Comparative genome analysis of Ralstonia solanacearum causing potato bacterial wilt in Korea

Abstract

Ralstonia solanacearum, causal agent of bacterial wilt, is one of the most destructive phytopathogen in the world. Soil-borne this bacterium invades plants mainly through the roots, colonizes and proliferates in the xylem, as the results plants wilt by blocking water. R. solanacearum has unusual broad host range over 450 plant species of 50 botanical families. This bacterium distributed worldwide encompassing tropical, subtropical, and temperate region. With these features, this species are very diverse and complex and call as pathogenic Ralstonia solanacearum species complex (RSSC). This study used 93 RSSC isolates collected from potato bacterial wilt (or brown rot) from 1998 to 2003 in Korea. To investigate the host specific factors by comparative genomic analyses, first, their properties were determined by analyzing of phylotype, biovar, and host range. Of the 93 isolates, 71 isolates determined to phylotype I and these divided into eight isolates of biovar 3 and sixty-three isolates of biovar 4. Twenty-two isolates determined to phylotype IV and these were all biovar 2. This phylotype-biovar classification was consistent with phylogenetic trees based on 16S rRNA and egl gene sequences, in which all biovar 3 and 4 isolates clustered to phylotype I, and all biovar 2 isolates clustered to phylotype IV. Korean phylotype IV isolates were distinct from phylotype I isolates pathologically as well as genetically, which was all phylotype IV isolates were nonpathogenic to peppers. In pathogenicity assays for host range determining, there were four pathotypes: (P) only pathogenic on potato, (PT) pathogenic on potato and tomato, (PTE) pathogenic on potato, tomato, and eggplant, and (PTEPe) pathogenic on all tested crops – potato, tomato, eggplant, and pepper. Based on this host range, twenty-five strains were selected and sequenced whole genome. The newly sequenced 25 Korean genomes were structurally compared with previously published nine genome data using ANI values, pair-wise and multiple genome alignment. As the results, genome sequences were usually conserved among the same phylotypes, but more divergent between phylotype I and IV. After that, to investigate candidate genes responsible for host specificity, functional genome comparisons were performed based on the host range by analyzing of pan-genome and type III secretion system effectors (T3Es). The same pathotype strains exhibited considerable gene repertoires for infection of tomato, eggplant, or pepper. In pan-genome analysis, total 127 genes present only in tomato nonpathogenic strains, 8 genes in tomato pathogenic strains, 5 genes from eggplant nonpathogenic strains, 7 genes from eggplant pathogenic strains, one gene from pepper nonpathogenic strains, and 34 genes from pepper pathogenic strains. In T3Es analysis, RipH3 and RipS3 were found only in the tomato pathogenic strains and RipAC were only in the eggplant pathogenic strains. This study showed that the host range of R. solanacearum required comprehensive actions of various virulence factors involving effectors, secretion systems, attachment, and enzymes, etc.