nodC Gene Research Articles

Diversity of rhizobia and non-rhizobia endophytes isolated from root nodules of Trifolium sp. growing in lead and zinc mine site Guelma, Algeria

High concentrations of heavy metals in mine soil disturb the interactions between legumes and microorganisms leading to select strains adapted to these specific conditions. In this work, we analyzed the diversity of fifty strains isolated from Trifolium sp. nodules growing on Pb-Zn mine soil, in the Northeastern of Algeria and highlighted their potential symbiotic traits. The phylogeny of the 16S rRNA gene sequences revealed a high bacterial diversity with a predominance of non-rhizobial endophytes. The identified isolates belong to the thirteen following genera Cupriavidus, Pseudomonas, Bacillus, Acinetobacter, Enterobacter, Roseomonas, Paracoccus, Frondihabitans, Microbacterium, Kocuria, Providencia, Micrococcus and Staphylococcus. Regarding rhizobial strains, only isolates affiliated to Rhizobium genus were obtained. The symbiotic gene nodC and the nitrogen fixation gene nifH present showed that Rhizobium isolates belonged to the symbiovar trifolii. In addition to bacterial, one yeast strain was isolated and identified as Rhodotorula mucilaginosa by sequencing the internal transcribed spacer (ITS) region.

Root endophyte-enhanced peanut-rhizobia interaction is associated with regulation of root exudates

Root exudates play a crucial role in the symbiosis between leguminous plants and rhizobia. Our previous studies have shown that a fungal endophyte Phomopsis liquidambaris promotes peanut-rhizobia nodulation and nitrogen fixation, but the underlying mechanism are largely unknown. Here, we explore the role of peanut root exudates in Ph. liquidambaris-mediated nodulation enhancement. We first collected root exudates from Ph. liquidambaris-inoculated and un-inoculated peanuts and determined their effects on rhizobial growth, biofilm formation, chemotaxis, nodC gene expression, and peanut nodulation. Our results found a positive effect of Ph. liquidambaris-inoculated root exudates on these characteristics of rhizobia. Next, we compared the root exudates profile of Ph. liquidambaris-inoculated and un-inoculated plants and found that Ph. liquidambaris altered the concentrations of phenolic acids, flavonoids, organic acids and amino acids in root exudates. Furthermore, the rhizobial chemotaxis, growth and biofilm formation in response to the changed compounds at different concentrations showed that all of the test compounds induced rhizobial chemotactic behavior, and organic acids (citric acid and oxalic acid) and amino acid (glutamate, glycine and glutamine) at higher concentrations increased rhizobial growth and biofilm formation. Collectively, our results suggest that root exudates alterations contribute to Ph. liquidambaris-mediated peanut-rhizobia nodulation enhancement.

Paraburkholderia spp. are the main rhizobial microsymbionts of Mimosa tenuiflora (Willd.) Poir. in soils of the Brazilian tropical dry forests (Caatinga biome)

Mimosa tenuiflora (Willd.) Poir. is widespread in southern and central American drylands, but little information is available concerning its associated rhizobia. Therefore, this study aimed to characterize M. tenuiflora rhizobia from soils of the tropical dry forests (Caatinga) in Pernambuco State, Brazil, at the molecular and symbiotic levels. Soil samples of pristine Caatinga areas in four municipalities were used to grow M. tenuiflora. First, the bacteria from root nodules were subjected to nodC/nifH gene amplification, and the bacteria positive for both genes had the 16S rRNA gene sequenced. Then, ten strains were evaluated using recA, gyrB, and nodC gene sequences, and seven of them had their symbiotic efficiency assessed. Thirty-two strains were obtained and 22 of them were nodC/nifH positive. Twenty strains clustered within Paraburkholderia and two within Rhizobium by 16S rRNA gene sequencing. The beta-rhizobia were similar to P. phenoliruptrix (12) and P. diazotrophica (8). Both alpha-rhizobia were closely related to R. miluonense. The recA + gyrB phylogenetic analysis clustered four and five strains within the P. phenoliruptrix and P. diazotrophica branches, respectively, but they were somewhat divergent to the 16S rRNA phylogeny. For Rhizobium sp. ESA 637, the recA + gyrB phylogeny clustered the strain with R. jaguaris. The nodC phylogeny indicated that ESA 626, ESA 629, and ESA 630 probably represented a new symbiovar branch. The inoculation assay showed high symbiotic efficiency for all tested strains. The results indicated high genetic diversity and efficiency of M. tenuiflora rhizobia in Brazilian drylands and included P. phenoliruptrix-like bacteria in the list of efficient beta-rhizobia in the Caatinga biome.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece

Phaseolus vulgaris (L.), commonly known as bean or common bean, is considered a promiscuous legume host since it forms nodules with diverse rhizobial species and symbiovars. Most of the common bean nodulating rhizobia are mainly affiliated to the genus Rhizobium, though strains belonging to Ensifer, Pararhizobium, Mesorhizobium, Bradyrhizobium, and Burkholderia have also been reported. This is the first report on the characterization of bean-nodulating rhizobia at the species and symbiovar level in Greece. The goals of this research were to isolate and characterize rhizobia nodulating local common bean genotypes grown in five different edaphoclimatic regions of Greece with no rhizobial inoculation history. The genetic diversity of the rhizobial isolates was assessed by BOX-PCR and the phylogenetic affiliation was assessed by multilocus sequence analysis (MLSA) of housekeeping and symbiosis-related genes. A total of fifty fast-growing rhizobial strains were isolated and representative isolates with distinct BOX-PCR fingerpriniting patterns were subjected to phylogenetic analysis. The strains were closely related to R. anhuiense, R. azibense, R. hidalgonense, R. sophoriradicis, and to a putative new genospecies which is provisionally named as Rhizobium sp. I. Most strains belonged to symbiovar phaseoli carrying the α-, γ-a and γ-b alleles of nodC gene, while some of them belonged to symbiovar gallicum. To the best of our knowledge, it is the first time that strains assigned to R. sophoriradicis and harbored the γ-b allele were found in European soils. All strains were able to re-nodulate their original host, indicating that they are true microsymbionts of common bean.

Characterization of Retama sphaerocarpa microsymbionts in Zaida lead mine tailings in the Moroccan middle Atlas

In the Moroccan Middle Atlas, the tailings rich in lead and other metal residues, in the abandoned Zaida mining district, represent a real threat to environment and the neighboring villages’ inhabitants’ health. In this semi-arid to arid area, phytostabilisation would be the best choice to limit the transfer of heavy metals to populations and groundwater. The aim of this work was to characterize the bacteria that nodulate Retama sphaerocarpa, spontaneous nitrogen fixing shrubby legume, native to the Zaida mining area, with great potential to develop for phytostabilisation. Forty-three bacteria isolated from root nodules of the plant were characterized. Based on REP-PCR and ARDRA, four strains were selected for further molecular analyzes. The 16S rRNA gene sequences analysis revealed that the isolated strains are members of the genus Bradyrhizobium, and the phylogenetic analysis of the housekeeping genes glnII, atpD, gyrB, rpoB, recA and dnaK individual sequences and their concatenation showed that the strains are close to B. algeriense RST89T and B. valentinum LmjM3T with similarity percentages of 89.07% to 95.66% which suggest that the newly isolated strains from this mining site may belong to a potential novel species. The phylogeny of the nodA and nodC genes showed that the strains belong to the symbiovar retamae of the genus Bradyrhizobium. These strains nodulate also R. monosperma, R. dasycarpa and Lupinus luteus.

Heavy metals multi-tolerant Bradyrhizobium isolated from mercury mining region in Algeria

Heavy metals accumulation in the environment has led to a decrease in the capacity of ecosystems to sustain life as human, animal and plant health is threatened. To remedy this problem, rhizoremediation has been suggested as a solution. Legumes and rhizobia symbiotic association has captivated attention due to its involvement in the restoration of heavy-metal-contaminated sites. Thus, the aim of this study was to isolate and characterize the strains nodulating Calicotome spinosa plant that naturally occurred in two Algerian mercury mines. Fifty-four bacterial strains were isolated, then grouped into sixteen distinct BOX-PCR patterns and were genetically identified as belonging to the Bradyrhizobium genus. The studied strains were able to induce nodules on Retama monosperma, R. reatam, Lupinus albus, while no nodulation was observed in Glycine max, their symbiotic capacity was confirmed by amplifying the nodC gene. The phylogenetic analysis based on the nodC has grouped this Bradyrhizobium strains to either symbiovar genistearum or retamae. The isolates revealed diversity in terms of NaCl; pH tolerance, and phosphate solubilization. Production of siderophores was negative for these strains. All the isolated Bradyrhizobium were tolerant to both Zn and Pb in contrast they were sensitive to Cu and Cd. Interestingly, 43% of strains were tolerant to high Hg levels. Hence, some strains displayed multiple tolerances to heavy metals. Therefore, this is the first time we identify Bradyrhizobium strains originating from a North African mercury mine. This study could help to select mercury and other heavy metal-tolerant rhizobia showing an interesting potential to be used as inoculants to remediate the heavy metal soil accumulation.

Rhizobial isolates in active layer samples of permafrost soil of Spitsbergen, Arctic

Twenty-nine strains were isolated from two samples of the permafrost active layer of the Spitsbergen archipelago. The estimated number of bacteria ranged from 4.0⋅104 to 1.7⋅107 CFU∙g-1. As a result of sequencing of the 16S rRNA (rrs) genes, the isolates were assigned to 13 genera belonging to the phyla Actinobacteria, Proteobacteria (classes α, β, and γ), Bacteroidetes, and Firmicutes. Six isolates belonged to the rhizobial genus Mesorhizobium (order Rhizobiales). A plant nodulation assay with seedlings of legume plants Astragalus norvegicus, A. frigidus, A. subpolaris and Oxytropis sordida, originated from Khibiny (Murmansk region, Russia) and inoculated with Mesorhizobium isolates, showed the inability of these strains to form nodules on plant roots. Symbiotic (sym) genes nodC and nodD were not detected in Mesorhizobium strains either.

Root Nodule Rhizobia From Undomesticated Shrubs of the Dry Woodlands of Southern Africa Can Nodulate Angolan Teak Pterocarpus angolensis, an Important Source of Timber.

Pterocarpus angolensis, a leguminous tree native to the dry woodlands of Southern Africa, provides valuable timber, but is threatened by land conversion and overharvesting while showing limited natural regeneration. Nitrogen-fixing root nodule symbionts that could improve establishment of young seedlings have not yet been described. Therefore, we investigated the ability of P. angolensis to form nodules with a diverse range of rhizobia. In drought-prone areas under climate change with higher temperatures, inoculants that are heat-tolerant and adapted to these conditions are likely to be of advantage. Sources of bacterial isolates were roots of P. angolensis from nurseries in the Kavango region, other shrubs from this area growing near Pterocarpus such as Indigofera rautanenii, Desmodium barbatum, Chamaecrista sp., or shrubs from drought-prone areas in Namaqualand (Wiborgia monoptera, Leobordea digitata) or Kalahari (Indigofera alternans). Only slight protrusions were observed on P. angolensis roots, from which a non-nodulating Microbacterium sp. was isolated. Rhizobia that were isolated from nodules of other shrubs were affiliated to Bradyrhizobium ripae WR4T, Bradyrhizobium spp. (WR23/WR74/WR93/WR96), or Ensifer/Mesorhizobium (WR41/WR52). As many plant growth-promoting rhizobacteria (PGPR), nodule isolates produced siderophores and solubilized phosphate. Among them, only the Bradyrhizobium strains nodulated P. angolensis under controlled conditions in the laboratory. Isolates were further characterized by multilocus sequence analysis and were found to be distant from known Bradyrhizobium species. Among additional reference species tested for nodulation on P. angolensis, Bradyrhizobium vignae 7-2T and Bradyrhizobium namibiense 5-10T from the Kavango region of Namibia as well as Bradyrhizobium elkanii LMG6234T and Bradyrhizobium yuanmingense LMG21728T induced nitrogen-fixing nodules, while Bradyrhizobium diazoefficiens USDA110T and Bradyrhizobium tropiciagri SEMIA6148T did not. This suggests a broad microsymbiont range from Bradyrhizobium japonicum and B. elkanii lineages. Phylogenetic analysis of nodC genes indicated that nodulating bradyrhizobia did not belong to a specific symbiovar. Also, for I. rautanenii and Wiborgia, nodule isolates B. ripae WR4T or Mesorhizobium sp. WR52, respectively, were authenticated. Characterization of symbionts inducing effective root nodules in P. angolensis and other shrubs from Subsahara Africa (SSA) give insights in their symbiotic partners for the first time and might help in future to develop bioinoculants for young seedlings in nurseries, and for reforestation efforts in Southern Africa.

Diverse Rhizobium strains isolated from root nodules of Trifolium alexandrinum in Egypt and symbiovars

Berseem clover (T. alexandrinum) is the main forage legume crop used as animal feed in Egypt. Here, eighty rhizobial isolates were isolated from root nodules of berseem clover grown in different regions in Egypt and were grouped by RFLP-16S rRNA ribotyping. Representative isolates were characterized using phylogenetic analyses of the 16S rRNA, rpoB, glnA, pgi, and nodC genes. We also investigated the performance of these isolates using phenotypic tests and nitrogen fixation efficiency assays. The majority of strains (<90%) were closely related to Rhizobium aegyptiacum and Rhizobium aethiopicum and of the remaining strains, six belonged to the Rhizobium leguminosarum genospecies complex and only one strain was assigned to Agrobacterium fabacearum. Despite their heterogeneous chromosomal background, most of the strains shared nodC gene alleles corresponding to symbiovar trifolii. Some of the strains closely affiliated to R. aegyptiacum and R. aethiopicum had superior nodulation and nitrogen fixation capabilities in berseem clover, compared to the commercial inoculant (Okadein®) and N-added treatments. R. leguminosarum strain NGB-CR 17 that harbored a nodC allele typical of symbiovar viciae, was also able to form an effective symbiosis with clover. Two strains with nodC alleles of symbiovar trifolii, R. aegyptiacum strains NGB-CR 129 and 136, were capable of forming effective nodules in Phaseolus vulgaris in axenic greenhouse conditions. This adds the symbiovar trifolii which is well-established in the Egyptian soils to the list of symbiovars that form nodules in P. vulgaris.

Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Bradyrhizobium hipponense sp. nov., isolated from Lupinus angustifolius growing in the northern region of Tunisia.

Strain aSej3T was isolated from a root nodule of a Lupinus angustifolius plant growing in Bizerte, Tunisia. 16S rRNA gene analysis placed this strain within the genus Bradyrhizobium. Multilocus sequence analysis (MLSA) including three housekeeping genes (glnII, gyrB and recA) grouped aSej3T together with Bradyrhizobium rifense CTAW71T, Bradyrhizobium cytisi CTAW11T, Bradyrhizobium ganzhouense RITF806T, Bradyrhizobium lupini USDA 3051T and Bradyrhizobium canariense BTA-1T. MLSA with five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed that this strain shares less than 93.5 % nucleotide identity with other type strains. Genome sequencing and inspection revealed a genome size of 8.83 Mbp with a G+C content of 62.8 mol%. Genome-wide average nucleotide identity and digital DNA-DNA hybridization values were below 87.5 and 36.2 %, respectively, when compared to described Bradyrhizobium species. Strain aSej3T nodulated L. angustifolius plants under axenic conditions and its nodC gene clustered within the genistearum symbiovar. Altogether, the phylogenetic data and the chemotaxonomic characteristics of this strain support that aSej3T represents a new species for which we propose the name Bradyrhizobium hipponense sp. nov. with the type strain aSej3T (=DSM 108913T=LMG 31020T).

Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., isolated from nodules of legumes indigenous to Western Australia.

The genus Bradyrhizobium is considered as the probable ancestor lineage of all rhizobia, broadly spread in a variety of ecosystems and with remarkable diversity. A polyphasic study was performed to characterize and clarify the taxonomic position of eight bradyrhizobial strains isolated from indigenous legumes to Western Australia. As expected for the genus, the 16S rRNA gene sequences were highly conserved, but the results of multilocus sequence analysis with four housekeeping genes (dnaK, glnII, gyrB and recA) confirmed three new distinct clades including the following strains: (1) WSM 1744T, WSM 1736 and WSM 1737; (2) WSM 1791T and WSM 1742; and (3) WSM 1741T, WSM 1735 and WSM 1790. The highest ANI values of the three groups in relation to the closest type strains were 92.4, 92.3 and 93.3 %, respectively, below the threshold of species circumscription. The digital DNA-DNA hybridization analysis also confirmed new species descriptions, with less than 52 % relatedness with the closest type strains. The phylogeny of the symbiotic gene nodC clustered the eight strains into the symbiovar retamae, together with seven Bradyrhizobium type strains, sharing from 94.2-98.1 % nucleotide identity (NI), and less than 88.7 % NI with other related strains and symbiovars. Morpho-physiological, phylogenetics, genomic and symbiotic traits were determined for the new groups and our data support the description of three new species, Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., with WSM 1744T (=CNPSo 4013T=LMG 31646T), WSM 1791T (=CNPSo 4014T=LMG 31647T) and WSM 1741T (=CNPSo 4020T=LMG 31651T) designated as type strains, respectively.

Phylogenetic diversity of bacterial strains from root nodules of legumes grown wild in Egypt

Abstract A total of 38 bacterial strains were isolated from root nodules of Lotus, Vicia, Trifolium, Lathyrus, Astragalus, and Hippocrepis plants grown-wild at different geographical locations in Egypt. Analysis of the almost complete sequence of the 16S rRNA gene from the bacterial strains showed that seventeen of them isolated from all the six plant species were members of the genus Rhizobium, six from Lotus, Vicia and Hippocrepis were Ensifer, and the remaining six strains from Lotus and Vicia were Neorhizobium. Sequencing of the symbiotic nodC gene from eight selected rhizobial strains confirmed their affiliation with rhizobial species. These results suggest the existence of high rhizobial diversity in wild-grown Egyptian legumes. Nodules from Lotus, Trifolium, and Astragalus also contained bacterial species of the genera Achromobacter, Ancylobacter, Bacillus, Cronobacter, Curtobacterium, Flavobacterium, Paenibacillus and Pseudomonas. These results provide information about the diversity of rhizobia associated with wild-grown legumes in Egypt, and enlarged the rhizobial spectrum of Lotus, Vicia, Trifolium, Lathyrus, Hippocrepis and Astragalus, which supported to the idea that rhizobial promiscuity could be an adaption strategy to diverse environments.

Plant growth-promoting bacteria isolated from wild legume nodules and nodules of Phaseolus vulgaris L. trap plants in central and southern Mexico

Central southern Mexico contains highly diverse legumes. In this study, nodule-associated bacteria (NAB) were isolated from wild legume nodules and from nodules on Phaseolus vulgaris plants used as a plant-trap in soils from the same areas as the wild legumes. The bacteria were identified through the 16S rRNA gene sequence analysis, tested for plant growth-promoting (PGP) activities and the production of antimicrobial compounds, and analyzed for potential nodulation by amplifying the nodC gene. Several genera with PGP activity were isolated from legume nodules, including Achromobacter, Acinetobacter, Bacillus, Brevibacillus, Brevibacterium, Burkholderia, Cupriavidus, Dyella, Ensifer, Enterobacter, Herbaspirillum, Kosakonia, Labrys, Microbacterium, Moraxella, Paraburkholderia, Pseudomonas, Rhizobium, Stenotrophomonas; and Aeromonas, Marinococcus Pseudarthrobacter and Pseudoxanthomonas were found in plant legume nodules for the first time. Pseudomonas was the most common bacteria, and Mimosa pudica was colonized by the largest number of genera (6 different genera). A Burkholderia strain from the Burkholderia cepacia complex and a firmicutes strain harbor the nodC gene, identifying them as potential novel nodulating bacteria and showing that most of the strains isolated in this study were NAB. The most frequent PGP activity identified among the strains isolated from wild legumes was IAA synthesis. Two bacteria, Stenotrophomonas sp. and Rhizobium sp., synthesized more than 250 μg/ml, which is more than the level of synthesis reported in this study for Azospirillum brasilense Sp7 (59.77 μg/ml). Nitrogen fixation and antimicrobial compound production were not common, but the production of siderophores was frequently found among all the strains. This study shows that diverse NAB with PGP activity are very common in the legume nodules from central southern Mexico.

Maize growth and yield promoting endophytes isolated into a legume root nodule by a cross-over approach

Rhizobia can colonize the roots of grasses in the Brazilian semiarid region; however, studies to isolate these bacteria for growth promotion of maize via unconventional isolation approaches have not been conducted. This study aimed to isolate, characterize (biochemically and molecularly) and assess the potential of cowpea nodulating/maize associated rhizobia for plant growth promotion. Extracts of surface-disinfected maize root were inoculated onto cowpea seeds in axenic conditions. Rhizobia were isolated and classified by sequencing of the 16 S rRNA and nodC genes, and recA and atpD were specifically assayed for Bradyrhizobium. The in vitro ability of rhizobia to produce auxin and siderophores, solubilize calcium phosphate, exhibit antagonism against the growth of the pathogen Fusarium verticillioides, and undergo endophytic colonization were evaluated. The ability of rhizobia to promote the growth of both potted cowpea and maize and to promote the growth and yield of maize under field conditions was also evaluated. Thirteen isolates were obtained, including six Rhizobium, four Agrobacterium, and three Bradyrhizobium isolates, which exhibited high genetic variability and low similarity with the type strains. All bacteria showed at least one plant growth promotion mechanism. Six bacteria stood out regarding the promotion of maize growth, and one bacterium showed the best performance in cowpea. The results of the field assay with maize indicated that Bradyrhizobium sp. ESA 114 and Rhizobium sp. ESA 116 induced the same maize grain yields as those observed in plants inoculated with the commercial strain of Azospirillum brasilense Ab-V5. Maize harbors a community of putative facultative endophytic rhizobia that are genetically diverse and have the potential to be used in inoculant production.

Genomic diversity of chickpea-nodulating rhizobia in Ningxia (north central China) and gene flow within symbiotic Mesorhizobium muleiense populations

Diversity and taxonomic affiliation of chickpea rhizobia were investigated from Ningxia in north central China and their genomic relationships were compared with those from northwestern adjacent regions (Gansu and Xinjiang). Rhizobia were isolated from root-nodules after trapping by chickpea grown in soils from a single site of Ningxia and typed by IGS PCR-RFLP. Representative strains were phylogenetically analyzed on the basis of the 16S rRNA, housekeeping (atpD, recA and glnII) and symbiosis (nodC and nifH) genes. Genetic differentiation and gene flow were estimated among the chickpea microsymbionts from Ningxia, Gansu and Xinjiang. Fifty chickpea rhizobial isolates were obtained and identified as Mesorhizobium muleiense. Their symbiosis genes nodC and nifH were highly similar (98.4 to 100%) to those of other chickpea microsymbionts, except for one representative strain (NG24) that showed low nifH similarities with all the defined Mesorhizobium species. The rhizobial population from Ningxia was genetically similar to that from Gansu, but different from that in Xinjiang as shown by high chromosomal gene flow/low differentiation with the Gansu population but the reverse with the Xinjiang population. This reveals a biogeographic pattern with two main populations in M. muleiense, the Xinjiang population being chromosomally differentiated from Ningxia-Gansu one. M. muleiense was found as the sole main chickpea-nodulating rhizobial symbiont of Ningxia and it was also found in Gansu sharing alkaline-saline soils with Ningxia. Introduction of chickpea in recently cultivated areas in China seems to select from alkaline-saline soils of M. muleiense that acquired symbiotic genes from symbiovar ciceri.

Novel putative rhizobial species with different symbiovars nodulate Lotus creticus and their differential preference to distinctive soil properties.

Phylogenetically diverse rhizobial strains endemic to Tunisia were isolated from symbiotic nodules of Lotus creticus, growing in different arid extremophile geographical regions of Tunisia, and speciated using multiloci-phylogenetic analysis as Neorhizobium huautlense (LCK33, LCK35, LCO42 and LCO49), Ensifer numidicus (LCD22, LCD25, LCK22 and LCK25), Ensifer meliloti (LCK8, LCK9 and LCK12) and Mesorhizobium camelthorni (LCD11, LCD13, LCD31 and LCD33). In addition, phylogenetic analyses revealed eight additional strains with previously undescribed chromosomal lineages within the genera Ensifer (LCF5, LCF6 and LCF8),Rhizobium (LCF11, LCF12 and LCF14) and Mesorhizobium (LCF16 and LCF19). Analysis using the nodC gene identified five symbiovar groups, four of which were already known. The remaining group composed of two strains (LCD11 and LCD33) represented a new symbiovar of Mesorhizobium camelthorni, which we propose designating as sv. hedysari. Interestingly, we report that soil properties drive and structure the symbiosis of L. creticus and its rhizobia.

Symbiotic, phenotypic and genotypic characterization of Bradyrhizobium sp. nodulating Spartium junceum L. from Bejaia, northeastern Algeria

Twenty-three bacterial strains were isolated from root nodules of Spartium junceum L., growing wild in four distinct geographic locations in Bejaia city (Northeastern Algeria). They were analyzed based on their symbiotic effectiveness, phenotypic properties and restriction fragment length polymorphism of the 16S rRNA gene. In addition, sequence analyses of the ribosomal gene, protein-coding housekeeping genes glnII and recA, and symbiotic gene (nodC) were used to clarifying their taxonomic and phylogenetic position. Plant infection tests revealed that all strains were able to form nodules on their original host plant, but with variable nodulation ability and symbiotic effectiveness. All strains were slow-growing, alkali-producing rhizobia in YEMA medium. According to phenotypic test results, the strains were grouped in four main clusters in a UPGMA dendrogram, while they were discriminated into 12 distinct genotypes by 16S rDNA-RFLP. Phylogenetic analyses of 16S rRNA genes and two concatenated housekeeping genes, glnII and recA, distributed the representative strains from each RFLP genotypes into two major clades within Bradyrhizobium genus. Some of these strains were closely related to B. retamae and B. japonicum whereas others represented four novel genospecies. Based on analysis of the symbiotic gene sequence (nodC), all strains were placed in the symbiovar retamae cluster except for SjBA12 which possibly constitutes a new symbiovar within the genus Bradyrhizobium.

Molecular, Physiological, and Symbiotic Characterization of Cowpea Rhizobia from Soils Under Different Agricultural Systems in the Semiarid Region of Brazil

The objectives of this study were to characterize the genetic diversity and evaluate the ability to tolerate stress as well as to assess the symbiotic efficiency of bacteria from cowpea nodules in agricultural soils with different uses in the semiarid region of Bahia state (Brazil). Soil samples were collected from six crop lands and one from the pristine Caatinga biome. After a trap-host experiment, the bacteria were isolated and culturally characterized. Isolates with typical characteristics of Bradyrhizobium were subjected to the nodC symbiotic gene amplification and those positive were evaluated by 16S-23S IGS-RFLP. Twenty-seven isolates belonging to different genetic clusters were selected for 16S-23S IGS sequencing. In additions, the selected bacteria were characterized biochemically and symbiotically. Among 420 characterized isolates, approximately 60% (251 isolates) displayed typical Bradyrhizobium cultural features. A total of 161, out of 251 isolates, showed positive amplification of the nodC gene fragment. The IGS-RFLP profiles analysis generated 33 groups and 27 were selected for further analysis. The fertility of the soils influenced the distribution of the isolates in the IGS-RFLP clusters. The bacteria were assigned to two genera, Bradyrhizobium and Microvirga, with 26 and 1 representative bacteria, respectively. Some isolates were able to tolerate NaCl as well as acidic and alkaline pH. In addition, isolates showed the abilities to produce biofilm under stress and to produce indole compounds, as well as efficient nodulation and nitrogen fixation. The isolates displayed great genetic, biochemical, and symbiotic variability with promising biotechnological potential.

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.