Biological nitrification inhibition (BNI)—is it a widespread phenomenon?

Regulating nitrification could be a key strategy in improving nitrogen (N) recovery and agronomic N-use efficiency in situations where the loss of N following nitrification is significant. A highly sensitive bioassay using recombinant luminescent Nitrosomonas europaea, has been developed that can detect and quantify the amount of nitrification inhibitors produced by plants (hereafter referred to as BNI activity). A number of species including tropical and temperate pastures, cereals and legumes were tested for BNI in their root exudate. There was a wide range in BNI capacity among the 18 species tested; specific BNI (AT units activity g⁻¹ root dry wt) ranged from 0 (i.e. no detectable activity) to 18.3 AT units. Among the tested cereal and legume crops, sorghum [Sorghum bicolor (L.)], pearl millet [Pennisetum glaucum (L.) R. Br.], and groundnut [Arachis hypogaea (L.)] showed detectable BNI in root exudate. Among pasture grasses, Brachiaria humidicola (Rendle) Schweick, B. decumbens Stapf showed the highest BNI capacity. Several high- and low-BNI genotypes were identified within the B. humidicola species. Soil collected from field plots of 10 year-old high-BNI genotypes of B. humidicola, showed a near total suppression (>90%) of nitrification; most of the soil inorganic N remained in the NH₄⁺ form after 30 days of incubation. In contrast, soils collected from low-BNI genotypes did not show any inhibitory effect; most of the soil inorganic N was converted to NO₃^– after 30 days of incubation. In both the high- and low-BNI genotypes, BNI was detected in root exudate only when plants were grown with NH₄⁺, but not when grown with NO₃^– as the sole source of N. BNI compounds when added to the soil inhibited nitrification and the relationship was linear (r ² = 0.92^**; n = 12). The BNI from high- and low-BNI types when added to N. europaea in pure culture, blocked both the ammonia monooxygenase (AMO) and the hydroxylamine oxidoreductase (HAO) pathways. Our results indicated that BNI capacity varies widely among and within species; and that some degree of BNI capacity is likely a widespread phenomenon in tropical pasture grasses. We suggest that the BNI capacity could either be managed and/or introduced into pastures/crops with an expression of this phenomenon, via genetic improvement approaches that combine high productivity along with some capacity to regulate soil nitrification process.     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

Responses to drought of five Brachiaria species. I. Biomass production,leaf growth,root distribution,water use and forage quality

The introduction of African grasses in Neotropical savannas has been a key factor to improve pasture productivity. We compared the response of five Brachiaria species to controlled drought (DT) in terms of biomass yield and allocation, pattern of root distribution, plant water use, leaf growth, nutrient concentration and dry matter digestibility. The perennial C₄ forage grasses studied were B. brizantha (CIAT 6780), B. decumbens (CIAT 606), B. dictyoneura (CIAT 6133), B. humidicola (CIAT 679) and B. mutica. Two DT periods, which mimic short dry spells frequent in the rainy season, were imposed by suspending irrigation until wilting symptoms appeared. They appeared after 14 days in B. brizantha, B. decumbens and B. mutica, and after 28 days in B. humidicola and B. dictyoneura. The impossed drought stress was mild and only the largest grass, B. brizantha, showed reduced (23%) plant yield. The other grasses were able to adjust growth and biomass allocation in response to DT leaving total plant yield relatively unaffected. Brachiaria mutica, had a homogeneous root distribution throughout the soil profile. In the other species more than 80% of root biomass was allocated within the first 30 cm of the soil profile. Brachiaria brizantha and B. decumbens had the lowest proportion of roots below 50 cm. Drought caused a general reduction in root biomass. The shoot:root ratio in B. mutica and B. humidicola increased in response to DT at the expense of a reduction in root yield down to 50 cm depth. Although the total water volume utilized under DT was similar among grasses, the rate of water use was highest (0.25 l day^–1) in B. brizantha, B. decumbens and B. mutica and lowest (0.13 l day^–1) in B. humidicola and B. dictyoneura. In all species leaf expansion was reduced by DT but it was rapidly reassumed after rewatering. Drought increased specific leaf mass (SLM) only in B. brizantha compensating for leaf area reduction, but leaf area ratio (LAR) was unaffected in all species. In almost all grasses DT increased leaf N and K concentration and in vitro dry matter digestibility. The results indicate that B. brizantha, B. decumbens and to a lesser extent, B. mutica are better adapted to short dry periods, whereas B. humidicola and B. dictyoneura are better adapted to longer dry periods.     

Who contributes more to N2O emission during sludge bio-drying with two different aeration strategies,nitrifiers or denitrifiers?

Global warming effects have drawn more and more attention to studying all sources and sinks of nitrous oxide (N₂O). Sludge bio-drying, as an effective sludge treatment technology, is being adopted worldwide. In this study, two aeration strategies (piles I and II) were compared to investigate the primary contributors to N₂O emission during sludge bio-drying through studying the evolution of functional genes involved in nitrification (amoA, hao, and nxrA) and denitrification (narG, nirS, nirK, norB, and nosZ) by quantitative PCR (qPCR). Results showed that the profile of N₂O emission can be divided into three stages, traditional denitrification contributed largely to N₂O emission at stage I (days 1–5), but N₂O emission mainly happened at stage II (days 5–14) due to nitrifier denitrification and NH₂OH accumulation by ammonia-oxidizing bacteria (AOB), accounting for 51.4% and 58.2% of total N₂O emission for piles I and II, respectively. At stage III (days 14–21), nitrifier denitrification was inhibited because sludge bio-drying proceeded mainly by the physical aeration, thus N₂O emission decreased and changed little. The improved aeration strategy availed pile I to reduce N₂O emission much especially at stages II and III, respectively. These results indicated that nitrifier denitrification by AOB and biological NH₂OH oxidation due to AOB made more contribution to N₂O emission, and aeration strategy was crucial to mitigate N₂O emission during sludge bio-drying.

     

Nitrification inhibitors effectively target N2O-producing Nitrosospira spp. in tropical soil

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N₂O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N₂O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N₂O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N₂O-producing AOB Nitrosospira with N₂O, NO₃⁻, NH₄⁺, water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO₃⁻, NH₄⁺ and pH; and AOA Nitrososphaera and NOB Nitrospira with NH₄⁺, which suggests different drivers. These results support the co-occurrence of non-N₂O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N₂O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.     

氮素类型和剂量对寒温带针叶林土壤N2O排放的影响

大气氮沉降输入会增加森林生态系统氮素有效性,进而改变土壤N_2O产生与排放,然而有关不同氮素离子(氧化态NO_3~--N与还原态NH_4~+-N)沉降对土壤N_2O排放的影响知之甚少。以大兴安岭寒温带针叶林为研究对象,构建了3种类型(NH_4Cl、KNO_3、NH_4NO_3)和4个施氮水平(0、10、20、40 kg N hm~(-2)a~(-1))的增氮控制试验,利用流动化学分析仪和静态箱-气相色谱法4次/月测定凋落物层和矿质层土壤无机氮含量、土壤-大气界面N_2O净交换通量以及相关环境因子,分析施氮类型和剂量对土壤氮素有效性、土壤N_2O通量的影响探讨氮素富集条件下土壤N_2O通量的环境驱动机制。结果表明:施氮类型和剂量均显著影响土壤无机氮含量,土壤NH_4~+-N的积累效应显著高于NO_3~--N。施氮一致增加寒温带针叶林土壤N_2O排放,NH_4NO_3促进效应最为明显,增幅为442%-677%,高于全球平均水平(134%)。土壤N_2O通量与土壤温度、凋落物层NH_4~+-N含量正相关,且随着施氮水平增加而增加。结果表明大气氮沉降短期内不会导致寒温带针叶林土壤NO_3~--N大量流失,但会显著促进土壤N_2O的排放。此外,外源性NH_4~+和NO_3~-输入对土壤N_2O排放的促进作用具有协同效应,在未来森林生态系统氮循环和氮平衡研究中应该区分对待。     

晋西北不同年限小叶锦鸡儿灌丛土壤氮矿化和硝化作用

利用PVC管顶盖埋管法研究了晋西北黄土高原区小叶锦鸡儿人工灌丛不同定植年限(5,10,20,30,40a)土壤氮矿化与硝化速率的动态和净矿化与硝化总量。结果表明,⑴小叶锦鸡儿灌丛土壤无机氮主要以NO_-~3-N形式存在,不同生长年限相同月份的土壤硝态氮(NO-3-N)含量分别是铵态氮(NH+4-N)含量的1.5—15.4倍;⑵土壤氮素硝化速率和矿化速率随生长年限延长而加快,30年生时达到高峰,数值达40.2,44.1 mg m~(-2)d~(-1)。从季节性变化看,7—8月份是硝化速率和矿化速率快速增长期,30年生小叶锦鸡儿灌丛土壤硝化速率和矿化速率分别达到86.9,93.1 mg m~(-2)d~(-1),显著高于其它生长年限(P0.05);(3)土壤氮素硝化与矿化总量同样随小叶锦鸡儿生长年限延长而增加,30年生时达到最高,与5年生相比,分别增加了3.7和3.1倍。(4)5—10月份小叶锦鸡儿生长期内,各年限土壤全氮量的2.3%被矿化成无机氮,其中87%最终被转化成NO-3-N形式存在于土体中。     

Dominance of bacterial ammonium oxidizers and fungal denitrifiers in the complex nitrogen cycle pathways related to nitrous oxide emission

Organic compounds and mineral nitrogen (N) usually increase nitrous oxide (N₂O) emissions. Vinasse, a by‐product of bio‐ethanol production that is rich in carbon, nitrogen, and potassium, is recycled in sugarcane fields as a bio‐fertilizer. Vinasse can contribute significantly to N₂O emissions when applied with N in sugarcane plantations, a common practice. However, the biological processes involved in N₂O emissions under this management practice are unknown. This study investigated the roles of nitrification and denitrification in N₂O emissions from straw‐covered soils amended with different vinasses (CV: concentrated and V: nonconcentrated) before or at the same time as mineral fertilizers at different time points of the sugarcane cycle in two seasons. N₂O emissions were evaluated for 90 days, the period that occurs most of the N₂O emission from fertilizers; the microbial genes encoding enzymes involved in N₂O production (archaeal and bacterial amoA, fungal and bacterial nirK, and bacterial nirS and nosZ), total bacteria, and total fungi were quantified by real‐time PCR. The application of CV and V in conjunction with mineral N resulted in higher N₂O emissions than the application of N fertilizer alone. The strategy of vinasse application 30 days before mineral N reduced N₂O emissions by 65% for CV, but not for V. Independent of rainy or dry season, the microbial processes were nitrification by ammonia‐oxidizing bacteria (AOB) and archaea and denitrification by bacteria and fungi. The contributions of each process differed and depended on soil moisture, soil pH, and N sources. We concluded that amoA‐AOB was the most important gene related to N₂O emissions, which indicates that nitrification by AOB is the main microbial‐driven process linked to N₂O emissions in tropical soil. Interestingly, fungal nirK was also significantly correlated with N₂O emissions, suggesting that denitrification by fungi contributes to N₂O emission in soils receiving straw and vinasse application.     

Simulated Nitrogen Deposition Reduces CH4 Uptake and Increases N2O Emission from a Subtropical Plantation Forest Soil in Southern China

To date, few studies are conducted to quantify the effects of reduced ammonium (NH₄ ⁺) and oxidized nitrate (NO₃ ⁻) on soil CH₄ uptake and N₂O emission in the subtropical forests. In this study, NH₄Cl and NaNO₃ fertilizers were applied at three rates: 0, 40 and 120 kg N ha⁻¹ yr⁻¹. Soil CH₄ and N₂O fluxes were determined twice a week using the static chamber technique and gas chromatography. Soil temperature and moisture were simultaneously measured. Soil dissolved N concentration in 0–20 cm depth was measured weekly to examine the regulation to soil CH₄ and N₂O fluxes. Our results showed that one year of N addition did not affect soil temperature, soil moisture, soil total dissolved N (TDN) and NH₄ ⁺-N concentrations, but high levels of applied NH₄Cl and NaNO₃ fertilizers significantly increased soil NO₃ ⁻-N concentration by 124% and 157%, respectively. Nitrogen addition tended to inhibit soil CH₄ uptake, but significantly promoted soil N₂O emission by 403% to 762%. Furthermore, NH₄ ⁺-N fertilizer application had a stronger inhibition to soil CH₄ uptake and a stronger promotion to soil N₂O emission than NO₃ ⁻-N application. Also, both soil CH₄ and N₂O fluxes were driven by soil temperature and moisture, but soil inorganic N availability was a key integrator of soil CH₄ uptake and N₂O emission. These results suggest that the subtropical plantation soil sensitively responses to atmospheric N deposition, and inorganic N rather than organic N is the regulator to soil CH₄ uptake and N₂O emission.     

A bioluminescence assay to detect nitrification inhibitors released from plant roots: a case study with Brachiaria humidicola

A bioluminescence assay using recombinant Nitrosomonas europaea was adopted to detect and quantify natural nitrification inhibitors in plant–soil systems. The recombinant strain of N. europaea produces a distinct two-peak luminescence due to the expression of luxAB genes, introduced from Vibrio harveyi, during nitrification. The bioluminescence produced in this assay is highly correlated with NO₂⁻ production (r ² = 0.94). Using the assay, we were able to detect significant amounts of a nitrification inhibitor produced by the roots of Brachiaria humidicola (Rendle) Schweick. We propose that the inhibitory activity produced/released from plants be termed ‘biological nitrification inhibition’ (BNI) to distinguish it from industrially produced inhibitors. The amount of BNI activity produced by roots was expressed in units defined in terms of the action of a standard inhibitor allylthiourea (AT). The inhibitory effect from 0.22 μM AT in an assay containing 18.9 mM of NH₄⁺ is defined as one AT unit of activity. A substantial amount of BNI activity was released from the roots of B. humidicola (15–25 AT unit g⁻¹ root dry wt day⁻¹). The BNI activity released was a function of the growth stage and N content of the plant. Shoot N levels were positively correlated with the release of BNI activity from roots (r ² = 0.76). The inhibitor/s released from B. humidicola roots suppressed soil nitrification. Additions of 20 units of BNI per gram of soil completely inhibited NO₃⁻ formation in a 55-day study and remained functionally stable in the soil for 50 days. Both the ammonia monooxygenase and the hydroxylaminooxidoreductase enzymatic pathways in Nitrosomonas were effectively blocked by the BNI activity released from B. humidicola roots. The proposed bioluminescence assay can be used to characterize and determine the BNI activity of plant roots, thus it could become a powerful tool in genetically exploiting the BNI trait in crops and pastures.     

Responses to drought of five Brachiaria species. II. Water relations and leaf gas exchange

Neotropical savannas are exposed to recurrent dry periods of varied duration, and forage grasses must be able to cope with such temporal stresses to maintain productive pastures. This study compared leaf water relations and net photosynthesis under drought of five perennial Brachiaria species: the tufted B. brizantha (CIAT 6780), the semi-stoloniferous B. decumbens (CIAT 606) and B. mutica, and the stoloniferous B. humidicola (CIAT 679) and B. dictyoneura (CIAT 6133). Plants of the five grasses were grown in large pots and subjected to drought by suspending watering until first wilting symptoms (14 days for B. brizantha, B. decumbens and B. mutica, and 29 days for B. humidicola and B. dictyoneura). Afterwards, they were re-watered and a second soil dry cycle was imposed. Time trends in leaf water potential (_l), relative water content (RWC), osmotic potential at full turgor (₀ ¹⁰⁰), stomatal conductance (Gs) and net photosynthesis (A) of stressed (DT) plants were compared to those of well-irrigated (CT) plants. Predawn _l in DT plants decreased to a minimum of –1.5 and –2.0 MPa in B. brizantha and B. mutica, compared to –2.5 to –3.0 MPa in B. decumbens, B. humidicola and B. dictyoneura. RWC decreased up to 50% in B. brizantha, compared to 75% in the other species. In B. humidicola, B. dictyoneura and in a lesser extent, B. decumbens, leaves of DT plants adjusted osmotically, by an apparent accumulation of nutrient solutes, at a rather constant ratio of turgid to dry weight of the tissue. Calculated osmotic adjustment ranged between 0.38 (B. decumbens) to 0.87 MPa (B. humidicola). This adjustment in ₀ ¹⁰⁰ was in some cases maintained 7 days after re-watering. In B. brizantha and B. mutica, Gs and A were significantly affected by drought, with maximum reduction percentages at the second drought period of 65 and 80%, respectively. The corresponding reduction in B. decumbens was 53 and 55%, respectively; whereas in B. humidicola and B. dictyoneura Gs and A were reduced less than 20%. In all species, re-watering allowed for the water relations (except ₀ ¹⁰⁰) and photosynthetic activity of leaves of DT plants to reach values comparable to those of CT plants. Results are discussed in term of root morphology and soil water extraction pattern, as well as leaf traits that may contribute to withstand drought under moderate soil water stress.     

Low Nitrification Rates in Acid Scots Pine Forest Soils Are Due to pH-Related Factors

In a previous study, ammonia-oxidizing bacteria (AOB)-like sequences were detected in the fragmentation layer of acid Scots pine (Pinus sylvestris L.) forest soils (pH 2.9–3.4) with high nitrification rates (>11.0 μg g⁻¹ dry soil week⁻¹), but were not detected in soils with low nitrification rates (<0.5 μg g⁻¹ dry soil week⁻¹). In the present study, we investigated whether this low nitrification rate has a biotic cause (complete absence of AOB) or an abiotic cause (unfavorable environmental conditions). Therefore, two soils strongly differing in net nitrification were compared: one soil with a low nitrification rate (location Schoorl) and another soil with a high nitrification rate (location Wekerom) were subjected to liming and/or ammonium amendment treatments. Nitrification was assessed by analysis of dynamics in NH₄ ⁺-N and NO₃ ⁻-N concentrations, whereas the presence and composition of AOB communities were assessed by polymerase chain reaction–denaturing gradient gel electrophoresis and sequencing of the ammonia monooxygenase (amoA) gene. Liming, rather than ammonium amendment, stimulated the growth of AOB and their nitrifying activity in Schoorl soil. The retrieved amoA sequences from limed (without and with N amendment) Schoorl and Wekerom soils exclusively belong to Nitrosospira cluster 2. Our study suggests that low nitrification rates in acidic Scots pine forest soils are due to pH-related factors. Nitrosospira cluster 2 detected in these soils is presumably a urease-positive cluster type of AOB.     

放牧对滇西北高原纳帕海沼泽化草甸湿地土壤氮转化的影响

选择位于滇西北高原纳帕海国际重要湿地内的典型沼泽化草甸湿地为研究对象,采用原位土柱室内控制实验法研究了放牧干扰(猪翻拱扰动和牲畜践踏)对沼泽化草甸湿地土壤氮转化的影响。研究结果表明,放牧活动显著提高了沼泽化草甸湿地表层土壤的容重和pH值,降低了土壤含水率、TOC、TN和NH_4~+-N含量,而对NO_3~--N含量影响不显著。放牧干扰下沼泽化草甸湿地土壤的矿化速率和硝化速率均表现为猪翻拱扰动样地(ZG)牲畜践踏样地(JT)对照样地(CK);表现为ZGJTCK。放牧干扰促进了沼泽化草甸湿地土壤的矿化和硝化作用,猪的翻拱活动比牲畜践踏活动对土壤氮矿化和硝化作用的促进作用更显著。放牧干扰下沼泽化草甸湿地土壤的反硝化速率表现为ZGCKJT,猪的翻拱活动促进了土壤N_2O气体的排放,而牲畜践踏活动抑制了土壤N_2O气体的排放。相关性分析表明,受放牧干扰的沼泽化草甸湿地土壤的矿化和硝化速率均与土壤容重、pH呈显著正相关,与土壤含水率、NH_4~+-N、TOC、TN含量呈显著负相关;反硝化速率与TOC含量呈显著负相关。     

Bacteria on leaves: a previously unrecognised source of N2O in grazed pastures

Nitrous oxide (N₂O) emissions from grazed pastures are a product of microbial transformations of nitrogen and the prevailing view is that these only occur in the soil. Here we show this is not the case. We have found ammonia-oxidising bacteria (AOB) are present on plant leaves where they produce N₂O just as in soil. AOB (Nitrosospira sp. predominantly) on the pasture grass Lolium perenne converted 0.02–0.42% (mean 0.12%) of the oxidised ammonia to N₂O. As we have found AOB to be ubiquitous on grasses sampled from urine patches, we propose a ‘plant'' source of N₂O may be a feature of grazed grassland.In terms of climate forcing, nitrous oxide (N₂O) is the third most important greenhouse gas (Blunden and Arndt, 2013). Agriculture is the largest source of anthropogenic N₂O (Reay et al., 2012) with about 20% of agricultural emissions coming from grassland grazed by animals (Oenema et al., 2005).Grazed grassland is a major source of N₂O because grazers harvest nitrogen (N) from plants across a wide area but recycle it back onto the pasture, largely as urine, in patches of very high N concentration. The N in urine patches is often in excess of what can be used by plants resulting in losses through leaching as nitrate, as N₂O and through volatilisation as ammonia (NH₃) creating a high NH₃ environment in the soil and plant canopy; an important point that we will return to later. The established wisdom is that N₂O is generated exclusively by soil-based microbes such as ammonia-oxidising bacteria (AOB). This soil biology is represented in models designed to simulate N₂O emissions and the soil is a target for mitigation strategies such as the use of nitrification inhibitors.We have previously shown that pasture plants can emit N₂O largely through acting as a conduit for emissions generated in the soil, which are themselves controlled to some degree by the plant (Bowatte et al., 2014). In this case the origin of the emission is still the soil microbes. However, AOB have been found on the leaves of plants, for example, Norway spruce (Papen et al., 2002; Teuber et al., 2007) and weeds in rice paddies (Bowatte et al., 2006), prompting us to ask whether AOB might be present on the leaves of pasture species and contribute to N₂O emissions as they do in soil.We looked for AOB on plants in situations where NH₃ concentrations were likely to be high, choosing plants from urine patches in grazed pastures and plants from pastures surrounding a urea fertiliser manufacturing plant. DNA was extracted from the leaves (including both the surface and apoplast) and the presence of AOB tested using PCR. AOB were present in all the species we examined—the grasses Lolium perenne, Dactylis glomerata, Anthoxanthum odoratum, Poa pratensis, Bromus wildenowii and legumes Trifolium repens and T. subterraneum.To measure whether leaf AOB produce N₂O, we used intact plants of ryegrass (L. perenne) lifted as cores from a paddock that had been recently grazed by adult sheep. The cores were installed in a chamber system designed to allow sampling of above- and belowground environments separately (Bowatte et al., 2014). N₂O emissions were measured from untreated (control) plants and from plants where NH₃ was added to the aboveground chamber and leaves were either untreated or sterilised by wiping twice with paper towels soaked in 1% hypoclorite (Sturz et al., 1997) and then with sterile water. We tested for the presence and abundance of AOB on the leaves by extracting DNA and using PCR and real-time PCR targeting the ammonia monoxygenase A (amoA) gene, which is characteristic of AOB. AOB identity was established using cloning and DNA sequencing. Further details of these experiments can be found in the Supplementary Information.The addition of NH₃ to untreated plants significantly stimulated N₂O emissions (P<0.001) compared with the controls; by contrast, the plants with sterilised leaves produced significantly less N₂O than controls (P<0.001) even with NH₃ added (Figure 1) providing strong evidence for emissions being associated with bacteria on the leaves. Control plants did emit N₂O suggesting there was either sufficient NH₃ available for bacterially generated emissions and/or other plant-based mechanisms were involved (Bowatte et al., 2014).Open in a separate windowFigure 1Effect of an elevated NH₃ atmosphere and surface sterilisation of leaves on leaf N₂O emissions measured over 1-h periods on three occasions during the day. Values are means (s.e.m.), where n=7.The major AOB species identified was Nitrosospira strain III7 that has been previously shown to produce N₂O (Jiang and Bakken, 1999). We measured 10⁹ AOB cells per m² ryegrass leaf, assuming a specific leaf area of 250 cm² g⁻¹ leaf.The rate of production of N₂O (0.1–0.17 mg N₂O-N per m² leaf area per hour) can be translated to a field situation using the leaf area index (LAI)—1 m² leaf per m² ground would be an LAI of 1. LAI in a pasture can vary from <1 to >6 depending on the management (for example, Orr et al., 1988). At LAI of 1, the AOB leaf emission rate would equate to a N₂O emission rate of about 0.1–0.3 mg N₂O-N per m² ground per hour. By comparison, the emission rates measured after dairy cattle urine (650 kg N ha⁻¹) was applied to freely and poorly drained soil were 0.024–1.55 and 0.048–3.33 mg N₂O-N per m² ground per hour, respectively (Li and Kelliher, 2005).The fraction of the NH₃ that was converted to N₂O by the leaf AOB was 0.02–0.42% (mean 0.12%). The mean value is close to that measured for Nitrosospira strains including strain III7 isolated from acidic, loamy and sandy soils where values ranged from 0.07 to 0.10% (Jiang and Bakken, 1999). This is good evidence that the AOB on leaves have the capacity to produce N₂O at the same rate as AOB in soils. We do not suggest that leaf AOB will produce as much N₂O as soil microbes; however, because leaf AOB have access to a source of substrate—volatilised NH₃—that is unavailable to soil microbes and may constitute 26% (Laubach et al., 2013) to 40% (Carran et al., 1982) of the N deposited in the urine, N₂O emissions from these aboveground AOB are additional to soil emissions. Further research is required to identify the situations in which leaf AOB contribute to total emissions and to quantify this contribution.     

The contribution of nitrogen transformation processes to total N2O emissions from soils used for intensive vegetable cultivation

The rapid expansion of intensively farmed vegetable fields has substantially contributed to the total N₂O emissions from croplands in China. However, to date, the mechanisms underlying this phenomenon have not been completely understood. To quantify the contributions of autotrophic nitrification, heterotrophic nitrification, and denitrification to N₂O production from the intensive vegetable fields and to identify the affecting factors, a ¹⁵N tracing experiment was conducted using five soil samples collected from adjacent fields used for rice-wheat rotation system (WF), or for consecutive vegetable cultivation (VF) for 0.5 (VF1), 6 (VF2), 8 (VF3), and 10 (VF4) years. Soil was incubated under 50% water holding capacity (WHC) at 25°C for 96 h after being labeled with ¹⁵NH₄NO₃ or NH ₄ ¹⁵ NO₃. The average N₂O emission rate was 24.2 ng N?kg^?1 h^?1 in WF soil, but it ranged from 69.6 to 507 ng N?kg^?1 h^?1 in VF soils. Autotrophic nitrification, heterotrophic nitrification and denitrification accounted for 0.3–31.4%, 25.4–54.4% and 22.5–57.7% of the N₂O emissions, respectively. When vegetable soils were moderately acidified (pH, 6.2 to ?≥?5.7), the increased N₂O emissions resulted from the increase of both the gross autotrophic and heterotrophic nitrification rates and the N₂O product ratio of autotrophic nitrification. However, once severe acidification occurred (as in VF4, pH?≤?4.3) and salt stress increased, both autotrophic and heterotrophic nitrification rates were inhibited to levels similar to those of WF soil. The enhanced N₂O product ratios of heterotrophic nitrification (4.84‰), autotrophic nitrification (0.93‰) and denitrification processes were the most important factors explaining high N₂O emission in VF4 soil. Data from this study showed that various soil conditions (e.g., soil salinity and concentration of NO ₃ ^- or NH ₄ ⁺ ) could also significantly affect the sources and rates of N₂O emission.     

Comparison of the efficiency of urea,aqueous ammonia and ammonium sulphate as nitrogen fertilizers

Summary Nitrogen-15 labelled urea, aqueous NH₃ and (NH₄)₂SO₄ were applied to soils contained in pots. The fertilizers were injected in 5 cm³ of solution, 3.5 cm below the soil surface, to simulate a fertilizer band in the field. Ryegrass (Lolium perenne) was planted, and several cuttings and roots were harvested. Efficiency was determined as the recovery of fertilizer-N in the plant tissues and soil.Total recovery varied from 94 to 100%. There was no significant difference between the total recovery of the 3 fertilizer forms, although recovery in the soil component was lower for (NH₄)₂SO₄ than for urea or NH₃. There was a significant difference in total recovery between soils due to the soil component. Only small amounts of¹⁵N were not recovered, whereas laboratory experiments reported elsewhere had demonstrated that substantial gaseous losses of N as N₂, N₂O and NO +NO₂ occurred in these soils during nitrification of added NH₃ fertilizer.     

Soil–atmosphere exchange of greenhouse gases in a Eucalyptus marginata woodland, a clover-grass pasture, and Pinus radiata and Eucalyptus globulus plantations

Soils provide the largest terrestrial carbon store, the largest atmospheric CO₂ source, the largest terrestrial N₂O source and the largest terrestrial CH₄ sink, as mediated through root and soil microbial processes. A change in land use or management can alter these soil processes such that net greenhouse gas exchange may increase or decrease. We measured soil–atmosphere exchange of CO₂, N₂O and CH₄ in four adjacent land‐use systems (native eucalypt woodland, clover‐grass pasture, Pinus radiata and Eucalyptus globulus plantation) for short, but continuous, periods between October 2005 and June 2006 using an automated trace gas measurement system near Albany in southwest Western Australia. Mean N₂O emission in the pasture was 26.6 μg N m⁻² h⁻¹, significantly greater than in the natural and managed forests (< 2.0 μg N m⁻² h⁻¹). N₂O emission from pasture soil increased after rainfall events (up to 100 μg N m⁻² h⁻¹) and as soil water content increased into winter, whereas no soil water response was detected in the forest systems. Gross nitrification through ¹⁵N isotope dilution in all land‐use systems was small at water holding capacity < 30%, and under optimum soil water conditions gross nitrification ranged between < 0.1 and 1.0 mg N kg⁻¹ h⁻¹, being least in the native woodland/eucalypt plantation < pine plantation < pasture. Forest soils were a constant CH₄ sink, up to −20 μg C m⁻² h⁻¹ in the native woodland. Pasture soil was an occasional CH₄ source, but weak CH₄ sink overall (−3 μg C m⁻² h⁻¹). There were no strong correlations (R < 0.4) between CH₄ flux and soil moisture or temperature. Soil CO₂ emissions (35–55 mg C m⁻² h⁻¹) correlated with soil water content (R < 0.5) in all but the E. globulus plantation. Soil N₂O emissions from improved pastures can be considerable and comparable with intensively managed, irrigated and fertilised dairy pastures. In all land uses, soil N₂O emissions exceeded soil CH₄ uptake on a carbon dioxide equivalent basis. Overall, afforestation of improved pastures (i) decreases soil N₂O emissions and (ii) increases soil CH₄ uptake.     

秸秆还田对玉米根际氨氧化微生物群落及红壤硝化潜势的影响

为探讨酸性红壤根际氨氧化微生物群落以及硝化作用对不同秸秆还田处理的响应,基于中国科学院鹰潭红壤生态实验站设置的秸秆还田长期试验平台(9年),采用荧光定量PCR和高通量测序技术,研究不同秸秆还田处理(不施肥(CK);氮磷钾肥(NPK);氮磷钾肥+秸秆(NPKS);氮磷钾肥+秸秆猪粪配施(NPKSM);氮磷钾肥+秸秆生物炭(NPKB))下玉米根际土壤氨氧化古菌(ammonia-oxidizing archaea, AOA)和细菌(ammonia-oxidizing bacteria, AOB)丰度和群落结构的变化,揭示了秸秆还田对根际氨氧化微生物群落结构和硝化潜势(potential nitrification activity, PNA)的影响机制。结果发现：相比CK和NPK处理,秸秆还田显著提高了土壤养分含量和硝化潜势,其中有机碳(SOC)、全氮(TN)、全磷(TP)、速效磷(AP)、速效钾(AK)、硝态氮(NO~-₃-N)和铵态氮(NH~+₄-N)含量显著增加,NPKSM处理对土壤肥力提升效果最佳。AOA的硝化潜势显著高于AOB,表明AOA...     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.     

Using nitrification inhibitors to mitigate agricultural N2O emission: a double‐edged sword?

Nitrification inhibitors show promise in decreasing nitrous oxide (N₂O) emission from agricultural systems worldwide, but they may be much less effective than previously thought when both direct and indirect emissions are taken into account. Whilst nitrification inhibitors are effective at decreasing direct N₂O emission and nitrate (NO₃^–) leaching, limited studies suggest that they may increase ammonia (NH₃) volatilization and, subsequently, indirect N₂O emission. These dual effects are typically not considered when evaluating the inhibitors as a climate change mitigation tool. Here, we collate results from the literature that simultaneously examined the effects of nitrification inhibitors on N₂O and NH₃ emissions. We found that nitrification inhibitors decreased direct N₂O emission by 0.2–4.5 kg N₂O‐N ha^?1 (8–57%), but generally increased NH₃ emission by 0.2–18.7 kg NH₃‐N ha^?1 (3–65%). Taking into account the estimated indirect N₂O emission from deposited NH₃, the overall impact of nitrification inhibitors ranged from ?4.5 (reduction) to +0.5 (increase) kg N₂O‐N ha^?1. Our results suggest that the beneficial effect of nitrification inhibitors in decreasing direct N₂O emission can be undermined or even outweighed by an increase in NH₃ volatilization.