The field study at the rice research station of Guangxi University, Nanning, China, was started in 2019. This location has a sub-tropical monsoon climate, with approximately 1,300 mm of annual rainfall and an average annual temperature of 24.6°C. The soil is defined as Ultisols (United States Department of Agriculture soil classification) and is acidic (pH 5.96). A soil test showed that the total nitrogen (TN) was 1.66 g kg^–1 and soil organic carbon (SOC) was 18.74 g kg^–1; other soil properties are provided in Table 1.

The dual cropping seasons ranged from March to July (early season) and July to November (late season). The experimental study was established in a randomized complete block design with three replications. Organic manure [cattle manure (CM) and poultry manure (PM)] and CF urea were used, and the combinations of treatment were: no N fertilizer control (Neg-Con); 100% CF in the form of urea (Pos-Con); 60% CM + 40% CF (High-CM); 30% CM + 70% CF (Low-CM); 60% PM + 40% CF (High-PM), and 30% PM + 70% CF (Low-PM). We grew the Zhenguiai, a broadly cultivated cultivar in Guangxi Province. This cultivar has a short growth duration (approximately 110–120 days) with a morphological structure and high grain filling rate (Li et al., 2006). Rice seeds were grown, and 25-days old seedlings of even size were transferred into the fields. Except for Neg-Con, which received no N fertilizer amendments, the recommended dose of N, P, and K at a rate of 150:75:150 (kg ha^–1) was applied to each plot (Sapkota et al., 2020). Table 2 shows the nutrient content of manure and the amount of each treatment. The potassium (KCl) and N fertilizers were delivered in three stages: 50% prior to transplantation, 30% during tillering, and the remaining 20% during the heading period. All phosphorous fertilizer was supplied as a base dose before transplanting. Furthermore, uniform flooding continued from seedling transplantation to rice physiological maturity. Normal agricultural practices, such as irrigation and insecticide application, were performed for all regimes.

At maturity, the rice was collected from the entire plot and the grain yields. The dry weight of the grain was calculated using an adjusted moisture content of 14% in rice grains.

The changes in soil biochemical characteristics and rice grain yields as affected by fertilization treatment were examined using the ANOVA method in Statistics 8.1 (Analytical Software Tallahassee, FL, United States). First, the data were subjected to standard tests to ensure they met the assumptions of normality. Furthermore, percentages data were arcsine transformed before analysis to normalize the variables. Tukey’s post hoc test was performed to compare means for the variables where the effects of treatments were significant. A Venn diagram was used to illustrate the number of related and distinctive OTUs in the samples and determine the similarity and coincide in the number of OTUs between the samples. Microbial alpha diversity indices, including Simpson’s and Shannon indices, were calculated using MOTHUR (Schloss et al., 2009). Species richness rarefaction curves were plotted against the series of data using the MicrobiomeAnalyst (Dhariwal et al., 2017). RDA was used to analyze the relationship strength between soil traits and soil bacterial diversity using the software package CANOCO5 (Microcomputer Power, Ithaca, NY, United States).

Co-fertilization of fields with manure and chemical nutrients significantly increased the SOC, TN, pH, MBC, and MBN compared to CF application: Pos-Con and Neg-Con (Table 3). The effect was greatest in all observed data when manure input was high; no substantial (P < 0.05) variations among CM and PM were observed. Across the seasons, the regimes revealed the same trend. Compared to Pos-Con, High CM and PM treatments increased SOC 26.3 and 31.2%, TN 25.3 and 25.6%, respectively. In addition, over the years, High-CM enhanced treatment increased soil MBN and MBC by 55 and 61%, respectively, relative to Pos-Con. Nevertheless, High CM was statistically (P < 0.05) comparable to High PM. Likewise, low manured input treatments also increased the measured soil biochemical traits as compared to control.

In addition, significant increases were measured for SOC, TN content, MBC, and MBN in the subsequent year; the average enhancement in SOC, TN, MBC, and MBN during 2020 was 26.4, 9.50, 8.9, and 22%, respectively, relative to 2019.

In this study, the co-applied manure and synthetic N significantly influenced the soil bacterial community composition and diversity (Figures 1A–H). The box plots based on the Simpson and Shannon index exhibited significant variations in bacterial α-diversity detected in the regimes. Co-fertilization of manure and CF resulted in the highest bacterial diversity and abundance relative to Pos-Con. In addition, the bacterial diversity and abundance were significantly higher in the High manured plots.

To determine rarefaction curves, richness, and diversity of bacteria, OTUs were identified at 97% of genetic similarity. The rarefaction curves exhibited that the sequencing effort was enough to describe most of the diversity in soil samples (Figure 2). The Venn diagram shows that in 2019, the number of unique OTUs in Neg-Con, Pos-Con, High-CM, Low-CM, High-PM, and Low-PM treatments was 309, 228, 229, 246, 241, and 275, correspondingly, and the number of shared OTUs was 1,100 (Figure 3). In 2020, the number of unique OTUs was 261, 339, 158, 146, 304, and 129, respectively, and the number of mutual OTUs was 584.

The analysis for bacterial community composition under combined manure and mineral fertilization indicated the presence of a total of 41 phyla, and the average relative abundance of 11 phyla exceeded 1% (Figures 4, 5). Compared to Pos-Con, the integrated application of manure with mineral fertilizer significantly increased the soil bacterial community composition. The top five dominant phyla in all treatments were Chloroflexi, Proteobacteria, Firmicutes, Acidobacteria, and Planctomycetes, which reported more than 70% of the relative abundance of the bacterial communities. Chloroflexi and Proteobacteria were the most abundant among all these dominant phyla in the High manured treatments compared to the Neg-Con and Pos-Con. Likewise, low CM and PM treatments also increased the abundance of Proteobacteria and Chloroflexi compared with control. Furthermore, Actinobacteria, Acidobacteria, and Firmicutes were the second most abundant phyla in the experiment across all treatments.

The cluster analysis (Figure 5A) demonstrates that bacterial communities in the six fertilization regimes are divided into main groups. One is made up of Neg-Con, the other is composed of High-CM, High-PM, Low-CM, Low-PM, and Pos-Con, indicating that bacterial communities in different fertilization regimes share a high similarity and the cluster. Moreover, the similarity among bacterial communities of all fertilization regimes is further proved by the NDMS analysis as shown in Figure 5B. In the two-dimensional NDMS plot, a small unweighted UniFrac distance indicates a similar bacterial community. As projected, soil samples in Neg-Con and Pos-Con are closely occurred and clustered and separated from other manured amendments along NMDS axes.

The joined manure and synthetic N fertilization considerably affected the rice yield in both years (Figure 6). Combined fertilization increased rice yield considerably across the years compared with control, and over both years, the regimes exhibited a similar trend. The Low PM treatment increased the grain yield by 17 and 32% in 2019 and 2020, correspondingly compared to the control. Nevertheless, there were no substantial differences (P < 0.05) in grain yield between the Low manured regimes. Likewise, High manured treatments also significantly enhanced rice yield relative to the Pos-Con. Significant enhancements in yield were noticed over the years, and the average rice yield was improved by 15% in 2020 relative to 2019.

Redundancy analysis was performed to determine the impact of different fertilization treatments on bacterial community composition and environmental factors and reveal the strength of the relationship between soil traits and soil bacterial community structure (Figure 5C). The RDA shows that the six treatments occurred in distant quadrants show that the treatments had a significant effect on the structure of soil microorganisms and soil properties. The RDA revealed that manure-treated treatments significantly affected soil bacterial community composition compared to non-manure treatments. Proteobacteria, Acidobacteria, and Chloroflexi were strongly associated with soil properties. The RDA outcomes demonstrated that combined organic and inorganic fertilizer application to the rice field has the most substantial influence on soil microbial community composition and environmental properties.

In this experiment, animal manure combined with CFs significantly increased soil qualitative traits (SOC, TN, MBC, MBN, and pH) compared with CF-only fertilization (Table 3). We detected that the biodegradation of animal manure gradually released plant nutrients to the soil and exhibited that increasing the rate of manure improves soil qualitative traits. Application of CFs reduced soil pH level, while joint manure amendments increased soil pH level significantly. Alike results were stated by Bhattacharyya et al. (2015), who concluded that continuous use of synthetic fertilizer causes soil acidification. Moreover, the decline in pH level is because the overuse of synthetic N forms H⁺ through nitrification in soil (Yang et al., 2018). Another possible reason for the lower pH resulting from the mineral N fertilization was the acidic nature of mineral N fertilizers, which could donate to the soil’s lower pH (Adekiya et al., 2020). Organic fertilizer application alters soil acidification because it frequently consists of enough basic cations and carbonate ions to neutralize soil acidity, explaining the increase in soil pH under manure amendment plots (Duruigbo et al., 2007).

The application of animal manure and CF meaningfully enhanced the SOC and TN, P and K status in the paddy soil (Table 3), which is in line with Wei et al. (2017). The main macronutrients (N, P, and K) are important for plant growth and production. Soil N, P, and K scarcity have a negative impact on soil fertility and crop yield (Kaur and Reddy, 2014; Verma et al., 2017). The substantial improvement of soil organic C (SOC) could be associated to the substantial effects of manure fertilization because the soil C variation rate is affected by direct carbon inputs from organic fertilizer and indirect carbon inputs from increased plant biomass return (Bitew and Alemayehu, 2017; Wang et al., 2021), such as crop and root residues. This is primarily due to the addition of manure (i.e., CM or poultry) having a significant effect on the soil TN, TP, and TK. This increment in nutrient content might also be related to incorporating manure remains, which, after decomposition, directly added nutrients to the soil (Zhang et al., 2019; Hou et al., 2022). Moreover, besides improving the physical and biochemical traits of soil, animal manure fertilization, steady releases of plant nutrients and prevent nutrient losses from the synthetic fertilizers by binding to plant nutrients and releasing them (Abedi et al., 2010; Ullah et al., 2020). Thus, the integrated application of manure and CFs improves CF use efficiency and, hence, decreases CF’s rate (Tilahun-Tadesse et al., 2013; Adekiya et al., 2019).

In the present research, organic fertilizer in conjunction with synthetic fertilizer, increased microbial biomass C and N (Table 3). Rises in MBC and MBN may have happened because manure enhanced the biogeochemical properties of the soil in the present study, leading to improved absorption of inorganic N by the plant (Ahmad et al., 2008). Another reason is that organic amendments may have boosted soil nutrient richness and crop biomass accumulation, resulting in enhanced crop residues (Akhtar et al., 2019). Such residues are useful for spreading soil bacteria and may promote the alteration of N and C (Lima et al., 2009).

In this experiment, the residual effects of manure have increased soil biochemical traits. Soil organic C, examined as a vital factor in evaluating soil health and quality, enhanced 26.4% in the residual effect of manure fertilization. A rise in SOC due to organic manure fertilization has a significant impact on soil productivity because SOC is the ultimate source of soil nutrients and soil microorganisms activity in the soil. Moreover, SOC also has a key role in enhancing soil quality, infiltration rate, water holding ability, porosity and aeration (Sarwar et al., 2008; Ali et al., 2020a). Moreover, manure contains macro-and micronutrients, such as N, K, P, Ca, and Mg (Abedi et al., 2010; Hafidi et al., 2012). In this study, a significant increase was noted in TN and MBN in the following year; the average enhancement in TN and MBN in 2020 was 9.5 and 22.2% compared with 2019. The observed residual effect of organic fertilizers or enhancement in nutrient contents of manured plots in our study might be due to the reason that soil nutrients contained in organic fertilizers are revered in soil for a longer period and released steadily, ensuring a long term residual influence, and to solubilization of plant nutrients from soil minerals due to the influence of organic manure organic acids (Sharma et al., 2013).

Microbes are a key index of soil quality (van Bruggen et al., 2015). The richness and biodiversity of the microbial population are crucial to soil integrity, performance, and soil sustainability; however, they are frequently reduced by conventional agricultural techniques (Zhao et al., 2014). In the current study, the fertilization treatments extensively affected the bacterial community structure and composition, and the soil bacterial communities were influenced by the application of organic amendments (Figures 1, 4). Manure and synthetic fertilizer co-application significantly increased the number of sequences and the species richness (i.e., ACE, Shannon, and Chao 1 index values) of the bacteria related to sole inorganic fertilizer (Table 4 and Figure 1). According to Dong et al. (2014), manure applications not only include a wider range of substrates for bacterial activity than synthetic fertilizers, and they also directly introduce microorganisms found in manure into the soil. Furthermore, Bacteria are broadly considered as critical mediators of the quick pathways of nutrient cycling in soil, and their growth rate is nearly ten times higher than that of fungi (Rousk and Bååth, 2007; Schmidt et al., 2014); thereby, bacterial communities can grow rapidly and diversify under manure addition.

In the current study, the bacterial community structure in synthetic fertilizers treatment and non-synthetic fertilizer treatment was similar; however, it changed following manure amendments treatment (Figure 4). This is probably because of short-term input of CFs did not considerably affect the soil pH. According to Ganzert et al. (2014), pH is the greatest important factor shaping the soil bacterial community. Furthermore, Geisseler and Scow (2014) stated that the response of CFs on soil microorganisms is carbon and pH-dependent. Organic amendments could enhance soil interspace and resource supply for bacterial growth (Li P. et al., 2020), thereby prompting microbe societies. Moreover, cluster analysis showed a substantial difference in bacterial community composition between manure treated and non-manure treatment (Figure 5A). This is primarily due to livestock manure containing more readily available microbial organic resources that can provide additional metabolic nutrients to soil microbes (Bei et al., 2018).

The integrated use of animal manure and CF increased the relative abundance of Proteobacteria, Chloroflexi, Firmicutes, Acidobacteria, and Planctomycetes, which described for more than 70% of the relative abundance of the soil bacterial communities (Figure 4). The relative abundance of the community’s dominant bacteria, Proteobacteria and a type of nutrient-rich bacteria are associated with soil properties (Merila et al., 2010; Luo et al., 2020). In our research, all soil chemical traits improved with manure fertilization (Table 3), promoting the growth of bacteria. Thus, the relative abundance of Acidobacteria, Chloroflexi, Proteobacteria, Firmicutes, and Planctomycetes, the dominant bacteria in the community, enhanced under the integration of organic and synthetic fertilizer treatments.

Application of organic and inorganic N significantly improved rice yield related to CF-only application in the current study. Manure application improves the physical and biogeochemical properties of the soil, resulting in increased plant growth and crop yield (Abedi et al., 2010; Hafidi et al., 2012). In this study, higher soil nutrients were observed under manured plots (Table 3), which, in turn, improved rice growth and biomass accumulation by providing adequate nutrients during the growth period. Moreover, The residual effect of 1 year (two consecutive seasons) of manure and CFs application also provided yield benefits. Significant increases in rice grain yield were observed between years, and the average rice yield was enhanced by 14% in the year 2020 relative to the year 2019. Person correlation analysis exhibited that grain yield was strongly correlated with SOC, TN, TP, and AK of soil (Table 4). This correlation shows that deficiency of anyone the three main nutrients (NPK) can decrease grain yield in flooded soil. The structural equation model result showed that differences in soil nutrients explained the greatest proportion of the variation in rice crop yield (Figure 7). These findings collectively show the importance of balanced and enough fertilization of organic and chemical N fertilizers for soil quality and sustainable grain yield of rice. According to Akhtar et al. (2019), changes in crop yield are closely related to soil biogeochemical characteristics and microbial biomass yield.

Manure fertilization can cause physicochemical variations in soil, resulting in changes in the bacterial community composition (Li et al., 2021). This study discovered that organic amendment treatments dramatically altered soil qualitative traits (Table 3). Moreover, Wu et al. (2020) reported that the structure and composition of the bacterial community were positively associated with soil C and N content. Figure 5C shows the relationship between the soil traits and the bacterial communities for the different fertilizer regimes. In this study, RDA showed that the organic amendments had significant effects on bacterial community and soil qualitative traits. The dominant bacteria at the phyla, notably Proteobacteria, Chloroflexi, and Acidobacteria, had a positive correlation with soil characteristics, but Proteobacteria had a strong correlation with SOC, TN, and pH. Bacterial growth is highly related to the type of fertilizer used, and controlling the type and ratio of animal fertilizer is an effective strategy for increasing bacterial growth. According to what has been discussed thus far, applying organic fertilizer combined with reduced inorganic fertilizers could provide a faster growth environment for soil microbes, improving the bacterial population structure and soil fertility.