Azolla  returning application Drives the Reshaping of Nitrogen-Fixing Microbial Communities in Paddy Soils to Enhance Fertilization and Reduce Emissions

Azolla returning application Drives the Reshaping of Nitrogen-Fixing Microbial Communities in Paddy Soils to Enhance Fertilization and Reduce Emissions | 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 Azolla returning application Drives the Reshaping of Nitrogen-Fixing Microbial Communities in Paddy Soils to Enhance Fertilization and Reduce Emissions Sufang Deng, Yanqiu Yang, Zhaoyang Ying This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8853068/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Azolla , a robust nitrogen (N)-fixing plant, can be efficiently and ecologically cultivated when incorporated into rice fields, while the mechanisms remain inadequately understood. This study systematically explored these mechanisms by assessing the effects of Azolla returning on soil nitrogen forms, ammonia volatilization, and microbial community responses across key rice growth stages. Methods The effects of Azolla incorporation were compared against rice monoculture and urea application at various stages of rice cultivation. The study monitored changes in soil nitrogen forms and ammonia volatilization. Furthermore, the response of the soil microbial community was examined, with a detailed analysis conducted during the grain-filling stage. Results Azolla returning significantly increased the content of total soil N, organic matter, available potassium and available phosphorus, while also elevating the soil pH. It substantially elevated ammonium nitrogen and microbial biomass nitrogen (peaking at grain-filling) compared to monoculture or urea, while significantly reducing nitrous oxide emissions, ammonia volatilization and maintaining nitrogen fertilizer content over time. Soil microbial community during the grain-filling stage indicated that urea fertilizer favored the enrichment of nitrifying bacteria ( Nitrospira and Nitrosomonas ) but also increased the abundance of pathogen Pseudomonas syringae . Azolla returning potentially reduced the abundance of pathogens while significantly promoting that of beneficial bacteria involved in N fixation and denitrification, including Azospirillum and Bacillus bataviensis . These enhanced soil nitrogen fixation and metabolic capacity. Conclusions This study is the first to track the dynamic characteristics and potential mechanisms behind the changes in nitrogen source during Azolla incorporation, a process that enhances soil fertility and reduces environmental emissions. It provides a theoretical foundation for the adoption of this sustainable practice. Azolla filiculoide Returning application Soil microbial Nitrogen-fixing Rice grain-filling stage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction As the population grows and dietary habits change, the tension between increasing grain production and reducing environmental costs is becoming more apparent. Rice ( Oryza sativa L.) is one of the most vital staple crops, and it provides sustenance for more than half of the world's population(Khush, 2005 ). By 2030, the global demand for rice is expected to reach 770 million tons – a 35% increase over the current levels – thus, placing immense pressure on the global production of rice(Qian et al., 2023 ). The demand for rice in China, a major producer and consumer, is expected to increase by 30% by 2030(Shen et al., 2013 ). However, rice paddies are a significant source of atmospheric methane (CH 4 ) and emissions of nitrous oxide (N 2 O), and they account for approximately 15%-20% of the global emissions of CH 4 (Butterbach-Bahl et al., 2022 ; Giltrap et al., 2020 ). Therefore, the large-scale cultivation of rice will exacerbate global warming. Amidst multiple pressures, including population growth, global warming and declining farmland quality, high-yield, low-consumption, green, and ecological cultivation models have been developed(Yuan et al., 2023 ). Azolla Lam. is an aquatic fern that coexists symbiotically with algae. It fixes atmospheric carbon dioxide (CO 2 ) to provide a source of carbon (C) for the cyanobacteria within its tissues(Talley et al., 1977 ). The fern and these microorganisms form a mutualistic symbiosis that enhances its ability to fix nitrogen (N). Owing to its rapid reproduction, high capacity to fix N and environmental friendliness, Azolla is commonly applied as a biofertilizer in paddy fields(Castro et al., 2003 ; Watanabe, 1982 ). It is widely used in rice production in many countries, including China, India, Ghana, Egypt and Italy(Nierzwicki-Bauer, 2018 ). Previous studies have shown that the application of Azolla into rice fields promotes N fixation and nutrient cycling, improves the efficiency of utilizing fertilizer, and reduces greenhouse gas emissions. Azolla application into rice fields has been shown to promote N fixation and nutrient cycling, improve the efficiency with which fertilizers are utilized, and reduce greenhouse gas emissions(Yao et al., 2018 ). For example, incorporating 30 t/hm² of Azolla before transplanting in a single rice season can replace 25–40% of the urea N while increasing yields by 10–15%(Malyan et al., 2021 ). In rice- Azolla -fish co-cultivation systems, the combined effects of Azolla and fish manure can reduce the use of chemical fertilizers by more than 50%, increase rice yields by 5.7%, and significantly mitigate pests, such as rice planthoppers, and diseases, such as sheath blight(Byrne and Chan, 2023 ). Cultivating Azolla in paddy fields absorbs N and phosphorus (P) from the surface water during the early stages of rice growth(Marzouk et al., 2024 ; Pereira, 2017 ). This reduces the runoff and volatilization of these nutrients, thereby promoting the development of rice in the later stages. Nitrogen is the most fundamental nutrient that affects the yield of rice. In rice production, the volatilization of ammonia is the main reason for the low utilization of N fertilizer and its loss in paddy fields(Fageria and Baligar, 2001 ). Cultivating Azolla in paddy fields significantly reduces the volatilization of ammonia from water bodies, thereby reducing the loss of N fertilizer by 15–30%, and mitigating greenhouse gas emissions from paddy fields(Yang et al., 2021 ). Previous studies have shown that incorporating 30 tons of fresh Azolla per ha of paddy field can replace 20% of the conventional N and potassium (K) fertilizers, while significantly increasing the yield of rice grain by 11.11%(Deng et al., 2022 ). However, the impact of the incorporation of Azolla on the microorganisms that fix N in the soil and the transformation of N in the soil remains unclear. This study compared the effects of Azolla incorporation on the forms of N in the soil (ammonium, microbial, and nitrate N) and the volatilization of ammonia from the soil at different stages of rice growth. The study analyzed the dynamic characteristics of changes in the forms of N after Azolla incorporation and explored the mechanisms of microbial responses that underly the processes of transforming N. This will provide a theoretical basis and practical guidance to formulate rational ecological planting strategies that involve the application of Azolla . Materials and Methods Cultivation of Azolla A total of 100 g of fresh Azolla filiculoide Lam. was placed in a 48-L plastic basin (60 cm *40 cm * 20 cm) and cultivated using 10 L of N-free culture medium (Zhejiang Agricultural 6302, Zhejiang, China). The medium formulation is shown in Table 1. A total of 0.5358 g of 99% pure 15 NH 2 CO 15 NH 2 (molecular weight 62.04 and N content 48.39%) was dissolved in distilled water, with 0.01% added by volume every 3 days. The plant was cultivated under sunlight at a temperature of 28–30°C for 4 weeks. At harvest, the Azolla was rinsed with distilled water to remove any surface contamination of the 15 N isotope and then air-dried for subsequent use. Rice cultivation following Azolla returning to the fields The study was conducted indoors at the institute of agricultural ecology, Fujian Academy of Agricultural Sciences, from April to December 2022 in Fuzhou City (Fujian, China, E119.333722, N26.132240, elevation 29.8m). The rice used in the study was variety “Jiafengyou No. 3.” The soil characteristics at the experimental site included contents of total N of 0.151%, available K (AK) of approximately 150 mg/kg, available P (AP) of 107.96 mg/kg, organic matter (OM) of 15.45 g/kg and a pH level of 5.4. Nutrient content and total 15 Nabundance in Azolla (dry basis) were as follows: total nitrogen 22.32 g·kg⁻¹, 15 N abundance 3.65%; urea abundance 10%. The following four treatments were established: rice and 15 N Azolla incorporation (RA); 15 N Azolla incorporation alone (A); rice alone (R); and rice and equivalent 15 N urea fertilizer (RU). Each treatment had three replicates, prior to the experiment, all treatments received chemical fertilizers at a rate of P 2 O 5 : K 2 O = 75:120 kg·hm -2 . with identical total phosphorus (12%) and potassium (60%) additions across treatments. Except for group R, all treatments received equivalent total exogenous nitrogen (180 kg·hm⁻²), where Azolla incorporation was calculated as nitrogen-equivalent to chemical nitrogen at 6000 kg·hm⁻² (dry weight basis). Specific exogenous nitrogen additions are detailed in Table 2. The experiment commenced in May 2022, soil was subjected to a 5-day flooding period to rice transplanting. The stages of rice growth were subsequently recorded at tillering and heading (T1, 31 days after transplanting), heading and flowering (T2, 49 days after transplanting), grain filling (T3, 65 days after transplanting) and maturity (T4, 133 days after transplanting). Soil samples collected from a depth of 0–20 cm during each stage, with seven samples taken per stage: four for soil biochemical analysis and three for soil microbial analysis. Nutrient analysis for the soil that rice was grown in The analysis of the contents of nutrients in the soil included the following: total N (TN); AP; AK; OM, and pH. A total of 300 g of soil was collected, dried naturally, and then sieved for each treatment group and time point. The content of TN in the soil was determined using the Kjeldahl method. The contents of AP and exchangeable K were measured using the molybdenum-antimony colorimetric method with 0.5 mol/L sodium bicarbonate (NaHCO 3 ) and ammonium acetate (NH 4 OAc) extraction, followed by flame photometry. The soil pH was assessed using a pH meter (soil-to-water ratio of 1:1). The potassium dichromate volumetric method with external heating was employed to determine the content of OM in the soil. Determination of the forms of nitrogen in the soil The contents of nitrate and ammonium N in the soil were determined using the potassium chloride (KCl) extraction method(Lu, 2000 ; Sparks et al., 2020 ). A total of 10.0 g of fresh soil sample was weighed into a clean 100 mL polyethylene centrifuge tube. A total of 50 mL of 2 mol/L KCl extraction solution (soil-to-liquid ratio of 5:1 [w/v]) was added to ensure that the soil was completely immersed. The tube was then placed in a constant temperature shaker and agitated at 25 ± 1°C and 200 rpm for 30 min. The shaken suspension was immediately filtered using a vacuum filtration apparatus. The filtrate was collected in polyethylene sample bottles that had been pretreated with dilute acid. The contents of nitrate and ammonium N in the filtrate were measured by spectrophotometry (Smart Chem ™200, AMS Allicance, USA). The content of nitrate N was determined using the cadmium column reduction method at 540 nm. The content of ammonium nitrogen (NH 4 ⁺-N) was measured using the salicylic acid–hypochlorite colorimetric method at 660 nm. N content (mg/kg) = (C*V*D)/(M*1,000) Eq. (1) where C is the instrumental nitrogen concentration (mg/L); V is the volume of extraction liquid (mL); D is the moisture correction factor (1 + soil moisture content), and M is the mass of the fresh soil sample (g). The microbial-bound N (MBN) was extracted using the chloroform fumigation method. A total of 20.0 g of fresh soil was weighed and subjected to vacuum fumigation with ethanol-free chloroform for 24 h at 25°C. Both the fumigated and non-fumigated (control) samples were extracted by shaking with 100 mL of 0.5 mol/L potassium sulfate (K 2 SO 4 ) at 250 rpm for 30 min. The filtrate was oxidatively digested with alkaline potassium persulfate at 120°C for 30 min, and the absorbance was then measured at 220 nm and 275 nm using UV spectrophotometry. The content of TN was calculated from the standard curve. The MBN was calculated using the formula: MBN = (N-fum - N-non)/0.54 Eq. (2) Determination of 15 N content and contribution rates in different soil nitrogen pools To track the dynamics of 15 N across various soil nitrogen pools following fertilizer application, initial soil samples were collected 6 days after fertilization (T0) to establish the baseline 15 N content for each treatment. The 15 N content of total soil nitrogen and of specific nitrogen forms was analyzed using stable isotope ratio mass spectrometry. For total soil 15 N analysis, one gram of air-dried soil (passed through a 60-mesh sieve) from each sampling point (T0–T4) was digested using a concentrated H 2 SO 4 –mixed accelerator (CuSO 4 : K 2 SO 4 = 10:1)–H 2 O 2 system. The digest was transferred to a Kjeldahl flask for distillation. The released ammonia nitrogen was trapped in a 2% (w/v) boric acid (H 3 BO 3 ) solution. This solution was then acidified to pH 3.5–4.0 with 0.05 mol/L H 2 SO 4 and concentrated to 3–5 mL in a 100°C water bath. After transfer to sealed ampoules, the total 15 N abundance was determined using a stable isotope ratio mass spectrometer (Thermo Fisher MAT 253). For the analysis of specific nitrogen forms, soil nitrate and ammonium were first extracted and then distilled separately. The distillates were similarly acidified and concentrated to 3–5 mL for the measurement of 15 N abundance in each nitrogen pool. Based on the measured 15 N abundance and the content of each nitrogen form, the contribution rate of the applied exogenous 15 N to soil ammonium nitrogen, nitrate nitrogen, and microbial biomass nitrogen at different rice growth stages was calculated. The proportions were determined as follows: $$ \text{Exogenous N utilization rate}\text{}（\text{%}）\text{=}\frac{\text{[}\sum \text{(}\text{Nitrogen }\text{c}\text{ontent}\text{×Atomic percentage of }\text{15}\text{N}\text{)}\text{]}}{\text{Exogenous N application}\text{×Atomic percentage of }\text{exogenous}\text{}\text{15}\text{N}}\text{×100}$$ $$ \text{P}\text{r}\text{o}\text{p}\text{o}\text{r}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{e}\text{x}\text{o}\text{g}\text{e}\text{n}\text{o}\text{u}\text{s} \text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n} \text{i}\text{n} \text{e}\text{a}\text{c}\text{h} \text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n} \text{f}\text{o}\text{r}\text{m} （\text{%}）=\frac{\text{Atomic percentage of }\text{}\text{sample}\text{}\text{15}\text{N}}{\text{Atomic percentage of }\text{}\text{fertilizer}\text{}\text{15}\text{N}}\times 100$$ $$ \text{P}\text{r}\text{o}\text{p}\text{o}\text{r}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{s}\text{o}\text{i}\text{l} \text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n} \text{b}\text{y} \text{f}\text{o}\text{r}\text{m} （\text{%}）=100-\text{P}\text{r}\text{o}\text{p}\text{o}\text{r}\text{t}\text{i}\text{o}\text{n} \text{o}\text{f} \text{e}\text{x}\text{o}\text{g}\text{e}\text{n}\text{o}\text{u}\text{s} \text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n} \text{b}\text{y} \text{f}\text{o}\text{r}\text{m} \left(\text{%}\right)$$ Volatilization of ammonia in the soil A static dark chamber and in situ sampling-gas chromatography (GC) were employed to detect the fluxes of the emissions of N₂O from the soil(Farquharson and Baldock, 2008 ). The chamber was constructed from an aluminum plate approximately 8 mm thick, with a base area of 0.08 m² buried 5 cm into the soil and sealed with water. Gas samples of 50 mL were extracted using a vacuum syringe into BD vacuum bottles at 0, 10, 20 and 30 min after the chamber had been sealed. The gas samples were analyzed using a GC (Agilent 7890A with an ECD detector; Agilent Technologies, Santa Clara, CA, USA) under the following conditions: Porapak Q column (65°C); carrier gas: N₂, flow rate: 30 mL/min. The flux calculation formula was as follows: F = ρ × (V/A) × (ΔC/Δt) × (273/T) Eq. (3) where ρ = 1.978 kg/m³ (N 2 O density); V = chamber volume (m³); A = base area (m²); ΔC/Δt = concentration change slope (ppmv/h), and T = average chamber temperature (K). Samples of the volatilized ammonia (NH 3 ) gas were collected in situ from paddy fields using the static absorption method. The capture device consisted of a gray-black, opaque polyvinyl chloride (PVC) pipe that had an inner diameter of 15 cm and was 35 cm high. Two sponges, each soaked in 15.0 mL of phosphoric acid/glycerol that was prepared by mixing 50.0 mL of 85.0% phosphoric acid (H 3 PO 4 ) with 40.0 mL of 99.0% glycerol (C 3 H 8 O 3 ) and then adjusting the volume to 1,000 mL, were placed in the PVC pipe. On the day that the basal rice fertilizer was applied, the device was inserted vertically into the soil between the rice rows, ensuring that the bottom of the device was tightly sealed against the water or soil surface. Monitoring began on the day of fertilization and continued until there was no significant difference in the rates of ammonia volatilization of all the fertilized treatments compared to the unfertilized control (CK) treatment. The sponge from the lower layer of the NH 3 capture device was transferred into a clean plastic bottle with a capacity of 500 mL. A volume of 100 mL of 1.0 mol/L KCl was added to fully submerge the sponge. The solution was shaken at a constant temperature of 25°C for 1 h, and the concentration of ammonium nitrogen in the extract was determined using a continuous flow analyzer (Smart Chem TM200, Thermofisher, USA). The ammonia volatilization flux was calculated based on the total NH₄⁺-N captured by the sponge, the soil area covered by the capture device and the exposure time using the following formula(Wang et al., 2002 ): F = (m / (A * t)) * 10 Eq. (4) where F represents the ammonia volatilization flux (kg N hm⁻² d⁻¹); m denotes the mass of NH 4 ⁺-N captured in the lower sponge layer (µg); A is the base area of the capture device (m²); t indicates the exposure time (d), and 10 serves as the unit conversion factor. Soil microbial DNA extraction and Illumina sequencing Samples collected during the period of highest soil N content were selected for 16S rDNA sequencing. A total of 0.5 g of fresh soil was stored at -80°C and placed in a pre-chilled mortar. After rapid freezing, the sample was grown to a particle size < 0.1 mm. The total soil DNA was extracted using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), and the quality and concentration of the DNA were assessed using a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified using PCR. The sequencing and bioinformatics analysis were performed by Fujian Manxiu Technology Co., Ltd (Fujian,China). on the Illumina NovaSeq platform. High-quality reads were clustered into operational taxonomic units (OTUs) at 97% similarity using UPARSE (version 7.1). Representative sequences from each OTU cluster were annotated against the RDP database to determine the species composition of each sample. The alpha-diversity indices (Chao 1, Shannon, observed species and phylogenetic diversity whole tree) were analyzed using Mothur software. A principal coordinate analysis (PCoA) based on the Bray–Curtis distances was then performed to identify the differences between groups. Statistical analysis All data are expressed as the mean ± standard error (s.e.). Soil physicochemical properties were analyzed using two-way analysis of variance (ANOVA) with SPSS 22.0 (IBM, Inc., Armonk, NY, USA). Microbial abundance comparisons between groups were performed using one-way ANOVA. P < 0.05 indicates a significant difference between the groups. Results Contents of the soil nutrient at different time periods Figure 1 shows the effects of incorporating Azolla on the contents of soil nutrients at different stages of rice cultivation. Significant increases in the content of soil TN at stages T2 and T4 were observed with both Azolla returning and urea fertilizer ( P < 0.05) (Fig. 1A). Concurrently, groups A, RA and RU all significantly increased the contents of OM and AK in the soil. During period T4, the RA group showed a more pronounced increase in the content of AK than the RU group (Fig. 1B-C). Both Azolla returning and the application of urea fertilizer reduced the content of AP in the soil. However, during period T4, the RA group had a significantly higher content of AP than the RU group (Fig. 1D). Azolla returning and the application of urea fertilizer both significantly increased the soil pH at stage T2 ( P < 0.05), which mitigated acidification of the soil (Fig. 1E). Nitrogen content in different forms in the soil at different stages Azolla returning to the field affected the N content of paddy soil in different forms at various time points. Compared with the R group, both the application of Azolla (RA group) and urea fertilizer (RU group) significantly increased the content of soil ammonium nitrogen (NH 4 ⁺-N). The RA group had significantly higher levels at the T2 stage. Additionally, the RA group significantly increased the content of soil nitrate N (NO 3 ⁻-N) during the stages T3 and T4. Azolla also increased the content of microbial biomass nitrogen (MBN) in the soil. From T2 to T4, Group RA exhibited significantly higher soil MBN than Group R and significantly exceeded Group RU ( P < 0.05). Notably, Group RA reached the highest MBN concentration during the T3 stage (Fig. 2). Proportion of different forms of 15 N in soil at different time periods 15 N tracing indicated that soil nitrogen was predominantly present as NO 3 ⁻-N and MBN, both of which remained relatively stable overall. In contrast, NH 4 ⁺-N declined to undetectable levels from the T3 period onward (Fig. 3A). A divergent pattern was observed only in A group, where the trends of all three nitrogen forms paralleled those in the R group: NO 3 ⁻-N and MBN were stable, while NH 4 ⁺-N dropped to 0% by T3. However, the abundance of NO 3 ⁻-N in A group was consistently higher than in R group. In RA group, NO 3 ⁻-N and NH 4 ⁺-N both showed an initial increase from T0 to T2, followed by a decrease, whereas MBN declined continuously. At T3, both NO 3 ⁻-N and MBN peaked before subsequently falling. In RU group, NO 3 ⁻-N and NH 4 ⁺-N first decreased, then increased to a peak at T3, before declining sharply. MBN exhibited a marked initial decrease and then stabilized. Overall, compared to RU group, RA group maintained more stable concentrations across the different nitrogen forms (Fig. 3). As shown in Fig. 4, the contribution rate of Azolla returning to soil NH 4 ⁺-N, NO 3 ⁻-N, and MBN was significantly higher ( P < 0.05) than that of urea-derived nitrogen at all rice growth stages. Specifically, Azolla returning contributed 67.67%–227.07%, 75.69%–172.31%, and 141.62%–213.74% more than urea nitrogen to these respective nitrogen pools. The contribution rate to soil NH 4 ⁺-N initially increased, peaked at the maximum tillering stage (T2), and then gradually declined to its lowest level at maturity. In contrast, the contribution to NO 3 ⁻-N exhibited a bimodal (“M”-shaped) trend, with peaks at the peak tillering (T1) and flowering (T3) stages. The contribution to MBN first decreased and then increased, reaching its highest point at T3 stage. Transformation of exogenous N in soil 15 N tracing revealed distinct transformation dynamics of applied nitrogen across treatments. In A group, conversion to nitrate nitrogen predominated over NH 4 ⁺-N from T0 to T2. In RA group, Azolla returning conversion to NO 3 ⁻-N and NH 4 ⁺-N increased to an initial peak at T1, then declined, with a sharper decrease for nitrate. Subsequently (T2–T4), conversion to ammonium rose sharply, peaked at T3, and became the dominant retained form. In RU group, urea nitrogen was rapidly converted to NO 3 ⁻-N and NH 4 ⁺-N by T1, then declined. Conversion rates increased again after T1, accelerating through T2 to a peak at T3 before a rapid decline. By the experiment's end, urea-derived ammonium was nearly undetectable. Comparatively, urea nitrogen in RA transformed faster and over a shorter duration than in RU group. Conversely, Azolla returning transformed more slowly but persistently than urea nitrogen. Furthermore, the presence of rice plants (RA vs. A) enhanced the conversion of Azolla - nitrogen into soil NH 4 ⁺-N, NO 3 ⁻-N and MBN. Volatilization of NH 3 and N 2 O gases at different time periods Figure 6 shows the results of soil NH 3 and greenhouse gas N 2 O volatilization in paddy fields at different stages after the application of N fertilizer or Azolla . The application of chemical fertilizer significantly increased the volatilization of NH 3 in the soil. During the T2–T4 period, the volatilization of NH 3 in the RU group were significantly higher than those in the other groups, with volatilization at T2 and T4 being significantly higher than in the RA group. The RA group only had significantly increased volatilization during the T2 period, with no significant differences from the A and R groups at other times. The application of chemical fertilizers significantly increased the volatilization of N 2 O. Throughout the T1, T3, and T4 stages of rice cultivation period, the volatilization of N 2 O in the RU group were significantly higher than in the other groups ( P < 0.05). Effects of Azolla returning to the fields on the soil microbial structure The content of soil N peaked at the grain filling (T3) stage, which prompted a microbial diversity analysis of the soil at this stage. Compared to the R group, the RA and RU groups exhibited increased alpha-diversity, with significant differences ( P < 0.05) observed in the Shannon, PD, Sobs and Chao1 indices. However, no significant difference was observed between the RA and RU groups. The PCoA analysis revealed a clear separation between the samples, which indicated that the incorporation of Azolla and the application of N fertilizer had significantly altered the structure of the soil microbial community (Fig. 7). Figure 8 shows the composition of soil microorganisms at the phylum and genus levels across the experimental groups during the T3 period. A total of 60 phyla and 642 genera were detected in the soil. The dominant phyla were Bacteroidetes (23.28%), Proteobacteria (15.07%), Firmicutes (12.42%), and Acidobacteria (11%). The dominant genera were Bacteroidetes vadinHA17 (3.83%), Chloroplast (2.86%), AKIW659 (2.05%), Citrifermentans (2.41%), and Geothrix (2.13%) (Fig. 5A-B). The abundance of Chloroplast (2.86%) may indicate the presence of plants or algae. To further investigate changes in the abundance of bacterial genera related to soil N, intergroup comparisons were conducted for the N fixing, nitrifying, and denitrifying genera. Compared to the R group, Azospirillum significantly increased in the A, RA, and RU groups, with the highest abundance observed in the RA group. Pseudomonas significantly increased in the RA and RU groups, while Bacillus significantly increased in the A and RU groups. Nitrospira significantly increased in the RU group, and Nitrosomonas significantly decreased in the RA group. Comparing the relative abundances of identifiable species within these genera against the R group, Bacillus bataviensis was significantly reduced in the RU group, with no significant differences in the A and RA groups. Bacillus megaterium significantly increased in the RA group, and Pseudomonas syringae significantly increased in the RU group ( P < 0.05) (Fig. 5C). Functional prediction of the key soil microbiota The functional prediction of soil microorganisms was performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software, which is based on marker gene sequences. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, abundance calculations were conducted for the pathways related to N fixation (Nitrogen metabolism, Alanine, aspartate and glutamate metabolism, and Lysine biosynthesis). As shown in the Fig. 9, the RA and RU groups had significantly higher levels of microbial abundance in the nitrogen metabolism pathway than the R group, with the RA group showing significantly higher abundance than the RU group. The RA group exhibited significantly higher predicted microbial abundance in the 'Alanine, aspartate and glutamate metabolism' and 'Lysine biosynthesis' pathways than the R group. Conversely, the RU group showed significantly lower abundance in the 'Lysine biosynthesis' pathway than the R group ( P < 0.05). Correlation analysis between the key microbiota and soil physicochemical properties The correlations between the significantly changed bacterial taxa (indicated in Fig. 5) and the soil physicochemical properties (soil nutrients, various N sources and N volatilization) are shown in Fig. 10. Azospirillum and Pseudomonas positively correlated with the TN, AK, (NH 4 ⁺-N) and nitrate nitrogen (NO 3 ⁻-N). Conversely, Nitrospira significantly negatively correlated with these indicators. The genus Nitrosomonas positively correlated with the NH 3 emissions and MBN. In contrast, species of Bacillus , including Bacillus bataviensis and Bacillus megaterium , significantly positively correlated with the N 2 O volatilization and NH 3 volatilization, conversely, it showed a significant positive correlation with MBN.. Pseudomonas syringae negatively correlated with the AP and positively correlated with the pH ( P < 0.05). Discussion Azolla returning application significantly increased various forms of nitrogen sources in the soil and suppressed volatilization Azolla is highly adaptable and fixes large quantities of N. The decomposition of its cellulose releases organic C, and certain organic acids are produced, such as citric acid and oxalic acid, which dissolve sequestered P and release mineral K(Nierzwicki-Bauer, 2018 ). Simultaneously, N fixation consumes H + , which helps to increase the soil pH and mitigate acidification. Previous studies found that the cultivation of Azolla in the paddy fields increased the contents of OM and TN in the soil by approximately 1%-2%. Previous studies have shown 100 kg of Azolla dry matter can be converted into 39 kg of OM in the soil within 1 year(Deng et al., 2022 ). Singh's pot simulation experiments demonstrated a trend of increase in the soil pH following the application of Azolla (Singh et al., 1981 ). This study also observed an increase in the content of TN in the soil and fertility following the application of Azolla . An increase in the pH was also noted. The increase in ammonium N probably stems from the enhanced soil microbial activity following application, which accelerates the mineralization of organic N into ammonium N. Additionally, Azolla itself may contain stored N that is gradually released during incorporation(Hasanah and Nabilah, 2024 ). The increase in the content of microbial N indicates enhanced microbial roles in N cycling. The microbiota participate in the transformation of soil N through processes, such as N fixation, ammonification, and nitrification(Verma and Patel, 2019 ). The Azolla application probably provided favorable conditions and abundant nutrients for microbial growth, thus, promoting proliferation and increasing the content of microbial N. However, Azolla incorporation may alter the soil aeration, or microbial metabolic activities may consume substantial oxygen, which creates relatively anoxic conditions that hinder nitrification(Yang et al., 2021 ). Consequently, this study found that Azolla application significantly increased the contents of ammonia N in the soil and microbial N while having little effect on nitrate N. Greenhouse gas emissions from farmland are significantly influenced by the methods and rates of fertilizer application(Qian et al., 2023 ). N 2 O emissions, an intermediate product of nitrification and denitrification processes, are particularly sensitive to the rates of application of N and water management practices. The amount of N 2 O emissions increased with higher rates of the application of N fertilizer. Azolla can serve as a source of N and can partially replace chemical N fertilizers and effectively reduce the N 2 O emissions(Verma and Patel, 2019 ). Simultaneously, Azolla primarily fixes N to produce ammonium N (NH 4 ⁺-N), thereby reducing the content of nitrate nitrogen (NO 3 ⁻-N). Previous studies have reported that cultivating Azolla in paddy fields significantly reduces the N 2 O fluxes during the early growth stages of rice(Yang et al., 2021 ). This reduction was attributed to the substantial lowering of the content of NO 3 ⁻-N in the soil by Azolla , which resulted in a decrease in the total N 2 O emissions of between 70.9% and 78.2%(Watanabe, 1982 ). Similarly, this study found that the RU group (applied N fertilizer) had a significantly increase in N 2 O emissions, whereas the RA group (applied Azolla ) significantly suppressed this phenomenon. The volatilization of NH 3 diminishes the soil fertility and impacts the atmospheric environment. The Azolla returning application probably reduced the NH 3 emissions by increasing the soil pH and content of OM. A higher pH favors the retention of ammonia, while the OM adsorbs ammonia gas, thus, reducing its volatilization(Abd El-Aal, 2022 ). Concurrently, the fixation of ammonia by soil microbes may have partially mitigated the NH 3 emissions. Azolla returning application regulated the structure of microbiota in the soil that fix nitrogen The grain-filling stage of rice growth and development is critical because the plant has high requirements for nutrients and water, while the soil environment and microbial activity are still active(Al-Tawaha et al., 2020 ). This study found that the content of OM in the soil and the capacity for microbial N fixation peaked during the T3 stage. Further analysis was conducted on the soil microbial structure and function prediction during this period. The results revealed that the Azolla returning application suppressed the abundance of nitrifying bacteria ( Nitrospira and Nitrosomonas ), whereas the application of urea fertilizer significantly increased the abundance of these genera. Nitrosomonas is a key genus in nitrification and primarily responsible for oxidizing ammonia (NH 3 ) to nitrite (NO 2 ⁻) and thus, completing the first stage of nitrification(Grunditz and Dalhammar, 2001 ). Nitrospira can then oxidize nitrite (NO 2 ⁻) to nitrate (NO 3 ⁻), which completes the second stage of nitrification(Daims et al., 2015 ). The application of urea fertilizer promotes the increase of both genera. In this study, the RU group significantly increased the abundance of these two genera, while the RA group significantly decreased it. Conversely, the abundance of certain probiotic genera that are can fix N and conduct denitrification increased significantly in the RA group. Azospirillum is a symbiotic nitrogen-fixing bacterium that converts atmospheric N gas (N 2 ) into ammonium N, which can be taken up by plants. This enhances the content of N in the soil. This genus also produces plant hormones, such as indoleacetic acid, that stimulate the development of roots and enhance the capacity to take up nutrients and water. Azospirillum also upregulates relevant gene expression and increases the content of nitrate in N fixation to further promote plant growth(Pelagio-Flores et al., 2025 ). Pseudomonas can produce plant hormones, such as auxin, dissolve phosphates, mineralize organic P and release insoluble K, thereby promoting plant growth(Weinmann, 2017 ). Bacillus decomposes organic N into ammonia (NH 4 ) and possesses properties antagonistic to plant pathogens. It can inhibit the growth and reproduction of plant pathogens and reduce the incidence of disease(Hlordzi et al., 2020 ). In particular, Bacillus bataviensis dissolves insoluble mineral K and converts it into water-soluble K that is available for the plant to take up and enhances the utilization of K in the soil(Basak et al., 2017 ). Bacillus megaterium transforms insoluble P in the soil into forms that are available to plants, thereby improving the utilization of P(Zhao et al., 2021 ). Consequently, the RA group exhibited a significant increase in the content of ammonium N along with elevated levels of readily available K and P. The functional prediction analysis revealed that the markedly increased beneficial bacteria in the RA group potentially promote N fixation-related metabolism (Nitrogen metabolism, Alanine, aspartate and glutamate metabolism, and Lysine biosynthesis). The microbial fixation of N in the soil is closely linked to the metabolism of amino acids. The nitrogen fixation product, ammonium (NH 4 ⁺), is integrated into amino acids via specific pathways and subsequently synthesized into proteins or other organic compounds that contain N(Pahari et al., 2021 ). This finding aligns with 15 N labeling studies, which revealed that urea nitrogen undergoes rapid conversion in soil with a short transformation process, whereas Azolla returning exhibits significantly slower conversion rates and a prolonged transformation duration. Furthermore, the introduction of plants (rice) accelerates the conversion of Azolla returning in soil. While the application of urea fertilizers enhances the soil nitrification capacity, it also readily leads to the emission of the greenhouse gas N 2 O and nitrogen volatilization(Qian et al., 2023 ). The excessive application of urea fertilizers may also increase the abundance of potential soil pathogens. For example, this study observed a significant increase in the bacterium Pseudomonas syringae within the RU group. This bacterium is widely present on plant surfaces and in soil and is capable of causing various diseases, such as leaf spot, wilt and canker. It primarily utilizes a type III secretion system (T3SS) to inject effector proteins, such as AvrE and HopZ1a, directly into the plant cells(Oh et al., 2007 ; Xie et al., 2021 ). These proteins interfere with plant immune responses and promote the colonization and growth of pathogens. It also produces various toxins, such as coronatine and mangotoxin, which disrupt the structure and function of plant cells, thus, facilitating infection(Dudnik and Dudler, 2014 ). The study also found that Azolla returning to fields promotes the growth of Pseudomonas while reducing the abundance of pathogens. This practice also mitigates the greenhouse gas emissions and nitrogen volatilization caused by the application of urea fertilizer. Conclusion Azolla returning to fields can effectively replace conventional nitrogen fertilizers, potentially enhance soil fertility by increasing the levels of available nitrogen, potassium and phosphorus, as well as organic matter, and increasing the soil pH. It notably increases the content of ammonium nitrogen (NH 4 ⁺-N) in the soil and the levels of microbial nitrogen (MBN). Simultaneously, it mitigates the volatization of the greenhouse gas N 2 O and the volatization of nitrogen (NH 3 ) caused by the application of nitrogen fertilizer. These beneficial effects stem from changes in the structure of the soil microbial community following the incorporation of Azolla . Returning Azolla to fields significantly increases the abundance of nitrogen-fixing and denitrifying bacteria, while reducing the levels of potential pathogenic microorganisms. This stimulates nitrogen fixation in the soil and amino acid metabolism. Therefore, returning Azolla to fields is an effective alternative to using urea fertilizers. Declarations CRediT authorship contribution statement Sufang Deng : Conceptualization, Data curation, Investigation, Methodology, Formal analysis, Writing the original draft. Yanqiu Yang : Conceptualization, Formal analysis, Investigation, Methodology, Data curation. Zhaoyang Ying : Conceptualization, Funding acquisition, Supervision, Methodology, Writing - review & editing. Declaration of Competing interest The authors declare that they have no competing interests or personal relationships that could have influenced reporting of the research findings. Acknowledgments This work was supported by ‌National Green Manure Industry Technology System (CARS-22)‌, ‌Fujian Provincial Natural Science Foundation Project (2025J01)‌, ‌Fujian Academy of Agricultural Sciences Youth Elite Project (YC2021007)‌, and ‌Fujian Provincial Public Welfare Research Institutes Basic Research Special Project – Public Welfare Competitive Project (2023R1063) in China. The authors would like to thank MogoEdit (htt ps://www.mogoedit.com) f or providing assistance in English language editing during the preparation of this manuscript. References Abd El-Aal AA (2022) Anabaena-azollae, significance and agriculture application: A case study for symbiotic cyanobacterium. Microbial syntrophy-mediated eco-enterprising, pp 1–14 Al-Tawaha ARMS, Singh S, Singh V, Kafeel U, Naikoo MI, Kumari A, Imran, Amanullah, Al-Tawaha AR, Qaisi AM (2020) Improving water use efficiency and nitrogen use efficiency in rice through breeding and genomics approaches. 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Agriculture, Ecosystems & Environment 307, 107236 Tables Table 1 Basic Composition of Nutrients for the Culture Medium Nutrients Concentration (mol/L) CaSO 4 2H 2 O 0.001 MgSO 4 7H 2 O 0.0017 KH 2 PO4 0.003 KCl 0.0003 EDTA-Na 2 1.1*10 − 5 FeSO 4 7H 2 O 1.0*10 − 5 H 3 BO 3 4.85*10 − 6 Na 2 MoO 4 2H 2 O 1.24*10 − 6 NaNO 3 0.0007 Table 2 Fertilizer content and exogenous nitrogen addition rate (kg/hm²) Treatment Group Urea Superphosphate Potassium chloride Azolla 15 N R 0 75 120 0 A 0 75 120 6000 RA 0 75 120 6000 RU 180 75 120 0 Supplementary Files Graphicabstract1.tif Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8853068","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592747257,"identity":"90f055b7-3cdc-4ea2-9eb4-350a9a45e7e4","order_by":0,"name":"Sufang Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYDCCA0BcAcT8zMyHHxCv5QwQS7azpRmQpsXgPI+CBFE6+A6wP5M4UHPHbvNhHgYDhhqbaIJaJA/wmEkcOPYsedth3gMPGI6l5TYQ0mJwgIdN+gPb4WSzw3wJBowNh4nRAnLYv8PJxs08BhJEamEwkzjYdtjOgJlYLZKHeYwtDvYdTpA4DAzkBGL8wne8/eGNA98O2/P3Hz784EONDWEtDMwQKhGsMoGgciRgT4riUTAKRsEoGGEAAFNsQqgpl3H7AAAAAElFTkSuQmCC","orcid":"","institution":"Fujian Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Sufang","middleName":"","lastName":"Deng","suffix":""},{"id":592747258,"identity":"11e92fc4-3338-4ceb-ac0d-a23b4437a0ca","order_by":1,"name":"Yanqiu Yang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yanqiu","middleName":"","lastName":"Yang","suffix":""},{"id":592747259,"identity":"5d120ba9-0663-4135-a912-d556c38594f3","order_by":2,"name":"Zhaoyang Ying","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhaoyang","middleName":"","lastName":"Ying","suffix":""}],"badges":[],"createdAt":"2026-02-11 14:55:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8853068/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8853068/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103505699,"identity":"4c31d860-b171-4f1b-a9a7-d4a621b73dbd","added_by":"auto","created_at":"2026-02-26 13:32:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180985,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eAzolla\u003c/em\u003e returning application on nutrients in the soil at different periods of rice growth. (A) Total nitrogen; (B) Organic matter; (C) Available potassium; (D) Available phosphorus; (E) Soil pH. T1, T2, T3, and T4, rice growth at tillering and heading, heading and flowering, grain filling and maturity stages, respectively. RA: rice and \u003cem\u003eAzolla\u003c/em\u003e incorporation; A: \u003cem\u003eAzolla\u003c/em\u003e application alone; R: rice alone; Ru: rice and equivalent urea fertilizer. Values are expressed as the mean ± SE (n\u003cem\u003e \u003c/em\u003e= 4). *, ** and *** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/81c3d4011dd5e3c849fe40ac.png"},{"id":103204500,"identity":"a611dfc2-ffe0-442e-85f9-450c8eb52526","added_by":"auto","created_at":"2026-02-23 07:02:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":132407,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eAzolla\u003c/em\u003e returning application on the content of nitrogen of different forms in the soil during rice growth period. (A) Soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e⁺-N); (B) Soil nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e⁻-N); (C) microbial biomass nitrogen (MBN). Values are expressed as the mean ± SE (n\u003cem\u003e \u003c/em\u003e= 4). *, ** and *** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/6b3a5a1b743f83b2bf0e7193.png"},{"id":103505537,"identity":"b318c456-88a5-44c4-b8ed-671984b0b6fb","added_by":"auto","created_at":"2026-02-26 13:31:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122338,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of \u003csup\u003e15\u003c/sup\u003eN in soil ammonium (NH\u003csub\u003e4\u003c/sub\u003e⁺-N), nitrate (NO\u003csub\u003e3\u003c/sub\u003e⁻-N), and microbial biomass nitrogen (MBN) under different treatments.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/67be9091e35a60eb0f20b5bb.png"},{"id":103505446,"identity":"8c36704e-f4b3-4363-b5ff-060f9a314ac8","added_by":"auto","created_at":"2026-02-26 13:31:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":87550,"visible":true,"origin":"","legend":"\u003cp\u003eContribution rates (%) of exogenous \u003csup\u003e15\u003c/sup\u003eN in soil ammonium (NH\u003csub\u003e4\u003c/sub\u003e⁺-N), nitrate (NO\u003csub\u003e3\u003c/sub\u003e⁻-N), and microbial biomass nitrogen (MBN) under different treatments. Bars with different letters indicate significant differences (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/d60ca0fae4dccabd13fad9c2.png"},{"id":103204504,"identity":"a63d1967-43d3-4936-8eea-83560a6d8bfb","added_by":"auto","created_at":"2026-02-23 07:02:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118483,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the forms and content of exogenous nitrogen in soil.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/c277cd207f14caa4441c78cf.png"},{"id":103505676,"identity":"88eb62f7-7666-4de5-865b-1d45dc09c396","added_by":"auto","created_at":"2026-02-26 13:32:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":65437,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eAzolla\u003c/em\u003e returning application on the rates of volatilization of NH\u003csub\u003e3\u003c/sub\u003e and N\u003csub\u003e2\u003c/sub\u003eO emissions in the soil during the rice growth period. Values are expressed as the mean ± SE (n\u003cem\u003e \u003c/em\u003e= 4). *, ** and *** indicate \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. NH\u003csub\u003e3\u003c/sub\u003e, ammonia; N\u003csub\u003e2\u003c/sub\u003eO, nitrous oxide.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/15b5152b4a02db31a3025e21.png"},{"id":103506105,"identity":"39c7edf5-19fe-4fff-b07d-95f22aaec42f","added_by":"auto","created_at":"2026-02-26 13:34:06","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":346905,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eAzolla\u003c/em\u003e returning application on Shannon index (A), phylogenetic diversity whole tree (B), Chao index (C), Observed species(D), and Principal coordinates analysis (E) of soil microbiota.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/a62bc1b463dba89c210361f1.png"},{"id":103505205,"identity":"cf4f3794-b580-4081-a831-af0243ed2fb8","added_by":"auto","created_at":"2026-02-26 13:27:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":200957,"visible":true,"origin":"","legend":"\u003cp\u003eSoil microbial composition at levels of phylum (A), genus (B) and relative abundance of the identified bacterial taxa with significant changes (C).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/da1a29e5dad7344e0bbe60b1.png"},{"id":103505921,"identity":"4341d1da-5a92-432c-afe8-f4526e20f9de","added_by":"auto","created_at":"2026-02-26 13:33:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":53191,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of microbiota in the nitrogen-fixing pathways.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/af0d72a617e2467f0bc38481.png"},{"id":103204508,"identity":"3809d0ff-d3f2-4704-a133-950617f015c4","added_by":"auto","created_at":"2026-02-23 07:02:14","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":183675,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heatmap of the soil physicochemical properties and significant changes in the bacterial taxa. *, ** and *** indicate significant differences between groups at the levels of \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, respectively.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/d37b6d29e4714c65477a8347.png"},{"id":106415088,"identity":"6a0e3855-404d-4953-8f97-7b8e53ccc696","added_by":"auto","created_at":"2026-04-08 10:32:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2291514,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/4e3263b0-d585-478d-8280-40a673905cd5.pdf"},{"id":103204510,"identity":"82054ba3-33eb-499e-8f1c-28ac9c0921b8","added_by":"auto","created_at":"2026-02-23 07:02:14","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6544408,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8853068/v1/535403e1bf32be31a9f03b11.tif"}],"financialInterests":"","formattedTitle":"Azolla returning application Drives the Reshaping of Nitrogen-Fixing Microbial Communities in Paddy Soils to Enhance Fertilization and Reduce Emissions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs the population grows and dietary habits change, the tension between increasing grain production and reducing environmental costs is becoming more apparent. Rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) is one of the most vital staple crops, and it provides sustenance for more than half of the world's population(Khush, \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e). By 2030, the global demand for rice is expected to reach 770\u0026nbsp;million tons \u0026ndash; a 35% increase over the current levels \u0026ndash; thus, placing immense pressure on the global production of rice(Qian et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The demand for rice in China, a major producer and consumer, is expected to increase by 30% by 2030(Shen et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, rice paddies are a significant source of atmospheric methane (CH\u003csub\u003e4\u003c/sub\u003e) and emissions of nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO), and they account for approximately 15%-20% of the global emissions of CH\u003csub\u003e4\u003c/sub\u003e (Butterbach-Bahl et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e; Giltrap et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the large-scale cultivation of rice will exacerbate global warming. Amidst multiple pressures, including population growth, global warming and declining farmland quality, high-yield, low-consumption, green, and ecological cultivation models have been developed(Yuan et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAzolla\u003c/em\u003e Lam. is an aquatic fern that coexists symbiotically with algae. It fixes atmospheric carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) to provide a source of carbon (C) for the cyanobacteria within its tissues(Talley et al., \u003cspan class=\"CitationRef\"\u003e1977\u003c/span\u003e). The fern and these microorganisms form a mutualistic symbiosis that enhances its ability to fix nitrogen (N). Owing to its rapid reproduction, high capacity to fix N and environmental friendliness, \u003cem\u003eAzolla\u003c/em\u003e is commonly applied as a biofertilizer in paddy fields(Castro et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e; Watanabe, \u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e). It is widely used in rice production in many countries, including China, India, Ghana, Egypt and Italy(Nierzwicki-Bauer, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Previous studies have shown that the application of \u003cem\u003eAzolla\u003c/em\u003e into rice fields promotes N fixation and nutrient cycling, improves the efficiency of utilizing fertilizer, and reduces greenhouse gas emissions. \u003cem\u003eAzolla\u003c/em\u003e application into rice fields has been shown to promote N fixation and nutrient cycling, improve the efficiency with which fertilizers are utilized, and reduce greenhouse gas emissions(Yao et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). For example, incorporating 30 t/hm\u0026sup2; of \u003cem\u003eAzolla\u003c/em\u003e before transplanting in a single rice season can replace 25\u0026ndash;40% of the urea N while increasing yields by 10\u0026ndash;15%(Malyan et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). In rice-\u003cem\u003eAzolla\u003c/em\u003e-fish co-cultivation systems, the combined effects of \u003cem\u003eAzolla\u003c/em\u003e and fish manure can reduce the use of chemical fertilizers by more than 50%, increase rice yields by 5.7%, and significantly mitigate pests, such as rice planthoppers, and diseases, such as sheath blight(Byrne and Chan, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cultivating \u003cem\u003eAzolla\u003c/em\u003e in paddy fields absorbs N and phosphorus (P) from the surface water during the early stages of rice growth(Marzouk et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pereira, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). This reduces the runoff and volatilization of these nutrients, thereby promoting the development of rice in the later stages.\u003c/p\u003e\n\u003cp\u003eNitrogen is the most fundamental nutrient that affects the yield of rice. In rice production, the volatilization of ammonia is the main reason for the low utilization of N fertilizer and its loss in paddy fields(Fageria and Baligar, \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). Cultivating \u003cem\u003eAzolla\u003c/em\u003e in paddy fields significantly reduces the volatilization of ammonia from water bodies, thereby reducing the loss of N fertilizer by 15\u0026ndash;30%, and mitigating greenhouse gas emissions from paddy fields(Yang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Previous studies have shown that incorporating 30 tons of fresh \u003cem\u003eAzolla\u003c/em\u003e per ha of paddy field can replace 20% of the conventional N and potassium (K) fertilizers, while significantly increasing the yield of rice grain by 11.11%(Deng et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the impact of the incorporation of \u003cem\u003eAzolla\u003c/em\u003e on the microorganisms that fix N in the soil and the transformation of N in the soil remains unclear. This study compared the effects of \u003cem\u003eAzolla\u003c/em\u003e incorporation on the forms of N in the soil (ammonium, microbial, and nitrate N) and the volatilization of ammonia from the soil at different stages of rice growth. The study analyzed the dynamic characteristics of changes in the forms of N after \u003cem\u003eAzolla\u003c/em\u003e incorporation and explored the mechanisms of microbial responses that underly the processes of transforming N. This will provide a theoretical basis and practical guidance to formulate rational ecological planting strategies that involve the application of \u003cem\u003eAzolla\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCultivation of\u003c/strong\u003e \u003cstrong\u003eAzolla\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 100 g of fresh \u003cem\u003eAzolla filiculoide\u003c/em\u003e Lam. was placed in a 48-L plastic basin (60 cm *40 cm * 20 cm) and cultivated using 10 L of N-free culture medium (Zhejiang Agricultural 6302, Zhejiang, China). The medium formulation is shown in Table\u0026nbsp;1. A total of 0.5358 g of 99% pure \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e2\u003c/sub\u003eCO\u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e2\u003c/sub\u003e (molecular weight 62.04 and N content 48.39%) was dissolved in distilled water, with 0.01% added by volume every 3 days. The plant was cultivated under sunlight at a temperature of 28\u0026ndash;30\u0026deg;C for 4 weeks. At harvest, the \u003cem\u003eAzolla\u003c/em\u003e was rinsed with distilled water to remove any surface contamination of the \u003csup\u003e15\u003c/sup\u003eN isotope and then air-dried for subsequent use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRice cultivation following\u003c/strong\u003e \u003cstrong\u003eAzolla\u003c/strong\u003e \u003cstrong\u003ereturning to the fields\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted indoors at the institute of agricultural ecology, Fujian Academy of Agricultural Sciences, from April to December 2022 in Fuzhou City (Fujian, China, E119.333722, N26.132240, elevation 29.8m). The rice used in the study was variety \u0026ldquo;Jiafengyou No. 3.\u0026rdquo; The soil characteristics at the experimental site included contents of total N of 0.151%, available K (AK) of approximately 150 mg/kg, available P (AP) of 107.96 mg/kg, organic matter (OM) of 15.45 g/kg and a pH level of 5.4. Nutrient content and total \u003csup\u003e15\u003c/sup\u003eNabundance in \u003cem\u003eAzolla\u003c/em\u003e (dry basis) were as follows: total nitrogen 22.32 g\u0026middot;kg⁻\u0026sup1;, \u003csup\u003e15\u003c/sup\u003eN abundance 3.65%; urea abundance 10%. The following four treatments were established: rice and \u003csup\u003e15\u003c/sup\u003eN \u003cem\u003eAzolla\u003c/em\u003e incorporation (RA); \u003csup\u003e15\u003c/sup\u003eN \u003cem\u003eAzolla\u003c/em\u003e incorporation alone (A); rice alone (R); and rice and equivalent \u003csup\u003e15\u003c/sup\u003eN urea fertilizer (RU). Each treatment had three replicates, prior to the experiment, all treatments received chemical fertilizers at a rate of P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e : K\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;=\u0026thinsp;75:120 kg\u0026middot;hm\u003csup\u003e-2\u003c/sup\u003e. with identical total phosphorus (12%) and potassium (60%) additions across treatments. Except for group R, all treatments received equivalent total exogenous nitrogen (180 kg\u0026middot;hm⁻\u0026sup2;), where \u003cem\u003eAzolla\u003c/em\u003e incorporation was calculated as nitrogen-equivalent to chemical nitrogen at 6000 kg\u0026middot;hm⁻\u0026sup2; (dry weight basis). Specific exogenous nitrogen additions are detailed in Table\u0026nbsp;2. The experiment commenced in May 2022, soil was subjected to a 5-day flooding period to rice transplanting. The stages of rice growth were subsequently recorded at tillering and heading (T1, 31 days after transplanting), heading and flowering (T2, 49 days after transplanting), grain filling (T3, 65 days after transplanting) and maturity (T4, 133 days after transplanting). Soil samples collected from a depth of 0\u0026ndash;20 cm during each stage, with seven samples taken per stage: four for soil biochemical analysis and three for soil microbial analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrient analysis for the soil that rice was grown in\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of the contents of nutrients in the soil included the following: total N (TN); AP; AK; OM, and pH. A total of 300 g of soil was collected, dried naturally, and then sieved for each treatment group and time point. The content of TN in the soil was determined using the Kjeldahl method. The contents of AP and exchangeable K were measured using the molybdenum-antimony colorimetric method with 0.5 mol/L sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e) and ammonium acetate (NH\u003csub\u003e4\u003c/sub\u003eOAc) extraction, followed by flame photometry. The soil pH was assessed using a pH meter (soil-to-water ratio of 1:1). The potassium dichromate volumetric method with external heating was employed to determine the content of OM in the soil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the forms of nitrogen in the soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe contents of nitrate and ammonium N in the soil were determined using the potassium chloride (KCl) extraction method(Lu, \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e; Sparks et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). A total of 10.0 g of fresh soil sample was weighed into a clean 100 mL polyethylene centrifuge tube. A total of 50 mL of 2 mol/L KCl extraction solution (soil-to-liquid ratio of 5:1 [w/v]) was added to ensure that the soil was completely immersed. The tube was then placed in a constant temperature shaker and agitated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 200 rpm for 30 min. The shaken suspension was immediately filtered using a vacuum filtration apparatus. The filtrate was collected in polyethylene sample bottles that had been pretreated with dilute acid. The contents of nitrate and ammonium N in the filtrate were measured by spectrophotometry (Smart Chem \u0026trade;200, AMS Allicance, USA). The content of nitrate N was determined using the cadmium column reduction method at 540 nm. The content of ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e⁺-N) was measured using the salicylic acid\u0026ndash;hypochlorite colorimetric method at 660 nm.\u003c/p\u003e\n\u003cp\u003eN content (mg/kg) = (C*V*D)/(M*1,000) Eq.\u0026nbsp;(1)\u003c/p\u003e\n\u003cp\u003ewhere C is the instrumental nitrogen concentration (mg/L); V is the volume of extraction liquid (mL); D is the moisture correction factor (1\u0026thinsp;+\u0026thinsp;soil moisture content), and M is the mass of the fresh soil sample (g).\u003c/p\u003e\n\u003cp\u003eThe microbial-bound N (MBN) was extracted using the chloroform fumigation method. A total of 20.0 g of fresh soil was weighed and subjected to vacuum fumigation with ethanol-free chloroform for 24 h at 25\u0026deg;C. Both the fumigated and non-fumigated (control) samples were extracted by shaking with 100 mL of 0.5 mol/L potassium sulfate (K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) at 250 rpm for 30 min. The filtrate was oxidatively digested with alkaline potassium persulfate at 120\u0026deg;C for 30 min, and the absorbance was then measured at 220 nm and 275 nm using UV spectrophotometry. The content of TN was calculated from the standard curve. The MBN was calculated using the formula:\u003c/p\u003e\n\u003cp\u003eMBN = (N-fum - N-non)/0.54 Eq.\u0026nbsp;(2)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN content and contribution rates in different soil nitrogen pools\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo track the dynamics of \u003csup\u003e15\u003c/sup\u003eN across various soil nitrogen pools following fertilizer application, initial soil samples were collected 6 days after fertilization (T0) to establish the baseline \u003csup\u003e15\u003c/sup\u003eN content for each treatment. The \u003csup\u003e15\u003c/sup\u003eN content of total soil nitrogen and of specific nitrogen forms was analyzed using stable isotope ratio mass spectrometry. For total soil \u003csup\u003e15\u003c/sup\u003eN analysis, one gram of air-dried soil (passed through a 60-mesh sieve) from each sampling point (T0\u0026ndash;T4) was digested using a concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026ndash;mixed accelerator (CuSO\u003csub\u003e4\u003c/sub\u003e : K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10:1)\u0026ndash;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e system. The digest was transferred to a Kjeldahl flask for distillation. The released ammonia nitrogen was trapped in a 2% (w/v) boric acid (H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e) solution. This solution was then acidified to pH 3.5\u0026ndash;4.0 with 0.05 mol/L H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated to 3\u0026ndash;5 mL in a 100\u0026deg;C water bath. After transfer to sealed ampoules, the total \u003csup\u003e15\u003c/sup\u003eN abundance was determined using a stable isotope ratio mass spectrometer (Thermo Fisher MAT 253). For the analysis of specific nitrogen forms, soil nitrate and ammonium were first extracted and then distilled separately. The distillates were similarly acidified and concentrated to 3\u0026ndash;5 mL for the measurement of \u003csup\u003e15\u003c/sup\u003eN abundance in each nitrogen pool. Based on the measured \u003csup\u003e15\u003c/sup\u003eN abundance and the content of each nitrogen form, the contribution rate of the applied exogenous \u003csup\u003e15\u003c/sup\u003eN to soil ammonium nitrogen, nitrate nitrogen, and microbial biomass nitrogen at different rice growth stages was calculated. The proportions were determined as follows:\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equa\" class=\"mathdisplay\"\u003e$$ \\text{Exogenous N utilization rate}\\text{}（\\text{%}）\\text{=}\\frac{\\text{[}\\sum \\text{(}\\text{Nitrogen }\\text{c}\\text{ontent}\\text{\u0026times;Atomic percentage of }\\text{15}\\text{N}\\text{)}\\text{]}}{\\text{Exogenous N application}\\text{\u0026times;Atomic percentage of }\\text{exogenous}\\text{}\\text{15}\\text{N}}\\text{\u0026times;100}$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equb\" class=\"mathdisplay\"\u003e$$ \\text{P}\\text{r}\\text{o}\\text{p}\\text{o}\\text{r}\\text{t}\\text{i}\\text{o}\\text{n} \\text{o}\\text{f} \\text{e}\\text{x}\\text{o}\\text{g}\\text{e}\\text{n}\\text{o}\\text{u}\\text{s} \\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n} \\text{i}\\text{n} \\text{e}\\text{a}\\text{c}\\text{h} \\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n} \\text{f}\\text{o}\\text{r}\\text{m} （\\text{%}）=\\frac{\\text{Atomic percentage of }\\text{}\\text{sample}\\text{}\\text{15}\\text{N}}{\\text{Atomic percentage of }\\text{}\\text{fertilizer}\\text{}\\text{15}\\text{N}}\\times 100$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\n\u003cdiv id=\"FileID_Equc\" class=\"mathdisplay\"\u003e$$ \\text{P}\\text{r}\\text{o}\\text{p}\\text{o}\\text{r}\\text{t}\\text{i}\\text{o}\\text{n} \\text{o}\\text{f} \\text{s}\\text{o}\\text{i}\\text{l} \\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n} \\text{b}\\text{y} \\text{f}\\text{o}\\text{r}\\text{m} （\\text{%}）=100-\\text{P}\\text{r}\\text{o}\\text{p}\\text{o}\\text{r}\\text{t}\\text{i}\\text{o}\\text{n} \\text{o}\\text{f} \\text{e}\\text{x}\\text{o}\\text{g}\\text{e}\\text{n}\\text{o}\\text{u}\\text{s} \\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n} \\text{b}\\text{y} \\text{f}\\text{o}\\text{r}\\text{m} \\left(\\text{%}\\right)$$\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eVolatilization of ammonia in the soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA static dark chamber and \u003cem\u003ein situ\u003c/em\u003e sampling-gas chromatography (GC) were employed to detect the fluxes of the emissions of N₂O from the soil(Farquharson and Baldock, \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). The chamber was constructed from an aluminum plate approximately 8 mm thick, with a base area of 0.08 m\u0026sup2; buried 5 cm into the soil and sealed with water. Gas samples of 50 mL were extracted using a vacuum syringe into BD vacuum bottles at 0, 10, 20 and 30 min after the chamber had been sealed. The gas samples were analyzed using a GC (Agilent 7890A with an ECD detector; Agilent Technologies, Santa Clara, CA, USA) under the following conditions: Porapak Q column (65\u0026deg;C); carrier gas: N₂, flow rate: 30 mL/min. The flux calculation formula was as follows:\u003c/p\u003e\n\u003cp\u003eF\u0026thinsp;=\u0026thinsp;\u0026rho; \u0026times; (V/A) \u0026times; (\u0026Delta;C/\u0026Delta;t) \u0026times; (273/T) Eq.\u0026nbsp;(3)\u003c/p\u003e\n\u003cp\u003ewhere \u0026rho;\u0026thinsp;=\u0026thinsp;1.978 kg/m\u0026sup3; (N\u003csub\u003e2\u003c/sub\u003eO density); V\u0026thinsp;=\u0026thinsp;chamber volume (m\u0026sup3;); A\u0026thinsp;=\u0026thinsp;base area (m\u0026sup2;); \u0026Delta;C/\u0026Delta;t\u0026thinsp;=\u0026thinsp;concentration change slope (ppmv/h), and T\u0026thinsp;=\u0026thinsp;average chamber temperature (K).\u003c/p\u003e\n\u003cp\u003eSamples of the volatilized ammonia (NH\u003csub\u003e3\u003c/sub\u003e) gas were collected \u003cem\u003ein situ\u003c/em\u003e from paddy fields using the static absorption method. The capture device consisted of a gray-black, opaque polyvinyl chloride (PVC) pipe that had an inner diameter of 15 cm and was 35 cm high. Two sponges, each soaked in 15.0 mL of phosphoric acid/glycerol that was prepared by mixing 50.0 mL of 85.0% phosphoric acid (H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) with 40.0 mL of 99.0% glycerol (C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) and then adjusting the volume to 1,000 mL, were placed in the PVC pipe. On the day that the basal rice fertilizer was applied, the device was inserted vertically into the soil between the rice rows, ensuring that the bottom of the device was tightly sealed against the water or soil surface. Monitoring began on the day of fertilization and continued until there was no significant difference in the rates of ammonia volatilization of all the fertilized treatments compared to the unfertilized control (CK) treatment. The sponge from the lower layer of the NH\u003csub\u003e3\u003c/sub\u003e capture device was transferred into a clean plastic bottle with a capacity of 500 mL. A volume of 100 mL of 1.0 mol/L KCl was added to fully submerge the sponge. The solution was shaken at a constant temperature of 25\u0026deg;C for 1 h, and the concentration of ammonium nitrogen in the extract was determined using a continuous flow analyzer (Smart Chem TM200, Thermofisher, USA).\u003c/p\u003e\n\u003cp\u003eThe ammonia volatilization flux was calculated based on the total NH₄⁺-N captured by the sponge, the soil area covered by the capture device and the exposure time using the following formula(Wang et al., \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e):\u003c/p\u003e\n\u003cp\u003eF = (m / (A * t)) * 10 Eq.\u0026nbsp;(4)\u003c/p\u003e\n\u003cp\u003ewhere F represents the ammonia volatilization flux (kg N hm⁻\u0026sup2; d⁻\u0026sup1;); m denotes the mass of NH\u003csub\u003e4\u003c/sub\u003e⁺-N captured in the lower sponge layer (\u0026micro;g); A is the base area of the capture device (m\u0026sup2;); t indicates the exposure time (d), and 10 serves as the unit conversion factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoil microbial DNA extraction and Illumina sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples collected during the period of highest soil N content were selected for 16S rDNA sequencing. A total of 0.5 g of fresh soil was stored at -80\u0026deg;C and placed in a pre-chilled mortar. After rapid freezing, the sample was grown to a particle size\u0026thinsp;\u0026lt;\u0026thinsp;0.1 mm. The total soil DNA was extracted using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany), and the quality and concentration of the DNA were assessed using a NanoDrop ND-2000 (Thermo Fisher Scientific, Waltham, MA, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified using PCR. The sequencing and bioinformatics analysis were performed by Fujian Manxiu Technology Co., Ltd (Fujian,China). on the Illumina NovaSeq platform. High-quality reads were clustered into operational taxonomic units (OTUs) at 97% similarity using UPARSE (version 7.1). Representative sequences from each OTU cluster were annotated against the RDP database to determine the species composition of each sample. The alpha-diversity indices (Chao 1, Shannon, observed species and phylogenetic diversity whole tree) were analyzed using Mothur software. A principal coordinate analysis (PCoA) based on the Bray\u0026ndash;Curtis distances was then performed to identify the differences between groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (s.e.). Soil physicochemical properties were analyzed using two-way analysis of variance (ANOVA) with SPSS 22.0 (IBM, Inc., Armonk, NY, USA). Microbial abundance comparisons between groups were performed using one-way ANOVA. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates a significant difference between the groups.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eContents of the soil nutrient at different time periods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 1 shows the effects of incorporating \u003cem\u003eAzolla\u003c/em\u003e on the contents of soil nutrients at different stages of rice cultivation. Significant increases in the content of soil TN at stages T2 and T4 were observed with both \u003cem\u003eAzolla\u003c/em\u003e returning and urea fertilizer (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1A). Concurrently, groups A, RA and RU all significantly increased the contents of OM and AK in the soil. During period T4, the RA group showed a more pronounced increase in the content of AK than the RU group (Fig.\u0026nbsp;1B-C). Both \u003cem\u003eAzolla\u003c/em\u003e returning and the application of urea fertilizer reduced the content of AP in the soil. However, during period T4, the RA group had a significantly higher content of AP than the RU group (Fig.\u0026nbsp;1D). \u003cem\u003eAzolla\u003c/em\u003e returning and the application of urea fertilizer both significantly increased the soil pH at stage T2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which mitigated acidification of the soil (Fig.\u0026nbsp;1E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNitrogen content in different forms in the soil at different stages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAzolla\u003c/em\u003e returning to the field affected the N content of paddy soil in different forms at various time points. Compared with the R group, both the application of \u003cem\u003eAzolla\u003c/em\u003e (RA group) and urea fertilizer (RU group) significantly increased the content of soil ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e⁺-N). The RA group had significantly higher levels at the T2 stage. Additionally, the RA group significantly increased the content of soil nitrate N (NO\u003csub\u003e3\u003c/sub\u003e⁻-N) during the stages T3 and T4. \u003cem\u003eAzolla\u003c/em\u003e also increased the content of microbial biomass nitrogen (MBN) in the soil. From T2 to T4, Group RA exhibited significantly higher soil MBN than Group R and significantly exceeded Group RU (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, Group RA reached the highest MBN concentration during the T3 stage (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProportion of different forms of\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003e15\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eN in soil at different time periods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN tracing indicated that soil nitrogen was predominantly present as NO\u003csub\u003e3\u003c/sub\u003e⁻-N and MBN, both of which remained relatively stable overall. In contrast, NH\u003csub\u003e4\u003c/sub\u003e⁺-N declined to undetectable levels from the T3 period onward (Fig.\u0026nbsp;3A). A divergent pattern was observed only in A group, where the trends of all three nitrogen forms paralleled those in the R group: NO\u003csub\u003e3\u003c/sub\u003e⁻-N and MBN were stable, while NH\u003csub\u003e4\u003c/sub\u003e⁺-N dropped to 0% by T3. However, the abundance of NO\u003csub\u003e3\u003c/sub\u003e⁻-N in A group was consistently higher than in R group. In RA group, NO\u003csub\u003e3\u003c/sub\u003e⁻-N and NH\u003csub\u003e4\u003c/sub\u003e⁺-N both showed an initial increase from T0 to T2, followed by a decrease, whereas MBN declined continuously. At T3, both NO\u003csub\u003e3\u003c/sub\u003e⁻-N and MBN peaked before subsequently falling. In RU group, NO\u003csub\u003e3\u003c/sub\u003e⁻-N and NH\u003csub\u003e4\u003c/sub\u003e⁺-N first decreased, then increased to a peak at T3, before declining sharply. MBN exhibited a marked initial decrease and then stabilized. Overall, compared to RU group, RA group maintained more stable concentrations across the different nitrogen forms (Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;4, the contribution rate of \u003cem\u003eAzolla\u003c/em\u003e returning to soil NH\u003csub\u003e4\u003c/sub\u003e⁺-N, NO\u003csub\u003e3\u003c/sub\u003e⁻-N, and MBN was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of urea-derived nitrogen at all rice growth stages. Specifically, \u003cem\u003eAzolla\u003c/em\u003e returning contributed 67.67%\u0026ndash;227.07%, 75.69%\u0026ndash;172.31%, and 141.62%\u0026ndash;213.74% more than urea nitrogen to these respective nitrogen pools. The contribution rate to soil NH\u003csub\u003e4\u003c/sub\u003e⁺-N initially increased, peaked at the maximum tillering stage (T2), and then gradually declined to its lowest level at maturity. In contrast, the contribution to NO\u003csub\u003e3\u003c/sub\u003e⁻-N exhibited a bimodal (\u0026ldquo;M\u0026rdquo;-shaped) trend, with peaks at the peak tillering (T1) and flowering (T3) stages. The contribution to MBN first decreased and then increased, reaching its highest point at T3 stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransformation of exogenous N in soil\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e15\u003c/sup\u003eN tracing revealed distinct transformation dynamics of applied nitrogen across treatments. In A group, conversion to nitrate nitrogen predominated over NH\u003csub\u003e4\u003c/sub\u003e⁺-N from T0 to T2. In RA group, \u003cem\u003eAzolla\u003c/em\u003e returning conversion to NO\u003csub\u003e3\u003c/sub\u003e⁻-N and NH\u003csub\u003e4\u003c/sub\u003e⁺-N increased to an initial peak at T1, then declined, with a sharper decrease for nitrate. Subsequently (T2\u0026ndash;T4), conversion to ammonium rose sharply, peaked at T3, and became the dominant retained form. In RU group, urea nitrogen was rapidly converted to NO\u003csub\u003e3\u003c/sub\u003e⁻-N and NH\u003csub\u003e4\u003c/sub\u003e⁺-N by T1, then declined. Conversion rates increased again after T1, accelerating through T2 to a peak at T3 before a rapid decline. By the experiment's end, urea-derived ammonium was nearly undetectable. Comparatively, urea nitrogen in RA transformed faster and over a shorter duration than in RU group. Conversely, \u003cem\u003eAzolla\u003c/em\u003e returning transformed more slowly but persistently than urea nitrogen. Furthermore, the presence of rice plants (RA vs. A) enhanced the conversion of \u003cem\u003eAzolla\u003c/em\u003e - nitrogen into soil NH\u003csub\u003e4\u003c/sub\u003e⁺-N, NO\u003csub\u003e3\u003c/sub\u003e⁻-N and MBN.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVolatilization of NH\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e3\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003eand N\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO gases at different time periods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 6 shows the results of soil NH\u003csub\u003e3\u003c/sub\u003e and greenhouse gas N\u003csub\u003e2\u003c/sub\u003eO volatilization in paddy fields at different stages after the application of N fertilizer or \u003cem\u003eAzolla\u003c/em\u003e. The application of chemical fertilizer significantly increased the volatilization of NH\u003csub\u003e3\u003c/sub\u003e in the soil. During the T2\u0026ndash;T4 period, the volatilization of NH\u003csub\u003e3\u003c/sub\u003e in the RU group were significantly higher than those in the other groups, with volatilization at T2 and T4 being significantly higher than in the RA group. The RA group only had significantly increased volatilization during the T2 period, with no significant differences from the A and R groups at other times. The application of chemical fertilizers significantly increased the volatilization of N\u003csub\u003e2\u003c/sub\u003eO. Throughout the T1, T3, and T4 stages of rice cultivation period, the volatilization of N\u003csub\u003e2\u003c/sub\u003eO in the RU group were significantly higher than in the other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of\u003c/strong\u003e \u003cstrong\u003eAzolla\u003c/strong\u003e \u003cstrong\u003ereturning to the fields on the soil microbial structure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe content of soil N peaked at the grain filling (T3) stage, which prompted a microbial diversity analysis of the soil at this stage. Compared to the R group, the RA and RU groups exhibited increased alpha-diversity, with significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) observed in the Shannon, PD, Sobs and Chao1 indices. However, no significant difference was observed between the RA and RU groups. The PCoA analysis revealed a clear separation between the samples, which indicated that the incorporation of \u003cem\u003eAzolla\u003c/em\u003e and the application of N fertilizer had significantly altered the structure of the soil microbial community (Fig.\u0026nbsp;7).\u003c/p\u003e\n\u003cp\u003eFigure 8 shows the composition of soil microorganisms at the phylum and genus levels across the experimental groups during the T3 period. A total of 60 phyla and 642 genera were detected in the soil. The dominant phyla were Bacteroidetes (23.28%), Proteobacteria (15.07%), Firmicutes (12.42%), and Acidobacteria (11%). The dominant genera were \u003cem\u003eBacteroidetes vadinHA17\u003c/em\u003e (3.83%), \u003cem\u003eChloroplast\u003c/em\u003e (2.86%), \u003cem\u003eAKIW659\u003c/em\u003e (2.05%), \u003cem\u003eCitrifermentans\u003c/em\u003e (2.41%), and \u003cem\u003eGeothrix\u003c/em\u003e (2.13%) (Fig.\u0026nbsp;5A-B). The abundance of \u003cem\u003eChloroplast\u003c/em\u003e (2.86%) may indicate the presence of plants or algae.\u003c/p\u003e\n\u003cp\u003eTo further investigate changes in the abundance of bacterial genera related to soil N, intergroup comparisons were conducted for the N fixing, nitrifying, and denitrifying genera. Compared to the R group, \u003cem\u003eAzospirillum\u003c/em\u003e significantly increased in the A, RA, and RU groups, with the highest abundance observed in the RA group. \u003cem\u003ePseudomonas\u003c/em\u003e significantly increased in the RA and RU groups, while \u003cem\u003eBacillus\u003c/em\u003e significantly increased in the A and RU groups. \u003cem\u003eNitrospira\u003c/em\u003e significantly increased in the RU group, and \u003cem\u003eNitrosomonas\u003c/em\u003e significantly decreased in the RA group. Comparing the relative abundances of identifiable species within these genera against the R group, \u003cem\u003eBacillus bataviensis\u003c/em\u003e was significantly reduced in the RU group, with no significant differences in the A and RA groups. \u003cem\u003eBacillus megaterium\u003c/em\u003e significantly increased in the RA group, and \u003cem\u003ePseudomonas syringae\u003c/em\u003e significantly increased in the RU group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;5C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional prediction of the key soil microbiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functional prediction of soil microorganisms was performed using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software, which is based on marker gene sequences. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, abundance calculations were conducted for the pathways related to N fixation (Nitrogen metabolism, Alanine, aspartate and glutamate metabolism, and Lysine biosynthesis). As shown in the Fig.\u0026nbsp;9, the RA and RU groups had significantly higher levels of microbial abundance in the nitrogen metabolism pathway than the R group, with the RA group showing significantly higher abundance than the RU group. The RA group exhibited significantly higher predicted microbial abundance in the 'Alanine, aspartate and glutamate metabolism' and 'Lysine biosynthesis' pathways than the R group. Conversely, the RU group showed significantly lower abundance in the 'Lysine biosynthesis' pathway than the R group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis between the key microbiota and soil physicochemical properties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe correlations between the significantly changed bacterial taxa (indicated in Fig.\u0026nbsp;5) and the soil physicochemical properties (soil nutrients, various N sources and N volatilization) are shown in Fig.\u0026nbsp;10. \u003cem\u003eAzospirillum\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e positively correlated with the TN, AK, (NH\u003csub\u003e4\u003c/sub\u003e⁺-N) and nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e⁻-N). Conversely, \u003cem\u003eNitrospira\u003c/em\u003e significantly negatively correlated with these indicators. The genus \u003cem\u003eNitrosomonas\u003c/em\u003e positively correlated with the NH\u003csub\u003e3\u003c/sub\u003e emissions and MBN. In contrast, species of \u003cem\u003eBacillus\u003c/em\u003e, including \u003cem\u003eBacillus bataviensis\u003c/em\u003e and \u003cem\u003eBacillus megaterium\u003c/em\u003e, significantly positively correlated with the N\u003csub\u003e2\u003c/sub\u003eO volatilization and NH\u003csub\u003e3\u003c/sub\u003e volatilization, conversely, it showed a significant positive correlation with MBN.. \u003cem\u003ePseudomonas syringae\u003c/em\u003e negatively correlated with the AP and positively correlated with the pH (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eAzolla\u003c/strong\u003e \u003cstrong\u003ereturning application significantly increased various forms of nitrogen sources in the soil and suppressed volatilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAzolla\u003c/em\u003e is highly adaptable and fixes large quantities of N. The decomposition of its cellulose releases organic C, and certain organic acids are produced, such as citric acid and oxalic acid, which dissolve sequestered P and release mineral K(Nierzwicki-Bauer, \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Simultaneously, N fixation consumes H\u003csup\u003e+\u003c/sup\u003e, which helps to increase the soil pH and mitigate acidification. Previous studies found that the cultivation of \u003cem\u003eAzolla\u003c/em\u003e in the paddy fields increased the contents of OM and TN in the soil by approximately 1%-2%. Previous studies have shown 100 kg of \u003cem\u003eAzolla\u003c/em\u003e dry matter can be converted into 39 kg of OM in the soil within 1 year(Deng et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Singh's pot simulation experiments demonstrated a trend of increase in the soil pH following the application of \u003cem\u003eAzolla\u003c/em\u003e(Singh et al., \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e). This study also observed an increase in the content of TN in the soil and fertility following the application of \u003cem\u003eAzolla\u003c/em\u003e. An increase in the pH was also noted. The increase in ammonium N probably stems from the enhanced soil microbial activity following application, which accelerates the mineralization of organic N into ammonium N. Additionally, \u003cem\u003eAzolla\u003c/em\u003e itself may contain stored N that is gradually released during incorporation(Hasanah and Nabilah, \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). The increase in the content of microbial N indicates enhanced microbial roles in N cycling. The microbiota participate in the transformation of soil N through processes, such as N fixation, ammonification, and nitrification(Verma and Patel, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003eAzolla\u003c/em\u003e application probably provided favorable conditions and abundant nutrients for microbial growth, thus, promoting proliferation and increasing the content of microbial N. However, \u003cem\u003eAzolla\u003c/em\u003e incorporation may alter the soil aeration, or microbial metabolic activities may consume substantial oxygen, which creates relatively anoxic conditions that hinder nitrification(Yang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, this study found that \u003cem\u003eAzolla\u003c/em\u003e application significantly increased the contents of ammonia N in the soil and microbial N while having little effect on nitrate N.\u003c/p\u003e\n\u003cp\u003eGreenhouse gas emissions from farmland are significantly influenced by the methods and rates of fertilizer application(Qian et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). N\u003csub\u003e2\u003c/sub\u003eO emissions, an intermediate product of nitrification and denitrification processes, are particularly sensitive to the rates of application of N and water management practices. The amount of N\u003csub\u003e2\u003c/sub\u003eO emissions increased with higher rates of the application of N fertilizer. \u003cem\u003eAzolla\u003c/em\u003e can serve as a source of N and can partially replace chemical N fertilizers and effectively reduce the N\u003csub\u003e2\u003c/sub\u003eO emissions(Verma and Patel, \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Simultaneously, \u003cem\u003eAzolla\u003c/em\u003e primarily fixes N to produce ammonium N (NH\u003csub\u003e4\u003c/sub\u003e⁺-N), thereby reducing the content of nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e⁻-N). Previous studies have reported that cultivating \u003cem\u003eAzolla\u003c/em\u003e in paddy fields significantly reduces the N\u003csub\u003e2\u003c/sub\u003eO fluxes during the early growth stages of rice(Yang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). This reduction was attributed to the substantial lowering of the content of NO\u003csub\u003e3\u003c/sub\u003e⁻-N in the soil by \u003cem\u003eAzolla\u003c/em\u003e, which resulted in a decrease in the total N\u003csub\u003e2\u003c/sub\u003eO emissions of between 70.9% and 78.2%(Watanabe, \u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e). Similarly, this study found that the RU group (applied N fertilizer) had a significantly increase in N\u003csub\u003e2\u003c/sub\u003eO emissions, whereas the RA group (applied \u003cem\u003eAzolla\u003c/em\u003e) significantly suppressed this phenomenon. The volatilization of NH\u003csub\u003e3\u003c/sub\u003e diminishes the soil fertility and impacts the atmospheric environment. The \u003cem\u003eAzolla\u003c/em\u003e returning application probably reduced the NH\u003csub\u003e3\u003c/sub\u003e emissions by increasing the soil pH and content of OM. A higher pH favors the retention of ammonia, while the OM adsorbs ammonia gas, thus, reducing its volatilization(Abd El-Aal, \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). Concurrently, the fixation of ammonia by soil microbes may have partially mitigated the NH\u003csub\u003e3\u003c/sub\u003e emissions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAzolla\u003c/strong\u003e \u003cstrong\u003ereturning application regulated the structure of microbiota in the soil that fix nitrogen\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe grain-filling stage of rice growth and development is critical because the plant has high requirements for nutrients and water, while the soil environment and microbial activity are still active(Al-Tawaha et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). This study found that the content of OM in the soil and the capacity for microbial N fixation peaked during the T3 stage. Further analysis was conducted on the soil microbial structure and function prediction during this period. The results revealed that the \u003cem\u003eAzolla\u003c/em\u003e returning application suppressed the abundance of nitrifying bacteria (\u003cem\u003eNitrospira\u003c/em\u003e and \u003cem\u003eNitrosomonas\u003c/em\u003e), whereas the application of urea fertilizer significantly increased the abundance of these genera. \u003cem\u003eNitrosomonas\u003c/em\u003e is a key genus in nitrification and primarily responsible for oxidizing ammonia (NH\u003csub\u003e3\u003c/sub\u003e) to nitrite (NO\u003csub\u003e2\u003c/sub\u003e⁻) and thus, completing the first stage of nitrification(Grunditz and Dalhammar, \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). \u003cem\u003eNitrospira\u003c/em\u003e can then oxidize nitrite (NO\u003csub\u003e2\u003c/sub\u003e⁻) to nitrate (NO\u003csub\u003e3\u003c/sub\u003e⁻), which completes the second stage of nitrification(Daims et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The application of urea fertilizer promotes the increase of both genera. In this study, the RU group significantly increased the abundance of these two genera, while the RA group significantly decreased it.\u003c/p\u003e\n\u003cp\u003eConversely, the abundance of certain probiotic genera that are can fix N and conduct denitrification increased significantly in the RA group. \u003cem\u003eAzospirillum\u003c/em\u003e is a symbiotic nitrogen-fixing bacterium that converts atmospheric N gas (N\u003csub\u003e2\u003c/sub\u003e) into ammonium N, which can be taken up by plants. This enhances the content of N in the soil. This genus also produces plant hormones, such as indoleacetic acid, that stimulate the development of roots and enhance the capacity to take up nutrients and water. \u003cem\u003eAzospirillum\u003c/em\u003e also upregulates relevant gene expression and increases the content of nitrate in N fixation to further promote plant growth(Pelagio-Flores et al., \u003cspan class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003ePseudomonas\u003c/em\u003e can produce plant hormones, such as auxin, dissolve phosphates, mineralize organic P and release insoluble K, thereby promoting plant growth(Weinmann, \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e decomposes organic N into ammonia (NH\u003csub\u003e4\u003c/sub\u003e) and possesses properties antagonistic to plant pathogens. It can inhibit the growth and reproduction of plant pathogens and reduce the incidence of disease(Hlordzi et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In particular, \u003cem\u003eBacillus bataviensis\u003c/em\u003e dissolves insoluble mineral K and converts it into water-soluble K that is available for the plant to take up and enhances the utilization of K in the soil(Basak et al., \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eBacillus megaterium\u003c/em\u003e transforms insoluble P in the soil into forms that are available to plants, thereby improving the utilization of P(Zhao et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Consequently, the RA group exhibited a significant increase in the content of ammonium N along with elevated levels of readily available K and P. The functional prediction analysis revealed that the markedly increased beneficial bacteria in the RA group potentially promote N fixation-related metabolism (Nitrogen metabolism, Alanine, aspartate and glutamate metabolism, and Lysine biosynthesis). The microbial fixation of N in the soil is closely linked to the metabolism of amino acids. The nitrogen fixation product, ammonium (NH\u003csub\u003e4\u003c/sub\u003e⁺), is integrated into amino acids via specific pathways and subsequently synthesized into proteins or other organic compounds that contain N(Pahari et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). This finding aligns with \u003csup\u003e15\u003c/sup\u003eN labeling studies, which revealed that urea nitrogen undergoes rapid conversion in soil with a short transformation process, whereas \u003cem\u003eAzolla\u003c/em\u003e returning exhibits significantly slower conversion rates and a prolonged transformation duration. Furthermore, the introduction of plants (rice) accelerates the conversion of \u003cem\u003eAzolla\u003c/em\u003e returning in soil.\u003c/p\u003e\n\u003cp\u003eWhile the application of urea fertilizers enhances the soil nitrification capacity, it also readily leads to the emission of the greenhouse gas N\u003csub\u003e2\u003c/sub\u003eO and nitrogen volatilization(Qian et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The excessive application of urea fertilizers may also increase the abundance of potential soil pathogens. For example, this study observed a significant increase in the bacterium \u003cem\u003ePseudomonas syringae\u003c/em\u003e within the RU group. This bacterium is widely present on plant surfaces and in soil and is capable of causing various diseases, such as leaf spot, wilt and canker. It primarily utilizes a type III secretion system (T3SS) to inject effector proteins, such as AvrE and HopZ1a, directly into the plant cells(Oh et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Xie et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). These proteins interfere with plant immune responses and promote the colonization and growth of pathogens. It also produces various toxins, such as coronatine and mangotoxin, which disrupt the structure and function of plant cells, thus, facilitating infection(Dudnik and Dudler, \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). The study also found that \u003cem\u003eAzolla\u003c/em\u003e returning to fields promotes the growth of \u003cem\u003ePseudomonas\u003c/em\u003e while reducing the abundance of pathogens. This practice also mitigates the greenhouse gas emissions and nitrogen volatilization caused by the application of urea fertilizer.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eAzolla\u003c/em\u003e returning to fields can effectively replace conventional nitrogen fertilizers, potentially enhance soil fertility by increasing the levels of available nitrogen, potassium and phosphorus, as well as organic matter, and increasing the soil pH. It notably increases the content of ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e⁺-N) in the soil and the levels of microbial nitrogen (MBN). Simultaneously, it mitigates the volatization of the greenhouse gas N\u003csub\u003e2\u003c/sub\u003eO and the volatization of nitrogen (NH\u003csub\u003e3\u003c/sub\u003e) caused by the application of nitrogen fertilizer. These beneficial effects stem from changes in the structure of the soil microbial community following the incorporation of \u003cem\u003eAzolla\u003c/em\u003e. Returning \u003cem\u003eAzolla\u003c/em\u003e to fields significantly increases the abundance of nitrogen-fixing and denitrifying bacteria, while reducing the levels of potential pathogenic microorganisms. This stimulates nitrogen fixation in the soil and amino acid metabolism. Therefore, returning \u003cem\u003eAzolla\u003c/em\u003e to fields is an effective alternative to using urea fertilizers.\u003c/p\u003e"},{"header":"Declarations","content":"\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSufang Deng\u003c/strong\u003e: Conceptualization, Data curation, Investigation, Methodology, Formal analysis, Writing the original draft. \u003cstrong\u003eYanqiu Yang\u003c/strong\u003e: Conceptualization, Formal analysis, Investigation, Methodology, Data curation. \u003cstrong\u003eZhaoyang Ying\u003c/strong\u003e: Conceptualization, Funding acquisition, Supervision, Methodology, Writing - review \u0026amp; editing.\u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of Competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests or personal relationships that could have influenced reporting of the research findings.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by \u0026zwnj;National Green Manure Industry Technology System (CARS-22)\u0026zwnj;, \u0026zwnj;Fujian Provincial Natural Science Foundation Project (2025J01)\u0026zwnj;, \u0026zwnj;Fujian Academy of Agricultural Sciences Youth Elite Project (YC2021007)\u0026zwnj;, and \u0026zwnj;Fujian Provincial Public Welfare Research Institutes Basic Research Special Project \u0026ndash; Public Welfare Competitive Project (2023R1063) in China. The authors would like to thank MogoEdit (htt\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eps://www.mogoedit.com) f\u003c/span\u003e\u003cspan address=\"http://ps://www.mogoedit.com) f\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eor providing assistance in English language editing during the preparation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd El-Aal AA (2022) Anabaena-azollae, significance and agriculture application: A case study for symbiotic cyanobacterium. Microbial syntrophy-mediated eco-enterprising, pp 1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Tawaha ARMS, Singh S, Singh V, Kafeel U, Naikoo MI, Kumari A, Imran, Amanullah, Al-Tawaha AR, Qaisi AM (2020) Improving water use efficiency and nitrogen use efficiency in rice through breeding and genomics approaches. 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Nat Reviews Earth Environ 4(10):716\u0026ndash;732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen J, Cui Z, Miao Y, Mi G, Zhang H, Fan M, Zhang C, Jiang R, Zhang W, Li H (2013) Transforming agriculture in China: From solely high yield to both high yield and high resource use efficiency. Global Food Secur 2(1):1\u0026ndash;8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh P, Panigrahi B, Satapathy K (1981) Comparative efficiency of Azolla, blue-green algae and other organic manures in relation to N and P availability in a flooded rice soil. Plant Soil 62(1):35\u0026ndash;44\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparks DL, Page AL, Helmke PA, Loeppert RH (2020) Methods of soil analysis, part 3: Chemical methods. Wiley\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalley SN, Talley BJ, Rains DW (1977) Nitrogen fixation by Azolla in rice fields. Genetic engineering for nitrogen fixation. Springer, pp 259\u0026ndash;281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma DK, Patel SK (2019) Cyanobacteria and Azolla in rice cultivation/improving biological N2-fixation system in rice. Biofertilizers and Biopesticides in Sustainable Agriculture. Apple Academic, pp 221\u0026ndash;236\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Liu X, Ju X, Zhang F (2002) Field in situ determination of ammonia volatilizatioon from soil: Venting method. Plant Nutr Fertilizer Sci (02), 205\u0026ndash;209\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe I (1982) Azolla\u0026mdash;Anabaena symbiosis\u0026mdash;its physiology and use in tropical agriculture. Microbiology of tropical soils and plant productivity. Springer, pp 169\u0026ndash;185\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinmann M (2017) Bio-effectors for improved growth, nutrient acquisition and disease resistance of crops\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Ding Y, Shao X, Yao C, Li J, Liu J, Deng X (2021) Pseudomonas syringae senses polyphenols via phosphorelay crosstalk to inhibit virulence. EMBO Rep 22(12), e52805\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang G, Ji H, Liu H, Feng Y, Zhang Y, Chen L, Guo Z (2021) Nitrogen fertilizer reduction in combination with Azolla cover for reducing ammonia volatilization and improving nitrogen use efficiency of rice. PeerJ 9, e11077\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao Y, Zhang M, Tian Y, Zhao M, Zeng K, Zhang B, Zhao M, Yin B (2018) Azolla biofertilizer for improving low nitrogen use efficiency in an intensive rice cropping system. Field Crops Res 216:158\u0026ndash;164\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan Z-F, Zhou Y, Chen Z, Tang X, Wang Y, Kappler A, Xu J (2023) Reduce methane emission from rice paddies by man-made aerenchymatous tissues. Carbon Res 2(1):17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Mao X, Zhang M, Yang W, Di HJ, Ma L, Liu W, Li B (2021) The application of Bacillus Megaterium alters soil microbial community composition, bioavailability of soil phosphorus and potassium, and cucumber growth in the plastic shed system of North China. Agriculture, Ecosystems \u0026amp; Environment 307, 107236\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \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 \u003cdiv class=\"SimplePara\"\u003eBasic Composition of Nutrients for the Culture Medium\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNutrients\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eConcentration (mol/L)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eCaSO\u003csub\u003e4\u003c/sub\u003e 2H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.001\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMgSO\u003csub\u003e4\u003c/sub\u003e 7H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.0017\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eKH\u003csub\u003e2\u003c/sub\u003ePO4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.003\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eKCl\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.0003\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eEDTA-Na\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.1*10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eFeSO\u003csub\u003e4\u003c/sub\u003e 7H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.0*10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eH\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e4.85*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNa\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e 2H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1.24*10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNaNO\u003csub\u003e3\u003c/sub\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.0007\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eFertilizer content and exogenous nitrogen addition rate (kg/hm\u0026sup2;)\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTreatment Group\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eUrea\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSuperphosphate\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cdiv class=\"SimplePara\"\u003ePotassium chloride\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eAzolla\u003c/span\u003e\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003csup\u003e15\u003c/sup\u003eN\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eR\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e120\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e120\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e6000\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRA\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e120\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e6000\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRU\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e180\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e75\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e120\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Azolla filiculoide, Returning application, Soil microbial, Nitrogen-fixing, Rice grain-filling stage","lastPublishedDoi":"10.21203/rs.3.rs-8853068/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8853068/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eAims\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAzolla\u003c/em\u003e, a robust nitrogen (N)-fixing plant, can be efficiently and ecologically cultivated when incorporated into rice fields, while the mechanisms remain inadequately understood. This study systematically explored these mechanisms by assessing the effects of \u003cem\u003eAzolla\u003c/em\u003e returning on soil nitrogen forms, ammonia volatilization, and microbial community responses across key rice growth stages.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effects of \u003cem\u003eAzolla\u003c/em\u003e incorporation were compared against rice monoculture and urea application at various stages of rice cultivation. The study monitored changes in soil nitrogen forms and ammonia volatilization. Furthermore, the response of the soil microbial community was examined, with a detailed analysis conducted during the grain-filling stage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAzolla\u003c/em\u003e returning significantly increased the content of total soil N, organic matter, available potassium and available phosphorus, while also elevating the soil pH. It substantially elevated ammonium nitrogen and microbial biomass nitrogen (peaking at grain-filling) compared to monoculture or urea, while significantly reducing nitrous oxide emissions, ammonia volatilization and maintaining nitrogen fertilizer content over time. Soil microbial community during the grain-filling stage indicated that urea fertilizer favored the enrichment of nitrifying bacteria (\u003cem\u003eNitrospira\u003c/em\u003e and \u003cem\u003eNitrosomonas\u003c/em\u003e) but also increased the abundance of pathogen \u003cem\u003ePseudomonas syringae\u003c/em\u003e. \u003cem\u003eAzolla\u003c/em\u003e returning potentially reduced the abundance of pathogens while significantly promoting that of beneficial bacteria involved in N fixation and denitrification, including \u003cem\u003eAzospirillum\u003c/em\u003e and \u003cem\u003eBacillus bataviensis\u003c/em\u003e. These enhanced soil nitrogen fixation and metabolic capacity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study is the first to track the dynamic characteristics and potential mechanisms behind the changes in nitrogen source during \u003cem\u003eAzolla\u003c/em\u003e incorporation, a process that enhances soil fertility and reduces environmental emissions. It provides a theoretical foundation for the adoption of this sustainable practice.\u003c/p\u003e","manuscriptTitle":"Azolla returning application Drives the Reshaping of Nitrogen-Fixing Microbial Communities in Paddy Soils to Enhance Fertilization and Reduce Emissions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 07:02:09","doi":"10.21203/rs.3.rs-8853068/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c316a486-f7cb-4a02-b3fd-d3be7fa6bfcc","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-04T05:02:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 07:02:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8853068","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8853068","identity":"rs-8853068","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}