| Gray mold caused by Botrytis cinerea has been prevalent in ginseng producing areas in China in recent years,resulting in rotting and shedding of leaves,stems,and seeds in the period of ginseng growing,seriously affecting the normal growth and seed development of ginseng plants,causing serious economic loss to ginseng planting industry.At present,the control of ginseng gray mold mainly depends on chemical pesticides.The abuse of fungicides led to the continuous emergence of fungicide resistance B.cinerea isolates,and the control efficacy of fungicides reduced significantly.In total,B.cinerea 102 isolates were purified from the main ginseng growing areas(MGGA)in China,the fungicide resistance of them to carbendazim,iprodione and pyrimethanil were examined in the present work.The research results have valuable reference for the overall understanding the fungicide resistance of B.cinerea isolates from MGGA in China and guiding to formulate scientific and reasonable grey mold control strategies.The mutation sites of β-tubulin gene and BoOS-1 gene in B.cinerea related to carbendazim and iprodione resistance were analyzed by DNA sequencing,respectively.Combined with the resistance phenotype of B.cinerea isolates,all the results providing a theoretical basis for further understanding the relationship between gene mutation and fungicide resistance of B.cinerea isolates.Furthermore,the differential expression of genes in resistant and sensitive isolates under pyrimethanil stress were studied by transcriptome sequencing technology,the results provided a theoretical basis for explaining the molecular mechanism of pyrimethanil resistance in B.cinerea.The full-text research results were listed as follows:(1)The fungicide resistance level of all B.cinerea isolates was examined by the mycelial growth rate method.The results showed that,the growth inhibition rate of all B.cinerea isolates on PDA medium containing 640μg·mL-1 carbendazim were no more than 50%,and the growth of part B.cinerea isolates even not inhibit,which indicated that carbendazim has lost its control efficacy on the current B.cinerea population from MGGA in China.For the resistance of B.cinerea isolates to iprodione,24 isolates were sensitive(23.53%),16 isolates were low resistance(15.69%),and 62 isolates were medium resistance(60.78%),high resistance isolate was not found.There were significant differences in the iprodione resistance of B.cinerea isolates from different MGGA.Iprodione resistance of B.cinerea isolates from Jilin Tonghua was the most sensitive(EC50=1.3344 μg·mL-1),and those from Xunke was the highest(EC50=3.1680μg·mL-1).For the resistance of B.cinerea isolates to pyrimethanil,28 isolates were sensitive(27.45%),3 isolates were moderate resistant(2.94%),and 71 isolates were high resistant(69.61%).The pyrimethanil resistance of B.cinerea isolates from Jingyu,Jilin was the highest(EC50=44.8734 μg·mL-1),and the pyrimethanil resistance of those from Jiayin of Heilongjiang was the lowest(EC50=1.2229 μg·mL-1).Correlation analysis showed that,B.cinerea isolates tested have no cross resistance to iprodione and pyrimethanil.(2)The β-tubulin and BcOS-1 gene related to carbendazim and iprodione resistance were amplified,respectively.Through blast with the nucleotide sequences of fungicide susceptible B.cinerea strain,we found that the codon 198 of β-tubulin gene in all B.cinerea isolates were mutated,and there are three mutation types were detected,including 32 strains of E198A mutation,69 strains of E198V mutation and 1 strain of E198K mutation.All B.cinerea isolates shown high resistance to carbendazim,indicating that the mutation at codon 198 of β-tubulin gene led to the resistance of B.cinerea to carbendazim.Except for 2 B.cinerea isolates have no mutation in BcOS-1 gene,three gene mutation types were detected on the other 100 B.cinerea isolates,including 47 strains of I365S mutation,23 strains of I365N mutation and 30 strains of Q369P+N373S mutation.Results indicated that,the iprodione resistance of B.cinerea isolates was closely related to the mutation of BcOS-1 gene,and the resistance of Q369P+N373S mutant was the highest.(3)The pyrimethanil resistant B.cinerea strain HRG21 and the pyrimethanil sensitive B.cinerea strain FSG43 treated 2 h(HM2 and FM2,respectively)and 6 h(HM6 and FM6,respectively)by pyrimethanil were performed transcriptome sequencing.B.cinerea strains treated with sterile water were used as blank control(HH2 and HH6,respectively).In total,158.87 Gb clean base was generated through Illumina sequencing.Under the conditions of padj≤0.05 and |log2FoldChange|≥1,1357 differential expression genes(DEGs)in HM2 vs FM2,1962 DEGs in HM6 vs FM6,687 DEGs in HM2 vs HH2,2820 DEGs in HM6 vs HH6,1786 DEGs in HM6 vs HM2,2467 DEGs in FM6 vs FM2,and 2047 DEGs in FM6 vs FH6 were identified.However,no DEGs were found in FM2 vs FH2.It was found that,most ABC transporter genes including BcatrB and BcatrO and MFS transporter genes including Bcmfs1 and BcmfsM2 were up-regulated in HRG21 and down-regulated in FSG43 after treatment with pyrimethanil.GO enrichment analysis results indicated that,the DEGs of resistant strain HRG21 before and after treated by pyrimethanil were mainly enriched in the pathways of cofactor binding,tetrapyrrole binding,iron ion binding,heme binding and oxidoreductase,while the DEGs of sensitive strain FSG43 were mainly enriched in the pathways of membrane composition,transmembrane transport,cofactor binding,coenzyme binding and transporter binding.The results of KEGG analysis showed that,some DEGs in each comparison group were involved in amino acid metabolism and ribosome-related pathways,and most of the DEGs involved in above metabolic pathways were down-regulated when treated by pyrimidine.Most of the DEGs involved in the metabolic pathway of ABC transporters were up-regulated in the HM2 vs HH2,HM2 vs FM2,HM6vsHH6,and HM6 vs FM6.Most of the DEGs involved in ABC transporter pathway in FM6 vs FH6 group were down-regulated. |
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