Co-incorporation of manure and inorganic fertilizer improves leaf physiological traits, rice production and soil functionality in a paddy field

The combined use of organic manure and chemical fertilizer (CF) is considered to be a good method for sustaining high crop yields and improving soil quality. We performed a field experiment in 2019 at the research station of Guanxi University, to investigate the effects of cattle manure (CM) and poultry manure (PM) combined with CF on soil physical and biochemical properties, rice dry matter (DM) and nitrogen (N) accumulation and grain yield. We also evaluated differences in pre-and post-anthesis DM and N accumulation and their contributions to grain yield. The experiment consisted of six treatments: no N fertilizer (T1), 100% CF (T2), 60% CM + 40% CF (T3), 30% CM + 70% CF (T4), 60% PM + 40% CF (T5), and 30% PM + 70% CF (T6). All CF and organic manure treatments provided a total N of 150 kg ha−1. Results showed that the treatment T6 increased leaf net photosynthetic rate (Pn) by 11% and 13%, chlorophyll content by 13% and 15%, total biomass by 9% and 11% and grain yield by 11% and 17% in the early and late season, respectively, compared with T2. Similarly, the integrated manure and CF treatments improved post-antheis DM accumulation and soil properties, such as bulk density, organic carbon, total N, microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) relative to the CF-only treatments. Interestingly, increases in post-anthesis DM and N accumulation were further supported by enhanced leaf Pn and activity of N-metabolizing enzyme during the grain-filling period. Improvement in Pn and N-metabolizing enzyme activity were due to mainly improved soil quality in the combined manure and synthetic fertilizer treatments. Redundancy analysis (RDA) showed a strong relationship between grain yield and soil properties, and a stronger relationship was noted with soil MBC and MBN. Conclusively, a combination of 30% N from PM or CM with 70% N from CF is a promising option for improving soil quality and rice yield.

. Changes in soil physical and checmial properties under the combined organic and inorganic N fertilization. Note: T 1 , no N fertilizer, T 2 100% CF, T 3 60% CM + 40% CF, T 4 30% CM + 70% CF, T 5 60% PM + 40% CF, T 6 30% PM + 70% CF, BD bulk density, SOC soil organic carbon, SOM soil organic matter, TN total nitrogen, AP available phosphorous, AK available potassium. Values followed by the same letters, within column, are not significantly different at p ≤ 0.05. Mean values (n = 3) ± SE. ns: non significant, *p < 0.05, **p < 0.05.  Accumulation and translocation of DM and N. Dry matter and N accumulation reflect plant growth and metabolic capacity and ultimately control the economic yield. In the present study, the accumulation of DM and N increased progressively with plant growth and attained maximum values at plant maturity. DM and N accumulation differed significantly among fertilizer treatments and seasons, as shown in Table 2. DM accumulation was 9% and 11% higher in T 6 than in T 2 at maturity in the early and late seasons, respectively, and N accumulation was 10% and 12% higher in T 6 . DM and N accumulation in T 6 were 22% and 20% higher in the late season than in the early season. DM and N did not differ significantly between T 4 and T 6 . The combined treatments T 3 and T 5 also improved DM and N accumulation but did not differ significantly from T 2 . The lowest values of DM and N accumulation were observed in non-N treated plots in both seasons. The combination treatments also showed the highest translocation rates of DM and N accumulated preanthesis (Table 5). Relative to non-N-treated plots, the combination treatments improved DM and N translocation significantly in both seasons.
Post-anthesis DM and N accumulation. The combination of manure and synthetic fertilizer significantly improved post-anthesis DM and N accumulation ( Table 2). Post-anthesis DM accumulation in the CFonly treatment (T 2 ) was 482 and 436 (g m −2 ) in the early and late seasons, respectively, and post-anthesis N accumulation in T 2 was 4.18 and 4.06 (g m −2 ). Post-anthesis DM accumulation was 9% and 14% higher in the T 6 combination treatment than in T 2 in the early and late seasons, respectively, and post-anthesis N accumulation was 10% and 13% higher. T 4 did not differ significantly from T 6 in post-anthesis DM and N accumulation. Treatments T 3 and T 5 also improved post-anthesis DM and N accumulation, and the lowest post-anthesis values were observed in non-N-treated plots.
Rice yield and yield components. Rice grain yield and yield attributes were significantly improved by the combination of organic manure and inorganic N fertilization (Table 3). However, there was no significant difference effects of different season on grain yield and yield components, with the exception of panicles number and 1000 grain weight. The T 6 treatment produced significantly higher panicle number (11% and 14%), grain filling percentage (5% and 7%), 1000-grain weight (6% and 9%), and grain yield (11% and 17%) compared with T 2 in the early and late seasons, respectively. These parameters did not differ significantly between T 4 and T 6 . The  Table 1. www.nature.com/scientificreports/ T 3 and T 5 combination treatments also had higher yields and yield components than non-N-treated plots. The lowest yield and yield components were observed in non-N-treated plots.

Relationships between leaf physiological traits and grain yield. Changes in leaf physiological
traits significantly affect the grain yield of rice. In the present study, linear regression analysis revealed highly significant and strong relationships between leaf physiological attributes and grain yield, as shown in  Fig. 6D) accumulated pre-anthesis were also positively related to grain yield. Finally, linear regression confirmed that post-anthesis DM accumulation was strongly positively correlated with grain yield. Therefore, improvements in post-anthesis DM accumulation contributed significantly to higher grain yield in rice.

Relationships of soil properties with N-metabolizing enzyme activities and grain yield. Changes
in above-average plant yields are highly dependent on fluctuations in soil quality and can be helpful in soil sustainability and stability. In this study, RDA revealed strong positive relationships of N metabolism enzyme activities and grain yield with soil properties and microbial activity (Fig. 7). Soil properties such as pH, SOC, TN, AP, MBC, and MBN showed strong correlations with plant biomass accumulation, rice grain yield and N metabolism enzyme activities, and photosynthetic efficiency during the grain-filling period. However, the correlation of MBN with N metabolism enzyme activity, plant N, and biomass accumulation was significantly higher under organic manure fertilization, presumably as a result of improved soil fertility.

Discussion
Soil physical and chemical properties were significantly improved by the combination of organic and inorganic N fertilization compared with the application of urea fertilizer alone ( Table 1). The combination treatment also resulted in lower soil BD. Reductions in soil BD in the combination treatments were due to the bulky nature of the organic manure, which prevented the soil from separating 34 . Moreover, the use of organic manures has been shown to promote soil aeration and enhance soil aggregation, which leads to a decline in soil BD 35 . Our outcomes are in consistent with Franzluebbers et al. 35 , who concluded that variation in SOC not only directly affects soil BD but also increases soil aggregation and healthy pore growth due to improved soil physicochemical and biological properties. Soil chemical properties such as soil pH, SOC, TN, AN, AK, and AP increased significantly with the combined application of organic and inorganic fertilizers. We noted that the decomposition of organic manure gradually  www.nature.com/scientificreports/ released nutrients into the soil, and increasing the amount of organic fertilizer from 30 to 60% improved soil chemical properties. The use of CF alone reduced soil pH, whereas the combination treatment increased soil pH considerably. Chemical N fertilization may have significantly reduced the exchangeable base cations in the soil, ultimately leading to a decline in soil pH. The use of synthetic N has also been shown to shift soils into the Al 3+ buffering stage 36 . Another possible explanation for increased soil pH in the combination treatments is that organic fertilizer contains enough basic cations and carbonate ions to neutralize the acidification effect 37,38 . Soil C is an important parameter for the evaluation of soil quality and fertility. The substantial improvements in SOC, TN and AN observed in this study may have resulted from both direct C and N inputs from the organic manure and indirect C and N inputs associated with greater crop biomass, such as roots and crop residues 38,39 . Soil organic C at any specific location depends mainly on the seasonal return and recycling of organic materials, roots, and shoot stubbles 40,41 . Our results are in agreement with Purakayastha et al. 42 , who reported that organic manure integrated with chemical fertilizer increased soil TN by 56-90% and SOC by 11-80% in the upper soil layer. The application of organic fertilizers may also have increased soil nutrient availability because the manure absorbed more leachate, improving soil water holding capacity, decreasing nutrient leaching, and ultimately increasing the availability of N, P, and K in the soil 43 .
Higher soil available P in the combination treatments is consistent with the results of P addition from chemical fertilizers, as plants typically use only a portion of the applied P 44 . Likewise, organic manure often provides a large amount of P to the soil and reduces the fixation of added P, resulting in enhanced competition of organic molecules with PO 4 3− ions for P retention sites in the manure treatments 45 . Higher available K in the combination treatments relative to the urea-only treatment may reflect exudation of organic acids during the decomposition process, which releases negative ions that have a preference for di-or trivalent cations (e.g., Al 3+ , Ca 2+ , and Mg 2+ ), leaving K + to be absorbed by negatively charged soil colloids 46 . This phenomenon can help to minimize K fixation and improve soil K availability.
Soil MBC and MBN reflect characteristics of the soil microbial community structure 47 . In the present study, organic manure in combination with CF significantly enhanced soil MBC and MBN (Fig. 1). Increases in MBC and MBN may have occurred because organic fertilizer improved the physicochemical and biological properties of the soil, leading to increased absorption and uptake of mineral N by the crop 48,49 . In addition, manure may have facilitated the conversion of mineral N to MBN and other N forms 50 . Another possible explanation is that the combination treatments may have improved soil fertility and rice biomass production (Tables 4, 5), leading to an increase in crop residues. Such residues are beneficial for the propagation of soil microbes and may therefore facilitate the conversion of C and N 51 . Furthermore, the integration of organic fertilizer with synthetic N is widely accepted as an efficient means of improving soil microbial activity, soil structure, aggregation, and water retention capacity 13,14 .
Leaf chlorophyll (Chl) content is widely used to assess plant photosynthetic health 52 . Chl synthesis and protein are also associated with leaf N concentration, and higher photosynthetic rates promote stem elongation, enhance leaf area expansion, and delay leaf senescence 53 . Leaf Chl content and Pn are directly related to N uptake 54 . Photosynthetic rate responds readily to N and water supply and is the key driver of plant production by enhancing  Table 1. www.nature.com/scientificreports/ growth and biomass 55,56 . In this work, leaf Pn and Chl content were highest in the integration treatments relative to the CF-only application during the grain-filling (Figs. 3 and 4). This increase may have been due to the fact that the combined application of manure and synthetic fertilizer increased soil fertility and quality (Table 4), decreasing the leaching of mineral elements from the upper soil layer and improving the physical structure of the soil, thereby increasingplant nutrient absorption 57 . Another possible reason for improvement in Pn and Chl content during the grain-filling period is the quicker release of nutrients from synthetic fertilizer and accompanied by the slow and steady release of nutrients from organic fertilizer across the entire growth period 20 . Pn and Chl a were always highest in T 6 and lowest in T 1 (Figs. 3 and 4). These findings highlight the importance of Chl a, as it is the primary photosynthetic pigment.
Several key enzymes such as NR, GS, and GOGAT play an important role in plant N assimilation 27 . In this study, higher N-metabolizing enzyme activity was observed in response to the combined application of manure and synthetic fertilizer (Fig. 4). This may have resulted from improved soil quality under the combination treatment (Table 5). Our findings indicated that the combination of organic manure and chemical fertilizer improved N accumulation in leaves more effectively than the traditional urea-only application, and this was necessary to provide adequate grain-filling substrate and promote superior grain yield 58 . Similarly, Gupta et al. 59 also reporteda strong relationship of soil N availibility and N absorption with the activities of key N assimilatory enzymes during grain filling. Our outcomes are consistent with those of Sun et al. 58 , who concluded that GS and GOGAT activities in flag leaves during the grain-filling period were strongly positively associated with grain yield and crop quality.
Delayed leaf senescence supports comparatively high photosynthetic activity and promotes maximum DM production and grain yield; it may be achieved by synchronizing soil N availability and plant N uptake during the grain-filling period 24,25 . In the present study, delayed leaf senescence, high photosynthetic efficacy, and enhanced N-metabolizing enzyme activity were observed during the grain-filling period in the combination treatments  . Changes in N metabolism enzyme activities at 4,1 4, and 24 days after anthesis during grain filling period, NR,GS and GOGAT at early season (A-C-E) and late season (B-D-F) in response to combined organic and inorganic N fertilizer application. Vertical bar represents the standard error of mean. Different letters above the column indicate statistical significance at the (P < 0.05). Note: NR-nitrate reductase, GS-glutamine synthetase, GOGAT-glutamine oxoglutarate aminotransferase. ns: non significant, *p < 0.05, **p < 0.05. For treatment details please see Table 1. www.nature.com/scientificreports/  www.nature.com/scientificreports/ yield. Consistent with our results, many authors have reported that increased N uptake leads to increases in Pn, overall photochemical efficiency of PSII, and leaf physiological activity; this delays leaf senescence in the late growth period and eventually enhances photosynthetic production during the grain-filling stage 59,60 . Cereal crop yields are strongly dependent on post-anthesis DM production and the translocation of DM accumulated prior to anthesis to grain 30 . Pal et al. 30 also concluded that the contribution to grain yield of DM production prior to anthesis ranged from 22 to 69%, depending on rice cultivar and the sowing time. Moreover, Wu et al. 32 stated that variation in rice yield between the early and late growing seasons could be explained primarily by the difference in post-anthesis DM production. In the current study, the combined treatments T 4 and T 6 had the highest values of DMT and accumulated pre-anthesis and post-anthesis DM production, as shown in Table 2. Highest DM and N accumulation under the combination of 30% manure and 70% inorganic fertilizer could be attributed to a high and continuous supply of nutrients due to improved soil fertility ( Table 1). The constant and steady release of N from the cattle and poultry manure, particularly during the grain-filling period, would have encouraged their use by the plant 26 .  www.nature.com/scientificreports/ Linear regression analysis showed that post-anthesis DM accumulation was more strongly positively correlated with grain yield (R 2 = 0.81**, Fig. 6A) than was DMT (R 2 = 0.71*, Fig. 6B). This finding underscored that both processes are important, but post-anthesis DM production plays a more important role in higher grain yield. Similarly, pre-and post-anthesis N accumulation were also highly positively correlated with grain yield: post-anthesis NA (R 2 = 0.73**, Fig. 6C) and NT (R 2 = 0.80**, Fig. 6D). The current study confirms that plants rely primarily on post-anthesis DM production and N accumulation for grain filling. Higher post-anthesis DM production and N accumulation in the combination treatments were due mainly to adequate availability of nutrients, which delayed leaf senescence and increased N remobilization.
Rice grain yield is determined by yield components, including the number of tillers, panicle length, and spikelets per panicle 32,61 . In the present study, the combination of manure and synthetic N fertilizer significantly improved rice growth, yield, and yield components compared to urea fertilization alone ( Table 3). The higher rice growth, yield, and yield traits under the combination treatments could be attributed mainly to a balanced and continuous supply of nutrients due to enhanced soil fertility (Table 4), which ultimately improved plant nutrient uptake and growth. The continued and slow release of N from organic manure, particularly during the grain-filling period, may have enabled its efficient utilization by the crop 41,62 . Moreover, the RDA showed that the x-axis explained 96.3% of the variation, and the y-axis explained 0.03%. It revealed significant positive correlations of grain yield, leaf physiological traits, N metabolism enzyme activities, and dry matter accumulation with all soil properties (Fig. 7).

Conclusion
Application of a combination of organic manure and chemical fertilizer enhanced soil physical and biochemical traits, leaf physiological activity, and rice yields compared with chemical fertilizer alone. The co-incorporation of manure and synthetic fertilizers significantly improved pre-and post-anthesis DM production and N accumulation compared with the application of urea alone. Improvements in DM production and N accumulation were due primarily to improved soil fertility and leaf physiological activity, including Pn, Chl, and the activities of N-metabolizing enzymes, which further increased DM production and N uptake. RDA revealed positive relationships between grain yield and soil properties (i.e., SOC, TN, AN, and AP), and a significantly higher correlation was observed between grain yield and soil MBC and MBN. The combination of organic manure and synthetic fertilizer in a 30:70 ratio is a beneficial and sustainable practice for rice production and soil quality improvement.  Table 4). The soil is classified as an ultisol based on USDA classification. It is slightly acidic with pH 5.94, soil organic carbon (SOC) 14.56 g kg −1 , total N (TN) 1.41 g kg −1 , available phosphorous (P) 23.12 mg kg −1 , available potassium (K) 233.33 mg kg −1 , and a high bulk density (BD) of 1.36 g cm −3 (Table 5).

Materials and methods
Experimental design and field management. The field experiment was performed in a randomized complete block design (RCBD) with three replicates and a plot size of 3.9 m × 6 m (23.4 m 2 ). Cattle manure (CM) and poultry manure (PM) were the organic fertilizers and urea was the chemical fertilizer (CF). The experiment www.nature.com/scientificreports/ consisted of six treatments, i.e., : no N fertilizer (T 1 ); 100% CF (T 2 ); 60% CM + 40% CF (T 3 ); 30% CM + 70% CF (T 4 ); 60% PM + 40% CF (T 5 ), and 30% PM + 70% CF (T 6 ). The noodle rice cultivar "Zhenguiai" was used as the test crop. Rice seeds were sown in an open filed in plastic seedling trays, and urea was applied to the nursery at the time of preparation. The 25 day-old seedlings were transplanted to the field, and two seedlings per hill . The plant-to-plant distance was 10 cm, the row-to-row distance was 30 cm, and the total number of plants in each plot was 780. The recommended dose of NPK was 150:75:150 (kg ha −1 ), and every plot received 175.5 g of P 2 O 5 from superphosphate, 365 g of KCl from potassium chloride, and 351 g of N from PM or CM and CF (urea) after proper N estimation. The net N, P, and K contents in the urea, superphosphate, and potassium chloride was 46%, 20%, and 60%, respectively. The chemical composition of the organic manure and the nutrient content and quantity for each treatment are shown in Tables 5 and 6. N and KCI were applied in three splits as a basal dose (60%), at the early tillering stage (20%), and at panicle initiation (20%). P was delivered as a basal dose one day before transplantation (Table 6).
Organic fertilizer, such as CM and PM were obtained from the cattle and poultry farms, located in the local area. Organic manure applied to plots 20 days prior to transplantation. The T 1 treatment received no N fertilizer but received P and K fertilizers at rates equal to those in N-treated plots. Standard flood water was provided at a depth of approximately 5 cm from transplant to physiological maturity. Normal agricultural practices were used for all treatments, including irrigation (about 5 cm flood water), insecticide application (chlorantraniliprole formulations sprayed at the recommended rate of 150 mL a.i. per ha), and herbicide application (paraquat at 10 gallons per acre).

Sampling and analysis.
Sampling and analysis of soil and manure. Subsamples of initial soil and organic fertilizers (CM and PM) were dried at room temperature and passed through a 2-mm sieve. Inaddition, three replicate soil samples were taken from the 0-20 cm depth after harvest in the early and late seasons, to assess changes in soil properties. Soil bulk density (BD) was determined by the core method as described by Grossman 63 , and used to calculate soil total porosity using Eq. (1) recomended by Hillel 64 :  Table 5. Physical and chemical properties of soil and manure before the experimentation. SOC soil organic carbon, SOM soil organic matter, N nitrogen, P phosphorous, K potassium, C: N carbon to nitrogen ratio. www.nature.com/scientificreports/ where BD is soil bulk density and PS is particle density, assumed to be 2.65 mg m −3 . Soil moisture content was measured as described by Ledieu et al 65 . Air-dried soil was passed through a 0.5-mm sieve, and the weight of the tin (g) was taken as W1. A 1 g soil sample was taken along with the tin and weighed as W2. The soil samples were dried in an oven for 2 h at 105 °C to obtain a constant weight as W3. Soil moisture content (%) was determined using Eq. (2): Soil organic carbon was measured using the oxidation method. Soil subsamples (0.5 g) were digested with 5 mL of 1 M K 2 Cr 2 O 7 − H 2 SO 4 and 5 mL of concentrated H 2 SO 4 and boiled at 175 °C for 5 min, accompanied by titration of FeSO 4 digests according to the method of Bao 66 . To measure total soil N content, 200 mg of soil was digested using the salicylic-acid sulfuric-acid hydrogen peroxide method of Ohyama et al. 67 , and TN was analyzed using the micro-Kjeldahl method of Jackson 68 . Total P was determined using the ascorbic acid described by Murphy 69 . Total K was measured by preparing a standard stock solution by dissolving KCI in distilled water and measuring TK at 7665 R wavelength with an atomic absorption spectrophotometer (Z-5300; Hitachi, Tokyo, Japan) after sample digestion. Soil organic matter (SOM) was calculated by multiplying the SOC by 1.72.
AN was estimated using the methods of Kostechkas and Marcinkevicinee 70 and Dorich and Nelson 71 . Soil AP was measured by the NaHCO 3 extraction method and analyzed by the Mo-Sb colorimetric procedure using a spectrophotometer (UV 2550, Shimadzu, Japan) by method of Bao 66 . Soil AK was assessed by the method of Knudsen et al. 72 , using normal 1 M NH 4 OAc. Soil pH was measured with a digital pH meter (Thunderbolt PHS-3C, Shanghai, China) after mixing the soil and organic manure with distilled water at a 1:2.5 (w/v) ratio for 1 h.
Soil microbial biomass. The fumigation extraction technique was used to determine MBC as described by Brookes et al. 73 , and MBN according to the procedure of Vaince et al. 74 . From the composite soil samples, we took 10 g of soil and divided it into similar halves. In a vacuum desiccator, 25 ml of ethanol-free CHCL 3 was put in petri dish to disinfect first half of the soil (5 g) for 24 h at room temperature (25 °C). The samples were placed in warm water at 80 °C after fumigation to eliminate fumes, and 20 ml of K 2 SO 4 (0.5 M) was then used to remove C and N from both the fumigated and non-fumigated soils. The filtrated samples were then processed on a TOC Analyzer (TNM1; Shimadzu) and subjected to Kjeldhal digestion in order to calculate total C (TC) and TN. Equation Accumulation and translocation of DM and N. Three replicate plants were collected at anthesis and at physiological maturity to measure DM and N accumulation. The rice plant was divided into leaves (leaf blade + leaf sheeth), stems, and panicles, and then oven-dried to constant weight at 65 °C. Rice plant sub samples were ground to powder, and total N was estimated using the micro-Kjeldhal method as described by Jackson 68 . Post-anthesis DM or N accumulation is considered to be the difference in aboveground accumulation between anthesis and maturity 30 . Assuming that all DM and N losses from the vegetative organs of the plant were transferred to the grains, N translocation (NT) and DM translocation (DMT) during the grain filling stage were measured according to the equations of Papakosta and Gagianas 77 : where DMa is the total aboveground DM accumulation at anthesis and DM stem,m , DM leaf,m , and DM chaff,m are the DM of leaves, stems, and chaff at maturity. NTa is the total aboveground N accumulation at anthesis, and NT stem,m , NT leaf,m , and NT chaff,m are the total N accumulation of stems, leaves, and chaff at physiological maturity.
Rice leaf net photosynthetic efficiency and chlorophyll content. To assess the process of leaf senescence during the reproductive phase, flag leaf chlorophyll content, photosynthetic rate (Pn), and days to maturity were also determined. Flag leaf Pn and Chl content (Chl a and Chl b) were measured at the grain-filling stage. Pn was measured on the completely expanded flag leaf using a portable photosynthesis instrument (LI-6400, LI-COR, Lincoln, NE, USA). Measurements were made on a sunny day from 10:00 a.m. to 12:00 p.m. The sampling conditions were light intensity 1200 µmol m −2 s −1 , air humidity 70%, CO 2 375 μmol mol −1 , and leaf temperature 28 °C.
To measure leaf chlorophyll content, 1 g of fresh leaf tissue was cut into small pieces, placed in a volumetric flask that contained 10 mL of 80% acetone solution as described in Porra et al. 78 , and stored in the dark for 24 h. The absorbance of the extracted solution was measured at 663 and 645 nm using a UV spectrophotometer (Infinite M200, Tecan, Männedorf, Switzerland) to estimate chlorophyll a and b content (mg g −1 ) using the equations described by Arnon 79 : Nitrogen metabolizing enzyme activities . Five flag leaves were collected from each treatment during the grain-filling period, immediately frozen in liquid N and stored at -80 °C for estimation of the activities of the N-metabolizing enzymes such as Nitrate reductase (NR), Glutamine synthetase (GS), and Glutamate synthase was extracted and measured using a Glutamate Synthase (GOGAT). NR was extracted and measured using a Nitrate Reductase (NR) assay Kit (BC0080, Solarbio, Beijing, China). Briefly, 0.1 g leaf samples were extracted in 1 ml extraction solution and the mixture was centrifuged at 4000 g for 10 min. The resulting supernatant was collected for further analysis. The absorbance at 520 nm was used for the calculation of NR activity. GS was extracted and measured using a Micro Glutamine Synthetase (GS) assay Kit (BC0915, Solarbio, Beijing, China). Briefly, 0.1 g leaf samples were thoroughly ground in liquid nitrogen and extracted with 1 mL extraction buffer. The mixture was centrifuged at 8000 g at 4 °C for 10 min. The supernatant after centrifuging was collected for activity measurement. The absorbance at 520 nm was used for the calculation of GS activity.
Glutamate synthase was extracted and measured using a Glutamate Synthase assay Kit (BC0070, Solarbio, Beijing, China). Briefly, 0.1 g leaf samples were extracted in 1 mL extraction buffer. The extraction mixture was centrifuged at 10,000 g at 4 °C for 10 min. The resulting supernatant was harvested and the absorbance at 340 nm was measured for the calculation of GOGAT activity.
Rice yield and yield attributes. Five central rows from each plot were selected at physiological maturity to measure rice growth, yield, and yield traits. Three hills were randomly selected at physiological maturity to measure plant height, panicle number, panicle length, spikelet number per panicle, grain filling percentage, and thousand-grain weight. The crop was harvested when almost all the heads showed a complete loss of green color. Grain yield (kg/ha) was measured from five central rows in each treatment and adjusted to 14% moisture content.
Statistical analysis. Analysis of variance (ANOVA) was performed with Statistics 8.1 software to examine variations in soil physical and biochemical properties, leaf physiological traits, rice grain yield, and yield components. Percentage data were arcsine transformed prior to analysis. Data from both seasons were used in the analysis in order to detect differences between seasons as well as fertilizer treatments. Treatment was considered to be a fixed effect, and season was considered to be a repeated measures factor and a fixed effect. The interaction between fertilizer treatment and season was also taken as a fixed effect, but the interaction of season and treatment with replication was taken as a random effect. Mean separation was performed using the least significant difference (LSD) method at p < 0.05. Linear regression analysis was performed to evaluate the relationship between grain yield and Pn, N-metabolizing enzyme activities, pre-and post-anthesis DM, and N accumulation. Redundancy analysis (RDA) was performed using Canoco version 5 (Cambridge University Press, Cambridge, UK). .  Table 6. Nutrient content and amount nutrient provided of each treatment and application time. N nitrogen, CF chemical fertilizer (urea), CM cattle manure, PM poultry manure, P 2 O 2 superphosphate, KCl potassium chloride.