José María Estavillo

Dicyandiamide and 3,4-dimethyl pyrazole phosphate decrease N2O emissions from grassland but dicyandiamide produces deleterious effects in clover.

The application of nitrogen fertilisers leads to different ecological problems such as nitrate leaching and the release of nitrogenous gases. N2O is a gas involved in global warming, therefore, agricultural soils can be regarded as a source of global warming. Soil N2O production comes from both the nitrification and denitrification processes. From an ecological viewpoint, using nitrification inhibitors with ammonium based fertilisers may be a potential management strategy to lower the fluxes of N2O, thus decreasing its undesirable effect. In this study, the nitrification inhibitors (NIs) dicyandiamide (DCD) and 3,4-dimethyl pyrazole phosphate (DMPP) have been evaluated as management tools to mitigate N2O emissions from mineral fertilisation and slurry application in grassland systems (experiments 1 and 2), and to assess the phytotoxic effect of these inhibitors per se on clover (experiment 3). Both nitrification inhibitors acted in maintaining soil nitrogen (N) in ammonium form, decreasing cumulative N2O emissions. DCD, but not DMPP, produced phytotoxic effects and yield reduction in white clover. A nutrient imbalance, which led to a senescence process visually observed as chlorosis and necrosis at the border of the leaves, was noted.

Increased emissions of nitric oxide and nitrous oxide following tillage of a perennial pasture

About 40% of the agricultural land in the European Union (EU) is grassland used for animal production. When grassland is tilled, organically bound carbon and nitrogen are released, providing substrates for nitrifying and denitrifying microorganisms. The aim of this study was to examine the immediate effects of tillage of a perennial grassland carried out on different dates, on the emissions of nitric oxide (NO) and nitrous oxide (N2O), monitored intensively over a 5-day period, in a humid, dairy farming area of northern Spain. Soil was tilled 12 days and 2 days prior to fertiliser application. Tillage, time of tillage, and N fertiliser application affected NO and N2O emissions. Tillage 12 days before the start of the flux measurements resulted in higher emissions than tillage one day before, the difference being related to differences in soil mineral N and water-filled pore space (WFPS). Emissions of NO peaked at a WFPS of 50–60%, while N2O fluxes peaked at 70–90% WFPS. Loss of N was greater as N2O than as NO. The total loss of N as N2O plus NO ranged from 0.027 kg N ha–1 in unfertilised plots to 0.56 kg N ha–1 in the tilled and N fertilised plot. Thereafter emissions decreased rapidly to low values. The results of this study indicate that tillage of perennial grassland may release large amounts of NO and N2O, the amounts also depending on moisture conditions and addition of N fertiliser. We suggest that in order to reduce such emissions, application of N fertiliser should not immediately follow tillage of perennial grassland, as there is an extra supply of N from mineralisation of organic matter at this time.

Short term effect of ploughing a permanent pasture on N2O production from nitrification and denitrification

Soils are an important source of N2O, which can be produced both in the nitrification and the denitrification processes. Grassland soils in particular have a high potential for mineralization and subsequent nitrification and denitrification. When ploughing long term grassland soils, the resulting high supply of mineral N may provide a high potential for N2O losses. In this work, the short-term effect of ploughing a permanent grassland soil on gaseous N production was studied at different soil depths. Fertiliser and irrigation were applied in order to observe the effect of ploughing under a range of conditions. The relative proportions of N2O produced from nitrification and denitrification and the proportion of N2 gas produced from denitrification were determined using the methyl fluoride and acetylene specific inhibitors. Irrespectively to ploughing, fertiliser application increased the rates of N2O production, N2O production from nitrification, N2O production from denitrification and total denitrification (N2O + N2). Application of fertiliser also increased the denitrification N2O/N2 ratio both in the denitrification potential and in the gaseous N productions by denitrification. Ploughing promoted soil organic N mineralization which led to an increase in the rates of N2O production, N2O production from nitrification, N2O production from denitrification and total denitrification (N2O + N2). In both the ploughed and unploughed treatments the 0–10 cm soil layer was the major contributing layer to gaseous N production by all the above processes. However, the contribution of this layer decreased by ploughing, gaseous N productions from the 10 to 30 cm layer being significantly increased with respect to the unploughed treatment. Ploughing promoted both nitrification and denitrification derived N2O production, although a higher proportion of N2O lost by denitrification was observed as WFPS increased. Recently ploughed plots showed lower denitrification derived N2O percentages than those ploughed before as a result of the lower soil water content in the former plots. Similarly, a lower mean nitrification derived N2O percentage was found in the 10–30 cm layer compared with the 0–10 cm.

Effect of N-(n-butyl) thiophosphoric triamide and 3,4 dimethylpyrazole phosphate on gaseous emissions from grasslands under different soil water contents.

The intensification of grassland systems is leading to serious environmental risks due to the large input of nitrogen (N) in fertilizers and the subsequent gaseous losses. Addition of nitrification inhibitors (NI) or urease activity inhibitors to fertilizers could reduce these losses to the atmosphere. In the present study, the effects of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) and the urease activity inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) were evaluated on NH3, N2O, NO, and CO2 emissions. Ammonium sulphate nitrate (ASN), urea and cattle slurry were applied at a rate of 70 kg N ha(-1) to a mixed clover-ryegrass sward in the Basque Country (northern Spain) under different soil water contents. NH3 and NO emissions were determined by photoacoustic and chemiluminescence respectively using an open chamber technique while N2O and CO2 emissions were measured by photoacoustic using a closed chamber technique. When the water filled pore space (WFPS) was under 60%, the application of NBPT reduced NO emissions a 34% on urea and an 18% on slurry, and the application of DMPP reduced them a 2% on ASN and a 4% on slurry. No significant effect was observed on NH3 losses. When WFPS was over 60%, no effect could be observed on NO and N2O emissions after the application of both inhibitors, but NH3 losses were reduced a 31% by NBPT when applied with the slurry. Carbon dioxide emissions were unaffected by the use of DMPP or NBPT at any soil water content. Neither grassland yield nor herbage N concentration were influenced by the application of both inhibitors.

Nitrification and denitrification derived N2O production from a grassland soil under application of DCD and Actilith F2

The relative contribution of nitrification and denitrification to N2O production was investigated by means of soil incubations with acetylene in a mixed clover/ryegrass sown sward 5 days after application of a mineral fertiliser (calcium ammonium nitrate) or an organic one (cattle slurry) with and without the addition of the nitrification inhibitor dicyandiamide (DCD) and the commercial slurry additive Actilith-F2. At this time, maximum field N2O emissions were taking place. N2O production by the slurry amended soil was twice as high as that of the mineral amended one. N2O came in a greater proportion from nitrification rather than from denitrification in the slurry treatment, while for the mineral fertilisation most N2O came from denitrification. The addition of DCD to slurry produced a decrease in N2O production both from nitrification and denitrification. No reduction in N2O losses was observed from addition of DCD to the mineral fertilisation, although DCD resulted effective in reducing the nitrification rate by 53% both in the slurry and the mineral fertilisation. Actilith F2 induced a high nitrification rate and N2O production from denitrification was reduced while that from nitrification was not.

Denitrification losses from a natural grassland in the Basque Country under organic and inorganic fertilization

Denitrification losses from a poorly drained clayey loamy soil under natural pasture were measured over a two-year period using the acetylene inhibition technique. Plots received two different applications of fertilizer as calcium ammonium nitrate or cow slurry (a total of 145–290 kg N ha−1 in 1991 and 120–240 kg in 1992). In the first year, N losses in the mineral treatments were about 4 times greater than losses in the slurry treatments. In the second year losses in the slurry treatments increased in such a way that losses in the higher slurry application became similar to those for the two mineral treatments. Soil nitrate was the factor producing differences between treatments. In this way, N mineralization in periods between fertilizations coinciding with high soil water contents was responsible in the second year for the increase in N losses in the slurry treatments. Denitrification rates greater than 0.1 kg N ha−1 day−1 occurred at soil water contents > 33 % (air filled porosity < 26 %) and soil nitrate contents > 1 mg N kg−1 dry soil. Spring and autumn were the seasons of highest risk of denitrification because of N fertilizations coinciding with periods of soil saturation with water. Winter losses were low, but this is a period when there is a risk of denitrification in wetter seasons, particularly for a slurry application management.

Biochar as a tool to reduce the agricultural greenhouse-gas burden – knowns, unknowns and future research needs

Agriculture and land use change has significantly increased atmospheric emissions of the non-CO2 green-house gases (GHG) nitrous oxide (N2O) and methane (CH4). Since human nutritional and bioenergy needs continue to increase, at a shrinking global land area for production, novel land management strategies are required that reduce the GHG footprint per unit of yield. Here we review the potential of biochar to reduce N2O and CH4 emissions from agricultural practices including potential mechanisms behind observed effects. Furthermore, we investigate alternative uses of biochar in agricultural land management that may significantly reduce the GHG-emissions-per-unit-of-product footprint, such as (i) pyrolysis of manures as hygienic alternative to direct soil application, (ii) using biochar as fertilizer carrier matrix for underfoot fertilization, biochar use (iii) as composting additive or (iv) as feed additive in animal husbandry or for manure treatment. We conclude that the largest future research needs lay in conducting life-cycle GHG assessments when using biochar as an on-farm management tool for nutrient-rich biomass waste streams.

Effects of cattle slurry and mineral N fertilizer applications on various components of the nitrogen balance of mown grassland

It is essential to establish more accurate N balances for different soil-plant systems in order to improve N use efficiency. In this study the N balance was studied in a poorly drained clayey loam soil under natural grassland supplied with either calcium ammonium nitrate or cattle slurry at two application rates. The aim was to determine the efficiency of the N applied and the factors which affect this efficiency. Mineralization-immobilization of N was calculated by balance between the quantified inputs and outputs of N. As N inputs increased, output via herbage yield was accompanied by an increase in apparent immobilization of N in the soil and by larger losses of N by denitrification. The difference between cattle slurry and N fertilizer was that the slurry behaved as a slow release fertilizer, its supply of mineral N being greater in the periods of time when fertilizer was applied a long time ago. Denitrification losses (up to 17% of the N applied) are suggested to be the main factor to mitigate in order to increase N use efficiency. A decrease in net mineralization (up to 136 kg N ha-1 year-1) was observed which was related to the mineral N application rate. There was evidence to suggest that this decrease was due both to the immobilization of the N applied and to a decrease in the rate of gross mineralization when mineral N was applied. Microbial biomass determinations could not explain the changes in the mineralization-immobilization equilibrium of N because of the great coefficients of variation for this determination (mean value of 18%). Nevertheless, it contributed to verify and explain some of the changes observed in this equilibrium.

Effect of Low Nitrate Supply on Nitrogen Fixation in Alfalfa Root Nodules Induced by Rhizobium meliloti Strains with Varied Nitrate Reductase Activity

Summary Eighteen strains of Rhizobium meliloti were screened for their nitrate reductase activity (EC 1.7.99.4), on the basis of which they can be classified into 5 groups. After being tested for this constitutive expression, six strains were grown in a medium with nitrate as the sole N source, under aerobic and anaerobic conditions. No correlation was found among constitutive, assimilatory and dissimilatory activities. After assessment of their effectiveness, three strains showing differences in their nitrate reductase expression were used to compare the effect of low nitrate treatment on nitrogen fixation. Nodule nitrate reductase activity increased significantly after 7 days, being related to induction of nitrate-reductase activity of bacteroids, but acetylene reduction was strongly inhibited within 3 days of treatment, irrespective of the infective strain. Moreover, nitrite was not detected throughout the treatment. These results do not support the «nitrite hypothesis» of nodular senescence in our experimental conditions.

Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato

Plant ammonium tolerance has been associated with the capacity to accumulate large amounts of ammonium in the root vacuoles, to maintain carbohydrate synthesis and especially with the capacity of maintaining high levels of inorganic nitrogen assimilation in the roots. The tricarboxylic acid cycle (TCA) is considered a cornerstone in nitrogen metabolism, since it provides carbon skeletons for nitrogen assimilation. The hypothesis of this work was that the induction of anaplerotic routes of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (NAD-ME) would enhance tolerance to ammonium nutrition. An experiment was established with tomato plants (Agora Hybrid F1) grown under different ammonium concentrations. Growth parameters, metabolite contents and enzymatic activities related to nitrogen and carbon metabolism were determined. Unlike other tomato cultivars, tomato Agora Hybrid F1 proved to be tolerant to ammonium nutrition. Ammonium was assimilated as a biochemical detoxification mechanism, thus leading to the accumulation of Gln and Asn as free amino acids in both leaves and roots as an innocuous and transitory store of nitrogen, in addition to protein synthesis. When the concentration of ammonium in the nutrient solution was high, the cyclic operation of the TCA cycle seemed to be interrupted and would operate in two interconnected branches to provide α-ketoglutarate for ammonium assimilation: one branch supported by malate accumulation and by the induction of anaplerotic PEPC and NAD-ME in roots and MDH in leaves, and the other branch supported by stored citrate in the precedent dark period.