Effects of Rhizobium and vesicular-arbuscular mycorrhiza inoculations on nodulation,symbiotic nitrogen fixation and soybean yield in subtropical-tropical fields

Summary Field experiments were carried out to determine the effects of single and mixed inoculations with Rhizobium and vesicular-arbuscular mycorrhiza (VAM) on nodulation, symbiotic N₂ fixation and yield of soybeans in six Taiwan subtropical-tropical sites. Inoculation with Rhizobium alone significantly increased nodulation, nodule weight and nitrogenase activity of nodules in three out of six experimental fields, and affected soybean yields in the range –13% to + 134%. Inoculation with VAM fungi alone did not have a significant effect on nodulation and nitrogenase activity. Mycorrhiza inoculation affected soybean yields in the range –13% to + 65%, but only the yield increases at one out of six sites with N application were statistically significant. Mixed inoculation with Rhizobium and mycorrhiza affected yields in the range –8% to + 145% A synergistic effect from mixed inoculation of Rhizobium-mycorrhiza on soybean yields was found in one out of six experimental fields. The yield response to N application (40 kg N ha^–1) in these six paddy-field trials was not significant. These results suggest that single or mixed inoculation of rhizobia can greatly assist soybean grain yields and can replace N fertilizers.     

Use of aliette,a basipetally translocated antimicrobial compound,to enhance rhizophere colonization and nodulation by root-nodule bacteria

Summary A method was developed to improve the colonizing ability of inoculated strains of root-nodule bacteria using aliette (aluminum tris-O-ethyl phosphonate), a basipetally translocated fungicide. Aliette applied to seeds of alfalfa inoculated with an aliette-resistant strain of Rhizobium meliloti increased the numbers of R. meliloti in the rhizosphere after 3 but not 37 days, increased the number of nodules, and with some seed treatments, increased the growth of alfalfa. The enhanced colonization by R. meliloti as a result of seed treatment with aliette lasted for at least 31 days for alfalfa, although plant weights did not increase, Colonization by R. meliloti was further enhanced if seeds and foliage were treated with the fungicide. Coating seeds or sparaying the foliage with aliette also increased the number and weight of nodules and nitrogenase activity in soybeans inoculated with an aliette-resistant strain of Bradyrhizobium japonicum. The stimulation of B. japonicum in the rhizosphere and of nodulation was evident with successive plantings of soybeans if the seeds for each planting were treated with the chemical, but aliette did not increase the yield of inoculated soybeans in the subsequent plantings. With only the seeds of the first planting of inoculated soybeans treated with aliette, the numbers of B. japonicum in the rhizosphere of subsequent plantings were only occasionally greater and the numbers of nodules on the later plantings were not increased. We suggest that root colonization, nodulation, and N₂ fixation by Rhizobium and Bradyrhizobium may be enhanced by the use of basipetally translocated antimicrobial compounds together with root-nodule bacteria that are resistant to those compounds.     

Response of soybean cultivars toward inoculation with three arbuscular mycorrhizal fungi and Bradyrhizobium japonicum in the alluvial soil

The aim of this study was to assess the comparative efficacy of three arbuscular mycorrhizal fungi (AMF) combined with cultivar specific Bradyrhizobium japonicum (CSBJ) in soybean under greenhouse conditions. Soybean seeds of four cultivars namely JS 335, JS 71-05, NRC 2 and NRC 7 were inoculated with three AM fungi (Glomus intraradices, Acaulospora tuberculata and Gigaspora gigantea) and CSBJ isolates, individually or in combination, and were grown in pots using autoclaved alluvial soil of a non-legume cultivated field of Ajmer (Rajasthan). Assessment of the data on nodulation, plant growth and seed yield revealed that amongst the single inoculations of three AMF, G. intraradices produced the largest increases in the parameters studied followed by A. tuberculata and G. gigantea indicating that plant acted selectively on AMF symbiosis. The dual inoculation with AMF + CSBJ further improved these parameters demonstrating synergism between the two microsymbionts. Among all the dual treatments, G. intraradices + B. japonicum brought about the largest increases in the studied characteristics particularly in seed weight per plant that increased up to 115.19%, which suggested that a strong selective synergistic relationship existed between AMF and B. japonicum. The cv. JS 335 exhibited maximum positive response towards inoculation. The variations in efficacy of different treatments with different soybean cultivars indicate the specificity of the inoculation response. These results provide a basis for selection of an appropriate combination of specific AMF and Bradyrhizobium which could further be utilized for verifying the symbiotic effectiveness and competitive ability of microsymbionts under field conditions of Ajmer region.     

Selection of type A and type B strains for improving symbiotic effectiveness on non-Rj and Rj4 genotype soybean varieties

The selection of effective rhizobia for higher efficiency nitrogen fixation is one of the most important steps for inoculant production. Therefore, this experiment was conducted to select the most effective type A and type B strains for specific Rj-gene harboring soybean varieties and to test the symbiotic effectiveness of selected strains on different Rj-gene harboring soybean varieties. Screening experiments using the specific soybean varieties were done with a completely randomized design and three replications in this study. Evaluation of the effective Myanmar Bradyrhizobium strains for plant growth, nodulation and N₂ fixation were studied in pot experiments using sterilized vermiculite in the Phytotron (controlled-environmental condition). Then, a pot experiment was conducted using Futsukaichi soil in the screen house (natural environmental condition). The N₂ fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and the relative ureide index method. In the first screening experiment, type A and type B strains with higher nitrogen fixation and proper nodulation on their respective soybean cultivars were selected for the next screening. In the second screening, Bradyrhizobium elkanii AHY3-1 (type A), Bradyrhizobium japonicum SAY3-7 (type A), B. elkanii BLY3-8 (type B) and B. japonicum SAY3-10 (type B) isolates, which showed higher nitrogen fixation and nodulation in Yezin-3 (Rj₄) and Yezin-6 (non-Rj), were selected for the next experiment. In the third screening experiment, SAY3-7 and BLY3-8, which had higher nitrogen fixing potential and proper nodulation, were selected as effective isolates. These two isolates were compatible with non-Rj and Rj₄ soybean varieties for nodulation and nitrogen fixation. Based on the results of the screening experiment, these two strains were tested for their symbiotic efficacy in Futsukaichi soil. This study shows that inoculation treatment of SAY3-7 and BLY3-8 significantly increased plant growth, nodulation, and N₂ fixation at the V6, R3.5 and R8 stages in Yezin-3 (Rj₄) and/or Yezin-6 (non-Rj), and the seed yield at R8 stage, in Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean varieties compared with the control treatment. It can be concluded that SAY3-7 and BLY3-8 are suitable for inoculant production because of their higher nitrogen fixation ability, proper nodulation and better productivity of Myanmar soybean cultivars.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Effects of co-inoculation of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 on plant growth,nodulation, nitrogen fixation,nutrient uptake,and yield of soybean in a field condition

ABSTRACT

Co-inoculation of nitrogen-fixing bacteria with plant growth-promoting bacteria has become more popular than single inoculation of rhizobia or plant-growth-promoting bacteria because of the synergy of these bacteria in increasing soybean yield and nitrogen fixation. This study was conducted to investigate the effects of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 co-inoculation on plant growth, nodulation, nitrogen fixation, nutrient uptake, and seed yield of the ‘Yezin-6’ soybean cultivar. Nitrogen fixation was measured using the acetylene reduction assay and ureide methods. Uptake of major nutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] was also measured. This study showed that single inoculation of SAY3-7 significantly increased shoot biomass; nodulation; Relative Ureide Index (RUI %), percent nitrogen derived from N fixation (% Ndfa); N, P, K, Ca, and Mg uptakes; during the later growth stages (R3.5 and R5.5), compared with control. These observations indicate that SAY3-7 is an effective N-fixing bacterium for the plant growth, nodulation, and nitrogen fixation with an ability to compete with native bradyrhizobia. Co-inoculation of SAY3-7 and P4 significantly improved nodule number; nodule dry weight; shoot and root biomass; N fixation; N, P, K, Ca, and Mg uptake; at various growth stages and seed yield in ‘Yezin-6’ soybean cultivar compared with the control, but not the single inoculation treatments. Significant differences in plant growth, nodulation, N fixation, nutrient uptake, and yield between co-inoculation and control, not between single inoculation and control, suggest that there is a synergetic effect due to co-inoculation of SAY3-7 and P4. Therefore, we conclude that Myanmar Bradyrhizobium strain SAY3-7 and P4 will be useful as effective inoculants in biofertilizer production in the future.     

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.     

Response of Soybean to dual Inoculation with Rhizobium japonicum and Phosphate dissolving Bacteria

A field experiment in silty clay soil was carried out to evaluate the effect of dual inoculation with Rhizobium japonicum and phosphate dissolving bacteria (PDB) on nitrogen and phosphorus uptakes as well as seed yield of soybean. Seed inoculation with PDB before planting did not induce marked enrichment of PDB counts in the rhizosphere. Inoculation with R. japonicum alone increased the dry weight, N-uptake and seed yield significantly. Further significant increases in P-uptake and seed yield were recorded by inoculation with rhizobia and fertilization with superphosphate. However, the inoculation with PDB in combination with R. japonicum in the presence of rock phosphate or superphosphate did not show significant increases in dry weight, N and P uptakes or seed yield as compared with the treatments inoculated with rhizobia alone.     

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%.     

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.     

Soybean preference for Bradyrhizobium japonicum for modulation

Abstract

Caldwell and Vest (1968) planted soybeans (Glycine max L. Merr.) with various genotypes at Beltsville, USA, without inoculating them with Bradyrhizobium japonicum, and showed that soybeans preferred certain serotypes of rhizobial strains for nodulation. Recently, the authors have reported that soybeans carrying nodulation-conditioning genes preferred appropriate strains showing specific behavior for nodulation (Ishizuka et al. 1991). For instance, nodulation of soybean cv. Hardee which carries the nodulation-conditioning genes, Rj ₂ and Rj ₃, does not occur with B. japonicum USDA122, USDA33, Is-1, etc. Nodulation of cv. Hill which carries the Rj ₄ gene, does not occur with B. japonicum USDA61, Is-21, etc. while A62-2 which carries a recessive gene rj ₁, does not nodulate with almost any of the strains of B. japonicum. Therefore, the B. japonicum strains can be classified into three nodulation types based on the compatibility with these Rj-cultivars, that is, type A strains which effectively nodulated both Rj ₂ Rj ₃-cultivars and Rj ₄-ones, type B strains which did not nodulate the Rj ₂ Rj ₃-cultivars and type C strains which did not nodulate the Rj ₄-cultivars. When the nodulation types of the isolates from nodules of field-grown soybeans were examined, it was suggested that the Rj ₂ Rj ₃-cultivars and Rj ₄-cultivars preferred the type C and type B strains, respectively (Ishizuka et al. 1991).     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

Bacillus amyloliquefaciens BNM122, a potential microbial biocontrol agent applied on soybean seeds,causes a minor impact on rhizosphere and soil microbial communities

The increase in soybean productivity has contributed to a greater use of agrochemicals, which cause major problems, such as soil and water pollution and reduction of biodiversity, and have a negative impact on non-target species. The development of microbial biocontrol agents for soybean diseases can help to reduce pesticide abuse. Bacillus amyloliquefaciens BNM122 is a potential microbial biocontrol agent able to control the damping-off caused by Rhizoctonia solani when inoculated in soybean seeds, both in a plant growth chamber and in a greenhouse. In this study, we report the effect of soybean seed treatments with strain BNM122 or with two fungicides (thiram and carbendazim) on the structure and function of the bacterial community that colonizes the soybean rhizosphere. Also, soybean root nodulation by Bradyrhizobium japonicum, mycorrhization by arbuscular mycorrhizal fungi and plant growth were evaluated. We used the r- and K-strategist concept to evaluate the ecophysiological structure of the culturable bacterial community, community-level physiological profiles (CLPP) in Biolog? EcoPlates to study bacterial functionality, and the patterns of 16S RNA genes amplified by PCR and separated by denaturing gradient gel electrophoresis (PCR–DGGE) to assess the genetic structure of the bacterial community. Neither the ecophysiological structure nor the physiological profiles of the soybean rhizosphere bacterial community showed important changes after seed inoculation with strain BNM122. On the contrary, seed treatment with fungicides increased the proportions of r-strategists and altered the metabolic profiles of the rhizosphere culturable bacterial community. The genetic structure of the rhizosphere bacterial community did not show perceptible changes between treated and non-treated seeds. Regarding the bacterial and fungal symbioses, seed treatments did not affect soybean nodulation, whereas soybean mycorrhization significantly decreased (P < 0.05) in plants obtained from seeds treated with strain BNM122 or with the fungicides. However, a higher negative effect was observed in plants which seeds were treated with the fungicides. Plant growth was not affected by seed treatments.It can be concluded that soybean seed treatment with B. amyloliquefaciens BNM122 had a lesser effect on soil microbial community than that with the fungicides, and that these differences may be attributed to the less environmental persistence and toxic effects of the strain, which deserve further studies in order to develop commercial formulations.     

Growth and yield of soybean varieties inoculated with <Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp in N-deficient calcareous soils

This research reports the effect of Bradyrhizobium spp strains on dry matter yield, nodulation and seed yield of soybean varieties grown in N-deficient soil in Uzbekistan in pot and field experiments, in 2001 and 2002. The results of experiments revealed that significant positive effects on growth, nodule number and yield of soybean were obtained after inoculation with Bradyrhizobium spp strains; and, generally, strains S62 and S63 were more effective than strain S61. The protein content of seeds also increased after inoculation. Both Orzu and Uzb2 soybean varieties gave a higher response than the Uzb6 variety.     

Effects of low temperature and shade on relationships between nodulation,vesicular-arbuscular mycorrhizal infection,and shoot growth of soybeans

The effects of low temperature and reduced light on a Glycine-Bradyrhizobium-Glomus spp. symbiosis were examined in pot experiments. Soybean plants, Glycine max L. Merr. cv. Tachiyutaka, were grown with N fertilization or inoculation with Bradyrhizobium japonicum plus P fertilization or inoculation with Glomus mosseae in the glasshouse. After the flowering stage, half the pots with soybean plants were subjected to low temperature (15°C 14h/13°C 10 h) with light reduced by shading. At 0, 7, 16, and 28 days after the application of the treatments, the growth, nodulation, vesicular-arbuscular mycorrhizal (VAM) infection and the N and P contents of the soybean plants were measured. In all symbiont-fertilization combinations, the low-temperature treatment reduced the production of dry matter by the soybeans. Nodulation (weight and number) was slightly reduced by this treatment but the proportion of larger nodules was increased. The root length infected by the VAM fungus was little affected by the low-temperature treatment. Both the nodule weight and the infected root length were linearly related to shoot dry weight regardless of treatment and of the symbiont-fertilization combination used. These results suggest that the growth of the symbionts on the root was in balance with the shoot growth of the host, irrespective of climatic conditions, and imply a considerable degree of host control. P inflows to root systems were greatly affected by low-temperature treatment regardless of the symbiont-fertilization combination. This suggests that a simple comparison of P inflows between mycorrhizal and non-mycorrhizal plants may give misleading information on the effects of low temperature or reduced light conditions on P uptake by mycorrhizal plants.     

Effect of soil physical properties on soybean nodulation and N2 fixation at the early growth stage in heavy soil field in Hachirougata Polder,Japan

We studied the effect of the soil physical properties on soybean nodulation and N₂ fixation in the heavy soil of an upland field (UF) and an upland field converted from a paddy field (UCPF) in the Hachirougata polder, Japan. Seeds of the soybean cultivar Ryuho were sown in each field with or without inoculation of Bradyrhizobium japonicum A1017. The soybean plants were sampled at 35 (V3) and 65 (Rl) d after sowing (DAS), and then nodulation and the percentage of N derived from N₂ fixation in the xylem sap were determined. The soil physical properties were different between UF and UCPF, especially the air permeability and soil water regime. Nodule growth was restricted in UCPF irrespective of rhizobial inoculation, though rhizobial infection was not inhibited by the unfavorable soil physical conditions. Soybean plant growth was closely related to the nodule mass and N₂ fixation activity, and the inoculation of a superior rhizobium strain was effective only at 35 DAS. These results indicate that soybean nodulation and N₂ fixation was considerably affected by the physical properties of heavy soil, and that it is important to maintain the N₂ fixation activity and inoculate the soybean plants with a superior rhizobium strain at a later growth stage in order to increase soybean production in heavy soil fields.     

Enhanced soybean (Glycine max L.) plant growth and nodulation by Bradyrhizobium japonicum-SB1 in presence of Bacillus thuringiensis-KR1

Abstract

Nodulation and subsequent nitrogen fixation are important factors that determine the productivity of soybean (Glycine max L.). The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote growth of soybean plants (variety VL Soya 2) under Jensen's tube and growth pouch conditions, when co-inoculated with Bradyrhizobium japonicum-SB1. Co-inoculation with Bacillus thuringiensis-KR1 (at a cell density of 10 cfu) provided the highest and most consistent increase in nodule number, shoot weight, root weight, root volume, and total biomass, over rhizobial inoculation and control, under both conditions. The results demonstrate the potential benefits of using nonrhizobial nodule occupants of wild legumes for the co-inoculation of soybean, with Bradyrhizobium japonicum-SB1, in order to achieve plant-growth promotion and increased nodulation.     

Effects of co-inoculation of Bradyrhizobium elkanii BLY3-8 and Streptomyces griseoflavus P4 on Rj4 soybean varieties

Co-inoculation of selected nitrogen-fixing bacteria with plant growth-promoting bacteria is the promising way for the improvement of soybean production through enhancing plant growth, nodulation, and N₂ fixation. Therefore, this experiment was conducted to study the effects of co-inoculation of Bradyrhizobium elkanii BLY3-8 with Streptomyces griseoflavus P4 on plant growth, nodulation, N₂ fixation, N uptake, and seed yield of Rj₄ soybean varieties. Two experiments with completely randomized design and three replicates were done in this study. N₂-fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and relative ureide method. In the first experiment, synergetic effect in N₂ fixation and nodulation was occurred in co-inoculation treatment (BLY3-8 + P4) in Yezin-3 and Fukuyutaka. Based on these results, co-inoculation effect of BLY3-8 and P4 was assessed on Yezin-3 and Fukuyutaka varieties at three different growth stages, using Futsukaichi soil under natural environmental conditions. This study shows that co-inoculation of BLY3-8 and P4 significantly increased N₂ fixation at V6 stage; plant growth, nodulation, N₂ fixation, and N uptake at R3.5 stage; and shoot growth, N uptake, and seed yield at R8 stage, in Rj₄ soybean varieties compared with the control. Significant difference in plant growth, nodulation, N₂ fixation, N uptake, and yield between co-inoculation and control, not between single inoculation and control, suggests that there is a synergetic effect due to co-inoculation of BLY3-8 and P4.     

Isolation of transposon Tn5-induced hydrophobic mutants of a Bradyrhizohium japonicum strain with improved competitive nodulation abilities

Abstract

Hydrophobic mutants of the Bradyrhizohium japonicum strain 138NR were obtained by transposon Tn5 mutagenesis followed by replica-plating on polystyrene plates. Fifteen mutants were isolated at a frequency of 10⁶. Gel-filtration analysis of the exopolysaccharides revealed that the hydrophobic mutants produced a significantly smaller amount of low-molecular-weight polysaccharides than the parent. Four of the isolated mutants formed a larger number of nodules on soybean (Glycine max L. Merr. cv. Tamahomare) than the parent, two were superior in symbiotic nitrogen fixation, and two were symbiotically defective on soybean. Competitive nodulation abilities of the mutants were examined by inoculating them to soybean with B. japonicum strain 123ET as a competitor and determining the nodule occupancy based on the antibiotic resistance. All the mutants tested except for the symbiotically defective ones were superior in their competitive nodulation ability to the parent strain. When inoculated 24 h before the inoculation with 123ET, the mutants exhibited an increased nodule occupancy (44–93% compared to 9% by the parent).     

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