A novel mutation Y192H in dihydroorotate dehydrogenase (DHODH) contributes high resistance to a novel chemical quinofumelin in Fusarium graminearum

Quinofumelin, developed by Mitsui Chemicals Agro, Inc., is a quinoline fungicide with strong activity against Fusarium graminearum , the causal agent of Fusarium head blight (FHB) in wheat. Our previous study identified dihydroorotate dehydrogenase (DHODH), a key enzyme in de novo pyrimidine biosynthesis, as the molecular target of quinofumelin. To assess resistance risk, eleven quinofumelin-resistant mutants were obtained through fungicide taming. These mutants showed markedly reduced mycelial growth, sporulation, perithecial formation, and virulence. Sequencing identified a substitution at codon 192 in FgDHODHⅡ. Site-directed mutagenesis confirmed that the Y192H mutation confers high resistance. Molecular docking, microscale thermophoresis (MST), and molecular dynamics (MD) simulations showed that Y192H substantially reduces quinofumelin binding affinity. These findings indicate a medium resistance risk of quinofumelin in F. graminearum . This study provides the first molecular insight into quinofumelin resistance in plant pathogenic fungi, advancing understanding of its mode of action and resistance mechanism. • First report of the resistance mechanism and risk assessment of the DHODH inhibitor quinofumelin in Fusarium graminearum. • The Y192H mutation in FgDHODHⅡ confers high resistance by markedly reducing quinofumelin binding affinity. • Assesses the resistance risk of quinofumelin in F. graminearum as medium. • Provides critical insights into the target-site resistance mechanism of quinofumelin, informing future strategies for fungicide management and DHODH inhibitor design.