Fungicide Resistance Of Botrytis Cinerea From Greenhouse Strawberries And Tomatoes In Hubei Province

The production of greenhouse strawberries and tomatoes has been a growing sector in Hubei Province.Gray mold,caused by Botrytis cinerea,is one of the most economically important diseases on both crops in greenhouse.At present,the control of gray mold largely relies on fungicides.However,B.cinerea is considered as a high-risk fungal pathogen for resistance development due to its high genetic plasticity and abundance of sporulation.Resistance to different chemical classes of fungicides has been reported in the pathogen from various crops worldwide,posing a threat to the sustainability of gray mold management.In order to identify potential resistance issues and make spray recommendations,isolates of B.cinerea collected from strawberry and tomato greenhouses in Hubei province were determined for their sensitivity to five commonly used fungicides.Resistance to carbendazim and cyprodinil was frequently detected in isolates from both strawberry and tomato greenhouses,while isolates with dual resistance to carbendazim and diethofencab were only frequently detected in strawberry greenhouses.Moreover,resistance to boscalid was only detected in resistant isolates from strawberry greenhouses.No resistance to fludioxonil was found in isolates from strawberry or tomato greenhouses.Resistance stability and fitness were examined for isolates with resistance to carbendazim,boscalid,or carbendazim/diethofencab/cyprodinil,in comparison with sensitive isolates.As a result,no difference in resistance status was observed after ten transfers on PDA medium without fungicides,indicating these resistances were genetically stable.Furthermore,there was no significant difference between most of the resistant isolates and sensitive isolates in mycelial growth rate,osmotic sensitivity to Na Cl,virulence,and sporulation in vitro and in vivo.The molecular basis of carbendazim,diethofencarb,and boscalid resistance was also investigated.The E198 V or E198 A mutation in the β-tubulin gene(TUB2)was found in isolates with carbendazim resistance only,whereas E198 K was found in isolates with resistance to carbendazim and diethofencarb.The H272 R mutation in the succinate dehydrogenase subunit B gene(Sdh B)was found in isolates with resistance to boscalid.These results demonstrated that fungicide resistance has developed in B.cinerea from strawberry and tomato greenhouses in Hubei province.No evidence was found for instability or fitness penalty at most of the parameters in the resistant phenotypes,indicating resistant population may be as competitive as sensitive population in the absence of fungicide selection pressure.In order to improve the efficiency of resistance detection,a loop-mediated isothermal amplification(LAMP)method was developed to facilitate the detection of resistance to methyl benzimidazole carbamate(MBC)and succinate dehydrogenase inhibitor(SDHI)fungicides in the field,based on the respective resistance mutations E198A/V/K and H272 R.This LAMP method was able to specifically identify the template DNA containing its corresponding mutation.Additionally,this LAMP assay was validated with 10-fold serial dilutions of template vectors,relative species of B.cinerea,and with resistant or sensitive genotype of B.cinerea at a large scale,indicating its good sensitivity,specificity and reproducibility.Above all,as part of the LAMP,the DNA extraction method was optimized for an improved efficiency and simplicity,which was better suited in a field condition.Considering its simplicity,rapidity,and high efficiency,the LAMP assay developed in this study is a promising tool for the rapid diagnosis of fungicide resistance in the field,and will contribute to the monitoring of resistance development to MBC and SDHI fungicides in the future,especially in the field and high-throughput tests.In order to explore the resistance mechanism of anilinopyrimidine(AP)fungicides,an isolate with resistance to AP fungicides was crossed with a sensitive isolate to construct the segregation population of resistance and sensitivity phenotypes.QTL-seq was then performed to investigate candidate genes associated with AP resistance.It was found that there were three quantitative trait loci(QTL)linked to AP resistance,locating on chromosome 6,9,and 11,respectively.After screening of these QTLs,29 single nucleotide polymorphisms(SNPs)in 11 genes were obtained.The SNPs were further narrowed down to seven in four genes by sequencing the same loci in the resistant and sensitive isolates from the field.The frequency of these SNPs in field resistant population was found to be over 50%,indicating a high correlation between these SNPs and AP resistance.The role of E407 K in Bcin16g00820 gene in AP resistance was further confirmed by genetic transformation.These results laid the foundation for the elucidation of the mechanism of AP resistance,and were useful for developing molecular markers for AP resistance detection.In conclusion,this study identified the resistance to four commonly used fungicides in B.cinerea from strawberry and tomato greenhouses in Hubei Province,established a LAMP assay for rapid detection of MBC and SDHI resistance in the field,found several SNPs related to AP resistance.All these results will contribute to the integrated management of gray mold in strawberry and tomato greenhouses in Hubei Province.