Development and application of an allele-specific PCR assay for detecting T409C mutation of cyp51 gene linked with tebuconazole resistance in Villosiclava virens (rice false smut)

Villosiclava virens is an ascomycete fungus (anamorph: Ustilaginoidea virens) recognized as one of the most economically important pathogens on rice in China that causes false smut disease. In our study, the V. virens cyp51 gene encoding the target protein for fungicide resistant to tebuconazole was cloned. Sequencing showed that fungal mutant strains resistant to tebuconazole harboured the T409C mutation in cyp51. A simple and rapid allele-specific PCR assay was developed to detect the T409C mutation. Among the isolates tested, a unique 391-bp fragment was amplified from resistant mutant strains with primer pair Uv137F1/Uv137DR, whereas no product was amplified from all sensitive isolates. Our study demonstrated that this allele-specific PCR assay is a rapid, simple and reliable method for identification of V. virens mutants resistant to tebuconazole.