Ecological and Evolutionary Insights into Xanthomonas citri Pathovar Diversity

Abstract

Citrus canker, caused by Xanthomonas citri pv. citri, is a serious disease of citrus plants worldwide. Earlier phylogenetic studies using housekeeping genes revealed that X. citri pv. citri is related to many other pathovars, which can be collectively referred as Xanthomonas citri pathovars (XCPs). From the present study, we report the genome sequences of 18 XCPs and compared them with four XCPs available in the public domain. In a tree based on phylogenomic marker genes, all the XCPs form a monophyletic cluster, suggesting their origin from a common ancestor. Phylogenomic analysis using the type strain further established that all the XCPs belong to one species. Clonal analysis of the core genome revealed the presence of two major lineages within this monophyletic cluster consisting of some clonal variants. Incidentally, the majority of these XCPs were first noticed in India, corroborating their clonal relationship and their common origin. Comparative analysis revealed an open pan-genome and the role of interstrain genomic flux of these XCPs since their diversification from a common ancestor. Even though there are wide variations in type III gene effectomes, we identified three core effectors which can be valuable in resistance-breeding programs. Overall, genomic examination of ecological relatives allowed us to dissect the tremendous genomic potential of X. citri species to rapidly evolve into specialized strains infecting diverse crop plants.IMPORTANCE Host specialization is one of the characteristic features of highly evolved pathogens such as the Xanthomonas group of phytopathogenic bacteria. Since the hosts involve staple crops and economically important fruits such as citrus, detailed understanding of the diversity and evolution of such strains infecting diverse plants is important for quarantine purposes. In the present study, we carried out genomic investigation of members of a phylogenetically and ecologically defined group of Xanthomonas strains pathogenic to diverse plants, including citrus. This group includes the oldest Xanthomonas pathovars and also recently emerged pathovars in a particular country where they are endemic. Our high-throughput genomic study has provided novel insights into the evolution of a unique lineage consisting of serious pathogens and their ecological relatives, suggesting the nature, scope, and pattern of rapid and recent diversification. Further, from the level of species to that of clonal variants, the study revealed interesting genomic patterns in diversification of a Xanthomonas lineage and perhaps will inspire careful study of the host range of the included pathovars.

Keywords: India; Xanthomonas; ecology; evolution; hypervariation; pathovar.

FIG 1

Maximum likelihood tree of different Xanthomonas species and pathovars infecting diverse hosts constructed using 28 reference genes with 500 bootstrap replications. The scale bar shows the number of nucleotide substitution per site. The “type strain,” X. citri pv. citri LMG 9322^T (Xct), is underlined. The strains in the green box consist of Xanthomonas pv. strains forming a clade with X. citri pv. citri LMG 9322 (denoted as XCPs). Abbreviations: Xvtw, X. campestris pv. vitiswoodrowii LMG 954; Xbuh, X. axonopodis pv. bauhiniae LMG 548; Xmar, X. axonopodis pv. martyniicola LMG 9049; Xctr, X. campestris pv. vitiscarnosae LMG 939; Xvt, X. campestris pv. viticola LMG 965; Xctf, X. campestris pv. vitistrifoliae LMG 940; Xkhy, X. axonopodis pv. khayae LMG 753; Xcj, P. cissicola LMG 21719; Xmlh, X. axonopodis pv. melhusii LMG 9050; Xcb, X. campestris pv. bilvae NCPPB 3213; Xazr, X. campestris pv. azadirachtae LMG 543; Xap, X. axonopodis pv. punicae LMG 859; Xdur, X. campestris pv. durantae LMG 696; Xct, X. citri pv. citri LMG 9322; Xcaj, X. axonopodis pv. cajani LMG 558; Xctl, X. axonopodis pv. clitoriae LMG 9045; Xmi, X. campestris pv. mangiferaeindicae LMG 941; Xcnt, X. campestris pv. centellae LMG 9044; Xgly, X. citri pv. glycines LMG 712; Xml, X. citri pv. malvacearum LMG 761; Xths, X. campestris pv. thespesiae LMG 9057; Xlen, X. campestris pv. leeana LMG 9048; Xf, X. fuscans pv. fuscans NCPPB 381; Xp, X. perforans 91-118; Xuv, X. euvesicatoria LMG27970; Xctm, X. axonopodis pv. citrumelo F1; Xlf, X. alfalfae subsp. alfalfae LMG 495; Xaxn, X. axonopodis DSM 3585; Xmh, X. axonopodis pv. manihotis LMG 784; Xoo, X. oryzae ATCC 35933; Xvc, X. vasicola NCPPB 2417; Xcs, X. cassavae CF BP 4642; Xca, X. campestris pv. campestris ATCC 33913; Xg, X. gardneri ATCC 19865; Xarb, X. arboricola pv. juglandis strain CF BP 2528; Xfr, X. fragariae LMG 25863. Except for the 18 genomes sequenced in-house (Table 2), all of the genomes were from databases.

FIG 2

XCP genealogy and graphical representation of recombinational events as inferred by ClonalFrameML tree. The major lineages obtained are designated ML-I and ML-II. The sublineages (SL-I, SL-II, SL-III, and SL-IV) are highlighted in green. Strains isolated from asterids are marked with a red star, and the remaining strains were isolated from rosids. Here, the variations detected by comparing each clade with its most recent common ancestor are depicted in the graph. Substitutions are represented by vertical lines and recombination events by dark blue horizontal bars. Light blue vertical lines represent the absence of substitutions, and white lines refer to nonhomoplasic substitutions. All other colors represent homoplastic substitutions, with increases in homoplasy associated with increases in the degree of redness (from white to red). The branches with ≥99 bootstrap values (obtained by the use of the initial PhyML tree) are marked by black stars.

FIG 3

Pan-genome analysis of XCPs. (a) Floral plot showing the number of unique genes in each XCP in the petals and the number of orthologous core sets in the center. (b) Pan-genome profile analysis showing the pan-genome curve generated by plotting the total number of distinct gene families in the pan-genome and the core genome against the number of genomes considered.

FIG 4

Distribution of COG-based functional categories of unique genes of the XCPs represented by bar graphs. Here, the x axis represents the number of genes and the y axis represents the functional categories. Each strain is represented by a colored box.

FIG 5

Comparison of nucleotide sequences of XCPs encoding LPS cassettes. LPS cassettes of ML-I and ML-II are indicated in purple and red boxes, respectively, and sublineages are also indicated. The color coding of ORFs indicates the levels of homology among different LPS cassettes. All maps are approximately to scale. Red ORFs represent transposable elements. Contig breaks are indicated by black diagonal lines.

FIG 6

Type III effectome among XCPs. The presence of a type III effector gene is indicated by blue coloring and its absence by white coloring, and effectors with incomplete sequences, a contig break, or a frameshift mutation resulting in a truncated gene product are indicated by gray coloring. A core set of T3Es is represented by red and a variable set by the yellow horizontal bar on the top.