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found in different N pools in both natural and man-made ecosystems and utilise previously published information and unpublished data to examine the potential advantages and limitations inherent in the application of the technique to study N₂ fixation by woody perennials.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.     

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.     

Characterization of fecal nitrogen forms produced by a sheep fed with <Superscript>15</Superscript>N labeled ryegrass

Little is known about nitrogen (N) forms in ruminant feces, although this information is important to understand N dynamics in agro-ecosystems. We fed ¹⁵N labeled ryegrass hay to a sheep and collected ¹⁵N labeled feces. Nitrogen forms in the feces were characterized by chemical extractions, solid-state cross polarization ¹⁵N nuclear magnetic resonance spectroscopy (SS CP/MAS ¹⁵N NMR) and Curie-point pyrolysis–gas chromatography/mass spectrometry (Cp Py-GC/MS). A 4 months incubation experiment was conducted to assess N release from the feces. Half of the fecal N could be ascribed to bacterial and endogenous debris and a third to undigested dietary N. About a tenth of the fecal N was mineralized during the incubation experiment. The ¹⁵N abundance of nitrate released during the incubation remained constant and close to the ¹⁵N abundance of the total feces N. The NMR analysis of the feces showed that most of the N was present in proteins, while some was present as heterocyclic N, amino acids and ammonium. The Cp Py-GC/MS analysis confirmed the presence of proteins, amino acids and heterocyclic N in the feces. Comparing these results to those obtained from the ¹⁵N labeled hay suggests that some N compounds present in the plant were not digested by the animal, and that the animal excreted de novo synthesized N compounds. The low content in ammonium and amino acids, the low rate of N release from these feces during the incubation and the relatively high fecal protein content, particularly the hard to mineralize undigested and microbially bound forms, can explain the low transfer of N from these feces to crops observed in a previous work.     

N<Subscript>2</Subscript>O emissions from grain cropping systems: a meta-analysis of the impacts of fertilizer-based and ecologically-based nutrient management strategies

Understanding how agricultural management practices impact nitrous oxide (N₂O) emissions is prerequisite for developing mitigation protocols. We conducted a meta-analysis on 597 pairwise comparisons (129 papers) to assess how management affects N₂O emissions. Pairwise comparisons of practices aimed at improving fertilizer use efficiency (39%) and tillage (30%) dominated the dataset, while ecologically-based nutrient management (ENM) practices constituted 15% of the pairs. In general, across management practices, the quantity of N added was a more significant driver of N₂O fluxes than was the form of N (fertilizer, legume biomass or animal manures). Manure interacted with soil texture so that in coarse soils, N₂O emissions from manures tended to be higher compared to inorganic N fertilizers. The studies of ENM strategies frequently involved over-application of N inputs in the ENM treatments. Cover crops reduced N₂O emissions compared to bare fallows. However, during the cash crop growing season, when differences in N added and N source were confounded, the extra N inputs from cover crops were significantly correlated with the differences in N₂O emissions between treatments with and without cover crops. Overall, in 38% of the data pairs, N₂O emissions were reduced with limited impacts on yields; in half of these pairs, yields were maintained or increased while in the other half they were reduced by only ≤10%. Knowledge gaps on mitigation of agricultural N₂O emissions could be addressed by applying an ecosystem-based, cross-scale perspective in conjunction with the N saturation conceptual framework to guide research priorities and experimental designs.     

Subsoil <Superscript>15</Superscript>N-N<Subscript>2</Subscript>O Concentrations in a Sandy Soil Profile After Application of <Superscript>15</Superscript>N-fertilizer

Studies on emissions of nitrous oxide (N₂O) from agricultural soils mostly focus on fluxes between the soil and the atmosphere or are limited to the atmosphere in the topsoil. However, in soils with shallow water tables, significant N₂O formation may occur closer to the groundwater. The aims of this study were (i) to determine the importance of subsoil N₂O formation in a sandy soil; and (ii) to obtain a quantitative insight in the contribution of subsoil N₂O to the overall losses of N₂O to the environment. We applied ¹⁵N labeled fertilizer at a rate of 5.22 kg ¹⁵N ha⁻¹; 50% as Ca(NO₃)₂ and 50% as NH₄Cl, on a mesic typic Haplaquod seeded with potatoes (Solanum tuberosum L.), and traced soil N₂O concentrations and fluxes over a one-year period. Throughout the year, total N₂O and the amount of ¹⁵N recovered in soil N₂O were highest in the subsoil, with a maximum concentration at 48 cm depth in mid-February of 19900 μl m⁻³ and 24 μg ¹⁵N m⁻³, respectively. The maximum concentration coincided with the highest water-filled pore space of 71%. The cumulative flux of N₂O was 446 g N₂O-N ha⁻¹, the recovery of ¹⁵N in this flux was 0.06%. During the summer, maximum fluxes followed high soil N₂O concentrations. During winter, no such relation was found. We concluded that the formation of N₂O was the highest in the subsoil, largely controlled by water-filled pore space rather than NO₃⁻ concentration or temperature. Although high subsoil N₂O concentrations did not lead to high surface fluxes of N₂O in the winter, artificial draining may lead to high indirect N₂O emissions through supersaturated drainage water.     

Nitrogen input, <Superscript>15</Superscript>N balance and mineral N dynamics in a rice–wheat rotation in southwest China

A field experiment and farm survey were conducted to test nitrogen (N) inputs, ¹⁵N-labelled fertilizer balance and mineral N dynamics of a rice–wheat rotation in southwest China. Total N input in one rice–wheat cycle averaged about 448 kg N ha⁻¹, of which inorganic fertilizer accounted for 63% of the total. The effects of good N management strategies on N cycling were clear: an optimized N treatment with a 27% reduction in total N fertilizer input over the rotation decreased apparent N loss by 52% and increased production (sum of grain yield of rice and wheat) compared with farmers’ traditional practice. In the ¹⁵N-labelled fertilizer experiment, an optimized N treatment led to significantly lower ¹⁵N losses than farmers’ traditional practice; N loss mainly occurred in the rice growing season, which accounted for 82% and 67% of the total loss from the rotation in farmers’ fields and the optimized N treatment, respectively. After the wheat harvest, accumulated soil mineral N ranged from 42 to 115 kg ha⁻¹ in farmers’ fields, of which the extractable soil NO₃ ⁻–N accounted for 63%. However, flooding soil for rice production significantly reduced accumulated mineral N after the wheat harvest: in the ¹⁵N experiment, farmers’ practice led to considerable accumulation of mineral N after the wheat harvest (125 kg ha⁻¹), of which 69% was subsequently lost after 13 days of flooding. Results from this study indicate the importance of N management in the wheat-growing season, which affects N dynamics and N losses significantly in the following rice season. Integrated N management should be adopted for rice–wheat rotations in order to achieve a better N recovery efficiency and lower N loss.     

Biological nitrogen fixation by soybean and fate of applied <Superscript>15</Superscript>N-fertilizer in succeeding wheat under conventional tillage and conservation agriculture practices

Four field experiments were conducted to investigate biological N₂ fixation (BNF) by irrigated soybean under conservation agriculture (CA) as compared with conventional tillage when crop residue (CR) is retained on the soil surface, and the fate of ¹⁵N-labelled fertilizer in succeeding wheat in the semi-arid subtropical soil. Comparable amounts of BNF by soybean were obtained using ¹⁵N isotope dilution and ¹⁵N natural abundance methods, suggesting that the latter, a less costly method could be employed to estimate BNF. Soybean could fix 61–125 kg N ha^?1 (52–85% of total N uptake), depending upon tillage and CR management. Significant increases in BNF by soybean were recorded when CR was retained on the soil surface of CA plots presumably due to better activity of rhizobia because of the relatively cooler rhizosphere environment. Recovery of applied fertilizer N in the soil–plant system at harvest of the wheat crop showed that 36–47% of it was utilized by the crop, 37–49% was left in the soil profile and 5–27% was lost (unrecovered fertilizer N). The recovery of fertilizer N in the soil profile revealed that the majority of it was present in the first 15 cm (54–61%), although downward movement of fertilizer N was also evident up to 120 cm soil depth. These results illustrate enormous benefits of CA practices with CR retained on soil surface on BNF in soybean, and similar patterns in N uptake and translocation from vegetative parts to grain and utilization of applied fertilizer N by wheat in both tillage systems.     

Effect of lime nitrogen on the efficiency of urea and other ammonium nitrogen fertilizers

Five pot experiments were conducted with wheat and rice in a net house to study the effect of lime nitrogen (LN, contains about 55% calcium cyanamide) amendment rates on the efficiency of urea, the recovery urea-¹⁵N, the efficiency of the three nitrogen fertilizers(NF), on the efficiency of urea in the three soils, and on NO ₃ ^- -N leaching from a flooded soil. A rate of LN-N of 5–8% of applied fertilizer N increased the recovery of labeled urea-N by 9.42%. The effect of LN on the efficiency of NF was urea > ammonium sulfate > ammonium chloride. Under flooded conditions, LN decreased NO ₃ ^- formation and leaching.Responses of several crops to LN amended fertilizers were also studied in field experiments. At equal NPK applications, the efficiency of basal applications to rice, wheat, corn, potatoes, soybean, peanut, grapes, peaches, melon and watermelon were bette r with LN than without. Efficiency with a basal fertilizer for rice or wheat with LN were the same as with the same fertilizer without LN applied in split applications.     

Quantification of symbiotic nitrogen fixation by <Emphasis Type="Italic">Elaeagnus angustifolia</Emphasis> L. on salt-affected irrigated croplands using two <Superscript>15</Superscript>N isotopic methods

Afforestation with fast growing N-fixing trees is an option for ecological restoration of highly-salinized irrigated croplands, but information about the N-fixing capability of trees on saline soils is sparse. The ¹⁵N-enrichment technique (¹⁵NET) and the A value (AV) method were used to quantify in lysimeters the proportion of atmospheric N₂ (%Ndfa) fixed by Elaeagnus angustifolia L., with a reference to non-N-fixing Gleditsia triacanthos L. and Ulmus pumila L. Twenty kg N ha⁻¹ of 5 atom %¹⁵N excess ammonium nitrate (35% N) was applied to 1-year-old trees in 2007 and 2-year-olds in 2008. Since this rate was insufficient for the older reference trees, 60 kg N ha⁻¹ was applied in 2008. With ¹⁵NET, the %Ndfa of E. angustifolia in 2007 was 79% when referenced against U. pumila and 68% against G. triacanthos. With the AV method, the %Ndfa of 2-year-old E. angustifolia was 80 and 68% when referenced against U. pumila and G. triacanthos, respectively. Over 2 years, E. angustifolia fixed 17 kg N ha⁻¹ when related to U. pumila and 14 kg N ha⁻¹ with G. triacanthos (assumed density: 5,000 trees ha⁻¹). N-fixing E. angustifolia has the potential to be self-sufficient in N when planted in the strongly saline soils.     

Nitrogen use efficiency of <Superscript>15</Superscript>N-labelled sheep manure and mineral fertiliser applied to microplots in long-term organic and conventional cropping systems

Nitrogen (N) utilisation by crops has to be improved to minimize losses to the environment. We investigated N use efficiency of animal manure and mineral fertiliser and fate of fertiliser N not taken up by crops in a conventional (CONMIN) and a bio-organic (BIOORG) cropping system of a long-term field experiment over three vegetation periods (winter wheat–soybean–maize). Microplots planted with wheat received a single application of ¹⁵N-labelled slurries (either urine or faeces labelled) or mineral fertiliser. At the end of each vegetation period we tested whether higher microbial activity and larger microbial biomass in BIOORG than CONMIN soils, and lower long-term N input level in BIOORG, affected use efficiency and fate of fertiliser N not taken up by crops. Recovery of ¹⁵N in wheat was 37%, 10% and 47% from urine, faeces and mineral fertiliser, respectively, and decreased strongly in the residual years. In total 41%, 15% and 50% of ¹⁵N applied as urine, faeces and mineral fertiliser was recovered by the three crops. ¹⁵N recovered from originally applied urine, faeces and mineral fertiliser in the topsoil (0–18 cm) at the end of the third vegetation period was 19%, 25% and 20%, respectively. Of urine-, faeces- and mineral fertiliser-¹⁵N, 40%, 61% and 29%, respectively, was not recovered by the three crops and in topsoil suggesting significant transport of ¹⁵N-labelled components to deeper soil layers. CONMIN and BIOORG differed neither in fertiliser N use efficiency by crops nor in ¹⁵N recovery in soil indicating insignificant difference in the turnover and utilization of the applied manure nitrogen in the conventional and the bio-organic cropping systems.     

Movement and distribution of fertilizer nitrogen as affected by depth of placement in wetland rice

Experiments were conducted to monitor the movement and distribution of ammonium-N after placement of urea and ammonium sulfate supergranules at 5, 7.5, 10, and 15 cm. By varying depths of fertilizer placement, it is possible to determine the appropriate depth for placement machines. There were no significant differences in grain yields with nitrogen placed 5 and 15 cm deep. However, grain yields were significantly higher with deep placement of nitrogen than with split application of the fertilizer. The lower yields with split-applied nitrogen were due to higher nitrogen losses from the floodwater. The floodwater with split application had 78–98µg N ml^–1 and that with deep-placed nitrogen had a negligible nitrogen concentration.Movement of NH ₄ ⁺ -N in the soil was traced for various depths after fertilizer nitrogen application. The general movement after deep-placement of the ammonium sulfate supergranules was downward > lateral > upward from the placement site. Downward movement was prevalent in the dry season: fertilizer placed at 5–7.5 cm produced a peak of NH ₄ ⁺ -N concentration at 8–12 cm soil depth; with placement at 15 cm, the fertilizer moved to 12–20 cm soil depth. Fertilizer placed at 10 cm tended to be stable. In the wet season, deep-placed N fertilizer was fairly stable and downward movement was minimal.A substantially greater percentage of plant N was derived from¹⁵N-depleted fertilizer when deep-placed in the reduced soil layer than that applied in split doses. The percent N recovery with different placement depths, however, did not vary from each other. The results suggest that nitrogen placement at a 5-cm soil depth is adequate for high rice yields in a clayey soil with good water control. In farmers' fields where soil and water conditions are often less than ideal, however, it is desirable to place nitrogen fertilizer at greater depths and minimize NH ₄ ⁺ -N concentration in floodwater.     

How much nitrogen is fixed by biological symbiosis in tropical dry forests? 1. Trees and shrubs

Despite the recognized importance of the process, estimates of the amount of nitrogen fixed by biological symbiosis in tropical dry forests are almost nonexistent. We estimated the nitrogen fixed annually by the leaves of trees and shrubs at sites regenerating for 16 and 38?years and in an old-growth dry forest using ¹⁵N abundance methodology. The total leaf biomass (1,824?C3,036?kg?ha^?1) and nitrogen contents (62?C90?kg?ha^?1) did not differ among the areas. In all of the areas, most of the leaf biomass belonged to legume plants, but the proportion of the N₂-fixing legumes decreased with increasing regeneration time. In the 16-year regenerating area, almost all of the N was in the leaves of the N-fixing Mimosa tenuiflora plants, but fixation was absent or very low as it was in the N-fixing species present in the 38-year regenerating area. In the old-growth Caatinga, all of the N-fixing species (M. tenuiflora, Piptadenia stipulacea and Anadenanthera colubrina) had large proportions (47?C62?%) of their N derived from atmospheric N₂, but the amount of fixed N (6?kg?ha^?1) was a small proportion of the total leaf N because these plant species were a small part of the vegetation. The total input of biologically fixed N to the old-growth forest was similar in magnitude to an estimate made for a humid tropical forest in Amazonia.     

Initial and residual effects of nitrogen fertilizers on grain yield of a maize/bean intercrop grown on a Humic Nitosol and the fate and efficiency of the applied nitrogen

Initial and residual effects of nitrogen (N) fertilizers on grain yield of a maize/bean intercrop grown on a deep, well-drained Humic Nitosol (66% clay, 3% organic carbon) were evaluated. Enriched (¹⁵N) N fertilizer was used to study the fate of applied N in two seasons: using urea (banded) at 50 kg N ha^–1 in one season, and¹⁵N-enriched urea (banded), calcium ammonium nitrate (CAN, banded), and urea supergranules (USG, point placement) were applied in the other season (different field) at 100 kg N ha^–1. Nitrogen fertilizer significantly (P = 0.05) increased equivalent maize grain yield in each season of application with no significant differences between N sources, i.e., urea, CAN, and USG. Profitmaximizing rates ranged from 75 to 97 kg N ha^–1 and value: cost ratios ranged from 3.0 to 4.8. Urea gave the highest value: cost ratio in each season. Most (lowest measurement 81%) of the applied N was accounted for by analyzing the soil (to 150 cm depth) and plant material. Measurements for urea, CAN, and USG were not significantly different. The high N measurements suggest low losses of applied N fertilizer under the conditions of the study. Maize plant recovery ranged from 35 to 55%; most of this N (51–65%) was in the grain. Bean plant recovery ranged from 8 to 20%. About 34–43% of the applied N fertilizer remained in the soil, and most of it (about 70%) was within the top soil layer (0–30 cm). However, there were no significant equivalent maize grain increases in seasons following N application indicating no beneficial residual effect of the applied fertilizers.     

Fallow replacement and alternative nitrogen management for reducing nitrate leaching in a semiarid region

A suite of ¹⁵N-based methodologies have been applied to quantify the transfer of symbiotically-fixed or legume N to succeeding non-legumes in crop sequences. The structure of these methods, their comparative advantages and efficacy are scrutinized in the present review. Methods are either direct or indirect, the former involving labelling of the legume with ¹⁵N₂, the substrate for symbiotic N₂ fixation, while in the indirect methods the legume is either enriched or depleted in ¹⁵N through addition of labelled fertilizer or is otherwise unamended at ¹⁵N natural abundance. The methods can be classified further according to their yield dependency, and whether they involve the relocation of residues or are located in situ as green manures or harvest residues. The greatest number of studies has involved the relocation of ¹⁵N-labelled plant materials produced artificially at locations remote from the actual crop sequence in the field. Depending on the physico-chemical properties of the residues and the environmental and edaphic conditions pertaining at the experimental site, the proportions of non-legume N in phase 2 of the rotation derived from the phase 1 legume have been quite variable. Nevertheless, there are many recorded instances where green manures and harvest residues represent significant sources of N in legume–non-legume crop sequences. The application of ¹⁵N-based methods has resulted in a better understanding of the relative contribution of below- and above-ground legume N in N transfer, and the efficacy of management practices such as incorporation of green manures or use of surface mulches.     

How much nitrogen is fixed by biological symbiosis in tropical dry forests? 2. Herbs

Although biological nitrogen fixation (BNF) is considered the main input of N in mature and regenerating native tropical vegetation, it has seldom been quantified. Biomass and N accumulation and fixation were determined for spontaneously occurring herbaceous species in caatinga areas in four regeneration stages (2, 17, 39 and >50?years after abandonment from agricultural use). BNF was estimated using the ¹⁵N natural-abundance method. The 2-year regeneration area had the highest total herb (6,355?kg?ha^?1) and legume (262?kg?ha^?1) biomass production, N stocks (82?kg?ha^?1) and fixed N (5.0?kg?ha^?1). N₂-fixing legumes (nine species in the sampled area) contributed over 97?% of legume biomass in all areas. Macroptilium gracile added the largest amount of N (3.9?kg?ha^?1 in the 2-year regeneration area) because of its large biomass production (205?kg?ha^?1), although it was not the species with the highest proportion of fixed N (76?%). All of the N₂-fixing species obtained large proportions of their N from symbiosis, most of them more than 50?%.However, the amounts of fixed N per unit area were relatively low (0.22?C5.00?kg?ha^?1) because the biomass of N₂-fixing species was always less than 5?% of the total herb biomass.     

Plastic-film mulch and fertilization rate affect the fate of urea-<Superscript>15</Superscript>N in maize production

We investigated the effects of interaction between plastic-film mulch and nitrogen (N) fertilization rate on the fate of fertilizer N in a ridge–furrow maize (Zea mays L.) cropping system. Three N levels (0, 138 and 207 kg ha^?1, abbreviated as N0, N1 and N2) were combined with plastic-film-mulching and no-mulching, successively in 2015 and 2016, at a cold semiarid site. Within each treated plot, a micro-plot was established to trace the fate of urea-N (only ¹⁵N-labeled in 2015). Averaging 2 years, increasing fertilization from N1 to N2 increased maize grain yield and total N uptake only in mulched soils. Mulch increased both maize grain yield and total N uptake more at N2 than at N1. In 2015, mulch increased the in-season fertilizer N uptake in maize by 53% at N1 but by 75% at N2; increasing N application from N1 to N2 enhanced the fertilizer N acquisition by 26% in non-mulched but by 45% in mulched plots. In 2016, similar effects of interaction existed between mulch and fertilization rate on the residual fertilizer N uptake by maize. Mulch enhanced fertilizer N availability in the topsoil relative to no mulch, responsible for the increased maize fertilizer N uptake in mulched treatments. Decreased in-season fertilizer N loss and transformation of urea N to the organic N in mulched soils were contributors to the increased fertilizer N availability, compared to non-mulched soils. We concluded that the effects of fertilization on maize total N uptake and fertilizer N recovery benefited from plastic-film mulch.     

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.     

Nitrogen leaching and plant uptake from controlled-release fertilizers

Controlled-release N fertilizers are commonly used in the production of container-grown ornamental crops, yet the relative effects of various nutrient sources on N leaching are not well known. A 27-week experiment was conducted to evaluate N leaching loss and plant growth following two applications of six controlled-release N fertilizers and one soluble N fertilizer to container-grownEuonymus patens Rehd. The controlled-release fertilizers evaluated were (noncoated) isobutylidene diurea, oxamide, urea formaldehyde, and (coated) Osmocote, Prokote Plus, and sulfur-coated urea. Of the fertilizers tested, the coated fertilizers generally out-performed the noncoated fertilizers in reducing N leaching losses, stimulating plant growth, and increasing tissue N concentrations. Low N concentrations in the leachate of some treatments indicated efficient nutrient use by the plant. In other treatments, low N concentrations in the leachate merely reflected incomplete N release from the fertilizer. A daily application of NH₄NO₃ resulted in a constant rate of N loss but was not the most effective in promoting growth. Plant growth, tissue N concentrations, and N leaching losses were all increased by doubling the fertilizer application rate from 1 kg N m^–3 to 2 kg N m^–3.     

Effects of type and amount of applied nitrogen fertilizer on nitrous oxide fluxes from intensively managed grassland

Five field experiments and one greenhouse experiment were carried out to assess the effects of nitrogen (N) fertilizer type and the amount of applied N fertilizer on nitrous oxide (N₂O) emission from grassland. During cold and dry conditions in early spring, emission of N₂O from both ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) containing fertilizers applied to a clay soil were relatively small, i.e. less than 0.1% of the N applied. Emission of N₂O and total denitrification losses from NO ₃ ^– containing fertilizers were large after application to a poorly drained sand soil during a wet spring. A total of 5–12% and 8–14% of the applied N was lost as N₂O and via denitrification, respectively. Emissions of N₂O and total denitrification losses from NH ₄ ⁺ fertilizers and cattle slurry were less than 2% of the N applied. Addition of the nitrification inhibitor dicyandiamide (DCD) reduced N₂O fluxes from ammonium sulphate (AS). However, the effect of DCD to reduce total N₂O emission from AS was much smaller than the effect of using NH ₄ ⁺ fertilizer instead of NO ₃ ^– fertilizer, during wet conditions. The greenhouse study showed that a high groundwater level favors production of N₂O from NO ₃ ^– fertilizers but not from NH ₄ ⁺ fertilizers. Inereasing calcium ammonium nitrate (CAN) application increased the emitted N₂O on grassland from 0.6% of the fertilizer application rate for a dressing of 50 kg N ha^–1 to 3.1% for a dressing of 300 kg N ha^–1. In another experiment, N₂O emission increased proportionally with increasing N rate. The results indicate that there is scope for reducing N₂O emission from grasslands by choosing the N fertilizer type depending on the soil moisture status. Avoiding excessive N application rates may also minimize N₂O emission from intensively managed grasslands.