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Plant growth-promoting rhizobacteria are bacteria that improve plant growth and reduce plant pathogen damages. In this study, 100 nodule bacteria were isolated from chickpea, screened for their plant growth-promoting (PGP) traits and then characterised by PCR-RFLP of 16 S rDNA. Results showed that most of the slow-growing isolates fixed nitrogen but those exhibiting fast-growth did not. Fourteen isolates solubilized inorganic phosphorus, 16 strains produced siderophores, and 17 strains produced indole acetic acid. Co-culture experiments identified three strains having an inhibitory effect against Fusarium oxysporum, the primary pathogenic fungus for chickpea in Tunisia. Rhizobia with PGP traits were assigned to Mesorhizobium ciceri, Mesorhizobium mediterraneum, Sinorhizobium meliloti and Agrobacterium tumefaciens. We noted that PGP activities were differentially distributed between M. ciceri and M. mediterraneum. The region of Mateur in northern Tunisia, with clay-silty soil, was the origin of 53% of PGP isolates. Interestingly, we found that S. meliloti and A. tumefaciens strains did not behave as parasitic nodule-bacteria but as PGP rhizobacteria useful for chickpea nutrition and health. In fact, S. meliloti strains could solubilize phosphorus, produce siderophore and auxin. The A. tumefaciens strains could perform the previous PGP traits and inhibit pathogen growth also. Finally, one candidate strain of M. ciceri (LL10)-selected for its highest symbiotic nitrogen fixation and phosphorus solubilization-was used for field experiment. The LL10 inoculation increased grain yield more than three-fold. These finding showed the potential role of rhizobia to be used as biofertilizers and biopesticides, representing low-cost and environment-friendly inputs for sustainable agriculture.

In this study, we explored the pathogenicity and phylogenetic position of Agrobacterium spp. strains isolated from crown gall tissues on annual, perennial, and ornamental plants in Iran. Of the 43 strains studied, 10 strains were identified as Allorhizobium vitis (formerly Agrobacterium vitis) using the species-specific primer pair PGF/PGR. Thirty-three remaining strains were studied using multilocus sequence analysis of four housekeeping genes (i.e., atpD, gyrB, recA, and rpoB), from which seven strains were identified as A. larrymoorei and one strain was identified as A. rubi (Rer); the remaining 25 strains were scattered within the A. tumefaciens species complex. Two strains were identified as genomospecies 1 (G1), seven strains were identified as A. radiobacter (G4), seven strains were identified as A. deltaense (G7), two strains were identified as A. nepotum (G14), and one strain was identified as “A. viscosum” (G15). The strains Rnr, Rnw, and Rew as well as the two strains OT33 and R13 all isolated from rose and the strain Ap1 isolated from apple were clustered in three atypical clades within the A. tumefaciens species complex. All but eight strains (i.e., Nec10, Ph38, Ph49, fic9, Fic72, R13, OT33, and Ap1) were pathogenic on tomato and sunflower seedlings in greenhouse conditions, whereas all but three strains (i.e., fic9, Fic72, and OT33) showed tumorigenicity on carrot root discs. The phylogenetic analysis and nucleotide diversity statistics suggested the existence of two novel genomospecies within the A. tumefaciens species complex, which we named “G19” and “G20.” Hence, we propose the strains Rew, Rnw, and Rnr as the members of “G19” and the strains R13 and OT33 as the members of G20, whereas the phylogenetic status of the atypical strain Ap1 remains undetermined.

International audience; Aims To identify the rhizobia nodulating Vicia sativa in Northwestern China and to estimate their geographic distribution. Methods Rhizobia trapped with V. sativa plants from soils at six sites in the northwest of China were classified into genotypes by PCR-based restriction fragment length polymorphism (RFLP) of 16S-23S rRNA intergenic spacer (IGS) and 16S rRNA genes, and phylogenetic analyses of housekeeping (16S rRNA, recA, atpD) and symbiotic genes were performed for the representative strains. Soil physicochemical characteristics were recorded and canonical correlation analysis was performed to examine the correlations between soil features and distribution of rhizobial genotypes. Results A total of 202 rhizobial isolates were discriminated by RFLP analyses into 15 IGS types and a single 16S rRNA type, which were identified as Rhizobium by 16S rRNA gene phylogeny and as four clusters by multilocus sequence analysis (MLSA). Cluster 1 covering 86 strains and 7 IGS types prevalent in all sites was identified as Rhizobium laguerreae; cluster 2 was R. sophorae with 18 strains in 2 IGS types found in the site Q-GD. Each of cluster 3 and cluster 4 contained three IGS types representing two novel Rhizobium genospecies specific to Gansu Province and Shanxi Province, respectively. Four nodC phylogenetic clades were defined among the isolates. Conclusions R. sophorae, R. laguerreae, and two novel Rhizobium genospecies with diverse symbiotic genotypes are associated with Vicia sativa L. in Northwest China. Their biogeographic patterns are mainly directed by soil pH and salinity. This is the first study on the diversity of rhizobia nodulating Vicia sativa in China.

It is of interest to investigate the diversity of arbuscular mycorrhizal fungi (AMF) associated with Vachellia tortillis ssp. raddiana to provide insights into the contribution of soil properties in shaping the AMF community composition and to define new strains for the production of inoculants under arid climate. A total of 230 symbiotic AMF clones associated with native V. tortilis roots growing in arid Tunisian soils were analyzed using nested PCR, cloning, and sequencing of the rDNA 18S region. The differential effects of soil properties on the diversity and community composition and distribution were also carried out. Phylogenetic analyses of four representative clones’ libraries grouped them into nine AMF operational taxonomic units (OTUs) among the Glomeraceae family. Two OTUs (OTU4 and OTU7) were separated from any AMF reference sequence and may constitute new species within the Glomeraceae. The distribution pattern under soil properties showed that high available phosphorus content in the soil and electric conductivity decrease AMF diversity and that the remaining soil parameters are correlated with the AMF community composition. Specific soil properties associated with the diverse relative distribution of AMF species, described for the first time in this work, may serve as a model for studying how spatially distributed AMF in a wide range of habitats may be utilized by a pioneering plant with a situation that is frequently occurring at reclaimed sites or in ameliorated soils when a new vegetation cover such as Vachellia sp. needs to be established.

To identify the rhizobia nodulating Astragalus sinicus in the acid soils at Xinyang, China and to estimate the effects of fertilization strategies on the rhizobial community. Soil samples were collected from treatments submitted to different fertilization strategies (chemical fertilizers, A. sinicus green manure, various input levels). Nodules obtained from the A. sinicus trapping plants grown in the soil samples were used for rhizobial isolation. The isolates were grouped into PCR-RFLP genotypes based on analyses of 16S rRNA gene and IGS, and representatives for each genotype were further subjected to phylogenetic analyses of housekeeping genes (16S rRNA gene, atpD, glnII, and rpoB), as well as symbiotic genes (nodC and nifH). Soil physicochemical characteristics were also estimated and redundancy analysis was performed to examine the relations between soil features and distribution of rhizobial genotypes across different fertilization treatments. A total of 257 rhizobial isolates were obtained and discriminated into 12 IGS types in a single 16S rRNA type by RFLP analyses. Further 16S rRNA gene sequence-based phylogeny and multilocus sequence analysis (MLSA) distinguished a minor group (10 strains, 4 IGS types) affiliated to Mesorhizobium huakuii and a main group (247 strains, 8 IGS types) to Mesorhizobium jarvisii, for which a novel symbiovar, sv. astragali, was described. The long-term input of A. sinicus green manure increased rhizobial diversity and modified rhizobial community structures, via the increase of organic matter and decrease of soil pH, available phosphate, available potassium, and total salt. M. jarvisii sv. nov. astragali was the predominant microsymbiont of A. sinicus in the tested acidic soils. The long-term input of green manure modified the soil features, enhancing the genetic diversity of rhizobia associated with A. sinicus and alternating the rhizobial community structure.

To investigate the succession of chickpea rhizobia over years and sampling sites. Rhizobia were sampled four times from nodules of chickpea grown in four sites of Mulei County and Qitai County in Xinjiang, China during 2009–2016. The recA genes of rhizobia representing different populations were phylogenetically compared for their genotypic differentiation. Soil properties were examined to explore their hypothetical incidence on the rhizobial succession. The 628 isolates were all identified as Mesorhizobium muleiense, belonging to 28 genotypes distinguished by recA phylogeny. For each site, 15, 17, 10 and 7 genotypes were detected during the whole sampling period; while 4, 15, 12 and 17 genotypes were evidenced in each of the 4 sampling years (2009, 2013, 2015 and 2016) from all the four sites. Four major genotypes (T12 through T15) were found throughout the four years in the four sites at various frequencies. While the other genotypes were site and/or year specific. Both increased genetic diversity as revealed by Shannon-Weaver index and shifts in the population composition as revealed by genotyping were detected in the chickpea rhizobial populations during the study period. Nitrogen content, available phosphorus and potassium, and pH were the main factors correlated to the genotype distribution of chickpea rhizobia in the soil over years. The genetic diversity of M. muleiense increased and the population composition shifted over years and at different sampling sites. The recA gene is an adequate molecular marker to estimate the intraspecific variation and succession of M. muleiense genotypes. The rhizobial genotype succession/evolution in the fields might be driven by the fluctuations of soil characteristics like nitrogen content, available phosphorus and potassium, and pH.

In this study, we explored the pathogenicity and phylogenetic position of

Minutes of the closed meeting of the ICSP Subcommittee on the Taxonomy of Rhizobia and Agrobacteria held by videoconference on 17 July 2019, and list of recent species.

With a view to introducing white lupin (Lupinus albus L.) for cultivation in Tunisian calcareous soils, compatible indigenous rhizobia for nitrogen-fixing symbiosis were investigated and characterized. Two L. albus varieties, Mekna and Lumen, were used to trap rhizobia in soil samples collected from 56 sites with high active lime contents (0–49%). Nodulation occurred in only 15 soils. The local variety, Mekna, developed significantly more root nodules and had a trapping capacity in more soils than the imported variety Lumen. A phylogenetic analysis based on the partial 16S-23S ribosomal RNA internal transcribed spacer region (ITS) and multi-locus sequence analysis (MLSA) of three chromosomal housekeeping genes, recA, atpD and dnaK, showed that strains were affiliated to Agrobacterium, Rhizobium, and Neorhizobium, with large internal diversity, including separate lineages. Infectivity tests highlighted some nodulation specificity at the plant variety level, since the strains originating from Mekna could only nodulate this variety, while strains trapped in Lumen could nodulate both varieties. When inoculated, almost all strains resulted in a significant increase in plant shoot dry weight on L. albus. Although Agrobacterium sp. strains isolated from L. albus could nodulate and had a plant growth promoting effect, no nodA and nodC genes could be amplified. This is discussed together with the absence of bradyrhizobia and the general infrequency of L. albus–nodulating rhizobia in Tunisian soils. The adapted and efficient rhizobial strains reported here were promising candidates for inoculant development and represent a contribution towards successful cultivation of L. albus in Tunisia, especially the most promising Mekna variety.

12 páginas, Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee onSystematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updatedminimal standards for the description of new species (and genera) in these groups. The essential requirements (minimalstandards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidencefor differentiation from other species based on genome sequence comparisons. It is also recommended that (3) geneticvariation within the species is documented with sequence data from several clearly different strains and (4) phenotypicfeatures are described, and their variation documented with data from a relevant set of representative strains. Furthermore,it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevantgene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a namethat conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication eitherdirectly in theInternational Journal of Systematic and Evolutionary Microbiologyor in a validation list when publishedelsewhere, and (8) deposition of the type strain in two international culture collections in separate countries

To describe the chickpea rhizobial diversity in Gansu Province of China, so far uninvestigated. Nodule bacteria were isolated from chickpea grown in Gansu and characterized by phylogenetic analysis of 16S rRNA gene, housekeeping genes (atpD, recA, and glnII), and symbiotic genes (nodC and nifH). The genetic differentiation and gene flow among the chickpea microsymbionts were estimated and considered in view of soil characteristics. A total of nineteen new isolates originating from alkaline-saline (pH 7.9–8.1) soils in Gansu formed two clades by housekeeping gene analyses; clade I (16 isolates) was identified as Mesorhizobium muleiense (97.7–98.7 % of similarities with the type strain CCBAU 83963T) and clade II (3 isolates) formed a separate group within Mesorhizobium branch, displaying 97.0 and 96.6 % similarities with the most related type strains M. temperatum SDW 018T and M. muleiense CCBAU 83963T, respectively. Their symbiotic genes (nodC and nifH) were highly similar (>98.0 %) with those described for chickpea microsymbionts in literature, i.e., M. muleiense, M. ciceri, and M. mediterraneum. The genetic differentiation and gene flow between the chickpea rhizobial groups supported our conclusions for species affiliation. M. muleiense and a novel Mesorhizobium genospecies, sharing similar symbiotic genes, are distributed in the alkaline-saline soils of Gansu and may be considered as the main and specific chickpea symbionts in the Northwest of China.

Rhizobia associated with white clover (Trifolium repens) grown in alkaline soils of China have never been investigated. This is the first survey to report of their genetic and biogeographical diversity. Nodule bacteria were isolated from white clover grown in alkaline soils (pH 8.18–8.99) in North and East China and were characterized by multilocus sequence analysis (MLSA) of the housekeeping genes (atpD, recA, and glnII), and phylogenies of 16S rRNA gene and symbiotic genes nodC and nifH. The biogeographic distribution of rhizobial species was analyzed in relation to the soil factors. A total of 83 new strains could be affiliated to Rhizobium that shared 100 % sequence similarity of 16S rRNA gene with R. leguminosarum, R. acidisoli, R. anhuiense, R. indigoferae, R. sophorae, and R. laguerreae. Three genospecies were further distinguished based on the housekeeping gene analysis among these new strains: R. anhuiense, R. leguminosarum, and a hypothetical novel Rhizobium genospecies. Highly conserved symbiotic genes corresponding to those of symbiovar trifolii in R. leguminosarum were observed among all the new strains. Unique rhizobial communities associated with white clover were detected in the tested alkaline soils, and soil characteristics such as pH and nutrient levels were estimated as the determinant factors. White clover established symbiosis with three Rhizobium genospecies harboring similar symbiotic genes in alkaline soils in China. Biogeographic pattern exists in clover rhizobia that was determined by the soil pH and nutrient levels.

To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

International audience; Three chickpea rhizobial strains (WYCCWR 10195(T)=S1-3-7, WYCCWR 10198=S1-4-3 and WYCCWR 10200=S1-5-1) isolated from Northwest China formed a group affiliated to Mesorhizobium based on 16S rRNA gene sequence comparison. To clarify their species status, multilocus sequence analysis and average nucleotide identity (ANI) values of whole genome sequences between the novel group and the type strains of the related species were further performed. Similarities of 95.7-96.6 % in the concatenated sequences of atpD-recA-glnII and 91.9-93.1 % of ANI values to the closest-related species Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium temperatum demonstrated the novel group a unique genospecies. The most abundant fatty acid in cells of WYCCWR 10195(T) were C-19( : 0) (cyclo omega 8c) (51.4 %), followed by C-18( : 1 omega 7c) 11-methyl (9.5 %) and C-16( : 0) (9.3 %). Its genome size was 6.37 Mbp, comprising 6633 predicted genes with a DNA G+C content of 61.9 mol%. The similarities of 99.0-99.8 % for the nodC gene and 98.3-99.44 % for the nifH gene to those of the chickpea rhizobial species and nodulation with Cicer arietinum L. confirmed the strains of the new genospecies as symbiovar ciceri. The weak utilization of most of the tested sugars/organic acids and non-utilization of l(+)-rhamnose, l-cysteine and l-glycine as sole carbon source, tolerance to 1 % (w/v) NaCl, resistance to 5 g mu ml(-1) chloromycetin and non-hydrolysis of l-tyrosine distinguished the novel group from the related species and supported this group as a novel species, for which the name Mesorhizobium wenxiniae sp. nov. is proposed, with WYCCWR 10195(T) (=S1-3-7=HAMBI 3692(T)=LMG 30254(T)) as the type strain.

International audience; Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S-23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S-23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with Rhizobium.

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N-2-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4%(w/v) NaCl and grew at 45 degrees C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.

Low Phosphorus availability in soil is worldwide a frequent major constraint for crops. Rhizobial strains, beneficial N2-fixing symbiotic partners of legumes, were reported to solubilize both organic and inorganic complex phosphates. Thus they may play a significant role as plant growth-promoting rhizobacteria (PGPR) in the biofertilization of crops. Natural rock phosphate may play a key role in sustainable agriculture, resulting in economic and environmental benefits. This work aims to evidence the capacity of rhizobial strains nodulating Vicia faba L. plants in Marrakech-Haouz region (Morocco) to solubilize Moroccan rock phosphate in vitro in either agar or liquid medium and to evaluate and compare the effect of these strains on growth and phosphorus uptake by V. faba plants of two Moroccan varieties (Defes and Aguadulce). Four rhizobial strains, contrasting for phosphate solubilization on agar medium, were further studied on liquid medium. RHOF147 and RHOF174 strains formed clear halos around colonies (1.37 cm and 1.2 respectively after 15 days); opposite RHOF170 and RHOF171 strains did not produce halo. The four strains were able to mobilize rock phosphate for growth in liquid medium (up to 0.59 mg/L of assimilated phosphorus). Lower pH values and most mobilized phosphorus production were registered for RHOF147 and RHOF174 strains. By screening the four rhizobial strains for growth on Chrome Azurol S (CAS) medium, only RHOF171 strain formed orange halo indicating its capacity to produce siderophores. Greenhouse experiments were undertaken including comparison of inoculated plants with these four rhizobial strains in the presence of rock phosphate as sole P source versus non-inoculated plants growing on KH2PO4 as sole P source (P-fertilized plants). Results show that the root length, the root dry weight and the ratio root /shoot dry weights were generally higher in the inoculated plants than in the P-fertilized plants, indicating the adaptation of the inoculated plants to phosphorus deficiency conditions. The effect of rock phosphate-solubilizing bacteria on plant growth depends on the symbiotic combination of the inoculated strain and the plant genotype. As for phosphorus uptake, rhizobial strains made phosphorus available to the plant at significant increased concentrations in some symbiotic combinations (e.g. Aguadulce-RHOF147). The use of selected symbiotic combinations having the capacity to solubilize rock phosphate may improve soil fertility and phosphorus availability to plants. (Resume d'auteur)

Legumes interact symbiotically with bacteria of the Rhizobiaceae to form nitrogen-fixing root nodules. We investigated the contribution of the three glutaredoxin (Grx)-encoding genes present in the Sinorhizobium meliloti genome to this symbiosis. SmGRX1 (CGYC active site) and SmGRX3 (CPYG) recombinant proteins displayed deglutathionylation activity in the 2-hydroethyldisulfide assay, whereas SmGRX2 (CGFS) did not. Mutation of SmGRX3 did not affect S. meliloti growth or symbiotic capacities. In contrast, SmGRX1 and SmGRX2 mutations decreased the growth of free-living bacteria and the nitrogen fixation capacity of bacteroids. Mutation of SmGRX1 led to nodule abortion and an absence of bacteroid differentiation, whereas SmGRX2 mutation decreased nodule development without modifying bacteroid development. The higher sensitivity of the Smgrx1 mutant strain as compared with wild-type strain to oxidative stress was associated with larger amounts of glutathionylated proteins. The Smgrx2 mutant strain displayed significantly lower levels of activity than the wild type for two iron-sulfur-containing enzymes, aconitase and succinate dehydrogenase. This lower level of activity could be associated with deregulation of the transcriptional activity of the RirA iron regulator and higher intracellular iron content. Thus, two S. meliloti Grx proteins are essential for symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and the regulation of iron metabolism.

In the present investigation, six Rhizobium strains isolated from Algerian soil were checked for their antimicrobial activity against Pseudomonas savastanoi, the agent responsible for olive knot disease. Rhizobium sp. ORN 24 and ORN 83 were found to produce antimicrobial activities against Pseudomonas savastanoi. The antimicrobial activity produced by Rhizobium sp. ORN24 was precipitable with ammonium sulfate, between 1,000 and 10,000 KDa molecular weight, heat resistant but sensitive to proteases and detergents. These characteristics suggest the bacteriocin nature of the antimicrobial substance produced by Rhizobium sp. ORN24, named rhizobiocin 24. In contrast, the antimicrobial activity produced by Rhizobium sp. ORN83 was not precipitable with ammonium sulfate; it was smaller than 1,000 KDa molecular weight, heat labile, and protease and detergent resistant. These characteristics could indicate the relationship between the antimicrobial substance produced by Rhizobium sp. ORN 83 and the “small” bacteriocins described in other rhizobia.

The aim of this study is to investigate the diversity of Retama raetam root-nodule bacteria isolated from arid regions of Tunisia. Twelve isolates, chosen as representative for different 16S rRNA gene patterns, were characterized by 16S rRNA gene sequencing and phenotypic analysis. Isolates were assigned to Sinorhizobium , Rhizobium and Agrobacterium . Symbiotic properties of Sinorhizobium and Rhizobium isolates showed a large diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Strain RK 22 identified as Rhizobium was the most effective isolate.

The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.

To serve as inoculants of legumes, nitrogen-fixing rhizobium strains should be competitive and tolerant of diverse environments. We hybridized the genomes of symbiotically efficient and salt tolerant Sinorhizobium inoculant strains onto the Sinorhizobium meliloti Rm1021 microarray. The number of variable genes, that is, divergent or putatively multiplied genes, ranged from 503 to 1556 for S. meliloti AK23, S. meliloti STM 1064 and S. arboris HAMBI 1552. The numbers of divergent genes affiliated with the symbiosis plasmid pSymA and related to DNA replication, recombination and repair were significantly higher than expected. The variation was mainly in the accessory genome, implying that it was important in shaping the adaptability of the strains.

The family Rhizobiaceae accommodates the seven genera Rhizobium, Neorhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), Shinella and Ciceribacter. However, several so-called Rhizobium species do not exhibit robust phylogenetic positions. Rhizobium is extremely heterogeneous and is in need of major revision. Therefore, a phylogenetic examination of the family Rhizobiaceae by multilocus sequence analysis (MLSA) of four housekeeping genes among 100 strains of the family was undertaken. Based on the results we propose the delineation of the new genus Pararhizobium in the Rhizobiaceae family, and 13 new species combinations: Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov.

We characterized 34 endophytic bacterial isolates associated to root nodules collected from spontaneous legumes in the arid zone of Tunisia by 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, whole cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 16S rDNA and 16S-23S rDNA internal transcribed spacer sequencing. Phylogenetically, these isolates belong to the branches containing the genera Inquilinus, Bosea, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Starkeya, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Ornithinicoccus, Bacillus, and Paenibacillus. These strains did not induce any nodule formation when inoculated on the wide host spectrum legume species M. atropurpureum (Siratro) and no nodA gene could be amplified by PCR. However, nifH sequences, most similar to those of Sinorhizobium meliloti, were detected within strains related to the genera Microbacterium, Agromyces, Starkeya and Phyllobacterium.

Taxonomy is the science that studies the relationships between organisms. It comprises classification, nomenclature, and identification. Modern bacterial taxonomy is polyphasic. This means that it is based on several molecular techniques, each one retrieving the information at different cellular levels (proteins, fatty acids, DNA...). The obtained results are combined and analysed to reach a "consensus taxonomy" of a microorganism. Until 1970, a small number of classification techniques were available for microbiologists (mainly phenotypic characterization was performed: a legume species nodulation ability for a Rhizobium, for example). With the development of techniques based on polymerase chain reaction for characterization, the bacterial taxonomy has undergone great changes. In particular, the classification of the legume nodulating bacteria has been repeatedly modified over the last 20 years. We present here a review of the currently used molecular techniques in bacterial characterization, with examples of application of these techniques for the study of the legume nodulating bacteria.Key words: polyphasic taxonomy, molecular characterization, bacteria, prokaryotes, legume nodulating bacteria, Rhizobium.

Summary Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0–0.25 m and 0.25–0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.

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.

Supplementary data; International audience; The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the "R. galegae complex" (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.

International audience; The genetic structure of rhizobia nodulating pea and lentil in Algeria, Northern Africa was determined. A total of 237 isolates were obtained from root nodules collected on lentil (Lens culinaris), proteaginous and forage pea (Pisum sativum) growing in two eco-climatic zones, sub-humid and semi-arid, in Eastern Algeria. They were characterised by PCR-restriction fragment length polymorphism (RFLP) of the 16S-23S rRNA intergenic region (IGS), and the nodD-F symbiotic region. The combination of these haplotypes allowed the isolates to be clustered into 26 distinct genotypes, and all isolates were classified as Rhizobium leguminosarum. Symbiotic marker variation (nodD-F) was low but with the predominance of one nod haplotype (g), which had been recovered previously at a high frequency in Europe. Sequence analysis of the IGS further confirmed its high variability in the studied strains. An AMOVA analysis showed highly significant differentiation in the IGS haplotype distribution between populations from both eco-climatic zones. This differentiation was reflected by differences in dominant genotype frequencies. Conversely, no host plant effect was detected. The nodD gene sequence-based phylogeny suggested that symbiotic gene diversity in pea and lentil nodulating rhizobial populations in Algeria was low compared to that reported elsewhere in the world.

Les rhizobia sont les bacteries qui forment des symbioses fixatrices d'azote avec des plantes de la famille des legumineuses. Suite a l'adoption de la taxonomie polyphasique comme critere de caracterisation, leur classification a subi de nombreux remaniements ces dernieres annees. Une revue de cette classification en six genres et 28 especes actuellement reconnus est presentee ici.

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.Key words: Crotalaria, Methylobacterium, rhizobium, symbiosis.

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$. Twenty strains were also studied by multilocus enzyme electrophoresis (MLEE) and the whole 16S rRNA gene was sequenced from two strains representing the two major clusters. These results, together with the previuously reported numerical taxonomy analysis, pulsed-field gel electrophoresis studies, DNA-DNA dot-blot hybridisation, genomic fingerprinting using repetitive sequence-basec PCR, DNA base composition analysis, DNA-DNA reassociation analysis, partial sequencing of the 16S rRNA gene and RFLP analysis of the amplified 16S rRNA gene, showed that all 30 strains belong to the genus #Sinorhizobium$. 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. (Résumé d'auteur)

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A ...

International audience; The diversity of rhizobia associated with introduced and native Acacia species in Algeria was investigated from soil samples collected across seven districts distributed in arid and semi-arid zones. The in vitro tolerances of rhizobial strains to NaCl and high temperature in pure culture varied greatly regardless of their geographical and host plant origins but were not correlated with the corresponding edaphoclimatic characteristics of the sampling sites, as clearly demonstrated by principal component analysis. Based on 16S rRNA gene sequence comparisons, the 48 new strains isolated were ranked into 10 phylogenetic groups representing five bacterial genera, namely, Ensifer, Mesorhizobium, Rhizobium, Bradyrhizobium, and Ochrobactrum. Acacia saligna, an introduced species, appeared as the most promiscuous host because it was efficiently nodulated with the widest diversity of rhizobia taxa including both fast-growing ones, Rhizobium, Ensifer, and Mesorhizobium, and slow-growing Bradyrhizobium. The five other Acacia species studied were associated with fast-growing bacterial taxa exclusively. No difference in efficiency was found between bacterial taxa isolated from a given Acacia species. The tolerances of strains to salinity and temperature remains to be tested in symbiosis with their host plants to select the most adapted Acacia sp.-LNB taxa associations for further revegetation programs.

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.

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.

In the context of an increasing utilization of the interspecific hybrid Acacia mangium × A. auriculiformis as a plantation tree in the tropical humid zone, its symbiotic characterization was carried out in comparison with that of its two parental species. Rhizobium strains of diverse geographical origins were isolated from root nodules of the hybrid and its parents. Almost all Acacia hybrid isolates were fast growing on yeast extract-mannitol medium, in contrast to those isolated from both parental species, which were mostly slow growing. The rhizobium strains were characterized through partial sequencing of the rRNA operon. In the phylogenetic tree, almost all strains isolated from the hybrid were grouped together in a clade close to Bradyrhizobium japonicum , while all strains isolated from both parental species were close to Bradyrhizobium elkanii . Inoculation experiments performed under in vitro or greenhouse conditions showed that all strains were infective with their original hosts but exhibited very variable degrees of effectivity according to the host plant tested. Thus, homologous strain-host associations were more effective than heterologous ones. This shows that there is still a high potential for isolating and testing new strains from hybrids to be used as inoculants in the context of large-scale afforestation programs.

The use of multilocus sequence analysis (MLSA) for the taxonomy of Bradyrhizobium was assessed. We compared partial sequences for atpD, recA, gyrB, rpoB and dnaK for a set of reference strains representing named species and genospecies, and a number of new isolates from Lupinus albus, Arachis hypogaea and Ornithopus compressus from Spain. The phylogenies of the individual genes were compared with previous DNA-DNA hybridization results. High hybridization values were well reflected, but intermediary hybridization values were less clearly apparent. However, the phylogeny of a concatenated dataset of the five genes did reflect all values and thus is more informative of overall genome similarity. Our results indicate that only for the genes gyrB, rpoB and dnaK there is a small gap between the interspecies sequence similarities and the intraspecies similarity, and therefore cut-off levels for species delineation cannot be set, although high sequence similarity (>99%) does permit identification. In a few instances, a reference strain did not group as expected for one of the five genes tested. This may be a result of horizontal gene transfer and recombination events occasionally involving housekeeping genes. This observation indicates it is best to consider more than one gene for taxonomic inferences. The majority of the new isolates from the three host species was identified as Bradyrhizobium canariense. Four strains from L. albus from Leon, Spain, formed a separate group close to Bradyrhizobium japonicum.

Chickpea is an important pulse crop grown mainly in the arid and semi-arid regions. The effect of water deficiency on nodulation, biomass production, and competition for nodule occupancy was evaluated in three different soils with two chickpea cultivars, Amdoun I and Chetoui. Two watering regimes were considered; a control that was irrigated three times per week and a water-deficient treatment that was irrigated only one time a week. Results showed that water deficiency significantly decreased the nodule number and the shoot dry weight for both cultivars. Root-nodule bacteria were isolated and characterized by PCR-RFLP of 16S rDNA and nif D-K intergenic spacer. The results show that water deficiency affects the diversity of nodulating rhizobia. The nodulation by Mesorhizobium mediterraneum was reduced while inefficient nodulation by Ensifer meliloti was favoured. In both treatments, chickpea was preferentially nodulated by nif D-K type N6. Analysis for NaCl tolerance showed that most of rhizobia nodulating chickpea under water deficiency are NaCl tolerant. Inoculation with the selected salt-tolerant strain of M. mediterraneum LILM10 increased significantly nodule number and grain yield in the field.

National audience; Plant symbioses are fundamental for ecosystem stability and sustainability. Introduction of legumes associated with microorganisms efficient in nitrogen fixation and promoting nutrient biodisponibility in limited-nutrient degraded sites is a key step for a successful plant establishment in stress situations such as those encountered in Mediterranean soils. A vegetation strategy based on microbial engineering of locally-adapted plants was investigated and tested in field conditions within the framework of calcareous quarry rehabilitation. Choice of symbiotic plants requires, first, a prospection of native legume species growing in relatively degraded sites in Mediterranean areas and easy to multiply, and second, an identification and production of efficient rhizobial partners. Plants produced in nursery and then transplanted into the quarry showed a good capacity for establishment after three years. Shrub species performed best with spontaneous mycorrhizal association and ability to enrich topsoil with organic matter; Les symbioses végétales sont une composante fondamentale de la stabilité et de la durabilité des écosystèmes. L’introduction de légumineuses associées à leurs auxiliaires microbiens fixateurs d’azote ou améliorant la biodisponibilité d’éléments nutritifs, dans des sites dégradés fortement appauvris en éléments nutritifs, est une condition primordiale pour réussir l’installation de plants en milieu particulièrement contraignant, comme le sont les sites de carrière de calcaire après exploitation sous climat méditerranéen. Une stratégie de végétalisation basée sur l’ingénierie microbiologique de plants adaptés aux conditions locales, a été élaborée puis testée sur le terrain en grandeur nature dans le cas particulier de carrières de granulats calcaires. Le choix des plants symbiotiques utilisables pour la végétalisation repose, d’une part, sur la prospection d’espèces de légumineuses repérées sur des sites assez dégradés en milieu méditerranéen, faciles à multiplier et, d’autre part, sur l’identification et la production de rhizobiums partenaires efficaces. Ces plants produits en pépinière puis transférés sur le terrain présentent après trois années une bonne capacité de croissance. Les espèces arbustives sont plus particulièrement performantes avec une capacité spontanée à s’associer aux mycorhizes et à favoriser l’enrichissement en matière organique de la surface du substrat.

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N(2)-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.

Taxonomy is the science that studies the relationships between organisms. It comprises classification, nomenclature, and identification. Modern bacterial taxonomy is polyphasic. This means that it is based on several molecular techniques, each one retrieving the information at different cellular levels (proteins, fatty acids, DNA...). The obtained results are combined and analysed to reach a "consensus taxonomy" of a microorganism. Until 1970, a small number of classification techniques were available for microbiologists (mainly phenotypic characterization was performed: a legume species nodulation ability for a Rhizobium, for example). With the development of techniques based on polymerase chain reaction for characterization, the bacterial taxonomy has undergone great changes. In particular, the classification of the legume nodulating bacteria has been repeatedly modified over the last 20 years. We present here a review of the currently used molecular techniques in bacterial characterization, with examples of application of these techniques for the study of the legume nodulating bacteria.

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped. (c) 2006 Elsevier B.V. All rights reserved.

International audience; We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega of- ficinalis and G. orientalis in northern regions so far