Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.)

The ability to recycle H₂ evolved by nitrogenase is thought to be of importance in increasing the efficiency of N₂ fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H₂ and in their relative efficiency of N₂ fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C₂H₂ reduction, H₂ evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup⁺) isolates did not exhibit higher dry weight or N content than those inoculated with Hup⁻ isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup⁻ isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup⁺ isolates. In some Hup⁺R. leguminosarum isolates, H₂ oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H₂ to Hup⁺ nodules whose supply of photosynthate had been removed by stem excision did not increase C₂H₂ reduction in either coupled or uncoupled types.     

Enrichment for Hydrogen-Oxidizing Acinetobacter spp. in the Rhizosphere of Hydrogen-Evolving Soybean Root Nodules

Field soybean plants were inoculated with Hup⁺ wild-type or H₂ uptake-negative (Hup⁻) mutants of Bradyrhizobium japonicum. For two consecutive summers we found an enrichment for acinetobacters associated with the surfaces of the H₂-evolving nodules. Soybean root nodules that evolved H₂ had up to 12 times more Acinetobacter spp. bacteria associated with their surfaces than did nodules incapable of evolving H₂. All of the newly isolated strains identified as Acinetobacter obtained from the surfaces of root nodules, as well as known established Acinetobacter strains, were capable of oxidizing H₂, a property not previously described for this alkane-degrading soil bacterium.     

Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium

Some Rhizobium bacteria have H₂-uptake (Hup) systems that oxidize H₂ evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars `JI1205' and `Alaska' produce high Hup (Hup⁺⁺) and moderate Hup (Hup⁺) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup⁺⁺ and Hup⁺ phenotypes in R. leguminosarum 128C53 were identified in 173 F₂-derived F₆ families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P ≤ 0.05) in 19 of 24 symbiotic combinations. Analyses of Hup phenotypes in F₆ families, the F₁ population, and two backcrosses suggested involvement of a single genetic locus. Three of the eight pairs of isolines were identified as being suitable for physiological studies, because the two lines in each pair showed similar growth, N assimilation, and flowering traits under nonsymbiotic conditions. Tests of those lines under N₂-dependent conditions with isogenic Hup⁺ and negligible Hup (Hup⁻) mutants of R. leguminosarum 128C53 showed that, in symbioses with Hup⁺ rhizobia, two out of three Hup⁺⁺ pea lines decreased N₂ fixation relative to Hup⁺ peas. In one of those cases, however, the Hup⁺⁺ plant line also decreased fixation by Hup⁻ rhizobia. When results were averaged across all rhizobia tested, Hup⁺ pea isolines had 8.2% higher dry weight (P ≤ 0.05) and fixed 12.6% more N₂ (P ≤ 0.05) than Hup⁺⁺ isolines. Pea lines described here may help identify host plant factors that influence rhizobial Hup activity and should assist in clarifying how Hup systems influence other physiological processes.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.     

Influence of Glycine spp. on Competitiveness of Bradyrhizobium japonicum and Rhizobium fredii

The displacement of indigenous Bradyrhizobium japonicum in soybean nodules with more effective strains offers the possibility of enhanced N₂ fixation in soybean (Glycine max (L.) Merr.). Our objective was to determine whether the wild soybean (G. soja Sieb. & Zucc.) genotype PI 468397 would cause reduced competitiveness of important indigenous B. japonicum strains USDA 31, 76, and 123 and thereby permit nodulation by Rhizobium fredii, the fast-growing microsymbiont of soybean. In an initial experiment, PI 468397 nodulated and fixed moderate amounts of N₂ with USDA 31 and 76 but, despite the formation of nodules, fixed essentially no N₂ with USDA 123. In contrast, PI 468397 formed a highly effective symbiosis with R. fredii strain USDA 193. In two subsequent experiments, Williams soybean and PI 468397 were grown in a pasteurized soil mixture or in soybean rhizobium-free soil and inoculated with both USDA 123 and USDA 193. In each experiment, more than 90% of the nodules of Williams contained USDA 123, while only a maximum of 2% were occupied with USDA 193. In contrast, in the two experiments, 16 and 11%, respectively, of the nodules produced on PI 468397 were occupied by USDA 123, while in both experiments 87% contained USDA 193. Thus, in relation to the cultivar Williams, which is commonly grown and used as a parent in soybean breeding programs in the United States, PI 468397 substantially reduced the competitive ability of B. japonicum strain USDA 123 in relation to R. fredii strain USDA 193.     

Symbiotic Bradyrhizobium japonicum Reduces N2O Surrounding the Soybean Root System via Nitrous Oxide Reductase

N₂O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N₂O uptake and conversion of ¹⁵N-N₂O into ¹⁵N-N₂. Free-living cells of USDA110 showed N₂O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N₂O reductase activity. When detached soybean nodules formed with USDA110 were fed with ¹⁵N-N₂O, they rapidly emitted ¹⁵N-N₂ outside the nodules at a ratio of 98.5% of ¹⁵N-N₂O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N₂O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N₂O (0.34 ppm). These results indicate that the conversion of N₂O to N₂ depends exclusively on the respiratory N₂O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N₂O.     

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.     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.     

Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation

The interaction between the ATP-dependent evolution of H₂ catalyzed by nitrogenase and the oxidation of H₂ via a hydrogenase has been postulated to influence the efficiency of the N₂-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H₂ in air and the H₂ produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N₂ fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H₂ in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C₂H₂ reduction and O₂ consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.

When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N₂ fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H₂-evolving strain 176A27 produced increases in plant dry weight and total N₂ fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N₂ fixation for nodulated legumes capable of reutilizing the H₂ evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H₂-recycling process in N₂-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

     

Evidence for a Third Uptake Hydrogenase Phenotype among the Soybean Bradyrhizobia

The existence of a hydrogen uptake host-regulated (Hup-hr) phenotype was established among the soybean bradyrhizobia. The Hup-hr phenotype is characterized by the expression of uptake hydrogenase activity in symbiosis with cowpea but not soybean. Uptake hydrogenase induction is not possible under free-living cultural conditions by using techniques developed for uptake hydrogenase-positive (Hup⁺) Bradyrhizobium japonicum. Hydrogen oxidation by Hup-hr phenotype USDA 61 in cowpea symbioses was significant because hydrogen evolution from nitrogen-fixing nodules was not detected. An examination for uptake hydrogenase activity in soybean and cowpea with 123 strains diverse in origin and serology identified 16 Hup⁺ and 28 Hup-hr phenotype strains; the remainder appeared to be Hup⁻. The Hup-hr phenotype was associated with serogroups 31, 76, and 94, while strains belonging to serogroups 6, 31, 110, 122, 123, and 38/115 were Hup⁺. Hup⁺ strains of the 123 serogroup typed positive with USDA 129-specific antiserum. The presence of the uptake hydrogenase protein in cowpea bacteroids of Hup⁺ strains was demonstrated with immunoblot analyses by using antibodies against the 65-kDa subunit of uptake hydrogenase purified from strain SR470. However, the hydrogenase protein of Hup-hr strains was not detected. Results of Southern hybridization analyses with pHU1 showed the region of DNA with hydrogenase genes among Hup⁺ strains to be similar. Hybridization was also obtained with Hup-hr strains by using a variety of cloned DNA as probes including hydrogenase structural genes. Both hydrogenase structural genes also hybridized with the DNA of four Hup⁻ strains.     

Role of N2 fixation in the soybean N credit in maize production

Many studies have shown that maize (Zea mays L.) requires less fertilizer N for optimum yield when grown in rotation with soybean [Glycine max (L.) Merr] than when grown in monoculture, which is referred to as the `soybean N credit' in the maize growing areas of the United States. Because the specific source of this soybean N credit is unclear, our objective was to determine the role of nodules and N₂ fixation as a contributing source of the soybean N credit. Our research approach was designed to separate the effect of symbiotic N₂ fixation from other rotational effects, as the treatments included: maize grown after nodulated (N₂ fixing) soybean and maize grown after non-nodulated (non N₂ fixing) soybean. A separate experiment examined maize grown after maize. For each previous crop, maize was grown the following year with varying rates of fertilizer applied N. In both years, the yield differences between nodulated and non-nodulated soybean as the previous crop were much smaller than the apparent yield decrease associated with continuous maize. Although small in magnitude, maize following non-nodulated soybean accumulated less total N, was paler in leaf color, and yielded less than maize following nodulated soybean in the more favorable year of 1999, while most of these differences were not observed in 2000. These findings indicate that soybean nodules and N₂ fixation, while having a certain role, are not the major determinants of the soybean N credit.     

Nitrate and flooding induce N<Subscript>2</Subscript>O emissions from soybean nodules

Nitrous oxide (N₂O) is one of the three main biogenic greenhouse gases (GHGs) and agriculture represents close to 30 % of the total N₂O net emissions. In agricultural soils, N₂O is emitted by two main microbial processes, nitrification and denitrification, both of which can convert synthetic nitrogen fertilizer into N₂O. Legume-rhizobia symbiosis could be an effective and environmental-friendly alternative to nitrogen fertilization and hence, to mitigate soil N₂O emissions. However, legume crops also contribute to N₂O emissions. A better understanding of the environmental factors involved in the emission of N₂O from nodules would be instrumental for mitigating the release of this GHG gas. In this work, in vivo N₂O emissions from nodulated soybean roots in response to nitrate (0, 1, 2 and 4 mM) and flooding have been measured. To investigate the contribution of rhizobial denitrification in N₂O emission from nodules, plants were inoculated with B. japonicum USDA110 and napA and nosZ denitrification mutants. The results showed that nitrate was essential for N₂O emissions and its concentration enhanced N₂O fluxes showing a statistical linear correlation, being the highest N₂O fluxes obtained with 4 mM nitrate. When inoculated plants grown with 4 mM nitrate were subjected to flooding, a 150- and 830-fold induction of N₂O emission rates from USDA110 and nosZ nodulated roots, respectively, was observed compared to non-flooded plants, especially during long-term flooding. Under these conditions, N₂O emissions from detached nodules produced by the napA mutant were significantly lower (p?<?0.05) than those produced by the wild-type strain (382 versus 1120 nmol N₂O h^?1 g^?1 NFW, respectively). In contrast, nodules from plants inoculated with the nosZ mutant accumulated statistically higher levels of N₂O compared to wild-type nodules (2522 versus nmol 1120 N₂O h^?1 g^?1 NFW, p?<?0.05). These results demonstrate that flooding is an important environmental factor for N₂O emissions from soybean nodules and that B. japonicum denitrification is involved in such emission.     

Effects of Bradyrhizobium japonicum and Soybean (Glycine max (L.) Merr.) Phosphorus Nutrition on Nodulation and Dinitrogen Fixation

Cells of Bradyrhizobium japonicum were grown in media containing either 1.0 mM or 0.5 μM phosphorus. In growth pouch experiments, infection of the primary root of soybean (Glycine max (L.) Merr.) by B. japonicum USDA 31, 110, and 142 was significantly delayed when P-limited cells were applied to the root. In a greenhouse experiment, B. japonicum USDA 31, 110, 122, and 142 grown with sufficient and limiting P were used to inoculate soybeans which were grown with either 5 μM or 1 mM P nutrient solution. P-limited cells of USDA 31 and 110 formed significantly fewer nodules than did P-sufficient cells, but P-limited cells of USDA 122 and 142 formed more nodules than P-sufficient cells. The increase in nodule number by P-limited cells of USDA 142 resulted in significant increases in both nodule mass and shoot total N. In plants grown with 1 mM P, inoculation with P-limited cells of USDA 110 resulted in lower total and specific nitrogenase activities than did inoculation with P-sufficient cells. Nodule numbers, shoot dry weights, and total N and P were all higher in plants grown with 1 mM P, and plants inoculated with USDA 31 grew poorly relative to plants receiving strains USDA 110, 122, and 142. Although the effects of soybean P nutrition were more obvious than those of B. japonicum P nutrition, we feel that it is important to develop an awareness of the behavior of the bacterial symbiont under conditions of nutrient limitation similar to those found in many soils.     

Expression of Chromosome-integrated Hydrogen-uptake Genes in Cicer-Rhizobium

Integration of H₂-uptake (hup) gene cosmid pHU52 into the chromosomal DNA, conferred H₂- uptake activity on the Hup^- Cicer-Rhizobium strain G36–84 in the free-living state and in nodules. In five transconjugants (G36–84:: Tn5:: pHU52) derepressed for hup gene expression, the specific Hup activity ranged from 158 to 256 nmole H₂ hr^-1 mg-¹ protein which was 42 to 64% lower than the activity obtained in transconjugant with pHU52 as an episome. Integration of the cosmid significantly improved the relative efficiency of symbiotic N₂-fixation by imparting H₂-recycling capability to Hup^- Cicer-Rhizobium. Demonstration of Hup activity in the nodules of field grown chickpea plants suggests that the integrated hup genes are stably maintained in natural environment     

The contribution of nitrogen by promiscuous soybeans to maize based cropping the moist savanna of Nigeria

Agronomic results indicate that maize grain yields generally are higher when the crop is planted following soybean than in continuous maize cultivation in the moist savanna agroecological zones of West Africa. Many factors have been hypothesized to explain this phenomenon, including enhanced N availability and the so-called `rotational effect'. There is, however, hardly any quantitative information on the residual N benefits of promiscuous soybeans to subsequent cereal crops grown in rotation with soybean. Three IITA promiscuous soybean breeding lines and two Brazilian soybean lines were grown in 1994 and 1995 at Mokwa in the southern Guinea savanna, Nigeria, to quantify the nitrogen contribution by soybeans to a succeeding crop of maize grown in rotation with soybean for two consecutive years, 1996 and 1997 using two methods of introducing ¹⁵N into soil (fresh ¹⁵N labelling and its residual ¹⁵N) and three maize cultivars (including one cultivar with high N use efficiency) used as reference plants. The nodulating soybeans fixed between 44 and 103 kg N ha^–1 of their total N and had an estimated net N balance input from fixation following grain harvest ranging from –8 to 43 kg N ha^–1. Results in 1996 and in 1997 showed that maize growing after soybean had significantly higher grain yield (1.2 – 2.3-fold increase compared to maize control) except for maize cultivar Oba super 2 (8644-27) (a N-efficient hybrid). The ¹⁵N isotope dilution method was able to estimate N contribution by promiscuous soybeans to maize only in the first succeeding maize crop grown in 1996 but not in the second maize crop in 1997. The first crop of maize grown after soybean accumulated an average between 10 and 22 kg N ha^–1 from soybean residue, representing 17–33% of the soybean total N ha^–1. The percentage ¹⁵N derived from residue recovery in maize grown after maize was influenced by the maize cultivars. Maize crop grown after the N-efficient hybrid cultivar Oba Super 2 (844-27) had similar ¹⁵N values similar to maize grown after soybeans, confirming the ability of this cultivar to use N efficiently in low N soil due to an efficient N translocation ability. The maize crop in 1997 grown after maize had lower ¹⁵N enrichment than that grown in soybean plots, suggesting that soybean residues contributed a little to soil available N and to crop N uptake by the second maize crop. The differential mineralization and immobilization turnover of maize and soybean residues in these soils may be important and N contribution estimates in longer term rotation involving legumes and cereals may be difficult to quantify using the ¹⁵N labelling approaches. Therefore alternative methods are required to measure N release from organic residues in these cropping systems.     

Autecology of Bradyrhizobium japonicum in soybean-rice rotations

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32×10⁵ g^–1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 ×10⁴, 1.26×10⁴ and 6.40×10² g^–1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N₂-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p<0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p<0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.     

Relationship between N(2)-Fixing Efficiency and Natural N Enrichment of Soybean Nodules

Non-nodular tissue of soybean (Glycine max L. Merrill) plants grown hydroponically in the absence of added N have a ¹⁵N abundance close to that of atmospheric N₂. In contrast, nodules are usually enriched in ¹⁵N. In this paper, we report measurements of the ¹⁵N abundance of foliar tissue and nodules of soybeans inoculated with 11 variably efficient strains of Rhizobum japonicum and grown hydroponically with no added N. The efficiency of the 11 symbioses varied over a wide range as judged by a 16-fold difference in N content. The degree of ¹⁵N enrichment of nodules was closely correlated with N₂-fixing efficiency (milligrams N fixed per milligram N in the nodules).

These results confirm prior preliminary data based on six variably efficient R. japonicum strains. The strong correlation between ^NN enrichment of soybean nodules and N₂-fixing efficiency is consistent with the hypothesis that new nodule tissue is synthesized from a pool of recently fixed N within the same nodule.

     

Inoculation with an enhanced N2‐fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max Merr.) response to elevated [CO2]

This study tested the hypothesis that inoculation of soybean (Glycine max Merr.) with a Bradyrhizobium japonicum strain (USDA110) with greater N₂ fixation rates would enhance soybean response to elevated [CO₂]. In field experiments at the Soybean Free Air CO₂ Enrichment facility, inoculation of soybean with USDA110 increased nodule occupancy from 5% in native soil to 54% in elevated [CO₂] and 34% at ambient [CO₂]. Despite this success, inoculation with USDA110 did not result in greater photosynthesis, growth or seed yield at ambient or elevated [CO₂] in the field, presumably due to competition from native rhizobia. In a growth chamber experiment designed to study the effects of inoculation in the absence of competition, inoculation with USDA110 in sterilized soil resulted in nodule occupation of >90%, significantly greater ¹⁵N₂ fixation, photosynthetic capacity, leaf N and total plant biomass compared with plants grown with native soil bacteria. However, there was no interaction of rhizobium fertilization with elevated [CO₂]; inoculation with USDA110 was equally beneficial at ambient and elevated [CO₂]. These results suggest that selected rhizobia could potentially stimulate soybean yield in soils with little or no history of prior soybean production, but that better quality rhizobia do not enhance soybean responses to elevated [CO₂].