Monitoring Of Resistance Of Botrytis Cinerea To Two Fungicides And Risk Assessment Of Resistance To Prochlora

Panax ginseng C.A.Mey.is an important traditional Chinese medicine.Gray mold caused by Botrytis cinerea is usually happened at ginseng producing areas of China in recent years,and threaten the yield and quality of cultivated P.ginseng seriously.At present,the control of ginseng gray mold mainly rely on chemical fungicides.However,fungicides abuse made ginseng B.cinerea isolates produced degrees of resistance to carbendazim,pyrimethanil,iprodione and other commonly used botryticides,which made the control efficacy of botryticides decreased significantly and even loss,the prevention and control of ginseng gray mold faces huge risk.So,in this study,the resistance levels and resistance frequencies of 102 strains of B.cinerea from ginseng producing areas to boscalid,pyraclostrobin and prochloraz were analyzed by mycelial growth rate method.Specific primer amplification method was used to analyze the base mutations of drug resistance genes,the types of base point mutations of fungicide resistance gene loci and their relationship with resistance levels and geographic origins were analyzed.The sensitivity baseline of B.cinerea to prochloraz was established by mycelial growth rate method.Prochloraz resistance mutants were obtained through chemical domestication and UV induction,their resistance mutation potential,stability,and biological characteristics were explored.The main results were summarized as follows:1.The resistance levels of ginseng B.cinerea isolates to boscalid and pyraclostrobin was analyzed,the cross-resistance of fungicides and the multi-resistance types of ginseng B.cinerea isolates were clarified.The resistance of B.cinerea isolates from ginseng producing areas of China to boscalid increased,the resistance frequency was 32.35%,the EC50 values ranged from 0.256 μg/mL to 235.868 μg/mL.The resistance frequency of ginseng B.cinerea isolates from Heilongjiang Province to boscalid was the highest,isolates from Jilin Province was medium,and isolates from Liaoning Province was the lowest.The general resistance frequency and resistance level of ginseng B.cinerea isolates to pyraclostrobin was high,the resistance frequency was 94.12%,the EC50 values ranged from 1.348μg/mL and 487.725 μg/mL.The resistance frequencies of ginseng B.cinerea isolates from Heilongjiang and Liaoning Province were 97.22%and 100%,respectively.Crossresistance analysis results shown that,no obvious cross-resistance were observed between boscalid and pyraclostrobin.Combined with the results of resistance monitoring of other agents,14%of the strains showed resistance to 5 agents,and 50%showed resistance to 4 agents.2.The base mutation types of boscalid-and pyraclostrobin-resistance genes in ginseng B.cinerea isolates of China and their relationship with resistance level and geographic origins were clarified.No base mutation in the succinate dehydrogenase gene sdhA and sdhD of ginseng B.cinerea isolates was observed.Four point mutations,G85A+I93V+M158V+V168I,were occurred simultaneously on the succinate dehydrogenase gene sdhC,no significant correlation with resistance levels and geographic origins were observed.Five types of base mutations,P225F,P225L,H272R,H272Y,and N230I,in the succinate dehydrogenase gene sdhB were found.Among of them,P225F has the highest correlation with boscalid resistance,the mutation have obvious geographic differences.P225L was also highly correlated with boscalid resistance,however,the mutation has no obvious geographic difference.H272R is not corelated with boscalid resistance,however,the mutation has obvious geographic differences.The frequencies of H272Y and N230I are low,only 1 from the 102 B.cinerea isolates was found,respectively.No mutation at position 129 of the cytochrome b gene cytb was found,the frequency of G143A was 97.06%.The resistance frequency of 99 B.cinerea isolates containing G143A mutation was 96.97%,isolates containing Bcbi-143/144 intron were sensitive to pyraclostrobin.3.The sensitivity baseline of B.cinerea to prochloraz was established,and the resistance risk of prochloraz used for ginseng gray mold control was evaluated.The sensitivity frequencies of 102 B.cinerea isolates to prochloraz were distributed continuously,the EC50 values were ranged from 0.0048 μg/mL to 0.0629 μg/mL.Based on the mean EC50 value of 0.018 μg/mL of the 89 ginseng B.cinerea isolates that satisfied normal distribution,the sensitive baseline of ginseng B.cinerea to prochloraz was established.Through fungicide domestication and UV induction in the laboratory,a total of 9 mutants could grew on prochloraz-containing plates were obtained.Among of them,3 were classified as prochloraz-sensitive and 6 were classified as prochloraz-resistant according to the established sensitive baseline.However,the 6 prochloraz-resistant mutants were instable,their growth rate,sporulation yield,and pathogenicity were lower than their wild parents,shown a significant low fitness in the ginseng B.cinerea population.Cross-resistance analyzing results indicated that,prochloraz had no significant cross resistance with boscalid,pyraclostrobin,iprodione and pyrimethanil.In brief,the resistance risk was low when forprochloraz used for ginseng grey mold control.