Optimization of combining controlled-release urea of different release period and normal urea improved rice yield and nitrogen use efficiency

ABSTRACT Controlled-release urea (CRU) and normal urea have been shown to improve yield and nitrogen use efficiency (NUE) in rice. However, the effects of combined fertilizers (CRU and normal urea, at different N ratios) on rice yield, temperature and solar radiation utilization efficiency (TSRUE) and NUEs remain unclear. In this study, CRU with release periods of 60 days and 100 days were mixed with urea at N ratios of 7:3, 6:4, 5:5, 4:6 and 3:7 and applied during the rice-growing season in a rice-wheat cropping system. Rice yield, dry matter accumulation (DMA), TSRUE and NUEs were investigated. The yields under one-time fertilization mode 2 (OFM2) and OFM8 were 5.36%-9.70% higher than that of farmer fertilization practices (FFP) on average across 2018 and 2019. The NUEs under OFM2 and OFM8 was 3.87%-35.56% and 6.26%-58.56% higher than that under FFP, respectively, across 2018 and 2019. Correlation analysis showed that TSRUE were significantly positively correlated with yield, DMA and NUEs. The higher TSRUE under OFM2 and OFM8 contributed to the synergistic improvement of yield and NUEs. Both OFM2 and OFM8 improved rice yields and NUEs compared to FFP while having lower fertilizer and labor costs; therefore, these treatments are worthy of promotion and application.


Introduction
Rice is the staple food for more than 70% of the total population in China, and it plays a very important role in China's food production and consumption (Che et al. 2015). Nitrogen (N) is a determining factor for crop growth and plays a vital role in maintaining rice production (Grant et al. 2012). More than 120 million tons of nitrogen fertilizer are applied worldwide every year, and the amount of nitrogen fertilizer consumed in China accounts for 30% of the total amount of nitrogen fertilizer applied worldwide (Peng et al. 2010). The traditional fertilization method includes three or four fertilizer applications and is time consuming, which seriously restricts the sustainable development of modern agriculture Zhao et al. 2021). Controlled-release urea (CRU) is beneficial because it releases nutrients slowly, is effective for a long period of time, and requires

Experimental location and weather conditions
The field trials for this study were performed at Shatou Experimental Farm (119°32ʹE, 32°18ʹN), Yangzhou University, Jiangsu Province, China, during the summer cropping season (between May and November) in 2018 and 2019. Yangzhou belongs to the transition zone from subtropical monsoon humid climate to temperate monsoon climate. The experiments were laid out in a completely randomized block design, with three replicates. The cropping system in this experiment was a wheat-rice rotation system. The field sites had a clay-loam soil with moderate fertility [organic matter, 21.01 g kg −1 ; total N, 1.45 g kg −1 ; alkaline hydrolysis nitrogen, 157.29 mg kg −1 ; available phosphorus (P), 18.77 mg kg −1 ; and available potassium (K), 138.65 mg kg −1 ] and a pH of 7.79. The mean temperature (Figure 1(a)) and sunshine hours (Figure 1(b)) during the rice-growing seasons of 2018 and 2019 were measured at a weather station close to the experimental site and are shown in Figure 1. The minimum (T min ), maximum (T max ), and mean (T mean ) temperatures; rainfall; mean relative humidity; and sunshine duration (SD) during the rice-growing seasons of 2018 and 2019 are shown in Supplementary Table S1.

Experimental design and field management
One japonica rice (Oryza sativa L.) variety which is widely grown in local production, Nanjing 9108 were used in this study. Plant height, growth period, amylose content, gel consistency of Nanjing 9108 was 94.6 cm, 153 d, 9.34%, 98 mm, respectively. Seeds were sown on plastic plates on 31 May in both 2018 and 2019, at a seeding rate of 120 g of dry seeds per plate. The seedlings were manually transplanted onto hills on 20 June. The area of each experimental plot was 20 m 2 , with 50 cm spaces between adjacent plots, and the hill spacing was 12 cm×30 cm, with four seedlings per hill. All the plots were separated by soil ridges (35 cm wide and 20 cm high) and covered with plastic film. Twelve fertilization treatments were applied: no N fertilization (0 N), typical farmer fertilization practices (FFP) (total N was 285 kg ha −1 ), and ten one-time basal fertilization modes (OFMs) ( Table 1). OFM1-OFM5 refer to a mixture of CRU with a release period of 60 days and normal urea at N (total N was 285 kg ha −1 ) ratios of 7:3, 6:4, 5:5, 4:6, and 3:7, respectively. OFM6-OFM10 refer to a mixture of CRU with a release period of 100 days and normal urea at N (total N was 285 kg ha −1 ) ratios of 7:3, 6:4, 5:5, 4:6, and 3:7, respectively. The FFP is to apply nitrogen fertilizer four times during the growth period of rice. Calcium  superphosphate (P 2 O 5 content: 12%) and potassium chloride (K 2 O content: 60%) were applied as basal fertilizers at rates of 150 kg P 2 O 5 ha −1 and 240 kg K 2 O ha −1 , respectively. Insect pests, pathogens, and weeds were controlled using common chemical treatments.

Plant sampling and data collection
At the maturity stage, the yield was determined from all rice plants in a 6.0 m 2 area of each plot (except plants on the edge of the area) and was calculated based on a standardized moisture content of 14%. The number of panicles per m 2 , number of spikelets per panicle, filled-grain percentage, and grain weight were determined from 50 plants (except plants on the edge of the plot) sampled randomly from each plot. The harvest index was calculated as the grain yield divided by the total dry matter produced aboveground. To record the total aboveground biomass, the sampled plants were dried at 105°C for 30 min to halt biological activity and then dried at 80°C to constant weight (DHG-9625A, Shanghai Yiheng Scientific Instruments Co., Ltd., Shanghai, China). The rice plants from six hills were sampled from each plot according to the average tiller numbers at the jointing stage (JS), heading stage (HS) and maturity stage (MS). The solar radiation and temperature data were downloaded from the China Meteorological Data Service Centre (http:// data.cma.cn/en/?r=site/index) and are provided in the supplementary Table S2. The methods for the calculating temperature and solar radiation utilization were those described by Xing et al. (2017). Total N analysis was conducted on plant samples collected at JS, HT and MS. The method of determining the N content was described by Zhao et al. (2021). Aspects of NUEs, such as N recovery efficiency (NRE), N agronomic efficiency (NAE), and N partial productivity (NPP), were calculated using the following formulas: NRE = (N up −N0 up )/FN, NAE = (GY−GY 0 )/FN, NPP (kg kg −1 ) = GY/FN, where TB up and TN up denote the total aboveground biomass and total aboveground N uptake, respectively; GY and GY 0 represent the grain yields in N-fertilized plots and N 0 plots, respectively; N up and N0 up denote the total aboveground N uptake in N-fertilized plots and N 0 plots, respectively; and FN denotes the total N application rate in N-fertilized plots.

Data analysis
The statistical analyses consisted of analyses of variance (ANOVAs). Means were compared by the Duncan's multiple range test at the 0.05 probability level. All statistical analyses were conducted using the SPSS software package (18.0; SPSS Inc., Chicago, IL, USA), and graphs were generated using Origin 8.0 (OriginLab, Hampton, MA, USA).

Grain yield and harvest index
The analysis of variance between years and among treatments showed that there were significant differences in yield between years and among treatments (Table 2). In both 2018 and 2019, the rice yield and harvest index first increased and then decreased with decreasing ratios of CRU to normal urea. The yield of OFM2 (CRU with a release period of 60 days and normal urea at an N ratio of 6:4) was the highest and was 1.03%-18.40% higher than those of OFM1, OFM3, OFM4 and OFM5 across two years. In 2018 and 2019, the yield of OFM8 (CRU with a release period of 100 days and normal urea with at an N ratio of 5:5) showed similar regularity (Table 2). We found that the yields of OFM2 and OFM8 were 3.88%-7.42% and 6.84%-11.97% higher than that of FFP, respectively, across 2018 and 2019. In terms of yield components, the number of panicles per m 2 of OFM5 was the highest in 2018 and 2019 and was 18.99% and 15.24% higher than that under FFP, respectively. The harvest index under OFM2 was significantly higher than that under FFP in 2018. There were no significant differences in harvest index between OFM8 and FFP in 2018 or 2019.

Dry matter accumulation (DMA)
The analysis of variance among years and treatments showed that the differences in DMA from sowing to JS, from JS to HS and from HS to MS were all significant (Table 3). At JS, the DMA under OFM4 was the highest and was 8.03%-70.65% higher than that under the other treatments. From JS to HS, the DMA under FFP was 24.62%-94.97% higher than that under the OFMs in which the release period of the CRU was 60 days. Among the OFMs with CRU with a release period of 100 days, OFM7 had the highest dry matter accumulation from JS to HS, which was 7.31%-52% higher than that of the other OFMs. From HS to MS in 2018 and 2019, the DMA under OFM1 was the highest and was 4.70%-53.62% higher than that under the other treatments (Table 3). A significant difference in DMA over the whole growth period between OFM8 and FFP was observed in 2018 and 2019 (p < 0.05). From sowing to MS, the DMA under OFM2 was 1.34% and 1.32% higher than that under FFP in 2018 and 2019, respectively, and the DMA under OFM8 was 8.35% and 8.31% higher than that under FFP in 2018 and 2019, respectively (Table 3). 0 N, FFP, OFM, represents no nitrogen application, farmers' fertilizer practice, one-time fertilization mode, respectively. OFM1-OFM5, represents the proportion of controlled-release urea (the release longevity was 60 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. OFM6-OFM10, represents the proportion of controlled-release urea (the release longevity was 100 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. NS, not significant at the P = 0.05 level; **, significant at the P = 0.01 level. Different letters indicate statistical significance at P = 0.05 within the same column, year, and variety.

Temperature and solar radiation utilization efficiency
The grain yield/EAT under OFM2 and OFM8 were the highest and were 3.89%-7.41% and 6.78%-11.98% higher than that under FFP across 2018 and 2019, respectively ( Figure 2). The grain yield/CSR under OFM2 and OFM8 were the highest and were 0.7%-0.86% and 7.69%-7.83% higher than that under FFP, respectively (Figure 2). At JS, the dry matter/EAT under OFM4 was 53.66%-55.56% higher than those under FFP. From HS to MS, the dry matter/EAT under OFM1 was 25%-30% higher than those under FFP (Table 4). Solar radiation utilization (SEU) was affected by the OFM and the year and was not affected by the interaction of treatment and year (Table 5). At JS, the dry matter/CSR (SEU) under OFM4 was 54.1%-55.56% higher than that under FFP in both 2018 and 2019. From HS to MS, no significant difference in dry matter/CSR was found between OFM1 and OFM2, and the dry matter/CSR under OFM2 was significantly higher than that under FFP. Similar trends were also found under OFM8; the dry matter/CSR (SEU) under OFM8 in 2018 and 2019 was 16.18% and 15.31% higher, respectively, than that under FFP (Table 5).

N accumulation and N uptake rate
The N accumulation under both OFM2 and OFM8 was significantly higher than that under FFP at JS. From JS to HS, N accumulation under OFM2 and OFM8 was 9.23% and 38.94% higher than that under FFP on average in 2018 and 2019, respectively. N accumulation under OFM2 and OFM8 was 1.6%-13.82% higher than that under FFP from 2018-2019 (Supplementary Table   Table 3. Effects of different fertilization modes on the dry matter accumulation (t ha −1 ) of rice in 2018 and 2019.
S2). N accumulation and the N uptake rate showed downward trends with the decrease in the ratio of CRU to urea in both 2018 and 2019. From sowing to MS, N accumulation under OFM2 and OFM8 was 14% and 13.32% higher than that under FFP on average from 2018-2019, respectively. The change trend for the N uptake rate was similar to that for N accumulation (Supplementary Table S2).

N use efficiency (NUE)
Nitrogen agronomy efficiency (NAE) first increased and then decreased with the decrease in the ratio of CRU to urea in 2018 and 2019. From OFM1 to OFM5, the NAE under OFM2 was the highest, and it was 8.29%-14.11% higher than that under FFP. From OFM6 to OFM10, OFM8 had the highest NAE, which was 14.53%-22.78% higher than that under FFP (Figure 3(a)). In 2018 and 2019, as the ratio of CRU to normal urea decreased, the N recovery efficiency (NRE) of each fertilization mode gradually declined. The NRE under OFM1, OFM2 and OFM3 were 7.98%-35.56% higher than that under FFP.
The NRE under OFM6 to OFM10 were 6.26%-58.56% higher than that under FFP (Figure 3(b)). Similar to the change in NAE, as the ratio of CRU to normal urea decreased, in 2018 and 2019, N partical productivity (NPP) first increased and then decreased. The NPP under OFM2 in 2018 and 2019 was 3.87% and 7.41% higher than that under FFP, respectively. The NPP under OFM8 in 2018 and 2019 was 6.78% and 11.97% higher than that under FFP, respectively (Figure 3(c)).

Correlation analyses
Grain yield, NAE, NRE, NPP, dry matter accumulation, N accumulation and N uptake rate were found to be significantly positively correlated with grain yield/EAT and grain yield/CSR, which implied that rice grain yield and NUEs were intricately related to multiple factors. Yield and NUEs were positively correlated with temperature and solar radiation utilization efficiency (Table 6). , represents the proportion of controlled-release urea (the release longevity was 60 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. OFM6-OFM10, represents the proportion of controlled-release urea (the release longevity was 100 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. Different lowercase letters indicate statistically significant differences among fertilization modes in 2018 according to a Duncan's multiple range test (P < 0.05). Different capital letters indicate statistically significant differences among fertilization modes in 2019 according to a Duncan's multiple range test (P < 0.05). The data are presented as mean ± SE (n = 3).

Effects of different fertilization modes on grain yield and dry matter accumulation
Our study indicates that one-time applications of CRU mixed with conventional urea achieved grain yields of 10.17 t/ha −12.82 t/ha across 2018-2019. Rice yield depends mainly on the number of panicles m -2 , number of spikelets per panicle, seed-setting rate, and 1000-grain weight (Xie et al. 2019). We found that mixed application of CRU and normal urea achieved the highest yields both in 2018 and 2019 (Table 2). Unlike with the application of conventional urea, after the application of CRU, which can release certain nutrients in the early stage of rice growth and facilitate the development of a suitable number of tillers, the sufficient supply of nutrients results in the formation of high-yield populations in the middle and late stages of growth; CRU helps to maintain leaf color, delay rice leaf senescence, promote grain filling, increase the number of grains per panicle, and thereby increase rice yield (Xie et al. 2006;Li et al. 2015;Wei et al. 2017). Studies have shown that compared with the application of CRU alone, the mixed application of CRU and normal urea can significantly increase rice yield and the number of effective panicles (Xue et al. 2018). We found similar results: the mixed application of slow-release fertilizer and urea increased the number of effective panicles by 3.42%-18.99% compared with that under FFP in 2018 and 2019 (Table 2). When CRU and conventional urea are applied as the basal fertilizer, the conventional urea provides an early N supply, which leads to tiller development and increases the number of effective panicles. Rice yield is the result of dry matter accumulation, transportation, distribution and 0 N, FFP, OFM, represents no nitrogen application, farmers' fertilizer practice, one-time fertilization mode, respectively. OFM1-OFM5, represents the proportion of controlled-release urea (the release longevity was 60 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. OFM6-OFM10, represents the proportion of controlled-release urea (the release longevity was 100 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. S-J, from sowing to jointing stage; J-H, from jointing stage to heading stage; H-M, from heading stage to maturity stage. NS, not significant at the P = 0.05 level; **, significant at the P = 0.01 level. Different letters indicate statistical significance at P = 0.05 within the same column, year, and variety.
transformation within the plant population. In our study, the dry matter accumulation under OFM2 and OFM8 was the highest, at 1.32%-8.35% higher than that under FFP from 2018-2019 (Table 3). Dry matter production and accumulation are the basis for high rice yields . The difference in dry matter accumulation is the most direct manifestation of rice population formation (Girsang et al. 2019). The application of CRU increased dry matter accumulation in rice plants. One possible reason for this effect is that the application of CRU enhanced the leaf area index and SPAD value in each phenological period and ensured that photosynthetic activity remained strong during the late growth period of rice, which promoted dry matter accumulation (Zhao et al. 2021).
Studies have also shown that the combined application of a certain proportion of normal urea at the same time as the one-time application of CRU results in significantly greater yields than the application of CRU alone (Zheng et al. 2016. However, Li et al. (2017) reported that a single basal application of CRU did not significantly affect the grain yield compared to that under a split application of normal urea in central China. These differences in results may be caused by the different environmental conditions, soil types and rice varieties in these studies. Environmental factors (temperature, moisture, pH) can affect the release of the nitrogen in CRU, which may be the reason why the number of spikelets per panicle under the one-time application of CRU was lower than that under FFP. Generally, moisture and temperature are the key factors that restrict the release of N by CRU (Grant et al. 2012). It has been reported that the nitrogen demand in rice is higher from the tillering stage to the milk stage but lower at the seedling stage and mature stage, forming an S-shaped curve . According to the current study, among the different N ratios of CRU to urea, OFM2 and OFM8 0 N, FFP, OFM, represents no nitrogen application, farmers' fertilizer practice, one-time fertilization mode, respectively. OFM1-OFM5, represents the proportion of controlled-release urea (the release longevity was 60 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. OFM6-OFM10, represents the proportion of controlled-release urea (the release longevity was 100 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. S-J, from sowing to jointing stage; J-H, from jointing stage to heading stage; H-M, from heading stage to maturity stage. NS, not significant at the P = 0.05 level; **, significant at the P = 0.01 level. Different letters indicate statistical significance at P = 0.05 within the same column, year, and variety.
were able to meet the fertilizer requirements of rice. The yield and dry matter accumulation under OFM2 and OFM8 were higher than that under FFP, which is in line with the trend of fertilizer requirements in rice. The release rate of nutrients supplied by CRU can be synchronized with the needs of rice growth and development to increase yield and NUE (Yang et al. 2021). However, the contents of soil N and the patterns of N release will be the focus of future research.

Effects of different fertilization modes on temperature and solar radiation utilization efficiency
Temperature and radiation are the main climatic factors that influence rice growth and yield (Deng et al. 2015). Temperature and solar radiation utilization efficiency (TSRUE) can be improved by changing the cultivation mode and adjusting cultivation management measures (Li et al. 2011). Xing et al. (2017) found that the yield/EAT, dry matter/EAT and SEU could contribute to high yields. Our results are consistent with this, and we found that the yield/EAT and SEU under OFM2 and OFM8 were 3.89%-11.98% and 0.7%-7.83% higher than those under FFP, respectively (Figure 2). A higher , represents the proportion of controlled-release urea (the release longevity was 60 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. OFM6-OFM10, represents the proportion of controlled-release urea (the release longevity was 100 days) and normal urea is 7:3, 6:4, 5:5, 4:6, 3:7, respectively. Different lowercase letters indicate statistically significant differences among fertilization modes in 2018 according to a Duncan's multiple range test (P < 0.05). Different capital letters indicate statistically significant differences among fertilization modes in 2019 according to a Duncan's multiple range test (P < 0.05). The data are presented as mean ± SE (n = 3).
utilization efficiency for environmental resources such as temperature and solar energy is closely related to higher grain yields and higher NUE in rice (Wassmann et al. 2009). Many studies have shown that radiation has a positive effect on the growth and development of rice, and the amount of solar radiation required by rice in different phenological periods is different (Islam and Morison 1992). It is interesting to note that the temperature and solar radiation utilization under OFM2 and OFM8 were higher than those under FFP from HS to MS. Ying et al. (1998) found that more EAT and solar radiation accumulation during the grain-filling period is beneficial to the production, accumulation and transformation of photosynthetic matter in rice and contributes to the formation of higher yields.

Effects of different fertilization modes on N accumulation and NUE
In the present study, at the same N application rates, the mixed urea treatments provided a consistent improvement in the N accumulation and NUE of rice compared with the normal urea treatments. Compared with the application of normal urea, the application of CRU improved N uptake and NUE Lu et al. 2016). The trend in NUEs followed a similar pattern as that in rice grain yield when the different treatments were compared with FFP from 2018 to 2019 (Supplementary Table S2, Figure 3). In this study, we found that the N accumulation and NUEs under OFM2 and OFM8 were higher than those under FFP (Supplementary Table S2, Figure 3). The rate of N absorption and utilization by rice is slower than the rate at which urea releases N, this discrepancy may be the main reason for the lower NUE when urea is applied (Ladha et al. 2005). The N supply under the urea treatments surpassed the N uptake of the rice plants before HS (Yang et al. 2021). The NAE is an important index that measures the response of yield to increases in the N application rate. In our study, the NAE was 11.07-25.41 kg kg −1 (Figure 3(a)). The NRE is another index that is used to express fertilizer N uptake efficiency. In the present study, we found that the combined application of CRU and normal urea improved the NRE by 6.26%-58.56% compared to that under FFP (Figure 3(b)). Studies have shown that the application of CRU can increase the NRE by 13.2-80.3% (Zheng et al. 2016. It has been reported that slow-release fertilizer can effectively reduce nitrogen losses through denitrification, NH 3 volatilization, leaching and surface runoff and thereby further improve nitrogen recovery efficiency (Mi et al. 2017;Sun et al. 2019). We found that the NPP under OFM2 and OFM8 was 3.87%-11.97% higher than that under FF (Figure 3(c)). The improvement in NUEs achieved through the combined application of CRU and normal urea may have occurred because the N released from the mixed fertilizer closely matches the N requirements of rice plants; this likely enhanced the activities of nitrogen transformation-related enzymes such as glutamine synthetase, glutamine 2-oxoglutarate transaminase and nitrate reductase in rice leaves (Yang et al. 2012). It has been reported that the release rate of N from CRU can basically be synchronized with the needs of rice growth and development; this synchronization promotes the absorption of N by rice and improves the NUE of rice (Geng et al. 2015;Yang et al. 2021). In this study, the nitrogen release rate after the combined application of CRU and normal urea met the N demands of rice throughout the growth period. Understanding the relationship between N uptake requirements and grain yield is essential for devising fertilizer management practices to optimize N fertilizer application and increase grain yield (Setiyono et al. 2010). The lowest N demand has been shown to occur during the earliest and latest growth periods, while plants need more N during the intermediate period of rapid growth (Overman and Scholtz 1999). Importantly, the combination of CRU and normal urea can be applied once, without the need for topdressing, which reduces labor costs and is feasible given the shortage of rural labor in China. Hence, synchronizing fertilizer input with crop requirements is very important for crop production. We speculate that under OFM2 and OFM8, urea provides the N required for the early growth of rice, and CRU provides the N in the middle and late growth stages; this process ultimately leads to a synergistic increase in rice yield and NUE. Additionally, the grain yield/EAT and grain yield/CSR were found to be significantly positively correlated with grain yield, NAE, NRE, NPP, dry matter accumulation, N accumulation and the N uptake rate (Table 6). Similarly, Tao et al. (2013) found a significant positive relation between radiation and yield. The TSRUE under OFM2 and OFM8 resulted in the synergistic improvement of yield and NUEs. Correlation analysis indicated that the grain yield/EAT and grain yield/CSR may be used as indicators of grain production.

Conclusion
The yield, TSRUE and NUEs under OFM2 (CRU with a release period of 60 days and normal urea at an N ratio of 6:4) and OFM8 (CRU with a release period of 100 days and normal urea with at an N ratio of 5:5) were higher than those under FFP. The N released under OFM2 and OFM8 met the N demands of rice and simultaneously achieved higher yields, higher NUEs and higher TSRUE. The higher TSRUE under OFM2 and OFM8 contributed to the synergistic improvement of yield and NUEs. These findings contribute to our understanding of the one-off application of the combination of CRU and normal urea to optimize N management in an economical and environmentally friendly way.