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.     

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.     

Field evaluation of N2 fixation and N partitioning in climbing bean (Phaseolus vulgaris L.) using 15N

Summary The common bean (Phaseolus vulgaris L.) is generally regarded as a poor N₂ fixer. This study assessed the sources of N (fertilizer, soil, and fixed N), N partitioning and mobilization, and soil N balance under field conditions in an indeterminate-type climbing bean (P. vulgaris L. cv. Cipro) at the vegetative, early pod-filling, and physiological maturity stages, using the A-value approach. This involved the application of 10 and 100 kg N ha^-1 of ¹⁵N-labelled ammonium sulphate to the climbing bean and a reference crop, maize (Zea mays L.). At the late pod-filling stage (75 days after planting) the climbing bean had accumulated 119 kg N ha^-1, 84% being derived from fixation, 16% from soil, and only 0.2% from the ¹⁵N fertilizer. N₂ fixation was generally high at all stages of plant growth, but the maximum fixation (74% of the total N₂ fixed) occurred during the interval between early (55 days after planting) and late podfilling. The N₂ fixed between 55 and 75 days after planting bas a major source (88%) of the N demand of the developing pod, and only about 11% was contributed from the soil. There was essentially no mobilization of N from the shoots or roots for pod development. The cultivation of common bean cultivars that maintain a high N₂-fixing capacity especially during pod filling, satisfying almost all the N needs of the developing pod and thus requiring little or no mobilization of N from the shoots for pod development, may lead to a net positive soil N balance.     

Symbiotic N2 fixation in 30 field-grown cowpea (Vigna unguiculata L. Walp.) genotypes in the Upper West Region of Ghana measured using 15N natural abundance

In this study, 30 cowpea genotypes were assessed for symbiotic N₂ fixation in 2005, and 15 of them were re-evaluated in 2006 using the ¹⁵N natural abundance technique. Shoot dry matter yield of cowpea genotypes increased significantly in cvs. Vuli-1, Glenda, IT93K-2045-29, IT90K-59, Omondaw, Apagbaala, and IT84S-2246 in 2005 producing about 3.0 to 3.6-fold more biomass relative to cv. Vallenga. In 2006, seven out of the 15 cowpea genotypes tested (namely, IT97K-499-39, TVu11424, Botswana White, IT84S-2246, Sanzie, Brown Eye, and Glenda) also produced more dry matter than cv. CH14. Shoot δ¹⁵N values ranged from −0.58‰ to 1.49‰ in 2005, and −1.51‰ to 1.40‰ in 2006, and these resulted in %Ndfa values of 63.5–86.7% and 56.2–96.3%, respectively. The amount of N-fixed was 49–178 kg N ha⁻¹ in 2005 and 62–198 kg N ha⁻¹ in 2006. Furthermore, there was a direct relationship between the level of symbiotic N nutrition and plant growth, and between grain yield and amount of N-fixed in 2005 and 2006. As a result, genotypes that fixed the most N also produced the largest biomass and the greatest amount of grain yield. The observed relationship between N₂ fixation and biomass confirmed our view that cowpea (and other grain legumes) can be concurrently selected for higher N₂ fixation, superior plant growth, and greater grain yield. The high levels of N-fixed by many of the cowpea genotypes in this study suggest that they can contribute large amounts of N to cropping systems in African agriculture.     

Genotypic variation for phosphorus uptake dinitrogen fixation in cowpea on low‐phosphorus soils of southern Cameroon

In cowpea, efficient N₂‐fixing genotypes are being selected to promote sustainable cropping systems in southern Cameroon (SC). However, N₂ fixation and growth of these genotypes are largely hampered by low levels of soil plant‐available P. To evaluate the genotypic variation in N₂ fixation and P uptake among cowpea (Vigna unguiculata L.) genotypes, field experiments were conducted over two years on two acid soils low in available P. The experiments were laid out in a split‐block design with four replications on typic (TK) and rhodic (RK) Kandiudult soils with seven cowpea genotypes. Phosphorus (P) fertilizers were applied on the main plots with 0 kg P, 30 kg P ha^–1 as triple superphosphate (TSP) and 90 kg P ha^–1 as Togo phosphate rock (PR). Nodule dry matter (DM), shoot DM, grain yield, and P uptake of cowpea significantly varied with site, P application, and genotype (p < 0.05). The N₂ fixation of the cowpea genotypes ranged from 29 to 51 kg N ha^–1 on both TK and RK soils and was significantly increased with P application. Significant genotypic variations in N₂ fixation were observed with superior ability of the genotypes IT89KD‐391 and IT90K‐59 to fix N₂. The harvest index (HI) did not significantly differ between soils and P application levels (p > 0.05). Four genotypes were selected to investigate root mechanisms responsible for efficient P acquisition in pot experiments. The results suggest that a better root infection by arbuscular mycorrhizal fungi (AMF) in genotype IT90K‐59 and root morphological and physiological characteristics in IT89KD‐391 were the most important factors for increasing P uptake.     

Determining Symbiotic Nitrogen Fixation in Dry Bean Cultivars Using Ureide Method and Isotope Dilution Techniques

Symbiotic nitrogen fixation (SNF) is an environmentally safe source of nitrogen (N) to the crop plants. In total, 12 dry bean (Phaseolus vulgaris L.) cultivars from pinto, navy, black, and kidney market classes were inoculated with rhizobia and grown in a greenhouse. SNF was estimated using isotope dilution technique and ‘ureide’ method. The amount of SNF ranged between 33 and 68 mg N plant^–1 when determined using ¹⁵N isotope dilution and followed the order: pinto > navy > black > kidney. Percent N derived from atmosphere (%Ndfa) significantly varied between 49% and 90% at V₃ and between 71% and 98% at R₂ stages. The outcomes of the experiment suggested that dry bean cultivars from different market classes have variable N₂ fixation ability, and fertilizer N required should be calculated according to their SNF potentials and N need of a specific market class or cultivar. Stable isotope dilution should be used as the standard procedure to estimate the SNF in dry bean.     

N supply by groundnuts to maize in a maize plus groundnut intercropping system,as affected by the genotype

Summary Identification of legume genotypes with high N-supplying ability is important in improving and sustaining the productivity of low-input cropping systems. Hence, ¹⁵N-aided studies were made to ascertain the relative N-supplying ability of some cultivars of groundnut, a widely grown tropical legume. The study was conducted outdoors in 1991 at Kamburupitiya, Sri Lanka, in tanks filled with 64 kg soil which had been tagged by incorporating ¹⁵N-labelled plant material. Maize cv. Badra was grown as a monocrop and as an intercrop with five genotypes of groundnut, X-14, MI-1, Red Spanish, ICGV 87127, and a non-nodulating line. All the nodulating genotypes derived over 90% of their N from the atmosphere. Significant genotypic differences in N₂ fixation were observed. X-14 fixed the highest amount (1.95 g plant^-1), while Red Spanish the lowest (0.88 g plant^-1). Intercropping of maize with nodulating groundnut significantly decreased the ¹⁵N atom excess of maize, depending on the genotype. However, this decrease did not appear to be related to the amount of N₂ fixed, based on aboveground material. The per cent N derived by maize from the intercropped groundnuts varied from 17% (X-14) to 39% (Red Spanish), indicating a marked genotypic variability in N-suppling ability. X-14, which fixed the largest amounts of N₂, grew most vigorously compared to other genotypes, causing a growth depression in the maize. The genotype that fixes the most N₂ may therefore not necessarily have the greatest N-supplying ability. The transfer of N from the legume and the consequent improvement of N nutrition in the associated cereal in low-fertility situations is therefore expected to be high when the growth of the legume is intermediate and does not suppress the growth of the cereal.     

Dynamics of nodulation,nitrogen fixation,nitrogen use and biomass yield over time in pot-grown Leucaena leucocephala (Lam.) de Wit

The dynamics of nodulation, N₂-fixation and N use in Leucaena leucocephala cv. K28 over time was investigated in a screenhouse at 4, 8, 12 and 16 months after planting (MAP) using the ¹⁵N-labelling method. Leucaena had a consistently increasing pattern of nodulation, dry biomass and nitrogen yield. A sharp rise in nodulation was observed between 12 and 16 MAP, whereas for biomass, N accumulation and N₂-fixation, and N₂-fixation, an upward surge occurred between 4 and 12 months. Nodulation, N accumulation, N₂-fixation and biomass yield all peaked at 16 MAP. Along with the steady increase in N₂-fixation throughout the 16-month growth period, the % N derived from the atmosphere rose from 17.9% to 61.5%, 70.1% and 74%, equivalent to 191, 1623, 2395 and 3385 mg N₂ fixed plant^-1 at 4, 8, 12 and 16 MAP, respectively. Nitrogen assimilation from soil and fertilizer decreased inversely to the increase in symbiotic nitrogen fixation with time.     

Evaluation of nitrogen fixation by different strains of theAzolla-Anabaena symbiosis in the presence of a high level of ammonium

Summary The proportion of N derived from N₂ fixation for 99 strains ofAzolla spp. (comprising all known species) in the presence of ammonium (40 mg/1) was assessed using a¹⁵N-dilution technique. The percentage of N derived from air varied from 29.5% to 79.9%. Although the N concentration ofAzolla spp. was not correlated with fertilizer N, it correlated fairly well with N₂ fixation. Regression analysis suggests that the N yield ofAzolla spp. is more dependent on N₂ fixation than on ammonium assimilation. The high correlation between N yield and isotopically determined, fixed N₂ indicates that the N yield could be used as a parameter in the selection ofAzolla spp. strains that are capable of maintaining high N₂ fixation in the presence of a high level of ammonium.     

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.     

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     

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.     

Effects of NPK fertilizer combinations on yield and nitrogen balance in sorghum or pigeonpea on a vertisol in the semi-arid tropics

A long-term experiment was carried out on a Vertisol from 1986 to 1992 to examine the combined effects of NPK fertilizers on yield using sorghum (Sorghum bicolor L. Moench cv. CSH 5) and short-duration pigeonpea (Cajanus cajan L. Millsp. cv. ICPL 87). The fertilizer treatments were as follows: 0 (no fertilization), N (150 kg N ha^-1 ), P (65.5 kg P₂O₅ ha^-1), K (124.5 kg K₂O ha^-1), and all possible combinations (NP, NK, PK, and NPK). In this study we continued this experiment during the period 1993 to 1994 and analyzed the crop yield response to fertilizers and the N balance. The amount of N derived from the atmosphere and fertilizer was estimated by the ¹⁵N natural abundance method and ^l5N isotope dilution method, respectively. A combined application of Nand P fertilizers gave the highest grain yield for the two crops under the 8th and 9th continuous croppings, unlike the application of K fertilizer. The values of total N for the two crops were significantly higher in the NP and NPK plots. These crops took up N mainly from soil. There was a significant positive relationship between the uptake of N_dff and N_dfs by each crop. Pigeonpea or sorghum took up more N from the soil in the N fertilizer plots than in the plots without N, suggesting that soil N fertility was enhanced and the amount of N supplied from soil increased in the plots with consecutive application of N fertilizer for 7 y. Even pigeonpea, which fixes atmospheric N inherently, needed N fertilizer to achieve high grain yield, suggesting that N fixation by the nodules was not always sufficient to meet the N requirements of the crop under these conditions. Although fertilizer N exerted a beneficial effect on plant growth and yield in the two crops, the values of fertilizer N recovery (FNR) by the two crops were considerably low. Therefore, it is suggested that the development of N fertilizer management which could maximize FNR of each crop should be promoted.     

A comparison of nitrogen fixation in genotypes of groundnut (Arachis hypogaea L.) using 15N-isotope dilution

Summary Nitrogen fixation in seven groundnut genotypes was measured by ¹⁵N-isotope dilution using a non-nodulating cultivar of groundnut as the nonfixing reference plant. Nitrogen fixation varied between 100 kg N ha^–1 in genotype J-11 and 153 kg N ha^–1 in Robut 33-1. The amount of plant-available soil N was small, so that 86%–92% of plant nitrogen was derived from N₂-fixation. Thus differences in N₂-fixation between genotypes closely reflected differences in their total N accumulation.ICRISAT Journal Article no. 600     

Quantitative analysis of the initial transport of fixed nitrogen in nodulated soybean plants using 15N as a tracer

The quantitative analysis of the initial transport of fixed isotope 15-nitrogen (¹⁵N) in intact nodulated soybean plants (Glycine max [L.] Merr. cv. Williams) was investigated at the vegetative stage (36 days after planting, DAP) and pod-filling stage (91 DAP) by the ¹⁵N pulse-chase experiment. The nodulated roots were exposed to N₂ gas labeled with a stable isotope ¹⁵N for 1 h, followed by 0, 1, 3 and 7 h of exposure with normal air. Plant roots and shoots were separated into three sections (basal, middle and distal parts) with the same length of the main stem or primary root. Approximately 80 and 92% of fixed N was distributed in the basal part of the nodulated roots at the vegetative and pod-filling stages by the end of 1 h of ¹⁵N₂ exposure, respectively. In addition, about 90% of fixed ¹⁵N was retained in the nodules and 10% was exported to root and shoot after 1 h of ¹⁵N₂ exposure at 91 DAP. The percentage distribution of ¹⁵N in the nodules at the pod-filling stage decreased from 90% to 7% during the 7 h of the chase period, and increased in the roots (14%), stems (54%), leaves (12%), pods (10%) and seeds (4%). The ¹⁵N distribution was negligible in the distal root segment, suggesting that N fixation activity was negligible and recycling fixed N from the shoot to the roots was very low in the initially short time of the experiment.     

Nitrogen use in maize-grain legume cropping systems in semi-arid Kenya

Locally suitable cultivars of maize, beans, and cowpeas were grown in field experiments for four seasons in semi-arid Kenya. For three seasons, the dry matter production and grain yield of maize and beans were not increased by N fertilizer additions up to 120 kg N ha^-1. Fertilizer recoveries measured by ¹⁵N isotope dilution techniques were low, less than 20%. Inoculated and uninoculated beans failed to fix N₂. By contrast the cowpea derived 50% of its N from fixation, equivalent to 197 kg N ha^-1. The N content of the grain generally exceeded 40 kg N ha^-1, and the N content of the seeds from the grain legumes were greater than those from the cereals. Large inputs of N fertilizer or N by fixation are required if maize-grain legume cropping system in semiarid Kenya are to be sustained in the long term.     

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.     

The use of nitrogen-15 natural abundance and nitrogen yield of non-nodulating isolines to estimate nitrogen fixation by soybeans (Glycine max L.) across three elevations

Summary Dissimilarities in soil N uptake between N₂-fixing and reference non-N₂-fixing plants can lead to inaccurate N₂ fixation estimates by N difference and ¹⁵N enrichment methods. The natural ¹⁵N abundance ( ¹⁵N) method relies on a stabilized soil ¹⁵N pool and may provide reliable estimates of N₂ fixation. Estimates based on the ¹⁵N and differences in N yield of nodulating and non-nodulating isolines of soybean were compared in this study. Five soybeans from maturity groups 00, IV, VI, and VIII and their respective non-nodulating isolines were grown at three elevations differing in ambient temperature and soil N availability. Despite large differences in phenological development and N yield between the non-nodulating isolines, the ¹⁵N values measured on seeds were relatively constant within a site. The ¹⁵N method consistently produced lower N₂ fixation estimates than the N difference method, but only in three of the 15 observations did they differ significantly. The average crop N derived from N₂ fixation across sites and maturity groups was 81% by N difference compared to 71% by ¹⁵N. The magnitude of difference between the two methods increased with increasing proportions of N derived from N₂ fixation. These differences between the two methods were not related to differences in total N across sites or genotypes. The low N₂ fixation estimates based on ¹⁵N might indicate that the nodulating isolines had assimilated more soil N than the non-nodulating ones. A lower variance indicated that the estimates by N difference using non-nodulating isolines were more precise than those by ¹⁵N. Since the differences between the estimates were large only at high N₂ fixation levels (low soil N availability), either method may be used in most situations when a non-nodulating isoline is used as the reference plant. The ¹⁵N method may have a comparative advantage over N difference and ¹⁵N enrichment methods in the absence of a suitable non-N₂-fixing reference plant such as a non-nodulating isoline.     

Cutting regime determines allocation of fixed nitrogen in white clover

Leguminous leys are important sources of nitrogen (N), especially in forage-based animal production and organic cropping. Models for estimating total N₂ fixation of leys—including below-ground plant-derived N (BGN)—are based on grazed or harvested leys. However, green manure leys can have different proportions of above-ground plant-derived N (AGN) and BGN when subjected to different cutting regimes. To investigate the effects of cutting on N distribution in white clover, a pot experiment was carried out using ¹⁵N techniques to determine N₂ fixation, N rhizodeposition and root C and N content of cut and uncut white clover (Trifolium repens L. cv. Ramona) plants. Percentage N derived from air (%Ndfa) was lower in uncut (63%) than in cut (72%) plants, but total Ndfa was not significantly affected by cutting. The higher reliance on N₂ fixation in cut plants was thus counterbalanced by lower biomass and total N content. With BGN taken into account, total plant-derived N increased by approximately 50% compared with AGN only. Cutting did not affect the proportion of BGN to standing shoot biomass N after regrowth, but decreased the proportion of BGN to total shoot biomass production during the entire growth period. Thus, estimates of N fixation in green manure leys should consider management practices such as cutting regime, as this can result in differences in above- and below-ground proportions of plant-derived N.