Functional Analysis Of BCIN16G00950 And BCIN16G00980 Genes And Development Of Fast Detection Kit For MBC Fungicide Resistance In Botrytis Cinerea

Gray mold is a fungal disease caused by Botrytis cinerea,which causes massive economic losses in production of vegetables and fruits.At present,chemical control is still the main measure to control this disease.B.cinerea is a high risk fungus which can develop fungicide resistance easily,thus it is very important to analyze the resistance mechanism and develop the detection kit for monitoring resistance.Anilinopyrimidine fungicides(APs)are efficient fungicide for gray mold control,but the resistance mechanism has not yet been elucidated.Benzimidazole fungicides(MBCs)are commonly used to control B.cinerea,but the resistance to MBCs has developed in practice.This study carried out experiments from two sides,exploring the functions of AP resistance-associated genes and development of rapid detection kits for monitoring MBC fungicide resistance.The following are major results:(1)AP resistance-associated genes may be used as molecular targets for resistance detection,providing scientific foundation for developing the rapid detection method for fungicide resistance.In previous studies,four resistance-associated genes were identified by using QTL-seq method.This study focused on two genes,Bcin16g00950 and Bcin16g00980.After treatment with cyprodinil for 1 h,2 h and 4 h,the expression level of Bcin16g00950 in the sensitive isolate firstly decreased and then increased,on the contrary in the resistant isolate,it was firstly increased and then decreased.To explore the biological functions of the Bcin16g00950 gene,it was knocked out using PEG mediated transformation of protoplasts.Results showed that the susceptibility of Bcin16g00950 knockout transformants to cyprodinil was similar with wild type isolate.There were no significant differences between the transformants and the wild type isolate in terms of mycelial growth,acid production,sporulation and pathogenicity.On the other hand,the expression dynamics of Bcin16g00980 gene in both sensitive and resistant isolates treated with cyprodinil was first decreased and then increased,but the expression level in the resistant isolate exceeded that of the control group after 4 h.The Bcin16g00980 gene was knocked out and complemented,it was found that the sensitivity to cyprodinil did not show significant difference among knockout transformants,complemented transformants and wild type isolate,indicating that this gene was not directly involved in the resistance regulatory network.There was also no significant difference among transformants and wild type isolate in other aspects,including mycelial growth,acid production,sporulation and pathogenicity.However,on the plates containing CR,the growth inhibition rate of the knockout transformants was decreased.Therefore,Bcin16g00980 played a role in the cell wall integrity pathway of B.cinerea.(2)Rapid and accurate detection of fungicide resistant is important for making scientific management strategies.In this study,we developed a lyophilized loop-mediated isothermal amplification(L-LAMP)assay to detect the MBC resistance of B.cinerea.The L-LAMP kit could be completed by adding template DNA and water,which largely simplified sample addition steps and improved the operability.As a result,it is convenient for local agency to use.The lyophilized kit still had feasibility compared with the original liquid system.DNA isolated from MBC resistant isolates of B.cinerea resulted in a fluorescent signal after L-LAMP amplification,but DNA from sensitive isolates and B.sinoallii,B.porri,S.sclerotiorum strains did not.The sensitivity of the L-LAMP protocol could reach 7.7 pg/μl,which is good enough for the crude DNA extracted from mycelium of symptomatic fruits.In addition,the lyophilized LAMP kits are stable for 3 months when stored at-20°C,4°C,25°C respectively and 1 month stored at 37°C.Overall,it is expected to be effectively used in practice.In conclusion,this study analyzed the function of AP resistance-associated candidate genes Bcin16g00950 and Bcin16g00980,and developed the L-LAMP detection kit for detection of MBC resistance.The results are of great significance for demonstrating the resistance mechanism of B.cinerea to APs and promoting rapid field detection of fungicide resistance in B.cinerea.