Homologous linkage groups were joined to form eight composite linkage groups
representing the eight chromosomes of M. sativa. The composite maps consist of
eight composite linkage groups with 243 SDAs from M. truncatula EST sequences, 38
SDAs from M. truncatula BAC clone sequences, and five SDAs from alfalfa genomic
SSRs. The total composite map length is 624 cM, with average marker density per
composite linkage group ranging from 1.5 to 4.4 cM, and an overall average
density of 2.2 cM. Segregation distortion was 10%, and distorted loci tended to
cluster on individual homologues of several linkage groups.
DOI: 10.1007/s00122-005-0038-8
2047. J Exp Bot. 2005 Sep;56(419):2443-52. Epub 2005 Aug 1.
Soluble methionine enhances accumulation of a 15 kDa zein, a methionine-rich

storage protein, in transgenic alfalfa but not in transgenic tobacco plants.
Amira G(1), Ifat M, Tal A, Hana B, Shmuel G, Rachel A.
Author information:
(1)Plant Science Laboratory, Migal-Galilee Technology Center, PO Box 831, Kiryat
Shmona, 11016, Israel.
With the general aim of elevating the content of the essential amino acid
methionine in vegetative tissues of plants, alfalfa (Medicago sativa L.) and
tobacco plants, as well as BY2 tobacco suspension cells, were transformed with a
beta-zein::3HA gene under the 35S promoter of cauliflower mosaic virus encoding a
rumen-stable methionine-rich storage protein of 15 kDa zein. To examine whether
soluble methionine content limited the accumulation of the 15 kDa zein::3HA,
methionine was first added to the growth medium of the different transgenic
plants and the level of the alien protein was determined. Results demonstrated
that the added methionine enhanced the accumulation of the 15 kDa zein::3HA in
transgenic alfalfa and tobacco BY2 cells, but not in whole transgenic tobacco
plants. Next, the endogenous levels of methionine were elevated in the transgenic
tobacco and alfalfa plants by crossing them with plants expressing the
Arabidopsis cystathionine gamma-synthase (AtCGS) having significantly higher
levels of soluble methionine in their leaves. Compared with plants expressing
only the 15 kDa zein::3HA, transgenic alfalfa co-expressing both alien genes
showed significantly enhanced levels of this protein concurrently with a
reduction in the soluble methionine content, thus implying that soluble
methionine was incorporated into the 15 kDa zein::3HA. Similar phenomena also
occurred in tobacco, but were considerably less pronounced. The results
demonstrate that the accumulation of the 15 kDa zein::3HA is regulated in a
species-specific manner and that soluble methionine plays a major role in the
accumulation of the 15 kDa zein in some plant species but less so in others.
DOI: 10.1093/jxb/eri237
2048. Chemosphere. 2006 Jan;62(4):616-25. Epub 2005 Jul 19.
Effects, transfer, and fate of RDX from aged soil in plants and worms.
Best EP(1), Geter KN, Tatem HE, Lane BK.
Author information:
(1)US Army Engineer Research and Development Center, Environmental Laboratory,
3909 Halls Ferry Road, Vicksburg, MS 39180, USA. beste@wes.army.mil
The objectives of this study were to provide data that can be used to predict
exposure-based effects of RDX in aged soil on multiple endpoint organisms
representing two trophic levels. These data can be used for defining criteria or
reference values for environmental management and conducting specific risk
assessment. Dose-response experiments formed the basis for the evaluation of
toxic effects and transfer of contaminants from soil into two trophic levels.
Long-term exposure tests were conducted to evaluate chronic, sublethal, toxicity
and transfer of aged soil-based explosives, with RDX as main contaminant. In
these tests, plants were exposed for 55 days in the greenhouse, biomass was
determined and residues of explosives parent compounds and RDX metabolites were
analyzed using HPLC techniques. Worms were exposed for 28 days (Eisenia fetida)
and 42 days (Enchytraeus crypticus) in the laboratory, biomass and number were
determined, and tissues were analyzed for explosives compounds. The plants
tolerated concentrations up to 1,540 mg RDX kg(-1) soil-DW. Biomass of Lolium
perenne was not significantly related to soil-RDX concentration, while biomass of
Medicago sativa significantly increased. No screening benchmark for RDX in soil
for plants was calculated, since concentrations up to 1,540 mg kg(-1) soil failed
to reduce biomass by 20% as required for a LOEC. RDX, RDX-metabolite MNX, and
accompanying HMX concentrations in plants were significantly related to
concentrations in soil after 55 days of exposure (RDX: R(2) = 0.77-0.89; MNX R(2)
= 0.53-0.77; HMX: R(2) = 0.67-0.71). The average bioconcentration factors (BCF)
were for RDX 17 in L. perenne and 37 in M. sativa, and for HMX 2 in L. perenne
and 44 in M. sativa. The worms also tolerated concentrations up to 1,540 mg RDX
kg(-1) soil-DW. Biomass of E. fetida adults decreased with soil-RDX
concentration, and a LOEC of 1,253 mg kg(-1) soil-DW was estimated. RDX
concentrations in E. fetida were significantly related to concentrations in soil
after 28-day exposure (R(2) = 0.88). The average BCF in E. fetida for RDX was 1.
Because in response to exposure to RDX-contaminated soil the RDX concentrations
in plants increased initially and decreased subsequently, while those in worms
increased continuously, RDX in worm tissues may accumulate to higher
concentrations than in plant tissues, regardless of the low average BCF for
worms.
2049. Mol Plant Microbe Interact. 2005 Jul;18(7):742-50.
GuaB activity is required in Rhizobium tropici during the early stages of

nodulation of determinate nodules but is dispensable for the Sinorhizobium
meliloti-alfalfa symbiotic interaction.
Collavino M(1), Riccillo PM, Grasso DH, Crespi M, Aguilar M.
Author information:
Universidad Nacional de La Plata, 1900-La Plata, Argentina.
The guaB mutant strain Rhizobium tropici CIAT8999-10T is defective in symbiosis

with common bean, forming nodules that lack rhizobial content. In order to
investigate the timing of the guaB requirement during the nodule formation on the
host common bean by the strain CIAT899-10.T, we constructed gene fusions in which
the guaB gene is expressed under the control of the symbiotic promoters nodA,
bacA, and nifH. Our data indicated that the guaB is required from the early
stages of nodulation because full recovery of the wild-type phenotype was
accomplished by the nodA-guaB fusion. In addition, we have constructed a guaB
mutant derived from Sinorhizobium meliloti 1021, and shown that, unlike R.
tropici, the guaB S. meliloti mutant is auxotrophic for guanine and induces
wild-type nodules on alfalfa and Medicago truncatula. The guaB R. tropici mutant
also is defective in its symbiosis with Macroptilium atropurpureum and Vigna
unguiculata but normal with Leucaena leucocephala. These results show that the
requirement of the rhizobial guaB for symbiosis is found to be associated with
host plants that form determinate type of nodules.
DOI: 10.1094/MPMI-18-0742
2050. Mol Plant Microbe Interact. 2005 Jul;18(7):694-702.
Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa

interactions.
Jensen JB(1), Ampomah OY, Darrah R, Peters NK, Bhuvaneswari TV.
Author information:
(1)Department of Biology, Faculty of Science, University of Tromsø, N-9037
Tromsø, Norway.
Erratum in
Mol Plant Microbe Interact. 2005 Nov;18(11):1243.
Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for
trehalose catabolism and are induced by trehalose but not by related structurally
similar disaccharides like sucrose or maltose. S. meliloti strains mutated in
either of these two genes were severely impaired in their ability to grow on
trehalose as the sole source of carbon. ThuA and ThuB show no homology to any
known enzymes in trehalose utilization. ThuA has similarity to proteins of
unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella
melitensis, and ThuB possesses homology to dehydrogenases containing the
consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the
root surface and in the infection threads but not in the symbiotic zone of the
nodules. Even though thuA and thuB mutants were impaired in competitive
colonization of Medicago sativa roots, these strains were more competitive than
the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing
nodules. Possible reasons for their increased competitiveness are discussed.
DOI: 10.1094/MPMI-18-0694
2051. Lett Appl Microbiol. 2005;41(2):112-8.
Comparison of primers for the detection of pathogenic Escherichia coli using

real-time PCR.
Barak JD(1), Sananikone K, Delwiche MJ.
Author information:
(1)Produce Safety and Microbiology Research Unit, USDA Agricultural Research
Service, Albany, CA 94710, USA. jbarak@pwusda.gov
AIMS: To evaluate PCR primers for the detection of pathogenic Escherichia coli in
a real-time PCR assay and determine their utility in produce irrigation water
testing.
METHODS AND RESULTS: Three previously published PCR primer sets and one set
designed for this study were tested for their ability to produce amplification
products for several pathogenic E. coli serotypes from whole cells as template.
Two of the previously published primer sets were chosen for real-time PCR
detection limit determination. The coneaeA and PEH detection limit of E. coli
O157:H7 was 10(0) and 10(1) CFU rxn(-1) in sterile water respectively. To detect
E. coli O157:H7 in sprout irrigation water, the water required dilution due to
PCR inhibitors. The detection limit of the coneaeA and PEH was 10(1) and between
10(2) and 10(3) CFU rxn(-1) in diluted sprout irrigation water respectively.
CONCLUSIONS: The primer set coneaeA was able to produce an amplification product
from each E. coli serotype, except O128:H7 and most sensitive for real-time PCR
detection of pathogenic E. coli in diluted sprout irrigation water.
SIGNIFICANCE AND IMPACT OF THE STUDY: The necessity of a dissociation analysis to
distinguish positive samples from those with fluorescence of random dsDNA
generation for real-time PCR in a complex background was established.
DOI: 10.1111/j.1472-765X.2005.01746.x
2052. J Basic Microbiol. 2005;45(4):251-6.
Selection of bacteria and plant seeds for potential use in the remediation of
diesel contaminated soils.
Al-Ghazawi Z(1), Saadoun I, Al-Shak'ah A.
Author information:
(1)Department of Civil Engineering, College of Engineering, Jordan University of
Science and Technology, Irbid-22110, Jordan.
Enumeration and recovery of the dominant bacteria from a chronically fuel

contaminated soil has been investigated. Bacterial counts from these polluted
soils ranged between 0.70x10(8) and 28.20x10(8) CFU/g soil. Three different types
of bacterial colonies have been recovered on the agar plates. Biochemical
examination of the recovered bacteria revealed that they mainly belonged to the
genus Pseudomonas, Micrococcus and Bacillus. Turbidity, cell biomass (dry weight
basis), and physical appearance determined the growth of these bacteria on
diesel. A noticeable decline in alfalfa (Medicago sativa) seeds germination of
15-30% was shown at 500 mg/kg diesel or higher. Under these contaminated
conditions, fescue grass (Cyndon dactylon) exhibited a higher viability than
alfalfa indicating that C. dactylon seeds are relatively tolerant to diesel and
can possibly be used in phytoremediation of diesel contaminated soils. Results of
diesel phyotoxicity to seed germination of these two plants were based on filter
paper media and therefore; should be considered as first indication only.
Extrapolation of such results to actual soil conditions should be cautiously
approached taking into account diesel sorption on soil and mechanisms of its
bioavailability.
Copyright (c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
DOI: 10.1002/jobm.200410503
2053. J Dairy Sci. 2005 Aug;88(8):3009-19.
Economic and environmental feasibility of a perennial cow dairy farm.
Rotz CA(1), Zartman DL, Crandall KL.
Author information:
(1)USDA/Agricultural Research Service, Pasture Systems and Watershed Management
Research Unit, University Park, PA 16802, USA. al.rotz@ars.usda.gov
More efficient and economical production systems are needed to improve the
sustainability of dairy farms. One concept to consider is using perennial cows.
Perennial cows are those that maintain a relatively high milk production for
>or=2 yr without going through the typical dry period followed by calving. Farm
records show that some cows have produced over 20 kg/d after 4 yr of continuous
lactation. A farm simulation model was used to evaluate the long-term
performance, environmental impact, and economics of a conceptual perennial cow
production system on a typical dairy farm in Pennsylvania. Compared with a
traditional 100-cow farm with replacement heifers produced on the farm, a
perennial herd of 100 cows and purchased replacements provided environmental
benefit but sustained a substantial economic loss. However, increasing the
perennial herd to 128 cows better utilized the feed produced on the farm.
Compared with the traditional 100-cow farm, use of the perennial 128-cow herd
reduced supplemental protein and mineral feed purchases by 38%, increased annual
milk sales by 21%, reduced nitrogen losses by 17%, maintained a phosphorus
balance, and increased annual net return to farm management by 3200 dollars. A
traditional 120-cow dairy farm with purchased replacements also used a similar
amount of farm-produced feed. Compared with this option, the farm with 128
perennial cows reduced protein and mineral feed purchases by 36%, maintained
similar annual milk sales, increased manure production by 7%, reduced N losses by
10%, and increased annual net return by 12,700 dollars. The economic feasibility
of the perennial-cow dairy farm was very sensitive to the milk production
maintained by the perennial herd and market prices for milk and perennial
replacement animals. The analysis was relatively insensitive to the assumed
useful life of perennial cows as long as they could be maintained in the herd for
at least 3 yr. Thus, a perennial cow production system can improve the economic
and environmental sustainability of a traditional dairy farm if a similar level
in annual milk production per cow can be maintained.
DOI: 10.3168/jds.S0022-0302(05)72981-7
2054. J Dairy Sci. 2005 Aug;88(8):2911-22.
A survey of selected heavy metal concentrations in Wisconsin dairy feeds.
Li Y(1), McCrory DF, Powell JM, Saam H, Jackson-Smith D.
Author information:
(1)Laboratory for Environmental Remediation, Institute of Geographical Sciences
and Natural Resources Research, Chinese Academy of Sciences, Beijing, PR China.
Heavy metals such as zinc (Zn), copper (Cu), chromium (Cr), arsenic (As), cadmium
(Cd), and lead (Pb) are potential bioaccumulative toxins of the dairy production
system. The heavy metal content of dairy feeds, however, remains poorly
documented, particularly in the United States. This survey determined the heavy
metal content of 203 typical dairy ration components sampled from 54 dairy farms
in Wisconsin. Lowest heavy metal concentrations were found in homegrown alfalfa
(Medicago sativa L.) hay and haylage, and corn (Zea mays L.) grain and silage.
Highest metal concentrations were found in purchased feeds, particularly mineral
supplements, and to a lesser extent corn- or soybean-based concentrates. Zinc and
Cu were found at the highest concentration in complete dairy (total mixed and
aggregated component) rations and reflected the deliberate addition of these
metals to meet animal nutrient requirements although more than half the farms fed
Cu and Zn above US recommended levels. Concentrations of Cr, As, Cd, and Pb were
present in much lower concentrations and decreased in the order Cr > As > Pb >
Cd. No complete Wisconsin dairy ration contained heavy metal concentrations above
US maximum acceptable concentrations and would be unlikely to induce any toxic
effects in dairy cattle. Concentrations of Cd in complete dairy rations were
closest to US maximum acceptable concentrations, suggesting the greatest
potential long-term risk to exceed US maximum acceptable concentrations if whole
farm levels of Cd were to increase in the future. With the exception of Pb, the
main sources of Zn, Cu, Cr, As, and Cd in the complete dairy feed ration
originated from imported feed. The continued importation of heavy metals in dairy
feed is likely to be associated with accumulation of these metals in soils where
manure is applied. Although the cycling of many heavy metals through the dairy
food chain will be limited by factors such as a soil's cation exchange capacity,
pH, salinity, and phytotoxicity of the metal, these may be less limiting for Cd.
It is important that sources of Cd in the dairy system are identified and
minimized to prevent problems associated with Cd accumulation in the dairy soil
system arising over the long-term.
DOI: 10.3168/jds.S0022-0302(05)72972-6
2055. J Dairy Sci. 2005 Aug;88(8):2860-9.
A meta-analysis on the relationship between intake of nutrients and body weight

with milk volume and milk protein yield in dairy cows.
Hristov AN(1), Price WJ, Shafii B.
Author information:
(1)Department of Animal and Veterinary Science, College of Agricultural and Life
Sciences, University of Idaho, Moscow 83844. ahristov@uidaho.edu
Previously observed strong relationships between dry matter (DM) intake and milk
yield in dairy cows were the basis for this meta-analysis aimed to determine the
influence of intake of specific dietary nutrients on milk yield and milk protein
yield in Holstein dairy cows. Diets (563) from feeding trials published in the
Journal of Dairy Science were evaluated for nutrient composition using 2 diet
evaluation programs. Intake of nutrients was estimated based on DM intake and
program-derived diet composition. Data were analyzed with and without the effect
of stage of lactation. Models based on intake of nutrients improved prediction of
milk yield and milk protein yield compared with DM intake alone. Intake of net
energy of lactation was the dominant variable in milk yield prediction models
derived from both diet evaluation models. Milk protein yield models also improved
prediction over the DM intake model. These models were dominated by ruminally
undegradable protein intake and included a number of energy-related intake
variables. In most models, incorporating stage of lactation improved the model
fit.
DOI: 10.3168/jds.S0022-0302(05)72967-2
2056. J Dairy Sci. 2005 Aug;88(8):2850-9.
Utilization of phosphorus in lactating cows fed varying amounts of phosphorus and

sources of fiber.
Wu Z(1).
Author information:
University Park 16802, USA. ziw1@psu.edu
This study was undertaken to determine the effect of dietary P content and fiber
source on P utilization. Four dietary treatments were formed in a 2 x 2 factorial
arrangement. The P content was 0.32 or 0.44%, and the fiber source was varied by
substituting 10% soyhulls for 6% alfalfa hay on a dry matter (DM) basis. Diets
also contained approximately 50% corn silage and alfalfa silage for all
treatments. The diets were fed to 32 early to midlactation Holsteins for 10 wk.
Fecal P excretion was estimated using indigestible acid detergent fiber marker
determined with 12-d in situ incubation and grab sampling. Milk yield was high,
averaging 43 kg/d across treatments, and 42.1 and 44.0 kg/d for the 0.32 and
0.44% P diets, respectively. Milk fat content was also high, averaging 3.68 and
4.12% for the 0.32 and 0.44% P diets, respectively. Milk protein yield averaged
1.240 and 1.323 kg/d. Differences in milk production were associated with 1.5
kg/d less DM intake for the lower P diets on average. Based on lactation
performance, 0.32% P appeared inadequate for this level of production, whereas
the calculated (National Research Council) requirement was 0.37%. Fecal P
concentration increased linearly with P intake, and based on this relationship,
reducing dietary P from 0.44 to 0.37% would reduce fecal P excretion by 12%.
Partial substitution of soyhulls for alfalfa hay did not affect feed intake or
milk production, but reduced fecal P excretion, partially because of increased P
apparent digestibility. The reduction in fecal P excretion resulting from reduced
P intake or substitution of soyhulls for alfalfa hay was apparently through
reductions in the regulated portion of fecal P. Cows producing 43 kg/d of milk
appeared to need > 0.32% P, whereas the requirement assessed from National
Research Council data was 0.37%. Using highly digestible nonforage fiber sources
in place of forage fiber sources in the diet may allow less P to be fed while
still meeting the requirement.
DOI: 10.3168/jds.S0022-0302(05)72966-0
2057. J Anim Sci. 2005 Aug;83(8):1967-71.
Effects of gamma-terpinene, terpinolene, alpha-copaene, and alpha-terpinene on

consumption of alfalfa pellets by sheep.
Estell RE(1), Fredrickson EL, Anderson DM, Remmenga MD.
Author information:
(1)ARS, USDA, Jornada Experimental Range, Las Cruces, NM 88003-8003, USA.
restell@nmsu.edu
Although plant secondary chemistry influences shrub consumption by free-ranging

ruminants, the effects of many specific compounds on herbivores have not been
examined. We conducted four experiments to examine effects of individual terpenes
on alfalfa pellet intake by lambs. Forty-five lambs were individually fed alfalfa
pellets sprayed with gamma-terpinene, terpinolene, alpha-copaene, or
alpha-terpinene at one of five concentrations in an ethanol carrier. Treatments
(0, 0.5, 1, 2, and 10x) were multiples of the concentration (x) of a specific
terpene on the leaf surface of Flourensia cernua (a low-preference shrub for
domestic ruminants). Terpenes were applied to alfalfa pellets (0.64
kg.lamb(-1).d(-1), DM basis), and consumption was measured during a 20-min
interval for 5 d. A day effect was detected for gamma-terpinene on intake (P <
0.001 for both linear and quadratic contrasts). No effect of terpinolene,
alpha-copaene, or alpha-terpinene on intake was detected in this study. None of
the terpenes tested was strongly related to intake of alfalfa pellets by lambs
under the conditions of this study.
DOI: 10.2527/2005.8381967x
2058. Plant Mol Biol. 2005 Jun;58(3):385-99.
Defensin gene family in Medicago truncatula: structure, expression and induction

by signal molecules.
Hanks JN(1), Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM.
Author information:
(1)Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO
63132, USA.
A large gene family encoding the putative cysteine-rich defensins was discovered
in Medicago truncatula. Sixteen members of the family were identified by
screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the
Institute for Genomic Research's (TIGR) M. truncatula gene index (MtGI version
7). Based on the comparison of their amino acid sequences, M. truncatula
defensins fell arbitrarily into three classes displaying extensive sequence
divergence outside of the eight canonical cysteine residues. The presence of
Class II defensins is reported for the first time in a legume plant. In silico as
well as Northern blot and RT-PCR analyses indicated these genes were expressed in
a variety of tissues including leaves, flowers, developing pods, mature seed and
roots. The expression of these genes was differentially induced in response to a
variety of biotic and abiotic stimuli. For the first time, a defensin gene
(TC77480) was shown to be induced in roots in response to infection by the
mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the
tissue-specific expression patterns of the cloned Def1 and Def2 genes differed
substantially between M. truncatula and M. sativa. Furthermore, the induction
profiles of the Def1 and Def2 genes in response to the signaling molecules methyl
jasmonate, ethylene and salicylic acid differed markedly between these two
legumes.
DOI: 10.1007/s11103-005-5567-7
2059. Chemosphere. 2005 Sep;60(9):1203-13. Epub 2005 Apr 9.
Selenium volatilization in vegetated agricultural drainage sediment from the San

Luis Drain, Central California.
Bañuelos GS(1), Lin ZQ, Arroyo I, Terry N.
Author information:
(1)USDA-ARS, Water Management Research Laboratory, Parlier, CA 93648, USA.
gbanuelos@fresno.ars.usda.gov
The presence of large amounts of Se-laden agricultural drainage sediment in the

San Luis Drain, Central California, poses a serious toxic threat to wildlife in
the surrounding environment. Effective management of the drainage sediment
becomes a practical challenge because the sediment is polluted with high levels
of Se, B, and salts. This two-year field study was conducted to identify the best
plant species that are salt and B tolerant and that have a superior ability of
volatilizing Se from drainage sediment. The drainage sediment was mixed with
clean soil, and vegetated with salado alfalfa (Medicago sativa 'salado'), salado
grass (Sporobulus airoides 'salado'), saltgrass-turf (Distichlis spp. 'NYPA
Turf'), saltgrass-forage (Distichlis spicata (L.) Greene), cordgrass (Spartina
patens 'Flageo'), Leucaenia (Leucaena leucocephola), elephant grass (Pennistum
purpureum), or wild type-Brassica (Brassica spp.). Results show that elephant
grass produced the greatest amount of biomass and accumulated highest
concentrations of B. Highest concentrations of Se, S, and Cl were observed in
wild-type Brassica. Biogenic volatilization of Se by plants and soil microbes was
greater in summer. Among the treatments, the mean daily rates of Se
volatilization (microg Se m(-2)d(-1)) were wild-type Brassica (39) >
saltgrass-turf (31) > cordgrass (27) > saltgrass forage (24) > elephant grass
(22) > salado grass (21) > leucaenia (19) > salado alfalfa (14) > irrigated bare
soil (11) > non-irrigated bare soil (6). Overall, rates of Se volatilization in
drainage sediment were relatively low due to high levels of sulfate. To manage Se
in drainage sediment by phytoremediation, the biological volatilization process
needs to be enhanced substantially under field conditions.
2060. Plant Physiol. 2005 Jul;138(3):1301-9.
Munich information center for protein sequences plant genome resources: a

framework for integrative and comparative analyses 1(W).
Schoof H(1), Spannagl M, Yang L, Ernst R, Gundlach H, Haase D, Haberer G, Mayer

KF.
Author information:
(1)Technische Universität München, Chair of Genome Oriented Bioinformatics,
Center of Life and Food Science, D-85354 Freising-Weihenstephan, Germany.
schoof@mpiz-koeln.mpg.de
With several plant genomes sequenced, the power of comparative genome analysis
can now be applied. However, genome-scale cross-species analyses are limited by
the effort for data integration. To develop an integrated cross-species plant
genome resource, we maintain comprehensive databases for model plant genomes,
including Arabidopsis (Arabidopsis thaliana), maize (Zea mays), Medicago
truncatula, and rice (Oryza sativa). Integration of data and resources is
emphasized, both in house as well as with external partners and databases. Manual
curation and state-of-the-art bioinformatic analysis are combined to achieve
quality data. Easy access to the data is provided through Web interfaces and
visualization tools, bulk downloads, and Web services for application-level
access. This allows a consistent view of the model plant genomes for comparative
and evolutionary studies, the transfer of knowledge between species, and the
integration with functional genomics data.
DOI: 10.1104/pp.104.059188
PMCID: PMC1176403
2061. Plant Physiol. 2005 Aug;138(4):2245-59. Epub 2005 Jul 8.
Metabolic engineering of isoflavonoid biosynthesis in alfalfa.
Deavours BE(1), Dixon RA.
Author information:
(1)Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma
73401, USA.
The potential health benefits of dietary isoflavones have generated considerable

interest in engineering the synthesis of these phytoestrogens into plants.
Genistein glucoside production (up to 50 nmol g(-1) fresh weight) was engineered
in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone
synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A
(4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated.
Although MtIFS1 was highly expressed in all organs examined, genistein
accumulation was limited to leaves. MtIFS1-expressing lines accumulated several
additional isoflavones, including formononetin and daidzein, in response to UV-B
or Phoma medicaginis, whereas the chalcone and flavanone precursors of these
compounds accumulated in control lines. Enhanced accumulation of the phytoalexin
medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing
plants. Microarray profiling indicated that MtIFS1 expression does not
significantly alter global gene expression in the leaves. Our results highlight
some of the challenges associated with metabolic engineering of plant natural
products, including tissue-specific accumulation, potential for further
modification by endogenous enzyme activities (hydroxylation, methylation, and
glycosylation), and the differential response of engineered plants to
environmental factors.
DOI: 10.1104/pp.105.062539
PMCID: PMC1183411
2062. J Exp Bot. 2005 Aug;56(418):2239-51. Epub 2005 Jul 4.
Cellular damage induced by cadmium and mercury in Medicago sativa.
Ortega-Villasante C(1), Rellán-Alvarez R, Del Campo FF, Carpena-Ruiz RO,

Hernández LE.
Author information:
Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain.
Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the

kinetics of diverse stress indexes. In the so-called beaker-size hydroponic
system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress
took place and increased over time, a linear response being observed with Cd but
not with Hg. To improve the sensitivity of the stress assays used, a micro-assay
system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of
metals, quantified as growth inhibition, was observed well before there was any
change in the non-protein thiol tissue concentration. When measured with
conventional techniques, oxidative stress indexes did not show significant
variation. To trace early and small plant responses to Cd and Hg, a microscopic
analysis with novel fluorescent dyes, which had not yet been exploited to any
significant extent for use in plants, was conducted. These fluorescent probes,
which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals
to be visualized in the roots of the alfalfa seedlings, were: (i)
2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane
that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium
iodide that marks nuclei of dead cells. Oxidative stress and cell death increased
after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased
acutely. This diminution might be the result of direct interaction of GSH/hGSH
with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore,
both Cd and Hg not only compromised severely the cellular redox homeostasis, but
also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine,
a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms
appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient
to promote cell death, and that other phytotoxic mechanisms might be involved.
DOI: 10.1093/jxb/eri223
2063. Virology. 2005 Sep 1;339(2):153-63.

Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus
infection.
Dong JL(1), Liang BG, Jin YS, Zhang WJ, Wang T.
Author information:
(1)State Key Laboratory of Agrobiotechnology, China Agricultural University, 2
Yuanmingyuan West Road, Haidian District, Beijing 100094, China.
dongjiangli@126.com
A critical factor in edible plant-derived vaccine development is adequate

expression of the exogenous antigens in transgenic plants. We synthesized a
codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus
and inserted it into the alfalfa genome using agrobacterium-mediated
transformation. As much as 0.28% of the total soluble protein of the
pBsVP6-transgenic alfalfa was sVP6. Female BALB/c mice were gavaged weekly with
10 mg of transgenic alfalfa extract containing 24 microg of sVP6 protein and 10
microg of CpG-rich oligodeoxynucleotides as mucosal adjuvant. Immunized mice
developed high titers of anti-VP6 serum IgG and mucosal IgA. Offspring of
immunized dams developed less severe diarrhea after challenge with simian
rotavirus SA-11, indicating that antibodies generated in the dams provided
passive heterotypic protection to the pups. These results suggest that oral
immunization with pBsVP6-transgenic alfalfa provides a potential means of
protecting children and young animals from severe acute rotavirus-induced
diarrhea.
DOI: 10.1016/j.virol.2005.06.004
2064. Mol Biol Evol. 2005 Oct;22(10):2084-9. Epub 2005 Jun 29.
MUSTANG is a novel family of domesticated transposase genes found in diverse

angiosperms.
Cowan RK(1), Hoen DR, Schoen DJ, Bureau TE.
Author information:
(1)McGill University, Biology Department, Montreal, Quebec H3A 1B1, Canada.
While transposons have traditionally been viewed as genomic parasites or "junk

DNA," the discovery of transposon-derived host genes has fueled an ongoing debate
over the evolutionary role of transposons. In particular, while mobility-related
open reading frames have been known to acquire host functions, the contribution
of these types of events to the evolution of genes is not well understood. Here
we report that genome-wide searches for Mutator transposase-derived host genes in
Arabidopsis thaliana (Columbia-0) and Oryza sativa ssp. japonica (cv. Nipponbare)
(domesticated rice) identified 121 sequences, including the taxonomically
conserved MUSTANG1. Syntenic MUSTANG1 orthologs in such varied plant species as
rice, poplar, Arabidopsis, and Medicago truncatula appear to be under purifying
selection. However, despite the evidence of this pathway of gene evolution,
MUSTANG1 belongs to one of only two Mutator-like gene families with members in
both monocotyledonous and dicotyledonous plants, suggesting that Mutator-like
elements seldom evolve into taxonomically widespread host genes.
DOI: 10.1093/molbev/msi202
2065. Yi Chuan. 2005 May;27(3):391-4.
[Genetic diversity analysis of alfalfa floral properties.].
Bi YF, He CG, Zhou H.
Genetic mutations of alfalfa floral properties,including calyx diameter, coronary

length, flower number per raceme, raceme number per twig, flower number per unit
area, percentage of flicked flowers, excreted nectar volume, sugar concentration
in nectar and contents of sucrose, fructose and glucose in nectar,have been
studied with both morphological markers and RAPD markers. The results showed that
the genetic variation of floral properties is from 0.80% to 92.30%, of which the
content of glucose was the most significant one with the variation from 0.01 to
0.53 mumol/L (p0.05), and the sugar concentration was the most insignificant one
(P>0.05). RAPD analysis showed that variances of genetic distances was from 0.21
to 0.35, among which the most significant one was WL323 and Shanbei, and the most
insignificant one was Derby and Prime. Thus it can be seen that genetic diversity
of floral properties among these varieties of alfalfas is abundant.
2066. Ann Allergy Asthma Immunol. 2005 Jun;94(6):A6.
Medicago sativa.
Weber RW(1).
Author information:
(1)National Jewish Medical & Research Center, 1400 Jackson Street, Room J326,
Denver, CO 80206, USA.
2067. Cell Cycle. 2005 Aug;4(8):1084-92. Epub 2005 Aug 18.
Arabidopsis anaphase-promoting complexes: multiple activators and wide range of

substrates might keep APC perpetually busy.
Fülöp K(1), Tarayre S, Kelemen Z, Horváth G, Kevei Z, Nikovics K, Bakó L, Brown

S, Kondorosi A, Kondorosi E.
Author information:
(1)Institut des Sciences du Végétal, Gif sur Yvette, France.
The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an

essential regulator of the cell cycle from metaphase until S phase in yeast and
metazoans. APC mediates degradation of numerous cell cycle-related proteins,
including mitotic cyclins and its activation and substrate-specificity are
determined by two adaptor proteins, Cdc20 and Cdh1. Plants have multiple APC
activators and the Cdh1-type proteins, in addition, are represented by two
subclasses, known as Ccs52A and Ccs52B. The Arabidopsis genome contains five
cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. In Schizosaccharomyces pombe,
expression of the three Atccs52 genes elicited distinct phenotypes supporting
nonredundant function of the AtCcs52 proteins. Consistent with these activities,
the AtCcs52 proteins were able to bind both to the yeast and the Arabidopsis
APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts were
present from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1
and ccs52A2 were from late M until early G2, suggesting consecutive action of
these APC activators in the plant cell cycle. The AtCcs52 proteins interacted
with different subsets of mitotic cyclins, in accordance with their expression
profiles, either in free- or CDK-bound forms. Expression of most APC subunits was
constitutive, whereas cdc27a and cdc27b, corresponding to two forms of apc3, and
ubc19 and ubc20 encoding E2-C type ubiquitin-conjugating enzymes displayed
differences in their cell cycle regulation. These data indicate the existence of
numerous APC(Cdc20/Ccs52/Cdc27) forms in Arabidopsis, which in conjunction with
different E2 enzymes might have distinct or complementary functions at distinct
stages of the cell cycle.
2068. J Biol Chem. 2005 Aug 26;280(34):30496-503. Epub 2005 Jun 21.
Structural elucidation of chalcone reductase and implications for deoxychalcone

biosynthesis.
Bomati EK(1), Austin MB, Bowman ME, Dixon RA, Noel JP.
Author information:
(1)Jack Skirball Chemical Biology and Proteomics Laboratory, Salk Institute for
Biological Studies, La Jolla, California 92037, USA
4,2',4',6'-Tetrahydroxychalcone (chalcone) and 4,2',4'-trihydroxychalcone

(deoxychalcone) serve as precursors of ecologically important flavonoids and
isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone
reductase; however, the identity of the chalcone reductase substrate out of the
possible substrates formed during the multistep reaction catalyzed by chalcone
synthase remains experimentally elusive. We report here the three-dimensional
structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose
the identity and binding mode of its substrate, namely the non-aromatized
coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of
the fully extended coumaryl-tetraketide thioester intermediate. In the absence of
a ternary complex, the quality of the refined NADP+-bound chalcone reductase
structure serves as a template for computer-assisted docking to evaluate the
likelihood of possible substrates. Interestingly, chalcone reductase adopts the
three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto
reductase fold is structurally distinct from all known ketoreductases of fatty
acid biosynthesis, which instead belong to the short-chain
dehydrogenase/reductase superfamily. The results presented here provide
structural support for convergent functional evolution of these two
ketoreductases that share similar roles in the biosynthesis of fatty
acids/polyketides. In addition, the chalcone reductase structure represents the
first protein structure of a member of the aldo/ketoreductase 4 family.
Therefore, the chalcone reductase structure serves as a template for the homology
modeling of other aldo/keto-reductase 4 family members, including the reductase
involved in morphine biosynthesis, namely codeinone reductase.
DOI: 10.1074/jbc.M502239200
PMCID: PMC2860619
2069. J Bacteriol. 2005 Jul;187(13):4562-72.
Two new Sinorhizobium meliloti LysR-type transcriptional regulators required for

nodulation.
Luo L(1), Yao SY, Becker A, Rüberg S, Yu GQ, Zhu JB, Cheng HP.
Author information:
York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA.
The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium

meliloti and its legume host alfalfa (Medicago sativa) depends on the timely
expression of nodulation genes that are controlled by LysR-type regulators.
Ninety putative genes coding for LysR-type transcriptional regulators were
identified in the recently sequenced S. meliloti genome. All 90 putative lysR
genes were mutagenized using plasmid insertions as a first step toward
determining their roles in symbiosis. Two new LysR-type symbiosis regulator
genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB
genes are expressed in free-living S. meliloti cells, but they are not required
for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only
white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant
were recovered from the white nodules, suggesting that the lsrA1 mutant was
blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and
elicited a mixture of pink and white nodules on alfalfa plants. These plants
exhibited lower overall nitrogenase activity than plants inoculated with the
wild-type strain, which is consistent with the fact that most of the alfalfa
plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1
mutant were recovered from both pink and white nodules, suggesting that lsrB1
mutants could be blocked at multiple points during nodulation. The identification
of two new LysR-type symbiosis transcriptional regulators provides two new
avenues for understanding the complex S. meliloti-alfalfa interactions which
occur during symbiosis.
DOI: 10.1128/JB.187.13.4562-4572.2005
PMCID: PMC1151776
2070. Epidemiol Infect. 2005 Jun;133(3):439-47.
Temporally distinct Escherichia coli 0157 outbreaks associated with alfalfa

sprouts linked to a common seed source--Colorado and Minnesota, 2003.
Ferguson DD(1), Scheftel J, Cronquist A, Smith K, Woo-Ming A, Anderson E, Knutsen

J, De AK, Gershman K.
Author information:
(1)Epidemic Intelligence Service, Colorado Department of Public Health and
Environment, Denver, CO 80246, USA. dayna.ferguson@state.co.us
Escherichia coli O157 outbreaks were identified in Minnesota in February 2003

involving seven persons and in Colorado in July 2003 involving 13 persons. Case
isolates from the two states had matching pulsed-field gel electrophoresis (PFGE)
patterns. Independent case-control studies linked infections in each outbreak
with eating alfalfa sprouts that were traced to the same seed distributor. The
Colorado sprouter reportedly complied with the Food and Drug Administration (FDA)
sprout guidance, whereas the Minnesota sprouter did not. These investigations
revealed that increased compliance with existing FDA guidance is needed and that
additional research is needed to improve the alfalfa seed decontamination
process. This reaffirms the FDA recommendation that raw alfalfa sprouts should be
considered potentially contaminated and avoided by persons at high-risk such as
the elderly, young children, and immunocompromised persons. PFGE played an
essential role in linking these two temporally and geographically distinct E.
coli O157 outbreaks.
PMCID: PMC2870267
2071. J Anim Sci. 2005 Jul;83(7):1616-24.
Changes in ruminal fermentation and protein degradation in growing Holstein

heifers from 80 to 250 kg fed high-concentrate diets with different
forage-to-concentrate ratios.
Rotger A(1), Ferret A, Calsamiglia S, Manteca X.
Author information:
(1)Departament de Ciència Animal i dels Aliments, Universitat Autònoma de
Barcelona, 08193 Bellaterra, Spain.
Six Holstein heifers (initial BW = 65.2 +/- 1.8 kg) fitted with ruminal cannulas
were used in a repeated measures trial to assess the effect of age and
forage-to-concentrate ratio on ruminal fermentation end products and in situ
degradation kinetics of four plant protein supplements (soybean meal, sunflower
meal, peas, and lupin seeds). Alfalfa hay also was incubated in situ to estimate
NDF degradation. Three experimental periods were conducted at 13, 27, and 41 wk
of age. Heifers were fed one of two diets, 12:88 vs. 30:70 forage-to-concentrate
ratio (DM basis), offered as total mixed ration on an ad libitum basis. Intakes
of DM, OM, CP, NDF, and ADG were not affected (P > or = 0.105) by diet. The 30:70
diet resulted in faster (P = 0.045) fluid passage rate and decreased (P = 0.015)
ammonia N concentration compared with the 12:88 diet, but no differences (P > or
= 0.244) were detected in ruminal pH and total VFA concentration between diets.
The rate of degradation and the effective degradability of N in protein
supplements was greater with the 30:70 diet for peas (P < or = 0.008) and lupin
seeds (P < or = 0.02), and in the 12:88 diet for sunflower meal (P < or = 0.06).
Degradation of NDF of alfalfa hay was low with both diets (18.5 and 23.7 % for
12:88 and 30:70, respectively); however, the rate and extent of DM and NDF
degradation were greater (P < or = 0.016) with the 30:70 diet, suggesting a
higher cellulolytic activity. Total VFA concentration and the proportion of
propionate increased (P < or = 0.035), and the acetate proportion decreased (P =
0.021) with age. Average pH, ammonia N concentration, and passage rates were not
affected (P > or = 0.168) by age. Degradation rate and effective degradability of
N of sunflower meal, peas, lupin seeds, and of DM of alfalfa hay increased (P <
or = 0.08) with age, but degradation kinetics of NDF of alfalfa hay was not
affected (P > or = 0.249). The increase in the rate and extent of N degradation
with age would suggest an increase in proteolytic activity, and the changes in
the fermentation pattern may reflect an increase in amylolytic activity caused
mainly by an increase in the gross intake of nonstructural carbohydrates and by
adaptation of ruminal microflora after long exposure to these nutrients.
DOI: 10.2527/2005.8371616x
2072. J Dairy Sci. 2005 Jul;88(7):2556-70.
Varying protein and starch in the diet of dairy cows. II. Effects on performance
and nitrogen utilization for milk production.
Ipharraguerre IR(1), Clark JH.
Author information:
(1)Department of Animal Sciences, University of Illinois, Urbana, 61801, USA.
ipharrag@uiuc.edu
The main objective of this experiment was to examine the effects of the
percentage and source of crude protein (CP) and the amount of starch in the diet
of dairy cows on the lactational performance and use of N for milk production.
Sixty multiparous Holstein cows were used in a 210-d lactational trial with a
completely randomized design with a 2 x 3 factorial arrangement of treatments.
Two sources of CP [solvent-extracted soybean meal (SBM) and a mixture of SBM and
a blend of animal-marine protein supplements plus ruminally protected Met (AMB)]
and 3 levels of dietary CP (means = 14.8, 16.8, and 18.7%) were combined into 6
treatments. On a dry matter (DM) basis, diets contained 25.0% corn silage, 20.0%
alfalfa silage, 10.0% cottonseed, 26.7 to 37.0% corn grain, and 4.8 to 13.5%
protein supplement, plus minerals and vitamins. Across the 210 d of lactation,
the productive response of dairy cows to the source of supplemental CP depended
on the concentration of CP in the diet. At 18.7% CP, cows fed SBM consumed more
DM and produced more milk, 3.5% fat-corrected milk, fat, and true protein, but
had lower efficiency of feed use and body condition score than cows fed AMB. At
16.8% CP, cows fed AMB produced more 3.5% fat-corrected milk, fat, and true
protein than cows fed SBM. At 14.8% CP, cows fed SBM consumed more DM but
produced less true protein and had lower feed efficiency than cows fed AMB.
Across CP sources, cows fed 14.8% CP produced less fat-corrected milk and true
protein than cows fed 16.8 and 18.7% CP. Across CP percentages, cows fed AMB
produced more fat-corrected milk per kilogram of DM consumed than cows fed SBM.
Despite these interactions, improvements in the gross efficiency of N use for
milk production were achieved through reductions in the intake of N independently
of the source of CP. Data suggest that the intake of N by high-producing dairy
cows that consume sufficient energy and other nutrients to meet their
requirements can be decreased to about 600 to 650 g daily if the source of RDP
and RUP are properly matched with the source and amount of carbohydrate in the
diet.
DOI: 10.3168/jds.S0022-0302(05)72932-5
2073. J Dairy Sci. 2005 Jul;88(7):2537-55.
Varying protein and starch in the diet of dairy cows. I. Effects on ruminal
fermentation and intestinal supply of nutrients.
Ipharraguerre IR(1), Clark JH, Freeman DE.
Author information:
(1)Department of Animal Sciences, University of Illinois, Urbana, 61801, USA.
ipharrag@uiuc.edu
The main objective of this experiment was to examine the effects of the
percentage and source of crude protein (CP) and the amount of starch in the diet
of dairy cows on ruminal fermentation, nutrient passage to the small intestine,
and nutrient digestibility. For this purpose, 6 multiparous Holstein cows
fistulated in the rumen and duodenum that averaged 73 d in milk were used in a 6
x 6 Latin square design with a 2 x 3 factorial arrangement of treatments. Two
sources of CP [solvent-extracted soybean meal (SBM) and a mixture of SBM and a
blend of animal-marine protein supplements plus ruminally protected Met (AMB)]
and 3 levels of dietary protein (about 14, 16, and 18%) were combined into 6
treatments. On a dry matter (DM) basis, diets contained 25% corn silage, 20%
alfalfa silage, 10% cottonseed, 26.7 to 37% corn grain, and 4 to 13.5% protein
supplement. Intakes and digestibilities in the rumen and total tract of DM,
organic matter, acid and neutral detergent fiber were unaffected by treatments.
Increasing dietary CP from 14 to 18% decreased the intake and apparent ruminal
and total tract digestion of starch, but increased the proportion of starch
consumed by the cows that was apparently digested in the small intestine. At 14%
CP, starch intake and total tract digestion were higher for the AMB diet than for
the SBM diet, but the opposite occurred at 16% CP. Across CP sources, increasing
CP in the diet from 14 to 18% increased the intakes of N and amino acids (AA),
and ruminal outflows of nonammonia N, nonammonia nonmicrobial N, each individual
AA except Met, total essential AA, and total AA. Across CP percentages, replacing
a portion of SBM with AMB increased the intake of Met and Val and decreased the
concentration of ammonia N in the rumen, but did not affect the intake of other
essential AA or the intestinal supply of any essential AA and starch. The ruminal
outflow of microbial N, the proportional contribution of Lys and Met to total AA
delivered to the duodenum, and milk yield were unaffected by treatments. Data
suggest that the intake of N by high-producing dairy cows that consume sufficient
energy and other nutrients to meet their requirements can be decreased to about
600 to 650 g daily without compromising the supply of metabolizable protein if
the source and amount of dietary CP and carbohydrate are properly matched.
DOI: 10.3168/jds.S0022-0302(05)72931-3
2074. J Dairy Sci. 2005 Jul;88(7):2487-97.
Effects of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester

on milk production and composition by Holstein cows.
St-Pierre NR(1), Sylvester JT.
Author information:
USA. st-pierre.8@osu.edu
The esterification of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) to isopropanol

(HMBi) decreases the rate and extent of its ruminal breakdown. The modes of
action of HMB and HMBi appear to be different. The quantification of the
production response to HMBi has not been done. The objectives of this study were
(1) to determine the lactation response to HMB, (2) to determine the lactation
response to HMBi, and (3) to evaluate whether the response to HMBi is affected by
HMB in the diet. Sixty-one Holstein cows (24 primiparous, 37 multiparous) were
assigned to 1 of 4 dietary treatments 21 to 28 d after calving. The base diet
consisted of [on a dry matter (DM) basis] 32.5% corn silage, 17.5% alfalfa hay,
10% whole cottonseed, and 40% of a pelleted concentrate made primarily of ground
corn, soybean meal, and blood meal, and was fed for 16 wk as a control diet. To
prepare the dietary treatments, the base diet was supplemented with 0.1% of diet
DM with HMB (treatment 2), with 0.15% HMBi (treatment 3), or with 0.045% HMB and
0.15% HMBi (treatment 4). Results showed a significant increase in milk yield
(2.9 kg/d), protein content (0.15%), protein yield (115 g/d), fat yield (165
g/d), and lactose yield (182 g/d) from HMBi. Supplementation of HMB had small and
nonsignificant effects on milk yield and composition. There were no significant
interaction effects of HMB with HMBi on any of the production traits measured in
this experiment. Plasma free Met as a proportion of essential amino acids was
increased by HMBi, but not by HMB. Dietary supplementation of HMBi increased
gross N efficiency expressed as the proportion of ingested N secreted in milk.
Consequently, HMBi significantly improved N efficiency.
DOI: 10.3168/jds.S0022-0302(05)72926-X
2075. Biotechnol Bioeng. 2005 Aug 20;91(4):447-59.
Industrial process proteomics: alfalfa protein patterns during wet fractionation

processing.
Incamps A(1), Hély-Joly F, Chagvardieff P, Rambourg JC, Dedieu A, Linares E,

Quéméneur E.
Author information:
(1)Viridis S.A., Aulnay-aux-Planches, F-51130 Val des Marais, France.
A proteome reference map of major soluble proteins from Medicago sativa (alfalfa)
leaves and stems has been established for the first time. Among 195 spots
analyzed by mass spectrometry and N-terminal Edman sequencing, 117 spots were
unambiguously identified, representing 87 different proteins. Of these 87
proteins, 13 proteins were directly identified from the partial genome of
Medicago sativa, 30 from expressed sequenced tags (ESTs) of the model legume
Medicago truncatula and 44 from closely relative species by a cross-species
protein identification method. The proteome map of Medicago sativa was then set
as a reference to study the major high protein content products that are
generated during the wet fractionation process of alfalfa green biomass. Using
two-dimensional electrophoresis, we studied the variation of the protein patterns
at different steps of the industrial-scale process. We clearly show that the
process induces significant changes including chemical modifications, proteolytic
events, and heat-shock protein responses. Strikingly, a certain level of cellular
regulation is conserved during biomass processing, as exemplified by the
induction of some heat shock proteins. Finally, all the results obtained in this
proteomic study may help to identify novel products and to improve process
designs in alfalfa biomass plants.
Copyright 2005 Wiley Periodicals, Inc.
DOI: 10.1002/bit.20520
2076. Vet Parasitol. 2005 Aug 10;131(3-4):267-82.
Effect of forage legumes and anthelmintic treatment on the performance,

nutritional status and nematode parasites of grazing lambs.
Marley CL(1), Fraser MD, Fychan R, Theobald VJ, Jones R.
Author information:
(1)Plant, Animal and Microbial Science Department, Institute of Grassland and
Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, UK.
christina.marley@bbsrc.ac.uk
Recent studies in New Zealand and the UK have shown that certain forages reduce
parasitic infection in sheep. The aim of this experiment was to investigate the
effects of legume forages compared to ryegrass on interactions between
production, nutritional status and nematodes in grazing lambs. Twenty-four male
lambs per forage treatment, half of which were treated with anthelmintics on Day
0, grazed monocultures of lucerne (Medicago sativa), red clover (Trifolium
pratense) and white clover (Trifolium repens) and were compared with lambs
grazing perennial ryegrass (Lolium perenne). Individual faecal egg counts (FEC)
and liveweight were determined every 7 days for 56 days, after which half the
lambs were slaughtered to determine total nematode intensities (TNI). Results
showed that lambs grazed on red or white clover, but not lucerne, had lower
pooled mean FEC and improved liveweight performance compared to lambs grazing
ryegrass. Lambs treated with anthelmintics had higher TNI compared to lambs not
treated, due to a trend for more adult nematodes in lambs grazing lucerne and
treated with anthelmintics than all other lambs, except those grazing red clover
and also given anthelmintics. Lambs grazing white clover tended to have fewer
adult nematodes than lambs grazing other forages. Examination of the nematode
species showed a change in female T. circumcincta occurred in all lambs following
anthelmintic treatment and that the forage species grazed by lambs affected
individual parasite species. Lambs grazing white clover had fewer male and adult
T. circumcincta compared to lambs grazing other forages, and lambs grazing
lucerne had fewer adult T. circumcincta compared to lambs grazing ryegrass or red
clover. Data on small intestine TNI showed that lambs grazing lucerne and given
anthelmintics had more male adult nematodes than other lambs, except those
grazing red clover and treated with anthelmintics. Results indicate that lucerne
and red clover both increase the re-infection of grazing lambs with
Trichostrongylus species compared to ryegrass following anthelmintic treatment.
In conclusion, legume forages have the potential to contribute to the control of
abomasal but not small intestine nematode parasites in finishing lamb systems.
DOI: 10.1016/j.vetpar.2005.04.037
[Dynamics of soil nitrogen, phosphorus and organic matter in alfalfa-crop rotated

farmland in semiarid area of Northwest China].
Wang J(1), Li F, Jia Y, Wang Y.
Author information:
(1)Department of Environmental Science, Northwest University, Xi'an 710069,
China. wangj@nwu.edu.cn
Pasture-crop rotation is regarded as a key planting system in the semiarid area

of China's Loess Plateau. This paper studied the dynamics of soil nitrogen,
phosphorus and organic matter within the two years of alfalfa-crop rotation. The
results showed that in comparing with continuous alfalfa planting, alfalfa-crop
rotation induced a decline of soil total nitrogen and organic matter contents by
5.4% and 19.5%, and 46.8% and 28.2%, respectively, in the first and second year,
but no significant difference was found in soil total phosphorus. Soil nutrient
availability was improved due to the rotation. In the first and second year of
rotation, soil nitrate nitrogen and available phosphorus contents increased by
15.5% and 159.1%, and 44.5% and 48.0%, respectively. Planting spring wheat could
maintain soil fertility, the second was fallow and planting potato, while
planting corn accelerated the depletion of soil total nitrogen, organic matter
and available phosphorus. As a result, in the alfalfa-crop rotation, planting
spring wheat could be more available to the maintenance of soil fertility.
[Alfalfa growth and its relation with soil water status in loess hilly and gully
region].
Cheng J(1), Wan H, Wang J.
Author information:
(1)Institute of Soil and Water Conservation, Chinese Academy of Sciences and
Ministry of Water Resources, Northwest Sci-Tech University of Agriculture and
Forestry, Yangling 712100, China. gyzcjm@ms.iswc.ac.cn
This paper studied the dynamics of alfalfa growth and its relation with the
excessive depletion and resumption of soil water in loess hilly and gully region.
The results showed that alfalfa could grow in this region for 10 years, being
most flourishing at the forth and fifth year, but declined gradually because of
the excessive depletion of soil water. From 0 to 35 cm above ground, the stem
biomass was higher than the leaf biomass, and the ratio of stem to leaf was
1.7:1; at 40 cm above ground, the biomass of stem and leaf was equal; and from 45
to 90 cm above ground, the leaf biomass was higher than the stem biomass, with
the ratio of leaf to stem 1.42:1. The soil dry layer was not distinct in first
two years of alfalfa growth, but getting thicker with time, being from 110 cm in
third year to 260 cm in seventh year, with a 4.6%-6.2% of soil water content. It
took 5 years for the natural resumption of soil water in degraded alfalfa
grassland, and soil water content was increased with time.
2079. Plant J. 2005 Jun;42(5):689-707.
Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing

transcription factor gene, increases cuticular wax accumulation and enhances
drought tolerance in transgenic alfalfa (Medicago sativa).
Zhang JY(1), Broeckling CD, Blancaflor EB, Sledge MK, Sumner LW, Wang ZY.
Author information:
(1)Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK
73401, USA.
The identification of leaf wax genes involved in stress tolerance is expected to

have great potential for crop improvement. Here we report the characterization of
a novel AP2 domain-containing putative transcription factor gene from the model
legume Medicago truncatula. The gene, designated WXP1, is able to activate wax
production and confer drought tolerance in alfalfa (Medicago sativa), the most
important forage legume species in the world and a close relative of M.
truncatula. The predicted protein of WXP1 has 371 aa; it is one of the longest
peptides of all the single AP2 domain proteins in M. truncatula. WXP1 is
distinctly different from the most studied genes in the AP2/ERF transcription
factor family such as AP2s, CBF/DREB1s, DREB2s, WIN1/SHN1 and GL15. Transcript
level of WXP1 is inducible by cold, abscisic acid and drought treatment mainly in
shoot tissues in M. truncatula. Overexpression of WXP1 under the control of the
CaMV35S promoter led to a significant increase in cuticular wax loading on leaves
of transgenic alfalfa. Scanning electron microscopy revealed earlier accumulation
of wax crystals on the adaxial surface of newly expanded leaves and higher
densities of wax crystalline structures on both adaxial and abaxial surfaces of
mature leaves. Gas chromatography-mass spectrometry analysis revealed that total
leaf wax accumulation per surface area increased 29.6-37.7% in the transgenic
lines, and the increase was mainly contributed by C30 primary alcohol. WXP1
overexpression induced a number of wax-related genes. Transgenic leaves showed
reduced water loss and chlorophyll leaching. Transgenic alfalfa plants with
increased cuticular waxes showed enhanced drought tolerance demonstrated by
delayed wilting after watering was ceased and quicker and better recovery when
the dehydrated plants were re-watered.
DOI: 10.1111/j.1365-313X.2005.02405.x
2080. Planta. 2005 Jul;221(5):667-74. Epub 2005 May 21.
LeRALF, a plant peptide that regulates root growth and development, specifically
binds to 25 and 120 kDa cell surface membrane proteins of Lycopersicon
peruvianum.
Scheer JM(1), Pearce G, Ryan CA.
Author information:
(1)Institute of Biological Chemistry, Washington State University, Pullman, WA
99164-6340, USA.
A photoaffinity analog of tomato leaf RALF peptide (LeRALF), (125)I-azido-LeRALF,

bound saturably to tomato suspension cultured cells in the dark in a classical
receptor binding assay. Classical kinetic analyses revealed that the analog
interacted with a single binding site on the surface of the cells with a KD of
0.8x10(-9) M, typical of known peptide hormone-receptor interactions in both
plants and animals. The (125)I-azido-LeRALF, when exposed to UVB light in the
presence of the cells, strongly labeled only two proteins of 25 kDa and 120 kDa,
with the 25 kDa protein being more strongly labeled than the 120 kDa protein. The
cell-surface localization of the interaction was indicated, as suramin, a known
inhibitor of peptide-receptor interactions, and native LeRALF peptide competed
with (125)I-azido-LeRALF labeling of both proteins. Two biologically inactive
LeRALF analogs were not competitors. Incubation of (125)I-azido-LeRALF with
suspension cultured cells in the dark, where it was fully active, could
subsequently be totally dissociated from cells by acid washes, indicating that it
was interacting at the cell surface and was not internalized. The
(125)I-azido-LeRALF-labeled 25 kDa and 120 kDa proteins could not be solubilized
from cell membranes by methods that release peripheral proteins, indicating that
they are integral membrane components. The cumulative kinetic and biochemical
evidence strongly indicates that the two proteins may be components of a LeRALF
receptor complex.
DOI: 10.1007/s00425-004-1442-z
2081. J Dairy Sci. 2005 Jun;88(6):2140-53.

Effects of a proteolytic feed enzyme on intake, digestion, ruminal fermentation,
and milk production.
Eun JS(1), Beauchemin KA.
Author information:
4B1, Canada.
The effects of exogenous proteolytic enzyme (EPE) on intake, digestibility,

ruminal fermentation, and lactational performance were determined using 8
lactating Holstein cows in a double 4 x4 Latin square experiment with a 2 x2
factorial arrangement of treatments. Diets based on barley silage and alfalfa hay
as the forage sources were formulated to maintain different forage to concentrate
ratios [60:40 vs. 34:66, dry matter (DM) basis]. Four dietary treatments were
tested: high forage (HF) without EPE (HF-EPE), HF with EPE (HF+EPE), low forage
(LF) without EPE (LF-EPE), and LF with EPE (LF+EPE). The EPE, which contained
proteolytic activity but negligible fibrolytic activity, was added to the
concentrate portion of the diets after pelleting at a rate of 1.25 mL/kg of DM.
Adding EPE to the diet increased total tract digestibilities of DM, organic
matter, N, acid detergent fiber, and neutral detergent fiber, with larger
increases in digestibility observed for cows fed LF+EPE. Effects of added EPE on
in vivo digestibility were consistent with improvements in gas production and
degradability of the individual components of the TMR observed in vitro. Ruminal
enzymic activities of xylanase and endoglucanase increased with addition of EPE
to the diet, which may have accounted for improvements in fiber digestion.
However, feeding EPE unexpectedly decreased feed intake of cows, which offset the
benefits of improved feed digestibility. Consequently, milk yield of cows fed
high or low forage diets decreased with adding EPE. Nevertheless, dairy
efficiency, expressed as milk/DM intake, was highest for the LF+EPE diet.
Addition of EPE to the diet increased milk fat and milk lactose percentages, but
decreased milk protein percentage of cows fed a low forage diet. For cows fed
high forage diets, EPE only increased milk lactose percentage. Efficiency of N
use for milk production was decreased for both the high and low forage diets when
EPE was added to the diet. Mean ruminal pH was lowered when EPE was added a low
forage diet, likely due to the increased degradation of forage and concentrate,
but there was no effect of EPE on rumen pH when cows were fed high forage diets.
Profiles of VFA and microbial yield were not affected by adding EPE to the diets.
Adding EPE to a total mixed ration containing alfalfa hay, barley silage, and
concentrate improved nutrient digestibility in the total tract, and the response
was maximized with a high concentrate diet. However, improvements in
digestibility were offset by decreased feed intake, likely due to increased
ruminal acidosis.
DOI: 10.3168/jds.S0022-0302(05)72890-3
2082. J Dairy Sci. 2005 Jun;88(6):2096-106.
Effects of parity and supply of rumen-degraded and undegraded protein on

production and nitrogen balance in Holsteins.
Flis SA(1), Wattiaux MA.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison, 53706, USA.
flis@whminer.com
Eight Holstein cows (4 primiparous and 4 multiparous) were used in a replicated 4
x 4 Latin square design to determine milk production response and N balance when
diets had no NRC-predicted excess of rumen-undegradable protein (RUP) or
rumen-degradable protein (RDP), 10% RUP excess, 10% RDP excess, or 10% excess of
both RUP and RDP. Diets were fed as a total mixed ration with (dry matter basis)
25% alfalfa silage, 25% corn silage, 19 to 21% corn grain, and varying
proportions of solvent soybean meal and expeller soybean meal as primary sources
of supplemental RDP and RUP, respectively. Milk yield and dry matter intake (DMI)
were recorded daily, and total collection of feces and urine was completed in the
last 3 d of each 21-d period. Dietary crude protein averaged 17.5 and 18.5% for
the recommended and excess RDP diets, respectively, and 17.3 and 18.4% for the
recommended and excess RUP diets, respectively. When cows were fed excess RUP
diets in the form of expeller soybean meal, DMI and milk production increased,
but the opposite was true when the diets contained excess RDP in the form of
solvent soybean meal. Milk composition was not affected by RDP, RUP, or by
parity, and there were no parity x RDP interactions for any of the measurements.
However, apparent digestibility of neutral detergent fiber, dry matter, and N
increased in multiparous cows but not in primiparous cows because of excess RUP.
The increase in the yield of milk N with excess RUP was not influenced by parity,
but multiparous cows retained more of the additional N apparently absorbed,
whereas primiparous cows excreted the additional apparently absorbed N in the
urine. Overall, the difference in urinary N due to parity (70 g/d) was about 4
times greater than the impact of dietary treatments (17 g/d). Our results suggest
that multiparous cows have either a much larger urea pool or a greater demand to
restore body protein mobilized earlier in lactation compared with primiparous
cows. Reduction in urinary N excretion in commercial dairy herds could be
obtained by separately balancing rations for first and later lactations.
DOI: 10.3168/jds.S0022-0302(05)72886-1
2083. FEMS Microbiol Lett. 2005 May 15;246(2):235-42.
RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and
transport genes in Sinorhizobium meliloti 2011.
Viguier C(1), O Cuív P, Clarke P, O'Connell M.
Author information:
(1)School of Biotechnology, National Centre for Sensor Research, Dublin City
University, Dublin 9, Ireland.
The genes encoding the biosynthesis and transport of rhizobactin 1021, a

siderophore produced by Sinorhizobium meliloti, are negatively regulated by iron.
Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the
iron responsive regulation of the biosynthesis and transport genes. Bioassay
analysis revealed that the siderophore is produced in the presence of iron in a
rirA mutant. RNA analysis and GFP fusions supported the conclusion that RirA is
the mediator of iron-responsive transcriptional repression of the two transcripts
encoding the biosynthesis and transport genes. RirA in S. meliloti appears to
fulfil the role often observed for Fur in other bacterial species. The regulator
was found to mediate the iron-responsive expression of two additional genes,
smc02726 and dppA1, repressing the former while activating the latter. The rirA
mutant nodulated the host plant Medicago sativa (alfalfa) and fixed nitrogen as
effectively as the wild type.
DOI: 10.1016/j.femsle.2005.04.012
2084. Immunol Cell Biol. 2005 Jun;83(3):229-38.
Transgenic plants for the production of veterinary vaccines.
Dus Santos MJ(1), Wigdorovitz A.
Author information:
(1)Veterinary and Agronomy Research Center, INTA-Castelar, Buenos Aires,
Argentina.
The expression of antigens in transgenic plants has been increasingly used in the
development of experimental vaccines, particularly oriented to the development of
edible vaccines. Hence, this technology becomes highly suitable to express
immunogenic proteins from pathogens. Foot and mouth disease virus, bovine
rotavirus and bovine viral diarrhoea virus are considered to be the most
important causative agents of economic loss of cattle production in Argentina,
and they are thus optimal candidates for alternative means of immunization. Here,
we present a review of our results corresponding to the expression of immunogenic
proteins from these three viruses in alfalfa transgenic plants, and we discuss
the possibility of using them for the development of plant-based vaccines.
DOI: 10.1111/j.1440-1711.2005.01338.x
2085. Plant Physiol Biochem. 2005 Mar;43(3):203-12. Epub 2005 Feb 25.
Molecular cloning and characterisation of a Rab-binding GDP-dissociation

inhibitor from Medicago truncatula.
Yaneva IA(1), Niehaus K.
Author information:
(1)Department of Genetics, Biology Faculty, University of Bielefeld, Post-Box
100131, 33501 Bielefeld, Germany. Ivanka.Yaneva@genetik.uni-bielefeld.de
We have isolated and sequenced the full-length cDNA of a GDP-dissociation

inhibitor (GDI) from the model legume Medicago truncatula L. The cDNA (MtGDI)
contains an open reading frame of 1335 bp, coding for a protein of 444 amino
acids with a calculated molecular mass of 49,785 kDa. The deduced amino acid
sequence shows significant homology to other plant GDIs, the highest homology
being found to GDI from the legume Cicer arietinum (96% identity). The MtGDI was
expressed as a N-terminal FLAG-fusion protein in Escherichia coli BL21 (DE3). Its
direct interaction with a small G protein of Rab type from Medicago sativa,
MsRab11f, was demonstrated in vitro by co-immunoprecipitation using a
peptide-specific antibody raised against MtGDI. The dissociation constant of the
MtGDI-MsRab11f complex (4 muM) was determined by a surface plasmon resonance
(SPR) assay. Real-time RT-PCR and Western blot analyses suggested that MtGDI is
ubiquitously expressed in M. truncatula. High levels of MtGDI mRNA were detected
in uninfected roots, leaves and root nodules. In etiolated seedlings and cell
cultures, the amount of MtGDI mRNA was much lower. In all tissues tested, the
peptide-specific anti-MtGDI antibody detected the expected 50 kDa protein in the
total protein extracts. MtGDI was found in the cytosol; however, a significant
fraction was associated with the intracellular membranes in seedlings and roots
indicating a membrane localisation of the protein. A second immunoreactive band
was detected in leaves suggesting that more than one GDI isoform exist in M.
truncatula.
DOI: 10.1016/j.plaphy.2005.01.019
2086. J Exp Bot. 2005 Jul;56(417):1779-84. Epub 2005 Apr 25.
P-deficiency increases the O2 uptake per N2 reduced in alfalfa.
Schulze J(1), Drevon JJ.
Author information:
(1)Institut für Agrikulturchemie der Georg-August-Universität Göttingen,
Carl-Sprengel-Weg 1, D-37075 Göttingen, Germany. jschulz2@gwdg.de
Nodulated alfalfa (Medicago sativa L. cv. Saranac) plants were grown in

hydroponics at P-sufficient and P-deficient supply levels. After 5 weeks of
growth, dry matter accumulation, nodulation, total N and P accumulation, as well
as 15N2 uptake, were measured. Moreover, the response of nodule O2-uptake to
raising external pO2 was determined in an open-flow measurement system and nodule
permeability was calculated. Plants in the P-deficient supply treatment had a
lower P concentration in all organs. In both treatments the highest P
concentration was found in nodules. In the P-deficient supply treatment plants
formed less dry matter, had a lower shoot/root ratio, less nodulation, decreased
total N accumulation, and lower 15N2 uptake per dry matter nodule. Nodules in the
P-deficient treatment were, on average, smaller and had a higher O2 uptake per N2
reduced, coinciding with increased nodule permeability and conductance. Thus
increased oxygen uptake appears to be a mechanism to adjust nodule metabolism to
P deficiency in indeterminate N2-fixing nodules such as in alfalfa, as has
previously been shown for determinate nodule forms.
DOI: 10.1093/jxb/eri166
2087. Biotechnol Lett. 2005 Mar;27(6):365-74.
Flavonoids: from cell cycle regulation to biotechnology.
Woo HH(1), Jeong BR, Hawes MC.
Author information:
(1)Department of Plant Sciences, Division of Plant Pathology and Microbiology,
University of Arizona, Tucson, AZ 85721, USA. hhwoo@email.arizona.edu
Flavonoids have been proposed to play diverse roles in plant growth and
development, including defense, symbiosis, pollen development and male fertility,
polar auxin transport, and protection against ultraviolet radiation. Recently, a
new role in cell cycle regulation has emerged. Genetic alteration of glucuronide
metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase
(PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In
alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like
compound that suppresses growth of cultured cells. The results are consistent
with the hypothesis that PsUGT1 functions by controlling cellular levels of a
factor controlling cell cycle (FCC).
DOI: 10.1007/s10529-005-1521-7
2088. J Dairy Sci. 2005 May;88(5):1807-14.
Digestion of feed amino acids in the rumen and intestine of steers measured using
a mobile nylon bag technique.
Taghizadeh A(1), Danesh Mesgaran M, Valizadeh R, Shahroodi FE, Stanford K.
Author information:
Mashad, Iran 91775-1163. ataghius2000@yahoo.com
The disappearance of dry matter (DM), crude protein (CP), and amino acids (AA) in
steers after rumen incubation and intestinal passage of alfalfa hay, barley hay,
corn silage, barley grain, corn grain, wheat bran, meat meal, fish meal,
cottonseed meal, and soybean meal were measured in 3 steers using a mobile nylon
bag technique. Ruminal degradation of individual AA differed between feedstuffs.
For barley hay and corn silage, the ruminal disappearance of total AA was higher
and lower than the other feedstuffs, respectively. The intestinal digestibility
of total AA in alfalfa hay was lower than the digestion of CP. The intestinal
digestibility of Arg and His was higher than that of total AA in alfalfa hay,
meat meal, cottonseed meal, soybean meal, barley hay, and wheat bran. In
addition, the intestinal digestibility of Lys was higher than that of total AA in
alfalfa hay, meat meal, cottonseed meal, soybean meal, barley hay, corn silage,
and wheat bran. The intestinal disappearance of CP in most cases was higher than
that of DM. The results indicated that feedstuffs with lower ruminal
disappearance of DM, CP, total AA, essential AA, and nonessential AA generally
had a higher intestinal disappearance, resulting in a relatively constant total
tract disappearance. These results could be used to improve the current system of
diet formulation in ruminants.
DOI: 10.3168/jds.S0022-0302(05)72855-1
2089. J Anim Sci. 2005 May;83(5):1142-52.
Ruminal in situ disappearance kinetics of dry matter and fiber in growing steers
for common crabgrass forages sampled on seven dates in northern Arkansas.
Ogden RK(1), Coblentz WK, Coffey KP, Turner JE, Scarbrough DA, Jennings JA,
Richardson MD.
Author information:
(1)Department of Animal Science, University of Arkansas Division of Agriculture,
Fayetteville, 72701, USA.
Southern crabgrass (Digitaria ciliaris [Retz.] Koel.) is often viewed as an

undesirable weed, largely because it encroaches upon field and forage crops,
gardens, and lawns. However, visual observations of livestock grazing
mixed-species pastures suggest that cattle seem to prefer crabgrass to many other
summer forages. The objectives of this study were to assess the nutritive value
of crabgrass sampled weekly between July 11, and August 22, 2001, and then to
determine ruminal in situ disappearance kinetics of DM and NDF for these
crabgrass forages. A secondary objective was to compare these kinetic estimates
with those of alfalfa (Medicago sativa L.), bermudagrass (Cynodon dactylon [L.]
Pers.), and orchardgrass (Dactylis glomerata L.) control hays. All forages were
evaluated in situ using five (383 +/- 22.7 kg) ruminally cannulated crossbred
(Gelbvieh x Angus x Brangus) steers. Whole-plant crabgrass exhibited more rapid
(P < or = 0.002) ruminal disappearance rates of DM (overall range = 0.069 to
0.084 h(-1)) than did bermudagrass (0.054 h(-1)) and orchardgrass (0.060 h(-1))
hays, but disappearance rates were slower (P < 0.001) for crabgrass than for
alfalfa hay (0.143 h(-1)). Effective ruminal disappearance of DM was greater (P <
0.001) for crabgrass (overall range = 69.3 to 75.4%) than for all the control
hays. Similarly, disappearance rates of NDF for crabgrass (overall range = 0.069
to 0.086 h(-1)) were more rapid (P < 0.001) than observed for bermudagrass and
orchardgrass hays; however, NDF in alfalfa disappeared at a faster (P < 0.001)
rate (0.107 h(-1)) than crabgrass. These results indicate that crabgrass offers
greater effective ruminal degradability of DM and NDF than orchardgrass or
alfalfa of moderate quality. More importantly, it potentially offers faster and
more extensive ruminal disappearance than perennial warm-season grasses typically
found throughout the southeastern United States, and it should likely support
improved performance by ruminant livestock in this region.
DOI: 10.2527/2005.8351142x
2090. J Anim Sci. 2005 May;83(5):1133-41.
Improving the nutritional value of oat hulls for ruminant animals with
pretreatment of a multienzyme cocktail: in vitro studies.
Yu P(1), McKinnon JJ, Christensen DA.
Author information:
Saskatoon, Saskatchewan S7N 5A8, Canada. yupe@sask.usask.ca
Relatively high amounts of hydroxycinnamic acid in oat hulls, mainly ferulic

acid, are believed to be inhibitory to digestion by ruminal microorganisms.
Ferulic acid is produced via the phenylpropanoid biosynthetic pathway and
covalently cross-linked to polysaccharides by ester bonds and to components of
lignin, mainly by ether bonds. Ferulic acid also forms dimers or trimers. As a
result, polysaccharides become extensively cross-linked by ferulate dimerization
or trimerization and incorporation into lignin. Previous studies have shown that
Aspergillus ferulic acid esterase and Trichoderma xylanase act synergistically to
release ferulic acid from feruloyl-polysaccharides in complex plant cell walls of
oat hulls. This activity opens the remainder of the polysaccharides to further
hydrolytic attack and facilitates the accessibility of the main polysaccharide
chain to cellulase, thereby increasing the release of reducing sugars. In Exp. 1,
the best multienzyme cocktail (ferulic acid esterase, xylanase, cellulase,
endo-glucanase [I, II], and beta-glucanase) was developed using an orthogonal
experimental design, L25 (5(6)), where L = orthogonal table; 6 = factors; 5 =
five levels of each; and 25 = experimental number, for further in situ and/or in
vivo study. In Exp. 2, in vitro biodegradation studies with a 3 x 2 x 4 factorial
arrangement of treatments were used to evaluate the responses of three
feedstuffs, oat hulls or standard references (wheat straw and alfalfa hay), two
particle sizes (1 mm and 250 microm), and four in vitro incubation treatments
with the best multienzyme cocktail developed in Exp. 1. Addition of the
multienzyme cocktail to the forages improved (P < 0.01) in vitro ruminal fluid
degradability. With respect to feedstuff, the order of response (P < 0.05) to the
treatments was oat hulls (+12% unit) > wheat straw (+5% unit) > alfalfa (+2%
unit). This multi-enzyme cocktail seems best suited for oat hulls containing
feruloyl ester bonds. In conclusion, data from this study suggest that the
addition of the multienzyme cocktail to poorly digestible feeds before feeding
enhanced degradation of DM.
DOI: 10.2527/2005.8351133x
2091. J Anim Sci. 2005 May;83(5):1075-87.
Splanchnic metabolism of nitrogenous compounds and urinary nitrogen excretion in

steers fed alfalfa under conditions of increased absorption of ammonia and
L-arginine supply across the portal-drained viscera.
Maltby SA(1), Reynolds CK, Lomax MA, Beever DE.
Author information:
(1)ARS, USDA, Beltsville, MD 20705, USA.
Effects of increased ammonia and/or arginine absorption across the portal-drained

viscera (PDV) on net splanchnic (PDV and liver) metabolism of nitrogenous
compounds and urinary N excretion were investigated in six catheterized Hereford
x Angus steers (501 +/- 1 kg BW) fed a 75% alfalfa:25% (as-fed basis)
corn-soybean meal diet (0.523 MJ of ME/[kg BW(0.75).d]) every 2 h without (27.0 g
of N/kg of dietary DM) and with 20 g of urea/kg of dietary DM (35.7 g of N/kg of
dietary DM) in a split-plot design. Net splanchnic flux measurements were
obtained immediately before beginning and ending a 72-h mesenteric vein infusion
of L-arginine (15 mmol/h). For 3 d before and during arginine infusion, daily
urine voided was measured and analyzed for N composition. Feeding urea increased
PDV absorption (P < 0.01) and hepatic removal (P < 0.01) of ammonia N, accounting
for 80% of increased hepatic urea N output (P < 0.01). Numerical increases in net
hepatic removal of AA N could account for the remaining portion of increased
hepatic urea N output. Arginine infusion increased hepatic arginine removal (P <
0.01) and hepatic urea N output (P < 0.03) and switched hepatic ornithine flux
from net uptake to net output (P < 0.01), but numerical changes in net hepatic
removal of ammonia and AA N could not account fully for the increase in hepatic
urea N output. Increases in urine N excretion equaled quantities of N fed as urea
or infused as arginine. Estimated salivary urea N excretion was not changed by
either treatment. Urea cycle regulation occurs via a complex interaction of
mechanisms and requires N sources other than ammonia, but the effect of increased
ammonia absorption on hepatic catabolism of individual AA in the present study
was not significant.
DOI: 10.2527/2005.8351075x
2092. J Anim Sci. 2005 May;83(5):1023-32.
Effects of short-term feed deprivation and melatonin implants on circadian

patterns of leptin in the horse.
Buff PR(1), Morrison CD, Ganjam VK, Keisler DH.
Author information:
(1)Department of Animal Sciences, University of Missouri, Columbia, 65211, USA.
Leptin is a protein hormone produced by adipose tissue that influences

hypothalamic mechanisms regulating appetite and energy balance. In species tested
thus far, including horses, concentrations of leptin increase as animal fat mass
increases. The variables and mechanisms that influence the secretion of leptin
are not well known, nor is it known in equine species how the secretion of leptin
is influenced by acute alterations in energy balance, circadian patterns, and/or
reproductive competence. Our objectives were to determine in horses: 1) whether
plasma concentrations of leptin are secreted in a circadian and/or a pulsatile
pattern; 2) whether a 48-h period of feed restriction would alter plasma
concentrations of leptin, growth hormone, or insulin; and 3) whether ovariectomy
and/or a melatonin implant would affect leptin. In Exp. 1, mares exposed to
ambient photoperiod of visible light (11 h, 33 min to 11 h, 38 min), received
treatments consisting of a 48-h feed restriction (RES) or 48 h of alfalfa hay fed
ad libitum (FED). Mares were maintained in a dry lot before sampling and were
tethered to a rail during sampling. Analyses revealed that leptin was not
secreted in a pulsatile manner, and that mean leptin concentrations were greater
(P < 0.001) in FED vs. RES mares (17.20 +/- 0.41 vs. 7.29 +/- 0.41 ng/mL). Plasma
growth hormone was pulsatile, and mean concentrations were greater in RES than
FED mares (2.15 +/- 0.31 vs. 1.08 +/- 0.31 ng/mL; P = 0.05). Circadian patterns
of leptin secretion were observed, but only in FED mares (15.39 +/- 0.58 ng/mL
for morning vs. 19.00 +/- 0.58 ng/mL for evening; P < 0.001). In Exp. 2, mares
that were ovariectomized or intact received either a s.c. melatonin implant or a
sham implant. Thereafter, blood was sampled at weekly intervals at 1000 and 1700.
Concentrations of leptin in samples collected at 1700 were greater (P < 0.001)
than in those collected at 1000 (28.24 +/- 1.7 vs. 22.07 +/- 1.7 ng/mL). Neither
ovariectomy nor chronic treatment with melatonin affected plasma concentrations
of leptin or the circadian pattern of secretion. These data provide evidence that
plasma leptin concentrations in the equine are sensitive to acute changes in
nutritional status and vary in a circadian pattern that is sensitive to fasting
but not to melatonin treatment or ovariectomy.
DOI: 10.2527/2005.8351023x
2093. Ann Bot. 2005 Jun;95(7):1237-45. Epub 2005 Apr 8.
Effects of burial in sand and water supply regime on seedling emergence of six
species.
Zheng Y(1), Xie Z, Yu Y, Jiang L, Shimizu H, Rimmington GM.
Author information:
(1)Laboratory of Quantitative Vegetation Ecology, Institute of Botany, Chinese
Academy of Sciences, Beijing, 100093, China. zhengyuanrun@hotmail.com
BACKGROUND AND AIMS: Air seeding has long been regarded as a quick and successful
measure for vegetation rehabilitation in China. However, seedling emergence of
often-used species including Agriophyllum squarrosum, Artemisia sphaerocephala,
Artemisia ordosica, Hedysarum fruticosum, Caragana korshinskii and Medicago
sativa is low. Experiments were conducted under controlled conditions to study
the effects of sowing depth and water supply on seedling emergence, in order to
understand the requirements for increasing seedling emergence.
METHODS: Seeds were exposed to different environments of burial and water supply
regimes in PVC pots (7 cm in diameter and 11 cm in height) under the same light
intensity and alternating temperature regimes in a growth chamber.
KEY RESULTS: Seedlings of three species (Agriophyllum squarrosum, Artemisia
sphaerocephala, Artemisia ordosica) with relatively light seeds emerged well at a
0.5 cm sowing depth under a 7.5 and 10 mm water supply regime. However, few
seedlings of these species emerged when the sowing depth was over 1 cm or when
water supply was 5 mm. Seedlings of Caragana korshinskii, Hedysarum fruticosum
and Medicago sativa emerged from sowing depths of 0.5-4 cm, 0.5-3 cm, and 0.5-4
cm, respectively, under both 7.5 and 10 mm water supply regimes. Under a 5 mm
water supply regime, seedlings of these species also emerged at over 1 cm sowing
depth. Seeds of all six species sown on the surface of sand did not germinate,
and seedlings did not emerge when they were sown at depths greater than 6 cm.
CONCLUSIONS: Based on these experiments, a 0.5 cm sowing depth resulted in the
highest seedling emergence and it is concluded that this is the optimal sowing
depth for seedling emergence of all six species.
DOI: 10.1093/aob/mci138
PMCID: PMC4246910
2094. Mol Biol Evol. 2005 Jun;22(6):1518-28. Epub 2005 Apr 6.
Dispersal and evolution of the Sinorhizobium meliloti group II RmInt1 intron in

bacteria that interact with plants.
Fernández-López M(1), Muñoz-Adelantado E, Gillis M, Willems A, Toro N.
Author information:
(1)Grupo de Ecología Genética, Departamento de Microbiología del Suelo y Sistemas
Simbióticos, Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científicas, calle Profesor Albareda 1, Granada, Spain.
Group II introns are both self-splicing RNAs and mobile retroelements found in
bacterial and archaeal genomes and in organelles of eukaryotes. They are thought
to be the ancestors of eukaryote spliceosomal introns and non-long terminal
repeat retrotransposons. We show here that RmInt1, a bacterial group II intron
first described in the nitrogen-fixing symbiont of alfalfa (Medicago sativa)
Sinorhizobium meliloti, is also present in other Sinorhizobium and Rhizobium
species. The intron-homing sites in these species are IS elements of the
ISRm2011-2 group as in S. meliloti, but ectopic insertion is also observed. We
present evidence that these related bacteria have acquired RmInt1 by vertical
inheritance from a common ancestor and by independent horizontal transfer events.
We also show that RmInt1 is mobile in related taxa of bacteria that interact with
plants and tends to evolve toward an inactive form by fragmentation, with loss of
the 3' terminus including the intron-encoded protein. Our results provide an
overview of the evolution and dispersion of a bacterial group II intron.
DOI: 10.1093/molbev/msi144
2095. Planta. 2005 Sep;222(1):70-9. Epub 2005 Apr 5.
Responsive modes of Medicago sativa proline dehydrogenase genes during salt

stress and recovery dictate free proline accumulation.
Miller G(1), Stein H, Honig A, Kapulnik Y, Zilberstein A.
Author information:
(1)Department of Plant Science, Tel Aviv University, Tel-Aviv 69978, Israel.
Free proline accumulation is an innate response of many plants to osmotic stress.

To characterize transcriptional regulation of the key proline cycle enzymes in
alfalfa (Medicago sativa), two proline dehydrogenase (MsPDH) genes and a partial
sequence of Delta (1) -pyrroline-5-carboxylate dehydrogenase (MsP5CDH) gene were
identified and cloned. The two MsPDH genes share a high nucleotide sequence
homology and a similar exon/intron structure. Estimation of transcript levels
during salt stress and recovery revealed that proline accumulation during stress
was linearly correlated with a strong decline in MsPDH transcript levels, while
Delta (1) -pyrroline-5-carboxylate synthetase (MsP5CS) and MsP5CDH steady-state
transcript levels remained essentially unchanged. MsPDH transcript levels
dramatically decreased in a fast, salt concentration-dependent manner. The extent
of salt-induced proline accumulation also correlated with salt concentrations.
Salt-induced repression of MsPDH1 promoter linked to the GUS reporter gene
confirmed that the decline in MsPDH transcript levels was due to less
transcription initiation. Contrary to the salt-dependent repression, a rapid
induction of MsPDH transcription occurred at a very early stage of the recovery
process, independently of earlier salt treatments. Hence our results suggest the
existence of two different regulatory modes of MsPDH expression; the repressing
mode that quantifies salt concentration in an as yet unknown mechanism and the
"rehydration"-enhancing mode that responds to stress relief in a maximal
induction of MsPDH transcription. As yet the components of salt sensing as well
as those that might interact with MsPDH promoter to reduce transcription are
still unknown.
DOI: 10.1007/s00425-005-1518-4
2096. Poult Sci. 2005 Mar;84(3):362-9.
Utilizing different ratios of alfalfa and layer ration for molt induction and
performance in commercial laying hens.
Donalson LM(1), Kim WK, Woodward CL, Herrera P, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station, Texas
77843-2472, USA.
Molting is a common practice used by the commercial egg industry to rejuvenate

flocks for a second or third laying cycle. During this time the hens rest from
production, and the reproductive organs are rejuvenated to increase production
and quality during the next laying cycle. Although feed withdrawal (FW) is the
most popular and effective method of molt induction, it has come under scrutiny
due to food safety issues and animal welfare issues. This study involved feeding
alfalfa mixed with layer ration at different ratios to hens to determine their
ability to induce molt. The treatment ratios were 100% alfalfa (A100), 90%
alfalfa and 10% layer ration (A90), and 70% alfalfa and 30% layer ration (A70).
In addition, a fully fed (FF) nonmolted control and a FW negative control were
used. Alfalfa is an insoluble, high fiber feedstuff with low metabolizable
energy. Egg production for A90 and FW treatments ceased completely by d 6,
whereas birds fed A100 and A70 ceased egg production by d 8. Ovary and oviduct
weight of hens fed all molting diets decreased (P < 0.05) by an average of 1.5 to
2.5% (BW basis) compared with FF control during the 9-d molt induction period. As
the percentage of layer ration increased, feed intake also increased and
percentage of BW loss decreased during the 9-d molt induction period. Hens molted
by FW lost an average of 25.8% BW, whereas A70 hens lost 18.9% BW. Nonmolted hens
(FF) and A70 treatment hens had significantly lower (P < 0.05) egg production
when compared with all other treatments over the 39-wk postmolt period. FF
treatment hens also had lower (P < 0.05) albumen heights when compared with all
other treatments. From these results, alfalfa or alfalfa mixed with layer ration
appears to be viable alternatives to conventional FW methods for the successful
induction of molt and retention of postmolt performance.
DOI: 10.1093/ps/84.3.362
2097. Plant Physiol. 2005 Apr;137(4):1420-34. Epub 2005 Mar 18.
Conservation of Arabidopsis flowering genes in model legumes.
Hecht V(1), Foucher F, Ferrándiz C, Macknight R, Navarro C, Morin J, Vardy ME,

Ellis N, Beltrán JP, Rameau C, Weller JL.
Author information:
(1)School of Plant Science, University of Tasmania, Hobart, Tasmania 7001,
Australia.
Erratum in
Plant Physiol. 2005 Aug;138(4):2417.
The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have
provided a wealth of information about genes and genetic pathways controlling the
flowering process, but little is known about the corresponding pathways in
legumes. The garden pea (Pisum sativum) has been used for several decades as a
model system for physiological genetics of flowering, but the lack of molecular
information about pea flowering genes has prevented direct comparison with other
systems. To address this problem, we have searched expressed sequence tag and
genome sequence databases to identify flowering-gene-related sequences from
Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated
corresponding sequences from pea by degenerate-primer polymerase chain reaction
and library screening. We found that the majority of Arabidopsis flowering genes
are represented in pea and in legume sequence databases, although several gene
families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL
FLOWER1 families, appear to have undergone differential expansion, and several
important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS
C clade, are conspicuously absent. In several cases, pea and Medicago orthologs
are shown to map to conserved map positions, emphasizing the closely syntenic
relationship between these two species. These results demonstrate the potential
benefit of parallel model systems for an understanding of flowering phenology in
crop and model legume species.
DOI: 10.1104/pp.104.057018
PMCID: PMC1088331
2098. Plant Physiol. 2005 Apr;137(4):1474-84. Epub 2005 Mar 18.
Aphid infestation causes different changes in carbon and nitrogen allocation in

alfalfa stems as well as different inhibitions of longitudinal and radial
expansion.
Girousse C(1), Moulia B, Silk W, Bonnemain JL.
Author information:
(1)Unité de Recherches de Zoologie, Institut National de la Recherche
Agronomique, F-86600 Lusignan, France. girousse@clermont.inra.fr
Alfalfa (Medicago sativa) stem elongation is strongly reduced by a pea aphid

(Acyrthosiphon pisum Harris) infestation. As pea aphid is a phloem feeder that
does not transmit virus or toxins, assimilate withdrawal is generally considered
as the main mechanism responsible for growth reduction. Using a kinematic
analysis, we investigated the spatial distributions of relative elemental growth
rates of control and infested alfalfa stems. The water, carbon, and nitrogen
contents per unit stem length were measured along the growth zone. Deposition
rates and growth-sustaining fluxes were estimated from these patterns. Severe
short-term aphid infestation (200 young adults over a 24-h period) induced a
strong and synchronized reduction in rates of elongation and of water and carbon
deposition. Reduced nitrogen content and associated negative nitrogen deposition
rates were observed in some parts of the infested stems, especially in the apex.
This suggested a mobilization of nitrogen from the apical part of the growth
zone, converted from a sink tissue into a source tissue by aphids. Calculation of
radial growth rates suggested that aphid infestation led to a smaller reduction
in radial expansion than in elongation. Together with earlier observations of
long-lasting effects of a short-term infestation, this supports the hypothesis
that in addition to nutrient withdrawal, a thigmomorphogenesis-like mechanism is
involved in the effect of aphid infestation on stem growth.
DOI: 10.1104/pp.104.057430
PMCID: PMC1088336
2099. J Dairy Sci. 2005 Apr;88(4):1399-403.
Subacute ruminal acidosis induces ruminal lipopolysaccharide endotoxin release

and triggers an inflammatory response.
Gozho GN(1), Plaizier JC, Krause DO, Kennedy AD, Wittenberg KM.
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, Canada R3T
2N2.
Subacute ruminal acidosis (SARA) was induced in 3 rumen fistulated Jersey steers
by offering them different combinations of wheat-barley pellets and chopped
alfalfa hay. Steers were offered 4, 5, and 6 kg/d of pelleted concentrate and 6,
5, and 4 kg/d of chopped alfalfa hay for diets 1, 2, and 3, respectively, during
5-d treatment periods and were fed chopped alfalfa hay between treatment periods.
Inducing SARA increased blood concentrations of haptoglobin and serum amyloid-A.
Dry matter intake of concentrate and hay decreased from d 1 to 5 in each period.
Subacute ruminal acidosis was induced in all steers during d 4 and 5 when
concentrate was fed, with ruminal pH remaining below 5.6 for an average of 187
and 174 min/d on these days. Lipopolysaccharide concentration increased
significantly during periods of grain feeding compared with times when only hay
was fed. Inducing SARA by feeding wheat-barley pellets activated a systemic
inflammatory response in the steers.
DOI: 10.3168/jds.S0022-0302(05)72807-1
2100. Mol Microbiol. 2005 Apr;56(1):68-80.
Importance of unusually modified lipid A in Sinorhizobium stress resistance and

legume symbiosis.
Ferguson GP(1), Datta A, Carlson RW, Walker GC.
Author information:
(1)Institute of Structural and Molecular Biology, School of Biological Sciences,
University of Edinburgh, Edinburgh, EH9 3JR, UK. G.Ferguson@ed.ac.uk
Sinorhizobium meliloti, a legume symbiont and Brucella abortus, a
phylogenetically related mammalian pathogen, both require their BacA proteins to
establish chronic intracellular infections in their respective hosts. The lipid A
molecules of S. meliloti and B. abortus are unusually modified with a
very-long-chain fatty acid (VLCFA; C > or = 28) and we discovered that BacA is
involved in this unusual modification. This observation raised the possibility
that the unusual lipid A modification could be crucial for the chronic infection
of both S. meliloti and B. abortus. We investigated this by constructing and
characterizing S. meliloti mutants in the lpxXL and acpXL genes, which encode an
acyl transferase and acyl carrier protein directly involved in the biosynthesis
of VLCFA-modified lipid A. Our analysis revealed that the unusually modified
lipid A is important, but not crucial, for S. meliloti chronic infection and that
BacA must have an additional function, which in combination with its observed
effect on the lipid A in the free-living form of S. meliloti, is essential for
the chronic infection. Additionally, we discovered that in the absence of VLCFAs,
S. meliloti produces novel pentaacylated lipid A species, modified with
unhydroxylated fatty acids, which are important for stress resistance.
DOI: 10.1111/j.1365-2958.2005.04536.x
2101. J Food Prot. 2005 Mar;68(3):557-61.
Combination of immunomagnetic separation with real-time PCR for rapid detection

of Salmonella in milk, ground beef, and alfalfa sprouts.
Mercanoglu B(1), Griffiths MW.
Author information:
(1)Hacettepe University, Faculty of Engineering, Food Engineering Department,
Beytepe, Ankara, Turkey.
A rapid, specific, and sensitive method for detecting Salmonella spp. in

pasteurized milk, ground beef, and alfalfa sprouts was developed. The method
combined immunomagnetic separation with a real-time PCR assay based on the
double-stranded DNA binding dye SYBR Green I. The primers used produced a product
with a melting temperature of 87+/-0.5 degrees C during the PCR assay by
amplifying a 284-bp sequence from the invasive gene (invA) of Salmonella. The
method was successful in detecting 20 Salmonella strains, but the expected PCR
product was not formed by any of 11 other bacterial strains. To test this
combined method for the monitoring of Salmonella, Salmonella enterica serotype
Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and
alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered
peptone water, cells were removed by immunomagnetic separation and DNA extracted
using the High Pure PCR template preparation kit. The DNA produced was used as a
template in the real-time PCR assay. When spiked pasteurized milk, ground beef,
and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of
1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13
h. These results indicate that the combination of immunomagnetic separation and
real-time PCR assay was a highly specific and sensitive method for the rapid
detection of Salmonella.
2102. J Agric Food Chem. 2005 Mar 23;53(6):2164-70.

Triterpene saponins from barrel medic (Medicago truncatula) aerial parts.
Kapusta I(1), Stochmal A, Perrone A, Piacente S, Pizza C, Oleszek W.
Author information:
ul. Czartoryskich 8, 24-100 Pulawy, Poland.
Triterpene saponins from Medicago truncatula aerial parts have been separated and
their structures determined by the extensive use of 1D- and 2D-NMR experiments
including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along
with ESIMS. Fifteen individual compounds were isolated that included seven
medicagenic acid and eight zanhic acid glycosides. Additionally, two
soyasapogenol B and soyasapogenol E glycosides were identified by MS/MS and TLC.
Four medicagenic acid glycosides (5, 11, 12, 14) and eight zanhic acid glycosides
(1-4, 6-9) are reported here for the first time. The common feature of M.
truncatula aerial part saponins is the (1-->3) linkage between the two glucose
units at C-3 of medicagenic and zanhic acids, which is different from that found
in alfalfa (Medicago sativa), where this linkage was always (1-->2). This may
suggest differences in glucosyltransferases between these two Medicago species.
DOI: 10.1021/jf048178i
Host effects of glandular-haired alfalfa on alfalfa weevil (Coleoptera:

Curculionidae) and potato leafhopper (Homoptera: Cicadellidae) populations in
Virginia.
Dellinger TA(1), Youngman RR, Laub CA, Brewster CC, Kuhar TP.
Author information:
(1)Department of Entomology, Virginia Polytechnic Institute and State University,
Blacksburg, VA 24061, USA.
Cultivars of glandular-haired alfalfa, Medicago sativa L., such as '54H69', are

currently available and marketed as being resistant to potato leafhopper,
Empoasca fabae (Harris). Between 2000 and 2002, studies were conducted to compare
the effects of '54H69' and a standard, nonglandular-haired alfalfa cultivar,
'Choice', on alfalfa weevil, Hypera postica (Gyllenhal), and potato leafhopper
populations at Campbell and Montgomery counties, Virginia. '54H69' had no effect
on alfalfa weevil populations. At each location, densities of alfalfa weevil in
'54H69' and 'Choice' were similar, but pest pressure was higher at Campbell Co.
than at Montgomery Co. and always exceeded the economic threshold before
insecticide was applied. Densities of potato leafhopper also did not differ
between '54H69' and 'Choice' in any year at the two locations. Insecticide
treatment effectively reduced potato leafhopper densities in the two cultivars,
although populations were below the economic threshold at both locations when the
insecticides were applied. Overall, postinsecticide treatment comparisons showed
that the densities of alfalfa weevil and potato leafhoppers were similar or
higher in untreated '54H69' compared with insecticide-treated 'Choice'. In
addition, there were no differences in seasonal dry yields between '54H69' and
'Choice' in any year at either location. Our results indicate that the
glandular-haired alfalfa '54H69' does not provide acceptable resistance to potato
leafhopper and also does not offer a yield advantage to growers in Virginia.
2104. Microbiology. 2005 Mar;151(Pt 3):975-83.
Two site-specific recombinases are implicated in phenotypic variation and

competitive rhizosphere colonization in Pseudomonas fluorescens.
Martínez-Granero F(1), Capdevila S, Sánchez-Contreras M, Martín M, Rivilla R.
Author information:
(1)Departamento de Biología, Universidad Autónoma de Madrid, Campus de
Cantoblanco, 28049 Madrid, Spain.
The biocontrol agent Pseudomonas fluorescens F113 undergoes phenotypic variation

during rhizosphere colonization, and this variation has been related to the
activity of a site-specific recombinase encoded by the sss gene. Here, it is
shown that a second recombinase encoded by the xerD gene is also implicated in
phenotypic variation. A putative xerD gene from this strain was cloned, and
sequence analysis confirmed that it encoded a site-specific recombinase of the
lambda integrase family. Mutants affected in the sss or xerD genes produced a
very low quantity of phenotypic variants compared to the wild-type strain, both
under prolonged cultivation in the laboratory and after rhizosphere colonization,
and they were severely impaired in competitive root colonization. Overexpression
of the genes encoding either recombinase resulted in a substantial increment in
the production of phenotypic variants under both culture and rhizosphere
colonization conditions, implying that both site-specific recombinases are
involved in phenotypic variation. Overexpression of the sss gene suppressed the
phenotype of a xerD mutant, but overexpression of the xerD gene had no effect on
the phenotype of an sss mutant. Genetic analysis of the phenotypic variants
obtained after overexpression of the genes encoding both the recombinases showed
that they carried mutations in the gacA/S genes, which are necessary to produce a
variety of secondary metabolites. These results indicate that the Gac system is
affected by the activity of the site-specific recombinases. Transcriptional
fusions of the sss and xerD genes with a promoterless lacZ gene showed that both
genes have a similar expression pattern, with maximal expression during
stationary phase. Although the expression of both genes was independent of
diffusible compounds present in root exudates, it was induced by the plant, since
bacteria attached to the root showed enhanced expression.
DOI: 10.1099/mic.0.27583-0
2105. J Anim Sci. 2005 Apr;83(4):940-7.
The effect of milk intake on forage intake and growth of nursing calves.
Abdelsamei AH(1), Fox DG, Tedeschi LO, Thonney ML, Ketchen DJ, Stouffer JR.
Author information:
(1)Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
Thirty-nine Holstein steer calves were assigned to one of five treatments at

birth and individually fed for 200 d with milk replacer reconstituted to equal
the fat and protein concentration of beef cow milk. Treatment levels were the
quantities of reconstituted milk fed per day based on lactation curves, which
were based on peak milk levels (PML) of 2.72, 5.44, 8.16, 10.88, and 13.6 kg/d,
respectively. In addition to reconstituted milk, chopped alfalfa hay was offered
ad libitum to allow for maximal voluntary forage consumption. All calves were fed
a high-energy diet postweaning until they reached a similar degree of fatness in
the 12th rib (4 to 5% chemical fat) as determined by ultrasound. There were
differences (P < 0.05) among groups in weaning weight, preweaning ADG, age, and
weight at slaughter. During the preweaning phase, there was a linear relationship
(P < 0.01) for daily milk and forage DE intake; however, DE intake per unit of BW
did not differ across treatments (P = 0.06). Increasing PML resulted in a linear
(P < 0.01) decrease in alfalfa hay intake in the preweaning phase, and G:F
increased quadratically (P < 0.01). During the postweaning phase, preweaning milk
intake had no meaningful effect on postweaning ADG, but overall ADG had a linear
relationship (P < 0.01) with preweaning milk level. There was no effect of PML on
the 12th-rib lipid percent, marbling score, or quality grade, but protein and fat
concentration in the carcass and empty BW increased linearly (P < 0.01) with PML.
The group fed at 2.72 kg/d PML was 58 kg lighter (P = 0.03) and required 34 d
more (P < 0.01) to reach the predetermined degree of fatness at slaughter than
the group fed at 13.6 kg/d PML, suggesting that increased milk production by the
dam can decrease the number of days to the slaughter weight at which a similar
rib lipid concentration is reached.
DOI: 10.2527/2005.834940x
2106. Poult Sci. 2005 Feb;84(2):185-93.
Reduction of Salmonella enterica serovar enteritidis colonization and invasion by

an alfalfa diet during molt in Leghorn hens.
Woodward CL(1), Kwon YM, Kubena LF, Byrd JA, Moore RW, Nisbet DJ, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, College Station, Texas
77843, USA.
The standard method for molting to stimulate multiple egg-laying cycles in laying
hens is feed deprivation. However, the physiological changes within hens caused
by feed deprivation increase susceptibility of the hens to Salmonella enterica
serovar Enteritidis (SE) infection. In an effort to develop an alternative method
to induce molting without increasing susceptibility to SE, an alfalfa diet was
compared with the standard molting method for the level of ovary regression and
SE colonization. Hens over 50 wk of age were divided into 3 treatment groups (12
hens/group): nonmolting by normal feeding (NM), molting by feed deprivation (M),
and molting by alfalfa diet (A). Individual hens on all treatments were
challenged orally with 10(5) cfu of SE on the fourth day after feed changes and
were analyzed for ovary weight and SE colonization or invasion in crop contents,
cecal contents, liver, spleen, and ovary on the ninth day. In 3 of the 4 trials,
there was a significant decrease in SE colonization of the crop between the
alfalfa diet (A) and the feed deprived molt (M). In most of the 4 trials, there
was a significant reduction in SE infected organs in birds fed the alfalfa diet
(A) compared with birds undergoing feed deprived molt (M). Most of the trials
showed no significant difference in overall SE between A and NM. Therefore, the
results of this study suggest that an alfalfa diet has the potential to be used
as an alternative method for forced molting, without increasing the incidence of
SE in eggs and internal organs.
DOI: 10.1093/ps/84.2.185
2107. Appl Microbiol Biotechnol. 2005 Mar;66(6):635-40. Epub 2004 Oct 23.
Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus

albus and Clostridium thermocellum.
Weimer PJ(1), Koegel RG, Lorenz LF, Frihart CR, Kenealy WR.
Author information:
(1)U.S. Dairy Forage Research Center, Agricultural Research Service, United
States Department of Agriculture, 1925 Linden Drive West, Madison, Wisconsin
53706, USA. pjweimer@facstaff.wisc.edu
Fermentation residues (consisting of incompletely fermented fiber, adherent

bacterial cells, and a glycocalyx material that enhanced bacterial adherence)
were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7
or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne
(Medicago sativa L.). The dried residue was able to serve as an effective
co-adhesive for phenol-formaldehyde (PF) bonding of aspen veneer sheets to one
another. Testing of the resulting plywood panels revealed that the adhesive,
formulated to contain 30% of its total dry weight as fermentation residue,
displayed shear strength and wood failure values under both wet and dry
conditions that were comparable with those of industry standards for PF that
contained much smaller amounts of fillers or extenders. By contrast, PF adhesives
prepared with 30% of dry weight as either unfermented lucerne fiber or
conventional fillers or extenders rather than as fermentation residues, displayed
poor performance, particularly under wet conditions.
DOI: 10.1007/s00253-004-1767-3
2108. Plant Physiol. 2005 Mar;137(3):1009-17. Epub 2005 Feb 25.
Crystal structures of alfalfa caffeoyl coenzyme A 3-O-methyltransferase.
Ferrer JL(1), Zubieta C, Dixon RA, Noel JP.
Author information:
(1)Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de
Biologie Structurale, 38027 Grenoble cedex 1, France.
Caffeoyl coenzyme A 3-O-methyltransferases (CCoAOMTs) are

S-adenosyl-l-methionine-dependent O-methyltransferases (OMTs) involved in lignin
biosynthesis. Plant CCoAOMTs belong to a distinct family of OMTs, more closely
related to the mammalian catechol OMTs than to other plant OMTs. The crystal
structure of alfalfa (Medicago sativa) CCoAOMT in complex with the reaction
products S-adenosine-l-homocysteine and feruloyl/sinapoyl CoAs presented here
belong to a structurally and mechanistically distinct family of plant small
molecule OMTs. These structures provide a new understanding of the substrate
preferences and the catalytic mechanism accompanying CCoAOMT-mediated
O-methylation of CoA-linked phenylpropanoid substrates.
DOI: 10.1104/pp.104.048751
PMCID: PMC1065401
2109. Vaccine. 2005 Mar 7;23(15):1844-6.

Immunoprotective properties of transgenic plants expressing E2 glycoprotein from
CSFV and cysteine protease from Fasciola hepatica.
Legocki AB(1), Miedzinska K, Czaplińska M, Płucieniczak A, Wedrychowicz H.
Author information:
(1)Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego
12/14, 61-704 Poznań, Poland. legocki@ibch.poznan.pl
Immune responses were elicited in laboratory animals after oral vaccination by

transgenic plants (lettuce and alfalfa) expressing the E2 glycoprotein of
Classical Swine Fever Virus (CSFV) or cysteine protease from Fasciola hepatica.
ELISA analyses demonstrated that the oral route is effective in inducing a
specific antibody response against these antigens in mice.
DOI: 10.1016/j.vaccine.2004.11.015
2110. Vaccine. 2005 Mar 7;23(15):1838-43.
Development of transgenic alfalfa plants containing the foot and mouth disease
virus structural polyprotein gene P1 and its utilization as an experimental
immunogen.
Dus Santos MJ(1), Carrillo C, Ardila F, Ríos RD, Franzone P, Piccone ME,
Wigdorovitz A, Borca MV.
Author information:
(1)Instituto de Virología S. Rivenson C.I.C.V.y A., INTA-Castelar, Hurlingham
(1712) Pcia. Buenos Aires, Argentina.
The use of transgenic plants as vectors for the expression of viral and bacterial
antigens has been increasingly tested as an alternative methodology for the
production of experimental vaccines. Here, we report the production of transgenic
alfalfa plants containing the genes encoding the polyprotein P1 and the protease
3C of foot and mouth disease virus (FMDV). The immunogenicity of the expressed
products was tested using a mouse experimental model. Parenterally immunized mice
developed a strong antibody response and were completely protected when
challenged with the virulent virus. This report demonstrates the possibility of
using transgenic plants to express polyprotein P1 and the protease 3C of FMDV and
their utilization as effective experimental immunogens.
DOI: 10.1016/j.vaccine.2004.11.014
2111. Phytochemistry. 2005 Mar;66(5):529-41.
Mass spectral characterization of fatty acid amides from alfalfa trichomes and
their deterrence against the potato leafhopper.
Ranger CM(1), Winter RE, Rottinghaus GE, Backus EA, Johnson DW.
Author information:
(1)Department of Entomology, University of Missouri, Columbia, MO 65211, USA.
ranger@aesop.rutgers.edu
A homologous series of N-(3-methylbutyl)amides of normal saturated C14, C15, C16,

C17 and C18 fatty acids were identified as major components of glandular trichome
extracts from Medicago sativa G98A, an alfalfa genotype resistant to the potato
leafhopper, Empoasca fabae. A second homologous series of
N-(2-methylpropyl)amides of C14 through C18 normal fatty acids were minor
components. Saturated free fatty acids C12, C13, C14, C15, C16, C17 and C18 were
present in trace amounts, as was the N-(3-methylbutyl)amide of linoleic acid
(C18:2). N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of C14 through C18
fatty acids, along with the N-(3-methylbutyl)amide of linoleic acid, were
synthesized and bioassayed for leafhopper deterrence by applying the compounds to
the surface of a sachet containing an artificial diet. Leafhoppers were then
offered a two-way choice between diet surfaces treated with the synthetic amides
or an untreated control. N-(3-methylbutyl)amides and N-(2-methylpropyl)amides of
C14 through C18 fatty acids did not deter leafhopper settling in a dose-dependent
fashion. In contrast, when tested singly, N-(3-methylbutyl)amide of linoleic acid
exhibited dose-dependent deterrence against leafhopper settling. Fatty acid
amides localized in alfalfa glandular trichomes likely contribute to leafhopper
resistance.
DOI: 10.1016/j.phytochem.2005.01.012
2112. New Phytol. 2005 Feb;165(2):559-65.
The Lipid A substructure of the Sinorhizobium meliloti lipopolysaccharides is

sufficient to suppress the oxidative burst in host plants.
Scheidle H(1), Gross A, Niehaus K.
Author information:
(1)University of Bielefeld, Department of Genetics, POB 100131, D-33501
Bielefeld, Germany.
Medicago sativa (alfalfa), Medicago truncatula and Nicotiana tabacum cell

suspension cultures, responding to elicitation with the production of reactive
oxygen species (ROS), were used to analyse the suppressor (and elicitor) activity
of lipopolysaccharides (LPS) of the symbiotic soil bacterium Sinorhizobium
meliloti. In order to identify the epitopes of the LPS molecule recognized by the
plant, S. meliloti mutants defective in LPS biosynthesis and hydrolytically
obtained Lipid A were analysed for biological activity. Lipopolysaccharides
isolated from Sinorhizobium meliloti mutants 6963 (altered core region) and L994
(no long-chain fatty acid) showed the same ability to suppress the oxidative
burst in host plant cell cultures as the wild-type LPS. Lipid A also displayed
the same suppressor activity. By contrast, rhizobial LPS, but not Lipid A, was
active as an inducer of the oxidative burst reaction in cell cultures of the
nonhost Nicotiana tabacum. In host plants of Sinorhizobium meliloti the Lipid A
part is sufficient to suppress the oxidative burst, but in non-host plants at
least some sugars of the LPS core region are required to induce defence
reactions.
DOI: 10.1111/j.1469-8137.2004.01214.x
Regulation of enteric endophytic bacterial colonization by plant defenses.
Iniguez AL(1), Dong Y, Carter HD, Ahmer BM, Stone JM, Triplett EW.
Author information:
(1)University of Wisconsin-Madison, Department of Agronomy, Madison 53706, USA.
Bacterial endophytes reside within the interior of plants without causing disease
or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae
342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella
enterica serovar Typhimurium (S. typhimurium), are human pathogens that
contaminate raw produce. Several lines of evidence are presented here to support
the hypothesis that plant defense response pathways regulate colonization by
endophytic bacteria. An ethylene-insensitive mutant of Medicago truncatula is
hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a
signal molecule for induced systemic resistance in plants, decreased endophytic
colonization in Medicago spp. This ethylene-mediated inhibition of endophytic
colonization was reversed by addition of the ethylene action inhibitor,
1-methylcyclopropene. Colonization of Medicago spp. by S. typhimurium also was
affected by exogenous ethylene. Mutants lacking flagella or a component of the
type III secretion system of Salmonella pathogenicity island 1 (TTSS-SPI1)
colonize the interior of Medicago spp. in higher numbers than the wild type.
Arabidopsis defense response-related genotypes indicated that only salicylic acid
(SA)-independent defense responses contribute to restricting colonization by
Kp342. In contrast, colonization by S. typhimurium is affected by both
SA-dependent and -independent responses. S. typhimurium mutants further
delineated these responses, suggesting that both flagella and TTSS-SPI1 effectors
can be recognized. Flagella act primarily through SA-independent responses
(compromising SA accumulation still affected colonization in the absence of
flagella). Removal of a TTSS-SPI1 effector resulted in hypercolonization
regardless of whether the genotype was affected in either SA-dependent or
SA-independent responses. Consistent with these results, S. typhimurium activates
the promoter of PR1, a SA-dependent pathogenesis-related gene, while S.
typhimurium mutants lacking the TTSS-SPI1 failed to activate this promoter. These
observations suggest approaches to reduce contamination of raw produce by human
enteric pathogens and to increase the number of growth-promoting bacteria in
plants.
DOI: 10.1094/MPMI-18-0169
Probing the Sinorhizobium meliloti-alfalfa symbiosis using temperature-sensitive

and impaired-function citrate synthase mutants.
Grzemski W(1), Akowski JP, Kahn ML.
Author information:
(1)Institute of Biological Chemistry, Washington State University, Pullman
99164-6340, USA.
To study the role of the decarboxylating leg of the bacterial TCA cycle in
symbiotic nitrogen fixation, we used DNA shuffling and localized random
polymerase chain reaction mutagenesis to construct a series of
temperature-sensitive and impaired-function mutants in the Sinorhizobium meliloti
Rm104A14 citrate synthase (gltA) gene. Reducing citrate synthase (CS) activity by
mutation led to a corresponding decrease in the free-living growth rate; however,
alfalfa plants formed fully effective nodules when infected with mutants having
CS activities as low as 7% of the wild-type strain. Mutants with approximately 3%
of normal CS activity formed nodules with lower nitrogenase activity and a mutant
with less than 0.5% of normal CS activity formed Fix- nodules. Two
temperature-sensitive (ts) mutants grew at a permissive temperature (25 degrees
C) with 3% of wild-type CS activities but were unable to grow on minimal medium
at 30 degrees C. Alfalfa plants that were inoculated with the ts mutants and
grown with a root temperature of 20 degrees C formed functional nodules with
nitrogenase activities approximately 20% of the wild type. When the roots of
plants infected with the ts mutants were transferred to 30 degrees C, the nodules
lost the ability to fix nitrogen over several days. Microscopic examination of
these nodules revealed the loss of bacteroids and senescence, indicating that CS
activity was essential for nodule maintenance.
DOI: 10.1094/MPMI-18-0134
2115. Ann Bot. 2005 Apr;95(5):863-8. Epub 2005 Feb 14.
The pattern of distribution of pectin, peroxidase and lignin in the middle

lamella of secondary xylem fibres in alfalfa (Medicago sativa).
Wi SG(1), Singh AP, Lee KH, Kim YS.
Author information:
(1)College of Agriculture and Life Sciences, Chonnam National University,
Yongbong-dong 300, Buk-gu, Gwangju, 500-757 Korea.
BACKGROUNDS AND AIMS: Information on the micro-distribution of lignin within the

middle lamella is only just beginning to emerge. This paper provides evidence of
marked heterogeneity in the micro-distribution of lignin, pectin, peroxidase and
hydrogen peroxide in the middle lamella of alfalfa (Medicago sativa).
METHODS: Specimens from alfalfa stems were collected and processed for
transmission electron microscopy. The middle lamella architecture was examined
prior to and during lignification, using transmission electron microscopy in
combination with pectin- and lignin-specific staining. In addition, immuno-gold
labelling of peroxidase and cytochemical localization of hydrogen peroxide (H2O2)
were undertaken.
KEY RESULTS: Lignin showed inhomogeneity in its distribution in the middle
lamella. It was found that the distribution of pectin was irregular and
corresponded to the pattern of deposited lignin. Additionally, a similarity in
the pattern of the deposited lignin to the pattern of distribution of peroxidase
and H2O2 was also observed.
CONCLUSIONS: Irregular distribution of pectin in the middle lamella may be
related to subsequent inhomegeneity in lignin in this region.
DOI: 10.1093/aob/mci092
PMCID: PMC4246741
2116. J Exp Bot. 2005 Mar;56(413):935-43. Epub 2005 Feb 14.
Developmental changes in shoot N dynamics of lucerne (Medicago sativa L.) in

relation to leaf growth dynamics as a function of plant density and hierarchical
position within the canopy.
Lemaire G(1), Avice JC, Kim TH, Ourry A.
Author information:
(1)INRA, Unité d'Ecophysiologie des Plantes Fourragères, F-86600 Lusignan,
France.
Erratum in
J Exp Bot. 2005 May;56(415):1437.
Shoot N concentration in plants decreases as they get bigger, due to the fact
that N accumulates less rapidly than dry matter in plants during the plant growth
process, leading to an allometric relationship between shoot N content (N(sh))
and shoot mass (W(sh)): N(sh)=a(W(sh))b. The results obtained on lucerne plants
growing either under controlled low density conditions or in dense stands under
field conditions show that the value of the allometric coefficient b that
represents the ratio between the relative N accumulation rate in shoots
[dN(sh)/(N(sh)dt)] and the relative growth rate [dW(sh)/(W(sh)dt)], decreases
from 0.88 for a low plant density to 0.72 for a dense stand. Therefore, the
fractional increase of shoot N per unit of shoot dry matter is lower when plants
are in competition for light in dense canopies. This decrease can be entirely
explained by the parallel decline in the leaf area per unit of shoot mass. Thus,
a remarkably constant linear relationship can be established between N(sh) and
leaf area (LA): N(sh)=1.7 g m(-2) LA, regardless of the conditions (low versus
high density, controlled versus field conditions). Moreover, in a field dense
stand, the comparison of plants with contrasting positions between the top and
the bottom of the canopy (dominant, intermediate or suppressed plants), also
shows that the difference in N(sh) at similar shoot mass is explained by the
proportion of leaf mass to shoot mass. These data support the idea that leaf
growth drives the dynamics of shoot N accumulation. These results also indicate
that competition for light among individual plants within a dense canopy induces
developmental changes in plant morphology (leaf:stem ratio) that explain the
differences observed in shoot N concentration. This last observation could be
extrapolated to multi-specific plant stands. Therefore, the sharing of N
resources among plant species could partially be the result of the sharing of
light within the canopy.
DOI: 10.1093/jxb/eri084
2117. J Exp Bot. 2005 Apr;56(414):1205-12. Epub 2005 Feb 14.
Cytoplasm and chloroplasts are not suitable subcellular locations for beta-zein
accumulation in transgenic plants.
Bellucci M(1), De Marchis F, Mannucci R, Bock R, Arcioni S.
Author information:
(1)Institute of Plant Genetics, Research Division of Perugia, CNR, via della
Madonna Alta, 130, 06128 Perugia, Italy. sergio.arcioni@igv.cnr.it.
Zeins, the main storage proteins of maize that accumulate in the endoplasmic
reticulum of the endosperm cells, are particularly interesting because they are
rich in the essential sulphur amino acids. Overexpression of certain zein genes
in plants such as alfalfa would be expected to improve the nutritional
characteristics of this crop. Recently, significant accumulation values have been
reached, but still far from those considered useful for nutritional purposes.
This study investigates whether targeting to compartments other than the
endoplasmic reticulum (cytosol and chloroplasts) could result in increasing
beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol,
the fragment which codes for the signal peptide has been removed. beta-zein has
also been targeted to alfalfa and tobacco chloroplasts by a transit peptide
signal. Both tobacco, as a model plant species, and alfalfa have been transformed
with the assembled constructs. An alternative route to accumulate beta-zein in
the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus,
the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein
gene in each different type of transformed plant was properly transcribed, as
determined by northern blot analysis, but no accumulation of beta-zein was
detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco
transformed plants. Therefore, it is concluded that chloroplasts and the cytosol
are not favourable subcellular locations for zein protein accumulation.
DOI: 10.1093/jxb/eri114
2118. J Environ Radioact. 2005;80(2):175-81.
Screening plant species native to Taiwan for remediation of 137Cs-contaminated

soil and the effects of K addition and soil amendment on the transfer of 137Cs
from soil to plants.
Chou FI(1), Chung HP, Teng SP, Sheu ST.
Author information:
(1)Nuclear Science and Technology Development Center, National Tsing Hua
University, Hsinchu, 300 Taiwan, R.O.C. fichou@mx.nthu.edu.tw
This study aims to screen plant species native to Taiwan that could be used to
eliminate (137)Cs radionuclides from contaminated soil. Four kinds of vegetables
and two kinds of plants known as green manures were used for the screening. The
test plants were cultivated in (137)Cs-contaminated soil and amended soil which
is a mixture of the contaminated one with a horticultural soil. The plant with
the highest (137)Cs transfer factor was used for further examination on the
effects of K addition on the transfer of (137)Cs from the soils to the plant.
Experimental results revealed that plants cultivated in the amended soil produced
more biomass than those in the contaminated soil. Rape exhibited the highest
production of aboveground parts, and had the highest (137)Cs transfer factor
among all the tested plants. The transfer of (137)Cs to the rape grown in the
soil to which 100 ppm KCl commonly used in local fertilizers had been added, were
restrained. Results of this study indicated that rape, a popular green manure in
Taiwan, could remedy (137)Cs-contaminated soil.
DOI: 10.1016/j.jenvrad.2004.10.002
2119. Biol Res. 2000;33(2):159-65.
Effect of nitric oxide and plant antioxidants on microsomal content of lipid

radicals.
Boveris AD(1), Galatro A, Puntarulo S.
Author information:
(1)Physical Chemistry, School of Pharmacy and Biochemistry, University of Buenos
Aires, Argentina.
The antioxidant ability of nitric oxide (NO) generated by a chemical donor and of
commercially available antioxidant preparations was assayed. SNAP
(S-Nitroso-N-acetylpenicilamine) was used as the NO donor, and Ginkgo biloba,
wheat and alfalfa preparations were tested. Lipid peroxidation was assayed by EPR
employing a reaction system consisting of rat liver microsomes, ADP, FeCl3, NADPH
and POBN in phosphate buffer, pH=7.4. In vitro NO exposure decreased microsomal
lipid peroxidation in a dose-dependent manner. The dose responsible for
inhibiting the microsomal content of lipid radical adducts by 50% (LD50) for SNAP
was 550 microM (NO generation rate 0.1 microM/min). The addition of 50 microM
hemoglobin to the incubation media prevented NO effect on lipid peroxidation. The
addition of an amount of the antioxidant preparations equivalent to the LD50
doses inhibited lipid peroxidation by 21, 15, and 33% for wheat, alfalfa, ginkgo
biloba preparations respectively in the presence of 550 microM SNAP. We detected
a decrease in the content of lipid radical adducts after simultaneous
supplementation, although it was less than 50%, even when LD50 doses of the
products were added. This suggests that NO and the natural antioxidants inhibit
lipid peroxidation by a mechanism that has both common and non-shared features.
2120. J Food Prot. 2005 Jan;68(1):40-8.
Effects of community versus single strain inoculants on the biocontrol of

Salmonella and microbial community dynamics in alfalfa sprouts.
Matos A(1), Garland JL.
Author information:
(1)US Department of Agriculture, Agricultural Research Service, Eastern Regional
Research Center, Wyndmoor, Pennsylvania 19038, USA. amatos@arserrc.gov
Potential biological control inoculants, Pseudomonas fluorescens 2-79 and

microbial communities derived from market sprouts or laboratory-grown alfalfa
sprouts, were introduced into alfalfa seeds with and without a Salmonella
inoculum. We examined their ability to inhibit the growth of this foodborne
pathogen and assess the relative effects of the inoculants on the alfalfa
microbial community structure and function. Alfalfa seeds contaminated with a
Salmonella cocktail were soaked for 2 h in bacterial suspensions from each
inoculant tested. Inoculated alfalfa seeds were grown for 7 days and sampled
during days 1, 3, and 7. At each sampling, alfalfa sprouts were sonicated for 7
min to recover microflora from the surface, and the resulting suspensions were
diluted and plated on selective and nonselective media. Total bacterial counts
were obtained using acridine orange staining, and the percentage culturability
was calculated. Phenotypic potential of sprout-associated microbial communities
inoculated with biocontrol treatments was assessed using community-level
physiological profiles based on patterns of use of 95 separate carbon sources in
Biolog plates. Community-level physiological profiles were also determined using
oxygen-sensitive fluorophore in BD microtiter plates to examine functional
patterns in these communities. No significant differences in total and mesophilic
aerobe microbial cell density or microbial richness resulting from the
introduction of inoculants on alfalfa seeds with and without Salmonella were
observed. P. fluorescens 2-79 exhibited the greatest reduction in the growth of
Salmonella early during alfalfa growth (4.22 log at day 1), while the market
sprout inoculum had the reverse effect, resulting in a maximum log reduction
(5.48) of Salmonella on day 7. Community-level physiological profiles analyses
revealed that market sprout communities peaked higher and faster compared with
the other inoculants tested. These results suggest that different modes of
actions of single versus microbial consortia biocontrol treatments may be
involved.
2121. Genetics. 2005 Apr;169(4):2371-82. Epub 2005 Jan 31.
A model selection-based interval-mapping method for autopolyploids.
Cao D(1), Craig BA, Doerge RW.
Author information:
(1)Department of Statistics, Purdue University, West Lafayette, Indiana 47907,
USA.
While extensive progress has been made in quantitative trait locus (QTL) mapping
for diploid species, similar progress in QTL mapping for polyploids has been
limited due to the complex genetic architecture of polyploids. To date, QTL
mapping in polyploids has focused mainly on tetraploids with dominant and/or
codominant markers. Here, we extend this view to include any even ploidy level
under a dominant marker system. Our approach first selects the most likely
chromosomal marker configurations using a Bayesian selection criterion and then
fits an interval-mapping model to each candidate. Profiles of the
likelihood-ratio test statistic and the maximum-likelihood estimates (MLEs) of
parameters including QTL effects are obtained via the EM algorithm. Putative QTL
are then detected using a resampling-based significance threshold, and the
corresponding parental configuration is identified to be the underlying parental
configuration from which the data are observed. Although presented via
pseudo-doubled backcross experiments, this approach can be readily extended to
other breeding systems. Our method is applied to single-dose restriction fragment
autotetraploid alfalfa data, and the performance is investigated through
simulation studies.
DOI: 10.1534/genetics.104.035410
PMCID: PMC1449588
2122. J Chem Ecol. 2004 Nov;30(11):2309-24.
Combined effects of elevated CO2 and herbivore damage on alfalfa and cotton.
Agrell J(1), Anderson P, Oleszek W, Stochmal A, Agrell C.
Author information:
(1)Department of Animal Ecology, Ecology Building Lund University, S-223 62 Lund,
Sweden. jep.agrell@zooekol.lu.se
We examined herbivore-induced responses of alfalfa (Medicago sativa) and cotton

(Gossypium hirsutum) under different CO2 conditions. Plants were grown under
ambient (350 ppm) or elevated (700 ppm) CO2 levels, and were either damaged or
undamaged by Spodoptera littoralis larvae. At harvest, growth of undamaged
(control) plants was determined, and foliar chemical composition of both
undamaged and damaged plants was analyzed. Cotton grew faster overall and showed
a greater increase in growth in response to CO2 enrichment than did alfalfa.
Elevated CO2 levels increased starch and decreased nitrogen levels in damaged
alfalfa and undamaged cotton plants. Alfalfa saponin levels were significantly
increased by elevated CO2 and damage. Regarding specific saponins, medicagenic
acid bidesmoside (3GlcA,28AraRhaXyl medicagenate) concentrations were reduced by
high CO2, whereas zanhic acid tridesmoside (3GlcGlcGlc,23Ara,28AraRhaXylApi Za)
levels were unaffected by the treatments. Soyasaponin I (3GlcAGalRha
soyasapogenol B) was only detected in minute amounts. Alfalfa flavonoid analyses
showed that total flavonoid levels were similar between treatments, although free
apigenin increased and apigenin glucoside
(7-O-[2-O-feruloyl-beta-D-glucuronopyranozyl
(1-->2)-O-8-D-glucuronopyranozyl]-4'-O-beta-D-glucuronopyranozide apigenin)
decreased in CO2-enriched plants. In cotton, herbivore damage increased levels of
total terpenoid aldehydes, gossypol, hemigossypolone, the heliocides H1 and H4,
but not H2 and H3, whereas CO2 enrichment had no effect. These results
demonstrate that combined effects of CO2 and herbivore damage vary between plant
species, which has implications for the competitive balance within plant
communities.
2123. Arch Insect Biochem Physiol. 2005 Feb;58(2):69-83.
Micro-injection of Lygus salivary gland proteins to simulate feeding damage in

alfalfa and cotton flowers.
Shackel KA(1), de la Paz Celorio-Mancera M, Ahmadi H, Greve LC, Teuber LR, Backus
EA, Labavitch JM.
Author information:
(1)Pomology Department, University of California, Davis 95616, USA.
Alfalfa and cotton flowers were pierced with small glass capillaries of an
overall size and shape similar to that of Lygus stylets, and injected with small
quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes.
Crude and partially purified protein solutions from Lygus heads and isolated
salivary glands showed substantial polygalacturonase (PG) activity, as has been
previously reported. Following injection with both crude and partially purified
protein solutions, as well as with pure fungal and bacterial PGs, flowers of both
alfalfa and cotton exhibited damage similar to that caused by Lygus feeding.
Injection with the same volume of a buffer control as well as a buffer control
containing BSA at a comparable protein concentration (approximately 6 microg/mL)
showed no symptoms. These results are consistent with a previously suggested
hypothesis that the extensive tissue damage caused by Lygus feeding is primarily
due to the action of the PG enzyme on the host tissue, rather than to mechanical
damage caused by the insect stylet. Substantial genotypic variation for a PG
inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest
that breeding and selection for increased native PGIP levels, or transformation
with genes encoding PGIP from other plant species, may be of value in obtaining
alfalfa and cotton varieties that are more resistant to Lygus feeding damage.
(c) 2005 Wiley-Liss, Inc.
DOI: 10.1002/arch.20033
2124. J Bacteriol. 2005 Feb;187(3):1161-72.
Sinorhizobium meliloti dctA mutants with partial ability to transport

dicarboxylic acids.
Yurgel SN(1), Kahn ML.

Author information:
(1)Institute of Biological Chemistry, Washington State University Pullman, WA
99164-6340, USA.
Sinorhizobium meliloti dctA encodes a transport protein needed for a successful

nitrogen-fixing symbiosis between the bacteria and alfalfa. Using the toxicity of
the DctA substrate fluoroorotic acid as a selective agent in an iterated
selection procedure, four independent S. meliloti dctA mutants were isolated that
retained some ability to transport dicarboxylates. Two mutations were located in
a region called motif B located in a predicted transmembrane helix of the protein
that has been shown in other members of the glutamate transporter family to be
involved in cation binding. A G114D mutation was located in the third
transmembrane helix, which had not previously been directly implicated in
transport. Multiple sequence alignment of more than 60 members of the glutamate
transporter family revealed a glycine at this position in nearly all members of
the family. The fourth mutant was able to transport succinate at almost wild-type
levels but was impaired in malate and fumarate transport. It contains two
mutations: one in a periplasmic domain and the other predicted to be in the
cytoplasm. Separation of the mutations showed that each contributed to the
altered substrate preference. dctA deletion mutants that contain the mutant dctA
alleles on a plasmid can proceed further in symbiotic development than null
mutants of dctA, but none of the plasmids could support symbiotic nitrogen
fixation, although they can transport dicarboxylates, some at relatively high
levels.
DOI: 10.1128/JB.187.3.1161-1172.2005
PMCID: PMC545702
2125. J Agric Food Chem. 2005 Jan 26;53(2):369-73.
Perchlorate accumulation in forage and edible vegetation.
Jackson WA(1), Joseph P, Laxman P, Tan K, Smith PN, Yu L, Anderson TA.
Author information:
(1)Department of Civil Engineering, Texas Tech University, Lubbock, Texas 79409,
USA. andrew.jackson@coe.ttu.edu
The accumulation of perchlorate in vegetation is becoming a concern, with

increasing numbers of sites reporting the presence of perchlorate in groundwater
and surface water. This study investigated potential perchlorate uptake and
distribution by a variety of forage and edible crops in both the laboratory and
the field. Perchlorate concentrations in soybean leaves grown in the greenhouse
were significantly higher than perchlorate concentrations in soybean seeds and
pods. Perchlorate concentrations in alfalfa grown in sand were significantly
lower than those in alfalfa grown in soil. The concentration of perchlorate in
tomato was lower in the fruit than the leaves. Commercially grown wheat and
alfalfa samples all contained perchlorate, 0.72-8.6 mg/kg of fresh weight (FW) in
the wheat stems, 0.71-4.4 mg/kg of FW in the wheat heads, and 2.9 mg/kg of FW in
alfalfa. All field garden samples tested (including cucumber, cantaloupe, and
tomato) that were irrigated with perchlorate-tainted water contained perchlorate
at various concentrations ranging from 0.040 to 1.65 mg/kg of FW.
Bioconcentration factors (BCF), ratios of plant fresh weight concentrations to
estimated or measured groundwater concentrations [(microg/kg of FW)/microg/L],
were all in the same order of magnitude ranging from 215 +/- 126 for wheat stems
to 233 +/- 264 for wheat heads and to 380 +/- 89 for alfalfa. BCF for garden
fruit samples were much lower (0.5-20). Results from this study highlight the
potential for perchlorate exposure by routes other than drinking water.
DOI: 10.1021/jf0493021
2126. J Dairy Sci. 2005 Feb;88(2):644-52.
Monensin for lactating dairy cows grazing mixed-alfalfa pasture and supplemented
with partial mixed ration.
Gallardo MR(1), Castillo AR, Bargo F, Abdala AA, Maciel MG, Perez-Monti H, Castro
HC, Castelli ME.
Author information:
(1)Estación Experimental INTA Rafaela, Argentina 2300.
mgallardo@rafaela.inta.gov.ar
The effect of monensin on milk production was evaluated in 58 lactating Holstein

cows (48 multiparous; 10 primiparous) grazing a mixed-alfalfa pasture and
supplemented with a partial mixed ration in a completely randomized design with
repeated measurements. Cows were paired by calving date, lactation number,
previous lactation milk production, body weight, and body condition score and
were assigned to one of 2 treatments: control or monensin. Cows on the monensin
treatment received 2 monensin controlled-release capsules (335 mg/d for 90 d),
one 30 d before the expecting calving date and the other 60 d after calving.
Short-term (0 to 150 d in milk) and long-term (305-d adjusted lactation) effects
of monensin were evaluated. Pasture (measured by difference between pre- and
postgrazing pasture mass), supplements, and total dry matter intake did not
differ between treatments and averaged 8.7, 14.1, and 22.9 kg/d, respectively. In
the short-term, monensin increased milk production (27.7 vs. 26.6 kg/d) and milk
protein yield (0.890 vs. 0.860 kg/d); milk fat yield was not affected (0.959
kg/d). Monensin decreased milk fat content (3.51 vs. 3.60%) with no changes in
milk protein content (3.25%). In the long term, milk production and milk protein
yield were also increased by monensin: 214 and 7 kg, respectively. Monensin
reduced the loss of body condition score and increased percentage of pregnancy at
first service (44.8 vs. 20.7%). Monensin improves production and reproduction
performance of dairy cows grazing a mixed-alfalfa pasture and supplemented with a
partial mixed ration.
DOI: 10.3168/jds.S0022-0302(05)72728-4
2127. J Dairy Sci. 2005 Feb;88(2):632-43.
Dry matter intake is decreased more by abomasal infusion of unsaturated free

fatty acids than by unsaturated triglycerides.
Litherland NB(1), Thire S, Beaulieu AD, Reynolds CK, Benson JA, Drackley JK.
Author information:
Previous experiments from our group have demonstrated that abomasal infusion of
unsaturated free fatty acids (FFA) markedly decreases dry matter intake (DMI) in
dairy cows. In contrast, experiments from other groups have noted smaller
decreases in DMI when unsaturated triglycerides (TG) were infused postruminally.
Our hypothesis was that unsaturated FFA would be more potent inhibitors of DMI
than an equivalent amount of unsaturated TG. Four Holstein cows in late lactation
were used in a single reversal design. Cows were fed a total mixed ration
containing (DM basis) 23% alfalfa silage, 23% corn silage, 40.3% ground shelled
corn, and 10.5% soybean meal. Two cows received soy FFA (UFA; 0, 200, 400, 600
g/d) and 2 received soy oil (TG) in the same amounts; cows then were switched to
the other lipid source. Cows were abomasally infused with each amount for 5-d
periods. The daily amount of lipid was pulse-dosed in 4 equal portions at 0600,
1000, 1700, and 2200 h; no emulsifiers were used and there was no sign of
digestive disturbance. Both lipid sources linearly decreased DMI, with a
significant interaction between lipid source and amount. Slope-ratio analysis
indicated that UFA were about 2 times more potent in decreasing DMI than were TG.
Decreased DMI led to decreased milk production. Milk fat content was increased
linearly by lipid infusion. Milk fat yield decreased markedly for UFA infusion
but was relatively unaffected by infusion of TG. Contents of short- and
medium-chain fatty acids in milk fat decreased as the amount of either infusate
increased. Contents of C(18:2) and C(18:3) in milk fat were increased linearly by
abomasal infusion of either fat source; cis-9 C(18:1) was unaffected. Transfer of
infused C(18:2) to milk fat was 35.6, 42.5, and 27.8% for 200, 400, and 600 g/d
of UFA, and 34.3, 39.6, and 34.0% for respective amounts of TG. Glucagon-like
peptide-1 (7-36) amide (GLP-1) concentration in plasma significantly increased as
DMI decreased with increasing infusion amount of UFA or TG. Plasma concentration
of cholecystokinin-octapeptide (CCK-8) was unaffected by lipid infusion. These
results indicate that unsaturated FFA reaching the duodenum are more potent
inhibitors of DMI than are unsaturated TG; the effect may be at least partially
mediated by GLP-1.
DOI: 10.3168/jds.S0022-0302(05)72727-2
2128. Altern Lab Anim. 2004 Oct;32(4):407-16.
Mitochondrial toxicity detected in a health product with a boar spermatozoan

bioassay.
Hoornstra D(1), Andersson MA, Johansson T, Pirhonen T, Hatakka M,

Salkinoja-Salonen MS.
Author information:
(1)Department of Applied Chemistry and Microbiology, University of Helsinki,
Finland.
Seaweed and organic alfalfa capsules sold as "health promoting" products had
repeatedly caused emesis in a consumer. Using the boar spermatozoan bioassay, the
capsule contents were found to contain a toxic substance that inhibited boar
sperm motility and depolarised mitochondria at low exposure concentrations of 10
microg/ml. The capsule also contained high amounts (10(5)-10(7) cfu/g), of
endospore-forming bacteria and Streptomyces-like bacteria. Bacteria from the
capsule produced toxic substances when cultured in the laboratory. Three
different toxic responses were provoked in the spermatozoa exposed to extracts
from the Streptomyces-like isolates: a) hyperpolarisation of the plasma membrane
and depolarisation of the mitochondria; b) depolarisation of mitochondria similar
to that caused by the capsule content extract; and c) motility inhibition, with
no observed change of any cytosolic transmembrane potential. Membrane potential
changes in the sperm cells exposed to the bacterial extracts were similar to
those provoked by exposure to valinomycin and bafilomycin A1, to nigericin, and
to oligomycin and ionomycin, respectively. Extracts prepared from Bacillus
isolated from the capsule non-specifically depolarised all the cellular
transmembrane potentials. The results demonstrate the potential value of a cell
toxicity assay with boar spermatozoa for detecting hazardous substances in
products intended for human consumption, without whole-animal exposure or using
fetal calf serum for cell cultures.
2129. Cell Mol Biol (Noisy-le-grand). 2004 Sep;50(6):773-81.
Endogenous protein kinase-C activity and phosphorylated proteins in messenger

ribonucleoprotein complexes of developing embryos of alfalfa.
Pramanik SK(1), Williams AL, Bewley JD.
Author information:
(1)Department of Botany, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Spramani@jewl.morgan.edu
Developing somatic and zygotic embryos of alfalfa (Medicago sativa L.) exhibited
endogenous protein kinase activity and protein acceptors of phosphate groups
using both cell-free translational extracts and
oligo(dT)-cellulose-column-purified mRNPs. The cell-free-translation extracts
from pre-cotyledonary-stage somatic embryos had approximately 50- and 100-fold
more protein kinase activity than cotyledonary-stage somatic and zygotic embryos.
Several polypeptides were phosphorylated; some of them were unique to the early
stage and some to the late-stage developing embryos. A 65 kDa protein was
phosphorylated heavily in pre-cotyledonary-stage somatic embryos. This
phosphorylated protein was comprised of three main components, two of which were
phosphorylated heavily. Heat-shock treated-embryos lost their exitant kinase
activity and at the same time another form of protein kinase activity was
activated which phosphorylated a novel 28 kDa protein. Endogenous protein kinase
activity was also observed within the mRNPs of polysomal and non-polysomal
fractions of developing embryos, and this phosphorylated only 65, 43 and 30 kDa
proteins within these fractions. A 30 kDa protein from the pre-cotyledonary-stage
somatic embryos showed a higher affinity for accepting phosphate groups than the
proteins from cotyledonary-stage somatic or zygotic embryos. The activity of
protein kinase was largely c-AMP-independent, but was dependent on Ca2+,
phospholipid and phorbol ester. The enzyme belongs to the protein kinase-C
family; the 65 kDa protein cross-reacts with antibodies made against protein
kinase-C (alpha- and beta-isoforms) and it may be an autophosphorylated protein.
2130. Plant J. 2005 Jan;41(2):257-68.
Signaling of phosphorus deficiency-induced gene expression in white lupin

requires sugar and phloem transport.
Liu J(1), Samac DA, Bucciarelli B, Allan DL, Vance CP.
Author information:
(1)Agronomy and Plant Genetics, University of Minnesota, St Paul, MN 55108, USA.
liuxx162@tc.umn.edu
Roots of phosphorus (P)-deficient white lupin exhibit striking changes in

morphology and gene expression. In this report we provide further insight into
genetic elements affecting transcription of P-deficiency-induced genes. Moreover,
we also show that sugars and photosynthates are integrally related to
P-deficiency-induced gene expression. White lupin phosphate transporter (LaPT1)
and secreted acid phosphatase (LaSAP1) promoter-reporter genes when transformed
into alfalfa, a heterologous legume, showed significant induction in roots
specifically in response to P-deficiency. In addition, both promoters were active
in nitrogen-fixing root nodules but not in ineffective nodules indicating a link
between P-deficiency and factors related to nitrogen fixation/metabolism. As
sugars play a role in signal transduction during nitrogen assimilation and are
required for effective nitrogen fixation, we tested the relationship of sugars to
P-deficiency-induced gene expression. Exogenous sucrose, glucose, and fructose
stimulated LaPT1 and LaSAP1 transcript accumulation in dark-grown P-sufficient
white lupin seedlings. Furthermore, in intact P-deficient white lupin plants,
LaPT1 and LaSAP1 expression in cluster roots was strikingly reduced in
dark-adapted plants with expression rapidly restored upon reexposure to light.
Likewise, interruption of phloem supply to P-deficient roots resulted in a rapid
decline in LaPT1 and LaSAP1 transcript accumulation. Similar results were also
obtained with a third lupin P-deficiency-induced gene encoding a putative
multidrug and toxin efflux protein (LaMATE). Taken together, our data show that
the regulation of P-deficiency-induced genes is conserved across plant species
and sugars/photosynthates are crucial for P-deficiency signal transduction.
DOI: 10.1111/j.1365-313X.2004.02289.x
2131. Microbiology. 2005 Jan;151(Pt 1):259-68.
Detailed studies of the binding mechanism of the Sinorhizobium meliloti

transcriptional activator ExpG to DNA.
Baumgarth B(1), Bartels FW, Anselmetti D, Becker A, Ros R.
Author information:
(1)Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, 33615
Bielefeld, Germany.
The exopolysaccharide galactoglucan promotes the establishment of symbiosis

between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti
2011 and its host plant alfalfa. The transcriptional regulator ExpG activates
expression of galactoglucan biosynthesis genes by direct binding to the expA1,
expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of
regulatory proteins. Analysis of target sequences of an ExpG(His)(6) fusion
protein in the exp promoter regions resulted in the identification of a binding
site composed of a conserved palindromic region and two associated sequence
motifs. Association and dissociation kinetics of the specific binding of
ExpG(His)(6) to this binding site were characterized by standard biochemical
methods and by single-molecule spectroscopy based on the atomic force microscope
(AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics
between the wild-type binding sequence and two mutated binding sites, leading to
a closer understanding of the ExpG-DNA interaction.
DOI: 10.1099/mic.0.27442-0
2132. Bioresour Technol. 2005 May;96(8):907-11.
Potential of alfalfa as an alternative molt induction diet for laying hens: egg
quality and consumer acceptability.
Landers KL(1), Howard ZR, Woodward CL, Birkhold SG, Ricke SC.
Author information:
(1)Department of Poultry Science, Texas A&M University, MS# 2472 TAMU, College
Station, TX 77843, USA.
Dietary molt induction to initiate additional egg laying cycles in commercial

laying hen flocks is a wide spread practice in the United States. Feed
deprivation is the most commonly used method but this practice has generated
several concerns which has lead to research for viable alternative approaches.
From a management standpoint a single ingredient molting diet consisting of high
fiber-low energy represents an easily adaptable diet for large laying hen
production units. Alfalfa meal is readily available in most commercial locations
and possesses many of the desirable properties of an ideal laying hen molt diet.
In the current study hens at a commercial laying facility were molted by both
alfalfa and feed deprivation. After the hens had reentered post-molt commercial
egg production, eggs were examined for egg quality performance. Egg shell
strength, albumen height, yolk height, weight, length, and yolk color were all
tested using various mechanical techniques. The eggs were also sampled for
testing by consumer sensory panels that assessed the desirability of the eggs'
color and flavor/texture. Eggs laid by hens molted by alfalfa had a significantly
lower (p<0.05) "a*" level of colorimetry. Eggs laid by hens molted with alfalfa
also exhibited significantly higher (p<0.05) egg weights and length. In the
consumer sensory test, there was no significant difference (p>0.05) in color or
flavor/texture scores in eggs from either feed deprived or alfalfa molted hens.
The consumer sensory and mechanical quality attributes indicates that alfalfa
shows promise as an alternative molt induction diet by providing a single diet
option for extending egg production into a second egg laying cycle.
DOI: 10.1016/j.biortech.2004.08.014
2133. DNA Seq. 2004 Aug;15(4):283-90.
Molecular cloning and sequence analysis of a novel chalcone synthase cDNA from
Ginkgo biloba.
Pang Y(1), Shen GA, Liu C, Liu X, Tan F, Sun X, Tang K.
Author information:
(1)State Key Laboratory of Genetic Engineering, School of Life Sciences,
Fudan-SJTU-Nottingham Plant Biotechnology R & D Center, Fudan University,
Shanghai 200433, P R China.
A chalcone synthase (CHS) gene was cloned from Ginkgo biloba for the first time
and it was also the first cloned gene involved in flavonoids metabolic pathway in
G. biloba. The full-length cDNA of G. biloba CHS (designated as Gbchs) was 1608bp
with poly(A) tailing and it contained a 1173bp open reading frame (ORF) encoding
a 391 amino acid protein. Gbchs was found to have extensive homology with those
of other plant chs genes via multiple alignments. The active sites of the CoA
binding, coumaroyl pocket and cyclization pocket in CHS protein of Medicago
sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the
three-dimensional structure of GbCHS strongly resembled that of M. sativa
(MsCHS2), implying GbCHS may have similar functions with MsCHS2. Phylogenetic
tree analysis revealed that GbCHS had closer relationship with CHSs from
gymnosperm plants than from other plants. Gbchs is a useful tool to study the
regulation of flavonoids metabolism in G. biloba.
2134. J Chem Ecol. 2004 Oct;30(10):1969-83.
Bioactivity of lipophilic metabolites from glandular trichomes of Medicago sativa

against the potato leafhopper.
Ranger CM(1), Winter RE, Rottinghaus GE, Backus EA, Ellersieck MR, Johnson DW.
Author information:
(1)Department of Entomology and Interdisciplinary Plant Group, 1-87 Agriculture
Building, University of Missouri, Columbia, Missouri 65211, USA.
ranger@aesop.rutgers.edu
Medicago sativa cv. G98A is highly resistant to the potato leafhopper,

Empoascafabae. Glandular trichome extracts from G98A were fractionated using
flash chromatography and tested for settling deterrency against the potato
leafhopper. A fraction of intermediate polarity exhibited strong, dose-dependent
deterrency when applied to the surface of an artificial diet sachet. Deterrency
was not detected, however, when the fraction was applied to the internal surface
of the sachet membrane (i.e., when contact was limited to only the leafhoppers'
stylets). Major components of the highly deterrent fraction, determined by gas
chromatography-mass spectrometry, were a homologous series of fatty acid amides
C(n)H(2n+1)NO (n = 19-23) and trace components were 12:0, 13:0, 14:0, 15:0, 16:0,
17:0, 18:0, and possibly 18:1 free fatty acids. Deterrency declined slightly, but
was still strong, after fatty acids were removed from crude extracts. When the
crude extracts were separated further, a fraction containing only the fatty acid
amides was also deterrent. Activity increased when this fraction was supplemented
with authentic (C12:0 through C18:0, and C18:1) free fatty acids. However, the
authentic free fatty acids were not deterrent when tested without the lipophilic
amides. Fatty acid amides and free fatty acids in trichomes of M. sativa G98A may
synergize and together function in deterring settling by the potato leafhopper.
2135. Environ Int. 2005 Jan;31(1):1-13.
Phytoremediation of aquaculture wastewater for water recycling and production of

fish feed.
Ghaly AE(1), Kamal M, Mahmoud NS.
Author information:
(1)Biological Engineering Department, Dalhousie University, P.O. Box 1000,
Halifax, Nova Scotia, Canada B3J 2X4. abdel.ghaly@dal.ca
Five plants were examined for their ability to remove nutrients from aquaculture
wastewater and suitability as fish feed: alfalfa, white clover, oat, fall rye,
barley. The seeds were first germinated in water in a hydroponic system, and the
plants were fed wastewater from Tilapia production facility. Clover and alfalfa
seeds were infected with fungus shortly after germination, and their roots were
completely destroyed by day 14. Oat, rye and barley had the fastest growth and
showed greater tolerance to fungal disease compared with alfalfa and clover.
Although substantial amounts of soluble and insoluble substances were released by
the seeds during the germination period, the plants were able to remove all the
pollutants in wastewater and significant portions of those released substances.
The total reductions in total solids, COD, NO3-N, NO2-N, phosphate and potassium
ranged from 54.7% to 91.0%, 56.0% to 91.5%, 82.9% to 98.1%, 95.9% to 99.5%, 54.5%
to 93.6% and 99.6% to 99.8%, respectively. Oat, barley and rye grow well in this
type of hydroponic system and can be used as a fish feed after being supplemented
with fat, Ca, Na, Mn and Fe. Oil seeds and the chlorides of these elements could
be added to these plants when formulating the fish feed. For a continuous
operation, a two-unit system could be configured to allow for one week
germination and one week cleaning and startup in one unit while the other unit is
in operation.
DOI: 10.1016/j.envint.2004.05.011
2136. J Immunol Methods. 2004 Nov;294(1-2):155-63. Epub 2004 Oct 4.
Immunoassay for biochanin A.
Lapcík O(1), Vítková M, Klejdus B, Al-Maharik N, Adlercreutz H.
Author information:
(1)Department of Chemistry of Natural Compounds, Institute of Chemical
Technology, Technická 5, 166 28 Praha 6, Czech Republic. oldrich.lapcik@vscht.cz
Two variants of immunoassay for the determination of biochanin A (5,7-dihydroxy

4'-methoxy isoflavone), i.e., a radioimmunoassay (RIA) and an indirect ELISA,
have been developed and evaluated. Both methods employ the same rabbit antiserum
to a 7-O-carboxymethyl-5-hydroxy-4'-methoxyisoflavone-bovine serum albumin (BSA)
conjugate. A 125I-labeled hapten-tyrosine methyl ester (TME) conjugate was used
as a radioligand for the RIA. The indirect ELISA uses immunogen-coated
microtitration plates and a peroxidase-labeled antirabbit Ig antibody. Both
methods are specific for biochanin A with a comparable sensitivity (3.1 pg/tube
for RIA; 5.3 pg/well for ELISA); however, their sensitivity to individual
cross-reactants differs. The main cross-reactants are sissotrin (the
cross-reactivity 15.7% for RIA; 120% for ELISA), 5-hydroxy, 4',7-dimethoxy
isoflavone (51.5% for RIA; 46.5% for ELISA), prunetin (4.5% for RIA; 5.0% for
ELISA), genistein (0.8% for RIA; 2.8% for ELISA) and formononetin (0.4% for RIA;
0.3% for ELISA). These methods were used for the analysis of biochanin A in
alfalfa and in several nonleguminous plants.
DOI: 10.1016/j.jim.2004.09.004
2137. J Bacteriol. 2005 Jan;187(1):376-81.
The katA catalase gene is regulated by OxyR in both free-living and symbiotic
Jamet A(1), Kiss E, Batut J, Puppo A, Hérouart D.
Author information:
(1)Laboratoire Interactions Plantes-Microorganismes et Santé Végétele, UMR, INRA,
Université de Nice Sophia-Antipolis, CNRS, 400 Route des Chappes, BP 167, F-06903
Sophia Antipolis Cedex, France.
The characterization of an oxyR insertion mutant provides evidences that katA,
which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not
only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti.
Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its
own expression in S. meliloti.
DOI: 10.1128/JB.187.1.376-381.2005
PMCID: PMC538821
2138. Mol Plant Microbe Interact. 2004 Dec;17(12):1294-305.
Localization of superoxide dismutases and hydrogen peroxide in legume root

nodules.
Rubio MC(1), James EK, Clemente MR, Bucciarelli B, Fedorova M, Vance CP, Becana
M.
Author information:
Superior de Investigaciones Cientificas, Apdo 202, 50080 Zaragoza, Spain.
Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to

O2 and H2O2 and thus represent a primary line of antioxidant defense in all
aerobic organisms. H2O2 is a signal molecule involved in the plant's response to
pathogen attack and other stress conditions as well as in nodulation. In this
work, we have tested the hypothesis that SODs are a source of H2O2 in
indeterminate alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The
transcripts and proteins of the major SODs of nodules were localized by in situ
RNA hybridization and immunogold electron microscopy, respectively, whereas H2O2
was localized cytochemically by electron microscopy of cerium-perfused nodule
tissue. The transcript and protein of cytosolic CuZnSOD are most abundant in the
meristem (I) and invasion (II) zones, interzone II-III, and distal part of the
N2-fixing zone (III), and those of MnSOD in zone III, especially in the infected
cells. At the subcellular level, CuZnSOD was found in the infection threads,
cytosol adjacent to cell walls, and apoplast, whereas MnSOD was in the
bacteroids, bacteria within infection threads, and mitochondria. The distinct
expression pattern of CuZnSOD and MnSOD suggests specific roles of the enzymes in
nodules. Large amounts of H2O2 were found at the same three nodule sites as
CuZnSOD but not in association with MnSOD. This colocalization led us to
postulate that cytosolic CuZnSOD is a source of H2O2 in nodules. Furthermore, the
absence or large reduction of H2O2 in nodule tissue preincubated with enzyme
inhibitors (cyanide, azide, diphenyleneiodonium, diethyldithiocarbamate) provides
strong support to the hypothesis that at least some of the H2O2 originates by the
sequential operation of an NADPH oxidase-like enzyme and CuZnSOD. Results also
show that there is abundant H2O2 associated with degrading bacteroids in the
senescent zone (IV), which reflects the oxidative stress ensued during nodule
senescence.
DOI: 10.1094/MPMI.2004.17.12.1294
2139. J Anim Sci. 2005 Jan;83(1):262-70.
Dry matter intake and digestion of alfalfa harvested at sunset and sunrise.
Burns JC(1), Mayland HF, Fisher DS.

Author information:
(1)ARS-USDA, and Crop Science Department, North Carolina State University,
Raleigh 27695, USA. joe_burns@ncsu.edu <joe_burns@ncsu.edu>
The preference exhibited by animals in selecting one feed over another is

important only if the preferred diet is consumed daily in larger quantities,
digested to a greater extent, or both. Six alfalfa (Medicago sativa L.) hays were
harvested in pairs at sunset (PM) and sunrise (AM) on consecutive days at three
harvest dates. A previous study of these hays demonstrated differences in
ruminant preference favoring PM harvests. This study evaluated the effects of
time of cutting and harvest date on voluntary DMI and nutrient digestibility. The
hays were field-cured, baled, and chopped before evaluation for intake and
digestibility. Studies were conducted for sheep (Ovis aries), goats (Capra
hircus), and cattle (Bos taurus). Goats, but not steers or sheep, demonstrated
differences in nutrient digestibility between PM- and AM-cut hays. Goats consumed
more PM than AM hay (2.97 vs. 2.83 kg/100 kg of BW; P = 0.07) and digested it to
a greater extent (0.710 vs. 0.696; P = 0.03), resulting in greater digestible DMI
(2.11 vs. 1.97 kg/100 kg of BW; P = 0.03). Sheep consumed (mean = 2.52 kg/100 kg
of BW; P = 0.59) and digested (mean = 0.681; P = 0.25) PM- and AM-cut hays
similarly. Steers consumed larger quantities of PM-than AM-cut hay (2.90 vs. 2.62
kg/100 kg of BW; P = 0.11), but digestion did not differ with cutting time (mean
= 0.660; P = 0.75). Difference values (composition of fed hay minus composition
of orts) indicated that sheep and goats selected from the feed offered similarly,
whereas steers selected differently. Difference values for CP averaged 94 and 101
g/kg for goats and sheep and 32 g/kg for steers (P < 0.01), and difference values
for NDF averaged 185 and 196 g/kg for goats and sheep and 73 g/kg for steers (P
<or= 0.01). Steer DMI and digestible DMI were associated with preference (r =
+0.83, P <or= 0.05; and r = +0.89, P <or= 0.05) and with coordinates for
preference criteria (dimension 1; r = +0.90, P <or= 0.05; and r = +0.89, P <or=
0.05) from a previous preference trial. Intake and digestion responses for goats
and sheep showed no relationship with the previous preference trial measurements.
For cattle and goats, the management strategy of mowing in the afternoon seems to
take advantage of small, but influential diurnal changes in the soluble
carbohydrate fraction and offers the potential to improve forage quality.
2140. Planta. 2005 Apr;221(1):28-38. Epub 2004 Dec 2.
Transcriptome analysis of alfalfa glandular trichomes.
Aziz N(1), Paiva NL, May GD, Dixon RA.
Author information:
Glandular trichomes are a major site of plant natural product synthesis and
accumulation for protection against insect predation. However, to date few
studies have attempted to obtain a global view of trichome gene expression. Two
contrasting approaches have been adopted to investigate genes expressed in
glandular trichomes from alfalfa (Medicago sativa L.). In the first approach,
5,674 clones from an alfalfa glandular trichome cDNA library were sequenced. The
most highly abundant expressed sequence tag (EST) corresponded to a lipid
transfer protein. The presence of ESTs corresponding to enzymes for all steps in
the biosynthesis of flavonoids suggests that these are important metabolites in
alfalfa trichome biology, as confirmed by histochemistry and metabolite
profiling. No ESTs corresponded to enzymes of cyclized terpenoid biosynthesis. In
a second approach, microarray analysis was used to compare levels of alfalfa
transcripts corresponding to 16,086 Medicago truncatula A17 genes in stems with
and without trichomes. This revealed over 1,000 genes with strong preferential
expression in the trichome fraction of the stem, 70% of which are of unknown
function. These define a class of genes that are not trichome-specific, since M.
truncatula A17 does not itself have glandular trichomes, but has potential
importance for trichome function within the stem.
DOI: 10.1007/s00425-004-1424-1
2141. Res Microbiol. 2004 Dec;155(10):855-60.
The symbiotic defect in a Sinorhizobium meliloti lipopolysaccharide mutant can be

overcome by expression of other surface polysaccharides.
Hozbor DF(1), Pich Otero AJ, Lodeiro AR, Del Papa MF, Pistorio M, Lagares A.
Author information:
Universidad Nacional de La Plata, Calles 47 y 115, 1900 La Plata, Argentina.
hozbor@biol.unlp.edu.ar
In this work we have examined the extent of functional complementation in

symbiosis among different Sinorhizobium meliloti surface polysaccharides
including lipopolysaccharide (LPS). We show that a symbiotic deficiency
associated with an LPS defect can be reversed by appropriate expression of other
surface polysaccharides such as galactoglucan (EPSII) and a particular form of
capsular polysaccharide (KdoPS). It is noteworthy that, while succinoglycan EPSI
and LPS cannot functionally substitute for each other, they can both be replaced
by the same common set of polysaccharides (i.e., EPSII/KdoPS). The complex
pattern of functional complementation in symbiosis among S. meliloti surface
polysaccharides was shown to be different in Medicago truncatula compared to that
previously reported for M. sativa.
DOI: 10.1016/j.resmic.2004.06.012
2142. Theor Appl Genet. 2005 Jan;110(2):219-27. Epub 2004 Nov 19.
Use of artificial environments to reproduce and exploit genotype x location

interaction for lucerne in northern Italy.
Annicchiarico P(1), Piano E.
Author information:
(1)Istituto Sperimentale per le Colture Foraggere, 29 viale Piacenza, 26900,
Lodi, Italy. bred@iscf.it
Genotype x environment interaction effects can be exploited by breeding for

specific adaptation to well-defined subregions within a target region. Previous
work showed that genotype x location interaction for dry matter (DM) yield of
lucerne (Medicago sativa L. subsp. sativa) cultivars in northern Italy is large
and associated with soil type and level of summer drought stress of locations,
suggesting the presence of two contrasting subregions. Thirteen farm landraces
collected across the region and four control varieties were evaluated for DM
yield in four artificial environments created at one site by the factorial
combination of soil type (sandy loam or silty clay) and drought stress level
(almost nil or high) for: (1) exploring the possibility to reproduce in
artificial environments the adaptation patterns occurring across the region; (2)
investigating the adaptation pattern of landraces and its relationship with
environmental factors at collecting sites; and (3) providing a preliminary
comparison of wide- versus specific-adaptation strategies based on yield gains
predicted from selection of populations. Different soils filled large
(24.0x1.6x0.8-m deep), bottomless containers in concrete. Water amounts were
controlled by irrigation under a moving rain shelter. Cultivars varied largely
for adaptation pattern across the artificial environments, mainly due to cultivar
x stress interaction. Better response to stress conditions of landraces was
closely associated with the level of summer drought at collecting sites (r=0.82),
highlighting the importance of evolutionary adaptation. The additive main effects
and multiplicative interaction-modelled responses of control cultivars
successfully reproduced those observed across locations, candidating the
artificial environments as a cheaper alternative to more selection locations when
breeding for wide or specific adaptation. The latter implied about 40-50% greater
estimated gains relative to breeding for wide adaptation.
DOI: 10.1007/s00122-004-1811-9
2143. J Food Prot. 2004 Nov;67(11):2488-95.
Strain-specific differences in the attachment of Listeria monocytogenes to

alfalfa sprouts.
Gorski L(1), Palumbo JD, Nguyen KD.
Author information:
(1)Produce Safety and Microbiology Research Unit, Agricultural Research Service,
U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710,
USA. lgorski@pw.usda.gov
Contamination of fresh produce with Listeria monocytogenes has resulted in

outbreaks of systemic listeriosis and febrile gastroenteritis. Recalls of alfalfa
sprouts have occurred due to contamination with L. monocytogenes. Alfalfa sprouts
were used as a preharvest model to study the interaction with this human
pathogen. Seventeen strains were assessed for their capacity to colonize alfalfa
sprouts, and strain-specific differences (not related to source, serotype, or
lineage) were revealed when the sprout irrigation water was changed daily. Two of
the strains colonized and attached to the sprouts very well, reaching levels of
more than 5 log CFU per sprout. The remaining strains varied in their final
levels on sprouts between less than 1 to 4.7 log CFU per sprout. All of the L.
monocytogenes strains grew to equivalent levels on the sprouts when the
irrigation water was not changed, suggesting the differences observed with
regular changing of the water resulted from differences in attachment. Further
analysis of the best colonizing strains indicated that only between 0.3 and 1 log
CFU per sprout could be removed by additional washing of the sprout, and the
presence of normal sprout bacteria did not compete with the L. monocytogenes
strains on the sprouts. The poorest colonizing strain was able to grow in the
irrigation water during the experiment but could not attach to the sprouts.
Microscopic examination of the sprouts with L. monocytogenes expressing the green
fluorescent protein indicated that L. monocytogenes was associated with the root
hairs of the sprouting alfalfa, with few to no cells visible elsewhere on the
sprout.
2144. Ecotoxicol Environ Saf. 2005 Feb;60(2):123-31.
The effect of Zn2+ on glucose 6-phosphate dehydrogenase activity from Bufo

arenarum toad ovary and alfalfa plants.
Fonovich de Schroeder TM(1).
Author information:
(1)Escuela de Ciencia y Tecnología, Universidad Nacional de General San Martín,
Alem 3901, 1653 Villa Ballester, Buenos Aires, Argentina.
teresa.fonovich@unsam.edu.ar
The effect of Zn2+ on glucose 6-phosphate dehydrogenase (G6PD) activity was

monitored in samples from Bufo arenarum toad ovary and alfalfa plants, in the
search for a possible new bioindicator able to detect levels of exposure through
contaminated soils, and also to elucidate possible similarities between the
enzyme from animal and plant tissues. The in vivo effect was evaluated after
exposure of the toads to the metal in Ringer solution during 30 days and after 10
days of treatment in 6 weeks old plants, cultured under laboratory conditions. In
vitro effects were measured in different extracts from control samples and
partially purified enzyme from ovarian tissue as well as in different extracts
from control alfalfa plants, by addition of the metal to the reaction mixture
containing the enzyme. G6PD from toad ovary was noncompetitively inhibited by
zinc both in vivo and in vitro, under all the experimental conditions studied. A
kinetic analysis of the enzyme activity showed that the Michaelis-Menten constant
(Km) was not modified, while maximal velocity (Vmax) decreased as the consequence
of treatment. It was not possible to obtain a dose-response curve for the effects
of Zn2+ on G6PD from alfalfa whole plants, measured in vivo or in vitro. Only
leaf extracts evidenced a possible relationship between treatment with the metal
and G6PD activity alteration. The results agree with a possible role for G6PD as
a biomarker of effect and exposure to Zn2+ in B. arenarum ovarian tissue but not
in alfalfa plants.
DOI: 10.1016/j.ecoenv.2004.07.008
2145. J Dairy Sci. 2004 Dec;87(12):4221-9.
Effects of replacing dietary starch with sucrose on ruminal fermentation and

nitrogen metabolism in continuous culture.
Vallimont JE(1), Bargo F, Cassidy TW, Luchini ND, Broderick GA, Varga GA.
Author information:
(1)Department of Dairy and Animal Science, The Pennsylvania State University,
University Park, 16802, USA.
A dual-effluent continuous-culture system was used to evaluate the effects of

partially replacing cornstarch with sucrose in a total mixed ration on ruminal
fermentation and N metabolism. The 4 treatments were 0 (control), 2.5, 5.0, and
7.5% sucrose and, respectively, 7.5 (control), 5.0, 2.5, and 0% cornstarch in a
total mixed ration containing 20% corn silage and 40% alfalfa silage. Fermenters
were fed 4 times a day during four 9-d periods with sampling beginning on d 6.
Replacing cornstarch with sucrose did not alter ruminal pH (5.97), total volatile
fatty acids (VFA) (104.4 mmol/L), or the acetate to propionate ratio (2.16);
however, branched-chain volatile fatty acids were higher for the control
treatment compared with the 7.5% sucrose treatment. Five hours postfeeding,
sucrose treatments significantly altered molar proportions of all volatile fatty
acids, and acetate-to-propionate and glucogenic-to-lipogenic ratios.
Digestibility of dry matter and N were not affected by treatment, but
digestibility of total non-structural carbohydrates was increased with sucrose
treatments. A quadratic effect was noted for neutral detergent fiber (NDF)
digestibility as sucrose replaced starch. A higher NDF digestibility (66.1 vs.
59.9%) was observed for the 7.5% sucrose treatment compared with the other 2
sucrose treatments. Levels of ammonia N were within an acceptable range to
support microbial protein synthesis and did not differ among treatments
(mean=9.23 mg/dL). Sucrose inclusion in the total mixed ration did not affect
bacterial N synthesis. Results indicate that (at the levels tested in this study)
inclusion of sucrose in the diet when rumen-degradable protein is adequate does
not affect ruminal fermentation.
DOI: 10.3168/jds.S0022-0302(04)73567-5
2146. J Dairy Sci. 2004 Dec;87(12):4178-81.
Feed efficiency of mid-lactation dairy cows fed yeast culture during summer.
Schingoethe DJ(1), Linke KN, Kalscheur KF, Hippen AR, Rennich DR, Yoon I.
Author information:
USA. david.schingoethe@sdstate.edu
Thirty-eight Holstein cows (26 multiparous and 12 primiparous), that averaged 105
d postpartum at the start of the experiment, were used to evaluate the feeding of
yeast culture (60 g/cow daily of Diamond V XP) on production efficiency during
hot summer weather. From early June until early September and after a 2-wk
covariate period, cows were fed a control diet without or with 60 g of yeast
culture/cow daily for 12 wk. Weekly daytime high temperatures in the free-stall
barn during the 12-wk period averaged 33 degrees C (28 to 39 degrees C). Total
mixed diets on a dry matter (DM) basis consisted of corn silage (28%), alfalfa
hay (21%), and a concentrate mix (51%) without or with the yeast culture added to
the total mixed ration at the time of feeding. Milk production (34.9 and 35.4
kg/d, for control and yeast culture treatment, respectively), 4% fat-corrected
milk (31.2 and 32.0 kg/d), energy-corrected milk (ECM; 33.4 and 34.2 kg/d), and
DM intake (23.1 and 22.1 kg/d) were similar for cows fed control and yeast
culture diets. Percentages of milk fat (3.34 and 3.41) and true protein (2.85 and
2.87) were similar for both diets. Feed efficiency defined as kilogram of
ECM/kilogram of DM intake was improved by 7% for cows fed the yeast culture. Body
weights and body condition scores were similar for both groups. The results
suggest that the yeast culture can improve feed efficiency of heat stressed dairy
cows in midlactation.
DOI: 10.3168/jds.S0022-0302(04)73561-4
2147. J Chem Ecol. 2004 Aug;30(8):1547-64.
Behavioral response of Lygus hesperus to conspecifics and headspace volatiles of

alfalfa in a Y-tube olfactometer.
Blackmer JL(1), Rodriguez-Saona C, Byers JA, Shope KL, Smith JP.
Author information:
(1)Western Cotton Research Laboratory, USDA-ARS, 4135 E. Broadway Rd., Phoenix,
Arizona 85040, USA. jblackmer@wcrl.ars.usda.gov
The western tarnished plant bug, Lygus hesperus Knight, feeds and develops on a
variety of weeds in the spring, with later generations moving to alfalfa and
cotton where severe damage to reproductive structures can occur. A synthetic
attractant for monitoring or mass-trapping L. hesperus, or the identification of
potential attractants for natural enemies, would be useful tools for integrated
pest management programs. Studies investigated the response of naive and
experienced fifth-instar and adult L. hesperus to odors associated with
conspecifics and alfalfa, Medicago sativa L. Fifth-instar L. hesperus responded
to all plant/insect combinations, whereas female L. hesperus only responded
preferentially to vegetative and flowering alfalfa where conspecifics had fed for
24-72 hr, and to vegetative alfalfa where conspecifics were added approximately
30 min before the test began. Males were not attracted to headspace volatiles
from any of the alfalfa treatments. Analysis of headspace volatiles showed that
(E)-2-hexanal, (Z)-3-hexen-l-ol, alpha-pinene, (Z)-3-hexenyl acetate,
(E)-2-hexenyl acetate, limonene, (Z)-ocimene, (E)-beta-ocimene, linalool,
(3E)-4,8-dimethyl-1,3,7-nonatriene, and (E, E)-alpha-farnesene are emitted from
both vegetative and flowering alfalfa. Indole and (3E,
7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene were only detected in flowering
alfalfa. Damage to alfalfa by L. hesperus increased emissions of (Z)-ocimene,
(E)-beta-ocimene, (E)-beta-caryophyllene, and (E, E)-alpha-farnesene, while
beta-pinene, myrcene, methyl salicylate, and (3E,
7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene were only detected from damaged
plants. Thus, individual or mixtures of these alfalfa volatiles may be useful as
attractants for capturing nymphs and adult females of L. hesperus in the field.
2148. Environ Pollut. 2005 Feb;133(3):455-65.
The effects of perennial ryegrass and alfalfa on microbial abundance and

diversity in petroleum contaminated soil.
Kirk JL(1), Klironomos JN, Lee H, Trevors JT.
Author information:
(1)Department of Environmental Biology, University of Guelph, Guelph, Ontario N1G
2W1, Canada.
Enhanced rhizosphere degradation uses plants to stimulate the rhizosphere

microbial community to degrade organic contaminants. We measured changes in
microbial communities caused by the addition of two species of plants in a soil
contaminated with 31,000 ppm of total petroleum hydrocarbons. Perennial ryegrass
and/or alfalfa increased the number of rhizosphere bacteria in the
hydrocarbon-contaminated soil. These plants also increased the number of bacteria
capable of petroleum degradation as estimated by the most probable number (MPN)
method. Eco-Biolog plates did not detect changes in metabolic diversity between
bulk and rhizosphere samples but denaturing gradient gel electrophoresis (DGGE)
analysis of PCR-amplified partial 16S rDNA sequences indicated a shift in the
bacterial community in the rhizosphere samples. Dice coefficient matrices derived
from DGGE profiles showed similarities between the rhizospheres of alfalfa and
perennial ryegrass/alfalfa mixture in the contaminated soil at week seven.
Perennial ryegrass and perennial ryegrass/alfalfa mixture caused the greatest
change in the rhizosphere bacterial community as determined by DGGE analysis. We
concluded that plants altered the microbial population; these changes were
plant-specific and could contribute to degradation of petroleum hydrocarbons in
contaminated soil.
DOI: 10.1016/j.envpol.2004.06.002
2149. Transgenic Res. 2004 Aug;13(4):349-61.
A comparison of constitutive promoters for expression of transgenes in alfalfa

(Medicago sativa).
Samac DA(1), Tesfaye M, Dornbusch M, Saruul P, Temple SJ.
Author information:
(1)USDA-ARS-Plant Science Research Unit, University of Minnesota, St. Paul, MN
55108, USA. dasamac@tc.umn.edu
The activity of constitutive promoters was compared in transgenic alfalfa plants

using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic
virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane
bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase
(gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV
promoter and all alfalfa cells assayed by in situ staining had high levels of
enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at
moderate levels, but the promoter was not active in stem pith cells, root
cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was
active primarily in vascular tissues throughout the plant. In leaves, GUS
activity driven by the CsVMV promoter was approximately 24-fold greater than the
activity from the 35S promoter and 38-fold greater than the activity from the
ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus
(FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco
(RbcS) promoter were used to control expression of a cDNA from Trichoderma
atroviride encoding an endochitinase (ech42). Highest chitinase activity in
leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42
transgene. Plants expressing the endochitinase were challenged with Phoma
medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot
of alfalfa. Although endochitinase activity in leaves of transgenic plants was
50- to 2650-fold greater than activity in control plants, none of the transgenic
plants showed a consistent increase in disease resistance compared to controls.
The high constitutive levels of both GUS and endochitinase activity obtained
demonstrate that the CsVMV promoter is useful for high-level transgene expression
in alfalfa.
2150. Bioresour Technol. 2005 Mar;96(5):565-70.
Alfalfa as a single dietary source for molt induction in laying hens.
Landers KL(1), Woodward CL, Li X, Kubena LF, Nisbet DJ, Ricke SC.
Author information:
77843, USA.
Molting is a process by which a hen's reproductive tract is rejuvenated prior to

the beginning of a laying cycle. This process is often artificially induced in
commercial settings in order to extend the productive life of a flock of hens.
The most common method for the induction of molt is feed withdrawal for a period
of several days. It has been noted that feed withdrawal, while effective in
inducing molt and allowing an adequate reproductive rest period for the hen, may
cause deleterious effects on the animal. This has prompted the investigation of
alternatives to feed deprivation for the induction of molt in commercial laying
hens. This study involved feeding alfalfa to hens to assess its ability to induce
molt. Results show that alfalfa meal and alfalfa pelleted diets were equally
effective as feed withdrawal in causing ovary weight regression in birds. Molted
hens induced by alfalfa diets exhibited postmolt levels of egg production over a
twelve week period that were similar to that of hens molted by feed withdrawal.
The postmolt eggs laid by hens molted by alfalfa were of comparable quality to
eggs from feed deprived hens. Alfalfa, a fibrous feed with low metabolizable
energy, may be provided to hens on an ad libitum basis for an effective molt
induction that retains comparable egg quality and production.
DOI: 10.1016/j.biortech.2004.06.013
2151. Environ Geochem Health. 2004 Jun-Sep;26(2-3):285-93.
Degradation of benzo[a]pyrene in soil with arbuscular mycorrhizal alfalfa.
Liu SL(1), Luo YM, Cao ZH, Wu LH, Ding KQ, Christie P.
Author information:
(1)Soil and Environment Bioremediation Research Centre, Institute of Soil
Science, Chinese Academy of Sciences, Nanjing 210008, China.
Mycorrhizal and non-mycorrhizal alfalfa (Medicago sativa) was grown in pots

containing soil artificially contaminated with various levels of benzo[a]pyrene
(B[a]P) (0, 1, 10 and 100 mg kg(-1)). Soil and plants were sampled after 30, 40,
50, 60 and 90 days and compared with unlanted pots. The percentage of mycorrhizal
root length colonized by Glomus caledoniun was not significantly affected by the
addition of B[a]P up to 10 mg kg(-1) but was significantly lower at 100 mg
kg(-1)B[a]P compared with low concentrations (p < 0.05). There was no difference
in soil polyphenol oxidase and dehydrogenase activity among the controls and
applications of 1 and 10 mg kg(-1) of B[a]P. However, enzyme activities were
significantly higher at 100 mg kg(-1) B[a]P compared with the other three
treatments, and there was no mycorrhizal effect. Over a period of 90 days the
concentration of B[a]P in soil in which alfalfa was grown was significantly lower
than in unplanted soil (p < 0.05). Degradation rates of B[a]P added at 1, 10 and
100 mg kg(-1) without G. caledonium were 76, 78 and 53%, and with mycorrhizal
inoculation were 86, 87 and 57%. The degradation rate in unplanted soil was
significantly lower than in planted soil, and was significantly higher in medium-
and low-B[a]P treatments than in the high B[a]P concentration tested. There is a
possibility of enhancement phytoremediation of PAHs in rhizosphere soil with
arbuscular mycorrhizal fungi.

2152. Genome. 2004 Oct;47(5):868-76.
Mining EST databases to resolve evolutionary events in major crop species.
Schlueter JA(1), Dixon P, Granger C, Grant D, Clark L, Doyle JJ, Shoemaker RC.
Author information:
(1)Interdepartmental Genetics, Department of Genetics Developmental and Cellular
Biology, Iowa State University, Ames, IA 50011, USA.
Using plant EST collections, we obtained 1392 potential gene duplicates across 8
plant species: Zea mays, Oryza sativa, Sorghum bicolor, Hordeum vulgare, Solanum
tuberosum, Lycopersicon esculentum, Medicago truncatula, and Glycine max. We
estimated the synonymous and nonsynonymous distances between each gene pair and
identified two to three mixtures of normal distributions corresponding to one to
three rounds of genome duplication in each species. Within the Poaceae, we found
a conserved duplication event among all four species that occurred approximately
50-60 million years ago (Mya); an event that probably occurred before the major
radiation of the grasses. In the Solanaceae, we found evidence for a conserved
duplication event approximately 50-52 Mya. A duplication in soybean occurred
approximately 44 Mya and a duplication in Medicago about 58 Mya. Comparing
synonymous and nonsynonymous distances allowed us to determine that most
duplicate gene pairs are under purifying, negative selection. We calculated
Pearson's correlation coefficients to provide us with a measure of how gene
expression patterns have changed between duplicate pairs, and compared this
across evolutionary distances. This analysis showed that some duplicates seemed
to retain expression patterns between pairs, whereas others showed uncorrelated
expression.
DOI: 10.1139/g04-047
2153. J Dairy Sci. 2004 Nov;87(11):3912-24.
Effects of alfalfa particle size and specific gravity on chewing activity,

digestibility, and performance of Holstein dairy cows.
Teimouri Yansari A(1), Valizadeh R, Naserian A, Christensen DA, Yu P, Eftekhari

Shahroodi F.
Author information:
(1)Department of Animal Science, Agricultural Faculty, Ferdowsi University of
Mashhad, Iran. astymori@yahoo.com <astymori@yahoo.com>
Two experiments were carried out to test the effects of alfalfa particle size and
functional specific gravity (FSG) on chewing activity, digestibility, rumen
kinetics, and production of lactating dairy cows fed corn silage based rations.
In experiment 1, water-holding capacity (WHC), insoluble dry matter, hydration
rate, and FSG changes were determined in alfalfa hay (varying in particle size)
and corn silage. Reduction of particle size increased bulk density, FSG, and the
rate of hydration, and decreased WHC of alfalfa. In experiment 2, 9 midlactation
Holstein dairy cows fed total mixed rations containing 3 sizes of alfalfa hay
(with geometric mean 7.83, 4.04, and 1.14 mm) were used in a replicated 3 x 3
Latin square design. The diets contained 20, 20, 35, 7, 7.5, 10, 0.3, 0.1, and
0.1% of DM alfalfa, corn silage, barley, soybean meal, beet pulp, wheat bran,
dicalcium phosphate, vitamin premix, and salt, respectively. The geometric means
(GM) of rations were 3.34, 2.47, and 1.66 mm in long, medium, and fine alfalfa
treatments, respectively. Reduction of particle size increased daily NDF intake
(kg), but decreased the proportion of physically effective factor (pef) and
physically effective NDF (peNDF) in the ingested rations. Reduction of particle
size increased the FSG of rations and intake of DM but reduced digestibility of
NDF and ash. Reduction of particle size decreased ruminal mean retention time
(RMRT), but increased the ruminal particulate passage rate. Milk and FCM yield
were not affected by treatments. The rumen pH, total chewing activity,
rumination, eating time, and milk fat were reduced as particle size decreased,
but milk protein increased. This study showed that reduction of forage particle
size increased bulk density, FSG, and hydration rate of alfalfa and was the most
influential factor affecting DMI, milk composition, and chewing behavior.
Reduction of forage particle size had minimal impact on digestibility and milk
production.
DOI: 10.3168/jds.S0022-0302(04)73530-4
Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in

Sinorhizobium meliloti increases its ability to nodulate alfalfa.
Ma W(1), Charles TC, Glick BR.
Author information:
(1)Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1,
Canada.
1-Aminocyclopropane-1-carboxylate (ACC) deaminase has been found in various plant

growth-promoting rhizobacteria, including rhizobia. This enzyme degrades ACC, the
immediate precursor of ethylene, and thus decreases the biosynthesis of ethylene
in higher plants. The ACC deaminase of Rhizobium leguminosarum bv. viciae 128C53K
was previously reported to be able to enhance nodulation of peas. The ACC
deaminase structural gene (acdS) and its upstream regulatory gene, a
leucine-responsive regulatory protein (LRP)-like gene (lrpL), from R.
leguminosarum bv. viciae 128C53K were introduced into Sinorhizobium meliloti,
which does not produce this enzyme, in two different ways: through a plasmid
vector and by in situ transposon replacement. The resulting ACC
deaminase-producing S. meliloti strains showed 35 to 40% greater efficiency in
nodulating Medicago sativa (alfalfa), likely by reducing ethylene production in
the host plants. Furthermore, the ACC deaminase-producing S. meliloti strain was
more competitive in nodulation than the wild-type strain. We postulate that the
increased competitiveness might be related to utilization of ACC as a nutrient
within the infection threads.
DOI: 10.1128/AEM.70.10.5891-5897.2004
PMCID: PMC522075
2155. Plant Physiol. 2004 Oct;136(2):3234-44. Epub 2004 Oct 1.
Cloning and characterization of red clover polyphenol oxidase cDNAs and

expression of active protein in Escherichia coli and transgenic alfalfa.
Sullivan ML(1), Hatfield RD, Thoma SL, Samac DA.
Author information:
(1)United States Dairy Forage Research Center, Agricultural Research Service,
United States Department of Agriculture, Madison, Wisconsin 53706, USA.
mlsulliv@wisc.edu
Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase
(PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and
ensiling results in browning of leaf tissues from activity of PPO on the
o-diphenols. In association with browning, leaf proteins remain undegraded during
ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases.
We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA
library. Sequence comparisons among the three red clover PPO clones indicated
they are 87% to 90% identical at the nucleotide level (80%-83% amino acid
identity). All three encode proteins predicted to localize to the chloroplast
thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is
expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in
leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A
portion of the expressed protein was soluble and functional in an assay for PPO
activity. We also expressed the red clover PPO cDNAs under the control of a
constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO
proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant
extract browning and quantitative assays of PPO activity. Proteolysis in leaf
extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the
presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red
clover PPO should prove an excellent model system to further characterize the red
clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in
forage plants.
DOI: 10.1104/pp.104.047449
PMCID: PMC523382
2156. J Dairy Sci. 2004 Jun;87(6):1840-7.
Effects of monensin on ruminal forage degradability and total tract diet

digestibility in lactating dairy cows during grain-induced subacute ruminal
acidosis.
Osborne JK(1), Mutsvangwa T, Alzahal O, Duffield TF, Bagg R, Dick P, Vessie G,

McBride BW.
Author information:
Canada N1G 2W1.
The effects of monensin premix supplementation on ruminal pH characteristics and

forage degradability, and total tract diet digestibility during grain-induced
subacute ruminal acidosis (SARA) in lactating dairy cows receiving a total mixed
ration were investigated. Six multiparous, rumen-fistulated Holstein cows were
used in a 2-treatment, 2-period (5 wk per period) crossover design. During wk 5
(d 29 to 35) of each period, SARA was induced using a grain challenge model, and
ruminal pH was measured continuously using indwelling pH probes. Ruminal
degradation of corn silage and alfalfa haylage was determined using the in situ
(nylon bag) technique, and total tract diet digestibility was determined by total
fecal collection during wk 5. Monensin supplementation did not affect dry matter
intake, milk yield, and composition, and ruminal pH characteristics under these
experimentally induced SARA conditions. Rates of ruminal forage fiber
degradability were similar between control and monensin-treated cows; however,
monensin supplementation increased total tract fiber digestion. This study
indicates that monensin altered total tract nutrient digestion by increasing
fiber digestion at postruminal sites.
DOI: 10.3168/jds.S0022-0302(04)73341-X
2157. J Dairy Sci. 2004 Jun;87(6):1815-9.
The energy content of wet corn distillers grains for lactating dairy cows.
Birkelo CP(1), Brouk MJ, Schingoethe DJ.
Author information:
(1)Department of Animal and Range Sciences, South Dakota State University,
Brookings 57007-0647, USA.
Forty-five energy balances were completed with 12 multiparous, lactating Holstein

cows in a study designed to determine the energy content of wet corn distillers
grains. Treatments were applied in a repeated switchback design and consisted of
total mixed diets containing 31.4% corn silage, 18.4% alfalfa hay, and either
30.7% rolled corn and 16.7% soybean meal or 17.0% rolled corn and 31.2% wet corn
distillers grains (dry matter basis). Replacement of corn and soybean meal with
wet corn distillers grains reduced dry matter intake 10.9% but did not affect
milk production. Neither digestible nor metabolizable energy were affected by
diet composition. Heat and milk energy output did not differ by diet, but body
energy retained was 2.8 Mcal/d less in cows fed the wet corn distillers grains
diet. Multiple regression estimates of maintenance metabolizable energy
requirement and partial efficiencies of metabolizable energy used for lactation
and body energy deposition did not differ by diet. Pooled estimates were 136.2,
0.66, and 0.85, kcal of metabolizable energy/ body weight0.75 per day,
respectively. Calculated by difference, wet corn distillers grains was estimated
to contain 4.09, 3.36, and 2.27 Mcal/kg of dry matter as digestible,
metabolizable, and lactational net energy, respectively. These energy estimates
were 7 to 11% and 10 to 15%, respectively, greater than those reported for dried
corn distillers grains by the 1989 and 2001 dairy NRC publications.
DOI: 10.3168/jds.S0022-0302(04)73338-X
2158. Plant Physiol. 2004 Oct;136(2):3276-83. Epub 2004 Sep 24.
Heavy metal stress. Activation of distinct mitogen-activated protein kinase

pathways by copper and cadmium.
Jonak C(1), Nakagami H, Hirt H.
Author information:
Sciences and Institute of Microbiology and Genetics, University of Vienna, Vienna
Biocenter, A-1030 Vienna, Austria. claudia.jonak@univie.ac.at
Excessive amounts of heavy metals adversely affect plant growth and development.
Whereas some regions naturally contain high levels of heavy metals, anthropogenic
release of heavy metals into the environment continuously increases soil
contamination. The presence of elevated levels of heavy metal ions triggers a
wide range of cellular responses including changes in gene expression and
synthesis of metal-detoxifying peptides. To elucidate signal transduction events
leading to the cellular response to heavy metal stress we analyzed protein
phosphorylation induced by elevated levels of copper and cadmium ions as examples
for heavy metals with different physiochemical properties and functions. Exposure
of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated
four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and
SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2,
MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions
induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK
specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3.
Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2).
These results suggest that plants respond to heavy metal stress by induction of
several distinct MAPK pathways and that excess amounts of copper and cadmium ions
induce different cellular signaling mechanisms in roots.
DOI: 10.1104/pp.104.045724
PMCID: PMC523386
2159. Curr Microbiol. 2004 Oct;49(4):295-9.
Nutritional requirements of Allisonella histaminiformans, a ruminal bacterium

that decarboxylates histidine and produces histamine.
Garner MR(1), Gronquist MR, Russell JB.
Author information:
(1)Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Histamine is an inflammatory agent that contributes to bovine laminitis. Cattle

fed silage-containing rations often have large populations of Allisonella
histaminiformans, but this obligate histidine-decarboxylating bacterium could not
be isolated from cattle fed timothy hay. The growth of A. histaminiformans was
stimulated by yeast extract, protein hydrolysates, and water-soluble extracts of
alfalfa or corn silage. Extracts of alfalfa were more potent than corn silage.
Because growth and histamine production were not stimulated by Casamino Acids or
a mixture of purified amino acids, it appeared that A. histaminiformans requires
peptides. The idea that A. histaminiformans requires peptides is consistent with
the observation that alfalfa silages often have a large amount of peptide
nitrogen.
DOI: 10.1007/s00284-004-4336-1
Distribution of alfalfa weevil (Coleoptera: Curculionidae) larvae among

postcutting locations.
Blodgett SL(1), Lenssen AW.
Author information:
(1)Department of Entomology, Montana State University, Bozeman, MT 59717, USA.
blodgett@montana.edu
A field study was conducted in 1996 and 1997 to determine survival and spatial
patterns of alfalfa weevil, Hypera postica (Gyllenhal), larvae within and between
windrows of swathed alfalfa, Medicago sativa L., forage as dry matter
concentration increased. After harvest, the percentage of live weevil larvae
residing in swathed forage decreased as swath dry matter percentage increased.
Conversely, larval populations in stubble between windrows increased with
increasing swath dry matter. Larval populations in stubble under windrowed forage
increased slightly as windrowed forage dry matter increased. Applications of
these results for the development of alfalfa harvest systems for improved
cultural control of alfalfa weevil are discussed.
2161. J Dairy Sci. 2004 Oct;87(10):3492-502.
Protein level for alfalfa and corn silage-based diets: II. Nitrogen balance and
manure characteristics.
Wattiaux MA(1), Karg KL.
Author information:
wattiaux@wisc.edu
This N balance study was completed with 48 multiparous Holstein cows (body weight
[BW] = 653 kg; days in milk = 89) blocked by calving date and assigned to a 2 x 2
factorial arrangement of dietary treatments. The total mixed ration included
alfalfa silage (AS) or corn silage (CS) as the primary forage source (41 and 14%
vs. 14 and 41% of diet dry matter (DM), respectively) and were formulated for
recommended (RP) or excessive (HP) amounts of rumen degradable protein (RDP) and
rumen undegradable protein (RUP) according to the guidelines of the National
Research Council (NRC). Crude protein (CP) averaged 16.5, 18.0, 16.4, and 17.3%
for the AS-RP; AS-HP; CS-RP; and CS-HP diet, respectively (DM basis). Regardless
of primary forage source, the reduction in dietary CP to the NRC guidelines
tended to improve milk yield (43.4 vs. 41.0 kg/d) but did not alter 3.5%
fat-corrected milk (37.0 kg/d) or milk true protein yield (1167 g/d). In this
trial, cows fed the CS-based diets consumed less DM than those fed the AS-based
diets in part because of rumen acidosis. The adverse effect of low rumen pH was
accompanied by an increase in urinary N (UN) as a percentage of N intake, but did
not alter milk yield. Notwithstanding partial confounding, fecal N (FN) was 49
g/d lower (213 vs. 164 g/d), UN was unchanged (229 g/d), but milk N tended to be
higher (194 vs. 206 g/d) when cows were fed the CS-based diets compared with
AS-based diets. Compared with the HP diets, cows fed the RP diets had similar FN
(189 g/d) and milk N (200 g/d), but UN and urine urea N were reduced by 41 g/d
(249 vs. 208 g/d) and 40 g/d (210 vs. 171 g/d), respectively. Fecal N
concentration was higher for CS-based diets, but urinary N concentration was
higher for AS-based diets. The reduction in dietary CP did not influence these
concentrations but lowered urine volume. The metabolic relationships between
energy and protein in determining the fate of excess dietary N (primarily in the
form of excess RUP in this trial) was illustrated by a 17% increase in the UN to
FN ratio for cows fed AS-HP compared with the AS-RP diet and a 42% increase in
the UN to FN ratio for CS-HP compared with CS-RP diet, when cows' energy status
was compromised because of rumen acidosis. In this trial, UN ranged from 150 to
320 g/d, and was best predicted as UN (g/d) = 0.0283 x BW (kg) x milk urea N
(mg/dL). The NRC protein guidelines should not be exceeded to avoid unnecessary
losses of manure N and, in particular, urine urea N.
DOI: 10.3168/jds.S0022-0302(04)73484-0
2162. J Dairy Sci. 2004 Oct;87(10):3480-91.
Protein level for alfalfa and corn silage-based diets: I. Lactational response
and milk urea nitrogen.
Wattiaux MA(1), Karg KL.
Author information:
wattiaux@wisc.edu
This study was designed to evaluate lactational responses of cows fed corn silage
(CS) or alfalfa silage (AS) as primary forage source when the diet was balanced
for recommended (RP) or excessive (HP) amounts of rumen degradable protein (RDP)
and undegradable protein (RUP) according to the recommendations of the National
Research Council (NRC). A second objective was to evaluate different sources of
variations in milk urea N (MUN). The total mixed rations included 55% forage on a
dry matter (DM) basis as either 14% CS and 41% AS or 14% AS and 41% CS. Diets
were offered to 48 multiparous Holstein cows (body weight = 652 kg) that were
assigned randomly to treatments arranged as a 2 x 2 factorial in 12 complete
blocks based on calving date. Data collected during wk 4 to 12 of lactation were
adjusted to those obtained from a pretreatment diet fed during wk 1 to 3. Crude
protein (CP) averaged 16.5, 18.0, 16.2, and 17.1% of DM in the AS-RP; AS-HP;
CS-RP; and CS-HP diets, respectively. Overall DM intake (DMI) was 1.5 kg/d lower
than predicted by NRC (24.6 vs. 26.1 kg/d), but 3.5% fat-corrected milk (FCM) was
higher than expected (46.1 vs. 45.0 kg/d). The responses to a reduction in
dietary protein were independent of primary forage source, except for milk true
protein (TP) percentage. Primary forage source did not influence DMI, 3.5% FCM,
TP yield, or MUN. However, compared with the AS-based diets, cows fed CS-based
diets produced more milk (49.0 vs. 46.4 kg/d), less fat (3.07% vs. 3.54% and 1500
vs. 1651 g/d), and tended to gain more body weight. There were no benefits to
feeding diets above NRC protein recommendations, regardless of forage source.
Reducing CP from 17.5 to 16.4% of diet DM did not alter milk yield (47.7 kg/d) or
milk TP yield (1293 g/d), but lowered N intake by 65 g/d (700 vs. 635 g/d) and
lowered MUN by 1 unit (12.7 vs. 11.7 mg/dL). A positive correlation between MUN
and production efficiency (3.5% FCM/DMI) on wk 3 of lactation suggested that body
protein mobilization might impact MUN in early lactation. The correlation between
MUN and DMI tended to be negative in wk 3, but was positive in wk 6 to 12 of
lactation. The same was true for the correlation between MUN and somatic cell
score. Regression analysis of the postpeak lactation data of this study indicated
that the expected MUN was essentially 12 mg/dL when NRC-predicted RDP and RUP
balances were 0 g/d, with a linear deviation of 0.1 and 0.03 mg/dL per 10 g of
change in RDP and RUP balance, respectively.
DOI: 10.3168/jds.S0022-0302(04)73483-9
2163. J Dairy Sci. 2004 Sep;87(9):2997-3009.
Effect of molasses supplementation on the production of lactating dairy cows fed

diets based on alfalfa and corn silage.
Broderick GA(1), Radloff WJ.
Author information:
(1)Agricultural Research Service, USDA US Dairy Forage Research Center, 1925
Linden Drive West, Madison, WI 53706, USA. gbroderi@wisc.edu
Adding sugar to the diet has been reported to improve production in dairy cows.
In each of 2 trials, 48 lactating Holsteins (8 with ruminal cannulas) were fed
covariate diets for 2 wk, blocked by days in milk into 12 groups of 4, and then
randomly assigned to diets based on alfalfa silage containing 4 levels of dried
molasses (trial 1) or liquid molasses (trial 2). In both studies, production data
were collected for 8 wk, ruminal samples were taken in wk 4 and 8, and
statistical models were used that included covariate means and block. In trial 1,
experimental diets contained 18% CP and 0, 4, 8, or 12% dried molasses with 2.6,
4.2, 5.6, or 7.2% total sugar. With increasing sugar, there was a linear increase
in dry matter intake (DMI), and digestibility of dry matter (DM) and organic
matter (OM), but no effect on yield of milk or protein. This resulted in linear
decreases in fat-corrected milk (FCM)/DMI and milk N/N-intake. There was a linear
decrease in urinary N with increasing sugar, and quadratic effects on milk fat
content, yield of fat and FCM, and ruminal ammonia. Mean optimum from these
quadratic responses was 4.8% total sugar in these diets. In trial 2, experimental
diets contained 15.6% crude protein (CP) and 0, 3, 6, or 9% liquid molasses with
2.6, 4.9, 7.4, or 10.0% total sugar, respectively. Again, there were linear
declines in FCM/DMI and milk N/N-intake with increasing sugar, but quadratic
responses for DMI, yield of milk, protein, and SNF, digestibility of neutral
detergent fiber and acid detergent fiber, milk urea, urinary excretion of purine
derivatives, and ruminal ammonia. Mean optimum from all quadratic responses in
this trial was 6.3% total sugar. An estimate of an overall optimum, based on
yield of fat and FCM (trial 1) and yield of milk, protein, and SNF (trial 2), was
5.0% total sugar, equivalent to adding 2.4% sugar to the basal diets. Feeding
more than 6% total sugar appeared to depress production.
DOI: 10.3168/jds.S0022-0302(04)73431-1
2164. Theor Appl Genet. 2004 Nov;109(8):1568-75. Epub 2004 Sep 15.
Population-based diallel analyses among nine historically recognized alfalfa

germplasms.
Segovia-Lerma A(1), Murray LW, Townsend MS, Ray IM.
Author information:
Identification of heterotic groups and patterns among breeding populations

provides fundamental information to help plant breeders more knowledgeably
manipulate heterosis. A diallel analysis was conducted among nine alfalfa (
Medicago sativa L.) germplasms, commonly referred to as African, Chilean,
Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian, and Turkistan, which
represent a significant proportion of the genetic diversity present in US
cultivars. Heterotic responses were determined by evaluating forage yield of the
germplasms and their 36 half-diallel hybrids in seeded plots that were harvested
five times in each of 2 years. Commercially acceptable yields were obtained from
some hybrids of unimproved parents, where at least one parent was adapted to the
study environment. Variation among crosses was attributed primarily to general
combining ability (GCA) effects; however, specific combining ability effects were
also significant. GCA estimates for African, Chilean and Peruvian were positive,
while those for Ladak, M. falcata, and M. varia were negative. Estimates for
variety heterosis effects were positive for Peruvian and M. falcata and negative
for Indian and M. varia. Significant mid-parent heterosis [(MPH) range of -21% to
55%] and high-parent heterosis [(HPH) range of -33% to 23%] was detected. M.
falcata hybrids exhibited the highest MPH values. However, this likely reflects
the poor yield of M. falcata per se in the study environment and consequently,
low MPH values. Peruvian hybrids demonstrated the highest cross mean performance,
significant positive MPH in all crosses, and positive HPH in five out of eight
crosses. The results indicate that Peruvian should be recognized as a heterotic
group. Alfalfa breeders may wish to explore opportunities for heterotic yield
gains that are likely to exist in hybrids between the Peruvian germplasm and
elite breeding populations, in particular, those adapted to the southwestern
United States. MPH results suggest that alfalfa breeders may have capitalized on
the heterotic response between Flemish and M. varia during past development of
alfalfa synthetics adapted to the central and northern latitudes of the United
States.
DOI: 10.1007/s00122-004-1784-8
2165. Arch Insect Biochem Physiol. 2004 Sep;57(1):1-14.
Characterization of triacylglycerols from overwintering prepupae of the alfalfa

pollinator Megachile rotundata (Hymenoptera: Megachilidae).
Buckner JS(1), Kemp WP, Bosch J.
Author information:
(1)Biosciences Research Laboratory, USDA-ARS, Fargo, North Dakota, USA.
bucknerj@fargo.ars.usda.gov
Alfalfa leafcutting bees, Megachile rotundata (F.), overwinter as prepupae. The

internal lipids were extracted from prepupae that had been wintered at 4 degrees
C for 7 months. Megachile rotundata prepupae possessed copious quantities of
internal lipids (20% of the fresh weight) that were extracted with CHCl3/methanol
(2:1). Transmission electron microscopy revealed that lipids were stored within
very large intracellular vacuoles. Separation by silica chromatography revealed
that 88% of the internal lipids were triacylglycerols. Ester derivatives of fatty
acids from triacylglycerol components were analyzed by gas chromatography-mass
spectrometry and 15 fatty acid constituents were identified. The majority (76%)
of the triacylglycerol fatty acids were unsaturated fatty acids. The major
triacylglycerol fatty acid constituent (30%) was the C16 monounsaturated fatty
acid, palmitoleic acid (16:1, hexadec-9-enoic acid), with substantial amounts of
linolenic acid (18:3, octadec-9,12,15-trienoic acid, 15%), palmitic acid (16:0,
hexadecanoic acid, 14%) and oleic acid (18:1, octadec-9-enoic acid, 13%).
Palmitoleic acid as the major fatty acid of an insect is an unusual occurrence as
well as the presence of the 16-carbon polyunsaturated fatty acids, 16:2 and 16:3.
The major intact triacylglycerol components were separated and identified by high
performance liquid chromatography-mass spectrometry. A complex mixture of
approximately 40 triacylglycerol components were identified and major components
included palmitoyl palmitoleoyl oleoyl glycerol, palmitoyl palmitoleoyl
palmitoleoyl glycerol, myristoyl palmitoleoyl palmitoleoyl glycerol, myristoleoyl
palmitoyl palmitoleoyl glycerol, and palmitoyl palmitoleoyl linolenoyl glycerol.
The function of these internal lipids and their relevance to winter survival and
post-wintering development of M. rotundata is discussed.
DOI: 10.1002/arch.20008
2166. Plant Physiol. 2004 Sep;136(1):2887-94. Epub 2004 Sep 3.

Microbial products trigger amino acid exudation from plant roots.
Phillips DA(1), Fox TC, King MD, Bhuvaneswari TV, Teuber LR.
Author information:
(1)Department of Agronomy and Range Science, University of California, Davis, CA
95616, USA. daphillips@ucdavis.edu
Plants naturally cycle amino acids across root cell plasma membranes, and any net
efflux is termed exudation. The dominant ecological view is that microorganisms
and roots passively compete for amino acids in the soil solution, yet the innate
capacity of roots to recover amino acids present in ecologically relevant
concentrations is unknown. We find that, in the absence of culturable
microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 microm)
exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago
truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial
products, which are produced by common soil microorganisms such as Pseudomonas
bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e.
exudation) of amino acids from roots of these four plant species. In alfalfa,
treating roots with 200 microm phenazine, 2,4-diacetylphloroglucinol, or
zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h.
Data from (15)N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid
uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under
normal conditions is a phenomenon that probably reflects both active manipulation
and passive uptake by microorganisms, as well as diffusion and adsorption to
soil, all of which help overcome the innate capacity of plant roots to reabsorb
amino acids. The importance of identifying potential enhancers of root exudation
lies in understanding that such compounds may represent regulatory linkages
between the larger soil food web and the internal carbon metabolism of the plant.
DOI: 10.1104/pp.104.044222
PMCID: PMC523350
2167. J Bacteriol. 2004 Sep;186(18):6042-9.
Sinorhizobium meliloti ExoR and ExoS proteins regulate both succinoglycan and
flagellum production.
Yao SY(1), Luo L, Har KJ, Becker A, Rüberg S, Yu GQ, Zhu JB, Cheng HP.
Author information:
York, 250 Bedford Park Blvd., West, Bronx, NY 10468, USA.
The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is

required for the formation of infection threads inside root hairs, a critical
step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two
bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction
of succinoglycan and a reduction in symbiosis. Systematic analyses of the
symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of
root hair colonization. In addition, both the exoR95 and exoS96 mutations caused
a marked reduction in the biosynthesis of flagella and consequent loss of ability
of the cells to swarm and swim. Succinoglycan overproduction did not appear to be
the cause of the suppression of flagellum biosynthesis. Further analysis
indicated that both the exoR95 and exoS96 mutations affected the expression of
the flagellum biosynthesis genes. These findings suggest that both the ExoR
protein and the ExoS/ChvI two-component regulatory system are involved in the
regulation of both succinoglycan and flagellum biosynthesis. These findings
provide new avenues of understanding of the physiological changes S. meliloti
cells go through during the early stages of symbiosis and of the signal
transduction pathways that mediate such changes.
DOI: 10.1128/JB.186.18.6042-6049.2004
PMCID: PMC515170
2168. J Bacteriol. 2004 Sep;186(18):5988-96.
The Sinorhizobium meliloti ABC transporter Cho is highly specific for choline and
expressed in bacteroids from Medicago sativa nodules.
Dupont L(1), Garcia I, Poggi MC, Alloing G, Mandon K, Le Rudulier D.
Author information:
(1)Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR6192
CNRS-INRA-Universitéde Nice Sophia Antipolis, Centre INRA Agrobiotech, 400 Route
des Chappes, BP167, 06903 Sophia Antipolis Cédex, France.
In Sinorhizobium meliloti, choline is the direct precursor of

phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family,
and glycine betaine, an important osmoprotectant. Moreover, choline is an
efficient energy source which supports growth. Using a PCR strategy, we
identified three chromosomal genes (choXWV) which encode components of an ABC
transporter: ChoX (binding protein), ChoW (permease), and ChoV (ATPase). Whereas
the best homology scores were obtained with components of betaine ProU-like
systems, Cho is not involved in betaine transport. Site-directed mutagenesis of
choX strongly reduced (60 to 75%) the choline uptake activity, and purification
of ChoX, together with analysis of the ligand-binding specificity, showed that
ChoX binds choline with a high affinity (KD, 2.7 microM) and acetylcholine with a
low affinity (KD, 145 microM) but binds none of the betaines. Uptake competition
experiments also revealed that ectoine, various betaines, and choline derivatives
were not effective competitors for Cho-mediated choline transport. Thus, Cho is a
highly specific high-affinity choline transporter. Choline transport activity and
ChoX expression were induced by choline but not by salt stress. Western blotting
experiments with antibodies raised against ChoX demonstrated the presence of ChoX
in bacteroids isolated from nitrogen-fixing nodules obtained from Medicago sativa
roots. The choX mutation did not have an effect on growth under standard
conditions, and neither Nod nor Fix phenotypes were impaired in the mutant,
suggesting that the remaining choline uptake system(s) still present in the
mutant strain can compensate for the lack of Cho transporter.
DOI: 10.1128/JB.186.18.5988-5996.2004
PMCID: PMC515146
2169. Mol Genet Genomics. 2004 Oct;272(3):235-46. Epub 2004 Sep 1.
Comparative mapping between Medicago sativa and Pisum sativum.
Kaló P(1), Seres A, Taylor SA, Jakab J, Kevei Z, Kereszt A, Endre G, Ellis TH,
Kiss GB.
Author information:
(1)Institute of Genetics, Agricultural Biotechnology Center, Szent-Györgyi A. u.
4, 2100 Gödöllo, Hungary.
Comparative genome analysis has been performed between alfalfa (Medicago sativa)
and pea (Pisum sativum), species which represent two closely related tribes of
the subfamily Papilionoideae with different basic chromosome numbers. The
positions of genes on the most recent linkage map of diploid alfalfa were
compared to those of homologous loci on the combined genetic map of pea to
analyze the degree of co-linearity between their linkage groups. In addition to
using unique genes, analysis of the map positions of multicopy (homologous) genes
identified syntenic homologs (characterized by similar positions on the maps) and
pinpointed the positions of non-syntenic homologs. The comparison revealed
extensive conservation of gene order between alfalfa and pea. However, genetic
rearrangements (due to breakage and reunion) were localized which can account for
the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these
genetic events and our increasing knowledge of the genomic structure of pea, it
was concluded that the difference in genome size between the two species (the pea
genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of
genome duplication in pea. The high degree of synteny observed between pea and
Medicago loci makes further map-based cloning of pea genes based on the genome
resources now available for M. truncatula a promising strategy.
DOI: 10.1007/s00438-004-1055-z
2170. J Dairy Sci. 2004 Aug;87(8):2563-70.
Microbial counts, fermentation products, and aerobic stability of whole crop corn
and a total mixed ration ensiled with and without inoculation of Lactobacillus
casei or Lactobacillus buchneri.
Nishino N(1), Wada H, Yoshida M, Shiota H.
Author information:
(1)Department of Animal Science, Faculty of Agriculture, Okayama University,
Okayama 700-8530, Japan. jloufeed@cc.okayama-u.ac.jp
Whole crop corn (DM 29.2%) and a total mixed ration (TMR, DM 56.8%) containing
wet brewers grains, alfalfa hay, dried beet pulp, cracked corn, soybean meal, and
molasses at a ratio of 5:1:1:1:1:1 on fresh weight basis, were ensiled with and
without Lactobacillus casei or Lactobacillus buchneri in laboratory silos. The
effects of inoculation on microbial counts, fermentation products, and aerobic
stability were determined after 10 and 60 d. Untreated corn silage was well
preserved with high lactic acid content, whereas large numbers of remaining
yeasts resulted in low stability on exposure to air. Inoculation with L. casei
suppressed heterolactic fermentation, but no improvements were found in aerobic
stability. The addition of L. buchneri markedly enhanced the aerobic stability,
while not affecting the DM loss and NH3-N production. Large amounts of ethanol
were found when the TMR was ensiled, and the content of ethanol overwhelmed that
of lactic acid in untreated silage. This fermentation was related to high yeast
populations and accounted for a large loss of DM found in the initial 10 d. The
ethanol production decreased when inoculated with L. casei and L. buchneri, but
the effects diminished at 60 d of ensiling. Inoculation with L. buchneri lowered
the yeasts in TMR silage from the beginning of storage; however, the populations
decreased to undetectable levels when stored for 60 d, regardless of inoculation.
No heating was observed in TMR silage during aerobic deterioration test for 7 d.
This stability was achieved even when a high population of yeasts remained and
was not affected by either inoculation or ensiling period. The results indicate
that inoculation with L. buchneri can inhibit yeast growth and improve aerobic
stability of corn and TMR silage; however, high stability of TMR silage can be
obtained even when no treatments were made and high population (>10(5) cfu/g) of
yeasts were detected.
DOI: 10.3168/jds.S0022-0302(04)73381-0
2171. J Dairy Sci. 2004 Aug;87(8):2495-505.
Replacing chopped alfalfa hay with alfalfa silage in barley grain and
alfalfa-based total mixed rations for lactating dairy cows.
Plaizier JC(1).
Author information:
(1)Department of Animal Science, University of Manitoba, Winnipeg, MB, Canada R3T
2N2. plaizier@ms.umanitoba.ca
The effects of replacing chopped alfalfa hay with alfalfa silage in a fine barley
grain and alfalfa-based total mixed ration (TMR) were evaluated. Diets contained
(dry matter basis) 53.0% commercial energy supplement, 10.3% commercial protein
supplement, and 9.7% corn silage. Diets varied in inclusion of chopped alfalfa
hay and alfalfa silage, and contained either 20.0% chopped alfalfa hay and 7.0%
alfalfa silage, 10.0% chopped alfalfa hay and 17.0% alfalfa silage, or 27.0%
alfalfa silage. Contents of crude protein, neutral detergent fiber (NDF), acid
detergent fiber, and minerals did not differ among diets. Replacing chopped
alfalfa hay with alfalfa silage decreased dietary dry matter, and increased
dietary soluble protein and physical effective NDF calculated as the proportion
of dietary NDF retained by the 8- and 19-mm screens of the Penn State Particle
Separator (peNDF(NDF)) from 13.3 to 15.6% DM. Replacing chopped alfalfa hay with
alfalfa silage did not affect dry matter intake, rumen pH, rumen volatile fatty
acids, blood lactate, milk fat, and milk protein percentage, but did decrease
blood glucose, tended to increase blood urea, and numerically decreased milk
yield and milk protein yield. A wider range in peNDF(NDF) and a higher inclusion
of corn silage might have resulted in greater differences in rumen fermentation
and milk production among diets. The pH of rumen fluid samples collected 4 h
after feeding varied from 5.90 to 5.98, and milk fat percentage varied from 2.50
to 2.60% among diets. These values suggest that mild subacute ruminal acidosis
was induced by all diets.
DOI: 10.3168/jds.S0022-0302(04)73374-3
2172. J Dairy Sci. 2004 Aug;87(8):2486-94.
Feeding lactose increases ruminal butyrate and plasma beta-hydroxybutyrate in

DeFrain JM(1), Hippen AR, Kalscheur KF, Schingoethe DJ.
Author information:
(1)Dairy Science Department, South Dakota State University, Brookings 57007, USA.
Ruminal fermentation of lactose increases molar proportions of butyrate, which is

metabolized by the ruminal epithelium to beta-hydroxybutyrate (BHBA). To
determine the effects of dietary whey, and specifically lactose, on
concentrations of ruminal and blood volatile fatty acids (VFA) and blood BHBA, 8
Holstein and 4 Brown Swiss multiparous cows (210 +/- 33 d in milk) were blocked
by breed and randomly assigned to one of three 4 x 4 Latin squares. Treatments
were control (CON; 7.1% of dietary dry matter [DM] as cornstarch), liquid whey
(WHEY; 9.4% of diet DM) containing 70% lactose on a DM basis, low lactose (LOLAC;
7.1% lactose), or high lactose (HILAC; 14.3% lactose). Diets contained 53% forage
as corn silage, alfalfa hay, and grass hay (DM basis) and a corn and soybean
meal-based concentrate. Average dietary percentage of crude protein and energy
density (Mcal/kg net energy for lactation) were 16.8 and 1.47, respectively.
Feeding lactose increased DM intake. Milk production and composition were not
affected by diet with the exception of decreased urea nitrogen in milk from cows
fed lactose. Greater proportions of ruminal propionate were observed in cows fed
CON relative to those fed WHEY and LOLAC. Increasing dietary lactose increased
proportions of ruminal butyrate and decreased acetate and branched-chain VFA.
Concurrent with the increase in ruminal butyrate concentrations, there was an
increase in plasma BHBA as lactose in the diet increased. Concentrations of VFA
in plasma were not affected by diet with the exception of the branched-chain VFA,
which were increased in cows fed LOLAC compared with WHEY. These data indicate
lactose fermentation increases proportions of ruminal butyrate and plasma BHBA in
lactating dairy cows; however, the observed increase in plasma BHBA is not
sufficient to subject cows to ketosis.
DOI: 10.3168/jds.S0022-0302(04)73373-1
2173. J Dairy Sci. 2004 Jul;87(7):2268-76.
Lactation performance of holstein cows fed fescue, orchardgrass, or alfalfa

silage.
Cherney DJ(1), Cherney JH, Chase LE.
Author information:
(1)Department of Animal Science, Cornell University, Ithaca, NY 14853, USA.
djc6@cornell.edu
Perennial grasses are increasingly being used as an integral part of nutrient

management plans, but fescue (Festuca arundinacea Schreb.) is often overlooked
because of perceived intake problems. A 30-d study was conducted to evaluate the
lactation performance of cows fed a fescue silage-based total mixed ration (TMR)
compared with orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa
L.) silage-based TMR, when forages are harvested at recommended neutral detergent
fiber (NDF) levels. Holstein cows (body weight [BW] = 627 +/- 66.0 kg, milk yield
= 40.9 +/- 6.93 kg/d, parity = 2.6 +/- 1.44, days in milk = 152 +/- 24.5) were
randomly assigned to treatment. Statistical design was a randomized complete
block with 10 cows per treatment. The 5 treatments consisted of TMR using
first-cutting alfalfa, and first- and second-cutting orchardgrass and tall fescue
silage. Diets were formulated to provide 0.95% of BW as forage NDF and contained
approximately 18% CP and 1.6 mcal/kg. This resulted in diets of about 30% NDF;
for a 612-kg cow, approximately 5.8 kg/d of forage NDF was fed. Second-cutting,
grass-based TMR had lower intake than alfalfa and first-cutting forage TMR. Cows
consuming second-cutting orchardgrass had lower milk production than did cows
consuming other forage TMR. Cows fed fescue TMR had higher milk production than
those fed orchardgrass. Indigestible residues were higher, and NDF
digestibilities were lower, in second-cutting forages vs. first-cutting forages,
likely contributing to the differences observed in intake and resulting
differences in milk production. Dairy cows consumed the first-cutting fescue TMR
readily and performed as well as those on alfalfa or first-cutting,
orchardgrass-based TMR in terms of lactation performance, but fescue and
orchardgrass rations will require more concentrate in the ration than alfalfa.
DOI: 10.3168/jds.S0022-0302(04)70047-8
2174. Shi Yan Sheng Wu Xue Bao. 2004 Jun;37(3):167-75.
[Intergeneric somatic hybridization between Astragalus adsurgens Pall and

Medicago sativa L].
Jin H(1), Jia JF, Hao JG.
Author information:
(1)Provincial Key Laboratory of Biotechnology, Northwest University, Xi'an
710069.
The protoplast fusion was induced between Astragalus adsurgens Pall and
Agrobacterium rhizogenes-transformed cell line of Medicago Sativa L. by use of
PEG method. Putative somatic hybrid cells were selected based on the observation
that only intergeneric hybrid cells continued to divide on the DPD medium without
any phytohormone while the cells and clusters derived from parental cells did not
survive further. After being cultured, the somatic hybrid calli were obtained.
Although both of the parental calli were not capable of regenerating , one of the
hydrid cell lines recovered the differentiation ability and produced small
shoots. Characterizations of the hybrid calli were carried out by examination of
chromsome number, opine synthetase assay, isoenzyme and RAPD analyses.
2175. Mycol Res. 2004 Jun;108(Pt 6):662-71.
Brefeldin A production by Phoma medicaginis in dead pre-colonized plant tissue: a

strategy for habitat conquest?
Weber RW(1), Stenger E, Meffert A, Hahn M.
Author information:
(1)Lehrbereich Biotechnologie, Universität Kaiserslautern, Paul-Ehrlich-Str. 23,
D-67663 Kaiserslautern, Germany. rwsweber@rhrk.uni-kl.de
Phoma medicaginis was isolated as the dominant endophyte from surface-sterilized

shoots of Medicago sativa and M. lupulina growing outdoors. Plants were either
symptomless or showed signs of infection in the shape of limited lesions which
sometimes contained melanized pycnidial initials. Rapid colonization of host
tissue and sporulation were observed within 9 d on dead plant material upon
incubation in a moist chamber. Such colonized material, but not freshly harvested
living tissue, contained brefeldin A (1.7 microg g(-1) D.W.). This toxin was also
produced in pure culture (20 mg l(-1)) and in artificially inoculated autoclaved
M. sativa stems (3 mg g(-1) D.W. =920 microg ml(-1)). The latter concentration of
brefeldin A should be similar to that produced within a fruiting lesion of P.
medicaginis and suppressed spore germination and growth of nine of 11 common
phylloplane fungi tested. This metabolite may thus have a function in substrate
defence after the switch from the endophytic to the saprotrophic period in the
life-cycle of P. medicaginis following the death of infected host tissue.
2176. Ying Yong Sheng Tai Xue Bao. 2004 May;15(5):803-8.
[Yield characteristics and genetic diversity of main alfalfa varieties in China].
Sun J(1), Wang Y.
Author information:
(1)Gansu Grassland Ecology Institute, College of Pastoral Agriculture Science and
Technology, Lanzhou University, Lanzhou 730020, China. sunjianhua@lzu.edu.cn
The dry matter (DM) yield and its characteristics of 28 alfalfa varieties were
evaluated in the 4th year under space sowing condition. The results showed that
there existed significant difference (P<0.01) in DM yield and its characteristics
among the varieties. The highest and higher DM yield were obtained form varieties
Xinjiang Deye (452.8 g x plant(-1)) and Gannong No. 3 (373.0 g x plant(-1)),
respectively, and the varieties with the highest height and branch/plant at the
3rd cutting were Gannong No. 3, Xinjiang Deye, Weixian, Tumu No. 2, Xinmu No. 1,
and Zhaodong. The correlation analysis between DM yield and its characteristics
indicated that the main factors affecting yield were the height and the
branch/plant. The genetic diversity of DM yield and its components in 28 alfalfa
varieties showed that the variations of DM yield and yield components of
within-variety were larger than those of among-variety in majority of test
varieties. The CV of all characteristics were presented in order of 3rd No.
branch/plant>DM yield/plant>1st No. branch/plant>2nd No. branch/plant>Spring
height>3rd height>2nd height>leaf width>budding height>leaf length>1st height.
2177. J Anim Sci. 2004 Jul;82(7):1986-96.
Effects of cereal grain supplementation on apparent digestibility of nutrients

and concentrations of fermentation end-products in the feces and serum of horses
consuming alfalfa cubes.
Hussein HS(1), Vogedes LA, Fernandez GC, Frankeny RL.
Author information:
(1)Department of Animal Biotechnology, University of Nevada-Reno, Reno, NV 89557,
USA. hhussein@agnt1.ag.unr.edu
Twenty geldings (five groups; similar age and BW) were used in a completely
randomized design experiment to determine effects of grain supplementation of an
alfalfa-cube diet on apparent nutrient digestibility and hindgut fermentation.
The geldings were housed individually, fed their diets in two equal meals (0600
and 1800), and adapted to five dietary treatments over 6 wk. The treatments were
alfalfa cubes (1% of BW; DM basis) without (control) or with one of four rolled
cereal grains (i.e., barley, corn, naked oats, or oats) to provide a target level
of 0.4% of BW as total nonstructural carbohydrates (TNC). Due to acute laminitis,
three geldings (one in the control group and two in the barley group) were
excluded. Because of this and multiple incidents of gas colic, TNC level was
decreased to 0.2% of BW to ensure the geldings' health throughout the adaptation
(7 d) and sample collection (5 d) periods. Grain intakes varied (P < 0.05) and
reflected the different TNC concentrations. Apparent digestibilities of DM, OM,
CP, NDF, ADF, and cellulose were not affected (P > 0.05) by grain supplementation
and averaged 63.2, 63.1, 79.5, 42.7, 39.9, and 50.3%, respectively. Regardless of
the source, grain supplementation increased (P < 0.05) apparent digestibility of
TNC (from 85.6 to 94.6%) and decreased (P < 0.05) fecal pH (from 7.04 to 6.74).
Fecal concentrations of total VFA (mg/g of DM) were greatest for the barley and
naked oats diets (averaging 11.73), intermediate for the oats diet (8.00), and
least for the control and corn diets (averaging 5.00; P < 0.05). Fecal
concentrations of lactate (microg/g of DM) were greatest for the barley diet
(254), intermediate for the oats diet (138), and least for the remaining diets
(averaging 100; P < 0.05). Fecal concentrations of NH3 N (mg/g of DM) were
greatest for the naked oats diet (1.68), intermediate for the barley and oats
diets (averaging 0.86), and least for the remaining diets (averaging 0.63; P <
0.05). Serum concentration of lactate was 46% higher (P < 0.05) for the control
than for the grain diets (averaging 0.05 mg/100 mL). Feeding barley, corn, naked
oats, and oats contributed to 13, 15, 8, and 20% higher (P < 0.05) serum NH3 N
concentrations than the control diet (0.25 mg/100 mL). Higher (P < 0.05) serum
concentrations of urea N (mg/100 mL) were detected for the control, barley, and
naked oats diets (averaging 25.28) than for the corn or oats diets (averaging
22.21). Results suggest that horses consuming alfalfa cubes could be supplemented
with rolled barley, corn, naked oats, or oats at levels not exceeding 0.2% of BW
as TNC without affecting nutrient digestion or overall health negatively.
DOI: 10.2527/2004.8271986x
Cytological, genetic, and molecular analysis to characterize compatible and

incompatible interactions between Medicago truncatula and Colletotrichum
trifolii.
Torregrosa C(1), Cluzet S, Fournier J, Huguet T, Gamas P, Prospéri JM,

Esquerré-Tugayé MT, Dumas B, Jacquet C.
Author information:
(1)UMR 5546 CNRS-UPS, Pô1e de Biotechnologie Végétale, 24 Chemin de Borde Rouge,
BP17 Auzeville, 31326 Castanet-Tolosan, France.
In this study, a new pathosystem was established using the model plant Medicago
truncatula and Colletotrichum trifolii, the causal agent of anthracnose on
Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and
F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1,
respectively. Symptom analysis and cytological studies indicated that resistance
of Jemalong was associated with a hypersensitive response of the plant. The two
selected lines were crossed, and inoculations with C. trifolii were performed on
the resulting F1 and F2 progenies. Examination of the disease phenotypes
indicated that resistance was dominant and was probably due to a major resistance
gene. Molecular components of the resistance were analyzed through macroarray
experiments. Expression profiling of 126 expressed sequence tags corresponding to
92 genes, which were selected for their putative functions in plant defense or
signal transduction, were compared in Jemalong and F83005.5 lines. A strong
correlation was observed between the number of up-regulated genes and the
resistance phenotype. Large differences appeared at 48 h postinoculation; more
than 40% of the tested genes were up-regulated in the Jemalong line compared with
only 10% in the susceptible line. Interestingly, some nodulin genes were also
induced in the resistant line upon inoculation with C. trifolii.
DOI: 10.1094/MPMI.2004.17.8.909
2179. Theor Appl Genet. 2004 Aug;109(4):775-82. Epub 2004 Aug 6.
Population structure and combining ability of diverse Medicago sativa germplasms.
Maureira IJ(1), Ortega F, Campos H, Osborn TC.
Author information:
(1)Plant Breeding and Plant Genetics Program and Department of Agronomy,
University of Wisconsin, Madison, WI 53706, USA.
Although unadapted germplasms have been used to improve disease and insect
resistance in alfalfa, there has been little effort to use these for improving
forage yield. We evaluated genetic diversity and combining ability among two
unadapted germplasms ( Medicago sativa ssp. sativa Peruvian and M. sativa ssp.
falcata WISFAL) and three Northern U.S. adapted alfalfa cultivars. Population
structure analyses indicated that the WISFAL and Peruvian germplasms were
genetically distinct from the cultivars, although Peruvian was relatively closer
to the cultivars. Peruvian and WISFAL germplasms were intermated to generate a
novel hybrid population. This population was crossed to the three cultivars as
testers, and the testcross progenies were evaluated for forage yield along with
the hybrid population, the original germplasms (Peruvian, WISFAL and cultivars),
testcrosses of Peruvian and WISFAL to the three cultivars and a three-way hybrid
of the cultivars. The experiment was carried out in the field in Temuco, Chile
and Arlington, Wisconsin, USA, and forage was harvested during two seasons.
Results from these evaluations showed that hybrids between the Peruvian x WISFAL
population and the cultivar testers yielded as much as the cultivar testers.
Heterosis was observed between Peruvian and WISFAL, and between these germplasms
and the cultivar testers, suggesting that each germplasm may contain different
favorable alleles. If Peruvian and WISFAL populations contain alleles at
different loci that complement cultivar testers, then combining and enriching
these alleles in a single population could result in improved combining ability
with alfalfa cultivars.
DOI: 10.1007/s00122-004-1677-x
2180. Plant Physiol. 2004 Aug;135(4):2055-67. Epub 2004 Aug 6.
Differential antifungal and calcium channel-blocking activity among structurally

related plant defensins.
Spelbrink RG(1), Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH.
Author information:
(1)The Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
Plant defensins are a family of small Cys-rich antifungal proteins that play
important roles in plant defense against invading fungi. Structures of several
plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants
in plant defensins that govern their antifungal activity and the mechanisms by
which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed
defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro,
and its antifungal activity is markedly reduced in the presence of Ca(2+). By
contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence
identity with MsDef1, lacks antifungal activity against F. graminearum.
Characterization of the in vitro antifungal activity of the chimeras containing
portions of the MsDef1 and MtDef2 proteins shows that the major determinants of
antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of
MsDef1. We further define the active site by demonstrating that the Arg at
position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we
have found for the first time, to our knowledge, that MsDef1 blocks the mammalian
L-type Ca(2+) channel in a manner akin to a virally encoded and structurally
unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar
MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the
L-type Ca(2+) channel. From these results, we speculate that the two unrelated
antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same
molecular target, whereas the two structurally related antifungal plant
defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in
fungi.
DOI: 10.1104/pp.104.040873
PMCID: PMC520777
2181. Environ Sci Technol. 2004 Jul 1;38(13):3581-6.
Selenium biotransformations in an insect ecosystem: effects of insects on

phytoremediation.
Vickerman DB(1), Trumble JT, George GN, Pickering II, Nichol H.
Author information:
(1)Department of Entomology, University of California at Riverside, Riverside,
California 92521, USA. danel.vickerman@ucr.edu
Comment in
Environ Sci Technol. 2004 Jul 1;38(13):240A.
Phytoremediation of selenium-contaminated soils may be influenced by higher

trophic levels including insects. We examined how selenium affects the behavior,
survival, and development of the wasp parasitoid Cotesia marginiventris,
parasitizing its natural host, the beet armyworm Spodoptera exigua, feeding on
alfalfa, Medicago sativa, irrigated with water containing selenate. X-ray
absorption spectroscopy was used to quantify the selenium chemical forms in each
trophic level. Alfalfa partially transformed selenate to organoselenium. S.
exigua contained only organoselenium, both directly absorbed from M. sativa and
transformed from selenate. C. marginiventris cocoons collected shortly after
larval emergence contained only organoselenium derived from the host. The
surprising finding of trimethylselenonium-like species in adult parasitoids and
the cocoons from which they emerged suggests that adults and pharates can
detoxify excess selenium through methylation and volatilization. Adult
parasitoids do not discriminate against selenium-containing alfalfa, even though
alfalfa generates selenium volatiles. Parasitoids raised on selenium-fed larvae
emerged later and pupae weighed less than their selenium-free counterparts. We
conclude therefore that C. marginiventris can be used to control S. exigua damage
to M. sativa being used to remove selenium from soils. Moreover, the presence of
such insects may improve phytoremediation by increasing biotransformation of
inorganic selenium and release of volatile selenium species.
2182. J Biol Chem. 2004 Oct 8;279(41):42552-9. Epub 2004 Aug 2.
Calcium/calmodulin up-regulates a cytoplasmic receptor-like kinase in plants.
Yang T(1), Chaudhuri S, Yang L, Chen Y, Poovaiah BW.
Collaborators: Poovaiah BW(2).
Author information:
(1)Center for Integrated Biotechnology and Department of Horticulture, Washington
State University, Pullman, Washington 99164-6414, USA.
(2)WA St U, Pullman
Calcium/calmodulin-dependent kinases play an important role in protein

phosphorylation in eukaryotes. However, not much is known about
calcium/calmodulin-dependent protein phosphorylation and its role in signal
transduction in plants. By using a protein-protein interaction-based approach, we
have isolated a novel plant-specific calmodulin-binding receptor-like cytoplasmic
kinase (CRCK1) from Arabidopsis thaliana, as well as its ortholog from Medicago
sativa (alfalfa). CRCK1 does not show high homology to
calcium/calmodulin-dependent protein kinases in animals. In contrast, it shows
high homology in the kinase domain to serine/threonine receptor-like kinases in
plants. However, it contains neither a transmembrane domain nor an extracellular
domain. Calmodulin binds to CRCK1 in a calcium-dependent manner with an affinity
of approximately 20.5 nm. The calmodulin-binding site in CRCK1 is located in
amino acids 160-183, which overlap subdomain II of the kinase domain. CRCK1
undergoes autophosphorylation in the presence of Mg2+ at the threonine
residue(s). The Km and Vmax values of CRCK1 for ATP are 1 microm and 33.6
pmol/mg/min, respectively. Calcium/calmodulin stimulates the kinase activity of
CRCK1, which increases the Vmax of CRCK1 approximately 9-fold. The expression of
CRCK1 is increased in response to stresses such as cold and salt and stress
molecules such as abscisic acid and hydrogen peroxide. These results indicate the
presence of a calcium/calmodulin-regulated receptor-like cytoplasmic kinase in
plants. Furthermore, these results also suggest that calcium/calmodulin-regulated
protein phosphorylation involving CRCK1 plays a role in stress signal
transduction in plants.
DOI: 10.1074/jbc.M402830200
2183. J Bacteriol. 2004 Aug;186(16):5460-72.
The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a
central role in Sinorhizobium meliloti gene expression.
Hoang HH(1), Becker A, González JE.
Author information:
Richardson, 75083-0688, USA.
Quorum sensing, a population density-dependent mechanism for bacterial

communication and gene regulation, plays a crucial role in the symbiosis between
alfalfa and its symbiont Sinorhizobium meliloti. The Sin system, one of three
quorum sensing systems present in S. meliloti, controls the production of the
symbiotically active exopolysaccharide EPS II. Based on DNA microarray data, the
Sin system also seems to regulate a multitude of S. meliloti genes, including
genes that participate in low-molecular-weight succinoglycan production,
motility, and chemotaxis, as well as other cellular processes. Most of the
regulation by the Sin system is dependent on the presence of the ExpR regulator,
a LuxR homolog. Gene expression profiling data indicate that ExpR participates in
additional cellular processes that include nitrogen fixation, metabolism, and
metal transport. Based on our microarray analysis we propose a model for the
regulation of gene expression by the Sin/ExpR quorum sensing system and another
possible quorum sensing system(s) in S. meliloti.
DOI: 10.1128/JB.186.16.5460-5472.2004
PMCID: PMC490858
2184. J Dairy Sci. 2004 May;87(5):1413-24.
Effects of fiber content and particle size of forage on the flow of microbial
amino acids from continuous culture fermenters.
Rodríguez-Prado M(1), Calsamiglia S, Ferret A.
Author information:
(1)Departament de Ciència Animal i dels Aliments, Universitat Autònoma de
Barcelona, Bellaterra, Spain.
Eight dual-flow continuous culture fermenters (1320 mL) were used in two 10-d
consecutive periods to study the effects of fiber content and particle size on
rumen fermentation, nutrient flow, and the profile and flow of amino acids (AA)
from microbial origin. Treatments were arranged in a 2 x 2 factorial; the main
factors were fiber content (high fiber [HF] = 67% alfalfa hay, 33% concentrate;
low fiber [LF] = 39% alfalfa hay, 61% concentrate) and forage stem particle size
(ground not to pass a 3-mm sieve = large size [LS]; ground to pass a 1-mm sieve =
small size [SS]). On the last day of the experiment, liquid- (LAB) and solid-
(SAB) associated bacteria were isolated from each fermenter for chemical
analysis. Microbial N and AA flows were estimated using LAB or SAB composition
data. Total volatile fatty acid concentration was higher for LF (122 mM) than for
HF (102 mM). The proportion of acetate was higher for HF (63.5%) than for LF
(58.2%). When SS was fed instead of LS, there was a reduction in the acetate
proportion (63.4% vs 58.3%, respectively) and in the acetate to propionate ratio
(3.13 vs. 2.41, respectively). Bacterial N flow was higher, and dietary N flow
was lower, in SS compared with LS when LAB were used for calculations. Efficiency
of microbial protein synthesis (EMPS) was affected by particle size, when LAB
were used for calculations, and by fiber content, when SAB were used for
calculations. The AA profile of LAB differed from SAB in 4 of 16 AA. The fiber
content and particle size had small effects on microbial AA profile. However, the
bacterial AA flow was higher in 13 of 16 AA, when SAB instead of LAB were used
for calculations. Estimates of bacterial N flow, EMPS, AA profile, and flows of
microbial origin were dependent on the type of microbial population used for
calculations. The representativeness of the microbial samples is essential for
the accurate prediction of the AA flow from microbial origin.
DOI: 10.3168/jds.S0022-0302(04)73290-7
2185. J Dairy Sci. 2004 May;87(5):1345-59.

Sources of variation in rates of in vitro ruminal protein degradation.
Broderick GA(1), Udén P, Murphy ML, Lapins A.
Author information:
WI 53706, USA. gbroderi@wisc.edu
Rates and extents of ruminal protein degradation for casein, solvent soybean meal
(SSBM), expeller soybean meal (ESBM), and alfalfa hay were estimated from net
appearance of NH3 and total amino acids in in vitro media containing 1 mM
hydrazine and 30 mg/L of chloramphenicol. Protein was added at 0.13 mg of N/mL of
medium, and incubations were conducted for 4 to 6 h, usually with hourly
sampling. Inocula were obtained from ruminally cannulated donor cows fed diets of
grass silage or alfalfa and corn silages plus concentrates. Preincubation or
dialysis of inocula was used to suppress background NH3 and total amino acids;
however, preincubation yielded more rapid degradation rates for casein and SSBM
and was used in subsequent incubations. Preincubation with added vitamins, VFA,
hemin, or N did not alter protein degradation. Protein degradation rates
estimated for SSBM, ESBM, and alfalfa were not different when computed from total
N release or N release in NH3 plus total amino acids, regardless of whether amino
acids were quantified using ninhydrin colorimetry or o-phthalaldehyde
fluorescence. Accounting for the release of peptide-N also did not affect
estimated degradation. However, casein degradation rates were more rapid when
using total N release or accounting for peptide-N, indicating significant
accumulation of small peptides during its breakdown. Rates also were more rapid
with inocula from lactating cows versus nonlactating cows with lower feed intake.
Protein degradation rates were different due to time after feeding: casein rate
was more rapid, but SSBM and ESBM rates were slower with inocula obtained after
feeding. Several characteristics of ruminal inoculum that influenced breakdown of
the rapidly degraded protein casein did not appear to have direct effects on
degradation of protein in soybean meal.
DOI: 10.3168/jds.S0022-0302(04)73284-1
2186. J Biotechnol. 2004 Aug 26;112(1-2):151-64.
The use of surface plasmon resonance (SPR) and fluorescence resonance energy
transfer (FRET) to monitor the interaction of the plant G-proteins Ms-Rac1 and
Ms-Rac4 with GTP.
Brecht M(1), Sewald K, Schiene K, Keen G, Fricke M, Sauer M, Niehaus K.
Author information:
(1)Faculty of Biology, Genetics, University of Bielefeld, D-33501 Bielefeld,
Germany.
Using an RT-PCR approach a cDNA clone, designated Ms-Rac4 and putatively coding
for a small GTPase was isolated from Medicago sativa. Ms-Rac4 and the earlier
described Ms-Rac1 [Mol. Gen. Genet. 263 (2000) 761] belong to the class of
GTP-binding Rho of plants (Rop) proteins. At the amino acid level they display
all conserved regions that are common to GTP-binding proteins. Phylogenetically
both are located in the group Ia, but within this group they are well-separated.
Computed structure models of both proteins revealed a high degree of structural
conservation. Particularly the switch I and switch II region are 100% conserved
between Ms-Rac1 and Ms-Rac4 and highly conserved as compared to other Rac-like
G-proteins. Both GTPases differ in structure within the fourth loop and the
fourth helix. GTP-binding properties of the heterologously expressed Ms-Rac1 and
Ms-Rac4 was shown by fluorescence resonance energy transfer (FRET) using mantGTP
and by surface plasmon resonance (SPR). By this method the specificity of the
G-protein/GTP interaction was shown and the inhibitory effect of GTP, EDTA and
Mg(2+) on the Ms-Rac1 and Ms-Rac4 binding to immobilized GTP was characterized.
Ms-Rac1 and Ms-Rac4 exhibited the same affinity to GTP and are similarly affected
by GTP, EDTA and Mg(2+). Thus, the predicted structural differences do not result
in different GTP-binding properties of Ms-Rac1 and Ms-Rac4.
DOI: 10.1016/j.jbiotec.2004.04.030
Isolation of salt-sensitive mutants from Sinorhizobium meliloti and

characterization of genes involved in salt tolerance.
Wei W(1), Jiang J, Li X, Wang L, Yang SS.
Author information:
(1)Department of Microbiology, College of Biological Sciences, China Agricultural
University, Beijing, PR China.
AIMS: The purpose of our research is to isolate salt-sensitive mutants and to

study the genes involved in salt tolerance of the salt-tolerant bacterium
Sinorhizobium meliloti 042BM.
METHODS: Wild type S. meliloti 042BM bacteria are able to grow at a NaCl
concentration of 0.6 mol l(-1). A transposon Tn5-1063a mutagenesis library of S.
meliloti 042BM was constructed and eight salt-sensitive mutants were isolated,
which were unable to growth on FY plates containing 0.4 mol l(-1) NaCl.
SIGNIFICANCE: Our interest is to provide information about the mechanism of salt
tolerance in bacteria by studying the genes involved in salt tolerance. Here,
seven different genes were identified. These genes include omp10 encoding a cell
outer membrane protein, relA encoding (p)ppGpp synthetase, greA encoding a
transcription cleavage factor, nuoL encoding NADH dehydrogenase I chain L
transmembrane protein, a putative nuclease/helicase gene and two unknown genes.
Based on these findings, we suggest that the regulation of salt tolerance of S.
meliloti 042BM is complex and on several levels.
DOI: 10.1111/j.1472-765X.2004.01577.x
2188. FEBS Lett. 2004 Jul 30;571(1-3):61-6.
NO-degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR-1a

gene expression in Mhb1-overproducing tobacco plants.
Seregélyes C(1), Igamberdiev AU, Maassen A, Hennig J, Dudits D, Hill RD.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, Manitoba,
Canada R3T 2N2.
Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been
shown to have reduced necrotic symptom development. Here, we show that this
altered pathogenic response is linked to a significant increase in the nitric
oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the
transgenic plants. Homogenates of HOT transgenic seedlings were also found to
have higher NO-scavenging activity than non-transformed ones. The NO-scavenging
properties of recombinant alfalfa class1 hemoglobin have been examined.
Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene
glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl
Superose columns. After the final purification step, the obtained preparations
were near homogeneous and had a molecular weight of 44 kDa determined by
size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a
dimer. The protein participated in NO-degradation activity with NAD(P)H as a
cofactor. After ion-exchange columns, addition of FAD was necessary for
exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging
activity of rMhb1, which is similar to that of barley hemoglobin, supports a
conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO
levels by scavenging NO formed during hypoxia, pathogen attack and other
stresses.
DOI: 10.1016/j.febslet.2004.06.055
2189. J Herb Pharmacother. 2003;3(2):69-90.
Alfalfa (Medicago sativa L.):a clinical decision support tool.
Basch E(1), Ulbricht C, Harrison M, Sollars D, Smith M, Dennehy C, Szapary P.
Author information:
(1)National Standard Research Collaboration. ebasch@naturalstandard.com
PMID: 15277068
2190. Phytochemistry. 2004 Jun;65(12):1709-20.
Proteomics of Medicago sativa cell walls.
Watson BS(1), Lei Z, Dixon RA, Sumner LW.
Author information:
(1)Plant Biology Division, The Samuel Roberts Noble Foundation, PO Box 2180,
Ardmore, OK 73402, USA.
A method for the sequential extraction and profiling by two-dimensional gel

electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is
described. Protein extraction included freezing, grinding in a sodium acetate
buffer, separation by filtration of cell walls from cytosolic contents, and
extensive washing. Cell wall proteins were then extracted sequentially with a
solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction
with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2
and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were
extracted from these two gels, digested with trypsin, and analyzed using
nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass
spectrometer (LC/MS/MS). More than 100 proteins were identified and used in
conjunction with the 2-DE profiles to generate proteomic reference maps for cell
walls of this important legume. Identified proteins include classical cell wall
proteins as well as proteins traditionally considered as non-secreted. Two unique
extracellular proteins were also identified.
DOI: 10.1016/j.phytochem.2004.04.026
2191. J Chem Ecol. 2004 May;30(5):927-43.
Glandular trichome extracts from Medicago sativa deter settling by the potato
leafhopper Empoasca fabae.
Ranger CM(1), Backus EA, Winter RE, Rottinghaus GE, Ellersieck MR, Johnson DW.
Author information:
(1)Department of Entomology and Interdisciplinary Plant Group, 1-87 Agriculture
Building, University of Missouri, Columbia, Missouri 65211, USA.
cmr0b2@mizzou.edu
Extracts of glandular trichomes from Medicago sativa were tested for their
ability to disrupt the settling behavior of the potato leafhopper, Empoasca
fabae. Erect and procumbent glandular trichomes were mechanically isolated from
stem sections of resistant genotype "G98A," and nonglandular trichomes were
collected from susceptible cultivar "Ranger." Isolated trichomes were extracted
with chloroform, acetone, and ethanol, and the resulting crude extracts were
applied to the surface of a sachet containing an artificial diet. Leafhoppers
were offered a two-way choice between crude trichome extracts from G98A and
Ranger. All three of G98A solvent extracts caused various degrees of diet
rejection, resulting in the crude Ranger trichome extracts being preferred over
G98A extracts. Overall, the fewest leafhoppers settled on the ethanolic extracts.
Additional bioassays documented a dose response associated with G98A ethanolic
extracts when compared with Ranger trichome extracts and a solvent control. No
difference in preference behavior was detected between Ranger trichome extracts
and a solvent control. Gas chromatography-mass spectrometry analysis revealed a
homologous series of nonvolatile fatty acid amides C(n)H(2n+1)NO (n = 19-23)
unique to G98A glandular trichome extracts.
2192. Ann Bot. 2004 Aug;94(2):311-21.
Influence of initial organic N reserves and residual leaf area on growth, N

uptake, N partitioning and N storage in alfalfa (Medicago sativa) during
post-cutting regrowth.
Meuriot F(1), Avice JC, Simon JC, Laine P, Decau ML, Ourry A.
Author information:
(1)UMR INRA/UCBN, Ecophysiologie Végétale, Agronomie & Nutritions N, C, S.
Institut de Biologie Fondamentale et Appliquée, Université, 14032 Caen Cedex,
France. avice@ibfa@unicaen.fr
BACKGROUND AND AIMS: The influence of initial residual leaf area and initial N
reserves on N uptake, final N distribution, and yield in alfalfa regrowing after
cutting, were studied.
METHODS: The effects of two levels of initial residual leaf area (plants cut to
15 cm, with (L+) or without (L-) their leaves) and two initial levels of N status
[high N (HN) or low N (LN)] on growth, N uptake and N partitioning, allocation
and storage after 29 d of post-cutting regrowth were analysed.
KEY RESULTS: During most of the regrowth period (8-29 d after the initial
harvest), HN and L+ plants had higher net N uptake rates than LN and L- plants,
respectively, resulting in a greater final mineral N uptake for these treatments.
However, the final partitioning of exogenous N to the regrowing shoots was the
same for all treatments (67 % of total exogenous N on average). Final shoot
growth, total plant N content, and N allocation to the different taproot N pools
were significantly lower in plants with reduced initial leaf area and initial N
reserve status.
CONCLUSIONS: Although both initial residual leaf area and initial N reserves
influenced alfalfa regrowth, the residual leaf area had a greater effect on final
forage production and N composition in the taproot, whereas the N uptake rate and
final total N content in plant were more affected by the initial N reserve status
than by the residual leaf area. Moreover, N storage as proteins (especially as
vegetative storage proteins, rather than nitrate or amino acids) in the taproot
allowed nitrate uptake to occur at significant rates. This suggests that protein
storage is not only a means of sequestering N in a tissue for further
mobilization, utilization for growth or tissue maintenance, but may also
indirectly influence both N acquisition and reduction capacities.
DOI: 10.1093/aob/mch144
PMCID: PMC4242169
2193. Chemosphere. 2004 Sep;56(10):957-65.
Implications of rhizospheric heavy metals and nutrients for the growth of alfalfa
in sludge amended soil.
Su DC(1), Wong JW, Jagadeesan H.
Author information:
(1)Department of Plant Nutrition, China Agricultural University, Beijing 100094,
PR China.
Rhizospheric distribution of nutrients and heavy metals in sludge amended soil

was investigated using the rhizobag technique to give an indication of the
release of metals from wastewater sludge. DTPA-extractable Zn, Cd, Ni and Mn, and
available P, K and NH4+-N in the rhizosphere were markedly depleted when soil was
amended with sludge. There was no conspicuous depletion or accumulation of
DTPA-extractable Cu in the rhizosphere when the soil was amended with sewage
sludge but DTPA-extractable Fe accumulated in the rhizosphere when the soil was
amended with increasing amounts of sludge. The pH value in the rhizosphere
increased with distance from the roots when soil was amended with larger amounts
of sludge. The exchangeable fraction of Cu in the rhizosphere was depleted
whether or not the soil was treated with sludge. Carbonate, oxide, organic and
residual fractions of Cu and Zn were depleted in the rhizosphere at a distance of
0-2 mm from the roots when soil was amended with 50% sludge. Application of
sewage sludge had a positive effect on alfalfa growth. With an increase in sludge
amounts, the concentrations of Fe, Cu and Zn in alfalfa shoots did not change.
Soil amendments with less than 25% sludge did not increase the availability or
mobility of heavy metals. The depletion in rhizospheric DTPA-extractable Zn, Cd
and Ni indicates that with the sole exception of Cu, release of metals from
sludge amended soil was very limited.

Computational identification and characterization of novel genes from legumes.
Graham MA(1), Silverstein KA, Cannon SB, VandenBosch KA.
Author information:
55108, USA.
The Fabaceae, the third largest family of plants and the source of many crops,
has been the target of many genomic studies. Currently, only the grasses surpass
the legumes for the number of publicly available expressed sequence tags (ESTs).
The quantity of sequences from diverse plants enables the use of computational
approaches to identify novel genes in specific taxa. We used BLAST algorithms to
compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean
(Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's
nonredundant and EST databases, and to the genomic sequences of rice (Oryza
sativa) and Arabidopsis. As a working definition, putatively legume-specific
genes had no sequence homology, below a specified threshold, to publicly
available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST
contigs were identified, of which less than three percent had clear homology to
previously characterized legume genes. As a first step toward predicting
function, related sequences were clustered to build motifs that could be searched
against protein databases. Three families of interest were more deeply
characterized: F-box related proteins, Pro-rich proteins, and Cys cluster
proteins (CCPs). Of particular interest were the >300 CCPs, primarily from
nodules or seeds, with predicted similarity to defensins. Motif searching also
identified several previously unknown CCP-like open reading frames in
Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M.
truncatula suggest that this family has evolved by local duplications and
divergent selection.
DOI: 10.1104/pp.104.037531
PMCID: PMC519039
2195. Arch Anim Nutr. 2004 Jun;58(3):255-63.
Effect of alfalfa meal in diets of laying quails on performance, egg quality and
some serum parameters.
Güçlu BK(1), Işcan KM, Uyanik F, Eren M, Ağca AC.
Author information:
(1)University of Erciyes, Faculty of Veterinary Medicine, Department of Animal
Nutrition and Nutritional Diseases, Kayseri, Turkey. berrinkg@hotmail.com
This study was conducted to investigate the effects of increasing levels of

alfalfa meal in the diet of laying quails on egg production, feed consumption,
feed efficiency, egg quality, egg yolk cholesterol and selected serum parameters.
In this study, 192, 10-week old quails (Coturnix coturnix japonica) were evenly
distributed to four groups with four replicates of 12 quails each. The control
group was fed a basal diet containing 0% alfalfa meal and the remaining groups
received 3, 6 or 9% alfalfa meal for 12 weeks. Live weight, feed consumption, and
egg production were recorded and feed efficiency were calculated. Eggs were
examined for interior and exterior quality and egg yolk cholesterol content. At
the end of the experiment, blood samples were collected and sera were analysed
for serum Ca, inorganic P (P(i)), Mg, triglycerides and total cholesterol. Any
level of alfalfa meal had no effect on live weight, egg production, feed
consumption, feed efficiency, egg weight, and egg yolk index. Six percent and 9%
alfalfa meal increased specific gravity of whole egg and eggshell thickness as
well as serum P(i) levels. Nine percent alfalfa meal reduced serum triglycerides,
total cholesterol levels and egg yolk cholesterol content. The results of this
experiment indicated that addition of 9% alfalfa meal into the laying quail diet
may improve eggshell quality and reduced serum triglycerides and serum and egg
yolk cholesterol without any adverse effect on performance.
DOI: 10.1080/00039420410001701350
2196. J Dairy Sci. 2004 Apr;87(4):1057-70.
Nitrogen balance, microbial protein production, and milk production in dairy cows
fed fodder beets and potatoes, or barley.
Eriksson T(1), Murphy M, Ciszuk P, Burstedt E.
Author information:
(1)Swedish University of Agricultural Sciences, Department of Animal Nutrition
and Management, Kungsängen Research Center, SE-753 23 Uppsala, Sweden.
Torsten.Eriksson@huv.slu.se
Fourteen multiparous midlactation dairy cows were used in a change-over

experiment with 3 periods and 3 diets to evaluate the effects of fodder beets and
potatoes on N metabolism, microbial protein production, and milk production. A
basal ration of alfalfa/grass silage offered ad libitum, 1 kg of grass hay and 1
kg of heat-treated rapeseed cake was supplemented with 5 kg DM of either rolled
barley/raw potatoes 80:20 (BAP), fodder beets/raw potatoes 80:20 (BEP) or rolled
barley (BA). Urine and feces were collected quantitatively from 8 cows and
ruminal samplings, and evacuations were performed on 4 cannulated cows. Intake
and production did not differ between BAP and BA, but the BEP diet lowered intake
of both silage and total ration by 0.9 kg DM. Daily yield of energy-corrected
milk (ECM) was decreased by 1.7 and 2.3 kg compared with BAP and BA,
respectively. Milk urea concentration was 1 mM lower with the BEP diet. The
proportion of feed N recovered in milk was 20 to 21% for all diets. With the BEP
diet, urinary N amount and proportion were reduced correspondingly to the lower
total N intake. Fecal N amount remained unchanged, and hence nitrogen apparent
digestibility decreased by 5 percentage units with the BEP diet. Microbial
protein production, assessed by allantoin excretion, tended to be highest with
the BAP diet. Acetate proportion of VFA was lowered by the BEP diet, while
proportions of propionate and butyrate both tended to increase. Different
fermentation patterns, probably related to differences in rumen microbiota, could
explain why changes in energetic efficiency and milk composition reported in the
literature did not occur in the actual experiment when roots replaced barley.
Compared with barley, roots appeared to have a greater negative effect on silage
intake in conjunction with a prewilted silage with high intake potential allowed
ad libitum and this decreased milk production by a magnitude corresponding to the
lower intake of ME.
DOI: 10.3168/jds.S0022-0302(04)73252-X
2197. Microbiology. 2004 Jul;150(Pt 7):2335-45.
FixJ-regulated genes evolved through promoter duplication in Sinorhizobium

meliloti.
Ferrières L(1), Francez-Charlot A, Gouzy J, Rouillé S, Kahn D.
Author information:
(1)Laboratoire des Interactions Plantes-Microorganismes, UMR 2594 INRA-CNRS,
Chemin de Borde-Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France.
The FixLJ two-component system of Sinorhizobium meliloti is a global regulator,

turning on nitrogen-fixation genes in microaerobiosis. Up to now, nifA and fixK
were the only genes known to be directly regulated by FixJ. We used a genomic
SELEX approach in order to isolate new FixJ targets in the genome. This led to
the identification of 22 FixJ binding sites, including the known sites in the
fixK1 and fixK2 promoters. FixJ binding sites are unevenly distributed among the
three replicons constituting the S. meliloti genome: a majority are carried
either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus
FixJ binding sites appear to be preferentially associated with the pSymA
replicon, which carries the fixJ gene. Functional analysis of FixJ targets led to
the discovery of two new FixJ-regulated genes, smc03253 and proB2. This
FixJ-dependent regulation appears to be mediated by a duplication of the whole
fixK promoter region, including the beginning of the fixK gene. Similar
duplications were previously reported for the nifH promoter. By systematic
comparison of all promoter regions we found 17 such duplications throughout the
genome, indicating that promoter duplication is a common mechanism for the
evolution of regulatory pathways in S. meliloti.
DOI: 10.1099/mic.0.27081-0
2198. Indian J Exp Biol. 2003 Oct;41(10):1198-204.
Isolation and symbiotic characterization of transposon Tn5-induced arginine

auxotrophs of Sinorhizobium meliloti.
Kumar A(1), Vij N, Randhawa GS.
Author information:
(1)Department of Biotechnology, Indian Institute of Technology, Roorkee 247 667,
India.
Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were

isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9.
Based on intermediate feeding studies, these mutants were designated as
argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH
mutants. The ornithine auxotrophs induced ineffective nodules whereas all other
arginine auxotrophs induced fully effective nodules on alfalfa plants. In
comparison to the parental strain induced nodule, only a few nodule cells
infected with rhizobia were seen in the nitrogen fixation zone of the nodule
induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the
nitrogen fixation zone of ornithine auxotroph induced nodule were mostly
spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone
of the parental strain induced nodule. These results indicate that ornithine or
an intermediate of ornithine biosynthesis, or a chemical factor derived from one
of these compounds is required for the normal development of nitrogen fixation
zone and transformation of rhizobial bacteria into bacteroids during symbiosis of
S. meliloti with alfalfa plants.
2199. Plant Physiol. 2004 Jul;135(3):1583-94. Epub 2004 Jul 2.
Proline betaine accumulation and metabolism in alfalfa plants under sodium

chloride stress. Exploring its compartmentalization in nodules.
Trinchant JC(1), Boscari A, Spennato G, Van de Sype G, Le Rudulier D.
Author information:
(1)Laboratoire de Biologie Végétale et Microbiologie, Centre National de la
Recherche Scientifique, Formation de Recherche en Evolution 2294, Université de
Nice-Sophia Antipolis, Faculté des Sciences, Parc Valrose, F-06108 Nice cedex 2,
France.
The osmoprotectant Pro betaine is the main betaine identified in alfalfa

(Medicago sativa). We have investigated the long-term responses of nodulated
alfalfa plants to salt stress, with a particular interest for Pro betaine
accumulation, compartmentalization, and metabolism. Exposure of 3-week-old
nodulated alfalfa plants to 0.2 m NaCl for 4 weeks was followed by a 10-, 4-, and
8-fold increase in Pro betaine in shoots, roots, and nodules, respectively.
Isotope-labeling studies in alfalfa shoots indicate that [14C]Pro betaine was
synthesized from l-[14C]Pro. [14C]Pro betaine was efficiently catabolized through
sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect
on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover.
Analysis of Pro betaine and Pro compartmentalization within nodules revealed that
4 weeks of salinization of the host plants induced a strong increase in cytosol
and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed
bacteroids reached 7.4 and 11.8 mm, respectively, compared to only 0.8 mm in
control bacteroids. Na+ content in nodule compartments was also enhanced under
salinization, leading to a concentration of 14.7 mm in bacteroids. [14C]Pro
betaine and [14C]Pro were taken up by purified symbiosomes and free bacteroids.
There was no indication of saturable carrier(s), and the rate of uptake was
moderately enhanced by salinization. Ultrastructural analysis showed a large
peribacteroid space in salt-stressed nodules, suggesting an increased turgor
pressure inside the symbiosomes, which might partially be due to an elevated
concentration in Pro, Pro betaine, and Na+ in this compartment.
DOI: 10.1104/pp.103.037556
PMCID: PMC519073
2200. Chemosphere. 2004 Aug;56(7):653-8.
Preliminary ecotoxicological characterization of a new energetic substance,

CL-20.
Gong P(1), Sunahara GI, Rocheleau S, Dodard SG, Robidoux PY, Hawari J.
Author information:
Royalmount Avenue, Montreal, Que., Canada.
A new energetic substance hexanitrohexaazaisowurtzitane (or CL-20) was tested for

its toxicities to various ecological receptors. CL-20 (epsilon-polymorph) was
amended to soil or deionized water to construct concentration gradients. Results
of Microtox (15-min contact) and 96-h algae growth inhibition tests indicate that
CL-20 showed no adverse effects on the bioluminescence of marine bacteria Vibrio
fischeri and the cell density of freshwater green algae Selenastrum capricornutum
respectively, up to its water solubility (ca. 3.6 mg l(-1)). CL-20 and its
possible biotransformation products did not inhibit seed germination and early
seedling (16-19 d) growth of alfalfa (Medicago sativa) and perennial ryegrass
(Lolium perenne) up to 10,000 mg kg(-1) in a Sassafras sandy loam soil (SSL).
Indigenous soil microorganisms in SSL and a garden soil were exposed to CL-20 for
one or two weeks before dehydrogenase activity (DHA) or potential nitrification
activity (PNA) were assayed. Results indicate that up to 10,000 mg kg(-1) soil of
CL-20 had no statistically significant effects on microbial communities measured
as DHA or on the ammonium oxidizing bacteria determined as PNA in both soils.
Data indicates that CL-20 was not acutely toxic to the species or microbial
communities tested and that further studies are required to address the potential
long-term environmental impact of CL-20 and its possible degradation products.
2201. Glycoconj J. 2004;20(5):331-8.
Identification of a biologically active component in minimally oxidized low

density lipoprotein (MM-LDL) responsible for aortic smooth muscle cell
proliferation.
Chatterjee S(1), Berliner JA, Subbanagounder GG, Bhunia AK, Koh S.
Author information:
(1)Department of Pediatrics, Lipid Signaling and Vascular Biology Laboratory,
Lipid Research-Atherosclerosis Division, Johns Hopkins University School of
Medicine, Baltimore, MD 21205, USA. schatte2@jhmi.edu
Although low concentrations (10 microg/ml) of oxidized LDL density lipoproteins

(Ox-LDL) and minimally modified LDL (MM-LDL) can stimulate the proliferation of
aortic smooth muscle cells the biologically active component responsible for this
phenomena has not been identified. Here we report that the
1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-4-phosphocholine (m/e594.3) (POVPC)
present in MM-LDL but not 1-palmitoyl-2-glutaryl-sn-glycero-3-phophochline
(m/e610.2)(PGPC) can stimulate the activity of UDP-galactose:glucosylceramide
(beta 1-->4) galactosyltransferase (GalT-2) and produce lactosyceramide (LacCer).
LacCer, in turn, generated superoxide radicals (O(2)(.-)). This is accompanied by
the phosphorylation/activation of a cytosolic transcriptional factor p(44) MAPK
and the subsequent proliferation of human aortic smooth muscle cells.
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor
of GalT-2, impaired the induction of GalT-2 activity, O(2)(.-)generation, and
cell proliferation. Thus POVPC may serve as a surrogate in MM-LDL mediated
induction of aortic smooth muscle cells (A-SMC) proliferation via GalT-2
activation. The LacCer produced as a consequence of GalT-2 activation may serve
as a lipid second messenger in the activation of an oxidant sensitive
transcriptional pahtway that ultimately leads to cell proliferation and may
contribute to the pathophysiology of atherosclerosis.
DOI: 10.1023/B:GLYC.0000033629.54962.68
2202. Mol Genet Genomics. 2004 Aug;272(1):57-66. Epub 2004 Jun 19.
A Rab-related small GTP binding protein is predominantly expressed in root

nodules of Medicago sativa.
Schiene K(1), Donath S, Brecht M, Pühler A, Niehaus K.
Author information:
(1)Biologie-Genetik, Universität Bielefeld, Postfach 100 131, 33501 Bielefeld,
Germany.
Rab-related small GTP-binding proteins are known to be involved in the regulation

of the vesicular transport system in eucaryotic cells. In this paper we report
the isolation of the cDNA clone MS- rab11f from Medicago sativa (alfalfa) root
nodules using a combination of RT-PCR and SSCP analysis. MS- rab11f shows high
homology to the Rab-related cDNA clone LJ- rab11f from Lotus japonicus root
nodules. The MS-Rab11F protein expressed in Escherichia coli was found to bind
GTP, confirming that the isolated cDNA indeed codes for a small GTP-binding
protein. Expression analysis by RT-PCR demonstrated that MS- rab11f is
preferentially expressed in root nodules of alfalfa. Using the cDNA-sequence of
MS-rab11f, a peptide-specific antibody was generated. Western blot analysis with
this antibody revealed that two Rab11F isoforms, designated MS-Rab11FA and
MS-Rab11FB, are found in M. sativa root nodules.
DOI: 10.1007/s00438-004-1029-1
2203. J Gen Virol. 2004 Jul;85(Pt 7):1825-32.
Protective lactogenic immunity conferred by an edible peptide vaccine to bovine

rotavirus produced in transgenic plants.
Wigdorovitz A(1), Mozgovoj M, Santos MJ, Parreño V, Gómez C, Pérez-Filgueira DM,

Trono KG, Ríos RD, Franzone PM, Fernández F, Carrillo C, Babiuk LA, Escribano JM,
Borca MV.
Author information:
(1)Instituto de Virología, CICV, INTA-Castelar, CC77, Morón 1708, Buenos Aires,
Argentina.
Vaccines produced in transgenic plants constitute a promising alternative to

conventional immunogens, presenting the possibility of stimulating secretory and
systemic immunity against enteric pathogens when administered orally. Protection
against enteric pathogens affecting newborn animals requires, in most cases, the
stimulation of lactogenic immunity. Here, the group presents the development of
an experimental immunogen based on expression of an immunorelevant peptide,
eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as
harbouring at least one neutralizing epitope as well as being responsible for the
adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently
expressed in transgenic alfalfa as a translational fusion protein with the highly
stable reporter enzyme beta-glucuronidase (betaGUS), which served as a carrier,
stabilized the synthesized peptide and facilitated screening for the higher
expression levels in plants. Correlation of expression of the eBRV4 epitope in
plants with those presenting the highest betaGUS activities was confirmed by a
Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in
plants was effective in inducing an anti-rotavirus antibody response in adult
female mice when administered either intraperitoneally or orally and, more
importantly, suckling mice born from immunized female mice were protected against
oral challenge with virulent rotavirus. These results demonstrate the feasibility
of inducing lactogenic immunity against an enteric pathogen using an edible
vaccine produced in transgenic plants.
DOI: 10.1099/vir.0.19659-0
2204. J Anim Sci. 2004 Jun;82(6):1781-7.
Pancreatic exocrine secretion and plasma concentration of some gastrointestinal

hormones in response to abomasal infusion of starch hydrolyzate and/or casein.
Swanson KC(1), Benson JA, Matthews JC, Harmon DL.
Author information:
(1)Department of Animal Sciences, University of Kentucky, Lexington 40546-0215,
USA.
Eight Angus steers (290 +/- 8 kg), surgically prepared with pancreatic
pouch-duodenal reentrant cannulas and abomasal infusion catheters were used in a
replicated 4 x 4 Latin square experiment to investigate the effects of abomasal
infusion of starch hydrolyzate (SH) and/or casein on pancreatic exocrine
secretion and plasma concentration of hormones. Steers were fed a basal diet of
alfalfa (1.2 x NEm) in 12 equal portions daily. Abomasal infusion treatments (6-L
total volume infused per day) were water (control), SH [2.7 g/(kg BW x d)],
casein [0.6 g/(kg BW x d)], and SH + casein. Periods were 3 d for adaptation and
8 d of full infusion. Pancreatic juice and jugular blood samples were collected
over 30-min intervals for 6 h on d 11. Weight and pH of pancreatic samples were
measured, and a 10% subsample was composited and frozen until analysis of total
protein and pancreatic enzyme activities. The remaining sample was returned to
the duodenum. Plasma was harvested and frozen until analyzed. Pancreatic juice
(67 mL/h) and protein (1.8 g/h) secretion rates were not affected by nutrient
infusion. There were SH x casein interactions for all pancreatic enzyme
secretions (U/h; alpha-amylase, P < 0.03; trypsin, P < 0.08; and chymotrypsin, P
< 0.03) and plasma insulin concentration (P < 0.10). Secretion of pancreatic
enzymes was increased by SH (trypsin) and casein (alpha-amylase, trypsin, and
chymotrypsin) but not when SH + casein were infused together. Glucose (P < 0.10)
and cholecystokinin octapeptide concentrations (CCK-8; P < 0.05) were increased
by SH, but glucagon was decreased (P < 0.10). Casein decreased (P < 0.10) plasma
CCK-8 concentrations. These data indicate that positive effects of postruminal
casein on enzyme secretion were inhibited by SH, emphasizing the complexity of
the regulatory mechanisms involved in dietary adaptation of pancreatic exocrine
secretion. Changes in hormone concentration may not relate directly to changes in
enzyme secretion.
DOI: 10.2527/2004.8261781x
2205. J Dairy Sci. 2004 Mar;87(3):652-64.
Effect of alfalfa forage preservation method and particle length on performance

of dairy cows fed corn silage-based diets and tallow.
Onetti SG(1), Reynal SM, Grummer RR.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison 53706-1284, USA.
A study was conducted to evaluate the effect of including alfalfa preserved

either as silage or long-stem or chopped hay on DMI and milk fat production of
dairy cows fed corn silage-based diets with supplemental tallow (T). Fifteen
Holstein cows that averaged 117 DIM were used in a replicated 5 x 5 Latin square
design with 21-d periods. Treatments (DM basis) were: 1) 50% corn silage:50%
concentrate without T (CS); 2) 50% corn silage:50% concentrate with 2% T (CST);
3) 25% corn silage:25% short-cut alfalfa hay:50% concentrate with 2% T (SAHT); 4)
25% corn silage:25% long-cut alfalfa hay:50% concentrate with 2% T (LAHT); and 5)
25% corn silage:25% alfalfa silage:50% concentrate with 2% T (AST). Cows were
allowed ad libitum consumption of a TMR fed 4 times daily. Diets averaged 16.4%
CP and 30.3% NDF. Including 2% T in diets with corn silage as the sole forage
source decreased DMI and milk fat percentage and yield. Replacing part of corn
silage with alfalfa in diets with 2% T increased milk fat percentage and yield.
The milk fat of cows fed CST was higher in trans-10 C18:1 than that of cows fed
diets with alfalfa. No effect of alfalfa preservation method or hay particle
length was observed on DMI and milk production. The milk fat percentage and yield
were lower, and the proportion of trans-10 C18:1 in milk fat was higher for cows
fed LAHT than for cows fed SAHT. Alfalfa preservation method had no effect on
milk fat yield. Ruminal pH was higher for cows fed alfalfa in the diets, and it
was higher for cows fed LAHT than SAHT. Feeding alfalfa silage or chopped hay
appears to be more beneficial than long hay in sustaining milk fat production
when 2% T is fed with diets high in corn silage. These results support the role
of trans fatty acids in milk fat depression.
DOI: 10.3168/jds.S0022-0302(04)73208-7
2206. Planta. 2004 Sep;219(5):807-18. Epub 2004 Jun 10.
Biochemical and molecular characterization of transgenic Lotus japonicus plants

constitutively over-expressing a cytosolic glutamine synthetase gene.
Ortega JL(1), Temple SJ, Bagga S, Ghoshroy S, Sengupta-Gopalan C.
Author information:
Higher plants assimilate nitrogen in the form of ammonia through the concerted
activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS
enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To
understand how modulation of GS activity affects plant performance, Lotus
japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV
35S promoter. The transformants showed increased GS activity and an increase in
GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing
gel electrophoresis and ion-exchange chromatography. The results showed the
presence of multiple GS isoenzymes in the different organs and the presence of a
novel isoform in the transgenic plants. The distribution of GS in the different
organs was analyzed by immunohistochemical localization. GS was localized in the
mesophyll cells of the leaves and in the vasculature of the stem and roots of the
transformants. Our results consistently showed higher soluble protein
concentration, higher chlorophyll content and a higher biomass accumulation in
the transgenic plants. The total amino acid content in the leaves and stems of
the transgenic plants was 22-24% more than in the tissues of the non-transformed
plants. The relative abundance of individual amino acid was similar except for
aspartate/asparagine and proline, which were higher in the transformants.
DOI: 10.1007/s00425-004-1292-8
PMCID: PMC3881563
2207. J Biomech Eng. 2004 Apr;126(2):146-51.
Effect of plant interaction on wind-induced crop motion.
Doaré O(1), Moulia B, de Langre E.
Author information:
(1)Département de mécanique, LadHyX, CNRS-Ecole Polytechnique, 91128, Palaiseau,
France.
Plant motion due to wind affects plant growth, a phenomenon called

thigmomorphogenesis. Despite intensive studies of the turbulence over plant
canopies, the study of plant motion induced by wind has often been limited to
individual trees or cereal plants. Few models of global canopy motions are
available. Moreover the numerical analysis of models that are based on individual
stems becomes time consuming when dealing with crops. A model of motion within
the canopies is proposed here using a wave propagation equation within a
homogenized continuous medium, and a forcing function representing turbulent
gusts advected over the canopy. This model is derived from a discrete model of a
set of plant shoots represented as individual oscillators, including elastic
contacts between shoots. Such contacts induce nonlinearities into the wave
equation. A new experimental method to measure stem dynamical properties and
elastic collision properties is presented with an illustration on alfalfa stems.
Results obtained modeling plant motions in an alfalfa crop are presented.
2208. Planta. 2004 Oct;219(6):925-35. Epub 2004 Jun 4.
The MsPRP2 promoter enables strong heterologous gene expression in a

root-specific manner and is enhanced by overexpression of Alfin 1.
Winicov I(1), Valliyodan B, Xue L, Hoober JK.
Author information:
(1)Department of Plant Biology, Arizona State University, Main Campus PO Box
871601, Tempe, AZ 85287-01601, USA. winicov@asu.edu
Promoter specificity and efficiency of utilization are essential for endogenous

and transgene expression. Selective root expression remains to be defined in
terms of both promoter elements and transcription factors that provide high
levels of ubiquitous expression. We characterized expression from the MsPRP2
promoter with the green fluorescent protein (GFP) reporter transgene in alfalfa
(Medicago sativa) and found that a promoter fragment (+1 to -652 bp) retained the
root and callus specificity of the endogenous MsPRP2 gene and hence this promoter
fragment contains elements necessary for root-specific expression. The strong
ubiquitous expression obtained from this promoter was comparable to that of the
CaMV 35S promoter in roots and was enhanced by transgenic overexpression of Alfin
1, a root- and callus-specific transcription factor in alfalfa. No transgenic
expression was obtained in leaves with this promoter in the presence or absence
of Alfin 1. The increased expression of GFP in alfalfa containing the Alfin 1
transgene confirms the function of Alfin 1 binding sites in the MsPRP2 promoter
fragment and also indicates that Alfin 1 concentrations are limiting for maximal
expression in calli and roots. These findings characterize the MsPRP2 promoter as
a novel root- and callus-specific promoter of plant origin that can be used as an
effective tool for strong root-directed gene expression. In addition, we have
demonstrated that the signal sequence of MsPRP2 can be used for efficient
secretion of transgene products from callus and roots.
DOI: 10.1007/s00425-004-1296-4
2209. Genetika. 2004 Apr;40(4):454-61.
[Instability of a cryptic plasmid in Sinorhizobium meliloti P108 during symbiosis

of it with alfalfa Medicago sativa].
Rumiantseva ML(1), Andronov EE, Sagulenko VV, Onishuk OP, Provorov NA, Simarov
BV.
Author information:
(1)All-Russia Research Institute of Agricultural Microbiology, St. Petersburg,
Pushkin, 196608 Russia. mroumiantseva@yandex.ru
Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains

SKhM1-188, DM7-R, and P108 as well as in their clones isolated from nodules of
alfalfa grown during a long-term microvegetation experiment (120 days) was
studied. The isolated clones of strains SKhM1-188 and DM7-R manifested stable
inheritance of plasmids, whereas 12.7-14.0% of clones with changed plasmid
profile were detected in a population of clones from strain P108. These
segregants were designated as P108c. Segregants P108c exhibited significantly
decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness
with respect to alfalfa, compared to the original strain P108. It was established
that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230
kb) of segregants P108c. It was concluded that genetic rearrangements are
possible in rhizobial clones that did not undergo structural transformation and
retained viability in the nodule during the natural vegetation period of alfalfa.
2210. Sci Total Environ. 2004 Jul 5;327(1-3):31-9.
Removal of azinphos methyl by alfalfa plants (Medicago sativa L.) in a soil-free

system.
Flocco CG(1), Carranza MP, Carvajal LG, Loewy RM, Pechén de D'Angelo AM,
Giulietti AM.
Author information:
(1)Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires, Junín 956, 6th Floor, C1113AAD Buenos
Aires, Argentina. flocco@ffyb.uba.ar
The objective of this study was to investigate the removal of azinphos methyl
assisted by alfalfa plants, with special emphasis on the effects of this compound
on some plant's physiological parameters. Hydroponic cultures of alfalfa
(Medicago sativa L., var Romagnola) were employed as a model system. These
cultures were exposed to a nutrient medium containing 10 mg/l of azinphos methyl.
A first-order kinetic approach was used to describe the removal of azinphos
methyl from the solution. After 20 days of culture, the initial amount of
azinphos methyl was reduced to non-detectable levels in the presence of plants.
In the absence of plants, 20% of azinphos methyl remained in the solution after
30 days of treatment. The half-life of the pesticide was reduced from 10.8 to 3.4
days in the presence of plants. The growth index of alfalfa plants exposed to
azinphos methyl was negatively affected. Chlorophyll contents were reduced after
24 h of treatment and thereafter the levels were comparable to that of control
plants. The peroxidase activity of alfalfa roots was not affected by the presence
of azinphos methyl. In conclusion, alfalfa plants were able to survive when
exposed to an effective concentration of 10 mg/l of azinphos methyl in the root
zone, with some alterations on their physiological parameters.
DOI: 10.1016/j.scitotenv.2003.08.024
2211. FEBS Lett. 2004 Jun 1;567(1):152-7.
Endoreduplication and activation of the anaphase-promoting complex during

symbiotic cell development.
Kondorosi E(1), Kondorosi A.
Author information:
(1)Institut des Sciences du Végétal, CNRS UPR 2355, Avenue de la Terrasse, 91198
Gif-sur- Yvette, France.
Postembryonic development of plant organs requires a constant interplay between

the cell cycle and the developmental programs. Upon endo- and exogenous signals,
plant cells can enter, exit or modify the cell cycle. Alteration of mitotic
cycles to endoreduplication cycles, where the genome is duplicated without
mitosis, is common in plants and may play a role in cell differentiation. The
switch from the mitotic to endocycles is regulated by Ccs52A, a plant orthologue
of the yeast and animal Cdhl proteins, acting as substrate-specific activator of
the anaphase-promoting complex E3 ubiquitin ligase. Here, several aspects of
endoreduplication are discussed with special attention on nitrogen-fixing nodule
development where endoreduplication is an integral part of symbiotic cell
differentiation.
DOI: 10.1016/j.febslet.2004.04.075
2212. Int J Food Microbiol. 2004 Jun 15;93(3):267-79.
Characterization of an E2-type colicin and its application to treat alfalfa seeds

to reduce Escherichia coli O157:H7.
Nandiwada LS(1), Schamberger GP, Schafer HW, Diez-Gonzalez F.
Author information:
(1)Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles
Avenue, St. Paul, MN 55108, USA.
Several outbreaks of Escherichia coli O157:H7 infections have been associated

with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was
capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was
mediated by the production of a colicin named Hu194. The objectives of this study
were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E.
coli O157:H7 strains, and to characterize this antimicrobial protein. Significant
reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains
43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and
after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU
g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a
concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains
43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve
as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon
were located on a 6 kb plasmid, and the sequence analysis revealed that this
colicin was an E-type DNAse. From the sequence data, the estimated molecular
masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0
and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with
other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster.
However, cross-immunity tests between E-group colicins suggested that Hu194
colicin was divergent from the previously characterized E2 colicins.
2213. Bioresour Technol. 2004 Sep;94(2):211-4.
Allelopathic potential of Citrus junos fruit waste from food processing industry.
Kato-Noguchi H(1), Tanaka Y.
Author information:
(1)Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa
University, Miki, Kagawa 761-0795, Japan. hisashi@ag.kagawa-u.ac.jp
The allelopathic potential of Citrus junos fruit waste after juice extraction was
investigated. Aqueous methanol extracts of peel, inside and seeds separated from
the fruit waste inhibited the growth of the roots and shoots of alfalfa (Medicago
sativa L.), cress (Lepidium sativum L.), crabgrass (Digitaria sanguinalis L.),
lettuce (Lactuca sativa L.), timothy (Pheleum pratense L.), and ryegrass (Lolium
multiflorum Lam.). The inhibitory activity of the peel extract was greatest and
followed by that of the inside and seed extracts in all bioassays. Significant
reductions in the root and shoot growth were observed as the extract
concentration was increased. The concentrations of abscisic
acid-beta-d-glucopyranosyl ester (ABA-GE) in peel, inside and seeds separated
from the C. junos fruit waste were determined, since ABA-GE was found to be one
of the main growth inhibitors in C. junos fruit. The concentration was greatest
in the peel, followed by the inside and seeds; there was a good correspondence
between these concentrations and the inhibitory activities of the extracts. This
suggests that ABA-GE may also be involved in the growth inhibitory effect of C.
junos waste. These results suggested that C. junos waste may possess allelopathic
potential, and the waste may be potentially useful for weed management.
Copyright 2004 Elsevier Ltd.
DOI: 10.1016/j.biortech.2003.12.007
Ovarian development and ovipositional preference of the western corn rootworm

(Coleoptera: Chrysomelidae) variant in east central Illinois.
Rondon SI(1), Gray ME.
Author information:
(1)Department of Crop Sciences, University of Illinois at Urbana-Champaign, 101
Turner Hall, 1102 South Goodwin Avenue, Urbana, IL 61801, USA.
The rotation of maize, Zea mays L., and soybean, Glycine max (L.) Merr., has been
the traditional cultural tactic to manage the western corn rootworm, Diabrotica
virgifera virgifera LeConte, in the Corn Belt. The reduced effectiveness of this
rotation as a pest management tool in east central Illinois, northern Indiana,
and southern Michigan can be explained by the shift in the ovipositional behavior
of the new variant of western corn rootworm. The objective of this study was to
evaluate the influence of maize, soybean, oat, Avena sativa L., stubble, and
alfalfa, Medicago sativa L., on the ovarian development and ovipositional
preferences of the variant western corn rootworm. Field research was conducted
near Urbana, IL, during 1998-2000. Gravid females were present throughout the
season in all crops, and due to the prolonged period in which western corn
rootworm females can lay eggs, none of the crops were immune from oviposition.
Results indicated that the western corn rootworm variant oviposits in maize,
soybean, oat stubble, and alfalfa In 1998 and 1999, maize was the preferred
oviposition site among crops; however, in 2000, maize, soybean, and oat stubble
treatments had similar densities of western corn rootworm eggs. Lack of
oviposition preference of the western corn rootworm variant demonstrated in this
experiment represents a reasonable explanation of why the effectiveness of the
rotation strategy to control western corn rootworm has diminished.
2215. J Food Prot. 2004 May;67(5):947-51.
Isolation, identification, and selection of lactic acid bacteria from alfalfa

sprouts for competitive inhibition of foodborne pathogens.
Wilderdyke MR(1), Smith DA, Brashears MM.
Author information:
(1)Department of Food Science and Technology, University of Nebraska, Lincoln,
Nebraska 68583-0919, USA.
Several studies have investigated the control of pathogens on alfalfa sprouts,

and some treatments have been shown to be effective in reducing pathogen
populations. However, control methods investigated thus far only provide pathogen
control at a given point in the sprouting process and can affect germination.
Competitive inhibition of pathogens with lactic acid bacteria might provide
pathogen control throughout the sprouting process and up to consumption. The
purpose of this study was to isolate and identify lactic acid bacteria from
alfalfa sprouts to inhibit the growth of foodborne pathogens. Fifty-eight lactic
acid bacteria isolates were obtained from alfalfa seeds and sprouts. These
isolates were evaluated for inhibitory action against Salmonella enterica,
Escherichia coli O157:H7, and Listeria monocytogenes by agar spot tests. All
pathogens were inhibited by 32 (55%) of the isolates, S. enterica by 56 (97%), E.
coli O157:H7 by 49 (84%), and L. monocytogenes by 41 (71%). The isolates were
identified by the Analytical Profile Index evaluation of carbohydrate
utilization. Isolates obtained from a sample of alfalfa seeds and identified as
Lactococcus lactis subsp. lactis showed zones of inhibition of 4.0 mm or greater
for all pathogens. One of these isolates, Lactococcus lactis subsp. lactis (L7),
and an isolate previously obtained, Pediococcus acidilactici (D3), were evaluated
for competitive inhibition of S. enterica, E. coli O157:H7, and L. monocytogenes
in deMan Rogosa Sharpe agar and broth. Pathogen populations were significantly
reduced by day 5. The selected isolates will be further evaluated in future
studies for inhibitory action toward S. enterica, E. coli O157:H7, and L.
monocytogenes during sprouting.
2216. J Bacteriol. 2004 Jun;186(11):3609-20.
The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II),

transcription of the sitABCD operon, encoding a metal-type transporter.
Chao TC(1), Becker A, Buhrmester J, Pühler A, Weidner S.
Author information:
(1)Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501
Bielefeld, Germany.
Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing

nodules on roots of specific legumes. In order to propagate in the soil and for
successful symbiotic interaction the bacterium needs to sequester metals like
iron and manganese from its environment. The metal uptake has to be in turn
tightly regulated to avoid toxic effects. In this report we describe the
characterization of a chromosomal region of S. meliloti encoding the sitABCD
operon and the putative regulatory fur gene. It is generally assumed that the
sitABCD operon encodes a metal-type transporter and that the fur gene is involved
in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was
found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA
promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately
by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the
fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated
when compared to the wild-type strain. Among the up-regulated genes only the
sitABCD operon could be associated with metal uptake. On the other hand, the
complete rhbABCDEF operon, which is involved in siderophore synthesis, was
identified among the down-regulated genes. Thus, in S. meliloti Fur is not a
global repressor of iron uptake. Under symbiotic conditions the sitA promoter was
strongly expressed and the S. meliloti sitA mutant exhibited an attenuated
nitrogen fixation activity resulting in a decreased fresh weight of the host
plant Medicago sativa.
DOI: 10.1128/JB.186.11.3609-3620.2004
PMCID: PMC415740
2217. Poult Sci. 2004 May;83(5):810-4.
Cholesterol levels and sensory characteristics of meat from broilers consuming

moderate to high levels of alfalfa.
Ponte PI(1), Mendes I, Quaresma M, Aguiar MN, Lemos JP, Ferreira LM, Soares MA,
Alfaia CM, Prates JA, Fontes CM.
Author information:
(1)CIISA-Faculdade de Medicina Veterinária, Pó1o Universitário do Alto da Ajuda,
Rua Prof. Cid dos Santos, 1300-477 Lisbon, Portugal.
There is an urgent need in industrialized societies to develop novel products
that can lower human dietary cholesterol intake. Dehydrated alfalfa is a good
source of hypocholesterolemic compounds such as saponins. Whether consumption of
alfalfa by chickens would decrease the cholesterol content of broiler meat
remains, however, to be established. We determined meat quality characteristics,
lipid and cholesterol contents, and consumer preference of broiler meat derived
from production systems based on restricted feeding of a commercial diet combined
with provision of free-choice dehydrated alfalfa. Results demonstrated that it
was possible to produce chicken breast meat with reduced cholesterol content. In
addition, total lipids in chicken meat were significantly decreased when a higher
level of restriction was applied. Members of a 50-person consumer panel preferred
meat from animals not consuming or consuming moderate levels of alfalfa. Those
members that preferred meat from animals consuming the higher percentage of
alfalfa identified taste as the primary attribute influencing that decision.
Together the results suggest that it is possible to develop novel broiler
production systems that will produce leaner meat that is acceptable to consumers
and has a reduced cholesterol content.
DOI: 10.1093/ps/83.5.810
New recombination methods for Sinorhizobium meliloti genetics.
House BL(1), Mortimer MW, Kahn ML.
Author information:
(1)Institute of Biological Chemistry, School of Molecular Biosciences, Washington
State University, Pullman, Washington 99164-6340, USA.
The availability of bacterial genome sequences has created a need for improved
methods for sequence-based functional analysis to facilitate moving from
annotated DNA sequence to genetic materials for analyzing the roles that
postulated genes play in bacterial phenotypes. A powerful cloning method that
uses lambda integrase recombination to clone and manipulate DNA sequences has
been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium
meliloti in two ways that increase the utility of the system. Adding plasmid oriT
sequences to a set of vehicles allows the plasmids to be transferred to S.
meliloti by conjugation and also allows cloned genes to be recombined from one
plasmid to another in vivo by a pentaparental mating protocol, saving
considerable time and expense. In addition, vehicles that contain yeast Flp
recombinase target recombination sequences allow the construction of deletion
mutations where the end points of the deletions are located at the ends of the
cloned genes. Several deletions were constructed in a cluster of 60 genes on the
symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification
pathway. The mutations do not affect the ability of the bacteria to form
nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.
PMCID: PMC404432
2219. Brain Res. 2004 Jun 4;1010(1-2):55-61.
Mediation of BMP7 neuroprotection by MAPK and PKC IN rat primary cortical

cultures.
Cox S(1), Harvey BK, Sanchez JF, Wang JY, Wang Y.
Author information:
(1)Neural Protection and Regeneration, National Institute on Drug Abuse, NIH,
5500 Nathan Shock Drive, Baltimore, MD 21224, USA.
We have previously demonstrated that pretreatment with bone morphogenetic protein

7 (BMP7), a trophic factor in the TGFbeta superfamily, reduces ischemia-induced
brain infarction induced by middle cerebral artery ligation in rats. Since the
mitogen-activated protein kinase (MAPK) pathway is involved in many
TGFbeta-mediated responses, we examined the interaction of BMP7 and MAPK in
primary cultures obtained from the cerebral cortex of E16-17 rat embryos. Lactate
dehydrogenase (LDH) in the media was used as an index of cell death. BMP7 did not
alter LDH levels at low concentration (1.25 nM), but exhibited increased cellular
toxicity at higher concentration (>12.5 nM). BMP7 at the low concentration
significantly attenuated H2O2-induced increases in LDH activity and decreases in
neuronal density. Pharmacological interactions were used to examine if MAPK was
involved in this response. BMP7-induced protection was antagonized by the p42,44
MAPK kinase inhibitors PD98059 and U0125. The p38 MAPK antagonist SB203580, and
their inactive analog SB202474, also attenuated BMP7-induced protection,
suggesting that the interaction with p38 MAPK is nonspecific. Previous studies
have indicated that SB202474 has inhibitory effects on other protein kinases. We
found that the protein kinase C inhibitor chelerythrine antagonized BMP7-induced
protection against H2O2. Western blot analysis indicated that BMP7 increased
phosphorylation of p42,44 MAPK and PKC. Taken together, our data suggest that
BMP7 is neuroprotective at low concentrations in primary cortical cell culture.
The protective effects of BMP7 may involve the activation of p42,44 MAPK and PKC.
Copyright 2004 Elsevier B.V.
DOI: 10.1016/j.brainres.2004.02.068
2220. Plant Cell Rep. 2004 Jul;22(12):959-66. Epub 2004 Apr 30.
Characterization of a multifunctional methyltransferase from the orchid Vanilla

planifolia.
Pak FE(1), Gropper S, Dai WD, Havkin-Frenkel D, Belanger FC.
Author information:
(1)Department of Plant Biology and Pathology, Rutgers University, 59 Dudley Road,
New Brunswick, NJ 08901, USA.
The final enzymatic step in the synthesis of the flavor compound vanillin
(4-hydroxy-3-methoxybenzaldehyde) is believed to be methylation of
3,4-dihydroxybenzaldehyde. We have isolated and functionally characterized a cDNA
that encodes a multifunctional methyltransferase from Vanilla planifolia tissue
cultures that can catalyze the conversion of 3,4-dihydroxybenzaldehyde to
vanillin, although 3,4-dihydroxybenzaldehyde is not the preferred substrate. The
higher catalytic efficiency of the purified recombinant enzyme with the
substrates caffeoyl aldehyde and 5-OH-coniferaldehyde, and its tissue
distribution, suggest this methyltransferase may primarily function in lignin
biosynthesis. However, since the enzyme characterized here does have
3,4-dihydroxybenzaldehyde-O-methyltransferase activity, it may be useful in
engineering strategies for the synthesis of natural vanillin from alternate
sources.
DOI: 10.1007/s00299-004-0795-x
2221. FEMS Microbiol Ecol. 2004 May 1;48(2):139-48. doi:

10.1016/j.femsec.2004.01.002.
Combined use of different Gfp reporters for monitoring single-cell activities of

a genetically modified PCB degrader in the rhizosphere of alfalfa.
Boldt TS(1), Sørensen J, Karlson U, Molin S, Ramos C.
Author information:
(1)Department of Environmental Chemistry and Microbiology, National Environmental
Research Institute, Roskilde, Denmark.
Single-cell localization and activity of Pseudomonas fluorescens F113, colonizing

alfalfa roots, were monitored using fusions of the Escherichia coli rrnBP1
ribosomal promoter and gfp genes encoding green fluorescent protein (Gfp) of
different stability. The monitoring systems permitted non-destructive in situ
detection of F113rifpcb cells on the entire root system grown in both the
presence and absence of 3-chlorobiphenyl (PCB-2). The root tip and sites of
lateral root emergence were found to be hotspots for fast-growing cells. In
addition, a reporter strain of P. fluorescens F113rifpcb for degradation of
chlorinated biphenyl was constructed, using another gfp fusion with the
meta-pathway Pm promoter from Pseudomonas putida (TOL plasmid). Expression of
this promoter, which is strongly induced by the PCB-2 degradation product,
3-chlorobenzoate, was tested in vitro and subsequently monitored in vivo on
alfalfa roots using the P. fluorescens F113rifpcb reporter. A small but distinct
fraction of the introduced bacteria activated the Pm promoter and thus appeared
to sense a PCB-2 degradation product in the alfalfa rhizosphere. The degrading
cells, which by design were identical to the sensing cells, were located in
distinct microcolonies on the root surface or in intercellular crevices between
the root epidermal cells. However, PCB-degrading cells were not observed in the
root areas containing fast-growing cells, indicating that PCB degradation was not
linked to high cellular activity.
DOI: 10.1016/j.femsec.2004.01.002
2222. J Food Prot. 2004 Apr;67(4):813-7.
Effect of alfalfa seed washing on the organic carbon concentration in chlorinated

and ozonated water.
Rajkowski KT(1), Rice EW.
Author information:
(1)U.S. Department of Agriculture, Agricultural Research Service, Eastern
Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038,
USA. krajkowski@errc.ars.usda.gov
The bioassays assimilable organic carbon (AOC) and coliform growth response are
better indexes than biological oxygen demand to determine water quality and
water's ability to support the growth of bacteria. Ozonated (5 mg/liter) and
chlorinated tap water were used to wash alfalfa seeds for 30 min. After washing
in the ozonated tap water, the AOC concentration increased 25-fold, whereas the
dissolved ozone decreased to undetectable levels. The AOC levels for the
chlorinated water after washing the seeds also increased. These increases are due
to ozone's strong oxidizing ability to break down refractory,
large-molecular-weight compounds, forming smaller ones, which are readily used as
nutrient sources for microorganisms. This same phenomenon was observed when using
ozone in the treatment of drinking water. The AOC value increased from 1,176 to
1,758 micrograms C-eq/liter after the reconditioned wastewater was ozonated. When
the ozonated wastewater was inoculated with Salmonella serotypes, the cells
survived and increased generation times were observed. The increased nutrients
would now become more readily available to any pathogenic microorganisms located
on alfalfa seed surface as seen with the increase in the inoculated levels of
Salmonella in the ozonated wastewater. If the washing process using ozonated
water is not followed by the recommended hypochlorite treatment or continually
purged with ozone, pathogen growth is still possible.
2223. Genetics. 2004 Mar;166(3):1463-502.
A sequence-based genetic map of Medicago truncatula and comparison of marker

colinearity with M. sativa.
Choi HK(1), Kim D, Uhm T, Limpens E, Lim H, Mun JH, Kalo P, Penmetsa RV, Seres A,
Kulikova O, Roe BA, Bisseling T, Kiss GB, Cook DR.
Author information:
(1)Department of Plant Pathology, University of California, Davis, California
95616, USA.
A core genetic map of the legume Medicago truncatula has been established by
analyzing the segregation of 288 sequence-characterized genetic markers in an
F(2) population composed of 93 individuals. These molecular markers correspond to
141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513
cM. In the case of EST-based markers we used an intron-targeted marker strategy
with primers designed to anneal in conserved exon regions and to amplify across
intron regions. Polymorphisms were significantly more frequent in intron vs. exon
regions, thus providing an efficient mechanism to map transcribed genes. Genetic
and cytogenetic analysis produced eight well-resolved linkage groups, which have
been previously correlated with eight chromosomes by means of FISH with mapped
BAC clones. We anticipated that mapping of conserved coding regions would have
utility for comparative mapping among legumes; thus 60 of the EST-based primer
pairs were designed to amplify orthologous sequences across a range of legume
species. As an initial test of this strategy, we used primers designed against M.
truncatula exon sequences to rapidly map genes in M. sativa. The resulting
comparative map, which includes 68 bridging markers, indicates that the two
Medicago genomes are highly similar and establishes the basis for a Medicago
composite map.
PMCID: PMC1470769
2224. Phytochemistry. 2004 Apr;65(7):837-46.
O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid

3-O-methyltransferase.
Kota P(1), Guo D, Zubieta C, Noel J, Dixon RA.
Author information:
Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic

acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin
biosynthesis, the present work demonstrates that alfalfa COMT methylates
benzaldehyde derivatives more efficiently than lignin pathway intermediates.
3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in
vitro substrates for OMT activity in extracts from developing alfalfa stems, and
these compounds were preferred over lignin pathway intermediates for
3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT
activity with benzaldehydes was strongly reduced in extracts from stems of
transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation
drastically affects lignin composition, it does not appear to significantly
impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed
site-directed mutants of COMT showed altered relative substrate preferences for
lignin precursors and benzaldehyde derivatives. Taken together, these results
indicate that COMT may have more than one role in phenylpropanoid metabolism (but
probably not in alfalfa), and that engineered COMT enzymes could be useful for
metabolic engineering of both lignin and benzaldehyde-derived flavors and
fragrances.
DOI: 10.1016/j.phytochem.2004.01.017
2225. J Anim Sci. 2004 Apr;82(4):1165-9.
Balance and serum concentration of biotin in sheep fed alfalfa meal-based diets
with increasing level of concentrate.
Peterson TE(1), McDowell LR, McMahon RJ, Wilkinson NS, Rosendo O, Seymour WM,
Henry PR, Martin FG, Shearer JK.
Author information:
(1)Department of Animal Sciences, University of Florida-IFAS, Gainesville 32611,
USA.
The objective of this study was to test the hypothesis that serum biotin
concentration and biotin balance (consumed - [urinary output + fecal output])
measured as total avidin-binding substances (biotin + biotin metabolites) are
responsive to changes in the proportions of dietary alfalfa meal and concentrate
fed to sheep. Eight sheep (initial BW = 40 kg) consumed a pelleted alfalfa
meal-based diet that had 95:5, 48:52, 23:77, or 9:91% alfalfa meal:concentrate
ratios (DM basis) in a replicated 4 x 4 Latin square design with 20-d periods (10
d of acclimation, 7 d of adaptation, and a 3-d collection period with jugular
blood drawn on the last day). Replacing alfalfa meal with concentrate in the
pelleted diets decreased dietary concentrations of biotin proportionally. As the
percentage of alfalfa meal in the diet decreased, there was a linear decrease in
daily DM intake (1,128 to 901 g of DMI/d; P < 0.01), with a linear (P < 0.01) and
quadratic (P < 0.01) increase in the apparent total-tract DM digestibility of
diets (51.0 to 80.0%). The biotin consumed decreased with alfalfa meal proportion
in the diet (linear, P < 0.01). Both fecal biotin concentration (linear, P <
0.01) and fecal biotin output (quadratic, P < 0.05) increased, reaching peaks at
23% alfalfa meal. Fecal biotin output was not correlated with biotin intake, DMI,
or intake of digestible DM. Mean urinary output, urinary biotin concentration,
urinary biotin output, and serum biotin concentration were not affected by
treatments. Means of biotin balance were negative and revealed the same trends
among treatments as did fecal output. Biotin balance was a quadratic (P < 0.05)
function of decreasing alfalfa meal in the diet, with more negative values at the
alfalfa meal:concentrate ratio of 23:77. Results suggest that the greatest
synthesis of biotin in the total digestive tract occurs with diets of either 52
or 77% concentrate for sheep; however, research addressing the significance of
biotin metabolites on biotin balance and plasma biotin pool is needed.
DOI: 10.2527/2004.8241165x
2226. FEMS Microbiol Lett. 2004 Apr 15;233(2):325-31.
Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a

cellulose binding site show synergistic effect on hydrolysis of native plant
cellulose.
Bae HJ(1), Turcotte G, Soo Kim Y, Vézina LP, Laberge S.
Author information:
(1)Département de Phytologie, Université Laval, Sainte-Foy, Que., Canada G1K 7P4.
baehyeunjong@hotmail.com
Hydrolysis of cellulose requires two different types of cellulases: exo- and

endocellulase. Here, we investigated for the hydrolysis of cellulose by two types
of cellulases, an endoglucanase (Cel5) from Ruminococcus albus fused with the
xylanase A cellulose binding domain II (CBM6) of Clostridium stercorarium and
Thermobifidus fusca E3, an exoglucanase (Cel6B). Cel5-CBM6 or Cel6B showed a
linear relationship between the production of soluble sugars and the incubation
time when native alfalfa cellulose was used as a substrate. Cel5-CBM6 produces
more soluble sugars than Cel6B and the hydrolysis of cellulose by a mixture of
the two enzymes produces substantially more (22%) soluble sugars than the total
amount produced by these enzymes individually. Although Cel5-CBM6 solubilized
high quantities of sugars from alfalfa cellulose, it did not significantly
decrease its crystallinity, while Cel6B decreased the crystallinity of cellulose
by 34%. When the two cellulases were combined, a decrease of more than 50% in the
content of crystalline cellulose was observed. The enzyme-gold labeling
experiments revealed that both enzymes showed a high affinity for all substrates.
Furthermore, simultaneous visualization of the enzyme-binding sites revealed the
preferred substrates in native lignocellulosic material. When plant cellulose was
pre-incubated with Cel5-CBM6, density of the gold labeling greatly increased
suggesting that preliminary exposure of lignocellulosic material to Cel5-CBM6 may
have enhanced the accessibility of the substrate to Cel5-CBM6 and Cel6B. This
result provides a plausible explanation for the observed endo/exo cellulase
synergism during hydrolysis.
DOI: 10.1016/j.femsle.2004.02.027
2227. Plant Physiol Biochem. 2004 Jan;42(1):73-9.
Purification of ENOD8 proteins from Medicago sativa root nodules and their
characterization as esterases.
Pringle D(1), Dickstein R.
Author information:
(1)Department of Bioscience and Biotechnology, Drexel University, Philadelphia,
PA 19104, USA.
ENOD8 proteins were purified from alfalfa (Medicago sativa) root nodules. After
extraction of ENOD8 proteins into an aqueous buffer, they were purified by
ammonium sulfate precipitation, concanavalin A Sepharose chromatography, and
ion-exchange chromatography. Purification was assessed by comparing silver
stained SDS-PAGE gels to Western blots developed with a highly specific ENOD8
antibody. Multiple ENOD8 proteins that co-purified were found. ENOD8 proteins
were found to have esterase activity, active on acetyl and butyrl esters but not
longer chain aliphatic esters. Thus, ENOD8 proteins are unlikely to be lipases.
Kinetic analysis showed that ENOD8 proteins esterase activity exhibited
Michaelis-Menten kinetics. Considering ENOD8 protein sequence similarity to an
exopolygalacturonase/EP4/iEP4 and lanatoside 15'-O-acetylesterase with the
results presented here predicts that ENOD8 substrates could be acetylated oligo-
or polysaccharides.
DOI: 10.1016/j.plaphy.2003.10.004
2228. Appl Biochem Biotechnol. 2004 Spring;113-116:1167-82.
Expression of UDP-glucose dehydrogenase reduces cell-wall polysaccharide

concentration and increases xylose content in alfalfa stems.
Samac DA(1), Litterer L, Temple G, Jung HJ, Somers DA.
Author information:
(1)USDA-ARS-Plant Science Research, 495 Borlaug Hall, 1991 Upper Buford Circle,
University of Minnesota, St. Paul, MN 55108, USA. dasamac@umn.edu
The primary cell-wall matrix of most higher plants is composed of large amounts
of uronic acids, primarily D-galacturonic acid residues in the backbone of pectic
polysaccharides. Uridine diphosphate (UDP)-glucose dehydrogenase is a key enzyme
in the biosynthesis of uronic acids. We produced transgenic alfalfa (Medicago
sativa) plants expressing a soybean UDP-glucose dehydrogenase cDNA under the
control of two promoters active in alfalfa vascular tissues. In initial
greenhouse experiments, enzyme activity in transgenic lines was up to seven-fold
greater than in nontransformed control plants; however, field-grown transgenic
plants had only a maximum of 1.9-fold more activity than the control. Cell-wall
polysaccharide content was lower and Klason lignin content was higher in
transgenics compared to the nontransformed control. No significant increase in
pectin or uronic acids in the polysaccharide fraction was observed in any line.
Xylose increased 15% in most transgenic lines and mannose concentration decreased
slightly in all lines. Because of the complexity of pectic polysaccharides and
sugar biosynthesis, it may be necessary to manipulate multiple steps in
carbohydrate metabolism to alter the pectin content of alfalfa.
2229. FEMS Microbiol Ecol. 2004 Apr 1;48(1):71-7. doi:

10.1016/j.femsec.2003.12.010.
Attachment to plant roots and nod gene expression are not affected by pH or
calcium in the acid-tolerant alfalfa-nodulating bacteria Rhizobium sp. LPU83.
Soto MJ(1), Dillewijn P, Martínez-Abarca F, Jiménez-Zurdo JI, Toro N.
Author information:
Experimental del Zaidín, Granada, Spain.
Soil acidification is one of the environmental factors that more strongly hampers
the establishment of an effective symbiotic interaction between rhizobia and
leguminous plants. Sinorhizobium meliloti and the acid-tolerant Rhizobium sp.
strain LPU83 are able to nodulate alfalfa plants at pH 5.6 but both exhibit a
delayed nodulation and a reduction in the number of elicited nodules. We show
here that the addition of calcium (Ca) has no positive effect on the nodulation
kinetics shown by LPU83 at low pH, but does retrieve the competition capacity of
S. meliloti strains in acidic media, likely by improving the ability of these
bacteria to attach to plant roots. In contrast, the attachment of the
acid-tolerant strain LPU83 to alfalfa roots is not greatly affected by pH or Ca
concentration. Media acidification impairs nod gene induction in different S.
meliloti strains but not in LPU83. However, the addition of Ca at low pH does not
affect neither nod gene expression in alfalfa-nodulating rhizobia (S. meliloti or
strain LPU83) nor the quality of nod gene inducers exudated by alfalfa plants, in
contrast to what has been reported previously. These data reveal differential
features among alfalfa-nodulating rhizobia and point out the adsorption of S.
meliloti to alfalfa roots as the major limiting step affecting its symbiotic
performance in acidic conditions.
DOI: 10.1016/j.femsec.2003.12.010
2230. Plant Physiol Biochem. 2004 Mar;42(3):181-6.
Immunolocalization of carbonic anhydrase and phosphoenolpyruvate carboxylase in

developing seeds of Medicago sativa.
Aivalakis G(1), Dimou M, Flemetakis E, Plati F, Katinakis P, Drossopoulos JB.
Author information:
(1)Laboratory of Plant Physiology and Morphology, Iera Odos 75, 11855, Athens,
Greece. gaivalakis@aua.gr
To investigate the role of carbonic anhydrase (CA; EC 4.2.1.1) and

phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) during Medicago sativa seed
development, the distribution of both proteins was examined using an
immunohistological approach. Both enzymes are co-localized in most ovular and
embryonic tissues. In early stages of seed development, both proteins were
abundant in embryo and integuments, while at subsequent stages both proteins are
accumulated in endosperm, nucellus and integuments. At late stages of seed
development when both endosperm and nucellus are degraded, significant
accumulation of both proteins was observed in the embryo proper. Chlorophyll was
found to accumulate in embryos after the heart stage and reached a maximum at
mature stage. It is suggested that CA and PEPC play a role in respiratory carbon
dioxide refixation while generating malate to support amino acid and/or fatty
acids biosynthesis.
DOI: 10.1016/j.plaphy.2004.01.006
2231. Plant Cell Rep. 2004 Jul;22(12):967-73. Epub 2004 Mar 23.
Altered susceptibility to infection by Sinorhizobium meliloti and Nectria

haematococca in alfalfa roots with altered cell cycle.
Woo HH(1), Hirsch AM, Hawes MC.
Author information:
(1)Department of Molecular, Cell, and Developmental Biology, University of
California, Los Angeles 90095, USA.
Most infections of plant roots are initiated in the region of elongation; the
mechanism for this tissue-specific localization pattern is unknown. In alfalfa
expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic
virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead
of 24 h, and border cell number is decreased by more than 99%. These plants were
found to exhibit increased root-tip infection by a fungal pathogen and reduced
nodule formation by a bacterial symbiont. Thus, the frequency of infection in the
region of elongation by Nectria haematocca was unaffected, but infection of the
root tip was increased by more than 90%; early stages of Sinorhizobium meliloti
infection and nodule morphology were normal, but the frequency of nodulation was
fourfold lower than in wild-type roots.
DOI: 10.1007/s00299-004-0787-x
2232. J Food Prot. 2004 Mar;67(3):561-6.
Changes in growth and antioxidant status of alfalfa sprouts during sprouting as

affected by gamma irradiation of seeds.
Fan X(1), Thayer DW, Sokorai KJ.
Author information:
(1)Food Safety, Intervention Technologies Research Unit, U.S. Department of
Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600
East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA. xfan@arserrc.gov
Viking 3000 alfalfa seeds irradiated with gamma rays to doses of 0, 1, 2, 3, or 4

kGy were sprouted and allowed to grow for up to 8 days at 23 degrees C.
Germination, growth (yield and length), antioxidant capacity, and ascorbic acid
(AA) were measured during sprouting. Results showed percent germination of the
seeds and the rates of growth of the sprouts were inversely related to the
radiation dose absorbed by the seeds. Both antioxidant capacity and AA content
expressed on a fresh weight basis decreased during growth of the sprouts. Sprouts
grown from irradiated seeds had greater antioxidant capacity and AA content on a
fresh weight basis than those grown from nonirradiated seeds. However, when the
nutritive values were expressed on a per gram of seed basis, irradiation had no
effect on the nutritive values of sprouts.
2233. J Biol Chem. 2004 Jun 25;279(26):26959-66. Epub 2004 Mar 19.
OMTK1, a novel MAPKKK, channels oxidative stress signaling through direct MAPK
interaction.
Nakagami H(1), Kiegerl S, Hirt H.
Author information:
(1)Institute of Microbiology and Genetics, Vienna Biocenter, University of
Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria.
In common with other eukaryotes, plants utilize mitogen-activated protein kinase

(MAPK) cascades to mediate responses to a wide variety of stimuli. In contrast to
other eukaryotes, plants have an unusually large number of MAPK components, such
as more than 20 MAPKs, 10 MAPK kinases (MAPKKs), and 60 MAPKK kinases (MAPKKKs)
in Arabidopsis (MAPK Group (2002) Trends Plant Sci. 7, 301-308). Presently it is
mostly unknown how MAPK signaling specificity is generated in plants. Here we
have isolated OMTK1 (oxidative stress-activated MAP triple-kinase 1), a novel
MAPKKK from alfalfa (Medicago sativa). In plant protoplasts, OMTK1 showed basal
kinase activity and was found to induce cell death. Among a panel of hormones and
stresses tested, only H(2)O(2) was found to activate OMTK1. Out of four MAPKs,
OMTK1 specifically activated MMK3 resulting in an increased cell death rate.
Pull-down analysis between recombinant proteins indicated that OMTK1 directly
interacts with MMK3 and that OMTK1 and MMK3 are part of a protein complex in
vivo. These results indicate that OMTK1 plays a MAPK scaffolding role and
functions in activation of H(2)O(2) -induced cell death in plants.
DOI: 10.1074/jbc.M312662200
2234. Physiol Plant. 2004 Jan;120(1):113-123.
Methyl jasmonate alters N partitioning, N reserves accumulation and induces gene

expression of a 32-kDa vegetative storage protein that possesses chitinase
activity in Medicago sativa taproots.
Meuriot F(1), Noquet C, Avice JC, Volenec JJ, Cunningham SM, Sors TG, Caillot S,
Ourry A.
Author information:
(1)UMR INRA UCBN 950 Physiologie et Biochimie Végétales, Institut de Recherche en
Biologie Appliquée, Université, 14032 Caen Cedex, France Department of Agronomy,
Purdue University, West Lafayette, IN 47907-1150, USA.
This study presents the effects of methyl jasmonate (MeJA) on growth, N uptake, N
partitioning, and N storage in taproots of non-nodulated alfalfa (cv. Lodi). When
compared to untreated plants, addition of 100 micro M MeJA to the nutrient
solution for 14 days reduced total growth and modified biomass partitioning
between shoots and roots in favour of taproots and lateral roots. MeJA decreased
N uptake (after 7 days) and increased N partitioning towards roots after 14 days.
This preferential N partitioning to roots was accompanied by increased N storage
in taproots as soluble proteins. Compared to total soluble proteins, VSP
accumulation occurred earlier (7 days), and was greater (2-fold increase) in
plants treated with 100 micro M MeJA. Steady-state transcript levels for two VSPs
(32 and 57 kDa) also increased markedly (about 4-fold) in roots of plants treated
with 100 micro M MeJA. This suggests that MeJA could act directly
(transcriptional regulation) or indirectly (via the changes of N partitioning
among alfalfa organs) on N storage as soluble proteins and in particular, VSPs.
Because the deduced amino acid sequence of the 32 kDa VSP clone reveals high
homology with Class III chitinases, we propose that the 32 kDa VSP may have a
role in pathogen defense, in addition to its function as a storage protein.
DOI: 10.1111/j.0031-9317.2004.0210.x
PMID: 15032883
2235. Plant Cell. 2004 Apr;16(4):933-44. Epub 2004 Mar 18.
A nonsymbiotic root hair tip growth phenotype in NORK-mutated legumes:

implications for nodulation factor-induced signaling and formation of a
multifaceted root hair pocket for bacteria.
Esseling JJ(1), Lhuissier FG, Emons AM.
Author information:
(1)Laboratory of Plant Cell Biology, Department of Plant Sciences, Wageningen
University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.
The Medicago truncatula Does not Make Infections (DMI2) mutant is mutated in the
nodulation receptor-like kinase, NORK. Here, we report that NORK-mutated legumes
of three species show an enhanced touch response to experimental handling, which
results in a nonsymbiotic root hair phenotype. When care is taken not to induce
this response, DMI2 root hairs respond morphologically like the wild type to
nodulation factor (NF). Global NF application results in root hair deformation,
and NF spot application induces root hair reorientation or branching, depending
on the position of application. In the presence of Sinorhizobium meliloti, DMI2
root hairs make two-dimensional 180 degrees curls but do not entrap bacteria in a
three-dimensional pocket because curling stops when the root hair tip touches its
own shank. Because DMI2 does not express the promoter of M. truncatula Early
Nodulin11 (ENOD11) coupled to beta-glucuronidase upon NF application, we propose
a split in NF-induced signaling, with one branch to root hair curling and the
other to ENOD11 expression.
DOI: 10.1105/tpc.019653
PMCID: PMC412867
2236. J Biol Chem. 2004 Jun 4;279(23):23933-41. Epub 2004 Mar 15.
Isolation and characterization of the early nodule-specific protein homologue

(Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex.
Arif SA(1), Hamilton RG, Yusof F, Chew NP, Loke YH, Nimkar S, Beintema JJ, Yeang
HY.
Author information:
(1)Rubber Research Institute of Malaysia, 50450 Kuala Lumpur, Malaysia.
sitiarija@lgm.gov.my
Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis

latex appeared to have been resolved with the discovery of a 43-kDa allergenic
latex protein that was a homologue to patatin. However, the low to moderate
prevalence of sensitization to the protein, designated Hev b 7, among
latex-allergic patients could not adequately explain the frequent observations of
the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic
protein of a similar molecular mass existed in Hevea latex. We report the
isolation and purification of a 42.98-kDa latex glycoprotein showing homology to
the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago
truncatula, and Glycine max. The protein is allergenic, being recognized by
immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope
resides on the carbohydrate moiety of the protein, and the presence of a similar
carbohydrate component on potato tuber patatin enables the latter to inhibit IgE
binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated
by reverse transcription-PCR and cloned. The protein predicted from the cDNA
sequence has 391 amino acids, the first 26 of which constitute a putative signal
peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its
isoelectric point is estimated at 5.0. The discrepancy between the predicted and
observed molecular mass might be due to glycosylation, for which three N-sites on
the protein are predicted. The purified protein showed lipase and esterase
activities and may be involved in plant defense.
DOI: 10.1074/jbc.M309800200
2237. Curr Microbiol. 2004 Jan;48(1):27-31.
Effects of DL-malate on in vitro forage fiber digestion by mixed ruminal

microorganisms.
Martin SA(1).
Author information:
(1)Department of Animal and Dairy Science, 312 Rhodes Center for Animal and Dairy
Science, The University of Georgia, Athens, GA 30602-2771, USA. scottm@uga.edu
The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM

DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay
and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM
DL-malate numerically increased propionate concentration, and 12 mM DL-malate
increased (P < 0.10) propionate. All three concentrations of DL-malate decreased
(P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay
fermentations, all three DL-malate concentrations increased (P < 0.05) propionate
and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM
DL-malate numerically increased in vitro dry matter disappearance. When mixed
ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa
hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate
increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate
ratio. DL-Malate treatment had little effect on in vitro dry matter
disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus
DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate
increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate
treatment consistently increased final pH values in fermentations that included
DL-lactic acid, there was not a corresponding increase in in vitro dry matter
disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch
culture incubations.
2238. Anticancer Res. 2004 Jan-Feb;24(1):227-33.
Effects of the Medicago scutellata trypsin inhibitor (MsTI) on cisplatin-induced

cytotoxicity in human breast and cervical cancer cells.
Lanza A(1), Tava A, Catalano M, Ragona L, Singuaroli I, Robustelli della Cuna FS,
Robustelli della Cuna G.
Author information:
(1)Divisione di Oncologia Medica, Fondazione Salvatore Maugeri, via Ferrata 8,
27100 Pavia, Italy. alanza@fsm.it
BACKGROUND: Snail medic (Medicago scutellata L.) seeds exhibit a significantly

higher content of a trypsin inhibitor than other Medicago species. This inhibitor
belongs to the Bowman-Birk family of serine protease inhibitors (BBI) and
exhibits a good sequence homology with the BBI from soybean, while presenting
some differences. It has been suggested that BBIs have antitumoral and
radio-protective activity.
MATERIALS AND METHODS: In order to assess whether the inhibitor from Medicago
scutellata (MsTI) seeds show similar properties to those of BBI from soybean with
respect to potentiation of cisplatin-induced cytotoxicity, we evaluated the
effects of MsTI on cisplatin-induced cell killing in MCF7 human breast carcinoma
cells and HeLa human cervical carcinoma cells.
RESULTS: The 24-hour treatment of MsTI in the cell culture medium decreased the
clonogenic survival of MCF7 and HeLa cells in a dose-dependent manner and
enhanced cisplatin-induced cytotoxicity. The presence of MsTI during the entire
incubation period reduced the D37 of cisplatin by 40% in both the cell lines.
CONCLUSION: MsTI could be an useful agent for the potentiation of
cisplatin-mediated cancer treatment.
2239. Mol Genet Genomics. 2004 May;271(4):416-25. Epub 2004 Mar 6.
Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis

with alfalfa.
Mitsui H(1), Sato T, Sato Y, Ito N, Minamisawa K.
Author information:
(1)Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku,
980-8577 Sendai, Japan. hmitsui@ige.tohoku.ac.jp
Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium. An S.

meliloti strain that is mutant for the rpoH(1) gene, which encodes a
sigma(32)-like protein, elicits the formation of ineffective nodules on the host
plant alfalfa. We characterized the rpoH(1) mutant for phenotypes related to
symbiosis. Alfalfa nodules formed by the rpoH(1) mutant exhibited greatly reduced
levels of acetylene reduction activity compared to the wild-type nodules. Whereas
intracellular colonization by rhizobia was observed in a zone just below the
apical meristem, we found ultrastructural abnormalities and signs of degeneration
of bacteroids within many host cells in the proximally adjacent zone. In the
proximal part of the nodule, only a few nodule cells contained bacteroids. In
contrast, the rpoH(1) mutant showed normal induction of nitrogen fixation gene
expression in microaerobic culture. These results suggest that the rpoH(1)
mutation causes early senescence of bacteroids during the endosymbiotic process,
but does not affect the invasion process or the synthesis of the nitrogenase
machinery. The rpoH(1) mutant exhibited increased sensitivity to various agents
and to acid pH, suggesting that RpoH(1) is required to protect the bacterial cell
against environmental stresses encountered within the host. Since RpoH(1) was
previously reported to be required for the synthesis of some heat shock proteins
(Hsps), we examined the transcription of several genes for Hsp homologs. We found
that transcription of groESL(5), lon, and clpB after heat shock was
RpoH(1)-dependent, and conserved nucleotide sequences were found in the -35 and
-10 regions upstream of the transcription start sites of these genes. Although
groESL(5) expression is almost completely dependent on RpoH(1), we found that a
groESL(5) mutant strain is still capable of normal symbiotic nitrogen fixation on
alfalfa.
DOI: 10.1007/s00438-004-0992-x
2240. Mol Plant Microbe Interact. 2004 Mar;17(3):235-44.
The typA gene is required for stress adaptation as well as for symbiosis of
Sinorhizobium meliloti 1021 with certain Medicago truncatula lines.
Kiss E(1), Huguet T, Poinsot V, Batut J.
Author information:
(1)Laboratoire des Interactions Plantes Microorganismes, Centre National de la
Recherche Scientifique (UMR 2594), Institut National de la Recherche Agronomique
(UMR 441), BP27, 31326 Castanet, Tolosan cedex, France.
In this article, we describe the typA gene of Sinorhizobium meliloti, the

orthologue of typA/bipA genes found in a wide range of bacteria. We found that
typA was required for survival of S. meliloti under certain stress conditions,
such as growth at low temperature or low pH and in the presence of sodium dodecyl
sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S.
meliloti typA mutants were cross-complemented, indicating that the two genes are
functionally equivalent. typA was indispensable for symbiosis on Medicago
truncatula Jemalong and F83005.5 and contributes to the full efficiency of
symbiosis on other host plant lines such as DZA315.16 or several cultivars of M.
sativa. Hence, the symbiotic requirement for typA is host dependent.
Interestingly, the symbiotic defect was different on Jemalong and F83005.5
plants, thus indicating that typA is required at a different stage of the
symbiotic interaction.
DOI: 10.1094/MPMI.2004.17.3.235
Triterpene saponins from aerial parts of Medicago arabica L.
Bialy Z(1), Jurzysta M, Mella M, Tava A.
Author information:
Czartoryskich 8, 24-100 Pulawy, Poland.
Eight major triterpene saponins have been isolated from the aerial parts of
Medicago arabica and their structures elucidated by FAB-MS and NMR analysis.
Three of them are new compounds and are identified as
3-O-(alpha-L-arabinopyranoside) bayogenin, 3-O-(alpha-L-arabinopyranosyl),
28-O-(beta-D-glucopyranoside) bayogenin, and
3-O-[alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl],
28-O-beta-D-glucopyranoside 2-beta-hydroxyoleanolic acid. Two saponins,
identified as 3-O-(alpha-L-arabinopyranoside) hederagenin and
3-O-(alpha-L-arabinopyranosyl), 28-O-(beta-D-glucopyranoside) hederagenin are
known compounds but not previously reported as saponin constituents of Medicago
species, while three other saponins, being mono- and bidesmosides of hederagenin,
have been previously isolated from roots of M. sativa.
DOI: 10.1021/jf030446+
2242. Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2730-5. Epub 2004 Feb 20.
Transition state stabilization by general acid catalysis, water expulsion, and

enzyme reorganization in Medicago savita chalcone isomerase.
Hur S(1), Newby ZE, Bruice TC.
Author information:
(1)Department of Chemistry and Biochemistry, University of California, Santa
Barbara, CA 93106, USA.
In aqueous solution, Medicago savita chalcone isomerase (CHI) enhances the

reaction rate for the unimolecular rearrangement of chalcone (CHN) into flavanone
by seven orders of magnitude. Conformations of CHN and their relative free
energies in water and CHI were investigated by the thermodynamic perturbation
method. In water, CHN adopts two conformations (I and II) with conformation I
being higher in energy than conformation II by 3 kcal/mol. Only I can give rise
to a near attack conformer (NAC) where the nucleophile O2' and the electrophile
C9 are placed in proximity. In CHI, I binds less tightly than II by approximately
2 kcal/mol, resulting in the free energy for NAC formation being approximately 2
kcal/mol higher in the enzyme than in water. This unfavorable feature in the
ground state of the CHI reaction requires the predominant catalytic advantage to
be taken in the step of NAC --> transition state (TS). From the molecular
dynamics simulations of apo-CHI, CHI complexed with CHN (CHI.CHN) and CHI.TS, we
found: (i) Lys-97-general-acid catalysis of the O2'(-) nucleophilic addition;
(ii) expulsion of three water molecules in the process of TS formation; (iii)
release of enzyme structural distortion on TS formation. In the conclusion, CHI's
remarkable efficiency of stabilizing the TS and its relatively poor ability in
organizing the ground state is compared with chorismate mutase whose catalytic
prowess, when compared with water, originates predominantly from the enhanced NAC
population at the active site.
DOI: 10.1073/pnas.0308264100
PMCID: PMC365689
Phenology of Lygus lineolaris (Hemiptera: Miridae) in Minnesota June-bearing

strawberries: comparison of sampling methods and habitats.
Wold SJ(1), Hutchison WD.
Author information:
(1)Department of Entomology, University of Minnesota, 219 Hodson Hall, 1980
Folwell Ave., St. Paul, MN 55108-6125, USA.
Field studies were conducted in southeastern Minnesota from 2000 to 2002 to

determine the phenology of Lygus lineolaris in various habitats and to compare
yellow and white sticky traps as a sampling method for adult L. lineolaris.
Strawberry fields were sampled for L. lineolaris adults using yellow sticky
traps, and nymphs were sampled using the standard white pan beat method. Adult L.
lineolaris abundance in alfalfa, an adjacent fence-row, and a wooded habitat were
also compared. The nonlinear relationship between cumulative trap catch and
cumulative degree-days was modeled with a two-parameter cumulative Weibull
function to predict early-season adult capture using yellow sticky traps. Adult
L. lineolaris were detected in bearing-year strawberries at the onset of
vegetative growth in all years. Yellow sticky traps caught significantly higher
densities of adult L. lineolaris than white sticky traps. The Weibull model
predicted 50% capture at 10 DD (>12.4 degrees C), which corresponds to the
vegetative strawberry growth stage. L. lineolaris nymphs were not detected until
the blossom stage. Alfalfa harbored significantly higher densities of L.
lineolaris than other habitats during early-season sampling (i.e., March-June).
Late-season sampling (July-September) revealed significantly higher densities in
bearing-year strawberries. These results suggest that monitoring at the onset of
vegetative growth, using yellow sticky traps, will be an efficient method for
detecting early L. lineolaris adult activity.
2244. Rev Argent Microbiol. 2003 Oct-Dec;35(4):193-7.
[Characterization of rhizobia causing nodules on leguminous trees native to

Uruguay using the rep-PCR technique].
Rodríguez A(1), Frioni L.
Author information:
(1)Departamento de Biología Vegetal-Microbiología, Facultad de Agronomía, Av.
Garzón 780, Montevideo, Uruguay. andrearb@fagro.edu.uy
Methods for identifying and following microorganisms in the environment such as

soils, water and plant association have been highly developed in recent years. In
this study, we used rep-PCR for the characterization of Rhizobium bacteria
isolated from legume trees native of Uruguay which permitted to follow them in
plant inoculation assays. Among the primers used, ERIC and BOX1AR, the latter
allowed the differentiation of isolates from different legumes and the finding of
a high level of homology among rhizobia that nodulate the same legume species.
Besides, it demonstrated that the profiles of the isolates recovered from nodules
of Acacia caven, were identical to the ones used as inoculants; this confirmed
the efficiency of these methods to follow these diazotrophs in the environment.
2245. J Anim Sci. 2004 Feb;82(2):487-94.
Effect of forage quality on digestion and performance responses of cattle to

supplementation with cooked molasses blocks.
Titgemeyer EC(1), Drouillard JS, Greenwood RH, Ringler JW, Bindel DJ, Hunter RD,
Nutsch T.
Author information:
66506-1600, USA. etitgeme@oznet.ksu.edu
We evaluated the effect of forage quality on response of cattle to

supplementation with cooked molasses blocks. In Exp. 1, 175 heifers had ad
libitum access to prairie hay (5.2% CP, DM basis). Treatments were a 2 x 3
factorial: supplementation with 0 or 1.96 kg/d of alfalfa DM, and supplementation
with no cooked molasses block or with a low-protein or a high-protein cooked
molasses block (14.4 and 27.5% CP, respectively, DM basis). There were no
significant interactions between alfalfa and cooked molasses block for intake or
gain. Forage intake and ADG were increased (P < 0.05) by alfalfa supplementation.
Heifers fed high-protein cooked molasses blocks gained more (P < 0.05) weight
than those fed low-protein cooked molasses blocks or no cooked molasses block.
Heifers fed high-protein cooked molasses blocks ate more (P < 0.05) forage than
those fed low-protein cooked molasses blocks, with heifers fed no cooked molasses
block being intermediate. In Exp. 2, responses to cooked molasses blocks
containing 33% CP (DM basis) were measured in 18 steers fed: 1) brome (8.4% CP),
2) alfalfa (19.2% CP), or 3) brome supplemented with 1.93 kg/d of alfalfa DM.
Forages were available ad libitum. Forage DM intake was not affected by cooked
molasses block and was greater (P < 0.05) for alfalfa than the alfalfa/brome mix,
which in turn was greater (P < 0.05) than brome. Digestibility of DM was greater
(P < 0.05) for alfalfa than brome or the alfalfa/brome mix and was not affected
by cooked molasses block supplementation. Supplementation with cooked molasses
blocks had only small effects on intake and digestion of medium- to high-quality
forages, but it improved gains and feed efficiencies of heifers fed prairie hay
ad libitum, with or without supplemental alfalfa.
DOI: 10.2527/2004.822487x
Effect of alkaline-stabilized biosolids on alfalfa molybdenum and copper content.
Stehouwer RC(1), Macneal KE.
Author information:
(1)Department of Crop and Soil Sciences, The Pennsylvania State University,
University Park, PA 16802-3504, USA. rcs15@psu.edu
Agricultural utilization of biosolids poses a potential risk to ruminant animals

due to transfer of Mo from biosolids to forage to the animal in amounts large
enough to suppress Cu uptake by the animal. Alkaline-stabilized biosolids (ASB)
must be given particular consideration in assessment of Mo risk because the high
pH of these biosolids could increase Mo and decrease Cu uptake by forage legumes.
In this 3-yr field experiment, ASB and ground agricultural limestone (AL) were
applied based on their alkalinity at rates equivalent to 0, 0.5, 1.0, and 2.0
times the lime requirement of the soil and alfalfa (Medicago sativa L.) was
grown. Alfalfa yield was similar with AL and ASB except in the second year when
ASB produced larger yields, apparently due to increased B availability with ASB.
Application of ASB did not detectably increase extractable soil Mo (0- to 15-cm
depth), but increased alfalfa Mo uptake in all cuttings with yield-weighted
uptake coefficients (UCs) of 8.07 and 7.11 following the first and second ASB
applications, respectively. Although ASB increased extractable soil Cu, and
alfalfa Cu content was greater with ASB than with AL, yield-weighted alfalfa Cu
to Mo ratio was decreased by ASB to levels near 3. These results suggest that ASB
may have a greater effect on Mo uptake and Cu to Mo ratio of forage legumes than
do other biosolids. Additional research is needed to determine implications of
larger Mo cumulative loading with ASB for Mo risk, particularly in the soil pH
range of 7 to 8.
2247. Planta. 2004 May;219(1):66-72. Epub 2004 Feb 12.
Class-1 hemoglobins, nitrate and NO levels in anoxic maize cell-suspension

cultures.
Dordas C(1), Hasinoff BB, Rivoal J, Hill RD.
Author information:
(1)Department of Plant Science, University of Manitoba, MB R3T 2N2, Winnipeg,
Canada.
Nitric oxide (NO) is a reactive gas involved in many biological processes of

animals, plants and microbes. Previous work has demonstrated that NO is formed
during hypoxia in alfalfa ( Medicago sativa L.) root cultures and that the levels
of NO detected are inversely related to the levels of expression of class-1
hemoglobin expressed in the tissue. The objectives of this study were: to examine
whether NO is produced in transgenic maize ( Zea mays L.) cell-suspension
cultures exposed to anoxic growth conditions; to determine whether a similar
relationship existed between a class-1 hemoglobin and the amount of NO detected
under these conditions; and, to estimate the route of formation and breakdown of
NO in the tissue. Maize cell-suspension cultures, transformed to express the
sense or antisense strands of barley hemoglobin were used to overexpress or
underexpress class-1 hemoglobin. A maize cell-suspension culture transformed with
an empty vector was used as a control. Up to 500 nmol NO (g FW)(-1) was detected
in maize cells exposed to low oxygen tensions for 24 h. The steady-state levels
of NO in the different cell lines under anoxic conditions had an inverse
relationship to the level of hemoglobin in the cells. There was no detectable NO
produced under aerobic growth conditions. Spectroscopic data demonstrated that
recombinant maize hemoglobin reacted with NO to form methemoglobin and NO(3)(-).
Nitrate was shown to be a precursor of NO in anoxic maize cell-suspension
cultures by using (15)NO(3)(-) and electron paramagnetic resonance spectroscopy,
suggesting that NO is formed via nitrate reductase during hypoxia. The results
demonstrate that NO is produced in plant tissues grown under low oxygen tensions
and suggest that class-1 hemoglobins have a significant function in regulating NO
levels.
DOI: 10.1007/s00425-004-1212-y
2248. Science. 2004 Feb 13;303(5660):950.
Society for Integrative and Comparative Biology meeting. Estrogen may disrupt
nitrogen fixation.
Pennisi E.
DOI: 10.1126/science.303.5660.950b
2249. Genome Res. 2004 Mar;14(3):459-62. Epub 2004 Feb 12.

Correct estimation of preferential chromosome pairing in autotetraploids.
Cao D(1), Osborn TC, Doerge RW.
Author information:
(1)Department of Statistics and Purdue University, West Lafayette, Indiana 47907,
USA.
In recent work, a statistical model was proposed for the purpose of estimating
parameters associated with quantitative trait locus (QTL) mapping and
preferential pairing within a polyploidy framework. The statistical model
contained several parameters that, when estimated from experimental data,
supplied information about QTL, including a preferential pairing factor. Among
the results reported were estimates of preferential pairing, many of which
indicated high levels of preferential pairing (p = 0.60) that were inconsistent
with biological expectations. By using the biological inconsistencies as our
motivation, we present a reformulated statistical method for estimating
preferential pairing, and use this method to reanalyze the same autotetraploid
alfalfa data and to conduct a simulation study. Our results directly contradict
the current findings of significant preferential pairing and affirm the
traditional view of random chromosome segregation in alfalfa.
DOI: 10.1101/gr.1596604
PMCID: PMC353233
2250. J Appl Genet. 2004;45(1):49-59.
Correlations and heritability of the characters determining the seed yield of the
long-raceme alfalfa (Medicago sativa L.).
Bodzon Z(1).
Author information:
(1)Plant Breeding and Acclimatization Institute, Radzików, Błonie, Poland.
z.bodzon@ihar.edu.pl
Phenotypic correlation coefficients and heritability of the characters

controlling seed yield of long-raceme forms of alfalfa was determined. It was
found that seed yield per plant, which was positively correlated with 10 out of
12 analysed characters, depended upon the number of pods per raceme and the
number of seeds per pod. Variability of these characters determined about 60% of
the variability of seed yield. Multiple linear regression and phenotypic
correlations show that simultaneous selection for increased pod number per raceme
and increased seed number per pod and raceme length resulted in enhanced seed
yield potential. The share of the additive genetic effects in the phenotypic
variance for number of pods per raceme was low and about 21-23%, while for number
of seeds per pod and per raceme amounted to about 50%. The expected genetic
progress in recombination breeding for number of seeds per pod and number of
seeds per raceme will be of medium magnitude, while one cannot expect any rapid
and considerable progress in the number of pods per raceme. Considering the high
positive correlation between raceme length and number of pods and seeds per
raceme, one should conclude that raceme length can be an important criterion in
selection of plants showing a high seed productivity.

2251. Plant Cell Rep. 2004 May;22(10):774-9. Epub 2004 Feb 10.
Kanamycin-resistant alfalfa has a point mutation in the 16S plastid rRNA.
Rosellini D(1), LaFayette PR, Barone P, Veronesi F, Parrott WA.
Author information:
(1)Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Università
degli Studi di Perugia, Perugia, Italy.
Genes conferring resistance to kanamycin are frequently used to obtain transgenic

plants as spontaneous resistance to kanamycin is not known to exist in higher
plants. Nevertheless, mutations conferring kanamycin resistance have been
identified in Chlamydomonas reinhardtii, raising the question as to why
kanamycin-resistant mutants have not been found in higher plants. While
attempting plastid transformation of alfalfa, we obtained non-transgenic but
kanamycin-resistant somatic embryos following 2 months of culture in the presence
of 50 mg l(-1) kanamycin. Sequencing of the plastid DNA region corresponding to
the decoding site of the 16S rRNA in ten independent resistant events revealed an
A to C transversion at position 1357 of the 16S plastid rDNA, the same site at
which an A to G conversion confers kanamycin resistance to C. reinhardtii by
reducing the ability of the antibiotic to bind to its target site. All plants
derived from the resistant embryos through additional cycles of somatic
embryogenesis in the absence of kanamycin retained the mutant phenotype,
suggesting that the mutation was homoplastomic. Resistant plants produced 85%
less biomass than controls; their leaves were chlorotic during early development
and over time slowly turned green. The absence of kanamycin- resistant mutants in
higher plants might be explained by the requirement for a regeneration system
capable of resulting in homoplastomic individuals, or it may be the result of the
detrimental effect of the mutation on the phenotype.
Copyright 2004 Springer-Verlag
DOI: 10.1007/s00299-004-0757-3
2252. FEMS Microbiol Lett. 2004 Feb 9;231(1):131-6.
The key Sinorhizobium meliloti succinoglycan biosynthesis gene exoY is expressed

from two promoters.
Cheng HP(1), Yao SY.
Author information:
(1)Biological Sciences Department, Lehman College, the City University of New
York, 250 Bedford Park Boulevard West, Bronx, NY 10468, USA.
haiping@lehman.cuny.edu
Bacterial exopolysaccharide, succinoglycan, plays an important role in eliciting

infection thread formation, which is a key step in the establishment of
Sinorhizobium meliloti-alfalfa (Medicago sativa) nitrogen fixing symbiosis. To
understand the regulatory mechanisms that control production of succinoglycan,
the expression of the key succinoglycan biosynthesis gene, exoY, was analyzed by
constructing a set of nested deletions of the exoY promoter region. Two exoY
promoters were identified based on the promoter activities and confirmed by
direct detection of the transcripts. The expression from both promoters was
induced in the exoR95 and exoS96 mutant backgrounds suggesting that both
promoters are regulated by the ExoR protein and the ExoS/ChvI two-component
signal transduction system. The identification of the exoY promoters provides
additional avenue for further analysis of the role of succinoglycan in S.
meliloti-alfalfa symbiosis.
DOI: 10.1016/S0378-1097(03)00952-2
PMCID: PMC3144747
2253. J Dairy Sci. 2004 Jan;87(1):139-45.
Effect of dietary phosphorus on performance of lactating dairy cows: milk

production and cow health.
Lopez H(1), Kanitz FD, Moreira VR, Wiltbank MC, Satter LD.
Author information:
The objective of this study was to measure cow response to feeding of two dietary
concentrations of P, one of which was close to recent National Research Council
requirements, and the other of which was well in excess of the requirement. Diets
containing 0.37 or 0.57% P (dry basis) were fed to Holstein cows for the first
165 d of lactation, and occasionally longer until cows were confirmed pregnant
approximately 60 d after insemination. At calving, cows were randomly assigned to
experimental diets. The number of cows completing a minimum of 165 d of lactation
was 123 for the 0.37 and 124 for the 0.57% P groups. Cows were housed in a
stanchion barn and fed one of two transition diets, each formulated to contain
one of the P treatments for the first 3 wk of lactation, and then cows were moved
to a free-stall barn where the experimental diets were group fed. Milk
production, milk fat, and milk protein averaged 35.1 kg/d, 3.92%, and 2.90% for
the 0.37% P diet, and 34.9 kg/d, 3.98%, and 2.91% for the 0.57% P diet. None of
these measures were different between treatments. Blood serum P concentrations on
d 50 and 100 of lactation averaged 6.1 and 6.2 mg/dL for the 0.37% P diet, and
6.8 and 6.9 mg/dL for the 0.57% P diet. No treatment differences were detected in
milk production, cow health, or body condition score.
DOI: 10.3168/jds.S0022-0302(04)73151-3
2254. J Dairy Sci. 2004 Jan;87(1):131-8.
Comparison of conventional linted cottonseed and mechanically delinted cottonseed

in diets for dairy cows.
Moreira VR(1), Satter LD, Harding B.
Author information:
(1)U.S. Dairy Forage Research Center, Madison, USDA-Agricultural Research
Service, USA.
Performance of lactating dairy cows fed diets containing either mechanically

delinted whole cottonseed (DWCS; 3.7% lint) or linted whole cottonseed (LWCS;
11.7% lint) was measured. Forty primiparous (86 +/- 39 d in milk) and 40
multiparous (88 +/- 30 d in milk) cows were fed a total mixed ration containing
13% (dry matter basis) DWCS or LWCS in two blocks of 112 d (n = 53 and 27,
respectively). Other total mixed ration ingredients (dry matter basis) were corn
silage (28.1%), alfalfa silage (23%), high moisture shelled corn (27.8%), soybean
meal (1.8%), expeller soybean meal (1.8%), blood meal (2%), and mineral-vitamin
supplements (2.5%). Dry matter intake and milk yield were measured daily and milk
composition every other week. Fecal grab samples were taken during wk 3 and 13 of
each block to estimate excretion of intact whole cottonseeds. Milk yield, 3.5%
fat-corrected milk, energy-corrected milk, milk composition and dry matter intake
were not affected by whole cottonseed source. Body condition score tended to
increase more with DWCS (0.22 vs. 0.11) for primiparous cows, although this was
not reflected in body weight change. Dry matter digestibilities, based on
indigestible ADF, were 63.5 and 64.8% for the DWCS and LWCS diets. It was
calculated that 2.5 and 1.5% of the consumed seeds were excreted as whole
cottonseeds in feces with the DWCS and LWCS diets, respectively. Although
statistically significant, treatment differences in the proportion of intact
seeds in the fecal DM would have little nutritional consequence. Mechanically
delinted WCS performed as well as LWCS for all of the cow performance and milk
composition variables measured.
DOI: 10.3168/jds.S0022-0302(04)73150-1
2255. J Dairy Sci. 2004 Jan;87(1):112-21.
Methane production by mixed ruminal cultures incubated in dual-flow fermentors.
Eun JS(1), Fellner V, Gumpertz ML.
Author information:
(1)Department of Animal Science, North Carolina State University, Raleigh 27695,
USA.
This study evaluated the effects of dilution rate and forage-to-concentrate ratio
on gas production by rumen microbes. Continuous cultures were used to monitor
methane production at three liquid dilution rates (3.2, 6.3, or 12.5%/h) and
three forage-to-concentrate ratios (70:30, 50:50, or 30:70). Filtered ruminal
contents were allowed 6 d of adaptation to diets followed by 7 d of data
collection. Forage consisted of pelleted alfalfa and the concentrate mix included
ground corn, soybean meal, and a mineral and vitamin premix. The experiment was
replicated in a split-plot design. Total volatile fatty acid production averaged
58.0 mmol/d and was not affected by treatment. Molar proportion of acetate
increased with increasing forage-to-concentrate ratio. Molar proportion of
propionate tended to decrease at dilution rate of 12.5%/h and increased with the
medium and low forage-to-concentrate ratio. Culture pH tended to be greater at a
dilution rate of 12.5%/h. Methane production that was calculated from
stoichiometric equations was not affected by treatments. However, methane
production based on methane concentration in fermentor headspace resulted in an
interaction effect of treatments. Stoichiometric equations underestimated methane
output at higher dilution rates and with high forage diets. Total diet
fermentability was lowest at dilution rate of 3.2%/h. Increasing dilution rates
increased microbial yield; increasing the proportion of concentrate improved
microbial efficiency. Dilution rate and forage-to-concentrate ratio altered the
partition of substrate by microbes. Methane production based on actual
concentrations differed from values estimated using stoichiometry of end-product
appearance.
DOI: 10.3168/jds.S0022-0302(04)73148-3
2256. J Dairy Sci. 2004 Feb;87(2):413-30.
Portal drained visceral flux, hepatic metabolism, and mammary uptake of free and
peptide-bound amino acids and milk amino acid output in dairy cows fed diets
containing corn grain steam flaked at 360 or steam rolled at 490 g/L.
Tagari H(1), Webb K Jr, Theurer B, Huber T, DeYoung D, Cuneo P, Santos JE, Simas
J, Sadik M, Alio A, Lozano O, Delgado-Elorduy A, Nussio L, Nussio C, Santos F.
Author information:
(1)Department of Animal Sciences, Faculty of Agriculture, The Hebrew University
of Jerusalem, Rehovot, Israel 76100. tagari@agri.huji.il
Objectives were to measure net fluxes of free (FAA) and peptide bound amino acids
(AA) (PBAA) across portal-drained viscera (PDV), liver, splanchnic, and mammary
tissues, and of milk AA output of lactating Holstein cows (n = 6, 109 +/- 9 d in
milk) as influenced by flaking density of corn grain. Cows were fed alfalfa-based
total mixed ration (TMR) containing 40% steam-flaked (SFC) or steam-rolled corn
(SRC) grain. The TMR were offered at 12-h intervals in a crossover design. Six
sets of blood samples were obtained from indwelling catheters in portal, hepatic,
and mammary veins and mesenteric or costoabdominal arteries every 2 h from each
cow and diet. Intake of dry matter (18.4 +/- 0.4 kg/d), N, and net energy for
lactation were not altered by corn processing. Milk and milk crude protein yields
(kg/12-h sampling) were 14.2 vs. 13.5 and 0.43 vs. 0.39 for cows fed SFC or SRC,
respectively. The PDV flux of total essential FAA was greater (571.2 vs. 366.4
g/12 h, SEM 51.4) in cows fed SFC. The PDV flux of total essential PBAA was 69.3
+/- 10.8 and 51.5 +/- 13.2 g/12 h for cows fed SFC and SRC, respectively, and
differed from zero, but fluxes of individual PBAA rarely differed between
treatments. Liver flux of essential FAA was greater in cows fed SRC, but only the
PBAA flux in cows fed SRC differed from zero. Splanchnic flux of FAA and PBAA
followed the pattern of PDV flux, but variation was greater. Mammary uptake (g/12
h) of total essential FAA was greater in cows fed SFC than SRC (224.6 vs. 198.3,
SEM 7.03). Mammary uptake of essential PBAA was 25.0 vs. 15.1, SEM 5.2, g/12 h
for cows fed SFC or SRC, respectively, and differed from zero in half of the
PBAA. Milk output of EAA was 187.8 vs 175.4, SEM 4.4 g/12 h in cows fed SFC and
SRC, respectively, and output of most essential AA consistently tended to be
greater in cows fed SFC. It is apparent that PBAA comprise a portion of total AA
flux across PDV and are affected by grain processing. Further, this pool supplies
an important component of AA taken up by the mammary gland. Quantifying the
contribution of PBAA may improve diet formulation with respect to intestinal
absorption and mammary uptake of AA.
DOI: 10.3168/jds.S0022-0302(04)73181-1
2257. J Dairy Sci. 2004 Feb;87(2):359-68.
Effect of low level monensin supplementation on the production of dairy cows fed
alfalfa silage.
Broderick GA(1).
Author information:
WI 53706, USA. gbroderi@dfrc.wisc.edu
Effectiveness of low level monensin supplementation on N utilization in lactating

dairy cows fed alfalfa silage was assessed using 48 multiparous Holsteins. Cows
were fed a covariate diet [% of dry matter (DM): 56% alfalfa silage, 39% ground
high moisture corn, 3% soybean meal, 1% ground corn, 1% vitamin-mineral
supplements] for 2 wk, then grouped by days in milk into blocks of 4. Cows were
randomly assigned within blocks to 1 of 4 diets that were fed for 10 wk: 1)
control (covariate diet), 2) control plus 3% fish meal (replacing DM from high
moisture corn), 3) monensin (10 mg/kg DM), and 4) monensin plus 3% fish meal.
Diets 1 and 3 averaged 16.7% crude protein (25% from free AA in alfalfa silage);
diets 2 and 4 averaged 18.5% crude protein. Monensin intake averaged 16 mg/d on
diets 1 and 2 (due to contamination) and 248 mg/d on diets 3 and 4. There was no
effect of fish meal or monensin on DM intake. However, weight gain and yield of
milk, protein, and SNF increased with fish meal feeding, indicating metabolizable
protein limited production. Feeding monensin increased blood glucose but reduced
yield of 3.5% fat-corrected milk, milk fat content and yield, and milk protein
content and yield. Apparent N efficiency was greatest on monensin (diet 3) but
lowest on monensin plus fish meal (diet 4). Fish meal reduced blood glucose
concentration and apparent N efficiency, and increased concentrations of milk and
blood urea. Monensin increased ruminal propionate concentration and decreased
concentration of acetate and butyrate and acetate:propionate in ruminally
cannulated cows fed the experimental diets. However, these changes were small,
suggesting that too little monensin was fed. Fish meal reduced ruminal total
amino acid (AA) but monensin did not alter ruminal NH(3) or total AA. Both fish
meal and monensin increased NH(3) formation from casein AA using ruminal inoculum
from the cannulated cows. There was no evidence from this trial that feeding 250
mg of monensin per day to lactating cows improved N utilization by reducing
ruminal catabolism of the large amounts of free AA in alfalfa silage.
DOI: 10.3168/jds.S0022-0302(04)73175-6
2258. FEMS Microbiol Ecol. 2004 Feb 1;47(2):215-22. doi: 10.1016/S0168-

6496(03)00259-9.
The effect of pH and a bacteriocin (bovicin HC5) on Clostridium sporogenes MD1, a

bacterium that has the ability to degrade amino acids in ensiled plant materials.
Flythe MD(1), Russell JB.
Author information:
(1)Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Fresh plant materials can be fermented and preserved as silage for cattle, but
clostridia that deaminate amino acids increase pH. If the pH of the silage rises,
spoilage microorganisms proliferate, and undesirable products accumulate.
Rod-shaped, anaerobic bacteria with spores were isolated from fresh alfalfa,
fresh corn, and silages. Strain MD1 had the highest specific activity of amino
acid deamination, and it was most closely related to Clostridium botulinum A and
B. However, because strain MD1 did not produce a toxin, it was classified as
Clostridium sporogenes. Washed cell suspensions of C. sporogenes MD1 had specific
activities as great as 690 nmol ammonia mg protein(-1) min(-1), and this rate did
not decrease until the pH was less than 4.5. Batch cultures of C. sporogenes MD1
did not initiate growth if the initial pH was less than 5.0, but continuous
cultures (0.1 h(-1) dilution rate) persisted until the pH in the culture vessel
was 4.6. When C. sporogenes MD1 was co-cultured with a bacteriocin-producing
Streptococcus bovis HC5, ammonia production was greatly reduced. The ability of
S. bovis HC5 to inhibit strain MD1 was pH-dependent. When the pH was 5.5 or less,
strain MD1 could no longer be detected. These latter results support the idea
that bacteriocin-producing bacteria may be used to improve silage quality.
DOI: 10.1016/S0168-6496(03)00259-9
Influence of organic and inorganic soil amendments on plant growth in crude

oil-contaminated soil.
White PM Jr(1), Wolf DC, Thoma GJ, Reynolds CM.
Author information:
(1)Department of Agronomy, Kansas State University, 2004 Throckmorton Plant
Sciences Center, Manhattan, KS 66506, USA. pmwhite@ksu.edu
Phytoremediation can be a viable alternative to traditional, more costly

remediation techniques. Three greenhouse studies were conducted to evaluate plant
growth with different soil amendments in crude oil-contaminated soil. Growth of
alfalfa (Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L.,
cultivar: Common), crabgrass (Digitaria sanguinalis, cultivar: Large), fescue
(Lolium arundinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium
multiflorum Lam., cultivar: Marshall) was determined in crude oil-contaminated
soil amended with either inorganic fertilizer, hardwood sawdust, papermill
sludge, broiler litter or unamended (control). In the first study, the addition
of broiler litter reduced seed germination for ryegrass, fescue, and alfalfa. In
the second study, bermudagrass grown in broiler litter-amended soil produced the
most shoot biomass, bermudagrass produced the most root biomass, and crabgrass
and bermudagrass produced the most root length. In the third study, soil amended
with broiler litter resulted in the greatest reduction in gravimetric total
petroleum hydrocarbon (TPH) levels across the six plant treatments following the
14-wk study. Ryegrass produced more root biomass than any other species when
grown in inorganic fertilizer- or hardwood sawdust + inorganic fertilizer-amended
soil. The studies demonstrated that soil amendments and plant species selection
were important considerations for phytoremediation of crude oil-contaminated
soil.
DOI: 10.1080/15226510309359044
2260. Planta. 2004 May;219(1):95-102. Epub 2004 Jan 22.
NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing

barley hemoglobin.
Igamberdiev AU(1), Seregélyes C, Manac'h N, Hill RD.
Author information:
(1)Department of Plant Science, University of Manitoba, R3T 2N2, Winnipeg,
Manitoba, Canada.
Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and
antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability
to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately
twice the NO conversion rate of either control or antisense lines under normoxic
conditions. Only the control line showed a significant increase in the rate of NO
degradation when placed under anaerobic conditions. The decline in NO was
dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent
rate being about 2.5 times faster than the NADPH-dependent rate. Most of the
activity was found in the cytosolic fraction of the extracts, while only small
amounts were found in the cell wall, mitochondria, and 105,000- g membrane
fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the
range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both). It
was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The
activity was strongly reduced by applying antibodies raised against recombinant
barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin
showed a 4-fold higher rate of NO metabolism as compared to non-transformed
cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing
hemoglobin, indicating that the presence of hemoglobin has an effect on these
ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root
extracts exhibited methemoglobin reductase activity, using either cytochrome c or
recombinant barley hemoglobin as substrates. There was a correspondence between
NO degradation and nitrate formation. The activity was eluted from a Superose 12
column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to
the size of the hemoglobin dimer. The results are consistent with an NO
dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein,
to metabolize NO to nitrate utilizing NADH as the electron donor.
DOI: 10.1007/s00425-003-1192-3
2261. Plant Physiol. 2004 Feb;134(2):595-604. Epub 2004 Jan 22.
Plant and bacterial symbiotic mutants define three transcriptionally distinct

stages in the development of the Medicago truncatula/Sinorhizobium meliloti
symbiosis.
Mitra RM(1), Long SR.
Author information:
(1)Department of Biological Sciences, Stanford University, Stanford, California
94305, USA.
In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes

a series of developmental changes simultaneously, creating a root nodule and
allowing bacterial entry and differentiation. Our studies of plant genes reveal
novel transcriptional regulation during the establishment of the symbiosis and
identify molecular markers that distinguish classes of plant and bacterial
symbiotic mutants. We have identified three symbiotically regulated plant genes
encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a
cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h
after exposure to the bacterial signal, Nod factor. The non-nodulating plant
mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and
MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We
examined the regulation of these two genes and three previously identified genes
(MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants
inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and
bacterial (exoA and exoH) mutants with defects in the initial stages of invasion
are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants
(fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7)
defective for nitrogen fixation induce the above genes. The bacA bacterial
mutant, which senesces upon deposition into plant cells, and two plant mutants
with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not
MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three
transcriptionally distinct developmental stages during invasion of M. truncatula
by S. meliloti.
DOI: 10.1104/pp.103.031518
PMCID: PMC344536
2262. J Environ Monit. 2004 Jan;6(1):38-47. Epub 2003 Dec 5.
Extractable copper, arsenic and antimony by EDTA solution from agricultural

Chilean soils and its transfer to alfalfa plants (Medicago sativa L.).
De Gregori I(1), Fuentes E, Olivares D, Pinochet H.
Author information:
(1)Laboratorio de Química Analítica y Ambiental, Instituto de Química,
Universidad Católica de Valparaíso, Avenida Brasil, 2950 Valparaíso, Chile.
idegrego@ucv.cl
Following our research on copper, arsenic and antimony in Chilean ecosystems, a

study to understand the mobility and transport of these elements from soil to
plants was carried out. So, the aim of this study, which follows on from the
previous work, was to demonstrate if the total concentrations of these elements
or their fractions extracted by 0.05 M EDTA pH 7 from different Chilean soils
correlate with the respective total concentrations in the edible tissue of
alfalfa plants collected simultaneously from 20 different sites affected or
unaffected by mining activities. The highest copper fractions extracted by EDTA
solutions were obtained in contaminated soils from the central region (41-69%);
however the northern soils presented the highest extractable fractions of arsenic
(9-34%). The antimony fraction was low in all soils (0.4-8.0%). Alfalfa plants
from all contaminated sites presented high copper, arsenic and antimony
concentrations (19-126 mg kg(-1), 5.7-16.3 mg kg(-1) and 0.16-1.7 mg kg(-1),
respectively). Statistically significant correlations were obtained between the
total contents of copper and arsenic and their respective extractable fractions
in soils. Good correlations were found between elements in alfalfa plants.
Correlations were also obtained between the total concentrations of three
elements in soils and in alfalfa plants. However, excepting for antimony in the
northern samples, higher correlation coefficients were evaluated when the
extractable fractions were considered. Samples from the north region presented
the highest copper transfer factor and the lowest for arsenic, in spite of the
high concentration of this metalloid extracted by EDTA solution in these soils.
There was not a clear trend on the transfer factor for antimony, probably due to
the low content of this element in alfalfa plants and/or the low recovery
obtained for this element by EDTA.
DOI: 10.1039/b304840c
2263. Planta. 2004 Apr;218(6):916-27. Epub 2004 Jan 13.
An auxin-inducible gene from loblolly pine (Pinus taeda L.) is differentially

expressed in mature and juvenile-phase shoots and encodes a putative
transmembrane protein.
Busov VB(1), Johannes E, Whetten RW, Sederoff RR, Spiker SL, Lanz-Garcia C,
Goldfarb B.
Author information:
(1)Department of Forestry, North Carolina State University, Raleigh, NC 27695,
USA.
We have isolated a gene from loblolly pine, 5NG4, that is highly and specifically
induced by auxin in juvenile loblolly pine shoots prior to adventitious root
formation, but substantially down-regulated in physiologically mature shoots that
are adventitious rooting incompetent. 5NG4 was highly auxin-induced in roots,
stems and hypocotyls, organs that can form either lateral or adventitious roots
following an auxin treatment, but was not induced to the same level in needles
and cotyledons, organs that do not form roots. The deduced amino acid sequence
shows homology to the MtN21 nodulin gene from Medicago truncatula. The expression
pattern of 5NG4 and its homology to a protein from Medicago involved in a
root-related process suggest a possible role for this gene in adventitious root
formation. Homology searches also identified similar proteins in Arabidopsis
thaliana and Oryza sativa. High conservation across these evolutionarily distant
species suggests essential functions in plant growth and development. A 38-member
family of genes homologous to 5NG4 was identified in the A. thaliana genome. The
physiological significance of this redundancy is most likely associated with
functional divergence and/or expression specificity of the different family
members. The exact biochemical function of the gene is still unknown, but
sequence and structure predictions and 5NG4::GFP fusion protein localizations
indicate it is a transmembrane protein with a possible transport function.
DOI: 10.1007/s00425-003-1175-4
2264. Environ Pollut. 2004;128(3):429-35.
Priming effects on PAH degradation and ecotoxicity during a phytoremediation

experiment.
Joner EJ(1), Hirmann D, Szolar OH, Todorovic D, Leyval C, Loibner AP.
Author information:
(1)LIMOS (Laboratoire des Interactions Microorganismes-Minéraux-Matière Organique
dans les Sols)-CNRS UMR 7131, Henri Poincaré University, Faculty of Science, PO
Box 239, F-54506 Vandoeuvre-les-Nancy Cedex, France. erik.joner@skogforsk.no
An experiment was conducted to distinguish priming effects from the effects of

phytoremediation of a creosote-polluted soil. The concentration of 13 polycyclic
aromatic hydrocarbons (PAHs), and their combined soil toxicity (using four
bioassays), was determined on recently excavated, homogenized soil and on such
soil subjected to a time-course phytoremediation experiment with lucerne. The
results showed a high priming effect, with minor positive and synergistic effects
of planting and fertilization on PAH degradation rates. At the end of the
experiment, PAH degradation reached 86% of the initial 519 mg PAHs kg(-1). Two of
the four toxicity tests (bioluminescence inhibition and ostracod growth
inhibition) corroborated the chemical data for residual PAHs, and indicated a
significant reduction in soil toxicity. We conclude that priming effects can
easily surpass treatment effects, and that an unintentional pre-incubation that
ignores these effects can jeopardize the full quantitative assessment of in situ
bioremediation of contaminated soil.
DOI: 10.1016/j.envpol.2003.09.005
2265. J Food Prot. 2004 Jan;67(1):212-4.

Simplified qualitative method for canavanine in seeds and sprouts.
Rajkowski KT(1).
Author information:
Research Center, Food Safety Intervention Technologies Research Unit, 600 East
Mermaid Lane, Wyndmoor, Pennsylvania 19038-8598, USA.
krajkowski@errc.ars.usda.gov
The major stored nitrogen compound in alfalfa seeds is canavanine. To identify

this nonprotein amino acid from seed extract and sprout water, a qualitative
micro-thin-layer chromatography method was developed. Successful separation and
identification was achieved using microsilica plates, a 70:30 ethyl alcohol-water
solvent system, and 1% ammonium disodium pentacyanoammineferrate II for color
development. This quick method was used to identify canavanine (sensitivity 50
microg) from irradiated and nonirradiated alfalfa and clover seed extracts and
alfalfa sprout water. Broccoli and radish seed extracts were negative for
canavanine. This simple method is useful to track the release and decrease of
canavanine in the sprout water.
2266. J Food Prot. 2004 Jan;67(1):46-52.
A rapid and automated fiber optic-based biosensor assay for the detection of
Salmonella in spent irrigation water used in the sprouting of sprout seeds.
Kramer MF(1), Lim DV.
Author information:
(1)Department of Biology, University of South Florida, 4202 East Fowler Avenue,
SCA110, Tampa, Florida 33620, USA. leverone@chuma.cas.usf.edu
Recent outbreaks of foodborne illness have been linked to the consumption of

contaminated sprouts. The spent irrigation water used to irrigate sprouts can
carry many microorganisms, including pathogenic strains of Escherichia coli and
Salmonella enterica. These pathogens are believed to originate from the seeds.
The U.S. Food and Drug Administration recommends that sprout producers conduct
microbiological testing of spent irrigation water from each production lot at
least 48 h after seeds have germinated. Microbial analysis for the detection of
Salmonella is labor-intensive and takes days to complete. A rapid and automated
fiber-optic biosensor assay for the detection of Salmonella in sprout rinse water
was developed in this study. Alfalfa seeds contaminated with various
concentrations of Salmonella Typhimurium were sprouted. The spent irrigation
water was assayed 67 h after alfalfa seed germination with the RAPTOR (Research
International, Monroe, Wash.), an automated fiber optic-based detector.
Salmonella Typhimurium could be positively identified in spent irrigation water
when seeds were contaminated with 50 CFU/g. Viable Salmonella Typhimurium cells
were also recovered from the waveguides after the assay. This biosensor assay
system has the potential to be directly connected to water lines within the
sprout-processing facility and to operate automatically, requiring manual labor
only for preventative maintenance. Therefore, the presence of Salmonella
Typhimurium in spent irrigation water could be continuously and rapidly detected
3 to 5 days before the completion of the sprouting process.
2267. Mol Plant Microbe Interact. 2004 Jan;17(1):16-26.
Expression of MsLEC1 transgenes in alfalfa plants causes symbiotic abnormalities.
Brill LM(1), Fujishige NA, Hackworth CA, Hirsch AM.
Author information:
(1)Department of Molecular, Cell and Developmental Biology, Molecular Biology
Institute, University of California, 405 Hilgard Avenue, Los Angeles, CA
90095-1606, USA.
Legume lectins have been proposed to have important symbiotic roles during
Rhizobium-legume symbioses. To test this hypothesis, the symbiotic responses of
transgenic alfalfa plants that express a portion of the putative alfalfa lectin
gene MsLEC1 or MsLEC2 in either the antisense or sense orientation were analyzed
following inoculation with wild-type Sinorhizobium meliloti 1021.
MsLEC1-antisense (LEC1AS) plants were stunted, exhibited hypernodulation, and
developed not only abnormally large nodules but also numerous small nodules, both
of which senesced prematurely. MsLEC2-antisense plants were intermediate in
growth and nodule number compared with LEC1AS and vector control plants. The
symbiotic abnormalities of MsLEC1-sense transgene plants were similar to but
milder than the responses shown by the LEC1AS plants, whereas MsLEC2-sense
transgene plants exhibited symbiotic responses that were identical to those of
vector and nontransgenic control plants. MsLEC1 mRNA accumulation was not
detected in nodule RNA by Northern blot analysis but was localized to alfalfa
nodule meristems and the adjacent cells of the invasion zone by in situ
hybridization; transcripts were also detected in root meristems. A similar
spatial pattern of MsLEC2 expression was found by using a whole-mount in situ
hybridization procedure. Moreover, mRNAs for an orthologous lectin gene (MaLEC)
were detected in white sweetclover (Melilotus alba) nodules and root tips.
DOI: 10.1094/MPMI.2004.17.1.16
2268. Prikl Biokhim Mikrobiol. 2003 Nov-Dec;39(6):681-8.
[Efficiency of alfalfa and reed in the phytoremediation of hydrocarbon polluted

soil].
Muratova AIu(1), Turkovskaia OV, Hubner T, Kuschk P.
Author information:
Academy of Sciences, Saratov, 410049 Russia.
The efficacy of plants as means of decontaminating hydrocarbon-polluted soil has

been studied. Ditch reed (Phragmites australis) and alfalfa (Medicago sativa)
markedly intensified processes of pollutant destruction, the effect being
particularly pronounced in the case of polycyclic aromatic hydrocarbons (PAHs).
Comparative analysis of microflora in soils (including those devoid of plants and
rhizosphere) demonstrated that, in addition to preventing the pollutant-induced
decrease in the amount of heterotrophic microorganisms, the plants stimulated
their development, significantly increasing the population of destructors.
Effects of plants on major physiological groups of soil microorganisms under
conditions of pollution were ambiguous. The rhizosphere consortium of alfalfa was
less susceptible to effects of pollutants than that of reed.
2269. J Hered. 2003 Nov-Dec;94(6):512-6.
Cytomixis in pollen mother cells of Medicago sativa L.
Bellucci M(1), Roscini C, Mariani A.
Author information:
(1)Institute of Plant Genetics - Research Division of Perugia, CNR, via della
Madonna Alta, 130, 06128 Perugia, Italy.
Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many
plant species, but not in Medicago sativa spp. In the present study we report the
identification of a few cytomictic alfalfa plants. Those plants, the "mother
plants," were selfed and crossed with a normal control plant. Microsporogenesis
analysis was performed on the mother plants, on the S(1) and F(1) plants, and on
controls. The S(1) and F(1) plants, like the mother plants, were found to be
cytomictic. Single or multiple chromatin bridges between two or more meiocytes
were observed almost exclusively in prophase I. Some completely empty meiocytes
were also observed. In addition to cytomixis, other meiotic abnormalities were
found. Control plants showed an almost regular meiosis. The highest values of
cytomixis were observed in the mother plants, and the lowest in their F(1)
progenies. Variability of cytomixis in the F(1) plants is probably due to a
heterozygotic condition of the parents for this trait. No significant correlation
was found between cytomixis and pollen viability, even if the cytomictic plants
showed low values of pollen viability.
2270. J Hered. 2003 Nov-Dec;94(6):435-41.
Willet M. Hays, great benefactor to plant breeding and the founder of our
association.
Troyer AF(1), Stoehr H.
Author information:
(1)Department of Crop Sciences, University of Illinois, 1102 South Goodwin Ave.,
Urbana, IL, USA. atroyer@uiuc.edu
Willet M. Hays was a great benefactor to plant breeding and the founder of the
American Genetic Association (AGA). We commemorate the AGA's centennial. We mined
university archives, U.S. Department of Agriculture (USDA) yearbooks, plant
breeding textbooks, scientific periodicals, and descendants for information.
Willet Hays first recognized the individual plant as the unit of selection and
started systematic pure-line selection and progeny tests in 1888. He developed
useful plant breeding methods. He selected superior flax (Linum usitatissimum
L.), wheat (Triticum vulgare L.), corn (Zea mays L.), barley (Hordeum vulgare
L.), and oat (Avena sativa L.) varieties, and discovered Grimm alfalfa (Medicago
sativa L.); all became commercially important. He initiated branch stations for
better performance testing. Willet Hays befriended colleagues in other
universities, in federal stations, in a London conference, and in Europe. He
gathered and spread the scientific plant breeding gospel. He also improved rural
roads and initiated animal breeding records and agricultural economics records.
He started the AGA in 1903, serving as secretary for 10 years. He became
assistant secretary of agriculture in 1904. He introduced the project system for
agricultural research. He authored or coauthored the Nelson Amendment, the
Smith-Lever Act, the Smith-Hughes Act, and the protocol leading to the United
Nations Food and Agriculture Organization-all involved teaching agricultural
practices that improved the world.
2271. BMC Plant Biol. 2003 Dec 19;3:9.
Construction of two genetic linkage maps in cultivated tetraploid alfalfa

(Medicago sativa) using microsatellite and AFLP markers.
Julier B(1), Flajoulot S, Barre P, Cardinet G, Santoni S, Huguet T, Huyghe C.
Author information:
(1)INRA, Unité de Génétique et d'Amélioration des Plantes Fourragères, 86600
Lusignan, France. julier@lusignan.inra.fr
BACKGROUND: Alfalfa (Medicago sativa) is a major forage crop. The genetic

progress is slow in this legume species because of its autotetraploidy and
allogamy. The genetic structure of this species makes the construction of genetic
maps difficult. To reach this objective, and to be able to detect QTLs in
segregating populations, we used the available codominant microsatellite markers
(SSRs), most of them identified in the model legume Medicago truncatula from EST
database. A genetic map was constructed with AFLP and SSR markers using specific
mapping procedures for autotetraploids. The tetrasomic inheritance was analysed
in an alfalfa mapping population.
RESULTS: We have demonstrated that 80% of primer pairs defined on each side of
SSR motifs in M. truncatula EST database amplify with the alfalfa DNA. Using a F1
mapping population of 168 individuals produced from the cross of 2 heterozygous
parental plants from Magali and Mercedes cultivars, we obtained 599 AFLP markers
and 107 SSR loci. All but 3 SSR loci showed a clear tetrasomic inheritance. For
most of the SSR loci, the double-reduction was not significant. For the other
loci no specific genotypes were produced, so the significant double-reduction
could arise from segregation distortion. For each parent, the genetic map
contained 8 groups of four homologous chromosomes. The lengths of the maps were
2649 and 3045 cM, with an average distance of 7.6 and 9.0 cM between markers, for
Magali and Mercedes parents, respectively. Using only the SSR markers, we built a
composite map covering 709 cM.
CONCLUSIONS: Compared to diploid alfalfa genetic maps, our maps cover about
88-100% of the genome and are close to saturation. The inheritance of the
codominant markers (SSR) and the pattern of linkage repulsions between markers
within each homology group are consistent with the hypothesis of a tetrasomic
meiosis in alfalfa. Except for 2 out of 107 SSR markers, we found a similar order
of markers on the chromosomes between the tetraploid alfalfa and M. truncatula
genomes indicating a high level of colinearity between these two species. These
maps will be a valuable tool for alfalfa breeding and are being used to locate
QTLs.
DOI: 10.1186/1471-2229-3-9
PMCID: PMC324403
2272. J Chem Ecol. 2003 Nov;29(11):2413-24.
Physiological assessment and path coefficient analysis to improve evaluation of

alfalfa autotoxicity.
Chon SU(1), Nelson CJ, Coutts JH.
Author information:
(1)Biotechnology Industrialization Center, Dongshin University, #880-4, Ansan-Ri
Noan-Myun, Naju-Si, Jeonnam 520-811, South Korea. chonsu@lycos.co.kr
Reseeding of alfalfa is affected until autotoxic chemicals break down or are

dispersed, often requiring a year or more. Bioassays of seed germination and
early seedling growth, on agar medium in petri dishes, were conducted to evaluate
autotoxic responses of 20 alfalfa germplasms to water-soluble extracts of alfalfa
leaf tissue. Root length, 120 hr after placing imbibed seed on agar, was more
sensitive to the autotoxin(s) than was hypocotyl length, germination speed, and
final germination percentage. Path coefficient analyses showed variation in root
length had 7-17 times more effect than variation in hypocotyl length in
determining autotoxic effects on total seedling length. Although variations in
seed size and germination rate were negatively associated (P < 0.05) with final
root length, the autotoxin had little effect on these factors relative to that on
root length. Germplasms in the control differed (P < 0.05) in root length,
requiring tolerance to be evaluated as percent of control. Germplasms, as percent
of control, differed significantly (P < 0.05) at extract concentrations of 1.0
and 4.0 g l(-1), but the range and LSD were more favorable for selection at 1.0 g
l(-1). Root length is appropriate for genetic assessments of tolerance to the
autotoxin when expressed as percent of control.
2273. FEMS Microbiol Lett. 2003 Dec 12;229(2):183-8.
Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be

efficient osmoprotectants in Sinorhizobium meliloti.
Barra L(1), Pica N, Gouffi K, Walker GC, Blanco C, Trautwetter A.
Author information:
(1)Osmorégulation chez les bactéries, CNRS UMR 6026, Université de Rennes I,
Campus de Beaulieu, Av. du Général Leclerc, 35042 Rennes, France.
Inactivation of the zwf gene in Sinorhizobium meliloti induces an osmosensitive

phenotype and the loss of osmoprotection by trehalose and sucrose, but not by
ectoine and glycine betaine. This phenotype is not linked to a defect in the
biosynthesis of endogenous solutes. zwf expression is induced by high osmolarity,
sucrose and trehalose, but is repressed by betaine. A zwf mutant is more
sensitive than its parental strain to superoxide ions, suggesting that glucose
6-phosphate dehydrogenase involvement in the osmotic response most likely results
from the production of reactive oxygen species during osmotic stress.

2274. Mikrobiologiia. 2003 Sep-Oct;72(5):633-8.
[The biological activity of the Sinorhizobium meliloti glucan].
Kosenko LV(1), Mikhalkiv LM, Krugova ED, Mandrovskaia NM, Zatovskaia TV, Kots'
SIa.
Author information:
(1)Zabolotnyi Institute of Microbiology and Virology, National Academy of
Sciences of Ukraine, ul. Zabolotnogo 154, Kiev, 03143 Ukraine.
The study of the effect of the periplasmic glucan isolated from the root-nodule
bacterium S. meliloti CXM1-188 on the symbiosis of another strain (441) of the
same root-nodule bacterium with alfalfa plants showed that this effect depends on
the treatment procedure. The pretreatment of alfalfa seedlings with the glucan
followed by their bacterization with S. meliloti 441 insignificantly influenced
the nodulation parameters of symbiosis (the number of root nodules and their
nitrogen-fixing activity) but induced a statistically significant increase in the
efficiency of symbiosis (expressed as the masses of the alfalfa overground parts
and roots). At the same time, the pretreatment of S. meliloti 441 cells with the
glucan brought about a considerable decrease in the nodulation parameters of
symbiosis (the number of the root nodules and their nitrogen-fixing activity
decreased by 2.5-11 and 7 times, respectively). These data suggest that the
stimulating effect of rhizobia on host plants may be due not only to
symbiotrophic nitrogen fixation but also to other factors. Depending on the
experimental conditions, the treatment of alfalfa plants with the glucan and
their bacterization with rhizobial cells enhanced the activity of peroxidase in
the alfalfa roots and leaves by 10-39 and 12-27%, respectively.
2275. J Anim Sci. 2003 Dec;81(12):3121-9.
Wet corn gluten feed and alfalfa hay combinations in steam-flaked corn finishing
cattle diets.
Sindt JJ(1), Drouillard JS, Titgemeyer EC, Montgomery SP, Coetzer CM, Farran TB,
Pike JN, Higgins JJ, Ethington RT.
Author information:
66506-1600, USA.
One finishing trial and one digestibility trial were used to evaluate wet corn
gluten feed (WCGF) and alfalfa hay (AH) combinations in steam-flaked corn (SFC)
finishing diets. In Exp. 1, 631 crossbred heifers (initial BW = 284 +/- 7.9 kg)
were fed SFC-based diets containing combinations of WCGF (25, 35, or 45% of diet
DM) and AH (2 or 6% of dietary DM) in a 2 x 3 factorial arrangement of
treatments. No interactions existed between WCGF and AH for heifer performance.
Increasing dietary WCGF linearly decreased gain efficiency (P < 0.01), dietary
NEg concentration (P < 0.05), and 12th-rib fat thickness (P = 0.10). Cattle fed
35% WCGF had the lowest occurrence of abscessed livers, resulting in a quadratic
response (P < 0.05) as dietary WCGF increased. In Exp. 2, 12 ruminally cannulated
Jersey steers (585 kg) were fed SFC-based diets containing combinations of WCGF
(25 or 45% of diet DM) and AH (0, 2, or 6% of diet DM) in an incomplete Latin
square design with a 2 x 3 factorial arrangement of treatments. Starch intake was
lower (P < 0.05), but NDF intake was greater (P < 0.05) as AH and WCGF increased
in the diet. Ruminal pH was increased by AH (linear, P < 0.05) and tended (P <
0.07) to increase with WCGF. Feeding 2% AH led to the greatest ruminal NH3 but
the lowest total VFA and propionate (quadratic, P < 0.05). Addition of AH to
diets containing 25% WCGF increased acetate to a greater extent than addition to
diets containing 45% WCGF (AH x WCGF interaction, P < 0.05). Feeding 45% WCGF
tended to increase passage rate (P = 0.17) and decrease (P < 0.05) total tract OM
digestibility but increase (P < 0.05) in situ degradation of DM from AH and WCGF.
Interactions between AH and WCGF existed (P < 0.05) for ruminal fluid volume
(quadratic effect of AH x WCGF level), in situ SFC degradation (linear effect of
AH x WCGF level), and in situ rate of WCGF DM disappearance (quadratic effect of
AH x WCGF level). We conclude that AH levels may be decreased when WCGF is added
to SFC diets as 25% or more of the dietary DM.
DOI: 10.2527/2003.81123121x
2276. J Dairy Sci. 2003 Nov;86(11):3611-9.
Effects of replacing chopped alfalfa hay with alfalfa silage in a total mixed
ration on production and rumen conditions of lactating dairy cows.
Calberry JM(1), Plaizier JC, Einarson MS, McBride BW.
Author information:
Canada N1G 2W1.
The effects of replacing chopped alfalfa hay with alfalfa silage in a total mixed
ration containing barley grain and corn silage on production and rumen conditions
were investigated. Cows received three diets that all contained (dry matter
basis) 38.5% barley grain-based energy supplement, 30.5% corn silage, 17.0%
protein supplement, and 4.2% sunflower seeds. One diet contained (dry matter
basis) 9.8% of chopped alfalfa hay and no alfalfa silage. One diet contained (dry
matter basis) 4.9% chopped alfalfa hay and 4.9% alfalfa silage. One diet
contained (dry matter basis) 9.8% of alfalfa silage and no chopped alfalfa hay.
Contents of crude protein, neutral detergent fiber, acid detergent fiber, and
starch, averaged across diets, were 16.7, 41.3, 21.1, and 24.4% DM, respectively,
and did not differ significantly among diets. Replacing chopped alfalfa hay with
alfalfa silage decreased the proportion of dietary DM passing through the 8-mm
screen of the Penn State Particle Separator from 61.9 to 55.2% dry matter and
significantly increased dietary physical effective NDF (peNDF) content,
calculated as the NDF retained by the two screens of the Penn State Particle
Separator, from 20.1 to 23.3% DM. Replacing chopped alfalfa hay with alfalfa
silage also reduced dietary DM content, increased rumen pH from 6.27 to 6.47,
reduced volatile fatty acid concentrations, numerically increased milk fat
concentration and milk fat yield. Milk yield, milk protein concentration, dry
matter intake, and rumen ammonia concentration were not affected.
DOI: 10.3168/jds.S0022-0302(03)73967-8
2277. Proc Biol Sci. 2003 Nov 7;270 Suppl 2:S209-12.
Facultative symbionts are associated with host plant specialization in pea aphid
populations.
Leonardo TE(1), Muiru GT.
Author information:
(1)Center for Population Biology, University of California at Davis, Davis, CA
95616, USA. teleonardo@ucdavis.edu
The pea aphid, Acyrthosiphon pisum, shows significant reproductive isolation and
host plant specialization between populations on alfalfa and clover in New York.
We examine whether specialization is seen in pea aphids in California, and
whether fitness on alternative host plants is associated with the presence of
bacterial symbionts. We measured the fitness of alfalfa- and clover-derived
aphids on both types of plants and found no evidence for specialization when all
aphid lineages were considered simultaneously. We then screened all aphids for
the presence of four facultative bacterial symbionts: PAR, PASS, PABS and PAUS.
Aphids with PAUS were host-plant specialized, having twice as many offspring as
other aphids on clover, and dying on alfalfa. Other aphids showed no evidence of
specialization. Additionally, aphids with PABS had 50% more offspring than aphids
with PASS when on alfalfa. Thus, specialist and generalist aphid lineages
coexist, and specialization is symbiont associated. Further work will resolve
whether PAUS is directly responsible for this variation in fitness or whether
PAUS is incidentally associated with host-plant specialized aphid lineages.
DOI: 10.1098/rsbl.2003.0064
PMCID: PMC1809968
2278. FEMS Microbiol Lett. 2003 Dec 5;229(1):23-30.
Phylogenetic analysis of fiber-associated rumen bacterial community and PCR

detection of uncultured bacteria.
Koike S(1), Yoshitani S, Kobayashi Y, Tanaka K.
Author information:
(1)Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
The fiber-associated rumen bacterial community was phylogenetically examined by

analysis of 16S rRNA gene (16S rDNA) sequences. Hay stems of orchardgrass and
alfalfa were incubated for 6 and 20 h, respectively in the rumen of two different
sheep, and total DNA was extracted from the incubated stems to clone bacterial
16S rDNAs using polymerase chain reaction (PCR). Of 91 such clones, 21 showed
more than 97% sequence similarity with known isolates, 32 clones had 90-97%
similarity with known sequences, and for the remaining 38 clones, the similarity
was less than 90%. The majority of clones fell into the
Cytophaga-Flavobacter-Bacteroides and low G+C Gram-positive bacterial phyla (43
and 44%, respectively). Prevotella-related and Butyrivibrio fibrisolvens-related
sequences formed large clusters in the phylogenetic tree. Unknown sequences were
found to form three unique clusters, one of which was suggested by
semi-quantitative PCR to be more prevalent in the rumen receiving a high alfalfa
diet.
2279. Crit Rev Food Sci Nutr. 2003;43(5):481-506.

Towards sustainable production of protein-rich foods: appraisal of eight crops
for Western Europe. PART II: Analysis of the technological aspects of the
production chain.
Dijkstra DS(1), Linnemann AR, van Boekel TA.
Author information:
(1)Product Design and Quality Management Group, Agrotechnology and Food Sciences,
Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Increased production of plant protein is required to support the production of

protein-rich foods which can replace meat in the human diet to reduce the strain
that intensive animal husbandry poses on the environment. The suitability of
lupin (Lupinus spp.), pea (Pisum sativum), quinoa (Chenopodium quinoa Willd.),
triticale (x Triticosecale), lucerne (Medicago sativa), grasses (Lolium and
Festuca spp.), rapeseed/canola (Brassica napus) and potato (Solanum tuberosum)
for protein production in Western Europe was studied on the basis of a
chain-approach. The technological aspects, which are considered in this paper,
are the processing methods, and the functional and nutritional properties of the
derived protein products. The overall evaluation of the technological prospects
of the eight crops as a protein source for Western Europe leads to the conclusion
that this part of the production chain is not decisive for that choice. Pea and
lupin have a slight advantage over the other crops, because their concentrates
and isolates are already commercially available.
DOI: 10.1080/10408690390246332
2280. Steroids. 2003 Dec;68(14):1147-55.
Synthesis of hapten and conjugates of coumestrol and development of immunoassay.
Lapcík O(1), Stursa J, Kleinová T, Vítková M, Dvoráková H, Klejdus B, Moravcová

J.
Author information:
(1)Department of Chemistry of Natural Compounds, Institute of Chemical
Technology, Technická 5, 166 28 Praha 6, Czech Republic. oldrich.lapcik@vscht.cz
3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a

partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized
with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by
column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR
data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective,
as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The
hapten was then conjugated to bovine serum albumin and used for immunization of
rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal
antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the
radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept:
140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a
panel isoflavonoid and lignan phytoestrogens was either negligible (e.g.
formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major
immunoreactive peak in HPLC fractions from an alfalfa extract had the same
retention time as coumestrol standard and represented 94.8% of the signal. The
remaining 5.2% of immunoreactivity was distributed between five minor peaks. We
conclude that after the validation for particular matrices, the method will be a
useful tool for analysis of coumestrol, especially in low volume and low
concentration samples.
2281. Am J Transplant. 2003 Dec;3(12):1608-9.
Acute renal transplant rejection possibly related to herbal medications.
Light TD(1), Light JA.
Author information:
(1)Section of Transplant, Department of Surgery, Washington Hospital Center,
Washington, DC, USA. timlightmd@aol.com
Use of herbal and alternative medications in the United States is increasing.

Many of these medications have unknown mechanisms of actions, and possible
metabolic interactions with prescribed medications. We report a case of late
acute rejection after exposure to two popular herbal medications.
2282. J Food Prot. 2003 Nov;66(11):2017-22.
Reduction of Salmonella enterica on alfalfa seeds with acidic electrolyzed

oxidizing water and enhanced uptake of acidic electrolyzed oxidizing water into
seeds by gas exchange.
Stan SD(1), Daeschel MA.
Author information:
(1)Department of Food Science and Technology, Oregon State University, 100
Wiegand Hall, Corvallis, Oregon 97331-8575, USA.
Alfalfa sprouts have been implicated in several salmonellosis outbreaks in recent

years. The disinfectant effects of acidic electrolyzed oxidizing (EO) water
against Salmonella enterica both in an aqueous system and on artificially
contaminated alfalfa seeds were determined. The optimum ratio of seeds to EO
water was determined in order to maximize the antimicrobial effect of EO water.
Seeds were combined with EO water at ratios (wt/vol) of 1:4, 1:10, 1:20, 1:40,
and 1:100, and the characteristics of EO water (pH, oxidation reduction potential
[ORP], and free chlorine concentration) were determined. When the ratio of seeds
to EO water was increased from 1:4 to 1:100, the pH decreased from 3.82 to 2.63,
while the ORP increased from +455 to +1,073 mV. EO water (with a pH of 2.54 to
2.38 and an ORP of +1,083 to +1,092 mV) exhibited strong potential for the
inactivation of S. enterica in an aqueous system (producing a reduction of at
least 6.6 log CFU/ml). Treatment of artificially contaminated alfalfa seeds with
EO water at a seed-to-EO water ratio of 1:100 for 15 and 60 min significantly
reduced Salmonella populations by 2.04 and 1.96 log CFU/g, respectively (P <
0.05), while a Butterfield's buffer wash decreased Salmonella populations by 0.18
and 0.23 log CFU/g, respectively. After treatment, EO water was Salmonella
negative by enrichment with or without neutralization. Germination of seeds was
not significantly affected (P > 0.05) by treatment for up to 60 min in
electrolyzed water. The uptake of liquid into the seeds was influenced by the
internal gas composition (air, N2, or O2) of seeds before the liquid was added.
2283. J Exp Bot. 2004 Jan;55(394):43-7. Epub 2003 Nov 17.
A Medicago sativa haem oxygenase gene is preferentially expressed in root

nodules.
Baudouin E(1), Frendo P, Le Gleuher M, Puppo A.
Author information:
(1)Laboratoire de Biologie Végétale et Microbiologie, CNRS FRE 2294, Université
de Nice-Sophia Antipolis, Parc Valrose, F-06108 Nice cédex 02, France.
Haem oxygenases (HO) are ubiquitous enzymes catalysing the oxidative degradation
of haem into biliverdin, iron and carbon monoxide. Whereas animal HOs participate
in multiple cellular functions including haemoglobin catabolism, antioxidant
defence and iron homeostasis, to date, plant HOs have so far only been involved
in phytochrome metabolism. The expression of the HO1 gene was studied in Medicago
sativa, especially during the interaction with its symbiotic partner,
Sinorhizobium meliloti. Transcript accumulation was higher in mature root nodules
than in roots and leaves and was correlated to HO1 protein immunodetection. The
analysis of HO1 expression following alfalfa root inoculation with S. meliloti
indicates that transcripts do not accumulate during the early steps of symbiosis,
but rather in the mature nodules. These results correlate with the expression of
the leghaemoglobin gene, which encodes the major haem-containing protein present
in the nodule. Contrary to its animal counterpart, alfalfa HO1 was not induced by
pro- oxidant compounds including H(2)O(2), paraquat and sodium nitroprusside,
suggesting that it is not involved in the antioxidant defence. The results
suggest that HO1 could play a role in the alfalfa mature nodule and its
involvement in leghaemoglobin metabolism is hypothesized.
DOI: 10.1093/jxb/erh020
2284. Pest Manag Sci. 2003 Nov;59(11):1179-90.
Chlorpyrifos residue levels in avian food items following applications of a

commercial EC formulation to alfalfa and citrus.
Brewer LW(1), McQuillen HL Jr, Mayes MA, Stafford JM, Tank SL.
Author information:
(1)Springborn Smithers Laboratories, PO Box 2005, Sisters, OR 97759, USA.
lbrewer@springborn.com
Two 10-day field residue studies were conducted to measure the amount of
chlorpyrifos residue found in typical avian food following applications of a
commercial 480 g liter(-1) EC (Lorsban 4E) at 1.1 kg AI ha(-1) (1 lb AI acre(-1))
to alfalfa and at 2.3 kg Al ha(-1) (2.0 lb AI acre(-1)) to citrus. Avian food
items used in these studies included: crickets (Acheta domestica (L)), earthworms
(Lumbricus terrestris L), darkling ground beetle larvae (Tenebrio molitor L),
seed heads (Triticum sp), and naturally occurring flying and ground-dwelling
insects. The studies incorporated a design involving three main study plots
placed within larger treated areas of an alfalfa crop and a mature orange grove.
The three main study plots represented three replications and each contained four
sub-plots. One sub-plot, on each study plot, was sampled on day 0 (2-h
post-application), day 1, day 5 and day 10 post-application. Chlorpyrifos
residues were present in all avian food sampled following the application;
however, residue levels were lower than estimated residue values typically used
by the US EPA to establish expected environmental concentration (EEC) used in
screening assessments of risk to terrestrial wildlife.
DOI: 10.1002/ps.752
2285. J Dairy Sci. 2003 Oct;86(10):3300-8.
Utilization of phosphorus in lactating cows fed varying amounts of phosphorus and

forage.
Wu Z(1), Tallam SK, Ishler VA, Archibald DD.
Author information:
University Park, PA 16802, USA. ziw1@psu.edu
The objective of this study was to determine the effect of dietary forage
proportion and P content on fecal P excretion. Four dietary treatments were
formed in a 2 x 2 factorial arrangement. The P content was 0.33 or 0.42%, and the
forage proportion was 48 or 58% on a dry matter (DM) basis. The neutral detergent
fiber content was 27 and 30% for the low and high forage diets, respectively. The
P amount was varied by using monosodium phosphate, and the forage amount by
changing the proportions of alfalfa silage and corn. The diets were fed to 44
midlactation Holsteins for 14 wk. Fecal P excretion was estimated using Cr marker
and grab sampling. Dietary P content did not affect DM intake, milk yield, or
milk composition. The P intake averaged 74 and 96 g/d and fecal P averaged 0.69
and 0.92% (DM basis) or 49 and 65 g/d for the low and high P diets, respectively.
Thus, reducing dietary P from 0.42 to 0.33% resulted in approximately 25% less
estimated fecal P excretion. Increasing dietary forage reduced milk yield (34.0
vs. 36.5 kg/d), but increased milk fat content (3.66 vs. 3.25%). Estimated
apparent digestibility of P tended to decrease (31.1 vs. 36.6%) when the forage
proportion increased, but most of the change occurred when the diets contained
the low amount of P. Overall, the effect of forage proportion on estimated fecal
P excretion was small when diets contained 48 or 58% forage, varied by alfalfa
silage. Phosphorus intake has a much larger impact on fecal P excretion than
forage proportion, and it does not seem necessary to adjust the dietary P content
according to the forage proportion to provide the same amount of absorbed P.
DOI: 10.3168/jds.S0022-0302(03)73931-9
2286. Phytochemistry. 2003 Nov;64(5):1013-21.
Profiling phenolic metabolites in transgenic alfalfa modified in lignin

biosynthesis.
Chen F(1), Duran AL, Blount JW, Sumner LW, Dixon RA.
Author information:
Parkway, Ardmore, OK 73401, USA. fchen@noble.org
Soluble phenolics, wall-bound phenolics and soluble and core lignin were analyzed
in transgenic alfalfa with genetically down-regulated O-methyltransferase genes
involved in lignin biosynthesis. High performance liquid chromatography and
principal component analysis were used to distinguish metabolic phenotypes of
different transgenic alfalfa genotypes growing under standard greenhouse
conditions. Principal component analysis of HPLC chromatograms did not resolve
differences in leaf metabolite profiles between wild-type and transgenic plants
of the same genetic background, although stem phenolic profiles were clearly
different between wild-type and transgenic plants. However, the analytical
methods clearly differentiated two non-transgenic alfalfa cultivars based on
either leaf or stem profiles. Metabolic profiling provides a useful approach to
monitoring the broader biochemical phenotypes of transgenic plants with altered
expression of lignin pathway enzymes.
2287. J Agric Food Chem. 2003 Oct 22;51(22):6521-9.
Soil amino acid composition quantified by acid hydrolysis and anion

chromatography-pulsed amperometry.
Martens DA(1), Loeffelmann KL.
Author information:
(1)Southwest Watershed Research Center, Agricultural Research Service, U.S.
Department of Agriculture, 2000 East Allen Road, Tucson, AZ 85719, USA.
dmartens@tucson.ars.ag.gov
Soil organic N accounts for 95-98% of the total soil N content with amino acids
(AAs) and amino sugars (ASs) identified as the major soil organic N compounds,
but traditional 6 M HCl with reflux or sealed digestions for 24 h and various
detection systems have accounted for only 30-40% of soil total N content as AA-N.
This study compared traditional HCl extraction methodology with methanesulfonic
acid (MSA) hydrolysis and nonderivatized AA and AS quantification by ion
chromatography with pulsed amperometric detection for determination of the AA
composition of plant litter and soils. MSA (4 M) gave AA-N recovery comparable to
or better than 6 M HCl for plant AA digestions (16 h, 121 degrees C, 104 kPa).
Use of 4 M MSA (0.5-1.5 h, 136 degrees C, 112 kPa) increased the total recovery
of organic N as AAs, ASs, and NH(4)(+) by 46% from soils (n = 22) compared with 6
M HCl (12 h, 110 degrees C, reflux) with a MSA recovery rate of 85.6% of the
total N content (n = 22 soils). The shorter MSA soil digestions (0.5-1.5 h)
suggested that the majority of soil organic N was not present as protein forms
found in plant litter analysis (16 h of digestion). MSA ion chromatographic
analysis for soil AA/AS composition is a robust nonderivatization method
requiring little sample preparation that can distinguish between small changes in
soil AA composition during one growing season due to vegetation and tillage
managements.
DOI: 10.1021/jf034422e
2288. Mol Plant Microbe Interact. 2003 Oct;16(10):884-92.
Identical accumulation and immobilization of sulfated and nonsulfated Nod factors

in host and nonhost root hair cell walls.
Goedhart J(1), Bono JJ, Bisseling T, Gadella TW Jr.

Author information:
(1)Laboratory for Molecular Biology, MicroSpectroscopy Center Wageningen,
Department of Plant Sciences, Wageningen University, Dreijenlaan 3, 6703 HA,
Wageningen, The Netherlands.
Nod factors are signaling molecules secreted by Rhizobium bacteria. These

lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can
elicit specific responses at subnanomolar concentrations on a compatible host.
How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor
analogs, we investigated whether sulfated and nonsulfated Nod factors were bound
and perceived differently by Medicago truncatula and Vicia sativa root hairs. The
bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair
deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM.
Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that
sulfated fluorescent Nod factors accumulate in the cell wall of root hairs,
whereas they are absent from the plasma membrane when applied at 10 nM. When the
fluorescent Nod factor distribution in medium surrounding a root was studied, a
sharp decrease in fluorescence close to the root hairs was observed, visualizing
the remarkable capacity of root hairs to absorb Nod factors from the medium.
Fluorescence correlation microscopy was used to study in detail the mobilities of
sulfated and nonsulfated fluorescent Nod factors which are biologically active on
M. truncatula and V. sativa, respectively. Remarkably, no difference between
sulfated and nonsulfated Nod factors was observed: both hardly diffuse and
strongly accumulate in root hair cell walls of both M. truncatula and V. sativa.
The implications for the mode of Nod factor perception are discussed.
DOI: 10.1094/MPMI.2003.16.10.884
2289. J Anim Sci. 2003 Oct;81(10):2628-38.
Screening of exogenous enzymes for ruminant diets: relationship between

biochemical characteristics and in vitro ruminal degradation.
Colombatto D(1), Morgavi DP, Furtado AF, Beauchemin KA.
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1.
colombat@agro.uba.ar
With the objective of developing a rational approach for the selection of feed
enzymes for ruminants, 22 commercial enzyme products were examined in terms of
protein concentration, enzymic activities on model substrates, and hydrolytic
capacity, the latter determined from the release of reducing sugars from alfalfa
hay and corn silage. An in vitro ruminal degradation assessment was carried out
using the same substrates, untreated or treated with the 22 enzyme products at
1.5 microL/g forage DM. Stepwise regressions were then performed to establish
relationships between these factors. Protein concentration and enzymic activities
explained at least 84% (P < 0.01) of the variation in the release of reducing
sugars from alfalfa and corn silage. Alfalfa DM degradation after incubation with
ruminal fluid for 18 h was positively related to xylanase activity (R2 = 0.29, P
< 0.01), but the same activity was negatively related to DM degradation of corn
silage (R2 = 0.19, P < 0.05). Protease activity explained a further 10% of the
alfalfa DM degradation (P < 0.10). Following sequential steps involving the
determination of rate and extent of DM and fiber degradation, the best candidates
for alfalfa and corn silage were selected. Enzyme products effective with alfalfa
hay seemed to exert part of their effect during the pretreatment period, whereas
enzymes effective with corn silage worked exclusively after ruminal fluid was
added. This finding suggests that different modes of action of exogenous enzymes
are attacking different substrates and may partly explain enzyme-feed
specificity. In alfalfa, it seems that effective enzymes work by removing
structural barriers that retard the microbial colonization of digestible
fractions, increasing the rate of degradation. In corn silage, effective enzymes
seem to interact with ruminal enzymes to degrade the forage more rapidly, which
is consistent with previous findings of synergism between exogenous and ruminal
enzymes.
DOI: 10.2527/2003.81102628x
2290. J Anim Sci. 2003 Oct;81(10):2617-27.
Effects of enzyme supplementation of a total mixed ration on microbial

fermentation in continuous culture, maintained at high and low pH.
Colombatto D(1), Hervás G, Yang WZ, Beauchemin KA.
Author information:
(1)Agriculture and Agri-Food Canada, Lethbridge, AB, Canada T1J 4B1.
colombat@agro.uba.ar
A dual-flow continuous culture system was used to investigate the effects of pH

and addition of an enzyme mixture to a total mixed ration (TMR) on fermentation,
nutrient digestion, and microbial protein synthesis. A 4 x 4 Latin square design
with a factorial arrangement of treatments was used, with four 9-d periods
consisting of 6 d for adaptation and 3 d for measurements. Treatments were as
follows: 1) high pH with control TMR, 2) high pH with TMR treated with enzyme, 3)
low pH with control TMR, and 4) low pH with TMR treated with enzyme. Ranges of pH
were 6.0 to 6.6 and 5.4 to 6.0 for high and low, respectively. Fermenters were
fed twice daily a TMR consisting of 30% alfalfa hay, 30% corn silage, and 40%
rolled corn (DM basis). The silage was milled fresh and the TMR was fed to the
fermenters in fresh form (64% DM). The enzyme mixture was a commercial product of
almost exclusive protease activity; it was applied daily to the fresh TMR and
stored at 4 degrees C for at least 12 h before feeding. Degradability of OM, NDF,
ADF, and cellulose was decreased (P < 0.05) by low pH. Hemicellulose and protein
degradation were not affected by pH. Enzyme addition increased (P < 0.01) NDF
degradability (by 43% and 25% at high and low pH, respectively), largely as a
result of an increase in hemicellulose degradation (by 79% and 51% at high and
low pH, respectively). This improvement was supported by an increase (P < 0.05)
in the xylanase and cellulase activities in the liquid phase of the fermenter
contents. Total VFA were decreased (P < 0.05) by low pH, but were not affected by
enzyme addition. Total bacterial numbers were increased (P < 0.03) at low pH and
tended (P < 0.13) to increase with enzyme addition. Cellulolytic bacteria in
effluent fluid were decreased (P < 0.02) at low pH but were unaffected by enzyme
addition. Despite a large increase (P < 0.001) in protease activity, protein
degradation was only numerically increased by enzyme addition. Microbial protein
synthesis was higher (P < 0.10) at high pH but was not affected by enzyme
addition. Methane production, expressed as a proportion of total gases, was
decreased (P < 0.001) at low pH but was not affected by enzyme addition. It is
concluded that it is possible to adapt the continuous culture system to use fresh
feeds instead of dried feeds. Overall, the results indicate that the enzyme
product used in this study has a potential to increase fiber degradability
without increasing methane production.
DOI: 10.2527/2003.81102617x
2291. Vet Res Commun. 2003 Sep;27 Suppl 1:643-6.
Effects of the dietary inclusion of vegetable fat and dehydrated alfalfa meal on
the technological properties of rabbit meat.
Cavani C(1), Betti M, Bianchi M, Petracci M.
Author information:
(1)Department of Food Science, Alma Mater Studiorum, University of Bologna,
Italy. cavani@foodsci.unibo.it
2292. J Environ Qual. 2003 Sep-Oct;32(5):1873-80.
On-farm assessment of biosolids effects on soil and crop tissue quality.
Shober AL(1), Stehouwer RC, Macneal KE.
Author information:
(1)Department of Plant and Soil Science, University of Delaware, Newark, DE
19717, USA. ashober@udel.edu
Agronomic use of biosolids as a fertilizer material remains controversial in part

due to public concerns regarding the potential pollution of soils, crop tissue,
and ground water by excess nutrients and trace elements in biosolids. This study
was designed to assess the effects of long-term commercial-scale application of
biosolids on soils and crop tissue sampled from 18 production farms throughout
Pennsylvania. Biosolids application rates ranged from 5 to 159 Mg ha(-1) on a dry
weight basis. Soil cores and crop tissue samples from corn (Zea mays L.), soybean
(Glycine spp.), alfalfa (Medicago sativa L.), orchardgrass (Dactylis spp.) hay,
and/or sorghum [Sorghum bicolor (L.) Moench] were collected for three years from
georeferenced locations at each farm. Samples were tested for nutrients, trace
elements, and other variables. Biosolids-treated fields had more post-growing
season soil NO3 and Ca and less soil K than control fields and there was some
evidence that soil P concentrations were higher in treated fields. The soil
concentrations of Cu, Cr, Hg, Mo, Mn, Pb, and Zn were higher in biosolids-treated
fields than in control fields; however, differences were < or = 0.06 of the USEPA
Part 503 cumulative pollutant loading rates (CPLRs). There were no differences in
the concentrations of measured nutrients or trace elements in the crop tissue
grown on treated or control fields at any time during the study. Commercial-scale
biosolids application resulted in soil trace element increases that were in line
with expected increases based on estimated trace element loading. Excess NO3 and
apparent P buildup indicates a need to reassess biosolids nutrient management
practices.
2293. J Am Mosq Control Assoc. 2001 Sep;17(3):206-8.
Effect of some animal feeds and oviposition substrates on Aedes oviposition in

ovitraps in Cairns, Australia.
Ritchie SA(1).
Author information:
(1)Tropical Public Health Unit, Queensland Health, PO Box 1103, Cairns 4870,
Queensland, Australia.
Animal feed pellets containing lupin seed or alfalfa were added to ovitraps set
in Cairns, Australia. Although they collected fewer Aedes eggs than Centers for
Disease Control enhanced ovitraps, they did outperform tap water alone. A wooden
tongue depressor collected comparable number of Aedes eggs as a Masonite board
and seed germination paper.
2294. Int J Food Microbiol. 2003 Nov 1;87(3):301-6.
Efficacy of chlorine and calcinated calcium treatment of alfalfa seeds and

sprouts to eliminate Salmonella.
Gandhi M(1), Matthews KR.
Author information:
(1)Department of Food Science, Cook College, Rutgers, The State University of New
Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520, USA.
The efficacy of a 20,000 ppm calcium hypochlorite treatment of alfalfa seeds

artificially contaminated with Salmonella was studied. Salmonella populations
reached >7.0 log on sprouts grown from seeds artificially contaminated with
Salmonella and then treated with 20,000 ppm Ca(OCl)(2). The efficacy of spray
application of chlorine (100 ppm) to eliminate Salmonella during germination and
growth of alfalfa was assessed. Alfalfa seed artificially contaminated with
Salmonella was treated at germination, on day 2 or day 4, or for the duration of
the growth period. Spray application of 100 ppm chlorine at germination, day 2,
or day 4 of growth was minimally effective resulting in approximately a 0.5-log
decrease in population of Salmonella. Treatment on each of the 4 days of growth
reduced populations of Salmonella by only 1.5 log. Combined treatment of seeds
with 20,000 ppm Ca(OCl)(2) and followed by 100 ppm chlorine or calcinated calcium
during germination and sprout growth did not eliminate Salmonella.
2295. Plant Physiol. 2003 Oct;133(2):773-82. Epub 2003 Sep 25.
Functional characterization and expression of a cytosolic iron-superoxide

dismutase from cowpea root nodules.
Moran JF(1), James EK, Rubio MC, Sarath G, Klucas RV, Becana M.
Author information:
Superior de Investigaciones Científicas, Apdo 202, 50080 Zaragoza, Spain.
An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization,

VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves.
The enzyme has two identical subunits of 27 kD that are not covalently bound.
Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid
sequence showed that VuFeSOD is synthesized as a precursor with seven additional
amino acids. The mature protein was overexpressed in Escherichia coli, and the
recombinant enzyme was used to generate a polyclonal monospecific antibody.
Phylogenetic and immunological data demonstrate that there are at least two types
of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean
(Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa
(Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also
contain FeSODs in the leaves and nodules, but the enzymes are presumably
localized to the chloroplasts and plastids. In contrast, immunoblots of the
soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule
sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot
analysis showed that the content of VuFeSOD protein increases in senescent
nodules with active leghemoglobin degradation, suggesting a direct or indirect
(free radical-mediated) role of the released Fe in enzyme induction. Therefore,
contrary to the widely held view, FeSODs in plants are not restricted to the
chloroplasts and may become an important defensive mechanism against the
oxidative stress associated with senescence.
DOI: 10.1104/pp.103.023010
PMCID: PMC219051
2296. J Dairy Sci. 2003 Sep;86(9):2940-8.
Fatty acid composition of mixed-rumen bacteria: effect of concentration and type

of forage.
Bas P(1), Archimède H, Rouzeau A, Sauvant D.
Author information:
(1)UMR de Physiologie de la Nutrition et Alimentation, INRA-INAPG, 16 rue Claude
Bernard, 75231 Paris Cedex 05, France. bas@inapg.inra.fr
The effects of concentration and type of forage in the diet on lipid content and
fatty acid (FA) composition of rumen bacteria were studied in 14 goats fitted
with duodenal cannulas. The goats were fed a complete maintenance diet containing
40, 70, or 100% chopped forage (dry matter basis) in two equal meals. Forage was
either corn stover or alfalfa hay. Microbial cell matter (MCM) was isolated by
differential centrifugation of duodenal contents. The FA content of the MCM
varied from 5 to 11% of DM and decreased with forage level in the diet. Main FA
in MCM were C18:0 and C16:0: together they accounted for 70% of total FA in MCM.
The mono-unsaturated FA and branched-chain FA (iso-FA and anteiso-FA) each
represented about 10% of FA by weight. The proportion of even-chain saturated FA
decreased and those of odd- and branched-chain FA increased with increasing
forage. With the corn stover-based diet even-chain saturated FA were lower than
with the alfalfa hay-based diet, whereas the unsaturated FA, odd-chain FA, and
branched-chain FA were higher. The neutral detergent fiber content of the diet
seemed to explain most of the variation associated with even-chain saturated FA,
and odd- and branched-chain FA. Our results suggest that, for diets not
supplemented with fat, mixed rumen bacteria accumulated energy reserves, by
increasing synthesis of either even-chain saturated FA, or saturated odd-chain FA
and saturated branched-chain FA.
DOI: 10.3168/jds.S0022-0302(03)73891-0
Within-field manipulation of potato leafhopper (Homoptera: Cicadellidae) and

insect predator populations using an uncut alfalfa strip.
Weiser LA(1), Obrycki JJ, Giles KL.
Author information:
(1)Department of Entomology, Iowa State University, Ames, IA 50011, USA.
weise023@tc.umn.edu
The effects of a 3-m wide uncut alfalfa strip on the within field distribution of
Empoasca fabae (Harris) (Homoptera: Cicadellidae) and several species of insect
predators were examined for 3 yr (1998-2000). The objectives were to determine
whether this uncut strip would serve as a trap crop for E. fabae and a refuge for
insect predators at first harvest. Empoasca fabae and insect predators in the
families Coccinellidae, Nabidae, Anthocoridae, Chrysopidae, and Hemerobiidae were
collected weekly using sweep samples and sticky traps from the uncut strips and
up to 40 m into the alfalfa regrowth. For 2 yr, both small- (0.34 ha) and
large-scale (approximately 11.3 ha) field trials showed higher numbers of E.
fabae in 73% of the uncut strips for 2-3 wk after harvest. Similarly, the number
of insect predators found within <50% of the uncut strips was also higher during
the first or second week after harvest. In 1999, however, we did not observe
higher numbers of E. fabae in the uncut strips. Differences may be because of
higher E. fabae population numbers in 1999 compared with 1998 and 2000. This
research provides alfalfa growers a potential cultural management technique for
E. fabae while conserving predatory insects.
2298. Oncogene. 2003 Sep 18;22(40):6231-42.
Inhibition of PI-3 kinase sensitizes human leukemic cells to histone deacetylase

inhibitor-mediated apoptosis through p44/42 MAP kinase inactivation and
abrogation of p21(CIP1/WAF1) induction rather than AKT inhibition.
Rahmani M(1), Yu C, Reese E, Ahmed W, Hirsch K, Dent P, Grant S.
Author information:
(1)Department of Medicine, Medical College of Viriginia, Virginia Commonwealth
University, Richmond, VA 23298, USA.
Effects of the PI-3 kinase inhibitor LY294002 (LY) have been examined in relation
to responses of human leukemia cells to histone deacetylase inhibitors (HDIs).
Coexposure of U937 cells for 24 h to marginally toxic concentrations of LY294002
(e.g., 30 microM) and sodium butyrate (SB; 1 mM) resulted in a marked increase in
mitochondrial damage (e.g., cytochrome c and Smac/DIABLO release, loss of
DeltaPsi(m)), caspase activation, and apoptosis. Similar results were observed in
Jurkat, HL-60, and K562 leukemic cells and with other HDIs (e.g., SAHA, MS-275).
Exposure of cells to SB/LY was associated with Bcl-2 and Bid cleavage, XIAP and
Mcl-1 downregulation, and diminished CD11b expression. While LY blocked
SB-mediated Akt activation, enforced expression of a constitutively active
(myristolated) Akt failed to attenuate SB/LY-mediated lethality. Unexpectedly,
treatment of cells with SB+/-LY resulted in a marked reduction in phosphorylation
(activation) of p44/42 mitogen-activated protein (MAP) kinase. Moreover, enforced
expression of a constitutively active MEK1 construct partially but significantly
attenuated SB/LY-induced apoptosis. Lastly, cotreatment with LY blocked
SB-mediated induction of p21(CIP1/WAF1); moreover, enforced expression of
p21(CIP1/WAF1) significantly reduced SB/LY-mediated apoptosis. Together, these
findings indicate that LY promotes SB-mediated apoptosis through an
AKT-independent process that involves MEK/MAP kinase inactivation and
interference with p21(CIP1/WAF1) induction.
DOI: 10.1038/sj.onc.1206646
2299. Br J Nutr. 2003 Sep;90(3):625-34.
Influence of diet on growth yields of rumen micro-organisms in vitro and in vivo:

influence on growth yield of variable carbon fluxes to fermentation products.
Blümmel M(1), Karsli A, Russell JR.
Author information:
(1)Institute for Animal Production in the Tropics and Subtropics (480),
University of Hohenheim, 70599 Stuttgart, Germany. m.blummel@cgiar.org
The efficiency of rumen microbial production (EMP) in vitro and in vivo was
examined for three roughages (lucerne (Medicago sativa L.) hay, oat (Avenia
sativa L.)-berseem clover (Trifolium alexandrinum cultivar BigBee) hay and maize
(Zea mays L.) crop residue (MCR)) and for five isonitrogenous (106 g crude
protein (Nx6.25)/kg) diets formulated from lucerne hay, oat-berseem clover hay,
MCR, soybean meal and maize grain to provide degradable intake protein for the
production of 130 g microbial protein/kg total digestible nutrients. EMP in vivo
was determined by intestinal purine recovery in sheep and ranged from 240 to 360
g microbial biomass/kg organic matter truly degraded in MCR and in one of the
diets respectively (P<0.05). EMP in vitro was estimated by the substrate degraded
: gas volume produced thereby (termed partitioning factor, PF (mg/ml)) at times
of estimated peak microbial production and after 16.0 and 24.0 h of incubation.
For the diets, PF values were significantly related to EMP in vivo at peak
microbial production (P=0.04), but not after 16.0 (P=0.08) and 24.0 h (P=0.66).
For roughages, PF values were significantly related to EMP in vivo only when
measured after 16.0 h (P=0.04). For MCR and diets, a close non-linear
relationship was found between PF values at peak microbial production and EMP in
vivo (R(2) 0.99, P<0.0001) suggesting a maximum EMP in vivo of 0.39. Low gas
production per unit substrate degraded (high PF) was associated with high EMP in
vivo. The in vitro study of the products of fermentation, short-chain fatty
acids, gases and microbial biomass (by purine analysis) after 16.0 h of
incubation showed very strong relationships (R(2)> or =0.89, P<0.0001) between
short-chain fatty acids, gases and gravimetrically measured apparent
degradability. Except for maize grain, the true degradability of organic matter
estimated by neutral-detergent solution treatment agreed with the sum of the
products of fermentation (R(2) 0.81, P=0.0004). After 16.0 h of incubation, the
synergistic effects of diet ingredient on diets were greater for microbial
biomass (18 %) than for short-chain fatty acids and gas production (7 %). It is
concluded that measurement of gas production only gives incomplete information
about fodder quality; complementation of gas measurements by true degradability
measurements is recommended.
2300. Plant Physiol. 2003 Oct;133(2):538-48. Epub 2003 Aug 21.

Altered life cycle in Arabidopsis plants expressing PsUGT1, a
UDP-glucuronosyltransferase-encoding gene from pea.
Woo HH(1), Faull KF, Hirsch AM, Hawes MC.
Author information:
(1)Division of Plant Pathology and Microbiology, Department of Plant Sciences,
University of Arizona, Tucson, AZ 85721, USA. hhwoo@ucla.edu
Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine
molecular mechanisms underlying developmental effects of a microsomal
UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1).
Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower
mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root
growth, and increased lateral root development. The timing of root emergence in
wild-type and antisense plants was correlated with the transient accumulation of
auxin at the site of root emergence. Cell suspension cultures derived from the
antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the
wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced
into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression
occurs in regions of active cell division. This includes the root cap and root
apical meristems, leaf primordia, tips of older leaves, and the transition zone
between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with
the expression of the auxin-responding reporter DR5::uidA. Co-expression of
DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed
that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in
endogenous auxin gradients in roots. Roots of ecotype Columbia expressing
CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic
acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7
weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct
this early senescence phenotype.
DOI: 10.1104/pp.103.026278
PMCID: PMC219030
2301. Plant J. 2003 Sep;35(6):763-70.
Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa

root cultures under hypoxic stress.
Dordas C(1), Hasinoff BB, Igamberdiev AU, Manac'h N, Rivoal J, Hill RD.
Author information:
(1)Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, R3T
2N2 Canada.
Transgenic alfalfa root cultures expressing sense and antisense barley hemoglobin
transcripts were examined under varying levels of atmospheric oxygen. Root
cultures overexpressing the hemoglobin gene (Hb+) maintained root growth when
placed under 3% oxygen, whereas control cultures or cultures underexpressing
hemoglobin (Hb-) experienced 30-70% declines in growth under the same conditions.
ATP levels and ATP/ADP ratios for Hb+ lines did not significantly differ in 40
and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb- lines
were significantly lower under 3% oxygen. Large increases in the production of
nitric oxide (NO) were measured in root cultures grown under hypoxic conditions
compared to aerobic conditions. The amount of NO accumulated in an Hb- line was
2.5-fold higher than that in the Hb+ line. Treatment of transgenic root lines
under 40% oxygen with NO resulted in significant declines in the ATP levels and
ATP/ADP ratio of an Hb- line and the control line, with no significant change in
an Hb+ line. The root cell structure of an Hb- line showed evidence of cell
breakdown under hypoxic growth, whereas an Hb+ line had no evidence of cell
breakdown under similar growth conditions. These results lead us to hypothesize
that NO is involved in the response of plants to hypoxia and that hemoglobin
modulates the levels of NO in the hypoxic cell.
2302. Ann Bot. 2003 Nov;92(5):679-88. Epub 2003 Sep 10.
Vertical leaf nitrogen distribution in relation to nitrogen status in grassland

plants.
Lötscher M(1), Stroh K, Schnyder H.
Author information:
(1)Lehrstuhl für Grünlandlehre, Technische Universität München, D-85350 Freising,
Germany. loetscher@wzw.tum.de
Vertical gradients of leaf nitrogen (N) per unit leaf area (NLA) are viewed as
plastic responses that optimize N utilization with respect to carbon
assimilation. However, it has been shown that plant species, sowing density and N
availability affect the steepness of the NLA gradient relative to the photon flux
density (PFD) gradient. This paper tests the hypothesis that such variation is
related to the N status of the plant. The N status was analysed using the concept
of the critical N concentration (Ncrit) in which shoot N per unit dry mass (NSM)
decreases with shoot mass, and a negative deviation of actual NSM from Ncrit
indicates N shortage in the plant. The hypothesis was tested with contrasting
grassland species Medicago sativa, Dactylis glomerata and Taraxacum officinale by
varying PFD and N availability, plant density and hierarchical positions of
individuals within stands. Combinations of all treatments showed a general
negative correlation between the N allocation coefficient (i.e. the slope of the
NLA-PFD relationship) and NSM for all three species. Thus, NLA, relative to PFD,
gradients became steeper with increasing shoot mass and increasing N shortage in
the plant. These data are consistent with the view that internal N availability
is an important factor in modifying the NLA gradient.
DOI: 10.1093/aob/mcg188
PMCID: PMC4244852
2303. Appl Environ Microbiol. 2003 Sep;69(9):5536-42.
Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa

sprouts: characterization, direct quantification, and in situ location of
transconjugant cells.
Mølbak L(1), Licht TR, Kvist T, Kroer N, Andersen SR.
Author information:
(1)Department of Environmental Chemistry and Microbiology, National Environmental
Research Institute, DK-4000 Roskilde, Denmark.
The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the
indigenous bacterial community on alfalfa sprouts was studied. Tagging with
fluorescent protein markers allowed direct quantification of the introduced donor
bacteria and of indigenous bacteria that had received the plasmids. The sprouts
were observed for 9 days; during this time alfalfa seeds, inoculated with donor
bacteria, developed to edible and subsequently decaying sprouts. The first
transconjugants were detected on day 6 after donor inoculation and occurred at
frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell
for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy
revealed that the sprouts were heavily colonized with donors and that most
transconjugants were located around the hypocotyl and root areas. Randomly
selected members of the indigenous bacterial community from both inoculated and
uninoculated sprouts, as well as a representative part of the community that had
received the plasmids, were characterized by polymorphisms of PCR-amplified
ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by
partial 16S rDNA sequencing. This showed that the initially dominating genera
Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully
developed sprouts. Transconjugants carrying either of the investigated plasmids
mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of
transconjugant cells did not reach detectable levels until 6 days after the onset
of germination, at which point these species constituted the majority of the
indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment
that allowed noteworthy frequencies of plasmid transfer from P. putida in the
absence of selective pressure that could favor the presence of the investigated
plasmids.
PMCID: PMC194921
2304. Reprod Nutr Dev. 2003 Mar-Apr;43(2):189-202.
Use of some novel alternative electron sinks to inhibit ruminal methanogenesis.
Ungerfeld EM(1), Rust SR, Burnett R.
Author information:
48824, USA.
Several compounds were evaluated in vitro as alternative electron sinks to

ruminal methanogenesis. They were incubated with ruminal fluid, buffer mixture,
and finely ground alfalfa hay for 24 h, at 0, 6, 12, and 18 mM initial
concentrations. The propionate enhancer oxaloacetic acid, the butyrate enhancer
beta-hydroxybutyrate, and the butyrate unsaturated analog 3-butenoic acid were
ineffective in decreasing methanogenesis. Nevertheless, beta-hydroxybutyrate
increased apparent fermentation of the alfalfa hay substrate from 58.0 to 63.4%,
and 3-butenoic acid seemed to increase it from 62.0 to 73.7%. Almost all of added
oxaloacetic acid disappeared during the incubation, while only between 30.3 and
53.4% of beta-hydroxybutyrate disappeared. The butyrate enhancers acetoacetate
and crotonic acid, and the butyrate unsaturated analog 2-butynoic acid, decreased
methanogenesis by a maximum of 18,9 and 9%, respectively. Crotonic acid at 18 mM
initial concentration seemed to increase the substrate apparent fermentation from
57.0 to 68.2%. Between 78.6 and 100% of acetoacetate disappeared during the
incubation. The propionate unsaturated analog propynoic acid, and the unsaturated
ester ethyl 2-butynoate, decreased methanogenesis by a maximum of 76 and 79%,
respectively. Less than 5% of propynoic acid disappeared. The substrate apparent
fermentation was decreased by propynoic acid from 62.0 to 57.4%, and seemed to
have been decreased by ethyl 2-butynoate from 62.0 to 29.3%. More accurate
measurements of the disappearance of some of the compounds studied are needed to
better understand how they are metabolized and how they affect fermentation.
2305. J Bacteriol. 2003 Sep;185(18):5602-10.
Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.
Wells DH(1), Long SR.
Author information:
(1)Department of Biological Sciences, Stanford University, Stanford, California
94305, USA.
The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa

requires complex physiological adaptation by both partners. One method by which
bacteria coordinately control physiological adaptation is the stringent response,
which is triggered by the presence of the nucleotide guanosine tetraphosphate
(ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the
ability of RNA polymerase (RNAP) to initiate and elongate transcription and
affect the affinity of the core enzyme for various sigma factors. An S. meliloti
relA mutant which cannot produce ppGpp was previously shown to be defective in
the ability to form nodules. This mutant also overproduces a symbiotically
necessary exopolysaccharide called succinoglycan. The work presented here
encompasses the analysis of suppressor mutants, isolated from host plants, that
suppress the symbiotic defects of the relA mutant. All suppressor mutations are
extragenic and map to either rpoB or rpoC, which encode the beta and beta'
subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal
that suppressor mutants can be divided into two classes; one is specific in its
effect on stringent response-regulated genes and shares striking similarity with
suppressor mutants of Escherichia coli strains that lack ppGpp, and another
reduces transcription of all genes tested in comparison to that in the relA
parent strain. Our findings indicate that the ability to successfully establish
symbiosis is tightly coupled with the bacteria's ability to undergo global
physiological adjustment via the stringent response.
PMCID: PMC193748
2306. Planta. 2003 Dec;218(2):276-87. Epub 2003 Aug 27.
The distributional changes and role of microtubules in Nod factor-challenged

Medicago sativa root hairs.
Weerasinghe RR(1), Collings DA, Johannes E, Allen NS.
Author information:
(1)Department of Botany, North Carolina State University, Box 7612, Raleigh, NC
27695-7612, USA.
The normal tip-growing pattern exhibited by root hairs of legumes is disrupted

when the hair is exposed to Nod factors generated by compatible bacteria capable
of inducing nodule formation. Since microtubules (MTs) play an important role in
regulating directionality and stability of apical growth in root hairs [T.N.
Bibikova et al. (1999) Plant J 17:657-665], we examined the possibility that Nod
factors might affect the MT distribution patterns in root hairs of Medicago
sativa L. We observed that Nod factor application caused rapid changes in the
pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min
after Nod factor application, first endoplasmic and then cortical MTs
depolymerised, initially at the proximal ends of cells. Twenty minutes after
exposure to Nod factors, a transverse band of microtubules was seen behind the
tip, while almost all other MTs had depolymerised. By 30 min, very few MTs
remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod
factors were applied in the presence of 10 microM oryzalin or 5 microM taxol, the
MTs appeared disintegrated while the morphological effects, such as bulging and
branching, became enhanced. Compared to the treatments with oryzalin or taxol
alone, the combinatory treatments exhibited higher growth rates. Since
microtubule reorganization is one of the earliest measurable events following Nod
factor application we conclude that microtubules have an important role in the
early phases of the signalling cascade. Microtubule involvement could be direct
or a consequence of Nod factor-induced changes in ion levels.
DOI: 10.1007/s00425-003-1097-1
2307. J Dairy Sci. 2003 Aug;86(8):2612-21.
Comparison of grass and legume silages for milk production. 2. In vivo and in
sacco evaluations of rumen function.
Dewhurst RJ(1), Evans RT, Scollan ND, Moorby JM, Merry RJ, Wilkins RJ.
Author information:
(1)Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth
SY23 3EB, UK. Richard.Dewhurst@bbsrc.ac.uk
Two experiments were conducted to investigate the basis for higher voluntary
intakes and increased alpha-linolenic acid content in milk from cows offered
clover silages. Six cows with rumen and duodenal cannulae were used in a
four-period changeover-design experiment. Cows received 8 kg/d of dairy
concentrate and had ad libitum access to one of six silage treatments: grass, red
clover, white clover, alfalfa, and 50/50 (dry matter basis) mixtures of grass
with red clover or white clover. The rumen fermentability of grass, red clover,
white clover, and grass/red clover silages was also evaluated in a nylon bag
study. Legume silages led to increased dry matter intake and milk production in
comparison with grass silage. There was no significant effect of legume silages
on rumen pH and volatile fatty acid concentrations, but a significant increase in
rumen ammonia concentration with the legume silages, reflecting their higher
protein content. The inclusion of white clover or alfalfa silage, but not red
clover silage, in diets led to an increase in molar proportions of isobutyric,
iso-valeric, and n-valeric acids in comparison with diets based on grass silage.
Rumen fill was significantly lower, and rumen passage rates were significantly
higher for cows offered alfalfa or white clover silages. However, the markedly
different particle size distribution of rumen contents with these feeds suggests
very different mechanisms for the high intake characteristics: high rates of
particle breakdown and passage with alfalfa, and high rates of fermentation and
passage with white clover. Microbial energetic efficiency (grams microbial N per
kilogram organic matter apparently digested in the rumen) was highest for cows
offered alfalfa silage, intermediate for clover silage, and lowest for cows
offered grass silage. These differences reflect the higher rumen outflow rates
for legume silages in comparison with grass silage. However, the effect of these
differences on N-use efficiency (feed to milk) was probably quite small in
comparison with effects of N intake. Although the biohydrogenation of
alpha-linolenic acid was still high for red clover silage (86.1% compared with
94.3% for grass silage), there was a 240% increase in the proportion of
alpha-linolenic acid passing through the rumen. This explains the increased
recovery of alpha-linolenic acid from feed into milk with diets based on red
clover silage.
DOI: 10.3168/jds.S0022-0302(03)73856-9
2308. Plant Cell Rep. 2003 Dec;22(5):328-37. Epub 2003 Aug 22.
Jellyfish green fluorescent protein as a useful reporter for transient expression

and stable transformation in Medicago sativa L.
Bellucci M(1), De Marchis F, Mannucci R, Arcioni S.
Author information:
(1)Institute of Plant Genetics, Research Division of Perugia, CNR, Via Madonna
Alta, 130, 06128 Perugia, Italy. michele.bellucci@igv.cnr.it
The aim of the experiments reported herein was to transiently test different gene
constructs using green fluorescent protein (GFP) as a reporter gene for a future
localization of the maize beta-zein in the chloroplast of alfalfa ( Medicago
sativa L.). The transient expression of two GFP genes was compared in alfalfa
leaves to determine which of these two mutants is the easier to detect. Based on
the intensity of fluorescence emitted, the GFP S65C gene was used to assemble a
chloroplast-targeted GFP to verify the efficiency of the transit peptide for
chloroplast targeting. A chloroplast-targeted fusion protein between beta-zein
and GFP was then assembled, and this protein was observed to accumulate in small
aggregates into the chloroplasts of transiently transformed cells. To the best of
our knowledge, this is the first report of the GFP S65C gene being used to obtain
transformed alfalfa plants expressing GFP.
DOI: 10.1007/s00299-003-0693-7
2309. Infect Immun. 2003 Sep;71(9):5306-13.
Comparative analysis of plant and animal models for characterization of

Burkholderia cepacia virulence.
Bernier SP(1), Silo-Suh L, Woods DE, Ohman DE, Sokol PA.
Author information:
(1)Department of Microbiology and Infectious Diseases, University of Calgary
Health Sciences Center, Calgary, Alberta T2N 4N1, Canada.
A simple alfalfa model was developed as an alternative infection model for

virulence studies of the Burkholderia cepacia complex. Symptoms of disease were
observed in wounded alfalfa seedlings within 7 days following inoculation of
10(1) to 10(5) CFU of most strains of the B. cepacia complex. Strains from seven
genomovars of the B. cepacia complex were tested for virulence in the alfalfa
model, and the degree of virulence was generally similar in strains belonging to
the same genomovar. Strains of Burkholderia multivorans and some strains of
Burkholderia stabilis did not cause symptoms of disease in alfalfa seedlings.
Representative strains were also tested for virulence using the rat agar bead
model. Most of the strains tested were able to establish chronic lung infections;
B. stabilis strains were the exception. Most of the strains that were virulent in
the alfalfa infection model were also virulent in the lung infection model. The
B. cepacia genomovar III mutants K56pvdA::tp and K56-H15 were significantly less
virulent in the alfalfa infection model than their parent strain. Therefore, this
alfalfa infection model may be a useful tool for assessing virulence of strains
of the B. cepacia complex and identifying new virulence-associated genes.
PMCID: PMC187319
Plant--rhizosphere-microflora association during phytoremediation of

PAH-contaminated soil.
Muratova A(1), Hūbner T, Tischer S, Turkovskaya O, Möder M, Kuschk P.
Author information:
Pr. Entuziastov, 410049 Saratov, Russia. ecbio@ibppm.sgu.su
The capability of plants to promote the microbial degradation of pollutants in

rhizosphere soil is a principal mechanism of phytoremediation of PAH-contaminated
soil. The formation of a specific rhizosphere microbocenosis with a high
degradative potential toward contaminants is largely determined by plant species.
The comparative PAH-degradation in unplanted soil and in soil planted with reed
(Phragmites australis) and alfalfa (Medicago sativa) was studied in pot
experiments during 2 years. Both alfalfa and reed successfully remediated
contaminated soil by degrading 74.5 and 68.7% of PAHs, respectively. The study of
the rhizosphere, rhizoplane, and unplanted-soil microflora in experimental pots
showed that alfalfa stimulated the rhizosphere microflora of PAH-contaminated
soil more effectively than did reed. Alfalfa clearly enhanced both the total
number of microorganisms (1.3 times, according to fluorescence microscopy data)
and the rate of the PAH-degrading population (almost seven times, according to
plate counting). The degradative potential of its rhizosphere microflora toward
PAHs was higher than the degradative activity of the reed rhizosphere. This study
provides relevant information for the successful application of alfalfa to
phytoremediate PAH-contaminated soil.
DOI: 10.1080/713610176
Ability of cold-tolerant plants to grow in hydrocarbon-contaminated soil.
Robson DB(1), Knight JD, Farrell RE, Germida JJ.
Author information:
(1)Department of Soil Science, University of Saskatchewan, 51 Campus Drive,
Saskatoon, Saskatchewan, S7N 5A8, Canada.
Phytoremediation of hydrocarbons in soil involves plants and their associated

microorganisms. Differences in environmental conditions and restrictions on
species importation mean that each country may need to identify indigenous plants
to use for phytoremedation. Screening plants for hydrocarbon tolerance before
screening for degradation ability may prove more economical than screening
directly for degradation. Thirty-nine cold-tolerant plants native, or exotic and
naturalized, in western Canada were assessed for their ability to survive in
crude oil-contaminated soil. Four naturalized grasses (i.e., Agropyron
pectiniforme, Bromus inermis, Phleum pratense, and Poa pratensis), three
naturalized legumes (i.e., Medicago sativa, Melilotus officinalis, and Trifolium
repens), two native forbs (i.e., Artemisia frigida and Potentilla pensylvanica),
one native grass (i.e., Bromus ciliatus) and two native legumes (i.e.,
Glycyrrhiza lepidota and Psoralea esculenta) exhibited phytoremediation
potential, based on survival. We determined the effect of increasing crude oil
concentrations on total and root biomass, and relative growth rate of those
species with the highest survival. The addition of 0.5%, 1%, and 5% (crude oil
wt/fresh soil wt) crude oil to soil significantly decreased both the total
biomass by at least 22% of the control and the relative growth rate of all
species except P. esculenta. Root biomass significantly decreased by at least 22%
with crude oil addition in all species except P. esculenta and A. frigida. Total
biomass production in contaminated soil had a significant negative correlation
with the relative growth rate in uncontaminated soil.
DOI: 10.1080/713610174
2312. Trends Plant Sci. 2003 Aug;8(8):365-8.
Ethylene signaling: the MAPK module has finally landed.
Chang C(1).
Author information:
(1)Department of Cell Biology and Molecular Genetics, HJ Patterson Hall,
University of Maryland, College Park, MD 20742-5815, USA. cc203@umail.umd.edu
Ethylene hormone responses are negatively regulated by the CTR1 protein, which
has similarity to mitogen-activated protein kinase kinase kinases (MAPKKKs).
Because of this similarity, it has long been speculated that ethylene signal
transduction involves a MAPK cascade. Now, a recent paper provides compelling
evidence for an ethylene-activated MAPK pathway. The implication is that CTR1 and
the newly identified MAPKK and MAPKs comprise a MAPK module that regulates
ethylene responses in plants.
DOI: 10.1016/S1360-1385(03)00156-0
2313. Rev Argent Microbiol. 2003 Apr-Jun;35(2):57-61.
[Growth and polysaccharide formation in Sinorhizobium meliloti strains in an

air-lift-type fermentor. Effect on nodulation velocity in alfalfa plants].
Lorda GS(1), Castaño RC, Pordomingo AB, Pastor MD, Balatti AP.
Author information:
(1)Facultad de Ciencias Exactas y Naturales, IMIQA, Universidad Nacional de La
Pampa, Av. Uruguay 151, 6300 Santa Rosa, La Pampa, Argentina.
lorda@exactas.unlpam.edu.ar
In this paper the influence of the exopolysaccharides produced by Sinorhizobium

meliloti strains on the nodulation rates in alfalfa plants has been considered.
The experiments were performed in a rotary shaker and in an air-lift type
fermentor. Different Sinorhizobium meliloti strains were used. Bacterial growth
rates were determined by viable cell counts. Exopolysaccharide concentration was
determined by precipitation with ethanol. It was observed that maximum cell
concentration was in the order of 1 x 10(10) cell/ml and exopolysaccharide
content was approximately 11 g/l. The experiments performed with alfalfa plants
in a controlled environment chamber showed that, when inoculation was carried out
with diluted suspensions (1/10), nodulation time was reduced from 10 to 4 days,
while the strains retained their symbiotic properties.
2314. Int J Food Microbiol. 2003 Sep 15;86(3):231-7.
Treatment of Escherichia coli O157:H7 inoculated alfalfa seeds and sprouts with
electrolyzed oxidizing water.
Sharma RR(1), Demirci A.
Author information:
(1)Department of Agricultural and Biological Engineering, Pennsylvania State
University, University Park, PA 16802, USA.
Electrolyzed oxidizing water is a relatively new concept that has been utilized
in agriculture, livestock management, medical sterilization, and food sanitation.
Electrolyzed oxidizing (EO) water generated by passing sodium chloride solution
through an EO water generator was used to treat alfalfa seeds and sprouts
inoculated with a five-strain cocktail of nalidixic acid resistant Escherichia
coli O157:H7. EO water had a pH of 2.6, an oxidation-reduction potential of 1150
mV and about 50 ppm free chlorine. The percentage reduction in bacterial load was
determined for reaction times of 2, 4, 8, 16, 32, and 64 min. Mechanical
agitation was done while treating the seeds at different time intervals to
increase the effectiveness of the treatment. Since E. coli O157:H7 was released
due to soaking during treatment, the initial counts on seeds and sprouts were
determined by soaking the contaminated seeds/sprouts in 0.1% peptone water for a
period equivalent to treatment time. The samples were then pummeled in 0.1%
peptone water and spread plated on tryptic soy agar with 5 microg/ml of nalidixic
acid (TSAN). Results showed that there were reductions between 38.2% and 97.1%
(0.22-1.56 log(10) CFU/g) in the bacterial load of treated seeds. The reductions
for sprouts were between 91.1% and 99.8% (1.05-2.72 log(10) CFU/g). An increase
in treatment time increased the percentage reduction of E. coli O157:H7. However,
germination of the treated seeds reduced from 92% to 49% as amperage to make EO
water and soaking time increased. EO water did not cause any visible damage to
the sprouts.
2315. Microbiol Res. 2003;158(2):151-61.
Rhizosphere microflora of plants used for the phytoremediation of

bitumen-contaminated soil.
Muratova A(1), Hübner T, Narula N, Wand H, Turkovskaya O, Kuschk P, Jahn R,
Merbach W.
Author information:
(1)Institute of Biochemistry & Physiology of Plants and Microorganisms RAS, 13
Pr. Entuziastov, 410049 Saratov, Russia. ecbio@ibppm.sgu.ru
The microbial communities and their degradative potential in rhizospheres of

alfalfa (Medicago sativa) and reed (Phragmites australis) and in unplanted soil
in response to bitumen contamination of soil were studied in pot experiments.
According to the results of fluorescence microscopy, over a period of 27 months,
bitumen contamination of soil reduced the total number of microorganisms more
significantly (by 75%) in unplanted than in rhizosphere soil (by 42% and 7% for
reed and alfalfa, respectively) and had various effects on some important
physiological groups of microorganisms such as actinomycetes as well as
nitrogen-fixing, nitrifying, denitrifying, ammonifying, phosphate-solubilizing,
sulphur-oxidizing, cellulolytic and hydrocarbon-degrading microorganisms. The
changes in the physiological structure of the microbial community under bitumen
contamination were found to hinge on not merely the presence of plants but also
their type. It was noted that the rhizosphere microflora of alfalfa was less
inhibited by hydrocarbon pollution and had a higher degradative potential than
the rhizosphere microflora of reed.
DOI: 10.1078/0944-5013-00187
The Sinorhizobium meliloti glycine betaine biosynthetic genes (betlCBA) are

induced by choline and highly expressed in bacteroids.
Mandon K(1), Osterås M, Boncompagni E, Trinchant JC, Spennato G, Poggi MC, Le

Rudulier D.
Author information:
(1)Laboratoire de Biologie végétale et Microbiologie, CNRS FRE2294, Université de
Nice-Sophia Antipolis, Faculté des Sciences, Parc Valrose, 06108 Nice Cédex 2,
France.
The symbiotic soil bacterium Sinorhizobium meliloti has the capacity to

synthesize the osmoprotectant glycine betaine from choline-O-sulfate and choline.
This pathway is encoded by the betICBA locus, which comprises a regulatory gene,
betI, and three structural genes, betC (choline sulfatase), betB (betaine
aldehyde dehydrogenase), and betA (choline dehydrogenase). Here, we report that
betICBA genes constitute a single operon, despite the existence of intergenic
regions containing mosaic elements between betI and betC, and betB and betA. The
regulation of the bet operon was investigated by using transcriptional lacZ
(beta-galactosidase) fusions and has revealed a strong induction by choline at
concentrations as low as 25 microM and to a lesser extent by choline-O-sulfate
and acetylcholine but not by osmotic stress or oxygen. BetI is a repressor of the
bet transcription in the absence of choline, and a nucleotide sequence of dyad
symmetry upstream of betI was identified as a putative betI box. Measurements of
intracellular pools of choline, well correlated with beta-galactosidase
activities, strongly suggested that BetI senses the endogenous choline pool that
modulates the intensity of BetI repression. In contrast to Escherichia coli, BetI
did not repress choline transport. During symbiosis with Medicago sativa, S.
meliloti bet gene expression was observed within the infection threads, in young
and in mature nodules. The existence of free choline in nodule cytosol,
peribacteroid space, and bacteroids was demonstrated, and the data suggest that
bet regulation in planta is mediated by BetI repression, as in free-living cells.
Neither Nod nor Fix phenotypes were significantly impaired in a betI::omega
mutant, indicating that glycine betaine biosynthesis from choline is not crucial
for nodulation and nitrogen fixation.
DOI: 10.1094/MPMI.2003.16.8.709
2317. J Dairy Sci. 2003 Jul;86(7):2428-37.
Milk production and composition from cows fed high oil or conventional corn at
two forage concentrations.
Whitlock LA(1), Schingoethe DJ, Hippen AR, Kalscheur KF, AbuGhazaleh AA.
Author information:
USA.
Twelve multiparous Holstein cows (63 +/- 24 d in milk) were used in a replicated
4 x 4 Latin square with 28-d periods to evaluate conventional and high oil corn
grains when fed at two different forage-to-concentrate ratios. Dietary treatments
consisted of conventional or high oil corn supplementing a diet with a 25:25:50
mixture of corn silage: alfalfa: concentrate mix, or a high forage diet with a
30:30:40 mixture of corn silage: alfalfa: concentrate mix. Dry matter intake
(28.1, 28.7, 26.9, and 26.2 kg/d for normal diets with conventional and high oil
corn, and high forage diets with conventional and high oil corn, respectively)
and milk yields (36.8, 37.2, 35.5, and 35.2 kg/d) were similar for conventional
and high oil corn diets and were lower with the high forage diet, regardless of
corn source. Milk fat concentrations were greater when cows were fed diets
containing 60% forage (4.03 vs. 3.88%, for the 60 and 50% forages, respectively),
but milk protein concentrations were not affected by forage content. Corn source
did not affect milk fat or protein concentrations. Long-chain fatty acid
concentrations, unsaturated fatty acid concentrations, and total 18:1 fatty acid
concentrations were greater when cows were fed high oil corn but were unaffected
by forage content of the diet. Concentrations of transvaccenic acid (0.58, 0.81,
0.62, and 0.69 g/100 g of fatty acids) and cis-9, trans-11 conjugated linoleic
acid (0.28, 0.39, 0.32, and 0.33 g/100 g of fatty acids) were greater when cows
were fed high oil compared with conventional corn when fed 50% forage but were
similar for both corn sources at 60% forage. Total n-3 fatty acids were not
affected by corn source or forage content. High forage diets decreased milk
production and increased milk fat concentration. Feeding high oil corn increased
concentrations of long-chain, unsaturated, transvaccenic, and conjugated linoleic
fatty acids in milk; however, production of transvaccenic and conjugated linoleic
acids were attenuated by high forage diet.
DOI: 10.3168/jds.S0022-0302(03)73837-5
2318. Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):10108-13. Epub 2003 Aug 4.
The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA
box-binding protein and a nuclear protein kinase in plants.
Bakó L(1), Umeda M, Tiburcio AF, Schell J, Koncz C.

Author information:
(1)Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10,
D-50829 Cologne (Köln), Germany.
The bacterial virulence protein VirD2 plays an important role in nuclear import
and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant,
animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2
interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of
cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates
the C-terminal regulatory domain of RNA polymerase II largest subunit, which can
recruit the TATA box-binding protein. VirD2 is found in tight association with
the TATA box-binding protein in vivo. These results indicate that recognition of
VirD2 is mediated by widely conserved nuclear factors in eukaryotes.
DOI: 10.1073/pnas.1733208100
PMCID: PMC187781
2319. Can J Microbiol. 2003 Apr;49(4):253-62.
Characterization of an antifungal soil bacterium and its antagonistic activities

against Fusarium species.
Chan YK(1), McCormick WA, Seifert KA.
Author information:
(1)Eastern Cereal and Oilseed Research Center, Agriculture and Agri-Food Canada,
Ottawa. chanyk@agr.gc.ca
Bacteria were isolated from a cultivated soil and screened for antagonistic
activity against Fusarium graminearum, a predominant agent of ear rot and head
blight in cereal crops. Based on its in vitro effectiveness, isolate D1/2 was
selected for characterization and identified as a strain of Bacillus subtilis by
phenotypic tests and comparative analysis of its 16S ribosomal RNA gene (rDNA)
sequence. It inhibited the mycelial growth of a collection of common fungal
phytopathogens, including eight Fusarium species, three other ascomycetes, and
one basidiomycete. The cell-free culture filtrate of D1/2 at different dilutions
was active against macroconidium germination and hyphal growth of F. graminearum,
depending on the initial macroconidium density. It induced the formation of
swollen hyphal cells in liquid cultures of this fungus grown from macroconidia. A
bioassay also demonstrated that D1/2 offered in planta protection against the
damping-off disease in alfalfa seedlings caused by F. graminearum, while the type
strain of B. subtilis was ineffective. Hence, B. subtilis D1/2 or its culture
filtrate has potential application in controlling plant diseases caused by
Fusarium.
DOI: 10.1139/w03-033
2320. J Food Prot. 2003 Jul;66(7):1253-9.
Industry practices and compliance with U.S. Food and Drug Administration
guidelines among California sprout firms.
Thomas JL(1), Palumbo MS, Farrar JA, Farver TB, Cliver DO.
Author information:
(1)California Department of Health Services, Food and Drug Branch, 601 North 7th
Street, Sacramento, California 94234, USA. jthomas2@dhs.ca.gov
Since 1995, raw vegetable sprouts have been implicated as the vehicle of
infection in 15 foodborne outbreaks involving Salmonella and 2 foodborne
outbreaks involving Escherichia coli O157:H7. To reduce the numbers of
sprout-related outbreaks, the U.S. Food and Drug Administration (FDA) published
Guidance for Industry: Reducing Microbial Food Safety Hazards for Sprouting Seeds
in 1999. Between October 2000 and April 2001, 61.5% (16 of 26) of the known
commercial sprout firms in California were enrolled in a survey to evaluate the
industry practices of California sprouting operations and to determine compliance
with FDA guidelines. A standardized questionnaire was used to collect data on
firm demographics and seed disinfection practices. Additionally, free chlorine
levels in seed disinfection solutions were measured, and 48-h spent irrigation
water samples were collected from each firm. The irrigation water was screened
for Salmonella and E. coli O157:H7 with FDA-recommended test kits. Free chlorine
levels in the treatment solutions ranged from 50 to 35,000 mg/liter (ppm), with a
median of 14,000 mg/liter (ppm). Free chlorine levels were higher for firms
producing alfalfa sprouts than for those producing only mung bean or soybean
sprouts (P=0.03). Levels of free chlorine tended to be higher for firms using a
calcium hypochlorite treatment solution than for firms using a sodium
hypochlorite treatment solution (P=0.067). All 32 irrigation water samples
screened for Salmonella tested negative. Of the irrigation water samples tested
for E. coil O157:H7, 75% (24 of 32) tested negative, and 25% (8 of 32) tested
presumptive positive. The eight presumptive positive samples were found to be
negative after further testing. These results indicate that producers of alfalfa
sprouts are generally achieving the FDA-recommended calcium hypochlorite level of
20,000 mg/liter (ppm), whereas mung bean sprout producers are not.
2321. J Food Prot. 2003 Jul;66(7):1158-65.
Reduction of Escherichia coli O157:H7 and Salmonella on laboratory-inoculated

alfalfa seed with commercial citrus-related products.
Fett WF(1), Cooke PH.
Author information:
Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038,
USA. wfett@arserrc.gov
Alfalfa sprouts contaminated with the bacterial pathogens Escherichia coli

O157:H7 and Salmonella have been the source of numerous outbreaks of foodborne
illness in the United States and in other countries. The seed used for sprouting
appears to be the primary source of these pathogens. The aim of this study was to
determine whether the efficacy of commercial citrus-related products for
sanitizing sprouting seed is similar to that of high levels of chlorine. Five
products (Citrex, Pangermex, Citricidal, Citrobio, and Environné) were tested at
concentrations of up to 20,000 ppm in sterile tap water and compared with
buffered chlorine (at 16,000 ppm). Alfalfa seeds were inoculated with four-strain
cocktails of Salmonella and E. coli O157:H7 to give final initial concentrations
of ca. 9.0 and 7.0 CFU/g, respectively. Treatments (10 min) with Citrex,
Pangermex, and Citricidal at 20,000 ppm and chlorine at 16,000 ppm produced
similar log reductions for alfalfa seed inoculated with four-strain cocktails of
E. coli O157:H7 and Salmonella (3.42 to 3.46 log CFU/g and 3.56 to 3.74 log
CFU/g, respectively), and all four treatments were significantly (P<0.05) more
effective than the control treatment (a buffer wash). Citrobio at 20,000 ppm was
as effective as the other three products and chlorine against Salmonella but not
against E. coli O157:H7. Environné was not more effective (producing reductions
of 2.2 to 2.9 log CFU/g) than the control treatment (which produced reductions of
2.1 to 2.3 log CFU/g) against either pathogen. None of the treatments reduced
seed germination. In vitro assays, as well as transmission electron microscopy,
confirmed the antibacterial nature of the products that were effective against
the two pathogens and indicated that they were bactericidal. When used at 20,000
ppm, the effective citrus-related products may be viable alternatives to chlorine
for the sanitization of sprouting seed pending regulatory approval.
2322. Mol Ecol. 2003 Aug;12(8):2187-99.
Inferences from mitochondrial DNA patterns on the domestication history of

alfalfa (Medicago sativa).
Muller MH(1), Prosperi JM, Santoni S, Ronfort J.
Author information:
(1)UMR Diversité et Génomes des Plantes Cultivées, Station de génétique et
amélioration des plantes, INRA, Domaine de Melgueil, 34130 Mauguio, France.
mullerm@ensam.inra.fr
The evolutionary history of a wild-cultivated complex is the outcome of different

factors, among which are the domestication of the cultivated form, its
geographical and demographic expansion, as well as gene flow between natural and
cultivated populations. To clarify this history for alfalfa (Medicago sativa), we
adopted a phylogeographical approach based on a sample of natural and cultivated
populations of the M. sativa species complex. This sample was characterized for
mtDNA variation through restriction fragment length polymorphisms. Twenty-two
mitotypes were identified in the whole data set (155 individuals). In the wild
pool from the presumed area of origin (Near East to Central Asia), the diversity
was high but the absence of geographical differentiation hinders a more precise
location of the centre of domestication. Within the cultivated alfalfa, the
geographical structure suggests strongly the existence of at least two
independent routes of dissemination of alfalfa from its centre of origin. In
particular, original mitotypes detected in some regions suggest that wild
populations not sampled in this study, and located outside the presumed area of
domestication, may have contributed to the diversity of the cultivated pool.
Finally, analysis of the mtDNA variation shows that natural populations collected
in Spain constitute an endemic wild pool now introgressed partially by cultivated
alfalfa.
Expression of anthocyanins and proanthocyanidins after transformation of alfalfa

with maize Lc.
Ray H(1), Yu M, Auser P, Blahut-Beatty L, McKersie B, Bowley S, Westcott N,

Coulman B, Lloyd A, Gruber MY.
Author information:
(1)Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science
Place, Saskatoon, Saskatchewan, Canada S7N 0X2.
Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were
introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the
flavonoid pathway and alter the composition of flavonoids produced in forage. Lc
constructs included a full-length gene and a gene with a shortened
5'-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa
foliage, but accumulation of red-purple anthocyanin was observed only under
conditions of high light intensity or low temperature. These stress conditions
induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic
alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc
transgene construct with a full-length 5'-untranslated region responded more
quickly to stress conditions and with a more extreme phenotype. High-performance
liquid chromatography analysis of field-grown tissue indicated that flavone
content was reduced in forage of the Lc transgenic plants. Leucocyanidin
reductase, the enzyme that controls entry of metabolites into the
proanthocyanidin pathway, was activated both in foliage and in developing seeds
of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated
in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and
C1 transgenic populations displayed no visible phenotypic changes, although these
transgenes were expressed at detectable levels. These results support the
emerging picture of Lc transgene-specific patterns of expression in different
recipient species. These results demonstrate that proanthocyanidin biosynthesis
can be stimulated in alfalfa forage using an myc-like transgene, and they pave
the way for the development of high quality, bloat-safe cultivars with ruminal
protein bypass.
PMCID: PMC167084
2324. J Anim Sci. 2003 Jul;81(7):1671-80.
Combinations of alfalfa hay and wet corn gluten feed in limit-fed growing diets
for beef cattle.
Montgomery SP(1), Drouillard JS, Sindt JJ, Farran TB, Pike JN, Trater AM, Coetzer
CM, LaBrune HJ, Hunter RD, Stocks RA.
Author information:
66506-1600, USA.
Two experiments were conducted to evaluate the effects of alfalfa hay (AH) and
wet corn gluten feed (WCGF) combinations on ADG and gain efficiency of cattle
limit-fed growing diets. In Exp. 1, crossbred beef steers (n = 220; initial BW =
262 kg) were limit-fed diets consisting of steam-flaked corn and 40% WCGF (DM
basis) with 0, 10, or 20% ground AH (0AH, 10AH, and 20AH, respectively). A fourth
diet containing 20% ground AH and steam-flaked corn served as a control. All
diets were fed once daily at 1.8% of BW (DM basis). Growing period ADG, gain
efficiency, and dietary NE calculated from performance data decreased linearly (P
< 0.01) with addition of AH to diets containing WCGF. Rate of DMI increased
linearly (P < 0.05) with AH addition to diets containing WCGF. Following the
growing period, steers were finished on a common diet offered ad libitum. Gain
efficiencies during the finishing period were higher (P < 0.05) for steers fed
the 20AH diet than for steers fed the control diet. In Exp. 2, crossbred beef
heifers (n = 339; initial BW = 277 kg) were limit-fed diets containing
steam-flaked corn with 10, 20, or 30% ground AH and 0, 40, or 68% WCGF in a 3 x 3
factorial arrangement, fed once daily at 1.6% of BW (DM basis). An AH x WCGF
interaction occurred (P < 0.05) for growing period ADG and gain efficiency.
Increasing AH or WCGF decreased cattle ADG, gain efficiency, and dietary NE with
the exception of heifers fed 30AH/40WCGF, which had ADG that did not differ (P >
0.10) from that of heifers fed 20AH/0WCGF or 30AH/0WCGF, and which had greater
gain efficiencies (P < 0.05) than heifers fed 30AH/0WCGF. Rate of DMI increased
linearly (P < 0.01) with increasing AH and decreased linearly (P < 0.01) with
increasing WCGF. Heifers were finished on diets containing 33% WCGF with 0 or
0.5% added urea (DM basis) offered ad libitum. Increasing WCGF in growing diets
tended (linear, P < 0.10) to increase finishing ADG and gain efficiency, whereas
increasing AH decreased (linear, P < 0.05) kidney, pelvic, and heart fat, and the
percentage of carcasses grading USDA Prime. Urea tended to increase ADG (P <
0.10), but decreased (P < 0.04) the percentage of carcasses grading USDA Choice.
Results suggest that the value of WCGF relative to steam-flaked corn in limit-fed
growing diets might be improved in diets containing 30% AH relative to diets
containing 10 or 20% AH.
DOI: 10.2527/2003.8171671x
Captures of western corn rootworm (Coleoptera: Chrysomelidae) adults with

Pherocon AM and vial traps in four crops in east central Illinois.
Rondon SI(1), Gray ME.
Author information:
(1)University of Illinois at Urbana-Champaign, Department of Crop Sciences,
AW-101 Turner Hall, 1102 South Goodwin Avenue, Urbana, IL 61801, USA.
It is hypothesized that the long-term rotation of maize (Zea mays L.) and soybean
(Glycine max L.) in east central Illinois has caused a significant change in the
ovipositional behavior of the western corn rootworm, Diabrotica virgifera
virgifera LeConte. Since the mid 1990s in east central Illinois, western corn
rootworm adults have been observed feeding on soybean foliage and also now use
soybean fields as egg laying sites. This behavioral adaptation has greatly
decreased the effectiveness of rotation as a pest management tactic. By using
Pherocon AM and vial traps, we evaluated the influence of maize, soybean, oat
stubble (Avena sativa L.), and alfalfa (Medicago sativa L.) on male and female
adult western corn rootworm densities from April 1998 through September 2000 near
Urbana, IL. Our results indicated that western corn rootworm adults are common
inhabitants of maize, soybean, oat stubble, and alfalfa. Trapping efforts with
both Pherocon AM (attractive) and vial traps (passive) revealed that initial
densities of both male and female western corn rootworm adults were greater in
maize. Soon after emergence, densities of females began to decline within maize
and increase in other crops (soybean, oat stubble, and alfalfa). Results from
this experiment support the hypothesis that variant western corn rootworm females
in east central Illinois are colonizing crops other than maize at densities of
potential economic importance. Those producers who choose to rotate maize with
soybean or alfalfa may remain at risk to economic larval injury to maize roots.
Potentially, oat stubble also may support levels of western corn rootworm females
resulting in sufficient oviposition to cause economic losses to rotated maize the
following season.
The probabilistic economic injury level: incorporating uncertainty into pest

management decision-making.
Peterson RK(1), Hunt TE.
Author information:
(1)Department of Entomology, 333 Leon Johnson Hall, Montana State University,
Bozeman, MT 59717-3020, USA. bpeterson@montana.edu
Despite the recognition that the economic injury level (EIL) is determined by
dynamic biological and economic parameters, which can be highly variable and
uncertain, there has been little effort to quantify uncertainty and to use
estimates of uncertainty in the determination of EILs. In this paper, we define
the probabilistic EIL (PEIL) and develop PEILs for two insect pest scenarios:
alfalfa weevil larvae, Hypera postica (Gyllenhal), on early bud-stage alfalfa,
and bean leaf beetle adults, Cerotoma trifurcata (Forster), on V1-stage soybean.
The PEIL is an EIL that reflects its probability of occurrence. The probability
of occurrence is determined by incorporating the uncertainty associated with the
input variables used to calculate the EIL. We used Monte Carlo simulation, a
random sampling technique in which each input variable in the model was sampled
repeatedly from a range of possible values based on probability distributions.
Each input variable's probability distribution was sampled such that the
distribution's shape was reproduced. Then, the variability for each input was
propagated into the output of the model so that the model output reflected the
probability of values that could occur. This represents the first use of the
Monte Carlo technique to determine EILs.
DOI: 10.1603/0022-0493-96.3.536
2327. J Dairy Sci. 2003 Jun;86(6):2178-92.
Comparison of the National Research Council-2001 model with the Dutch system
(DVE/OEB) in the prediction of nutrient supply to dairy cows from forages.
Yu P(1), Christensen DA, McKinnon JJ.
Author information:
Saskatoon, SK, S7N 5A8, Canada. yupe@sask.usask.ca
The objective of this study was to compare the DVE/OEB system (DVE = truly
absorbed protein in the small intestine; OEB = degraded protein balance) and the
NRC-2001 model in the prediction of supply of protein to dairy cows from selected
forages: alfalfa (Medicago sativa L. cv. Pioneer and Beaver) and timothy (Phleum
pratense L. cv. Climax and Joliette). Comparisons were made in terms of 1)
ruminally synthesized microbial CP, 2) truly absorbed protein in the small
intestine, and 3) degraded protein balance. In addition, the effects of variety
and cutting stage of the selected forages on the potential nutrient supply to
dairy cows were also studied. The results showed that the predicted values from
the DVE/OEB system and the NRC-2001 model had significant correlations with high
R (>0.96) values. However, using the DVE/OEB system, the overall average
microbial protein supply based on available energy was 12% higher, and the truly
absorbed protein in the small intestine was 15% lower than that predicted by the
NRC-2001 model. The difference was also found in the prediction of the degraded
protein balances, which was 11% higher based on data from the NRC-2001 model.
These differences are due to considerably different factors used in calculations
in the two models, although both are based on similar principles. This indicates
that a further refinement is needed for a modern protein evaluation and
prediction system. In addition, this study showed that the two alfalfa varieties
studied (Pioneer vs. Beaver) had no effect, but cutting stage had a profound
influence on ruminally synthesized microbial CP (93, 96, 86 g/kg DM at stage of
early bud, late bud, and early bloom, respectively) and truly absorbed intestinal
protein predicted by the DVE/OEB system (80, 79, 67 g/kg DM at stage of early
bud, late bud, and early bloom, respectively). With timothy, both variety (Climax
vs. Joliette) and cutting stage had significant impacts on the potential protein
supply predicted by both models. The potential protein supply (DVE or MP) to the
dairy cow from Climax timothy was higher than that from Joliette timothy (DVE: 46
vs. 32 g/kg DM; MP: 61 vs. 38 g/kg DM). With increasing stage of cutting, the
potential protein supply (DVE or MP) was reduced (DVE: 53, 39, 25 g/kg DM; MP:
62, 51, 36 g/kg DM at stage of joint, prebloom head, and full head,
respectively).
DOI: 10.3168/jds.S0022-0302(03)73808-9
2328. J Dairy Sci. 2003 Jun;86(6):2154-62.
Effects of a polymer-coated urea product on nitrogen metabolism in lactating

Holstein dairy cattle.
Galo E(1), Emanuele SM, Sniffen CJ, White JH, Knapp JR.
Author information:
(1)Department of Animal Science, University of Vermont, Burlington 05405, USA.
The purpose of this study was to evaluate the impact of polymer-coated urea on
nitrogen retention, rumen microbial growth, and milk production and composition.
Coated urea (CU) that is more slowly hydrolyzed to ammonia than unprotected urea
could potentially be used more efficiently by rumen microorganisms. Eight cows
were offered each of three diets in a randomized crossover design. Each treatment
period consisted of a 14-d adjustment period and a 5-d collection period. Diets
were formulated to maintain milk production while reducing plasma urea nitrogen
concentrations and urinary nitrogen excretion. Diets consisted of corn silage,
mixed grass/legume haylage, chopped alfalfa hay, corn meal, protein, vitamin and
mineral supplements, in a total mixed ration and fed ad libitum. The diets
contained 17.9%, 18.1%, and 16.4% CP and 0, 0.77%, and 0.77% CU (dry matter
basis) and are denoted as CP18-CU, CP18+CU, and CP16+CU, respectively. Individual
feed intakes were measured, and total fecal, and urine collections were
conducted. Cows were milked twice daily at 0500 and 1700 h, and the milk sampled
for composition and milk urea N analysis. Dry matter intake averaged 23.5 +/- 0.2
kg/d and was not altered by diet. Also, milk fat and true protein were not
altered by diet and averaged 3.72 and 3.07%, respectively. Milk yield was highest
for diets CP18-CU and CP18+CU. Significant differences were observed in N intake
and excretion in urine, feces, and milk between dietary treatments. Cows fed
CP16+CU consumed 11% less N than in CP18-CU. Cows fed CP18+CU showed the highest
excretion of N in urine, and together with CP16+CU, the lowest N excretion in
feces. Nitrogen excretion in milk was lower for cows fed CP16+CU. Calculated N
balance was not significantly different between diets nor was it significantly
different from zero. Efficiency of N capture in milk protein as a function of N
intake was higher for animals on CP16+CU. Urinary excretion of purine derivatives
was not different between diets, and estimated microbial CP was also similar.
Coated urea was not effective at reducing nitrogen excretion by dairy cattle.
DOI: 10.3168/jds.S0022-0302(03)73805-3
2329. Plant Mol Biol. 2003 May;52(1):121-33.
The over-expression of an alfalfa RING-H2 gene induces pleiotropic effects on

plant growth and development.
Karlowski WM(1), Hirsch AM.
Author information:
California, 405 Hilgard Avenue, Los Angeles, CA 90095-1606, USA.
w.karlowski@gsf.de
The alfalfa MsRH2-1 gene encodes a small protein with a RING-H2 motif and an
N-terminal transmembrane domain. The transcript can be found in all tested plant
organs, but roots and nodules show the highest levels of RH2-1 mRNA accumulation.
Promoter-GUS fusion studies demonstrate that the activity of this gene is closely
correlated with development of lateral roots (in alfalfa and Arabidopsis) and
symbiotic nodules (in alfalfa). Although antisense-expressing alfalfa plants did
not show a significantly different phenotype from the control plants, by
contrast, when the level of the MsRH2-1 RNA was raised by introducing the coding
part of the gene under the control of the CaMV promoter, both the transgenic
alfalfa and Arabidopsis lines exhibited dramatic alterations in plant morphology,
including shorter stature, increased apical dominance, leaf hyponasty, and
inhibition of leaf venation and lateral root development. Moreover, nodulation of
transgenic alfalfa roots was delayed and partially inhibited, and some of the
Arabidopsis lines showed abnormal floral development. The nature of pleiotropic
developmental phenotypes suggests a hormonal basis. The possible connection
between MsRH2-1 function and substrate specific degradation via the ubiquitin
pathway involved in auxin signaling is discussed.
2330. J Bacteriol. 2003 Jul;185(13):3853-62.
Striking complexity of lipopolysaccharide defects in a collection of

Sinorhizobium meliloti mutants.
Campbell GR(1), Sharypova LA, Scheidle H, Jones KM, Niehaus K, Becker A, Walker
GC.
Author information:
Although the role that lipopolysaccharide (LPS) plays in the symbiosis between
Sinorhizobium meliloti and alfalfa has been studied for over a decade, its
function in this process remains controversial and poorly understood. This is
largely due to a lack of mutants affected by its synthesis. In one of the
definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber,
and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of
mutants with putative LPS defects, judged them to be symbiotically proficient on
Medicago sativa, and concluded that LPS might not have a symbiotic function in S.
meliloti. The mutations in these strains were never characterized at the
molecular level nor was the LPS from most of them analyzed. We have transduced
these mutations from the Rm2011 background from which they were originally
isolated into the sequenced strain Rm1021 and have characterized the resulting
strains in greater detail. We found the LPS from these mutants to display a
striking complexity of phenotypes on polyacrylamide electrophoresis gels,
including additional rough LPS bands and alterations in the molecular weight
distribution of the smooth LPS. We found that some of the mutants contain
insertions in genes that are predicted to be involved in the synthesis of
carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE. The
majority, however, code for proteins predicted to be involved in a wide variety
of functions not previously recognized to play a role in LPS synthesis, including
a possible transcription elongation factor (GreA), a possible queuine synthesis
protein, and a possible chemotaxis protein. Furthermore, using more extensive
assays, we have found that most of these strains have symbiotic deficiencies.
These results support more recent findings that alterations in LPS structure can
affect the ability of S. meliloti to form an effective symbiosis.
PMCID: PMC161594
2331. J Environ Qual. 2003 May-Jun;32(3):916-26.
Nitrate exported in drainage waters of two sprinkler-irrigated watersheds.
Cavero J(1), Beltrán A, Aragüés R.
Author information:
(1)Dep. Genética y Producción Vegetal, Estación Experimental de Aula Dei (CSIC),
Laboratorio de Agronomía y Medio Ambiente (DGA-CSIC), Apdo. 202, 50080 Zaragoza,
Spain. jcavero@eead.csic.es
Nitrate contamination of surface waters has been linked to irrigated agriculture

across the world. We determined the NO3-N loads in the drainage waters of two
sprinkler-irrigated watersheds located in the Ebro River basin (Spain) and their
relationship to irrigation and N management. Crop water requirements, irrigation,
N fertilization, and the volume and NO3-N concentration of drainage waters were
measured or estimated during two-year (Watershed A; 494 irrigated ha) and
one-year (Watershed B; 470 irrigated ha) study periods. Maize (Zea mays L.) and
alfalfa (Medicago sativa L.) were grown in 40 to 60% and 15 to 33% of the
irrigated areas, respectively. The seasonal irrigation performance index (IPI)
ranged from 92 to 100%, indicating high-quality management of irrigation.
However, the IPI varied among fields and overirrigation occurred in 17 to 44% of
the area. Soil and maize stalk nitrate contents measured at harvest indicated
that N fertilizer rates could be decreased. Drainage flows were 68 mm yr(-1) in
Watershed A and 194 mm yr(-1) in Watershed B. Drainage NO3-N concentrations were
independent of drainage flows and similar in the irrigated and nonirrigated
periods (average: 23-29 mg L(-1)). Drainage flows determined the exported mass of
NO3-N, which varied from 18 (Watershed A) to 49 (Watershed B) kg ha(-1) yr(-1),
representing 8 (Watershed A) and 22% (Watershed B) of the applied fertilizer plus
manure N. High-quality irrigation management coupled to the split application of
N through the sprinkler systems allowed a reasonable compromise between
profitability and reduced N pollution in irrigation return flows.
2332. ScientificWorldJournal. 2001 Sep 27;1 Suppl 2:707-13.
Nitrogen use and carbon sequestered by corn rotations in the northern corn belt,
U.S.
Pikul JL Jr(1), Schumacher TE, Vigil M.
Author information:
(1)USDA-ARS, Northern Grain Insects Research Laboratory, Brookings, SD 57006,
USA. jpikul@ngirl.ars.usda.gov
Diversified crop rotation may improve production efficiency, reduce fertilizer

nitrogen (N) requirements for corn (Zea mays L.), and increase soil carbon (C)
storage. Objectives were to determine effect of rotation and fertilizer N on soil
C sequestration and N use. An experiment was started in 1990 on a Barnes clay
loam (U.S. soil taxonomy: fine-loamy, mixed, superactive, frigid Calcic
Hapludoll) near Brookings, SD. Tillage systems for corn-soybean ( Glycine max
[L.] Merr.) rotations were conventional tillage (CS) and ridge tillage (CSr).
Rotations under conventional tillage were continuous corn (CC), and a 4-year
rotation of corn-soybean-wheat ( Triticum aestivum L.) companion-seeded with
alfalfa ( Medicago sativa L.)-alfalfa hay (CSWA). Additional treatments included
plots of perennial warm season, cool season, and mixtures of warm and cool season
grasses. N treatments for corn were corn fertilized for a grain yield of 8.5 Mg
ha(-1) (highN), of 5.3 Mg ha(-1) (midN), and with no N fertilizer (noN). Total
(1990-2000) corn grain yield was not different among rotations at 80.8 Mg ha(-1)
under highN. Corn yield differences among rotations increased with decreased
fertilizer N. Total (1990-2000) corn yields with noN fertilizer were 69 Mg ha-1
under CSWA, 53 Mg ha(-1) under CS, and 35 Mg ha(-1) under CC. Total N attributed
to rotations (noN treatments) was 0.68 Mg ha(-1) under CSWA, 0.61 Mg ha(-1) under
CS, and 0.28 Mg ha(-1) under CC. Plant carbon return depended on rotation and N.
In the past 10 years, total C returned from aboveground biomass was 29.8 Mg
ha(-1) under CC with highN, and 12.8 Mg ha(-1) under CSWA with noN. Soil C in the
top 15 cm significantly increased (0.7 g kg(-1)) with perennial grass cover,
remained unchanged under CSr, and decreased (1.7 g kg(-1)) under CC, CS, and
CSWA. C to N ratio significantly narrowed (-0.75) with CSWA and widened (0.72)
under grass. Diversified rotations have potential to increase N use efficiency
and reduce fertilizer N input for corn. However, within a corn production system
using conventional tillage and producing (averaged across rotation and N
treatment) about 6.2-Mg ha(-1) corn grain per year, we found no gain in soil C
after 10 years regardless of rotation.
DOI: 10.1100/tsw.2001.90
2333. ScientificWorldJournal. 2001 Oct 3;1 Suppl 2:181-6.
Nitrogen fertilizer rate and crop management effects on nitrate leaching from an
agricultural field in central Pennsylvania.
Fox RH(1), Zhu Y, Toth JD, Jemison JM Jr, Jabro JD.
Author information:
(1)Department of Crop and Soil Sciences, Penn State University, University Park,
PA 16802, USA. rhf@psu.edu
Eighteen pan lysimeters were installed at a depth of 1.2 m in a Hagerstown silt

loam soil in a corn field in central Pennsylvania in 1988. In 1995, wick
lysimeters were also installed at 1.2 m depth in the same access pits. Treatments
have included N fertilizer rates, use of manure, crop rotation (continuous corn,
corn-soybean, alfalfa-corn), and tillage (chisel plow-disk, no-till). The
leachate data were used to evaluate a number of nitrate leaching models. Some of
the highlights of the 11 years of results include the following: 1) growing corn
without organic N inputs at the economic optimum N rate (EON) resulted in NO3--N
concentrations of 15 to 20 mg l(-1) in leachate; 2) use of manure or previous
alfalfa crop as partial source of N also resulted in 15 to 20 mg l(-1) of NO3--N
in leachate below corn at EON; 3) NO3--N concentration in leachate below alfalfa
was approximately 4 mg l(-1); 4) NO3--N concentration in leachate below soybeans
following corn was influenced by fertilizer N rate applied to corn; 5) the mass
of NO3--N leached below corn at the EON rate averaged 90 kg N ha(-1) (approx. 40%
of fertilizer N applied at EON); 6) wick lysimeters collected approximately 100%
of leachate vs. 40-50% collected by pan lysimeters. Coefficients of variation of
the collected leachate volumes for both lysimeter types were similar; 7) tillage
did not markedly affect nitrate leaching losses; 8) tested leaching models could
accurately predict leachate volumes and could be calibrated to match nitrate
leaching losses in calibration years, but only one model (SOILN) accurately
predicted nitrate leaching losses in the majority of validation treatment years.
Apparent problems with tested models: there was difficulty estimating sizes of
organic N pools and their transformation rates, and the models either did not
include a macropore flow component or did not handle macropore flow well.
DOI: 10.1100/tsw.2001.91
2334. Am J Physiol Endocrinol Metab. 2003 Oct;285(4):E708-17. Epub 2003 Jun 3.
High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP
kinase.
McGinn S(1), Saad S, Poronnik P, Pollock CA.
Author information:
(1)Renal Research Group, Kolling Institute, Royal North Shore Hospital,
University of Sydney, New South Wales 2065, Sydney, Australia.
The mitogen-activated protein (MAP) kinases contribute to altered cell growth and
function in a variety of disease states. However, their role in the endothelial
complications of diabetes mellitus remains unclear. Human endothelial cells were
exposed for 72 h to 5 mM (control) or 25 mM (high) glucose or 5 mM glucose plus
20 mM mannitol (osmotic control). The roles of p38 and p42/44 MAP kinases in the
high glucose-induced growth effects were determined by assessment of
phosphorylated MAP kinases and their downstream activators by Western blot and by
pharmacological inhibition of these MAP kinases. Results were expressed as a
percentage (means +/- SE) of control. High glucose increased the activity of
total and phosphorylated p38 MAP kinase (P < 0.001) and p42/44 MAP kinase (P <
0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the
antiproliferative but not the hypertrophic effects associated with high-glucose
conditions. Transforming growth factor (TGF)-beta1 increased the levels of
phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the
antiproliferative effects of this cytokine. The high glucose-induced increase in
phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1
neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P
< 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity,
and therefore inhibition of p38 MAP kinase had no influence on these growth
responses. Blockade of p42/44 MAP kinase had no effect on the changes in
endothelial cell growth induced by either high glucose or hyperosmolarity. High
glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells,
but only p38 MAP kinase mediated the antiproliferative growth response through
the effects of autocrine TGF-beta1. High glucose-induced endothelial cell
hypertrophy was independent of activation of the MAP kinases studied. In
addition, these effects were independent of any increase in osmolarity associated
with high-glucose exposure.
DOI: 10.1152/ajpendo.00572.2002
2335. J Dairy Sci. 2003 May;86(5):1858-63.
Modification of the Penn State forage and total mixed ration particle separator
and the effects of moisture content on its measurements.
Kononoff PJ(1), Heinrichs AJ, Buckmaster DR.
Author information:
The Penn State Particle Separator has led to widespread measurement of forage and
total mixed ration (TMR) particle size. However, a large proportion of small
particles may pass through both sieves when a TMR is analyzed, and field research
has suggested that both shaking frequency and sample dry matter may affect the
results. The objectives of this project were to test the effects of an additional
sieve with a smaller aperture size, shaking frequency, and sample moisture
content on results obtained. A sieve was constructed out of wire with a nominal
size aperture of 1.18 mm. Samples of alfalfa haylage, corn silage, and a TMR were
shaken at frequencies of 0.9, 1.1, and 1.6 Hz with a 17-cm stroke length.
Reducing shaking frequency to 0.9 Hz resulted in more material being retained on
the 19.0-mm sieve for all sample types, increasing the geometric mean. Increasing
frequency to 1.6 Hz did not affect the geometric mean, but did result in a
greater amount of corn silage falling through the 1.18-mm sieve. For alfalfa
haylage, moisture content between 57.4 and 35.6% did not affect results; however,
for corn silage, less moisture increased the percentage of particles less than
1.18 mm and decreased the geometric mean. For both sample types, further drying
caused a greater proportion of small particles and a smaller geometric mean. We
suggest using a third sieve and shaking at 1.1 Hz or greater with a stroke length
of 17 cm when using the Penn State Particle Separator to analyze forage particle
size.
DOI: 10.3168/jds.S0022-0302(03)73773-4
2336. Environ Pollut. 2003;124(3):419-28.
The response of some common Egyptian plants to ozone and their use as
biomonitors.
El-Khatib AA(1).
Author information:
(1)Department of Botany, Faculty of Science, 82524 Sohag, Egypt.
aaelkhatib@yahoo.com
Relative sensitivity of five common Egyptian plant species namely, Senecio

vulgaris, Malva parviflora, Sonchus oleraceus, Medicago sativa and Melilotus
indicus to elevated levels of ozone has been studied. The plants were exposed to
charcoal filtered air (CFA) and different levels of O3 (50 and 100 ppb) for 5 h
per day. The studied parameters were recorded for five consecutive days after
fumigation. The foliar injury varied significantly among species in a
dose-dependent manner. Severe injury symptoms were recorded on the leaves of M.
sativa. With the exception of M. parviflora, all species exhibited significant
increases in the percentage reduction of the above-ground dry weight as a result
of reductions in both leaf and stem dry weights. M. sativa showed a marked
reduction in its relative growth rate at elevated levels of O3. The extent of
chlorophyll a destruction was higher in both M. sativa and S. oleraceus than in
the other species tested. No differences in the sensitivity of chlorophylls a+b
and carotenoids to ozone levels were recorded in this work. Percentage reduction
of ascorbic acid was higher in M. sativa and S. oleraceus, compared with the
other species studied. With respect to relative percentages of proline, there was
a significant difference in the responses of plants to ozone. According to the
ozone resistance (R%), measured as relative growth rate, the test species were
arranged in the descending order: M. parviflora>M. Indicus>S. Vulgaris>S.
Oleraceus>M. sativa. In M. sativa, both determinant and correlation coefficients
are well reflected in the relationship between its physiological response, its
performance and ozone levels, supporting its recommendation as a candidate for
biomonitoring in Egypt.
2337. J Exp Bot. 2003 Jul;54(388):1721-30. Epub 2003 May 13.
Oxygen deficiency affects carbohydrate reserves in overwintering forage crops.
Bertrand A(1), Castonguay Y, Nadeau P, Laberge S, Michaud R, Bélanger G, Rochette

P.
Author information:
Canada, 2560 Hochelaga Blvd., Sainte-Foy, Québec, Canada G1V 2J3.
bertranda@agr.gc.ca
Anaerobic conditions developing under an ice cover affect winter survival and
spring regrowth of economically important perennial crops. The objective was to
compare, during a prolonged period of low (<2%) O2 at low temperature, the
concentration of carbohydrates of four plant species contrasting in their
resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago
sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and
cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing
oxygen deficiency stress by enclosing potted plants in gas-tight bags in late
autumn for overwintering in an unheated greenhouse. Timothy was previously
reported to be more resistant to oxygen deficiency than the three other species.
Non-structural carbohydrates increased and remained at a higher concentration in
timothy than in the other three species under low O2 concentration.
Concentrations of sucrose, fructose, glucose, and fructans increased in response
to oxygen deficiency in timothy, whereas the concentration of soluble sugars
decreased under the same conditions in lucerne, red clover, and cocksfoot. The
gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response
to low oxygen concentration in oxygen deficiency-sensitive lucerne while it
remained unchanged in the oxygen deficiency-resistant timothy. It is concluded
that timothy maintains higher carbohydrate reserves under oxygen deficiency, a
specific feature that could favour its winter survival and spring regrowth.
DOI: 10.1093/jxb/erg182
2338. J Food Prot. 2003 May;66(5):767-74.
Chemical and irradiation treatments for killing Escherichia coli O157:H7 on

alfalfa, radish, and mung bean seeds.
Bari ML(1), Nazuka E, Sabina Y, Todoriki S, Isshiki K.
Author information:
(1)Food Hygiene Team, National Food Research Institute, Kannondai-2-1-12, Tsukuba
305-8642, Japan.
In this study, the effectiveness of dry-heat treatment in combination with

chemical treatments (electrolyzed oxidizing [EO] water, califresh-S, 200 ppm of
active chlorinated water) with and without sonication in eliminating Escherichia
coli O157:H7 on laboratory-inoculated alfalfa, radish, and mung bean seeds was
compared with that of dry-heat treatment in combination with irradiation
treatment. The treatment of mung bean seeds with EO water in combination with
sonication followed by a rinse with sterile distilled water resulted in
reductions of approximately 4.0 log10 CFU of E. coli O157:H7 per g. whereas
reductions of ca. 1.52 and 2.64 log10 CFU/g were obtained for radish and alfalfa
seeds. The maximum reduction (3.70 log10 CFU/g) for mung bean seeds was achieved
by treatment with califresh-S and chlorinated water (200 ppm) in combination with
sonication and a rinse. The combination of dry heat, hot EO water treatment, and
sonication was able to eliminate pathogen populations on mung bean seeds but was
unable to eliminate the pathogen on radish and alfalfa seeds. Other chemical
treatments used were effective in greatly reducing pathogen populations on radish
and alfalfa seeds without compromising the quality of the sprouts, but these
treatments did not result in the elimination of pathogens from radish and alfalfa
seeds. Moreover, a combination of dry-heat and irradiation treatments was
effective in eliminating E. coli O157:H7 on laboratory-inoculated alfalfa,
radish, and mung bean seeds. An irradiation dose of 2.0 kGy in combination with
dry heat eliminated E. coli O157:H7 completely from alfalfa and mung bean seeds,
whereas a 2.5-kGy dose of irradiation was required to eliminate the pathogen
completely from radish seeds. Dry heat in combination with irradiation doses of
up to 2.0 kGy did not unacceptably decrease the germination percentage for
alfalfa seeds or the length of alfalfa sprouts but did decrease the lengths of
radish and mung bean sprouts.
2339. Health Phys. 2003 May;84(5):637-41.
Effect of soil amendments on radiocesium transfer to alfalfa.
Ioannides KG(1), Papachristodoulou CA, Mertzimekis TJ, Tzialla CE.
Author information:
(1)Nuclear Physics Laboratory, The University of Ioannina, 451 10 Ioannina,
Greece. kioannid@cc.uoi.gr
Greenhouse experiments were carried out to evaluate the effect of different

soil-based countermeasures on radiocesium transfer to Medicago saliva (alfalfa)
grown on artificially contaminated loamy-clayey soil. Various rates of potassium,
ammonium, and Prussian Blue supplements were applied, and the uptake of
radiocesium by control and treated alfalfa plants was monitored during four
growth periods. Transfer factors ranging between 0.06 and 0.02 were determined
for control plants. Application of potassium at rates higher than 0.1 meq per 100
g soil was found to suppress radiocesium uptake, the effect being more pronounced
at increasing fertilization rates. On the contrary, soil treatment with ammonium
enhanced the bio-accumulation of radiocesium, indicating that Cs+ ions,
previously unavailable to plant roots, were released from soil particles.
Prussian Blue supplements had practically no effect on soil-to-alfalfa transfer
of the radionuclide.
2340. Plant Physiol. 2003 May;132(1):311-7.
Nod factor and elicitors activate different phospholipid signaling pathways in

suspension-cultured alfalfa cells.
den Hartog M(1), Verhoef N, Munnik T.
Author information:
(1)Swammerdam Institute for Life Sciences, Department of Plant Physiology,
University of Amsterdam, Kruislaan 318, The Netherlands.
Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp.

bacteria to elicit Nod responses on their legume hosts. One of the earliest
responses is the formation of phosphatidic acid (PA), a novel second messenger in
plant cells. Remarkably, pathogens have also been reported to trigger the
formation of PA in nonlegume plants. To investigate how host plants can
distinguish between symbionts and pathogens, the effects of Nod factor and
elicitors (chitotetraose and xylanase) on the formation of PA were investigated
in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be
synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via
phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a
strategy involving differential radiolabeling with [(32)P]orthophosphate was used
to determine the contribution of each pathway to PA formation. In support, PLD
activity was specifically measured by using the ability of the enzyme to transfer
the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod
factor, chitotetraose, and xylanase induced the formation of PA and its
phosphorylated product DAG pyrophosphate within 2 min of treatment. However,
whereas phospholipase C and DAG kinase were activated during treatment with all
three different compounds, PLD was only activated by Nod factor. No evidence was
obtained for the activation of phospholipase A(2).
DOI: 10.1104/pp.102.017954
PMCID: PMC166976
2341. J Dairy Sci. 2003 Apr;86(4):1445-57.
The effect of reducing alfalfa haylage particle size on cows in early lactation.
Kononoff PJ(1), Heinrichs AJ.
Author information:
The objective of this experiment was to evaluate effects of reducing forage

particle size on cows in early lactation based on measurements of the Penn State
Particle Separator (PSPS). Eight cannulated, multiparous cows averaging 19 +/- 4
d in milk and 642 +/- 45 kg BW were assigned to one of two 4 x 4 Latin Squares.
During each of the 23-d periods, animals were offered one of four diets, which
were chemically identical but included alfalfa haylage of different particle
size; short (SH), mostly short (MSH), mostly long (MLG), and long (LG).
Physically effective neutral detergent fiber (peNDF) was determined by measuring
the amount of neutral detergent fiber retained on a 1.18 mm screen and was
similar across diets (25.7, 26.2, 26.4, 26.7%) but the amount of particles >19.0
mm significantly decreased with decreasing particle size. Reducing haylage
particle size increased dry matter intake linearly (23.3, 22.0, 20.9, 20.8 kg for
SH, MSH, MLG, LG, respectively). Milk production and percentage fat did not
differ across treatments averaging 35.5 +/- 0.68 kg milk and 3.32 +/- 0.67% fat,
while a quadratic effect was observed for percent milk protein, with lowest
values being observed for LG. A quadratic effect was observed for mean rumen pH
(6.04, 6.15, 6.13, 6.09), while A:P ratio decreased linearly (2.75, 2.86, 2.88,
2.92) with decreasing particle size. Total time ruminating increased
quadratically (467, 498, 486, 468 min/d), while time eating decreased linearly
(262, 253, 298, 287 min/d) with decreasing particle size. Both eating and
ruminating per unit of neutral detergent fiber intake decreased with reducing
particle size (35.8, 36.7, 44.9, 45.6 min/kg; 19.9, 23.6, 23.5, 23.5 min/kg).
Although chewing activity was closely related to forage particle size, effects on
rumen pH were small, indicating factors other than particle size are critical in
regulating pH when ration neutral detergent fiber met recommended levels. Feeding
alfalfa haylage based rations of reduced particle size resulted in animals
consuming more feed but did not affect milk production.
DOI: 10.3168/jds.S0022-0302(03)73728-X
2342. J Dairy Sci. 2003 Apr;86(4):1382-97.
Effects of forage particle size, forage source, and grain fermentability on

performance and ruminal pH in midlactation cows.
Krause KM(1), Combs DK.
Author information:
(1)Department of Dairy Science, University of Wisconsin-Madison, 53706, USA.
Our study investigated the effects of, and interactions between, forage particle
size, level of dietary ruminally fermentable carbohydrate (RFC), and level of
dietary starch on performance, chewing activity, and ruminal pH for dairy cows
fed one level of dietary NDF. Twelve cows (48 DIM) were assigned to six
treatments in a replicated 6 x 6 Latin square. Treatments were arranged in an
incomplete 2 x 2 x 2 factorial design. Factors were: dry cracked shelled corn
(DC, low RFC) or ground high-moisture corn (HMC; high RFC), finely chopped or
coarse silage, and alfalfa silage as the only forage or a 50:50 ratio (DM basis)
of alfalfa and corn silage. Diets combining HMC with only alfalfa silage were not
included in the experiment. Diets were fed for ad libitum intake as a TMR with a
concentrate:forage ratio of 61:39. Diets based on only alfalfa silage and diets
based on a mix of alfalfa and corn silage averaged 18.6 and 15.8% CP, 25.8 and
24.7% NDF, 17.7 and 14.8% ADF, and 29.1 and 37.3% starch, respectively. Mean
particle sizes were 5.3, 2.7, 5.6, and 2.8 mm for coarse alfalfa, fine alfalfa,
coarse corn silage, and fine corn silage, respectively. Decreasing forage
particle size decreased DMI (23.3 vs. 21.6 kg) and organic matter intake (22.0
vs. 20.2 kg). Increasing RFC decreased DMI (22.8 vs. 21.0 kg) and organic matter
intake (21.5 vs. 20.0 kg). Decreasing forage particle size increased
energy-corrected milk for alfalfa based diets (34.9 vs. 37.4 kg). Percentage of
milk fat decreased with decreasing forage particle size (3.07 vs. 2.90%) and
increased level of RFC (3.04 vs. 2.57%). Percentage of protein increased when
corn silage partially replaced alfalfa silage (2.84 vs. 2.90%) but decreased when
HMC replaced DC (2.90 vs. 2.84%). Apparent total tract digestibility of DM (66.7
vs. 68.5%), OM (65.9 vs. 70.7%), and starch (88.9 vs. 93.4%) increased when level
of RFC was increased. Increasing level of RFC decreased mean ruminal pH from 5.82
to 5.67 and decreased minimum pH. Hours per day at which pH was <5.8, and area
<5.8, increased when corn silage partially replaced alfalfa silage (2.6 vs. 4.4 h
and 8.9 h x pH vs. 11.4 h x pH) and decreased further when level of RFC was
increased (4.4 vs. 6.4 h and 11.4 h x pH vs. 14.3 h x pH). Decreasing forage
particle size in HMC diets increased hours and area <5.8, but for DC diets, the
effect of forage particle size depended on forage source. Interactions were found
between level of physically effective fiber, forage source, and level of RFC on
production and pH, complicating the inclusion of these effects in dairy ration
formulation and evaluation.
DOI: 10.3168/jds.S0022-0302(03)73722-9
2343. J Dairy Sci. 2003 Apr;86(4):1370-81.
Effects of varying dietary protein and energy levels on the production of

Broderick GA(1).
Author information:
Linden Drive West, Madison 53706, USA. glenb@dfrc.wisc.edu
Forty-five multiparous and 18 primiparous Holstein cows were fed three levels of
crude protein (CP), each at three levels of neutral detergent fiber (NDF), to
identify optimal dietary CP and energy. Cows were blocked by parity and days in
milk into seven groups of nine and randomly assigned to an incomplete 9 x 9 Latin
square trial with four, 4-wk periods. Diets were formulated from alfalfa and corn
silages, high-moisture corn, soybean meal, minerals, and vitamins. Forage was 60%
alfalfa and 40% corn silage on all diets; NDF contents of 36, 32, and 28% were
obtained by feeding 75, 63, and 50% forage, respectively. Dietary CP contents of
15.1, 16.7, and 18.4% were obtained by replacing high-moisture corn with soybean
meal. Production data were from the last 2 wk of each period. Spot fecal and
urine samples were collected from 36 cows to estimate N excretion using fecal
indigestible acid detergent fiber (ADF) and urinary creatinine as markers. There
were no interactions (P > or = 0.08) between dietary CP and NDF for any trait;
thus, effects of CP were not confounded by NDF or vice versa. Intake of DM and
fat yield were lower on 15.1% CP than at higher CP. There were linear increases
in milk urea and urinary N excretion and linear decreases in N efficiency with
increasing CP. Increasing CP from 15.1 to 18.4% reduced milk N from 31 to 25% of
dietary N, increased urinary N from 23 to 35% of dietary N, and reduced fecal N
from 45 to 41% of dietary N. Decreasing NDF gave linear increases in BW gain,
yield of milk, protein, true protein, lactose, and SNF, and milk/DM intake and
milk N/N intake, and linear decreases in milk urea. However, fat yield was lower
on 28% than 32% NDF. Reducing NDF from 36 to 28% increased purine derivative
excretion by 19%, suggesting increased microbial protein. Increasing CP by adding
soybean meal to diets fed cows averaging 34 kg/d of milk increased intake and fat
yield but depressed N efficiency. Increasing dietary energy by reducing forage
improved milk yield and efficiency and decreased excretion of environmentally
labile urinary N.
DOI: 10.3168/jds.S0022-0302(03)73721-7
2344. J Dairy Sci. 2003 Apr;86(4):1341-53.
Effects of increasing levels of refined cornstarch in the diet of lactating dairy

cows on performance and ruminal pH.
Krause KM(1), Combs DK, Beauchemin KA.
Author information:
Our study investigated the effect of a linear increase in level of ruminally

fermentable carbohydrate, at a constant level of dietary starch and fiber, on
performance, microbial N yield, chewing activity, and ruminal pH of midlactation
dairy cows. Eight cows (53 DIM) were assigned to four treatments in a double 4 x
4 Latin square. Diets consisted of increasing levels of refined cornstarch (0,
5.9, 11.9, and 17.9% of diet dry matter) replacing dry cracked, shelled corn so
that increasing amounts of dietary starch originated from refined cornstarch.
Corn gluten feed was used to balance diets for similar NDF content. The four
diets averaged 17.9% CP, 27.2% NDF, 18.7% ADF, and 31.1% starch (dry matter
basis). Diets were fed for ad libitum intake and had a forage to concentrate
ratio of 40:60. Forage was coarsely chopped (13.7 mm mean particle size) alfalfa
silage. Daily dry matter intake averaged 26.0 kg and tended (P = 0.08) to
increase quadratically with increasing level of refined cornstarch. Milk
production averaged 38.9 kg/d and milk fat percentage tended (P = 0.08) to
decrease linearly, whereas percentage of protein increased quadratically, with
increasing level of refined cornstarch. Yield of components and energy corrected
milk was similar across diets. Total tract digestibility of starch increased
linearly from 85.1% to 92.4% with increasing level of refined cornstarch.
Microbial yield was unaffected by diet and averaged 371.1 g N/d. Time spent
eating decreased linearly from 329 to 308 min/d when level of refined cornstarch
was increased, but rumination time was unaffected. Ruminal concentration and
proportion of acetate decreased linearly while concentration and proportion of
propionate increased linearly with increasing level of refined cornstarch. Mean
ruminal pH, time spent below pH 5.8 (h), and area below pH 5.8 (h x pH units/d)
were unaffected by level of refined cornstarch and averaged 5.97, 8.4, and 2.9,
respectively. Increasing the level of carbohydrates fermented in the rumen by
replacing dry cracked corn with refined cornstarch (up to 57% of dietary starch)
did not compromise rumen fermentation or affect performance of midlactation dairy
cows.
DOI: 10.3168/jds.S0022-0302(03)73719-9
2345. J Dairy Sci. 2003 Apr;86(4):1306-14.

Responses of primiparous and multiparous Holstein cows to additional energy from
fat or concentrate during summer.
Drackley JK(1), Cicela TM, LaCount DW.
Author information:
drackley@uiuc.edu
Supplemental fat has been advocated for use during hot weather and often
increases milk yield of cows past peak production when energy intake should not
be limiting. Relative responses of primiparous and multiparous cows to
supplemental fat or isocaloric addition of concentrates under hot weather
conditions have not been determined. Nine multiparous and nine primiparous
Holstein cows (154 and 167 d in milk, respectively) were used in a replicated 3 x
3 Latin square design with 28-d periods. Diets were 1) control (35% alfalfa
silage, 25% corn silage, and 40% concentrate, dry matter [DM] basis); 2) control
plus 3% fat (HF); and 3) high concentrate ([HC] 15% alfalfa silage, 25% corn
silage, and 60% concentrate). Diets were isonitrogenous; diets HF and HC were
isocaloric (1.60 Mcal of net energy for lactation [NE(L)] per kilogram DM) and
higher energy than the control (1.52 Mcal/kg). No parity x diet interactions
approached significance. DM intake (DMI) was greater when cows were fed HC than
when they were fed HF (21.0, 20.1, and 21.3 kg/d for control, HF, and HC,
respectively); intake of NE(L) tended to be increased only for HC. Milk yield was
increased by higher-energy diets, but milk fat content was decreased. Milk total
protein content was decreased by HF and increased by HC. Yield of
solids-corrected milk (SCM) was not different among diets. Efficiency of milk
production, expressed either as total milk solids yield per kilogram of DMI or as
kilograms of SCM per megacalorie of NE(L) intake, was greater for HF than for HC.
Plasma glucose was higher after feeding for cows fed HC; plasma nonesterified
fatty acids were greater for HF. Respiration rate and rectal temperature were
greater for HC than for HF. Regardless of parity, increased energy density from
either fat or concentrate increased milk yield in midlactation cows, but diets
caused energy to be partitioned differently among milk components and body
storage. Supplemental rumen-active fat had modest advantages over additional
starch-based concentrate during summer heat conditions.
DOI: 10.3168/jds.S0022-0302(03)73714-X
2346. J Chem Ecol. 2003 Feb;29(2):303-20.
Herbivore-induced responses in alfalfa (Medicago sativa).
Agrelli J(1), Oleszek W, Stochmal A, Olsen M, Anderson P.
Author information:
(1)Department of Animal Ecology, Lund University, SE-223 62 Lund, Sweden.
jep.agrell@zooekol.lu.se
The herbivore-induced response of alfalfa (Medicago sativa) was examined through

assays with Spodoptera littoralis larvae and analyses of important secondary
substances. In food preference experiments, larvae preferred young undamaged
alfalfa plants over plants that had been damaged by feeding larvae 5 and 7 days
earlier. while no difference in feeding preferences could be detected 1. 9. and
14 days after damage. This suggests a peak in the herbivore induced resistance of
alfalfa approximately one week after initial damage. The induced resistance in
young plants was also shown to be systemic, while older flowering plants failed
to show increased resistance after defoliation. Larvae gained weight slower and
had lower pupal mass when fed damaged alfalfa than when fed undamaged alfalfa.
Levels of total saponins were increased in foliage of damaged alfalfa, and
detailed analyses of specific saponin components revealed doubled concentrations
of 3GlcA,28AraRhaXyl medicagenate (medicagenic acid bidesmoside) and 3GlcAGalRha
soyasapogenol B (soyasaponin I). Levels of the flavonoid apigenin (as free
aglycone) also were increased in herbivore damaged plants. The herbivore-induced
response of alfalfa was significantly weaker than that of cotton: S. littoralis
larvae given a choice of undamaged cotton and undamaged alfalfa preferred to feed
on cotton, whereas preferences shifted towards alfalfa when plants were damaged.
2347. Bull Environ Contam Toxicol. 2003 May;70(5):1036-44.
Effects of zinc upon tolerance and heavy metal uptake in alfalfa plants (Medicago
sativa).
Peralta-Videa JR(1), Gardea-Torresdey JL, Walton J, Mackay WP, Duarte-Gardea M.
Author information:
(1)Environmental Science and Engineering, Ph.D. Program, University of Texas at
El Paso, El Paso, TX 79968, USA.
DOI: 10.1007/s00128-003-0087-0
Selenium and sulfur accumulation and soil selenium dissipation in planting of

four herbaceous plant species in soil contaminated with drainage sediment rich in
both selenium and sulfur.
Wu L(1), Guo X, Bañuelos GS.
Author information:
(1)Department of Environmental Horticulture, University of California, Davis,
California 95616, USA.
Four selenium (Se) nonaccumulator plant species, including a forage grass

species, Tall Fescue (Festuca arundinacea Schreb.), a forage legume species,
Alfalfa (Medicago sativa L.), a wetland species, Rush (Juncus tenuis Wild.), and
a dry-land alkaline soil species, Saltgrass (Distichlis spicata L.), were grown
in soil contaminated by agricultural drainage sediment having elevated levels of
Se and sulfur (S). The above-ground plant tissues were consecutively harvested
five times and examined for Se and S accumulation. Plant tissue Se concentrations
ranged from 23.0 mg kg-1 to 8.3 mg kg-1. Tissue S concentrations ranged from 3239
mg kg-1 to 7034 mg kg-1. Both tissue Se and S concentrations were significantly
different between harvests, species, and species/harvest interactions. Total Se
accumulation by the plant biomass harvested ranged from 0.3 to 1.3 mg per soil
column and total S accumulations ranged from 87.5 to 321.1 mg per soil column.
The reduction in the percentage of total soil Se after 24 weeks growth of the
plant species ranged from 12.0% in the Tall Fescue planting to 17.3% in the Rush
planting. Over 90% of the soil Se losses were unidentified losses and leaching of
Se was prevented. The accumulations of Se and S in the plant biomass were very
small compared with the total soil Se and S losses, but substantial amounts of
total soil Se (12.0 to 15.0%) and S (28.0 to 50.9%) inventories were dissipated
by the growing and harvesting of the plants. The soil S concentration was several
hundred times higher than the soil Se concentration, but Se accumulation by the
plants and Se dissipation from the soil were not impaired by the high level of
soil sulfur. For natural grassland habitat restoration, such as at the Kesterson
Wildlife Refuge in the Central Valley of California, or for restoration of
large-scale Se contaminated agricultural lands, Se nonaccumulator plant species
are favorable candidates, because the possibility of introducing Se toxicity into
the food chain can be minimized.
DOI: 10.1080/16226510390856457
2349. J Exp Bot. 2003 May;54(386):1481-7.
Suppression of arbuscular mycorrhizal colonization and nodulation in split-root

systems of alfalfa after pre-inoculation and treatment with Nod factors.
Catford JG(1), Staehelin C, Lerat S, Piché Y, Vierheilig H.
Author information:
(1)CRBF, Faculté de Foresterie et de Géomatique, Université Laval, Pavillon C-E.
Marchand, Ste-Foy G1K 7P4, Québec, Canada.
Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and
nodule-inducing rhizobia. The existing nodules systemically suppress subsequent
nodule formation in other parts of the root, a phenomenon termed autoregulation.
Similarly, mycorrhizal roots reduce further AMF colonization on other parts of
the root system. In this work, split- root systems of alfalfa (Medicago sativa)
were used to study the autoregulation of symbiosis with Sinorhizobium meliloti
and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation
systemically influences AMF root colonization and vice versa. Nodules on one half
of the split-root system suppressed subsequent AMF colonization on the other
half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced
nodule formation on the other side. An inhibition effect was also observed with
Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S.
meliloti systemically suppressed both nodule formation and AMF colonization. The
application of Nod factors, however, did not influence the allocation of (14)C
within the split-root system, excluding competition for carbohydrates as the
regulatory mechanism. These results indicate a systemic regulatory mechanism in
the rhizobial and the arbuscular mycorrhizal association, which is similar in
both symbioses.
2350. J Dairy Sci. 2003 Mar;86(3):944-53.
Milk conjugated linoleic acid response to fish oil supplementation of diets

differing in fatty acid profiles.
AbuGhazaleh AA(1), Schingoethe DJ, Hippen AR, Kalscheur KF.
Author information:
USA.
The objective of this experiment was to examine the effect of feeding fish oil
(FO) along with fat sources that varied in their fatty acid compositions (high
stearic, high oleic, high linoleic, or high linolenic acids) to determine which
combination would lead to maximum conjugated linoleic acid (cis-9,trans-11 CLA)
and transvaccenic acid (TVA) concentrations in milk fat. Twelve Holstein cows
(eight multiparous and four primiparous cows) at 73 (+/- 32) DIM were used in a 4
x 4 Latin square with 4-wk periods. Treatment diets were 1) 1% FO plus 2% fat
source high in stearic acid (HS), 2) 1% FO plus 2% fat from high oleic acid
sunflower seeds (HO), 3) 1% FO plus 2% fat from high linoleic acid sunflower
seeds (HLO), and 4) 1% FO plus 2% fat from flax seeds (high linolenic; HLN).
Diets formulated to contain 18% crude protein were composed of 50% (dry basis)
concentrate mix, 25% corn silage, 12.5% alfalfa haylage, and 12.5% alfalfa hay.
Milk production (35.8, 36.3, 34.9, and 35.0 kg/d for diets 1 to 4) was similar
for all diets. Milk fat percentages (3.14, 2.81, 2.66, and 3.08) and yields
(1.13, 1.02, 0.93, and 1.08 kg/d) for diets 1 to 4 were lowest for HLO. Milk
protein percentages (3.04, 3.03, 3.10, and 3.08) and dry matter intake (DMI)
(25.8, 26.0, 26.2, and 26.2 kg/d) for diets 1 to 4 were similar for all diets.
Milk cis-9,trans-11 CLA concentrations (0.70, 1.04, 1.70, and 1.06 g/100 g fatty
acids) for diet 1 to 4 and yields (7.7, 10.7, 15.8, and 11.3 g/d) for diets 1 to
4 were greatest with HLO and were least with HS. Milk cis-9,trans-11 CLA
concentrations and yields were similar for cows fed the HO and the HLN diets.
Similar to milk cis-9,trans-11 CLA, milk TVA concentration (1.64, 2.49, 3.74, and
2.41 g/100 g fatty acids) for diets 1 to 4 was greatest with the HLO diet and
least with the HS diet. Feeding a high linoleic acid fat source with fish oil
most effectively increased concentrations and yields of milk cis-9,trans-11 CLA
and TVA.
DOI: 10.3168/jds.S0022-0302(03)73677-7
2351. J Dairy Sci. 2003 Mar;86(3):835-43.
Effects of feeding dairy cows protein supplements of varying ruminal

degradability.
Reynal SM(1), Broderick GA.
Author information:
(1)Department of Dairy Science, University of Wisconsin, Madison, WI, USA.
Twenty-five (10 ruminally cannulated) Holstein cows averaging 82 +/- 34 d in milk

were assigned to 5 x 5 Latin squares (21-d periods) and fed diets supplemented
with one of four different proteins to assess effects on production, ruminal
metabolism, omasal flow of N fractions, and degradation rates of protein
supplements. Total mixed diets contained (dry matter basis) 44% corn silage, 22%
alfalfa silage, 2% urea, and 31% concentrate. Five concentrate mixes were fed:
31% high-moisture shelled corn (HMSC; basal); 9% solvent soybean meal (SSBM), 22%
HMSC; 10% expeller soybean meal (ESBM), 21% HMSC; 5.5% blood meal (BM), 25.5%
HMSC; and 7% corn gluten meal (CGM), 24% HMSC. Diets averaged, respectively,
15.8, 19.1, 19.7, 20.3, and 19.3% crude protein. Feeding the basal diet reduced
intake and yield of milk, fat-corrected milk (FCM), and all milk components
compared to the protein-supplemented diets. Milk yield was higher for cows fed
ESBM and CGM, fat yield was higher for cows fed SSBM and CGM, but FCM and protein
yields were not different among cows fed supplemental protein. Based on omasal
sampling, mean in vivo estimates of ruminal degradation rate for the crude
protein in SSBM, ESBM, BM, and CGM was, respectively, 0.417, 0.179, 0.098, and
0.051/h (computed using passage rates observed for the small particle phase; mean
= 0.14/h), and 0.179, 0.077, 0.042, and 0.026/h (computed using a passage rate of
0.06/h). The in vivo degradation rate computed for SSBM at a passage rate =
0.06/h was similar to that estimated using the inhibitor in vitro method.
However, in vivo degradation rates computed at passage rate = 0.06/h for ESBM,
BM, and CGM were about two, four, and three times more rapid than those estimated
by inhibitor in vitro. Experimental proteins fed in this trial will be used as
standards for developing in vitro methods for predicting rates of ruminal protein
degradation.
DOI: 10.3168/jds.S0022-0302(03)73666-2
2352. Emerg Infect Dis. 2003 Apr;9(4):474-9.
Alfalfa seed decontamination in a Salmonella outbreak.
Gill CJ(1), Keene WE, Mohle-Boetani JC, Farrar JA, Waller PL, Hahn CG, Cieslak
PR.
Author information:
(1)Oregon Department of Human Services, Portland, Oregon, USA.
Based on in vitro data, the U.S. Food and Drug Administration recommends chemical
disinfection of raw sprout seeds to reduce enteric pathogens contaminating the
seed coats. However, little is known about the effectiveness of decontamination
at preventing human disease. In 1999, an outbreak of Salmonella enterica serotype
Mbandaka occurred in Oregon, Washington, Idaho, and California. Based on
epidemiologic and pulsed-field gel electrophoresis evidence from 87 confirmed
cases, the outbreak was linked to contaminated alfalfa seeds grown in
California's Imperial Valley. Trace-back and trace-forward investigations
identified a single lot of seeds used by five sprout growers during the outbreak
period. Cases of salmonellosis were linked with two sprout growers who had not
employed chemical disinfection; no cases were linked to three sprout growers who
used disinfection. This natural experiment provides empiric evidence that
chemical disinfection can reduce the human risk for disease posed by contaminated
seed sprouts.
DOI: 10.3201/eid0904.020519
PMCID: PMC2957971
2353. Pest Manag Sci. 2003 Apr;59(4):437-42.
Can field-scale habitat diversification enhance the biocontrol potential of

spiders?
Samu F(1).
Author information:
(1)Plant Protection Institute, Hungarian Academy of Sciences, PO Box 102
Budapest, H-1525 Hungary. samu@julia-nki.hu
The study investigated the effect of strip management in alfalfa on the abundance
and diversity of spiders. In strip management, narrow strips were left unmown in
the crop, which were then shifted at next mowing. In an experimental field, out
of the six 50 x 50-m blocks, three received strip management and three were
managed traditionally (ie the whole block was mown). Our main hypothesis was that
unmown strips will contain a more diverse and abundant spider assemblage, which
will increase spider numbers in the mown parts of the crop. Over the 3 years of
the study, unmown strips contained a spider assemblage of more than 50% higher
abundance than in control alfalfa. Species diversity was also greater in the
strips, and the presence of specific indicator species could be shown. We found
that five dominant species comprised over 75% of spiders in the strips, and they
were the same species as those that are dominant in alfalfa, where they represent
85% of all spiders. In contrast, a neighbouring meadow, which was a control in
the study, also had high diversity, but different dominant species. The main
result of the experiment was negative in that elevated abundance of spiders in
unmown strips did not raise spider numbers in the mown strips of alfalfa, where
abundance was only marginally higher than in the control alfalfa. We can
speculate that better ecological conditions in unmown strips attract and conserve
spiders from neighbouring areas and from the crop at mowing. We suggest that if
diversification is more interspersed within the crop, we have a higher chance of
increasing the number of natural enemies and bringing them closer to pests, so
that they can interact more intensively.
DOI: 10.1002/ps.635
Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium

meliloti.
Abbas BA(1), Vineetha KE, Prasad CK, Vij N, Hassani R, Randhawa GS.
Author information:
(1)Department of Biosciences & Biotechnology, Indian Institute of Technology,
Roorkee 247 667, India.
Twenty one cysteine and 13 methionine auxotrophs of Sinorhizobium meliloti Rmd201

were obtained by random mutagenesis with transposon Tn5. The cysteine auxotrophs
were sulfite reductase mutants and each of these auxotrophs had a mutation in
cysI/cysJ gene. The methionine auxotrophs were metA/metZ, metE and metF mutants.
One hundred per cent co-transfer of Tn5-induced kanamycin resistance and
auxotrophy from each Tn5-induced auxotrophic mutant indicated that each mutant
cell most likely had a single Tn5 insertion. However, the presence of more than
one Tn5 insertions in the auxotrophs used in our study cannot be ruled out. All
cysteine and methionine auxotrophs induced nodules on alfalfa plants. The nodules
induced by cysteine auxotrophs were fully effective like those of the parental
strain-induced nodules, whereas the nodules induced by methionine auxotrophs were
completely ineffective. The supplementation of methionine to the plant nutrient
medium completely restored symbiotic effectiveness to the methionine auxotrophs.
These results indicated that the alfalfa host provides cysteine but not
methionine to rhizobia during symbiosis. Histological studies showed that the
defective symbiosis of methionine auxotrophs with alfalfa plants was due to
reduced number of infected nodule cells and incomplete transformation of
bacteroids.
Symbiotic characterization of isoleucine+valine and leucine auxotrophs of

Hassani R(1), Prasad CK, Vineetha KE, Vij N, Singh P, Sud R, Yadav S, Randhawa
GS.
Author information:
Roorkee 247 667, India.
Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti

Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by
screening of Tn5 derivatives on minimal medium supplemented with modified
Holliday pools. Based on intermediate feeding, intermediate accumulation and
cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated
as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively.
Symbiotic properties of all ilvD mutants with alfalfa plants were similar to
those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-.
Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair
curling and infection thread formation. The ilvC mutants were capable of curling
root hairs but did not induce infection thread formation. All leucine auxotrophs
were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not
restore symbiotic effectiveness to the auxotrophs. Histological studies revealed
that the nodules induced by the leucine auxotrophs did not develop fully like
those induced by the parental strain. The nodules induced by leuB mutants were
structurally more advanced than the leuC/leuD mutant induced nodules. These
results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may
have a role in symbiosis. The position of ilv genes on the chromosomal map of S.
meliloti was found to be near ade-15 marker.
2356. Cell Biol Int. 2003;27(3):257-9.
Involvement of MAP kinase SIMK and actin cytoskeleton in the regulation of root
hair tip growth.
Samaj J(1), Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I,

Lenart P, Salaj J, Volkmann D, Bögre L, Baluska F, Hirt H.
Author information:
(1)Institute of Microbiology and Genetics, Vienna Biocenter, Dr Bohrgasse 9, 1030
Vienna, Austria.
2357. Planta. 2003 Apr;216(6):993-1002. Epub 2002 Dec 18.
The mode of action of cell wall-degrading enzymes and their interference with Nod
factor signalling in Medicago sativa root hairs.
Carden DE(1), Felle HH.
Author information:
(1)Institut für Allgemeine Botanik und Pflanzenphysiologie,
Justus-Liebig-Universität Giessen, Senckenbergstrasse 17-21, 35390 Giessen,
Germany.
Medicago sativa L. (alfalfa) root hairs respond to Nod factors
[NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion
fluxes. Protoplasts prepared from these cells using the cell wall-digesting
enzymes pectolyase and cellulase do not, or to a rather small extent, respond to
Nod factors. In an effort to understand this activity loss we analyzed the mode
of action of both enzymes with respect to their effects on the root hairs as well
as their interference with the Nod factor response. (i) In the presence of the
enzymes, Nod factor at saturating concentrations neither depolarized the plasma
membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes,
Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s,
pectolyase depolarized the plasma membrane, alkalized the external space,
acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii)
Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the
cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike
pectolyase, the cellulase response was only weakly refractory to a second
addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase
inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while
cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects
similar to those of untreated enzymes. We conclude that the effects shown do not
originate from enzymatic activity, but from interactions of the proteins with
cell wall or plasma membrane constituents. It is further concluded that these
enzymes (pectolyase more so than cellulase) trigger defense-related signal
pathways, which makes protoplasts prepared with such enzymes unsuitable for
studies of symbiotic or defense-related signalling.
DOI: 10.1007/s00425-002-0952-9
[Simulation alfalfa growth in Wulanbuhe sandy region].
Bai W(1), Bao X.
Author information:
(1)Laboratory of Quantitative Vegetation Ecology, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093. bwming@ns.ibcas.ac.cn
Based on the theories of accumulated temperature and on the physio-ecological

characteristics of Medicago sativa, a simulation model for its growth under soil
water stress in arid sandy region was developed. The model was mainly composed of
four modules: the stage module of growth, the dynamic module of leaf area index,
the accumulated module of dry matter, and the distributive module of dry matter.
After simulating and calculating, the model could be used to predict the growing
progress and dynamic changes of leaf area and yield for herbage in sandy region.
The result shows that the application of the model to production is usually
effective.
2359. J Agric Food Chem. 2003 Apr 9;51(8):2193-9.
Evaluation of the estrogenic effects of legume extracts containing

phytoestrogens.
Boué SM(1), Wiese TE, Nehls S, Burow ME, Elliott S, Carter-Wientjes CH, Shih BY,
McLachlan JA, Cleveland TE.
Author information:
(1)Agricultural Research Service, Southern Regional Research Center, U.S.
Department of Agriculture, New Orleans, Louisiana 70179, USA.
sboue@nola.srrc.usda.gov
Seven legume extracts containing phytoestrogens were analyzed for estrogenic

activity. Methanol extracts were prepared from soybean (Glycine max L.), green
bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean
sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover
blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and
red clover blossom showed significant competitive binding to estrogen receptor
beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent
MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and
sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell
proliferation above levels observed with estradiol. The pure estrogen antagonist,
ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an
ER-related signaling pathway was involved. The ER subtype-selective activities of
legume extracts were examined using transiently transfected human embryonic
kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist
activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell
proliferation, the active components in kudzu root extract were determined to be
the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These
results show that several legumes are a source of phytoestrogens with high levels
of estrogenic activity.
DOI: 10.1021/jf021114s
2360. Genome. 2003 Feb;46(1):51-8.
AFLP-based assessment of genetic diversity among nine alfalfa germplasms using

bulk DNA templates.
Segovia-Lerma A(1), Cantrell RG, Conway JM, Ray IM.
Author information:
(1)Facultad de Ciencias Agrícolas y Forestales, Universidad Autónoma de
Chihuahua, Km. 2 1/2 Carretera Delicias-Rosales, Ciudad Delicias, Chihuahua, C.P.
33000, México.
Improving commercial utilization of perennial Medicago collections requires

developing approaches that can rapidly and accurately characterize genetic
diversity among large numbers of populations. This study evaluated the potential
of using amplified fragment length polymorphism (AFLP) DNA markers, in
combination with DNA bulking over multiple genotypes, as a strategy for
high-throughput characterization of genetic distances (D) among alfalfa (Medicago
sativa L.) accessions. Bulked DNA templates from 30 genotypes within each of nine
well-recognized germplasms (African, Chilean, Flemish, Indian, Ladak, Medicago
sativa subsp. falcata, Medicago sativa subsp. varia, Peruvian, and Turkistan)
were evaluated using 34 primer combinations. A total of 3754 fragments were
identified, of which 1541 were polymorphic. The number of polymorphic fragments
detected per primer combination ranged from 20 to 85. Pairwise D estimates among
the nine germplasms ranged from 0.52 to 1.46 with M. sativa subsp. falcata being
the most genetically dissimilar. Unweighted pair-group method arithmetic average
(UPGMA) analysis of the marker data produced two main clusters, (i) M. sativa
subsp. sativa and M. sativa subsp. varia, and (ii) M. sativa subsp. falcata.
Cluster-analysis results and D estimates among the Chilean, Peruvian, Flemish,
and M. sativa subsp. varia germplasms supported the hypothesis that Peruvian was
more similar to original Spanish introductions into Central and South America
than Chilean. Hierarchical arrangement of the nine germplasms was supported by
their respective geographic, subspecific, and intersubspecific hybrid origins.
Subsets of as few as seven highly informative primer pairs were identified that
produced comparable D estimates and similar heirarchical arrangements compared
with the complete dataset. The results indicate that use of primer-pair subsets
for AFLP analysis of bulk DNA templates could serve as a high-throughput system
for accurately characterizing genetic diversity among large numbers of alfalfa
populations.
DOI: 10.1139/g02-100
2361. Protoplasma. 2003 Mar;220(3-4):179-87.
Structural changes induced by NaCl in companion and transfer cells of Medicago

sativa blades.
Boughanmi N(1), Michonneau P, Verdus MC, Piton F, Ferjani E, Bizid E,

Fleurat-Lessard P.
Author information:
(1)Laboratoire de Biologie et Physiologie Cellulaires, Faculté des Sciences,
Université de Bizerte, Bizerte.
Medicago sativa var. Gabes is a perennial glycophyte that develops new shoots
even in high salinity (150 mM NaCl). In the upper exporting leaves, K(+) is high
and Na(+) is low by comparison with the lower leaves, where Na(+) accumulation
induces chlorosis after 4 weeks of NaCl treatment. By secondary ion mass
spectroscopy, a low Na(+)/K(+) ratio was detected in the phloem complex of blade
veins in these lower leaves. By transmission electron microscopy, the
ultrastructural features were observed in the phloem complex. In the upper leaves
of both control and NaCl-treated plants, companion cells in minor veins were
found to be transfer cells. These cells may well be involved in the intravenous
recycling of ions and in Na(+) flowing out of exporting leaves. Under the effect
of NaCl, companion cells in the main veins develop transfer cell features, which
may favor the rate of assimilate transport from exporting leaves toward
meristems, allowing the positive balance necessary for the survival in salt
conditions. These features no longer assist the lower leaves when transfer cells
are necrotized in both minor and main veins of NaCl-treated plants. As transfer
cells are the only degenerating phloem constituent, our observations emphasize
their role in controlling nutrient (in particular, Na(+)) fluxes associated with
the stress response.
DOI: 10.1007/s00709-002-0043-6
2362. Plant Mol Biol. 2003 Mar;51(4):555-66.
Ankyrin protein kinases: a novel type of plant kinase gene whose expression is
induced by osmotic stress in alfalfa.
Chinchilla D(1), Merchan F, Megias M, Kondorosi A, Sousa C, Crespi M.
Author information:
(1)Institut des Sciences du Végétal, Centre National de la Recherche
Scientifique, F-91198 Gif-sur-Yvette Cedex, France.
Interaction between Medicago spp. and Sinorhizobium meliloti leads to the

development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel
type of plant protein kinase containing a N-terminal region with an ankyrin
domain, was identified and shown to be expressed both in S. meliloti-infected and
spontaneous nodules in alfalfa. This gene is not exclusively associated to
nodulation since its expression was detected in other plant organs. Several genes
coding for ankyrin protein kinases (APKs) were detected in various plants and
animals. Three closest A. thaliana homologues of Msapk1 were identified in
databases and two of them were shown to express differentially in various organs
using gene-specific RT-PCR. In contrast, Southern analysis suggests that a
single-copy gene exists in diploid M. truncatula. By screening a M. truncatula
BAC library the Mtapk1 genomic region was isolated and sequenced. Two
neighbouring genes showing homologies to previously identified sequences in data
banks were detected in the vicinity of the Mtapk1 gene and compared to similar
regions of the three Atapk genes. The distribution of exons/introns was the same
for all expressed genes of both species although Mtapk1 contained larger introns.
Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting
from three hours up to two days of treatment. These data suggest that Msapk1,
involved in alfalfa osmotic stress responses, belongs to a novel class of plant
protein kinases.
2363. Mol Plant Microbe Interact. 2003 Mar;16(3):217-25.
Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago

sativa symbiosis and their crucial role during the infection process.
Jamet A(1), Sigaud S, Van de Sype G, Puppo A, Hérouart D.
Author information:
(1)Laboratoire de Biologie Végétale et Microbiologie, CNRS FRE 2294, Université
de Nice Sophia-Antipolis, Parc Valrose, 06108 Nice cedex 2, France.
Sinorhizobium meliloti possesses three distinct catalases to cope with oxidative

stress: two monofunctional catalases (KatA and KatC) and one bifunctional
catalase-peroxydase (KatB). The katB gene is constitutively expressed during
growth in batch culture and is not induced under oxidative stress conditions. In
contrast, the expression of katA and katC genes is mainly regulated at the
transcription level in these conditions. A differential expression of kat genes
was observed during the development of the nodule. A high expression of katA gene
was detected in bacteroids, suggesting that the nitrogen-fixation process induces
a strong oxidative stress. In contrast, bacteria express katB and katC genes and
not the H2O2-inducible katA gene in infection threads despite the detection of
H2O2 around the bacteria. A katB katC double mutant nodulated poorly and
displayed abnormal infection. After nonefficient release into plant cells,
bacteria failed to differentiate into bacteroids and rapidly underwent
senescence. Our results indicate that these two catalases are essential for the
establishment of the symbiosis. They also suggest that the bacteria are in a
nonexponential growth phase in infection threads and corroborate previous studies
on the growth rate of bacteria inside the plant.
DOI: 10.1094/MPMI.2003.16.3.217
2364. J Dairy Sci. 2003 Feb;86(2):630-43.
Effects of particle size of alfalfa-based dairy cow diets on chewing activity,

ruminal fermentation, and milk production.
Beauchemin KA(1), Yang WZ, Rode LM.
Author information:
(1)Livestock Sciences Section, Research Centre Agriculture and Agri-Food Canada,
Lethbridge, AB, T1J 4B1, Canada. beauchemin@agr.gc.ca
Effects offorage particle size measured as physically effective NDF and ratio of
alfalfa silage to alfalfa hay of diets on feed intake, chewing activity, particle
size reduction, salivary secretion, ruminal fermentation, and milk production of
dairy cows were evaluated using a 4 x 4 Latin square design with a 2 x 2
factorial arrangement of treatments. The diets consisted of 60% barley-based
concentrate and 40% forage, comprised either of 50:50 or 25:75 of alfalfa
silage:alfalfa hay, and alfalfa hay was either chopped or ground. Various methods
were used to determine physically effective NDF content of the diets. Cows
surgically fitted with ruminal and duodenal cannulas were offered ad libitum
access to these total mixed diets. The physically effective NDF content of the
diets was significantly lower when measured using the Penn State Particle
Separator than when measured based on particles retained on 1.18-mm screen.
Intake of DM was increased by increasing the ratio of silage to hay but was not
affected by physically effective NDF content of diets. Eating time (hours per
day) was not affected by the physically effective NDF content of diets, although
cows spent more time eating per unit of DM or NDF when consuming high versus low
alfalfa hay diets. Ruminating time (hours per day) was increased with increased
physically effective NDF content of the diets. Rumen pH was affected more by
changing dietary particle size than altering the ratio of silage to hay. Feeding
chopped hay instead of ground hay improved ruminal pH status: time during which
ruminal pH was above 6.2 increased and time during which ruminal pH was below 5.8
decreased. Milk production was increased by feeding higher concentrations of
alfalfa silage due to increased DM intake, but was not affected by dietary
particle size. Feed particle size, expressed as mean particle length or
physically effective NDF was moderately correlated with ruminating time but not
with eating time. Although physically effective NDF and chewing time were not
correlated to mean rumen pH, they were negatively correlated to the area between
the curve and pH 5.8, indicating a positive effect on reducing the risk of
acidosis. Milk fat content was correlated to rumen pH but not to physically
effective NDF or chewing activity. These results indicate that increasing
physically effective NDF content of the diets increased chewing activity and
improved rumen pH status but had limited effect on milk production and milk fat
content.
DOI: 10.3168/jds.S0022-0302(03)73641-8
2365. J Dairy Sci. 2003 Feb;86(2):593-609.
Effects of tallow in diets based on corn silage or alfalfa silage on digestion

and nutrient use by lactating dairy cows.
Ruppert LD(1), Drackley JK, Bremmer DR, Clark JH.
Author information:
Six multiparous Holstein cows (average 31 days in milk; 36.3 kg/d of milk) fitted
with ruminal cannulas were used in a 6 x 6 Latin square with 21-d periods to
investigate the effects of diets that varied in forage source and amount of
supplemental tallow. Isonitrogenous diets in a 2 x 3 factorial arrangement were
based on either high corn silage (40:10 corn silage to alfalfa silage, % of dry
matter) or high alfalfa silage (10:40 corn silage to alfalfa silage, % of dry
matter) and contained 0, 2, or 4% tallow. Intakes of dry matter and total fatty
acids were lower when cows were fed the high corn silage diet. Tallow
supplementation linearly decreased dry matter intake. Milk yield was unaffected
by diet; yields of milk fat and 3.5% fat-corrected milk were higher for the high
alfalfa silage diet but were unaffected by tallow. Milk fat percentage was higher
for the high alfalfa silage and tended to decrease when tallow was added to the
high corn silage diet. Contents of trans-C18:1 isomers in milk fat were increased
by high corn silage and tallow, and tended to be increased more when tallow was
fed in the high corn silage diet. Ruminal pH and acetate:propionate were lower
when high corn silage was fed. Ruminal acetate:propionate decreased linearly as
tallow increased; the molar proportion of acetate was decreased more when tallow
was added to the high corn silage diet. Ruminal liquid dilution rates were higher
for the alfalfa silage diet; ruminal volume and solid passage rates were similar
among diets. Total tract apparent digestibilities of dry matter, organic matter,
crude protein, starch, energy, and total fatty acids were unaffected by diet.
Digestibilities of neutral detergent fiber, acid detergent fiber, hemicellulose,
and cellulose were lower when high corn silage was fed. The high alfalfa silage
diet increased intakes of metabolizable energy and N, and increased milk energy
and productive N. Tallow decreased the amount of N absorbed but had few other
effects on utilization of energy or N. Tallow linearly increased concentrations
of nonesterified fatty acids and cholesterol in plasma; cholesterol was increased
by high alfalfa silage. Overall, forage source had more pronounced effects on
production and metabolism than did tallow supplementation. Few interactions
between forage source and tallow supplementation were detected except that
ruminal fermentation and milk fat content were affected more negatively when
tallow was fed in the high corn silage diet.
DOI: 10.3168/jds.S0022-0302(03)73638-8
2366. J Dairy Sci. 2003 Feb;86(2):576-85.
Effect of feeding supplemental fibrolytic enzymes or soluble sugars with malic

acid on milk production.
Vicini JL(1), Bateman HG, Bhat MK, Clark JH, Erdman RA, Phipps RH, Van Amburgh
ME, Hartnell GF, Hintz RL, Hard DL.
Author information:
(1)Animal Agriculture Business, Monsanto Co., St. Louis, MO 63198, USA.
john.l.vicini@monsanto.com
Two trials were conducted to evaluate effects of feeding supplemental fibrolytic

enzymes or soluble sugars and malic acid on milk production. In trial 1, 257 cows
at four sites were fed a basal diet consisting of no more than 60% of forage DM
as corn silage and less than 40% as alfalfa hay. Cows were assigned randomly
within site, parity, and two stages of lactation to: 1) control; 2) enzyme A; 3)
enzyme B; and 4) soluble sugars and malic acid. There was a 14-d pretreatment and
an 84-d treatment period. Enzyme solutions were sprayed on either the forage
component or the TMR each day while mixing feed. Trial 2 was similar, except 122
cows at one site in the United Kingdom were fed diets containing forage that was
75% corn silage and 25% grass silage, and all cows began the study between 25 to
31 DIM. Mean milk productions for 233 cows that completed trial 1 were 32.9,
32.5, 32.4, and 32.9 kg/d for control, enzyme A, enzyme B, and soluble sugars and
malic acid, respectively. Mean milk productions for 116 cows that completed trial
2 were 28.2, 27.9, 28.8, and 28.4 kg/d, respectively. In vitro analyses of the
activities of enzyme solutions indicated that all major cellulose and
hemicellulose degrading activities were present; however, the pH optima
(approximate pH = 4 to 5) were more acidic, and the temperature optimum
(approximately 50 degrees C) was greater than normal pH and temperature in the
rumen. If fibrolytic activity in the rumen is a major mechanism of action of
supplemental fibrolytic enzymes, it appears that considerable activity of these
preparations was lost due to conditions in the rumen. In conclusion, feeding
supplemental fibrolytic enzymes or malic acid with soluble sugars had no effect
on milk production under the conditions used in this study.
DOI: 10.3168/jds.S0022-0302(03)73636-4
2367. J Dairy Sci. 2003 Feb;86(2):557-64.
Effect of quantity, quality, and length of alfalfa hay on selective consumption

by dairy cows.
Leonardi C(1), Armentano LE.
Author information:
Twenty-four lactating Holstein cows were used in a replicated 6 x 6 Latin square

design. Experimental periods were 6 or 7 d. Cows were housed in tie-stalls, and
diets were fed ad libitum twice daily at 1100 and 1600 h. Diets contained 60%
concentrate and either 40% alfalfa hay or 20% alfalfa hay and 20% alfalfa silage
(dry matter basis). The effect of quantity, quality, and length of hay on sorting
behavior was determined. Treatments consisted of 20% lower or higher quality long
alfalfa hay, 20% lower or higher quality chopped alfalfa hay, and 40% lower or
higher quality chopped alfalfa hay. Variation of sorting among cows was also
determined. Particle size distribution of samples of as-fed total mixed rations
and orts were determined using the Wisconsin particle size separator. Screens
have square holes with diagonals of 26.9, 18, 8.98, 5.61, and 1.65 mm (screens Y1
to Y5, respectively). Sorting was calculated as the actual intake of each
fraction expressed as a percentage ofthe predicted intake. Increasing the
proportion of dry hay increased sorting. Quality of alfalfa hays that were
offered did not affect sorting activity. Feeding long alfalfa hay increased
selective consumption of fine particles. However, feeding long alfalfa hay also
increased intake of longer particles because a higher percentage of long
particles was offered. Across treatments, animals consistently sorted against
longer particles in favor of finer particles. In particular, intake of Y1 as a
percentage of the predicted intake was the most variable. Average Y1 intake,
across the six treatments for each cow, was between 60 and 70% of predicted
intake for four cows, 71 to 80% for 11 cows, 81 to 90% for five cows, 91 to 100%
for two cows, and 101 to 110% for two cows. On one diet a cow failed to consume
any of the Y1 portion of the total mixed ration. This variation among animals in
sorting of very long feed particles may have practical significance.
DOI: 10.3168/jds.S0022-0302(03)73634-0
2368. Oecologia. 2003 Mar;135(1):67-77. Epub 2003 Jan 30.
Molecular and carbon isotopic composition of leaf wax in vegetation and aerosols
in a northern prairie ecosystem.
Conte MH(1), Weber JC, Carlson PJ, Flanagan LB.
Author information:
(1)Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic
Institution, Woods Hole, MA 02543, USA. mconte@whoi.edu
We measured the molecular and carbon isotopic composition of major leaf wax
compound classes in northern mixed mesic prairie species (Agropyron smithii,
Stipa viridula, Bouteloua gracilis, Tragopogon dubius) and in selected crops
(Triticum aestivum, Brassica napus, Hordeum vulgare, Medicago sativa) of southern
Alberta and also in aerosols collected 4 m above the prairie canopy. Our aims
were to better constrain the wax biosynthetic carbon isotopic fractionation
relative to the plant's carbon isotopic discrimination and to quantitatively
assess the correspondence between wax composition in vegetation and in boundary
layer aerosols. Wax molecular composition of the C(3)prairie species and bulked
vegetation was characterized by high abundance of C(28) n-alkanol and C(31)
n-alkane compounds whereas the C(4) species B. gracilis had several co-dominant
n-alkanol and n-alkane compounds. Wax molecular composition of crop species
differed significantly from that of prairie vegetation and was often dominated by
a single compound. Results indicate that leaf wax isotopic composition is
quantitatively related to the plant's carbon isotopic discrimination. Although
species variations were evident, n-alcohol, n-acid and n-alkane wax compounds
were on average depleted in (13)C by approximately 6.0+/-1 per thousand relative
to total plant carbon. The magnitude of the depletion in wax delta(13)C was
unaffected by environmental factors which altered photosynthetic carbon isotopic
discrimination. No consistent difference in the magnitude of wax biosynthetic
fractionation was observed between C(3) and C(4) species, indicating that
photosynthetic pathway has little influence on the isotopic fractionation of wax
during biosynthesis. The isotopic composition of ablated waxes in aerosols
collected above the canopy was similar to that of the grassland vegetation but
the molecular composition differed significantly and indicated that the source
"footprint" of the ablated leaf wax particles we sampled in boundary layer air
masses was of a regional or larger spatial scale.
DOI: 10.1007/s00442-002-1157-4
2369. J Biol Chem. 2003 May 23;278(21):18945-52. Epub 2003 Mar 19.
Stress-induced protein phosphatase 2C is a negative regulator of a

mitogen-activated protein kinase.
Meskiene I(1), Baudouin E, Schweighofer A, Liwosz A, Jonak C, Rodriguez PL,

Jelinek H, Hirt H.
Author information:
(1)Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohrgasse 9,
A-1030 Vienna, Austria. irute@gem.univie.ac.at
Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal
transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an
unusually large group of 69 different PP2C genes. At present, little is known
about the functions and substrates of plant PP2Cs. We have previously shown that
MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated
protein kinase (MAPK) pathways in yeast and plants. In this report, we provide
evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK
(SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that
directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is
inactivated through threonine dephosphorylation of the pTEpY motif, which is
essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK
depends on the catalytic activity of MP2C. A comparison of MP2C with two other
PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have
similar activities toward casein as a substrate, only MP2C is able to
dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C
interacts directly with SIMK, the MAPK was identified as an interacting partner
of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in
a complex in vivo and shows direct binding to SIMK in vitro in protein
interaction assays. Wound-induced MP2C expression correlates with the time window
when SIMK is inactivated, corroborating the notion that MP2C is involved in
resetting the SIMK signaling pathway.
DOI: 10.1074/jbc.M300878200
2370. Plant Physiol. 2003 Mar;131(3):1091-103.
The Medicago species A2-type cyclin is auxin regulated and involved in meristem
formation but dispensable for endoreduplication-associated developmental
programs.
Roudier F(1), Fedorova E, Lebris M, Lecomte P, Györgyey J, Vaubert D, Horvath G,

Abad P, Kondorosi A, Kondorosi E.
Author information:
(1)Institut des Sciences du Végétale, Centre National de la Recherche Scientiique
Unité Propre de Recherche, Gi-sur-Yvette, France.
Phytohormones as well as temporal and spatial regulation of the cell cycle play a
key role in plant development. Here, we investigated the function and regulation
of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root
developmental programs: in primary and secondary root development, nodule
development, and nematode-elicited gall formation. Using transgenic plants
carrying the Medsa;cycA2;2 promoter-beta-glucuronidase gene fusion, in
combination with other techniques, cycA2;2 expression was localized in meristems
and proliferating cells in the lateral root and nodule primordia. Rapid induction
of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle
activation of differentiated cells developing to nodule primordia. Surprisingly,
cycA2;2 was repressed in the endoreduplicating, division-arrested cells both
during nodule development and formation of giant cells in nematode-induced galls,
indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles.
Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein
levels and had no apparent phenotype. In contrast, antisense expression of
cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in
the formation or activity of apical meristems. Expression of cycA2;2 was
up-regulated by auxins, as expected from the presence of auxin response elements
in the promoter. Moreover, auxin also affected the spatial expression pattern of
this cyclin by shifting the cycA2;2 expression from the phloem to the xylem
poles.
DOI: 10.1104/pp.102.011122
PMCID: PMC166874
Stability of saponins in alcoholic solutions: ester formation as artifacts.
Tava A(1), Mella M, Bialy Z, Jurzysta M.
Author information:
(1)Istituto Sperimentale per le Colture Foraggere, viale Piacenza 29, 26900 Lodi,
Italy. aldotava@katamail.com
Saponins containing a free carboxylic group in the molecule give the

corresponding esters as artifacts when stored for a long time in alcoholic
solutions. Two saponins from Medicago sativa L., chosen on the basis of their
different positions of the carboxylic group in the molecule, were refluxed with
methanol and ethanol under neutral conditions. 3,28-di-O-glu medicagenic acid
possesses a carboxylic group on the triterpenic moiety, whereas soyasaponin I, a
glycoside of soyasapogenol B, has a glucuronic acid unit as the first sugar
linked to the triterpene structure. Artifacts were quantified by HPLC. The peaks
identified as the corresponding esters were examined during boiling from 1 h to 5
days. Quantitative results indicated that the carboxylic group on the sugar
moiety, as for soyasaponin I, is more reactive than that on the triterpenic
structure, as for 3,28-di-O-glu medicagenic acid. Saponins having the free
carboxylic groups create enough acidity in their alcoholic solutions to catalyze
the formation of the corresponding esters.
DOI: 10.1021/jf020996s
2372. Plant J. 2003 Mar;33(6):1037-49.
Antisense inhibition of NADH glutamate synthase impairs carbon/nitrogen

assimilation in nodules of alfalfa (Medicago sativa L.).
Cordoba E(1), Shishkova S, Vance CP, Hernández G.
Author information:
(1)Centro de Investigación sobre Fijación de Nitrógeno, UNAM, Ap. Postal 565-A,
Cuernavaca, Mor. México.
Legumes acquire significant amounts of nitrogen for growth from symbiotic

nitrogen fixation. The glutamine synthetase (GS)/NADH-dependent glutamate
synthase (NADH-GOGAT) cycle catalyzes initial nitrogen assimilation. This report
describes the impact of specifically reducing nodule NADH-GOGAT activity on
symbiotic performance of alfalfa (Medicago sativa L.). Four independent
transgenic alfalfa lines, designated GA89, GA87, GA88, and GA82 (for
GOGATantisense), containing an antisense NADH-GOGAT cDNA fragment under the
control of the soybean leghemoglobin (lbc3) promoter were evaluated. The GA
plants were fertile and showed normal growth in non-symbiotic conditions. The
NADH-GOGAT antisense transgene was heritable and the T1 plants showed phenotypic
alterations - similar to primary transformants. Clonally propagated plants were
inoculated with Sinorhizobium meliloti after rooting and the symbiotic phenotype
was analyzed 21 days post-inoculation. Nodules of each GA line had reduced
NADH-GOGAT activity, ranging from 33 to 87% of control plants, that was
accompanied by comparable decreases in RNA and protein. Plants from the GA89
line, with the lowest NADH-GOGAT activity (c. 30%), presented a strikingly
altered symbiotic phenotype: concomitantly activities of key enzyme for carbon
and nitrogen assimilation decreased; nodule amino acids and amides were reduced
while sucrose accumulated. Antisense GOGAT plants were chlorotic, reduced in
fresh weight, and had a lower N content than control plants. Photosynthesis was
also impaired in antisense plants. Specifically, reducing NADH-GOGAT in nodules
resulted in plants having impaired nitrogen assimilation and altered
carbon/nitrogen metabolic flux.
2373. J Comp Physiol B. 2003 Mar;173(2):141-8. Epub 2003 Feb 8.
How important is milk for near-weaned red kangaroos ( Macropus rufus) fed
different forages?
Munn AJ(1), Dawson TJ.
Author information:
(1)School of Biological, Earth and Environmental Sciences, University of New
South Wales, Sydney, NSW 2052, Australia. a.munn@unsw.edu.au
Red kangaroos (Macropus rufus) are large (>20 kg) herbivorous marsupials common
to the arid and semi-arid regions of inland Australia, where drought is frequent.
Young-at-foot (YAF) red kangaroos are the age/size class usually most affected by
drought. Kangaroos at this YAF stage are making the transition from a milk-based
diet to one of herbivory and an inability to adequately digest high-fibre feeds
may contribute to their high mortalities during drought. We examined the role of
milk in the nutrition of YAF red kangaroos fed forages of different fibre content
and evaluated it as an extra energy and/or nitrogen source. Milk intake had
little impact on the digestion of herbage by YAF red kangaroos fed low-fibre
chopped lucerne (alfalfa) hay. Organic matter (OM) intake was 210+/-20 g day(-1)
and 228+/-22 g day(-1), respectively, by YAF fed lucerne and lucerne with milk.
Apparent digestibility of lucerne OM was ca. 55%, regardless of milk intake. Fed
lucerne, with and without milk, YAF sustained growth rates of ca. 45 g day(-1).
Conversely, even with a milk supplement, YAF red kangaroos ingested only 90+/-11
g day(-1) of high-fibre chopped oaten hay, of which they digested only ca. 36%.
Despite milk intake, YAF fed chopped oaten hay lost between 0 and 75 g body mass
day(-1) and were in negative nitrogen balance (-0.40+/-0.11 g N day(-1)). On all
diets nitrogen loss was primarily as endogenous nitrogen (urinary and faecal)
rather than as dietary nitrogen. Endogenous nitrogen losses were elevated in YAF
fed chopped oaten hay, primarily as non-dietary faecal nitrogen. Overall, when
high-quality feed was available, YAF were not markedly dependent on milk.
However, YAF fed poor-quality chopped oaten hay would require up to 540 ml
day(-1) of late-stage kangaroo milk to attain intakes of energy and nitrogen, and
hence growth rates, comparable with those YAF fed lucerne.
DOI: 10.1007/s00360-002-0319-3
2374. Appl Environ Microbiol. 2003 Mar;69(3):1783-90.
Kinetics and strain specificity of rhizosphere and endophytic colonization by

enteric bacteria on seedlings of Medicago sativa and Medicago truncatula.
Dong Y(1), Iniguez AL, Ahmer BM, Triplett EW.
Author information:
(1)Department of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive,
Madison, WI 53706, USA.
The presence of human-pathogenic, enteric bacteria on the surface and in the

interior of raw produce is a significant health concern. Several aspects of the
biology of the interaction between these bacteria and alfalfa (Medicago sativa)
seedlings are addressed here. A collection of enteric bacteria associated with
alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella
enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize,
Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their
abilities to colonize the rhizosphere and the interior of the plant were
compared. These strains differed widely in their endophytic colonization
abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst
colonizers, respectively. The abilities of the pathogens were between those of K.
pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors
of the seedlings in high numbers with an inoculum of 10(2) CFU, although
infection characteristics were different for each strain. For most strains, a
strong correlation between endophytic colonization and rhizosphere colonization
was observed. These results show significant strain specificity for plant entry
by these strains. Significant colonization of lateral root cracks was observed,
suggesting that this may be the site of entry into the plant for these bacteria.
At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was
colonized in higher numbers on the rhizosphere and in the interior by a
Salmonella endophyte than was the wild-type host. Endophytic entry of M.
truncatula appears to occur by a mechanism independent of the symbiotic
infections by Sinorhizobium meliloti or mycorrhizal fungi.
PMCID: PMC150109
2375. Genome Biol. 2003;4(2):R15. Epub 2003 Jan 31.
Transcriptome analysis of Sinorhizobium meliloti during symbiosis.
Ampe F(1), Kiss E, Sabourdy F, Batut J.
Author information:
(1)Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR
215 Centre National de la Recherche Scientifique - Institut National de la
Recherche Agronomique, BP27-31326 Castanet-Tolosan cedex, France.
BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the
root nodules, as a result of an elaborated developmental program involving the
two partners. In order to contribute to a more global view of the genetics
underlying this plant-microbe symbiosis, we have mined the recently determined
Sinorhizobium meliloti genome sequence for genes potentially relevant to
symbiosis. We describe here the construction and use of dedicated nylon
macroarrays to study simultaneously the expression of 200 of these genes in a
variety of environmental conditions, pertinent to symbiosis.
RESULTS: The expression of 214 S. meliloti genes was monitored under ten
environmental conditions, including free-living aerobic and microaerobic
conditions, addition of the plant symbiotic elicitor luteolin, and a variety of
symbiotic conditions. Five new genes induced by luteolin have been identified as
well as nine new genes induced in mature nitrogen-fixing bacteroids. A bacterial
and a plant symbiotic mutant affected in nodule development have been found of
particular interest to decipher gene expression at the intermediate stage of the
symbiotic interaction. S. meliloti gene expression in the cultivated legume
Medicago sativa (alfalfa) and the model plant M. truncatula were compared and a
small number of differences was found.
CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome
analysis of the model rhizobium S. meliloti, the present work has highlighted the
differential expression of several classes of genes during symbiosis. These genes
are related to invasion, oxidative stress protection, iron mobilization, and
signaling, thus emphasizing possible common mechanisms between symbiosis and
pathogenesis.
PMCID: PMC151305
2376. J Dairy Sci. 2003 Jan;86(1):336-43.
The effect of treating alfalfa with Lactobacillus buchneri 40788 on silage

fermentation, aerobic stability, and nutritive value for lactating dairy cows.
Kung L Jr(1), Taylor CC, Lynch MP, Neylon JM.
Author information:
(1)Delaware Agricultural Experiment Station, Department of Animal and Food
Science, College of Agriculture and Natural Resources, University of Delaware,
Newark, 19717-1303, USA. lkung@udel.edu
Lactobacillus buchneri 40788 and enzymes (beta-glucanase, alpha-amylase,

xylanase, and galactomannase) were applied to chopped alfalfa (39% DM) to study
their effects on the fermentation and nutritive value of the silage. Alfalfa was
treated with nothing, or L. buchneri 40788, for a final application rate of 1 x
10(5), 5 x 10(5), or 1 x 10(6) cfu/g of fresh forage and ensiled in laboratory
silos for 2, 4, 8, and 56 d. Treatment with L. buchneri 40788 had few effects on
the end products of fermentation through 8 d of ensiling. However, after 56 d of
ensiling, treated silages had a higher pH (4.55 vs. 4.38) and higher
concentrations of acetic acid (6.40 vs. 4.24%), propionic acid (0.18 vs. 0.06%),
and ammonia-N (0.35 vs. 0.29%) when compared to untreated silage. Lactic acid was
also numerically lower in treated (3.51%) than untreated (4.12%). Silages treated
with the moderate and highest dose of L. buchneri 40788 also resulted in greater
recoveries of DM than did untreated silage. Alfalfa (43% DM) was also untreated
or treated with a commercial application of L. buchneri 40788 (4 x 10(5) cfu/g, a
commercial dose) in farm-scale bag silo. Holstein cows were fed a diet comprised
of 32% untreated or treated alfalfa silage, 11% corn silage, 5% chopped alfalfa
hay, and 52% of concentrate (DMB) for a 6-wk treatment period. Dry matter intake
and milk composition were unaffected by treatment, but cows fed silage treated
with L. buchneri 40788 produced 0.8 kg more milk than did cows fed untreated
silage. Treated silage had a higher concentration of acetic acid (5.67 vs. 3.35%)
but lower lactic acid (3.50 vs. 4.39%) than untreated silage. When exposed to
air, the total mixed ration containing treated alfalfa silage remained stable for
100 h, whereas the ration containing untreated silage spoiled after 68 h.
Treating alfalfa silage with L. buchneri 40788 increased the concentration of
acetic acid, and when the silage was combined into a total mixed ration and fed
to lactating cows, it improved the aerobic stability of the ration and increased
milk production.
DOI: 10.3168/jds.S0022-0302(03)73611-X
2377. Eur J Biochem. 2003 Jan;270(2):261-9.
Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals

preferential tip localization of the protein in root hairs.
Rodríguez-Llorente ID(1), Pérez-Hormaeche J, Dary M, Caviedes MA, Kondorosi A,

Ratet P, Palomares AJ.
Author information:
(1)Departamento de Microbiología y Parasitología, Facultad de Farmacia,
Universidad de Sevilla, Spain.
Tip growth is a specialized type of polar growth where new cell wall is deposited
in a localized region of the cell, the growing tip. These cells show a
characteristic zonation, with a high accumulation of secretory vesicles
containing cell wall components at the tip, followed by an organelle-enriched
zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our
laboratory, specifically expressed during the interaction of this plant with its
symbiotic partner Sinorhizobium meliloti and which might participate in tip
growth processes during symbiosis. We have used MsPG3-GFP fusions to study in
vivo protein transport processes and localization during root hair growth.
Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots'
following a protocol developed for this study and also tested by transient
expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP
fusion protein in the tip of the growing root hair at different developmental
stages was found, confirming the delivery of MsPG3 to the newly synthesized cell
wall. This indicates that this protein may participate in tip growth processes
during symbiosis and, in addition, that this fusion could be a useful tool to
study this process in plants.
2378. J Exp Bot. 2003 Mar;54(384):1085-91.
Leghaemoglobin oxygenation gradients in alfalfa and yellow sweetclover nodules.
Denison RF(1), Okano Y.
Author information:
(1)Agronomy and Range Science, University of California, 1 Shields Ave, Davis, CA
95616, USA. rfdenison@ucdavis.edu
Respiration in support of N(2) fixation by rhizobia in legume root nodules

depends on an adequate supply of O(2), but excessive O(2) can damage nitrogenase,
the key enzyme. The movement of O(2) into and within the nodule is driven by
gradients in the concentration of O(2) or in the oxygenation of the O(2)-carrier,
leghaemoglobin. Steeper gradients may increase flux to the sites of respiration,
but gradients also raise the possibility of inadequate O(2) in some nodule zones
and excessive O(2) in others. No detailed study of O(2) gradients in the interior
of nodules has been published previously. Spectral changes in leghaemoglobin with
oxygenation, previously used to measure the average O(2) status of the nodule
interior, were used to map longitudinal gradients in O(2) and in respiratory
capacity in the elongated nodules of alfalfa (Medicago sativa L.) and sweetclover
(Melilotus officinalis L.). Variability among nodules under air in the magnitude
and direction of internal O(2) gradients was seen in both species. Despite
consistently higher respiratory capacity near the meristematic tip, a majority of
nodules had higher O(2) towards the tip than towards the base. These results
contrast with a previous report, apparently based on limited data, but they are
consistent with anatomical and tracer studies showing higher gas permeability
near the tip.
2379. Indian J Exp Biol. 2002 Jul;40(7):755-64.
Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and

vitamins in symbiosis.
Randhawa GS(1), Hassani R.
Author information:
Roorkee 247 667, India. sharnfbs@iitr.ernet.in
Rhizobia require the availability of 20 amino acids for the establishment of

effective symbiosis with legumes. Some of these amino acids are synthesized by
rhizobium, whereas the remaining are supplied by the host plant. The supply from
plant appears to be plant-type specific. Alfalfa provides arginine, cysteine,
isoleucine, valine and tryptophan, and cowpea and soybean provide histidine. The
production of ornithine and anthranilic acid, the intermediates in the
biosynthetic pathways of arginine and tryptophan, respectively, seems to be
essential for effective symbiosis of Sinorhizobium meliloti with alfalfa. The
expression of ilvC gene of S. meliloti is required for induction of nodules on
the roots of alfalfa plants. An undiminished metabolic flow through the rhizobial
pathways for the synthesis of purines and pyrimidines and the synthesis of
biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be
requirements for normal symbiosis. To the best of our knowledge, this is the
first review article on the role of rhizobial biosynthetic pathways of amino
acids, nucleotide bases and vitamins in rhizobium-legume symbiosis. The
scientific developments of about 35 years in this field have been reviewed.
2380. J Food Prot. 2003 Feb;66(2):208-14.
Efficacy of electrolyzed oxidizing water in inactivating Salmonella on alfalfa

seeds and sprouts.
Kim C(1), Hung YC, Brackett RE, Lin CS.
Author information:
(1)Department of Food Science and Technology, College of Agricultural and
Environmental Sciences, University of Georgia, Griffin, Georgia 30223-1797, USA.
Studies have demonstrated that electrolyzed oxidizing (EO) water is effective in

reducing foodborne pathogens on fresh produce. This study was undertaken to
determine the efficacy of EO water and two different forms of chlorinated water
(chlorine water from Cl2 and Ca(OCl)2 as sources of chlorine) in inactivating
Salmonella on alfalfa seeds and sprouts. Tengram sets of alfalfa seeds inoculated
with a five-strain cocktail of Salmonella (6.3 x 10(4) CFU/g) were subjected to
90 ml of deionized water (control), EO water (84 mg/liter of active chlorine),
chlorine water (84 mg/liter of active chlorine), and Ca(OCl)2 solutions at 90 and
20,000 mg/liter of active chlorine for 10 min at 24 +/- 2 degrees C. The
application of EO water, chlorinated water, and 90 mg/liter of Ca(OCl)2 to
alfalfa seeds for 10 min reduced initial populations of Salmonella by at least
1.5 log10 CFU/g. For seed sprouting, alfalfa seeds were soaked in the different
treatment solutions described above for 3 h. Ca(OCl)2 (20,000 mg/liter of active
chlorine) was the most effective treatment in reducing the populations of
Salmonella and non-Salmonella microflora (4.6 and 7.0 log10 CFU/g, respectively).
However, the use of high concentrations of chlorine generates worker safety
concerns. Also, the Ca(OCl)2 treatment significantly reduced seed germination
rates (70% versus 90 to 96%). For alfalfa sprouts, higher bacterial populations
were recovered from treated sprouts containing seed coats than from sprouts with
seed coats removed. The effectiveness of EO water improved when soaking
treatments were applied to sprouts in conjunction with sonication and seed coat
removal. The combined treatment achieved 2.3- and 1.5-log10 CFU/g greater
reductions than EO water alone in populations of Salmonella and non-Salmonella
microflora, respectively. This combination treatment resulted in a 3.3-log10
CFU/g greater reduction in Salmonella populations than the control (deionized
water) treatment.
2381. J Food Prot. 2003 Feb;66(2):182-7.
Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia
coil O157:H7, Salmonella, and Listeria with BAX, and evaluation of this
polymerase chain reaction-based system with experimentally contaminated samples.
Strapp CM(1), Shearer AE, Joerger RD.
Author information:
Delaware 19717, USA.
BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used
to survey retail sprouts and mushrooms for contamination with Escherichia coli
O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or
E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples
screened. L. monocytogenes was detected in one sprout sample, and seven
additional sprout samples tested positive for the genus Listeria. BAX also
detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays
(0.7%) failed to amplify control DNA, and therefore reagent failures and the
inhibition of PCR by plant compounds were rare. The sensitivity of the detection
system was evaluated by assaying samples inoculated with 10 CFU of each of the
pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E.
coli O157:H7, and two sets of 130 samples were experimentally contaminated with
Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended
on the protocols used for inoculation and culturing. Inoculation of samples with
approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5%
for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The
corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7
detection rate was 100% for mushrooms but only 48.6% for sprouts when standard
BAX culture protocols were used. The substitution of an overnight incubation in
modified E. coli medium for the 3-h brain heart infusion incubation increased the
rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts.
The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight
culture were used for the inoculation. Test sensitivity is therefore influenced
by the type of produce involved and is probably related to the growth of
pathogens in the resuscitation and enrichment media.
2382. J Food Prot. 2003 Feb;66(2):175-81.
Inactivation of Escherichia coli O157:H7 and Salmonella by gamma irradiation of

alfalfa seed intended for production of food sprouts.
Thayer DW(1), Rajkowski KT, Boyd G, Cooke PH, Soroka DS.
Author information:
(1)Food Safety Intervention Technologies Research Unit, U.S. Department of
Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600
East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA. dthayer@arserrc.gov
Inonizing irradiation was determined to be a suitable method for the inactivation

of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the
production of food sprouts. The radiation D (dose resulting in a 90% reduction of
viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on
alfalfa seeds were higher than the D-values for their inactivation on meat or
poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds
was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for
vegetable-associated isolates were not significantly different. The D-values for
nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55
+/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the
relatively high D-values were not due to the low moisture content or the low
water activity of the seed. The D-values for Salmonella on alfalfa seeds from two
different sources did not differ significantly, even though there were
significant differences in seed size and water activity. The increased moisture
content of the seed after artificial inoculation did not significantly alter the
D-value for the inactivation of Salmonella. The results of this study demonstrate
that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing
radiation, which will permit satisfactory commercial yields of sprouts from
alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.
2383. Int J Food Microbiol. 2003 May 15;82(3):245-53.
Isolation of Salmonella from alfalfa seed and demonstration of impaired growth of

heat-injured cells in seed homogenates.
Liao CH(1), Fett WF.
Author information:
Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA.
cliao@arserrc.gov
Three major foodborne outbreaks of salmonellosis in 1998 and 1999 were linked to
the consumption of raw alfalfa sprouts. In this report, an improved method is
described for isolation of Salmonella from alfalfa seed lots, which had been
implicated in these outbreaks. From each seed lot, eight samples each containing
25 g of seed were tested for the presence of Salmonella by the US FDA
Bacteriological Analytical Manual (BAM) procedure and by a modified method
applying two successive pre-enrichment steps. Depending on the seed lot, one to
four out of eight samples tested positive for Salmonella by the standard
procedure and two to seven out of eight samples tested positive by the modified
method. Thus, the use of two consecutive pre-enrichment steps led to a higher
detection rate than a single pre-enrichment step. This result indirectly
suggested that Salmonella cells on contaminated seeds might be injured and failed
to fully resuscitate in pre-enrichment broth containing seed components during
the first 24 h of incubation. Responses of heat-injured Salmonella cells grown in
buffered peptone water (BPW) and in three alfalfa seed homogenates were
investigated. For preparation of seed homogenates, 25 g of seeds were homogenized
in 200 ml of BPW using a laboratory Stomacher and subsequently held at 37 degrees
C for 24 h prior to centrifugation and filtration. While untreated cells grew at
about the same rate in BPW and in seed homogenates, heat-injured cells (52
degrees C, 10 min) required approximately 0.5 to 4.0 h longer to resuscitate in
seed homogenates than in BPW. This result suggests that the alfalfa seed
components or fermented metabolites from native bacteria hinder the repair and
growth of heat-injured cells. This study also shows that an additional
pre-enrichment step increases the frequency of isolation of Salmonella from
naturally contaminated seeds, possibly by alleviating the toxic effect of seed
homogenates on repair or growth of injured cells.
2384. Indian J Exp Biol. 2002 Sep;40(9):981-8.
Nitrogen control of bacterial signal production in Rhizobium meliloti-alfalfa

symbiosis.
Dusha I(1).
Author information:
(1)Institute of Genetics, Biological Research Center, Hungarian Academy of
Sciences, Szeged, P.O. Box 521, H-6701 Hungary. dushai@nucleus.szbk.u-szeged.hu
Under nitrogen-depleted conditions nitrogen-fixing soil bacteria of the family

Rhizobiaceae are able to induce symbiotic nodules on the roots of leguminous
plants where bacteroids convert atmospheric nitrogen to ammonia. The presence of
exogenous nitrogen source inhibits the development and the functioning of
bacterium-plant symbiosis. Earlier experiments demonstrated that nitrate
inhibited all stages of symbiotic interaction, affecting primarily the host
functions. The investigation of the possible involvement of the microsymbiont in
nitrogen regulation showed that two signalling steps were controlled by ammonium.
The synthesis of the first bacterial signal, the Nod factor was repressed by
ammonium. The nitrogen signal is conveyed to nodulation (nod) genes by the
general nitrogen regulatory (ntr) system and by the nodD3-syrM self-amplifying
system. The fine control also involves a negative regulatory factor, ntrR. When
ntrR is mutated, more efficient nodule formation and nitrogen fixation is
observed in symbiosis with alfalfa even in the presence of ammonium. The
biosynthesis of the second bacterial signal succinoglycan is also controlled by
ammonium. SyrM, a common regulatory factor for nod and exo gene expression, may
contribute to the adjustment of the amount of succinoglycan and the ratio of its
biologically active form.
2385. FEMS Microbiol Lett. 2003 Jan 21;218(1):65-70.
Mutations in sit B and sit D genes affect manganese-growth requirements in

Platero RA(1), Jaureguy M, Battistoni FJ, Fabiano ER.
Author information:
(1)Departamento de Bioquímica, Instituto de Investigaciones Biológicas Clemente
Estable, Ministerio de Educación y Cultura, Av Italia 3318, Montevideo 11600,
Uruguay.
Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based

on their inability to grow on rich medium supplemented with the metal chelator
ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds
or siderophores as iron sources. Tagged loci of these mutants were identified as
sit B and sit D genes. These genes encode components of an ABC (ATP-binding
cassette) metal-type permease in several Gram-negative bacteria. In this work,
the phenotypes of these two mutants were compared with those of two
siderophore-mediated iron transport mutants. The results strongly implicate a
role of the sit genes in manganese acquisition when this metal is limiting in S.
meliloti.
2386. Theor Appl Genet. 2002 Jun;104(8):1283-1289. Epub 2002 Apr 19.
Isolation from alfalfa of resistance gene analogues containing nucleotide binding

sites.
Cordero C(1), Skinner Z.
Author information:
(1)Genetics Program and USDA-ARS, Throckmorton Hall, Kansas State University,
Manhattan, KS 66506-5501, USA.
Cloned resistance (R) genes from a broad range of plant species are known to
share similarities in DNA sequence and structural motifs. Degenerate
oligonucleotide primers designed from conserved regions of the nucleotide binding
site (NBS), common to many R genes, were used to amplify the NBS regions from
genomic DNA from alfalfa ( Medicago sativa L). Sequence comparisons of the
amplified fragments indicated that at least 18 families of NBS-containing R genes
are present in alfalfa. Comparisons to R genes from other species suggested a
polyphyletic origin of these gene families. Using the same degenerate primers,
PCR analysis of cDNA prepared from a plant not challenged with a pest or pathogen
revealed that many of the NBS-containing gene families were transcribed actively.
Amplification of NBS regions from other Medicago species showed the presence of
some NBS-containing genes not present in alfalfa. These results indicate that the
NBS-containing R genes comprise a large gene family in Medicago, at least some of
which are transcribed in healthy plants, and that different Medicago species
carry unique NBS genes.
DOI: 10.1007/s00122-001-0821-0
PMID: 12582582
Detection of Salmonella by indicator agar media and PCR as affected by alfalfa

seed homogenates and native bacteria.
Liao CH(1), Shollenberger LM.
Author information:
Research Center, Wyndmoor, PA 19038, USA. cliao@arserrc.gov
AIMS: To investigate and prevent the undesirable effect of native bacteria and
alfalfa seed homogenates on detection of Salmonella in alfalfa seeds by indicator
agar media and polymerase chain reaction (PCR).
METHODS AND RESULTS: The relative sensitivity of five indicator agar media,
including modified semisolid RV (MSRV), xylose-lysine-Tergitol 4 (XLT4), Hektoen
enteric agar (HEA), brilliant green agar (BGA) and bismuth sulphite agar (BSA),
for detection of Salmonella in the presence of a large number of native bacteria
from alfalfa seeds was examined. The detection limit as measured by the ratio
between the numbers of native bacteria and Salmonella was estimated to be 10(6)
to 1 for MSRV and 10(3) to 1 for XLT4, HEA, BGA or BSA. Presence of alfalfa seed
homogenates markedly reduced the sensitivity of Salmonella detection by PCR. The
minimal number of Salmonella detectable by PCR was determined to be 1-10 and
100-1000 CFU in the absence and presence of seed homogenate, respectively.
Application of anti-Salmonella immunomagnetic beads permitted detection of 2-5
CFU of heat-injured cells in 25 g of seeds within 24 h by PCR.
CONCLUSIONS: The MSRV medium is more sensitive than other indicator agars for
detecting a small number of motile Salmonella in samples containing a large
number of native bacteria. Application of immunomagnetic beads eliminates the
PCR-inhibitory activity of seed homogenates and improves the detection of
Salmonella in inoculated seeds.
SIGNIFICANCE AND IMPACT: The results generated from this study will aid the seed
distributors, sprout growers and public health officials to identify and recall
the Salmonella-contaminated seed lots to be used for sprout production.
2388. Mol Plant Microbe Interact. 2003 Jan;16(1):83-91.
Salicylic acid inhibits indeterminate-type nodulation but not determinate-type

nodulation.
van Spronsen PC(1), Tak T, Rood AM, van Brussel AA, Kijne JW, Boot KJ.
Author information:
(1)Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. spronsen@rulbim.leidenuniv.nl
LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial

symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating
plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an
18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes
to signaling in indeterminate-type nodulation. In a working hypothesis, we
speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable
to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid
(the octadecanoid pathway). Because salicylic acid (SA) is known to interfere
with oxylipin signaling, we tested whether nodulation of vetch could be affected
by addition of 10(-4) M SA. This concentration completely blocked nodulation of
vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of
18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act
systemically, and only biologically active SA derivatives were capable of
inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae
association with vetch roots. In contrast, addition of SA to Lotus japonicus (a
determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit
nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants
showed the same inhibiting response toward SA, whereas SA did not inhibit the
nodulation of other determinate-type nodulating plants. SA may be a useful tool
for studying fundamental differences between signal transduction pathways of
indeterminate- and determinate-type nodulating plants.
DOI: 10.1094/MPMI.2003.16.1.83
2389. Appl Environ Microbiol. 2003 Feb;69(2):926-32.
Terminal restriction fragment length polymorphism data analysis for quantitative

comparison of microbial communities.
Blackwood CB(1), Marsh T, Kim SH, Paul EA.
Author information:
(1)Center for Microbial Ecology and Department of Crop and Soil Sciences,
Michigan State University, East Lansing, Michigan 48824, USA.
blackwoc@ba.ars.usda.gov
Comment in
Appl Environ Microbiol. 2003 Oct;69(10):6342; author reply 6342-3.
Terminal restriction fragment length polymorphism (T-RFLP) is a

culture-independent method of obtaining a genetic fingerprint of the composition
of a microbial community. Comparisons of the utility of different methods of (i)
including peaks, (ii) computing the difference (or distance) between profiles,
and (iii) performing statistical analysis were made by using replicated profiles
of eubacterial communities. These samples included soil collected from three
regions of the United States, soil fractions derived from three agronomic field
treatments, soil samples taken from within one meter of each other in an alfalfa
field, and replicate laboratory bioreactors. Cluster analysis by Ward's method
and by the unweighted-pair group method using arithmetic averages (UPGMA) were
compared. Ward's method was more effective at differentiating major groups within
sets of profiles; UPGMA had a slightly reduced error rate in clustering of
replicate profiles and was more sensitive to outliers. Most replicate profiles
were clustered together when relative peak height or Hellinger-transformed peak
height was used, in contrast to raw peak height. Redundancy analysis was more
effective than cluster analysis at detecting differences between similar samples.
Redundancy analysis using Hellinger distance was more sensitive than that using
Euclidean distance between relative peak height profiles. Analysis of Jaccard
distance between profiles, which considers only the presence or absence of a
terminal restriction fragment, was the most sensitive in redundancy analysis, and
was equally sensitive in cluster analysis, if all profiles had cumulative peak
heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a
sensitive method of differentiating between microbial communities when the
optimal statistical method is used for the situation at hand. It is recommended
that hypothesis testing be performed by redundancy analysis of
Hellinger-transformed data and that exploratory data analysis be performed by
cluster analysis using Ward's method to find natural groups or by UPGMA to
identify potential outliers. Analyses can also be based on Jaccard distance if
all profiles have cumulative peak heights greater than 10,000 fluorescence units.
PMCID: PMC143601
2390. J Am Chem Soc. 2003 Feb 12;125(6):1472-3.
Enzymes do what is expected (chalcone isomerase versus chorismate mutase).
Hur S(1), Bruice TC.
Author information:
(1)Department of Chemistry and Biochemistry, University of California, Santa
Barbara, California 93106, USA.
Madicago sativa chalcone isomerase (CI) catalyzes the isomerization of chalcone

to flavanone, whereas E. coli chorismate mutase (CM) catalyzes the pericyclic
rearrangement of chorismate to prephenate. Covalent intermediates are not formed
in either of the enzyme-catalyzed reactions, K(M) and k(cat) are virtually the
same for both enzymes, and the rate constants (k(o)) for the noncatalyzed
reactions in water are also the same. This kinetic identity of both the enzymatic
and the nonenzymatic reactions is not shared by a similarity in driving forces.
The efficiency (DeltaG(o)() - DeltaG(cat)()) for the CI mechanism involves
transition-state stabilization through general-acid catalysis and freeing of
three water molecules trapped in the E.S species. The contribution to lowering
DeltaG(cat)() by an increase in near attack conformer (NAC) formation in E.S as
compared to S in water is not so important. In the CM reaction, the standard free
energy for NAC formation in water is 8.4 kcal/mol as compared to 0.6 kcal/mol in
E.S. Because the value of (DeltaG(o)() - DeltaG(cat)()) is 9 kcal/mol, the
greater percentage of NACs accounts for approximately 90% of the kinetic
advantage of the CM reaction. There is no discernible transition-state
stabilization in the CM reaction. These results are discussed. In anthropomorphic
terms, each enzyme has had to do what it must to have a biologically relevant
rate of reaction.
DOI: 10.1021/ja0293047
2391. J Biol Chem. 2003 Apr 11;278(15):12946-54. Epub 2003 Feb 3.
Sinorhizobium meliloti acpXL mutant lacks the C28 hydroxylated fatty acid moiety
of lipid A and does not express a slow migrating form of lipopolysaccharide.
Sharypova LA(1), Niehaus K, Scheidle H, Holst O, Becker A.
Author information:
(1)Institute of Genetics, Biology VI, University of Bielefeld, Postfach 100131,
Bielefeld D-33501, Germany. Larissa.Sharpova@Genetik.Uni-Bielefeld.DE
Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer

membrane of Gram-negative bacteria. Lipid A of all Rhizobiaceae is acylated with
a long fatty acid chain, 27-hydroxyoctacosanoic acid. Biosynthesis of this long
acyl substitution requires a special acyl carrier protein, AcpXL, which serves as
a donor of C28 (omega-1)-hydroxylated fatty acid for acylation of rhizobial lipid
A (Brozek, K.A., Carlson, R.W., and Raetz, C. R. (1996) J. Biol. Chem. 271,
32126-32136). To determine the biological function of the C28 acylation of lipid
A, we constructed an acpXL mutant of Sinorhizobium meliloti strain 1021.
Gas-liquid chromatography and mass spectrometry analysis of the fatty acid
composition showed that the acpXL mutation indeed blocked C28 acylation of lipid
A. SDS-PAGE analysis of acpXL mutant LPS revealed only a fast migrating band,
rough LPS, whereas the parental strain 1021 manifested both rough and smooth LPS.
Regardless of this, the LPS of parental and mutant strains had a similar sugar
composition and exposed the same antigenic epitopes, implying that different
electrophoretic profiles might account for different aggregation properties of
LPS molecules with and without a long acyl chain. The acpXL mutant of strain 1021
displayed sensitivity to deoxycholate, delayed nodulation of Medicago sativa, and
a reduced competitive ability. However, nodules elicited by this mutant on roots
of M. sativa and Medicago truncatula had a normal morphology and fixed nitrogen.
Thus, the C28 fatty acid moiety of lipid A is not crucial, but it is beneficial
for establishing an effective symbiosis with host plants. acpXL lies upstream
from a cluster of five genes, including msbB (lpxXL), which might be also
involved in biosynthesis and transfer of the C28 fatty acid to the lipid A
precursor.
DOI: 10.1074/jbc.M209389200
Herbicide loading to shallow ground water beneath Nebraska's Management Systems

Evaluation Area.
Spalding RF(1), Watts DG, Snow DD, Cassada DA, Exner ME, Schepers JS.
Author information:
(1)Dep. of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583-0915,
USA. rspalding1@unl.edu
Better management practices can counter deterioration of ground water quality.

From 1991 through 1996 the influence of improved irrigation practices on ground
water pesticide contamination was assessed at the Nebraska Management Systems
Evaluation Area. Three 13.4-ha corn (Zea mays L.) fields were studied: a
conventional furrow-irrigated field, a surge-irrigated field and a center
pivot-irrigated field, and a center pivot-irrigated alfalfa (Medicago sativa L.)
field. The corn fields received one identical banded application of Bicep
(atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4,-diamine] +
metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)
acetamidel) annually; the alfalfa field was untreated. Ground water samples were
collected three times annually from 16 depths of 31 multilevel samplers. Six
years of sample data indicated that a greater than 50% reduction in irrigation
water on the corn management fields lowered average atrazine concentrations in
the upper 1.5 m of the aquifer downgradient of the corn fields from approximately
5.5 to <0.5 microg L(-1). Increases in deethylatrazine (DEA;
2-chloro-4-amino-6-isopropylamino-s-triazine) to atrazine molar ratios indicated
that reducing water applications enhanced microbial degradation of atrazine in
soil zones. The occurrence of peak herbicide loading in ground water was
unpredictable but usually was associated with heavy precipitation within days of
herbicide application. Focused recharge of storm runoff that ponded in the
surge-irrigated field drainage ditch, in the upgradient road ditch, and at the
downgradient end of the conventionally irrigated field was a major mechanism for
vertical transport. Sprinkler irrigation technology limited areas for focused
recharge and promoted significantly more soil microbial degradation of atrazine
than furrow irrigation techniques and, thereby, improved ground water quality.
2393. Wei Sheng Wu Xue Bao. 2001 Apr;41(2):127-32.
[A sinorhizobium fredll strain that effectively nodulate Medicago sativa].
Zhang H(1), Zhang H, Li X, Yang S.
Author information:
(1)Department of Microbiology, College of Biological Sciences, China Agricultural
University, Beijing 100094, China.
Sinorhizobium fredii 042BS was isolated from root nodules of alfalfa (Medicago
sativa) from Xinjiang Region. Nodulation experiments showed that both soybean and
alfalfa were nodulated by 042BS effectively. The 16S rDNA PCR-RFLP analysis was
carried out by four restriction endonucleases, and the restriction maps of strain
042BS were identical with those of S. fredii USDA205. The DNA G + C mol% of
strain 042BS was 60.0. The DNA homology between 042BS and S. fredii USDA205 and
USDA191 were 84.9% and 89.6%, respectively. To prove the capability of 042BS to
nodulate both soybean and alfalfa, constitutively expressed green fluorescence
protein gene(gfp) was introduced to 042BS, and the recombinant strain 042BSG was
obtained. The reisolates from nodules of the soybean and alfalfa inoculated with
042BSG were observed using the confocal laser-scanning microscope, and the
expressions of gfp were detected, respectively. 042BS showed various nodulation
capacities with different alfalfa cultivars used.
2394. J Anim Sci. 2002 Dec;80(12):3301-6.
Effects of four mono- and sesquiterpenes on the consumption of alfalfa pellets by

sheep.
Estell RE(1), Fredrickson EL, Anderson DM, Havstad KM, Remmenga MD.
Author information:
(1)USDA/ARS Jornada Experimental Range, Las Cruces, NM 88003-8003, USA.
restell@nmsu.edu
Effects of individual terpenes on alfalfa pellet intake by lambs were examined in

four experiments. Forty-five lambs (nine lambs/treatment) were individually fed
alfalfa pellets sprayed with either camphene, myrcene, caryophyllene oxide, or
beta-pinene at one of five concentrations (one terpene per experiment).
Treatments (0, 0.5, 1, 2, and 10x) were multiples of the concentration (x) of a
specific terpene in tarbush. Terpenes were applied to alfalfa pellets (0.64 kg x
lamb(-1) x d(-1), DM basis), and consumption was measured during a 20-min
interval for 5 d. Lambs were maintained and fed alfalfa pellets in one group
(except during 20-min tests) at a mean total daily intake of 4.7% of BW (DM
basis). Camphene and caryophyllene oxide tended to decrease intake (linear
contrasts were P = .0651 and P = .0504, respectively), whereas myrcene and
beta-pinene exerted no effect on the consumption of alfalfa pellets by lambs.
Camphene and caryophyllene oxide may be involved in the differential herbivory of
individual tarbush plants by livestock.
2395. J Food Prot. 2003 Jan;66(1):44-51.
Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts

and effects on sensory quality of sprouts.
Wade WN(1), Scouten AJ, McWatters KH, Wick RL, Demirci A, Fett WF, Beuchat LR.
Author information:
(1)Center for Food Safety and Department of Food Science and Technology,
University of Georgia, 1109 Experiment Street, Griffin, Georgia 30223-1797, USA.
A study was done to determine the efficacy of aqueous ozone treatment in killing
Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in
populations of naturally occurring aerobic microorganisms on sprouts and changes
in the sensory quality of sprouts were also determined. The treatment (10 or 20
min) of seeds in water (4 degrees C) containing an initial concentration of 21.8
+/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05)
reduction in populations of L. monocytogenes. The continuous sparging of seeds
with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for
20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2
min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/-
0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05)
reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to
populations detected on sprouts treated with water. Treatments (2 min) with up to
23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce
populations of aerobic naturally occurring microorganisms. The continuous
sparging of sprouts with ozonated water for 5 to 20 min caused significant
reductions in L. monocytogenes and natural microbiota compared to soaking in
water (control) but did not enhance the lethality compared to the sprouts not
treated with continuous sparging. The treatment of sprouts with ozonated water
(20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the
sensory quality during subsequent storage at 4 degrees C for 7 to 11 days.
Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed
physical damage, fungal and bacterial growth, and biofilm formation that provide
evidence of factors contributing to the difficulty of killing microorganisms by
treatment with ozone and other sanitizers.
2396. J Food Prot. 2003 Jan;66(1):13-7.
Alfalfa sprouts and Salmonella Kottbus infection: a multistate outbreak following

inadequate seed disinfection with heat and chlorine.
Winthrop KL(1), Palumbo MS, Farrar JA, Mohle-Boetani JC, Abbott S, Beatty ME,
Inami G, Werner SB.
Author information:
(1)California Department of Health Services, Division of Communicable Disease
Control, 2151 Berkeley Way, Berkeley, California 94704, USA. kwinthro@dhs.ca.gov
Raw sprouts have been implicated in a number of foodborne disease outbreaks.

Because contaminated seeds are usually responsible, many sprout producers attempt
to disinfect seeds before germination and detect sprout contamination during
production. In March 2001, we detected an increased number of Salmonella serotype
Kottbus isolates in California. Overall, we identified 31 cases from three
western states. To identify the cause, we conducted a case-control study with the
first 10 identified case-patients matched to 20 controls by age, sex, and
residential area. Our case-control study found illness to be statistically
associated with alfalfa sprout consumption. The traceback investigation
implicated a single sprouter, where environmental studies yielded Salmonella
Kottbus from ungerminated seeds and floor drains within the production facility.
Pulsed-field gel electrophoresis patterns of all patient, seed, and floor drain
Salmonella Kottbus isolates were indistinguishable. Most implicated sprouts were
from seeds that underwent heat treatment and soaking with a 2,000-ppm sodium
hypochlorite solution rather than the Food and Drug Administration
(FDA)-recommended 20,000-ppm calcium hypochlorite soak. Other implicated seeds
had been soaked in a calcium hypochlorite solution that, when tested, measured
only 11,000 ppm. The outbreak might have been averted when screening tests of
sprout irrigation water detected Salmonella in January; however, confirmatory
testing of these samples was negative (but testing improperly utilized
refrigerated irrigation water). Producers should use the enrichment broth of
positive screening samples, not refrigerated irrigation water, for confirmatory
testing. Until other effective disinfection technologies are developed, producers
should adhere to FDA recommendations for sprout seed disinfection.
2397. Appl Environ Microbiol. 2003 Jan;69(1):548-53.
Growth dynamics of Salmonella enterica strains on alfalfa sprouts and in waste

seed irrigation water.
Howard MB(1), Hutcheson SW.
Author information:
(1)Department of Cell Biology and Molecular Genetics, University of Maryland,
College Park, Maryland 20742, USA.
Alfalfa sprouts and other seed sprouts have been implicated in numerous outbreaks
of salmonellosis. The source of these epidemics appears to have been low-level
contamination of seeds by Salmonella bacteria that developed into clinically
significant populations during the seed germination process. To test the
possibility that Salmonella enterica strains carry host range determinants that
allow them to grow on alfalfa, strains isolated from alfalfa or other sources
were surveyed for their ability to grow on germinating alfalfa seeds. An S.
enterica serovar Cubana strain originally isolated from contaminated alfalfa
sprouts multiplied most rapidly during the initial 24 h of the seed germination
process. Germinating alfalfa seeds supported the multiplication of S. enterica
cells prior to the emergence of the root radicle at 72 h. Thereafter, much lower
rates of multiplication were apparent. The ability of S. enterica to grow on
germinating alfalfa seeds was independent of the serovar, isolation source, or
virulence of the strain. Isolates obtained from alfalfa attained population
levels similar to those observed for strains isolated from contaminated meat
products or stools. Each of the strains could be detected in the waste irrigation
water, with populations being strongly correlated with those detected on the
germinating alfalfa seeds. The S. enterica strains were capable of utilizing the
waste irrigation water as a sole carbon and nitrogen source. S. enterica strains
thus appear to grow saprophytically on soluble organics released from seeds
during early phases of germination. The ability to detect S. enterica in the
waste irrigation water early in the germination process indicates that this
method may be used as a simple way to monitor the contamination of sprouts during
commercial operations.
PMCID: PMC152435
2398. J Dairy Sci. 2002 Dec;85(12):3420-9.
The proportion of the diet to which fibrolytic enzymes are added affects nutrient
digestion by lactating dairy cows.
Bowman GR(1), Beauchemin KA, Shelford JA.
Author information:
(1)Faculty of Agricultural Science, University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z4.
Eight lactating Holstein cows, four with ruminal cannulas, were used in a
duplicated 4 x 4 Latin square design to investigate a fibrolytic enzyme product
characterized by xylanase and cellulase activities (Promote N.E.T. Agribrands
International, St. Louis, MO). The diet consisted of concentrate containing
rolled barley and supplement, barley silage and alfalfa haylage (55% to 45% DM
basis, forage to concentrate ratio) and differed in enzyme application: 1)
control, 2) enzyme applied to concentrate (45% of TMR), 3) enzyme applied to
supplement (4% of TMR), and 4) enzyme applied to premix (0.2% of TMR). All diets
that were supplemented with the enzyme product delivered about 1.0 grams per cow
per day. Digestibility of OM, NDF and ADF in the total tract was increased in
comparison to the control when enzymes were added to the entire concentrate.
Enzyme treatments that were applied to a smaller portion of the diet showed only
numerical increases in digestibility over the control. However, there was an
increase in microbial N synthesis for cows fed enzymes added to the premix. The
effects of enzyme supplementation on milk production and composition were not
statistically significant, but cows receiving the enzyme product added to the
concentrate had a numerically higher FCM compared to the control cows. These
results indicate that enzyme supplementation increases total tract digestibility
of organic matter and fiber. The proportion of the diet to which the enzyme is
applied must be maximized to ensure a beneficial response.
DOI: 10.3168/jds.S0022-0302(02)74430-5
2399. J Dairy Sci. 2002 Dec;85(12):3403-10.
A pelleted combination of raw soyhulls and condensed corn steep liquor for
DeFrain JM(1), Shirley JE, Titgemeyer EC, Park AF, Ethington RT.
Author information:
66506, USA.
An experiment was conducted to evaluate the effect of a pelleted combination of

raw soyhulls and condensed corn steep liquor on performance and plasma
metabolites when fed to replace a portion of the grain and forage in diets for
lactating dairy cows. The raw soybean hull-corn steep liquor pellet (SHSL)
contained 24.2% crude protein, 8.7% rumen undegradable protein, 28.9% acid
detergent fiber, 36.7% neutral detergent fiber, and 2.5% ether extract (% of dry
matter, [DM]). Eighteen multiparous Holstein cows were assigned to one of three
diets in a replicated 3 x 3 Latin square design with 28-d periods. Cows were
blocked by pretreatment body weight and energy-corrected milk (ECM) and assigned
to control, SHSL (20.7% of diet DM), or pelleted raw soybean hulls ([PSH] 14.3%
of diet DM). The SHSL product replaced 6.2% alfalfa hay, 3.7% corn silage, 6.6%
corn, and 3.3% soybean meal (SBM), and 1.7% expeller SBM replaced solvent SBM in
order to maintain dietary levels of rumen undegradable protein. PSH replaced 6.2%
alfalfa hay, 3.7% corn silage, and 5.1% corn. Diet crude protein (%) and energy
density (Mcal/kg NEL) were 16.6 and 1.64, 16.3 and 1.65, 17.1 and 1.63 for
control, SHSL, and PSH, respectively. Cows fed PSH consumed more DM than cows fed
control, with the intake of cows fed SHSL being intermediate. SHSL and PSH
increased ECM, milk protein, and solids-notfat and showed higher concentrations
of milk and plasma urea N and total alpha-amino N in plasma than the control
diet. Furthermore, feeding SHSL tended to improve the ratio of ECM to DM intake.
There was no effect of diet on concentrations of total essential and nonessential
amino acids in plasma. These production data suggest SHSL can replace a portion
of the forage, grain, and SBM in diets for lactating dairy cows without
decreasing lactational performance.
DOI: 10.3168/jds.S0022-0302(02)74428-7
2400. J Dairy Sci. 2002 Dec;85(12):3304-13.
Effects of a subacute ruminal acidosis model on the diet selection of dairy cows.
Keunen JE(1), Plaizier JC, Kyriazakis L, Duffield TF, Widowski TM, Lindinger MI,
McBride BW.
Author information:
(1)Department of Animal and Poultry Science, University of Guelph, Guelph,
Ontario, Canada N1G2W1.
Two experiments were conducted to study the effects of a subacute ruminal

acidosis (SARA) model on diet choice in dairy cows. In the first experiment, 25%
of the ad libitum dry matter intake (DMI) of the total mixed ration (TMR) was
replaced with wheat-barley pellets (WBP, 50% ground wheat, 50% ground barley).
Rumen pH was measured continuously via in-dwelling probes in 4 mid to late
lactation cows. This diet change reduced rumen pH by 0.14 +/- 0.02 pH units (mean
+/- SE) and increased time below pH 6.0, from 319 +/- 36 min(-1) to 641 +/- 36
min(-1). Hence, the nutritional model successfully induced SARA. The second
experiment determined if inducing SARA increases the feed preference for long
alfalfa hay compared with alfalfa pellets. The 2 wk of inducing SARA were
separated by 1 control wk. Four cows on either SARA and control diets were given
a choice of 2 feeds, 2 times per d, for 30 min. The preference ratios (PR =
Amount of Hay consumed/Amount of Hay + Pellets consumed) for alfalfa hay during
two SARA weeks was greater (0.85 +/- 0.03) compared with the control week (0.60
+/- 0.03). In SARA weeks, average rumen pH was 0.23 +/- 0.03 units lower, and
time below pH 6.0 and 5.6 was higher compared to control. These results suggest
that when given a choice of feeds, dairy cows alter their diet selection to
attempt to attenuate SARA.
DOI: 10.3168/jds.S0022-0302(02)74419-6
2401. Reprod Nutr Dev. 2002 Jul-Aug;42(4):295-305.
Influence of the load of nylon capsules on their passage through the digestive
tract and specific gravity.
Trináctý J(1), Homolka P, Richter M, Sustala M, Vymetal A.
Author information:
(1)Research Institute of Animal Nutrition, Ltd., 691 23 Pohorelice, Czech
Republic. trinacty@vuvz.cz
The passage and specific gravity of nylon capsules were evaluated in five trials.
In individual trials, different lactating cows were fed the same diet consisting
of maize silage, alfalfa hay and concentrate. In each trial the different feeds
(or no feed) were used to fill the capsules. The capsules were made of nylon
cloth (42 microm pore size, 10 mm external diameter). The different weights of
the load (L1-L5) were obtained using a combination of 2 and 3 mm stainless steel
balls. The highest recovery of the capsules was obtained with the L3 and L4 loads
(91.4 and 92.3%, resp.). After 14 hours of incubation in the rumen, the
calculated values of functional specific gravity of the capsules ranged from 0.92
to 2.05 g x cm(-3). It was concluded that L3 (one 2 mm and one 3 mm ball) was the
suitable weight of the load.
Characterization of rhizobia that nodulate legume species of the genus Lespedeza

and description of Bradyrhizobium yuanmingense sp. nov.
Yao ZY(1), Kan FL, Wang ET, Wei GH, Chen WX.
Author information:
(1)College of Biological Sciences, China Agricultural University, Beijing 100094,
Legume species belonging to the genus Lespedeza are annual or perennial herb or
shrub plants that grow in the northern hemisphere. They are known for the
formation of root nodules, but little information is available about their
microsymbionts. In this study, 58 root-nodule isolates from Lespedeza spp.,
obtained from China and the USA, were characterized using numerical taxonomic
analysis of phenotypic features, SDS-PAGE analysis of whole-cell proteins,
DNA-DNA hybridization, 16S rRNA gene sequence analysis and cross-nodulation with
selected legume species. From the results generated using these approaches, it
was concluded that Lespedeza spp. were promiscuous hosts for rhizobia. Four main
clusters of bacteria, which included 35 of the strains isolated from Lespedeza
spp., were defined upon numerical taxonomic analysis; these groups corresponded
to those determined from analyses of protein electrophoretic and DNA-DNA
hybridization data. The four clusters were found to define strains belonging to
one of four species, Sinorhizobium saheli, Bradyrhizobium japonicum,
Bradyrhizobium elkanii or a novel species of the genus Bradyrhizobium. The
strains of B. japonicum and B. elkanii were all from the USA soil samples, and
their representative strains could not nodulate soybean. The seven strains found
to represent the novel Bradyrhizobium sp. were from China. These were
differentiated from recognized species of the genus Bradyrhizobium by all of the
taxonomic methods used here; hence, it is proposed that the novel strains
isolated from Lespedeza spp. represent a novel species of the genus
Bradyrhizobium, Bradyrhizobium yuanmingense. The type strain of the novel
species, CCBAU 10071(T) (= CFNEB 101(T)), formed ineffective nodules on Medicago
sativa and Melilotus albus but did not nodulate soybean. The other 23 bacterial
strains isolated from Lespedeza spp. were found to form single branches or small
groups (two to three strains) that were related to Bradyrhizobium, Mesorhizobium,
Rhizobium and Sinorhizobium spp. on the basis of numerical taxonomic analysis,
indicating the possibility that other rhizobial species are also associated with
Lespedeza spp.
DOI: 10.1099/00207713-52-6-2219
2403. Microbiol Res. 2002;157(4):283-92.
Colony variation in Sinorhizobium meliloti inoculant strain U 45.
Bloem JF(1), Botha WJ, Law IJ, Steyn PL.
Author information:
(1)ARC-Plant Protection Research Institute, Private Bag X 134, Pretoria 0001,
Republic of South Africa.
A culture of Sinorhizobium meliloti strain U 45, maintained on yeast

extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and
non-absorbing (W1) colonies when grown on YM medium containing Congo red. The
original freeze-dried (FD) culture formed gummy (G), white (W2) and small red
(R2) colony types on the above medium. All colonies were stable except G, which
segregated into G and W2-like types. Immune diffusion patterns of all colony
types were identical. The W1 colony type dominated R1 when a 1:1 combination was
sub-cultured on YM agar. The parent cultures and their variants exhibited a range
of N2-fixing effectiveness and competitiveness when inoculated onto two cultivars
of Medicago sativa. Variant R2 from the FD culture was ineffective on both
cultivars. Genomic DNA fingerprinting with insertion elements ISRm3 and
ISRm2011-2 suggested that transposition of these elements was not a cause of
variation, but a DNA band was absent in the profiles of two out of three W2-like
colonies. Protein profile comparisons showed high similarity (r = 0.98) between
the colony types when grown in YM broth. When grown on Tryptone-Yeast extract
medium, variants from the FD and agar-maintained cultures formed separate
clusters with r = 0.79. Polymerase chain reaction fingerprinting using
repetitive, site-directed and arbitrary primers failed to differentiate the
variants. The results emphasize the need to monitor culture variability to
maintain the quality of legume inoculants.
DOI: 10.1078/0944-5013-00158
2404. J Gen Appl Microbiol. 1998 Feb;44(1):1-9.
Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine

and its related compounds by ruminal bacteria, protozoa and their mixture.
Amin MR(1), Onodera R.
Author information:
(1)Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science,
Miyazaki University, Miyazaki 889-21, Japan.
An in vitro study was conducted to examine the effects of salinomycin (SL) and
vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic
acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen
bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen
microorganisms were collected from fistulated goats fed lucerne cubes (Medicago
sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were
anaerobically incubated at 39 degrees C for 12 h. Phe and some other related
compounds in both supernatants and microbial hydrolysates of the incubations were
analyzed by HPLC. When PPY was used as a substrate, it completely disappeared
without additives and converted mainly to Phe and PAA on the average by 396 and
178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the
12 h incubation period. The rate of disappearance showed no significant
differences between the microbial suspensions with and without SL and B(6) during
the incubation period. The production of Phe from PPY with SL was enhanced
(p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production
from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP,
respectively, during the 12 h incubation period. On the other hand, with B(6),
the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and
11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation
period. When PAA added as a substrate was incubated in the incubation medium
without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted
mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP,
respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15
and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6
was almost the same as that without B(6) in B and BP suspensions but tended to be
enhanced by more than 9% in P suspensions during the 12 h incubation period. The
production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B,
P and BP, respectively, during the 6 h incubation period, but the inhibition was
weakened during the 12 h incubation period, whereas Phe production from PAA with
B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When
Phe was added as a substrate, the net Phe disappearance without additives was
549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205
and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL
was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe
with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The
production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B,
P and BP, respectively. On the other hand, with B(6), PAA production from Phe was
enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these
results, it seems that SL inhibited Phe disappearance and enhanced the synthesis
of Phe from PPY, though not from PAA, and accumulated free Phe in the medium,
whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could
provide additional amino N for animals.
PMID: 12501287
2405. J Food Prot. 2002 Dec;65(12):1903-8.
Composition and physiological profiling of sprout-associated microbial

communities.
Matos A(1), Garland JL, Fett WF.
Collaborators: Garland JL(2).
Author information:
Regional Research Center, Wyndmoor, Pennsylvania 19038, USA. amatos@arserrc.gov
(2)NASA KSC
The native microfloras of various types of sprouts (alfalfa, clover, sunflower,

mung bean, and broccoli sprouts) were examined to assess the relative effects of
sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and
inoculation with sprout-derived inocula) on the microbial community structure of
sprouts. Sprouts were sonicated for 7 min or hand shaken with glass beads for 2
min to recover native microfloras from the surface, and the resulting suspensions
were diluted and plated. The culturable fraction was characterized by the density
(log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g.,
microbial richness and evenness) of colonies on tryptic soy agar plates incubated
for 48 h at 30 degrees C. The relative similarity between sprout-associated
microbial communities was assessed with the use of community-level physiological
profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources.
Aerobic plate counts of 7.96 +/- 0.91 log CFU/g of sprout tissue (fresh weight)
were observed, with no statistically significant differences in microbial cell
density, richness, or diversity due to sprout type, sprout-growing facility, or
seed lot. CLPP analyses revealed that the microbial communities associated with
alfalfa and clover sprouts are more similar than those associated with the other
sprout types tested. Variability among sprout types was more extensive than any
differences between microbial communities associated with alfalfa and clover
sprouts from different sprout-growing facilities and seed lots. These results
indicate that the subsequent testing of biocontrol agents should focus on similar
organisms for alfalfa and clover, but alternative types may be most suitable for
the other sprout types tested. The inoculation of alfalfa sprouts with
communities derived from various sprout types had a significant,
source-independent effect on microbial community structure, indicating that the
process of inoculation alters the dynamics of community development regardless of
the types of organisms involved.
2406. J Dairy Sci. 2002 Nov;85(11):3000-7.
Influence of particle size on the effectiveness of the fiber in alfalfa silage.
Clark PW(1), Armentano LE.
Author information:
(1)Department of Animal and Food Science, University of Wisconsin-River Falls,
River Falls 54022-5001, USA. perry.w.clark@uwrf.edu
The objective was to determine the influence of alfalfa silage particle length on
milk yield, milk composition, and chewing activity. Sixteen multiparous lactating
Holsteins were used in each of two separate feeding trials over a 2-yr period
providing two repetitions. Each trial was based on 4 x 4 Latin squares with 21-d
periods. All four diets (2-yr average, dry basis) contained a basal level of
10.7% neutral detergent fiber from corn silage and 2.3% neutral detergent fiber
from chopped alfalfagrass hay. One of the diets served as a low forage, low fiber
control and contained only the basal forages. The other three diets contained an
additional 8.6% neutral detergent fiber from coarser alfalfa silage, finely
rechopped alfalfa silage or an equal mixture ofthe two. An increase in the forage
content above the basal amount using alfalfa silage increased 4% fat-corrected
milk yield, milk fat yield and concentration, eating time, and total chewing
time. Dry matter intake was not influenced by diet. Linear increases in
rumination and total chewing times were observed as the mean particle size of the
alfalfa silage increased from finer to coarser. There was no linear effect of
alfalfa silage particle size on milk yield, 4% fat-corrected milk yield, dry
matter intake, or milk composition.
DOI: 10.3168/jds.S0022-0302(02)74385-3
2407. J Bacteriol. 2003 Jan;185(1):325-31.
Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti.
Marketon MM(1), Glenn SA, Eberhard A, González JE.
Author information:
Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a

symbiotic relationship with alfalfa plants. This invasion process requires the
synthesis, by S. meliloti, of at least one of the two symbiotically important
exopolysaccharides, succinoglycan and EPS II. We have previously shown that the
sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in
the symbiotic process. Here we show that the sinRI locus exerts one level of
control through regulation of EPS II synthesis. Disruption of the autoinducer
synthase gene, sinI, abolished EPS II production as well as the expression of
several genes in the exp operon that are responsible for EPS II synthesis. This
phenotype was complemented by the addition of acyl homoserine lactone (AHL)
extracts from the wild-type strain but not from a sinI mutant, indicating that
the sinRI-specified AHLs are required for exp gene expression. This was further
confirmed by the observation that synthetic palmitoleyl homoserine lactone
(C(16:1)-HL), one of the previously identified sinRI-specified AHLs, specifically
restored exp gene expression. Most importantly, the absence of symbiotically
active EPS II in a sinI mutant was confirmed in plant nodulation assays,
emphasizing the role of quorum sensing in symbiosis.
PMCID: PMC141839
2408. Phytochemistry. 2003 Jan;62(1):53-65.
Structural and compositional modifications in lignin of transgenic alfalfa

down-regulated in caffeic acid 3-O-methyltransferase and caffeoyl coenzyme A
3-O-methyltransferase.
Marita JM(1), Ralph J, Hatfield RD, Guo D, Chen F, Dixon RA.
Author information:
(1)US Dairy Forage Research Center, USDA-Agricultural Research Service, 1925
Linden Drive West, Madison, WI 53706-1108, USA.
Isolated lignins from alfalfa deficient in caffeic acid 3-O-methyltransferase
contained benzodioxanes resulting from the incorporation of the novel monomer,
5-hydroxyconiferyl alcohol. Due to the high level incorporated into the soluble
lignin fraction and the use of sensitive NMR instrumentation, unique structural
features were revealed. A new type of end-unit, the 5-hydroxyguaiacyl glycerol
unit, was identified. It was possible to establish that coniferyl alcohol,
sinapyl alcohol, and the novel 5-hydroxyconiferyl alcohol can cross-couple with
the 5-hydroxyguaiacyl units that are formed in the lignin, the latter giving rise
to extended chains of benzodioxane units. There is also evidence that
5-hydroxyconiferyl alcohol couples with normal (guaiacyl or syringyl) lignin
units. Lignin in the alfalfa deficient in caffeoyl CoA 3-O-methyltransferase was
structurally similar to the control lignin but the transgenic exhibited a
dramatic decrease in lignin content (approximately 20%) and modest increase in
cellulose (approximately 10%) reflecting a 30% increase in cellulose:lignin
ratio. The compositional changes in both transgenics potentially allow enhanced
utilization of alfalfa as a major forage crop by increasing the digestibility of
its stem fraction.
2409. Genome Res. 2002 Dec;12(12):1974-81.
A unified framework for mapping quantitative trait loci in bivalent tetraploids

using single-dose restriction fragments: a case study from alfalfa.
Ma CX(1), Casella G, Shen ZJ, Osborn TC, Wu R.
Author information:
(1)Department of Statistics, University of Florida, Gainesville, Florida 32611,
USA.
The development of statistical methodologies for quantitative trait locus (QTL)

mapping in polyploids is complicated by complex polysomic inheritance. In this
article, we propose a statistical method for mapping QTL in tetraploids
undergoing bivalent formation at meiosis by using single-dose restriction
fragments. Our method is based on a unified framework, one that uses chromosome
bivalent pairing configuration and gametic recombination to discern different
mechanisms of gamete formation. Our bivalent polyploid model can not only provide
a simultaneous estimation of the linkage and chromosome pairing configuration-a
cytological parameter of evolutionary and systematic interest-but also enhances
the precision of estimating QTL effects and position by correctly characterizing
gene segregation during polyploid meiosis. By using our method and a linkage map
constructed in a previous study, we successfully identify several QTL affecting
winter hardiness in bivalent tetraploid alfalfa. Moreover, our results reveal
significant preferential chromosome pairing at meiosis in an F1 hybrid
population, which indicates the importance of reassessing the traditional view of
random chromosome segregation in alfalfa.
DOI: 10.1101/gr.320202
PMCID: PMC187580
2410. J Anim Sci. 2002 Nov;80(11):3016-20.
Effect of exogenous fibrolytic enzyme on ruminal fermentation and digestibility

of alfalfa and rye-grass hay fed to lambs.
Pinos-Rodríguez JM(1), González SS, Mendoza GD, Bárcena R, Cobos MA, Hernández A,
Ortega ME.
Author information:
(1)Instituto de Investigación de Zonas Desérticas, Universidad Autónoma de San
Luis Potosí, San Luis Potosí, México. jpinos@uaslp.mx
This experiment was carried out to study the effect of a directly fed exogenous
fibrolytic enzyme on intake and digestion of DM, OM, protein, NDF, ADF, and
hemicellulose of alfalfa and ryegrass hay by sheep. Four diets were randomly
assigned to four ruminally cannulated lambs using a 4 x 4 Latin square design,
repeated in time, with a factorial arrangement (2 x 2) of diets: 1) alfalfa hay;
2) alfalfa hay + exogenous fibrolytic enzymes (enzyme); 3) ryegrass hay; and 4)
ryegrass hay + enzyme. Lambs consumed more DM and OM from alfalfa than from
ryegrass hay (P < 0.001). The ADF intake was not different between the hays, but
NDF intake was lower for alfalfa (P < 0.001). For both hays, the enzyme increased
intake of DM (P < 0.01), as well as OM and CP (P < 0.05); however, NDF and ADF
intake were not changed. Alfalfa hay had higher apparent digestibility of DM, OM,
and CP (P < 0.001), but lower digestibility for NDF, ADF, and hemicellulose. The
enzyme increased apparent digestibility of CP, hemicellulose (P < 0.05), and NDF
(P < 0.10) for alfalfa. Also, for both hays, the enzyme improved N balance
because lambs retained more N (P < 0.05). The enzyme increased (P < 0.05) total
VFA concentration (3 and 6 h) for both hays. Results from this trial indicate
that directly fed exogenous fibrolytic enzymes may change ruminal fermentation,
intake, and digestibility of forages with different nutritive value.
2411. J Anim Sci. 2002 Nov;80(11):2989-95.
Digestibility and dry matter intake of diets containing alfalfa and kenaf.
Phillips WA(1), Ra SC, Fitch JQ, Mayeux HS.
Author information:
(1)USDA-ARS Grazinglands Research Laboratory, El Reno, OK 73036, USA.
bphillip@grl.ars.usda.gov
Two experiments were conducted to determine the dietary value of pellets

containing kenaf (Hibiscus cannabinus cv. 'Everglade 41') hay. Averaged across
both experiments, kenaf pellets contained 82.6% kenaf hay, 16.6% liquid molasses,
and 0.8% mineral oil. The chemical composition of the kenaf pellet was 12.6%
crude protein (CP), 41.2% neutral detergent fiber (NDF), and 14.4% acid detergent
fiber (ADF). In Exp. 1 (digestion and N balance trial), 18 lambs (body weight
[BW] = 36.4 kg) were blocked by BW. Lambs were randomly assigned within a block
to Diet 1 (59.5% corn and 40.5% alfalfa pellet), Diet 2 (59.7% corn, 28.4%
alfalfa pellets, and 11.9% kenaf pellets), or Diet 3 (59.6% corn, 16.5% alfalfa
pellets, and 23.9% kenaf pellets). Diets were formulated so that CP was the
first-limiting nutrient. Each diet was limit-fed at 2.4% of BW. Replacing alfalfa
pellets with kenaf pellets tended to decrease (P = 0.10) CP and ADF intakes, but
increased (P = 0.01) DM digestibility. Diet had no effect (P = 0.33) on N
balance. In Exp. 2 (dry matter [DM] intake trial), 32 lambs (BW = 30.4 kg) were
blocked by gender and BW. Within a block, lambs were randomly assigned to one of
four diets in a 2 x 2 factorial arrangement. Main effects were hay (bermudagrass
or fescue) and supplemental protein source (kenaf or alfalfa pellets). Lambs were
housed in individual pens with ad libitum access to the assigned hay.
Supplemental protein was fed (185 g of DM) once daily. Hay intake was measured
weekly for 8 wk. Lambs consumed more (P = 0.002) fescue than bermudagrass hay
(743 vs 621 g/ d). Lambs fed fescue hay gained weight more rapidly (P = 0.001)
than lambs fed bermudagrass hay (120 vs 72 g/d). Hay intake and ADG were similar
(P = 0.90) for lambs fed alfalfa or kenaf pellets. Kenaf hay mixed with molasses
and mineral oil can be formed into a pellet. In the diets used in this
experiments, kenaf pellets can replace alfalfa pellets in diets fed to lambs
without altering forage intake, gain, or N retention.
2412. J Anim Sci. 2002 Nov;80(11):2960-6.
Matua bromegrass hay for mares in gestation and lactation.
Guay KA(1), Brady HA, Allen VG, Pond KR, Wester DB, Janecka LA, Heninger NL.
Author information:
(1)Department of Animal and Food Sciences, Texas Tech University, Lubbock 79409,
USA.
Matua bromegrass hay (Bromus willdenowii Kunth) is a high quality forage, but its
value for mares during gestation and lactation is not well known. Intake, rate of
passage, performance, and reproduction by gestating and lactating Quarter Horse
mares fed the hay was investigated. In this experiment, 12, 2- to 12-yr-old
gravid mares (mean BW = 553 kg, SD = 36) were fed Matua hay (CP = 11.5%) or
alfalfa hay (Medicago sativa L.) (CP = 15.4%) for variable days prepartum (mean
59.9 d; SD = 23.5) and for 70 d postpartum. Matua and alfalfa hay were fed as the
roughage portion of the diet with a grain supplement. Mares, blocked by age,
expected date of foaling, and BW, were assigned randomly within blocks to
treatments (six mares per treatment). Forage type did not affect intake,
gestation length, birth weight, number of foals, foal weight gain, day of first
postpartum ovulation, cycles per conception, or pregnancy rate at 70 d. On d 1,
milk from mares fed alfalfa hay contained less (P < 0.03) CP than milk from mares
fed Matua hay. Milk CP decreased (P < 0.01) in all mares over time. In a separate
experiment, voluntary intake and rate of passage of Matua (CP = 15.5%), alfalfa
(CP = 24.9%), and Timothy (Phleum pratense L.) (CP = 4.1%) hays were determined
in nine 2-yr-old pregnant mares (mean BW = 447 kg; SD = 21). Diets were 100%
forage. Timothy hay did not meet CP requirements for mares. Voluntary intake of
alfalfa hay was higher (P < 0.01) than Matua hay. Intake of Timothy hay was lower
(P < 0.01) than the mean of alfalfa and Matua hay. Rate of passage offorage was
measured by passage of Cr-mordanted fiber. Passage rate and retention time did
not differ between Matua and alfalfa hay; however, the retention times of Matua
and alfalfa hays were shorter (P < 0.01) than for Timothy hay. Our results
indicate that Matua hay is a forage that can be used safely for mares during
gestation and early lactation and for their young foals.
2413. Plant Cell Physiol. 2002 Nov;43(11):1351-8.
Expression of symbiotic and nonsymbiotic globin genes responding to

microsymbionts on Lotus japonicus.
Uchiumi T(1), Shimoda Y, Tsuruta T, Mukoyoshi Y, Suzuki A, Senoo K, Sato S, Kato

T, Tabata S, Higashi S, Abe M.
Author information:
(1)Department of Chemistry and BioScience, Kagoshima University, Kagoshima,
890-0065 Japan. uttan@sci.kagoshima-u.ac.jp
Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and
nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb
genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima.
Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression
were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream
region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with
specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1,
2, 3) were expressed specifically and strongly in root nodules. The expression of
LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant,
whereas low level expression was detected in seedlings by RT-PCR. This suggests
that sym-Hbs may have another unknown function besides being oxygen transporter
for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1,
examined with RT-PCR, was detected at low level in root, leaf and stem. The
expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in
roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the
nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by
Glomus sp. R10.
2414. Eukaryot Cell. 2002 Aug;1(4):626-33.
Colletotrichum trifolii TB3 kinase, a COT1 homolog, is light inducible and

becomes localized in the nucleus during hyphal elongation.
Chen C(1), Dickman MB.
Author information:
(1)Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska
68583, USA.
Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease

of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus
(TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has
been isolated in our laboratory and appears to be associated with hyphal
elongation and branching. In this report we show that light treatment rapidly
induces TB3 expression and hyphal branching frequency. Western analysis showed
TB3 localization in both the cytoplasm and nucleus, but not in membranes.
Moreover, indirect immunofluorescence indicated that TB3 levels were most
abundant in the nucleus. To further evaluate the subcellular distribution of TB3,
a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that
the cellular location of TB3 changed during fungal growth and development.
Consistent with previous observations, TB3 was localized in both the cytoplasm
and the nucleus but was preferentially localized in the nucleus during extended
hyphal growth. The amino terminus of TB3 contains two relatively long
polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts
can activate transcription. These results suggest that TB3 may be positioned in a
signaling cascade regulating proper hyphal growth and development by functioning
as a transcription factor.
PMCID: PMC117997
2415. Can J Microbiol. 2002 Sep;48(9):765-71.
Identification and cloning of the bacterial nodulation specificity gene in the

Sinorhizobium meliloti--Medicago laciniata symbiosis.
Barran LR(1), Bromfield ES, Brown DC.
Author information:
(1)Soils and Crops Development Centre, Agriculture and Agri-Food Canada, 2560
Hochelaga Blvd., Sainte-Foy, QC G1V 2J3, Canada. barranlr@em.agr.ca
Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation

specific, nodulating only with a restricted range of Sinorhizobium meliloti.
e.g., strain 102L4, but not with most strains that nodulate Medicago sativa
(alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the
S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and
to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment
from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of
M. laciniata. Nucleotide sequencing revealed that this fragment contained
nodABCIJ genes whose overall arrangement was similar to those found in strains
RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of
the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in
the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave
mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only
subclones of the 11-kb DNA fragment containing a functional nodC gene from strain
102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The
practical implications of these findings are discussed in the context of the
development of a specific M. sativa - S. meliloti combination that excludes
competition for nodulation by bacterial competitors resident in soil.
2416. Antonie Van Leeuwenhoek. 2002 Aug;81(1-4):607-16.
The influence of the symbiotic plasmid pRL1JI on the distribution of GM rhizobia

in soil and crop rhizospheres, and implications for gene flow.
Clark IM(1), Mendum TA, Hirsch PR.
Author information:
(1)Agriculture and the Environment Division, IACR-Rothamsted, Harpenden,
Hertfordshire, UK.
The distribution of two genetically modified Rhizobium leguminosarum strains was

investigated in the field. One, RSM2004, released in 1987, carries a TnS marker
on its conjugative symbiotic plasmid (pSym). The second, CT0370, released at the
same site in 1994, has a gusA gene integrated into its chromosome but no pSym.
Plate counts indicated that the CT0370 population became established at a higher
level than RSM2004. However, when peas, alfalfa and barley were grown, RSM2004
was found to outnumber CT0370 on all roots and by 100-fold on pea. Although the
transfer of pSym from RSM2004 to CT0370 could be detected on plates and in
microcosm studies with high inoculum densities, no transfer was detected in the
field. Subsequent transfer of pSym from RSM2004 to CT0370 demonstrated that it
conferred an advantage in the rhizosphere. In addition to increasing host
fitness, plasmids may transfer, or mobilise other genetic elements, to other
bacteria. This is more likely in sites such as the rhizosphere, where cells are
active and numbers are high. The distribution of pSym and other genetic elements
associated with rhizobia, in bacterial sub-populations from the soil and roots of
the different plants, was investigated using PCR. The genetic elements studied
were: ISRm3, an insertion element from Sinorhizobium meliloti; pSB 102, a broad
host range mer plasmid; the Rhizobium nodC gene (carried on pSym) and plasmid
replication origins repCI and repCII. As expected, ISRm3 was detected in
rhizoflora cultured from alfalfa but not the other plants. The mer gene was
ubiquitous but the transfer region of pSB 102 was not detected. The nodC and both
repC primers amplified products from all the plants, giving further evidence for
the occurrence of plasmids originating from Rhizobium in the rhizoflora of
non-host plants. Despite the abundance of elements associated with transferable
plasmids in rhizobia, none was detected in either inoculant strain.
2417. J Bacteriol. 2002 Dec;184(24):7042-6.
Analysis of infection thread development using Gfp- and DsRed-expressing

Gage DJ(1).
Author information:
(1)Department of Molecular and Cell Biology, University of Connecticut, Storrs
06269, USA. gage@uconnvm.uconn.edu
Sinorhizobium meliloti growth inside infection threads was monitored after

inoculation of alfalfa with red- or green-tagged bacteria. Most threads were
populated with single bacterial types. Mixed infections were present but gave
mixed nodules less often than expected. These patterns are explained by a model
describing bacterial growth during infection.
PMCID: PMC135452
2418. Mol Plant Microbe Interact. 2002 Oct;15(10):1008-13.
The endosymbiosis-induced genes ENOD40 and CCS52a are involved in

endoparasitic-nematode interactions in Medicago truncatula.
Favery B(1), Complainville A, Vinardell JM, Lecomte P, Vaubert D, Mergaert P,

Kondorosi A, Kondorosi E, Crespi M, Abad P.
Author information:
(1)Unité Interactions Plantes-Microorganismes et Santé Végétale, INRA, 123,
Antibes, France.
Plants associate with a wide range of mutualistic and parasitic biotrophic

organisms. Here, we investigated whether beneficial plant symbionts and
biotrophic pathogens induce distinct or overlapping regulatory pathways in
Medicago truncatula. The symbiosis between Sinorhizobium meliloti and this plant
results in the formation of nitrogen-fixing root nodules requiring the activation
of specific genes in the host plant. We studied expression patterns of
nodule-expressed genes after infection with the root-knot nematode Meloidogyne
incognita. Two regulators induced during nodule organogenesis, the early nodulin
gene ENOD40 involved in primordium formation and the cell cycle gene CCS52a
required for cell differentiation and endoreduplication, are expressed in galls
of the host plant. Expression analysis of promoter-uidA fusions indicates an
accumulation of CCS52a transcripts in giant cells undergoing endoreduplication,
while ENOD40 expression is localized in surrounding cell layers. Transgenic
plants overexpressing ENOD40 show a significantly higher number of galls. In
addition, out of the 192 nodule-expressed genes tested, 38 genes were upregulated
in nodules at least threefold compared with control roots, but only two genes,
nodulin 26 and cyclin D3, were found to be induced in galls. Taken together,
these results suggest that certain events, such as endoreduplication,
cell-to-cell communication with vascular tissues, or water transport, might be
common between giant cell formation and nodule development.
DOI: 10.1094/MPMI.2002.15.10.1008
2419. Cell Cycle. 2002 May-Jun;1(3):187-92.
A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed

by ectopic expression of mitotic cyclin B2.
Giménez-Abián JF(1), Weingartner M, Binarova P, Clarke DJ, Anthony RG, Calderini

O, Heberle-Bors E, Moreno Díaz de la Espina S, Bögre L, De la Torre C.
Author information:
(1)Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid,
Spain.
Comment in
Cell Cycle. 2002 May-Jun;1(3):176-7.
DNA topoisomerase II is required for mitotic chromosome condensation and

segregation. Here we characterize the effects of inhibiting DNA topoisomerase II
activity in plant cells using the non-DNA damaging topoisomerase II inhibitor
ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured
alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led
to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry
into mitosis, increasing the frequency of cells having a pre-prophase band of
microtubules, a marker of late G2 and prophase, and delaying the activation of
cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint
in plant cells that is activated in the absence of topoisomerase II activity. To
determine whether the checkpoint-induced delay was a result of reduced
cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed.
Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and
correspondingly, reduced the frequency of interphase cells with a pre-prophase
band. These data provide evidence that plant cells possess a topoisomerase
II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK
activation and entry into mitosis, and that is overridden by raising the level of
CDK activity through the ectopic expression of a plant mitotic cyclin.
The breast cancer resistance protein protects against a major chlorophyll-derived
dietary phototoxin and protoporphyria.
Jonker JW(1), Buitelaar M, Wagenaar E, Van Der Valk MA, Scheffer GL, Scheper RJ,
Plosch T, Kuipers F, Elferink RP, Rosing H, Beijnen JH, Schinkel AH.
Author information:
(1)Division of Experimental Therapy, The Netherlands Cancer Institute, 1066 CX
Amsterdam, The Netherlands.
The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding

cassette family of drug transporters and confers resistance to various anticancer
drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to
the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe,
sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs
in various plant-derived foods and food supplements. Bcrp1 transports
pheophorbide a and is highly efficient in limiting its uptake from ingested food.
Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria.
Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which
is structurally related to pheophorbide a, were increased 10-fold.
Transplantation with wild-type bone marrow cured the protoporphyria and reduced
the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans
or animals with low or absent BCRP activity may be at increased risk for
developing protoporphyria and diet-dependent phototoxicity and provide a striking
illustration of the importance of drug transporters in protection from toxicity
of normal food constituents.
DOI: 10.1073/pnas.202607599
PMCID: PMC137771
A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains

associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to
pathogenesis.
Silo-Suh L(1), Suh SJ, Sokol PA, Ohman DE.
Author information:
(1)Department of Microbiology and Immunology, Medical College of Virginia Campus
of Virginia Commonwealth University, Richmond 23298-0678, USA.
A sensitive plant infection model was developed to identify virulence factors in

nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains
isolated from cystic fibrosis (CF) patients with chronic pulmonary disease.
Nontypeable strains with defects in lipopolysaccharide O-side chains are common
to CF and often exhibit low virulence in animal models of infection. However,
1,000 such bacteria were enough to show disease symptoms in the alfalfa
infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were
tested for alfalfa seedling infection. Although defects in the global regulators
Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR
reduced the infection frequency by >50%. A defect in alginate biosynthesis
resulted in plant disease with >3-fold more bacteria per plant, suggesting that
alginate overproduction attenuated bacterial growth in planta. FRD1 derivatives
lacking AlgT, a sigma factor required for alginate production, were reduced >50%
in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1,
besides alginate, important for pathogenesis. In contrast, in a non-CF strain,
PAO1, an algT mutation did not affect its virulence on alfalfa. Conversely, PAO1
virulence was reduced in a mucA mutant that overproduced alginate. These
observations suggested that mucoid conversion in CF may be driven by a selection
for organisms with attenuated virulence or growth in the lung, which promotes a
chronic infection. These studies also demonstrated that the wounded alfalfa
seedling infection model is a useful tool to identify factors contributing to the
persistence of P. aeruginosa in CF.
DOI: 10.1073/pnas.242343999
PMCID: PMC137779
2422. J Bacteriol. 2002 Dec;184(23):6681-9.
A Sinorhizobium meliloti lipopolysaccharide mutant altered in cell surface

sulfation.
Keating DH(1), Willits MG, Long SR.
Author information:
(1)Howard Hughes Medical Institute. Department of Biology, Stanford University,
Stanford, California 94305, USA.
The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the
nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange
for plant photosynthates. Nodule development requires a bacterial signal referred
to as Nod factor, which in Sinorhizobium meliloti is a beta-(1,4)-linked tetramer
of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the
nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate
modification requires the action of three gene products: nodH, which catalyzes
the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate,
3'-phosphoadenosine-5'-phosphosulfate. It was previously reported that S.
meliloti cell surface polysaccharides are also covalently modified by sulfate in
a reaction dependent on NodPQ. We have further characterized this unique form of
bacterial carbohydrate modification. Our studies have determined that one of the
nodPQ mutant strains used in the initial study of sulfation of cell surface
harbored a second unlinked mutation. We cloned the gene affected by this mutation
(referred to as lps-212) and found it to be an allele of lpsL, a gene previously
predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL
encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212
mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure
that was reduced in sulfate modification in vitro and in vivo. Finally, we
determined that the lps-212 mutation resulted in a reduced ability to elicit the
formation of plant nodules and by altered infection thread structures that
aborted prematurely.
PMCID: PMC135449
2423. Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet. 2001;66(2a):519-24.
Effects of neem-based and chemical insecticides on some arthropods in alfalfa.
Yardim EN(1), Ozgen I, Kulaz H.

Author information:
(1)Yuzuncu Yil Universitesi, Ziraat Fakultesi, Bitki Koruma Bolumu, 65080, Van,
Turkey.
In this study, we tested the effects of two different doses of neem

(azadirachtin) on the alfalfa weevil, Hypera variabilis Hbst. (Coleoptera:
Curculionidae), aphids and predators (Coccinellids, anthocorids, chrysopids and
spiders) and compared the effects with those of a broad spectrum insecticide
(malathion) in a controlled experiment in 1998 and 1999. Malathion treatments
reduced alfalfa weevil densities significantly (p < 0.01) by 77.9 and 87.4% in
1998 and 1999, respectively. Alfalfa weevil numbers in the low dose neem-treated
plots were lower by 45.2% in 1998 and 50.2% in 1999. There were 45.3% fewer
alfalfa weevils in the high dose neem-treated plots in 1999. In 1998, aphid
numbers were reduced by 11.1, 25.3 and 41% in the low dose neem-treated plots,
the malathion-treated plots and the high dose neem-treated plots, respectively.
Aphids were more abundant in the malathion-treated plots, whereas they were fewer
in the neem-treated plots compared to the control in 1999. The treatments did not
have any significant effects on the total numbers of predators.
2424. Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet. 2001;66(2a):513-8.
A comparison of recommended and reduced insecticide regimes in alfalfa.
Yardim EN(1), Ozgen I, Kulaz H.
Author information:
(1)Yuzuncu Yil Universitesi, Ziraat Fakultesi, Bitki Koruma Bolumu, 65080, Van,
Turkey.
The objective of this study was to evaluate the effects of the recommended rates
of insecticides applied at two different population levels of the alfalfa weevil,
Hypera variabilis Hbst. (Coleoptera: Curculionidae) and the reduced levels of
insecticides on alfalfa weevil, predators and yield in 1998 and 1999. More
specific objective was to search for options to reduce insecticide usage while
implementing an effective pest control, thereby developing more economically and
environmentally sound alfalfa production systems. The treatments used included:
i) recommended rate insecticide (malathion) at population level of 5
weevils/sweep, ii) recommended rate at population level of 10 weevils/sweep, iii)
50% of recommended rate at population level of 10 weevils/sweep, iv) stripe spray
of 50% rate at population level of 10 weevils/sweep, where insecticides were
applied only on 0.5 m stripes in plots while the next 0.5 m stripes were not
treated and v) control. The results indicated that the recommended rate
insecticide application at 5 weevils/sweep level provided the highest reduction
in the weevil densities in both years. No significant differences occurred among
the weevil densities in the plots received the recommended rate, the 50% rate and
the stripe spray insecticide treatments at 10 weevils/sweep level. Predators were
in general more abundant in the insecticide treated plots compared to the
control. The insecticide applications, except the stripe spray in 1999, led to
increases in yields.
2425. Phytochemistry. 2002 Nov;61(6):657-67.

Monolignol biosynthesis in microsomal preparations from lignifying stems of
Guo D(1), Chen F, Dixon RA.
Author information:
Parkway, Ardmore, Oklahoma 73401, USA.
Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.)

contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic
acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine
(SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl
alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl
aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of
sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was
5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric
acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of
monolignol precursors was observed in microsomal preparations from transgenic
alfalfa down-regulated in COMT expression. In most microsomal preparations, the
level of the metabolic conversions was independent of added recombinant COMT.
Taken together, the data provide only limited support for the concept of
metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.
[Root system development ability of several alfalfa cultivars in the hilly and
valley regions of Loess Plateau].
Guo Z(1), Zhang Z, Xiao J, Hou F, Lu N.
Author information:
(1)Chinese Academy of Sciences, College of Pastoral Agriculture Science and
Technology, Lanzhou University, Gansu Grassland Ecological Research Institute,
Lanzhou.
A field study was conducted in the hilly and valley regions of Loess Plateau in
Gansu Province to analyze the root system development ability of several alfalfa
(Medicago sativa) cultivars. Eight introduced cultivars and one local cultivar
were arranged in the same site. The secondary roots of alfalfa cultivars grew
mainly from the taproot underground 10-20 cm depth, and there were no secondary
roots below 50 cm depth. The total amount of secondary roots of Sandili and
Ameristand 201 was the biggest, and that of Longdong alfalfa was the lowest. The
volume and biomass of different cultivars were decreased from 0-10 cm depth to
below 50 cm depth, except those of Sandili and Ameristand 201 at 20-30 cm depth
was bigger than at 10-20 cm depth. The biomass of Ameristand 201 was 170.54 g and
bigger than that of others, and that of Longdong alfalfa, which was 22.4% of
Ameristand 201, was the lowest. According to cluster analysis on 9 alfalfa
cultivars, Sandili, Ameristand 201 and Amerigraze 401 were suitable for planting
on the hills and valleys of Loess Plateau due to their root system development
ability.
[Temporal pattern of arthropod community on cultivated alfalfa grassland].
Liu C(1), Wang W, Wu X, Shang J.
Author information:
(1)Department of Plant Protection, Gansu Agricultural University, State Key
Laboratory of Arid Agroecology, Lanzhou University, Lanzhou.
liuchzh@public.lz.gs.cn
The variation of species and diversities of the arthropod community on cultivated

alfalfa grassland was investigated and studied. Principal component analysis and
optimal sorting method were used to study its temporal patterns. The results
showed that the difference and regularity of species richness, and the diversity
and evenness of the arthropod community at different time were obvious. The
numbers of the species of insect pests and their natural enemies were the most in
the last ten days of June, but the peak period of individual numbers of the
natural enemies was later than that of insect pests. The main factors and
temporal patterns of arthropod community in the course of seasons were obvious.
Based on the results, the temporal patterns of arthropod community on cultivated
alfalfa grassland could be divided into four stages: before the first ten days of
May, the middle ten days of May to the first ten days of June, the middle ten
days of June to the middle ten days of July, and after the middle ten days of
July. Considering the climatic and phenological development, the characteristics
of the pests and the natural enemies occurring in each stage were analyzed, and
the corresponding management tactics were discussed.
[Response of alfalfa seed to stress storage conditions].
Li C(1), Wang Y, Zhu T, Yu L.
Author information:
(1)College of Pastoral Agriculture Science and Technology, Lanzhou University,
Gansu Grassland Ecological Research Institute, Lanzhou. licj68@public.lz.gs.cn
The seed germination rate, seed mortality, seedling length, and infection rate of
alfalfa (Medicago sativa L. cv. 'Longdong') were measured at constant temperature
20 degrees C every 60 days during one year storage period after inoculated or no
inoculated by Fusarium avenaceum under room temperature (RT), 35 degrees C, and
35 degrees C and +10% seed moisture content (SMC) conditions. Field emergence
rates of seeds under above treatments were also observed, and seed-borne fungi
were detected under the conditions mentioned above and controlled deterioration
(CD) as well. The results showed that the percentage of isolated alfalfa
seed-borne fungi was increased from 10% under room temperature and 35 degrees C
to 29% under 35 degrees C + 10% SMC. Disease resistance was declined, and seed
mortality and seedling infection rate under 35 degrees C + 10% SMC were
significantly higher than those under room temperature and 35 degrees C
respectively (P < 0.05). The seed germination rate and field emergence rate were
also decreased significantly (P < 0.05). Seedling shoot and root length under 35
degrees C + 10% SMC were significantly less than those under RT and 35 degrees C
respectively (P < 0.05). The percentages of both seed-borne fungi isolated and
field emergence were decreased, and that of seedling infection was increased with
storage period extending from 60 to 360 days. Compared to no inoculated control,
the percentage of seed germination, seedling shoot and root length were
decreased, and seed mortality and seedling infection rate were increased after
inoculated by F. avenaceum.
2429. J Dairy Sci. 2002 Oct;85(10):2650-61.
Effects of pretrial milk yield on responses of feed intake, digestion, and

production to dietary forage concentration.
Voelker JA(1), Burato GM, Allen MS.
Author information:
(1)Department of Animal Science, Michigan State University, East Lansing
48824-1225, USA.
The relationships between pretrial milk yield and effects of dietary

forage-to-concentrate ratio on dry matter intake (DMI), digestion, and milk yield
were evaluated using 32 Holstein cows in a crossover design with two 16-d
periods. Cows were 197 +/- 55 (mean +/- SD) days in milk at the beginning of the
experiment. Milk yield averaged 33.9 kg/d and ranged from 16.5 to 55.0 kg/d for
the 4 d before initiation of treatments. Treatments were diets with
forage-to-concentrate ratios of 67:33 and 44:56. Forages were alfalfa silage and
corn silage, each at 50% of forage dry matter (DM). Neutral detergent fiber (NDF)
concentrations of high-forage and low-forage diets were 30.7 and 24.3% of DM,
respectively. Dry matter intake was 1.7 kg/d higher for cows fed the low-forage
diet. Milk yield was 2.3 kg/d greater on low forage than on high forage, but 3.5%
fat-corrected milk yield and yield of milk fat were not different between
treatments. Individual DMI response to the low-forage diet relative to the
high-forage diet (low-high) was positively and linearly related to pretrial
fat-corrected milk yield, but fat-corrected milk yield response demonstrated a
quadratic relationship with pretrial fat-corrected milk yield. Milk yield
responded more positively to low forage among low- and high-producing cows than
among moderate-producing cows. Energy partitioned to body reserves and to milk,
and passage rate of indigestible NDF, also responded to dietary forage level in
quadratic relationships with pretrial milk energy output. Individual responses of
intake, production, and fiber digestion to a change in forage-to-concentrate
ratio were dependent on production level.
DOI: 10.3168/jds.S0022-0302(02)74350-6
2430. J Dairy Sci. 2002 Oct;85(10):2603-8.
Effects of Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live

cells on in vitro mixed ruminal microorganism fermentation.
Lynch HA(1), Martin SA.
Author information:
(1)Department of Animal and Dairy Science, The University of Georgia, Athens
30602-2771, USA.
The objective of this study was to examine the effects of a Saccharomyces

cerevisiae live cell product and a S. cerevisiae culture product on the in vitro
mixed ruminal microorganism fermentation of ground corn, soluble starch, alfalfa
hay, and Coastal bermudagrass hay. In the presence of ground corn, neither
concentration (0.35 or 0.73 g/L) of S. cerevisiae culture nor live cells had any
effect on final pH, H2, CH4, propionate, or butyrate. The S. cerevisiae culture
had no effect on acetate, but both concentrations of S. cerevisiae live cells
decreased acetate and the acetate:propionate ratio. When soluble starch was the
substrate, both concentrations of S. cerevisiae live cells and 0.73 g/L of S.
cerevisiae culture decreased the acetate:propionate ratio. Although the treatment
effects were not statistically significant, both concentrations of live cells and
0.73 g/L of the culture decreased lactate concentrations compared with the
control incubations. When alfalfa hay served as the substrate, neither the S.
cerevisiae culture nor the live cells had an effect on propionate, butyrate, or
the acetate:propionate ratio. Both concentrations of S. cerevisiae culture
decreased the final pH and in vitro dry matter disappearance, and the 0.73 g/L
treatment decreased the amount of acetate. However, both treatments of S.
cerevisiae live cells increased final pH and decreased acetate and in vitro dry
matter disappearance. Neither yeast treatment had much effect on the Coastal
bermudagrass hay fermentations. In general, both S. cerevisiae supplements seemed
to have similar effects on the mixed ruminal microorganism fermentation.
DOI: 10.3168/jds.S0022-0302(02)74345-2
2431. Environ Pollut. 2002;120(2):339-53.
Egyptian plant species as new ozone indicators.
Madkour SA(1), Laurence JA.
Author information:
(1)Department of Agricultural Botany, Faculty of Agriculture, Alexandria
University, Damanhour, Egypt.
The aim of this study was to test and select one or more highly sensitive,
specific and environmentally successful Egyptian bioindicator plants for ozone
(O3). For that purpose more than 30 Egyptian species and cultivars were subjected
to extensive screening studies under controlled environmental and pollutant
exposure conditions to mimic the Egyptian environmental conditions and O3 levels
in urban and rural sites. Four plant species were found to be more sensitive to
O3 than the universally used O3-bioindicator, tobacco Bel W3, under the Egyptian
environmental conditions used. These plant species, jute (Corchorus olitorius
c.v. local), clover (Trifolium alexandrinum L. c.v. Masry), garden rocket (Eruca
sativa c.v. local) and alfalfa (Medicago sativa L. c.v. local), ranked in order
of decreasing sensitivity, exhibited typical O3 injury symptoms faster and at
lower 03 concentrations than Bel W3. Three variables were tested in search of a
reliable tool for the diagnosis and prediction of O3 response prior to the
appearance of visible foliar symptoms: pigment degradation, stomatal conductance
(g(s)) and net photosynthetic CO2 assimilation (Pnet). Pigment degradation was
found to be unreliable in predicting species sensitivity to O3. Evidence
supporting stomatal conductance involvement in 03 tolerance was found only in
tolerant species. A good correlation was found between g(s), restriction of O3
and CO2 influx into the mesophyll tissues, and Pnet. Changes in Pnet seemed to
depend largely on fluctuations in g(s).
2432. Amino Acids. 2002;22(2):167-77.
Aromatic amino acid biosynthesis and production of related compounds from

p-hydroxyphenylpyruvic acid by rumen bacteria, protozoa and their mixture.
Khan RI(1), Onodera R, Amin MR, Mohammed N.
Author information:
(1)Laboratory of Animal Nutrition and Biochemistry, Faculty of Agriculture,
Miyazaki University, Miyazaki-shi, Japan. rikhanah@bdonline.com
Aromatic amino acid biosynthesis and production of related compounds from

p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and
their mixture (BP) in an in vitro system were quantitatively investigated.
Microbial suspensions prepared from mature, fistulated goats fed Lucerne (
Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at
39 degrees C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and
other related compounds in both supernatants and hydrolyzates of all incubations
were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared
HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h
incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than
those in B and BP, respectively. Appreciable amounts of Phe (3-12% of the
disappeared HPY) and Trp (2-10% of the disappeared HPY) were also produced from
HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp
synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe
and Trp from HPY in BP was higher than those in B plus P. A large amount of
p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from
HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid
produced from HPY in P was 1.6 times higher than that in B. Considerable amounts
of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2-6% of the
disappeared HPY) were produced only in B.
DOI: 10.1007/s007260200005
2433. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14578-83. Epub 2002 Oct 16.
Bottlenecks for metabolic engineering of isoflavone glycoconjugates in

Arabidopsis.
Liu CJ(1), Blount JW, Steele CL, Dixon RA.
Author information:
In view of their perceived chemopreventive activities against hormone-dependent

cancers, cardiovascular disease, and postmenopausal ailments, there is
considerable interest in engineering plants to contain isoflavone phytoestrogens.
However, attempts to date have only resulted in low levels of isoflavone
accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) into
Arabidopsis thaliana leads to accumulation of low levels of genistein glycosides.
Leaves of wild-type A. thaliana contain high levels of similar conjugates of the
flavonols quercetin and kaempferol, which could be increased by threefold on
introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were
not increased by expressing both IFS and alfalfa chalcone isomerase, but levels
of flavonol conjugates were reduced to a greater extent than could be accounted
for by flux into isoflavone. Introduction of IFS into the tt6/tt3 double mutant
blocked in flavonol, and anthocyanin synthesis resulted in high levels of
genistein. The bottleneck for constitutive isoflavone production in Arabidopsis
is, therefore, competition for flavanone between IFS and endogenous flavonol
synthesis, and the flavonol pathway is reciprocally but disproportionately
affected by IFS.
DOI: 10.1073/pnas.212522099
PMCID: PMC137925
2434. Cell Mol Biol Lett. 2002;7(2B):745-51.
Microsatellite markers discriminating accessions within collections of plant

genetic resources.
Kraic J(1), Gregová E, Jomová K, Hudcovicová M.
Author information:
(1)Research Institute of Plant Production, Bratislavská cesta 122, 92168
Piestany, Slovakia.
The reliability of microsatellite analyses for discriminating between plant

accessions maintained in collections of genetic resources was tested for 53
accessions of barley, 65 of soybean, 49 of chickpea, and 19 of alfalfa. The
specific primer pairs used in this study were based on microsatellite DNA
sequences surrounded by perfect dinucleotide and imperfect trinucleotide tandem
repeat units. The evaluated polymorphic information content, diversity index, and
probabilities of identity indicate that there is value in the application of SSR
analyses in barley, soybean, and chickpea genetic resource management. Variation
between alfalfa genotypes was not revealed at the five analyzed microsatellite
loci.
2435. Plant Physiol. 2002 Oct;130(2):649-56.
L-Ascorbic acid is accumulated in source leaf phloem and transported to sink

tissues in plants.
Franceschi VR(1), Tarlyn NM.
Author information:
(1)School of Biological Sciences, Washington State University, Pullman, WA
99164-4236, USA. vfrances@mail.usu.edu
L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and
transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact
plants of three different species, autoradiographs and HPLC analysis demonstrated
that AsA was accumulated into phloem and transported to root tips, shoots, and
floral organs, but not to mature leaves. AsA was also directly detected in
Arabidopsis sieve tube sap collected from an English green aphid (Sitobion
avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa
with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA,
lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold
increase of AsA in untreated sink tissues of the same plant. The amount of AsA
produced in treated leaves and accumulated in sink tissues was proportional to
the amount of GAL-L applied. Studies of the ability of organs to produce AsA from
GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and
much lower AsA turnover rate than sink tissues. The results indicate AsA
transporters reside in the phloem, and that AsA translocation is likely required
to meet AsA demands of rapidly growing non-photosynthetic tissues. This study
also demonstrates that source leaf AsA biosynthesis is limited by substrate
availability rather than biosynthetic capacity, and sink AsA levels may be
limited to some extent by source production. Phloem translocation of AsA may be
one factor regulating sink development because AsA is critical to cell
division/growth.
DOI: 10.1104/pp.007062
PMCID: PMC166594
2436. Acta Biol Hung. 2002;53(3):317-24.
Intra- and interspecific molecular polymorphism of thrips species.
Bayar K(1), Törjék O, Kiss E, Gyulai G, Heszky L.
Author information:
(1)Department of Plant Protection, Szent István University, Gödöllö, Hungary.
bayar@fau.gau.hu
Molecular polymorphism of six species of Thysanoptera of both sexes, collected

from different locations and host plants in Hungary was studied by using RAPD-PCR
technique. The specimens were classified according to sampling sites (Gödöllö,
Nagykovácsi and Valkó), host plants (Lathyrus tuberosus, Medicago sativa,
Taraxacum officinale, Trifolium pratense), sexes, and larvae in case of
Aeolothrips intermedius. On the basis of the total of 103 fragments generated by
15 RAPD primers the genetic distances were calculated by cluster analysis using
simple matching method. The dendrogram resulted in two main groups:
Aeolothripidae (Aeolothrips intermedius) and Thripidae (Frankliniella intonsa,
Kakothrips robustus, Odontothrips confusus, Thrips dilatatus and T. tabaci).
Within the family Thripidae two subgroups were observed including (i) F. intonsa,
T. dilatatus and T. tabaci, and (ii) K. robustus and O. confusus. Two
population-specific and one sex-linked fragments were identified by the RAPD
primers, OPQ 14, NO11 and OPA08, respectively.
DOI: 10.1556/ABiol.53.2002.3.8
2437. J Dairy Sci. 2002 Sep;85(9):2396-402.
The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed
ratio to lactating dairy cows.
Kung L Jr(1), Cohen MA, Rode LM, Treacher RJ.
Author information:
(1)Delaware Agricultural Experiment Station, College of Agriculture & Natural
Resources, University of Delaware, Newark 19717-1303, USA. lkung@udel.edu
We investigated the effect of spraying different combinations of fibrolytic

enzymes onto forages on their nutritive value for lactating cows. Holstein cows
were fed a TMR consisting of 30% corn silage, 15% alfalfa hay, and 55%
concentrate (dry matter basis). During a 12-wk treatment period, the forages were
treated with no enzymes (control), cellulase D and sultanas B, or cellulase D and
xylanase C. Enzymes were diluted in water and sprayed onto the forages while
mixing. Both combinations of enzymes supplied similar amounts of fibrolytic
activity based on classical enzyme assays conducted at 50 degrees C. Cows fed
forages treated with cellulase D and xylanase B tended to produce more 3.5% FCM
(+2.5 kg/d) than did cows fed the untreated forages. Dry matter intake, milk
production, milk fat, and milk protein were unaffected by treatment. In vitro
production of gas from forages treated with enzymes was greater than from
untreated forage, but 96-h volatile fatty acid production was not different among
treatments. With an alternative enzyme assay based on the depolymerization of
dyed substrate at 40 degrees C, activity of xylanase C was greatest at a pH of
6.5 but was substantially reduced as the pH of the assay was decreased. In
contrast, xylanase B showed highest activity at pH 5 and enzyme activity was
twice that of xylanase B at pH 5.5 and 6. Overall, the results of this study
provide more evidence that fibrolytic enzymes can be used to improve milk
production in lactating cows.
DOI: 10.3168/jds.S0022-0302(02)74321-X
2438. J Dairy Sci. 2002 Sep;85(9):2266-76.
Fatty acid profiles of milk and rumen digesta from cows fed fish oil, extruded
soybeans or their blend.
AbuGhazaleh AA(1), Schingoethe DJ, Hippen AR, Kalscheur KF, Whitlock LA.
Author information:
USA.
Four fistulated primiparous cows (two Holstein and two Brown Swiss) averaging 102
DIM were used in a 4 x 4 Latin square with 3-wk periods to determine the effect
of feeding fish oil, extruded soybeans, or their combination on fatty acid
profiles of milk and rumen digesta. Experimental diets consisted of: 1) control
diet; 2) a diet with 2% (DM basis) added fat from menhaden fish oil; 3) a diet
with 2% added fat from extruded soybeans; and 4) a diet with 1% added fat from
fish oil and 1% fat from extruded soybeans. All diets consisted of 25% corn
silage, 25% alfalfa hay, and 50% concentrate. Milk yields (28.6, 29.7, 29.2, and
28.1 kg/d for control, fish oil, extruded soybeans, and combination diets,
respectively) were similar for all fat supplements and control. Milk fat and
protein percentages (3.49, 3.08; 3.25, 2.96; 3.47, 3.01; 3.48, 2.99 for diets 1,
2, 3, and 4, respectively) were not affected by fat supplements compared with
control. Dry matter intake (23.0, 21.6, 22.7, and 21.6 kg/d) was reduced when
diets containing fish oil were fed. Concentrations of conjugated linoleic acid
[CLA; cis-9, trans-11 CLA, 0.40, 0.88, 0.87, and 0.80 g/100 g fatty acids (FA)]
and transvaccenic acid (TVA, 1.02, 2.34, 2.41, and 2.06 g/100 g of FA) were
increased in milk fat by all fat supplements, with no differences in milk CLA and
TVA observed among fat supplements. As with milk fat, proportions of ruminal CLA
(0.09, 0.26, 0.18, and 0.21 g/100 g of FA) and TVA (2.61, 4.56, 4.61, and 4.39
g/100 g of FA) increased with fat supplements. The effects of fat supplements on
ruminal TVA and CLA concentrations were also reflected in rumen FA-salts, free
fatty acids, and neutral lipids. The higher TVA to CLA ratio in the rumen
compared with milk indicated that fat supplements increased milk CLA
concentration mainly by increasing ruminal production of TVA, which also implied
the significant role that mammary delta-9 desaturase plays in milk CLA
concentrations.
DOI: 10.3168/jds.S0022-0302(02)74306-3
2439. Proteomics. 2002 Sep;2(9):1288-303.
Evaluation of proteome reference maps for cross-species identification of

proteins by peptide mass fingerprinting.
Mathesius U(1), Imin N, Chen H, Djordjevic MA, Weinman JJ, Natera SH, Morris AC,
Kerim T, Paul S, Menzel C, Weiller GF, Rolfe BG.
Author information:
(1)Genomic Interactions Group, Research School of Biological Sciences, Australian
National University, Canberra, Australia.
We tested whether proteome reference maps established for one species can be used
for cross-species protein identification by comparing two-dimensional protein gel
patterns and protein identification data of two closely related bacterial strains
and four plant species. First, proteome profiles of two strains of the fully
sequenced bacterium Sinorhizobium meliloti were compared as an example of close
relatedness, high reproducibility and sequence availability. Secondly, the
proteome profiles of three legumes (Medicago truncatula, Melilotus alba and
Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to
test cross-species similarities. In general, we found stronger similarities in
gel patterns of the arrayed proteins between the two bacterial strains and
between the plant species than could be expected from the sequence similarities.
However, protein identity could not be concluded from their gel position, not
even when comparing strains of the same species. Surprisingly, in the bacterial
strains peptide mass fingerprinting was more reliable for species-specific
protein identification than N-terminal sequencing. While peptide masses were
found to be unreliable for cross-species protein identification, we present
useful criteria to determine confident matching against species-specific
expressed sequence tag databases. In conclusion, we present evidence that
cautions the use of proteome reference maps and peptide mass fingerprinting for
cross-species protein identification.
DOI: 10.1002/1615-9861(200209)2:9<1288::AID-PROT1288>3.0.CO;2-H
2440. Arch Tierernahr. 2001;55(4):351-69.
Effect of starch application into the proximal duodenum of ruminants on starch

digestibility in the small and total intestine.
Matthé A(1), Lebzien P, Hric I, Flachowsky G, Sommer A.
Author information:
(1)Institute of Animal Nutrition, Federal Agricultural Research Centre,
Bundesallee 50, D-38116 Braunschweig, Germany.
Four Slovakian Black-and-white bulls (LW 410 +/- 12 kg; Exp. 1) and four
Slovakian Black-and-white non lactating dairy cows (LW 475 +/- 14 kg; Exp. 2)
with permanent ruminal cannulas, duodenal T-cannulas and ileal re-entrant
cannulas were used in a 4 x 4 Latin square design to determine the postruminal
capacity of starch digestion. In Exp. 1 bulls received 5.4 kg DM from corn silage
and 3.6 kg DM from alfalfa hay, in Exp. 2 cows consumed only 2.1 kg DM corn
silage and 1.9 kg DM alfalfa hay. Additionally, either 750 or 1500 g (Exp. 1) or
resp. 1000 or 2000 g (Exp. 2) gelatinized corn or wheat starch per animal and day
were applied as pulse doses or as infusion into the proximal duodenum. In both
experiments the duodenal and ileal nutrient flow, as well as the faecal excretion
without starch application, were measured in a pre-period. After starting starch
application ileal digesta and faeces were sampled over 120 h after 9 or 23 days
of adaptation respectively. Cr2O3 was used as a flow marker. It was shown, that
the capacity of starch utilisation in the small intestine was limited. The effect
of different doses of bypass-starch was more pronounced than the effect of
different starch sources. Starch digestibility decreased with increasing amounts
of starch in the intestine (Exp. 1: corn starch: from 74.3 to 68.0%, P < 0.001;
wheat starch: from 76.7 to 67.4%, P < 0.001; Exp. 2: corn starch: from 71.4 to
50.3%. P < 0.001; wheat starch: from 73.8 to 53.1%, P < 0.001). Corn starch was
0.6 to 2.4% units (P < 0.05) and 2.4 to 2.8% units (P < 0.001) less digested than
wheat starch in Exp. 1 and Exp. 2, respectively.
Differences in attachment of Salmonella enterica serovars and Escherichia coli

O157:H7 to alfalfa sprouts.
Barak JD(1), Whitehand LC, Charkowski AO.
Author information:
(1)Produce Safety and Microbiological Research, Western Regional Research Center,
USDA Agricultural Research Service, Albany, California 94710, USA.
jbarak@pw.usda.gov
Numerous Salmonella enterica and Escherichia coli O157:H7 outbreaks have been
associated with contaminated sprouts. We examined how S. enterica serovars, E.
coli serotypes, and nonpathogenic bacteria isolated from alfalfa sprouts grow on
and adhere to alfalfa sprouts. Growth on and adherence to sprouts were not
significantly different among different serovars of S. enterica, but all S.
enterica serovars grew on and adhered to alfalfa sprouts significantly better
than E. coli O157:H7. E. coli O157:H7 was essentially rinsed from alfalfa sprouts
with repeated washing steps, while 1 to 2 log CFU of S. enterica remained
attached per sprout. S. enterica Newport adhered to 3-day-old sprouts as well as
Pantoea agglomerans and 10-fold more than Pseudomonas putida and Rahnella
aquatilis, whereas the growth rates of all four strains throughout seed sprouting
were similar. S. enterica Newport and plant-associated bacteria adhered 10- to
1,000-fold more than E. coli O157:H7; however, three of four other E. coli
serotypes, isolated from cabbage roots exposed to sewage water following a spill,
adhered to sprouts better than E. coli O157:H7 and as well as the Pseudomonas and
Rahnella strains. Therefore, attachment to alfalfa sprouts among E. coli
serotypes is variable, and nonpathogenic strains of E. coli to be used as
surrogates for the study of pathogenic E. coli may be difficult to identify and
should be selected carefully, with knowledge of the biology being examined.
PMCID: PMC126431
2442. J Bacteriol. 2002 Oct;184(20):5686-95.
Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production

of novel N-acyl homoserine lactones.
Marketon MM(1), Gronquist MR, Eberhard A, González JE.
Author information:
Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing

symbiosis with the legume Medicago sativa. Recent work has identified a pair of
genes, sinR and sinI, which represent a potential quorum-sensing system and are
responsible for the production of N-acyl homoserine lactones (AHLs) in two S.
meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus
and show that these genes are responsible for the synthesis of several long-chain
AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl
HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel
structures. This is the first report of AHLs having acyl chains longer than 14
carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR
disruption results in only basal levels of the AHLs. Moreover, the same sinI and
sinR mutations also lead to a decrease in the number of pink nodules during
nodulation assays, as well as a slight delay in the appearance of pink nodules,
indicating a role for quorum sensing in symbiosis. We also show that sinI and
sinR mutants are still capable of producing several short-chain AHLs, one of
which was identified as octanoyl HL. We believe that these short-chain AHLs are
evidence of a second quorum-sensing system in Rm1021, which we refer to here as
the mel system, for "S. meliloti."
PMCID: PMC139616
2443. J Bacteriol. 2002 Oct;184(20):5625-32.
Deficiency of a Sinorhizobium meliloti BacA mutant in alfalfa symbiosis

correlates with alteration of the cell envelope.
Ferguson GP(1), Roop RM 2nd, Walker GC.
Author information:
(1)Biology Department, Massachusetts Institute of Technology, 77 Massachusetts
Avenue, Cambridge, Massachusetts 02139, USA.
The BacA protein is essential for the long-term survival of Sinorhizobium

meliloti and Brucella abortus within acidic compartments in plant and animal
cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have
an increased resistance to bleomycin, it was hypothesized that BacA was a
transporter of bleomycin and bleomycin-like compounds into the bacterial cell.
However, our finding that the S. meliloti bacA mutant also has an increased
sensitivity to detergents, a hydrophobic dye, ethanol, and acid pH supported a
model in which BacA function affects the bacterial cell envelope. In addition, an
S. meliloti lpsB mutant that is defective at a stage in infection of the host
similar to that found for a bacA mutant is also sensitive to the same agents, and
the carbohydrate content of its lipopolysaccharide (LPS) is altered. However,
analysis of crude preparations of the bacA mutant LPS suggested that, unlike that
for LpsB, BacA function did not affect the carbohydrate composition of the LPS.
Rather, we found that at least one function of BacA is to affect the distribution
of LPS fatty acids, including a very-long-chain fatty acid thought to be unique
to the alpha-proteobacteria, including B. abortus.
PMCID: PMC139620
2444. Planta. 2002 Sep;215(5):735-44. Epub 2002 Jul 3.
Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot.
Tanaka R(1), Ikeda M, Funatsuki K, Yukioka H, Hashimoto Y, Fujimoto S, Takata M,

Katoh K, Konno H.
Author information:
(1)Aburahi Laboratories, Shionogi & Co., Ltd., Gotanda, Koka, Shiga, Japan.
reiji.tanaka@shionogi.co.jp
Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus

of the polygalacturonic acid in pectic molecules, releasing galacturonic acid. We
cloned the cDNA of exo-PGase purified from cell homogenates of
suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells. The nucleotide
sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a
391-amino-acid polypeptide. Sequence homology research showed 97.9% identity to
the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to
the ENOD8 gene product of alfalfa ( Medicago sativa). However, no significant
similarity was found to known PGases. The Southern hybridization pattern
indicated that this exo-PGase protein is a member of a small-sized gene family.
Predominant expression of the exo-PGase gene was detected by in situ
hybridization and immunohistochemistry in the root apical meristem and in the
elongation region, but not in the root cap. A cross-immunoresponse with
anti-exo-PGase also occurred in the root nodule meristem of alfalfa. These
results suggest that this exo-PGase plays a role in the degradation of pectic
molecules during root development.
DOI: 10.1007/s00425-002-0807-4
Glycine-rich proteins encoded by a nodule-specific gene family are implicated in

different stages of symbiotic nodule development in Medicago spp.
Kevei Z(1), Vinardell JM, Kiss GB, Kondorosi A, Kondorosi E.
Author information:
(1)Institut des Sciences du Végétal, CNRS UPR 2355, Gif-sur-Yvette, France.
Four genes encoding small proteins with significantly high glycine content have
been identified from root nodules of Medicago sativa. All of these proteins as
well as their Medicago truncatula homologues carried an amino terminal signal
peptide and a glycine-rich carboxy terminal domain. All except nodGRP3 lacked the
characteristic repeat structure described for cell wall and stress
response-related glycine-rich proteins (GRP). Expression of these GRP genes was
undetectable in flower, leaf, stem, and hypocotyl cells, whereas expression was
highly induced during root nodule development, suggesting that GRP genes act as
nodulins. Moreover, none of these nodule-expressed GRP genes were activated by
hormones or stress treatments, which are inducers of many other GRPs. In
Rhizobium-free spontaneous nodules and in nodules induced by a noninfective
mutant strain of Sinorhizobium meliloti, all these genes were repressed, while
they were induced in Fix- nodules, unaffected in bacterial infection, but halted
in bacteroid differentiation. These results demonstrated that bacterial infection
but not bacteroid differentiation is required for the induction of the
nodule-specific GRP genes. Differences in kinetics and localization of gene
activation as well as in the primary structure of proteins suggest nonredundant
roles for these GRPs in nodule organogenesis.
DOI: 10.1094/MPMI.2002.15.9.922
2446. EMBO J. 2002 Sep 16;21(18):4831-40.
Induction of COX-2 by LPS in macrophages is regulated by Tpl2-dependent CREB

activation signals.
Eliopoulos AG(1), Dumitru CD, Wang CC, Cho J, Tsichlis PN.
Author information:
(1)Kimmel Cancer Center, Department of Microbiology and Immunology, Thomas
Jefferson University, Philadelphia, PA 19107, USA. ptsichlis@lifespan.org
Macrophage activation by bacterial lipopolysaccharide (LPS) promotes the

secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha
(TNF-alpha) and interleukin-1beta (IL-1beta), and of secondary mediators, such as
leukotrienes and prostaglandins (PGs). Mice lacking the gene encoding the
serine/threonine protein kinase Tpl2/Cot produce low levels of TNF-alpha in
response to LPS because of an ERK-dependent post-transcriptional defect, and they
are resistant to LPS/D-galactosamine-induced endotoxin shock. In this study we
demonstrate that prostaglandin E2 and its regulatory enzyme, COX-2, are also
targets of Tpl2-transduced LPS signals in bone marrow-derived mouse macrophages.
Thus, LPS-stimulated Tpl2(-/-) macrophages express low levels of COX-2 and PGE2,
compared with wild-type Tpl2(+/+) cells. The ability of Tpl2 to regulate COX-2
expression depends on ERK signals that activate p90Rsk and Msk1, which in turn
phosphorylate CREB, a key regulator of COX-2 transcription. These data identify
physiological targets of Tpl2 signaling downstream of ERK and further implicate
Tpl2 in the pathophysiology of inflammation.
PMCID: PMC126277
2447. Eur J Biochem. 2002 Sep;269(18):4656-65.
Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase, a

xanthophyll cycle enzyme, is regulated by membrane fluidity in model lipid
bilayers.
Latowski D(1), Kruk J, Burda K, Skrzynecka-Jaskier M, Kostecka-Gugała A, Strzałka
K.
Author information:
(1)Department of Plant Physiology and Biochemistry, The Jan Zurzycki Institute of
Molecular Biology and Biotechnology, Jagiellonian University, Kraków, Poland.
This paper describes violaxanthin de-epoxidation in model lipid bilayers.

Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with
monogalactosyldiacylglycerol were found to be a suitable system for studying this
reaction. Such a system resembles more the native thylakoid membrane and offers
better possibilities for studying kinetics and factors controlling de-epoxidation
of violaxanthin than a system composed only ofmonogalactosyldiacylglycerol and is
commonly used in xanthophyll cycle studies. The activity of violaxanthin
de-epoxidase (VDE) strongly depended on the ratio of monogalactosyldiacylglycerol
to PtdCho in liposomes. The mathematical model of violaxanthin de-epoxidation was
applied to calculate the probability of violaxanthin to zeaxanthin conversion at
different phases of de-epoxidation reactions. Measurements of deepoxidation rate
and EPR-spin label study at different temperatures revealed that dynamic
properties of the membrane are important factors that might control conversion of
violaxanthin to antheraxanthin. A model of the molecular mechanism of
violaxanthin de-epoxidation where the reversed hexagonal structures (mainly
created by monogalactosyldiacylglycerol) are assumed to be required for
violaxanthin conversion to zeaxanthin is proposed. The presence of
monogalactosyldiacylglycerol reversed hexagonal phase was detected in the
PtdCho/monogalactosyldiacylglycerol liposomes membrane by 31P-NMR studies. The
availability of violaxanthin for de-epoxidation is a diffusion-dependent process
controlled by membrane fluidity. The significance of the presented results for
understanding themechanism of violaxanthin de-epoxidation in native thylakoid
membranes is discussed.
2448. Plant Physiol. 2002 Sep;130(1):415-21.
Short-term boron deprivation inhibits endocytosis of cell wall pectins in

meristematic cells of maize and wheat root apices.
Yu Q(1), Hlavacka A, Matoh T, Volkmann D, Menzel D, Goldbach HE, Baluska F.
Author information:
(1)Institute of Botany, Rheinische Friedrich-Wilhelms-University of Bonn,
Kirschallee 1, D-53115 Bonn, Germany.
By using immunofluorescence microscopy, we observed rapidly altered distribution

patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat
(Triticum aestivum) root apices. This response was shown for homogalacturonan
pectins characterized by a low level (up to 40%) of methylesterification and for
rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron
deprivation, abundance of these pectins rapidly increased in cell walls, whereas
their internalization was inhibited, as evidenced by a reduced and even blocked
accumulation of these cell wall pectins within brefeldin A-induced compartments.
In contrast, root cells of species sensitive to the boron deprivation, like
zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell
wall pectins into brefeldin A compartments and do not show accumulation of
pectins in their cell walls under boron deprivation. For maize and wheat root
apices, we favor an apoplastic target for the primary action of boron
deprivation, which signals deeper into the cell via endocytosis-mediated pectin
signaling along putative cell wall-plasma membrane-cytoskeleton continuum.
DOI: 10.1104/pp.006163
PMCID: PMC166573
Alfalfa weevil (Coleoptera: Curculionidae) larval sampling: comparison of

shake-bucket and sweep-net methods and effect of training.
Hoff KM(1), Brewer MJ, Blodgett SL.
Author information:
(1)Department of Renewable Resources, Entomology, University of Wyoming, Laramie
82071, USA.
Two field-sampling methods, shake-bucket and sweep-net, were compared for use in
monitoring alfalfa weevil, Hypera postica (Gyllenhal), larvae in alfalfa hay of
the high plains and intermountain region of North America. In this region,
alfalfa grows to sufficient height to use both methods before the more damaging
late instars peak in abundance. Both methods also were compared with extracting
larvae by using Berlese funnels in the laboratory. The shake-bucket method was
more sensitive in detecting small larvae (first and second instars) than large
larvae (third and fourth instars), and the sweep-net method detected a lower
proportion of small larvae. The number of larvae collected with the shake-bucket
method was strongly correlated with number of larvae recovered from Berlese
funnels (total larval counts, R2 = 0.85). Correlation of the sweep-net samples
with the Berlese extraction was also significant but less strong (R2 = 0.56). In
addition, sampler instruction was evaluated to determine whether demonstration
training affects performance of inexperienced samplers using the two
field-sampling methods. Training did not significantly change sampler performance
in using the shake-bucket but did increase the number of large larvae when using
the sweep-net. In addition, less variability was associated with the shake-bucket
sampling method than with the sweep-net method for samplers who only had access
to written sampling instructions. Therefore, when estimation of small larval
abundance is desired for economic decision-making and sampling is performed by
people with little or infrequent sampling experience, such as growers, the
shake-bucket method is the preferred field-sampling method in the high plains and
intermountain region of North America Sweep-net sampling is more variable than
shake-bucket sampling, although demonstration training improves the usefulness of
the sweep-net.
2450. J Dairy Sci. 2002 Aug;85(8):1988-99.
Whole linted cottonseed as a forage substitute: fiber effectiveness and digestion

kinetics.
Harvatine DI(1), Winkler JE, Devant-Guille M, Firkins JL, St-Pierre NR, Oldick
BS, Eastridge ML.
Author information:
USA.
Six ruminally and duodenally cannulated Holstein cows were used in a 6 x 6 Latin
square design to 1) evaluate the potential interaction in effectiveness of
neutral detergent fiber (NDF) from whole cottonseed (WCS) when it was substituted
for forage NDF (FNDF) and fed with ground (G) or steam-flaked (SF) corn and 2) to
determine whether the kinetic properties of NDF digestion further clarify the
effectiveness of WCS. The six dietary treatments were: forage control with G corn
(21% FNDF), 5% WCS with G or SF corn (18% FNDF), 10% WCS with G or SF corn (15%
FNDF), and 15% WCS with G corn (12% FNDF). Based on chewing activity, the NDF
from WCS was estimated to be 84% (SE = 36%) as effective as alfalfa silage NDF.
Decreasing passage and digestion rates of potentially digestible NDF with
increasing WCS increased the evacuated pool size of ruminal DM, apparently
explaining the similar ruminal mat consistency among treatments. Measures of
effectiveness ofWCS treatments did not interact with corn source. Fluid dilution
rate was estimated based on a two-compartment model describing Co dilution, but
no treatment differences were detected. There was a strong linear bias for
estimates of ruminal NDF digestibility based on a single compartment model using
the digestion rate of potentially digestible NDF and the passage rate of either
indigestible NDF or digestible NDF when compared with NDF digestibility
calculated using duodenal flows. Although further verification is needed, these
digestion and passage kinetics help explain why WCS are effective at stimulating
chewing during eating and rumination.
DOI: 10.3168/jds.S0022-0302(02)74275-6
2451. J Dairy Sci. 2002 Aug;85(8):1958-68.
Effects of particle size of alfalfa-based dairy cow diets on site and extent of
digestion.
Yang WZ(1), Beauchemin KA, Rode LM.
Author information:
(1)Livestock Sciences Section, Research Centre Agriculture and Agri-Food Canada,
Lethbridge, AB.
Effects of ratio of alfalfa silage to alfalfa hay and forage particle size on
nutrient intakes, site of digestion, rumen pools, and passage rate of ruminal
contents were evaluated in a 4 x 4 Latin square design with a 2 x 2 factorial
arrangement of treatments. The diets consisted of 60% barley-based concentrate
and 40% forage made up either of 50:50 or 25:75 of alfalfa silage:alfalfa hay and
alfalfa hay was either chopped or ground. Lactating dairy cows surgically fitted
with ruminal and duodenal cannulas were used and offered ad libitum access to a
total mixed ration. Intakes of nutrients were increased by increasing ratio of
silage to hay but were not affected by particle size of forage. Change in ratio
of silage to hay of diets did not affect site and extent of digestion. However,
increased forage particle size of the diets improved digestibility of fiber and N
in the total tract, and as well as digestibility of organic matter, starch, and
acid detergent fiber in the intestine. There was a shift of starch digestion from
the rumen to the intestine when forage particle size was increased, although
total digestion of starch was not changed. Ruminal microbial protein synthesis
and microbial efficiency also improved with increasing forage particle size. Cows
fed ground hay versus chopped hay had significantly lower rumen wet mass
regardless of the ratio of silage to hay. Reduced forage particle size also
lowered ruminal nutrient pool size for cows fed the high silage diet. Ruminal
passage rates of liquid and solid were decreased by reducing the ratio of silage
to hay, and retention time of solids in the total tract was shortened by reducing
forage particle size. These results indicate that manipulating ratio of silage to
hay in the diets of dairy cows changed feed intake but had little effect on
digestion. In contrast, increased forage particle size in dairy cow diets
improved fiber digestion and microbial protein synthesis in the rumen, and
shifted starch digestion from the rumen to the intestine. Dietary particle size,
expressed as physically effective neutral detergent fiber, was a reliable
indication of ruminal microbial protein synthesis and nutrient digestion.
DOI: 10.3168/jds.S0022-0302(02)74272-0
2452. J Dairy Sci. 2002 Aug;85(8):1947-57.
Effects of forage particle size and grain fermentability in midlactation cows.

II. Ruminal pH and chewing activity.
Author information:
Our study investigated the effects of, and interactions between, level of dietary
ruminally fermentable carbohydrate (RFC) and forage particle size on rumen pH and
chewing activity for dairy cows fed one level of dietary NDF. Also, correlations
between intake, production, chewing, and ruminal pH parameters were investigated.
Eight cows (61 days in milk) were assigned to four treatments in a double 4 x 4
Latin square. Treatments were arranged in a 2 x 2 factorial design; finely
chopped alfalfa silage (FS) and coarse alfalfa silage (CS) were combined with
concentrates based on either dry, cracked-shelled corn (DC; low RFC) or ground,
high-moisture corn (HMC; high RFC). Diets were fed ad libitum as a total mixed
rations with a concentrate:forage ratio of 60:40. Diets averaged 18.7% crude
protein, 24.0% neutral detergent fiber, 18.3% , acid detergent fiber and 27.4%
starch on a DM basis. Mean particle size of the four diets were 6.3, 2.8, 6.0,
and 3.0 mm for DCCS, DCFS, HMCCS, and HMCFS, respectively. Decreasing forage
particle size decreased ruminal pH from 6.02 to 5.81, and increasing level of RFC
decreased pH from 5.99 to 5.85. Minimum daily ruminal pH decreased from 5.66 to
5.47 when level of RFC was increased, and decreased from 5.65 to 5.48 when forage
particle size decreased. Time below pH 5.8 per day increased from 7.4 h to 10.8 h
when level of RFC increased, and increased from 6.4 h to 11.8 h when forage
particle size was decreased. Area below 5.8 showed the same relationship with RFC
and forage particle size. Also, forage particle size affected the postprandial pH
pattern. Cows spent more time eating when fed CS compared with FS (274 vs. 237
min/d), and time spent eating decreased when level of RFC was increased (271 vs.
241 min/d). Decreasing forage particle size decreased time spent ruminating (485
vs. 320 min/d), rumination periods (15.3 vs. 11.7), and duration of rumination
periods (29 vs. 26 min). Increasing level of RFC increased time spent ruminating
per kg NDF intake (68.5 vs. 79.5 min/kg). Milk fat percentage was correlated to
mean ruminal pH (r = 0.41), time spent below pH 5.8 (r = -0.55), and area below
5.8 (r = -0.57), but not to intake or chewing variables. DMI of particles
retained on a screen equivalent in size to the top screen of the Penn State
particle separator was the intake parameter explaining most of the variation in
mean ruminal pH (r = 0.27) and was correlated to time spent ruminating (r = 0.61)
and chewing (r = 0.61).
DOI: 10.3168/jds.S0022-0302(02)74271-9
2453. J Dairy Sci. 2002 Aug;85(8):1936-46.
Effects of forage particle size and grain fermentability in midlactation cows. I.

Milk production and diet digestibility.
Author information:
Our study investigated the effects of, and interactions between, level of dietary
ruminally fermentable carbohydrate (RFC) and forage particle size on milk
production, nutrient digestibility, and microbial protein yield for dairy cows
fed one level of dietary NDF. Eight cows (61 days in milk) were assigned to four
treatments in a double 4 x 4 Latin square. Treatments were arranged in a 2 x 2
factorial design; finely chopped alfalfa silage (FS) and coarse alfalfa silage
(CS) were combined with concentrates based on either dry cracked shelled corn
(DC; low RFC) or ground high-moisture corn (HMC; high RFC). Diets were fed ad
libitum as a total mixed rations with a concentrate to forage ratio of 61:39.
Diets based on DC had a predicted NEL content of 1.73 Mcallkg dry matter (DM),
while HMC diets contained 1.80 Mcal/kg DM. Diets averaged 18.7% CP, 24.0% NDF,
18.3% ADF, and 27.4% starch on a DM basis. Mean particle size of the four diets
was 6.3, 2.8, 6.0, and 3.0 mm for DCCS, DCFS, HMCCS, and HMCFS, respectively.
Increasing level of RFC decreased dry matter intake (DMI) from 25.0 to 23.8 kg/ d
and organic matter intake from 22.3 to 21.1 kg/d, but intake was not affected by
particle size. Milk production averaged 44.0 and 26.8 kg/d solids corrected milk
(SCM) and was not affected by diet, but increasing level of RFC tended to
increase milk yield. Efficiency of milk production, expressed as SCM/DMI,
increased from 1.06 to 1.14 when level of RFC was increased. Milk composition or
yield of milk components was not affected by diet, and averaged 3.53% fat, 3.11%
protein, 1.55 kg/d fat, and 1.36 kg/d protein. Total tract digestibility of DM
and OM increased from 71.4 to 73.0% and 72.4 to 76.1% for DM and OM,
respectively, when level of RFC was increased. Total tract digestibility of fiber
was unaffected by diet, but total tract starch digestibility increased from 93.1
to 97.4% when HMC replaced DC. Total urinary excretion of the purine derivatives
uric acid and allantoin increased from 415 to 472 mmol/d when level of RFC was
increased, and calculated microbial N supply increased from 315 to 365 g/d. When
expressed as per kilogram of digestible OMI, increasing level of RFC tended to
increase microbial N supply (20.4 vs. 22.2 g/kg). Cow productivity was not
affected by forage particle size and ruminally fermentable carbohydrates in this
study.
DOI: 10.3168/jds.S0022-0302(02)74270-7
2454. Plant J. 2002 Sep;31(5):629-38.
Opposite changes in membrane fluidity mimic cold and heat stress activation of
distinct plant MAP kinase pathways.
Sangwan V(1), Orvar BL, Beyerly J, Hirt H, Dhindsa RS.
Author information:
(1)Department of Biology, McGill University, 1205 Avenue Docteur Penfield,
Montreal, Quebec, H3A 1B1, Canada.
Mitogen-activated protein kinases (MAPKs) appear to be ubiquitously involved in
signal transduction during eukaryotic responses to extracellular stimuli. In
plants, no heat shock-activated MAPK has so far been reported. Also, whereas cold
activates specific plant MAPKs such as alfalfa SAMK, mechanisms of such
activation are unknown. Here, we report a heat shock-activated MAPK (HAMK)
immunologically related to ERK (Extracellular signal-Regulated Kinase)
superfamily of protein kinases. Molecular mechanisms of heat-activation of HAMK
and cold-activation of SAMK were investigated. We show that cold-activation of
SAMK requires membrane rigidification, whereas heat-activation of HAMK occurs
through membrane fluidization. The temperature stress- and membrane
structure-dependent activation of both SAMK and HAMK is mimicked at 25 degrees C
by destabilizers of microfilaments and microtubules, latrunculin B and oryzalin,
respectively; but is blocked by jasplakinolide, a stabilizer of actin
microfilaments. Activation of SAMK or HAMK by temperature, chemically modulated
membrane fluidity, or by cytoskeleton destabilizers is inhibited by blocking the
influx of extracellular calcium. Activation of SAMK or HAMK is also prevented by
an antagonist of calcium-dependent protein kinases (CDPKs). In summary, our data
indicate that cold and heat are sensed by structural changes in the plasma
membrane that translates the signal via cytoskeleton, Ca2+ fluxes and CDPKs into
the activation of distinct MAPK cascades.
2455. Plant J. 2002 Sep;31(5):615-27.
Genotype-dependent transcriptional activation of novel repetitive elements during

cold acclimation of alfalfa (Medicago sativa).
Ivashuta S(1), Naumkina M, Gau M, Uchiyama K, Isobe S, Mizukami Y, Shimamoto Y.
Author information:
(1)Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo
060-8589, Japan. sergey@affrc.go.jp
In a search for cold-regulated genes that are differentially expressed in alfalfa

genotypes of contrasting freezing tolerance, we screened 1036 arrayed cDNA
clones. The screening resulted in isolation of cDNA clones, which demonstrated
dramatic differences in expression between hardy and un-hardy alfalfa varieties.
Detailed analysis revealed that these cDNAs represent parts of novel non-coding
repetitive elements carrying long-terminal repeats (LTR) and other
retroelement-like features. Despite strong expression under low temperatures, DNA
templates remained highly methylated, and a drug-induced decrease in methylation
did not activate transcription under normal temperatures. We identified that
these repetitive elements represent a large family and could insert into, or be
adjacent to, the unrelated polyprotein sequences of putative retrotransposons.
These retrotransposons also showed low temperature-induced transcriptional
activation; however, this activation was not genotype-dependent. The
retroelements described in this study are the first retroelement characterized in
the Medicago genus. Furthermore, they represent the only known example of
genotype-specific cold-induced transcriptional activation of multiple copies of a
repetitive element whose expression is associated with a genotype difference in
cold acclimation.

2456. J Dairy Sci. 2002 Jul;85(7):1894-901.
Ryegrass or alfalfa silage as the dietary forage for lactating dairy cows.
Broderick GA(1), Koegel RG, Walgenbach RP, Kraus TJ.
Author information:
(1)Agricultural Research Service, USDA US Dairy Forage Research Center, Madison
53706, USA. glenb@dfrc.wisc.edu
Renewed interest exists in using grass forages to dilute the higher crude protein
(CP) and lower digestible fiber present in legumes fed to lactating dairy cows. A
3 x 3 Latin square feeding study with 4-wk periods was conducted with 24 Holstein
cows to compare ryegrass silage, either untreated control or macerated
(intensively conditioned) before ensiling, with alfalfa silage as the sole
dietary forage. Ryegrass silages averaged [dry matter (DM) basis] 18.4% CP, 50%
neutral detergent fiber (NDF), and 10% indigestible acid detergent fiber (ADF)
(control) and 16.6% CP, 51% NDF, and 12% indigestible ADF (macerated). Alfalfa
silage was higher in CP (21.6%) and lower in NDF (44%) but higher in indigestible
ADF (26%). A lower proportion of the total N in macerated ryegrass silage was
present as nonprotein N than in control ryegrass and alfalfa silages. Diets were
formulated to contain 41% DM from either rye-grass silage, or 51% DM from alfalfa
silage, plus high moisture corn, and protein concentrates. Diets averaged 17.5%
CP and 28 to 29% NDF. The shortfall in CP on ryegrass was made up by feeding 7.6%
more soybean meal. Intake and milk yields were similar on control and macerated
ryegrass; however, DM intake was 8.3 kg/d greater on the alfalfa diet. Moreover,
feeding the alfalfa diet increased BW gain (0.48 kg/d) and yield of milk (6.1
kg/d), FCM (6.8 kg/d), fat (0.26 kg/d), protein (0.25 kg/d), lactose (0.35 kg/d),
and SNF (0.65 kg/d) versus the mean of the two ryegrass diets. Both DM efficiency
(milk/DM intake) and N efficiency (milk-N/N-intake) were 27% greater, and
apparent digestibility was 16% greater for DM and 53% greater for NDF and ADF, on
the ryegrass diets. However, apparent digestibility of digestible ADF was greater
on alfalfa (96%) than on ryegrass (average = 91%). Also, dietary energy content
(estimated as net energy of lactation required for maintenance, milk yield, and
weight gain) per unit of digested DM was similar for all three diets. Results of
this trial indicated that, relative to ryegrass silage, feeding alfalfa silage
stimulated much greater feed intake, which supported greater milk production.
DOI: 10.3168/jds.S0022-0302(02)74264-1
2457. J Dairy Sci. 2002 Jul;85(7):1767-76.
Efficacy of carbohydrate sources for milk production by cows fed diets based on
alfalfa silage.
Broderick GA(1), Mertens DR, Simons R.
Author information:
(1)Agricultural Research Service, USDA US Dairy Forage Research Center, Madison,
WI 53706, USA. glenb@dfrc.wisc.edu
The effectiveness of three carbohydrate sources, high-moisture ear corn (HMEC),

cracked shelled corn (CSC), and a 50:50 mixture of HMEC plus dried citrus pulp
(DCP), fed with or without supplemental rumen-undegraded protein as expeller
soybean meal (ESBM), was assessed in 48 multiparous dairy cows. All diets
contained (dry mater [DM] basis) 50% alfalfa silage, 10% ryegrass silage, 28%
NDF, and one of six concentrates: A) 38% HMEC; B) 38% CSC; C) 19% DCP plus 19%
HMEC; D) 27% HMEC plus 12% ESBM; E) 27% CSC plus 12% ESBM; or F) 13% DCP, 13%
HMEC, and 12% ESBM. Diets A, B, and C averaged 19% crude protein, of which 53%
was nonprotein nitrogen (NPN), and diets D, E, and F averaged 22% crude protein,
of which 40% was NPN. Cows were fed a high-energy covariate diet for 2 wk,
blocked into eight groups of six, based on covariate protein yield, then randomly
assigned to diets that were fed for 12 wk. Feeding ESBM increased DM intake,
yields of milk, fat-corrected milk, fat, protein, SNF, and milk and blood urea
concentration and decreased weight loss. There were no production differences
between HMEC and CSC. However, DM intake, yields of milk, fat-corrected milk,
fat, protein, lactose, SNF, and milk SNF content all were lower on the diets
containing DCP versus HMEC and CSC. A 6 x 6 Latin square trial conducted at the
same time with six ruminally cannulated cows showed similar effects of diet on DM
intake and milk production. Ruminal ammonia was elevated by ESBM but not ruminal
total amino acids and branched-chain volatile fatty acids. Ruminal propionate was
highest on HMEC diets and lowest on DCP diets; acetate, butyrate and
acetate-to-propionate ratio were lowest on HMEC diets and highest on DCP diets.
These results indicated that, compared to HMEC and CSC, feeding the pectin-rich
carbohydrate source DCP altered ruminal fermentation but depressed intake and
milk production in lactating cows.
DOI: 10.3168/jds.S0022-0302(02)74251-3
Diversity of Sinorhizobium meliloti from the Central Asian Alfalfa Gene Center.
Roumiantseva ML(1), Andronov EE, Sharypova LA, Dammann-Kalinowski T, Keller M,

Young JP, Simarov BV.
Author information:
(1)Research Institute for Agricultural Microbiology, St.-Petersburg-Pushkin 8,
196608, Russia. genet@yandex.ru
Sinorhizobium meliloti was isolated from nodules and soil from western
Tajikistan, a center of diversity of the host plants (Medicago, Melilotus, and
Trigonella species). There was evidence of recombination, but significant
disequilibrium, between and within the chromosome and megaplasmids. The most
frequent alleles matched those in the published genome sequence.
PMCID: PMC124126
Effect of a Sinorhizobium meliloti strain with a modified putA gene on the

rhizosphere microbial community of alfalfa.
van Dillewijn P(1), Villadas PJ, Toro N.
Author information:
(1)Grupo de Ecología Genética, Estación Experimental del Zaidín, Consejo Superior
de Investigaciones Científicas, 18008 Granada, Spain.
The success of a rhizobial inoculant in the soil depends to a large extent on its
capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti
strain with enhanced competitiveness for nodule occupancy, was recently
constructed by introducing a plasmid containing an extra copy of a modified putA
(proline dehydrogenase) gene. This strain and M401, a control strain carrying the
same plasmid without the modified gene, were used as soil inoculants for alfalfa
in a contained field release experiment at León, Spain. In this study, we
determined the effects of these two strains on the indigenous microbial
community. 16S rRNA genes were obtained from the rhizosphere of alfalfa
inoculated with strain M403 or strain M401 or from noninoculated plants by
amplification of DNA from soil with bacterial group-specific primers. These genes
were analyzed and compared by restriction fragment length polymorphism and
temperature gradient gel electrophoresis. The results allowed us to differentiate
between alterations in the microbial community apparently caused by inoculation
and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa
plants and by the environment. Only moderate inoculation-dependent effects could
be detected, while the alfalfa plants appeared to have a much stronger influence
on the microbial community.
PMCID: PMC124105
2460. J Bacteriol. 2002 Sep;184(18):5067-76.
A LuxR homolog controls production of symbiotically active extracellular

polysaccharide II by Sinorhizobium meliloti.
Pellock BJ(1), Teplitski M, Boinay RP, Bauer WD, Walker GC.
Author information:
Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing

soil bacterium Sinorhizobium meliloti is required for efficient invasion of root
nodules on the host plant alfalfa. Any one of three S. meliloti polysaccharides,
succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and
extension (root nodule invasion) on alfalfa. Of these three polysaccharides, the
only symbiotically active polysaccharide produced by S. meliloti wild-type strain
Rm1021 is succinoglycan. The expR101 mutation is required to turn on production
of symbiotically active forms of EPS II in strain Rm1021. In this study, we have
determined the nature of the expR101 mutation in S. meliloti. The expR101
mutation, a spontaneous dominant mutation, results from precise, reading
frame-restoring excision of an insertion sequence from the coding region of expR,
a gene whose predicted protein product is highly homologous to the Rhizobium
leguminosarum bv. viciae RhiR protein and a number of other homologs of Vibrio
fischeri LuxR that function as receptors for N-acylhomoserine lactones (AHLs) in
quorum-sensing regulation of gene expression. S. meliloti ExpR activates
transcription of genes involved in EPS II production in a density-dependent
fashion, and it does so at much lower cell densities than many quorum-sensing
systems. High-pressure liquid chromatographic fractionation of S. meliloti
culture filtrate extracts revealed at least three peaks with AHL activity, one of
which activated ExpR-dependent expression of the expE operon.
PMCID: PMC135333
2461. J Agric Food Chem. 2002 Aug 28;50(18):5063-9.
Characterization and identification of alfalfa and red clover dietary supplements

using a PCR-based method.
LeRoy A(1), Potter E, Woo HH, Heber D, Hirsch AM.
Author information:
California, Los Angeles, 90095-1606, USA.
The use of herbal remedies is very popular in the United States, with >80 million
people buying plant-derived preparations that are often highly degraded or
potentially contaminated with nonefficacious plant material. A method utilizing
DNA-based markers to identify highly fragmented or powdered plant material sold
as botanicals in dietary supplements has been developed. By incorporating and
streamlining a repair reaction that utilized fill-in and ligation reactions
before the PCR steps, it was possible to amplify highly degraded or sheared DNA
isolated from powdered plant material removed from over-the-counter capsules. The
primers for the internal transcribed spacer (ITS) region of nuclear ribosomal DNA
generate a PCR fragment compatible with the sizes of the repaired DNA. Moreover,
a large data set in Genbank facilitated subsequent analysis. This method is a
relatively rapid and simple system to facilitate the authentication, as well as
the monitoring, of the purity of botanicals in dietary supplements, even those
that are improperly dried or stored.
2462. J Food Prot. 2002 Aug;65(8):1259-66.
Assessment of the potential for Listeria monocytogenes survival and growth during
alfalfa sprout production and use of ionizing radiation as a potential
intervention treatment.
Schoeller NP(1), Ingham SC, Ingham BH.
Author information:
(1)Department of Food Science, University of Wisconsin, Madison 53706, USA.
Alfalfa seeds (Australian, nondormant, nonscarified) were treated with 20,000 ppm
active chlorine, sprouted in canning jars for 5 days, and packaged and stored at
5 degrees C for up to 9 days. Seeds or sprouts were inoculated with a
three-strain cocktail of Listeria monocytogenes at one of three points during the
process-day 0 (before 24-h aqueous seed soak), day 1 (after 24-h aqueous seed
soak), or day 5 (after sprouting, before prepackaging 10 ppm chlorine rinse)--or
control (no inoculum), and the ability of the inoculum to survive and grow was
evaluated. Total bacterial numbers on uninoculated seeds increased dramatically
during the first 24-h the seeds were soaked, from 3.5 to ca. 8.0 log CFU/g, and
remained at this level during refrigerated storage. When the seeds were
inoculated with a cocktail of L. monocytogenes (log 5 CFU/10 ml) on day 0 or 1,
the population of the pathogen increased dramatically, to within 1 to 2 logs of
the total, and remained high during refrigerated storage. When sprouted seeds
were inoculated with L. monocytogenes later in the process (day 5), the inoculum
survived but did not grow more than ca. 1 log CFU/g, regardless of whether the
inoculation level in each jar was low (10(3)) or high (10(5)). Irradiation of
sprouts with beta radiation at 3.3 or 5.3 kGy, but not 1.5 kGy, was effective at
eliminating L. monocytogenes from inoculated sprouts (6 log CFU/g) without
causing noticeable changes in appearance or odor. In summary, L. monocytogenes
can grow on sprouts during production, can survive on refrigerated sprouts, and
may be eliminated on sprouts with beta radiation.
2463. Crit Rev Food Sci Nutr. 2002 Jul;42(4):377-401.
Toward sustainable production of protein-rich foods: appraisal of eight crops for

Western Europe. Part I. Analysis of the primary links of the production chain.
Linnemann AR(1), Dijkstra DS.
Author information:
(1)Product Design and Quality Management Group, Food Technology and Nutritional
Sciences, Wageningen University, The Netherlands.
anita.linnemann@ift.fdsci.wag-ur.nl
Increased production of plant protein is required to support the production of

protein-rich foods that can replace meat in the human diet to reduce the strain
that intensive animal husbandry poses to the environment. The suitability of
lupin (Lupinus spp.), pea (Pisum sativum), quinoa (Chenopodium quinoa Willd.),
triticale (x Triticosecale), lucerne (Medicago sativa), grasses (Lolium and
Festuca spp.), rapeseed/canola (Brassica napus), and potato (Solanum tuberosum)
for protein production in Western Europe was studied on the basis of a chain
approach. The aspects considered are the familiarity of farmers with the
cultivation of the crop, prospects for rapid crop improvement, protein production
(kg/ha), protein quality (absence of unwanted substances) and familiarity with
the usage for human food in Western Europe. Pea, lucerne, and grasses are the
most promising, fair prospects are foreseen for lupin, triticale, rapeseed, and
potato, whereas the possibilities for quinoa are judged to lag far behind.
Estimated protein production for pea, lucerne, and grasses is 1250, 2500, and
2500 kg/ha, respectively.
DOI: 10.1080/20024091054193
2464. Electrophoresis. 2002 Jun;23(11):1642-51.
Profiling isoflavonoids found in legume root extracts using capillary

electrophoresis.
Baggett BR(1), Cooper JD, Hogan ET, Carper J, Paiva NL, Smith JT.
Author information:
(1)Department of Physical Sciences, Southeastern Oklahoma State University, P.O.
Box 4025-Campus, Durant, OK 74701-0609, USA.
Legumes such as alfalfa (Medicago sativa L.), barrel medic (Medicago truncatula),
white sweet clover (Melilotus alba) and fenugreek (Trigonella graecum), normally
accumulate (-)-medicarpin and its malonated glucose conjugate as natural
inhibitors of fungal pathogens. These plants also accumulate the biosynthetic
precursor formononetin as well as the malonated glycoside. We were interested in
developing a robust high-throughput method to quantitate the levels of these two
isoflavonoids, both free and conjugated, in legume root extracts, for use in
screening for mutant plants accumulating altered levels of these compounds.
Capillary electrophoresis was examined as an alternative to current
high-performance liquid chromatography (HPLC) methods to generate isoflavonoid
profiles. The developed assay used micellar electrokinetic capillary
chromatography (MEKC) to provide the required selectivity in complex root
extracts. The addition of 1,2-hexanediol to the sodium dodecyl sulfate (SDS)
electrolyte provided improved resolution of adjacent isoflavonoids. We examined
the role of several factors including sample preparation, buffer composition,
buffer pH, and organic component in the injected sample. The use of capillaries
with longer path lengths were also examined to increase sensitivity. A comparison
of results obtained using MEKC and HPLC showed good correlation in the relative
amounts of the isoflavonoids studied.
DOI: 10.1002/1522-2683(200206)23:11<1642::AID-ELPS1642>3.0.CO;2-Y
2465. Plant Physiol. 2002 Aug;129(4):1807-19.
The Role of auxin, pH, and stress in the activation of embryogenic cell division
in leaf protoplast-derived cells of alfalfa.
Pasternak TP(1), Prinsen E, Ayaydin F, Miskolczi P, Potters G, Asard H, Van

Onckelen HA, Dudits D, Fehér A.
Author information:
(1)Laboratory of Cell Division and Differentiation, Institute of Plant Biology,
Biological Research Centre, H-6701 Szeged, Hungary.
Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago

sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10
microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in
different cell types. Cells exposed to high 2,4-D concentration remain small with
dense cytoplasm and can develop into proembryogenic cell clusters, whereas
protoplasts cultured at low auxin concentration elongate and subsequently die or
form undifferentiated cell colonies. Fe stress applied at nonlethal
concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the
development of the embryogenic cell type. Although cytoplasmic alkalinization was
detected during cell activation of both types, embryogenic cells could be
characterized by earlier cell division, a more alkalic vacuolar pH, and
nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells.
Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM
2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in
nonembryogenic cell-type formation. The level of endogenous indoleacetic acid
(IAA) increased transiently in all protoplast cultures during the first 4 to 5 d,
but an earlier peak of IAA accumulation correlated with the earlier activation of
the division cycle in embryogenic-type cells. However, this IAA peak could also
be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid.
Based on the above data, we propose the involvement of stress responses,
endogenous auxin synthesis, and the establishment of cellular pH gradients in the
formation of the embryogenic cell type.
DOI: 10.1104/pp.000810
PMCID: PMC166769
2466. J Environ Qual. 2002 Jul-Aug;31(4):1339-48.
Growth of alfalfa in sludge-amended soils and inoculated with rhizobia produced

in sludge.
Rebah FB(1), Prévost D, Tyagi RD.
Author information:
(1)Université du Quebec, Institut national de la recherche scientifique
(INRS-Eau), Sainte Fov, QC, Canada.
The efficiency of rhizobial inoculants produced in wastewater sludge used as a

growth medium and as a carrier was compared with that of inoculants produced in
yeast mannitol broth (YMB) medium and by using peat as a carrier. Alfalfa
(Medicago sativa L.) plants were inoculated with solid and liquid Sinorhizobium
meliloti inoculants and grown in pots containing two soil types (Kamouraska clay
soil and Saint-André sandy soil). The effect of various levels of sludge
amendment (60 and 120 kg N/ha) and nitrogen fertilizer (60 kg N/ ha) was also
studied. The sludge-based inoculants showed the same symbiotic efficiency
(nodulation and plant yield) as YMB-based inoculants. The inoculation increased
the nodulation indexes from 4-6 to 8-12, and the rhizobial number from 10(3)
(uninoculated soils) to 10(6)-10(7) cells/g in inoculated soils. However, the
shoot dry weights and the nitrogen contents were not increased significantly by
the inoculation. Applying sludge as an amendment enhanced the rhizobial number in
soils from 10(3) to 10(4) cells/g and improved significantly the plant growth
(shoot dry weights and nitrogen contents). This improvement increased with sludge
rate and with the cut (three cuts). Compared with sludge, N fertilizer gave lower
plant yields. The nodulation was not affected by sludge and N-fertilizer
application. The texture and physico-chemical properties of soil were found to
affect the yield and nitrogen content of the plants. In this study, macroelements
and heavy metals were at acceptable levels and were not considered to be negative
factors.
2467. Environ Pollut. 2002;119(3):291-301.
Effect of mixed cadmium, copper, nickel and zinc at different pHs upon alfalfa
growth and heavy metal uptake.
Peralta-Videa JR(1), Gardea-Torresdey JL, Gomez E, Tiemann KJ, Parsons JG,

Carrillo G.
Author information:
(1)Environmental Science and Engineering PhD Program, University of Texas at El
Paso, 79968, USA.
Alfalfa plants were grown in soil-pots contaminated with a mixture of Cd(II),

Cu(II), Ni(II), and Zn(II), (at 50 mg/kg each) at pHs of 4.5, 5.8, and 7.1. The
plants were fertilized using a nutrient solution, which was adjusted
appropriately to the same pH. Plants in the control treatment were grown in the
absence of the heavy metals mixture. The growth of the control plants was the
same at the three pHs studied and the heavy metal stressed plants also showed
similar behavior at each pHs. There were statistically significant differences
(P<0.05) between the shoot length of the control treatment plants and the length
of plants grown in the presence of the heavy metal mixture. Under the effects of
the heavy metal mixture, nickel was the most accumulated element in the shoot
tissue, with 437, 333, and 308 ppm at pH 7.1, 5.8, and 4.5, respectively. Cadmium
was found to be second in accumulated concentrations with 202 ppm, 124 ppm, and
132 ppm at pH 7.1, 5.8, and 4.5, respectively, while zinc was third, followed by
copper. The maximum relative uptakes (element in plant/element in
soil-water-solution) were found to be 26 times for nickel, 23 times for cadmium,
12 times for zinc. and 6 times for copper. We considered these relations as
indicative of the ability of alfalfa plants to take up elements from a soil
matrix contaminated with a mixture of cadmium, copper, nickel, and zinc.
2468. J Clin Microbiol. 2002 Aug;40(8):3109.
Eventual management of sprout-transmitted salmonellosis.
Struijk CB, Mossel DA.
Comment on
J Clin Microbiol. 2001 Oct;39(10):3461-5.
PMCID: PMC120693
2469. J Exp Bot. 2002 Aug;53(375):1831-2.
Dodder infection induces the expression of a pathogenesis-related gene of the

family PR-10 in alfalfa.
Borsics T(1), Lados M.
Author information:
(1)Agricultural Biotechnology Center, PO Box 411, H-2101 Gödöllõ, Hungary.
borsics@abc.hu
A full-length cDNA, PPRG2, representing a gene highly expressed in dodder

(Cuscuta trifolii Bab et. Gibs)-infected alfalfa (Medicago sativa L.) stems was
isolated by differential screening. The predicted protein contains 157 amino
acids and belongs to the PR-10 family of the pathogenesis-related genes with
putative ribonuclease activities. Northern hybridizations showed that PPRG2 is
transcribed in root and crops of uninfected alfalfa and is induced not only by
dodder attack but also by bacterial infections and a large variety of
environmental stresses.
2470. Biochem Cell Biol. 2002;80(3):363-78.
Dynamics of histone acetylation in vivo. A function for acetylation turnover?
Waterborg JH(1).
Author information:
(1)Division of Cell Biology and Biophysics, School of Biological Sciences,
University of Missouri-Kansas City, 64110, USA. WaterborgJ@umkc.edu
Histone acetylation, discovered more than 40 years ago, is a reversible

modification of lysines within the amino-terminal domain of core histones.
Amino-terminal histone domains contribute to the compaction of genes into
repressed chromatin fibers. It is thought that their acetylation causes localized
relaxation of chromatin as a necessary but not sufficient condition for processes
that repackage DNA such as transcription, replication, repair, recombination, and
sperm formation. While increased histone acetylation enhances gene transcription
and loss of acetylation represses and silences genes, the function of the rapid
continuous or repetitive acetylation and deacetylation reactions with half-lives
of just a few minutes remains unknown. Thirty years of in vivo measurements of
acetylation turnover and rates of change in histone modification levels have been
reviewed to identify common chromatin characteristics measured by distinct
protocols. It has now become possible to look across a wider spectrum of
organisms than ever before and identify common features. The rapid turnover rates
in transcriptionally active and competent chromatin are one such feature. While
ubiquitously observed, we still do not know whether turnover itself is linked to
chromatin transcription beyond its contribution to rapid changes towards hyper-
or hypoacetylation of nucleosomes. However, recent experiments suggest that
turnover may be linked directly to steps in gene transcription, interacting with
nucleosome remodeling complexes.
2471. Biochem Cell Biol. 2002;80(3):279-93.
Kinetic analysis of histone acetylation turnover and Trichostatin A induced

hyper- and hypoacetylation in alfalfa.
Waterborg JH(1), Kapros T.
Author information:
(1)Division of Cell Biology and Biophysics, School of Biological Sciences,
University of Missouri, Kansas City 64110, USA. WaterborgJ@umkc.edu
Dynamic histone acetylation is a characteristic of chromatin transcription. The

first estimates for the rate of acetylation turnover of plants are reported,
measured in alfalfa cells by pulse, pulse-chase, and steady-state acetylation
labeling. Acetylation turnover half-lives of about 0.5 h were observed by all
methods used for histones H3, H4, and H2B. This is consistent with the rate at
which changes in gene expression occur in plants. Treatment with histone
deacetylase inhibitor Trichostatin A (TSA) induced hyperacetylation at a similar
rate. Replacement histone variant H3.2, preferentially localized in highly
acetylated chromatin, displayed faster acetyl turnover. Histone H2A with a low
level of acetylation was not subject to rapid turnover or hyperacetylation.
Patterns of acetate labeling revealed fundamental differences between histone H3
versus histones H4 and H2B. In H3, acetylation of all molecules, limited by
lysine methylation, had similar rates, independent of the level of lysine
acetylation. Acetylation of histones H4 and H2B was seen in only a fraction of
all molecules and involved multiacetylation. Acetylation turnover rates increased
from mono- to penta- and hexaacetylated forms, respectively. TSA was an effective
inhibitor of alfalfa histone deacetylases in vivo and caused a doubling in
steady-state acetylation levels by 4-6 h after addition. However,
hyperacetylation was transient due to loss of TSA inhibition. TSA-induced
overexpression of cellular deacetylase activity produced hypoacetylation by 18 h
treatment with enhanced acetate turnover labeling of alfalfa histones. Thus,
application of TSA to change gene expression in vivo in plants may have
unexpected consequences.
2472. Physiol Plant. 2002 Aug;115(4):531-540.
Effects of water stress on antioxidant enzymes of leaves and nodules of

transgenic alfalfa overexpressing superoxide dismutases.
Rubio MC(1), González EM, Minchin FR, Webb KJ, Arrese-Igor C, Ramos J, Becana M.
Author information:
Superior de Investigaciones Científicas, Apdo 202,E-50080 Zaragoza, Spain
Departamento de Ciencias del Medio Natural, Universidad Pública de Navarra,
E-31006 Pamplona, Spain Institute of Grassland and Environmental Research, Plas
Gogerddan, Aberystwyth, SY23 3EB, UK.
The antioxidant composition and relative water stress tolerance of nodulated

alfalfa plants (Medicago sativa L. x Sinorhizobium meliloti 102F78) of the elite
genotype N4 and three derived transgenic lines have been studied in detail. These
transgenic lines overproduced, respectively, Mn-containing superoxide dismutase
(SOD) in the mitochondria of leaves and nodules, MnSOD in the chloroplasts, and
FeSOD in the chloroplasts. In general for all lines, water stress caused moderate
decreases in MnSOD and FeSOD activities in both leaves and nodules, but had
distinct tissue-dependent effects on the activities of the peroxide-scavenging
enzymes. During water stress, with a few exceptions, ascorbate peroxidase and
catalase activities increased moderately in leaves but decreased in nodules. At
mild water stress, transgenic lines showed, on average, 20% higher photosynthetic
activity than the parental line, which suggests a superior tolerance of
transgenic plants under these conditions. However, the untransformed and the
transgenic plants performed similarly during moderate and severe water stress and
recovery with respect to important markers of metabolic activity and of oxidative
stress in leaves and nodules. We conclude that the base genotype used for
transformation and the background SOD isozymic composition may have a profound
effect on the relative tolerance of the transgenic lines to abiotic stress.
PMID: 12121459
2473. Ann Bot. 2002 Apr;89(4):391-400.
In vivo characterization of the effects of abscisic acid and drying protocols

associated with the acquisition of desiccation tolerance in alfalfa (Medicago
sativa L.) somatic embryos.
Sreedhar L(1), Wolkers WF, Hoekstra FA, Bewley JD.
Author information:
(1)Department of Botany, University of Guelph, Ontario, Canada.
Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some
tolerance to desiccation at the cotyledonary stage of development (22 d after
plating), additional culturing in 20 microm abscisic acid (ABA) for 8 d induced
greater desiccation tolerance, as determined by increased germination. Compared
with fast drying, slow drying of the ABA-treated embryos improved desiccation
tolerance. However, slow drying of non-ABA-treated embryos led to the complete
loss of germination capacity, while some fast-dried embryos survived. An electron
paramagnetic resonance spin probe technique and in vivo Fourier transform
infrared microspectroscopy revealed that cellular membrane integrity and
a-helical protein secondary structure were maintained during drying in embryos
cultured in media enriched with 20 microM ABA, but not in embryos cultured in the
absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to
sucrose ratios, an increased proportion of beta-sheet protein secondary
structures and broad membrane phase transitions extending over a temperature
range of more than 60 degrees C, suggestive of irreversible phase separations.
The spin probe study showed evidence of imbibitional damage, which could be
alleviated by prehydration in humid air. These observations emphasize the
importance of appropriate drying and prehydration protocols for the survival and
storage of somatic embryos. It is suggested that ABA also plays a role in
suppressing metabolism, thus increasing the level of desiccation tolerance; this
is particularly evident under stressful conditions such as slow drying.
PMCID: PMC4233871
2474. Bioresour Technol. 2002 Jul;83(3):189-94.
Effect of carbon source on compost nitrogen and carbon losses.
Barrington S(1), Choinière D, Trigui M, Knight W.
Author information:
(1)Department of Agricultural and Biosystems Engineering, McGill University, Ste
Anne de Bellevue, Que., Canada. barrington@macdonald.mcgill.ca
The effect of C source on N losses by volatilization during composting was

measured using four bulking agents, each at three humidity levels and composted
in duplicate under passive and active aeration. The bulking agents were pine
shavings alone and corrected with soybean, chopped grass hay alone and corrected
with urea, long (unchopped) wheat straw and chopped oat straw. The readily
available C of each bulking agent was determined by analyzing for BOD5. In 105 l
laboratory vessels, the bulking agents were mixed with liquid swine manure and
tap water for a C/N of 20 and three humidity levels of 60%, 65% and 70%. While
being aerated actively or passively, the mixtures were composted for 21 days.
Their initial and final C and N contents were measured to conduct a mass balance
analysis and calculate C and N losses. C and N losses were compared to bulking
agent BOD5. N losses were compared to C losses. The humidity level and aeration
regime had no effect on C and N losses but the N losses were correlated to C
losses and only the C losses could be correlated to the BOD5 of the bulking
agent. Thus, the N losses are related not only to the availability of C but also
to the extent of composting. A relationship established between N and C losses
indicated that 85% of the initial total N of the compost was available for
microbial degradation and that 70% of the available C was lost as CO2 during the
immobilization process.
2475. EMBO J. 2002 Jul 1;21(13):3296-306.
Involvement of the mitogen-activated protein kinase SIMK in regulation of root

hair tip growth.
Samaj J(1), Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I,
Lenart P, Salaj J, Volkmann D, Bögre L, Baluska F, Hirt H.
Author information:
(1)Institute of Microbiology and Genetics, Vienna Biocenter, University of
Vienna, Dr Bohrgasse 9, A-1030 Vienna, Austria.
Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the

actin cytoskeleton in yeast and animals. We have analyzed the function of the
stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK
is predominantly nuclear. During root hair formation, SIMK was activated and
redistributed from the nucleus into growing tips of root hairs possessing dense
F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK
relocation to the nucleus. Conversely, upon actin stabilization with
jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm.
Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in
a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by
the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126
inhibited targeted vesicle trafficking and polarized growth of root hairs. In
contrast, overexpression of gain-of-function SIMK induced rapid tip growth of
root hairs and could bypass growth inhibition by UO 126. These data indicate that
SIMK plays a crucial role in root hair tip growth.
DOI: 10.1093/emboj/cdf349
PMCID: PMC126098
2476. Nature. 2002 Jun 27;417(6892):962-6.
A receptor kinase gene regulating symbiotic nodule development.
Endre G(1), Kereszt A, Kevei Z, Mihacea S, Kaló P, Kiss GB.
Author information:
Sciences, H-6701 Szeged, PO Box 521, Hungary.
Comment in
Nature. 2002 Jun 27;417(6892):910-1.
Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil

bacteria generally known as rhizobia. Metabolites exuded by the plant root
activate the production of a rhizobial signal molecule, the Nod factor, which is
essential for symbiotic nodule development. This lipo-chitooligosaccharide signal
is active at femtomolar concentrations, and its structure is correlated with host
specificity of symbiosis, suggesting the involvement of a cognate perception
system in the plant host. Here we describe the cloning of a gene from Medicago
sativa that is essential for Nod-factor perception in alfalfa, and by genetic
analogy, in the related legumes Medicago truncatula and Pisum sativum. The
identified 'nodulation receptor kinase', NORK, is predicted to function in the
Nod-factor perception/transduction system (the NORK system) that initiates a
signal cascade leading to nodulation. The family of 'NORK
extracellular-sequence-like' (NSL) genes is broadly distributed in the plant
kingdom, although their biological function has not been previously ascribed. We
suggest that during the evolution of symbiosis an ancestral NSL system was
co-opted for transduction of an external ligand, the rhizobial Nod factor,
leading to development of the symbiotic root nodule.
DOI: 10.1038/nature00842
2477. J Dairy Sci. 2002 May;85(5):1183-90.
Response of forage fiber degradation by ruminal microorganisms to branched-chain

volatile fatty acids, amino acids, and dipeptides.
Yang CM(1).
Author information:
(1)Applied Animal Science Department, National I-Lan Institute of Technology,
Taiwan, ROC. cmyang@ilantech.edu.tw
This study evaluated the effect of branched-chain volatile fatty acids (VFA;
isobutyric acid, isovaleric acid), amino acids (valine, leucine), and dipeptides
(valine-valine, leucine-leucine) on neutral detergent fiber (NDF) degradation by
rumen microorganisms in vitro. The CP (%) and in situ NDF degradation rate (%/h)
for alfalfa, bermudagrass, and pangolagrass hays, and napiergrass silage were
17.2 and 7.5, 4.7 and 3.1, 8.3 and 5.3, and 9.6 and 3.4, respectively. In vitro
NDF digestibility was the lowest for bermudagrass; alfalfa and napiergrass were
the highest. When the incubation contained more ammonia initially,
digestibilities increased, but relative differences among forages were unchanged.
Adding branched-chain VFA (2 mM) to incubations increased digestibilities more
than controls on 15 out of 16 occasions. The effectiveness varied with isoacids
and forages used. Amino acid (2 mM) or dipeptide (1 mM) addition consistently
increased digestibility over controls. Amino acids further increased
digestibility over corresponding isoacids on 14 occasions. Improvement in
digestibility over control by leucine appeared to be greater than that by valine.
Digestibilities with dipeptides were always greater than those with isoacids,
except for one case. Dipeptide addition further increased digestibility
significantly over corresponding amino acids on only six occasions, while percent
improvement in digestibility numerically by dipeptides occurred in 10 cases.
Valine-valine seemed to exert different effect than leucine-leucine, depending on
initial ammonia availability. The results indicate that dipeptides could be more
effective than isoacids and amino acids in improving NDF digestion. Forages with
high CP content or rapid NDF degradation rate appeared to respond to additives to
smaller degrees.
DOI: 10.3168/jds.S0022-0302(02)74181-7
2478. J Dairy Sci. 2002 May;85(5):1079-84.
Milk protein response to abomasal or mesenteric vein essential amino acid

infusion in lactating dairy cows.
Aikman PC(1), Reynolds CK, Humphries DJ, Beever DE, MacRae JC.
Author information:
(1)Centre for Dairy Research, Department of Agriculture, The University of
Reading, UK.
Previous experiments performed at our location suggested that the milk protein
response to infusions of mixed essential amino acids (EAA) was greater when these
were supplied via intravenous rather than abomasal or duodenal routes. However,
as far as we are aware there have been no direct comparisons of the milk protein
response to site of EAA provision in the same animals. Our objective was to
directly compare the milk protein responses when cows were given mixtures of EAA
provided via abomasal or mesenteric vein infusions. Four multiparous, ruminally
cannulated, multicatheterized Holstein x Friesian cows averaging 18 wk postpartum
were fed dehydrated alfalfa, grass silage, and low protein (11.9% CP, DM basis)
concentrates at 30, 20, and 50%, respectively, of dry matter (DM) offered. Total
mixed rations (14% crude protein, DM basis) were fed daily as 3 equal meals at
8-h intervals and 95% of ad libitum DM intake. The experimental design was a
single reversal with two 10-d infusion periods separated by a 10-d rest period.
Each infusion period consisted of 4-d control infusions into the mesenteric vein
(saline at 2 ml/min) and abomasum (water at 9 L/d) followed by 6-d infusion of a
mixture of EAA equal to 400 g of milk protein/d into the abomasum or mesenteric
vein. Control infusions continued into the site not receiving EAA. Intake of DM
(20.3 kg/d), milk yield (28.9 kg/d), and milk fat concentration (41.2 g/kg) were
not affected by EAA infusions. Milk protein concentration (33.4 vs 34.6 g/kg) and
output (938 vs. 982 g/d) were increased and milk lactose concentration was
decreased (46.6 vs. 46.1 g/kg) by EAA, but the responses were not affected by
infusion site. Recovery of EAA as increased milk protein output (10.9%) was
similar for intravascular and abomasally infused EAA in these cows, but these
responses were low compared with published effects of postruminal casein
infusion.
DOI: 10.3168/jds.S0022-0302(02)74168-4
2479. J Agric Food Chem. 2002 Jul 3;50(14):3938-42.
Selenium accumulation in beef: effect of dietary selenium and geographical area

of animal origin.
Hintze KJ(1), Lardy GP, Marchello MJ, Finley JW.
Author information:
(1)Department of Animal and Range Sciences, North Dakota State University, Fargo,
North Dakota 58105, USA.
Selenium (Se) is an essential nutrient with multiple human health benefits; the
single most important dietary source of Se is beef. The Se content of beef
varies, and cattle fed a high selenium diet may have Se concentrations in beef
that are well above average. Such beef is potentially a unique supplemental
source of dietary Se. To examine factors affecting Se accumulation in beef, 16
steers (initial wt 374.4 +/- 33.7 kg) were taken from seleniferous or
nonseleniferous areas and fed in a 2 x 2 factorial design with diets high or
moderate in Se (11.9 or 0.62 mg Se/kg diet). Diets contained 50% alfalfa, 25%
wheat, and 25% corn on a dry matter basis. All dietary Se was from agricultural
products, and Se in the high Se diet was primarily from high Se wheat and alfalfa
hay. A loin muscle biopsy was taken at the start of the trial to determine
initial Se content of beef. Steers were slaughtered after 14 weeks of the trial,
and edible carcass (round, sirloin, shoulder clod, and ribeye) and organ samples
were collected. Diets did not affect growth or feed intake (P > 0.05), and Se
toxicity signs were not observed. Different cuts of meat had similar Se
concentrations, and the Se content of all cuts was increased by both high dietary
Se and high Se background. Except for liver and kidney, Se in tissues was
increased by seleniferous background (P < 0.02) and high dietary Se (P < 0.001).
Kidney Se concentrations of animals fed the high Se diet were lowest in animals
from seleniferous areas (P = 0.04), suggesting a possible adaptation to the high
Se diet. These results demonstrate that cattle fed diets high in Se from
agricultural products will accumulate substantial amounts of Se in the beef
without developing signs of Se toxicity and that prior Se status regulates Se
accumulation in some organs. They further demonstrate that management practices
may be altered so as to make beef a significant source of dietary Se.
2480. Adv Exp Med Biol. 2002;505:51-60.
Flavonoids: signal molecules in plant development.
Wo HH(1), Kuleck G, Hirsch AM, Hawes MC.
Author information:
(1)Department of Molecular, Cellular, and Developmental Biology, Molecular
Biology Institute, University of California, Los Angeles 90095-1606, USA.
2481. Adv Exp Med Biol. 2002;505:23-39.
Signalling in arbuscular mycorrhiza: facts and hypotheses.
Vierheilig H(1), Piché Y.
Author information:
(1)Centre de Recherche en Biologic Forestère, Pavillon C.- E.- Marchand,
Université Laval, Québec, Canada.
Influence of alfalfa cultivar on suitability of Acyrthosiphon kondoi (Homoptera:

Aphididae) for survival and development of hippodamia convergens and Coccinella
septempunctata (Coleoptera: Coccinellidae).
Giles KL(1), Berberet RC, Zarrabi AA, Dillwith JW.
Author information:
(1)Department of Entomology and Plant Pathology, Oklahoma State University,
Stillwater 74078-3033, USA. kgiles@okstate.edu
Hippodamia convergens Guérin-Méneville and Coccinella septempunctata L.

(Coleoptera: Coccinellidae) larvae were supplied daily with 1, 2, 4, or 16 mg of
Acyrthosiphon kondoi Shinji (Homoptera: Aphididae) reared on one of two
susceptible ('OK08' or 'CUF-101') or one resistant ('54H55') alfalfa cultivar
(IMedicago sativa L.) . Hippodamia convergens survived to the adult stage when
supplied with > or = 1 mg of A. kondoi per day from both susceptible and
aphid-resistant cultivars, whereas C. septempunctata required > or = 2 mg of A.
kondoi per day (from each cultivar) for survival to the adult stage. For both H.
convergens and C. septempunctata, no consistent differences in survivorship or
developmental times were observed between predator larvae supplied with
increasing daily levels of A. kondoi from susceptible (OKO8 orCUF-101) versus
resistant (54H55) cultivars. Additionally, alfalfa cultivar had no indirect
influence on adult weight of H. convergens or C. septempunctata. Results from our
study suggest that the resistant alfalfa cultivar (54H55) would have little to no
effect on the nutritional value of A. kondoi for ladybeetle predators.
2483. Eur J Biochem. 2002 Jun;269(12):2842-50.
Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea
leaves.
Gavidia I(1), Pérez-Bermúdez P, Seitz HU.
Author information:
(1)Center of Plant Molecular Biology (ZMBP), University of Tübingen, Germany.
Isabel.gavidia@uv.es
The aldo-keto reductase (AKR) superfamily comprises proteins that catalyse mainly
the reduction of carbonyl groups or carbon-carbon double bonds of a wide variety
of substrates including steroids. Such types of reactions have been proposed to
occur in the biosynthetic pathway of the cardiac glycosides produced by Digitalis
plants. Two cDNAs encoding leaf-specific AKR proteins (DpAR1 and DpAR2) were
isolated from a D. purpurea cDNA library using the rat Delta4-3-ketosteroid
5beta-reductase clone. Both cDNAs encode 315 amino acid proteins showing 98.4%
identity. DpAR proteins present high identities (68-80%) with four Arabidopsis
clones and a 67% identity with the aldose/aldehyde reductase from Medicago
sativa. A molecular phylogenetic tree suggests that these seven proteins belong
to a new subfamily of the AKR superfamily. Southern analysis indicated that DpARs
are encoded by a family of at most five genes. RNA-blot analyses demonstrated
that the expression of DpAR genes is developmentally regulated and is restricted
to leaves. The expression of DpAR genes has also been induced by wounding,
elevated salt concentrations, drought stress and heat-shock treatment. The
isolated cDNAs were expressed in Escherichia coli and the recombinant proteins
purified. The expressed enzymes present reductase activity not only for various
sugars but also for steroids, preferring NADH as a cofactor. These studies
indicate the presence of plant AKR proteins with ketosteroid reductase activity.
The function of the enzymes in cardenolide biosynthesis is discussed.
2484. Arch Microbiol. 2002 Jul;178(1):36-44. Epub 2002 Apr 23.
Involvement of the Sinorhizobium meliloti leuA gene in activation of nodulation

genes by NodD1 and luteolin.
Sanjuán-Pinilla JM(1), Muñoz S, Nogales J, Olivares J, Sanjuán J.
Author information:
Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain.
The role of leucine biosynthesis by Sinorhizobium meliloti in the establishment

of nitrogen-fixing symbiosis with alfalfa ( Medicago sativa) was investigated.
The leuA gene from S. meliloti, encoding alpha-isopropylmalate synthase, which
catalyses the first specific step in the leucine biosynthetic pathway, was
characterized. S. melilotiLeuA(-) mutants were Leu auxotrophs and lacked
alpha-isopropylmalate synthase activity. In addition, leuA auxotrophs were unable
to nodulate alfalfa. Alfalfa roots did not seem to secrete enough leucine to
support growth of leucine auxotrophs in the rhizosphere. Thus, this growth
limitation probably imposes the inability to initiate symbiosis. However, in
addition to the leucine auxotrophy, leuA strains were impaired in activation of
nodulation genes by the transcriptional activator NodD1 in response to the plant
flavone luteolin. By contrast, nod gene activation by NodD3, which does not
involve plant-derived inducers, was unaffected. Our results suggest that a
leucine-related metabolic intermediate may be involved in activation of
nodulation genes by NodD1 and luteolin. This kind of control could be of
relevance as a way to link bacterial physiological status to the response to
plant signals and initiation of symbiosis.
DOI: 10.1007/s00203-002-0421-7
2485. Plant J. 2002 Jun;30(6):699-709.
Plant RanGAPs are localized at the nuclear envelope in interphase and associated
with microtubules in mitotic cells.
Pay A(1), Resch K, Frohnmeyer H, Fejes E, Nagy F, Nick P.
Author information:
(1)Plant Biology Institute, Biological Research Center, H-6701 Szeged, PO Box
521, Hungary.
In animals and yeast, the small GTP-binding protein Ran has multiple functions -
it is involved in mediating (i) the directional passage of proteins and RNA
through the nuclear pores in interphase cells; and (ii) the formation of spindle
asters, the polymerization of microtubules, and the re-assembly of the nuclear
envelope in mitotic cells. Nucleotide binding of Ran is modulated by a series of
accessory proteins. For instance, the hydrolysis of RanGTP requires stimulation
by the RanGTPase protein RanGAP. Here we report the complementation of the yeast
RanGAP mutant rna1 with Medicago sativa and Arabidopsis thaliana cDNAs encoding
RanGAP-like proteins. Confocal laser microscopy of Arabidopsis plants
overexpressing chimeric constructs of GFP with AtRanGAP1 and 2 demonstrated that
the fusion protein is localized to patchy areas at the nuclear envelope of
interphase cells. In contrast, the cellular distribution of RanGAPs in
synchronized tobacco cells undergoing mitosis is characteristically different.
Double-immunofluorescence shows that RanGAPs are co-localized with spindle
microtubules during anaphase, with the microtubular phragmoplast and the surface
of the daughter nuclei during telophase. Co-assembly of RanGAPs with tubulin
correlates with these in vivo observations. The detected localization pattern is
consistent with the postulated function of plant RanGAPs in the regulation of
nuclear transport during interphase, and suggests a role for these proteins in
the organization of the microtubular mitotic structures.
2486. Physiol Plant. 2001 Nov;113(3):424-429.
The effect of AOA on ethylene and polyamine metabolism during early phases of
somatic embryogenesis in Medicago sativa.
Huang XL(1), Li XJ, Li Y, Huang LZ.
Author information:
(1)School of Life Sciences, Department of Biology, Zhongshan University,
Guangzhou 510275, China.
Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC),

1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were
simultaneously investigated during the early phases of alfalfa somatic
embryogenesis. These included the period of induction and subculture of callus,
and 3- and 7-day suspension cultures for the induction of somatic embryogenesis.
The polyamines contained in the embryogenic callus were found to include
putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was
much less than that of Put and Spd. There was a dramatic increase in MACC after
induction of embryogenesis, and ACC levels were lower in somatic embryos than in
embryogenic callus. Induction of embryogenesis for 3 days increased the levels of
ACC and polyamines to a maximum level, and these then reduced as the
embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during
the induction. This indicated that both high levels of ACC and polyamines might
be a prerequisite for early differentiation during the induction of the
embryogenesis. Thus, there appears not to be competition between polyamine
biosynthesis and ethylene biosynthesis at least during the induction of somatic
embryogenesis, because both the polyamines and ACC were simultaneously increased
during the induction period. Conversion of ACC into MACC and the maintenance of a
relatively high level of polyamines, especially Spd, appear to be important for
further development of the embryos. When aminooxylvinylglycine (AOA) was added at
the initiation of the callus subculture, it had no significant effect on the
callus growth, the ethylene production and ACC level of the callus. However, AOA
increased the numbers of the embryos accompanying an increase in Spd level and
S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could
be associated with Spd increase rather than with the effect of ethylene
biosynthesis.
PMID: 12060289
2487. Mol Plant Microbe Interact. 2002 Jun;15(6):529-39.
Phylogeny and genomic organization of the TIR and non-tIR NBS-LRR resistance gene
family in Medicago truncatula.
Zhu H(1), Cannon SB, Young ND, Cook DR.
Author information:
(1)Department of Plant Pathology, University of California, Davis 95616, USA.
Sequences homologous to the nucleotide binding site (NBS) domain of

NBS-leucine-rich repeat (LRR) resistance genes were retrieved from the model
legume M. truncatula through several methods. Phylogenetic analysis classified
these sequences into TIR (toll and interleukin-1 receptor) and non-TIR NBS
subfamilies and further subclassified them into several well-defined clades
within each subfamily. Comparison of M. truncatula NBS sequences with those from
several closely related legumes, including members of the tribes Trifoleae,
Viceae, and Phaseoleae, reveals that most clades contain sequences from multiple
legume species. Moreover, sequences from species within the closely related
Trifoleae and Viceae tribes (e.g., Medicago and Pisum spp.) tended to be
cophyletic and distinct from sequences of Phaseoleae species (e.g., soybean and
bean). These results suggest that the origin of major clades within the NBS-LRR
family predate radiation of these Papilionoid legumes, while continued
diversification of these sequences mirrors speciation within this legume
subfamily. Detailed genetic and physical mapping of both TIR and non-TIR NBS
sequences in M. truncatula reveals that most NBS sequences are organized into
clusters, and few, if any, clusters contain both TIR and non-TIR sequences.
Examples were found, however, of physical clusters that contain sequences from
distinct phylogenetic clades within the TIR or non-TIR subfamilies. Comparative
mapping reveals several blocks of resistance gene loci that are syntenic between
M. truncatula and soybean and between M. truncatula and pea.
DOI: 10.1094/MPMI.2002.15.6.529
2488. Folia Microbiol (Praha). 2002;47(2):167-70.
Isozyme variation in Verticillium dahliae isolates from Crete.
Ligoxigakis EK(1), Fragkiadakis GA, Manganaris AG, Vakalounakis DJ,

Thanassoulopoulos KK.
Author information:
(1)National Agricultural Research Foundation, Plant Protection Institute,
Heraklio, Crete, Greece.
Fifteen isolates of Verticillium dahliae (eight of race 1, seven of race 2; most

from the island of Crete, Greece) were examined for isozyme and molecular
variation. Among the isozyme banding patterns (zymograms) of six enzymes that
were "activity-stained" after electrophoresis in 9% polyacrylamide gels,
differences were observed in diaphorase, alpha-esterase, peroxidase and
superoxide dismutase; 2, 2, 3 and 5 different types of zymograms were recorded,
respectively. The zymograms could not be correlated with either race 1 or 2.
However, all six isolates originating from the Oropedio (plateau) area of Lasithi
(Crete) showed an esterase zymogram clearly distinguishable from the other
isolates. No differences were observed when staining for acid phosphatase or
aspartate aminotransferase ('glutamic-oxaloacetic transaminase'). Furthermore,
electrophoresis of random-amplified polymorphic DNA (RAPD) in 2% agarose gels
showed that three race-2 isolates from Oropedio of Lasithi could also be
distinguished by the RAPD pattern generated with primer OPA-1. The variation
observed possibly represents adaptation of V. dahliae to the Oropedio
environment.
2489. J Bacteriol. 2002 Jul;184(13):3466-75.
Identification of two quorum-sensing systems in Sinorhizobium meliloti.
Marketon MM(1), González JE.
Author information:
Sinorhizobium meliloti is a free-living soil bacterium which is capable of

establishing a symbiotic relationship with the alfalfa plant (Medicago sativa).
This symbiosis involves a network of bacterium-host signaling, as well as the
potential for bacterium-bacterium communication, such as quorum sensing. In this
study, we characterized the production of N-acyl homoserine lactones (AHLs) by
two commonly used S. meliloti strains, AK631 and Rm1021. We found that AK631
produces at least nine different AHLs, while Rm1021 produces only a subset of
these molecules. To address the difference in AHL patterns between the strains,
we developed a novel screening method to identify the genes affecting AHL
synthesis. With this screening method, chromosomal groEL (groELc) was shown to be
required for synthesis of the AHLs that are unique to AK631 but not for synthesis
of the AHLs that are made by both AK631 and Rm1021. We then used the screening
procedure to identify a mutation in a gene homologous to traM of Agrobacterium
tumefaciens, which was able to suppress the phenotype of the groELc mutation. A
traR homolog was identified immediately upstream of traM, and we propose that its
gene product requires a functional groELc for activity and is also responsible
for inducing the synthesis of the AHLs that are unique to AK631. We show that the
traR/traM locus is part of a quorum-sensing system unique to AK631 and propose
that this locus is involved in regulating conjugal plasmid transfer. We also
present evidence for the existence of a second quorum-sensing system, sinR/sinI,
which is present in both AK631 and Rm1021.
PMCID: PMC135146
2490. Bull Environ Contam Toxicol. 2002 Jul;69(1):74-81.
Potential of alfalfa plant to phytoremediate individually contaminated

montmorillonite-soils with cadmium(II), chromium(VI), copper (II), nickel(II),
and zinc(II).
Peralta-Videa JR(1), Gardea-Torresdey JL, Gomez E, Tiemann KJ, Parsons JG, de la

Rosa G, Carrillo G.
Author information:
(1)Environmental Science and Egineering Program, University of Texas at El Paso,
79995, USA.
DOI: 10.1007/s00128-002-0012-y
2491. Appl Environ Microbiol. 2002 Jun;68(6):3114-20.
Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on

alfalfa sprouts.
Charkowski AO(1), Barak JD, Sarreal CZ, Mandrell RE.
Author information:
Wisconsin 53706, USA.
Sprout producers have recently been faced with several Salmonella enterica and
Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to
sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were
inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa
seeds or other environmental sources and sprouted to examine growth of these
human pathogens in association with sprouting seeds. S. enterica strains grew an
average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7
strains grew significantly less, an average of 2.3 log(10). The initial S.
enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature
significantly affected the levels of both S. enterica and E. coli O157:H7 on the
sprouts and in the irrigation water, while the frequency of irrigation water
replacement affected only the levels of E. coli O157:H7. Colonization of
sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7
strains transformed with a plasmid encoding the green fluorescent protein was
examined with fluorescence microscopy. Salmonella serovar Newport colonized both
seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only
sprout roots.
PMCID: PMC123925
2492. Mol Plant Microbe Interact. 2002 May;15(5):456-62.
Roles for riboflavin in the Sinorhizobium-alfalfa association.
Yang G(1), Bhuvaneswari TV, Joseph CM, King MD, Phillips DA.
Author information:
(1)Department of Agronomy and Range Science, University of California, Davis
95616, USA.
Genes contributing to riboflavin production in Sinorhizobium meliloti were

identified, and bacterial strains that overproduce this vitamin were constructed
to characterize how additional riboflavin affects interactions between alfalfa
(Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were
found in three separate linkage groups and designated as ribBA, ribDribC, and
ribH for their similarities to Esch

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