Eur J Plant Pathol (2012) 133:631–644
DOI 10.1007/s10658-012-9942-3
Evaluation of resistance to Phomopsis stem blight
(caused by Diaporthe toxica) in Lupinus albus
Raymond B. Cowley & Gavin J. Ash &
John D. I. Harper & David J. Luckett
Accepted: 9 January 2012 / Published online: 8 February 2012
# KNPV 2012
Abstract Historically, in Australia, Phomopsis stem
blight in Lupinus albus crops is rare. However, in 2004
an outbreak of this disease occurred in southern New
South Wales affecting stems, pods and seeds of the
cultivar Kiev-Mutant. This virulent outbreak represents
a potential threat to the Australian lupin industry. The
current research was therefore initiated to optimise dis-
ease screening protocols for evaluation of the disease.
Screening for resistance to Phomopsis stem blight is
important because stubble is a potential high source of
inoculum in no-tillage systems, and grazing of Phomop-
sis infected stubble can cause lupinosis in stock animals.
A single spore isolate of the fungal pathogen Diaporthe
toxica collected from the 2004 outbreak was used to
assess the levels of disease resistance in stem tissue in
L. albus cultivars in some experiments and in other
experiments field-infected stubble was used as inoculum.
R. B. Cowley (*) : D. J. Luckett
EH Graham Centre for Agricultural Innovation (an alliance
between NSW Department of Primary Industries and Charles
Sturt University), NSW Department of Primary Industries,
Pine Gully Road,
Wagga Wagga, NSW 2650, Australia
e-mail: raymond.cowley@dpi.nsw.gov.au
G. J. Ash : J. D. I. Harper
EH Graham Centre for Agricultural Innovation (an alliance
between NSW Department of Primary Industries and
Charles Sturt University), School of Agricultural & Wine
Sciences, Charles Sturt University,
Locked Bag 588,
Wagga Wagga, NSW 2678, Australia
Resistance of current L. albus cultivars, breeding lines,
and landraces to D. toxica was assessed in both glass-
house and field experiments in 2007 and 2008. The
results showed that resistance existed in some cultivars
and several germplasm accessions. Environmental
effects and possible differences in pathogen race struc-
ture lead to some poor correlations of resistance ratings
between different experiments (ranging from r0−0.489
to 0.800, depending on the experiment and tissue
assessed). In field experiments consistent expression of
the disease was dependent on rainfall. Screening in an
irrigated disease nursery improved the methodology.
Nevertheless, results suggest that it will be possible to
develop new L. albus varieties that are resistant to Pho-
mopsis stem blight should the disease become wide-
spread in south-eastern Australia.
Keywords Phomopsis leptostromiformis . Broad-leaf
lupin
Introduction
Lupinus albus is an important legume crop in higher
rainfall regions in New South Wales (NSW), Australia,
with a production forecast of ~51 KT in 2011, but
production has reached 100 KT in previous years
(www.pulseaus.com.au accessed 1 Aug. 2011). This
study was prompted by an outbreak of Phomopsis stem
blight caused by Diaporthe toxica in southern NSW in
2004 which resulted in stock losses (Cowley et al 2010).
632
Previously D. toxica was regarded as only a minor
pathogen of L. albus in Australia, although it is a
common pathogen of L. angustifolius (Cowling et al
1986; Sweetingham et al. 1998). The outbreak
caused concern about future breeding efforts in
L. albus, and raised the question of whether exist-
ing host resistance was breaking down (Wood and
Allen 1980; Sweetingham et al. 1998). L. albus
cultivar Ultra has been reported resistant to Phomopsis
stem blight (Wood and Allen 1980) and the cultivar
Kiev-Mutant is reported to have useful resistance
under Australian commercial growing conditions
(Sweetingham et al. 1998), however, the stability
of this resistance is uncertain (Cowley et al. 2010).
Identifying new sources of genetic resistance, and
pyramiding existing disease resistance genes into elite
backgrounds, are key aims in plant breeding programs.
This relies on suitable screening protocols being estab-
lished to reliably screen large numbers of host genotypes
against the pathogen of concern (Russell 1978; Tivoli et
al. 2006). Resistance to Pleiochaeta setosa (Pleiochaeta
Root Rot) has been identified in L. albus breeding lines
and landraces and has been incorporated into modern
cultivars (Luckett et al. 2009; Wunderlich et al. 2008).
Two QTLs for resistance to Colletotrichum lupini (An-
thracnose) have been identified in Ethiopian L. albus
landraces (Phan et al. 2007) and transferred into modern
cultivars (Adhikari et al. 2009). However, because Pho-
mopsis stem blight was not considered a threat, limited
breeding effort has been directed at finding resistance to
D. toxica and transferring resistance genes into modern
L. albus varieties.
The specific aims of this study were: to deter-
mine the most suitable glasshouse disease screen-
ing method for Phomopsis stem blight resistance in
L. albus; to compare glasshouse to field screening; and
to evaluate a range of L. albus germplasm for resistance
to D. toxica.
Method and materials
All the experiments described were designed in R (R
Development Core Team 2010) using the DiGGer soft-
ware package in R (Cullis et al. 2006) (software avail-
able from http://www.austatgen.org/files/software/
downloads). Data analysis was undertaken using
ASReml in R (Butler et al. 2009). A list of genotypes
tested in this study is shown in Table 1.
Eur J Plant Pathol (2012) 133:631–644
Development of glasshouse screening methodologies
Experiment 1 Five parents of existing mapping popu-
lations were used—Kiev-Mutant, Rosetta and three
germplasm accessions P27174, P27593 and P28096.
These were inoculated by either stabbing with tooth-
picks colonised by D. toxica, injecting conidia into the
stems, or by spraying conidia onto the plants 42 days
after sowing. Sixteen seeds of each accession were sown
into 30 cm plastic pots in sandy loam. They were
thinned to 10 plants per pot 21 days after sowing. In
total there were 30 genotype × treatment combinations,
replicated three times in a randomised complete block.
At sowing, the pots were treated with Group G lupin
inoculant (Becker Underwood Pty Ltd, Somersby,
NSW, Australia). Liquid fertiliser (Thrive, Yates Pty
Ltd, Sydney, Australia) was applied as required, and
the pots were kept well-watered.
The toothpicks were prepared using a method modi-
fied from Keeling (1982). Briefly, boiled toothpicks
were autoclaved in 200 ml screw top jars. Sterile potato
dextrose broth (Sigma P6685) was added to the jar so
that about 1 cm of broth remained in the bottom of the jar
after the toothpicks had become saturated. Several agar
plugs from the advancing edge of a culture of D. toxica
isolate DAR80114 (Living Culture Collection, NSW
Department of Primary Industries, Orange, Australia)
were added to the jar, sealed, and incubated at 20°C with
a 12-h light period in a culture room. Uninoculated
toothpicks were used as a control.
A spore suspension of D. toxica isolate DAR80114
was prepared as described in Cowley et al. (2010),
adjusted to 107 spores per ml, and sprayed to runoff
using an atomiser. The remaining spore suspension
was injected into the stem (0.05μl) of each plant
between the 2nd and 3rd internodes using a gauge
22 continuous flow hypodermic needle. Sterile dis-
tilled water of the same volume was injected into
the stems of the plants in the control pots using a clean
needle.
Lesion length (mm) was recorded 70 days after
sowing on the stems of all plants in each pot. The
plants were maintained until maturity and senescence
and the number of plants in each pot that produced
visible pycnidia was recorded, giving an incidence of
pycnidia formation on mature plant stems. The lesion
length data was square-root transformed prior to
analysis.
Eur J Plant Pathol (2012) 133:631–644 633

Table 1 List of Lupinus albus and L. angustifolius genotypes Table 1 (continued)

tested for resistance to Diaporthe toxica in the experiments
Line Country Type Experiment
presented in this study. The country of origin for landraces
of Origin
indicates were the line was initially collected, for breeding lines
where created, and for cultivars the country of release. Experi- P27664 Turkey Landrace 3
ment 1 assessed glasshouse screening techniques, experiments 2
P27840 Syria Landrace 2,3
and 3 were glasshouse screening, and experiments 4 and 5 were
field screening P28096 Syria Landrace 1,2,3
P28199 Algeria Landrace 3
Line Country Type Experiment P28233 Ethiopia Landrace 3
of Origin
P28507 Ethiopia Landrace 2,3
75A:258 Morocco L. angustifolius 2,4,5 P28552 Ethiopia Landrace 3
breeding line P28561 Ethiopia Landrace 3
97B031-3 Australia Breeding line 3 P28573 Ethiopia Landrace 3
98B001-5-5 Australia Breeding line 2,4,5 P28752 Ukraine Cultivar 3
AMIGA Chile Cultivar 3 P28753 Ukraine Cultivar 3
ANDROMEDA Australia Cultivar 2,3,4,5 P28754 Ukraine Cultivar 3
ASTRA France Cultivar 2,3 P28974 Russian Breeding line 3
DANJA Australia L. angustifolius 2,4,5 Federation
cultivar P28975 Ukraine Breeding line 3
ESTA-1 South Africa Cultivar 3 P28978 Ukraine Breeding line 3
FP21 Germany Breeding line 3 P28979 Ukraine Breeding line 3
HAMBURG Germany Cultivar 2,3,4,5 P28980 Russian Breeding line 3
IDA Germany Cultivar 3 Federation
JINDALEE Australia L. angustifolius 5 P28981 Russian Breeding line 3
cultivar Federation
KIEV-MUTANT Ukraine Cultivar 1,2,3,4,5 P28983 Spain Breeding line 3
LA300-SD France Breeding line 3 P28984 Spain Breeding line 3
LAGO-AZZURRO Australia Cultivar 3 P28985 Italy Breeding line 3
LUBLANC-1 France Cultivar 3 P28986 Germany Breeding line 3
LUCKY-1 France Cultivar 3 P28989 Greece Landrace 3
LUTOP-1 France Cultivar 3 P28990 Morocco Landrace 2,3
LUXOR Australia Cultivar 2,3,4,5 P28991 Israel Landrace 2,3
MADEIRA Portugal Cultivar 3 P28993 Sudan Breeding line 3
MAGNA Australia Cultivar 2,3 P28994 Ukraine Breeding line 3
MINIBEAN Australia Cultivar 2,3 P28995 Russian Breeding line 3
Federation
MINORI Germany Cultivar 3 P28996 Spain Landrace 3
MULTOLUPA-2 Germany Cultivar 3 P28997 Spain Landrace 3
NEULAND Germany Cultivar 2,3 P28998 Spain Landrace 3
P20913 Egypt Cultivar 3 P28999 Poland Breeding line 3
P25758 Greece Landrace 2,3 P29000 Netherlands Breeding line 3
P25863 United Breeding line 3 P29002 USA Landrace 3
Kingdom
P26734 Hungry Landrace 3 P29003 Argentina Breeding line 3
P26777 Greece Landrace 3 P29005 Turkey Landrace 3
P26791 Syria Cultivar 3 P29017 Poland Breeding line 3
P27154 Spain Landrace 3 P29021 Germany Breeding line 3
P27172 Ethiopia Landrace 3 P29022 Germany Breeding line 2
P27174 Ethiopia Landrace 1,2,3 P29029 Germany Breeding line 2
P27277 Italy Landrace 3 ROSETTA Australia Cultivar 1,2,3,4,5
P27279 Italy Landrace 3 START Russian Cultivar 2,3
Federation
P27393 Syria Landrace 3 TYPTOP Chile Cultivar 3
P27433 Syria Landrace 3 ULTRA Germany Cultivar 2,3,4,5
P27441 Syria Landrace 3 UNICROP Australia L. angustifolius 2,4
P27593 Portugal Landrace 1,2,3 cultivar
P27662 Turkey Landrace 3 VLADIMIR Russian Cultivar 3
Federation
P27663 Turkey Landrace 3
634 Eur J Plant Pathol (2012) 133:631–644

Table 1 (continued) Unicrop and Danja are susceptible (Shankar et al.

Line Country Type Experiment 1996). Fifteen seeds were sown into each pot. No inoc-
of Origin ulant or fertiliser was added to the pots as the glasshouse
WALAB2008 Australia Breeding line 2,3,4,5
component of this experiment ceased after 24 days. The
WK134 Australia Breeding line 3 pots were well watered for the duration of the experi-
WK147 Australia Breeding line 3 ment. The plants were inoculated by placing approxi-
WK163 Australia Breeding line 3 mately 5 g of macerated infected stubble on the soil
WK172 Australia Breeding line 3 surface of each pot. The stubble was collected from the
WK188 Australia Breeding line 2,3,4,5 same field in Eurongilly, NSW, where a field disease
WK212 Australia Breeding line 3 screening experiment was conducted (see below). Pre-
WK236 Australia Breeding line 2,3,4,5
liminary experiments (data not shown) indicated that
WK255 Australia Breeding line 2,4,5
placing infected stubble collected from an infected field
WK262 Australia Breeding line 2,4,5
WK263 Australia Breeding line 2,4,5
resulted in high disease pressure. Latent stem infection
WK264 Australia Breeding line 2,4,5 (Cowling et al. 1996; Shankar et al. 1996) was assessed
WK266 Australia Breeding line 2,4,5 on excised stem portions 24 days after sowing as de-
WK268 Australia Breeding line 2,4,5 scribed by Cowley et al. (2010). Briefly, the internodes
WK271 Australia Breeding line 2,4,5 between the cotyledon and the first leaf were excised,
WK278 Australia Breeding line 2,4,5 surface sterilised before being rinsed in sterile distilled
WK279 Australia Breeding line 2,4,5 water and placed onto sterile filter paper in Petri dishes.
WK281 Australia Breeding line 2,4,5 The dishes were sealed in plastic bags in groups of 20 in
WK290 Australia Breeding line 2,3,4,5
a randomised design. The stem pieces were allowed to
WK292 Australia Breeding line 2,4,5
dry in the dishes for 7 days before being moistened with
WK302 Australia Breeding line 2,3,4,5
WK320 Australia Breeding line 2,3,4,5
3 ml sterile distilled water. The dishes were re-sealed in
WK325 Australia Breeding line 3 plastic bags and incubated on the laboratory bench at
WK326 Australia Breeding line 2,4,5 room temperature for a further 14 days. The treatment of
WK338 Australia Breeding line 2,3,4,5 the stem pieces was to simulate crop maturity and the
WK339 Australia Breeding line 2,4,5 onset of the saprophytic phase of pathogen after summer
XA100 France Cultivar 3 rain (Wood 1986). The stem portions were scored for the
MP selectionsa Australia Mapping 5 presence of symptoms using a 0 to 5 scale (where 00 no
(n022) population
disease, 50 stem portion completely diseased and pyc-
a
subset of non-bitter lines from a mapping population (Kiev- nidia formed; Shankar et al. 1996).
Mutant × P27174, as described in Phan et al 2007). Only non-
bitter, low alkaloid, L. albus can be sown in field trials in NSW
in order to prevent bitter contamination of breeding populations
and commercial seed lots (Richards et al 2008)
Experiment 3 An experiment was conducted in the
glasshouse to screen a genetically diverse set of L.
albus genotypes (Raman et al. 2008) for stem resis-
tance, using either externally-applied spores (spray
Glasshouse screening for resistance to D. toxica treatment), or injected spores. In this experiment, 95
L. albus genotypes were sown on 20 October 2008 in
Experiment 2 Forty-two genotypes of L. albus (11 sandy loam in 175-mm diameter pots in an
cultivars, 21 breeding genotypes, and 10 landraces) evaporative-cooled glasshouse at Wagga Wagga.
and three L. angustifolius genotypes were sown in These genotypes were the same set used to assess
175 mm diameter pots containing sandy-loam in a genetic diversity in L. albus breeding material using
randomised glasshouse trial in Wagga Wagga, NSW, SSR and DArT molecular markers (Raman et al.
Australia with four replicates to assess latent stem 2008). The experiment consisted of a split-plot design
infection to D. toxica. The L. angustifolius genotypes of three replicates sown in a 20×30 pot array. Each
were included as standards: 75A:258 is the major replicate (20×10) contained two main-blocks of 100
source of Phomopsis stem blight resistance in modern pots (10×10 array). Each main-block was split into
L. angustifolius varieties (Yang et al. 2002), while two sub-blocks (5×10 array). Inoculation treatments
Eur J Plant Pathol (2012) 133:631–644 635

were assigned to the sub-blocks. There were four sub- The experiment consisted of 30 genotypes (6 culti-
blocks in each replicate. vars, 21 breeding lines, plus three L. angustifolius gen-
Two inoculation treatments were used: either a otypes included as controls). Entries were sown as 10 m
conidial spore suspension of 5×106 spores per ml long single rows with four replicates in an 8×15 grid.
was sprayed to runoff and the plants enclosed by a Disease incidence relied on natural infection and suit-
large plastic sheet to maintain a dew period for 48 h or able environmental conditions. No supplementary inoc-
a 0.05μl aliquot of the prepared spore suspension was ulum or irrigation was provided. At maturity (12
injected into each stem between the 2nd and 3rd December 2007), 15 plants were randomly selected
internodes (as described above). Both inoculation from each row and assessed for Phomopsis stem blight
treatments were performed on the same day, 40 days using the 0 to 5 scale described by Cowley et al. (2010).
after sowing. Lesion length (mm) was measured Phomopsis pod blight was scored on each plant as the
28 days after inoculation. Incidence of pycnidia for- incidence of pods per plant with visual disease symp-
mation (presence/absence) was visually assessed toms (i.e. a presence/absence score).
98 days after sowing. Lesion length data was square-
root transformation before analysis.
Experiment 5 In 2008, 52 L. albus genotypes (6 culti-
Field screening for resistance to D. toxica vars, 21 breeding genotypes, a subset of 22 low-alkaloid
recombinant-inbred lines from a mapping population
Experiment 4 A replicated disease screening experi- (Phan et al. 2007), and three L. angustifolius genotypes)
ment was sown on 8 June 2007 at Eurongilly, NSW were sown in the field on 18 July 2008 in 10 m rows at
(−34.930°; 147.767°) in a field which had a history of a Wagga Wagga, NSW (−35.050º; 147.350º). The experi-
severe D. toxica epidemic on L. albus in 2005. Despite ment was irrigated using in-line drippers from August to
the field having a cereal crop in 2006, dry seasonal November as required. Weather conditions for Wagga in
conditions through 2006 meant that considerable 2008 are shown in Fig. 1. The trial was sown in a 6×40
infected lupin stubble remained on the soil surface in array. Most genotypes were sown with five replicates.
2007. During this experiment in 2007 there was low The three L. angustifolius genotypes and seven L. albus
rainfall during August, September and October (14.6, genotypes (Kiev-Mutant, Ultra, Hamburg, Rosetta, An-
17.4 and 19.8 mm respectively, Fig. 1). dromeda, WALAB2008 and WK264) were replicated
twice with 10 rows each across the whole experiment.
Eurongilly 2007
Ave. Maximum Temperature
Due to limited seed supply, each mapping population
40 40
Ave. Minimum Temperature
S own S c ored
entry was sown with two replicates. The DiGGer soft-
30 30 ware was used to produce a spatially efficient experi-
20 20
mental design incorporating the unequal replication.
A susceptible disease spreader, Kiev-Mutant, was
Temperature (oC)

10 10
Rainfall (mm)

sown in every third row. Ninety days after sowing the

0 0 experiment was inoculated by stabbing plants in the
Wagga Wagga 2008
spreader rows with phomopsis-inoculated toothpicks
40 40
as described above. The toothpicks were inoculated
Sown Inoculated Scored
30 30 with isolate DAR80114 and inserted into the stem just
below the developing pods on 14 October 2008 and
20 20
left in place for the duration of the trial. Approximate-
10 10 ly 6–10 plants were inoculated per 10 m spreader row,
0 0
with 120 spreader rows inoculated throughout the
trial. This method was designed to mimic a late-
Jan

Feb
Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

season phomopsis epidemic.

Fig. 1 Weather conditions for the field screening experiments The disease level was scored at harvest by recording
conducted at Eurongilly in 2007 and Wagga Wagga in 2008.
the incidence of infection in stems and pods in the
The arrows indicate when each trial was sown and scored.
Weather data was sourced from archived files at http://www. central 5-m section of each row, and expressed as a
weatherzone.com.au/ proportion of the total number of plants in the section.
636 Eur J Plant Pathol (2012) 133:631–644

Results 1.0 KIEV-MUTANT

ROSETTA
P27593
Glasshouse screening methodology

Incidence of Pycnidia Formation

0.8
KIEV-MUTANT
In this experiment the genotype × inoculation effects P27174
ROSETTA
P28096
were significant (P<0.001). The L. albus landraces P27593
P27593
0.6 P28096
P28096 and P27174 had the least disease when conid- P27174

ia of D. toxica were applied to the stem surface (pre- ROSETTA

KIEV-MUTANT
dicted mean square-root lesion length 0.46 and 1.51
0.4
respectively, Fig. 2). Kiev-Mutant, Rosetta and
P27593 were not significantly different when inocu- LSD5%

lated by spraying conidia (ranging from 3.12 to 3.84).

0.2 P27174
The toothpick method, the most aggressive and most Inoculation method
damaging of the three inoculation methods, resulted in Inject
Spray
the largest stem lesions and the highest incidence of 0.0
P28096 Toothpick

plants developing visible pycnidia (Fig. 3). However, 5 10 15

genotypes P27174 and P28096 showed significantly
less incidence of pycnidia formation compared with
the other genotypes (Fig. 3). The injection conidia into
stems method also had high incidence of pycnidia
formation but smaller lesions than the toothpick meth-
od. The five genotypes were very similar in their mean
responses for the toothpick and needle inoculation
methods. The spray inoculation treatment gave the
greatest discrimination between genotypes with
Rosetta and Kiev-Mutant being the most susceptible.
P27174 and P28096 were resistant.
Glasshouse screening
In the first screening experiment (Experiment 2) the
effect of genotype on latent stem infection was signif-
icant (P<0.001) and genotype-predicted means ranged
Control:
Inoculation: Spray
Control:
Inoculation: Inject
Lesion length (sqaure-root)
Lesion Length (square-root)
Fig. 3 Phomopsis stem blight severity (caused by Diaporthe
toxica) on Lupinus albus in glasshouse trial scored as lesion
length 28 days after inoculation using three inoculation methods
and the incidence of pycnidia formation scored on mature plants
at harvest
from 1.61 to 3.60 (Fig. 4). The most resistant genotype
was P28096, confirming the resistance of this geno-
type to Phomopsis stem blight in the previous exper-
iment (Fig. 3, spray treatment). Although the narrow-
leaf lupin breeding line 75A:258 was included as it is
highly resistant to D. toxica (Shankar et al. 1996) it
was amongst the least resistant lines in this experiment
(predicted mean 3.50) and was not significantly dif-
ferent to the two other L. angustifolius control geno-
types known to be susceptible to D. toxica, cultivars
Danja and Unicrop (Shankar et al. 1996).
Spray
In the second screening experiment (Experiment
Inject
LSD5%
3) the effect of genotype, inoculation and their

Control: Toothpick
15 Inoculation: Toothpick interaction were significant (P <0.001) on lesion
length score. For incidence of pycnidia formation
the effect of genotype was significant (P<0.001)
10
and the genotype by inoculation interaction was
significant (P<0.05). For the lesion length score
several landraces were resistant to Phomopsis stem
5
blight with the spray inoculation method. The
most resistant genotypes were breeding lines
P28979, P28978, P28975 all from the Ukraine
KIEV-MUTANT P27174 P27593 P28096 ROSETTA
(predicted means of 0.80, 0.82 and 0.96, respec-
Fig. 2 Effect of inoculation treatment on Phomopsis Stem tively, Fig. 5). It is possible that the same genetic
Blight of Lupinus albus stems caused by Diaporthe toxica control is responsible for resistance in these three
Eur J Plant Pathol (2012) 133:631–644 637

Fig. 4 Latent Phomopsis

stem blight (caused by Dia-
porthe toxica) on excised
stems of Lupinus albus. 4

Latent Phomopsis stem blight score

Bars shown in white are L.
LSD5%
angustifolius included as
control lines
3

P28096
P28507
MAGNA
WK188
P28991
START
ROSETTA
LUXOR
P27840
WK338
WK339
WK302
ASTRA
MINIBEAN
WK290
ANDROMEDA
P29022
ULTRA
WK292
P25758
WK281
KIEV-MUTANT
WK263
WK326
P27174
WK278
WK268
P28990
WK262
WK255
WK266
P27593
98B001-5-1
WK320
HAMBURG
WK236
NEULAND
98B001-5-5
WK264
DANJA
WK279
WK271
75A-258
UNICROP
P29029
breeding lines. P28096 (landrace from Syria) and and P27662 were also susceptible (9.45, 9.33 and
P27664 (landrace from Turkey) were also resistant 8.75, respectively). The predicted mean of Kiev-
(predicted means of 1.02 and 1.09, respectively, Mutant and Rosetta were 2.68 and 6.35, respec-
Fig. 5). The most susceptible genotypes were tively. All of the L. albus cultivars grown commer-
breeding lines P28999 and P29017 from Poland cially in Australia had similar incidences of pycnidia
(10.53 and 10.02, respectively). Lucky-1, Start formation in the spray treatment (ranging from 0.32 to

Inject conidia
15
Square-root transformed lesion length (mm)

LSD5%

10

Spray conidia
15
LSD5%

10

5
WK320
P28978
ULTRA

P28979
P28507
P28561
P28096
WK212
P26734
XA100
P28975
WK147

WK338
P28754
P27433
MAGNA

FP21

IDA
WALAB2008

MADEIRA

P28984
P27441
P25758
WK236

WK172
WK325
WK290
P28994
P28752

WK188
MINORI
P27279
P28980
P28573
P28552
P28753

P28996
P28985
AMIGA

ASTRA
LUBLANC-1

LUTOP-1
WK134

P28989
P27664
ESTA-1
P28986
P27174
P28997
P27840
WK163

P20913
P28199
HAMBURG

MINIBEAN

VLADIMIR
P27393
P28981
NEULAND

P27593
P29021
P28995
TYPTOP
WK302
P27154
LUXOR
P27172
P28993
P28983
P28998
97B031-3
P27277
P28974
P27663
ROSETTA
P28233
P29000

P25863

P29005
START
P28990
P28991
P26791
ANDROMEDA

KIEV-MUTANT

MULTOLUPA-2

LA300-SD

LUCKY-1
P29017
P29003
P29002
P27662
P28999
LAGO-AZZURRO

Fig. 5 Lesion length of Phomopsis stem blight (Diaporthe toxica) in glasshouse screening experiment of Lupinus albus comparing the
inoculation methods of injecting conidia into stems, versus spraying conidia onto stems. Certain key lines are shown with white bars
638 Eur J Plant Pathol (2012) 133:631–644

0.46, Fig. 6), but varied in their lesion length scores using Pearson’s product moment correlation coeffi-
(ranging from 1.45 to 6.35) with Andromeda and Ultra cient (r 00.23, P < 0.05). Landraces P28974 and
having the smallest lesion length. Landraces P26734, P26791 were resistance and moderately resistant to
P27840, P28507 and P28980 had the lowest incidence Phomopsis stem blight when the spores were applied
of pycnidia formation (<0.10, Fig. 6) and may represent externally (1.59 and 2.88, respectively). In contrast
a potential source of resistance to Phomopsis stem blight. when the spores were applied internally via injection
The two inoculation treatments in this experiment they were the most susceptible genotypes tested (11.92
were contrasting in that the spray method applied and 13.75, respectively, Fig. 5).
spores to the exterior of the plant and epidermal resis-
tance mechanisms (if they exist) may affect the out- Field screening
come. In the method by injecting conidia into stems,
the spores are introduced into the interior of the plant Dry conditions during August to October 2007 (Fig. 1)
thereby bypassing any epidermal influence. This tech- restricted plant growth and disease development in
nique was of interest because hail damage of an Experiment 4. Nevertheless, significant differences
L. albus crop in 2004 had helped contribute to a severe (P<0.001) in Phomopsis stem and pod blight were
Phomopsis epidemic (Cowley et al. 2010). One of the found. Most of the cultivars included in this experi-
aims of this experiment was to determine if resistance ment had similar levels of Phomopsis stem blight,
to D. toxica can be found in internal tissues, that is, based on a 0 to 5 score (ranging from 2.52 to 2.76,
once epidermal resistance is bypassed. This situation Fig. 7), with Hamburg being the exception (stem score
is likely to occur following a wounding event such as of 1.80). WALAB2008, WK339, WK278 and WK320
hail or insect damage. D. toxica spores are normally were the best performing breeding lines under the test
wind and water dispersed (Wood 1986). conditions with the lowest stem disease scores (stem
No genotypes were identified that had resistance to score <1.84, Fig. 7). With the L. angustifolius lines
Phomopsis stem blight when the spores were applied Unicrop had the highest Phomopsis stem blight sever-
sub-epidermally by injecting conidia into the stems ity followed by Danja and 75A:258 (stem scores of
(Fig. 5). There was, however, a moderate but signifi- 2.74, 2.65 and 2.12, respectively). Lines WK320,
cant correlation between the two inoculation methods WK236, Ultra and WK278 had the lowest incidence
of Phomopsis pod blight (0.18, 0.20, 0.21, and 0.21,
0.8 Key to symbols START
respectively). A group of sister lines, that is selections
Breeding lines
Cultivars
from the same cross—WK262, WK263, WK264 and
Landraces
WK266—had the highest incidence of infected pods
per plant (pod infection incidence >0.40, Fig. 7), con-
Incidence of pycnidia formation

0.6 firming previous glasshouse screening observations

(Cowley et al. 2010).
ULTRA
Environmental conditions for field screening in
0.4
KIEV-MUTANT
ANDROMEDA
LUXOR 2008, Experiment 5, were conducive for disease de-
HAMBURG
velopment (Fig. 1). Supplementary irrigation was re-
ROSETTA

P28096 P27593
quired throughout August to October. Several storm
WALAB2008 P27174
NEULAND
events in November aided the spread of the pathogen.
0.2 P25758 Genotype differences in both Phomopsis stem and pod
P27664 MADEIRA
P28980
blight were significant (P<0.001). Rosetta was resis-
WK236
P28507 LSD5%
tant to Phomopsis stem blight in this experiment (pre-
P27840

0.0
P26734
dicted mean incidence 0.17, Fig. 8). The breeding
2 4 6 8 10
lines WK266, WK279 and WK188 also had low stem
Lesion length (mm) infection incidence (0.11, 0.17 and 0.19 respectively).
The most resistant genotype was a mapping popula-
Fig. 6 Lesion length of Phomopsis stem blight (Diaporthe
toxica) and incidence of pycnidia formation in a glasshouse
tion line MP-A-153 (0.10). Luxor was the poorest
screening experiment of Lupinus albus inoculated by spraying performing cultivar tested (predicted mean incidence
conidia 0.41). The eight most susceptible genotypes were all
Eur J Plant Pathol (2012) 133:631–644 639

Fig. 7 Phomopsis pod and Pod

0.6
stem blight of Lupinus albus
genotypes scored at harvest

0.5
in a replicated field disease LSD5%
nursery at Eurongilly, NSW

0.4
in 2007. L. angustifolius
genotypes are shown as

Disease severity scores

0.3
white bars

0.2
Stem

3.0
LSD5%

2.5
2.0
1.5

WALAB2008

ANDROMEDA
WK339
WK278
HAMBURG
WK320
WK236
WK271
WK326
75A-258
98B001-5-5
WK279
WK290
WK281
KIEV-MUTANT
ULTRA
ROSETTA
WK268
UNICROP
DANJA
WK302

WK255
WK338
LUXOR
WK292
WK188
WK263
WK266
WK264
WK262
mapping population lines (predicted means >0.54). Jindalee (moderately resistant) and Danja (susceptible,
Breeding lines WK271, WK320, WK236 and Shankar et al. 1996) had predicted mean incidences for
WALAB2008 were also susceptible (0.47, 0.49, 0.53 Phomopsis stem blight of 0.41 and 0.40, and for Pho-
and 0.53, respectively). Kiev-Mutant had a predicted mopsis pod blight 0.38 and 0.33, respectively (Fig. 8).
mean incidence of 0.25 for stem infection. Rosetta was
the genotype most resistant to Phomopsis pod blight
(predicted mean incidence 0.13, Fig. 8). The correla- 0.7
tion between stem infection and pod infection was Key to symbols
Breeding line
significant (r00.82, P<0.001). Cultivar
0.6 Mapping pop.
The predicted mean incidence for the selected map- L. angusifolius
Pod infection incidence

ping population entries ranged from 0.10 to 0.58 for

75A:258
Phomopsis stem blight, and 0.12 to 0.71 for Phomop- 0.5
sis pod blight implying that the population is segre-
gating for resistance in both stems and pods. Further 0.4
work is required to determine the nature of the genetic JINDALEE

resistances that exist in the population. HAMBURG DANJA

0.3
In the field screening conducted in 2008, the L.
angustifolius breeding line 75A:258, included as a ANDROMEDA LUXOR

“highly resistant” control (Shankar et al 1996), per- 0.2 KIEV-MUTANT

LSD5%
formed poorly and had a greater disease incidence than ULTRA
ROSETTA
the other L. angustifolius controls and significantly 0.1
higher disease incidence than Rosetta (P < 0.05). 0.1 0.2 0.3 0.4 0.5 0.6 0.7
75A:258 was replicated in 10 rows throughout the ex- Stem infection incidence
periment and had a predicted mean incidence for Pho-
Fig. 8 Phomopsis pod and stem blight caused Diaporthe toxica
mopsis stem blight of 0.59 and Phomopsis pod blight of in an irrigated field disease nursery at Wagga Wagga, NSW, in
0.51 (Fig. 8). The other L. angustifolius controls, 2008
640 Eur J Plant Pathol (2012) 133:631–644

Correlations between glasshouse screening and field for both stem and pod scores in either field screening
screening experiments experiment.

The Pearson product-moment correlation between the Sources of resistance

two glasshouse screening experiments was poor (r0
0.11) and was not significant. For these two experi-
ments the inoculum source varied, as did the methods
used to assess disease response (Table 2). In the first
glasshouse screening experiment infected stubble was
used, whereas the second glasshouse screening exper-
iment was inoculated with a single spore isolate.
The different environments and potentially differ-
ent isolate combinations in the field disease screening
may partially explain the negative correlation between
the stem scores in the two experiments (r0-0.46, P<
0.05). Also, the experiment conducted in 2008 used
spreader rows to aid pathogen dispersal, whereas the
experiment in 2007 relied on natural inoculum. Both
experiments experienced low rainfall during the spring
growing season (August–October, Fig. 1) and only the
experiment conducted in 2008 was provided with sup-
plementary irrigation.
The correlations between the two glasshouse
screening experiments and the two field screening
experiments were varied. There was significant corre-
lation (P<0.001) between the first glasshouse experi-
ment and the stem and pod scores in field screening in
2008 (r00.47 and r00.61, respectively), and no cor-
relation with field screening in 2007. The second
glasshouse experiment had no significant correlation
The glasshouse and field screening reported here
showed that Rosetta has useful resistance under field
conditions, although may succumb to Phomopsis stem
blight when disease pressure is high. The resistance of
Ultra was confirmed and a further 17 genotypes were
identified as being potential new resistance donors for
breeding (Table 3).
Discussion
The objective of this research was to evaluate screen-
ing methodology to determine if genetic variability
existed in L. albus germplasm to Phomopsis stem
blight caused by D. toxica and to identify genotypes
with superior resistance that maybe useful in resis-
tance breeding.
A useful disease screening method for breeding must
be reliable and give consistent results (Tivoli et al.
2006). A large and variable contribution from uncon-
trolled environmental factors may not be acceptable
(Luckett et al. 2008). In this study both glasshouse and
field screening was attempted. In all cases, there were
genotypic differences but there was not always agree-
ment between experiments of resistance rankings due to

Table 2 Summary of details for each experiment reported in this study on Phomopsis stem blight caused by Diaporthe toxica in
Lupinus albus

Experiment Location Year Lines Inoculation method Pathogen source Scoring method

1 Glasshouse 2007 5 Spray DAR80114 a Length; pycnidia incidence b

Toothpick DAR80114 Length; pycnidia incidence

Injection DAR80114 Length; pycnidia incidence
c d
2 Glasshouse 2007 45 Infected stubble Eurongilly 0–5 scale
3 Glasshouse 2008 95 Spray DAR80114 Length; pycnidia incidence
Injection DAR80114 Length; pycnidia incidence
4 Field 2007 30 None (natural) Unknown 0–5 scale
5 Field 2008 52 Spreader rows (with toothpicks) DAR80114 plus natural Incidence
a
Single spore isolate of Diaporthe toxica sourced from infected seed from Tarcutta NSW (Cowley et al. 2010)
b
Incidence of pycnidia on mature stems was scored as a proportion of plants in each experimental unit (pots or rows) with obvious
pycnidia
c
Stubble sourced from site of Experiment 4
d
0–5 scale presented in Cowley et al. (2010)
Eur J Plant Pathol (2012) 133:631–644 641

Table 3 Lupinus albus lines resistant to Phomopsis stem blight caused by Diaporthe toxica

Lupin line Synonym or cross Country of origin Type Other reported disease resistance

ANDROMEDA (Kiev-Mutant/P27174) Australia Cultivar Colletotrichum lupini (Adhikari et al. 2009)

LUBLANC-1 Intervarietal selection France Cultivar
MADEIRA Portugal Cultivar
P27664 FRA6707B Turkey Landrace
P27840 SYR6728B Syria Landrace Pleiochaeta setosa (Luckett et al. 2009)
P28096 SYR6728B Syria Landrace Pleiochaeta setosa (Luckett et al. 2009)
P28507 ETH065 Ethiopia Landrace Colletotrichum lupini (Adhikari et al. 2009)
P28754a BORKI Ukraine Cultivar
P28974 KVIR1597 Russian Federation Breeding line
P28975 KVIR1640 Ukraine Breeding line
P28978 KVIR2238 Ukraine Breeding line
P28979 KVIR2240 Ukraine Breeding line
P28980a KVIR2499 Russian Federation Breeding line
P28986 BR36415 Germany Breeding line
ULTRA Germany Cultivar
WALAB2008 (Kiev-Mutant/P27174) Australia Cultivar Colletotrichum lupini (Adhikari et al. 2009)
WK147 Australia Breeding line Pleiochaeta setosa (unpublished data)
WK320 Australia Breeding line Pleiochaeta setosa (unpublished data)
a
Genotype found to contain a non-pauper allele for low alkaloid (data not shown)

the different environments in each experiment. Certain
genotypes (most of the “P” lines in this study) that had
been examined in glasshouse experiments could not be
studied in the field because they contained alleles for
high alkaloid content or alleles other than those used in
commercial crops to condition the low-alkaloid trait
(Lin et al. 2009). These undesirable genes could have
spread by pollen movement to neighbouring commer-
cial L. albus crops (Richards et al. 2008).
The totally natural infection in the drought-affected
year 2007 (without irrigation) may not have reflected
true genotype response to disease pressure. Conse-
quently, disease screening in drought conditions may
not be sufficient for reliable, repeatable and efficient
screening for plant breeding purposes. As a result
spreader rows and irrigation were employed in 2008,
in which 10 of the top 12 genotypes for Phomopsis
pod blight resistance were either breeding lines or
released cultivars. Also in field screening in 2008, 17
of the 19 most susceptible lines were from an unse-
lected mapping population (Phan et al. 2007).
The entries from the mapping population (Kiev-
Mutant ×P27174, Phan et al. 2007) were included in
this experiment to gauge whether or not the population
is segregating for either Phomopsis stem blight or
Phomopsis pod blight, or both. The mapping popula-
tion is segregating for low-alkaloid alleles causing the
selection of mapping population entries to be biased.
Nevertheless the spread of the mapping population
entries (ranging from 0.10 to 0.69 for stems and 0.12
to 0.71 for pods) implies that it is segregating for
resistance in both stems and pods. The mapping pop-
ulation is segregating for pod resistance but not leaf
resistance (Cowley et al. 2011). Further work is re-
quired to determine the nature of genetic resistances in
the population.
There were differences in the inoculum sources
used in the various experiments, although an attempt
was made in most cases to use the isolate originating
from Tarcutta, NSW, or the single spore isolate from
Tarcutta, DAR80114 (Cowley et al. 2010). This isolate
has been demonstrated as capable of causing Phomop-
sis blight in leaves, stems and pods of L. albus geno-
types (Cowley et al. 2010). The rationale was to
determine if the fungus had overcome the long-term
moderate resistance that had existed in L. albus culti-
vars in Australia (Sweetingham et al. 1998). Based on
the results of this study, cultivars Rosetta and Ultra
642
would be recommended for growing in south-eastern
Australia in areas where Phomopsis stem blight may be
prevalent, although, in growing seasons and environ-
ments with high disease pressure, genetic resistance
within Rosetta may be overcome. Also, results from
field screening in drought-affected seasons may induce
increased susceptibility of Rosetta. The interaction be-
tween disease and moisture stress needs further research.
For routine disease screening for breeding, field evalua-
tion requires suitable environmental conditions to ex-
press disease reliably (Wood 1986) and is aided by the
addition of irrigation.
Several potentially new sources of resistance to
Phomopsis stem blight using the Tarcutta isolate were
identified in this study. Genotypes P28096, P27840,
WK147 and WK320 are also resistant to Pleiochaeta
Root Rot, caused by the fungal pathogen Pleiochaeta
setosa (Kirchn.) S. Hughes (Luckett et al. 2009). An-
dromeda, P28507 and WALAB2008 have resistance
to Colletotrichum lupini (Bondar) Nirenberg (Adhikari
et al. 2009). It has not been established whether these
genotypes have genes for resistance against multiple
pathogens.
The genotypes P28980 and P28754 are resistant to
Phomopsis stem blight but have been found to contain
non-pauper genes for controlling the low-alkaloid trait
in domesticated L. albus (unpublished data). Care
should be exercised when using these lines in breeding
with genotypes containing the pauper gene. Such
crosses will result in high alkaloid progeny (Lin et
al. 2009) and may lead to bitter contamination in
breeding populations and commercial crops (Richards
et al. 2008).
Although resistance to externally applied spores
was identified, no lines were found to have resistance
to D. toxica when the spores were applied internally,
in this study via injection or stabbing with inoculated
toothpicks. This strongly implies that all the L. albus
genotypes tested are vulnerable to Phomopsis stem
blight if infection coincides with a wounding event,
such as hail or possibly insect damage. Some geno-
types, however, were markedly more susceptible to
internally applied spores, in particular genotypes
P26791, P28974 and P28991. The outbreak of Pho-
mopsis stem blight in 2004 (Cowley et al. 2010) that
prompted this study followed a late-season hail event.
The highly resistant L. angustifolius breeding line
75A:258 (Shankar et al. 1996) was included in three
experiments reported in this paper as a control line. In
Eur J Plant Pathol (2012) 133:631–644
each of the experiments it was susceptible to Phomop-
sis stem and pod blight when challenged with the
Tarcutta isolate of D. toxica. This line has been
used extensively in breeding for Phomopsis resis-
tant L. angustifolius cultivars (Shankar et al. 2002)
and has been used to develop molecular markers
for disease resistance using microsatellite-anchored
fragment length polymorphism (MFLP) (Yang et al.
2002). 75A:258 contains a single, dominant gene
(named Phr1) for strong resistance to Phomopsis
and may be subject to unlinked modifier genes
(Shankar et al. 2002). It would appear from these
results that the resistance provided by Phr1 has
been overcome by the Tarcutta isolate of the pathogen
in L. angustifolius.
In future L. albus breeding work the method recom-
mended for routine glasshouse screening for disease
resistance to Phomopsis stem blight is to use spray
inoculation and to score lesion length. This differs from
reports of the established protocols for assessing
Phomopsis stem blight in L. angustifolius which exam-
ined latent stem infection using excised stem portions
(Cowling et al 1996; Shankar et al 1996; Williamson
et al 1991). With L. angustifolius the pathogen invades
stem tissue whilst the plant is growing and forms coral-
loid hyphae structures under the epidermis (Williamson
and Sivasithamparam 1994; Williamson et al. 1991).
These appear to remain more or less in stasis until plant
maturity and senescence, when the fungus proliferates
on the stubble in response to rainfall (Williamson et al.
1991). The lengthy latent phase in L. angustifolius is
generally symptomless (Cowling et al. 1984; Wood and
Sivasithamparam 1989). The latent infection structures
vary greatly in size and number and have been used to
discriminate between resistant and susceptible L. angus-
tifolius genotypes (Shankar et al. 1996; Williamson et
al. 1991). In contrast to L. angustifolius, it has not been
established whether a lengthy latent phase occurs when
L. albus tissue is colonised with D. toxica. Using tooth-
pick inoculations on L. albus, L. angustifolius, L. luteus
and L. mutabilis, Jaarsveld and Knox-Davies (1974)
noted that only L. albus developed symptoms while
green. In glasshouse trials, Kochman and Kubicka
(1974) were able to infect L. albus stems with droplets
containing only five spores. When assessing the forma-
tion of coralloid hyphae, Williamson et al. (1991) failed
to find coralloid hyphae on the small number of L. albus
plants tested. We have observed and reported here
lesions developing on green stems of L. albus after
Eur J Plant Pathol (2012) 133:631–644
inoculating with conidia, which supports previous find-
ings (Cowley et al 2010). This study did not examine
coralloid hyphae structures because lesion length meas-
urements were quick and easy to undertake.
This study highlights the importance of stem resis-
tance and the development of a stem-based screening
protocol to screen breeding material. Stem resistance is
important because stem pieces are the largest component
of L. albus stubbles, and if contaminated stubble is
grazed by sheep in significant quantities sheep poisoning
by lupinosis becomes a serious risk (Allen 1986). In
addition, stubble is the main source of spores for future
infection, so any reduction in disease development on
stems and subsequently on stubble due to genetic resis-
tance can be beneficial to subsequent or neighbouring
crops (Cowling et al. 1986). The problem of a stubble
as a source of spores is especially important in
minimum tillage situations where stubble may be
left on the soil surface for several years and can
sporulate freely. Work is continuing to uncover the
genetic control of resistance to Phomopsis blight in both
stems and pods of L. albus, and to find molecular
markers, so that marker-assisted selection can be used
by L. albus breeders in the future.
Acknowledgements Mr Tom Brabbin is thanked for the use
of his farm to screen for Phomopsis stem and pod blight, and the
supply of infected lupin stubble used in glasshouse screening.
David Roberts and Mark Richards are thanked for technical
assistance. Dr Neil Coombes is thanked for assistance with
experimental design.
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