Study On The Resistance Mechanism Of Botrytis Cinerea To Propamidine

Propamidine is a novel structure and has independent intellectual property rights of alkyamidine fungicide.It has strong inhibition on a variety of pathogenic fungi,especially on Botrytis cinerea with different resistance background,and the EC₅₀is only 0.87μg/m L,its resistance risk level is low.Twelve highly resistant propamidine mutants of B.cinerea were obtained through laboratory acclimation.Whole genome resequencing showed that,compared with the parent strain B05.10,there were 32 common SNPS in 12 proamidine highly resistant strains.The mutation sites were distributed in exons,introns,intergene and UTR regions,among which there were 9 common non-synonymous single point mutations located in the exon region.The mutant genes mentioned above are related to various metabolic pathways in organisms,and target gene mutation is the main mechanism that causes pathogen to produce high level of resistance to fungicides.Therefore,we speculate that the development of the high level of resistance to proamidine in B.cinerea may be caused by the above gene mutation.Based on this,In this study,BCIN-16g04050（A）,Bcnew1（C）,BCIN-12g06020（F）,BCIN＿01g04190（H）,BCIN＿14g05250（K）,BCIN＿12g03280（L）,Bcdsf2（M）and BCIN＿01g05760（N）and Bcpmr1（Q）genes with common SNP loci located in the exon region were replaced bidirectional between resistant and sensitive strains.In order to reveal the molecular mechanism of propamidine resistance of B.cinerea and provide theoretical basis for further analysis of the molecular mechanism of propamidine inhibition and its scientific and rational use,the changes of sensitivity to propamidine and other biological characteristics between the parent strain and gene replacement mutant were compared.The main results are as follows:（1）Nine single point mutant gene replacement vectors located in the exon region were constructed by fusion PCR（Double-joint PCR）.（2）PEG-mediated protoplast transformation was used to replace 9 genes from resistant strains to sensitive strains,and the genes were screened by 50μg/m L hydomycin resistance plate,PCR and sequencing.Nine replacement mutants A40,C18,F21,H51,K53,L24,M20,N11,Q27 were obtained successfully.Sensitivity and phenotypic analysis showed that the EC₅₀values of the nine mutant strains ranged from 2.08 to 19.80μg/m L,and the resistance ratio of the nine mutant strains ranged from 1.73 to 16.50 times compared with their parents.According to Hou Jun’s classification criteria for the resistance level in B.cinerea,one medium-level drug-resistant mutant F21,three low-level drug-resistant mutants K53,M20,Q27,and five sensitive mutants were obtained.At the same time,the homologous gene of the sensitive strain was replaced into the resistant strain,and nine replacement mutants A7,C2,F33,H17,K3,L8,M41,N34,Q16were obtained successfully.The EC₅₀values of the nine mutants ranged from 68.91-160.16μg/m L.The resistance ratio was 57.43-133.46 times.The resistance ratio of the mutant was140.73 times lower than that of the original resistant strain R19.F33 EC₅₀was 59.19μg/m L,which was 59.19%lower than that of R19.The resistance ratio decreased from 140.73 to57.43.These results suggest that the BCIN-12g06020 gene plays a key role in the development of high levels of resistance to propamidine by B.cinerea.（3）Compared with the parent strain B05.10,it was found that the mycelial growth,pathogenicity and spore germination rate of the mutant C18 and F21 decreased,but sporulation yield increased significantly.The mycelium growth and pathogenicity of mutants M20 and Q27 were decreased.The pathogenicity of mutant N11 was significantly decreased.Correspondingly,the biological characters of the replacement mutants C2,F33,M41,N34and Q16 obtained from R19 were reversed.These results indicated that the SNP loci in Bcnew1（C）,BCIN-12g06020（F）,Bcdsf2（M）,BCIN＿01g05760（N）and Bcpmr1（Q）of the9 genes had a significant effect on the biological characters of B.cinerea.In summary,this study identified the influence of 9 common SNP sites located on exons of resistant strains on the resistance of B.cinerea,in which the BCIN-12g06020 gene encodes the putative plasma membrane H⁺-ATPase that is also considered to be an important target for broad-spectrum antifungal drug development due to its functional importance and species specificity.Therefore,we believe that this gene point mutation is a major factor in the development of resistance to propamidine in B.cinerea.The superposition effect of other common SNP sites can also cause the resistance of B.cinerea to propamidine,and ultimately lead to the high level of resistance of B.cinerea to propamidine.In addition,the decline of biological fitness of B.cinerea may be caused by the joint action of several SNP loci,among which the SNP loci of BCIN-12g06020（F）gene had a significant effect on biological traits of the parent strain,and BCIN＿01g05760（N）gene was closely related to the pathogenicity of B.cinerea.