Manure Applications Combined With Chemical Fertilizer Improves Soil Functionality, Microbial Biomass and Rice Production in a Paddy Field

Synthetic fertilizer with organic fertilizer (OF) is an approach for the improvement of soil health and quality without compromising crop yield. Therefore, a two-year eld experiment was conducted to explore optimal chemical fertilizer (CF) management strategies in the context of OF, such as cattle manure (CM) and poultry manure (PM) fertilization to Ultisol soil to improve soil microbial biomass production, enzyme activities and nutrient contents, as well as grain yield of rice. A total of six treatments in the following combinations were used: i.e., T 1 — CF 0 ; T 2 —100% CF; T 3 —60% CM + 40% CF; T 4 —30% CM + 70%CF; T 5 — 60% PM + 40% CF, and T 6 —30% PM + 70% CF. Results showed that the combined fertilization signicantly increased soil enzymatic activities such as soil invertase, acid phosphatase, urease, catalase, (cid:0) -glucosidase, and cellulase as compared to sole CF application. Similarly, the integrated manure and inorganic fertilizers led to signicant increases in soil microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), soil pH, soil organic carbon (SOC), total nitrogen (TN), available nitrogen (AN), available phosphorous (AP) and grain yield of rice. Average increases in soil MBC, MBN, SOC AN, and AP in the 0–20 cm soil depth were 62.2%, 54.5%, 29.2%, 17.4%, and 19.8%, respectively, across the years in treatment T 3 compared with T 2 . Interestingly, the linear regression analysis displayed that soil enzymatic activities were highly positively correlated with MBC and MBN. Furthermore, the PCA exhibited that the improved soil enzyme activities and microbial biomass production played a key role in the higher grain yield of rice. Overall, the results of this study demonstrate that the combined use of CF and OF in paddy soil could be benecial for the farmers in southern China by improving soil functionality and yield of rice on a sustainable basis.


Introduction
Appropriate management strategies are important for maintaining soil health and ensuring the long-term sustainability of agricultural production (Ramesh et al., 2009;Li et al., 2015). Intensive agriculture has resulted in a clear decline in soil quality over the last two decades, which is a major issue for long-term agricultural production and food security (Agegnehu et  Chen et al., 2014). Farmers treat CFs as the most reliable and e cient way to improve crop production and revenue, without seeing the effects on soil or environmental protection (Yan et al., 2013;Qiao et al., 2012). Furthermore, the excessive use of mineral nitrogen (N) and phosphorous (P) also decline the soil microbial population and increase soil acidity (Zhang et al., 2015;Luo et al., 2020). Hence, full knowledge of soil's response to CFs additions is critical to protect and even improve soil health, particularly in paddy elds.
Recently, there is an increasing demand for agricultural output not only to reach high-quality standards but also to adopt eco-friendly techniques. Several researchers have recommended that OF application meet the requirements of sustainable farming, and thus organic fertilizers have a clear advantage over For example, cellulases and glucosidase are a class of hydrolytic enzymes generated by soil microorganisms that decompose polysaccharides (Deng and Tabatabai, 1994). Soil available and total P contents are related to the change in soil phosphatase activity, and more loss in P content was noted in paddy elds (Wang et al., 2012 (Raiesi and Beheshti, 2014;Medeiris et al., 2015). However, little is known about the effect of combined organic and inorganic N fertilization in speci c prorations on soil enzymatic activity, microbial biomass production and grain yield of rice under paddy elds condition.
In an agroecosystem, the relationships between soil enzyme activities, microbial biomass, and soil quality traits are crucial. Therefore, the purpose of this study was to investigate how different forms of manure, such as cattle and chicken manure in various proportions combined with synthetic fertilizer (urea) in uence soil enzymatic activities and their relationships with soil quality, microbial biomass production, and rice yield under paddy eld. We collected the soil samples from a 4-seasons (2 years) continued fertilizers trial on a rice paddy soil treated by combined organic and inorganic fertilizers. The soil enzyme activities (invertase, urease, catalase, -glucosidase, acid phosphatase and cellulase), microbial indicators (MBC and MBN), soil environmental factors (pH value, SOC, TN, AN, AP, and AK contents) and grain yield of rice were investigated. The aims of this work are: (1) to evaluate the effects of different types and ratios of OF and CF treatments on the enzyme activities, microbial biomass production and soil quality; (2) to analyze the correlations among the enzyme activities, microbial production, and soil fertility and; (3) to expose how the changes of soil nutrients and enzymes activities in uences rice yields.

Site description
The ongoing eld experiment at the rice experimental station of Guangxi University (22°49′12″ N, 108°19′11″ E), China was initiated in 2019. This site experiences a subtropical monsoon climate, with a total annual rainfall of 1398 mm, and an average temperature of 24.8°C (Fig. 1). The soil is classi ed as Ultisols (USDA soil classi cation), which is slightly acidic with a pH of 5.96. A soil test revealed that the SOC was 18.75 g kg − 1 , and total nitrogen (TN) was 1.64 g kg − 1 ; details of other nutrients are shown in Table 1. Note: SOC-soil organic carbon, SOM-soil organic matter, N-nitrogen, P-phosphorous, Kpotassium, C: N-carbon to nitrogen ratio.

Experimental design
The double rice-growing season experiment (the early season runs from (March to July) and the late season runs from (July to November)) was arranged in a randomized complete block design having three replications. The plot size was 3.9 m × 6 m (23.4m 2 ). Organic manure (PM and CM) and CF (urea) was used in this study, and the treatments combination were: T 1 -CF 0 ; T 2 -100% CF; T 3 -60% CM + 40% CF; T 4 -30% CM + 70%CF; T 5 -60% PM + 40% CF, and T 6 -30% PM + 70% CF used. The rice seeds were germinated in plastic trays, and the uniform-sized 25 days old seedlings were transferred into the rice eld. The recommended dose of NPK (150:75:150) was used for each treatment, except T 1 . The nutrient contents of OF and the quantity of all treatments are shown in Table 2. The N and K fertilizers were delivered in three splits: 50% was applied initially before transplanting, 30% at tillering, and 20% at the heading stage. All superphosphate fertilizer was applied before transplanting as a basal dose. In addition, even ooding was maintained from transplantation to physiological maturity. During the entire season, standard farming practices, including irrigation and insecticides application was performed the same for all treatments.

Soil properties
Soil samples were obtained by a core sampler at depth (0-20 cm) from each treatment after the lateseason rice harvest in 2019-2020. Soil sampling was done at different points and then mix to make a composite sample, and divide into two parts, one for the measurements of soil nutrients, and the other was stored at 4℃ for the determination of soil enzymes Soil organic C was measured by the K 2 Cr 2 O 7 -H 2 SO 4 oxidation method followed by titration (Wang et al., 2003). For soil total nitrogen (TN) analysis, 200 mg of the samples were processed using the salicylic acid-sulfuric acid-hydrogen peroxide method de ned by Ohyama et al. (1991), and total N was measured using the micro-Kjeldahl technique according to Jackson (1956). The other chemical traits, including soil pH, available N (AN), available P (AP), and available K (AK), were measured using the methods de ned by Lu (2000).

Soil enzyme activities
The determination of soil enzymatic activity was performed by the described procedure of Jin et al. activity was checked in a 5 g fresh soil sample by using 10% of urea solution as substrate, and was incubated for one day at 37℃ with 5 mL of citrate solution at pH 6.7 and 5 mL of the substrate, and then ltered. One ml ltrate was treated with 4 mL of sodium phenol solution and 3 mL of 0.9% sodium hypochlorite solution. The ammonium released from urea hydrolysis was measured using an ultraviolet spectrometer subsystem at 578 nm.
Soil invertase activity was measured using 8% glucose solution as the substrate. A 5-g fresh soil sample was incubated with 15 mL of the substrate, 5 mL of 0.2 M phosphate buffer (pH 5.5), and 5 drops of toluene for one day at 37°C. After incubation, the mixture was ltered (Whatman 2V) immediately, and a 1-mL aliquot was reacted with 3 mL of 3, 5-dinitrylaslicylate in a volumetric ask and heated for 5 min.
When cool, the soil solution in the ask was measured in a UVS at 508 nm. The soil acid phosphatase activity was measured in one g sample using p-nitrophenyl phosphate disodium as substrate, followed by incubation in a modi ed universal buffer (acid phosphatase: pH 6.5) for 1 h at 37°C. Cellulase was measured following the method of Pancholy and Rice (1973). Reducing sugars were produced when soil samples were incubated at 37°C with carboxymethyl cellulase. Soil β-glucosidase activity was determined by the colorimetric method described by Eivazi and Tabatabai (1994). Brie y, 1 g of soil was incubated with the substrate at pH 6.0 and 37°C. After 1 h, 0.5 M CaCl2 and pH 12.0 modi ed universal buffer were added to extract p-nitrophenol. The amount of p-nitrophenol released by glycosidases was determined calorimetrically at 410 nm.

Rice grain yield
Rice plants from the whole plot area were harvested at maturity to measure the rice grain yield adjusted to 14% moisture content.

Statistical analysis
One-way analysis of variance (ANOVA) test was applied to study the effects of organic and inorganic fertilizers on soil enzymatic activities, biochemical traits, and grain yield of rice Tukey's posthoc test was used to compare multiple means for the variables where effects of experimental factors were signi cant. Principal component analysis (PCA) was used to test the difference among treatments for studied variables (Canoco5). Relationships between soil enzyme activities, chemical properties, and grain yield were studied using redundancy analysis (RDA). Statistical analyses were performed with SPSS for Window Software v. 19.

Soil Chemical properties
The effects of co-application of manure and synthetic fertilizer on soil chemical attributes are shown in Table 3. Co-application of OF and CF signi cantly enhanced the soil pH compared with soil chemical N fertilization (T 2 ). In 2019 and 2020, the soil pH of the T 3 and T 5 treatments was considerably (P < 0.05) greater compared with the rest of the treatments. Soil pH showed the same pattern across years, and an average increase in soil pH by 5.5% and 5.3% was exhibited in the T 3 and T 5 regimes, respectively, compared with the control (T 2 ). The other combined treatments also had signi cantly higher soil pH during both years compared with T 2 . Note: T 1 -control, 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, SOC-soil organic carbon, TN-total nitrogen, AN-available nitrogen, APavailable phosphorous, AK-available potassium. Values followed by the same letters, within column, are not signi cantly different at p ≤ 0.05.
The positive effects of OF and CF treatments on SOC content are shown in Table 3. SOC content increased signi cantly under integrated treatments during both years. In both years, the SOC content was maximum in the T 3 and T 5 treatments than in the rest of the treatments. Furthermore, the SOC content among the different treatments showed the same pattern across years, and average increases in SOC content were 29.2% and 29.8% in the T 3 and T 5 treatment, respectively, compared with control. Compared with control, the T 4 and T 6 treatments also showed signi cantly higher SOC content. Moreover, substantial enhancements were detected in SOC content during the second year, the average increase in SOC content in the year 2020 was 27.2% compared with the year 2019.
The difference in soil total N (TN) content at the 0-20 cm soil depth is shown in Table 3. Soil TN content was signi cantly increased (P < 0.05) in the combined treatments compared with CT in both years. The TN content of all treatments showed similar patterns across years. The average increases in TN were 26.3%, and 25.9 %, respectively, in the T 3 and T 5 treatment compared with control. Similarly, the T 4 and T 6 treatments also had considerably superior soil total N content compared with Control. Furthermore, signi cant improvements were observed in TN content during the seceding year, the average increment in TN content in the year 2020 was 9.55% compared with the year 2019.
The variations in the soil available N, P, and K content in the top layer are shown in Table 3. The coapplied organic and mineral N fertilization considerably (P < 0.05) improved soil available N, P, and K content across years compared with control (T 2 ). The AN, AP, and AK content of all treatments showed similar patterns across years, and the average increases in AN, AP, and AK were 17.02% and 19.84%, respectively, in the T 3 and T 5 treatment compared with control. However, AN, AP, and AK content did not signi cantly differ (P < 0.05) between the treatments T 3 and T 5 . The N, P and K content was also considerably greater in the rest of the integrated treatments compared with CT. Moreover, signi cant developments were noted in AN, AP, and AK content during the following year, and the average increase in AN and AP content in the year 2020 was 12.28% and 16.96%, compared with the year 2019.

Soil microbial biomass
Soil MBC and MBN were signi cantly different at the 0-20 cm soil depth among the treatments in both years as shown in Fig. 2. The integrated use of manure with urea (CF) signi cantly enhanced the soil MBC and MBN, compared to urea-only (T 2 ). Across the years the treatments showed the same behaved.
Averaged across the years, the combined treatment T 3 enhanced soil MBC and MBN by 62% and 54%, respectively, compared with T 2 . But, T 3 was statistically (P < 0.05) similar to T 5 . Likewise, the joint treatments T 4 and T 6 also signi cantly enhanced soil MBC and MBN compared with sole urea fertilization. In-addition, signi cant improvements in soil MBC and MBN were observed among the different years, and the average soil MBC and MBN was increased by 9.2% and 21%, respectively, in 2020 compared with 2019.

Soil phosphatases and catalase activity
The activities of phosphatases (POH) and catalase (CAT) soil enzymes at the 0-20 cm soil depth are shown in Fig. 3. The co-applied organic and inorganic N fertilization treatments signi cantly affected soil POH and CAT activity in 2019 and 2020. The POH and CAT activity were considerably (P < 0.05) higher in the T 3 and T 5 treatments compared with the other treatments. Patterns of POH and CAT activity among the treatments were similar across years. Average across the years, POH activity 7.45 and 9.21 (mg phenol g − 1 h − 1 ) were recorded in T 3 and T 5 , respectively, and average CAT activity 19.99 and 21.33 (0.1 mol KMnO4 g − 1 soil h − 1 ) was noted in T 3 and T 5 , respectively. The POH and CAT activity of the T 3 and T 5 treatment was increased by 43 & 48%, and 35 & 39%, respectively, compared with T 2 averaged across years. The rest of the combined treatments also had signi cantly higher POH and CAT activity compared with control. Furthermore, signi cantly enhances were observed in POH and CAT activity during the seceding year in 2020, compared with 2019.

Soil urease and cellulase activity
The combined fertilization signi cantly enhanced the activity of cellulase and urease in both years (Fig. 4). Soil urease and cellulase enzyme activity was considerably (P < 0.05) superior in the T 5 treatment than in all other treatments. However, treatments T 3 were signi cantly (P < 0.05) similar to T 5 . Patterns of urease and cellulase activity among all the treatments were similar across years. Average across the years, urease activity 1.15 and 1.19 (NH3-N g − 1 day − 1 ) were noted in T 3 and T 5 , respectively, and average cellulase activity 2.35 and 2.54 (mg glucose g − 1 day − 1 ) was noted in T 3 and T 5 , respectively. The urease and cellulase activity of the T 5 treatment was 52% and 48% higher, respectively, compared with control (T 2 ) averaged across years. The other combined treatments, such as T 4 and T 6 had signi cantly higher urease and cellulase activity compared with CT. Moreover, signi cantly enhances were observed in POH and CAT activity during the seceding year in 2020, compared with 2019.

Soil -glucosidase and acid invertase activity
The in uence of joint use of manure and synthetic fertilizers on soil glucosidase and invertase activity is shown in Fig. 5. During 2019 and 2020, glucosidase and invertase activity were higher in the combined treatment compared with sole urea application. Furthermore, patterns of glucosidase and invertase activity among treatments were similar across years. Average across the years, maximum soil glucosidase activity 15.14 and 14.45 (µg p-nitrophenol g − 1 soil) were observed in T 3 and T 5 , respectively, and invertase activity 44.54 and 43.71 (mg glucose g − 1 24h − 1 ) was observed in T 3 and T 5 , respectively.
The glucosidase and invertase activity of the T 5 treatment was 62% and 58% higher, respectively, compared with control averaged across. But, T 3 was noted non-signi cant with T 5 . Similarly, the T 4 and T 6 also had notably higher glucosidase and invertase activity compared with control. Moreover, substantially enhances were detected in POH and CAT activity during the year 2020, compared with 2019.

Grain yield
The combined organic-inorganic fertilizers application signi cantly affected the grain yield of rice in both 2019 and 2020 (Fig. 7). Compared with control, co-applied N fertilization considerably improved the grain yield of rice across years, and the treatments showed a similar pattern across years. The grain yield of the T 6 treatment was enhanced by 15% and 33% during 2019 and 2020, respectively, relative to control.
However, there were no signi cant (P < 0.05) differences in grain yield among the T 4 and T 6 treatments.
Similarly, the combined treatments T3 and T5 also signi cantly improved grain yield, compared with control. Signi cant improvements in grain yield were observed among the different years, and the average grain yield was increased by 14% in 2020 compared with 2019. The PCA revealed a signi cant difference in soil biochemical properties, enzyme activity, and rice grain yield between the various combined OF and CF treatments (Fig. 9). All the variables for soil biochemical attributes, soil enzyme activities, and rice grain yield were grouped into 4 well-differentiated classes. The rst group contained the sample from T1, the second group from T 2 , the third form T 4 and T 6 , and the fourth from T 3 and T 6 . The cumulative variance of contribution reached 99.3% (PCA 1 explained 95.7%, and PCA 2 explained 3.6%) over the years. Furthermore, the analysis showed that rice grain yield, MBC, MBN, SOC, TN, AN, AP, pH, glucoside, cellulase, acid invertase, acid POH, CAT, and urease were highly affected by the co-applied of both OF and CF fertilization treatment.

Discussion
The

Soil properties
Soil qualitative traits (i.e., SOC, TN, AN, AK, AP and pH) were signi cantly enhanced in integrated organic and inorganic N fertilization compared with soil urea fertilization in the present study (Table 3). We found that the decomposition of organic fertilizer slowly released nutrients to the soil and demonstrate that rising the manure level from 30-60% enhanced soil chemical attributes. In this work, sole urea application decreased pH, while integrated organic and inorganic N fertilization treatments signi cantly increased soil pH. A feasible reason for this is that farmyard manure, such as cattle or chicken manure in uences soil acidi cation because it often contains su cient basic cations and carbonate ions that C cycling and accumulation also plays role in higher C in this study. The RDA analysis in this study showed that the soil enzyme activities, MBC and SOC are strongly associated with each other.
In addition, soil nutrients, N, Kand P were signi cantly improved under combined treatments in this study (Table 3). This is mainly due to the addition of organic fertilizer such as CM or PM had a positive effect on the soil N, P and K content. This might be allied with the incorporation of organic fertilizers remains, which directly added nutrients into the soil after decomposition ( Moreover, organic fertilizers application provides a great amount of P to the soil, and decreases the xation of delivered P in the soil, resulting in the enhanced competition of organic molecules with PO 4 3− ions for P retention sites (Xie et al., 1991). The higher AK under combined treatments could be associated with the release of organic acids in decomposition, which generate negative electron charges in the soil with a preference for di or tri valent cations, such as Al3+, Ca2+, and Mg2+, leaving K + to be absorbed by negatively charged soil colloids (Timsina et al., 2006). This process could help to decrease K xation and improve its accessibility in soil. Furthermore, organic manure fertilization could also enhance the population of soil microbes and the activity of enzymes about nutrient transformation, therefore

Soil microbial biomass
The rate of microbial biomass C and N production re ects the features of the microbial community composition and structure (Norris and Congreves, 2018). In the present study, organic manure along with CF signi cantly enhanced the production of MBC and MBN (Fig. 2). The increments in MBC and MBN may be associated with organic fertilizer, along with CF fertilization increased the physicochemical and biological traits of soil, leading to an increase in the absorption and uptake of mineral N by the crop (Ahmad et al., 2008;Mahmood et al., 2017). In addition, manure promotes the conservation of mineral N to microbial N and other forms (Fan et al., 2017). Another possible explanation is that the combined organic amendments fertilization might improve the soil fertility and rice biomass production in the present study (Tables 4-5) ultimately lead to increase crop residues. This so useful for the spread of soil microorganisms, and thereby facilitates the conversion of carbon and nitrogen (Lima et al., 2009). Furthermore, the enhancement in soil enzymatic activities also a reason for higher C and N production under combined treatments. More importantly, our result showed that the combined application of manure and mineral N fertilization at the ratio of (30% OM + %70 CF) and (60% OF + 40% CF) signi cantly enhanced soil enzyme activities during both years, and high ratio of organic manure supplementation caused higher enzyme activity . This was associated with the enhanced inputs of organic substrates that promote microbial population and growth as well as and enzyme synthesis. Alike outcomes were also found from the study of Marcote et al. (2001). In addition, the RDA analysis exhibited a very strong and signi cant relationship between soil enzyme activity and soil properties (Fig. 7). (2012) reported that CFs application may decline the activities of soil enzymes associated to C, N and P cycling, maybe because enzymes are not required by microorganisms to obtain N and P nutrients.

Rice grain yield
Combined manure and mineral N fertilization signi cantly improved the grain yield of rice compared with that of sole inorganic fertilizer (Fig. 6). The enhancements in grain yield could be due to the enhanced soil fertility and functionality under treatment combinations, which ultimately increased crop growth, biomass and yield accumulation by providing su cient nutrients throughout the crop growing period. Furthermore, the RDA analysis showed that the soil biochemical and enzyme activates in the current study were strongly related to the grain yield of rice (Fig. 7). Hence, changes in cereal grain yield are closely

Conclusion
Our ndings indicated that the continuous application of OF and CF signi cantly improved soil qualitative traits, including pH, SOC, TN, AN, AP, MBC and MBN compared with sole urea (CF) fertilization.
The combined use of both fertilizers led to higher soil enzymatic activities (i.e., soil acid phosphatase, urease, catalase, invertase, glucosidase, and cellulase). Improvements in these soil enzymatic activities further improved soil microbial biomass production and grain yield of rice. Moreover Tables Tables 4 and 5 are not available with this version. Figure 1 Mean monthly rainfall and air temperature during the experimental years      Changes in grain yield of rice as in uenced by different combined organic and inorganic N fertilization.

Figures
The mean comparison was made using the least signi cant differences (LSD) test for treatments and seasons means based on the LSD test at 5%. Different letters within the line are not signi cantly different at p < 0.05. Note: For treatments combination please see Fig. 2.

Figure 9
Principal components analysis of all soil enzymes, soil chemical properties, and rice grain yield under combined organic and inorganic N fertilization at 0-20cm soil depth over the two-year study. Note: GYgrain yield, POH-phosphatase, MBN-microbial biomass carbon, MBN-microbial biomass nitrogen, SOC-soil organic carbon, TN-total nitrogen, AN-available nitrogen, AP-available phosphorous, AKavailable potassium. For treatments combination please see Fig. 2.