Ontogenic variation in nitrogen fixation and accumulation of nitrogen in mungbean,blackgram, cowpea,and groundnut

Ontogenic variations in N₂ fixation and accumulation of N by the mungbean (Vigna radiata L. Wilczek), blackgram (Vigna mungo L. Hepper), cowpea (Vigna unguiculata L. Walp.), and groundnut (Arachis hypogaea L.) were studied by a ¹⁵N-dilution technique. Pots filled with 7 kg of red yellow podzolic soil were used. Samples were taken 20, 40, 60, and 80 days after emergence which approximately corresponded to preflowering, flowering, early/mid-pod filling and late pod filling stages, respectively. During early growth (up to 40 days after emergence), the carryover of seed N accounted for a considerable fraction of the total plant N in the legumes, the highest being in the groundnut. With a correction for carryover, the groundnut derived over 45% of its N content from the atmosphere 20 days after emergence whereas the corresponding figures were 33% for the blackgram and about 28% for the cowpea and mungbean. Between flowering and early pod fill, there was a rapid increase in N₂ fixation in all legumes except in groundnut which showed highest fixation from 60 to 80 days after emergence. In the mungbean, N₂ fixation and uptake of soil N were insignificant 60 days after emergence while in other legumes these processes continued beyond this time. All legumes derived about 90% of their N from atmosphere by 80 days after emergence. However, due to considerable interspecific differences in total N yield the final amount of N₂ fixed showed an appreciable variation among legumes. It was highest in the groundnut (443 mg N plant^-1) followed by the cowpea (385), blackgram (273), and mungbean (145), respectively. The groundnut maintained nodules until the late pod filling stage while in other legumes, nodules senesced progressively following the mid-pod filling stage. During pod filling there was a net mobilization of N from vegetative tissues to developing pods in the mungbean, which amounted to about 20% of N in seeds. This mobilization was not evident in other legumes.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Nitrogen fixation and beneficial effects of some grain legumes and green-manure crops on rice

Studies were conducted on paddy soils to ascertain N₂ fixation, growth, and N supplying ability of some green-manure crops and grain legumes. In a 60-day pot trial, sunhemp (Crotalaria juncia) produced a significantly higher dry matter content and N yield than Sesbania sesban, S. rostrata, cowpeas (Vigna unguiculata), and blackgram (V. mungo), deriving 91% of its N content from the atmosphere. Dry matter production and N yield by the legumes were significantly correlated with the quantity of N₂ fixed. In a lowland field study involving sunhemp, blackgram, cowpeas, and mungbean, the former produced the highest stover yield and the stover N content, accumulating 160–250 kg N ha^-1 in 60 days, and showed great promise as a biofertilizer for rice. The grain legumes showed good adaptability to rice-based cropping systems and produced a seed yield of 1125–2080 kg ha^-1, depending on the location, species, and cultivar. Significant inter- and intraspecific differences in the stover N content were evident among the grain legumes, with blackgram having the highest N (104–155 kg N ha^-1). In a trial on sequential cropping, the groundnut (Arachis hypogaea) showed a significantly higher N₂ fixation and residual N effect on the succeeding rice crop than cowpeas, blackgram, mungbeans (V. radiata), and pigeonpeas (Cajanus cajan). The growth and N yield of the rice crop were positively correlated with the quantity of N₂ fixed by the preceding legume crop.     

Estimating N2 fixation by field-grown lupins (Lupinus angustifolius L.) using soil and plant 15N enrichment

Summary Biological N₂ fixation was estimated in a field experiment following the addition of NH₄Cl or KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). A model of total N and ¹⁵N accumulation in lupins and decreasing ¹⁵N enrichment in the KCl-extractable soil-N pool (0–0.15 m depth) was used to estimate the proportion of N in lupins derived from biological N₂ fixation. Estimates of N₂ fixation derived from the model were compared with ¹⁵N isotope-dilution estimates obtained using canola, annual ryegrass, and wheat as nonfixing reference plants. Biomass, total N accumulation, or ¹⁵N enrichment in the lupin and reference crops did not differ whether NH _inf4 ^sup+ or NO _inf3 ^sup- was added as the labelled inorganic-N source. The decrease in soil ¹⁵N enrichment was described by first-order kinetics, whereas total N and ¹⁵N accumulation in the lupins were described by logistical equations. Using these equations, the uptake of soil N by lupins was estimated and was then used to calculate fixed N₂. Estimates of N₂ fixation derived from the model increased from 0 at 50 days after sowing to a maximum of 0.79 at 190 days after sowing. Those based on the ¹⁵N enrichment of the NO _inf3 ^sup- pool were 10% higher than those based on the mineral-N pool. ¹⁵N isotope-dilution estimates of N₂ fixation ranged from 0.37 to 0.55 at 68 days after sowing and from 0.71 to 0.77 at 190 days after sowing. Reference plant-derived values of N₂ fixation were all higher than modelled estimates during the early states of growth, but were similar to modelled estimates at physiological maturity. The use of the model to estimate N₂ derived from the atmosphere has the intrinsic advantage that the need for a non-fixing reference plant is avoided.     

Effect of nitrogen application on the A N value of soil

Pot experiments were conducted with two soils, from Rottenhaus and Seibersdorf in Austria, to ascertain whether the rate of fertilizer N application and the test crop would influence the amount of N available in the soil as assessed by the A-value method. ¹⁵N-labelled fertilizer was applied at rates of 10, 25, 40, 60, and 100 mg N kg^-1 soil, corresponding approximately to 20, 50, 80, 120 and 200 kg N ha^-1 respectively, and two crop species, barley (Hordeum vulgareL.) and non-nodulating soybean (Glycine max L.) were used to determine the soil A _N value under the various fertilizer regimes. The results showed that the Rottenhaus soil had a higher A _N value than the Seibersdorf soil, suggesting that the former was more fertile than the latter. The A _N values of both soils were significantly affected by the level of N application. When grown in the same soil, the two test crops showed significantly different fertilizer use efficiency and per cent N derived from fertilizer when the rate of N application exceeded 20 kg ha^-1. Thus, the A _N value as determined by the two test crops differed significantly for the same soil when the rate of N application was greater than 20 kg/ha. The difference was greater when the soil fertility level was high. The dependence of the A _N value on the level of N application and the species of crop seriously compromises the suitability of this method for determining plant-associated N₂ fixation. Hence, considerable caution is required when using this method to estimate plant-associated N₂ fixation.     

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Summary Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using ¹⁵N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N₂-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with ¹⁵N by adding cellulose and (¹⁵NH₄)₂SO₄. The results of these studies showed fixation values of 6%–32% when estimated by ¹⁵N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of ¹⁵N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N₂.     

Effect of successive cuttings on uptake and partitioning of 15N among plant parts of Leucaena leucocephala

Summary We studied the effect of three successive cuttings on N uptake and fixation and N distribution in Leucaena leucocephala. Two isolines, uninoculated or inoculated with three different Rhizobium strains, were grown for 36 weeks and cut every 12 weeks. The soil was labelled with 50 ppm KNO₃ enriched with 10 atom % ¹⁵N excess soon after the first cutting. Except for the atom % ¹⁵N excess in branches of K28 at the second cutting, both the L. leucocephala isolines showed similar patterns of total N, fixed N₂, and N from fertilizer distribution in different parts of the plant at each cutting. The Rhizobium strain did not influence the partitioning of ¹⁵N among the different plant parts. Significant differences in ¹⁵N enrichment occurred in different parts. Live nodules of both isolines showed the lowest atom % ¹⁵N excess values (0.087), followed by leaves (0.492), branches (0.552), stems (0.591), and roots (0.857). The roots contained about 60% of the total plant N and about 70% of the total N derived from fertilizer over the successive cuttings. The total N₂ fixed in the roots was about 60% of that fixed in the whole plant, while the shoots contained only 20% of the fixed N₂. We conclude that N reserves in roots and nodules constitute another N source that must be taken into account when estimating fixed N₂ or the N balance after pruning or cutting plants. ¹⁵N enrichment declined up to about fivefold in the reference and the N₂-fixing plants over 24 weeks following the ¹⁵N application. The proportion and the amounts of N derived from fertilizer decreased, while the amount derived from N₂ fixation increased with time although its proportion remained constant.     

Landscape-scale estimates of dinitrogen fixation by Pisum sativum by nitrogen-15 natural abundance and enriched isotope dilution

Topography and slope position influence the soil and environmental factors that affect N₂ fixation by legumes. The present study was conducted to (1) estimate N₂ fixation by field peas in a gently rolling farm field using the natural ¹⁵N abundance and the ¹⁵N-enriched isotope dilution techniques and (2) identify soil and environmental factors that influence N₂ fixation at the landscape scale. Whereas soil available water capacity, available NH _inf4 ^sup+ , total crop yield, and percent N derived from N₂ fixation (% Ndfa) estimated using enriched N were significantly affected by landform patterns, soil NO _inf3 ^sup- levels, seed yield, and the % Ndfa estimated using natural abundance did not follow landform patterns. The % Ndfa using natural abundance was correlated with NH _inf4 ^sup+ but not with available soil water, pH, electrical conductivity, NO _inf3 ^sup- , or particle size. Estimates of the % Ndfa using enriched ¹⁵N ranged from 0 to 92.8%. The highest median value (68.6%) for % Ndfa using enriched N occurred on the divergent footslopes, with the lowest value (28.1%) on the convergent shoulders. Estimates of % Ndfa using natural abundance ranged from 13.2% to 96.9%. Smaller fluctuations during the growing season in the ¹⁵N of the available N pool may have resulted in less variability for % Ndfa using natural abundance compared to enriched ¹⁵N. Despite similar mean values for % Ndfa using natural abundance (44.5) and enriched ¹⁵N (49.6), no significant correlation between the two estimates was found. These results suggest that although topography may exert gross controls on N₂ fixation, large variations in N₂ fixation at the microsite level may preclude correlations between individual estimates and limit detection of landscape scale patterns of N₂ fixation.Contribution No. R754 of the Saskatchewan Center of Soil Research     

Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas

The intact nodules attached to the upper part of soybean roots were exposed to ¹⁵N₂ and the incorporation of ¹⁵N into various soluble nitrogen constituents was investigated. Results indicated that ammonia, a primary product of N₂ fixation, was located in more than two compartments. Ammonia reduced from N₂ gas seemed to be incorporated firstly into glutamine especially amido-group nitrogen. Newly fixed nitrogen was secondly incorporated into glutamic acid and alanine in this sequence. These results suggested that fixed ammonia was assimilated by glutamine synthetase/glutamate synthase pathway. Turn-over rate of allantoin plus allantoic acid and serine was relatively high, although apparently these compounds were not primary products of newly fixed ammonia. ¹⁵N content of allantoin was always higher than that of allantoic acid. ¹⁵N incorporation to aspartic acid and asparagine was relatively slow, especially in early period. In bacteroid fraction there is much amount of ammonia comparing with other compounds, while allantoin and asparagine were presented exclusively in cytosol. ¹⁵N was incorporated into nitrate within a few minutes especially in bacteroids.     

Low levels of ferredoxin, ATP and leghemoglobin contribute to limited N2 fixation of peas (Pisum sativum L.) and alfalfa (Medicago sativa L.) under S deficiency conditions

Sulphur (S) has become a major limiting factor for plant production in industrial as well as in remote industrial rural areas. Limitation of S can reduce legume N₂ fixation by affecting nodule development and function. In pot experiments with pea (Pisum sativum L.) and alfalfa (Medicago sativa L.), we investigated the influence of S on growth, ferredoxin, ATP and leghemoglobin concentrations. Addition of 200 mg S pot⁻¹ increased yield of shoots, roots and nodules of both plant species significantly. However, the influence of S on nodule yield formation was most pronounced. Pea and alfalfa roots were found to have higher S concentrations than shoots and being up to 2.9 times the S concentration in the shoots of peas under S-sufficient conditions. Sulphur addition also increased N₂ fixation significantly. The ferredoxin concentration in bacteroids of root nodules of pea was increased significantly by S only 10 weeks after planting and in bacteroids of root nodules of alfalfa 10 and 17 weeks after planting, while on per pot base the amounts of ferredoxin were higher throughout the experimental period of time. The ATP concentration of bacteroids of root nodules of both plant species as well as of mitochondria of root nodules of pea were significantly higher with optimum S supply. The effects of S deficiency on N₂ fixation are likely to be caused by the shortage of ferredoxin and ATP. The amount of leghemoglobin was reduced in comparison to nodules of the S-sufficient plants.     

Influence of mycorrhiza vs. soluble phosphate on growth,nodulation, and N2 fixation (15N) in alfalfa under different levels of water potential

Summary The legume Medicago sativa (+Rhizobium melilott) was grown under controlled conditions to study the interactions between soluble P in soil (four levels), or a mycorrhizal inoculum, and the degree of water potential (four levels) in relation to plant development and N₂ fixation. ¹⁵N-labelled ammonium sulphate was added to each pot for a qualitative estimate of N₂ fixation, in order to rank the effects of the different treatments.Dry-matter yield, nutrient content and nodulation increased with the amount of plant-available P in the soil, and decreased as the water stress increased, for each P-level. The mycorrhizal effect on dry matter, N yield, and on nodulation was little affected by the water potential. Since P uptake was affected by the water content in mycorrhizal plants, additional mechanisms, other than those mediated by P, must be involved in the mycorrhizal activity.There was a positive correlation between N yield and nodulation for the different P levels and the mycorrhizal treatment at all water levels. A high correlation between plant unlabelled N content and atom% ¹⁵N excess was also found for all levels of P. In mycorrhizal plants, however, the correlation between unlabelled N yield and ¹⁵N was lower. This suggests that mycorrhiza supply plants with other N sources in addition to those derived from the improvement on N₂ fixation.     

Estimating N2 fixation by Sesbania rostrata and S. cannabina (syn. S. aculeata) in lowland rice soil by the 15N dilution method

Summary A field experiment in concrete-based plots was conducted to estimate the contribution of N derived from air (Ndfa) or biological N₂ fixation in Sesbania rostrata and S. cannabina (syn. S. aculeata), using various references, by the ¹⁵N dilution method. The two Sesbania species as N₂-fixing reference plants and four aquatic weed species as non-N₂-fixing references were grown for 65 days after sowing in two consecutive crops, in the dry and the wet seasons, under flooded conditions. Soil previously labeled with ¹⁵N at 0.26 atom % ¹⁵N excess in mineralizable N was further labeled by ammonium sulfate with 3 and 6 atom % ¹⁵N excess. The results showed that ¹⁵N enrichment of soil NH ₄ ⁺ -N dropped exponentially in the first crop to half the original level in 50 days while in the second crop, it declined gradually to half the level in 130 days. The decline in ¹⁵N enrichment, in both N₂-fixing and non-fixing species, was also steeper in the first crop than in the second crop. Variations in ¹⁵N enrichment among non-fixing species were smaller in the second crop. The ratio of the uptake of soil N to that of fertilizer N in N₂-fixing and non-fixing species was estimated by the technique of varying the ¹⁵N level. In the second crop, this ratio in non-fixing species was higher than that in N₂-fixing species. Comparable estimates of % Ndfa were obtained by using ¹⁵N enrichment of various non-fixing species. There was also good agreement between the estimates obtained by using ¹⁵N enrichment of non-fixing species and those by using soil NH ₄ ⁺ -N, particularly in the second crop. By 25 days after sowing, the first crop of both Sesbania spp. had obtained 50% of total N from the atmosphere and the second crop had obtained 75%. The contribution from air increased with the age of the plant and ranged from 70% to 95% in 45–55 days. S. rostrata fixed substantially higher amounts of N₂ due to its higher biomass production compared with S. cannabina. Mathematical considerations in applying the ¹⁵N dilution method are discussed with reference to these results.     

N2 fixation in two Sesbania species and its transfer to rice (Oryza sativa L.) as revealed by 15N technology

Summary We used ¹⁵N technology to investigate N₂ fixation by Sesbania speciosa and Sesbania rostrata and its transfer to a lowland rice crop after incorporation of the Sesbania spp. into soil as green manure. During the first 50 days after establishment in November–December 1989, S. speciosa and S. rostrata produced 1126 and 923 kg dry matter ha^-1 respectively. They gathered 31 and 23 kg N ha^-1 respectively, of which 62%±5% and 55%±3% respectively, came from N₂ fixation. Both these species produced a greater biomass during September–October 1989, with S. rostrata producing more than S. speciosa. These results reflected differential responses by the plants to different day lengths at different times of the year. Furthermore, the dry matter yield and %N of ¹⁵N-labelled S. speciosa were smaller than those of the unlabelled plants, possibly due to inhibition of N₂ fixation in root nodules by the chemical N fertilizers added during labelling. These differences were not so pronounced in the stem-nodulated S. rostrata. The increased grain yield of rice fertilized with N in the form of chemical fertilizer or green manure was a result of an increased number of panicles per hill. The rice crop manured with S. speciosa produced a lower grain yield, with a lower grain weight than that manured with S. rostrata. This was due to a low uptake of soil N by rice manured with S. speciosa. Recovery of N from the green manure in rice straw with S. speciosa was significantly higher than from rice manured with S. rostrata, because of the higher applied N uptake by rice manured with the former.     

Time-course of nitrogen fixation in two bambara groundnut (Vigna subterranea L. Verdc.) cultivars

TheA-value method, involving the application of a higher¹⁵N rate to a reference non-N₂-fixing plant, was used to assess the magnitude of N₂ fixation in two bambara groundnut cultivars at four growth stages [vegetative, 0–47 days after planting (DAP); early pod-filling, 47–99 DAP; mid-pod-filling, 99–120 DAP; physiological maturity, 120–148 DAP). The cultivars were Ex-Ada, a bunchy type, and CS-88-11, a slightly spreading type. They were grown on a loamy sand. Uninoculated Ex-Ada and CS-88-11 were used as reference plants to measure the N₂ fixed in the inoculated bambara groundnuts. In this greenhouse study, soil was the major source of N in bambara groundnuts during vegetative growth, and during this period it accounted for over 80% of the N accumulaed in the plants. However, N₂ fixation became the major source of plant N during reproductive growth. There were significant differences between the two cultivars in the ability to fix N₂, and at physiological maturity, almost 75% of the N in CS-88-11 was derived from the atmosphere compared to 55% in Ex-Ada. Also, the total N fixed in CS-88-11 at physiological maturity was almost double that in Ex-Ada. Our data indicate that the higher N₂ fixation in CS-88-11 was due to two factors, a higher intensity of N₂ fixation and a longer active period of N₂ fixation. The results also suggest that bambara groundnut genotypes could be selected for higher N₂ fixation in farining systems.     

The role of arbuscular mycorrhiza in legume symbiotic performance

Legumes may respond to non-rhizobial inoculants such as arbuscular mycorrhizal (AM) fungi either through an effect on plant growth or, in addition, through an effect on the function of the legume-Rhizobium symbiosis. We have examined the literature where the application of ¹⁵N isotope dilution methodology permits the effect of indigenous AM and AM inoculants to be quantitatively separated into plant-growth-mediated and biological N₂ fixation (BNF)-mediated components. These studies clearly demonstrate the beneficial effects that both indigenous and inoculated AM have on legume growth, N uptake and the proportional dependence of the legume on atmospheric N₂. While the published data allow an assessment of various biological, edaphic and environmental factors that affect the response of various legumes to AM inoculation, they also highlight the paucity of quantitative field data and the lack of understanding of the interaction of legume genotype with AM species with respect to legume symbiotic performance.     

Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.     

Field measurements of nitrogen fixation in leguminous trees used in agroforestry systems: Influence of 15N-labeling approaches and reference trees

Appropriate ¹⁵N-labeling methods are crucial for estimating N₂-fixation in trees used in agroforestry systems. A 4-year field experiment was conducted on an Alfisol in Southwestern Nigeria to compare the estimates of N₂ fixed in Leucaena leucocephala, using two non-N₂-fixing leguminous trees, Senna siamea and S. spectabilis, as reference plants and three different methods of introducing ¹⁵N into soil. The atom % ¹⁵N uptake pattern (as reflected in the leaves) was identical in both N₂- and non-N₂-fixing tree species irrespective of the ¹⁵N-application method. There was a significant decline in atom % ¹⁵N excess in the leaves of L. leucocephala (from 0.266 to 0.039), S. siamea (0.625 to 0.121), and S. spectabilis (from 0.683 to 0.118) from the first sampling 12 months after planting and the second sampling 18 months after sampling. From the second harvest in 1991 until the end of the experiment (fifth) harvest in 1993, however, the atom ¹⁵N % excess decline in leaves of the three species was less pronounced and depended on the method of ¹⁵N application. In those plants to which the tracer was applied once at planting, the ¹⁵N decline was steady between the second and the last prunings. In the split-application treatment, the atom ¹⁵N % excess increased slightly at the third pruning and decreased during the subsequent two prunings. The reference tree and the method of ¹⁵N application influenced the estimated proportion of N derived from atmospheric N₂ by L. leucocephala, calculated as 73 and 64%, corresponding to 119 and 98 kg N ha^-1 of N₂ fixed per 6 months, when S. spectabilis and S. siamea were used as reference trees, respectively. The approach by which ¹⁵N-labeled fertilizer was applied to the soil in three splits gave slightly higher estimates of N derived from the atmosphere but this was of little agronomic significance because total N₂ fixed was similar for all methods.     

Natural N abundances in a tallgrass prairie ecosystem exposed to 8-y of elevated atmospheric CO2

After 8-y of elevated CO₂, we previously detected greater amounts of total soil nitrogen, suggesting that rates of ecosystem N flux into or out of tallgrass prairie had been altered. Denitrification and associative N fixation rates are the two primary biological processes that are known to control N loss and accumulation in tallgrass prairie soil. Therefore, our objective was to assess the natural abundance of plant and soil ¹⁵N isotopes as a cumulative index of potential change in efflux or influx of N into and out of the tallgrass prairie after 8-y of exposure to elevated CO₂. Aboveground plant delta ¹⁵N values of Andropogon gerardii were close to zero and more positive as a result of elevated CO₂, but whole-soil values at the 5-30 cm depth were significantly reduced (6.8 vs 7.3; P<0.05) under elevated CO₂-chamber (EC) relative to ambient CO₂- chamber (AC). Total, aboveground plant biomass, root-in-growth, extractable N, microbial biomass N, and soil pools collectively exhibited a range of delta ¹⁵N values from −2.8 to 7.3. Measurements of surface soil ¹⁵N indicate that a change in N inputs and outputs has occurred as a result of elevated atmospheric CO₂. In addition to possible changes in denitrification and N₂ fixation, other sources of N such as the re-translocation of N to the surface from deeper soil layers are needed to explain how soil N accrues in surface soils as a consequence of elevated CO₂. Our results support the notion that C accrual may promote N accrual, possibly driven by high plant and microbial N demand amplified by soil N limitation.     

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon (C)-rich organic matter plays a vital role in sustaining soil fertility. However, it remains poorly understood whether dinitrogen (N₂) fixation occurs in support of soil heterotrophic respiration. In this study, ¹⁵N₂-tracing indicated that strong N₂ fixation occurred during heterotrophic respiration of carbon-rich glucose. Soil organic ¹⁵N increased from 0.37 atom% to 2.50 atom% under aerobic conditions and to 4.23 atom% under anaerobic conditions, while the concomitant CO₂ flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions. Soil N₂ fixation was completely absent in soils replete with inorganic N, although soil N bioavailability did not alter soil respiration. High-throughput sequencing of the 16S rRNA gene further indicated that: i) under aerobic conditions, only 15.2% of soil microbiome responded positively to glucose addition, and these responses were significantly associated with soil respiration and N₂ fixation and ii) under anaerobic conditions, the percentage of responses was even lower at 5.70%. Intriguingly, more than 95% of these responses were originally rare with < 0.5% relative abundance in background soils, including typical N₂-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N₂-fixing heterotrophs such as Sporosarcina, Agromyces, and Sedimentibacter. These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers. Our study indicates that N₂ fixation could be closely associated with microbial turnover of soil organic C when available in excess.     

Arbuscular mycorrhizae affect the N and C economy of nodulated Phaseolus vulgaris (L.) during NH4 nutrition

In the symbiosis between nodulated legume roots and arbuscular mycorrhizal (AM) fungi, the C and N economy can be influenced by the source of N-supply from either AM-derived NH₄⁺ uptake or nodule-derived biological nitrogen fixation (BNF). This relationship was investigated in terms of NH₄⁺ supply and BNF by the two symbionts. Nodulated Phaseolus vulgaris seedlings with and without AM, were hydroponically grown with either 0 N or 1 mM NH₄⁺ supply. Plants were harvested at 30 days after emergence and measurements were taken for biomass, N₂ fixation, photosynthesis, CO₂ and O₂ root respiration, calculated C and N economy. AM roots had higher NH₄⁺ uptake and this was associated with the suppression of BNF and nodule growth. The higher NH₄⁺ uptake in AM roots occurred with lower root maintenance respiration, compared to when N was derived from BNF. There was also an increase in the below-ground sink strength of NH₄⁺ fed AM roots compared to NH₄⁺ fed non-AM roots, as evidenced by the increases in root CO₂ and O₂ respiration and photosynthetic stimulation. These results indicate that although the AM root had higher total below-ground respiratory costs during NH₄⁺ nutrition, there were lower respiratory C costs associated with N derived from AM symbionts in comparison to N from BNF.