Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR–RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR–RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N₂-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.     

Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China

As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

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.     

Symbiotic efficiency and phylogeny of the rhizobia isolated from Leucaena leucocephala in arid–hot river valley area in Panxi, Sichuan, China

In search of effective nitrogen-fixing strains for inoculating Leucaena leucocephala, we assessed the symbiotic efficiency of 41 rhizobial isolates from root nodules of L. leucocephala growing in the arid–hot river valley area in Panxi, China. The genetic diversity of the isolates was studied by analyzing the housekeeping genes 16S rRNA and recA, and the symbiotic genes nifH and nodC. In the nodulation and symbiotic efficiency assay, only 11 of the 41 isolates promoted the growth of L. leucocephala while the majority of the isolates were ineffective in symbiotic nitrogen fixation. Furthermore, one fourth of the isolates had a growth slowing effect on the host. According to the 16S rRNA and recA gene analyses, most of the isolates were Ensifer spp. The remaining isolates were assigned to Rhizobium, Mesorhizobium and Bradyrhizobium. The sequence analyses indicated that the L. leucocephala rhizobia had undergone gene recombination. In contrast to the promiscuity observed as a wide species distribution of the isolates, the results implied that L. leucocephala is preferentially nodulated by strains that share common symbiosis genes. The symbiotic efficiency was not connected to chromosomal background of the symbionts and isolates carrying a similar nifH or nodC showed totally different nitrogen fixation efficiency.     

Alkalinity of Lanzarote soils is a factor shaping rhizobial populations with Sinorhizobium meliloti being the predominant microsymbiont of Lotus lancerottensis

Lotus lancerottensis is an endemic species that grows widely throughout Lanzarote Island (Canary Is.). Characterization of 48 strains isolated from root nodules of plants growing in soils from eleven locations on the island showed that 38 isolates (79.1%) belonged to the species Sinorhizobium meliloti, whereas only six belonged to Mesorhizobium sp., the more common microsymbionts for the Lotus. Other genotypes containing only one isolate were classified as Pararhizobium sp., Sinorhizobium sp., Phyllobacterium sp. and Bradyrhizobium-like. Strains of S. meliloti were distributed along the island and, in most of the localities they were exclusive or major microsymbionts of L. lancerottensis. Phylogeny of the nodulation nodC gene placed the S. meliloti strains within symbiovar lancerottense and the mesorhizobial strains with the symbiovar loti. Although strains from both symbiovars produced effective N₂-fixing nodules, S. meliloti symbiovar lancerottense was clearly the predominant microsymbiont of L. lancerottensis. This fact correlated with the better adaptation of strains of this species to the alkaline soils of Lanzarote, as in vitro characterization showed that while the mesorhizobial strains were inhibited by alkaline pH, S. meliloti strains grew well at pH 9.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical–ecological–climatic regions of India are nodulated by Bradyrhizobium yuanmingense

Vigna mungo, Vigna radiata and Vigna unguiculata are important legume crops cultivated in India, but little is known about the genetic resources in native rhizobia that nodulate these species. To identify these bacteria, a core collection of 76 slow-growing isolates was built from root nodules of V. mungo, V. radiata and V. unguiculata plants grown at different sites within three agro-ecological-climatic regions of India. The genetic diversity of the bacterial collection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNA fragments of the 16S–23S rDNA intergenic spacer (IGS) region, and the symbiotic genes nifH and nodC. One rDNA IGS type grouped 91% of isolates, but more diversity was found at the symbiotic loci (17 symbiotic genotypes). Overall, no host plant specificity was shown, the three host plant species sharing common bradyrhizobial genotypes that represented 62% of the collection. Similarly, the predominant genotypes were found at most sampling sites and in all agro-ecological-climatic regions. Phylogenies inferred from IGS sequencing and multi-locus sequence analysis of the dnaK, glnII and recA genes indicated that all isolates but one were clustered with the Bradyrhizobium yuanmingense species. The nifH phylogeny also grouped the different nif haplotypes within a cluster including B. yuanmingense, except for one infrequent nif haplotype which formed a new lineage within the Bradyrhizobium genus. These results may reflect a long history of co-evolution between B. yuanmingense and Vigna spp. in India, while intra-species polymorphism detected in the symbiotic loci may be linked with the long history of diversification of B. yuanmingense coinciding with that of its host legumes.     

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.     

Genetic diversity and symbiotic evolution of rhizobia from root nodules of Coronilla varia

Ninety symbiotic rhizobial isolates from root nodules of Coronilla varia growing in the Shaanxi province of China were characterized. Combined with the results of RFLP patterns, six genotypes were defined among the rhizobial strains and they were divided into three genomic genera. These included Mesorhizobium sp., M. alhagi, M. amorphae, M. metallidurans/M. gobiense as the dominant group (86.7%), and Rhizobium yanglingense and Agrobacterium tumefaciens as the minor groups, according to analysis of the corresponding 16S rRNA, nodC and nifH genes. Five nodC types, which mainly grouped into the Mesorhizobium genus, were obtained from all the isolates examined, implying that nodC genes probably occurred from the native habitat through lateral transfer and long-term adaptation, finally evolving toward M. alhagi. Four different nifH types, displaying obvious differences compared to those of 16S rRNA and nodC, implied that possible lateral transfer of the symbiotic genes occurred between different genera. The association between soil components and the genetic diversity of the rhizobial population demonstrated that combined genotypes were positively correlated with the pH of soil samples.     

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.     

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

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.     

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.     

Multilocus sequence analyses reveal several unnamed Mesorhizobium genospecies nodulating Acacia species and Sesbania sesban trees in Southern regions of Ethiopia

Leguminous trees play an important role in agroforestry in Ethiopia, but studies of their rhizobial symbionts are scarce. In earlier studies, we surveyed natural nodulation of native leguminous trees growing in different agro-ecological zones in Southern Ethiopia, isolated 400 rhizobia, and characterized them based on different phenotypic and genotypic methods. In the present study we characterized 18 strains belonging to the genus Mesorhizobium, isolated from nodules of Acacia abyssinica, A. senegal, A. tortilis and Sesbania sesban. Phylogenetic analysis of nearly full-length 16S rRNA gene grouped the test strains into three distinct clades separated from all currently recognized Mesorhizobium species. Three divergent strains formed separate branches while the other 15 strains formed three distinct groups, genospecies I-III. Grouping of the isolates under study based on the house-keeping genes recA, gyrB, rpoB and gltA were consistent and in agreement with that of 16S rRNA. Similarly phylogenetic relationships based on the symbiosis-related genes nodC, nodA and nifH were generally similar to those shown by the core genes, suggesting that these Acacia and Sesbania symbionts have a long history of separate evolution within Mesorhizobium. Cross inoculation experiments demonstrated a large variation in the ability of the test strains to elicit effective nodules. The Sesbania isolates, occupying a distinct clade in the nodC phylogenetic tree, formed effective nodules only with this host legume. The study strongly suggests that this collection of Mesorhizobium strains comprises several new species, and also indicates the role of the symbiotic genes in determining the host range of these bacteria.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Physiological characterization of four model Lotus diploid genotypes: L. japonicus (MG20 and Gifu), L. filicaulis, and L. burttii under salt stress

The genus Lotus comprises a heterogeneous group of annual and perennial species. Lotus japonicus (with MG20 and Gifu ecotypes) has been adopted as one of the model legumes in genetic and genomic studies. Other Lotus species, such us Lotus burttii and Lotus filicaulis, have also been used in genetic and genomic studies because of their capacity to produce fertile progenies in crosses with L. japonicus. In the present work, physiological responses to salt stress in four Lotus genotypes were evaluated on the basis of growth and associated parameters, such as photosynthesis, ions, relative water content, oxidative damage and antioxidant system responses, using two NaCl levels applied by acclimation for up to 28 and 60 d. Growth responses varied with plant developmental stage in the four Lotus genotypes. L. japonicus MG20 was found to be a salt-tolerant genotype, mainly when exposed to salt stress at the young plant stage. The capacity of Lj MG20 to sustain growth under salt stress was correlated with enhancement of Superoxide dismutase and Glutathione reductase activities, as well as with increases in total and reduced glutathione content and lower Na⁺ accumulation in leaves. These results suggest that enhancement of antioxidant responses in Lj MG20 contributed to improve salt stress tolerance at early stages. On the other hand, after long-term high NaCl stress treatment, L. filicaulis exhibited lower biomass reduction, lower oxidative damage and Na⁺ accumulation in leaves than the control treatment; hence, this genotype was considered salt-tolerant. These apparently ambiguous results remark that salt tolerance, as a development-related process, was differentially expressed among the Lotus genotypes and depended on stress duration and plant phenological stage.     

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name “vachelliae” is proposed.     

Genetic diversity and distribution of Bradyrhizobium and Azorhizobium strains associated with the herb legume Zornia glochidiata sampled from across Senegal

Herb legumes have great potential for rehabilitation of semi-arid degraded soils in Sahelian ecosystems as they establish mutualistic symbiosis with N₂-fixing rhizobia. A phylogenetic analysis was performed for 78 root nodule bacteria associated with the common Sahelian herb legume Zornia glochidiata Reichb ex DC in Senegal. Based on ITS (rDNA16S-23S) and recA sequences, these strains were shown to belong to the two genera Bradyrhizobium and Azorhizobium. Strains of this latter, although frequent, formed small and ineffective nodules and suggested a parasitism rather than a symbiotic association. A potential negative effect of Azorhizobium on Zornia growth was tested for when inoculated alone or in association with a Bradyrhizobium strain. Bradyrhizobium isolates were distributed in four groups. Groups A and B were two sister clades in a larger monophyletic group also including Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense, and Bradyrhizobium japonicum. Strains of cluster D fell in a sister clade of the photosynthetic Bradyrhizobium sp. group, including ORS278, whereas group C appeared to be divergent from all known Bradyrhizobium clusters. Amplified fragment length polymorphism (AFLP) clustering was congruent with ITS and recA phylogenies, but displayed much more variability. However, within the main Bradyrhizobium clades, no obvious relationship could be detected between clustering and geographical origin of the strains. Each sub-cluster included strains sampled from different locations. Conversely, Azorhizobium strains showed a tendency in the phylogeny to group together according to the site of sampling. The predominance of ineffective Azorhizobium strains in the nodules of Zornia roots, the large Bradyrhizobium genetic diversity and the geographical genetic diversity pattern are explored.