Frequency and genetic variability of the avirulence gene AvrLm4-7 among Leptosphaeria maculans isolates collected in Oklahoma, USA

Abstract

Blackleg, caused by Leptosphaeria maculans, is an important disease of canola managed primarily through host-plant resistance. Fungal avirulence (AvrLm) genes code for effectors recognized by major host-plant resistance (Rlm) genes. AvrLm4-7 is predominant in the pathogen population in Oklahoma and Kansas, USA, and interacts with the Rlm4 and Rlm7 resistance genes. High-resolution melting analysis and sequencing were used to evaluate the genetic variability of AvrLm4-7 in avirulent (A) and virulent (a) reference isolates, and in isolates (n=67) collected in Oklahoma from 2014 to 2017. Melting analysis of PCR products from the central primers (493 bp) and from new upstream external primers (105 bp) produced melting profiles that differed among avirulent (AvrLm4-AvrLm7 (A4A7)) and virulent (avrLm4-AvrLm7 (a4A7) and (avrLm4-avrLm7 (a4a7)) pathogen genotypes. Genotypic classification by melting analysis was supported by pathogenicity scores on seedling cotyledons of susceptible, Rlm4, and Rlm7 differentials. All of the local isolates were a4A7 genotypes. In the sequences of a4A7 reference isolates, all showed the expected mutation at bp 358 of C to G resulting in virulence on Rlm4, and silent mutations of either C to T at bp 256 or A to G at bp 134. These two haplotypes displayed unique melting profiles from the central primers in 64% and 36% of local isolates, respectively. Only the central primers produced pathotype-specific melting curves. Sustainable deployment of Rlm7 canola is needed for effective management of blackleg in the region. High-resolution melting will be useful to monitor allelic variability in AvrLm4-7 and for rapid detection of a4a7 strains.