Symbiotic N nutrition, bradyrhizobial biodiversity and photosynthetic functioning of six inoculated promiscuous-nodulating soybean genotypes

Six promiscuous soybean genotypes were assessed for their ability to nodulate with indigenous root-nodule bacteria in Ghana, with Bradyrhizobium japonicum WB74 serving as positive control. Although the results revealed free nodulation of all six genotypes in both inoculated and uninoculated plots, there was a marked effect of inoculation on photosynthetic rates and whole-plant C. Inoculation also increased stomatal conductance in TGx1485-1D, TGx1448-2E, TGx1740-2F and TGx1445-3E, leading to significantly elevated transpiration rates in the last two genotypes, and a decrease in TGx1485-1D, TGx1440-1E and Salintuya-1, resulting in reduced leaf transpiration and decreased C accumulation. Nodulation, total plant biomass, plant N concentration and content also increased and ∂¹⁵N of the six genotypes, except for TGx1448-2E decreased. Significantly higher %Ndfa resulted in all the soybean genotypes tested (except for TGx1485-1D), and the symbiotic N yield in TGx1740-2F and TGx1448-2E doubled. PCR-RFLP revealed 18 distinct IGS types present in root nodules of the six promiscuous soybean genotypes, with IGS type II being isolated from all six genotypes, followed by IGS types X and XI from five out of the six genotypes. Marked differences in strain IGS type symbiotic efficiency were revealed. For example, as sole nodule occupant, IGS type XI produced high symbiotic N in TGx1445-3E, but low amounts in TGx1448-2E. Inoculated Salintuya-1, which trapped nine strain IGS types in its root nodules, was the most promiscuous genotype, but produced less symbiotic N compared to genotypes with fewer strains in their root nodules.     

Uricase from soybean root nodules: Purification,properties, and comparison with the enzyme from cowpea

A 45-fold purification of uricase (urate:O₂ oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 ± 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed K_m values of 10 and 31 μm for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (K_i = 10 μm). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of M_r 50,000 at pH 7.5.     

Preparation and properties of mitochondria from cowpea nodules

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O₂ uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD⁺-malic enzyme which was 12-fold lower in the mitochondria from nodules.     

Diurnal variation in the functioning of cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of nodules of cowpea (Vigna unguiculata [L.] Walp), maintained under conditions of a 12-hour day at 30°C and 800 to 1,000 microeinsteins per square meter per second (photosynthetically active radiation) and a 12-hour night at 20°C, showed a marked diurnal variation with the total electron flux through the enzyme at night being 60% of that in the photoperiod. This diurnal pattern was, however, due to changes in hydrogen evolution. The rate of nitrogen fixation, measured by short-term ¹⁵N₂ assimilation or estimated from the difference in hydrogen evolution in air or Ar:O₂ (80:20; v/v), showed no diurnal variation. Carbon dioxide released from nodules showed a diurnal variation synchronized with that of nitrogenase functioning and, as a consequence, the apparent `respiratory cost' of nitrogen fixation in the photoperiod was almost double that at night (9.74 ± 0.38 versus 5.70 ± 0.90 moles CO₂ evolved per mole N₂ fixed). Separate carbon and nitrogen balances constructed for nodules during the photoperiod and dark period showed that, at night, nodule functioning required up to 40% less carbohydrate to achieve the same level of nitrogen fixation as during the photoperiod (2.4 versus 1.4 moles hexose per mole N₂ fixed).

Stored reserves of nonstructural carbohydrate of the nodule only partly satisfied the requirement for carbon at night, and fixation was dependent on continued import of translocated assimilates at all times. Measurements of the soluble nitrogen pools of the nodule together with ¹⁵N studies indicated that, both during the day and night, nitrogenous products of fixation were effectively translocated to all organs of the host plant despite low rates of transpiration at night. Reduced fluxes of water through the plant at night were apparently counteracted by increased concentration of nitrogen, especially as ureides, in the xylem stream.

     

Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O₂ (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O₂ showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O₂ treatments up to 16 d after sowing. Thereafter the Ar: O₂ treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO ₃ ^- (0.1–0.2 mM) to Ar: O₂-grown seedlings overcame the abovementioned consequences of N₂ deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O₂ treatment on nodule structure and senescence. Treatment of Ar: O₂-grown seedlings with NO ₃ ^- greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO ₃ ^- treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO ₃ ^- . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from ¹⁵N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase     

Effect of temperature on nitrogenase functioning in cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30°C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20°C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30°C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15°C to a maximum around 35°C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38°C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35°C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15°C and 47°C.     

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb³⁺) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb³⁺ to oxyferrous (Lb²⁺ · O₂) or deoxyferrous (Lb²⁺) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O₂ the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb³⁺ reduced) than in the presence of O₂; this phenomenon was probably because O₂ competes with Lb³⁺ for reductant, thus generating activated O₂ species. The flavin-mediated reduction of Lb³⁺ did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb³⁺ directly. The NAD(P)H + flavin system also reduced the complexes Lb³⁺ · nicotinate and Lb³⁺ · acetate to Lb²⁺ · O₂, Lb²⁺ or Lb²⁺ · nicotinate, depending on the concentrations of ligands and of O₂. In the presence of 200 M nitrite most Lb remained as Lb³⁺ in aerobic conditions but the nitrosyl complex (Lb²⁺ · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb³⁺ at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb³⁺ in vivo.Abbreviations and Terminology FLbR ferric leghemoglobin reductase - Hb²⁺ /Hb³⁺ hemoglobin containing Fe²⁺ /Fe²⁺ - Lb²⁺ /Lb³⁺ leghemoglobin containing Fe²⁺ /Fe³⁺ - Lb³⁺ · nicotinate/acetate Lb in which nicotinate or acetate are complexed to Lb³⁺ - Lb²⁺ · O₂/CO/NO/nicotinate Lb in which O₂, CO, NO or nicotinate are complexed to Lb²⁺ - Rfl riboflavin - SOD superoxide dismutase (EC 1.15.1.1) Published as Paper No. 9237, Journal Series, Nebraska Agricultural Research DivisionWe thank M.B. Crusellas for his skillful drawings. M. Becana thanks the Spanish Ministry of Education and Science/Fulbright Commission for financial support.     

Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules

An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves. The enzyme has two identical subunits of 27 kD that are not covalently bound. Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids. The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody. Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants. An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules. The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids. In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol. Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction. Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence.     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

Influence of fall armyworm previous experience with soybean genotypes on larval feeding behavior

Previous experience on host plants can modify insect feeding behavior. Because insect habituation and induction of preference to host plants are variable across species of plants and insects, it is necessary to investigate each insect-plant interaction to determine whether this phenomenon occurs or not in the system. In this study we investigated the potential occurrence of habituation and induction of preference in fall armyworm (FAW) Spodoptera frugiperda to soybean genotypes. Neonate FAW larvae reared on artificial diet were divided into four treatment groups and fed for one generation with either the resistant soybeans PI 227687 or IAC 100 or the susceptible soybeans BRS Valiosa RR or IGRA RA 626 RR. Biological parameters of FAW were recorded. Eggs obtained from FAW of each genotype group were separated, and the newly hatched larvae were fed on the same genotypes experienced by their parents for additional 8 days. FAW larval preference and leaf area consumed were evaluated in choice feeding assays with the four soybean genotypes within a 24-h period. Genotypes PI 227687 and IAC 100 negatively affected FAW development, demonstrating they are FAW-resistant. FAW larvae exposed to both resistant genotypes consumed more foliage of genotype IGRA RA 626 RR in the choice assays, whereas larvae reared on both susceptible genotypes did not show any preference. From our preliminary study, FAW does not show habituation and induction of preference toward the experienced soybean genotypes. The importance of our findings to host plant resistance and insect-plant biology fields is discussed.     

Fe chelates from compost microorganisms improve Fe nutrition of soybean and oat

Iron availability to plants is often limited when soil pH is 7 or higher. In C rich, but Fe limiting environments, microorganisms may produce organic chelators that complex Fe and increase its availability to plants. Seedlings of soybean (Glycine max L.) and oat (Avena sativa L.) plants, with Fe-efficient or inefficient uptake mechanisms, were grown in an Fe free nutrient solution at pH 7.5. Experiments (using a complete factorial design) were conducted in which these seedlings were transferred to a fresh nutrient solution and treated with Fe sources (FeCl₃, FeEDDHA, and Fe complexed with chelators produced by compost microorganisms (CCMs) after their enrichment in an Fe free, glucose medium), Fe concentrations (0 and 6.7 M) and antibiotic (0 and 100 mg streptomycin L^-1). Dry weight of soybean plants and Fe uptake were significantly (P 0.05) higher when Fe was supplied as ⁵⁹FeCCM than as⁵⁹ FeCl₃ and similar to when Fe was supplied as⁵⁹ FeEDDHA. Dry weight of the Fe-inefficient Tam 0-312 oat cultivar was also significantly higher when Fe was supplied as FeCCM. Fe uptake by oat, when supplied as ⁵⁹FeCCM, was twice that for⁵⁹ FeEDDHA and ⁵⁹FeCl₃. Chlorophyll concentration in plants supplied with FeCCM and FeEDDHA was significantly greater (P 0.05) than in minus Fe control plants and in FeCl₃ supplied plants. Activities of catalase and peroxidase, measured as indicators of Fe nutrition in soybean and oats, were generally increased when Fe was supplied with FeCCM as compared to the other Fe sources. The experimental conditions in which the CCMs were produced are similar to those in soil after amendment with manures or other readily available organic materials. These CCMs can bind with Fe, even under slightly alkaline conditions, and effectively improve Fe nutrition of soybean and oat.     

Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules

miRNAs are ～21 nt non-coding RNAs and play important roles in plant development and response to stress. Symbiotic nitrogen fixation (SNF) is agronomically important for reducing the need of nitrogen fertilizers. The soybean root nodule is the place where SNF takes place. To identify miRNAs that are possibly involved in nitrogen fixation in soybean functional nitrogen-fixing nodules, a small library of RNAs was constructed from the functional nodules harvested 28 days after inoculation with rhizobium. Thirty-two small RNA sequences were identified as belonging to 11 miRNA families. Eight miRNAs are conserved across plant species, twenty are specific to soybean, and the four remaining miRNAs are novel. Expression analysis revealed that miRNAs were differentially expressed in the different tissues. Combinatorial miRNA target prediction identified genes that are involved in multiple biological processes. The results suggest that miRNAs play critical and diverse roles in SNF, nutrient acquisition, and plant development.     

Purification and properties of asparagine synthetase from soybean root nodules

Asparagine synthetase was purified 240-fold from soybean (Glycine max (L.) Merr.) root nodules with a final recovery of 5% using Reactive Blue 2-crossed linked Agarose affinity gel chromatography. High levels of sulfhydryl protectants were required and the inclusion to glycerol and substrates in the extraction buffer helped to stabilize the enzyme. The final preparation had a specific activity of 3.77 mkat/kg protein when assayed at 30°C and was free of contaminating asparaginase activity. The enzyme had a broad pH maximum around pH 8.0 and apparent K_m values for the substrates aspartate, Mg · ATP, and glutamine were 1.24 mM, 0.076 mM and 0.16 mM, respectively. Ammonium ion could partially replace glutamine as the nitrogen donor. Initial velocity patterns yielded parallel inverse plots with all substrate pairs suggesting an overall ping-pong reaction mechanism. Product inhibition patterns provided evidence that glutamine was the first substrate to bind to the enzyme and asparagine was the last product released.     

Efficiency of nodule initiation in cowpea and soybean

When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts.     

Isolation of plant-growth-promoting Bacillus strains from soybean root nodules

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.     

Effects of validamycin A,a potent trehalase inhibitor,and phytohormones on trehalose metabolism in roots and root nodules of soybean and cowpea

Trehalose, a common microbial disaccharide, has been reported to be toxic to plants, and plant trehalase has therefore been hypothesized to function as a detoxifying enzyme. To test this, aseptically grown soybean (Glycine max L. Merr.) plantlets were supplied with trehalose. The plants accumulated trehalose only when validamycin A, a potent trehalase inhibitor, was added as well. Under these conditions, they accumulated trehalose to up to 8% of the dry weight in their primary leaves without any detectable impairment of growth or health. We have previously shown that in soybean nodules, trehalose is generated by the symbiotic bacteria, and trehalase is strongly induced. However, direct exposure of plants to trehalose did not affect their trehalase activity, whereas a treatment with auxin strongly increased it, indicating that the enzyme level is regulated by hormones rather than by its substrate. Addition of validamycin A to nodules caused an increase in the amount of trehalose and a decrease in the sucrose and starch pools, but nitrogen fixation was not affected. Similar results were obtained with cowpea (Vigna unguiculata L.) plantlets and nodules. These results indicate that plant trehalase is functional in metabolizing trehalose from exogenous and endogenous sources, even though the disaccharide has no obvious toxic effects.Abbreviations ABA abscisic acid - ARA acetylene-reduction activity (assay for nitrogenase) - 2,4-D 2,4-dichlorophenoxyacetic acid - DW dry weight - FW fresh weight - GA₃ gibberellic acid - -NAA, -NAA -,-naphthaleneacetic acid We are indebted to Prof. Dr. W. Broughton (University of Geneva, Switzerland) for kindly providing us cowpea seeds and the symbiont strain Rhizobium sp. NGR 234. Validamycin A was a gift of Dr. J.-P. Métraux, Ciba, Basel. This work was supported by the Swiss National Foundation.     

Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences.     

Elevated CO2 induces differences in nodulation of soybean depending on bradyrhizobial strain and method of inoculation

High CO₂ has been shown to increase plant growth and to affect symbiotic activity in many legumes species, including soybean (Glycine max [L.] Merr.). In order to assess the interaction between elevated CO₂ and rhizobial symbionts on soybean growth and nodulation, we combined the effects of CO₂ with those of different bradyrhizobial strains and methods of inoculation. Soybean seeds were sown in agricultural soil in pots and inoculated with three strains of Bradyrhizobium japonicum (5Sc2 and 12NS14 indigenous to Quebec soils, and 532c, a reference strain), the inoculum being either applied directly to the seed or incorporated into the soil. Plants were grown in growth chambers (22/17ºC) for 6 weeks, under either near ambient (400 μmol mol^?1) or elevated (800 μmol mol^?1) concentrations of CO_2. Elevated CO₂ increased mass (63%) and number (50%) of soybean nodules, particularly medium and large, allowed a deeper nodule development, and increased shoot dry weight (+30%), shoot C uptake (+33%) and shoot N uptake (+78%), compared to ambient CO₂. The two indigenous strains induced more medium and large nodules under elevated CO₂ than the reference strain and showed the greatest increases in shoot dry weight. Soil inoculation induced higher number of small nodules than seed inoculation, specifically for the two indigenous strains, but did not affect plant growth parameters. We conclude that soybean yield enhancements due to elevated CO₂ are associated with the production of large and medium-size nodules and a deep nodulation, that the two indigenous strains better respond to elevated CO₂ than the reference strain, and that the method of inoculation has little influence on this response.     

A possible new nucleoside diphosphatase from the cytosol of soybean and alfalfa nodules

Nucleoside diphosphatase activity has been found in the cytosol of soybean (Glycine max) and alfalfa (Medicago satavia) nodules. The enzyme differs from other diphosphatases in that it does not exhibit a strong preference for one nucleoside diphosphate over another. Very little, if any, diphosphatase activity was detected in root extracts of alfalfa and soybean seedlings.