Systematic and Applied Microbiology 29 (2006) 315–332

ARTICLE IN PRESS

Systematic and Applied Microbiology 29 (2006) 315–332 www.elsevier.de/syapm

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants Paˆmela Mennaa,b,c, Mariangela Hungriaa,d,Ã, Fernando G. Barcellosa,d, Eliane V. Bangele, Pablo N. Hessf,g, Esperanza Martıńez-Romeroh aEmbrapa Soja, Cx. Postal 231, 86001-970, Londrina, PR, Brazil bDept. Microbiology, Universidade Estadual de Londrina cCAPES, Cx. Postal 365, 70359-970, Brasilia, DF, Brazil dCNPq, SEPN 509, Bloco ‘‘A’’, Ed. Nazirl, 70. 750-501, Brasilia, DF, Brazil eFEPAGRO, Rua Gonçalves Dias 579, 90130-060, Porto Alegre, RS, Brazil fLaborato´rio Nacional de Computaçaõ Cientı´fica, Rua Getu´lio Vargas 333, CEP 25651-071, Petro´polis, RJ, Brazil gDept. Genetics, Universidade Federal do Rio de Janeiro hCentro de Ciencias Geno´micas, UNAM, Ave. Universidad s/n, Col. Chamilpa, Cuernavaca, Morelos, Mexico

Received 7 November 2005

Abstract

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes. r 2006 Elsevier GmbH. All rights reserved.

Keywords: Azorhizobium; Biological nitrogen ﬁxation; Bradyrhizobium; Burkholderia; Methylobacterium; Rhizobium; Sinorhizobium; 16S rRNA gene

ÃCorresponding author. Embrapa Soja, Cx. Postal 231, 86001-970, Londrina, PR, Brazil. Tel.: +55 43 33716206; fax: +55 43 33716100. E-mail address: [email protected] (M. Hungria).

Introduction Rhizobium Collections no. 443 in the WFCC World Data Center on Microorganisms), at the Fundaçaõ Divergence within the family Leguminosae (Fabaceae Estadual de Pesquisa Agropecua´ria [11]. in the USA) is estimated to have occurred over 65 Searching for the most effective rhizobial strains is a million years ago, such that over 18,000 species are now time-consuming process involving the production of classified into around 650 genera, occupying nearly all thousands of rhizobial cultures, and many greenhouse terrestrial biomes [18,34]. The wide use of legumes as experiments and field trials. In the case of soybean food crops, forages and green manures is mainly (Glycine max) alone, the benefits resulting from the use associated with their ability to establish symbiotic of inoculants with selected superior strains is equivalent associations with stem and root-nodulating N2-fixing to about US$ 3 billion per cropping season, that bacteria, collectively called rhizobia [2]. These bacteria otherwise would be spent on the purchase, transporta- are among the most intensely studied groups of tion and application of N-fertilizers [19]. However, microorganisms [38] mainly due to their potential to despite the considerable effort expended in selecting replace N-fertilizers, with emphasis on their key role in effective strains for almost a hundred legumes, their achieving sustainability of tropical N-poor soils. genetic characterization and taxonomic position is still Nodules generally occur in the subfamilies Mimosoi- poor. In this context, this study aimed at gaining better deae and Papilionoideae, and are rare in the Caesalpi- understanding of phylogenetic relationships, based on nioideae; recent information indicates that about 3000 the sequencing of the whole 16S rRNA gene, of 68 elite taxa are capable of nodulating and 400 taxa are not; rhizobial strains recommended for 64 legumes in Brazil. however, information is lacking for nearly 40% of the genera [21]. Until the early 1980s, all bacteria isolated from root nodules were classified into the genus Material and methods Rhizobium, and speciation was based on nodulation with certain host plants, establishing the ‘‘cross-inocula- Strains used tion group’’ concept [13]. After that, numerical taxonomy considering several properties led to the definition Sixty-eight strains from the Brazilian (SEMIA) of a new genus, Bradyrhizobium, and the renaming of culture collection of rhizobia were selected. Table 1 some other species [22]. Particularly in the last decade, provides information of the strains, as well as of the host ribosomal sequences with emphasis on the 16S rDNA plants from which they were isolated and for which they have become the basis of bacterial molecular phylogeny are recommended. In total the strains represent 94 and taxonomy [14] leading to the description of new recommendations for commercial inoculants. Strains rhizobial genera and species. Currently, rhizobia are were provided by FEPAGRO and their purity was positioned in four deep branches, Azorhizobium, Bra- verified on yeast extract-mannitol agar (YMA) medium dyrhizobium, Mesorhizobium, and Rhizobium-Sinorhizo- [45] containing Congo red (0.00125%). Stocks were bium-Allorhizobium, and non-symbiotic relatives within prepared on YMA and kept at 70 1C (under 30% of those branches may indicate common ancestry for glycerol) for long-term storage and at 4 1C as source rhizobial species and other parasitic or soil-borne cultures. bacteria [14,48]. However, in comparison to the number of nodulating-legume species, very few rhizobial species have been described. Morpho-physiological characterization In Brazil, economical and agronomic benefits have been achieved with several legumes due to research Colony morphology (color, mucosity, diameter, transparency, borders, elevation) and acid/alkaline efforts focused on N2 fixation. Since 1975, the government demands that inoculants commercialized in the reaction were evaluated as described previously [45] country contain only strains recommended by Brazilian after 7 days of growth in the dark, at 28 1C, on YMA public research institutions. To enforce the recommen- containing either Congo red or bromothymol blue dation, the RELARE ( ¼ Rede de Laborato´rios para a (0.00125%) as a pH-change indicator. Recomendaçaõ de Estirpes de Rhizobium) was created in 1985, by the initiative of the Microbial Resources Centre DNA extraction Network (MIRCEN), establishing a net of laboratories with the objective of identifying the most effective Total genomic DNA of each strain was extracted rhizobial strains for each legume species. Since then, the from bacterial batch cultures grown in YM broth until maintenance of the strains and their distribution to the late exponential phase (109 cells mL1). Extraction of inoculant industry has been a responsibility of the DNA was performed as described before [12] and to ‘‘Rhizobium Culture Collection SEMIA’’ (Seçaõde obtain clean DNA samples the extraction procedure Microbiologia Agrıćola) (IBP World Catalogue of included the addition, for each 400 mL of bacteria ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 317 g II II IV II IV IV II II III IV IV III IV Basis e f IAC IV Agrobiologia Agrobiologia Agrobiologia Agrobiologia Embrapa Agrobiologia Agrobiologia Embrapa Agrobiologia Agrobiologia Embrapa Agrobiologia Agrobiologia Cerrados Agrobiologia Embrapa Agrobiologia Institution recommendding d,e 3187, MAR 11 Other designa- tions 9961 9960 9965 6144 SMS 400, USDA 6163 BR 3607 Embrapa 6156 CPAC-IJ, DF F-26157 Embrapa Cerrados6152 IV BR 28016156 BR 1602 CPAC-IJ, DF F-2 Embrapa Embrapa6158 Cerrados Embrapa III CPAC 42 Embrapa Cerrados III 6164 BR 3608 Embrapa SEMIA number 6391 BR 36246390 BR 3614 Embrapa Embrapa 6160 BR 5610 6420 BR 3617, LMG 64246425 CPAC L36 CIAT 2380 Embrapa Cerrados Embrapa Cerrados IV IV c environment wood food, medicine, wood manure, forage manure, forage, food Environment, chemical, toxins, wood Grain Food, medicine, forage, Tree TreeTree Environment, wood 6387Tree Environment, wood BR 3609, LMG 6164Tree Environment, wood BR 3608, LMG 6387 Environment, chemical, BR 3609 LMG 9961 Embrapa Forage (tropical), Forage, environmentForage (tropical) 6440 Forage environment, MGAP 13Green manure EPAMIG/Embrapa Green Cover manure crop, green Cover crop, green Forage (tropical) Forage, environment 6146 BR 1808 Embrapa Main use in Brazil Applications worldwide c herb Descriptor climbing tree climbing tree climbing tree climbing tree climbing shrub or tree climbing herb non-climbing herb or shrub non-climbing herb climbing or non- climbing herb or shrub non-climbing herb Perennial non- b Aeschynomeneae Annual non-climbing Tribe b Subfamily Mimosoideae AcacieaeMimosoideae Perennial non- AcacieaeMimosoideae Perennial non- AcacieaeMimosoideae Perennial non- AcacieaeMimosoideae Perennial non- Ingeae Papilionoideae Papilionoideae Aeschynomeneae Perennial non- Papilionoideae Phaseoleae Annual/perennial, Papilionoideae PhaseoleaePapilionoideae Phaseoleae Perennial climbing or Annual/perennial, Papilionoideae Phaseoleae Perennial climbing or h h , h h cia , ´ nio cia h ´ cia ´ h h h h lia ´ h , pau preto h h o de negro c h ˜ h o de porco ˜ cia ´ Forage peanut, amendoim forrageiro languette, pois piante, choncho, conchitas, centrosema Calopo, calopogoˆ Jackbean, swordbean, horsebean, pois de sabre, dolique, cocorico, haba criolla, feija Rain tree, woman’s tongue, bois noir, baile de caballero, lengua de mujer, coraça Green wattle, aca da Austra Mangium, silk leaf acacia, akasia, aca Peanut, groundnut, cacahoute, amendoim Pigeonpea, Congo pea, pois cajan, frijol de arbol, lenteja, guandu Black wattle, acacia negra Earpod wattle, blackwattle, aca faveiro aca Some common names mangium (L.) Information about the strains recommended for the use in Brazilian commercial inoculants and which DNA was sequenced in this study a,b spp. Centrosema, fleur (L.) Arachis pintoi Krapov. and W. Gregory Centrosema Calopogonium spp. Canavalia ensiformis DC. Albizia lebbeck (L.) Benth. Acacia decurrens Willd. Acacia mangium Willd. Arachis hypogaea L. Cajanus cajan Millsp. Acacia mearnsii De Wild. Table 1. Plant species Acacia auriculiformis Benth. ARTICLE IN PRESS 318 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 g IV II II II III Basis e f Agrobiologia Embrapa Agrobiologia Agrobiologia Agrobiologia Agrobiologia FEPAGRO/UFRGS IV Institution recommendding d,e I28 Other designa- tions 3100, Nit 27A3 64126053 BR 8005 TAL 827, UMKL Embrapa 6145 BR 20016156 CPAC-IJ, DF F-2 Embrapa 6156 Embrapa Cerrados IV CPAC-IJ, DF F-26158 Embrapa6145 Cerrados IV CPAC 426319 BR 20016101 NC 92, Embrapa SMS Cerrados 561 BR 8404 Embrapa 6028 IV IAC TAL 569, MAR Embrapa 472 FEPAGRO/UFRGS II II SEMIA number c wood chemical, wood, forage, toxins firewood firewood firewood, toxins food, forage wood medicine GrainTree Food, forage, medicineTree 396 Environment, medicine, TAL 1148, USDA Environment, food, Green manure Fibre, environment, Green manure Fibre, environment, Green manure Fibre, environment, Green manure Environment, chemical, TreeForage (tropical) Environment, Environment, chemical, forage, Forage (tropical) Environment, forage 656Forage (tropical) Environment, forage SEMIA original 6208Forage FEPAGRO/UFRGS (tropical) II Environment, forage CIAT 2372 696 CEPEC CB 627 III FEPAGRO/UFRGS III Main use in Brazil Applications worldwide c Descriptor herb climbing tree herb or shrub herb herb herb or shrub herb climbing tree climbing herb or shrub climbing herb or shrub climbing herb or shrub climbing herb or shrub b Tribe b Subfamily Papilionoideae CicereaePapilionoideae Phaseoleae Annual non-climbing Papilionoideae Perennial Phaseoleae non- Perennial climbing Papilionoideae CrotalarieaePapilionoideae Annual non-climbing CrotalarieaePapilionoideae Annual non-climbing Phaseoleae Annual non-climbing Papilionoideae IndigofereaePapilionoideae Annual non-climbing DalbergieaePapilionoideae Perennial non- Desmodieae Perennial non- Papilionoideae DesmodieaePapilionoideae Perennial non- Desmodieae Perennial non- Papilionoideae Desmodieae Perennial non- Mimosoideae Ingeae Tree 6159 BR 4406 III h o h ˜ - ´ h h h h ode h h ˜ dio dio ´ ´ , h h , sombreiro h , jacaranda h ,guar h h dio dio c ´ ´ h Pigeon wings, pois sauvage, blue pea, azulejo, campanilla, conchita blanca o azul, zapatico de la reina, clitoria Sunnhemp, cascavelle, grand sonnette, grand tcha- tcha, crotalaria Sunnhemp, cascavelle, grand sonnette, grand tcha- tcha, crotalaria Spanish clover; wild peanut, colle-colle, herbe gallon, cepa de caballo, collant, pega pega; desmo Butterfly pea tree, faveira, palheteira, palheiro Desmodium ovalifolium desmodium, desmo Silverleaf desmodium, desmo Rattle Box, showy rattlepod, cascavelle jaune, sonnette, maraquita, crotalaria Clusterbean, guar bean, guar gum, feija guarda Brazilian rosewood, jacaranda, palisandre, cabiuńa Beggarlice, copal de coche, pega pega, zarza blanca, amor seco, desmo garbanzo, gra bico Some common names da-Bahia ) L. Chickpea, pois chiche, (L.) (L.) (D. R. a,b (Jacq.) Roth (Miller) DC. Clitoria ternatea L. Crotalaria juncea L. Crotalaria juncea L. Desmodium incanum canum) Clitoria fairchildiana Howard Desmodium heterocarpon DC. subsp. Ovalifolium (Prain) Ohashi Desmodium uncinatum DC. Taubert Crotalaria spectabilis Cyamopsis tetragonoloba Dalbergia nigra (Vell. Conc.) Benth. Desmodium intortum Urban Table 1. ( continued Plant species Cicer arietinum ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 319 III III IV III IV IV II IV Embrapa FEPAGRO/UFRGS II Agrobiologia Embrapa Agrobiologia Embrapa Agrobiologia Embrapa Agrobiologia Embrapa Agrobiologia Embrapa Agrobiologia Agrobiologia Agrobiologia Embrapa Agrobiologia 422, NA 630 10132 10132 6150 SMS 300 IAC501950795080 29W, BR 29 CPAC 15, DF 24 CPAC 7 II Embrapa Cerrados FEPAGRO/UFRGS IV IV Embrapa Cerrados IV 6169 BR 5612 Embrapa 6169 BR 5612 61566158 CPAC-IJ, DF F-2662 Embrapa Cerrados CPAC 42 III 695 CB 188 Embrapa QA Cerrados 922, E 85, SU III FEPAGRO/UFRGS II 6159 BR 4406 6159 BR 4460 Environment, wood, food 6100 BR 5609 medicine environment, food, medicine, toxins Environment, medicine, pasture, wood, food Environment,medicine, toxin, wood Environment, medicine, toxin, wood Tree Environment, food 6069 DF 10, BR 414 Embrapa Cerrados IV Tree Tree TreeTree Environment, wood 6100Forage (tropical) Forage, environment BR 5609 6149Tree Embrapa CB 627, SMS 138 IACGrain Environment, wood 6168 Food, medicineForage II (tropical) BR Forage, 8801, environment, LMG 587Forage (tropical) Forage, chemical, BR 96Tree FEPAGRO/UFRGS IV Environment, food 6168 BR 8801, LMG climbing shrub, tree climbing tree climbing tree climbing tree climbing tree herb climbing tree non-climbing herb non-climbing herb climbing or non- climbing herb climbing shrub, tree Perennial non- climbing tree Perennial non- Perennial non- Perennial non- Mimosoideae Mimoseae Perennial non- Mimosoideae Ingeae MimosoideaePapilionoideae Ingeae PhaseoleaeMimosoideae Perennial, non- Ingeae Papilionoideae Phaseoleae Perennial climbing Papilionoideae RobinieaePapilionoideae Perennial non- Phaseoleae Annual climbing or Papilionoideae IndigofereaePapilionoideae Annual/perennial, Phaseoleae Annual/pere-nnial, Mimosoideae Mimoseae Perennial non- h h ; h , , ´ va ´ va h h , , , , h h h ˜ h h h h , xia ´ h ´ h , timbau , timbou h , suina h h h h , amendoim h h ba , anileira h fera da serra h ´ ˜ phant, ctia ´ h ´ le ´ guanacaste, orelha- de-elefante Wild tamarind, guash, guache, guaje, guaje blanco, leucena Jumbie bean, lead tree, leucaena, cowbush, bois bourro, Galactia, guatabito, frijolillo, gala Grow stick, cacahuananche, madricacao, gliricidia Soybean, soya bean, Manchurian bean, soja Hairy indigo, anil bravo Mulungu, cristi galli, mulungu Albizia falcataria, molucca albizia, batai wood, sau, malesia, albizia Black bean antaque, pois antaque, bonavist, lablab Devil’s ear, earpod tree, elephant’s ear, monkey ear, bois tanniste rouge, oreille d’e Earpod tree, oreja de negro, timbo suina de veado tamboril do campo orelha de negro indigo timbau´va Timbor, tamboril timbau´ gala orelha de macaco (L.) (Miq.) Martius Leucaena diversifolia (Schldl.) Benth. Leucaena leucocephala Galactia striata (Jacq.) Urban Gliricidia sepium (Jacq.) Walp. Glycine max Indigofera hirsuta L. Merr. Enterolobium timbouva Erythrina verna Vell. Conc. Falcataria moluccana Barneby and Grimes Lablab purpureus (L.) Sweet Enterolobium cyclocarpum (Jacq.) Griseb. Enterolobium contortisiliquum (Vell. Conc.) Morong ARTICLE IN PRESS 320 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 g IV II III III III II Basis e f Agrobiologia Agrobiologia Embrapa Cerrados IV Agrobiologia Agrobiologia Institution recommendding Embrapa Agrobiologia Embrapa Agrobiologia i i d,e Other designa- tions BR 3446 BR 3446 i i 6382 BR 3405 Embrapa 938 Hansen inoculant656 FEPAGRO/UFRGS III SEMIA original FEPAGRO/UFRGS103 II SEMIA original134 FEPAGRO/UFRGS II 135 SEMIA original6384 SEMIA FEPAGRO/UFRGS original IV FEPAGRO/UFRGS IV 6167 BR 3452 Embrapa SEMIA number 6070 DF 15 c Forage, environment 816Forage, environment 830 SEMIA originalgreen manure, medicine, FEPAGRO/UFRGStoxin, food IV Hansen inoculant FEPAGRO/UFRGS II medicine, food Forage, environment, medicine, food Forage, environment, medicine, food Forage (tropical) ForageForage (subtropical) Forage (subtropical) 658Green manure Forage, environment, CB 376Forage (tropical) Forage, environment, Forage (tropical) FEPAGRO/UFRGS Forage, III environmentForage 6149(subtropical) CB 627, SMS 138Forage IAC(subtropical) TreeTree II Environment, wood 6383 Environment, wood BR 3407 6166 Tree Embrapa Environment, wood 6165 BR 3454 Embrapa Main use in Brazil Applications worldwide c herb climbing herb, shrub climbing herb, shrub herb non- climbing, herb non-climbing herb herb climbing herb climbing shrub or tree climbing shrub or tree climbing shrub or tree Descriptor Perennial non- Perennial non- b Tribe b Subfamily Papilionoideae CrotalarieaePapilionoideae Loteae Annual non- climbing Papilionoideae Loteae Papilionoideae CytiseaePapilionoideae Annual non- climbing PhaseoleaePapilionoideae Perennial Phaseoleae climbing or Papilionoideae Perennial Trifolieae climbing or Papilionoideae Annual non-climbing TrifolieaeMimosoideae Perennial Mimoseae non- Mimosoideae Perennial non- Mimoseae Perennial non- Mimosoideae Mimoseae Perennial non- o ˜ 0 h h h o o ˜ ˜ h h foot , sansa h 0 , narrow h ´ h h h h , unha de h h h ¸ o c , espinheiro , sabia h h Abaracaatinga, bracaatinga, paracaatinga, bracatinga Miles lotononis, lotononis Bur medic, purple medick, California burclover, luzerne, clover, trevo carretilha Cat’s clover, birds Lotus tenuis trefoil, birds Egyptian lupine, chochos, lupino blanco, tremozo, tremoc Macrotyloma, perennial horse, macrotiloma Purple bean conchito, siratro Alfalfa, lucerne, luzerne, alfafa Mimosa, jupinuan, mimosa cassie blanc, graines de lin, granadillo bobo, leucena trefoil, cornicha do campo gata Some common names foot trefoil, broadleaf trefoil, cornicha Mimosa, jurema preta ) L. White lupine, a,b L. Benth. (E. (L. tenuis Mimosa scabrella Benth. Lotononis bainesii Baker Lotus corniculatus L. Lotus glaber Miller Willd.) Lupinus albus Medicago polymorpha Meyer) Verdc. Macroptilium atropurpureum (DC.) Urban Macrotyloma axillare Medicago sativa L. Mimosa acutistipula Mimosa caesalpiniifolia Benth. Plant species Table 1. ( continued (Lam.) De Wit v. Cunningham ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 321 II II II II IV II III FEPAGRO/UFRGS IV Agrobiologia FEPAGRO/UFRGS II UFC III Embrapa Agrobiologia FEPAGRO/UFRGS IV FEPAGRO/UFRGS IV Agrobiologia UFC II Agrobiologia Agrobiologia Agrobiologia Agrobiologia 422, NA 630 832.55 9993 14 14 832.59 3007 B 11A 6402 BR 5404 LMG 9994 Embrapa 6148 SMS 303 IAC II 2081 EEL 1285 EPAGRI IV 656695 SEMIA original FEPAGRO/UFRGS III E85, QA 922, SU 6398 BR 4812, UFC 6175 DF Q-16420 Embrapa Cerrados III 6401 BR 3617 LMG 9965 Embrapa BR 5401, LMG 6158 CPAC 42 Embrapa Cerrados6192 IV 222 SEMIA original FEPAGRO/UFRGS II SU 329,222 TA-1, NA 222 SU 329, TA-1, NA TA 1, CB 1165 FEPAGRO/UFRGS IV food chemical Food, forage environment forage, medicine manure, forage chemical, forage Forage, environment, medicine Forage, environment, medicine Forage, environment, medicine Forage (tropical) Forage, environment, TreeTree Environment, woodTree Wood, environment, 6394Grain BR Wood, 4103 environmentTree 6398 Embrapa Green manure BR Environment, 4812 wood UFC Green manure, forage, 6161Tree BR 4002, PRJ B4 Embrapa Environment, woodTree 6160Green manure BR Environment, 5610 wood, Environment, green Embrapa Forage (tropical), Forage, environmentTree 6155Forage BR 502(subtropical) Environment, wood, Forage (subtropical) Embrapa Forage (subtropical) herb climbing shrub or tree climbing tree climbing tree herb climbing, tree, shrub herb climbing tree climbing shrub, tree climbing, shrub climbing herb or shrub climbing tree climbing, herb climbing herb climbing, herb Annual, climbing, Papilionoideae Phaseoleae Perennial climbing Papilionoideae SophoreaeMimosoideae Mimoseae Perennial non- Mimosoideae Perennial non- MimoseaePapilionoideae Vicieae Perennial non- Mimosoideae MimoseaePapilionoideae Perennial non- PhaseoleaeCaesalpinioideae Perennial climbing Caesalpinieae Perennial non- Papilionoideae RobinieaePapilionoideae Perennial non- Phaseoleae Perennial non- Papilionoideae Aeschynomeneae Perennial non- Papilionoideae DalbergieaePapilionoideae Perennial non- TrifolieaePapilionoideae Perennial non- TrifolieaePapilionoideae Trifolieae Perennial non- Annual non- , h h h h , ´ h h h h , , , h h neo h h , tipu ´ h h h , tento , carvoeiro h h ba h ´ h , ajusta ,anga h h , angico , caniveteiro h h ´ h h h h ´ ´ o-mucuna ˜ Velvet-, Bengal bean, Portuguese coffee, black mu-cuna, cafe Brazilii, pois mascate, feija Piptadenia, jurema branca Mesquite, cashaw, bayarone, epinard bayahonda blanca, algaroba Acacia, cibil, cumand, sesbania alverja, guisante, ervilha Subclover, subterranean clover, trevo subterraˆ Ormosia Dutch, ladino, whiteclover, trebol blanco, trevo branco Piptadenia, pau jacare Tropical kudzu, grande feuille, puero, cudzu tropical lucerne, stylosanthes, estilosantes Tipa, palo mortero, tipa blanca, tipuana Red clover, trebol de los prados, trebol rojo, trevo vermelho arapaçu Glycine, perennial soybean, soja perene macanaı contas mucuna-preta jacare kudzu Velame, veludo, taxi- branco icarape tipa- branca L. Field pea, petit pois, L. spp. Stylo, Brazilian Vogel Piper (Benth.) Stizolobium aterrimum and Tracy Piptadenia stipulacea Ducke Trifolium subterraneum Prosopis juliflora (Sw.) DC. Sesbania virgata (Cav.) Pers. Pisum sativum Ormosia nitida Vogel Trifolium repens L. Piptadenia gonoacantha (Martius) Macbr. Pueraria phaseoloides (Roxb.) Benth. Sclerolobium paniculatum Stylosanthes Tipuana tipu (Benth.) Kuntze Trifolium pratense L. Neonotonia wightii (Wight and Arn.) Lackey ARTICLE IN PRESS 322 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 ; o ˜ g www. . ; ); UFC IV Basis ´ e a, Agricultura y ´ f Brookfield, USA); PRF , FEPAGRO/UFRGS III Agrobiologia Institution recommendding www.hear.org/pier/species/ ; www.rain-tree.com/plants.htm Collection, Harpenden, UK); SEMIA ; iology, Kuala Lumpur, Malasya); UMR d,e A); Nit (Nitragin, Inc. RCR 3824 Other designa- tions Rhizobium www.arboretos.cnpm.embrapa.br/faz_sm/especies.html ; dica, Brazil); CB (Commonwealth Scientific and Industrial ´ 60026145 CB 756, TAL 309, BR 2001 Embrapa 2051384 SEMIA original FEPAGRO/UFRGS II SEMIA original FEPAGRO/UFRGS II SEMIA number c Forage, environment, medicine Forage, environment, medicine ria dos Cerrados. Embrapa Cerrados, Planaltina, Brazil); NA (New South Wales Dept. ´ ria de Minas Gerais); IAC (Instituto Agronoˆmico de Campinas); FEPAGRO (Fundaça www.fao.org/ag/agp/agpc/doc/Gbase/latinsearch.htm ´ www.newcrops.uq.edu.au/listing/clitoriaternatea.htm ; ; Forage (subtropical) Grain Grain, forage 662 CB 188 FEPAGRO/UFRGS III Forage (subtropical) Main use in Brazil Applications worldwide umbuzeiro.cnip.org.br/db/forrag/vernac.shtml ; ). o de Microbiologia do Solo, IAC, Campinas, Brazil); SU (The University of Sydney, Sidney, Australia); TA ˜ 3 c lia, University of Queensalnd, St. Lucia, Australia); RCR (Rothamsted ´ Annual climbing or herb climbing, non- climbing herb non- climbing herb Descriptor b ria); EPAMIG (Empresa de Pesquisa Agropecua ´ Tribe and actas of RELARE (unpublished). juazeiro.cnip.org.br/edalcin/arvores/ Collection, Soil Productivity Research Laboratory, Marondera, Zimbabwe; also called SPRL); MGAP (Ministerio de Ganaderı [11] b www.ipef.br/identificacao/nativas/detalhes.asp?codigo=65 www.fao.org/ag/AGA/AGAP/FRG/AFRIS/Data/215.HTM ; Rhizobium ; . Subfamily Papilionoideae TrifolieaePapilionoideae Vicieae Annual non- climbing Papilionoideae Phaseoleae Annual/pere-nnial, [11] , St. Paul, USA); USDA (United States Department of Agriculture, Beltsville, USA). . h ria); UFRGS (Universidade Federal do Rio Grande do Sul); CEPEC (Centro de Pesquisas do Cacau); IAPAR (Instituto Agronoˆmico do Parana ´ do a Agropecuaria – INTA – Castelar, Argentina); H (Embrapa Cerrados, Planaltina, Brazil); LMG (Laboratorium voor Microbiologie, Universiteit Gent, Gent, ´ ´ ). [21] ´ cola, FEPAGRO, Porto Alegre, Brazil); SMS (Seça ´ omiu o-de- ˜ ˜ h Rhizobium h , feija , feija c h h Cowpea, black eye pea, barbati, boeme, pois manger cochon, caupi corda Some common names France, arveja, ervilhaca Arrowleaf clover, trevo yuchi, trevo vesiculoso ) o, Embrapa Soja, Londrina, Brazil); QA (Queensland Austra ˜ a,b L. Common vetch, pois Savi Feija ´ o de Microbiologia Agrı ˜ Taxonomy based on ILDIS Culture collections: ATCC (The American Type Culture Collection, Manassas, USA); BR (Brazil, Embrapa Agrobiologia, Serope Common names used in Brazil. Information obtained from the sites cited on ( Plant species for which the strain is commercially recommended. Basis for strain recommendation: (I) selection in glass tubes or bags under axenic conditions; (II) selection in jars under axenic conditions; (III) selection in non-sterile soil; (IV) field experiments. Information obtained from the following sites: After Hungria and Araujo (1995); FEPAGRO Embrapa (Empresa Brasileira de Pesquisa Agropecua Different numbers in FEPAGRO a b c d e f g h i Estadual de Pesquisa(Universidade Agropecua Federal do Ceara Vicia sativa Vigna unguiculata (L.) Walp. Plant species Table 1. ( continued Trifolium vesiculosum (Tasmania Dept. of Agriculture, The DepartmentTropical of Agricultural Primary Legumes Industries, Project, Water University and of Environment, Hawaii, Tasmanian Paia, State USA); Government UMKL Agency, (University Tasmania, of Australia); Malaya TAL – (NifTAL, Kuala Nitrogen Lumpur, Fixation Dept. by of Genetics and Cellular B (University of Minnesota www.arvores.brasil.nom.br/listacient.htm Research Organization – CSIRO,(Instituto Canberra, Nacional Australia); de CIAT Tecnologı Belgium); (Centro MAR Internacional (Marondera, de Grasslands Agricultura Tropical, Cali, Colombia); DF (Distrito Federal, Embrapa Cerrados, Planaltina, Brazil); E of Agriculture, NSW Dept. of Primary Industries – Agriculture, Victoria, Australia); NC (North Caroline, University of North Caroline, Raleigh, US Pesca, Laboratorio de Microbiologia y Suelos, Montevideo, Uruguay) CPAC (Centro de(Parana Pesquisa Agropecua (Seça ildis.org/LegumeWeb/6.00/taxa/1621.shtml ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 323 resuspended in TE 50/20, of 50 mL of 10% SDS, 5 mLof sequence of the 16S rRNA gene, five reactions were proteinase-K (20 mg mL1), 10 mL of lysozyme carried out, with the following primers: fD1, Y2 [54] (5 mg mL1), and 1 mL of RNAse (20 mg mL1). After (target gene region in E. coli K12 362-339) and primers two steps of purification with ethanol at 99.5% and at designed by Prof. Leonardo M. Cruz (Dept. of 70%, the pellet was resuspended in 50 mL of TE 10/1 to Biochemistry, UFPR, Curitiba, PR, Brazil): 362f (target estimate the concentration of the DNA. Samples were region 339–362) (50-CTCCTACGGGAGGCAGCAGT- then diluted to 20 ng of DNA mL1 and were kept at GGGG-30), 786f (target region 764-786) (50-CGAAA- 20 1C. GCGTGGGGAGCAAACAGG-30) and 1203f (target region 1179-1203) (50-GAGGTGGGGATGACGTCA- 0 Amplification of the DNA region coding for the 16S AGTCCTC-3 ). The same program was used with all primers, as follows: denaturation at 95 1C for 2 min; rRNA gene and purification of the PCR-products 30 cycles of denaturation at 95 1C for 10 s, 50 1C for 4 s, and extension at 60 1C for 4 min; final soak The DNA of each bacterial strain was amplified with at 4 1C. the universal primers fD1 and rD1 described previously After amplification, to 20 mL of each sample (7 mLof [49] (target gene regions in Escherichia coli strain K12), each PCR reaction+13 mL of milli-Q water) were added fD1 (8-27) and rD1 (1525–1541). Each replicate 2 mL of sterile ammonium acetate (7.5 M) and 65 mLof contained, in a volume of 50 mL: dNTPs (300 mMof 99.5% ethanol (room temperature). The plate was each); PCR-buffer (Tris-base 20 mM pH 8.4 and KCl sealed, homogenized and centrifuged at 4000 rpm for 50 mM); MgCl (1.5 mM); primers (15 pmol of each); 2 45 min. The supernatant was discarded and the plate Taq DNA polymerase (1.2 U); DNA (20 ng) and DMSO was inverted on absorbent paper to dry. After drying, (5%). The reaction was carried out in an MJ Research the pellet received 165 mL of freshly prepared 70% Inc. PTC 200 thermocycler, using an initial cycle of ethanol, the plate was sealed, homogenized, centrifuged denaturation at 95 1C for 2 min; 30 cycles of denatura- again at 4000 rpm for 10 min, and the supernatant was tion at 94 1C for 15 s, 93 1C for 45 s, annealing at 55 1C discarded. The plate was inverted on absorbent paper for 45 s, and extension at 72 1C for 2 min; a final and centrifuged at 300 rpm for 25 s. The pellet was dried extension cycle of 72 1C for 5 min. at room temperature for 30 min, or at 37 1C for 15 min, For the purification of the PCR-products, 48 mLof resuspended in 7 ml of milli-Q water or in buffer (70% each PCR reaction were added to each 300 mL-capacity formamide, 1 mM EDTA), and submitted for sequen- well of a Nunc U96 MicroWellTM Plate. Each well also cing analysis in a MegaBACE 1000 DNA Analysis received 5 mL of sterile ammonium acetate (7.5 M) and System (Amersham Biosciences). In general, the electro- 165 mL of 99.5% ethanol (room temperature). The plate phoresis parameters used were: sample injection voltage, was sealed, homogenized and the mixture was centri- 1 kV; sample injection time, 40 s; run voltage, 5 kV; run fuged at 4000 rpm for 45 min. The supernatant was time, 240 min. discarded and the plate was inverted on absorbent paper The high-quality sequences obtained for each strain to dry. After drying, the pellet received 150 mL of freshly were assembled into contigs using the programs phred prepared 70% ethanol, the plate was sealed and the [8,9] phrap (www.phrap.org) and Consed [15]. Se- suspension was homogenized and centrifuged again at quences confirmed in the 30 and 50 directions were 4000 rpm for 10 min. The supernatant was discarded as submitted to the GenBank database (http:// described previously and the plate was inverted on www.ncbi.nlm.nih.gov/blast) to seek significant absorbent paper and centrifuged at 300 rpm for 25 s. The alignments. Accession numbers from AY904726 to pellet was dried at room temperature for 30 min, or at AY904789 were given to the 16S rRNA sequences 37 1C for 15 min, followed by the addition of 15 mLof of 64 strains (Table 2). The sequences of the four milli-Q water, homogenized, and kept at 4 1C. After strains recommended for the soybean crop were 24 h, the concentration of the samples was verified in reported before [7] and were also confirmed and used 1.5% agarose gels, adjusted to 40 ng DNA mL1 and in this study: B. japonicum strains SEMIA 5079 kept at 20 1C. (AF234888) and SEMIA 5080 (AF234889), and B. elkanii strains SEMIA 587 (AF234890) and SEMIA Sequencing analysis of the 16S rRNA gene 5019 (AF237422).

The PCR-reactions were carried out in 96-well-full- skirt-PCR microplates. Puriﬁed PCR products of each Phylogeny based on the 16S rRNA gene and bacterium culture (80 ng per reaction) received a mixture diversity of rhizobia of 3 mL of dye (DYEnamic ET terminator reagent premix for the MegaBACE, Amersham Biosciences), The sequences obtained were aligned and compared and 3 pmol of each primer. To obtain the complete to those of the following type/reference strains ARTICLE IN PRESS 324 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332

Table 2. Acid/alkaline reaction in yeast extract-mannitol agar (YMA) medium, accession number of the 16S rRNA sequence, original host and proposed taxonomic position of 68 strains ofﬁcially recommended for the use in inoculants for 64 legume species in Brazil

SEMIA strain Reaction in Original host Source of the Proposed taxonomic Gene Bank Access no. YMA strain position

103 Neutral Medicago polymorpha Brazil Sinorhizobium meliloti AY904726 134 Acid Medicago sativa Brazil Sinorhizobium meliloti AY904727 135 Acid Medicago sativa Brazil Sinorhizobium meliloti AY904728 222 Acid Trifolium Australia Rhizobium AY904729 subterraneum leguminosarum 384 Acid Vicia sp. Brazil Rhizobium etli AY904730 396 Acid Not known USA Mesorhizobium ciceri AY904731 587 Alkaline Glycine max Brazil Bradyrhizobium AF234890 a elkanii 656 Alkaline Neonotonia wightii Brazil Bradyrhizobium sp. AY904732 658 Alkaline Lotononis bainesii South Africa Methylobacterium sp. AY904733 662 Alkaline Vigna unguiculata Australia Bradyrhizobium AY904734 elkanii 695 Alkaline Neonotonia wightii Australia Bradyrhizobium AY904735 elkanii 696 Alkaline Desmodium uncinatum Austra´lia Bradyrhizobium AY904736 elkanii 816 Acid Lotus corniculatus Brazil Mesorhizobium sp. AY904737 830 Acid Not known USA Mesorhizobium sp. AY904738 938 Alkaline Lupinus albus USA Bradyrhizobium AY904739 elkanii 2051 Acid Trifolium vesiculosum Brazil Rhizobium AY904740 leguminosarum 2081 Acid Trifolium pratense Brazil Rhizobium AY904741 leguminosarum 3007 Acid Pisum sativum Mexico Rhizobium AY904742 leguminosarum 5019 Alkaline Glycine max Brazil Bradyrhizobium AF237422 a elkanii 5079 Alkaline Glycine max Brazil Bradyrhizobium AF234888 a japonicum 5080 Alkaline Glycine max Brazil Bradyrhizobium AF234889 a japonicum. 6002 Alkaline Vigna unguiculata Zimbabwe Bradyrhizobium AY904743 japonicum 6028 Alkaline Desmodium uncinatum Zimbabwe Bradyrhizobium AY904744 elkanii 6053 Alkaline Clitoria ternatea Malaysia Bradyrhizobium AY904745 elkanii 6069 Alkaline Leucaena leucocepha- Brazil Bradyrhizobium AY904746 la elkanii 6070 Acid Leucaena leucocepha- Brazil Rhizobium sp. AY904747 la 6100 Alkaline Falcataria moluccana Brazil Bradyrhizobium AY904748 elkanii 6101 Alkaline Dalbergia nigra Brazil Bradyrhizobium AY904749 elkanii 6144 Alkaline Arachis hypogaea Zimbabwe Bradyrhizobium sp. AY904750 6145 Alkaline Arachis hypogaea Libia Bradyrhizobium sp. AY904751 6146 Alkaline Centrosema sp. Brazil Bradyrhizobium AY904752 elkanii 6148 Alkaline Neonotonia wightii Brazil Bradyrhizobium sp. AY904753 6149 Alkaline Galactia striata Australia Bradyrhizobium AY904754 elkanii ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 325

Table 2. (continued )

SEMIA strain Reaction in Original host Source of the Proposed taxonomic Gene Bank Access no. YMA strain position

6150 Alkaline Acacia mearnsii Brazil Bradyrhizobium AY904755 elkanii 6152 Alkaline Calopogonium sp. Brazil Bradyrhizobium AY904756 japonicum 6155 Alkaline Stylosanthes sp. Brazil Bradyrhizobium AY904757 japonicum 6156 Alkaline Crotalaria spectabilis Brazil Bradyrhizobium sp. AY904758 6157 Alkaline Cajanus cajan Brazil Bradyrhizobium AY904759 elkanii 6158 Alkaline Crotalaria spectabilis Brazil Bradyrhizobium AY904760 elkanii 6159 Alkaline Enterolobium Brazil Bradyrhizobium AY904761 ellipticum elkanii 6160 Alkaline Albizia lebbeck Brazil Bradyrhizobium sp. AY904762 6161 Acid Prosopis juliﬂora Brazil Sinorhizobium sp. AY904763 6163 Alkaline Acacia mearnsii Brazil Bradyrhizobium sp. AY904764 6164 Alkaline Acacia mearnsii Brazil Bradyrhizobium sp. AY904765 6165 Acid Mimosa scabrella Brazil Rhizobium sp. AY904766 6166 Alkaline Mimosa Brazil Burkholderia sp. AY904767 caesalpiniifolia 6167 Alkaline Mimosa Brazil Burkholderia sp. AY904768 caesalpiniifolia 6168 Acid Gliricidia sepium Brazil Rhizobium sp. AY904769 6169 Alkaline Falcataria moluccana Brazil Bradyrhizobium AY904770 elkanii 6175 Alkaline Pueraria phaseoloides Brazil Bradyrhizobium AY904771 elkanii 6192 Alkaline Tipuana tipu Brazil Bradyrhizobium sp. AY904772 6208 Alkaline Desmodium Colombia Bradyrhizobium AY904773 heterocarpon elkanii 6319 Alkaline Arachis sp. Bolivia Bradyrhizobium sp. AY904774 6382 Neutral Mimosa Brazil Burkholderia sp. AY904775 caesalpiniifolia 6383 Acid Mimosa Brazil Rhizobium sp. AY904776 caesalpiniifolia 6384 Alkaline Mimosa obovata Brazil Bradyrhizobium AY904777 elkanii 6387 Alkaline Acacia auriculiformis Brazil Bradyrhizobium AY904778 elkanii 6390 Acid Acacia decurrens Brazil Burkholderia cepacia AY904779 6391 Alkaline Acacia auriculiformis Brazil Bradyrhizobium sp. AY904780 6394 Alkaline Ormosia nitida Brazil Burkholderia cepacia AY904781 6398 Acid Piptadenia stipulacea Brazil Burkholderia sp. AY904782 6401 Alkaline Sesbania virgata Brazil Azorhizobium sp. AY904783 6402 Alkaline Sesbania virgata Brazil Azorhizobium sp. AY904784 6412 Neutral Clitoria fairchildiana Brazil Burkholderia sp. AY904785 6420 Alkaline Acacia mangium Brazil Bradyrhizobium sp. AY904786 6424 Alkaline Centrosema pubescens Brazil Bradyrhizobium AY904787 elkanii 6425 Alkaline Centrosema pubescens Brazil Bradyrhizobium AY904788 elkanii 6440 Alkaline Arachis pintoi Brazil Bradyrhizobium sp. AY904789

aThe nucleotides conﬁrmed a previous submission of our group [7]. ARTICLE IN PRESS 326 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332

(accession numbers of the GenBank Data Library in Results parentheses): Azorhizobium caulinodans USDA 4892T T (X67221); Bradyrhizobium betae PL74H1 (AY372184); Morpho-physiological characterization of SEMIA T Bradyrhizobium canariense BC-C2 (AY577427); Bra- strains dyrhizobium elkanii USDA 76T (U35000); Bradyrhizo- bium japonicum USDA 6T (U69638); Bradyrhizobium T Seventeen strains showed acid reactions on YMA liaoningense USDA 3622 (AF208513); Burkholderia medium (Table 2), and in general they were character- cepacia ATCC 53867T (AY741356); Burkholderia T ized by medium to high production of mucus (data not graminis C4D1 M (U96939); Burkholderia sp. TJ182 shown). Forty-eight strains showed alkaline reactions on (AJ505301); Burkholderia sp. BR 3405 (AY773186); YMA, most with low to medium production of mucus Burkholderia sp. BR 3407 (AY773186); Burkholderia (data not shown). In relation to the other morphological sp. tpig4.4 (AY691396); Burkholderia sp hpud10.4 parameters evaluated (color, transparency, borders, (AY691394); Mesorhizobium ciceri USDA 3383T T elevation), no consistent patterns emerged within the (U07934); Mesorhizobium loti USDA 3471 (X67229); acid- or alkali-producing group (data not shown). Only Methylobacterium nodulans ORS 2060T (AF220763); T three strains, SEMIA 103, SEMIA 6382 and SEMIA Rhizobium etli CFN 42 (U28916); Rhizobium legumi- 6412 showed neutral reaction on YMA (Table 2), all of nosarum USDA 2370T (U29386); Rhizobium rhizogenes T which with medium production of mucus and colonies ATCC 11325 (D14501.1); Rhizobium tropici CIAT of 2–3 mm in diameter after 7 days. 899T (U89832); Sinorhizobium fredii USDA 205T (X67231); Sinorhizobium meliloti USDA 1002T (X67222). Azospirillum brasilense strain A154 Phylogeny based on the 16S rRNA gene and (DQ104848) was used as an outgroup reference. diversity of rhizobia Multiple alignments were performed with ClustalX version 1.83 [43]. Phylogenetic trees were generated Fig. 1 shows the phylogenetic tree obtained with the using MEGA version 3.1 [25] with default para- 16S rRNA aligned sequences of the 68 rhizobial strains, meters, K2P distance model [24] and the Neighbor- as well as of the type and reference strains used in this Joining algorithm [35]. Azospirillum brasilense strain study; A. brasiliense strain A154 as used as an outgroup A154 was used as an outgroup for 16S rDNA reference. Strains were grouped into nine phylogenetic phylogenies. Statistic support for tree nodes was branches or well-supported main clusters, with some evaluated by bootstrap [10] analyses with 2000 sam- subclusters. We have defined that strains differing in plings [17]. more than 1.03% (15) nucleotides from the closest type strain could represent new species and were therefore designated in this paper as ‘‘sp.’’ (Table 2). In the first phylogenetic branch (I) strain SEMIA 658 PCR-amplification of the DNA region coding for the was clustered with Metylobacterium nodulans ORS nodB, nodC and nifH genes 2060T with a bootstrap support of 99% (Fig. 1). The second phylogenetic branch (II) clustered 42 Bradyrhi- The DNAs of the bacteria showing similarity with the zobium strains with a bootstrap support of 99%, and genera Methylobacterium and Burkholderia were used two main clusters (II.1 and II.2) were observed (Fig. 1). for amplification of the regions coding for the genes Cluster II.1 included 28 SEMIA strains together with nodB, nodC and nifH. B. elkanii type strain USDA 76T and reference strain For the amplification of the nodB gene region, the SEMIA 587. Within this cluster were strains isolated primers used were nodB3f [50] and nodCRR [39]. from legumes of the following subfamilies and tribes: Each replicate contained, in a volume of 50 mL: dNTPs Papilionoideae (Aechynomeneae, Crotalarieae, Cyti- (200 mM of each); PCR-buffer (Tris-base 20 mM pH 8.4 seae, Dalbergieae, Desmodieae, Phaseoleae) and Mimo- and KCl 50 mM); MgCl2, (2.4 mM); primers (30 pmol soideae (Acacieae, Ingeae, Mimoseae). Three out of the of each); Taq DNA polymerase (1.5 U); DNA (40 ng). four strains from this study that had been isolated from The reaction was carried out with 30 cycles of tribe Desmodieae, as well as one from the Dalbergieae, denaturation at 94 1C for 90 s, annealing at 67 1C for and the only strain from the Cytiseae were within this 30 s, and extension at 72 1C for 80 s; and a final branch. The SEMIA strains positioned in the cluster II.1 extension cycle of 72 1C for 3 min. are officially recommended for 34 host legume species, The nodC amplification was performed with primers with some (SEMIAs 6160, 6100, 6169, 6387 and 6149) nodCIf and nodCp8, used for Bradyrhizobium,as being the most effective for two different legume species, described previously [40]. For the DNA region coding SEMIA 6145 and 6159 the most effective for three and for nifH, amplification was performed with primers SEMIA 6158 the most effective for four different host nifHF and nifHI [26]. species. Strains from this phylogenetic branch differing ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 327

Fig. 1. Phylogenetic tree based on the 16S rRNA sequences of 68 strains, N2-fixing symbionts isolated from 46 legumes and officially recommended for the use in Brazilian commercial inoculants. GeneBank accession numbers of SEMIA strains (Table 2) and of type strains (material and methods) are given in the text. Phylogenetic trees were generated using MEGA version 3.1 with default parameters, K2P distance model and the Neighbor-Joining algorithm. ARTICLE IN PRESS 328 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 in more than 15 bases from B. elkanii USDA 76T were Strains SEMIA 6402 and 6401, isolated from stem nominated as Bradyrhizobium sp. (Table 2). nodules of Sesbania virgata (Table 1), were clustered Phylogenetic branch II.2 grouped 13 SEMIA with Azorhizobium caulinodans type strain in phyloge- with type strains of B. japonicum, B. liaoningense, netic branch VIII (Fig. 1). However, both SEMIAs B. canariense and B. betae with a bootstrap support of differed in more than 2.07% nucleotides from the type 99% (Fig. 1). The majority (38%) was isolated from strain, therefore at this moment they are nominated as legumes of the Papilionoideae tribe Phaseoleae, followed Azorhizobium sp., as they might represent another by 23% from the Mimosoideae tribe Acacieae; promis- species (Table 2). cuity of host species was also observed within this group Finally, the most divergent group of strains fit into (Fig. 1, Table 1). The strains differing in more than phylogenetic branch IX, also with bootstrap support of 1.03% of bases of Bradyrhizobium species positioned in 99% (Fig. 1). This branch included seven SEMIA this cluster were nominated as Bradyrhizobium sp. strains, all isolated in Brazil, with type and reference Four SEMIA strains fit into phylogenetic branch III, strains belonging to the genus Burkholderia. Two of Rhizobium tropici-R. rhizogenes (Agrobacterium), with subclusters were defined, and one isolated strain, a bootstrap support of 99% (Fig. 1). The comparison of SEMIA 6398. Subcluster IX.1 included strain SEMIA the four SEMIA with the type strains showed higher 6390 and SEMIA 6394 with Burkholderia cepacia similarity of bases with R. rhizogenes ATCC 11325T, but ATCC53867T, while strains SEMIAs 6167, 6382, 6166, differing in 0.76–1.34% nucleotides. The highest blast and SEMIA 6412 were joined to Burkholderia sp. strain was with another strain (163C) of R. rhizogenes TJ 182 with a bootstrap support of 92% (Fig. 1, Tables (AY206687), isolated from tumors of Prunus 1and2). At the SEMIA collection, strains SEMIA 6382 persica, and high similarity was also observed with and SEMIA 6383 should be the same as BR 3405 and strain p1-7 (AY206687), isolated from nodules of BR 3407, respectively, however, differences in some common beans (Phaseolus vulgaris) and classified nucleotides were observed when compared with pre- as R. lusitanum. Finally, a lower but still high similarity viously deposited sequences (AY773185 and AY773186, of nucleotides was observed with several R. tropici respectively). We still have to compare the strains in strains isolated from common bean, some of them from relation to other characteristics. Brazil. The R. leguminosarum type strain was positioned in phylogenetic branch IV, together with four SEMIA Amplification of the DNA of Methylobacterium and strains, with a bootstrap support of 99% (Fig. 1). Burkholderia strains with primers for the nod and nif Strain SEMIA 384, symbiont of Vicia sativa and gene regions isolated in Brazil, was highly related to R. etli type strain in phylogenetic branch V, but with the The DNA of eight SEMIA strains classified as lowest bootstrap support from this study (85%) Burkholderia and Methylobacterium were used for the (Fig. 1, Table 1); due to the symbiotic properties, amplification with primers for the nod and nif regions. further investigation of the plasmids and other genes of Using the primers reported to amplify nodB genes of SEMIA 384 should be performed to confirm its Rhizobium, a PCR-product of about 300 bp was taxonomic position. obtained exclusively with Burkholderia sp. SEMIA Sinorhizobium species were positioned in cluster VI 6398 (Table 3). For the nodC gene, using a set of (Fig. 1). Medicago is the host genus of SEMIA strains primers that resulted in a product of 243 bp in 135, 134 and 103, and the strains were highly related to Bradyrhizobium strains [40], amplification was obtained S. meliloti USDA 1002T (Fig. 1, Table 1). Contrarily, with Burkholderia sp. strains SEMIA 6398 and 6412, strain SEMIA 6161 from Prosopis was distinct from with B. cepacia strains SEMIA 6390 and SEMIA 6394 type strains of S. meliloti (1.17% nucleotides) and and with Methylobacterium sp. SEMIA 658 (Table 3). from S. fredii (1.72% nucleotides); it might represent The primers used to detect nifH resulted in products another species, thus was nominated as Sinorhizobium between 780 and 890 bp in analyses of several rhizobial sp. (Table 2). strains [26] and the PCR-products of Methylobacterium Three SEMIA strains were positioned in Mesorhizo- and both B. cepacia had approximately 700 bp, while the bium phylogenetic branch VII, with a bootstrap products of Burkholderia sp. SEMIAs 6166, 6167 and support of 99%. Strains SEMIA 816 and 830 are 6382, all isolated from Mimosa caesalpiniifolia, had highly related (99.5% of similarity of bases); but about 1500 bp (Table 3); amplification with those differed considerably from type strain of M. loti and primers was not observed with strains SEMIA 6398 might represent another species (Table 2). SEMIA 396 is and 6412 (Table 3). Our goal is now to sequence the a symbiont of Cicer arietinum and was highly PCR-products obtained aiming at better understand the related (99.6%) to the M. ciceri type strain (Fig. 1, evolutionary relationships of symbiotic genes in tropical Tables 1 and 2). rhizobia. ARTICLE IN PRESS P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332 329

Table 3. Amplificationa of nif and nod genes of SEMIA B. elkanii SEMIA 6160, recognized as the most effective strains classified in the genera Methylobacterium and Burkhol- for both Albizia lebbeck (Mimosoideae, Ingeae) and deria Sclerolobium paniculatum (Caesalpinioideae, Caesalpi- nieae), SEMIA 6156, isolated from Crotalaria spect- SEMIA strain nifH nodB nodC abilis, and identified as the most effective for five species 658 + + (C. spectabilis, C. juncea, Cajanus cajan, Canavalia 6390 + + ensiformis, Indigofera hirsuta), and B. japonicum SEMIA 6394 + + 656, recommended for plants of the subfamily Papilio- 6166 + noideae: Desmodium (Desmodieae), Macroptilium (Pha- 6167 + seoleae), and Neonotonia (Phaseoleae). 6382 + R. etli species has been reported as the main symbiont 6398 ++of common beans in the centers of origin of this legume: 6412 + Mesoamerica [28,37] and Northern [4] and Southern [1] a(+) amplified and () not amplified with the primers described in Andean South America. However, recent reports the material and methods section. indicate a wide distribution of R. etli associated with common bean in Brazil [16] and this study extends the host specificity including the Brazilian strain SEMIA Discussion 384 from Vicia sativa. Symbionts of Medicago isolated from subtropical Brazil were confirmed as S. meliloti, Nitrogen is often the most limiting nutrient for plant but SEMIA 6161 from tropical Prosopis juliflora growth worldwide. The situation is especially critical in differed considerably from both S. meliloti and S. fredii the tropics, where the usually low levels of soil organic type strains, which suggests that it may represent a new matter have resulted in the depletion of this nutrient. In Sinorhizobium species. addition, the high cost of N-fertilizers in countries like Four SEMIA strains were clustered with plant- Brazil has resulted in the need for actions emphasizing pathogenic non-N2-fixing agrobacteria. It has been biological nitrogen fixation (BNF) [19,20]. Successful known that Agrobacterium spp. share several character- approaches begin with long-term programs of rhizobia istics and are genetically closely related to some selection, and the identification of elite strains for each rhizobial species (R. tropici, Rhizobium genomic species legume host of interest. Countries differ in their policies Q, R. galegae, R. huautlense, and Allorhizobium un- concerning the commercialization of rhizobial inocu- dicola) [27,42,48,53,55]. Consequently, based on 16S lants, and in Brazil they must contain elite strains rRNA gene sequences, agrobacteria were recently evaluated and recommended by an official committee of reclassified into the genus Rhizobium [53].N2-fixing rhizobiologists [20]. The Brazilian Rhizobium Culture rhizobia resembling agrobacteria were isolated from Collection (SEMIA) was created in 1985 by the root nodules of Acacia spp. [23] and common bean [29] Microbial Resources Centre Network (MIRCEN), with in Africa, but the isolates were not able to maintain the the purpose of maintain the recommended rhizobial symbiotic effectiveness. However, isolates from soybean strains and distribute the cultures to the inoculant nodules in Paraguay [5] and the isolates obtained from industry [20]. Nowadays, SEMIA strains are classified as Mimosa scabrella, Gliricida sepium, and Leucaena R. meliloti, R. leguminosarum, B. japonicum, Bradyrhi- leucocephala in this study showed effectiveness and zobium sp., R. fredii,orR. loti [11] based exclusively on genetic stability of symbiotic properties. In the future, their ability to produce alkaline or acid reaction in YM efforts should focus in understanding the evolution and medium and on the cross-inoculation group [13,45]. ecological importance of these effective tropical rhizobia Therefore, although the SEMIA collection is a reservoir closely related to agrobacteria. of rhizobia resulting from decades of selection, the Putative new species were also observed in two other genetic knowledge about the strains in the collection is genera. Strains SEMIAs 816 and 830, symbionts of very poor. Lotus corniculatus, isolated in Brazil and USA, respec- Phylogeny of the 68 SEMIA strains, the great tively, differed from type strain of Mesorhizobium loti by majority from Brazil, was based on the sequencing of 1.65% and 2.07% nucleotides, respectively. Also two the 16S rRNA, as this gene has become the method of strains isolated from stem nodules of Sesbania virgata in choice for tracing bacterial phylogenies and defining Brazil, SEMIAs 6401 and 6402, differed by more than taxonomy [14,49]. Many SEMIA strains have a broad 30 nucleotides from type strain of Azorhizobium host range, and apparently we found no evidence of caulinodans. evolutionary correlation with the host plants, similarly The majority of the strains from this study (42) were to other results obtained with tropical rhizobia [31]. classified into the genus Bradyrhizobium and two Some of the strains were very effective in fixing N2 major subclusters were observed. The first one included with legumes of distinct tribes and even subfamilies, e.g., B. elkanii USDA 76T and symbionts of all three ARTICLE IN PRESS 330 P. Menna et al. / Systematic and Applied Microbiology 29 (2006) 315–332

Leguminosae subfamilies and several tribes. Type variability in relation to the host plant, to the ribosomal strains of B. japonicum, B. liaoningense and B. canariense 16S gene, and probably to the nif and nod genes among were clustered into the second subcluster, and the burkholderia capable of nodulating legumes than similarity of the sequences of these three species has previously thought [6,32]. Further sequencing of nod been previously reported [47,51,55]. Although high and nif PCR-products may help to clarify the origin of diversity in morphological, physiological, and genetic those genes in burkholderia. properties within Bradyrhizobium strains has been It has been suggested that tropical rhizobia are poorly reported, the differences are not reflected in diversity documented [55] although reports indicate that rhizobia of the 16S rRNA genes [3,5,30,44,46,51,52]. However, diversity may be greater in tropical than in temperate the results obtained in our study show novel variability regions [33]. The strains from this study have been in the 16S rRNA genes within Bradyrhizobium. selected for higher efficiency of N2 fixation with several Strain SEMIA 658 isolated from Lotononis bainesii in legume hosts and are recognized as the most effective for South Africa and effective under Brazilian conditions, their host legumes in Brazil. Host specificity of several was clustered with the type strain of the a-proteobacter- strains was very low, as they were capable of nodulating ium Methylobacterium nodulans, isolated from Crolalar- legumes of different tribes and subfamilies. Host ia podocarpa in Senegal [36,41]. The strains studied by specificity was not related to 16S rRNA, therefore Sy et al. [41] showed similarity of nodA genes with evolution of ribosomal and symbiotic genes may have Bradyrhizobium, which was suggestive of horizontal been diverse. The promiscuous nature of some strains, gene transfer. SEMIA 658 should be the same as CB the wide-range of symbiotic associations with a- and 376, cited as belonging to the genus Methylobacterium b-proteobacteria, and the detection of several putative [41] and in our study amplification was achieved with new species emphasize the great diversity of rhizobial the primers designed for the nodC region of Bradyrhi- strains that still remains to be discovered in the tropics. zobium. Seven strains from Brazil were grouped with b-Proteobacteria of the genus Burkholderia.N2-fixing Acknowledgments symbiotic associations of burkholderia with legume plants, preferentially from the Mimosoideae, have been The authors thank Ligia Maria O. Chueire (Embrapa reported [6,32]. In our study, SEMIA 6394 isolated from Soja) for help in several steps of this work, and to Dr. Ormosia nitida (Papilionoideae), and SEMIA 6390, Maria Monteros (University of Georgia, USA) for isolated from Acacia decurrens (Mimosoideae), showed helpful discussion. Research described herein was higher similarity of nucleotides with Burkholderia partially supported by CNPq (Conselho Nacional de T cepacia ATCC53867 . Three other strains (SEMIA Desenvolvimento Cientı´fico e Tecnolo´gico, Brazil; 6382, 6167, 6166) isolated from Mimosa caesalpiniifolia PRONEX, Instituto do Mileˆnio, Edital Universal and were highly similar with each other in the 16S rRNA Produtividade em Pesquisa) and by ANPII (Associaçaõ analysis, but we were unable to get amplification of Nacional dos Produtores e Importadores de Inocu- those strains with the nodB or nodC primers. However, lantes, Brazil). P. Menna received a fellowship from DNA amplification with primers for the nifH region of CAPES and F.G. Barcellos and M. Hungria from these three strains produced PCR-products of similar CNPq. Finally, the authors wish to emphasize the size (1500 bp), but differed from the products obtained relevant studies of Dr. Sergio M. Faria (Embrapa with the other SEMIA classified as Burkholderia Agrobiologia, Serope´dica, Brazil), who selected most (700 bp). Strain SEMIA 6412 isolated from Clitoria of the tree legume rhizobia. fairchildiana (Papilionoideae) was grouped in the same cluster with these three strains and amplified with the nodC primers. The grouping of these four strains was References genetically closer to the diazotrophic Burkholderia strain TJ 182, isolated from Mimosa diplotricha in Taiwan [6]. [1] O.M. Aguilar, M.V. Lo´pes, P.M. Riccillo, R.A. Gonza´lez, Finally, SEMIA 6398 from Piptadenia gonoacantha M. Pagano, D.H. Grasso, A. Pu¨hler, G. Favelukes, (Mimosoideae) occupied an isolated position in the Prevalence of the Rhizobium-etli like allele in genes coding cluster and the strain did not amplify with nifH primers, for 16S rRNA among the indigenous rhizobial popula- but PCR-products were obtained with both nodB tions found associated with wild beans from the Southern Andes in Argentina, Appl. Environ. Microbiol. 64 (1998) primers of common bean rhizobia and nodC primers 3520–3524. of soybean bradyrhizobia. It is also noteworthy that [2] O.N. Allen, E.K. Allen, The Leguminosae: A Source PCR-products obtained with nodC primers varied in size Book of Characteristics, Uses, and Nodulation, Univ. among the strains, and were different from those Wisconsin Press, Madison, USA, 1981. obtained with Bradyrhizobium by Sterner and Parker [3] L.L. Barrera, M.E. Trujillo, M. Goodfellow, F.J. Garcıá, [40]. The results obtained in our study indicate higher I. Hernańdez-Lucas, G. Da´vila, P. van Berkum, ARTICLE IN PRESS P. 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