Rhizobium Leguminosarum Biovar Viciae Research Articles

NoiD, a DedA membrane protein required for homeostasis maintaining of Rhizobium leguminosarum biovar viciae during symbiosis with Pisum sativum

NoiD, a DedA membrane protein required for homeostasis maintaining of Rhizobium leguminosarum biovar viciae during symbiosis with Pisum sativum

Enhancement of drought tolerance in diverse Vicia faba cultivars by inoculation with plant growth-promoting rhizobacteria under newly reclaimed soil conditions

Water deficit has devastating impacts on legume production, particularly with the current abrupt climate changes in arid environments. The application of plant growth-promoting rhizobacteria (PGPR) is an effective approach for producing natural nitrogen and attenuating the detrimental effects of drought stress. This study investigated the influence of inoculation with the PGPR Rhizobium leguminosarum biovar viciae (USDA 2435) and Pseudomonas putida (RA MTCC5279) solely or in combination on the physio-biochemical and agronomic traits of five diverse Vicia faba cultivars under well-watered (100% crop evapotranspiration [ETc]), moderate drought (75% ETc), and severe drought (50% ETc) conditions in newly reclaimed poor-fertility sandy soil. Drought stress substantially reduced the expression of photosynthetic pigments and water relation parameters. In contrast, antioxidant enzyme activities and osmoprotectants were considerably increased in plants under drought stress compared with those in well-watered plants. These adverse effects of drought stress reduced crop water productivity (CWP) and seed yield‐related traits. However, the application of PGPR, particularly a consortium of both strains, improved these parameters and increased seed yield and CWP. The evaluated cultivars displayed varied tolerance to drought stress: Giza-843 and Giza-716 had the highest tolerance under well-watered and moderate drought conditions, whereas Giza-843 and Sakha-4 were more tolerant under severe drought conditions. Thus, co-inoculation of drought-tolerant cultivars with R. leguminosarum and P. putida enhanced their tolerance and increased their yield and CWP under water-deficit stress conditions. This study showed for the first time that the combined use of R. leguminosarum and P. putida is a promising and ecofriendly strategy for increasing drought tolerance in legume crops.

Optimizing the growth of forage and grain legumes on low pH soils through the application of superior Rhizobium leguminosarum biovar viciae strains

AbstractClimate variability and current farming practices have led to declining soil fertility and pH, with a heavy reliance on fertilizers and herbicides. The addition of forage and grain legumes to farming systems not only improves soil health but also increases farm profitability through nitrogen (N) fertilizer cost offsets. However, the formation of effective symbioses between legumes and rhizobia can be unreliable and is considered at risk when combined with dry sowing practices such as those that have been designed to obviate effects of climate change. This research was initiated to improve the robustness of the legume/rhizobia symbiosis in low pH, infertile and dry soils. Production from two cultivars of field pea (Pisum sativum) and two species of vetch (Vicia spp.), and symbiotic outcomes when inoculated with a range of experimental rhizobial strains (Rhizobium leguminosarum biovar viciae), was assessed in broad acre field trials which simulated farmer practice. New rhizobia strains increased nodulation, N fixation, produced more biomass and higher seed yield than comparator commercial strains. Strain WSM4643 also demonstrated superior survival when desiccated compared to current commercial strains in the laboratory and on seed when delivered as inoculant in peat carriers. WSM4643 is a suitable prospect for a commercial inoculant in Australia and other agricultural areas of the world where growing peas and vetch on soils generally considered problematic for this legume/rhizobia symbiosis. A particular advantage of WSM4643 may be that it potentiates sowing inoculated legumes into dry soil, which is a contemporary response by farmers to climate variation.

Sourcing Rhizobium leguminosarum biovar viciae strains from Mediterranean centres of origin to optimize nitrogen fixation in forage legumes grown on acid soils

AbstractOver the last three decades, farming systems in Europe and Australia have seen a decline in legume plantings, leading to reduced soil carbon and fertility, and an increase in plant disease, reliance on industrial nitrogen fertilizer and herbicides. In Australia, one reason for this decline has been the movement towards sowing crops and forages into dry soil, before the opening rains, as a consequence of climate variability. This practice predicates against the survival of rhizobial inoculants, and hence generates uncertainty about legume performance. The research reported here was initiated to improve the robustness of a specific forage legume/rhizobia symbiosis to increase nitrogen fixation in low pH, infertile soils. Rhizobial strains (Rhizobium leguminosarum biovar viciae) from Pisum sativum L. were sourced from acid soils in southern Italy and southern Australia. Strains were evaluated for N fixation on the forage legumes P. sativum, Vicia sativa and Vicia villosa, then for survival and persistence in acid soils (pHCa 4.6). Fourteen of the strains produced a higher percentage of nitrogen derived from the atmosphere (%Ndfa) compared to commercial comparator strain SU303 (<78%). Twenty‐two strains survived sufficiently into the second season to form more nodules than SU303, which only achieved 3% of plants nodulated. Elite strains WSM4643 and WSM4645 produced six times more nodulated plants than SU303 and had significantly higher saprophytic competence in acid soil. These strains have the ability to optimize symbiotic associations with field peas and vetch in soils with low fertility, carbon and pH that are restrictive to the current commercial strain SU303.

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Didymella pinodes Affects N and P Uptakes and Their Efficiencies in a Tripartite Mutualism of Pea

In pea (Pisum sativum L.) production, Didymella pinodes (Berk. & A. Bloxam) Petr. is the most damaging aerial pathogen globally. In two completely randomized pot experiments with four replicates, we studied the effects of D. pinodes infection interaction with three symbiotic treatments (Rhizobium leguminosarum biovar viciae, arbuscular mycorrhizal fungi (AMF) and co-inoculation of both) and a non-symbiotic control on one or two pea cultivars. Grain yield and yield components of pea, uptakes and physiological efficiencies of N and P and nitrogen fixation were recorded. The results show that there were significant interaction effects among treatments. Therefore, productivity of crops and their uptakes and efficiencies of N and P are dependent on plant health conditions, effectiveness of microbial symbionts and response of pea genotypes. For cv. Protecta inoculated with both symbionts, pathogen infection compared to healthy plants significantly enhanced P acquisition. Overall, plants inoculated with rhizobia alone had higher grain yield by 20–30% and nitrogen fixation by 20–25% than in dual symbiosis independent of plant health conditions. In conclusion, aerial pathogen, pea genotypes and microbial symbionts interactions modified N and P uptake and their efficiencies, which can lead to improving final grain yield quantity and quality in a sustainable farming system.

Towards a characterisation of the wild legume bitter vetch (Lathyrus linifolius L. (Reichard) Bässler): heteromorphic seedgermination, root nodule structure and N-fixing rhizobial symbionts.

Lathyrus linifolius L. (Reichard) Bässler (Fabiaceae, bitter vetch) is a nitrogen (N) fixing species. A coloniser of low nutrient (N) soils, it supports biodiversity such as key moth and butterfly species, and its roots are known for their organoleptic and claimed therapeutic properties. Thus, the species has high potential for restoration, conservation, novel cropping and as a model species. The last because of its genetic synteny with important pulse crops. However, regeneration and functional attributes of L. linifolius remain to be characterised. Seeds of L. linifolius were characterised using physical, colorimetric and chemical data. Ultrastructural and functional characterisation of the N-fixing root nodules included immunolabelling with nifH protein antibodies (recognising the N-fixing enzyme, nitrogenase). Endosymbiotic bacteria were isolated from root nodules and characterised phylogenetically using 16S rRNA, nodA and nodD gene sequences. L. linifolius yielded heteromorphic seed of distinct colour classes: green and brown. Seed morphotypes had similar C:N ratios and were equally germinable (ca. 90%) after scarification at differing optimal temperatures (16 and 20°C). Brown seeds were larger and comprised a larger proportion of the seed batch (69%). L. linifolius root nodules appeared indeterminate in structure, effective (capable of fixing atmospheric N) and having strains very similar to Rhizobium leguminosarum biovar viciae. The findings and rhizobial isolates have potential application for ecological restoration and horticulture using native seeds. Also, the data and rhizobial resources have potential applications in comparative and functional studies with related and socio-economically important crops such as Pisum, Lens and Vicia.

Population, Symbiotic Effectiveness, and Protein Profile Patterns of Indigenous Rhizobium leguminosarum biovar viciae to Korean Soils

Some symbiotic characteristics of native Korean Rhizobium leguminosarum biovar viciae were analysed to get some informations desirable for cultivation of hairy vetch (Vicia villosa Roth) using its symbiont in Korea. The size of indigenous populations of R. leguminosarum biovar viciae was higher in seven upland soils showing 1.7✕102~5.8✕104 cells g ‧ soil-1, which appeared to be 10% and 37% higher for cultivated and uncultivated soils of hairy vetch, respectively, than seven paddy soils with 1.7✕102~1.7✕104 cells g ‧ soil-1. In symbiotic potentials, however, the yields of hairy vetch treated with 10-fold-diluted (10-1) inoculum and 1000-fold-diluted (10-3) one was 11.2% and 8.8% more, respectively, in paddy than upland. Hairy vetch inoculated with either strain KHR 106 from Sacheon or strain KHR 120 from Yesan among native Korean R. leguminosarum biovar viciae isolates was of similar yield increment of 16% (p <0.05) in upland soils with native R. leguminosarum biovar viciae of 5.8✕10 cells g ‧ soil-1. In case of coinoculation of the two strains, however, the yields was not significantly increased. In especial, isolate KHR 106, KHR 120, and KHR 122 from Suwon, which has also good symbiotic effectiveness, showed different protein profile patterns each other. As a result, hairy vetch is possibly able to use atmospheric nitrogen through symbiotic relationship with diverse native R. leguminosarum biovar viciae in Korean arable lands. For safe and good production of hairy, however, the use of superior strains with high symbiotic effectiveness and competitiveness will be desirable. Protein profile patterns of three R. leguminosarum bv. viciae isolates in the automatic gel electrophoresis using Experion Pro260 (Bio-Rad). Here strain for lane 1 is KHR 106; lane 2 for KHR 120; lane 3 for KHR 122, which were isolated from hairy vetch nodules (a) and examined for the symbiotic ability (b).

Carboxymethyl cyclosophoraoses as a flexible pH-responsive solubilizer for pindolol

In the present study, cyclosophoraoses (CyS) (β-1,2 linked cyclic glucans, with glucopyranose units ranging from 17 to 23) isolated from Rhizobium leguminosarum biovar viciae VF-39 were modified with carboxymethyl (CM) groups, and the pH-sensitive complexation of CM CyS with pindolol was investigated. The solubility of pindolol increased 32-fold by its complexation with 5mM CM CyS at pH 10, whereas it shows no significant change at pH 3. Pindolol, a β-adrenergic blocking agent, has a hydrophobic nature at non-ionized state, and CM CyS could solubilize efficiently pindolol in a high alkaline solution. The carboxymethylation of flexible CyS allows them to present a more suitable cavity for the hydrophobic pindolol at pH 10, which is differentiated from CM β-cyclodextrin (β-CD). It can be interpreted as that the anionic repulsion effectively modulates the flexible and distorted conformation of CyS rather than rigid annular shape of β-CD. Resultingly, the highly solubilized CM CyS/pindolol complex was characterized by UV–vis, T1 relaxation, ROESY, DOSY NMR spectroscopy, FT-IR spectroscopy, SEM, and molecular modeling studies. The antioxidant activity of pindolol was also improved 260% in the complex compared to free pindolol. The use of flexible host molecules with pH-responsive substituents would be applied in the development of smart systems for sensing or in biomedical fields.

Glutathione affects the transport activity of Rhizobium leguminosarum 3841 and is essential for efficient nodulation.

As glutathione (GSH) plays an essential role in growth and symbiotic capacity of rhizobia, a glutathione synthetase (gshB) mutant of Rhizobium leguminosarum biovar viciae 3841 (Rlv3841) was characterised. It fails to efficiently utilise various compounds as a sole carbon source, including glucose, succinate, glutamine and histidine, and shows 60%–69% reduction in uptake rates of glucose, succinate and the non-metabolisable substrate α-amino isobutyric acid. The defect in glucose uptake can be overcome by addition of exogenous GSH, indicating GSH, but not its bacterial synthesis, is required for efficient transport. GSH is not involved in the regulation of the activity of Rlv3841's transporters via the global regulator of transport, PtsNTR. Although lack of GSH reduces transcription of the branched amino acid transporter, this was not the case for all uptake transport systems, for example, the amino acid permease. This suggests GSH alters activity and/or assembly of transport systems by an unknown mechanism. In interaction with plants, the gshB mutant is not only severely impaired in rhizosphere colonisation, but also shows a 50% reduction in dry weight of plants and nitrogen-fixation ability. This reveals that changes in GSH metabolism affect the bacterial–plant interactions required for symbiosis.

Responsiveness to inoculation and application of micronutrient fertilizers in common vetch (Vicia sativa L.) new varieties at seed production

The responsiveness of common vetch varieties to inoculation with Rhizobium leguminosarum biovar-viciae of different genotypes and applications of molybdenum and boron micronutrients has been revealed. It is shown that the effect of interactions between molybdenum and boron on the plants at the joint application is nonadditive. The artificial colonization of seeds by the complementary strains of root nodule bacteria and the molybdenum treatment are expedient to increase the efficiency of the symbiotic interactions between rhizobia and common vetch varieties. Inoculation with strain no. 145 and supplementation with molybdenum improved the seed yields of the common vetch Valentina and Lugovskaya-98 varieties by 20% and 17%, respectively.

Arabinose and protocatechuate catabolism genes are important for growth of Rhizobium leguminosarum biovar viciae in the pea rhizosphere.

Background and aimsTo form nitrogen-fixing nodules on pea roots, Rhizobium leguminosarum biovar viciae must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness.MethodsSignature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres. Candidate mutants were assessed relative to controls for growth in minimal medium, growth in pea rhizospheres and for infection of peas in mixed inoculants. Mutated genes were identified by DNA sequencing and confirmed by transduction.ResultsOf 5508 signature-tagged mutants, microarrays implicated 50 as having decreased rhizosphere fitness. Growth tests identified six mutants with rhizosphere-specific phenotypes. The mutation in one of the genes (araE) was in an arabinose catabolism operon and blocked growth on arabinose. The mutation in another gene (pcaM), encoding a predicted solute binding protein for protocatechuate and hydroxybenzoate uptake, decreased growth on protocatechuate. Both mutants were decreased for nodule infection competitiveness with mixed inoculants, but nodulated peas normally when inoculated alone. Other mutants with similar phenotypes had mutations predicted to affect secondary metabolism.ConclusionsCatabolism of arabinose and protocatechuate in the pea rhizosphere is important for competitiveness of R.l. viciae. Other genes predicted to be involved in secondary metabolism are also important.

Population structure of root nodulating Rhizobium leguminosarum in Vicia cracca populations at local to regional geographic scales

The genetic diversity and population structure of about 350 Rhizobium leguminosarum biovar viciae isolates from Vicia cracca were analysed. A hierarchical sampling design was used covering three regions, one region in Belgium and two in France, in which multiple local V. cracca populations were sampled. Rhizobium isolates were genotyped using RAPD and by sequencing two chromosomal housekeeping genes (glnII and recA) and one plasmid-borne gene (nodC). Twenty-six nodC types and sixty-seven chromosomal types were identified, many of which appeared to be regional or local endemics. We found strong genetic differentiation both among V. cracca populations that are separated by only a few kilometres, and among regions that are 50 to 350km apart. Despite significant plasmid exchange, chromosomal and nod types were similarly structured among host populations and regions. We found two lineages of which one prevailed in the Belgian region while the other dominated the French regions. Although a significant correlation between genetic differentiation and geographic distance was found, it is deemed more likely that the observed biogeographic patterns are rather due to coevolutionary interactions and environmental pressures. Furthermore, the impact of recombination on the chromosomal differentiation was found to be considerable.

Enhanced solubility of galangin based on the complexation with methylated microbial cyclosophoraoses

Methylated cyclosophoraoses (M-Cys) were synthesized by reaction using dimethyl sulfate with native Cys (unbranched cyclic β-1,2-d-glucans) isolated from Rhizobium leguminosarum biovar viciae VF-39. Its structure was proven using nuclear magnetic resonance (1H NMR) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Based on the enhanced hydrophobicity by methylation of Cys, we investigated the inclusion property with the water-insoluble flavonoid, galangin, through a phase solubility study using ultraviolet–visible spectroscopy. The solubility of galangin was enhanced 5.6-fold according to the added concentrations (1 mM) of M-Cys, compared to the 1.9-fold and 3.4-fold enhancements by β-Cyclodextrin (β-CD) and heptakis (2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD), respectively. M-Cys was also shown to have the highest binding constant (5,534 M−1) with galangin among the tested host molecules (β-CD, DM-β-CD, Cys, and M-Cys). From this result, we can infer that the complex of galangin with M-Cys is more stable than any of the other host molecules. The continuous variation method showed that the galangin/M-Cys complex was suitable for 1:1 stoichiometry. The formation of the complex was confirmed with 1H NMR, FT-IR, differential scanning calorimetry, and scanning electron microscopy. Furthermore, the hypothetical molecular model of 1:1 galangin/M-Cys complex was suggested by molecular docking simulations. The cytotoxicity to the human cervical adenocarcinoma cell lines was enhanced by the galangin/M-Cys complex compared with free galangin. The obtained results indicate that M-Cys can be utilized as an effective complexing agent for galangin.

Genetic diversity of resident soil rhizobia isolated from nodules of distinct hairy vetch (Vicia villosa Roth) genotypes

Hairy vetch (Vicia villosa Roth, HV) is widely grown as a legume cover crop throughout the U.S.A., with biological nitrogen fixation (BNF) through symbiosis with Rhizobium leguminosarum biovar viciae (Rlv) being one of the most sought after benefits of its cultivation. This study determined if HV cultivation history and plant genotype affect genetic diversity of resident Rlv. Soil samples were collected from within farmers’ fields at Graham, Cedar Grove and Ivanhoe sites in North Carolina and pairs of genetically similar hairy vetch genotypes used as trap hosts. A total of 519 Rlv strains were isolated from six paired field soils, three with and three without histories of HV cultivation. A total of 46 strains failed to PCR-amplify the nifH gene; however nodC PCR amplification of these nifH-negative strains resulted in amplification of 22 of the strains. Repetitive element polymerase chain reaction (rep-PCR) with BOX-A1R primer and redundancy analysis showed rhizobial diversity to vary greatly within and between fields, with over 30 BOX banding patterns obtained across the six fields. Cluster analysis of BOX-PCR banding patterns resulted in 36 genetic groups of Rlv at a similarity level of 70%, with 15 of the isolates from fields with HV history not belonging to any of the clusters. Site was found to be the main driver of isolate diversity overall, explaining 57%, of the total variation among rhizobia occupying HV nodules, followed by history of hairy vetch cultivation. Evidence of a HV host genotype influence on the populations of rhizobia that infect hairy vetch was also observed, with plant genotype explaining 12.7% of the variation among all isolates. Our results show that second to site, HV cultivation history was the most important driver of rhizobial nodule community structure and increases the genetic diversity of resident Rlv in soils.

Identification of a NodD repressible gene adjacent to &amp;lt;italic&amp;gt;nodM&amp;lt;/italic&amp;gt; in &amp;lt;italic&amp;gt;Rhizobium leguminosarum&amp;lt;/italic&amp;gt; biovar &amp;lt;italic&amp;gt;viciae&amp;lt;/italic&amp;gt;

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3' end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.

Mutation of the sensor kinase chvG in Rhizobium leguminosarum negatively impacts cellular metabolism, outer membrane stability, and symbiosis.

Two-component signal transduction systems (TCS) are a main strategy used by bacteria to sense and adapt to changes in their environment. In the legume symbiont Rhizobium leguminosarum biovar viciae VF39, mutation of chvG, a histidine kinase, caused a number of pleiotropic phenotypes. ChvG mutants are unable to grow on proline, glutamate, histidine, or arginine as the sole carbon source. The chvG mutant secreted smaller amounts of acidic and neutral surface polysaccharides and accumulated abnormally large amounts of poly-ß-hydroxybutyrate. Mutation of chvG caused symbiotic defects on peas, lentils, and vetch; nodules formed by the chvG mutant were small and white and contained only a few cells that had failed to differentiate into bacteroids. Mutation of chvG also destabilized the outer membrane of R. leguminosarum, resulting in increased sensitivity to membrane stressors. Constitutive expression of ropB, the outer membrane protein-encoding gene, restored membrane stability and rescued the sensitivity phenotypes described above. Similar phenotypes have been described for mutations in other ChvG-regulated genes encoding a conserved operon of unknown function and in the fabXL genes required for synthesis of the lipid A very-long-chain fatty acid, suggesting that ChvG is a key component of the envelope stress response in Rhizobium leguminosarum. Collectively, the results of this study demonstrate the important and unique role the ChvG/ChvI TCS plays in the physiology, metabolism, and symbiotic competency of R. leguminosarum.

Response to salinity stress of Rhizobium leguminosarum bv. viciae strains in the presence of different legume host plants

We investigated the effect of residual salts from the previous summer’s irrigation on two non-irrigated cover crops—broad bean and common vetch—and on their rhizobial symbiontics. Before sowing, seeds were inoculated with a salt-tolerant strain and a salt-sensitive strain of Rhizobium leguminosarum biovar viciae. An increase in the electrical conductivity of the saturated-soil extract from 2.0 dS m−1 to 6.0 dS m−1 caused a severe reduction of broad bean biomass, while growth of common vetch was almost unaffected by the salinity level. Our results clearly indicate that common vetch as a cover crop may increase the availability of nitrogen in soil more than broad bean also in saline environments. A high ability of both inoculated strains to nodulate Vicia plants was observed under controlled conditions. In contrast, inoculated strains were not able to nodulate plants in salinized soils, showing lower competitiveness compared to natural rhizobial population. Indeed, the new isolates from nodules were salt-sensitive when tested in vitro. The complex interaction among bacteria, plant and soil determined the survival of endogenous salt sensitive rhizobial strains and limited the success of inoculation with exogenous salt resistant rhizobia.

Nitrous oxide emissions from grain legumes as affected by wetting/drying cycles and crop residues

Grain legume production with rhizobial inoculation has drawn attention not only because of the economic value of nitrogen (N) fixation by grain legumes, but also because of the concern that N2 fixation by grain legumes may enhance emissions of nitrous oxide (N2O), a powerful greenhouse gas. However, the relationship between N2O emissions and biological N2 fixation by grain legumes is not well understood. The objective of this study was to quantify N2O emissions associated with N2 fixation by grain legumes as affected by wetting/drying cycles and crop residues. Two grain legumes, lentil (Lens esculenta Moench) and pea (Pisum sativum L.), either inoculated with two Rhizobium leguminosarum biovar viciae strains, 99A1 and RGP2, respectively, or fertilized with 15N-labeled fertilizer were grown in a controlled environment under three wetting/drying cycles. Profile N2O concentrations and surface N2O emissions were measured from the soil–plant systems, which were compared with those from a cereal, spring wheat (Triticum aestivum L. ac. Barrie). After harvest, crop residues were incorporated into soils that were seeded to spring wheat to evaluate the effect of crop residues on N2O emissions. Results indicated that: (1) inoculating grain legumes with non-denitrifying rhizobia did not enhance N2O emissions and the presence of grain legumes did not increase N2O emissions compared with the cereal crop, and (2) profile N2O accumulation and surface emissions were not related to the type of crop residues added to the soil, but related to the residual N applied previously as N fertilizer. This suggests that N2O emissions are not directly related to biological N2 fixation by grain legumes, and on a short time scale, N rich residues of N2-fixing crops have a limited impact on N2O emissions compared with N fertilization.

Field response of legumes to inoculation with plant growth-promoting rhizobacteria

Carrier-based (soil/FYM, 1:1) plant growth-promoting rhizobacteria (PGPR) isolates (Bacillus subtilis, Klebsiella planticola and Proteus vulgaris) were tested individually and in combination with Bradyrhizobium japonicum and Rhizobium leguminosarum biovar viciae under field conditions on soybean and lentil crops, respectively, under field conditions. Inoculation of soybean (Glycine max) cv. Pusa 22 with B. subtilis produced maximum nodule number, mass and nitrogenase activity (acetylene reduction activity, ARA) followed by B. japonicum (SB 271). Maximum soybean yield was registered with the coinoculation of B. japonicum and B. subtilis over an uninoculated control. Maximum nodulation in the lentil (Lens culinaris) cv. L 4147 was obtained with a combination of R. leguminosarum (L-12-87) and P. vulgaris inoculation followed by a single inoculation with Rhizobium and B. subtilis. None of the PGPR isolates either singly or in coinoculation with R. leguminosarum could significantly influence the yield of the lentil crop.