Phylogenetic analysis of Xanthomonas species based upon 16S-23S rDNA intergenic spacer sequences

Phylogenetic relationships of 17 species of Xanthomonas were assessed based on analysis of 16S-23S rDNA intergenic spacer (ITS) sequences; a higher level of resolution was obtained than that revealed by 16S rDNA analysis. ITS sequences varied in size from 492 to 578 nt within the genus and the similarity among sequences ranged from 63 to 99%. Major differences were found for the hyacinthi group, which included Xanthomonas albilineans, Xanthomonas hyacinthi, Xanthomonas sacchari, Xanthomonas theicola and Xanthomonas translucens. A common ITS structure with tRNAAla and tRNAlle embedded within the sequence was found for all ITS sequences of Xanthomonas species and for Stenotrophomonas maltophilia. These tRNAs were highly conserved and divided the ITS sequence into three regions (ITS1, ITS2 and ITS3). ITS1 and ITS2 sequences of Xanthomonas species showed mean similarities of 87.1 and 86.8%, respectively, and differences consisted of substitution and addition/deletion of 1-5 nt. ITS2 showed remarkable variation in sequence length: most species had an ITS2 of 19-20 nt, whereas a long insertion of 51-56 nt was found in Xanthomonas codiaei, X. hyacinthi, Xanthomonas melonis, X. theicola and X. translucens. For ITS3 the most striking alteration was seen in X. hyacinthi, which showed a large deletion of 44 nt. The ITS phylogenetic tree grouped Xanthomonas species into six major clusters. Cluster I included Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas bromi, Xanthomonas campestris, X. campestris pv. gardneri, Xanthomonas cassavae, X. codiaei, Xanthomonas cucurbitae, Xanthomonas fragariae, Xanthomonas hortorum, X. melonis, Xanthomonas oryzae, Xanthomonas pisi, Xanthomonas vasicola and Xanthomonas vesicatoria. The species X. albilineans, X. sacchari, X. hyacinthi, X. theicola and X. translucens represented distinct clusters (II-VI). Topology of the 16S-23S rDNA ITS phylogenetic tree was very similar to that of the 16S rDNA tree reported previously, but more clusters were discriminated because of the higher level of diversity among the ITS sequences (16.2%) compared with the 16S rDNA sequences (1.8%).