Journal of Integrative Agriculture

Coupling of reduced inorganic fertilizer with plant-based organic fertilizer as a promising

fertilizer management for colored rice in tropical regions
ZHAO Ting-cheng1*, HE Ai-bin1*, Mohammad Nauman Khan1, YIN Qi1, SONG Shao-kun1, NIE

Li-xiao1#
1
Research Center for Physiology and Ecology and Green Cultivation of Tropical Crops, College of
Tropical Crops, Hainan University, Haikou 570228, P.R.China

Abstract Colored rice is a kind of high-quality, high-added-value rice that has attracted increasing
attention in recent years. The use of the large amount of inorganic nitrogen fertilizer in rice fields results
in low fertilizer use efficiency and high environmental pollution. Organic fertilizer is a promising way to
improve soil quality and sustain high yields. However, most studies focus on the effect of animal-based
organic fertilizers. The effect of different ratios of plant-based organic fertilizer substitution with
inorganic fertilizer on grain yield and quality of colored rice has rarely been reported. A two-year field
experiment was conducted to study the effects of replacing inorganic N fertilizers with plant-based
organic fertilizers on the yield, NUE, and anthocyanin content of colored rice in tropical regions. Field
experiments were conducted to examine the effects of partial substitution of inorganic fertilizers with
organic fertilizers on grain yield, nitrogen use efficiency and anthocyanin content in grains of two colored
rice in 2020 and 2021. Experimental treatments included no nitrogen fertilization (T1), 100% inorganic
nitrogen fertilizer (T2), 30% inorganic nitrogen fertilizer substitution with plant-based organic fertilizer
(T3), 60% inorganic nitrogen fertilizer substitution with plant-based organic fertilizer (T4), and 100%
plant-based organic fertilizer (T5). The total nitrogen provided to all the treatments was 120 kg ha-1
except for T1. Our results showed that the T3 treatment enhanced the grain yield and anthocyanin
content of colored rice by increasing nitrogen use efficiency compared with T2. On average, grain
yields were increased by 9 and 8%. Anthocyanin content increased by 16 and 10% of two colored rice
under T3 across two years, respectively, as compared with T2. Further research on the residual effect
of partial substitution of inorganic fertilizers showed that substitution of inorganic fertilizer with
plant-based organic fertilizer improved soil physio-chemical properties and thus increased the rice grain
yield in the subsequent seasons. The highest grain yield of subsequent rice was observed under the
T5 treatment. Our results suggested that the application of plant-based organic fertilizers sustained the
production of colored rice with high anthocyanin content in tropical regions, which was beneficial in
reconciling the relationship between rice production and environmental protection.
Keywords: colored rice, organic fertilizer, soil quality, grain yield, anthocyanin1

1. Introduction
Rice is one of the world’s most important food crops, accounting for 25% of the world's total calorie
intake, and 60% of the Chinese population takes rice as their main food source (FAO 2004; Yuan 2014;
Kusano et al. 2015). With the development of the economy and the improvement of people's living
standards, the planting structure of rice has also changed, and it tends to develop rice with high
value-added and high economic benefits (Chen et al. 2021). The demand for rice with unique qualities

Received 6 January, 2023 Accepted 6 April, 2023

#
Correspondence, NIE Li-xiao, Tel: +86-898-66279257, Email: lxnie@hainanu.edu.cn
*
These authors contributed equally to this work.
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 Journal of Integrative Agriculture
and nutritional value is gradually increasing worldwide. Colored rice is a kind of high-quality,
high-added-value rice that has attracted increasing attention in recent years. For colored rice, it has
been reported that the main substance of phenolic compounds is anthocyanin, which are a major active
component for antioxidants (Iqbal et al. 2005; Zhang et al. 2006; Yawadio et al. 2007). The effects of
anthocyanins include improving iron-deficiency anemia, enhancing the body’s antioxidant, anti-cancer,
and anti-allergic capabilities, and preventing atherosclerosis and some diseases associated with
abnormal glucose metabolism. Studies have provided conclusive evidence for its role in chronic
hyperglycemia disease etiology (Yawadio et al. 2007; Krishnan et al. 2021).
With the continuous promotion of modern agricultural concepts, implementing high-quality and
sustainable development is the inevitable direction of agricultural development (Liu et al. 2020).
However, farmers are accustomed to relying on high levels of inorganic nitrogen (N) to increase crop
yields in China, making Chinese nitrogen fertilizer consumption the largest in the world (FAO 2014).
The application of nitrogen fertilizer in China was 33.6 Tg in 2013, accounting for 33% of the total
nitrogen fertilizer consumption worldwide. In addition, rice yield growth has slowed significantly and
even stagnated in many regions of China in the past 10-20 years, despite high nitrogen inputs (Grassini
et al. 2013; Zhao et al. 2015). Nitrogen input eventually reaches a point where it stops increasing crop
yield, and excessive N input results in most of the N being lost to the surrounding environment and in low
nitrogen use efficiency (NUE), as well as soil acidification, greenhouse gas emissions, and groundwater
nitrate pollution (Galloway et al. 2008; Ju et al. 2009; Chen et al. 2010; Zhang et al. 2017). Therefore,
an effective fertilizer management method is urgently needed to improve soil fertility and the yield and
quality of rice.
The application of organic fertilizers has been neglected due to the obvious effect of increasing
production, while organic fertilizers are released slowly, and there are insufficient sources of nutrients
(Geng et al. 2019). To achieve sustainable agricultural development, green and organic agriculture
must be pursued as the development direction of modern agriculture (Kilcher 2007). Previous studies
have shown that organic fertilizers are a key strategy for improving soil structure and nutrient content to
achieve sustainable crop growth and high economic benefits (Cui et al. 2021; Zhang et al. 2022).
Organic fertilizer application. Long-term organic fertilizer application maintains the soil organic matter at
a relatively stable level, improves soil quality, and increases soil organic matter content. Long-term
organic fertilizer application maintains the soil organic matter at a relatively stable level, improves soil
quality, and increases stress resistance in farmland (Xu et al. 2021; Asghar et al. 2022). Studies have
shown that soil microorganisms are closely related to the content of nutrients such as soil organic carbon.
The presence of organic matter can provide microorganisms with nutrients required for life activities,
thereby promoting the development of microbial communities (Lazcano et al. 2013, Liu et al. 2022).
The release of organic fertilizers is slow, and the effect of increasing crop yields cannot be directly
reflected. Still, long-term use of organic fertilizers can improve plant growth and crop quality (Ye et al.
2020). Partial replacement of inorganic fertilizers with organic fertilizers significantly improved NUE in
rice and maintained grain yield by establishing a more balanced source-sink relationship during the
grain-filling phase (Zhang et al. 2018; Pan et al. 2022).
At present, most studies focus on the effect of animal-based organic fertilizers. However, the effect
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of different ratios of plant-based organic rather than inorganic fertilizer on grain yield and the
anthocyanin content of colored rice has rarely been reported. In this scenario, a two-year field
experiment was conducted to study the effects of replacing inorganic N fertilizers with plant-based
organic fertilizers on the yield, NUE, and anthocyanin content of colored rice in the tropical region.
2. Materials and methods
2.1. Site description

The study was conducted in Nanbao town (19°64′N，109°62′E), Lingao City, Hainan Province, China,
during the rice growing in 2020 and 2021. Hainan Province is located in the tropical region. The
typical rice growing season in the experimental site is from the end of February to November. The
experiment was conducted on August 2, 2020 (the first season) and February 24, 2021 (the second
season).
The pH, total nitrogen (N), available phosphorus, potassium, and total organic carbon in the top 20
cm of soil were 6.17, 1.90g kg-1, 62.59 mg kg−1, 38.86 mg kg−1, and 0.89%, respectively, in 2020, and
they were 6.12, 1.10 g kg-1, 60.58 mg kg−1, 42.55 mg kg−1, and 1.14% in 2021.
2.2. Experimental design

Youxianghongdao (YXHD) and Suixiangheinuo (SXHN), two main locally grown colored rice varieties,
were adopted in this study. YXHD is normally used to produce wine, while SXHN is sticky rice, mostly
used for making traditional Chinese rice pudding. Besides, both are also used for domestic cooking
purposes.
The experiments were arranged in a randomized complete block design (RCBD) with four
repetitions. The plot area was 15 m2 (3 m×5 m) (Fig. 1). The treatments included no N fertilization
(T1), 100% inorganic N fertilizer (T2, the representative of inorganic fertilization), 70% inorganic N
fertilizer+30% organic fertilizer (T3), 40% inorganic N fertilizer+60% organic fertilizer (T4), and 100%
organic fertilizer (T5). The total N rate for all treatments (except for T1) was 120 kg N ha-1 over two
years. During all treatments, phosphorus (P) and potassium (K) were applied at 60 kg ha-1 as P2O5 and
100 kg ha-1 as K2O, respectively. Nitrogen fertilizer was applied in three splits (basal fertilizer: tillering
fertilizer: grain-filling fertilizer=1:1:1), and P and K fertilizers were applied once as basal fertilizers. The
application amounts of organic fertilizer in T3-T5 were calculated based on the N content of organic
fertilizer, which was consistent with the inorganic fertilizer treatment (T2). To make the P and K rates
consistent with T2, calcium superphosphate and potassium chloride, respectively, were supplemented to
T3 to T5. The total application rates of organic fertilizer of T3-T5 were 1800, 3600, and 6000 kg ha-1,
respectively. The organic fertilizer used in this study was plant-based organic fertilizer manufactured by
CNOOC Fudao (Hainan) Chemical Co., Ltd., and the contents of N, P2O5, and K2O were 2.02, 1.9, and
1.69%, respectively. Organic fertilizer was incorporated into the soil during land preparation.
Pregerminated seeds were sown in nurseries on August 2, 2020 and February 24, 2021. The
transplanting dates were August 23, 2020 and March 21, 2021. All plots were plowed and puddled
before transplanting. Seedlings were transplanted into the paddy soil with a hill spacing of 20×20 cm,
having 3 seedlings per hill. A shallow water layer (3-5 cm) was maintained during the whole rice growth
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period. Careful management was carried out to fully control diseases and pests, including hanging bird
protection nets and laying down anti-rat mulch film at maturity to avoid crop yield losses.

Fig. 1 Field trial design (photographed at heading-early grain-filling stage). T1, no N fertilization; T2, 100% inorganic N

fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40% inorganic N fertilizer+60% organic fertilizer; T5,

100% organic fertilizer. YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo, respectively.

After harvesting colored rice SXHN and YXHD, the ridges of previous plots were maintained to
study the residual effect of partial substitution of inorganic fertilizer. All plots were uniformly planted with
the YXHD after the plot was re-leveling to a hill spacing of 20 cm×20 cm, with 3 seedlings per hill. Only
inorganic fertilizers were applied in all the plots. The rates were the same as 60 kg N ha-1, 60 kg P2O5
ha-1, and 100 kg K2O ha-1 for all treatments (of N:P:K) in all plots, and only the N fertilizer was applied as
basal fertilizer, tillering fertilizer, and grain-filling fertilizer in a ratio of 1:1:1. Phosphorus and potassium
fertilizer were once used as basal fertilizer. The sources of N, P, and K were urea (46.4% N), calcium
superphosphate (12.0% P2O5), and potassium chloride (60.0% K2O), respectively.
Soil physical and chemical properties Soil samples were collected after rice flowering. A five-point
sampling method was adopted to collect 1-20 cm of topsoil. Soil samples were refrigerated and
transported back to the laboratory in an ice box after contaminants, such as leaves, roots, and stones,
were removed. After that, each soil sample was divided into two parts, and one part was used to detect
the physical and chemical properties of the soil after air drying and screening, such as soil organic matter.
The other part was stored in a 4°C refrigerator to determine the soil's other physical and chemical
properties. The measurement methods of basic soil physical and chemical indices are as follows:
(1) Soil water content and electrical conductivity: A soil multiparameter tester (SYS-TRD) was used for
field measurements.
(2) pH: Deionized water with the CO2 removed was used, the water and soil ratio was mixed at 1:1, and
the mixture was stirred thoroughly. The fully stirred water and soil mixture were centrifuged. After
centrifugation, the pH of the upper layer solution was measured by a pH meter (Leici, Shanghai).
(3) Organic matter (TOC): The oil bath temperature was 180°C, boiled for 5 min, the soil organic matter
was oxidized with potassium dichromate-sulfuric acid solution, and then the excess potassium
dichromate was determined by titration with standard 0.2 M ferrous sulfate. The amount of potassium
dichromate consumed was used to calculate soil organic matter content (Strickland and Sollin 1987).
The reaction during oxidation and titration is as follows:
2K2Cr2O7+8H2SO4+3C→2K2SO4+2Cr2(SO4)3+3CO2+8H2O
K2Cr2O7+6FeSO4+7H2SO4→K2SO4+Cr2(SO4)3+3Fe2(SO4)3+7H2O
(4) Total Nitrogen (TN): Total nitrogen was analyzed using the Kjeldahl method (Bremner and Tabatabai
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1972). Digesting with concentrated sulfuric acid accelerates the decomposition of organic matter with
the help of catalysts (K2SO4, CuSO4, Se) so that organic nitrogen is converted into ammonia in the
solution. Finally, the distilled ammonia is determined by titration.
(5) Available phosphorus (AP): measured by molybdenum blue spectrophotometry (Liao et al. 2018).
(6) Quick-acting potassium (AK): detected by a flame photometer (FAPRC 2015).
(7) Cation exchange capacity (CEC): measured by the BaCl2-MgSO4 method (Hendershot and
Duquette 1986).
Plant sampling and analysis Six plants of rice were sampled at the middle tillering stage (MT),
panicle initiation stage (PI), full heading stage (HD), and physiological maturity stage (PM) for growth
analysis. The plants were washed, and the number of tillers (including main shoots and tillers) and
plant height were recorded. In addition, leaf SPAD value at MT, PI, HD, and PM was determined.
After removing the roots, the plants were divided into leaves, stems, and panicles (HD and PM) and
dried at 70°C to a constant weight.

Nitrogen accumulation and utilization At MT, PI, HD, and PM, grain sample and straw were
oven-dried at 70°C to constant weight and ground to powder (<0.149 mm fragments) for total N (TN)
analysis. The N concentration of the samples was analyzed using the micro-Kjeldahl method. Plant
N uptake (PNU) was calculated by multiplying dry matter yield by TN. The agronomic nitrogen use
efficiency (AEN), the ratio of the difference between the grain yield of plants with and without N
fertilization and the N rate, that is, the increase in grain yield per unit of N application, was calculated.
The N recovery efficiency (REN, %) described the nitrogen use efficiency. The fertilizer- N Partial factor
productivity (PFPN) and the ratio of N fertilization rice yield to N rate were calculated. AEN, REN, and
PFPN were calculated as follows.
AEN=Grain yield increase/Fertilizer N input
REN=100×N uptake from fertilizer N/Fertilizer N input
PFPN=Grain yield/Fertilizer N input
Grain yield and yield components At the maturity stage, 6 hills of rice samples were taken from each
plot to determine the 1000-grain weight, effective panicle number, spikelets per panicle (filled grain,
half-filled grain, and unfilled grain), filled grain rate, and harvest index. Through hand-threshing, all
spikelets were submerged into tap water to separate the filled grains from the others (half-filled spikelets
and unfilled spikelets). Then, a 3-m2 area in the center of each plot was selected as a production
testing area, and the production was manually harvested and measured. The threshed grains were
naturally sun-dried, an artificial air separator removed the impurities, and empty grains. Grain yield was
adjusted to a moisture content of 0.14 g H2O g-1 FW.

Anthocyanin analysis At the maturity stage, 30 normal rice panicles were randomly taken from each
plot, and after natural shade drying, they were artificially threshed and screened for filled grains. Then,
the grains were dehulled, and 10 g of completely unpolished rice was collected, crushed, and ground
using a hybrid ball mill at a 30.0 frequency s-1 for 1 minute (MM400, RETSCH, Germany). Afterward,
0.1 g of the ground sample from each treatment was stored in a self-seal bag. A total of 1 mL of extract
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(mixture of hydrochloric acid and ethanol) was mixed with the ground sample, and the slurry was well
homogenized and transferred to an EP tube. After the EP tube was tightly covered, it was extracted at
75°C in a constant temperature water bath pot for 20 min and then centrifuged for 10 min under a
centrifugal force of 8000 g min-1 at room temperature. The supernatant was taken to be measured.
Following the Kit's instructions for determining plant anthocyanin content by Suzhou Comin
Biotechnology Co., Ltd., sampling and determination were repeated three times.

2.4. Weather data collection

Meteorological data, including solar radiation, temperature, and precipitation, were collected from a
weather station (Qi Weather Station, Insentek, China) near the experimental field. Total precipitation,
average daily solar radiation, and daily mean temperature during the rice growing seasons in 2020 and
2021 are shown in Fig. 2. The total rainfall in the first season was 480.6 mm, which was 11.8% lower
than that in 2021 (544.9 mm). The average daily solar radiation in 2020 was 10.34 MJ m-2 d-1, 42.8%
lower than in 2021 (18.07 MJ m-2 d-1). While the daily mean temperatures in 2020 and 2021 were 25.1
and 27.7°C, respectively.

Fig. 2 Daily mean temperature (A and D), rainfall (B and E), and solar radiation (C and F) during the rice-growing season

at Nanbao town, Lingao City of Hainan Province, China, in 2020 (A, B and C) and 2021 (D, E and F).

2.5. Data analysis

Data were analyzed by analysis of variance using Statistix 9.0. Differences between treatments were
separated at probability levels of 0.05, 0.01, and 0.001 using the LSD (Least Significant Difference) test.
The data was graphically represented with Sigmaplot 12.0.

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3. Results
3.1. Soil properties

Different proportions of organic fertilizer substitution for inorganic fertilizers had significant effects on the
physical and chemical properties of total soil nitrogen (TN), alkaline hydrolyzable nitrogen (AN),
available phosphorus (AP), available potassium (AK), pH, electrical conductivity (EC), and soil organic
carbon (SOC), and the trend was consistent between the two years (Table 1). Compared to T2, the soil
TN, AN, AP, AK, pH, EC and SOC increased by 24, 11, 8, 39, 5, 46, and 19% under T5 in 2020,
respectively, and 15, 18, 18, 24, 6, 25 and 22% in 2021. The soil TN, AP, AK, SOC, CEC, and SBD were
not significantly different between T4 and T5. In addition, compared to T2, the soil physical and
chemical properties under T3 and T4 were improved. Finally, the lowest soil physical and chemical
property indices were observed under T1 (non-N treated). And there was no interaction between
treatment and year (Table 1).

Table 1 Changes in soil physical and chemical properties under substituting inorganic fertilizer with plant-based organic

fertilizer1)

Alkaline Available Available EC Soil CEC Soil

Total N pH
Year Treatment2) N (mg P (mg K (mg (µs organic (cmol bulk
(g kg-1) value
kg-1) kg-1) kg-1) cm-1) carbon (+) density

(%) kg-1) (g cm-3)

2020 T1 0.94 c 100.00 b 53.97 c 20.45 c 5.78 52.68 0.98 d 13.19 1.48 a

T2 1.01 c 105.98 b 55.93 bc 22.51 c b

5.74 c57.23 1.05 c c16.77 1.45 ab

T3 1.16 b 115.55 a 58.12 ab 26.79 b b

5.84 c70.66 1.15 b bc
19.09 1.40 ab

T4 1.21 116.68 a 58.58 a 27.68 ab ab

5.91 b
73.79 1.19 ab ab
21.40 1.37 b

T5 ab
1.25 a 118.07 a 60.25 a 31.36 a ab
6.00 b
83.92 1.24 a a
22.19 1.36 b

Mean 1.11 B 111.25 B 57.37 A 25.76 A a

5.85 a
67.62 1.12 B a
18.53 1.41 A

2021 T1 1.09 d 124.74 c 48.06 b 13.96 d A

5.31 A
55.93 1.11 c A
8.55 c 1.49 a

T2 1.11 131.49 c 48.38 b 14.70 cd c5.32 d

62.81 1.18 c 11.07 1.48 ab

T3 cd
1.19 155.82 b 54.16 a 16.04 bc c5.48 c72.37 1.29 b c17.69 1.42 bc

T4 bc
1.24 133.99 b 55.74 a 16.67 ab b
5.54 b
75.87 1.34 ab b
18.34 1.39 c

T5 ab
1.28 a 145.19 a 57.09 a 18.16 a b
5.66 ab
78.39 1.44 a ab
20.74 1.37 c

Mean 1.18 A 140.75 A 52.69 A 15.91 B a

5.46 a
69.07 1.27 A a
15.28 1.43 A

ANOVA Y * ** ** *** B
*** A
ns * A
ns ns

T *** *** *** *** *** *** *** *** ***

Y×T ns ns ns *
ns ns ns ns ns

1)
EC and CEC represent electrical conductivity and cation exchange capacity, respectively.
2)
T1, no N fertilization; T2, 100% inorganic N fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40%

inorganic N fertilizer+60% organic fertilizer; T5, 100% organic fertilizer.

Different lowercase letters between different treatments in the same year and different uppercase letters in the same

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 Journal of Integrative Agriculture
columns represent significance at 0.05 level according to the Least Significant Difference (LSD) test.

3.2. SPAD value

Partial organic fertilizer substitution for inorganic fertilizers significantly affected rice leaf SPAD value.
The leaf SPAD value under T3 was higher than that of the other treatments at each growth stage (Fig. 3).
The SPAD value of leaves showed a gradual downward trend from MT to PM, and the same trends were
observed in 2020 and 2021. The leaf SPAD values of T3 and T2 were significantly higher than those of
T1, T4, and T5 at each growth stage. Furthermore, the leaf SPAD value of T3 was significantly higher
than that of T2 at the heading and maturity stage; however, there was no significant difference between
T2 and T3 at the middle tillering and young panicle differentiation stages, and it was consistent in the
2020 and 2021. When compared with T2, The leaf SPAD values of T3 were increased by 2.5 and 1.4%
at the physiological maturity stage in 2020 and 2021. In the whole growth period of rice, the decline
rate of the leaf SPAD value of rice in the treatments with organic fertilizer (T3, T4, and T5) was relatively
gentle compared with that in the other treatments (T1 and T2).

Fig. 3 Changes in leaf SPAD value under substitution of inorganic fertilizer with a plant-based organic fertilizer in 2020

(A and B) and 2021 (C and D). T1, no N fertilization; T2, 100% inorganic N fertilizer; T3, 70% inorganic N fertilizer+30%

organic fertilizer; T4, 40% inorganic N fertilizer+60% organic fertilizer; T5, 100% organic fertilizer. YXHD and SXHN

represent the varieties of Youxianghongdao and Suixiangheinuo, respectively.

3.3. Plant height, tiller or panicle number and aboveground biomass

Partial organic fertilizer substitution for inorganic fertilizers significantly affected plant height, the number
of tillers or panicles, and aboveground biomass. The same trends were observed between the two
varieties and two years (Figs. 4 and 5). Plant height at MT and PI under different treatments was in the
sequence of T2=T3>T4= T5>T1, while at HD and PM, the sequence was T3=T2=T4>T5>T1, and the
results were consistent in the two years (Figs. 4-A and B, 5-A and B). In addition, the average plant
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height of SNHN was significantly higher than that of YXHD, and the average plant height in 2020 was
higher than that of 2021. The same results were observed in both varieties. Moreover, the tiller
number of each cultivar at MT and PI under different treatments was in the sequence of
T2=T3>T4=T5>T1, while the panicle number under T3 at HD and PM was higher than that of the other
treatments compared to T2, the panicle numbers of colored rice under T3 at HD and PM were increased
by 2.7 and 8.5% respectively, in 2020 , and by 6.7 and 7.7% in 2021 (Figs. 4-C and D, 5-C and D). The
tiller number and panicle number in each treatment showed a downward trend from MT to PM, and the
results were consistent in 2020 and 2021. Furthermore, the aboveground biomass of T3 in 2020 was
significantly higher than that of T2. Compared to T2, the aboveground biomass of YXHD and SXHN
under T3 increased by 3.9 and 10.4% in 2020. However, there was no significant difference between
T2 and T3 in 2021.

Fig. 4 Effects of substitution of inorganic fertilizer with plant-based organic fertilizer on plant height (A & B), tiller (C and
D), and aboveground biomass (E and F) in 2020 (A-F). Error bars are mean±SE. T1, no N fertilization; T2, 100% N
inorganic fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40% inorganic N fertilizer+60% organic
fertilizer; T5, 100% organic fertilizer. MT, PI, HD, and PM represent the middle tillering, panicle initiation, heading, and
physiological maturity stages, respectively. YXHD and SXHN represent the varieties of Youxianghongdao and
Suixiangheinuo, respectively. Different lowercase letters at the same stage represent a significant difference at a 0.05
level.

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Fig. 5 Effects of substitution of inorganic fertilizer with plant-based organic fertilizer on plant height (A and B), tiller (C

and D), and aboveground biomass (E and F) in 2021 (A-F). Error bars are mean±SE. T1, no N fertilization; T2, 100%

inorganic N fertilizer; T3, 70% N inorganic fertilizer+30% organic fertilizer; T4, 40% inorganic N fertilizer+60% organic

fertilizer; T5, 100% organic fertilizer. MT, PI, HD, and PM represent the middle tillering, panicle initiation, heading, and

physiological maturity stages, respectively. YXHD and SXHN represent the varieties of Youxianghongdao and

Suixiangheinuo, respectively. Different lowercase letters at the same stage represent a significant difference at 0.05,

according to the Least Significant Difference (LSD) test.

3.4. Nitrogen accumulation and utilization

Partial organic fertilizer substitution for inorganic fertilizers significantly impacted nitrogen accumulation,
AEN, REN, and PFPN at each growth stage. The highest values of these parameters were observed in
T3 across the two varieties and two years (Table 2). The nitrogen accumulation of T3 at MT, PI, HD,
and MS was significantly higher than in the other treatments, consistent between two years. Compared
with T2, the nitrogen accumulation of YXHN and SXHN in T3 increased by 9 and 8% in 2020 and by 11
and 8% in 2021. Moreover, compared with T2, the AEN, REN, and PFPN of YXHN at T3 increased in
2020 by 15, 18, and 5%, respectively, and SXHN increased by 45, 25, and 5% in 2021; the AEN, REN,
and PFPN of YXHN at T3 increased by 35, 18, and 8%, respectively, and SXHN by 28, 18, and 7%. In
addition, there was no interaction in AEN, REN, and PFPN among treatment, variety, and year, except that
REN was significantly influenced by the two-way interaction between treatments and year, and the
three-way interaction between season, treatment, and variety significantly influenced PFPN.

Table 2 Effects of substitution of inorganic fertilizer with plant-based organic fertilizer on nitrogen
accumulation of rice at different growth stages and nitrogen use efficiencies1)

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Nitrogen uptakes in different growth stages (kg
AE N RE N PFPN
Year Variety2) Treatment3) ha-1)4)
(kg kg-1) (%) (kg kg-1)
MT PI HD PM
2020 YXHD T1 8.00 d 19.06 e 44.61 e 59.67 e / / /
T2 16.75 b 42.32 b 85.23 b 109.96 b 11.82 b 41.91 b 37.26 b
T3 19.63 a 45.53 a 95.34 a 119.77 a 13.56 a 49.25 a 39.00 a
T4 17.06 b 36.70 c 77.76 c 98.94 c 7.27 c 32.72 c 32.71 c
T5 14.71 c 31.50 d 71.30 d 72.73 d 3.36 d 10.89 d 28.80 d
Mean 15.23 B 31.77 B 66.43 B 91.94 B 9.00 A 33.69 A 34.44 B
SXHN T1 12.37 e 25.45 e 45.06 e 68.33 e / / /
T2 19.95 b 56.79 b 107.24 b 126.83 b 10.92 b 48.74 b 37.93 b
T3 21.90 a 62.31 a 118.16 a 141.23 a 15.83 a 60.75 a 42.85 a
T4 18.26 c 50.98 c 96.94 c 108.45 c 8.05 c 33.43 c 35.07 c
T5 16.65 d 47.74 d 83.10 d 88.58 d 4.60 d 16.87 d 31.61 d
Mean 17.82 A 45.17 A 84.48 A 106.90A 9.85 A 39.95 A 36.86 A
2021 YXHD T1 8.77 d 20.61 c 40.95 e 73.59 e / / /
T2 16.11 b 45.60 a 108.66 b 137.74 b 9.98 b 53.46 b 42.68 b
T3 20.77 a 51.48 a 125.71 a 149.01 a 13.52 a 62.85 a 46.21 a
T4 15.96 bc 37.94 b 90.20 c 116.25 c 7.07 bc 35.55 c 39.76 c
T5 14.16 c 32.21 b 76.75 d 92.56 d 4.63 c 15.81 d 37.32 d
Mean 15.15 B 37.56 B 88.45 B 113.83 B 8.80 A 41.92 B 41.49 B
SXHN T1 13.16 c 20.05 e 52.00 e 78.19 e / / /
T2 18.23 b 52.69 b 130.38 b 154.74 b 11.36 ab 63.79 b 45.99 ab
T3 21.06 a 61.94 a 142.58 a 167.05 a 14.53 a 74.05 a 49.17 a
T4 17.99 b 46.91 c 112.05 c 125.99 c 8.04 b 39.84 c 42.68 b
T5 17.98 b 44.48 d 93.04 d 101.48 d 3.28 c 19.41 d 37.92 c
Mean 17.68 A 45.21 A 106.01 A 125.49 A 9.30 A 49.27 A 43.94 A

ANOVA Y Ns ns *** ***

ns *** ***

V *** *** *** ***

ns *** ***

T *** *** *** *** *** *** ***

Y×V ns ***
ns ns ns ns ns
V×T ** *** *** **
ns ns ns
Y×T *** * *** ***
ns **
ns
Y×V×T ns ns *** ***
ns *** ***

1)
AEN, agronomic N use efficiency; REN, apparent uptake efficiency of fertilizer N; PFPN, partial factor productivity of

fertilizer N. 2)
YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo, respectively.
3)
T1, no N fertilization; T2, 100% inorganic N fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40%

inorganic N fertilizer+60% organic fertilizer; T5, 100% organic fertilizer.

4)
MT, PI, HD and PM represent the middle tillering stage, panicle initiation stage, heading stage, and physiological

maturity stage, respectively.

Data followed by different lower-case letters among different treatments at the same variety and different upper-case

letters within one column at the same year denote significant differences at the 5% level according to the Least Significant

Difference (LSD) test.

3.5. Grain yield and its components

11
 Journal of Integrative Agriculture

Partial organic fertilizer substitution for inorganic fertilizers significantly affected grain yield and its
components. The yield gradually decreased with the increase in the application proportion of organic
fertilizer. The performance of the two varieties in both years was consistent (Table 3). Compared to T2,
grain yields of YXHD and SXHN under T3 were increased by 5 and 8%, respectively, in 2020 and by 13
and 7% in 2021. This was mainly due to the higher panicle number and total above-ground biomass.
The effective panicle number of YXHD under T3 was significantly higher than that under the other
treatments in 2020; however, there were no significant differences among all treatments in 2021. The
effective panicle number of SXHN was not significantly different between T2 and T3; however, the
effective panicle number of T3 was significantly higher than that of T4 and T5, which was consistent in
the two years. In addition, the number of spikelets per panicle was not significantly different between T2
and T3 across the two varieties and two years, except for that of SXHN in 2020. The number of
spikelets per panicle in T2 and T3 was significantly higher than in T4 and T5. In contrast, the
differences in the filled grain rate, 1000-grain weight, and harvest index among all treatments were
insignificant.
The average grain yields of YXHD (3.98 t ha-1) and SXHN (4.08 t ha-1) in 2020 were significantly
lower than those in 2021 (4.77 and 5.05 t ha-1) (Table 3). And no interaction was observed in grain
yield, panicles m-2, spikelets per panicle, filled grain rate, and harvest index between treatment and
variety, treatment and season, and treatment and variety and year, except for 1000-grain weight and
harvest index between year and treatment.

12
 Journal of Integrative Agriculture
Table 3 Effects of substitution of inorganic fertilizer with plant-based organic fertilizer on grain yield and
its components of colored rice
Grain Spikelets Filled
Panicles 1000-grain Harvest
Year Variety1) Treatment2) yield Spikelets/panicle grain
(no. m-2) weight (g) index
(t ha-1) (m-2×103) rate

2020 YXHD T1 3.05 e 129.2 e 149.3 a 19.3 c (%)

76.6 b 20.1 ab 0.50 ab
T2 4.47 b 168.9 b 151.2 a 25.5 a 79.2 ab 20.2 ab 0.51 a
T3 4.68 a 187.2 a 135.1 b 25.3 a 81.2 a 20.5 a 0.51 a
T4 3.93 c 154.0 c 142.3 ab 21.9 b 78.8 ab 20.5 a 0.46 b
T5 3.46 d 140.1 d 142.5 ab 19.9 bc 78.6 ab 19.9 b 0.49 ab
Mean 3.98 A 155.9 B 144.1 A 22.4 A 78.9 A 20.2 B 0.49 A
SXHN T1 3.24 e 168.2 c 101.2 a 17.0 b 69.6 b 25.7 b 0.43 a
T2 4.55 b 195.1 ab 100.9 a 19.6 a 75.2 ab 26.4 ab 0.43 a
T3 5.14 a 207.1 a 96.2 a 19.9 a 79 a 26.8 a 0.45 a
T4 4.21 b 184.7 bc 104.0 a 19.2 a 75.3 ab 26.4 ab 0.44 a
T5 3.79 d 178.2 bc 105.0 a 18.6 a 72.2 b 25.5 b 0.45 a
Mean 4.08 A 186.6 A 101.4 B 18.8 B 74.2 B 26.2 A 0.44 B
2021 YXHD T1 3.92 e 179.2 b 125.9 b 22.4 c 78.5 b 20.1 ab 0.45 a
T2 5.12 b 201.1 ab 142.4 a 28.4 a 80.2 ab 20.2 ab 0.41 b
T3 5.55 a 215.6 a 134.4 ab 29.0 a 84.0 a 20.5 a 0.43 ab
T4 4.77 c 197.9 ab 131.1 ab 25.9 b 80.9 a 20.5 a 0.43 ab
T5 4.48 d 191.7 ab 132.6 ab 25.3 b 80.2 ab 19.9 b 0.45 a
Mean 4.77 B 197.1 A 133.3 A 26.2 A 80.8 A 20.2 B 0.43 A
SXHN T1 4.16 e 149.0 b 125.7 ab 18.6 d 78.6 b 25.7 b 0.44 a
T2 5.52 b 189.6 a 122.8 ab 23.3 a 80.9 ab 26.4 ab 0.40 a
T3 5.90 a 205.2 a 116 b 23.7 a 83.4 a 26.8 a 0.40 a
T4 5.12 c 162.5 b 131.9 a 21.4 b 81.7 ab 26.4 ab 0.42 a
T5 4.55 d 151.1 b 132.3 a 19.9 c 80.8 ab 25.5 b 0.43 a
Mean 5.05 A 171.5 B 125.7 A 21.4 B 81.6 A 26.2 A 0.42 B
ANOVA Y *** *** *** *** *** *** ***

V ***
ns *** *** ** *** ***

T *** ***
ns *** ** ***
ns
Y×V ns *** *** * **
ns **

V×T ns ns ns *
ns ns ns
Y×T ns ns ns ns ns * *

Y×V×T ns ns ns ns ns ns ns
1)
YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo, respectively.
2)
T1, no N fertilization; T2, 100% inorganic N fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40%

inorganic N fertilizer+60% organic fertilizer; T5, 100% organic fertilizer.

Data followed by different lower-case letters among treatments of the same variety and upper-case letters within one

column at the same year denote significant differences at the 5% level according to the LSD test. *** **
, and * represents

the significant difference at the 0.1, 1, and 5% level according to the Least Significant Difference (LSD) test, respectively;

ns represents no significant difference.

3.6. Anthocyanin analysis

13
 Journal of Integrative Agriculture
With the increase in the proportion of organic fertilizer replacing inorganic fertilizer, the grain anthocyanin
contents of YXHD and SXHN showed a trend of increasing first and then decreasing in the two years
(Fig. 6). In addition, in YXHD, the grain anthocyanin content of YXHD was not significantly different
between T2 and T3; however, the grain anthocyanin content of YXHD under T3 was significantly higher
than that under T2. Moreover, the average grain anthocyanin contents of SXHN in 2020 and 2021 were
1044.4 and 393.1 μg g-1, respectively, which were significantly higher than those of YXHD (186.7 and
144.3 μg g-1). In addition, rice average grain anthocyanin content in 2020 was significantly higher than
in 2021.

Fig. 6 Effects of substitution of inorganic fertilizer with plant-based organic fertilizer on grain anthocyanin content of

YXHD and SXHN in 2020 (A) and 2021 (B). Error bars are mean±SE. T1, no N fertilization; T2, 100% inorganic N

fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40% inorganic N fertilizer+60% organic fertilizer; T5,

100% organic fertilizer. YXHD and SXHN represent the varieties of Youxianghongdao and Suixiangheinuo, respectively.

Different lowercase letters between different treatments at the same variety represent a significant difference at 0.05,

according to the Least Significant Difference (LSD) test. The break up is 250-900 and 210-290 in Fig. 6-A and B,

respectively.

3.7. Grain yield and its components of rice in the subsequent season

The application of organic fertilizer significantly improved the yield of subsequent rice and increased with
the increase of the ratio of organic fertilizers to inorganic fertilizers (Table 4). Compared to T2, rice
yields in the subsequent season under T5 treatment increased by 11% (2020) and 23% (2021),
respectively. The main reason for the increase in grain yield was that the aboveground biomass and
effective panicle number of subsequent rice under T5 were higher than those under T2. Among them,
the effective panicle number of the subsequent rice under the treatment of T5 was significantly higher
than that of T2 in 2020. Still, there was no significant difference between the two treatments in 2021.
The number of spikelets per panicle and the seed setting rate of subsequent rice under T5 were
significantly higher than those under T3. Other than that, the treatments had no significant differences
in 1000-grain weight and harvest index.

Table 4 Effects of substituting inorganic fertilizer with plant-based organic fertilizer on rice grain yield
and its components in the subsequent season

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 Journal of Integrative Agriculture

Treat- Grain yield Panicles Spikelets/pa Spikelets Filled grain 1000-grain Harvest
Year
Ment 1)
(t ha )
-1
m -2
nicle (m ×10 )
-2 3
rate (%) weight (g) index

2020 T1 5.18 b 238.6 b 123.1 a 29.2 a 77.7 a 20.5 a 0.43 a

T2 5.30 ab 233.3 b 126.6 a 29.5 a 78.9 a 20.5 a 0.45 a

T3 5.59 ab 239.6 b 135.7 a 32.6 a 77.0 a 20.1 a 0.47 a

T4 5.61 ab 253.1 ab 125.0 a 31.5 a 77.5 a 20.7 a 0.47 a

T5 5.88 a 262.5 a 124.6 a 32.7 a 79.5 a 20.4 a 0.46 a

Mean 5.51 A 245.4 A 127.0 A 31.1 A 78.1 A 20.5 A 0.46 B

2021 T1 4.75 c 190.6 a 153.3 ab 28.9 bc 71.8 b 20.6 a 0.50 a

T2 4.63 c 179.2 a 155.7 ab 27.6 c 74.6 ab 20.3 a 0.51 a

T3 4.91 bc 205.2 a 141.6 b 28.8 bc 76.6 a 20.1 a 0.52 a

T4 5.25 ab 205.2 a 155.7 ab 31.2 ab 75.3 ab 20.1 a 0.54 a

T5 5.70 a 203.1 a 164.9 a 33.4 a 76.5 a 20.0 a 0.53 a

Mean 5.05 A 196.7 B 154.2 A 30.0 A 74.9 A 20.2 A 0.52 A

ANOVA Y Ns *
ns ns ns ns **

T *** *
ns **
ns ns ns

Y×T Ns ns ns ns ns ns ns
1)
T1, no N fertilization; T2, 100% inorganic N fertilizer; T3, 70% inorganic N fertilizer+30% organic fertilizer; T4, 40%

inorganic N fertilizer+60% organic fertilizer; T5, 100% organic fertilizer. YXHD and SXHN represent the varieties of

Youxianghongdao and Suixiangheinuo, respectively.

Data followed by different lower-case letters among treatments at the same variety and upper-case letters within one

column at the same year denote significant differences at the 5% level according to the LSD test. , , and * represents
*** **

the significant difference at the 0.1, 1 and 5% level according to the Least Significant Difference (LSD) test, respectively;

ns represents no significant difference.

4. Discussion
4.1. Organic fertilizers promoted the sustainable supply of soil nutrients and improved
subsequent rice yields

Soil physio-chemical properties and nutritional status were significantly improved with the application of
organic fertilizers, which significantly increased the yield of subsequent rice crop in T5 treatment (Tables
1 and 4). Generally, the soil in Hainan province is acidic, with low organic matter content and weak
nutrient supply and retention capabilities, which reflects the soil conditions in tropical areas (Zhang et al.
2003). Usually, in Hainan province, rice has been intensively rotated with melons and vegetables,
which require more nutrients, accelerating the mineralization and decomposition of soil organic matter
(Luo and Luo 2001; Li et al. 2007; Jiang et al. 2019). This process exhausts soil organic matter and
depletes most soil nutrients, reducing soil fertility for future sustainable crop production. While organic
fertilizers can have long-term effects on soil nutrient content, which reflects the amount of nutrients

15
 Journal of Integrative Agriculture
available to crops in the current season and contributes to improved nutrient uptake in subsequent rice
seasons (Gutser et al. 2005; Cela et al. 2011; Peng et al. 2023). These findings support our results that
T5 outyielded T2, as shown in Table 4.

4.2. Organic-inorganic fertilizer system enhanced nitrogen accumulation and utilization

Coupling of 30% inorganic nitrogen with organic fertilizer (T3) significantly increased nitrogen
accumulation, REN, AEN, and PFPN compared with the application of only inorganic fertilizer (T2) (Table
2). On the one hand, this may be because inorganic fertilizers release nutrients rapidly, and organic
fertilizers release nutrients slowly and steadily, which results in more nitrogen in inorganic fertilizers
being absorbed and utilized by rice (Roba 2018). Applying organic fertilizers improves the ability to
preserve soil nutrients and eliminates the downward movement of minerals, thereby reducing nitrogen
leaching (Cai et al. 2016). In addition, partial organic fertilizer substitution for inorganic fertilizer can
improve nitrogen use efficiency compared to applying only inorganic fertilizer, which may be related to
improving soil quality and increasing nitrogen retention capacity (Mehasen et al. 2012) was consistent
with our result. Studies have shown that partial organic fertilizer substitution for inorganic fertilizers
improved the characteristics of the soil nitrogen supply, increased the content of soil mineral nitrogen,
promoted the rapid growth of microorganisms, and correspondingly improved the ability of
microorganisms to maintain nitrogen nutrients (Baaru et al. 2007; Ma et al. 2010). Moreover, some
studies have also reported that organic fertilizers combined with nitrogen fertilizers could balance crop
nutrient supply and demand and improve plant nutrient assimilation and nitrogen use (Liu et al. 2008;
Shang et al. 2014). Therefore, 30% organic fertilizer substitution for inorganic fertilizer was a highly
efficient nitrogen management model for colored rice.

4.3. Synergetic effects of organic-inorganic fertilizers on grain yield and anthocyanin

content in colored rice

In the present study, 30% inorganic N fertilizer substitution with organic fertilizer had a significant effect
on grain yield and grain anthocyanin content of colored rice; T3 treatment had the best performance
(Table 3; Fig. 6). It significantly improved root morphological traits (root length, surface area, diameter,
and volume), which was consistent with the results of Iqbal (2019) and Hayatu (2022). This may be
related to the significantly higher nitrogen accumulation and nitrogen use efficiency with the T3 treatment
than in the other treatments (Table 3). It has been documented that grain anthocyanin accumulation in
colored rice was regulated by grain nitrogen content (Chen et al. 2016). In addition, organic nitrogen
application upregulated gene expression in anthocyanin synthesis (Fongfon et al. 2021). However,
compared to T2, both the yield and the grain anthocyanin content of the T5 treatment showed a
downward trend. With the application of an augmented amount of organic nutrients substituting
nutrients in inorganic fertilizers showed a downward trend in the number of effective panicles per unit
area. This may be due to the fact that the more organic fertilizers in the soil, the limited release of
nutrients in the soil during decomposition, which affects the roots development and rice production (Xu
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 Journal of Integrative Agriculture
et al. 2008). Nitrogen from organic fertilizers often shows little effect on crop growth in the season of
application due to the slow-release characteristics of organic fertilizers (Gutser et al. 2005). In addition,
polysaccharides such as hemicellulose and cellulose in plant-based organic fertilizers and increased
levels of aromatic compounds in organic fertilizers decelerated the mineralization process (Sradnick and
Feller 2020).
The grain anthocyanin of SXHN in 2020 was significantly higher than those in 2021 (Fig. 6). Low
temperature and short periods of sunshine were beneficial for grain anthocyanin accumulation in colored
rice (Zhang 2006). Studies have shown that light conditions regulate the expression of anthocyanin
synthesis genes, and weak light conditions decrease the synthesis of grain anthocyanin (Albert et al.
2009). The solar radiation and daily average temperature during the grain-filling period of 2020 were
significantly lower than those of 2021 (Fig. 2). Therefore, the difference in grain anthocyanin of SXHN
between the two years may be related to temperature and solar radiation. In addition, the average grain
yield of YXHD (3.98 t ha-1) and SXHN (4.08 t ha-1) in 2020 was significantly lower than that in 2021 (4.77
and 5.05 t ha-1, respectively) (Table 3). It was reported that low solar radiation during the rice growth
stage could interfere with the re-accumulation of photosynthetic products, decreasing grain yield (Mauro
et al. 2011; Liu et al. 2019). In this study, the total solar radiation increased by 66.1% in 2021
compared to 2020, and the filled grain rate in 2021 was significantly higher than that in 2020 (Fig. 2;
Table 3). Therefore, the differences in grain yield of the two varieties between the two years may be
closely related to the difference in solar radiation.

5. Conclusion

In terms of grain yield, grain anthocyanin content, and N use efficiency of colored rice in the current
season under organic fertilizer incorporation, the best performance was observed under T3 treatment.
While partial substitution of inorganic fertilizer with organic fertilizer improved soil physio-chemical
properties and thus increased the rice grain yield in the subsequent seasons. The highest grain yield of
subsequent rice was observed under the T5 treatment. Results suggested that the application of
plant-based organic fertilizers sustained the production of colored rice in tropical regions. However, it is
still unknown how long the residual effect of organic fertilizer last, and the mechanisms underlying the
residual effect of plant-based organic fertilizer, especially the microbial mechanism, should be addressed
in the future.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (32060430 and
31971840) and the Research Initiation Fund of Hainan University, China (KYQD (ZR) 19104).

Declaration of competing interest

The authors declare that they have no conflict of interest.

17
 Journal of Integrative Agriculture
References

Albert N W, Lewis D H, Zhang H, Irving L J, Jameson P E, Davies K M. 2009. Light-induced vegetative

anthocyanin pigmentation in Petunia. Journal of Experimental Botany, 60, 2191-2202.
Asghar W, Akça M O, Akça H, Tarf O J, Kataoka R, Turgay O C. 2022. Alternative strategies to synthetic
chemical fertilizers, revitalization of soil quality for sustainable agriculture using organic-based
approaches. In: Gupta V K, ed., New and Future Developments in Microbial Biotechnology and
Bioengineering. Elsevier, The Netherlands. pp. 1-30.
Baaru M W, Mungendi D N, Bationo A, Verchot L, Waceke W. 2007. Soil microbial biomass carbon and
nitrogen as influenced by organic and inorganic inputs at Kabete, Kenya. In: Bationo A, Waswa B,
Kihara J, Kimetu J, ed., Advances in Integrated Soil Fertility Management in Sub-Saharan Africa:
Challenges and Opportunities. Springer, Netherlands Dordrecht. pp. 827-832.
Bremner J M, Tabatabai M A. 1972. Use of an ammonia electrode for determination of ammonium in
Kjeldahl analysis of soils. Communications in Soil Science and Plant Analysis, 3, 159-165.
Cai A, Xu H, Shao X, Zhu P, Zhang W, Xu M, Murphy D V. 2016. Carbon and nitrogen mineralization in
relation to soil particle-size fractions after 32 years of chemical and manure application in a
continuous maize cropping system. PLoS ONE, 11, e0152521.
Cela S, Santiveri F, Lloveras J. 2011. Residual effects of pig slurry and mineral nitrogen fertilizer on
irrigated wheat. European Journal of Agronomy, 34, 257-262.
Chen B, Ao H J, Zeng X S. 2021. Analysis of the changes in sown area and yield of rice in China from
2009 to 2018. Journal of Hunan Agricultural University, 47, 495-500.
Chen L, Huang Y, Xu M, Cheng Z, Zhang D, Zheng J. 2016. iTRAQ-based quantitative proteomics
analysis of black rice grain development reveals metabolic pathways associated with anthocyanin
biosynthesis. PLoS ONE, 11, e0159238.
Chen X, Wo F, Chen C, Fang K. 2010. Seasonal changes in the concentrations of nitrogen and
phosphorus in farmland drainage and groundwater of the Taihu Lake region of China. Environmental
Monitoring and Assessment, 169, 159-168.
Cui X, Guo L, Li C, Liu M, Wu G, Jiang G. 2021. The total biomass nitrogen reservoir and its potential of
replacing chemical fertilizers in China. Renewable and Sustainable Energy Reviews, 135, 110215.
FAO (Food and Agriculture Organization of the United Nations). 2004. Rice and Human Nutrition. Food
and Nutrition Division.
FAO (Food and Agriculture Organization of the United Nations). 2018.
Fongfon S, Prom-U-Thai C, Pusadee T, Jamjod S. 2021. Responses of purple rice genotypes to nitrogen
and zinc fertilizer application on grain yield, nitrogen, zinc, and anthocyanin concentration. Plants, 10,
1717.
FAPRC (Forestry Administration of the People's Republic of China). 2015. LY/T 1234-2015
Determination of Potassium in Forest Soil. China Standards Press, China.
Galloway J N, Townsend A R, Erisman J W, Bekunda M, Cai Z, Freney J R, Martinelli L A, Seitzinger S P,
Sutton M A. 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential
solutions. Science, 320, 889-892.
18
 Journal of Integrative Agriculture
Geng Y, Cao G, Wang L, Wang S. 2019. Effects of equal chemical fertilizer substitutions with organic
manure on yield, dry matter, and nitrogen uptake of spring maize and soil nitrogen distribution. PLoS
ONE, 14, e0219512.
Grassini P, Eskridge K M, Cassman K G. 2013. Distinguishing between yield advances and yield
plateaus in historical crop production trends. Nature Communications, 4, 2918.
Gutser R, Ebertseder T, Weber A, Schraml M, Schmidhalter U. 2005. Short‐term and residual
availability of nitrogen after long‐term application of organic fertilizers on arable land. Journal of
Plant Nutrition and Soil Science, 168, 439-446.
Hayatu N G, Liu Y R, Han T F, Daba N A, Zhang L, Shen Z, Li J W, Muazu H, Lamlom S F, Zhang H M.
2022. Carbon sequestration rate, nitrogen use efficiency and rice yield responses to long-term
substitution of chemical fertilizer by organic manure in a rice-rice cropping system. Journal of
Integrative Agriculture.
Hendershot W H, Duquette M. 1986. A simple barium chloride method for determining cation exchange
capacity and exchangeable cations. Soil Science Society of America Journal, 50, 605-608.
Iqbal A, He L, Khan A, Wei S, Akhtar K, Ali I, Ullah S, Munsif F, Zhao Q, Jiang L. 2019. Organic manure
coupled with inorganic fertilizer: An approach for the sustainable production of rice by improving soil
properties and nitrogen use efficiency. Agronomy, 9, 651.
Iqbal S, Bhanger M I, Anwar F. 2005. Antioxidant properties and components of some commercially
available varieties of rice bran in Pakistan. Food Chemistry, 93, 265-272.
Jiang S, Zhang R, Zhang W, Xu A, Zhang H, Xie L, Ji H. 2019. Spatial and temporal variation of soil
organic matter and cause analysis in Hainan Island in resent 30 years. Scientia Agricultura Sinica, 52,
1032-1044.
Ju X T, Xing G X, Chen X P, Zhang S L, Zhang L J, Liu X J, Cui Z L, Yin B, Christie P, Zhu Z L, Zhang F S.
2009. Reducing environmental risk by improving N management in intensive Chinese agricultural
systems. Proceedings of the National Academy of Sciences of the United States of America, 106,
3041-3046.
Kilcher L. 2007. How organic agriculture contributes to sustainable development. Journal of Agricultural
Research in the Tropics and Subtropics, Supplement, 89, 31-49.
Krishnan V, Rani R, Awana M, Pitale D, Kulshreshta A, Sharma S, Bolinedi H, Singh A, Songh B, Singh A
K, Praveen S. 2021. Role of nutraceutical starch and proanthocyanidins of pigmented rice in
regulating hyperglycemia: Enzyme inhibition, enhanced glucose uptake and hepatic glucose
homeostasis using in vitro model. Food Chemistry, 335, 127505.
Kusano M, Yang Z, Okazaki Y, Nakabayashi, R, Fukushima A, Saito K. 2015. Using metabolomic
approaches to explore chemical diversity in rice. Molecular Plant, 8, 58-67.
Lazcano C, Gómez-Brandón M, Revilla P, Domínguez J. 2013. Short-term effects of organic and
inorganic fertilizers on soil microbial community structure and function. Biology and Fertility of Soils,
49, 723-733.
Li Z P, Wu X C, Chen B Y. 2007. Changes in transformation of soil organic carbon and functional
diversity of soil microbial community under different land use patterns. Agricultural Sciences in China,
6, 1235-1245.
19
 Journal of Integrative Agriculture
Liao H, Zhang Y, Zuo Q, Du B, Chen W, Wei D, Huang Q. 2018. Contrasting responses of bacterial and
fungal communities to aggregate-size fractions and long-term fertilizations in soils of northeastern
China. Science of the Total Environment, 635, 784-792.
Liu H W, Zhang X K, Zhang G Z, Kou X C, Liang W J. 2022. Partial organic substitution weakens the
negative effect of chemical fertilizer on soil micro-food webs. Journal of Integrative Agriculture, 21,
3037-3050.
Liu J, Xie Q, Shi Q, Li M. 2008. Rice uptake and recovery of nitrogen with different methods of applying
15N-labeled chicken manure and ammonium sulfate. Plant Production Science, 11, 271-277.
Liu K, Yang R, Lu J, Wang X, Lu B, Tian X, Zhang Y. 2019. Radiation use efficiency and source-sink
changes of super hybrid rice under shade stress during grain-filling stage. Agronomy Journal, 111,
1778-1798.
Liu Y, Sun D, Wang H, Wang X, Yu G, Zhao X. 2020. An evaluation of China's agricultural green
production: 1978-2017. Journal of Cleaner Production, 243, 118483.
Ma S C, Li F M, Xu B C, Huang Z B. 2010. Effect of lowering the root/shoot ratio by pruning roots on
water use efficiency and grain yield of winter wheat. Field Crops Research, 115, 158-164.
Mauro R P, Occhipinti A, Longo A M G, Mauromicale G. 2011. Effects of shading on chlorophyll content,
chlorophyll fluorescence and photosynthesis of subterranean clover. Journal of Agronomy and Crop
Science, 197, 57-66.
Mehasen S A S, Gebaly S G, Seoudi O A. 2012. Effectiveness of organic and inorganic fertilization in
presence of some growth regulators on productivity and quality of Egyptian cotton. Asian Journal of
Biological Sciences, 5, 171-182.
Pan Y, Guo J, Fan L, Ji Y, Liu Z, Wang F, Pu Z, Ling N, Shen Q, Guo S. 2022. The source-sink balance
during the grain filling period facilitates rice production under organic fertilizer substitution. European
Journal of Agronomy, 134, 126468.
Peng G, Zhang T, Lei X Y, Cui X W, Lu Y X, Fan P F, Long S P, Huang J, Gao J S, Zhang Z H, Zhang H M.
2023. Improvement of soil fertility and rice yield after long-term application of cow manure combined
with inorganic fertilizers. Journal of Integrative Agriculture.
Roba T B. 2018. Review on: The effect of mixing organic and inorganic fertilizer on productivity and soil
fertility. Open Access Library Journal, 5, e4618.
Shang Q, Gao C, Yang X, Wu P, Ling N, Shen Q, Guo S. 2014. Ammonia volatilization in Chinese double
rice-cropping systems: a 3-year field measurement in long-term fertilizer experiments. Biology and
Fertility of Soils, 50, 715-725.
Sradnick A, Feller C. 2020. A typological concept to predict the nitrogen release from organic fertilizers in
farming systems. Agronomy, 10, 1448.
Strickland T C, Sollins P. 1987. Improved method for separating light- and heavy-fraction organic
material from soil. Science Society of America Journal, 51, 1390-1393.
Luo W, Luo W. 2001. Farming system reform of rice field in Hainan. Journal of Hainan University, 19,
71-75.
Xu F, Liu Y, Du W, Li C, Xu M, Xie T, Yin Y, Guo H. 2021. Response of soil bacterial communities,
antibiotic residuals, and crop yields to organic fertilizer substitution in North China under wheat-maize
20
 Journal of Integrative Agriculture
rotation. Science of the Total Environment, 785, 147248.
Xu M G Li D C Li J M. 2008. Effects of organic manure application combined with chemical fertilizers on
nutrients absorption and yield of rice in Hunan of China. Scientia Agricultura Sinica, 41, 3133-3139.
Yawadio R, Tanimori S, Morita N. 2007. Identification of phenolic compounds isolated from pigmented
rices and their aldose reductase inhibitory activities. Food Chemistry, 101, 1616-1625.
Ye L, Zhao X, Bao E, Li J, Zou Z, Cao K. 2020. Bio-organic fertilizer with reduced rates of chemical
fertilization improves soil fertility and enhances tomato yield and quality. Scientific Reports, 10, 1-11.
Yuan L. 2014. Development of hybrid rice to ensure food security. Rice Science, 21, 1-2.
Zhang H, Zhang G L, Qi Z P, Zhao Y G. 2003. Systematic evaluation of soil quality status at farm scale in
tropical regions. Journal of Soil Science, 02, 186-193.
Zhang M W, Guo B J, Zhang R F, Chi J W, Wei Z C, Xu Z H, Zhang Y, Tang X J. 2006. Separation,
purification and identification of antioxidant compositions in black rice. Agricultural Sciences in China,
5, 431-440
Zhang M, Tian Y, Zhao M, Yin B, Zhu Z. 2017. The assessment of nitrate leaching in a rice-wheat rotation
system using an improved agronomic practice aimed to increase rice crop yields. Agriculture,
Ecosystems & Environment, 241, 100-109.
Zhang M, Yao Y, Tian Y, Ceng K, Zhao M, Yin B. 2018. Increasing yield and N use efficiency with organic
fertilizer in Chinese intensive rice cropping systems. Field Crops Research, 227, 102-109.
Zhang Y J, Gao W, Luan H A, Tang J W, Li R N, Li M Y, Zhang H Z, Huang S W. 2022. Effects of a
decade of organic fertilizer substitution on vegetable yield and soil phosphorus pools, phosphatase
activities, and the microbial community in a greenhouse vegetable production system. Journal of
Integrative Agriculture, 21, 2119-2133.
Zhao M, Tian Y, Ma Y, Zhang M, Yao Y, Xiong Z, Yin B, Zhu Z. 2015. Mitigating gaseous nitrogen
emissions intensity from a Chinese rice cropping system through an improved management practice
aimed to close the yield gap. Agriculture, Ecosystems & Environment, 203, 36-45.

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