Multilocus sequence analysis of root nodule isolates from Lotus arabicus (Senegal), Lotus creticus, Argyrolobium uniflorum and Medicago sativa (Tunisia) and description of Ensifer numidicus sp. nov. and Ensifer garamanticus sp. nov.

Nine isolates from Argyrolobium uniflorum, Lotus creticus , Medicago sativa (Tunisia) and Lotus arabicus (Senegal) were analysed by multilocus sequence analysis (MLSA) of five housekeeping genes (recA, atpD, glnA, gltA and thrC), the 16S rRNA gene and the nodulation gene nodA. Analysis of the individual and concatenated gene sequences demonstrated that the nine new strains constituted three stable, well-supported (bootstrap and gene sequence similarity values) monophyletic clusters, A, B and C, all belonging to the branch of the genus Ensifer, regardless of the phylogenetic reconstruction method used (maximum likelihood, maximum-parsimony, neighbour-joining). The three groups were further characterized by API 100 auxanographic tests, host specificity and nodA gene sequence analysis. On the basis of these data, clusters A and C are suggested as representing two novel species within the genus Ensifer, for which the names Ensifer numidicus sp. nov. (type strain ORS 1407(T)=LMG 24690(T)=CIP 109850(T)) and Ensifer garamanticus sp. nov. (type strain ORS 1400(T)=LMG 24692(T)=CIP 109916(T)) are proposed. The cluster B strains were assigned to Ensifer adhaerens genomovar A.

Multilocus sequence analysis of bradyrhizobia isolated from Aeschynomene species in Senegal.

This study reports the multilocus sequence analysis (MLSA) of nine house-keeping gene fragments (atpD, dnaK, glnA, glnB, gltA, gyrB, recA, rpoB and thrC) on a collection of 38 Bradyrhizobium isolated from Aeschynomene species in Senegal, which had previously been characterised by several phenotypic and genotypic techniques, allowing a comparative analysis of MLSA resolution power for species delineation in this genus. The nifH locus was also studied to compare house-keeping and symbiotic gene phylogenies and obtain insights into the unusual symbiotic properties of these Aeschynomene symbionts. Phylogenetic analyses (maximum likelihood, Bayesian) of concatenated nine loci produced a well-resolved phylogeny of the strain collection separating photosynthetic bradyrhizobial strains (PB) from non-photosynthetic bradyrhizobial (NPB) ones. The PB clade was interpreted as the remains an expanding ancient species that presently shows high diversification, giving rise to potential new species. B. denitrificans LMG8443 and BTAi1 strains formed a sub-clade that was identified as recently emerging new species. Congruence analyses (by Shimodaira-Hasegawa (S-H) tests) identified three gene-fragments (dnaK, glnB and recA) that should be preferred for MLSA analyses in Bradyrhizobium genus. The nine loci or nifH phylogenies were not correlated with the unusual symbiotic properties of PB (nod-dependent/nod-independent). Advantages and drawbacks of MLSA for species delineation in Bradyrhizobium are discussed.

Phylogenetic and symbiotic characterization of rhizobial bacteria nodulating Argyrolobium uniflorum in Tunisian arid soils.

Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tunisia were characterized by phenotypic features, RFLP, and sequencing of PCR-amplified 16S rRNA genes. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at temperatures up to 40 degrees C. Phylogenetically, the new isolates were grouped in the genera Sinorhizobium (27), Rhizobium (13), and Agrobacterium (2). Except for the 2 Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among them. Sinorhizobium sp. strains STM 4034, STM 4036, and STM 4039, forming the most effective symbiosis, are potential candidates for inoculants in revegetalisation programs.

Phenotypic and genotypic diversity of Genista saharae microsymbionts from the infra-arid region of Tunisia.

AIMS: Genista saharae, indigenous of Sahara, is a spontaneous shrub that plays an important ecological role for the preservation and fertility of poor and eroded soils. This legume has not been examined for its root nodule bacteria. The taxonomic diversity of bacteria from root nodules of G. saharae growing in the infra-arid region of Tunisia was investigated. METHODS AND RESULTS: A total of 28 bacterial strains isolated from root nodules of G. saharae grown in Tunisian soil were characterized using a polyphasic approach including phenotypic characteristics, PCR-RFLP of 16S rDNA and 16S rRNA gene sequencing. It was found that new isolates are diverse and affiliated to Ensifer (75%), Rhizobium (10%) and Phyllobacterium (15%). The Phyllobacterium isolates lacked the capacity for nodule formation on this plant. CONCLUSIONS: Genista saharae formed nodules with diverse rhizobia in Tunisian soils. Furthermore, our results support the presence of non-nodulating commensal strains (Phyllobacterium) in legumes nodule. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the characterization of G. saharae microsymbionts in Tunisia.

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis.

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.

Phenotypical and genotypical characteristics of root-nodulating bacteria isolated from annual Medicago spp. in Soummam Valley (Algeria).

AIMS: In the framework of agro-pastoral system management using local annual medics coupled with their native root-nodulating bacteria to extend pasture zones, increase forage yields and improve ovine and bovine breeding in Algeria, we investigated diversity of rhizobia from annual Medicago spp. (Medicago arabica, Medicago polymorpha, Medicago minima and Medicago orbicularis). METHODS AND RESULTS: Ten nodulating-isolates were characterized by morphological, cultural, physiological and biochemical features, SDS-PAGE analysis and PCR-RFLP of 16S rDNA. The results show some degree of genetic diversity among the isolates; three can be affiliated to Sinorhizobium meliloti, one to Rhizobium galegae and six were separate. CONCLUSIONS: Local annual medics would have a high degree of specificity in their symbiotic interaction. Furthermore, our results support the presence of Rh. galegae in the Mediterranean region. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is a preliminary step towards selection of efficient symbiotic Medicago-rhizobia to develop inoculants for management of agro-pastoral systems using local annual medics in Algeria.

Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to alpha- and beta-Proteobacteria.

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.

DNA-DNA hybridization study of Bradyrhizobium strains.

DNA-DNA hybridizations were performed between Bradyrhizobium strains, isolated mainly from Faidherbia albida and Aeschynomene species, as well as Bradyrhizobium reference strains. Results indicated that the genus Bradyrhizobium consists of at least 11 genospecies, I to XI. The genospecies formed four subgeneric groups that were more closely related to each other (>40% DNA hybridization) than to other genospecies (<40% DNA hybridization): (i) genospecies I (Bradyrhizobium japonicum), III (Bradyrhizobium liaoningense), IV and V; (ii) genospecies VI and VIII; (iii) genospecies VII and IX; and (iv) genospecies II (Bradyrhizobium elkanii), X and XI. Photosynthetic Aeschynomene isolates were found to belong to at least two distinct genospecies in one subgeneric group. DNA-DNA hybridization data are compared with data from amplified fragment length polymorphism analysis and 165-23S rDNA spacer sequence analysis.

[ Nodulation of certain legumes of the genus Crotalaria by the new species Methylobacterium]. / Certaines légumineuses du genre Crotalaria sont spécifiquement nodulées par une nouvelle espèce de Methylobacterium.

We studied a collection of 126 rhizobial isolates from eight species of Crotalaria (C. comosa, C. glaucoides, C. goreensis, C. hyssopifolia, C. lathyroides, C. perrottetii, C. podocarpa, and C. retusa) growing in Senegal. Nodulation and nitrogen-fixation tests on nine Crotalaria species revealed two specificity groups within the genus Crotalaria. Group I consists of plants solely nodulated by very specific fast-growing strains. Group II plants are nodulated by slow-growing strains similar to promiscuous Bradyrhizobium spp. strains already reported to nodulate many tropical legumes. SDS-PAGE studies showed that slow-growing strains grouped with Bradyrhizobium while fast-growing strains constituted a homogeneous group distinct from all known rhizobia. Amplified ribosomal DNA restriction analysis (ARDRA) of 10 representative strains of this group using four restriction enzymes showed a single pattern for each enzyme confirming the high homogeneity of group I. The 16S rDNA sequence analysis revealed that this specific group belonged to the genus Methylobacterium, thus constituting a new branch of nodulating bacteria.

Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes.

Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the alpha subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that "M. nodulans" is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that "M. nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.

Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata.

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.

Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses.

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species.

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

Polyphasic characterization of rhizobia that nodulate Phaseolus vulgaris in West Africa (Senegal and Gambia).

Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).

Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System.

Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished. These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti. The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris. S. meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp. (Hedysarum coronarium). However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH. The genus Rhizobium contained a single cluster containing Rhizobium sp. (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense. R. tropici and R. hainanense exhibited similarity to Agrobacterium biovar 2, whilst R. galegae was similar to Agrobacterium biovar 1. R. giardinii appeared unique, with comparatively little similarity to the other species. Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied. Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum. Bradyrhizobium liaoningense and Bradyrhizobium sp. (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B. japonicum, whilst a subgroup of M. tianshanense appeared identical to Bradyrhizobium sp. (Arachis hypogaea).

Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov., isolated from leguminous trees in Sudan and Kenya.

SDS-PAGE of total bacterial proteins was applied to the classification of 25 Sudanese and five Kenyan strains isolated from the root nodules of Acacia senegal and Prosopis chilensis. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridization, genomic fingerprinting using repetitive sequence-based PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus Sinorhizobium. Two of the strains grouped with Sinorhizobium saheli and seven with Sinorhizobium terangae, while the rest did not cluster with any of the established species. The majority of the strains formed two phenotypically and genotypically distinct groups and we therefore propose that these strains should be classified as two new species, Sinorhizobium arboris sp. nov. and Sinorhizobium kostiense sp. nov.

Photosynthetic bradyrhizobia from Aeschynomene spp. are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group.

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.

Diversity of bradyrhizobia from 27 tropical Leguminosae species native of Senegal

We isolated 71 slow-growing bacterial strains from nodules of 27 native leguminous plants species in Senegal (West-Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia playing an ecological role and having agronomic potential in arid regions. The isolates were characterised by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA and comparative SDS-PAGE of whole-cell proteins; reference strains of the different known rhizobial species and groups were included as references. We conclude that these nodule isolates are diverse, and form several phylogenetic subgroups inside Bradyrhizobium. Nodulation tests performed on 5 plant species demonstrated host specificity among the strains studied.

Allorhizobium undicola gen. nov., sp. nov., nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal.

A group of nodule isolates from Neptunia natans, an indigenous stemnodulated tropical legume found in waterlogged areas of Senegal, was studied. Polyphasic taxonomy was performed, including SDS-PAGE of total proteins, auxanography using API galleries, host-plant specificity, PCR-RFLP of the internal transcribed spacer region between the 16S and the 23S rRNA coding genes, 16S rRNA gene sequencing and DNA-DNA hybridization. It was demonstrated that this group is phenotypically and phylogenetically separate from the known species of Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, Bradyrhizobium and Azorhizobium. Its closest phylogenetic neighbour, as deduced by 16S rRNA gene sequencing, is Agrobacterium vitis (96.2% sequence homology). The name Allorhizobium undicola gen. nov., sp. nov., is proposed for this group of bacteria, which are capable of efficient nitrogen-fixing symbiosis with Neptunia natans, and the type strain is ORS 992T (= LMG 11875T).

Characterization of tropical tree rhizobia and description of Mesorhizobium plurifarium sp. nov.

A collection of strains isolated from root nodules of Acacia species in Senegal was analysed previously by electrophoresis of total cell protein, auxanographic tests, rRNA-DNA hydridization, 16S rRNA gene sequencing, DNA base composition and DNA-DNA hybridization [de Lajudie, P., Willems, A., Pot, B. & 7 other authors (1994). Int J Syst Bacteriol 44, 715-733]. Strains from Acacia were shown to belong to two groups, Sinorhizobium terangae, and a so-called gel electrophoretic cluster U, which also included some reference strains from Brazil. Further taxonomic characterization of this group using the same techniques plus repetitive extragenic palindromic-PCR and nodulation tests is presented in this paper. Reference strains from Sudan and a number of new rhizobia isolated from nodules of Acacia senegal, Acacia tortilis subsp. raddiana and Prosopis juliflora in Senegal were included. As a result of this polyphasic approach, the creation of a new species, Mesorhizobium plurifarium, is proposed for a genotypically and phenotypically distinct group corresponding to the former cluster U and containing strains isolated from Acacia, Leucaena, Prosopis and Chamaecrista in West Africa (Senegal), East Africa (Sudan) and South America (Brazil). The type strain of Mesorhizobium plurifarium ORS 1032 has been deposited in the LMG collection as LMG 11892.