Mutations in Cyp51 of Venturia inaequalis and their effects on DMI sensitivity

DMIs (sterol demethylation inhibitors) provide an important mode of action for control of Venturia inaequalis. They target the enzyme 14a-demethylase, encoded by the Cyp51 gene, which acts on an essential precursor for ergosterol biosynthesis. One mechanism, which leads to reduced DMI sensitivity of fungi, is based on mutations in Cyp51. In contrast to other phytopathogenic fungi, only little information is available on changes in the Cyp51 gene of V. inaequalis and their potential influence on DMI sensitivity. In this study, 126 monoconidial isolates were generated from field samples of V. inaequalis collected in apple orchards in different countries in 2018 and 2019. The Cyp51 gene of all isolates was sequenced. Furthermore, a new liquid culture method for V. inaequalis was established, which provides strongly sporulating cultures as a basis for the sensitivity tests. The sequence analysis revealed 15 different point mutations, and each of them resulted in an amino acid exchange in the 14α-demethylase. For some of them, corresponding mutations had been reported in other plant pathogens and shown to influence DMI sensitivity. We measured fungal growth in microtiter-plate tests to evaluate the influence of the amino acid changes in the Cyp51 gene on the sensitivity of the isolates towards the DMIs mefentrifluconazole, difenoconazole, and myclobutanil. While some mutations (e.g. those in the YGYG region 443–446) showed little impact on DMI sensitivity, others caused higher EC50 values, in particular Y133F and M141T. The sensitivity reducing effects differed for the three DMIs and were most pronounced for myclobutanil and less for mefentrifluconazole. The variability in the DMI sensitivity in each Cyp51 haplotype suggests the presence of additional resistance mechanisms caused by genetic background.