Phylogeny and taxonomy of rhizobia

Rhizobia are bacteria that form nitrogen-fixing nodules on the roots, or occasionally the shoots, of legumes. There are currently more than a dozen validly named species, but the true number of species is probably orders of magnitude higher. The named species are listed and briefly discussed. Sequences of the small subunit ribosomal RNA (SSU or 16S rRNA) support the well-established subdivision of rhizobia into three genera: Rhizobium, Bradyrhizobium, and Azorhizobium. These all lie within the alpha subdivision of the Proteobacteria, but on quite distinct branches, each of which also includes many bacterial species that are not rhizobia. It has been clear for several years that Rhizobium, on this definition, is still too broad and is polyphyletic: there are many non-rhizobia within this radiation. Recently, therefore, it has been suggested that this genus should be split into four genera, namely Rhizobium (R. leguminosarum, R. tropici, R. etli), Sinorhizobium (S. fredii, S. meliloti, S. teranga, S. saheli), Mesorhizobium (M. loti, M. huakuii, M ciceri, M. tianshanense, M. mediterraneum), and a fourth, unnamed, genus for the current R. galegae. The evidence and pros and cons are reviewed.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

An attempt at taxonomical characterization of some Rhizobial species by intact cell MALDI mass spectrometry

In the present study an attempt was made to exploit the benefit of intact cell MALDI mass spectrometry (ICM-MS) in bringing out similarities and differences among some Rhizobium species and a species of Agrobacterium based on specific mass:charge (m/z) values. Rhizobium species isolated from the root nodules of selected leguminous plants were analysed by ICM-MS. The spectra were acquired in the range of 500–10,000 Da yielding several peaks specific to each species. The peaks obtained corresponded to the respective bacterial cell surface molecules which were desorbed during matrix-assisted laser desorption ionization. The number of peaks were more in the range of 500–1200 Da. Dice similarity coefficient analysis of m/z values indicated that Rhizobium species isolated from Trigonella foenum-graecum and Pisum arvense showed more similarity than any other species. Agrobacterium species did show a few common m/z values in comparison with other Rhizobium species. This clearly shows that Agrobacterium is closely related to Rhizobium. Eventually, ICM-MS technique offers clear, distinct, and consistent results for replicates, in less than an hour’s time, therefore this technique has high potential in molecular taxonomy.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New taxonomic markers for identification of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> and discrimination between closely related species

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)     

Complete sequence of a Rhizobium plasmid carrying genes necessary for symbiotic association with the plant host

The soil bacteria rhizobia have the capacity to establish nitrogen-fixing symbiosis with their leguminous host plants. In most Rhizobium species the genes for nodule development and nitrogen fixation have been localized on large indigenous plasmids that are transmissible, allowing lateral transfer of symbiotic functions. A recent paper reports on the complete sequencing of the symbiotic plasmid pNGR234a from Rhizobium species NGR234⁽¹⁾, revealing not only putative new symbiotic genes but also possible mechanisms for evolution and lateral dispersal of symbiotic nitrogen-fixing abilities among rhizobia.     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}     

The effect of aluminium on growth of and nitrogen fixation in vegetable soybean germplasm

Vegetable soybeam germplasm was screened for its tolerance to 0, 50 and 100 μM Al in solution culture. Plants were inoculated with prescreened acid-Al tolerantBradyrhizobium japonicum strain USDA 110 and a localRhizobium isolate SM867. Aluminum concentrations of 0, 50, and 100 μM affected the root lengths of all germplasm lines in the first few weeks of their growth. At 100 μM, the plants had severely stunted roots throughout the growing period of 35 days, but at 50 μM the initial stunting of the roots was overcome after the third week of growth, and there were no significant differences between the root lengths of these plants and of the controls. The appearance of the first nodule was delayed for 2–3 and 4–5 days at 50 μM and 100 μM Al, respectively. There was a significant reduction in nodule numbers and acetylene reduction activity (ARA) at 100 μM Al. At 50 μM Al, even though the number of nodules was decreased significantly, nodules were larger in size, so there was no significant reduction in nodule fresh weight and ARA. No significant differences in nitrogen fixing abilities of the soybean lines were observed between the twoRhizobium strains. Germplasm line Kahala showed the greatest tolerance to 50 μM Al, and Kahala, Kim and Wolverine tolerated 100 μM Al better than other germplasm lines.     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

Coculture of Pachyrhizus erosus (L.) hairy roots with Rhizobium spp.

Summary We have established an in vitro system for the induction and study of nodulation in Pachyrhizus erosus (jicama) via a hairy root-Rhizobium coculture. In vitro-grown P. erosus plantlets were infected with Agrobacterium rhizogenes (ATCC No. 15834) and two hairy root lines were established. Hairy roots were grown in a split-plate system in which compartment I (CI) contained MS medium with nitrogen and different sucrose levels (0–6%), while CII held MS medium without nitrogen and sucrose. Nodule-like structures developed in transformed roots grown in CI with 2–3% surcose, inoculated with Rhizobium sp. and transferred to CII. Nodule-like structures that developed from hairy roots lacked the rigid protective cover observed in nodules from plants grown in soil. Western blot analysis of nodules from hairy roots and untransformed roots (of greenhouse-grown jicama) showed expression of glutamine synthetase leghemoglobin and nodulins. Leghemoglobin was expressed at low levels in hairy root nodules.     

Efficient micropropagation of Robinia ambigua var. idahoensis (Idaho Locust) and detection of genomic variation by ISSR markers

Robinia ambigua var. idahoensis, presumably originated from interspecific hybridization of R. pseudoacacia L. and R. hispida L., is a multipurpose tree. Several reports have showed that in vitro micropropagation is a feasible method to produce large quantities of ‘clonal’ plants from R. pseudoacacia, however, no information is available on micropropagation of R. ambigua or the other assumed parental species, R. hispida. Here, we report on a tissue culture system for efficient micropropagation of R. ambigua plants by enhanced branching of axillary buds taken from a single branch of a donor tree. The culture system consists of sequential use of three media, namely, the bud-induction medium (MS medium supplemented with 0.8–1.4 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA), elongation medium (MS medium added with 0.35–0.5 mg l⁻¹ 6-BA, 0.05–0.08 mg l⁻¹ NAA and 0.07–0.1 mg l⁻¹ GA) and root-induction medium (1/4 MS medium fortified with 1.7–2.5 mg l⁻¹ IAA and 0.1–0.5 mg l⁻¹ IBA). In addition, we investigated the genetic stability (relative to the donor plant) of a sample of 41 morphologically normal plants randomly taken from ca. 13,000 micropropagated plants, by using the inter-simple sequence repeat (ISSR) marker with 32 selected primers. We found that of the 226 reproducible bands scored, 24 were polymorphic (10.62%), thus pointing to the occurrence, though at a relatively low level compared with an earlier study on R. pseudoacacia, of genomic variation in these micropropagated plants. Further sequencing on seven loci underlying the variations showed that two had significant homology to known or predicted plant genes.     

The presence and possible significance of cross-reactive antigens inRhizobium — legume associations

Soluble antigens of threeRhizobium species were compared by the agar-gel double-diffusion technique with those of eight legumes representing compatible and non-compatible hosts. Cross-reactive antigens were found between all the legume hosts and the three rhizobia. These common antigens among hosts and bacteria were not related to the specificity of compatibleRhizobium-legume associations. The cross-reactive antigens were absent between rhizobia and eight non-legume plants tested, but present between five out of eleven gramnegative phytopathogenic bacteria and legumes. The potential significance of cross-reactive antigens betweenRhizobium and legume hosts in nodule development is discussed.     

Genetic diversity of rhizobia associated with <Emphasis Type="Italic">Vicia faba</Emphasis> in three ecological regions of China

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA–DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nodulation competitiveness in the Rhizobium-legume symbiosis

Successful nodulation of legumes by rhizobia is a complex process that, in the open field, depends on many different environmental factors. Generally, legume productivity in an agricultural field may be improved by inoculation with selected highly effective N₂-fixing root nodule bacteria. However, field legume inoculation with Rhizobium and Bradyrhizobium spp. has often been unsuccessful because of the presence in the soil of native strains that compete with the introduced strain in nodule formation on the host plants. This ability to dominate nodulation is termed competitiveness and is critical for the successful use of inoculants.The author is with the Departmentode Microbiologia del Suelo y Sistemas Simbioticos, Estation Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, C/Professor Albareda 1, 18008 Granada, Spain     

Discovery of a life history shift: precocious flowering in an introduced population of Prosopis

The genus Prosopis contains many valuable, long-lived, multi-purpose legume trees, some of which are also invasive species. The time of first flowering is important for increasing production of sweet, protein-rich pods in plantations, especially under short rotations, and affects the rate of spread as a weed. Trees generally begin flowering at 3–5 years of age. However, seedlings from a small seedlot collected from an introduced population in southern Mauritania were observed to begin flowering at 3–4 months after germination. This is unknown in the native range of any Prosopis species, and appears to represent a major evolutionary event triggered after naturalization. This paper reports a detailed investigation with seed collected from Aleg, Brakna region, Mauritania, in 1998 (Prosopis sp. ‘Aleg’). Two experiments were established, in glasshouses at Cirad, Montpellier, France, and Coventry University, UK. Flowering began at the two sites 104 and 169 days after sowing, respectively, and 97.5% of plants had flowered at Coventry after 306 days. Flowers produced abundant pollen with 50–60% viability indicated by FDA staining and 40–50% germination on an artificial medium. No pods were formed. All plants analysed were diploid. Morphologically, these juvenile plants were similar to American species of section Algarobia, and exhibited leaf characters typical of P. pallida. However, due to the large variation in morphology within this section of the genus, further studies are required to confirm the actual species. This is the first record of precocious flowering in Prosopis, and has been found only in a single, introduced population. Implications for genetic improvement of tropical Prosopis, and particularly the spread of these species as invasive weeds, are discussed. Such a life history shift in the increased reproductive ability of a species following introduction, with potentially significant environmental effects, may not be restricted to Prosopis and merits further detailed investigation.     

Contrasting effects of invasive plants in plant–pollinator networks

The structural organization of mutualism networks, typified by interspecific positive interactions, is important to maintain community diversity. However, there is little information available about the effect of introduced species on the structure of such networks. We compared uninvaded and invaded ecological communities, to examine how two species of invasive plants with large and showy flowers (Carpobrotus affine acinaciformis and Opuntia stricta) affect the structure of Mediterranean plant–pollinator networks. To attribute differences in pollination to the direct presence of the invasive species, areas were surveyed that contained similar native plant species cover, diversity and floral composition, with or without the invaders. Both invasive plant species received significantly more pollinator visits than any native species and invaders interacted strongly with pollinators. Overall, the pollinator community richness was similar in invaded and uninvaded plots, and only a few generalist pollinators visited invasive species exclusively. Invasive plants acted as pollination super generalists. The two species studied were visited by 43% and 31% of the total insect taxa in the community, respectively, suggesting they play a central role in the plant–pollinator networks. Carpobrotus and Opuntia had contrasting effects on pollinator visitation rates to native plants: Carpobrotus facilitated the visit of pollinators to native species, whereas Opuntia competed for pollinators with native species, increasing the nestedness of the plant–pollinator network. These results indicate that the introduction of a new species to a community can have important consequences for the structure of the plant–pollinator network.     

The Chloroplast trnT–trnF Region in the Seed Plant Lineage Gnetales

The trnT–trnF region is located in the large single-copy region of the chloroplast genome. It consists of the trnL intron, a group I intron, and the trnT–trnL and trnL–trnF intergenic spacers. We analyzed the evolution of the region in the three genera of the gymnosperm lineage Gnetales (Gnetum, Welwitschia, and Ephedra), with especially dense sampling in Gnetum for which we sequenced 41 accessions, representing most of the 25–35 species. The trnL intron has a conserved secondary structure and contains elements that are homologous across land plants, while the spacers are so variable in length and composition that homology cannot be found even among the three genera. Palindromic sequences that form hairpin structures were detected in the trnL–trnF spacer, but neither spacer contained promoter elements for the tRNA genes. The absence of promoters, presence of hairpin structures in the trnL–trnF spacer, and high sequence variation in both spacers together suggest that trnT and trnF are independently transcribed. Our model for the expression and processing of the genes tRNA^Thr(UGU), tRNA^Leu(UAA), and tRNA^Phe (GAA) therefore attributes the seemingly neutral evolution of the two spacers to their escape from functional constraints. [Reviewing Editor: Debashish Bhattacharya]     

蚕豆根瘤菌接种效应研究

为发掘和利用青海冷凉地区蚕豆优良的根瘤菌种质资源,确定根瘤菌的接种效应。将分离、纯化、分子鉴定的16株蚕豆根瘤菌通过盆栽回接试验的方法进行筛选。结果表明,筛选出6株根瘤菌,它们与青海13号蚕豆共生匹配效果较好,共生固氮能力强,促进蚕豆生长效果明显。