Planting Density and Variety Modulated Root and Leaf Characteristics to Improve Grain Yield of Spring Maize

Qinglong Yang^1,2, Xiwen Shao¹, Yujun Cao², Yanjie Lv², Zhiming Liu², Wenhua Xu³, Lichun Wang^2* and Yongjun Wang^1,2*

¹College of Agronomy, Jilin Agricultural University, Changchun 130118, China

²Institute of Agricultural Resources and Environment, Jilin Academy of Agricultural Sciences/State Engineering Laboratory of Maize, Changchun 130033, China

³CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China

^*For correspondence: wlc1960@163.com; yjwang2004@126.com

Received 28 July 2020; Accepted 13 August 2020; Published 10 December 2020

Abstract

To better understand the accumulation and transport of substances under different planting densities, the adaptation of maize root and leaf in response to increasing planting densities was investigated. In this two-year filed study, three maize varieties, Fumin108 (FM), Xianyu335 (XY) and Dika159 (DK), were sown under three different planting densities: 15,000 (D₁), 60,000 (D₂) and 90,000 plants ha^-1 (D₃) during 2018 and 2019. Increase in planting density gradually increased leaf area index along with reduced leaf area and net photosynthetic rate of individual leaves. In the 0–20 cm soil layer, the average root dry matter decreased by 55.88 and 80.92%, and the average root number decreased by 31.18 and 38.71% under D₂ and D₃, respectively, compared with D₁. With increase in planting density, yield and dry matter per plant of maize gradually decreased while yield and dry matter per ha was increased with increase in D₁-D₂ density and then flattened in D₂-D₃ density. Compared with D₁, two-year average yield per plant was decreased by 34.10 and 51.87% under D₂ and D₃, respectively. The difference in the number of roots of XY, FM and DK were not significant, so change in variety did not alleviate the decrease in the number of roots. At higher planting densities (above D₂), the increase in density did not increase per ha grain yield. In conclusion, the suitable plant density was about 60,000 plants ha^-1 to harvest more yield of spring maize while density higher than that reduced leaf area and photosynthesis per plant. Moreover, leaf area, root number and net photosynthesis per plant was higher in lower planting density coupled with overall less yield on ha basis and thus seemed wastage of soil nutrients and light resources. © 2021 Friends Science Publishers

Keywords: Grain yield; Leaf source; Maize variety; Planting density; Root source

Introduction

One half of the increase in maize (Zea mays L.) production has been attributed to improved fertilizers, farmland management, and cultivation techniques, while the other half increase has been attributed to heterosis (Yang et al. 2019). However, 35 to 40% of the increase in maize yield has been due to genetic improvement in China. Improved cultivation techniques and field management models have played a major role in improving maize production in China (Dai 2000). Among them, increasing planting density is one of the key management practices. Increasing the planting density usually increases maize grain yield until an optimum number of plants per unit area is reached (Duvick 2005; Turgut et al. 2010). However, after reaching the optimum density, the grain yield decreases as the density increases (Zhang et al. 2019). With increasing planting density per plant yield and biomass decreases (Maddonni and Otegui 2006). Therefore, determining the optimal planting density will facilitate the early realization of high-yield maize cultivation. High-density and ultra-high-density planting helped achieve higher maize yields (Zhang et al. 2019). Tokatlidis and Koutroubas (2004) conducted field experiments and argued that increase in modern maize yield is dependent on an increase in density rather than an increase in yield per plant. The source-sink ratio of maize varies with planting density, and the coordination between source and sink organs is directly related to crop yield. Source and sink are closely linked to each other; size of source and its ability to accumulate and distribute substances directly affect sink formation and enrichment (Oorbessy et al. 2016). To explore the effect of source organs on the coordinated growth of source and sink under different planting densities is conducive to identify ways to increase maize yield.

Sources are organs that synthesize and provide nutrients for plant growth. There are three types of sources: leaf sources, stem and sheath sources, and root sources. The former two are the photosynthetic sources and the latter is the nutrition source of crops. Leaves are the main source organs, and about 95% of the grain yield comes from organic compounds, such as carbohydrates and proteins, synthesized via photosynthesis (Fang et al. 2018). Within a certain range, the photosynthetic intensity of crops positively correlates with leaf area index (LAI) (Yan et al. 2019). Therefore, the amount of green leaf area significantly affects leaf photosynthetic capacity, which in turn determines crop dry matter accumulation and grain yield (Jiang et al. 2000). Reasonable utilization of group light energy is the basis of dry matter accumulation, and the flatness of maize leaves is an important criterion to measure the quality of maize itself. Leaf is the main source organ in maize, where the topmost leaf is compact, and the bottommost leaf is flattened to help absorb more light energy. An increase in group leaf area was partly due to the increase in density; larger group leaf area helped achieve high yield (Liu et al. 2000). Therefore, understanding in source-sink relationship is important to improve yield in maize.

The development of roots, an important organ that absorbs nutrients and water, is closely related to the growth of aboveground parts and the formation of grain "sink" (Santiago et al. 2019). Grain yield formation stage is a critical stage for plant nutrient absorption. Nitrogen (N) absorbed by plants after silking accounts for more than 60% of the total nitrogen absorbed during the entire growth period. Nitrogen absorbed is related to higher dry matter accumulation efficiency and an abundant supply of root assimilation during the filling period. As plant density increases, the interaction between roots of the neighboring plants has a greater influence on grain formation. Any impact on dry matter distribution and nutrient absorption significantly affects the change in yield (Yang et al. 2020). Studies have positively correlated root biomass with green leaf area (Ogawa et al. 2005). Further studies on the effects of root interaction on resource distribution, capture, and utilization during grain formation are necessary. This will help breeders to develop high-yielding maize and agronomists to efficiently use resources to increase yield.

Due to the difficulty in sampling and determination of root system, studies have so far focused on yield and photosynthetic performance to evaluate the effects of sources on physiological characteristics of maize. However, research on the interaction between roots of the same variety in a group and its effect on resource distribution and mineral absorption and utilization is relatively less. To fill this knowledge gap, this experiment was conducted to evaluate the influence of leaf source and root source on yield of divergent spring maize varieties under high-medium-low planting density.

Materials and Methods

Experimental site

The experiment was conducted at the Jilin Academy of Agricultural Sciences, Qian’an County (N: 45°01, E: 124°02). The area was located in a semi-arid region with a continental monsoon climate in the mid-temperate zone, sufficient light and heat resources, and an average frost-free period of 146 days. The maize growing seasons in 2018 and 2019 (May 13 to October 8, 2018; May 12 to October 9, 2019) had total precipitation of 407.90 and 506.60 mm, a daily average temperature of 21.09 and 20.53°C and an effective accumulated temperature of 1656.55 and 1592.10°C, respectively.

Experimental design

In this two-year field study, three maize varieties [Fumin108 (FM), Xianyu335 (XY) and Dika159 (DK)] were sown under three different planting densities i.e., 15,000 (D₁), 60,000 (D₂), 90,000 plants ha^-1 (D₃) during 2018 and 2019. Wide and narrow row planting (70 cm, 40 cm) was adopted, and the soil was covered with degradable plastic film. Experiment was conducted under randomized complete block design with factorial arrangement. Each treatment was composed of three replicates with net plot size of 20 m × 10 m. All the plots were supplied with nitrogen (N, 280 kg ha^-1), phosphorus (P₂O₅, 123 kg ha^-1), and potassium (K₂O, 127 kg ha^-1). Total phosphorus (P), potassium (K) fertilizers, and half of nitrogen (N) fertilizer were applied at pre-sowing, and the remaining N fertilizer was top-dressed at six-leaf stage (V6). Irrigation was carried out on all test points to ensure that the water is non-restrictive. Recommended pesticides available in market were sprayed to control pests and diseases while weeds were controlled manually.

Analysis of soil samples

Samples of soil from the surface layer (0–20 cm) soils were collected at random in triplicate at maturity. The soil was divided into 2 sub-samples after sieving it to < 5 mm. A part of the sample was used to determine the composition of soil N (NO₃^--N and NH₄⁺-N) and the soil water content using a standard gravimetric method, whereas the other part was air-dried for analysis of total N. The moisture content of soil was dried at 105°C to a constant weight. NH₄⁺-N and NO₃^--N contents were measured by AAIII continuous flow auto-analyzer. The organic matter content of the soil was determined by the potassium dichromate oxidation-colorimetric method (China Soil Science Association Agricultural Chemistry Committee 1983). The total N content of the soil was determined with a Hanon K9860 Kjeldahl analyzer (Lu 2000).

Measurement of plant parameters

The green leaf length and width of three plants with different treatments were measured at twelfth-leaf stage (V12), tasseling stage (VT), 20 days after flowering (R20), and 40 days after flowering (R40).

Leaf area index (LAI) = leaf area per plant (m²) × number of plants per unit land area (plant) / land area (m²).

At twelfth-leaf stage (V12), tasseling stage (VT), and 20 d (R20) after flowering, different parameters including photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO₂ concentration (Ci) were measured using a portable LI-6400 photosynthesis meter around 10 noon on a sunny day. Three replicates were maintained per treatment. Chlorophyll fast-phase fluorescence kinetic parameters were measured using a Handy PEA (Hansatedi Company) at twelfth-leaf stage (V12), tasseling stage (VT), and 20 days after flowering (R20), and three replicates were maintained per treatment.

Maize plants were sampled at tasseling (VT) and physiological maturity (R6) (Han et al. 2014; Jia et al. 2018a). The dry matter accumulation was determined after drying the plant parts at 80°C to a constant weight. The nitrogen content of the plant was determined by an AAIII continuous flow analyzer (Yang et al. 2019). All the ears in the middle 3 rows of each plot were harvested at physiological maturity used to determine grain yield and yield components, which including kernel number, and 1000-kernel weight. The kernels were separated from the cob by hand and air dried to determine the yield, which was expressed at 14% moisture content.

Statistical analysis

The data were prepared using Sigma Plot 10.0 and Microsoft Excel 2010. DPS 15.10 software was used to perform two-way analysis of variance (ANOVA) and means were separated using Duncan’s New Multiple Range (DMNR) test at a probability level of 0.05. Moreover, Microsoft Excel program was used for graphical presentation of data.

Results

Effects of variety and planting density on maize leaf source

Leaf area index of maize was significantly different between varieties and planting densities (Table 1). The leaf area index of each treatment reached the maximum at tasseling (VT) and then gradually decreased. As the planting density increased from low (D₁) to high (D₃), leaf area index gradually increased while leaf area per plant decreased. Under D₁ planting density, there was little difference in leaf area index among varieties; however, the difference in leaf area index among varieties was significant at D₂ and D₃ densities (Table 1). The maize variety XY showed intolerance to densities. Compared with other varieties, the leaf area index of XY decreased as the density increase (Table 1).

At tasseling and maturity, planting density had a significant effect on leaf nitrogen content of a single plant. Leaf nitrogen content of a single plant of maize gradually decreased as the density increased (Table 2). At the tasseling stage, compared with D₁ (1.50 g), the two-year average maize leaf nitrogen content of D₂ and D₃ decreased by 9.4 and 35.3%, respectively (Table 2). At maturity, compared with D₁ (0.77 g), the two-year average maize leaf nitrogen content of D₂ and D₃ decreased by 37.3 and 51.0%, respectively (Table 2).

The single leaf net photosynthetic rate decreased with increase in density at twelfth-leaf stage (V12), VT, and 20 days after flowering (R20) stage. Compared with D₁, the average net photosynthetic rate of D₂ and D₃ decreased by 7.5 and 12.1%, respectively, at the V12 stage and decreased by 1.0 and 44.5%, respectively, at the VT stage (Table 3). At the V12 and VT stage, the effects of variety, density, and variety × density on net photosynthetic rate were significant (Table 4). The effects of variety and variety × density on net photosynthetic rate reached a significant level after 20 days of flowering (Table 4). The effect of density on net photosynthetic rate first increased and then decreased with growth. At the V12 and R20 stages, GS, Ci, and Tr showed no significant differences between the three densities. During the VT stage, GS, Ci, and Tr decreased as the density increase (Table 3).

The maximum fluorescence (Fm') under light-adapted state at twelfth-leaf stage (V12) first increased and then decreased with increase in density, except for XY at D₃ (Table 5). The Fm' and actual photochemical efficiency (ΦPSII) under photoadaptation at the VT stage first increased and then flattened with increase in density. The electron transfer rate (ETR) increased with increase in density at VT stage and was significantly different between the three densities (Table 5). The Fm', ΦPSII, and ETR in the light-adapted state at 20 days after flowering (R20) increased with increase in density; however, the differences between the three densities were not significant (Table 5). Density had a significant effect on the ΦPSII at the VT stage (Table 4).

The maximum variable fluorescence (Fv) and maximum fluorescence (Fm) in the dark-adapted state increased with increase in density at V12, VT, and R20 (Table 6). Significant differences were observed in the maximum photochemical efficiency (Fv/Fm) between low density (D₁) and higher densities (D₂ and D₃) during V12 and R20 stages, and the difference between D₂ and D₃ was not significant. These findings indicate that within a certain density range, the maximum photochemical efficiency gradually increased with increase in density and then flattened (Table 6). The effect of density on the maximum photochemical efficiency (Fv/Fm) was significant during the V12, VT, and R20 stages (Table 4).

Table 1: Effect of planting density on LAI of three spring maize varieties in 2019

Varieties	Planting densities	V12	VT	R20	R40
Xianyu335	D1	1.03e	1.04c	0.89c	0.70c
	D2	4.13c	4.54b	4.18b	3.48b
	D3	5.42b	5.71a	5.19a	4.25a
Fumin108	D1	1.02e	1.14c	0.98c	0.77c
	D2	3.66d	4.51b	4.03b	3.24b
	D3	5.71ab	5.89a	5.41a	4.41a
Dika159	D1	1.11e	1.03c	0.90c	0.70c
	D2	3.68d	4.29b	3.76b	3.06b
	D3	5.86a	5.56a	5.01a	4.11a

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

LAI= Leaf area index; V12= Twelfth-leaf stage; VT= Tasseling stage; R20= 20 days after flowering; R40= 40 days after flowering; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Table 2: Effect of planting density on leaf nitrogen content of three spring maize varieties

Varieties	Planting densities	VT (g)		R6 (g)
		2018	2019	2018	2019
Xianyu335	D1	1.31bc	1.58ab	0.84a	0.79ab
	D2	0.83d	1.48ab	0.48bc	0.52ce
	D3	1.03bd	1.05c	0.32c	0.30ef
Fumin108	D1	1.42b	1.69ab	0.62ab	0.83a
	D2	1.98a	1.51ab	0.49bc	0.75ac
	D3	0.99bd	0.84c	0.38c	0.59bd
Dika159	D1	1.28bd	1.72a	0.79a	0.74ac
	D2	0.96bd	1.39b	0.45bc	0.20f
	D3	0.94cd	0.98c	0.31c	0.36df

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

VT= Tasseling stage; R6= Physiological maturity; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Effect of variety and planting density on maize roots

With increase in plant density, both root dry matter and root number gradually decreased (Table 7). In the 0–20 cm soil layer, compared with D₁ (43.95 g), the average root dry weight of the tasseling stage (VT) and maturity (R6) under D₂ (17.74 g) and D₃ (8.79 g) decreased by 59.3 and 83.2%, respectively. Compared with D₁ (94), the average number of roots of VT and R6 under D₂ (64) and D₃ (56) decreased by 32.1 and 40.8%, respectively. Compared with D₁ (0.59 g), the average root nitrogen content of VT and R6 under D₂ (0.21 g) and D₃ (0.10 g) decreased by 64.2 and 83.9%, respectively (Table 7). The effects of variety, density, and variety × density on root dry weight were significant at VT and R6 stages. Density showed a significant effect on the number of maize roots at VT and R6 stages (Table 4).

In the 0–20 soil layer, the soil nitrate nitrogen (NO₃^-) and ammonium nitrogen (NH₄⁺) under D₁ were more than D₂ and D₃ densities at the maturity stage of both years (Table 8). Compared with D₁ (3.72 mg kg^-1), the two-year average soil NH₄⁺ content of D₂ and D₃ decreased by 9.08 and 19.25%, respectively. However, NO₃^- content first decreased and then increased as the density increased. Compared with D₁ (18.40 mg kg^-1), the two-year average soil NO₃^- content of D₂ and D₃ decreased by 52.3 and

Table 3: Effect of planting density on photosynthetic parameters of leaves of three maize varieties at different stages in 2019

Stage	Varieties	Planting densities	Pn (m^2 s)^-1	Gs mol (m^2 s)^-1	Ci mmol mol^-1	Tr mmol (m^2 s)^-1
V12	Xianyu335	D1	42.14ab	0.350cd	113.53b	7.77ab
		D2	43.01a	0.435bc	122.24ab	7.76ab
		D3	45.63a	0.691a	160.38a	8.90a
	Fumin108	D1	44.94a	0.509b	137.44ab	8.18a
		D2	45.58a	0.680a	159.72a	8.89a
		D3	37.27bc	0.365cd	120.83ab	7.13ab
	Dika159	D1	44.80a	0.521b	143.39ab	8.04ab
		D2	33.26c	0.284d	138.37ab	7.31ab
		D3	33.06c	0.293d	115.66b	6.26b
VT	Xianyu335	D1	9.39ab	0.451ab	20.81ab	10.87a
		D2	8.52b	0.376bc	19.13bc	9.81ab
		D3	5.50cd	0.203ef	18.93bc	6.42ef
	Fumin108	D1	10.54a	0.487a	17.13cd	10.73a
		D2	8.92ab	0.366cd	16.18cd	8.99bc
		D3	4.88cd	0.213ef	16.95cd	6.33f
	Dika159	D1	6.40c	0.286de	23.68a	7.65de
		D2	8.64b	0.339cd	14.84d	8.21cd
		D3	4.23d	0.155f	19.20bc	4.97g
R20	Xianyu335	D1	19.64bc	0.319a	63.87d	3.84ab
		D2	15.35c	0.232bd	68.89d	3.19bc
		D3	28.24a	0.241bc	94.63c	4.09a
	Fumin108	D1	27.36a	0.175d	131.08a	2.57c
		D2	30.58a	0.287ab	112.53b	3.90ab
		D3	26.13ab	0.205cd	131.31a	3.15bc
	Dika159	D1	29.69a	0.240bc	125.92a	3.54ab
		D2	27.16a	0.262ac	114.04b	3.33ac
		D3	31.71a	0.289ab	107.77b	4.10a

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

V12= Twelfth-leaf stage; VT= Tasseling stage; R20= 20 days after flowering; Pn= Net photosynthetic rate; Gs= Stomatal conductance; Ci= Intercellular CO₂ concentration; Tr= Transpiration rate; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

35.3%, respectively (Table 8). Compared with D₁ (1.70 g kg^-1), the two-year average of total nitrogen content of D₂ and D₃ decreased by 18.41 and 8.72%, respectively. Compared with D₁ (3.5%), the two-year average soil organic matter of D₂ and D₃ was decreased by 12.5 and 5.2%, respectively (Table 8).

Effect of variety and planting density on dry matter accumulation and yield of maize

Table 4: Statistical summary of Pn, ΦPSII, Fv / Fm, root dry weight, number of roots, dry matter per plant, dry matter per ha, yield per ha and HI of three maize varieties grown under different planting densities

Measurement index	Origin of variance	V12		VT		R20		R6
		F value	p value	F value	p value	F value	p value	F value	p value
Pn	Variety (A)	12.9035	0.0005	7.8798	0.0041	11.5948	0.0008	-	-
	Density (B)	7.4639	0.0051	48.3917	0.0000	2.8546	0.0871	-	-
	A×B	8.3261	0.0008	4.2014	0.0163	3.6701	0.0264	-	-
ΦPSII	Variety (A)	2.2355	0.1393	1.0235	0.3817	0.9230	0.4175	-	-
	Density (B)	1.1532	0.3405	14.5188	0.0003	3.4550	0.0566	-	-
	A×B	0.5257	0.7184	1.0334	0.4204	0.4235	0.7895	-	-
Fv / Fm	Variety (A)	3.6883	0.0482	0.4142	0.6677	0.4442	0.6490	-	-
	Density (B)	7.7151	0.0045	25.2743	0.0000	8.7284	0.0027	-	-
	A×B	0.9034	0.4852	0.5006	0.7357	1.0177	0.4277	-	-
Root dry weight	Variety (A)	-	-	20.0982	0.0000	-	-	5.6183	0.0142
	Density (B)	-	-	131.349	0.0000	-	-	78.3639	0.0000
	A×B	-	-	13.2659	0.0001	-	-	1.6680	0.2064
Number of roots	Variety (A)	-	-	2.4197	0.1207	-	-	9.3944	0.0020
	Density (B)	-	-	20.8556	0.0000	-	-	36.2259	0.0000
	A×B	-	-	0.6874	0.6111	-	-	0.7429	0.5766
Dry matter per plant	Year (A)	-	-	0.012	0.913	-	-	23.827	0.0000
	Variety (B)	-	-	1.445	0.250	-	-	12.041	0.0001
	Density (C)	-	-	125.48	0.000	-	-	332.81	0.0000
	A×B	-	-	0.303	0.741	-	-	4.3528	0.0207
	A×C	-	-	3.346	0.047	-	-	3.7819	0.0329
	B×C	-	-	1.423	0.247	-	-	0.7560	0.5611
	A×B×C	-	-	7.523	0.000	-	-	1.5199	0.2183
Dry matter per ha	Year (A)	-	-	2.4835	0.1243	-	-	11.471	0.002
	Variety (B)	-	-	0.8165	0.4505	-	-	11.020	0.000
	Density (C)	-	-	426.811	0.0000	-	-	257.773	0.000
	A×B	-	-	3.2535	0.0509	-	-	1.251	0.299
	A×C	-	-	8.4501	0.0010	-	-	1.003	0.377
	B×C	-	-	2.5349	0.0580	-	-	1.960	0.123
	A×B×C	-	-	5.6637	0.0013	-	-	0.391	0.813
Yield per ha	Year (A)	-	-	-	-	-	-	45.5231	0.0000
	Variety (B)	-	-	-	-	-	-	1.9575	0.1568
	Density (C)	-	-	-	-	-	-	145.2296	0.0000
	A×B	-	-	-	-	-	-	3.7212	0.0346
	A×C	-	-	-	-	-	-	0.7767	0.4679
	B×C	-	-	-	-	-	-	3.0202	0.0311
	A×B×C	-	-	-	-	-	-	2.3357	0.0752
HI	Year (A)	-	-	-	-	-	-	2.9833	0.0932
	Variety (B)	-	-	-	-	-	-	1.0152	0.3731
	Density (C)	-	-	-	-	-	-	4.0032	0.0275
	A×B	-	-	-	-	-	-	13.3025	0.0001
	A×C	-	-	-	-	-	-	5.9217	0.0062
	B×C	-	-	-	-	-	-	10.2722	0.0000
	A×B×C	-	-	-	-	-	-	5.5553	0.0015

V12= Twelfth-leaf stage; VT= Tasseling stage; R20= 20 days after flowering; R6= Physiological maturity; Pn= Net photosynthetic rate; ΦPSII= Actual photochemical efficiency; Fv/Fm= Maximum photochemical efficiency; HI= Harvest index

At the VT and R6 stages, dry matter per plant decreased significantly with increase in planting density while dry matter per ha first increased and then flattened (Table 9). At the VT stage, the two-year average dry matter per plant under D₂ and D₃ decreased by 26.72 and 44.27%, respectively, compared with D₁. At the R6 stage, the two-year average dry matter per plant under D₂ and D₃ decreased by 36.90 and 54.30%, respectively, compared with D₁. At the VT stage, the two-year average dry matter per ha of D₂ and D₃ increased by 198.81 and 238.83%, respectively, compared with D₁. At the R6 stage, the two-year average dry matter per ha under D₂ and D₃ increased by 152.4 and 174.2%, respectively, compared with D₁ (Table 9). At R6 stage, year and variety showed significant effects on dry matter per plant and dry matter per ha. Density significantly affected dry matter accumulation per plant and dry matter per ha at VT and R6 stages (Table 4).

At the maturity stage (R6), the two-year average yield per ha under D₂ and D₃ increased by 73.27 and 79.91%, respectively, compared with D1 (Table 10). Compared with D₁, the two-year average grains per ear under D₂ and D₃ was decreased by 2.6 and 10.9%, respectively (Table 10). Compared with D₁, the two-year average ears per unit area under D₂ and D₃ increased by 74.3 and 133.0%, respectively. Compared with D₁, the two-year average 1000-kernel weight under D₂ and D₃ was decreased by 2.8 and 13.9%, respectively (Table 10). Compared with D₁, the two-year average harvest index under D₂ and D₃ was increased by 3.9 and 5.1%, respectively (Table 10). The effects of year and density on yield per ha were significant while the effect of variety was not significant at the maturity stage. The effects of year, variety, and density on yield per plant were significant at the maturity stage. Density had a significant effect on harvest index (Table 4).

Discussion

Table 5: Effect of planting density on fluorescence and light response index of plant leaves of three maize varieties in 2019

Varieties	Planting densities	V12			VT			R20
		Fm’	ΦPSII	ETR	Fm’	ΦPSII	ETR	Fm’	ΦPSII	ETR
Xianyu335	D1	84.33a	0.833a	3.50b	63b	0.775bc	2.39e	85.00ac	0.43ab	1.54b
	D2	88.67a	0.857a	3.78ab	100a	0.854a	2.75d	97.33ac	0.51ab	1.82ab
	D3	57.00cd	0.793a	3.67ab	117a	0.866a	3.76a	108.00a	0.54a	2.10a
Fumin108	D1	43.67d	0.795a	3.51b	61b	0.752c	2.68d	82.67bc	0.42ab	1.50b
	D2	64.67bc	0.803a	3.71ab	97a	0.837a	3.11c	91.33ac	0.45ab	1.71ab
	D3	63.00bc	0.792a	3.78ab	108a	0.856a	3.77a	92.67ac	0.45ab	1.80ab
Dika159	D1	77.33ab	0.817a	3.62ab	74b	0.813ab	3.13bc	73.67c	0.38b	1.42b
	D2	93.00a	0.875a	4.04a	103a	0.842a	3.36b	94.67ac	0.49ab	1.89ab
	D3	91.67a	0.850a	3.99ab	106a	0.851a	3.76a	98.67ab	0.52a	2.09a

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

V12= Twelfth-leaf stage; VT= Tasseling stage; R20= 20 days after flowering; Fm’= Maximum fluorescence; ΦPSII = Actual photochemical efficiency; ETR = Electron transfer rate; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Table 6: Effect of planting density on fluorescence dark response index of plant leaves of three maize varieties in 2019

Varieties	Planting densities	V12			VT			R20
		Fv	Fm	Fv/Fm	Fv	Fm	Fv/Fm	Fv	Fm	Fv/Fm
Xianyu335	D1	64.00df	68.33d	0.832c	72b	86bc	0.840b	51.00bc	94.67b	0.54c
	D2	71.00cd	83.00cd	0.855bc	102a	114a	0.895a	64.33ab	109.33ab	0.59ab
	D3	82.33bc	92.67bc	0.888ab	116a	129a	0.901a	72.00a	117.33a	0.61a
Fumin108	D1	49.33f	67.00d	0.830c	64b	74c	0.854b	53.67bc	96.33b	0.56bc
	D2	65.00de	78.00cd	0.834c	99a	110a	0.899a	62.33ac	107.00ab	0.58ac
	D3	95.00ab	110.67ab	0.860bc	107a	119a	0.899a	62.33ac	108.67ab	0.57ac
Dika159	D1	53.67ef	66.67d	0.832c	72b	84bc	0.861b	50.33c	94.00b	0.54c
	D2	97.33a	108.67ab	0.895ab	95a	106ab	0.893a	59.67ac	103.67ab	0.57ac
	D3	109.67a	120.33a	0.912a	107a	118a	0.902a	68.67a	114.67a	0.60ab

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

V12= Twelfth-leaf stage; VT= Tasseling stage; R20= 20 days after flowering; Fv = Maximum variable fluorescence; Fm = Maximum fluorescence; Fv/Fm = Maximum photochemical efficiency; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Table 7: Effect of planting density on root dry weight, root number and nitrogen content of 20 × 20 × 20 cm volume in 2019

Varieties	Planting densities	VT			R6
		Root dry weight (g)	Number of roots	Nitrogen content (g)	Root dry weight (g)	Number of roots	Nitrogen content (g)
Xianyu335	D1	23.57c	87a	0.48b	35.99b	78b	0.47a
	D2	13.28de	58bc	0.18cd	14.08cd	51d	0.15b
	D3	5.90f	48c	0.08d	8.00d	48d	0.07b
Fumin108	D1	38.29b	97a	0.54b	53.11a	101a	0.65a
	D2	20.75c	76ab	0.27c	20.62c	62bd	0.22b
	D3	10.56df	59bc	0.12cd	12.40cd	55d	0.14b
Dika159	D1	55.76a	95a	0.78a	56.94a	105a	0.63a
	D2	17.86cd	59bc	0.21cd	19.80cd	77bc	0.24b
	D3	5.99ef	64bc	0.06d	9.91cd	59cd	0.10b

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

VT= Tasseling stage; R6= Physiological maturity; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Table 8: Effect of planting density on soil nutrient status of 0-20 cm soil layer at maize maturity stage

Varieties	Planting densities	NH4^+-nitrogen (mg kg^-1)		NO3^--nitrogen (mg kg^-1)		Total nitrogen (g kg^-1)		Soil organic matter (%)
		2018	2019	2018	2019	2018	2019	2018	2019
Xianyu335	D1	2.23cd	6.77a	21.53a	27.63a	1.61ab	2.34a	2.97bd	5.41a
	D2	2.88a	4.20bc	13.37d	10.79c	1.36b	1.30a	3.39ab	3.00b
	D3	1.93cd	4.36bc	19.89b	7.04d	1.45ab	1.58a	3.46a	3.66ab
Fumin108	D1	1.93cd	4.83b	19.77b	13.53b	1.76a	1.40a	3.01ad	3.31ab
	D2	2.33bc	3.90cd	5.24f	8.16d	1.31b	1.46a	2.81cd	3.27b
	D3	2.02cd	3.43d	5.77ef	12.79b	1.42ab	1.42a	2.81cd	3.32ab
Dika159	D1	2.68ab	3.85cd	17.28c	10.62c	1.69ab	1.41a	3.26ac	2.94b
	D2	1.80d	4.16c	6.49e	8.23d	1.50ab	1.40a	2.65d	3.17b
	D3	1.86d	4.04cd	13.54d	12.49b	1.48ab	1.97a	2.78d	3.79ab

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-11

Planting density is an important factor that improves root and canopy conditions and affects the group photosynthetic system, and increase density is the easiest way to improve yield, because within a certain period of time, it is difficult to increase the crop's yield potential through breeding (Xu et al. 2017). Root is the nutrient source while leaf is the photosynthetic source of the crop. These two sources are indispensable and contribute differently to yield (Liu et al. 2018). Increase in density causes a series of changes in the root and leaf sources of maize plants that help adapt to changes in the external environment. Studies have pointed out that nitrogen uptake in plants is determined by the size of the root system. Longer root system increases surface area of the root, which helps the plant to absorb more nitrogen (Zhu et al. 2016; Jia et al. 2020). The change in planting density changed the environmental conditions of maize at various growth stages. This affected growth and development of the root system, which in turn promoted nitrogen absorption, assimilation, and distribution in maize (Shi et al. 2016; Jia et al. 2018). In this study, increase in planting density of maize reduced the number of roots and the dry weight of roots. The replacement of varieties in cultivation did not alleviate the reduction in number of roots. Increase in planting density also reduced the soil nitrate nitrogen and ammonium nitrogen residues, which improved the nutrient use efficiency. Soil nitrate content had the highest nutrient use efficiency at medium planting density (D₂). Higher the density, higher the nutrient use efficiency; however, this theory holds true only within a certain density range. Nitrogen content of the root system clearly indicates that the increase in density reduced the absorption and utilization of nutrients per plant in maize. In the planting density range of D₁–D₂, the nutrient utilization efficiency of a single plant decreased, whereas of the group improved. At higher densities (above D₂), the number of roots significantly reduced and nutrient absorption and utilization by a single plant got restricted, which resulted in a decrease in nutrient utilization efficiency of the group.

Table 9: Effect of planting density on plant dry matter accumulation at flowering and mature stages in three spring maize varieties

Year	Varieties	Planting densities	VT Dry matter per plant (g)	R6 Dry matter per plant (g)	VT Dry matter per ha (kg ha^-1)	R6 Dry matter per ha (kg ha^-1)
2018	Xianyu335	D1	235.47a	592.52a	3438.33e	8887.75c
		D2	119.70d	403.59c	7302.00d	24215.60a
		D3	137.45cd	271.01de	11976.40a	24391.20a
	Fumin108	D1	187.27b	497.93b	2839.33e	7468.95c
		D2	163.60bc	298.54d	10148.00bc	17912.20b
		D3	112.80d	225.49e	10551.00bc	20294.10b
	Dika159	D1	188.27b	471.97b	2747.33e	7079.60c
		D2	158.17bc	325.72d	9661.33c	19543.20b
		D3	121.90d	224.53e	11259.00ab	20208.00b
2019	Xianyu335	D1	199.33ab	595.27a	2990.00c	8929.05c
		D2	175.33bc	389.43b	10520.00a	23366.00ab
		D3	117.00d	305.89cd	10530.00a	27530.40a
	Fumin108	D1	228.33a	629.19a	3425.00c	9437.80c
		D2	149.67c	351.80bc	8980.00b	21108.20b
		D3	102.00d	268.02d	9180.00ab	24121.80ab
	Dika159	D1	208.00a	597.45a	3120.00c	8961.70c
		D2	147.33c	359.30bc	8840.00b	21557.80b
		D3	102.33d	250.47d	9210.00ab	22542.60b

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

VT= Tasseling stage; R6= Physiological maturity; D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Table 10: Effect of planting density on yield and related traits of spring maize varieties

Year	Varieties	Planting densities	Number of grains per ear	Ears per unit area (ear ha^- 1)	1000-grainweight (g)	Grain yield (kg ha^-1)	HI (%)
2018	Xianyu335	D1	568ab	29000cd	395.87a	6660.59de	50.47c
		D2	558ac	58500b	388.84a	12774.28ab	60.30ab
		D3	423d	85000a	336.60b	12849.23ab	59.91ab
	Fumin108	D1	551abc	36500c	347.83b	6899.54d	62.62a
		D2	594a	59000b	343.18b	12269.09ac	57.30b
		D3	502bc	81000a	318.80bc	13263.52a	56.97b
	Dika159	D1	567ab	26500d	339.92b	5327.13e	46.02d
		D2	543ac	59000b	342.52b	10916.48c	58.59b
		D3	489cd	82500a	293.58c	11594.72bc	57.83b
2019	Xianyu335	D1	591a	41026d	410.39a	9668.53c	50.52d
		D2	572ab	66667c	391.01ab	14892.06ab	59.21ac
		D3	487c	73504bc	347.93cd	12920.24b	58.92ac
	Fumin108	D1	523ac	44444d	417.19a	9237.41c	59.37ac
		D2	574ab	64957c	377.40b	13366.73b	53.42cd
		D3	516bc	83761ab	326.98d	13671.51b	56.59bd
	Dika159	D1	489c	41026d	410.35a	7842.52c	65.43a
		D2	531ac	66667c	409.65a	13509.27b	58.70ac
		D3	508bc	90598a	372.18bc	16254.92a	61.08ab

Values followed by different small letters in the same column are significantly different from each other at P ≤ 0.05

D₁ = 15000 plants ha ^-1; D₂ = 60000 plants ha ^-1; D₃ = 90000 plants ha ^-1

Root system of a plant influences growth and development of the aboveground parts. In maize, the moisture and nutrient absorption capacity of the root system depends on the size and distribution in the soil and on the photosynthetic supply from aboveground parts. In turn, the root system provides the inorganic nutrients required for leaf growth and photosynthesis (Lu et al. 2017). Studies have found close interaction between roots and leaves of maize. Leaf area is closely related to root dry weight and total root absorption area (Chilundo et al. 2017; Liu et al. 2018). Additionally, leaf number and photosynthetic capacity are important parameters to determine yield (Zhang et al. 2017). In this experiment, with increase in density, plant leaf area and photosynthesis and fluorescence decreased, which resulted in a decrease in dry matter accumulation. Increase in density resulted in smaller leaves with lesser surface area for photosynthesis, the main source of material accumulation. Increase in density decreased stomatal conductance and intercellular CO₂ concentration, which significantly reduced the photosynthetic rate per unit time. Additionally, the decrease in leaf nitrogen content affected the leaf photosynthetic rate, which decreased plant dry matter accumulation. Significant difference was observed in dry matter per ha accumulation between D₁ and D₂ and not between D₂ and D₃. This finding indicates that in the low to medium density range (D1–D2), the increase in density reduced photosynthesis and dry matter accumulation of each plant; however, it increased dry matter accumulation of the group. At higher planting densities (above D₂), photosynthesis of a single plant played a major role in dry matter per ha accumulation, and therefore, the difference in dry matter per ha accumulation with increase in density was insignificant.

Planting density is one of the important factors that influence grain yield in maize and use of an optimal planting density is the best way to obtain high yield (Nyakudya and Stroosnijder 2014). In our study, medium (D₂) planting density significantly increased the yield per ha compared to low planting density (D₁). Medium (D₂) and high (D₃) planting densities showed no significant difference between each other in grain yield. These findings indicate that within the range D₁–D₂, increase in density significantly increased maize yield; however, further increase in density in the range D₂–D₃ did not increase the yield. In maize, number of ears per unit area is the main factor that contributes to yield increase. In the present study, in the range D₁–D₂, grains per ear and 1000-kernel weight remained almost the same; however, ears per unit area increased significantly with increase in density. In the range D₂–D₃, the differences in grains per ear, 1000-kernel weight, and ears per unit area were significant. The effects of grains per ear and 1000-kernel weight on yield may have predominated in the range D₂–D₃. Although ears per unit area increased, grains per ear and 1000-kernel weight decreased significantly with increase in density in the range D₂–D₃ with no increase in yield. Increase in density increased harvest index in the density range D₁–D₂. However, at low density, the proportion of total grains in total dry matter was relatively small and the transfer of photosynthetic products to the grains was low. These products remained concentrated in the stalks and leaves and resulted in waste of photosynthetic products. Therefore, increase in density increased harvest index. However, no significant increase was observed in the harvest index with increase in planting density from D₂ to D₃. Therefore, the planting density should be increased considering the local conditions.

In the current study, we studied the changes in root system in the 0–20 cm soil layer; however, there is a lack of research on deeper roots. In future, we will have to systematically explore the effects of variety and density on microbial diversity and nutrient absorption and utilization in the deep root soil.

Conclusion

Increase in planting density reduced the root number and root dry weight of individual plants and all three varieties showed similar decrease in root number, which limited soil nutrient absorption and utilization. Increase in planting density weakened individual plant photosynthetic ability, while increased population dry matter accumulation. In conclusion, all three maize varieties harvested higher grain yield under planting density of 60,000 plants ha^-1 and density lower than that could cause wastage of soil and light resources.

Acknowledgment

We acknowledge the financial supports of the National Key Research and Development Program of China under Grant No. 2016YFD0300103 and the National Natural Science Foundation of China under Grant No. 31701349.

Author Contributions

Lichun Wang and Yongjun Wang conceived and designed the experiments; Qinglong Yang performed the experiments; Qinglong Yang, Xiwen Shao and Wenhua Xu analyzed the data; Yujun Cao, Yanjie Lv and Zhiming Liu contributed reagents/materials/analysis tools; Qinglong Yang wrote the paper.

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