Soil carbon and nitrogen regulation using organic and inorganic fertilizers for leaf physiological activity, grain yield and nutritional quality improvements in rice

Organic fertilizers are widely used in agriculture production and change the soil carbon (C) and nitrogen (N) contents, thus improving crop production. The increased amount of soil C and N exhibit a greater potential to improve the leaf physiological activity, yields and grain quality of rice by improving soil fertility indices. To understand the relationship between soil C and N contents with leaf physiological activity and grain quality; organic fertilizers (i.e., cattle manure (CM) and poultry manure (PM)) coupled with chemical fertilizer (CF) was applied at the different proportion. The recommended rate of N 150 (kg ha −1 ) was provided from manure and CF using six treatments, 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 soil organic C (SOC), total N (TN), leaf net photosynthetic rate (Pn), SPAD values, grain yield and grain quality attributes were signicantly increased with combined organic and inorganic fertilizer application. Averaged over the years, T 6 treatment signicantly improved SOC, TN, Pn, starch content (SC), amylose content (AC), and grain yield by 16%, 12%, 9%, 7%, 12%, and 24%, respectively, compared to CF-only. However, no signicant differences among T 4 and T 6 were observed for studied parameters. In addition, the linear regression exhibited that SOC (R 2 = 0.70** & R 2 =0.50*) and TN (R 2 = 0.62* & R 2 =0.58*) were positively correlated with grain SC and AC, respectively. Likewise, Pn (R 2 = 0.51* & R 2 =0.62*) were also positively associated with SC and AC, respectively. The correlation analysis showed that improving SOC and TN played a key role in enhancing leaf physiological activity and grain nutritional quality. Thus, the combination of organic and inorganic fertilizers at a 30:70 ratio is a promising option for the improvement of soil fertility and CF—chemical fertilizer (urea), CM—cattle manure, PM—poultry manure, P 2 O 2 —superphosphate, KCl—potassium chloride.


Introduction
Improving crop production and quality to feed the global rising population and sustain high living standards has become a great challenge, recently (Fischer et al., 2014). After green revaluation, the modern farming system had played a crucial role in feeding the world's population (Jensen et al., 2011) but relied on the usage of heavy chemical fertilizers (CF) (Chen et al., 2014;Zhang et al., 2016). However, the usage of CF had led to a signi cant decrease in soil quality and is a major concern for sustainable agricultural productivity and crop quality (Sarma et al., 2017;Agegnehu et al., 2014). In addition, the extreme N application causes signi cant environmental problems, such as increased greenhouse gas emission, soil acidi cation, and groundwater pollution (Mahajan et al., 2008;Cai et al., 2018). Application of N fertilizer for a long time has also decreased soil microbial diversity, and make compaction of arable soils, thereby suppressed plant growth and crop production Yue et al., 2015). In China, it has been documented that the CF application rates are usually far higher than the crop requirements (Ju et al., 2009). It is perceived by the farmers treat CFs are the most reliable and effective way to improve crop production and their revenue (Yan et al., 2013;Qiao et al., 2012), but have totally ignored the ill effects on soil and environment by using the N fertilizers continuously. For example, in winter wheat and summer maize rotation, the annual fertilization of mineral N (550-600 kg N ha −1 ) was almost three times greater than the minimum fertilization rate i.e., about 180 kg N ha −1 (Zhao et al., 2006;Cui et al., 2008).
Consequently, a huge amount of the applied nutrients, particularly N lost to the environment and have negative effects.
Organic fertilizers, such as farmyard manure (FYM), green manure, straw and compost are an alternative source of fertilizers that can provide various bene ts over CF, such as improved soil health (Thangarajan et al., 2013), increased soil fertility (Iqbal et al., 2019a;Ali et al., 2020a), maintain soil quality and health (Xie et al., 2014;Ali et al., 2020b), and in particular, similar or even greater crop grain yields in some cases (Iqbal et al., 2020b;Seufert et al., 2012). Moreover, organic manure fertilization could provide all essential nutrients required for plant growth and is also considered eco-friendly (Ullah 2020; Gu et al., 2015). But organic manure is quite low in nutrients content and its releasing capability is also low to meet plant demands in a short time, therefore the sole fertilization of manure cannot meet the usual demands of plants nutrients. Therefore, the organic fertilizers if coupled with mineral fertilizer, can be considered a better approach to enhance soil quality on a sustainable base and crop productivity than sole CF application Bandyopadhyay et al., 2010). However, gaps still exist in the knowledge presently available about the responses of soil chemical properties and leaf physiological traits with grain quality attributes and grain yield of rice using the various proportion of organic and inorganic N fertilization.
Rice is one of the major stable crops in China with a per capita consumption of 100 to 120 kg year −1 , which is almost 2.2 times the world average (FAO, 2016;Ding and Zhai, 2013). Rice grain quality determination can judge by four components, i.e., appearance, cooking, milling and nutritional quality (Li et al., 2003;Stockdale et al., 2001). Environmental factors, such as temperature, precipitation, ecological zone, and amount and type of fertilization signi cantly affecting rice grain quality traits (Iqbal et al., 2020;Patindol et al., 2015). Most importantly, the accumulation of starch content in the grain is closely related to the rate and timing of N fertilization which can be improved by using the optimum rate of N (Yang et al., 2020;Zhou et al., 2020). In a conventional agricultural system, the excessive use of N is not suitable for the accumulation of grain starch and therefore does not result in high-quality rice grains . This may be due to the starch mobilizing enzyme activities, which is severally affected by N fertilization (Tang et al., 2019;Iqbal et al., 2020). Moreover, earlier ndings have also shown that rice grain lling rate (starch accumulation) is strongly associated with the rice grain processing and appearance qualities (Shen et al., 1997;Jin et al., 2000;Sun and Yang, 2008). However, we do not yet have full knowledge of the mechanism for linking soil properties to grain quality characteristics under the joint application of manure and mineral N fertilizers. Therefore, in conventional farming systems, improving the quality of grain is very important by adjusting the proportion of organic and inorganic fertilizer.
It is well documented that fertilization improves the grain yield, however, most of the studies were conducted on a weight basis of organic fertilizers rather than the speci c rate and N concentration along with mineral N fertilizer (Zhang et al., 2015;Arif et al., 2015). Therefore, the primary mechanism of N on a concentration basis remains unknown. Moreover, there is a lack of information on the role and relationship of soil chemical and leaf physiological characteristics to the quality and yield of rice. So, it was expected from the present study that organic N along with synthetic N fertilization could improve soil health, which in turn will have a signi cant role in higher leaf physiological activity, rice yield and grain qualitative traits. The aims of this experiment are: (1) to explore the combined effect of organic and inorganic N on soil quality; (2) to evaluate the integrated effect of organic and mineral fertilizers on leaf physiological activity, rice yields and grain qualitative traits; (3) To examine the relationship among soil quality, leaf physiological activity and grain nutritional quality.

Site description
Field experiment at the research farm of Guangxi University, China was conducted in 2019 and 2020 during early season runs (from March-July) and the late season (from July-November). This site experiences a subtropical monsoon climate, with a total annual rainfall of 1398 mm, and an average annual temperature of 24.8°C ( Figure 1). The soil is classi ed as Ultisols (USDA soil classi cation), which is slightly acidic with a pH of 5.96. The soil physico-chemical properties indicated that the soil contained 18.75 g kg −1 SOC and 1.64 g kg −1 total nitrogen (TN), with other details shown in Table 1. Note: SOC-soil organic carbon, SOM-soil organic matter, N-nitrogen, P-phosphorous, Kpotassium, C: N-carbon to nitrogen ratio.
The rice seeds were germinated in plastic trays, and the uniform-sized 25 days old seedlings were transferred into the rice eld. The nutrient contents of organic fertilizers and the quantity of all treatments are shown in Table 2. The N and K fertilizers were applied in three splits: 50% initially before transplanting, 30% at tillering, and 20% at the heading stage. All phosphate fertilizer was applied before transplanting as a basal dose. Flooding was maintained from transplantation to physiological maturity.
During the entire rice growth period, standard agronomic practices, including irrigation and pesticide application was performed the same for all amendments. Samples were obtained by a core sampler at a depth (0-20 cm) from each treatment immediately after the late-season rice harvest in 2019-2020. Soil sampling was done at ve different points and then mix to make a composite sample, and divide into two parts, one part was used for the measurements of soil nutrients, and the other was stored at 4℃ for molecular analysis.
Soil organic C was determined by the K 2 Cr 2 O 7 -H 2 SO 4 oxidation method (Wang et al., 2003). For soil TN analysis, 200 mg of the samples were processed using the salicylic acid-sulfuric acid-hydrogen peroxide method (Ohyama et al. 1991), and total N was measured using the micro-Kjeldahl technique according to Jackson (1956).

Leaf physiological traits
The Pn and SPAD value was found at tillering, heading and ripening stage during both years. From every plot, fully expanded ag leaves were targeted for Pn and SPAD value assessment. Portable photosynthesis (Li-6400, Li-COR Inc., Lincoln, NE, USA) system was used for Pn measurement on a shiny day: air humidity (70%), light intensity (1200 µmol m −2 s −1 ), CO 2 (375 µmol mol −1 ), and temperature (28°C). A SPAD meter (SPAD-502, Minta Camera Co., Osaka, Japan) was used to take SPAD values of the 4 uppermost leaves on each plant at tillering, heading and ripening stage.

Rice grain yield, grain nutritional and noodles appearance traits
Rice plants from the whole plot area were harvested at maturity to measure the grain yield of rice and accommodated to 14% moisture content. For AC and SC concentration, rice grains were ground using a mortar, and the powder was then degreased two times with anhydrous ether. The total grain SC and AC were determined via the dual-wavelength iodine binding method as suggested by He (1985) and Zhu et al. (2008). Grain crude protein content was measured as the product of grain N contents and 5.75 (protein conversion coe cient) according to Fujihara et al. (2008). The value of dissolved starch was measured according to the method of Weiguo et al. (2005). The noodles expansion rate and evenness were determined according to the procedure as suggested by Shaomao and Mao, (2004) and Guo et al. (2016), respectively.

Statistical analysis
The collected data on soil chemical traits, leaf physiology, yield, and grain qualitative attributes were analyzed according to the ANOVA techniques relevant to RCB design using Statistics 8.1 software. The data were rst checked for a normal test. Furthermore, data in percentage were arcsine transformed to normalize the variables before analysis. The analysis was performed combined over the years, to detect changes between years as well as in fertilizer treatments. Fertilizer treatments were considered as a xed factor, years were considered as a repetitive measured factor and also a xed effect. Likewise, the interaction among fertilizer treatments and the years was taken as a xed effect. But, the interaction of years and treatment with replications was taken as a random effect. The means were separated using Tukey's test at p < 0.05. Linear regression analysis was performed to evaluate the relationship of SOC, TN, Pn, and SPAD value with grain SC and AC.

Soil Chemical properties
The effects of combined organic and inorganic N fertilization treatments on soil chemical attributes are shown in Figure 2. The co-applied fertilizers signi cantly increased SOC and soil organic matter (SOM) during both years. In both years, the SOC and SOM content were maximum in T 3 and T 5 treatments than in the rest of the treatments. Furthermore, the SOC and SOM contents among the different treatments showed the same pattern across years, and average increases in SOC and SOM content were 29.3% and 29.3% in the T 3 treatment, respectively, compared with control (T 2 ). But, T 5 was non-signi cant (P < 0.05) with T 3 . Compared with control, the combined treatments, such as T 4 and T 6 showed signi cantly higher SOC and SOM content. Moreover, substantial enhancements were detected in SOC and SOM 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 TN and available N (AN) content at the 0-20 cm soil depth is shown in Figure 3. Soil TN and AN contents were considerably improved in the combined treatments compared with CT in both years. The average increases in TN and AN were 24.2%, and 17.02%, respectively, in the T 3 treatment compared with control. But, T 5 was similar (P < 0.05) non-signi cant with T 3 . Likewise, the integrated treatments like T 4 and T 6 had considerably higher soil TN and AN contents compared to control.
Furthermore, signi cant improvements were observed in TN and AN contents during the seceding year. The average increment in TN and AN contents in the year 2020 was 9.55% and 12.28%, respectively, which was higher than the year 2019.

Soil Leaf physiological traits
Leaf photosynthetic rate (Pn) and SPAD values at the tillering, heading, and ripening stage was signi cantly affected by N amendments during the year 2019 and 2020 ( Fig. 4 & 5). Both traits exhibited a quadratic trend across growth, with higher values at heading and lower values at the ripening stage in both years. Further, the Pn and SPAD values showed a similar trend across the years. Average across the years, at the tillering stage, the Pn and SPAD were 20% and 23%, respectively, higher in T 2 compared to T 1 , while at the heading and ripening stage, Pn and SPAD were signi cantly higher in T 6 and T 4 compared with other treatments. Average increases across the growth stages and years in Pn and SPAD were 16% and 18%, respectively, in T 6 treatment compared to T 1 . Furthermore, the joint N treatments, such as T 3 and T 5 also improved leaf Pn and SPAD values.

Rice grain nutritional and appearance qualities
Grain nutritive quality is a primary feature of rice, such as starch, amylose and protein content. The collective use of organic and mineral fertilizers had signi cantly improved rice grain SC, AC and PC in both years 2019 and 2020 (Table 3). The integrated treatment i.e., T 6 signi cantly enhanced the SC, AC and PC by 6.4%, 12.7% and 12.2%, respectively, compared to T 2 . But, the treatment T 4 and T 6 were statistically (P<0.05) non-signi cantly different from each other. Similarly, the co-applied organic and synthetic N treatments i.e., T 3 and T 5 also signi cantly increased the nutritive traits of grain compared to sole urea. The lowest content of SC, AC and PC were noted in control plots during both years.
The in uence of joint manure and synthetic N fertilizer application on rice noodle appearance traits are presented in Table 3. The combined fertilization had no signi cant effects on starch dissolving value, noodles expansion rate and evenness.

Rice grain yield
The combined organic-inorganic fertilizer application signi cantly affected the grain yield of rice in both 2019 and 2020 (Table 3). Compared with control (T 2 ), the co-applied N fertilization considerably improved the grain yield of rice 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, such as T 5 and T 3 also signi cantly increased grain yield, compared to 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 to 2019.

Relationship of the soil chemical traits with grain starch and amylose contents
Rice grain starch and amylose content were strongly reliant on soil parameters. This was con rmed by linear regression analysis, which showed that the grain SC was positively correlated with SOC content (R 2 =0.70** Fig. 6 [A]) and TN (R 2 =0.62* Fig. 6 [C]). Similarly, the AC showed a positive relationship with SOC (R 2 =0.52* Fig. 6 [B]) and TN (R 2 =0.58* Fig. 6 [D]). The leaf physiological traits, such as Pn and SPAD values were correlated with SC and AC. The positive correlation was observed of SC with Pn (R 2 =0.51* Fig. 7 [A]) and SPAD (R 2 =0.60* Fig. 7 [C]); as well as of AC and SC with Pn (R 2 =0.62* Fig. 7 [B]) and SPAD (R 2 =0.63* Fig. 7 [D]). Such results showed that the improvements in soil SOC, TN and leaf physiological activity directly affect the starch and amylose accumulation in rice grain.

Discussion
The present agricultural system is massively dependent on CFs, which have adversely affected soil quality, environment and cereal yields as well as grains quality. The improvement of soil fertility and cereal yields and its quality on a sustainable basis is novel research these days (Adekiya et al., 2019;Iqbal et al., 2019). Therefore, the in uence of a cumulative application of manure and mineral fertilizers on soil chemical properties. Leaf physiological traits, grain yield and its qualitative traits of rice were undertaken in eld studies to devise a sound strategy for improvement in these indicators on a sustainable basis.

Soil quality
The collective use of organic and inorganic N signi cantly increased the SOC, SOM, TN and AN concentration in the soil as compared with sole urea application (Fig. 2 & 3). The decomposition of organic fertilizer slowly released nutrients to the soil and exhibited that rising the organic fertilizer ratio from 30-60% had enhanced the soil health. Soil organic C is a vital indicator of soil health and fertility (Stockmann et al., 2013;Johanne's et al., 2017), soil having greater SOC would have better health. A signi cant improvement in SOC content was noted under combined treatments compared with control (Fig. 2). The enhancement in SOC and SOM could be explained by two possible mechanisms, i.e., the direct addition of C by adding organic fertilizer and improvement in rhizosphere quality by increased root exudate as a result of improved plant root activities (Bitew and Alemayehu, 2017). In fact, the SOC at any certain position strongly relies on the annual organic turnover (i.e., plant root exudates, root and shoot stubbles, and their recycling) (Adekiya et al., 2019;Biratu et al., 2019). In accordance with our nding, Purakayastha et al., (2008), documented that manure signi cantly improves soil C in the top layer.
The co-applied fertilization of manure and mineral fertilizers signi cantly improved the content of total and available N (Fig. 3). This is mainly due to the addition of organic fertilizer such as CM or PM, which had a positive effect on the soil N and other nutrients. This might be related to the direct addition of N when the organic fertilizer was added to the soil had increased the TN, but its decomposition had increased the AN (Wei et al., 2013). Another possible explanation for enhancements in soil nutrients might be related to the ability of organic fertilizers in reducing the leaching of nutrients and thus had a greater capability of absorbing these leachates. The greater absorbing capability is due to improvement in water holding capacity, a decline in nutrients leaching, and consequently greater availability of NPK (Adekiya et al., 2019;Murmu et al., 2013). Our results are also in line with Purakayastha et al. (2008), who concluded that organic manure coupled with mineral fertilizer improves soil organic C by 11-80% and soil total N by 56-90% in the upper layer.

Leaf physiological activity
The chlorophyll content directly affects the plant photosynthetic activities (Wittmann et al., 2001), and hence crop production. Photosynthesis is the key driver of crop production by increasing plant growth and dry matter accumulation as well as displayed a strong response to N-uptake and water (Wittmann et al., 2001). Leaf Pn and SPAD values were noted higher under co-applied manure and mineral N fertilizers treatments compared with the sole synthetic N fertilization (Fig. 3). This increment might be due to improvement in soil health and fertility, declines in nutrients leaching downside (Geng et al., 2019) due to increased organic and inorganic fertilization. We con rmed that the combined manure and mineral N improved soil fertility (Fig. 2 & 3), which eventually increased the root's ability to absorb more water and nutrients, lead to higher stomatal conductance and CO 2 xing (Iqbal et al. 2019). The higher Pn and SPAD values of the plant at heading and ripening stages under combined fertilization treatment could be due to faster release of nutrient from mineral fertilizers and slow and steady releases of the plant needed nutrient from manure upon decomposition (Iqbal et al., 2020;Ali et al., 2020).

Rice grain yield
The integrated use of manure and synthetic N signi cantly increased the grain yield of rice compared with that of sole synthetic fertilizer (Table 3). The improvements in grain yield could be owing to the enhanced soil fertility and functionality under mutual treatments, which ultimately increased crop growth, biomass and yield. Our ndings are also in agreement with the results of  and Akhtar et al. (2018) who reported that cereals grain yield is closely associated to soil properties and fertilization application. Similarly, Mangalassery et al. (2018) concluded that the continued application of organic manure coupled with mineral fertilizers improves the biomass and rice yield.

Rice grain nutritional and appearance traits
The concentration of SC and AC strongly affects the eating and cooking quality of rice noodles (Adu-Kwarteng et al., 2003). The combined (organic fertilizer and CF) fertilization considerably improved the AC and PC of grain compared with sole CF fertilization ( Table 3). The observed improvement in PC and AC under integrated amendments explores that both fertilizers deliver enough amount of nutrients (macro + micro), mainly N, which is essential for growth and development for rice. Kumar et al. (2007), nding are in agreements with our nding, and had reported that co-application of inorganic and organic fertilizers enhance grain quality and AC by 7% than sole inorganic fertilizer. Similarly, Sravan and Sing (2019) concluded that the higher grain protein content of rice was associated with the improvement of soil fertility. Moreover, the manure coupled mineral fertilizers did not signi cantly in uence rice noodle physical traits, such as starch dissolving value, expansion rate, and evenness (Table 3). In general, rice grain and noodle physical quality do not indicate variability in the short-time intake under manure fertilization. However, the combined organic and inorganic amendment improves the physical quality of rice grain, which is also supported by earlier researchers (Kesarwani et al., 2016).
Soil quality and leaf physiological traits had affected the grain nutritional quality. The correlation analysis showed a strong relationship between soil properties and rice grain starch and amylose (Fig. 5 &  6). Changes in soil supply C and N and the improvement in the potential and regulation of source-sinktranslocation might be the possible explanation for this shift, which had a positive impact on the grain lling process and grain quality . The leaf Pn and chlorophyll content showed a signi cant and positive relation with grain quality (Fig. 6). The higher leaf Pn and SPAD values were primarily due to the enhanced soil health under integrated organic and inorganic armaments (Fig. 2 & 3), which eventually increased root growth, nutrients and water uptake. Such improvement had increased the leaf physiological activity. Moreover, the combined treatments prolonged N supply and leaf physiological activity during the ripening period; and thus, might have enhanced the accumulation capacity of the "source", and ultimately improved the grain lling process.

Conclusion
In this study, the combined application of manure and synthetic fertilizers signi cantly improved the soil fertility, leaf physiological activity, rice yield and grain quality attributes. Higher SOC, TN, Pn, AC and PC, as well as grain yield, were observed in the treatments as compared to sole urea application during both years. Improvements in leaf Pn, SPAD value, grain yield and grain nutritional characteristics were primarily due to enhanced soil quality under manure application. In addition, according to the linear regression analysis, the nutritional characteristics of rice grain are strongly dependent on soil fertility and leaf physiological activity. It is concluded that the combined application of manure and chemical fertilizers at 30:70 ratio is the best strategy for producing higher rice yields both quantitatively and qualitatively along with improvement in soil fertility.  Figure 1 Mean monthly rainfall and air temperature during the experimental years.

Figure 2
Changes in soil organic carbon (g kg-1) and soil organic matter (g kg-1) as in uenced by combined organic and inorganic N fertilization. Means sharing the same letter within a category are nonsigni cantly different from each other using the Tukey HSD test at P < 5%. **= signi cant at 1%, and ns= non-signi cant. For treatments combination please see Table 3.

Figure 3
Changes in soil total nitrogen (g kg-1) and soil available nitrogen (mg kg-1) as in uenced by combined organic and inorganic N fertilization. The mean comparison was made using Tukey tests for treatments and years and the lettering was on basis of Tukey HSD test at 5% using simple effect. **= signi cant at 1%, respectively and ns= non-signi cant. For treatments combination please see Table 3.

Figure 4
Changes in leaf net photosynthetic rate as in uenced by combined organic and inorganic N fertilization. The mean comparison was made using Tukey tests for treatments and years and the lettering was on Page 22/24 basis of Tukey HSD test at 5% using simple effect. **, *= signi cant at 5% and 1%, respectively and ns= non-signi cant. For treatments combination please see Table 3.

Figure 5
Changes in leaf SPAD values as in uenced by combined organic and inorganic N fertilization. The mean comparison was made using Tukey tests for treatments and years and the lettering was on basis of Tukey HSD test at 5% using simple effect. **, *= signi cant at 5% and 1%, respectively and ns= nonsigni cant. For treatments combination see please Table 3.

Figure 7
Linear regression of starch and amylose content with net photosynthetic rate (Pn) and SPAD values *= signi cant at 1%. n= 6.