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International Journal of Systematic Evolutionary Microbiology International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4441–4448 DOI 10.1099/ijsem.0.000591 Bradyrhizobium viridifuturi sp. nov., encompassing nitrogen-fixing symbionts of legumes used for green manure and environmental services Luisa Caroline Ferraz Helene,1,2 Jakeline Renata Marc¸on Delamuta,1,2 Renan Augusto Ribeiro,3 Ernesto Ormen˜o-Orrillo,4 Marco Antonio Rogel,5 Esperanza Martı´nez-Romero5 and Mariangela Hungria1,2,3 Correspondence 1Embrapa Soja, C.P. 231, 86001-970, Londrina, Parana´, Brazil Mariangela Hungria 2Universidade Estadual de Londrina, Dept. of Microbiology, C.P. 10.011, 86057-970, Londrina, [email protected]; Parana´, Brazil [email protected]; 3Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, SHIS QI 1 Conjunto B – Blocos [email protected] A, B, C e D, Lago Sul, 71605-001, Brası´lia, Distrito Federal, Brazil 4Universidad Nacional Agraria La Molina, Av. La Molina s/n La Molina, Lima, Peru 5Centro de Ciencias Geno´micas, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos, Mexico Symbiotic nitrogen-fixing bacteria, commonly called rhizobia, are agronomically important because they can provide significant amounts of nitrogen to plants and help in recovery of impoverished soils and improvement of degraded environments. In recent years, with advances in molecular techniques, several studies have shown that these bacteria have high levels of genetic diversity, resulting in taxonomic reclassifications and descriptions of new species. However, despite the advances achieved, highly conserved 16S ribosomal genes (16S rRNA) do not elucidate differences between species of several genera, including the genus Bradyrhizobium. Other methodologies, such as multilocus sequence analysis (MLSA), have been used in such cases, with good results. In this study, three strains (SEMIAs 690T, 6387 and 6428) of the genus Bradyrhizobium, isolated from nitrogen-fixing nodules of Centrosema and Acacia species, without clear taxonomic positions, were studied. These strains differed from genetically closely related species according to the results of MLSA of four housekeeping genes (dnaK, glnII, gyrB and recA) and nucleotide identities of the concatenated genes with those of related species ranged from 87.8 % to 95.7 %, being highest with Bradyrhizobium elkanii. DNA–DNA hybridization (less than 32 % DNA relatedness) and average nucleotide identity values of the whole genomes (less than 90.5 %) indicated that these strains represented a novel species, and phenotypic traits were determined. Our data supported the description of the SEMIA strains as Bradyrhizobium viridifuturi sp. nov., and SEMIA 690T (5CNPSo 991T5C 100aT5BR 1804T5LMG 28866T), isolated from Centrosema pubescens, was chosen as type strain. Biological nitrogen fixation has been recognized for over Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA 130 years as a key process for environmental sustainability, hybridization; MLSA, multilocus sequence analysis. but lately it has become the subject of increased interest, The GenBank/EMBL/DDBJ accession numbers for the dnaK gene with an emphasis on the symbioses with legumes, as a T sequences of B. viridifuturi SEMIA 690 , SEMIA 6387 and SEMIA means of increasing nutrient and energy balances in agri- 6428 are KR149128–KR149130; those for the glnII sequences of culture, in environmentally beneficial reforestation efforts SEMIA 690T, SEMIA 6387 and SEMIA 6428 are KR149131– KR149133; those for the gyrB sequences of SEMIA 690T, SEMIA and in the mitigation of greenhouse-gas emissions (e.g. 6387 and SEMIA 6428 are KR149134–KR149136; those for the Hungria et al., 2005, 2013; Ormen˜o-Orrillo et al., 2013). recA sequences of SEMIA 690T, SEMIA 6387 and SEMIA 6428 are However, although our knowledge of the rhizobia– KR149140–KR149142; those for the nifH sequences of SEMIA 690T, legume symbioses is rapidly increasing, driven largely by SEMIA 6387 and SEMIA 6428 are KR149137–KR149139. ‘omics’ studies, we are still far from fully understanding Three supplementary tables and six supplementary figures are available this biological process, which has evolved over millions with the online Supplementary Material. of years. A good example is our still poor knowledge of Downloaded from www.microbiologyresearch.org by 000591 G 2015 IUMS Printed in Great Britain 4441 IP: 189.172.145.110 On: Mon, 13 Mar 2017 19:59:35 L. C. F. Helene and others the phylogeny and taxonomy of the genus Bradyrhizobium, & Sokal, 1973) algorithm and the Jaccard coefficient considered to be the ancestor of all rhizobia (Norris, 1965; (Jaccard, 1912) with 3 % tolerance. The three strains had Lloret & Martı´nez-Romero, 2005; Germano et al., 2006; similarities higher than 72 % among themselves and less Menna et al., 2006; Binde et al., 2009; Menna & Hungria, than 66 % with closely related species (Fig. S1, available 2011; Delamuta et al., 2013). One limitation in defining in the online Supplementary Material). species within the genus Bradyrhizobium is that 16S Phylogenetic trees were reconstructed with 16S rRNA gene rRNA genes are highly conserved. On the other hand, the sequences obtained from the GenBank database (accession multilocus sequence analysis (MLSA) technique, applied numbers are given on the trees and in Supplementary Table to concatenated housekeeping genes, has greatly clarified phylogenetic relationships and elucidated novel species S1) of the strains from this study and 29 species of the within the genus (e.g. Menna et al., 2009; Delamuta et al., genus Bradyrhizobium. Xanthobacter autotrophicus Py2 2013; Dura´n et al., 2014a, b; Parker & Rousteau, 2014). was used as outgroup. The MEGA 6.0 program (Tamura New insights into the evolution of the symbiosis with et al., 2013) was used to generate the alignments and Bradyrhizobium have also been obtained by the analysis of phylogenies, using maximum-likelihood (ML) (Felsenstein, nodulation and nitrogen-fixation genes (Menna & Hungria, 1981) and neighbour-joining (NJ) (Saitou & Nei, 1987) 2011; Parker & Rousteau, 2014; Zhang et al., 2014). algorithms and Tamura–Nei distance (Tamura & Nei, 1993). Statistical support for the trees was assessed by boot- Leguminous species used as green manure, for reforesta- strapping (Felsenstein, 1985) with 1000 replicates. NJ and tion and for remediation of degraded areas are key for ML reconstructions gave similar results; therefore, only sustainability, considering that the global loss of fertile the ML phylogram is presented. The SEMIA strains were soil has been estimated at 24 billion tonnes per year, positioned in the B. elkanii clade, and their closest neigh- adversely affecting 1.5 billion people (United Nations, T bours were Bradyrhizobium neotropicale, Bradyrhizobium 2015). The three strains (SEMIA 690 , SEMIA 6387 and jicamae and Bradyrhizobium erythrophlei (Fig. 1). SEMIA 6428) used in this study were isolated from different sites in Brazil from legumes used for those three purposes, The MLSA approach using housekeeping concatenated Centrosema pubescens, Acacia auriculiformis and Acacia genes has been successfully used to define many groups saligna, respectively. SEMIA 690T was isolated at the Insti- of the genus Bradyrhizobium (Menna et al., 2009; Rivas tuto de Pesquisas e Experimentac¸a˜oAgropecua´ria do et al., 2009; Chang et al., 2011; Delamuta et al., 2012, Centro-Sul (IPEACS), Rio de Janeiro, Brazil, and SEMIA 2013; Dura´n et al., 2014a, b). MLSA phylograms were 6387 and SEMIA 6428 by Dr Sergio M. Faria at the Embrapa reconstructed as described for the 16S rRNA gene with Agrobiologia, Serope´dica, Rio de Janeiro, Brazil. The strains four housekeeping genes (dnaK, glnII, gyrB and recA, acces- are recognized as the most effective for fixing nitrogen with sion numbers in Supplementary Table S1) and it clearly the legumes from which they were isolated; they have been separated the three strains from this study into a single authorized for inclusion in commercial inoculants for their and consistent group with 100 % bootstrap support, iso- respective host legumes by the Ministry of Agriculture in lated from all described species of the genus Bradyrhizo- Brazil since 1994 (MAPA, 2011). bium (Fig. 2). Their closest neighbours were B. elkanii The strains of members of the genus Bradyrhizobium used in and Bradyrhizobium pachyrhizi. Each individual phyloge- this study have been deposited at the Diazotrophic and Plant netic tree reconstructed with each of the four housekeeping Growth Promoting Bacteria Culture Collection of Embrapa genes supported the distinctiveness between the cluster of Soja (WFCC Collection # 1213, WDCM Collection # 1054), SEMIA strains and the other species of the genus Bradyrhi- located at Londrina (State of Parana´, Brazil) and at the zobium. In all four trees, the strains were clustered with Center for Genomic Sciences Culture Collection (Cuernavaca, high bootstrap support and the most closely related species Mexico). They are also deposited at the Fundac¸a˜oEstadualde were B. pachyrhizi, Bradyrhizobium ferriligni and B. elkanii Pesquisa Agropecua´ria (FEPAGRO; WDCM # 443, Porto (Figs S2–S5). The three reconstructed with three genes + + Alegre, Rio Grande do Sul), Embrapa Agrobiologia (WDCM (glnII gyrB recA) also supported the proposal of the # 364, Serope´dica, Rio de Janeiro) Culture Collections. novel species (Fig. S6). Unless otherwise indicated, strains were grown on yeast Nucleotide identities of every analysed gene and of conca- extract–mannitol agar (YMA) medium at 28 8C(Vincent,
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