Biological nitrogen fixation by two tropical forage legumes assessed from the relative ureide abundance of stem solutes: 15N calibration of the technique in sand culture

The use of the relative ureide abundance (RUA) in the sap of mainly tropical ureide-producing legumes as a means to estimate the contribution of biological nitrogen fixation (BNF) is potentially an useful technique as it does not require the use of reference plants or additions of ¹⁵N-labelled fertilizer, and the analyses necessitate only relatively simple equipment. However, one problem in the application of the technique arises from the difficulty of obtaining sap samples from such legumes, especially small-stemmed forage legumes under field conditions. This study was conducted to investigate the possibility of using RUA in hot-water extracts of the stems of two forage legumes, Desmodium ovalifolium and a Centrosema hybrid, to estimate the contribution of BNF. In this case only ureide and nitrate are analysed to calculate RUA (100 × ureide-N/(ureide-N + nitrate-N)). The technique was calibrated with the ¹⁵N isotope dilution technique in sand culture where the plants were fed with 5 different levels of nitrate (0, 12.5, 25, 50 and 100 mg N pot^-1). Despite the fact that in many stem extracts more than 90% of the N was neither nitrate or ureide, the colorimetric techniques utilised proved reliable and relatively immune to interference from other solutes in the extracts. One problem with the use of the ¹⁵N dilution technique to calibrate the RUA technique is that the former gives an integrated estimate of the BNF contribution since planting (or between harvests) and the latter is a point estimate at the time of sampling. This was overcome by using a `plant to plant simulation technique' where estimates of BNF are calculated from the daily accumulation of total N and the labelled N derived from the growth medium by the legumes using a curve-fitting strategy. These estimates of BNF for the days when stem extracts were analysed for nitrate and ureide showed linear correlations (r ² = 0.82 and 0.90 for the D. ovalifoliumand Centrosema hybrid, respectively). This indicated that RUA of stem extracts of these two legumes was a reliable indicator of the BNF contribution, at least under controlled conditions.     

Assessment of biological nitrogen fixation

The four commonly used methods for measuring biological nitrogen fixation (BNF) in plants are: the total nitrogen difference (TND) method, acetylene reduction assay (ARA) technique, xylem-solute (or ureide production) method and the use of¹⁵N labelled compounds.The TND method relies on a control non-N₂-fixing plant to estimate the amount of N absorbed by the fixing plant from soil. It is one of the simplest and least expensive methods, but works best under low soil N conditions. The ARA technique measures the rate of acetylene conversion to ethylene by the N₂-fixing enzyme, nitrogenase. The ethylene produced can then be converted into N₂ fixed, using a conversion ratio, originally recommended as 3. Although the method is inexpensive and highly sensitive, its major disadvantages are, the short-term nature of the assays, the doubtful validity of always using a conversion ratio of 3 and the auto-inhibition of acetylene conversion to ethylene. The ARA technique is therefore not a method of choice for measuring BNF.The xylem-solute technique can be used to measure BNF for those species that produce significant quantities of ureide as product of BNF. Although simple and relatively inexpensive, it is an instantaneous assay and also needs to be calibrated against a known method. The most serious limitation is, that only a small proportion of N₂-fixing plants examined are ureide exporters, and the method is therefore not widely applicable.The¹⁵N methods, classified into the isotope dilution and A-value methods, appear to be the most accurate, but also the most expensive. They involve labelling soil with¹⁵N fertilizer and using a non-N₂-fixing reference plant to measure the¹⁵N/¹⁴N ratio in the soil. The¹⁵N isotope dilution approach is both operationally and mathematically simpler than the A-value approach. To limit potential errors in the selection of reference crops, it is recommended to use¹⁵N labelled compounds or soil labelling methods that result in the slow release of¹⁵N or the slow decline of¹⁵N/¹⁴N ratio in the soil. Additionally, the use of several reference plants rather than a single one can improve the accuracy of the results.     

Estimation of the contribution of biological N2 fixation to twoPhaseolus vulgaris genotypes using simulation of plant nitrogen uptake from15N-labelled soil

A technique for the application of the¹⁵N isotope dilution technique for the quantification of plant associated biological nitrogen fixation (BNF) was tested and applied to quantify the BNF contribution to two genotypes ofPhaseolus vulgaris. The technique makes use of sequential measurements of the¹⁵N enrichment of soil mineral N, and the uptake of labelled N by the N₂-fixing plant, to simulate its uptake of soil N (the soil to plant simulation technique). The test was made with two non-N₂-fixing crops (non-nodulating beans and wheat) and two bean genotypes (PR 923450 and Puebla 152), at two levels of N fertilizer addition (10 and 40 kg N ha^–1), to compare the actual N uptake with that simulated from the soil and crop¹⁵N data. The simulation of the soil N uptake by the non-nod bean crop using this soil to plant simulation technique underestimated by 20 to 30% the true N uptake, suggesting that the mineral N extracted from soil samples taken from the 0–15cm layer had a higher¹⁵N enrichment than that N sampled by the roots of this crop. In the case of the wheat crop the simulation resulted in a much greater underestimation of actual N uptake. In general the results using this technique suggested that BNF inputs to the bean cultivars was higher than would be expected from the nodulation and acetylene reduction data, except for the early PR beans in the 40 kg N ha^–1 treatment. In this case the total N and simulated soil N accumulation were well matched suggesting no BNF inputs. An allied technique (the plant to plant simulation technique) was proposed where the¹⁵N enrichrnent of soil mineral N was simulated from the data for total N and labelled N accumulation taken from sequential harvests of either of the non-N₂ -fixing control crops. This was then utilized in combination with the labelled N uptake data of the other crop to simulate its soil N uptake. However, the results using either technique indicated that the wheat and non-nod or nodulating beans exploited pools of N in the soil with completely different¹⁵N enrichments probably due to differences in exploitation of the soil N with depth.     

Use of the 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials

Biological nitrogen fixation (BNF) associated with trees and shrubs plays a major role in the functioning of many ecosystems, from natural woodlands to plantations and agroforestry systems, but it is surprisingly difficult to quantify the amounts of N₂ fixed. Some of the problems involved in measuring N₂ fixation by woody perennials include: (a) diversity in occurrence, and large plant-to-plant variation in growth and nodulation status of N₂-fixing species, especially in natural ecosystems; (b) long-term, perennial nature of growth and the seasonal or year-to-year changes in patterns of N assimilation; and (c) logistical limitations of working with mature trees which are generally impossible to harvest in their entirety. The methodology which holds most promise to quantify the contributions of N₂ fixation to trees is the so-called `¹⁵N natural abundance' technique which exploits naturally occurring differences in ¹⁵N composition between plant-available N sources in the soil and that of atmospheric N₂. In this review we discuss probable explanations for the origin of the small differences in ¹⁵N abundance found in different N pools in both natural and man-made ecosystems and utilise previously published information and unpublished data to examine the potential advantages and limitations inherent in the application of the technique to study N₂ fixation by woody perennials. Calculation of the proportion of the plant N derived from atmospheric N₂ (%Ndfa) using the natural abundance procedure requires that both the ¹⁵N natural abundance of the N derived from BNF and that derived from the soil by the target N₂-fixing species be determined. It is then assumed that the ¹⁵N abundance of the N₂-fixing species reflects the relative contributions of the N derived from these two sources. The ¹⁵N abundance of the N derived from BNF (B) can vary with micro-symbiont, plant species/provenance and growth stage, all of which create considerable difficulties for its precise evaluation. If the%Ndfa is large and the ¹⁵N abundance of the N acquired from other sources is not several ¹⁵N units higher or lower than B, then this can be a major source of error. Further difficulties can arise in determining the ¹⁵N abundance of the N derived from soil (and plant litter, etc.) by the target plant as it is usually impossible to predict which, if any, non-N₂-fixing reference species will obtain N from the same N sources in the same proportions with the same temporal and spatial patterns as the N₂-fixing perennial. The compromise solution is to evaluate the ¹⁵N abundance of a diverse range of neighbouring non-N₂-fixing plants and to compare these values with that of the N₂-fixing species and the estimate of B. Only then can it be determined whether the contribution of BNF to the target species can be quantified with any degree of confidence. This review of the literature suggests that while the natural abundance technique appears to provide quantitative measures of BNF in tree plantation and agroforestry systems, particular difficulties may arise which can often limit its application in natural ecosystems.     

Towards a Revised Coefficient for Estimating N2O Emissions from Legumes

The Intergovernmental Panel on Climate Change (IPCC) standard methodology to conduct national inventories of soil N₂O emissions is based on default (or Tier I) emission factors for various sources. The objective of our study was to summarize recent N₂O flux data from agricultural legume crops to assess the emission factor associated with rhizobial nitrogen fixation. Average N₂O emissions from legumes are 1.0 kg N ha⁻¹ for annual crops, 1.8 kg N ha⁻¹ for pure forage crops and 0.4 kg N ha⁻¹ for grass legume mixes. These values are only slightly greater than background emissions from agricultural crops and are much lower that those predicted using 1996 IPCC methodology. These field flux measurements and other process-level studies offer little support for the use of an emission factor for biological N fixation (BNF) by legume crops equal to that for fertiliser N. We conclude that much of the increase in soil N₂O emissions in legume crops may be attributable to the N release from root exudates during the growing season and from decomposition of crop residues after harvest, rather than from BNF per se. Consequently, we propose that the biological fixation process itself be removed from the IPCC N₂O inventory methodology, and that N₂O emissions induced by the growth of legume crops be estimated solely as a function of crop residue decomposition using an estimate of above- and below-ground residue inputs, modified as necessary to reflect recent findings on N allocation.     

Effect of K on nitrogen fixation of intercrop groundnut and the competition between intercrop groundnut and maize

Application of adequate level of K has shown to improve the competitive ability of the legume in legume/grass mixtures. However, the effect of K on the competitive ability of grain legumes in legume/cereal intercropping systems has not been adequately studied. Hence, studies were made to ascertain if the effects of K could be exploited in improving the performance of groundnut (Arachis hypogaea L.) cv. No. 45 when intercropped with maize (Zea mays L.) cv. Badra. The study was conducted at the Faculty of Agriculture, University of Ruhuna, Kamburupitiya, Sri Lanka in 1988 in basins filled with 36 kg of soil. It involved establishing maize and groundnut as monocrops and as intercrops at three K levels viz. 0, 20 and 40 mg of K kg^–1 of soil. Monocrop maize and groundnut had 2 and 5 plants/basin, respectively while the intercrop had 1 maize plant and 3 groundnut plants/basin. The soil used was Red Yellow Podzolic which was tagged by incorporating¹⁵N-labelled plant material. When grown as a monocrop, K had no effect on the percent N derived from atmosphere, amount of N₂ fixed, dry matter production, pod yield and total N content of groundnut. However, when intercropped with maize lack of K application affected the above parameters significantly which was overcome by improving K level. Thus, the optimum level of K for groundnut was greater when intercropped than monocropped. A significant interaction between K level and cropping system was evident with regard to N₂ fixation, pod yield and total dry matter production of groundnut. Intercrop maize derived 30–35% of its N content from the associated groundnut plants which amounted to 13–22 mg N/plant. The amount of N supplied by groundnut to associated maize plant was not affected by K level. It appears that there is scope for alleviating growth depression of the legume component in legume/cereal intercropping systems by developing appropriate K fertilizer practices.     

Nitrogen fixation of and N transfer from cowpea, mungbean and groundnut when intercropped with maize

Grain legumes are used widely in intercropping systems. However, quantitative and comparative data available as to their N₂ fixation and N beneficial effect on the companion crop in intercropping systems are scarce. Hence, studies were conducted to ascertain the above when cowpea (Vigna unguiculata L.), mungbean (Vigna radiata L.) and groundnut (Arachis hypogaea L.) were intercropped with maize. The study was¹⁵N-aided and made outdoors in basins (30 L) filled with 38 kg of soil.¹⁵N labelling was effected by incorporating¹⁵N-tagged plant material or applying¹⁵N-labelled fertilizer along with sucrose to stabilize¹⁵N enrichment in the soil during the experimental period. Intercropped groundnut fixed the highest amount of nitrogen from the atmosphere (i.e. 552 mg plant^–1), deriving 85% of its N from the atmosphere. Intercropped cowpea and mungbean fixed 161 and 197 mg N plant^–1, obtaining 81% and 78% of their N content from the atmosphere, respectively. The proportion of N derived by maize from the associated legume varied from 7-11% for mungbean, 11–20% for cowpea and 12–26% for groundnut which amounted to about 19–22, 29–45 and 33–60 mg N maize plant^–1, respectively. The high nitrogen fixation potential of groundnut in dual stands and its relatively low harvest index for N have apparently contributed to greater N-benefical effect on the associated crop.     

Field application of the15N isotope dilution technique for the reliable quantification of plant-associated biological nitrogen fixation

To apply the isotope dilution (ID) technique, it is necessary to grow the N₂-fixing crop in a soil where the mineral N is labelled with¹⁵N. Normally the N₂-fixing crop and a suitable non-N₂-fixing control crop are grown in the same labelled soil and the¹⁵N enrichment of the control crop is assumed to be equal to the¹⁵N enrichment of the nitrogen (N) derived from the soil in the N₂-fixing crop. In this case the proportion of unlabelled N being derived from the air via biological N₂ fixation (BNF) in the N₂-fixing crop will be proportional to the dilution of the enrichment of the N derived from the labelled soil.To label the soil, the technique most often used is to add a single addition of¹⁵N-labelled N fertilizer shortly before, at, or shortly after, the planting of the crops. Data in the literature clearly show that this technique results in a rapid fall in the¹⁵N enrichment of soil mineral N with time. Under these conditions, if the control and the N₂-fixing crops have different patterns of N uptake from the soil they will inevitably obtain different¹⁵N enrichments in the soil-derived N. In this case the isotope dilution technique cannot be applied, or if it is, there will be an error introduced into, the estimate of the contribution of N derived from BNF.Several experiments are described which explore different strategies of application of the ID technique to attempt to attenuate the errors involved. The results suggest that it is wise to use slow-release forms of labelled N, or in some cases, multiple additions, to diminish temporal changes in the¹⁵N enrichment of soil mineral N. The use of several control crops produces a range of different estimates of the BNF contributions to the N₂-fixing crops, and the extent of this range gives a measure of the accuracy of the estimates. Likewise the use of more than one¹⁵N enrichment technique in the same experiment will also give a range of estimates which can be treated similarly. The potential of other techniques, such as sequential harvesting of both control and test crops, are also discussed.     

A comparison between legume technologies and fallow, and their effects on maize and soil traits, in two distinct environments of the West African savannah

Legume–maize rotation and maize nitrogen (N)-response trials were carried out simultaneously from 1998 to 2004 in two distinct agro-ecological environments of West Africa: the humid derived savannah (Ibadan) and the drier northern Guinea savannah (Zaria). In the N-response trial, maize was grown annually receiving urea N at 0, 30, 60, 90 and 120 kg N ha⁻¹. In Ibadan, maize production increased with N fertilization, but mean annual grain yield declined over the course of the trial. In Zaria, no response to N treatments was observed initially, and an increase in the phosphorus (P) and sulphur (S) fertilizer application rate was required to increase yield across treatments and obtain a response to N applications, stressing the importance of non-N fertilizers in the savannah. In the rotation trial, a 2-year natural fallow–maize rotation was compared with maize rotated with different legume types: green manure, forage, dual-purpose, and grain legumes. The cultivation of some legume types resulted in a greater annual maize production relative to the fallow–maize combination and corresponding treatments in the N-response trial, while there was no gain in maize yield with other legume types. Large differences in the residual effects from legumes and fallow were also observed between sites, indicting a need for site-specific land management recommendations. In Ibadan, cultivation of maize after the forage legume (Stylosanthes guianensis) achieved the highest yield. The natural fallow–maize rotation had improved soil characteristics (Bray-I P, exchangeable potassium, calcium and magnesium) at the end of the trial relative to legume–maize rotations, and natural fallow resulted in higher maize yields than the green manure legume (Pueraria phaseoloides). In Zaria, maize following dual-purpose soybean achieved the highest mean yield. At both sites, variation in aboveground N and P dynamics of the legume and fallow vegetation could only partly explain the different residual effects on maize.     

The effect of grazing intensity and the presence of a forage legume on nitrogen dynamics in Brachiaria pastures in the Atlantic forest region of the south of Bahia, Brazil

It has been shown that with careful grazing management and addition of Pand K, but not N, fertilisers Brachiaria pastures are ableto maintain sustainable live weight gains over many years. However, standardon-farm practice, which generally involves high stocking rates, leads after afew years to pasture decline due mainly to N deficiency for grass regrowth. Togenerate an understanding of the mechanism of pasture decline and possiblemanagement options to mitigate this process, a study was performed in theAtlantic forest region of the south of Bahia state to study the N dynamics inpastures of Brachiaria humidicola subject to threedifferent stocking rates of beef cattle, with and without the presence of theforage legume Desmodium ovalifolium. Despite the fact thatthe C:N ratio of the deposited litter was high (60 to 70) the rate ofdecomposition was very rapid (k –0.07 gg^–1 day^–1) and annual rates of Nturnover through the litter pathway were between 105 and 170 kg Nha^–1 year^–1. In the grass-onlypasturesas stocking rate increased from 2 to 3 head ha^–1, N recycledinthe litter decreased by 11%, but a further increase to 4 headha^–1 decreased N recycling by 30% suggesting thatbeyonda certain critical level higher grazing stocking rates would lead to pasturedecline if there was no N addition. High stocking rates decreased theproportionof the legume in the sward, but at all rates the concentration of N in both thegreen and dead grass in the forage on offer and in the litter was higher in themixed sward. The presence of the legume caused a decrease in the C:N ratio ofthe microbial biomass while both soil N mineralisation and nitrificationincreased. This increased rate of turnover of the microbial biomass and thecontribution of N₂ fixation to the legume resulted in largeincreasesin the N recycled via litter deposition ranging from 42 to 155 kg Nha^–1 year^–1.     

Quantification of nitrogen inputs from biological nitrogen fixation to whole farm nitrogen budgets of two dairy farms in Atlantic Canada

Legume biological N fixation (BNF) is a large source of uncertainty in farm N budgets. This study sought to quantify the BNF-N input to two whole farm nitrogen budgets and establish a simple and accurate method for incorporating BNF values as inputs in whole farm N budgets. Nitrogen inputs and outputs as well as flows of N between animal and crop production components were determined for a dairy farm in New Brunswick (NB) and Prince Edward Island (PE) over a two year period. The ¹⁵N natural abundance method was used to determine the %N derived from the atmosphere (%Ndfa) through BNF at both sites. Red clover (Trifolium pratense) at the PE site derived 77 % of its N from BNF and alfalfa (Medicago sativa) collected at both the PE and NB farms derived 72 % of its N from BNF. Total BNF-N present in legume biomass from mixed forage fields measured with the ¹⁵N natural abundance method ranged from 39 to 116 kg N ha^?1 year^?1. A legume dry matter conversion model adjusted with %Ndfa and %N of red clover and alfalfa samples from both farm sites was selected to estimate BNF-N inputs from mixed forage fields on the farms. Averaged across the entire cropland area at each farm site, the BNF-N inputs ranged from 27 to 52 kg N ha^?1 year^?1. The farmgate BNF-N inputs are low in comparison to other studies, possibly due to low legume contents in forage fields. BNF accounted for 18–29 % of farmgate N inputs at the farms. Surpluses of N found at both farm sites ranged from 98 to 135 kg N ha^?1 year^?1, typical to the whole farm N budgets of similar dairy farms.     

Nitrogen fertilizer in leucaena alley cropping. II. residual value of nitrogen fertilizer and leucaena residues

Legume residues have been credited with supplying mineral nitrogen (N) to the associated cereal crop and improving soil fertility in the long term. Few studies using¹⁵N have reported the fate of legume N and fertilizer N in the presence of legume residues in soil-plant systems over periods of two years or longer. A field experiment was conducted in microplots to evaluate: (1) the residual value of the¹⁵N added in leucaena residues; (2) the residual value of fertilizer¹⁵N applied in the presence of unlabelled leucaena residues in the first year to maize over three subsequent years; and (3) the long-term fate of residual fertilizer and leucaena¹⁵N in a leucaena alley cropping system.There was a significant increase in maize production over three subsequent years after addition of leucaena residues. The residual effect of fertilizer N increased maize yield in the second year when N fertilizer was applied at 36 kg N ha^–1 in the first year in the presence of leucaena residues. Of the leucaena¹⁵N applied in the first year, the second, third and fourth maize crop recovered 2.6%, 1.8% and 1.4%, respectively. The corresponding values for the residual fertilizer¹⁵N were 0.7%, 0.4% and 0.3%. About 12–14% of the fertilizer¹⁵N added in the first year was found in the 200 cm soil profile over the following three years. This differed from the 38–41% of leucaena¹⁵N detected in the soil over the same period. Most of the residual fertilizer and leucaena¹⁵N in the soil was immobilized in the top 25 cm with less than 1% leached below 100 cm. More than 36% of the leucaena¹⁵N and fertilizer¹⁵N added in the first year was apparently lost from the soil-plant system in the first two years. No further loss of the residual leucaena and fertilizer¹⁵N was detected after two years.     

Growth, composition, biological N2 fixation and nutrient uptake of a leguminous cover crop mixture and the effect of their removal on field nitrogen balances and nitrate leaching risk

Cover crops (CC) are an important source of nitrogen (N) in organic farming systems. Only few data are available about the effect of management activities (liquid slurry amendments, crop residue management) on growth, nutrient uptake and biological N₂ fixation (BNF) of a CC mixture. Furthermore, little information is available about the effect of CC harvesting on nutrient flows, nitrate leaching risk and soil mineral N supply of the succeeding main crop. The objectives of the presented field trials were (1) to measure the impact of organic manuring (straw residues and liquid slurry applications) on growth, composition, and BNF of a CC mixture with legumes and oil radish as components; (2) to determine the effect of CC species composition on nutrient content and uptake (N, P, K, Mg); and (3) to evaluate the effect of CC removal on field N balances and nitrate leaching risk. A CC mixture with legumes and non-legumes was able to compensate for many environmental and cultivation effects by influencing the competitive ability of the partners. For example, an increase of soil N supply due to additions of slurry or removal of cereal straw promoted growth of non-legumes at the expense of the legumes, resulting in N shortage at the end of the growing period, as shown by lower N contents and a wider C/N ratio of the non-legume partner. Low N availability at the beginning of the CC growth enhanced legume growth and/or reduced non-legume growth, resulting in a higher N supply in later periods of CC growth. A high legume percent composition within a CC mixture increases overall N content in the aboveground biomass and the N content of non-legumes within the mixture, and decreases the C/N ratio. Large amounts of nutrients were removed from the field by the harvesting of the CC aboveground biomass, significantly reducing the nitrate leaching risk. However, a reduction of the nitrate leaching risk was found only on fields where the green manure was incorporated in autumn.     

Fate of Fertilizer15N applied as urea and ammonium sulphate in opium poppy (Papaver somniferum L.) grown under greenhouse conditions

Laboratory incubation and greenhouse experiments were conducted to investigate the comparative effectiveness of urea and ammonium sulphate in opium poppy (Papaver somniferum L.) using¹⁵N dilution techniques. Fertilizer treatments were control (no N), 600 mg N pot^–1 and 1200 mg N pot^–1 (12 kg oven dry soil) applied as aqueous solution of urea or ammonium sulphate. Fertilizer rates, under laboratory incubation study were similar to that under greenhouse conditions. A fertilizer¹⁵N balance sheet reveals that N recovery by plants was 28–39% with urea and 35–45% with ammonium sulphate. Total recovery of¹⁵N in soil-plant system was 77–82% in urea. The corresponding estimates for ammonium sulphate were 89–91%. Consequently the unaccounted fertilizer N was higher under urea (18–23%) as compared to that in ammonium sulphate (9–11%). The soil pH increased from 8.2 to 9.4 with urea whereas in ammonium sulphate treated soil pH decreased to 7.3 during 30 days after fertilizer application. The rate of NH₃ volatilization, measured under laboratory conditions, was higher with urea as compared to the same level of ammonium sulphate. The changes in pH of soil followed the identical trend both under laboratory and greenhouse conditions.     

Detectability of15N-depleted fertilizer N in soil and plant tissue during a corn-rye crop rotation

Use of¹⁵N-depleted fertilizer materials have been primarily limited to fertilizer recovery studies of short duration. The objective of this study was to determine if¹⁵N-depleted fertilizer N could be satisfactorily used as a tracer of residual fertilizer N in plant tissue and various soil N fractions through a corn (Zea mays L.) -winter rye (Secale cereale L.) crop rotation. Nitrogen as¹⁵N-depleted (NH₄)₂SO₄ was applied at five rates (0, 84, 168, 252, and 336 kg N ha^–1) to corn. Immediately following corn harvest a winter rye cover crop treatment was initiated. Residual fertilizer N was easily detected in the soil NO ₃ ^- -N fraction following corn harvest (140-d after application). Low levels of exchangeable NH ₄ ⁺ -N (<2.5 mg kg^–1) did not permit accurate isotope-ratio analysis. Fertilizer-derived N recovered in the soil total N fraction following corn harvest was detectable in the 0 to 30-cm depth at each N rate and in the 30 to 60 and 60 to 90-cm depths at the 336 kg ha^–1 N rate. Atom %¹⁵N concentrations in the nonexchangeable NH ₄ ⁺ -N fraction did not differ from the control at each N rate. Nitrogen recovery by the winter rye cover crop reduced residual soil NO ₃ ^- -N levels below the 10 kg ha^–1 level needed for accurate isotope-ratio analysis. Atom %¹⁵N concentrations in the soil total N fraction (approximately one yr after application) were indistinguishable from the control plots below the 168, 252, and 336 kg ha^–1 N rate at the 0 to 30, 30 to 60, and 60 to 90-cm depths, respectively. Recovery of residual fertilizer N by the winter rye cover crop was verified by measuring significant decreases in atom %¹⁵N concentrations in rye tissue with increasing N rates. The greatest limitation to the use of¹⁵N-depleted fertilizer N as a tracer of residual fertilizer N in a corn-rye crop rotation appears to be its detectibility from native soil N in the total N pool.Research partially supported by grants from the National Fertilizer and Environmental Research Center/TVA and the Virginia Division of Soil and Water Conservation.     

Grain legume rotation benefits to maize in the northern Guinea savanna of Nigeria: fixed-nitrogen versus other rotation effects

The yield increases often recorded in maize following grain legumes have been attributed to fixed-N and ‘other rotation’ effects, but these effects have rarely been separated. Field trials were conducted between 2003 and 2005 to measure these effects on maize following grain legumes in the northern Guinea savanna of Nigeria. Maize was grown on plots previously cultivated to two genotypes each of soybean (TGx 1448-2E and SAMSOY-2) and cowpea (IT 96D-724 and SAMPEA-7), maize, and natural fallow. The plots were split into four N fertilizer rates (0, 30, 60 and 90 kg N ha⁻¹) in a split plot design. The total effect was calculated as the yield of maize following a legume minus the yield following maize, both without added N and the rotation effect was calculated as the difference between rotations at the highest N fertilizer rate. The legume genotypes fixed between 14 and 51 kg N ha⁻¹ of their total N and had an estimated net N balance ranging from −29.8 to 9.5 kg N ha⁻¹. Positive N balance was obtained only when the nitrogen harvest index was greater than the proportion of N derived from atmosphere. The results also indicated that the magnitude of the fixed-N and other rotation effects varied widely and were influenced by the contributions of the grain legumes to the soil N-balance. In general, fixed-N effects ranged from 124 to 279 kg ha⁻¹ while rotation effects ranged between 193 and 513 kg ha⁻¹. On average, maize following legumes had higher grain yield of 1.2 and 1.3-fold compared with maize after fallow or maize after maize, respectively.     

Sugarcane residue management and grain legume crop effects on N dynamics,N losses and growth of sugarcane

To reduce greenhouse gas emissions farmers are being encouraged not to burn sugarcane residues. An experiment was set up in NE Thailand, where sugarcane residues of the last ratoon crop were either burned, surface mulched or incorporated and subsequently the field left fallow or planted to groundnut or soybean. The objectives of the current experiment were to evaluate the residual effects of these treatments during the following new sugarcane crop on (i) microbial and mineral N dynamics, (ii) performance of sugarcane and (iii) effectiveness of recycled legume residues compared to mineral N fertilizer on N use efficiencies, ¹⁵N recovery in the system and in soil particle size and density fractions (using ¹⁵N labelled legume residues and fertilizer). The millable cane and sugar yield were positively affected by sugarcane residue mulching and incorporation compared to burning suggesting microbial remobilization of previously immobilized N. Residual effects of legumes increased sugarcane tillering and yield (127 and 116 Mg ha⁻¹ for groundnut and soybean, respectively) compared to the fallow treatment without N fertilizer (112 Mg ha⁻¹). Soybean residues of higher C:N ratio (33:1) and lignin content (13%) compared to groundnut residues (21:1 C:N, 5% lignin) decomposed slower and improved N synchrony with cane N demand. This led to a better conservation of residue N in the system with proportionally less ¹⁵N losses (15–17%) compared to the large losses from groundnut residues (50–57%) or from mineral N fertilizer (50–63%). ¹⁵N recoveries in soil were larger from residues (41–80%) than from fertilizer (30%) at final harvest. Recycled legume residues were able to substitute basal fertilizer N application but not topdressing after 6 months.     

Denitrification, leaching and immobilisation of applied 15N following legume and grass pastures in a semi-arid climate in Australia

Four consecutive ¹⁵N mass balance experiments lasting 18 months from February 1993 to August 1994 were carried out to assess the fate of applied ¹⁵N at 3 sites after 4 years of lucerne or snail medic and 20 years of Mitchell grass/naturalised medic pastures respectively in the Roma district of Queensland, Australia. ¹⁵N loss via denitrification was estimated from the difference between the recovery of applied Br(100kg Br/ha) and that of applied ¹⁵N(40kg N/ha) in the top 250mm at the end of each mass balance experiment. From February to August 1993, denitrification losses were 12–38% of applied ¹⁵N. N losses increased to 36–51% during August to November 1993, responding to the higher rainfall during this period. With even more rainfall during the period between November 1993 and March 1994, N losses were estimated to be 16–23%while displacement of ¹⁵N below 250 mm was 74–81%. When rainfall was much less between March 1994 and August 1994, N losses of only 15–19% of the applied ¹⁵N occurred at the 3 sites. It was found that although rainfall was the dominant factor controlling denitrification of the applied ¹⁵N, soil available carbon (C) (measured as water-soluble C) and the quantity of nitrate available were also important for soils already containing a considerable quantity of organic matter and N from residues of pasture legumes. This revised version was published online in June 2006 with corrections to the Cover Date.     

The application of isotopic (32P and 15N) dilution techniques to evaluate the interactive effect of phosphate-solubilizing rhizobacteria, mycorrhizal fungi and Rhizobium to improve the agronomic efficiency of rock phosphate for legume crops

A pot experiment was designed to evaluate the interactive effects of multifunctional microbial inoculation treatments and rock phosphate (RP) application on N and P uptake by alfalfa through the use of ¹⁵N and ³²P isotopic dilution approaches. The microbial inocula consisted of a wild type (WT) Rhizobium meliloti strain, the arbuscular mycorrhizal (AM) fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, and a phosphate solubilizing rhizobacterium (Enterobacter sp.). Inoculated microorganisms were established in the root tissues and/or in the rhizosphere soil of alfalfa plants (Medicago sativa L.). Improvements in N and P accumulation in alfalfa corroborate beneficial effects of Rhizobium and AM interactions. Inoculation with selected rhizobacteria improved the AM effect on N or P accumulation in both the RP-added soil and in the non RP-amended controls. Measurements of the ¹⁵N/¹⁴N ratio in plant shoots indicate an enhancement of the N₂ fixation rates in Rhizobium-inoculated AM-plants, over that achieved by Rhizobium in non-mycorrhizal plants. Whether or not RP was added, AM-inoculated plants showed a lower specific activity (³²P/³¹P) than did their comparable non-mycorrhizal controls, suggesting that the plant was using otherwise unavailable P sources. The phosphate-solubilizing, AM-associated, microbiota could in fact release phosphate ions, either from the added RP or from the indigenous ``less-available' soil phosphate. A low Ca concentrations in the test soil may have benefited P solubilization. Under field conditions, the inoculation with AM fungi significantly increased plant biomass and N and P accumulation in plant tissues. Phosphate-solubilizing rhizobacteria improved mycorrhizal responses in soil dually receiving RP and organic matter amendments. Organic matter addition favoured RP solubilization. This, together with a tailored microbial inoculation, increased the agronomic efficiency of RP in the test soil that was Ca deficient at neutral pH.     

6. Ammonium dynamics of puddled soils in relation to growth and yield of lowland rice

The release of non-exchangeable (fixed) NH ₄ ⁺ and the importance of exchangeable NH ₄ ⁺ at transplanting (initial exchangeable NH ₄ ⁺ ) for rice (Oryza sativa L.) growth was studied in representative lowland rice soils of the Philippines.The experiments showed that initial exchangeable ammonium behaved like fertilizer N and thus may serve as a valuable guideline for nitrogen fertilizer application rates when calculated on a hectare basis. By using the¹⁵N tracer technique it was found that nonexchangeable ammonium in soil may contribute to the nitrogen supplying capacity of lowland rice soils. Fixation and release of NH ₄ ⁺ seem to be more dependent on the form of clay minerals than on clay content. In soils rich in vermiculite non-exchangeable ammonium should be considered together with other available N sources such as exchangeable ammonium for N fertilizer recommendations for lowland rice.