Journal of Integrative Agriculture

Combined application of organic fertilizer and chemical fertilizer alleviates the

kernel position effect in summer maize by promoting post-silking nitrogen
uptake and dry matter accumulation
ZHAI Li-chao1*, ZHANG Li-hua1, CUI Yong-zeng1, ZHAI Li-fang2, ZHENG Meng-jing1, YAO Yan-rong1,
ZHANG Jing-ting1, HOU Wan-bin3, WU Li-yong4, JIA Xiu-ling1*
1
Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Science/Key Laboratory
of Crop Cultivation Physiology and Green Production of Hebei Province, Shijiazhuang 050035,
P.R.China
2 Hebei Science and Technology Innovation Service Center, Shijiazhuang 050051, P.R.China
3
Yongnian Agricultural Technology Extension Center, Handan 050036, P.R.China
4
Ningjin Agricultural Technology Extension Center, Xingtai 050036, P.R.China

Abstract Adjustment of agronomic measures to alleviate the kernel position effect in maize is
important for ensuring high yields. In order to clarify whether combined application of organic fertilizer
and chemical fertilizer can alleviate the kernel position effect of summer maize, field experiments were
conducted during 2019 and 2020 growing seasons, and the following five treatments were assessed: CF,
100% chemical fertilizer; OFCF1, 15% organic fertilizer+85% chemical fertilizer; OFCF2, 30% organic
fertilizer+70% chemical fertilizer; OFCF3, 45% organic fertilizer+55% chemical fertilizer; and OFCF4,
60% organic fertilizer+40% chemical fertilizer. Compared with the CF treatment, the OFCF1 and
OFCF2 treatments significantly alleviated the kernel position effect by increasing the weight ratio of
inferior kernels to superior kernels and reducing the weight gap between the superior and inferior kernels.
These effects were largely due to improved the filling and starch accumulation of inferior kernel.
However, there was no obvious difference in kernel position effect among plants treated with CF, OFCF3,
or OFCF4 in most cases. Leaf area indexes, post-silking photosynthetic rates, and net assimilation rates
were higher in plants treated with OFCF1 or OFCF2 than in those treated with CF, reflecting enhanced
photosynthetic capacity and improved post-silking dry matter accumulation (DMA) in plants treated with
OFCF1 or OFCF2. Compared with the CF treatment, the OFCF1 and OFCF2 treatments increased
post-silking N uptake by 66.3 and 75.5%, respectively, which was the major factor driving post-silking
photosynthetic capacity and DMA. Moreover, the increases in root DMA and zeatin riboside content
observed following the OFCF1 and OFCF2 treatments resulted in decreased root senescence, which is
associated with increased post-silking N uptake. Analyses showed that post-silking N uptake, DMA,
and grain yield in summer maize were negatively correlated with the kernel position effect. In
conclusion, the combined application of 15–30% organic fertilizer and 70–85% chemical fertilizer
alleviated the kernel position effect in summer maize by improving post-silking N uptake and DMA.
These results provide new insight into how CAOFCF could be used to improve maize productivity.
Keywords: chemical fertilizer, dry mater accumulation, kernel position effect, N uptake, organic
fertilizer1

1. Introduction
Kernel position effect is common in maize ear (i.e., the inferior kernels located in the upper part of ear

Received 14 February, 2023 Accepted 29 March, 2023

#
Correspondence ZHAI Li-chao, Tel: +86-311-87670620, E-mail: zhailichao@163.com; JIA Xiu-ling, Tel: +86-311-87670620,
E-mail: jiaxiuling2013@126.com
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 Journal of Integrative Agriculture
had slower filling rate, insufficient filling and lower final grain weight, compared to that of the superior
kernels located in the middle and lower part of ear) (Xu et al. 2015). Unreasonable cultural practices or
abiotic stress usually exacerbate this kernel position effect, and even lead to the abortion of inferior
kernel, which seriously restrict the improvement of maize grain yield potential (Shen et al. 2018; Yan et al.
2018). Therefore, it is of great significance to explore cultural practices to alleviate kernel position
effect of maize in high-yielding and high- efficiency production.
Among the cultural practices in maize production, optimizing fertilizer management is a key
measure to regulate grain filling and grain yield of maize (Mueller and Vyn 2018; Wu et al. 2022). Since
organic fertilizers have advantage over chemical fertilizers in regulating soil quality, applying organic
fertilizer in field crops production is gaining more attention in recent years in China (Fang et al. 2021;
Ren et al. 2021; Hayatu et al. 2022). However, if organic fertilizer is applied alone, it may be hard to
meet the nutrients requirement of crop plants due to its slow-release characteristic of nutrients, and
finally affect the crop yield. In this context, combined application of organic fertilizer and chemical
fertilizer (CAOFCF) is considered an effective fertilization measure for sustainable crop production.
Numerous studies have reported that CAOFCF is beneficial to crop yield improvement (Lv et al.
2020; Xu et al. 2021; Aluoch et al. 2022; Liu H W et al. 2022; Zhai et al. 2022; Zhang W T et al. 2022).
However, most of these studies suggested that the increase in crop yield under CAOFCF treatments
was closely related to the change in soil properties, there is a lack of information on in-depth and
systematic analysis of the mechanism of yield improvement from the perspective of plant physiology,
especially from the perspective of kernel development. It is acknowledged that increasing plant dry
matter accumulation (DMA), especially the post-silking DMA, is an important guarantee for good grain
filling and higher grain yield of maize (Ning et al. 2013; Yang et al. 2020; Wang et al. 2021). Compared
with applying chemical fertilizer alone, previous studies have demonstrated that CAOFCF can effectively
promote crop DMA under the same N rate (Li et al. 2018; Zhai et al. 2022). Saikia et al. (2015) reported
that CAOFCF enhanced wheat carbon assimilation by improving photosynthetic rate during reproductive
stages, and Zhai et al. (2022) also found the CAOFCF is beneficial to the post-silking leaf area duration
and photosythettic capacity of maize. Therefore, the higher DMA under CAOFCF treatments may be
associated with its enhanced photosynthetic capacity. In addition, increasing post-silking N uptake is
essential to maintain higher productivity of maize (Mu et al. 2015; Cao et al. 2021), because post-silking
N accumulation is associated with grean leaf persistence, which in turn affect the photosynthetic
capacity and biomass production. Huang et al. (2022) suggested that organic nitrogen substituting
30% chemical N rate significantly increased the N uptake of banana. Similarly, the improvement in N
uptake in cereal crops have also been observed under reasonable CAOFCF treatments, including maize,
wheat, and rice (Wang et al. 2022; Zhai et al. 2022; Fan et al. 2023). Recently, increasing evidences
have shown that CAOFCF was beneficial to mitigate the soil reactive N losses by affecting the
conversion of N in the soil (Xia et al. 2017; Tang et al. 2019; Wei et al. 2021; Li Y W et al. 2022), which
can increase the soil N availability and facilitate N uptake by crop plants. Moreover, a strong root system
is essential for plant nutrient uptake. A number of studies have confirmed that the higher nutrients
accumulation in plants and crop productivity under CAOFCF treatments is associated with the improved
root system (Chen et al. 2021; Ramesha et al. 2021). Therefore, the improved root system driven by
organic fertilizer application may be the key to the higher plant N uptake.
It is generally known that post-silking assimilate accumulation is the main material source for grain
filling of maize. The poor grain filling of inferior kernel resulted by the insurffficent assimilate supply is a
major reason for the aggravated kernel position effect of maize (Shen et al. 2018). Thus, maintaining
higher post-silking photosynthetic capacity and DMA by regulating post-silking N uptake may be the key
to alleviate the kernel position effect of maize. In this study, we hypothesize that combined application
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 Journal of Integrative Agriculture
of organic fertilizer and chemical fertilizer with a reasonable proportion was conducive to alleviate the
kernel position effect by improving the filling of inferior kernels, which is closely related to its role in
regulating post-silking N uptake and DMA. To test this hypothesis, a 2-year field experiment was
conducted with the objectives to (1) evaluate the impact of CAOFCF on grain filling and carbohydrate
accumulation in the inferior and superior kernels, and on the kernel position effect in summer maize; (2)
investigate the effect of CAOFCF on the post-silking N uptake, photosynthetic capacity, and DMA; and (3)
analyze the relationships of the kernel position effect with post-silking N uptake, DMA, and grain yield in
summer maize.

2. Materials and methods

2.1. Experimental site and meteorological condition

Field experiments were conducted during the 2019 and 2020 growing seasons at the Scientific
Observing and Experimental Station of Crop Cultivation in the North China Plain, Institute of Cereal and
Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, Hebei Province (37°95′N,
114°71′E). This experimental site is characterized by a warm temperate sub-humid continental
monsoon climate. The mean annual air temperature is 12.8°C and the mean precipitation level is 484
mm (rainfall plus snowfall). The primary soil in the area is meadow cinnamon soil, with the soil nutrients
in the upper 0–20-cm soil profile comprising 1.57% organic matter, 1.04 g kg−1 total N, 80 mg kg−1
available N, 21.4 mg kg−1 available phosphorus (P), and 113.9 mg kg−1 available potassium (K) before
the sowing of 2019 growing season. The daily meteorological data for the two growing seasons are
shown in Fig. 1.

2.2. Experimental design and management

The maize cultivar Yudan 9953 (YD9953), which is widely cultivated in the North China Plain, was used
as material for the field experiments, in which a random block design was employed. There were five
treatments: CF, chemical NPK fertilizer; OFCF1, co-application of 15% OF and 85% CF; OFCF2,
co-application of 30% OF and 70% CF; OFCF3, co-application of 45% OF and 55% CF; OFCF4,
co-application of 60% OF and 40% CF. All treatments included 225 kg ha−1 N, 120 kg ha−1 P2O5, and
120 kg ha−1 K2O (i.e., the total N, P2O5, and K2O provided by the chemical fertilizer and organic fertilizer
remained constant for all treatments). The organic fertilizer used in this study was produced from cow
manure and contained 45% organic matter, 2.48% N, 0.27% P2O5, and 0.96% K2O. All CAOFCF
treatments in this study is actually replacing partial chemical fertilizer by organic fertilizer with equal N
rate. First, the application amount of organic fertilizer and chemical N fertilizer can be calculated
according to the principle of equal N rate substitution; second, the P2O5 and K2O amount contained in
the organic fertilizer can be also calculated according to its nutrients content; third, the actual application
amount of P2O5 or K2O chemical fertilizer is 120 kg ha-1 minus its amount contained in organic fertilizer.
Detailed information of the calculated application amount of fertilizers for all treatments is shown in Table
1. Each treatment was repeated with three times, and the experimental plot size was 10 m× 8 m. The
row spacing in each plot was 60 cm and there was a 1.5-m-wide buffer zone between plots. In 2019
and 2020 growing seasons, seeds were sown to create a planting density of 7.50×104 plants ha-1, all
plots received a basal application of 135 kg ha−1 N, 120 kg ha−1 P2O5, and 120 kg ha−1 K2O, with 90 kg
ha−1 of N topdressing applied at the V8 (eight-leaf) stage. All organic fertilizer was applied as basal
fertilizer. After the harvest of winter wheat, the wheat straws was crushed and returned to the field, and
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 Journal of Integrative Agriculture
the basal organic fertilizer and chemical fertilizer were manually spread on the surface of each
experimental plot, then a rotary tiller with suppressive function was used to incorporate the fertilizers into
the soil at a depth of 20 cm. Other field management procedures were the same as those used by local
smallholder farmers.

2.3. Sampling and measurements

Grain filling and the kernel position effect At the silking stage, 80 uniform plants (i.e., plants with
similar plant height, ear height, stem diameter, and silking time) from each plot were tagged. Five ears
from each tagged plant were sampled every 5 days from pollination until physiological maturity. A total
of 100 kernels were removed from both the top (inferior kernels, i.e., kernels of the upper third of maize
ears) and middle (superior kernels, i.e., kernels of the middle third of maize ears) part of the ears. After
the 100-kernel fresh weight was measured, each sample was dried at 70° to a constant weight. The
grain filling process was fitted to Richard’s (1959) growth equation, as described by Zhu et al. (1988):
A
W 
1  e 
B  Ct 1 / D eq. (1)

The grain filling rate was calculated as the derivative of Eq. (1)

ACe B Ct
G eq. (2)
 
D 1
B Ct
D 1 e D

where W is the kernel weight (mg), A is the final grain weight (mg), t is the time after anthesis (days), and
B–D are coefficients determined by regression.
In this study, we used the weight ratio of inferior kernels to superior kernels and the weight gap
between the superior and inferior kernels as indexes to evaluate the kernel position effect in maize.
These parameters were calculated using the equations below:

IKW
WR  eq. (3)
SKW

WG  SKW  IKW eq. (4)

where WR represents the weight ratio of inferior kernels to superior kernels and WG is the weight gap
between the superior and inferior kernels; IKW and SKW represent the inferior kernel weight and the
superior kernel weight, respectively. The lower the WR value or higher the WG value, the greater the
position effect.
Kernel starch and soluble sugar content At physiological maturity, five ears were taken from each
plot, inferior and superior kernel were manually separated and then grind the kernels into power for the
measurement of starch and soluble sugar content. Starch content and sugar content of maize kernel
were measured by using acid hydrolysis method and anthrone colorimetry (Li and Li 2013), respectively.
FlexStation 3 MultiMode Microplate Reader (Molecular Devices LLC.3860 N First Street San Jose, CA
95134, USA) was used to determine the absorption value of sample and blank control at 620 nm
wavelength, and then the starch and sugar contents were calculated according to these absorption
values.
Plant DMA and N accumulation At silking and physiological maturity, five similar plants were selected
from the middle of each plot. The plants were separated into stalks, leaves, sheaths, tassels, and ears,
which were oven dried to measure the dry weight of each part and the DMA.
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 Journal of Integrative Agriculture
Post-silking DMA=Total DMA at physiological maturity−Total DMA at silking.
After measuring the DMA, samples from each part were milled to a fine powder. Total N was
extracted with H2SO4-H2O2 and analyzed for total N content (%) with a continuous-flow autoanalyzer
(AutoAnalyzer 3; Bran+Luebbe, Noderstedt, Germany).
Post-silking N uptake (g/plant)=Total N accumulation at physiological maturity−N accumulation at silking
Leaf area index (LAI), Pn, and net assimilation rate (NAR) Before the V6 stage, five representative
plants were marked in each plot. Leaf length and maximum leaf width of all green leaves were
measured at the V6, V12, R1, R3, and R6 stages. Leaf area and LAI were calculated as follows:
Leaf area=Leaf length×Leaf maximum width×0.75
LAI=(Leaf area per plant×Plant number per plot)/Plot area
Pn was measured at silking and 25 days after pollination (DAP) in 2019. Pn was measured at
silking, 25 DAP, and 45 DAP in 2020. Measurements were taken from five randomly selected ear
leaves on sunny days between 10:00 and 11:30 using a Li-6400 portable photosynthesis system
(LI-COR Biosciences, Lincoln, NE, USA). NAR (g m−2 d−1) was calculated using the equation below
(Power et al. 1967):

 ln L 2  ln L1    W 2  W1  
NAR     eq. (5)
  L 2  L1    t 2  t1  
where L1 and L2 are single plant leaf areas from the maize cultivars at t1 and t2, respectively; and W1 and
W2 are the above-ground dry matter weights at t1 and t2, respectively.
Root DMA and hormone content Plant roots were collected from a soil depth of 0–40 cm. At the
mid-grain filling stage, three similar plants were selected from the middle of each plot for root sampling.
Boundaries were defined separately based on the area occupied by each plant (half of the distance
between each row on either side of the base of each plant, and half of the distance between each plant
in a row). Soil and root samples were collected from each of the following layers: 0–10, 10–20, and
20–40 cm. Samples from each layer were numbered and placed in a separate nylon bag; the roots were
then washed and oven dried to measure root dry matter. Fresh root samples were also collected from
three plants to measure root hormone content. Abscisic acid (ABA) and zeatin riboside (ZR) content
were measured using high-performance liquid chromatography, as described by Han et al. (2011).
Grain yield At physiological maturity, all plants in a 4 m×2 m area containing the middle rows of each
plot were harvested, and the grain yield was determined under the condition of 14% grain water content.

2.4. Data analyses

Statistical analyses were performed using SPSS software (ver. 19.0; SPSS Inc., Chicago, IL, USA) and
graphs were analyzed using SigmaPlot (ver. 12.0; Systat, Inc., San Jose, CA, USA) or Microsoft Excel
software (ver. 2016; Microsoft Corp., Redmond, WA, USA). One-way analysis of variance followed by
Tukey’s test, and treatment means were compared by computing least significant differences (LSDs) to
identify significant differences at the 0.05 probability level.

3. Results
3.1. Kernel weight gap and weight ratio

In both growing seasons, the weight gap between superior and inferior kernels was significantly lower in
OFCF1 and OFCF2 plants than CF plants (Fig. 2-A). OFCF1 and OFCF2 treatments reduced the
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 Journal of Integrative Agriculture
kernel weight gap by 59.2 and 43.7%, respectively, compared with the CF treatment. Similarly,
CAOFCF treatments significantly affected the weight ratio of inferior kernels to superior kernels in both
growing seasons (Fig. 2-B). In the 2019 growing season, the OFCF1, OFCF2, OFCF3, and OFCF4
treatments increased the weight ratio of inferior kernels to superior kernels by 15.5, 10.4, 7.3, and 9.7%,
respectively, compared with the CF treatment; significant differences from the CF treatment were
observed for OFCF1, OFCF2, and OFCF4. In the 2020 growing season, the OFCF1, OFCF2, OFCF3,
and OFCF4 treatments significantly increased the weight ratio of inferior kernels to superior kernels by
8.1, 8.1, 10.5, and 7.0%, respectively, compared with the CF treatment.

3.2. Grain filling and grain-filling rate

The grain filling process in summer maize was affected by CAOFCF treatments, especially for the
inferior kernels (Fig. 3). In 2019, differences were observed for inferior and superior kernel weights
from 30 and 40 DAP, respectively; differences in kernel weights between CAOFCF plants and CF plants
increased gradually over time. The OFCF1, OFCF2, OFCF3, and OFCF4 treatments increased the
final inferior kernel weights by 24.3, 20.2, 9.7, and 4.0%, respectively, compared with CF treatment.
The final superior kernel weights in OFCF1 and OFCF2 plants were 7.8 and 7.7% greater than those in
CF plants, respectively, but OFCF4 treatment reduced kernel weights by 5.7%, compared with CF
treatment. The maximum filling rate for inferior kernels in CF plants was observed 15 DAP; however,
CAOFCF treatments delayed the maximum filling rate for inferior kernels by 5 days. OFCF1, OFCF2,
OFCF3, and OFCF4 increased the filling rate for inferior kernels by 24.9, 20.7, 9.7, and 3.7%,
respectively, compared with CF. For superior kernels, all treatments resulted in maximum grain filling
rates 20 DAP. The filling rates of superior kernels in OFCF1, OFCF2, and OFCF3 plants were 7.9, 7.9,
and 0.4% greater than those in CF plants, respectively, but CFOF4 treatment decreased the grain filling
rate by 5.5% compared with CF treatment.
In 2020, CAOFCF treatments increased inferior kernel weights from 40 DAP compared with CF
treatment. The final inferior kernel weights in OFCF1, OFCF2, OFCF3, and OFCF4 plants were 8.5, 8.1,
9.1, and 5.1% greater than those in CF plants, respectively. However, no significant differences in
superior kernel weights were observed during the grain filling process. For inferior kernels, all
treatments resulted in maximum grain filling rates 25 DAP. The grain filling rates of inferior kernels in
OFCF1, OFCF2, OFCF3, and OFCF4 plants were 8.9, 8.4, 8.9, and 5.3% greater than in CF plants,
respectively. For superior kernels, maximum grain filling rates were observed in OFCF1, OFCF2, and
OFCF3 plants at 25 DAP, but in CF and OFCF4 plants at 20 DAP. For superior kernels, there were no
obvious differences in grain filling rates among the treatments.

3.3. Starch and soluble sugar content

As shown in Fig.4, the starch and soluble sugar content in superior kernel were higher than that in
inferior kernel under each treatment. In 2019, compared with CF, OFCF1 and OFCF2 increased the
starch content in inferior kernel and superior kernel by 7.4%, 7.7% (P<0.05) and 2.4%, 3.2%,
respectively; OFCF1 and OFCF2 increased the soluble sugar content in inferior and superior kernel by
11.2%, 10.8% (P<0.05) and 6.3%, 5.3%, respectively. However, the difference in starch and soluble
sugar content among CF, OFCF3, and OFCF4 was not obvious in most cases.
In 2020, the starch content in inferior kernel under OFCF1, OFCF2, and OFCF3 were 5.6, 4.7, and
3.3% higher than that of CF (P<0.05); the difference in starch content in superior kernel among CF,
OFCF1, OFCF2, and OFCF3 was not obvious. However, OFCF4 significantly decreased the starch
content in inferior and superior kernel, compared to that of CF. Compared with CF, OFCF1 and OFCF2
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 Journal of Integrative Agriculture
increased the soluble sugar content in inferior kernel by 10.5 and 17.2% (P<0.05), respectively; and
OFCF1, OFCF2, and OFCF3 increased the soluble sugar content in superior kernel by 19.4, 16.6, and
9.2% (P<0.05), respectively. The difference in soluble sugar content in inferior and superior kernel
between CF and OFCF4 was not significant.

3.4. LAI, post-silking Pn, and NAR

CAOFCF had considerable effects on LAI, post-silking Pn, and NAR in summer maize (Fig. 5). For all
treatments, LAI was maximal at the R1 stage, and decreased gradually thereafter. In the 2019 growing
season, the maximum LAI of CF plants was significantly greater than that of all CAOFCF treatment
plants, but decreased rapidly during the grain filling period. The LAIs of OFCF1, OFCF2, and OFCF3
plants were greater than those of CF plants at the R3 and R6 stages, and significant differences were
observed at the R6 stage (P<0.01). In the 2020 growing season, the LAIs of OFCF1, OFCF2, and
OFCF3 plants were greater than those of CF plants after the V12 stage; significant differences were
observed at the V12, R1, and R6 stages (P<0.05). In both growing seasons, the Pn was greater in
OFCF1 and OFCF2 plants than CF plants after anthesis; however, significant differences were only
observed in OFCF2 plants at the M-GF and L-GF stages (P<0.05). In the 2019 growing season,
OFCF1, OFCF2, and OFCF3 plants exhibited increases in post-silking NAR of 6.8, 7.6, and 21.7%,
respectively, compared with CF plants; however, there was no obvious difference between CF and
OFCF4 plants. In the 2020 growing season, OFCF2 plants exhibited a significant increase in NAR, of
6.9%, compared with CF plants; however, there was no obvious difference among CF, OFCF1, and
OFCF4 plants. The NAR of OFCF3 plants was significantly lower than that of CF plants.

3.5. Post-silking N uptake

Post-silking N uptake was significantly affected by the CAOFCF treatments (Table 2). In the 2019
growing season, the OFCF1, OFCF2, OFCF3, and OFCF4 treatments increased post-silking N uptake
by 78.2, 111.3, 110.9, and 34.5%, respectively, compared with the CF treatment. The contribution of
post-silking N uptake to grain N was also significantly greater for the CAOFCF treatments than the CF
treatment. In 2020, the post-silking N uptake of OFCF1 and OFCF2 plants increased by 54.4 and
39.7%, respectively, compared with CF plants. However, the OFCF3 and OFCF4 treatments
significantly reduced post-silking N uptake. Similarly, the contribution of post-silking N uptake to grain
N increased in OFCF1 and OFCF2 plants, but decreased in OFCF3 and OFCF4 plants, compared with
CF plants. In addition, tthe interaction between year and treatment had a significant effect on
post-silking N uptake (P<0.01).

3.6. Plant DMA, grain yield, and yield components

Pre-silking DMA, post-silking DMA, total DMA, and grain yield per unit area are presented in Table 3.
Compared with the CF treatment, pre-silking DMA was significantly increased by the OFCF1 treatment
(8.42%) but decreased by the OFCF2, OFCF3, and OFCF4 treatments (P<0.05). The OFCF1, OFCF2,
and OFCF3 treatments increased post-silking DMA and total DMA by 40.4, 33.6, 44.6% and 26.1, 12.9,
17.5%, respectively, compared with the CF treatment (P<0.05). However, the OFCF4 treatment
significantly decreased post-silking DMA and total DMA, by 8.62 and 8.17%, respectively. The grain
yield of OFCF1 and OFCF2 were always significantly higher than that of CF and OFCF4 in both growing
seasons. No difference in ear numbers was observed among treatments, with the exception of OFCF4
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 Journal of Integrative Agriculture
was significantly lower than CF in 2019. In both growing seasons, the thousand kernel weight of
OFCF1 and OFCF2 were significantly higher than that of CF, OFCF3, and OFCF4. In 2019, the kernels
per ear of OFCF2 was higher than other treatments, but no significant difference was found among
treatments in 2020. The interaction between year and treatment had no significant effect on pre-silking
DMA, post-silking DMA, total DMA, grain yield, and yield components.

3.7. Root DMA and hormone content

CAOFCF had a significant effect on root DMA in the 0–40-cm soil layer (P<0.05; Fig. 6). In both
growing seasons, root DMA in OFCF1, OFCF2, and OFCF3 plant samples from the 0–10-, 10–20-, and
20–40-cm soil layers was greater than root DMA in corresponding CF plant samples; we observed no
significant difference between CF and OFCF4 plant samples. The OFCF1, OFCF2, and OFCF3
treatments increased root DMA in the 0–40-cm soil layer by 27.2, 27.3, and 24.7%, respectively,
compared with CF.
Statistical analyses showed that the ZR and ABA content and ZR/ABA ratio in roots were
significantly affected by the CAOFCF treatments (P<0.05; Fig. 7). The OFCF1 and OFCF2 treatments
increased the ZR content by 60.9 and 34.6%, respectively, compared with the CF treatment; however,
there was little difference in ZR content between the CF and OFCF3 plants. ABA content was
increased in OFCF1 plants, but decreased in OFCF2 and OFCF3 plants, compared with CF plants.
The ZR/ABA ratio was significantly increased in OFCF1 and OFCF2 plants compared with CF plants
(P<0.05).

3.8. Correlation analysis

As shown in Fig. 8, WR was positively correlated with post-silking N uptake and DMA (Fig. 8-A,
P=0.0392, R2=0.4310; Fig. 8-C, P=0.0389, R2=0.3611). By contrast, WG was negatively correlated
with post-silking N uptake and DMA (Fig. 8-B, P=0.0435, R2=0.4176; Fig. 8-D, P=0.0497, R2=0.3250).
Similarly, there was a significant positive correlation between grain yield and WR (P=0.04397,
R2=0.4295), and grain yield was negatively correlated with WG (P=0.0235, R2=0.3250).

4. Discussion
4.1. CAOFCF alleviates the kernel position effect in summer maize

Kernel position effect is common in maize cultivars. However, there exist difference in kernel position
effect among maize cultivars (Calvo-Brenes et al. 2019), which is mainly determined by the genetic
characteristics of cultivars. Alleviating the kernel position effect is important for increasing maize yields
(Yang and Li 2012). Generally, inferior kernels are more susceptible to modification than superior ones
(Zhai et al. 2020), and the inferior kernels of maize are more susceptible than superior kernels to abiotic
stress during the grain filling period (Shen et al. 2018; Yan et al. 2018). Our results showed that,
compared with the CF treatment, the OFCF1 and OFCF2 treatments significantly reduced WG and
increased WR (Fig. 2), suggesting that CA of 15–30% OF with 70–85% CF alleviated the kernel position
effect in summer maize, largely due to a significant increase in the weight of inferior kernels. As is
known to all, grain filling directly affects the final kernel weight of cereal crops (Li et al. 2020; Zhai et al.
2020; Wu et al. 2022). Compared with the CF treatment, the OFCF1 and OFCF2 treatments
significantly increased kernel weights by improving the grain filling rate from 30 DAP (Fig. 3). These
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kernel weight increases were more marked in inferior than superior kernels. Generally, the starch
content in maize grains accounts for more than 70% of the total grain weight, the grain filling process of
maize kernels is mainly a process of starch synthesis and accumulation (Zhang et al. 2007; Xu et al.
2015). The present result showed that OFCF1 and OFCF2 increased the starch content in inferior
kernels more than that in superior kernels (Fig. 4-A and C), which implied that OFCF1 and OFCF2 can
increase the carbohydrate allocation to inferior kernels, compared to that of CF. Moreover, adequate
post-silking assimilates accumulation is important for grain filling and ensuring normal kernel weights
(Wang et al. 2021; Li R F et al. 2022; Liu G Z et al. 2022). Generally, the assimilates unloading ability of
superior kernel is higher than that of inferior kernels (Shen et al. 2022), and the sink of superior kernel is
also larger than that of inferior kernels (Xu et al. 2013). These intrinsic characteristics make the
superior kernels have certain advantage over inferior kernels in assimilate allocation (Fan et al. 2011).
When the post-silking assimilate supply is low, inferior kernels cannot obtain sufficient assimilates, which
leads to poor grain filling (Yang and Li 2012). In this study, the OFCF1, OFOCF2, and OFCF3
treatments significantly increased post-silking DMA compared with the CF treatment (Table 3), reflecting
more post-silking assimilates were accumulated. Therefore, the increase in post-silking assimilate
accumulation and allocation of assimilates to inferior kernel may be the direct reason for the alleviated
kernel position effect under OFCF1 and OFCF2 treatments.

4.2. Effect of CAOFCF on post-silking N uptake in summer maize

Increasing post-silking N uptake by optimizing N fertilizer management could increase maize yields
(Chen et al. 2022; Guo et al. 2022). Recent research has shown that combined application of organic
fertilizer and chemical fertilizer has been proven to be an effective strategy for N management, and has
been applied to various crops (Zhang et al. 2018, 2021; He et al. 2022). In this study, the OFCF1 and
OFCF2 treatments increased total plant N uptake, mainly due to a significant increase in post-silking N
uptake (Table 2). Therefore, substituting 15–30% CF with OF containing an equivalent quantity of N
enhanced post-silking N uptake. Generally, improving soil N availability or reducing N loss can increase
plant N uptake (Liang et al. 2022). In many studies, CAOFCF reduced N leaching loss or mitigated
reactive N loss (Lv et al. 2020; Liu et al. 2021; He et al. 2022; Li Y W et al. 2022), suggesting that
CAOFCF may increase the N in soil available to plants. Tang et al. (2022) reported that CAOFCF can
provide a carbon source for microorganisms that regulate soil N cycling, thereby reducing soil N loss.
Therefore, CAOFCF treatments may improve post-silking N uptake partly by decreasing the loss of soil
N.
Roots are the most important organs involved in N uptake in crops, and root size and vigor are
important for efficient nutrient uptake (Peng et al. 2010; Zhang B C et al. 2022). We found that the
OFCF1 and OFCF2 treatments significantly increased root DMA (Fig. 6). Therefore, OFCF1 and
OFCF2 may promote root growth, which is beneficial for plant N uptake (Chen et al. 2021; Zhai et al.
2022). Moreover, root cytokinin content is an important physiological index of root senescence (Artner
and Benkova 2019), it is usually negatively correlated with root senescence. At the mid-grain filling stage,
the OFCF1 and OFCF2 treatments in this study significantly increased root ZR content (Fig. 7),
indicating that CAOFCF could alleviate post-silking root senescence. However, Liu et al. (2011) found
that adding OF had no obvious effect on root senescence in maize. This discrepancy may be explained
by the different cropping systems and organic fertilizer used. The increased post-silking N uptake
exhibited by OFCF1 and OFCF2 plants may be linked to the alleviation of root senescence and
improved root growth in summer maize.

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 Journal of Integrative Agriculture
4.3. Effect of CAOFCF on the post-silking LAI and photosynthetic capacity of summer
maize

Maintaining higher post-silking LAIs and photosynthetic capacity are important for ensuring higher grain
yields in maize (Qin et al. 2013; Li Y Y et al. 2022; Ren et al. 2022; Yue et al. 2022). The OFCF1 and
OFCF2 treatments in this study increased post-silking LAIs and reduced the decline of post-silking LAIs
(Fig. 5-A). These observations are consistent with results reported by Pan et al. (2022) and suggest
that the OFCF1 and OFCF2 treatments alleviated leaf senescence. Post-silking N uptake is a key factor
influencing senescence in maize (Zhang et al. 2019). Our results showed OFCF1 and OFCF2
treatments increased post-silking N uptake and leaf N content, which is crucial for maintaining higher
post-silking LAIs. Because sufficient leaf area is necessary for DMA in maize (Li Y Y et al. 2022), the
improved post-silking DMA stimulated by CAOFCF treatments is partly attributable to higher post-silking
LAIs.
Photosynthetic capacity influences assimilate accumulation in maize (Zhai et al. 2020; Yue et al.
2022). Post-silking photosynthetic capacity is particularly important for supplying the assimilates used
in grain filling (Li R F et al. 2022; Li Y T et al. 2022). The present result showed that OFCF1 and OFCF2
treatments significantly increased post-silking Pn and NAR (Fig. 5-B and C). These findings are
consistent with similar studies on wheat (Saikia et al. 2015), and suggest that CAOFCF may improve
post-silking photosynthetic capacity. Meanwhile, these studies showed that the higher photosynthetic
capacity under CAOFCF treatments is associated with its role in regulating root growth. Because crop
root and canopy photosynthesis are mutually beneficial relationship, thus the root growth is closely
related to canopy photosynthetic capacity (Yousefzadeh-Najafbadi and Ehsanzadeh 2021). Moreover,
adjusting leaf N content may enhance crop photosynthetic capacity (Fernandez et al. 2022; Qu et al.
2022). Our results showed that OFCF1 and OFCF2 treatments significantly increased leaf N content,
compared to that of CF. Given that maize leaf N content is positively correlated with photosynthetic
capacity (Zhang et al. 2019), the increased leaf N content may explain the increased photosynthetic
capacity of OFCF1 and OFCF2 plants.

4.4. Correlation analysis between kernel position effect and post-silking N uptake, DMA,
and grain yield

Correlation analysis revealed that WR was positively correlated with post-silking N uptake and DMA
(P<0.05), while WG was negatively correlated with post-silking N uptake and DMA (P<0.05; Fig. 8).
These findings suggest that the kernel position effect is negatively correlated with post-silking N uptake
and DMA. Yang and Li (2012) found that poor grain filling of inferior kernels, caused by an insufficient
supply of assimilates, was the main factor exacerbating the kernel position effect in maize. Therefore,
ensuring sufficient post-silking assimilate supply is key to alleviating the kernel position effect. Generally,
increased post-silking N uptake alleviates plant senescence and increases the photosynthetic capacity
of maize (Zhang et al. 2019), and increased photosynthetic capacity usually results in greater assimilate
accumulation (Li Y T et al. 2022), which is beneficial to grain filling in inferior kernels and alleviates the
kernel position effect. Moreover, the result confirmed that the kernel position effect was negatively
correlated with grain yield, which suggested that alleviating kernel position effect is an important
breakthrough point to improve grain yield of summer maize.

5. Conclusion
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 Journal of Integrative Agriculture
Our results showed that, compared with CF treatment, the OFCF1 and OFCF2 treatments alleviated the
kernel position effect in summer maize by stimulating grain filling and starch accumulation in inferior
kernels. Suppression of the kernel position effect by the OFCF1 and OFCF2 treatments was closely
associated with improved post-silking N uptake and DMA, which in turn resulted from the robust root
system and increased post-silking photosynthetic capacity. Therefore, CA of 15–30% OF and 70–85%
CF alleviated the kernel position effect in summer maize by increasing post-silking N uptake and DMA.
This combined fertilizer application approach may improve sustainable maize production.

Acknowledgements

This study was financially supported by HAAFS Science and Technology Innovation Special Project
(2022KJCXZX-LYS-9), the Natural Science Foundation of Hebei Province, China (C2021301004), and
the Key Research and Dvelopment Program of Hebei Province, China (20326401D).

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Fig. 1 Daily mean temperature and precipitation during 2019 and 2020 growing seasons.

Fig. 2 Kernel weight gap between superior kernel and inferior kernel (A and C) and weight ratio of inferior to superior
kernel (B and D) of summer maize under different treatments. Different lowercase letters above a column indicate
significant differences among treatments (P<0.05).

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Fig. 3 Effect of CAOFCF on dynamic change of kernel weight (A, C, E, and G and the grain filling rate (B, D, F, and H) of
inferior and superior kernels in summer maize.

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Fig. 4 Effect of CAOFCA treatments on the kernel starch content (A and C) and soluble sugar content (B and D) in
inferior and superior kernels. Different small letters above the bars representing the same kernel type indicate significant
difference among treatments at P<0.05.

Fig. 5 Effect of CAOFCF on the leaf area index (A and D), post-silking photosynthetic rate (B and E), and net
assimilation rate (C and F) of summer maize. AS, anthesis stage; M-GF, mid-grain filling stage; L-GF, late-grain filling

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stage. Different lowercase letters above a column indicate significant differences (P<0.05) among treatments. , P<0.05;
*

**
, P<0.01 (significant differences among treatments).

Fig. 6 Effect of CAOFCF treatment on root dry matter accumulation in different soil layers (0–40 cm). Different letters
above the bars within the same soil layer indicate significant differences (P<0.05) among treatment.

Fig. 7 Effect of CAOFCF treatment on the ZR (A) and ABA (B) content and ZR/ABA (C) of roots. Different letters above
the bars indicate significant differences (P<0.05) among treatments.

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Fig. 8 Relationships of WR with post-silking N uptake, post-silking DMA, and grain yield (A, C, and E, respectively);
relationships of WG with post-silking N uptake, post-silking DMA, and grain yield (B, D, and F, respectively).

20
Table 1 Detailed description of the amount of organic fertilizer and chemical fertilizer applied in all treatments

Chemical fertilizer (kg ha-1)

Treatment Organic fertilizer （kg ha )
-1

Urea (N) Calcium superphosphate (P2O5) Potassium chloride (K2O)

CK 0 489 261 200

OFCF1 1361 416 253 178
OFCF2 2722 342 245 156
OFCF3 4083 269 237 135
OFCF4 5444 196 229 113
The organic fertilizer used in this study was produced from cow manure and contained 45% organic matter. The N content in urea is 46%, the P2O5 content in calcium superphosphate
is 46%, and the K2O content in potassium chloride is 60%.

Table 2 Post-silking N uptake and its contribution to grain N for different treatments

N content at silking (g/plant) N content at maturity (g/plant) Post-silking N uptake

Ratio of
post-silking Contribution
Year Treatment Amount
Leaf Stem+sheath Husk+cob Total Leaf Stem+sheath Husk+cob Grain Total N uptake to to grain N
(g/plant)
total N (%)
uptake (%)
CF 1.128 a 1.300 b 0.020 a 2.448 b 0.365 c 0.464 c 0.108 c 2.228 bc 3.164cd 0.716d 0.226 c 0.322 c
OFCF1 1.136 a 1.709 a 0.020 a 2.865 a 0.675 a 0.703 a 0.155 a 2.609a 4.141a 1.276bc 0.308 b 0.489 b
2019 OFCF2 1.019 b 1.053 c 0.014b 2.086 c 0.512 b 0.519 b 0.123b 2.445ab 3.599bc 1.513ab 0.421 a 0.619 a
OFCF3 0.997 b 1.027 c 0.011 c 2.035 c 0.552 b 0.588 b 0.143 a 2.619a 3.903 ab 1.868a 0.479 a 0.713 a
OFCF4 1.028b 1.109 bc 0.016b 2.153 c 0.398 c 0.507 b 0.097 c 2.075c 3.077 d 0.963cd 0.313 b 0.464 b
CF 0.951a 1.005 a 0.13 ab 2.086 a 0.508 b 0.881 b 0.108b 2.014b 3.510 b 1.425b 0.406 b 0.708 b
OFCF1 0.973 a 0.834 b 0.145 a 1.951 ab 0.620 a 0.903 a 0.122 a 2.508a 4.153a 2.201a 0.530 a 0.877 a
2020 OFCF2 0.982 a 0.892 b 0.108 ab 1.982a 0.592 a 0.877 a 0.109b 2.395a 3.972a 1.991a 0.501 a 0.831 a
OFCF3 0.895 b 0.821 b 0.097b 1.813ab 0.521 b 0.502 c 0.096bc 1.762c 2.880c 1.067c 0.370 b 0.605 c
OFCF4 0.881 b 0.726 c 0.101b 1.708b 0.527 b 0.498 c 0.095 c 1.754c 2.875c 1.167bc 0.406 b 0.665 bc
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 ANOVA P-value
Year (Y) 0.001 <0.001 <0.001 <0.001 0.001 <0.001 0.001 <0.001 ns 0.003 0.002 <0.001

Treatment (T) ns <0.001 0.039 <0.001 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 0.015 0.022

Y×T 0.624 <0.001 0.214 0.002 0.001 0.003 0.033 <0.001 <0.001 <0.001 0.007 0.007
Different lowercase letters within a column representing the same year indicate significant differences (P<0.05) among treatments. ns, not significant

Table 3 Effect of CAOFCF treatments on dry matter accumulation, grain yield, and yield components of summer maize

Pre-silking DMA Total DMA Post-silking DMA Grain yield Ear number Thousand-kernel
Year Treatments Kernels per ear
(t ha )
−1
(t ha )
−1
(t ha )
−1
(t ha )-1
(×10 ha ）
4 -1
weight (g)

CF 7.85±0.15 b 17.58±1.05 b 9.73±1.05 b 9.46±0.19 b 7.42±0.18 a 270.5±3.6 b 556.2±7.1 b

OFCF1 8.49±0.13 a 22.12±0.78 a 13.63±0.78 a 10.66±0.15 a 7.27±0.18 ab 303.0±2.8 a 568.2±5.0 b
2019 OFCF2 6.90±0.19 c 20.00±0.24 a 13.10±0.24 a 10.48±0.17 a 7.29±0.13 ab 294.4±6.3 a 595.5±1.4 a
OFCF3 6.65±0.09 c 20.86±0.52 a 14.21±0.52 a 9.51±0.26 b 7.17±0.08 ab 259.8±5.6 b 551.5±8.3 b
OFCF4 7.04±0.21 c 15.67±0.56 b 8.63 ± 0.56b 9.60±0.30 b 6.92±0.03 b 270.2±14.5 b 569.2±5.6 b
CF 8.53±0.16 b 19.10±1.14 cd 10.57±0.14 b 9.01±0.19 b 7.11±0.18 a 331.5±2.7 b 406.4±11.5 a
OFCF1 9.27±0.15 a 24.15±0.85 a 14.88±0.85 a 10.16±0.17 a 7.43±0.17 a 338.7±1.0 a 433.8±9.9 a

2020 OFCF2 7.39±0.20 c 21.42±0.26 bc 14.03±0.26 a 9.81±0.10 a 7.30±0.19 a 343.1±2.6 a 425.9±12.6a

OFCF3 7.10±0.09 c 22.25±0.56 ab 15.16±0.56 a 9.79±0.12 a 7.15±0.34 a 330.5±3.1 b 402.1±14.6 a

OFCF4 8.09±0.24 b 18.01±0.64 d 9.92±0.34 b 8.98±0.10 b 7.25±0.42 a 326.8±1.2 b 387.6±17.4 a

P-value for
Year (Y) <0.001 0.001 0.032 <0.001 0.001 <0.001 <0.001
Treatment (T) <0.001 <0.001 <0.001 <0.001 ns <0.001 ns
Y×T ns ns ns ns ns ns ns
Different lowercase letters within a column representing the same year indicate significant differences (P<0.05) among treatments. ns, not significant.

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