One-time application of controlled-release blended
fertilizer increases rice yield and nitrogen utilization by
optimizing root morphological trait distribution and
nitrogen uptake

Yuhui Wang, Shen Gao, Jie Sun, Bin He, Wenjun He, Weike Tao, Xin'ao Tang, Zhi Geng, Zhengyang Wu, Weiwei Li, et al.

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To cite this version:

Yuhui Wang, Shen Gao, Jie Sun, Bin He, Wenjun He, et al. One-time application of controlled-release blended fertilizer increases rice yield and nitrogen utilization by optimizing root morphological trait distribution and nitrogen uptake. The Crop Journal, 2025, 13 (4), pp.1234-1235. 10.1016/j.cj.2025.04.008 . hal-05067544v2

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# One-time application of controlled-release blended fertilizer increases rice yield and nitrogen utilization by optimizing root morphological trait distribution and nitrogen uptake

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Yuhui Wang<sup>a,c</sup>, Shen Gao<sup>a</sup>, Jie Sun<sup>a</sup>, Bin He<sup>a</sup>, Wenjun He<sup>d</sup>, Weike Tao<sup>a</sup>, Xin'ao Tang<sup>a</sup>, Zhi Geng<sup>a</sup>, Zhengyang Wu<sup>a</sup>, Weiwei Li<sup>a,b</sup>, Zhengrong Jiang<sup>a,c</sup>, Zhenghui Liu<sup>a</sup>, Yanfeng Ding<sup>a</sup>, Soulaiman Sakr<sup>c</sup>, Pengfu Hou<sup>b,*</sup>, Ganghua Li<sup>a,*</sup>

## ARTICLE INFO

**Article history:**

Received 26 February 2025

Revised 15 April 2025

Accepted 22 April 2025

Available online 10 May 2025

**Keywords:**

Rice (*Oryza sativa* L.)

Controlled release blended fertilizer

Root

Yield

Nitrogen utilization

## ABSTRACT

One-time application of controlled-release blended fertilizer (CRBF, a mixture of five nitrogen (N) fertilizers in a certain ratio) can achieve high yield and N use efficiency (NUE) in rice (*Oryza sativa* L.). However, the effects of CRBF with one-time application on root spatial distribution and physiological characteristics remain unclear. We measured the effects of CRBF with one-time application on rice yield, NUE, root morphology and growth, and N uptake capacity in field and root box experiments. Six N treatments were set up: no nitrogen (N₀), high-yield three-split application of urea as a control (CK), urea (U) with broadcast, U with side-deep fertilization, CRBF with broadcast, and CRBF with side-deep fertilization. Our findings showed that root characters were positively correlated with yield and NUE. Compared to CK and U treatments, CRBF with one-time applications increased root characters (including root biomass, root N uptake, root activity, and the expression level of ammonium transporters) at tillering and heading stages. The root length, surface area and volume in the 0–10 cm soil layer enhanced under CRBF with one-time applications at tillering stage, and in the 0–20 cm soil layer at the heading stage. This contributed a 5.96%–39.40% and 3.69%–16.87% increase in plant dry matter accumulation and N uptake, and a 2.08%–18.28% and 14.60%–149.57% increase in yield and NUE, in 2022 and 2023, respectively. Taken together, our findings showed that one-time application of CRBF could increase rice yield and NUE by optimizing the root morphology distribution and N uptake.

© 2025 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

## 1. Introduction

Rice (*Oryza sativa* L.) sustains more than half of the world's population as a staple food, particularly in Asia. Fertilizer application is essential for increasing crop yields, and N fertilizer, as one of the key inputs for rice cultivation, can significantly improve grain yield [1,2]. However, excessive or inappropriate N fertilizer application may result in N losses through run-off, leaching, ammonia volatilization, and N₂O emissions, leading to reduced N use effi-

ciency (NUE) and exacerbating the global greenhouse effect and other environmental issues [3]. To meet these challenges, innovative N management strategies are urgently needed. One-time application of controlled-release blended fertilizer (CRBF), obtained by combining several types of controlled-release N fertilizers (CRNFs) and granular urea in fixed proportions, is an attractive N management approach to increasing rice yield and NUE while reducing environmental pollution [4–6].

Root is the primary organ responsible for nutrient uptake in crops [7]. Root traits (biomass and morphology) and their distribution in different soil layers influence crop yield and NUE. The formation and characteristics of root morphology, such as length, surface area, and volume, and its spatial distribution are regulated

<footnote><sup>a</sup>Sanya Institute of Nanjing Agriculture University, Jiangsu Collaborative Innovation Center for Modern Crop Production, Key Laboratory of Crop Physiology Ecology and Production Management, Nanjing Agricultural University, Nanjing 211800, Jiangsu, China</footnote>

<footnote><sup>b</sup>Key Laboratory of Agro-Environment in Downstream of Yangze Plain, Ministry of Agriculture and Rura Affairs, Nanjing 210014, Jiangsu, China</footnote>

<footnote><sup>c</sup>Institut Agro, University of Angers, INRAE, IRHS, SFR QUASAV, Angers 49000, France</footnote>

<footnote><sup>d</sup>Taizhou Academy of Agricultural Sciences, Taizhou 317000, Zhejiang, China</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: pengfuhou100smby@163.com (P. Hou), lgh@njau.edu.cn (G. Li).</footnote>
by inorganic N availability in various soil layers [8–10]. Liu et al. [9] introduced the study of wheat root traits and N uptake and showed that NUE was higher in plants with shallower root angles, more numerous roots, and finer root diameters. Grain yield and NUE of rice were highly dependent on root morphology [11,12]. Root morphology in different soil layers was influenced by soil N content [13,14]. Ammonium shaped root architecture by regulating auxin and reactive oxygen species, limiting root elongation and stimulating the development of lateral roots or the expansion of root hairs [15]. In contrast, nitrate positively influenced lateral root initiation and elongation through the involvement of auxin and gibberellins [16]. Vertical distribution of roots was necessary for improving rice yield and NUE [8,17]. Increasing the proportion of roots in the 10–20 cm soil layer at the maturity stage increased rice yield [18]. Rice root distribution and their morphological traits determined their N uptake and utilization capacity, influencing shoot growth, N accumulation, NUE and grain yield [7]. In flooded paddy soils, the primary form of N absorbed by rice is ammonium, which is mediated by ammonium transporters (OsAMTs) [19,20]. These transporters influence the root's response to ammonium through apoplast acidification and auxin transport activation [21]. In rice, OsAMTs are the main ammonium transporters, containing 11 putative transmembrane-spanning domains with OsAMT1.1, OsAMT1.2 and OsAMT1.3 playing major roles [19,22]. Among them, OsAMT1.1 was highly expressed in roots, accounting for 25% of the total ammonium absorbed by roots, and mediated ammonium transport from root to the shoot [23]. Stimulating the high expression of ammonium transporters was crucial for improving N uptake in rice roots, which in turn offered a viable way to achieve higher rice yield and improved NUE [12,24,25].

Both the application methods and types of N fertilizers shape the soil N content and distribution in rice production [26,27], thereby influencing root growth and distribution [7]. For different types of N fertilizer, Liao et al. [28] believed that the polypeptide urea can increase root biomass and root length density at essential growth stages of rice, and promote root growth. The application of organic N fertilizer resulted in higher root length and weight density in rice [29]. In terms of application methods, Mumtahina et al. [30] found that deep placement of N fertilization altered root growth, increased root distribution at the fertilization site, accumulated greater root surface area and root diameter, and promoted plant N uptake. Therefore, it is feasible to construct an efficient N-utilizing rice root system by optimizing N fertilizer application methods and types.

The use of CRNFs promoted the root growth of rice seedlings after sowing [31], increased root activity after heading, and delayed root senescence [32]. CRNFs combined with one-time application, such as root-zone one-time fertilization technology, optimized rice root morphology, increased root length, surface area and volume, and synergistically fulfilled the aims of producing high yield with optimal NUE [33]. Research on increasing rice yield and NUE by one-time application of CRBF has focused on the fate of N in paddy fields and on rice shoot growth and development [5,34,35]. However, how to promote plant N uptake and increase rice yield and NUE by regulating the spatial distribution of roots under one-time application of CRBF is still not clear.

The objectives of this study were to measure (1) the effects of one-time application of CRBF on morphological traits distribution of rice roots; (2) the responses of root N uptake capacity, plant dry matter accumulation, rice yield and NUE to one-time application of CRBF; and (3) the effects of root morphological traits distribution on rice yield and NUE. The experimental strategy was to measure these effects in field and root box experiments.

# 2. Materials and methods

## 2.1. Experiment site and weather

A field experiment was conducted in Danyang, Jiangsu, China (32°00′N, 119°32′E) during the 2021 growth season and a two-year root box experiment was conducted in an artificial climate chamber during the 2022 and 2023 growth seasons. A local high-yield rice cultivar "Ningxiangjing 9" developed by Nanjing Agricultural University and commonly used in rice production systems in the lower Yangtze River region of China from 2021 to 2023 was used. The soil analysis before planting revealed the following characteristics: organic carbon content of 15.32 g kg⁻¹, total N of 1.10 g kg⁻¹, available phosphorus of 13.40 mg kg⁻¹, available potassium of 198.70 mg kg⁻¹, and pH of 5.54. The weather in 2021 is described in Fig. S1.

## 2.2. Experiment design and growth conditions

The field experiment was conducted with four treatments: i) N₀, calculated NUE without N fertilizer; ii) CK, a control; iii) B-CRBF, CRBF was applied as a basal fertilizer by broadcast; iv) S-CRBF, CRBF was applied as a basal fertilizer by side-deep fertilization. For broadcast, N fertilizer was one-time spread on the soil surface while transplanting rice seedlings. For side-deep fertilization, a 5 cm deep trench was created 5 cm adjacent to the rice seedlings and N fertilizer was uniformly applied into the trench in a single application. Each treatment was applied with three replicates. CK was a three-split application of urea (basal, tillering, and panicle fertilizer, 3:2:5), which represented the local conventional high-yield fertilization method. CRBF is a N fertilizer with N content of 41.5% that was made by mixing the following five N fertilizers in a certain proportion: three types of poly-coated urea (43% N) with different release rates (2-month polymer-coated urea, 3-month polymer-coated urea, 4-month polymer-coated urea), sulfur-coated urea (37% N) and granular urea (46% N). CRBF released N gradually and evenly during the whole rice growth stage, showing two N release peaks at 0–20 d and 50–60 d after applied in the soil [36]. The N rate was 210 kg ha⁻¹ for all treatments except for N₀. Superphosphate served as the source of phosphorus, and potassium chloride as the potassium fertilizer. All treatments were supplied with 42 kg P₂O₅ ha⁻¹ and 84 kg K₂O ha⁻¹ as basal fertilizers. Sowing was performed on May 26 and rice seedling was machine-transplanted on June 11, with a transplanting density of 30 cm × 11 cm. Harvest was conducted on October 23. Each plot covered an area of 133 m² (19 m × 7 m) and was bordered by ridges, with plastic film used for isolation to prevent water and nutrient exchange between plots. During the whole rice growth stage, flooded water was mostly maintained by irrigation at a depth of 5 ± 2 cm on top of the soil until physiological maturity except for midseason aerations. All other agronomic practices adhered to regional standards for high-yield rice cultivation management.

The LK-1250 Plant Root Box Imaging System (Eco-mind Technology Co., Ltd., Beijing, China) was used to culture rice and observe the root system (Fig. S2A). The self-assembling root box (60 cm in height, 30 cm in length and 10 cm in width) was assembled with transparent and black acrylic plates. A transparent acrylic plate was used on the front of the root box for root observation, and the rest was black for light shielding. The transparent surface of the root box was tilted 45° downward and placed on the shelf. During the culture, the transparent surface was shielded by a black light-shielding panel. (Fig. S2BC). The root box was filled with long-term paddy soil that was naturally air-dried, manually
crushed and then sieved through a 1 cm mesh as the growth medium. Each root box contained approximately 19 kg of dry soil. Before transplanting, the soil was watered thoroughly and a 1 cm water layer was maintained, and plants were grown in the artificial climate chamber with an average day/night temperature of 30/25 °C, a 12-h light/dark cycle with a daytime light intensity of 500 µmol m⁻² s⁻¹. During rice growth, a 5 cm water depth was maintained, with re-irrigation occurring when the soil was nearly dried to allow alternate wetting and drying.

The root box experiment was conducted with six treatments: i) N₀; ii) CK; iii) B-U, normal urea (U) was applied by broadcast; iv) S-U, U was applied by side-deep fertilization; v) B-CRBF; vi) S-CRBF. U was normal urea, with a N content of 46%. All fertilizers and fertilization methods were consistent with those used in the field experiment. The N rate was 0.9 g plant⁻¹ for all treatments except for N₀, calculated based on the soil weight ratio from the field experiment's fertilization rates. The application rates of P₂O₅, and K₂O were determined using the same method. The specific fertilization managements about the field and root box experiments were shown in Tables S1 and S2. Each treatment was conducted with thirty replicates. Rice seedlings were cultured in conventional rice seedling medium in seedling trays. After 15 d of sowing, at a leaf age of 3.1, the seedlings were transplanted into the root box, with one seedling per root box. All other agronomic practices were consistent with those used in the field experiment.

The field experiment evaluated the effect of one-time application of CRBF on rice yield and NUE in actual rice production by comparing CRBF (broadcasting and side-deep fertilization) with CK (rice conventional high-yield N fertilizer application method). In the root box experiment, U treatment (broadcasting and deep lateral fertilization) was added under controlled and stable conditions to better explain the effects of CRBF under one-time application on root development.

## 2.3. Sampling and measurements

### 2.3.1. Yield, plant N uptake and NUE

In the field experiment, each plot was machine-harvested at maturity stage to measure the grain yield. In the root box experiment, five plants were sampled from the root box, selected based on the average panicles, to measure the grain yield at maturity stage. Grain yield corrected to a 14.5% standard japonica rice moisture level.

Five representative plants (only aboveground part) were chosen for sampling based on the average tillering number at maturity stage in the field experiment. In the root box experiment, the plant samples (both aboveground part and root) were collected at tillering, heading and maturity stages. At each stage, five representative plants were chosen for sampling based on the average tillering number. The drying process for the plant samples involved pre-drying at 105 °C for 30 min, followed by drying at 80 °C to a stable weight. After weighing the dried plant samples (plant aboveground and root dry matter accumulation, t ha⁻¹ for the field experiment and g plant⁻¹ for the root box experiment), they were ground and passed through a 2-mm sieve. The N content (%) of the plant samples was determined with a C/N analyzer. Plant N uptake (kg ha⁻¹; mg plant⁻¹) and NUE (%) were calculated by formula (1) and formula (2), respectively.

$$ \text{Plant N uptake} = \text{PDW} \times \text{N content (\%)} \quad (1) $$

$$ \text{NUE (\%)} = (N_n - N_0/N_r) \times 100 \quad (2) $$

where PDW (kg ha⁻¹; g plant⁻¹) is the plant dry matter weight of different treatments at different stages; N_n is the plant N aboveground uptake at maturity; N_0 is the plant aboveground N uptake of N_0 treatment at maturity; N_r is N application rate.

### 2.3.2. Root morphological trait distribution

At the tillering and heading stages, rice root images acquired in the top four 10-cm soil layers were used to measure root morphological traits in different soil layers, including root length, surface area, and volume were assessed using rhizoTrak according to Möller et al. [37].

### 2.3.3. Root samples and analysis

The fresh roots of five representative plants were excavated from each root box at the tillering and heading stages. Roots were encased in 40-mesh nylon mesh bags and washed with a continuous stream of water and the collected root samples from different treatments were mixed and impurities removed for measurement of root physiological indicators. Subsamples of 5 g were frozen in liquid nitrogen for measuring expression of ammonium transporters.

Totally 0.5 g of clean and fresh white root were selected from the above root samples for determining root activity. Five replicates were measured for each treatment. Root activity was quantified using the TTC method as described by He et al. [38].

Total RNA was extracted from rice roots at the tillering and heading stages using the RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Co., Ltd., Beijing, China). Approximately 1.0 µg of the extracted RNA was converted into cDNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech, Co., Ltd., Nanjing, Jiangsu, China). Quantitative RT-PCR (qPCR) was performed on the QuantStudio 6 Flex System (Thermo Fisher Scientific, Waltham, MA, USA) using the ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Co., Ltd., Nanjing, Jiangsu, China). OsACTIN1 (Os03g0718100) and OsUbiquitin (Os03g0234200) served as reference genes for normalizing gene expression, and the data from OsUbiquitin were shown in Luo et al. [22]. The relative gene expression levels under various treatments were compared to the control (CK) at different stages using the 2⁻⁰⁻⁰ΔΔCT method, and specific primers (OsUbiquitin, different ammonium transporters: OsAMT1.1, OsAMT1.2, and OsAMT1.3) listed in Table S3 were employed for these analyses.

## 2.4. Data statistical analysis

Standard ANOVA and Duncan's multiple range test (P < 0.05) were applied for statistical evaluation using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Graphs were created using the GraphPad Prism 10.1.2 (San Diego, CA, USA). Correlation analysis was performed using the "corrplot" R package.

# 3. Results

## 3.1. Yield and plant dry matter accumulation

In the 2021 field experiment, the yields of B-CRBF and S-CRBF reached 10.99 t ha⁻¹ and 11.23 t ha⁻¹, respectively, which were 2.91% and 5.11% higher than that of CK (Fig. 1A). The field and root box experiment showed consistent trends in yield (Fig. 1B, C). Year, fertilization method, and fertilizer type affected rice yield, whereas no interaction was observed between fertilization method and fertilizer type. The average yields in 2022 and 2023 under B-CRBF and S-CRBF were 43.05 g plant⁻¹ and 43.58 g plant⁻¹, respectively, higher than those under B-U and S-U, with increases of 16.86% and 18.28%, and there were no differences compared to CK. There were no differences in yield between fertilization methods within the same fertilizer type. The trends were consistent across the two years.

Fertilizer type and fertilization method influenced plant aboveground dry matter accumulation during essential growth stages (Table 1). The dry matter accumulation under CRBF with
<figure><img src="image_6.png" /><figcaption>**Fig. 1.** Effects of different treatments on rice yield in field (A) and root box (B, C) experiments from 2021 to 2023. CK, three-split application of urea; B-U, urea under one-time broadcast; S-U, urea under one-time side-deep fertilization; B-CRBF, controlled release blended fertilizer under one-time broadcast; S-CRBF, controlled release blended fertilizer under one-time side-deep fertilization; F, fertilization methods; T, fertilizer types; \*, *P* < 0.05; \*,\*, *P* < 0.01; ns, no significant difference. Error bars indicate standard errors of replicates. Different letters indicate a significant difference (P < 0.05).</figcaption></figure>

side-deep fertilization was higher than that under U treatment and CK by 20.70% and 21.75% at tillering stage, respectively, but showed no difference from B-CRBF. The remaining treatments did not differ. At the heading and maturity stages, CRBF with one-time applications showed higher dry matter accumulation than the U treatment but showed no differences from CK. No differences were found among fertilization methods within the same fertilizer type. The trends were consistent across years. The ANOVA further revealed that year influenced dry matter accumulation at the tillering and maturity stages, fertilization method affected dry matter accumulation at the tillering stage, and fertilizer type affected dry matter accumulation at all essential growth stages and no interaction effects were observed between fertilization method and fertilizer type.

## 3.2. Plant N uptake and NUE

Year, fertilization method, and fertilizer type affected N uptake and NUE at essential growth stages and there was no interaction between fertilization method and fertilizer type (Table 1). At the tillering stage, plant N uptake under CRBF with side-deep fertilization increased more than under other treatments. At the heading stage, CRBF with one-time applications showed no differences

compared to CK but outperformed U with one-time application. Plant N uptake under CRBF with side-deep fertilization was higher than that with broadcast. At the maturity stage, plant N uptake followed the trend: CRBF with side-deep fertilization > CRBF with broadcast > CK > U with side-deep fertilization > U with broadcast. CRBF with side-deep fertilization increased N uptake by 11.98% and 5.68% compared to CK and CRBF with broadcast, respectively. U with both fertilization methods showed no differences but showed lower N uptake than CK and CRBF treatments.

In the root box experiment, the NUE exhibited a pattern consistent with plant N uptake at the maturity stage (Table 1). CRBF with both fertilization methods increased NUE by 135.3% and 21.97% compared to U treatments and CK, respectively. NUE under CRBF with side-deep fertilization was higher than that under CRBF with broadcast. The same trend was observed for NUE of one-time application of CRBF in the field experiment in 2021 (Fig. S3).

## 3.3. Root biomass and root N uptake

Only year and fertilizer type affected root biomass at the tillering and heading stages (Fig. 2). Fertilizer type influenced root N uptake at tillering and heading stages, while year only had an effect at the tillering stage. Fertilization methods had no effect, and no
<table><caption>**Table 1**
Effects of different treatments on plant dry matter accumulation, N uptake, and NUE in 2022 and 2023.</caption><thead><tr><th rowspan="2">Year</th><th rowspan="2">Fertilization method</th><th rowspan="2">Fertilizer type</th><th colspan="3">Dry matter accumulation (g plant<sup>-1</sup>)</th><th rowspan="2">N uptake (mg plant<sup>-1</sup>)</th><th rowspan="2">Heading</th><th rowspan="2">Maturity</th><th rowspan="2">NUE (%)</th></tr><tr><th>Tillering</th><th>Heading</th><th>Maturity</th></tr></thead><tbody><tr><td rowspan="5">2022</td><td rowspan="3">Broadcast</td><td>CK</td><td>3.91 ± 0.19 b</td><td>47.70 ± 3.35 a</td><td>75.37 ± 1.44 a</td><td>122.72 ± 5.14 b</td><td>780.34 ± 8.78 ab</td><td>845.52 ± 9.02 c</td><td>38.68 ± 1.00 c</td></tr><tr><td>U</td><td>3.87 ± 0.22 b</td><td>39.76 ± 0.59 b</td><td>65.68 ± 0.70 b</td><td>117.65 ± 7.52 b</td><td>571.77 ± 13.98 c</td><td>652.37 ± 9.93 d</td><td>17.22 ± 1.10 d</td></tr><tr><td>CRBF</td><td>4.60 ± 0.22 ab</td><td>46.54 ± 1.53 a</td><td>74.59 ± 5.01 a</td><td>134.67 ± 10.12 b</td><td>777.29 ± 4.82 b</td><td>882.93 ± 6.28 b</td><td>42.84 ± 0.70 b</td></tr><tr><td rowspan="2">Side-deep fertilization</td><td>U</td><td>4.11 ± 0.33 b</td><td>40.24 ± 1.05 b</td><td>67.07 ± 2.09 b</td><td>122.14 ± 5.00 b</td><td>582.83 ± 20.22 c</td><td>666.25 ± 22.58 d</td><td>18.76 ± 2.51 d</td></tr><tr><td>CRBF</td><td>5.10 ± 0.50 a</td><td>47.45 ± 1.36 a</td><td>77.25 ± 1.03 a</td><td>162.13 ± 15.42 a</td><td>809.73 ± 12.98 a</td><td>929.46 ± 10.36 a</td><td>48.01 ± 1.15 a</td></tr><tr><td rowspan="5">2023</td><td rowspan="3">Broadcast</td><td>CK</td><td>5.86 ± 0.39 b</td><td>50.49 ± 3.04 a</td><td>100.01 ± 3.07 a</td><td>175.76 ± 2.93 b</td><td>706.30 ± 4.44 ab</td><td>1028.36 ± 22.63 c</td><td>46.33 ± 2.51 c</td></tr><tr><td>U</td><td>5.79 ± 0.33 b</td><td>39.84 ± 2.54 b</td><td>88.23 ± 4.34 b</td><td>175.93 ± 12.54 b</td><td>482.01 ± 19.79 c</td><td>828.72 ± 32.15 d</td><td>24.14 ± 3.57 d</td></tr><tr><td>CRBF</td><td>6.24 ± 0.20 ab</td><td>51.07 ± 2.26 a</td><td>100.98 ± 0.85 a</td><td>192.31 ± 6.98 b</td><td>702.90 ± 20.92 b</td><td>1102.61 ± 11.30 b</td><td>54.58 ± 1.26 b</td></tr><tr><td rowspan="2">Side-deep fertilization</td><td>U</td><td>5.94 ± 0.21 b</td><td>40.26 ± 3.75 b</td><td>90.22 ± 2.61 b</td><td>181.86 ± 3.56 b</td><td>494.28 ± 20.20 c</td><td>863.29 ± 34.10 d</td><td>27.98 ± 3.79 d</td></tr><tr><td>CRBF</td><td>6.80 ± 0.24 a</td><td>51.98 ± 3.46 a</td><td>104.61 ± 2.27 a</td><td>211.26 ± 7.81 a</td><td>741.63 ± 9.32 a</td><td>1168.89 ± 24.89 a</td><td>61.94 ± 2.77 a</td></tr><tr><td rowspan="4">ANOVA</td><td>Year</td><td>**</td><td>ns</td><td>**</td><td>**</td><td>**</td><td>**</td><td>**</td><td>**</td></tr><tr><td>F</td><td>*</td><td>ns</td><td>ns</td><td>**</td><td>**</td><td>**</td><td>**</td><td>**</td></tr><tr><td>T</td><td>**</td><td>ns</td><td>**</td><td>**</td><td>**</td><td>**</td><td>**</td><td>**</td></tr><tr><td>F × T</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr></tbody></table>

CK, three-split application of urea; U, urea; CRBF, controlled release blended fertilizer; NUE, nitrogen use efficiency; F, fertilization methods; T, fertilizer types; *, P < 0.05; **, P < 0.01; ns, no significant difference. Different letters indicate a significant difference at P < 0.05.

interactions were observed. At the tillering stage, CRBF with one-time applications increased root biomass by 32.15% and 32.55% and root N uptake by 52.66% and 34.62% compared to U treatments and CK, respectively. At the heading stage, CRBF with both one-time applications increased root biomass and root N uptake by 46.25% and 69.17% compared to U treatments, with no significant differences observed between CRBF treatments and CK.

### 3.4. Root morphological trait distribution

Rice root was primarily distributed in the 0–30 cm soil layer at the tillering stage (Fig. 3) and in the 0–40 cm soil layer at the heading stage (Fig. 4). The distribution of root length, surface area, and volume across different soil layers was influenced by the treatments, with consistent trends observed over two years. At the tillering stage, differences in root morphology among treatments were mainly concentrated in the 0–20 cm soil layer. In the 0–10 cm layer, CRBF with one-time applications significantly increased root length, surface area, and volume compared to other treatments. S-CRBF displayed significantly higher root volume than B-CRBF. In the 10–20 cm layer, B-U treatment exhibited significantly greater root length, while CK and B-U had significantly higher root surface area and volume than other treatments. At the heading stage, CRBF with one-time applications significantly increased root length, surface area, and volume in the 0–10 cm soil layer compared to other treatments. Once again, S-CRBF showed significantly higher values than B-CRBF. In the 10–20 cm layer, B-CRBF exhibited significantly greater root length, surface area, and volume than other treatments, while S-CRBF showed no significant differences from CK but significantly increased these parameters compared to U treatment. In the 20–30 cm layer, B-CRBF had greater root length, surface area, and volume than other treatments. In 2022, S-CRBF showed no differences from CK but increased root surface area compared to U treatment. However, in 2023, S-CRBF reduced root length and surface area compared to CK, with no differences in root volume. In the 30–40 cm soil layer, root length under B-CRBF showed no differences from CK and B-U but significantly decreased under S-CRBF. S-U exhibited the lowest root length, which was not different from S-CRBF. For root surface area and volume, CRBF with one-time applications showed no differences from other treatments.

### 3.5. Root activity and the relative expression of OsAMT1s

Year, fertilization method, and fertilizer type affected root activity during the tillering and heading stages and there was no interaction between fertilization method and fertilizer type (Fig. 5A). Compared to CK and U treatments, CRBF under both fertilization methods increased root activity, with increases of 42.44% and 57.04% at the tillering stage, and 30.97% and 68.43% at the heading stage, respectively.

qPCR results showed that in the root, CRBF under two one-time application methods up-regulated the expression level of two ammonium transporters: OsAMT1.1 and OsAMT1.2 at the tillering and heading stage compared to CK and U treatments, whereas there was no difference in the expressions of OsAMT1.3 in root at tillering and heading stages (Fig. 5B).

### 3.6. Relationship between root traits, yield and NUE

The correlation analysis between the root traits (root morphological traits, including root length, surface area, and volume) distribution, root biomass, root N uptake, root activity, and root OsAMT1s relative expression) at the tillering (Fig. 6A) and heading (Fig. 6B) stages and yield and NUE was conducted combined with 2-year data in root box experiment. A positive relationship was
<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Effects of different treatments on rice root biomass and N uptake at tillering and heading stage in 2022 and 2023. CK, three-split application of urea; B-U, urea under one-time broadcast; S-U, urea under one-time side-deep fertilization; B-CRBF, controlled release blended fertilizer under one-time broadcast; S-CRBF, controlled release blended fertilizer under one-time side-deep fertilization; F, fertilization methods; T, fertilizer types. *, P < 0.05; **, P < 0.01; ns, no significant difference. Error bars indicate standard errors of replicates. Different letters indicate a significant difference (P < 0.05) in the same stage.</figcaption></figure>

observed between yield and NUE and root morphological traits in the 0–10 cm soil layer at tillering and the 0–20 cm soil layer at heading. However, no correlations were observed between yield and NUE and root morphological traits in the 10–40 cm soil layer at the tillering stage or in the 20–40 cm soil layer at the heading stage. At the tillering stage, root biomass, root N uptake, root activity, and the expression levels of OsAMT1.1 and OsAMT1.2 were positively associated with both yield and NUE. The yield was positively correlated only with root biomass, root N accumulation, and root activity, while NUE remained positively correlated with root biomass, root N uptake, root activity, and the expression levels of OsAMT1.1 and OsAMT1.2 at the heading stage.

# 4. Discussion

## 4.1. Distribution of root morphological traits under one-time application of CRBF

The root system is a key regulator of rice growth and development [25]. Over 80% of the rice roots are localized within the soil tillage layer, which serves as the primary zone for nutrient absorption. The distribution of root is tightly related to rice yield and NUE [7,18]. Great number of researchers supported that promoting a higher proportion of rice root in the 10–20 cm soil layer can lead to increased rice yield [18,39]. In rice production, N fertilizers are often used to regulate the temporal and spatial distribution of soil inorganic N, thereby regulating the rice root distribution. Our results revealed that at the tillering stage, the root morphological traits (length, surface area and volume) of the surface soil (0–10 cm soil layer) were enhanced under CRBF with one-time applications, while there was no significant difference in the deep soil

(20–40 cm soil layer) (Fig. 3) and the root length, surface area and volume in the tillage layer (0–20 cm soil layer) were increased at the heading stage (Fig. 4). Such root morphological behavior may be related to the NH₄-N content of the soil. NH₄-N is the main form of inorganic N supply in paddy soil [40], and rice is an ammonium-preferred crop [41,42]. Soil NH₄-N content affected rice root morphology, especially the root length and root number [10,15,22]. A certain concentration of NH₄-N can promote the elongation of the rice root [43]. Indeed, the absorption of NH₄ will enhance ability of root cells to divide and expand and increase the root, and at the same time, it can increase the surface area of the root and then promote the root's ability to absorb [15,44]. Our results indicated that under both one-time applications, CRBF led to a significant rise in NH₄-N content within the 0–20 cm soil layer at tillering and heading stages, but no substantial differences were found among treatments in the 20–40 cm layer. (Fig. S4). The sustained release of N from CRBF created a stable soil N supply environment under CRBF with one-time applications [34]. In addition, soil NH₄-N was concentrated in the 0–20 cm soil layer by reducing N losses through leaching, runoff, and ammonia volatilization [4]. Rice root can use its morphological plasticity to adapt to the characteristic distribution of soil NH₄-N content under CRBF with one-time applications [45]. This adaptation resulted in root being more developed in the 0–20 cm soil layer, with increased root length, surface area, and volume concentrated in soil layers with elevated NH₄-N content.

Jia et al. [46] and Nacry et al. [47] found that soil inorganic N levels are related to the content of root hormones such as auxin, cytokinins, and strigolactones. Root hormones can promote root growth and alter root morphology. For instance, auxin and cytokinin work together to regulate the size of root meristem to maintain
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Root morphological traits distribution in different soil layer under different treatments at tillering stage in 2022 and 2023. CK, three-split application of urea; B-U, urea under one-time broadcast; S-U, urea under one-time side-deep fertilization; B-CRBF, controlled release blended fertilizer under one-time broadcast; S-CRBF, controlled release blended fertilizer under one-time side-deep fertilization. Error bars indicate standard errors of replicates. Different letters indicate a significant difference (*P* < 0.05) in the same soil layer.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Root morphological traits distribution in different soil layer under different treatments at heading stage in 2022 and 2023. CK, three-split application of urea; B-U, urea under one-time broadcast; S-U, urea under one-time side-deep fertilization; B-CRBF, controlled release blended fertilizer under one-time broadcast; S-CRBF, controlled release blended fertilizer under one-time side-deep fertilization. Error bars indicate standard errors of replicates. Different letters indicate a significant difference (*P* < 0.05) in the same soil layer.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Effects of different treatments on rice root activity (A) and OsAMT1s relative expression (B) at tillering and heading stage in 2022 and 2023. CK, three-split application of urea; B-U, urea under one-time broadcast; S-U, urea under one-time side-deep fertilization; B-CRBF, controlled release blended fertilizer under one-time broadcast; S-CRBF, controlled release blended fertilizer under one-time side-deep fertilization; F, fertilization methods; T, fertilizer types; *, *P* < 0.05; **, *P* < 0.01; ns, no significant difference. Error bars indicate standard errors of replicates. Different letters indicate a significant difference (*P* < 0.05) in the same stage.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 6.** Correlation analysis illustrates the relationships in the yield, NUE, root morphological traits in different soil layers, the relative expression of OsAMT1s, root growth and root activity at tillering (A) and heading (B) stage. NUE, nitrogen use efficiency; RLO_10, root length in 0–10 cm soil layer; RS0_10, root surface area in 0–10 cm soil layer; RV0_10, root volume in 0–10 cm soil layer; RL10_20, root length in 10–20 cm soil layer; RS10_20, root surface area in 10–20 cm soil layer; RV10_20, root volume in 10–20 cm soil layer; RL20_40, root length in 20–40 cm soil layer; RS20_40, root surface area in 20–40 cm soil layer; RV20_40, root volume in 20–40 cm soil layer. *, *P* < 0.05; **, *P* < 0.01.</figcaption></figure>
root growth while also controlling root length and the number of root tips, thereby regulating N uptake [46,48–50]. Auxin promotes the formation of lateral root primordia and root elongation [51,52]. The finding that CRBF application increased root IAA content at the tillering and heading stages compared with U treatment shows that increasing soil NH₄-N content induced a higher level of root IAA, improving rice root morphological characteristics and vertical distribution (Fig. S5). The plasticity of the root morphology improved the vertical distribution of the root, leading to a more concentrated distribution in soil layers with higher NH₄-N content, which enhance the plant N uptake to a certain extent.

## 4.2. Optimized root traits increased rice yield and NUE

Root growth is critical for N uptake from the soil. Increased root length, surface area, and volume in the soil result in increased root-soil contact area. This increased contact allows the root to capture more soil N to satisfy their growth needs, which is beneficial for the rice aboveground dry matter accumulation and ultimately contributes to higher yields [12,53]. Previous research provided little insight into the interactions between rice root morphology and physiology, and root characteristics under CRBF with one-time applications, in relation to yield and NUE. Under CRBF with one-time applications, rice root morphology was improved at key growth stages (tillering and heading stages), and root physiological activity was increased, leading to increased root biomass, root N uptake, root length, root surface area, and root volume (Figs. 2–4). Root activity and the expression levels of two ammonium transporters (OsAMT1.1 and OsAMT1.2) were significantly increased (Fig. 5). Root morphological and physiological traits were associated with high grain yield [12,24,25,54]. Here, the increase in root morphological traits in the 0–10 cm soil layer at the tillering stage and in the 0–20 cm soil layer at the heading stage contributed to yield improvement, thereby enhancing NUE (Fig. 6). Rice N use depends not only on soil N availability but also on root distribution and the root N uptake capacity [8,12]. According to the findings of Li et al. [55], CRBF increased the soil inorganic N content through its slow N release characteristic, thereby increasing wheat root growth (including root length, surface area, and volume), and enabling a greater portion of the roots to develop in soil layers with higher inorganic N content. This resulted in higher wheat yield and N accumulation. These results indicated that CRBF can improve crop yield and plant N uptake by strengthening the correspondence between soil inorganic N distribution and root morphological traits distribution. Deng et al. [56] identified a strong positive correlation between rice root in the 0–20 cm soil layer and yield. Our study showed that CRBF with one-time applications increased root length, surface area, and volume in the 0–20 cm soil layer, which enhanced plant aboveground dry matter accumulation and N uptake, contributing to improved grain yield and NUE. Side-deep fertilization of CRBF strengthened root spatial adaptability. By deeply applying CRBF in the root zone, the resulting localized high-N environment triggered a root chemotactic response, further boosted root development, and improved root morphology, thereby achieving higher NUE than broadcast of CRBF [10,45,57].

Root activity and the expression levels of ammonium transporters (OsAMT1s) were closely related to yield and NUE (Fig. 6). This indicated that yield and NUE increased as root physiological activity improved. In line with this, root activity and physiological traits are crucial for crop yield and nutrient uptake, with improved root functionality facilitating rice growth and promoting yield and NUE [58,59]. Soil N levels significantly affect the activity and physiological characteristics of rice roots [18,54]. Although recent data from Qi et al. [60] found that a combination of CRNF and normal urea can improve rice root activity, nothing is known about how

one-time fertilization of CRBF affects rice root activity. CRBF improved root activity at the early (tillering) stage and delayed root senescence after heading and promoted root activities (Fig. 5A). Rice roots take up most of the NH₄ from the soil via ammonium transporters, including OsAMT1.1, OsAMT1.2, and OsAMT1.3 [19,61]. High expression of root ammonium transport-related genes can promote N uptake and NUE in rice [62]. CRBF can up-regulate the expression level of OsAMT1.1 and OsAMT1.2 at the tillering and heading stages to enhance N uptake (Fig. 5B). N fertilizer application can increase soil NH₄-N concentration, thereby improving root activity and showing the high expression level of OsAMTs [24,62]. These results were consistent with our findings that compared with CK, CRBF with one-time applications improved the root's ability to absorb N by increasing root activity and up-regulating the expression level of root OsAMT1s genes because of the increase in soil NH₄-N concentration during essential growth stages, thereby promoting the plant aboveground dry matter accumulation and N uptake during the critical growth stages, and ultimately increasing yield and NUE. Using one-time application of CRBF as an optimized N management strategy, improved root morphology and root physiological traits in rice contribute to high yield and high NUE.

## 5. Conclusions

One-time application of CRBF is a high-yield and high-efficiency N management strategy for rice. This approach significantly influenced rice yield, NUE, plant aboveground growth, root growth, root distribution, and root physiological characteristics. Compared to other treatments, one-time application of CRBF increased root biomass and root N uptake and increased root length, surface area, and volume in the 0–10 cm soil layer at the tillering stage and in the 0–20 cm soil layer at the heading stage, optimizing the spatial distribution of root morphology. It improved root activity and increased the expression of ammonium transporters in root, increasing rice yield and NUE. Further research is needed to explore the physiological and molecular mechanisms underlying the improvements in rice yield and NUE under multi-variety, multi-location validation conditions. This would provide deeper insights into the role of a one-time application of CRBF in optimizing rice productivity and NUE.

### CRediT authorship contribution statement

**Yuhui Wang:** Writing – original draft, Visualization, Investigation, Data curation, Conceptualization. **Shen Gao:** Investigation, Data curation, Writing – original draft. **Jie Sun:** Visualization, Investigation, Data curation. **Bin He:** Investigation, Formal analysis, Data curation. **Wenjun He:** Investigation, Data curation. **Weike Tao:** Investigation, Visualization. **Xin'ao Tang:** Visualization, Investigation. **Zhi Geng:** Methodology, Validation. **Zhengyang Wu:** Investigation, Validation. **Weiwei Li:** Conceptualization, Supervision. **Zhengrong Jiang:** Writing – review & editing, Conceptualization. **Zhenghui Liu:** Conceptualization, Supervision. **Yanfeng Ding:** Supervision, Funding acquisition. **Soulaiman Sakr:** Writing – review & editing, Conceptualization. **Pengfu Hou:** Project administration, Funding acquisition, Conceptualization, Supervision. **Gang-hua Li:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgments

The research was supported by the National Key Research and Development Program of China (2023YFD2301304, 2022YFD2301404-4, 2023YFD2302600, and 2022YFE0116200), Open Subjects of the Key Laboratory of Agro-Environment in the Middle and Lower Yangtze River Plain, Ministry of Agriculture and Rural Affairs of China (2023F12), National Natural Science Foundation of China (32301964), and Sanya Yazhou Bay Science and Technology City Project (SKJC-2023-02-004).

## Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2025.04.008.

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