Nitrogen-induced acidification increases soil organic carbon accrual by promoting particulate organic carbon and microbial necromass under long-term experiment in the paddy soils of East China

Nitrogen-induced acidification increases soil organic carbon accrual by promoting particulate organic carbon and microbial necromass under long-term experiment in the paddy soils of East China | 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 Nitrogen-induced acidification increases soil organic carbon accrual by promoting particulate organic carbon and microbial necromass under long-term experiment in the paddy soils of East China Zhaoming Chen, Qiang Wang, Jinchuan Ma, Feng Wang, Junwei Ma, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5562758/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted 5 You are reading this latest preprint version Abstract Background and aims Nitrogen (N) addition can substantially affect soil carbon cycling in agroecosystems. Microbial necromass carbon (MNC) is widely recognized as a key contributor to soil organic C (SOC) fractions. However, the mechanisms underlying the responses of MNC and SOC fractions to N fertilization in paddy soils remain unclear. Methods A field experiment with four N rates, namely, 0, 300, 450, and 600 kg N ha –1 yr –1 was conducted to determine the effects of N addition on SOC fractions, soil microbial necromass carbon (MNC), enzyme activity, and microbial biomass in paddy soils with rice–wheat rotation. Results N addition increased SOC and POC concentrations by 2.88–8.41% and 14.6–41.2%, respectively, but did not affect MAOC. The ratio of MAOC to POC was reduced by N addition, indicating that N addition decreased SOC stability. N addition increased MNC concentration by 7.32–22.5% and its contribution to SOC by 4.14–13.7%. The activity of β-1,4- N -acetyl-glucosaminidase was decreased, while the activities of leucine amino peptidase and acid phosphatase were increased under P addition. Structural equation modeling and random forest revealed that N-induced decrease in soil pH promoted the accrual of MNC by increasing root and microbial biomass, consequently improving POC. Conclusions POC is likely more vulnerable to N addition than MAOC. N-induced acidification is the primary driver for promoting SOC accrual by increasing POC in paddy soils. Nitrogen fertilization Mineral-associated organic carbon Microbial necromass Particulate organic carbon Paddy soil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The continuous increase in SOC sequestration is important for alleviating global climate change because SOC is the largest C pool in terrestrial ecosystems (Lal 2018 ; Lugato et al. 2021 ; Sun et al. 2024 ). Rice is a primary food product worldwide, especially in China, India, and Southeast Asia (Bräuer et al. 2013 ; Tian et al. 2015 ). The global stock of SOC in paddy fields is approximately 8.5 Pg of carbon in the topsoil layer, accounting for 47.2% of the total SOC storage in the 1-m depth (Liu et al. 2021 ). Hence, even a slight change in paddy SOC stocks under N addition substantially affects climate change (Pan et al. 2004 ). Therefore, understanding the mechanisms underlying SOC sequestration in paddy soils is important in the context of agricultural C neutrality. Nitrogen (N) addition is generally considered an important practice for improving crop productivity in agroecosystems (Zhang et al. 2012 ). N addition increases crop yields and affects SOC cycling in cropland soils owing to the high relevance of soil C and N cycles (Kicklighter et al. 2019 ; Hu et al. 2022b ; Hu et al. 2023 ; Zhou et al. 2023a ), given that N fertilization has the potential to enhance SOC accrual (Sun et al. 2023 ). N fertilization increases plant aboveground and root biomass by enhancing soil N availability, resulting in the increased plant C incorporation into the soil and SOC accumulation (Ye et al. 2018 ; Chen et al. 2020 ). In general, N addition-induced soil acidification can inhibit the microbial decomposition of SOC, resulting in SOC accumulation (Lu et al. 2021 ). Conversely, other studies have suggested that N addition decreases SOC concentration due to increased positive priming effect of SOC (Zheng et al. 2023 ; Liu et al. 2025 ). Therefore, it is crucial to reveal the mechanisms behind SOC dynamics following long-term N addition in paddy soils. SOC is a complex continuum consisting of gradually decomposing plant compounds and microbial residues (Lehmann and Kleber 2015 ; Lavallee et al. 2020 ). Considering their different turnover rates and functions, the SOC pools are generally divided into two fractions, that is, particulate and mineral-associated organic C, namely, POC and MAOC, respectively (Lugato et al. 2021 ; Georgiou et al. 2022 ). The fractionation method provides more mechanistic information than that of the entire SOC component (Lavellee et al. 2020; Yu et al. 2022 ; Zhang et al. 2024a ). POC is a temporary SOC reservoir that consists primarily of partly decomposed fragments (Craig et al. 2021 ; Angst et al. 2023 ). In contrast, MAOC is composed of relatively small and recognizable compounds that interact with mineral particles (von Lützow et al. 2007 ; Sokol et al. 2018 ; Wu et al. 2023 ). The mean turnover time (129 vs. 23 years) in MAOC is greater than that in POC (Zhou et al. 2024 ). Therefore, POC is more susceptible to agricultural management practices such as N fertilization than MAOC (Chen et al. 2021 ). Tang et al. ( 2023 ) conducted a meta-analysis, and who reported that N addition promoted POC by 16.4% and MAOC by 3.7%. However, some researchers found that N addition increased POC, but decreased MAOC (Sun et al. 2023 ). Therefore, it is necessary to investigate the factors controlling the response of the two SOC fractions to N addition in paddy fields. Microbial necromass C (MNC), including dead microbial residues and by-products, is an important component of SOC formation and accrual (Liang et al. 2017 ; 2019 ; Whalen et al. 2022 ). Microorganisms can convert plant residues into their own biomass and accumulate in soils as necromasses via a microbial carbon pump, including microbial catabolism and anabolism (Zhu et al. 2020 ). In theory, N fertilization can affect MNC formation and accumulation by altering microbial trials, and thereby changing POC and MAOC accumulation (Hu et al. 2022a ). For example, N addition reduces the total microbial, bacterial, and fungal biomass (Zhang et al. 2018 ), which may restrict MNC formation and accumulation. In contrast, N fertilization can significantly increase MNC concentration by 12% by increasing microbial biomass (Zhou et al. 2023b ). Therefore, it is imperative to understand the effects of N addition on MNC formation and sequestration in paddy soils. Recent paradigms suggest that microbial necromass is not only a crucial contributor to the formation of MOAC but also to that of POC (Bian et al. 2024 ; Jia et al. 2025 ; Yang et al. 2025 ). Given that microbial necromass plays a vital role in MAOC formation and accumulation due to its easy association with soil minerals (Sokol et al. 2019b ; Lavallee et al. 2020 ). Meanwhile, evidence has shown that microbial necromass also contributes to POC accumulation (Hu et al. 2025 ; Jia et al. 2025 ). Because particulate organic matter serves a hotspot for microbial turnover driving the formation and accumulation of microbial necromass (Li et al. 2024 ). Yet, it remains unclear how N addition affect the linkages of MNC with SOC fractions in paddy soils. Long-term N addition experiments can deepen our understanding of the effects of N on SOC sequestration in paddy soils. Here, we set up a long-term experiment with four N rates, that is, 0, 300, 450, and 600 kg N ha –1 yr –1 in 2011 in paddy soils with a rice–wheat rotation. Taking advantage of this field experiment, we assessed the responses of POC, MAOC, MNC, extracellular enzyme activity, and microbial biomass to long-term N addition. Our objectives were: 1) to identify the effects of N addition on SOC fractions and MNC in paddy soils; and 2) to investigate how N-induced acidification influence SOC accrual by combing microbial traits, soil chemical properties and root biomass. We hypothesized that 1) N fertilization would increase POC, resulting from an increase in root biomass and a decrease in microbial decomposition induced by N fertilization; 2) N addition would not affect MAOC because of its longer turnover and lower sensitivity to environmental disturbance; and 3) N fertilization would increase MNC and its contribution to SOC persistence due to enhanced microbial biomass. Methods and materials Experimental design and sampling The field experiment with rice–wheat rotation was started in 2011 at the experimental farm of Zhejiang Academy of Agricultural Sciences in Shaoxing City (119°54´E, 31°16´N), Zhejiang Province, China (Fig. 1 ). The tested soil belonged to the Gleyi–Stagnic Anthrosol category. The original soil chemical properties (0–20 cm) were pH, 6.27; SOC, 13.0 g kg –1 ; total N (TN), 1.45 g kg –1 . Four treatments were designed in randomized field plots (6 m × 5 m) with three replicates. The treatments consisted of N fertilizer application during the rice/wheat season at rates of 0, 150, 225, and 300 kg N ha –1 , that is 0 (N0), 300 (N1), 450 (N2), and 600 kg N ha –1 yr –1 (N3). In both seasons, N fertilizer (urea, 46%N) was applied at a rate of 50% during the sowing stage and the remaining 50% during the booting stage. In all plots, P fertilizer was applied as basal fertilizer at rate of 170 kg P ha –1 , and K fertilizer was applied at rate of 100 kg K ha –1 in both of rice and wheat seasons. All the field management practices followed those used by the local farmers. After the wheat had been harvested, root sample was collected from a 0–20 cm soil layer in a randomly subplot (1 m × 1 m) in each plot, and immediately rinsed with clean water. The clean root sample was dried at 70°C to reach a stable weight. Root biomass was estimated as the sum of the rice and wheat root biomasses. Topsoil (0–20 cm) was collected in May 2023 after the wheat had been harvested. Five soil cores were sampled from each plot at random locations and homogenized as a single sample. The soil sample from each plot was separated into three portions. One subsample was air-dried for determination of soil physiochemical properties, including SOC fractions and amino sugars. One was immediately used for soil mineral N and enzyme activity analysis; and the third soil subsample was stored at − 60°C for assessment of microbial traits. Soil property analysis The concentrations of SOC and TN were measured by an elemental analyzer (Elementar, Vario Max, Germany). Soil available N (NH 4 + -N and NO 3 − –N) was measured using an auto-analyzer after extracting with 1 M KCl solution (Bran Lubbe, Norderstedt, Germany). Soil available phosphorus (AP, Olsen P) was extracted with HCl-NH 4 F and determined using ICP-MS (Optima 2000DV, Waltham, USA). Soil pH was measured using a pH electrode (S400-K, Mettler-Toledo, Switzerland). Soil organic carbon fractionation The SOC pool was divided into POC and MAOC using particle size fractionation (Chen et al. 2017 ). Air-dried soil (20 g) was dispersed in sodium hexametaphosphate solution (100 mL, 5% w/v). The dispersed soil sample was rinsed onto a 53-µm sieve and deionized water was used to wash the soil sample until the water was clear. The fraction on the sieve was collected as the POC fraction and the fraction passing through the sieve was collected as the MAOC fraction. The POC and MAOC fractions were dried at 60°C, and then ground. Organic C concentrations in the POC and MAOC fractions were measured by an elemental analyzer (Elementar, Vario Max, Germany). Soil enzyme activity assay The activities of the soil extracellular β-N-acetyl-glucosaminidase (NAG), leucine aminopeptidase (LAP), β-1, 4-glucosidase (BG), and acid phosphatase (ACP) were determined according to Luo et al. ( 2022 ). Briefly, soil samples (1 g) were mixed with 100 mL acetate buffer (50 mM). NAG, BG, and ACP activities were measured using substrates labeled with 4-methylumbelliferyl (MUB), whereas a substrate labeled with 7-amino-4-methylcoumarin (MUC) was used to determine LAP activity. Soil slurry (200 µL) and substrate (200 µM, 50 µL) were placed into a 96-well microplate for each sample. A microplate reader was used to quantify fluorescence at 360 nm excitation and 460 nm emission. The activities of soil extracellular enzymes were calculated based on MUB or MUC standard curves. PLFA analysis Soil microbial biomass PLFAs concentration was measured using the method described by Chen et al. ( 2017 ). Freeze-dried soils were extracted with a mixture containing chloroform, citrate buffer and methanol (1:0.8:2 v/v/v). A silica column was used to separate the phospholipids. After that, they transformed into fatty acid methyl esters. We determined the methyl esters using an Agilent GC combined with MIDI identification software 4.5 (MIDI Sherlock, Newark, DE, USA). An internal standard (19:0) was used to quantify the PLFA concentration. The representative PLFAs of bacteria, fungi and actinomycete were identified according to previous studies (Zelles, 1999 ; Fierer et al., 2003 ; Chen et al., 2017 ; Joergensen, 2021 ). Their specific PLFAs are shown in Table S1 . Amino sugar analysis Soil amino sugar concentration was determined according to Zhang and Amelung ( 1996 ). A soil sample (< 0.15 mm) was hydrolyzed for 8 h at 105°C using HCl (6 M, 10 mL). After cooling, the hydrolysate was purified by filtration, centrifugation, and drying under N gas. The dried residue was derivatized using a derivatization reagent and dissolved in dichloromethane. The derivatives were determined using an Agilent GC. The soil MNC (FNC + BNC) contents were estimated using the methods used by Hu et al. ( 2024 ). Statistical analysis The effects of N fertilization on soil chemical properties (pH, SOC, TN, AN, AP, POC, and MAOC), root biomass, extracellular enzyme activities (NAG, LAP, BG, and ACP), and soil microbial communities, that is, total PLFAs, fungi, bacteria, and actinomycetes were examined using analysis of variance (ANOVA). Pearson’s correlations were used to assess the relationships between amino sugars, MNC, and environmental factors. Random forest (RF) analysis was used to detect the relative importance of soil parameters for POC, MAOC, and MNC concentrations using R. We estimated the significance of each predictor using the “rfPermute” package in R. A structural equation model (SEM) was applied to assess the direct and indirect effects of N addition on MNC, POC and MAOC using AMOS 22.0 (Smallwaters Corporation, IL, USA). A prior SEM model was constructed base on the ecological hypotheses, assuming that N addition indirectly impact SOC fractions through influencing soil biotic and abiotic properties, and root biomass (Fig. S1 ). Microbial biomass and root biomass were indicated by the total microbial PLFAs and total crop root biomass in the model, respectively. All the data analyses were performed using SPSS software (SPSS 22.0, Chicago, IL, USA). Results Soil chemical properties and root biomass N fertilization increased SOC and POC concentrations by 2.87–8.41% and 14.6–41.2%, respectively, compared to N0 (Fig. 2 ). The soil MAOC concentration increased by 12.4% in N1 compared to that in N0. However, no significant changes were noted among the N0, N1, and N2 treatments. N addition, except for N1, significantly decreased the MAOC: POC ratio relative to that of the N0 treatment. Long-term N fertilization reduced soil pH from 6.39 in N0 to 5.50 in HN (Table 1 ). Soil AN concentration in N2 and N3 were significantly higher than those in N0. Long-term N addition decreased the soil AP concentration compared to N0. Root biomass increased with increasing the addition rate of N fertilizer ( P < 0.05). N addition increased rice and wheat root biomass by 40.7–53.1% and 142–249%, respectively, compared to N0. Table 1 Soil chemical properties and crop root biomass under nitrogen addition Treatment pH TN (g kg –1 ) AN (mg kg –1 ) AP (mg kg –1 ) Rice root biomass (kg ha –1 ) Wheat root biomass (kg ha –1 ) N1 6.39 ± 0.04 a 1.43 ± 0.03 b 15.1 ± 0.7 c 44.3 ± 2.9 a 1336 ± 93 b 228 ± 49 c N2 6.20 ± 0.07 b 1.61 ± 0.06 ab 17.9 ± 3.9 bc 30.6 ± 3.1 b 2597 ± 224 a 552 ± 86 b N3 5.82 ± 0.10 c 1.70 ± 0.07 a 23.5 ± 1.2 a 38.4 ± 6.0 b 2643 ± 153 a 764 ± 115 a N4 5.50 ± 0.05 d 1.71 ± 0.10 a 20.3 ± 0.1 ab 34.5 ± 3.5 b 2759 ± 200 a 797 ± 160 a TN, total nitrogen; AN, available nitrogen; AP, available phosphorus. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha –1 yr –1 , respectively. Different letters indicate significant differences among different treatments at P < 0.05. Soil amino sugar and microbial necromass The total soil amino sugar concentration increased with increasing the addition rate of N fertilizer ( P < 0.05; Fig. 3 a). Compared to N0, N addition enhanced the concentrations of glucosamine (GlcN), galactosamine (GalN), and MurA by 6.61–21.2%, 9.68–22.1% and 10.6–28.6%, respectively (Fig. 3 b, c, and d). The MNC, FNC, and BNC concentrations were 1.07–1.22, 1.06–1.21, and 1.11–1.29 times greater in N-fertilized than in N0, respectively (Figs. 4 a, b, and c). Higher N addition (N2 and N3) significantly increased the MNC and BNC contributions to SOC by 9.70–13.1% and 15.0–19.3%, respectively, compared to N0. The contribution of FNC to SOC was 1.12 times higher in N3 than in N0 but was similar among N0, N1, and N2. Soil enzyme activity and microbial biomass Long-term N fertilization decreased soil NAG activity by 21.3–27.0%, but increased soil LAP and ACP activities by 32.6–103% and 36.3–50.7%, respectively, compared to N0 (Fig. 5 ). N fertilization increased total PLFAs by 29.3–60.9% and bacterial PLFAs by 32.5–66.1% relative to the N0 treatment (Fig. 6 a and b). High and medium levels of N fertilization significantly increased fungal abundance (Fig. 6 c). The abundance of actinomycetes was only increased by medium N fertilization (N2), and no significant change was found between the N0, N1, and N3 treatments (Fig. 6 d). The fungal/bacterial PLFAs ratio was reduced by N fertilization (Fig. S2 b). This indicated that N addition changed the soil microbial community composition. Relationship between SOC fractions, MNC, and environmental factors Random forest (RF) analysis was performed to determine the most important abiotic and biotic factors predicting POC, MAOC, and MNC accumulation. This explained 70.92%, 37.80%, and 78.79% of the total variation in the POC, MAOC, and MNC concentrations, respectively (Fig. 7 ). Soil pH was an important factor in predicting the POC and MNC concentrations (Figs. 7 a and c). POC was significantly affected by MNC, SOC, microbial biomass, and root biomass. MAOC was significantly affected by root biomass, MNC, and bacterial biomass. MNC were significantly affected by microbial biomass, POC, SOC, and root biomass. The SEM results showed that the model explained 80%, 43%, and 94% of the total variance in POC, MAOC, and MNC, respectively (Fig. 8 ). N addition negatively affected soil pH but positively affected soil TN, root biomass, and MNC. Soil pH indirectly affected MNC by influencing root and microbial biomass. Root biomass directly affected MNC, which is related to POC. Soil microbial biomass directly and negatively affected the MAOC. Discussion Effects of N addition on soil organic carbon Our data indicated that the SOC concentration significantly enhanced by N fertilization (Fig. 1 ). This is consistent with the increase in SOC concentrations reported in previous meta-analyses (Chen et al. 2018 ; Ni et al. 2022 ; Yang et al. 2022a ). The balance between the plant C input and SOC loss plays a crucial role in the accrual of SOC (Xu et al. 2021 ; Liu et al. 2023b ). In our study, rice and wheat straw were both removed from the experimental plots. Therefore, crop-root-derived organic C may be the primary C source for SOC accumulation. Plant roots may play a more important role in SOC formation and accrual than the aboveground biomass (Sokol and Bradford 2019; Sokol et al. 2019a; Xu et al. 2022 ). Increased root biomass resulted from N addition owing to enhanced soil N availability under sufficient soil P and K (Table 1 ). This led to increased root-derived C incorporation into the soil, promoting SOC formation and accrual (Xu et al. 2021 ; Yang et al. 2022a ; Cai et al. 2023 ; Hu et al. 2024 ). However, N addition may not change root biomass under P limitation (Chen et al. 2020 ; Liu et al. 2023b ). In these soils, N addition increases SOC concentration, which is coincide with a decrease in soil microbial activity (Liu et al. 2023b ; Qi et al. 2023 ). Alternatively, SOC loss via soil respiration is a vital factor controlling SOC content (Yang et al. 2022b ). A 1413-paired observation was used to assess the response of soil respiration to N input (Yang et al. 2022b ). This suggested that soil microbial activity was reduced by N fertilization, indicating that N input reduced the loss of SOC and increased the sequestration of SOC (Yang et al. 2022b ). Soil acidification can constrain microbial activity and metabolism, consequently reducing SOC decomposition and increasing SOC accumulation (Malik et al. 2018 ). In the present study, soil pH was negatively correlated with SOC concentration (Fig. S3). This indicated that N-induced soil acidification was the main driver of SOC sequestration (Xu et al. 2021 ). Similarly, a study from field experiment showed that the SOC concentration increased with decreasing soil pH induced by N fertilization in paddy soils (Sun et al. 2023 ). The increase in root C and the decrease in SOC loss suggest that N addition promotes SOC accrual in paddy fields. Effects of N addition on SOC physical fractions In accordance with previous studies (Sun et al. 2023 ; Tang et al. 2023 ), we observed that N addition enhanced POC concentration. The positive responses of POC to N fertilization have been attributed to higher plant C inputs with N fertilization (Ye et al. 2018 ; Rocci et al. 2021 ). POC primarily consists of plant materials that are partially decomposed (Lavallee et al. 2020 ; Keller et al. 2022). POC concentration was positively correlated with root biomass (Fig. S4), indicating that the increased root biomass induced by N input promoted the POC pool in the paddy soils. Root biomass contributes more to the POC pool than aboveground biomass (Ye et al. 2018 ; Püspök et al. 2022 ). Soil microbial community activity plays a crucial role in determining the quantity of POC because of its easy microbial decomposition of POC (Cotrufo et al. 2019 ). Although the soil microbial biomass increased, soil NAG activity decreased with N addition (Figs. 5 a and 6 ). The enhancement of the POC pool is partly explained by the inhibition of POC decomposition caused by N-induced soil acidification. This is in accordance with findings from a semi-arid steppe ecosystem, where N addition decreased microbial activity and increased POC accumulation (Averill and Waring 2018 ; Ye et al. 2018 ). MAOC is more critical in maintaining SOC than POC because of its slower turnover (Lugato et al. 2021 ; Zhou et al. 2024 ). The microbial conversion of POC into MAOC plays a vital role in the preservation of SOC (Witzgall et al. 2021 ; Niu et al. 2024 ). Therefore, an increase in MAOC can reduce soil C emissions and promote SOC persistence (Liu et al. 2022 , 2023a ; Zhou et al. 2024 ). Nevertheless, our study has shown that N fertilization did not affect MAOC, which is similar with previous study (Rocci et al. 2021 ). Similar to POC, MAOC formation and persistence are affected by plant and microbial traits (Georgiou et al. 2022 ; Li et al. 2024 ). MNC makes a crucial contribution to the formation and accrual of MAOC (Sokol and Bradford 2019; Sokol et al. 2019a, 2022 ). However, MAOC was negatively correlated with MNC in our study (Fig. S4). In addition, the SEM results showed that MNC were not correlated with MAOC. Microbial necromass is not a key factor in regulating MAOC accumulation. Zhu et al. ( 2024 ) found that mineral preservation plays a vital role in MAOC accumulation. N-induced soil acidification can stimulate the release of base cations from soil minerals, thereby reducing MAOC stability and accumulation (Püspök et al. 2022 ; Sun et al. 2023 ). Previous studies have revealed that POC is more vulnerable to N fertilization than MOAC (Wu et al. 2023 ). Our results showed that POC increased but MOAC was not affected by N input (Fig. 1 ). Moreover, the POC proportion in SOC increased but proportion of MAOC decreased by N addition (Fig. S1 ). POC, rather than MAOC, was the main contributor to the preservation of the SOC pool in paddy fields with long-term N addition. The RF results demonstrated that POC was the most vital factor affecting SOC accumulation (Fig. S5). Furthermore, some studies have reported different responses of POC and MAOC fractions to N fertilization in rice paddy soils (Sun et al. 2023 ). They found that N enrichment increased soil POC concentrations, but decreased MAOC concentrations. The MAOC: POC ratio decreased with N addition, indicating that N addition decreased SOC functionality. Similar results were reported by Tang et al. ( 2023 ). Given that POC has a shorter turnover time and lower resistance to environmental disturbances than MAOC (Wu et al. 2023 ). Therefore, our findings suggest that long-term N addition reduces the stability of the SOC pool, increasing the vulnerability of SOC to future global change. Effects of N addition on microbial necromass carbon Microbial necromasses are one of the most important components of the stable SOC pools (Liang et al. 2017 ; Wang et al. 2021 ). MNC accounted for 28.9–32.8% of SOC, of which FNC accounted for 21.6–24.1% of SOC (Fig. 4 ). These results are similar to those of previous studies on paddy fields (Chen et al. 2021 ). Both of the total MNC concentration and its contribution to SOC increased by N addition, which is consistent with two global meta-analyses (Hu et al. 2022a ; Zhou et al. 2023b ). The increase in MNC with N addition can be explained as follows. First, N addition affects crop root biomass, which stimulates soil microbial proliferation and enhances its biomass, ultimately accumulating more microbial necromass in soils (Hu et al. 2023 ; Yang et al. 2024 ). Roots play a more crucial role in the formation and accumulation of MNC than the aboveground biomass (Jia et al. 2022 ; Zhao et al. 2024 ). Second, N addition increases soil N availability, which inhibits microbial necromass decomposition because microbial necromass is considered an N-rich compound (Wang et al. 2020 ). As expected, a negative correlation is found between NAG and MNC (Fig. S5). Therefore, the decreased the soil NAG activity caused by N addition may reduce the microbial decomposition of MNC and promoted MNC accumulation (Ma et al., 2023). Third, N addition increases the microbial C-use efficiency (Feng et al. 2022 ), which can promote microbial biomass and enhance MNC accumulation (Duan et al. 2023 ). Our findings showed that MNC significantly and directly affected POC accumulation (Figs. 8 and S5), indicating that the enhancement of POC partly contributed to the increase in MNC under N addition. Generally, soil microbial necromass is preferentially occluded in MAOC (Xuan et al. 2024 ). However, some studies have shown that MNC can contribute to approximately 40% of the POC in paddy soils (Bian et al. 2024 ), suggesting that MNC should be considered as an important contributor to POC formation. Soil microorganisms can decompose and use plant debris for their growth in POC and accumulate as necromass within it (Li et al. 2023 ). Therefore, the POC surface can serve as a hotspot for microbial growth and proliferation, driving necromass formation and accumulation (Li et al. 2024 ). Contrary to POC, our data revealed that MAOC accrual was not driven by MNC under N addition (Fig. 8 and S5). Increased soil pH under N addition could weaken mineral protection of MNC (Chen et al. 2020 ; Sun et al. 2023 ), although N addition significantly increased MNC concentration, resulting in balance of MAOC. Moreover, recent study has shown that mineral protection is a more important role in MAOC formation and accrual than microbial necromass (Zhu et al. 2024 ). In the future, we will focus on the responses of unprotected and protected MNC to N addition, which could help better understand the dynamics of SOC fractions under long-term N addition in paddy soils. Conclusion This study has provided empirical evidence of the mechanisms that promote SOC persistence and change SOC functionality following long-term N fertilization in paddy soils. SOC concentration was increased by N addition, primarily because of enhanced POC rather than MAOC. The POC was significantly increased by N addition owing to the increase in root biomass and microbial necromass. Meanwhile, N addition enhanced the contribution of microbial necromass to total SOC accrual, which was mainly attributed to the enhancement of soil microbial biomass. Collectively, soil pH is the most important factor regulating POC and MNC accumulation, indicating that N-induced acidification drives SOC formation and persistence in paddy soils. Taken together, combining soil chemical properties, plant input, and microbial traits should contribute to our understanding of how N fertilization affects SOC formation and sequestration in rice–wheat rotation. Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 08 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted Reviewers agreed at journal 26 Mar, 2025 Reviewers invited by journal 26 Mar, 2025 Editor assigned by journal 25 Mar, 2025 First submitted to journal 25 Mar, 2025 Editorial decision: Minor revisions 27 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5562758","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434187375,"identity":"760a1b23-1e6d-4e0d-973a-799b4b34c4eb","order_by":0,"name":"Zhaoming Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhaoming","middleName":"","lastName":"Chen","suffix":""},{"id":434187376,"identity":"6789c497-ef15-4ebf-92f6-6b67414a4cce","order_by":1,"name":"Qiang Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wang","suffix":""},{"id":434187377,"identity":"27017326-56d0-434a-9428-930f68723245","order_by":2,"name":"Jinchuan Ma","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinchuan","middleName":"","lastName":"Ma","suffix":""},{"id":434187378,"identity":"5e69d13b-c8b6-4ddd-82d9-1013b249758b","order_by":3,"name":"Feng Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Wang","suffix":""},{"id":434187379,"identity":"f1028591-70f4-4e6c-8154-86688994d396","order_by":4,"name":"Junwei Ma","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Ma","suffix":""},{"id":434187380,"identity":"3e61cf38-2f09-4b7b-b815-ccb72fa8a037","order_by":5,"name":"Jing Ye","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Ye","suffix":""},{"id":434187381,"identity":"898f348d-1525-4f08-84ca-730c90691563","order_by":6,"name":"Ping Zou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Zou","suffix":""},{"id":434187382,"identity":"4e1019ff-1c59-4131-a5d8-1f7c9f833906","order_by":7,"name":"Wanchun Sun","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Wanchun","middleName":"","lastName":"Sun","suffix":""},{"id":434187383,"identity":"5fd91307-b7a1-4bcb-aa1f-4586e365c008","order_by":8,"name":"Qiaogang Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYLCCDyTrYJxBshZmHpKUG9xI3vza5o+dvHwD88MPDDV3iNGSVmadw5NsuOEAm7EEw7FnxGjJMTPOkWBm3MDAYMbA2HCYSC0WBvX28xvYvxGtxfgxQ8LhxIYDPETaInnmWRljz4HjyRsO8xRLJBwjQgvf8eTNH378qbad396+8cOHGiK0KBxgMJMAs5iBOIGwBgYG+QYGY9LTyygYBaNgFIwsAAA6lzli0afDWAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Qiaogang","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-12-02 08:34:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5562758/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5562758/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07551-y","type":"published","date":"2025-06-08T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79323069,"identity":"74746c68-0b9e-4208-9f69-154a3b54a2e8","added_by":"auto","created_at":"2025-03-27 04:50:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42800,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the experimental site in this study.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/2c6c49887db7623da245f1d1.jpg"},{"id":79323072,"identity":"32f444cf-808b-4b91-86d9-d0562acd4ca5","added_by":"auto","created_at":"2025-03-27 04:50:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38125,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of soil organic carbon (a), particulate organic carbon (b) and mineral-associated organic carbon (c), and ratio of mineral-associated organic carbon to particulate organic carbon (d) in all treatments. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e–1\u003c/sup\u003e yr\u003csup\u003e–1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/bf0cff1e8b6ea48de1971db3.jpg"},{"id":79323070,"identity":"2fe67d26-4a10-4280-b4d2-daf6bcedb889","added_by":"auto","created_at":"2025-03-27 04:50:45","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39203,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of total amino sugars (a), glucosamine (b), galactosamine (c) and murA (d) in all treatments. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e–1\u003c/sup\u003e yr\u003csup\u003e–1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/e65f1558b0b6e67cf5c2e6fb.jpg"},{"id":79323564,"identity":"03d09f50-fc2c-46cf-a6b4-b684d2827900","added_by":"auto","created_at":"2025-03-27 04:58:45","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":82886,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of MNC (a, b, c) and their contributions to soil organic carbon (d, e, f) in all treatments. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e–1\u003c/sup\u003e yr\u003csup\u003e–1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/470862a2dd80092feb77ff14.jpg"},{"id":79323568,"identity":"d49c69b6-3134-41ef-93ba-52b510174091","added_by":"auto","created_at":"2025-03-27 04:58:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37338,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of the soil extracellular β-N-acetyl-glucosaminidase (a), leucine aminopeptidase (b), β-1, 4-glucosidase (c) and ACP (d) in all treatments. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e–1\u003c/sup\u003e yr\u003csup\u003e–1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/6a42f4ec3f012b7907efd584.jpg"},{"id":79323902,"identity":"3547649c-47f1-4092-9106-d51d2ae627d9","added_by":"auto","created_at":"2025-03-27 05:06:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39011,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of total (a), bacterial (b), fungal (c) and actinoacteiral phospholipid fatty acids (d) in all treatments. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e–1\u003c/sup\u003e yr\u003csup\u003e–1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/cd8fad632494aa7f99d47fef.jpg"},{"id":79323079,"identity":"edd641a9-7a21-4fdf-bedb-5da4df07417f","added_by":"auto","created_at":"2025-03-27 04:50:46","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":127677,"visible":true,"origin":"","legend":"\u003cp\u003eRelative importance of soil abiotic and biotic properties controlling particulate organic carbon (a), mineral-associated organic carbon (b) and MNC (c) by the percentage increase of the mean squared error (%IncMSE) using Random Forest analysis. SOC, soil organic carbon; TN, total nitrogen; AN, available nitrogen; AP, available phosphorus; POC, particulate organic carbon; MAOC, mineral-associated organic carbon; MNC, microbial necromass carbon; NAG, β-N-acetyl-glucosaminidase; LAP, leucine aminopeptidase; BG, β-1, 4-glucosidase; ACP, acid phosphatase; PLFAs, phospholipid fatty acids. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/de88340bb8bf60a7adc20d9f.jpg"},{"id":79323090,"identity":"f75d7a01-5119-46ec-8fe6-667a00f597a6","added_by":"auto","created_at":"2025-03-27 04:50:46","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":56079,"visible":true,"origin":"","legend":"\u003cp\u003eSEM of the effects of N addition on microbial necromass carbon, particulate organic carbon and mineral-associated organic carbon in soils. The red and blue solid arrows indicate positive and negative relationships, respectively. Dotted line arrows indicate no significant relationships. Numbers next to the arrows are 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.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/d3569656a5d7966b8d29c648.jpg"},{"id":84242623,"identity":"a9ac0b24-46ae-4fc2-98dd-d2ee9faf3ec4","added_by":"auto","created_at":"2025-06-09 16:10:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1114437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/11939fc3-4da8-4d05-8b1f-c94893b1cf6d.pdf"},{"id":79323078,"identity":"bf1539c5-cebc-46ba-a4d4-1d376184d206","added_by":"auto","created_at":"2025-03-27 04:50:46","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":857188,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5562758/v1/4470d6f517b8791894cd058b.docx"}],"financialInterests":"","formattedTitle":"Nitrogen-induced acidification increases soil organic carbon accrual by promoting particulate organic carbon and microbial necromass under long-term experiment in the paddy soils of East China","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe continuous increase in SOC sequestration is important for alleviating global climate change because SOC is the largest C pool in terrestrial ecosystems (Lal \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lugato et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Rice is a primary food product worldwide, especially in China, India, and Southeast Asia (Br\u0026auml;uer et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tian et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The global stock of SOC in paddy fields is approximately 8.5 Pg of carbon in the topsoil layer, accounting for 47.2% of the total SOC storage in the 1-m depth (Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hence, even a slight change in paddy SOC stocks under N addition substantially affects climate change (Pan et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Therefore, understanding the mechanisms underlying SOC sequestration in paddy soils is important in the context of agricultural C neutrality.\u003c/p\u003e \u003cp\u003eNitrogen (N) addition is generally considered an important practice for improving crop productivity in agroecosystems (Zhang et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). N addition increases crop yields and affects SOC cycling in cropland soils owing to the high relevance of soil C and N cycles (Kicklighter et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), given that N fertilization has the potential to enhance SOC accrual (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). N fertilization increases plant aboveground and root biomass by enhancing soil N availability, resulting in the increased plant C incorporation into the soil and SOC accumulation (Ye et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In general, N addition-induced soil acidification can inhibit the microbial decomposition of SOC, resulting in SOC accumulation (Lu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, other studies have suggested that N addition decreases SOC concentration due to increased positive priming effect of SOC (Zheng et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Therefore, it is crucial to reveal the mechanisms behind SOC dynamics following long-term N addition in paddy soils.\u003c/p\u003e \u003cp\u003eSOC is a complex continuum consisting of gradually decomposing plant compounds and microbial residues (Lehmann and Kleber \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lavallee et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Considering their different turnover rates and functions, the SOC pools are generally divided into two fractions, that is, particulate and mineral-associated organic C, namely, POC and MAOC, respectively (Lugato et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Georgiou et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The fractionation method provides more mechanistic information than that of the entire SOC component (Lavellee et al. 2020; Yu et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). POC is a temporary SOC reservoir that consists primarily of partly decomposed fragments (Craig et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Angst et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In contrast, MAOC is composed of relatively small and recognizable compounds that interact with mineral particles (von L\u0026uuml;tzow et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sokol et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The mean turnover time (129 vs. 23 years) in MAOC is greater than that in POC (Zhou et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, POC is more susceptible to agricultural management practices such as N fertilization than MAOC (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Tang et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) conducted a meta-analysis, and who reported that N addition promoted POC by 16.4% and MAOC by 3.7%. However, some researchers found that N addition increased POC, but decreased MAOC (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, it is necessary to investigate the factors controlling the response of the two SOC fractions to N addition in paddy fields.\u003c/p\u003e \u003cp\u003eMicrobial necromass C (MNC), including dead microbial residues and by-products, is an important component of SOC formation and accrual (Liang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Whalen et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Microorganisms can convert plant residues into their own biomass and accumulate in soils as necromasses via a microbial carbon pump, including microbial catabolism and anabolism (Zhu et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In theory, N fertilization can affect MNC formation and accumulation by altering microbial trials, and thereby changing POC and MAOC accumulation (Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). For example, N addition reduces the total microbial, bacterial, and fungal biomass (Zhang et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which may restrict MNC formation and accumulation. In contrast, N fertilization can significantly increase MNC concentration by 12% by increasing microbial biomass (Zhou et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Therefore, it is imperative to understand the effects of N addition on MNC formation and sequestration in paddy soils. Recent paradigms suggest that microbial necromass is not only a crucial contributor to the formation of MOAC but also to that of POC (Bian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Given that microbial necromass plays a vital role in MAOC formation and accumulation due to its easy association with soil minerals (Sokol et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Lavallee et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Meanwhile, evidence has shown that microbial necromass also contributes to POC accumulation (Hu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Jia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Because particulate organic matter serves a hotspot for microbial turnover driving the formation and accumulation of microbial necromass (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Yet, it remains unclear how N addition affect the linkages of MNC with SOC fractions in paddy soils.\u003c/p\u003e \u003cp\u003eLong-term N addition experiments can deepen our understanding of the effects of N on SOC sequestration in paddy soils. Here, we set up a long-term experiment with four N rates, that is, 0, 300, 450, and 600 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e yr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in 2011 in paddy soils with a rice\u0026ndash;wheat rotation. Taking advantage of this field experiment, we assessed the responses of POC, MAOC, MNC, extracellular enzyme activity, and microbial biomass to long-term N addition. Our objectives were: 1) to identify the effects of N addition on SOC fractions and MNC in paddy soils; and 2) to investigate how N-induced acidification influence SOC accrual by combing microbial traits, soil chemical properties and root biomass. We hypothesized that 1) N fertilization would increase POC, resulting from an increase in root biomass and a decrease in microbial decomposition induced by N fertilization; 2) N addition would not affect MAOC because of its longer turnover and lower sensitivity to environmental disturbance; and 3) N fertilization would increase MNC and its contribution to SOC persistence due to enhanced microbial biomass.\u003c/p\u003e"},{"header":"Methods and materials","content":"\u003cp\u003eExperimental design and sampling\u003c/p\u003e \u003cp\u003eThe field experiment with rice\u0026ndash;wheat rotation was started in 2011 at the experimental farm of Zhejiang Academy of Agricultural Sciences in Shaoxing City (119\u0026deg;54\u0026acute;E, 31\u0026deg;16\u0026acute;N), Zhejiang Province, China (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tested soil belonged to the Gleyi\u0026ndash;Stagnic Anthrosol category. The original soil chemical properties (0\u0026ndash;20 cm) were pH, 6.27; SOC, 13.0 g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e; total N (TN), 1.45 g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFour treatments were designed in randomized field plots (6 m \u0026times; 5 m) with three replicates. The treatments consisted of N fertilizer application during the rice/wheat season at rates of 0, 150, 225, and 300 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, that is 0 (N0), 300 (N1), 450 (N2), and 600 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e yr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (N3). In both seasons, N fertilizer (urea, 46%N) was applied at a rate of 50% during the sowing stage and the remaining 50% during the booting stage. In all plots, P fertilizer was applied as basal fertilizer at rate of 170 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and K fertilizer was applied at rate of 100 kg K ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in both of rice and wheat seasons. All the field management practices followed those used by the local farmers.\u003c/p\u003e \u003cp\u003eAfter the wheat had been harvested, root sample was collected from a 0\u0026ndash;20 cm soil layer in a randomly subplot (1 m \u0026times; 1 m) in each plot, and immediately rinsed with clean water. The clean root sample was dried at 70\u0026deg;C to reach a stable weight. Root biomass was estimated as the sum of the rice and wheat root biomasses. Topsoil (0\u0026ndash;20 cm) was collected in May 2023 after the wheat had been harvested. Five soil cores were sampled from each plot at random locations and homogenized as a single sample. The soil sample from each plot was separated into three portions. One subsample was air-dried for determination of soil physiochemical properties, including SOC fractions and amino sugars. One was immediately used for soil mineral N and enzyme activity analysis; and the third soil subsample was stored at \u0026minus;\u0026thinsp;60\u0026deg;C for assessment of microbial traits.\u003c/p\u003e \u003cp\u003eSoil property analysis\u003c/p\u003e \u003cp\u003eThe concentrations of SOC and TN were measured by an elemental analyzer (Elementar, Vario Max, Germany). Soil available N (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026ndash;N) was measured using an auto-analyzer after extracting with 1 M KCl solution (Bran Lubbe, Norderstedt, Germany). Soil available phosphorus (AP, Olsen P) was extracted with HCl-NH\u003csub\u003e4\u003c/sub\u003eF and determined using ICP-MS (Optima 2000DV, Waltham, USA). Soil pH was measured using a pH electrode (S400-K, Mettler-Toledo, Switzerland).\u003c/p\u003e \u003cp\u003eSoil organic carbon fractionation\u003c/p\u003e \u003cp\u003eThe SOC pool was divided into POC and MAOC using particle size fractionation (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Air-dried soil (20 g) was dispersed in sodium hexametaphosphate solution (100 mL, 5% w/v). The dispersed soil sample was rinsed onto a 53-\u0026micro;m sieve and deionized water was used to wash the soil sample until the water was clear. The fraction on the sieve was collected as the POC fraction and the fraction passing through the sieve was collected as the MAOC fraction. The POC and MAOC fractions were dried at 60\u0026deg;C, and then ground. Organic C concentrations in the POC and MAOC fractions were measured by an elemental analyzer (Elementar, Vario Max, Germany).\u003c/p\u003e \u003cp\u003eSoil enzyme activity assay\u003c/p\u003e \u003cp\u003eThe activities of the soil extracellular β-N-acetyl-glucosaminidase (NAG), leucine aminopeptidase (LAP), β-1, 4-glucosidase (BG), and acid phosphatase (ACP) were determined according to Luo et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, soil samples (1 g) were mixed with 100 mL acetate buffer (50 mM). NAG, BG, and ACP activities were measured using substrates labeled with 4-methylumbelliferyl (MUB), whereas a substrate labeled with 7-amino-4-methylcoumarin (MUC) was used to determine LAP activity. Soil slurry (200 \u0026micro;L) and substrate (200 \u0026micro;M, 50 \u0026micro;L) were placed into a 96-well microplate for each sample. A microplate reader was used to quantify fluorescence at 360 nm excitation and 460 nm emission. The activities of soil extracellular enzymes were calculated based on MUB or MUC standard curves.\u003c/p\u003e \u003cp\u003ePLFA analysis\u003c/p\u003e \u003cp\u003eSoil microbial biomass PLFAs concentration was measured using the method described by Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Freeze-dried soils were extracted with a mixture containing chloroform, citrate buffer and methanol (1:0.8:2 v/v/v). A silica column was used to separate the phospholipids. After that, they transformed into fatty acid methyl esters. We determined the methyl esters using an Agilent GC combined with MIDI identification software 4.5 (MIDI Sherlock, Newark, DE, USA). An internal standard (19:0) was used to quantify the PLFA concentration. The representative PLFAs of bacteria, fungi and actinomycete were identified according to previous studies (Zelles, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Fierer et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Joergensen, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Their specific PLFAs are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAmino sugar analysis\u003c/p\u003e \u003cp\u003eSoil amino sugar concentration was determined according to Zhang and Amelung (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). A soil sample (\u0026lt;\u0026thinsp;0.15 mm) was hydrolyzed for 8 h at 105\u0026deg;C using HCl (6 M, 10 mL). After cooling, the hydrolysate was purified by filtration, centrifugation, and drying under N gas. The dried residue was derivatized using a derivatization reagent and dissolved in dichloromethane. The derivatives were determined using an Agilent GC.\u003c/p\u003e \u003cp\u003eThe soil MNC (FNC\u0026thinsp;+\u0026thinsp;BNC) contents were estimated using the methods used by Hu et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"407\" height=\"82\"\u003e\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe effects of N fertilization on soil chemical properties (pH, SOC, TN, AN, AP, POC, and MAOC), root biomass, extracellular enzyme activities (NAG, LAP, BG, and ACP), and soil microbial communities, that is, total PLFAs, fungi, bacteria, and actinomycetes were examined using analysis of variance (ANOVA). Pearson\u0026rsquo;s correlations were used to assess the relationships between amino sugars, MNC, and environmental factors. Random forest (RF) analysis was used to detect the relative importance of soil parameters for POC, MAOC, and MNC concentrations using R. We estimated the significance of each predictor using the \u0026ldquo;rfPermute\u0026rdquo; package in R. A structural equation model (SEM) was applied to assess the direct and indirect effects of N addition on MNC, POC and MAOC using AMOS 22.0 (Smallwaters Corporation, IL, USA). A prior SEM model was constructed base on the ecological hypotheses, assuming that N addition indirectly impact SOC fractions through influencing soil biotic and abiotic properties, and root biomass (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Microbial biomass and root biomass were indicated by the total microbial PLFAs and total crop root biomass in the model, respectively. All the data analyses were performed using SPSS software (SPSS 22.0, Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSoil chemical properties and root biomass\u003c/p\u003e \u003cp\u003eN fertilization increased SOC and POC concentrations by 2.87\u0026ndash;8.41% and 14.6\u0026ndash;41.2%, respectively, compared to N0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The soil MAOC concentration increased by 12.4% in N1 compared to that in N0. However, no significant changes were noted among the N0, N1, and N2 treatments. N addition, except for N1, significantly decreased the MAOC: POC ratio relative to that of the N0 treatment. Long-term N fertilization reduced soil pH from 6.39 in N0 to 5.50 in HN (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Soil AN concentration in N2 and N3 were significantly higher than those in N0. Long-term N addition decreased the soil AP concentration compared to N0. Root biomass increased with increasing the addition rate of N fertilizer (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). N addition increased rice and wheat root biomass by 40.7\u0026ndash;53.1% and 142\u0026ndash;249%, respectively, compared to N0.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSoil chemical properties and crop root biomass under nitrogen addition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTN\u003c/p\u003e \u003cp\u003e(g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAN\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAP\u003c/p\u003e \u003cp\u003e(mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRice root biomass\u003c/p\u003e \u003cp\u003e(kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWheat root biomass\u003c/p\u003e \u003cp\u003e(kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1336\u0026thinsp;\u0026plusmn;\u0026thinsp;93 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e228\u0026thinsp;\u0026plusmn;\u0026thinsp;49 c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2597\u0026thinsp;\u0026plusmn;\u0026thinsp;224 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e552\u0026thinsp;\u0026plusmn;\u0026thinsp;86 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2643\u0026thinsp;\u0026plusmn;\u0026thinsp;153 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e764\u0026thinsp;\u0026plusmn;\u0026thinsp;115 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2759\u0026thinsp;\u0026plusmn;\u0026thinsp;200 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e797\u0026thinsp;\u0026plusmn;\u0026thinsp;160 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eTN, total nitrogen; AN, available nitrogen; AP, available phosphorus. N0, N1, N2 and N3 stand for N addition rates of 0, 300, 450 and 600 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e yr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, respectively. Different letters indicate significant differences among different treatments at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSoil amino sugar and microbial necromass\u003c/p\u003e \u003cp\u003eThe total soil amino sugar concentration increased with increasing the addition rate of N fertilizer (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Compared to N0, N addition enhanced the concentrations of glucosamine (GlcN), galactosamine (GalN), and MurA by 6.61\u0026ndash;21.2%, 9.68\u0026ndash;22.1% and 10.6\u0026ndash;28.6%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c, and d). The MNC, FNC, and BNC concentrations were 1.07\u0026ndash;1.22, 1.06\u0026ndash;1.21, and 1.11\u0026ndash;1.29 times greater in N-fertilized than in N0, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, and c). Higher N addition (N2 and N3) significantly increased the MNC and BNC contributions to SOC by 9.70\u0026ndash;13.1% and 15.0\u0026ndash;19.3%, respectively, compared to N0. The contribution of FNC to SOC was 1.12 times higher in N3 than in N0 but was similar among N0, N1, and N2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil enzyme activity and microbial biomass\u003c/p\u003e \u003cp\u003eLong-term N fertilization decreased soil NAG activity by 21.3\u0026ndash;27.0%, but increased soil LAP and ACP activities by 32.6\u0026ndash;103% and 36.3\u0026ndash;50.7%, respectively, compared to N0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). N fertilization increased total PLFAs by 29.3\u0026ndash;60.9% and bacterial PLFAs by 32.5\u0026ndash;66.1% relative to the N0 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and b). High and medium levels of N fertilization significantly increased fungal abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). The abundance of actinomycetes was only increased by medium N fertilization (N2), and no significant change was found between the N0, N1, and N3 treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The fungal/bacterial PLFAs ratio was reduced by N fertilization (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). This indicated that N addition changed the soil microbial community composition.\u003c/p\u003e \u003cp\u003eRelationship between SOC fractions, MNC, and environmental factors\u003c/p\u003e \u003cp\u003eRandom forest (RF) analysis was performed to determine the most important abiotic and biotic factors predicting POC, MAOC, and MNC accumulation. This explained 70.92%, 37.80%, and 78.79% of the total variation in the POC, MAOC, and MNC concentrations, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Soil pH was an important factor in predicting the POC and MNC concentrations (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and c). POC was significantly affected by MNC, SOC, microbial biomass, and root biomass. MAOC was significantly affected by root biomass, MNC, and bacterial biomass. MNC were significantly affected by microbial biomass, POC, SOC, and root biomass.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SEM results showed that the model explained 80%, 43%, and 94% of the total variance in POC, MAOC, and MNC, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). N addition negatively affected soil pH but positively affected soil TN, root biomass, and MNC. Soil pH indirectly affected MNC by influencing root and microbial biomass. Root biomass directly affected MNC, which is related to POC. Soil microbial biomass directly and negatively affected the MAOC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEffects of N addition on soil organic carbon\u003c/p\u003e \u003cp\u003eOur data indicated that the SOC concentration significantly enhanced by N fertilization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This is consistent with the increase in SOC concentrations reported in previous meta-analyses (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ni et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The balance between the plant C input and SOC loss plays a crucial role in the accrual of SOC (Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In our study, rice and wheat straw were both removed from the experimental plots. Therefore, crop-root-derived organic C may be the primary C source for SOC accumulation. Plant roots may play a more important role in SOC formation and accrual than the aboveground biomass (Sokol and Bradford 2019; Sokol et al. 2019a; Xu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Increased root biomass resulted from N addition owing to enhanced soil N availability under sufficient soil P and K (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This led to increased root-derived C incorporation into the soil, promoting SOC formation and accrual (Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Cai et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, N addition may not change root biomass under P limitation (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). In these soils, N addition increases SOC concentration, which is coincide with a decrease in soil microbial activity (Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Qi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlternatively, SOC loss via soil respiration is a vital factor controlling SOC content (Yang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). A 1413-paired observation was used to assess the response of soil respiration to N input (Yang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). This suggested that soil microbial activity was reduced by N fertilization, indicating that N input reduced the loss of SOC and increased the sequestration of SOC (Yang et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Soil acidification can constrain microbial activity and metabolism, consequently reducing SOC decomposition and increasing SOC accumulation (Malik et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, soil pH was negatively correlated with SOC concentration (Fig. S3). This indicated that N-induced soil acidification was the main driver of SOC sequestration (Xu et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, a study from field experiment showed that the SOC concentration increased with decreasing soil pH induced by N fertilization in paddy soils (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The increase in root C and the decrease in SOC loss suggest that N addition promotes SOC accrual in paddy fields.\u003c/p\u003e \u003cp\u003eEffects of N addition on SOC physical fractions\u003c/p\u003e \u003cp\u003eIn accordance with previous studies (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we observed that N addition enhanced POC concentration. The positive responses of POC to N fertilization have been attributed to higher plant C inputs with N fertilization (Ye et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Rocci et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). POC primarily consists of plant materials that are partially decomposed (Lavallee et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Keller et al. 2022). POC concentration was positively correlated with root biomass (Fig. S4), indicating that the increased root biomass induced by N input promoted the POC pool in the paddy soils. Root biomass contributes more to the POC pool than aboveground biomass (Ye et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; P\u0026uuml;sp\u0026ouml;k et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil microbial community activity plays a crucial role in determining the quantity of POC because of its easy microbial decomposition of POC (Cotrufo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although the soil microbial biomass increased, soil NAG activity decreased with N addition (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The enhancement of the POC pool is partly explained by the inhibition of POC decomposition caused by N-induced soil acidification. This is in accordance with findings from a semi-arid steppe ecosystem, where N addition decreased microbial activity and increased POC accumulation (Averill and Waring \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ye et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMAOC is more critical in maintaining SOC than POC because of its slower turnover (Lugato et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The microbial conversion of POC into MAOC plays a vital role in the preservation of SOC (Witzgall et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Niu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, an increase in MAOC can reduce soil C emissions and promote SOC persistence (Liu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, our study has shown that N fertilization did not affect MAOC, which is similar with previous study (Rocci et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar to POC, MAOC formation and persistence are affected by plant and microbial traits (Georgiou et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). MNC makes a crucial contribution to the formation and accrual of MAOC (Sokol and Bradford 2019; Sokol et al. 2019a, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, MAOC was negatively correlated with MNC in our study (Fig. S4). In addition, the SEM results showed that MNC were not correlated with MAOC. Microbial necromass is not a key factor in regulating MAOC accumulation. Zhu et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that mineral preservation plays a vital role in MAOC accumulation. N-induced soil acidification can stimulate the release of base cations from soil minerals, thereby reducing MAOC stability and accumulation (P\u0026uuml;sp\u0026ouml;k et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies have revealed that POC is more vulnerable to N fertilization than MOAC (Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our results showed that POC increased but MOAC was not affected by N input (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, the POC proportion in SOC increased but proportion of MAOC decreased by N addition (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). POC, rather than MAOC, was the main contributor to the preservation of the SOC pool in paddy fields with long-term N addition. The RF results demonstrated that POC was the most vital factor affecting SOC accumulation (Fig. S5). Furthermore, some studies have reported different responses of POC and MAOC fractions to N fertilization in rice paddy soils (Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They found that N enrichment increased soil POC concentrations, but decreased MAOC concentrations. The MAOC: POC ratio decreased with N addition, indicating that N addition decreased SOC functionality. Similar results were reported by Tang et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Given that POC has a shorter turnover time and lower resistance to environmental disturbances than MAOC (Wu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, our findings suggest that long-term N addition reduces the stability of the SOC pool, increasing the vulnerability of SOC to future global change.\u003c/p\u003e \u003cp\u003eEffects of N addition on microbial necromass carbon\u003c/p\u003e \u003cp\u003eMicrobial necromasses are one of the most important components of the stable SOC pools (Liang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MNC accounted for 28.9\u0026ndash;32.8% of SOC, of which FNC accounted for 21.6\u0026ndash;24.1% of SOC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results are similar to those of previous studies on paddy fields (Chen et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Both of the total MNC concentration and its contribution to SOC increased by N addition, which is consistent with two global meta-analyses (Hu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). The increase in MNC with N addition can be explained as follows. First, N addition affects crop root biomass, which stimulates soil microbial proliferation and enhances its biomass, ultimately accumulating more microbial necromass in soils (Hu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Roots play a more crucial role in the formation and accumulation of MNC than the aboveground biomass (Jia et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Second, N addition increases soil N availability, which inhibits microbial necromass decomposition because microbial necromass is considered an N-rich compound (Wang et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As expected, a negative correlation is found between NAG and MNC (Fig. S5). Therefore, the decreased the soil NAG activity caused by N addition may reduce the microbial decomposition of MNC and promoted MNC accumulation (Ma et al., 2023). Third, N addition increases the microbial C-use efficiency (Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which can promote microbial biomass and enhance MNC accumulation (Duan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur findings showed that MNC significantly and directly affected POC accumulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S5), indicating that the enhancement of POC partly contributed to the increase in MNC under N addition. Generally, soil microbial necromass is preferentially occluded in MAOC (Xuan et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, some studies have shown that MNC can contribute to approximately 40% of the POC in paddy soils (Bian et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), suggesting that MNC should be considered as an important contributor to POC formation. Soil microorganisms can decompose and use plant debris for their growth in POC and accumulate as necromass within it (Li et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the POC surface can serve as a hotspot for microbial growth and proliferation, driving necromass formation and accumulation (Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Contrary to POC, our data revealed that MAOC accrual was not driven by MNC under N addition (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and S5). Increased soil pH under N addition could weaken mineral protection of MNC (Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), although N addition significantly increased MNC concentration, resulting in balance of MAOC. Moreover, recent study has shown that mineral protection is a more important role in MAOC formation and accrual than microbial necromass (Zhu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the future, we will focus on the responses of unprotected and protected MNC to N addition, which could help better understand the dynamics of SOC fractions under long-term N addition in paddy soils.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study has provided empirical evidence of the mechanisms that promote SOC persistence and change SOC functionality following long-term N fertilization in paddy soils. SOC concentration was increased by N addition, primarily because of enhanced POC rather than MAOC. The POC was significantly increased by N addition owing to the increase in root biomass and microbial necromass. Meanwhile, N addition enhanced the contribution of microbial necromass to total SOC accrual, which was mainly attributed to the enhancement of soil microbial biomass. Collectively, soil pH is the most important factor regulating POC and MNC accumulation, indicating that N-induced acidification drives SOC formation and persistence in paddy soils. Taken together, combining soil chemical properties, plant input, and microbial traits should contribute to our understanding of how N fertilization affects SOC formation and sequestration in rice\u0026ndash;wheat rotation.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research was supported by funds from the Key Research and Development Program of Zhejiang Province (2023C02005; 2023C02015) and the National Key Research and Development Program of China (2023YFD1902904).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAngst G, Mueller KE, Castellano MJ, Vogel C, Wiesmeier M, Mueller CW (2023) Unlocking complex soil systems as carbon sinks: multi-pool management as the key. 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Geophysical Research Letter 51: e2024GL108466.\u003c/li\u003e\n\u003cli\u003eZhu X, Jackson RD, DeLucia EH, Tiedje JM, Liang C (2020) The soil microbial carbon pump: From conceptual insights to empirical assessments. Global Change Biology 26: 6032-6039.\u003c/li\u003e\n\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":"Nitrogen fertilization, Mineral-associated organic carbon, Microbial necromass, Particulate organic carbon, Paddy soil","lastPublishedDoi":"10.21203/rs.3.rs-5562758/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5562758/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and aims\u003c/h2\u003e \u003cp\u003eNitrogen (N) addition can substantially affect soil carbon cycling in agroecosystems. Microbial necromass carbon (MNC) is widely recognized as a key contributor to soil organic C (SOC) fractions. However, the mechanisms underlying the responses of MNC and SOC fractions to N fertilization in paddy soils remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA field experiment with four N rates, namely, 0, 300, 450, and 600 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e yr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e was conducted to determine the effects of N addition on SOC fractions, soil microbial necromass carbon (MNC), enzyme activity, and microbial biomass in paddy soils with rice\u0026ndash;wheat rotation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eN addition increased SOC and POC concentrations by 2.88\u0026ndash;8.41% and 14.6\u0026ndash;41.2%, respectively, but did not affect MAOC. The ratio of MAOC to POC was reduced by N addition, indicating that N addition decreased SOC stability. N addition increased MNC concentration by 7.32\u0026ndash;22.5% and its contribution to SOC by 4.14\u0026ndash;13.7%. The activity of β-1,4-\u003cem\u003eN\u003c/em\u003e-acetyl-glucosaminidase was decreased, while the activities of leucine amino peptidase and acid phosphatase were increased under P addition. Structural equation modeling and random forest revealed that N-induced decrease in soil pH promoted the accrual of MNC by increasing root and microbial biomass, consequently improving POC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePOC is likely more vulnerable to N addition than MAOC. N-induced acidification is the primary driver for promoting SOC accrual by increasing POC in paddy soils.\u003c/p\u003e","manuscriptTitle":"Nitrogen-induced acidification increases soil organic carbon accrual by promoting particulate organic carbon and microbial necromass under long-term experiment in the paddy soils of East China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 04:50:41","doi":"10.21203/rs.3.rs-5562758/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-27T00:24:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-26T09:10:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-26T02:59:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-03-25T06:35:47+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revisions","date":"2025-01-27T11:15:09+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":"b818d259-637e-49c7-95e1-d4b2deeb0bc5","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:04:08+00:00","versionOfRecord":{"articleIdentity":"rs-5562758","link":"https://doi.org/10.1007/s11104-025-07551-y","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-06-08 15:57:49","publishedOnDateReadable":"June 8th, 2025"},"versionCreatedAt":"2025-03-27 04:50:41","video":"","vorDoi":"10.1007/s11104-025-07551-y","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07551-y","workflowStages":[]},"version":"v1","identity":"rs-5562758","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5562758","identity":"rs-5562758","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}