Molecular and biological characterization of Sclerotinia sclerotiorum resistant to the anilinopyrimidine fungicide cyprodinil

Cyprodinil belongs to the chemical class of anilinopyrimidines fungicides. In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to cyprodinil was determined using 100 strains collected from the fields in Jiangsu Province of China. The EC50 (50% effective concentration) values ranged from 0.0636–0.8163 μg/ml with a mean value of 0.1869 (±0.1118) ug/ml for mycelial growth. Nine cyprodinil-resistant mutants (Range of resistance factor: 20.22–271.59) were obtained from sensitive strains exposed on PDA medium amended with cyprodinil and the resistance was stable after their ten transfers on PDA without the fungicide or stored at 4 °C for two months. There was positive cross-resistance between cyprodinil and pyrimethanil but not to fludioxonil, dimetachlone, procymidone, carbendazim and boscalid in S. sclerotiorum. Compared with the parental strains, all of the nine cyprodinil-resistant mutants decreased in sclerotial production. The dry weight of mycelia, pathogenicity and cell membrane permeability of most resistant mutants decreased. The mycelial growth, oxalic acid content, and the response to various stress for resistant mutants were almost the same as the sensitive parental strains. Sequencing alignment results showed that there was no alteration of amino acid in cystathionine γ-synthase (MetB) and cystathionine β-lyase (MetC) between cyprodinil-resistant mutants and their sensitive parental strains, which indicated that MetB or MetC was not the molecular target of cyprodinil in S. sclerotiorum. The addition of amino acids L-methionine, L-cystine or L-cysteine decreased the inhibition of cyprodinil against mycelial growth of S. sclerotiorum, which indicated that cyprodinil could not only inhibited methionine biosynthesis but also suppressed cystine and cysteine biosynthesis. These results will contribute to evaluating the resistance risk of cyprodinil for management of the plant diseases of Sclerotinia stem rot caused by S. sclerotiorum and further increase our understanding about the mode of action of cyprodinil.