Genomic sequence analysis reveals diversity of Australian Xanthomonas species associated with bacterial leaf spot of tomato, capsicum and chilli

Abstract

Background: The genetic diversity in Australian populations of Xanthomonas species associated with bacterial leaf spot in tomato, capsicum and chilli were compared to worldwide bacterial populations. The aim of this study was to confirm the identities of these Australian Xanthomonas species and classify them in comparison to overseas isolates. Analysis of whole genome sequence allows for the investigation of bacterial population structure, pathogenicity and gene exchange, resulting in better management strategies and biosecurity.

Results: Phylogenetic analysis of the core genome alignments and SNP data grouped strains in distinct clades. Patterns observed in average nucleotide identity, pan genome structure, effector and carbohydrate active enzyme profiles reflected the whole genome phylogeny and highlight taxonomic issues in X. perforans and X. euvesicatoria. Circular sequences with similarity to previously characterised plasmids were identified, and plasmids of similar sizes were isolated. Potential false positive and false negative plasmid assemblies were discussed. Effector patterns that may influence virulence on host plant species were analysed in pathogenic and non-pathogenic xanthomonads.

Conclusions: The phylogeny presented here confirmed X. vesicatoria, X. arboricola, X. euvesicatoria and X. perforans and a clade of an uncharacterised Xanthomonas species shown to be genetically distinct from all other strains of this study. The taxonomic status of X. perforans and X. euvesicatoria as one species is discussed in relation to whole genome phylogeny and phenotypic traits. The patterns evident in enzyme and plasmid profiles indicate worldwide exchange of genetic material with the potential to introduce new virulence elements into local bacterial populations.

Keywords: CAZymes; Cell wall degrading enzymes; Pan genome; Secretion system.

Fig. 1

Phylogeny of Australian and Genbank genomes of a) X. euvesicatoria, b) X. perforans and c) X. vesicatoria based on core genome alignments generated by the Roary program. Australian strains are indicated by BRIP and DAR collection prefixes and highlighted; all others are public genomes of related species. Type strains are indicated in bold and branch support values are displayed to clade level (measured with the Shimodaira-Hasegawa test within FastTree). Branch length is indicated by the scale bar

Fig. 2

A heat map and dendrogram of average nucleotide identity (ANI) between 147 Xanthomonas genomes. The coloured bars represent the species as indicated in the SNP phylogeny and supported ANI values shown here. Xanthomonas perforans strains of X. euvesicatoria are indicated separately to highlight ANI differences. ANI is depicted as the colour gradient indicated by the legend: darker = 1 (100% ANI), lighter = 0.88 (88% ANI)

Fig. 3

Cluster matrix of 147 Xanthomonas genomes with dendrogram based on homologue presence (dark) and absence (light). Species groupings are indicated with coloured bars as determined by phylogeny and ANI. Xanthomonas perforans strains of X. euvesicatoria are indicated separately to highlight homologue differences. The four Australian strains most closely related to X. arboricola are designated in the text as an uncharacterised Xanthomonas species

Fig. 4

Pie plots of gene content in core, soft core, shell and cloud genomes describing the pan genome for X. euvesicatoria, X. perforans, X. vesicatoria and X. gardneri. The core genome is defined as genes present in 99–100% of strains; soft core, shell and cloud genomes are defined as 95–99%, 15–95% and 0–15% respectively. Number of genomes in each pan genome is indicated as ‘n’

Fig. 5

Presence/ absence matrix with dendrogram of effectors identified in 147 Xanthomonas genomes. Effector presence is indicated by colour as described in the legend (presence = blue, absence = red). Names and Genbank numbers of identified effectors are listed vertically. Species groupings as determined by phylogeny and ANI are indicated by the horizontal coloured bar as follows: X. euvesicatoria; orange, X. perforans; pink, X. vesicatoria; blue, X. gardneri; dark green, X. arboricola; green, Xanthomonas sp.; light green

Fig. 6

Cluster matrix and dendrogram based on number of CAZyme families identified in 147 Xanthomonas genomes. Number of CAZyme families present in each strain is indicated by the red-blue scale of the figure legend (17 families = red, 1 = blue, 0 = white). CAZyme families are listed vertically. Horizontal coloured bars represent species as indicated by SNP phylogeny and ANI. Xanthomonas perforans strains of X. euvesicatoria are indicated separately to highlight differences in cazyme profile