tic Interactions Between Strains of Rhizobium phaseoli and Cultivars of Phaseolus vulgaris L.' R, S. Pacovsky, H. G. Bayne, and G. J. Bethlenfalvay ABSTRACT Several cultivar-strain combinations ofthe Phaseolus vulgaris L.-Rhizabium phaseol association were evaluated for Ny fixation, 8, and primary (biomass) production. Plants were 0.60 indicating a loss of reducing power effective strains Suggests that energy losses throu Tation may limit the productivity of the Phaseolus Rhi symbiosis. Assimilation of N by the symbiotic assoc response to Ny fixation tivar effects, Analysis of variance showed rignificant differences due to both symbionts in most growth parameters measured. However, there was no significant correlation (P< 0.05) be- tween N assimilation and the estimated rates of Ny fix the various effective host-endophyte combi ssimilated Nisa better, more direct measure of symbiotic efficiency than the estimate of N-fixation rate by C,H, and Hy production, and. the merits of different growth parameters as measures of Sym- biotic effectiveness are compared and discussed. Additional index words: Bean, Hydrogen evolution, fixation, Nodulation, Nitrogenase, Nitrogen assimi ni effectiveness (Natman, 1954) of symbiotic Nz fixation is expressed in terms of enhanced owth and nutrition of the host plant and has been Finked vo genetic differences among strains of Rt cobiun and compatible legume hosts, Strain charac- teristics of R. phaseoli (Burton et al., 1954) influence the response of Phaseolus vulgaris L. to inoculation. Host genotypic factors affecting nodulation and nod- ule activity have recentiy been identified in pulse leg- umes (Holl, 1975; Graham and Rosas, 1977; Wes- termann and Kolar, 1978; Zary et al., 1978; Graham, 1981), Screening of rhizobia for efficiency in Ny fix: ation has el othe isolation of superior strains of japonicum (Caldwell and Vest, 1970), R. leguninosa- rum, (Nelson and Child, 1981), the chickpea miscel- lany (Minguez and Ruiz-Argueso, 1980), and thizobia colonizing peanuts (Wynne et al., 1980). Host-endophyte interactions have been investi- fated in some major field crops but only recently al,, 1982). Variability in field responses to nodulation in P, vulgaris has been ascribed to unsatisfactory host- microsymbiont interactions (Graham, 1981), com- petition between R. phaseoli strains (Das and Bhaduri, 1974), and in temperate climates, to the environment (Sprent, 1982). A screening program for efficient strains under controlled contions is therefore de- sirable (Date, 1976). Considerable scope exists for “Fan fm he eae Kon eee Cee, gael try eke eh Fee e a A nl “pene SSSR ae eR ARE rept ete ach cf my Sa at en se De Sper alata oh ed tee ca Meenas tare ie 101 selecting and modiying the yenowypes of bath Pv gars and fe phaseol @prenty 1985). However, eff ent strains must be matched with compatible hosts and the resulting association selected for suitable e vironments (Rys and Bonish, 1981) to achieve sur perior Nr-fixing associations’ The purpose of this Study was to evaluate several cultivar-strain coml nations of the P. vulgaris XR. phaseoli symbiosis, and to measure the effect of Ne fixation on N assimilation and primary production. MATERIALS AND METHODS Biological Materials Seeds of P. vulgaris L. evs. Red Kidney, Toperop, and Dwarf Horticultural (obiained from Burpee Co., Warmin- ister, PA 18974) were weighed and seleeted within n row range (0.6 to 0.7 g for Red Kidney, 0.4 to 0.5 g for Toperop, and 0.5 to 0°6 g for Dwarf Horticultural eul- vars). Seeds were surface sterilized successively in 70% (¥/ 4) ethanol (I min), 0.1% (w/s) HgCl, (10 min), and 0.01 N HCI (10 min) with a thorough wash between and after treatments, and were germinated for 3 days at 28°C. Three seedlings were aseptically transferred to pots containing 600 cm? of autoclaved perlite. Two plants per pot were removed after 1 week with the remalning plants selected to produce a uniform stand Cultures of R phaseoli strains 127K 14, 127K46, 127K 48, 127K59 originally supplied by J. C. Burton, Nitragin Co., Milwaukee, W1, 58209, and strains 2667, 2668, RCR3610, CIAT632, TAL182 obtained originally from H. H. Keyser USDA, Beltvlie, MD 20705) were maintained om yeas ‘mannitol agar slants at 4°C. Individual strains were cul {ured separately in Vincent's (1970) yeast-mannitol broth prior tofnoculation, and dilution plate counts of viable cells Mere made. To eliminate differences in nodblation due to Variable Rhizobiom number, pote were inoculated with Saturating number of cells {pproximately 10") of the ap- roprate strain at planting and 7 days afler planting to Insure a high number of viable cell were present in the inert potting media during the inital perso of infection Controls were given 5 mL-of sterile Vincents medium t0 compensate foPN input due to the nutrient broth Growth Conditions A total of 120 plants were grown in a greenhouse in Albany, Calif, between July and September 1981, Plants of each of the three cultivars were inoculated with one of the nine strains of R, phaseoli, or were left uninoculated as con. trols. ‘The experiment was replicated four times, one rep- lication per week, at L-week intervals. Contamination be- tween pots was minimized by placing them on a wide wire mesh sereen above a corrugated fiberglass catchment sys- tem which was sterilized three times a week by spraying with 70% (v/v) ethanol. Temperature and relative humidity varied from day to day within the day/night ranges of 20/32"C and 55/89%, respectively. Photosynthetic photon flux density (PPFD) varied between 450 and 900 mol m-? s-! on cloudy or sunny days. Pots were rotated twice weekly to minimize102 CROP SCIENCE, VOL. 24, JANUARY-PEBRUARY 1984 ‘Table 1. Tukey's Multiple Comparison Tests for plant and Nrfixation characteristics among Rhicobium strains combined over three "Phaseolus vulgons cultivar, Bhizobium sain ‘Assimilated? Plantdry we Nedaledry wt CHlprodueed__H, evolved SNA Re rrwplant ‘eplant plant somal mol Hy pmolNe lane ‘ant temdeby ‘TALIS? 650° 2ase 02356 zioob oab 14.9 ede ost be imiKse Ssab Tote ase Bale aaa 1 abe Osta tanks S58b 208be oaite Tate Boe Lise Bante crates B38 216 020080 Baba Sitbe 228 Osbe doer Sb 205be ond tase 92be sede base iaikes 534d 2p be Oat a 2a tga Testbed 0800 LIKS8 su2b tase oats ise tsa M3ae bate ReROEIO ae2e sed 20s Tease hae Ieee O54 288 22d ted Stas. Biss ood 208T : Conerot doa ioe oor 0a bod oor . “afeens having common letnr within a cola are not sgafcatly diferent athe 3% evel of significance. { Repeecets total Nena sed 1 Spie nodl acivty(SNAI was calculated Rint eftclency (RE) was caleaated asl ~ (H,evolved GH, produced) positional effects. An additional 20 uninoculated control Plants served to indicate if chance contamination occurred Among treatments. Pots were watered to field capacity 5 daysa week with a nutrient solution (pH 6.4) consisting of 1.5 mM CaCl, 0.5, mM KH,PO,, 0.29 mM K,SO,, and 0.25 mM MgSO. Mi- Cronutrients were according to Johnson etal. (1967) at one- {quarter strength and included 0.6 uM CoCl, and 20 uM Fe Aipplied as FeEDDA (ferric ethylenediamine dido-hydrox: YyphenyDacetic acid), One day a week the pots were flushed wrth deionized water. Harvest was planned to coincide with, high rates of nodule activity (Bethlenfalvay and Phillips, 1977b). Plants were harvested over a 3-day period during the 5th week after planting, Assays Nodule activity Pate, 1977) of excised roots (ATP-de- pendent Ty protuction and C3H reduetion) was deter Pepe as described previously (Bethienfalvay and Philips, TD7fa), withthe folowing modifications Euhyene analyses ttre bude with a Varian Model 1400 gas chromatograph Npulpped with aflame ionization detector using a 0.82 % <1 eee Sites see column filed swith 80 t0 100 mesh Poropak N. Hydrogen was measured with a Hewlet-Pack- Ted hodel 5880 gas chromatograph equipped with ather- ful conductivity detector using # 0.32 % 183 cm column flisd with 60 to BO-nesh molecular sieve 5A. Helium served ie the earrer gas for Cel and Ne for Ha, both ata flow a G80 mbymin. Oven temperatures were 85°C for Chicana 30°C for Ha. Nitrogen fixation was estimated fom Calledependent CH, production and Hy-evolution SaacNe fixed ™ (Cally produced ~ Hy evolved)/S. The Maldive efficiency (RE) of electron transfer to Ny via nitro- Fone was calculated as RE 1 ~ (Hy evolved/CaH, pro- uced) according to Schubert and Evans (1970) ‘anc N content was determined by Kjeldahl digestion (ausoe,Octal Analytical Chem. 1980). Assimilated N was (itinced asthe difference between total Nand seed N fy meights of plant parts were measured after drying a 80°C for 2 days "The experiment was of completely random design with 0 culsear X strain combinations (including uninoculated conteols) arranged over an area of homogencous growth SOnuitions. The expesiment was repeated four times. Ad- ional controts used to check contamination, were inter- Spereed among the experimental plans, An analysis of va- Bice was eared out to determine the effect of sta Elida, or strain. cultivar interactions on symbiotic IC. produced — 1, evolvedahg nodule dry wth, growth (Dann and Clarke, 1974), Linear regression analysis wrerperformed according to Draper ana Smith (1905) Taping the correlation beoween Nesation and the serch sate of Ne fation, Comparisons of significant Glnteences among cults or stra for airetenssured weve made by Tukey's multiple comparison tes (1958), RESULTS Effect of Rhizobium Strain on Host Plant Mean fresh weight for the seeds of the Red Kidney, Dwarf Horticultural, and Topcrop cultivars were 0.65, 0.55, and 0.45 g, respectively. Mean N content for the seeds of the Red Kidney, Dwarf Horticultural, wert Topcrop cultivars were 188, 17.1, and 14.4 mg N, respectively. ‘Nitrogen fixation by the nine strains of R. phaseoli was the only source of N for N assimilation by the host (Table 1), as the N content of control plants was escentially unchanged from that found in the seeds. Al strains were capable of nodulation, even though one of the strains (2668) was ineffective (Table 1). Relatively large amounts of Hz were produced by the other eight effective strains, and approximately half Of the electron flow to nitrogenase was apparently sed to reduce H? to H, (Table 1). None of the con- trol plants were nodulated "An analysis of variance (Table 2) showed that the main effects due to strain were significant (P <0.001) for all plant and Ne-fixation parameters. Estimated Nz fixation (as defined in Methods) was significantly correlated with assimilated N (0.91**, significant at the 0.01 level of probability), N content (0.89%*), nodule dry weight (0.93**), plant dry weight (0.75%), and GsH, reduction (0.99**) at the 0.05(*, significant at the 0.05 level of probability) and 0.01(#*) levels. Correlations between the plant parameters and Calls reduction or estimated Ne-fixation calculated from H, and CoH, data showed no significant differences (P°>0.05) between these two measures of nodule ac tivity (0.89** and 0.90**, respectively). ‘Comparison of significant differences within the plant parameters du to strain effects by Tukey's mul Hip comparion est (Table 1) revealed no significant {E> 0.09) differences between the uninoculated con-PACOVSKY ET AL: RIIZOBIUM-PHASEOLUS SYMBIOSIS 108 Table2. Analysis of variance for assimilated N, plant and nodule dry wt, and the nodule activity parameters ‘ease of waration at Assimilated N Plant dry wt SNA RE, Strain) 8 e219 Loe paneer Seog oni Gultvaetoy 2 "400° one 036 ‘08 oir sxe 18 ies O18 i 2 ‘ooo? Enor % nie 013 130 159 0.008 Differences were significant atthe 005 nd 0.001 lvl, reepetivey, ‘Table 3. Tukey's Multiple Comparison Tests for plant and Nyfixation characteristics among Phaseolus vulgaris cultivars combined ‘over nine fhizebium phaseol strains and the control Cativar AssimletedN} —Plantdry wt Noduledrywe Ny fbationt SNAG eA ‘aelant wolant ‘plant nol fmol ‘Gianesy (ead) ‘Toperop 06 186 02040 asp Ret Kidney Boe 2h 20a tab Dinar Horticleral Boab ine O16 Hoe * Means having common letters within a column are not significantly different atthe 5% level { Represent al N lee sed Win calivare inoculated with elective stain of phase ‘Dinitogenfzation was neat aH IC, produced ~ H,eveved) theplant 4 Specie Nodule Activity (SNA) was ealclated as[GyHl, produced ~ H evolve) nodule dy wth J euintiveEiiclency INE} wes clelatedas[l"~ WlgcvelvelG, produced trol and plants inoculated with strain 2668, with the exception of nodule weight. The correlation between assimilated N and estimated No fixation was highly significant with the ineffective strain and control in- cluded but were not correlated among the effective strains (Fig. 1). Among the effective strains, TAL182 fas superior and strain RCR8610 was least effective in providing N to the plant, even though both strains had similar rates of estimated No fixation (Fig. 1) Host plants grown in association with the remaining, strains assimilated amounts of N which were not sig nificantly different, even though there were signif cant differences (P < 0.05) for the estimated rates of Ne fixation (Fig. 1) between strains. Effect of Cultivar on Symbiotic Characteristics Host genotype markedly influenced most of the symblouic characteristics (able 2), but no significant ifference (P > 0.05) in estimated No fixation due to cultivar was found (Table 3). Dwarf Horticultural had lower dry weights, nodule weights, and assimilated N, while having significantly higher SNA than the other two cultivars. There were no significant (P > 0.05) host X strain interactions (Table 2) indicating that no specific combination was superior over the broad range of all treatments. DISCUSSION ‘The efficiency of No fixation is often evaluated by relating the rate of C,H, reduction to other processes in a symbiotic legume association, such as ATP-de- pendent Hy evolution (Schubert and Evans, 1976) or CO» fixation (Bethlenfalvay and Phillips, 19770). ‘These measures of Ng fixation efficiency express tran- sient cost-benefit relationships and do not consider other important aspects of the interaction between the symbionts, such as the transfer and utilization of fixed N by the host plant. The most meaningful mea- sures of symbiotic efectiveness, N to C balance and association biomass (Phillips, 1980), are influenced by Ny FIXATION/PLANT (mol Nz /PLANT:HOUR) ao CO N ASSIMILATED (mg/plont) Relationship between assimilated N and N, fx trains of R. pheseoli and an uninoculated control. Means and LSD were combined over three cul tion was estimated as defined in Table 8. rs. Nitrogen fa the host’s capability to assimilate N fixed in the root nodule. Assimilation, in turn, depends on total N in- put, integrated over the entire time span of effective nodule activity. However, the CaHz reduction and Hy evolution assays provide only estimates of nitrogen- ase activity and are thus ill adapted to estimate total 'N input, especially if restricted to a single measure- ment. ‘The present data illustrate the points made above. Comparison of the estimated Ne-fixation rate and the assimilated N of the association shows that the two expressions do not always correspond (Fig. 1). Amon, the cultivars asociated with effective strains, those with the highest (TAL182) or lowest (RCR3610) lev- els of assimilated N did not have the highest {CIAT682) or lowest (2667) rates of estimated Ns yxation (Fig. 1). Although assimilated-N levels in the TALI82 and RCR361 associations were signifi-104 CROP SCIENCE, VOL. 24, JANUARY-FEBRUARY 1984 cantly different, their CzH¢ and Hy-production rates were not (Table 1). Conversely, assimilated-N levels of the CIAT632 and 2667 associations were alike, while their estimated Ny-fixation rates were signi cantly different. This observation can be attributed to differences in the intensity of Ne fixation at the time of measurement and to different time spans of effective Nz reduction in different associations. This lack of correlation between assimilated N and esti- mated Ne-fixation rate (Fig. 1) does not diminish the value of the CsH,-reduction assay in measuring nod- ule activity per se, but points to its inadequacy as a measure of symbiotic efectivencs, unless continuous or repeated readings, permitting an integration of the results, can be made, Its concluded that an evale uation of R. phaseoli strains for symbiotic effectiveness based on C,Hy reduction is meaningful only in con- junction with an N-assimilation determination. Specific nodule activity (SNA) is even more mis leading than the estimated Ne-fixation rate as an in- dicator of the effect of nodule activity on the host. Nodule weight is a host-plant parameter. There was, however, little relationship between nodule weight and other measures of plant development (Table 1). ‘A ranking of the values for SNA showed no rela tionship to the ranking of values for assimilated N (Table'l). Although frequently used in the literature, we consider SNA to be a parameter which should be used with caution in describing either the symbiotic ‘effectiveness of the association or the rate of Nz fix- ation. While the range of cultivars used in this study was limited, main effects due to the host plant were still significant (Tables 2 and 3). Values for assimilated N, averaged over the eight associations infected with effective R. phaseoli strains, were significantly differ- ent for the cultivars Topcrop and Dwarf Horticul- tural, even though estimated Ne-fixation rates were not significantly different (Table 3). This finding can be best ascribed to a property of the host which af- fects the transfer of N from endophyte to host, the gubsequent incorporation of N by the plant andthe loss of N through root exudation or photorespira- tion, even though variations in estimated Nz fixation rates not detected by our sampling method cannot, bbe excluded as a cause. Low nodule weights in Dwarf Horticultural did not affect the estimated Ne-fixation rates per plant, but significantly increased the SNA value (Table 3). “The copious production of Hg by all of the effective strains suggested that energy losses through Hz ev- olution may limit the productivity of the Phaseolus- Rhizobium symbiosis. Relative efficiencies (RE) for the eight effective strains (Table 1) indicated that more than 40% of the available electron flow to nitrogen- ase was used to reduce H* to He, thus significantly curtailing the production of NH,*. By this criterion (Schubert and Evans, 1976), these strains could be considered inefficient at No fixation, as their RE val- ues were all approximately 0.50 (Table 1). Under these conditions the H,-evolution assay appears to be 1g comparisons of nodule activity between strains with similar relative efficien- ies. “The capability for H, uptake, currently unreported in R. phaseoli, would increase the RE. and may en- hance. symbiotic effectiveness (Zablotowicz ct al., 1980). In the absence of native strains capable of Hy uptake, efficient strains might be engineered by in- troducing the uptake hydrogenase gene on recom- binant plasmids through genetic transformation (Sel- varaj and Iyer, 1981). Thus inoculation of beans with a R. phaseoli strain capable of recycling Hz might in- ‘crease the productivity of the association, ACKNOWLEDGMENTS We thank Leo Oceguera (sponsored by the Am. Chem, Soc. under the $.E.£.D. project) for general help with the experiment, W. C. Montague for the Kjeldahl N deter minations, and M. Weaver, M. Firestone, D. T. Wester~ ‘mann, and P. H. Graham for reviewing the manuscript. REFERENCES tical Chemists, 1980. Offical 'A.O.A.C. Washington D.C. p. 1. Association of Official methods of analysis 18th 1% 2, Bethlenfalvay, G.}, and D.A, Philips. 19772. Ontogenic in- teractions bet ch photon a gym nsrogen Sion in legumes. Pant Physiol, 0:419-421 3 \G--- 1977b, Photosynthesis and symbiotic nitro en fnation jn Phaseolus vudgors Lp. 401-408, In A. Ho Erender fed} Genetic engineering for trogen fixation. Plenum 1977. ‘The effect of light intensity on the sn of COy and Ny redaction in Bum satu. Plant Physiol bO-410-421 5. Burton, Cy ON. Allen, and K.G. Berge ‘Of bean Phasola calgon: Lt inoculation with mixtores of hecave and inllctve rhiobia. Proc. Soll Se'Soc: Am is156=139. 6, Caldwell, BE. and G, Vest, 1970. Effects off {Gam strains on soybean yields. Crop Set. 10:19 7. Dan, SN and PAN. Badr. 1974 Hom Razobi between Phases vdgars and Rhzoven phase Proc. Indian ‘Read. 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ABSTRACT The brown midrib (bm3) gene associated with low lign ‘corn (Zea mays L.) was evaluated for is effects on yield and yield ‘components in Hawall Fifteen bm} hybrids representing & parent diallel and their 15 isogenic normal counterpart hyb ‘were planted as paired rows in two trials. Vields of the bm3 hybrids were significantly lower than normals in both trial, with average reductions of 20% (range 7 to 29%) for grain and 176 (range 8 to 25%) for stover. Traits also affected negatively ‘by bm$ included kernel number per row (12%): filled eat length (10%), ear height (—7%), plant height (~5%), and Give other ‘measured traits. Correlation matrices of yields and all measured ‘alts revealed few consistent differences among normal and m3 hybrids. Grain yields correlated highly with leaf area index for ‘normal hybrida, but not for the isogenic bmd hybrids, Mean jae values for general and specific combining sbility (CCA, were significant for grain and stover yields and plant height, and GCA effects were significant for kernel number per row and ‘ar length. The GCA/SCA ratios were higher for bm? hybrids virtually excluded inthis study aa case forthe yeh reduction, tnd role of colored lignins tn reducing photoryathates is pore folted. Additional index words: Diallel analysis, General combining ability, Grain yield, Lignin, Silage yield, Specific combining abil ity, Zea mays Le HE mutant gene brown midrib-3 (m3) reduces dramatically the lignin contents of vegetative parts of corn (Zea mays L.) (11,15). Stover lignin re- ductions of up to 40% (16) were associated with in- creases of 10% or more in IVDMD (in vitro dry mat- ter disappearance) and with significant increases in the feed value of 6m3 corn silage (1,6). Other brown midrib mutants also lowered lignin, but 63 was the most effective (11). Increased body weight gains and milk production were reported on bm3 vs. normal shed with the approval ofthe Director ofthe Hawaii tn "Tropical Agriculture and Human Resources ax Journal Series no. 2799. Received 13 May 1985. Assistant professor, Dongguk Univ., Seoul, Kores, and pro- fessor of horticulture and genetics, Uni of Hawai, Honolulus HI 96822. corn silage (5,7). Animals were found to have greater dry matter intakes and feed efficiencies on the brown midrib stover or silage. ‘The brown midrib trait has not attracted com- mercial interest despite its nutritional advantages. Decreased yields and increased lodging have been the evident reasons. Frenchick et al. (7) reported a 16.6% reduction in silage yield of a bm3 hybrid vs. a normal that was nearly tsogenetic (16.1 Mg/ha or t/ha vs. 19. Mg/ha). Lowered grain yields of three bm3 hy- brids vs. their normal counterparts at an interme- diate stage in inbred conversion were observed by ‘Tuand Bauman (18). The three hybrids ranged from 17.6 to 23.5% yield reduction due to the mutant gene, with greater reduction at 54 000 plants/ha than at 32.000 plants/ha, The data suggested that the bm3 effect might be reduced by further backerossing, since more advanced conversions showed less reduction (24%) than earlier conversions (32%). Recent studies of Miller et al. (13, 14) showed a 23% reduction in rain yield and 10% reduction in stover yield of hy- rids based on first-generation inbreds, and 19% re- duction in dry matter accumulation of the inbreds. ‘The present study was conducted to determine the ‘effect of bm3 on grain and silage yields and on yield components, using isogenic hybrids drawn from dial- lel crosses. The study was designed to assess the sibility of identifying superior hybrids for the bm3 mutant, and for estimating combining ability for higher yield in the presence of this low-lignin gene. MATERIALS AND METHODS ‘Twelve inbreds and composites were converted in Hawaii to the m3 gene with five or more backcrosses, The gene was derived from H55bm3.X H49bm3 from the Maize Genetics Coop. Six of the most vigorous inbreds were cho- sen as parents for isogenic bm3 and normal diallels, includ- ing three tropical inbreds—Hi27, Hi28, and Hi34—and three temperate inbreds—B37, Mol7, and Oh645. All six inbreds resulted from earlier conversions in Hawai to genes, for resistance to MMV virus, common rust, and Northern, leaf blight (3,4,12). Yield trials of the 15 hybrids were planted on the Wai- manalo Research Station of the Univ. of Hawaii on 12 Aug.