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Bradyrhizobium Tropiciagri Sp. Nov. and Bradyrhizobium Embrapense Sp. Nov., Nitrogen- Fixing Symbionts of Tropical Forage Legume

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Bradyrhizobium Tropiciagri Sp. Nov. and Bradyrhizobium Embrapense Sp. Nov., Nitrogen- Fixing Symbionts of Tropical Forage Legume International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4424–4433 DOI 10.1099/ijsem.0.000592 Bradyrhizobium tropiciagri sp.nov.and Bradyrhizobium embrapense sp. nov., nitrogen- fixing symbionts of tropical forage legumes Jakeline Renata Marc¸on Delamuta,1,2 Renan Augusto Ribeiro,3 Ernesto Ormen˜o-Orrillo,4 Marcia Maria Parma,5 Itamar Soares Melo,5 Esperanza Martı´nez-Romero6 and Mariangela Hungria1,2,3 Correspondence 1Embrapa Soja, C.P. 231, 86001-970 Londrina, Parana´, Brazil Mariangela Hungria 2Universidade Estadual de Londrina, Department of Microbiology, C.P. 10.011, 86057-970 [email protected] Londrina, Parana´, Brazil or 3Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, SHIS QI 1 Conjunto B, [email protected] Blocos 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 5Embrapa Meio Ambiente, C.P. 69, 13820-000 Jaguariu´na, Sa˜o Paulo, Brazil 6Centro de Ciencias Geno´micas, Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Morelos, Mexico Biological nitrogen fixation is a key process for agricultural production and environmental sustainability, but there are comparatively few studies of symbionts of tropical pasture legumes, as well as few described species of the genus Bradyrhizobium, although it is the predominant rhizobial genus in the tropics. A detailed polyphasic study was conducted with two strains of the genus Bradyrhizobium used in commercial inoculants for tropical pastures in Brazil, CNPSo 1112T,isolated from perennial soybean (Neonotonia wightii), and CNPSo 2833T, from desmodium (Desmodium heterocarpon). Based on 16S-rRNA gene phylogeny, both strains were grouped in the Bradyrhizobium elkanii superclade, but were not clearly clustered with any known species. Multilocus sequence analysis of three (glnII, gyrB and recA) and five (plus atpD and dnaK) housekeeping genes confirmed that the strains are positioned in two distinct clades. Comparison with intergenic transcribed spacer sequences of type strains of described species of the genus Bradyrhizobium showed similarity lower than 93.1 %, and differences were confirmed by BOX-PCR analysis. Nucleotide identity of three housekeeping genes with type strains of described species ranged from 88.1 to 96.2 %. Average nucleotide identity of genome sequences showed values below the threshold for distinct species of the genus Bradyrhizobium (,90.6 %), and the value between the two strains was also below this threshold (91.2 %). Analysis of nifH and nodC gene sequences positioned the two strains in a clade distinct from other species of the genus Bradyrhizobium. Morphophysiological, genotypic and genomic data supported the description of two novel species in the genus Bradyrhizobium, Bradyrhizobium tropiciagri sp. nov. (type strain CNPSo 1112T5SMS 303T5BR 1009T5SEMIA 6148T5LMG 28867T)andBradyrhizobium embrapense sp.nov.(type strain CNPSo 2833T5CIAT 2372T5BR 2212T5SEMIA 6208T5U674T5LMG 2987). Biological nitrogen fixation, performed by prokaryotes, mostly bacteria, with the ability to convert atmospheric Abbreviations: ANI, average nucleotide identity; ITS, intergenic transcribed spacer; ML, maximum-likelihood; MLSA, multilocus nitrogen into ammonia and thereafter other nitrogen com- sequence analysis; NJ, neighbour-joining. pounds that can be assimilated by plants, stands out as a key process for agricultural production and environmental The GenBank/EMBL/DDBJ accession numbers for the nifH, dnaK and nodC gene sequences of B. embrapense CNPSo 2833T and the nodC sustainability (Hungria et al., 2005). The most effective gene sequence of B. tropiciagri CNPSo 1112T are KP234518, contribution occurs when the process is performed by bac- KP234519, KP234521 and KP234520, respectively. teria collectively known as rhizobia in symbiosis with legu- Four supplementary figures are available with the online Supplementary minous plants (Ormen˜o-Orrillo et al., 2013). Brazil is a Material. major producer of several grain legumes of economic Downloaded from www.microbiologyresearch.org by 4424 000592 G 2015 IUMS Printed in Great Britain IP: 189.172.145.110 On: Mon, 13 Mar 2017 19:55:49 Bradyrhizobium tropiciagri and B. embrapense spp. nov. Table 1. Strains used in this study Strain Other strain names Original Geographical Reference(s) host species origin B. tropiciagri sp. nov. SMS 303T, BR 1009T, SEMIA 6148T, Neonotonia wightii Brazil Delamuta et al. (2012) CNPSo 1112T LMG 28867T B. embrapense sp. nov. CIAT 2372T, BR 2212T, SEMIA 6208T, U674T Desmodium Colombia Delamuta et al. (2012); CNPSo 2833T LMG 29087 heterocarpon Menna et al. (2009a) B. elkanii USDA 76T ATCC 49852T, DSM 11554T, NBRC 14791T, Glycine max USA Kuykendall et al. (1992) LMG 6134T, CNPSo 62T B. pachyrhizi PAC 48T LMG 24246T, CECT 7396T, CNPSo 2077T Pachyrhizus erosus Costa Rica Ramı´rez-Bahena et al. (2009) B. jicamae PAC 68T LMG 24556T, CECT 7395T, CNPSo 2076T Pachyrhizus erosus Honduras Ramı´rez-Bahena et al. (2009) B. lablabi CCBAU 23086T LMG 25572T, HAMBI 3052T, CNPSo 2585T Lablab purpureus China Chang et al. (2011) B. retamae Ro19T LMG 27393T, CECT 8261T, CNPSo 2586T Retama monosperma Morocco Guerrouj et al. (2013) importance such as soybean [Glycine max (L.) Merr.], but for forage legumes, despite their key role in nitrogen others are variously employed as green manures, in for- cycling, helping to maintain soil fertility and contributing estry, and for pastures, among other uses, all contributing to animal nutrition. Due to the relevance of strains to the improvement of soil quality and fertility (Hungria CNPSo 1112T and CNPSo 2833T as commercial inoculants et al., 2005; Ormen˜o-Orrillo et al., 2013). Several efficient for tropical forages, we performed a polyphasic analysis to rhizobial strains for this broad range of applications have determine their taxonomic positions. been isolated from Brazilian soils and are available for Strains used in this study are listed in Table 1. Strains CNPSo use in commercial inoculants; the great majority belong T T 1112 and CNPSo 2833 are deposited at the Diazotrophic to the genus Bradyrhizobium (Binde et al., 2009; Menna and Plant Growth Promoting Bacteria Culture Collection et al., 2006, 2009a, b; Roma Neto et al., 2010). In the last of Embrapa Soja (WFCC collection # 1213 and WDCM decade, our research groups have reported several studies collection # 1054), located at Londrina, State of Parana´, showing high levels of genetic diversity among indigenous Brazil, and at the Center for Genomic Sciences Culture tropical rhizobia, including novel species and several Collection (Cuernavaca, Mexico), in addition to other inter- groups that may represent novel species (Binde et al., national collections. Unless otherwise indicated, strains were 2009; Dall’Agnol et al., 2013, 2014; Delamuta et al., 2012, grown on yeast extract-mannitol agar (YMA) medium at 2013; Germano et al., 2006; Menna et al., 2006, 2009a, b; 28 8C (Vincent, 1970). Stock cultures were maintained on Ribeiro et al., 2009, 2012; Roma Neto et al., 2010). YMA at 4 8C, while long-term preservation was performed The bradyrhizobial strains used in this study, CNPSo 1112T in 30 % glycerol at 280 and 2150 8C, or by lyophilization. T and CNPSo 2833 (Table 1), were identified as forming A BOX-PCR genomic fingerprint was generated as described independent branches in a previous multilocus sequence previously (Kaschuk et al., 2006), using the closest species analysis (MLSA) phylogeny (Delamuta et al., 2012; Menna based on the 16S rRNA gene phylogeny. One cluster included et al., 2009a). These strains are effective symbionts of tropi- T T T strains CNPSo 1112 and CNPSo 2833 and the type strains cal pasture legumes. Strain CNPSo 1112 was isolated from of Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi, perennial soybean [Neonotonia wightii (Wight & Arn.) J. A. with a similarity level of 81 % (Fig. S1, available in the Lackey; formerly classified as Glycine wightii ] by researchers online Supplementary Material). Another cluster including of the Instituto Agronoˆmico de Campinas (Collection SMS, the type strains of Bradyrhizobium jicamae and Sec¸a˜o de Microbiologia do Solo), State of Sa˜o Paulo, Brazil, Bradyrhizobium lablabi joined at 84 % similarity. These and has been used in commercial inoculants for this legume two clusters were 75.5 % similar among each other. The since 1994. Neonotonia wightii is a perennial forage from type strain of Bradyrhizobium retamae occupied an isolated Africa that grows well in several tropical countries including T position in relation to the other species, with a similarity Brazil. Strain CNPSo 2833 was isolated from Desmodium level of 73.2 % (Fig. S1). Although BOX-PCR is suitable heterocarpon (L.) DC. subsp. ovalifolium (Prain) for revealing prokaryotic diversity, the results show that, H. Ohashi (former Desmodium ovalifolium Merr.) by within the B. elkanii superclade, the diversity is apparently researchers of the International Center for Tropical Agricul- lower than in other rhizobial superclades, especially those ture (CIAT), Colombia, and it has been used in commercial of fast growers, as observed previously (Menna et al., 2009b). inoculants for this legume in Brazil since 1988. The use of commercial rhizobial inoculants should be emphasized, For the 16S rRNA gene analyses, sequences were retrieved but globally is often concentrated in a small number of from the GenBank database and accession numbers are legumes (Hungria et al., 2005),
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