Applied Soil Ecology 165 (2021) 103966

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Applied Soil Ecology

journal homepage: www.elsevier.com/locate/apsoil

Chemical fertilizer reduction with organic fertilizer effectively improve soil

fertility and microbial community from newly cultivated land in the Loess
Plateau of China

Jianqiao Han a,b, Yunyun Dong a,b, Man Zhang a,b,*

a Institute of Soil and Water Conservation, Northwest A&F University, Yangling,

Shanxi 712100, PR China
b Institute of Soil and Water Conservation, Chinese Academy of Sciences and
Ministry of water Resources, Yangling, Shanxi 712100, PR China

A R T I C L E I N F O

Keywords: Organic fertilizer Soil fertility

Microbial community Newly cultivated soil Loess Plateau
A B S T R A C T

Improving the soil quality of cultivated land generated by the Gully Land
Consolidation Project could promote sustainable economic and ecological
development in the hilly gully area of the Loess Plateau, China. A pot
experiment with maize crop was conducted to assess the effects of different
organic amendments and chemical fertilizer reduction on soil fertility and the
microbial community in newly cultivated land, of which the amount
of chemical fertilizer applied was half of the local conventional maize field (90
kg ha 1 of CO(NH2)2, 45 kg ha 1
of P2O5, and 60 kg ha 1 of K2O). The experiment involved six treatments: bare
soil (CK), only maize (M), maize
+ chemical fertilizer (MF), maize + chemical fertilizer + organic fertilizer
(MFO), maize + chemical fertilizer + biochar (MFB), and maize + chemical
fertilizer + polyacrylamide (MFP). The results are as follows: (1) compared with
the MF treatment, the MFO treatment exhibited the best effect on improving
soil physicochemical prop- erties, such as electrical conductivity (EC), soil
organic carbon (SOC), nitrate nitrogen (NO -N), total nitrogen (TN), available
phosphorus (AP) and available potassium (AK), followed by MFB and MFP. (2)
The MFO treatment significantly (P < 0.05) improved the Species richness and
Shannon indices of the bacteria, but its effects on fungal diversity index are
not significant. (3) The MFO treatment increased the relative abundance of
Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteria and Gemmatimonadetes
and decreased the relative abundance of Sordariomycetes and Agaricomycetes, and
it also increased the functional abundance of Amino acid metabolism, Membrane
transport, Xenobiotics biodegradation and metabolism, Undefined Sapro- trophs and
Arbuscular Mycorrhizal Fungi. (4) The TN and EC were the main driving
factors of bacterial com- munity structural variation, while TN and NO -N were
the main factors for variation of fungal community structure. (5) The fresh
weight and dry weight of maize increased significantly (P < 0.05) under all
amendment treatments, among which the MFO treatment had the greatest effect. In
conclusion, organic fertilizer application is the most effective measure for
rapidly improving soil fertility and microbial community structure. Our findings
have valuable implications for agricultural land productivity, food security and
long-term sustainability of the environment.

1. Introduction

Abundant farmland and sustainable crop production is needed to feed the ever-
growing population. However, the agriculture productiv- ity is consistently on
decline due to severe vegetation degradation and soil erosion in the Chinese
Loess Plateau (Wang et al., 2016; Zhao et al.,
2019). Especially from 2000 to 2008, the huge high-quality farmland decreased
in the Chinese Loess Plateau due to the Green for Grain Project and rapid
urbanization (Lü et al., 2012; Zhou et al., 2019). To
increase cultivated farmland, the Gully Land Consolidation Project, with a total
construction area of 33,700 ha was implemented in Yan’an city located in the
middle of the Chinese Loess Plateau from 2013 to 2017 (Zhao et al., 2019).
However, the newly cultivated soil sourced from deep soil of the slopes has low
economic benefit for farmers due to poor fertility (Liu et al., 2013).
Therefore, improvement in the field managing technologies is urgently needed to
increase soil fertility and thereby increase crop productivity in such newly
cultivated soil.
Numerous studies have shown that balanced application of inorganic

* Corresponding author at: No.26 Xinong Road, Institute of Soil and Water
Conservation, Northwest A&F University, Yangling, Shanxi 712100, PR China.
E-mail address: zmcocoo0203@nwsuaf.edu.cn (M. Zhang).

https://doi.org/10.1016/j.apsoil.2021.103966
Received 21 October 2020; Received in revised form 9 February 2021; Accepted 24
February 2021
Available online 10 March 2021
0929-1393/© 2021 Elsevier B.V. All rights reserved.
J. Han et al.
Applied Soil Ecology 165 (2021) 103966

and organic amendments can increase SOM and maintain soil produc- tivity
(Arancon et al., 2006; Blair et al., 2006; Gong et al., 2009). The reduction of
chemical fertilization and increase of organic nutrients sources into the soil
is a sustainable approach for improving soil phys- ical, chemical and biological
properties (Girmay et al., 2008; Zhang et al., 2015). In recent years,
such practices are very important to enhance soil fertility and crop
productivity in soils with intrinsically low soil fertility. Biochar, it has been
reported that have many positive ef- fects on improving soil quality. As has
been demonstrated, biochar could improve soil nutrient retention, water holding
capacity, soil pH, EC and SOC (Ghezzehei et al., 2014). The combination of biochar
and fertilizer could increase soil C stability in soil with low native
organic matter contents (Madiba et al., 2016; Fahad et al., 2016). Therefore,
biochar could act as a soil conditioner, enhances plant growth by supplying
nutrients efficiently and increases crop yields (Spokas et al., 2012).
Moreover, soil microbial biomass and activity significantly increase with biochar
application (Domene et al., 2015; Bashir et al., 2019). In addi- tion, Organic
fertilizer, also has a remarkable effect on SOC, soil bulk density and crop yield
(KhHM and Fadni, 2013). Soil P, K, and organic matter increase linearly with the
rates of cattle manure application (Schlegel, 1992). Combined application of
organic and inorganic fer- tilizers could accelerate the microbial growth, alter
the structure of the soil microbial community and increase the enzyme activity
(Lazcano et al., 2013). Whether the chemical fertilizers are applied or not,
organic fertilizer application has positive effects on bacterial and fungal
di- versity (Kamaa et al., 2011). Polyacrylamide (PAM), which is an
important water-soluble polymer, has been widely used in soil in recent years.
PAM application obviously improves the soil structure, soil organic
matter and soil moisture (He et al., 2008). The number of cultivable
heterotrophic bacteria increases significantly after adding PAM to the soil
(Kay-Shoemake et al., 1998). Furthermore, PAM has been proven to facilitate the
survival and growth of specific fungal and bacterial species (Caesar-TonThat et
al., 2008).
Soil microbial communities are important indicators of soil quality (Schloter et
al., 2018; García-Delgado et al., 2019). Soil microbes have a critical role in
soil organic matter decomposition and nutrient cycling in ecosystems, determining
the nutrient utilization efficiency (Kallenbach et al., 2016; Cui et al., 2019).
Moreover, soil microbes, which directly or indirectly participate in the process
of soil material flow and energy flow, have obvious effects on the
maintenance and recovery of land productivity (Reinhart et al., 2016;
George et al., 2019). Thus, improving the soil nutrient and microbial
community is a key challenge in improving the utilization rate of newly cultivated
land.
From what has been discussed above, few studies have been con- ducted on
the combined effect of different organic amendments and chemical fertilizer
reduction on the newly land, especially on their
27.95¢¢ E). The soil used in this experiment was collected from the newly
cultivated land in Yan’an city, Shanxi Province. Soil samples were
thoroughly mixed, air dried, and sieved through a 2 mm sieve. The soil properties
are shown in Table 1. The maize cultivar was Shandan 650, which were sown in
plastic basins with an inner diameter of 25 cm at the bottom, an inner diameter of
30 cm at the top and a height of 28 cm. The fertilizer and 25 kg of soil were mixed
evenly and placed into the basins. Local traditional measures were used to
manage these pots. The PAM, white granular polyacrylamide, was purchased from
Dongying Huaye New Material Co., Ltd. The organic fertilizer was pig manure
compost obtained from farmers. Biochar was generated from apple tree branches
subjected to 600 ◦C under anaerobic conditions and ground into a 1 mm
sieve. Chemical fertilizers were urea (46% N), calcium superphosphate (16% P2O5)
and potassium sulfate (52% K2O), which were provided by Shaanxi Yixin
Biotechnology Development Co., Ltd. The properties of biochar are shown in Table
1.

2.2. Experimental design and sampling

The dosage of chemical fertilizer reduction were 90 kg ha 1 of CO (NH2)2, 45 kg

ha 1 of P2O5, and 60 kg ha 1 of K2O, which is half that of the local conventional
maize field. Three replicates of six treatments were included in this study:
blank soil (CK), only maize (M), maize + chemical fertilization (MF), maize +
chemical fertilization + organic fertilization (MFO), maize + chemical
fertilization + biochar (MFB), and maize + chemical fertilization +
polyacrylamide (MFP). Consistent chemical fertilizers were used in these
treatments to reflect the effects of
organic fertilization, biochar and PAM. Details of the experimental design
are presented in Table 2.
Three replicate soil samples were collected prior to the harvest of the maize
plants at each treatment. Soil collected from six sampling point (0–20 cm) were
mixed as the soil samples for each replicate. Half of soil samples were air dried
to measure the physical and chemical properties, the remaining soil from each
replicate was sealed in a polythene plastic bag and stored in a 80 ◦C
refrigerator for microbial analysis.
The maize was harvested on October 20, 2019. First, the whole maize plant was cut
into small sections with scissors and then placed into a file bag to obtain the
fresh weight. Finally, the file bag was placed into an oven in the laboratory
and dried at 85 ◦C to obtain the dry weight.

2.3. Analysis of soil physicochemical properties

Soil pH was measured using Potentiometric method with a ratio of

1:2.5 (w/v). The soil EC was measured by electromagnetic induction (EM) using
a EM-38 meter (Brevik et al., 2004). The SOC was measured using the dichromate
oxidation technique (Sanmanee and Suwannaoin,
+
application under the Gully Land Consolidation Project. Herein, we
conducted a pot experiment to assess the effects of different organic
amendments combined with reduced chemical fertilizer on soil physi- cochemical
properties, maize biomass and microbial community. The objectives of this study
were to (1) asses the effects of different organic amendments plus chemical
fertilizer reduction on soil nutrients, crop biomass, microbial diversity and
composition, and microbial commu- nity structure (bacteria and fungi) and (2)
identify the relationship be- tween the soil physicochemical properties and
the communities of bacterial and fungal. These results are valuable for the
efficient utili- zation of new land and food security and provide support for
the long- term sustainability of the environment.

2. Materials and methods

2.1. Experimental detail

2009). The NO3 -N and NH4 -N were measured using an AA3 continuous-
flow autoanalyzer (Dafner, 2015). The TN was measured using an automatic
Kjeldahl instrument (Dinuccio et al., 2010). The AP was determined according
to the Olsen method (Iatrou et al., 2014). The AK was measured in 1.0 M NH4OAc
extracts by flame photometry (Pan et al., 2019).

Table 1
The basic properties of soil and biochar used in the field experiment.

Items
Soil Biochar
Soil organic carbon (SOC, g kg 1) 1.28
567.36
Total nitrogen (TN, g kg 1) 0.21
9.72
Available phosphorus (AP, mg kg 1) 3.30
19.33
Available potassium (AK, mg kg 1) 66.42
39.51
1
Nitrate nitrogen (NO3 -N, mg kg
) 1.05 5.33
A maize pot experiment on soil organic amendments was conducted
from April 28 to October 20, 2019 in a Laboratory at Northwest A&F University,
located in Yangling, Shaanxi (34◦ 16¢ 56.24¢¢ N, 108◦ 4¢
Ammonium nitrogen (NH+-N, mg kg 1) 0.27
2.46
pH (H2O) 8.97
9.81
 electrical conductivity (EC, μS cm ←1) 152.74
179.64

2
J. Han et al.
Applied Soil Ecology 165 (2021) 103966

Table 2
Detailed design of each test treatment.
Treatment Crop Biochar (t/hm2) Polyacrylamide
(kg/hm2) Organic fertilizer (t/hm2) Fertilizer
P2O5 (kg/hm2) CO(NH2)2 (kg/hm2) K2O (kg/hm2) CK
– – –
– –
– –
M Maize – –
– –
– –
MF Maize – –
– 45
90 60
MFO Maize – –
15 45
90 60
MFP Maize – 10
– 45
90 60
MFB Maize 15 –
– 45
90 60

2.4. DNA extraction, amplification, sequencing

First, the soil DNA was extracted from 0.25 g of frozen soil using the PowerSoil
DNA Isolation Kit (MoBio Laboratories). The concentration and purity of the
extracted DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo
Scientific, Waltham, MA, USA), and the DNA quality was checked with 1%
agarose gel electrophoresis. The resulting purified DNA was stored at 80 ◦C
until further assaying and processing. Then, a fragment of the bacterial 16S
rRNA gene (V3–V4 region) was amplified by PCR with the universal primers
338F (5¢- ACTCCTACGGGAGGCAGCA-3¢) and 806R (5¢-GGACTACHVG
GGTWTCTAAT-3¢). The ITS1 region of the fungal 18S rRNA gene was amplified using
the primers ITS5-1737F (5¢-GGAAGTAAAAGTCGTAA- CAAGG-3¢) and ITS2-2043R (5¢-
GCTGCGTTCTTCATCGATGC-3¢). The first round of the PCR amplification was performed
in a total volume of
50 μl, which contained 10 μl of buffer, 0.2 μl of Q5 High-Fidelity DNA
Polymerase, 10 μl of High GC Enhancer, 1 μl of dNTP, 10 μM of each primer and 60
ng of genome DNA. The thermal cycling conditions were as follows: initial
denaturation at 95 ◦C for 5 min, followed by 15 cycles at 95 ◦C for 1 min, 50 ◦C
for 1 min and 72 ◦C for 1 min, with a final extension at 72 ◦C for 7 min. The
PCR products from the first step of the PCR were purified with VAHTSTM DNA Clean
Beads. A second round of PCR was then performed in a 40 μl reaction that
contained 20 μl of 2 ´ Phμsion HF MM, 8 μl of ddH2O, 10 μM of each primer and 10
μl of the PCR products from the first step. The thermal cycling conditions were as
follows: initial denaturation at 98 ◦C for 30 s, followed by 10 cycles at
98 ◦C for 10 s, 65 ◦C for 30 s min and 72 ◦C for 30 s, with a final extension
at 72 ◦C for 5 min. The PCR products obtained from the two rounds of PCR were
quantified with Quant-iT™ dsDNA HS Reagent and pooled together. Finally, high-
throughput sequencing analysis of the bacterial rRNA genes was performed with
the purified pooled sample using the Illumina HiSeq 2500 platform (2 ´ 250
paired ends) at Biomarker Technologies Corporation, Beijing, China. All raw
sequencing data were deposited into the NCBI Sequence Read Archive
under accession numbers PRJNA682543 (Fungi) and PRJNA682542 (Bacteria).

2.5. Sequencing data processing

Quality filtering of the raw tags was performed under specific

filtering conditions to obtain high-quality clean tags according to the QIIME
1.9.1 quality-control process (Deng et al., 2019). The sequences with the same
barcode were sorted into the same sample. Bacterial and fungal tags were
compared with those in reference databases (Gold database,
http://drive5.com/uchime/uchime_download.html, and Unite database,
https://unite.ut.ee/, respectively) using the UCHIME algorithm to detect
chimera sequences, and the chimera sequences were removed (Caporaso et al., 2010).
Then, the effective tags were obtained. The remaining sequences were clustered
with UPARSE software and assigned to OTUs at similarities of 97% (Edgar,
2013). The bacterial taxonomic identity was determined using the Silva 123 SSU
Ref data- base (http://www.arb-silva.de) via the RDP classifier, and fungi
were identified using the Unite v.7 database (https://unite.ut.ee/) with the
BLAST tool and QIIME software (http://qiime.org/index.html). Finally, a total of
1,269,795 bacterial sequences (on average, 133,663 per
sample) and 1,289,291 fungal sequences (on average, 135,714 per sample) were
obtained (Table S1). Relatively high Good’s coverage values ranging from
0.998 to 0.999 were obtained (Table S1). Among them, the average lengths of
the sequences of the bacteria and fungi were 421 bp and 258 bp, respectively.
To better analyze the potential functional contributions of the observed
microorganisms in the soils under the different improvement treatments, we used
16S rRNA gene sequences to calculate the metabolic cycles and pathways with the
Kyoto Encyclopedia of Genes and Ge- nomes (KEGG) and the Tax4fun package in R
3.4.3. This technique maps partial 16S rRNA gene sequences to SILVA (SILVA SSU Ref
NR database release 125 and KEGG database release 87.0) reference phylogenies
to predict potential functional profiles with QIIME 1.9.1. After the func-
tional assignment, the KEGG pathways were summarized in R with the Tax4Fun
package (Aßhauer et al., 2015). In addition, the level 1-to-level
3 subsystem of the KEGG orthologous database was downloaded for further
analysis. Meanwhile, we classified the fungal OTUs into different functional groups
according to the method of Eldridge and Delgado- Baquerizo (2018).

2.6. Statistical analysis

All statistical analyses were conducted in R software version 3.4.3. One-way

analysis of variance was used to evaluate the effects of the different
improvement measures on the soil physicochemical properties and microbial
diversity. We used Duncan’s test (P < 0.05) and the duncan.test function in
the AGRICOLAE package for multiple compari- sons. Logarithmic transformation of
the data was necessary to improve normality (Shapiro-Wilk test) and
homogeneity (Bartlett’s test). In addition, we used principal coordinate
analysis (PCoA) to assess the differences in improvement measures based on
Bray-Curtis distance at the OTU level, and then the significance of the
differences was tested with permutational multivariate analysis of variance
(PERMANOVA). The similarity percentages of microbial community composition be-
tween the different treatments were calculated by Bray-Curtis dissimi-
larities. A Bray-Curtis distance-based Mantel test was used to determine the
relationship between microbial community compositions and soil properties at the
OTU level. Finally, we identified the main driving factors of changes in
microbial community structure with redundancy analysis (RDA) and multivariate
regression tree (MRT) analysis.

3. Results

3.1. Variations in the maize biomass and soil physicochemical characteristics

Compared with M treatment, all fertilization treatments significantly increased

the maize biomass, whether wet weight or dry weight (Table 3). During
the growing season, the fresh weight and dry weight of maize under the all
organic amendment treatments were significantly greater than that of MF
treatment. The fresh weight of the MFO and MFB were significantly higher than
MFP treatment. Furthermore, the dry weight under MFO was significantly higher
than the MFB and MFP treatment (P < 0.001).

3
J. Han et al.
Applied Soil Ecology 165 (2021) 103966

Table 3
Maize biomass of different measure in pot experiment. Values for individual
treatments are the means of the three replicate soil (mean ± standard error).

M MF
MFO MFB MFP
F P

Fresh weight (g) 67.89 ± 6.44d 113.57 ± 7.53c

403.98 ± 0.83a 393.33 ± 3.57a 371.7 ± 5.8b
935.20 <0.001
Dry weight (g) 13.24 ± 1.91d 26.83 ± 0.57c
72.86 ± 1.65a 51.32 ± 0.85b 50.41 ± 0.71b
342.59 <0.001

Different letters indicate significant difference between treatments.

The effects of various fertilization strategies during the experiment period

on the soil physicochemical properties were summarized in Table 4.
Statistical analysis showed that all treatments did not signifi- cantly change
soil pH in the short term (P > 0.05). Organic fertilizer addition
significantly increased the soil EC (P < 0.05). The MFO and MFB treatments
significantly increased the soil SOC, especially in MFB

Table 5
Soil microbial α-diversities among the different treatments. Results is reported as
the mean ± SE (n = 3).

Species richness ACE Chao1

Shannon

Bacteria
soil (P < 0.05). Soil NO -N content was significantly increased by
organic fertilizer additions (P < 0.05). There was no significant differ-
CK 1510.33 ± 17.9
d
1547.24 ± 16.11
b
1563.4 ± 19.76 c 5.84 ± 0.01
b
ence of soil NH+-N content between the various treatments of soil (P >
M 1553 ± 30.51 c 1591.44 ± 21.83
a
1597.5 ± 22.79
b
5.87 ± 0.02
b
0.05). Soil TN contents were significantly increased under MFO and MFB
treatments (P < 0.01), while changed little under MFP treatment. Be- sides,
the soil AP and AK under MFO treatment were higher than that
under other treatments, AP content under all fertilization treatment was
MF 1586.67 ± 38.8 bc
MFO 1690.33 ± 15.95
a
1612.69 ± 28.42 a
1642.71 ± 11.69
a
1623.5 ± 35.93 a
1648.31 ± 10.62
a
5.85 ± 0.07 b
6.18 ± 0.03
a
significantly (P < 0.01) higher than that unfertilized treatments.
MFB 1599.67 ± 4.73
b
MFP 1599.67 ± 1.53 b
1616.75 ± 4.45 a 1619.49 ± 4.54
a
1616.26 ± 3.72 a 1620.72 ± 6.35 a
5.88 ± 0.04
b
5.90 ± 0.02 b
3.2. Microbial community

3.2.1. Richness and diversity

F 13.23 12.51 7.97
13.32
P <0.001 <0.001 0.002
<0.001

Fungi
The effects of different fertilization strategies on richness and di- versity
of bacteria and fungi are briefly shown in Table 5. ACE and Chao
CK 363 ± 56.2 a 375.48 ± 61.8 a 377.69 ± 62.25 a
3.92 ± 0.3 a
1 indices were employed as richness indices and the diversity indices
M 357.33 ± 90.53
a
382.98 ± 70.93 a 393.27 ± 68.51
a
3.53 ± 0.7 a
were estimated by the Shannon index. The organic amendment treat-
ments significantly increased the bacteria species richness compared
MF 457 ± 98.15 a 479.14 ± 95.44 a 484.05 ± 96.85 a
3.34 ± 1.38 a
with the M and CK treatments (P < 0.001). The bacteria species richness
under the MFO, MFB and MFP treatments were 6.53% (P < 0.05), 0.82%
MFO 377.67 ± 9.61 a 401.79 ± 7.78 a 408.84 ± 7.93 a 3.66
± 0.16
a
and 0.82% higher than that under MF, respectively (Table 5). Although no
profound effects on ACE and Chao1 indices of bacteria were observed between four
treatments with fertilization (MF, MFO, MFB and MFP),
MFB 448.67 ± 91.76
a
MFP 408.67 ± 11.59 a
470.51 ± 96.93 a 471.22 ± 92.99
a
425.4 ± 10.12 a 435.55 ± 14.76 a
3.86 ± 0.48
a
3.74 ± 0.2 a
they were significantly higher than the CK treatment. And the Shannon index
under the three treatments was 5.64% (P < 0.05), 0.85% and
0.51% higher than that under MF, respectively. However, for fungi, there
were no notable effects on richness and diversity index among different
fertilization strategies.

3.2.2. Community composition

PCoA and PERMANOVA analysis showed that the community com- positions of the
soil bacteria and fungi were changed by the organic amendments (Fig. 1). For
the bacterial community structure, the first and second principal coordinates
explained 40.47% and 17.49%, respectively, of the differences among the six
treatments (Fig. 1a). For the fungal community structure, the first and
second principal co- ordinates explained 23.90% and 15.00%, respectively, of the
differences among the treatments (Fig. 1b). Both the bacterial and fungal
commu- nity structures under MFO treatment were significantly different from the
other treatments (P < 0.001). The SIMPER analysis indicate that
F 1.14 1.27 1.23
0.30
p 0.39 0.34 0.35
0.90

Different letters indicate significant difference between treatments.

Betaproteobacteria, Alphaproteobacteria, Subgroup_6, Actinobacteria,

Gemmatimonadetes, Thermoleophilia, Blastocatellia, and Gammapro- teobacteria
contributed 67.83% of the differences in bacterial phyla composition among the
different treatments (Table S2a), and Sordar- iomycetes, Agaricomycetes,
Dothideomycetes, and Eurotiomycetes contributed 53.08% of the differences in
fungal class composition (Table S2b).
The Relative abundance of the bacterial (a) and fungal (b) taxa at the class level
was detailed in Fig. 2. Compared with the MF treatment, MFO treatment improved the
relative abundance of Gammaproteobacteria, Betaproteobacteria and
Alphaproteobacteria by 31.34%, 24.58% and
21.84%, respectively, while significantly reduced the relative

Table 4
Soil properties of different measure in pot experiment. Values for individual
treatments are the means of the three replicate soil (mean ± standard error).
— 1 + 1
1
1 1
Treatment pH EC (μS cm 1) SOC (g
kg 1) NO3 N (mg kg
) NH4 -N (mg kg
) TN (g kg
) AP (mg kg
) AK (mg kg )

CK 8.97 ± 0.04 b 152.74 ± 1.51 b 1.28 ± 0.08

c 1.05 ± 0.06 ab 0.27 ± 0.04 a 0.21 ± 0.02 d
3.3 ± 0.2 d 66.42 ± 48.79 b M 8.97 ± 0.13 b
129.47 ± 24.01 b 1.24 ± 0.02 c 0.54 ± 0.01 bc 0.23 ±
0.17 a 0.25 ± 0.01 c 3.26 ± 1 d 111.25 ±
4.52 b MF 9.1 ± 0.01 a 107.78 ± 3.72 b
1.35 ± 0.06 c 0.18 ± 0.03 c 0.29 ± 0.04 a 0.26
± 0.01 c 8.88 ± 0.51 b 78.18 ± 3.99 b MFO 8.99
± 0.04 b 211.56 ± 69.22 a 1.74 ± 0.04 b 1.21 ± 0.73 a
0.23 ± 0.01 a 0.36 ± 0.03 a 12.86 ± 1.97 a 384.88
± 32.04 a MFB 8.94 ± 0.03 b 113.85 ± 5.17 b
2.67 ± 0.14 a 0.42 ± 0.15 c 0.28 ± 0.01 a 0.29
± 0.01 b 7.11 ± 1.22 bc 81.95 ± 3.18 b MFP 8.96
± 0.08 b 115.37 ± 5.82 b 1.25 ± 0.11 c 0.57 ± 0.02 bc
0.25 ± 0.04 a 0.27 ± 0.01 bc 6.35 ± 0.14 c 76.42 ±
3.95 b
F 2.13 5.10
129.84 4.94 0.36
37.43 35.49 80.08
P 0.13 0.01
<0.01 0.01 0.86
<0.01 <0.01 <0.01

Different letters indicate significant difference between treatments.

4
J. Han et al.
Applied Soil Ecology 165 (2021) 103966

Fig. 1. Principal coordinate analysis (PCoA) of overall bacterial (A) and fungal
(B) community composition based on the OTU level for control (CK), maize (M),
maize + chemical fertilizer (MF), maize + chemical fertilizer + organic
fertilizer (MFO), maize + chemical fertilizer + biochar (MFB), and maize +
chemical fertilizer + polyacrylamide (MFP) treatments.

(a)
100%

80%

60%

40%

20%

0%

other Blastocatellia KD4-96

Nitrospira Thermoleophilia bacterium Sphingobacteriia Holophagae Cytophagia
Actinobacteria Acidimicrobiia Gammaproteobacteria Deltaproteobacteria
Gemmatimonadetes Subgroup_6
Betaproteobacteria
Alphaproteobacteria
(b)
100%

80%

60%

40%

20%

0%

other Leotiomycetes Mortierellomycotina_cls_Incertae_sedis Eurotiomycetes

Dothideomycetes Sordariomycetes Agaricomycetes

Fig. 2. Relative abundance of the bacterial (a) and fungal (b) taxa at the
class level. “Other” includes classes with <1% average relative abundance.
Values for individual treatments are the means of the three replicate soil
samples.

abundances of Blastocatellia and Subgroup_6 by 52.67% and 44.03%, respectively

(Fig. 2a). In addition, the relative abundance of Betapro- teobacteria also
improved under the MFB and MFP treatments.
For fungi, MFB and MFP improved the relative abundances of
Dothideomycetes and Eurotiomycetes to some extent, and all the MFO treatment
reduced the relative abundances of Agaricomycetes and Sor- dariomycetes compared
with the other treatments (Fig. 2b). Surpris- ingly, the relative
abundance of Eurotiomycetes under the MFO treatment was 3527.91% higher than
that under MF (P < 0.05), while no significant differences were found under MFB
and MFP treatments.

3.3. Bacterial potential functionality and fungal functional guild analysis

Tax4Fun was used to predict OTU-based bacterial function in the soil from different
treatments. In this study, there were a total of 344 groups at the level 3
KEGG orthologues (Table S3). More than 96% of the bacteria could be
categorized into the functional classes of Metabolism, Genetic Information
Processing, Human Diseases, Cellular Processes, Environmental Information
Processing, and Organismal Systems (Fig. 3a). Among them, the top six
most abundant bacterial functional pathways occur in Global and Overview
maps, Carbohydrate meta- bolism, Amino acid metabolism, Signal transduction,
Membrane trans- port and Cellular community – prokaryotes (Fig. 3a). The MFO, MFB
and MFP treatments all improved the abundance of the Membrane transport
and Cellular community – prokaryotes pathways. Compared with that under MF, the
abundance of the Membrane transport pathway increased by 7.52% and 5.99% under
the MFO and MFP treatments, respectively, and the abundance of the Cellular
community – prokaryotes pathway increased by 3.89% under the MFB treatment. In
addition, Global and overview maps were the largest metabolism functional
class, but all organic amendments reduced its abundance. Compared with MFB and
MFP, MFO significantly reduced the abundance of the Metabolic path- ways,
Biosynthesis of secondary metabolites, Biosynthesis of antibiotics and Carbon
metabolism pathways (Fig. S1).
The effect of different organic amendments differed on the KEGG category
abundance of fungal functional groups (Fig. 3b). Approxi- mately 50% of the
fungal OTUs were assigned to different functional groups, of which including
Undefined Saprotroph, Animal pathogen, Endomycorrhizal Fungi, Dung Saprotroph, and
Plant Pathogen were the main functional groups. Further analysis revealed that
MFO is superior to the other organic amendment treatments in terms of improving
the abundance of the Undefined Saprotroph and Arbuscular Mycorrhizal Fungi groups
by comparison with MF treatment, while the MFB and MFP treatments were superior to
MFO in improving the abundance of the Endomycorrhizal Fungi, Plant Pathogen and
Animal Pathogen groups. Compared with the MF treatment, the abundance of
Arbuscular Mycorrhizal Fungi increased by 242.9% and 214.3% under the MFO and MFP
treatments, respectively.

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J. Han et al.
Applied Soil Ecology 165 (2021) 103966

Fig. 3. The predicted KEGG category abundances for the bacterial community (a)
and fungal functional groups (b).

3.4. Driving factors of microbial community variation

The Mantel test revealed that NO -N, TN, EC, AP, and AK were significantly
correlated with bacterial community composition, while TN, AK, and NO -N were
remarkable correlated with fungal community composition (Table 6). The RDA of
the environmental characteristics associated with the bacterial and fungal
communities showed that the selected environmental factors explained 50.84% of
the bacterial com- munity structure variation and 55.35% of the fungal community
struc- ture variation (Fig. 4). Furthermore, the microbial communities were
further analyzed by MRA analysis to identify the main environmental factors
driving the soil bacterial and fungal communities (Fig. 5). The main driving
factor of the bacterial community was TN content, and its
threshold was 0.24 g⋅kg 1. However, when the TN content was greater
than or equal to 0.24 g⋅kg 1, the bacterial community structure was mainly
affected by EC, with a threshold of 150.20 μS⋅cm 1. Moreover,

Table 6
Mantel test results for the correlation between community composition and
environmental variables for bacteria and fungi based on OUT.
Parameters Bacterial composition Fungal
composition

r P r
P

pH 0.002 0.490 0.087 0.228

EC 0.511 0.001
0.228 0.064
SOC 0.114 0.210
0.087 0.288
NO- -N 0.427 0.013 0.189
0.044
NH+-N 0.252 0.052 0.107
0.239
TN 0.708 0.001 0.308
0.032
AP 0.354 0.006
0.200 0.063
AK 0.703 0.001 0.348
0.023

The bold fonts indicate significance at the 0.05, 0.01 or 0.001 probability
levels.

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J. Han et al.
Applied Soil Ecology 165 (2021) 103966

Fig. 4. Redundancy analysis (RDA) of the bacterial (a) and fungal (b)
communities with environmental properties.

Fig. 5. Multivariate regression tree analysis discerns the effects of

environmental parameters on bacterial (a) and fungal (b) community
structure in different treatments.

the main driving factor of the fungal community was also TN content, and its
threshold was 0.31 g⋅kg 1. However, when the TN content was less than 0.31
g⋅kg 1, the fungal community structure was mainly
Higher biomass in organic amendments plots mostly might be due to the positive
effects on crop N uptake through improved N fertilizer use ef- ficiency in soils.
The available nutrient contents and C input rates con-
affected by NO -N, and its threshold was 0.535 mg⋅kg
1. In general, the
tained within organic fertilizer and biochar could be responsible for such
TN was the strongest predictor of the both variations in the soil bacterial and
fungal communities.

4. Discussion

4.1. Effects of chemical fertilizer reduction with different organic amendments

on maize biomass and soil properties

Application of organic amendments and reduction of inorganic fer- tilizer are

economically feasible and environmentally sound approaches to develop
sustainable agriculture. The results of this study demon- strated that the
different organic amendments coupled with an appro- priate reduction in
chemical fertilizer inputs greatly impacted the maize biomass and soil
physicochemical properties in newly cultivated soil. Through a 15-year field
experiment, Zhang et al. (2009) also showed that a suitable combined application
of chemical fertilizer and straw or cow manure could increase crop yield, as
well as maintain soil fertility and soil buffering capacity. Furthermore,
incorporation of organic amendments can improve chemical fertilizer
utilization efficiency.
short-term increase in crop yields (Bˇrendova´ et al., 2012; Farrell et al.,
2014; Qaswar et al., 2020). Other possible major reasons for increase in maize
biomass in organic amendments plots include the effects on soil physio-chemical
properties. Hijbeek et al. (2017) and Cai et al. (2019) also reported that C
input increased the nutrient availability by improving soil physical and
chemical properties. In our results, appli- cation of organic fertilizer and
biochar significantly increased the SOC content, and the EC under MFO was 96.3%
(P < 0.05) higher than that under the MF treatment (Table 4). In previous
studies, animal residues mixed with mineral nitrogen fertilizers could increase
the EC due to the high salt content in livestock manure (Hao and Chang, 2003;
Mahmoud et al., 2009). Furthermore, application of organic fertilizer increased
NO -N, TN, AP and AK compared with chemical fertilization alone in this study
(Table 4), which was also consistent with previous studies (Bednarek et al.,
2012; Lee et al., 2013; Zhang et al., 2013). Surprisingly, the soil pH was
significantly decreased by organic amendments treat- ments compared to MF
treatment, which is not consistent with previous results. This discrepancy might
be attributed to variance in the soil type, as proposed by Wei et al. (2017), who
observed that organic fertilizer

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Applied Soil Ecology 165 (2021) 103966

could decrease the soil pH in alkaline soils while increasing the soil pH in acidic
soils. Except for increasing soil TN content, as a polymer soil improver,
PAM could also enhance grain yield by altering soil structural stability by
increasing the soil water-holding capacity (Busscher et al.,
2005). From the overall results, the application of organic fertilizer has the
best effect on improving soil physicochemical properties compared with biochar and
PAM. However, the improvement effect of organic amendments on soil quality is
long-term and sustainable, and long-term monitoring of the effect is needed.

4.2. Effects of chemical fertilizer reduction with organic amendments on

microbial community structures and functional profiles

4.2.1. Microbial community structures and driving factors

Soil microbial activity as an important regulatory factor of SOC and nutrient
cycle has an indispensable role in the process of SOC degra- dation. Organic
amendments could increase the SOC content, and pro- vide ample carbon source
for microbial activity, and improved the microbial activity and microbial
biomass (Rui et al., 2009). Under the condition of chemical fertilizer reduction,
organic amendments could appropriately adjust soil C:N ratio and improve the
utilization efficiency of organic matter, thus stimulate the activity of soil
microbes and in- crease beneficial microbial biomass (Ye et al., 2015). The
results showed that the soil bacteria community structure was significantly
changed by the different organic amendments on the basis of inorganic
fertilizer reduction (Fig. 1 and Table 5), in agreement with previous
studies (Chaudhry et al., 2012; Zeng et al., 2015; Pan et al., 2020). A short-term
field experiment by Lazcano et al. (2013) showed that partial replace- ment of
inorganic fertilizer by vermicompost or manure had a signifi- cant positive
impact on soil microbial activity and maintained nutrient supply at similar
levels with inorganic fertilizer. In this study, organic fertilizer significantly
increased the species richness, Chao 1 and Shan- non index values of the
bacteria compared to other treatments. Zeng et al. (2007) and Dong et al. (2014)
demonstrated organic fertilizers may not only provide a greater diversity of
substrates than inorganic fertil- izers for microbial activity but they also
directly introduce microor- ganisms which occur naturally in the organic
fertilizers into the soil. In addition, Lin et al. (2019) also found that adverse
soil environmental conditions can aggravate competition and cooperation among
microbial communities, and the application of organic materials provides a large
number of nutrient sources for these heterotrophic bacteria, thus
reducing competition among bacterial communities. Different from the bacterial
community, the fungi abundance was little changed by different treatment
(Table 5), which is inconsistent with the results re- ported by Wang et al.
(2017). Studies have shown that fungi were found to be more suitable for growing
in the acidic soil (Sun et al., 2016). The soil in this study was weakly
alkaline, which would lead to significant growth inhibition of fungi. In
addition, this may be due to the “prefer- ence” effect of the application of
organic amendments on certain fungal groups, which consumes a large amount of
nutrients and thus inhibits the growth of other groups. Long-term application
of organic materials is conducive to the transformation of soil from
“fungicidal” to “bacte- riological” (Ling et al., 2016). Moreover, generally
bacteria are regarded as important mediators of the rapid pathways of nutrient
cycling in soil, and their growth tends to be approximately 10-fold greater than
that of fungi (Rousk and Bååth, 2007; Schmidt et al., 2014); thus, bacterial
community can faster growth under organic addition and increase their diversity.
It can be seen that the growth of bacteria and fungi is closely related to the
type of fertilizer, and regulating the type and proportion of fertilizer is an
effective strategy to coordinate the growth from different types of
microorganisms, and thus improve soil biological fertility.
The addition of biochar and PAM increased the species richness, ACE and Chao 1
index values of the bacteria. The numerous voids in biochar can provide a place
for bacteria to grow and reproduce without being affected by competitors (He et
al., 2007). Moreover, PAM could provide an appropriate environment for microbial
growth (Kay-Shoemake et al.,
1998). The organic fertilizer with a large number of microorganisms, could be
effectively improved the microbial community structure through changing the
ratio of bacteria to fungi in the soil (Kamaa et al.,
2011). Furthermore, the application of organic fertilizer to the soil
increased the content of soil organic matter, which is rich in nutrients such
as carbon, nitrogen, phosphorus and potassium, thus providing a good growth
environment for microorganisms and ultimately promoting the diversity of the
microbial community (KhHM and Fadni, 2013).
Intriguingly, the fungi community structures were little changed by different
organic treatment, which was inconsistent with previous studies. Ding et
al. (2017) reported that manure decreased fungal gene abundances, which
indicated that the soil type could be a major reason for the disparities.
Previous studies also showed that the addition of biochar significantly
increased the densities of bacteria but reduced the densities of fungi (Zhang et
al., 2017; Yao et al., 2017). However, rele- vant studies have found that organic
fertilizers can increase the biomass and proportion of fungi in the soil microbial
community, which is also not consistent with our results (Lee et al., 2013),
which may be caused by different organic fertilizers types, crop species and
soil environment conditions.
The application of organic fertilizers increased the bacteria relative abundance
of Alphaproteobacteria, Betaproteobacteria and Gammap- roteobacteria (Ding et al.,
2016), which is consistent with our results. The relative abundance of the
dominant bacteria in the community, proteobacteria and a kind of nutrient-rich
bacteria, is related to the soil physical and chemical properties (Fierer et al.,
2007; Shen et al., 2013). All soil nutrients under the MFO treatment were at a
high level (except
NH+-N) in this study (Table 4), promoting the growth of proteobacteria.
Therefore, the relative abundances of Betaproteobacteria, Alphapro- teobacteria
and Gammaproteobacteria, the dominant bacteria in the community, increased
under the combination of pig fertilizer and chemical fertilizer in this
study. In addition, the MFO treatment reduced the relative abundances of
Blastocatellia and Subgroup_6, which is inconsistent with previous research.
The study has showed that appli- cation of organic fertilizer could increase
a large number of eutrophic bacteria (Fierer et al., 2007). As we know,
bacteria are the most important decomposers in soil, long-term organic
fertilizations caused the enrichment of specific bacteria that could
efficiently utilize these nutrients (Wang et al., 2017), which will lead to
competition between Blastocatellia and Subgroup_6 and other bacteria.
It was shown that the relative abundance of Eurotiomycetes under the MFO
treatment was significantly higher than that under the other treatments.
Eurotiomycetes could be found on all types of decaying material, and the
organic fertilizer used in this study was pig manure, which provided a suitable
physiological environment for the growth and reproduction of Eurotiomycetes (Al-
Mazroui and Al-Sadi, 2015). In addition, Eurotiomycetes are associated with
some important physio- logical functions, such as beneficial secondary metabolic
processes and important food fermentation processes (Geiser et al., 2006),
which is also consistent with the conclusion that organic fertilizer
promoted microbial metabolism in this study.
From what has been discussed above, the combined application of pig manure
and reduced chemical fertilizers could provide a better growth environment for
bacteria, thus improving the microbial com- munity structures.

4.2.2. Microbial functional profile

In this study, the addition of biochar significantly improved the abundance of
the Signal transduction and Cellular community – pro- karyotes groups. After
biochar application, an improved soil environ- ment facilitates soil enzyme
activity and microbial activity as well as ecosystem material circulation,
energy flow and information transfer processes in the ecosystem (Liao et al.,
2016). The combination of PAM and chemical fertilizer significantly increased the
abundance of the Dung Saprotroph and Endomycorrhizal Fungi groups
because the growth of the Saprotroph and Symbiotroph groups requires a great
deal

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J. Han et al.
Applied Soil Ecology 165 (2021) 103966

of nutrients, such as carbon sources, nitrogen sources and inorganic salt

significantly increased the contents of EC, SOC, NO -N, TN, AP
+ +
vitamins, and PAM enhanced the soil adsorption of NH4 , K , NO3 , and
3
and AK.
PO4
(Cline et al., 2018).
(2) The diversity and richness of the bacterial community increased
It is worth noting that the combination of pig manure and chemical fertilizer
had the greatest effect on the microbial community function in all fertilization
treatments, significantly increasing the abundance of the Amino acid metabolism,
Membrane transport, Xenobiotics biodegrada- tion and metabolism, Undefined
Saprotroph and Arbuscular Mycor- rhizal Fung. Since the metabolism of amino
acids, carboxylic acids, polymers and amines is more sensitive to the
application of organic fertilizers than to that of sugars and phenols, the
combination of pig manure and chemical fertilizer increased the abundance of the
Amino acid metabolism (Yu-Hong et al., 2011). Arbuscular Mycorrhizal Fungi not only
cause a series of physiological changes in plant growth but also directly or
indirectly affect the ecological environment of soil micro- domains (Hawksworth
and Rossman, 1997). They were greatly affected by fertilizer management, with the
combination of organic fertilizer and chemical fertilizer promoting their growth
and improving their diversity index values, which is in accordance with the
results of this paper (Kamaa et al., 2011).

4.3. Relationship between the microbial community and soil environment factors

In this experiment, the main driving factors for the bacterial com- munity
were TN, and EC, while the main driving factors of the fungal community
were TN and NO3 -N. It was demonstrated that TN was significantly correlated
with the ammonia-oxidizing bacterial commu- nity and was one of the main
factors driving community change (Qu et al., 2016; Yang et al., 2017).
Bacteria can synthesize biological
macromolecules with important physiological functions in the body such as
proteins and nucleotides after absorbing and utilizing nitrogen in the soil
(Sarathchandra et al., 2001). Moreover, as the dominant taxa in the bacterial
community, the relative abundances of Actinobacteria, Alphaproteobacteria and
Gammaproteobacteria were positively corre- lated with the soil nitrogen pool,
and thus TN drives changes in the bacterial community (Nemergut et al., 2010).
Soil EC reflects the level of water-soluble salt content, which was also one
of the main factors affecting the change in bacterial flora (Qu et al.,
2016). As one of the dominant bacterial taxa, changes in Gemmatimonadetes
will cause major changes in the bacterial community. The soil EC was
significantly positively correlated with the abundance of Gemmatimonadetes and
thus drives structural changes in the bacteria (Kim et al., 2016). TN is not
only the main driving factor for bacterial communities but also for fungal
communities. NO3 -N is a nitrogen form that is easily used and is
the main factor causing the variation in soil fungal community structure
(Zhong et al., 2015). In this experiment, NO -N was positively corre- lated
with the change in the fungal community, which indicates that fungi may
participate in the nitrification process of soil nitrogen. Pre- vious studies
have shown that pH is the most significant factor deter- mining the soil
bacterial community composition (Zhao et al., 2015; Kim et al., 2016). The driving
effect of pH was not observed due to the lack of significant changes in the pH
in each treatment in our work. More attention should be paid to the
changes in pH associated with the application of organic fertilizer in the
future.

5. Conclusions, limitations and further research

The results from a pot experiment with maize crop concluded that the effects
of different organic amendments and chemical fertilizer reduction on soil
fertility and the microbial community in newly culti- vated land. The specific
conclusions are as follows:

(1) The soil physicochemical properties were improved by the different

organic amendments and chemical fertilizer reduction. Compared with MF treatment,
the addition of organic fertilizer
under each organic amendment, while the diversity and richness of fungal
community did not change significantly. Among them, the organic fertilizer had
the most significant influence on the community structure of bacteria and
fungi. It increased the relative abundance of the dominant bacteria of
Betaproteobac- teria, Alphaproteobacteria, Gammaproteobacteria and Gemma-
timonadetes and reduced the relative abundance of the dominant fungi of
Sordariomycetes and Agaricomycetes. In addition, the improvements in the
functional abundance of Amino acid meta- bolism, Membrane transport, Xenobiotics
biodegradation and metabolism, Undefined Saprotrophs and Arbuscular Mycorrhizal
Fungi under the combination of organic fertilizer and chemical fertilizer
reduction treatment were superior to those under the other fertilization
treatments.
(3) TN and EC were the main driving factors of bacterial community structural
variation, while TN and NO -N were the main driving factors of fungal community
structural variation.
(4) The fresh and dry weights of maize were significantly increased by the
different organic amendments, among which the organic fertilizer had the
greatest effect on the fresh and dry weight of maize.

Overall, under the contradictory background of the rapid improve- ment of

soil fertility and reduction of fertilizer pollution from nonpoint sources, the
combined application of organic fertilizer and chemical fertilizer reduction was
an effective measure for improving soil fertility, improving microbial community
structure and promoting corn yield.
Time-scale for benefits of different organic amendments under field conditions
is a critical factor needs to be taken into consideration before recommendation
of organic fertilizer for soil management at farm levels. Lack of organic
fertilizer-based long term field experiments warrants a cautious approach to test
the organic fertilizer efficacy for soil fertility management. Although our a
growing season for maize-based study also reflects the promising potential of
organic fertilizer in integrative nutrient management for productivity of
cereal-based cropping systems from newly cultivated land in the Loess Plateau of
China. However, we stress on careful and critical assessment of organic
fertilizer integration into current farm management practices using long-term
farmers’ participatory field experiments to evaluate organic fertilizer usefulness
as sustainable nutrient management strategy in arid and semi-arid agro- ecosystems.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.apsoil.2021.103966.

Declaration of competing interest

The authors declared that they have no conflicts of interest to this work.
We declare that we have no financial and personal relationships with other people
or organizations that can inappropriately influence our work, there is no
professional or other personal interest of any nature or kind in any product,
service and/or company that could be construed as influencing the position
presented in, or the review of, the manuscript entitled.

Acknowledgements

This work was supported by the National Key Research and Devel- opment Program
of China [grant number 2017YFC0504703], the Na- tional Natural Science
Foundation of China [grant numbers 41807067,
41771558, 41601321], Youth Talent Lift Project of China Association for Science
and Technology [grant number 2019-2021QNRC001], and the Water and Soil
Conservancy Science Plan in Shaanxi Province of

9
J. Han et al.
Applied Soil Ecology 165 (2021) 103966

China [grant number 2017sbkj-01].

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