Studies On Risk Of Resistance To Fludioxonil And Fluazinam In Botrytis Cinerea

Tomato gray mold is a worldwide disease, causing serious reduction of output of tomato. The control of gray mould relies on application of chemical fungicides. But the Botrytis cinerea developed resistance to those common fungicides, such as carbendazim, procymidone, diethofencarb and pyrimethanil, etc., resulting in the great decrease in control efficacy and severe losses of tomato. Fludioxonil and fluazinam are the novel fungicides with new modes of action to B.cinerea, they were developed by the Swiss Syngenta and Ishihara Sangyo Kaisha Ltd., Japan, respectively. So far they have not been used widely in China. To learify risk of resistance of B.cinerea to fludioxonil and fluazinam, we undertook the studies on testing the biological activity of fludioxonil and fluazinam against B.cinerea, detection of sensitivity of B.cinerea to fludioxonil and fluazinam, analysis of the cross resistance relationship between fludioxonil and the other fungicides with different action modes to B.cinerea, the potential of acquiring the resistant mutants in laboratory, the biological characteristics of the resistant mutants, and the mechanism of fludioxonil-resistant mutants, the influence of consecutive application of fludioxonil in fields on variation of sensitivity to fludioxonil of B.cinerea on tomato. The main results are listed as follows:1. Protective action and curative action of fludioxonil and fluazinam against tomato gray mold were determined by detached leaves test. The protective effect and curative effect of fludioxonil were 87.55% and 96.23% at the concentration of 2μg/mL and 20μg/mL, respectively. The protective effect and curative effect of fluazinam were 82.10% and 96.06% at the concentration of 2μg/mL and 200μg/mL, respectively. Fludioxonil has both protective effect and curative effect against tomato gray mold, while the main effect of fluazinam to tomato gray mold is protection.2. The sensitivity of 106 isolates of B.cinerea collected from different regions of Hebei and Shandong province to fludioxonil and fluazinam was tested by measuring mycelial growth rate. The results show that the B.cinerea is highly sensitive to fludioxonil, with EC50 value ranging from 0.0039 to 0.0449μg/mL. The sensitivity to fluazinam is quite different, with EC50 value ranging from 0.0016 to 0.0531μg/mL. The linear regression analysis of EC50 value among the different fungicides showed that there was no positively correlated cross-resistance among fludioxonil, fluazinam, SYP-Z048, boscalid or pyrimethanil.3. Six fludioxonil-resistant mutants were obtained from two parental isolates through ultraviolet irradiation, with resistant levels ranged from 319 to 10000 times, and the resistance mutation frequency of 3.13×10^-7. Two fludioxonil-resistant mutants were obtained from thirty parental isolates through adaption test with the resistance level of 46 times and >150000 times, the resistance frequency is 0.3175%. While no fluazinam-resistant mutants were acquired under the same conditions. The resistance of seven mutants can be inherited steadily, however, the resistance of one mutant obtained through ultraviolet irradiation disappeared after subcultured for 11 generations on PDA plate. It was found that the resistant mutants significantly decreased in the mycelial growth rate, sporulation, sclerotial production and pathogenicity, compared with their parental isolates.4. All the fludioxonil-resistant mutants were extremely sensitive to high concentration of NaCl on PDA. Resistant mutants obtained through ultraviolet irradiation were extremely sensitive to high concentration of glucose agar, while the resistant mutants obtained through fungicide taming were not sensitive to high osmotic pressure medium.5. We investigated the molecular mechanism of resistance of B.cinerea to fludioxonil premarily. Through PCR, the fragment of histidine kinase gene of the sensitive and resistant mutants was obtained with the sequence ranging from 918 to 981 bp. Through DNA sequencing, the difference was found between the mutant Rla-j and parental isolate in the base sequence of histidine kinase gene, the G2205 coding serine was changed into A2205 coding asparagine in Rla-j. There was no difference in the fragment of histidine kinase gene was observed between the other resistant mutants and their parental strains.6. A fungicide spraying programming was conducted in tomato greenhouse where fludioxonil was sprayed at the recommended rate for 7 times regularly. The results showed that the B.cinerea was still sensitive to fludioxonil after 7 continuous sprays, with EC50 value ranging from 0.0047 to 0.0462μg/mL. Indicated that the resistance of B.cinerea to fludioxonil was difficult to form after sprayed alone.Based on the study results mentioned above, we conclude that a low or moderate risk of resistance to fludioxonil is supposed to exist in B.cinerea in fields.