Novel A576T or M626I substitutions in the HAMP5 domain of histidine kinase FfOs1 alter ligand-binding pocket to mediate fludioxonil resistance in Fusarium falciforme.

Fusarium falciforme, one of the predominant causal agents of Fusarium root rot (FRR), poses a severe threat to sustainable soybean production worldwide. Fludioxonil (Flu) exhibits high antifungal activity against F. falciforme. In this study, the baseline sensitivity of 60 wild-type isolates to Flu was determined to have a mean EC₅₀ of 0.0401 ± 0.0114 μg/mL, with a skewed distribution. Three FluHR mutants generated via in vitro repetitive exposure to Flu (mutation frequency: 0.6%) exhibited heritable Flu resistance. All FluHR mutants exhibited significantly reduced mycelial growth, conidiation, and pathogenicity. Sequence alignment results showed that two novel amino acid substitutions (FfOs1A576T and FfOs1M626I) were located in the highly conserved HAMP5 domain, and independently conferred high-level Flu resistance (RF > 2000) as validated of genetic methods. Molecular docking results revealed that the binding affinities of Flu to FfOs1A576T or FfOs1M626I were significantly reduced compared with those of the corresponding FfOs1 from the FluS parental strain. Furthermore, no cross-resistance was observed between Flu and other fungicides with different modes of action. All the FluR mutants displayed pronounced hypersensitivity to osmotic stress agents and a significant reduction in intracellular glycerol accumulation. Collectively, this study is the first to identify two novel substitutions, A576T and M626I, in the HAMP5 domain of FfOs1 that individually confer Flu resistance while substantially impairing asexual reproduction, virulence, and osmoregulatory capacity in F. falciforme.