Introduction
Maize
is an important crop worldwide for grain, economic and feed production (Zhang et al. 2015; Kumar and Singh, 2017).
Heilongjiang province is China's main maize producing area. In recent years,
with the adjustment of the national planting structure, the area of maize in
Heilongjiang province has been continuously reduced, with a reduction of 34
million ha in two years. With the continuous reduction of the maize planting
area, the total maize production in Heilongjiang province reached 61.88 million
tons, making outstanding contributions to China's food security and playing a
pivotal role in China's food security (FAO 2017). At present, worldwide maize
production is facing problems such as insufficient planting densities and
excessive application of N fertilizer. Studies have shown that the planting
density of maize in the United States is 85,000~100,000 plants ha-1
(Pei et al. 2017), whereas in
Heilongjiang province, it is typically 45,000~60,000 plants ha-1.
Therefore, increasing the planting density can enhance the maize yield in China
if optimum planting density exists (Shi et
al. 2016; Ning et al. 2017).
Exceeding the optimum planting density will not only inhibit the growth of
maize per plant but also increase the risk of lodging, leading to difficulty in
harvesting and decline in yield (Haegele et
al. 2014).
Nitrogen (N) is a major factor restricting crop
yields. Since the 19th century, the amount of N applied to maize
under traditional cultivation has been approximately 130 kg ha-1,
and the application rate of N is currently twice these historic levels (Zhang et al. 2011). Excessive application of N
fertilizer reduces the utilization rate and increases the risk of groundwater
pollution (Ye et al. 2016). At the
same time, such application increases the height of the crops, which causes the
plant to stretch continuously at the base and results in plants which easily fall over. Excessive N fertilizer also
accelerates its absorption and transport by crops, leading to premature ageing
(Zhu et al. 2016).
Studies have shown that maize stems and roots have a
"flow" function in the source-sink system, playing an important role
in water and nutrient absorption, synthesis and transport (Wang et al. 2004). Endogenous hormones in the
root system regulate the relationship between roots and shoots and play roles
in improving crop quality. Previous studies have shown that the self-regulation
of endogenous hormone levels in crops can regulate the growth and development
of plants and the differentiation of tissues and organs (Kiba et al. 2011).
Cai et al. (2012) reported that appropriate planting
density and reasonable N fertilizer application can facilitate the growth and
development of maize. Density has a significant effect on the number of spikes
per unit area of maize. The amount of nitrogen applied has a significant effect
on the number of effective panicles and 100-grain weight of maize. Increasing
the planting density or the application rate of N fertilizer decreased the
lodging resistance of maize but increased the stem rate, grain weight and
100-grain quality (Deng et al. 2017;
Piao et al. 2017).
Lodging is a serious
obstacle to normal growth and yields in maize production. An annual loss of 5–25%
of maize production is caused by lodging, and every 1% increase in lodging will
cause a decrease of 108 kg•hm-2 in yield (Norboerg et al. 1988). Studies have shown that
the lodging of maize stems is related to morphological indexes, such as plant
height, ear height and length of internode elongation (Ma et al. 2014), and is closely related to stem cellulose,
hemicellulose and lignin. When lodging occurs, the normal canopy structure of
maize is destroyed, resulting in decreased photosynthesis and grain yield,
reduced crop quality, and difficult harvesting (Li et al. 2017).
In previous studies, a
series of experiments were performed on the effects of N fertilizer or planting
density on the lodging of maize stems and the morphological shape and
mechanical strength of stems (Gou et al.
2007; Bian et al. 2017; Xue et al. 2017; Yu et al. 2019). Research on the physiological indexes of maize stems,
the composition of xylem sap and its relationship with stem lodging under
different N fertilizers and planting densities has rarely been reported. The
purpose of this study was to investigate the effects of a reasonable nitrogen
application rate and planting density on the physiological characteristics of
stems and root sap and the lodging resistance of spring maize stalks in
Heilongjiang province and at the same time, the objective was to provide a
theoretical and experimental basis for achieving lodging resistance and high
yield of Heilongjiang spring maize with reasonable nitrogen fertilization and
planting density.
Materials
and Methods
Table 1: Daily mean values of the
weather variables at the experimental site during the six months of the maize
growing season in 2016 and 2017
|
Month |
Average temperature (°C) |
Precipitation (mm) |
Sunshine (h) |
|||
|
2016 |
2017 |
2016 |
2017 |
2016 |
2017 |
|
|
April |
8.0 |
17.6 |
15.2 |
74.1 |
219.10 |
246.9 |
|
May |
16.0 |
22.7 |
106.8 |
27.5 |
183.00 |
282.5 |
|
June |
20.1 |
25.2 |
206.1 |
49.5 |
238.10 |
244.8 |
|
July |
24.3 |
30.6 |
44.2 |
16.9 |
246.20 |
302.7 |
|
August |
23.2 |
28.6 |
31.7 |
54.4 |
283.70 |
204.8 |
|
September |
17.1 |
23.5 |
70.3 |
35.8 |
152.40 |
211 |
|
Total |
18.1 |
24.7 |
474.3 |
258.2 |
1322.50 |
1492.7 |
Site description
and weather data
This
experiment was carried out at the A Cheng experimental base, Northeast
Agricultural University, Heilongjiang province, China (45º 42’ N, 126º 36’ E).
The soil was a typical black soil. It contained 28.35 g kg-1 organic
matter, 1.55 g kg-1 total nitrogen, 24.92 mg kg-1
available nitrogen, 59.58 mg kg-1 available phosphorus and 219.5 mg
kg-1 available potassium. Meteorological data during the maize
growth cycle were provided by the Harbin Academy of Agricultural Sciences
(Table 1). The test variety 'Nonghua 101' was provided by Beijing Golden
Nonghua Seed Industry Technology Co., Ltd. The experiment was conducted using a
randomized block design with two factors. The tested nitrogen treatments were
100 (N1), 200 (N2) and 300 kg ha-1 (N3) and the planting densities
were 6.75 (D1), 8.25 (D2) and 9.75 (D3) million plants ha-1. Before sowing, 100 kg ha-1
of phosphate fertilizer (superphosphate) and 100 kg ha-1 potassium
fertilizer (potassium sulfate) were released as base fertilizer and applied to the ridge side (depth: 10 cm). The N fertilizer (urea, nitrogen
content approximately 48%) was equally divided into two soil applications as
base fertilizer before sowing and the other as top dressing before the ridge
was closed.
The test plot had 10 rows with 8 m in length and with
row spacing of 65 cm and the area was 52 m2. In the small section
and the repeating section, a walkway with a width of 50 cm was arranged, and a
protective buffer line with a width of 1 m was established around the plots.
The other management measures were the same as those applied in high-yield
fields. The trial was planted on April 25, 2016 and
April 27, 2017 and harvested on September 28, 2016 and September 29, 2017 in
the two study years.
Data collection
and analyses
Stem lodging
rate
At
the time of harvesting, the central three rows of each treatment were selected
and the number of lodgings was counted. The lodging rate is the ratio of the
number of lodgings to the total number of plants.
Stem physiological
index
In
the elongation stage (July 5), the tasseling stage (July 25), the early filling
stage (August 3) and the milk stage (August 24), a standard scrubbing method was
used to determine the lignin, cellulose and hemicellulose content of the third
internode of maize. This procedure was repeated three times for each indicator for each
treatment and the average was taken.
Key lignin
synthesis enzymes
The phenylalanine ammonia
lyase (PAL) activity was determined
(Heinzmann and Seitz 1974); the tyrosine ammonia lyase (TAL) activity was
performed (Khan et al. 2003); the
method for determining 4-coumaric acid: Co A ligase (4CL) activity was
described (Knobloch and Hahlbrock, 1975) and the cinnamyl-alcohol dehydrogenase
(CAD) activity was determined (Morrison et
al. 1994).
Root wound
fluid collection
Root
wound fluid was collected during the elongation stage (July 5), tasseling stage
(July 25), early filling stage (August 3), and milk stage (August 24). The
collection time was from 5:00 pm to 5:00 a.m. the next day. A test tube was
filled with moderately dry, absorbent cotton (approximately 2/3 of the volume
of the finger tube). The plants were quickly cut with scissors at the 3rd
stem section, the stems were rinsed with deionized water, the tubes were fixed
on the residual stems with plastic wrap and collection was performed for 12 h.
Endogenous hormones
Three samples were
selected from the top of the maize plant to the third stem section, frozen in
liquid nitrogen for 30 min and stored in a -40°C refrigerator. The contents of
auxin (IAA), gibberellin (GA), cytokinin (CTK) and abscisic acid (ABA) were determined by Shanghai
Ji Ning Industrial Co., Ltd. with an enzyme-linked immunosorbent assay.
Yield
During the harvest stage,
each group of 4 rows and 5 rows of each plot were selected as the actual
harvest, and the whole spike was harvested to calculate the average single ear
quality. Twenty uniform ears were selected to determine the average single ear
quality. Ears were brought inside for air drying, and the number of ears per
row, number of rows of grains and number of grains per ear were determined.
After threshing, the moisture content of the grain and the 1000-grain weight
were measured. Actual yield (kg ha-1, 14% water content) = measured
maize ear quality (kg)/measured area (m2) × seed yield × 15 × 666.7
m2 × (1 - grain moisture content)/0.86.
Data analysis
According
to the analysis of variance, data were statistically analysed following
standard methods using Microsoft Excel 2010 and SPSS 12.0. Differences between
treatments were determined by a posteriori Tukey’s test at P < 0.05.
Results
Cellulose contents
The
lodging resistance of maize is related to the physiological characteristics of
stem development. When the content of cellulose, hemicellulose and lignin in a
unit volume of stem was high, the degree of lignification was high, the
mechanical properties were good and the lodging rate was low. The cellulose
content of the differently treated maize stems showed a curve with a single
peak. As the growth period progressed, the cellulose content of the stem first
increased and then decreased, reaching a maximum at the early filling stage (Fig.
1).
The cellulose content of the stem for D2N2 was lower
than other treatments at the elongation stage. The cellulose content under the
D1N3 and D2N2 treatments was significantly higher than under the other
treatments during the tasseling stage and early filling stage. Compared with
the D1N1 and D3N3, we found D1N3 treatment increased the cellulose content by
5.3%, 51.61% and 49.71%, 33.36% during the various stages, respectively.
Compared to the D1N1 and D3N3 treatments, the D2N2 treatment increased the
cellulose content by 0.39%, 45.39% and 36.14%, 9.69%, respectively. The maximum
cellulose content at the tasseling stage was obtained with D1N2, and the
maximum cellulose content values at the early filling stage and milk stage was
obtained with D1N3 and D1N1, respectively.
Hemicellulose
content
The change in hemicellulose
content in stems treated with different N fertilizer rates was similar to
cellulose contents. As the growth period progressed, the hemicellulose content
of stems increased first and then decreased %20711-720_files/image002.png)
Fig. 1: Effects of N fertilizer
and planting density on the cellulose content of maize
%20711-720_files/image004.png)
Fig. 2: Effects of N fertilizer and planting density on the hemicellulose content of maize
and
the hemicellulose content of stems reached its maximum value at the early
filling stage. Except during the early filling stage, the maximum hemicellulose
content in the remaining periods was obtained with D1N1 (Fig. 2).
At the early filling stage, the maximum hemicellulose
content of the stem was obtained with D1N3, and D1N2 and D2N1 that were 24.36,
23.12, 16.09 and 14.94% higher than D1N1 and D3N3, respectively. At the same
planting density, the hemicellulose content decreased with increasing nitrogen
application rates.
Lignin content
The lignin content of each
treatment peaked at the early filling stage. During the tasseling stage, the
lignin content of maize stems showed the trend D1N2>D3N3>D3N2, and the
maximum value was obtained 44.87 mg g-1 for D1N2 treatment. In the
early filling stage, the lignin content of maize stems in each treatment showed
this trend D2N3> D1N2>D3N3, reaching a maximum of 50.05 mg g-1
under D2N3. The rest of the treatments did not show significant differences in
lignin levels in these two periods (Fig. 3).
%20711-720_files/image006.png)
Fig. 3: Effects of N fertilizer
and planting density on the lignin content of maize
Key lignin
synthesis enzymes activities of maize
As
the growth period progressed, the PAL TAL and 4CL activity of the third
internode of each treated maize plant gradually decreased. Except during the
elongation stage, the D1N1 treatment did not result in activities that were
significantly higher than other treatments. At the tasseling and the early
filling stages, the PAL activity reached its maximum under the D1N3 treatment.
The enzyme activity of each treatment showed this trend D1N3>D2N1>D2N2.
In the milk stage, the difference in PAL activity between the treatments was
not significant.
The TAL activity of each treatment was lower than PAL
activity. With advancing growth stage, the TAL activity of each treatment
increased slightly at the early filling stage while the CAD activity decreased
gradually, and each treatment increased the CAD activity during the milk stage.
The treatments showed this trend D1N2>D1N3>D2N2 for CAD activity. The
result indicated the CAD activity was significantly enhanced with increased
planting density and decreased nitrogen fertilization, which promoted the
synthesis of lignin. The 4CL activity of each treatment decreased rapidly after
the elongation stage. At the early filling stage, the 4CL activity reached its
maximum under D2N2 treatment, and this value was significantly higher than in
the other treatments (Fig. 4).
Correlation
analysis between lignin content and lignin synthetase activity
Correlation
analysis showed that the lignin content of maize stems was significantly
negatively correlated with the stem lodging rate. When the lignin content was
high, the lodging rate of the maize stem was low and the lodging resistance was
strong. There were significant positive correlations between lignin content and
PAL activity, TAL activity and 4CL activity with correlation coefficients of
0.82, 0.52 and 0.78, respectively and were significantly negatively correlated
with CAD activity (Table 2).
Endogenous hormones
in root bleeding sap
The root system is an important
site of endogenous hormone synthesis and transformation. The balance of hormone
levels between roots and crowns is particularly important for maize growth and
morphogenesis. The four endogenous
hormones IAA, ABA, GA and CTK exhibited different trends during the growth
period (Table 3).
%20711-720_files/image008.png)
Fig. 4: Effects of N fertilizer and
planting density on the activities of key lignin synthesis enzymes of maize,
PAL, TAL, CAD and 4CL in the years 2016 and 2017
Table 2: Correlation analysis between lignin content and
lignin synthetase activity in the elongation stage of maize
|
Year |
PAL activity |
TAL activity |
CAD activity |
4CL activity |
Lodging percentage |
|
2016 |
0.817** |
0.507** |
-0.437* |
0.778** |
-0.470* |
|
2017 |
0.899** |
0.310 |
0.470 |
0.823** |
-0.491* |
Note: * and **
indicate significance at 0.05 and 0.01 probability levels, respectively
The
peak of IAA flow occurred during the early filling stage, the peak of GA and
CTK flow occurred during the tasseling stage and the lowest value of ABA
occurred during the early filling stage. Except for ABA, the trend of each
hormone flow was similar under different treatments in different periods.
Taking the early filling stage as an example, at the
same planting density level, the flow rates of IAA, GA and CTK increased
significantly with increasing nitrogen application rates. The peak flow rate
reached under the D2N2 treatment, which was 203.92, 168.13, 25.81 and 22.83%, as
well as 68.16 and 22.14% higher than under D1N1 and D3N3, respectively.
Table 3: Effects of N fertilizer
and planting density on endogenous hormones in root bleeding sap of maize
|
Year |
Treatment |
Elongation stage |
Tasselling stage |
Early filling stage |
Milk stage |
||||||||||||
|
IAA |
ABA |
GA |
CTK |
IAA |
ABA |
GA |
CTK |
IAA |
ABA |
GA |
CTK |
IAA |
ABA |
GA |
CTK |
||
|
|
D1N1 |
113.00±0.91e |
14.82±0.06c |
33.41±0.18g |
132.89±2.97g |
122.13±2.57g |
12.21±0.15ab |
45.53±3.88h |
142.75±5.81e |
134.55±3.69g |
9.22±0.75b |
40.29±0.14g |
125.88±2.53g |
124.05±2.57g |
11.29±0.09c |
20.99±0.75f |
103.34±1.51d |
|
|
D1N2 |
130.96±3.66d |
16.55±0.13a |
36.99±0.47e |
147.33±3.18ef |
138.67±0.78f |
12.16±0.67ab |
53.62±3.24fg |
188.23±3.63d |
166.81±9f |
8.93±0.33b |
42.30±0.55e |
174.06±2.53f |
158.40±4.76e |
12.59±0.1a |
25.88±1.32de |
146.42±6.06b |
|
|
D1N3 |
135.40±4.62d |
14.68±0.05c |
37.29±0.49e |
149.31±4.7de |
168.35±12.46e |
12.69±0.05a |
61.24±2.6de |
200.06±6.87c |
325.41±9.44d |
12.91±1.87a |
43.95±0.74d |
182.91±1.94e |
179.56±14.33d |
12.37±0.04b |
27.34±1.33cd |
157.68±11.94b |
|
|
D2N1 |
138.57±5.07c |
12.81±0.51d |
38.36±0.25d |
154.70±3.47cd |
146.32±1.73f |
12.56±0.11ab |
57.30±2.03ef |
187.49±8.05d |
193.22±15.37e |
6.19±1.06c |
46.16±0.46c |
188.20±1.7d |
185.23±10.48d |
11.08±0.14d |
26.60±0.63de |
148.64±6.63b |
|
2016 |
D2N2 |
165.18±1.7a |
12.28±0.08e |
54.62±1a |
170.60±3.14a |
260.15±8.12a |
11.24±0.06c |
76.13±4.62a |
242.75±3.71a |
408.93±9.19a |
5.06±0.34c |
50.69±0.99a |
211.68±1.54a |
223.74±6.87a |
11.22±0.04c |
36.36±0.24a |
192.00±8.15a |
|
|
D2N3 |
156.73±7.59ab |
15.40±0.48b |
39.30±0.7c |
162.15±4.74b |
226.86±6.95c |
11.18±0.07c |
69.40±3.21bc |
221.04±4.48b |
380.9±10.33ab |
6.27±0.05c |
48.94±0.48b |
195.51±2.81c |
211.12±3.45bc |
12.63±0.03a |
32.39±0.99b |
183.95±10.25a |
|
|
D3N1 |
158.86±5.09ab |
14.57±0.14c |
42.23±0.32b |
166.23±0.54ab |
241.39±5.94b |
12.27±0.12ab |
71.57±1.99ab |
235.79±5.88a |
386.80±5.03ab |
7.81±0.63b |
49.37±0.33b |
204.62±1.58b |
216.91±3.95ab |
12.41±0.05b |
33.68±0.35b |
189.73±6.59a |
|
|
D3N2 |
149.39±1.06b |
12.67±0.04de |
38.90±0.13cd |
155.64±4.29c |
207.85±9.14d |
12.12±0.05b |
65.38±2.65cd |
210.65±2.67c |
360.52±9.54c |
8.93±0.8b |
48.51±0.33b |
196.66±2.03c |
202.00±0.17c |
11.28±0.08c |
28.94±0.58c |
178.70±10.76a |
|
|
D3N3 |
121.41±1.7d |
12.47±0.08de |
34.97±0.36f |
142.59±2.3f |
127.73±4.94g |
11.24±0.52c |
48.95±4.69gh |
179.65±7.47d |
152.51±2.05f |
8.02±0.15b |
41.27±0.24f |
173.31±1.44f |
143.31±0.78f |
11.29±0.07c |
25.09±1.88e |
121.29±1.09c |
|
|
D1N1 |
122.93 ±1.44f |
15.62±0.35c |
34.69±0.12g |
143.40±3.08f |
133.44± 2.75h |
13.33±0.13ab |
47.06±3.96h |
150.50±5.3g |
145.29±2.89g |
10.39±0.83b |
41.54±0.18f |
136.26±2.56f |
135.97±2.52f |
12.53±0.13bc |
22.24±0.54f |
113.97±1.82d |
|
|
D1N2 |
140.51 ±1.61d |
17.53±0.13a |
38.07±0.48e |
158.67±2.55de |
151.15±2.5fg |
13.30±0.57ab |
54.98±3.12fg |
197.93±2.54e |
175.40±10.73f |
10.08±0.38b |
43.53±1.07e |
185.26±2.03e |
168.98±7.03d |
13.83±0.11a |
26.90±1.22de |
156.79±5.07b |
|
|
D1N3 |
142.77 ±5.11cd |
15.89±0.07c |
38.43±0.44de |
158.60±5.55de |
176.81±9.4e |
13.67±0.23a |
62.32±2.53de |
209.91±5.71cd |
336.15±9.56d |
13.92±1.7a |
44.89±0.61d |
193.84±1.66d |
190.76±14.48c |
13.73±0.2a |
28.35±1.29cd |
168.18±14.34b |
|
|
D2N1 |
150.33 ±5.81c |
13.74±0.36d |
39.49±0.27cd |
165.12±3.46cd |
156.83±2.78f |
13.69±0.23a |
57.87±0.54ef |
199.55±5.56de |
207.18±27.2e |
7.83±0.86c |
47.33±0.45c |
196.24±2.97d |
196.31±10.78c |
12.38±0.39c |
27.51±0.9de |
160.60±7.45b |
|
2017 |
D2N2 |
176.54 ±2.02a |
13.33±0.13d |
56.56±1.49a |
180.26±5.69a |
275.71±11.02a |
12.61±0.24b |
77.11±4.82a |
247.60±9.97a |
419.96±10.98a |
6.25±0.32d |
51.62±1.08a |
222.71±2.05a |
232.17±6.61a |
12.50±0.13bc |
37.62±0.3a |
203.12±9.29a |
|
|
D2N3 |
169.83 ±6.58ab |
16.50±0.43b |
40.46±1.1c |
172.15±4.51bc |
233.75±8.72c |
12.57±0.3b |
68.62±3.16bc |
228.75±7.07b |
394.25±7.15b |
7.46±0.26cd |
49.82±0.31b |
206.99±3.09c |
221.49±4.72ab |
13.72±0.12a |
33.34±0.89b |
194.30±10.06a |
|
|
D3N1 |
170.23 ±2.99ab |
15.60±0.26c |
43.50±0.65b |
176.54±0.92ab |
252.29±4.94b |
13.62±0.2a |
72.64±1.88ab |
241.54±9.79a |
398.86±5.92b |
9.19±0.28b |
50.15±0.33b |
213.52±0.86b |
226.92±7.03ab |
13.79±0.18a |
34.60±0.44b |
202.34±8.69a |
|
|
D3N2 |
163.42 ±7.17b |
13.72±0.1d |
39.85±0.27c |
166.24±5.07cd |
217.39±6.25d |
13.61±0.18a |
66.47±3.13cd |
214.56±2.54c |
372.18±10.74c |
10.38±0.62b |
49.37±0.23b |
206.63±2.18c |
213.84±2.37b |
12.93±0.48b |
29.81±0.86c |
188.47±11.08a |
|
|
D3N3 |
131.34±4.37e |
13.54±0.07d |
35.98±0.44f |
154.80±5.86e |
140.74±4.43gh |
12.47±1.11b |
50.04±4.43gh |
185.87±5.02f |
162.33±0.98fg |
9.74±0.66b |
42.32±0.27f |
185.03±3.07e |
154.28±4.03e |
12.60±0.1bc |
26.01±2.19e |
133.79±0.41c |
Note: Different lowercase letters
after the same row show significant differences in the same period (P ˂ 0.05). D1N1 (6.75 million
plants ha-1+100 kg N ha-1), D1N2 (6.75 million plants ha-1+200
kg N ha-1), D1N3 (6.75 million plants ha-1+300 kg N ha-1),
D2N1 (8.25 million plants ha-1+100 kg N ha-1), D2N2 (8.25
million plants ha-1+200 kg N ha-1), D2N3 (8.25 million
plants ha-1+300 kg N ha-1), D3N1 (9.75 million plants ha-1+100
kg N ha-1), D3N2 (9.75 million plants ha-1+200 kg N ha-1),
D3N3 (9.75 million plants ha-1+300 kg N ha-1). The same
below
Endogenous hormone ratios in root bleeding sap
Different
N fertilizer density treatments not only affected the endogenous hormone flow
in the root wound fluid of maize but also affected the ratio of endogenous
hormones. Different N fertilizer treatments have different effects on the ratio
of endogenous hormones, changing the balance between hormones. As fertilization
rates increased, the ratios of IAA/ABA, GA/ABA, and CTK/ABA first increased and then decreased and both were
peak at the initial stage of grain filling stage (Table 4).
At the same planting density level, the ratios of
IAA/ABA, GA/ABA and CTK/ABA increased significantly with
increasing nitrogen application rates. The D2 planting density and N1 nitrogen
application treatments showed that the ratios of IAA/ABA, GA/ABA and CTK/ABA were 11.03–31.7, 2.4–7.6, and 12.09–31.05,
respectively. Under N2 treatment, the ratios of IAA/ABA, GA/ABA, and CTK/ABA were 13.42–80.95, 3.24–10.05 and 13.89–41.93,
respectively. Under N3 treatment, the ratios of IAA/ABA, GA/ABA and CTK/ABA were 10.31–60.75, 2.55–7.81 and 10.53–31.19,
respectively.
Correlation
analysis between endogenous hormones and fibre traits
Correlation
analysis showed that IAA was significantly positively correlated with stem
cellulose, hemicellulose and lignin. There was a significant negative
correlation between ABA and stem cellulose, hemicellulose and lignin. There was
no significant correlation between GA and CTK and stalk fibre
traits. These results indicated that increasing IAA can promote the synthesis
of stem cellulose, hemicellulose and lignin. Measures can be taken in
production to increase the concentration of IAA, decrease the concentration of
ABA, enhance stalk fibre traits, and reduce the stalk lodging rate (Table 5).
Yield and yield
components
The
maize yield increased significantly after treatment with N fertilizer density,
reaching a maximum of 9321.21 kg ha-1 under D2N2 treatment, which
was 5.14% and 59.01% higher than D1N1 and D3N3, respectively. The difference
reached a significant level. In terms of factors, after the treatment of N
fertilizer density, the effect on the number of ear rows was not significant,
but the number of grains and 100-grain weight increased significantly (Table
6).
Correlation analysis between cellulose, hemicellulose,
lignin content and lodging resistance of maize in milk stage
Correlation
analysis showed that the cellulose content of maize stems was significantly
positively correlated with the hemicellulose and lignin contents and
significantly negatively correlated with the lodging rate. This indicates that
the stem cellulose and hemicellulose are closely related to the lodging
resistance of the stem. When the cellulose and hemicellulose content is high,
the maize stem has strong lodging resistance (Table 7).
Table 4: Effects of N fertilizer
and planting density on endogenous hormones ratios in root bleeding sap of
maize
|
Year |
Treatment |
Elongation stage |
Tasseling stage |
Early filling stage |
Milk stage |
||||||||
|
IAA/ABA |
GA/ABA |
CTK/ABA |
IAA/ABA |
GA/ABA |
CTK/ABA |
IAA/ABA |
GA/ABA |
CTK/ABA |
IAA/ABA |
GA/ABA |
CTK/ABA |
||
|
|
D1N1 |
7.59±0.11f |
2.25±0.02e |
8.97±0.24e |
10.09±0.11f |
3.73±0.36f |
11.53±0.32e |
14.67±1.46g |
4.39±0.34de |
13.72±1.27e |
10.98±0.18f |
1.86±0.08g |
9.15±0.07f |
|
|
D1N2 |
7.89±0.2f |
2.24±0.02e |
8.90±0.23e |
11.62±0.89e |
4.43±0.48e |
15.51±0.95d |
18.68±0.58g |
4.74±0.24d |
19.52±0.97d |
12.58±0.48e |
2.06±0.09f |
11.63±0.39e |
|
|
D1N3 |
9.10±0.34e |
2.54±0.03d |
10.17±0.34d |
13.08±0.78d |
4.82±0.2de |
15.74±0.33d |
25.49±2.98f |
3.45±0.45e |
14.38±2.25e |
14.52±1.21d |
2.21±0.1e |
12.75±0.92d |
|
|
D2N1 |
11.03±0.32c |
3.00±0.13b |
12.09±0.57b |
11.69±0.04e |
4.56±0.12e |
15.02±0.49d |
31.70±4.76e |
7.60±1.34b |
31.05±5.84b |
16.72±0.84c |
2.40±0.07d |
13.42±0.57d |
|
2016 |
D2N2 |
13.42±0.18a |
4.45±0.06a |
13.89±0.22a |
23.47±0.86a |
6.78±0.44a |
21.13±0.93a |
80.95±4.27a |
10.05±0.88a |
41.93±2.71a |
19.95±0.57a |
3.24±0.02a |
17.12±0.78a |
|
|
D2N3 |
10.31±0.71d |
2.55±0.04d |
10.53±0.11d |
19.97±0.77b |
6.21±0.3ab |
19.70±0.22b |
60.75±1.19b |
7.81±0.05b |
31.19±0.7b |
16.71±0.26c |
2.56±0.07c |
14.56±0.83c |
|
|
D3N1 |
10.98±0.21c |
2.90±0.02c |
11.41±0.14c |
19.68±0.32b |
5.83±0.12bc |
18.89±0.47b |
49.75±4.12c |
6.35±0.56c |
26.33±2.43c |
17.48±0.38bc |
2.71±0.02b |
15.29±0.56bc |
|
|
D3N2 |
12.07±0.45b |
3.07±0.02b |
12.28±0.36b |
17.10±0.61c |
5.40±0.2cd |
17.11±0.51c |
40.54±2.85d |
5.46±0.53cd |
22.15±2.18cd |
17.90±0.14b |
2.56±0.05c |
15.83±0.86b |
|
|
D3N3 |
9.66±0.11e |
2.81±0.03c |
11.44±0.25c |
11.46±0.84e |
4.37±0.59e |
15.97±0.94d |
19.01±0.48g |
5.14±0.12d |
21.61±0.53d |
12.69±0.02e |
2.22±0.15e |
10.74±0.03e |
|
|
D1N1 |
7.88±0.26f |
2.22±0.05f |
9.19±0.27e |
10.01±0.15f |
3.53±0.32e |
11.29±0.29e |
14.05±1.34f |
4.01±0.33e |
13.17±1.15e |
10.86±0.3e |
1.78±0.06h |
9.10±0.23f |
|
|
D1N2 |
8.02±0.06f |
2.17±0.04f |
9.05±0.1e |
11.38±0.57e |
4.14±0.38d |
14.90±0.68cd |
17.39±0.52f |
4.32±0.27de |
18.40±0.92d |
12.21±0.41d |
1.94±0.1g |
11.34±0.42de |
|
|
D1N3 |
8.98±0.3e |
2.42±0.03e |
9.98±0.31d |
12.94±0.86d |
4.56±0.13cd |
15.35±0.22cd |
24.33±2.28e |
3.25±0.36f |
14.07±1.81e |
13.89±0.99c |
2.07±0.08ef |
12.25±1.09cd |
|
|
D2N1 |
10.94±0.41c |
2.87±0.07bc |
12.02±0.42b |
11.46±0.15e |
4.23±0.04d |
14.58±0.34d |
26.64±4.01e |
6.10±0.66bc |
25.31±3.25bc |
15.85±0.64b |
2.22±0.13de |
12.98±0.71c |
|
2017 |
D2N2 |
13.25±0.27a |
4.24±0.09a |
13.53±0.37a |
21.86±0.82a |
6.11±0.27a |
19.62±0.45a |
67.29±3.28a |
8.28±0.59a |
35.69±1.56a |
18.57±0.55a |
3.01±0.04a |
16.26±0.9a |
|
|
D2N3 |
10.30±0.64cd |
2.45±0.04e |
10.43±0.08d |
18.59±0.27b |
5.46±0.34b |
18.21±0.89b |
52.89±1.07b |
6.69±0.22b |
27.79±1.39b |
16.15±0.48b |
2.43±0.08bc |
14.16±0.61b |
|
|
D3N1 |
10.91±0.31c |
2.79±0.02c |
11.32±0.25c |
18.52±0.16b |
5.33±0.07b |
17.73±0.63b |
43.40±0.87c |
5.46±0.18c |
23.24±0.74c |
16.45±0.34b |
2.51±0.03b |
14.67±0.46b |
|
|
D3N2 |
11.91±0.44b |
2.90±0.04b |
12.12±0.43b |
15.97±0.25c |
4.89±0.28bc |
15.77±0.21c |
35.89±1.21d |
4.77±0.28d |
19.94±1.09d |
16.56±0.75b |
2.31±0.03cd |
14.58±0.7b |
|
|
D3N3 |
9.70±0.29d |
2.66±0.03d |
11.44±0.48c |
11.35±1.18e |
4.05±0.69de |
14.97±1.14cd |
16.71±1.08f |
4.36±0.29de |
19.04±0.97d |
12.25±0.27d |
2.07±0.18ef |
10.62±0.08e |
Note: Different lowercase letters after the same row show significant
differences in the same period (P ˂
0.05). D1N1 (6.75 million plants ha-1+100 kg N ha-1),
D1N2 (6.75 million plants ha-1+200 kg N ha-1), D1N3 (6.75
million plants ha-1+300 kg N ha-1), D2N1 (8.25 million
plants ha-1+100 kg N ha-1), D2N2 (8.25 million plants ha-1+200
kg N ha-1), D2N3 (8.25 million plants ha-1+300 kg N ha-1), D3N1 (9.75
million plants ha-1+100 kg N ha-1), D3N2 (9.75 million
plants ha-1+200 kg N ha-1), D3N3 (9.75 million plants ha-1+300
kg N ha-1)
Table 5: Correlation
Analysis between Endogenous Hormones and Fibre Traits
|
Year |
Traits |
IAA |
ABA |
GA |
CTK |
|
2016 |
Cellulose |
0.530** |
-0.591** |
0.002 |
0.184 |
|
Hemicellulose |
0.456** |
-0.599** |
0.046 |
0.092 |
|
|
Lignin |
0.380* |
-0.632** |
-0.122 |
-0.021 |
|
|
2017 |
Cellulose |
0.529** |
-0.602** |
-0.003 |
0.205 |
|
Hemicellulose |
0.460** |
-0.616** |
0.049 |
0.105 |
|
|
Lignin |
0.385* |
-0.635** |
-0.107 |
-0.002 |
Note: * and ** indicate significance at 0.05 and
0.01 probability levels, respectively
Table 6: Effects of N fertilizer and
density on yield and yield components of maize
|
Year |
Treatment |
Ear rows |
Row grains |
Grain number per spike |
100-grain weight (g) |
Theoretic Yield (kg hm-2) |
|
2016 |
D1N1 |
13ab |
42.75a |
555.75a |
37.42b |
8865.91a |
|
D1N2 |
14.5a |
38ab |
551a |
36.69b |
8293.77a |
|
|
D1N3 |
13ab |
35.5ab |
461.5ab |
38.04b |
8138.39ab |
|
|
D2N1 |
13.5ab |
31b |
418.5abc |
41.98a |
6966.23ab |
|
|
D2N2 |
12b |
37.75ab |
453ab |
40.35a |
9321.21a |
|
|
D2N3 |
13ab |
37.5ab |
487.5ab |
40.69a |
9184.83a |
|
|
D3N1 |
14ab |
32b |
448ab |
37.88b |
8998.32ab |
|
|
D3N2 |
13ab |
31.25b |
406.25bc |
38.40b |
8854.79ab |
|
|
D3N3 |
13ab |
23.75c |
308.75c |
38.39b |
5862.53b |
|
|
2017 |
D1N1 |
15ab |
32a |
489.25a |
33.39b |
8732.68cd |
|
D1N2 |
16.5a |
29.5ab |
488.88a |
35ab |
8992.71cd |
|
|
D1N3 |
15ab |
32.75a |
466.25a |
34.69ab |
8249.50d |
|
|
D2N1 |
15.5ab |
29.5ab |
468a |
33.26b |
9912.66ab |
|
|
D2N2 |
14b |
33.5a |
469a |
34.82ab |
10831.57a |
|
|
D2N3 |
15ab |
30.75ab |
469.5a |
32.8b |
8973.68cd |
|
|
D3N1 |
16ab |
30ab |
495a |
32.98b |
9992.68ab |
|
|
D3N2 |
15ab |
31ab |
461.5a |
34.87ab |
9637.91bc |
|
|
D3N3 |
15ab |
24.75b |
380.75b |
37.32a |
6602.17e |
Note: Different letters in the
same column indicate significant differences (P < 0.05). D1N1 (6.75 million plants ha-1+100 kg N ha-1),
D1N2 (6.75 million plants ha-1+200 kg N ha-1), D1N3 (6.75
million plants ha-1+300 kg N ha-1), D2N1 (8.25 million
plants ha-1+100 kg N ha-1), D2N2 (8.25 million plants ha-1+200
kg N ha-1), D2N3 (8.25 million plants ha-1+300 kg N ha-1),
D3N1 (9.75 million plants ha-1+100 kg N ha-1), D3N2 (9.75
million plants ha-1+200 kg N ha-1), D3N3 (9.75 million
plants ha-1+300 kg N ha-1)
Correlation
analysis of root bleeding sap and its components with yield
There
was a significant positive correlation between GA and CTK in the root wound fluid of maize during the four growth stages of
maize. CTK (0.99**, 0.98**) had the greatest
correlation with yield during the elongation stage and milk stage. GA (0.98**)
had the highest correlation with yield during the tasseling stage. There was no
significant correlation between IAA and ABA and yield (Table 8).
Discussion
The
lignin, cellulose and hemicellulose in the stem are closely related to the
lodging resistance (Jung et al.
2015). Kamran et al. (2018) found
that high cellulose content increases the strength of the stem and enhances its
lodging resistance. Zhang et al.
(2019) found that extremely significant differences in stem lignin content
among varieties with different lodging resistance. The lignin content of stems
with strong lodging resistance is significantly higher than that of varieties
that are prone to lodging. Barrière et al.
(2010) showed a significant negative correlation between stem lignin content
and actual lodging rates and a significant positive correlation between stem
lignin content and flexural strength. Nitrogen fertilization and planting
density treatment significantly increased the cellulose, hemicellulose and
lignin content in maize internodes. The results showed that as the growth
period progressed, the cellulose content in the stem first increased and then
decreased, and each treatment showed maximum cellulose values at the early
filling stage. The D1N3 and D2N2 treatments resulted in significantly higher
cellulose contents than did the other treatments during the heading and the early
filling stage. This result indicated that increasing nitrogen fertilization and
establishing a reasonable planting density can increase the cellulose content
in the stem, but a high planting density will reduce the cellulose content. The
PAL, TAL, CAD and 4CL are key enzymes for lignin synthesis in grasses.
Correlation analysis showed significant positive correlations between lignin
content and PAL activity, TAL activity and 4CL activity (correlation
coefficients were 0.817, 0.507 and 0.778, respectively), which were all
significantly negatively correlated with CAD activity. Higher PAL, TAL and 4CL
activities contribute to the synthesis and accumulation of lignin.
Table 7: Correlation analysis between
cellulose, hemicellulose and lignin contents and lodging resistance of maize in
the milk stage
|
Year |
Cellulose content |
Hemicellulose content |
Lignin content |
|
2016 |
-0.804** |
0.060 |
-0.375 |
|
2017 |
0.102 |
-0.803** |
0.004 |
Note: * and **
indicate significance at 0.05 and 0.01 probability levels, respectively
Table 8: Correlation analysis of root bleeding sap and its
components with yield
|
Year |
Traits |
IAA |
ABA |
GA |
CTK |
|
2016 |
Elongation stage |
0.559 |
-0.186 |
0.802** |
0.985** |
|
Tasseling stage |
0.665 |
-0.342 |
0.978** |
0.935** |
|
|
Early filling stage |
0.641 |
-0.192 |
0.903** |
0.801** |
|
|
Milk stage |
0.504 |
-0.201 |
0.944** |
0.983** |
|
|
2017 |
Elongation stage |
0.704* |
-0.136 |
0.681* |
0.674* |
|
Tasseling stage |
0.650 |
0.237 |
0.656 |
0.545 |
|
|
Early filling stage |
0.504 |
-0.616 |
0.749* |
0.453 |
|
|
Milk stage |
0.663 |
-0.092 |
0.609 |
0.618 |
Note: * and ** indicate significance at 0.05 and 0.01 probability levels, respectively
The inorganic ions and water absorbed by the roots
from underground, in addition to supplying the growth and development of the
roots, also flow to the shoots through the xylem. It is now known that IAA,
ABA, GA3 and CTK can be synthesized in the roots and play an important role in
regulating the growth of roots and information exchange between root and crown
(Locher and Pilet, 1994). Tian et al.
(2008) found that maize roots have a threshold for the concentration of IAA and
that its high concentrations inhibit root growth. The study showed that
endogenous hormones and yield were significantly correlated at each measurement
period. The concentration of IAA in the wound fluid varied significantly with
different nitrogen fertilization and planting density treatments. The results
of this study showed that during the same growth period, the concentrations of
IAA, GA3 and CTK first increased and then
decreased, reaching maximum levels under the D2N2 treatment. Ma et al. (2019)
and Chen et al. (2012) also showed that shows that there is a certain correlation
between hormone content and lodging resistance of stem. The ABA concentration
decreased first and then increased. Correlation analysis showed that IAA had a
significant positive correlation with fibre content, such as cellulose, and ABA
showed a significant negative correlation with the same, indicating that a high
concentration of IAA can increase the cellulose content, thus increasing the
lodging resistance of stems. In this study, the concentration of the IAA under
the D2N2 treatment was the highest, the concentration of ABA was lowest and the
lodging resistance was the best. Swarup et al. (2008) pointed out that
the polar transport of auxin can regulate the expression of expansion and cell
wall relaxant, increase the expansibility of cell wall rapidly and regulate
cell growth. Our study also indirectly shows that auxin may promote the growth
of cell wall in the later period of corn growth, and then improve the lodging
resistance of corn stalk. The
present study results areconsistent with previous results indicate that the N fertilizer rate and planting
density in the D2N2 treatment are the most suitable for maize growth with good
resistance to lodging and high yield.
Maize yield is associated with a variety of traits,
most of which can be increased by increasing the planting density and
increasing the number of effective spikes (Asimet al. 2013). Reasonable densification is a key strategy to achieve
large-scale yield increases in spring maize in Heilongjiang province and
previous studies have suggested that increasing planting density will result in
a decrease in grain number per spike and 100-grain weight, and the number of
kernels affected by environmental impacts will be greater (Cui et al. 2015). The results of this
experiment showed that the effect on ear rows was not significant, but the
number of grains and 100-grain weight increased significantly. Under the
interaction between N fertilizer and planting density treatments, the density
increased, the number of ear rows, the number of rows and the 100-grain quality
all increased first and then decreased. Increasing the density increases the
number of effective spikes, hence, the yield results showed this trend D2 >
D3 > D1. As planting density increased, maize production first increased and
then declined, indicating that excessive planting density leads to a decline in
maize production. The analysis showed a significant positive correlation
between GA3 and CTK and yield. Therefore, in addition to increasing maize yield by
increasing planting density, high yields were achieved by appropriate increase
in the concentrations of GA3 and CTK. In this experiment, the maximum yield of maize was
obtained under D2N2 with 82,500 plants ha-1, which is basically
consistent with previous studies (Liu et
al. 2012).
Conclusion
The
cellulose content of stems was negatively correlated with the lodging rate in
maize. Nitrogen application significantly increased the cellulose and
hemicellulose contents of the stem. At the same time, the activity of CAD was
significantly improved, the lignin content increased, and the stem lodging rate
lowered; thus, the lodging resistance of maize was enhanced. Appropriate
nitrogen fertilization and planting density treatment also significantly
improved the endogenous hormone levels, which beneficially regulated the
relationship between root and crown, affected the physiological activity of the
aboveground parts, and ultimately increased the yield of maize. The maximum
yield 9321.21 kg ha-1 was obtained with the combination of 8.25
million plants ha-1 and 200 kg nitrogen ha-1(D2N2).
Acknowledgments
We thank the anonymous reviewers and the editor for their
valuable comments and suggestions, which greatly contributed to the improvement
of this paper. This research was funded by National Key R&D Program of
China (2016YFD0300103, 2017YFD0300506), Heilongjiang Provincial Funding for
National Key R&D Programs of China (GX18B029) and “Academic Backbone”
Project of Northeast Agricultural University (17XG23).
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