Fludioxonil resistance in Lasiodiplodia theobromae: HOG pathway-associated alterations, fitness costs, and cross-resistance.

Abstract

Lasiodiplodia theobromae is a destructive avocado pathogen. Fludioxonil exhibits strong activity against this fungus, yet its resistance risk and resistance-associated molecular changes remain insufficiently characterized. The baseline sensitivity of 100 field isolates collected in Yunnan Province, China, was established, with EC₅₀ values ranging from 0.0102 to 0.0652 μg/mL in a unimodal distribution; thus, no naturally resistant subpopulation was detected within the sampled population. Twelve phenotypically stable, highly resistant mutants (RF > 4166.67) were generated by repeated in vitro exposure. Nine of the 12 mutants carried sequence alterations in LtOS1, LtOS4, and/or LtOS5, including frameshifts, premature stop codons, and amino acid substitutions, whereas three resistant mutants carried no detectable mutations in the four examined HOG pathway genes. Most resistant mutants exhibited fitness penalties, including reduced growth on one or more media, decreased conidiation and virulence, altered stress tolerance, and impaired carbon-source utilization. Resistant mutants also accumulated substantially less intracellular glycerol than their parental isolates after fludioxonil or NaCl treatment, a pattern consistent with altered HOG-mediated osmoregulation. Fludioxonil sensitivity was strongly positively correlated with iprodione sensitivity, whereas no statistically detectable correlations were observed with metconazole, pydiflumetofen, or fluazinam in the tested panel. Collectively, these findings establish a regional baseline sensitivity for L. theobromae and show that high-level laboratory resistance is associated with HOG pathway-related genetic and physiological alterations and substantial, genotype-dependent fitness costs.