Hydrogen metabolism and nitrogen fixation of soybean (Glycine max. L.) inoculated with different strains ofRhizobium spp.

Summary Hydrogenase activities and N₂-fixing capacities of soybean nodules (Glycine max. cv. Hodgson), inoculated with strains ofBradyrhizobium japonicum andRhizobium fredii from different geographical regions, were measured after 35 days of culture under controlled conditions. Of the strains tested, 47% induced nodules with bacteroids which recycled H₂. The data obtained suggest that H₂-recycling ability is not a major factor influencing early N₂-fixation which depends essentially on the precocity and intensity of the initial nodulation.     

Nitrogen fixation and CO2 exchange in soybeans (Glycine max L.) inoculated with mixed cultures of different microorganisms

N₂ fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of biological nitrogen in plants as determined by the ¹⁵N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g^-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N₂ fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.     

Occurrence and nature of mixed infections in nodules of field-grown soybeans (Glycine max)

Summary Mixed infections of Bradyrhizobium japonicum strains in early and late nodules of four soybean cultivars were studied in a field soil. Nodule occupants were identified by immunofluorescence using serogroup specific antibodies prepared against B. japonicum strains USDA 110, USDA 123, and USDA 138. Double infection was determined directly by combined examination of the same microscopic field by fluorescence and phase contrast microscopy. Double strain occupancy was observed consistently, and its occurrence did not differ substantially in pouch, soil pot, and field experiments, ranging in incidence from 12% to 32%. No significant differences in the incidence or nature of double infection could be attributed to cultivar, seed inoculation, or plant maturity. Strains reactive to strain USDA 123-fluorescent antibody were dominant in both singly and doubly infected nodules irrespective of cultivar, plant age, or seed inoculation with strain USDA 110.Paper no. 15092 in the Scientific Journal Series of the Minnesota Agricultural Experiment Station, St. Paul     

Effect of some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean [Glycine max. (L) Merr.]

Several commercial fungicide seed treatments were evaluated for their possible effect on the survival of Bradyrhizobium japonicum on seeds and on the nodulation and yield of soybeans in a greenhouse and a field experiment. quinolate Pro (carbendazim and oxine copper), Vitavax 200FF (carboxin and thiram), and Monceren (pencycuron) had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybeans. They can thus be considered compatible with soybean seed inoculation. Germipro UFB (carbendazim and iprodione), Apron 35J (metalaxyl), and Tachigaren (hymexazol) decreased B. japonicum survival and the nodulation and yield of soybeans and thus cannot be considered compatible with soybean seed inoculation.     

Differential effect of various endomycorrhizal fungi on nodulating ability of green gram by Bradyrhizobium sp. (Vigna) strain S24

Bradyrhizobium sp. (Vigna) strain S24 interacted differentially with eight vesicular-arbuscular mycorrhizal (VAM) fungi and caused significant variations in nodulation and growth parameters of green gram. Coinoculation with Scutellospora calospora resulted in the highest nitrogenase activity and dry biomass. The nodulation competitiveness of strain S24 was significantly higher (60–63%) in the presence of Glomus mosseae, G. fasciculatum and Scutellospora calospora when compared to treatment with single inoculation of S24 (51%). Percentage VAM colonization was higher in treatments having higher nocule occupancy of introduced strain (S24). Received: 26 June 1995     

Relative efficiency,ureide transport and harvest index in soybeans inoculated with isogenic HUP mutants of Bradyrhizobium japonicum

Summary Differences between isogenic uptake hydrogenase (HUP) mutants of Bradyrhizobium japonicum in terms of nodule efficiency, N₂ fixation and N incorporation into various plant parts were studied in a monoxenic greenhouse experiment in order to confirm previous results with soybeans and beans inoculated with various HUP⁺ and HUP^– strains. The HUP+ revertant PJ17-1 of a HUP^– mutant (PJ17) of strain USDA DES 122 showed a completely restored relative efficiency (100% versus 78±2% for the HUP^– mutant), higher nodule efficiency (N₂ fixed per g nodules), higher ureide-N transport rates, higher N contents in pods and higher N harvest indices. All these observations confirm previous experiments with HUP⁺ and HUP^– strains.     

Field performance of three Bradyrhizobium japonicum strains with two soybean (Glycine max. L.) cultivars

Summary A field experiment was condutced in a clay loam soil to study the performance of three Bradyrhizobium japonicum strains; USDA 110, USDA 138 and TAL 379, in relation to their N₂-fixing potential and competitiveness on two soybean cultivars (Clark and Calland). Inoculation of soybean cultivars with these strains, either singly or in combination, induced significant increases in plant dry weight, N₂ fixation and seed yields. However, no significant differences were found between the rhizobial strains and/or their mixtures in N₂ fixation and increased seed yield for both cultivars. The two soybean cultivars gave similar responses to inoculation. No significant differences in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant. The competition between strains for nodulation was assessed. Strain USDA 110 was the most competitive, followed by USDA 138. Strain TAL 379 was always less competitive on both cultivars. The incidence of double-strain occupancy of nodules varied from 8% to 40%.     

Influence of methyl isocyanate on the growth,ATP levels,and nitrogen fixation of Azotobacter chroococcum

Summary Growth, ATP levels, and N₂-fixing activity of Azotobacter chroococcum culture exposed to 500 g ml^-1 of methyl isocyanate were investigated. In methyl isocyanate-exposed cells, the lag phase was extended up to 16 h but the control cells started to multiply immediately. ATP levels and N₂-fixing activity as determined by acetylene reduction assay were markedly reduced in the treated cells.     

The use of nitrogen-15 natural abundance and nitrogen yield of non-nodulating isolines to estimate nitrogen fixation by soybeans (Glycine max L.) across three elevations

Summary Dissimilarities in soil N uptake between N₂-fixing and reference non-N₂-fixing plants can lead to inaccurate N₂ fixation estimates by N difference and ¹⁵N enrichment methods. The natural ¹⁵N abundance ( ¹⁵N) method relies on a stabilized soil ¹⁵N pool and may provide reliable estimates of N₂ fixation. Estimates based on the ¹⁵N and differences in N yield of nodulating and non-nodulating isolines of soybean were compared in this study. Five soybeans from maturity groups 00, IV, VI, and VIII and their respective non-nodulating isolines were grown at three elevations differing in ambient temperature and soil N availability. Despite large differences in phenological development and N yield between the non-nodulating isolines, the ¹⁵N values measured on seeds were relatively constant within a site. The ¹⁵N method consistently produced lower N₂ fixation estimates than the N difference method, but only in three of the 15 observations did they differ significantly. The average crop N derived from N₂ fixation across sites and maturity groups was 81% by N difference compared to 71% by ¹⁵N. The magnitude of difference between the two methods increased with increasing proportions of N derived from N₂ fixation. These differences between the two methods were not related to differences in total N across sites or genotypes. The low N₂ fixation estimates based on ¹⁵N might indicate that the nodulating isolines had assimilated more soil N than the non-nodulating ones. A lower variance indicated that the estimates by N difference using non-nodulating isolines were more precise than those by ¹⁵N. Since the differences between the estimates were large only at high N₂ fixation levels (low soil N availability), either method may be used in most situations when a non-nodulating isoline is used as the reference plant. The ¹⁵N method may have a comparative advantage over N difference and ¹⁵N enrichment methods in the absence of a suitable non-N₂-fixing reference plant such as a non-nodulating isoline.     

Effect of high temperature on plasmid curing of Rhizobium spp. in relation to nodulation of pigeonpea [Cajanus cajan (L.) Millsp.]

Summary Fifty-six isolates of Rhizobium and Bradyrhizobium spp. (Cajanus) were studied for their plasmid profile and N₂-fixation efficacy. One to three plasmids were reproducibly detected in all the Rhizobium spp. strains but no plasmid was detected in the Bradyrhizobium spp. strains. Rhizobium sp. strain P-1 was mutagenized by Tn5 and three nod^– and six nod⁺fix^– were screened for symbiotic parameters. Neomycin-sensitive mutants were isolated by elevated temperatrue (40°C) from tranconjugants carrying Tn5 insertions. The high temperature cured these mutants from the single large plasmid present in the parent strain P-1. All these cured mutants were nod^–, indicating that the genes for nodulation were present on this plasmid, which is readily cured at a high temperature (40°C). The high temperature in the semi-arid zones of Haryana could be responsible for the low nodulation of pigeonpea because the plasmid carrying the nodulation genes is cured at 40°–45°C giving rise to non-nodulating mutants.     

Effect of high temperature on growth,nitrogen fixation,and chlorophyll content of five species of Azolla Anabaena symbiosis

Summary Cultures of Azolla sp. ST-SI, A. microphylla BR-GI, A. mexicana BR-GL, A. caroliniana WT-V, and A. filiculoides BR -H were grown in N-free International Rice Research Institute growth medium in the glasshouse at 38±1 °C (day) and 25±1 °C (night) under a light intensity of 350 Em²s^–1 for 27 days. Biomass, chlorophyll contents and nitrogenase activity (acetylene reduction assay) were recorded on the 19th and 27th day. For comparison the same parameters were studied in Azolla spp. under normal growth conditions at 26±1 °C (day) and 19±1 °C (night). Azolla sp. STSI and A. microphylla BR-GI had produced a larger biomass by the 19th and the 27th day of incubation than A. caroliniana WTV and A. filiculoides which showed poor growth. Under normal growth conditions A. caroliniana WTV and A. filiculoides BRH produced less biomass than the other Azolla spp. cultures tested. A. mexicana BR-GL had a higher total chlorophyll content in both incubation periods than A. caroliniana WT-V and A. filiculoides BR-H. The N content was high in Azolla sp. ST-SI, A. microphylla BR-GI, and A. mexicana BR-GL compared with the low N content of A. filiculoides BR-H and A. caroliniana WT-V. At the higher temperature (38±1 °C/25±1 °C) Azolla sp. ST-SI and A. microphylla BR-GI consistently showed a higher growth rate than A. filiculoides BR-H and A. caroliniana WTV, while the growth rate of A. mexicana BR-GL was intermediate.The study was carried out at C.F. Kettering Research Laboratory, Yellowsprings, OH - 45387, USA     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

Rapid and reversible nitrate inhibition of nodule growth and N2 fixation activity in soybean (Glycine max (L.) Merr.)

Nodulated soybean (Glycine max. (L) Merr. cv. Williams) plants were hydroponically cultured, and various combinations of 1-week culture with 5 or 0 mm nitrate were applied using 13-d-old soybean seedlings during three successive weeks. The treatments were designated as 0-0-0, 5-5-5, 5-5-0, 5-0-0, 5-0-5, 0-5-5, and 0-0-5, where the three sequential numbers denote the nitrate concentration (mm) applied in the first-second-third weeks. The size of the individual nodule was measured periodically using a slide caliper. All the plants were harvested after measurement of the acetylene reduction activity (ARA) at the end of the treatments. In the 0-0-0 treatment, the nodules grew continuously during the treatment period. Individual nodule growth was immediately suppressed after 5 mm nitrate supply. However, the nodule growth rapidly recovered by changing the 5 mm nitrate solution to a 0 mm nitrate solution in the 5-0-0 and 5-5-0 treatments. In the 5-0-5 treatment, nodule growth was completely inhibited in the first and the third weeks with 5 mm nitrate, but the nodule growth was enhanced in the second week with 0 mm nitrate. The nodule growth response to 5 mm nitrate was similar between small and large size nodules. After the 5-5-5, 5-0-5, 0-0-5, and 0-5-5 treatments, where the plants were cultured with 5 mm nitrate in the last third week, the ARA per plant was significantly lower compared with the 0-0-0 treatment. On the other hand, the ARA after the 5-0-0 and 5-5-0 treatments was relatively higher than that after the 0-0-0 treatment, possibly due to the higher photosynthate supply associated with the vigorous vegetative growth of the plants supplemented with nitrate nitrogen. It is concluded that both soybean nodule growth and N₂ fixation activity sensitively responded to the external nitrate level, and that these parameters were reversibly regulated by the current status of nitrate in the culture solution, possibly through sensing of the nitrate concentration in roots and / or nodules.     

Molybdenum reserves of seed,and growth and N2 fixation by Phaseolus vulgaris L.

Summary Plants grown from seed with high (1.5–7.3 g Mo seed^-1) and low (0.07–1.4 g Mo seed^-1) Mo contents were grown in the presence and absence of Mo in growth media (perlite) or in a flowing-solution culture, in a controlled environment. Neither the high (1.5 g Mo seed^-1) nor the low (0.1 g Mo seed^-1) Mo content in seed from a small-seeded genotype (BAT 1297) was able to prevent Mo deficiency (reduced shoot, root and nodule dry weight, N₂ fixation and seed production) in growth media without an external supply of Mo, whereas both the high (7.3 g Mo seed^-1) and the low (0.07 g Mo seed^-1) contents in seed were able to prevent Mo deficiency in a large-seeded genotype (Canadian Wonder). Responses to Mo treatment by the Two genotypes were inconsistent between the growth media and solution culture experiments. Seed with a large Mo content (3.5 g Mo seed^-1) from the Canadian Wonder genotype was unable to prevent Mo deficiency (reduced shoot and nodule dry weight and N₂-fixation) in a solution culture without an external source of Mo, whereas both the large (1.7 g Mo seed^-1) and the small (0.13 g Mo seed^-1) contents in seed prevented a deficiency in BAT 1297. Growing plants from seed with a small Mo content, without additional Mo, reduced the seed Mo content by 83–85% and seed production by up to 38% in both genotypes. Changes in seed size and increases in shoot, root and nodule dry weight occurred, but varied with the genotype and growth conditions. These effects were also observed in some cases where plants were grown with additional Mo, demonstrating that the amount of Mo in the seed sown can influence plant nutrition irrespective of the external Mo supply. Nodule dry weight, total N content of shoots and seed production were improved by using seed with a small Mo content (1.64–3.57 g Mo seed^-1) on acid tropical soils in Northern Zambia. Plants of both the large- and small-seeded genotypes grown from seed with a small Mo content (<1.41 g Mo seed^-1) had a smaller nodule weight, accumulated less N and produced less seed. The viability of seed with a small Mo content was lower (germination up to 50% less) than that of seed with a large Mo content.     

Variation in nitrogen fixation among populations ofFrankia sp. andCeanothus sp. in actinorhizal association

Summary Wildland shrub improvement is needed for sound range and disturbed land revegetation practice. The possibility of selecting superior N₂-fixingFrankia-Ceanothus spp. actinorhizal associations was examined. Greenhouse tests were used to expose various soil-borne microsymbiont andCeanothus sp. population accessions in reciprocal combination. The acetylene reduction rate was used as a measure of N₂-fixation capacity. There was no significant interaction between host and microsymbiont regardless of source for all variables measured. The acetylene reduction rate, nodule number and mass, plant biomass, and root: shoot ratio were significantly different among soil sources. The acetylene reduction rate was not significantly different amongCeanothus sp. accessions. Neither was it strongly correlated with other variables. It was concluded that the N₂-fixation rate is more a function ofFrankia sp. than the hostCeanothus sp. in actinorhizal associations. It appears possible to select soil sources with superior N₂-fixing microsymbiont populations.The use of trade or firm names in this paper is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.This article was produced by government employees and is in the public domain and not subject to copyright. It may be freely reprinted with customary crediting of the source.     

Rhizobium sp. exopolysaccharide production,nodulation, and acetylene reduction activity (N2-fixation) in pigeon pea (Cajanus cajan L.)

Summary There was no correlation between the quantity of exopolysaccharide produced and acetylene reduction activity by Rhizobium spp. or by Bradyrhizobium spp. (Cajanus). The exopolysaccharide-defective mutants of Rhizobium sp. strain P 116 either failed to nodulate or showed a decrease in effectiveness. The deficiency in exopolysaccharide production was corrected by the addition of purified exopolysaccharide from the parent strain, or from Bradyrhizobium sp. strain P 149 or S24 isolated from pigeonpea (Cajanus cajan) and mungbean (Vigna radiata), respectively. However, the nodules so formed were not fully effective compared to those formed by the parent strain.     

Evaluation of 15N-isotope dilution for measurement of nitrogen fixation in chickpea (Cicer arietinum L.)

Summary N accumulation, nodulation, and acetylene reduction activity were measured at frequent intervals during the growth of two chickpea genotypes, and N₂ fixation was estimated by an isotope-dilution method, using safflower as a non-N₂-fixing reference. Safflower was more efficient at N uptake than both the chickpea genotypes for at least the first 50 days and thus could not be used as an accurate reference control. We recommend that further work should employ non-nodulatiog genotypes of chickpea as reference plants and use slow-release forms of 15N fertilizer. Direct genotype comparison by isotope dilution estimated that genotype K 850 fixed 16–18 kg ha^–1 more N than G 130, and this difference was supported by the greater nodule mass and acetylene reduction activity in the K 850 cultivar. Inoculation with an ineffective chickpea Rhizobium sp. led to 69% nodulation on cultivar K 850 but only 33% on G 130. While nodule weight, N uptake, and acetylene reduction activity decreased with inoculation in K 850, the isotope dilutions were similar for both inoculation treatments. The lack of a significant effect on N₂ fixation was ascribed to the partial success of inoculant establishment.Published as Journal Article No. JA 692 of the International Crops Research Institute for the Semi Arid Tropics, Patancheru, A.P. 502324, India     

Effect of CO2-free air treatment on nitrogen fixation of soybeans inoculated with Bradyrhizobium japonicum and Rhizobium fredii

Soybean (Glycine max L. Merr.) cultivars Akisirome and Peking were inoculated with Bradyrhizobium japonicum Is-21 and Rhizobium fredii USDA 194, respectively, and were grown in cylindrical pots containing sterilized vermiculite which were aerated with CO₂-free air or ambient air to study the effects of CO₂ deficiency in the rhizosphere on plant growth, nodulation, and nitrogen fixation. The repressive effects of CO₂-free air treatment were more conspicuous in Peking than Akisirome, and nodule number, nodule mass, amount of biologically fixed N and plant growth of Peking were reduced remarkably by the CO₂-free air treatment.

Acetylene reduction activity (ARA) of Peking inoculated with USDA 194 and Akisirome inoculated Is-21 was assayed in the absence and presence of CO₂, ARA of Akisirome was not affected by the absence of CO₂, while that of Peking decreased drastically.

Based on these results, it was concluded that nitrogen fixation by Akisirome inoculated with B. japonicum Is-21 was not appreciably influenced by CO₂ deficiency in the rhizosphere, while that of Peking inoculated with R. fredii USDA 194 was severely repressed, and the decrease was estimated to be due to both the reduction of the nodule mass and specific nitrogen-fixing activity.     

Effect of nitrogen on the yield response of Pennisetum americanum,Triticum aestivum and Zea mays to inoculation with Azospirillum brasilense under temperate conditions

Summary A nitrate-respiring strain, a denitrifying strain, and a non-nitrogen-fixing strain of Azospirillum brasilense were compared for their effect on the growth of pearl millet (Pennisetum americanum), wheat (Triticum aestivum) and maize (Zea mays) under temperate conditions in nitrogen-limited pot cultures. Increases in yield of Z. mays shoots occurred with all three strains when inoculation coincided with the addition of low levels of combined nitrogen. The inoculation of A. brasilense did not show any effect on the yield of P. americanum and T. aestivum. Increased numbers of A. brasilense became associated with Z. mays roots following the addition of low levels of combined nitrogen. Low and very variable rates of acetylene reduction activity were observed from excised roots of inoculated Z. mays plants without preincubation. Results indicate that inoculation of cereals with A. brasilense under temperate conditions has only a limited effect on plant growth.     

Effectiveness of foreign bradyrhizobia strains in enhancing nodulation, dry matter and seed yield of soybean (Glycine max L.) cultivars in Nigeria

 Field experiments were conducted to investigate the performance of three soybean cultivars with five foreign bradyrhizobia strains in different regions. The experiments at the two sites were designed with soybean (Glycine max L.) cultivars as the main factor and bradyrhizobia strains (USDA 136, TAL 122, USDA 6, TAL 377 and TAL 102) as the sub-factor. The experiments were arranged in randomised complete block design with four replications. Results show that nodule number, nodule dry weight and shoot dry weight, total N and seed yield were significantly increased when soybean cultivars were inoculated with foreign bradyrhizobia in two locations in the south east of Nigeria. At 63 days after planting the percentage increase in nodule number and dry weight after inoculation of soybean cultivars with bradyrhizobia strains ranged from 71 to 486% and from 0 to 200%, respectively. The percentage increase in shoot dry matter, %N and total N after bradyrhizobia inoculation ranged between 2–130%, 18–62% and 35–191%, respectively at Awka, and at the Igbariam site the percentage increase in shoot dry weight, %N and total N ranged between 3–76%, 0–43% and 19–125%, respectively. Seed yields after bradyrhizobia inoculation of soybean cultivar TGX 1485–1D at Igbariam ranged between 1.20 and 2.18 t ha^–1 against the uninoculated plants, which had seed yields of 1.05 t ha^–1. The poorest yield response after inoculation with bradyrhizobia strains was observed in soybean cultivar M-351, with a seed yield ranging from 0.60 to 0.98 t ha^–1. The fact that foreign bradyrhizobia strains were more effective than the indigenous strains for all the parameters studied suggests that there is a need to use bradyrhizobia inoculants for increased soybean production in Nigeria. The variations in the strain performance with the different soybean cultivars at the two sites, emphasises the need for careful Bradyrhizobium spp. strain selection. The fact that inoculation response was cultivar- and site-specific suggests that strategies for improving inoculation response in soybean cultivars should also consider the soil environment where the soybean is to be produced. Received: 25 May 1999