Symbiotic Genes nodC Research Articles

Genetic Characterization of Rhizobium spp. Strains in an Organic Field Pea (Pisum sativum L.) Field in Lithuania.

Biological nitrogen fixation in legume plants depends on the diversity of rhizobia present in the soil. Rhizobial strains exhibit specificity towards host plants and vary in their capacity to fix nitrogen. The increasing interest in rhizobia diversity has prompted studies of their phylogenetic relations. Molecular identification of Rhizobium is quite complex, requiring multiple gene markers to be analysed to distinguish strains at the species level or to predict their host plant. In this research, 50 rhizobia isolates were obtained from the root nodules of five different Pisum sativum L. genotypes ("Bagoo", "Respect", "Astronaute", "Lina DS", and "Egle DS"). All genotypes were growing in the same field, where ecological farming practices were applied, and no commercial rhizobia inoculants were used. The influence of rhizobial isolates on pea root nodulation and dry biomass accumulation was determined. 16S rRNA gene, two housekeeping genes recA and atpD, and symbiotic gene nodC were analysed to characterize rhizobia population. The phylogenetic analysis of 16S rRNA gene sequences showed that 46 isolates were linked to Rhizobium leguminosarum; species complex 1 isolate was identified as Rhizobium nepotum, and the remaining 3 isolates belonged to Rahnella spp., Paenarthrobacter spp., and Peribacillus spp. genera. RecA and atpD gene analysis showed that the 46 isolates identified as R. leguminosarum clustered into three genospecies groups (B), (E) and (K). Isolates that had the highest influence on plant dry biomass accumulation clustered into the (B) group. NodC gene phylogenetic analysis clustered 46 R. leguminosarum isolates into 10 groups, and all isolates were assigned to the R. leguminosarum sv. viciae.

Different species of Bradyrhizobium from symbiovars genistearum and retamae nodulate the endemic Retama dasycarpa in the High Atlas Mountains.

Retama dasycarpa is an endemic Retama species native to the cold semi-arid bioclimates of the High Atlas Mountains in Morocco. In this work, we analyzed the diversity of the microsymbionts nodulating this plant and their different phenotypic and symbiotic characteristics. Phylogenetic analysis of the 16S rRNA gene revealed that the tested isolates clustered in the Bradyrhizobium genus. Multilocus sequence analyses of four housekeeping genes (recA, gyrB, glnII and atpD) for 12 selected strains grouped them into four clusters close to B. lupini USDA 3051T, B. frederickii CNPSo 3446T, B. valentinum LmjM3T and B. retamae Ro19T. The individual phylogenies of these core genes and the symbiotic genes nodC, nodA and nifH were congruent. These isolates showed a broad host range, being able to nodulate different legume hosts, such as R. sphaerocarpa, R. monosperma, Lupinus luteus, Cytisus grandiflorus andChamaecytisus albidus, but not Phaseolus vulgaris or Glycine max. They all had a similar metabolic capacity, using the majority of the carbohydrates and amino acids tested as sole sources of carbon and nitrogen. Furthermore, out of the 12 selected strains, some displayed plant growth-promoting features, with six of them solubilizing phosphate and three of them producing siderophores. The present work provides, for the first time, a detailed description about the microsymbionts associated with the endemic legume R. dasycarpa.

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.

Phylogenetic Analyses of Rhizobia Isolated from Nodules of Lupinus angustifolius in Northern Tunisia Reveal Devosia sp. as a New Microsymbiont of Lupin Species

Thirty-two bacterial isolates were obtained from root nodules of Lupinus angustifolius growing in Northern Tunisia. Phylogenetic analyses based on recA and gyrB partial gene sequences grouped the strains into six clusters: four clusters belonged to the genus Bradyrhizobium (22 isolates), one to Microvirga (8 isolates) and one to Devosia (2 isolates), a genus that has not been previously reported to nodulate lupin. Representative strains of each group were further characterized. Multi-Locus Sequence Analysis (MLSA) based on recA and glnII gene sequences separated the strains within the genus Bradyrhizobium into four divergent clusters related to B. canariense, B. liaoningense, B. lupini, and B. algeriense, respectively. The latter might constitute a new Bradyrhizobium species. The strains in the Microvirga cluster showed high identity with M. tunisiensis. The Devosia isolates might also represent a new species within this genus. An additional phylogenetic analysis based on the symbiotic gene nodC affiliated the strains to symbiovars genistearum, mediterranense, and to a possibly new symbiovar. These results altogether contributed to the existing knowledge on the genetic diversity of lupin-nodulating microsymbionts and revealed a likely new, fast-growing, salt-tolerant rhizobial species within the genus Devosia as a potentially useful inoculant in agricultural practices or landscape restoration.

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

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

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

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

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

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

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.

Bradyrhizobium niftali sp. nov., an effective nitrogen-fixing symbiont of partridge pea [Chamaecrista fasciculata (Michx.) Greene], a native caesalpinioid legume broadly distributed in the USA.

Information about the symbionts of legumes of the Caesalpinioideae subfamily is still limited, and we performed a polyphasic approach with three Bradyrhizobium strains-CNPSo 3448T, CNPSo 3394 and CNPSo 3442-isolated from Chamaecrista fasciculata, a native legume broadly distributed in the USA. In the phylogenetic analysis of both the 16S rRNA gene and the intergenic transcribed spacer, the CNPSo strains were clustered within the Bradyrhizobium japonicumsuperclade. Multilocus sequence analysis with six housekeeping genes-glnII, gyrB, recA, rpoB, atpD and dnaK-indicated that Bradyrhizobium diazoefficiens is the closest species, with 83 % of nucleotide identity. In the genome analyses of CNPSo 3448T, average nucleotide identity and digital DNA-DNA hybridization results confirmed higher similarity with B. diazoefficiens, with values estimated of 93.35 and 51.50 %, respectively, both below the threshold of the same species, confirming that the CNPSo strains represent a new lineage. BOX-PCR profiles indicated high intraspecific genetic diversity between the CNPSo strains. In the analyses of the symbiotic genes nodC and nifH the CNPSo strains were clustered with Bradyrhizobium arachidis, Bradyrhizobium forestalis, Bradyrhizobium cajani, Bradyrhizobium kavangense and Bradyrhizobium vignae, indicating a different phylogenetic history compared to the conserved core genes. Other physiological (C utilization, tolerance to antibiotics and abiotic stresses), chemical (fatty acid profile) and symbiotic (nodulation host range) properties were evaluated and are described. The data from our study support the description of the CNPSo strains as the novel species Bradyrhizobiumniftali sp. nov., with CNPSo 3448T (=USDA 10051T=U687T=CL 40T) designated as the type strain.

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.

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia.

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.IMPORTANCELupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia.

Biodiversity and biogeography of rhizobia associated with common bean (Phaseolus vulgaris L.) in Shaanxi Province

The biodiversity and biogeography of rhizobia associated with bean in Shaanxi Province were investigated. A total of 194 bacterial isolates from bean nodules collected from 13 sampling sites were characterized based on phylogenetic analyses of the 16S rRNA gene, the housekeeping genes recA, glnII and atpD, and the symbiotic genes nodC and nifH. Fifteen genospecies belonging to the genera Rhizobium, Agrobacterium, Ensifer, Bradyrhizobium and Ochrobactrum were defined among the isolates, with Rhizobium sp. II, Agrobacterium sp. II, E. fredii and R. phaseoli being the dominant groups. Four symbiotic gene lineages corresponding to Rhizobium sp. I, Rhizobium sp. II, R. phaseoli and B. liaoningense were detected in the nodC and nifH sequence analyses, indicating different origins for the symbiotic genes and their co-evolution with the chromosome of the bacteria. Moreover, the Ensifer isolates harbored symbiotic genes closely related to bean-nodulating Pararhizobium giardinii, indicating possible lateral gene transfer from Rhizobium to Ensifer. Correlation of rhizobial community composition with moisture, temperature, intercropping, soil features and nutrients were detected. All the results demonstrated a great diversity of bean rhizobia in Shaanxi that might be due to the adaptable evolution of the bean-nodulating rhizobia subjected to the diverse ecological conditions in the area.

Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils

Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963T in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms.

Eco-geographical diversity of cowpea bradyrhizobia in Senegal is marked by dominance of two genetic types

The genetic diversity of native cowpea rhizobia originating from 60 sites across four eco-geographic zones in Senegal was studied. More than 300 cowpea nodules were analyzed by PCR-RFLP of the 16S–23S rDNA InterGenic Spacer region (IGS). Alignments of IGS sequences indicated that all genotypes were grouping within the Bradyrhizobium genus. The geographical distribution showed that apart from five IGS types, the others were specifically found in only one region. The diversity was significantly higher in the Senegal River valley zone, which presents lower mean annual rainfalls and slightly alkaline soils. Interestingly, two IGS types dominated the Senegalese rhizobial collection, one IGS type (VI) was found on more than half of the nodules collected in the northern Senegal River valley while another IGS type (I) was recovered from the great majority of nodules in the three other regions sampled. Two representative strains from each of these two dominant types were isolated and further analyzed. Multi Locus Sequence Analyses using 6 housekeeping genes indicate that they belong to a new Bradyrhizobium species closely related to B. yuanmingense. Phylogenetic analyses of 2 symbiotic genes nodC and nifH show that they are clustered with B. arachidis. Physiological tests on these strains have shown that under laboratory conditions, the growth of the IGS type VI strains was slightly less affected by a higher osmotic strength in the medium and to alkaline pH, which corroborates the soil physico-chemical parameters.

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.

Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium)

We investigated the diversity of rhizobia isolated from different indigenous legumes in Flanders (Belgium). A total of 3810 bacterial strains were analysed originating from 43 plant species. Based on rep-PCR clustering, 16S rRNA gene and recA gene sequence analysis, these isolates belonged to Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium. Of the genera encountered, Rhizobium was the most abundant (62%) and especially the species Rhizobium leguminosarum, followed by Ensifer (19%), Bradyrhizobium (14%) and finally Mesorhizobium (5%). For two rep-clusters only low similarity values with other genera were found for both the 16S rRNA and recA genes, suggesting that these may represent a new genus with close relationship to Rhodopseudomonas and Bradyrhizobium. Primers for the symbiotic genes nodC and nifH were optimized and a phylogenetic sequence analysis revealed the presence of different symbiovars including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Moreover, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria, respectively. Discriminant and MANOVA analysis confirmed the correlation of symbiosis genes with certain bacterial genera and less with the host plant. Multiple symbiovars can be present within the same host plant, suggesting the promiscuity of these plants. Moreover, the ecoregion did not contribute to the separation of the bacterial endosymbionts. Our results reveal a large diversity of rhizobia associated with indigenous legumes in Flanders. Most of the legumes harboured more than one rhizobial endosymbiont in their root nodules indicating the importance of including sufficient isolates per plant in diversity studies.

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.