Soil N2O and N2 emissions during anaerobic soil disinfestation period in a greenhouse vegetable production system: quantified by in situ 15N labeling method

Soil N2O and N2 emissions during anaerobic soil disinfestation period in a greenhouse vegetable production system: quantified by in situ 15N labeling method | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Soil N2O and N2 emissions during anaerobic soil disinfestation period in a greenhouse vegetable production system: quantified by in situ 15N labeling method Xue Li, Jin Li, Yingying Wang, Ronghua Kang, Keping Sun, Kai Huang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4091615/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Oct, 2024 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Background and Aims: Greenhouse vegetable production (GVP) is expanding worldwide. The high application of nitrogen (N) fertilizers has caused soil diseases and nitrate residue. Farmers usually adopt anaerobic soil disinfestation (ASD), involving organic carbon addition, extensive irrigation, plastic films laying, and greenhouse sealing during the summer fallow. These conditions may promote denitrification, causing nitrous oxide (N 2 O) and dinitrogen (N 2 ) emissions. However, this is rarely reported. Methods: We used ¹⁵N labeling for in situ monitoring of N₂O and N₂ emissions during ASD in a GVP system in Shouguang, Northern China. Two treatments were implemented: conventional organic fertilization (Fertilizer) and a control (No-fertilizer), with continuous monitoring over 14 days. Results: Within 14 days, cumulative gaseous N emissions in Fertilizer and No-fertilizer treatments were 0.82, 0.47 kg N ha -1 for N 2 O, and 40.7 and 25.5 kg N ha -1 for N 2 , respectively. Organic fertilization significantly increased N 2 O and N 2 emission. From days 1–6, the predominant gaseous N was N 2 , with an N 2 O/ (N 2 O + N 2 ) ratio (R N2O ) between 0.007 and 0.015. From days 7–14, N 2 O proportion increased, with R N2O ranging from 0.21 to 0.75. Isotopic information showed that denitrification contributed to 48.9%–51.2% and 27.1%–36.7% of total N 2 O and N 2 emissions. The structural equation model showed that high soil temperature during ASD significantly reduced N 2 O emissions. Conclusion: Our findings emphasize the importance of N 2 emissions in N loss and provide a basis for studying the fate of N, as well as developing measures to reduce N 2 O emissions within GVP systems. 15N labeling greenhouse vegetable anaerobic soil disinfestation dinitrogen emission in situ N2O/ (N2O + N2) ratio Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Global greenhouse vegetable production (GVP) systems are rapidly expanding, with an area exceeding 4 million ha in China, accounting for over 80% of the global total (Fei et al., 2018 ; Qasim et al., 2021 ). For maximizing yield and profits, GVP systems in China are often overfertilized (Agostini et al., 2010 ). Surveys indicate that the annual nitrogen (N) fertilizer application rates in China’s GVP systems typically exceed 2000 kg N ha − 1 , with some regions surpassing 3000 kg N ha − 1 (e.g., Guo et al., 2016; Ju et al., 2006 ; Liu et al., 2008 ; Yang et al., 2016 ; Yu et al., 2010 ), leding to soil acidification (Guo et al., 2010 ; Lv et al., 2020 ), soil-borne diseases (Huang et al., 2016 ), and a significant surplus of N (Ju et al., 2004 , 2006 ). Studies have shown that the residual nitrate (NO 3 − ) in the 2 m soil layer after crop harvesting in GVP systems exceeded 500 kg N ha − 1 (Chen et al., 2004 ; Zhou et al., 2006 , 2010 ). To address soil degradation, anaerobic soil disinfestation (ASD) is widely adopted during the summer fallow period (Di Gioia et al., 2016 ; Zhao et al., 2021b ). This approach involves saturating the soil through irrigation, covering the soil with plastic film, and adding organic carbon (C) (Di Gioia et al., 2017 ). Additionally, greenhouses are sealed to enhance the internal temperature and accelerate the decomposition of organic C, creating an anaerobic environment and eliminating soil diseases and pests (Momma et al., 2013 ; Zhu et al., 2012 ). During ASD, the high-temperature and anaerobic environment favor the reduction of accumulated NO₃⁻ in the soil through denitrification, resulting in gaseous N, including N₂O and N₂ emissions (Charles et al., 2017 ). N 2 O is a potent greenhouse gas that has been identified as a major contributor to stratospheric ozone depletion (IPCC, 2021 ). By contrast, N 2 is an inert, environmentally benign gas; however, its emission represents the final step of N cycling in terrestrial ecosystems. This process is crucial for closing N cycles and maintaining N balance in ecosystems. If the gaseous N is primarily N 2 O, it will accelerate global warming and ozone layer degradation, whereas if it is primarily N 2 , it will be environmentally neutral. Studies on N 2 O emissions from GVP systems have mainly focused on the growing season, with relatively limited research on the microbial processes involved (e.g., Liu & Qiu, 2016; Min et al., 2021 ; Yao et al., 2019 ; Zhang et al., 2019 ). For N 2 , the difficulties in directly measuring soil N 2 emissions (Groffman et al., 2006 ) have resulted in the absence of in-situ research measuring N 2 emission rates and distinguishing the associated microbial processes within the GVP system. This absence contributes to considerable uncertainty when assessing N loss pathways in the GVP system and estimating N 2 emissions in specific regions. Commonly used methods for measuring N 2 emissions include the acetylene inhibition method (Yoshinari et al., 1997 ), gas flow soil core method (Butterbach-Bahl et al., 2002 ; Wang et al., 2011 , 2013 ), and 15 N labeling method (Morse & Bernhardt, 2013 ; Yang et al., 2014 ). However, the incomplete inhibition of reductases by using the acetylene inhibition method can lead to underestimated N 2 emissions. (Felber et al., 2012 ; Qin et al., 2012 ; Yu et al., 2010 ). The gas flow soil core method requires a long duration to create an N 2 -free environment, often exceeding 20 h, during which N 2 emissions cannot be quantified (Butterbach-Bahl et al., 2002 ; Wang et al., 2011 ). Among these methods, only the 15 N labeling technique enables the in-situ measurement of N 2 emissions and differentiation of microbial processes responsible for gaseous N production (Stevens et al., 1997 ). This method involves adding a certain amount of 15 N-enriched nitrogenous compounds to the soil and analyzing their fate after a certain period, achieving tracing and source analysis purposes (Templer et al., 2012 ). With the development of stable isotope technology, improvements in instrument detection techniques, and cost reduction, the 15 N labeling method has become widely used (He et al., 2011 ; Kulkarni et al., 2014 ; Li et al., 2002 ; Sgouridis & Ullan, 2015; Yang et al., 2014 ). Additionally, studies have used the laboratory-derived ratio of N 2 O / (N 2 O + N 2 ) (R N2O ) along with in situ measured N 2 O flux to estimate N 2 flux (Saggar et al., 2013 ; Wang et al., 2020 ). However, owing to the complexity of field environments, the R N2O obtained in the laboratory can not represent the actual values in fields (Kulkarni et al., 2015 ; Well et al., 2019 ). The range of R N2O in terrestrial ecosystems is wide (Schlesinger, 2009 ), but in situ measurements of R N2O in GVP systems have not been conducted. Thus, whether the R N2O values in the laboratory are applicable for estimating in situ N 2 emissions from GVP systems remains to be verified. In this study, we conducted in situ measurements of soil N 2 O and N 2 emissions during the ASD period in a typical GVP system in Shouguang, Shandong Province, China, using the 15 N labeling method. The research objectives were to (1) explore the emission characteristics of N 2 O and N 2 , (2) distinguish the microbial processes responsible for N 2 O and N 2 production, (3) analyze the influence of environmental factors on N 2 O and N 2 emissions, and (4) evaluate the fate of N in the soil. 2. Materials and Methods 2.1 Study Site The experimental site was located in Zhaili Village, Luocheng Street, Shouguang City, Shandong Province, China (E118°42' 4.5″, N 36°55' 26.4″), an area with a warm, temperate, continental monsoon climate. The average temperature in 2021 was 14.4°C, with precipitation of 895.4 mm and 2306.9 h of sunshine (Shouguang Statistical Yearbook, 2021). The soil in this area is classified as a Cambisol, with organic C, nitrate N, and ammonium N contents of 16.6 g C kg − 1 , 44.4 mg kg − 1 , and 6.7 mg kg − 1 , respectively, in the 0–10 cm soil layer, and a pH value of 7.7. The experiments were conducted in a greenhouse that had been in use for five years and was oriented in an east-west direction. Typically, in this greenhouse, two seasons of crops, tomatoes and cucumbers, had been grown each year. Farmers usually close greenhouses to conduct ASD during the summer fallow period in July and August. 2.2 Field 15 N Labeling Experiment The period of the experiment was from July 20, 2021, to August 2, 2021. The experiment used two treatments: regular fertilization (Fertilizer) and no fertilization control (No-fertilizer), with five replicates for each treatment. In the Fertilizer treatment, commercial organic fertilizer was uniformly applied on July 19, 2021, at a rate of 280 kg N ha − 1 and 2829 kg C ha − 1 . After fertilization, both treatment areas were plowed and drip-irrigated. Plastic film was used to cover the soil after irrigation to prevent water evaporation. Subsequently, ten gas-sampling static chambers were randomly placed, with five in the Fertilizer area and five in the No-fertilizer treatment area. The static chambers were 30 cm long, 30 cm wide, and 10 cm high, with a water-filled groove (3 cm wide, 3 cm deep) around the base. There was a three-way valve at the top for collecting the gas samples. A grid with 36 small compartments was placed inside the static chamber, each measuring 5 cm × 5 cm, to facilitate the addition of K 15 NO 3 . A solution of K 15 NO 3 (99 15 N atom%) at a rate of 0.3 g (equivalent to 33 kg N ha − 1 ) was uniformly injected into each static chamber. An equal-sized static chamber base was placed next to each gas-sampling static chamber to collect the soil samples. The same method was used to inject equal amounts of K 15 NO 3 into the soil sample base (Fig S1). After the soil was labeled, gas samples were collected three times every day. Because of the high daytime temperature inside the greenhouse, which was not conducive to sampling, gas collection times were set at 5:00, 6:00, and 19:30 daily for 14 d to obtain detectable 15 N-N 2 signals. Before the first sampling each day, the chambers were placed on the bases, and the grooves of the bases were sealed with water. The chamber was opened after the third sampling period. During sampling, a 100 mL syringe was connected to the three-way valve outlet at the top of the chamber, the three-way valve was opened, and the syringe plunger was moved five times to evenly mix the gases inside the chamber. Subsequently, 100 mL gas was collected using a syringe, and the three-way valve was switched to allow the collected gas to be transferred into a 150 mL gas bag. Balancing the pressures inside and outside the chamber was achieved by injecting 100 mL air into the chamber after gas collection. Additionally, soil samples from the 0–10 cm layer were collected every 2 d (Fig S1). We used soil temperature and moisture probes (TMS4, TOMST, Czech Republic) to monitor soil (5 cm depth), temperature (°C), and moisture (volumetric water content, %) simultaneously. The soil volumetric water content was further converted to soil water-filled pore space (WFPS) by using the following formula: WFPS = [soil volumetric water content / (1 - (soil bulk density / 2.65)] × 100%, where 2.65 g cm − 3 is the assumed soil particle density, and soil bulk density is 1.4 g cm − 3 . 2.3 Analysis of N 2 O and Flux Calculation We measured N 2 O concentration by using a gas chromatograph (GC2014, Shimadzu, Japan). The N 2 O flux (F N2O , µg m − 2 h − 1 ) was calculated based on the linear change in N 2 O concentration over time using the following formula: F N2O = (dc/dt) × ρN 2 O × V/A (1) In Eq. (1), the N 2 O flux comprises flux from both the 15 N-labeled source and unlabeled sources; dc/dt is the rate of change in N 2 O concentration over time determined using linear regression; ρN 2 O is the density of N 2 O at standard conditions; and V and A are the volume (0.009 m 3 ) and base area (0.09 m 2 ) of the static chamber, respectively. The 15 N abundance of N 2 O was determined using a combined system comprising a continuous-flow isotope ratio mass spectrometry (IRMS, IsoPrime 100, Cheadle, UK), low-temperature focusing unit (Trace Gas Preconcentrator, IsoPrince Limited), and 112-position autosampler (Gilson GX-271, Dunstable, UK). The peak areas of 44 N 2 O, 45 N 2 O, and 46 N 2 O and the ratios 45 R ( 45 N 2 O/ 44 N 2 O) and 46 R ( 46 N 2 O/ 44 N 2 O) were measured using IRMS. Studies have indicated that in high-abundance 15 N labeling experiments, isotope fractionation can be ignored (Yang et al., 2014 ). Therefore, the 15 N abundance in N 2 O was calculated using formula (2), assuming 17 R ( 17 O/ 16 O) = 3.8861×10 − 4 and 18 R ( 18 O/ 16 O) = 2.0947×10 − 3 . The 15 N 2 O flux (F 15 N 2 O) was estimated from the difference in 15 N abundance in the samples collected at different times and the total N 2 O flux (Eq. 3). Eq. 4 was used to estimate the N 2 O flux produced by denitrification processes (Buchen et al., 2016 ; Yang et al., 2014 ). \({ }^{15}{\text{X}}_{\text{N}2\text{O}}\) = 100 \(\times \frac{{ }^{45}\text{R} + 2 \times { }^{46}\text{R} -{ }^{17}\text{R}-2 \times { }^{18}\text{R}}{2 + 2 \times { }^{45}\text{R} + 2 \times { }^{46}\text{R}}\) (2) F 15 N 2 O= \(\frac{{\text{C}}_{\text{N}2\text{O}\text{t}}{\times }^{15}{{\text{X}}_{\text{N}2\text{O}\text{t}}}^{ }- {\text{C}}_{\text{N}2\text{O}0} {\times }^{15}{\text{X}}_{\text{N}2\text{O}0}}{{\text{C}}_{\text{N}2\text{o}\text{t} -} {\text{C}}_{\text{N}2\text{O}0}}\) \(\times {\text{F}}_{\text{N}2\text{O}}\) (3) where \({\text{C}}_{\text{N}2\text{O}0}\) and \({\text{C}}_{\text{N}2\text{O}\text{t}}\) represent the N 2 O concentrations at 5:00 and 19:30, respectively, in the gas samples collected daily, and 15 \({{\text{X}}_{\text{N}2\text{O}0}}^{ }\) and 15 \({{\text{X}}_{\text{N}2\text{O}\text{t}}}^{ }\) represent the 15 N abundances in the N 2 O samples at 5:00 and 19:30, respectively. FN 2 O denitrification = \(\frac{{\text{F}}^{15}{\text{N}}_{2}\text{O}}{{ }^{15}\text{X}{ }_{{{\text{N}\text{O}}_{3}}^{-}}}\) (4) Where \({ }^{15}\text{X}{ }_{{{\text{N}\text{O}}_{3}}^{-}}\) represents the 15 N abundance of NO 3 − in the soil. 2.4 Analysis of N 2 and Flux Calculation 15 N abundance in N 2 was determined using the aforementioned trace gas pre-concentration system and isotope ratio mass spectrometry (TG-IRMS). The peak areas of 28 N 2 , 29 N 2 , and 30 N 2 , as well as the ratios 29 R ( 29 N 2 / 28 N 2 ) and 30 R ( 30 N 2 / 28 N 2 ) in the sample, were measured using a mass spectrometer. The 15 N mole fraction ( 15 X N2 ) and 15 N flux (F 15 N 2 ) of N 2 were calculated using Equations (5) and (6), respectively. Subsequently, the N 2 flux produced by denitrification (FN 2 -denitrification) was calculated using Eq. (7). Assuming that all N 2 was derived from the reduction of N 2 O, total N 2 flux (FN 2 -total) was estimated using Eq. (8) (Yang et al., 2014 ). 15 X N2 = \(\frac{{ }^{29}\text{R} + 2{\times }^{ 30}\text{R}}{2 + 2 {\times }^{ 29}\text{R} + 2 {\times }^{30}\text{R}}\) (5) F 15 N2 = (d 15 X N2 /dt) × ρN 2 × V/A (6) ρN 2 is the density of N 2 at standard conditions; V and A are the volume (0.009 m 3 ) and base area (0.09 m 2 ) of the static box. FN 2 − denitrification = \(\frac{{\text{F}}^{15}{\text{N}}_{2}}{{ }^{15}\text{X}{ }_{{{\text{N}\text{O}}_{3}}^{-}}}\) (7) FN 2 − total = \(\frac{{\text{F}}^{15}{\text{N}}_{2}}{{ }^{15}\text{X}{ }_{\text{N}2\text{O}}}\) (8) 2.5 Soil Parameter Analysis The soil samples were sieved through a 2 mm mesh. A portion of the sieved soil was used to determine the total N (TN) content by using an elemental analyzer (Elementar Analysen Systeme, GmbH, Germany), and another 10 g soil was extracted with 100 ml 2 M KCl solution, and the mixture was shaken for 1 h. The extract was frozen for preservation and analyzed for ammonium nitrogen (NH 4 + -N), nitrate nitrogen (NO 3 − -N), and nitrite nitrogen (NO 2 − -N) content by using a fully automatic discrete chemical analyzer (Smartchem 200, Westco Scientific Instruments, Inc., Italy). The soil total organic carbon (TOC) content was determined using a TOC/TN analyzer (Analytic Jean, Germany). The 15 N enrichment of NO 3 − in the soil extract was determined using the denitrifying bacterial method with pseudomonas aureofaciens . The specific method was activating the bacterial strain in 50 mL culture medium, transferring it to a 500 mL Erlenmeyer flask with fresh culture medium, and cultivating it at a constant temperature of 28°C for 4 d to amplify the denitrifying bacteria and deplete the NO 3 − . The bacterial liquid was then purified by centrifugation at 15°C and 4000 rpm, and the resulting colonies were re-cultured, mixed, and dispensed into clean headspace vials, each containing approximately 3–6 mL of bacterial liquid. The vials were promptly sealed and purged with high-purity N 2 to remove any N 2 O produced from background NO 3 − in the culture medium. Soil extract samples were then added to headspace vials and allowed to react overnight at 28°C to ensure the complete conversion of NO 3 − to N 2 O. The N 2 O generated from the conversion was measured for 15 N enrichment by using TG-IRMS. 2.6 Statistical Analyses This study used R (4.1.1) and RStudio for statistical analysis and data visualization. A piecewise structural equation model (SEM) was constructed to assess the direct and indirect effects of key factors on N 2 O, N 2 , and R N2O . Piecewise, the SEM also reported a marginal R 2 (R 2 M, the proportion of variance explained by fixed effects). The analysis was conducted using the "piecewiseSEM" package (Lefcheck, 2016), with linear mixed-effects models matching the "lme" function of the "nlme" package. Four indicators were selected to represent a good model fit: low Fisher’s C value, non-significant P values (> 0.05), low Akaike Information Criterion (AIC), and Bayesian Information Criterion (BIC) values. All data analyses were performed at a significance level of P < 0.05. 3. Results 3.1 Soil Physical-chemical Parameters During the experiment, the soil temperature at a depth of 5 cm ranged between 30°C and 45°C, with an average of 39.1°C (Fig. 1 a). The mean WFPS in the 0–10 cm soil layer was 96% for the Fertilizer treatment and 100% for the No-fertilizer treatment (Fig. 1 b). In the Fertilizer treatment, the average concentrations of NO 3 − , NO 2 − , NH 4 + , and TOC in the 0–10 cm soil were 56.6, 0.2, and 8.6 mg N kg − 1 and 79.5 mg C kg − 1 , respectively, and in the No-fertilizer treatment, these concentrations were 58.0, 0.2, and 6.9 mg N kg − 1 and 60.8 mg C kg − 1 (Fig. 1 c, d, e, f). 3.2 Soil N 2 O and N 2 Emissions On day 1 of the experiment, the N 2 O fluxes for the Fertilizer and No-fertilizer treatments reached peak values, 0.42 and 0.18 kg N ha − 1 d − 1 , respectively. Subsequently, the N 2 O fluxes decreased and remained relatively low from days 2 to 14. During this period, the N 2 O fluxes under the Fertilizer treatment ranged from 0.02 to 0.06 kg N ha − 1 d − 1 , averaging 0.03 kg N ha − 1 d − 1 , and under the No-fertilizer treatment, from 0.02 to 0.03 kg N ha − 1 d − 1 , averaging 0.02 kg N ha − 1 d − 1 (Fig. 2 ). During the 14 days period, the total N 2 O emissions for the Fertilizer and No-fertilizer treatments were 0.82 and 0.47 kg N ha − 1 , with associated 15 N 2 O emissions totaling 0.14 and 0.07 kg N ha − 1 , respectively (Fig. 3 ). In the Fertilizer treatment, the peak N 2 flux was on day 2 (12.4 kg N ha − 1 d − 1 ), gradually decreasing to an average of 2.9 kg N ha − 1 d − 1 . In the No-fertilizer treatment, the N 2 flux gradually increased after the start of the experiment, peaked on day 3 (6.9 kg N ha − 1 d − 1 ), and then gradually decreased to an average of 1.8 kg N ha − 1 d − 1 . Over the 14 d period, the total N 2 emissions for the Fertilizer and No-fertilizer treatments were 40.7 and 25.5 kg N ha − 1 , with the associated 15 N 2 emissions totaling 4.3 and 1.9 kg N ha − 1 , respectively (Fig. 3 ). 3.3 N 2 O/(N 2 O + N 2 ) ratio During the experiment, the average R N2O for the Fertilizer and No-fertilizer treatments was 0.28 and 0.32, respectively, with no significant difference between the treatments except on day 13. In the first 6 days, both treatments exhibited low R N2O values, with the Fertilizer treatment ranging from 0.007 to 0.015 (average 0.024) and the No-fertilizer treatment ranging from 0.006 to 0.087 (average 0.026). On day 7, R N2O began to increase. From days 7 to 14, R N2O levels ranged from 0.21 to 0.75 (average 0.46) for the the Fertilizer treatment and from 0.29 to 0.73 (average 0.53) for the No-fertilizer treatment. 3.4 15 N Atom%-excess and Biological Processes During the experiment, sufficient labeled 15 NO 3 − substrate was present in the soil for both treatments. The 15 N abundance of NO 3 − in the Fertilizer treatment decreased gradually from 34.4–7.2%, and that in the No-fertilizer treatment decreased from 30.4–9.6%. From days 1 to 3, the 15 N abundance of NO 3 − in the Fertilizer treatment was higher than that in the No-fertilizer treatment but decreased to below that of the No-fertilizer treatment in subsequent days. Similar trends were observed for the 15 N abundance of N 2 O in both treatments. In the Fertilizer treatment, the 15 N abundance of N 2 O decreased from 26.3–0.88%, and in the No-fertilizer treatment, it decreased from 32.7–0.91% (Fig. 4 ). When calculating the N 2 emissions, we assumed that the generated N 2 originated from the reduction of N 2 O; hence, the 15 N enrichment of N 2 was consistent with that of N 2 O. Based on the 15 N abundances of NO 3 − , N 2 O, and N 2 , we estimated the emissions of N 2 O and N 2 derived from the denitrification processes, denoted as D N2O and D N2 , respectively. During the first 1–2 days of the experiment, N 2 O was primarily derived from the denitrification process, with its contribution decreasing thereafter. N 2 produced by denitrification reached its peak on the second day and then gradually decreased. In the Fertilizer treatment, D N2O was 0.45 kg N ha − 1 , 51.2% of the total N 2 O emissions, and in the No-fertilizer treatment, it was 0.23 kg N ha − 1 , 48.9% of the total N 2 O emissions. For N 2 , in the Fertilizer treatment, D N2 was 14.4 kg N ha − 1 , 36.7% of the total N 2 emissions, and in the No-Fertilizer treatment, it was 6.9 kg N ha − 1 , 27.1% of the total N 2 emissions (Fig. 5 ). 3.5 15 N Recovery Rate From July 20, 2021, to September 5, 2021, the total 15 N recovery rates for the N pools in the Fertilizer and No-fertilizer treatments were 76.0% and 70.0%, respectively. After the addition of organic fertilizer, the N residue increased, primarily in the form of NO 3 − . Under conventional fertilization conditions, the losses of 15 N 2 and 15 N 2 O accounted for 13.6% and 0.5% of the added 15 N, respectively. Under no fertilization conditions, the losses of 15 N 2 and 15 N 2 O accounted for 6.3% and 0.2% of the added 15 N, respectively (Fig. 6 ). 3.6 Relationships Among Gaseous N Rates, Ratios, and Soil Properties The piecewise SEM indicated that the application of organic fertilizer and soil environmental factors accounted for 55%, 13%, and 30% of the variance in N 2 O, N 2 , and R N2O , respectively. The N 2 O emission rate was mainly influenced by soil temperature and NO 2 − content, showing a significant negative correlation with soil temperature ( P < 0.001) and a significant positive correlation with NO 2 − content ( P < 0.01). The N 2 emission rate was significantly negatively correlated with the NO 3 − content ( P < 0.05), and the N 2 emission rate directly affected R N2O ( P < 0.001; Fig. 5 ). Additionally, Pearson partial correlation coefficients and multiple stepwise regression models were used for N 2 O and N 2 emissions and for D N2O and D N2 to further investigate the influence of soil environmental factors. The results revealed that N 2 O emissions, D N2O , and D N2 were significantly negatively correlated with soil temperature, whereas R N2O was significantly positively correlated with soil NO 3 − content (Table 1 ). Table 1 Pearson partial correlation coefficients and multiple stepwise regression models for N 2 O, N 2 fluxes and N 2 O, N 2 from denitrification and the R N2O (dependent variables), as a function of soil variables (regressors) Treatment Dependent variables Pearson’s R correlation coefficients of regressors Regression models NO 3 − NH 4 + NO 2 − NH 4 + /NO 3 − TOC Temperature WFPS F N 2 O -0.22 0.20 0.79*** 0.37* -0.35* -0.68*** 0.38 Y = 0.70 + 0.44 N 2 O − + 0.14 NH 4 + /NO 3 − − 0.01 TOC − 0.02T N 2 -0.32 -0.22 0.02 -0.06 -0.32 -0.20 -0.11 - D N2O -0.20 0.22 0.80*** 0.38* -0.32 -0.66*** 0.34 Y = 0.63 + 0.42 NO 2 − + 0.17 NH 4 + /NO 3 − − 0.02 T D N2 -0.36* -0.04 0.48** 0.18 -0.45** -0.56** 0.14 Y = 9.43 − 0.33 NO 3 − + 2.66 N 2 O − − 0.06 TOC − 0.13 T N N 2 O 0.11 -0.07 0.24 -0.12 -0.37* -0.65*** 0.10 Y = 0.49 − 0.001 TOC − 0.01 T N 2 -0.28 -0.29 -0.08 -0.14 -0.17 -0.02 0.26 - D N2O 0.08 -0.07 0.32 -0.11 -0.38* -0.65*** 0.13 Y = 0.58 − 0.001 TOC − 0.01 T D N2 -0.11 -0.23 0.13 -0.16 -0.39* -0.53** 0.25 Y = 5.08 − 0.02 TOC − 0.01 T F + N R N2O 0.31* 0.07 -0.10 -0.07 0.08 0.03 0.01 Y = 0.04 + 0.01 NO 3 − Nitrate, DOC, ammonium, soil temperature, and soil water content were considered independent variables (X), whereas N 2 , N 2 O, D N2 , D N2O , and R N2O were considered dependent variables (Y). D N2O and D N2 are the N 2 O and N 2 fluxes generated during denitrification, respectively. F represents conventional fertilization treatment; N represents control treatment without fertilization. F + N indicates that the data were combined for all treatments. * P < 0.05 level of significance; ** P < 0.01 level of significance; *** P < 0.001 level of significance. 4. Discussion 4.1 N 2 O and N 2 Emissions In contrast with most studies on N 2 O emissions in GVP systems during the growing season, we investigated the characteristics of N 2 O emissions during the ASD period. On day 1 of ASD, the N 2 O flux reached its peak, consistent with the results of our automatic monitoring during the previous year's ASD period at the same site (Li et al., 2024 ) and the findings of Qasim et al. ( 2022 ). Different from the typical pattern of N 2 O flux reaching its peak two to three weeks after fertilization during the growing season, the high-temperature and high-WFPS soil environment during the ASD period favors the rapid mineralization of organic fertilizers, leading to an earlier peak in N 2 O emissions (He et al., 2009 ). In our study, the average N 2 O fluxes for the Fertilizer and No-fertilizer treatments were 0.03 and 0.02 kg N ha − 1 d − 1 . These values were lower than those reported during ASD by Qasim et al. ( 2022 ) (0.1–0.5 kg N ha − 1 d − 1 ) and Zhao et al. ( 2021b ) (0.4–1.6 kg N ha − 1 d − 1 ). This difference is probably due to the higher amount of organic C added during ASD compared to our experiment. Zhao et al. ( 2021b ) added 12.6 t C ha − 1 of corn straw and 2.7 t C ha − 1 of chicken manure, and Qasim et al. ( 2022 ) added 8.8 t C ha − 1 of rice husks and 4.2 t C ha − 1 of chicken manure, far exceeding the 2.8 t C ha − 1 added in our study. To the best of our knowledge, this study represents the first in-situ measurement of soil N 2 emissions in the GVP system. We found that fertilization significantly promoted N 2 emissions, consistent with the findings of Chen et al. ( 2019 ) and Roobroeck et al. ( 2010 ). In contrast with N 2 O emissions, N fertilizer application did not immediately lead to a peak in N 2 emissions. Dendooven & Anderson ( 1994 ) suggested that the lag phase between the N 2 O and N 2 peaks was due to the different synthesis times of the enzymes involved in the production and consumption of N 2 O under anaerobic conditions. Another reason might be that different from N 2 O, NO 3 − preferentially accepts electrons (Firestone & Tiedje, 1979 ). In our study, the average N 2 emission rates for the Fertilizer and No-fertilizer treatments were 2.9 and 1.8 kg N ha − 1 d − 1 , respectively, higher than the average N 2 emission rate (approximately 0.6 kg N ha − 1 d − 1 ) observed in Shouguang GVP soil under the incubator conditions of 25°C and 80% WHC (Zhao et al., 2021a ). This is mainly attributed to the addition of urea in that study, which lacked C sources and limited the occurrence of denitrification process (Senbayram et al., 2012 ). Pan et al. ( 2022 ) collected N 2 emission data from studies that used the 15 N labeling method, and they found that the N 2 fluxes in farmland, grassland, forest, and marsh were in the range of 0.06–0.6, 0.2–2.4, 0.2–4.1, and 0.1–0.6 kg N ha − 1 d − 1 , respectively. Our study revealed that the highest N 2 flux during the ASD period in the GVP system (12.4 kg N ha − 1 d − 1 ), a peak significantly higher than that in other ecosystems. 4.2 Microbial Processes Responsible for N 2 O and N 2 Production By applying the 15 N isotope tracing technique, we distinguished the microbial processes contributing to soil N 2 O emissions in the GVP system through in situ monitoring for the first time. The results revealed that N 2 O derived from denitrification accounted for 51.2% and 41.9% of the total N 2 O emissions in the Fertilizer and No-fertilizer treatments, respectively. Soil temperature was significantly negatively correlated with D N2O (Table 1 ). The reason for this result may be that from day 2 of the experiment, the soil temperature exceeded 35°C, inhibiting denitrification (Lai & Denton, 2017 ; Tan et al., 2020 ). Yu ( 2023 ) also found that the N 2 O produced by denitrification showed an initial increase followed by a decrease with increasing temperature, with the peak occurring at 25°C or 30°C in an incubator experiment. In that study, the N 2 O generated by nitrification and co-denitrification increased with temperatures ranging from 5°C to 35°C. We speculated that N 2 O also originated from nitrification and co-denitrification in addition to denitrification processes in our study. Previous incubator experiments have shown that nitrification and denitrification are the main sources of N 2 O in acidic and alkaline GVP soils, respectively. The soil in the Shouguang GVP system is alkaline, and under incubator conditions with temperatures ranging from 15°C to 35°C and a WFPS of 65%, the denitrification only account for 15–21% of total N 2 O emissions (Duan et al., 2019 ). In our study, WFPS was above 95% (Fig. 1 b) but had no significant impact on N 2 O emissions (Table 1 ). Studies have suggested that when soil WFPS exceeds 60%, N 2 O is primarily produced through denitrification (Davidson et al., 2000 ; Pilegaard, 2013 ), but many studies refute this view. Sgouridis & Ullah ( 2015 ) conducted in situ 15 N labeling experiments on various terrestrial soils in the United Kingdom and found that denitrification rates in organic soils decreased as the WFPS increased beyond 60%. Other investigations have indicated that nitrification processes continue to occur in soil conditions between 85% and 100% WFPS, contributing between 33% and 53% of N 2 O emissions. (Liu et al., 2017 ). Johannes et al. ( 2016 ) conducted incubator experiments on subtropical pasture soils in Australia, and after 6 d of incubator under 100% WFPS conditions, nitrification processes contributed more to N 2 O emissions than denitrification processes did; they also suggested that under flooded conditions, NH 4 + oxidation could still occur if denitrification processes were inhibited. Studies have also shown that co-denitrification occurs in grasslands (Laughlin & Stevens, 2002 ; Selbie et al., 2015 ) and croplands (Long et al., 2013 ). Under the same NO 3 − substrate concentration conditions, the potential for N 2 O production through co-denitrification (2 mol NO 3 − produces 2 mol N 2 O) is twice that of denitrification (2 mol NO 3 − produces 1 mol N 2 O). Spott & Florian ( 2011 ) added NH 2 OH to agricultural soil extracts and found that > 98% of N 2 O was produced by co-denitrification. Through incubator studies on forest soils, Li et al. ( 2021 ) discovered that 10.6% and 30.7% of the total N 2 O emissions were generated via co-denitrification. Li et al. ( 2020 ) conducted a 15 NO 3 − addition experiment and found that co-denitrification processes contributed N 2 O emissions from 1.8–34.2%. We used the 15 N labeling technique to quantify the N 2 emissions originating from denitrification processes. In our study, N 2 produced through denitrification accounted for 36.7% and 27.1% of the total N 2 emissions in the Fertilizer and No-fertilizer treatments, respectively. As the experiment progressed, D N2 gradually decreased, showing a significant negative correlation with soil temperature (Table 1 ). Similar to our speculation regarding D N2O , we speculated that the inhibition of denitrification processes occurred when soil temperature exceeded 35°C (Lai & Denton, 2017 ; Tan et al., 2020 ; Yu, 2023 ). Additionally, a significant negative correlation was observed between D N2 and TOC content (Table 1 ). A reason for this result might be that a higher C/N ratio in the soil increases the microbial assimilation of N (Aulakh et al., 2001 ). Another reason is that a lower N content may not fulfill microbial demand, and the reduced N consumption caused by net N fixation leads to a decrease in nitrification and denitrification rates (Liu et al., 2017 ). Other studies have also reported a negative correlation between C/N and the potential for microbial processes such as denitrification (Klemedtsson et al., 2005 ; Ollinger et al., 2002 ; Sgouridis & Ullah, 2015 ; Ullah & Moore, 2009 , 2011 ). We found that, in addition to the denitrification processes, co-denitrification accounted for 63.3% and 72.9% of N 2 in the Fertilizer and No-fertilizer treatments, respectively, over the ASD period. Co-denitrification, a biological process that occurs under moderate-to-high pH conditions (> 6), is responsible for producing N 2 . Selbie et al. ( 2015 ) conducted in situ 15 N labeling experiments in grasslands and found that over 98% of N 2 emissions were attributed to co-denitrification. Laughlin & Stevens ( 2002 ) demonstrated that after applying 15 N-labeled NH 4 NO 3 to grassland soil, 92% of N 2 emissions resulted from co-denitrification, with 8% from denitrification. Incubator experiments of Li et al. ( 2021 ) and Xi et al. ( 2016 ) on forest soils in Northeast China, involving anaerobic conditions and 15 N labeling, have revealed contributions of co-denitrification to N 2 ranging from 4.9–14.4%. Additionally, anaerobic ammonia oxidation (ANAMMOX) is also an important mechanism for N 2 production. However, research has suggested that ANAMMOX is primarily present in paddy fields and wetland soils, and its specific genetic potential is relatively low, contributing little to N 2 emissions (Shen et al., 2014 ; Ligi et al., 2015 ). Our study only estimated the N 2 emissions originating from N 2 O reduction, excluding the N 2 produced from the ANAMMOX process. Consequently, we may have underestimated be a certain degree of underestimation in the total N 2 emissions. Further research is still necessary to quantify the N 2 emissions from the ANAMMOX process. In summary, in addition to denitrification, other processes in the GVP system should be considered when examining N 2 emissions. 4.3 N 2 O/(N 2 O + N 2 ) Ratio Because of the difficulty in directly measuring N 2 , researchers have estimated soil N 2 emissions at site, regional, or global scales by combining R N2O values with in-situ measurements of soil N 2 O emissions (Saggar et al., 2013 ; Schlesinger, 2009 ; Wang et al., 2020 ). However, R N2O values have significantly differed in different soil types. Schlesinger ( 2009 ) collected R N2O data from the literature and calculated average values for denitrification processes in terrestrial, farmland, and wetland areas as 0.49, 0.37, and 0.082, respectively. Based on our study, the average R N2O values for the Fertilizer and No-fertilizer treatments were 0.28 and 0.32, respectively, during the ASD period in the GVP system. Different soil physicochemical properties and microbial communities lead to varying control factors for R N2O (Senbayram et al., 2012 ). Sgouridis & Ullan (2015) found that R N2O concentrations in natural and unmanaged ecosystems ranged from 0.01 to 0.07. They observed that organic soils were mainly influenced by soil temperature and pH, deciduous forests were affected by soil moisture; mixed forests were influenced by soil organic C and pH; semi-improved grasslands were affected by soil organic C; and improved grasslands were affected by soil bulk density, organic C, moisture, and pH. The in-situ labeling experiment conducted by Pan et al. ( 2022 ) revealed a significant correlation between R N2O and soil temperature and moisture in cron field. Xi et al. ( 2022 ) observed that R N2O in forest soils was mainly influenced by soil moisture through in-situ monitoring. Our study found a significant positive correlation between R N2O and soil NO 3 − content (Table 1 ), consistent with the findings of Saggar et al. ( 2013 ) and Scheer et al. ( 2016 ). The reason for this relationship is primarily due to the inhibition of N 2 O reductase activity at high NO 3 − concentrations. Additionally, microorganisms obtain more energy from reducing NO 3 − than N 2 O, so high NO 3 − concentrations are more favorable for N 2 O production rather than N 2 O reduction. (Senbayram et al., 2012 ; van Cleemput, 1998 ; Weier et al., 1993 ). Studies have mostly aimed to reveal the variations in R N2O among different soils, not the temporal variability of R N2O . Our study found a significant difference in R N2O between the first 6 days and the subsequent 8 days (with average values of 0.024 and 0.026 for the Fertilizer and No-fertilizer treatments in the first 6 days, and 0.46 and 0.53, respectively, in the subsequent 8 days). In an in-situ labeling experiment, Liu et al. ( 2022 ) reported that R N2O during the maize season was much higher than that during the wheat season (0.49 vs. 0.02) in the same field. Wang et al. ( 2020 ) found in their maize soil incubator experiment that the average R N2O value was 0.31 within two months after fertilization, and the average R N2O value for other time periods was 0.05. Stevens & Laughlin ( 1998 ) reported that the R N2O levels immediately increased from 0.05 to 0.8 three days after fertilizing grassland. Studies have shown that the temporal variability in R N2O is influenced by various factors. For instance, Clemens et al. ( 2020 ) estimated the global terrestrial ecosystem R N2O and found that short-term changes in R N2O are influenced by factors such as water content, temperature, O 2 , soil texture, pH, NO 3 − , and organic C. Using the DAMM model, Wang et al. ( 2020 ) concluded that the temporal variability of R N2O is mainly affected by soil NH 4 + and NO 3 − contents, as well as temperature and moisture. In addition to the influence of the soil type and temporal scale, the method used to determine N 2 affects R N2O (Sgouridis et al., 2016 ). Xi et al. ( 2022 ) synthesized the literature and found significant variations in R N2 O values measured by different methods in forest soils (ranging from 0.01 to 1, with an average of 0.3). The highest value was obtained by the acetylene inhibition method, and the lowest value was obtained by the 15 N labeling method. Similarly, Clemens et al. ( 2020 ), in their study of global terrestrial ecosystem R N2O , found that R N2O calculated using the 15 N labeling method was significantly lower than that using the acetylene inhibition method. This difference is due to systemic limitations, such as incomplete inhibition of reductase in the acetylene inhibition method, which can lead to an underestimation of N 2 emissions and denitrification potential and, thus, an overestimation of R N2O (Felber et al., 2012 ; Qin et al., 2012 ; Yu et al., 2010 ). In summary, different soil types, timescales, and measurement methods can affect R N2O . Thus, estimating N 2 emissions based on average R N2O values involves significant uncertainties. Further research into the relationship between R N2O and environmental factors is necessary to improve the accuracy of estimating N 2 emissions from terrestrial ecosystems (Clemens et al., 2020 ; Liu et al., 2022 ). The environmental conditions of the GVP system that we studied were unique, and the response of gaseous N to environmental factors differed from those in the literature. Therefore, it is not recommended to use constant R N2O values derived from observational datasets or existing models to estimate its N 2 emissions. 4.4 N Losses Gaseous N loss, particularly the emission of N 2 , has long been considered the primary pathway for N loss in farmlands (Zhao et al., 2012 ). However, owing to the difficulty of directly measuring soil N 2 emissions, few studies have investigated the proportion of gaseous N in N losses within the GVP system. Our study revealed that the primary form of gaseous N in the GVP during the ASD period was N 2 . The total 15 N 2 emissions ranged from 6.3–13.6% of the added 15 N, and 15 N 2 O emissions accounted for 0.2–0.5% of the added 15 N. These findings are similar to the gaseous N pathways reported in other studies. In a wheat-maize rotation system in northern China, the emissions of N 2 and N 2 O during the growing season accounted for 4.1–10% and 0.4–0.7% of N fertilizer application, respectively (Chen et al., 2019 ). In rice planting systems in southern China, the N 2 produced by denitrification accounted for 10.2–13.5% of the N fertilizer application, and N 2 O accounted for 0.09–0.2% (Xia et al., 2020 ). In our study, during the ASD period, 7–14% of the added N was emitted as N 2 + N 2 O, and 62–64% remained in the soil primarily as NO 3 − . The unrecovered 24–30% of N may leach into the deeper soil layers. Based on our previous monitoring of NO emissions and NH 3 volatilization at this site, the emissions of these two gases were relatively small, accounting for 5% and 6% of N 2 O emissions, respectively. Ti et al. ( 2015 ) conducted a meta-analysis of N fate in GVP system and estimated that the proportions of N in N 2 + NOx, soil retention, plant uptake, leaching and runoff, and NH 3 volatilization were 33%, 31%, 23%, 12%, and 1% of the added N, respectively. Our findings differ from theirs, possibly due to the absence of crop planting during the observed ASD period and the use of different methods for measuring N 2 emissions in our study (their study used the balance method) (Groffman et al., 2006 ). Hence, further research should aim to validate the N loss pathways in GVP systems using various methods. Notably, implementing reasonable measures to reduce gaseous N loss and improve N fertilizer use efficiency is necessary. 5. Conclusion In the agricultural ecosystems, accurate estimation of N 2 emissions is crucial for reducing the loss of available N and formulating effective strategies to mitigate N 2 O emissions. This study is the first to quantify N 2 emissions in the GVP system by in situ 15 N labeling method. The results indicate that the predominant gaseous N released from the soil during the ASD period was N 2 , with relatively low N 2 O emissions. The total 15 N 2 emissions accounted for 6.3–13.6% of the added 15 N, and 15 N 2 O emissions represented 0.2–0.5% of the added 15 N. Furthermore, our study emphasized the importance of microbial processes other than denitrification in N 2 O and N 2 emissions. During the ASD period, N 2 O and N 2 originating from denitrification processes accounted for 48.9–51.2% and 27.1–36.7%, respectively. However, we were unable to differentiate the microbial processes in detail. Further research should employ dual labeling with 15 NH 4 + and 15 NO 3 − combined with incubator experiments to explore more detail microbial processes that contribute to N 2 O and N 2 production in the GVP system. Additionally, we observed a significant variability in R N2O during the ASD period. Hence, we advise against estimating GVP system N 2 emissions using constant R N2O values obtained from observations in other locations or existing models. Declarations Author Contributions Y.T.F and X.L designed the study. X.L, J.L, Y.Y.W and K.P.S performed the experiment. Data analysis was conducted by X.L, J.L, and K.H. The paper was written by X.L with contribution from other co-authors. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements The work was financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences [grant number XDA28020302], the National Key Research and Development Program of China (2023YFD1500802; 2023YFD1501400), the National Natural Science Foundation of China [grant number 42177214], the Shandong Provincial Natural Science Foundation [grant number ZR2023YQ030], the Liaoning Vitalization Talents Program [grant number XLYC1902016, XLYC2203058], the Taishan Scholars [R.K. and Z.Q.], and the China Postdoctoral Science Foundation [grant number 2023M733675]. We also acknowledge support from the K.C. Wong Education Foundation (Y.F.) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Z.Q.). References Agostini F, Tei F, Silgram M, Farneselli M, Benincasa P, Aller M (2010) Decreasing nitrate leaching in vegetable crops with better N management. In: Lichtfouse E (ed) Genetic Engineering, Biofertilisation, Soil Quality and Organic Farming. Springer Netherlands, Dordrecht, pp 147–200 Aulakh MS, Khera TS, Doran JW, Bronson KF (2001) Denitrification, N 2 O and CO 2 fluxes in rice-wheat cropping system as affected by crop residues, fertilizer N and legume green manure. Biol Fertil Soils 34:375–389 Buchen C, Lewicka-Szczebak D, Fuss R, Helfrich M, Flessa H, Well R (2016) Fluxes of N 2 and N 2 O and contributing processes in summer after grassland renewal and grassland conversion to maize cropping on a Plaggic Anthrosol and a Histic Gleysol. Soil Biol Biochem 101:6–19 Butterbach-Bahl K, Willibald G, Papen H (2002) Soil core method for direct simultaneous determination of N 2 and N 2 O emissions from forest soils. Plant Soil 240:105–116 Charles A, Rochette P, Whalen JK, Angers DA, Chantigny MH, Bertrand N (2017) Global nitrous oxide emission factors from agricultural soils after addition of organic amendments: a meta-analysis. Agr Ecosyst Environ 236:88–98 Chen Q, Zhang XS, Zhang HY, Christie P, Li XL, Horlacher D, Liebig H (2004) Evaluation of current fertilizer practice and soil fertility in vegetable production in the Beijing region. Nutr Cycl Agrosyst 69:51–58 Chen T, Oenema O, Li J, Misselbrook T, Dong W, Qin S, Yuan H, Li X, Hu C (2019) Seasonal variations in N 2 and N 2 O emissions from a wheat–maize cropping system. Biol Fertil Soils 55:539–551. https://doi.org/10.1007/s00374-019-01373-8 Clemens S, Kathrin F, David EP, Klaus B (2020) Estimating global terrestrial denitrification from measured N 2 O:(N 2 O + N 2 ) product ratios. Curr Opin Environ Sustain 47:72–80. https://doi.org/10.1016/j.cosust.2020.07.005 Davidson EA, Keller M, Erickson HE, Verchot LV, Veldkamp E (2000) Testing a conceptual model of soil emissions of nitrous and nitric oxides: using two functions based on soil nitrogen availability and soil water content, the hole-in-the-pipe model characterizes a large fraction of the observed variation of nitric oxide and nitrous oxide emissions from soils. Bioscience 50(8):667–680 Dendooven L, Anderson JM (1994) Dynamics of reduction enzymes involved in the denitrification process in pasture soil. Soil Biol Biochem 26:1501–1506. https://doi.org/10.1016/0038-0717(94)90091-4 Di Gioia F, Ozores-Hampton M, Hong J, Kokalis-Burelle N, Albano J, Zhao X, Black Z, Gao Z, Moore K, Swisher M, Guo H, Rosskopf EN, Wilson C, Thomas J (2016) The effects of anaerobic soil disinfestation on weed and nematode control, fruit yield, and quality of Florida fresh-market tomato. HortScience 51:703–711. https://doi.org/10.21273/hortsci.51.6.703 Di Gioia F, Ozores-Hampton M, Zhao X, Thomas J, Wilson P, Li Z, Hong J, Albano J, Swisher M, Rosskopf E (2017) Anaerobic soil disinfestation impact on soil nutrients dynamics and nitrous oxide emissions in fresh-market tomato. Agric Ecosyst Environ 240:194–205. https://doi.org/10.1016/j.agee.2017.02.025 Duan P, Song Y, Li S, Xiong Z (2019) Responses of N 2 O production pathways and related functional microbes to temperature across greenhouse vegetable field soils. Geoderma 355:113904 Fei C, Zhang S, Liang B, Li J, Jiang L, Xu Y, Ding X (2018) Characteristics and correlation analysis of soil microbial biomass phosphorus in greenhouse vegetable soil with different planting years. Acta Agric Boreali-sinica 33(1):195–202 Felber R, Conen F, Flechard CR, Neftel A (2012) Theoretical and practical limitations of the acetylene inhibition technique to determine total denitrification losses. Biogeosciences 9(10):4125–4138 Firestone MK, Tiedje JM (1979) Temporal change in nitrous oxide and dinitrogen from denitrification following onset of anaerobiosis. Appl Environ Microbiol 38:673–679 Groffman PM, Altabet MA, Böhlke JK, Butterbach-Bahl K, David MB, Firestone MK, Giblin AE, Kana TM, Nielsen LP, Voytek MA (2006) Methods for measuring denitrification: Diverse approaches to a difficult problem. Ecol Appl 16:2091–2122 Guo J, Liu X, Zhang Y, Shen J, Han W, Zhang W, Christie P, Goulding KWT, Vitousek PM, Zhang F (2010) Significant acidification in major Chinese croplands. Science 327:1008–1010 Guo J (2016) Inputs of irrigation water, fertilizers, pesticides to and life cycle assessment of environment impacts from typical greenhouse vegetable production systems (In Chinese). PhD thesis. China Agricultural University He H, Lv H, Zhang W, Hou S, Zhang X (2011) A liquid chromatographic/mass spectrometric method to evaluate 13 C and 15 N incorporation into soil amino acids. J Soils Sediments 11:731–740 He F, Jiang R, Chen Q, Zhang F, Su F (2009) Nitrous oxide emissions from an intensively managed greenhouse vegetable cropping system in Northern China. Environ Pollut 157(5):1666–1672 Huang X, Liu L, Wen T, Zhang J, Shen Q, Cai Z (2016) Reductive soil disinfestations combined or not with Trichoderma for the treatment of a degraded and Rhizoctonia solani infested greenhouse soil. Sci Hortic 206:51–61. https://doi.org/10.1016/j.scienta.2016.04.033 IPCC (2021) In: Masson-Delmotte V et al (eds) The Physical Science Basis. Cambridge Univ. Press Johannes F, Clemens S, David WR, Heather VM, Alice S, Daniel IW, Peter RG (2016) Denitrification losses from an intensively managed sub-tropical pasture – Impact of soil moisture on the partitioning of N 2 and N 2 O emissions. Soil Biol Biochem 92:58–66. https://doi.org/10.1016/j.soilbio.2015.09.016 Ju X, Liu X, Zhang F, Roelcke M (2004) Nitrogen fertilization, soil nitrate accumulation, and policy recommendations in several agricultural regions of China. Ambio 33(6):300–305. https://doi.org/10.1579/0044-7447-33.6.300 Ju X, Kou C, Zhang F, Christle P (2006) Nitrogen balance and groundwater nitrate contamination: Comparison among three intensive cropping systems on the North China Plain. Environ Pollut 143(1):117–125 Klemedtsson L, Von Arnold K, Weslien P, Gundersen P (2005) Soil CN ratio as a scalar parameter to predict nitrous oxide emissions. Global Change Biol 11:1142–1147 Kulkarni MV, Burgin AJ, Groffman PM, Yavitt JB (2014) Direct flux and 15 N tracer methods for measuring denitrification in forest soils. Biogeochemistry 117:359–373. https://doi.org/10.1007/s10533-013-9876-7 Kulkarni MV, Groffman PM, Yavitt JB, Goodale CL (2015) Complex controls of denitrification at ecosystem; landscape and regional scales in northern hardwood forests. Ecol Model 298:39–52 Lai TV, Denton MD (2017) N 2 O and N 2 emissions from denitrification respond differently to temperature and nitrogen supply. J S Sediment 18(4):1–10 Laughlin RJ, Stevens RJ (2002) Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Sci Soc Am J 66:1540–1548 Li J, Kang R, Yu H, Wang Y, Yao M, Fang Y (2021) Effects of soil moisture on micribial processes of soil nitrogen gases production under anaerobic conditions. Chin J Appl Ecol 32(6):9. https://doi.org/10.13287/j.1001-9332.202106.010 Li X, Nishio T, Uemiya Y, Inubushi K (2002) Gaseous losses of applied nitrogen from a cron field determined by 15 N abundance of N 2 and N 2 O. Commun Soil Sci Plant Anal 33(15–18):2715–2727. https://doi.org/10.1081/CSS-120014475 Li X, Quan Z, Huang K, Kang R, Su C, Liu D, Ma J, Chen X, Fang Y (2024) High soil nitrous oxide emissions from a greenhouse vegetable production system in Shouguang, Northern China. Atmos Environ 319:120264. https://doi.org/10.1016/j.atmosenv.2023.120264 Li Y, Wang S, Li H, Fang Y, Yang L, Su F (2020) Contribution of co-denitrification to nitrous oxide production from subsurface wastewater infiltration system. Ecol Eng 151:105854. https://doi.org/10.1016/j.ecoleng.2020.105854 Ligi T, Truu M, Oopkaup K, Nõlvak H, Mander Ü, Mitsch W, Truu J (2015) The genetic potential of N 2 emission via denitrification and ANAMMOX from the soils and sediments of a created riverine treatment wetland complex. Ecol Eng 80:181–190. https://doi.org/10.1016/j.ecoleng.2014.09.072 Liu Y, Wang R, Pan Z, Zheng X, Wei H, Zhang H, Quan M, Fang Z, Ju Y X (2022) Quantifying in situ N 2 fluxes from an intensively managed calcareous soil using the 15 N gas-flux method. J Integr Agric 21(9):2750–2766. https://doi.org/10.1016/j.jia.2022.07.016 Liu R, Hayden HL, Suter H, Hu H, Lam SK, He J, Mele PM, Chen D (2017) The effect of temperature and moisture on the source of N 2 O and contributions from ammonia oxidizers in an agricultural soil. Biol Fertil Soils 53:141–152. https://doi.org/10.1007/s00374-016-1167-8 Liu S, Lin F, Wu S, Ji C, Sun Y, Jin Y, Li S, Li Z, Zou J (2017) A meta-analysis of fertilizer‐induced soil NO and combined with N 2 O emissions. Glob Change Biol 23:2520–2532. http://doi.org/10.1111/gcb.13485 Liu Z, Jiang L, Zhang W, Zheng F, Wang Mei, Lin H (2008) Evolution of fertilization rate and variation of soil nutrient contents in greenhouse vegetable cultivation in Shandong (In Chinese). Acta Pedol Sin 45(2):296–303 Long A, Heitman J, Tobias C, Philips R, Song B (2013) Co-occurring anammox, denitrification, and codenitrification in agricultural soils. Appl Environ Microbiol 79:168–176. https://doi.org/10.1128/aem.02520-12 Lv H, Zhao Y, Wang Y, Wan L, Wang J, Butterbach-Bahl K, Lin S (2020) Conventional flooding irrigation and over fertilization drives soil pH decrease not only in the top but also in subsoil layers in solar greenhouse vegetable production systems. Geoderma 363:114156. https://doi.org/10.1016/j.geoderma.2019.114156 Min J, Sun H, Kronzucker H, Wang Y, Shi W (2021) Comprehensive assessment of the effects of nitrification inhibitor application on reactive nitrogen loss in intensive vegetable production systems. Agric Ecosyst Environ 307:107227 Momma N, Kobara Y, Uematsu S, Kita N, Shinmura A (2013) Development of biological soil disinfestations in Japan. Appl Microbiol Biotechnol 97:3801–3809. https://doi.org/10.1007/s00253-013-4826-9 Morse JL, Bernhardt ES (2013) Using 15 N tracers to estimate N 2 O and N 2 emissions from nitrification and denitrification in coastal plain wetlands under contrasting land-uses. Soil Biol Biochem 57:635–643 Ollinger S, Smith M, Martin M, Hallett R, Goodale C, Aber J (2002) Regional variation in foliar chemistry and N cycling among forests of diverse history and composition. Ecology 83:339–355 Pan Z, Wang R, Liu Y, Wang L, Zheng X, Yao Z, He H, Zhang X (2022) Characteristics of N 2 and N 2 O fluxes from a cultivated black soil: A case study through in situ measurement using the 15 N gas flux method. Agriculture 12:1664. https://doi.org/10.3390/agriculture12101664 Pilegaard K (2013) Processes regulating nitric oxide emissions from soils. Philosophical Trans Royal Soc B 368(1621):20130126. https://doi.org/10.1098/rstb.2013.0126 Qasim W, Wan L, Lv H, Zhao Y, Hu J, Meng F, Lin S, Butterbach-Bahl K (2022) Impact of anaerobic soil disinfestation on seasonal N 2 O emissions and N leaching in greenhouse vegetable production system depends on amount and quality of organic matter additions. Sci Total Environ 830:154673 Qasim W, Xia L, Lin S, Wan Li, Zhao Y, Butterbach-Bahl K (2021) Global greenhouse vegetable production systems are hotspots of soil N 2 O emissions and nitrogen leaching: A meta-analysis. Environ Pollut 272:116372 Qin S, Hu C, Oenema O (2012) Quantifying the underestimation of soil denitrification potential as determined by the acetylene inhibition method. Soil Biol Biochem 47:14–17 Roobroeck D, Butterbach-Bahl K, Brüggemann N, Boeckx P (2010) Dinitrogen and nitrous oxide exchanges from an undrained monolith fen: short-term responses following nitrate addition. Eur J Soil Sci 61:662–670. https://doi.org/10.1111/j.1365-2389.2010.01269.x Saggar S, Jha N, Deslippe J, Bolan NS, Luo J, Giltrap DL, Kim DG, Zaman M, Tillman RW (2013) Denitrification and N 2 O:N 2 production in temperate grasslands: Processes; measurements; modelling and mitigating negative impacts. Sci Total Environ 465:173–195 Scheer C, Meier R, Bruggemann N, Grace PR, Dannenmann M (2016) An improved 15 N tracer approach to study denitrification and nitrogen turnover in soil incubations. Rapid Commun Mass Spectrom 30:2017–2026 Schlesinger WH (2009) On the fate of anthropogenic nitrogen. Proc. Natl. Acad. Sci. USA 106:203–208 Selbie DR, Lanigan GJ, Laughlin RJ, Di HJ, Moir JL, Cameron KC, Clough TJ, Watson CJ, Grant J, Somers C, Richards KG (2015) Confirmation of co-denitrification in grazed grassland. Sci Rep 5:1–9 Senbayram M, Chen R, Budai A, Bakken L, Dittert K (2012) N 2 O emission and the N 2 O/(N 2 O + N 2 ) product ratio of denitrification as controlled by available carbon substrates and nitrate concentrations. Agr. Ecosyst. Environ. 2012, 147, 4–12 Sgouridis F, Ullah S (2015) Relative magnitude and controls of in situ N 2 and N 2 O fluxes due to denitrification in natural and seminatural terrestrial ecosystems using 15 N tracers. Environ Sci Technol 49(24):14110–14119. https://doi.org/10.1021/acs.est.5b03513 Sgouridis F, Stott A, Ullah S (2016) Application of the 15 N gas-flux method for measuring in situ N 2 and N 2 O fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique. Biogeosciences 13:1821–1835 Shen J, Xu Z, He J (2014) Frontiers in the microbial processes of ammonia oxidation in soils and sediments. J Soils Sediments 14:1023–1029. 10.1007/s11368-014-0872-x Shougung Statistical, Yearbook (2021) China Statistics, Beijing (in Chinese). Spott O, Florian S (2011) Formation of hybrid N 2 O in a suspended soil due to co-denitrification of NH 2 OH. J Plant Nutr Soil Sci 174:554–567 Stevens RJ, Laughlin RJ (1998) Measurement of nitrous oxide and dinitrogen emissions from agricultural soils. Nutr Cycl Agrosyst 52:131–139 Stevens RJ, Laughlin RJ, Burns LC, Arah JRM, Hood RC (1997) Measuring the contributions of nitrification and denitrification to the flux of nitrous oxide from soil. Soil Biol Biochem 29:139–151 Tan E, Zou W, Zheng Z, Yan X, Du M, Hsu T, Tian L, Middelburg J, Trull TW, Kao S (2020) Warming stimulates sediment denitrification at the expense of anaerobic ammonium oxidation. Nat Clim Change 10(4):349–355 Templer PH, Mack MC, Chapin FS, Christenson LM, Compton JE, Crook HD, Currie WS, Curtis CJ, Dail DB, D’antonio CM, Emmett BA, Epstein HE, Goodale CL, Gundersen P, Hobbie SE, Holland K, Hooper DU, Hungate BA, La-montagne S, Nadelhoffer KJ, Osenberg CW, Perakis SS, Schleppi P, Schimel J, Schmidt IK, Sommerkorn M, Spoelstra J, Tietema A, Wessel WW, Zak DR (2012) Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15 N tracer field studies. Ecology 93:1816–1829 Ti C, Luo Y, Yan X (2015) Characteristics of nitrogen balance in open-air and greenhouse vegetable cropping systems of China. Environ Sci Pollut Control Ser 22(23):18508–18518 Ullah S, Moore TR (2011) Biogeochemical controls on methane, nitrous oxide, and carbon dioxide fluxes from deciduous forest soils in eastern Canada. J Geophys Res Biogeosci 116:G03010 Ullah S, Moore TR (2009) Soil drainage and vegetation controls of nitrogen transformation rates in forest soils, southern Quebec. J Geophys Research-Biogeosciences 114:G01014 van Cleemput O (1998) Subsoils: chemo- and biological denitrification, N 2 O and N 2 emissions. Nutr Cycl Agroecosyst 52:187–194 Wang R, Pan Z, Zheng X, Ju X, Yao Z, Butterbach-Bahl K, Zhang C, Wei H, Huang B (2020) Using field-measured soil N 2 O fluxes and laboratory scale parameterization of N 2 O/(N 2 O + N 2) ratios to quantify field-scale soil N 2 emissions. Soil Biol Biochem 148:107904. https://doi.org/10.1016/j.soilbio.2020.107904 Wang R, Willibald G, Feng Q, Zheng X, Liao T, Brüggemann N, Butterbach-Bahl K (2011) Measurement of N 2 , N 2 O, NO, and CO 2 emissions from soil with the Gas-Flow-Soil-Core technique. Environ Sci Technol 45:6066–6072. https://doi.org/10.1021/es1036578 Wang R, Feng Q, Liao T, Zheng X, Butterbach-Bahl K, Zhang W, Jin C (2013) Effects of nitrate concentration on the denitrification potential of a calcic cambisol and its fractions of N 2 , N 2 O and NO. Plant Soil 363:175–189 Weier KL, Doran JW, Power JF, Walters DT (1993) Denitrification and the dinitrogen/nitrous oxide ratio as affected by soil water, available carbon, and nitrate. Soil Sci Soc Am J 57(1):66–72. https://doi.org/10.2136/sssaj1993.03615995005700010013x Well R, Burkart S, Giesemann A, Grosz B, Koester JR, Lewicka-Szczebak D (2019) Improvement of the 15 N gas flux method for in situ measurement of soil denitrification and its product stoichiometry. Rapid Commun Mass Spectrom 33:437–448 Xi D, Bai R, Zhang L, Fang Y (2016) Contribution of anammox to nitrogen removal in two temperate forest soils. Appl Environ Microbiol 82(15):4602–4612. https://doi.org/10.1128/AEM.00888-16 Xi D, Fang Y, Zhu W (2022) Spatial variations of soil N 2 and N 2 O emissions from a temperate forest: quantified by the in situ 15 N Labeling Method. Forests 13:1347. https://doi.org/10.3390/f13091347 Xia L, Li X, Ma Q, Lam SK, Wolf B, Kiese R, Butterbach-Bahl K, Chen D, Li Z, Yan X (2020) Simultaneous quantification of N 2 , NH 3 and N 2 O emissions from a flooded paddy field under different N fertilization regimes. Glob Chang Biol 26(4):2292–2303. https://doi.org/10.1111/gcb.14958 Yang WH, McDowell AC, Brooks PD, Silver WL (2014) New high precision approach for measuring 15 N-N 2 gas fluxes from terrestrial ecosystems. Soil Biol Biochem 69:234–241 Yang L, Huang B, Mao M, Yao L, Niedermann S, Hu W, Chen Y (2016) Sustainability assessment of greenhouse vegetable farming practices from environmental, economic, and socio-institutional perspectives in China. Environ Sci Pollut Res 23(17):17287–17297 Yao Z, Yan G, Wang R, Zheng X, Liu C, Butterbach-Bahl K (2019) Drip irrigation or reduced N-fertilizer rate can mitigate the high annual N 2 O + NO fluxes from Chinese intensive greenhouse vegetable systems. Atmos Environ 212:183–193 Yoshinari T, Hynes R, Knowles R (1997) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol Biochem 9:177–183 Yu K, Seo DC, DeLaune RD (2010) Incomplete acetylene inhibition of nitrous oxide reduction in potential denitrification assay as revealed by using 15 N-nitrate tracer. Commun Soil Sci Plant Anal 41(18):2201–2210 Yu H (2023) Temperature Sensitivity of gaseous nitrogen losses in forest soils (in Chinese). PhD thesis. Chinese Academy of Sciences Yu H, Li T, Zhang X Nutrient budget and soil nutrient status in greenhouse system. Agr Sci China 43(3):514–522 Zhang Y, Wang H, Maucieri C, Liu S, Zou J (2019) Annual nitric and nitrous oxide emissions response to biochar amendment from an intensive greenhouse vegetable system in southeast China. Sci Hortic-Amsterdam 246:879–886 Zhao X, Zhou Y, Wang S, Xing G, Shi W, Xu R, Zhu Z (2012) Nitrogen balance in a highly fertilized rice-wheat double-cropping system in southern China. Soil Sci Soc Am J 76:1068–1078. https://doi.org/10.2136/sssaj2011.0236 Zhao X, Liu D, Huang K, Quan Z, Huang B, Fang Y, Chen X (2021a) N 2 O and N 2 emission characteristics from maize and greenhouse vegetable soils in Northern China. Chin J Appl Ecol 32(9):3204–3212. https://doi.org/10.13287/j.1001-9332.202109.007 Zhao Y, Lin S, Li W, Qasim W, Hu J, Xue T, Lv H, Butterbach-Bahl K (2021b) Anaerobic soil disinfestation with incorporation of straw and manure significantly increases greenhouse gases emission and reduces nitrate leaching while increasing leaching of dissolved organic N. Sci Total Environ 785:147307 Zhou J, Chen Z, Liu X, Zhai B, Powlson DS (2010) Nitrate accumulation in soil profiles under seasonally open ‘sunlight greenhouses’ in northwest China and potential for leaching loss during summer fallow. Soil Use Manag 26(3):332–339. https://doi.org/10.1111/j.1475-2743.2010.00284.x Zhou J, Chen Z, Tang L, Zhai B, Xu A (2006) Changes of the contents of mineral nitrogen and their accumulation in soils under greenhouse cultivation (In Chinese with English abstract). Plant Nutr Fertilizer Sci 12:675–680 Zhu T, Zhang J, Cai Z (2012) Effect of organic material amendment on vegetable soil nitrate content and nitrogenous gases emission under flooding condition. Chin J Appl Ecol 23:109–114 Cite Share Download PDF Status: Published Journal Publication published 31 Oct, 2024 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 02 Jun, 2024 Reviewers agreed at journal 13 Mar, 2024 Reviewers invited by journal 13 Mar, 2024 Editor invited by journal 13 Mar, 2024 Editor assigned by journal 13 Mar, 2024 First submitted to journal 13 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4091615","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":279381739,"identity":"3a1f5823-5dfc-454f-b038-f131a01bc869","order_by":0,"name":"Xue Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYBACPmYQWSFBghY2sJYzJGkBEYxtJOhgYGPnMZP4OM9Cnp+B+eEHhpo7xDiMLU1y5jYJw5kNbMYSDMeeEaOF+Zg07zYJxg0HGMwYGBsOE6OFsU367xwJ+/0H2L8RqwVoC2ODROIGBh6ibWFLtuw5JpE84zBPsUTCMSK08POfMbzxo6bOtr+9feOHDzVEaEEAUJwmkKJhFIyCUTAKRgFuAADYtSxQZnqyvgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3070-8996","institution":"Institute of Applied Ecology Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Xue","middleName":"","lastName":"Li","suffix":""},{"id":279381740,"identity":"22b63976-d14d-4f7c-895d-2132fbd52713","order_by":1,"name":"Jin Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Li","suffix":""},{"id":279381741,"identity":"4328835f-c95c-4e12-a945-254f314c0ede","order_by":2,"name":"Yingying Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Wang","suffix":""},{"id":279381742,"identity":"c724d1c0-5c64-4f0a-b64d-126b40f66db2","order_by":3,"name":"Ronghua Kang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ronghua","middleName":"","lastName":"Kang","suffix":""},{"id":279381743,"identity":"8e17c6ae-3faa-46fd-b94d-e808a7582198","order_by":4,"name":"Keping Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Keping","middleName":"","lastName":"Sun","suffix":""},{"id":279381744,"identity":"09d6ad77-d766-4992-91b8-46fe37e991ad","order_by":5,"name":"Kai Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Huang","suffix":""},{"id":279381745,"identity":"aaaa084f-fd2f-4882-aa16-95cf824ebd6f","order_by":6,"name":"Shuo Fang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Fang","suffix":""},{"id":279381746,"identity":"5accc617-c0aa-48fb-a43d-72a375dd8ce7","order_by":7,"name":"Xin Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":279381747,"identity":"f4e908c7-92a8-4aaa-a832-bff3662cf1d5","order_by":8,"name":"Zhi Quan","email":"","orcid":"https://orcid.org/0000-0003-4918-7382","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhi","middleName":"","lastName":"Quan","suffix":""},{"id":279381748,"identity":"02ddd84d-cdd4-4890-9a94-ecaba3d3cbd5","order_by":9,"name":"Yunting Fang","email":"","orcid":"https://orcid.org/0000-0001-7531-546X","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yunting","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2024-03-13 10:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4091615/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4091615/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-024-07014-w","type":"published","date":"2024-10-31T16:20:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52931156,"identity":"bea2bdbb-55ec-4b97-a0b0-c48a2787f571","added_by":"auto","created_at":"2024-03-18 20:16:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnvironment factors of Fertilizer and No-fertilizer area during the observation period from July 20, 2021, to August 9, 2021. a\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e, Daily mean soil temperature and moisture at 5 cm depth. \u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee, f\u003c/strong\u003e concentrations of nitrate, nitrous oxide, ammonium, and total organic carbon at 0–10 cm soil depth. Error bars indicate standard errors. F represents conventional fertilization treatment; N represents control treatment without fertilization.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/e9e3535fe6a7fbfeac358827.png"},{"id":52931159,"identity":"b131e579-b374-4f60-afff-9d21a37c0f48","added_by":"auto","created_at":"2024-03-18 20:16:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDynamical of soil N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO fluxes and N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO/(N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO+N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) ratio of Fertilizer and No-fertilizer area during the observation period from July 20, 2021, to August 9, 2021. a\u003c/strong\u003e represents N\u003csub\u003e2\u003c/sub\u003eO fluxes. \u003cstrong\u003eb\u003c/strong\u003e represents N\u003csub\u003e2\u003c/sub\u003e fluxes. \u003cstrong\u003ec\u003c/strong\u003e represents ratio of N\u003csub\u003e2\u003c/sub\u003eO to N\u003csub\u003e2\u003c/sub\u003eO plus N\u003csub\u003e2\u003c/sub\u003e fluxes (RN\u003csub\u003e2\u003c/sub\u003eO). Error bars indicate standard errors. F represents conventional fertilization treatment; N represents control treatment without fertilization.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/fb2b84c3de865ba581a787c5.png"},{"id":52931162,"identity":"50e4644b-db21-484b-961e-dd593a744cd6","added_by":"auto","created_at":"2024-03-18 20:16:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCumulative emissions of N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO (a), N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e (b), \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO (c), \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e (d) from Fertilizer and No-fertilizer areas from July 20, 2021, to July 30, 2021.\u003c/strong\u003e F represents conventional fertilization treatment; N represents control treatment without fertilization.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/d37cd02ec5b93f65279e273a.png"},{"id":52931452,"identity":"ff0e6944-9b63-48d6-bb06-2e5420f1dd05","added_by":"auto","created_at":"2024-03-18 20:24:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125327,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN atom%-excess in soil NO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003csup\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO.\u003c/strong\u003e F and N represent the conventional fertilization and control treatment without fertilization, respectively. N\u003csub\u003e2\u003c/sub\u003eO is the N\u003csub\u003e2\u003c/sub\u003eO production (F\u003csub\u003eN2O\u003c/sub\u003e) calculated based on N\u003csub\u003e2\u003c/sub\u003eO samples at three time points. Due to our assumption that N\u003csub\u003e2\u003c/sub\u003e production was entirely derived from the reduction of N\u003csub\u003e2\u003c/sub\u003eO, the \u003csup\u003e15\u003c/sup\u003eN atom%-excess in N\u003csub\u003e2\u003c/sub\u003e is consistent with N\u003csub\u003e2\u003c/sub\u003eO and not shown in the figure.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/af15d8e8696597c6497c5dfa.png"},{"id":52931453,"identity":"1db56669-b98c-42c1-8b66-bd441a951d5a","added_by":"auto","created_at":"2024-03-18 20:24:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative contribution rate of different microbial pathways to N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO and N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e production.\u003c/strong\u003e F represents conventional fertilization treatment; N represents control treatment without fertilization.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/61dd5f4867d93c471147b47b.png"},{"id":52931158,"identity":"b572d0ab-9686-432a-8370-639f69059a52","added_by":"auto","created_at":"2024-03-18 20:16:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":50237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN recovery rate in different nitrogen pools of Fertilizer and No-fertilizer treatment.\u003c/strong\u003e F represents conventional fertilization treatment; N represents control treatment without fertilization. Residual N does not include NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/3e366a5bb3239dbf6098672c.png"},{"id":52931161,"identity":"9093318b-56bb-442b-abc3-de5fb886fd8d","added_by":"auto","created_at":"2024-03-18 20:16:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":183040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural equation model fitted with range-standardized coefficients.\u003c/strong\u003e Solid lines indicate that the driver influences the likelihood of the model via a χ2 Likelihood ratio test. Dashed paths indicate no detectable influence of the driver (\u003cem\u003eP\u003c/em\u003e\u0026gt; 0.05). Standardized coefficients are presented for each path.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/4e0c1c9b1d452536276db89e.png"},{"id":68207145,"identity":"1a129266-b5fa-47a3-9d3f-1a3e2d2ce838","added_by":"auto","created_at":"2024-11-04 16:35:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2174505,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4091615/v1/24877f63-58d8-45ea-a6fb-ce411eb03d6f.pdf"}],"financialInterests":"","formattedTitle":"Soil N2O and N2 emissions during anaerobic soil disinfestation period in a greenhouse vegetable production system: quantified by in situ 15N labeling method","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlobal greenhouse vegetable production (GVP) systems are rapidly expanding, with an area exceeding 4\u0026nbsp;million ha in China, accounting for over 80% of the global total (Fei et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qasim et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). For maximizing yield and profits, GVP systems in China are often overfertilized (Agostini et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Surveys indicate that the annual nitrogen (N) fertilizer application rates in China\u0026rsquo;s GVP systems typically exceed 2000 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with some regions surpassing 3000 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (e.g., Guo et al., 2016; Ju et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), leding to soil acidification (Guo et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lv et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), soil-borne diseases (Huang et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and a significant surplus of N (Ju et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Studies have shown that the residual nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) in the 2 m soil layer after crop harvesting in GVP systems exceeded 500 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). To address soil degradation, anaerobic soil disinfestation (ASD) is widely adopted during the summer fallow period (Di Gioia et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). This approach involves saturating the soil through irrigation, covering the soil with plastic film, and adding organic carbon (C) (Di Gioia et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, greenhouses are sealed to enhance the internal temperature and accelerate the decomposition of organic C, creating an anaerobic environment and eliminating soil diseases and pests (Momma et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). During ASD, the high-temperature and anaerobic environment favor the reduction of accumulated NO₃⁻ in the soil through denitrification, resulting in gaseous N, including N₂O and N₂ emissions (Charles et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO is a potent greenhouse gas that has been identified as a major contributor to stratospheric ozone depletion (IPCC, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). By contrast, N\u003csub\u003e2\u003c/sub\u003e is an inert, environmentally benign gas; however, its emission represents the final step of N cycling in terrestrial ecosystems. This process is crucial for closing N cycles and maintaining N balance in ecosystems. If the gaseous N is primarily N\u003csub\u003e2\u003c/sub\u003eO, it will accelerate global warming and ozone layer degradation, whereas if it is primarily N\u003csub\u003e2\u003c/sub\u003e, it will be environmentally neutral. Studies on N\u003csub\u003e2\u003c/sub\u003eO emissions from GVP systems have mainly focused on the growing season, with relatively limited research on the microbial processes involved (e.g., Liu \u0026amp; Qiu, 2016; Min et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yao et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For N\u003csub\u003e2\u003c/sub\u003e, the difficulties in directly measuring soil N\u003csub\u003e2\u003c/sub\u003e emissions (Groffman et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) have resulted in the absence of in-situ research measuring N\u003csub\u003e2\u003c/sub\u003e emission rates and distinguishing the associated microbial processes within the GVP system. This absence contributes to considerable uncertainty when assessing N loss pathways in the GVP system and estimating N\u003csub\u003e2\u003c/sub\u003e emissions in specific regions.\u003c/p\u003e \u003cp\u003eCommonly used methods for measuring N\u003csub\u003e2\u003c/sub\u003e emissions include the acetylene inhibition method (Yoshinari et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), gas flow soil core method (Butterbach-Bahl et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u003csup\u003e15\u003c/sup\u003eN labeling method (Morse \u0026amp; Bernhardt, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the incomplete inhibition of reductases by using the acetylene inhibition method can lead to underestimated N\u003csub\u003e2\u003c/sub\u003e emissions. (Felber et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The gas flow soil core method requires a long duration to create an N\u003csub\u003e2\u003c/sub\u003e-free environment, often exceeding 20 h, during which N\u003csub\u003e2\u003c/sub\u003e emissions cannot be quantified (Butterbach-Bahl et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Among these methods, only the \u003csup\u003e15\u003c/sup\u003eN labeling technique enables the in-situ measurement of N\u003csub\u003e2\u003c/sub\u003e emissions and differentiation of microbial processes responsible for gaseous N production (Stevens et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This method involves adding a certain amount of \u003csup\u003e15\u003c/sup\u003eN-enriched nitrogenous compounds to the soil and analyzing their fate after a certain period, achieving tracing and source analysis purposes (Templer et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). With the development of stable isotope technology, improvements in instrument detection techniques, and cost reduction, the \u003csup\u003e15\u003c/sup\u003eN labeling method has become widely used (He et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kulkarni et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sgouridis \u0026amp; Ullan, 2015; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, studies have used the laboratory-derived ratio of N\u003csub\u003e2\u003c/sub\u003eO / (N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003e) (R\u003csub\u003eN2O\u003c/sub\u003e) along with in situ measured N\u003csub\u003e2\u003c/sub\u003eO flux to estimate N\u003csub\u003e2\u003c/sub\u003e flux (Saggar et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, owing to the complexity of field environments, the R\u003csub\u003eN2O\u003c/sub\u003e obtained in the laboratory can not represent the actual values in fields (Kulkarni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Well et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The range of R\u003csub\u003eN2O\u003c/sub\u003e in terrestrial ecosystems is wide (Schlesinger, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), but in situ measurements of R\u003csub\u003eN2O\u003c/sub\u003e in GVP systems have not been conducted. Thus, whether the R\u003csub\u003eN2O\u003c/sub\u003e values in the laboratory are applicable for estimating in situ N\u003csub\u003e2\u003c/sub\u003e emissions from GVP systems remains to be verified.\u003c/p\u003e \u003cp\u003eIn this study, we conducted in situ measurements of soil N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions during the ASD period in a typical GVP system in Shouguang, Shandong Province, China, using the \u003csup\u003e15\u003c/sup\u003eN labeling method. The research objectives were to (1) explore the emission characteristics of N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e, (2) distinguish the microbial processes responsible for N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e production, (3) analyze the influence of environmental factors on N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions, and (4) evaluate the fate of N in the soil.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Site\u003c/h2\u003e \u003cp\u003eThe experimental site was located in Zhaili Village, Luocheng Street, Shouguang City, Shandong Province, China (E118\u0026deg;42' 4.5\u0026Prime;, N 36\u0026deg;55' 26.4\u0026Prime;), an area with a warm, temperate, continental monsoon climate. The average temperature in 2021 was 14.4\u0026deg;C, with precipitation of 895.4 mm and 2306.9 h of sunshine (Shouguang Statistical Yearbook, 2021). The soil in this area is classified as a Cambisol, with organic C, nitrate N, and ammonium N contents of 16.6 g C kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 44.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 6.7 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, in the 0\u0026ndash;10 cm soil layer, and a pH value of 7.7. The experiments were conducted in a greenhouse that had been in use for five years and was oriented in an east-west direction. Typically, in this greenhouse, two seasons of crops, tomatoes and cucumbers, had been grown each year. Farmers usually close greenhouses to conduct ASD during the summer fallow period in July and August.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Field \u003csup\u003e15\u003c/sup\u003eN Labeling Experiment\u003c/h2\u003e \u003cp\u003eThe period of the experiment was from July 20, 2021, to August 2, 2021. The experiment used two treatments: regular fertilization (Fertilizer) and no fertilization control (No-fertilizer), with five replicates for each treatment. In the Fertilizer treatment, commercial organic fertilizer was uniformly applied on July 19, 2021, at a rate of 280 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2829 kg C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After fertilization, both treatment areas were plowed and drip-irrigated. Plastic film was used to cover the soil after irrigation to prevent water evaporation. Subsequently, ten gas-sampling static chambers were randomly placed, with five in the Fertilizer area and five in the No-fertilizer treatment area. The static chambers were 30 cm long, 30 cm wide, and 10 cm high, with a water-filled groove (3 cm wide, 3 cm deep) around the base. There was a three-way valve at the top for collecting the gas samples. A grid with 36 small compartments was placed inside the static chamber, each measuring 5 cm \u0026times; 5 cm, to facilitate the addition of K\u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e. A solution of K\u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e (99 \u003csup\u003e15\u003c/sup\u003eN atom%) at a rate of 0.3 g (equivalent to 33 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was uniformly injected into each static chamber. An equal-sized static chamber base was placed next to each gas-sampling static chamber to collect the soil samples. The same method was used to inject equal amounts of K\u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e into the soil sample base (Fig S1).\u003c/p\u003e \u003cp\u003eAfter the soil was labeled, gas samples were collected three times every day. Because of the high daytime temperature inside the greenhouse, which was not conducive to sampling, gas collection times were set at 5:00, 6:00, and 19:30 daily for 14 d to obtain detectable \u003csup\u003e15\u003c/sup\u003eN-N\u003csub\u003e2\u003c/sub\u003e signals. Before the first sampling each day, the chambers were placed on the bases, and the grooves of the bases were sealed with water. The chamber was opened after the third sampling period. During sampling, a 100 mL syringe was connected to the three-way valve outlet at the top of the chamber, the three-way valve was opened, and the syringe plunger was moved five times to evenly mix the gases inside the chamber. Subsequently, 100 mL gas was collected using a syringe, and the three-way valve was switched to allow the collected gas to be transferred into a 150 mL gas bag. Balancing the pressures inside and outside the chamber was achieved by injecting 100 mL air into the chamber after gas collection. Additionally, soil samples from the 0\u0026ndash;10 cm layer were collected every 2 d (Fig S1).\u003c/p\u003e \u003cp\u003eWe used soil temperature and moisture probes (TMS4, TOMST, Czech Republic) to monitor soil (5 cm depth), temperature (\u0026deg;C), and moisture (volumetric water content, %) simultaneously. The soil volumetric water content was further converted to soil water-filled pore space (WFPS) by using the following formula: WFPS = [soil volumetric water content / (1 - (soil bulk density / 2.65)] \u0026times; 100%, where 2.65 g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e is the assumed soil particle density, and soil bulk density is 1.4 g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Analysis of N\u003csub\u003e2\u003c/sub\u003eO and Flux Calculation\u003c/h2\u003e \u003cp\u003eWe measured N\u003csub\u003e2\u003c/sub\u003eO concentration by using a gas chromatograph (GC2014, Shimadzu, Japan). The N\u003csub\u003e2\u003c/sub\u003eO flux (F\u003csub\u003eN2O\u003c/sub\u003e, \u0026micro;g m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was calculated based on the linear change in N\u003csub\u003e2\u003c/sub\u003eO concentration over time using the following formula:\u003c/p\u003e \u003cp\u003eF\u003csub\u003eN2O\u003c/sub\u003e = (dc/dt) \u0026times; ρN\u003csub\u003e2\u003c/sub\u003eO \u0026times; V/A (1)\u003c/p\u003e \u003cp\u003eIn Eq.\u0026nbsp;(1), the N\u003csub\u003e2\u003c/sub\u003eO flux comprises flux from both the \u003csup\u003e15\u003c/sup\u003eN-labeled source and unlabeled sources; dc/dt is the rate of change in N\u003csub\u003e2\u003c/sub\u003eO concentration over time determined using linear regression; ρN\u003csub\u003e2\u003c/sub\u003eO is the density of N\u003csub\u003e2\u003c/sub\u003eO at standard conditions; and V and A are the volume (0.009 m\u003csup\u003e3\u003c/sup\u003e) and base area (0.09 m\u003csup\u003e2\u003c/sup\u003e) of the static chamber, respectively.\u003c/p\u003e \u003cp\u003eThe \u003csup\u003e15\u003c/sup\u003eN abundance of N\u003csub\u003e2\u003c/sub\u003eO was determined using a combined system comprising a continuous-flow isotope ratio mass spectrometry (IRMS, IsoPrime 100, Cheadle, UK), low-temperature focusing unit (Trace Gas Preconcentrator, IsoPrince Limited), and 112-position autosampler (Gilson GX-271, Dunstable, UK). The peak areas of \u003csup\u003e44\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO, \u003csup\u003e45\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO, and \u003csup\u003e46\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO and the ratios \u003csup\u003e45\u003c/sup\u003eR (\u003csup\u003e45\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO/\u003csup\u003e44\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO) and \u003csup\u003e46\u003c/sup\u003eR (\u003csup\u003e46\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO/\u003csup\u003e44\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO) were measured using IRMS. Studies have indicated that in high-abundance \u003csup\u003e15\u003c/sup\u003eN labeling experiments, isotope fractionation can be ignored (Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, the \u003csup\u003e15\u003c/sup\u003eN abundance in N\u003csub\u003e2\u003c/sub\u003eO was calculated using formula (2), assuming \u003csup\u003e17\u003c/sup\u003eR (\u003csup\u003e17\u003c/sup\u003eO/\u003csup\u003e16\u003c/sup\u003eO)\u0026thinsp;=\u0026thinsp;3.8861\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e and \u003csup\u003e18\u003c/sup\u003eR (\u003csup\u003e18\u003c/sup\u003eO/\u003csup\u003e16\u003c/sup\u003eO)\u0026thinsp;=\u0026thinsp;2.0947\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e. The \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO flux (F\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO) was estimated from the difference in \u003csup\u003e15\u003c/sup\u003eN abundance in the samples collected at different times and the total N\u003csub\u003e2\u003c/sub\u003eO flux (Eq.\u0026nbsp;3). Eq.\u0026nbsp;4 was used to estimate the N\u003csub\u003e2\u003c/sub\u003eO flux produced by denitrification processes (Buchen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\({ }^{15}{\\text{X}}_{\\text{N}2\\text{O}}\\)\u003c/span\u003e \u003c/span\u003e= 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times \\frac{{ }^{45}\\text{R} + 2 \\times { }^{46}\\text{R} -{ }^{17}\\text{R}-2 \\times { }^{18}\\text{R}}{2 + 2 \\times { }^{45}\\text{R} + 2 \\times { }^{46}\\text{R}}\\)\u003c/span\u003e\u003c/span\u003e (2)\u003c/p\u003e \u003cp\u003eF\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO= \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\text{C}}_{\\text{N}2\\text{O}\\text{t}}{\\times }^{15}{{\\text{X}}_{\\text{N}2\\text{O}\\text{t}}}^{ }- {\\text{C}}_{\\text{N}2\\text{O}0} {\\times }^{15}{\\text{X}}_{\\text{N}2\\text{O}0}}{{\\text{C}}_{\\text{N}2\\text{o}\\text{t} -} {\\text{C}}_{\\text{N}2\\text{O}0}}\\)\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times {\\text{F}}_{\\text{N}2\\text{O}}\\)\u003c/span\u003e\u003c/span\u003e (3)\u003c/p\u003e \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{C}}_{\\text{N}2\\text{O}0}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\text{C}}_{\\text{N}2\\text{O}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e represent the N\u003csub\u003e2\u003c/sub\u003eO concentrations at 5:00 and 19:30, respectively, in the gas samples collected daily, and \u003csup\u003e15\u003c/sup\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\text{X}}_{\\text{N}2\\text{O}0}}^{ }\\)\u003c/span\u003e\u003c/span\u003eand \u003csup\u003e15\u003c/sup\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({{\\text{X}}_{\\text{N}2\\text{O}\\text{t}}}^{ }\\)\u003c/span\u003e\u003c/span\u003erepresent the \u003csup\u003e15\u003c/sup\u003eN abundances in the N\u003csub\u003e2\u003c/sub\u003eO samples at 5:00 and 19:30, respectively.\u003c/p\u003e \u003cp\u003eFN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003edenitrification\u003c/sub\u003e = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\text{F}}^{15}{\\text{N}}_{2}\\text{O}}{{ }^{15}\\text{X}{ }_{{{\\text{N}\\text{O}}_{3}}^{-}}}\\)\u003c/span\u003e\u003c/span\u003e (4)\u003c/p\u003e \u003cp\u003eWhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({ }^{15}\\text{X}{ }_{{{\\text{N}\\text{O}}_{3}}^{-}}\\)\u003c/span\u003e\u003c/span\u003erepresents the \u003csup\u003e15\u003c/sup\u003eN abundance of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the soil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of N\u003csub\u003e2\u003c/sub\u003e and Flux Calculation\u003c/h2\u003e \u003cp\u003e \u003csup\u003e15\u003c/sup\u003eN abundance in N\u003csub\u003e2\u003c/sub\u003e was determined using the aforementioned trace gas pre-concentration system and isotope ratio mass spectrometry (TG-IRMS). The peak areas of \u003csup\u003e28\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e, \u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e, and \u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e, as well as the ratios \u003csup\u003e29\u003c/sup\u003eR (\u003csup\u003e29\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e/\u003csup\u003e28\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e) and \u003csup\u003e30\u003c/sup\u003eR (\u003csup\u003e30\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e/\u003csup\u003e28\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e) in the sample, were measured using a mass spectrometer. The \u003csup\u003e15\u003c/sup\u003eN mole fraction (\u003csup\u003e15\u003c/sup\u003eX\u003csub\u003eN2\u003c/sub\u003e) and \u003csup\u003e15\u003c/sup\u003eN flux (F\u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e) of N\u003csub\u003e2\u003c/sub\u003e were calculated using Equations (5) and (6), respectively. Subsequently, the N\u003csub\u003e2\u003c/sub\u003e flux produced by denitrification (FN\u003csub\u003e2\u003c/sub\u003e-denitrification) was calculated using Eq.\u0026nbsp;(7). Assuming that all N\u003csub\u003e2\u003c/sub\u003e was derived from the reduction of N\u003csub\u003e2\u003c/sub\u003eO, total N\u003csub\u003e2\u003c/sub\u003e flux (FN\u003csub\u003e2\u003c/sub\u003e-total) was estimated using Eq.\u0026nbsp;(8) (Yang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003csup\u003e15\u003c/sup\u003eX\u003csub\u003eN2\u003c/sub\u003e = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{ }^{29}\\text{R} + 2{\\times }^{ 30}\\text{R}}{2 + 2 {\\times }^{ 29}\\text{R} + 2 {\\times }^{30}\\text{R}}\\)\u003c/span\u003e\u003c/span\u003e(5)\u003c/p\u003e \u003cp\u003eF\u003csup\u003e15\u003c/sup\u003e\u003csub\u003eN2\u003c/sub\u003e = (d\u003csup\u003e15\u003c/sup\u003eX\u003csub\u003eN2\u003c/sub\u003e /dt) \u0026times; ρN\u003csub\u003e2\u003c/sub\u003e\u0026times; V/A (6)\u003c/p\u003e \u003cp\u003eρN\u003csub\u003e2\u003c/sub\u003e is the density of N\u003csub\u003e2\u003c/sub\u003e at standard conditions; V and A are the volume (0.009 m\u003csup\u003e3\u003c/sup\u003e) and base area (0.09 m\u003csup\u003e2\u003c/sup\u003e) of the static box.\u003c/p\u003e \u003cp\u003eFN\u003csub\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;denitrification\u003c/sub\u003e = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\text{F}}^{15}{\\text{N}}_{2}}{{ }^{15}\\text{X}{ }_{{{\\text{N}\\text{O}}_{3}}^{-}}}\\)\u003c/span\u003e\u003c/span\u003e (7)\u003c/p\u003e \u003cp\u003eFN\u003csub\u003e2\u0026thinsp;\u0026minus;\u0026thinsp;total\u003c/sub\u003e = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{{\\text{F}}^{15}{\\text{N}}_{2}}{{ }^{15}\\text{X}{ }_{\\text{N}2\\text{O}}}\\)\u003c/span\u003e\u003c/span\u003e (8)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Soil Parameter Analysis\u003c/h2\u003e \u003cp\u003eThe soil samples were sieved through a 2 mm mesh. A portion of the sieved soil was used to determine the total N (TN) content by using an elemental analyzer (Elementar Analysen Systeme, GmbH, Germany), and another 10 g soil was extracted with 100 ml 2 M KCl solution, and the mixture was shaken for 1 h. The extract was frozen for preservation and analyzed for ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N), nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N), and nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) content by using a fully automatic discrete chemical analyzer (Smartchem 200, Westco Scientific Instruments, Inc., Italy). The soil total organic carbon (TOC) content was determined using a TOC/TN analyzer (Analytic Jean, Germany). The \u003csup\u003e15\u003c/sup\u003eN enrichment of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the soil extract was determined using the denitrifying bacterial method with \u003cem\u003epseudomonas aureofaciens\u003c/em\u003e. The specific method was activating the bacterial strain in 50 mL culture medium, transferring it to a 500 mL Erlenmeyer flask with fresh culture medium, and cultivating it at a constant temperature of 28\u0026deg;C for 4 d to amplify the denitrifying bacteria and deplete the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. The bacterial liquid was then purified by centrifugation at 15\u0026deg;C and 4000 rpm, and the resulting colonies were re-cultured, mixed, and dispensed into clean headspace vials, each containing approximately 3\u0026ndash;6 mL of bacterial liquid. The vials were promptly sealed and purged with high-purity N\u003csub\u003e2\u003c/sub\u003e to remove any N\u003csub\u003e2\u003c/sub\u003eO produced from background NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the culture medium. Soil extract samples were then added to headspace vials and allowed to react overnight at 28\u0026deg;C to ensure the complete conversion of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to N\u003csub\u003e2\u003c/sub\u003eO. The N\u003csub\u003e2\u003c/sub\u003eO generated from the conversion was measured for \u003csup\u003e15\u003c/sup\u003eN enrichment by using TG-IRMS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analyses\u003c/h2\u003e \u003cp\u003eThis study used R (4.1.1) and RStudio for statistical analysis and data visualization. A piecewise structural equation model (SEM) was constructed to assess the direct and indirect effects of key factors on N\u003csub\u003e2\u003c/sub\u003eO, N\u003csub\u003e2\u003c/sub\u003e, and R\u003csub\u003eN2O\u003c/sub\u003e. Piecewise, the SEM also reported a marginal R\u003csup\u003e2\u003c/sup\u003e (R\u003csup\u003e2\u003c/sup\u003eM, the proportion of variance explained by fixed effects). The analysis was conducted using the \"piecewiseSEM\" package (Lefcheck, 2016), with linear mixed-effects models matching the \"lme\" function of the \"nlme\" package. Four indicators were selected to represent a good model fit: low Fisher\u0026rsquo;s C value, non-significant \u003cem\u003eP\u003c/em\u003e values (\u0026gt;\u0026thinsp;0.05), low Akaike Information Criterion (AIC), and Bayesian Information Criterion (BIC) values. All data analyses were performed at a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Soil Physical-chemical Parameters\u003c/h2\u003e\n \u003cp\u003eDuring the experiment, the soil temperature at a depth of 5 cm ranged between 30\u0026deg;C and 45\u0026deg;C, with an average of 39.1\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). The mean WFPS in the 0\u0026ndash;10 cm soil layer was 96% for the Fertilizer treatment and 100% for the No-fertilizer treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the Fertilizer treatment, the average concentrations of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, and TOC in the 0\u0026ndash;10 cm soil were 56.6, 0.2, and 8.6 mg N kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 79.5 mg C kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, and in the No-fertilizer treatment, these concentrations were 58.0, 0.2, and 6.9 mg N kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 60.8 mg C kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, d, e, f).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Soil N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e Emissions\u003c/h2\u003e\n \u003cp\u003eOn day 1 of the experiment, the N\u003csub\u003e2\u003c/sub\u003eO fluxes for the Fertilizer and No-fertilizer treatments reached peak values, 0.42 and 0.18 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Subsequently, the N\u003csub\u003e2\u003c/sub\u003eO fluxes decreased and remained relatively low from days 2 to 14. During this period, the N\u003csub\u003e2\u003c/sub\u003eO fluxes under the Fertilizer treatment ranged from 0.02 to 0.06 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, averaging 0.03 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and under the No-fertilizer treatment, from 0.02 to 0.03 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, averaging 0.02 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). During the 14 days period, the total N\u003csub\u003e2\u003c/sub\u003eO emissions for the Fertilizer and No-fertilizer treatments were 0.82 and 0.47 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with associated \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions totaling 0.14 and 0.07 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn the Fertilizer treatment, the peak N\u003csub\u003e2\u003c/sub\u003e flux was on day 2 (12.4 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), gradually decreasing to an average of 2.9 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In the No-fertilizer treatment, the N\u003csub\u003e2\u003c/sub\u003e flux gradually increased after the start of the experiment, peaked on day 3 (6.9 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and then gradually decreased to an average of 1.8 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Over the 14 d period, the total N\u003csub\u003e2\u003c/sub\u003e emissions for the Fertilizer and No-fertilizer treatments were 40.7 and 25.5 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, with the associated \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e emissions totaling 4.3 and 1.9 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 N\u003csub\u003e2\u003c/sub\u003eO/(N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003e) ratio\u003c/h2\u003e\n \u003cp\u003eDuring the experiment, the average R\u003csub\u003eN2O\u003c/sub\u003e for the Fertilizer and No-fertilizer treatments was 0.28 and 0.32, respectively, with no significant difference between the treatments except on day 13. In the first 6 days, both treatments exhibited low R\u003csub\u003eN2O\u003c/sub\u003e values, with the Fertilizer treatment ranging from 0.007 to 0.015 (average 0.024) and the No-fertilizer treatment ranging from 0.006 to 0.087 (average 0.026). On day 7, R\u003csub\u003eN2O\u003c/sub\u003e began to increase. From days 7 to 14, R\u003csub\u003eN2O\u003c/sub\u003e levels ranged from 0.21 to 0.75 (average 0.46) for the the Fertilizer treatment and from 0.29 to 0.73 (average 0.53) for the No-fertilizer treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 \u003csup\u003e15\u003c/sup\u003eN Atom%-excess and Biological Processes\u003c/h2\u003e\n \u003cp\u003eDuring the experiment, sufficient labeled \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e substrate was present in the soil for both treatments. The \u003csup\u003e15\u003c/sup\u003eN abundance of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the Fertilizer treatment decreased gradually from 34.4\u0026ndash;7.2%, and that in the No-fertilizer treatment decreased from 30.4\u0026ndash;9.6%. From days 1 to 3, the \u003csup\u003e15\u003c/sup\u003eN abundance of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the Fertilizer treatment was higher than that in the No-fertilizer treatment but decreased to below that of the No-fertilizer treatment in subsequent days. Similar trends were observed for the \u003csup\u003e15\u003c/sup\u003eN abundance of N\u003csub\u003e2\u003c/sub\u003eO in both treatments. In the Fertilizer treatment, the \u003csup\u003e15\u003c/sup\u003eN abundance of N\u003csub\u003e2\u003c/sub\u003eO decreased from 26.3\u0026ndash;0.88%, and in the No-fertilizer treatment, it decreased from 32.7\u0026ndash;0.91% (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). When calculating the N\u003csub\u003e2\u003c/sub\u003e emissions, we assumed that the generated N\u003csub\u003e2\u003c/sub\u003e originated from the reduction of N\u003csub\u003e2\u003c/sub\u003eO; hence, the \u003csup\u003e15\u003c/sup\u003eN enrichment of N\u003csub\u003e2\u003c/sub\u003e was consistent with that of N\u003csub\u003e2\u003c/sub\u003eO. Based on the \u003csup\u003e15\u003c/sup\u003eN abundances of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, N\u003csub\u003e2\u003c/sub\u003eO, and N\u003csub\u003e2\u003c/sub\u003e, we estimated the emissions of N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e derived from the denitrification processes, denoted as D\u003csub\u003eN2O\u003c/sub\u003e and D\u003csub\u003eN2\u003c/sub\u003e, respectively. During the first 1\u0026ndash;2 days of the experiment, N\u003csub\u003e2\u003c/sub\u003eO was primarily derived from the denitrification process, with its contribution decreasing thereafter. N\u003csub\u003e2\u003c/sub\u003e produced by denitrification reached its peak on the second day and then gradually decreased. In the Fertilizer treatment, D\u003csub\u003eN2O\u003c/sub\u003e was 0.45 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 51.2% of the total N\u003csub\u003e2\u003c/sub\u003eO emissions, and in the No-fertilizer treatment, it was 0.23 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 48.9% of the total N\u003csub\u003e2\u003c/sub\u003eO emissions. For N\u003csub\u003e2\u003c/sub\u003e, in the Fertilizer treatment, D\u003csub\u003eN2\u003c/sub\u003e was 14.4 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 36.7% of the total N\u003csub\u003e2\u003c/sub\u003e emissions, and in the No-Fertilizer treatment, it was 6.9 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 27.1% of the total N\u003csub\u003e2\u003c/sub\u003e emissions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 \u003csup\u003e15\u003c/sup\u003eN Recovery Rate\u003c/h2\u003e\n \u003cp\u003eFrom July 20, 2021, to September 5, 2021, the total \u003csup\u003e15\u003c/sup\u003eN recovery rates for the N pools in the Fertilizer and No-fertilizer treatments were 76.0% and 70.0%, respectively. After the addition of organic fertilizer, the N residue increased, primarily in the form of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. Under conventional fertilization conditions, the losses of \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO accounted for 13.6% and 0.5% of the added \u003csup\u003e15\u003c/sup\u003eN, respectively. Under no fertilization conditions, the losses of \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e and \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO accounted for 6.3% and 0.2% of the added \u003csup\u003e15\u003c/sup\u003eN, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Relationships Among Gaseous N Rates, Ratios, and Soil Properties\u003c/h2\u003e\n \u003cp\u003eThe piecewise SEM indicated that the application of organic fertilizer and soil environmental factors accounted for 55%, 13%, and 30% of the variance in N\u003csub\u003e2\u003c/sub\u003eO, N\u003csub\u003e2\u003c/sub\u003e, and R\u003csub\u003eN2O\u003c/sub\u003e, respectively. The N\u003csub\u003e2\u003c/sub\u003eO emission rate was mainly influenced by soil temperature and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content, showing a significant negative correlation with soil temperature (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a significant positive correlation with NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The N\u003csub\u003e2\u003c/sub\u003e emission rate was significantly negatively correlated with the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and the N\u003csub\u003e2\u003c/sub\u003e emission rate directly affected R\u003csub\u003eN2O\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, Pearson partial correlation coefficients and multiple stepwise regression models were used for N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions and for D\u003csub\u003eN2O\u003c/sub\u003e and D\u003csub\u003eN2\u003c/sub\u003e to further investigate the influence of soil environmental factors. The results revealed that N\u003csub\u003e2\u003c/sub\u003eO emissions, D\u003csub\u003eN2O\u003c/sub\u003e, and D\u003csub\u003eN2\u003c/sub\u003e were significantly negatively correlated with soil temperature, whereas R\u003csub\u003eN2O\u003c/sub\u003e was significantly positively correlated with soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePearson partial correlation coefficients and multiple stepwise regression models for N\u003csub\u003e2\u003c/sub\u003eO, N\u003csub\u003e2\u003c/sub\u003e fluxes and N\u003csub\u003e2\u003c/sub\u003eO, N\u003csub\u003e2\u003c/sub\u003e from denitrification and the R\u003csub\u003eN2O\u003c/sub\u003e (dependent variables), as a function of soil variables (regressors)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTreatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDependent variables\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003ePearson\u0026rsquo;s R correlation coefficients of regressors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRegression models\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTOC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWFPS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.37*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.35*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.68***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;0.70\u0026thinsp;+\u0026thinsp;0.44 N\u003csub\u003e2\u003c/sub\u003eO\u003csup\u003e\u0026minus;\u003c/sup\u003e + 0.14 NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026minus;\u0026thinsp;0.01 TOC \u0026minus;\u0026thinsp;0.02T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003eN2O\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.80***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.66***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;0.63\u0026thinsp;+\u0026thinsp;0.42 NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e + 0.17 NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026minus;\u0026thinsp;0.02 T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003eN2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.36*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.48**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.45**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.56**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;9.43\u0026thinsp;\u0026minus;\u0026thinsp;0.33 NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e + 2.66 N\u003csub\u003e2\u003c/sub\u003eO\u003csup\u003e\u0026minus;\u003c/sup\u003e \u0026minus;\u0026thinsp;0.06 TOC \u0026minus;\u0026thinsp;0.13 T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.37*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.65***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;0.49\u0026thinsp;\u0026minus;\u0026thinsp;0.001 TOC \u0026minus;\u0026thinsp;0.01 T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003eN2O\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.38*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.65***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;0.58\u0026thinsp;\u0026minus;\u0026thinsp;0.001 TOC \u0026minus;\u0026thinsp;0.01 T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003csub\u003eN2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.39*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.53**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;5.08\u0026thinsp;\u0026minus;\u0026thinsp;0.02 TOC \u0026minus;\u0026thinsp;0.01 T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eF\u0026thinsp;+\u0026thinsp;N\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003csub\u003eN2O\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.31*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eY\u0026thinsp;=\u0026thinsp;0.04\u0026thinsp;+\u0026thinsp;0.01 NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003eNitrate, DOC, ammonium, soil temperature, and soil water content were considered independent variables (X), whereas N\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003eO, D\u003csub\u003eN2\u003c/sub\u003e, D\u003csub\u003eN2O\u003c/sub\u003e, and R\u003csub\u003eN2O\u003c/sub\u003e were considered dependent variables (Y). D\u003csub\u003eN2O\u003c/sub\u003e and D\u003csub\u003eN2\u003c/sub\u003e are the N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e fluxes generated during denitrification, respectively. F represents conventional fertilization treatment; N represents control treatment without fertilization. F\u0026thinsp;+\u0026thinsp;N indicates that the data were combined for all treatments.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"10\"\u003e*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level of significance; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 level of significance; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 level of significance.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.1 N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e Emissions\u003c/h2\u003e \u003cp\u003eIn contrast with most studies on N\u003csub\u003e2\u003c/sub\u003eO emissions in GVP systems during the growing season, we investigated the characteristics of N\u003csub\u003e2\u003c/sub\u003eO emissions during the ASD period. On day 1 of ASD, the N\u003csub\u003e2\u003c/sub\u003eO flux reached its peak, consistent with the results of our automatic monitoring during the previous year's ASD period at the same site (Li et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and the findings of Qasim et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Different from the typical pattern of N\u003csub\u003e2\u003c/sub\u003eO flux reaching its peak two to three weeks after fertilization during the growing season, the high-temperature and high-WFPS soil environment during the ASD period favors the rapid mineralization of organic fertilizers, leading to an earlier peak in N\u003csub\u003e2\u003c/sub\u003eO emissions (He et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In our study, the average N\u003csub\u003e2\u003c/sub\u003eO fluxes for the Fertilizer and No-fertilizer treatments were 0.03 and 0.02 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These values were lower than those reported during ASD by Qasim et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (0.1\u0026ndash;0.5 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and Zhao et al. (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) (0.4\u0026ndash;1.6 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This difference is probably due to the higher amount of organic C added during ASD compared to our experiment. Zhao et al. (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) added 12.6 t C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of corn straw and 2.7 t C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of chicken manure, and Qasim et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) added 8.8 t C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of rice husks and 4.2 t C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of chicken manure, far exceeding the 2.8 t C ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e added in our study.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this study represents the first in-situ measurement of soil N\u003csub\u003e2\u003c/sub\u003e emissions in the GVP system. We found that fertilization significantly promoted N\u003csub\u003e2\u003c/sub\u003e emissions, consistent with the findings of Chen et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Roobroeck et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In contrast with N\u003csub\u003e2\u003c/sub\u003eO emissions, N fertilizer application did not immediately lead to a peak in N\u003csub\u003e2\u003c/sub\u003e emissions. Dendooven \u0026amp; Anderson (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) suggested that the lag phase between the N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e peaks was due to the different synthesis times of the enzymes involved in the production and consumption of N\u003csub\u003e2\u003c/sub\u003eO under anaerobic conditions. Another reason might be that different from N\u003csub\u003e2\u003c/sub\u003eO, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e preferentially accepts electrons (Firestone \u0026amp; Tiedje, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1979\u003c/span\u003e). In our study, the average N\u003csub\u003e2\u003c/sub\u003e emission rates for the Fertilizer and No-fertilizer treatments were 2.9 and 1.8 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, higher than the average N\u003csub\u003e2\u003c/sub\u003e emission rate (approximately 0.6 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) observed in Shouguang GVP soil under the incubator conditions of 25\u0026deg;C and 80% WHC (Zhao et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). This is mainly attributed to the addition of urea in that study, which lacked C sources and limited the occurrence of denitrification process (Senbayram et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Pan et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) collected N\u003csub\u003e2\u003c/sub\u003e emission data from studies that used the \u003csup\u003e15\u003c/sup\u003eN labeling method, and they found that the N\u003csub\u003e2\u003c/sub\u003e fluxes in farmland, grassland, forest, and marsh were in the range of 0.06\u0026ndash;0.6, 0.2\u0026ndash;2.4, 0.2\u0026ndash;4.1, and 0.1\u0026ndash;0.6 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Our study revealed that the highest N\u003csub\u003e2\u003c/sub\u003e flux during the ASD period in the GVP system (12.4 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), a peak significantly higher than that in other ecosystems.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Microbial Processes Responsible for N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e Production\u003c/h2\u003e \u003cp\u003eBy applying the \u003csup\u003e15\u003c/sup\u003eN isotope tracing technique, we distinguished the microbial processes contributing to soil N\u003csub\u003e2\u003c/sub\u003eO emissions in the GVP system through in situ monitoring for the first time. The results revealed that N\u003csub\u003e2\u003c/sub\u003eO derived from denitrification accounted for 51.2% and 41.9% of the total N\u003csub\u003e2\u003c/sub\u003eO emissions in the Fertilizer and No-fertilizer treatments, respectively. Soil temperature was significantly negatively correlated with D\u003csub\u003eN2O\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The reason for this result may be that from day 2 of the experiment, the soil temperature exceeded 35\u0026deg;C, inhibiting denitrification (Lai \u0026amp; Denton, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tan et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Yu (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also found that the N\u003csub\u003e2\u003c/sub\u003eO produced by denitrification showed an initial increase followed by a decrease with increasing temperature, with the peak occurring at 25\u0026deg;C or 30\u0026deg;C in an incubator experiment. In that study, the N\u003csub\u003e2\u003c/sub\u003eO generated by nitrification and co-denitrification increased with temperatures ranging from 5\u0026deg;C to 35\u0026deg;C.\u003c/p\u003e \u003cp\u003eWe speculated that N\u003csub\u003e2\u003c/sub\u003eO also originated from nitrification and co-denitrification in addition to denitrification processes in our study. Previous incubator experiments have shown that nitrification and denitrification are the main sources of N\u003csub\u003e2\u003c/sub\u003eO in acidic and alkaline GVP soils, respectively. The soil in the Shouguang GVP system is alkaline, and under incubator conditions with temperatures ranging from 15\u0026deg;C to 35\u0026deg;C and a WFPS of 65%, the denitrification only account for 15\u0026ndash;21% of total N\u003csub\u003e2\u003c/sub\u003eO emissions (Duan et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, WFPS was above 95% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) but had no significant impact on N\u003csub\u003e2\u003c/sub\u003eO emissions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Studies have suggested that when soil WFPS exceeds 60%, N\u003csub\u003e2\u003c/sub\u003eO is primarily produced through denitrification (Davidson et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pilegaard, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), but many studies refute this view. Sgouridis \u0026amp; Ullah (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) conducted in situ \u003csup\u003e15\u003c/sup\u003eN labeling experiments on various terrestrial soils in the United Kingdom and found that denitrification rates in organic soils decreased as the WFPS increased beyond 60%. Other investigations have indicated that nitrification processes continue to occur in soil conditions between 85% and 100% WFPS, contributing between 33% and 53% of N\u003csub\u003e2\u003c/sub\u003eO emissions. (Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Johannes et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) conducted incubator experiments on subtropical pasture soils in Australia, and after 6 d of incubator under 100% WFPS conditions, nitrification processes contributed more to N\u003csub\u003e2\u003c/sub\u003eO emissions than denitrification processes did; they also suggested that under flooded conditions, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e oxidation could still occur if denitrification processes were inhibited. Studies have also shown that co-denitrification occurs in grasslands (Laughlin \u0026amp; Stevens, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Selbie et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and croplands (Long et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Under the same NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e substrate concentration conditions, the potential for N\u003csub\u003e2\u003c/sub\u003eO production through co-denitrification (2 mol NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e produces 2 mol N\u003csub\u003e2\u003c/sub\u003eO) is twice that of denitrification (2 mol NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e produces 1 mol N\u003csub\u003e2\u003c/sub\u003eO). Spott \u0026amp; Florian (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) added NH\u003csub\u003e2\u003c/sub\u003eOH to agricultural soil extracts and found that \u0026gt;\u0026thinsp;98% of N\u003csub\u003e2\u003c/sub\u003eO was produced by co-denitrification. Through incubator studies on forest soils, Li et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) discovered that 10.6% and 30.7% of the total N\u003csub\u003e2\u003c/sub\u003eO emissions were generated via co-denitrification. Li et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) conducted a \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e addition experiment and found that co-denitrification processes contributed N\u003csub\u003e2\u003c/sub\u003eO emissions from 1.8\u0026ndash;34.2%.\u003c/p\u003e \u003cp\u003eWe used the \u003csup\u003e15\u003c/sup\u003eN labeling technique to quantify the N\u003csub\u003e2\u003c/sub\u003e emissions originating from denitrification processes. In our study, N\u003csub\u003e2\u003c/sub\u003e produced through denitrification accounted for 36.7% and 27.1% of the total N\u003csub\u003e2\u003c/sub\u003e emissions in the Fertilizer and No-fertilizer treatments, respectively. As the experiment progressed, D\u003csub\u003eN2\u003c/sub\u003e gradually decreased, showing a significant negative correlation with soil temperature (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Similar to our speculation regarding D\u003csub\u003eN2O\u003c/sub\u003e, we speculated that the inhibition of denitrification processes occurred when soil temperature exceeded 35\u0026deg;C (Lai \u0026amp; Denton, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tan et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yu, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, a significant negative correlation was observed between D\u003csub\u003eN2\u003c/sub\u003e and TOC content (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A reason for this result might be that a higher C/N ratio in the soil increases the microbial assimilation of N (Aulakh et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Another reason is that a lower N content may not fulfill microbial demand, and the reduced N consumption caused by net N fixation leads to a decrease in nitrification and denitrification rates (Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Other studies have also reported a negative correlation between C/N and the potential for microbial processes such as denitrification (Klemedtsson et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Ollinger et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Sgouridis \u0026amp; Ullah, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ullah \u0026amp; Moore, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe found that, in addition to the denitrification processes, co-denitrification accounted for 63.3% and 72.9% of N\u003csub\u003e2\u003c/sub\u003e in the Fertilizer and No-fertilizer treatments, respectively, over the ASD period. Co-denitrification, a biological process that occurs under moderate-to-high pH conditions (\u0026gt;\u0026thinsp;6), is responsible for producing N\u003csub\u003e2\u003c/sub\u003e. Selbie et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) conducted in situ \u003csup\u003e15\u003c/sup\u003eN labeling experiments in grasslands and found that over 98% of N\u003csub\u003e2\u003c/sub\u003e emissions were attributed to co-denitrification. Laughlin \u0026amp; Stevens (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) demonstrated that after applying \u003csup\u003e15\u003c/sup\u003eN-labeled NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e to grassland soil, 92% of N\u003csub\u003e2\u003c/sub\u003e emissions resulted from co-denitrification, with 8% from denitrification. Incubator experiments of Li et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and Xi et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) on forest soils in Northeast China, involving anaerobic conditions and \u003csup\u003e15\u003c/sup\u003eN labeling, have revealed contributions of co-denitrification to N\u003csub\u003e2\u003c/sub\u003e ranging from 4.9\u0026ndash;14.4%. Additionally, anaerobic ammonia oxidation (ANAMMOX) is also an important mechanism for N\u003csub\u003e2\u003c/sub\u003e production. However, research has suggested that ANAMMOX is primarily present in paddy fields and wetland soils, and its specific genetic potential is relatively low, contributing little to N\u003csub\u003e2\u003c/sub\u003e emissions (Shen et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ligi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Our study only estimated the N\u003csub\u003e2\u003c/sub\u003e emissions originating from N\u003csub\u003e2\u003c/sub\u003eO reduction, excluding the N\u003csub\u003e2\u003c/sub\u003e produced from the ANAMMOX process. Consequently, we may have underestimated be a certain degree of underestimation in the total N\u003csub\u003e2\u003c/sub\u003e emissions. Further research is still necessary to quantify the N\u003csub\u003e2\u003c/sub\u003e emissions from the ANAMMOX process. In summary, in addition to denitrification, other processes in the GVP system should be considered when examining N\u003csub\u003e2\u003c/sub\u003e emissions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3 N\u003csub\u003e2\u003c/sub\u003eO/(N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003e) Ratio\u003c/h2\u003e \u003cp\u003eBecause of the difficulty in directly measuring N\u003csub\u003e2\u003c/sub\u003e, researchers have estimated soil N\u003csub\u003e2\u003c/sub\u003e emissions at site, regional, or global scales by combining R\u003csub\u003eN2O\u003c/sub\u003e values with in-situ measurements of soil N\u003csub\u003e2\u003c/sub\u003eO emissions (Saggar et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Schlesinger, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, R\u003csub\u003eN2O\u003c/sub\u003e values have significantly differed in different soil types. Schlesinger (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) collected R\u003csub\u003eN2O\u003c/sub\u003e data from the literature and calculated average values for denitrification processes in terrestrial, farmland, and wetland areas as 0.49, 0.37, and 0.082, respectively. Based on our study, the average R\u003csub\u003eN2O\u003c/sub\u003e values for the Fertilizer and No-fertilizer treatments were 0.28 and 0.32, respectively, during the ASD period in the GVP system. Different soil physicochemical properties and microbial communities lead to varying control factors for R\u003csub\u003eN2O\u003c/sub\u003e (Senbayram et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Sgouridis \u0026amp; Ullan (2015) found that R\u003csub\u003eN2O\u003c/sub\u003e concentrations in natural and unmanaged ecosystems ranged from 0.01 to 0.07. They observed that organic soils were mainly influenced by soil temperature and pH, deciduous forests were affected by soil moisture; mixed forests were influenced by soil organic C and pH; semi-improved grasslands were affected by soil organic C; and improved grasslands were affected by soil bulk density, organic C, moisture, and pH. The in-situ labeling experiment conducted by Pan et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) revealed a significant correlation between R\u003csub\u003eN2O\u003c/sub\u003e and soil temperature and moisture in cron field. Xi et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed that R\u003csub\u003eN2O\u003c/sub\u003e in forest soils was mainly influenced by soil moisture through in-situ monitoring. Our study found a significant positive correlation between R\u003csub\u003eN2O\u003c/sub\u003e and soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e content (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), consistent with the findings of Saggar et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Scheer et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The reason for this relationship is primarily due to the inhibition of N\u003csub\u003e2\u003c/sub\u003eO reductase activity at high NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations. Additionally, microorganisms obtain more energy from reducing NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e than N\u003csub\u003e2\u003c/sub\u003eO, so high NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentrations are more favorable for N\u003csub\u003e2\u003c/sub\u003eO production rather than N\u003csub\u003e2\u003c/sub\u003eO reduction. (Senbayram et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; van Cleemput, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Weier et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1993\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies have mostly aimed to reveal the variations in R\u003csub\u003eN2O\u003c/sub\u003e among different soils, not the temporal variability of R\u003csub\u003eN2O\u003c/sub\u003e. Our study found a significant difference in R\u003csub\u003eN2O\u003c/sub\u003e between the first 6 days and the subsequent 8 days (with average values of 0.024 and 0.026 for the Fertilizer and No-fertilizer treatments in the first 6 days, and 0.46 and 0.53, respectively, in the subsequent 8 days). In an in-situ labeling experiment, Liu et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that R\u003csub\u003eN2O\u003c/sub\u003e during the maize season was much higher than that during the wheat season (0.49 vs. 0.02) in the same field. Wang et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found in their maize soil incubator experiment that the average R\u003csub\u003eN2O\u003c/sub\u003e value was 0.31 within two months after fertilization, and the average R\u003csub\u003eN2O\u003c/sub\u003e value for other time periods was 0.05. Stevens \u0026amp; Laughlin (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) reported that the R\u003csub\u003eN2O\u003c/sub\u003e levels immediately increased from 0.05 to 0.8 three days after fertilizing grassland. Studies have shown that the temporal variability in R\u003csub\u003eN2O\u003c/sub\u003e is influenced by various factors. For instance, Clemens et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) estimated the global terrestrial ecosystem R\u003csub\u003eN2O\u003c/sub\u003e and found that short-term changes in R\u003csub\u003eN2O\u003c/sub\u003e are influenced by factors such as water content, temperature, O\u003csub\u003e2\u003c/sub\u003e, soil texture, pH, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e, and organic C. Using the DAMM model, Wang et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) concluded that the temporal variability of R\u003csub\u003eN2O\u003c/sub\u003e is mainly affected by soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e contents, as well as temperature and moisture.\u003c/p\u003e \u003cp\u003eIn addition to the influence of the soil type and temporal scale, the method used to determine N\u003csub\u003e2\u003c/sub\u003e affects R\u003csub\u003eN2O\u003c/sub\u003e (Sgouridis et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Xi et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) synthesized the literature and found significant variations in R\u003csub\u003eN2\u003c/sub\u003eO values measured by different methods in forest soils (ranging from 0.01 to 1, with an average of 0.3). The highest value was obtained by the acetylene inhibition method, and the lowest value was obtained by the \u003csup\u003e15\u003c/sup\u003eN labeling method. Similarly, Clemens et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), in their study of global terrestrial ecosystem R\u003csub\u003eN2O\u003c/sub\u003e, found that R\u003csub\u003eN2O\u003c/sub\u003e calculated using the \u003csup\u003e15\u003c/sup\u003eN labeling method was significantly lower than that using the acetylene inhibition method. This difference is due to systemic limitations, such as incomplete inhibition of reductase in the acetylene inhibition method, which can lead to an underestimation of N\u003csub\u003e2\u003c/sub\u003e emissions and denitrification potential and, thus, an overestimation of R\u003csub\u003eN2O\u003c/sub\u003e (Felber et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Qin et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn summary, different soil types, timescales, and measurement methods can affect R\u003csub\u003eN2O\u003c/sub\u003e. Thus, estimating N\u003csub\u003e2\u003c/sub\u003e emissions based on average R\u003csub\u003eN2O\u003c/sub\u003e values involves significant uncertainties. Further research into the relationship between R\u003csub\u003eN2O\u003c/sub\u003e and environmental factors is necessary to improve the accuracy of estimating N\u003csub\u003e2\u003c/sub\u003e emissions from terrestrial ecosystems (Clemens et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The environmental conditions of the GVP system that we studied were unique, and the response of gaseous N to environmental factors differed from those in the literature. Therefore, it is not recommended to use constant R\u003csub\u003eN2O\u003c/sub\u003e values derived from observational datasets or existing models to estimate its N\u003csub\u003e2\u003c/sub\u003e emissions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.4 N Losses\u003c/h2\u003e \u003cp\u003eGaseous N loss, particularly the emission of N\u003csub\u003e2\u003c/sub\u003e, has long been considered the primary pathway for N loss in farmlands (Zhao et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, owing to the difficulty of directly measuring soil N\u003csub\u003e2\u003c/sub\u003e emissions, few studies have investigated the proportion of gaseous N in N losses within the GVP system. Our study revealed that the primary form of gaseous N in the GVP during the ASD period was N\u003csub\u003e2\u003c/sub\u003e. The total \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e emissions ranged from 6.3\u0026ndash;13.6% of the added \u003csup\u003e15\u003c/sup\u003eN, and \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions accounted for 0.2\u0026ndash;0.5% of the added \u003csup\u003e15\u003c/sup\u003eN. These findings are similar to the gaseous N pathways reported in other studies. In a wheat-maize rotation system in northern China, the emissions of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO during the growing season accounted for 4.1\u0026ndash;10% and 0.4\u0026ndash;0.7% of N fertilizer application, respectively (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In rice planting systems in southern China, the N\u003csub\u003e2\u003c/sub\u003e produced by denitrification accounted for 10.2\u0026ndash;13.5% of the N fertilizer application, and N\u003csub\u003e2\u003c/sub\u003eO accounted for 0.09\u0026ndash;0.2% (Xia et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, during the ASD period, 7\u0026ndash;14% of the added N was emitted as N\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003eO, and 62\u0026ndash;64% remained in the soil primarily as NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e. The unrecovered 24\u0026ndash;30% of N may leach into the deeper soil layers. Based on our previous monitoring of NO emissions and NH\u003csub\u003e3\u003c/sub\u003e volatilization at this site, the emissions of these two gases were relatively small, accounting for 5% and 6% of N\u003csub\u003e2\u003c/sub\u003eO emissions, respectively. Ti et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) conducted a meta-analysis of N fate in GVP system and estimated that the proportions of N in N\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;NOx, soil retention, plant uptake, leaching and runoff, and NH\u003csub\u003e3\u003c/sub\u003e volatilization were 33%, 31%, 23%, 12%, and 1% of the added N, respectively. Our findings differ from theirs, possibly due to the absence of crop planting during the observed ASD period and the use of different methods for measuring N\u003csub\u003e2\u003c/sub\u003e emissions in our study (their study used the balance method) (Groffman et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Hence, further research should aim to validate the N loss pathways in GVP systems using various methods. Notably, implementing reasonable measures to reduce gaseous N loss and improve N fertilizer use efficiency is necessary.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn the agricultural ecosystems, accurate estimation of N\u003csub\u003e2\u003c/sub\u003e emissions is crucial for reducing the loss of available N and formulating effective strategies to mitigate N\u003csub\u003e2\u003c/sub\u003eO emissions. This study is the first to quantify N\u003csub\u003e2\u003c/sub\u003e emissions in the GVP system by in situ \u003csup\u003e15\u003c/sup\u003eN labeling method. The results indicate that the predominant gaseous N released from the soil during the ASD period was N\u003csub\u003e2\u003c/sub\u003e, with relatively low N\u003csub\u003e2\u003c/sub\u003eO emissions. The total \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003e emissions accounted for 6.3\u0026ndash;13.6% of the added \u003csup\u003e15\u003c/sup\u003eN, and \u003csup\u003e15\u003c/sup\u003eN\u003csub\u003e2\u003c/sub\u003eO emissions represented 0.2\u0026ndash;0.5% of the added \u003csup\u003e15\u003c/sup\u003eN. Furthermore, our study emphasized the importance of microbial processes other than denitrification in N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions. During the ASD period, N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e originating from denitrification processes accounted for 48.9\u0026ndash;51.2% and 27.1\u0026ndash;36.7%, respectively. However, we were unable to differentiate the microbial processes in detail. Further research should employ dual labeling with \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e combined with incubator experiments to explore more detail microbial processes that contribute to N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e production in the GVP system. Additionally, we observed a significant variability in R\u003csub\u003eN2O\u003c/sub\u003e during the ASD period. Hence, we advise against estimating GVP system N\u003csub\u003e2\u003c/sub\u003e emissions using constant R\u003csub\u003eN2O\u003c/sub\u003e values obtained from observations in other locations or existing models.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.T.F and X.L designed the study. X.L, J.L, Y.Y.W and K.P.S performed the experiment. Data analysis was conducted by X.L, J.L, and K.H. The paper was written by X.L with contribution from other co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences [grant number XDA28020302], the National Key Research and Development Program of China (2023YFD1500802; 2023YFD1501400), the National Natural Science Foundation of China [grant number 42177214], the Shandong Provincial Natural Science Foundation [grant number ZR2023YQ030], the Liaoning Vitalization Talents Program [grant number XLYC1902016, XLYC2203058], the Taishan Scholars [R.K. and Z.Q.], and the China Postdoctoral Science Foundation [grant number 2023M733675]. We also acknowledge support from the K.C. Wong Education Foundation (Y.F.) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (Z.Q.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgostini F, Tei F, Silgram M, Farneselli M, Benincasa P, Aller M (2010) Decreasing nitrate leaching in vegetable crops with better N management. In: Lichtfouse E (ed) Genetic Engineering, Biofertilisation, Soil Quality and Organic Farming. Springer Netherlands, Dordrecht, pp 147\u0026ndash;200\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAulakh MS, Khera TS, Doran JW, Bronson KF (2001) Denitrification, N\u003csub\u003e2\u003c/sub\u003eO and CO\u003csub\u003e2\u003c/sub\u003e fluxes in rice-wheat cropping system as affected by crop residues, fertilizer N and legume green manure. Biol Fertil Soils 34:375\u0026ndash;389\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchen C, Lewicka-Szczebak D, Fuss R, Helfrich M, Flessa H, Well R (2016) Fluxes of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO and contributing processes in summer after grassland renewal and grassland conversion to maize cropping on a Plaggic Anthrosol and a Histic Gleysol. Soil Biol Biochem 101:6\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eButterbach-Bahl K, Willibald G, Papen H (2002) Soil core method for direct simultaneous determination of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions from forest soils. Plant Soil 240:105\u0026ndash;116\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCharles A, Rochette P, Whalen JK, Angers DA, Chantigny MH, Bertrand N (2017) Global nitrous oxide emission factors from agricultural soils after addition of organic amendments: a meta-analysis. Agr Ecosyst Environ 236:88\u0026ndash;98\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Q, Zhang XS, Zhang HY, Christie P, Li XL, Horlacher D, Liebig H (2004) Evaluation of current fertilizer practice and soil fertility in vegetable production in the Beijing region. Nutr Cycl Agrosyst 69:51\u0026ndash;58\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Oenema O, Li J, Misselbrook T, Dong W, Qin S, Yuan H, Li X, Hu C (2019) Seasonal variations in N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions from a wheat\u0026ndash;maize cropping system. Biol Fertil Soils 55:539\u0026ndash;551. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00374-019-01373-8\u003c/span\u003e\u003cspan address=\"10.1007/s00374-019-01373-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClemens S, Kathrin F, David EP, Klaus B (2020) Estimating global terrestrial denitrification from measured N\u003csub\u003e2\u003c/sub\u003eO:(N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003e) product ratios. Curr Opin Environ Sustain 47:72\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cosust.2020.07.005\u003c/span\u003e\u003cspan address=\"10.1016/j.cosust.2020.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavidson EA, Keller M, Erickson HE, Verchot LV, Veldkamp E (2000) Testing a conceptual model of soil emissions of nitrous and nitric oxides: using two functions based on soil nitrogen availability and soil water content, the hole-in-the-pipe model characterizes a large fraction of the observed variation of nitric oxide and nitrous oxide emissions from soils. Bioscience 50(8):667\u0026ndash;680\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDendooven L, Anderson JM (1994) Dynamics of reduction enzymes involved in the denitrification process in pasture soil. Soil Biol Biochem 26:1501\u0026ndash;1506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0038-0717(94)90091-4\u003c/span\u003e\u003cspan address=\"10.1016/0038-0717(94)90091-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Gioia F, Ozores-Hampton M, Hong J, Kokalis-Burelle N, Albano J, Zhao X, Black Z, Gao Z, Moore K, Swisher M, Guo H, Rosskopf EN, Wilson C, Thomas J (2016) The effects of anaerobic soil disinfestation on weed and nematode control, fruit yield, and quality of Florida fresh-market tomato. HortScience 51:703\u0026ndash;711. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21273/hortsci.51.6.703\u003c/span\u003e\u003cspan address=\"10.21273/hortsci.51.6.703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Gioia F, Ozores-Hampton M, Zhao X, Thomas J, Wilson P, Li Z, Hong J, Albano J, Swisher M, Rosskopf E (2017) Anaerobic soil disinfestation impact on soil nutrients dynamics and nitrous oxide emissions in fresh-market tomato. Agric Ecosyst Environ 240:194\u0026ndash;205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.agee.2017.02.025\u003c/span\u003e\u003cspan address=\"10.1016/j.agee.2017.02.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuan P, Song Y, Li S, Xiong Z (2019) Responses of N\u003csub\u003e2\u003c/sub\u003eO production pathways and related functional microbes to temperature across greenhouse vegetable field soils. Geoderma 355:113904\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFei C, Zhang S, Liang B, Li J, Jiang L, Xu Y, Ding X (2018) Characteristics and correlation analysis of soil microbial biomass phosphorus in greenhouse vegetable soil with different planting years. Acta Agric Boreali-sinica 33(1):195\u0026ndash;202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelber R, Conen F, Flechard CR, Neftel A (2012) Theoretical and practical limitations of the acetylene inhibition technique to determine total denitrification losses. Biogeosciences 9(10):4125\u0026ndash;4138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFirestone MK, Tiedje JM (1979) Temporal change in nitrous oxide and dinitrogen from denitrification following onset of anaerobiosis. Appl Environ Microbiol 38:673\u0026ndash;679\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroffman PM, Altabet MA, B\u0026ouml;hlke JK, Butterbach-Bahl K, David MB, Firestone MK, Giblin AE, Kana TM, Nielsen LP, Voytek MA (2006) Methods for measuring denitrification: Diverse approaches to a difficult problem. Ecol Appl 16:2091\u0026ndash;2122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo J, Liu X, Zhang Y, Shen J, Han W, Zhang W, Christie P, Goulding KWT, Vitousek PM, Zhang F (2010) Significant acidification in major Chinese croplands. Science 327:1008\u0026ndash;1010\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo J (2016) Inputs of irrigation water, fertilizers, pesticides to and life cycle assessment of environment impacts from typical greenhouse vegetable production systems (In Chinese). PhD thesis. China Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe H, Lv H, Zhang W, Hou S, Zhang X (2011) A liquid chromatographic/mass spectrometric method to evaluate \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN incorporation into soil amino acids. J Soils Sediments 11:731\u0026ndash;740\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe F, Jiang R, Chen Q, Zhang F, Su F (2009) Nitrous oxide emissions from an intensively managed greenhouse vegetable cropping system in Northern China. Environ Pollut 157(5):1666\u0026ndash;1672\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang X, Liu L, Wen T, Zhang J, Shen Q, Cai Z (2016) Reductive soil disinfestations combined or not with Trichoderma for the treatment of a degraded and Rhizoctonia solani infested greenhouse soil. Sci Hortic 206:51\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2016.04.033\u003c/span\u003e\u003cspan address=\"10.1016/j.scienta.2016.04.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIPCC (2021) In: Masson-Delmotte V et al (eds) The Physical Science Basis. Cambridge Univ. Press\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohannes F, Clemens S, David WR, Heather VM, Alice S, Daniel IW, Peter RG (2016) Denitrification losses from an intensively managed sub-tropical pasture \u0026ndash; Impact of soil moisture on the partitioning of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions. Soil Biol Biochem 92:58\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2015.09.016\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2015.09.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJu X, Liu X, Zhang F, Roelcke M (2004) Nitrogen fertilization, soil nitrate accumulation, and policy recommendations in several agricultural regions of China. Ambio 33(6):300\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1579/0044-7447-33.6.300\u003c/span\u003e\u003cspan address=\"10.1579/0044-7447-33.6.300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJu X, Kou C, Zhang F, Christle P (2006) Nitrogen balance and groundwater nitrate contamination: Comparison among three intensive cropping systems on the North China Plain. Environ Pollut 143(1):117\u0026ndash;125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKlemedtsson L, Von Arnold K, Weslien P, Gundersen P (2005) Soil CN ratio as a scalar parameter to predict nitrous oxide emissions. Global Change Biol 11:1142\u0026ndash;1147\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulkarni MV, Burgin AJ, Groffman PM, Yavitt JB (2014) Direct flux and \u003csup\u003e15\u003c/sup\u003eN tracer methods for measuring denitrification in forest soils. Biogeochemistry 117:359\u0026ndash;373. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10533-013-9876-7\u003c/span\u003e\u003cspan address=\"10.1007/s10533-013-9876-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulkarni MV, Groffman PM, Yavitt JB, Goodale CL (2015) Complex controls of denitrification at ecosystem; landscape and regional scales in northern hardwood forests. Ecol Model 298:39\u0026ndash;52\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai TV, Denton MD (2017) N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions from denitrification respond differently to temperature and nitrogen supply. J S Sediment 18(4):1\u0026ndash;10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaughlin RJ, Stevens RJ (2002) Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Sci Soc Am J 66:1540\u0026ndash;1548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Kang R, Yu H, Wang Y, Yao M, Fang Y (2021) Effects of soil moisture on micribial processes of soil nitrogen gases production under anaerobic conditions. Chin J Appl Ecol 32(6):9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13287/j.1001-9332.202106.010\u003c/span\u003e\u003cspan address=\"10.13287/j.1001-9332.202106.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Nishio T, Uemiya Y, Inubushi K (2002) Gaseous losses of applied nitrogen from a cron field determined by \u003csup\u003e15\u003c/sup\u003eN abundance of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO. Commun Soil Sci Plant Anal 33(15\u0026ndash;18):2715\u0026ndash;2727. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1081/CSS-120014475\u003c/span\u003e\u003cspan address=\"10.1081/CSS-120014475\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Quan Z, Huang K, Kang R, Su C, Liu D, Ma J, Chen X, Fang Y (2024) High soil nitrous oxide emissions from a greenhouse vegetable production system in Shouguang, Northern China. Atmos Environ 319:120264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.atmosenv.2023.120264\u003c/span\u003e\u003cspan address=\"10.1016/j.atmosenv.2023.120264\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Wang S, Li H, Fang Y, Yang L, Su F (2020) Contribution of co-denitrification to nitrous oxide production from subsurface wastewater infiltration system. Ecol Eng 151:105854. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoleng.2020.105854\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoleng.2020.105854\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLigi T, Truu M, Oopkaup K, N\u0026otilde;lvak H, Mander \u0026Uuml;, Mitsch W, Truu J (2015) The genetic potential of N\u003csub\u003e2\u003c/sub\u003e emission via denitrification and ANAMMOX from the soils and sediments of a created riverine treatment wetland complex. Ecol Eng 80:181\u0026ndash;190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoleng.2014.09.072\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoleng.2014.09.072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Wang R, Pan Z, Zheng X, Wei H, Zhang H, Quan M, Fang Z, Ju Y X (2022) Quantifying in situ N\u003csub\u003e2\u003c/sub\u003e fluxes from an intensively managed calcareous soil using the \u003csup\u003e15\u003c/sup\u003eN gas-flux method. J Integr Agric 21(9):2750\u0026ndash;2766. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jia.2022.07.016\u003c/span\u003e\u003cspan address=\"10.1016/j.jia.2022.07.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu R, Hayden HL, Suter H, Hu H, Lam SK, He J, Mele PM, Chen D (2017) The effect of temperature and moisture on the source of N\u003csub\u003e2\u003c/sub\u003eO and contributions from ammonia oxidizers in an agricultural soil. Biol Fertil Soils 53:141\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00374-016-1167-8\u003c/span\u003e\u003cspan address=\"10.1007/s00374-016-1167-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Lin F, Wu S, Ji C, Sun Y, Jin Y, Li S, Li Z, Zou J (2017) A meta-analysis of fertilizer‐induced soil NO and combined with N\u003csub\u003e2\u003c/sub\u003eO emissions. Glob Change Biol 23:2520\u0026ndash;2532. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1111/gcb.13485\u003c/span\u003e\u003cspan address=\"10.1111/gcb.13485\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Jiang L, Zhang W, Zheng F, Wang Mei, Lin H (2008) Evolution of fertilization rate and variation of soil nutrient contents in greenhouse vegetable cultivation in Shandong (In Chinese). Acta Pedol Sin 45(2):296\u0026ndash;303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong A, Heitman J, Tobias C, Philips R, Song B (2013) Co-occurring anammox, denitrification, and codenitrification in agricultural soils. Appl Environ Microbiol 79:168\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aem.02520-12\u003c/span\u003e\u003cspan address=\"10.1128/aem.02520-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv H, Zhao Y, Wang Y, Wan L, Wang J, Butterbach-Bahl K, Lin S (2020) Conventional flooding irrigation and over fertilization drives soil pH decrease not only in the top but also in subsoil layers in solar greenhouse vegetable production systems. Geoderma 363:114156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.geoderma.2019.114156\u003c/span\u003e\u003cspan address=\"10.1016/j.geoderma.2019.114156\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin J, Sun H, Kronzucker H, Wang Y, Shi W (2021) Comprehensive assessment of the effects of nitrification inhibitor application on reactive nitrogen loss in intensive vegetable production systems. Agric Ecosyst Environ 307:107227\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMomma N, Kobara Y, Uematsu S, Kita N, Shinmura A (2013) Development of biological soil disinfestations in Japan. Appl Microbiol Biotechnol 97:3801\u0026ndash;3809. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00253-013-4826-9\u003c/span\u003e\u003cspan address=\"10.1007/s00253-013-4826-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorse JL, Bernhardt ES (2013) Using \u003csup\u003e15\u003c/sup\u003eN tracers to estimate N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions from nitrification and denitrification in coastal plain wetlands under contrasting land-uses. Soil Biol Biochem 57:635\u0026ndash;643\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOllinger S, Smith M, Martin M, Hallett R, Goodale C, Aber J (2002) Regional variation in foliar chemistry and N cycling among forests of diverse history and composition. Ecology 83:339\u0026ndash;355\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan Z, Wang R, Liu Y, Wang L, Zheng X, Yao Z, He H, Zhang X (2022) Characteristics of N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO fluxes from a cultivated black soil: A case study through in situ measurement using the \u003csup\u003e15\u003c/sup\u003eN gas flux method. Agriculture 12:1664. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agriculture12101664\u003c/span\u003e\u003cspan address=\"10.3390/agriculture12101664\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePilegaard K (2013) Processes regulating nitric oxide emissions from soils. Philosophical Trans Royal Soc B 368(1621):20130126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rstb.2013.0126\u003c/span\u003e\u003cspan address=\"10.1098/rstb.2013.0126\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQasim W, Wan L, Lv H, Zhao Y, Hu J, Meng F, Lin S, Butterbach-Bahl K (2022) Impact of anaerobic soil disinfestation on seasonal N\u003csub\u003e2\u003c/sub\u003eO emissions and N leaching in greenhouse vegetable production system depends on amount and quality of organic matter additions. Sci Total Environ 830:154673\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQasim W, Xia L, Lin S, Wan Li, Zhao Y, Butterbach-Bahl K (2021) Global greenhouse vegetable production systems are hotspots of soil N\u003csub\u003e2\u003c/sub\u003eO emissions and nitrogen leaching: A meta-analysis. Environ Pollut 272:116372\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin S, Hu C, Oenema O (2012) Quantifying the underestimation of soil denitrification potential as determined by the acetylene inhibition method. Soil Biol Biochem 47:14\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoobroeck D, Butterbach-Bahl K, Br\u0026uuml;ggemann N, Boeckx P (2010) Dinitrogen and nitrous oxide exchanges from an undrained monolith fen: short-term responses following nitrate addition. Eur J Soil Sci 61:662\u0026ndash;670. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2389.2010.01269.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2389.2010.01269.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaggar S, Jha N, Deslippe J, Bolan NS, Luo J, Giltrap DL, Kim DG, Zaman M, Tillman RW (2013) Denitrification and N\u003csub\u003e2\u003c/sub\u003eO:N\u003csub\u003e2\u003c/sub\u003e production in temperate grasslands: Processes; measurements; modelling and mitigating negative impacts. Sci Total Environ 465:173\u0026ndash;195\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScheer C, Meier R, Bruggemann N, Grace PR, Dannenmann M (2016) An improved \u003csup\u003e15\u003c/sup\u003eN tracer approach to study denitrification and nitrogen turnover in soil incubations. Rapid Commun Mass Spectrom 30:2017\u0026ndash;2026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlesinger WH (2009) On the fate of anthropogenic nitrogen. Proc. Natl. Acad. Sci. USA 106:203\u0026ndash;208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelbie DR, Lanigan GJ, Laughlin RJ, Di HJ, Moir JL, Cameron KC, Clough TJ, Watson CJ, Grant J, Somers C, Richards KG (2015) Confirmation of co-denitrification in grazed grassland. Sci Rep 5:1\u0026ndash;9\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenbayram M, Chen R, Budai A, Bakken L, Dittert K (2012) N\u003csub\u003e2\u003c/sub\u003eO emission and the N\u003csub\u003e2\u003c/sub\u003eO/(N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2\u003c/sub\u003e) product ratio of denitrification as controlled by available carbon substrates and nitrate concentrations. Agr. Ecosyst. Environ. 2012, 147, 4\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSgouridis F, Ullah S (2015) Relative magnitude and controls of in situ N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO fluxes due to denitrification in natural and seminatural terrestrial ecosystems using \u003csup\u003e15\u003c/sup\u003eN tracers. Environ Sci Technol 49(24):14110\u0026ndash;14119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.est.5b03513\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.5b03513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSgouridis F, Stott A, Ullah S (2016) Application of the \u003csup\u003e15\u003c/sup\u003eN gas-flux method for measuring in situ N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO fluxes due to denitrification in natural and semi-natural terrestrial ecosystems and comparison with the acetylene inhibition technique. Biogeosciences 13:1821\u0026ndash;1835\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen J, Xu Z, He J (2014) Frontiers in the microbial processes of ammonia oxidation in soils and sediments. J Soils Sediments 14:1023\u0026ndash;1029. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11368-014-0872-x\u003c/span\u003e\u003cspan address=\"10.1007/s11368-014-0872-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShougung Statistical, Yearbook (2021) China Statistics, Beijing (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpott O, Florian S (2011) Formation of hybrid N\u003csub\u003e2\u003c/sub\u003eO in a suspended soil due to co-denitrification of NH\u003csub\u003e2\u003c/sub\u003eOH. J Plant Nutr Soil Sci 174:554\u0026ndash;567\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStevens RJ, Laughlin RJ (1998) Measurement of nitrous oxide and dinitrogen emissions from agricultural soils. Nutr Cycl Agrosyst 52:131\u0026ndash;139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStevens RJ, Laughlin RJ, Burns LC, Arah JRM, Hood RC (1997) Measuring the contributions of nitrification and denitrification to the flux of nitrous oxide from soil. Soil Biol Biochem 29:139\u0026ndash;151\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan E, Zou W, Zheng Z, Yan X, Du M, Hsu T, Tian L, Middelburg J, Trull TW, Kao S (2020) Warming stimulates sediment denitrification at the expense of anaerobic ammonium oxidation. Nat Clim Change 10(4):349\u0026ndash;355\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTempler PH, Mack MC, Chapin FS, Christenson LM, Compton JE, Crook HD, Currie WS, Curtis CJ, Dail DB, D\u0026rsquo;antonio CM, Emmett BA, Epstein HE, Goodale CL, Gundersen P, Hobbie SE, Holland K, Hooper DU, Hungate BA, La-montagne S, Nadelhoffer KJ, Osenberg CW, Perakis SS, Schleppi P, Schimel J, Schmidt IK, Sommerkorn M, Spoelstra J, Tietema A, Wessel WW, Zak DR (2012) Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of \u003csup\u003e15\u003c/sup\u003eN tracer field studies. Ecology 93:1816\u0026ndash;1829\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTi C, Luo Y, Yan X (2015) Characteristics of nitrogen balance in open-air and greenhouse vegetable cropping systems of China. Environ Sci Pollut Control Ser 22(23):18508\u0026ndash;18518\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUllah S, Moore TR (2011) Biogeochemical controls on methane, nitrous oxide, and carbon dioxide fluxes from deciduous forest soils in eastern Canada. J Geophys Res Biogeosci 116:G03010\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUllah S, Moore TR (2009) Soil drainage and vegetation controls of nitrogen transformation rates in forest soils, southern Quebec. J Geophys Research-Biogeosciences 114:G01014\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Cleemput O (1998) Subsoils: chemo- and biological denitrification, N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions. Nutr Cycl Agroecosyst 52:187\u0026ndash;194\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Pan Z, Zheng X, Ju X, Yao Z, Butterbach-Bahl K, Zhang C, Wei H, Huang B (2020) Using field-measured soil N\u003csub\u003e2\u003c/sub\u003eO fluxes and laboratory scale parameterization of N\u003csub\u003e2\u003c/sub\u003eO/(N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;N\u003csub\u003e2)\u003c/sub\u003e ratios to quantify field-scale soil N\u003csub\u003e2\u003c/sub\u003e emissions. Soil Biol Biochem 148:107904. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2020.107904\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2020.107904\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Willibald G, Feng Q, Zheng X, Liao T, Br\u0026uuml;ggemann N, Butterbach-Bahl K (2011) Measurement of N\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003eO, NO, and CO\u003csub\u003e2\u003c/sub\u003e emissions from soil with the Gas-Flow-Soil-Core technique. Environ Sci Technol 45:6066\u0026ndash;6072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/es1036578\u003c/span\u003e\u003cspan address=\"10.1021/es1036578\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Feng Q, Liao T, Zheng X, Butterbach-Bahl K, Zhang W, Jin C (2013) Effects of nitrate concentration on the denitrification potential of a calcic cambisol and its fractions of N\u003csub\u003e2\u003c/sub\u003e, N\u003csub\u003e2\u003c/sub\u003eO and NO. Plant Soil 363:175\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeier KL, Doran JW, Power JF, Walters DT (1993) Denitrification and the dinitrogen/nitrous oxide ratio as affected by soil water, available carbon, and nitrate. Soil Sci Soc Am J 57(1):66\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2136/sssaj1993.03615995005700010013x\u003c/span\u003e\u003cspan address=\"10.2136/sssaj1993.03615995005700010013x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWell R, Burkart S, Giesemann A, Grosz B, Koester JR, Lewicka-Szczebak D (2019) Improvement of the \u003csup\u003e15\u003c/sup\u003eN gas flux method for in situ measurement of soil denitrification and its product stoichiometry. Rapid Commun Mass Spectrom 33:437\u0026ndash;448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXi D, Bai R, Zhang L, Fang Y (2016) Contribution of anammox to nitrogen removal in two temperate forest soils. Appl Environ Microbiol 82(15):4602\u0026ndash;4612. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00888-16\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00888-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXi D, Fang Y, Zhu W (2022) Spatial variations of soil N\u003csub\u003e2\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions from a temperate forest: quantified by the in situ \u003csup\u003e15\u003c/sup\u003eN Labeling Method. Forests 13:1347. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/f13091347\u003c/span\u003e\u003cspan address=\"10.3390/f13091347\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia L, Li X, Ma Q, Lam SK, Wolf B, Kiese R, Butterbach-Bahl K, Chen D, Li Z, Yan X (2020) Simultaneous quantification of N\u003csub\u003e2\u003c/sub\u003e, NH\u003csub\u003e3\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions from a flooded paddy field under different N fertilization regimes. Glob Chang Biol 26(4):2292\u0026ndash;2303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/gcb.14958\u003c/span\u003e\u003cspan address=\"10.1111/gcb.14958\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang WH, McDowell AC, Brooks PD, Silver WL (2014) New high precision approach for measuring \u003csup\u003e15\u003c/sup\u003eN-N\u003csub\u003e2\u003c/sub\u003e gas fluxes from terrestrial ecosystems. Soil Biol Biochem 69:234\u0026ndash;241\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Huang B, Mao M, Yao L, Niedermann S, Hu W, Chen Y (2016) Sustainability assessment of greenhouse vegetable farming practices from environmental, economic, and socio-institutional perspectives in China. Environ Sci Pollut Res 23(17):17287\u0026ndash;17297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Z, Yan G, Wang R, Zheng X, Liu C, Butterbach-Bahl K (2019) Drip irrigation or reduced N-fertilizer rate can mitigate the high annual N\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;NO fluxes from Chinese intensive greenhouse vegetable systems. Atmos Environ 212:183\u0026ndash;193\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshinari T, Hynes R, Knowles R (1997) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol Biochem 9:177\u0026ndash;183\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu K, Seo DC, DeLaune RD (2010) Incomplete acetylene inhibition of nitrous oxide reduction in potential denitrification assay as revealed by using \u003csup\u003e15\u003c/sup\u003eN-nitrate tracer. Commun Soil Sci Plant Anal 41(18):2201\u0026ndash;2210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H (2023) Temperature Sensitivity of gaseous nitrogen losses in forest soils (in Chinese). PhD thesis. Chinese Academy of Sciences\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu H, Li T, Zhang X Nutrient budget and soil nutrient status in greenhouse system. Agr Sci China 43(3):514\u0026ndash;522\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang H, Maucieri C, Liu S, Zou J (2019) Annual nitric and nitrous oxide emissions response to biochar amendment from an intensive greenhouse vegetable system in southeast China. Sci Hortic-Amsterdam 246:879\u0026ndash;886\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Zhou Y, Wang S, Xing G, Shi W, Xu R, Zhu Z (2012) Nitrogen balance in a highly fertilized rice-wheat double-cropping system in southern China. Soil Sci Soc Am J 76:1068\u0026ndash;1078. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2136/sssaj2011.0236\u003c/span\u003e\u003cspan address=\"10.2136/sssaj2011.0236\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Liu D, Huang K, Quan Z, Huang B, Fang Y, Chen X (2021a) N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emission characteristics from maize and greenhouse vegetable soils in Northern China. Chin J Appl Ecol 32(9):3204\u0026ndash;3212. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13287/j.1001-9332.202109.007\u003c/span\u003e\u003cspan address=\"10.13287/j.1001-9332.202109.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Lin S, Li W, Qasim W, Hu J, Xue T, Lv H, Butterbach-Bahl K (2021b) Anaerobic soil disinfestation with incorporation of straw and manure significantly increases greenhouse gases emission and reduces nitrate leaching while increasing leaching of dissolved organic N. Sci Total Environ 785:147307\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Chen Z, Liu X, Zhai B, Powlson DS (2010) Nitrate accumulation in soil profiles under seasonally open \u0026lsquo;sunlight greenhouses\u0026rsquo; in northwest China and potential for leaching loss during summer fallow. Soil Use Manag 26(3):332\u0026ndash;339. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1475-2743.2010.00284.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1475-2743.2010.00284.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J, Chen Z, Tang L, Zhai B, Xu A (2006) Changes of the contents of mineral nitrogen and their accumulation in soils under greenhouse cultivation (In Chinese with English abstract). Plant Nutr Fertilizer Sci 12:675\u0026ndash;680\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu T, Zhang J, Cai Z (2012) Effect of organic material amendment on vegetable soil nitrate content and nitrogenous gases emission under flooding condition. Chin J Appl Ecol 23:109\u0026ndash;114\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"15N labeling, greenhouse vegetable, anaerobic soil disinfestation, dinitrogen emission, in situ N2O/ (N2O + N2) ratio","lastPublishedDoi":"10.21203/rs.3.rs-4091615/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4091615/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and Aims: \u003c/strong\u003eGreenhouse vegetable production (GVP) is expanding worldwide. The high application of nitrogen (N) fertilizers has caused soil diseases and nitrate residue. Farmers usually adopt anaerobic soil disinfestation (ASD), involving organic carbon addition, extensive irrigation, plastic films laying, and greenhouse sealing during the summer fallow. These conditions may promote denitrification, causing nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) and dinitrogen (N\u003csub\u003e2\u003c/sub\u003e) emissions. However, this is rarely reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe used ¹⁵N labeling for in situ monitoring of N₂O and N₂ emissions during ASD in a GVP system in Shouguang, Northern China. Two treatments were implemented: conventional organic fertilization (Fertilizer) and a control (No-fertilizer), with continuous monitoring over 14 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWithin 14 days, cumulative gaseous N emissions in Fertilizer and No-fertilizer treatments were 0.82, 0.47 kg N ha\u003csup\u003e-1\u003c/sup\u003e for N\u003csub\u003e2\u003c/sub\u003eO, and 40.7 and 25.5 kg N ha\u003csup\u003e-1\u003c/sup\u003e for N\u003csub\u003e2\u003c/sub\u003e, respectively. Organic fertilization significantly increased N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emission. From days 1–6, the predominant gaseous N was N\u003csub\u003e2\u003c/sub\u003e, with an N\u003csub\u003e2\u003c/sub\u003eO/ (N\u003csub\u003e2\u003c/sub\u003eO + N\u003csub\u003e2\u003c/sub\u003e) ratio (R\u003csub\u003eN2O\u003c/sub\u003e) between 0.007 and 0.015. From days 7–14, N\u003csub\u003e2\u003c/sub\u003eO proportion increased, with R\u003csub\u003eN2O\u003c/sub\u003e ranging from 0.21 to 0.75. Isotopic information showed that denitrification contributed to 48.9%–51.2% and 27.1%–36.7% of total N\u003csub\u003e2\u003c/sub\u003eO and N\u003csub\u003e2\u003c/sub\u003e emissions. The structural equation model showed that high soil temperature during ASD significantly reduced N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our findings emphasize the importance of N\u003csub\u003e2\u003c/sub\u003e emissions in N loss and provide a basis for studying the fate of N, as well as developing measures to reduce N\u003csub\u003e2\u003c/sub\u003eO emissions within GVP systems.\u003c/p\u003e","manuscriptTitle":"Soil N2O and N2 emissions during anaerobic soil disinfestation period in a greenhouse vegetable production system: quantified by in situ 15N labeling method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-18 20:16:11","doi":"10.21203/rs.3.rs-4091615/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-06-03T03:13:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-14T01:21:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-14T01:19:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2024-03-14T00:35:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-13T22:28:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2024-03-13T06:08:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"81dd2503-1949-4ded-936b-2197aa1c98ec","owner":[],"postedDate":"March 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T16:27:03+00:00","versionOfRecord":{"articleIdentity":"rs-4091615","link":"https://doi.org/10.1007/s11104-024-07014-w","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2024-10-31 16:20:17","publishedOnDateReadable":"October 31st, 2024"},"versionCreatedAt":"2024-03-18 20:16:11","video":"","vorDoi":"10.1007/s11104-024-07014-w","vorDoiUrl":"https://doi.org/10.1007/s11104-024-07014-w","workflowStages":[]},"version":"v1","identity":"rs-4091615","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4091615","identity":"rs-4091615","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}