Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum

Pyrimethanil is an anilinopyrimidine (AP) fungicide that is highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In this study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC₅₀) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC₅₀ values was unimodal but with a long right tail. The mean ± SD EC₅₀ value was 0.137 ± 0.046 μg/ml, and the minimum and maximum were 0.073 and 0.436 μg/ml, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 μg/ml decreased methionine production in the mycelia by 21.6% and reduced the activity of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive subculturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for four generations, and the resistance factors ranged from 69 to 3,421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl₂, Congo red, or sodium dodecyl sulfate. The exogenous addition of methionine into PDA partially alleviated pyrimethanil toxicity to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.