Effects of Nitrification Inhibitor Application on Methanogenic and Methanotrophic Activity in Paddy Soil under Water-Saving Irrigation

Effects of Nitrification Inhibitor Application on Methanogenic and Methanotrophic Activity in Paddy Soil under Water-Saving Irrigation | 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 Article Effects of Nitrification Inhibitor Application on Methanogenic and Methanotrophic Activity in Paddy Soil under Water-Saving Irrigation Wenli Cui, Wenjun Jin, Ning Zhou, ZongXiang Zhang, Zhaorong Dong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7218479/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 The nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) modulates methane (CH4) emissions in rice paddies by altering soil biogeochemistry and microbial functionality. This study elucidates the interactive effects of DMPP and water management on methanogen (mcrA) and methanotroph (pmoA) gene abundances across rice growth stages using a rhizobox system. Results revealed that alternate wetting-drying (A) and wetting irrigation (W) suppressed methanogen activity pre-panicle fertilization, whereas DMPP application post-panicle fertilization reduced mcrA abundance by 197–569% across treatments. Methanotroph activity peaked under continuous flooding (C) without DMPP, while DMPP enhanced pmoA abundance by 23.7–35.1% under A. Soil organic carbon (SOC) positively correlated with methanogens, whereas methanotrophs exhibited inverse relationships with SOC and dissolved organic carbon (DOC). These findings demonstrate that optimized water regimes coupled with DMPP application can mitigate CH₄ emissions by restructuring microbial communities and carbon dynamics. The application of 3,4-dimethylpyrazole phosphate (DMPP) can affect paddy soil methane emissions through soil and microbial properties. However, little is know about the effects of DMPP on methanogens and methanotrophs in rice soils under different water management practices. This study utilized the rhizobox method to quantify the gene abundances of these microbial communities across various growth stages. The results showed that water management practices significantly influenced the community structures and gene abundances of methanogens and methanotrophs. Under alternate wetting and drying (A) and continuous flooding (W), methanogen growth was either inhibited or promoted without DMPP application before and after panicle fertilization. However, DMPP application significantly suppressed methanogen activity after panicle fertilization. For methanotrophs, DMPP application enhanced pmoA gene abundance under A, while W and continuous saturation (C) without DMPP promoted methanotroph growth. Additionally, DMPP markedly altered the relationships between SOC, DOC, and the community structures of methanogens and methanotrophs. Methanogens exhibited a significant positive correlation with SOC, while methanotrophs showed negative correlations with both SOC and DOC. Overall, this study demonstrates that water management practices and DMPP application significantly affect methanogen and methanotroph communities in rice soils, with these changes closely linked to soil physicochemical properties. Earth and environmental sciences/Biogeochemistry Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Microbiology Nitrification inhibitor Water-saving irrigation Methanogens Methanotrophs Paddy soil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Rice paddy soils are recognized as significant sources of atmospheric methane (CH 4 ), contributing substantially to global warming [ 1 – 2 ] . CH 4 production in these ecosystems is driven by anaerobic conditions and the availability of organic C, with methanogens decomposing organic matter to produce CH₄. Water management practices, such as continuous flooding, create anaerobic environments conducive to CH₄ production, whereas water-saving irrigation can mitigate emissions. Nitrification inhibitors like DMPP inhibit the conversion of NH₄⁺-N to NO₃⁻-N, indirectly affecting CH₄ dynamics. Methane monooxygenase, responsible for CH₄ oxidation, shares structural similarities with ammonia monooxygenase, leading to competitive interactions between NH₄⁺ and CH₄. Studies have shown that nitrification inhibitors influence CH₄ emissions by modulating methanogen and methanotroph activity. For instance, DMPP application during later rice growth stages may enhance methanotroph abundance, thereby promoting CH₄ oxidation and reducing emissions. Methanogens thrive in anaerobic environments, and oxygen availability directly impacts their community structure and metabolic activity. Periodic drainage during rice cultivation improves soil aeration, inhibiting methanogens while promoting methanotrophs. Methanotrophs, aerobic bacteria utilizing CH₄ as their sole carbon and energy source, oxidize approximately 80% of CH₄ produced in rice paddies, with only a minor fraction escaping to the atmosphere. Water-saving irrigation enhances soil aeration, favoring methanotroph activity and reducing CH₄ emissions. Soil organic carbon (SOC) and dissolved organic carbon (DOC) are critical determinants of microbial activity and CH₄ emissions. SOC serves as the primary substrate for methanogens, while DOC, though a minor fraction of total soil carbon, plays a vital role in nutrient provision. Water-saving irrigation can reduce DOC content, particularly in low-precipitation years, further influencing CH₄ dynamics. This study systematically investigated the effects of water management and DMPP application on methanogen and methanotroph communities in rice soils. By analyzing SOC and DOC interactions with microbial communities, we provide insights into sustainable agricultural practices for mitigating CH₄ emissions. Materials and Methods 1.1 Experimental site The experimental site was located at the Agricultural Excellence Garden Experimental Base of Anhui Agricultural University (31°87′N, 117°25′E). The soil used in this study was obtained from a rice-wheat rotation field at the Guohe Experimental Station of Anhui Agricultural University (31°48′N, 117°23′E), featuring a soil type of sandy loam and gleyed paddy soil. Soil samples were collected during the wheat maturity stage, specifically from the 0–20 cm soil layer, which were then sieved through a 4 mm mesh and homogenized. Nine pre-experimental soil samples were randomly selected and divided into three groups for different purposes: three samples were stored in a − 80°C freezer for soil DNA analysis; another three were kept in a 4°C refrigerator for assessing soil microbial activity; and the remaining three samples were air-dried for determining physicochemical properties and conducting mineralization experiments. The basic physicochemical properties of the soil prior to the experiment are presented in Table 1 . Table 1 Soil physicochemical properties before experiment TN (g kg − 1 ) NH 4 + –N (mg kg − 1 ) NO 3 − –N (mg kg − 1 ) NO 2 − –N (mg kg − 1 ) AK (mg kg − 1 ) AP (mg kg − 1 ) SOC (g kg − 1 ) DOC (mg kg − 1 ) pH EC (µs cm − 1 ) Soil before testing 1.56 5.91 9.35 0.07 201.67 39.24 11.98 71.96 5.32 118.28 Note: TN: total nitrogen; NH 4 + –N: soil ammonium content; NO 3 – –N: soil nitrate content; NO 2 – –N: soil nitrite content; AK: available potassium; AP, available phosphorus; OC: organic carbon; DOC, soil dissolved organic carbon; EC: electrical conductivity. 1.2 Experimental Design This experiment takes Zhendao 18 (japonica rice Oryza sativa L. subsp. japonica) as the research object, and the seedling cultivation method is dry cultivation. Select seedlings with consistent growth and transplant them manually, with 3 seedlings transplanted from each root box. Sow and raise seedlings on May 12, 2021, transplant seedlings on June 23, and harvest rice grains on October 6. Water management is described in section 2.2.2. Based on the local soil source conditions, apply 300 N kg hm-2 of pure nitrogen in three stages, with a base fertilizer: tiller fertilizer: ear fertilizer ratio of 4:3:3. Apply 135 P2O5 kg hm-2 of pure phosphorus and 270 K2O kg hm-2 of pure potassium, both as base fertilizers mixed into the soil at once. Diseases and pests are controlled according to the field conditions. This experiment adopts the root box method, and the root box design is shown in Fig. 1 . The root box is made of PVC pipe and is divided into two parts. The periphery is the non rhizosphere area, and the middle part is the rhizosphere area. The non rhizosphere and rhizosphere soil are separated by a 30 µm nylon mesh, which can effectively prevent the root system from crossing without affecting water and nutrient exchange. Based on the preliminary investigation data of soil bulk density in the 0-20cm soil layer of paddy fields (taken from fields near the cultivation soil) by the research team, the soil bulk density in the root box was determined to be 1.11 g cm-3 (n = 14). Based on the soil bulk density, calculate the mass of soil in the 0-20cm soil layer, fill the mixed soil into the root box, and compact it to the corresponding soil bulk density. Bury the root box in a 20cm deep soil layer to prevent drastic changes in soil temperature inside the root box. Use 40 root boxes for each treatment. To prevent the impact of rainfall on the experiment, plastic film is used to construct rain blocking facilities above the experimental area. This study adopts a two factor experimental design. Factor 1 is water treatment, and three types of water treatments were set up in the experiment, namely continuous flooding irrigation (C), wetting irrigation (W), and alternative wetting and drying irrigation (A). Combining the research methods of Carrijo, Li Yongsong, Xu Fenfen, etc., the following three types of water were set for continuous flooding irrigation. Except for the drying of soil moisture during the roasting period and one week before harvest, the soil water layer was maintained at around 3cm for the rest of the time. Moist irrigation involves keeping the soil moist for the rest of the time, except during the roasting period and one week before harvest. In order to better control the moisture state of moist irrigation, the soil water level should be kept between − 1-1cm, that is, when the water level drops to -1cm, water should be poured to 1cm, and when the soil moisture freely dries to -1cm, water should be poured again. This cycle is repeated. Alternating wet dry irrigation begins after the rice seedlings turn green, maintaining the soil water level between − 10cm and 3cm. When the water level drops to -10cm, water up to 3cm, wait for the water to freely dry up to -10cm, and then water up to 3cm again. This cycle continues. The soil water level is observed by burying a water level tube in the root box. Factor 2 is whether to apply 3,4-dimethylpyrazole phosphate (DMPP). DMPP is provided by Shanghai Xianding Biotechnology Co., Ltd., with a purity of ≥ 97%, and is calculated as 97% when used. The DMPP treatment was divided into two groups, one group was treated with DMPP in stages along with base fertilizer, tiller fertilizer, and ear fertilizer, while the other group was not treated with DMPP. The application rate of DMPP is 2% of nitrogen fertilizer, which meets the experimental requirements. 1.3 Sampling and Indicator Measurement Soil sampling and pretreatment were carried out on the 14th day after each fertilization and at the maturity stage, respectively. Samples were taken on the 14th day after rice transplantation (14th day after applying base fertilizer and greening stage), 28th day (14th day after applying tillering fertilizer and tillering stage), 67th day (14th day after applying panicle fertilizer and heading stage), and 105th day (maturity stage). Randomly select five replicates for backup each time, and conduct destructive sampling on at least three of them. When taking soil samples, distinguish between rhizosphere and non rhizosphere soils. Take the soil sample back to the laboratory, sieve it through a 2mm sieve, remove impurities, and mix well. Divide the mixed soil sample into three parts, with one part stored at -80 ℃ for soil microbial functional gene analysis; Part of it is stored in a 4 ℃ refrigerator for testing soil microbial activity and soil physicochemical properties; Part of it is air dried and used for soil physical and chemical property testing. According to Bao Shidan's "Soil Agrochemical Analysis", soil organic carbon is determined by potassium dichromate volumetric method. Soil water-soluble organic carbon was extracted by 0.5 mol L-1 K2SO4 solution (soil water ratio 1:2.5) and determined by potassium dichromate digestion method. Soil DNA was extracted using the Fast DNA SPIN kit for soil (USA). According to the reagent kit manual, weigh 0.5 g of soil sample frozen at -80 ℃ and complete the DNA extraction of the sample on an ultra clean workbench. The extracted DNA sample is stored in an ultra-low temperature freezer at -80 ℃. Quantitative analysis was performed on the functional genes (mcrA) and methane oxidizing bacteria (pmoA) of methanogens using a fluorescence quantitative PCR instrument (Stratagene Mx3000P, Agilent, USA). Primer information is shown in Table 2 . Fluorescence quantitative PCR reaction system for all functional genes: SYBR ® Premix Ex Taq ™ II (TakaRa, Japan) 10 µ L, primer 0.5 µ L each, DNA extraction 0.5 µ L; Add sterile water to 20 µ L. PCR program: 95 ℃ for 10 minutes; 95 ℃ for 20 seconds, 60 ℃ for 30 seconds (45 cycles); 4 ℃, ∞. The qPCR amplification efficiency of mcrA and pmoA functional genes was 97%. Table 2 Information of primers Target gene Primer sequence (5'~3') Amplification length mcrA F:5'GGTGGTGTMGGATTCACACARTAYGC 3' 467 R: 5' TTCATTGCRTAGTTWGGRTAGTT 3' pmoA F: 5' GGNGACTGGGACTTCTGG 3' 510 R: 5' CCGGMGCAACGTCYTTACC 3' 1.4 Statistical analysis Data processing utilized Excel 2023, SPSS 27, and R 4.1.2. Microbial community structures were analyzed at family-level taxonomy after removing OTUs with < 1% abundance. Treatment effects were assessed via ANOVA with Tukey's post-hoc test (α = 0.05). Correlations between SOC/DOC and microbial indices were evaluated using Pearson coefficients. Results 2.1 Abundance of methane producing and oxidizing bacteria in rice soil As shown in Fig. 2 , 14 days after the application of basal fertilizer, under the condition of no DMPP, the abundance of mcrA gene in W treatment was significantly higher than that in A treatment. In treatments A and W, the application of DMPP reduced the abundance of the mcrA gene, while in treatment W, DMPP inhibited the growth of the mcrA gene, with an inhibition rate of 28.82%; In treatment C, the application of DMPP increased the abundance of mcrA gene by 21.04%. For the rhizosphere soil 14 days after application of tillering fertilizer, water management had a significant effect on the abundance of mcrA genes in the rhizosphere soil without DMPP, with the order of mcrA gene abundance being C > W > A. The application of DMPP significantly promoted the growth of mcrA gene in rhizosphere soil treated with A, while inhibiting the growth of mcrA gene in rhizosphere soil treated with C, with an inhibition rate of 12.50%. In the W treatment, the application of DMPP increased the abundance of mcrA gene by 24.78%. For the rhizosphere soil 14 days after applying ear fertilizer, under the condition of not applying DMPP, water management has a significant effect on the abundance of mcrA genes in the rhizosphere soil, with the order of mcrA gene abundance being W > A > C; The abundance of mcrA gene in W treatment was the highest, with W treatment having a higher abundance of mcrA gene than A treatment by 20.25% and C treatment by 197.60%, respectively. In treatment A, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 493.42%; In the W treatment, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 569.74%; In treatment C, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 197.09%. As shown in Fig. 3 , 14 days after the application of basal fertilizer, under the condition of no DMPP, the abundance of pmoA genes in W treatment was significantly higher than that in C treatment, and the order of pmoA gene abundance was W > A > C. In treatments A and C, the application of DMPP significantly increased the abundance of pmoA genes, while in treatment W, DMPP inhibited the growth of pmoA genes with an inhibition rate of 12.78%. For the rhizosphere soil 14 days after application of tillering fertilizer, under the condition of no DMPP application, the abundance of pmoA genes in treatment A was significantly lower than that in treatment W and C, and the order of pmoA gene abundance was C > W > A. The application of DMPP significantly promoted the growth of pmoA gene in A treatment. The application of DMPP significantly inhibited the growth of pmoA genes in W and C treatments, with inhibition rates of 27.36% and 81.05%, respectively. For the rhizosphere soil 14 days after applying panicle fertilizer, under the condition of not applying DMPP, the abundance of pmoA genes in treatment A was significantly lower than that in treatment W and C, and the order of pmoA gene abundance was C > W > A. In treatment A, the application of DMPP significantly increased the abundance of pmoA genes in the rhizosphere. After the application of DMPP, there was no significant difference between W treatment and C treatment. W treatment promoted the growth of pmoA gene, while C treatment inhibited the growth of pmoA gene, with an inhibition rate of 13.00%. 2.2 Community Structure of Methane Producing and Oxidizing Bacteria in Rice Soil In order to ensure the reliability and accuracy of the analysis results, based on previous research, OTUs with abundance values lower than 1% of the total sequencing amount of the entire sample were removed, and then the differences in the number of classification units among different samples were compared by the classification level of each family. As shown in Fig. 5 − 2, at the level of family classification, six major methane producing bacteria were detected after applying basal fertilizer, tillering fertilizer, and ear fertilizer. They were ranked by their proportion as Methanosaetaceae, Methanosarcinaceae, Methanocellaceae, Methanoregiaceae, Methanomasiliicoccaceae, and Methanobacteraceae, among which Methanosaetaceae, Methanosarrcinaceae, and Methanocellaceae accounted for over 20%. In addition, there is another type of methanogenic bacteria belonging to the Methanomicrobiales order that has not been classified by comparison. As shown in Fig. 4 -a, in treatment A, the application of DMPP had inhibitory effects on Methanosaetaceae and Methanobacteriaceae, with inhibition rates of 6.30% and 8.89%, respectively. It had a promoting effect on Methanosarcinaceae, Methanocellaceae, Methanoregularity, and Methanomasilicaceae, with enhancement rates of 13.87%, 4.68%, 18.54%, and 20.41%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanolegulaceae, Methanomassilicoccaceae, and Methanobacteriaceae, with inhibition rates of 17.55%, 12.99%, and 74.17%. It had a promoting effect on Methanosaetaceae, Methanosarcinaceae, and Methanocellaceae, with enhancement rates of 1.52%, 11.40%, and 1.52%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosaetaceae, Methanosarcinaceae, and Methanobacteriaceae, with inhibition rates of 8.47%, 2.14%, and 121.45%. It had a promoting effect on Methanoceraceae, Methanomasiliicoccaceae, and Methanocellaceae, with enhancement rates of 22.68%, 6.37%, and 5.02%. As shown in Fig. 4 -b, in treatment A, the application of DMPP had inhibitory effects on Methanoceraceae and Methanobacteriaceae, with inhibition rates of 4.12% and 35.66%, and promoting effects on Methanosaetaceae, Methanoarcinaceae, Methanocellaceae, and Methanomasilicaceae, with enhancement rates of 9.17%, 12.63%, 1.09%, and 22.10%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanoceraceae and Methanomasiliicoccaceae, with inhibition rates of 24.49% and 59.99%, and promoting effects on Methanosaetaceae, Methanoarcinaceae, Methanocellaceae, and Methanobacteriaceae, with enhancement rates of 3.67%, 1.28%, 29.06%, and 29.95%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosaetaceae and Methanomasiliicoccaceae, with inhibition rates of 44.05% and 33.58%, respectively. It had a promoting effect on Methanosarcinaceae, Methanocellaceae, Methanoregularity, and Methanobacteriaceae, with enhancement rates of 11.96%, 14.55%, 29.43%, and 65.78%, respectively. As shown in Fig. 4 -c, in treatment A, the application of DMPP had inhibitory effects on Methanosaetaceae, Methanosarcinaceae, and Methanocellaceae, with inhibition rates of 9.08%, 10.47%, and 10.71%, respectively. It had a promoting effect on Methanoregularity, Methanobacteriaceae, and Methanomasilicaceae, with enhancement rates of 1.04%, 48.79%, and 90.53%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanosarcinaceae and Methanoshellaceae, with inhibition rates of 1.22% and 20.53%, respectively. It had a promoting effect on Methanosarcinaceae, Methanosauriculaceae, Methanosobacteriaceae, and Methanosassilicosaceae, with enhancement rates of 17.84%, 4.26%, 15.54%, and 25.50%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosarcinaceae, Methanoshellaceae, and Methanosobacteriaceae, with inhibition rates of 4.14%, 16.96%, and 15.24%, respectively. It had a promoting effect on Methanosataceae, Methanosegulaceae, and Methanosassilicosaceae, with enhancement rates of 13.23%, 35.88%, and 1.06%, respectively. In order to ensure the reliability and accuracy of the analysis results, based on previous research, OTUs with abundance values lower than 1% of the total sequencing amount of the entire sample were removed, and then the differences in the number of classification units among different samples were compared by the classification level of each family. As shown in Fig. 6 − 2, at the level of family classification, three types of methane oxidizing bacteria were detected after applying basal fertilizer, tillering fertilizer, and ear fertilizer. They were ranked by their proportion as follows: Methylocystaceae Methylococcaceae、Candidatus Methylumidiphilus。 As shown in Fig. 5 -a, 14 days after the application of basal fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylocystiaceae and Candidatus Methyluminophilus, with inhibition rates of 13.97% and 67.20%, respectively. It had a promoting effect on Methylocystiaceae, with an increase rate of 23.74%; In the W treatment, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 2.54% and 13.45%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 4.60%; In treatment C, the application of DMPP had inhibitory effects on Methylocystaceae, Methylocccaca, and Candidatus Methyluminophilus, with inhibition rates of 5.11%, 3.31%, and 4.47%, respectively. As shown in Fig. 5 -b, 14 days after the application of tillering fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 1.83% and 10.03%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 2.79%; In the W treatment, the application of DMPP had an inhibitory effect on Methylococcaceae with an inhibition rate of 5.44%, and a promoting effect on Methylococcaceae and Candidatus Methyluminophilus with an increase rate of 7.22% and 32.89%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 12.82% and 1.74%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 17.89%. As shown in Fig. 5 -c, 14 days after applying ear fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 10.11% and 7.02%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 5.47%; In the W treatment, the application of DMPP had an inhibitory effect on Methylocystiaceae with an inhibition rate of 26.79%, and a promoting effect on Methylocystiaceae and Candidatus Methyluminophilus with an increase rate of 35.08% and 5.39%, respectively; In treatment C, the application of DMPP had an inhibitory effect on Methylococcaceae with an inhibition rate of 5.52%, and a promoting effect on Methylococcaceae and Candidatus Methyluminophilus with an increase rate of 6.89% and 101.63%, respectively. 2.3 Correlation analysis between methane producing bacteria, oxidizing bacteria, SOC, and DOC As shown in Fig. 6 , there are differences in the correlation between rhizosphere soil SOC, DOC, and mcrA gene abundance under the conditions of applying and not applying DMPP. Under the condition of not applying DMPP, the SOC and DOC of rhizosphere soil were positively correlated with the abundance of mcrA gene after applying basal fertilizer; After applying tillering fertilizer, the soil SOC in the rhizosphere was positively correlated with the abundance of mcrA gene, while DOC was negatively correlated with the abundance of mcrA gene; After applying panicle fertilizer, the soil SOC and DOC in the rhizosphere were positively correlated with the abundance of mcrA gene. Under the condition of DMPP application, there is a positive correlation between soil organic carbon (SOC) and mcrA gene abundance in the rhizosphere soil after basal fertilizer application, while there is a negative correlation between soil organic carbon (DOC) and mcrA gene abundance; After applying tillering fertilizer, the soil SOC and DOC in the rhizosphere were negatively correlated with the abundance of mcrA gene; After applying panicle fertilizer, the soil SOC and DOC in the rhizosphere were positively correlated with the abundance of mcrA gene. As shown in Fig. 7 , there are differences in the correlation between rhizosphere soil SOC, DOC, and pomA gene abundance under the conditions of applying and not applying DMPP. Under the condition of not applying DMPP, the abundance of SOC, DOC, and pomA genes in rhizosphere soil was positively correlated after applying basal fertilizer; After applying tillering fertilizer, the soil SOC in the rhizosphere was positively correlated with the abundance of pomA genes, while DOC was negatively correlated with the abundance of pomA genes; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying panicle fertilizer. Under the condition of DMPP application, the abundance of soil organic carbon (SOC), dissolved organic carbon (DOC), and pomA genes in the rhizosphere soil was positively correlated after the application of basal fertilizer; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying tillering fertilizer; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying panicle fertilizer. Discussions 3.1 Effects of DMPP on methanogenic bacteria under different moisture management Field management affects the community structure and composition of methane producing bacteria in rice soil, with water management being particularly critical. Alternating wet dry irrigation increases soil permeability, reduces anaerobic environment, and inhibits the growth of methane producing bacteria; Wet irrigation promotes beneficial microbial activity by improving soil aeration and physicochemical properties [ 33 – 36 ] . Long term flooding irrigation significantly increases the abundance of mcrA genes in methane producing bacteria, especially during the jointing stage [ 37 – 38 ] .Unlike previous findings, we found that, under the condition of not applying DMPP, both treatment A and treatment W were able to inhibit the growth of methane-producing bacteria before the application of panicle fertilizer, suggesting that the combined effects of these two treatments may offer a more pronounced inhibition of methane-producing bacteria.. Treatment A had the highest mcrA gene abundance after applying panicle fertilizer, and treatment W also had the highest mcrA gene abundance after applying panicle fertilizer. Moreover, treatment W had the highest mcrA gene abundance after applying panicle fertilizer throughout the entire rice growth period; In the C treatment, the abundance of mcrA gene was highest after applying tiller fertilizer, and lowest after applying panicle fertilizer. There is controversy over the effects of nitrification inhibitors on methane producing bacteria. Studies have shown that DCD first promotes and then inhibits the growth of methane producing bacterial communities, while the combined application of CP continuously inhibits the growth of methane producing bacterial communities [ 10 ] . Liu Zhaobing et al. [ 39 ] found that during the peak tillering stage of rice, the application of DCD significantly increased the abundance of methane producing bacterial communities, while the application of CP significantly reduced the abundance of methane producing bacterial communities. In this study, after the application of DMPP, the abundance of mcrA gene reached its maximum after the application of tillering fertilizer, decreased to its minimum after the application of panicle fertilizer, and the C treatment showed the largest decrease, indicating that nitrification inhibitors have a significant inhibitory effect on methane producing bacteria after the application of tillering fertilizer. Methanogenic bacteria are influenced by environmental factors such as soil physical and chemical properties. For example, in nitrogen enriched environments, an increase in litter can slow down the limiting effect of carbon on microorganisms and enhance the activity of methanogenic bacteria [ 19 ] . Chen Zhongyun [ 40 ] , Yuan Jing et al. [ 31 ] found that different types of organic fertilizers can significantly alter the dominant microbial community structure of methane producing bacteria, leading to changes in community composition. This study found that six major methane producing bacteria were detected at the family level, with Methanosaetaceae being the main community. The relative abundance changes of Methanosaetaceae varied at different treatment and fertilization stages, reflecting the complex impact of field management measures on methane producing bacterial communities. When DMPP was not applied, Methanosaetaceae showed a decrease followed by an increase during the growth phase in treatment A, a sustained decrease in treatment W, and an increase followed by a decrease in treatment C. After the application of DMPP, the trend of Methanosaetaceae changes was similar in each treatment, but the specific abundance values were different, indicating that nitrification inhibitors and irrigation methods jointly affect the methanogenic bacterial community. In addition, Methanosarcinaceae and Methanoshellaceae also play an important role in different treatment and fertilization stages, and their relative abundance changes further confirm the profound impact of field management on the community structure of methane producing bacteria. 3.2 Effect of DMPP on methane oxidizing bacteria under different water management CH 4 oxidation is the result of the activity of methane oxidizing bacteria under aerobic conditions, mainly occurring in the soil surface and rhizosphere oxidation microdomains. Soil moisture status is one of the important factors affecting the potential for CH 4 oxidation in rice fields [ 41 ] . Before CH 4 is emitted into the atmosphere, most of it is oxidized by methane oxidizing bacteria at the root soil interface and soil water interface [ 3 ] . Alternating wet dry irrigation enhances soil permeability, increases surface oxygen, and promotes the survival of methane oxidizing bacteria; The moist irrigation water layer is relatively thin, which improves soil aeration and enhances microbial activity; However, continuous flooding is not conducive to the growth of methane oxidizing bacteria [ 42 ] . Yue Jin et al. [ 43 ] found that the number of methane oxidizing bacteria gradually increased after rice cultivation and peaked in June before decreasing. Peng Dengyun et al. [ 38 ] found that in flood irrigation treatment, the abundance of pmoA genes reached its peak at the jointing stage, while in controlled irrigation treatment, the abundance of pmoA genes reached its peak at the tillering stage. During the tillering and milk ripening stages, the abundance of pmoA genes in irrigated paddy soil was higher than that in flooded irrigated paddy soil. This study found that under the condition of not applying DMPP, treatment A inhibited the growth of methane oxidizing bacteria, and the abundance of pmoA gene reached its maximum value after applying base fertilizer; Both W and C treatments promote the growth of methane oxidizing bacteria, and the abundance of pmoA genes reaches its maximum value after applying panicle fertilizer. Moreover, the C treatment has the highest pmoA gene abundance throughout the entire rice growth period after applying panicle fertilizer. During the growth period of rice, the application of DCD in mulching rice fields initially inhibited and then promoted the methane oxidizing bacterial community, while the application of CP exhibited a sustained promotion followed by some inhibition on the growth of the methane oxidizing bacterial community [ 10 ] . Research by Liu Zhaobing et al. [ 39 ] has shown that during the peak tillering stage of rice, the application of DCD significantly reduces the abundance of methane oxidizing bacterial communities, while the application of CP significantly increases the abundance of methane oxidizing bacterial communities. This study found that under the condition of DMPP application, treatment A had the highest pmoA gene abundance after applying base fertilizer, while it had the lowest after applying tiller fertilizer; The abundance of pmoA gene was highest in the W and C treatments after applying panicle fertilizer, while it was lowest in both treatments after applying tillering fertilizer, and the C treatment had the lowest abundance throughout the entire rice growth period. At the scientific level, this study detected three major methane oxidizing bacteria in each treatment. Three major methane oxidizing bacteria were detected in different treatments. Under the condition of no DMPP application, Methylocystiaceae was the main community in treatments A and W, while Methylocystiaceae was the main community in the early stage of treatment C. Under the condition of DMPP application, the community structure of methane oxidizing bacteria in each treatment changed with the fertilization stage. Methane oxidizing bacteria are influenced by environmental factors such as soil physical and chemical properties. Relevant studies have shown that soil moisture content is the main factor in CH 4 oxidation, and low soil moisture can cause water stress and affect microbial activity, thereby affecting CH 4 oxidation. 3.3 Effects of organic carbon on methane producing and oxidizing bacteria The effects of soil organic carbon (SOC) and water-soluble organic carbon (DOC) on methane producing and methane oxidizing bacteria are complex. Soil organic carbon, as an important component of soil ecosystems, provides abundant substrates for methane producing bacteria and is an important material basis for methane production. Methanogenic bacteria, as a strictly anaerobic microbial group, utilize these organic carbon sources to generate methane gas through a series of biochemical reactions. The increase in soil organic carbon content usually promotes the activity of methanogenic bacteria, thereby increasing methane emissions. Related studies have shown that water-soluble organic carbon, as a part of soil organic carbon that is easily utilized by microorganisms, is also of great significance for methane oxidizing bacteria. Water soluble organic carbon provides the necessary carbon source and energy for methane oxidizing bacteria, promoting their growth and activity. Increasing the content of water soluble organic carbon helps to enhance the oxidation ability of methane oxidizing bacteria, thereby reducing methane emissions [ 44 – 48 ] . Water management is one of the important factors affecting the variation of soil organic carbon (SOC) content in paddy fields. It changes crop growth status by altering soil environment and physicochemical properties, thereby affecting soil microbial community structure and ultimately altering SOC content [ 49 ] . This study found a significant positive correlation between methane producing bacteria and soil environmental factors such as organic carbon, as well as between the abundance of mcrA gene and SOC. Under the conditions of DMPP application, SOC had a significant impact on the community structure of methanogenic bacteria in treatment A, while DOC had a significant impact on the community structure of methanogenic bacteria in treatment C. The relationship between methane oxidizing bacteria and soil environmental factors, and the negative correlation between pmoA gene abundance and soil SOC and DOC. Under the condition of not applying DMPP, SOC has a significant impact on the community structure of methane oxidizing bacteria in treatment A, DOC has a significant impact on the community structure of methane oxidizing bacteria in treatment C, and DOC has a significant impact on the community structure of methane oxidizing bacteria in treatment W. Under the condition of DMPP application, SOC has a significant impact on the community structure of methane oxidizing bacteria in treatment A, as evidenced by signifianct increasein pmoA gene abundance and mcrA gene abundance after applying panicle fertilizer. Conclusion Coupling alternate wetting-drying with DMPP application reduces methanogen activity by 197–570% post-panicle while enhancing methanotrophs by 23–35%. These practices disrupt SOC-driven methanogenesis and promote CH₄ oxidation, offering a dual strategy for emission mitigation. Future studies should quantify long-term carbon sequestration under these regimes. Declarations CRediT authorship contribution statement Wenli Cui:Investigation, Writing-original draft, Writing-review &editing.Wenjun Jin:Methodology,Data curation,Formal analysis. Ning Zhou: Data curation. ZongXiang Zhang: Visualization. Zhaorong Dong: Conceptualization. He Song: Funding acquisition, Supervision.Xiaoxiao Li: Writing - review & editing. Funding Declaretion This work was supported by the Open Fund Project of Anhui Provincial Key Laboratory of Farmland Ecological Conservation and Pollution Prevention and Control (FEPP202202); Research and development project on high-yield cultivation technology of climate smart rice wheat system along the Huai River (FYHT20230036). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ ence the work reported in this paper. Author Introduction: Cui Wenli, born in 1983, holds a PhD and is an associate researcher. He mainly engages in research on crops cultivation and quality improvement, E-mail： [email protected] . Jin Wenjun,PhD, Crop Institute of Anhui Academy of Agricultural Sciences,E-mail： [email protected] Ning Zhou，graduate student，E-mail： [email protected] Zhang，graduate student，E-mail： [email protected] Dong，graduate student，E-mail： [email protected] Song，graduate student，E-mail： [email protected] Corresponding autho r:Li Xiaoxiao, female, Han, PhD, lecturer, mainly engaged in high-yield and high-efficiency cultivation of rice, E-mail： [email protected] . References Qin Yanmei. 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Research progress on influencing factors of methane emissions from paddy fields [J]. Chin. J. Agricultural Meteorol. 14 (4), 48–53 (1993). Xingjian, S. & Mingxing, W. Shen Renxing The emission pattern of methane from paddy fields [J]. Adv. Earth Sci. , (5): 23–36. (1993a). Xingjian, S. & Mingxing, W. Chen Dezhang, etc Transport of CH 4 in Rice Fields [J]. Adv. Earth Sci. , (5): 13–21. (1993b). Wu Ne, H. et al. Microbial effects of rice field water management and straw returning on methane emissions [J]. J. Agricultural Eng. 32 (2), 69–76 (2016). Dengyun, P. & Shihong, Y. Li Weizheng, etc The effect of biochar application on methane producing and oxidizing bacteria in water-saving irrigated rice fields [J] Water saving irrigation, (5): 54–59. (2022). Liu Zhaobing, J., Xionghui, P. & Hua etc The effect of nitrogen application rate and inhibitor ratio on long-term greenhouse gas emissions of double cropping rice [J] Journal of Ecological Environment, 19 (04): 919–925. (2010). Chen Zhongyun, M. et al. Study on the correlation between the number of methane oxidizing bacteria and methane producing bacteria in different paddy soils and methane emissions [J]. Chin. J. Ecol. 21 (9), 1498–1505 (2001). Li, X. et al. Effects of Water Management on CH 4 Emissions during Rice Growth [J]. J. Soil Sci. , (2): 238–242. (2007). Liu Jinjian. Study on methane emissions from double cropping red soil rice fields in Hunan Province under long-term different fertilization systems [D] (Nanjing Agricultural University, 2008). Yue Jin, H., Guohong, L. & Wei etc The relationship between CH 4 and N 2 O emissions and microbial communities in paddy soil under different water management conditions [J]. J. Appl. Ecol. , (12): 2273–2277. (2003). Dong Linlin, Y. & Hao Yu Dongsheng, etc Comparative Study on Carbon Sequestration Effects of Yellow River Irrigation in Different Types of Soil [J]. J. Soil Sci. 48 , 922–930 (2011). Li Quanqi, C. & Yuhai Yu Shunzhang, etc Dynamic changes in nutrient content of topsoil in farmland under cover and irrigation conditions [J]. J. Soil Water Conserv. 20 , 37–40 (2006). Kelliher, F. M., Condron, L. M., Cook, F. J. & Black, A. Sixty years of seasonal irrigation affects carbon storage in soils beneath pasture grazed by sheep. Agr Ecosyst. Environ[J] . 148 , 29–36 (2012). Houlbrooke, D. J., Littlejohn, R. P., Morton, J. D. & Paton, R. J. Effect of irrigation and grazing animals on soil quality measurements in the North Otago Rolling Downlands of New Zealand. Soil. Use Manage[J] . 24 , 416–423 (2008). Presley, D. R., Ransom, M. D., Kluitenberg, G. J. & Finnell, P. R. Effects of thirty years of irrigation on the genesis and morphology of two semiarid soils in Kansas. Soil. Sci. Soc. Am. J[J] . 68 , 1916–1926 (2004). Li, F. & Guangshuai, Z. Li Yunsheng, etc Research progress on the impact of irrigation on soil organic carbon in farmland [J]. J. Ecol. Environ. 21 , 1905–1910 (2012). Additional Declarations No competing interests reported. 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7218479","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":506194675,"identity":"191e32db-4c0d-4f62-9c8a-598659bf27cc","order_by":0,"name":"Wenli Cui","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenli","middleName":"","lastName":"Cui","suffix":""},{"id":506194676,"identity":"7507af72-ae00-4dc8-91a8-e958bec83e15","order_by":1,"name":"Wenjun Jin","email":"","orcid":"","institution":"Crop Institute of Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wenjun","middleName":"","lastName":"Jin","suffix":""},{"id":506194677,"identity":"b59a1454-12e4-488f-a0a0-0a380d8ef989","order_by":2,"name":"Ning Zhou","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Zhou","suffix":""},{"id":506194678,"identity":"6c91e7f1-785b-4fe0-a0db-33f17f4cb9e4","order_by":3,"name":"ZongXiang Zhang","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"ZongXiang","middleName":"","lastName":"Zhang","suffix":""},{"id":506194679,"identity":"5f20f1cf-e876-494f-b8c6-e856a09e0fce","order_by":4,"name":"Zhaorong Dong","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhaorong","middleName":"","lastName":"Dong","suffix":""},{"id":506194680,"identity":"d8f79e31-8bc5-4013-bd56-a64d2627a025","order_by":5,"name":"He Song","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Song","suffix":""},{"id":506194681,"identity":"0835c983-5a37-49a6-9096-7c4a31832885","order_by":6,"name":"Xiaoxiao Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACNvnDBw58MJCol2dmPkCcFj4JtsSDMypsEgzb2xKI0yInwaN8mOdMWgLDmTMGRDpMuofhAG/b4TzGGTkfb7xhsJPTbSCkRebsgQOSbYeL2SVyN1vOYUg2NjtASAtDXsIBw7bDjI0zcrdJ8zAcSNxGWEuOwYFEoJaGGznPiNQiAdRy4ExaYsOZM2xEauE5lnCwocLGGBjIxpZzDIjwi3x78+HPfwwk5IBR+fDGmwo7OYJaUIAED5FRg6yFVB2jYBSMglEwIgAAATxH2aCC67kAAAAASUVORK5CYII=","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoxiao","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-26 04:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7218479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7218479/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90313131,"identity":"d4cf707e-ea91-482d-8061-c53c225f2a33","added_by":"auto","created_at":"2025-09-01 10:05:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96376,"visible":true,"origin":"","legend":"\u003cp\u003eDesign drawing of root box\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/a8d9f921ea8fdd6a95eb7cd9.png"},{"id":90315679,"identity":"76137ce9-5104-49c3-be01-d0fa995170c3","added_by":"auto","created_at":"2025-09-01 10:13:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146812,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DMPP under different water management on the abundance of mcrA in rhizosphere soil after applying basal fertilizer, tillering fertilizer, and panicle fertilizer(A: alternate wetting and drying irrigation; W: wetting irrigation; C: continuous flooding irrigation; D: DMPP; Dates presented as means ± standard error.Different lowercase letters indicate significant difference at 0.05 level among treatments.)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/820028d687674895e0c64ea0.jpeg"},{"id":90313132,"identity":"b6a8839c-d970-4da0-988b-482f18d51a5e","added_by":"auto","created_at":"2025-09-01 10:05:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188447,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of DMPP under different water management on the abundance of pmoA in rhizosphere soil after applying basal fertilizer, tillering fertilizer, and panicle fertilizer(A: alternate wetting and drying irrigation; W: wetting irrigation; C: continuous flooding irrigation; D: DMPP; Dates presented as means ± standard error.Different lowercase letters indicate significant difference at 0.05 level among treatments.)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/8b8433097ab50562f7fc694e.jpeg"},{"id":90313138,"identity":"b1b5f6d4-7254-48b6-b710-79413a4cdb8c","added_by":"auto","created_at":"2025-09-01 10:05:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267326,"visible":true,"origin":"","legend":"\u003cp\u003eMethanogenic community structures from paddy soil at family levelr(A: alternate wetting and drying irrigation; W: wetting irrigation; C: continuous flooding irrigation; D: DMPP)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/ed4d4f2211158bd46245c0bd.jpeg"},{"id":90313134,"identity":"f20ae773-ba00-45eb-a70c-8d2db3c6fb7b","added_by":"auto","created_at":"2025-09-01 10:05:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":213036,"visible":true,"origin":"","legend":"\u003cp\u003eMethanogenic community structures from paddy soil at family level(A: alternate wetting and drying irrigation; W: wetting irrigation; C: continuous flooding irrigation; D: DMPP)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/a483d5a3b0f93c4e6c4d743f.jpeg"},{"id":90313146,"identity":"1435286f-9e73-4e75-9247-d77a5f82d1a9","added_by":"auto","created_at":"2025-09-01 10:05:48","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":426502,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between\u003cem\u003e mcr\u003c/em\u003eA, SOC, and DOC under the condition of DMPP and no DMPP application (A: after applying basal fertilizer; B: after applying tillering fertilizer; C: after applying panicle fertilizer)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/c26cbd35fff8576f7d2cc2cf.jpeg"},{"id":90313153,"identity":"c7841408-6d4a-4434-88bb-fcb3192bd0be","added_by":"auto","created_at":"2025-09-01 10:05:48","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":431931,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between \u003cem\u003epom\u003c/em\u003eA, SOC, and DOC under the condition of DMPP and no DMPP application (A: after applying basal fertilizer; B: after applying tillering fertilizer; C: after applying panicle fertilizer)\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/9c465aceddddc094e3563d95.jpeg"},{"id":93169409,"identity":"03f758c9-81b5-4233-8d0c-9d765f252441","added_by":"auto","created_at":"2025-10-09 18:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2659253,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7218479/v1/f8f340d7-b1a6-4977-9ea2-0970f858e408.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Nitrification Inhibitor Application on Methanogenic and Methanotrophic Activity in Paddy Soil under Water-Saving Irrigation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice paddy soils are recognized as significant sources of atmospheric methane (CH\u003csub\u003e4\u003c/sub\u003e), contributing substantially to global warming \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. CH\u003csub\u003e4\u003c/sub\u003e production in these ecosystems is driven by anaerobic conditions and the availability of organic C, with methanogens decomposing organic matter to produce CH₄. Water management practices, such as continuous flooding, create anaerobic environments conducive to CH₄ production, whereas water-saving irrigation can mitigate emissions. Nitrification inhibitors like DMPP inhibit the conversion of NH₄⁺-N to NO₃⁻-N, indirectly affecting CH₄ dynamics. Methane monooxygenase, responsible for CH₄ oxidation, shares structural similarities with ammonia monooxygenase, leading to competitive interactions between NH₄⁺ and CH₄. Studies have shown that nitrification inhibitors influence CH₄ emissions by modulating methanogen and methanotroph activity. For instance, DMPP application during later rice growth stages may enhance methanotroph abundance, thereby promoting CH₄ oxidation and reducing emissions.\u003c/p\u003e\u003cp\u003eMethanogens thrive in anaerobic environments, and oxygen availability directly impacts their community structure and metabolic activity. Periodic drainage during rice cultivation improves soil aeration, inhibiting methanogens while promoting methanotrophs. Methanotrophs, aerobic bacteria utilizing CH₄ as their sole carbon and energy source, oxidize approximately 80% of CH₄ produced in rice paddies, with only a minor fraction escaping to the atmosphere. Water-saving irrigation enhances soil aeration, favoring methanotroph activity and reducing CH₄ emissions. Soil organic carbon (SOC) and dissolved organic carbon (DOC) are critical determinants of microbial activity and CH₄ emissions. SOC serves as the primary substrate for methanogens, while DOC, though a minor fraction of total soil carbon, plays a vital role in nutrient provision. Water-saving irrigation can reduce DOC content, particularly in low-precipitation years, further influencing CH₄ dynamics.\u003c/p\u003e\u003cp\u003eThis study systematically investigated the effects of water management and DMPP application on methanogen and methanotroph communities in rice soils. By analyzing SOC and DOC interactions with microbial communities, we provide insights into sustainable agricultural practices for mitigating CH₄ emissions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1 Experimental site\u003c/h2\u003e\u003cp\u003eThe experimental site was located at the Agricultural Excellence Garden Experimental Base of Anhui Agricultural University (31\u0026deg;87\u0026prime;N, 117\u0026deg;25\u0026prime;E). The soil used in this study was obtained from a rice-wheat rotation field at the Guohe Experimental Station of Anhui Agricultural University (31\u0026deg;48\u0026prime;N, 117\u0026deg;23\u0026prime;E), featuring a soil type of sandy loam and gleyed paddy soil. Soil samples were collected during the wheat maturity stage, specifically from the 0\u0026ndash;20 cm soil layer, which were then sieved through a 4 mm mesh and homogenized. Nine pre-experimental soil samples were randomly selected and divided into three groups for different purposes: three samples were stored in a \u0026minus;\u0026thinsp;80\u0026deg;C freezer for soil DNA analysis; another three were kept in a 4\u0026deg;C refrigerator for assessing soil microbial activity; and the remaining three samples were air-dried for determining physicochemical properties and conducting mineralization experiments. The basic physicochemical properties of the soil prior to the experiment are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSoil physicochemical properties before experiment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTN \u003c/p\u003e\u003cp\u003e(g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u0026ndash;N\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026ndash;N\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026ndash;N\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAK\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAP\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSOC\u003c/p\u003e\u003cp\u003e(g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eDOC\u003c/p\u003e\u003cp\u003e(mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eEC\u003c/p\u003e\u003cp\u003e(\u0026micro;s cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoil before testing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e201.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e39.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e11.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e71.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e118.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"11\"\u003eNote: TN: total nitrogen; NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u0026ndash;N: soil ammonium content; NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u0026ndash;N: soil nitrate content; NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u0026ndash;N: soil nitrite content; AK: available potassium; AP, available phosphorus; OC: organic carbon; DOC, soil dissolved organic carbon; EC: electrical conductivity.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2 Experimental Design\u003c/h2\u003e\u003cp\u003eThis experiment takes Zhendao 18 (japonica rice Oryza sativa L. subsp. japonica) as the research object, and the seedling cultivation method is dry cultivation. Select seedlings with consistent growth and transplant them manually, with 3 seedlings transplanted from each root box. Sow and raise seedlings on May 12, 2021, transplant seedlings on June 23, and harvest rice grains on October 6. Water management is described in section 2.2.2. Based on the local soil source conditions, apply 300 N kg hm-2 of pure nitrogen in three stages, with a base fertilizer: tiller fertilizer: ear fertilizer ratio of 4:3:3. Apply 135 P2O5 kg hm-2 of pure phosphorus and 270 K2O kg hm-2 of pure potassium, both as base fertilizers mixed into the soil at once. Diseases and pests are controlled according to the field conditions.\u003c/p\u003e\u003cp\u003eThis experiment adopts the root box method, and the root box design is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The root box is made of PVC pipe and is divided into two parts. The periphery is the non rhizosphere area, and the middle part is the rhizosphere area. The non rhizosphere and rhizosphere soil are separated by a 30 \u0026micro;m nylon mesh, which can effectively prevent the root system from crossing without affecting water and nutrient exchange.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the preliminary investigation data of soil bulk density in the 0-20cm soil layer of paddy fields (taken from fields near the cultivation soil) by the research team, the soil bulk density in the root box was determined to be 1.11 g cm-3 (n\u0026thinsp;=\u0026thinsp;14). Based on the soil bulk density, calculate the mass of soil in the 0-20cm soil layer, fill the mixed soil into the root box, and compact it to the corresponding soil bulk density. Bury the root box in a 20cm deep soil layer to prevent drastic changes in soil temperature inside the root box. Use 40 root boxes for each treatment. To prevent the impact of rainfall on the experiment, plastic film is used to construct rain blocking facilities above the experimental area.\u003c/p\u003e\u003cp\u003eThis study adopts a two factor experimental design. Factor 1 is water treatment, and three types of water treatments were set up in the experiment, namely continuous flooding irrigation (C), wetting irrigation (W), and alternative wetting and drying irrigation (A). Combining the research methods of Carrijo, Li Yongsong, Xu Fenfen, etc., the following three types of water were set for continuous flooding irrigation. Except for the drying of soil moisture during the roasting period and one week before harvest, the soil water layer was maintained at around 3cm for the rest of the time. Moist irrigation involves keeping the soil moist for the rest of the time, except during the roasting period and one week before harvest. In order to better control the moisture state of moist irrigation, the soil water level should be kept between \u0026minus;\u0026thinsp;1-1cm, that is, when the water level drops to -1cm, water should be poured to 1cm, and when the soil moisture freely dries to -1cm, water should be poured again. This cycle is repeated. Alternating wet dry irrigation begins after the rice seedlings turn green, maintaining the soil water level between \u0026minus;\u0026thinsp;10cm and 3cm. When the water level drops to -10cm, water up to 3cm, wait for the water to freely dry up to -10cm, and then water up to 3cm again. This cycle continues. The soil water level is observed by burying a water level tube in the root box.\u003c/p\u003e\u003cp\u003eFactor 2 is whether to apply 3,4-dimethylpyrazole phosphate (DMPP). DMPP is provided by Shanghai Xianding Biotechnology Co., Ltd., with a purity of \u0026ge;\u0026thinsp;97%, and is calculated as 97% when used. The DMPP treatment was divided into two groups, one group was treated with DMPP in stages along with base fertilizer, tiller fertilizer, and ear fertilizer, while the other group was not treated with DMPP. The application rate of DMPP is 2% of nitrogen fertilizer, which meets the experimental requirements.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.3 Sampling and Indicator Measurement\u003c/h2\u003e\u003cp\u003eSoil sampling and pretreatment were carried out on the 14th day after each fertilization and at the maturity stage, respectively. Samples were taken on the 14th day after rice transplantation (14th day after applying base fertilizer and greening stage), 28th day (14th day after applying tillering fertilizer and tillering stage), 67th day (14th day after applying panicle fertilizer and heading stage), and 105th day (maturity stage). Randomly select five replicates for backup each time, and conduct destructive sampling on at least three of them. When taking soil samples, distinguish between rhizosphere and non rhizosphere soils. Take the soil sample back to the laboratory, sieve it through a 2mm sieve, remove impurities, and mix well. Divide the mixed soil sample into three parts, with one part stored at -80 ℃ for soil microbial functional gene analysis; Part of it is stored in a 4 ℃ refrigerator for testing soil microbial activity and soil physicochemical properties; Part of it is air dried and used for soil physical and chemical property testing. According to Bao Shidan's \"Soil Agrochemical Analysis\", soil organic carbon is determined by potassium dichromate volumetric method. Soil water-soluble organic carbon was extracted by 0.5 mol L-1 K2SO4 solution (soil water ratio 1:2.5) and determined by potassium dichromate digestion method.\u003c/p\u003e\u003cp\u003eSoil DNA was extracted using the Fast DNA SPIN kit for soil (USA). According to the reagent kit manual, weigh 0.5 g of soil sample frozen at -80 ℃ and complete the DNA extraction of the sample on an ultra clean workbench. The extracted DNA sample is stored in an ultra-low temperature freezer at -80 ℃. Quantitative analysis was performed on the functional genes (mcrA) and methane oxidizing bacteria (pmoA) of methanogens using a fluorescence quantitative PCR instrument (Stratagene Mx3000P, Agilent, USA). Primer information is shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Fluorescence quantitative PCR reaction system for all functional genes: SYBR \u0026reg; Premix Ex Taq \u0026trade; II (TakaRa, Japan) 10 \u0026micro; L, primer 0.5 \u0026micro; L each, DNA extraction 0.5 \u0026micro; L; Add sterile water to 20 \u0026micro; L. PCR program: 95 ℃ for 10 minutes; 95 ℃ for 20 seconds, 60 ℃ for 30 seconds (45 cycles); 4 ℃, \u0026infin;. The qPCR amplification efficiency of mcrA and pmoA functional genes was 97%.\u003c/p\u003e\u003cp\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\u003cp\u003eInformation of primers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget gene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer sequence (5'~3')\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmplification length\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003emcrA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eF:5'GGTGGTGTMGGATTCACACARTAYGC 3'\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e467\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR: 5' TTCATTGCRTAGTTWGGRTAGTT 3'\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003epmoA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eF: 5'\u003c/em\u003e GGNGACTGGGACTTCTGG \u003cem\u003e3'\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e510\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eR: 5'\u003c/em\u003e CCGGMGCAACGTCYTTACC \u003cem\u003e3'\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e1.4 Statistical analysis\u003c/h2\u003e\u003cp\u003eData processing utilized Excel 2023, SPSS 27, and R 4.1.2. Microbial community structures were analyzed at family-level taxonomy after removing OTUs with \u0026lt;\u0026thinsp;1% abundance. Treatment effects were assessed via ANOVA with Tukey's post-hoc test (α\u0026thinsp;=\u0026thinsp;0.05). Correlations between SOC/DOC and microbial indices were evaluated using Pearson coefficients.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Abundance of methane producing and oxidizing bacteria in rice soil\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 14 days after the application of basal fertilizer, under the condition of no DMPP, the abundance of mcrA gene in W treatment was significantly higher than that in A treatment. In treatments A and W, the application of DMPP reduced the abundance of the mcrA gene, while in treatment W, DMPP inhibited the growth of the mcrA gene, with an inhibition rate of 28.82%; In treatment C, the application of DMPP increased the abundance of mcrA gene by 21.04%.\u003c/p\u003e\u003cp\u003eFor the rhizosphere soil 14 days after application of tillering fertilizer, water management had a significant effect on the abundance of mcrA genes in the rhizosphere soil without DMPP, with the order of mcrA gene abundance being C\u0026thinsp;\u0026gt;\u0026thinsp;W\u0026thinsp;\u0026gt;\u0026thinsp;A. The application of DMPP significantly promoted the growth of mcrA gene in rhizosphere soil treated with A, while inhibiting the growth of mcrA gene in rhizosphere soil treated with C, with an inhibition rate of 12.50%. In the W treatment, the application of DMPP increased the abundance of mcrA gene by 24.78%.\u003c/p\u003e\u003cp\u003eFor the rhizosphere soil 14 days after applying ear fertilizer, under the condition of not applying DMPP, water management has a significant effect on the abundance of mcrA genes in the rhizosphere soil, with the order of mcrA gene abundance being W\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;\u0026gt;\u0026thinsp;C; The abundance of mcrA gene in W treatment was the highest, with W treatment having a higher abundance of mcrA gene than A treatment by 20.25% and C treatment by 197.60%, respectively. In treatment A, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 493.42%; In the W treatment, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 569.74%; In treatment C, the application of DMPP significantly inhibited the abundance of mcrA gene, with an inhibition rate of 197.09%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 14 days after the application of basal fertilizer, under the condition of no DMPP, the abundance of pmoA genes in W treatment was significantly higher than that in C treatment, and the order of pmoA gene abundance was W\u0026thinsp;\u0026gt;\u0026thinsp;A\u0026thinsp;\u0026gt;\u0026thinsp;C. In treatments A and C, the application of DMPP significantly increased the abundance of pmoA genes, while in treatment W, DMPP inhibited the growth of pmoA genes with an inhibition rate of 12.78%.\u003c/p\u003e\u003cp\u003eFor the rhizosphere soil 14 days after application of tillering fertilizer, under the condition of no DMPP application, the abundance of pmoA genes in treatment A was significantly lower than that in treatment W and C, and the order of pmoA gene abundance was C\u0026thinsp;\u0026gt;\u0026thinsp;W\u0026thinsp;\u0026gt;\u0026thinsp;A. The application of DMPP significantly promoted the growth of pmoA gene in A treatment. The application of DMPP significantly inhibited the growth of pmoA genes in W and C treatments, with inhibition rates of 27.36% and 81.05%, respectively.\u003c/p\u003e\u003cp\u003eFor the rhizosphere soil 14 days after applying panicle fertilizer, under the condition of not applying DMPP, the abundance of pmoA genes in treatment A was significantly lower than that in treatment W and C, and the order of pmoA gene abundance was C\u0026thinsp;\u0026gt;\u0026thinsp;W\u0026thinsp;\u0026gt;\u0026thinsp;A. In treatment A, the application of DMPP significantly increased the abundance of pmoA genes in the rhizosphere. After the application of DMPP, there was no significant difference between W treatment and C treatment. W treatment promoted the growth of pmoA gene, while C treatment inhibited the growth of pmoA gene, with an inhibition rate of 13.00%.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Community Structure of Methane Producing and Oxidizing Bacteria in Rice Soil\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to ensure the reliability and accuracy of the analysis results, based on previous research, OTUs with abundance values lower than 1% of the total sequencing amount of the entire sample were removed, and then the differences in the number of classification units among different samples were compared by the classification level of each family. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2, at the level of family classification, six major methane producing bacteria were detected after applying basal fertilizer, tillering fertilizer, and ear fertilizer. They were ranked by their proportion as Methanosaetaceae, Methanosarcinaceae, Methanocellaceae, Methanoregiaceae, Methanomasiliicoccaceae, and Methanobacteraceae, among which Methanosaetaceae, Methanosarrcinaceae, and Methanocellaceae accounted for over 20%. In addition, there is another type of methanogenic bacteria belonging to the Methanomicrobiales order that has not been classified by comparison.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-a, in treatment A, the application of DMPP had inhibitory effects on Methanosaetaceae and Methanobacteriaceae, with inhibition rates of 6.30% and 8.89%, respectively. It had a promoting effect on Methanosarcinaceae, Methanocellaceae, Methanoregularity, and Methanomasilicaceae, with enhancement rates of 13.87%, 4.68%, 18.54%, and 20.41%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanolegulaceae, Methanomassilicoccaceae, and Methanobacteriaceae, with inhibition rates of 17.55%, 12.99%, and 74.17%. It had a promoting effect on Methanosaetaceae, Methanosarcinaceae, and Methanocellaceae, with enhancement rates of 1.52%, 11.40%, and 1.52%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosaetaceae, Methanosarcinaceae, and Methanobacteriaceae, with inhibition rates of 8.47%, 2.14%, and 121.45%. It had a promoting effect on Methanoceraceae, Methanomasiliicoccaceae, and Methanocellaceae, with enhancement rates of 22.68%, 6.37%, and 5.02%.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-b, in treatment A, the application of DMPP had inhibitory effects on Methanoceraceae and Methanobacteriaceae, with inhibition rates of 4.12% and 35.66%, and promoting effects on Methanosaetaceae, Methanoarcinaceae, Methanocellaceae, and Methanomasilicaceae, with enhancement rates of 9.17%, 12.63%, 1.09%, and 22.10%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanoceraceae and Methanomasiliicoccaceae, with inhibition rates of 24.49% and 59.99%, and promoting effects on Methanosaetaceae, Methanoarcinaceae, Methanocellaceae, and Methanobacteriaceae, with enhancement rates of 3.67%, 1.28%, 29.06%, and 29.95%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosaetaceae and Methanomasiliicoccaceae, with inhibition rates of 44.05% and 33.58%, respectively. It had a promoting effect on Methanosarcinaceae, Methanocellaceae, Methanoregularity, and Methanobacteriaceae, with enhancement rates of 11.96%, 14.55%, 29.43%, and 65.78%, respectively.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e-c, in treatment A, the application of DMPP had inhibitory effects on Methanosaetaceae, Methanosarcinaceae, and Methanocellaceae, with inhibition rates of 9.08%, 10.47%, and 10.71%, respectively. It had a promoting effect on Methanoregularity, Methanobacteriaceae, and Methanomasilicaceae, with enhancement rates of 1.04%, 48.79%, and 90.53%, respectively; In the W treatment, the application of DMPP had inhibitory effects on Methanosarcinaceae and Methanoshellaceae, with inhibition rates of 1.22% and 20.53%, respectively. It had a promoting effect on Methanosarcinaceae, Methanosauriculaceae, Methanosobacteriaceae, and Methanosassilicosaceae, with enhancement rates of 17.84%, 4.26%, 15.54%, and 25.50%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methanosarcinaceae, Methanoshellaceae, and Methanosobacteriaceae, with inhibition rates of 4.14%, 16.96%, and 15.24%, respectively. It had a promoting effect on Methanosataceae, Methanosegulaceae, and Methanosassilicosaceae, with enhancement rates of 13.23%, 35.88%, and 1.06%, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn order to ensure the reliability and accuracy of the analysis results, based on previous research, OTUs with abundance values lower than 1% of the total sequencing amount of the entire sample were removed, and then the differences in the number of classification units among different samples were compared by the classification level of each family. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2, at the level of family classification, three types of methane oxidizing bacteria were detected after applying basal fertilizer, tillering fertilizer, and ear fertilizer. They were ranked by their proportion as follows: Methylocystaceae Methylococcaceae、Candidatus Methylumidiphilus。\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-a, 14 days after the application of basal fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylocystiaceae and Candidatus Methyluminophilus, with inhibition rates of 13.97% and 67.20%, respectively. It had a promoting effect on Methylocystiaceae, with an increase rate of 23.74%; In the W treatment, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 2.54% and 13.45%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 4.60%; In treatment C, the application of DMPP had inhibitory effects on Methylocystaceae, Methylocccaca, and Candidatus Methyluminophilus, with inhibition rates of 5.11%, 3.31%, and 4.47%, respectively.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-b, 14 days after the application of tillering fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 1.83% and 10.03%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 2.79%; In the W treatment, the application of DMPP had an inhibitory effect on Methylococcaceae with an inhibition rate of 5.44%, and a promoting effect on Methylococcaceae and Candidatus Methyluminophilus with an increase rate of 7.22% and 32.89%, respectively; In treatment C, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 12.82% and 1.74%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 17.89%.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e-c, 14 days after applying ear fertilizer, in treatment A, the application of DMPP had inhibitory effects on Methylococcaceae and Candidatus Methyluminophilus, with inhibition rates of 10.11% and 7.02%, respectively. It had a promoting effect on Methylococcaceae, with an increase rate of 5.47%; In the W treatment, the application of DMPP had an inhibitory effect on Methylocystiaceae with an inhibition rate of 26.79%, and a promoting effect on Methylocystiaceae and Candidatus Methyluminophilus with an increase rate of 35.08% and 5.39%, respectively; In treatment C, the application of DMPP had an inhibitory effect on Methylococcaceae with an inhibition rate of 5.52%, and a promoting effect on Methylococcaceae and Candidatus Methyluminophilus with an increase rate of 6.89% and 101.63%, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Correlation analysis between methane producing bacteria, oxidizing bacteria, SOC, and DOC\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, there are differences in the correlation between rhizosphere soil SOC, DOC, and mcrA gene abundance under the conditions of applying and not applying DMPP. Under the condition of not applying DMPP, the SOC and DOC of rhizosphere soil were positively correlated with the abundance of mcrA gene after applying basal fertilizer; After applying tillering fertilizer, the soil SOC in the rhizosphere was positively correlated with the abundance of mcrA gene, while DOC was negatively correlated with the abundance of mcrA gene; After applying panicle fertilizer, the soil SOC and DOC in the rhizosphere were positively correlated with the abundance of mcrA gene. Under the condition of DMPP application, there is a positive correlation between soil organic carbon (SOC) and mcrA gene abundance in the rhizosphere soil after basal fertilizer application, while there is a negative correlation between soil organic carbon (DOC) and mcrA gene abundance; After applying tillering fertilizer, the soil SOC and DOC in the rhizosphere were negatively correlated with the abundance of mcrA gene; After applying panicle fertilizer, the soil SOC and DOC in the rhizosphere were positively correlated with the abundance of mcrA gene.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, there are differences in the correlation between rhizosphere soil SOC, DOC, and pomA gene abundance under the conditions of applying and not applying DMPP. Under the condition of not applying DMPP, the abundance of SOC, DOC, and pomA genes in rhizosphere soil was positively correlated after applying basal fertilizer; After applying tillering fertilizer, the soil SOC in the rhizosphere was positively correlated with the abundance of pomA genes, while DOC was negatively correlated with the abundance of pomA genes; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying panicle fertilizer. Under the condition of DMPP application, the abundance of soil organic carbon (SOC), dissolved organic carbon (DOC), and pomA genes in the rhizosphere soil was positively correlated after the application of basal fertilizer; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying tillering fertilizer; There is a negative correlation between the abundance of SOC, DOC, and pomA genes in the rhizosphere soil after applying panicle fertilizer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussions","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Effects of DMPP on methanogenic bacteria under different moisture management\u003c/h2\u003e\u003cp\u003eField management affects the community structure and composition of methane producing bacteria in rice soil, with water management being particularly critical. Alternating wet dry irrigation increases soil permeability, reduces anaerobic environment, and inhibits the growth of methane producing bacteria; Wet irrigation promotes beneficial microbial activity by improving soil aeration and physicochemical properties \u003csup\u003e[\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Long term flooding irrigation significantly increases the abundance of mcrA genes in methane producing bacteria, especially during the jointing stage \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.Unlike previous findings, we found that, under the condition of not applying DMPP, both treatment A and treatment W were able to inhibit the growth of methane-producing bacteria before the application of panicle fertilizer, suggesting that the combined effects of these two treatments may offer a more pronounced inhibition of methane-producing bacteria.. Treatment A had the highest mcrA gene abundance after applying panicle fertilizer, and treatment W also had the highest mcrA gene abundance after applying panicle fertilizer. Moreover, treatment W had the highest mcrA gene abundance after applying panicle fertilizer throughout the entire rice growth period; In the C treatment, the abundance of mcrA gene was highest after applying tiller fertilizer, and lowest after applying panicle fertilizer.\u003c/p\u003e\u003cp\u003eThere is controversy over the effects of nitrification inhibitors on methane producing bacteria. Studies have shown that DCD first promotes and then inhibits the growth of methane producing bacterial communities, while the combined application of CP continuously inhibits the growth of methane producing bacterial communities \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Liu Zhaobing et al. \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e found that during the peak tillering stage of rice, the application of DCD significantly increased the abundance of methane producing bacterial communities, while the application of CP significantly reduced the abundance of methane producing bacterial communities. In this study, after the application of DMPP, the abundance of mcrA gene reached its maximum after the application of tillering fertilizer, decreased to its minimum after the application of panicle fertilizer, and the C treatment showed the largest decrease, indicating that nitrification inhibitors have a significant inhibitory effect on methane producing bacteria after the application of tillering fertilizer.\u003c/p\u003e\u003cp\u003eMethanogenic bacteria are influenced by environmental factors such as soil physical and chemical properties. For example, in nitrogen enriched environments, an increase in litter can slow down the limiting effect of carbon on microorganisms and enhance the activity of methanogenic bacteria \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Chen Zhongyun \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, Yuan Jing et al. \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e found that different types of organic fertilizers can significantly alter the dominant microbial community structure of methane producing bacteria, leading to changes in community composition. This study found that six major methane producing bacteria were detected at the family level, with Methanosaetaceae being the main community. The relative abundance changes of Methanosaetaceae varied at different treatment and fertilization stages, reflecting the complex impact of field management measures on methane producing bacterial communities.\u003c/p\u003e\u003cp\u003eWhen DMPP was not applied, Methanosaetaceae showed a decrease followed by an increase during the growth phase in treatment A, a sustained decrease in treatment W, and an increase followed by a decrease in treatment C. After the application of DMPP, the trend of Methanosaetaceae changes was similar in each treatment, but the specific abundance values were different, indicating that nitrification inhibitors and irrigation methods jointly affect the methanogenic bacterial community. In addition, Methanosarcinaceae and Methanoshellaceae also play an important role in different treatment and fertilization stages, and their relative abundance changes further confirm the profound impact of field management on the community structure of methane producing bacteria.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Effect of DMPP on methane oxidizing bacteria under different water management\u003c/h2\u003e\u003cp\u003eCH\u003csub\u003e4\u003c/sub\u003e oxidation is the result of the activity of methane oxidizing bacteria under aerobic conditions, mainly occurring in the soil surface and rhizosphere oxidation microdomains. Soil moisture status is one of the important factors affecting the potential for CH\u003csub\u003e4\u003c/sub\u003e oxidation in rice fields \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Before CH\u003csub\u003e4\u003c/sub\u003e is emitted into the atmosphere, most of it is oxidized by methane oxidizing bacteria at the root soil interface and soil water interface \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Alternating wet dry irrigation enhances soil permeability, increases surface oxygen, and promotes the survival of methane oxidizing bacteria; The moist irrigation water layer is relatively thin, which improves soil aeration and enhances microbial activity; However, continuous flooding is not conducive to the growth of methane oxidizing bacteria \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eYue Jin et al. \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e found that the number of methane oxidizing bacteria gradually increased after rice cultivation and peaked in June before decreasing. Peng Dengyun et al. \u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e found that in flood irrigation treatment, the abundance of pmoA genes reached its peak at the jointing stage, while in controlled irrigation treatment, the abundance of pmoA genes reached its peak at the tillering stage. During the tillering and milk ripening stages, the abundance of pmoA genes in irrigated paddy soil was higher than that in flooded irrigated paddy soil. This study found that under the condition of not applying DMPP, treatment A inhibited the growth of methane oxidizing bacteria, and the abundance of pmoA gene reached its maximum value after applying base fertilizer; Both W and C treatments promote the growth of methane oxidizing bacteria, and the abundance of pmoA genes reaches its maximum value after applying panicle fertilizer. Moreover, the C treatment has the highest pmoA gene abundance throughout the entire rice growth period after applying panicle fertilizer.\u003c/p\u003e\u003cp\u003eDuring the growth period of rice, the application of DCD in mulching rice fields initially inhibited and then promoted the methane oxidizing bacterial community, while the application of CP exhibited a sustained promotion followed by some inhibition on the growth of the methane oxidizing bacterial community \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Research by Liu Zhaobing et al. \u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e has shown that during the peak tillering stage of rice, the application of DCD significantly reduces the abundance of methane oxidizing bacterial communities, while the application of CP significantly increases the abundance of methane oxidizing bacterial communities. This study found that under the condition of DMPP application, treatment A had the highest pmoA gene abundance after applying base fertilizer, while it had the lowest after applying tiller fertilizer; The abundance of pmoA gene was highest in the W and C treatments after applying panicle fertilizer, while it was lowest in both treatments after applying tillering fertilizer, and the C treatment had the lowest abundance throughout the entire rice growth period.\u003c/p\u003e\u003cp\u003eAt the scientific level, this study detected three major methane oxidizing bacteria in each treatment. Three major methane oxidizing bacteria were detected in different treatments. Under the condition of no DMPP application, Methylocystiaceae was the main community in treatments A and W, while Methylocystiaceae was the main community in the early stage of treatment C. Under the condition of DMPP application, the community structure of methane oxidizing bacteria in each treatment changed with the fertilization stage.\u003c/p\u003e\u003cp\u003eMethane oxidizing bacteria are influenced by environmental factors such as soil physical and chemical properties. Relevant studies have shown that soil moisture content is the main factor in CH\u003csub\u003e4\u003c/sub\u003e oxidation, and low soil moisture can cause water stress and affect microbial activity, thereby affecting CH\u003csub\u003e4\u003c/sub\u003e oxidation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Effects of organic carbon on methane producing and oxidizing bacteria\u003c/h2\u003e\u003cp\u003eThe effects of soil organic carbon (SOC) and water-soluble organic carbon (DOC) on methane producing and methane oxidizing bacteria are complex. Soil organic carbon, as an important component of soil ecosystems, provides abundant substrates for methane producing bacteria and is an important material basis for methane production. Methanogenic bacteria, as a strictly anaerobic microbial group, utilize these organic carbon sources to generate methane gas through a series of biochemical reactions. The increase in soil organic carbon content usually promotes the activity of methanogenic bacteria, thereby increasing methane emissions. Related studies have shown that water-soluble organic carbon, as a part of soil organic carbon that is easily utilized by microorganisms, is also of great significance for methane oxidizing bacteria. Water soluble organic carbon provides the necessary carbon source and energy for methane oxidizing bacteria, promoting their growth and activity. Increasing the content of water soluble organic carbon helps to enhance the oxidation ability of methane oxidizing bacteria, thereby reducing methane emissions \u003csup\u003e[\u003cspan additionalcitationids=\"CR45 CR46 CR47\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWater management is one of the important factors affecting the variation of soil organic carbon (SOC) content in paddy fields. It changes crop growth status by altering soil environment and physicochemical properties, thereby affecting soil microbial community structure and ultimately altering SOC content \u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis study found a significant positive correlation between methane producing bacteria and soil environmental factors such as organic carbon, as well as between the abundance of mcrA gene and SOC. Under the conditions of DMPP application, SOC had a significant impact on the community structure of methanogenic bacteria in treatment A, while DOC had a significant impact on the community structure of methanogenic bacteria in treatment C. The relationship between methane oxidizing bacteria and soil environmental factors, and the negative correlation between pmoA gene abundance and soil SOC and DOC. Under the condition of not applying DMPP, SOC has a significant impact on the community structure of methane oxidizing bacteria in treatment A, DOC has a significant impact on the community structure of methane oxidizing bacteria in treatment C, and DOC has a significant impact on the community structure of methane oxidizing bacteria in treatment W. Under the condition of DMPP application, SOC has a significant impact on the community structure of methane oxidizing bacteria in treatment A, as evidenced by signifianct increasein \u003cem\u003epmoA\u003c/em\u003e gene abundance and \u003cem\u003emcrA\u003c/em\u003e gene abundance after applying panicle fertilizer.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCoupling alternate wetting-drying with DMPP application reduces methanogen activity by 197\u0026ndash;570% post-panicle while enhancing methanotrophs by 23\u0026ndash;35%. These practices disrupt SOC-driven methanogenesis and promote CH₄ oxidation, offering a dual strategy for emission mitigation. Future studies should quantify long-term carbon sequestration under these regimes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenli Cui:Investigation, Writing-original draft, Writing-review \u0026amp;editing.Wenjun Jin:Methodology,Data curation,Formal analysis. Ning Zhou: Data curation. ZongXiang Zhang: Visualization. Zhaorong Dong: Conceptualization. He Song: Funding acquisition, Supervision.Xiaoxiao Li: Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Open Fund Project of Anhui Provincial Key Laboratory of Farmland Ecological Conservation and Pollution Prevention and Control (FEPP202202); Research and development project on high-yield cultivation technology of climate smart rice wheat system along the Huai River (FYHT20230036).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ ence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Introduction:\u0026nbsp;\u003c/strong\u003eCui Wenli, born in 1983, holds a PhD and is an associate researcher. He mainly engages in research on crops \u0026nbsp;cultivation and quality improvement, E-mail： [email protected].\u003c/p\u003e\n\u003cp\u003eJin Wenjun,PhD, Crop Institute of Anhui Academy of Agricultural Sciences,E-mail：[email protected]\u003c/p\u003e\n\u003cp\u003eNing Zhou，graduate student，E-mail：[email protected] Zhang，graduate student，E-mail：[email protected] Dong，graduate student，E-mail：[email protected] Song，graduate student，E-mail：[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding autho\u003c/strong\u003er:Li Xiaoxiao, female, Han, PhD, lecturer, mainly engaged in high-yield and high-efficiency cultivation of rice, E-mail： [email protected] .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eQin Yanmei. \u003cem\u003eStudy on Greenhouse Gas (CH\u003c/em\u003e\u003csub\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sub\u003e \u003cem\u003eand N\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eO) Emissions from Rice Fields and Vegetable Fields under Conventional and Organic Production Methods [D]\u003c/em\u003e (Nanjing Agricultural University, 2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Lihong, N. et al. 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Environ.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 1905\u0026ndash;1910 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Nitrification inhibitor, Water-saving irrigation, Methanogens, Methanotrophs, Paddy soil","lastPublishedDoi":"10.21203/rs.3.rs-7218479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7218479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) modulates methane (CH4) emissions in rice paddies by altering soil biogeochemistry and microbial functionality. This study elucidates the interactive effects of DMPP and water management on methanogen (mcrA) and methanotroph (pmoA) gene abundances across rice growth stages using a rhizobox system. Results revealed that alternate wetting-drying (A) and wetting irrigation (W) suppressed methanogen activity pre-panicle fertilization, whereas DMPP application post-panicle fertilization reduced mcrA abundance by 197\u0026ndash;569% across treatments. Methanotroph activity peaked under continuous flooding (C) without DMPP, while DMPP enhanced pmoA abundance by 23.7\u0026ndash;35.1% under A. Soil organic carbon (SOC) positively correlated with methanogens, whereas methanotrophs exhibited inverse relationships with SOC and dissolved organic carbon (DOC). These findings demonstrate that optimized water regimes coupled with DMPP application can mitigate CH₄ emissions by restructuring microbial communities and carbon dynamics.\u003c/p\u003e\u003cp\u003eThe application of 3,4-dimethylpyrazole phosphate (DMPP) can affect paddy soil methane emissions through soil and microbial properties. However, little is know about the effects of DMPP on methanogens and methanotrophs in rice soils under different water management practices. This study utilized the rhizobox method to quantify the gene abundances of these microbial communities across various growth stages. The results showed that water management practices significantly influenced the community structures and gene abundances of methanogens and methanotrophs. Under alternate wetting and drying (A) and continuous flooding (W), methanogen growth was either inhibited or promoted without DMPP application before and after panicle fertilization. However, DMPP application significantly suppressed methanogen activity after panicle fertilization. For methanotrophs, DMPP application enhanced \u003cem\u003epmoA\u003c/em\u003e gene abundance under A, while W and continuous saturation (C) without DMPP promoted methanotroph growth. Additionally, DMPP markedly altered the relationships between SOC, DOC, and the community structures of methanogens and methanotrophs. Methanogens exhibited a significant positive correlation with SOC, while methanotrophs showed negative correlations with both SOC and DOC. Overall, this study demonstrates that water management practices and DMPP application significantly affect methanogen and methanotroph communities in rice soils, with these changes closely linked to soil physicochemical properties.\u003c/p\u003e","manuscriptTitle":"Effects of Nitrification Inhibitor Application on Methanogenic and Methanotrophic Activity in Paddy Soil under Water-Saving Irrigation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 10:05:43","doi":"10.21203/rs.3.rs-7218479/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":"7e5482a5-14c1-438e-8367-07e8b587c5b7","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53765445,"name":"Earth and environmental sciences/Biogeochemistry"},{"id":53765446,"name":"Biological sciences/Ecology"},{"id":53765447,"name":"Earth and environmental sciences/Ecology"},{"id":53765448,"name":"Earth and environmental sciences/Environmental sciences"},{"id":53765449,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-10-09T18:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 10:05:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7218479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7218479","identity":"rs-7218479","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}