agronomy

Article
Effects of Manure and Chemical Fertilizer on Bacterial
Community Structure and Soil Enzyme Activities in
North China
Zhiping Liu 1 , Wenyan Xie 1 , Zhenxing Yang 1 , Xuefang Huang 2, * and Huaiping Zhou 1

1 College of Resources and Environment, Shanxi Agricultural University, Taiyuan 030031, China;
liuzhiping@sxau.edu.cn (Z.L.); xwy6018060@163.com (W.X.); yangzhenxing@sxau.edu.cn (Z.Y.);
huaipingzhou@126.com (H.Z.)
2 Shanxi Institute of Organic Dryland Farming, Shanxi Agricultural University, Taiyuan 030031, China
* Correspondence: hxuefang@sxau.edu.cn

Abstract: The application of organic fertilizer affects soil microbes and enzyme activities. In this study,
we explored the effects of various long-term different fertilization treatments (manure, M; chemical
fertilizer, NP; manure + chemical fertilizer, MNP; and no fertilizer, CK) on bacterial community
structure and soil sucrase, urease, and alkaline phosphatase activities in Shaping, Hequ, China.
High-throughput sequencing was used to amplify the third to the fourth hypervariable region
of the 16S ribosomal RNA for analysis of the bacterial community structure. Enzyme activities
were determined by colorimetry. Soil treated with MNP had the highest bacterial Abundance-
based Coverage Estimator index and enzyme activities. The principal coordinates analysis results



showed significant differences among the various fertilization treatments (p < 0.001). Proteobacteria,


Actinobacteria, Acidobacteria, Gemmatimonadetes, and Chloroflexi were consistently dominant in
Citation: Liu, Z.; Xie, W.; Yang, Z.; all soil samples. The redundancy analysis and Monte Carlo permutation tests showed that the soil
Huang, X.; Zhou, H. Effects of
bacterial communities were significantly correlated with alkali-hydrolyzable nitrogen, organic matter,
Manure and Chemical Fertilizer on
urease, and alkaline phosphatase. Our results reveal the fundamentally different effects that organic
Bacterial Community Structure and
and inorganic fertilizers have on soil bacterial communities and their functions.
Soil Enzyme Activities in North
China. Agronomy 2021, 11, 1017.
Keywords: bacterial community; long-term fertilization; soil enzyme activities; high-throughput
https://doi.org/10.3390/agronomy
11051017
sequencing

Academic Editor: Florian Wichern

Received: 24 March 2021 1. Introduction

Accepted: 13 May 2021 Soil microorganisms and enzyme activities are important indicators in the charac-
Published: 20 May 2021 terization of soil fertility and these play a vital role in soil material transformation and
energy flow [1,2]. Soil bacteria are one of the largest functional microbial subgroups in soil,
Publisher’s Note: MDPI stays neutral participating in soil respiration, nutrient transformation, organic matter decomposition,
with regard to jurisdictional claims in and other processes [3,4]. Soil enzymes secreted by microorganisms play critical roles in
published maps and institutional affil- nutrient cycling (such as nitrogen fixation, phosphorus adsorption and desorption, potas-
iations.
sium release, etc.), as well as soil structure maintenance and crop production [5,6]. Organic
matter in soil serves as a substrate for a diverse set of soil microorganisms, promoting
enzymatic activity [2]. Previous studies have shown that soil microorganisms and enzyme
activities are highly sensitive to changes in soil properties, including pH, the degree of
Copyright: © 2021 by the authors. organic matter, moisture content, the presence of mineral substances, etc. Furthermore, soil
Licensee MDPI, Basel, Switzerland. microorganisms are also regarded as vital factors driving the production and turnover of
This article is an open access article extracellular enzymes and their activities in soil [5,7]. Studies showed that the physical
distributed under the terms and and chemical properties of soil could be largely influenced by fertilization management
conditions of the Creative Commons
practices [8,9].
Attribution (CC BY) license (https://
Fertilization is an important measure in agricultural production, which can improve
creativecommons.org/licenses/by/
soil nutrients and increase crop yield. Recently, a large number of chemical fertilizers
4.0/).

Agronomy 2021, 11, 1017. https://doi.org/10.3390/agronomy11051017 https://www.mdpi.com/journal/agronomy

Agronomy 2021, 11, 1017 2 of 14

have been imported into farmland to ensure food supply, and the result was an imbalance
in soil nutrients, as well as a series of environmental problems [8,10]. Manure, however,
could partly prevent many environmental problems caused by chemical fertilizers and also
improve crop quality. It might be due to the fact that numerous beneficial microorganisms
and organic materials enter soil through manure application, leading to changes in the soil
environment that include differences in enzyme activity. Thus, it is necessary to investigate
the soil’s bacterial community structure and soil enzymatic activity. One of the main chal-
lenges in microbiological research is to improve the identification of bacteria. Traditional
methods have various defects. For instance, the plating method can only reflect culturable
microorganisms that account for only about 1% of the total population [11,12]. Further-
more, neither single-strand conformation polymorphism (SSCP) nor terminal restriction
fragment length polymorphism (T-RFLP) are suitable for bacterial analysis because they
only capture the dominant community members as selected by their PCR primers [13].
High-throughput sequencing technology, however, has facilitated bacterial research, al-
lowing for huge amounts of information to be obtained, and for the bacterial diversity
in the environment to be fully reflected [14]. In this study, we explored the effects of the
long-term application of manure and chemical fertilizer on bacterial community structure
with high-throughput sequencing technology, and we studied the soil’s enzymatic activities
by colorimetry in North China. The objectives of this study were as follows: (1) to elucidate
the influences of different fertilization on bacterial community structure and soil enzyme
activities, (2) and to search for the most suitable fertilization method.

2. Materials and Methods

2.1. Field Description and Experimental Design
The fertilization experiment was established in a maize field in Shaping, Hequ, China
(39◦ 120 18” N, 111◦ 150 41” E). The altitude is 1089 m, with a frost-free period of about
140 days. The average temperature is 8.8 ◦ C with an effective accumulative temperature of
3000–3360 ◦ C per year. The experimental site has a light loam loess, classified as Hapli-Ustic
Cambosols in the World Reference Base for Soil Resources (WRB) [15,16]. Analysis of soil
samples taken from the experimental area showed that the basic physical and chemical
properties of the surface 0–20 cm of soil before planting were as follows: organic matter
(OM), 5.64 g/kg; total nitrogen (TN), 0.45 g/kg; total phosphorus (TP), 1.23 g/kg; alkali-
hydrolyzable nitrogen (AN), 34.90 mg/kg; available phosphorus (AP), 2.69 mg/kg; cation
exchange capacity (CEC), 6.97 cmol/kg; pH, 8.2.
The long-term fertilization trial began in 1988, and it lasted for 31 years. From 1988 to
2008, millet (Panicum miliaceum L.) and potatoes (Solanum tuberosum L.) were rotated; from
2009 to 2013, maize (Zea mays L.) was continuously cropped; from 2014 to 2017, potatoes
and millet were rotated; and since 2018, potatoes and maize have been periodically rotated.
The samples were obtained on 25 July 2019, when the crop was maize. The four treatments
were arranged in a randomized block design with three replications, with each plot being
24 m2 . The four treatments were (1) M—manure, 22,500 kg/ha; (2) NP—N: 120 kg/ha,
P2 O5 : 75 kg/ha; (3) MNP—manure, 22,500 kg/ha, N: 120 kg/ha, P2 O5 : 75 kg/ha; and
(4) CK—control treatment without fertilizer. Nitrogen was applied in the form of urea,
while phosphorus was applied as superphosphate. The manure was made up of local
cow dung with an average N content of 3.64 g/kg, P2 O5 content of 2.46 g/kg, and K2 O
content of 7.87 g/kg. The fertilization details are shown in Table 1. All of the fertilizers
were applied as basal fertilizers before planting.

2.2. Soil Sampling and Analysis of Soil Properties

Soil were sampled from five points in each plot and mixed into one sample. There
were 12 composite samples in total. After removing visible stone and debris, samples were
collected in sterile plastic bags in an ice-box and transported to the laboratory. One part was
used for analyzing physicochemical properties and enzyme activities after sieved through
Agronomy 2021, 11, 1017 3 of 14

a 2 mm screen, and the other part was stored in −80°C refrigerator for DNA extraction
and sequencing.

Table 1. Experimental treatments and fertilization.

Chemical Fertilizer Manure Total Nutrient

Treatments (kg/hm2 ) (kg/hm2 ) (kg/hm2 )
N P2 O5 K2 O N P2 O5 K2 O N P2 O5 K2 O
Manure (M) 0 0 0 80 55 180 80 55 180
Chemical fertilizer (NP) 120 75 0 0 0 0 120 75 0
Manure + Chemical
120 75 0 80 55 180 200 130 180
fertilizer (MNP)
No fertilizer (CK) 0 0 0 0 0 0 0 0 0

2.3. Soil Physicochemical Analytical Procedures

Soil TN, TP, and total potassium (TK) were analyzed using a Vario Max element
analyzer (Elementar Vario PYRO cube and Isoprime100, Hanau, Germany). Soil pH
was determined at a soil/water ratio of 1:2.5 with a pH meter. OM was determined by
K2 Cr2 O7 oxidation method. Soil AN was determined by the alkaline hydrolysis diffusion
method. AP was determined by NaHCO3 -extracted method. Available potassium (AK)
was analyzed by CH3 COONH4 -extracted method. The details can be found from the
reference [17].

2.4. Soil Enzyme Activities’ Determination

In this study, 3, 5-dinitrosalicylic acid colorimetry was used for sucrase (SUC) activity,
and the weight of glucose (mg) in 1 g of soil after incubation for 24 h was used to represent
SUC activity. The urease (URE) activity was indicated by sodium phenol-sodium hypochlo-
rite colorimetric method, and the weight of NH3 -N (mg) in 1 g soil after incubation for
24 h was used to represent the URE activity. The alkaline phosphatase (ALP) activity was
represented by the phenyl disodium phosphate method. Detailed procedures for the three
enzymes can be found from the reference [18].

2.5. Soil DNA Extraction and High-Throughput Sequencing

Total DNA of each sample was extracted form 0.5 g soil using the Fast DNA SPIN
Isolation Kit (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s in-
structions. The bacterial third to the fourth hypervariable region of the 16S ribosomal RNA
(16S rRNA) was amplified using primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and
806R (5’-GGACTACHVGGGTWTCTAAT-3’). Specific barcodes of 7-bp were incorporated
into the primers for multiplex sequencing. The first round PCR reactions were performed
in a total volume of 50 µL mixture containing 10 µL of 5×Q5 Reaction Buffer, 10 µL of
5 × Q5 High-Fidelity GC Enhancer, 1 µL of dNTPs, 1 µL of each primer (10 µM), 0.2 µL of
Q5 High-Fidelity DNA Polymerase (Transgen, Beijing, China), 2 µL of temple DNA and
24.8 µL of ddH2 O. PCR process were as follows: 5 min of initial denaturation at 95 ◦ C,
followed by 15 cycles of denaturation at 95 ◦ C for 1 min, annealing at 50 ◦ C for 1 min and
extension at 72 ◦ C for 1 min, and a final extension of 7 min at 72 ◦ C.
PCR products from the first PCR step were purified through VAHTSTM DNA Clean
Beads (Vazyme, Nanjing, China). A second round PCR was then performed in a 40 µL
reaction system which contained 20 µL 2 × Phusion High-Fidelity Master Mix (New
England Biolabs, Boston, MA, USA), 8 µL ddH2 O, 10 µM of each primer and 10 µL PCR
products from the first step. PCR process was as follows: an initial denaturation at 98 ◦ C for
30 s, followed by 10 cycles of denaturation at 98 ◦ C for 10 s, annealing at 65 ◦ C for 30 s and
extension 72 ◦ C for 30 s, and a final extension at 72 ◦ C for 5 min. Finally, all PCR products
were quantified by Quant-iT™ dsDNA HS Reagent (Thermo Fisher, Waltham, MA, USA)
and pooled together. High-throughput sequencing analysis of bacterial 16S rRNA genes
Agronomy 2021, 11, 1017 4 of 14

was performed with the purified and pooled sample using the Illumina Hiseq 2500 platform
(2 × 250 paired ends) at Biomarker Technologies Corporation (Beijing, China).

2.6. Bioinformatics Analysis

The Quantitative Insights Into Microbial Ecology (QIIME, version 1.8.0) pipeline
was employed to process the sequencing data [19]. Briefly, raw sequencing reads with
exact matches to the barcodes were assigned to respective samples and identified as valid
sequences. The low-quality sequences were removed. Paired-end reads were assembled
using FLASH (version 1.2.7) [20]. After chimeras’ detection with UCHIME (version 4.2),
the remaining high-quality sequences were clustered into operational taxonomic units
(OTUs) at a 97% sequence identity threshold by UCLUST [21]. A representative sequence
was selected from each OTU using default parameters. OTU taxonomic classification was
conducted by BLAST searching the representative sequences set against the Silva Database
(version 132) [22]. An OTU table was further generated to record the taxonomy of the
OTUs and the abundance of each OTU in each sample. OTUs containing less than 0.001%
of total sequences across all samples were discarded. To minimize sequencing depth
differences across samples, an averaged, rounded, and rarefied OTU table was generated
by averaging 100 evenly resampled OTU subsets under the 90% minimum sequencing
depth for further analysis.
Sequence data analyses were mainly performed using QIIME and R (version 3.1.2,
vegan package) [23,24]. The Abundance-based Coverage Estimator (ACE) and Shannon
index were calculated based on the OTU table using QIIME. β-diversity analysis was
performed to investigate the bacterial communities’ structural variation across samples
using principal coordinate analysis (PCoA), which was based on the OTU level compo-
sitional profiles [25]. The significance of bacteria structure differentiation among groups
was assessed by permutational multivariate analysis of variance (PERMANOVA) [26] with
the distance legend of 0.01. The taxonomic compositions and abundances were visualized
using MEGAN [27]. A Venn diagram was generated to visualize the shared and unique
OTUs among groups based on the occurrence of OTUs across groups regardless of their
relative abundances [28]. Redundancy analysis (RDA) and Monte Carlo permutation tests
were carried out using Canoco for Windows 4.5 (Cabiy Information Techonogy Co., LTD.,
Shanghai, China). The species compositional similarity was analyzed by unweighted
pair-group method with arithmetic mean (UPGMA) with R software.

2.7. Statistical Analysis

Statistical analyses were carried out using a one-way analysis of variance (ANOVA)
procedure in the SPSS software (IBM SPSS Statistics version 22) to check the normal dis-
tribution and homoscedasticity, and to detect differences in soil parameters, soil enzyme
activities, the bacterial relative abundance, α-diversity indices, etc. Significant differ-
ences between data were determined with the least significant difference (LSD) test at the
p = 0.05 level.

3. Results
3.1. Soil Physicochemical Characteristics
As shown in Table 2, long-term fertilization significantly promoted soil TP, AN and
AP (p < 0.05) compared to the control. The TN level was the highest in treatment MNP,
followed by treatment M, and then treatment NP, while M was not significantly different
from MNP. The TP value was the highest in treatment MNP, followed by treatment NP and
treatment M. Again, however, there was no significant difference between NP and MNP.
Soil pH (H2 O) values decreased with the application of fertilizers, although no significant
differences were observed. F- and p-values of the factors can be found in Table S1.
Agronomy 2021, 11, 1017 5 of 14

Table 2. Soil physicochemical properties under different treatments.

AN AP AK OM TN TP TK
Treatments pH
(mg/kg) (mg/kg) (mg/kg) (g/kg) (g/kg) (g/kg) (g/kg)
M 8.40 ± 0.23 a 63.09 ± 3.06 ab 22.53 ± 1.73 a 244.78 ± 16.67 a 11.76 ± 2.26 a 0.81 ± 0.10 a 0.80 ± 0.03 b 12.79 ± 0.65 a
NP 8.51 ± 0.13a 59.15 ± 7.35 b 14.40 ± 5.52 b 71.27 ± 4.25 c 7.82 ± 0.57 b 0.74 ± 0.14 ab 0.84 ± 0.04 ab 12.58 ± 0.34 a
MNP 8.40 ± 0.18 a 74.55 ± 4.55 a 26.90 ± 2.80 a 223.90 ± 3.04 b 12.27 ± 1.14 a 0.85 ± 0.12 a 0.85 ± 0.12 a 12.36 ± 0.74 a
CK 8.59 ± 0.10 a 37.98 ± 1.23 c 5.10 ± 0.70 c 60.01 ± 8.30 c 6.52 ± 1.48 b 0.56 ± 0.06 b 0.66 ± 0.03 c 12.78 ± 0.17 a
Note: M: manure; NP: NP chemical fertilizer; MNP: manure + NP chemical fertilizer; CK: no fertilizer. AN: alkali-hydrolyzable Nitrogen;
AP: available phosphorus; AK: available potassium; OM: organic matter; TN: total nitrogen; TP: total phosphorus; TK: total potassium;
Different letters in the same column indicate significant differences (ANOVA followed by LSD post hoc test, n = 3, p < 0.05, average value,
SD standard deviation).

3.2. Bacterial α-Diversity

After the removal of chimeras, a total number of 748,247 sequences were obtained
in this study, which are available through the NCBI Sequence Read Archive (Accession:
PRJNA706481). The OTU numbers in the samples did not significantly increase with
the number of sequences sampled and the curve gradually flattened out (Supplementary
Figure S1), indicating that the sample size was large enough and that the library could
satisfactorily characterize the bacterial communities. The ACE and Shannon indices under
different treatments were estimated. As shown in Table 3, the ACE was the highest
in treatment MNP with a combination of manure and chemical fertilizer, followed by
treatments M, NP, and CK, respectively. The Shannon index in the M treatment was
significantly higher than in the CK treatment. However, no significant differences were
observed between the other treatments.

Table 3. α-diversity indices of the bacteria under different treatments.

Treatments ACE Shannon

M 1828.12 ± 6.06 b 6.57 ± 0.01 a
NP 1769.66 ± 7.89 c 6.53 ± 0.02 ab
MNP 1867.82 ± 8.81 a 6.55 ± 0.05 ab
CK 1665.51 ± 5.54 d 6.48 ± 0.02 b
Note: M: manure; NP: NP chemical fertilizer; MNP: manure + NP chemical fertilizer; CK: no fertilizer. Different
letters in the same column indicate significant differences (ANOVA followed by LSD post hoc test, n = 3, p < 0.05,
average value, SD standard deviation).

3.3. Soil Bacterial Community Composition

As shown in Figure 1, 1701 OTUs were shared by different treatments, accounting
for 23.4% of the total OTU of all the samples. Numbers of specific OTUs of treatments M,
Agronomy 2021, 11, x 6 of 17
NP, MNP, and CK were 1, 0, 25, and 0, respectively. The combined application of manure
and chemical fertilizer notably increased the number of unique OTUs in treatment MNP
compared with the other treatments.

Figure 1. Venn Diagram showing unique and overlapped OTUs between different fertilization treat-
ments.
FigureM: manure;
1. Venn NP:showing
Diagram NP chemical
unique fertilizer; MNP:
and overlapped manure
OTUs + NP
between chemical
different fertilizer; CK: no fertilizer.
fertilization
treatments. M: manure; NP: NP chemical fertilizer; MNP: manure + NP chemical fertilizer; CK: no
fertilizer.

3.4. Effect of Different Treatments on Soil Bacterial Communities

PCoA was used to compare the similarity of soil bacterial communities among dif-
Agronomy 2021, 11, 1017 6 of 14

3.4. Effect of Different Treatments on Soil Bacterial Communities

PCoA was used to compare the similarity of soil bacterial communities among differ-
ent treatments at the OTU level. As shown in Figure 2, the plot identified two principal
component factors related to the percentage abundance of groups, explaining 63.91% (PC1)
and 7.99% (PC3) of the total variation. PCoA showed that the four treatments could be
separated clearly. The three repeats of each treatment were clustered together, showing
good repeatability. Samples of treatment NP were parted from the other treatments by
the first axis, while samples of treatments NP and CK were separated from samples of
treatments M and MNP by the second axis. Samples of treatments M and MNP tended to
cluster together, which could also be seen in the clustering tree (Supplementary Figure S2).
In Figure 3, PERMANOVA showed significant differences in the bacterial community
structures among treatments M, NP, MNP, and CK at the OTU level (p < 0.001).

3.5. Taxonomic Composition Analysis at the Phylum and Genus Level

The taxonomic distributions of bacterial communities were evaluated at different
levels of classification. As shown in Figure 4, Proteobacteria, Actinobacteria, Acidobacteria,
Gemmatimonadetes, and Chloroflexi were the most abundant phyla, accounting for ap-
proximately 90% of the bacteria detected in all 12 soil samples. Compared with treatment
CK, the percentage of Proteobacteria increased by 2.49% in treatment M, 0.08% in treatment
NP, and 1.94% in treatment MNP. The percentage of Gemmatimonadetes increased in
treatments M, NP, and MNP by 0.29%, 1.58%, and 0.45%, respectively. However, compared
Agronomy 2021, 11, x 7 of 17
with CK, the relative abundance of Chloroflexi decreased in treatments M, NP, and MNP
by 0.97%, 0.53%, and 1.11%, respectively. The bacterial difference significance analysis can
be found in Table S2.

Figure2.2.PCoA
Figure PCoA ofofthe
thebacterial community
bacterial compositions
community in soil underin
compositions different treatments
soil under based on
different OTUs. M: manure;
treatments based on
NP: chemical fertilizer; MNP: manure + chemical fertilizer; CK: no fertilizer.
OTUs. M: manure; NP: chemical fertilizer; MNP: manure + chemical fertilizer; CK: no fertilizer.
Agronomy 2021, 11, 1017 7 of 14
Agronomy 2021, 11, x 8 of 17

Figure PERMANOVA
Figure 3. PERMANOVA analysis
analysis under under
different different
treatments. treatments.
M: manure; M: manure;
NP: chemical fertilizer; NP:
MNP: manure chemical fertilizer;
+ chem-
ical fertilizer; CK: no fertilizer.
MNP: manure + chemical fertilizer; CK: no fertilizer.
3.5. Taxonomic Composition Analysis at the Phylum and Genus Level
As shown in Figure 5, at genus level, the top ten genera accounted for about 32%
The taxonomic distributions of bacterial communities were evaluated at different lev-
of the total generaels
and belongedAsto
of classification. four
shown phyla:
in Figure Gemmatimonadetes,
4, Proteobacteria, Acidobacteria, Pro-
Actinobacteria, Acidobacteria,
Gemmatimonadetes, and Chloroflexi were the most abundant phyla, accounting for ap-
teobacteria, and Actinobacteria. Compared with CK, the percentage of MND1 decreased
proximately 90% of the bacteria detected in all 12 soil samples. Compared with treatment
by 1.42% in treatment M,percentage
CK, the 1.56% in treatment increased
of Proteobacteria NP, and by2.26%
2.49% in in treatment
treatment M, 0.08%MNP,
in treat-respectively.
ment NP, and 1.94% in treatment MNP. The percentage of Gemmatimonadetes increased
The relative abundance of RB41 decreased by 0.23%, 0.86%, and 0.85% in treatments M,
in treatments M, NP, and MNP by 0.29%, 1.58%, and 0.45%, respectively. However, com-
NP, and MNP, respectively.
pared with CK,On the contrast,
the relative abundance ofthe percentage
Chloroflexi decreased of Arthrobacter
in treatments M, NP, andincreased by
0.03% in treatmentMNPM,by0.88%
0.97%, 0.53%, and 1.11%, respectively.
in treatment NP, andThe bacterial
0.29% indifference
treatment significance
MNP, anal-
respectively.
ysis can be found in Table S2.
The relative abundance of Steroidobacter increased by 0.60%, 0.21%, and 0.74% in treatments
Agronomy 2021, 11, x 9 of 17
M, NP, and MNP, respectively. The bacterial difference significance analysis can be found
in Table S3.

Figure
Figure 4. Taxonomic
4. Taxonomic composition
composition and abundanceand abundance
distribution of bacteriadistribution of bacteria at the phylum level under dif-
at the phylum level
under different treatments. M: manure; NP: chemical fertilizer; MNP: manure + chemical fertilizer;
ferent
CK: treatments. M: manure; NP: chemical fertilizer; MNP: manure +
no fertilizer. chemical fertilizer; CK: no fertilizer.
As shown in Figure 5, at genus level, the top ten genera accounted for about 32% of
the total genera and belonged to four phyla: Gemmatimonadetes, Acidobacteria, Proteo-
bacteria, and Actinobacteria. Compared with CK, the percentage of MND1 decreased by
1.42% in treatment M, 1.56% in treatment NP, and 2.26% in treatment MNP, respectively.
The relative abundance of RB41 decreased by 0.23%, 0.86%, and 0.85% in treatments M,
Agronomy 2021, 11, 1017 8 of 14
Agronomy 2021, 11, x 10 of 17

Figure 5. Taxonomic composition and abundance distribution of bacteria at the genus level under different treatments.
M:Figure 5. Taxonomic
manure; NP: composition
chemical fertilizer; MNP: manure +and abundance
chemical fertilizer; CK:distribution
of bacteria at the genus level under
no fertilizer.
different treatments. M: manure; NP: chemical fertilizer; MNP: manure + chemical fertilizer; CK:
3.6. Effect of Different Treatments on Soil Enzyme Activities
no fertilizer.
Fertilization significantly increased soil enzyme activities compared with CK (Table
4). Three enzyme activities were the highest in MNP treatment, in which the nutrients
3.6. Effect of Different Treatments
were likewise the moston Soil Enzyme
abundant, followedActivities
by the M and NP treatments. There was no
significant difference in the
Fertilization significantly increased soil activities of the three soilactivities
enzyme enzymes between treatments
compared withM and
CK (Table 4).
NP, but they were slightly higher in the M treatment than in the NP treatment. Compared
Three enzyme activities were the highest in MNP treatment, in which the
with CK, SUC activity increased in treatments M, NP, and MNP by 124.1%, 80.9%, and nutrients were
likewise the most145.6%,
abundant, followed
respectively. by the
The increasing M and
range of URENPandtreatments. There was
ALP were 63.9–109.7% no significant
and 4.11–
67.9%.
difference in the activities of the three soil enzymes between treatments M and NP, but
they were slightly
Tablehigher indifferent
4. Effects of the M treatment
treatments thanactivities.
on soil enzyme in the NP treatment. Compared with
CK, SUC activity increased in treatments M,
SUC Activity URE NP, and MNP by 124.1%,
Activity 80.9%, and 145.6%,
ALP Activity
Treatments
(mg·glucose·grange
respectively. The increasing −1·
24h−1) of URE (mg· NH3-N·
and g−1·24h
ALP −1)
were (mg phenol·gand
63.9–109.7% −1·
24h−14.11–67.9%.
)
M 19.55 ± 3.227 ab 1.983 ± 0.021 b 1.820 ± 0.080 b
NP 15.78 ± 1.520 b 1.900 ± 0.290 b 1.404 ± 0.153 bc
Table
MNP
4. Effects of different treatments on soil enzyme
21.42 ± 0.708 a
activities.
2.537 ± 0.218 a 2.264 ± 0.278 a
CK 8.723 ± 0.083 c 1.210 ± 0.128 c 1.348 ± 0.016 c
Note: M: manure; SUC Activity
NP: NP chemical fertilizer; MNP: manure + NP chemical URE Activity
fertilizer; ALPletters
CK: no fertilizer. Different Activity
Treatments
in the same column indicate (mg ·Glucose
significant ·g−1 ·(ANOVA
differences 24 h−1 ) followed
(mg·NH by LSD
3 -N · −1hoc
post · 24 h −1n) = 3,(mg
test, p < Phenol
0.05, ·g−1 ·24
average h−1 )
value, SD standard deviation).
M 19.55 ± 3.227 ab 1.983 ± 0.021 b 1.820 ± 0.080 b
NP 15.78 ± 1.520 b 1.900 ± 0.290 b 1.404 ± 0.153 bc
MNP 21.42 ± 0.708 a 2.537 ± 0.218 a 2.264 ± 0.278 a
CK 8.723 ± 0.083 c 1.210 ± 0.128 c 1.348 ± 0.016 c
Note: M: manure; NP: NP chemical fertilizer; MNP: manure + NP chemical fertilizer; CK: no fertilizer. Different
letters in the same column indicate significant differences (ANOVA followed by LSD post hoc test, n = 3, p < 0.05,
average value, SD standard deviation).

3.7. Correlations
We performed RDA to understand the correlations between phylum-level bacterial
community composition and soil properties and that of enzyme activities. These eight
environmental variables together explained 82.9% of the bacterial community variances.
In addition, the first axis of the RDA accounted for a higher variation of 62.9%, whereas
the second axis accounted for a lower of 20.0% (Figure 6a). The bacterial community
composition had significant correlation with AN (F = 10.892, p = 0.002) and OM (F = 10.847,
p = 0.002).
Agronomy 2021, 11, 1017 9 of 14
Agronomy 2021, 11, x 12 of 17

Figure 6. RDA depicting the relationship between the soil properties, the phylum-level soil bacteria and soil enzyme activi-
Figure 6. RDA depicting the relationship between the soil properties, the phylum-level soil bacteria and soil enzyme ac-
ties. (a) The
tivities. relationship
a. The between
relationship between soil bacterial
soil community
bacterial communitycomposition
composition and properties.
and properties.(b)
b. The
The relationship
relationship between
between soil
soil
enzyme
enzyme activities and properties. c. The relationship between soil bacterial community composition and enzyme activities.
activities and properties. (c) The relationship between soil bacterial community composition and enzyme activities.

Figure 6b illustrated the relationship between enzyme activities and soil characteristics.
4. Discussion
The first and second axes explained 91.2% and 3.0% of the total change of the soil enzyme
4.1. Effects of Treatments on the Soil Physicochemical Properties
activities. The enzyme activities showed significant correlation with AP (F = 44.136,
In theand
p = 0.002) present
AN (Fstudy, no pfertilizer
= 34.551, = 0.002).had been applied in treatment CK for 31 years,
leading
RDA to the lowestphylum-level
between chemical properties.
bacterialLong-term
community fertilization
composition improved
and soilsoil nutrients
enzyme ac-
including AN, AP, AK, OM, TN, and TP. In this study, fertilization lowered soil
tivities was illustrated in Figure 6c and soil enzyme activities were taken as explanatory pH, which
was consistent
variables. withand
The first thesecond
findings
axesofexplained
Wang, who reported
64.9% and 8.9%a decline
of the in soil
total pH with
changes of the
application of fertilizer
bacterial community. The[29]. pH was
bacterial regarded composition
community as one of theshowed
main factors related
significant to fertili-
correlation
zation
with management.
URE (F = 15.559,Ap large number
= 0.002) and ALPof studies have pshown
(F = 14.798, that soil pH decreased with
= 0.002).
excessive application of chemical N fertilizer, resulting in soil acidification [30,31]. In this
study, no significant differences were observed in these treatments, which might be due
to the fact that the calcareous soils containing higher carbonates to buffer soil pH [32].
Agronomy 2021, 11, 1017 10 of 14

4. Discussion
4.1. Effects of Treatments on the Soil Physicochemical Properties
In the present study, no fertilizer had been applied in treatment CK for 31 years,
leading to the lowest chemical properties. Long-term fertilization improved soil nutrients
including AN, AP, AK, OM, TN, and TP. In this study, fertilization lowered soil pH,
which was consistent with the findings of Wang, who reported a decline in soil pH with
the application of fertilizer [29]. pH was regarded as one of the main factors related to
fertilization management. A large number of studies have shown that soil pH decreased
with excessive application of chemical N fertilizer, resulting in soil acidification [30,31]. In
this study, no significant differences were observed in these treatments, which might be
due to the fact that the calcareous soils containing higher carbonates to buffer soil pH [32].

4.2. Effects of Treatments on the Bacterial Diversity and Community Composition

Alpha diversity analysis showed significant difference (p < 0.05) of ACE index under
different fertilization measures, and these were consistent with the previous results [8].
The combination of manure and chemical fertilizer significantly increased the bacterial
ACE index compared with the other fertilization measures. The main reason was that
organic materials themselves contain a variety of nutrient elements, which could increase
the exogenous carbon, promote the formation of aggregate structure, and provide good
conditions for the growth and reproduction of bacteria [33].
PCoA and PERMANOVA showed significant differences (p < 0.001) in bacterial
community structure under different fertilization treatments, consistent with the results
of Liang, who documented a clear separation of bacteria under different fertilization
modes [34]. Fertilization directly affected the soil’s bacterial community structure by chang-
ing nutrients in the soil and by further impacting the biological activity of bacteria in soil.
Additionally, the change of soil pH caused by fertilization had an indirect effect on the bac-
terial community structure. This change might be due to the fact that the optimal pH range
for bacterial growth and reproduction was narrow [35]. The application of manure could
cause significant differences on bacterial community structures compared with chemical
fertilizer [36].
The availability of soil nutrients was usually linked with the transformation from
oligotrophic microorganisms to copiotrophic microorganisms. For example, a high nutrient
availability in soil promoted the growth and reproduction of copiotrophic microorganisms,
while in nutrient-restricted soil environments, the number of oligotrophic microorganisms
that grow slowly would increase [37,38]. Proteobacteria, Actinobacteria, Acidobacteria,
Gemmatimonadetes, and Chloroflexi were the most abundant phyla, accounting for ap-
proximately 90% of the total bacteria, consistent with the previous results reported in
different systems [39,40]. Among these phyla, Proteobacteria is considered to be a kind
of copiotrophic microorganism widely existing in soil and whose abundance is relatively
higher in treatments with manure. Most of the taxa within Acidobacteria are thought
to be slow growing, oligotrophic microorganisms [37]. In the present study, the relative
abundance of Acidobacteria was higher in CK without fertilizer than in NP and MNP by
2.16% and 2.34%, respectively, which indicated that Acidobacteria was more suitable to
nutrient-restricted soil environments [38]. Gemmatimonadetes is dominant in soils with
low water content [41]. The NP treatment in this study seemed to be more suitable for its
survival. Chloroflexi is considered an oligotrophic bacteria in the soil and prefers to decom-
pose some refractory organic compounds [37]. In the present study, the relative abundance
of Chloroflexi decreased in treatments with fertilizer compared with CK. However, the
abundance was relatively higher in treatments M and MNP, compared with treatment NP.
Most of dominant genera are widely distributed in the environment. For example,
Sphingomonas has been found in both aqueous (both fresh- and seawater) and terrestrial
habitats, plant root systems, and others. MND1 can be detected more frequently in soil,
aquatic and subsurface ecosystems [40]. Arthrobacter is commonly found in soils, the aerial
surfaces of plants, and wastewater sediments [41]. Some of the genera have the ability
Agronomy 2021, 11, 1017 11 of 14

to use organic matter to grow and reproduce. For example, Sphingomonas distributes
widely in the environment due to its ability to use a wide range of organic compounds
and to grow and survive in low-nutrient conditions. Arthrobacter can degrade unusual
and polymeric compounds and plays an important role in biodegrading agrochemicals
and pollutants. In the present study, the relative abundance of these genera tended to
be higher in treatments with manure than the others, which was consistent with these
properties. In the previous study, Steroidobacter was reported to be a complex organic
compound degrading bacteria [42], whose abundance was the highest in MNP treatment,
followed by M treatment, and then by NP treatment in this study. On the contrary, the
abundance of RB41 was the highest in CK treatment, consistent with Yan who observed the
highest relative abundance of RB41 in abandoned farmland compared to other treatments
with fertilizer [43].

4.3. Effect of Different Treatments on Soil Enzyme Activities

Enzymatic activity in soil is largely related to the microorganisms present, to available
nutrients and to root exudates, as well [44,45]. Compared with CK, fertilization significantly
increased the activities of SUC, URE, and ALP (p < 0.05). With the increase of nutrients
input, soil enzyme activities gradually increased. Enzyme activities were the highest in
treatment MNP. The results indicated that soil enzyme activity was directly affected by the
amount of fertilizer applicated. Previous studies have shown that manure application was
of paramount importance to improve soil enzyme activities, which significantly increased
enzyme activities more than that of chemical fertilizer application [46].

4.4. Relationship between Soil Bacteria Community Composition and Environmental Factors and
Soil Enzyme Activities
SUC, URE, and ALP were mainly involved in soil’s carbon, nitrogen, and phosphorus
cycles, which were not only limited by soil nutrients, but also affected by other soil
properties. In this study, AP and AN were two significant factors related to the changes
of enzyme activities, which were mainly caused by fertilization regimes. Part of the
soil enzymes are derived from the decomposed materials of bacterial cell exudates and
residues, and in turn, soil bacterial diversity is greatly affected by soil enzyme activities [47].
Moreover, the composition of the bacterial community was influenced by its environment,
including soil properties. Furthermore, through the RDA analysis, we found that AN,
OM, URE, and ALP were main factors attributed to bacterial community structure in this
region. This might be due to OM-provided substrates and energy for bacterial reproduction
and metabolism [2]. Alkali-hydrolyzable nitrogen and carbon were coupled to participate
in many bacterial metabolic processes. Previous studies also reported that microbial
community composition had a significant correlation with URE [47].
In the present study, MNP treatment had the best effect on the improvement of
bacterial diversity and enzyme activity, not only because of the maximum input of nitrogen,
phosphorus, and potassium, but also because of the key advantages of combining of manure
and chemical fertilizer application. Admittedly, manure contains a variety of nutrient
elements, which can increase the input of exogenous carbon, promote the formation of
aggregate structures, reduce soil bulk density, enhance the activity of microorganisms,
promote the growth of crop roots and above-ground parts, and even increase the activity
of soil enzymes [48–50]. However, a single application of manure alone is not conducive
to the growth of crops, possibly because the slow and delayed release of nutrients in
organic fertilizer cannot meet the needs of crops for nutrients in time [51]. Chemical
fertilizers can provide quick nutrients, but studies have shown that a single application
of chemical fertilizer can easily lead to soil degradation and is not suitable for the growth
and reproduction of microorganisms. By contrast, manure has a good buffering ability
to protect crops from adverse fluctuations and enhance the anti-interference ability of the
soil [52,53]. Thus, manure and chemical fertilizer cooperation can foster strengths and
circumvent weaknesses of either technique, and this can provide a more balanced supply
of nutrients, just as it can also promote the growth of crops more effectively.
Agronomy 2021, 11, 1017 12 of 14

5. Conclusions
Various degrees of difference in bacterial community structures, soil enzyme activities
and soil properties were observed with manure, chemical fertilizers, or both. As a whole,
the combination of manure and chemical fertilizer showed great advantages, which could
be a promising method in agricultural production. The proportion of the combination,
however, has yet to be explored for different crops in the future.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/

10.3390/agronomy11051017/s1, Figure S1: Rarefraction Curve. M: manure; NP: chemical fertilizer;
MNP: manure + chemical fertilizer; CK: no fertilizer. Figure S2: UPGMA clustering tree. M: manure;
NP: chemical fertilizer; MNP: manure + chemical fertilizer; CK: no fertilizer. The diatance equals to
0.01. Table S1: F and p-value of soil properties between groups from the ANOVA. Table S2: Difference
analysis of bacteria at phylum level. Table S3: Difference analysis of bacteria at genus level.
Author Contributions: Conceptualization: H.Z.; Writing-Original draft preparation: Z.L. and W.X.;
Resources: Z.Y. Supervision: X.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Key R&D plan of Shanxi Province (201703D211002-3), Key
R&D plan of Shanxi Province (201703D211002-4), Applied and Basic Research Program of Shanxi
Province (201901D211557), Applied Basic Research Program of Shanxi Academy of Agricultural
Sciences (YBSJJ2012).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original sequence data are available through the NCBI Sequence
Read Archive (Accession: PRJNA706481).
Acknowledgments: We thank Biomarker Technologies Corporation for assistance in bioinformatics
analysis.
Conflicts of Interest: The authors declare no conflict of interest.

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