P. De Lajudie

Polyphasic Taxonomy of Rhizobia: Emendation of the Genus Sinorhizobium and Description of Sinorhizobium meliloti comb. nov., Sinorhizobium saheli sp. nov., and Sinorhizobium teranga sp. nov.

A total of 80 bacterial strains isolated from different Sesbania and Acacia species growing in various sites in Senegal (West Africa) were compared with 35 reference strains of Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium species and with 33 representative strains of the different groups of Brazilian isolates described on the basis of the results of a numerical analysis of the whole-cell protein patterns obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fifty-two strains could be placed in three protein electrophoretic clusters, two of which were different from the clusters containing various reference or representative strains, while 30 other strains could not be placed in any group. The strains belonging to the three clusters were studied by determining their nodulation host ranges and their morphological, physiological, and auxanographic characteristics. Representative strains of the three clusters were also genotypically characterized by determining their DNA base compositions, by performing DNA-DNA and DNA-rRNA hybridization experiments, and by determining their 16S rRNA gene sequences. Our results showed that two of the clusters identified on the basis of SDS-PAGE data are genotypically and phenotypically distinct groups that belong on the Rhizobium meliloti-Rhizobium fredii rRNA branch. The third cluster is localized on the Rhizobium loti rRNA branch in the vicinity of Rhizobium huakuii and contains strains isolated in Africa, in Brazil, and in New Zealand from different leguminous species. On the basis of the results of the present study, we propose to emend the genus Sinorhizobium and to reclassify R. meliloti as Sinorhizobium meliloti comb. nov. In addition, two new species, Sinorhizobium saheli and Sinorhizobium teranga, are proposed for isolates from Senegal.

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).

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.

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.

Nitrogen-fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to α- and β-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 α‐class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the β‐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.

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).

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.

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.

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).

Taxonomy and phylogeny of diazotrophs

The biodiversity of nitrogen-fixing organisms is huge. Taxonomic and phylogenetic research is needed to structure this diversity, to facilitate communication among scientists, and to increase our understanding of the evolution and biology of diazotrophs. Molecular tools for taxonomic and biodiversity studies of diazotrophic rhizobia, frankiae, cyanobacteria and bacilli are presented in sections 2 to 5. Sections 6 to 9 focus on problems with genus and species assignment.