The duration of intensive vegetable cultivation regulates the fates of accumulated nitrate under reductive soil disinfestation

The duration of intensive vegetable cultivation regulates the fates of accumulated nitrate under reductive soil disinfestation | 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 The duration of intensive vegetable cultivation regulates the fates of accumulated nitrate under reductive soil disinfestation Huimin Zhang, Jing Wang, Nyumah Fallah, Yves Uwiragiye, Yinfei Qian, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5860188/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Apr, 2025 Read the published version in Plant and Soil → Version 1 posted 6 You are reading this latest preprint version Abstract Purpose Reductive soil disinfestation (RSD) can remove over-accumulated nitrate (NO 3 − -N) from topsoil in intensive vegetable fields via elevating NO 3 − -N consumption processes. The duration of intensive vegetable cultivation may affect the relative importance of these consuming processes of NO 3 − -N during RSD treatment by altering topsoil properties. However, it remains elusive how the duration of intensive vegetable cultivation affects the fates of topsoil NO 3 − -N during RSD treatment. Methods Here, a soil column experiment labeled with K 15 NO 3 was conducted to investigate the effects of different cultivation ages (5, 10, 20 and 30 years) of intensive vegetables on the fates of topsoil NO 3 − -N under RSD treatment. Results The results showed that more than 91.8% of the added 15 NO 3 − -N in topsoil was removed by RSD treatment, regardless of cultivation years. There was a trade-off between denitrification and NO 3 − -N leaching into the subsoil, both of which together accounted for 85.5–97.1% of the added 15 NO 3 − -N, regardless of cultivation years. The proportion of gaseous 15 N loss via denitrification to added 15 NO 3 − -N (P denitrification ) initially increased from 5 to 10 years of cultivation, and then decreased with further cultivation ages, but the trend was reversed for the proportion of leaching of 15 NO 3 − -N into the subsoil to added 15 NO 3 − -N (P leaching ). The structural equation model revealed that the initial soil carbon/nitrogen ratio had an indirect positive effect on P denitrification by driving the initial nirK abundance under RSD treatment. Conclusion Overall, our results highlight the critical role of using RSD in removing accumulated NO 3 − -N from the topsoil with its fates of a trade-off between P denitrification and P leaching as ages of intensive vegetable cultivation. Intensive vegetable cultivation Reductive soil disinfestation NO3−-N fates NO3−-N removal NO3−-N leaching Denitrification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Intensive vegetable cultivation has been one of the most important agricultural sectors in China because vegetables are high-priced cash crops with short growing cycles leading to high returns (Carter et al. 2012 ; Wang et al. 2021 ). It routinely receives large amounts of nitrogen (N) fertilizers to ensure high yield and quality, with an average N application rate of 1252 kg N ha − 1 year − 1 (Qasim et al. 2021 ). However, the sparse root systems of vegetables are often associated with low N use efficiency (< 50%; Ti et al. 2015 ; Valenzuela 2024 ). As a consequence, large amounts of nitrate (NO 3 − -N) are retained in soils (Clément et al. 2019 ; Bai et al. 2020 ). For example, Bai et al. ( 2020 ) found that accumulated NO 3 − -N in 0–2 m soil profiles averaged 1814 kg N ha − 1 in solar greenhouse vegetable production after five years of cultivation. Accumulated NO 3 − -N is vulnerable to losses via denitrification, runoff, and leaching, causing N-related environmental pollution (Stark and Richards 2008 ; Zhang et al. 2015 ). Therefore, there has been an increased focus on development of strategies to eliminate accumulated NO 3 − -N from soils under intensive vegetable cultivation. One of the potential pathways to eliminate accumulated NO 3 − -N is reductive soil disinfestation (RSD), originally developed to suppress soil-borne pathogens, particularly for intensive cropping systems with continuous monocultures (Lamers et al. 2014 ; Meng et al. 2022 ). RSD, that involves incorporation of organic materials, irrigation to soil saturation, and mulching the soil surface with plastic film, probably creates a unique circumstance for removal of topsoil NO 3 − -N (Momma et al. 2013 ; Meng et al. 2022 ). Such a strong reductive environment in soils under RSD would favor the occurrence of denitrification and dissimilatory nitrate reduction to ammonium (DNRA; Luvizotto et al. 2019; Li et al. 2022 ). Meanwhile, an abundant supply of carbon (C) under RSD would stimulate microbial demand for N, thereby compelling microorganisms to assimilate NO 3 − -N (Cao et al. 2021 ; Elrys et al. 2022 ). Finally, RSD would facilitate the migration of NO 3 − -N into the deep soil due to the irrigation (Bar-Yosef 2008 ). Our recent study has demonstrated that nearly 100% of added 15 NO 3 − -N in topsoil could be removed by denitrification and leaching into the subsoil under RSD in intensive vegetable systems (Zhang et al. 2023 ). However, it remains largely unknown whether the effects of RSD on the fates of accumulated NO 3 − -N of the topsoil vary with ages of intensive vegetable cultivation. Resolving this issue is critical for predicting the effectiveness of RSD in removing NO 3 − -N and the resulting environmental consequences under different cultivation ages. With increasing ages of intensive vegetable cultivation, soil properties could be significantly changed (Zhao et al. 2011 ; Li et al. 2019 ). For example, there were two contrasting trends with a gradual decline in soil bulk density (BD) and pH over cultivation years and an initial rise in soil organic carbon (SOC) and total N (TN) contents followed by either a gradual decline or stabilization (Li et al. 2019 ). Such different trends of soil properties would make it more complicated to predict the effects of RSD on the fates of NO 3 − -N in the topsoil under different ages of intensive vegetable cultivation. Given that the fates of removed 15 NO 3 − -N under RSD were regulated by topsoil pH, dissolved organic C, and bacterial abundance (Zhang et al. 2023 ), it could be expected that ages of intensive vegetable cultivation might affect the fates of accumulated NO 3 − -N of topsoil in response to RSD. However, this important aspect has not been studied so far. Here, based on a soil column experiment using the 15 N tracing technique in intensive vegetable lands with four different plantation ages (5, 10, 20, and 30 years), we aimed to investigate the effects of plantation ages on the fates of accumulated NO 3 − -N in topsoil during RSD. We hypothesized that accumulated NO 3 − -N in the topsoil would be completely removed by RSD treatment, irrespective of plantation ages, while its fates would depend on plantation age-induced changes in soil properties. Materials and methods Site description and sample collection The sampled site was located in Zibo City (118°24’E, 36°52’N) of Shandong Province, China. The site represents an important vegetable-growing region in China. The climate is characterized as temperate monsoon climate, with a mean annual precipitation of 650 mm and a mean annual air temperature of 14.2°C. Intensive vegetable fields have been developed at the expense of nearly half of wheat-maize rotation lands over the past three decades, on which zucchini ( Cucurbita pepo L.) have been cultivated during two growing seasons per year. Approximately 4.5 t ha -1 of air-dried chicken manure, equivalent to 135 kg N, 135 kg calcium superphosphate (P 2 O 5 ) and 90 kg potassium chloride (K 2 O), was applied to the soils once a year. Chemical fertilizers at rates of 360 kg N ha -1 year -1 , 570 kg P 2 O 5 ha -1 year -1 and 225 kg K 2 O ha -1 year -1 were also added to the soils. Irrigation water was supplied by sprinkling irrigation in these intensive vegetable fields. In November 2020, soil samples were collected from 0-20 cm (topsoil) and 20-50 cm (subsoil) depths in the intensive vegetable fields with four different establishment periods (5, 10, 20, and 30 years, represented as Y5, Y10, Y20, and Y30, respectively). Five soil cores from five plots (1 m × 1 m), constructed at 15 m intervals, were randomly selected in each intensive vegetable field and then homogenized into a single composite sample per field corresponded to a certain soil depth. The fresh soils for each layer were thoroughly mixed, sieved through a 2-mm-mesh sieve, and stored at 4°C for use in a 15 N-labeling column experiment. Basic soil characteristics are provided in Table S1. Sugarcane bagasse (extracted from Saccharum officinarum L. ) used in the RSD treatment was dried and crushed (particle size < 2 mm). The total C content, TN content, and C/N ratio of sugarcane bagasse were 458.1 g C kg -1 , 6.9 g N kg -1 , and 66.4, respectively. 15 N labeling column experiment Soil columns made of polyvinyl chloride (inner diameter: 10 cm, length: 53 cm) were set up in triplicate for the experiment. A soil column (50 cm long) was packed with the 20 cm topsoil and 30 cm subsoil according to their respective BD. All columns with soils inside were treated using the RSD method. Specifically, the sugarcane bagasse was added to the topsoil at a rate of 0.1 g kg -1 soil and well-mixed with the topsoil. Then, the topsoil was irrigated to saturation and covered with lids. Two holes of the upper lid were opened to flush the headspace of the columns with N 2 until no soil surface was exposed to air. Before irrigation to saturation, 200 mL of K 15 NO 3 solution was added to each topsoil at a rate of 50 mg N kg -1 soil enriched with 15 N at 50 atom%, using a four-needle injection technique (He et al. 2022). After a three-week incubation at 30°C, the subsoil was divided into 20-30 cm, 30-40 cm, and 40-50 cm. The topsoil and each subsoil samples were subsequently collected (4 cultivated ages × 4 layers × 3 replicates) to determine concentrations and isotopic compositions of ammonium (NH 4 + -N), NO 3 - -N, insoluble organic nitrogen (ION), and TN at the end of RSD treatment for quantifying the fates of NO 3 - -N. Soil physicochemical properties such as pH, electrical conductivity (EC), and soil organic carbon (SOC) content of the topsoil, as well as dissolved organic nitrogen (DON) content of the entire soil profile, were measured after RSD treatment. Microbial properties in the topsoil after RSD treatment were also determined as follows: abundances of bacteria, fungi, nitrifiers and denitrifiers targeting bacterial (16s rRNA), fungal (ITS1), ammonia monooxygenase ( amoA ), nitrite reductase ( nirK and nirS ), and nitrous oxide reductase ( nosZ ) genes, respectively. Determination of soil physicochemical properties Soil pH was determined by a S220 meter (Mettler Toledo, Shanghai, China) using a soil-water solution of 1:2.5 (w/v). Soil EC was measured using a 1:5 (w/v) soil-water ratio with a S230 meter (Mettler, Shanghai, China). Soil texture was analyzed by a laser grain-size analyzer (Beckman Coulter, Brea, CA, USA). Soil samples were extracted with 1 mol/L potassium chloride (KCl) solution at a soil-solution ratio of 1:5 (w/v) to determine the concentrations of mineral N. To analyse ION, the following procedure was carried out: residual soil was washed using deionized water to remove inorganic N after KCl extraction, oven-dried at 60°C to a constant weight, and ground to pass through a 0.15-mm sieve. The semi-micro Kjeldahl digestion method described by Bremner (1960) was employed to determine soil ION and TN contents. The SOC concentration was determined by the potassium dichromate volumetric method (Bremner and Jenkinson 2010). The total dissolved nitrogen (TDN) concentration was determined using an Analyzer Multi N/C (Analytic Jena, Jena, Germany), and the DON concentration was calculated as the difference between TDN and mineral N. The isotopic compositions of NH 4 + -N, NO 3 - -N, ION, and TN were determined using an Isotope-Ratio Mass Spectrometry system (Europa Scientific Integra, Crewe, UK). 15 N recovery in DON pool was calculated as the difference between 15 N recovery in TN pool and other N pools including mineral N and ION. To quantify the proportion of gaseous N loss via denitrification to added 15 NO 3 - -N (P denitrification ), we hypothesized that the non-recovery of 15 N-labeled compounds resulted from denitrification ( 15 N-balance method; Nieder et al. 1989). Soil DNA extraction and real-time PCR assay A FastDNA SPIN Kit (MP Biomedicals, Santa Ana, CA, USA) was used for soil DNA extraction. The DNA was dissolved in 100 μL of elution buffer, and its quality and concentration were measured by a DS-11 spectrophotometer (Denovix, Wilmington, DE, USA). The abundances of bacteria (16s rRNA), fungi (ITS), ammonia-oxidizing archaea (AOA amoA ), ammonia-oxidizing bacteria (AOB amoA ), and denitrifiers ( nirK , nirS and nosZ ) were detected using the QuantStudio 3 Real-Time PCR system with 96-well plates (Applied Biosystems, USA). Standard curves were created after ten-fold serial dilutions of plasmid DNA (target gene). The amplification efficiencies ranged between 99.95% and 99.97%, and the correlation coefficients (R 2 ) were > 0.97. All the primers and thermal conditions used in the present study are listed in Table S2. All measurements were performed in triplicate. For each reaction, 10 μL of SYBR Green premix Taq (2×, TaKaRa, Japan), 1 μL of each forward and reverse primers (10 μM), 6 μL of sterilized water, and 2 μL of template DNA were used. Data calculation and statistical analyses Statistical analyses were conducted using IBM SPSS 25.0 (SPSS Inc., USA) and Origin Pro 8.5 (OriginLab, Northampton, MA, USA). T-test was used to assess RSD treatment effects on soil properties in the same soil layer for each cultivation year. One-way analysis of variance (ANOVA) and the least significant difference (LSD) test ( p < 0.05) were used to assess the variance of mineral N and DON contents after RSD treatment among different soil depths for each cultivation year. Pearson correlation and regression analyses were used to explore the relationships between the soil properties and fates of NO 3 - -N, including P denitrification and the proportion of leaching of 15 NO 3 - -N into the subsoil to added 15 NO 3 - -N (P leaching ). The relative influence of soil properties on P denitrification was examined by Variable Importance Projection (VIP) in SIMCA 13.0 (Umetrics, Malmo, Sweden). A cutoff of 1.0 was set to differentiate between important and non-essential variables. The structural equation modeling (SEM) was carried out using AMOS 22.0 (Amos Development Corporation, Meadville, PA, USA) to determine the direct and indirect effects of the initial C/N ratio, initial abundance of nirK and pH after RSD treatment (pH t ) of topsoil on the P denitrification across different cultivation ages. Results Soil properties Implementation of RSD significantly increased topsoil pH and the SOC concentration while significantly declined topsoil EC and the TN concentration, regardless of cultivation duration (Fig. 1 a-d). Abundances of bacteria, AOB, and denitrifiers ( nirK, nirS, and nosZ ) in the topsoil were enhanced by RSD for all cultivation years, although not always significantly (Fig. 1 e, h-k). In contrast, there was no clear pattern for fungi, AOA, and the ratio of ( nirK + nirS ) to nosZ between RSD-treated and untreated topsoils across all cultivation years (Fig. 1 f, g, and l). Prior to RSD, the NO 3 − -N concentration of topsoil was significantly greater than that of each layer of subsoil (Fig. 2 a-d), regardless of cultivation duration. For all cultivation years, implementation of RSD resulted in a sharp decline in NO 3 − -N concentration of topsoil with the corresponding increase in NO 3 − -N concentration of each layer of subsoil. Among the subsoil layers, the highest NO 3 − -N concentration was found in 20–30 cm after RSD (Fig. 2 a-d). Meanwhile, RSD significantly increased the NH 4 + -N concentration of the entire soil profile in all cultivation years (Fig. 2 e-h). In most cases, the DON concentration was significantly enhanced by RSD throughout the entire soil profile across all cultivation years, except for Y20 (Fig. 2 i-l). Fates of added NO-N Following the implementation of RSD, a significant 15 N enrichment in the NO 3 − -N pool was observed in each subsoil across all cultivation years, indicating a downward shift of added 15 NO 3 − -N from topsoil to subsoil (Fig. S1 a). Likewise, both ION and NH 4 + -N pools in the entire soil profile were noticeably enriched by 15 N for all cultivation years following the implementation of RSD, indicating the conversion of added 15 NO 3 − -N to IO 15 N and 15 NH 4 + -N, respectively (Fig. S1 b and c). Overall, 0.02‰, 0.2%, 0.06‰, and 9.2% of added 15 NO 3 − -N remained in topsoil after the implementation of RSD in Y5, Y10, Y20, and Y30, respectively (Fig. 3 ). The main fates of added 15 NO 3 − -N following the implement of RSD were denitrification and leaching to the subsoil, across cultivation ages. P denitrification accounted for 67.9%, 88.7%, 56.2%, and 39.9% in the Y5, Y10, Y20 and Y30, respectively (Fig. 3 ). Correspondingly, P leaching accounted for 26.8%, 8.4%, 36.4%, and 45.7%, respectively (Fig. 3 ). P leaching was significantly and negatively correlated with P denitrification after RSD ( p < 0.01; Fig. 4 e), indicating a trade-off between P denitrification and P leaching . In contrast, other fates including transformations of added 15 NO 3 − -N to IO 15 N, 15 NH 4 + -N, and DO 15 N only contributed to 2.6–7.5% of added 15 NO 3 − -N in the entire soil profile across cultivation ages (Fig. 3 ). Factors affecting the fates of added NO-N The initial abundance of nirK , initial C/N ratio, and pH t of topsoil were identified to be the most important factors influencing P denitrification (Fig. 4 a). Specifically, P denitrification was significantly and positively correlated with the initial abundance of nirK , initial C/N ratio, and pH t of topsoil ( p < 0.05; Fig. 4 b-d). However, P leaching was negatively and positively correlated with topsoil BD and the amount of irrigation water during RSD, respectively ( p < 0.05; Fig. S2). The SEM revealed that the initial C/N ratio of topsoil had an indirect positive effect on P denitrification by driving the initial nirK abundance of topsoil under RSD treatment. Moreover, pH t of topsoil promoted P denitrification during RSD treatment (Fig. 5 ). Discussion Application of high rates of N fertilizers has led to serious soil NO 3 − -N accumulation in intensive vegetable cultivation of China (Qasim et al. 2021 ). A recent meta-analysis showed that soil NO 3 − -N accumulation at 0-100 cm was 504 kg N ha − 1 for intensive vegetable soils in China (Bai et al. 2021 ). The residual NO 3 − -N may be lost through runoff, leaching, and N gaseous (i.e., N 2 O and NO) emission, posing a threat to the environment (Husain et al. 2019 ; Liang et al. 2020 ; Tang et al. 2022 ). Previously, we found that RSD application could effectively remove accumulated NO 3 − -N of topsoil in representative intensive vegetable lands in China (Zhang et al. 2023 ). However, whether the duration of intensive vegetable cultivation affects its ability to remove NO 3 − -N is not resolved. Here, using a 15 N tracing column experiment, we showed that RSD application could remove more than 91.8% of added 15 NO 3 − -N in topsoil across cultivation duration, aligned with our first half of the hypothesis (Fig. 3 ). Such a finding, along with our previous results (Zhang et al. 2023 ), demonstrated a powerful and universal role of RSD application in removing over-accumulated NO 3 − -N in soils used for intensive vegetable cultivation. We further determined the fates of removed NO 3 − -N by RSD application and found that denitrification and leaching into the subsoil were primarily responsible for its removal over the cultivation duration. RSD was characterized by irrigation to soil saturation, covering with plastic film, and addition of C source (Butler et al. 2014 ; Ji et al. 2022 ). It was, therefore, likely that RSD might have provided the drive for downward migration and strong reductive circumstances, ultimately promoting leaching and denitrification, respectively. It is worth noting that strong reductive circumstances, coupled with the presence of C source, should also stimulate DNRA and immobilization of NO 3 − -N, in addition to denitrification (Qiu et al. 2013 ; Putz et al. 2018 ; Cheng et al. 2022 ). However, our results showed that DNRA and immobilization of NO 3 − -N only accounted for less than 0.07% and 3.0% of the added 15 NO 3 − -N in the entire soil profile across cultivation ages, respectively (Fig. 3 ). In contrast to denitrification, that represents N losses to the environment, DNRA is a N-conserving process that can convert mobile NO 3 − -N to relatively immobile NH 4 + -N (Silver et al. 2001 ; Cheng et al. 2022 ). Our results found that denitrification was one of the two major fates of removed NO 3 − -N, whereas DNRA was negligible in RSD-treated soils across cultivation ages. It has been recently surmised that importance of DNRA relative to denitrification in soils increases with a higher C/NO 3 − -N ratio (Espenberg et al. 2024 ). Similarly, lots of previous work showed that conditions with increased availability of electron donor (C) and limitation of electron acceptor (NO 3 − -N) favoured DNRA over denitrification (Van Den Berg et al. 2015 ; Putz et al. 2018 ; Pandey et al. 2020 ). It was proposed that the lower limit of the C/NO 3 − -N ratio threshold for DNRA was 10–15 (Stremińska et al. 2012 ; Chen et al. 2022 ). Although our RSD treatment with easily decomposable organic amendment might have increased soil C availability, serious accumulation of soil NO 3 − -N inevitably resulted in a low C/NO 3 − -N ratio of < 0.1, causing a dominance of denitrification over DNRA. In contrast, it is surprising that soil microbial immobilization of NO 3 − -N in the present study was not substantially stimulated by the addition of sugarcane bagasse with a C/N ratio of 66.4. This was not consistent with previous findings that the addition of organic materials with a high C/N ratio (> 18) generally stimulates soil microbial immobilization of NO 3 − -N (Cheng et al. 2017 ; Chen et al. 2024 ). The low microbial immobilization of NO 3 − -N could be due to rapid denitrification taking precedence over microbial immobilization of NO 3 − -N, thereby limiting the amount of NO 3 − -N available for immobilization (Ragab et al. 1988). Alternatively, the presence of NH 4 + -N probably inhibited microbial immobilization of NO 3 − -N, considering the preferential use of NH 4 + -N over NO 3 − -N (Azam and Ifzal 2006 ; Ma et al. 2021 ). Our results showed that RSD significantly increased the NH 4 + -N concentration of the entire soil profile in all cultivation years. It was thus likely that the enhanced NH 4 + -N concentration under RSD might have supressed microbial immobilization of NO 3 − -N. This was supported by our recent study, which revealed that microbial immobilization of NO 3 − -N gradually decreased with increasing NH 4 + -N concentration under increased C availability (Chen et al. 2024 ). Although the ability to remove NO 3 − -N and subsequent dominant fates were independent of cultivation duration following the implementation of RSD, the relative importance of both fates changed over the cultivation years, validating our second half of the hypothesis. Denitrification is a biochemical process mediated by denitrifiers ( nirK, nirS and nosZ ; Yan et al. 2024 ). P denitrification initially increased from 5 to 10 years of cultivation, and then decreased with cultivation years during RSD. The SEM revealed that the initial C/N ratio of topsoil had an indirect positive effect on P denitrification by driving the initial nirK abundance of topsoil under RSD treatment, whereas pH t of topsoil promoted P denitrification directly during RSD treatment (Fig. 5 ). A high C/N ratio in soil may strengthen enzymatic activities associated with denitrification, such as the nitrite reductase ( nirK ), owing to the ample availability of C source, thereby further promoting denitrification (Lan et al. 2023). A previous study also found that the absolute abundance of the nirK gene gradually increased with the increase of soil C/N ratio (Zhi and Ji 2014 ). In addition, stronger denitrification often occurs at a higher pH value of the soil under anaerobic conditions (Šimek and Cooper 2002 ; Pan et al. 2022 ). It is thus likely that increased soil pH is probably responsible for enhanced denitrification following RSD. In contrast, leaching is considered as a physical process, and thus can be regulated by soil BD, porosity, texture, irrigation water, etc. (Young et al. 2021 ; Hafshejani et al. 2024 ). Our results indeed found that P leaching was negatively correlated with topsoil BD (Fig. S2a), in agreement with other studies (Celik et al. 2017 ; Khan et al. 2017 ). This was because increased BD can lead to smaller porosity with greater penetration resistance (Unger et al. 1996). In addition, we found a positive relationship between the volume of irrigation water and P leaching (Fig. S2b), corroborating with the finding of Zhu et al. ( 2022 ), showing that higher irrigation levels could trigger more substantial leaching of soil NO 3 − -N. In spite of a powerful and universal role of RSD application in removing over-accumulated NO 3 − -N, the dominant fates of denitrification and leaching into subsoil inevitably caused N-related losses and environmental pollution. It was not viable to remove accumulated NO 3 − -N at the expense of enhanced N 2 O emission and NO 3 − -N leaching during RSD treatment. Thus, future studies are needed to investigate the conditions (i.e. quantity and types of organic materials) that can promote the conversion of accumulated NO 3 − -N to NH 4 + -N or organic N when applying RSD into the intensive vegetation cultivation soils. For instance, the addition of organic materials with higher C/N ratios could cause higher microbial immobilization of NO 3 − -N but lower N 2 O emission in soils (Chen et al. 2013 ; Cheng et al. 2017 ). Furthermore, biochar amendment has been proposed as a promising strategy for mitigating N 2 O emission from denitrification due to its liming effect and high surface area (Nelissen et al. 2014 ; Lehmann et al. 2021 ). Conclusions Our results demonstrated a powerful and universal role of RSD application in removing over-accumulated NO 3 − -N from the topsoil in intensive vegetable lands across cultivation years. We found that denitrification and leaching into subsoil were the main fates of removed NO 3 − -N, exhibiting a trade-off in all cultivation years. The duration of intensive vegetable cultivation affected the two fates of topsoil NO 3 − -N during RSD treatment. We proposed that cultivation age-induced changes in initial soil C/N ratio had a positive effect on P denitrification by driving the initial nirK abundance under RSD treatment, while pH t had a direct positive effect on P denitrification . Notably, denitrification and NO 3 − -N leaching into subsoil signified the N losses and environmental pollution during RSD treatment. For better management of NO 3 − -N in intensive vegetable production systems when utilizing RSD treatment, future research should focus on: (1) how to mitigate soil N 2 O emission from denitrification and NO 3 − -N leaching; (2) how to improve soil N conservation processes, including microbial NO 3 − -N immobilization and DNRA. Overall, our research provided a new perspective and feasible technology for utilizing RSD to remove accumulated NO 3 − -N in intensive vegetable topsoil. Declarations Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgements: This work was financially supported by the National Natural Science Foundation of China (grant numbers 42425705, 42122055 and 41977032), Jiangsu Carbon Peak Carbon Neutrality Science and Technology Innovation Fund (BE2022308), the Innovative and Entrepreneurial Talents Project of Jiangxi Province (grant number S2021DQKJ0001) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (grant number KYCX23_1704). References Azam F, Ifzal M (2006) Microbial populations immobilizing NH 4 + -N and NO 3 – -N differ in their sensitivity to sodium chloride salinity in soil. Soil Biol Biochem 38:2491–2494 Bai XL, Gao JJ, Wang SC, Cai HM, Chen ZJ, Zhou JB (2020) Excessive nutrient balance surpluses in newly built solar greenhouses over five years leads to high nutrient accumulations in soil. 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ISME J 9:2153–2161 Wang XZ, Dou ZX, Shi XJ, Zou CQ, Liu DY, Wang ZY, Guan XL, Sun YX, Wu G, Zhang BG, Li JL, Liang B, Tang L, Jiang LH, Sun ZM, Yang JG, Si DX, Zhao H, Liu B, Zhang W, Zhang F, Zhang FS, Chen XP (2021) Innovative management programme reduces environmental impacts in Chinese vegetable production. Nat Food 2:47–53 Yan SJ, Liu YL, Revillini D, Delgado-Baquerizo M, van Groenigen KJ, Shang ZY, Zhang X, Qian HY, Deng AX, Smith P, Ding YF, Zhang WJ (2024) Synergistic effect of elevated CO 2 and straw amendment on N 2 O emissions from a rice-wheat cropping system. Biol Fertil Soils 1–13 Young MD, Ros GH, de Vries W (2021) Impacts of agronomic measures on crop, soil, and environmental indicators: A review and synthesis of meta-analysis. 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Agric Ecosyst Environ 335:108005 Supplementary Files SupplementaryMaterial.docx Supplementary Table 2 Supplementary Figure 2 Cite Share Download PDF Status: Published Journal Publication published 05 Apr, 2025 Read the published version in Plant and Soil → Version 1 posted Editorial decision: Major revisions 10 Mar, 2025 Reviewers agreed at journal 24 Jan, 2025 Reviewers invited by journal 24 Jan, 2025 Editor invited by journal 21 Jan, 2025 Editor assigned by journal 20 Jan, 2025 First submitted to journal 19 Jan, 2025 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. 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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-5860188","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406609292,"identity":"de67d633-8f88-4d2e-97a5-c9a6d81a8cc0","order_by":0,"name":"Huimin Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Zhang","suffix":""},{"id":406609293,"identity":"b0ccb3dd-c8d5-404e-8d94-a572e8c26fc8","order_by":1,"name":"Jing Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Wang","suffix":""},{"id":406609294,"identity":"f116afe2-7a36-4ff6-9b30-a485718be050","order_by":2,"name":"Nyumah Fallah","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nyumah","middleName":"","lastName":"Fallah","suffix":""},{"id":406609295,"identity":"09eedbe6-28bc-4f36-bcaf-8dc13474e25a","order_by":3,"name":"Yves Uwiragiye","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yves","middleName":"","lastName":"Uwiragiye","suffix":""},{"id":406609296,"identity":"4d27d025-7764-4723-a0f3-d61918531a0d","order_by":4,"name":"Yinfei Qian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yinfei","middleName":"","lastName":"Qian","suffix":""},{"id":406609297,"identity":"0f7b3dac-d29b-479f-8216-fec231c36162","order_by":5,"name":"Cheng Yi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACHhBhw8DYAKLZiNeSRrqWw1AOMVoMjp89+Ljg1/k85tk9Bgwfyg4z8M9uIKDlTF6y8cy+28WMc84YMM44d5hB4s4BAlpu8JhJ8/bcTmyckWPAzNt2mMFAIoGgFvPfvD3nIFr+EqnFjJnnxwGIFkZitEieyTGW5m1IBmpJKzjYcy6dR+IGAS18x88Yfub5Y5e4cUbyxgc/yqzl+GcQ0KJwAEgwtjEwGDYwMIDYPPjVA4F8A4j8A2QQVDoKRsEoGAUjFgAAhv1F2M2orA8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0559-1319","institution":"Nanjing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Yi","suffix":""},{"id":406609298,"identity":"2ac2253f-8818-4d52-bfeb-f5af3b8c1416","order_by":6,"name":"Maoheng zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maoheng","middleName":"","lastName":"zhang","suffix":""},{"id":406609299,"identity":"7e1ce91d-6187-457f-9d73-8fb7bcba499a","order_by":7,"name":"Zucong Cai","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zucong","middleName":"","lastName":"Cai","suffix":""},{"id":406609300,"identity":"0dcb161c-102e-40b5-9b0d-9bc4f71e0091","order_by":8,"name":"Christoph Müller","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Christoph","middleName":"","lastName":"Müller","suffix":""}],"badges":[],"createdAt":"2025-01-19 15:20:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5860188/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5860188/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07420-8","type":"published","date":"2025-04-05T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74934056,"identity":"bb06fe43-ec08-437b-a6fd-3c2346208ea9","added_by":"auto","created_at":"2025-01-28 13:03:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35851,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of physicochemical (a-d) and microbial (e-l) properties in the topsoil by RSD under different years of intensive vegetable cultivation. Error bars represent the standard deviations from the mean (n = 3). Some standard deviations are not visible because they are very small. Different letters indicate significant differences (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05) between untreated and RSD-treated soils for the same age of intensivevegetable cultivation. Soils in Y5, Y10, Y20 and Y30 treatments were collected from intensive vegetable fields cultivated for 5, 10, 20 and 30 years, respectively. EC, electrical conductivity; TN, total nitrogen; SOC, soil organic carbon; AOA, ammonia-oxidizing archaea; AOB, ammonia-oxidizing bacteria; \u003cem\u003enirK\u003c/em\u003e and \u003cem\u003enirS\u003c/em\u003e, nitrite reductases; \u003cem\u003enosZ\u003c/em\u003e, nitrous oxide reductase; RSD, reductive soil disinfestation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/a4178b20492620d661fae089.png"},{"id":74934057,"identity":"660ebd33-fc7c-4fa4-a18f-c8c95476eb48","added_by":"auto","created_at":"2025-01-28 13:03:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53330,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (a-d), NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (e-h) and DON (i-l) concentrations at various soil depths by RSD under different years of intensive vegetable cultivation. Error bars represent the standard deviations from the mean (n = 3). Some standard deviations are not visible because they are very small. Different lowercase and capital letters indicate significant differences (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05) between untreated and RSD-treated soils and among different depths, respectively. Soils in Y5, Y10, Y20 and Y30 treatments were collected from intensive vegetable fields cultivated for 5, 10, 20 and 30 years, respectively. DON, dissolved organic nitrogen; RSD, reductive soil disinfestation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/914d2c898c044d530621f38f.png"},{"id":74935675,"identity":"45810462-6c52-458e-88a8-b6ebfd1a17b9","added_by":"auto","created_at":"2025-01-28 13:19:23","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":306156,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic abstract describing the total and relative fates of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (% of applied \u003csup\u003e15\u003c/sup\u003eN) during RSD with different years of intensive vegetable cultivation. Soils in Y5, Y10, Y20 and Y30 treatments were collected from intensive vegetable fields cultivated for 5, 10, 20 and 30 years, respectively. SON, soil organic nitrogen; ION, insoluble organic nitrogen; DON, dissolved organic nitrogen; DNRA, dissimilatory nitrate reduction to ammonium; RSD, reductive soil disinfestation.\u003c/p\u003e","description":"","filename":"3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/e01afe76401115f122cb7fdb.jpeg"},{"id":74936949,"identity":"91776ab4-07f9-4645-af6a-7d4f6c991fc8","added_by":"auto","created_at":"2025-01-28 13:27:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20283,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The variable importance of the projection (VIP, bars) values of influence factors of the proportion of gaseous \u003csup\u003e15\u003c/sup\u003eN loss via denitrification (P\u003csub\u003edenitrification\u003c/sub\u003e). Linear relationships between P\u003csub\u003edenitrification\u003c/sub\u003e and the (b) initial abundance of \u003cem\u003enirK\u003c/em\u003e, (c) initial C/N ratio, (d) pH\u003csub\u003et\u003c/sub\u003e and (e) P\u003csub\u003eleaching\u003c/sub\u003e. In panel (a), all the variables were ranked and the straight dashed line indicates a threshold above which variables are considered to be important for interpretation purpose. \u003cem\u003enirK\u003c/em\u003e, nitrite reductase; C/N, soil organic carbon to total nitrogen; pH\u003csub\u003et\u003c/sub\u003e, pH after RSD treatment; P\u003csub\u003eleaching\u003c/sub\u003e, the proportion of leaching of \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N into the subsoil to the added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N; BD, bulk density.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/c321885f212cc2a2c2b0f347.png"},{"id":74934063,"identity":"4dcb67de-e420-45c5-a6d3-dca530fb5854","added_by":"auto","created_at":"2025-01-28 13:03:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":174641,"visible":true,"origin":"","legend":"\u003cp\u003eStructural equation model (SEM) describing multivariate effects of topsoil properties on the proportion of gaseous \u003csup\u003e15\u003c/sup\u003eN loss via denitrification to the added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (P\u003csub\u003edenitrification\u003c/sub\u003e) under RSD across different cultivation ages. The solid blacklines represent significant positive relationships. Numbers next to arrows represent standardized path coefficients (*, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). The strength of the relationships is proportional by the arrow and line widths. R\u003csup\u003e2\u003c/sup\u003e values indicate the proportion of the variance explained by endogenous variables. Goodness-of-fit statistics are shown underneath the modeling frame. RSD, reductive soil disinfestation; C/N, soil organic carbon to total nitrogen; \u003cem\u003enirK\u003c/em\u003e, nitrite reductase; pH\u003csub\u003et\u003c/sub\u003e, pH after RSD treatment; c\u003csup\u003e2\u003c/sup\u003e, chi-square; df, degree of freedom; \u003cem\u003ep\u003c/em\u003e, probability level; RMSEA, the root mean square error of approximation.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/80e95585ce1ad826c7c1132e.png"},{"id":80082404,"identity":"4d6658cb-9b99-429f-b0c6-346bf4402932","added_by":"auto","created_at":"2025-04-07 16:08:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1281986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/2b729f20-0531-4ec4-bab1-6e22fdbab1f4.pdf"},{"id":74935421,"identity":"03f033fc-a316-484a-af23-048c266bea9d","added_by":"auto","created_at":"2025-01-28 13:11:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":178824,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e\n\u003cp\u003eSupplementary Figure 2\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5860188/v1/001b19cca0e2be67a64aafb2.docx"}],"financialInterests":"","formattedTitle":"The duration of intensive vegetable cultivation regulates the fates of accumulated nitrate under reductive soil disinfestation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntensive vegetable cultivation has been one of the most important agricultural sectors in China because vegetables are high-priced cash crops with short growing cycles leading to high returns (Carter et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It routinely receives large amounts of nitrogen (N) fertilizers to ensure high yield and quality, with an average N application rate of 1252 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Qasim et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the sparse root systems of vegetables are often associated with low N use efficiency (\u0026lt;\u0026thinsp;50%; Ti et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Valenzuela \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As a consequence, large amounts of nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) are retained in soils (Cl\u0026eacute;ment et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Bai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, Bai et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in 0\u0026ndash;2 m soil profiles averaged 1814 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in solar greenhouse vegetable production after five years of cultivation. Accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N is vulnerable to losses via denitrification, runoff, and leaching, causing N-related environmental pollution (Stark and Richards \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, there has been an increased focus on development of strategies to eliminate accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N from soils under intensive vegetable cultivation.\u003c/p\u003e \u003cp\u003eOne of the potential pathways to eliminate accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N is reductive soil disinfestation (RSD), originally developed to suppress soil-borne pathogens, particularly for intensive cropping systems with continuous monocultures (Lamers et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). RSD, that involves incorporation of organic materials, irrigation to soil saturation, and mulching the soil surface with plastic film, probably creates a unique circumstance for removal of topsoil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (Momma et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Such a strong reductive environment in soils under RSD would favor the occurrence of denitrification and dissimilatory nitrate reduction to ammonium (DNRA; Luvizotto et al. 2019; Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Meanwhile, an abundant supply of carbon (C) under RSD would stimulate microbial demand for N, thereby compelling microorganisms to assimilate NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (Cao et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Elrys et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Finally, RSD would facilitate the migration of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N into the deep soil due to the irrigation (Bar-Yosef \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Our recent study has demonstrated that nearly 100% of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in topsoil could be removed by denitrification and leaching into the subsoil under RSD in intensive vegetable systems (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, it remains largely unknown whether the effects of RSD on the fates of accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of the topsoil vary with ages of intensive vegetable cultivation. Resolving this issue is critical for predicting the effectiveness of RSD in removing NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and the resulting environmental consequences under different cultivation ages.\u003c/p\u003e \u003cp\u003eWith increasing ages of intensive vegetable cultivation, soil properties could be significantly changed (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, there were two contrasting trends with a gradual decline in soil bulk density (BD) and pH over cultivation years and an initial rise in soil organic carbon (SOC) and total N (TN) contents followed by either a gradual decline or stabilization (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Such different trends of soil properties would make it more complicated to predict the effects of RSD on the fates of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the topsoil under different ages of intensive vegetable cultivation. Given that the fates of removed \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N under RSD were regulated by topsoil pH, dissolved organic C, and bacterial abundance (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), it could be expected that ages of intensive vegetable cultivation might affect the fates of accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of topsoil in response to RSD. However, this important aspect has not been studied so far.\u003c/p\u003e \u003cp\u003eHere, based on a soil column experiment using the \u003csup\u003e15\u003c/sup\u003eN tracing technique in intensive vegetable lands with four different plantation ages (5, 10, 20, and 30 years), we aimed to investigate the effects of plantation ages on the fates of accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in topsoil during RSD. We hypothesized that accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the topsoil would be completely removed by RSD treatment, irrespective of plantation ages, while its fates would depend on plantation age-induced changes in soil properties.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eSite description and sample collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sampled site was located in Zibo City (118\u0026deg;24\u0026rsquo;E, 36\u0026deg;52\u0026rsquo;N) of Shandong Province, China. The site represents an important vegetable-growing region in China. The climate is characterized as temperate monsoon climate, with a mean annual precipitation of 650 mm and a mean annual air temperature of 14.2\u0026deg;C. Intensive vegetable fields have been developed at the expense of nearly half of wheat-maize rotation lands over the past three decades, on which zucchini (\u003cem\u003eCucurbita pepo\u003c/em\u003e L.) have been cultivated during two growing seasons per year. Approximately 4.5 t ha\u003csup\u003e-1\u003c/sup\u003e of air-dried chicken manure, equivalent to 135 kg N, 135 kg calcium superphosphate (P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) and 90 kg potassium chloride (K\u003csub\u003e2\u003c/sub\u003eO),\u0026nbsp;was\u0026nbsp;applied to the soils\u0026nbsp;once a year.\u0026nbsp;Chemical fertilizers\u0026nbsp;at rates of 360 kg N ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eyear\u003csup\u003e-1\u003c/sup\u003e, 570 kg P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eyear\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand 225 kg K\u003csub\u003e2\u003c/sub\u003eO ha\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eyear\u003csup\u003e-1\u003c/sup\u003e were also added to the soils. Irrigation water was supplied by sprinkling irrigation in these intensive vegetable fields.\u003c/p\u003e\n\u003cp\u003eIn November 2020, soil samples were collected from 0-20 cm (topsoil) and 20-50 cm (subsoil) depths in the intensive vegetable fields with four different establishment periods (5, 10, 20, and 30 years, represented as Y5, Y10, Y20, and Y30, respectively). Five soil cores from five plots (1 m \u0026times; 1 m), constructed at 15 m intervals, were randomly selected in each intensive vegetable field and then homogenized into a single composite sample per field corresponded to a certain soil depth. The fresh soils for each layer were thoroughly mixed, sieved through a 2-mm-mesh sieve, and stored at 4\u0026deg;C for use in a \u003csup\u003e15\u003c/sup\u003eN-labeling column experiment. Basic soil characteristics are provided in Table S1. Sugarcane bagasse (extracted from \u003cem\u003eSaccharum officinarum L.\u003c/em\u003e)\u0026nbsp;used in the RSD treatment was dried and crushed (particle size \u0026lt; 2 mm).\u0026nbsp;The total\u0026nbsp;C content, TN content, and C/N ratio of sugarcane bagasse were 458.1 g C kg\u003csup\u003e-1\u003c/sup\u003e, 6.9 g N kg\u003csup\u003e-1\u003c/sup\u003e, and 66.4, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e15\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eN labeling column experiment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil columns made of polyvinyl chloride (inner diameter: 10 cm,\u0026nbsp;length: 53 cm) were set up in triplicate for the experiment. A soil column (50 cm long) was packed with the 20 cm topsoil and 30 cm subsoil according to their respective BD.\u003c/p\u003e\n\u003cp\u003eAll columns with soils inside were treated using the RSD method. Specifically, the sugarcane bagasse was added to the topsoil at a rate of 0.1 g kg\u003csup\u003e-1\u003c/sup\u003e soil and well-mixed with the topsoil. Then, the topsoil was irrigated to saturation and covered with lids. Two holes of the upper lid were opened to flush the headspace of the columns with N\u003csub\u003e2\u003c/sub\u003e until no soil surface was exposed to air. Before irrigation to saturation, 200 mL of K\u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e solution was added to each topsoil at a rate of 50 mg N kg\u003csup\u003e-1\u003c/sup\u003e soil enriched with \u003csup\u003e15\u003c/sup\u003eN at 50 atom%, using a four-needle injection technique (He et al. 2022). After a three-week incubation at 30\u0026deg;C, the subsoil was divided into 20-30 cm, 30-40 cm, and 40-50 cm. The topsoil and each subsoil samples were subsequently collected (4 cultivated ages \u0026times; 4 layers \u0026times; 3 replicates) to determine concentrations and isotopic compositions of ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N), NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, insoluble organic nitrogen (ION), and TN at the end of RSD treatment for quantifying the fates of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N. Soil physicochemical properties such as pH, electrical conductivity (EC), and soil organic carbon (SOC) content of the topsoil, as well as dissolved organic nitrogen (DON) content of the entire soil profile, were measured after RSD treatment. Microbial properties in the topsoil after RSD treatment were also determined as follows: abundances of bacteria, fungi, nitrifiers and denitrifiers targeting bacterial (16s rRNA), fungal (ITS1), ammonia monooxygenase (\u003cem\u003eamoA\u003c/em\u003e), nitrite reductase (\u003cem\u003enirK\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;nirS\u003c/em\u003e), and nitrous oxide reductase (\u003cem\u003enosZ\u003c/em\u003e) genes, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of soil physicochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil pH was determined by a S220 meter (Mettler Toledo, Shanghai, China) using a soil-water solution of 1:2.5 (w/v). Soil EC was measured using a 1:5 (w/v) soil-water ratio with a S230 meter (Mettler, Shanghai, China). Soil texture was analyzed by a laser grain-size analyzer (Beckman Coulter, Brea, CA, USA). Soil samples were extracted with 1 mol/L potassium chloride (KCl) solution at a soil-solution ratio of 1:5 (w/v) to determine the concentrations of mineral N. To analyse ION, the following procedure was carried out: residual soil was washed using deionized water to remove inorganic N after KCl extraction, oven-dried at 60\u0026deg;C to a constant weight, and ground to pass through a 0.15-mm sieve. The semi-micro Kjeldahl digestion method described by Bremner (1960) was employed to determine soil ION and TN contents. The SOC concentration was determined by the potassium dichromate volumetric method (Bremner and Jenkinson 2010). The total dissolved nitrogen (TDN) concentration was determined using an Analyzer Multi N/C (Analytic Jena, Jena, Germany), and the DON concentration was calculated as the difference between TDN and mineral N. The isotopic compositions of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, ION, and TN were determined using an Isotope-Ratio Mass Spectrometry system (Europa Scientific Integra, Crewe, UK). \u003csup\u003e15\u003c/sup\u003eN recovery in DON pool was calculated as the difference between \u003csup\u003e15\u003c/sup\u003eN recovery in TN pool and other N pools including mineral N and ION. To quantify the proportion of gaseous N loss via denitrification to added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (P\u003csub\u003edenitrification\u003c/sub\u003e), we hypothesized that the non-recovery of \u003csup\u003e15\u003c/sup\u003eN-labeled compounds resulted from denitrification (\u003csup\u003e15\u003c/sup\u003eN-balance method; Nieder et al. 1989).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil DNA extraction and real-time PCR assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA FastDNA SPIN Kit (MP Biomedicals, Santa Ana, CA, USA) was used for soil DNA extraction. The DNA was dissolved in 100 \u0026mu;L of elution buffer, and its quality and concentration were measured by a DS-11 spectrophotometer (Denovix, Wilmington, DE, USA). The abundances of bacteria (16s rRNA), fungi (ITS), ammonia-oxidizing archaea (AOA \u003cem\u003eamoA\u003c/em\u003e), ammonia-oxidizing bacteria (AOB \u003cem\u003eamoA\u003c/em\u003e), and denitrifiers (\u003cem\u003enirK\u003c/em\u003e, \u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e) were detected using the QuantStudio 3 Real-Time PCR system with 96-well plates (Applied Biosystems, USA). Standard curves were created after ten-fold serial dilutions of plasmid DNA (target gene). The amplification efficiencies ranged between 99.95% and 99.97%, and the correlation coefficients (R\u003csup\u003e2\u003c/sup\u003e) were \u0026gt; 0.97. All the primers and thermal conditions used in the present study are listed in Table S2. All measurements were performed in triplicate. For each reaction, 10 \u0026mu;L of SYBR Green premix Taq (2\u0026times;, TaKaRa, Japan), 1 \u0026mu;L of each forward and reverse primers (10 \u0026mu;M), 6 \u0026mu;L of sterilized water, and 2 \u0026mu;L of template DNA were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData calculation and statistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using IBM SPSS 25.0 (SPSS Inc., USA) and Origin Pro 8.5 (OriginLab, Northampton, MA, USA). T-test was used to assess RSD treatment effects on soil properties in the same soil layer for each cultivation year. One-way analysis of variance (ANOVA) and the least significant difference (LSD) test (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) were used to assess the variance of mineral N and DON contents after RSD treatment among different soil depths for each cultivation year. Pearson correlation and regression analyses were used to explore the relationships between the soil properties and fates of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, including P\u003csub\u003edenitrification\u003c/sub\u003e and the proportion of leaching of \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N into the subsoil to added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N (P\u003csub\u003eleaching\u003c/sub\u003e). The relative influence of soil properties on P\u003csub\u003edenitrification\u0026nbsp;\u003c/sub\u003ewas examined by Variable Importance Projection (VIP) in SIMCA 13.0 (Umetrics, Malmo, Sweden). A cutoff of 1.0 was set to differentiate between important and non-essential variables. The structural equation modeling (SEM) was carried out using AMOS 22.0 (Amos Development Corporation, Meadville, PA, USA) to determine the direct and indirect effects of the initial C/N ratio, initial abundance of \u003cem\u003enirK\u003c/em\u003e and pH after RSD treatment (pH\u003csub\u003et\u003c/sub\u003e) of topsoil on\u0026nbsp;the P\u003csub\u003edenitrification\u003c/sub\u003e across different cultivation ages.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSoil properties\u003c/h2\u003e \u003cp\u003eImplementation of RSD significantly increased topsoil pH and the SOC concentration while significantly declined topsoil EC and the TN concentration, regardless of cultivation duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-d). Abundances of bacteria, AOB, and denitrifiers (\u003cem\u003enirK, nirS, and nosZ\u003c/em\u003e) in the topsoil were enhanced by RSD for all cultivation years, although not always significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, h-k). In contrast, there was no clear pattern for fungi, AOA, and the ratio of (\u003cem\u003enirK\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003enirS\u003c/em\u003e) to \u003cem\u003enosZ\u003c/em\u003e between RSD-treated and untreated topsoils across all cultivation years (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, g, and l).\u003c/p\u003e \u003cp\u003ePrior to RSD, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N concentration of topsoil was significantly greater than that of each layer of subsoil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-d), regardless of cultivation duration. For all cultivation years, implementation of RSD resulted in a sharp decline in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N concentration of topsoil with the corresponding increase in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N concentration of each layer of subsoil. Among the subsoil layers, the highest NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N concentration was found in 20\u0026ndash;30 cm after RSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-d). Meanwhile, RSD significantly increased the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration of the entire soil profile in all cultivation years (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-h). In most cases, the DON concentration was significantly enhanced by RSD throughout the entire soil profile across all cultivation years, except for Y20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei-l).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFates of added NO-N\u003c/h3\u003e\n\u003cp\u003eFollowing the implementation of RSD, a significant \u003csup\u003e15\u003c/sup\u003eN enrichment in the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N pool was observed in each subsoil across all cultivation years, indicating a downward shift of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N from topsoil to subsoil (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Likewise, both ION and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N pools in the entire soil profile were noticeably enriched by \u003csup\u003e15\u003c/sup\u003eN for all cultivation years following the implementation of RSD, indicating the conversion of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to IO\u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, respectively (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb and c). Overall, 0.02\u0026permil;, 0.2%, 0.06\u0026permil;, and 9.2% of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N remained in topsoil after the implementation of RSD in Y5, Y10, Y20, and Y30, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The main fates of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N following the implement of RSD were denitrification and leaching to the subsoil, across cultivation ages. P\u003csub\u003edenitrification\u003c/sub\u003e accounted for 67.9%, 88.7%, 56.2%, and 39.9% in the Y5, Y10, Y20 and Y30, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Correspondingly, P\u003csub\u003eleaching\u003c/sub\u003e accounted for 26.8%, 8.4%, 36.4%, and 45.7%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). P\u003csub\u003eleaching\u003c/sub\u003e was significantly and negatively correlated with P\u003csub\u003edenitrification\u003c/sub\u003e after RSD (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), indicating a trade-off between P\u003csub\u003edenitrification\u003c/sub\u003e and P\u003csub\u003eleaching\u003c/sub\u003e. In contrast, other fates including transformations of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to IO\u003csup\u003e15\u003c/sup\u003eN, \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and DO\u003csup\u003e15\u003c/sup\u003eN only contributed to 2.6\u0026ndash;7.5% of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the entire soil profile across cultivation ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFactors affecting the fates of added NO-N\u003c/h3\u003e\n\u003cp\u003eThe initial abundance of \u003cem\u003enirK\u003c/em\u003e, initial C/N ratio, and pH\u003csub\u003et\u003c/sub\u003e of topsoil were identified to be the most important factors influencing P\u003csub\u003edenitrification\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Specifically, P\u003csub\u003edenitrification\u003c/sub\u003e was significantly and positively correlated with the initial abundance of \u003cem\u003enirK\u003c/em\u003e, initial C/N ratio, and pH\u003csub\u003et\u003c/sub\u003e of topsoil (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). However, P\u003csub\u003eleaching\u003c/sub\u003e was negatively and positively correlated with topsoil BD and the amount of irrigation water during RSD, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. S2).\u003c/p\u003e \u003cp\u003eThe SEM revealed that the initial C/N ratio of topsoil had an indirect positive effect on P\u003csub\u003edenitrification\u003c/sub\u003e by driving the initial \u003cem\u003enirK\u003c/em\u003e abundance of topsoil under RSD treatment. Moreover, pH\u003csub\u003et\u003c/sub\u003e of topsoil promoted P\u003csub\u003edenitrification\u003c/sub\u003e during RSD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eApplication of high rates of N fertilizers has led to serious soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N accumulation in intensive vegetable cultivation of China (Qasim et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). A recent meta-analysis showed that soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N accumulation at 0-100 cm was 504 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for intensive vegetable soils in China (Bai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The residual NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N may be lost through runoff, leaching, and N gaseous (i.e., N\u003csub\u003e2\u003c/sub\u003eO and NO) emission, posing a threat to the environment (Husain et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Previously, we found that RSD application could effectively remove accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N of topsoil in representative intensive vegetable lands in China (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, whether the duration of intensive vegetable cultivation affects its ability to remove NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N is not resolved. Here, using a \u003csup\u003e15\u003c/sup\u003eN tracing column experiment, we showed that RSD application could remove more than 91.8% of added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in topsoil across cultivation duration, aligned with our first half of the hypothesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such a finding, along with our previous results (Zhang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), demonstrated a powerful and universal role of RSD application in removing over-accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in soils used for intensive vegetable cultivation.\u003c/p\u003e \u003cp\u003eWe further determined the fates of removed NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N by RSD application and found that denitrification and leaching into the subsoil were primarily responsible for its removal over the cultivation duration. RSD was characterized by irrigation to soil saturation, covering with plastic film, and addition of C source (Butler et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ji et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It was, therefore, likely that RSD might have provided the drive for downward migration and strong reductive circumstances, ultimately promoting leaching and denitrification, respectively. It is worth noting that strong reductive circumstances, coupled with the presence of C source, should also stimulate DNRA and immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, in addition to denitrification (Qiu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Putz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, our results showed that DNRA and immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N only accounted for less than 0.07% and 3.0% of the added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the entire soil profile across cultivation ages, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to denitrification, that represents N losses to the environment, DNRA is a N-conserving process that can convert mobile NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to relatively immobile NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N (Silver et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results found that denitrification was one of the two major fates of removed NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, whereas DNRA was negligible in RSD-treated soils across cultivation ages. It has been recently surmised that importance of DNRA relative to denitrification in soils increases with a higher C/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N ratio (Espenberg et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, lots of previous work showed that conditions with increased availability of electron donor (C) and limitation of electron acceptor (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) favoured DNRA over denitrification (Van Den Berg et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Putz et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pandey et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It was proposed that the lower limit of the C/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N ratio threshold for DNRA was 10\u0026ndash;15 (Stremińska et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although our RSD treatment with easily decomposable organic amendment might have increased soil C availability, serious accumulation of soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N inevitably resulted in a low C/NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N ratio of \u0026lt;\u0026thinsp;0.1, causing a dominance of denitrification over DNRA. In contrast, it is surprising that soil microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in the present study was not substantially stimulated by the addition of sugarcane bagasse with a C/N ratio of 66.4. This was not consistent with previous findings that the addition of organic materials with a high C/N ratio (\u0026gt;\u0026thinsp;18) generally stimulates soil microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (Cheng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The low microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N could be due to rapid denitrification taking precedence over microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, thereby limiting the amount of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N available for immobilization (Ragab et al. 1988). Alternatively, the presence of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N probably inhibited microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, considering the preferential use of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N over NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (Azam and Ifzal \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our results showed that RSD significantly increased the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration of the entire soil profile in all cultivation years. It was thus likely that the enhanced NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration under RSD might have supressed microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. This was supported by our recent study, which revealed that microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N gradually decreased with increasing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N concentration under increased C availability (Chen et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the ability to remove NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and subsequent dominant fates were independent of cultivation duration following the implementation of RSD, the relative importance of both fates changed over the cultivation years, validating our second half of the hypothesis. Denitrification is a biochemical process mediated by denitrifiers (\u003cem\u003enirK, nirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e; Yan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). P\u003csub\u003edenitrification\u003c/sub\u003e initially increased from 5 to 10 years of cultivation, and then decreased with cultivation years during RSD. The SEM revealed that the initial C/N ratio of topsoil had an indirect positive effect on P\u003csub\u003edenitrification\u003c/sub\u003e by driving the initial \u003cem\u003enirK\u003c/em\u003e abundance of topsoil under RSD treatment, whereas pH\u003csub\u003et\u003c/sub\u003e of topsoil promoted P\u003csub\u003edenitrification\u003c/sub\u003e directly during RSD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). A high C/N ratio in soil may strengthen enzymatic activities associated with denitrification, such as the nitrite reductase (\u003cem\u003enirK\u003c/em\u003e), owing to the ample availability of C source, thereby further promoting denitrification (Lan et al. 2023). A previous study also found that the absolute abundance of the \u003cem\u003enirK\u003c/em\u003e gene gradually increased with the increase of soil C/N ratio (Zhi and Ji \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, stronger denitrification often occurs at a higher pH value of the soil under anaerobic conditions (Šimek and Cooper \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is thus likely that increased soil pH is probably responsible for enhanced denitrification following RSD.\u003c/p\u003e \u003cp\u003eIn contrast, leaching is considered as a physical process, and thus can be regulated by soil BD, porosity, texture, irrigation water, etc. (Young et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hafshejani et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our results indeed found that P\u003csub\u003eleaching\u003c/sub\u003e was negatively correlated with topsoil BD (Fig. S2a), in agreement with other studies (Celik et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This was because increased BD can lead to smaller porosity with greater penetration resistance (Unger et al. 1996). In addition, we found a positive relationship between the volume of irrigation water and P\u003csub\u003eleaching\u003c/sub\u003e (Fig. S2b), corroborating with the finding of Zhu et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), showing that higher irrigation levels could trigger more substantial leaching of soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N.\u003c/p\u003e \u003cp\u003eIn spite of a powerful and universal role of RSD application in removing over-accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, the dominant fates of denitrification and leaching into subsoil inevitably caused N-related losses and environmental pollution. It was not viable to remove accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N at the expense of enhanced N\u003csub\u003e2\u003c/sub\u003eO emission and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N leaching during RSD treatment. Thus, future studies are needed to investigate the conditions (i.e. quantity and types of organic materials) that can promote the conversion of accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N or organic N when applying RSD into the intensive vegetation cultivation soils. For instance, the addition of organic materials with higher C/N ratios could cause higher microbial immobilization of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N but lower N\u003csub\u003e2\u003c/sub\u003eO emission in soils (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, biochar amendment has been proposed as a promising strategy for mitigating N\u003csub\u003e2\u003c/sub\u003eO emission from denitrification due to its liming effect and high surface area (Nelissen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lehmann et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results demonstrated a powerful and universal role of RSD application in removing over-accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N from the topsoil in intensive vegetable lands across cultivation years. We found that denitrification and leaching into subsoil were the main fates of removed NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, exhibiting a trade-off in all cultivation years. The duration of intensive vegetable cultivation affected the two fates of topsoil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N during RSD treatment. We proposed that cultivation age-induced changes in initial soil C/N ratio had a positive effect on P\u003csub\u003edenitrification\u003c/sub\u003e by driving the initial \u003cem\u003enirK\u003c/em\u003e abundance under RSD treatment, while pH\u003csub\u003et\u003c/sub\u003e had a direct positive effect on P\u003csub\u003edenitrification\u003c/sub\u003e. Notably, denitrification and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N leaching into subsoil signified the N losses and environmental pollution during RSD treatment. For better management of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in intensive vegetable production systems when utilizing RSD treatment, future research should focus on: (1) how to mitigate soil N\u003csub\u003e2\u003c/sub\u003eO emission from denitrification and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N leaching; (2) how to improve soil N conservation processes, including microbial NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N immobilization and DNRA. Overall, our research provided a new perspective and feasible technology for utilizing RSD to remove accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in intensive vegetable topsoil.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003ch2\u003eAcknowledgements:\u003c/h2\u003e \u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (grant numbers 42425705, 42122055 and 41977032), Jiangsu Carbon Peak Carbon Neutrality Science and Technology Innovation Fund (BE2022308), the Innovative and Entrepreneurial Talents Project of Jiangxi Province (grant number S2021DQKJ0001) and Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (grant number KYCX23_1704).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAzam F, Ifzal M (2006) Microbial populations immobilizing NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e-N differ in their sensitivity to sodium chloride salinity in soil. 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Agric Ecosyst Environ 335:108005\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":"Intensive vegetable cultivation, Reductive soil disinfestation, NO3−-N fates, NO3−-N removal, NO3−-N leaching, Denitrification","lastPublishedDoi":"10.21203/rs.3.rs-5860188/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5860188/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eReductive soil disinfestation (RSD) can remove over-accumulated nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) from topsoil in intensive vegetable fields via elevating NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N consumption processes. The duration of intensive vegetable cultivation may affect the relative importance of these consuming processes of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N during RSD treatment by altering topsoil properties. However, it remains elusive how the duration of intensive vegetable cultivation affects the fates of topsoil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N during RSD treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHere, a soil column experiment labeled with K\u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e was conducted to investigate the effects of different cultivation ages (5, 10, 20 and 30 years) of intensive vegetables on the fates of topsoil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N under RSD treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that more than 91.8% of the added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N in topsoil was removed by RSD treatment, regardless of cultivation years. There was a trade-off between denitrification and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N leaching into the subsoil, both of which together accounted for 85.5\u0026ndash;97.1% of the added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, regardless of cultivation years. The proportion of gaseous \u003csup\u003e15\u003c/sup\u003eN loss via denitrification to added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (P\u003csub\u003edenitrification\u003c/sub\u003e) initially increased from 5 to 10 years of cultivation, and then decreased with further cultivation ages, but the trend was reversed for the proportion of leaching of \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N into the subsoil to added \u003csup\u003e15\u003c/sup\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N (P\u003csub\u003eleaching\u003c/sub\u003e). The structural equation model revealed that the initial soil carbon/nitrogen ratio had an indirect positive effect on P\u003csub\u003edenitrification\u003c/sub\u003e by driving the initial \u003cem\u003enirK\u003c/em\u003e abundance under RSD treatment.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverall, our results highlight the critical role of using RSD in removing accumulated NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N from the topsoil with its fates of a trade-off between P\u003csub\u003edenitrification\u003c/sub\u003e and P\u003csub\u003eleaching\u003c/sub\u003e as ages of intensive vegetable cultivation.\u003c/p\u003e","manuscriptTitle":"The duration of intensive vegetable cultivation regulates the fates of accumulated nitrate under reductive soil disinfestation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 13:03:18","doi":"10.21203/rs.3.rs-5860188/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-03-11T00:48:39+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-01-24T10:48:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-24T10:40:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-01-21T08:20:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-21T03:19:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-01-19T10:20:00+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":"bed2bc82-a4f2-4d08-8e5c-1695c98205c2","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-07T16:07:16+00:00","versionOfRecord":{"articleIdentity":"rs-5860188","link":"https://doi.org/10.1007/s11104-025-07420-8","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-04-05 15:57:18","publishedOnDateReadable":"April 5th, 2025"},"versionCreatedAt":"2025-01-28 13:03:18","video":"","vorDoi":"10.1007/s11104-025-07420-8","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07420-8","workflowStages":[]},"version":"v1","identity":"rs-5860188","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5860188","identity":"rs-5860188","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}