G550R Mutation of Histidine Kinase LtOs1 Confers High Fludioxonil Resistance in Lasiodiplodia theobromae from Avocado

Lasiodiplodia theobromae is a destructive phytopathogenic fungus causing severe diseases across numerous crops, notably avocado dieback. While the phenylpyrrole fungicide fludioxonil (Flu) exhibits high efficacy against L. theobromae, its underlying resistance risk remains elusive. In the current study, the frequency distribution of Flu EC 50 (mean EC 50 = 0.0110 μg/mL) was established for 46 field isolates. In vitro -generated Flu-resistant mutants showed severe fitness penalties. Sequence analysis identified a novel LtOs1 G550R substitution in the conserved HAMP 4 domain of LtOs1. Targeted gene replacement confirmed that this mutation confers high-level Flu resistance. Molecular docking revealed that the G550R substitution drastically weakened Flu-LtOs1 binding affinity. Furthermore, no cross-resistance was observed between Flu and six other fungicides. Ultimately, LtOs1 G550R -mediated resistance disrupted intracellular glycerol accumulation and osmoregulation. These findings elucidate the molecular basis of Flu resistance and associated fitness costs in L. theobromae, providing vital insights for sustainable management in avocado orchards.