A Six-Year Investigation of the Dynamics of Avirulence Allele Profiles, Blackleg Incidence, and Mating Type Alleles ofLeptosphaeria maculansPopulations Associated with Canola Crops in Manitoba, Canada

Abstract

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is one of the most economically important diseases of canola (Brassica napus, oilseed rape) worldwide. This study assessed incidence of blackleg, the avirulence allele, and mating type distributions of L. maculans isolates collected in commercial canola fields in Manitoba, Canada, from 2010 to 2015. A total of 956 L. maculans isolates were collected from 2010 to 2015 to determine the presence of 12 avirulence alleles using differential canola cultivars and/or PCR assays specific for each avirulence allele. AvrLm2, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm11, and AvrLmS were detected at frequencies ranging from 97 to 33%, where the AvrLm1, AvrLm3, AvrLm9, AvrLepR1, and AvrLepR2 alleles were the least abundant. When the race structure was examined, a total of 170 races were identified among the 956 isolates, with three major races, AvrLm-2-4-5-6-7-11, AvrLm-2-4-5-6-7-11-S, and Avr-1-4-5-6-7-11-(S) accounting for 15, 10, and 6% of the total fungal population, respectively. The distribution of the mating type alleles (MAT1-1 and MAT1-2) indicated that sexual reproduction was not inhibited in any of the nine Manitoba regions in any of the years L. maculans isolates were collected.

Generation and Characterization of a Virulent Leptosphaeria maculans Isolate Carrying a Mutated AvrLm7 Gene Using the CRISPR/Cas9 System

“…A L. maculans isolate (DS103), termed UMAvr7, was originally identified in a previous study during a canola disease survey in Manitoba, Canada ( Fernando et al, 2018 ). PCR analysis of this isolate, applied to determine the Avr gene profile, indicated that only the AvrLm5 , AvrLm6 , and AvrLm4-7 genes could be positively amplified.…”

“…A L. maculans isolate (DS103) (hereafter named UMAvr7) collected in a previous study, and carrying the AvrLm5-6-7 gene, was identified ( Fernando et al, 2018 ). The isolate was grown on V8 agar juice medium amended with 0.0035% (w/v) streptomycin sulfate under continuous florescent light at room temperature for one week, before being subjected to a second round of single-pycnidiospore isolation to obtain a pure culture.…”

“…A collection of 123 B. napus genotypes, consisting of varieties and advanced breeding lines, were selected for the pathogenicity test using the umavr7 isolate under greenhouse conditions ( Fernando et al, 2018 ). These B. napus genotypes included winter types and semi-winter types and were derived from the major canola growing areas of China.…”

“…A total of 180 Avr gene races were identified from 964 isolates, with three major races observed: AvrLm-2-4-5-6-7 , AvrLm2-4-5-6-7-S , and AvrLm-1-4-5-6-7-11-(S) . Very few isolates carried a single Avr gene conferring virulence to most commercial canola cultivars ( Fernando et al, 2018 ). However, a L. maculans isolate (DS103) with few Avr genes, termed UMAvr7, was identified and carried avrLm1-2-3-4-9-11-LepR1-LepR2-S-AvrLm5-6-7 , which is virulent on B. napus genotypes with different resistance genes (except Rlm7 ).…”

Remarkable recent changes in genetic diversity of the avirulence geneAvrStb6in global populations of the wheat pathogenZymoseptoria tritici

“…Often, these have a fairly narrow geographical area coverage. For example, studies into the changes in Avr gene prevalence in the fungal pathogen Leptosphaeria maculans of Brassica crops over time have focussed on single countries such as Canada (Fernando et al, 2018) or Australia (Van de Wouw et al, 2018). On the other hand, studies with a global geographical scope often do not consider the impact of sampling time on changes in Avr factor prevalence.…”