氮肥在水稻根际的硝化-反硝化损失的评价

稻田土壤的硝化一反硝化作用可以发生在:(1)土壤表面的氧化层和土表以下的还原层;(2)水稻根际的氧化层和其外的还原层。前者已有很多研究资料,而后者则还缺乏直接的测定。     

稻田反硝化速率测定方法研究进展

反硝化作用是淹水稻田肥料氮损失的主要途径之一。采用合适的反硝化测定方法是开展稻田反硝化作用研究的前提。然而，由于反硝化过程主要产物N₂的大气背景值较高，以及反硝化作用具有高度时空异质性，淹水稻田反硝化作用损失氮量难以准确量化一直是阻碍科学评价稻田气态氮损失的关键难题。本文综述了研究稻田反硝化作用的4种方法（乙炔抑制法、¹⁵N同位素示踪法、密闭培养-氦气环境法和N₂/Ar比值-膜进样质谱法），分析了这些方法各自的优缺点和适用性，并提出了稻田反硝化研究的参考建议，以期推动稻田反硝化的研究。     

区分土壤中硝化与反硝化对N2O产生贡献的方法

土壤是产生N₂O的最主要来源之一。硝化和反硝化反应是产生N₂O的主要机理，由于硝化和反硝化微生物同时存在于土壤中，因而硝化和反硝化作用能同时产生N₂O。N₂O的来源可通过使用选择性抑制剂，杀菌剂以及加入的标记底物确定。通过对生成N₂O反应的每一步分析，主要从抑制反应发生的催化酶和细菌着手，总结了测量区分硝化、反硝化和DNRA反应对N₂O产生的贡献方法。并对¹⁵N标记底物法，乙炔抑制法和环境因子抑制法作了详细介绍。     

一种直接测定硝化—反硝化气体的15N示踪—质谱法

本文对¹⁵N示踪—质谱法的可靠性进行了检验。结果表明,在不同的¹⁵N丰度气体样品的测定中,用两种方法(反硝化作用源的¹⁵N丰度法和气样的¹⁵N丰度法)计得的反硝化损失量基本一致,故建立起来的¹⁵N示踪—质谱法是可靠的。该方法的测定偏差随气样¹⁵N丰度的降低而增大。此外,回收率结果表明,(N₂+N₂O+NO_x)-¹⁵N累积释放量占加入NO₃-¹⁵N量的94.1％。因此,这一方法可用于直接测定氮肥的硝化—反硝化损失的研究中。     

土壤静电场对水稻生长发育的影响

稻田在淹水期间,水层的氧化还原电位明显高于土壤耕层,二者的电位差一般为0.2—0.6伏。于天仁等研究过水稻土淹水期间氧化还原电位在剖面上的分布^[1]。     

水稻生育期内红壤稻田氨氧化微生物数量和硝化势的变化

利用荧光定量PCR(Real-timePCR)技术,通过特异引物检测amoA基因拷贝数分析了水稻不同生育期红壤稻田土壤中氨氧化细菌(Ammonia oxidizing bacteria,AOB)和氨氧化古菌(Ammonia oxidizing archaea,AOA)的数量变化,并测定了土壤潜在硝化势。结果显示:红壤稻田土壤中AOA数量显著高于AOB,二者比例在1.6～120.7之间;红壤稻田根层土中AOA数量显著高于表土,随水稻生长根层和表土中AOA数量均逐渐增加,且根层土中增加幅度更大;在水稻生长前期表土中AOB数量较多,孕穗期后根层土中AOB数量显著增加且高于表土。水稻生长期内土壤潜在硝化势也具有逐渐增加趋势,且根层土潜在硝化势增加幅度更大。根层土中潜在硝化势与AOB和AOA数量均呈显著正相关,而表土中潜在硝化势只与AOA数量存在显著正相关。研究表明,红壤稻田土壤中AOA数量更为丰富,且与硝化作用的关联程度更为密切,证实了氨氧化微生物在红壤稻田土壤微生物组成及其生态系统功能中的重要性。     

水分调控对水稻根际土壤反硝化作用的影响

以水稻农田生态系统为研究对象,采用室内盆栽试验,研究干湿交替、浅水层连续灌溉以及控水3种水分管理模式引起的水分变化对水稻根际土壤反硝化作用过程的影响。结果表明:浅水层连续灌溉模式下的反硝化强度、反硝化速率、反硝化势的平均值为2.19 mg/（kg·d）,118.54 mmol/（m²·d）,28.42 mol/（m²·d）,而干湿交替模式以及控水模式下,反硝化强度平均值仅为连续灌溉模式的64.40%,52.34%,反硝化速率平均值为连续灌溉模式的69.02%,59.73%,反硝化势平均值为连续灌溉模式的77.39%,81.43%,即3种水分管理模式下,水稻根际土壤反硝化强度、反硝化势以及反硝化速率均表现为连续灌溉 > 干湿交替 > 控水模式。随着水稻的生长,3种水分管理模式下的水稻根际土壤反硝化强度、反硝化势以及反硝化速率均呈现递减趋势,表现为分蘖期 > 孕穗期 > 成熟期;相关分析表明,根际土壤反硝化强度、反硝化势及反硝化速率与系统中NO₃^-浓度有显著相关性,由此可见,3种水分模式下,水分及其植物生长导致底物NO₃^-浓度的差异是影响水稻根系土壤反硝化作用过程的因子。     

稻田中西吡的硝化抑制效应

稻田施用氮肥,由于硝化和反硝化作用交替进行,引起脱氮损失或淋失,氮素利用率很低。     

模拟条件下土壤硝化作用及硝化微生物对不同水分梯度的响应

以江苏滨海县一植稻土壤为研究对象,在微宇宙培养条件下设置了不同水分处理(最大持水量的30%、60%、90%和淹水2 cm深),研究了硝化作用及硝化微生物对水分变化的响应特征。结果表明:淹水处理显著降低了土壤的氧化还原电位(Eh),但所有处理土壤Eh变化范围为330~500 m V,土壤整体处于氧化态。在每7天向土壤加入10 mg kg-1NH+4-N的连续培养过程中,各个水分处理均观察到明显的NH+4-N降低和NO-3-N累积的现象,60%WHC处理下土壤硝态氮累积最显著和迅速,90%WHC处理次之,随培养时间延长,30%WHC和淹水处理也观察到明显的硝化作用。淹水处理中氨氧化细菌(Ammonia-oxidizing bacteria,AOB)的数量显著高于非淹水处理,且淹水处理中AOB在DGGE图谱上的条带更加清晰明亮,而氨氧化古菌(Ammonia-oxidizing archaea,AOA)的群落组成和数量在不同水分处理间无明显变化。表明该土壤中AOB对水分条件变化响应灵敏,是该土壤的硝化作用、尤其是淹水条件下硝化作用发生的主要原因。     

水分管理对水稻籽粒硒积累及根际土壤细菌群落多样性的影响

采用盆栽试验方法,研究不同水分管理方式对水稻根际土壤硒组分、籽粒硒积累以及根际土壤细菌群落多样性的影响。结果表明：在水稻的各生育期,好氧和干湿交替较淹水灌溉一定程度上提高了土壤pH和氧化还原电位（Eh）,土壤可溶性和可交换态硒含量增加,从而提高了土壤硒的有效性。水稻成熟后,不同部位的含硒量由高到低依次为根（1.38~2.22 mg·kg^-1）、叶（0.55~0.85 mg·kg^-1）、茎（0.53~0.61 mg·kg^-1）和籽粒（0.15~0.53 mg·kg^-1）。水稻籽粒含硒量以干湿交替灌溉最高,淹水灌溉最低,二者含硒量差异达显著水平。干湿交替灌溉的水稻产量显著高于常规淹水灌溉,且较淹水灌溉提高了7.83%,较好氧灌溉提高13.51%。水稻根际土壤优势菌为变形菌门、绿弯菌门、拟杆菌门、酸杆菌门、Patescibacteria和芽单胞菌门,变形菌门是不同水分管理方式下水稻根际土壤中丰度最高的细菌,水分管理措施显著影响其丰富度,干湿交替和好氧灌溉中变形菌门的丰富度明显高于淹水灌溉。从纲水平看,Gammaproteobacteria的丰度与土壤有效硒含量呈正相关,故Gammaproteobacteria丰度的增加可能是土壤硒生物有效性增加的另一个重要原因。综上,干湿交替灌溉不但能提高水稻产量和稻米硒含量,且较正常淹水管理节约用水,在水稻生产中,是一种优先推荐的水分管理方法。     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

Denitrification losses from puddled rice soils in the tropics

Summary Although denitrification has long been considered a major loss mechanism for N fertilizer applied to lowland rice (Oryza sativa L.) soils, direct field measurements of denitrification losses from puddled rice soils in the tropics have only been made recently. This paper summarizes the results of direct measurement and indirect estimation of denitrification losses from puddled rice fields and reviews the status of research methodology for measurement of denitrification in rice fields. The direct recovery of (N₂+N₂O)-¹⁵N from ¹⁵N-enriched urea has recently been measured at sites in the Philippines, Thailand, and Indonesia. In all 12 studies, recoveries of (N₂+N₂O)-¹⁵N ranged from less than 0.1 to 2.2% of the applied N. Total gaseous N losses, estimated by the ¹⁵N-balance technique, were much greater, ranging from 10 to 56% of the applied urea-N. Denitrification was limited by the nitrate supply rather than by available C, as indicated by the values for water-soluble soil organic C, floodwater (nitrate+nitrite)-N, and evolved (N₂+N₂O)-¹⁵N from added nitrate. In the absence of runoff and leaching losses, the amount of (N₂+N₂O)-¹⁵N evolved from ¹⁵N-labeled nitrate was consistently less than the unrecovered ¹⁵N in ¹⁵N balances with labeled nitrate, which presumably represented total denitrification losses. This finding indicates that the measured recoveries of (N₂+N₂O)-¹⁵N had underestimated the denitrification losses from urea. Even with a probable two-or threefold underestimation, direct measurements of (N₂+N₂O)-¹⁵N failed to confirm the appreciable denitrification losses often estimated by the indirect difference method. This method, which determines denitrification losses by the difference between total ¹⁵N loss and determined ammonia loss, is prone to high variability. Measurements of nitrate disappearance and ¹⁵N-balance studies suggest that nitrification-denitrification occurs under alternate soil drying and wetting conditions both during the rice cropping period and between rice crops. Research is needed to determine the magnitude of denitrification losses when soils are flooded and puddled for production of rice.     

Evidence of N<Subscript>2</Subscript>O emission and gaseous nitrogen losses through nitrification-denitrification induced by rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A greenhouse experiment was conducted with a specially designed apparatus consisting of an upper and lower chamber where the treatment with rice was carried out (treatment 1). The apparatus also had a single chamber where treatment 2, without rice plants, was carried out. The scope of this study was to elucidate the influence of rice plant growth on gaseous N losses as N₂ and N₂O produced by nitrification-denitrification in a flooded soil fertilized with (NH₄)₂SO₄ (with 56.50 atom% ¹⁵N). Gas samples were withdrawn weekly and analyzed for (N₂ + N₂O)-¹⁵N losses by mass spectrometer and for N₂O by gas chromatograph. The gaseous (N₂ + N₂O)-¹⁵N losses of the treatment with rice plants were significantly (P =0.01) higher than those of the treatment without rice plants, as were the amounts of N₂O emitted. Rice plants facilitate the efflux of N₂ and N₂O from soil to atmosphere, as about half of the total gaseous ¹⁵N loss as N₂ and N₂O was found in the upper chamber. The proportion of N₂O-¹⁵N to (N₂ + N₂O)-¹⁵N in the upper chamber was 10.56%, much higher than that of the lower chamber in treatment 1 and the headspace of treatment 2.     

Influence of carbon substrates and moisture regime on nitrogen fixation in paddy soils

The influence of several carbon sources on heterotrophic N₂ fixation in four paddy soils under flooded and nonflooded conditions was investigated by ¹⁵N-tracer technique. Greater N₂ fixation occurred in submerged soils amended with cellulose and rice straw, the former being superior. Addition of sucrose, glucose and malate in that order stimulated N₂ fixation in submerged alluvial soil, while sucrose alone enhanced N₃ fixation in laterite soil. In submerged acid soils none of these C sources stimulated N₂ fixation. Nonflooded conditions favoured N₂ fixation in alluvial and acid saline soils amended with cellulose, sucrose and glucose.     

Measurement of coupled nitrification-denitrification in paddy fields affected by Terrazole,a nitrification inhibitor

Coupled nitrification-denitrification and potential denitrification were measured as ¹⁵N₂O and ¹⁵N₂ evolution rates in ammonium sulphate-treated rice soils with or without Terrazole [5-ethoxy-3 (trichloromethyl) 1,2,3 Thiadizole] under laboratory and field conditions. The greatest coupled nitrification-denitrification activity was found after drying and rewetting the soil, with maximum values of 322 ng N cm^–2 h^–1 in the laboratory and 90.8 ng N cm^–2 h^–1 in the field. These ¹⁵N₂O + ¹⁵N₂ evolution rates were about 10 times lower than potential denitrification in these soils. These results and the observed decrease in ¹⁵N₂O + ¹⁵N₂ evolution rate in soils treated with Terrazole (60% under laboratory conditions and 52% under field conditions) indicate that denitrification was limited by coupled nitrification-denitrification activity. Oxygen and previous addition of ammonium sulphate appear to control the rate of ¹⁵N₂O + ¹⁵N₂ evolution in ammonium sulphate-fertilised soils.     

Influences of organic debris and rice root on the nitrogen fixation in the submerged soil

Abstract

A series of experiments has been conducted on the N2 fixation in the paddy soils by the authors (1–4). The amount of organic substrates for microorganisms and the degree of reduction of the soil are found to be two major factors affecting the N₂-fixing activity of the heterotrophic microorganisms in the submerged soil. Organic debris, rice root and their neighboring soils are identified to be the important micro-sites for the heterotrophic N₂ fixers. The organic debris and the rice root are considered to play dual roles by supplying the organic substances; (1) increase of the population of the heterotrophic N₂ fixers—the amount of nitrogenase, (2) preparation of the reduced conditions favorable for the nitrogenase activity.

However, it is not yet clearly known which of these two roles of the organic substrates is more essential to the N₂-fixing activity in the paddy soil. In addition, it is expected that there must be some differences between the organic debris and rice root in their contribution to the N₂ fixation in the paddy soil.

An experiment was carried out to clarify these problems. Moist soil sample was collected from the plough layer of the paddy field at Central Agric. Exp. Sta. in Konosu City, Saitama Pref., passed through a 5 mm sieve and placed in pots (3 kg moist soil/pot). Ammonium sulfate, calcium superphosphate, and potassium chloride at the rate of 0.4-0.4-0.4 (N-P₂O₅-K₂0) g/pot were incorporated into the soil 7 days before transplanting. Split application of ammonium sulfate at the rates of 0.2 and 0.4 g N/pot were also incorporated at 30 and 41 days after transplanting respectively. These pots were divided into three series; planted (P-series), non-planted (N-series), and non-planted and applied with organic manure (O-series). In case of O-series, 60 g of fairly rotted organic manure was applied to each pot. Each pot of P-series was planted with two 4O-day-old seedlings of rice plant at 7 days after submergence. The Nseries was regarded as a control. Each series was not replicated in this preliminary experiment.     

Evolution of dinitrogen and nitrous oxide from the soil to the atmosphere through rice plants

Summary It is commonly assumed that a large fraction of fertilizer N applied to a rice (Oryza sativa L.) field is lost from the soil-water-plant system as a result of denitrification. Direct evidence to support this view, however, is limited. The few direct field, denitrification gas measurements that have been made indicate less N loss than that determined by ¹⁵N balance after the growing season. One explanation for this discrepancy is that the N₂ produced during denitrification in a flooded soil remains trapped in the soil system and does not evolve to the atmosphere until the soil dries or is otherwise disturbed. It seems likely, however, that N₂ produced in the soil uses the rice plants as a conduit to the atmosphere, as does methane. Methane evolution from a rice field has been demonstrated to occur almost exclusively through the rice plants themselves. A field study in Cuttack, India, and a greenhouse study in Fort Collins, Colorado, were conducted to determine the influence of rice plants on the transport of N₂ and N₂O from the soil to the atmosphere. In these studies, plots were fertilized with 75 or 99 atom % ¹⁵N-urea and ¹⁵N techniques were used to monitor the daily evolution of N₂ and N₂O. At weekly intervals the amount of N₂+N₂O trapped in the flooded soil and the total-N and fertilized-N content of the soil and plants were measured in the greenhouse plots. Direct measurement of N₂+N₂O emission from field and greenhouse plots indicated that the young rice plant facilitates the efflux of N₂ and N₂O from the soil to the atmosphere. Little N gas was trapped in the rice-planted soils while large quantities were trapped in the unplanted soils. N losses due to denitrification accounted for only up to 10% of the loss of added N in planted soils in the field or greenhouse. The major losses of fertilizer N from both the field and greenhouse soils appear to have been the result of NH₃ volatilization.     

Liming reduces nitrogen uptake from chemical fertilizer but increases that from straw in a double rice cropping system

Liming materials are widely applied to alleviate soil acidification and increase rice yield in acidic soils, but their effects on nitrogen (N) use efficiency are still unclear. Here, we conducted a field-, pot-, and micro-plot experiment to investigate how the application of slaked lime (i.e., Ca(OH)₂) affects the fate of chemical fertilizer-N and straw-N in a double rice cropping system. In the field experiment, liming increased grain yield and N uptake by an average of 9.0% and 10.6%, respectively. In contrast, CaCl₂ application did not affect rice yield and N uptake, suggesting that the effects of lime application were not related to the addition of Ca²⁺. Results from a ¹⁵N tracer experiment (i.e., ¹⁵N-labeled urea and straw) indicated that liming reduced N uptake from fertilizer (−5.7%), but increased N uptake from straw (+31.3%). Liming also reduced soil retention of both urea- and straw-N and increased their loss rates. Taken together, our results indicate that although liming increases rice yield and N uptake, it lowers the use efficiency of fertilizer N and facilitates N losses. In addition, our results emphasize the need for long-term studies on the impact of liming on soil N dynamics in paddy soils.     

Fate of 15N Derived from Composts and Urea In Soils under Different Long-term N Management In Pot Experiments

Understanding of dynamics of N derived from organic N sources in soil is required for the development of sustainable agricultural systems. The aim of this paper is to compare, using pot experiments, the fate of N from urea (UF) and organic N sources such as rice straw compost (RC) and cattle compost (CC) using ¹⁵N labeled materials in paddy soil planted with rice. Two soils with a history of long-term applications of chemical fertilizers (LTCN) and organic N sources, i.e. straw compost +soybean cake, (LTON), were also compared. Nitrous oxide emissions were monitored during the growing period. Yield and N uptake of rice were higher in LTON soil than LTCN soil with no significant interaction with N applications. Chemical fertilizer increased yield and N uptake with a recovery rate by rice of 36 to 45%. Nitrogen recovery from RC and CC by rice was less than 10%. When recovery of N in soil was included with that recovered in the plant, 70% and 61% of applied N in the UF treatment was recovered from the LTCN and LTON soils, respectively. In comparison, more than 95% of applied N was recovered in the plant and soil for the RC and CC treatments. There was a sharp increase in N₂O emission during the aerated period in nonplanted pots regardless of whether supplemental N was added, and this was associated with the increase in NO₃- in soil solution at 0.5 cm depth. There was a much lower N₂O emission in planted pots than nonplanted pots with no significant difference among the LTCN and LTON soils or the N treatments. The results indicated that the application of organic N source provided lower N supply to the plant than urea, but also could reduce N loss because of higher retention in the soil. Long-term continuous application of organic N sources enlarged the labile N pool without increasing N₂O emission. Nitrous oxide emission was important during the mid-season aerated period from pot experiments and was partly related to the concentration of NO₃- and the rate of nitrification.     

Application of membrane inlet mass spectrometry to directly quantify denitrification in flooded rice paddy soil

Denitrification has long been considered a major mechanism of N loss when N fertilizer is applied to flooded rice paddies. However, the direct determination of denitrification in soils is almost impossible because of the high atmospheric background of dinitrogen (N₂). Dissolved N₂ in a small water sample can be rapidly and precisely measured through membrane inlet mass spectrometry (MIMS). This study is the first to directly measure N₂ flux through MIMS in flooded rice paddy plots that received different amounts of urea. Ammonia (NH₃) volatilization was measured simultaneously to verify whether NH₃ volatilization and denitrification are complementary loss mechanisms. The average cumulative N₂–N loss measured by MIMS 21 days after fertilization was 4.7?±?1.7 % of the applied N, which was within the range of the reported values obtained by cumulative recovery of (N₂ + N₂O)–¹⁵N and ¹⁵N-balance technique. Underestimation or overestimation of denitrification can be prevented in MIMS given that N₂ can be measured directly without ¹⁵N-labeled fertilizer. A good positive correlation was found between the dissolved in situ N₂ concentrations of floodwater and the denitrification rates of intact soil cores. Urea incorporation reduced NH₃ volatilization unlike surface broadcasting. However, urea incorporation significantly increased cumulative N₂–N loss during the 21 days after fertilization. Correlation analysis showed that nitrate (NO₃ ^?–N) concentration in floodwater could be the primary restricting factor for soil denitrification in the experimental field. Results suggest that MIMS is a promising technique for the measurement of denitrification in a flooded rice paddy.