Effects of Planting Density, Levels, and Forms of Nitrogen Application on the Yield and Nitrogen Utilization of Wheat following Rice in East China

Abstract

A major challenge is to achieve the goal of synergistically increasing grain yield and nitrogen use efficiency in wheat production. Many studies have focused on one aspect of cultivation such as fertilizer management, suitable planting density, and straw returning. However, there are few studies on the effect of integrated cultivation practices on yield and nitrogen absorption and utilization of wheat. A field experiment to investigate the characteristics was conducted across two years using Yangmai 16 and Yangmai 20 with five cultivation practices including nitrogen blank area (NB), local practices (LP), nitrogen reduction (NR), planting density reduction and nitrogen reduction (DN), and organic fertilizer (OF). As compared with LP, the DN treatment improved the yield (+4.54%), recovery efficiency of N fertilizer (+5.59%), N partial factor productivity (+15.28%), agronomic N use efficiency (+21.43%), physiological N use efficiency (+14.90%), and nitrogen harvest index (+6.45%). All previous indices were increased by 16.84%, 28.18%, 19.59%, 45.81%, 13.96%, and 3.37% under the OF treatment, as compared with LP. The DN and OF significantly improved nitrogen use efficiency, photosynthetic characteristics, dry matter accumulation, root total and active absorbing surface area, root oxidation activity, nitrogen accumulation, nitrogen harvest index, and nitrogen transportation in various organs. The results suggest that integrated cultivation practices are beneficial to achieve high yield and high nitrogen use efficiency through improving the agronomic performance and root physiological characteristics.

1. Introduction

Wheat (Triticum aestivum L.) is the staple food for one-third of the world’s population [1]. China is a major wheat producer and consumer in the world and increasing grain yield is important in ensuring food security in China and around the world [2]. China’s wheat planting area decreased from 0.25 million hectares to 0.23 million hectares between 2015 and 2020, while the yield increased from 1.33 million tons to 1.34 million tons in the same period [3]. The rice–wheat rotation area in East China is one of China’s most important grain production and high-yielding areas [4]. Increased wheat yield is primarily due to the use of gene editing technology; breeding of new cultivars [5,6]; and development of monoculture cultivation practices, such as sowing date, fertilizer application, and precision regulation of cover [7,8,9]. However, wheat production has also formed a high input, high output, and low efficiency of the ‘two high and one low’ production pattern, which places a great burden on sustainable agricultural development [10,11]. The achievement of synergistic improvement of yield and resource utilization efficiency is a key issue in the field of wheat cultivation research at home and abroad.

Nitrogen fertilizer plays an important role in crop yield increase [12]. Statistics show that 3.81 billion tons of nitrogen fertilizer is applied globally to all crops, of which one-third is for wheat alone [13]. Farmers’ customary nitrogen application methods are a common cultivation practice to maintain wheat grain yield. However, owing to the rapid dissolution of urea, single application may affect soil nitrogen availability at the late growth stage of wheat, thus limiting photosynthetic efficiency and post-anthesis assimilate accumulation [14,15]. Compared with farmers’ customary nitrogen application methods, reasonable nitrogen application (appropriate nitrogen reduction [16], adjustment of base and topdressing ratio [17], and precise management of nitrogen fertilizer [18], among others) could not only promote the formation of high yield of wheat, but also meet the demand of wheat for nutrients in the later stage of growth, thereby improving nitrogen use efficiency.

The application of chemical fertilizer significantly reduced soil organic matter compared with organic fertilizer, limiting the potential of wheat production [19]. It is difficult to achieve a higher and more stable yield of wheat with nitrogen fertilizer alone. In recent years, the effect of combined application of organic and inorganic fertilizers on wheat yield and nitrogen use efficiency has become a research hotspot [20]. Studies have shown that the fertilizer application pattern of ‘chemical fertilizer + organic fertilizer’ could improve wheat yield and nitrogen use efficiency [21]. This may be the result of the coordination between the slow release at the early stage and the long time of fertilizer effect of organic fertilizer [22], as well as the strong nutrient and rapid effect [23] of chemical fertilizer.

Optimizing planting density is one of the important cultivation practices to improve yield per unit area and nitrogen use efficiency [24]. Planting density largely determines the population dynamics of wheat, thus further affecting environmental factors of light, moisture, and nutrients. Studies have shown that an appropriate increase in planting density can promote root length, root volume, and canopy photosynthetic characteristics, thereby increasing yield and nitrogen use efficiency [25]. However, too high of a planting density will affect grain filling and reduce yield [26]. Therefore, having a reasonable planting density can promote the coordinated development of individuals and groups, so that the potential of wheat production can be fully utilized.

However, most of them focus on the formation of high yield and high efficiency in single cultivation practices and few studied the physiological mechanism of high yield of wheat and nitrogen efficient absorption and utilization under integrated cultivation practices. In this study, Yangmai 16 and Yangmai 20 were used in the field experiment. Five different cultivation practices including nitrogen blank area, local practices, nitrogen reduction, planting density reduction and nitrogen reduction, and organic fertilizer were set up in the field environment to evaluate their integrated effects on the yield formation and nitrogen accumulation and distribution of wheat following rice. This study was to establish a practical guidance for wheat high yield and efficient utilization of nitrogen in East China.

2. Materials and Methods

2.1. Plant Materials and Growing Conditions

The experiment was conducted in two consecutive wheat growing seasons from 2019 to 2021 at the research farm of Yangzhou University, Jiangsu Province, China (32°30′ N, 119°25′ E). The previous crop in the field was rice, while the current test crop is wheat. The soil textural class was sandy loam. The topsoil contained 2.02% organic matter, 105.0 mg·kg⁻¹ available nitrogen, 34.2 mg·kg⁻¹ available phosphorus, and 68.0 mg·kg⁻¹ available potassium. The spring wheat cultivars Yangmai 16 and Yangmai 20 were used as test crops. For optimal crop growth, weeds, insects, and diseases were controlled by either chemical or manual methods. Figure 1 shows the average precipitation, air temperature and sunshine hours during the wheat growing seasons from the nearby weather station.

2.2. Treatment

The experiment was laid in a split-plot design with three replications. The treatment was the main plot factor, while the cultivar was the sub-plot factor. The plot size was 30 m², the ploughing depth was 10 cm, and the irrigation mode was conventional irrigation. A single application of fertilizer was performed at the rates of 90 kg·ha⁻¹ of phosphorus (calcium superphosphate, 12% P₂O₅) and 90 kg·ha⁻¹ of potassium (potassium chloride, 60% K₂O) before sowing. Ditches were constructed around the experimental plots to prevent fertilizer and water from mixing. Ridges were constructed between blocks to prevent the overlap of water–fertilizer into different blocks. Five treatments, including a nitrogen blank area (NB), local practices (LP), nitrogen reduction (NR), planting density reduction and nitrogen reduction (DN), and organic fertilizer (OF), are shown in

Table 1. Cultivation practices for different treatments.

Treatment †	N Application		Basic Seedling (104·ha−1)	Basic Application of Organic Fertilizer (kg·ha−1)
	Overall Amount (kg·ha−1)	Proportion
NB	0	0	240	0
LP	240	6:1:3	240	0
NR	216	5:1:2:2	240	0
DN	216	5:1:2:2	192	0
OF	216	5:1:2:2	192	1800

^† NB, LP, NR, DN, and OF indicate nitrogen blank area, local practices, nitrogen reduction, planting density reduction and nitrogen reduction, and organic fertilizer, respectively.

.

In the NB treatment, there was no nitrogen fertilizer. Sowing was carried out in strips with a 30 cm row spacing using three-leaf stage seedlings at a rate of 240 × 10⁴ ha in NB, LP, and NR treatments. Population density was lowered to 192 × 10⁴ ha in DN and OF treatments. In the LP treatment, nitrogen application (240 kg·ha⁻¹ pure nitrogen) was split into three applications (ratio of 6:1:3): 1 day before sowing, four-leaf stage, and jointing stage (leaf age 2.5 leaves) (Table 1). In the NR, DN, and OF treatments, the total nitrogen application rate (pure nitrogen) was 216 kg·ha⁻¹ and nitrogen application was split into four applications (ratio of 5:1:2:2): 1 day before sowing, four-leaf stage, jointing stage (leaf age 2.5 leaves), and booting stage (leaf age 1.2 leaves). OF treatment base fertilizer increased organic fertilizer to 1800 kg·ha⁻¹ (organic matter content 45%, containing N 2.1%).

2.3. Sampling and Measurements

Three plants of uniform growth were taken from each treatment at jointing, booting, anthesis, and maturity, in that order. Deep soil blocks (20 cm × 20 cm × 20 cm) were dug around each plant. The roots were carefully rinsed with a hydropneumatic elutriation device (Gillison’s Cultivar Fabrications, MI, Benzonia). The root oxidation activity (ROA) was determined by the naphthalene amine method [27]. The total absorption surface area and active absorption area of the root system were determined by the methylene blue dip method [28].

The photosynthetic characteristics of wheat flag leaf were measured by the portable photosynthesis instrument LI-6400 (LI-COR, Lincoln, NE, USA) at the jointing, booting, anthesis, and milky-ripe stages. The sample chamber CO₂ concentration was 380 μmol·mol⁻¹, the flow rate was 400 μmol·m⁻²·s⁻¹, and the leaf temperature was 25 °C. Ten fully expanded uppermost leaves were measured per plot.

Twenty representative plants were selected from each plot and divided into leaves, stems, and ears (after heading), at jointing, booting, anthesis, and maturity. They were dried at 105 °C for 30 min and dried at 75 °C to a constant weight, and the dry matter was weighed. Some of the dried samples were crushed and sieved and accurately weighed. The samples were placed in a catalyst and concentrated sulfuric acid, and then boiled in the digester at 420 °C for about 1.5 h. After being green for clarification, the samples were cooled to normal temperature and transferred to the FOSS automatic nitrogen analyzer (Kjeltec^TM 8400, FOSS, Denmark) to determine the nitrogen content of the plants.

A 1 m² quadrat was selected for yield calculations and three 1 m uniform sample areas were selected for each plot, at maturity. Twenty panicles were randomly selected for grain count investigation. The grains were harvested and threshed manually and weighed after natural drying. The yield and grain weight were calculated and expressed at 13% moisture content.

2.4. Data Calculation and Analysis

Microsoft Excel 2019 and IBM SPSS Statistics (version 26; IBM company, New York, USA) were used for data processing. The plots were generated using Origin 2021 software. Rstudio software (Corrplot, version 3.5.1, https://cran.r-project.org, accessed on 25 August 2021) was used to calculate the Pearson correlation coefficient and perform graphing. Treatments were compared using the least significant difference test at the 5% probability level. The data are represented by two study years and cultivars. In this study, the effects of comprehensive cultivation practices on nitrogen transport in various organs at the filling stage and nitrogen accumulation in various organs at the flowering stage and maturity stage were elaborated for the second season (

Table A1. Contribution of nitrogen transfer in various organs to grains during the grain filling stage in 2020–2021.

Cultivar	Treatment †	Stem + Sheath		Leaf		Glume + Spike
		N Remobilization (kg·ha−1)	Contribution to N in Grains (%)	N Remobilization (kg·ha−1)	Contribution to N in Grains (%)	N Remobilization (kg·ha−1)	Contribution to N in Grains (%)
Yangmai 16	LP	30.28 b ‡	27.70 a	39.30 c	36.00 ab	12.75 a	11.63 a
	NR	34.86 ab	32.08 a	33.39 d	30.79 c	14.33 a	13.16 a
	DN	31.24 b	26.49 a	43.97 b	37.55 a	14.39 a	12.18 a
	OF	41.60 a	30.01 a	49.32 a	35.61 b	18.50 a	13.32 a
Yangmai 20	LP	35.77 b	27.55 a	51.49 a	39.70 a	12.22 b	9.41a
	NR	36.68 b	29.67 a	45.44 b	36.77 a	8.35 c	6.76 b
	DN	36.32 b	27.98 a	47.46 b	36.61 a	11.83 bc	9.12 a
	OF	46.69 a	30.51 a	55.33 a	36.28 a	15.89 a	10.37 a

^† LP, NR, DN, and OF indicate local practices, nitrogen reduction, planting density reduction and nitrogen reduction, and organic fertilizer, respectively. ^‡ Different letters indicate statistically significant differences at the p = 0.05 level within the same column and cultivar.

and Figure A2).

The calculation of nitrogen-related indexes was based on methods by [29,30,31].

Nitrogen transportation at the grain filling stage (kg·ha⁻¹) = Nitrogen accumulation in vegetative organs at anthesis − Nitrogen accumulation in vegetative organs at maturity stage;

(1)

Nitrogen translocation contribution rate to grain at grain filling stage (%) = Nitrogen translocation at grain filling stage/Grain yield × 100%;

(2)

RE_N (%) = [N uptake in N application plots − N uptake in N omission plots (kg)]/N rate (kg) × 100;

(3)

PFP_N (kg·kg⁻¹) = Grain yield (kg)/N rate (kg);

(4)

AE_N (kg·kg⁻¹) = [Grain yield in N application plots − Grain yield in N omission plots (kg)]/N rate (kg);

(5)

PE_N (kg·kg⁻¹) = [Grain yield in N application plots − Grain yield in N omission plots (kg)]/[N uptake in N application plots − N uptake in N omission plots (kg)].

(6)

3. Results

3.1. Nitrogen Use Efficiency and Nitrogen Harvest Index

With the introduction of individual cultivation practices, the recovery efficiency of N fertilizer (RE_N), N partial factor productivity (PFP_N), agronomic N use efficiency (AE_N), physiological N use efficiency (PE_N), and nitrogen harvest index (NHI) of the two cultivars increased in each treatment. Compared with local practices (LP), RE_N, PFP_N, AE_N, PE_N, and NHI were significantly higher in planting density reduction and nitrogen reduction (DN) and organic fertilizer (OF), while nitrogen reduction (NR) increased the RE_N of the two cultivars by 3.55% on average. When compared with DN, OF treatment increased the RE_N of the two cultivars by 28.17% on average. The average PFP_N, AE_N, PE_N, and NHI of the two cultivars were consistent with the overall trend in RE_N (except that AE_N and PE_N decreased under NR) (

Table 2. Effects of integrated cultivation practices on nitrogen use efficiency and nitrogen harvest index.

Year/Cultivar	Treatment †	REN ‡ (%)	PFPN (kg·kg−1)	AEN (kg·kg−1)	PEN (kg·kg−1)	NHI
2019–2020/Yangmai 16	LP	34.29 b §	24.33 b	10.01 b	29.17 b	0.67 b
	NR	35.51 b	25.32 bc	9.42 b	26.47 b	0.69 ab
	DN	36.53 b	27.41 b	11.51 b	31.53 b	0.74 ab
	OF	45.27 a	34.18 a	18.28 a	40.54 a	0.75 a
2019–2020/Yangmai 20	LP	33.78 b	29.06 c	11.63 bc	34.42 ab	0.74 c
	NR	34.78 b	29.67 c	10.30 c	29.79 b	0.74 bc
	DN	36.68 b	32.94 b	13.58 b	37.20 ab	0.76 b
	OF	47.98 a	38.18 a	18.81 a	39.19 a	0.79 a
2020–2021/Yangmai 16	LP	31.65 c	25.03 c	10.50 b	33.18 b	0.67 c
	NR	34.23 b	26.38 c	10.24 b	29.94 b	0.67 c
	DN	33.44 bc	29.49 b	13.34 b	39.79 ab	0.73 b
	OF	43.64 a	36.18 a	20.04 a	45.92 a	0.76 a
2020–2021/Yangmai 20	LP	38.09 b	30.84 c	12.19 c	32.13 bc	0.71 c
	NR	38.19 b	31.82 c	11.10 c	29.15 c	0.71 c
	DN	38.87 b	36.11 b	15.40 b	39.58 ab	0.74 b
	OF	49.64 a	42.08 a	21.36 a	43.13 a	0.77 a

^† LP, NR, DN, and OF indicate local practices, nitrogen reduction, planting density reduction and nitrogen reduction, and organic fertilizer, respectively. ^‡ RE_N, PFP_N, AE_N, PE_N, and NHI indicate recovery efficiency of N fertilizer, N partial factor productivity, agronomic N use efficiency, physiological N use efficiency, and nitrogen harvest index, respectively. ^§ Different lowercase letters in the same column represent the same variety and the difference between different treatments in the same year is significant (p < 0.05).

).

3.2. Root Oxidation Activity (ROA)

The ROA of the two cultivars in each growth stage, except for the jointing stage, increased as follows: OF > DN > LP > NR. With the introduction of single cultivation technology, the ROA gradually increased across all growth stages. The ROA of the two cultivars under NR and DN treatments increased and decreased to varying degrees throughout the growth stage, but the overall difference is not significant. The ROA of the two cultivars was significantly greater in OF treatment than in LP, at the jointing, booting, and anthesis stages. There was no significant difference in ROA among different cultivation treatments at maturity (Figure 2). Because the change trend of root total absorbing surface area and root active absorbing surface area is consistent with ROA, only ROA is used to represent the results of the root physiological characteristics. Root total absorbing surface area and root active absorbing surface area are shown in Figure A1.

3.3. Photosynthetic Characteristics at Different Growth Stages

The net photosynthetic rate (P_n) of the two cultivars was as follows: OF > DN > LP > NR. Compared with LP, NR treatment decreased P_n of the two cultivars by 4.54%, 4.91%, 3.03%, and 7.91% on average, respectively, at all growth stages. Compared with LP, the DN treatment increased P_n of the two cultivars by 7.83%, 7.48%, 8.88%, and 7.74% on average, respectively, at all growth stages. Compared with DN, OF treatment increased P_n of the two cultivars by 17.68%, 17.54%, 15.63%, and 18.29% on average, respectively (Figure 3).

3.4. Dry Matter Accumulation at Different Growth Stages

The dry matter accumulation of the two cultivars in each treatment showed OF > DN > LP > NR (except the jointing stage). NR reduced dry matter accumulation of the two cultivars in each stage. With the introduction of single cultivation technology, dry matter accumulation increased gradually in each growth stage. Compared with LP, OF treatment increased dry matter accumulation of the two cultivars by 11.00% on average at maturity. When compared with DN, OF treatment increased dry matter accumulation of the two cultivars by 10.08% on average at maturity (Figure 4).

3.5. Nitrogen Accumulation at Different Growth Stages

The nitrogen accumulation of the two cultivars was significantly greater in NR, DN, and OF treatments than in LP at the jointing stage. The nitrogen accumulation of the two cultivars was significantly greater in OF than in LP at the booting stage. NR treatment reduced nitrogen accumulation in the two cultivars in every stage. Compared with LP, OF treatment increased nitrogen accumulation of the two cultivars by 10.19% on average at maturity. Compared with DN, OF treatment increased nitrogen accumulation of the two cultivars by 12.92% on average at maturity. The increases in nitrogen accumulation at anthesis, similar to those in maturity, were more associated with the improvement in cultivation practices (Figure 5).

3.6. Yield, Yield Component, and Correlation Analysis

The two cultivars under the OF treatment had the highest yield in all treatments; the average yields of Yangmai 16 and Yangmai 20 over two years were 7.23 t·ha⁻¹ and 8.08 t·ha⁻¹, respectively. The NR treatment reduced the yield of both cultivars. Compared with LP, DN treatment increased the grain yield of the two cultivars by 4.54% on average. Compared with DN, OF treatment increased the grain yield of the two cultivars by 16.84% on average. The increase in yield was due to a synchronous increase in the number of panicles, spikelets per panicle, and grain weight, based on yield components’ analysis. The average number of panicles, spikelets per panicle, and grain weight of the two varieties under OF treatment were 546.48 × 10⁴·ha⁻¹, 39.71, and 38.72 mg, respectively. Compared with LP, OF treatment increased the average number of panicles, spikelets per panicle, and grain weight by 13.73%, 5.12%, and 3.61%, respectively (Figure 6). At anthesis, P_n, dry matter accumulation, nitrogen accumulation, nitrogen translocation (except glume + panicle axis), RE_N, PFP_N, AE_N, PE_N, and NHI were all positively correlated with grain yield and its components (Figure 7). There was a significant positive correlation between root physiological characteristics and nitrogen uptake (Figure A3). There were significant differences in yield and yield components among treatments (

Table A2. Analysis of variance of yield and main measurement characteristics.

Yield and Characteristics	Year (Y)	Cultivar (C)	Treatment (T)	Y * C	Y * T	C * T	Y * C * T
Actual yield	** ‡	**	**	ns	ns	*	ns
Number of panicles	ns	**	**	ns	ns	ns	ns
Spikelets per panicle	**	**	**	ns	ns	ns	ns
Grain weight	**	**	**	**	**	*	**
REN †	ns	**	**	**	ns	ns	ns
PFPN	*	**	**	ns	ns	ns	ns
AEN	ns	**	**	ns	ns	ns	ns
PEN	ns	ns	**	ns	ns	ns	ns
Root active absorbing surface area at JS	ns	**	**	ns	ns	ns	ns
Root active absorbing surface area at BS	**	**	**	ns	ns	ns	ns
Root active absorbing surface area at AS	**	**	**	ns	ns	ns	ns
Root active absorbing surface area at GFS	**	ns	**	ns	**	ns	ns
Root total absorbing surface area at JS	**	**	**	**	ns	ns	ns
Root total absorbing surface area at BS	**	**	**	**	ns	*	ns
Root total absorbing surface area at AS	**	**	**	ns	ns	ns	ns
Root total absorbing surface area at GFS	**	**	**	ns	ns	ns	ns
ROA at JS	**	**	**	*	ns	ns	ns
ROA at BS	**	**	**	ns	ns	ns	ns
ROA at AS	**	**	**	ns	*	ns	ns
ROA at MA	**	**	**	ns	ns	ns	ns
Pn at JS	**	**	**	**	ns	**	ns
Pn at BS	**	**	**	**	**	**	ns
Pn at AS	ns	**	**	**	**	**	ns
Pn at GFS	**	**	**	**	**	**	ns
Dry matter accumulation at JS	**	**	**	**	**	**	ns
Dry matter accumulation at BS	**	**	**	**	**	**	ns
Dry matter accumulation at AS	**	**	**	ns	**	ns	ns
Dry matter accumulation at MA	**	**	**	*	**	ns	**
Nitrogen accumulation at JS	**	**	**	ns	ns	ns	ns
Nitrogen accumulation at BS	**	**	**	ns	ns	*	ns
Nitrogen accumulation at AS	**	**	**	ns	ns	ns	ns
Nitrogen accumulation at MA	**	**	**	**	ns	ns	ns

^† RE_N, recovery efficiency of N fertilizer; PFP_N, N partial factor productivity; AE_N, agronomic N use efficiency; PE_N, physiological N use efficiency; NHI, nitrogen harvest index; JS, jointing stage; BS, booting stage; AS, anthesis stage; GFS, grain filling stage; MA, maturity; ROA, root oxidation activity; P_n, photosynthetic rate. ^‡, *, **, and ns indicate significant differences at p ≤ 0.05, p < 0.01, and p > 0.05, respectively.

).

4. Discussion

4.1. Effects of Integrated Cultivation Practices on Photosynthetic Characteristics and Dry Matter Accumulation

Previous studies have shown that farmers’ customary nitrogen application methods could cause total nitrogen leaching [32]. Reasonable management of nitrogen fertilizer, such as combined application of organic and inorganic fertilizers [29], as well as adjustment of base and topdressing ratio [27], can reduce total nitrogen leaching. In this study, compared with LP, the application of pre-nitrogen postponing cultivation techniques (NR, DN, and OF treatments) and the combination of ‘organic fertilizer + inorganic fertilizer + density reduction’ (OF treatment) laid the foundation for the continuous supply of nitrogen fertilizer in the late growth stage of wheat (Table 1).

Nitrogen plays an important role in improving photosynthetic characteristics and dry matter production of wheat flag leaves [33,34]. At the same time, the improvement in photosynthetic characteristics will be conducive to grain filling in the late growth stage of wheat [35,36]. In this study, NR, DN, and OF treatments increased the absorption of nitrogen in wheat at the late growth stage (especially OF treatment), so that the photosynthetic rate decreased slowly at the late growth stage, thereby increasing dry matter accumulation and promoting wheat filling (Figure 3 and Figure 4). This may be the reason the photosynthetic rate and dry matter accumulation of NR treatment did not decrease significantly and the photosynthetic rate and dry matter accumulation of DN and OF treatments increased significantly. The results of the correlation analysis also confirm this (Figure 7).

4.2. Effects of Integrated Cultivation Practices on Root Growth

The root is the main place for wheat to absorb nutrients. The root development degree of wheat indicates the ability of wheat to absorb nutrients [37]. Good root physiological characteristics are an important basis for efficient use of nutrients by wheat. Different nitrogen supply levels have great plasticity on crop root physiological characteristics [38,39]. Higher ROA, root total absorption area, and root active absorption area could promote nutrient uptake by roots [40]. In this study, the physiological characteristics of wheat roots (root total absorbing surface area, root active absorbing surface area, and ROA) were significantly enhanced with the increase in nitrogen supply level (the application of pre-nitrogen backward cultivation technology and the slow release of organic fertilizer) (Figure 2 and Figure A1). This shows that, under different nitrogen supply levels, wheat can regulate root physiological characteristics, thereby obtaining more nitrogen and a higher yield (Figure A3).

So, what role do the morphological characteristics of wheat roots play in the process of nitrogen absorption and utilization? Do cultivation practices affect root morphological characteristics? Xu et al. [41] proposed that the effects of different straw and nitrogen levels on yield and nitrogen use efficiency of winter wheat were mainly through regulating the growth and distribution of roots in the soil, especially in the lower soil layer. Liu et al. [42] showed that optimizing nitrogen fertilizer application could improve nitrogen absorption capacity by increasing root weight density, thereby increasing yield. However, less research has been carried out on the effects of integrated cultivation practices on root morphological characteristics. Therefore, agronomic traits of roots play an important role in yield and nutrient uptake, and further research is needed.

4.3. Effects of Integrated Cultivation Practices on Grain Yield and Nitrogen Uptake and Utilization

It is a challenge for modern cultivation practices to achieve high grain yield while also achieving efficient nitrogen absorption and utilization. Even though many single cultivation technologies, such as density regulation [43,44], fertilizer application [45,46], and improvement to sowing mode [47], have focused on improving grain yield and nitrogen use efficiency, few studies have evaluated the effects of comprehensive cultivation practices, including nitrogen fertilizer application, density regulation, and organic fertilizer application combinations. It is worth noting that reducing nitrogen input and planting density generally reduces the yield by reducing the nitrogen absorption and spike number. However, excessive nitrogen fertilizer input and higher planting density can also reduce the yield by affecting grain filling [26]. Our previous study found that an appropriate reduction of planting density and nitrogen fertilizer input was beneficial to improve the canopy structure of wheat, obtain a higher tiller heading rate, and then increase the yield [48]. In this study, DN treatment obtained a synergistic increase in yield and nitrogen use efficiency when compared with LP, which could be attributed to the higher planting density of LP and the lower nitrogen use efficiency. With the introduction of single cultivation technology, the grain yield and nitrogen use efficiency of OF treatment were significantly increased compared with those of DN treatment (Table 2 and Figure 6).

Previous studies on the relationship between wheat yield and nitrogen uptake and utilization found that, under the conditions of high yield and high N efficiency, wheat could achieve higher nitrogen accumulation at maturity and it was concentrated in grains (higher nitrogen harvest index) [49]. In this study, we observed that grain nitrogen accumulation was higher under integrated cultivation (Figure A2), which was closely related to nitrogen accumulation during the whole growth period and nitrogen translocation during grain filling (Figure 5 and Table A1). Increasing nitrogen accumulation and nitrogen transportation can promote the formation of high yield and high efficiency [50]. In addition, correlation analysis showed that nitrogen accumulation and nitrogen translocation from different parts to grain (except spikelet + rachis) were positively correlated with yield and its components (Figure 7). Therefore, the results suggest that promoting nitrogen transport could be beneficial to increase wheat yield and nitrogen use efficiency under the premise of high nitrogen accumulation.

5. Conclusions

The integrated cultivation practices for wheat following rice through improvement in controlling basic seedling, reduced nitrogen application, applying N at a later growth stage, and combined organic and inorganic fertilizer could achieve the dual goal of increasing yield and nitrogen efficiency. The above superior performance of Yangmai 16 and Yangmai 20 under integrated cultivation practices with basic seedlings 192 × 10⁴·ha⁻¹, nitrogen application of 216 kg·hm⁻², and on this basis with organic fertilizer 1 800 kg·ha⁻¹ was attributed to the efficient nitrogen absorption and utilization, enhanced leaf photosynthetic characteristics and dry matter accumulation, and vigorous root physiological activity. This study aimed to establish a practical guidance for high yield (7.66 t·ha⁻¹, on average) and efficient utilization of nitrogen (RE_N 46.63%, PFP_N 37.66 kg·kg⁻¹, AE_N 19.62 kg·kg⁻¹, and PE_N 42.19 kg·kg⁻¹, on average) of wheat following rice under OF treatment in East China. The mechanism underlying the efficient nitrogen absorption and utilization for high grain yield and high nitrogen use efficiency merits further investigation.