A Novel Field Resistance Mechanism: Two Structural Variations in FgOs1 Confer Ultrahigh Fludioxonil Resistance in Fusarium graminearum without Disrupting FgOs1 Transcription or Translation

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), reduces the wheat yield and quality. Fludioxonil, a phenylpyrrole fungicide, is registered in China for FHB control. In 2024, two structurally resistant variants in FgOs1 were identified from field isolates ZJ-44 and ZJ-78 in Hangzhou, Zhejiang, China. Gene editing and structural analysis revealed deletions spanning HAMP2–HAMP3 and HisKA–HATPase_c domains, conferring ultrahigh resistance (EC 50 > 100 μg/mL; RF > 4000) without disrupting FgOs1 expression. Molecular docking suggested these deletions reduce fludioxonil binding affinity. The mutants exhibited fitness costs, including reduced asexual/sexual reproduction, pathogenicity, and increased osmotic sensitivity. Glycerol accumulation was significantly lower under fludioxonil treatment and osmotic stress. No cross-resistance was observed with phenamacril, pyraclostrobin, pydiflumetofen, carbendazim, or tebuconazole; however, positive cross-resistance occurred with dimethachlon. These findings reveal a novel resistance mechanism in F. graminearum, where loss of specific FgOs1 domains confers fludioxonil resistance, while compromising fungal fitness.