cotyledon assay as a rapid and reliable method of screening for resistance against sclerotinia...

Cotyledon assay as a rapid and reliable methodof screening for resistance against Sclerotinia sclerotiorumin Brassica napus genotypes

Harsh GargA, K. SivasithamparamB, S. S. BangaC and M. J. BarbettiA,D,E

ASchool of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia,

35 Stirling Highway, Crawley, WA 6009, Australia.BSchool of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, The University

of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.CDepartment of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University,

Ludhiana 141004, India.DDepartment of Agriculture and Food Western Australia, Baron-Hay Court, South Perth, WA 6151, Australia.ECorresponding author. Email: [email protected]

Abstract. Sclerotinia sclerotiorum is amajor pathogenofmanycrops, includingoilseed rape (Brassicanapus), and there iskeen interest worldwide to identify Brassica genotypes with resistance to this pathogen. However, field testing to identifyresistance in B. napus germplasm is expensive, time-consuming and at times unreliable due to variability in fieldenvironmental conditions and plant architecture. To address this, we aimed to examine the feasibility of utilisingfor B. napus a cotyledon test already developed for Sclerotinia disease on legumes. Initially, cotyledons of 32 B. napusgenotypes were drop-inoculated using macerated mycelium (104mycelial fragments/mL) under controlled environmentalconditions. Significant differences were recorded betweenB. napus genotypes, and the experiment was repeated twice usinggenotypes selected from the first experiment. Certain genotypes responded with a distinct hypersensitive reaction (lesions<1mm diameter), either always (cv. Mystic) or frequently (cv. Charlton), which is the first report of this phenomenon in theB. napus--S. sclerotiorum pathosystem. Responses of genotypes between the three screening experiments were significantlyand positively correlated. Results obtained in the first experiment were compared with those from our earlier field screeningfor stem rot that utilised the same strain of S. sclerotiorum and the same B. napus genotypes. In particular, there was asignificant positive correlation (r = 0.62, P < 0.01) between published field data for stem rot and our cotyledon test resultsacrossgenotypes in common.This indicates the usefulnessof this cotyledonassay toprovide a relatively reliable indicationoffield performance of genotypes. We believe that this is the first report demonstrating that a cotyledon assay can besuccessfully applied to rapidly differentiate the reactions of B. napus genotypes against S. sclerotiorum.

Additional keywords: disease screening methodology, white mould.

Introduction

Sclerotinia stem rot, caused by the fungus Sclerotiniasclerotiorum is one of the most serious and damaging diseasesof oilseed rape (Brassica napus) (McCartney et al. 1999) andyield losses as high as 24% have been recorded under Australianconditions (Hind-Lanoiselet 2004). Various methods used formanaging S. sclerotiorum include cultural control, chemicalcontrol and varietal resistance (Bardin and Huang 2001).Chemical control in managing this disease is often ineffective,largely due to difficulty in timing the application with the releaseof ascospores (Bolton et al. 2006), especially in Australia wherepetal infection can be a poor indicator of subsequent steminfection levels (Hind et al. 2003). Cultural practices tend toavoid or reduce the severity of Sclerotinia stem rot, but noneeffectively control S. sclerotiorum on their own. Selection of hostresistance is the only economic and sustainable means ofmanaging this disease (Zhao et al. 2004). However, host

resistance works best when used in conjunction with thefollowing cultural practices: (i) practices involving croprotation (Williams and Stelfox 1980); (ii) practices thatincrease row spacing and decrease seeding rate (Hoes andHuang 1975); and (iii) practices that discourage apothecialproduction and ascospore release, such as maintaining highirrigation to increase rotting of sclerotia (Teo et al. 1989) orburning of crop residues (Hind-Lanoiselet et al. 2005).

Field evaluation of Sclerotinia stem rot for selection ofresistant cultivars often provides highly variable results as theresponses of various genotypes are heavily dependent upon theenvironment (Abawi and Grogan 1979). Moreover, diseasepressure may not be uniform in field situations, which furthercomplicates the phenotypic classification of host genotypes. Inaddition, under field conditions oilseed Brassica genotypes maydiffer in their plant architecture and maturity, which frequentlyresults in measuring disease escape rather than physiological

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Australasian Plant Pathology, 2008, 37, 106--111 www.publish.csiro.au/journals/app

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resistance in field screening experiments (Phillips et al. 1990). Incontrast to field screening, resistance against S. sclerotiorum ingreenhouse or laboratory evaluation is more likely to be duesolely to physiological resistance, with little chance ofinvolvement of disease escape mechanisms, as demonstratedpreviously for soybean and/or other non-Brassica hosts byGrau and Bissonette (1974), Nelson et al. (1991) and Vuonget al. (2004).

Various controlled environment screening methods havebeen used to evaluate resistance in oilseed rape. Theseinclude cut petiole inoculation (Zhao et al. 2004; Bradleyet al. 2006), detached leaf inoculation (Bailey 1987; Bradleyet al. 2006), and an oxalic acid assay (Bradley et al. 2006).Although the petiole inoculation method (Zhao et al. 2004;Bradley et al. 2006) has been reported to be a goodmethod to useto compare the level of resistance against S. sclerotiorum, thereis not always a good correlation with results obtained from fieldscreening. For example, Kim et al. (2000) reported thatresistance ratings for soybean cultivars having intermediatereactions to S. sclerotiorum were inconsistent across differenttests. Similarly, while Sun (1995) reported consistent genotypeperformance for resistant and susceptible spring type B. napusaccessions, they found that accessions with intermediate ratingsvaried depending upon the test method utilised. While Bradleyet al. (2006) reported that disease reactionofBrassicagenotypesfrom a petiole inoculation method were negatively correlatedwith yield (P = 0.038; r = --0.58), results obtained from detachedleaf and oxalic acid tests were not correlated with field results.Even for soybean genotypes, only moderate correlation valueswere reported between screening methods using excised leafinoculations, detached leaf and oxalic acid assays and fieldreactions (Wegulo et al. 1998; Kim et al. 1999).

A cotyledon test has been used by some researchers toidentify resistance to S. sclerotiorum in genotypes of soybean(e.g. Grau and Bissonette 1974; Hartman et al. 2000; Kim et al.2000; Kull et al. 2003) and alfalfa (Pratt and Rowe 1998). Kimet al. (2000) reported that responses in greenhouse experimentsto agar plugs or oat seed infestedwith themyceliumapplied ontocotyledons were correlated with field screening results forsoybean. Clearly, there is potential to develop a cotyledontest method for B. napus genotypes, which can also addressmany of the constraints listed above. Furthermore, a morereliable screening technique for the B. napus--S. sclerotiorumpathosystem is needed, which can rapidly predict the responsesof genotypes in the field while producing consistent resultsacross repeated experiments. To address these concerns weaimed to examine the feasibility of utilising a cotyledon testto predict the field reaction of B. napus genotypes againstS. sclerotiorum.

Materials and methods

S. sclerotiorum isolate

A single isolate of S. sclerotiorum (MBRS1; University ofWestern Australia Culture Collection No. WUF2004.1) wasused. This isolate was collected from Mount Barker ResearchStation in Western Australia (WA) in 2004 and this same isolatehad been used previously by Li et al. (2006) to evaluate theresponses of B. napus andB. juncea genotypes to S. sclerotiorum

in the field in WA. This isolate was selected on the basis of itsvirulence and the fact that it was isolated from a site where therewas significant disease.

Test conditions

All screening test lines were on B. napus genotypes grown in13.7� 6.6� 4.9 cmtrays, eachhavingeightcells andcontainingasoillesscompostmixture.Groupsof four trayswereplaced in10-Lplastic storage boxes (34� 13� 23 cm). Three seeds of eachgenotype were sown in each cell and thinned to a singleseedling per cell after emergence. A complete randomisedblock design was utilised with four replications and two plantsper genotype per replication. All experiments were conductedunder controlled environment growth room conditions of18� 1�C during the day and 14� 1�C at night, with lightintensity of 150mE/m2.s. Seedlings were grown untilcotyledons were fully expanded, equivalent to growth stage1.00 on the scale given by Sylvester-Bradley and Makepeace(1984).

Genotypes tested

Three separate experiments were conducted using the cotyledoninoculationmethod. In experiment 1, 11B. napusgenotypes fromChina and 21 B. napus genotypes from Australia, as listed inTable 1, were tested to represent various levels of field resistanceor susceptibility identified by Li et al. (2006). Seed was obtainedfrom Australia and China through an Australian Centre forInternational Agricultural Research collaborative program.Twelve of these same genotypes were selected on the basis ofperformance in experiment 1 for use in experiment 2, with thegenotypes ranging from most resistant to most susceptible to thedisease, and retested in experiment 2 to confirm the results ofexperiment 1. In experiment 3, six of the same genotypes fromexperiments 1 and 2 were further selected on the basis of theirperformance in experiments 1 and 2 in order to confirm thereliability of the genotype responses to S. sclerotiorum.

Inoculum production

A single sclerotium of S. sclerotiorum isolate MBRS1 wassurface sterilised in 1% (v/v) sodium hypochlorite and 70%ethanol for 4min followed by two washes in sterile distilledwater for 1min as described by Clarkson et al. (2003). Thesclerotium was cut in half and placed on potato dextrose agar(PDA). S. sclerotiorum was subcultured and maintained in anincubator at 20�C on PDA. Seven agar plug discs (each 5mm2 indiameter) were cut from the actively growing margin of a 3-day-old colony and transferred to a 250-mL flask containing 75mL ofa sterilised liquid medium (potato dextrose broth 24 g, peptone10 g, H2O 1L). Flasks were rotated on an Innova 2300 platformshaker (New Brunswick Scientific, Edison, NJ) at 120 rpm/min.After 3 days, colonies of S. sclerotiorum were harvested andwashed twice with sterilised deonised water. The fungal matsobtained were transferred to ~125mL of the same liquid mediumand mycelia macerated in a Breville food grinder for 3min. Themacerated mycelial suspension was then filtered through fourlayers of cheese cloth and the concentration was adjusted to104 fragments/mL using a haemocytometer (SUPERIOR, Berlin,Germany) with the same liquid medium.

Cotyledon assay for Sclerotinia in Brassica Australasian Plant Pathology 107

Inoculations

Inoculations were carried out when cotyledons were 10 days old.A total of four droplets of mycelial suspension of 10mL weredeposited on every seedling using a micropipette, with a singledrop on each cotyledon lobe. While inoculating, the mycelialsuspension was shaken regularly to maintain the homogenousmixture of themycelial suspension. A 2.5-cm-deep layer of waterwas added at the bottom of the boxes to helpmaintain a high levelof humidity. In addition, a very fine mist of water was sprayedboth over cotyledons and on the inside of the container lids.Together, these procedures allowed maintenance of a relativehumidity level of at ~100% within the boxes. Followinginoculation, the boxes were placed for 2 days under thebenches in the controlled environment room and maintained ata low light intensity of ~13mE/m2.s and then returned to theoriginal light intensity and relative humidity conditions describedabove for the remainder of each experiment.

Disease assessment

Typical hypersensitive and/or necrotic and water soaked lesionswere apparent by 1--2 days post-inoculation (dpi). At 4 dpi, boxcovers were removed and lesions assessed on the basis of lesiondiameter (mm).All lesion diametersweremeasured using a linearruler.

Data analyses

The lesion rating data from experiment 1 were analysed by singlefactor ANOVA using GENSTAT (9th edition, Lawes AgriculturalTrust). Fisher’s least significant differences (l.s.d.) at P= 0.05was used to calculate the differences between the genotypes.Regression analysis was undertaken using GENSTAT to determinethe relationship between the cotyledon responses obtained withthe stem lesion lengths with those found for the same genotypeswhen tested earlier by Li et al. (2006) underWA field conditions.The relationships between experiments 1, 2 and 3 were assessedby computing correlation coefficients using the data analysisfunction in Microsoft Excel.

Results

Experiments 1, 2 and 3

Experiment 1 was the initial screening of 32 genotypes,experiment 2 was a confirmation test of experiment 1 onselecting 12 of these genotypes, while experiment 3 wasagain a confirmation trial but restricted to six genotypes.Typical necrotic and/or water-soaked lesions appeared oncotyledons of susceptible genotypes inoculated withS. sclerotiorum (Fig. 1). The type, size and severity oflesions on cotyledons varied between the genotypes, rangingfrom small, necrotic hypersensitive lesions <1.6mm, to theother extreme where entire cotyledons collapsed and werecovered with white cottony mycelial growth. It is noteworthythat hypersensitive lesions <1mm were observed on genotypesMystic and Charlton. There were significant differences(P� 0.001) between the different genotypes in relation toseverity of cotyledon lesions by 4 dpi (Table 1). GenotypesMystic and Charlton from Australia with mean lesiondiameters� 1.6 and Zhongyou-za No.8 and Ding 474 fromChina with mean lesion diameters� 3.8 were found to be themost resistant. In contrast, Australian genotypes TQ055-02W2,AV-Sapphire, Surpass 400 and Rivette were found to be themost susceptible. All had mean lesion diameters� 9.1.Generally, where lesions were in the 11--13mm diameterrange the cotyledons totally collapsed from diseasedevelopment.

Although there were small differences in lesion size onindividual genotypes across the different experiments, therelative rankings of genotypes were similar across the threeexperiments, with Mystic and Charlton being the mostresistant, AV-Sapphire and Rivette being very susceptible andRQ001-02M2 having an intermediate reaction (Table 2).

There was significant positive correlation between thecotyledon lesion diameter across the genotypes betweenexperiments 1 and 2 (r= 0.92, P< 0.001, n= 12); betweenexperiments 1 and 3 (r= 0.93, P < 0.001, n = 12); andbetween experiments 2 and 3 (r= 0.96, P < 0.001, n= 6).

Table 1. Reaction to Sclerotinia sclerotiorum of 32 Brassica napus

genotypes from Australia and China in terms of stem lesion length 3

weeks after inoculation in field screening (data taken fromLi et al. 2006)

and in terms of the diameter of lesions 4 days after inoculation on the

cotyledons in experiment 1 undertaken under controlled environment

conditions

Genotypes Origin Field stem test Cotyledon test

Fan168 China 3.15 4.98

RR002 Australia 3.48 4.79

AG-Spectrum Australia 3.65 4.34

Oscar Australia 4.10 6.87

Lantern Australia 4.12 6.57

Fan 028 China 4.77 4.37

Zhongyou-za No.8 China 4.79 2.84

BST7-02M2 Australia 4.87 7.09

Zhongshu-ang No.4 China 5.07 6.35

Ding474 China 5.25 2.81

Mystic Australia 5.53 0.58

RQ011 Australia 5.78 3.81

Charlton Australia 5.80 1.60

Ding110 China 5.89 3.78

P617 China 6.00 5.72

AG-Outback Australia 6.02 7.90

Fan 023 China 6.02 6.83


P3083 China 6.23 5.95

Yu 178 China 6.23 7.57

AV-Sapphire Australia 6.62 9.37

Surpass 400 Australia 6.65 9.13

Tranby Australia 6.8 7.96

Qu1104 China 6.93 7.02

Skipton Australia 7.10 8.40

Trigold Australia 7.18 7.10

Rainbow Australia 7.20 6.39



RQ001-02M2 Australia 8.69 8.31

TQ05502W2 Australia 9.34 12.82

Rivette Australia 10.39 9.63

Significance P< 0.001l.s.d. (P= 0.05) 1.47

108 Australasian Plant Pathology H. Garg et al.

Correlation of experiment 1 cotyledon test resultswith field ratings of Li et al. (2006)

There was significant positive correlation of genotype ratings oflesions on cotyledons in experiment 1 (4 dpi) with stem lesionlength in the previous field trial (3 weeks post-inoculation)published by Li et al. (2006) (r= 0.62, P < 0.001, n= 32)(Fig. 2). This relationship accounted for ~38% of the totalvariation and it was noted that the two most resistantgenotypes identified in the cotyledon test both showedintermediate reactions in the field stem test where there was noartificial control of environmental conditions.

Discussion

We believe that this is the first report in which a cotyledonassay has been utilised to differentiate and characterise theresponses of any genotypes of Brassica species toS. sclerotiorum. The varying lesion size on cotyledonsbetween the different genotypes was found to be a reliableindicator of B. napus genotype resistance to S. sclerotiorum.

The cotyledon inoculation technique was successful indifferentiating the reaction of the B. napus genotypesagainst S. sclerotiorum. Typical necrotic and water soakedlesions appeared on susceptible genotypes as early as 1--2 dpi.Some of the most susceptible genotypes were covered withwhite mycelial growth by 4 dpi. More resistant genotypesshowed a small lesion confined to the size of the inoculumdroplet. The most resistant genotypes showed only very smallnecrotic flecks depicting a hypersensitive lesion that were alsoalways contained within the confines of the inoculated area.This is the first report of a hypersensitive reaction in thisB. napus--S. sclerotiorum pathosystem. The absence of ahypersensitive reaction in the field, despite expression ofresistance, may be due to uncontrolled environmentalconditions, which may be more conducive for diseasedevelopment in the field.

The high degree of reliability of the cotyledon assay wasevident from the high correlation coefficient values betweenexperiments 1, 2, and 3 (all with r values > 0.90). Althoughthere were some small differences in absolute lesion sizes for oneor more of the same genotypes across different experiments, the

Table 2. Reaction to Sclerotinia sclerotiorum of 12 Brassica napus

genotypes from Australia and China in terms of the diameter of

lesions 4 days after inoculation on the cotyledons in experiments 1, 2,

and 3 undertaken under controlled environment conditions

Genotypes not tested are indicated by dashes

Genotype Expt 1 Expt 2 Expt 3

Fan168 4.98 5.65 --

AG-Spectrum 4.34 6.15 --

Zhongyou-za No.8 2.84 4.87 --

Mystic 0.58 1.54 3.36

Charlton 1.58 1.07 2.16

P617 5.72 7.00 5.71

P3083 5.95 7.54 --

AV-Sapphire 9.37 9.90 10.36

Tranby 7.96 6.31 --

RR005 8.00 9.03 --

RQ001-02M2 8.31 7.34 6.33

Rivette 9.63 9.68 10.34

Significance 0.001 0.001 0.001

l.s.d. (P= 0.05) 1.47 1.93 2.55

0

2

4

6

8

10

12

5 10 15

Lesion diameter (mm) in cotyledon test

Ste

m l

esio

n le

ngth

(cm

) in

fiel

d te

st

Fig. 2. Relationship between stem lesion length (cm) caused by Sclerotinia

sclerotiorum in the field with diameter of lesions (mm) on cotyledons in

experiment 1. The stem lesion length was assessed 3 weeks after inoculation

and this data taken from Li et al. (2006), while the lesion size on cotyledons

was assessed 4 days after inoculation. Thirty-two Brassica napus genotypes

from Australia and China were investigated. y= 0.8848x�2� 10�14;(r= 0.62, P< 0.001, n= 32); R2 = 0.38.

1 2 3 4 5 6

Fig. 1. Typical hypersensitive (far right cotyledon) and/or necrotic and water-soaked lesions of various diameters as assessed

4 days post-inoculation following inoculation of Brassica napus cotyledons with washed macerated mycelium of Sclerotinia

sclerotiorum. The genotypes represented, in order from the left, are as follows: 1, TQ055-02W2; 2, Rivette; 3, RR005; 4, P617;

5, Ding474; and 6, Charlton.


overall relative rankings of the genotypes against S. sclerotiorumshowed similar trends with Mystic and Charlton being the mostresistant, AV-Sapphire and Rivette being very susceptible andRQ001-02M2 having an intermediate reaction. Althoughgenotypes with intermediate reactions were difficult tocharacterise in some previous studies on soybean conductedby Kull et al. (2003) and by Sun (1995), we found that evengenotypes with intermediate reaction to S. sclerotiorum couldreadily and consistently be characterised with our cotyledonassay. Moreover, even where cotyledon assays had beenutilised previously on other non-Brassica crops and comparedwith other methods of assay, correlation between the cotyledonassay and other methods such as the cut stemmethod for soybean(r= 0.54, P� 0.05) and dry bean (r= 0.54, P� 0.05) were muchless significant compared with our study.

The cotyledon assay developed in the present study is anefficient, rapid and inexpensive method of screening B. napusgenotypes for resistance to S. sclerotiorum. It takes only a total of16 days to assess the responses of genotypes using this methodcompared with up to 3--4months when other methods such as thestem inoculation technique are utilised. Although there aredetached leaf assays on B. napus that also are rapid, theseoften have poor correlation with field performance of the samegenotypes (Bradley et al. 2006). It is apparent that a large numberof genotypes can be subjected to preliminary screening using ourcotyledon assay in a comparatively small time frame.

It is noteworthy that genotype performance againstS. sclerotiorum in the cotyledon assay was significantly andpositively correlated with stem lesion ratings from fieldscreening of the same genotypes. However, this relationship wasnot perfect, perhaps due to that fact that field screening tests ofoilseed Brassica genotypes can include components of diseaseescape rather than physiological resistance (Phillips et al. 1990),primarily due to differences between genotypes in plantarchitecture and maturity (Phillips et al. 1990). Despite thisimperfection, the commonality between the cotyledon and fieldtests we found confirmed the viability of utilising the cotyledonassayasareliableandrapidmethodfor initialcharacterisationofB.napusgenotype responses toS. sclerotiorum.WhileBradley et al.(2006) compared different controlled environment screeningmethods with field test results and found that the petioleinoculation method was significantly correlated with yield ofB. napus cultivars, ours is the first report in which a cotyledonassay has been successfully utilised to differentiate B. napusgenotype responses to S. sclerotiorum in a similar way to thatobtained for stem lesions under field testing.

Many of the previous methods employed for screeninggenotypes for resistance to S. sclerotiorum have utilisedcolonised agar plugs as the form of inoculum. However, use ofcolonised agar plugs has limitations. For example, oncemycelium is applied with an appropriate source of energy(e.g. PDA), then the pathogen ramifies so rapidly that it doesnot give adequate time for the host plant to fully engage defenceresponses, making resistant genotypes difficult to distinguish. Inaddition, the actual amountof hyphal inoculumused for screeningcan vary when using colonised agar plugs as a food base and as acarrier of the pathogen. Additionally, asynchronous initiation oflesion development can occurwhen colonised agar plugs are usedas the inoculum source (Chun et al. 1987). Although macerated

mycelium of S. sclerotiorum has been used previously by otherson non-cotyledon tissues [e.g. by Chen and Wang (2005) onsoybean],we found that quantification of themycelial suspensionin our cotyledon assay ensured uniform application of inoculumacross test genotypes. Moreover, in our cotyledon assay, anyinterference and/or variability in the genotype responses fromtoxic metabolites released by S. sclerotiorum in liquid growthmedia would have been reduced by the fact that we washed themycelial mats with deionised water before maceration andresuspended them in the liquid medium.

In conclusion, this cotyledon assay can be used to rapidly,reliably and cheaply characterise B. napus genotype responses toS. sclerotiorum andour results suggest that the technique could beuseful across a wide diversity of germplasm.While the cotyledonassay could best be utilised for initial screening of large genotypepopulations, the fact that our assay relates well to results obtainedwith field screening indicates that this cotyledon assay can hastenthe development of new B. napus cultivars with improved levelsof resistance against S. sclerotiorum. However, it still remains tobe shown that this technique will characterise genotypereactions against S. sclerotiorum in other cultivated Brassicaspecies, e.g. B. juncea, and in wild and/or weedy Brassica andcrucifer germplasm, which may have different sizes, types orshapes of cotyledons.

Acknowledgements

The first author gratefully acknowledges the financial assistance of the

Australian Centre for International Agricultural Research by way of a John

Allwright PhD fellowship. We thank Dr Hua Li for assistance with

development of this cotyledon technique and Dr Caixia Li for provision of

the isolate of Sclerotinia sclerotiorum used in this study and for advice on

statistical analysis.

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http://www.publish.csiro.au/journals/app

cotyledon assay as a rapid and reliable method of screening for resistance against sclerotinia...

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