Cyclobutrifluram resistance in Fusarium oxysporum: risk assessment and mechanistic characterization.

Soybean root rot (SRR) is a major soil-borne disease that severely undermines global soybean yields. Fusarium oxysporum ranks among the primary causal agents of this disease in China. Strong inhibitory activity against F. oxysporum has been demonstrated by cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI); however, its resistance risk and underlying mechanisms have yet to be elucidated. In the present study, we evaluated the baseline sensitivity of 104 field isolates of F. oxysporum to cyclobutrifluram. The mean EC50 was 0.0580 μg/mL, with a unimodal frequency distribution, indicating no naturally occurring resistant subpopulations. Through fungicide adaption, we obtained 12 resistant mutants from six parental strains. These mutants displayed significantly reduced fitness compared to their wild-type parents, suggesting a considerable fitness cost for high-level resistance. Cross-resistance assays revealed that cyclobutrifluram exhibits positive cross-resistance only with another SDHI fungicide, pydiflumetofen, but not with non-SDHIs like fludioxonil, pyraclostrobin, carbendazim, or difenoconazole. Sequencing identified two distinct point mutations: ten mutants carried the H248Y substitution in FoSdhB, while two mutants harbored the D133N substitution in FoSdhD. To confirm causality, we generated site-directed mutants via protoplast transformation and verified that both FoSdhB-H248Y and FoSdhD-D133N confer resistance to cyclobutrifluram. Collectively, our findings indicate that F. oxysporum carries a moderate risk of resistance development to cyclobutrifluram, and the resistance is mediated by specific mutations in the SDH subunits. This work provides a crucial theoretical foundation for rational deployment of cyclobutrifluram in integrated disease management and for designing strategies to delay resistance evolution in field populations.