Eur J Pathol (2015) 143:527–541 DOI 10.1007/s10658-015-0696-6

Virulence differences among Sclerotinia sclerotiorum isolates determines host cotyledon resistance responses in genotypes

Xin Tian Ge & Ming Pei You & Martin J. Barbetti

Accepted: 19 June 2015 /Published online: 1 July 2015 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2015

Abstract Differences in Sclerotinia rot (SR) disease se- (e.g., B. napus Charlton against the ‘’ and verity, caused by two categorized pathotypes and one MBRS1 isolates; B. napus Oscar against the WW4 iso- more recent isolate of S. sclerotiorum andmeasuredin late). These findings highlight the value from using terms of cotyledon lesion diameter, were studied across pathotypes of different physiological specialization in diverse Brassicaceae hosts to characterize host response screening programs to identify host resistance that is du- and pathogen virulence. There were significant differences rable across multiple pathotypes. Distinct host resistance (P ≤0.001) between genotypes, isolates and a significant symptom types were reported for the first time on some genotype x isolate interaction. The mean diameter of genotypes against isolate WW4; including a distinct yel- cotyledon lesions ranged from 5 mm in the most resistant low halo observed around lesions on B. napus RQ001, genotypes (e.g., Brassica juncea Ringot I and Seeta) to≥ indicative of leaf senescence involved in programmed cell 13.6 mm in the most susceptible genotypes (e.g., death (PCD); a distinct dark brown margin observed B. tournefortii Wild turnip #1 and #2, irio around lesions on R. sativus, indicative of a hypersensitive London rocket Wild #1 and #2, and B. nigra 4381). response (HR); and the HR ‘flecking’ on Sinapis alba Responses, in at least one experiment for some Concerta and B. juncea Seeta. That WW4 was the most B. juncea (e.g., Seeta, Ringot I) and Raphanus sativus pathogenic isolate for genotypes such as B. juncea (e.g., Colonel) genotypes, were generally highly resistant Hetianyoucai and B. napus Oscar that showed high level irrespective of the isolate used, making them ideal sources resistance to the ‘Cabbage’ isolate and intermediate resis- of resistance to exploit for developing new varieties with tance to MBRS-1, dispels previously held views that more effective resistance to SR across multiple pathotypes WW4 was a largely avirulent pathotype of little conse- of this pathogen. In contrast, some other genotypes quence. Rather, isolate WW4 offers unique opportunities showed significant isolate dependency, with high levels to investigate HR and PCD host resistance responses to of resistance against one isolate (e.g., B. napus Charlton S. sclerotiorum in Brassicaceae. against the WW4 isolate; B. napus Oscar against the ‘ ’ Cabbage isolate) but quite susceptible to other isolates Keywords Sclerotinia sclerotiorum . Sclerotinia rot ...... : : Brassicaceae Crucifer Raphanus Brassica Radish X. T. Ge M. P. You M. J. Barbetti Oilseed rape . Mustard . Host resistance School of Plant Biology, Faculty of Science, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Introduction M. P. You : M. J. Barbetti (*) The UWA Institute of Agriculture, Faculty of Science, The University of Western Australia, Crawley, WA6009, Australia Sclerotinia sclerotiorum (Lib) de Bary causes devastat- e-mail: [email protected] ing and severe yield-limiting diseases worldwide for 528 Eur J Plant Pathol (2015) 143:527–541 many agriculturally and horticulturally important spe- identification of hosts with durable resistance may only cies, in particular Brassicaceae (Saharan et al. 2008). be possible if several pathotypes are used to screen for This includes sclerotinia rot (SR), particularly on oilseed resistance (Garg et al. 2010a;Geetal.2012; Uloth et al. rape (Brassica napus L.) and mustard (B. juncea (L.) 2013, 2014). Studying the interaction of a range of Czern.) (Barbetti and Khangura. R 2000; Delourme cruciferous host species to multiple pathotypes of et al. 2011; Singh et al. 2011), and in crops S. sclerotiorum is also important because it not only such as cabbage (B. oleracea L. var. capitata (L.) Alef.), enhances our understanding of resistance and increases Chinese cabbage (B. rapa L. var. pekinensis (Lour.) the possibility of identifying resistant hosts to individual Kitam.), radish (Raphanus sativus L.), broccoli pathotypes, but more importantly, allows opportunities (B. oleracea L. var. italica Plenck), turnip (B. rapa L. to identify hosts with resistance against multiple var. glabra Regel), swede or rutabaga (B. napus L. var. pathotypes (Ge et al. 2012;Gargetal.2010a;Uloth naprobrassica (L.) Reichenb.), and kale (B. oleracea L. et al. 2013, 2014). While various Brassicaceae geno- var. acephala Bailey) (Kim et al. 2003; Pedras and types have been studied for resistance to S. sclerotiorum, Ahiahonu 2004). The fact that S. sclerotiorum exhibits fewer studies have assessed resistance when challenged little host specificity to Brassicaceae, and infects almost with multiple isolates. For example, while Kim and Cho all species, has constrained selection for locating disease (2003) studied pathogenicity in cruciferous crops using resistance such as the hypersensitive response (HR) and six randomly picked S. sclerotiorum isolates, they found programmed cell death (PCD) among Brassicaceae no significant differences in the susceptibility of the host species (McCartney 2000; Purdy 1979). to these different isolates. In contrast, Ge et al. (2012) In addition to the lack of host specificity by showed that 53 isolates could be clustered into 8 distinct S. sclerotiorum, infection with different S. sclerotiorum pathotypes, while Clarkson et al. (2008, 2013)showed pathotypes or isolates can result in range of virulence the wide diversity of S. sclerotiorum from agricultural and/or pathogenicity, including variations in disease crops and/or wild hosts. Such studies suggest that progress and severity (Garg et al. 2010a;Geetal. utilising randomly picked and uncharacterized isolates 2012). The molecular basis of this variation is uncertain may not be the most effective approach for screening and may come from various S. sclerotiorum genetic for resistance as this can confound interpretation of determinants (Harel et al. 2006; Jurick and Rollins the host resistance reactions, even where the host 2007; Rahmanpour et al. 2010). Many studies report reactions are similar across isolates. Screening multi- tremendous pathogenic and phylogenetic variation in ple hosts using multiple pathotypes is the best ap- S. sclerotiorum populations. For example, in Ontario proach for efficient screening for resistance against Canada, studies have shown that field populations of S. sclerotiorum. S. sclerotiorum on oilseed rape are genetically hetero- Although absolute or near-absolute resistance to geneous (Kohn et al. 1990, 1991). In south-east S. sclerotiorum is considered by some to be not feasible Australia, S. sclerotiorum isolates collected from oilseed (e.g., Saharan et al. 2008), it is not impossible as dem- rape crops also showed high levels of genotypic diver- onstrated by Garg et al. (2010b). In contrast, screen- sity (Sexton et al. 2006); and within a particular geo- ing for varying levels of partial resistance, while of graphic region, oilseed rape can host different higher occurrence (e.g., Li et al. 2006, 2007, 2009; S. sclerotiorum pathotypes (Ge et al. 2012). High vari- Uloth et al. 2013, 2014), requires a screening method ation in aggressiveness between isolates of that is consistent in terms of host resistance across S. sclerotiorum has also been observed on common bean multiple experiments (Garg et al. 2010b;Lietal. (Phaseolus vulgaris L.) (Otto-Hanson et al. 2011)and 2006; Nelson et al. 1991; Prajapati et al. 2005;Uloth on sunflower (Helianthus annuus L.) (Ekins et al. 2007). et al. 2013). Resistance screening against As S. sclerotiorum frequently exhibits little host S. sclerotiorum in B. napus and/or B. juncea commonly specificity, identification of resistant genotypes may involves utilization of a field stem inoculation test orig- best be achieved by screening genotypes from diverse inally developed by Buchwaldt et al. (2005) that, with cruciferous species rather than a single species (Saharan some modification, has proved very successful in iden- et al. 2008; Uloth et al. 2013, 2014). In addition, as tifying resistance that will be effective in commercial polymorphisms in S. sclerotiorum may be responsible crops (e.g., Li et al. 2006, 2007, 2009;Gargetal.2010b; for pathogenic and phylogenetic differences, Ge et al. 2012; Uloth et al. 2014). While field Eur J Plant Pathol (2015) 143:527–541 529 evaluations of SR were generally considered the most whether selected isolates representing distinct pathotype appropriate and reliable technique for evaluating stem groups is a more efficient and effective means for resis- resistance against S. sclerotiorum, expression of host tance screening than utilizing a single isolate; and, 3) to resistance can be highly variable, particularly where determine whether host resistance expression to fluctuating environmental conditions influence the re- S. sclerotiorum is expressed as programed cell death sponse of individual Brassicaceae (Uloth et al. 2015a) (PCD) and HR on some Brassicaceae. and similarly for infection and disease development in other crops, for example lettuce (Young et al. 2004) genotypes. For this reason, controlled-environment Materials and methods screening methods have been widely used to assess physiological resistance to S. sclerotiorum across a S. sclerotiorum isolates range of different crop types. Further, not all Brassicaceae genotypes can be assessed using the field Three isolates of S. sclerotiorum were used in this study, stem technique as some genotypes have phenotypes that namely: MBRS1, WW4 and ‘Cabbage’. MBRS1 had are either not amenable to field stem inoculation or been extensively used previously for resistance screen- where the disease occurrence in the field is not related ing of B. juncea and B. napus genotypes in the glass- to infection of the stem, such as those that remain in a house and/or the field (Li et al. 2006, 2007, 2009; Garg rosette stage for extended periods; or where disease et al. 2010a;Geetal.2012; Uloth et al. 2013, 2014; occurs at the seedling stage when S. sclerotiorum also Barbetti et al. 2014). WW4 has previously been utilized causes damping-off (Laemmlen 2001). For in field stem and cotyledon tests (e.g., Ge et al. 2012 and Brassicaceae, this led to development of a cotyledon Garg et al. 2010a,respectively).The‘Cabbage’ isolate, assay for screening large numbers of B. napus geno- has not been studied previously, MBRS1 was collected types challenged by various isolates of S. sclerotiorum in 2004 from the Mt Barker area in the southern agri- (Garg et al. 2008, 2010a). As all Brassicaceae genotypes cultural region and WW4 was collected from the have similar cotyledon structures, although they can Walkway area of the northern agricultural region of vary significantly in terms of their size (Uloth et al. Western Australia (Garg et al. 2010a;Geetal.2012). 2014), this cotyledon assay has the potential to be ap- In 2009, the isolate ‘Cabbage’ was isolated from severe- plied much more broadly to other Brassica genotypes. ly diseased cabbage grown in the Perth Metropolitan This was recently demonstrated by Uloth et al. (2014) vegetable-growing region of Western Australia. All iso- who used a cotyledon assay to assess selections of lates had been stored as air-dried sclerotia at 4 °C and Brassica carinata A. Braun., B. incana, B. juncea, B. were only subcultured a single time to produce fresh napus, and B. napus introgressed with B. carinata, B. sclerotia during this storage period to maintain via- nigra, B. oleracea, B. rapa L. var. rosularis Tsen & Lee bility and virulence. In all previous screening trials, (Herk),B.rapaL. var. chinensis (L.) Hanelt,B. MBRS1 showed very high virulence levels (Li et al. tournefortii, Raphanus raphanistrum L., R. sativus, 2006, 2007, 2009;Gargetal.2010a;Geetal.2012; and Sinapis arvensis L. against a single isolate of Barbetti et al. 2014), in contrast to WW4 that was S. sclerotiorum. the least virulent isolate of eight tested isolates by In response to the challenges outlined above, the Garg et al. (2010a). All three isolates have been overall objective of the current paper is to assess the confirmed as S. sclerotiorum (Garg et al. 2010a; effect of contrasting pathotypes of S. sclerotiorum [viz. Ge et al. 2012; Ge et al. unpublished) and MBRS1 two S. sclerotiorum pathotypes from B. napus with very and WW4 have been categorized as separate and different virulence, and an isolate from an infected cab- distinct pathotypes, pathotype 76 for MBRS1 and bage ( var. capitata) devastated by pathotype 36 for WW4 (Ge et al. 2012). SR], on the severity of S. sclerotiorum disease develop- ment in cotyledons across a diverse range of Inoculum production Brassicaceae species. Specific objectives of the paper include determining: 1) how virulence differences Dry-stored sclerotia at room temperature (approx. among S. sclerotiorum isolates determines host cotyle- 22 °C) for each of the three S. sclerotiorum isolates were don resistance responses in Brassicaceae genotypes; 2) surface sterilized, cut in half, sub-cultured on potato 530 Eur J Plant Pathol (2015) 143:527–541 dextrose agar (PDA). In preparing for inoculation, iso- cultured on PDA at 20 °C. The plugs were transferred lates were cultured using methods described by Garg to 250 mL flasks containing 100 mL of sterilized liquid et al. (2008, 2010a, b). In brief, eight agar plug discs medium (potato dextrose broth 24 g, peptone 10 g, H2O (each 5 mm diameter) were cut from the actively grow- 1 l). The flasks were then incubated on an Innova® 2300 ing margin of three day-old colonies of each isolate platform shaker (New Brunswick Scientific,

Table 1 Genus and species, common name, genotype and Number Genus/species Common name Genotype (and origin) origin for 38 Brassicaceae genotypes used in one or more 1 Brassica carinata Ethiopian mustard ATC-94011 (Australia) of Experiments 1, 2, 3 2 Brassica carinata Ethiopian mustard ATC-04129 (Australia) 3 Brassica carinata Ethiopian mustard ATC-94024 (Australia) 4 Brassica juncea Indian mustard JM-06020 (Australia) 5 Brassica juncea Indian mustard JM-18 (Australia) 6 Brassica juncea Indian mustard JN-006 (Australia) 7 Brassica juncea Indian mustard Berry (China) 8 Brassica juncea Indian mustard Datonghuangyoucai (China) 9 Brassica juncea Indian mustard Hetianyoucai (China) 10 Brassica juncea Indian mustard Ringot I (China) 11 Brassica juncea Indian mustard Xinyou 9 (China) 12 Brassica juncea Indian mustard Yilihuang (China) 13 Brassica juncea Indian mustard GM-1 (India) 14 Brassica juncea Indian mustard Seeta (India) 15 Brassica juncea Indian mustard Sej-2 (India) 16 Brassica napus Oilseed rape Charlton (Australia) 17 Brassica napus Oilseed rape GSL −1 (India) 18 Brassica napus Oilseed rape Oscar (Australia) 19 Brassica napus Oilseed rape RQ-001 (Australia) 20 Brassica nigra Black mustard 4381 (Australia) 21 Brassica nigra Black mustard ATC-91105 (Australia) 22 Brassica nigra Black mustard ATC-94745 (Australia) 23 Brassica tournefortii Wild turnip Wild turnip #1 (Australia) 24 Brassica tournefortii Wild turnip Wild turnip #2 (Australia) 25 Crambe abyssinica Abyssinian kale Shuie (Western Australia) 26 Camelina sativa False flax R4175-01 W2 (Australia) 27 vesicaria ssp.sativa Rucola MJB-black seed #1A (Australia) 28 ssp.sativa Rucola MJB-brown seed #1 (Australia) 29 Eruca vesicaria ssp.sativa Rucola MJB-brown seed #2 (Australia) 30 Eruca vesicaria ssp.sativa Rucola MJB-yellow seed #1 (Australia) 31 Raphanus sativus Oil radish Adagio (Europe) 32 Raphanus sativus Oil radish Pegletta (Europe) 33 Raphanus sativus Oil radish Colonel (Europe) 34 Raphanus sativus Oil radish Boss (Europe) 35 Raphanus sativus Vegetable radish White Icicle (Australia) 36 Sinapis alba White mustard Concerta (Europe) 37 London rocket Wild #1 (Western Australia) 38 Sisymbrium irio London rocket Wild #2 (Western Australia) Eur J Plant Pathol (2015) 143:527–541 531

Connecticut, USA) at 120 rpm min−1. After 3 days, the experiment 2 to confirm the major host resistance ex- S. sclerotiorum fungal mats were harvested, washed pressions across the three experiments (Table 4). twice with sterilized liquid medium, re-suspended in 125 ml of liquid medium and macerated using a Breville® food grinder for 45 s. The macerated mycelial Test conditions suspension was then filtered through three layers of cheesecloth. The macerated mycelia were adjusted to In all three experiments, seed of each genotype was and re-suspended at 1.5×104 fragments ml−1 in the sown in 30×20×7 cm trays holding 40 cells filled with same liquid medium using a haemocytometer UWA potting mix consisting of: 2.5 m3 fine composted (SUPERIOR®, Berlin, Germany). Three separate exper- pine bark, 1 m3 coco peat, 5 m3 brown river sand, 10 kg iments were undertaken using the above three isolates. slow release fertilizer Osmoform® NXT 22 N+2.2

P2O5 +9.1 K2O+1.2 Mg+trace elements (Everris International B.V.), 10 kg Dolomite (CalMag®), 5 kg Genotypes gypsum clay breaker, 5 kg extra fine limestone, 4 kg iron hepta sulphate, and 1 kg iron chelate. Potting mix Previous studies of S. sclerotiorum utilizing a cotyledon was pasteurized at 63 °C for 30 min. Each tray was test have either been constrained to B. napus species placed in a 34×23×13 cm plastic storage container. Five (Garg et al. 2008, 2010a), or only very recently extended seeds of each genotype (except B. tournefortii Gouan., to a wider range of Brassicaceae hosts (Uloth et al. Brassica nigra (L.) W.Koch in Roehl. and S. irio L.) 2014). Details of all 38 Brassicaceae genotypes across were sown in each cell and thinned to three seedlings per seven different genera and 10 different species used are cell after emergence. For B. tournefortii,andS. irio and provided in Table 1. In experiment 1, 10 genotypes were B. nigra genotypes, 10 seeds of each genotype were used (Table 2); in experiment 2, 36 genotypes were sown in each cell and thinned to five seedlings after utilized that included five genotypes from experiment emergence as germination percentage was lower for 1 for comparison between experiments (Table 3); while these three species. Seedlings were maintained under in experiment 3, 22 genotypes were selected, including controlled growth room conditions with 12/12 h day/ eight genotypes from experiment 1 and 19 from night at 20 °C, and a light intensity of 385 μMol m−2 s−1.

Table 2 Experiment 1: diameter of lesions recorded 72 h after inoculation of the cotyledons of 10 Brassicaceae species genotypes with three different isolates of Sclerotinia sclerotiorum. Rank order numbers in parentheses represent lesion diameter from smallest (1) to largest

Genotype Isolate Mean

‘Cabbage’ MBRS-1 WW4

Adagio (R. sativus) 11.89(4) 7.80(5) 8.91(8) 9.53(6) ATC-94011 (B. carinata) 13.60(5) 9.30(6) 3.99(1) 8.97(4) ATC-94129 (B. carinata) 16.09(9) 9.64(9) 14.65(10) 13.46(10) Boss (R. sativus) 10.47(3) 5.59(1) 6.16(5) 7.41(1) Charlton (B. napus) 16.14(10) 9.95(10) 5.35(3) 10.48(7) Colonel (R. sativus) 10.06(2) 6.65(3) 5.61(4) 7.44(2) Datonghuangyoucai (B. juncea) 14.00(6) 9.35(7) 9.26(9) 10.87(9) Pegletta (R. sativus) 10.01(1) 6.12(2) 7.56(7) 7.90(3) RQ001 (B. napus) 15.73(8) 9.48(8) 6.55(6) 10.53(8) ‘Unknown’ (Crambe abyssinicia) 15.32(7) 7.43(4) 4.96(2) 9.24(5) Mean 13.33 8.13 7.30

Significance of genotypes P <0.001; l.s.d. (P ≤0.05)=1.58 VR=11.0 Significance of isolates P <0.001; l.s.d. (P ≤0.05)=0.86 VR=13.2 Significance of genotypes×isolates P<0.001;l.s.d.(P ≤0.05)=2.73 VR=5.5 532 Eur J Plant Pathol (2015) 143:527–541

Table 3 Experiment 2: Diameter of lesions recorded 72 h after inoculation of the cotyledons of 36 Brassicaceae species genotypes with three different isolates of Sclerotinia sclerotiorum. Rank order numbers in parentheses represent lesion diameter from smallest (1) to largest

Genotype Isolate

‘Cabbage’ MBRS-1 WW4 Mean

4381 (B. nigra) 17.00(33) 15.25(32) 10.75(29) 14.33(30) Adagio (R. sativus) 11.56(16) 10.44(24) 6.94(18) 9.65(17) ATC-91105 (B. nigra) 7.19(7) 7.38(8) 5.25(8) 6.60(6) ATC-94745 (B. nigra) 8.69(13) 9.13(15) 4.25(1) 7.35(11) Berry (B. juncea) 7.00(4) 6.38(4) 5.63(11) 6.33(4) Boss (R. sativus) 8.69(13) 6.13(2) 4.75(2) 6.52(5) Carinata 94024 (B. carinata) 17.00(33) 11.19(25) 7.00(19) 11.73(25) Concerta (Sinapis alba) 15.56(24) 10.06(21) 12.19(30) 12.60(28) Datonghuangyoucai (B. juncea) 15.26(22) 9.67(18) 6.38(12) 10.43(20) GM-1 (B. juncea) 8.00(12) 6.81(6) 5.43(10) 6.75(8) GSL-1 (B. napus) 7.75(10) 9.25(16) 5.00(6) 7.33(10) Hetianyoucai (B. juncea) 7.88(11) 8.44(12) 7.13(22) 7.81(13) JM-06020 (B. juncea) 15.88(25) 7.94(10) 8.44(26) 10.75(24) JM-18 (B. juncea) 7.00(4) 9.88(20) 7.00(19) 7.96(14) JN-006 (B. juncea) 7.25(8) 8.95(14) 4.94(4) 7.04(9) London rocket Wild #1 (Sisymbrium irio) 17.00(33) 13.98(28) 14.44(33) 15.14(33) London rocket Wild #2 (Sisymbrium irio) 17.00(33) 17.00(33) 7.00(19) 13.67(29) MJB black seed #1A (Eruca vesicaria ssp.sativa) 11.81(18) 12.8(27) 7.44(24) 10.68(22) MJB-brown seed #1 (Eruca vesicaria ssp. sativa) 17.00(33) 12.56(26) 6.50(13) 12.02(27) MJB-brown seed #2 (Eruca vesicaria ssp. sativa) 9.63(15) 15.95(30) 6.56(15) 10.71(23) MJB yellow seed #1 (Eruca sativa) 12.81(19) 10.25(22) 6.69(17) 9.92(19) Oscar (B. napus) 6.88(3) 8.13(11) 7.50(25) 7.50(12) Pegletta (R. sativus) 14.25(21) 7.77(9) 6.56(15) 9.53(16) R4175-01 W2 (Camelina sativa) 17.00(33) 10.25(22) 8.69(27) 11.98(26) Ringot I (B. juncea) 7.44(9) 6.19(3) 4.94(4) 6.19(2) RQ001 (B. napus) 15.31(23) 9.5(17) 6.5(13) 10.44(21) Seeta (B. juncea) 6.00(1) 5.50(1) 5.00(6) 5.50(1) Sej-2 (B. juncea) 7.06(6) 6.56(5) 5.25(8) 6.29(3) WhiteIcicle(R. sativus) 11.69(17) 7.25(7) 9.16(28) 9.37(15) Wild turnip #1 (B. tournefortii) 17.00(33) 15.78(30) 13.94(32) 15.57(32) Wild turnip #2 (B. tournefortii) 16.75(26) 15.24(29) 12.88(31) 14.96(31) Xinyou 9 (B. juncea) 12.88(20) 9.67(18) 7.13(22) 9.89(18) Yilihuang (B. juncea) 6.5(2) 8.75(13) 4.75(2) 6.67(7) Mean 11.24 10.00 7.33

Significance of genotypes P<0.001;l.s.d.(P ≤0.05)=0.56 VR=233.80 Significance of isolates P<0.001;l.s.d.(P ≤0.05)=0.17 VR=1171.58 Significance of genotypes×isolates P<0.001;l.s.d.(P ≤0.05)=0.96 VR=37.94

Inoculation procedure on the Sylvester-Bradley and Makepeace scale (Sylvester-Bradley and Makepeace 1984)] they were When seedlings had cotyledons fully expanded at inoculated, as described by Garg et al. (2008, 2010a), 12 days after sowing [equivalent to growth stage 1.00 but with some minor modification. Briefly, a single Eur J Plant Pathol (2015) 143:527–541 533

Table 4 Experiment 3: Diameter of lesions recorded 72 h after inoculation of the cotyledons of 22 Brassicaceae species genotypes with three different isolates of Sclerotinia sclerotiorum. Rank order numbers in parentheses represent lesion diameter from smallest (1) to largest

Genotype Isolate Mean

‘Cabbage’ MBRS-1 WW4

Adagio (R. sativus) 9.97(13) 7.6(10) 7.02(18) 8.20(11) ATC-94011(B. carinata) 12.03(17) 10.95(21) 4.69(8) 9.22(17) ATC-94129 (B. carinata) 8.65(9) 9.52(18) 17.00(22) 11.72(22) ATC-91105 (B. nigra) 8.16(6) 7.69(11) 5.72(12) 7.19(8) ATC-94745 (B. nigra) 10.89(16) 9.49(17) 4.52(7) 8.30(12) Berry (B. juncea) 9.12(11) 6.35(7) 6.24(14) 7.24(9) Boss (R. sativus) 7.50(5) 2.00(1) 1.25(1) 3.58(1) Charlton (B. napus) 15.69(21) 9.67(19) 4.42(5) 9.93(19) Concerta (Sinapis alba) 16.01(22) 11.13(22) 6.11(13) 11.08(21) Datonghuangyoucai (B. juncea) 13.57(19) 7.72(12) 6.50(17) 9.26(18) GM-1 (B. juncea) 8.20(8) 6.27(5) 5.00(11) 6.49(6) GSL-1 (B. napus) 9.55(12) 8.55(14) 4.50(6) 7.54(10) Hetianyoucai (B. juncea) 6.47(2) 6.53(9) 6.26(15) 6.42(5) JM-06020 (B. juncea) 14.10(20) 9.24(16) 8.44(19) 10.6(20) JM-18 (B. juncea) 10.28(14) 8.66(15) 6.38(16) 8.44(14) JN-006 (B. juncea) 8.92(10) 6.26(4) 4.72(9) 6.63(7) Pegletta (R. sativus) 6.47(2) 6.19(3) 12.58(21) 8.41(13) Ringot I (B. juncea) 6.31(1) 6.15(2) 4.38(4) 5.61(2) RQ001 (B. napus) 12.37(18) 10.18(20) 4.76(10) 9.10(16) Seeta (B. juncea) 6.53(4) 6.33(6) 4.25(3) 5.70(3) WhiteIcicle(R. sativus) 10.81(15) 7.76(13) 8.56(20) 9.04(15) Yilihuang (B. juncea) 8.19(7) 6.35(7) 4.13(2) 6.22(4) Mean 9.99 7.75 6.25

Significance of genotypes P<0.001;l.s.d.(P ≤0.05)=0.64 VR=137.2 Significance of isolates P<0.001;l.s.d.(P ≤0.05)=0.24 VR=604.7 Significance of genotypes×isolates P<0.001;l.s.d.(P ≤0.05)=1.11 VR=14.6 droplet of mycelia suspension (8 μl) was deposited on 2010a) had shown that this technique was reliable in each lobe of each cotyledon using a micropipette as determining the level of resistance in B. napus described by Garg et al. 2008, 2010a). However, for genotypes. B. tournefortii, S. irio and B. nigra genotypes, each half of cotyledon was inoculated with a single inoculum Disease assessment drop in the centre of the cotyledon because of their smaller cotyledon size. The mycelia suspension was At 72 h post-inoculation (72 hpi), container lids were shaken regularly to maintain a homogenous suspension removed and lesions assessed on the basis of lesion state during inoculation. After inoculation, groups of diameter (mm) as described by Garg et al. (2008). two trays, together containing the test genotypes, were Briefly, all lesion diameters were measured; in the case placed in separate 30-l clear plastic storage boxes with that lesion area had expanded to the cotyledon edge, the lids to maintain high humidity. Black plastic sheeting lesion diameter was recorded as 17 mm which was the was placed on top of the plastic storage boxes to reduce length of the biggest cotyledon in this study. In addition, − − the light intensity to~10 μMol m 2 s 1 for 72 h prior to any unusual or distinct response on cotyledons was disease assessment. Previous studies (Garg et al. 2008, noted at both 48 and 72hpi. 534 Eur J Plant Pathol (2015) 143:527–541

Statistical design and analyses Expression of resistance in tested Brassicaceae genotypes A randomized block design was utilised with ge- notypes (10)×isolates (3)×replicates (4)=120 pots There was a wide range in terms of the level of for experiment 1; genotypes (36)×isolates (3)×rep- resistance/susceptibility across test Brassicaceae geno- licates (4)=408 pots for experiment 2; and geno- types, ranging from lesions of only 4 mm diameter on types (22)×isolates (3)×replicates (4)=264 pots for the most resistant genotypes to lesions that spanned the experiment 3. For each replication of each geno- width of the cotyledon (≥17 mm) on the most suscepti- type there were three seedlings, viz. 12 cotyledon ble genotypes. Across two or more repeated experi- lobes, with the mean lesion diameter across the 12 ments, genotypes such as R. sativus Boss (Tables 2, 3 lobes representing the individual score for each and 4)andB. juncea Seeta (Tables 3 and 4). overall replication for each genotype. Analyses of variance showed consistently high levels of resistance; while were conducted using Genstat (14th edition, Lawes across a single experiment other genotypes showed Agricultural Trust). Treatment means were com- resistance expressions that were independent of pared using Fisher’s least significant difference S. sclerotiorum isolate used; as was the case for (l.s.d.) to test significant differences between ge- R. sativus Colonel (Table 2), B. juncea Ringot I notypes, isolates and genotype x isolate interac- (Table 4) in a single experiment. Some slightly less tions. Genotypes were ranked according to their resistant genotypes such as B. nigra ACT-91105 still meansinrelationtolesiondiameterinallthree showed a consistent and isolate-independent response experiments. Additional comparisons of the rela- (Tables 3 and 4). In contrast, even some genotypes that tionship between experiments were assessed by showed high levels of resistance against one isolate computing Pearson correlation coefficients. (e.g., B. napus Charlton against the WW4 isolate), were quite susceptible to other isolates. For example, B. napus Charlton against the ‘Cabbage’ and MBRS1 isolates (Tables 2 and 4). Similarly, B. napus Oscar Results showed a high level of resistance to the ‘Cabbage’ isolate, but was very susceptible to the WW4 isolate Genotypes, isolates and genotype-isolate interactions (Table 3). These results suggested there was significant isolate dependency in the response of such genotypes. There were significant differences between genotypes Some genotypes with lower, but still significant levels of (P≤0.001) in terms of cotyledon lesion diameter; sig- resistance such genotypes as B. carinata ATC-94011 nificant differences between isolates (P≤0.001) in rela- (Tables 2 and 4) and B. nigra ATC-94745,(Tables 3 tion to their pathogenicity; and significant isolate x and 4)showedmuchhigherlevelsofrelativeresistance genotype interactions (P≤0.001) throughout the three rankings against WW4 than the other two isolates experiments. Tested genotypes in common across ex- and were also clearly isolate-dependent in terms of periments generally performed consistently in terms of relative resistance rankings. The most susceptible cotyledon lesion diameter across two or more of the genotypes were B. tournefortii Wild turnip #1 and three experiments (Tables 2, 3 and 4). Wild turnip #2, S. irio London rocket Wild #1 and For experiments 1, 3, and 2, as the numbers of London rocket Wild #2, and B. nigra 4381, all with test genotypes increased from 10 to 22 to 34, re- lesions >13.6 mm in experiment 2 (Table 3). spectively, so did the isolate variance ratios (VR= There were differences in host response across the 113.2 for experiment 1, 604.7 for experiment 3, and different experiments. For example, in experiment 2, the 1171.6 for experiment 2, respectively) (Tables 2, 3 repeated host genotypes from experiment 1 showed on and 4).Thehostgenotypevarianceratioswere11.0 average larger lesion sizes, where the most susceptible for experiment 1, 137.1 for experiment 3, 233.8 for genotypes, viz. B. nigra 4381, B. carinata 94024, Eruca experiment 2; while the genotype×isolate interac- vesicaria (L.) Cav. ssp. sativa (Mill.) Thell. MJB-brown tion variance ratios were 5.5 for experiment 1, 14.6 seed #1, E. vesicaria ssp. sativa MJB-brown seed #1, for experiment 3, and 37.7 for experiment 2 S. irio London rocket Wild #1 and Wild #2, Camelina (Tables 2, 3 and 4). sativa (L.) Crantz. R4175-01 W2, and B. tournefortii Eur J Plant Pathol (2015) 143:527–541 535

Wild turnip #1 all had lesion sizes reaching or exceeding and 72 hpi. Symptoms included the expected ‘normal’ cotyledon diameters (≥17 mm) when challenged with at lesions (Fig. 1a), including those on highly susceptible least one of the isolates. genotypes where the ‘normal’ lesions quickly expanded to the cotyledon edge (Fig. 1b)evenby48hpi. Variation in pathogen isolate virulence However, there were also atypical symptoms observed, including a distinct HR lesion, either extending beyond There were significant differences in overall virulence the site of original inoculation droplet (Fig. 1c)or among isolates; with ‘Cabbage’ the most virulent and contained within the site of inoculation droplet WW4 was the least virulent across the three experiments (Fig. 1d) at 72 hpi; and also highly distinctive lesions when the genotype x isolate interaction is ignored. with a yellow halo indicative of leaf senescence in- However, there were significant genotype×isolate inter- volved in PCD that were observed in some genotypes actions (P<0.001 in three experiments) and specific in response to isolate WW4 (Figs. 1e, f, g, h). In contrast genotypes were highly susceptible to specific isolates to lesions produced by the ‘Cabbage’ and MBRS1 as indicated above (Tables 2, 3 and 4). This is best isolates where symptoms were characterized by ‘nor- illustrated by the observation that WW4 was the least mal’ lesions that quickly expanded beyond the limit of virulent isolate overall across most genotypes, but was the inoculated area, the WW4 isolate induced lesions highly virulent on R. sativus Pegletta in experiment 3 that were distinctly different and showed three important (Table 4), and on B. juncea Hetianyoucai, in experi- different features as displayed in Fig. 1. Initially, when ments 2 and 3, compared with isolates MBRS1 and R. sativus Pegletta (genotype displayed in Fig. 1c)and ‘Cabbage’. Thus, even though WW4 was the least vir- Boss (genotype displayed in Fig. 1d) were challenged ulent overall it still had the ability to be a very highly with WW4, they developed HR on the cotyledons. virulent on some such specific genotypes. Although R. sativus Pegletta expressed a HR both within and ‘Cabbage’ was the most virulent isolate overall in ex- outside of the inoculation droplet (Fig. 1c)while periment 2 (Table 3), MBRS-1 was most virulent isolate R. sativus Boss expressed a HR that was contained for some genotypes such as E. vesicaria ssp. sativa MJB within the diameter of the initial inoculation droplet brown seed #2 (Table 3). This particular genotype was (Fig. 1d). In contrast, when challenged with MBRS1 one of the most susceptible against MBRS1, but showed and ‘Cabbage’ isolates, these same genotypes devel- a middle order genotype resistance ranking for the other oped ‘normal’ lesions (Figs. 1a, b and 2a). Secondly, two isolates (Table 2). when B. napus RQ001 was challenged with WW4, a Based on comparisons between overall mean lesion distinctive and extensive yellow halo developed around size for each isolate across the three experiments, in each and every lesion (Fig. 1e). All three E. vesicaria experiments 1 and 3, MBRS-1 and WW4 showed sim- ssp. sativa genotypes not only showed extreme suscep- ilar overall mean virulence across the two experiments tibility to isolates MBRS1 and ‘Cabbage’ with pale with mean lesion diameters of 8.13 and 7.75, respec- yellow lesions formed when challenged by these iso- tively (Tables 2 and 4). This was in contrast to experi- lates, but also within the inoculum-applied area ment 2 where ‘Cabbage’ and MBRS-1 demonstrated displaying a distinctly pink colouration by 48 hpi somewhat similar overall mean virulence with mean (Fig. 1f). The highly resistant B. juncea Seeta (Fig. 1g) lesion diameters of 11.24 and 10.0, respectively, com- challenged by MBRS1 and S. alba Concerta challenged pared to 7.33 with WW4 (Table 3). In general, differ- by WW4 (Fig. 1h) displayed distinct HR ‘flecking’ type ences in the overall mean virulence values were most lesions with slow lesion development. In a comparative pronounced between WW4 and ‘Cabbage’ isolates and experiment where B. napus Charlton and B. napus less so, but still significant between MBRS 1 and RQ001 were challenged by WW4, ‘normal’ lesions ‘Cabbage’ isolates. were observed when B. napus Charlton was inoculated with WW4 (Fig. 2a), while distinctive yellow halo in- Lesion symptoms dicative of leaf senescence involved in PCD was ob- served when B. napus RQ001 was inoculated with While lesion size was measured at 72 hpi, lesion symp- WW4 Fig. 2b); but no symptoms were observed when tom development was noted at both 48 and 72 hpi, with B. napus Charlton or RQ001 were inoculated with plain several distinct lesion symptom types evident by at 48 liquid media (control) (Fig. 2c, d). 536 Eur J Plant Pathol (2015) 143:527–541

Fig. 1 Examples of different host responses observed for geno- for R. sativus Boss challenged with WW4 at 72hpi. e Yellow halo types challenged with one or more isolates of Sclerotinia indicative of leaf senescence involved in PCD for B. napus RQ001 sclerotiorum. a Normal lesions for B. napus Charlton challenged challenged with WW4 at 72hpi. f Pink colouration of inoculation with MBRS-1 at 48hpi. b Highly susceptible for B. carinata ATC- droplet on top of the lesion for Eruca vesicaria ssp. sativa MJB- 94129 challenged with the ‘Cabbage’ isolate at 48hpi. c Hyper- black seed #1A challenged with ‘Cabbage’ isolate at 48hpi. g HR sensitive response (HR) extending beyond the site of the inocula- ‘flecking’ type lesion observed for B. juncea Seeta challenged with tion droplet observed for R. sativus Pegletta challenged with MBRS1 at 72hpi. h HR ‘flecking’ type lesion observed for Sinapis WW4 at 72hpi. d HR lesion within the site of inoculation droplet alba Concerta challenged with WW4 at 48hpi

Fig. 2 Inoculation with the WW4 isolate of Sclerotinia in PCD observed when B. napus RQ001 inoculated with WW4. c sclerotiorum and control comparison with plain liquid media on No symptom observed when B. napus Charlton inoculated with to B. napus Charlton and B. napus RQ001 at 72 hpi. (a) ‘Normal’ plain liquid media (control). d No symptom observed when lesion observed when B. napus Charlton inoculated with WW4. B. napus RQ001 inoculated with plain liquid media (control) (b) Distinctive yellow halo indicative of leaf senescence involved Eur J Plant Pathol (2015) 143:527–541 537

Correlation of genotype responses across experiments that this is the first report of both a HR lesion in some R. sativus genotypes around the cotyledon inoculation There was a significant positive correlation (r=0.84, site and the HR ‘flecking’ observed in S. alba and P<0.001, n=15, Fig. 3) between experiments 1 and 2 B. juncea. With isolate WW4, lesions developed very in relation to lesion size for five genotypes in common slowly, allowing a broader range of different symptom against three S. sclerotiorum isolates. A significant pos- expression such as the HR in R. sativus and the exten- itive correlation (r=0.82, P<0.001, n=24, Fig. 4)was sive yellow halo around lesions on B. napus RQ001. also observed between experiments 1 and 3 in relation to Such symptoms were in distinct contrast to the highly lesion size for eight genotypes in common against the virulent ‘Cabbage’ and MBRS-1 isolates, which only three S. sclerotiorum isolates. Finally, there was a sig- generated ‘normal’ macerated lesions that expanded nificant positive correlation (r=0.84, P<0.001, n=57, rapidly within 72 hpi. Uloth et al. (2013)reportedblack Fig. 5) between experiments 2 and 3 in relation to lesion ‘flecking’ on the surface of the cotyledon in the area of size for 19 genotypes in common against three the inoculation droplet, indicating an HR on B. oleracea S. sclerotiorum isolates. var. italica Prophet, similar to that previously reported in B. napus Charlton when challenged with S. sclerotiorum (Garg et al. 2010a). There are two main S. sclerotiorum Discussion virulence factors for most Brassicaceae : the se- cretion of oxalic acid (Uloth et al. 2015b); and hydro- This study highlights the wide-ranging and variable lytic enzymes that together work in concert to bring responses in terms of resistance in diverse about the maceration of plant tissues and subsequent Brassicaceae genotypes against S. sclerotiorum. The necrosis (Collmer and Keen 1986). Successful distinct host resistance symptom types reported for the S. sclerotiorum infection only develops when many first time on some genotypes against isolate WW4 are individual infection sites coalesce (Sutton and Deverall noteworthy. For example, a distinct yellow halo was 1983). Oligogalacturonides released from the plant cell observed around lesions on B. napus RQ001, which is wall by the enzymatic activity of endo- indicative of leaf senescence involved in PCD, while a polygalacturonases have been shown to act as endoge- distinct dark brown margin was observed around lesions nous elicitors of the HR (Davis et al. 1986). Future on R. sativus, indicative of a HR. The HR ‘flecking’ studies are needed to determine if such compounds are response to WW4 was also observed on S. alba involved in the HR observed in the current study, as it Concerta and B. juncea Seeta. We believe this unique remains unclear whether the reduced virulence in WW4 symptom of a yellow halo to be the first report of a on some specific genotypes relates to reduced secretion reaction of leaf senescence in these Brassicaceae genera of either of these two virulence/pathogenicity factors involving PCD (Greenberg 1997;vanDoornand (Saharan et al. 2008). WW4 offers a unique opportunity Woltering 2004; Yen and Yang 1998). We also believe not only to identify particular genotype resistant

Fig. 3 Correlation between 18.00 experiments 1 and 2 in terms of y = 0.9946x + 0.1052 16.00 overall mean values for diameter R² = 0.71 of cotyledon lesions (mm) at 72 14.00 hpi across five genotypes in 12.00 common against three isolates of Sclerotinia sclerotiorum 10.00 (r=0.84, P<0.001, n=15) 8.00 6.00 4.00 2.00

Cotyledon lesion diameterlesion (Expt Cotyledon 2) 0.00 0.00 5.00 10.00 15.00 20.00 Cotyledon lesion diameter (Expt 1) 538 Eur J Plant Pathol (2015) 143:527–541

Fig. 4 Correlation between 18 experiments 1 and 3 for overall 16 mean values of diameter of y = 0.9165x - 0.394 cotyledon lesions (mm) across 14 R² = 0.68 eight genotypes 72 hpi to three 12 isolates of S. sclerotiorum (r=0.82, P<0.001, n=24) 10 8 6 4 2

Cotyledon lesion diameter (Expt 3) 0 0 5 10 15 20 Cotyledon lesion diameter (Expt 1) responses that are only observable when triggered by a ‘Cabbage’ and MBRS1 isolates). Similarly, B. napus less virulent pathotype. Moreover, this isolate can facil- Oscar showed a high level of resistance to the itate the further study of Brassicaceae-S. sclerotiorum ‘Cabbage’ isolate, but was very susceptible to the interactions in R. sativus, B. napus, S. alba and WW4 isolate. These findings highlight the value from B. juncea because disease severity and development using pathotypes of different physiological specializa- are moderated such that differentiation of responses is tion in screening programs to identify host resistance possible with selected genotypes of these four species. that is effective across multiple pathotypes. Further, It is noteworthy that some genotypes of B. juncea most molecular and histology studies to date have fo- (e.g., Seeta, Ringot I) and R. sativus (e.g., Colonel) cused on the host resistance mechanism to showed a consistent and more resistant host response S. sclerotiorum per se and without including the influ- irrespective of the S. sclerotiorum isolate used, making ence of pathogen virulence on disease progress and host them ideal sources of resistance to exploit to develop responses. Including distinct pathotypes of new varieties with more effective resistance to SR across S. sclerotiorum in such future studies could provide a multiple pathotypes of this pathogen In contrast, there much broader understanding as to how different was significant isolate dependency in the response of pathotypes contribute to disease development, as well some genotypes to different isolates. For example, while as the molecular differences associated with the various some genotypes showed high levels of resistance pathotypes and the respective host defences when chal- against one isolate (e.g., B. napus Charlton against the lenged with different pathotypes. Assessing disease de- WW4 isolate), the same genotypes were quite suscepti- velopment, while studying molecular differences in the ble to other isolates (e.g., B. napus Charlton against the host-pathogen interaction over a range of pathotypes,

Fig. 5 Correlation between 18 experiments 2 and 3 for overall 16 y = 0.8485x + 1.7247 mean values of diameter of R² = 0.70 cotyledon lesions (mm) 72 hpi 14 across 19 genotypes to three 12 isolates of S. sclerotiorum (r=0.84, P<0.001, n=57) 10 8 6 4 2

Cotyledon lesionCotyledon diameter (Expt 2) 0 0 5 10 15 20 Cotyledon lesion diameter (Expt 3) Eur J Plant Pathol (2015) 143:527–541 539 could provide a better understanding of processes and generally isolate/pathotype-host combinations do not mechanisms associated with pathogenesis and should invoke either a HR or PCD response because of strong help in the development of new cultivars with durable pathotype virulence. Furthermore, unless there is only disease resistance to a wide range of pathotypes of one or more prevailing and/or highly virulent S. sclerotiorum. pathotypes of S. sclerotiorum (e.g., MBRS1; Ge et al. In terms of level of virulence, the ‘Cabbage’ and 2012) against which resistance is specifically sought, MBRS1 isolates were similar and more virulent than ideally different pathotypes representing the widest pos- WW4. However, there were exceptions, such as sible physiological specialization should be included to B. juncea JM-06020 that was relatively more resistant screen for resistance. It is noteworthy that as the num- to MBRS1 compared to a highly susceptible reaction bers of test genotypes increased from 10 to 22 to 34, for when challenged by the ‘Cabbage’ isolate. Overall, experiments 1, 3, and 2, respectively, so did the isolate WW4 was the least virulent pathotype, possibly a con- variance ratios, a strong indication that experiments with sequence of its reduced ability to infect, colonize, and larger numbers of test genotypes can best differentiate kill cells in susceptible host tissue (Saharan et al. 2008). responses across different pathogen isolates or However, it was the most pathogenic isolate for geno- pathotypes. While it is recognised that use of multiple types such as B. juncea Hetianyoucai and B. napus pathotypes would be a challenging task with current Oscar which showed high levels of resistance to the methodologies where each plant is individually hand- ‘Cabbage’ isolate and intermediate resistance to inoculated, screening against diverse pathotypes will al- MBRS1. This is in contrast to other reports that WW4 low oilseed and vegetable Brassicaceae breeding pro- is largely an avirulent pathotype of little consequence grams to develop new varieties with appropriate resis- (Garg et al. 2010a). Both Garg et al. (2010a)andGe tances against the prevailing pathotypes of S. sclerotiorum et al. (2012) demonstrated that isolates derived from particularly where the level of pathotype-independent Walkway, Geraldton, Western Australia (viz. WW1, resistance is insufficient to manage SR. WW2, WW3 (pathotype10; Ge et al. 2012), WW4 (pathotype36; Ge et al. 2012) were less virulent than Acknowledgements Xintian Ge is the recipient of an In- isolates derived from the Mt. Baker region of Western ternational Postgraduate Research Scholarship, The Univer- sity of Western Australia, a scholarship from Kunming Floral Australia (viz. MBRS1 (pathotype76; Ge et al. 2012), World Bio-Tech Co. Ltd., Kunming, Peoples Republic of China, MBRS2, MBRS3, MBRS5 (pathotype76; Ge et al. and ‘top-up’ funding by the Institute of Agriculture at the Univer- 2012), suggesting that a ‘regional effect’ may contribute sity of Western Australia. We appreciate the operational funding toward aggressiveness of a particular geographical pop- support for this research provided by the Australia Research Council and the Department of Agriculture and Food Western ulation. However, recent studies by Uloth et al. (2015a) Australia (Project LP100200113, ‘Factors responsible for host have shown that one of these same isolates from the resistance to the pathogen Sclerotinia sclerotiorum for developing same Walkway region, (viz. isolate WW3 that is usually effective disease management in vegetable Brassicas’); and the designated as low in virulence), could cause as much Australian Centre for International Agricultural Research and the Grains Research and Development Corporation, Canberra, along disease on B. carinata stems as MBRS1 but only at with the School of Plant Biology, The University of Western higher temperature conditions of 28/24 °C. This is par- Australia, for additional operational funding this work. We grate- ticularly interesting as the octal code (i.e., octal nomen- fully acknowledge the provision of half the salary of Martin clature, as developed by Goodwin et al. 1990 for char- Barbetti during the early part of these studies by the Department of Agriculture and Food Western Australia. Exceptional technical acterizing pathotypes) for WW4 in Ge et al. (2012)is support is acknowledged from Mr Robert Creasy and Mr Bill 36. This octal code number indicates that such a Piasini in the UWA Plant Growth Facilities. pathotype has wide virulence across several host differ- entials used to delineate pathotypes. For example, pathotypes 00, 10 and 24 are virulent to 0, 1, and 2 of References the host differential set genotypes, respectively; while pathotype 36 is virulent across four separate host differ- entials that were susceptible (Ge et al. 2012). Isolate Barbetti, M. J., & Khangura. R., (2000). Fungal diseases of canola WW4 in combination with some of the hosts tested, in Western Australia. Bulletin 4406, Agriculture Western Australia. 15pp. offers unique opportunities to investigate HR and PCD Barbetti, M. J., Banga, S. K., Fu, T. D., Li, Y.C., Singh, D., Liu, S. host resistance responses in Brassicaceae genotypes as Y., Ge, X. T., & Banga, S. S. (2014). Comparative genotype 540 Eur J Plant Pathol (2015) 143:527–541

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