Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

ABSTRACTAcquired resistance is a threat for antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms, as well as the highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complexIII of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely-used strobilurins which the respiratory complex by binding to the ubiquinol oxidation site (Qo site). We identified the G37V change, within the cytochrome b Qi site, as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. In addition, we identified a negative cross-resistance between isolates harboring G37V or G143A, a Qo site change previously selected by strobilurins. Moreover, double mutants were less resistant to both QiIs and QoIs compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides, and provides new perspectives for the management of QiIs.Originality-Significance StatementThe highly adaptive traits of pathogens render evolutionary outcomes of antifungal treatments difficult to predict.We used directed evolution to assess the risk of resistance to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici.We identified a target modification as the most likely resistance mechanism to be selected.This change triggered high fenpicoxamid resistance and halved the activity of the target enzyme despite no significant penalty for in vitro growth.This work supports the use of experimental evolution as a method to predict adaptation to fungicides and provides important information for the management of QiIs.