Nitrogen fixation and growth of soybean as influenced by varying the methods of inoculation with Bradyrhizobium japonicum

The effects of inoculating soil with a water suspension of Bradyrhizobium japonicum (i) at seeding, (ii) 7, or (iii) 14 days after planting (DAP), (iv) seed slurry inoculation and (v) seed slurry supplemented with postemergence inoculation of a water suspension of Bradyrhizobium at 7 or (vi) 14 DAP, on nodulation, N₂ fixation and yield of soybean (Glycine max. [L.] Merrill) were compared in the greenhouse. The ¹⁵N isotope dilution technique was used to quantify N₂ fixed at flowering, early pod filling and physiological maturity stages (36, 52 and 70 DAP, respectively). On average, the water suspension inoculation formed the greatest number of nodules, and seed plus postemergence inoculation formed slightly more nodules than the seed-only inoculated plants (27, 19 and 12 nodules/plant respectively at physiological maturity). Seed slurry inoculation followed by postemergence inoculation at 14 DAP gave the highest nodule weight, with the plants fixing significantly more (P<0.05) N₂ (125 mg N plant⁻¹ or 56% N) than any other treatment (mean, 75 mg plant⁻¹ or 35% N). However, the higher N₂ fixation was not translated into higher N or dry matter yields. Estimates of N₂ fixed by the ostemergence Bradyrhizobium inoculations as well as plant yield were not significantly different from those of the seed slurry inoculation. Thus, delaying inoculation (e.g., by two weeks as in this study) did not reduce the symbiotic ability of soybean plants.     

Nitrogen metabolism and seed composition as influenced by foliar boron application in soybean

The physiological effects of foliar boron application (FB) on nitrogen metabolism and seed composition have not been well established in soybean [(Glycine max(L.)Merr.)]. Therefore, the effect of FB on nitrogen metabolism and seed composition was investigated. Nitrate assimilation was evaluated by measuring nitrate reductase activity (NRA) and nitrogen fixation was evaluated by measuring nitrogenase activity and natural abundance of ¹⁵N/¹⁴N. NRA were significantly (P?≤?0.05) higher in plants that received FB than the control plants. Higher rate of FB (One application of four times of commercial rate) inhibited nitrogen fixation as measured by natural abundance of ¹⁵N/¹⁴N ratio, but increased NRA. The higher activities of NR and nitrogenase by FB were accompanied with a higher B concentration in leaves. The significant (P?<?0.0001) enrichment of ¹⁵N/¹⁴N, accompanied with a higher rate of FB, suggested a possible mechanism where nitrate assimilation may compensate for the decrease in nitrogen fixation. FB increased seed protein by 13.7% and oleic acid by 30.9% compared to the control plants. This alteration was accompanied by a higher B concentration in leaves and seed. The results suggest that FB affects nitrogen metabolism and alters seed compositions, especially protein and unsaturated fatty acids.     

Growth and yield of groundnut (Arachis hypogaea L.) as influenced by weed management practices and Rhizobium inoculation

Groundnut (Arachis hypogaea L.) productivity in India is low, because of many problems beset in its cultivation. One of the serious problems are weeds. Groundnut yield losses due to weeds have been estimated as high as 24 to 70 percent. This has created a scope for using herbicides in groundnut crop. A field investigation was carried out during kharif (rainy) season of 2001-2002 on a sandy loam soil at College Agronomy Farm, B.A. College of Agriculture, Gujarat Agricultural University, Anand, India to study the effect of weed management practices and Rhizobium inoculation on growth and yield of groundnut (Arachis hypogaea L.). Ten weed control treatments, comprising four treatments of sole application of fluchloralin, pendimethalin, butachlor and metolachlor, respectively each applied at 1.0 kg ha(-1); four treatments comprising of an application of the same herbicides at the same levels coupled with one hand weeding at 30 DAS; one weed-free treatment (hand weedings at 15, 30, 45 DAS); and one unweeded control. All 10 treatmets were combined with and without Rhizobium inoculation (i.e. a total of 20 treatment combinations) under a factorial randomized complete block design (FRBD) with four replications. Minimum weed dry matter accumulation (70 kg/ha) with higher weed control efficiency (90.70%) was recorded under an integrated method i.e. pendimethalin at 1.0 kg ha(-1) + hand weeding at 30 DAS, which also resulted in maximum pod yield (1773.50 kg ha(-1)). This treatment was comparable to fluchloralin applied at 1.0 kg ha(-1) combined with hand- weeding at 30 DAS. Weedy conditions in the unweeded control treatment reduced pod yield by 29.90-35.95% as compared to integrated method. Significantly higher pod yield was obtained with Rhizobium inoculation than the mean value of all treatments without inoculation. For most agronomical parameters examined, Rhizobium inoculation and weed control treatments were independent in their effect.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum.

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

Composted sawdust as a carrierfor Bradyrhizobium,Rhizobium and Azospirillum in crop inoculation

Sawdust was composted by inoculation with a cellulose-decomposing fungus (Cephalosporium sp.) and an N₂-fixing bacterium (Azospirillum brasilense). The product was investigated as a possible carrier for Bradyrhizobium, Rhizobium and Azospirillum. The simple technology and composition of the carrier supported good growth and survival of the investigated strains. Yield increases following crop inoculation with the carrier containing the Bradyrhizobium/Rhizobium/Azospirillum mixture were observed with soybean (34–62%), groundnuts (4–39%), lucerne (24–82%) and a grass mixture of bird's foot trefoil and ryegrass (20–21%).     

Effects of exogenous nitrogen-compounds on the concentrations of allantoin and various constituents in several organs of soybean plants

The effects of nitrogen compounds supplied to culture solutionson the concentrations of allantoin and various constituentsin several organs of soybean plants A62-1 (nodulating variety)were studied to elucidate the symbiotic relation. A62-1 plantsbearing well developed nodules accumulated a large quantityof allantoin in the upper stems, roots, developing leaves, podsand maturing seeds in the reproductive stage. However, the additionof N lowered the allantoin accumulation without changing thesoluble Kjeldahl-N concentration in any organs of the host plants.Also addition of N increased the amino-N concentration in upperstems and roots in contrast with the lack of change in developingleaves, pods and maturing seeds. The decrease of allantoin accumulationwas parallel with the weakened formation of nodules. The additionof N also scarcely affected the concentrations of reducing sugarand sugar in the upper stems. There were few differences inthe concentrations of allantoin, amino-N and soluble Kjeldahl-Namong nodules attached to the A62-1 plants grown in variousamounts of nitrate. Statistical calculations showed that the allantoin concentrationin A62-1 plants was correlated negatively with N applicationand positively with nodule weights. Significant levels of bothcorrelation coefficients were attained in the reproductive stage.Thevariation in allantoin concentration in A62-1 plants was notcorrelated with that in the sugar/soluble Kjeldahl-N ratio andthe reducing sugar/soluble Kjeldahl-N ratio. A large quantity of nitrate added to the nutrient solutionsof the A62-2 (non-nodulating variety) plants elicited only asmall amount of allantoin accumulation in the upper stems, witha consequent increase in the concentrations of amino-N and solubleKjeldahl-N and a decrease in the concentrations of sugar andreducing sugar. (Received August 25, 1976; )     

Studies on the nodulation of strain-specific resistant pea lines using single,mixed and delayed inoculation by Rhizobium leguminosarum

Symbiotic interactions between peas and Rhizobium leguminosarum were investigated by inoculating four pea lines, three of which are strain-specific resistant to the European strain 311d, with various combinations of two strains of Rhizobium, 311d and Tom⁺⁺. The strains were almost equally good to infect the susceptible European cultivar Hero when added singly inoculated. After mixed inoculation (1:1 proportion) strain analysis by ELISA revealed that the nodules were preferentially formed by 311d, although some Tom⁺⁺ nodules were also found mainly on the upper part of the root. Our conclusion is that Tom⁺⁺ is less compatible in comparison with 311d. In addition, we found that as the Hero plants emerged, they were becoming more resistant towards infection with not adapted bacteria. The strain-specific resistant lines from Afghanistan belong to two different systems: Afgh. I, completely resistant to 311d and highly nodulating with Tom⁺⁺, and Afgh. III, incompletely resistant to 311d and poorly nodulating with Tom⁺⁺. Mixed inoculations resulted in nodule depressions, as compared to single inoculations with Tom⁺⁺ ranging from 87% to 14%. The ability of 311d to block infection sites on the roots were found to depend on the degree of symbiotic adaptation between Afgh. I and Tom⁺⁺, respectively Afgh. III and Tom⁺⁺. Strain analysis after double strain inoculation of Afgh. I plants revealed that some nodules were induced by strain 311d. Thus, the presence of Tom⁺⁺ in this case influences the degree of host resistance. However, in Afgh. III plants the resistance towards nodulation were unaffected by the presence of Tom⁺⁺. We suggest that the degree of symbiotic adaptation may change the barrier of resistance towards infection.     

Nodulation,accumulation and redistribution of nitrogen in soybean (Glycine max (L.) Merrill) as influenced by seed inoculation and scheduling of irrigation

Summary A field experiment was conducted on soybean (Glycine max (L.) Merrill) with a view to find out the effect of seed inoculation and scheduling of irrigation on nodulation, accumulation and re-distribution of nitrogen in plant tops and soil. The eight treatment combinations consists of two seed inoculations,viz. uninoculated and inoculated with rhizobium culture, and four irrigation schedules,viz. irrigation water to the cumulative pan evaporation (IW/CPE) ratio of 0.5, 0.7, 0.9 and a control (rainfed). Seed inoculation by, rhizobium culture increased the number, dry-weight and N content of nodules per plant. Inoculation of seeds also increased the N accumulation rate in plant top and it was 2.48 kg/ha/day during the flower-initiation to the pod-initiation stage (30–60 days interval). At harvest, 32.2, 47.8 and 26.2 kg N/ha was re-distributed from the stems, leaves and pods-wall of inoculated plants to the grains, respectively. A total of 186.5 kg N/ha was harvested and 64.7 kg N/ha, was accumulated in soil under the inoculated condition.Scheduling of irrigation at 0.7 IW/CPE proved better, than other irrigation schedules and helped in increasing the nodulation, nitrogen accumulation and grain yield. As compared to control, 8.4, 17.8 and 18.4 kg more of N/ha was redistributed from the stems, leaves and pods-wall respectively when the irrigations were scheduled at 0.7 IW/CPE ratio. Under this irrigation schedule the total N harvest was 200.1 kg/ha while the total N increased by 55.9 kg over that present in soil at the time of sowing.     

Genetic diversity and structure of Rhizobium leguminosarum populations associated with clover plants are influenced by local environmental variables

The identification and conservation of indigenous rhizobia associated with legume plants and their application as biofertilizers is becoming an agricultural worldwide priority. However, little is known about the genetic diversity and phylogeny of rhizobia in Romania. In the present study, the genetic diversity and population composition of Rhizobium leguminosarum symbiovar trifolii isolates from 12 clover plants populations located across two regions in Romania were analyzed. Red clover isolates were phenotypically evaluated and genotyped by sequencing 16S rRNA gene, 16S-23S intergenic spacer, three chromosomal genes (atpD, glnII and recA) and two plasmid genes (nifH and nodA). Multilocus sequence typing (MLST) analysis revealed that red clover plants are nodulated by a wide genetic diversity of R. leguminosarum symbiovar trifolii sequence types (STs), highly similar to the ones previously found in white clover. Rhizobial genetic variation was found mainly within the two clover populations for both chromosomal and plasmid types. Many STs appear to be unique for this region and the genetic composition of rhizobia differs significantly among the clover populations. Furthermore, our results showed that both soil pH and altitude contributed to plasmid sequence type composition while differences in chromosomal composition were affected by the altitude and were strongly correlated with distance.     

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841–852; H.P. Spaink et al., Nature 354: 125–130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.     

Ureide metabolism in leaves of nitrogen-fixing soybean plants

In leaf pieces from nodulated soybean (Glycine max [L] Merr cv Maple Arrow) plants, [¹⁴C]urea-dependent NH₃ and ¹⁴CO₂ production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH₃ per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH₃ and CO₂ production from the utilization of [2-¹⁴C] allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH₃ production. The almost complete sensitivity of NH₃ and CO₂ production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH₃ and two molecules of CO₂.     

Enhancement of nitrogen fixation in lentil,faba bean,and soybean by dual inoculation with Rhizobia and mycorrhizae

The combined effect of Vesicular Arbuscular Mycorrhizae (VAM) and Rhizobium on the cold season legumes, lentil and faba bean, as well as on summer legume, soybean, were studied in soils with low indeginous VA mycorrhizal spores. Inoculation of the plant with VA mycorrhizal fungi increased the level of mycorrhizal root infection of lentil, faba bean and soybean. The inoculation with Rhizobium had no significant effect on VA mycorrhizal infection percent, but VA mycorrhizal inoculation increased nodulation of the three legumes. The inoculation with Rhizobium alone significantly increased plant dry weight and N content of lentil and faba bean as well as seed yield of soybean. VA mycorrhizal inoculation also significantly increased plant dry weight and phosphorus content of the plants as did fertilization with superphosphate. Rock phosphate fertilization, however, had no significant effect on plant growth or phosphorus uptake. The addition of rock phosphate in combination with VA mycorrhizal inoculation significantly increased plant dry weight and P uptake of the plants. The dual inoculation with both rhizobia and mycorrhizae induced more significant increases in plant dry weight, N and P content of lentil and faba bean as well as seed yield of soybean than inoculation with either VA mycorrhizae or Rhizobium alone.     

Yield and composition of soybean seed as a function of potassium supply

Summary A reduction in K supply to soybean plants to deficiency levels during both vegetative and reproductive development resulted in reductions not only in yield, but also in oil and K concentrations in the seed and a concomittant increase in seed protein concentration. Correlations between mean fruit yield and oil, protein and K concentrations, over a wide range of K regimes, were 0.97, −0.94 and 0.98, respectively. When K supply was increased well above the level necessary to produce maximum yields,i.e. luxury consumption, there was no significant change in K concentration in the seed, indicating a high degree of control in the movement of K to the developing seed under high K regimes. When the K supply to the plant was limiting, the rate of accumulation of oil and carbohydrate fractions, but not of seed protein, declined during the latter part of podfilling. This resulted in a fall in the C/N ratio in the non-structural seed components during this part of seed development. Depriving plants of K only during seed development had no effect on seed composition or yield, whereas resupplying K to deficient plants after anthesis resulted in almost the same seed composition and yield as that which occurred with control plants. Possible mechanisms whereby K deficiency influences soybean seed composition and yield are discussed in terms of movement of carbohydrate and nitrogen to the seed. We suggest that potassium-deficient soils are likely to produce crops with low yields and low seed oil levels; the crop may respond to K fertilizers as late as anthesis.     

Diurnal variation of root respiration rates and nitrate uptake as influenced by nitrogen supply

Root respiration rates of Lolium multiflorum supplied with nitrate or ammonium were measured continuously during several days (Exp. A). Net uptake rate of nitrate was similarly measured by an ion selective nitrate electrode in a system of flowing nutrient solution (Exp. B). Diurnal variation of in vitro nitrate reductase activity and nitrate content of tops and roots were determined (Exp. C). Two levels of irradiance were applied throughout, with day:night of 16:8 h. Root respiration rates showed diurnal patterns, most pronounced in the nitrate treatment, with two peaks appearing about 6 and 16 h after commencement of the photoperiod. Respiration rates were highest in the nitrate treatment and at high irradiance. Respiration rates fell after removal of nitrogen, particularly in the nitrate supplied plant and at high irradiance. Net uptake rate of nitrate exhibited diurnal patterns, often with two peaks occurring at the same times as those of respiration rates. In vitro nitrate reductase activity of tops increased steeply 16 h after commencement of the photoperiod and remained at the high level during the following 8 h of darkness. Nitrate content of tops was highest during the 8 h dark period and fell at the start of the photoperiod. Possible controlling systems of the apparent coincidences of diurnal variation rates, net nitrate uptake and nitrate reduction are discussed.     

Growth, metabolism and growth efficiency of a euphausiid crustacean Euphausia pacifica in the southern Japan Sea, as influenced by temperature

Growth (assessed from intermolt period and molt increment) andmetabolism (oxygen consumption) of the post-larva of Euphausiapacifica from the southern Japan Sea were determined at sevengraded temperatures ranging from 1 to 25°C. The intermoltperiod shortened progressively as temperature increased from1 to 20°C, but an effect of temperature on molt-to-moltgrowth increment was not seen. Oxygen consumption rates wereaccelerated by the increase in temperature up to 20°C. Beyond20°C, E.pacifica exhibited reduced oxygen consumption anddied within 1 day without molting. After removing the effectof body size, the relationships between growth rate and temperature,and between oxygen consumption rate and temperature, were established.The carbon budget was calculated as a function of temperature.Because of differential effects of temperature on growth andmetabolism, the net growth efficiency [K₂ growthx100/(growth+metabolism)]changed with temperature. The optimum temperature at which E.pacificaattained the maximum K₂ was 11.4°C, which was derived fromcalculation of cumulative carbon invested in growth and metabolismin this animal. In an alternative method, the optimum temperaturewas obtained mathematically by solving a set of differentialequations. The biological and ecological significance of theoptimum temperature which leads to the maximum K₂ is discussed.     

Growth and nutrient status of citrus plants as influenced by mycorrhiza and phosphorus application

To test the hypothesis that high levels of soluble phosphate applied in combination with VAM fungi, to citrus plants, can cause growth depression even in the absence of other limiting factors, and also to test if rock phosphate, under these conditions, may be a satisfactory P source, a greenhouse experiment was conducted using sterilized soil with four levels of phosphate (0, 50, 100 and 200 ppm P) supplied either as soluble P or as rock phosphate. Citrus seedlings were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus etunicatum or left uninoculated. Six months after the start of the experiment, the plants were harvested and shoot dry weight, P and K uptake, root colonization and the number of spores in 50 cm³ of soil were determined. Significant increases were found in dry matter yields and in P and K contents, due to VAM fungus inoculation, at the zero and 50 ppm soluble P levels and at all rock phosphate levels. At 100 ppm soluble P, the development of VAM plants was equilvalent to that of non-VAM plants, and at 200 ppm, growth was significantly less than that of non-VAM plants. Root colonization and sporulation were reduced at higher P levels. The absolute growth depression of VAM plants at the higher P level was likely due to P toxicity. In addition, high leaf P and K concentrations may have interfered with carbohydrate distribution and utilization in these symbioses. Rock phosphate may be used with VAM citrus to substitute for medium amounts of soluble phosphate.