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.     

Phaseolus vulgaris is nodulated by the symbiovar viciae of several genospecies of Rhizobium laguerreae complex in a Spanish region where Lens culinaris is the traditionally cultivated legume

Phaseolus vulgaris and Lens culinaris are two legumes with different distribution centers that were introduced in Spain at different times, but in some regions L. culinaris has been traditionally cultivated and P. vulgaris did not. Here we analysed the rhizobia isolated from nodules of these two legumes in one of these regions. MALDI-TOF MS analysis showed that all isolated strains matched with Rhizobium laguerreae and the phylogenetic analysis of rrs, atpD and recA genes confirmed these results. The phylogenetic analysis of these core genes allowed the differentiation of several groups within R. laguerreae and unexpectedly, strains with housekeeping genes identical to that of the type strain of R. laguerreae presented some differences in the rrs gene. In some strains this gene contains an intervening sequence (IVS) identical to that found in Rhizobium strains nodulating several legumes in different geographical locations. The atpD, recA and nodC genes of all isolated strains clustered with those of strains nodulating L. culinaris in its distribution centers, but not with those nodulating P. vulgaris in theirs. Therefore, all these strains belong to the symbiovar viciae, including those isolated from P. vulgaris, which in the studied region established effective symbiosis with the common endosymbiont of L. culinaris, instead to with its common endosymbiont, the symbiovar phaseoli. These results are particularly interesting for biogeography studies, because they showed that, due its high promiscuity degree, P. vulgaris is able to establish symbiosis with local symbiovars well established in the soil after centuries of cultivation with other legumes.     

Characterization of Bradyrhizobium strains indigenous to Western Australia and South Africa indicates remarkable genetic diversity and reveals putative new species

Bradyrhizobium are N₂-fixing microsymbionts of legumes with relevant applications in agricultural sustainability, and we investigated the phylogenetic relationships of conserved and symbiotic genes of 21 bradyrhizobial strains. The study included strains from Western Australia (WA), isolated from nodules of Glycine spp. the country is one genetic center for the genus and from nodules of other indigenous legumes grown in WA, and strains isolated from forage Glycine sp. grown in South Africa. The 16S rRNA phylogeny divided the strains in two superclades, of B. japonicum and B. elkanii, but with low discrimination among the species. The multilocus sequence analysis (MLSA) with four protein-coding housekeeping genes (dnaK, glnII, gyrB and recA) pointed out seven groups as putative new species, two within the B. japonicum, and five within the B. elkanii superclades. The remaining eleven strains showed higher similarity with six species, B. lupini, B. liaoningense, B. yuanmingense, B. subterraneum, B. brasilense and B. retamae. Phylogenetic analysis of the nodC symbiotic gene clustered 13 strains in three different symbiovars (sv. vignae, sv. genistearum and sv. retamae), while seven others might compose new symbiovars. The genetic profiles of the strains evaluated by BOX-PCR revealed high intra- and interspecific diversity. The results point out the high level of diversity still to be explored within the Bradyrhizobium genus, and further studies might confirm new species and symbiovars.     

The Mimosoid tree Leucaena leucocephala can be nodulated by the symbiovar genistearum of Bradyrhizobium canariense

Leucaena leucocephala is a Mimosoid legume tree indigenous to America that has spread to other continents, although it is not still present in some European countries such as Portugal. Nevertheless, we found that this legume can be nodulated in this country by slow-growing rhizobial strains which were identified as Bradyrhizobium canariense trough the analysis of the core genes recA and glnII. The analysis of the symbiotic gene nodC showed that these strains belong to the symbiovar genistearum, which commonly nodulates Genistoid legumes. Although two strains nodulating L. leucocephala in China and Brazil were classified within the genus Bradyrhizobium, they belong to undescribed species and to the symbiovars glycinearum and tropici, respectively. Therefore, we report here for the first time the ability of L. leucocephala to establish symbiosis with strains of B. canariense sv genistearum confirming the high promiscuity of L. leucocephala, that allows it to establish symbiosis with rhizobia native to different continents increasing its invasiveness potential.     

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.     

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.     

Adaptability to local conditions and phylogenetic differentiation of microsymbionts of TGx soybean genotypes in the semi-arid environments of Ghana and 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.     

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

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

Molecular phylogeny of Bradyrhizobium bacteria isolated from root nodules of tribe Genisteae plants growing in southeast Poland

The phylogeny of 16 isolates from root nodules of Genista germanica, Genista tinctoria, Cytisus ratisbonensis, and Cytisus scoparius growing in southeast Poland was estimated by comparative sequence analysis of core (16S rDNA, atpD, glnII, recA) and symbiosis-related (nodC, nodZ, nifH) genes. All the sequences analyzed placed the studied rhizobia in the genus Bradyrhizobium. Phylogenetic analysis of individual and concatenated housekeeping genes showed that the Genisteae microsymbionts form a homogeneous group with Bradyrhizobium japonicum strains. The phylogeny of nodulation and nitrogen fixation genes indicated a close relationship of the examined rhizobia with B. japonicum, Bradyrhizobium canariense, Bradyrhizobium cytisi, Bradyrhizobium rifense and Bradyrhizobium lupini strains infecting other plants of the tribe Genisteae. For the first time, the taxonomic position of G. germanica and C. ratisbonensis rhizobia, inferred from multigenic analysis, is described. The results of the phylogenetic analysis based on the protein-coding gene sequences presented in this study also indicate potential pitfalls concerning the choice of marker and reference strains, which may lead to conflicting conclusions in species delineation.     

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

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

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of <Emphasis Type="Italic">Glycine max</Emphasis> Grown in Hebei Province,China

A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S–23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline–saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.     

Phylogenies of symbiotic genes of Bradyrhizobium symbionts of legumes of economic and environmental importance in Brazil support the definition of the new symbiovars pachyrhizi and sojae

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil (Arachis hypogaea, Acacia auriculiformis, Glycine max, Lespedeza striata, Lupinus albus, Stylosanthes sp. and Vigna unguiculata) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades—B. japonicum and B. elkanii—with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII-recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi, the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium.     

Microsymbionts of Phaseolus vulgaris in acid and alkaline soils of Mexico

In order to investigate bean-nodulating rhizobia in different types of soil, 41 nodule isolates from acid and alkaline soils in Mexico were characterized. Based upon the phylogenetic studies of 16S rRNA, atpD, glnII, recA, rpoB, gyrB, nifH and nodC genes, the isolates originating from acid soils were identified as the phaseoli symbiovar of the Rhizobium leguminosarum-like group and Rhizobium grahamii, whereas the isolates from alkaline soils were defined as Ensifer americanum sv. mediterranense and Rhizobium radiobacter. The isolates of “R. leguminosarum” and E. americanum harbored nodC and nifH genes, but the symbiotic genes were not detected in the four isolates of the other two species. It was the first time that “R. leguminosarum” and E. americanum have been reported as bean-nodulating bacteria in Mexico. The high similarity of symbiotic genes in the Rhizobium and Ensifer populations showed that these genes had the same origin and have diversified recently in different rhizobial species. Phenotypic characterization revealed that the “R. leguminosarum” population was more adapted to the acid and low salinity conditions, while the E. americanum population preferred alkaline conditions. The findings of this study have improved the knowledge of the diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico.     

Polyphasic characterization of rhizobia isolated from Leucaena leucocephala from Panxi, China

Leucaena leucocephala was introduced into Panxi, Sichuan, China, in the 1980s and 1990s for afforestation and preventing water loss and soil erosion in this area. The co-introduction of rhizobial symbionts of introduced plants has drawn attention since they may influence local soil communities. We studied the phylogenetic position of the L. leucocephala isolates and assessed if the rhizobia were introduced together with the host to Panxi, Sichuan, China. The glnII and atpD genes of fifteen representative isolates were sequenced and analyzed, and applied multilocus sequence analyses in which the housekeeping genes recA, glnII and atpD were included. Furthermore, we estimated the within species diversity directly with 23S rDNA and IGS RFLP and indirectly through phenotypic analysis of forty L. leucocephala isolates. The isolates represented seven species and 38 diversified strains in the genera Ensifer, Mesorhizobium, Bradyrhizobium and Rhizobium. The within species diversity of the Ensifer isolates was large, proposing a potential to occupy novel niches. There was not conclusive evidence to show that any of the strains would have been co-introduced with L. leucocephala. On the contrary, we came to a conclusion that the possible introduction should not be inferred from sequence data alone.     

Distribution and biodiversity of rhizobia nodulating Chamaecrista mimosoides in the Shandong peninsula of china

Chamaecrista mimosoides is an annual herb legume widely distributed in tropical and subtropical Asia and Africa. It may have primitive and independently-evolved root nodule types but its rhizobia have not been systematically studied. Therefore, in order to learn the diversity and species affinity of its rhizobia, root nodules were sampled from C. mimosoides plants growing in seven geographical sites along the coast line of Shandong Peninsula, China. A total of 422 rhizobial isolates were obtained from nodules, and they were classified into 28 recA haplotypes. By using multilocus sequence analysis of the concatenated housekeeping genes dnaK, glnII, gyrB, recA and rpoB, the representative strains for these haplotypes were designated as eight defined and five candidate novel genospecies in the genus Bradyrhizobium. Bradyrhizobium elkanii and Bradyrhizobium ferriligni were predominant and universally distributed. The symbiotic genes nodC and nifH of the representative strains showed very similar topology in their phylogenetic trees indicating their co-evolution history. All the representative strains formed effective root nodules in nodulation tests. The correlation between genospecies and soil characteristics analyzed by CANOCO software indicated that available potassium (AK), organic carbon (OC) and available nitrogen (AN) in the soil samples were the main factors affecting the distribution of the symbionts involved in this current study. The study is the first systematic survey of Chamaecrista mimosoides-nodulating rhizobia, and it showed that Chamaecrista spp. were nodulated by bradyrhizobia in natural environments. In addition, the host spectrum of the corresponding rhizobial species was extended, and the study provided novel information on the biodiversity and biogeography of rhizobia.     

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).     

Cytisus villosus from Northeastern Algeria is nodulated by genetically diverse Bradyrhizobium strains

Fifty-one rhizobial strains isolated from root nodules of Cytisus villosus growing in Northeastern Algeria were characterized by genomic and phenotypic analyses. Isolates were grouped into sixteen different patterns by PCR-RAPD. The phylogenetic status of one representative isolate from each pattern was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and one symbiotic gene (nodC). Analysis of 16S rRNA gene sequences showed that all the isolates belonged to the genus Bradyrhizobium. Phylogenetic analyses based on individual or concatenated genes glnII, recA, and atpD indicated that strains cluster in three distinct groups. Ten out of the sixteen strains grouped together with Bradyrhizobium japonicum, while a second group of four clustered with Bradyrhizobium canariense. The third group, represented by isolates CTS8 and CTS57, differed significantly from all other bradyrhizobia known to nodulate members of the Genisteae tribe. In contrast with core genes, sequences of the nodC symbiotic gene from all the examined strains form a homogeneous group within the genistearum symbiovar of Bradyrhizobium. All strains tested nodulated Lupinus angustifolius, Lupinus luteus, and Spartium junceum but not Glycine max. From these results, it is concluded that C. villosus CTS8 and CTS57 strains represent a new lineage within the Bradyrhizobium genus.     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Unexpectedly Diverse Mesorhizobium Strains and Rhizobium leguminosarum Nodulate Native Legume Genera of New Zealand, while Introduced Legume Weeds Are Nodulated by Bradyrhizobium Species

The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.     

Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries

Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidic and degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a poor acid savanna soil from Venezuela was characterized, and the members of the collection were compared to other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulating Centrosema in American countries were closely related to different species of the genus Bradyrhizobium. However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that they formed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineages that were divergent among themselves and with respect to those formed by Centrosema strains isolated in other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed for the first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhizobium. The nodC genes of the Centrosema strains were divergent among themselves and with respect to the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume. According to these results, the currently known Centrosema-nodulating strains represent several new putative species and symbiovars of the genus Bradyrhizobium.