Estimation of cadmium load on Japanese farmland associated with the application of chemical fertilizers and livestock excreta

It is very important to use ¹⁵N labeled nitrogen gas (¹⁵N₂) in studies on biological nitrogen fixation. For example, ¹⁵N₂ is necessary for direct measurement of the amount of fixed nitrogen, and is useful for studies on the assimilation and transport of fixed nitrogen. However, ¹⁵N₂ is sometimes troublesome to deal with for the following reasons. Decline in the ¹⁵N content of ¹⁵N₂ gas may occur as a result of contamination with atmospheric N₂ during the storage period or application to nitrogen fixing organisms. Also, the ¹⁵N₂ gas provided commercially in a glass bottle or gas cylinder is technically and economically not convenient for experiments employing small amounts of ¹⁵N₂. Moreover, purification of ¹⁵N₂ gas is necessary for biological research, since contamination with the oxidized forms of nitrogen represses biological nitrogen fixation to a certain extent. A simple method for the preparation, purification and storage of ¹⁵N₂ gas for biological nitrogen fixation studies, was therefore devised.     

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.     

Evaluation of isotopic dilution method for measuring N2 fixation in Azolla: Comparison with other methods 1

An isotopic dilution method that overcomes the drawbacks of commonly used methods for measuring N₂ fixation by aquatic N‐fixers such as Azolla pinnata‐Anabaena azollae association (Azolla) is presented. The method was compared with ¹⁵N₂ gas (while maintaining CO₂) and the difference methods of measuring N₂ fixation. The isotopic dilution method was used for two conditions: a. For ¹⁵N‐free growth medium, Azolla was pre‐enriched with ¹⁵N, and N₂ fixation was determined by measuring the dilution of ¹⁵N in the tissue. b. For the growth medium containing N, N₂ fixation was determined by providing ¹⁵N enriched ammonium sulfate in the growth medium and measuring ¹⁵N to ¹⁴N ratio in the tissue. An airtight chamber, necessary for ¹⁵N₂ gas and acetylene reduction methods, was not representative of the growing environment of Azolla. Temperature in the airtight chamber was far from uniform and CO₂ was rapidly depleted. The isotopic dilution method is simpler, relatively inexpensive, subject to fewer errors and applicable to more diverse conditions, and yet was as accurate as ¹⁵N₂‐gas method.     

Nitrogen fixation as influenced by moisture content,ammonium sulphate and organic sources in a paddy soil

The effect of moisture and (NH₄)₂SO₄ on N₂ fixation in a paddy soil was investigated employing C₂H₂ reduction assay and ¹⁵N-tracers. N₂ fixation was negligible under nonflooded conditions. Soil submergence accelerated N₂ fixation; with a further increase in N₂ fixation when the flooded soil was incubated under an Ar atmosphere. Rice straw additions to both moist and flooded soils enhanced N₂ fixation. N₂-ase activity in the soil decreased with increasing concentration of added N although complete suppression of the activity was not evident even at concentrations as high as 160–320 parts/10⁶ N. A similar trend of inhibition by N was also noticed in soils amended with glucose or cellulose in combination with N. However, the inhibitory effect of N decreased with increased incubation of soil except at 320 parts/10⁶ N.     

Carbon and oxygen controls on N2O and N2 production during nitrate reduction

Here we provide evidence that the form of carbon compound and O₂ concentration exert an inter-related regulation on the production and reduction of N₂O in soil. 6.7 mM d-glucose, 6.7 mM D-mannitol, 8 mM L-glutamic acid or 10 mM butyrate (all equivalent to 0.48 g C l⁻¹) were applied to slurries of a sandy loam soil. At the start of the experiment headspace O₂ concentrations were established at ∼2%, 10% and 21% O₂ v/v for each C treatment, and 2 mM K¹⁵NO₃ (25 atom % excess ¹⁵N) was applied, enabling quantification of ¹⁵N-N₂ production, ¹⁵N-(N₂O-to-N₂) ratios and DNRA. The form of C compound was most important in the initially oxic (21% O₂ v/v) soils, where addition of butyrate and glutamic acid resulted in greater N₂O production (0.61 and 0.3 μg N₂O-N g⁻¹ soil for butyrate and glutamic acid, respectively) than the addition of carbohydrates (glucose and mannitol). Although, there was no significant effect of C compound at low initial O₂ concentrations (∼2% O₂ v/v), production of ¹⁵N-N₂ was greatest where headspace O₂ concentrations were initially, or fallen to, ∼2% O₂ v/v, with greatest reduction of N₂O and lowering ¹⁵N-(N₂O-to-N₂) ratios (∼0-0.27). This may reflect that the effect of C is indirect through stimulation of heterotrophic respiration, lowering O₂ concentrations, providing sub-oxic conditions for dissimilatory nitrate reduction pathways. Addition of carbohydrates (glucose and mannitol) also resulted in greatest recovery of ¹⁵N in NH₄⁺ from applied ¹⁵N-NO₃⁻, indicative of the occurrence of DNRA, even in the slurries with initial 10% and 21% O₂ v/v concentrations. Our ¹⁵N approach has provided the first direct evidence for enhancement of N₂O reduction in the presence of carbohydrates and the dual regulation of C compound and O₂ concentration on N₂O production and reduction, which has implications for management of N₂O emissions through changing C inputs (exudates, rhizodeposition, residues) with plant species of differing C traits, or through plant breeding.     

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.     

Modelling nitrogen fixation of pea ( Pisum sativum L.)

Abstract

Nitrogen fixation was simulated for a leafless variety (Delta) of pea (Pisum sativum L.) in central Sweden. It is assumed that N₂ fixation is basically proportional to root biomass, but limited by high root N or low substrate carbon concentrations. Input data on root carbon and nitrogen were estimated from observations of above-ground biomass and nitrogen. The simulated N₂ fixation was compared with estimated values from observations using the ¹⁵N labelling technique. Test data were taken from pea monocultures and pea-oat mixtures with varying pea biomass levels during 1999. Simulated within-season accumulated N₂ fixation correlated to the estimated N₂ fixation with a correlation coefficient (R ²) of 0.74. For seasonal simulations, the predictability was higher (R ²=0.93). Two alternative non-dynamic models, estimating seasonal N₂ fixation as proportional to above-ground biomass and above-ground N, respectively, gave lower predictability (R ²=0.83 and 0.80, respectively). The models were also applied to a second year (1998) and two other sites by comparison with accumulated N₂ fixation estimated by the Difference method. A halved specific N₂ fixation rate (expressed per unit of root biomass) in 1999, compared with 1998, corresponded to essentially dryer and warmer soil conditions during 1999. It was indicated that the variations in soil moisture were more important than soil temperature. It was concluded that the abiotic responses might be of great importance for modelling N₂ fixation rate under different soil conditions.     

Influence of carbon substrates and moisture regime on nitrogen fixation in paddy soils

The influence of several carbon sources on heterotrophic N₂ fixation in four paddy soils under flooded and nonflooded conditions was investigated by ¹⁵N-tracer technique. Greater N₂ fixation occurred in submerged soils amended with cellulose and rice straw, the former being superior. Addition of sucrose, glucose and malate in that order stimulated N₂ fixation in submerged alluvial soil, while sucrose alone enhanced N₃ fixation in laterite soil. In submerged acid soils none of these C sources stimulated N₂ fixation. Nonflooded conditions favoured N₂ fixation in alluvial and acid saline soils amended with cellulose, sucrose and glucose.     

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 biological N2 fixation by a tropical legume tree using the non-nodulating phenophase as the reference in the 15N natural abundance method

The non-nodulating phenophase of a legume tree was tested as a non-N₂-fixing reference in application of the ¹⁵N natural abundance method for estimating the N₂ fixation. We applied this method to study the effects of three pruning intensities, complete pruning every 6 months (T-6), ca. 50% pruning every 3 months (P-3) and intact control (C), on N₂ fixation in Erythrina lanceolata (Papilionaceae) planted as shade and support trees for vanilla (Vanilla planifolia) in a subhumid tropical site in Quepos, Costa Rica. We measured nodulation and N₂ fixation for 12 months. The trees under the C regime nodulated abundantly during the rainy season vegetative growth but did not nodulate during the dry season and flowering. A linear regression (r²=0.76) was observed between the nodule biomass and δ¹⁵N values under the C regime, suggesting quite a stable specific N₂ fixation rate. The stable δ¹⁵N values throughout the year in the non-N₂-fixing Morus nigra (Moraceae) growing on the same soil indicated that the temporal variation in the plant available soil ¹⁵N was low. We used the intercept term of the regression (δ¹⁵N=3.5 when nodule biomass was 0) as the non-N₂-fixing δ¹⁵N value when estimating the percentage of N fixed from atmosphere out of tree total N. The percentage varied from 0 during the driest period to 53% during rainy season. Pruning reduced rainy season nodulation under the T-6 and P-3 regimes almost to nil, and the δ¹⁵N values were high. Our results suggest that the conditions for using the non-nodulating phenophase as the non-N₂-fixing reference required in the ¹⁵N natural abundance method were fulfilled. The C trees showed a clear phenological cycle in N₂ fixation, while pruning severely disturbed the N₂ fixation under the T-6 and P-3 regimes, indicating that E. lanceolata is better suited for agroforestry systems in which the trees are not managed by periodic prunings.     

N2 fixation and growth of legumes as affected by sulphur fertilization

The influence of three sulphur application rates in combination with two nitrogen application rates on N₂ fixation and growth of different legumes was investigated. N was applied as N-labelled ¹⁵NH₄ ¹⁵NO₃. The ¹⁵N isotope dilution technique was used to estimate N₂ fixation. At both N increments dry matter yield was highest with high S supply. Independently of the N supply, the high S application rate resulted in a significantly higher N accumulation, which was mainly caused by a higher N₂ fixation rate. With the grain legumes the weight of nodules was increased by the high S application rate. The higher number of nodules per pot with optimum S supply was the result of a better root growth. Rates of acetylene reduction correlated significantly with S supply.     

Pesticides and nitrogen fixation in a paddy soil

The influence of six pesticides, applied singly or in combination, on ¹⁵N₂ incorporation and C₂H₂ reduction in a submerged paddy soil was studied under laboratory conditions. While the application of diazinon had no marked effect, benomyl, carbofuran, parathion, nitrofen and γ-HCH, at concentrations close to recommended field application rates (5μg ^?1) significantly stimulated N₂ fixation. Synergistic stimulatory effects of the pesticides on N₂ fixation were evident particularly in combinations of carbofuran with benomyl, nitrofen and γ-HCH. On the contrary, diazinon slightly retarded the stimulatory effect of benomyl and carbofuran. Results indicated that the differential effects of pesticides on N₂ fixation could be attributed partly to fluctuations in the population of certain groups of N₂ fixers in submerged soil.     

Pesticides and heterotrophic nitrogen fixation in paddy soils

The effect of three pesticides, benomyl, carbofuran and gamm-BHC at 5 parts 10⁶, rates equivalent to recommended field levels, on the heterotrophic N₂ fixation in five air-dried, cellulose-amended, submerged tropical soils was invevstigated employing ¹⁵N tracer technique under laboratory conditions. Addition of benomyl, a carbamate fungicide, to alluvial, laterite and two acid sulphate soils resulted in significant increases in N₂ fixation, while carbofuran, a methylcarbamate insecticide, exerted a stimulatory effect on N₂ fixation in alluvial, alterite and acid saline soils. Gamma-BHC, a chlorinated hydrocarbon insecticide, stimulated N₂ fixation in alluvial and acid sulphate pokkali solis, while considerable inhibition of N₂ fixation was evident inother soils. Results showed differential responses of specific groups of N₂-fixing organisms to the pesticides depending ont he soil type.     

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 symbiotic nitrogen fixation in an established pasture using acetylene reduction and a 15N method

The C₂H₂ reduction (A.R.) assay was investigated for quantitative measurement of symbiotic N₂ fixation in established legume-based pastures under field conditions.It was found that the rate of C₂H₄ production was relatively constant for approx 6 h. For an accurate estimation of the N₂ fixing activity, many field samples are required to overcome the errors due to the inherent spatial distribution of white clover (Trifolium repens L.) within the pasture. Furthermore, it is necessary to physically integrate the day-to-day and diurnal variations in the N₂ fixing activity of the legume to obtain a reliable estimate of the rate of C₂H₄ production and hence symbiotic N₂ fixation.The A.R. assay and an ¹⁵N technique were compared for measuring symbiotic N₂ fixation in established pastures. A 3 h incubation in the A.R. assay gave the best estimate of symbiotic N₂ fixation relative to the ¹⁵N technique. The 1 h incubation over-estimated and the 6 h incubation under-estimated the rate of symbiotic N₂ fixation.     

Measurement of asymbiotic N2 fixation in Australian agriculture

A wide range of bacteria capable of nitrogen fixation (free-living and associative) can be found in all agricultural soils across Australia, however measurement of their effectiveness in N₂ fixation has proved to be problematic because rates are low compared to symbiotic systems and quantitative methodologies barely adequate. It is generally believed that associative N₂ fixation rates may be greater than free-living N₂ fixation rates in ecosystems where grasses (including cereals) dominate, although this has not been unequivocally proven. Conditions promoting asymbiotic N₂ fixation are reduced availability of oxygen, high temperature and soil water, and large amounts of microbially available C in the soil. The most direct measure of N₂ fixation, incorporation of ¹⁵N₂, has rarely been used in undisturbed systems, and we can find no examples of its field application in Australia. Nitrogen balance calculations, based on long-term changes in total soil N of systems and crop N removal, have been used to infer asymbiotic N₂ fixation, but do not measure it directly. Such N balance studies can thus only give an indication of potential asymbiotic N₂ fixation over long periods of time, but cannot confirm it. There are no robust N balances published for Australian ecosystems. The acetylene reduction assay for nitrogenase activity has been used in Australia to study responses of both free-living and associative N₂ fixation systems to regulating factors. These studies have highlighted the importance of C supply, high soil water content and temperature in increasing asymbiotic N₂ fixation in soils. However significant methodological limitations do not allow field scale quantification using this assay. On balance we would concur with the authors of several earlier global reviews of this topic and conclude that (in Australia) contributions of nitrogen to crop growth from asymbiotic N₂ fixation are likely to be <10 kg N ha^?1 y^?1 and generally not of agronomic significance under low rainfall conditions. In tropical environments where higher rainfall and temperatures coincide, rates are likely to be greater if soil mineral N is low and carbon substrates are available for N₂ fixing microorganisms. If asymbiotic N₂ fixation is to be encouraged or profitably managed, there is a need for more reliable field measurement and a combination of methodologies including ¹⁵N might provide more definitive quantitative indications.     

Growth and Nitrogen Fixation in Soybean as Affected by Phosphorus Fertilizer and Sheep Manure using 15N Isotopic Dilution

A field experiment was conducted to study the effect of adding different phosphorus (P) fertilizer levels [0, 40, and 80 kg phosphorus pentoxide (P₂O₅) ha^?1 (abbreviated as P0, P1, and P2, respectively)] and rates of sheep manure (M) [0, 20, and 40 ton ha^?1 (abbreviated as M0, M1, and M2, respectively)] on growth and nitrogen (N₂) fixation of soybean (Glycine max L.). Sorghum bicolor L. was employed as a reference crop to evaluate N₂ fixation using the ¹⁵N-isotpic dilution technique. Results showed that addition of P fertilizer or sheep manure had positive effects on dry-matter production, N accumulation, and seed yield. Such effects were more pronounced when adding sheep manure and P together than adding separately. Solely P fertilizer had a small impact on N₂ fixation. A tangible increase in the amounts of N₂ fixed due to manure addition occurred. The efficient use of N fertilizer (%NUE) increased significantly as the result of adding a high level of P fertilizer. However, a drastic decrease in %NUE was observed when sheep manure was added solely or in combination with P fertilizer. From productivity and ecological standpoints, P2M1 and P2M2 surpassed the other treatments in showing greater grain yield and greater N₂ fixation. However, considering the high cost of sheep manure, P2M1 was the optimal treatment for improving growth and N₂ fixation in soybean plants with minimal manure consumption. In conclusion, the integrated use of manure and P fertilizer could be considered a useful agricultural practice for improving the performance of soybean plants grown in an Aridisol. Their beneficial effects were mainly attributed to the enhancement of N₂ fixation through root growth and soil property improvements besides being a source of P and other nutrients that are essential for N₂-fixation process.     

Host-rhizobia interaction for effective inoculation: evaluation of the potential use of the ureide assay to monitor the symbiotic performance of tepary bean (Phaseolus acutifolius A. Gray)

Domesticated and wild-type tepary beans (Phaseolus acutifolius A. Gray) were grown with or without inoculation with rhizobia in pots under bacteriologically controlled conditions in a temperature-controlled glasshouse. Seeds were inoculated with a mixture of seven strains isolated from nodules collected from domesticated field-grown tepary bean in Arizona, USA, or with a commercial inoculant strain for Phaseolus vulgaris (CC511). Different degrees of plant reliance upon N₂ fixation for growth were generated by supplying the inoculated plants throughout growth with nutrients containing a range of concentrations of ¹⁵N-labeled NO₃ (0, 1, 2, 5 or 10 mM). An uninoculated treatment that received 10 mM ¹⁵N-labeled NO₃ was included to provide data for plants solely dependent upon NO₃ for growth. Six weeks after sowing, shoots were harvested for dry matter determination and subsequent ¹⁵N analysis, root-bleeding xylem sap was collected, and nodulation assessed. With regard to shoot biomass production, domesticated lines were more responsive to inoculation, but less responsive to applied N than wild types. All inoculated plants were nodulated, but the field isolates from tepary bean were more effective in N₂ fixation than strain CC511. It was concluded that tepary bean requires a specific inoculant to benefit from fixation of atmospheric N₂. Xylem sap samples were analysed for ureides (allantoin and allantoic acid), amino acid content (α-amino-N), and NO₃ concentration. The amount of ureide-N present in xylem sap was expressed as a percentage of total solute N, described as the relative abundance of ureide-N (RUN), for each N treatment and was compared to the proportion of plant N derived from N₂ fixation (%Ndfa) calculated using a ¹⁵N dilution technique. The RUN values ranged from 8% for saps collected from uninoculated plants provided with 10 mM NO₃ in the nutrient solution (%Ndfa=0) to 86-91% for nodulated plants grown in the absence of externally supplied NO₃ (%Ndfa=100). These data indicated that ureides were the principal product of N₂ fixation exported from the nodules to the shoot in xylem sap. Since RUN values were closely related to %Ndfa, it was proposed that N-solute analysis of xylem sap could provide a valuable analytical tool to monitor the symbiotic performance of tepary bean.     

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.     

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.