Optimization And Application Of Suspension Array For The Detection Of Multiple Resistance Of Botrytis Cinerea In Field

Gray mold caused by the plant pathogen Botrytis cinerea is a destructive and ubiquitous disease.The control of gray mold mainly depends on using different chemical fungicides.However,the high rate of reproduction and genetic variation of B.cinerea,along with the abuse of fungicides,has resulted in increasing resistance of B.cinerea in the worldwide.Therefore,a high-throughput method for detection multiple fungicides resistance of field strains was needed.The suspension array established in the previous study can meet this demand of high-throughput but with a problem of insufficient detection range.In this study,we intend to extend the detection range,and then apply the optimized method to detect the field strains in Hebei Province.Through the combination of molecular method and mycelial growth assay,the fields could receive suitable fungicide treatments against B.cinerea.The main results are as follows:（1）The method established in previous study can only detect 8 genotypes of 4fungicide-resistance genes.In this study,after the optimization of conditions and the screening of TAG-ASPE primers,new detection codons were successfully added into the suspension array.The detected genotypes show below:Ben A-E198（GAG）,Ben A-E198A（GCG）,Ben A-E198V（GTG）,Ben A-E198K（AAG）,Ben A-F200（TTC）,Ben A-F200Y（TAC）,Sdh B-H272（CAC）,Sdh B-H272R（TCG）,Sdh B-H272Y（TAC）,Bc OS1-I365（ATC）,Bc OS1-I365N（AAC）,Bc OS1-I365S（AGC）,Bc OS1-Q369（CAG）,Bc OS1-Q369P（CCG）,Bc OS1-N373（AAC）,Bc OS1-N373S（AGC）,erg27-F412（TTC）,erg27-F412S（TCC）.The optimized suspension array can comprehensively cover the common fungicide-resistance related genotypes and provide a platform for monitoring multiple resistance of Botrytis cinerea in the field.（2）Total 103 strains from 2016 to 2017 in Hebei Province were detected by the optimized suspension array method.The results indicated that the resistance frequencies of the Ben A,Sdh B,Bc OS1,erg27 were 100%,4.85%,75.73%,0% respectively.Isolates with multiple fungicide resistance represented the major part of the populations（98.06%）.Isolates with dual,triple,quadruple fungicide-resistance were 13.59%,83.50%,0.97%respectively,and type X（Car^RDie^RBos^SIpr^RFen^S）isolates accounted for the majority.This indicated that the situation of multiple resistance in Hebei province was serious.（3）In order to provide a reasonably fungicide application,mycelial growth assay was used to test the sensitivity of strains.The E198A/K/V mutations were associated with HR,while one F200Y mutant was MR to carbendazim.All isolates with E198K mutation showed HR to diethofencarb,and the isolates with E198V mutations were MR or HR to diethofencarb.H272R mutation in Sdh B were determined as LR to boscalid.Isolates with mutations at codons 365,369 and 373 in Bc OS1 showed LR and MR to iprodione.Through the changes in resistance levels of different genotypes from 2016to 2017,we found that the EC₅₀ of Ben A-E198K/V,Bc OS1-I365S and Bc OS1-Q369P+N373S mutants increased during 2016 to 2017,which was driven by the continuous use of fungicides.According to these results,carbendazim is thus not recommended for controlling Gray mold in Hebei province in China.The application of diethofencarb,iprodione and boscalid should be prudent.Rather,fenhexamid is suggested.Overall,the suspension array with detection range of 18 common fungicides-related genotypes,can provide a molecular detection platform for the monitoring of B.cinerea in the field.Combined with our observations,a combination method of high-throughput suspension array and mycelial growth assay was suggested to accurately monitor the multiple resistance of B.cinerea in the field.