Breakdown of Rlm3 resistance in the Brassica napusLeptosphaeria maculans pathosystem in western Canada

Abstract

Blackleg disease, caused by the fungal pathogen Leptosphaeria maculans, is a serious disease of Brassica napus. The disease is mainly controlled by genetic resistance and crop rotation. However, L. maculans has displayed a high evolutionary potential to overcome major resistance genes in B. napus. This study aimed to analyze the major-gene and adult-plant resistance (APR) of Canadian B. napus varieties/lines (accessions) and the avirulence allele frequency in L. maculans populations in western Canada. For resistance identification, a set of L. maculans isolates with known avirulence genes were used to characterize major resistance (R) genes in 104 Canadian B. napus accessions and 102 seed samples collected from growers’ fields; with 104 B. napus accessions further evaluated for APR under controlled conditions. In addition, avirulence genes of 300 L. maculans isolates collected from infected canola stubbles in growers’ fields were determined by cotyledon inoculation and gene-specific PCR assays. The results indicated that R genes were present in the majority of these B. napus accessions, with the Rlm3 gene being predominant while other R genes were rarely detected. APR was identified in more than 50 % of the accessions. Predominance of Rlm3 in 102 seed samples from growers’ fields suggested Rlm3-carrying B. napus varieties were currently widely used in western Canada. Avirulence allele frequency identification of field L. maculans isolates revealed the scarcity of the avirulence allele towards Rlm3, AvrLm3. This indicated the breakdown of Rlm3 resistance, which could be due to the over use of this single resistance gene in Canadian B. napus germplasm.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Ansan-Melayah, D., Balesden, M. H., Delourme, R., Pilet, M. L., Renard, M., Tanguy, X., et al. (1998). Genes for race specific resistance against blackleg disease in Brassica napus L. Plant Breeding, 117, 373–378.

    Article  Google Scholar 

  2. Balesdent, M. H., Attard, A., Ansan-Melayah, D., Delourme, R., Renard, M., & Rouxel, T. (2001). Genetic control and host range of avirulence towards Brassica napus cvs. Quinta and Jet Neuf in Leptosphaeria maculans. Phytopathology,, 91, 70–76.

    CAS  Article  Google Scholar 

  3. Balesdent, M. H., Attard, A., Kuhn, M. L., & Rouxel, T. (2002). New avirulence genes in the phytopathogenic fungus Leptosphaeria maculans. Phytopathology, 92, 1122–1133.

    CAS  Article  PubMed  Google Scholar 

  4. Balesdent, M. H., Barbetti, M. J., Hua, L., Sivasithamparam, K., Gout, L., & Rouxel, T. (2005). Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates. Phytopathology, 95, 1061–1071.

    CAS  Article  PubMed  Google Scholar 

  5. Balesdent, M. H., Louvard, K., Pinochet, X., & Rouxel, T. (2006). A large-scale survey of races of Leptosphaeria maculans occurring on oilseed rape in France. European Journal of Plant Pathology, 114, 53–65.

    Article  Google Scholar 

  6. Balesdent, M. H., Fudal, I., Ollivier, B., Bally, P., Grandaubert, J., Eber, F., et al. (2013). The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards brassica Rapa. New Phytologist, 198, 887–898.

    CAS  Article  PubMed  Google Scholar 

  7. Brun, H., Chevre, A. M., Fitt, B. D. L., Powers, S., Besnard, A. L., Ermel, M., et al. (2010). Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus. New Phytologist, 185, 285–299.

    Article  PubMed  Google Scholar 

  8. Calderon, C., Ward, E., Freeman, J., Foster, S. J., & Mccartney, H. A. (2002). Detection of airborne inoculum of Leptosphaeria maculans and pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays. Plant Pathology, 51, 303–310.

    CAS  Article  Google Scholar 

  9. Chen, Y., & Fernando, W. G. D. (2006). Prevalence of pathogenicity groups of Leptosphaeria maculans in western Canada and North Dakota, USA. Canadian Journal of Plant Pathology, 1, 533–539.

    Article  Google Scholar 

  10. Chèvre, A. M., Eber, F., This, P., Barret, P., Tanguy, X., Brun, H., et al. (1996). Characterization of brassica nigra chromosomes and of blackleg resistance in B. napusB. nigra addition lines. Plant Breeding, 115, 113–118.

    Article  Google Scholar 

  11. Chèvre, A. M., Barret, P., Eber, F., Dupuy, P., Brun, H., Tanguy, X., et al. (1997). Selection of stable Brassica napus-B. juncea recombinant lines resistant to blackleg (Leptosphaeria maculans). Theoretical and Applied Genetics, 95, 1104–1111.

    Article  Google Scholar 

  12. Delourme, R., Pilet-Nayel, M. L., Archipiano, M., Horvais, R., Tanguy, X., Rouxel, T., et al. (2004). A cluster of major specific resistance genes to Leptosphaeria maculans in Brassica napus. Phytopathology, 94, 578–583.

    CAS  Article  PubMed  Google Scholar 

  13. Delourme, R., Chèvre, A. M., Brun, H., Rouxel, T., Balesdent, M. H., Dias, J. S., et al. (2006). Major gene and polygenic resistance to Leptosphaeria maculans in oilseed rape (Brassica napus). European Journal of Plant Pathology, 114, 41–52.

    Article  Google Scholar 

  14. Delourme, R., Brun, H., Ermel, M., Lucas, M. O., Vallee, P., Domin, C., et al. (2008). Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location. Annals of Applied Biology, 153, 259–269.

  15. Delourme, R., Bousset, L., Ermel, M., Duffé, P., Besnard, A. L., Marquer, B., et al. (2014). Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape. Infection, Genetics and Evolution, 27, 490–499.

    CAS  Article  PubMed  Google Scholar 

  16. Dilmaghani, A., Balesdent, M. H., Didier, J. P., Wu, C., Davey, J., Barbetti, M. J., et al. (2009). The Leptosphaeria maculans -leptosphaeria biglobosa species complex in the American continent. Plant Pathology, 58, 1044–1058.

    Article  Google Scholar 

  17. Dilmaghani, A., Gout, L., Moreno-Rico, O., Dias, J. S., Coudard, L., Castillo-Torres, N., et al. (2013). Clonal populations of Leptosphaeria maculans contaminating cabbage in Mexico. Plant Pathology, 62, 520–532.

    Article  Google Scholar 

  18. Eber, F., Lourgant, K., Brun, H., Lode, M., Huteau, V., Coriton, O., et al. (2011). Analysis of Brassica nigra chromosomes allows identification of a new effective Leptosphaeria maculans resistance gene introgressed in Brassica napus. Proceeding of the 13th International rapeseed congress, Prague 5–9 June.

  19. Ferreira, M. E., Rimmer, S. R., Williams, P. H., & Osborn, T. C. (1995). Mapping loci controlling brassica resistance to L. maculans under different screening conditions. Phytopathology, 85, 213–217.

    CAS  Article  Google Scholar 

  20. Fitt, B. D. L., Brun, H., Barbetti, M. J., & Rimmer, S. R. (2006). World-wide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). European Journal of Plant Pathology, 114, 3–15.

    Article  Google Scholar 

  21. Flor, H. H. (1971). Current status of the gene-for-gene concept. Annual Review of Phytopathology, 9, 275–296.

    Article  Google Scholar 

  22. Fudal, I., Ross, S., Gout, L., Blaise, F., Kuhn, M. L., Eckert, M. R., et al. (2007). Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map-based cloning of AvrLm6. Molecular Plant-Microbe Interactions, 20, 459–470.

    CAS  Article  PubMed  Google Scholar 

  23. Fudal, I., Ross, S., Brun, H., Besnard, A. L., Ermel, M., Kuhn, M. L., et al. (2009). Repeat-induced point mutation (RIP) as an alternative mechanism of evolution towards virulence in Leptosphaeria maculans. Molecular Plant-Microbe Interactions, 22, 932–941.

    CAS  Article  PubMed  Google Scholar 

  24. Ghanbarnia, K., Fernando, W. G. D., & Crow, G. (2011). Comparison of disease severity and incidence at different growth stages of naturally infected canola plants under field conditions by pycnidiospores of phoma lingam as a main source of inoculum. Canadian Journal of Plant Pathology, 33(3), 355–363.

    Article  Google Scholar 

  25. Ghanbarnia, K., Lydiate, J. L., Rimmer, S. R., Li, G., Kutcher, H. R., & Larkan, N. J. (2012). Genetic mapping of the Leptosphaeria maculans avirulance gene corresponding to the LepR1 resistance gene of Brassica napus. Theoretical Applied Genetics, 124, 505–513.

    CAS  Article  PubMed  Google Scholar 

  26. Ghanbarnia, K., Fudal, I., Larkan, N. J., Links, M. G., Balesdent, M. H., Profotova, B., et al. (2015). Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2, using an intraspecific comparative genomics approach. Molecular Plant Pathology, 6, 1–11.

  27. Gout, L., Eckert, M., Rouxel, T., & Balesdent, M. H. (2006a). Genetic variability and distribution of mating-type alleles in field populations of Leptosphaeria maculans from France. Applied and Environmental Microbiology, 72, 185–191.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Gout, L., Fudal, I., Kuhn, M. L., Blaise, F., Eckert, M., Cattolico, L., et al. (2006b). Lost in the middle of nowhere: the AvrLm1 avirulence gene of the dothideomycete Leptosphaeria maculans. Molecular Microbiology, 60, 67–80.

    CAS  Article  PubMed  Google Scholar 

  29. Gugel, P. K., & Petrie, G. A. (1992). History, occurrence, impact and control of blackleg of rapeseed. Canadian Journal of Plant Pathology, 14, 36–45.

    Article  Google Scholar 

  30. Guo, X. W., Fernando, W. G. D., & Entz, M. (2005). Effects of crop rotation and tillage on blackleg disease of canola. Canadian Journal of Plant Pathology, 27, 53–57.

    Article  Google Scholar 

  31. Huang, Y. J., Qi, A., King, G. J., & Fitt, B. D. L. (2014). Assessing quantitative resistance against Leptosphaeria maculans (phoma stem canker) in Brassica napus (oilseed rape) in young plants. PloS One, 9(1), e84924.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Jestin, C., Lodé, M., Vallée, P., Domin, C., Falentin, C., Horvais, R., et al. (2011). Association mapping of quantitative resistance for Leptosphaeria maculans in oilseed rape (Brassica napus L.). Molecular Breeding, 27, 271–287.

    Article  Google Scholar 

  33. Jestin, C., Bardol, N., Lodé, M., Duffé, P., Domin, C., Vallée, P., et al. (2015). Connected populations for detecting quantitative resistance factors to Phoma stem canker in oilseed rape (Brassica napus L.). Molecular Breeding, 35, 167.

  34. Keri, M., Kutcher, H. R., & Rimmer, S. R. (2001). Virulence of isolates of Leptosphaeria maculans from western Canada on Brassica napus differentials. Canadian Journal of Plant Pathology, 23, 199.

    Google Scholar 

  35. Kiyosawa, S. (1982). Genetics and epidemiological modeling of breakdown of plant disease resistance. Annual Review of Phytopathology, 20, 93–117.

    Article  Google Scholar 

  36. Kutcher, H. R., van den Berg, C. G. J., & Rimmer, S. R. (1993). Variation in pathogenicity of Leptosphaeria maculans on brassica spp. based on cotyledon and stem reactions. Canadian Journal of Plant Pathology, 15, 253–258.

    Article  Google Scholar 

  37. Kutcher, H. R., Keri, H., Mclaren, D. L., & Rimmer, S. R. (2007). Pathogenic variability of Leptosphaeria maculans in western Canada. Canadian Journal of Plant Pathology, 393, 388–393.

    Article  Google Scholar 

  38. Kutcher, H. R., Balesdent, M. H., Rimmer, S. R., Rouxel, T., Chèvre, A. M., Delourme, R., et al. (2010). Frequency of avirulence genes in Leptosphaeria maculans in western Canada. Canadian Journal of Plant Pathology, 32, 77–85.

    CAS  Article  Google Scholar 

  39. Kutcher, H. R., Fernando, W. G. D., Turkington, T. K., & McLaren, D. L. (2011). Best management practices for blackleg disease of canola. Prairie Soils & Crops Journal, 4, 122–134.

    Google Scholar 

  40. Kutcher, H. R., Brandt, S. A., Smith, E. G., Ulrich, D., Malhi, S. S., & Johnston, A. M. (2013). Blackleg disease of canola mitigated by resistant cultivars and four-year crop rotations in western Canada. Canadian Journal of Plant Pathology, 35, 209–221.

    Article  Google Scholar 

  41. Larkan, N. J., Lydiate, D. J., Parkin, I. A., Nelson, M. N., Epp, D. J., Cowling, W. A., et al. (2013). The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1. New Phytologist, 197, 595–605.

    CAS  Article  PubMed  Google Scholar 

  42. Larkan, N. J., Ma, L., & Borhan, M. H. (2015). The Brassica napus receptor-like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus. Plant Biotechnology Journal, 13, 983–992.

    CAS  Article  PubMed  Google Scholar 

  43. Leflon, M., Brun, H., Eber, F., Delourme, R., Lucas, M. O., Vallée, P., et al. (2007). Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from brassica Rapa into B. napus. Theoretical and Applied Genetics, 115, 897–906.

    CAS  Article  PubMed  Google Scholar 

  44. Liban, S. H., Cross, D. J., Fernando, W. G. D., Peng, G., & Kutcher, H. R. (2013). Practical genomics: geographic mapping of the race structure in the Canadian Leptosphaeria maculans pathogen population. Acta Phytopathologica Sinica, 43, S282.

    Google Scholar 

  45. Long, Y., Wang, Z., Sun, Z., Fernando, W. G. D., McVetty, B. E. P., & Li, G. (2011). Identification of two blackleg resistance genes and fine mapping of one of these two genes in a Brassica napus canola variety ‘surpass 400’. Theoretical and Applied Genetics, 122, 1223–1231.

    Article  PubMed  Google Scholar 

  46. Marcroft, S. J., Elliott, V. L., Cozijnsen, A. J., Salisbury, P. A., Howlett, B. J., & Van de Wouw, P. (2012a). Identifying resistance genes to Leptosphaeria maculans in Australian Brassica napus varieties based on reactions to isolates with known avirulence genotypes. Crop Pasture Science, 63, 338–350.

    CAS  Article  Google Scholar 

  47. Marcroft, S. J., Van de Wouw, A. P., Salisbury, P. A., Potter, T. D., & Howlett, B. J. (2012b). Effect of rotation of canola (Brassica napus) varieties with different complements of blackleg resistance genes on disease severity. Plant Pathology, 61, 934–944.

    CAS  Article  Google Scholar 

  48. Mayerhofer, R., Bansal, V. K., Thiagarajah, G. R., Stringam, G. R., & Good, A. G. (1997). Molecular mapping of resistance to Leptosphaeria maculans in Australian varieties of Brassica napus. Genome, 40, 294–301.

    CAS  Article  PubMed  Google Scholar 

  49. Parlange, F., Daverdin, G., Fudal, I., Kuhn, M. L., Balesdent, M. H., Blaise, F., et al. (2009). Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change. Molecular Microbiology, 71, 851–863.

    CAS  Article  PubMed  Google Scholar 

  50. Pietravalle, S., Lemarié, S., & van den Bosch, F. (2006). Durability of resistance and cost of virulence. European Journal of Plant Pathology, 114(1), 107–116.

    Article  Google Scholar 

  51. Pilet, M. L., Delourme, R., Foisset, N., & Renard, M. (1998). Identification of loci contributing to quantitative field resistance to blackleg disease, causal agent Leptosphaeria maculans (desm.) Ces. et de not., in winter rapeseed (Brassica napus L.). Theoretical and Applied Genetics, 96, 23–30.

    Article  Google Scholar 

  52. Pilet, M. L., Duplan, G., Archipiano, M., Barret, P., Baron, C., Horvais, R., et al. (2001). Stability of QTL for field resistance to blackleg across two genetic backgrounds in oilseed rape. Crop Science, 41, 197–205.

    CAS  Article  Google Scholar 

  53. Plissonneau, C., Daverdin, G., Ollivier, B., Blaise, B., Degrave, A., Fudal, I., et al. (2015) A game of hide and seek between avirulence genes AvrLm4-7 and AvrLm3 in Leptosphaeria maculans. New Phytologist.

  54. Pongam, P., Osborn, T. C., & Williams, P. H. (1998). Genetic analysis and identification of amplified fragment length polymorphism markers linked to the alm1 avirulence gene of Leptosphaeria maculans. Phytopathology, 88, 1068–1072.

    CAS  Article  PubMed  Google Scholar 

  55. Rimmer, S. R. (2006). Resistance genes to Leptosphaeria maculans in Brassica napus. Canadian Journal of Plant Pathology, 28, S288–S297.

    CAS  Article  Google Scholar 

  56. Rimmer, S. R., & van den Berg, C. G. J. (1992). Resistance of oilseed brassica spp. to blackleg caused by Leptosphaeria maculans. Canadian Journal of Plant Pathology, 14, 56–66.

    Article  Google Scholar 

  57. Rouxel, T., Penaud, A., Pinochet, X., Brun, H., & Gout, L. (2003a). A 10-year survey of populations of Leptosphaeria maculans in France indicates a rapid adaptation towards the Rlm1 resistance gene of oilseed rape. European Journal of Plant Pathology, 109, 871–881.

    CAS  Article  Google Scholar 

  58. Rouxel, T., Willner, E., Coudard, L., & Balesdent, M. H. (2003b). Screening and identification of resistance to Leptosphaeria maculans (stem canker) in Brassica napus accessions. Euphytica, 133, 219–231.

    CAS  Article  Google Scholar 

  59. Sprague, S. J., Balesdent, M. H., Brun, H., Hayden, H. L., Marcroft, S. J., Pinocher, X., et al. (2006). Major gene resistance in Brassica napus (oilseed rape) is overcome by changes in virulence of populations of Leptosphaeria maculans in France and Australia. European Journal of Plant Pathology, 114, 33–44.

    Article  Google Scholar 

  60. Stachowiak, A., Olechnowicz, J., Jedryczka, M., Rouxel, T., Balesdent, M. H., Happstadius, I., et al. (2006). Frequency of avirulence alleles in field populations of Leptosphaeria maculans in Europe. European Journal of Plant Pathology, 114(1), 67–75.

    Article  Google Scholar 

  61. Van de Wouw, A. P., Marcroft, S. J., Barbetti, M. J., Hua, L., Salisbury, P. A., Gout, L., et al. (2009). Dual control of avirulence in Leptosphaeria maculans towards a Brassica napus variety with ‘sylvestris-derived’ resistance suggests involvement of two resistance genes. Plant Pathology, 58, 305–313.

    Article  Google Scholar 

  62. Van de Wouw, A. P., Cozijnsen, A. J., Hane, J. K., Brunner, P. C., McDonald, B. A., Oliver, R. P., et al. (2010). Evolution of linked avirulence effectors in Leptosphaeria maculans is affected by genomic environment and exposure to resistance genes in host plants. PLoS Pathogens, 6, 1–15.

    Google Scholar 

  63. Van de Wouw, A. P., Lowe, R. G. T., Elliott, C. E., Dubois, D. J., & Howlett, B. J. (2014a). An avirulence gene, AvrLmJ1, from the blackleg fungus, Leptosphaeria maculans, confers avirulence to Brassica juncea varieties. Molecular Plant Pathology, 15, 523–530.

    Article  PubMed  Google Scholar 

  64. Van de Wouw, A. P., Marcroft, S. J., Ware, A., Lindbeck, K., Khangura, R., & Howlett, B. J. (2014b). Breakdown of resistance to the fungal disease, blackleg, is averted in commercial canola (Brassica napus) crops in Australia. Field Crops Research, 166, 144–151.

    Article  Google Scholar 

  65. West, J. S., Kharbanda, P. D., Barbetti, M. J., & Fitt, B. D. L. (2001). Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe. Plant Pathology, 50, 10–27.

    Article  Google Scholar 

  66. Williams, P. H., & Delwiche, P. A. (1979). Screening for resistance to blackleg of crucifers in the seedling stage. In In: Proc eucarpia conference on the breeding of cruciferous crops, Wageningen (pp. 164–170.). The Netherlands pp.

  67. Yu, F., Lydiate, D. J., & Rimmer, S. R. (2005). Identification of two novel genes for blackleg resistance in Brassica napus. Theoretical and Applied Genetics, 110, 969–979.

    CAS  Article  PubMed  Google Scholar 

  68. Yu, F., Lydiate, D. J., Hahn, K., Kuzmicz, S., Hammond, C., & Rimmer, S. R. (2007). Identification and mapping of a novel blackleg resistance locus LepR4 in the progenies from Brassica napus × B. rapa subsp. sylvestris. Proceedings of the 12th IRC, Wuhan, PRC.

  69. Yu, F., Lydiate, D. J., & Rimmer, S. R. (2008). Identification and mapping of a third blackleg resistance locus in Brassica napus derived from B. rapa subsp. Sylvestris. Genome, 51, 64–72.

    CAS  Article  PubMed  Google Scholar 

  70. Zhu, B., & Rimmer, S. R. (2003). Inheritance of resistance to L. maculans in two accessions of B. napus. Canadian Journal of Plant Pathology, 3, 98–103.

    Article  Google Scholar 

Download references

Acknowledgments

This work was funded by Canola Agronomic Research Program (CARP) of the Canola Council of Canada. Xuehua Zhang is supported by China Scholarship Council (CSC). The authors wish to thank Dr. Angela Van de Wouw at the University of Melbourne for providing differential isolates for R gene characterization. The authors thank Paula Parks, Sakaria Liban, Besrat Demoz and Jaqueline Huzar Novakowiski for technical assistance. We are grateful to Drs. Carrie Selin and Shuanglong Huang for internal review of the manuscript. We acknowledge several seed companies and research institutions for providing canola seeds for this study.

Author information

Affiliations

Authors

Corresponding author

Correspondence to W. G. Dilantha Fernando.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, X., Peng, G., Kutcher, H.R. et al. Breakdown of Rlm3 resistance in the Brassica napusLeptosphaeria maculans pathosystem in western Canada. Eur J Plant Pathol 145, 659–674 (2016). https://doi.org/10.1007/s10658-015-0819-0

Download citation

Keywords

  • Brassica napus
  • Leptosphaeria maculans
  • R genes
  • Adult plant resistance
  • Avirulence genes
  • Canola
  • Blackleg
  • Rlm
  • Avr
  • Canada