nodC Gene Sequences Research Articles

Ecological adaptation and phylogenetic analysis of microsymbionts nodulating Polhillia, Wiborgia and Wiborgiella species in the Cape fynbos, South Africa

Polhillia, Wiborgia and Wiborgiella species are shrub legumes endemic to the Cape fynbos of South Africa. They have the ability to fix atmospheric N2 when in symbiosis with soil bacteria called ‘rhizobia’. The aim of this study was to assess the morpho-physiological and phylogenetic characteristics of rhizobia associated with the nodulation of Polhillia, Wiborgia and Wiborgiella species growing in the Cape fynbos. The bacterial isolates from root nodules consisted of a mixture of fast and intermediate growers that differed in colony shape and size. The isolates exhibited tolerance to salinity (0.5–3% NaCl) and pH (pH 5–10) and different antibiotic concentrations, and could produce 0.51 to 51.23 µg mL−1 of indole-3-acetic acid (IAA), as well as solubilize tri-calcium phosphate. The ERIC-PCR results showed high genomic diversity in the rhizobial population and grouped them into two major clusters. Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, nifH and nodC gene sequences revealed distinct and novel evolutionary lineages related to the genus Rhizobium and Mesorhizobium, with some of them being very close to Mesorhizobium australicum. However, the phylogenetic analysis of glnII and nifH genes of some isolates showed incongruency.

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

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

Mesorhizobium sanjuanii sp. nov., isolated from nodules of Lotus tenuis in the saline-alkaline lowlands of Flooding Pampa, Argentina.

Two rhizobial strains, BSA136T and BSA150, related to the genus Mesorhizobium were isolated from root nodules of Lotus tenuis grown in saline-alkaline lowlands soil from Argentina. These strains showed different repetitive element palindromic PCR fingerprinting patterns but shared more than 99 % sequence similarity for both 16S rRNA and recA genes. Despite the symbiotic nodC gene sequences of our strains being related to the canonical Lotus biovar species comprising Mesorhizobium loti and Mesorhizobium japonicum, the 16S rRNA phylogenetic marker suggests that their taxonomical identities are closely related to Mesorhizobium helmanticense, Mesorhizobium metallidurans, Mesorhizobium thianshanense, Mesorhizobium gobiense and Mesorhizobium tarimense. Multilocus sequence analysis performed with seven housekeeping genes confirmed that BSA136T belongs to a separate clade within the genus Mesorhizobium. The results of comparisons for in silico DNA-DNA hybridization and average nucleotide identity indexes between the genomes of BSA136T and closest-related Mesorhizobium species were below the threshold for species delineation. Phenotypic features differentiated BSA136T from its closest-related species. On the basis of our results, BSA136T and BSA150 can be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium sanjuanii sp. nov. is hereby proposed. The type strain of this species is BSA136T (=CECT 9305T=LMG 30060T), for which the draft genome sequence is available.

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.

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.

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.

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.

Bradyrhizobium tropiciagri sp. nov. and Bradyrhizobium embrapense sp. nov., nitrogen-fixing symbionts of tropical forage legumes.

Biological nitrogen fixation is a key process for agricultural production and environmental sustainability, but there are comparatively few studies of symbionts of tropical pasture legumes, as well as few described species of the genus Bradyrhizobium, although it is the predominant rhizobial genus in the tropics. A detailed polyphasic study was conducted with two strains of the genus Bradyrhizobium used in commercial inoculants for tropical pastures in Brazil, CNPSo 1112T, isolated from perennial soybean (Neonotonia wightii), and CNPSo 2833T, from desmodium (Desmodium heterocarpon). Based on 16S-rRNA gene phylogeny, both strains were grouped in the Bradyrhizobium elkanii superclade, but were not clearly clustered with any known species. Multilocus sequence analysis of three (glnII, gyrB and recA) and five (plus atpD and dnaK) housekeeping genes confirmed that the strains are positioned in two distinct clades. Comparison with intergenic transcribed spacer sequences of type strains of described species of the genus Bradyrhizobium showed similarity lower than 93.1 %, and differences were confirmed by BOX-PCR analysis. Nucleotide identity of three housekeeping genes with type strains of described species ranged from 88.1 to 96.2 %. Average nucleotide identity of genome sequences showed values below the threshold for distinct species of the genus Bradyrhizobium ( < 90.6 %), and the value between the two strains was also below this threshold (91.2 %). Analysis of nifH and nodC gene sequences positioned the two strains in a clade distinct from other species of the genus Bradyrhizobium. Morphophysiological, genotypic and genomic data supported the description of two novel species in the genus Bradyrhizobium, Bradyrhizobium tropiciagri sp. nov. (type strain CNPSo 1112T = SMS 303T = BR 1009T = SEMIA 6148T = LMG 28867T) and Bradyrhizobium embrapense sp. nov. (type strain CNPSo 2833T = CIAT 2372T = BR 2212T = SEMIA 6208T = U674T = LMG 2987).

Rhizobial strains isolated from nodules of Medicago marina in southwest Spain are abiotic-stress tolerant and symbiotically diverse

The isolation and characterisation of nitrogen-fixing root nodule bacteria from Medicago marina, a tolerant legume species, were studied in two areas from southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS, including the partial sequence of the housekeeping gene glnII and the symbiotic gene nodA of some representatives), physiological (utilisation of sole carbon sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis, M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M. marina nodules belonged to Ensifer meliloti, except for one strain that belonged to E. medicae. To determine the nodulation range of M. marina, 10 different Ensifer species were tested for their ability to nodulate on this plant. E. kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most of the M. marina-nodulating strains showed tolerance to stress factors and all of them shared the presence of a gene similar to cadA, a gene that encodes for a PIB-type ATPase, which is a transporter belonging to the large superfamily of ATP-driven pumps involved in the transport of metals across cell membranes.

Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan=4200m2) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5kb) and whole ribosomal DNA (5kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.

Mesorhizobium qingshengii sp. nov., isolated from effective nodules of Astragalus sinicus

In a study on the diversity of rhizobia isolated from root nodules of Astragalus sinicus, five strains showed identical 16S rRNA gene sequences. They were related most closely to the type strains of Mesorhizobium loti, Mesorhizobium shangrilense, Mesorhizobium ciceri and Mesorhizobium australicum, with sequence similarities of 99.6-99.8%. A polyphasic approach, including 16S-23S intergenic spacer (IGS) RFLP, comparative sequence analysis of 16S rRNA, atpD, glnII and recA genes, DNA-DNA hybridization and phenotypic tests, clustered the five isolates into a coherent group distinct from all recognized Mesorhizobium species. Except for strain CCBAU 33446, from which no symbiotic gene was detected, the four remaining strains shared identical nifH and nodC gene sequences and nodulated with Astragalus sinicus. In addition, these five strains showed similar but different fingerprints in IGS-RFLP and BOX-repeat-based PCR, indicating that they were not clones of the same strain. They were also distinguished from recognized Mesorhizobium species by several phenotypic features and fatty acid profiles. Based upon all the results, we suggest that the five strains represent a novel species for which the name Mesorhizobium qingshengii sp. nov. is proposed. The type strain is CCBAU 33460(T) (=CGMCC 1.12097(T)=LMG 26793(T)=HAMBI 3277(T)). The DNA G+C content of the type strain is 59.52 mol% (Tm).

Phylogenetic multilocus sequence analysis identifies seven novel Ensifer genospecies isolated from a less-well-explored biogeographical region in East Africa

The diversity of 71 rhizobial strains belonging to the genus Ensifer, isolated from root nodules of woody legumes growing in southern Ethiopia, was studied using multilocus sequence analysis (MLSA) and phenotypic approaches. Phylogenetic analyses based on core genes revealed that 43 strains were clustered in seven distinct and consistent positions (genospecies I-VII), while another 25 strains were also distinct but were discrepant in their placement on the different gene trees. The remaining three strains occupied the same phylogenetic branches as defined Ensifer species and thus were not distinct. Irrespective of their chromosomal background, the majority of the test strains were highly related with respect to their nifH and nodC gene sequences, suggesting that these symbionts might have acquired these genes recently from a common origin. On the nifH phylogenetic tree, the branch containing the test strains and reference species isolated from woody legumes in Africa was clearly separate from those isolated outside the continent, suggesting that these symbionts have a long history of separate evolution within Ensifer for this gene. A cross-inoculation study showed that our strains were capable of eliciting effective nodulation on the homologous host and on other host species. This suggests a potential to improve nitrogen fixation by selecting for broad-host-range inoculants. Our study confirms the presence of a wide diversity of Ensifer in East Africa and, while contributing to the general knowledge of the biodiversity within the genus, also highlights the need to focus on previously less-well-explored biogeographical regions to unravel as-yet-unidentified rhizobial resources.

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.

Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea

Five strains isolated from root nodules of Lablab purpureus and Arachis hypogaea grown in the Anhui and Sichuan provinces of China were classified as members of the genus Bradyrhizobium. These strains had identical 16S rRNA gene sequences which shared 99.48 %, 99.48 % and 99.22 % similarity with the most closely related strains of Bradyrhizobium jicamae PAC68(T), Bradyrhizobium pachyrhizi PAC48(T) and Bradyrhizobium elkanii USDA 76(T), respectively. A study using a polyphasic approach, including 16S rRNA gene RFLP, IGS-RFLP, BOX-PCR, comparative sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) and the recA, atpD and glnII genes, DNA-DNA hybridization and phenotypic tests, showed that the five strains clustered into a coherent group that differentiated them from all recognized species of the genus Bradyrhizobium. Sequencing of nifH and nodC genes and cross-nodulation tests showed that the representative strains CCBAU 23086(T), CCBAU 23160 and CCBAU 61434, isolated from different plants, had identical nifH and nodC gene sequences and were all able to nodulate Lablab purpureus, Arachis hypogaea and Vigna unguiculata. Based upon these results, the name Bradyrhizobium lablabi sp. nov. is proposed for this novel species and strain CCBAU 23086(T) ( = LMG 25572(T) = HAMBI 3052(T)) is designated as the type strain. The DNA G+C mol% is 60.14 (T(m)).

Mesorhizobium alhagi sp. nov., isolated from wild Alhagi sparsifolia in north-western China

Eleven strains that formed symbiotic root nodules on Alhagi sparsifolia, designated previously as genospecies II, were identified as a new lineage of Mesorhizobium (Alphaproteobacteria) that could be differentiated from all previously recognized species of the genus Mesorhizobium by using 16S rRNA gene sequences (<97.8 % similarity), DNA-DNA hybridization (<45 %), dnaJ, dnaK, recA, glnA, nifH, nodA and nodC gene sequences, fatty acid profiles (C(18 : 1)omega7c, 35 %;11-methyl C(18 : 1)omega7c, 30 %) and numerical taxonomy. These strains are therefore considered to represent a novel species, for which the name Mesorhizobium alhagi sp. nov. is proposed, with isolate CCNWXJ12-2(T) (=ACCC 15461(T)=HAMBI 3019(T)) as the type strain.

Rhizobium mesosinicum sp. nov., isolated from root nodules of three different legumes

Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

Diversity of Rhizobial Bacteria Isolated from Nodules of the Gypsophyte Ononis tridentata L. Growing in Spanish Soils

The aim of this work is to describe the diversity and phylogeny of rhizobial bacteria associated to nodules of Ononis tridentata L. in different geographical regions of Spain. Twenty-two bacterial isolates were characterized using several molecular techniques (16S amplified ribosomal deoxyribonucleic acid restriction analysis, fingerprinting, and sequencing) and phylogenies were inferred from their 16S and nodC gene sequences. Phylogenetically, the isolates grouped with the genera Rhizobium, Mesorhizobium, Phylobacterium, and Bosea. The nodC gene, essential for nodulation, was detected for the first time in isolates close to the genera Bosea and Phyllobacterium. The bacteria isolated showed a high diversity at the genus, species, and strain level regardless of the geographical origin of the host plant. This is the first report describing bacteria associated to nodules of O. tridentata. This shrub legume is highly prized for the revegetation of gypsum soils in semiarid Mediterranean areas. Our molecular description of bacteria associated to this legume improves the current understanding of the ecology of this plant species. Our findings have implications for formulating suitable bacterial inocula to recover gypsum ecosystems.

Mesorhizobium albiziae sp. nov., a novel bacterium that nodulates Albizia kalkora in a subtropical region of China

A novel Mesorhizobium group associated with Albizia kalkora [Wang et al. (2006), Syst Appl Microbiol 29, 502-517] was further characterized. The seven strains in this group showed similar protein patterns and were different from defined Mesorhizobium species in SDS-PAGE of whole-cell proteins. The representative strain CCBAU 61158(T) formed a novel Mesorhizobium lineage in phylogenetic analyses of 16S rRNA, atpD, glnII and nifH genes. However, its nodC gene sequence was more similar to that of Rhizobium gallicum R602sp(T) than to those of Mesorhizobium species. DNA-DNA relatedness between CCBAU 61158(T) and reference strains of defined Mesorhizobium species was lower than 34.1 %. These results indicated that this Mesorhizobium group was a unique genomic species. The subtropical distribution, host origin, PCR-RFLP patterns of 16S rRNA genes, fatty acid profile and a series of phenotypic characteristics could be used as distinctive features of this group. This group is therefore proposed as a novel species, Mesorhizobium albiziae sp. nov., with CCBAU 61158(T) (=LMG 23507(T)=USDA 4964(T)) as the type strain. Strain CCBAU 61158(T) could form effective nodules on Albizia julibrissin, Glycine max, Leucaena leucocephala and Phaseolus vulgaris.

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer's weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer's weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.