| Gray mold,caused by Botrytis cinerea Pers.Fr.is one of the most destructive diseases worldwide.More than 200 plant species are susceptible to B.cinerea,particularly fruits,vegetables,and ornamental flowers.Application of fungicides is one of major approaches for management of this disease.However,B.cinerea is a high-risk fungal pathogen in the development of fungicide resistance.The spread of B.cinerea populations resistant to multiple fungicides with different modes of action(e.g.benzimidazoles,carbamates,dicarboximides,anilinepyrimidines,and hydroxyanilide fungicides)has greatly reduced the usefulness of these compounds worldwide and even leads to control failures.Benzothiostrobin,is a novel strobilurin fungicide developed by Central China Normal University with a broad-spectrum fungicidal activity.In order to provide insights into the scientific application of benzothiostrobin to control gray mold,the biological activities,resistant risk and resistant mechanism of B.cinerea to benzothiostrobin were studied.The results were listed as follows.1.Benzothiostrobin can strongly inhibit the mycelial growth and spore germination of B.cinerea,and it exhibited greater antifungal activity than azoxystrobin against B.cinerea.2.Benzothiostrobin could penetrate from the upper surface to the lower surface of the leaf,but systemic translocation activity was very poor.It showed great protective effect in controlling gray mold.Over 95%disease control was obtained with benzothiostrobin at 25 ?g/mL.3.The baseline sensitivity of 100 B.cinerea strains showed the EC50 values for benzothiostrobin inhibiting mycelial growth of B.cinerea ranged from 0.0033 to 0.3921 ?g/mL with a mean of 0.0859 ± 0.0788 ?g/mL and the frequency distribution of EC50 values was unimodal.4.Benzothiostrobin had positive cross-resistance with other QoIs,including azoxystrobin,kresoxim-methyl and pyraclostrobin,but had no cross-resistance with other types of fungicide.5.The benzothiostrobin-resistant strains showed comparable fitness in mycelial growth,sporulation and virulence to that of benzothiostrobin-sensitive strains,and the mutation G143A in Cyt b confers the benzothiostrobin-resistance.In summary,the risk of B.cinerea developing resistance to benzothiostrobin was assessed as high.In order to delay the development of resistance,benzothiostrobin should be used as one component of the mixture for disease management.Fludioxonil,a phenylpyrrole fungicide,is currently one of the effective fungicides to control gray mold.In 2012,it is registered to control gray mold in China.However,field strains lowly resistant to fludioxonil were already reported in several foreign countries.In this study,fludioxonil-sensitivity shifts of B.cinerea isolates were monitored during 2012-2014 in the regions of the two provinces,Jiangsu and Shandong in China,where had history of fludioxonil application.Biochemical and molecular characterization of fludioxonil-mutants were studied.This will provide important information for the scientific application of fludioxonil and enhance our understanding of resistance mechanism of B.cinerea to fludioxonil.The results were listed as follows.1.During 2012-2014,fludioxonil-resistant strains were not detected in Jiangsu Province,but we detected fludioxonil-resistant strains in Shouguang and Weifang regions of Shandong Province with resistance frequencies of 10.67%,17.21%,respectively,for LR phenotype and 2.67%,1.30%,respectively,for HR phenotype.This is the first report that Botrytis cinerea strains highly resistant to fludioxonil were detected in field.2.Six fludioxonil-resistant mutants were obtained by fungicide training in the laboratory.Fludioxonil resistance of these mutants was stable and the level of fludioxonil-resistance,as indicated by the RF values,ranged from 34.38 to>10000.3.All these field and laboratory resistant mutants showed reduced fitness in mycelial growth,sporulation and virulence,and increased sensitivity to osmotic stress(e.g.,0.8 M glucose,0.5 M sorbitol,0.5 M NaCl,0.5 M KCl,0.2 M CaCl2,and 0.3 M MgCl2).4.When treated with 1 ?g/mL of fludioxonil for 4 h,glycerol concentrations and expression levels of Bchogl in the sensitive isolates increased significantly,while those in the field and laboratory resistant mutants increased slightly.5.Mutations in the Bosl gene were detected in the field and laboratory fludioxonil-HR mutants,but there are some differences.Mutations in the field mutants were all located in the HAMP domains of N-terminal region,mutations in the laboratory mutants,however,were distributed in HAMP domains or in the HATPase c domain of C-terminal region.In summary,it is concluded that mutations in the two-component histidine kinase Bosl confer high resistance to fludioxonil.It is speculated that these mutations in Bos1 suppress the level of activation of HOG-MAPK pathway in resistant mutants under fludioxonil stress,which made the glycerol content of these mutants to remain relatively stable,and then the resistant mutants could survive in condition of fludioxonil treatment.The emergence of fludioxonil-resistance in the field warned us that the current applied technology of fludioxonil should be change,and it can be used as one component of the mixture or fungicides with different modes of action should be alternatively used to delay the development of resistance.The highly specific mode of action fungicide has a high risk of rapid selection of resistant genotypes among pathogen populations after application a few years.Thus,we evaluated interactions between fungicides against B.cinerea as a first step toward assessing the potential for the development of synergistic mixtures that could provide satisfactory disease control while also aiding in resistance management.The results were listed as follows.1.The ratio of the dual mixture of benzothiostrobin with A at 1:1 revealed the highlight synergistic effect with a SR value of 3.2.The ratio of the dual mixture of benzothiostrobin with B at 1:4 revealed the highlight synergistic effect with a SR value of 2.5.2.All these 3 fungicides and 2 mixtures were highly effective in controlling gray mold and the 2 mixtures were more effective.At 25 ?g/mL,the disease control of both the 2 mixtures was 100%.3.The 2 mixtures had good inhibitory activity against B.cinerea strains which were resistant to multiple fungicides.Thus,this study provided 2 potential dual mixtures for management of multidrug-resistance in B.cinerea,but their practical values still need to be further evaluated through field-efficacy trials. |
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