Molecular characterization and detection of overexpressed C-14 alpha-demethylase-based DMI resistance in Cercospora beticola field isolates

Cercospora beticola strains were isolated from sugar beet fields in N. Greece heavily sprayed with triazoles. Two resistant phenotypes with resistance factors ranging from 18 to 25 and 65 to 115 were identified. Epoxiconazole-resistant isolates were less sensitive to the triazole flutriafol but not to the benzimidazole benomyl and the carboxamide boscalid. No fitness penalties were associated with resistance mutations in the highly resistant phenotype and most isolates retained their resistance levels even after four generations on fungicide free medium. The C-14 alpha-demethylase gene (CYP51) was isolated for the first time in C. beticola. Sequence comparison between resistant and sensitive strains revealed three mutations in only two out of five of the resistant isolates. However, 3-dimensional model analysis showed that these mutations are unlikely to interfere with fungicide binding. Study of transcriptional levels of the C-14 alpha-demethylase gene showed that overexpression was strongly associated with the highly DMI-resistant phenotype. A neutral mutation observed only in highly resistant isolates permitted the development of a PCR–RFLP molecular resistance diagnostic.