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 stemblight in Lupinus albus crops is rare. However, in 2004an outbreak of this disease occurred in southern NewSouth Wales affecting stems, pods and seeds of thecultivar Kiev-Mutant. This virulent outbreak representsa potential threat to the Australian lupin industry. Thecurrent research was therefore initiated to optimise dis-ease screening protocols for evaluation of the disease.Screening for resistance to Phomopsis stem blight isimportant because stubble is a potential high source ofinoculum 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 Diaporthetoxica collected from the 2004 outbreak was used toassess the levels of disease resistance in stem tissue inL. albus cultivars in some experiments and in otherexperiments field-infected stubble was used as inoculum.

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. Theresults showed that resistance existed in some cultivarsand several germplasm accessions. Environmentaleffects and possible differences in pathogen race struc-ture lead to some poor correlations of resistance ratingsbetween different experiments (ranging from r0−0.489to 0.800, depending on the experiment and tissueassessed). In field experiments consistent expression ofthe disease was dependent on rainfall. Screening in anirrigated disease nursery improved the methodology.Nevertheless, results suggest that it will be possible todevelop 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-leaflupin

Introduction

Lupinus albus is an important legume crop in higherrainfall regions in New South Wales (NSW), Australia,with a production forecast of ~51 KT in 2011, butproduction has reached 100 KT in previous years(www.pulseaus.com.au accessed 1 Aug. 2011). Thisstudy was prompted by an outbreak of Phomopsis stemblight caused by Diaporthe toxica in southern NSW in2004 which resulted in stock losses (Cowley et al 2010).

Eur J Plant Pathol (2012) 133:631–644DOI 10.1007/s10658-012-9942-3

R. B. Cowley (*) :D. J. LuckettEH Graham Centre for Agricultural Innovation (an alliancebetween NSW Department of Primary Industries and CharlesSturt University), NSW Department of Primary Industries,Pine Gully Road,Wagga Wagga, NSW 2650, Australiae-mail: raymond.cowley@dpi.nsw.gov.au

G. J. Ash : J. D. I. HarperEH Graham Centre for Agricultural Innovation (an alliancebetween NSW Department of Primary Industries andCharles Sturt University), School of Agricultural & WineSciences, Charles Sturt University,Locked Bag 588,Wagga Wagga, NSW 2678, Australia

Previously D. toxica was regarded as only a minorpathogen of L. albus in Australia, although it is acommon pathogen of L. angustifolius (Cowling et al1986; Sweetingham et al. 1998). The outbreakcaused concern about future breeding efforts inL. albus, and raised the question of whether exist-ing host resistance was breaking down (Wood andAllen 1980; Sweetingham et al. 1998). L. albuscultivar Ultra has been reported resistant to Phomopsisstem blight (Wood and Allen 1980) and the cultivarKiev-Mutant is reported to have useful resistanceunder Australian commercial growing conditions(Sweetingham et al. 1998), however, the stabilityof this resistance is uncertain (Cowley et al. 2010).

Identifying new sources of genetic resistance, andpyramiding existing disease resistance genes into elitebackgrounds, are key aims in plant breeding programs.This relies on suitable screening protocols being estab-lished to reliably screen large numbers of host genotypesagainst the pathogen of concern (Russell 1978; Tivoli etal. 2006). Resistance to Pleiochaeta setosa (PleiochaetaRoot Rot) has been identified in L. albus breeding linesand landraces and has been incorporated into moderncultivars (Luckett et al. 2009; Wunderlich et al. 2008).Two QTLs for resistance to Colletotrichum lupini (An-thracnose) have been identified in Ethiopian L. albuslandraces (Phan et al. 2007) and transferred into moderncultivars (Adhikari et al. 2009). However, because Pho-mopsis stem blight was not considered a threat, limitedbreeding effort has been directed at finding resistance toD. toxica and transferring resistance genes into modernL. 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 inL. albus; to compare glasshouse to field screening; andto evaluate a range of L. albus germplasm for resistanceto D. toxica.

Method and materials

All the experiments described were designed in R (RDevelopment 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 usingASReml in R (Butler et al. 2009). A list of genotypestested in this study is shown in Table 1.

Development of glasshouse screening methodologies

Experiment 1 Five parents of existing mapping popu-lations were used—Kiev-Mutant, Rosetta and threegermplasm accessions P27174, P27593 and P28096.These were inoculated by either stabbing with tooth-picks colonised by D. toxica, injecting conidia into thestems, or by spraying conidia onto the plants 42 daysafter sowing. Sixteen seeds of each accession were sowninto 30 cm plastic pots in sandy loam. They werethinned to 10 plants per pot 21 days after sowing. Intotal there were 30 genotype × treatment combinations,replicated three times in a randomised complete block.At sowing, the pots were treated with Group G lupininoculant (Becker Underwood Pty Ltd, Somersby,NSW, Australia). Liquid fertiliser (Thrive, Yates PtyLtd, Sydney, Australia) was applied as required, andthe pots were kept well-watered.

The toothpicks were prepared using a method modi-fied from Keeling (1982). Briefly, boiled toothpickswere autoclaved in 200 ml screw top jars. Sterile potatodextrose broth (Sigma P6685) was added to the jar sothat about 1 cm of broth remained in the bottom of the jarafter the toothpicks had become saturated. Several agarplugs from the advancing edge of a culture of D. toxicaisolate DAR80114 (Living Culture Collection, NSWDepartment of Primary Industries, Orange, Australia)were added to the jar, sealed, and incubated at 20°Cwitha 12-h light period in a culture room. Uninoculatedtoothpicks were used as a control.

A spore suspension of D. toxica isolate DAR80114was prepared as described in Cowley et al. (2010),adjusted to 107 spores per ml, and sprayed to runoffusing an atomiser. The remaining spore suspensionwas injected into the stem (0.05μl) of each plantbetween the 2nd and 3rd internodes using a gauge22 continuous flow hypodermic needle. Sterile dis-tilled water of the same volume was injected intothe stems of the plants in the control pots using a cleanneedle.

Lesion length (mm) was recorded 70 days aftersowing on the stems of all plants in each pot. Theplants were maintained until maturity and senescenceand the number of plants in each pot that producedvisible pycnidia was recorded, giving an incidence ofpycnidia formation on mature plant stems. The lesionlength data was square-root transformed prior toanalysis.

632 Eur J Plant Pathol (2012) 133:631–644

Table 1 List of Lupinus albus and L. angustifolius genotypestested for resistance to Diaporthe toxica in the experimentspresented in this study. The country of origin for landracesindicates were the line was initially collected, for breeding lineswhere created, and for cultivars the country of release. Experi-ment 1 assessed glasshouse screening techniques, experiments 2and 3 were glasshouse screening, and experiments 4 and 5 werefield screening

Line Countryof Origin

Type Experiment

75A:258 Morocco L. angustifoliusbreeding line

2,4,5

97B031-3 Australia Breeding line 3

98B001-5-5 Australia Breeding line 2,4,5

AMIGA Chile Cultivar 3

ANDROMEDA Australia Cultivar 2,3,4,5

ASTRA France Cultivar 2,3

DANJA Australia L. angustifoliuscultivar

2,4,5

ESTA-1 South Africa Cultivar 3

FP21 Germany Breeding line 3

HAMBURG Germany Cultivar 2,3,4,5

IDA Germany Cultivar 3

JINDALEE Australia L. angustifoliuscultivar

5

KIEV-MUTANT Ukraine Cultivar 1,2,3,4,5

LA300-SD France Breeding line 3

LAGO-AZZURRO Australia Cultivar 3

LUBLANC-1 France Cultivar 3

LUCKY-1 France Cultivar 3

LUTOP-1 France Cultivar 3

LUXOR Australia Cultivar 2,3,4,5

MADEIRA Portugal Cultivar 3

MAGNA Australia Cultivar 2,3

MINIBEAN Australia Cultivar 2,3

MINORI Germany Cultivar 3

MULTOLUPA-2 Germany Cultivar 3

NEULAND Germany Cultivar 2,3

P20913 Egypt Cultivar 3

P25758 Greece Landrace 2,3

P25863 UnitedKingdom

Breeding line 3

P26734 Hungry Landrace 3

P26777 Greece Landrace 3

P26791 Syria Cultivar 3

P27154 Spain Landrace 3

P27172 Ethiopia Landrace 3

P27174 Ethiopia Landrace 1,2,3

P27277 Italy Landrace 3

P27279 Italy Landrace 3

P27393 Syria Landrace 3

P27433 Syria Landrace 3

P27441 Syria Landrace 3

P27593 Portugal Landrace 1,2,3

P27662 Turkey Landrace 3

P27663 Turkey Landrace 3

Table 1 (continued)

Line Countryof Origin

Type Experiment

P27664 Turkey Landrace 3

P27840 Syria Landrace 2,3

P28096 Syria Landrace 1,2,3

P28199 Algeria Landrace 3

P28233 Ethiopia Landrace 3

P28507 Ethiopia Landrace 2,3

P28552 Ethiopia Landrace 3

P28561 Ethiopia Landrace 3

P28573 Ethiopia Landrace 3

P28752 Ukraine Cultivar 3

P28753 Ukraine Cultivar 3

P28754 Ukraine Cultivar 3

P28974 RussianFederation

Breeding line 3

P28975 Ukraine Breeding line 3

P28978 Ukraine Breeding line 3

P28979 Ukraine Breeding line 3

P28980 RussianFederation

Breeding line 3

P28981 RussianFederation

Breeding line 3

P28983 Spain Breeding line 3

P28984 Spain Breeding line 3

P28985 Italy Breeding line 3

P28986 Germany Breeding line 3

P28989 Greece Landrace 3

P28990 Morocco Landrace 2,3

P28991 Israel Landrace 2,3

P28993 Sudan Breeding line 3

P28994 Ukraine Breeding line 3

P28995 RussianFederation

Breeding line 3

P28996 Spain Landrace 3

P28997 Spain Landrace 3

P28998 Spain Landrace 3

P28999 Poland Breeding line 3

P29000 Netherlands Breeding line 3

P29002 USA Landrace 3

P29003 Argentina Breeding line 3

P29005 Turkey Landrace 3

P29017 Poland Breeding line 3

P29021 Germany Breeding line 3

P29022 Germany Breeding line 2

P29029 Germany Breeding line 2

ROSETTA Australia Cultivar 1,2,3,4,5

START RussianFederation

Cultivar 2,3

TYPTOP Chile Cultivar 3

ULTRA Germany Cultivar 2,3,4,5

UNICROP Australia L. angustifoliuscultivar

2,4

VLADIMIR RussianFederation

Cultivar 3

Eur J Plant Pathol (2012) 133:631–644 633

Glasshouse screening for resistance to D. toxica

Experiment 2 Forty-two genotypes of L. albus (11cultivars, 21 breeding genotypes, and 10 landraces)and three L. angustifolius genotypes were sown in175 mm diameter pots containing sandy-loam in arandomised glasshouse trial in Wagga Wagga, NSW,Australia with four replicates to assess latent steminfection to D. toxica. The L. angustifolius genotypeswere included as standards: 75A:258 is the majorsource of Phomopsis stem blight resistance in modernL. angustifolius varieties (Yang et al. 2002), while

Unicrop and Danja are susceptible (Shankar et al.1996). Fifteen seeds were sown into each pot. No inoc-ulant or fertiliser was added to the pots as the glasshousecomponent of this experiment ceased after 24 days. Thepots were well watered for the duration of the experi-ment. The plants were inoculated by placing approxi-mately 5 g of macerated infected stubble on the soilsurface of each pot. The stubble was collected from thesame field in Eurongilly, NSW, where a field diseasescreening experiment was conducted (see below). Pre-liminary experiments (data not shown) indicated thatplacing infected stubble collected from an infected fieldresulted in high disease pressure. Latent stem infection(Cowling et al. 1996; Shankar et al. 1996) was assessedon excised stem portions 24 days after sowing as de-scribed by Cowley et al. (2010). Briefly, the internodesbetween the cotyledon and the first leaf were excised,surface sterilised before being rinsed in sterile distilledwater and placed onto sterile filter paper in Petri dishes.The dishes were sealed in plastic bags in groups of 20 ina randomised design. The stem pieces were allowed todry in the dishes for 7 days before being moistened with3 ml sterile distilled water. The dishes were re-sealed inplastic bags and incubated on the laboratory bench atroom temperature for a further 14 days. The treatment ofthe stem pieces was to simulate crop maturity and theonset of the saprophytic phase of pathogen after summerrain (Wood 1986). The stem portions were scored for thepresence of symptoms using a 0 to 5 scale (where 00 nodisease, 50 stem portion completely diseased and pyc-nidia formed; Shankar et al. 1996).

Experiment 3 An experiment was conducted in theglasshouse to screen a genetically diverse set of L.albus genotypes (Raman et al. 2008) for stem resis-tance, using either externally-applied spores (spraytreatment), or injected spores. In this experiment, 95L. albus genotypes were sown on 20 October 2008 insandy loam in 175-mm diameter pots in anevaporative-cooled glasshouse at Wagga Wagga.These genotypes were the same set used to assessgenetic diversity in L. albus breeding material usingSSR and DArT molecular markers (Raman et al.2008). The experiment consisted of a split-plot designof three replicates sown in a 20×30 pot array. Eachreplicate (20×10) contained two main-blocks of 100pots (10×10 array). Each main-block was split intotwo sub-blocks (5×10 array). Inoculation treatments

Table 1 (continued)

Line Countryof Origin

Type Experiment

WALAB2008 Australia Breeding line 2,3,4,5

WK134 Australia Breeding line 3

WK147 Australia Breeding line 3

WK163 Australia Breeding line 3

WK172 Australia Breeding line 3

WK188 Australia Breeding line 2,3,4,5

WK212 Australia Breeding line 3

WK236 Australia Breeding line 2,3,4,5

WK255 Australia Breeding line 2,4,5

WK262 Australia Breeding line 2,4,5

WK263 Australia Breeding line 2,4,5

WK264 Australia Breeding line 2,4,5

WK266 Australia Breeding line 2,4,5

WK268 Australia Breeding line 2,4,5

WK271 Australia Breeding line 2,4,5

WK278 Australia Breeding line 2,4,5

WK279 Australia Breeding line 2,4,5

WK281 Australia Breeding line 2,4,5

WK290 Australia Breeding line 2,3,4,5

WK292 Australia Breeding line 2,4,5

WK302 Australia Breeding line 2,3,4,5

WK320 Australia Breeding line 2,3,4,5

WK325 Australia Breeding line 3

WK326 Australia Breeding line 2,4,5

WK338 Australia Breeding line 2,3,4,5

WK339 Australia Breeding line 2,4,5

XA100 France Cultivar 3

MP selectionsa

(n022)Australia Mapping

population5

a subset of non-bitter lines from a mapping population (Kiev-Mutant × P27174, as described in Phan et al 2007). Only non-bitter, low alkaloid, L. albus can be sown in field trials in NSWin order to prevent bitter contamination of breeding populationsand commercial seed lots (Richards et al 2008)

634 Eur J Plant Pathol (2012) 133:631–644

were assigned to the sub-blocks. There were four sub-blocks in each replicate.

Two inoculation treatments were used: either aconidial spore suspension of 5×106 spores per mlwas sprayed to runoff and the plants enclosed by alarge plastic sheet to maintain a dew period for 48 h ora 0.05μl aliquot of the prepared spore suspension wasinjected into each stem between the 2nd and 3rdinternodes (as described above). Both inoculationtreatments were performed on the same day, 40 daysafter sowing. Lesion length (mm) was measured28 days after inoculation. Incidence of pycnidia for-mation (presence/absence) was visually assessed98 days after sowing. Lesion length data was square-root transformation before analysis.

Field screening for resistance to D. toxica

Experiment 4 A replicated disease screening experi-ment was sown on 8 June 2007 at Eurongilly, NSW(−34.930°; 147.767°) in a field which had a history of asevere D. toxica epidemic on L. albus in 2005. Despitethe field having a cereal crop in 2006, dry seasonalconditions through 2006 meant that considerableinfected lupin stubble remained on the soil surface in2007. During this experiment in 2007 there was lowrainfall during August, September and October (14.6,17.4 and 19.8 mm respectively, Fig. 1).

The experiment consisted of 30 genotypes (6 culti-vars, 21 breeding lines, plus three L. angustifolius gen-otypes included as controls). Entries were sown as 10 mlong single rows with four replicates in an 8×15 grid.Disease incidence relied on natural infection and suit-able environmental conditions. No supplementary inoc-ulum or irrigation was provided. At maturity (12December 2007), 15 plants were randomly selectedfrom each row and assessed for Phomopsis stem blightusing the 0 to 5 scale described by Cowley et al. (2010).Phomopsis pod blight was scored on each plant as theincidence of pods per plant with visual disease symp-toms (i.e. a presence/absence score).

Experiment 5 In 2008, 52 L. albus genotypes (6 culti-vars, 21 breeding genotypes, a subset of 22 low-alkaloidrecombinant-inbred lines from a mapping population(Phan et al. 2007), and three L. angustifolius genotypes)were sown in the field on 18 July 2008 in 10 m rows atWagga Wagga, NSW (−35.050º; 147.350º). The experi-ment was irrigated using in-line drippers from August toNovember as required. Weather conditions for Wagga in2008 are shown in Fig. 1. The trial was sown in a 6×40array. Most genotypes were sown with five replicates.The three L. angustifolius genotypes and seven L. albusgenotypes (Kiev-Mutant, Ultra, Hamburg, Rosetta, An-dromeda, WALAB2008 and WK264) were replicatedtwice with 10 rows each across the whole experiment.Due to limited seed supply, each mapping populationentry was sown with two replicates. The DiGGer soft-ware was used to produce a spatially efficient experi-mental design incorporating the unequal replication.

A susceptible disease spreader, Kiev-Mutant, wassown in every third row. Ninety days after sowing theexperiment was inoculated by stabbing plants in thespreader rows with phomopsis-inoculated toothpicksas described above. The toothpicks were inoculatedwith isolate DAR80114 and inserted into the stem justbelow the developing pods on 14 October 2008 andleft in place for the duration of the trial. Approximate-ly 6–10 plants were inoculated per 10 m spreader row,with 120 spreader rows inoculated throughout thetrial. This method was designed to mimic a late-season phomopsis epidemic.

The disease level was scored at harvest by recordingthe incidence of infection in stems and pods in thecentral 5-m section of each row, and expressed as aproportion of the total number of plants in the section.

Tem

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(o C)

Rai

nfal

l (m

m)

0

10

20

30

40

0

10

20

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derocSnwoS

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Apr

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Sown Inoculated Scored

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Fig. 1 Weather conditions for the field screening experimentsconducted at Eurongilly in 2007 and Wagga Wagga in 2008.The arrows indicate when each trial was sown and scored.Weather data was sourced from archived files at http://www.weatherzone.com.au/

Eur J Plant Pathol (2012) 133:631–644 635

Results

Glasshouse screening methodology

In this experiment the genotype × inoculation effectswere significant (P<0.001). The L. albus landracesP28096 and P27174 had the least disease when conid-ia of D. toxica were applied to the stem surface (pre-dicted mean square-root lesion length 0.46 and 1.51respectively, Fig. 2). Kiev-Mutant, Rosetta andP27593 were not significantly different when inocu-lated by spraying conidia (ranging from 3.12 to 3.84).The toothpick method, the most aggressive and mostdamaging of the three inoculation methods, resulted inthe largest stem lesions and the highest incidence ofplants developing visible pycnidia (Fig. 3). However,genotypes P27174 and P28096 showed significantlyless incidence of pycnidia formation compared withthe other genotypes (Fig. 3). The injection conidia intostems method also had high incidence of pycnidiaformation but smaller lesions than the toothpick meth-od. The five genotypes were very similar in their meanresponses for the toothpick and needle inoculationmethods. The spray inoculation treatment gave thegreatest discrimination between genotypes withRosetta and Kiev-Mutant being the most susceptible.P27174 and P28096 were resistant.

Glasshouse screening

In the first screening experiment (Experiment 2) theeffect of genotype on latent stem infection was signif-icant (P<0.001) and genotype-predicted means ranged

from 1.61 to 3.60 (Fig. 4). The most resistant genotypewas 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 ishighly resistant to D. toxica (Shankar et al. 1996) itwas 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, cultivarsDanja and Unicrop (Shankar et al. 1996).

In the second screening experiment (Experiment3) the effect of genotype, inoculation and theirinteraction were significant (P<0.001) on lesionlength score. For incidence of pycnidia formationthe effect of genotype was significant (P<0.001)and the genotype by inoculation interaction wassignificant (P<0.05). For the lesion length scoreseveral landraces were resistant to Phomopsis stemblight with the spray inoculation method. Themost resistant genotypes were breeding linesP28979, P28978, P28975 all from the Ukraine(predicted means of 0.80, 0.82 and 0.96, respec-tively, Fig. 5). It is possible that the same geneticcontrol is responsible for resistance in these three

Lesi

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ngth

(sqa

ure-

root

)

5

10

15

KIEV-MUTANT P27174 P27593 P28096 ROSETTA

LSD5%

Control: SprayInoculation: SprayControl: InjectInoculation: InjectControl: ToothpickInoculation: Toothpick

Fig. 2 Effect of inoculation treatment on Phomopsis StemBlight of Lupinus albus stems caused by Diaporthe toxica

Lesion Length (square-root)

Inci

denc

e of

Pyc

nidi

a F

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atio

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P27593P27593

P27593

P28096

P28096

P28096

ROSETTA

ROSETTA

ROSETTA

LSD5%

Inoculation methodInjectSprayToothpick

Fig. 3 Phomopsis stem blight severity (caused by Diaporthetoxica) on Lupinus albus in glasshouse trial scored as lesionlength 28 days after inoculation using three inoculation methodsand the incidence of pycnidia formation scored on mature plantsat harvest

636 Eur J Plant Pathol (2012) 133:631–644

breeding lines. P28096 (landrace from Syria) andP27664 (landrace from Turkey) were also resistant(predicted means of 1.02 and 1.09, respectively,Fig. 5). The most susceptible genotypes werebreeding lines P28999 and P29017 from Poland(10.53 and 10.02, respectively). Lucky-1, Start

and P27662 were also susceptible (9.45, 9.33 and8.75, respectively). The predicted mean of Kiev-Mutant and Rosetta were 2.68 and 6.35, respec-tively. All of the L. albus cultivars grown commer-cially in Australia had similar incidences of pycnidiaformation in the spray treatment (ranging from 0.32 to

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Fig. 5 Lesion length of Phomopsis stem blight (Diaporthe toxica) in glasshouse screening experiment of Lupinus albus comparing theinoculation methods of injecting conidia into stems, versus spraying conidia onto stems. Certain key lines are shown with white bars

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Fig. 4 Latent Phomopsisstem blight (caused by Dia-porthe toxica) on excisedstems of Lupinus albus.Bars shown in white are L.angustifolius included ascontrol lines

Eur J Plant Pathol (2012) 133:631–644 637

0.46, Fig. 6), but varied in their lesion length scores(ranging from 1.45 to 6.35) with Andromeda and Ultrahaving the smallest lesion length. Landraces P26734,P27840, P28507 and P28980 had the lowest incidenceof pycnidia formation (<0.10, Fig. 6) and may representa potential source of resistance to Phomopsis stem blight.

The two inoculation treatments in this experimentwere contrasting in that the spray method appliedspores to the exterior of the plant and epidermal resis-tance mechanisms (if they exist) may affect the out-come. In the method by injecting conidia into stems,the spores are introduced into the interior of the plantthereby bypassing any epidermal influence. This tech-nique was of interest because hail damage of anL. albus crop in 2004 had helped contribute to a severePhomopsis epidemic (Cowley et al. 2010). One of theaims of this experiment was to determine if resistanceto D. toxica can be found in internal tissues, that is,once epidermal resistance is bypassed. This situationis likely to occur following a wounding event such ashail or insect damage. D. toxica spores are normallywind and water dispersed (Wood 1986).

No genotypes were identified that had resistance toPhomopsis stem blight when the spores were appliedsub-epidermally by injecting conidia into the stems(Fig. 5). There was, however, a moderate but signifi-cant correlation between the two inoculation methods

using Pearson’s product moment correlation coeffi-cient (r00.23, P<0.05). Landraces P28974 andP26791 were resistance and moderately resistant toPhomopsis stem blight when the spores were appliedexternally (1.59 and 2.88, respectively). In contrastwhen the spores were applied internally via injectionthey were the most susceptible genotypes tested (11.92and 13.75, respectively, Fig. 5).

Field screening

Dry conditions during August to October 2007 (Fig. 1)restricted plant growth and disease development inExperiment 4. Nevertheless, significant differences(P<0.001) in Phomopsis stem and pod blight werefound. Most of the cultivars included in this experi-ment had similar levels of Phomopsis stem blight,based on a 0 to 5 score (ranging from 2.52 to 2.76,Fig. 7), with Hamburg being the exception (stem scoreof 1.80). WALAB2008, WK339, WK278 and WK320were the best performing breeding lines under the testconditions with the lowest stem disease scores (stemscore <1.84, Fig. 7). With the L. angustifolius linesUnicrop had the highest Phomopsis stem blight sever-ity followed by Danja and 75A:258 (stem scores of2.74, 2.65 and 2.12, respectively). Lines WK320,WK236, Ultra and WK278 had the lowest incidenceof Phomopsis pod blight (0.18, 0.20, 0.21, and 0.21,respectively). A group of sister lines, that is selectionsfrom the same cross—WK262, WK263, WK264 andWK266—had the highest incidence of infected podsper plant (pod infection incidence >0.40, Fig. 7), con-firming previous glasshouse screening observations(Cowley et al. 2010).

Environmental conditions for field screening in2008, Experiment 5, were conducive for disease de-velopment (Fig. 1). Supplementary irrigation was re-quired throughout August to October. Several stormevents in November aided the spread of the pathogen.Genotype differences in both Phomopsis stem and podblight were significant (P<0.001). Rosetta was resis-tant to Phomopsis stem blight in this experiment (pre-dicted mean incidence 0.17, Fig. 8). The breedinglines WK266, WK279 and WK188 also had low steminfection incidence (0.11, 0.17 and 0.19 respectively).The most resistant genotype was a mapping popula-tion line MP-A-153 (0.10). Luxor was the poorestperforming cultivar tested (predicted mean incidence0.41). The eight most susceptible genotypes were all

Lesion length (mm)

Inci

denc

e of

pyc

nidi

a fo

rmat

ion

0.0

0.2

0.4

0.6

0.8

2 4 6 8 10

ANDROMEDAHAMBURG

MADEIRA

P25758

P26734

P27174

P27593

P27840

START

ULTRA

WK236

LUXOR

NEULAND

P27664

P28096

P28507

ROSETTA

WALAB2008

KIEV-MUTANT

P28980LSD5%

Key to symbolsBreeding linesCultivarsLandraces

Fig. 6 Lesion length of Phomopsis stem blight (Diaporthetoxica) and incidence of pycnidia formation in a glasshousescreening experiment of Lupinus albus inoculated by sprayingconidia

638 Eur J Plant Pathol (2012) 133:631–644

mapping population lines (predicted means >0.54).Breeding lines WK271, WK320, WK236 andWALAB2008 were also susceptible (0.47, 0.49, 0.53and 0.53, respectively). Kiev-Mutant had a predictedmean incidence of 0.25 for stem infection. Rosetta wasthe genotype most resistant to Phomopsis pod blight(predicted mean incidence 0.13, Fig. 8). The correla-tion between stem infection and pod infection wassignificant (r00.82, P<0.001).

The predicted mean incidence for the selected map-ping population entries ranged from 0.10 to 0.58 forPhomopsis stem blight, and 0.12 to 0.71 for Phomop-sis pod blight implying that the population is segre-gating for resistance in both stems and pods. Furtherwork is required to determine the nature of the geneticresistances that exist in the population.

In the field screening conducted in 2008, the L.angustifolius breeding line 75A:258, included as a“highly resistant” control (Shankar et al 1996), per-formed poorly and had a greater disease incidence thanthe other L. angustifolius controls and significantlyhigher disease incidence than Rosetta (P<0.05).75A:258 was replicated in 10 rows throughout the ex-periment and had a predicted mean incidence for Pho-mopsis stem blight of 0.59 and Phomopsis pod blight of0.51 (Fig. 8). The other L. angustifolius controls,

Jindalee (moderately resistant) and Danja (susceptible,Shankar et al. 1996) had predicted mean incidences forPhomopsis stem blight of 0.41 and 0.40, and for Pho-mopsis pod blight 0.38 and 0.33, respectively (Fig. 8).

Stem infection incidence

Pod

infe

ctio

n in

cide

nce

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.1 0.2 0.3 0.4 0.5 0.6 0.7

DANJA

JINDALEE

KIEV-MUTANT

LUXOR

ROSETTAULTRA

75A:258

LSD5%

HAMBURG

ANDROMEDA

Key to symbolsBreeding lineCultivarMapping pop.L. angusifolius

Fig. 8 Phomopsis pod and stem blight caused Diaporthe toxicain an irrigated field disease nursery at Wagga Wagga, NSW, in2008

Dis

ease

sev

erity

sco

res

LSD5%

Pod

0.2

0.3

0.4

0.5

0.6

1.5

2.0

2.5

3.0

WA

LAB

2008

WK

339

WK

278

HA

MB

UR

GW

K32

0W

K23

6W

K27

1W

K32

675

A-2

5898

B00

1-5-

5W

K27

9W

K29

0W

K28

1K

IEV

-MU

TA

NT

ULT

RA

RO

SE

TT

AW

K26

8U

NIC

RO

PD

AN

JAW

K30

2A

ND

RO

ME

DA

WK

255

WK

338

LUX

OR

WK

292

WK

188

WK

263

WK

266

WK

264

WK

262

LSD5%

Stem

Fig. 7 Phomopsis pod andstem blight of Lupinus albusgenotypes scored at harvestin a replicated field diseasenursery at Eurongilly, NSWin 2007. L. angustifoliusgenotypes are shown aswhite bars

Eur J Plant Pathol (2012) 133:631–644 639

Correlations between glasshouse screening and fieldscreening experiments

The Pearson product-moment correlation between thetwo glasshouse screening experiments was poor (r00.11) and was not significant. For these two experi-ments the inoculum source varied, as did the methodsused to assess disease response (Table 2). In the firstglasshouse screening experiment infected stubble wasused, 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 screeningmay partially explain the negative correlation betweenthe stem scores in the two experiments (r0-0.46, P<0.05). Also, the experiment conducted in 2008 usedspreader rows to aid pathogen dispersal, whereas theexperiment in 2007 relied on natural inoculum. Bothexperiments experienced low rainfall during the springgrowing season (August–October, Fig. 1) and only theexperiment conducted in 2008 was provided with sup-plementary irrigation.

The correlations between the two glasshousescreening experiments and the two field screeningexperiments 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 in2008 (r00.47 and r00.61, respectively), and no cor-relation with field screening in 2007. The secondglasshouse experiment had no significant correlation

for both stem and pod scores in either field screeningexperiment.

Sources of resistance

The glasshouse and field screening reported hereshowed that Rosetta has useful resistance under fieldconditions, although may succumb to Phomopsis stemblight when disease pressure is high. The resistance ofUltra was confirmed and a further 17 genotypes wereidentified as being potential new resistance donors forbreeding (Table 3).

Discussion

The objective of this research was to evaluate screen-ing methodology to determine if genetic variabilityexisted in L. albus germplasm to Phomopsis stemblight caused by D. toxica and to identify genotypeswith superior resistance that maybe useful in resis-tance breeding.

A useful disease screening method for breeding mustbe 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 andfield screening was attempted. In all cases, there weregenotypic 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 inLupinus 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

2 Glasshouse 2007 45 Infected stubble c Eurongilly 0–5 scale d

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 obviouspycnidiac Stubble sourced from site of Experiment 4d 0–5 scale presented in Cowley et al. (2010)

640 Eur J Plant Pathol (2012) 133:631–644

the different environments in each experiment. Certaingenotypes (most of the “P” lines in this study) that hadbeen examined in glasshouse experiments could not bestudied in the field because they contained alleles forhigh alkaloid content or alleles other than those used incommercial crops to condition the low-alkaloid trait(Lin et al. 2009). These undesirable genes could havespread by pollen movement to neighbouring commer-cial L. albus crops (Richards et al. 2008).

The totally natural infection in the drought-affectedyear 2007 (without irrigation) may not have reflectedtrue genotype response to disease pressure. Conse-quently, disease screening in drought conditions maynot be sufficient for reliable, repeatable and efficientscreening for plant breeding purposes. As a resultspreader rows and irrigation were employed in 2008,in which 10 of the top 12 genotypes for Phomopsispod blight resistance were either breeding lines orreleased cultivars. Also in field screening in 2008, 17of 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 inthis experiment to gauge whether or not the population

is segregating for either Phomopsis stem blight orPhomopsis pod blight, or both. The mapping popula-tion is segregating for low-alkaloid alleles causing theselection of mapping population entries to be biased.Nevertheless the spread of the mapping populationentries (ranging from 0.10 to 0.69 for stems and 0.12to 0.71 for pods) implies that it is segregating forresistance in both stems and pods. The mapping pop-ulation is segregating for pod resistance but not leafresistance (Cowley et al. 2011). Further work is re-quired to determine the nature of genetic resistances inthe population.

There were differences in the inoculum sourcesused in the various experiments, although an attemptwas made in most cases to use the isolate originatingfrom Tarcutta, NSW, or the single spore isolate fromTarcutta, DAR80114 (Cowley et al. 2010). This isolatehas 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 todetermine if the fungus had overcome the long-termmoderate resistance that had existed in L. albus culti-vars in Australia (Sweetingham et al. 1998). Based onthe results of this study, cultivars Rosetta and Ultra

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)

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would be recommended for growing in south-easternAustralia in areas where Phomopsis stem blight may beprevalent, although, in growing seasons and environ-ments with high disease pressure, genetic resistancewithin Rosetta may be overcome. Also, results fromfield screening in drought-affected seasons may induceincreased 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 theaddition of irrigation.

Several potentially new sources of resistance toPhomopsis stem blight using the Tarcutta isolate wereidentified in this study. Genotypes P28096, P27840,WK147 and WK320 are also resistant to PleiochaetaRoot Rot, caused by the fungal pathogen Pleiochaetasetosa (Kirchn.) S. Hughes (Luckett et al. 2009). An-dromeda, P28507 and WALAB2008 have resistanceto Colletotrichum lupini (Bondar) Nirenberg (Adhikariet al. 2009). It has not been established whether thesegenotypes have genes for resistance against multiplepathogens.

The genotypes P28980 and P28754 are resistant toPhomopsis stem blight but have been found to containnon-pauper genes for controlling the low-alkaloid traitin domesticated L. albus (unpublished data). Careshould be exercised when using these lines in breedingwith genotypes containing the pauper gene. Suchcrosses will result in high alkaloid progeny (Lin etal. 2009) and may lead to bitter contamination inbreeding populations and commercial crops (Richardset al. 2008).

Although resistance to externally applied sporeswas identified, no lines were found to have resistanceto D. toxica when the spores were applied internally,in this study via injection or stabbing with inoculatedtoothpicks. This strongly implies that all the L. albusgenotypes tested are vulnerable to Phomopsis stemblight if infection coincides with a wounding event,such as hail or possibly insect damage. Some geno-types, however, were markedly more susceptible tointernally applied spores, in particular genotypesP26791, P28974 and P28991. The outbreak of Pho-mopsis stem blight in 2004 (Cowley et al. 2010) thatprompted this study followed a late-season hail event.

The highly resistant L. angustifolius breeding line75A:258 (Shankar et al. 1996) was included in threeexperiments reported in this paper as a control line. In

each of the experiments it was susceptible to Phomop-sis stem and pod blight when challenged with theTarcutta isolate of D. toxica. This line has beenused extensively in breeding for Phomopsis resis-tant L. angustifolius cultivars (Shankar et al. 2002)and has been used to develop molecular markersfor disease resistance using microsatellite-anchoredfragment length polymorphism (MFLP) (Yang et al.2002). 75A:258 contains a single, dominant gene(named Phr1) for strong resistance to Phomopsisand may be subject to unlinked modifier genes(Shankar et al. 2002). It would appear from theseresults that the resistance provided by Phr1 hasbeen overcome by the Tarcutta isolate of the pathogenin L. angustifolius.

In future L. albus breeding work the method recom-mended for routine glasshouse screening for diseaseresistance to Phomopsis stem blight is to use sprayinoculation and to score lesion length. This differs fromreports of the established protocols for assessingPhomopsis stem blight in L. angustifolius which exam-ined latent stem infection using excised stem portions(Cowling et al 1996; Shankar et al 1996; Williamsonet al 1991). With L. angustifolius the pathogen invadesstem tissue whilst the plant is growing and forms coral-loid hyphae structures under the epidermis (Williamsonand Sivasithamparam 1994; Williamson et al. 1991).These appear to remain more or less in stasis until plantmaturity and senescence, when the fungus proliferateson the stubble in response to rainfall (Williamson et al.1991). The lengthy latent phase in L. angustifolius isgenerally symptomless (Cowling et al. 1984; Wood andSivasithamparam 1989). The latent infection structuresvary greatly in size and number and have been used todiscriminate between resistant and susceptible L. angus-tifolius genotypes (Shankar et al. 1996; Williamson etal. 1991). In contrast to L. angustifolius, it has not beenestablished whether a lengthy latent phase occurs whenL. albus tissue is colonised with D. toxica. Using tooth-pick inoculations on L. albus, L. angustifolius, L. luteusand L. mutabilis, Jaarsveld and Knox-Davies (1974)noted that only L. albus developed symptoms whilegreen. In glasshouse trials, Kochman and Kubicka(1974) were able to infect L. albus stems with dropletscontaining only five spores. When assessing the forma-tion of coralloid hyphae, Williamson et al. (1991) failedto find coralloid hyphae on the small number of L. albusplants tested. We have observed and reported herelesions developing on green stems of L. albus after

642 Eur J Plant Pathol (2012) 133:631–644

inoculating with conidia, which supports previous find-ings (Cowley et al 2010). This study did not examinecoralloid 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 screeningprotocol to screen breeding material. Stem resistance isimportant because stem pieces are the largest componentof L. albus stubbles, and if contaminated stubble isgrazed by sheep in significant quantities sheep poisoningby lupinosis becomes a serious risk (Allen 1986). Inaddition, stubble is the main source of spores for futureinfection, so any reduction in disease development onstems and subsequently on stubble due to genetic resis-tance can be beneficial to subsequent or neighbouringcrops (Cowling et al. 1986). The problem of a stubbleas a source of spores is especially important inminimum tillage situations where stubble may beleft on the soil surface for several years and cansporulate freely. Work is continuing to uncover thegenetic control of resistance to Phomopsis blight in bothstems and pods of L. albus, and to find molecularmarkers, so that marker-assisted selection can be usedby L. albus breeders in the future.

Acknowledgements Mr Tom Brabbin is thanked for the useof his farm to screen for Phomopsis stem and pod blight, and thesupply of infected lupin stubble used in glasshouse screening.David Roberts and Mark Richards are thanked for technicalassistance. Dr Neil Coombes is thanked for assistance withexperimental design.

References

Adhikari, K. N., Buirchell, B. J., Thomas, G. J., Sweetingham,M. W., & Yang, H. (2009). Identification of anthracnoseresistance in Lupinus albus L. and its transfer from land-races to modern cultivars. Crop & Pasture Science, 60,472–479.

Allen, J. G. (1986). Lupinosis, a review. In, Proceedings of theFourth International Lupin Conference, pp. 173–187. Ger-aldton, Western Australia: International Lupin Association.

Butler, D. G., Cullis, B. R., Gilmour, A. R., & Gogle, B. J. (2009).ASReml-R reference manual, release 3. Technical SeriesQE02001, Queensland Department of Primary Industries.

Cowley, R. B., Ash, G., Harper, J. D. I., & Luckett, D. J. (2010).Evidence that Diaporthe toxica infection of Lupinus albusis an emerging concern for the Australian lupin industry.Australasian Plant Pathology, 39, 146–153.

Cowley, R. B., Ash, G.J., Harper, J.D.I., Smith, A.B., Cullis, B.R., & Luckett, D.J. (2011). Application of multi-phaseexperiments in plant pathology to identify genetic

resistance to Diaporthe toxica in Lupinus albus. Euphytica,Published online 14 September 2011. DOI 10.1007/s10681-011-0529-4

Cowling, W. A., Wood, P. M., & Brown, A. G. P. (1984). Use ofa paraquat-diquat herbicide for the detection of Phomopsisleptostromiformis infection in lupins. Australasian PlantPathology, 13, 45–46.

Cowling, W. A., Allen, J. G., Wood, P. M., & Hamblin, J.(1986). Phomopsis-resistant lupins - breakthrough towardsthe control of lupinosis. Journal of Agriculture of WesternAustralia, 27, 43–46.

Cowling, W. A., Sweetingham, M. W., & Shankar, M. (1996).Development of a rapid screening test for Phomopsis re-sistance in lupins. Research and Development Final Report(Grains Research and Development Corporation);DAW307WR.

Cullis, B. R., Smith, A. B., & Coombes, N. E. (2006). On thedesign of early generation variety trials with correlateddata. Journal of Agricultural, Biological, and Environmen-tal Statistics, 11, 381–393.

R Development Core Team (2010). R: A language and environ-ment for statistical computing. R Foundation for StatisticalComputing, Vienna, Austria. ISBN 3-900051-07-0, URLhttp://www.R-project.org.

Jaarsveld, A. B. V., & Knox-Davies, P. S. (1974). Resistance oflupins to Phomopsis leptostromiformis. Phytophylactica, 6,55–60.

Keeling, B. L. (1982). A seedling test for resistance to soybeanstem canker caused by Diaporthe phaseolorum var. cauli-vora. Phytopathology, 72, 807–809.

Kochman, J., & Kubicka, H. (1974). Aggressiveness and path-ogenicity of Phomopsis leptostromiformis (Kuhn) Bubakand development of the process of infection caused by thefungus. Acta Agrobotanica, 27, 5–17.

Lin, R., Renshaw, D., Luckett, D., Clements, J., Yan, G., Adhikari,K., et al. (2009). Development of a sequence-specific PCRmarker linked to the gene “pauper” conferring low-alkaloidsin white lupin (Lupinus albus L.) for marker assisted selec-tion. Molecular Breeding, 23, 153–161.

Luckett, D. J., Cowley, R. B., Richards, M. F., & Roberts, D. M.(2008). Improved methodology for screening for resistanceto Pleiochaeta setosa root rot in Lupinus albus. In J. A. Palta& J. Berger (Eds.), 12th international lupin conference (pp.447–450). Fremantle: International Lupin Association.

Luckett, D. J., Cowley, R. B., Richards, M. F., & Roberts, D. M.(2009). Breeding Lupinus albus for resistance to the rootpathogen Pleiochaeta setosa. European Journal of PlantPathology, 125, 131–141.

Phan, H. T. T., Ellwood, S. R., Adhikari, K., Nelson, M. N., &Oliver, R. P. (2007). The first genetic and comparative mapof White Lupin (Lupinus albus L.): Identification of QTLsfor anthracnose resistance and flowering time, and a locusfor alkaloid content. DNA Research, 14, 59–70.

Raman, R., Luckett, D. J., & Raman, H. (2008). Estimation ofgenetic diversity in albus lupins (Lupinus albus L.) usingDArT and genic markers. In J. A. Palta & J. Berger (Eds.),12th international lupin conference (pp. 236–241). Fre-mantle: International Lupin Association.

Richards, M. F., Luckett, D. J., Cowley, R. B., & Roberts, D. M.(2008). Controlling bitter-seed contamination in the NSWLupinus albus industry. In J. A. Palta & J. Berger (Eds.),

Eur J Plant Pathol (2012) 133:631–644 643

12th international lupin conference (pp. 275–278). Freman-tle: International Lupin Association.

Russell, G. E. (1978). Plant breeding for pest and diseaseresistance. London: Butterworths & Co (Publishers) Ltd.

Shankar, M., Cowling, W. A., & Sweetingham, M. W. (1996).The expression of resistance to latent stem infection byDiaporthe toxica in narrow-leafed lupin. Phytopathology,86, 692–697.

Shankar, M., Sweetingham, M. W., & Cowling, W. A. (2002).Identification of alleles at two loci controlling resistance toPhomopsis stem blight in narrow-leafed lupin (Lupinusangustifolius L.). Euphytica, 125, 35–44.

Sweetingham, M. W., Jones, R. A., & Brown, A. G. (1998).Diseases and pests. In J. S. Gladstones, C. Atkins, & J.Hamblin (Eds.), Lupins as crop plants: Biology, productionand utilization (pp. 263–289). Wallingford: CabInternational.

Tivoli, B., Baranger, A., Avila, C. M., Banniza, S., Barbetti, M.,Chen, W. D., et al. (2006). Screening techniques and sour-ces of resistance to foliar diseases caused by majornecrotrophic fungi in grain legumes. Euphytica, 147,223–253.

Williamson, P. M., & Sivasithamparam, K. (1994). Factorsinfluencing the establishment of latent infection ofnarrow-leafed lupins by Diaporthe toxica. Australian Jour-nal of Agricultural Research, 45, 1387–1394.

Williamson, P. M., Sivasithamparam, K., & Cowling, W. A.(1991). Formation of subcuticular coralloid hyphae byPhomopsis leptostromiformis upon latent infection ofnarrow-leafed lupins. Plant Disease, 75, 1023–1026.

Wood, P. (1986). Epidemiology of Phomopsis leptostromifor-mis. In, Proceedings of the Fourth International LupinConference, pp. 220–229. Geraldton, Western Australia:International Lupin Association.

Wood, P. M., & Allen, J. G. (1980). Control of ovine lupinosis:Use of a resistant cultivar of Lupinus albus - cv. Ultra.Australian Journal of Experimental Agriculture and AnimalHusbandry, 20, 316–318.

Wood, P., & Sivasithamparam, K. (1989). Diaporthe woodii (ana-morph Phomopsis leptostromiformis)—A toxigenic fungusinfecting cultivated lupins.Mycopathologia, 105, 79–86.

Wunderlich, N., Ash, G. J., Harper, J. D. I., Cowley, R. B., &Luckett, D. J. (2008). Penetration and symptom develop-ment of Pleiochaeta root rot in susceptible and resistantLupinus albus cultivars. Australasian Plant Pathology, 37,387–391.

Yang, H., Shankar, M., Buirchell, B. J., Sweetingham, M. W.,Caminero, C., & Smith, P. M. C. (2002). Development ofmolecular markers using MFLP linked to a gene conferringresistance toDiaporthe toxica in narrow-leafed lupin (Lupinusangustifolius L.). Theoretical and Applied Genetics, 105,265–270.

644 Eur J Plant Pathol (2012) 133:631–644