Differentiating Indigenous Soybean Bradyrhizobium and Rhizobium spp. of Indian Soils

Soybean is extensively cultivated worldwide and is the largest source of biologically fixed nitrogen among legumes. It is nodulated by both slow and fast growing rhizobia. Indigenous soybean rhizobia in Vertisols of central India were assessed for utilization of 35 carbon sources and intrinsic resistance to 19 antibiotics. There was greater utilization of trehalose and raffinose by fast growers (87 and 73 % by fast vs. 35 and 30 % by slow growers); but slow growers had higher ability to utilize glucosamine (75 % by slow vs. 33 % by fast growers). A larger proportion of slow growers were resistant to vancomycin, polymyxin-B and rifampicin (70, 65 and 55 %) compared to fast growers (13, 7 and 7 % each). Among the two 16S rRNA sequence types in the slow growers, those belonging to Bradyrhizobium spp. utilized glucosamine while those belonging to Rhizobium radiobacter did not. All the fast growers had 16S rRNA homology to R. radiobacter and majority could not utilize glucosamine. It is suggested that during initial isolations and screening of rhizobia in strain selection programmes, using carbon sources like glucosamine and antibiotics like vancomycin, polymyxin-B and rifampicin in the media may provide a simple way of distinguishing Bradyrhizobium strains from R. radiobacter among the slow growers.     

Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal

A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.     

Novel root nodule bacteria belonging to the genus Caulobacter

Aims: Aim of this study is to determine the genetic variation of rhizobia associated with horse gram [Macrotyloma uniflorum (Lam.) Verdc.] plants grown in different regions of Andhra Pradesh, India. Methods and Results: Four representative isolates having most representative characters from the previous characterization were selected for 16S rRNA sequence. The sequences were submitted to the NCBI GenBank and Ribosomal Database Project (RDP). The isolates HGR‐4, 6 and 13 showed more than 99% homology between them and they were grouped with Rhizobium reference strains where as the isolate HGR‐25 showed 87·1, 87·4 and 87·2% homology with the isolates HGR‐4, 6 and 13, respectively, and were grouped with reference strains for Caulobacter. The nodulation ability of these isolates on horse gram was confirmed by inoculation tests. Conclusions: The isolate HGR‐25 was identified as Caulobacter isolated from the plants growing in soil samples collected from Khareemnagar district, Andhra Pradesh, India. Inoculation tests revealed that Caulobacter formed nodules on horse gram. It was also confirmed by RDP. Significance and Impact of the Study: This is the first report that a legume was nodulated by a member of the genus Caulobacter, which belongs to the family Caulobacteriaceae in the order Caulobacterales of Alphaproteobacteria.     

An assessment of a method based on intrinsic antibiotic resistance for identifying Rhizobium strains

A method based on intrinsic antibiotic resistance (IAR) for identifying large numbers of Rhizobium strains was assessed and found to be unsatisfactory for R. phaseoli and isolates from Cicer arietinum (Rhizobium spp.). Our data showed that the number of different IAR patterns always exceeded the number of strains tested. With 90 nodule isolates from plants inoculated with a mixture of three strains of R. Phaseoli, the technique gave 18 different resistance patterns. When 24 strains of Rhizobium spp., each replicated three times, were examined 68 different resistance patterns were obtained. Single colony isolates from one strain also gave several different IAR patterns. All strains tested with fluorescent“ antibody were readily identified. Attempts to obtain correct strain identification with IAR by simplifying the scoring systems or allowing up to two differences in the resistance patterns were unsuccessful. We were unable to define the source of this variation although incubation time and inoculum concentration were shown to affect the IAR patterns     

Phenotypic characteristics of root-nodulating bacteria isolated from Acacia spp. grown in Libya

Thirty isolates of root-nodulating bacteria obtained from Acacia cyanophylla, A. karroo, A. cyclops, A. tortilis (subsp.raddiana), Faidherbia albida and Acacia sp., grown in different regions of Libya, were studied by performing numerical analysis of 104 characteristics. Three fast- and one slow-growing reference strains from herbaceous and woody legumes were included. Five distinct clusters were formed. The fast-growing reference strains were separated from the isolates whereas the slow-growing was included in cluster 4. With the exception of one cluster, the majority of clusters were formed regardless of the host plant or site of origin. Based on plant tests, generation times, acid production and carbon utilization the isolates were diverse (fast and slow-growing isolates). Like slow-growing isolates, most of the fast-growing isolates appeared to be non-specific, nodulated many species from the same genus notably F. albida, known to nodulate only with slow-growing strains. Most clusters grew at temperatures 35 °C and 37 °C; some grew at temperatures above 40 °C. The majority of isolates grew at acid and alkaline pH and only one isolate grew below pH 4. Most isolates were able to utilize many amino acids as nitrogen sources and to reduce nitrate. Urea was hydrolysed by all clusters. Monosaccharides and polyols were used by slow and fast-growing isolates as the only carbon sources whereas assimilation of disaccharides varied: Some isolates, like slow-growing isolates, failed to utilize these carbon sources. Most isolates were unable to utilize polysaccharides. Regarding tolerance to NaCl on agar medium, the majority of isolates were unable to grow at a concentration of 2% NaCl, but some were highly resistant and there was one isolate which grew at 8% NaCl. Most isolates were resistant to heavy metals and to antibiotics.     

Genetic Characterization of Soybean Rhizobia in Paraguay

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

The phenotypic,phylogenetic and symbiotic characterization of rhizobia nodulating Lotus sp. in Tunisian arid soils

Fifteen bacterial isolates, representatives of different 16S rRNA-RFLP genomogroups which were isolated from root nodules of Lotus creticus and L. pusillus growing in the arid areas of Tunisia were characterized by phenotypic features and 16S rDNA sequences. Phenotypically, all isolates are fast growers with the ability to grow at a pH between 5.5 and 9. Most of the tested isolates tolerate NaCl concentrations from 1.39 to 3.48 %. Phylogenetically, the studied isolates are affiliated into the genera: Sinorhizobium (5 strains), Rhizobium (2 strains), and Mesorhizobium (4 strains). The 16S rDNA sequences of Tunisian Lotus sp. nodule isolates: LAC7511, LAC733, and Mesorhizobium alhagi (Alhagi sparsifolia symbiont) shared 100 % identical nucleotides similar to the 16S rDNA sequences of LAC831, LAC814 and Mesorhizobium temperatum CCNWSX0012-2 (Astragalus adsurgens symbiont). Non-nodulating bacteria, considered as endophytes of Lotus sp. nodules, were also found in our studies and they were classified into the genera: Phyllobacterium (2 strains), Starkeya (1 strain) and Pseudomonas (1 strain). Except for these four endophytic Lotus sp. bacteria, all other strains under investigation induce nodules on Lotus sp., but they differ in the number of induced root nodules and the effectiveness of atmospheric nitrogen fixation. The Sinorhizobium sp., Mesohizobium sp. and Lotus sp. nodule isolates, forming the most effective symbiosis with the plant host, are potential candidates for inoculants in revegetation programs.     

Identification of Rhizobium strains on antibiotic concentration gradients

Antibiotic concentration gradients were used to characterise several cultures of R. phaseoli and Rhizobium spp. (isolated from Cicer arietinum) by differences in intrinsic antibiotic resistance (IAR). Differentiation between cultures was facilitated by use of cluster analyses. The method permitted 15/16 cultures of R. phaseoli to be distinguished on 14 antibiotics. Two cultures which exhibited similar IAR patterns were shown to be the same strain obtained from different collections. The validity of the technique for strain identification was demonstrated by fluorescent antibody tests which gave corresponding identity for 50 nodule isolates from plants inoculated with a mixture of three strains of R. phaseoli. The method was less suitable for characterising cultures of the slow-growing Rhizobium spp. because several antibiotics produced growth lacking a clearly defined boundary between resistance and susceptibility. Although 15/16 cultures of Rhizobium spp. could be differentiated, several isolates were distinguishable only by a difference on a single antibiotic. Similarity between stock cultures and derivative nodule isolates indicated that IAR on gradient plates was a stable property unaffected by plant passage.     

Genetic Diversity of Rhizobia Nodulating Arachis hypogaea L. in Central Argentinean Soils

The diversity of thirty-nine isolates from peanut plants growing at fourteen different sites in the Argentinean province of Córdoba was examined by rep-PCR, RFLP of PCR amplified 16S rRNA gene and complete sequencing of ribosomal genes. The genomic analysis of the peanut isolates indicated that each group encompasses heterogeneity among their members, having distinct rep fingerprints and 16S rRNA alleles. Complete sequencing of 16S rRNA demonstrated that native peanut rhizobia from Córdoba soils representative of the slow and fast growers are phylogenetically related to Bradyrhizobium japonicum and Bradyrhizobium sp. and Rhizobium giardinii and R. tropici species, respectively. The nodC gene sequence analysis showed phylogenetic similarity between fast grower peanut symbionts and Rhizobium tropici.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Crossing the Limits of Rhizobium Existence in Extreme Conditions

An ecological survey was conducted to characterize 5000 Rhizobium sp. sesbania strains of diverse geographical origin, isolated from the root nodules of Sesbania aculeata growing in neutral (pH 7) and alkaline (pH 8.5 and above) soils. The rhizobia from the alkaline soil showed significantly higher salt tolerance than those isolated from neutral soil. Upper limits of stress survival of rhizobial isolates, Rhizobium sp. NBRI0102 sesbania selected from neutral soil, and Rhizobium sp. NBRI2505 sesbania selected from alkaline soil, were studied under free living conditions. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 10% and 28% salt (NaCl, wt/vol) for up to 18 h of incubation at 30°C. Growth of Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania at pH 7, 11, and 12 was identical, except for a lag period of about 10 h in the growth of Rhizobium sp. NBRI0102 sesbania at pH 11 and 12, as compared with pH 7. Rhizobium sp. NBRI0102 sesbania and Rhizobium sp. NBRI2505 sesbania survived at 50°C and 65°C, in YEB at pH 7 for up to 4 and 2 h, respectively. To our knowledge, this is the first report of rhizobia demonstrating survival of Rhizobium sp. NBRI2505 sesbania, estimated by counting viable cells, to such extreme conditions of salt and temperature, individually. In contrast to Rhizobium sp. NBRI0102 sesbania, high temperature was tolerated efficiently by Rhizobium sp. NBRI2505 sesbania, in the presence of salt at higher pH. Our results suggest that the possession of the trait of high salt tolerance might be of some evolutionary significance for the survival of rhizobia in alkaline soils, at high pH and temperature. Received: 23 May 2000 / Accepted: 26 June 2000     

Root nodulation studies inAeschynomene aspera

Summary Fifteen isolates of nodule bacteria were isolated from root and stem nodules ofAeschynomene aspera and they were characterized as Rhizobium by well known laboratory tests. All these isolates together with other efficient strains of known rhizobia belonging to different cross-inoculation groups were evaluated for their nodulation abilities onAeschynomene aspera, Cajanus cajan (pigeon pea),Cicer arietinum (chickpea),Pisum sativum (pea),Trifolium repens (clover),Medicago sativa (lucerne),Lens culinaris (lentil),Glycine max (soybean),Vigna sinensis (cowpea),Vigna radiata (mung bean),Vigna mungo (urd bean) andArachis hypogea (peanut). The results demonstrated that Rhizobium fromAeschynomene could form nodules only on its homologous host (Aeschynomene) but not on other legumes tested. Secondly, none of the rhizobia of other cross-inoculation groups could nodulateA. aspera.     

Hsp12p and PAU genes are involved in ecological interactions between natural yeast strains

The coexistence of different yeasts in a single vineyard raises the question on how they communicate and why slow growers are not competed out. Genetically modified laboratory strains of Saccharomyces cerevisiae are extensively used to investigate ecological interactions, but little is known about the genes regulating cooperation and competition in ecologically relevant settings. Here, we present evidences of Hsp12p‐dependent altruistic and contact‐dependent competitive interactions between two natural yeast isolates. Hsp12p is released during cell death for public benefit by a fast‐growing strain that also produces a killer toxin to inhibit growth of a slow grower that can enjoy the benefits of released Hsp12p. We also show that the protein Pau5p is essential in the defense against the killer effect. Our results demonstrate that the combined action of Hsp12p, Pau5p and a killer toxin is sufficient to steer a yeast community.     

Host/parasite relationships between tomato and pathogenic isolates of Verticillium

A close relationship was established between the virulence in the field of six isolates of Verticillium and their ability to penetrate and colonize sterile tomato seedlings grown in culture. The highly pathogenic species V. albo-atrum and V. tricorpus rapidly colonized host tissue in culture, host reactions being absent or only slight. V. nigrescens and V. nubilum, mild pathogens, penetrated sterile roots more slowly and caused host reactions. The variation in pathogenicity in the field between two isolates of V. dahliae suggests that they are different physiological strains, but they induced no difference during the first stages of invasion-reaction of sterile seedlings. Hyaline variants of all these isolates were less pathogenic than the original parent types. Variations in temperature from 25° C. (near optimum for the growth of both tomato plants and the fungi) caused changes in host reactions. Ability to penetrate was not affected within the pH range 4.0–8.0, but at extreme values (3.2, 9.4 and 10.0) all isolates entered without any host reaction. Variations in nitrogen supply to the pathogens induced modifications in their ability to penetrate, whereas changes in supply of nitrogen to the seedlings had no apparent effect upon host/parasite relations. The effects of simultaneous contact of non-pathogenic and pathogenic isolates with seedling roots suggested that resistance of host tissue was controlled by the growing tip.     

Chemotaxis of Rhizobium SP.S2 Towards Cajanus cajan Root Exudate and Its Major Components

The chemotactic response of Rhizobium sp. S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined. Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained. The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin. A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated. The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established. Received: 18 September 1998 / Accepted: 22 October 1998     

Ecology of Hup+ Rhizobium strains of cow pea miscellany: native frequency and competence

Rhizobium strains nodulating summer legumes cow pea [Vigna unguiculata (L.)], green gram [V. radiata (L.) (Wilczek)], black gram [V. mungo (L.) (Hepper)] and cluster bean [Cyamopsis tetragonoloba (L.) (Taub)] and a winter legume chick pea [Cicer arietinum (L.)] were surveyed in the Northern Plains of India and screened for hydrogenase activity to determine distribution of Hup character in the native ecosystem. It was observed that 56% of the Rhizobium strains of summer legumes were Hup⁺ whereas that of the winter legume, chick pea, were all Hup^-. Ex planta acetylene reduction activity was observed in most of the Hup⁺ but not in the Hup^- strains of any of the host species. In summer legume, mixed inoculation of Hup⁺ and Hup^- strains, under sterilized as well as unsterilized soil conditions, showed that the host species were predominantly nodulated with Hup⁺ strain.     

Functional expression of Escherichia coli fhuA gene in Rhizobium spp. of Cajanus cajan provides growth advantage in presence of Fe3+: ferrichrome as iron source

Cajanus cajan rhizobial isolates were found to be unable to utilize iron bound to ferrichrome, desferrioxamine B or rhodotorulic acid, all being hydroxamate type siderophores. A broad host range expression vector containing the Escherichia coli fhuA gene, encoding the outer membrane receptor for Fe-ferrichrome, was constructed. The plasmid construct (pGR1), designed to express fhuA under the lac promoter of E. coli, complemented E. coli MB97 ΔfhuA mutant for ferri-ferrichrome utilization and also allowed Rhizobium spp. ST1 and Rhizobium spp. IC3123 to grow using iron bound to ferrichrome. Sensitivity to the antibiotic albomycin, transported via the FhuA receptor, was found in case of MB97 as well as rhizobial transformants harboring pGR1. The rhizobial transformants expressing fhuA showed growth stimulation when co-inoculated with Ustilago maydis, a fungal species known to produce ferrichrome under iron starved conditions. Growth stimulation was also observed in the presence of externally supplied ferrichrome. The significance of these findings in terms of the potential for improving the survivability of rhizobial bioinoculant strains in natural soils is discussed.     

Effects of Salt and pH Stress on Temperature-Tolerant Rhizobium sp. NBRI330 Nodulating Prosopis juliflora

A study was conducted to examine the growth response of a rhizobial strain Rhizobium sp. NBRI330 isolated from root nodules of Prosopis juliflora growing in alkaline soil. The strain had the ability to nodulate P. juliflora. Nursery grown plants inoculated with Rhizobium sp. NBRI330 had 60.6% higher plant dry weight, as compared with uninoculated plants. The individual stress survival limit of a rhizobial strain Rhizobium sp. NBRI330 isolated from alkaline soil in a medium containing 32% (wt/vol) salt was 8 h, and at 55°C up to 3 h. The length of Rhizobium sp. NBRI330 in salt-stressed cells increased significantly to 3.04 μm from 1.75 μm of non-stressed control cells. On the contrary, the length of pH-stressed cells declined to 1.40 μm. Compared with non-stressed control rod-shaped cells, the shape of temperature-stressed cells changed to spherical, of 0.42 μm diameter. High temperature (45°C) was tolerated efficiently by Rhizobium sp. NBRI330 in the presence of salt at pH 12, as compared with pH 7. Received: 13 September 1999 / Accepted: 14 October 1999     

Rhizobium qilianshanense sp. nov., a novel species isolated from root nodule of Oxytropis ochrocephala Bunge in China

During a study of the diversity and phylogeny of rhizobia isolated from root nodules of Oxytropis ochrocephala grown in the northwest of China, four strains were classified in the genus Rhizobium on the basis of their 16S rRNA gene sequences. These strains have identical 16S rRNA gene sequences, which showed a mean similarity of 94.4 % with the most closely related species, Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have less than 88.1 and 88.7 % similarity with the defined species of Rhizobium, respectively. The genetic diversity revealed by ERIC-PCR fingerprinting indicated that the isolates correspond to different strains. Strain CCNWQLS01^T contains Q-10 as the predominant ubiquinone. The major fatty acids were identified as feature 8 (C18: 1ω7c and/or C18: 1ω6c; 67.2 %). Therefore, a novel species Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01^T (= ACCC 05747^T = JCM 18337^T) is designated as the type strain.