Molecular Mechanisms Of Botrytis Cinerea From Field And Lab-induced Strains Resistance To Boscalid

Chemical control is the main means of crop disease control,but the issues of pathogen resistance are getting more serious,which leads to the decline of field efficacy.Therefore,it is necessary to perform the risk assessment of pathogen resistance in lab to fungicides before new pesticides are applied,in order to guide farmers to use pesticides scientifically.It has been reported that there are genetic differences between the resistance evolution mechanism of plant pathogens in lab and the field.The reason for this difference may be from mutagenic factors,environmental conditions and other biological factors.Therefore,it is urgent to study the lab resistance mechanism of plant pathogens and select the induction methods of pesticide-resistant mutants which are the same or similar to those of resistant strains from field,so as to improve the reliability and accuracy of lab resistance risk assessment of fungicides.In this study,the molecular mechanism of resistance between lab and field was studied by using Botrytis cinerea as the tested pathogen and boscalid in succinate dehydrogenase inhibitor(SDHIs)fungicides as target agent.The resistant mutants of B.cinerea in lab to boscalid were obtained by various induction methods.B.cinerea from field was collected and isolated from strawberry planting fields with serious grey mold,and the sensitivity of B.cinerea to boscalid was determined.The molecular resistance mechanisms of resistant strains from field and lab of B.cinerea were compared and analyzed,and the resistant mutants similar to or consistent with those of resistant strains from field were obtained.The purpose of this study is to provide a suitable induction method of lab resistance mutants for the fungicide resistance risk assessment.1.64 B.cinerea strains were isolated from Shandong strawberry planting fields,of which 59 strains showed resistance,the resistance frequency was 92.2%,and 26 strains showed highly resistance,indicating that B.cinerea has a very serious resistance problem to boscalid in the field.2.It has been reported that the resistance of B.cinerea to boscalid is mainly related to the mutation of the target gene and the increase of transporter efflux activity,that is,the overexpression of transporter gene.Among the SDHB subunits of the field strains,the most common mutation is that proline mutated to phenylalanine(P225F),of which one strain was sensitive and the others showed varying levels of resistance.There were also mutations at position 272 from histidine to tyrosine or arginine(H272Y/R)and at position 230 from asparagine to isoleucine(N230I)in SDHB subunits,while 12 strains had no amino acid point mutations in SDHB subunits.However,there was no point mutation in the SDHC subunit of the field strains,and a new mutation was found in the SDHD subunit from two resistant strains(J2-10/J2-11),which is at position 9,mutated from valine to alanine(V9A).The expression of transporter gene and target gene in field strains was detected by real-time fluorescence quantitative PCR.Among them,47% of the strains show significantly up-regulated expression of BcAtrB gene in ABC transporter,61% of the strains show significantly up-regulated expression of BcmfsM2 gene in MFS transporter which speculated that the overexpression of BcAtrB and BcmfsM2 gene was related to the resistance of B.cinerea.However,the number of strains with significantly up-regulated expression of target genes was significantly less than transporter genes,the up-regulated expression rates of BcSdhB,BcSdhC and BcSdhD genes were 23%,22% and 13%,respectively.It was speculated that the overexpression of target genes was not the main reason for the resistance of strains to SDHIs.3.In the lab,15 chemical domesticated mutants had no amino acid mutations in all subunits.Among the 21 mutants obtained by UV mutagenesis,the most common mutation is that proline mutated to phenylalanine at position 225(P225F),accounting for 38%,followed by histidine to tyrosine or arginine at position272(H272Y/R)and asparagine to isoleucine at position 230(N230I),accounting for 33% and 14%,respectively.In addition,3 strains had no amino acid point mutations in SDHB subunits.All 36 lab-induced mutants have no point mutations in SDHC and SDHD subunits.Real-time quantitative PCR was used to detect the differences in the expression of transporter genes and target genes in lab-induced mutants.Among them,the mutants with significantly up-regulated expression of BcAtrB gene in ABC transporter accounted for 81%,while the mutants with significantly up-regulated expression of target genes BcSdhB,BcSdhC and BcSdhD accounted for 17%,31% and 33%,respectively.4.The fitness of B.cinerea in field and lab was analyzed.The results showed that:(1)the mycelial growth rate of 11 lab-induced mutants was lower than that of standard strains,but there was no significant difference in mycelial growth rate between field strains and lab-induced mutants with different resistance levels.(2)the spore production of field strains was negatively correlated with their resistance level,and the spore production of lab-induced mutants was lower than that of standard strains.However,there was no significant difference in spore production between field strains and lab-induced mutants with different resistance levels.(3)there was no significant difference in pathogenicity between field strains and lab-induced mutants with different resistance levels.In summary,the fitness of the strains was not affected by the resistance levels.