Discovery of globally rare CYP51 mutations associated with azole resistance in Iranian Zymoseptoria tritici isolates.

backgroundSeptoria tritici blotch, caused by Zymoseptoria tritici, is a major wheat disease worldwide. Demethylation inhibitor (DMI) fungicides, which target the sterol 14α-demethylase enzyme encoded by the CYP51 gene, remain central to disease control. In Iran, propiconazole is extensively applied, raising concerns about the evolution of resistance. This study investigated potential CYP51-mediated resistance mechanisms in Iranian Z. tritici populations.

resultsTwenty-eight isolates collected from six major wheat-producing provinces were assessed for propiconazole sensitivity using a microdilution assay and were classified as sensitive, tolerant, or resistant based on IC₅₀ values. Sequencing of the CYP51 coding region in representative isolates revealed several amino acid substitutions. Two novel mutations (G450R and G516D) were identified, together with rare variants previously reported at low global frequencies (E454K and L4V) and well-established resistance-associated changes such as Y461S and ΔY459/G460. These mutations defined seven haplotypes with variable resistance phenotypes. To validate these findings, we also performed whole-genome sequencing (WGS) on representative Iranian isolates. The WGS results were fully consistent with targeted sequencing, confirming the robustness of CYP51 mutation detection. Gene expression analysis showed inducible CYP51 upregulation in the most resistant isolate. Structural modelling using both homology-based and AlphaFold2 predictions indicated that the novel substitutions may alter surface electrostatics or cavity properties of the enzyme, potentially affecting fungicide binding.

conclusionsThis study documents the emergence of novel resistance-associated mutations in Iranian Z. tritici populations, expanding the spectrum of known CYP51 variants. The findings highlight the interplay between coding mutations, regulatory changes, and structural flexibility in shaping fungicide resistance. An integrated approach combining phenotypic assays, sequencing, expression analysis, and structural modelling provides a robust framework for monitoring resistance and informing sustainable fungicide use in wheat disease management.