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Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal
System. Appl. Microbiol. 25, 572–583 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal Samba Ndao Sylla1, Ramatoulaye Thiaba Samba2, Marc Neyra2, Ibrahima Ndoye1, Eric Giraud3, Anne Willems4, Philippe de Lajudie3, and Bernard Dreyfus3 1
Université Cheikh Anta Diop, F.S.T., Dakar, Sénégal Laboratoire de Microbiologie, IRD, Dakar, Sénégal 3 LSTM, UMR1063, IRD/CIRAD/INRA/ENSA-M, Montpellier, France 4 Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium 2
Received: October 14, 2002
Summary A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus erinaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S–23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S–23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorhizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrhizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrhizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins. Key words: Bradyrhizobium – Rhizobium – Pterocarpus spp. – phylogeny – diversity – 16SrDNA sequence – 16S–23SrDNA IGS sequence
Introduction Pterocarpus erinaceus and Pterocarpus lucens are tree species belonging to the family Leguminosae, subfamily Fabaceae [1]. They are endemic to the sahelian arid and soudanean semi arid tropical regions respectively. They have high socio-economic and ecological importance as sources of forage and wood for local populations and might improve N2 status of soil, due to their nitrogen fixing symbiosis with rhizobia [25, 38]. The natural populations of Pterocarpus erinaceus and P. lucens are disappearing from their natural areas and yet their cultivation is included in reforestation programs in Senegal. Inoculation by rhizobium is necessary to improve plant growth at the nursery stage and transplantation success. In order to optimise biological nitrogen fixation efficiency, the biodiversity of their associated rhizobia has to be exam-
ined. The wide distribution of Pterocarpus erinaceus and P. lucens in diverse ecological arid and semi-arid conditions [11, 25] suggested that rhizobia associated with Pterocarpus spp. may be heterogeneous, similar to what was described for rhizobia isolated from many tropical tree legume species. Early studies showed that Acacia spp. and other woody legumes are nodulated by fast-growing and/or slow-growing Rhizobium spp. [2, 8, 39]. More recent numerical phenotypic and genotypic analyses revealed a large genetic diversity among rhizobia isolated from several tree legume species, and several new species were described [5, 6, 19, 20, 23, 26, 30, 50]. To date nodule-forming bacteria on legumes are classified in more than 30 validly published species in 10 genera eight of which belonging to the α-Proteobacteria Rhizobium, 0723-2020/02/25/04-572 $ 15.00/0
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal
Bradyrhizobium, Mesorhizobium, Sinorhizobium, Azorhizobium, Allorhizobium [48, 49], Methylobacterium [36], Blastobacter [40] and most surprisingly two of which belonging to the β-Proteobacteria Ralstonia [4] and Burkholderia sp. [24]. Thus rhizobia form a phylogenetically heterogeneous group, and their taxonomy is being reexamined. Rhizobia are interspersed with clinical bacteria, like Afipia, and other phytopathogenic bacteria, like Mycoplana and Bartonella [45, 47]. Until now, the taxonomic status of published strains isolated from various legumes is not clarified [7, 9, 21, 23]. Recently, Young et al. [49] proposed to group Rhizobium, Allorhizobium and Agrobacterium in one revised genus Rhizobium. Bradyrhizobium contains three named species B. japonicum, B. elkanii and Bradyrhizobium liaoningense [17, 46], but Willems et al. [44] identified eight other potential genospecies among isolates from Aeschynomene, A. albida and other small legumes native of Senegal. In a previous study, based on nodulation plant test, antibody tests and growth characteristics, Sylla et al. [37] revealed a great diversity of indigenous rhizobia isolated from P. erinaceus and P. lucens in Senegal. In this paper, a polyphasic approach combining phenotypic and genotypic methods was used to further investigate molecular diversity of these rhizobial isolates by comparative SDS-PAGE analysis of proteins and 16S rDNA gene and 16S–23S rDNA IGS sequencing.
Material and Methods Phenotypic Characterization Strains used. Bacterial strains used in this study and their sources are listed in Table 1. Rhizobial strains were isolated from naturally occurring Pterocarpus root nodules [37]. The nodules were washed and immersed in 0.1% HgCl2 for 5 min.; after this, the nodules were manipulated aseptically. Each nodule was rinsed eight times in sterile water and crushed in a drop of sterile water. The resulting suspension was streaked onto yeast mannitol agar (YMA) plates [43], and isolated colonies appeared after incubation for 2 days (fast growing) and more than 3 days (slow growing) at 30 °C. Slow growing and fast growing status of 3 representative strains of each group was confirmed by measuring generation time as described by Jordan [15]. Pure cultures were obtained after single colonies were streaked two or three times. Isolates were checked for nodulation on their original host plants, and stored at –80 °C in YM adjusted to 20% (v/v) glycerol [37]. PAGE of total bacterial proteins. A selection of 28 fast growers and 22 slow growers Pterocarpus rhizobial new isolates were analysed and compared to 67 reference strains. For comparison, type and representative strains of the different species of Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium, were included. Strains were grown in Roux flasks at 28 °C for 48 h (fast growers) or 72 h (slow growers), on TY medium containing (g/l): tryptone (Oxoid), 5; Yeast extract, 0.75; KH2PO4, 0.454; Na2HPO4,12 H2O, 2.388; agar (Lab M), 20; pH 6.8–7. Whole-cell protein extracts were prepared and SDS-PAGE was performed as previously described [6]. The normalised densitometric traces of the protein electrophoretic patterns were grouped by performing a numerical analysis using the Gel Compar 2.2 software package [42]. The
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similarity between all pairs of traces was expressed by the Pearson product moment correlation (r) converted for convenience to percent value [33]. Genotypic characterization Bacterial strains. Seven rhizobial strains (ORS196, ORS199, ORS204, ORS206, ORS207, ORS214 and ORS217) were selected as representatives of different clusters and subclusters defined from numerical analysis of the protein patterns. Culture conditions and DNA isolation. Strains were grown at 28 °C for 48 to 72 hours on yeast extract mannitol agar YMA. DNA was purified from cells treated with sodium dodecyl sulfate (1% [wt/vol])-Pronase (50 g/ml), and then subjected to serial extractions with phenol chloroform (1:1 [vol/vol]) and precipitation with ethanol and NaCl as described by MartinezRomero et al. [18]. After precipitation, purified DNA was washed three times in ethanol, dried, dissolved overnight at 4 °C in TE buffer, and treated (60 min, 37 °C) with RNAse (50 µg/ml final concentration). The control of the DNA quality and the calculation of DNA concentration were performed as described by Henydrickx et al. [14]. PCR amplification. PCR was performed in a Perkin Elmer 2400 Thermal cycler in a 25 µl (total volume) reaction mixture containing 50 ng of bacterial genomic DNA extract; 2.5 mM of each deoxynucleotide triphosphate (Pharmacia Biotech), 10 mM Tris-HCl, (pH 9 at room temperature); 50 mM KCl; 1.5 mM MgCl2; 20 mM of each primers and 1 U of Taq polymerase, (Eurogentec Bel SA). A negative control where water was substituted to DNA was included in every PCR run. 16S rDNA PCR amplification was done using primers FGPS 6 [27] and FGPS 1509 [27] to the following program : initial denaturation at 94 °C for 5 min, followed by 35 cycles consisting of a 30 sec. denaturation at 94 °C, 30 sec. at annealing temperature of 60 °C, and a 2 min. primer extension at 72 °C. Final elongation was performed at 72 °C for 7 min. 16S–23S rDNA PCR amplification was done using primers FGPS1490-72 [28], and FGPL132-38 [32] to the following program profiles: initial denaturation at 95 °C for 5 min, followed by 35 cycles consisting of a 30 sec. denaturation at 95 °C, 1 min at annealing temperature of 55 °C, and a 1 min primer extension at 72 °C. Final elongation was performed at 72 °C for 3 min. PCR amplification was controlled by horizontal electrophoresis in 1% SIGMA Type II agarose gels. DNA molecularweight-marker (Smart Ladder) was used for gel calibration. Electrophoreses were carried out at 100 V for 30 min. The amplified product was stained 30 min in an aqueous solution of ethidium bromide (1 µg/ml) and photographed under UV illumination with Gel Doc (BIO-RAD) software. PCR products were purified using QIAquick PCR purification kit (Quiagen) following the manufacturer’s instructions. DNA sequencing. The purified PCR products corresponding either to 16 S rDNA fragment or 16–23S rDNA IGS were sequenced using primers listed in Table 2. The ABI Prism Big Dye Terminator Cycle sequence DNA Kit (Applied Biosystems, Forster City, California) was used. Sequencing reaction was performed by PCR amplification in a final volume of 20 µl according to Applied Biosystems protocol. After heating to 96 °C for 3 min, the reaction was cycled as follows: 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 4 min at 60 °C (2400 thermal cycler Perkin Elmer). Sequencing reactions were analysed on an Applied Biosystems model 310 DNA sequencer (Perkin Elmer Co.). Sequence analysis. Nucleotide sequences were analysed and corrected with Autoassembler software on the basis of electrophoregrams. The double strands were aligned in the consensus sequences by using the algorithm blast and the closely relat-
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Table 1. Nomenclature of strains used. Strains and (growth caracteristics)
LMG Code
Pant of isolation
Geographical origin
Fast growing isolates from Pterocarpus spp. ORS 207 ORS 210 LMG 15199 ORS 211 LMG 15200 ORS 212 ORS 214 LMG 15203 ORS 215 LMG 15204 ORS 217 LMG 15206 ORS 220 LMG 15208 ORS 227 LMG 15214 ORS 230 LMG 15216 ORS 231 LMG 15217 ORS 235 ORS 239 LMG 15222 ORS 240 LMG 15223 ORS 242 LMG 15225 ORS 243 LMG 15226 ORS 246 LMG 15227 ORS 247 LMG 15228 ORS 248 LMG 15229 ORS 251 LMG 15230 ORS 252 ORS 253 LMG 15232 ORS 255 LMG 15234 ORS 256 ORS 257 LMG 15236 ORS 259 LMG 15237 ORS 260 ORS 261 LMG 15239
P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens
Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal
Slow growing isolates from Pterocarpus spp. ORS 196 LMG 15188 ORS 198 ORS 199 LMG 15190 ORS 200 ORS 201 LMG 15192 ORS 203 LMG 15193 ORS 204 LMG 15194 ORS 206 LMG 15196 ORS 208 ORS 221 LMG 15209 ORS 222 ORS 223 LMG 15211 ORS 226 LMG 15213 ORS 228 LMG 15215 ORS 232 ORS 233 ORS 238 ORS 241 LMG 15224 ORS 244 ORS 245 ORS 263 ORS 264
P. lucens P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens
Sahelian zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal
Reference strains used for numerical analysis of protein patterns (SDS-PAGE) Agrobacterium (separate taxa) NCPPB 1771 LMG 233A Agrobacterium biovar 1 B6T B2 A ATCC 19358T
LMG 187TA LMG 268A LMG 140TA
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal Table 1. (Continued). Strains and (growth caracteristics)
LMG Code
Agrobacterium biovar 2 ATCC 13335
LMG 156TA
Agrobacterium rhizogenes ATCC 11325T
LMG 150TA
Agrobacterium vitis PAN AG 61
LMG 257A
Azorhizobium caulinodans (fast growing) FY 12 LMG 11352 ORS 478 LMG 11820 ORS 571T LMG 6465T ORS 486? LMG11822
Pant of isolation
Geographical origin
Sesbania rostrata Sesbania rostrata Sesbania rostrata Sesbania rostrata
Senegal Senegal Senegal Senegal
Bradyrhizobium japonicum NZP 5533 NZP 5549T Cb 756 NZP 2314 NZP 2309
LMG 6136 LMG 6138T LMG 8319 LMG 6129 LMG 6128
Glycine max Glycine hispida
USA Japon
Bradyrhizobium elkanii NZP 5531T NZP 5532
LMG 6134T LMG 6135
Glycine max Glycine max
USA USA
Bradyrhizobium sp. (Gr. 1ND,Gr.12FM.) Br. 29 Br. 6011 Br. 6009 Br 8402 INPA 260 MAR 411 ORS 184 ORS 188 ORS 347 ORS 306 ORS Pa 44
LMG 9520 LMG 9514 LMG 9512 LMG 10015 LMG 1090 LMG 14300 LMG 10723 LMG 10727 LMG 10298 LMG 11797 LMG 11928
Lonchocarpus costatus Lonchocarpus costatus Dalbergia riparia Dalbergia riparia
Brazil Brazil Brazil Brazil Brazil
Acacia albida Acacia albida Aeschynomene indica Aeschynomene indica Prosopis africana
Senegal Senegal Senegal Senegal
Mesorhizobium sp. cicer (fast growing) IC-60 LMG 14995
Cicer arietinum
Spain
Mesorhizobium mediterraneum (fast growing) UPM Ca 142 LMG 14990
Cicer arietinum
Spain
Rhizobium galeagae (fast growing) HAMBI 540T LMG 6214 HAMBI 1147 LMG 6215
Galegae orientalis Galegae orientalis
Finland Finland
Rhizobium leguminosarum (fast growing) biovar viciae NZP 561 LMG 6122 biovar viciae ATCC 10004T LMG 8817 biovar phaseoli ATCC 14482 LMG 8819
Pisum sativum Phaseolus L.
Rhizobium tropici (fast growing) CFN 299 LMG 9517
Phaseolis vulgaris
Brazil
Rhizobium sp. (fast growing) (Gr.5, Gr.7 et Gr.15 FM.) Br 809 LMG 9950 Br. 8803 LMG 10022 INPA 95A LMG 10134 INPA 133B LMG 10062
Leucaena leucocephala Glyricidia sepium Leucaena pulvurulenta Leucaena leucocephala
Brazil Brazil Brazil Brazil
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Table 1. (Continued). Strains and (growth caracteristics)
LMG Code
Pant of isolation
Geographical origin
Lotus corniculatus Lotus marccanus
New Zealand Maroc
Mesorhizobium plurifarium (fast growing) INPA 129 A LMG 6123 NZP 2037 LMG 6123 ORS 1004 LMG 7848 ORS 1014t2 LMG 7849 ORS 1029 LMG 11889 ORS 1040 LMG 11898 ORS 1032T LMG 11892T
Lotus divaricatus Lotus divaricatus Acacia senegal Acacia senegal Acacia senegal Acacia senegal Acacia senegal
New Zealand New Zealand Senegal Senegal Senegal Senegal Senegal
Mesorhizobium ciceri (fast growing) UPM Ca 7T LMG 14989 IC 2091 LMG 15086
Cicer arietinum Cicer arietinum
Spain Spain
Mesorhizobium huakuii (fast growing) IAM 14158T LMG 14107
Astragalus sinicus
China
Sesbania pachycarpa Sesbania grandiflora Sesbania cannabina Sesbania cannabina Prosopis juliflora
Senegal Senegal Senegal Senegal Senegal
Acacia laeta Acacia senegal
Senegal Senegal
Acacia raddiana
Senegal
Medicago sativa Medicago sativa
Australia Australia
Mesorhizobium loti (fast growing) NZP 2213 LMG 6125 NZP 2230 LMG 6126 USDA 208 LMG 6219
Mesorhizobium huakuii (fast growing) UPM Ca142 LMG14990 Sinorhizobium fredii (fast growing) USDA 191 LMG 6217 USDA 205T LMG 8317T Sinorhizobium saheli (fast growing) ORS 600 LMG 11864 (YM) ORS 611 LMG 8310 ORS 609T LMG 7837Td ORS 609t2 LMG 8309 ORS 12 = Pj12 LMG 7835 Sinorhizobium terangae (fast growing) ORS 1009 LMG 7834 ORS 1013 LMG 7844T1 ORS 1025 LMG 11885 ORS 1045 LMG 11901 Sinorhizobium meliloti (fast growing) NZP 4009 LMG 6130 NZP 4027T LMG 6133
ATCC, American type Culture collection. Rockville, Md.; Br. Strain from CNPBS/EMBRAPA, Centro Nacional de Pesquisa em Biologia do solo, Seropedica, Rio de Janeiro, Brasiliera de Pesquisa Agropecaria; CDC, Centers for Desease Control, Atlanta, Ga.; CFN, Centro de investigacion sobre Fijacion de Nitrogeno, Universidad Nacional Autonomade Mexico, cueranavaca, Mexico; HAMBI, Culture Collection of the Departement of Microbiology, University of Helsinki, Finland; IAM Institute of Applied Microbiology, the University of Tokyo, Japan; IC et UPM, Departemento de Microbiologia, E.T.S. de Ingenieros Agronomos, Universidad Politecnica, Madrid, Spain; INPA, National Institute of Amazonia Reserch, Manaus Brazil; LMG, Collection of Bacteria of the Laboratorium voor microbiologie, University of Ghent, Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, U.K.; NZP, Culture Collection of the Departement for Scientific Research and Industrial, Biochemistry Division, Palmerson North, New Zealand; ORS , Collection de l'Institut Français de Recherche Scientifique pour le Développement en Coopération, dakar, Sénégal; pan, Panagopoulos, Crete, greece; USDA, united States Departement of Agriculture, Beltsville, Md.
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Table 2. PCR and sequencing primers used in this study. Primer name
Primer sequence (5′ – 3′)
Target Gene
Reference
FGPS6 FGPS 1509 16S 370f 16S-1080rev 16S 870f 16S 1924 rev FGPL 132-38 FGPS 1490-72
(5′-GGA-GAG-TTA-GAT-CTT-GGC-TCA-G-3′) (5′-AAG-GAG-GGG-ATC-CAG-CCG-CA-3′) (5′-GGC-AGC-AGT-GGG-GAA-TAT-TG-3′) (5′-GGG-ACT-TAA-CCC-AAC-ATC-T-3′) (5′-CCT-GGG-GAG-TAC-GGT-CGC-AAG-3′) (5′-GGC-ACG-AAG-TTA-GCC-GGG-GC-3′) (5′-CCGGGTTTCCCCATTCGG-3′) (5′-TGCGGCTGGATCCCCTCCTT-3′)
16SrDNA 16SrDNA 16SrDNA 16SrDNA 16SrDNA 16SrDNA IGS rDNA IGS rDNA
Normand et al. (1992) Normand et al. (1992) Sy et al. (2001) Sy et al. (2001) Sy et al. (2001) Sy et al. (2001) Ponsonnet et al. (1994) Normand et al. (1996)
ed sequences found were included in the following analyses. Phylogenetic analysis was inferred by using the CLUSTAL X program of the GCG (Genetics Computer Group, Wisconsin). Matrix pairwise comparisons were corrected for multiple base substitutions by the two-parameter method of Kimura [16]. Phylogenetic trees were constructed using the neighbour-joining [34] method. A bootstrap confidence analysis was performed on 1000 replicates to determine the reliability of the distance tree topology obtained [10]. Graphic representation of the resulting trees was done using njplot [31] software.
Results Numerical analysis of proteins patterns. We analysed 50 protein profiles from fast-growing and slow-growing nodule isolates from P. erinaceus and P. lucens. Fig. 1 shows a simplified dendrogram including bacterial isolates from Pterocarpus spp. and their closest neighbours as references. Results show that at a coefficient similarity of 81%, Pterocarpus rhizobial strains are heterogeneous and form seven groups (I to VII). Nineteen slow-growing strains constitute the Cluster I, where we distinguish four subclusters (Ia, Ib, Ic, Id). Subcluster Ia includes four new isolates and one Bradyrhizobium sp. (A. albida) strain (ORS 184) published by Dupuy et al. [9]. Subcluster Ib consists of four Pterocarpus strains and one strain of Bradyrhizobium sp. (ORS 188) identified by Dupuy et al. [9]. Subcluster Ic consists of eight new isolates and includes the type strain of Bradyrhizobium japonicum (NZP 5549T). Subcluster Id consists of new isolates from both Pterocarpus species. A total number of 31 fast-growing strains constituted clusters II, IV, V, VI, VII and numerous separate strains. Cluster II consists of four Pterocarpus spp. new isolates and includes Rhizobium sp. strain Br. 809 identified by Moreira et al. [23], as separate from all described species. Cluster III consists of six new isolates from Pterocarpus spp not related to any reference strain. Cluster IV consists of new isolates from Pterocarpus spp. and includes type strains of Mesorhizobium plurifarium (ORS1032T) and Mesorhizobium huakuii (IAM14158 T). Clusters V, VI and VII are small groups each consisting of a few set of Pterocarpus strains. 16S rDNA gene sequence analysis. The 16S rDNA sequences generated were deposited in the Genbank database to search for significant 16S rDNA alignments.
The complete 16S rDNA sequences of seven strains ORS 196, ORS 199, ORS 204, ORS 206, ORS 207, ORS 214 and ORS 217 received respectively accession numbers AF514792, AF514794, AF5147916, AF5147918, AF514800, AF5801 and AF514802. The 16S rDNA gene sequences (> 1460 bp) were compared to those of members of Azorhizobium, Bradyrhizobium, Methylobacterium, Mesorhizobium, Rhizobium, Sinorhizobium species and also to members of Afipia, Agrobacterium, Blastobacter, Nitrobacter, Rhodopseudomonas, and Xanthobacter, and a phylogeny tree was inferred (Fig. 2.). The sequence divergence obtained by comparing 16S rDNA gene among the strains ORS 196, ORS 199, ORS 204, ORS 206, Bradyrhizobium elkanii, B. japonicum, B. liaoningense, B. sp. (Lupinus), Blastobacter denitrificans, Nitrobacter winogradski Rhodopseudomonas palustris was less than 0.2% (99.8% of sequence similarities). This degree of similarity is very low to distinguish phylogenetic placement of taxa. Similarly, sequences divergence within the group of Mesorhizobium species group including ORS 207 showed to be less than 0.2% and also the group constituted by ORS 214, ORS 217, Rhizobium species and Agrobacterium rhizogenesT appear to be highly similar. The reconstruction of phylogenetic tree based on 16S rDNA sequence analysis (Fig. 2) separated the seven Pterocarpus isolates into three major groups: Rhizobium spp. strains; Mesorhizobium spp strains; Bradyrhizobium elkanii and B. japonicum strains. Except the group of B. japonicum, the separate groups of strains were supported by significant bootstraap values. Inside these clusters, ORS 214 and ORS 217 are situated in the vicinity of Rhizobium tropici IIB; ORS 207, groups close to Mesorhizobium plurifarium; ORS 199 forms a branch with Blastobacter denitrificans, Bradyrhizobium liaoningense and B. sp. (Lupinus) as closest neighbour; the slow growing strains ORS 204 and ORS 196 are positioned in the branch of B. elkanii; the slow growing strain ORS 206 is located in the vicinity of Bradyrhizobium japonicum. 16S–23S IGS rDNA sequencing. The 16S–23S IGS rDNA sequences generated for Bradyrhizobia strains ORS 196, ORS 199, ORS 204 and ORS 206 were deposited to the Genbank database and received respectively accession numbers AF514793, AF514795, AF514797 and AF514799. The IGS sequences obtained varied in length from 890 to 1054 nucleotides. To precise phylogenetic diversity of the Bradyrhizobium sp. (Pterocarpus) strains,
Fig. 1. Dendrogram showing the relationship between the electrophoretic protein patterns from Pterocarpus spp. rhizobium strains and reference strains. The mean correlation coefficient ® was calculated by unweighted average pairs grouping method (UPGMA). Reference strains are limited to the closest neighbours to new isolates: ORS184; ORS188; ORS306; ORS347; ORS1032T; ORS1270; NZP2314; NZP5531T; NZP5533; NZP5549T; NZP5532; Br29; Br809; CB756; ATCC11325T; IAM14158; and UPMCa142.
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Fig. 2. Neighbor joining dendrogram showing the sequence similarities of 16S rDNA gene sequences of new isolated strains (bold style) and some related organisms. The length of the aligned sequences was >1460 bases and bootstrap values were calculated from 1000 trees. The levels of support for the presence of nodes above a value of 60% ar eindicated.
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Fig. 3. Neighbor joining dendrogram shwoing the sequence similarities of 16S–23S rDNA internal gene spacer sequences of Bradyrhizobium strains isolated from Ptercarpus trees (bold style) and some related organisms. Roman numbers correspond to Bradyrhizobium genospecies as described by Willems et al. (2001). The length of the aligned sequences was >890 bases. Bootstrap values were calculated from 1000 trees and the levels of support for the presence of nodes above a value of 60% are indicated.
we performed sequence alignments of Pterocarpus spp. strains and compared them with those of members of Bradyrhizobium species available in Genbank database. We included recent Bradyrhizobium genospecies reported by van Berkum et al. [41] and by Willems et al. [44]. The IGS sequence of Blastobacter denitrificans (AF 338176) and BTAi1 (AF338169) were also added. All IGS sequences determined comprised two tRNA genes, for tRNAIle and tRNAAla that were located in the middle to first half of the spacer region. This structure is similar to that reported for the majority of Gram-negative bacteria [12]. The dendrogram obtained by neighbor-joining analysis of the similarity matrix is shown in Fig. 3.
ORS 196 and ORS 204 grouped in a major cluster with strains of Bradyrhizobium elkanii as closest neighbour and strains of genospecies VII [44]. Relatively little variation in the IGS sequence (98–99%) similarities was found between the different members of this cluster. The sequence similarities between ORS 196, ORS 204, B. elkanii (type strain) and members of genospecies II and VII was supported with high values from bootstrap analysis (Fig. 3). ORS 206 was on the Bradyrhizobium liaoningense and Bradyrhizobium japonicum branch. IGS sequence similarities founded between ORS 206 and these reference strains were respectively 98.9 and 98.5%. In Fig. 3, the Pterocarpus isolates, ORS 206 was together with
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal
members of Bradyrhizobium liaoningense and Bradyrhizobium japonicum but their phylogenetic relationships are not well supported by bootstrap values. ORS 199 was inside the group consisting of Bradyrhizobium genospecies V [44]. IGS sequence divergence values calculation revealed that Pterocarpus isolates have 99–99.5 % similaries with members of genospecies V described by Willems et al. [44]. The phylogenetic position of ORS 199 within the members of genospecies V is supported by significant bootstrap values.
Discussion A total of fifty strains isolated from Pterocarpus erinaceus and P. lucens nodules in Senegal was examined. Among these strains we previously distinguished two types of rhizobia: fast growing and slow growing ones. Here, we further characterized them using phenotypic and genotypic approaches to determine their phylogenetic diversity. As a grouping method, we used SDS-PAGE of total protein profiles comparison and included the results in the large database available in our research group. Results confirmed our previous classification based on growth rate [37]. Consistent with the results reported on tropical rhizobia isolated from Acacia (Faidherbia) albida [9] and from various leguminous trees [6, 22, 26, 50], we found a considerable heterogeneity within both fast- and slow-growing nodule isolates from Pterocarpus spp. Slow growing strains form one major cluster (cluster I) subdivided into four subclusters. This major cluster corresponds to Bradyrhizobium. The fast growing rhizobia grouped in six electrophoretic clusters and several strains were separate. Except ORS 207 grouping with Mesorhizobium plurifarium and Mesorhizobium huakuii, the fast growing rhizobia of our collection clustered apart from described strains suggesting that they represent new groups. To further describe and differentiate the new groups obtained by SDS-PAGE, we performed genotypic investigations across the entire 16S rDNA gene and the 16S–23S rDNA IGS region by sequence analysis. Rhizobial strains representative for seven SDS-PAGE groups or subgroups were selected for 16S rDNA sequence analysis. Results corroborated those obtained previously. The slow-growing strains clustered with either B. japonicum type strain (cluster Ic) or closely related to B. elkanii. In reconstruction derived from 16S rDNA sequence divergence, one of the slow growing rhizobia (ORS 199) isolated from Pterocarpus spp. grouped with Bradyrhizobium spp. and Blastobacter denitrificans. This result related those of van Berkum and Eardly [40] which showed high identity in 16S rDNA sequences between the type strain of B. denitrificans IFAM 1005 (LMG8443) and several bradyrhizobial isolates. From this work, authors demonstrated that B. denitrificans could well form a symbiotic relationship with A. indica and suggest that the type strain for B. denitrificans be transferred to an existing genus or become the type strain for a new genus. The fast growing rhizobial strains ORS 217 and ORS 214, belonging respectively to the electrophoretic groups II and III are closely related to Rhizobi-
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um tropici IIb. The fast growing strain ORS 207 grouping in electrophoretic cluster IV close to Mesorhizobium huakii and M. plurifarium, has a 16SrDNA sequence close to M. plurifarium. The phylogenetic results obtained by 16SrDNA sequence analysis confirms those of protein patterns numerical analysis, but internal relationships within the different clusters are not clearly elucidate. Haukka et al. [13] have previously noted the limited use of 16SrDNA sequence at species and subspecies level for fast growing rhizobia, a group of organisms where lateral gene lateral is known to occur and the 16S rDNA genes show relatively little divergence. Similarly for bradyrhizobia, this study and others [3, 44] demonstrate that 16SrDNA sequence provide little phylogenetic depth. According to these authors, the spacer DNA region between the 16S and 23S rDNA in bacteria generally shows much more variation, both in length and sequence leading to a higher resolution in the characterization of bradyrhizobia strains. For these reason we extended the genomic investigation by performing the 16S–23S IGS rDNA sequence analysis of slow growing strains classified as Bradyrhizobium sp. Genotypic characterization confirmed the affiliation of strains ORS 204 and ORS 196, both representative strains of electrophoretic group IIIc, to B. elkanii. ORS 206 was identified as Bradyrhizobium japonicum. ORS 199 appear to be on Bradyrhizobium genospecies V. The position of ORS 199 strain was different to those obtained by 16SrDNA sequence analysis. Van Berkum and Eardly [40], reported a similar case where relative placements of members within group 2b of the α-Proteobacteria differed between reconstruction derived from 16S rDNA and those from IGS rDNA region sequence. It represent a case to elucidate, and alternative approaches, for example DNA-DNA hybridization areneeded to assess relationships. Despite this case, we consider that there is good agreement between the three approaches adopted. Consistent with the recent recommendations of the ad hoc committee for the re-evaluation of the species definition in bacteriology [35], we characterized Pterocarpus rhizobia diversity using various techniques which show great promise in bacterial systematic approaches. We thus may conclude that rhizobial strains associated with Pterocarpus erinaceus and P. lucens are diverse. Rhizobial strains were here affiliated to Mesorhizobium plurifarium, Rhizobium tropici, Bradyrhizobium japonicum and B. elkanii. Other strains not related to any of the described rhizobial species remain unaffiliated. An important remark is that among the new isolates, Bradyrhizobium and Rhizobium genus were the most represented, while Mesorhizobium was represented by only one strain isolated from P. erinaceus in Sudanean zone; Azorhizobium and Sinorhizobium were not represented among the new isolates. The predominance of slow growing strains in tropical legumes has already been observed by Norris [29] and is supported by our study. From many tropical Legumes, Clitoria, Dimorphandra, Chamaescrista, Prosopis, Acacia, Albizia, Calliandra, Enterolobium, Pittecellobium, Swartzia, Machaerium, Derris, Lonchocarpus and Sesbania, slow and fast growing strains were isolated and described [23]. There is no clear relation between phylogenetic positions of the strains and their ecogeographical
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origins (sudanean region and sahelian region) or plant host (P. erinaceus and P. lucens) of isolation. The new strains could be mostly related to those isolated from other tropical tree species like A. albida and from herbaceous legumes like Aeschynomene spp. in Senegal. Moreira et al. [23], using SDS PAGE protein profiles numerical analysis, found fast growing strains from Leucaena together with slow growing isolates from Melanoxylon, Abrus and Albizia. In a previous work, Sylla et al. [37] showed that both Pterocarpus erinaceus and P. lucens and also Acacia albida were nodulated by most of their symbionts. Moreover, we showed a great variability for these strains to fix effectively N2 in symbiosis, and Bradyrhizobium spp. strains (B. elkanii, B. japonicum or others) are the most efficient strains [37]. Some of the strains studied here were all found in α-Proteobacteria, closely related to several Rhizobial and Bradyrhizobial described species. However, others form groups a part from known species and may constitute new species. DNA-DNA hybridisation is needed to precise the taxonomical status of these unclassified strains. Acknowledgements This work received financial support of the fund for scientific research FNRAA (ref. 28/AP01SS310800). Dr. S.N.S. is grateful to IFS for individual research grant (ref. 2259-D). Part of this research was carried out in the framework of subcontract INCOENRICH.
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Corresponding author: Samba Ndao Sylla, Université Cheikh Anta Diop, F.S.T., Departement de B.V., B.P. 5005, Dakar, Sénégal Tel.: ++221 849 3322; Fax: ++221 832 1675; e-mail: [email protected]
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal Samba Ndao Sylla1, Ramatoulaye Thiaba Samba2, Marc Neyra2, Ibrahima Ndoye1, Eric Giraud3, Anne Willems4, Philippe de Lajudie3, and Bernard Dreyfus3 1
Université Cheikh Anta Diop, F.S.T., Dakar, Sénégal Laboratoire de Microbiologie, IRD, Dakar, Sénégal 3 LSTM, UMR1063, IRD/CIRAD/INRA/ENSA-M, Montpellier, France 4 Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium 2
Received: October 14, 2002
Summary A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus erinaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S–23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S–23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorhizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrhizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrhizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins. Key words: Bradyrhizobium – Rhizobium – Pterocarpus spp. – phylogeny – diversity – 16SrDNA sequence – 16S–23SrDNA IGS sequence
Introduction Pterocarpus erinaceus and Pterocarpus lucens are tree species belonging to the family Leguminosae, subfamily Fabaceae [1]. They are endemic to the sahelian arid and soudanean semi arid tropical regions respectively. They have high socio-economic and ecological importance as sources of forage and wood for local populations and might improve N2 status of soil, due to their nitrogen fixing symbiosis with rhizobia [25, 38]. The natural populations of Pterocarpus erinaceus and P. lucens are disappearing from their natural areas and yet their cultivation is included in reforestation programs in Senegal. Inoculation by rhizobium is necessary to improve plant growth at the nursery stage and transplantation success. In order to optimise biological nitrogen fixation efficiency, the biodiversity of their associated rhizobia has to be exam-
ined. The wide distribution of Pterocarpus erinaceus and P. lucens in diverse ecological arid and semi-arid conditions [11, 25] suggested that rhizobia associated with Pterocarpus spp. may be heterogeneous, similar to what was described for rhizobia isolated from many tropical tree legume species. Early studies showed that Acacia spp. and other woody legumes are nodulated by fast-growing and/or slow-growing Rhizobium spp. [2, 8, 39]. More recent numerical phenotypic and genotypic analyses revealed a large genetic diversity among rhizobia isolated from several tree legume species, and several new species were described [5, 6, 19, 20, 23, 26, 30, 50]. To date nodule-forming bacteria on legumes are classified in more than 30 validly published species in 10 genera eight of which belonging to the α-Proteobacteria Rhizobium, 0723-2020/02/25/04-572 $ 15.00/0
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal
Bradyrhizobium, Mesorhizobium, Sinorhizobium, Azorhizobium, Allorhizobium [48, 49], Methylobacterium [36], Blastobacter [40] and most surprisingly two of which belonging to the β-Proteobacteria Ralstonia [4] and Burkholderia sp. [24]. Thus rhizobia form a phylogenetically heterogeneous group, and their taxonomy is being reexamined. Rhizobia are interspersed with clinical bacteria, like Afipia, and other phytopathogenic bacteria, like Mycoplana and Bartonella [45, 47]. Until now, the taxonomic status of published strains isolated from various legumes is not clarified [7, 9, 21, 23]. Recently, Young et al. [49] proposed to group Rhizobium, Allorhizobium and Agrobacterium in one revised genus Rhizobium. Bradyrhizobium contains three named species B. japonicum, B. elkanii and Bradyrhizobium liaoningense [17, 46], but Willems et al. [44] identified eight other potential genospecies among isolates from Aeschynomene, A. albida and other small legumes native of Senegal. In a previous study, based on nodulation plant test, antibody tests and growth characteristics, Sylla et al. [37] revealed a great diversity of indigenous rhizobia isolated from P. erinaceus and P. lucens in Senegal. In this paper, a polyphasic approach combining phenotypic and genotypic methods was used to further investigate molecular diversity of these rhizobial isolates by comparative SDS-PAGE analysis of proteins and 16S rDNA gene and 16S–23S rDNA IGS sequencing.
Material and Methods Phenotypic Characterization Strains used. Bacterial strains used in this study and their sources are listed in Table 1. Rhizobial strains were isolated from naturally occurring Pterocarpus root nodules [37]. The nodules were washed and immersed in 0.1% HgCl2 for 5 min.; after this, the nodules were manipulated aseptically. Each nodule was rinsed eight times in sterile water and crushed in a drop of sterile water. The resulting suspension was streaked onto yeast mannitol agar (YMA) plates [43], and isolated colonies appeared after incubation for 2 days (fast growing) and more than 3 days (slow growing) at 30 °C. Slow growing and fast growing status of 3 representative strains of each group was confirmed by measuring generation time as described by Jordan [15]. Pure cultures were obtained after single colonies were streaked two or three times. Isolates were checked for nodulation on their original host plants, and stored at –80 °C in YM adjusted to 20% (v/v) glycerol [37]. PAGE of total bacterial proteins. A selection of 28 fast growers and 22 slow growers Pterocarpus rhizobial new isolates were analysed and compared to 67 reference strains. For comparison, type and representative strains of the different species of Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium, were included. Strains were grown in Roux flasks at 28 °C for 48 h (fast growers) or 72 h (slow growers), on TY medium containing (g/l): tryptone (Oxoid), 5; Yeast extract, 0.75; KH2PO4, 0.454; Na2HPO4,12 H2O, 2.388; agar (Lab M), 20; pH 6.8–7. Whole-cell protein extracts were prepared and SDS-PAGE was performed as previously described [6]. The normalised densitometric traces of the protein electrophoretic patterns were grouped by performing a numerical analysis using the Gel Compar 2.2 software package [42]. The
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similarity between all pairs of traces was expressed by the Pearson product moment correlation (r) converted for convenience to percent value [33]. Genotypic characterization Bacterial strains. Seven rhizobial strains (ORS196, ORS199, ORS204, ORS206, ORS207, ORS214 and ORS217) were selected as representatives of different clusters and subclusters defined from numerical analysis of the protein patterns. Culture conditions and DNA isolation. Strains were grown at 28 °C for 48 to 72 hours on yeast extract mannitol agar YMA. DNA was purified from cells treated with sodium dodecyl sulfate (1% [wt/vol])-Pronase (50 g/ml), and then subjected to serial extractions with phenol chloroform (1:1 [vol/vol]) and precipitation with ethanol and NaCl as described by MartinezRomero et al. [18]. After precipitation, purified DNA was washed three times in ethanol, dried, dissolved overnight at 4 °C in TE buffer, and treated (60 min, 37 °C) with RNAse (50 µg/ml final concentration). The control of the DNA quality and the calculation of DNA concentration were performed as described by Henydrickx et al. [14]. PCR amplification. PCR was performed in a Perkin Elmer 2400 Thermal cycler in a 25 µl (total volume) reaction mixture containing 50 ng of bacterial genomic DNA extract; 2.5 mM of each deoxynucleotide triphosphate (Pharmacia Biotech), 10 mM Tris-HCl, (pH 9 at room temperature); 50 mM KCl; 1.5 mM MgCl2; 20 mM of each primers and 1 U of Taq polymerase, (Eurogentec Bel SA). A negative control where water was substituted to DNA was included in every PCR run. 16S rDNA PCR amplification was done using primers FGPS 6 [27] and FGPS 1509 [27] to the following program : initial denaturation at 94 °C for 5 min, followed by 35 cycles consisting of a 30 sec. denaturation at 94 °C, 30 sec. at annealing temperature of 60 °C, and a 2 min. primer extension at 72 °C. Final elongation was performed at 72 °C for 7 min. 16S–23S rDNA PCR amplification was done using primers FGPS1490-72 [28], and FGPL132-38 [32] to the following program profiles: initial denaturation at 95 °C for 5 min, followed by 35 cycles consisting of a 30 sec. denaturation at 95 °C, 1 min at annealing temperature of 55 °C, and a 1 min primer extension at 72 °C. Final elongation was performed at 72 °C for 3 min. PCR amplification was controlled by horizontal electrophoresis in 1% SIGMA Type II agarose gels. DNA molecularweight-marker (Smart Ladder) was used for gel calibration. Electrophoreses were carried out at 100 V for 30 min. The amplified product was stained 30 min in an aqueous solution of ethidium bromide (1 µg/ml) and photographed under UV illumination with Gel Doc (BIO-RAD) software. PCR products were purified using QIAquick PCR purification kit (Quiagen) following the manufacturer’s instructions. DNA sequencing. The purified PCR products corresponding either to 16 S rDNA fragment or 16–23S rDNA IGS were sequenced using primers listed in Table 2. The ABI Prism Big Dye Terminator Cycle sequence DNA Kit (Applied Biosystems, Forster City, California) was used. Sequencing reaction was performed by PCR amplification in a final volume of 20 µl according to Applied Biosystems protocol. After heating to 96 °C for 3 min, the reaction was cycled as follows: 30 cycles of 30 s at 96 °C, 30 s at 55 °C, and 4 min at 60 °C (2400 thermal cycler Perkin Elmer). Sequencing reactions were analysed on an Applied Biosystems model 310 DNA sequencer (Perkin Elmer Co.). Sequence analysis. Nucleotide sequences were analysed and corrected with Autoassembler software on the basis of electrophoregrams. The double strands were aligned in the consensus sequences by using the algorithm blast and the closely relat-
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Table 1. Nomenclature of strains used. Strains and (growth caracteristics)
LMG Code
Pant of isolation
Geographical origin
Fast growing isolates from Pterocarpus spp. ORS 207 ORS 210 LMG 15199 ORS 211 LMG 15200 ORS 212 ORS 214 LMG 15203 ORS 215 LMG 15204 ORS 217 LMG 15206 ORS 220 LMG 15208 ORS 227 LMG 15214 ORS 230 LMG 15216 ORS 231 LMG 15217 ORS 235 ORS 239 LMG 15222 ORS 240 LMG 15223 ORS 242 LMG 15225 ORS 243 LMG 15226 ORS 246 LMG 15227 ORS 247 LMG 15228 ORS 248 LMG 15229 ORS 251 LMG 15230 ORS 252 ORS 253 LMG 15232 ORS 255 LMG 15234 ORS 256 ORS 257 LMG 15236 ORS 259 LMG 15237 ORS 260 ORS 261 LMG 15239
P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens
Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal
Slow growing isolates from Pterocarpus spp. ORS 196 LMG 15188 ORS 198 ORS 199 LMG 15190 ORS 200 ORS 201 LMG 15192 ORS 203 LMG 15193 ORS 204 LMG 15194 ORS 206 LMG 15196 ORS 208 ORS 221 LMG 15209 ORS 222 ORS 223 LMG 15211 ORS 226 LMG 15213 ORS 228 LMG 15215 ORS 232 ORS 233 ORS 238 ORS 241 LMG 15224 ORS 244 ORS 245 ORS 263 ORS 264
P. lucens P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. erinaceus P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens P. lucens
Sahelian zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sudanean zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal Sahelian zone, Senegal
Reference strains used for numerical analysis of protein patterns (SDS-PAGE) Agrobacterium (separate taxa) NCPPB 1771 LMG 233A Agrobacterium biovar 1 B6T B2 A ATCC 19358T
LMG 187TA LMG 268A LMG 140TA
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal Table 1. (Continued). Strains and (growth caracteristics)
LMG Code
Agrobacterium biovar 2 ATCC 13335
LMG 156TA
Agrobacterium rhizogenes ATCC 11325T
LMG 150TA
Agrobacterium vitis PAN AG 61
LMG 257A
Azorhizobium caulinodans (fast growing) FY 12 LMG 11352 ORS 478 LMG 11820 ORS 571T LMG 6465T ORS 486? LMG11822
Pant of isolation
Geographical origin
Sesbania rostrata Sesbania rostrata Sesbania rostrata Sesbania rostrata
Senegal Senegal Senegal Senegal
Bradyrhizobium japonicum NZP 5533 NZP 5549T Cb 756 NZP 2314 NZP 2309
LMG 6136 LMG 6138T LMG 8319 LMG 6129 LMG 6128
Glycine max Glycine hispida
USA Japon
Bradyrhizobium elkanii NZP 5531T NZP 5532
LMG 6134T LMG 6135
Glycine max Glycine max
USA USA
Bradyrhizobium sp. (Gr. 1ND,Gr.12FM.) Br. 29 Br. 6011 Br. 6009 Br 8402 INPA 260 MAR 411 ORS 184 ORS 188 ORS 347 ORS 306 ORS Pa 44
LMG 9520 LMG 9514 LMG 9512 LMG 10015 LMG 1090 LMG 14300 LMG 10723 LMG 10727 LMG 10298 LMG 11797 LMG 11928
Lonchocarpus costatus Lonchocarpus costatus Dalbergia riparia Dalbergia riparia
Brazil Brazil Brazil Brazil Brazil
Acacia albida Acacia albida Aeschynomene indica Aeschynomene indica Prosopis africana
Senegal Senegal Senegal Senegal
Mesorhizobium sp. cicer (fast growing) IC-60 LMG 14995
Cicer arietinum
Spain
Mesorhizobium mediterraneum (fast growing) UPM Ca 142 LMG 14990
Cicer arietinum
Spain
Rhizobium galeagae (fast growing) HAMBI 540T LMG 6214 HAMBI 1147 LMG 6215
Galegae orientalis Galegae orientalis
Finland Finland
Rhizobium leguminosarum (fast growing) biovar viciae NZP 561 LMG 6122 biovar viciae ATCC 10004T LMG 8817 biovar phaseoli ATCC 14482 LMG 8819
Pisum sativum Phaseolus L.
Rhizobium tropici (fast growing) CFN 299 LMG 9517
Phaseolis vulgaris
Brazil
Rhizobium sp. (fast growing) (Gr.5, Gr.7 et Gr.15 FM.) Br 809 LMG 9950 Br. 8803 LMG 10022 INPA 95A LMG 10134 INPA 133B LMG 10062
Leucaena leucocephala Glyricidia sepium Leucaena pulvurulenta Leucaena leucocephala
Brazil Brazil Brazil Brazil
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Table 1. (Continued). Strains and (growth caracteristics)
LMG Code
Pant of isolation
Geographical origin
Lotus corniculatus Lotus marccanus
New Zealand Maroc
Mesorhizobium plurifarium (fast growing) INPA 129 A LMG 6123 NZP 2037 LMG 6123 ORS 1004 LMG 7848 ORS 1014t2 LMG 7849 ORS 1029 LMG 11889 ORS 1040 LMG 11898 ORS 1032T LMG 11892T
Lotus divaricatus Lotus divaricatus Acacia senegal Acacia senegal Acacia senegal Acacia senegal Acacia senegal
New Zealand New Zealand Senegal Senegal Senegal Senegal Senegal
Mesorhizobium ciceri (fast growing) UPM Ca 7T LMG 14989 IC 2091 LMG 15086
Cicer arietinum Cicer arietinum
Spain Spain
Mesorhizobium huakuii (fast growing) IAM 14158T LMG 14107
Astragalus sinicus
China
Sesbania pachycarpa Sesbania grandiflora Sesbania cannabina Sesbania cannabina Prosopis juliflora
Senegal Senegal Senegal Senegal Senegal
Acacia laeta Acacia senegal
Senegal Senegal
Acacia raddiana
Senegal
Medicago sativa Medicago sativa
Australia Australia
Mesorhizobium loti (fast growing) NZP 2213 LMG 6125 NZP 2230 LMG 6126 USDA 208 LMG 6219
Mesorhizobium huakuii (fast growing) UPM Ca142 LMG14990 Sinorhizobium fredii (fast growing) USDA 191 LMG 6217 USDA 205T LMG 8317T Sinorhizobium saheli (fast growing) ORS 600 LMG 11864 (YM) ORS 611 LMG 8310 ORS 609T LMG 7837Td ORS 609t2 LMG 8309 ORS 12 = Pj12 LMG 7835 Sinorhizobium terangae (fast growing) ORS 1009 LMG 7834 ORS 1013 LMG 7844T1 ORS 1025 LMG 11885 ORS 1045 LMG 11901 Sinorhizobium meliloti (fast growing) NZP 4009 LMG 6130 NZP 4027T LMG 6133
ATCC, American type Culture collection. Rockville, Md.; Br. Strain from CNPBS/EMBRAPA, Centro Nacional de Pesquisa em Biologia do solo, Seropedica, Rio de Janeiro, Brasiliera de Pesquisa Agropecaria; CDC, Centers for Desease Control, Atlanta, Ga.; CFN, Centro de investigacion sobre Fijacion de Nitrogeno, Universidad Nacional Autonomade Mexico, cueranavaca, Mexico; HAMBI, Culture Collection of the Departement of Microbiology, University of Helsinki, Finland; IAM Institute of Applied Microbiology, the University of Tokyo, Japan; IC et UPM, Departemento de Microbiologia, E.T.S. de Ingenieros Agronomos, Universidad Politecnica, Madrid, Spain; INPA, National Institute of Amazonia Reserch, Manaus Brazil; LMG, Collection of Bacteria of the Laboratorium voor microbiologie, University of Ghent, Belgium; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, U.K.; NZP, Culture Collection of the Departement for Scientific Research and Industrial, Biochemistry Division, Palmerson North, New Zealand; ORS , Collection de l'Institut Français de Recherche Scientifique pour le Développement en Coopération, dakar, Sénégal; pan, Panagopoulos, Crete, greece; USDA, united States Departement of Agriculture, Beltsville, Md.
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Table 2. PCR and sequencing primers used in this study. Primer name
Primer sequence (5′ – 3′)
Target Gene
Reference
FGPS6 FGPS 1509 16S 370f 16S-1080rev 16S 870f 16S 1924 rev FGPL 132-38 FGPS 1490-72
(5′-GGA-GAG-TTA-GAT-CTT-GGC-TCA-G-3′) (5′-AAG-GAG-GGG-ATC-CAG-CCG-CA-3′) (5′-GGC-AGC-AGT-GGG-GAA-TAT-TG-3′) (5′-GGG-ACT-TAA-CCC-AAC-ATC-T-3′) (5′-CCT-GGG-GAG-TAC-GGT-CGC-AAG-3′) (5′-GGC-ACG-AAG-TTA-GCC-GGG-GC-3′) (5′-CCGGGTTTCCCCATTCGG-3′) (5′-TGCGGCTGGATCCCCTCCTT-3′)
16SrDNA 16SrDNA 16SrDNA 16SrDNA 16SrDNA 16SrDNA IGS rDNA IGS rDNA
Normand et al. (1992) Normand et al. (1992) Sy et al. (2001) Sy et al. (2001) Sy et al. (2001) Sy et al. (2001) Ponsonnet et al. (1994) Normand et al. (1996)
ed sequences found were included in the following analyses. Phylogenetic analysis was inferred by using the CLUSTAL X program of the GCG (Genetics Computer Group, Wisconsin). Matrix pairwise comparisons were corrected for multiple base substitutions by the two-parameter method of Kimura [16]. Phylogenetic trees were constructed using the neighbour-joining [34] method. A bootstrap confidence analysis was performed on 1000 replicates to determine the reliability of the distance tree topology obtained [10]. Graphic representation of the resulting trees was done using njplot [31] software.
Results Numerical analysis of proteins patterns. We analysed 50 protein profiles from fast-growing and slow-growing nodule isolates from P. erinaceus and P. lucens. Fig. 1 shows a simplified dendrogram including bacterial isolates from Pterocarpus spp. and their closest neighbours as references. Results show that at a coefficient similarity of 81%, Pterocarpus rhizobial strains are heterogeneous and form seven groups (I to VII). Nineteen slow-growing strains constitute the Cluster I, where we distinguish four subclusters (Ia, Ib, Ic, Id). Subcluster Ia includes four new isolates and one Bradyrhizobium sp. (A. albida) strain (ORS 184) published by Dupuy et al. [9]. Subcluster Ib consists of four Pterocarpus strains and one strain of Bradyrhizobium sp. (ORS 188) identified by Dupuy et al. [9]. Subcluster Ic consists of eight new isolates and includes the type strain of Bradyrhizobium japonicum (NZP 5549T). Subcluster Id consists of new isolates from both Pterocarpus species. A total number of 31 fast-growing strains constituted clusters II, IV, V, VI, VII and numerous separate strains. Cluster II consists of four Pterocarpus spp. new isolates and includes Rhizobium sp. strain Br. 809 identified by Moreira et al. [23], as separate from all described species. Cluster III consists of six new isolates from Pterocarpus spp not related to any reference strain. Cluster IV consists of new isolates from Pterocarpus spp. and includes type strains of Mesorhizobium plurifarium (ORS1032T) and Mesorhizobium huakuii (IAM14158 T). Clusters V, VI and VII are small groups each consisting of a few set of Pterocarpus strains. 16S rDNA gene sequence analysis. The 16S rDNA sequences generated were deposited in the Genbank database to search for significant 16S rDNA alignments.
The complete 16S rDNA sequences of seven strains ORS 196, ORS 199, ORS 204, ORS 206, ORS 207, ORS 214 and ORS 217 received respectively accession numbers AF514792, AF514794, AF5147916, AF5147918, AF514800, AF5801 and AF514802. The 16S rDNA gene sequences (> 1460 bp) were compared to those of members of Azorhizobium, Bradyrhizobium, Methylobacterium, Mesorhizobium, Rhizobium, Sinorhizobium species and also to members of Afipia, Agrobacterium, Blastobacter, Nitrobacter, Rhodopseudomonas, and Xanthobacter, and a phylogeny tree was inferred (Fig. 2.). The sequence divergence obtained by comparing 16S rDNA gene among the strains ORS 196, ORS 199, ORS 204, ORS 206, Bradyrhizobium elkanii, B. japonicum, B. liaoningense, B. sp. (Lupinus), Blastobacter denitrificans, Nitrobacter winogradski Rhodopseudomonas palustris was less than 0.2% (99.8% of sequence similarities). This degree of similarity is very low to distinguish phylogenetic placement of taxa. Similarly, sequences divergence within the group of Mesorhizobium species group including ORS 207 showed to be less than 0.2% and also the group constituted by ORS 214, ORS 217, Rhizobium species and Agrobacterium rhizogenesT appear to be highly similar. The reconstruction of phylogenetic tree based on 16S rDNA sequence analysis (Fig. 2) separated the seven Pterocarpus isolates into three major groups: Rhizobium spp. strains; Mesorhizobium spp strains; Bradyrhizobium elkanii and B. japonicum strains. Except the group of B. japonicum, the separate groups of strains were supported by significant bootstraap values. Inside these clusters, ORS 214 and ORS 217 are situated in the vicinity of Rhizobium tropici IIB; ORS 207, groups close to Mesorhizobium plurifarium; ORS 199 forms a branch with Blastobacter denitrificans, Bradyrhizobium liaoningense and B. sp. (Lupinus) as closest neighbour; the slow growing strains ORS 204 and ORS 196 are positioned in the branch of B. elkanii; the slow growing strain ORS 206 is located in the vicinity of Bradyrhizobium japonicum. 16S–23S IGS rDNA sequencing. The 16S–23S IGS rDNA sequences generated for Bradyrhizobia strains ORS 196, ORS 199, ORS 204 and ORS 206 were deposited to the Genbank database and received respectively accession numbers AF514793, AF514795, AF514797 and AF514799. The IGS sequences obtained varied in length from 890 to 1054 nucleotides. To precise phylogenetic diversity of the Bradyrhizobium sp. (Pterocarpus) strains,
Fig. 1. Dendrogram showing the relationship between the electrophoretic protein patterns from Pterocarpus spp. rhizobium strains and reference strains. The mean correlation coefficient ® was calculated by unweighted average pairs grouping method (UPGMA). Reference strains are limited to the closest neighbours to new isolates: ORS184; ORS188; ORS306; ORS347; ORS1032T; ORS1270; NZP2314; NZP5531T; NZP5533; NZP5549T; NZP5532; Br29; Br809; CB756; ATCC11325T; IAM14158; and UPMCa142.
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Fig. 2. Neighbor joining dendrogram showing the sequence similarities of 16S rDNA gene sequences of new isolated strains (bold style) and some related organisms. The length of the aligned sequences was >1460 bases and bootstrap values were calculated from 1000 trees. The levels of support for the presence of nodes above a value of 60% ar eindicated.
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Fig. 3. Neighbor joining dendrogram shwoing the sequence similarities of 16S–23S rDNA internal gene spacer sequences of Bradyrhizobium strains isolated from Ptercarpus trees (bold style) and some related organisms. Roman numbers correspond to Bradyrhizobium genospecies as described by Willems et al. (2001). The length of the aligned sequences was >890 bases. Bootstrap values were calculated from 1000 trees and the levels of support for the presence of nodes above a value of 60% are indicated.
we performed sequence alignments of Pterocarpus spp. strains and compared them with those of members of Bradyrhizobium species available in Genbank database. We included recent Bradyrhizobium genospecies reported by van Berkum et al. [41] and by Willems et al. [44]. The IGS sequence of Blastobacter denitrificans (AF 338176) and BTAi1 (AF338169) were also added. All IGS sequences determined comprised two tRNA genes, for tRNAIle and tRNAAla that were located in the middle to first half of the spacer region. This structure is similar to that reported for the majority of Gram-negative bacteria [12]. The dendrogram obtained by neighbor-joining analysis of the similarity matrix is shown in Fig. 3.
ORS 196 and ORS 204 grouped in a major cluster with strains of Bradyrhizobium elkanii as closest neighbour and strains of genospecies VII [44]. Relatively little variation in the IGS sequence (98–99%) similarities was found between the different members of this cluster. The sequence similarities between ORS 196, ORS 204, B. elkanii (type strain) and members of genospecies II and VII was supported with high values from bootstrap analysis (Fig. 3). ORS 206 was on the Bradyrhizobium liaoningense and Bradyrhizobium japonicum branch. IGS sequence similarities founded between ORS 206 and these reference strains were respectively 98.9 and 98.5%. In Fig. 3, the Pterocarpus isolates, ORS 206 was together with
Phenotypic and Genotypic Diversity of Rhizobia Nodulating Pterocarpus erinaceus and P. lucens in Senegal
members of Bradyrhizobium liaoningense and Bradyrhizobium japonicum but their phylogenetic relationships are not well supported by bootstrap values. ORS 199 was inside the group consisting of Bradyrhizobium genospecies V [44]. IGS sequence divergence values calculation revealed that Pterocarpus isolates have 99–99.5 % similaries with members of genospecies V described by Willems et al. [44]. The phylogenetic position of ORS 199 within the members of genospecies V is supported by significant bootstrap values.
Discussion A total of fifty strains isolated from Pterocarpus erinaceus and P. lucens nodules in Senegal was examined. Among these strains we previously distinguished two types of rhizobia: fast growing and slow growing ones. Here, we further characterized them using phenotypic and genotypic approaches to determine their phylogenetic diversity. As a grouping method, we used SDS-PAGE of total protein profiles comparison and included the results in the large database available in our research group. Results confirmed our previous classification based on growth rate [37]. Consistent with the results reported on tropical rhizobia isolated from Acacia (Faidherbia) albida [9] and from various leguminous trees [6, 22, 26, 50], we found a considerable heterogeneity within both fast- and slow-growing nodule isolates from Pterocarpus spp. Slow growing strains form one major cluster (cluster I) subdivided into four subclusters. This major cluster corresponds to Bradyrhizobium. The fast growing rhizobia grouped in six electrophoretic clusters and several strains were separate. Except ORS 207 grouping with Mesorhizobium plurifarium and Mesorhizobium huakuii, the fast growing rhizobia of our collection clustered apart from described strains suggesting that they represent new groups. To further describe and differentiate the new groups obtained by SDS-PAGE, we performed genotypic investigations across the entire 16S rDNA gene and the 16S–23S rDNA IGS region by sequence analysis. Rhizobial strains representative for seven SDS-PAGE groups or subgroups were selected for 16S rDNA sequence analysis. Results corroborated those obtained previously. The slow-growing strains clustered with either B. japonicum type strain (cluster Ic) or closely related to B. elkanii. In reconstruction derived from 16S rDNA sequence divergence, one of the slow growing rhizobia (ORS 199) isolated from Pterocarpus spp. grouped with Bradyrhizobium spp. and Blastobacter denitrificans. This result related those of van Berkum and Eardly [40] which showed high identity in 16S rDNA sequences between the type strain of B. denitrificans IFAM 1005 (LMG8443) and several bradyrhizobial isolates. From this work, authors demonstrated that B. denitrificans could well form a symbiotic relationship with A. indica and suggest that the type strain for B. denitrificans be transferred to an existing genus or become the type strain for a new genus. The fast growing rhizobial strains ORS 217 and ORS 214, belonging respectively to the electrophoretic groups II and III are closely related to Rhizobi-
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um tropici IIb. The fast growing strain ORS 207 grouping in electrophoretic cluster IV close to Mesorhizobium huakii and M. plurifarium, has a 16SrDNA sequence close to M. plurifarium. The phylogenetic results obtained by 16SrDNA sequence analysis confirms those of protein patterns numerical analysis, but internal relationships within the different clusters are not clearly elucidate. Haukka et al. [13] have previously noted the limited use of 16SrDNA sequence at species and subspecies level for fast growing rhizobia, a group of organisms where lateral gene lateral is known to occur and the 16S rDNA genes show relatively little divergence. Similarly for bradyrhizobia, this study and others [3, 44] demonstrate that 16SrDNA sequence provide little phylogenetic depth. According to these authors, the spacer DNA region between the 16S and 23S rDNA in bacteria generally shows much more variation, both in length and sequence leading to a higher resolution in the characterization of bradyrhizobia strains. For these reason we extended the genomic investigation by performing the 16S–23S IGS rDNA sequence analysis of slow growing strains classified as Bradyrhizobium sp. Genotypic characterization confirmed the affiliation of strains ORS 204 and ORS 196, both representative strains of electrophoretic group IIIc, to B. elkanii. ORS 206 was identified as Bradyrhizobium japonicum. ORS 199 appear to be on Bradyrhizobium genospecies V. The position of ORS 199 strain was different to those obtained by 16SrDNA sequence analysis. Van Berkum and Eardly [40], reported a similar case where relative placements of members within group 2b of the α-Proteobacteria differed between reconstruction derived from 16S rDNA and those from IGS rDNA region sequence. It represent a case to elucidate, and alternative approaches, for example DNA-DNA hybridization areneeded to assess relationships. Despite this case, we consider that there is good agreement between the three approaches adopted. Consistent with the recent recommendations of the ad hoc committee for the re-evaluation of the species definition in bacteriology [35], we characterized Pterocarpus rhizobia diversity using various techniques which show great promise in bacterial systematic approaches. We thus may conclude that rhizobial strains associated with Pterocarpus erinaceus and P. lucens are diverse. Rhizobial strains were here affiliated to Mesorhizobium plurifarium, Rhizobium tropici, Bradyrhizobium japonicum and B. elkanii. Other strains not related to any of the described rhizobial species remain unaffiliated. An important remark is that among the new isolates, Bradyrhizobium and Rhizobium genus were the most represented, while Mesorhizobium was represented by only one strain isolated from P. erinaceus in Sudanean zone; Azorhizobium and Sinorhizobium were not represented among the new isolates. The predominance of slow growing strains in tropical legumes has already been observed by Norris [29] and is supported by our study. From many tropical Legumes, Clitoria, Dimorphandra, Chamaescrista, Prosopis, Acacia, Albizia, Calliandra, Enterolobium, Pittecellobium, Swartzia, Machaerium, Derris, Lonchocarpus and Sesbania, slow and fast growing strains were isolated and described [23]. There is no clear relation between phylogenetic positions of the strains and their ecogeographical
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origins (sudanean region and sahelian region) or plant host (P. erinaceus and P. lucens) of isolation. The new strains could be mostly related to those isolated from other tropical tree species like A. albida and from herbaceous legumes like Aeschynomene spp. in Senegal. Moreira et al. [23], using SDS PAGE protein profiles numerical analysis, found fast growing strains from Leucaena together with slow growing isolates from Melanoxylon, Abrus and Albizia. In a previous work, Sylla et al. [37] showed that both Pterocarpus erinaceus and P. lucens and also Acacia albida were nodulated by most of their symbionts. Moreover, we showed a great variability for these strains to fix effectively N2 in symbiosis, and Bradyrhizobium spp. strains (B. elkanii, B. japonicum or others) are the most efficient strains [37]. Some of the strains studied here were all found in α-Proteobacteria, closely related to several Rhizobial and Bradyrhizobial described species. However, others form groups a part from known species and may constitute new species. DNA-DNA hybridisation is needed to precise the taxonomical status of these unclassified strains. Acknowledgements This work received financial support of the fund for scientific research FNRAA (ref. 28/AP01SS310800). Dr. S.N.S. is grateful to IFS for individual research grant (ref. 2259-D). Part of this research was carried out in the framework of subcontract INCOENRICH.
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Corresponding author: Samba Ndao Sylla, Université Cheikh Anta Diop, F.S.T., Departement de B.V., B.P. 5005, Dakar, Sénégal Tel.: ++221 849 3322; Fax: ++221 832 1675; e-mail: [email protected]