Population Structure Of Rasltonia Solanacearum Isolated From Guangdong Province And Genomic Analysis Of Race4 Strain YC45

Bacterial wilt,caused by Ralstonia solanacearum（Smith）Yabuuchi is one of the most devastating diseases in the world.Bacterial wilt is one of the most important diseases on crops in Guangdong province.For the present,the results of the study on R.solanacearum isolated from Guangdong province makes it difficult to develop control methods and bacterial wilt-resistance breeding.The aims of this study were to investigate the occurrence of bacterial wilt,analyze biological characteristics of R.solanacearum strains from different hosts collected in Guangdong,and analyze the difference between YC45,which isolated from a special host kencur（Kacmpferia galangal）in Guangdong,and other R.solanacearum strains using comparative genomics.The results are as follow.（1）The occurrence of bacterial wilt and the damages of bacterial wilt to crops were figure out,and the geographical distributions of bacterial wilt of 15 host plants were made clear.A total of 382 strains of R.solanacearum of 15 host plants were isolated and preserved.Bacterial wilt in Cucurbita maxima,Ageratum conyzoides and Pogostemon cablin in Guangdong is caused by R.solanacearum by using isolation of pathogen,16 S rDNA sequencing and proved by the rule of Koch.All 24 strains isolated from C.maxima belong to phylotype I,race 1 and biovar 3 or 4.These isolates clustered into sequevars 17,45 and 56.Eight strains isolated from A.conyzoides belong to phylotype I,race 4 and biovar 3.These isolates clustered into sequevars 17,18 and 45.Three strains isolated from P.cablin belong to phylotype I,race 1 and biovar 3.These isolates clustered into sequevars 17 and 44.Sequevar 45 was identified in China for the first time in this study,and sequevar 56 is a novel sequevar that has not been described previously.This is the first report of C.maxima bacterial wilt caused by R.solanacearum in the world,first report of A.conyzoides bacterial wilt caused by R.solanacearu in China,and the first report of P.cablin bacterial wilt caused by R.solanacearum in Guangdong province.（2）The population of R.solanacearum strains obtained from 16 different host plants and geographical areas in Guangdong province,China was characterized for biological characteristics.The results show that,of the 110 strains tested,only one strain belonged to biovar 1,two strains belonged to biovar 2,fifty-five（50% of the tested strains）strains belonged to biovar 3 and fifty-two（47.3% of the tested strains）strains belonged to biovar 4.Only two strains from potato are phylotype II strains,while other 108 strains are phylotype I strains.One hundred and ten strains were clustered into eleven sequevars 1,13,14,15,17,18,34,44,45,48 and 57,respectively.Sequevar 57 is a new sequevar.Sequevars 15,34 and 44 were distributed widespread in east,west,north and central areas of Guangdong.Sequevars 13,14,17,45 and 48 were distributed in west and central areas.Sequvars 1 and 18 were only found in central area.The new sequevar 57 was found in west area.Only sequevar 15,34 and 44 were found in east and north.Different sequevars strains were found on the same host.Eight sequevars were found on tomato,six sequevars were found on potato,five sequevars were found on kencur,four sequevars were found on eggplant and pepper,three sequevars were found on peanut,two sequevars were found on ginger,tobacco,water spinach and P.cablin,one sequevar was found on pumpkin,Chrysanthemum,sweet potato,A.conyzoides,eucalypt and mulberry.Different pathogenicity types were found in different sequevars.（3）The genome sequence of YC45 was determined using the PacBio RS and Illumina MiSeq 2000 platforms.The assembly is 5,732,909 bp in sizewith a GC content of 67.09%.A circular chromosome was 3,788,542 bp in size（GenBank no.CP011997）while a megaplasmid 2,008,422 in size（GenBank no.CP011998）.The genome contains 12 rRNAs,58 tRNAs and 1 ncRNA,two intact prphages.A total of 4917 genes were predicted,including 4,621 CDS,243 pseudo genes and 116 frameshifted genes.There were 121 specific genes in YC45.A total of 2192 CDS in chromosome and 930 CDS in megaplasmid were annotated in the GO database.A total of 2742 proteins in chromosome were successful classified into 22 COG functional categories,while 2742 proteins in megaplasmid were successful classified into 21 COG functional categories.1454 genes are involved in six biological pathways.The results of genome comparative analysis show that the relationship of YC45 and other strains in the same phylotype was close and the relationship between YC45 and strains in other phylotypes was distant.The thirty virulence factor genes and thirty-one core T3 Es genes are conserved among the phylotype I strains,while significant variations were found among YC45 and phylotype II,III and IV strains.（4）The specific gene ACH51₀7235 mutant of R.solanacearum YC45 was constructed using genetic homologous recombination.Compared with the wild type YC45,the colony of mutant YC45ΔACH51₀7235 became significantly smaller and non-mobility,extracellular polysaccharide secretion decreased significantly,biofilm formation was the same as wild type YC45,growth rate increases both in rich medium and Boucher’s minimal medium.YC45ΔACH51₀7235 was nonpathogenic on R.kaempferiae.Gene ACH51₀7235 play an important role in the growth,extracellular polysaccharide secretion and pathogenesis of R.solanacearum YC45.