Molecular Mechanism Underlying Resistance Variation to the Novel Agrochemical Quinofumelin in <i>Fusarium graminearum</i>

Quinofumelin, a quinoline-based fungicide, demonstrated potent antifungal activity against Fusarium graminearum, the pathogen responsible for Fusarium head blight (FHB) in wheat. Previously, we elucidated that quinofumelin targets dihydroorotate dehydrogenase (DHODH), an indispensable enzyme in the de novo pyrimidine biosynthesis pathway. To explore its resistance mechanism, five FgDHODHII site-directed mutants with distinct genotypes were generated using the homologous recombination strategy. Sensitivity assays identified varying resistance levels: FgDHODHII-A94V remained sensitive to quinofumelin, FgDHODHII-D155T conferred low-level resistance, FgDHODHII-N281A conferred moderate-level resistance, and FgDHODHII-V179E/V179D conferred high-level resistance. Quinofumelin inhibition was reversed by the addition of uridine monophosphate (UMP), uridine or uracil. Although the mutants did not exhibit growth defects, quinofumelin-resistant mutants displayed reduced sporulation and virulence. Quinofumelin showed cross-resistance to ipflufenoquin, but no cross-resistance to other common fungicides (phenamacril, carbendazim, tebuconazole, pydiflumetofen, prothioconazole, fluopyram, benzovindiflupyr, pyraclostrobin and azoxystrobin). Molecular docking, molecular dynamics (MD) simulations and microscale thermophoresis (MST) revealed that D155T, V179E, V179D and N281A mutations altered the binding mode of quinofumelin, resulting in diminished binding affinity. This study provides key insights into its mode of action and resistance mechanism of quinofumelin, facilitating its application in sustainable FHB management.