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

<i>Fusarium graminearum</i>, 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 (EC₅₀ > 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 <i>F. graminearum</i>, where loss of specific FgOs1 domains confers fludioxonil resistance, while compromising fungal fitness.