Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 01 Feb 2019 21:12:22 Phylogenetic analysis of Xanthomonas based on partial rpoB gene sequences and species differentiation by PCR-RFLP Mariana Ferreira-Tonin, 1 Ju ´ lio Rodrigues-Neto, 1 Ricardo Harakava 2 and Suzete Aparecida Lanza Deste ´ fano 1 Correspondence Suzete Aparecida Lanza Deste ´ fano suzete@biologico.sp.gov.br 1 Laborato ´ rio de Bacteriologia Vegetal, Instituto Biolo ´ gico, Campinas, SP, Brazil 2 Laborato ´ rio de Bioquı ´mica Fitopatolo ´ gica, Instituto Biolo ´ gico, Sa ˜ o Paulo, SP, Brazil The rpoB gene was evaluated as an alternative molecular marker for the differentiation of Xanthomonas species and in order to understand better the phylogenetic relationships within the genus. PCR-RFLP experiments using HaeIII allowed differentiation of Xanthomonas species, particularly those that affect the same plant host such as Xanthomonas albilineans and X. sacchari, pathogenic to sugar cane, Xanthomonas cucurbitae and X. melonis, which cause disease in melon, and Xanthomonas gardneri, X. vesicatoria and X. euvesicatoria/X. perforans, pathogenic to tomato. Phylogenetic relationships within the genus Xanthomonas were also examined by comparing partial rpoB gene sequences (612 nt) and the Xanthomonas species were separated into two main groups. Group I, well supported by bootstrap values of 99 %, comprised X. euvesicatoria, X. perforans, X. alfalfae, X. citri, X. dyei, X. axonopodis, X. oryzae, X. hortorum, X. bromi, X. vasicola, X. cynarae, X. gardneri, X. campestris, X. fragariae, X. arboricola, X. cassavae, X. cucurbitae, X. pisi, X. vesicatoria, X. codiaei and X. melonis. Group II, again well supported by bootstrap values of 99 %, comprised X. albilineans, X. sacchari, X. theicola, X. translucens and X. hyacinthi. The rpoB gene sequence similarity observed among the species in this study ranged from 87.8 to 99.7 %. The results of PCR-RFLP of the rpoB gene indicated that this technique can be used for diagnosis and identification of most Xanthomonas strains, including closely related species within the genus. However, species that showed identical profiles could be differentiated clearly only by sequence analysis. The results obtained in our phylogenetic analysis suggested that the rpoB gene can be used as an alternative molecular marker for genetic relatedness in the genus Xanthomonas. The results of PCR-RFLP of the rpoB gene indicate that this technique can be used for diagnosis and identification of closely related species within the genus, representing a rapid and inexpensive tool that can be easily standardized between laboratories. INTRODUCTION The genus Xanthomonas comprises a large number of bacterial species with different physiological and pathological char- acteristics responsible for diseases in many economically important crops, including rice, beans, cassava, tomato, wheat, citrus, crucifers and many others. Xanthomonas species have been subjected to several taxonomic studies using biochemical tests (Van den Mooter & Swings, 1990), pathogenicity tests on host range (Berthier et al., 1993), SDS-PAGE (Vauterin et al., 1991) and analysis of fatty acids (Yang et al., 1993). However, such techniques are time-consuming, as they require days or weeks of manipulative procedures for their evaluation. Molecular studies have also been performed, including DNA– DNA hybridization (Vauterin et al. , 1992, 1995), analysis of 16S rRNA gene sequences (Moore et al., 1997), PCR- RFLP analysis of rpfB and atpD genes (Simo ˜es et al., 2007), analysis of the sequence of the 16S–23S rRNA intergenic spacer region (Gonc ¸alves & Rosato, 2002) and the gyrB gene (Parkinson et al., 2009), comparison of repetitive extragenic palindromic PCR (rep-PCR) profiles (Rademaker et al. , 2005) and multilocus sequence analysis (MLSA) (Young et al. , 2008, 2010), and these techniques have shown significant results for the differentiation of species of Xanthomonas. The rpoB gene has been used as another molecular marker for identification, differentiation and phylogenetic analysis The GenBank/EMBL/DDBJ accession numbers for the rpoB sequences determined in this study are GU074401–GU074403, HM469984–HM470000 and JF409701–JF409706. Two supplementary figures and two supplementary tables are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1419–1424 DOI 10.1099/ijs.0.028977-0 028977 G 2012 IUMS Printed in Great Britain 1419