Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

Abstract

Background: The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats.

Results: The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole.

Conclusions: Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in conjunction with CGH analysis of a larger set of strains, revealed differences great enough to consider reclassification of the R. solanacearum species complex into three species. The data are still too fragmentary to link genomic classification and phenotypes, but these new genome sequences identify a pan-genome more representative of the diversity in the R. solanancearum species complex.

Figure 1

Number of genes in the R. solanacearum pan-genome.

Figure 2

Synteny in the R. solanacearum species complex. Figure 2A shows the percentage of CDS in synteny in each strain compared with strain GMI1000. Figure 2B shows the mean number of CDS in 1 synton compared with GMI1000. Results for chromosome and megaplasmids are depicted in black and grey, respectively.

Figure 3

Multiple genome alignment for strains GMI1000, CMR15, PSI07 and CFBP2957. Fine colored lines represent rearrangements or inversions relative to the GMI1000 genome. Chromosomes are separated from megaplasmids by the thick red line.

Figure 4

Localization of the principal Genomic Islands detected in strains GMI1000, CMR15, CFBP2957 and PSI07. From inside to outside: genomic islands from GMI1000 (in blue), from CFBP2957 (in purple), from CMR15 (in orange) and from PSI07 (in red). Chromosomes and megaplasmids are represented in green and yellow, respectively.

Figure 5

Principal Component Analysis of six R. solanacearum genomes, performed from a two dimensional matrix, combining genomes and metabolic pathways. Individual points represent genomes, and colored vectors symbolize completion of some pathways (number of reaction for pathway x in a given organism/total number of reactions in the same pathway x defined in the MetaCyc database) in the data (see method section). Pathways with similar completions (vectors with similar orientation) have been clustered and drawn in a same color. Thus, genomes can be associated with their representative and characteristic groups of metabolic pathways (i.e. vectors pointing in their direction). The corresponding pathway functions are listed in Suppl.Table S4.A.

Figure 6

Circular representation of plasmids pRSC35 and pRSI13. A: pRSC35, B: pRSI13; in blue: Type IV Secretion System; in green: proteins involved in DNA metabolism; in purple: toxin/antitoxin systems; in red: proteins of unknown function; in orange: the metalloprotease gene of pRSC35.

Figure 7

Phylogeny based on pairwise comparison of average nucleotide identity (ANI). Strains grouped with an ANI value >95% are in the same color. Bootstrap values are indicated in blue. ANI analyses were conducted using perl scripts (Konstantinidis and Tiedje, 2004).

Figure 8

Hierarchical clustering of 51 R. solanacearum strains from CGH results. The six sequenced strains are lined by a thin black label. Bootstrap values are figured by grayscale circles.