PCR-RFLP detection of the E198A mutation conferring resistance to benzimidazoles in field isolates of Monilinia laxa from Greece

Abstract

Sensitivity to benzimidazoles in isolates of the brown rot pathogen (Monilinia laxa) collected from stone fruit in central and northern Greece was evaluated and the molecular basis for resistance was investigated. M. laxa isolates were classified as benzimidazole – sensitive (S) or highly resistant (HR) based on their sensitivity profiles to carbendazim. Thirty seven percent of the isolates belonged to the HR phenotype, carried no apparent fitness penalties and exhibited resistance factor values (based on EC50 values) greater than 500. Highly resistant isolates were also less sensitive to the benzimidazoles benomyl and thiophanate-methyl but more sensitive to the N-phenylcarbamate diethofencarb and the benzamide zoxamide compared to isolates belonging to the S phenotype. Fungitoxicity tests with fungicides belonging to other chemical classes revealed no cross resistance relationships between benzimidazoles and the dicarboximide iprodione, the phenylpyrrole fludioxonil, the hydroxyanilide fenhexamid, the carboxamide boscalid, the triazole tebuconazole and the strobilurin-type fungicide pyraclostrobin, indicating that a target site modification is probably responsible for the resistant phenotypes observed. Comparison of the β-tubulin gene DNA sequences between resistant and sensitive isolates revealed a point mutation resulting from the E198A substitution of the corresponding protein in all HR isolates tested. An Eco31I restriction site in the β-tubulin gene, which was destroyed in HR M. laxa isolates, allowed the development of a PCR-RFLP diagnostic for the detection of the E198A resistance mutation.