Divergent roles of AaOS1 and AaOS2 in procymidone resistance and pathogenicity of Alternaria alternata from tobacco.

Tobacco brown spot disease (TBSD), is a severe leaf disease caused by Alternaria alternata, and its management heavily relies on dicarboximide fungicides. This study analyzed procymidone, a dicarboximide fungicide, resistance in 96 strains of A. alternata isolated from tobacco in Guizhou Province. Results revealed a resistance frequency of 66.67 %, with low-resistance (Pro) and high-resistance (Pro) strains accounting for 54.17 % and 12.50 %, respectively. Compared to procymidone-sensitive strains, Pro isolates suffered significant fitness penalties, including impaired mycelial growth, spore production, and germination, despite retaining pathogenicity. Resistant strains also exhibited significantly enhanced sensitivity to NaCl, SDS, and Congo red. Sequencing analysis showed that Pro strain GZA-28 existed G305K + M306I mutations in AaOS1 may potentially impact its function, and the molecular dynamics simulation further verified this, as evidenced by the reduced HAMP binding free energy from-249.78 kcal/mol in sensitive strains to-240.84 kcal/mol in GZA-28, resulting abnormal signal transduction function and endowing with procymidone resistance. Functional analysis of the HOG-MAPK pathway components AaOS1 and AaOS2 revealed divergent roles: AaOS1 deletion primarily enhanced DCFs resistance with moderate fitness costs, while AaOS2 deletion caused severe growth, sporulation, and virulence defects alongside resistance, highlighting AaOS2's critical role in basic physiology. This study integrates field resistance prevalence, fitness costs, a novel resistance mechanism (AaOS1 G305K/M306I), osmotic stress linkage, and the distinct functions of AaOS1/AaOS2, offering vital insights for understanding and managing procymidone resistance in TBSD.