Alterations in CYP51 of Cercospora beticola and their effects on DMI sensitivity

Cercospora leaf spot disease (CLS) caused by the ascomycete Cercospora beticola is the most widespread fungal leaf disease in sugar beet. Fungicides of two active ingredient classes, quinone-outside inhibitors (QoIs) and demethylation inhibitors (DMIs), were important tools for CLS control. Over the years, C. beticola has become resistant to QoIs and a sensitivity shift has been reported for DMIs. In this study the mechanisms causing variation in DMI sensitivity in C. beticola isolates from Europe were analyzed. The CYP51 mutations I387M, Y464S, and L144F were detected in many isolates and most isolates carried the L144F in combination with mutation I309T. Furthermore, single isolates with other mutation combinations have been found. Wildtype isolates were found in low frequency in all European countries. Isolates that contained L144F showed higher EC₅₀ values than those without L144F. Ranges of EC₅₀ values of different CYP51 haplotypes were overlapping, an indication that other resistance mechanisms are present. Mutation L144F is more frequently encoded by codon TTC (96%) than by TTT (4%), and the usage of codon TTC was correlated with increased EC₅₀ values, this being more pronounced for difenoconazole than for mefentrifluconazole. In addition, it could be observed that the usage of codon GAG for E at amino acid position 170, instead of GAA, was more frequently found in isolates with a higher adaptation compared with haplotypes that did not contain L144F. Overall, GAA was present in 67% of all isolates and GAG in 33%, with an unequal distribution within the haplotypes. These data indicate that target site mutations, especially L144F haplotypes, influence DMI sensitivity, and that in L144F haplotypes, L144F codon usage might be responsible for variations within L144F haplotypes. The codon usage for E170 may influence sensitivity and increase EC₅₀ variation of wildtype isolates and isolates with “weak” mutations, but not in L144F haplotypes.