Molecular Study And Effects Of Exogenous Compounds On The Virulence Factors Of Ralstonia Solanacearum

Ralstonia solanacearum is one of the ten most important plant pathogens in the world.It is considered to be a species complex due to the broad host range,wide geographical distribution,and extensive genetic diversity.At present,R.solanacearum strains are spreading from the lowlands to the highlands and cold areas in China.The whole genome sequencing provided the basic to explain the genetic and pathogenic characteristics.The measures used to control bacterial wilt caused by R.solanacearum mainly include the use of chemical pesticides,cultivating resistant varieties,biological control,and agricultural control.Because there are many problems in the chemical measures like the noneffective result and the environment pollution,natural active compounds which are more effective and friendly to environment are developed to control this disease.Besides,the virulence variation of R.solanacearum affected by the long-term use of different control agents is unknown,which catches our attention.This paper analyzed the virulence genes of the strain based on its complete genome sequence accomplished here and studied the effects on the expression of virulence genes by the long-term stress produced by applying streptomycin and scopoletin,which provided the basis for studying the relationship between ecological adaptability and pathogen traits such as population diversity and pathogen virulence variability and a reliable theoretical basis for scientific control of bacterial wilt in the future.The main results of this study are as follows:1.Accomplished the whole genome sequencing,and analyzed the virulence genes of R.solanacearum strain CQPS-1,a strain isolated from wilted tobacco plants in Chongqing.Whole genome was performed using single molecule real-time sequencing（SMRT）on the Pac Bio RS II platform.The completed genome of R.solanacearum strain CQPS-1 was 5.89 Mb（G+C%,66.84%）,contained one circular chromosome（3.83 Mb）and one megaplasmid（2.06 Mb）.5229 CDS were predicted and annotated.We also performed a pan-genome analysis of R.solanacearum strains.Firstly,we compared strain CQPS-1 to four phylotype I strains（GMI1000,YC45,Y45,FQY₄）,finding 16 specific gene families in strain CQPS-1,which contained 442 genes.Then,strain CQPS-1 as phylotype I strain was compared with other 4 phylotype strains（PO82,CFBP2957,CMR15,and PSI07）.From the result,we knew that the number of CQPS-1-specific gene families was 49,included 625 genes.Compared with strain GMI1000,type III secretion system（T3SS）gene cluster（hrp gene cluster）and most virulence genes were conserved.However,some genes coding core type III effectors（T3Es）like Rip AA were significantly variable.2.Valuated the variation of genes coding pathogenic factors of R.solanacearum in China.Using strain CQPS-1 as the reference genome,genomic of 106 strains collected from all tobacco-growing areas in China were resequenced by Hi Seq 4000 sequencing technology（Illumina）.Then the genome sequences were used for analyzing the variation of genes coding pathogenic factors.The results showed that about 64,741 SNPs and 2,977 In Dels were found from all 106 strains,and 371 SV mutations were obtained for each strain.After analyzing the variation of virulence genes,we knew that there were more types of In Del mutations in the T3 Es than in the other type virulence genes.In addition,the coding sequence of Rip AA mutated small among all strains,which only had one SNP variation.3.Determined the effects on the growth,antibacterial activity,and the pathogenicity of R.solanacearum by streptomycin and scopoletin.The results showed that the two exogenous compounds（streptomycin and scopoletin）had effect on the size of R.solanacearum colony.The IC50 of streptomycin and scopoletin were 0.38 mg/L and 133.87 mg/L respectively measured by turbidimeter test method.Then,the mediums with streptomycin and scopoletin were prepared for long-term serial passage experiment to observe the changes in the pathogenicity of strain.After 30 times and 60 times of serial passage experiment,the pathogenicity of strain was evaluated on tobacco.The long-term treatment by the streptomycin could increase the pathogenicity to some degree,while the scopoletin was not obvious.The results of serial passage experiments by scopoletin showed that there was no significant difference between the treatment and the water control when treated 30 times,and also no significant difference among the treatment,the solvent control,the water control when treated 60 times.4.Studied the expression of virulence genes in R.solanacearum influenced by streptomycin and scopolamine.Treated by streptomycin for a long time,the transcriptional regulator phc A of the strain significantly increased.In addition,the extracellular polysaccharide biosynthetic gene eps R increased a lot in the early stage of treatment,but the difference was not found in the later stage of treatment.In the early stage of scopoletin treatment,two genes increased a lot,contained the type III transcriptional regulator hrp G,and the key virulence gene peh S.After the long-term treatment,the effect on these selected virulence genes was not significant.In summary,based on the whole genome sequence,this study analyzed the differences of virulence genes in R.solanacearum strain CQPS-1,and found that some genes were significantly mutated,such as Rip AA.More In Dels were found in the gene of T3 Es than other virulence genes when comparing the 106 strains.In addtion,the growth of the strain was inhibited by exogenous compounds（streptomycin and scopoletin）.After serial passage experiments,the effect of different exogenous compounds on pathogenicity and virulence genes were different.