Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum

In the current study, sensitivity distribution of Sclerotinia sclerotiorum populations to fluazinam was determined using 103 strains collected from the fields of Jiangsu Province of China in 2016-2017 and the resistance risk of fluazinam was assessed. The average EC50 (50% effective concentration) values and MIC (minimum inhibitory concentration) values of 103 S. sclerotiorum strains against fluazinam were 0.0073 +/- 0.0045 mu g/ml and < 0.3 mu g/ml for mycelial growth, respectively. Nine mutants with low resistance level were obtained from wild type sensitive strains exposed on PDA medium amended with fluazinam and the resistance was stable after their ten transfers on PDA without the fungicide. Compared with the parental strains, the nine fluazinam-resistant mutants decreased in mycelial growth, sclerotial production, pathogenicity and were more sensitive to 0.7 M NaCl. In addition, cell membrane permeability of resistant mutants was higher than that of their parental strains. Cross resistance assay showed that there was no cross-resistance between fluazinam and fludioxonil, dimetachlone, prochloraz, tebuconazole, azoxystrobin, or procymidone in S. sclerotiorum. The above results indicated that there was a low resistance risk for fluazinam in S. sclerotiorum. However, the sensitivity of all fluazinam-resistant mutants to fludioxonil decreased. Sequencing alignment results showed that there were no mutations in the two-component histidine kinase gene (Shk1) of the resistant mutants and the expression levels of Shk1 of three resistant mutants were significantly up-regulated while others were almost the same as their parental strains. These results will contribute to evaluating the resistance risk of fluazinam for management of diseases caused by S. sclerotiorum and further increase our understanding about the mode of action of fluazinam.