Overcoming challenges to developing varieties resistant to Sclerotinia - managing pathogen variation Photos: Caixia Li
Lupin Sclerotina patches Oilseed Rape Sclerotina patches
Photos: Cai Xia Li - unpublished
Disease Management Challenges Cultural control Persistent nature of resting sclerotia Wide host range Minimum tillage deep burial not possible Chemical control Difficult to time fungicide application with time of ascospore release Ascospore release does not always result in significant disease Resistant varieties Offers the only economic/sustainable method of control
FOCUS ON DEVELOPING SCREENING PROTOCOLS Field stem inoculation: Developed for screening genotypes under field conditions at the flowering stage [based on Buchwaldt et al. (2005) and adapted by Cai Xia Li et al. (2006)] Cai Xia Li et al. unpublished
FOCUS ON DEVELOPING SCREENING PROTOCOLS Field stem inoculation: Stem lesion length relates well to plant death Percentage of plant death 120 100 80 60 40 20 0 y = 3.193x + 25.928 R 2 = 0.6382 0 5 10 15 20 25 Stem lesion length (cm) Cai Xia Li et al. 2006 - Aust J Agric Sci 57: 1131-1135 Cai Xia Li et al. 2007 - Aust J Agric Sci 58: 1198-1203
FOCUS ON DEVELOPING SCREENING PROTOCOLS Field stem inoculation: Time of disease assessment resolves challenge of different genotype maturities Stem lesion length at 1 wai (cm) 6 5 4 3 2 1 0 y = 0.0359x - 1.3057 R 2 = 0.3824 0 50 100 150 Time of inoculation (days) Stem lesion length at 2 wai (cm) 10 8 6 4 2 0 y = 0.0326x + 1.4645 R 2 = 0.0977 0 50 100 150 Time of inoculation (days) BUT - No effect of flowering time if wait for 3 weeks post-inoculation to assess disease i.e., The impact of different flowering times rendered insignificant when assessment of stem inoculation is delayed until 3 wks post-inoculation Cai Xia Li et al. 2006 - Aust J Agric Sci 57: 1131-1135 Cai Xia Li et al. 2007 - Aust J Agric Sci 58: 1198-1203
FOCUS ON DEVELOPING MORE RAPID SCREENING PROTOCOLS Glasshouse cotyledon test: Fast - already used for Sclerotinia on legumes (Grau and Bissonette, 1974) Repeatable - responses on B. napus repeatable between expts (Garg et al 2008) Reliable indicator of field performance B. napus only (Garg et al 2008) Highly susceptible Sclerotinia cotyledon inoculation test range of host responses Highly resistant Garg et al. 2008 - Australas Pl Path 37: 106-111
FOCUS ON DEVELOPING MORE RAPID SCREENING PROTOCOLS Glasshouse cotyledon test: Trying to adapt this to wider range of crucifers and Sclerotinia pathotypes Better distinguish resistance in some B. juncea genotypes Better distinguish resistance in some other Brassicas Xin Tian Ge et al. unpublished data
EXCELLENT FIELD STEM RESISTANCE FOUND IN ACIAR GERMPLASM Best = B. napus ZY006 (China) (stem lesion length <0.45cm) Others excellent = B. napus 06-6-3792 & ZY004 (China) RT108 (Australia) B. juncea JM06018 & JM06006 (Australia) B. juncea-2 (China) Caixia Li et al. unpublished Cai Xia Li et al. 2006 - Aust J Agric Sci 57: 1131-1135 Cai Xia Li et al. 2007 - Aust J Agric Sci 58: 1198-1203 Cai Xia Li et al. 2009 - Australas Pl Path 38: 149-152
OUTSTANDING FIELD STEM RESISTANCE FOUND IN WILD BRASSICAS Introgression lines developed following hybridization of three wild crucifers (viz. Erucastrum cardaminoides, Diplotaxis tenuisiliqua and E. abyssinicum) with B. napus, B. juncea. B. rapa, B. nigra Stem lesion length (cm) 3 wai 0 10 20 30 40 aciara ACIAR b c d B. e juncea f x gwiild hspecies i Bj wild species crosses [aciar] ACIAR" [a] "(B. juncea x D. tenuisiliqua) x B. juncea" [b] "(B. juncea x E. abyssinicum) x B. juncea" [c] "(E. cardaminoides x B. nigra) x B.juncea" [d] "(E. cardaminoides x B. rapa) x B.juncea" [e] "B. juncea x D. tenuisiliqua [f] "B. juncea x E. abyssinicum" [g] "B. napus x E. cardaminoides" [h] "(E. cardaminoides x B. nigra) x B. nigra" [i] " E. cardaminoides x B. nigra" [j] " E. cardaminoides x B. rapa" Garg et Garg al. Field et al. Crops 2010 Res - Field 2010 Crops Res 117: 51 58
Wild Brassicas 98% of progenies of the selected resistant plants exhibited consistent responses with stem lesion length <1.0 cm in subsequent experiments Suggests a very high transmission frequency of the gene(s) governing resistance Garg et al. 2010 - Field Crops Res 117: 51 58
Impact of this research for Australia 1. Now have a reliable field stem inoculation test - one that differentiates resistance across diverse germplasm under Australian conditions 2. Now have high level host resistance is now available for oilseed Brassica breeding programs in Australia - now evaluating new B. napus and B. juncea breeding populations from India and China 3. Now have a cotyledon test developed for rapid growth room screenings for B. napus genotypes - now trying to extend this to other Brassicas 4. Future prospect for using host resistance as a critical component of Sclerotinia management in Australia is, for the first time, a real possibility
BUT There remains one big challenge!! PHYSIOLOGICAL SPECIALIZATION OF THE PATHOGEN Field expression of stem resistance between genotypes varied between regions, and in particular between countries Cotyledon studies showed genotype-pathotype interactions - e.g., Garg et al. Eur J Plant Path 2010 Became clear that there was physiological specialization in Sclerotinia - i.e., different pathotypes or strains can cause different host reactions Consideration: mostly single pathogen isolates used to identify resistance - consequence of the costs and resources needed for field screening Garg et al. Eur J Plant Path 2010 Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
1. Selected 14 genotypes to make potential host differential set - field tested these against 3 historical isolates of S. sclerotiorum Field experiment : Stem lesion length (cm) on 9 B. napus and 5 B. juncea genotypes inoculated with 3 historical Sclerotinia isolates at 3 weeks post-inoculation (Numbers in parenthesis are genotype rank orders for resistance - 1 most resistant and 14 most susceptible) Cultivar Species Origin Isolate MBRS1 MBRS5 WW3 Mean 06-6-3792 B. napus China 12.1(11) 7.8(6) 0.8(3) 6.9(6) 06-p71-2 B. napus China 15.513) 10.2(10) 2.3(12) 9.3(13) ZY004 B. napus China 5.5(2) 1.7(2) 0.9(6) 2.7(2) ZY006 B. napus China 2.2(1) 0.2(1) 0.5(1) 1.0(1) Zhongyou 821 B. napus China 11.5(7) 7.1(4) 2.7(13) 7.1(7) Rivette B. napus Australia 11.6(8) 14.3(13) 1.8(11) 9.2(12) RT108 B. napus Australia 9.0(4) 5.3(3) 1.7(10) 5.3(3) Mystic B. napus Australia 8.7(3) 7.9(7) 0.7(2) 5.8(4) Charlton B. napus Australia 11.9(9) 11.8(11) 0.8(5) 8.2(9) Brassica juncea #2 B. juncea China 10.9(6 ) 14.8(14) 1.6(9) 9.1(11) Montara B. juncea China 19.0(14) 13.8(12) 1.0(7) 11.3(14) Xinyou9 B. juncea China 9.5(5) 7.7(5) 0.8(3) 6.0(5) JM06006 B. juncea Australia 13.2(12) 8.4(8) 1.3(8) 7.6(8) JM06018 B. juncea Australia 12.7(10) 9.8(9) 4.2(14) 8.9(10) Mean 10.9 8.6 1.5 Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
2. Second larger field experiment where selected best 4 B. napus and 4 B. juncea genotypes from the first field experiment and tested these against 50 S. sclerotiorum isolates Able to show: i), that 3 B. napus and 3 B. juncea genotypes were suitable as host differentials to characterize S. sclerotiorum isolates ii), then used these 6 host differentials to delineate 8 distinct pathotypes of S. sclerotiorum WE DID THIS AS FOLLOWS: Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
3. Need host differentials with a bimodal frequency distribution of lesion size i.e., two clear possible modes, one susceptible and the other resistant (Goodwin et al. 1990) e.g., B. juncea Montara and B. napus 06-P71-2 Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
3. Need host differentials with a bimodal frequency distribution of lesion size Some clearly NOT suitable, for example, B. juncea Xinyou 9 and B. napus 06-6-3792 Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
4. Pathotypes of Sclerotinia as determined by disease severity (assessed as stem lesion length), on 3 differential accessions each of B. napus and B. juncea (0 = resistance reaction of differential) and 1 = susceptible reaction of differential) B. juncea JM06006 B. juncea Montara B. juncea JM06018 B. napus 06-p71-2 B. napus Charlton B. napus Mystic Pathotype octal code Isolates belonging to each pathotype code Octal digits assigned as follows: 000 = 0; 001 = 1; 010 = 2; 011 = 3; 100 = 4; 101 = 5; 110 = 6; 111 = 7 (Goodwin et al., 1990)] Pathotype frequency (%) 0 0 0 0 0 0 00 UWA7S1, UWA8S1, UWA8S2, UWA9S1, UWA9S4 9.4 0 0 1 0 0 0 10 WW3 1.9 0 1 0 1 0 0 24 UWA8S5 1.9 0 1 1 1 1 0 36 UWA3S5, WW4 3.8 1 0 1 1 1 0 56 UWA1S1 1.9 1 1 1 1 0 0 74 UWA7S2 1.9 1 1 1 1 1 0 76 MBRS1, MBRS5, UWA10S1, UWA10S4, UWA11S1, UWA11S2, UWA11S3, UWA11S4, UWA11S5, UWA12S3, UWA12S4, UWA13S1, UWA13S3, UWA13S7, UWA13S8, UWA1S2, UWA1S3, UWA1S4, UWA1S5, UWA2S1, UWA2S2, UWA2S3, UWA3S1, UWA3S2, 73.6 UWA3S4, UWA4S1, UWA5S2, UWA5S3, UWA6S1, UWA6S2, UWA6S3, UWA6S4, UWA7S3, UWA7S4, UWA7S5, UWA8S3, UWA8S4, UWA9S2, UWA9S3, UWA9S5 1 1 1 1 1 1 77 UWA10S2, UWA5S1 3.8 Robustness of Sclerotinia isolate allocations into pathotypes confirmed by cluster analyses with PRIMER V.6 (Clarke 1993; Clarke et al. 2006) Xin Tian Ge et al. 2012 Field Crops Res 22: 248-58
Identification of host differential set provides a means to: - identify and monitor current and new pathotype distributions - identify resistance(s) against the predominant pathotype(s) - combine resistances to different pathotypes in future cultivars Photos: Caixia Li
Acknowledgements AUSTRALIA University of Western Australia - Xin Tian Ge, Cai Xia Li, Yu Pin Li, Zhi Jian Wan, Ming Pei You, Patrick Finnegan University of Melbourne - Alison Gurung, Phillip Salisbury, INDIA Punjab Agric University - Surinder Banga, Harsh Garg, Shashi Banga, Prabhjodh Sandhu FINANCIAL ASSISTANCE Grains Research and Development Corporation Australia Centre of International Agriculture Research (ACIAR) Australian Research Council Australian Govt via Postgraduate scholarship support Kunming Floral World Bio-Tech Co, scholarship support, China Department of Plant Breeding and Genetics, Punjab Agricultural University Institute of Agriculture and School of Plant Biology, UWA