A large European diversity panel reveals complex azole fungicide resistance gains of a major wheat pathogen

Fungicide resistance in crop pathogens poses severe challenges to sustainable agriculture. Demethylation inhibitors (DMIs) are critical for controlling crop diseases but face rapid resistance gains. Even though the main molecular basis of resistance is well established, field surveys have repeatedly revealed alternative resistance mechanisms. The European continent has seen rapid and heterogeneous gains in azole resistance. Here, we establish a large genome panel to dissect the genetic architecture of emerging resistance in the major wheat pathogen <i>Zymoseptoria tritici</i>. The European diversity panel spans 15 sampling years and 27 countries for a total of 1,394 sequenced and phenotyped strains. Using two complementary assays to quantify resistance levels of each strain, we captured fine-grained shifts in DMI resistance over space and time. We conducted genome-wide association studies based on a comprehensive set of genotyping approaches for six DMIs. We mapped a vast array of loci encoding target functions, but also diverse channel, kinase, phosphotransferase, oxidoreductase, and monooxygenase functions. The substantial scope in genetic mechanisms underpinning DMI resistance significantly expands our mechanistic understanding of how continent-wide resistance arises in fungal pathogens over time. Diversification of the <i>Cyp51</i> coding sequence was particularly striking with new resistant haplotypes emerging with complex configurations and geographic patterns. Finally, we functionally assessed the impact of resistance-associated synonymous mutations in <i>Cyp51</i> in a sensitive and resistant background. However, no significant contribution was detectable, suggesting that associations were caused by linkage disequilibrium. This study provides expansive new insights into fungicide resistance gains of crop pathogens relevant for future resistance management strategies.IMPORTANCESustainable food production requires the management of disease agents attacking crops. Application of antifungal compounds is among the key elements of pathogen containment; however, resistance can rise rapidly, leaving crop plants vulnerable to resurgent pathogen attacks. Most research has focused on establishing the molecular basis of fungicide resistance, studying mutant strains under laboratory conditions. How fungicide resistance is gained in agricultural fields over space and time remains poorly understood. Our work introduces the largest yet systematic collection of strains of a crop pathogen to assess how resistance mutations arose against a major class of fungicides. Tracking the fungal wheat pathogen, <i>Zymoseptoria tritici</i>, reveals heterogeneous gains in resistance over the past decades across the European continent. Using association mapping techniques and functional validation, we disentangled a complex set of mutations contributing to resistance. Unraveling the complexity of concurrent resistance mutation gains helps focus resistance management practices in agriculture.