Preincubation of Bradyrhizobium japonicum with Genistein Accelerates Nodule Development of Soybean at Suboptimal Root Zone Temperatures

In the soybean (Glycine max [L.] Merr.) N2-fixing symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development, especially at temperatures below 17[deg]C. A step in the infection process that occurs within the first 24 h is particularly sensitive to suboptimal RZT. The first phase in the establishment of the soybean-Bradyrhizobium japonicum symbiosis is the exchange of recognition molecules. The most effective plant-to-bacterium signal is genistein. Binding of genistein to B. japonicum activates many of the B. japonicum nod genes. To our knowledge, the potential of sub-optimal RZT to disrupt this interorganismal signaling has not previously been investigated. Controlled environment experiments were conducted to determine whether the preincubation of B. japonicum with genistein increases soybean nodulation and N2 fixation at suboptimal RZT and whether the time between inoculation and root-hair curling is shortened by genistein application. The results of these experiments indicated that (a) genistein application increased soybean nodulation at suboptimal RZTs (17.5 and 15[deg]C) but not at the optimal RZT (25[deg]C); (b) the period between inoculation and root-hair curling was shortened by inoculation with bradyrhizobia preincubated with genistein; (c) at 17.5 and 15[deg]C RZT, the onset of N2 fixation occurred earlier in plants that received genistein-treated bradyrhizobia than in plants inoculated with untreated bradyrhizobia; (d) over the tested concentration range, genistein application at 15 to 20 [mu]M was the most effective in stimulating nodulation; and (e) between 25 and 15[deg]C, as RZT decreased, there was an increase in the nodulation-stimulating potential of genistein.     

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.     

Inoculation of soybean (Glycine max. (L.) Merr.) with genistein-preincubated Bradyrhizobium japonicum or genistein directly applied into soil increases soybean protein and dry matter yield under short season conditions

In short-season soybean production areas, low soil temperature is the major factor limiting plant growth and yield. The decreases in soybean yield at low temperatures are mainly due to nitrogen limitation. Genistein, the most effective plant-to-bacterium signal in the soybean (Glycine max (L.) Merr.) nitrogen fixation symbiosis, was used to pretreat Bradyrhizobium japonicum. We have previously reported that this increased soybean nodulation and nitrogen fixation in growth chamber studies. 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 grain yield and protein yield in short season areas. The results of these experiments indicated that genistein-preincubated bradyrhizobia increased the grain yield and protein yield of AC Bravor, the later maturing of the two cultivars tested. Genistein without B. japonicum, applied onto seeds in the furrow at the time of planting also increased both grain and protein yield by stimulation of native soil B. japonicum. Interactions existed between genistein application and soybean cultivars, and indicated that the cultivar with the greatest yield potential responded more to genistein addition.     

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.     

Plant growth promoting rhizobacteria accelerate nodulation and increase nitrogen fixation activity by field grown soybean [Glycine max (L.) Merr.] under short season conditions

A 3 × 2 × 2 factorial field experiment, organized in a randomized complete block split-plot with four replications, was conducted in 1994 to evaluate the effect of two plant growth-promoting rhizobacteria (PGPR) strains (Serratia liquefaciens 2-68 or Serratia proteamaculans 1-102) on nodulation, nitrogen fixation, and total nitrogen yield by two soybean cultivars in a short season area. The experiments were conducted at the Emile A. Lods Research Centre, McGill University, Macdonald Campus, Montreal, Canada, and performed at two adjacent sites. One site was fumigated with methyl bromide (50 g m^-2). Another site was kept unfumigated. Co-inoculation of soybean with B. japonicum and PGPR increased soybean nodulation and hastened the onset of nitrogen fixation, when the soils were still cool. Total fixed N, fixed N as a percentage of total plant N, and protein and N yield were also increased by PGPR inoculation. AC Bravor tended to be more responsive to both PGPR treatments for total fixed N and N yields than Maple Glen, suggesting that inoculation with PGPR was more effective for cultivars with higher yield potentials.     

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     

Restriction of Nodulation by Bradyrhizobium japonicum Is Mediated by Factors Present in the Roots of Glycine max

Reciprocal grafting experiments done using soybean plant introduction genotypes indicated that restriction of nodulation by Bradyrhizobium japonicum is determined by the genotype of the root and is dependent on plant growth temperature. Microscopic analyses indicated that the soybean plant introduction genotypes restrict nodulation of B. japonicum at symbiotic stages which occur both before and after the formation of nodule primordia.     

Effects of delayed soil and foliar N fertilization on yield and N2 fixation of soybean

Summary Field experiments were conducted to determine the effects of the amount, time and method of fertilizer N application on the efficiency of N uptake, N₂ fixatio and yield of soybean. Soil and foliar fertilizer N, applied during the pod-filling stage were absorbed by plants with equal and high efficiency, compared to an appreciably lower utilization efficiency for N applied before seedling emergence. These results reveal that the soybean roots were active in N uptake during these late stages of growth. Nitrogen fertilization during pod-filling resulted in significant yield increases over the control treatment which received an early application of 20 Kg N/ha. Seed yield increases were, however, more pronounced than total dry matter yield, and virtually all of the late-applied N was translocated into the pods. Nitrogen fixation in soybean was not influenced by the application of 40 kg N/ha to plants as soil or foliar N during the pod-filling stage. However, 80 kg N/ha supplied during pod-filling as 40 kg soil plus 40 kg foliar N/ha significantly reduced the amount of N₂ fixed. The results obtained in these studies suggest that inadequate N supply during pod-filling limited soybean yields, and that by the judicious application of fertilizer N during the late stages of growth, it was possible to enhance soybean yields without necessarily inhibiting N₂ fixation.     

Nitrogen fixation (N-15 dilution) with soybeans under Thai field conditions. IV. Effect of N addition and Bradyrhizobium japonicum inoculation in soils with indigenous B. japonicum populations

The effects of Bradyrhizobium japonicum inoculation and pre-plant additions of N fertilizer on soybean ( Glycine max L. Merrill) yields and levels of N₂ fixation were studied under field conditions at two sites in Thailand. Bacterial inoculants were composed of B. japonicum strains selected for high N₂ fixation levels in Thai soils. Nitrogen fertilizer addition rates used were from 0 to 250 kg N/ha in 50 kg N/ha increments. At the Chiang Mai site in northern Thailand, bacterial inoculation increased nodule weights on plants receiving 100 kg N/ha or less. Increases in nodule parameters due to inoculation were evident at 45 d after planting (DAP) but disappeared by 60 DAP. Addition of N fertilizers decreased the incidence of nodulation and sap ureide contents and decreased the contribution of N₂ fixation to the N content of plants at maturity as measured by N-15 isotope dilution methods. At the Kampang Saen site in central Thailand, bacterial inoculation had significant positive effects on nodule numbers and weights, ARA, sap ureide contents and levels of N₂ fixed as measured by N-15 isotope dilution methods. Addition of N fertilizers at this site also reduced the effectiveness of N₂-fixing symbioses. It was concluded that small additions of N fertilizer added before planting did not significantly decrease N₂ fixation levels, but did have a significant positive effect on plant growth. Larger N additions would reduce N₂ fixation levels in excess of the benefits of adding more N in chemical form.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

Phosphorus and Nitrogen Interactions in Field-Grown Soybean as Related to Genetic Attributes of Root Morphological and Nodular Traits

Two field experiments with different soybean (Glycine max L.) materials were conducted to investigate the interactions between phosphorus (P) and nitrogen (N) as related to the genetic attributes of root morphological and nodular traits. In experiment one, 13 cultivated soybean varieties were grown in a field with relatively low soil P and N availability. P application with 160 kg P/hm2 as triple superphosphate produced a significant simultaneous increase in the content of both P and N in shoot, demonstrating positive P and N interactions. The addition of P also increased root dry weight, root nodule number, nodule mass, nodule size, and nodulation index, but decreased root length and root surface area, indicating that P may affect N nutrition in soybean through a number of root morphological and nodular traits. Interestingly,like P content, N content appeared to be more correlated with root morphological traits (root weight, root length, and root surface area) than with root nodular traits (nodule number, nodule size, nodule mass, and nodulation index) at both P levels, implying that N taken up by the roots may contribute more to the plant N status than biological N2 fixation under the present experimental conditions. In experiment two, 57 soybean lines of a recombinant inbred line (RIL) population derived from a cross between a cultivated variety and a wild genotype were grown on another field site with moderately sufficient P and N levels to further characterize the genetic attributes of root morphological and nodular traits and their relationships with P and N interactions. The results indicated that all morphological and nodular traits measured continually segregated in the RIL population with a normal distribution of the phenotypic values, indicating that these traits are possibly controlled by quantitative trait loci (QTLs). Genetic analysis revealed that all these root traits had relatively low heritabilities (h2b=74.12, 70.65, 73.76, 56.34, 52.59, and 52.24 for root weight, root length,root surface area, nodule number, nodule mass, and nodule size, respectively), suggesting that root morphology and nodule formation are influenced greatly by environmental factors. Correlation analysis of the RILs showed that shoot N content was significantly correlated with P content, confirming positive P×N interactions. Similar to experiment one, shoot N content was only significantly correlated with root morpho logical traits, but not with root nodular traits, again denoting the fact that the N status in soybean could be attributed more to N uptake from the soil than to biological N2 fixation under the present experimental conditions.     

Inhibition of N2 fixation in soybean is associated with elevated ureides and amino acids

Decreased N2 fixation in soybean (Glycine max) L. Merr. during water deficits has been associated with increases in ureides and free amino acids in plant tissues, indicating a potential feedback inhibition by these compounds in response to drought. We evaluated concentrations of ureides and amino acids in leaf and nodule tissue and the concurrent change in N2 fixation in response to exogenous ureides and soil-water treatments for the cultivars Jackson and KS4895. Exogenous ureides applied to the soil and water-deficit treatments inhibited N2 fixation by 85% to 90%. Mn fertilization increased the apparent catabolism of ureides in leaves and hastened the recovery of N2 fixation following exogenous ureide application for both cultivars. Ureides and total free amino acids in leaves and nodules increased during water deficits and coincided with a decline in N2 fixation for both cultivars. N2 fixation recovered to 74% to 90% of control levels 2 d after rewatering drought-stressed plants, but leaf ureides and total nodule amino acids remained elevated in KS4895. Asparagine accounted for 82% of the increase in nodule amino acids relative to well-watered plants at 2 d after rewatering. These results indicate that leaf ureides and nodule asparagine do not feedback inhibit N2 fixation. Compounds whose increase and decrease in concentration mirrored the decline and recovery of N2 fixation included nodule ureides, nodule aspartate, and several amino acids in leaves, indicating that these are potential candidate molecules for feedback inhibition of N2 fixation.     

Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase is essential for symbiosis with soybean and contains a novel metal-binding domain.

The Bradyrhizobium japonicum hemA gene product delta-aminolevulinic acid (ALA) synthase is not required for symbiosis of that bacterium with soybean. Hence, the essentiality of the subsequent heme synthesis enzyme, ALA dehydratase, was examined. The B. japonicum ALA dehydratase gene, termed hemB, was isolated and identified on the basis of its ability to confer hemin prototrophy and enzyme activity on an Escherichia coli hemB mutant, and it encoded a protein that was highly homologous to ALA dehydratases from diverse organisms. A novel metal-binding domain in the B. japonicum ALA dehydratase was identified that is a structural composite of the Mg(2+)-binding domain found in plant ALA dehydratases and the Zn(2+)-binding region of nonplant ALA dehydratases. Enzyme activity in dialyzed extracts of cells that overexpressed the hemB gene was reconstituted by the addition of Mg2+ but not by addition of Zn2+, indicating that the B. japonicum ALA dehydratase is similar to the plant enzymes with respect to its metal requirement. Unlike the B. japonicum hemA mutant, the hemB mutant strain KP32 elicited undeveloped nodules on soybean, indicated by the lack of nitrogen fixation activity and plant hemoglobin. We conclude that the hemB gene is required for nodule development and propose that B. japonicum ALA dehydratase is the first essential bacterial enzyme for B. japonicum heme synthesis in soybean root nodules. In addition, we postulate that ALA is the only heme intermediate that can be translocated from the plant to the endosymbiont to support bacterial heme synthesis in nodules.     

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     

Relationship between Ureide N and N(2) Fixation, Aboveground N Accumulation, Acetylene Reduction, and Nodule Mass in Greenhouse and Field Studies with Glycine max L. (Merr)

The relationship between ureide N and N₂ fixation was evaluated in greenhouse-grown soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) and in field studies with soybean. In the greenhouse, plant N accumulation from N₂ fixation in soybean and lima bean correlated with ureide N. In soybean, N₂ fixation, ureide N, acetylene reduction, and nodule mass were correlated when N₂ fixation was inhibited by applying KNO₃ solutions to the plants. The ureide-N concentrations of different plant tissues and of total plant ureide N varied according to the effectiveness of the strain of Bradyrhizobium japonicum used to inoculate plants. The ureide-N concentrations in the different plant tissues correlated with N₂ fixation. Ureide N determinations in field studies with soybean correlated with N₂ fixation, aboveground N accumulation, nodule weight, and acetylene reduction. N₂ fixation was estimated by ¹⁵N isotope dilution with nine and ten soybean genotypes in 1979 and 1980, respectively, at the V9, R2, and R5 growth stages. In 1981, we investigated the relationship between ureide N, aboveground N accumulation, acetylene reduction, and nodule mass using four soybean genotypes harvested at the V4, V6, R2, R4, R5, and R6 growth stages. Ureide N concentrations of young stem tissues or plants or aboveground ureide N content of the four soybean genotypes varied throughout growth correlating with acetylene reduction, nodule mass, and aboveground N accumulation. The ureide-N concentrations of young stem tissues or plants or aboveground ureide-N content in three soybean genotypes varied across inoculation treatments of 14 and 13 strains of Bradyrhizobium japonicum in 1981 and 1982, respectively, and correlated with nodule mass and acetylene reduction. In the greenhouse, results correlating nodule mass with N₂ fixation and ureide N across strains were variable. Acetylene reduction in soybean across host-strain combinations did not correlate with N₂ fixation and ureide N. N₂ fixation, ureide N, acetylene reduction, and nodule mass correlated across inoculation treatments with strains of Bradyrhizobium spp. varying in effectiveness on lima beans. Our data indicate that ureide-N determinations may be used as an additional method to acetylene reduction in studies of the physiology of N₂ fixation in soybean. Ureide-N measurements also may be useful to rank strains of B. japonicum for effectiveness of N₂ fixation.     

Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants

Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO(3) (-) application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3). Nitrate application markedly decreased isoflavonoid concentrations in non-inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO(3) (-) compared with the other root-half receiving 0 millimolar NO(3) (-). High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO(3) (-) was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO(3) (-) on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO(3) (-). These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO(3) (-) on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO(3) (-) also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development.     

Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum

Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre-treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin-inducible gene expression and auxin transport. However, use of a genistein-hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS-silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation.     

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.     

Evaluation of commercial soybean inoculants from Argentina

Eighteen samples of soybean inoculants representative of the major manufacturing companies in Argentina were purchased from the market and evaluated using plate counts, most probable number (MPN) of Bradyrhizobium japonicum on plants and time of nodule appearance. One or two B. japonicum isolates per product were isolated and typed by analysis of their DNA patterns. The Log10 numbers of B. japonicum obtained were in the range of 0 to 6/soybean seed, with only two products above 1 × 106 bacteria/seed. Of 18 products, 17 were contaminated, and of these 14 contained more contaminants than B. japonicum. The time of nodule appearance varied between 8 and 16 days, indicating a great difference in microbial activity between products. The strains were found to be similar to USDA 138 (five isolates), E45-INTA Argentina (two isolates), USDA 142 (four isolates) and E4-INTA (one isolate). Thus, even if most of the typed strains are considered as good N2-fixing strains, the average quality of the analysed samples was low, and could not support efficient inoculation of soybean.