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.     

Nitrogen turnover rates in a riparian fen determined by 15N dilution

Summary Gross rates of N mineralization, assimilation, nitrification, and NO _in3 ^sup- reduction were determined in soil from a wet riparian fen by 1-day incubations of soil cores and slurries with ¹⁵N-labelled substrates. N mineralization transformed 0.1% of the total organic N pool daily in the soil cores, of which 25% was oxidized through autotrophic nitrification and 53%–70% was incorporated into microorganisms. N mineralization and nitrification were markedly inhibited below 5 cm in soil depth. At least 80% of the NO _in3 ^sup- reduction in aerated cores occurred through dissimilatory processes. Dissimilatory reduction to NH _in4 ^sup+ (DNRA) occurred only below 5 cm in depth. The results show that NH _in4 ^sup+ oxidation was limited by available substrate and was itself a strong regulator of NO _in3 ^sup- -reducing activity. NO _in3 ^sup- reduction was significantly increased when the soil was suspended under anaerobiosis; adding glucose to the soil slurries increased NO _in3 ^sup- reduction by 2.4–3.7 times. Between 3% and 9% (net) of the added NO _in3 ^sup- was reduced through DNRA in the soil slurries. The highest percentage was observed in soil samples from deeper layers that were pre-incubated anaerobically.     

Hyphal transport by a vesicular-arbuscular mycorrhizal fungus of N applied to the soil as ammonium or nitrate

Summary Transport of N by hyphae of a vesicular-arbuscular mycorrhizal fungus was studied under controlled experimental conditions. The N source was applied to the soil as ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- . Cucumis sativus was grown for 25 days, either alone or in symbiosis with Glomus intraradices, in containers with a hyphal compartment separated from the root compartment by a fine nylon mesh. Mineral N was then applied to the hyphal compartment as ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- at 5 cm distance from the root compartment. Soil samples were taken from the hyphal compartment at 1, 3 and 5 cm distance from the root compartment at 7 and 12 days after labelling, and the concentration of mineral N in the samples was measured from 2 M KCl extracts. Mycorrhizal colonization did not affect plant dry weight. The recovery of ¹⁵N in mycorrhizal plants was 38 or 40%, respectively, when ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- was applied. The corresponding values for non-mycorrhizal plants were 7 and 16%. The higher ¹⁵N recovery observed in mycorrhizal plants than in non-mycorrhizal plants suggests that hyphal transport of N from the applied ¹⁵N sources towards the host plant had occurred. The concentration of mineral N in the soil of hyphal compartments was considerably less in mycorrhizal treatments than in controls, indicating that the hyphae were able to deplete the soil for mineral N.     

Isotope 15N enrichment of soil-ammonium N as a reference to estimate N2 fixation by stem and root-inoculated S. rostrata

Summary A pot experiment in the greenhouse was conducted to compare the contribution of N derived from the atmosphere or from biological N₂ fixation by Sesbania rostrata inoculated with Azorhizobium caulinodans, applied either to roots or to roots and stems (single or multiple stem inoculation). Two subsequent crops were grown for 50 days under flooded conditions. N derived from air was estimated by ¹⁵N dilution using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N and of Echinochloa crusgalli as the non-N₂-fixing reference datum and compared with estimates obtained by the N-difference method. The first crop was grown to stabilize the ¹⁵N into the soil organic N fraction. The ¹⁵N enrichment of soil NH _inf4 ^sup+ -N in the second crop declined slowly. The extractability ratio (¹⁵N enrichment of extractable soil N to ¹⁵N enrichment of total soil N) decreased from 4.8 to 4.1 50 days after planting. The enrichment of soil NH _inf4 ^sup+ -N was comparable to that of E. crus-galli, resulting in similar estimates of N derived from air when either soil NH _inf4 ^sup+ -N or enrichment of E. crus-galli was used as a non-fixing reference. The N-difference method did not always provide reliable estimates of N derived from air; percentages ranged from 75 to more than 80 by 50 days after planting in both crops and did not differ among treatments. The study demonstrates the potential of using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N as a non-N₂-fixing reference for reliable BNF estimates of crops in lowland puddled soil.     

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.     

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.     

Growth and nitrogen nutrition of maize (Zea mays L.) in soil treated with the nitrification-inhibiting insecticide Baythroid

A pot experiment was conducted to compare the uptake and dry matter production potential of NH _inf4 ^sup+ and NO _inf3 ^sup- and to study the effect of Baythroid, a contact poison for several insect pests of agricultural crops, on growth and N uptake of maize (Zea mays L.). Nitrogen was applied as (¹⁵NH₄)₂SO₄, K¹⁵NO₃, or ¹⁵NH₄NO₃ and in one treatment Baythroid was combined with ¹⁵NH₄NO₃. Source of N had, in general, a nonsignificant effect on dry matter and N yield, but uptake of NO _inf3 ^sup- was significantly higher than that of NH _inf4 ^sup+ when both N sources were applied together. Substantial loss of N occurred from both the sources, with NH _inf4 ^sup+ showing greater losses. Baythroid was found to have a significant positive effect on dry matter yield of both root and shoot; N yield also increased significantly. Uptake of N from both the applied and native sources increased significantly in the presence of Baythroid and a substantial added nitrogen interaction (ANI) was determined. The positive effect of Baythroid was attributed to: (1) a prolonged availability of NH _inf4 ^sup+ due to inhibition of nitrification, (2) an increased availability of native soil N through enhanced mineralization, and (3) an enhanced root proliferation.     

Immobilization of ammonium and nitrate and their interaction with native N in three Illinois Mollisols

Summary Three Illinois Mollisols were incubated for 2 weeks at 25°C after treatment with different amounts of glucose and/or ¹⁵N-labelled (NH₄)₂SO₄ or ¹⁵N-labelled KNO₃. The objectives were: (1) to compare the immobilization and interaction of NH _inf4 ^sup+ –N and NO _inf3 ^sup- –N with the native soil N, and (2) to study the relationship between immobilization of applied N and the added N interaction. As determined, immobilized N refers to forms not extractable with 2 MKCl (immobilized ¹⁵N+clay-fixed ¹⁵NH _inf4 ^sup+ ). In all cases, both NH _inf4 ^sup+ –N and NO _inf3 ^sup- –N were actively immobilized and transformed into organic forms in the presence of glucose. In the absence of glucose, a higher proportion of NH _inf4 ^sup+ than NO _inf3 ^sup- was recovered in organic forms. Although the three soils differed considerably in the amounts of applied N immobilized, similar trends in N immobilization were observed. A positive added N interaction occurred with all soils, the magnitude increasing with the rate of N addition. In the absence of glucose, higher added N interactions were obtained for NH _inf4 ^sup+ than NO _inf3 ^sup- , whereas there was very little difference between NH _inf4 ^sup+ and NO _inf3 ^sup- in the presence of glucose. The results indicate that under conditions of rapid immobilization (e.g., in the presence of glucose), NH _inf4 ^sup+ and NO _inf3 ^sup- will show comparable interaction with the native soil N, whereas in unamended soil, the extent of this interaction will be greater with NH _inf4 ^sup+ than with NO _inf3 ^sup- . Significant correlations were observed between applied N immobilized and the added N interaction only in one soil having a high initial mineral N content.     

Extractability of 15N-labeled corn-shoot tissue in a sandy and a clay soil by 0.01 MCaCl2 method in laboratory incubation experiments

Management of N fertilization depends not only on the mineral N measured at the beginning of the growing season but also on the status of the low-molecular-weight organic-N fraction. Our study was conducted to analyze how much of the ¹⁵N applied in labeled cornshoot tissue would be recovered in 0.01 M CaCl₂-extractable ¹⁵N fractions and wheter a decrease in the CaCl₂-extractable ¹⁵N fraction quantitatively followed the trend in net mineralization of the ¹⁵N applied in corn-shoot tissue during an incubation period. The effects of adding ¹⁵N-labeled young corn-shoot tissue to a sandy soil and a clay soil were investigated for 46 days in an aerobic incubation experiment at 25°C. The application of 80 mg N kg^-1 soil in the form of labeled corn-shoot tissue (24.62 mg ¹⁵N kg^-1 soil) resulted in a significant initial increase, followed by a decrease the labeled organic-N fraction in comparison with the untreated soils during the incubation. The labeled organic-N fraction was significantly higher in the sandy soil than in the clay soil until the 4th day of incubation. The decrease in labeled organic N in the sandy soil resulted in a subsequent increase in ¹⁵NO _inf3 ^sup- during the incubation. Ammonification of applied plant N resulted in a significant increase in the 1 M HCl-extractable non-exchangeable ¹⁵NH _inf4 ^sup+ fraction in the clay soik, owing to the vermiculite content. The ¹⁵N recovery was analyzed by the 0.01 M CaCl₂ extraction method; at the beginning of the incubation experiment, recovery was 37.0% in the sandy soil and 36.7% in the clay soil. After 46 days of incubation, recovery increased to 47.2 and 43.8% in the sandy and clay soils, respectively. Net mineralization of the ¹⁵N applied in corn-shoot tissue determined after the 46-day incubation was 6.60 mg ¹⁵N kg^-1 soil (=34.9% of the applied organic ¹⁵N) and 4.37 mg ¹⁵N kg^-1 soil (=23.1% of the applied organic ¹⁵N) in the sandy and the clay soils, respectively. The decrease in the labeled organic-N fraction extracted by 0.01 M CaCl₂ over the whole incubation period was 3.14 and 2.33 mg ¹⁵N kg^-1 soil in the sandy and clay soil, respectively. These results indicate that net mineralization of ¹⁵N was not consistent with the decrease in the labeled organic-N fraction. This may have been due to the inability of 0.01 M CaCl₂ to extract or desorb all of the applied organic ¹⁵N that was mineralized during the incubation period.     

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.     

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%.     

Alder-poplar associations: Determination of plant nitrogen sources by isotope techniques

Summary Three¹⁵N isotopic dilution methods (¹⁵N natural abundance, labelled mineral fertilizer, and organic matter) were used to determine the proportion of N derived from different available sources in seedlines ofAlnus glutinosa andPopulus nigra planted together or in monoculture under natural climatic conditions. The proportion of N derived from N₂ fixation in associated alders was appreciably higher than that determined in monoculture. The reduction of soil N uptake by associated alders contributed to an increase in total plant N and biomass production in associated poplars. When slightly N-labelled organic matter (alder leaf litter) was incorporated into the soil, 10–15% of its initial N content was recovered in poplar tissues, showing that this N source makes an important contribution to the N yield of associated non-fixing plants. There were no significant differences between the results obtained by¹⁵N natural abundance and those obtained by labelled fertilizer methods, suggesting that the ¹⁵N method could be used to evaluate annual N budgets in natural ecosystems.     

Effects of ammonium and nitrate on mineralization of nitrogen from leguminous residues

A laboratory incubation experiment was conducted to compare the effects of NH _inf4 ^sup+ and NO _inf3 ^sup- on mineralization of N from ¹⁵N-labelled vetch (Vicia villosa Rotn) in an Illinois Mollisol, and to determine the effect of a nitrification inhibitor (nitrapyrin) on mineralization of vetch N when used with NH _inf4 ^sup+ . The addition of either NH _inf4 ^sup+ or NO _inf3 ^sup- (100 and 200 mg N kg^-1 soil) significantly increased mineralization of vetch N during incubation for 40 days. The effect was greater with NH _inf4 ^sup+ than with NO _inf3 ^sup- , and a further increase occurred in the presence of nitrapyrin (10 mg kg^-1 soil). The addition of NO _inf3 ^sup- retarded the nitrification of NH _inf4 ^sup+ -N derived from vetch.     

NATURAL ABUNDANCES OF 15NITROGEN AND 13CARBON INDICATIVE OF GROWTH AND N2 FIXATION IN POTASSIUM FED LENTIL GROWN UNDER WATER STRESS

Dual natural abundance analysis of ¹⁵Nitrogen (N) and ¹³Carbon (C) isotopes in lentil plants subjected to different soil moisture levels and rates of potassium (K) fertilizer were determined to assess crop performance variability in terms of growth and N₂-fixation (Ndfa). The δ¹⁵N values in lentils ranged from +0.67 to +1.36‰; whereas, those of the N₂-fixed and reference plant were ?0.45 and +2.94‰, respectively. Consequently, the Ndfa% ranged from 45 and 65% of total plant N uptake. Water stress reduced Δ¹³C values. However, K fertilization enhanced whole plant Δ¹³C along with dry matter yield and N₂-fixation. The water stressed plants amended with K fertilizer seemed to be the best treatment because of its highest pod yield, high N balance, and N₂-fixation with low consumption of irrigation water. This illustrates the ecological and economical importance of K fertilizer in alleviating water stress occurring during the post-flowering period of lentil.     

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.     

Estimating symbiotic N2 fixation in Robinia pseudoacacia

Estimating symbiotic di‐nitrogen (N₂) fixation is challenging, especially when working with woody N₂ fixers in field trials. Fortunately, isotope methods based on ¹⁵N natural abundance or on ¹⁵N artificial enrichment (dilution method) make it possible to estimate the proportion of nitrogen derived from the atmosphere (Ndfa) in N₂‐fixing species. These methods have been extensively used in the field for herbaceous species, much less for tree species such as alder and acacia, and rarely for black locust (Robinia pseudoacacia). The objectives of this study were to characterize the fixation potential of black locust in a plantation by using the two ¹⁵N isotope methods in the field, and to document values of isotope fractionation occurring during N₂ fixation (the B value). B values were estimated both by growing trees on an N‐free medium in controlled conditions (B_lab) and by making Ndfa calculated with the natural abundance method converge with Ndfa calculated with the ¹⁵N dilution method in the field (B_field). The two methods gave consistent estimates of the B value. B values ranging between –1.4 and –3.2‰ were found, varying with the age of the plant material. Up to 76% of the N in the black locust trees came from the atmosphere, representing more than 45 kg N ha^?1 over five years and confirming that black locust may be well adapted to N‐poor soils.     

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.     

Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system

Below-ground transfer of nitrogen (N) fixed by legume trees to associated non-N₂-fixing crops has received little attention in agroforestry, although the importance of below-ground interactions is shown in other ecosystems. We used ¹⁵N natural abundance to estimate N transfer from the legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. to C₄ grass Dichanthium aristatum (Poir.) C.E. Hubb. in a silvopastoral system, where N was recycled exclusively by below-ground processes and N₂ fixation by G. sepium was the sole N input to the system. Finding a suitable reference plant, a grass without contact with tree roots or litter, was problematic because tree roots invaded adjacent grass monocrop plots and soil isotopic signature in soil below distant grass monocrops differed significantly from the agroforestry plots. Thus, we used grass cultivated under greenhouse conditions in pots filled with agroforestry soil as the reference. A model of soil ¹⁵N fractionation during N mineralization was developed for testing the reliability of that estimate. Experimental and theoretical results indicated that 9 months after greenhouse transplanting, the percentage of fixed N in the grass decreased from 35% to <1%, due to N export in cut grass and dilution of fixed N with N taken up from the soil. The effect of soil ¹⁵N fractionation on the estimate of the reference value was negligible. This indicates that potted grass is a suitable reference N transfer studies using ¹⁵N natural abundance. About one third of N in field-grown grass was of atmospheric origin in agroforestry plots and in adjacent D. aristatum grassland invaded by G. sepium roots. The concentration of fixed N was correlated with fine root density of G. sepium but not with soil isotopic signature. This suggests a direct N transfer from trees to grass, e.g. via root exudates or common mycorrhizal networks.     

Nitrogen accumulation in three legumes and two cereals with emphasis on estimation of N2 fixation in the legumes by the natural 15N-abundance technique

Summary N accumulation and natural ¹⁵N abundance in three legumes (groundnuts, cowpeas, and soybeans) and in two cereals (sorghum and maize) were investigated over two seasons in Alfisols with and without N fertilization. Using the N uptake and natural ¹⁵N abundance of non-nodulating plants as the indication of N derived from soil and fertilizer, the per cent N derived from atmospheric N₂ was calculated for nodulated plants. In the first experiment, the groundnut genotype contained 85% atmosphere-derived N, but the percentage decreased with N application. Estimates of atmosphere-derived N by the N-difference and ¹⁵N-abundance techniques gave identical results. The percentages of atmosphere-derived N estimated by the two methods at different stages of groundnut growth were also similar. In the second experiment, atmosphere-derived N was estimated in plants grown with 0–200 kg ha^-1 applied N. The estimated atmosphere-derived N ranged from 42% to 61% for groundnuts from 33% to 77% for cowpeas, and from 24% to 48% for soybeans, depending on the amount of N applied. Inoculation with a Bradyrhizobium strain increased the percentage of atmospherederived N in soybean plants grown without any fertilizer N. The natural ¹⁵N abundance of sorghum and maize was very close to that of the non-nodulating groundnut, suggesting that these cereals can be used as reference plants in the estimation of atmosphere-derived N by the natural ¹⁵N-abundance method.ICRISAT Journal Article No. 876     

Phytotron studies to compare nitrogen losses from corn-planted soil by the 15-N balance or direct dinitrogen and nitrous oxide measurements

Summary Containers filled with soil mixed with potassium nitrate highly enriched in ¹⁵N were planted with corn (Zea mays L.) and kept in a phytotron under controlled conditions for 79 days. Soil water content was normally maintained at exactly 60% water-holding capacity (–33 kPa), but it was increased several times to 85% (–5 kPa) for short periods to favour denitrification. The soil headspace was sealed from the phytotron atmosphere and aerated by a continuous stream of air. Nitrous oxide emission was measured by estimating the N₂O concentration differences in the air entering and leaving the containers. Emission of N₂ was estimated by mass spectroscopy from changes in the N₂ composition in the temporarily enclosed soil headspace. Both methods were carefully checked for accuracy by different tests. At specific times during the experiment the distribution of ¹⁵N between plants and soil was determined and a ¹⁵N balance established. Emission of N gases peaked at times of increased water content and reached maxima of 149 and 142 g N pot^–1 day^–1 for N₂O and N₂, respectively. While N losses of 5% ± 2% were indicated by the ¹⁵N balance, only 1.1% ± 0.3% loss from 2.7 g applied N was estimated from the N₂O and N₂ measurements after 79 days. Possible reasons for these differences are discussed.