Differential sensitivity to DMI fungicides in Cercospora beticola is mediated by a novel target site modification and C-14 alpha-demethylase overexpression

Sensitivity assessment of 300 Cercospora beticola isolates collected from North Greece revealed that 38 % of the population was highly resistant to at least one of the demethylase inhibitors (DMIs) difenoconazole, epoxiconazole and flutriafol. Resistance factors greater than 50, 100 and 100 were calculated for the most resistant C. beticola isolates to flutriafol, epoxiconazole and difenoconazole, respectively. DMI-resistant isolates carried fitness penalties in terms of mycelial growth, sporulation and pathogenicity, probably accounting for their low frequencies in the field. Pearson correlation analysis revealed a positive cross-resistance relationship between all three DMIs tested suggesting the existence of a target-gene (CbCyp51) related resistance mechanism in the observed phenotypes. Sequence analysis of the CbCyp51 gene from sensitive and DMI-resistant isolates revealed a novel mutation (E149K) present only in C. beticola isolates resistant to all three fungicides tested. To the best of the author's knowledge, this is the first report of this target-site mutation associated with high resistance levels to all flutriafol, epoxiconazole and difenoconazole fungicides. No target-site mutations were found in the rest of DMI-resistance phenotypes. A compound-specific induced CbCyp51 overexpression was observed in C. beticola isolates with specific resistance to flutriafol, epoxiconazole, and difenoconazole, while no indication of constitutive overexpression was found. Overall, the results of the present study provide insights on the mechanisms responsible for the occurrence of high DMI-resistance in the field and a basis for establishing effective strategies for the control of C. beticola in sugar beet fields in Greece.