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Actually, in Republic of Congo, rhizobia have poorly phenotypically and biochemically characterized. This study aimed to characterize native rhizobia. Rhizobia strains were isolated using nodule roots collected on Milletia laurentii, Acacia spp., Albizia lebbeck, and Vigna unguiculata. The strains isolated were characterized microbiologically, biochemically, physiologically, and molecularly identified using 16S rRNA method. The results reported in this study are only for six strains of all 77 isolated: RhA1, RhAc4, RhAc15, RhAc13, RhW1, and RhV3. All native strains were positive to urease activity, negative to cellulase and pectinase activity except for one isolate that showed a positive cellulase activity. Moreover, isolates have grown at 12% of NaCl. On different effects of temperatures, isolates were able to grow up to 44°C and showed good growth at pH from 7 to 9 and the ability to use ten different carbon hydrates sources. The strains were identified as Rhizobium tropici, Rhizobium sp., Mesorhizobium sp. Bradyrhizobium yuanmingense and Bradyrhizobium elkanii. The phylogenetically analysis of the 16S rRNA genes, using a clustering method, allowed us to have a history that is both ancient and stable of four clades among genes with similar patterns. Expanding our awareness of the new legume-rhizobia will be a valuable resource for incorporating an alternative nitrogen fixation approach to consolidate the growth of legumes. These germs can be used in Congolese agriculture to improve yield of crops.

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

The study of the nitrogen fixation and phylogenetic diversity of nodule microsymbionts of grain legumes in many parts of the globe is often carried out in order to identify legume-rhizobia combinations for agricultural sustainability. Several reports have therefore found that rhizobial species diversity is shaped by edapho-climatic conditions that characterize different geographic locations, suggesting that rhizobial communities often possess traits that aid their adaptation to their habitat. In this study, the soybean-nodulating rhizobia from semi-arid savannahs of Ghana and South Africa were evaluated. The authenticated rhizobial isolates were highly diverse based on their colony characteristics, as well as their BOX-PCR profiles and gene sequences. In the 16S rRNA phylogeny, the isolates were placed in the different clades Bradyrhizobium iriomotense and Bradyrhizobium jicamae together with two superclades Bradyrhizobium japonicum and Bradyrhizobium elkanii. The multilocus (atpD, glnII, gyrB, recA) phylogenetic analyses indicated the dominance of Bradyrhizobium diazoefficiens and putative new Bradyrhizobium species in the semi-arid Ghanaian region. The phylogenetic analyses based on the symbiotic genes (nifH and nodC) clustered the test isolates into different symbiovars (sv. glycinearum, sv. retame and sv. sojae). Principal component analysis (PCA) showed that soil factors played a significant role in favoring the occurrence of soybean-nodulating microsymbionts in the tested local conditions. The results suggested that isolates had marked local adaptation to the prevailing conditions in semi-arid regions but further studies are needed to confirm new Bradyrhizobium species.

Vigna minima is a climbing annual plant widely distributed in barren wilderness, grass land, and shrub bush of China and other countries such as Japan. However, the rhizobia nodulating with this plant has never been systematically studied. In order to reveal the biodiversity of nodulating rhizobia symbiosis with V. minima, a total of 874 rhizobium isolates were obtained from root nodules of the plant spread in 11 sampling sites of Shandong Peninsula, China, and they were designated as 41 haplotypes in the genus Bradyrhizobium based upon recA sequence analyses. By multilocus sequence analysis (MLSA) of five housekeeping genes (dnaK, glnII, gyrB, recA, and rpoB), the 41 strains representing different recA haplotypes were classified into nine defined species and nine novel genospecies. Bradyrhizobium elkanii, Bradyrhizobium ferriligni, and Bradyrhizobium pachyrhizi were the predominant and universally distributed groups. The phylogeny of symbiotic genes of nodC and nifH showed similar topology and phylogenetic relationships, in which all the representative strains were classified into two clades grouped with strains nodulating with Vigna spp., demonstrating that Vigna spp. shared common nodulating groups in the natural environment. All the representative strains formed nodules with V. minima in a nodulation test performed in green house conditions. The correlation between V. minima nodulating rhizobia and soil characteristics analyzed by CANOCO indicates that available nitrogen, total nitrogen, and organic carbon in the soil samples were the main factors affecting the distribution of rhizobia isolated in this study. This study systematically uncovered the biodiversity and distribution characteristics of V. minima nodulating rhizobia for the first time, which provided novel information for the formation of the corresponding rhizobium community.

In natural plant populations, a fine-scale spatial genetic structure (SGS) can result from limited gene flow, selection pressures or spatial autocorrelation. However, limited gene flow is considered the predominant determinant in the establishment of SGS. With limited dispersal ability of bacterial cells in soil and host influence on their variety and abundance, spatial autocorrelation of bacterial communities associated with plants is expected. For this study, we collected genetic data from legume host plants, Chamaecrista fasciculata, their Bradyrhizobium symbionts and rhizosphere free-living bacteria at a small spatial scale to evaluate the extent to which symbiotic partners will have similar SGS and to understand how plant hosts choose among nodulating symbionts. We found SGS across all sampled plants for both the host plants and nodulating rhizobia, suggesting that both organisms are influenced by similar mechanisms structuring genetic diversity or shared habitat preferences by both plants and microbes. We also found that plant genetic identity and geographic distance might serve as predictors of nodulating rhizobia genetic identity. Bradyrhizobium elkanii was the only type of rhizobia found in nodules, which suggests some level of selection by the host plant.

We report here the annotated draft genome sequence of the rhizobium strain BR 2003. This strain is able to establish symbiosis and to fix nitrogen with a broad range of leguminous species. The estimation of the average nucleotide identity confirmed the strain as a member of Bradyrhizobium elkanii.

Organisms rely on symbiotic associations for metabolism, protection, and energy. However, these intimate partnerships can be vulnerable to exploitation. What prevents microbial mutualists from parasitizing their hosts? In legumes, there is evidence that hosts have evolved sophisticated mechanisms to manage their symbiotic rhizobia, but the generality and evolutionary origins of these control mechanisms are under debate. Here, we focused on the symbiosis between Parasponia hosts and N2-fixing rhizobium bacteria. Parasponia is the only non-legume lineage to have evolved a rhizobial symbiosis and thus provides an evolutionary replicate to test how rhizobial exploitation is controlled. A key question is whether Parasponia hosts can prevent colonization of rhizobia under high nitrogen conditions, when the contribution of the symbiont becomes nonessential. We grew Parasponia andersonii inoculated with Bradyrhizobium elkanii under four ammonium nitrate concentrations in a controlled growth chamber. We measured shoot and root dry weight, nodule number, nodule fresh weight, nodule volume. To quantify viable rhizobial populations in planta, we crushed nodules and determined colony forming units (CFU), as a rhizobia fitness proxy. We show that, like legumes and actinorhizal plants, P. andersonii is able to control nodule symbiosis in response to exogenous nitrogen. While the relative host growth benefits of inoculation decreased with nitrogen fertilization, our highest ammonium nitrate concentration (3.75 mM) was sufficient to prevent nodule formation on inoculated roots. Rhizobial populations were highest in nitrogen free medium. While we do not yet know the mechanism, our results suggest that control mechanisms over rhizobia are not exclusive to the legume clade.

Cowpea (Vigna unguiculata L. Walp.) is indigenous to Africa, and highly valued for its N2-fixing trait and the nutritional attributes of its grain and leaves. The species’ ability to establish effective symbiosis with diverse rhizobial populations gives it survival and growth advantage in N-limited environments. To explore the functional diversity and phylogenetic positions of rhizobia nodulating cowpea in Africa, nodules were collected from various cowpea varieties grown in soils from the Guinea savanna and Sudano-sahelian agroecologies of Northern Ghana, and from the lowveld and middleveld areas of Mpumalanga Province in South Africa. Box-PCR profiling and multilocus sequence analysis revealed the presence of diverse microsymbionts responsible for cowpea nodulation across the study sites. BOX-PCR amplifications yielded variable band sizes, ranging from 618 bp to 5354 bp, which placed the isolates in six major clusters (Cluster A–F). Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, rpoB, nifH and nodC genes revealed the presence of diverse Bradyrhizobium sp. closely related to Bradyrhizobium daqingense, Bradyrhizobium subterraneum, Bradyrhizobium yuanmingense, Bradyrhizobium embrapense, Bradyrhizobium pachyrhizi, Bradyrhizobium elkanii and novel Bradyrhizobium species in the soils studied, a finding that could be attributed to the unique edapho-climatic conditions of the contrasting environments. The test isolates exhibited distinct symbiotic efficiencies, and also induced variable (p ≤ 0.001) photosynthetic rates, leaf transpiration, total chlorophyll and shoot biomass accumulation on cowpea (their homologous host). Canonical correspondence analysis showed that the distribution of these microsymbionts was influenced by the concentrations of macro- and micronutrients in soils. The pairwise genetic distances derived from phylogenies and nodule functioning showed significant (p Bradyrhizobium symbiosis.

Some bacteria collectively known as rhizobia can establish symbiotic relationships and the N2-fixation process with several legumes used as green manure, in pastures and for wood production. Symbionts belonging to the genus Bradyrhizobium are predominant in the tropics, and an increasing number of studies report high genetic diversity within the genus. We performed a polyphasic study with two strains belonging to the genus Bradyrhizobium – SEMIA 6399T and SEMIA 6404–isolated from root nodules of Deguelia costata (syn. Lonchocarpus costatus), an important legume native to eastern Brazil. In general, sequences of the 16S rRNA gene were highly conserved in members of the genus Bradyrhizobium , and the two strains were positioned in the Bradyrhizobium elkanii superclade, sharing 100 % nucleotide identity with Bradyrhizobium embrapense , Bradyrhizobium erythrophlei and Bradyrhizobium viridifuturi . However, multilocus sequence analysis with four housekeeping genes (dnaK, glnII, gyrB and recA) confirmed that the two strains belong to a distinct clade, sharing from 87.7 to 96.1 % nucleotide identity with related species of the genus Bradyrhizobium , being most closely related to B. viridifuturi . Average nucleotide identity of genome sequences between SEMIA 6399T and related species was lower than 92 %, below the threshold of species circumscription. nifH phylogeny clustered the SEMIA strains in a clade separated from other species of the genus Bradyrhizobium , and the nodD phylogeny revealed that SEMIA 6399T presents a more divergent sequence. Other phenotypic and genotypic traits were determined for the new group, and our data support the description of the SEMIA strains as representatives of Bradyrhizobium mercantei sp. nov.; SEMIA 6399T (=CNPSo 1165T=BR 6010T=U675T=LMG 30031T) was chosen as the type strain.

Reforestation of native Acacia confusa Merr. on landslide areas in Taiwan is important for agroforestry and soil conservation. To ensure high survival and growth vigor, A. confusa seedlings must develop a strong root system. Inoculating of acacia with symbiotic nitrogen-fixing bacteria (NFB) may ameliorate the problems associated with soil nutrient deficiency on landslide sites. In this study, under plastic house condition, a NFB was isolated from the root nodules of native A. confusa and identified as Bradyrhizobium elkanii, and its effects on growth, root system morphology and pullout resistance of acacia seedlings were investigated. Our results revealed that the growth of inoculated seedlings is significantly more vigor than that of the noninoculated controls. The enhancements in height, tap root length, shoot biomass and root biomass were 40, 100, 140 and 130%, respectively. Also, inoculated seedlings had significantly longer total root length (150%), larger external root surface area (130%), larger root volume (70%), and more root tip number (60%) than the controls. Moreover, the inoculated seedlings developed significantly stronger root functional traits, that is, root density (130%), root length density (60%) and specific root length (60%), than the controls. Consistently, the root pullout resistance of inoculated seedlings was significantly higher than that of the noninoculated ones. These results demonstrate that B. elkanii is an effective nitrogen-fixing bacterium capable of enhancing growth, root development and pullout resistance of A. confusa. Key words: Fabaceae, inoculation, nodules, pullout resistance, root morphology.

Understanding the factors that influence the diversity of soybean-nodulating rhizobia is important before doing inoculation. Since studies about this topic in tropical regions are limited, this could lay the groundwork for related research particularly on Bradyrhizobium elkanii. To determine the genetic diversity of B. elkanii in different regions, we conducted Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequence analysis of 16S rRNA gene, internal transcribed spacer (ITS) region and rpoB gene. Also, sequence analysis of symbiotic nifD and nodD1 genes was conducted. Analysis of the rpoB gene revealed a higher genetic diversity than the ITS region, and possible endemic B. elkanii strains were observed. Meanwhile, no variation was detected among the strains in both nifD and nodD1 phylogenies. Through rpoB gene analysis, variations in the ITS-rpoB type of B. elkanii strains were distinguished and differentiated with that of the closest reference strains. We identified potential soybean inoculants which possess symbiotic efficiency regardless of the Rj genotypes used, suggesting broad host-range of the strains. We show how the genetic diversity of soybean-nodulating B. elkanii strains in subtropical and tropical regions might be influenced by temperature and soil pH and, provided some insights between the symbiotic genes and Rj genotypes.

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some Bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain Bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

Due to its high capacity for nitrogen fixation, strain SEMIA 938 is used in commercial inoculants for lupins in Brazil. Its genome was estimated at 8,780,064 bp and indicates that it belongs to the Bradyrhizobium elkanii species, while the analysis of nodulation genes classifies the strain in the symbiovar sojae.

In the N2-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S–23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S–23S rRNA region yielded single polymorphic bands of varying lengths (573–1298 bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard’s similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.

The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an “eco-friendly agricultural practice”. Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea ( Vigna unguiculata ) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense ; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi . Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium : strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi . Genome average nucleotide identity and DNA–DNA reassociation confirmed the genomic identification of B . yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nod C and nif H gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Root-nodule bacteria were isolated from wild legumes growing in the Kavango region, Namibia. Using a polyphasic approach, four strains belonging to the genus Bradyrhizobium (WR4T, WR87, T10 and T12) were further characterized to clarify the taxonomic status of this group. On the basis of 16S rRNA gene sequences, the four strains showed highest similarity to Bradyrhizobium elkanii USDA 76T (99.9 %), Bradyrhizobium pachyrhizi PAC48T (identical) and to Bradyrhizobium brasilense UFLA03-321T (identical). Multilocus sequence analysis of concatenated glnII-recA-gyrB-dnaK-rpoB sequences and comparison of the intergenic transcribed spacer (ITS) sequences confirmed that the novel group belongs to a distinct lineage of the genus Bradyrhizobium , with

Biological nitrogen fixation is a key process for agricultural production and environmental sustainability, but there are comparatively few studies of symbionts of tropical pasture legumes, as well as few described species of the genus Bradyrhizobium, although it is the predominant rhizobial genus in the tropics. A detailed polyphasic study was conducted with two strains of the genus Bradyrhizobium used in commercial inoculants for tropical pastures in Brazil, CNPSo 1112T, isolated from perennial soybean (Neonotonia wightii), and CNPSo 2833T, from desmodium (Desmodium heterocarpon). Based on 16S-rRNA gene phylogeny, both strains were grouped in the Bradyrhizobium elkanii superclade, but were not clearly clustered with any known species. Multilocus sequence analysis of three (glnII, gyrB and recA) and five (plus atpD and dnaK) housekeeping genes confirmed that the strains are positioned in two distinct clades. Comparison with intergenic transcribed spacer sequences of type strains of described species of the genus Bradyrhizobium showed similarity lower than 93.1 %, and differences were confirmed by BOX-PCR analysis. Nucleotide identity of three housekeeping genes with type strains of described species ranged from 88.1 to 96.2 %. Average nucleotide identity of genome sequences showed values below the threshold for distinct species of the genus Bradyrhizobium ( nifH and nodC gene sequences positioned the two strains in a clade distinct from other species of the genus Bradyrhizobium. Morphophysiological, genotypic and genomic data supported the description of two novel species in the genus Bradyrhizobium, Bradyrhizobium tropiciagri sp. nov. (type strain CNPSo 1112T = SMS 303T = BR 1009T = SEMIA 6148T = LMG 28867T) and Bradyrhizobium embrapense sp. nov. (type strain CNPSo 2833T = CIAT 2372T = BR 2212T = SEMIA 6208T = U674T = LMG 2987).

Nodulation symbiosis is a key component of natural ecosystems, and the nitrogen-fixing ability of such symbioses can have a significant impact on the restoration of degraded ecosystems. Here, we investigated the diversity of legume-rhizobium symbioses, compared the N2-fixing efficiency of legume-rhizobium symbioses, and clarified the relationship of rhizobia, host species, and microhabitat. Eighteen potential N2-fixing species were surveyed, and only nine of them were found to be nodulated. Most of wild legumes were found to be associated with rhizobia belonging to the genus Bradyrhizobium, except for Callerya nitida, which formed associations with the genus Mesorhizobium. δ15N signatures of Derris fordii were significantly closer to zero than all the other plants tested, suggesting that the D. fordii-ISA2507 (Bradyrhizobium elkanii) symbiosis might have the highest N2-fixing efficiency of all the associations. D. fordii and its rhizobial symbionts may have potential for application in ecological restoration and agricultural production. Host species selection was the major factor, and there was no significant effect of slope position on nodulation-legume symbioses in our study area, in which altitudes ranged from 287 m to 521 m at the small scale. The Shannon diversity and the evenness of N2-fixing species (nodulated legumes) were significantly positively correlated (P

Rhizobia induce root nodules and fix atmospheric N2 for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume–bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of Dalbergia odorifera seedlings that were uninoculated or inoculated with Bradyrhizobium elkanii H255, Rhizobium multihospitium–like HT221, or Burkholderia pyrrocinia–like H022238, under two growth media (nitrogen [N] supplied soil or N omitted potting mix). Seedlings inoculated with Bradyrhizobium H255 had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the 15N natural abundance method, it was shown that the inoculated plants had significantly higher N2 fixation efficiency (48−57%) and specific nodule activity (269−313 μg N mg-1 of dry weight [dwt] nodule) compared to the uninoculated plants (203 μg N mg-1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (P

Desmodium spp. are leguminous plants belonging to the tribe Desmodieae of the subfamily Papil-ionoideae. They are widely distributed in temperated and subtropical regions and are used as forageplants, for biological control, and in traditional folk medicine. The genus includes pioneer species thatresist the xerothermic environment and grow in arid, barren sites. Desmodium species that form nitrogen-fixing symbiosis with rhizobia play an important role in sustainable agriculture. In Argentina, 23 nativespecies of this genus have been found, including Desmodium incanum. In this study, a total of 64 D.incanum-nodulating rhizobia were obtained from root nodules of four Argentinean plant populations.Rhizobia showed different abiotic-stress tolerances and a remarkable genetic diversity using PCR fin-gerprinting, with more than 30 different amplification profiles. None of the isolates were found at morethan one site, thus indicating a high level of rhizobial diversity associated with D. incanum in Argentineansoils. In selected isolates, 16S rDNA sequencing and whole-cell extract MALDI TOF analysis revealedthe presence of isolates related to Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobiumyuanmingense, Bradyrhizobium liaoningense, Bradyrhizobium denitrificans and Rhizobium tropici species.In addition, the nodC gene studied in the selected isolates showed different allelic variants.Isolates were phenotypically characterized by assaying their growth under different abiotic stresses.Some of the local isolates were remarkably tolerant to high temperatures, extreme pH and salinity,which are all stressors commonly found in Argentinean soils. One of the isolates showed high tolerance totemperature and extreme pH, and produced higher aerial plant dry weights compared to other inoculatedtreatments. These results indicated that local isolates could be efficiently used for D. incanum inoculation., Instituto de Biotecnologia y Biologia Molecular, Universidad Nacional del Litoral

Made available in DSpace on 2018-12-11T17:10:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The genus Bradyrhizobium comprises bacteria with the ability to form nitrogen-fixing symbioses with legumes. They are of great interest in agriculture, as well as for the production of biopolymers such as polyhydroxyalkanoates. Here, we report the draft genome assembly of Bradyrhizobium elkanii TnphoA 33 comprising 9 Mb, 1,124 contigs, and 9,418 open reading frames. Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias UNESP-Universidade Estadual Paulista University Medical Center Utrecht Departamento de Biologia Aplicada à Agropecuária Faculdade de Ciências Agrárias e Veterinárias UNESP- Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Tecnologia Laboratorio Multiusuario de Sequenciamento em Larga Escala e Expressao Genica UNESP-Universidade Estadual Paulista Departamento de Tecnologia Faculdade de Ciências Agrárias e Veterinárias UNESP-Universidade Estadual Paulista Departamento de Biologia Aplicada à Agropecuária Faculdade de Ciências Agrárias e Veterinárias UNESP- Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias Departamento de Tecnologia Laboratorio Multiusuario de Sequenciamento em Larga Escala e Expressao Genica UNESP-Universidade Estadual Paulista

Root nodule bacteria were isolated from Centrolobium paraense Tul. grown in soils from the Amazon region, State of Roraima (Brazil). 16S rRNA gene sequence analysis of seven strains (BR 10247T, BR 10296, BR 10297, BR 10298, BR 10299, BR 10300 and BR 10301) placed them in the genus Bradyrhizobium with the closest neighbours being the type strains of Bradyrhizobium paxllaeri (98.8 % similarity), Bradyrhizobium icense (98.8 %), Bradyrhizobium lablabi (98.7 %), Bradyrhizobium jicamae (98.6 %), Bradyrhizobium elkanii (98.6 %), Bradyrhizobium pachyrhizi (98.6 %) and Bradyrhizobium retamae (98.3 %). This high similarity, however, was not confirmed by the intergenic transcribed spacer (ITS) 16S–23S rRNA region sequence analysis nor by multi-locus sequence analysis. Phylogenetic analyses of five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EK05T ( = LMG 24129T) to be the most closely related type strain (95.7 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [major components being C16 : 0 and summed feature 8 (18 : 1ω6c/18 : 1ω7c)], DNA G+C content, slow growth rate and carbon compound utilization patterns, supported the placement of the novel strains in the genus Bradyrhizobium . Results of DNA–DNA relatedness studies and physiological data (especially carbon source utilization) differentiated the strains from the closest recognized species of the genus Bradyrhizobium . Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) placed the novel species in a new branch within the genus Bradyrhizobium . Based on the current data, these seven strains represent a novel species for which the name Bradyrhizobium neotropicale sp. nov. is proposed. The type strain is BR 10247T ( = HAMBI 3599T).

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.

Isolados de actinomicetos e de fungos micorrízicos arbusculares foram avaliados em sistemas de inoculação conjunta in vivo, em plantas de soja inoculadas com rizóbios, com a finalidade de se observar interações microbianas. A pesquisa foi realizada no Centro Nacional de Pesquisa de Agrobiologia (Embrapa CNPAB). Foram utilizados, como substrato, solo da série Itaguaí (textura média) misturado com areia de rio na proporção 1:1 (v:v), com adição de isolados de actinomicetos (ACT-78 e ACT-370); estirpes de rizóbios (Bradyrhizobium elkanii - BR 29 e Bradyrhizobium japonicum - BR 33) e fungos micorrízicos arbusculares (Gigaspora margarita e Glomus clarum). Os resultados revelam que a inoculação com os fungos micorrízicos arbusculares influenciou de modo expressivo as variáveis de crescimento, nodulação e densidade de actinomicetos na rizosfera de soja, que apresentaram valores maiores do que as plantas sem o inóculo fúngico. Houve efeitos sinergísticos e antagônicos dos fungos micorrízicos arbusculares com os actinomicetos e com rizóbios. Actinomycete isolates and arbuscular mycorrhizal fungi were evaluated in vivo using jointly inoculation systems with soybean plants inoculated with rhizobia, with the aim to observe microbial interactions. The study was carried out at the National Center of Agrobiologic Research (Embrapa CNPAB). Soil of the Itaguai series (medium texture) was used as substrate mixed with river sand in proportion 1:1 (v:v), adding actinomycete isolates (ACT 78 and ACT 370), rhizobia strains (Bradyrhizobium elkanii - BR 29 and Bradyrhizobium japonicum - BR 33), and arbuscular mycorrhizal fungi (Gigaspora margarita and Glomus clarum). The results revealed the effect of the arbuscular mycorrhizal fungi addition with significant and expressive influence on growth parameters, nodulation and density of actinomycete isolates on the soybean rhizosphere which showed greater values compared with fungi uninoculated plants. Synergistic and antagonistic effects were observed between the arbuscular mycorrhizal fungi with actinomycetes isolates and rhizobia strains.

Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a critical role in sustainable agriculture because it reduces the need for external nitrogen input. One remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both inter- and intra-species levels and takes place at multiple phases of the interaction, ranging from initial bacterial infection and nodulation to late nodule development associated with nitrogen fixation. Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this report, we describe fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4 allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus within a 57-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.

Background and aims Bradyrhizobium japonicum and Bradyrhizobium elkanii dominated soybean nodules in temperate and subtropical regions in Nepal, respectively, in our previous study. The aims of this study were to reveal the effects of temperature on the nodulation dominancy of B. japonicum and B. elkanii and to clarify the relationship between the effects of temperature and the climate-dependent distribution of Bradyrhizobium species.

We investigated the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia and their geographical distribution in the United States using nine soil isolates from eight states. The bradyrhizobia were inoculated on three soybean Rj genotypes (non- Rj , Rj 2 Rj 3 , and Rj 4 ). We analyzed their genetic diversity and community structure by means of restriction fragment length polymorphisms of PCR amplicons to target the 16S-23S rRNA gene internal transcribed spacer region, using 11 USDA Bradyrhizobium strains as reference strains. We also performed diversity analysis, multidimensional scaling analysis based on the Bray-Curtis index, and polar ordination analysis to describe the structure and geographical distribution of the soybean-nodulating bradyrhizobial community. The major clusters were Bradyrhizobium japonicum Bj123, in the northern United States, and Bradyrhizobium elkanii , in the middle to southern regions. Dominance of bradyrhizobia in a community was generally larger for the cluster belonging to B. elkanii than for the cluster belonging to B. japonicum . The indigenous American soybean-nodulating bradyrhizobial community structure was strongly correlated with latitude. Our results suggest that this community varies geographically.

O objetivo deste trabalho foi avaliar a atividade de enzimas fermentativas em raízes e nódulos de soja (Glycine max) sob condições de hipóxia e pós-hipóxia. Os genótipos Fundacep 53 RR e BRS Macota foram cultivados em vasos de 3L contendo vermiculita como substrato em associação com Bradyrhizobium elkanii em casa de vegetação. No estádio R2, o sistema radicular das plantas foi submetido à hipóxia pelo encaixe de um segundo vaso sem perfurações e inundado com solução nutritiva diluída a 1/3 da concentração original, por 8 dias. Posteriormente, foram retirados os vasos sem perfurações para avaliação da recuperação. O material vegetal foi coletado no 2º, 4º, 6º e 8º dias após a inundação ou recuperação, juntamente com os controles para a dosagem da atividade das enzimas álcool desidrogenase (ADH), piruvato descarboxilase (PDC) e lactato desidrogenase (LDH) em raízes e nódulos. Os dados foram submetidos à ANOVA e comparados pelo teste de Tukey a 5% de probabilidade. As atividades da ADH, PDC e LDH foram fortemente estimuladas com a inundação, especialmente em nódulos, e diminuíram com a recuperação, em ambos os genótipos. Fundacep 53 RR respondeu de forma mais efetiva aos efeitos dos tratamentos. This study aimed to evaluate the activity of fermentative enzymes in soybean (Glycine max) roots and nodules under hypoxia and post-hypoxia conditions. The soybean genotypes Fundacep 53 RR and BRS Macota associated with Bradyrhizobium elkanii were grown in 3L pots containing vermiculite as substrate under greenhouse conditions. Root system of plants at the R2 stage was subjected to hypoxia by blocking drainage of the pot, by fitting a second one without holes and flooded with nutrient solution diluted one-third of the original. The flooding of the plants was carried out for eight days and after, the pot without drilling was removed for recovery assessment. Non-waterlogged plants were kept as control. Activities of the fermentation enzymes alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC) and lactate dehydrogenase (LDH) were quantified in roots and nodules collected at 2, 4, 6 and 8 days after flooding or recovery. Data were analyzed by ANOVA and compared by the Tukey test (P ≤ 0,05). Activities of ADH, PDC and LDH were hardly stimulated during flooding, especially in nodules, in both genotypes and decreased with recovery. Fundacep 53 RR responded more effectively to treatment effects.

In smallholder farms of Côte d'Ivoire, particularly in the northeast of the country, Cajanus cajan (pigeonpea) has become an important crop because of its multiple beneficial facets. Pigeonpea seeds provide food to make ends meet, are sold on local markets, and aerial parts serve as forage for animals. Since it fixes atmospheric nitrogen in symbiosis with soil bacteria collectively known as rhizobia, C. cajan also improves soil fertility and reduces fallow time. Yet, seed yields remain low mostly because farmers cannot afford chemical fertilizers. To identify local rhizobial strains susceptible to be used as bio-inoculants to foster pigeonpea growth, root nodules were collected in six fields of three geographically distant regions of Côte d'Ivoire. Nodule bacteria were isolated and characterized using various molecular techniques including matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) and DNA sequencing. These molecular analyses showed that 63 out of 85 nodule isolates belonged to two major clades of bradyrhizobia, one of which is known as the Bradyrhizobium elkanii super clade. Phylogenies of housekeeping (16S-ITS-23S, rpoB) and symbiotic (nifH) genes were not always congruent suggesting that lateral transfer of nitrogen fixation genes also contributed to define the genome of these bradyrhizobial isolates. Interestingly, no field-, plant-, or cultivar-specific effect was found to shape the profiles of symbiotic strains. In addition, nodule isolates CI-1B, CI-36E, and CI-41A that belong to distinct species, showed similar symbiotic efficiencies suggesting that any of these strains might serve as a proficient inoculant for C. cajan.

Bradyrhizobium elkanii UASWS1016 has been isolated from a wet oxidation sewage plant in Italy. Fully equipped for ammonia assimilation, heavy metal resistances, and aromatic compounds degradation, it carries a large type IV secretion system, specific of plant-associated microbes. Deprived of toxins, it could be considered for agricultural and environmental uses.

We report here the draft genome sequences of Bradyrhizobium elkanii strains BLY3-8 and BLY6-1, which are incompatible with Rj 3 genotype soybean cultivars. The genome sequences of these strains will be useful to identify a causal gene for this incompatibility.

Bradyrhizobium elkanii UASWS1015 was isolated from a sewage plant in Switzerland. Its genome indicates that it is fully equipped for ammonia assimilation and aromatic compound degradation, and it displays a large type IV secretion system, which characterizes plant-associated microbes. Totally deprived of toxins, it could be considered for agricultural and environmental uses.

Soybean [ Glycine max (L.) Merr.] is the most important legume cropped worldwide, with an economic value attributed mainly to the high protein content of the grain, which, in turn, demands a heavy supply of nitrogen. An environmentally friendly source of nitrogen at low cost to farmers is represented by the biological fixation of atmospheric nitrogen that takes place is association with symbiotic bacteria. For the soybean, the association occurs mainly with bacteria belonging to the species Bradyrhizobium japonicum and Bradyrhizobium elkanii , but our knowledge about gene expression associated with the symbiosis is still poor. This work—part of an effort by the Soybean Genome Consortium (Genosoja)—was conducted with the aim of achieving better understanding about the functionality of the genes expressed in soybean roots in the early stages of the interaction with B. japonicum . The study was performed with soybean plants growing under greenhouse conditions, inoculated (or not) with strain CPAC 15 (=SEMIA 5079) of B. japonicum , which is broadly used in Brazilian commercial inoculants. Total RNA was extracted, the mRNA was isolated and a subtractive library was constructed. Sequencing analysis generated 4,621,072 reads, which were assembled in 3,776 sequences, defined as the differentially expressed sequences of the inoculated versus non-inoculated treatments. The sequences were categorized according to their molecular function and grouped with representatives of antioxidant activities, molecular transduction, transporters and enzyme regulation activities, but with an emphasis on catalytic and molecular binding/signaling

The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

Twenty-seven and twenty-four soybean rhizobial isolates from Java and Sumatra, respectively,were characterized. Based on cross-inoculation, eight isolates from Java and nine from Sumatra could be grouped as soybean specific rhizobial species , while 19 isolates from Java and 15 from Sumatra were promiscuous. ARDRA of intergenic spacer region of 16S-23S rDNA showed that the isolates from Java were different from those from Sumatra. Six soybean specific isolates from Java and one from Sumatra were in the same cluster with the reference strain, Bradyrhizobium japonicum USDA 110, thus could be classified as B . japonicum . One soybean specific isolate from Java’s has a distinct position, while the other soybean specific isolate from Java was placed in another group dominated by isolates from Sumatra. The nineteen promiscuous isolates from Java were clustered in a different group. This group, together with the isolate with distinct position and the other group that were dominated by isolates from Sumatra, were distinct from B. japonicum USDA 110. Therefore it is tempting to speculate that they represent indigenous soybean rhizobial strains. Based on complete sequencing of the amplified 16S rDNA of 21 selected isolates, these isolates could be divided into three groups consisting of twelve Bradyrhizobium elkanii , eight Bradyrizobium japonicum and one Sinorhizobium fredii . Most of the B. elkanii strains were isolated from acid soils at Sitiung, West-Sumatra, while only two isolates were obtained from Java. Four isolates from Java, two isolates from Sitiung, and two isolates from Bukit Tinggi were identified as B. japonicum . One isolate from Java with a distinct position on the ARDRA was identified as S. fredii .

Bacteria belonging to the genus Bradyrhizobium are capable of establishing symbiotic relationships with a broad range of plants belonging to the three subfamilies of the family Leguminosae ( = Fabaceae), with the formation of specialized structures on the roots called nodules, where fixation of atmospheric nitrogen takes place. Symbiosis is under the control of finely tuned expression of common and host-specific nodulation genes and also of genes related to the assembly and activity of the nitrogenase, which, in Bradyrhizobium strains investigated so far, are clustered in a symbiotic island. Information about the diversity of these genes is essential to improve our current poor understanding of their origin, spread and maintenance and, in this study, we provide information on 40 Bradyrhizobium strains, mostly of tropical origin. For the nodulation trait, common (nodA), Bradyrhizobium-specific (nodY/K) and host-specific (nodZ) nodulation genes were studied, whereas for fixation ability, the diversity of nifH was investigated. In general, clustering of strains in all nod and nifH trees was similar and the Bradyrhizobium group could be clearly separated from other rhizobial genera. However, the congruence of nod and nif genes with ribosomal and housekeeping genes was low. nodA and nodY/K were not detected in three strains by amplification or hybridization with probes using Bradyrhizobium japonicum and Bradyrhizobium elkanii type strains, indicating the high diversity of these genes or that strains other than photosynthetic Bradyrhizobium must have alternative mechanisms to initiate the process of nodulation. For a large group of strains, the high diversity of nod genes (with an emphasis on nodZ), the low relationship between nod genes and the host legume, and some evidence of horizontal gene transfer might indicate strategies to increase host range. On the other hand, in a group of five symbionts of Acacia mearnsii, the high congruence between nod and ribosomal/housekeeping genes, in addition to shorter nodY/K sequences and the absence of nodZ, highlights a co-evolution process. Additionally, in a group of B. japonicum strains that were symbionts of soybean, vertical transfer seemed to represent the main genetic event. In conclusion, clustering of nodA and nifH gives additional support to the theory of monophyletic origin of the symbiotic genes in Bradyrhizobium and, in addition to the analysis of nodY/K and nodZ, indicates spread and maintenance of nod and nif genes through both vertical and horizontal transmission, apparently with the dominance of one or other of these events in some groups of strains.

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium , including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii , Bradyrhizobium elkanii , B. japonicum , and B. yuanmingense , demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.

A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S–23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.

Summary Although some sugarcane cultivars may benefit substantially from biological nitrogen fixation (BNF), the responsible bacteria have been not identified yet. Here, we examined the active diazotrophic bacterial community in sugarcane roots from Africa and America by reverse transcription (RT)-PCR using broad-range nifH-specific primers. Denaturing gradient gel electrophoresis (DGGE) profiles obtained from sugarcane showed a low diversity at all sample locations with one phylotype amounting up to 100% of the nifH transcripts. This major phylotype has 93.9–99.6% DNA identity to the partial nifH sequence from a strain affiliated with Rhizobium rosettiformans. In addition, nifH transcripts of this phylotype were also detected in spruce roots sampled in Germany, where they made up 91% of nifH transcripts detected. In contrast, in control soil or shoot samples two distinct nifH transcript sequences distantly related to nifH from Sulfurospirillum multivorans or Bradyrhizobium elkanii, respectively, were predominant. These results suggest that R. rosettiformans is involved in root-associated nitrogen fixation with sugarcane and spruce, plants that do not form root–nodule symbioses.

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.

Soybean plants establish symbiotic relationships with soil rhizobia which form nodules on the plant roots. Nodule formation starts when the plant roots exudate isoflavonoids that induce nod gene expression of a specific Bradyrhizobium. We examined the specific indigenous rhizobia that form nodules with the soybean cultivars Peking and Tamahomare in different soils. PCR-RFLP analysis targeted to the 16S-23S rRNA gene internal transcribed spacer (ITS) region of the bacterial type of each root nodule showed that Bradyrhizobium japonicum (USDA110-type) and Bradyrhizobium elkanii (USDA94-type) had high compatibility with the Tamahomare and Peking cultivars, respectively. We grew 93 recombinant inbred lines (RIL) of soybean seeds derived from the cross between Peking and Tamahomare in three different field soils and identified the indigenous rhizobia nodulating each line using the same PCR-RFLP analysis. QTL analysis identified one QTL region in chromosome-18 with a highly significant additive effect that controls compatibility with both B. japonicum USDA110 and B. elkanii USDA94. We also measured the amount of daidzein and genistein secretion from roots of the 93 RILs by HPLC analysis. QTL analysis showed one QTL region in chromosome-18 controlling genistein secretion from roots and coinciding with that regulating compatibility of specific indigenous rhizobia with soybean. The amount of genistein may be a major regulatory factor in soybean-rhizobium compatibility.

The diversity among 269 rhizobia isolated from naturally occurring root nodules of soybean collected from two different agro-ecological regions of India, based on RFLP and sequences of the intergenic spacer (IGS) between the 16S and 23S rRNA genes, growth rate, and indole acetic acid production, revealed their significant, site-dependent genomic diversity. Among these bacteria, nine IGS genotypes were identified with two endonucleases. They were distributed into five divergent lineages by sequence analysis of each IGS representative strain, i.e., (1) comprising IGS genotypes I, II, III, and reference Bradyrhizobium yuanmingense; (2) with genotype IV and strains of unclassified bradyrhizobia genomic species; (3) including genotypes V, VI, and Bradyrhizobium liaoningense; (4) with IGS genotype VII and Bradyrhizobium elkanii strains; and (5) comprising IGS genotypes VIII, IX, and different Ensifer genus bacteria. Host-specificity test revealed that all rhizobia-nodulated soybean and cowpea and only part of them formed nodules on Arachis hypogeae and Cajanus cajan. The great diversity of soybean nodulators observed in this study emphasises that Indian soil is an important reservoir of nitrogen-fixing rhizobia.

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.

Polyphasic characterization of 54 indigenous mung bean (Vigna radiata L.) rhizobia from different geographic regions of China was determined by analyzing the variability of 16S rRNA gene RFLP, 16S–23S rRNA gene Intergenetic Spacer (IGS) RFLP, G-C rich RAPD and phenotype assays. Based on these characteristics, mung bean rhizobia were clustered into four groups. Group I comprised 16 slow-growing isolates from a variety of geographic regions. This group was genetically distinct from Bradyrhizobium japonicum and Bradyrhizobium liaoningense, and may represent a new species. Group II was composed of 18 isolates, which could be sub-divided into two sub-groups that were respectively related to B. japonicum and B. liaoningense. Group III comprised 12 isolates from South China and clustered together with Bradyrhizobium elkanii. Group IV formed a miscellany of 8 fast-growing isolates variously related to the genera Sinorhizobium, Rhizobium and Mesorhizobium.

We investigated the genetic diversity and field distribution of indigenous soybean-nodulating rhizobia in the Okinawa islands, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of their 16S rDNA regions and 16S–23S rDNA internal transcribed spacer (ITS) regions. We collected rhizobia from four sites on Okinawa Island, one on Miyako Island and one on Ishigaki Island, isolating 360 strains from soybean cultivars with three Rj-genotypes, Akishirome or Bragg (non-Rj), CNS (Rj 2 Rj 3) and Hill or Fukuyutaka (Rj 4), planted with soil from each field site. Phylogenies of all isolates were analyzed by PCR-RFLP with Bradyrhizobium and Sinorhizobium USDA strains as reference strains. The PCR-RFLP analysis of 16S rDNA and the ITS regions revealed the dominance of Bradyrhizobium elkanii strains in acidic Okinawa soils, Miyako soil and Ishigaki soil, and the dominance of Sinorhizobium fredii strains in alkaline Okinawa soils. To detect phaAB genes, which are componen...

The Araucaria Forest is a sub-type of the Atlantic Forest, dominated by Araucaria angustifolia, which is considered an endangered species. The understory has a high diversity of plant species, including several legumes. Many leguminous plants nodulate with rhizobia and fix atmospheric nitrogen, contributing to forest sustainability. This work aimed at bacteria isolation and phenotypic characterization from the root nodules of legumes occurring in Araucaria Forests, at Campos do Jordão State Park, Brazil. Nodule bacteria were isolated in YMA growth media and the obtained colonies were classified according to their growth characteristics (growth rate, color, extra cellular polysaccharide production and pH change of the medium). Data were analyzed by cluster and principal components analysis (PCA). From a total of eleven collected legume species, nine presented nodules, and this is the first report on nodulation of five of these legume species. Two hundred and twelve bacterial strains were isolated from the nodules, whose nodule shapes varied widely and there was a great phenotypic richness among isolates. This richness was found among legume species, individuals of the same species, different nodule shapes and even among isolates of the same nodule. These isolates could be classified into several groups, two up to six according to each legume, most of them different from the used growth standards Rhizobium tropici, Bradyrhizobium elkanii and Burkholderia sp. There is some evidence that these distinct groups may be related to the presence of Burkholderia spp. in the nodules of these legumes.

†Background and Aims Rhizobitoxine, produced by the legume symbiont Bradyrhizobium elkanii, inhibits cystathionine-b-lyase (EC 4.4.1.8) in methionine biosynthesis and 1-aminocyclopropane-1-carboxylate synthase (ACC) in ethylene biosynthesis. Rhizobitoxine production by B. elkanii enhances nodulation of host legumes via the inhibition of ethylene synthesis, but causes foliar chlorosis in susceptible soybeans, though how it does so remains to be investigated. The aim of this study was to examine the physiological basis of rhizobitoxineinduced chlorosis in soybeans. †Methods Wild-type B. elkanii and a rhizobitoxine-deficient mutant were inoculated in Glycine max ‘Lee’. Thirty days after inoculation, the upper parts of soybean shoots were analysed for amino acid contents. Chlorotic soybeans inoculated with wild-type B. elkanii were treated with methionine and ACC to assess the effects of the chemicals on the chlorosis. †Key Results Chlorotic upper shoots of soybeans inoculated with wild-type B. elkanii had a lower methionine content and higher accumulation of the methionine precursors than those with the rhizobitoxine-deficient mutant. In addition, the foliar chlorosis was alleviated by the application of methionine. †Conclusions Rhizobitoxine-induced chlorosis occurs in coincidence with methionine deficiency as a result of cystathione-b-lyase inhibition during methionine biosynthesis.

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean ( Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N 2 -fixation genes (PCR-RFLP and sequencing of nodY-nodA , nodC , and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum . Another one (CPAC 402) was identified as an indigenous Sinorhizobium ( Ensifer ) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.

Bradyrhizobium pachyrhizi PAC48 T has been isolated from a jicama nodule in Costa Rica. The draft genome indicates high similarity with that of Bradyrhizobium elkanii . Several coding sequences (CDSs) of the stress response might help in survival in the tropics. PAC48 T carries nodD1 and nodK , similar to Bradyrhizobium ( Parasponia ) ANU 289 and a particular nodD2 gene.

Symbiotic nitrogen-fixing bacteria, commonly called rhizobia, are agronomically important because they can provide significant amounts of nitrogen to plants and help in recovery of impoverished soils and improvement of degraded environments. In recent years, with advances in molecular techniques, several studies have shown that these bacteria have high levels of genetic diversity, resulting in taxonomic reclassifications and descriptions of new species. However, despite the advances achieved, highly conserved 16S ribosomal genes (16S rRNA) do not elucidate differences between species of several genera, including the genus Bradyrhizobium. Other methodologies, such as multilocus sequence analysis (MLSA), have been used in such cases, with good results. In this study, three strains (SEMIAs 690T, 6387 and 6428) of the genus Bradyrhizobium, isolated from nitrogen-fixing nodules of Centrosema and Acacia species, without clear taxonomic positions, were studied. These strains differed from genetically closely related species according to the results of MLSA of four housekeeping genes (dnaK, glnII, gyrB and recA) and nucleotide identities of the concatenated genes with those of related species ranged from 87.8 % to 95.7 %, being highest with Bradyrhizobium elkanii. DNA–DNA hybridization (less than 32 % DNA relatedness) and average nucleotide identity values of the whole genomes (less than 90.5 %) indicated that these strains represented a novel species, and phenotypic traits were determined. Our data supported the description of the SEMIA strains as Bradyrhizobium viridifuturi sp. nov., and SEMIA 690T ( = CNPSo 991T = C 100aT = BR 1804T = LMG 28866T), isolated from Centrosema pubescens, was chosen as type strain.