Comparative effects of app1ication of coated and non-coated urea in clayey and sandy paddy soi1 microcosms examined by the 15N tracer technique

Abstract

¹⁵N assimilation was studied in bacteroid and cytosol fractions of soybean nodules. In the first experiment, after exposing the intact nodules to ¹⁵N₂ for 5 min and 10 min, most of the fixed ¹⁵N was detected in cytosol fraction. In cytosol fraction, ¹⁵N content of glutamine was the highest and followed by glutamic acid, alanine, and allantoin in this sequence, whereas, in bacteroid fraction, glutamic acid showed the highest ¹⁵N content and alanine and glutamine followed. In the second experiment, ¹⁵N assimilation of various ¹⁵N-labeled compounds in the separated bacteroid and cytosol fractions was investigated. In the separated bacteroid fraction which was fed with ¹⁵NH₄, ¹⁵N was incorporated very rapidly into glutamic acid, alanine, and aspartic acid, but very slowly into glutamine.

From these results, it was suggested that most of the fixed ammonia was exported to cytosol and assimilated via glutamine synthetase to glutamine, then via glutamate synthase to glutamic acid, and from these compounds various nitrogenous compounds were formed, but in bacteroids glutamate dehydrogenase and alanine dehydrogenase played an important role in the assimilation of fixed ammonia though quantitatively the contribution to ammonia assimilation in nodules was much less compared with cytosol.     

The fate of markerAzospirillum lipoferum inoculated into rice and its effect on growth,yield and N2 fixation of plants studied by acetylene reduction,15N2 feeding and15N dilution techniques

Summary A spontaneous mutant ofAzospirillum lipoferum, resistant to streptomycin and rifampicin, was inoculated into the soil immediately before and 10 days after transplanting of rice (Oryza sativa L.). Two rice varieties with high and low nitrogen-fixing supporting traits, Hua-chou-chi-mo-mor (Hua) and OS4, were used for the plant bacterial interaction study. The effect of inoculation on growth and grain and dry matter yields was evaluated in relation to nitrogen fixation, by in situ acetylene reduction assay,¹⁵N₂ feeding and¹⁵N dilution techniques. A survey of the population of marker bacteria at maximum tillering, booting and heading revealed poor effectivety. The population of nativeAzospirillum followed no definite pattern. Acetylene-reducing activity (ARA) did not differ due to inoculation at two early stages but decreased in the inoculated plants at heading. In contrast, inoculation increased tiller number, plant height of Hua and early reproductive growth of both varieties. Grain yield of both varieties significantly increased along with the dry matter. Total N also increased in inoculated plants, which was less compared with dry matter increase.¹⁵N₂ feeding of OS4 at heading showed more¹⁵N₂ incorporation in the control than in the inoculated plants. The ARA,¹⁵N and N balance studies did not provide clear evidence that the promotion of growth and nitrogen uptake was due to higher N₂ fixation.     

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.     

Sesbania spp., Aeschynomene indica and Crotalaria spp. are amide-exporters

Abstract

Extract

Leguminous plants consist of two groups, amide-exporting and ureide-exporting plants. The former legumes export a large fraction of fixed-N in the form of amides (asparagine and glutamine), and the latter legumes in the form of ureides (allantoic acid and allantoin). Another characteristic of the nodules is the enrichment in ¹⁵N. There are two types of legumes: one characterized by the enrichment with ¹⁵N in N₂-fixing nodules, in contrast to the other where the enrichment does not occur. The first investigation by Shearer et al. (1982) suggested that the nodules exporting fixed-N in the form of ureides were enriched in ¹⁵N unlike those exporting it in the form of amides. Soybeans, mungbean, and cowpea belong to the former group and groundnut, alfalfa, white clover to the latter. Although pea and faba bean were first classified into the latter group (Shearer et al. 1982), a recent investigation (Yoneyama 1988) showed that these nodules were also enriched in ¹⁵N.     

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.     

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.     

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 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.     

Soil N Losses by Denitrification Evaluated Using the 15N Tracer Method

Molecular nitrogen (N₂) and nitrous oxide (N₂O) generated by denitrification increase N losses in the soil–plant system. This study aimed to quantify N₂ and N₂O from potassium nitrate (K¹⁵NO₃) applied to soils with different textures and moisture contents in the absence and presence of a source of carbon (C) using the ¹⁵N tracer method. In the three soils used (sandy texture (ST), sandy clay loam texture (SCLT), and clayey texture (CT)), three moisture contents were evaluated (40%, 60%, and 80% of the water holding capacity (WHC)) with (D⁺) and without (D^?) dextrose added. The treatments received 100 mg N kg^?1 (KNO₃ with 23.24 atom% ¹⁵N). N₂ emissions occurred in all of the treatments, but N₂O emissions only occurred in the D⁺ treatment, showing increases with increasing moisture content. SCLT with 80% WHC in the D⁺ treatment exhibited the highest accumulated N emission (48.26 mg kg^?1). The ¹⁵N balance suggested trapping of the gases in the soil.     

Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

15N isotope dilution methodology for evaluating the dynamics of biologically fixed N in legume-non-legume associations

We examined the theoretical basis for estimating the transfer of N₂ fixed by legumes to companion cereals or grasses in intercropping or pasture systems using ¹⁵N isotope dilution methodology. A method was developed to calculate the symbiotic dependence of the legume in a mixed stand based on ¹⁵N enrichment of the associated non-legume and the estimate of fixed N transfer. Published field data were used to illustrate the application of the method. Complementary treatments for verifying N transfer and options for increasing the accuracy of estimates of N transfer are discussed.     

NH3 Volatilization,N2O Emission and Microbial Biomass Turnover from 15N-Labeled Manure Under Laboratory Conditions

On irrigated agricultural soils from semi-arid and arid regions, ammonia (NH₃) volatilization and nitrous oxide (N₂O) emission can be a considerable source of N losses. This study was designed to test the capture of ¹⁵N loss as NH₃ and N₂O from previous and recent manure application using a sandy, calcareous soil from Oman amended one or two times with ¹⁵N labeled manure to elucidate microbial turnover processes under laboratory conditions. The system allowed to detect ¹⁵N enrichments in evolved N₂O-N and NH₃-N of up to 17% and 9%, respectively, and total N, K₂SO₄ extractable N and microbial N pools from previous and recent ¹⁵N labeled manure applications of up to 7%, 8%, and 15%. One time manured soil had higher cumulative N₂O-N emissions (141 µg kg^?1) than repeatedly manured soil with 43 µg kg^?1 of which only 22% derived from recent manure application indicating a priming effect.     

A GC/MS method for the assessment of N and C incorporation into soil amino acid enantiomers

Identifying the transformation process of amino acid enantiomers was essential to probe into the fate, turnover and aging of soil nitrogen due to their important roles in the biogeochemical cycling. If this can be achieved by differentiating between the newly biosynthesized and the inherent compounds in soil, then the isotope tracer method can be considered most valid. We thereby developed a gas chromatography/mass spectrometry (GC/MS) method to trace the ¹⁵N or ¹³C isotope incorporation into soil amino acid enantiomers after being incubated with ¹⁵NH₄⁺ or U-¹³C-glucose substrates. The most significant fragments (F) as well as the related minor ions were monitored by the full scan mode and the isotope enrichment in amino acids was estimated by calculating the atom percentage excess (APE). ¹⁵NH₄⁺ incorporation was evaluated according to the relative abundance increase of m/z F+1 to F for neutral and acidic amino acids and F+2 to F (mass 439) for lysine. The assessment of ¹³C enrichment in soil amino acids was more complicated than that of ¹⁵N due to multi-carbon atoms in amino acid molecules. The abundance ratio increment of m/z F+n to F (n is the original skeleton carbon number in each fragment) indicated the direct conversion from the added glucose to amino acids, but the total isotope incorporation from the added ¹³C can only be calculated according to all target isotope fragments, i.e. the abundance ratio increment summation from m/z (Fa+1) through m/z (Fa+T) represented the total incorporation of the added ¹³C (Fa is the fragment containing all original skeleton carbons and T is the carbon number in the amino acid molecule). This method has a great advantage especially for the evaluation of high-abundance isotope enrichment in organic compounds compared with GC/C/IRMS. And in principle, this technique is also valid for amino acids besides enantiomers if stereoisomers are not concerned. Our assessment approach could shine a light on investigating the biochemical mechanism of microbial transformation of N and C in soils of terrestrial ecosystem.     

Assimilation of ammonia by the azolla‐anabaena symbiosis

¹⁵N‐labeled ammonia was rapidly assimilated by Azolla caroliniana and incorporated into plant material even though sustained growth of the fern‐algae symbiosis cannot be maintained with ammonia as nitrogen source. During ammonia uptake, the nitrogenous pool of the fern rapidly increases and contains large amounts of free ammonia and glutamine. N₂ fixation activity of the algal symbiont declines during assimilation of ammonia, but it is restored to a high level upon transfer of plants to nitrogen‐free media, as the pool ammonia content decreases. During growth of the fern on N₂, the algal symbiont supplies ammonia in a manner permitting sustained growth of the plant. Exogenous ammonia, therefore, appears to interrupt regulation of inorganic nitrogen metabolism of the plant‐algal symbiosis.     

Dynamics of 15N-labeled ammonium sulfate in various inorganic and organic soil fractions of wetland rice soils

Summary The dynamics of basally applied ¹⁵N-labeled ammonium sulfate in inorganic and organic soil fractions of five wetland rice soils of the Philippines was studied in a greenhouse experiment. Soil and plant samples were collected and analyzed for ¹⁵N at various growth stages. Exchangeable NH₄ ⁺ depletion continued after 40 days after transplanting (DAT) and corresponded with increased nitrogen uptake by rice plants. Part of the applied fertilizer was fixed by 2:1 clay minerals, especially in Maligaya silty clay loam, which contained beidellite as the dominant clay mineral. After the initial fixation, nonexchangeable ¹⁵N was released from 20 DAT in Maligaya silty clay loam, but fixation delayed fertilizer N uptake from the soil. Part of the applied N was immobilized into the organic fraction. In Guadalupe clay and Maligaya silty clay loam, immobilization increased with time while the three other soils showed significant release of fertilizer N from the organic fraction during crop growth. Most of the immobilized fertilizer N was recovered in the nondistillable acid soluble (alpha-amino acid + hydrolyzable unknown-N) fraction at crop maturity. Between 61% and 66% of applied N was recovered from the plant in four soils while 52% of fertilizer N was recovered from the plant in Maligaya silty loam. Only 20% – 30% of the total N uptake at maturity was derived from fertilizer N. Nmin (mineral N) content of the soil before transplanting significantly correlated with N uptake. Twenty-two to 34% of applied N was unaccounted for possibly due to denitrification and ammonia volatilization.     

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 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.     

Technique of soil sample pretreatment for analysis of 15N:14N isotope ratio

Abstract

The technique of simultaneous quantitative determination of mineral N soil forms (nitrates, exchangeable and non‐exchangeable ammonium, and total amount of these compounds) and sample pretreatment for the analysis of ¹⁵N:¹⁴N ratio is suggested. The technique is based on the selective association of NH₄ ⁺‐ions into indophenol complex and subsequent ethyl‐acetate extraction of this complex from solution. The mineralization of indophenol is carried out in alkaline medium with simultaneous NH₃ distillation into H₂SO₄ titrant. The application of given technique allows us to shorten significantly the time required for analysis and to increase the accuracy of analytical determination.     

Nitrogen fixation by intact grass-soil cores using 15N2 and acetylene reduction

To determine N₂ fixation by intact grass-soil cores, samples were collected from 25 sites in central Texas during the summer. Three cores (32 cm² each) were extracted immediately adjacent to one another from single grass clumps or sods. Two of these cores were incubated under 10% C₂H₂ in air and the third core was incubated for 12 h in an atmosphere with 10% ¹⁵N₂ enrichment. Following incubation with ¹⁵N₂ the same core was assayed for rate of C₂H₂ reduction (AR). Rates of AR were generally low and quite variable (0–7.6 μmol C₂H₄ core^?1 day^?1). ¹⁵N₂ was incorporated into root and shoot tissues within 12–24 h. Extrapolated values of N₂ fixation based on ¹⁵N₂ incorporation ranged from 0 to 20 kg N ha^?1100 day^?1. The ratio of C₂H₂ reduced (μ mol C₂H₄ core^?1 day^?1) to N₂ fixed (μ mol N₂ fixed core^?1 day^?1) was highly variable ranging from 0 to 12. This study confirmed that N₂ is fixed in the rhizosphere of grasses grown in Texas through the use of ¹⁵N₂ and demonstrated that incorporation of fixed N into shoots was relatively rapid.     

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.