Rapid detection of Leptosphaeria maculans avirulence gene AvrLm4-7 conferring the avirulence/virulence specificity on Brassica napus using a tetra-primer ARMS-PCR

Abstract

Leptosphaeria maculans is the causal agent of blackleg disease in canola (Brassica napus), resulting in significant yield loss in canola fields worldwide. AvrLm4-7 is an avirulence effector gene in L. maculans, and a single nucleotide mutation at codon 358 is responsible for the absence of the AvrLm4 allele. A tetra-primer amplification refractory mutation system-PCR assay (ARMS-PCR) was developed to rapidly differentiate the AvrLm4AvrLm7 and avrLm4AvrLm7 genes of L. maculans isolates, which differ by a single point mutation. By this approach, we were able to amplify distinct PCR products to infer the gene of the tested isolates. These results were also confirmed through phenotyping, using the cotyledon inoculation test and two canola genotypes with the corresponding resistance genes. The tetra-primer ARMS-PCR assay developed in this study is a simple, rapid, and useful protocol to identify the AvrLm4-7 alleles in L. maculans isolates. This assay has potential applications in the selection of resistant canola cultivars as part of broader antifungal strategies.