Downloaded from www.microbiologyresearch.org by IP: 54.90.167.105 On: Tue, 03 May 2016 18:15:58 Burkholderia silvatlantica sp. nov., a diazotrophic bacterium associated with sugar cane and maize L. Perin, 1,2 3 L. Martı ´nez-Aguilar, 3 3 G. Paredes-Valdez, 3 J. I. Baldani, 2 P. Estrada-de los Santos, 3 V. M. Reis 2 and J. Caballero-Mellado 3 Correspondence J. Caballero-Mellado jesuscab@cifn.unam.mx 1,2 Universidade Federal Rural do Rio de Janeiro, km 45 1 and Embrapa Agrobiology, km 47 2 , BR 465, C.P. 74505, Serope ´ dica, Rio de Janeiro, Brazil 3 Centro de Ciencias Geno ´ micas, Universidad Nacional Auto ´noma de Me ´ xico, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico In a previous study, nitrogen-fixing isolates were recovered from the rhizosphere of maize and from surface-sterilized leaves of sugar cane cultivated in Rio de Janeiro, Brazil. On the basis of 16S rRNA gene sequence similarities, these isolates were identified as belonging to the genus Burkholderia, and whole-cell-protein profiles demonstrated that they are closely related to each other. In the present study, novel isolates were recovered from the roots of different sugar-cane varieties cultivated in diverse geographical regions of Brazil. Twenty-one nitrogen-fixing isolates were analysed using polyphasic taxonomy criteria, including DNA–DNA relatedness, 16S rRNA gene sequence similarities, fatty acid profiles, whole-cell-protein patterns and multilocus enzyme electrophoresis profiles, as well as morphological, physiological and biochemical characterization. The analysis confirmed that these isolates belong to a novel species within the genus Burkholderia, for which the name Burkholderia silvatlantica sp. nov. is proposed. The type strain, SRMrh-20 T (=LMG 23149 T =ATCC BAA-1244 T ), was isolated from the rhizosphere of maize var. Avantis A2345 cultivated in Serope ´ dica, Rio de Janeiro. The genus Burkholderia was created in 1992 to include seven species transferred from Pseudomonas, with Burkholderia cepacia as the type species (Yabuuchi et al., 1992). Coenye & Vandamme (2003) reported that the genus Burkholderia included over 30 species, but N 2 -fixing species were poorly represented in the genus until recently. Nitrogen fixation, the reduction of atmospheric N 2 to ammonia, was reported only for Burkholderia vietnamiensis (Gillis et al., 1995) of all the species of the genus. Nevertheless, a study by Estrada-de los Santos et al. (2001) showed that Burkholderia could be a genus rich in plant-associated nitrogen-fixers. In that study, Burkholderia kururiensis (Zhang et al., 2000) was identified as a diazotrophic species, and many N 2 -fixing isolates recovered from different plants (maize, coffee and sorghum) or their rhizospheres have subsequently been classified within novel Burkholderia species, including Burkholderia unamae (Caballero-Mellado et al., 2004), Burkholderia xenovorans (Goris et al., 2004) and Burkholderia tropica (Reis et al., 2004). Concomitantly, two nodulating N 2 -fixing strains recovered from legume plants were assigned to the genus Burkholderia according to their 16S rRNA gene sequences (Moulin et al., 2001), and later they were formally classified as Burkholderia phymatum and Burkholderia tuberum (Vandamme et al., 2002). The ability of several as yet unnamed strains to form nodules on Macroptilium atropurpureum, on Mimosa spe- cies (including Mimosa pigra) and on other mimosoid legumes probably indicates that these micro-organisms represent novel Burkholderia species (Barrett & Parker, 2005, 2006; Chen et al., 2005). It is worth noting that the diazotrophic Burkholderia species, with the exception of B. vietnamiensis, comprise a group of closely related spe- cies that are distant from the group of opportunistic patho- gens referred to as the Burkholderia cepacia complex, which includes B. vietnamiensis (Coenye & Vandamme, 2003). In the present study, a polyphasic approach was undertaken to determine the taxonomic status of diazotrophic isolates tentatively designated as the Burkholderia NAR group. These include isolates from sugar-cane and maize plants reported by Perin et al. (2006) together with novel strains isolated from other sugar-cane varieties grown in different geogra- phical regions of Brazil. A detailed analysis confirmed that these isolates belong to a novel species within the genus Burkholderia. 3These authors contributed equally to this work. Abbreviations: ARDRA, amplified rDNA restriction analysis; MLEE, multilocus enzyme electrophoresis. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SRMrh-20 T , SRCL-18, SRMrh-85 and PPCR-2 are AY965240–AY965243, respectively. A dendrogram derived from MLEE and SDS-PAGE showing whole-cell protein profiles for the novel strains, N 2 -fixing Burkholderia species and B. sacchari are available as supplementary figures in IJSEM Online. 64362 G 2006 IUMS Printed in Great Britain 1931 International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1931–1937 DOI 10.1099/ijs.0.64362-0