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 Zymoseptoria tritici. 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 Cyp51 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 Cyp51 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.