nodC Gene Research Articles

Phylogenetic diversity of Bradyrhizobium strains nodulating Calicotome spinosa in the Northeast of Algeria

Fifty-two slow-growing strains were isolated from root nodules of Calicotome spinosa grown in the Northeast of Algeria and grouped in 24 rep-PCR clusters. One representative strain for each profile was further phylogenetically characterized. The nearly complete 16S rRNA gene sequence indicated that all strains were affiliated to Bradyrhizobium. Multi-Locus Sequence Analysis (MLSA) of the atpD, glnII and recA genes and of the 16S-23S rRNA internal transcribed spacer (ITS) showed that these strains formed four divergent clusters: one close to Bradyrhizobium canariense and Bradyrhizobium lupini and three others separate from all the described species, representing three putative new Bradyrhizobium species. A phylogenetic analysis based on the nodC gene sequence affiliated the strains to either of the two symbiovars, genistearum or retamae.

Definition of two new symbiovars, sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities.Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).

Bradyrhizobium algeriense sp. nov., a novel species isolated from effective nodules of Retama sphaerocarpa from Northeastern Algeria

We have characterized genetic, phenotypic and symbiotic properties of bacterial strains previously isolated from nitrogen-fixing nodules of Retama sphaerocarpa from Northern Algeria. Phylogenetic analyses of 16S rRNA genes and three concatenated housekeeping genes, recA, atpD and glnII, placed them in a new divergent group that is proposed to form a new Bradyrhizobium species, Bradyrhizobium algeriense sp. nov. (type strain RST89T, LMG 27618 and CECT 8363). Based on these phylogenetic markers and on genomic identity data derived from draft genomic sequences, Bradyrhizobium valentinum LmjM3T, Bradyrhizobium lablabi CCBAU 23086T, Bradyrhizobium retamae Ro19T, and Bradyrhizobium jicamae PAC68T are the closest relatives of B. algeriense RST89T, with sequence identities of 92–94% and Average Nucleotide Identities (ANIm) under 90%, well below the 95–96% species circumscription threshold. Likewise, a comparison of whole-cell proteomic patterns, estimated by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometric analysis, yielded almost identical spectra between B. algeriense strains but significant differences with B. valentinum, Bradyrhizobium paxllaeri, Bradyrhizobium icense, B. lablabi, B. jicamae and B. retamae. A phylogenetic tree based on symbiotic gene nodC revealed that the B. algeriense sequences cluster with sequences from the Bradyrhizobium symbiovar retamae, previously defined with B. retamae strains isolated from Retama monosperma. B. algeriense strains were able to establish effective symbioses with Retama raetam, Lupinus micranthus, Lupinus albus and Genista numidica, but not with Lupinus angustifolius or Glycine max.

Genomic insight into the taxonomy of Rhizobium genospecies that nodulate Phaseolus vulgaris

Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species.

Bacteria related to Bradyrhizobium yuanmingense from Ghana are effective groundnut micro-symbionts

The identification of locally-adapted rhizobia for effective inoculation of grain legumes in Africa’s semiarid regions is strategic for developing and optimizing cheap nitrogen fixation technologies for smallholder farmers. This study was aimed at selecting and characterising effective native rhizobia, from Ghanaian soils for groundnut (Arachis hypogaea L.) inoculation. From surface-disinfected root nodules of cowpea and groundnut plants grown on farmers’ fields, 150 bacterial isolates were obtained, 30 of which were eventually found to nodulate groundnut plants. After testing the symbiotic potential of these isolates on groundnut on sterilized substrate, seven of them, designated as KNUST 1001–1007, were evaluated in an open field pot experiment using 15N-labelled soil. Although 15N dilution analyses did not indicate differences among treatments in the proportion of nitrogen (N) derived from the atmosphere (%Ndfa), all seven strains increased total N derived from N2 fixation by inoculated groundnut plants as compared to the non-inoculated control. Inoculation with KNUST 1002 led to total N accumulation as high as that of the groundnut reference strain 32H1. Genetic characterisation of the isolates by sequence analysis of 16S rRNA gene, 16S – 23S rRNA intergenic transcribed spacer (ITS) region and nodC gene revealed that isolates KNUST 1003 and 1007 were related to Rhizobium tropici, a common bean symbiont. The other five isolates, including KNUST 1002 belonged to the Bradyrhizobium genus, being closely related to Bradyrhizobium yuanmingense. Therefore, this study revealed novel native Ghanaian rhizobia with potential for the development of groundnut inoculants.

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N2) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.

Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of Paraburkholderia

The Chapada Diamantina in NE of Brazil is a biodiversity hotspot and a center of radiation for many Neotropical legume genera, such as Calliandra and Mimosa. The present study aimed to evaluate nodulation in Calliandra species endemic to various environments, and to characterize the diversity of their symbiotic rhizobia using housekeeping (16S rRNA, recA) and plasmid-borne, symbiosis-related (nifH and nodC) genes. The nodulation ability of selected isolates was assessed. All of the 126 bacterial isolates from 18 Calliandra species collected in six different vegetation types were identified as Paraburkholderia according to their housekeeping and symbiosis gene phylogenies. They were grouped in seven clades in relation to the dominant vegetation type in their native environments. The majority, particularly those from highland “campo rupestre” vegetation, were similar to Paraburkholderia nodosa, but had nodC genes identical to the Mimosa symbiont Paraburkholderia tuberum sv. mimosae. The other smaller groups were related to Paraburkholderia diazotrophica and Paraburkholderia sabiae, and some single strains were not close to any known species. The symbionts of Calliandra spp. in NE Brazil are Paraburkholderia strains closely-related to Mimosa symbionts from the same region. NE Brazil is a reservoir of symbiotic Paraburkholderia that have an affinity for genera in the Mimosoid clade.

Characterization and molecular diversity of Iranian rhizobia isolated from faba bean

The diversity and phylogeny of 30 rhizobia isolated from nodules of faba bean plants grown on 5 geographic regions located in the East Azerbaijan province of Iran were examined using rep-PCR fingerprinting, sequence analysis of 16S rRNA accompanied with nodC genes. Based on cluster analysis of rep-PCR fingerprints, faba bean rhizobia isolates were differentiated into five clusters (A to E) at 80% similarity level. The cophenetic correlation coefficient for the dendrogram obtained from the combined dataset of BOX and ERIC primers was 0.942. The percentage of polymorphic loci was 59.2% using the BOX-PCR primer and 67.3% using the ERIC-PCR primers. The data obtained by rep-PCR fingerprinting showed high apparent correlation between genetic diversity and geographical origin of the isolates. The phylogenetic analysis based on 16S rRNA and nodC sequences showed that representative isolates were closely related to R. leguminosarum bv. viciae and R. fabae. To the best of our knowledge, this is first report of isolation and characterization of R. fabae from Iran.

Vicia faba L. in the Bejaia region of Algeria is nodulated by Rhizobium leguminosarum sv. viciae, Rhizobium laguerreae and two new genospecies

Fifty-eight rhizobial strains were isolated from root nodules of Vicia faba cv. Equina and Vicia faba cv. Minor by the host-trapping method in soils collected from eleven sites in Bejaia, Eastern Algeria. Eleven genotypic groups were distinguished based on the combined PCR/RFLP of 16S rRNA, 16S–23S rRNA intergenic spacer and symbiotic (nodC and nodD-F) genes and further confirmed by multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and the nodulation genes nodC and nodD. Of the 11 genotypes, 5 were dominant and 2 were the most represented. Most of the strains shared high nodD gene sequence similarity with Rhizobium leguminosarum sv. viciae; their nodC sequences were similar to both Rhizobium leguminosarum and Rhizobium laguerreae. Sequence analyses of the 16S–23S rRNA intergenic spacer showed that all the new strains were phylogenetically related to those described from Vicia sativa and V. faba in several African, European, American and Asian countries, with which they form a group related to Rhizobium leguminosarum. Phylogenetic analysis based on MLSA of 16S rRNA, recA, atpD and rpoB genes allowed the affiliations of strain AM11R to Rhizobium leguminosarum sv. viciae and of strains EB1 and ES8 to Rhizobium laguerreae. In addition, two separate clades with <97% similarity may represent two novel genospecies within the genus Rhizobium.

Rhizobia inhabiting nodules and rhizosphere soils of alfalfa: A strong selection of facultative microsymbionts

Alfalfa (Medicago sativa L.) is an important leguminous forage crop, which is hypothesized to select compatible rhizobial partners from rhizosphere to form nitrogen-fixing nodules. Here we investigated the diversity of rhizobia from both nodules and rhizosphere of alfalfa from farmlands and natural ecosystems along the Yarlung Zangbo River valley in the Tibetan Plateau. 581 nodule isolates were characterized using BOX-A1R PCR fingerprinting, sequences of rpoB and nodulation gene nodC. They belong to four rpoB haplotypes of Sinorhizobium meliloti (rpoB-I, 473 isolates; rpoB-II, 95; rpoB-III, 1; rpoB-IV, 10), Sinorhizobium medicae (1 isolate) and Rhizobium sp. (1 isolate). Re-inoculation experiments of the 32 representative strains suggested a great variation in their symbiotic performance on alfalfa. High-throughput sequencing of rpoB for 34 rhizosphere samples uncovered that the predominant rpoB-I in nodules also dominated among fourteen rpoB haplotypes of S. meliloti in rhizosphere soils. In addition to S. meliloti, more than 40 rhizobial species documented as microsymbionts of other legumes were also identified. Although a very low level of nucleotide diversity for S. meliloti was found among sampling sites, multivariate statistical analysis demonstrated a significant differentiation of total rhizobial community between farmlands and natural ecosystems. Moreover, edaphic conditions, especially pH and nitrogen content, were revealed as important deterministic factors shaping the observed beta-diversity of rhizobial community. Taken together, these results demonstrated that the rhizospheric pool of rhizobial germplasms was significantly affected by land tillage compared to that of natural ecosystems, while a predominant population of S. meliloti was selected by alfalfa.

Genetic diversity and nitrogen fixation of mesorhizobia symbionts of New Zealand endemic Sophora species

ABSTRACTForty-four rhizobial isolates from New Zealand endemic Sophora species growing in natural conditions were characterised and the effectiveness of selected isolates with regard to growth and N2 fixation of different Sophora species was tested. Sequences for the housekeeping genes (concatenated recA, gln11 and rpoB) of the isolates were diverse but sequences for their symbiosis genes (nifH and nodC) were similar. Generally, isolates from the same field site showed similar housekeeping gene sequences. All isolates were Mesorhizobium. Twenty-six of the isolates aligned with Mesorhizobium species previously isolated from New Zealand native Sophora on the basis of their housekeeping gene sequences, but 18 isolates separated into two groups and a pair of isolates clearly separated from all Mesorhizobium type strains. DNA–DNA hybridisations indicated that isolates in the two groups could belong to two novel Mesorhizobium species. Rhizobial isolates with different housekeeping gene sequences (but similar symbiosis gene sequences) gave similar increases in dry weight and total N content of four Sophora species under glasshouse conditions.

Burkholderia species associated with legumes of Chiapas, Mexico, exhibit stress tolerance and growth in aromatic compounds

Leguminous plants have received special interest for the diversity of β-proteobacteria in their nodules and are promising candidates for biotechnological applications. In this study, 15 bacterial strains were isolated from the nodules of the following legumes: Indigofera thibaudiana, Mimosa diplotricha, Mimosa albida, Mimosa pigra, and Mimosa pudica, collected in 9 areas of Chiapas, Mexico. The strains were grouped into four profiles of genomic fingerprints through BOX-PCR and identified based on their morphology, API 20NE biochemical tests, sequencing of the 16S rRNA, nifH and nodC genes as bacteria of the Burkholderia genus, genetically related to Burkholderia phenoliruptrix, Burkholderia phymatum, Burkholderia sabiae, and Burkholderia tuberum. The Burkholderia strains were grown under stress conditions with 4% NaCl, 45°C, and benzene presence at 0.1% as the sole carbon source. This is the first report on the isolation of these nodulating species of the Burkholderia genus in legumes in Mexico.

Chickpea (Cicer arietinum) is nodulated by unexpected wide diversity of Mesorhizobium species in Eastern Algeria

ABSTRACTUsing a local Algerian cultivar of chickpea, from 9 sites in Eastern Algeria, along a sub-humid to arid gradient, we isolated 60 nodulating isolates, among which 19 were assigned to Mesorhizobium species and 41 to Ensifer meliloti. Trapping revealed great differences among sites for their ability to induce nodules on plant, but no correlation with chemical and physical characterizations of soil samples could be found. Based on 16S rRNA gene sequences, 16S-23S IGS, recA, nodC and nifH sequences, Mesorhizobium isolates were assigned to at least 5 different species, among which one had never been described as nodulating chickpea before. The symbiotic diversity is conversely low, and shared by several species, reflecting horizontal gene transfer. The ability of Mesorhizobium genus to recombine and exchange symbiotic clusters among different species must play a great role in this ability to be associated with chickpea. The question to what extent the symbiotic species would end in this genus is discussed.

Bradyrhizobium sacchari sp. nov., a legume nodulating bacterium isolated from sugarcane roots.

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280T), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303T. Average nucleotide identity (ANI) analyses confirmed that BR 10280T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303T, but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

Nitrogen-fixing rhizobial strains isolated from Desmodium incanum DC in Argentina: Phylogeny, biodiversity and symbiotic ability

Desmodium spp. are leguminous plants belonging to the tribe Desmodieae of the subfamily Papilionoideae. They are widely distributed in temperated and subtropical regions and are used as forage plants, for biological control, and in traditional folk medicine. The genus includes pioneer species that resist 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 native species 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 fingerprinting, with more than 30 different amplification profiles. None of the isolates were found at more than one site, thus indicating a high level of rhizobial diversity associated with D. incanum in Argentinean soils. In selected isolates, 16S rDNA sequencing and whole-cell extract MALDI TOF analysis revealed the presence of isolates related to Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium yuanmingense, 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 to temperature and extreme pH, and produced higher aerial plant dry weights compared to other inoculated treatments. These results indicated that local isolates could be efficiently used for D. incanum inoculation.

Agrobacterium salinitolerans sp. nov., a saline-alkaline-tolerant bacterium isolated from root nodule of Sesbania cannabina.

Two Gram-staining-negative, aerobic bacteria (YIC 5082T and YIC4104) isolated from root nodules of Sesbania cannabina grown in a high-salt and alkaline environment were identified as a group in the genus Agrobacterium because they shared 100 and 99.7 % sequence similarities of 16S rRNA and recA+atpD genes, respectively. These two strains showed 99.2/100 % and 93.9/95.4 % 16S rRNA and recA+atpD gene sequence similarities to Agrobacterium radiobacter LMG140T and Agrobacterium. pusense NRCPB10T, respectively. The average nucleotide identities (ANI) of genome sequences were 89.95 % or lower between YIC 5082T and the species of the genus Agrobacterium examined. Moreover, these two test strains formed a unique nifH lineage deeply separated from other rhizobia. Although the nodC gene was not detected in YIC 5082T and YIC4104, they could form effective root nodules on S. cannabina plants. The main cellular fatty acids in YIC 5082T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C19 : 0cyclo ω8c, summed feature 2 (C12 : 0 aldehyde/unknown equivalent chain length 10.9525) and C16 : 0. The DNA G+C content of YIC 5082T was 59.3 mol%. The failure to utilize d-sorbitol as a carbon source distinguished YIC 5082T from the type strains of related species. YIC 5082T could grow in presence of 5.0 % (w/v) NaCl and at a pH of up to 10.0. Based on results regarding the genetic and phenotypic properties of YIC 5082T and YIC4104 the name Agrobacterium salinitolerans sp. nov. is proposed and YIC 5082T (=HAMBI 3646T=LMG 29287T) is designed as the type strain.

Diversity and plant growth promoting properties of rhizobia isolated from root nodules of Ononis arvensis

This is the first report describing isolates from root nodules of Ononis arvensis (field restharrow). The aim of this investigation was to describe the diversity, phylogeny, and plant growth promoting features of microsymbionts of O. arvensis, i.e., a legume plant growing in different places of the southern part of Poland. Twenty-nine bacterial isolates were characterized in terms of their phenotypic properties, genome fingerprinting, and comparative analysis of their 16S rRNA, nodC and acdS gene sequences. Based on the nodC and 16S rRNA gene phylogenies, the O. arvensis symbionts were grouped close to bacteria of the genera Rhizobium and Mesorhizobium, which formed monophyletic clusters. The acdS gene sequences of all the isolates tested exhibited the highest similarities to the corresponding gene sequences of genus Mesorhizobium strains. The presence of the acdS genes in the genomes of rhizobia specific for O. arvensis implies that these bacteria may promote the growth and development of their host plant in stress conditions. The isolated bacteria showed a high genomic diversity and, in the BOX-PCR reaction, all of them (except three) exhibited DNA fingerprints specific only for them. Our studies showed that restharrow isolates formed effective symbiotic interactions with their native host (O. arvensis) and Ononis spinosa but not with Trifolium repens and Medicago sativa belonging to the same tribe Trifolieae as Ononis species and not with Lotus corniculatus, representing the tribe Loteae.

Phylogenetically diverse group of native bacterial symbionts isolated from root nodules of groundnut (Arachis hypogaea L.) in South Africa

Groundnut is an economically important N​2-fixing legume that can contribute about 100–190kgNha−1 to cropping systems. In this study, groundnut-nodulating native rhizobia in South African soils were isolated from root nodules. Genetic analysis of isolates was done using restriction fragment length polymorphism (RFLP)-PCR of the intergenic spacer (IGS) region of 16S-23S rDNA. A total of 26 IGS types were detected with band sizes ranging from 471 to 1415bp. The rhizobial isolates were grouped into five main clusters with Jaccard's similarity coefficient of 0.00–1.00, and 35 restriction types in a UPGMA dendrogram. Partial sequence analysis of the 16S rDNA, IGS of 16S rDNA-23S rDNA, atpD, gyrB, gltA, glnII and symbiotic nifH and nodC genes obtained for representative isolates of each RFLP-cluster showed that these native groundnut-nodulating rhizobia were phylogenetically diverse, thus confirming the extent of promiscuity of this legume. Concatenated gene sequence analysis showed that most isolates did not align with known type strains, and may represent new species from South Africa. This underscored the high genetic variability associated with groundnut Rhizobium and Bradyrhizobium in South African soils, and the possible presence of a reservoir of novel groundnut-nodulating Bradyrhizobium and Rhizobium in the country.

Genetic diversity of symbiotic Paraburkholderia species isolated from nodules of Mimosa pudica (L.) and Phaseolus vulgaris (L.) grown in soils of the Brazilian Atlantic Forest (Mata Atlântica).

Some species of the genus Paraburkholderia that are able to nodulate and fix nitrogen in symbiosis with legumes are called β-rhizobia and represent a group of ecological and biotechnological importance. We used Mimosa pudica and Phaseolus vulgaris to trap 427 rhizobial isolates from rhizospheric soil of Mimoseae trees in the Brazilian Atlantic Forest. Eighty-four representative strains were selected according to the 16S rRNA haplotypes and taxonomically characterized using a concatenated 16S rRNA-recA phylogeny. Most strains were assembled in the genus Paraburkholderia, including Paraburkholderia sabiae and Pa. nodosa. Mesorhizobium (α-rhizobia) and Cupriavidus (β-rhizobia) were also isolated, but in smaller proportions. Multilocus sequence analysis and BOX-PCR analyses indicated that six clusters of Paraburkholderia represent potential new species. In the phylogenetic analysis of the nodC gene, the majority of the strains were positioned in the same groups as in the 16S rRNA-recA tree, indicative of stability and vertical inheritance, but we also identified horizontal transfer of nodC in Pa. sabiae. All α- and β-rhizobial species were trapped by both legumes, although preferences of the host plants for specific rhizobial species have been observed.

Bradyrhizobium centrolobii and Bradyrhizobium macuxiense sp. nov. isolated from Centrolobium paraense grown in soil of Amazonia, Brazil.

Thirteen Gram-negative, aerobic, motile with polar flagella, rod-shaped bacteria were isolated from root nodules of Centrolobium paraense Tul. grown in soils from the Amazon region of Brazil. Growth of strains was observed at temperature range 20-36 °C (optimal 28 °C), pH ranges 5-11 (optimal 6.0-7.0), and 0.1-0.5%NaCl (optimal 0.1-0.3%). Analysis of 16S rRNA gene placed the strains into two groups within Bradyrhizobium. Closest neighbouring species (98.8%) for group I was B. neotropicale while for group II were 12 species with more than 99% of similarity. Multi-locus sequence analysis (MLSA) with dnaK, glnII, recA, and rpoB confirmed B. neotropicale BR 10247T as the closest type strain for the group I and B. elkanii USDA 76T and B. pachyrhizi PAC 48T for group II. Average Nucleotide Identity (ANI) differentiated group I from the B. neotropicale BR 10247T (79.6%) and group II from B. elkanii USDA 76T and B. pachyrhizi PAC 48T (88.1% and 87.9%, respectively). Fatty acid profiles [majority C16:0 and Summed feature 8 (18:1ω6c/18:1ω7c) for both groups], DNA G + C content, and carbon compound utilization supported the placement of the novel strains in the genus Bradyrhizobium. Gene nodC and nifH of the new strains have in general low similarity with other Bradyrhizobium species. Both groups nodulated plants from the tribes Crotalarieae, Dalbergiae, Genisteae, and Phaseoleae. Based on the presented data, two novel species which the names Bradyrhizobium centrolobii and Bradyrhizobium macuxiense are proposed, with BR 10245T (=HAMBI 3597T) and BR 10303T (=HAMBI 3602T) as the respective-type strains.