Biological characteristics and molecular mechanisms of Fusarium graminearum resistance to a novel myosin I inhibitor ZJS178

BACKGROUND Fusarium head blight (FHB), primarily caused by F. graminearum , is a major global threat to cereal crops, resulting in significant yield losses and mycotoxin contamination. Resistance to current fungicides is a growing challenge, underscoring the need for novel inhibitors. In this study, we introduce ZJS178, a novel myosin I inhibitor, and provide a comprehensive evaluation of biological characteristics and molecular mechanisms of F. graminearum resistance to ZJS178. RESULTS Baseline sensitivity analysis revealed EC 50 values of ZJS178 against mycelial growth in 120 isolates ranged from 0.0066 to 0.0364 μg mL −1 (mean 0.0157 ± 0.0016 μg mL −1 ). Of the 46 ZJS178‐resistant mutants generated via fungicide adaptation, 12 were randomly selected for analysis, all demonstrating stably inherited resistance. Most mutants exhibited biological fitness comparable to parental strains in vegetative growth, asexual reproduction, virulence, deoxynivalenol production, and stress tolerance. Positive cross‐resistance occurred between ZJS178 and phenamacril but not with fludioxonil, tebuconazole, carbendazim, azoxystrobin, or chlorothalonil in the tested strains. Sequencing identified resistance‐linked FgMyoI mutations (e.g., S418N/A577T for low resistance, S217L for moderate, Y409F/E420G/E420K/C423R for high resistance), which were further confirmed through genetic validation. CONCLUSION ZJS178 exhibits significant inhibitory effect against F. graminearum and novel mutations (Y409F, C423R) in FgMyoI suggest distinct binding mechanisms for ZJS178 and phenamacril, demonstrating significant potential for application against FHB in field. However, as most ZJS178‐resistant mutants show comparable fitness to wild‐type strains, proactive resistance management is critical for sustained efficacy.