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Applied Soil Ecology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Applied Soil Ecology

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

Long-term application of manure over plant residues mitigates acidification,

builds soil organic carbon and shifts prokaryotic diversity in acidic Ultisols
Guiping Yea, b, Yongxin Lina, Deyan Liua, , Zengming Chena, Jiafa Luoc , Nanthi Boland,
Fan Jianbo , Weixin Dinga,
a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
AgResearch Limited, Ruakura Research Centre, Hamilton 3240, New Zealand
dGlobal Center for Environmental Remediation, University of Newcastle, Newcastle, NSW 2308, Australia

ARTICLE INFO ABSTRACT

Keywords: Addition of organic materials is believed to be a feasible practice for mitigating Ultisols acidification and loss of soil
Long-term fertilization organic carbon (SOC). However, how organic materials mitigate acidification, affect SOC content and aggregation
Organic materials and shift microbial community structure requires further investigation. In this study, we used high throughput
Soil organic carbon
sequencing of microbial DNA to evaluate the relationships between soil properties, aggregation and prokaryotic
Soil aggregation
communities in soil subjected to 27 years of inorganic and organic fertilization. The field experiment included
Prokaryotic community
seven treatments: no fertilization (control), inorganic NPK fertilizer (I), inorganic NPK fertilizer plus liming (CaCO3)
(IL), and inorganic NPK fertilizer plus peanut straw (IPS), rice straw (IRS), radish (IR), or pig manure (IPM).
Amendment with NPK fertilizer plus pig manure more effectively increased soil pH, SOC, total nitrogen (TN),
available phosphorus (AP) and dissolved organic carbon (DOC) compared with NPK fertilizer plus crop residues.
IPM also increased the mass proportion of large macroaggregates (> 2000 m) from 7.8% in the control to 30.6%
while it reduced effective diffusion coefficient of oxygen (DCo) from 12.58 × 10ÿ6 m2 s in the control to 2.81 ×1 10ÿ 6
m2 s 1
. Application of pig manure increased prokaryotic diversity and altered pro
karyotic community structure, while crop residues did not. Soil pH was the predominant factor influencing
prokaryotic community structure. Bacillales and Clostridiales accounted for 47.5% and 21.4%, respectively of the
indicator species in the HDI and the relative abundances of them were increased, compared with the other
treatments. Furthermore, the relative abundances of Bacillales and Clostridiales were correlated with SOC, TN,
AP and DOC, and negatively with DCo in the soil. Overall, our results suggest that application of NPK fertilizer
plus pig manure rather than crop residues enhanced soil pH, improved SOC content and aggregation, increased
prokaryotic diversity and altered community structure of prokaryote after 27-year fertilization.

1. Introduction hilly Ultisols regions, and generally monocropped on a large scale.

Ultisols are widely distributed throughout tropical and subtropical regions
(Alvear et al., 2005; Hauser et al., 2006), representing 1105 million ha and
accounting for 8% of the world's ice-free land (Lal, 2004). The Ultisols region
is under great pressure, in order to meet an increasing demand for food (Zhang
and He, 2004). Moreover, Ultisols are important, typical agricultural soils and
support 22.5% of the population in China (Lou et al., 2003). However, even
with abundant water and heat content, the productivity of Ultisols is low, mainly
due to its low fertility and strong acidity (Liu and He, 1991; Clair and Lynch,
2010). Peanut (Arachis hypogaea L.) is a predominant upland crop in
However, consecutive monoculturing has, in a decrease in crop yield and
quality as well as an increase in disease occurrence (Larkin, 2003; Liu et al.,
2015).
To improve crop yield in Ultisols regions, the annual application rate of
inorganic fertilizer has increased from 90 to 300 kg N haÿ1 in the past three
decades. Excessive loading of inorganic N fertilizers has resulted in serious
soil degradation in the form of soil acidification and losses of organic carbon
(Guo et al., 2010), subsequently reducing the use efficiency of inorganic
fertilizers (Horrigan et al., 2002). New strategies at preventing soil degradation
caused by long-term application of inorganic fertilizers are therefore urgently
required.

Corresponding authors.
E-mail addresses: dyliu@issas.ac.cn (D. Liu), wxding@issas.ac.cn (W. Ding).

https://doi.org/10.1016/j.apsoil.2018.09.008
Received 23 April 2018; Received in the revised form 27 August 2018; Accepted 11 September
2018 0929-1393/ © 2018 Elsevier BV All rights reserved.

Please cite this article as: Ye, G., Applied Soil Ecology, https://doi.org/10.1016/j.apsoil.2018.09.008
Machine Translated by Google
G. Ye et al.
Adoption of management practices on agricultural lands and degraded
soils would enhance soil organic carbon (SOC) and nutrients content,
available water holding capacity, and soil aggregation (Manna et al.,
2005; Lal, 2006). It has been suggested that a combined application of
inorganic fertilizers with organic materials (eg crop residues and stock
manure) can efficiently improve soil properties (Reeves, 1997; Lv et al.,
2011) and maintain or increase soil productivity (Ai et al. , 2015). Liming
is also an effective practice for increasing soil pH (Li et al., 2014).
However, soil physiochemical and biological properties respond differently
to different management practices (Sun et al., 2015). A decrease in soil
pH was observed under addition of wheat straw (Hazarika et al., 2009)
due to the generation of acidic compounds during cellulose degradation
(Leschine, 1995; Kato et al., 2005). In contrast, Materechera and
Mkhabela (2002) found that the manure amendment increased the pH of
acid soils by buffering of carboxyl, phenolic hydroxyl groups and
bicarbonates in the manure. Iyamuremye and Dick (1996) suggested that
either wheat straw or manure amend ment could increase soil pH in
Ultisols, with greater increases observed under manure addition.
Meanwhile, it has also been suggested that SOC is more efficiently
increased with application of manure compared to crop straw (Mando et
al., 2005; He et al., 2015). Despite these findings, the potential effects of
management practices on soil properties require further investigation.
Application of organic and inorganic fertilizers was reported to affect
the diversity, composition, abundance, and functioning of soil
microorganisms (Marschner et al., 2003; Cederlund et al., 2014; Kumar
et al., 2017). However, the amendment of various organic materials might
exert different impacts on microbial diversity. The addition of manure,
alone or in combination with inorganic fertilizer, was found to increase
soil bacterial diversity (Enwall et al., 2007), while rice straw application in
paddy fields did not (Ahn et al., 2012). A decrease in bacterial diversity
has also been observed under long-term inorganic fertilization (Ramirez
et al., 2010; Chen et al., 2016). Furthermore, short-term application of
lime after long-term inorganic fertilization was found to increase bacterial
diversity in an acid soil (Xun et al., 2016). However, long-term application
of lime experiments was required since it takes a long time for the
microbial community structure to stabilize (Reardon et al., 2014).
Long-term fertilization can also alter the community structure of
microbes by regulating various soil physiochemical properties. Soil pH
(Enwall et al., 2007; Rousk et al., 2010; Sun et al., 2015), nutrients
(Ramirez et al., 2010; Xun et al., 2016) or aggregation (Davinic et al.,
2012 ; Zhang et al., 2015) were found to influence microbial community
structure greatly, although which variables played a key role is still
debated. Lentendu et al. (2014) suggested that microbial community
composition is influenced more by organic manure than inorganic fer
tilizer, since soil physiochemical properties were changed more vigor
ously by application of organic manure. Moreover, application of or ganic
manure might select for different types of microorganisms than
amendments with inorganic fertilizers. For example, the abundance of
Actinobacteria was found to decrease under manure amendment but
increase under inorganic N application (Peacock et al., 2001). In contrast,
organic manure application stimulated certain microbial groups such as
Firmicutes, Proteobacteria and Zygomycota, which are known to prefer
nutrient-rich environments (Francioli et al., 2016). More in investigations
are required to understand how long-term application of inorganic and
organic fertilizer influences microbial groups in Ultisols.
In order to monitor changes in soil properties in Ultisols, a long term
(27 years) field experiment examining the effects of organic and inorganic
fertilization was implemented. In this study, high-throughput sequencing
was used to evaluate the diversity and composition of microbial
communities under these treatments. The objectives were to (1)
investigate the effect of long-term application of inorganic and organic
fertilizers on soil properties, aggregates, and the overall prokaryotic
community; and (2) evaluate the key factors influencing prokaryotic
diversity and community structure.
2
Applied Soil Ecology xxx (xxxx) xxx–xxx
2. Materials and methods
2.1. Field experiment
The study site was located at the Yingtan Red Soil Ecology
Experimental Station, Chinese Academy of Sciences, Yujiang county,
Jiangxi Province, China (28°15ÿ20ÿN, 116°55ÿ30ÿ E). The region has a
typical subtropical monsoon climate, with a mean annual temperature of
17.6 °C and mean annual precipitation of 1795 mm for the last 30 years.
The soil, derived from quaternary red clay, is classified as Typic Plinthudult
(Ultisols) based on the US Department of Agriculture soil taxonomy. The
surface soil (0–20 cm) contains 41.2% clay, 33.2% silt and 25.6% sand.
The field experiment was commenced in April 1988 and consisted of
three replicates of seven treatments: no fertilization (control), inorganic
NPK fertilizer (I), inorganic NPK fertilizer plus liming (CaCO3) (IL), and
inorganic NPK fertilizer plus peanut straw ( IPS), rice straw (IRS), radish
(IR), or pig manure (IPM). A randomized block design was used to al
locate the different treatments to field plots. The size of each plot was
34.6 m2 with concrete walls embedded 100 cm into the soil between
individual plots. The field was cultivated with peanut monocropping in the
summer and left fallow in winter. Annual application rates of fer tilizer N,
P and K in the I and IL treatments were 120 kg N haÿ1 as urea, 30 kg P
haÿ1 as calcium magnesium phosphate, and 90 kg K haÿ1 as potassium
chloride, respectively . Under the IPS, IRS, IR and IPM treatments, 30%
of the inorganic fertilizer N was replaced with organic N. Pig manure was
stockpiled on a concrete slab for 3 months before application. The
properties of the organic materials are shown in Table S1. The amounts
of total N, P and K were the same in all the treatments (Table S2).
Amounts of P and K in the applied organic materials were generally less
than the prescribed doses; therefore, application of the organic materials
soils were supplemented with calcium magnesium phosphate and
potassium chloride, depending on their P and K content.
Under IL treatment, CaCO3 was applied at 1500 kg haÿ1 yrÿ1 . The
fertilizer and organic materials (crop residues were cut into 5 cm lengths)
were evenly spread onto the soil surface by hand and im mediately
incorporated into the plowed soil (0–20 cm) by tillage before sowing.
Peanut (Ganhua 5) was sown manually on April 10 each year, two seeds
per hole, with 20 cm plant-to-plant spacing and 30 cm row to-row spacing.
2.2. Soil sampling and analysis
Plow layer (0–20 cm) soil samples were collected on 13 December
2014. Ten intact soil cores (10-cm diameter) were randomly collected
from each plot and combined to form one composite sample. Soil samples
were transported to the laboratory from the experimental site in a constant
temperature box with ice. After visible stones and plant residues were
carefully removed with forceps, moist soil samples were gently broken
apart along natural-break points and passed through an 8-mm sieve.
After thorough mixing, the fresh soil samples were im mediately divided
into three subsamples. One subsample was sieved to < 2 mm to reduce
heterogeneity, then stored at 80 °C for DNA extraction; the second was
air-dried for analysis of soil properties; and the third was stored at 4 °C
for laboratory incubation and wet-sieving for analysis of water-stable
aggregates.
Soil pH was measured with a glass electrode using a 1:5 soil-water
ratio. Concentrations of SOC and total nitrogen (TN) were determined
using the wet oxidation redox titration method and micro-Kjeldahl method,
respectively (Lu, 2000). Soil dissolved organic carbon (DOC) was
extracted by shaking fresh soil (equivalent to 10 g oven-dried) with 50 ml
deionized water for 30 min on an end-over-end shaker at 25 °C.
After extraction, samples were then centrifuged at 10000 rpm for 10 min
at 4 °C and the supernatants filtered through a 0.45-ÿm mem brane filter
(Whatman, Clifton, NJ, USA). DOC in the extracts was analyzed on a
Shimadzu C analyzer (TOC-VCPH, Shimadzu, Kyoto,
Machine Translated by Google
G. Ye et al.
Japan). Soil available phosphorus (AP) was extracted with 0.0125 M
H2SO4 in 0.05 M HCl and determined using the molybdenum blue
methods. Available potassium (AK) in the soil was extracted with 1 M
ammonium acetate and determined using flame photometry (FP640,
INASA, China).
Six undisturbed soil core samples (100-cm3 cylinder) were obtained
from each replicate plot and used to measure water retention curves
with a ceramic pressure plate at equilibrium matrix potentials of 0.1,
0.2, 1, 3.5, 6, 10, 33, 50, 100, 200, 500, and
1500 kPa in a pressure chamber. The effective diffusion coefficient of
1
oxygen in the soil (DCo, m2 s ) was calculated as follows (Aachib
et al., 2004):
2 Clara, CA, USA). Separation was conducted using a 177/149-ÿm (80/
= 1/ KH× DQ
DCo NDQ ×+
[ ao× × pa p wo w ] (1)
where N is the soil porosity; Dao is the free diffusion coefficient of
1
oxygen in air at 20 °C (1.8 × 10ÿ5 m2 s ); KH is Henry's equilibrium
constant at 20 °C (0.03); Dwo is the free diffusion coefficient of oxygen in
1
water at 20 °C (2.2 × 10ÿ9 m2 s ); Qa and Qw are the proportions of
soil porosity occupied by water and water, respectively (Qa + Qw = 1);
and p is the power constant (3.4). Soil porosity (N) was calculated as
follows:
N=1/0
where is the soil bulk density (g cmÿ3 ); and 0 is the soil particle
density (g cmÿ3 ). The proportion of soil porosity occupied by water
(Qw) was calculated as follows:
Qw = × w / N
where w is the soil gravimetric moisture content (cm3 gÿ1 ).
2.3. Soil fractionation
Water-stable aggregates in the soil were separated using the wet sieving
protocol described by Elliott (1986). Briefly, 100 g (on an oven dried basis) of
moist soil was submerged in deionized water for 5 min
on top of a 2000-ÿm sieve at room temperature. The sieve was manually
moved up and down 3 cm, and repeated 50 times within 2 min. The
fraction remaining on the 2000-ÿm sieve was collected in a pre-weighed
aluminum pan, then the water and soil that passed through the sieve
were poured through a 250-ÿm sieve, and the sieving procedure was
repeated. The water plus < 250 m soil fraction was poured through a
53-ÿm sieve, and the sieving procedure was repeated. To obtain a soil
fraction with a particle size of < 53 m, the remaining suspension was
poured into 250-mL centrifuge tubes and centrifuged at 5000 rpm for
30 min at 4 °C. The pellets were then re-suspended in deionized water
and re-centrifuged three times under the same conditions. Accord ingly, >
2000 m, 250–2000 m, 53–250 m, and the < 53 m frac tions were separated.
An aliquot of each fraction was used to determine
the moisture content, and a second subsample dried at 50 °C for de
termination of the organic C content. In this study, we did not do sand
corrections since sand grains can be completely embedded within large
aggregates and sand is considered as a legitimate component of the
macroaggregates as suggested by Zhang et al. (2013b).
The mean weight diameter (MWD, m) of the soil was calculated
using the following formula (Kemper and Rosenau, 1986):
n
MWD x W =_ ii
i= 1
where xi represents the mean diameter of each size of aggregate; and Wi
represents the weight percentage of size i of the aggregates.
2.4. Laboratory incubation
Ten grams (on an oven-dried basis) of bulk soil from each plot was
placed in a 150-ml glass jar and adjusted to a water holding capacity of
Applied Soil Ecology xxx (xxxx) xxx–xxx
60% (Cai et al., 2001). The jars were covered with plastic wrap with
needle-punctured holes to maintain aerobic conditions, and incubated
in the dark at 25 °C. During incubation, soil moisture was adjusted
every other day by adding deionized water (to maintain the initial
weight) with mini-pipettes. Carbon dioxide (CO2) emission rates were
measured at 0.5, 1, 2, 3, 5, 7, 9, 11, 14, 17, 20, 24, and 30 days after
beginning of incubation. To measure the CO2 flux, each jar was sealed
using an airtight butyl rubber stopper perforated with centered Perspex
tubes for sampling. Immediately after closure and again after 6 h, 1 ml
of headspace gas was sampled using an airtight syringe for measure ment of
CO2 concentrations using a gas chromatograph equipped with a
thermal conductivity detector operated at 60 °C (Agilent 7890, Santa
100 mesh) Chromosorb 102 column (Advanced Minerals, Santa Barbara,
CA, USA) at 40 °C. The carrier gas (H2) flow rate was 80 ml minÿ1
and the temperature of the injecting port was 100 °C. Carbon dioxide
gas standards were supplied by the National Research Center for Certified
Reference Materials, Beijing, China. CO2 fluxes were calculated
using a linear model from the change of CO2 concentration in the jar
over a 6-h period with an average chamber temperature of 25 °C.
Specific C mineralization rates in the soil were calculated as follows (Yu
et al., 2012b):
(2)
Specific C mineralization rate=C mineralization rate/SOC content (5)
2.5. DNA extraction and high-throughput sequencing
(3)
Soil nucleic acid was extracted from fresh soil (equivalent to 0.5 g
oven-dried) using the Fast DNA Spin Kit for Soil (MP Biomedicals, CA,
USA) according to the manufacturer's protocol. The quality and abun dance
of the extracted DNA was measured by gel electrophoresis (0.8%
agarose) and NanoDrop spectrophotometer (NanoDrop Technologies
Inc., Wilmington, DE).
Universal primers 515F (5ÿ-GTGCCAGCMGCGCGCGGTAA-3ÿ) and
909R (5ÿ- CCCCGYCAATTCMTTTRAGT -3ÿ) with 12-nt unique barcodes
were used to amplify the V4-V5 hypervariable region of the 16S rRNA
gene for high-throughput sequencing using a Miseq sequencer
(Caporaso et al., 2011). The PCR mixture (25 l) contained 1 × PCR
buffer, 1.5 mM MgCl2, each deoxynucleoside triphosphate at 0.4 M,
each primer at 1.0 M, 0.5 U of Ex Taq (TaKaRa, Dalian) and 10 ng soil
genomic DNA. The PCR amplification program included initial dena turation
at 94 °C for 3 min followed by 30 cycles of 94 °C for 40 s, 56 °C
for 60 s, and 72 °C for 60 s, and final extension at 72 °C for 10 min. Two
PCR reactions were conducted per sample and combined after PCR
amplification. PCR products were subjected to electrophoresis using
1.0% agarose gel and appropriate bands excised and purified using a
SanPrep DNA Gel Extraction Kit (Sangon Biotech, China, Cat# SK8132),
then quantified with Nanodrop. All samples were pooled together on a
molar proportional basis. Sequencing samples were prepared using a
TruSeq DNA kit according to the manufacturer's instructions. The
purified library was diluted, denatured, re-diluted and mixed with PhiX
(equal to 30% of final DNA amount) according to the Illumina library
preparation protocol then applied to an Illumina Miseq system for se quencing
with the Reagent Kit v2 2 × 250 bp according to the manu facturer.
Paired-end raw reads were initially merged using FLASH (version
1.2.7) (Magoÿ and Salzberg, 2011), in which forward and reverse reads
of same sequence with at least 10 bp overlap and no base mismatches
(4)
were combined as single sequence. Sequence data were then processed
using QIIME Pipeline-Version 1.7.0 (http://qiime.org/). All sequences
reads were trimmed and assigned to each sample based on their bar code.
High-quality sequences (length > 300 bp, without an ambiguous
base 'N', and with an average base quality score > 30) were used for
downstream analysis. Sequences were clustered into operational taxo nomic
units (OTUs) at a 97% identity threshold using the Uparse software (Uparse
v7.0.1001) (Edgar, 2013), and aligned gene sequences
3
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G. Ye et al. Applied Soil Ecology xxx (xxxx) xxx–xxx

15
used for chimera checking using the Uchime algorithm (Edgar et al.,
2011). All samples were randomly resampled to 19,000 reads which
were approximately the lowest sequencing depth across all samples.
Two diversity indices, the Chao 1 index (Chao, 1984) and Faith's index 12

of phylogenetic diversity (Faith's PD) (Faith, 1992), were used to

compare soil bacterial alpha diversity. Taxonomy was assigned using y = 17250 e-0.059 x
R2 = 0.938
the Ribosomal Database Project classifier (Wang et al., 2007). All 9
P < 0.0001
sequences have been deposited in the DNA Data Bank of Japan under the
accession number DRA006143.

6
2.6. Data analysis and statistics

Statistical analyzes were carried out using the software package

3
SPSS 20.0. The Kolmogorov-Smirnov test was applied for data normality
assessment prior to analysis of variance (ANOVA), which were
then ln-transformed to improve the normality of data distribution if
5 10 15 20 25 30 35
necessary. Differences in soil properties and microbial
community composition were analyzed using one-way analysis of variance Mass proportion of large macroaggregates (%)

(ANOVA) followed by the least significant difference (LSD) test
for multiple comparisons at P < 0.05 (unless otherwise stated).
Regression analyzes were used to determine the relationships between
the mass proportion of large macroaggregates and DCo. Pearson analysis
was used to extensively investigate the correlations between soil
variables and the prokaryotic diversity indices or the relative abun dance
of indicator species.
R software package (version 3.1.2) with the “vegan” package was
utilized to conduct the following analyzes unless otherwise stated. first,
hierarchical clustering was determined by the unweighted pair group
method with an arithmetic mean (UPGMA) (Milligan, 1985), using
weighted UniFrac distances. As best-fit model, linear redundancy analysis
(RDA) was carried out to determine the effect of soil properties on
the prokaryotic community and was plotted by Origin 8.5 software. The
Forward-selection procedure during redundancy analysis was used to
select the soil physicochemical variables with the greatest significant
influence on soil microbial community. The Monte Carlo test with 999
permutations under a reduced model gave the P value associated with
the effect of soil variables on microbial composition. Only soil variables
that were significantly (P < 0.05) correlated with the RDA model were
selected. Finally, a Mantel test was used to determine the soil physi
cochemical variables that significantly correlated with soil microbial
community. Indicator species analysis was carried out using “in
discspecies” package (Cáceres and Legendre, 2009) in R and visualized
using the Cytoscape software (Version 3.2.1) (Shannon et al., 2003).
3. Results
3.1. Soil characteristics
The highest soil pH of 6.59 was observed under IL treatment, and
was higher (P < 0.01) than that under other treatments (Table 1).
Fig. 1. Relationship between the mass proportion of large macroaggregates and
effective diffusion coefficients of oxygen in the soil.
Long-term (27 years) application of inorganic NPK fertilizer also in
creased soil pH (P < 0.01) from 4.96 in the control to 5.20. Compared
with the I treatment, inorganic NPK fertilizer plus pig manure treatment
(IPM) further increased soil pH while combined application of in organic
NPK plus crop residues (IPS, IRS and IR) did not. Compared
with the control, fertilization increased SOC and TN contents, with
highest increases of 44.3% and 44.1%, respectively, under HDI treat
ment. Fertilization increased AP concentration, with the highest con
centration of 156.73 mg P kgÿ1 under IPM treatment. There were no
significant differences in concentrations of SOC, TN and AP between I
and IL treatment. The highest DOC concentration was observed under
IPM treatment, and was higher (P < 0.01) than that under all other
treatments, while no remarkable differences were observed among the
other treatments. The DCo in the control soil was estimated at
12.58 × 10ÿ6 m2 s 1 , which was higher than that under all other
1
treatments, while the lowest value of 2.81 × 10ÿ6 m2 s was observed
under HDI treatment. Moreover, DCo was found to be negative
(P < 0.01) correlated with the mass proportion of large macro aggregates
(Fig. 1).
The sum of the mass of large macroaggregates (> 2000 m), small
macroaggregates (250–2000 m), microaggregates (53–250 m) and
the free silt plus clay fraction (< 53 m) accounted for 98.2–100% of
the bulk soil in all the treatments. Under IPM treatment, the MWD was
increased by 103% compared with the control (Table 2), while the mass
proportion of large macroaggregates increased (P < 0.01) from 7.8%
in the control to 30.6%. In contrast, the mass proportion of small
macroaggregates and microaggregates decreased under HDI treatment.
Except for IRS treatment, the remaining fertilizer treatments had no

Table 1
Effect of long-term (27 years) application of inorganic and organic fertilizers on soil properties.
Treatment pH SOC TN AP AK DOC DCo

–1
(1:5 H2O) (g C kgÿ1 ) (g N kgÿ1 ) (mg P kgÿ1 ) (mg K kgÿ1 ) (mg C kgÿ1 ) (×10–6 m2 s )

Control 4.96d‡ 5.69d 0.59d 5.25d 84.65c 4.27b 12.58a

I 5.20c 6.44c 0.70c 15.00bc 177.59b 4.38b 10.71b
IL 6.59a 6.32c 0.72c 16.40b 203.10a 5.33b 9.95b
IPS 5.13c 6.62bc 0.78b 10.30c 178.02b 4.35b 9.96b
IRS 5.16c 7.13b 0.82ab 10.78c 206.69a 5.83b 8.20c
IR 5.17c 6.76bc 0.82ab 11.63bc 204.84a 5.63b 9.70bc
HDI 5.58b 8.21a 0.85a 156.73a 170.77b 12.25a 2.81d

ÿ
Abbreviations: SOC, soil organic carbon; TN, total nitrogen; AP, available phosphorus; AK, available potassium; DOC, dissolved organic carbon; DCo, effective
diffusion coefficient of oxygen.
Values with in a column followed by the same letter are not significantly different at P 0.05.

4
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G. Ye et al. Applied Soil Ecology xxx (xxxx) xxx–xxx

Table 2
Effect of long-term application of inorganic and organic fertilizers on the mass proportion and organic C concentration of aggregates.

Treatment MWD† (ÿm) Proportion (%) Organic C (g C kgÿ1 )

LM SM m SC LM SM m SC

Control 944c‡ 7.80c 45.33a 25.55a 21.32a 5.87c 6.01d 5.16b 5.74d
I 968c 8.20c 45.75a 24.65a 21.40a 6.49b 6.60cd 5.56ab 6.09cd
IL 972c 8.15c 46.47a 23.82a 21.55a 6.34b 6.61cd 5.58ab 6.09cd
IPS 1014bc 9.14bc 45.69a 24.71a 20.47a 6.46b 7.23bc 5.69ab 6.38bc
IRS 1155b 11.84b 46.84a 20.54bc 20.78a 6.65b 7.96ab 5.88a 6.33bc
IR 1022bc 9.36bc 45.58a 23.45ab 21.62a 6.64b 7.46bc 5.66ab 6.29bc
HDI 1913a 30.63a 30.93b 18.52c 19.93a 9.54a 8.61a 6.08a 6.92a

Abbreviations : MWD, mean weight diameter; LM, large macroaggregates; SM, small macroaggregates; m, microaggregates; SC, silt plus clay fraction. Values
with in a column followed by the same letter are not significantly different at P 0.05.

160000 900
significant effect on the mass proportion of large macroaggregates
Chao1
a
compared with the control. All fertilizer treatments increased the or ganic C Faith's PD
content in large macroaggregates, and organic fertilizer treatments also a

increased the organic C content in small macroaggregates ab 800

140000 ab
and the silt plus clay fraction. Only the IPM and IRS treatments caused b ab
b b
an increase in the organic C content of the microaggregates compared b b
with the control.
b b b Faith's
PD
Chao1
120000 700
b
3.2. Mineralization rates of soil organic C

Compared with the control, fertilization increased the SOC mineral

100000 600
alization rates (Table 3). The C mineralization rate under HDI treatment
was 4.81 mg C kgÿ1 dÿ1, which was lower than that under NPK plus
crop residue treatments (IPS, IRS and IR). The specific mineralization
rate of SOC, expressed as the mineralization rate per unit SOC, ranged 80000 500
from 0.59 to 0.80 mg C gÿ1 SOC dÿ1 under all treatments. The lowest Control I IL IPS IRS IR HDI

specific C mineralization rate was observed with IPM treatment, and

Fig. 2. Comparison of the estimated Chao 1 index and Faith's index of phylo
was lower (P < 0.01) than that under all other treatments. in contrast,
genetic diversity of the 16S rRNA gene libraries from high-throughput sequencing
inorganic NPK plus crop residue treatments (IPS, IRS and IR) increased analysis. All indices were calculated using the subset of 19,000 se quences per
(P < 0.01) the specific C mineralization rate compared with the control. sample. Vertical bars denote the standard errors of the mean
(n = 3). Pairs of bars with the same letter are not significantly different at
P 0.05.
3.3. Prokaryotic communities
1.2
Control
The highest prokaryotic diversity and richness was observed under I
MWD
IPM treatment, and was higher than that under the control treatment SOC AP IL
0.8
(Fig. 2). In contrast, no obvious differences were observed among any TN DOC IPS
other treatments. The Chao1 and Faith's PD indices were correlated IRS
IR
with SOC, AP, DOC and MWD, and negatively correlated with DCo 0.4
HDI
(Table S3). Hierarchical cluster analysis indicated high dissimilarity of
prokaryotic communities between the control and IL or IPM treatments
0.0
(Fig. S1). In contrast, prokaryotic communities were similar and clustered
together among control, I, IPS, IRS and IR treatments. Red dundancy (9.86%)
RDA2

analysis confirmed these results, showing that the prokar yotic communities -0.4 pH

were different under IL or IPM treatment compared

Table 3 -0.8
DCo
Effect of long-term (27 years) application of inorganic and organic fertilizers on
organic C mineralization rates and specific C mineralization.
-1.2
Treatment C mineralization rate -0.6 -0.3 0.0 0.3 0.6 0.9 1.2
Specific C mineralization rate
RDA1 (23.07%)
(mg C kgÿ1 dÿ1 ) (mg C gÿ1 SOC dÿ1 )
Fig. 3. A redundancy analysis plot depicting the correlation between prokaryotic
Control 4.01e† 0.71cd community structure and soil properties. SOC, soil organic carbon; TN,
I 4.39d 0.68d total nitrogen; AP, available phosphorus; DOC, dissolved organic carbon; DCo,
IL 4.69cd 0.74bc effective diffusion coefficient of oxygen; MWD, mean weight diameter.
IPS 5.20b 0.79ab
IRS 5.66a 0.79a
IR 5.39ab 0.80a with the other treatments (Fig. 3). Coat tests showed that soil pH was
HDI 4.81c 0.59e the primary influencing factor of prokaryotic communities (P = 0.001),
while AP, DOC, DCo and MWD also influenced the community structure
Values with in a column followed by the same letter are not significantly (P = 0.020–0.048) (Table 4).
different at P 0.05.

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G. Ye et al. Applied Soil Ecology xxx (xxxx) xxx–xxx

Table 4
Mantel test correlations between prokaryotic community structure and soil
physicochemical properties.
Soil variables r P
pH 0.830
SOC 0.250
TN 0.156
AP 0.338
AK 0.093
DOC 0.302
DCo 0.304
MWD 0.282
Abbreviations : SOC, soil organic carbon; TN, total nitrogen; AP, available
phosphorus; AK, available potassium; DOC, dissolved organic carbon; DCo,
effective diffusion coefficient of oxygen; MWD, mean weight diameter.
The dominant phyla across all the treatments were Crenarchaeota,
Acidobacteria, Proteobacteria, Chloroflexi, Cyanobacteria and
Actinobacteria, which together accounted for more than 68% of the total
prokaryotic sequences (Table S4). For further analysis of the high throughput
sequencing data, we calculated the relative percentage of
variations in dominant phylum (relative abundance > 1%) under IL
and IPM treatment compared with the control, since only the community
structure under these treatments was different from the control.
The relative abundances of Proteobacteria and Actinobacteria increased,
while those of Chloroflexi and AD3 decreased under IL and IPM treatment
compared with the control (Fig. 4). In addition, the relative
abundance of Crenarchaeota, Gemmatimonadetes and Bacteroidetes in
creased under IL treatment by 51.1%, 87.4% and 155.5%, respectively,
while the relative abundance of Planctomycetes decreased by 17.8%.
The lowest relative abundance of Acidobacteria (9.5%) was observed
under IPM treatment, and was lower than that under the control
treatment. In contrast, the highest relative abundance of Firmicutes was
found under IPM treatment, increasing (P < 0.01) by 406% compared
with the control, mainly due to the increased abundance of Bacilles
(the dominant order of the phylum Firmicutes). The relative abundance
of Bacilles was higher (P < 0.01) under IPM treatment than all other
treatments (Fig. S2), and was 4.69-fold higher than that under the
control treatments.
Indicator species analyzes (genera) were conducted to identify
0.001 prokaryotic genera that were specifically associated with IPM treatment
0.052
and were presented in Fig. 5 and their taxonomic assignments are listed
0.105
0.020 in Table S5. The most abundant indicator species belongs to the order
0.220 Bacillales and accounted for 47.5% of all indicator species, with Clos
0.044 tridiales and Burkholderiales accounting for 21.4% and 18.0%, respec
0.035
tively. Like Bacilles, the relative abundance of Clostridiales under IPM
0.048
treatment was also higher (P < 0.01) than that under all other treat ments
(Fig. S2). In contrast, fertilization had no effect on the relative
abundance of Burkholderiales, compared with the control. The relative
abundances of Bacillales and Clostridiales were correlated with SOC TN,
AP, DOC and MWD, and negatively correlated with DCo (Table S6).
4. Discussion
4.1. Effect of long-term fertilization on soil properties
Acidification is one of the most serious problems associated with
agricultural production in Ultisols, reducing the availability of nutrients,
especially P, and increasing losses in nutrients and biodiversity
(Hartman et al., 2008; Hooper et al., 2012). Guo et al. (2010) reported
that red and yellow soils, which are the most acidic in southern China,
have acidified further since the 1980s, with relative declines in pH of
0.23 and 0.30 in cereal and cash crop systems, respectively, due to high
N fertilizer input and the uptake and removal of base cations by crops.
In contrast, in this study, long-term (27 years) application of inorganic
NPK fertilizer increased soil pH, probably due to the addition of calcium
magnesium phosphate (Liu et al., 2015). Compared with the I treat ment,
the highest increase in soil pH was found with liming (IL), a
conventional practice for alleviating soil acidification in the world (Li
et al., 2014). The application of inorganic fertilizer plus pig manure also

(a) Relative percentage variation compared with Control (%) (b) 95% confidence intervals (c)

Crenarchaeota IL HDI

Acidobacteria 14

14

Proteobacteria

12
Chloroflex
12

Cyanobacteria

Actinobacteria 10

10

AD3

Verrucomicrobia 8

Gemmatimonadetes

Firmicutes 6

Bacteroidetes

4
Planctomycetes
4

-90 -45 0 45 90 135 360 420 -3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3

IL HDI
Difference between proportions (%)
Fig. 4. Percentage variations in the relative abundances of dominant prokaryotic phyla (relative abundance > 1%) under inorganic fertilizer plus liming (IL) and
inorganic fertilizer plus pig manure (IPM) treatment relative to the control (a). Significant changes in the dominant phylum under IL (b) and IPM (c) treatment
relative to the control according to the response ratio method at a 95% confidence interval (CI). If error bars of the 95% CI range of the response ratio of a specific
phylum overlap with zero, this represents no significant effect on this specific phylum. If the 95% CI range is greater or less than zero, this represents a significant
positive or negative effects, respectively.

6
Machine Translated by Google
G. Ye et al.
Fig. 5. Indicator species under inorganic fertilizer plus pig manure (IPM) treatment. Circles represent genera and the size of each circle denotes its relative abun
dance. Pet chart summarizing the percentage of each indicator species. Taxonomic levels: c, class; oh, orders.
increased soil pH, but amendment with inorganic fertilizer plus crop
residues did not, compared to I treatment (Table1). It is likely that the
increase in soil pH under IPM treatment was due to the high con
centrations of carbonates and bicarbonates in the manure (Whalen
et al., 2000; Materechera and Mkhabela, 2002) since CaCO3 is generally
added to pig diets to lower internal heat. Another possibility is that
composted manure contains considerable amounts of humic- and fulvic
type substances with functional groups such as carboxyl and phenolic
hydroxyl groups, which chelate protons and aluminum at their natural
pH values, causing the solution of aluminum hydroxide and the release
of an equivalent amount of hydroxyls to buffer soil acidity (Wong et al.,
1998; Materechera and Mkhabela, 2002). Iyamuremye and Dick (1996)
also suggested that the increase of soil pH by organic amendments was
due to the ligand exchange between organic acids and hydroxyl groups
of A1 or Fe hydrous oxides.
Repeated application of manure more effectively increased SOC,
TN, and AP contents compared with the I treatment. Under IPM treat
ment, the increase in SOC was unlikely to have been caused by a
reduction in SOC content under I treatment, since SOC levels were pre
viously found to increase by 13.2% compared with the control due to
enhanced crop production and increased input of crop residues such as
roots, stubble and exudates into the soil (Paustian et al., 1997). A
previous study showed that as much as 40% of plant photosynthates are
deposited in the soil as sugars, organic acids and larger organic com
pounds (Kumar et al., 2006). However, in this study, combined application
of inorganic fertilizer plus peanut straw, rice straw or radish had
limited effects on SOC even though the C input was higher than that
under HDI treatment (Table S2). In general, organic compounds in crop
residues are mainly in the form of hydrophilic fractions of humic sub
stances, which are more easily degraded by microbes (Spaccini et al.,
2000, Xu et al., 2017b). In contrast, manure contains a larger proportion
of recalcitrant organic compounds (Drinkwater et al., 1998; Xu
et al., 2017a) compared to crop residues. Three-month composting was
found to reduce the proportion of labile, hydrophilic and plant-derived
organic compounds, resulting in the accumulation of more stable hy
drophobic moieties including lignin-derived phenols and microbial derived
carbohydrates (Said-Pullicino et al., 2007). After being
7
Applied Soil Ecology xxx (xxxx) xxx–xxx
incorporated into the soil, these stable hydrophobic moieties are not
usually utilized by microbes, and become the main source of re-calcitrant
organic compounds in the soil (Spaccini et al., 2000; Yu et al.,
2012b). In addition, long-term application of manure was found to
increase the content of non-crystalline Fe in Ultisols (Zhang et al.,
2013a), in turn providing an extensive surface area and increasing the
chelation capacity of organic biomolecules to form metastable and in
intermediate complexes between organic compounds and minerals (Berhe
et al., 2012). Overall, our results suggest that amendment with pig
manure is more effective than the use of crop residues in increasing soil
pH and organic carbon content.
Application of pig manure increased the organic C content in bulk
soil and all aggregates, and promoted the formation of large macro
aggregates (Table 2), however, amendment with crop residues, except
IRS treatment, had no effect on aggregation. Manure amendment more
effectively increased organic C content in all aggregates, compared with
crop residues addition. Tong et al. (2014) observed a similar result
wherein 17-year application of manure more effectively increased the
organic C content in all aggregates as well as the mass proportion of
macroaggregates compared to straw amendment. It has been suggested
that increased organic C in the microaggregates and silt plus clay
fraction accentuates the formation of macroaggregates (Yu et al.,
2012a; Das et al., 2014). Manure was also found to have a more
beneficial effect on the formation of particulate organic matter (POM)
compared to crop residues (Mando et al., 2005), since amended manure
preferentially accumulates in the free POM fraction by forming intra POM
on an aggregate scale (Kong et al., 2005). In turn, increased POM
promotes the formation of macroaggregates, accordingly enhancing
the physical protection of organic C within aggregates (Puget et al.,
2000; Micah and Rice, 2004).
In this study, the DCo was lower under IPM treatment than any
other treatments (Table 1), and was negatively correlated with the mass
proportion of large macroaggregates (Fig. 1). Previous studies suggest
that increased organic C in microaggregates (free or within
macroaggregates) is due partly to an increase in pore-filling organic
matter (POM or amorphous) (Zhuang et al., 2008; Yu et al., 2012a).
This, in turn, reduces pore connectivity, increases water retention and
Machine Translated by Google
G. Ye et al.
decreases the DCo in the soil, accordingly promoting the formation of anaerobic microsites
(Schjønning et al., 2003). We found that the specific C mineralization rate in IPM-amended
soil was lower than that in the control and inorganic fertilizer treatments (Table 3). Similarly,
Bidisha et al. (2010) reported that 20-year addition of farmyard manure at 60 t haÿ1 yrÿ1
decreased the specific C mineralization rate compared with inorganic fertilizer. In contrast,
we found that inorganic fertilizer plus crop residue treatments increased the C mineralization
rate and specific C mineralization rate compared with the control. Soil aeration conditions
are known to play an important role in the storage and turnover of organic C (Sundh et al.,
1997, Keiluweit et al., 2017). This is due to the fact that organic C is more efficiently
decomposed by aerobic than by facultative aerobic and/or obligate anaerobic microorganisms
(Ding and Sun, 2005). In addition, the accumulation rate of organic C is higher in soil with
an oxygen concentration 10% because the oxidation of labile organic C is slowed (Zibilske
and Bradford, 2007). Thus, it is likely that the decrease in DCo in the soil reduced the rate
of organic C decomposition under IPM treatment, favoring organic C accumulation.
4.2. Effect of long-term fertilization on prokaryotic communities
High microbial diversity and functional redundancy are believed to be beneficial in
maintaining a healthy soil ecosystem (Bhat, 2013; Chen et al., 2016). Soil pH was frequently
reported to predominantly influence microbial diversity from agricultural soils (Sun et al.,
2015; Zhalnina et al., 2015; Shen et al., 2016). However, in this study, liming to Ultisols
failed to change prokaryotic diversity and richness (Fig. 2).
Thus, merely increasing soil pH by liming might not be enough to im prove soil prokaryotic
diversity. In contrast, application of pig manure enhanced prokaryotic diversity and richness
which could be mainly attributed to the increase of soil nutrients (SOC, AP and DOC) and/
or soil aggregation (Table S3). Sessitsch et al. (2001) further suggested that high availability
of nutrients in soil could stimulate bacterial growth and increase bacterial diversity. These
findings suggest that the application of inorganic fertilizer plus pig manure has the ability to
improve soil microbial diversity.
In contrast to prokaryotic diversity, prokaryotic community structure was primarily
regulated by soil pH in the test soil, which is consistent with numerous previous findings
from agricultural soils (Enwall et al., 2007; Rousk et al., 2010; Sun et al., 2010; Sun et al. .,
2015). Indeed, soil pH could directly influence bacterial communities since the growth
tolerances to pH of most of bacterial taxa were generally narrow (Rousk et al., 2010).
However, in addition to soil pH, soil nutrients (AP and DOC) and physical behavior (DCo
and MWD) also made significant contributions to prokaryotic communities in this study
(Table 4). Fertilization has been found to alter bacterial community structure by changing
soil nutrients (Ramirez et al., 2010; Sun et al., 2015; Xun et al., 2016) or soil aggregation
(Zhang et al., 2015, 2017) in previous studies. Compared with the I treatment, liming and
pig manure altered prokaryotic community structure, while plant residues did not (Fig. 3).
This might be mainly attributed to the fact that amended plant residues did not change soil
properties, especially soil pH, AP, DOC, MWD and DCo.
We found that Bacilles and Clostridiales accounted for most of the
indicator species in the IPM treatment (Fig. 5) and were correlated with soil nutrients (SOC,
TN, AP and DOC) and aggregates (MWD) while negative with DCo in the soil (Table S6).
Bacillales and Clostridiales have been proposed to be copiotrophic bacteria (Fierer et al.,
2007), thus it is likely that increasing soil nutrients in the HDI could stimulate their growth.
In addition, the spatial distribution, functioning and diversity of bacterial populations at the
microscale have been found to be affected by the size and stability of aggregates through
changing factors such as predation pressure, water potential and oxygen availability (Kong
et al., 2005). ; Hansel et al., 2008; Kong et al., 2011). Bacillales and Clostridiales are gram-
positive bacteria, Bacillales belonging to the fa cultative anaerobes and Clostridiales being
strictly anaerobic (Lauga
8
Applied Soil Ecology xxx (xxxx) xxx–xxx
et al., 2013), and both of them increased under anoxic incubation compared with oxic
incubation (Angel and Conrad, 2013). Further more, in the North China Plain, Bacilles
became dominant in compost treated soil due to a significant increase in the mass proportion
of macroaggregates (Feng et al., 2015; Zhang et al., 2015). Thus, the long term application
of pig manure decreased the DCo due to the increased mass proportion of large
macroaggregates, accordingly increasing the relative abundances of Bacillales and
Clostridiales. Moreover, Bacillales and Clostridiales secrete enzymes (Govindasamy et al.,
2010; Kamada et al., 2013), of which polyphenol oxidase, peroxidase and lipases promote
the transformation of organic materials into humus (Gupta et al., 2004; Sinsabaugh, 2010 ).
The increase in Bacillales is also a fa vorable biocontrol method against plant pathogens,
acting as an tagonist against plant pathogens and insect pests, and thereby stimulating
plant host defense mechanisms (Sun et al., 2013; Abdallah et al., 2016) . Thus, the
increased abundance of Bacillales and Clostridiales under IPM treatment might be beneficial
to soil fertility and plant growth in Ultisols.
5. Conclusions
Long-term (27 years) amendment with inorganic NPK fertilizer plus pig manure
increased soil pH, SOC, TN and AP in Ultisols. The resultant SOC increase accelerated the
formation of large macroaggregates under IPM treatment but not under NPK fertilizer plus
crop residue treat ments. Amendment with pig manure increased prokaryotic diversity and
altered prokaryotic community structure, while plant residues did not. Soil pH was the key
factor for regulating prokaryotic community structures in the studied soils. Bacillales and
Clostridiales accounted for most of indicator species in the HDI, and might be beneficial to
soil fertility and plant growth. These results suggest that application of NPK fertilizer
combined with pig manure rather than plant residues is an effective way to mitigate
acidification, building up organic C, accel erating the formation of large macroaggregates,
enhancing prokaryotic diversity and regulating prokaryotic community structure.
Acknowledgments
This work was financially supported by the Chinese Academy of Sciences
(XDB15020100) and the National Natural Science Foundation of China (41471207,
31561143011).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https:// doi.org/10.1016/
j.apsoil.2018.09.008.
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