Development of a specific marker for detection of a functional AvrLm9 allele and validating the interaction between AvrLm7 and AvrLm9 in Leptosphaeria maculans.

Blackleg, which is caused by the fungus Leptosphaeria maculans (L. maculans), is a major disease of canola in western Canada and worldwide. Long-term use of one source of resistance could cause the breakdown of its effectiveness. Therefore, appropriate use of R genes is very important, and knowledge about the distribution of avirulence genes is a prerequisite for effectively deploying resistance. Of the 14 avirulence genes identified in L. maculans, AvrLm5 and AvrLm9 were recognized as the two alleles of the same gene based on two single nucleotide polymorphisms, C85T and G164A/C. In this study, a specific marker was developed to identify AvrLm5 and AvrLm9 based on two single nucleotide polymorphisms, C85T and G164A/C, which are responsible for the function of AvrLm9. The specific marker can be used to discriminate the AvrLm9 from avrLm9 accurately in L. maculans isolates, which is consistent with inoculation tests in isolates without AvrLm4-7. This specific marker was used to screen 1229 isolates collected from fields in the years 2014 through 2016 in Manitoba. From 68 to 84% of the isolates were found to contain the AvrLm9 allele; while 4-7% of them were avirulent on the variety Goéland with Rlm9 loci. Furthermore, no isolates having both AvrLm9 and AvrLm7 were detected using a cotyledon test, while 67% to 84% of isolates contained both avirulence genes via PCR detection, implying suppression of AvrLm9 by AvrLm7. In addition, avirulence gene profiles of the other 10 avirulence alleles were examined with the 1229 isolates using cotyledon tests or PCR amplifications. Taken together, this research enables the fast identification of AvrLm5/9, provides the Avr genes' landscape of western Canada and elaborates the relationship between AvrLm9 and AvrLm7 using isolates from grower fields.