Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles

Abstract

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.

Figure 1. Genome alignment.

Conserved synteny lineplot between R. solanacearum PSI07 (as reference), R. syzygii R24 and BDB R229, in the chromosome (1A) and the megaplasmid (1B). Strand inversions are lined in blue.

Figure 2. Number of genes in the species complex pan-genome.

From inside to outside: Core Genome, Dispensable Genome, Specific Genome at the phylotype level (blue: phylotype I and III; green: phylotype II; red: phylotype IV), and Specific Genome at the strain level.

Figure 3. Localization of specific genes in the PSI07, R. syzygii and the BDB genomes.

For each figure, the two inner circles represent the localization of genomic islands and a graphical representation of the genome (green = chromosome, yellow = megaplasmid) A: Genome of R. solanacearum strain PSI07. From inside to outside: (1) PSI07 CDS absent in both BDB and R. syzygii, (2) PSI07 CDS absent in R. syzygii only, (3) PSI07 CDS absent in the BDB only. B: Genome of R. syzygii strain R229. From inside to outside: (1) R. syzygii CDS absent in PSI07, (2) R. syzygii CDS absent in PSI07 but present in BDB. C: Genome of the BDB strain R24. From inside to outside: (1) BDB CDS absent in PSI07, (2) BDB CDS absent in PSI07 but present in R. syzygii.

Figure 4. Average Nucleotide Identity (ANI) pairwise comparisons among sequenced strains in the R. solanacearum species complex.

ANI was calculated using the method of Konstaninidis and Tiedje . Strains with ANI values over 95% are considered to belong in the same species. Red text denotes strains belonging to proposed Ralstonia haywardii nov. species, Blue text denotes strains proposed to be retained in Ralstonia solanacearum, Green text denotes strains belonging to proposed Ralstonia sequeirae nov. species.