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Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

Tobacco (Nicotiana tabacum L.) bacterial wilt caused by Ralstonia solanacearum was widespread in recent years, resulting in a great damage in tobacco production. Biological control is an effective measurement for the bacterial wilt, however, related work has not been researched deeply. By screening the biocontrol microorganisms in Wenshan tobacco growing fields in Yunnan province, we discovered a Bacillus amyloliquefaciens strain Y4 and a Pseudomonas sp. strain Y8 that could significantly suppress the growth of R. solanacearum in the culture. The occurrence of wilt disease by field application of Y4 and/or Y8 was 3–4 folds lower than that of the untreatment at different tobacco growth stages. The microbial composition analysis showed that the relative abundance of the Enterobacteriaceae, Sphingomonadaceae, Caulobacteraceae, Sphingobacteriaceae, Flavobacteriacea, Streptomycetaceae, and Burkholderoaceae were significantly increased in the rhizosphere soils of infected tobacco. Moreover, field application of Y4 or Y8 significantly changed the rhizosphere soil microbiota. The relative abundance of the Lactobacillus was increased, however, that of R. solanacearum, Pseudoxanthomonas mexicana, Sphingomonas koreensis, Variovorax paradoxus, and Bradyrhizobium elkanii were decreased. Our results indicate that biocontrol microorganisms reduce plant disease occurrence by changing their microbiota in rhizosphere soil.

Nitrogen (N) and phosphorus (P) are nutrients that frequently limit plant performance and crop yield. Currently, awareness of the importance of plant-microbe interactions in plant nutrient acquisition and growth promotion persist, particularly under stressful conditions, such as those resulting from P scarcity. The aim of the present study was to evaluate the effects of P starvation on the influence of arbuscular mycorrhiza (AM) symbiosis on N 2 -fixing capacities, plant growth, nutrient uptake and photosynthetic performance of soybean plants at flowering and grain filling stages. To this end, soybean ( Glycine max ) plants were nodulated with Bradyrhizobium elkanii and inoculated or not with the AM fungus Glomus macrocarpum . The +AM and −AM plants were grown under low P conditions (50 μM), and a control treatment with −AM plants under sufficient P concentration (500 μM) was also performed (−AM + P). Plants were grown until the beginning of the flowering and another set till the grain filling stage. The results showed that mycorrhization improved soybean plant performance under P scarcity compared with non-AM associated plants under the same conditions. AM positively influenced growth and plant nodulation at both flowering and grain-filling stages, without significant changes in plant P content. AM symbiosis increased nitrogenase activity, P content in nodules and leaf N content, the maximum carboxylation rate of Rubisco and reduced the metabolic limitation of photosynthesis under P starvation, which indicated the AM stimulus to symbiotic N 2 fixation (SNF) and photosynthesis. Under these conditions, mycorrhizal symbiosis could not completely meet soybean P demand compared with well-nourished soybean plants, which produced higher plant and nodules biomass. In conclusion, AM association in nodulated soybean plants is likely an important strategy for maintaining SNF functioning, conferring increased plant growth and better N status under P scarcity conditions and significantly attenuating the negative effects of low Pi in nodulated-soybean plants.

Controlled-release phosphate carriers based on hydrocalumite-like Layered Double Hydroxides (Ca-Al-LDHs) have been synthesized to serve as a P-source for Bradyrhizobium elkanii growth. [Ca-Al]-LDHs loaded with different phosphate contents were prepared by structural reconstruction (SR) and ion exchange (IE) and characterized by powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), adsorption experiments, solid-state nuclear magnetic resonance (NMR) and thermal analysis (TG/DTG). [Ca-Al-PO 4 3 − ]-LDHs comprising similar crystalline phases were formed regardless the phosphate uptake route. However, phosphate loadings were found to be higher in [Ca-Al-PO 4 3 − ]-LDH obtained by IE rather than by SR. This was related to differences in specific surface area between precursors used in each route. [Ca-Al]-LDH IE-33.10 mM significantly increased the growth of Bradyrhizobium elkanii , a nitrogen-fixing symbiotic bacterium widely used in crop (mainly soybean) plantations. Once [Ca-Al-PO 4 3 − ]-LDHs are eco-friendly materials, they have enormous potential to be used in leguminous crops and raise agriculture production.

Flavonoids produced by roots are secondary metabolites involved in the induction of nod genes in rhizobial cells and nodule organogenesis. We describe the development and quality control of a new formulation for soybean (Glycine max (L.) Merr.) based on Bradyrhizobium elkanii cells grown in a culture medium containing isoflavones. As opposed to B. japonicum, the performance of B. elkanii pre-incubated with isoflavones is unknown. Its evaluation implied in vitro, greenhouse, and field research trials. Greenhouse and in vitro treatments comprised: seeds inoculated with a traditional inoculant based on B. elkanii; seeds inoculated with a new formulation based on isoflavones-cultivated B. elkanii cells, the exogenous application of isoflavones into the growth substrate, and the non-inoculated control. Field treatments included non-inoculated controls with and without N-fertilizer and seeds inoculation with the traditional or the new formulation. No changes in quality parameters (bacterial survival on both seeds and liquid formulation) were observed, as compared with the traditionally produced bradyrhizobial cells. Also, improved plant response (nodulation, plant growth, and seed yield) was observed when seeds were inoculated with the new formulation. The exogenous application of isoflavones directly into the substrate had deleterious effects on soybean growth and nodulation; however, the increased values of agronomical parameters in field conditions show that the addition of isoflavones during the industrial fermentation of B. elkanii is a better option over the common bradyrhizobial inoculant.

Nodulation symbiosis is a key component of natural ecosystems, and the nitrogen-fixing ability of such symbioses can have a significant impact on the restoration of degraded ecosystems. Here, we investigated the diversity of legume-rhizobium symbioses, compared the N2-fixing efficiency of legume-rhizobium symbioses, and clarified the relationship of rhizobia, host species, and microhabitat. Eighteen potential N2-fixing species were surveyed, and only nine of them were found to be nodulated. Most of wild legumes were found to be associated with rhizobia belonging to the genus Bradyrhizobium, except for Callerya nitida, which formed associations with the genus Mesorhizobium. δ15N signatures of Derris fordii were significantly closer to zero than all the other plants tested, suggesting that the D. fordii-ISA2507 (Bradyrhizobium elkanii) symbiosis might have the highest N2-fixing efficiency of all the associations. D. fordii and its rhizobial symbionts may have potential for application in ecological restoration and agricultural production. Host species selection was the major factor, and there was no significant effect of slope position on nodulation-legume symbioses in our study area, in which altitudes ranged from 287 m to 521 m at the small scale. The Shannon diversity and the evenness of N2-fixing species (nodulated legumes) were significantly positively correlated (P

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.

The 15N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N2 fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the 15N abundance of the N derived from N2 fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched 15N ammonium sulphate was added. The %Ndfa was then used with the 15N natural abundance data of the nodulated soybean and non-N2-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘Bs’) was higher. The differences in ‘Bs’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.

Polyphasic characterization of 54 indigenous mung bean (Vigna radiata L.) rhizobia from different geographic regions of China was determined by analyzing the variability of 16S rRNA gene RFLP, 16S–23S rRNA gene Intergenetic Spacer (IGS) RFLP, G-C rich RAPD and phenotype assays. Based on these characteristics, mung bean rhizobia were clustered into four groups. Group I comprised 16 slow-growing isolates from a variety of geographic regions. This group was genetically distinct from Bradyrhizobium japonicum and Bradyrhizobium liaoningense, and may represent a new species. Group II was composed of 18 isolates, which could be sub-divided into two sub-groups that were respectively related to B. japonicum and B. liaoningense. Group III comprised 12 isolates from South China and clustered together with Bradyrhizobium elkanii. Group IV formed a miscellany of 8 fast-growing isolates variously related to the genera Sinorhizobium, Rhizobium and Mesorhizobium.

Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process by inhibiting ACC (1-aminocyclopropane-1-carboxylate) synthase in the ethylene biosynthesis of host roots. B. elkanii rtxA and rtxC genes are required for rhizobitoxine production. In particular, rtxC gene is involved in the desaturation of dihydrorhizobitoxine into rhizobitoxine. A legume with a mutated ethylene receptor gene produced markedly higher numbers of rhizobial infection threads and nodule primordia. Thus, endogenous ethylene in legume roots negatively regulates the formation of nodule primordia, which is overcome by rhiozbitoxine. Although a plant pathogen Burkholderia andropogonis has been known to produce rhizobitoxine, the genome sequence of Xanthomonas oryzae showed the existence of a putative rhizobitoxine transposon in the genome. The cumulative evidence suggests that rhizobitoxine-producing bacteria modulate plant-microbe interactions via ethylene in the rhizosphere and phyllosphere environments. In addition, rhizobitoxine-producing capability might be utilized as tools in agriculture and biotechnology.

Biosolubilization of rock phosphate (RP) using a Penicillium spp ., an Aspergillus spp ., Pleurotus ostreatus , Bradyrhizobium elkanii SEMIA 5019 and their fungal–rhizobial biofilms was investigated. Eppawala Rock Phosphate (ERP, total P concentration 17.6%), a RP from a deposit in Sri Lanka was used. Penicillium spp.– B elkanii SEMIA 5019 biofilm released the highest amount of P from the ERP with the highest P release-to-P uptake ratio. The P release of Penicillium spp. alone was significantly lower than that of its biofilm. Similarly, P. ostreatus – B. elkanii SEMIA 5019 biofilm showed a higher P release than P. ostreatus alone. However, P. ostreatus alone or its biofilm showed lower P release-to-P uptake ratios indicating relatively higher P uptake compared to the P release. The Aspergillus spp ., showed a moderate P release. Large bradyrhizobial cell clusters attached to the mycelial mat of Penicillium spp. and P. ostreatus were observed under light microscope after 12 and 25 days of incubation, respectively. The present study, identified an effective method of fungal–rhizobial biofilm mediated solubilization of RP.

Four decades of soybean [Glycine max (L.) Merr.] cultivation in South Africa has resulted in the establishment of populations of bradyrhizobia against which the recently introduced inoculant strain CB 1809 must compete. Serological and DNA fingerprinting methods were used to study the diversity of nodule isolates from soils at Bergville, Koedoeskop and Morgenzon. Dominant serogroups included Bradyrhizobium elkanii serotype 76 at Bergville (67%), Bradyrhizobium japonicum serotype 123 at Morgenzon (81%) and B. japonicum serotype 135 at Koedoeskop (100%). Their origin is unknown as they do not correspond in serotype to strains used in previous inoculants. A small percentage of isolates from Bergville (13%) and Morgenzon (16%) were serologically homologous to strain WB 1 (serotype 31/76), applied for two decades before CB 1809 (serotype 122). Nitrogen-fixing effectiveness of CB 1809 was superior to 60% of the isolates tested from Bergville and Morgenzon, but similar to 73% of the Koedoeskop isolates. Seed and liquid-in-furrow application methods increased CB 1809 nodule occupancy at least three-fold above background levels at Bergville (pH 5.16) and Morgenzon (pH 6.33). Inoculation did not, however, increase CB 1809 nodule occupancy at Koedoeskop (pH 7.76), possibly because alkaline soil conditions favoured the serotype 135 population predominant at this site.

Bacteria were isolated from root-nodules collected from indigenous legumes at 38 separate locations in the Gascoyne and Pilbara regions of Western Australia. Authentication of cultures resulted in 31 being ascribed status as root-nodule bacteria based upon their nodulation of at least one of eight indigenous legume species. The authenticated isolates originated from eight legume genera from 19 sites. Isolates were characterised on the basis of their growth and physiology; 20 isolates were fast-growing and 11 were slow-growing (visible growth within 3 and 7d, respectively). Fast-growers were isolated from Acacia, Isotropis, Lotus and Swainsona, whilst slow-growers were from Muelleranthus, Rhynchosia and Tephrosia. Indigofera produced one fast-growing isolate and seven slow-growing isolates. Three indigenous legumes (Swainsona formosa, Swainsona maccullochiana and Swainsona pterostylis) nodulated with fast-growing isolates and four species (Acacia saligna, Indigofera brevidens, Kennedia coccinea and Kennedia prorepens) nodulated with both fast- and slow-growing isolates. Swainsona kingii did not form nodules with any isolates. Fast-growing isolates were predominantly acid-sensitive, alkaline- and salt-tolerant. All slow-growing isolates grew well at pH 9.0 whilst more than half grew at pH 5.0, but all were salt-sensitive. All isolates were able to grow at 37deg;C. The fast-growing isolates utilised disaccharides, whereas the slow-growing isolates did not. Symbiotic interactions of the isolates were assessed on three annual, one biennial and nine perennial exotic legume species that have agricultural use, or potential use, in southern Australia. Argyrolobium uniflorum, Chamaecytisus proliferus, Macroptilium atropurpureum, Ononis natrix, Phaseolus vulgaris and Sutherlandia microphylla nodulated with one or more of the authenticated isolates. Hedysarum coronarium, Medicago sativa, Ornithopus sativus, Ornithopus compressus, Trifolium burchellianum, Trifolium polymorphum and Trifolium uniflorum did not form nodules. Investigation of the 31 authenticated isolates by polymerase chain reaction with three primers resulted in the RPO1 primer distinguishing 20 separate banding patterns, while ERIC and PucFor primers distinguished 26 separate banding patterns. Sequencing the 16S rRNA gene for four fast- and two slow-growing isolates produced the following phylogenetic associations; WSM1701 and WSM1715 (isolated from Lotus cruentus and S. pterostylis, respectively) displayed 99% homology with Sinorhizobium meliloti, WSM1707 and WSM1721 (isolated from Sinorhizobium leeana and Indigofera sp., respectively) displayed 99% homology with Sinorhizobium terangae, WSM1704 (isolated from Tephrosia gardneri) shared 99% sequence homology with Bradyrhizobium elkanii, and WSM1743 (isolated from Indigofera sp.) displayed 99% homology with Bradyrhizobium japonicum.

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the 15N natural abundance technique, it is necessary to determine the 15N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N2-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with 15N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the 15N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ15N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (Bs) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (Bwp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.

Natural genetic diversity and symbiotic status were studied among 41 root nodule bacteria from ecologically diverse sites in Kenya having no history of growing domesticated legumes, but where species of Acacia and Sesbania sesban grow naturally. Isolates originated either from nodules of field plants (trees and shrubs) or from trap host plants grown in field-collected soil. The host plants were Acacia mellifera , Acacia nilotica , Acacia nubica , Acacia polyacantha , Acacia seyal , Acacia tortilis , Acacia xanthophloea , and Faidherbia (Syn. Acacia ) albida , Prosopis juliflora , S. sesban , Macroptilium atropurpureum , Phaseolus vulgaris and Vigna unguiculata . Two molecular methods were used to characterize these strains; PCR–RFLP of the 16S rRNA gene (12 distinct genotypes) and sequence analysis of a 230 bp fragment of this gene (10 genotypes) with the former showing better discriminatory power. The two methods were in good agreement in delineating five bacterial genera: Agrobacterium , Bradyrhizobium , Mesorhizobium , Rhizobium and Sinorhizobium . Bradyrhizobium strains were all alkali-producing, exhibiting very slow to intermediate growth; Mesorhizobium strains were acid-producing with intermediate to fast growth; Rhizobium and Sinorhizobium were acid-producing with fast to very fast growth, and Agrobacterium strains were acid-producing and showed very fast growth. PCR–RFLP analysis further revealed strains corresponding to the species Bradyrhizobium elkanii , Bradyrhizobium japonicum , Rhizobium leguminosarum and Rhizobium tropici IIB, as well as others characterized to genus level only. Apparent diversity varied from site to site and was greatly influenced by host of isolation. For example, five rhizobial genotypes (four genera) were found using five different trap host species in a single Acacia tortilis woodland site, thus reaffirming occurrence of a heterogeneous population regardless of their nodulating ability with the native legume species. Bradyrhizobium spp. ( F. albida ), known for their preference of F. albida in tropical African soils, were abundant and widespread mainly because F. albida was the most frequently used trap host. Twenty Kenyan and eight reference strains were tested on tree species that are a priority for Kenyan use; these showed considerable diversity in symbiotic characters. Whereas Acacia species and F. albida showed variation in symbiotic effectiveness with test strains, S. sesban nodulated most effectively only with its homologous strains. Agrobacterium -allied strains failed to nodulate any of the host species tested. The high diversity (rhizobial genotypes, host affinities and their symbiotic effectiveness) found in the Kenyan sites will require rigorous selection for host×strain×site combinations in order to obtain effective and competitive inoculants for legume trees and crops in rehabilitation and agroforestry programmes.

Summary A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus erinaceus 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 Mesorhizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrhizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrhizobium 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.

Symbiotic efficiency of soybean Glycine max L. cv. Doko nodulated by BR-33 (CB 1809) strain of Bradyrhizobium japonicum or BR-29 (29W) strain of B. elkanii was investigated under two light intensities: full sunlight or 30% of full sunlight. Plants were harvested during pod-filling and at maturity. Soybeans cultivated under full sunlight and inoculated with strain BR-33 showed higher shoot dry mass and nitrogen accumulation, nitrogenase activity, ureide-N in the xylem sap and grain yield than plants inoculated with strain BR-29, confirming the superior symbiotic effectiveness of strain BR-33. In contrast, soybean submitted to shading treatment showed no significant differences regardless of the strain used as inoculant. When submitted to shading, plants inoculated with BR-29 strain showed an increase in most of the variables observed while plants inoculated with strain BR-33 were not significantly affected by the level of shading. The results suggest that strain efficiency used as an inoculant for soybean can be different in plants cultivated either in the greenhouse or in the field. This can help to explain why the potential of hydrogenase activity to improve symbiotic effectiveness is still a matter of controversy when comparing results from different experiments.

Bacterial strains isolated from root nodules on Acacia mangium at various locations in Indonesia were characterized by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA and by an intergenic repetitive extragenic palindromic (REP) sequence. The RFLP-16S rDNA analysis used four restriction endonucleases classified into seven Sau3AI, eight TaqI, nine HinfI, and nine MspI groups. Analysis of the amplified REP-PCR products showed intra-specific variations in the rhizobial genomic DNA. From the data obtained, 10 of the 15 strains isolated from A. mangium were classified as Bradyrhizobium. These isolates clustered with B. elkanii, and were predominant in the A. mangium nodulation systems. Partial 16S rDNA sequence analysis of some representative strains demonstrated that the RFLP clusters were stable and phylogenetically relevant.

Certain strains of Bradyrhizobium japonicum and B. elkanii produce a polysaccharide within the root nodules of their legume host, soybean. These nodule polysaccharides (NPSs) were isolated and characterized. The NPS produced by B. elkanii strains proved to be identical in glycosyl composition and linkages to the extracellular polysaccharide (EPS) of this species indicating that the NPS and EPS for B. elkanii have identical structures (W.F. Dudman, Carbohydr. Res., 66 (1978) 9-23), [formula: see text] However, the structure of the NPS from B. japonicum proved to be quite different from that of its EPS. Methylation analysis of this NPS showed that it consists of 3-linked Gal, 3-linked Rha, 2,4-linked Rha, 4-linked Rha, and terminal 2-O-methyl GlcA in a 1:1:1:1:1 ratio. Stereochemical configurations of the glycosyl residues were determined by the preparation and analysis of trimethylsilyl (Me3Si) (-)-2-butyl glycosides. NMR spectroscopy (both 1H and 13C) showed that the Gal residue is alpha-linked, while all the other glycosyl residues are beta-linked. Oligosaccharides produced by periodate oxidation-Smith degradation were purified, as were oligosaccharides produced by partial acid hydrolysis. Characterization of the Smith degradation products by methylation analysis. NMR spectroscopy, electrospray-mass spectrometry, and characterization of the partial acid hydrolysate oligosaccharides showed that the repeating oligosaccharide unit of the NPS has the structure, [formula: see text]