Bradyrhizobium Liaoningense Research Articles

Ameliorating the drought stress tolerance of a susceptible soybean cultivar, MAUS 2 through dual inoculation with selected rhizobia and AM fungus

BackgroundDrought stress is currently the primary abiotic stress factor for crop loss worldwide. Although drought stress reduces the crop yield significantly, species and genotypes differ in their stress response; some tolerate the stress effect while others not. In several systems, it has been shown that, some of the beneficial soil microbes ameliorate the stress effect and thereby, minimizing yield losses under stress conditions. Realizing the importance of beneficial soil microbes, a field experiment was conducted to study the effect of selected microbial inoculants namely, N-fixing bacteria, Bradyrhizobium liaoningense and P-supplying arbuscular mycorrhizal fungus, Ambispora leptoticha on growth and performance of a drought susceptible and high yielding soybean cultivar, MAUS 2 under drought condition.ResultsDrought stress imposed during flowering and pod filling stages showed that, dual inoculation consisting of B. liaoningense and A. leptoticha improved the physiological and biometric characteristics including nutrient uptake and yield under drought conditions. Inoculated plants showed an increased number of pods and pod weight per plant by 19% and 34% respectively, while the number of seeds and seed weight per plant increased by 17% and 32% respectively over un-inoculated plants under drought stress condition. Further, the inoculated plants showed higher chlorophyll and osmolyte content, higher detoxifying enzyme activity, and higher cell viability because of less membrane damage compared to un-inoculated plants under stress condition. In addition, they also showed higher water use efficiency coupled with more nutrients accumulation besides exhibiting higher load of beneficial microbes.ConclusionDual inoculation of soybean plants with beneficial microbes would alleviate the drought stress effects, thereby allowing normal plants’ growth under stress condition. The study therefore, infers that AM fungal and rhizobia inoculation seems to be necessary when soybean is to be cultivated under drought or water limiting conditions.

Exploring potential soybean bradyrhizobia from high trehalose-accumulating soybean genotypes for improved symbiotic effectiveness in soybean.

Drought is the most important factor limiting the activity of rhizobia during N-fixation and plant growth. In the present study, we isolated Bradyrhizobium spp. from root nodules of higher trehalose-accumulating soybean genotypes and examined for moisture stress tolerance on a gradient of polyethylene glycol (PEG 6000) amended in yeast extract mannitol (YEM) broth. In addition, the bradyrhizobial strains were also evaluated for symbiotic effectiveness on soybean. Based on 16S rDNA gene sequences, four bradyrhizobial species were recovered from high trehalose-accumulating genotypes, i.e., two Bradyrhizobium liaoningense strains (accession number KX230053, KX230054) from EC 538828 and PK-472, respectively, one Bradyrhizobium daqingense (accession number KX230052) from PK-472, and one Bradyrhizobium kavangense (accession number MN197775) from Valder genotype having low trehalose. These strains, along with two native strains, viz., Bradyrhizobium japonicum (JF792425), Bradyrhizobium liaoningense (JF792426), and one commercial rhizobium, were studied for nodulation, leghaemoglobin, and N-fixation abilities on soybean under sterilized sand microcosm conditions in a completely randomized design. Among all the strains, D-4A (B. daqingense) followed by D-4B (B. liaoningense) was found to have significantly higher nodulation traits and acetylene reduction assay (ARA) activity when compared to other strains and commercial rhizobia. The bradyrhizobia isolates showed plant growth promotion traits such as indole acetic acid (IAA), exopolysaccharide (EPS), and siderophore production, phosphate-solubilizing potential, and proline accumulation. The novel species B. daqingense was reported for the first time from Indian soil and observed to be a potential candidate strain and should be evaluated for conferring drought tolerance in soybean under simulated stress conditions.

Unique Rhizobial Communities Dominated by Bradyrhizobium liaoningense and Bradyrhizobium ottawaense were Found in Vegetable Soybean Nodules in Osaka Prefecture, Japan.

Vegetable soybean (Glycine max [L.]) is mainly consumed in Asian countries, but has recently attracted attention worldwide due to its high nutritional value. We aimed to identify the indigenous rhizobia of vegetable soybean in Yao City, Osaka Prefecture, Japan, and to clarify the relationships between the rhizobial community and soil environmental factors. Soil samples were collected from 12 vegetable soybean cultivation fields under two different conditions (six greenhouses and six open fields) in Yao City with different varieties of vegetable soybean. A total of 217 isolates were obtained from the nodules and clustered into nine operational taxonomic units (OTUs) with 97% homology based on the 16S-23S rRNA internal transcribed spacer (ITS) region. A phylogenetic ana-lysis showed that OTUs were closely related to Bradyrhizobium liaoningense, B. ottawaense, B. elkanii, and other Bradyrhizobium species and were dominant in this order. B. liaoningense was widely found in sampled sites and accounted for 50.7% of all isolates, while B. ottawaense was mostly limited to open fields. This rhizobial community differed from Japanese soybean rhizobia, in which B. diazoefficiens, B. japonicum, and B. elkanii were dominant. These results imply the characteristic differences among host plants or regional specialties. A non-metric multidimensional scaling (NMDS) ana-lysis revealed the significant impact of soil pH and the contents of Ca, Mg, Mn, total nitrogen (TN), and total carbon (TC) on the distribution of rhizobia. B. liaoningense was detected in soils with a neutral pH, and high TN and low Mn contents increased its abundance. The present study provides novel insights into Japanese rhizobia and potentially novel resources for sustainable agriculture.

Genetic and Physiological Characterization of Soybean-Nodule-Derived Isolates from Bangladeshi Soils Revealed Diverse Array of Bacteria with Potential Bradyrhizobia for Biofertilizers.

Genetic and physiological characterization of bacteria derived from nodules of leguminous plants in the exploration of biofertilizer is of paramount importance from agricultural and environmental perspectives. Phylogenetic analysis of the 16S rRNA gene of 84 isolates derived from Bangladeshi soils revealed an unpredictably diverse array of nodule-forming and endosymbiotic bacteria-mostly belonging to the genus Bradyrhizobium. A sequence analysis of the symbiotic genes (nifH and nodD1) revealed similarities with the 16S rRNA gene tree, with few discrepancies. A phylogenetic analysis of the partial rrn operon (16S-ITS-23S) and multi-locus sequence analysis of atpD, glnII, and gyrB identified that the Bradyrhizobium isolates belonged to Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense species. In the pot experiment, several isolates showed better activity than B. diazoefficiens USDA110, and the Bho-P2-B2-S1-51 isolate of B. liaoningense showed significantly higher acetylene reduction activity in both Glycine max cv. Enrei and Binasoybean-3 varieties and biomass production increased by 9% in the Binasoybean-3 variety. Tha-P2-B1-S1-68 isolate of B. diazoefficiens significantly enhanced shoot length and induced 10% biomass production in Binasoybean-3. These isolates grew at 1-4% NaCl concentration and pH 4.5-10 and survived at 45 °C, making the isolates potential candidates for eco-friendly soybean biofertilizers in salty and tropical regions.

EFFECT OF INOCULATING Bradyrhizobium liaoningense ON GROWTH, CHLOROPHYLL CONCENTRATION, NODULATION AND YIELD OF SOYA BEANS

Soya bean is important staple food crops in Western Kenya. Legumes play a significant role in agriculture by fixing nitrogen. Currently Kenya is experiencing low yield of soya beans. Approximately 0.8 t/ha is under production and this is due to low soil fertility. This has posed a threat to agricultural productivity. Symbiotic rhizobia inoculation increases crop yields through biological nitrogen fixation. However, the potential of Bradyrhizobium liaoningense in improving yield and productivity of soya beans has not been documented. Rhizobia bacteria are known to improve growth and yield of several other crops but the effect of inoculating soya beans with B. liaoningense on growth, chlorophyll concentration, nodulation and yields has not been determined. This study was conducted at Maseno University under greenhouse conditions. Previously purified local isolates of B. liaoningense (S3) from wild soya beans plants were used to inoculate the soya bean seeds. 4.5 litre plastic pots were filled with 7kg of top soil. Ten seeds of Soya beans coated with Bradyrhizobium liaoningense inoculants were sown in each pot except the control. The seeds were treated with the isolates inoculants as: un-inoculated (control), 1.07x107 cfu/ml, 1.19x107 cfu/ml, 1.31x107 cfu/ml and 2.67x107 cfu/ml inoculation of B. liaoningense. The experiment was laid out as Completely Randomised Design (CRD). The treatments were replicated three times. After two weeks of germination, the seedlings were thinned to three plants per pot. Watering was done daily with 200 ml of water per pot up to the end of the experiment. Data on plant height, number of leaves, leaf area, shoot and root fresh weight and dry weight, chlorophyll concentration, number of nodules and number of pods were determined. Data was subjected to Analysis of Variance (ANOVA). Treatments means were separated using Fisher’s Least Significant Difference at (P = 0.05). Soya bean growth, chlorophyll concentration, number of nodules and number of pods after inoculation treatments were significantly different among the treatments (P ≤ 0.05). Plant height, number of leaves and leaf area were highest in 2.67 x 107 cfu/ml of B. liaoningense inoculation followed by 1.31x 107 cfu/ml inoculation, 1.19x107 cfu/ml, 1.07x107 cfu/ml and lowest in un-inoculated. Inoculation at 2.67 x 107 cfu/ml significantly increased soya bean root and shoot fresh weight and dry weights. Chlorophyll concentration was found to be highest in 2.67 x 107 cfu/ml treatment and lowest in un-inoculated control treatment. Inoculation at 2.67 x 107 cfu/ml increased the number of nodules of soya beans. Number of pod per plant were highest at 2.67 x 107 cfu/ml treatment and lowest in un-inoculated (control treatment). These findings showed that B. liaoningense inoculation was effective in enhancing growth, chlorophyll synthesis, formation of root nodules and pods in soya beans. From the study it can be recommended that B. liaoningense from wild soya bean may be used as a biofertiliser to improve productivity of soya beans by smallholder farmers in Kenya.

Dual inoculation with rhizobia and arbuscular mycorrhizal fungus improves water stress tolerance and productivity in soybean

A glasshouse experiment was conducted to understand the influence of rhizobia, Bradyrhizobium liaoningense and an arbuscular mycorrhizal fungus, Ambispora leptoticha on growth and yield performance of soybean cultivars, MAUS 2 and MAUS 212 (drought susceptible) and DSR 12 (drought tolerant) under different levels of moisture stress conditions. Three different water levels [100%, 75% and 50% FC] with three microbial inocula (B. liaoningense, A. leptoticha and B. liaoningense + A. leptoticha) served as main treatments and the water level treatments were imposed 31–45 days after sowing. Various growth, physiological and microbial parameters were recorded besides analysing the samples for nutrients. The results showed that the plant growth parameters like biovolume index, total dry biomass, root volume and seed yield were significantly higher in dual inoculated plants in all the cultivars at all the moisture levels. There was an increase in mean seed weight by 88% and dry biomass by 58% in dual inoculated plants compared to uninoculated plants. Further physiological parameters and nutrients level improved significantly with dual inoculation under stress condition. However significant difference in recorded parameters was not observed in plants grown at 100% and 75% FC suggesting dual inoculation of B. liaoningense + A. leptoticha is beneficial when stress is severe in all the 3 cultivars. The microbial and nutrient parameters followed the same trend. Further of the two drought susceptible cultivars, MAUS 2 performed better compared to MAUS 212 in all the studied parameters under stress with dual inoculation. The results therefore signify the importance of rhizobia and mycorrhizal inoculation for imparting stress tolerance in soybean.

Exploring apoplast reorganization in the nodules of Lotus corniculatus L. growing on old Zn–Pb calamine wastes

Nodulation and symbiotic nitrogen fixation are important factors that determine legume growth. A pot experiment was carried out to determine the effects of Zn–Pb contamination on nodule apoplast (cell walls and intercellular spaces) of bird's foot trefoil (Lotus corniculatus L.) that spontaneously colonized old calamine wastes. The plants were grown in pots filled with sterile calamine substrate (M, metal treated) or expanded clay (NM, untreated) and inoculated with calamine-derived Lotus-nodulating Bradyrhizobium liaoningense. Apoplast reorganization in the nodules was examined using specific dyes for cellulose, pectin and lignin detection, and immuno-histochemical techniques based on monoclonal antibodies against xyloglucan (Lm25), pectins (Jim5 and Jim7), and structural proteins (arabinogalactan protein - Lm14 and extensin - Jim12). Microscopic analysis of metal-treated nodules revealed changes in the apoplast structure and composition of nodule cortex tissues and infected cells. Wall thickening was accompanied by intensified deposition of cellulose, xyloglucan, esterified pectin, arabinogalactan protein and extensin. The metal presence redirected also lignin and suberin deposition in the walls of the nodule cortex tissues.Our results showed reorganization of the apoplast of cortex tissues and infected cells of Lotus nodules under Zn–Pb presence. These changes in the apoplast structure and composition may have created actual barriers for the toxic ions. For this reason, they can be regarded as an element of legume defense strategy against metal stress that enables effective functioning of L. corniculatus-rhizobia symbiosis on Zn–Pb polluted calamine tailings.

Bacterial Diversity in a Sri Lankan Geothermal Spring Assessed by Culture-Dependent and Culture-IndependentApproaches.

Hot springs harbour diverse and interesting groups of microorganisms adapted to extreme conditions. However, due to limitations in the culture-dependent approach, most of such thermophiles remain uncultured and unexplored. Hence, this study was conducted to gain a comprehensive understanding of the bacterial diversity of Mahapelessa hot spring, Sri Lanka using both culture-dependent and culture-independent approaches. Thein situtemperature of the water sample was 44.5°C and thepH was 8.14. 16S rRNA Sanger sequencing of DNA extracted from the 18 bacterial isolates revealed the presence of eight genera belonging to two phyla: Proteobacteria (84%) and Firmicutes (16%) and the most abundant genus being Klebsiella. A total of 23 bacterial phyla representing 80 classes, 43 orders, 123 families, 205 genera and 83 species were detected by 16S rRNA V3-V4 region by amplicon metagenome sequencing of DNA extracted from water samples, where the most abundant phylum was the Proteobacteria (57.39%), followed by Firmicutes (23.7%) and Chloroflexi (4.14%). The three phyla Actinobacteria, Planctomycetes and Bacteroidetes were also detected less than 3% in abundance while 4.48% of bacteria could not be fit into any known phylum. The most abundant genera were Burkholderia (14.87%), Desulfotomaculum (7.23%) and Stenotrophomonas (6.1%). Four strictly anaerobic bacteria, Anaerosolibacter carboniphilus (0.71%), Bellilinea caldifistulae (0.04%), Salimesophilobacter vulgaris (0.1%), Anaerobacterium chartisolvens (0.12%); two potential plant growth-promoting bacteria, Azospirillum halopraeferens (0.04%) and Bradyrhizobium liaoningense (0.16%) and one potential alkali tolerant and sulphate-reducing bacterium,Desulfovibrio alkalitolerans (0.45%) were recorded. Pigmentiphaga sp. was isolated from Mahapelessa hot spring and to the best of our knowledge, this is the first record of this genus from a hot spring. This study gives insight into the vast bacterial diversity present in the Mahapelessa hot spring from the culture-independent approach which could not be identified using standard culturing techniques.

Nitrogen-fixing rhizobial strains isolated from Desmodium incanum DC in Argentina: Phylogeny, biodiversity and symbiotic ability

Desmodium spp. are leguminous plants belonging to the tribe Desmodieae of the subfamily Papilionoideae. They are widely distributed in temperated and subtropical regions and are used as forage plants, for biological control, and in traditional folk medicine. The genus includes pioneer species that resist the xerothermic environment and grow in arid, barren sites. Desmodium species that form nitrogen-fixing symbiosis with rhizobia play an important role in sustainable agriculture. In Argentina, 23 native species of this genus have been found, including Desmodium incanum. In this study, a total of 64 D. incanum-nodulating rhizobia were obtained from root nodules of four Argentinean plant populations. Rhizobia showed different abiotic-stress tolerances and a remarkable genetic diversity using PCR fingerprinting, with more than 30 different amplification profiles. None of the isolates were found at more than one site, thus indicating a high level of rhizobial diversity associated with D. incanum in Argentinean soils. In selected isolates, 16S rDNA sequencing and whole-cell extract MALDI TOF analysis revealed the presence of isolates related to Bradyrhizobium elkanii, Bradyrhizobium japonicum, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense, Bradyrhizobium denitrificans and Rhizobium tropici species. In addition, the nodC gene studied in the selected isolates showed different allelic variants.Isolates were phenotypically characterized by assaying their growth under different abiotic stresses. Some of the local isolates were remarkably tolerant to high temperatures, extreme pH and salinity, which are all stressors commonly found in Argentinean soils. One of the isolates showed high tolerance to temperature and extreme pH, and produced higher aerial plant dry weights compared to other inoculated treatments. These results indicated that local isolates could be efficiently used for D. incanum inoculation.

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48T and B. liaoningense USDA 3622T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.

Genetic Characterization of Soybean Rhizobia Isolated from Different Ecological Zones in North-Eastern Afghanistan.

Seventy rhizobial isolates were obtained from the root nodules of two soybean (Glycine max) cultivars: Japanese cultivar Enrei and USA cultivar Stine3300, which were inoculated with different soil samples from Afghanistan. In order to study the genetic properties of the isolates, the DNA sequences of the 16S rRNA gene and symbiotic genes (nodD1 and nifD) were elucidated. Furthermore, the isolates were inoculated into the roots of two soybean cultivars, and root nodule numbers and nitrogen fixation abilities were subsequently evaluated in order to assess symbiotic performance. Based on 16S rRNA gene sequences, the Afghanistan isolates obtained from soybean root nodules were classified into two genera, Bradyrhizobium and Ensifer. Bradyrhizobium isolates accounted for 54.3% (38) of the isolates, and these isolates had a close relationship with Bradyrhizobium liaoningense and B. yuanmingense. Five out of the 38 Bradyrhizobium isolates showed a novel lineage for B. liaoningense and B. yuanmingense. Thirty-two out of the 70 isolates were identified as Ensifer fredii. An Ensifer isolate had identical nodD1 and nifD sequences to those in B. yuanmingense. This result indicated that the horizontal gene transfer of symbiotic genes occurred from Bradyrhizobium to Ensifer in Afghanistan soil. The symbiotic performance of the 14 tested isolates from the root nodules of the two soybean cultivars indicated that Bradyrhizobium isolates exhibited stronger acetylene reduction activities than Ensifer isolates. This is the first study to genetically characterize soybean-nodulating rhizobia in Afghanistan soil.

Pongamia pinnata inoculated with Bradyrhizobium liaoningense PZHK1 shows potential for phytoremediation of mine tailings.

Mine tailings contain high concentrations of metal contaminants and only little nutrients, making the tailings barren for decades after the mining has been terminated. Effective phytoremediation of mine tailings calls for deep-rooted, metal accumulating, and soil fertility increasing plants with tolerance against harsh environmental conditions. We assessed the potential of the biofuel leguminous tree Pongamia pinnata inoculated with plant growth promoting rhizobia to remediate iron-vanadium-titanium oxide (V-Ti magnetite) mine tailing soil by pot experiment and in situ remediation test. A metal tolerant rhizobia strain PZHK1 was isolated from the tailing soil and identified as Bradyrhizobium liaoningense by phylogenetic analysis. Inoculation with PZHK1 increased the growth of P. pinnata both in V-Ti magnetite mine tailings and in Ni-contaminated soil. Furthermore, inoculation increased the metal accumulation capacity and superoxide dismutase activity of P. pinnata. The concentrations of Ni accumulated by inoculated plants were higher than the hyperaccumulator threshold. Inoculated P. pinnata accumulated high concentration of Fe, far exceeding the upper limit (1000mgkg-1) of Fe in plant tissue. In summary, P. pinnata-B. liaoningense PZHK1 symbiosis showed potential to be applied as an effective phytoremediation technology for mine tailings and to produce biofuel feedstock on the marginal land.

Functional characterization of a csoR-cueA divergon in Bradyrhizobium liaoningense CCNWSX0360, involved in copper, zinc and cadmium cotolerance.

Random mutagenesis in a symbiotic nitrogen-fixing Bradyrhizobium liaoningense CCNWSX0360 (Bln0360) using Tn5 identified five copper (Cu) resistance-related genes. They were functionally sorted into three groups: transmembrane transport (cueA and tolC); oxidation (copA); and protection of the membrane barrier (lptE and ctpA). The gene cueA, together with the upstream csoR (Cu-sensitive operon repressor), constituted a csoR-cueA divergon which plays a crucial role in Cu homeostasis. Deletion of cueA decreased the Cu tolerance of cells, and complementation of this mutant restored comparable Cu resistance to that of the wild-type. Transcriptional and fusion expression analysis demonstrated that csoR-cueA divergon was up-regulated by both the monovalent Cu+ and divalent Zn2+/Cd2+, and negatively regulated by transcriptional repressor CsoR, via a bidirectional promoter. Deletion of csoR renders the cell hyper-resistant to Cu, Zn and Cd. Although predicted to encode a Cu transporting P-type ATPase (CueA), cueA also conferred resistance to zinc and cadmium; two putative N-MBDs (N-terminal metal binding domains) of CueA were required for the Cu/Zn/Cd tolerance. Moreover, cueA is needed for nodulation competitiveness of B. liaoningense in Cu rich conditions. Together, the results demonstrated a crucial role for the csoR-cueA divergon as a component of the multiple-metal resistance machinery in B. liaoningense.

Inoculation with Native Bradyrhizobia Strains Improved Growth of Cowpea Plants Cultivated on a Saline Soil

ABSTRACTSelection of osmotolerant rhizobia gained importance because of increasing soil salinity in many regions. A field experiment in the Cauto Valley was conducted to determine the effect of seed inoculation with two native strains of Bradyrhizobium sp. on biological nitrogen (N)-fixation of cowpea (Vigna unguiculata) on a salt affected soil. Inoculation with both strains resulted in higher numbers of nodules, specific N-fixation, and yields of cowpeas than the control without inoculation. The strain VIBA-1 (Bradyrhizobium liaoningense) was partly superior to VIBA-2 (Bradyrhizobium yuanmingense). This study indicates that the strains inoculated are competitive against other natural strains and able to nodulate cowpea.

Nodulation Characterization and Proteomic Profiling of Bradyrhizobium liaoningense CCBAU05525 in Response to Water-Soluble Humic Materials.

The lignite biodegradation procedure to produce water-soluble humic materials (WSHM) with a Penicillium stain was established by previous studies in our laboratory. This study researched the effects of WSHM on the growth of Bradyrhizobium liaoningense CCBAU05525 and its nodulation on soybean. Results showed that WSHM enhanced the cell density of CCBAU05525 in culture, and increased the nodule number, nodule fresh weight and nitrogenase activity of the inoculated soybean plants. Then the chemical compounds of WSHM were analyzed and flavonoid analogues were identified in WSHM through tetramethyl ammonium hydroxide (TMAH)-py-GC/MS analysis. Protein expression profiles and nod gene expression of CCBAU05525 in response to WSHM or genistein were compared to illustrate the working mechanism of WSHM. The differently expressed proteins in response to WSHM were involved in nitrogen and carbon metabolism, nucleic acid metabolism, signaling, energy production and some transmembrane transports. WSHM was found more effective than genistein in inducing the nod gene expression. These results demonstrated that WSHM stimulated cell metabolism and nutrient transport, which resulted in increased cell density of CCBAU05525 and prepared the bacteria for better bacteroid development. Furthermore, WSHM had similar but superior functions to flavone in inducing nod gene and nitrogen fixation related proteins expression in CCBAU05525.

Phenotypic and molecular differences among rhizobia that nodulate Phaseolus lunatus in the Supe valley in Peru

A collection of 16 bacterial strains isolated from root nodules of Lima bean (Phaseolus lunatus L.) in the Supe valley of Peru were characterised using phenotypic and molecular methods. The isolates were clustered into fast-growing, alkalinising and extra-alkalinising slow-growing isolates with marked morphological differences, according to the rate of growth and alkalinisation of yeast extract mannitol (YEM) medium. Fast-growing isolates were salt tolerant and sensitive to 40 °C, while alkalinising slow-growing isolates behaved oppositely. Extra-alkalinising slow-growing isolates were sensitive to 8 °C and 40 °C and tolerated 1 % NaCl. Fast-growing isolates also showed higher indole-3-acetic acid (IAA) production and tri-calcium phosphate solubilisation than the alkalinising slow-growing isolates. Half of the isolates were able to nodulate Phaseolus vulgaris and Vigna unguiculata. Individually, BOX-PCR, ERIC-PCR, and REP-PCR fingerprints patterns resulted in ten profiles from 16 isolates and they were clustered into three profile groups that correspond to the clusters obtained by YEM medium alkalinisation. Analysis of 16S rRNA gene sequences revealed that fast-growing isolates showed 99.7 % sequence identity with Rhizobium mesosinicum CCBAU 25010T and Rhizobium alamii GBV016T. Alkalinising slow-growing isolates were related to both Bradyrhizobium yuanmingense CCBAU10071T and Bradyrhizobium liaoningense 2281T, with 99.8 % sequence identity, and extra-alkalinising slow-growing isolates had 100 % sequence identity to both Bradyrhizobium paxllaeri LMTR 21T and Bradyrhizobium icense LMTR 13T.

Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen fixing bacterium from root nodules of soybeans in Canada.

Sixteen strains of symbiotic bacteria from root nodules of Glycine max grown in Ottawa, Canada, were previously characterized and placed in a novel group within the genus Bradyrhizobium. To verify their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences that were 99.79 % similar to the closest relative, Bradyrhizobium liaoningense LMG 18230T. Phylogenetic analysis of concatenated atpD, glnII, recA, gyrB, rpoB and dnaK genes divided the 16 strains into three multilocus sequence types that were placed in a highly supported lineage distinct from named species of the genus Bradyrhizobium consistent with results of DNA–DNA hybridization. Based on analysis of symbiosis gene sequences (nodC and nifH), all novel strains were placed in a phylogenetic group with five species of the genus Bradyrhizobium that nodulate soybeans. The combination of phenotypic characteristics from several tests including carbon and nitrogen source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain OO99T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium ottawaense sp. nov. is proposed, with OO99T ( = LMG 26739T = HAMBI 3284T) as the type strain. The DNA G+C content is 62.6 mol%.

Genetic Divergence of Bradyrhizobium Strains Nodulating Soybeans as Revealed by Multilocus Sequence Analysis of Genes Inside and Outside the Symbiosis Island

The genus Bradyrhizobium has been considered to be a taxonomically difficult group. In this study, phylogenetics and evolutionary genetics analyses were used to investigate divergence levels among Bradyrhizobium strains nodulating soybeans in China. Eleven genospecies were identified by sequence analysis of three phylogenetic and taxonomic markers (SMc00019, thrA, and truA). This was also supported by analyses of eight genes outside the symbiosis island ("off-island" genes; SMc00019, thrA, truA, fabB, glyA, phyR, exoN, and hsfA). However, seven genes inside the symbiosis island ("island" genes; nifA, nifH, nodC, nodV, fixA, trpD, and rhcC2) showed contrasting lower levels of nucleotide diversity and recombination rates than did off-island genes. Island genes had significantly incongruent gene phylogenies compared to the species tree. Four phylogenetic clusters were observed in island genes, and the epidemic cluster IV (harbored by Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium huanghuaihaiense, Bradyrhizobium liaoningense, Bradyrhizobium daqingense, Bradyrhizobium sp. I, Bradyrhizobium sp. III, and Bradyrhizobium sp. IV) was not found in Bradyrhizobium yuanmingense, Bradyrhizobium sp. II, or Bradyrhizobium elkanii. The gene flow level of island genes among genospecies is discussed in the context of the divergence level of off-island genes.

Co-introduction of exotic rhizobia to the rhizosphere of the invasive legume Acacia saligna, an intercontinental study

Invasive woody legumes have profound impacts in the nitrogen content and cycling of invaded ecosystems due to the ability to enter into symbiosis with nitrogen-fixing bacteria. In spite of the relevance of this symbiosis, the identity and origin of the symbionts involved in invasion are not well understood. We conducted a study to assess the diversity of symbiotic root-nodulating bacteria associated with the invasive Acacia saligna, in newly colonized areas in Portugal and Australia. BOX-PCR was used to discriminate the isolated bacteria and 16S rRNA and nifD genes were sequenced to identify the different isolates and their geographic origin. Bradyrhizobium and Mesorhizobium nodulated A. saligna in Australia while only Bradyrhizobium spp. were found in Portugal. The dominant strains nodulating A. saligna were related to Bradyrhizobium liaoningense and Bradyrhizobium canariense. Co-occurring Acacia longifolia and A. saligna in Australia harbor different rhizobial communities. As an example, we found Mesorhizobium sp. and Phyllobacterium trifolii in A. saligna and A. longifolia respectively, being this the first report for this association. The analysis of the phylogeographic marker nifD clustered most of the sequences obtained in this study with sequences of Australian origin, indicating that exotic bradyrhizobia might have been co-introduced with A. saligna in Portugal. This result highlights the risks of introducing exotic inoculants that might facilitate the invasion of new areas and alter native soil bacterial communities, hindering the recovery of ecosystems.

Effects of rhizobial inoculation, cropping systems and growth stages on endophytic bacterial community of soybean roots

The effects of rhizobial inoculation, soybean-maize intercropping and plant growth stages on the diversity of soybean root endophytic bacteria were evaluated. PCR-based terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes and analyses of cloned partial 16S rRNA gene libraries were used to study the endophytic bacterial communities. The root endophytic bacterial populations during the whole growing season was significantly affected by the three factors analyzed, with plant growth and rhizobial inoculation showing the highest and lowest impact. The alphaproteobacteria and betaproteobacteria were the most predominant endophytes of soybean roots. Some undefined bacterial lineages were also found, indicating that the root endophytic populations might constitute an important source for the isolation of novel bacteria. Bradyrhizobium liaoningense (used as inoculant) occupied most of the soybean nodules formed and Sinorhizobium americanum-related bacteria were detected as the main root endophytes. Results also suggest that bacteria might be transferred from nodules to roots (as endophytic bacteria) when the seeds become mature, which might be a possible process in the life cycle of rhizobia. Rhizobial inoculation and intercropping practices could increase the diversity of the soybean root endophytes, which might have a positive influence on the growth of soybean plants.