Study On Fungicide Resistance Monitoring And Resistance Mechanism Of Strawberry Botrytis Cinerea In Chongqing

Gray mold caused by Botrytis cinerea is one of the most common diseases in strawberry production and storage,which seriously affects the yield and quality of strawberry.Due to the relatively few strawberry varieties resistant to gray mold in production and the limitations of biological control due to changes in the natural environment,so chemical control is still the main control method for strawberry gray mold.However,with the long-term use of fungicides on a large scale and the characteristics of extensive hosts,rapid reproduction and strong genetic plasticity of B.cinerea,which has developed resistance to a variety of fungicides with different modes of action.Therefore,this paper monitored the resistance of 142 strains of strawberry B.cinerea isolated from Chongqing from 2021 to 2022,clarified the occurrence of strawberry B.cinerea resistance,and studied the molecular resistance mechanism and biological characteristics of resistant strains.Finally,the resistance mechanism of cyprodinil resistant strains was explored.The main results are as follows:1.In order to clarify the resistance of strawberry B.cinerea to commonly used fungicides in Chongqing,the minimum inhibitory concentration（MIC）method was used to monitor the fungicide resistance of 142 strains of strawberry B.cinerea isolated from Beibei,Changshou and Shapingba district of Chongqing from 2021 to 2022.The results showed that the resistance frequencies of B.cinerea to boscalid（Bos）,carbendazim（Car）,quinone outside inhibitors（Qo Is,pyraclostrobin and azoxystrobin）,dicarboximides（DCFs,procymidone and iprodione）were 92.25%,88.03%,78.17%and51.41%,respectively.No fludioxonil strain was detected.The resistance of strawberry B.cinerea isolated from different regions was different.The strains isolated from Beibei and Shapingba had higher resistance frequency to carbendazim,while the strains from Changshou had higher resistance frequency to quinone outside inhibitors,but all strains isolated from the three regions showed higher resistance frequency to boscalid.At the same time,the resistance phenotypes of strawberry B.cinerea were also classified,and there were 8 resistance phenotypes.Among them Car ^RQo Is ^RDCFs ^RBos ^R,Car ^RQo Is ^RBos ^Rand Car ^RBos ^Rwere the three most resistant phenotypes,with frequencies of 40.85%,21.13%and 17.61%,respectively.The above results showed that strawberry B.cinerea in Chongqing had developed serious resistance to commonly used fungicides,and there was multiple resistance.2.In order to clarify the molecular resistance mechanism of resistant strains of B.cinerea in Chongqing,the resistant strains of each type of fungicides were randomly selected to sequence and compare the target sites of fungicides.The results showed that two mutation modes E198V and E198A were detected on theβ-tubulin of carbendazim resistant strains,among which E198A was the main mutation type,accounting for75.0%.In dicarboximide resistant strains,three mutation modes I365S,I365N and Q369P+N373S were detected on two-component histidine kinase,among which I365N was the main mutation type,accounting for 52.1%.Only one mutation G143A was detected on cytochrome b in quinone outside inhibitors resistant strains.Among the boscalid resistant strains,amino acid mutations were detected on the B subunit of succinate dehydrogenase in 45 strains.There were three mutation modes:P225F,N230I,and H272R,of which P225F was the main mutation type,accounting for 53.3%.Molecular docking results showed that the mutations of P225F and H272R in B subunit could change the binding pocket structure of succinate dehydrogenase and weaken the interaction between boscalid and the amino acids in the binding pocket,resulting in the resistance of B.cinerea to boscalid.In addition,8 strains did not detect mutation sites on the B subunit,and their C and D subunits were sequenced.5 strains only detected amino acid mutations in the C subunit,and only one mutation mode was G85A+I93V+M158V+V168I.Molecular docking results showed that only the 85th amino acid of the four amino acids mutated in the C subunit was close to the binding site,but the amino acid mutation did not directly affect the binding of boscalid,while the other three amino acids were relatively far away from the binding site,and the mutation did not affect the binding of boscalid.The expression of ABC transporter Bcatr B was determined in the above 8 strains with no mutation site in B subunit.The results showed that the expression of Bcatr B in 7 of the 8 resistant strains increased significantly after boscalid treatment.The above results showed that the mutation of amino acid at the target site of fungicide was the main reason for the resistance of B.cinerea to carbendazim,quinone outside inhibitors and dicarboximide fungicides.The resistance of B.cinerea to boscalid was not only related to the mutation of amino acid at the target site,but also closely related to the increase of ABC transporter Bcatr B expression.3.In order to clarify the biological characteristics of strawberry B.cinerea resistant strains,the biological characteristics of three resistant phenotypes(Car ^HRBos ^HR,Car ^HRQo Is ^LRBos ^HR,Car ^HRQo Is ^HRDCFs ^MRBos ^HR)resistant strains and sensitive strains were determined.The results showed that the colony diameter and spore production of Car ^HRQo Is ^LRBos ^HRand Car ^HRQo Is ^HRDCFs ^MRBos ^HRresistant strains were significantly higher than those of sensitive strains in the range of osmotic pressure of 10-20 g/L and p H of 7-8.There was no significant difference between Car ^HRBos ^HRstrains and sensitive strains.There was no significant difference in spore germination and pathogenicity between resistant and sensitive strains.The above results showed that the sensitivity of Car ^HRQo Is ^LRBos ^HRand Car ^HRQo Is ^HRDCFs ^MRBos ^HRresistant strains to osmotic pressure and p H decreased compared with sensitive strains.4.In order to further explore the resistance mechanism of B.cinerea to cyprodinil,10 resistant strains were obtained by fungicide domestication,and the gene sequences and expression levels of cystathionineγsynthetase（CGS）and cystathionineβlyase（CBL）,two key enzymes in the methionine synthesis pathway,were determined.No mutation sites were detected on the resistant strains CGS and CBL.After fungicide treatment,the expression levels of CGS and CBL in 2 and 4 resistant strains decreased significantly respectively,and the expression levels of the remaining resistant strains and sensitive strains did not change significantly.Cystathionineγsynthase and cystathionineβlyase may not be the target sites of cyprodinil acting on B.cinerea.Transcriptome sequencing was performed on resistant and sensitive strains,and GO and KEGG enrichment analysis was performed on differentially expressed genes（DEGs）.The results showed that under the condition of no fungicide treatment,the resistant strains were compared with the sensitive strains.The genes related to the nuclear and carbohydrate metabolic pathways were mainly up-regulated,and the genes related to the ribonucleoprotein complex biosynthesis,RNA metabolism,RNA binding,and tryptophan metabolic pathways were mainly down-regulated.After fungicide treatment,compared with the sensitive strains,the genes related to heme binding,monooxygenase activity and oxidoreductase activity were mainly up-regulated,and the genes related to zinc ion transport,secondary transmembrane transporter,phosphate binding and amino acid metabolism were mainly down-regulated.The above results indicate that genes related to heme binding,monooxygenase activity,oxidoreductase activity,and carbohydrate metabolic pathway in B.cinerea may be involved in regulating the sensitivity of B.cinerea to cyprodinil.Finally,the biological characteristics of the resistant strains obtained by fungicide domestication were determined.The results showed that the mycelial growth rate and sporulation of the resistant strains were significantly lower than those of the sensitive strains,and the sensitivity to different stress factors decreased,but the spore germination and pathogenicity were not significantly different from those of the sensitive strains.The results of genetic stability of resistance showed that the EC₅₀value and resistance ratio of resistant strains decreased gradually with the increase of transfer generation,and the resistance could not be inherited stably.The results of cross-resistance determination showed that cyprodinil had positive cross-resistance with pyrimethanil,a fungicide with the same mode of action,and no cross-resistance with other types of fungicides.