High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea

Field Crops Research 117 (2010) 51–58

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High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea Harsh Garg a, Chhaya Atri b, Prabhjodh S. Sandhu b, Balvir Kaur b, Michael Renton a,c, Shashi K. Banga b, Hardeep Singh b, Charandeep Singh b, Martin J. Barbetti a,d,*, Surinder S. Banga b a

School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, 141004 Punjab, India c CSIRO Sustainable Ecosystems, Floreat, WA 6014, Australia d Department of Agriculture and Food Western Australia, Baron-Hay Court, South Perth, WA 6151, Australia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 September 2009 Received in revised form 11 December 2009 Accepted 29 January 2010

Sclerotinia rot caused by the fungus Sclerotinia sclerotiorum is one of the most serious and damaging diseases of oilseed rape and there is keen worldwide interest to identify Brassica genotypes with resistance to this pathogen. Complete resistance against this pathogen has not been reported in the field, with only partial resistance being observed in some Brassica genotypes. Introgression lines were developed following hybridization of three wild crucifers (viz. Erucastrum cardaminoides, Diplotaxis tenuisiliqua and E. abyssinicum) with B. napus or B. juncea. Their resistance responses were characterized by using a stem inoculation test. Seed of 54 lines of B. napus and B. juncea obtained from Australia, India and China through an Australian Centre for International Agricultural Research (ACIAR) collaboration programme were used as susceptible check comparisons. Introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum had much higher levels (P < 0.001) of resistance compared with the ACIAR germplasm. Median values of stem lesion length of introgression lines derived from the wild species were 1.2, 1.7 and 2.0 cm, respectively, as compared with the ACIAR germplasm where the median value for stem lesion length was 8.7 cm. This is the first report of high levels of resistance against S. sclerotiorum in introgression lines derived from E. cardaminoides, D. tenuisiliqua and E. abyssinicum. The novel sources of resistance identified in this study are a highly valuable resource that can be used in oilseed Brassica breeding programmes to enhance resistance in future B. napus and B. juncea cultivars against Sclerotinia stem rot. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Erucastrum cardaminoides Diplotaxis tenuisiliqua E. abyssinicum Brassicas Introgression lines Resistance White mold

1. Introduction Sclerotinia disease, caused by the fungal pathogen Sclerotinia sclerotiorum, is a serious threat to oilseed rape production with substantial yield losses recorded worldwide including India, Europe, China, North America and Australia (Li et al., 1999; McCartney et al., 1999; Sprague and Stewart-Wade, 2002; Hind et al., 2003; Koch et al., 2007; Malvarez et al., 2007; Singh et al., 2008). Various methods used for managing Sclerotinia disease include cultural control, chemical control and varietal resistance (Bardin and Huang, 2001). The persistent nature of sclerotia and the wide host range of this pathogen from taxonomically diverse

* Corresponding author at: School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 64883924; fax: +61 8 64887077. E-mail address: [email protected] (M.J. Barbetti). 0378-4290/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.fcr.2010.01.013

hosts (over 408 plant species) generally render cultural practices such as crop rotation to be ineffective (Williams and Stelfox, 1980; Boland and Hall, 1994). Further, disease management through chemical control is also largely ineffective due to difficulty in timing the fungicide application with the release of ascospores (Bolton et al., 2006). Host resistance offers the only economic and sustainable method for effectively managing this disease (Zhao et al., 2004; Li et al., 2006). While partial resistance against this pathogen has been observed in certain genotypes of sunflower (Helianthus annuus) (Godoy et al., 2005), beans (Phaseolus coccineus) (Gilmore et al., 2002), peas (Pisum sativum) (Porter et al., 2009), peanut (Arachis hypogea) (Cruickshank et al., 2002), or soybean (Glycine max) (Hartman et al., 2000), complete resistance has not been reported in the field. Partial resistance was also identified in some of the Brassica napus and, to a lesser extent B. juncea, genotypes from China (Li et al., 1999, 2006, 2008; Zhao et al., 2004), Australia (Li et al., 2006, 2008) and India (Singh et al., 2008). Although, a

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H. Garg et al. / Field Crops Research 117 (2010) 51–58

significant number of at least partially resistant genotypes have been identified, breeding to increase the levels of resistance against Sclerotinia disease, in B. napus and/or B. juncea has been ineffective. This is mainly because resistance to S. sclerotiorum in existing cultivars of Brassica and in other cultivated germplasm appears to be of a complex nature, i.e., it can either be monogenic and/or polygenic depending on the different plant species and materials under investigation (Abawi et al., 1978; Baswana et al., 1991; Zhao and Meng, 2003; Zhao et al., 2006). Genotypes with higher levels of resistance are urgently required for inclusion in oilseed Brassica breeding programmes to enhance the level of field resistance in cultivated species such as B. napus and B. juncea. Lack of effective resistance to Sclerotinia disease in cultivated species has stimulated the interest of researchers towards exploitation of wild crucifer species to diversify the existing gene pool. Higher levels of resistance against Sclerotinia have already been reported in the secondary gene pool of bean (Abawi et al., 1978; Gilmore et al., 2002; Schwartz et al., 2006), wild Helianthus species (Seiler, 1992; Gulya et al., 2009) and in a Pisum core collection (Porter et al., 2009). Several successful attempts have been reported to introgress the resistance from the secondary gene pool of bean (Phaseolus vulgaris) into the cultivated bean species through interspecific hybridization followed by backcrossing (e.g., Schwartz et al., 2006; Singh et al., 2009). Introgression of genomic segments responsible for resistance against Sclerotinia from wild to cultivated species of sunflower has been attempted in the past (e.g., Ronicke et al., 2004). Despite the Brassicaceae family comprising of a wide array of different species, to date, it appears that only two wild crucifers, Capsella bursa-pastoris (Chen et al., 2007) and Erucastrum gallicum (Lefol et al., 1997a; Seguin-Swartz and Lefol, 1999), have been previously reported to show high levels of resistance against Sclerotinia disease. Although introgressive hybrids were successfully obtained between different Brassica (B. rapa and B. napus) species and Capsella bursa-pastoris (Chen et al., 2007), it remains to be confirmed if the introgression of resistance against S. sclerotiorum from E. gallicum into cultivated species has in fact been accomplished (Lefol et al., 1997a,b; Seguin-Swartz and Lefol, 1999). There remains substantial potential both to identify wild crucifers with high levels of resistance to Sclerotinia disease and for its successful introgression to the cultivated species. Three wild crucifers, viz. Erucastrum cardaminoides, Diplotaxis tenuisiliqua and E. abyssinicum, have been identified with very high levels of resistance against S. sclerotiorum (S.S. Banga, unpublished data). The aim of this study was to introgress the genomic segments responsible for resistance against S. sclerotiorum from these three wild species into the cultivated germplasm. This paper reports the results of screening highly fertile (S4/S5) introgression lines of B. juncea and B. napus derived from three different wild Brassica species, viz. E. cardaminoides, D. tenuisiliqua and E. abyssinicum, against S. sclerotiorum.

Fig. 1. The general breeding scheme used to introgress segments of the genome from wild species Erucastrum cardaminoides into cultivated lines of Brassica juncea.

Fig. 2. The schematic representation for developing introgression lines by crossing Brassica napus with Erucastrum cardaminoides.

nigra Chandra et al. (2004), B. napus/E. cardaminoides, B. juncea/D. tenuisiliqua, and B. juncea/E. abyssinicum. Chromosome doubling was achieved using colchicine in cross combinations involving monogenomic Brassica species to restore seed fertility. In crosses of wild crucifers with digenomics, chromosome doubling was not required as these were partially fertile. The synthetic amphiploids or the trigenomic hybrids were subsequently used as pollen/seed parents to hybridize with cultivated digenomics, B. juncea or B. napus (Figs. 1–3). This was followed by three to four generations of selfing using the single pod descent method. Special attempts were

2. Materials and methods 2.1. Plant materials 2.1.1. Experiment 1 Introgression lines, carrying alien genomic segments, were developed for B. juncea and B. napus by introgression from three wild crucifers viz. E. cardaminoides (2n = 18), D. tenuisiliqua (2n = 18) and E. abyssinicum (2n = 32). The general outlines of breeding schemes to introgress genomic segments from wild crucifers into these cultivated species are detailed in Figs. 1–3. These lines were developed by S. S. Banga ([email protected]) and co-workers, Punjab Agricultural University from identified wild crucifers by synthesizing intergeneric hybrids, E. cardaminoides/B. rapa Chandra et al. (2004), E. cardaminoides/B.

Fig. 3. The schematic representation for the development of introgression lines from wild crucifers Erucastrum abyssinicum and Diplotaxis tenuisiliqua.

H. Garg et al. / Field Crops Research 117 (2010) 51–58 Table 1 Reactions against Sclerotinia sclerotiorum of different cross combinations of wild crucifers with cultivated species (Brassica rapa, B. nigra, B. juncea and B. napus) and ACIAR Brassica napus and B. juncea germplasm in relation to stem lesion length (cm) 3 weeks after inoculation. Cross combinations

Number of genotypes tested

Median value of stem lesion length (cm)

E. cardaminoides  B. rapaa E. cardaminoides  B. nigraa (E. cardaminoides  B. rapa)  B. juncea (E. cardaminoides  B. nigra)  B. juncea (E. cardaminoides  B. nigra)  B. nigra B. napus  E. cardaminoides B. juncea  D. tenuisiliqua (B. juncea  D. tenuisiliqua)  B. juncea B. juncea  E. abyssinicum (B. juncea  E. abyssinicum)  B. juncea

5 367 331 1154 96 105 109 21 227 334

1.8 2.0 1.3 1.6 1.5 2.5 1.2 1.0 1.7 2.9

ACIAR

1080

8.7

a Selfed progenies of up to five generation (S4) were evaluated and in other cross combinations selfed progenies of up to four generations followed by backcross (BC1S4) were evaluated.

made to select plants showing higher degree of pollen stainability and self-seed set to initiate next round of selfing and selection. Selfed progenies of either up to four (BC1S4) or five (S5) generations of all the 10 cross combinations (Table 1) were involved in experiment 1 for identifying their reactions against S. sclerotiorum. The numbers of genotypes evaluated in each cross combination are shown in Table 1. These genotype were selected randomly from the populations (BC1S4/S5) derived from the cross combinations involving wild species and cultivated germplasm. 2.1.2. Susceptible check comparison genotypes Fifty-four germplasm lines of B. napus and B. juncea obtained from Australia, India and China through an Australian Centre for International Agricultural Research (ACIAR) collaboration programme were used as susceptible comparison genotypes to the introgression lines derived from three wild crucifers. These materials were included as susceptible check comparisons from genotypes of B. napus and B. juncea and these included some of the most resistant Brassica lines identified in the current ACIAR disease screening programme (Li et al., 2008). 2.1.3. Experiment 2 Introgression lines that showed stem lesion lengths <1 cm in first experiment were re-inoculated at late flowering stage above the site of the first inoculation in order to confirm the resistance observed in experiment 1. The second experiment was performed approximately 4 weeks after the initial inoculation (data not presented). 2.1.4. Experiment 3 Introgression lines with stem lesion length, <1 cm in experiment 1 were selected for further evaluation in 2008–2009. Three genotypes of B. napus and B. juncea, viz. JLM 298, PBR 91 and GSC 5, were used as susceptible check comparisons during screening undertaken to identify resistance responses (data not presented). 2.2. Field experimental site All the introgression lines were tested under field conditions at the field site of the Punjab Agricultural University, India during December to February in 2007–2008 and again in 2008–2009 (Figs. 1–3). For the purpose of screening, 20 seeds per introgression line were hand sown in single rows of 2 m length and with 0.6 m between rows. Plants were not thinned after germination.

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2.3. S. sclerotiorum isolate A single isolate of S. sclerotiorum was collected from infested B. juncea at the field site of the Punjab Agricultural University, where significant disease on Brassica lines is frequently observed during flowering. Surface sterilization of a field collected sclerotium was done in 50% (v/v) sodium hypochlorite and 70% ethanol for 4 min with agitation, followed by two washes in sterile distilled water for 1 min as described by Clarkson et al. (2003) and Li et al. (2006). The sclerotium was then cut in half, placed on potato dextrose agar (PDA) and incubated at 20 8C with 12/12 h light/dark (Clarkson et al., 2003). Sclerotia subsequently produced were then harvested from the incubated plates after 4 weeks, air dried overnight at 15 8C and finally stored at 4 8C, for future use. 2.4. S. sclerotiorum inoculations Resistance responses of all introgression lines were evaluated by using the stem inoculation test as described by Buchwaldt et al. (2005) and Li et al. (2006). Briefly, 10 plants in each test line were randomly picked and were inoculated at the flowering stage when 50% of the plants in the rows had at least one opened flower. For each plant, a single agar plug disc (5 mm diameter) was cut from the actively growing margin of a 3-day-old colony on a glucoserich medium (peptone 10 g, glucose 20 g, agar 18 g, KH2PO4 0.5 g, H2O 1 L, adjusted to pH 4.0 with HCl before autoclaving) and an inoculum plug was wrapped onto the first internode above the middle node of each stem using Parafilm1. 2.5. Disease assessment All disease assessments were made at 3 weeks after inoculation (wai) as impact of different times of flowering of Brassica genotypes for determining physiological resistance is insignificant at this particular time of disease assessment (Li et al., 2007). Stem lesion length was measured with a ruler and vernier callipers were used to measure stem diameters of all inoculated plants as done in earlier studies by Li et al. (2006, 2007). 2.6. Resistance categories Introgression lines were categorized into five different classes based on their resistant responses, namely highly resistant (HR), resistant (R), moderately resistant (MR), susceptible (S) and highly susceptible (HS) with stem lesion lengths ranging from 0 to <2.5; 2.5 to <5.0; 5.0 to <7.5; 7.5 to 10.0 and >10.0 cm, respectively. 2.7. Data analysis The R statistical (version 2.8.0) programme was used to identify significant differences in resistance responses between introgression lines. Significant differences in relation to stem lesion length between different wild species and ACIAR germplasm were evaluated using the non-parametric Kruskal–Wallis test. The Mann–Whitney U-test (Wilcoxon–Mann–Whitney test) was used to compare significant differences with respect to stem lesion length between all the wild species taken together and the ACIAR germplasm. The Wilcoxon test was also used for pair-wise comparison of different wild species (E. cardaminoides, E. abyssinicum and D. tenuisiliqua) and to compare different cross combinations within a wild species, e.g., comparison of the cross of E. cardaminoides and B. rapa with E. cardaminoides and B. nigra. Proportions of different resistant categories as described above were calculated using R statistical within every cross combination and the significance of differences in these proportions were tested using the Chi-square test. Non-parametric tests were used because

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Fig. 4. An example of reactions of introgression lines against Sclerotinia sclerotiorum developed from wild crucifers Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum in relation to stem lesion length assessed 3 weeks after inoculation.

the data was clearly not normally distributed and standard transformations failed to normalise it. 3. Results 3.1. Resistance responses of introgression lines derived from three wild species (E. cardaminoides, D. tenuisiliqua and E. abyssinicum) and of ACIAR germplasm 3.1.1. Experiment 1 The Kruskal–Wallis test indicated significant differences (P < 0.001) between introgression lines derived from three wild species and the ACIAR germplasm in relation to the stem lesion length 3 wai. Introgression lines derived from D. tenuisiliqua were highly resistant with a median value for stem lesion length of 1.2 cm as compared with wild species E. cardaminoides and E. abyssinicum (median values = 1.7 and 2.0 cm, respectively; Figs. 4– 6). The range of stem lesion lengths for introgression lines derived from three wild species viz. E. cardaminoides, D. tenuisiliqua and E. abyssinicum varied from 0 to 20, 0.2 to 12.0 and 0 to 25 cm, respectively (Figs. 5 and 6). The median value for stem lesion length of ACIAR germplasm was very high (i.e., 8.7 cm as compared to introgression lines) with stem lesion lengths ranging up to 47.8 cm, which also confirms the high level of pathogenicity of the isolate of S. sclerotiorum utilized for this study. Significant differences were also observed (P < 0.001) between different crosses involving the wild species with the ACIAR germplasm. The most resistant cross was (B. juncea  D. tenuisiliqua)  B. juncea with a median value for stem lesion length of 1.0 cm (Fig. 7). The maximum median value for stem lesion length in the (B. juncea  E. abyssinicum)  B. juncea cross combination was 2.9 cm, which is much smaller than in the ACIAR germplasm which had a median value of 8.7 cm (Table 1). 3.1.2. Experiment 2 All introgression lines with a stem lesion length of <1.0 cm were re-inoculated at the late flowering stage above the site of inoculation for the first experiment. Overall, genotypes showed reasonably consistent results between experiments 1 and 2 (significant linear relationship; P < 0.001), confirming the physiological resistance identified in experiment 1.

3.1.3. Experiment 3 As determined by the Kruskal–Wallis test, there were significant overall differences (P < 0.001) between the introgression lines derived from three different wild crucifers and with the susceptible check comparison genotypes, in relation to stem lesion lengths 3 wai. The mean stem lesion length of genotypes used as susceptible check comparisons in experiment 3 was 4.0 cm with a range varying from 0 to 15.5 cm. However, mean values for stem lesion length for introgression lines developed from the wild crucifers E. cardaminoides, D. tenuisiliqua and E. abyssinicum were much lower, viz. 0.42, 0.36 and 0.36 cm, with ranges for stem lesion length of 0–1.5, 0–1.2 and 0–1.2 cm, respectively. The highly reduced stem lesion lengths recorded on the introgression lines were in accordance with what

Fig. 5. Box plot representation of resistance responses against Sclerotinia sclerotiorum of ACIAR Brassica napus and B. juncea germplasm and introgression lines derived from wild crucifers Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum in relation to stem lesion length assessed 3 weeks after inoculation. The central bar represents the median value and each box corresponds to the range between 25th and 75th percentiles. Whiskers from the boxes include the lowest and highest values. A, B and C represent introgression lines developed from E. cardaminoides, D. tenuisiliqua and E. abyssinicum, respectively.

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(P < 0.001) and D. tenuisiliqua and E. abyssinicum (P < 0.001) with respect to stem lesion length 3 wai as observed in experiment 1. However, no significant differences were found when responses of introgression lines in relation to stem lesion length were compared in experiment 3, which was expected as only those genotypes whose stem lesion length were <1.0 cm in experiment 1 were evaluated in this experiment. Individuals from each cross combination (involving wild species with cultivated species), along with introgression lines developed solely from the three wild species, were divided into different resistance categories (HR, R, MR, S, HS) based on their responses to S. sclerotiorum. The percent proportions of every resistant category between different cross combinations and introgression lines were significantly different (P < 0.001) as calculated using the Chi-square test (Fig. 8; Table 2). 3.3. Comparison of different cross combinations within each wild species/effect of second cross species

Fig. 6. A magnified scale view of resistance responses against Sclerotinia sclerotiorum of ACIAR Brassica napus and B. juncea germplasm and introgression lines derived from wild crucifers Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum by box plot representation when range of stem lesion length was restricted to 0–2.0 cm as assessed 3 weeks after inoculation. The central bar, lower bar of the boxes and the lower whisker represent the median value, 25th percentile and the minimum value of the stem lesion length, respectively. A, B and C represent introgression lines developed from E. cardaminoides, D. tenuisiliqua and E. abyssinicum, respectively.

was expected, as only introgression lines with stem lesion lengths <1.0 cm in experiment 1 were selected for testing in experiment 3. Moreover, 98% of the genotypes selected in experiment 3 exhibited consistent results with stem lesion length <1.0 cm, confirming the high level of resistance in the introgression lines derived from the three wild crucifers as well as reliability of the screening technique used in this study. 3.2. Comparison of introgression lines derived from three wild species Significant differences were observed between the introgression lines developed from the wild species E. cardaminoides and D. tenuisiliqua (P = 0.003), E. cardaminoides and E. abyssinicum

The cross combinations within the wild species E. cardaminoides, D. tenuisiliqua and E. abyssinicum were significantly different (P < 0.05) in relation to stem lesion length at 3 wai. However, while crosses involving B. nigra or B. rapa with the wild species E. cardaminoides (E. cardaminoides  B. nigra, E. cardaminoides  B. rapa) were not significantly different from each other, significant differences were observed when these same crosses were further back-crossed with B. juncea, i.e. [(E. cardaminoides  B. nigra)  B. juncea and (E. cardaminoides  B. rapa)  B. juncea] (Table 3). However, proportion of the plants in the highly resistant (HR) category was similar in all the crosses mentioned above (Table 2). 3.4. Correlation between stem lesion length and stem diameter Positive significant correlation between stem lesion length and stem diameter (r = 0.05, P < 0.001, n = 5040) was observed in experiment 1. However, the value of this Pearson correlation coefficient was so low that this relationship between stem lesion length and stem diameter was of very little importance. Similar results were also obtained when stem lesion length was compared with stem diameter in experiment 3 (r = 0.0015, P < 0.001, n = 1135).

Fig. 7. A box plot representation against Sclerotinia sclerotiorum of resistance responses of ACIAR Brassica napus and B. juncea germplasm and different cross combinations involving wild crucifers Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum with cultivated species of B. rapa, B. nigra, B. juncea and B. napus in relation to stem lesion length assessed at 3 weeks after inoculation. The central bar, boxes represent the median value, 25–75th percentile and whiskers the full range of responses.

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Fig. 8. The percent distribution of resistance responses against Sclerotinia sclerotiorum of introgression lines developed from wild crucifers Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum and ACIAR Brassica napus and B. juncea germplasm in relation to stem lesion length assessed at 3 weeks after inoculation. HR, R, MR, S and HS represent highly resistant, resistant, moderately resistant, susceptible and highly susceptible categories with stem lesion length ranging from 0 to <2.5; 2.5 to <5.0; 5.0 to <7.5; 7.5 to 10.0 and >10.0 cm, respectively.

4. Discussion Introgression lines developed from the three different wild crucifers (viz. E. cardaminoides, D. tenuisiliqua and E. abyssinicum) showed extremely high levels of resistance against S. sclerotiorum as compared to the currently available B. napus and B. juncea germplasm. It is noteworthy that 98% of the progenies of the selected resistant plants (experiment 3) exhibited consistent responses with stem lesion length <1.0 cm. This demonstrates a very high transmission frequency of the gene(s) governing

resistance. Such high transmission frequency would be improbable if introgression was incomplete or if there were one or more addition chromosomes. Maximum transmission frequency of addition chromosomes cannot be more than 0.5% through the female gamete and is much less through male gamete as reported previously in Brassicas (Hua et al., 2006) and in other cultivated crops (Chetelat et al., 1998; Shigyo et al., 1998; Becerra LopezLavalle and Brubaker, 2007). Furthermore, selfed progenies of up to four generations (BC1S4) of each different cross combination involving wild and cultivated species were used in our study to evaluate their responses against Sclerotinia disease. The probability of transmission of the alien chromosome is reduced with each selfed generation. This coupled with very high male and female fertility of the introgression lines justifies our contention of stable introgression of the gene(s) governing resistance into the lines used for evaluation in our study. We believe that the very high levels of resistance identified in these introgression lines against S. sclerotiorum is the first example of such resistance being reported anywhere among oilseed Brassicas. Only one B. napus line from China, viz. ZY006, with mean stem lesion length <0.45 cm in a similar stem inoculation test, had been previously identified as having high levels of resistance among cultivated genotypes of oilseed Brassica (Li et al., 2008). Progeny of a single cross between B. napus and Capsella bursa-pastoris were the next most resistant genotypes previously reported with mean stem lesion length of 1.3 cm (Chen et al., 2007). The next most resistant B. napus genotypes that have been previously reported included 06-6-3792 (China), ZY004 (China) and RT 108 (Australia) with mean stem lesion lengths of <3.0 cm (Li et al., 2008) and also Zhongyou 821 (He et al., 1987; Li et al., 1999). In addition, the levels of resistance reported previously in B. juncea were, in particular, far lower, e.g., B. juncea JM06018 and JM 06006 with mean stem lesion lengths of 4.8 cm (Li et al., 2008) as compared with B. napus genotypes. Our study, in contrast,

Table 2 Resistance responses against Sclerotinia sclerotiorum of different cross combinations of wild crucifers with cultivated species of Brassica rapa, B. nigra, B. juncea and B. napus and ACIAR Brassica napus and B. juncea germplasm. Cross

HR

R

MR

S

VS

Total

E. cardaminoides  B. rapaa E. cardaminoides  B. nigraa (E. cardaminoides  B. rapa)  B. juncea (E. cardaminoides  B. nigra)  B. juncea (E. cardaminoides  B. nigra)  B. nigra B. napus  E. cardaminoides B. juncea  D. tenuisiliqua (B. juncea  D. tenuisiliqua)  B. juncea B. juncea  E. abyssinicum (B. juncea  E. abyssinicum)  B. juncea

4 (80.0) 261 (71.1) 256(77.3) 895 (77.6) 61 (63.5) 66 (62.9) 80 (73.4) 18 (85.7) 142 (62.6) 162 (48.5)

0 (0) 80(21.8) 55(16.6) 170 (14.7) 18 (18.8) 38 (36.2) 17 (15.6) 2 (9.5) 45 (19.8) 70 (21.0)

0 (0) 15(4.1) 15(4.5) 58 (5.0) 9 (9.4) 1 (1.0) 6 (5.5) 1 (4.8) 27 (11.9) 45 (13.5)

1 (20) 6(1.6) 3(0.9) 22 (1.9) 5 (5.2) 0 (0) 3 (2.8) 0 (0) 8 (3.5) 32 (9.6)

0 (0) 5 (1.4) 2(0.6) 9 (0.8) 3 (3.1) 0 (0) 3 (2.8) 0 (0) 5 (2.2) 25 (7.5)

5 367 331 1154 96 105 109 21 227 334

ACIAR

232 (21.5)

162 (15)

118 (10.9)

69 (6.4)

499 (46.2)

1080

HR, R, MR, S and HS stand for highly resistant, resistant, moderately resistant, susceptible and highly susceptible categories with stem lesion length (cm) ranging from 0 to <2.5; 2.5 to <5.0; 5.0 to <7.5; 7.5 to 10.0 and >10.0 cm, respectively. Values shown represent total number of genotypes of each cross combination for each resistance category while figures in parenthesis represent their percent proportions. a Selfed progenies of up to five generations (S5) were evaluated and in other cross combinations selfed progenies of up to four generations followed by backcross (BC1S4) were evaluated.

Table 3 Comparison of different crosses within each wild species Erucastrum cardaminoides, Diplotaxis tenuisiliqua and Erucastrum abyssinicum. Combination 1

Combination 2

Significance

E. cardaminoides  B. nigra (E. cardaminoides  B. nigra)  B. juncea (E. cardaminoides  B. nigra)  B. juncea (E. cardaminoides  B. rapa)  B. juncea (B. juncea  D. tenuisiliqua)  B. juncea (B. juncea  E. abyssinicum)  B. juncea

E. cardaminoides  B. rapa (E. cardaminoides  B. rapa)  B. juncea E. cardaminoides  B. nigra E. cardaminoides  B. rapa B. juncea  D. tenuisiliqua B. juncea  E. abyssinicum

ns s s ns s s

(P < 0.05) ‘s’ and ‘ns’ represent significant and non-significant differences, respectively, between crosses in relation to stem lesion length assessed 3 weeks after inoculation with Sclerotinia sclerotiorum.

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identified 60% and 3.8% of the genotypes with mean stem lesion lengths <3.0 and <0.45 cm, respectively, among introgression lines involving the cross combinations of three wild crucifers with B. juncea and B. napus, signifying the outstanding levels of resistance present in these genotypes. Median values for stem lesion length for introgression lines derived from D. tenuisiliqua, E. cardaminoides and E. abyssinicum were 1.2, 1.7 and 2.0 cm, respectively, as compared to 8.7 cm for the ACIAR genotypes. Clearly, all three wild crucifers used in this study had extremely high levels of resistance against S. sclerotiorum. Even though the wild crucifer E. gallicum had been previously reported to have better resistance to Sclerotinia disease as compared to cultivated lines of Brassica species (Lefol et al., 1997a; Seguin-Swartz and Lefol, 1999), it was only evaluated using a cut leaf bioassay with ascospores as the inoculation source. The efficacy of cut leaf bioassays or detached leaf methods remains debatable, as this method correlates poorly with the field performance of the Brassica genotypes (Bradley et al., 2006; C-X. Li and M.J. Barbetti, unpublished). However, our study utilized a field stem inoculation technique across two consecutive years, a methodology shown to be reliable and repeatable in differentiating oilseed B. napus genotypes responses to Sclerotinia disease (Li et al., 2004, 2006; Buchwaldt et al., 2005). Consistent resistant reactions were observed in introgression lines across experiments 1 and 2 and also across experiments 1 and 3, confirming the very high level of physiological resistance identified in the developed introgression lines. Previous studies have reported that some of the morphological traits such as stem diameter and growth stage of canola can affect the reaction of Brassica genotypes to Sclerotinia disease (Zhao et al., 2004; Zhao and Wang, 2004; Li et al., 2006). While our studies showed some relationship between stem lesion length and stem diameter in both experiments 1 and 3, this relationship was weak and not considered meaningful. This finding, taken in conjunction with the consistent results between experiments 1 and 2, which were performed at two different stages of flowering, is indicative that high levels of resistance identified in these introgression lines were not related to these morphological traits. The novel sources of resistance identified in our study are highly valuable genetic resources that can be utilized in oilseed Brassica breeding programmes to increase resistance in new cultivars against Sclerotinia disease. The present study constitutes the first report of successful introgression of very high levels of resistance against S. sclerotiorum from three wild crucifers, namely E. cardaminoides, D. tenuisiliqua and E. abyssinicum, into cultivated Brassicas. Future work will focus on the identification and mapping of the genes governing resistance against S. sclerotiorum. Acknowledgments Ms Harsh Garg gratefully acknowledges the financial assistance of the Australian Centre for International Agricultural Research, Canberra, Australia, by way of a John Allwright PhD fellowship. IPR of the introgression lines used in the studies vests exclusively with Punjab Agricultural University, Ludhiana, India. References Abawi, G.S., Provvidenti, R., Crosier, D.C., Hunter, J.E., 1978. Inheritance of resistance to white mold disease in Phaseolus coccineus. J. Hered. 69, 200–202. Bardin, S.D., Huang, H.C., 2001. Research on biology and control of Sclerotinia disease in Canada. Can. J. Plant Pathol. 23, 88–98. Baswana, K.S., Rastogi, K.B., Sharma, P.P., 1991. Inheritance of stalk rot resistance in cauliflower (Brassica oleracea var. Botrytis L.). Euphytica 57, 93–96. Becerra Lopez-Lavalle, L.A., Brubaker, C.L., 2007. Frequency and fidelity of alien chromosome transmission in Gossypium hexaploid bridging populations. Genome 50, 479–491.

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