Whole Genome Analysis Of Ralstonia Solanacearum FJAT-91 And Function Study On The Extracellular Polysaccharide

Bacterial wilt is a devastating soil-borne disease caused by Ralstonia solanacearum,which is the second most serious bacterial disease causing crop economic losses.The pathogen is widely distributed throughout the world and can infect more than hundreds plant species belonging to over 50 families.It is widely distributes in tropic,semitropics and temperate zones.There are many virulence factors in R.solanacearum,including extracellular polysaccharides,cell wall degrading enzymes,and effector proteins.As more and more R.solanacearum strains have been sequenced in whole genomes,which makes it possible to analyze pathogenic factors and pathogenesis of Rsolanacearum from gene levels.Furthermore,it provides new ways and methods for controlling bacterial wilt,and lays a theoretical foundation for the prevention and control of bacterial wilt.The whole genome sequencing of R.solanacearum FJAT-91 was completed and de novo splicing was performed using Velvet and ABySS.The total length of the FJAT-91 genome was 5.60 Mb which contained a chromosome with a size of 3.69 Mb and a large plasmid with a size of 1.91 Mb.The GC content of genome was 66.85%.There were 5166 total predicted genes in FJAT-91 genome,of which 4870 were coding genes and the gene density was 85.83%.By tRNA prediction and rRNA homology alignment,57 tRNA and 12 rRNA were found in the whole genome of FJAT-91.Comparative genomic analysis of FJAT-91 and 9 other R.solanacearum strains that the whole genome sequencing was completed was performed.Phylogenetic tree was constructed using 16S rRNA.The result showed that FJAT-91 belonged to phylotype I,and was closest to strains FJAT-1458 and GMI1000.The physiological functions of extracellular polysaccharides on the pathogenesis of bacterial wilt were studied by constructing a mutant with synthesis of extracellular polysaccharides deficiency.Firstly,we cloned the homologous arm of epsD from the genome of R.solanacearum FJAT-91,then inserted into suicide plasmid pK18mobsacB.Secondly,the Gm gene was inserted into homologous arm to obtain recombinant plasmid pK18-epsD.Thirdly,the recombinant plasmid was transformed into R.solanacearum FJAT-91 competent cells.The epsD gene deletion mutant was constructed by homologous recombination.Finally,we detected the differences of the biological characteristics of mutant strains and wild-type strains.The main results of this study are as follows:Compared with the wild-type strains,we found that the mutant strains showed different features.1.The colony and cell morphologies of FJAT-91 ?epsD were different from the original strain FJAT-91.2.The yield of extracellular polysaccharides was decreased;3.The abilities of swimming motility and swarming motility was significantly reduced;4.The colonization ability in roots and stems of tomato was significantly decreased;5.Had the Attenuation Index(AI)with 0.905 and identified as a nonpathogenic strain;6.The pathogenicity test revealed that mutant had no pathogenicity to tomato,and the virulence of the reverse mutation could be restored.