Variability in yield and yield component responses to genistein pre-incubated Bradyrhizobium japonicum by soybean [Glycine max (L.) Merr] cultivars

The sub-tropical legume, soybean [Glycine max (L.) Merr.], has lower grain yields at low temperatures, mainly due to reduced nitrogen fixation. The isoflavone genistein has been identified as one of the major compounds in soybean seed and root extracts responsible for inducing the expression of the B. japonicum nod genes. A 2-year field study was conducted in 1997 and 1998 with 11 soybean cultivars recommended for Québec, and representing a range of yield potentials and maturity groups. The objective of this study was to assess the variability among soybean cultivar maturity groups in terms of response to genistein application under Canadian short season and cool-spring conditions. The experiments were organized in a randomized complete block design with three replications. The two genistein treatments included B. japonicum inoculant pre-incubated with 20 m genistein and B. japonicum inoculant only. The inoculants were applied into the furrow at the time of planting. The results of this study showed that genistein pre-incubated B. japonicum increased soybean grain yield and protein content over two years. In 1998, pod number per plant^–1 and seed number plant^–1 were also clearly increased. When 20 m genistein was applied in 1998, cultivars in the late maturity group had 28 and 70% more shoot and total protein content, respectively, than the early maturity groups with or without genistein, or the late maturity without genistein, in 1998. There was no interaction between genistein application and soybean cultivar in this study, indicating that both early and late maturing cultivars responded similarly to genistein pretreated inocula. Pre-incubation of B. japonicum with genistein can increase N₂ fixation potential in short season areas. Key words: Soybean, cultivars, genistein, yield, and yield components     

Preincubation of B. japonicum cells with genistein reduces the inhibitory effects of mineral nitrogen on soybean nodulation and nitrogen fixation under field conditions

Genistein is the major root produced isoflavonoid inducer of nod genes in the symbiosis between B. japonicum and soybean plants. Reduction in the isoflavonoid content of the host plants has recently been suggested as a possible explanation for the inhibition of mineral nitrogen (N) on the establishment of the symbiosis. In order to determine whether genistein addition could overcome this inhibition, we incubated B. japonicum cells (strain 532C) with genistein. Mineral N (in the form of NH₄NO₃) was applied at 0, 20 and 100 kg ha^-1. The experiments were conducted on both a sandy-loam soil and a clay-loam soil. Preincubation of B. japonicum cells with genistein increased soybean nodule number and nodule weight, especially in the low-N-containing sandy-loam soil and the low N fertilizer treatment. Plant growth and yield were less affected by genistein preincubation treatments than nitrogen assimilation. Total plant nitrogen content was increased by the two genistein preincubation treatments at the early flowering stage. At maturity, shoot and total plant nitrogen contents were increased by the 40 μM genistein preincubation treatment at the sandy-loam soil site. Total nitrogen contents were increased by the 20 μM genistein preincubation treatment only at the 0 and 20 kg ha^-1 nitrate levels in clay-loam soil. Forty μM genistein preincubation treatment increased soybean yield on the sandy-loam soil. There was no difference among treatments for 100-seed weight. The results suggest that preincubation of B. japonicum cells with genistein could improve soybean nodulation and nitrogen fixation, and at least partially overcome the inhibition of mineral nitrogen on soybean nodulation and nitrogen fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inoculation of pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and hesperetin or application of naringenin and hesperetin directly into soil increased pea nodulation under short season conditions

In short season areas, low soil temperature is the major limiting factor for symbiotic nitrogen fixation of legume. One greenhouse and four field experiments were conducted in 1999 to determine whether the pre-incubation of Rhizobium leguminosarum bv. viceae with hesperetin and naringenin or application of these compounds onto the seed surface or into the seed furrow at the time of planting can increase pea nodulation and final grain yield. The results from these experiments clearly indicated that application of naringenin and hesperetin by either pre-incubating R. leguminosarum bv. viceae prior to inoculation of plant or directly applying onto the seed surface or into seed furrow at the time of planting can increase pea nodulation, and plant pod numbers. Interactions existed between symbiotic signal compounds and pea cultivars or R. leguminosarum bv. viceae strains. However, there was no impact on the final grain yield by the treatments from the field experiments. The effects of these treatments on the final grain yield have to be farther tested.     

Application of genistein to inocula and soil to overcome low spring soil temperature inhibition of soybean nodulation and nitrogen fixation

In the soybean (Glycine max. (L.) Merr)– Bradyrhizobium japonicum symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development by disruption of the interorganismal signal exchange between the host plant and bradyrhizobia. Two field experiments were conducted on two adjacent sites in 1994 to determine whether the incubation of B. japonicum with genistein prior to application as an inoculant, or genistein, without B. japonicum, applied onto seeds in the furrow at the time of planting, increased soybean nodulation, N fixation, and total N yield. The results of these experiments indicated that genistein application increased nodule number and nodule dry matter per plant and hastened the onset of N fixation during the early portion of the soybean growing season, when the soils were still cool. Because these variables were improved, total fixed. N, fixed N as a percentage of total plant N, and N yield increased due to genistein application. The interaction between genistein application and soybean cultivars indicated that genistein application was more effective on N-stressed plants.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Application of gibberellic acid to the surface of soybean seed (t Glycine max (L.) Merr.) and symbiotic nodulation,plant development,final grain and protein yield under short season conditions

In short-season soybean production areas, low soil temperature is the major factor limiting soybean establishment, nodulation and nitrogen fixation. Gibberellic acid (GA) pretreatment of crop seeds can overcome low soil temperature inhibition of seed germination and seedling development. However, previous studies have found that the application of GAs decreased legume nodulation and nitrogen fixation under optimal growth conditions. A field experiment was conducted under short season conditions in eastern Canada to determine whether the application of GA₃ to soybean seed could accelerate germination, and increase plant nodulation and nitrogen fixation. The results indicated that GA₃ application accelerated seedling emergence but decreased plant nodulation and nitrogen accumulation at early plant growth stages. However, these initial negative effects were overcome as the plants developed. Gibberellic acid applied to soybean seed at the time of planting did not influence final grain and protein yield.     

Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha^-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the cowpea cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.     

The Use of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> to Enhance Growth and Yield of Soybean in Calcareous Soil in Uzbekistan

In this work the effect of inoculation with Bradyrhizobium japonicum S2492 on soybean (Glycine max (L) Merr) growth, nodulation and yield in nitrogen-deficient soil of Uzbekistan was studied. The field experiments were carried out in Tashkent Province of Uzbekistan in a randomized complete block design with four replicates of each treatment. The results revealed positive effects on growth, nodule number and yields of soybean after inoculation with B. japonicum S2492. The yield of soybean varieties was 48% higher for inoculated than for uninoculated plants. The effect of the inoculation was specific for variety but not for growth type. The protein and oil contents of seeds also increased after inoculation. It was concluded that B. japonicum S2492 can be considered as a biofertilizer for increasing the productivity of soybean in nitrogen-deficient soils in Uzbekistan.     

The influence of soybean planting density on dinitrogen fixation and yield

We investigated the effect of planting density on soybean (Glycine max (L.) Merr.) yield in glasshouse and field experiments. Because net canopy photosynthesis increases with increasing plant density, we hypothesized that increasing planting density would result in increasing rates of dinitrogen fixation in soybeans and higher yields per unit land area.In glasshouse studies, Wayne variety soybeans were planted in 10-cm diameter pots, 1 plant pot^-1 in matrices of 10-, 15-, 20-, 25-, or 30-cm equidistant intervals. Bradyrhizobium japonicum inoculum was added to half of the plants in each treatment. Replicate measurements of total stem height, internode lengths, leaf mass, stem mass, root mass, nodule number, nodule mass, and nitrogenase activity were obtained at 3, 6, and 9 weeks post-emergence. Fruits were harvested and counted at week 14. As planting density increased there were (1) altered morphology and growth rates, (2) increased apparent specific nodule activity (SNA), (3) decreased nodule number and mass, and (4) nearly constant fruit and seed production/plant. Expressed on a unit area basis, nitrogen influx and yield increased geometrically as planting density increased, with maximum values observed for 10-cm plantings.Field studies of Wayne, Stein, Williams, and Gold Harvest soybean varieties were made in 1985. Plots were established containing 100 plants spaced at 10-, 20-, and 30-cm distances. Measurements made during the growing season and at harvest established the same relative trends identified from the glasshouse studies. Increasing plant densities resulted in higher yields per unit land. Varietal differences were almost significant.     

Morphological and physiological response of soybean treated with the microsymbiont Bradyrhizobium japonicum pre-incubated with genistein

Genistein, a major root-secreted isoflavone of soybean (Glycine max (L.) Merr), is critical for the legume-Bradyrhizobium symbiosis as it induces several bacterial nod-gene systems. An experiment with soybean grown under salt stress was conducted to evaluate the effect of exogenous genistein addition to the Bradyrhizobium culture medium on subsequent nodulation, nitrogen fixation and selected plant physiological attributes. Five day-old plants (in pots) were inoculated with a liquid B. japonicum broth culture and irrigated with B&D solution containing either 0, 25, 50 and 100 mM NaCl. Four weeks after inoculation, maximum photochemical efficiency of PSII (Fv/Fm), photosynthetic rate, stomatal conductance, and transpiration rate were measured. Number of nodules per plant and apparent nitrogen fixation (as acetylene reduction activity) were determined. Salt stress decreased nodule number/plant and nitrogenase activity/plant and induced large changes of both photosynthetic parameters and antioxidant enzyme activity, compared to the control, genistein reversed the effect in each level of salinity tested. Moreover, pre-treatment of the microsymbiont with genistein enhanced maximum photochemical efficiency, photosynthetic rate, stomatal conductance and transpiration rate, while the enzymatic activities of catalase, superoxide dismutase and peroxidase in leaves and roots were not affected. It can be concluded that preincubation of the B. japonicum inoculant with genistein probably contributed towards growth in soybean via enhancement of nodulation and nitrogen fixation under both normal and salt stress conditions.     

Nitrogen fixation by soybeans (Glycine max L.) in Zambia

Soybeans (Glycine max L.) are being introduced as a cash crop to small scale farmers in Zambia for rotation in their farming systems. The objectives of this study were to compare and select the most approriate non-fixing reference crop for estimating N₂ fixation by soybeans and assess yields and N₂ fixation of soybeans in Zambia. Nitrogen isotope dilution techniques using¹⁵N-labelled organic or inorganic materials were utilized. Two nonnodulating soybean cultivars, Clark RJ1 and N77 or in their absence Pearl millet (Panicum glaucum L.) were judged to be appropriate reference crops. A local soybean fixing cultivar (Glycine max L. cv. Magoye) rated highest among three cultivars tested for its ability to support symbiotic N₂ fixation byB. japonicum under the experimental conditions. Values of percent N derived from atomosphere for this cultivar were in the order of 65 to 70%.deceased.Contribution no R531 of the Saskatchewan Institute of Pedology. Present address (REK): Esso Chemical Canada, P.O. Box 3010, Lethbridge, Alberta Canada T1J 4A9.     

Pre-incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures

Jasmonic acid (JA) and methyl jasmonate, collectively known as jasmonates, are naturally occurring in plants; they are important signal molecules involved in induced disease resistance and mediate many physiological activities in plants. We studied the effect of JA and its methyl ester, methyl jasmonate (MeJA), on the induction of nod genes in Bradyrhizobium japonicum GG4 (USDA3) carrying a plasmid with a translational fusion between B. japonicum nodY and lacZ of Escherichia coli, and the expression activity was measured by β-galactosidase activity. Both JA and MeJA strongly induced the expression of nod genes. They have little or no deleterious effects on the growth of B. japonicum cells, while genistein (Gen) showed inhibitory effects. We further studied the effect of JA- and MeJA-induced B. japonicum on soybean nodulation and nitrogen fixation under optimal (25°C) and suboptimal (17°C) root zone temperature (RZT) conditions. B. japonicum cells were grown in liquid yeast extract mannitol media and induced with a range of Gen, JA, and MeJA concentrations, including a treatment control with no inducer added. Soybean seedlings were grown at 25 or 17°C RZT with a constant air temperature (25°C) and inoculated, at the vegetative cotyledonary stage, with various B. japonicum induction treatments. Addition of Gen or jasmonates to B. japonicum, prior to inoculation, enhanced nodulation, nitrogen fixation, and plant growth at suboptimal RZT conditions. A higher concentration of Gen was inhibitory at 25°C, while this same concentration was stimulatory at 17°C. Interestingly, pre-incubation of B. japonicum with JA and MeJA enhanced soybean nodulation and nitrogen fixation under both optimal and suboptimal RZTs. We show that jasmonates are thus a new class of signaling molecules in the B. japonicum-soybean symbiosis and that pre-induction of B. japonicum with jasmonates can be used to enhance soybean nodulation, nitrogen fixation, and early plant growth.     

Application of plant growth-promoting rhizobacteria to soybean (Glycine max [L.] Merr.) increases protein and dry matter yield under short-season conditions

We previously reported that application of plant growth-promoting rhizobacteria (PGPR) increased soybean growth and development and, specifically, increased nodulation and nitrogen fixation over a range of root zone temperatures (RZTs) in controlled environment studies. In order to expand on the previous studies, field experiments were conducted on two adjacent sites, one fumigated with methyl bromide and one nonfumigated, in 1994. Two experiments were conducted at each site, one involving combinations of two soybean cultivars and two PGPR strains, the other involving the same factors, but also in combination with two strains Bradyrhizobium japonicum. Soybean grain yield and protein yield were measured. The results of these experiments indicated that co-inoculation of soybean with B. japonicum and Serratia liquefaciens 2-68 or Serratia proteamaculans 1-102 increased soybean grain yield, protein yield, and total plant protein production, compared to the nontreated controls, in an area with low spring soil temperatures. Interactions existed between PGPR application and soybean cultivar, suggesting that PGPRs applied to cultivars with higher yield potentials were more effective. PGPRs applied to the rhizosphere without addition of B. japonicum also increased only leaf area and seed number at the fumigated site. Overall, inoculation of soybean plants with PGPRs in the presence of B. japonicum increased soybean grain yield, grain protein yield, and total plant protein production under short season conditions.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Genistein accumulation in soybean (Glycine max [L.] Merr.) root systems under suboptimal root zone temperatures

Genistein, as a plant-to-bacteria signal, plays an importantrole in the establishment of the soybean (Glycine max [L.] Merr.)-Bradyrhizobiumjaponicum nitrogen-fixing symbiosis. It is essential to thedevelopment of effective root nodules and responsible for inducingthe nod genes of B. japonicum. Because sub-optimal root zonetemperature (RZT) delays infection and early nodule development,and decreases plant nodule number, and genistein addition overcomessome of this, it is reasonable to hypothesize that suboptimalRZT disrupts the inter-organismal signal exchange by inhibitinggenistein synthesis. Four experiments were conducted to testthese hypotheses. The results of these studies indicated that:(1) when soybean plants were germinated and maintained at RZTsranging from 13 to 17C, root genistein concentration and contentper plant were lower than those of plants with roots maintainedat RZTs above 17C; (2) when plants were germinated at an optimalRZT (25 C) then transferred to RZTs below 17C, and acclimatedfor a few days, root genistein concentration and content perplant were higher than those of plants with roots maintainedeither at optimal RZT, or transferred to RZT above 17 C, althoughby the end of the experiment, the genistein concentration ofroot systems at below 17C RZT appeared to be declining to valuesbelow those of plants with above 17 C RZT; (3) the root genisteinconcentration increased before the onset of nitrogen fixationand decreased thereafter; and (4) part of the effect of RZTson genistein content per plant root system was from reductionsin genistein concentration at lower RZT5, and part was due todecreased plant root growth. Key words: Genistein, Glycine max, suboptimal temperature     

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 g ml^-1. Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30–70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - SBA soybean agglutinin     

Establishment of Bradyrhizobium japonicum for soybean by inoculation of a preceding wheat crop

On fields with no history of soybean (Glycine max (L.) Merr.) production, inoculation alone is often inadequate to provide for adequate nodulation the first time this crop is grown. The objective of this study was to determine if inoculation of spring wheat (Triticum aestivum L.) seed with Bradyrhizobium japonicum would lead to an increase of B. japonicum numbers in the soil, and improve nodulation of a subsequent soybean crop. In the greenhouse, wheat seed inoculation increased B. japonicum numbers from undetectable numbers to >9000 g^–1 of soil, whereas the numbers of introduced B. japonicum declined in unseeded pots. In the field, inoculation of wheat seed increased B. japonicum numbers in the soil from undetectable levels to >4000 g^–1 the following year. When soybean seed was inoculated, but grown in soil devoid of B. japonicum, nodules formed only near the point of seed placement. The heaviest nodulation, and widest distribution of nodules in the topsoil were found whenB. japonicum was established the year before by wheat seed inoculation, plus soybean seed inoculation. Wheat seed inoculation the year before growing soybean, combined with proper soybean seed inoculation, should provide for abundant nodulation the first time soybean is grown on a field.