Estimates of seasonal nitrogen fixation of annual subterranean clover-based pastures using the15N natural abundance technique

Annual pasture legumes play a key role in ley farming systems of southern Australia, providing biologically fixed nitrogen (N) to drive the production of the pastures as well as subsequent crops grown in rotation. Seasonal inputs of biologically fixed N in shoot biomass of the subterranean clover (Trifolium subterraneum) component of grazed annual pastures were assessed using the¹⁵N natural abundance technique and appropriately timed sampling of herbage dry matter (DM) for N accumulation. At three study sites spanning a gradient across the Western Australian wheatbelt from 300 to 600 mm annual rainfall the performance of the clover and non-legume herbs and grasses was examined as paired comparisons involving two management treatments expected to give contrasting effects on pasture productivity, botanical composition and N₂ fixation. The proportion of clover N derived from atmospheric N₂ fixation (%Ndfa) ranged from 65 to 95% across sites, treatments and sampling times. Amounts of fixed N accumulated in clover shoot biomass ranged from 50 to 125 kg ha⁻¹, and paralleled trends in clover production. Substantial increases in pasture production in high yielding treatments generally occurred without decrease in %Ndfa, suggesting that N₂ fixation was essentially non-limiting to performance of the clover component. Seasonal profiles for accumulation of fixed N were skewed towards the late winter and spring period, particularly in low plant density pastures following a cereal crop. There were seasonal, site and treatment-specific effects on the proportion of clover and non-legume pasture components and consequently clover yield and N₂ fixation were variably affected by competition from non-legume species.     

The effect of a single application of cow urine on annual N2 fixation under varying simulated grazing intensity, as measured by four 15N isotope techniques

The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated over 12 months using mowing for defoliation. A single application of urine (equivalent to 746 kg N ha^–1), was applied in late spring to plots immediately after light and moderately-severe defoliation (35 mm and 85 mm cutting heights, respectively) treatments were imposed. Estimates of percentage clover N derived from N₂ fixation (%Ndfa) were compared by labelling the soil with ¹⁵N either by applying a low rate of ¹⁵N-labelled ammonium sulphate, immobilising ¹⁵N in soil organic matter, adding ¹⁵N to applied urine, or by utilising the small differences in natural abundance of ¹⁵N in soil. Urine application increased annual grass production by 85%, but had little effect on annual clover production. However, urine caused a marked decline in %Ndfa (using an average of all ¹⁵N methods) from 84% to a low of 22% by 108 days, with recovery to control levels taking almost a year. As a result, total N fixed (in above ground clover herbage) was reduced from 232 to 145 kg N ha^–1 yr^–1. Moderately–severe defoliation had no immediate effect on N₂ fixation, but after 108 days the %Ndfa was consistently higher than light defoliation during summer and autumn, and increased by up to 18%, coinciding with an increase in growth of weeds and summer-grass species. Annual N₂ fixation was 218 kg N ha^–1 yr^–1 under moderately-severe defoliation compared to 160 kg N ha^–1 yr^–1 under light defoliation. Estimates of %Ndfa were generally similar when ¹⁵N-labelled or immobilised ¹⁵N were used to label soil regardless of urine and defoliation severity. The natural abundance technique gave highly variable estimates of %Ndfa (–56 to 24%) during the first 23 days after urine application but, thereafter, estimates of %Ndfa were similar to those using ¹⁵N-labelling methods. In contrast, in urine treated plots the use of ¹⁵N-labelled urine gave estimates of %Ndfa that were 20–30% below values calculated using conventional ¹⁵N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in ¹⁵N distribution with depth. This study shows that urine has a prolonged effect on reducing N₂ fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N₂ fixation.     

Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization

Effects of rate of nitrogen (N) fertilizer and stocking rate on production and N₂ fixation by white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L.) were determined over 5 years in farmlets near Hamilton, New Zealand. Three farmlets carried 3.3 dairy cows ha^–1 and received urea at 0, 200 or 400 kg N ha^–1 yr^–1 in 8–10 split applications. A fourth farmlet received 400 kg N ha^–1 yr^–1 and had 4.4 cows ha^–1.There was large variation in annual clover production and total N₂ fixation, which in the 0 N treatment ranged from 9 to 20% clover content in pasture and from 79 to 212 kg N fixed ha^–1 yr^–1. Despite this variation, total pasture production in the 0 N treatment remained at 75–85% of that in the 400 N treatments in all years, due in part to the moderating effect of carry-over of fixed N between years.Fertilizer N application decreased the average proportion of clover N derived from N₂ fixation (P_N; estimated by ¹⁵N dilution) from 77% in the 0 N treatment to 43–48% in the 400 N treatments. The corresponding average total N₂ fixation decreased from 154 kg N ha^–1 yr^–1 to 39–53 kg N ha^–1 yr^–1. This includes N₂ fixation in clover tissue below grazing height estimated at 70% of N₂ fixation in above grazing height tissue, based on associated measurements, and confirmed by field N balance calculations. Effects of N fertilizer on clover growth and N₂ fixation were greatest in spring and summer. In autumn, the 200 N treatment grew more clover than the 0 N treatment and N₂ fixation was the same. This was attributed to more severe grazing during summer in the 0 N treatment, resulting in higher surface soil temperatures and a deleterious effect on clover stolons.In the 400 N treatments, a 33% increase in cow stocking rate tended to decrease P_N from 48 to 43% due to more N cycling in excreta, but resulted in up to 2-fold more clover dry matter and N₂ fixation because lower pasture mass reduced grass competition, particularly during spring.     

Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Nitrogen cycling in low input legume-based agriculture,with emphasis on legume/grass pastures

Low input legume-based agriculture exists in a continuum between subsistence farming and intensive arable and pastoral systems. This review covers this range, but with most emphasis on temperate legume/grass pastures under grazing by livestock. Key determinants of nitrogen (N) flows in grazed legume/grass pastures are: inputs of N from symbiotic N₂ fixation which are constrained through self-regulation via grass/legume interactions; large quantities of N cycling through grazing animals with localised return in excreta; low direct conversion of pasture N into produce (typically 5–20%) but with N recycling under intensive grazing the farm efficiency of product N: fixed N can be up to 50%; and regulation of N flows by mineralisation/immobilisation reactions. Pastoral systems reliant solely on fixed N are capable of moderate-high production with modest N losses e.g. average denitrification and leaching losses from grazed pastures of 6 and 23 kg N ha^–1 yr^–1. Methods for improving efficiency of N cycling in legume-based cropping and legume/grass pasture systems are discussed. In legume/arable rotations, the utilisation of fixed N by crops is influenced greatly by the timing of management practices for synchrony of N supply via mineralisation and crop N uptake. In legume/grass pastures, the spatial return of excreta and the uptake of excreta N by pastures can potentially be improved through dietary manipulation and management strategies. Plant species selection and plant constituent modification also offer the potential to increase N efficiency through greater conversion into animal produce, improved N uptake from soil and manipulation of mineralisation/immobilisation/nitrification reactions.     

Prospects and problems of simple linear models for estimating symbiotic N2 fixation by crop and pasture legumes

Symbiotic dinitrogen (N₂) fixation of crop and pasture legumes is a critical component of agricultural systems, but its measurement is expensive and labour intensive. Simple models which can provide approximations based on crop or pasture dry matter production would be useful for agrononomists and those interested in regional nitrogen (N) cycle fluxes. We investigate meta analysis of published data on legume shoot dry matter production, shoot %N and legume %N fixed (%Ndfa) and look for relationships among these, as a possible way of providing useful approximations of N₂ fixation. We restricted our analysis to Australian studies where we have ready access to the primary data and where cultivars, management and climate are more constrained compared to a universal dataset. Regression analysis between shoot dry matter and amounts of shoot N₂ fixed were strong for all crop and pasture legumes with significant differences in slope and intercept values being obtained between pastures and crops, and between chickpea (Cicer arietinium) and all other crop and pasture legumes. Annual pasture legumes showed the strongest linear relationship between N₂ fixation and shoot dry matter and had the greatest slope (20.2–24.3 kg N₂ fixed/t), compared to 18.7 kg N₂ fixed/t for the perennial pasture legume lucerne (alfalfa, Medicago sativa), and between 10.7 to 23.0 kg N₂/t for crop legumes, depending upon species. It was recognised that the use of such shoot-based relationships would underestimate the total amounts of N₂ fixed since the contributions of fixed N present in, or derived from, roots and nodules are not included. Furthermore there needs to be careful consideration of the validity of an intercept term, which might reflect suppression of N₂ fixation at low dry matter and high soil mineral N availability, or possibly the use of non-linear regression. For chickpea crops grown in north-eastern Australia, multiple regression indicated that N₂ fixation was much more closely correlated with %Ndfa than dry matter production. Evidence presented also indicated that %Ndfa of other crops and lucerne in this region may similarly be influenced by soil mineral N. The regression approach presented provides a statistical basis to approximate N₂ fixation in the first instance. This work highlights some of the dangers of fitting single regressions to aggregated datasets and using these to approximate symbiotic N₂ fixation. The analysis indicates that where pasture legumes are grown in mixtures with non-legumes, and driven to high dependence on N₂ fixation, simple linear regressions may be quite useful, provided that possible differences between species are investigated as the slopes of the regressions between these can be quite different. For crop legumes, where low dependence on N₂ fixation can occur at higher mineral N availability, there is a need to carefully consider the intercept term, obtain estimates of mineral N availability, and/or resort to non-linear models. The gross generalisations presented in scatter plots cannot be reliably applied any more specifically, even within the datasets from which they were generated, and in some cases even within legume species between regions. They cannot substitute for direct measurement where any certainty is required under a particular set of defined conditions.     

Key processes of the nitrogen cycle in an irrigated and a non-irrigated grazed pasture

The paper presents integrated measurements of N fixation, net mineralisation, pasture yield and change in soil mineral N over a 12 month period for dairy pastures on a sandy loam soil in the South East of South Australia. The two adjacent pastures studied were an irrigated perennial white clover-ryegrass and an annual non-irrigated subterranean clover with mixed annual grasses. This produced the most comprehensive mineral N balance reported for grazed pastures, to the authors' knowledge, allowing calculation of gaseous and leaching losses of N (210 kg ha^–1 in the irrigated and paddock and 81 kg ha^–1 in the non irrigated paddock) primarily from urine patches. In both paddocks these losses were about three times the N yield in milk (61 and 28 kg N ha^–1 respectively) and were replenished by biological N fixation (294 and 100 kg N ha^–1). However, mineralisation of soil organic N, excretal N and pasture residues (687 and 438 kg N ha^–1) was the major source of mineral N for cycling and losses. The results demonstrate the enormous impact of pasture management on N fluxes and reinforce the importance of livestock urine on the magnitude of N fluxes including gaseous and leaching losses.     

Nitrogen inputs and losses from New Zealand dairy farmlets, as affected by nitrogen fertilizer application: year one

Inputs and losses of nitrogen (N) were determined in dairy cow farmlets receiving 0, 225 or 360 kg N ha^-1 (in split applications as urea) in the first year of a large grazing experiment near Hamilton, New Zealand. Cows grazed perennial ryegrass/white clover pastures all year round on a free-draining soil. N₂ fixation was estimated (using ¹⁵N dilution) to be 212, 165 and 74 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. The intermediate N rate had little effect on clover growth during spring but favoured more total pasture cover in summer and autumn, thereby reducing overgrazing and resulting in 140% more clover growth during the latter period.Removal of N in milk was 76,89 and 92 kg N ha^-1 in the 0, 225 and 360 N treatments, respectively. Denitrification losses were low (7–14 kg N ha^-1 yr^-1), increased with N application, and occurred predominantly during winter. Ammonia volatilization was estimated by micrometeorological mass balance at 15, 45 and 63 kg N ha^-1 yr^-1 in the 0, 225 and 360 N treatments, respectively. Most of the increase in ammonia loss was attributed to direct loss after application of the urea fertilizer.Leaching of nitrate was estimated (using ceramic cup samplers at 1 m soil depth, in conjunction with lysimeters) to be 13, 18 and 31 kg N ha^-1 yr^-1 in a year of relatively low rainfall (990 mm yr^-1) and drainage (170–210 mm yr^-1). Drainage was lower in the N fertilized treatments and this was attributed to enhanced evapotranspiration associated with increased grass growth.Nitrate-N concentrations in leachates increased gradually over time to 30 mg L^-1 in the 360 N treatment whereas there was little temporal variation evident in the 0 (mean 6.4 mg L^-1) and 225 (mean 10.1 mg L^-1) N treatments. Thus, the 360 N treatment had a major effect by greatly reducing N₂ fixation and increasing N losses, whereas the 225 N treatment had little effect on N₂ fixation or on nitrate leaching. However, these results refer to the first year of the experiment and further measurements over time will determine the longer-term effects of these treatments on N inputs, transformations and losses.     

Growing vetches (Vicia villosa Roth) in irrigated cotton systems: inputs of fixed N, N fertiliser savings and cotton productivity

We compared symbiotic N₂ fixation by winter forage legumes (clovers, medics and vetches) using the ¹⁵N natural abundance technique in three experiments. Vetches (Vicia spp.) were the most productive legumes, and woollypod vetch fixed (shoot+root) up to 265 kg N ha^–1 (mean 227 kg N ha^–1) during a 4–5 months period over winter and early spring. Balansa and Berseem clovers, and Gama medic were highly productive in the first experiment, but fixed significantly less N than woollypod vetch in the second experiment. A 6-year study (1997–2003) compared cotton (Gossypium hirsutum L.) systems with and without vetch, or with faba beans (Vicia faba L.) to assess the effects of these crops on cotton production. Woollypod vetch was grown either between annual cotton crops, or between wheat (Triticum aestivumL.) and cotton crops. Vetch added 230 kg N ha^–1 (174 kg fixed N ha^–1) to the soil when incorporated as a green manure. Faba bean shoot residues and nodulated roots contributed 108 kg fixed N ha^–1 to the soil, following the removal of 80 kg N ha^–1 in the harvested seed (meaned over three crops). Lablab (Lablab purpureus L. – summer-growing and irrigated) added 277 kg N ha^–1 (244 kg fixed N ha^–1) before incorporation as a green manure in the first year of the experiment. The economic optimum N fertiliser rate for each cropping system was determined every second year when all systems were sown to cotton. Cotton following cotton required 105 kg fertiliser N ha^–1, but only 40 kg N ha^–1 when vetch was grown between each cotton crop. Cotton following wheat required 83 kg fertiliser N ha^–1 but no N fertiliser was needed when vetch was grown after wheat (the highest yielding system). Cotton following faba beans also required no N fertiliser. The vetch-based systems became more N fertile over the course of the experiment and produced greater lint yields than the comparative non-legume systems, and required less N fertiliser. While no cash flow was derived from growing vetch, economic benefits accrued from enhanced cotton yields, reduced N fertiliser requirements and improved soil fertility. These findings help explain the rotational benefits of vetches observed in other regions of the world.     

Biological nitrogen fixation in mixed legume/grass pastures

Biological nitrogen fixation (BNF) in mixed legume/grass pastures is reviewed along with the importance of transfer of fixed nitrogen (N) to associated grasses. Estimates of BNF depend on the method of measurement and some of the advantages and limitations of the main methods are outlined. The amounts of N fixed from atmospheric N₂ in legume/grass pastures throughout the world is summarised and range from 13 to 682 kg N ha^-1 yr^-1. the corresponding range for grazed pastures, which have been assessed for white clover pastures only, is 55 to 296 kg N ha^-1 yr^-1.Biological nitrogen fixation by legumes in mixed pastures is influenced by three primary factors; legume persistence and production, soil N status, and competition with the associated grass(es). These factors and the interactions between them are discussed. Legume persistence, production and BNF is also influenced by many factors and this review centres on the important effects of soil moisture status, soil acidity, nutrition, and pests and disease.Soil N status interacts directly with BNF in the short and long term. In the short-term, increases in soil inorganic N occurs during dry conditions and where N fertiliser is used, and these will reduce BNF. In the long-term, BNF leads to accumulation of soil N, grass dominance, and reduced BNF. However, cyclical patterns of legume and grass dominance can occur due, at least in part, to temporal changes in plant-available N levels in soil. Thus, there is a dynamic relationship between legumes and grasses whereby uptake of soil N by grass reduces the inhibitory effect of soil N on BNF and competition by grasses reduces legume production and BNF. Factors affecting the competition between legumes and grasses are considered including grass species, grazing animals, and grazing or cutting management.Some fixed N is transferred from legumes to associated grasses. The amount of N transferred below-ground, predominantly through decomposition of legume roots and nodules, has been estimated at 3 to 102 kg N ha^-1 yr^-1 or 2 to 26% of BNF. In grazed pasture, N is also transferred above-ground via return in animal excreta and this can be of a similar magnitude to below-ground transfer.Increased BNF in mixed legume/grass pastures is being obtained through selection or breeding of legumes for increased productivity and/or to minimise effects of nutrient limitations, low soil moisture, soil acidity, and pests and disease. Ultimately, this will reduce the need to modify the pasture environment and increase the role of legumes in low-input, sustainable agriculture.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

Nitrogen cycling in an acid forest ecosystem in the Netherlands under increased atmospheric nitrogen input

Within a long-term research project studying the biogeochemical budget of an oak-beech forest ecosystem in the eastern part of the Netherlands, the nitrogen transformations and solute fluxes were determined in order to trace the fate of atmospherically deposited NH₄ ⁺ and to determine the contribution of nitrogen transformations to soil acidification.The oak-beech forest studied received an annual input of nitrogen via throughfall and stemflow of 45 kg N ha^–1 yr^–1, mainly as NH₄ ⁺, whereas 8 kg N ha^–1 yr^–1 was taken up by the canopy. Due to the specific hydrological regime resulting in periodically occurring high groundwater levels, denitrification was found to be the dominant output flux (35 kg N ha^–1 yr^–1). N₂0 emmission rate measurements indicated that 57% of this gaseous nitrogen loss (20 kg N ha^–1 yr^–1) was as N₂O. The forest lost an annual amount of 11 kg N ha^–1 yr^–1 via streamwater output, mainly as N0₃ ^–.Despite the acid conditions, high nitrification rates were measured. Nitrification occurred mainly in the litter layer and in the organic rich part of the mineral soil and was found to be closely correlated with soil temperature. The large amount of NH₄ ⁺ deposited on the forest floor via atmospheric deposition and produced by mineralization was to a large extent nitrified in the litter layer. Almost no NH₄ ⁺ reached the subsurface soil horizons. The N0₃ ^– was retained, taken up or transformed mainly in the mineral soil. A small amount of N0₃ ^– (9 kg N ha^–1 yr^–1) was removed from the system in streamwater output. A relatively small amount of nitrogen was measured in the soil water as Dissolved Organic Nitrogen.On the basis of these data the proton budget of the system was calculated using two different approaches. In both cases net proton production rates were high in the vegetation and in the litter layer of the forest ecosystem. Nitrogen transformations induced a net proton production rate of 2.4 kmol ha^–1 yr^–1 in the soil compartment.     

Nitrogen fixation and nitrogen balance studies in Rhizobium-VA mycorrhiza inoculated legume-grass association by15N isotope dilution technique under a field condition

A mixed pasture comprising of buffel grass and a legume siratro was studied under field condition for a two-year period to know the fodder yield increase, nitrogen fixation and nitrogen balance with and without the inoculation of VA mycorrhiza to grass and Rhizobium to legume component.¹⁵N dilution technique was followed using labelled ammonium sulphate. The data showed that during the first year of the above study combined inoculation of VA mycorrhiza and Rhizobium to grass and legume respectively significantly increased the total dry matter (DM) (23,900 kg ha^–1 yr^–1) and total N content (308 kg ha^–1 yr^–1) of the mixed pasture over the uninoculated mixture. However, the above increase due to combined inoculation was maximum during second year with respect to DM yield (28,200 kg ha^–1 yr^–1), but the total N harvested through grass-legume mixture was comparatively lower than the first year (297 kg ha^–1 yr^–1). The amount of biologically fixed N was highest in the first year (79 kg ha^–1 yr^–1) and showed a very drastic reduction at the end of second year (39 kg ha^–1 yr^–1). A positive nitrogen balance was observed in the grass-legume mixture irrespective of inoculation of VA mycorrhiza and/or Rhizobium.     

Exchange of N-gases at the Höglwald Forest – A summary

During 4 years continuous measurements of N-trace gas exchange were carried out at the forest floor-atmosphere interface at the Höglwald Forest that is highly affected by atmospheric N-deposition. The measurements included spruce control, spruce limed and beech sites. Based on these field measurements and on intensive laboratory measurements of N₂-emissions from the soils of the beech and spruce control sites, a total balance of N-gas emissions was calculated. NO₂-deposition was in a range of –1.6 –2.9 kg N ha^–1 yr^–1 and no huge differences between the different sites could be demonstrated. In contrast to NO₂-deposition, NO- and N₂O-emissions showed a huge variability among the different sites. NO emissions were highest at the spruce control site (6.4–9.1 kg N ha^–1 yr^–1), lowest at the beech site (2.3–3.5 kg N ha^–1 yr^–1) and intermediate at the limed spruce site (3.4–5.4 kg N ha^–1 yr^–1). With regard to N₂O-emissions, the following ranking between the sites was found: beech (1.6–6.6 kg N ha^–1 yr^–1) >> spruce limed (0.7–4.0 kg N ha^–1 yr^–1) > spruce control (0.4–3.1 kg N ha^–1 yr^–1). Average N-trace gas emissions (NO, NO₂, N₂O) for the years 1994–1997 were 6.8 kg N ha^–1 yr^–1 at the spruce control site, 3.6 kg N ha^–1 yr^–1 at the limed spruce site and 4.5 kg N ha^–1 yr^–1 at the beech site. Considering N₂-losses, which were significantly higher at the beech (12.4 kg N ha^–1 yr^–1) than at the spruce control site (7.2 kg N ha^–1 yr^–1), the magnitude of total gaseous N losses, i.e. N₂-N + NO-N + NO₂-N + N₂O-N, could be calculated for the first time for a forest ecosystem. Total gaseous N-losses were 14.0 kg N ha^–1 yr^–1 at the spruce control site and 15.5 kg N ha^–1 yr^–1 at the beech site, respectively. In view of the huge interannual variability of N-trace gas fluxes and the pronounced site differences in N-gas emissions it is concluded that more research is needed in order to fully understand patterns of microbial N-cycling and N-gas production/emission in forest ecosystems and mechanisms of reactions of forest ecosystems to the ecological stress factor of atmospheric N-input.     

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Effect of nitrogen fertilization and cropping system of the reference crop on estimation of N2 fixation by vetch using15N methodology

This study was conducted to examine the effects of varying N rates and cropping systems (mixedversus pure stand) on the suitability of oats (Avena sativa L.) for estimating N₂ fixed in sequentially harvested vetch (Vicia sativa L.) over two growing seasons (1984–85 and 1985–86). The N rates were, 20 and 100 kg N ha^–1 in 1984–85 and 15 and 60 kg N ha^–1 in 1985–86. In the 1984–85 season, vetch at maturity derived 76 and 63% N from fixation at the high and low N rates respectively. The corresponding values for the second season were 66 and 42%. Except in the 1985–86 season when some significantly higher values of % N₂ fixed were estimated by using the reference crop grown at the higher (A-value approach) than at the lower N rate (isotope-dilution approach), both approaches resulted in similar measurements of N₂ fixed. In the 1984–85 season, similar values of N₂ fixed were obtained using either the pure or mixed stand oats reference crops. Although in the 1985–86 season, the mixed reference crop occasionally estimated lower % N₂ fixed than pure oats, total N₂ fixed estimates were always similar (P<0.05). Thus, in general, N fertilization and cropping system of the reference crop did not significantly influence estimates of N₂ fixation.     

Land-use change effects on soil C and N transformations in soils of high N status: comparisons under indigenous forest, pasture and pine plantation

Globally, land-use change is occurring rapidly, and impacts on biogeochemical cycling may be influenced by previous land uses. We examined differences in soil C and N cycling during long-term laboratory incubations for the following land-use sequence: indigenous forest (soil age = 1800 yr); 70-year-old pasture planted after forest clearance; 22-year-old pine (Pinus radiata) planted into pasture. No N fertilizer had been applied but the pasture contained N-fixing legumes. The sites were adjacent and received 3–6 kg ha^–1 yr^–1volcanic N in rain; NO₃ ^–-N leaching losses to streamwater were 5–21 kg ha^–1 yr^–1, and followed the order forest < pasture = pine. Soil C concentration in 0–10 cm mineral soil followed the order: pasture > pine = forest, and total N: pasture > pine > forest. Nitrogen mineralization followed the order: pasture > pine > forest for mineral soil, and was weakly related to C mineralization. Based on radiocarbon data, the indigenous forest 0–10 cm soil contained more pre-bomb C than the other soils, partly as a result of microbial processing of recent C in the surface litter layer. Heterotrophic activity appeared to be somewhat N limited in the indigenous forest soil, and gross nitrification was delayed. In contrast, the pasture soil was rich in labile N arising from N fixation by clover, and net nitrification occurred readily. Gross N cycling rates in the pine mineral soil (per unit N) were similar to those under pasture, reflecting the legacy of N inputs by the previous pasture. Change in land use from indigenous forest to pasture and pine resulted in increased gross nitrification, net nitrification and thence leaching of NO₃ ^–-N.     

Mineralization of nitrogen in long-term pasture soils: effects of management

A field incubation technique with acetylene to inhibit nitrification was used to estimate net N mineralization rates in some grassland soils through an annual cycle. Measurements were made on previously long-term grazed pastures on a silty clay loam soil in S.W. England which had background managements of +/– drainage and +/– fertilizer (200 kg N ha^–1 yr^–1). The effect of fertilizer addition on mineralization during the year of measurement was also determined. Small plots with animals excluded, and with herbage clipped and removed were used as treatment areas and measurements were made using an incubation period of 7 days at intervals of 7 or 14 days through the year. Soil temperature, moisture and mineral N contents were also determined. Mineralization rates fluctuated considerably in each treatment. Maximum daily rates ranged from 1.01 to 3.19 kg N ha^–1, and there was substantial net release of N through the winter period (representing, on average, 27% of the annual release). Changes in temperature accounted for 35% of the variability but there was little significant effect of soil moisture. Annual net release of N ranged from 135 kg ha^–1 (undrained soil, no previous or current fertilizer) to 376 (drained soil, +200 kg N ha^–1 yr^–1 previous and current fertilizer addition). Addition of fertilizer N to a previously unfertilized sward significantly increased the net release of N but there was no immediate effect of withholding fertilizer on mineralization during the year in which measurements were made.     

A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation

N₂ fixation in lines of Phaseolus vulgaris was measured by ¹⁵N-isotope dilution to determine whether a programme of crossing and recurrent selection had resulted in enhanced nitrogen fixation. In field experiments on an isohyperthermic Aquic Hapludoll soil the amount of N₂ fixed by the different lines ranged from 18 to 36 kg ha^–1 (32 to 47% of plant N) in 56 days. The quantity of N₂ fixed and the proportion of plant N derived from fixation was not significantly greater in the lines selected for N₂ fixation (RIZ lines) than parental lines. Total shoot N ranged from 53 to 77 kg ha^–1 and partitioning of N to pods differed from 28 to 52% among the lines which all had similar growth habit and duration. Nodulation patterns were also distinct. Nodules formed early (10 to 15 plant^–1 at 13 days) in many lines, and smallest amounts of fixation were observed in those lines which nodulated slowly and did not form substantial nodule mass until after 40 days. The screening criteria used in the selection of the RIZ lines had been largely indirect with other factors such as disease resistance also being included. Progress for increasing N₂ fixation over good-fixing parental lines such as BAT76 was not significant and it is recommended that more attention be paid to early nodulation, to the use of soils with lower available N and to inter-crossing of lines having different good N₂ fixation traits in order to further enhance the potential for N₂ fixation in beans.     

Animal treading during wet soil conditions reduces N2 fixation in mixed clover-grass pasture

A field experiment was carried out over 12 months to determine the effect of animal treading on N₂ fixation in a mixed white clover-ryegrass pasture. The experimental site was defoliated by mowing for the duration of the study. A single treading event of moderate or severe pugging intensity was initiated in plots during wet spring conditions by using dairy cows at varying stocking rates (4.5 cows 100 m⁻² for 1.5 or 2.5 h, respectively). Inputs of dung and urine onto the plots was avoided by overnight housing of the cows and interception of excreta during the pugging event. Soil air-filled porosity decreased from 21% in the non-pugged control to 15–16% in pugged treatments by day 3. Bulk density of soil was not significantly affected by pugging. Soil inorganic N concentration increased in pugged treatments, and was 4-fold greater on day 28 in severely pugged plots compared to non-pugged plots. White clover plant density and plant size was markedly lower in pugged treatments (up to 85% and 72% reduction, respectively under severe pugging). White clover growth was most affected during the first 156 days after pugging (up to 90% decrease under severe pugging), leading to an annual clover dry matter production loss of 9% and 52%, respectively. The proportion of clover N derived from atmospheric N₂ (%Ndfa; estimated by ¹⁵N dilution) was initially reduced (to a lower limit of 43%) by severe pugging (days 28–71) before recovery to control levels (90%) by day 91. Annual N₂ fixation in clover herbage decreased significantly from 76 kg N ha⁻¹ yr⁻¹ in the non-pugged control, to 66 and 36 kg N ha⁻¹ yr⁻¹ under moderate and severe pugging, respectively. Most of this difference was evident within the first 156 days after pugging. Our data indicates that the major loss in fixed N₂ input under pugging was due to reduced clover growth and production resulting from pugging damage and loss of residual white clover biomass by hoof action.