Characterization of extrachromosomal replicons present in the extended host range Rhizobium sp. LPU83

In several rhizobia, bacteria that inhabit the soil in free-living conditions and associate in symbiosis with the root of legumes as nitrogen-fixing organisms, plasmid DNA can constitute a high percentage of the genome. We have characterized acid-tolerant isolates of rhizobia-here represented by the strain Rhizobium sp. LPU83-that have an extended nodulation-host range including alfalfa, the common bean, and Leucena leucocephala. In this study we analyzed the plasmids of R. sp. LPU83 in order to characterize their role in the evolution of Medicago symbionts and their involvement in symbiotic behavior. The pLPU83a plasmid was found to be transmissible with no associated phenotypic traits. The symbiotic plasmid pLPU83b could be transferred at very low frequencies under laboratory conditions only when pLPU83a was present; could restore nodulation to a strain cured of its symbiotic plasmid, S. meliloti A818; but could not restore the full nitrogen fixation associated with alfalfa.     

Isolation of Insertion Sequence ISRLdTAL1145-1 from a Rhizobium sp. (Leucaena diversifolia) and Distribution of Homologous Sequences Identifying Cross-Inoculation Group Relationships

Insertion sequence (IS) element ISRLdTAL1145-1 from Rhizobium sp. (Leucaena diversifolia) strain TAL 1145 was entrapped in the sacB gene of the positive selection vector pUCD800 by insertional inactivation. A hybridization probe prepared from the whole 2.5-kb element was used to determine the distribution of homologous sequences in a diverse collection of 135 Rhizobium and Bradyrhizobium strains. The IS probe hybridized strongly to Southern blots of genomic DNAs from 10 rhizobial strains that nodulate both Phaseolus vulgaris (beans) and Leucaena leucocephala (leguminous trees), 1 Rhizobium sp. that nodulates Leucaena spp., 9 R. meliloti (alfalfa) strains, 4 Rhizobium spp. that nodulate Sophora chrysophylla (leguminous trees), and 1 nonnodulating bacterium associated with the nodules of Pithecellobium dulce from the Leucaena cross-inoculation group, producing distinguishing IS patterns for each strain. Hybridization analysis revealed that ISRLdTAL1145-1 was strongly homologous with and closely related to a previously isolated element, ISRm USDA1024-1 from R. meliloti, while restriction enzyme analysis found structural similarities and differences between the two IS homologs. Two internal segments of these IS elements were used to construct hybridization probes of 1.2 kb and 380 bp that delineate a structural similarity and a difference, respectively, of the two IS homologs. The internal segment probes were used to analyze the structures of homologous IS elements in other strains. Five types of structural variation in homolog IS elements were found. The predominate IS structural type naturally occurring in a strain can reasonably identify the strain's cross-inoculation group relationships. Three IS structural types were found in Rhizobium species that nodulate beans and Leucaena species, one of which included the designated type IIB strain of R. tropici (CIAT 899). Weak homology to the whole IS probe, but not with the internal segments, was found with two Bradyrhizobium japonicum strains. The taxonomic and ecological implications of the distribution of ISRLdTAL1145-1 are discussed.     

Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N₂-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.     

Multiple host-specificity loci of the broad host-range Rhizobium sp. NGR234 selected using the widely compatible legume Vigna unguiculata

Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to nod-box-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.     

Diversity of endophytic bacteria associated with nodules of two indigenous legumes at different altitudes of the Qilian Mountains in China

A total of 201 endophytic root nodule-associated bacteria collected from two legumes indigenous to different Qilian Mountain altitudes (Hexi Corridor) were characterized through 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, 16S rRNA gene sequence analysis, and enterobacterial repetitive intergenic consensus-PCR clustering. The isolates phylogenetically belonged to 35 species in the Phyllobacterium, Ensifer, Rhizobium, Microvirga, Sphingomonas, Paracoccus, Mycobacterium, Paenibacillus, Cohnella, Sporosarcina, Bacillus, Staphylococcus, Brevibacterium, Xenophilus, Erwinia, Leclercia, Acinetobacter, and Pseudomonas genera. Phylogenetic nodA sequence analysis showed higher similarity to Sinorhizobium meliloti with strains related to the Rhizobium, Sinorhizobium, and Acinetobacter genera. Sequence analysis of the nifH gene revealed that the strains belonging to Xenophilus, Acinetobacter, Phyllobacterium, and Rhizobium had genes similar to those of Mesorhizobium and Sinorhizobium. The results indicated that horizontal gene transfer could have occurred between rhizobia and non-rhizobial endophytes. Canonical correspondence analysis revealed that altitude and host plant species contributed more to the bacterial endosymbiont separation than other ecological factors. This study provided valuable information on the interactions between symbiotic bacteria, non-symbiotic bacteria and their habitats, and thus provided knowledge on their genetic diversity and ecology.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Mineral Soils as Carriers for Rhizobium Inoculants

Mineral soil-based inoculants of Rhizobium meliloti and Rhizobium phaseoli survived better at 4°C than at higher temperatures, but ca. 15% of the cells were viable at 37°C after 27 days. Soil-based inoculants of R. meliloti, R. phaseoli, Rhizobium japonicum, and a cowpea Rhizobium sp. applied to seeds of their host legumes also survived better at low temperatures, but the percent survival of such inoculants was higher than peat-based inoculants at 35°C. Survival of R. phaseoli, R. japonicum, and cowpea rhizobia was not markedly improved when the cells were suspended in sugar solutions before drying them in soil. Nodulation was abundant on Phaseolus vulgaris derived from seeds that had been coated with a soil-based inoculant and stored for 165 days at 25°C. The increase in yield and nitrogen content of Phaseolus angularis grown in the greenhouse was the same with soil-and peat-based inoculants. We suggest that certain mineral soils can be useful and readily available carriers for legume inoculants containing desiccation-resistant Rhizobium strains.     

Congo Red Absorption by Rhizobium leguminosarum

Congo red absorption is generally considered a contraindication of Rhizobium. However, R. leguminosarum takes up the dye on yeast extract-mannitol agar. The uptake of congo red varies among strains of R. leguminosarum, as shown elsewhere with strains of R. trifolii and R. meliloti. Congo red absorption does not distinguish rhizobia from other bacteria, but may be useful as a strain marker.     

Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha- and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals. Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of the extremely large megaplasmid pNGR234b (86), and some snapshot genome information was made available earlier (91); however, the use of pyrosequencing methods greatly facilitated this process. We report here the genome sequence of NGR234 that is able to nodulate more than 120 genera of legumes and the nonlegume Parasponia andersonii (69). It seems likely that the vast richness of secretory systems might be a major key to the broad host range.     

Diversity of endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.     

Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains

Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical.     

Rhizobium croatiense sp. nov. and Rhizobium redzepovicii sp. nov., two new species isolated from nodules of Phaseolus vulgaris in Croatia

Phaseolus vulgaris is a legume indigenous to America which is nodulated by strains of genus Rhizobium in Croatia. Four of these strains, 13T^T, 9T, 18T^T and 8Z are phylogenetically close to the species from the Rhizobium leguminosarum phylogenetic complex in the 16S rRNA gene analysis. The results of both the analyses of the concatenated recA and atpD genes and whole genomes revealed that the strains 13T^T and 9T clustered with Rhizobium sophoriradicis CCBAU 03470^T and the strains 18T^T and 8Z with Rhizobium ecuadorense CNPSO 671^T. Whole genome average nucleotide identity blast (ANIb) and dDDH values between the strains 13T^T and the type strain of R. sophoriradicis and between the strains 18T^T and the type strain of R. ecuadorense were lower than 95% and 70%, respectively, which are the threshold values recommended for bacterial species differentiation. These results combined with those of chemotaxonomic and phenotypic analyses support the affiliation of these strains to two novel species within the genus Rhizobium for which we propose the names Rhizobium croatiense sp. nov. 13T^T (=LMG 32397^T, = HAMBI 3740^T) as type strain and Rhizobium redzepovicii sp. nov. 18T^T (=LMG 32398^T, = HAMBI 3741^T) as type strain.     

Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov

In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA–DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732^T = LMG 26835^T = HAMBI 3286^T) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729^T = LMG 26833^T = HAMBI 3287^T). They had a DNA G + C mol% (T_m) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.     

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

Bacteria belonging to the genera Rhizobium, Mesorhizobium, Sinorhizobium, Bradyrhizobium, and Azorhizobium (collectively referred to as rhizobia) grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodule cells of legume plants. The interactions between several rhizobial species and their host plants have become models for this type of nitrogen-fixing symbiosis. Temperate legumes such as alfalfa, pea, and vetch form indeterminate nodules that arise from root inner and middle cortical cells and grow out from the root via a persistent meristem. During the formation of functional indeterminate nodules, symbiotic bacteria must gain access to the interior of the host root. To get from the outside to the inside, rhizobia grow and divide in tubules called infection threads, which are composite structures derived from the two symbiotic partners. This review focuses on symbiotic infection and invasion during the formation of indeterminate nodules. It summarizes root hair growth, how root hair growth is influenced by rhizobial signaling molecules, infection of root hairs, infection thread extension down root hairs, infection thread growth into root tissue, and the plant and bacterial contributions necessary for infection thread formation and growth. The review also summarizes recent advances concerning the growth dynamics of rhizobial populations in infection threads.     

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.     

Geographically structured genetic variation in the Medicago lupulina–Ensifer mutualism

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large‐scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome‐wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. and <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> are predominant in root nodules of <Emphasis Type="Italic">Vigna angularis</Emphasis>, a native legume crop in the subtropical region of China

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.