Resolving the taxonomic and phylogenetic status of plant pathogenic bacteria has been challenging due to the existence of numerous pathovars and species complexes [1]. For example, there are more than 50 pathovars in Pseudomonas syringae and more than 140 pathovars in Xanthomonas spp. [2][3] [4] [5]. In particular, the taxonomic status of several strains assigned to species like X. axonopodis , X.campestris and X. albilineans remain controversial [6] [7] [8], while there is also a call to assign a separate genus status to Xanthomonas albilineans [8]. Stenotrophomonas maltophilia (Sma) is another interesting member of family Xanthomonadaceae , which is not a plant pathogen, but is taxonomically and phylogenetically very closely related to Xanthomonas [9]. Originally, S.maltophilia was classified as Pseudomonas maltophilia , re-classified as Xanthomonas maltophilia [10] [11], and later upgraded to a novel genus and named Stenotrophomonas maltophilia [12]. Considering the ecological and economical importance of these xanthomonads, resolving their true relationship or taxonomic status is necessary to understanding their evolution.

Molecular phylogenetics and genome-derived taxonomic criteria are revolutionizing the tree of life in general and bacterial taxonomy in particular [13] [14], the foremost development being the use of 16S rRNA typing to reveal the novelty of a species [15]. However, considering the low rate of evolution of the 16SrRNA gene, and the low resolution at infra-species level, this focus has shifted to using protein-coding genes involved in housekeeping functions [15] [16]. This has led to phylogenomics studies in search of new marker genes for bacterial taxonomy (phylogenetic) studies [17] [18]. One such promising marker is the RNA polymerase beta-subunit ( rpoB ) encoding gene, because its average nucleotide similarity between two bacterial strains was found to correlate with DNA-DNA hybridization (DDH), the traditional gold standard for assigning novel species [18]. Complete rpoB gene sequencing has been proposed as a supplement to DDH [18], and for species delineation, rpoB gene sequence similarityof ≤ 97.7 % was found to be significantly correlated with DDH value <70 %. Similarly, complete rpoB gene sequencesimilarity <85.5 % delineated genera [18]. Complete rpoB gene sequencing is emerging as one of the powerful marker genes, owing to its large size (4 kb) and immunity to horizontal gene transfer, as reported in a recent phylogenomic study [17]. In the present study we have investigated the phylogenetic and taxonomic relationship among various xanthomonads using the complete rpoB gene sequence.

Results and Discussion:

Whole-genome sequence of 17 strains of Xanthomonas and 3 strains of Stenotrophomonas are available in the NCBI genome database [19]. These Xanthomonas strains belong to what are classically defined as eight species or nine pathovars, based on host/tissue specificities. Among the three Stenotrophomonas strains, two belong to species S. maltophilia and one to an unassigned species. The rpoB gene is around 4.1 kb, except in X.fuscans strains (Table 1). Interestingly, the relatively small of size of 3. 7 kb in Xf strain 10535 and 2.9 kb in Xf strain 11122 is apparently due to frame-shift mutation in this gene. It is present as a single copy gene in all the sequenced Xanthomonas genomes. Its G+C content is typical of Xanthomonas/Stenotrophomonas , which is 66% (Table 1). A phylogenetic tree based on complete rpoB gene sequence-data of Xanthomonas and Stenotrophomonas strains is shown in Figure 1. The tree is robust with high bootstrap values for all the groupings. As expected the three Stenotrophomonas strains make one phylogenetic group, and the other phylogenetic group consists of all Xanthomonas strains except X. albilineans, which distinctly separates from other groups. In the Xanthomonas strains, Xoc and Xoo strains make the first sub-group, the Xcm and Xcv strains make the second sub-group, the third sub-group includes the pathovars Xac, Xp, Xav and Xf, and the Xcc strains make the fourth sub-group. Interestingly, the novel Xanthomonas species, Xg and Xv, form independent lineages without any relatives.

Table 1. List of Xanthomonas and Stenotrophomonas studied and their rpoB gene sequence information.

S. No Strain Abbreviation Gene size (bp) G+C %
1. X. oryzae pv. oryzae KACC10331 XooK 4152 61.8%
2. X. oryzae pv. oryzae MAFF 311018 XooM 4152 61.8%
3. X. oryzae pv. oryzae PXO99A XooP 4152 61.7%
4. X. oryzae pv. oryzicola BLS256 Xoc 4152 61.8%
5. X. campestris pv. vasculorum NCPPB702 Xcv 4152 61.3%
6. X. campestris pv. musacearum NCPPB4381 Xcm 4152 61.3%
7. X. axonopodis pv. citri str. 306 Xac 4152 62.5%
8. Xanthomonas axonopodis pv. vesicatoria str. 85-10 Xav 4152 62.5%
9. X.fuscans subsp. aurantifolii str. ICPB10535 Xf 10535 3750 63%
10. X. fuscans subsp. aurantifolii str. ICPB 11122 Xf 11122 2943 62.8%
11. X. campestris pv. campestris str. B100 XccB 4164 62.3%
12. X. campestris pv. campestris str. ATCC 33913 XccA 4164 62.3%
13. X. campestris pv. campestris str. 8004 Xcc8 4164 62.3%
14. X. perforans str. 91-118 Xp 4152 62.4
15. X. gardneri str. 101 Xg 4152 61.9
16. X. vesicatoria str.1111 Xv 4152 62.6
17. Xanthomonas albilineans Xalb 4152 61.9%
18. Stenotrophomonas maltophilia K279a SmaK 4167 64.1%
19. Stenotrophomonas maltophilia R551-3 SmaR 4155 63.9%
20. Stenotrophomonas SKA14 SKA14 4155 64.1%

The rpoB gene identity among different Xanthomonas and Stenotrophomonas strains is shown in Table 2. This study validates the right taxonomic status between Xanthomonas oryzae pathovars, (Xoo and Xoc) as they also share 99% sequence identity (well above the cutoff for novel species assignment, which is ≤ 97.7% [18]). However, the species status assigned to the other monocot pathovars, Xcv and Xcm, is under debate, and there has been a call to assign novel species status ( X vasicola ) to these strains [20]. In fact this study also supports assigning novel species status to Xcv and Xcm and removing the species X. campestris designation, as the rpoB gene sequence identity with the Xcc ATCC33913, the type strain of the species X campestris as well as the the pathovar Xanthomonas campestris pv. campestris , is only 95%. This is well below the cutoff for novel species assignment, which is ≤ 97.7% [18]. Similarly, rpoB gene sequence comparison supports classification of two groups of tomato and pepper pathogens in to novel species as X. vesicatoria and X. gardneri [21] [22]. Both Xv and Xg share only 95-96% sequence identities with rest of Xanthomonas strains. This is below the cutoff proposed for novel species assignment, which is ≤ 97.7% [18].

Fig. 1: Phylogenetic tree of Xanthomonas and Stenotrophomonas strains based on the complete rpoB gene sequence

One controversial topic has been the relationship between the X. axonopodis pathovars, Xac and Xav. Although DNA-DNA hybridization studies suggested that they belong to the same species, and were rightly classified as X. axonopodis pathovars [21], other researchers went ahead and classified them into different species, applying more stringent conditions, with Xav becoming Xanthomonas euvesicatoria [22]. In this case also, the rpoB gene similarity clearly suggests that Xac and Xav are synonymous, as their similarity is 98% (above the cutoff for novel species assignment, which is ≤ 97.7% [18]). Interestingly, some of the Xanthomonas axonopodis pv. vesicatoria strains (ATCC BAA-983T = NCPPB 4321T) were assigned as novel species, i.e., Xanthomonas perforans (Xp) [22]. Again, the rpoB gene comparison clearly indicates that the classification is incorrect as Xav and Xp share 99% sequence identity (well above the cutoff for novel species assignment, which is ≤ 97.7% [18]). Similarly, rpoB gene sequence comparison shows that X. fuscans has been incorrectly assigned a separate species status [23], and that it belongs to the species X. axonopodis , as the sequence similarity with other axonopodis strains is 98-99% (above the cutoff for novel species assignment, which is ≤ 97.7% [18]).

Table 2. Complete rpoB gene identity among different Xanthomonas and Stenotrophomonas strains

Xanthomonas albineans forms a distinct lineage in the phylogenetic tree based on rpoB gene sequence (Fig 1), and has been rightly assigned to a novel species taxonomically, which is supported by rpoB sequence similarity of only 88% with other Xanthomonas strains (well below the cutoff for novel species assignment, which is ≤ 97.7% [18]). Surprisingly, there have also been attempts/suggestions to put it into a novel genus [8]. The rpoB similarity values here support the argument that X. albilineans is indeed a distinct species but not a novel genus, because the sequence similarity of its rpoB gene with that of the sequenced Xanthomonas strains is 88% (which is well above the cutoff for novel genus assignment based on rpoB gene sequence, which is <85.5% [18]). Similarly, Stenotrophomonas maltophilia was originally classified as Pseudomonas maltophilia[10], then re-classified as Xanthomonas maltophilia, only to be again classified to a novel genus i.e. Stenotrophomonas maltophilia[11][12]. However our study suggest that Stentrophomonas was rightly classified as, and is synonymous to Xanthomonas, because the sequence similarity of its rpoB gene with that of sequenced Xanthomonas strains is 91% (which is well above the cutoff for novel genus assignment, which is <85.5% [18]). In fact, the rpoB gene of Stenotrophomonas strains show more (91%) sequence identity with all the sequenced Xanthomonas strains than X. albilineans, which shows less (88%) sequence identity with all other sequenced Xanthomonas strains.

Overall, our study reveals the power of rpoBgene sequencing in resolving the phylogeny and taxonomy of xanthomonads. The first insight being that X. axonopodisspecies members, Xac and Xav are indeed pathovars of same species, as they had been originally classified. The rpoB gene sequence comparisons also support classification of Xcm/Xcv as novel species ie X.vasicola, and also of X. albilineans as its present novel species of Xanthomonas,but not as a novel genus. The most surprising revelation is that Stenotrophomonas is synonymous to Xanthomonas, as previously classified by the Swings group [11]. The highlight of this study is that using the complete rpoBgene sequence information is useful for the phylogenetic and taxonomic studies of existing and novel strains of Xanthomonas.

We would like to point out the fact the rpoBgene sequence constitutes only a part of the valuable (genomic) data, and that it is not the final say on the phylogenetic and taxonomic status of xanthomonad strains, particularly, when complete genome sequencing is becoming more economical, and with the availability of highly robust genome based criterions for phylogeny and taxonomy like Average Nucleotide Identity [24] and Digital DNA-DNA hybridization [25]. However, rpoBgene information is also promising as a simple, supplementary and valuable aid in attempts towards understanding the phylogenetic and taxonomic relationship of a complex group of plant-associated bacteria like Xanthomonas.

Materials and Methods:

Complete rpoBgene sequences were retrieved from the respective genome of Xanthomonas and Stenotrophomonas strains available at NCBI [19]. Phylogenetic tree was built using MEGA5 [26] using NJ method, with 1000 bootstrap replicates. Sequence comparisons were obtained by NCBI BlastN [27].


We thank Drs. BD Shenoy, Suresh Korpole, A. Pinnaka for their comments and discussions on the manuscript.

Funding Information

This work was financially supported by Council for Scientific and Industrial Research (CSIR-IMTECH). VS is currently a research intern under Diamond Jubilee Scheme of CSIR at IMTECH.

Competing Interests

The authors have declared that no competing interests exist.