Retama species growing in different ecological–climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological–climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes (dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.     

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.     

Multilocus sequence analysis of the genus Bradyrhizobium

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 León, Spain, formed a separate group close to Bradyrhizobium japonicum.     

Characterization of Bradyrhizobium species isolated from root nodules of Cytisus villosus grown in Morocco

From a total of 73 bacterial strains isolated from root nodules of Cytisus villosus grown in soils of the central-western region of the Moroccan Rif, 68 strains clustered into 19 repetitive extragenic palindromic (REP)-polymerase chain reaction (PCR) groups. The nearly complete 16S rRNA gene sequence from each strain showed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis showed that 11 out of the 19 strains belong to Bradyrhizobium canariense and that another three strains were Bradyrhizobium japonicum. The remaining five strains represented new lineages within the genus Bradyrhizobium since they were not identified with any previously described species. Sequencing of the symbiotic nodC and nifH genes from each bradyrhizobial strain revealed they were all similar to those of the strains included in biovar genistearum.     

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.     

Diverse bacteria isolated from root nodules of wild Vicia species grown in temperate region of China

In the present study, a total of 154 bacterial strains isolated from nodules of eighteen Vicia species mainly grown in the temperate Chinese provinces were characterized by ARDRA, ITS PCR–RFLP, BOX-PCR, sequencing of 16S rDNA, nodC, nifH, atpD and glnII, and nodulation tests. The results demonstrated that most of the R. leguminosarum strains were effective microsymbionts of the wild Vicia species, while genomic species related to Rhizobium gallicum, Mesorhizobium huakuii, Ensifer meliloti and Bradyrhizobium spp. were symbiotic bacteria occasionally nodulating with Vicia species. In addition, fourteen strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, as well as 22 strains of R. leguminosarum might be nodule endophytes without symbiotic genes. Diverse symbiotic gene lineages were found among the test strains and a strong association was found among the symbiotic gene types and genomic species, indicating the absence of lateral gene transfer. These results greatly enlarged the rhizobial spectrum of Vicia species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range

Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.     

Lentil (Lens culinaris Medik.) nodulates with genotypically and phenotypically diverse rhizobia in Ethiopian soils

Forty-eight lentil-nodulating rhizobia were isolated from soil samples collected from diverse agro-ecological locations in Ethiopia, and characterized based on 76 phenotypic traits. Furthermore, 26 representative strains were selected and characterized using multilocus sequence analyses (MLSA) of core (16S rRNA, recA, atpD, glnII and gyrB) and symbiotic (nodA and nifH) genes. Numerical analysis of phenotypic characteristics showed that the 48 test strains fell into three major distinct clusters. The phylogenetic trees based on 16S rRNA genes showed that they belong to the Rhizobium genus. Our phylogenetic reconstruction based on combined gene trees (recA, atpD and glnII) supported three distinct sub-lineages (Clades I–III). While genospecies I and II could be classified with Rhizobium etli and Rhizobium leguminosarum, respectively, genospecies III, might be an unnamed genospecies within the genus Rhizobium. Phylogenetic reconstruction based on the symbiosis-related genes supported a single cluster, indicating differences in the evolutionary histories between chromosomal and symbiotic genes. Overall, these results confirmed the presence of a great diversity of lentil-nodulating Rhizobium species in Ethiopia, inviting further exploration. Moreover, the differences in symbiotic effectiveness of the test strains indicated the potential for selecting and using them as inoculants to improve the productivity of lentil in the country.     

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.The GenBank accession numbers for the sequences reported in this paper are as follows: nodC, DQ139776–DQ139781; nodA, DQ135897, Q135898; nodZ, DQ135899–DQ135903.     

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).     

Isolation and diversity analysis of resistance gene analogues (RGAs) from cultivated and wild strawberries

Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria × ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry.Communicated by M.-A. Grandbastien     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.