July 2020. Horticultural Plant Journal, 6 (4): 223–230.

Horticultural Plant Journal

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Soil Fertility, Microbial Biomass, and Microbial Functional

Diversity Responses to Four Years Fertilization in an Apple
Orchard in North China
Zhanling Zhu a,1, Yan Bai b,1, Minglu Lv a, Ge Tian a, Xin Zhang a, Li Li b, Yuanmao Jiang a,∗, and
Shunfeng Ge a,∗
a State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an, Shandong
271018, China
b National Agro-technical Extension and Service Center, Beijing 100125, China

Received 10 October 2019; Received in revised form 25 November 2019; Accepted 7 April 2020
Available online 10 June 2020

A B S T R A C T

Soil microbial communities play an essential role in maintaining soil fertility and are considered as ecological indicators to evaluate soil
health. In the present study, we examined the influence of almost 4 years of fertilization [no fertilizer (CK), nitrogen alone (N), nitrogen, phos-
phorus and potassium chemical fertilizer (NPK), organic manure (M), nitrogen plus organic manure (NM), and NPK plus organic manure (NPKM)]
on soil fertility and the functional diversity of soil microbial communities in an apple orchard. Compared to CK, fertilization increased soil
organic carbon, total nitrogen, and available nutrients, but reduced soil pH in N and NPK treatments. The highest microbial biomass carbon and
nitrogen, most probable number of actinomycetes, bacteria, and fungi occurred in the NPKM treatment. The average well color development
(AWCD) values followed the order of NPKM > M> NPK and NM > CK and N. The Shannon index in organic manure treatments were signifi-
cantly higher than in control and in treatments without organic manure. The principal component analysis showed that manure treatment
was significantly separated from other treatments. These results indicated that organic manure applied alone or in combination with chemical
fertilizers would increase soil fertility and functional diversity of soil microbial communities. Moreover, applying balanced N, P, K fertilizer in
combination with organic manure was found to be superior to the use of a single fertilizer in improving soil microbial community quality.

Keywords: apple orchard; fertilization; soil fertility; soil microbial community; functional diversity

1. Introduction tal factors and human activities (application of fertilizers, pes-

Soil microbes are important components of the soil ecosys-
tem and their diverse functions play key roles in many biochem-
ical cycles. The soil microbial community is a sensitive indicator
of soil quality and also a bio-indicator of soil ecosystem sustain-
ability. Its diversity and activity is associated with environmen-
ticides, and other agricultural activities) (Tamilselvi et al., 2015).
Nutrient management significantly affects soil microbial abun-
dance, diversity and activity under long-term fertility manage-
ment (Chinnadurai et al., 2014; Gao et al., 2015; Wang et al., 2019a).
At present, excess nitrogen and low amount of manure are gen-

∗
Corresponding authors. Tel.: +86 538 8249778.
E-mail addresses: ymjiang@sdau.edu.cn; ymjiang@sdau.edu.cn
1
These authors contributed equally to this work.
Peer review under responsibility of Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese
Academy of Agricultural Sciences (CAAS)

https://doi.org/10.1016/j.hpj.2020.06.003
2468-0141/© 2020 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural
Sciences (CAAS). Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC
BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)
224
eral practice for most economic crops in China, such as apple.
Nitrogen (N) fertilizer is commonly used at levels as high as 700–
800 kg · hm−2 in apple orchards in China, however, organic fertil-
izer is only used at a maximum of 8 000 kg · hm−2 (Ge et al., 2018a).
Therefore, continuous monitoring of these soils is necessary to
develop appropriate indicators for sustainable fruit production.
Functional diversity of soil microbial community can be mea-
sured by the Biolog EcoPlatesTM method (Choi and Dobbs, 1999;
Hu and Wang, 2007; Li et al., 2012). This method can distin-
guish bacterial communities and can be used to describe tempo-
ral changes in physiological characteristics of different environ-
ments (Li et al., 2012). Some researchers have mentioned that the
Biolog EcoPlatesTM method is not valid to measure the diversity of
soil microbial community alone, however it gives a comprehen-
sive indication of the functional diversity of the soil microbiota
(Sun et al., 2010; Kumar et al., 2017; Zhu et al., 2017).
Continuous fertilization, in particular the use of organic ma-
nure, can improve soil organic carbon, total nitrogen, microbial
biomass and functional diversity of soil microbial communities
and increase soil productivity (Lv et al., 2011; Chinnadurai et al.,
2014; Tamilselvi et al., 2015). However, Leroy et al. (2008) found
that long-term fertilization, especially with N fertilizer, can ac-
celerate soil acidification and reduce base saturation, cation ex-
change capacity, soil aggregation, water-holding capacity, and soil
microbial activity, but increase soil bulk density and compaction,
thereby decreasing soil productivity. Sun et al. (2010) found that
application of chemical fertilizers together with organic fertil-
izer could increase microbial utilization rate of carbon sources
in a red soil, whereas, applying simply N, phosphorus (P) and
potassium (K) fertilizer significantly decreased the utilization
rate. However, Luo et al. (2009) found that applying chemical fer-
tilizer can increase microbial utilization rate of carbon sources
in a paddy soil. Therefore, different soil types, fertilization con-
ditions, tillage methods, and crop types have various effects
on the soil microbial community structure, quantity and activ-
ity (Zhang et al., 2014b; Xia et al., 2019). In turn, different soil
microbial communities have different effects on physical and
chemical properties of the soil, and crop growth, production and
quality.
Apple cropping system in China is one of the most intensive
cultivation systems and consumes most of the chemical fertil-
izers used in agriculture Zhu et al. (2018). However, many re-
searchers reported the decline in fruit quality and soil produc-
tivity in a number of long-term intensive apple cropping systems
due to the excessive usage of fertilizer (Liu et al., 2010; Gao et
al., 2012; Qiu et al., 2013; Zhao et al., 2013; Ge et al., 2018b; Wang
et al., 2020). Moreover, information on long-term effect of using
chemical fertilizers with or without organic manure in an inten-
sive apple cropping system on soil fertility and temporal shift
in functional diversity of the microbial community is limited.
Thus, the present study was undertaken in a long-term fertil-
ization experiment which started in 2015 with apple cropping
system in North China. Our objectives were to determine the
effects of chemical fertilizers (alone or in combination with or-
ganic manure) on the soil fertility and functional diversity of soil
microbes.
Zhanling Zhu et al.
2. Materials and methods
2.1. Experimental site
The experimental station is located in Jiangyu town, Linqu
City, Shandong Province, China. This region has a semi-humid
continental monsoon climate with a mean annual precipitation
of 710 mm and a mean annual temperature of 12.4 °C. The long-
term fertilization experiment started in 2015, the apple orchard
was dominated by 2-year-old Fuji/M9.T337. The plant density
was 2 500 · hm−2 , and the plants were spaced at 1 m in rows
and 4 m between the rows. The soil is classified as Hapli-Udic
Cambisol (FAO Classification), and consisted of 16.7% sand, 58.4%
silt and 24.9% clay at depth of 0–30 cm. In 2016, the contents
of soil organic carbon, total nitrogen, total phosphorus and to-
tal potassium in the initial soil plow layer (0–30 cm depth) were
7.21, 0.45, 0.27, and 10.1 g · kg−1 , respectively. The available nitro-
gen, phosphorus, and potassium contents were 76.22, 19.64, and
65.93 mg · kg−1 , respectively. The soil pH (H2 O) was 6.26.
2.2. Experimental design
There were six treatments with the same N application rate:
no fertilizer (CK), nitrogen only (N), nitrogen, phosphorus and
potassium (NPK), organic manure (M), nitrogen plus organic ma-
nure (NM), and NPK plus organic manure (NPKM). Treatments
were arranged in a randomized complete block design with three
replicates. The plot size was 60 m2 (6 m × 10 m), and included 16
apple trees. During the year of 2015–2018, the detailed fertiliza-
tion information of the six treatments was shown in Table 1. Urea,
calcium magnesium phosphate and potassium sulfate were ap-
plied as nitrogen, phosphorus and potassium chemical fertiliz-
ers. A total of 60% of N, P and K fertilizers and 100% of organic
manure were applied as base fertilizers after fruit harvest and
the remaining fertilizer was applied as a top-dressing in June.
In the M treatment, the amount of N from organic manure was
equal to that from NPK and the N treatment, without regard to P
and K. In the NM and NPKM treatments, N from organic manure
and chemical fertilizer were 40% and 60% respectively. In the or-
ganic manure-treated treatments, the organic manure used was
pig manure, and the average content of C, total N, total P, and
total K are 239, 21.2, 24.5, 17.2 g · kg−1 , respectively. Weeds were
controlled by artificial weeding during the growing season.
2.3. Soil sampling and analyses
After almost 4 years of fertilization (October 13, 2018), three
replicate rhizospheric soil (soil adhering to roots) samples were
collected from 10 locations at a depth of 10–25 cm (plow layer) per
plot, and put together in a sterile plastic bag from each plot. Hu-
midified soil was sifted (2 mm openings) and stored at 4 °C; bio-
chemical analysis was performed within 14 days. A portion (25%)
of the soil samples were air-dried and stored at room tempera-
ture for chemical analysis.
The physical and chemical properties of the soil were mea-
sured. Soil pH was measured with glass electrode in a 1:2.5
soil/water suspension Bao (2005). Soil organic C was determined
Soil Fertility, Microbial Biomass, and Microbial Functional Diversity Responses to Four Years Fertilization in an Apple Orchard
Table 1 Fertilizer application rate in different treatments in 2015–2018 kg • hm−2
Treatment Chemical fertilizer
N P2 O5
CK 0 0
N 200 0 0
NPK 200 100
M 0 0 0
NM 120 0 0
NPKM 120 150
by a K2 CrO7 –H2 SO4 oxidation procedure and TN by the Kjeldahl
method Bao (2005). Soil available N (AN) was determined by us-
ing a micro-diffusion technique after alkaline hydrolysis (Wang et
al., 2001). Soil available P (AP) and available K (AK) were extracted
with sodium bicarbonate and ammonium acetate (Lu, 1999). Mi-
crobial biomass C was determined by chloroform fumigation ex-
traction method and microbial biomass N was calculated as re-
ported by Ross (1990). Most probable number of bacteria (MPN-B),
actinomycetes (MPN-A) and fungi (MPN-F) were calculated with
Blodget method (Blodget, 2006).
2.4. Functional diversity of soil microbial community
Functional diversity of soil microbial community was ana-
lyzed by Biolog EcoPlatesTM (Biolog Inc., CA, USA). Each plate
had 96 wells, containing three replications of 31 different car-
bon sources plus control. The 31 substrates are divided into six
categories (Choi and Dobbs, 1999): carbohydrates (D-cellobiose, i-
erythritol, d-galactonic acid γ -lactone, N-acetyl-D–gluco-samine,
glucose-1-phosphate, β-methyl-d-glucoside, D,L-d-llycerol phos-
phate, α-d-lactose, d-mannitol, and d-xylose), amino acids (L-
arginine, l-asparagine, glycyl-l-glutamic acid, l-phenylalanine,
L–serine, and l-threonine), carboxylic acids (γ -hydroxybutyric
acid, α-ketobutyric acid, d-galacturonic acid, d-glucosaminic
acid, itaconic acid, d-malic acid, and pyruvatic acid methyl es-
ter), polymers (α-cyclodextrin, glycogen, Tween 40, and Tween
80), amines (phenyl ethylamine and putrescine), and phenolic
compounds (2-hydroxybenzoic acid and 4-hydroxybenzoic acid).
Fresh soil, equivalent to 10 g dry weight, was mixed with 100 mL
distilled water in a 250 mL flask and then shaken for 30 min at
250 r · min−1 . Final suspension was diluted to 10−3 and inocu-
lated in a Biolog EcoPlatesTM . The plates were incubated at 25 °C
and the absorbance of each well color resulting from the carbon
utilization of the microorganism was measured at 590 nm after
incubation at 0, 24, 48, 72, 96, 120, 144, and 168 h. The data were
recorded using Microlog 4.01 (Biolog Inc., CA, USA). The average
well color development (AWCD) for all carbon sources was calcu-
lated to assess total microbial activity (Li et al., 2012).
 centrations. The soil pH of all the six fertilization treatments was
AWCD = (C − R )/31
where C is the optical density (OD) value of the reaction wells, R
is the OD value of the control well.
The functional diversity of soil microbial communities was ex-
pressed by the Shannon (H) and McIntosh (U) indices, which were
derived from absorbance obtained from the Biolog EcoPlatesTM .
The Shannon index (H) is sensitively affected by species rich-
225
Organic manure (The amount of
N from organic manure)
K2 O
0 0
0
200 0
9 435 (200)
3 775 (80)
300 3 775 (80)
ness of microbial community; the McIntosh index (U) is one of
the measurements of species evenness of the community.
2.5. Principal component analysis
Principal component analysis (PCA) based on Pearson correla-
tion matrix was performed to determine the microbial commu-
nity functions at the various fertilization treatments. Substrate
utilization assay data were analyzed after substrates were di-
vided into six groups and the average absorbance per category
was calculated. All meaningful loadings (> 0.5) were included and
considered significant in the interpretation of principal compo-
nents. In most cases principal component 1 (PC1) and principal
component 2 (PC2) would account for most of the variance. Bi-
plots were constructed to interpret the analysis, with the original
variables drawn as vectors to summarize the correlation between
the variable and both illustrated axes.
2.6. Statistical analysis
Significant differences between fertilization treatments were
analyzed by a one-way ANOVA, followed by Tukey HSD Post Hoc
test at P < 0.05 using SPSS 19.0.
3. Results
3.1. Soil organic carbon, total nitrogen, microbial biomass
carbon, and biological properties
After almost 4 years of fertilization, soil organic carbon (SOC),
total nitrogen (TN), pH and available nutrients were all changed
(Table 2). SOC and TN content in all treatments showed the in-
crease pattern: the NPKM treatment showed the highest value,
followed by NM and NPK treatment. N and M treatment had
lower values, but they still exceeded control. The highest soil
available-N, P, and K contents were found in the NPKM treat-
ment, which were 1.69, 7.11 and 1.49 times of control. Compared
with other treatments, application of P and K fertilizers signifi-
cantly increased soil available phosphorus and potassium con-
slightly lower (5.88–6.24) than control (6.26). Compared with con-
trol, soil pH decreased in the treatments without organic manure
(N and NPK treatment) by 0.35 and 0.38, respectively, and in NM
and NPKM by 0.16 and 0.14. However, no significant differences
in soil pH were observed between the treatments with organic
manure (M, NM and NPKM treatment) and control.
Soil microbial biomass carbon (MBC) and soil microbial
biomass nitrogen (MBN) were significantly different among the
226 Zhanling Zhu et al.

Table 2 Chemical properties of the soil under different treatments in the long-term fertilization experiment
Treatment SOC/(g • kg−1 ) TN/(g • kg−1 ) AN/(mg • kg−1 ) AP/(mg • kg−1 ) AK/(mg • kg−1 ) pH
CK 7.24 ± 0.12 e 0.93 ± 0.04 d 69.85 ± 2.45 e 4.81 ± 0.03 e 78.24 ± 2.67 d 6.26 ± 0.09 a
N 8.26 ± 0.08 d 0.96 ± 0.02 c 88.12 ± 3.24 bc 9.97 ± 0.61 d 79.56 ± 3.54 d 5.91 ± 0.08 b
NPK 9.46 ± 0.16 bc 1.01 ± 0.06 b 96.54 ± 4.28 b 19.56 ± 0.84 b 97.41 ± 1.99 bc 5.88 ± 0.04 b
M 9.11 ± 0.14 c 0.99 ± 0.02 bc 80.56 ± 4.17 cd 15.47 ± 0.49 c 102.35 ± 6.31 b 6.24 ± 0.11 a
NM 10.08 ± 0.20 b 1.03 ± 0.03 b 109.42 ± 6.61 ab 15.58 ± 0.64 c 110.57 ± 7.09 ab 6.10 ± 0.07 a
NPKM 10.67 ± 0.09 a 1.07 ± 0.03 a 118.31 ± 5.48 a 34.22 ± 1.15 a 116.54 ± 6.44 a 6.12 ± 0.06 a
Note: All values represent means ± standard error (n = 3), different letters within a column indicate statistically significant difference between treatments
at P < 0.05.

Fig. 1 Changes of soil biological properties under different treatments in the long-term fertilization experiment
All values represent means ± standard error (n = 3), different letters indicate statistically significant difference between treatments at
P < 0.05.

treatments (Fig. 1, A, B). MBC and MBN were significantly im-

proved in all fertilization treatments. The NPKM treatment had
the highest values of MBC and MBN, which were 377.28 and
35.98 μg . kg−1 respectively, followed by the NPK treatment. The
N treatment had the lowest one. MBC in the M and N treatments
were not significantly different, but they were significantly lower
than other fertilizer treatments. There was no significant differ-
ence for MBN between NPK, M, and NM treatments, but they were
significantly higher than those of N treatment and significantly
lower than those of NPKM treatment.
Among all treatments, MPN-B was the highest in NPKM treat-
ment, and MPN-F was the lowest in the NPK treatment (Fig. 1,
C). Fertilization increased MPN-A, MPN-B and MPN-F values, with
the largest increase in NPKM and NM treatments. Compared with Fig. 2 Changes in average well color development (AWCD)
the control, MPN-A, MPN-B and MPN-F in the NPKM treatment over time of the soil under different treatments in the
was significantly increased by 4.12%, 7.08%, and 10.44%, respec- long-term fertilization experiment
tively. All values represent means ± standard error (n = 3). Different
letters within the same incubation time indicate statistically
3.2. Dynamics of average well color development significant difference between treatments at P < 0.05.

Average well color development (AWCD) changes can distin-

guish the differences among soil microbial community carbon
metabolism capacity. Microbial utilization rate of carbon sources
increased in all treatments over time (Fig. 2). Between 0 and 24 h,
the AWCD value was less than 0.1, which meant most of the car-
bon sources were not utilized; between 24 and 96 h, AWCD val-
ues increased rapidly, indicating soil microbiota started using car-
bon sources; after 96 h, AWCD increased slowly and plateaued
at 120 h, implying maximal carbon source utilization. At 120 h,
the highest soil microbial community carbon metabolism capac-
ity appeared in NPKM treatment. The AWCD values in N alone
were the lowest among all the treatments up to 96 h. Then, there
was a slight increase after 96 h compared to control and finally
occupied the lowest value similar to the control at 120 h. This
might due to the low soil pH at that time in the N alone treatment
(Table 2), which inhibited microorganism growth.
3.3. Overall utilization of six kinds of carbon sources in
different treatments
After almost 4 years of fertilization, the overall utilization of
six carbon sources differed among the treatments (Table 3). The
consumption of amino acids and carbohydrates was found to be
Soil Fertility, Microbial Biomass, and Microbial Functional Diversity Responses to Four Years Fertilization in an Apple Orchard 227
Table 3 The overall utilization of carbon sources in different treatments in the long-term fertilization experiment
Treatment Carbohydrates Amines Amino acids Carboxilic acids Polymers Phenolic compounds
CK 0.46 ± 0.01 e 0.32 ± 0.01 d 0.41 ± 0.01 c 0.33 ± 0.01 d 0.48 ± 0.01 d 0.44 ± 0.01 b
N 0.58 ± 0.02 d 0.30 ± 0.01 d 0.21 ± 0.00 d 0.32 ± 0.01 d 0.37 ± 0.01 e 0.39 ± 0.02 bc
NPK 0.49 ± 0.02 e 0.45 ± 0.01 c 1.18 ± 0.06 a 0.50 ± 0.01 ab 0.97 ± 0.05 a 0.41 ± 0.03 b
M 0.74 ± 0.04 c 0.83 ± 0.03 a 0.92 ± 0.01 b 0.51 ± 0.02 ab 0.66 ± 0.03 c 0.46 ± 0.03 b
NM 0.89 ± 0.03 b 0.44 ± 0.01 c 0.96 ± 0.03 b 0.54 ± 0.01 a 0.67 ± 0.03 c 0.41 ± 0.01 b
NPKM 0.94 ± 0.02 a 0.66 ± 0.02 b 1.21 ± 0.04 a 0.41 ± 0.02 c 0.78 ± 0.04 b 0.59 ± 0.01 a
Note: All values represent means ± standard error (n = 3), different letters within a column indicate statistically significant difference between treatments
at P < 0.05.

Table 4 Diversity indexes of soil microorganisms at 120 h in

different treatments in the long-term fertilization experiment
Treatment Shannon index (H) McIntosh index (U)
CK 2.38 ± 0.05 d 0.992 ± 0.01 abc
N 2.35 ± 0.11 d 0.995 ± 0.02 a
NPK 3.60 ± 0.08 c 0.993 ± 0.01 ab
M 3.96 ± 0.10 b 0.989 ± 0.03 bc
NM 3.67 ± 0.15 c 0.987 ± 0.02 c
NPKM 4.10 ± 0.16 a 0.985 ± 0.02 d
Note: All values represent means ± standard error (n = 3), different let-
ters within a column indicate statistically significant differences between
treatments at P < 0.05.

the highest in NPKM and NM treatments, respectively. Besides

amino acids and carbohydrates, amines were also a main car-
bon source in M treatment. The dominate carbon sources were
significantly different between two non-organic manure-treated
treatments; amino acids and polymers utilizing microbes were
dominant in the NPK treatment, whereas, the main carbon source
was carbohydrates in N treatment. Except for NPKM treatment,
the utilization of phenolic compounds did not differ significantly
between the other treatments.
3.4. Diversity indexes of soil microorganisms
Diverse indexes were obtained by calculating the AWCD value
at 120 h (Table 4). The Shannon index (H) was higher in the or-
ganic manure-treated treatments (M, NM and NPKM) than control
and chemical fertilizer treatments. However, the McIntosh index
(U) decreased in the treatment with organic fertilizer, especially
in NPKM treatment. The McIntosh index of the nitrogen alone
treatment was the highest whereas the Shannon index were the
lowest.
3.5. Principal component analysis of soil microbial biomass
carbon metabolism
A principal component analysis (PCA) biplot showed distinct
differences in carbon source utilization patterns of soil microbes
in the six fertilization treatments (Fig. 3). The first and second
principal components (PC1 and PC2) of 120 h data account for
40.4% and 18.5% of the variance of the data, respectively. The
treatments were divided into four groups based on the PCA
scores. On the PC1 axis, organic manure-treated and the NPK
treatment localized on the positive end, and N and control local-
ized on the negative end. On the PC2 axis, three organic manure-
treated treatments and control localized on the positive end, and
the other treatments localized on the negative end.
Fig. 3 Principal component analysis based on 120 h Biolog
data of soil microbial biomass carbon metabolism under
different treatments in the long-term fertilization experiment
Values in parentheses indicate percentage contribution of PC1
and PC2.
4. Discussion
4.1. Effects of different fertilization treatments on soil chemical
properties
After almost 4 years of fertilization, the SOC and TN increased
in all fertilization treatments, and that the SOC and TN of organic
fertilizer increased more than that of chemical fertilizer. Appli-
cation of organic manure fertilizer can increase SOC, but the ef-
fects of chemical fertilizer on SOC remain unclear. Some stud-
ies showed that applying N, P, and K simultaneously, in pairs,
or alone all promoted plant root growth, increased the organic
matter input in the rhizosphere, and thus increased SOC levels
(Tan et al., 2014). However, other studies have demonstrated that
SOC is lower in chemical fertilizer-treated soil than non-fertilizer-
treated soil (Zhang et al., 2014a). This might be because the ap-
plication of chemical fertilizers affected soil microbe quantity
and activity and affected bio-degradation of the organic carbon
sources (Gao et al., 2015). We found that the SOC content was the
highest in the NPKM treatment, which was consistent with the
results of Kumar et al. (2017). Similar to SOC, TN content was also
the highest in NPKM treatment, which may be partly due to the
slow release of manure-N (Bhandari et al., 2002).
Guo et al. (2010) showed that long-term and excessive use of
inorganic fertilizer, especially N fertilizer, can lead to soil acidifi-
cation. Most N fertilizers contain NH4 + and during oxidation of
NH4 + , the H+ is released into the soil, so long-term N fertiliza-
228 Zhanling Zhu et al.

tion also can lead to cation losses in the soil. In our study, the pH 4.2. Effects of different fertilization treatments on the
of organic manure-treated soil decreased but the decrease was functional diversity of soil microbial communities
smaller than in the CK and chemical fertilizer treatment. This
The AWCD value is an important index of microbial functional
is because application of organic manure can neutralize H+ re-
diversity, because it represents the ability of soil microorganisms
leased from the soil acidification process and enhance the reten-
to utilize different carbon sources. Different incubation time were
tion of soil nutrients to prevent further cation losses (Cai et al.,
employed in previous studies, such as Luo et al. (2009) used the
2015).
AWCD value at 96 h and 72 h after incubation in a study of red
We found that soil MBC and MBN contents were significantly
soil. In our study, we used the AWCD value at 120 h because, at
increased in the long-term NPK treatment and the NPK plus
this time, the AWCD value reached a plateau in all treatments.
organic manure treatment. Kumar et al. (2017) also concluded
Our results concerning microbial utilization of carbon sources
that applying simultaneously chemical and organic fertilizers
among different fertilization patterns were consistent with those
improved SOC, nutrient availability and water retaining capac-
of Kumar et al. (2017) and Sun et al. (2010), but not consistent
ity, and also improved soil microbial activity. However, the N
with the results of Luo et al. (2009). The consistent point of our
alone and M alone just slightly increased MBC and MBN con-
study and that of Luo et al. (2009) is the increased microbial uti-
tent in this study. This maybe because long-term fertilization
lization of carbon sources in the chemical fertilizer plus organic
with N alone leads to imbalanced soil nutrients, and then the
manure treated-soils. However, Luo et al. (2009) showed that fer-
root growth was inhibited and their residues were also decreased
tilization with N alone significantly increased the microbial uti-
(Shahid et al., 2016; Wang et al., 2019b). Meanwhile, long-term
lization rate of carbon sources, whereas, it was decreased in this
fertilization with N alone can accelerate the decomposition of
study. Applying organic manure or organic manure plus chem-
the soil organic carbon (Gao et al., 2015). Furthermore long-term
ical fertilizer enhances the carbon utilization capacity and also
N fertilization increases the soil acidification process, however
forms a unique and beneficial carbon utilization method for mi-
optimal growth conditions for most soil microbes include neu-
croorganisms that prefer amino acids and carbohydrates as the
tral pH, so soil acidification may be main reason for low MBC
carbon source. As a result, long-term organic fertilization in apple
and MBN content in the long-term fertilization treatment with
orchards can provide the optimal conditions for the soil microbial
N alone. The effect of the M treatment on soil MBC and MBN im-
community and maintain a high level of functional community
provement was not as significant as that of the NPK treatment.
diversity.
These results are inconsistent with those of Lv et al. (2011) and
Organic manure and organic manure plus chemical fertiliza-
Sun et al. (2010) on sterile paddy soil and red soil, but consis-
tion have similar carbon sources, because either the soil micro-
tent with the results of Kumar et al. (2017) on Aeric Endoaquept
bial community introduced by organic fertilization use similar
soil. These different conclusions may be caused by different soil
carbon sources, or introduction of organic manure changes the
pH Li et al. (2005) showed that balanced chemical fertilizer is fa-
physical and chemical properties of the soil and enhances the
vorable for the synthesis of cellular components of microorgan-
carbon source utilization ability. In the long-term N alone treat-
isms. This may be the probable reason why soil MBC content was
ment, the number of favorable species increased and the pattern
higher in NPK treatment than in M alone treatment in the present
of carbon utilization also changed towards carbohydrates. This
study.
might be due to the root exudates and soil carbon content. Plant
Some studies have demonstrated that the number of bacteria
root exudates provide carbon source for soil microbes, and other
were positively correlated with the content of soil organic matter
secondary metabolites can shape on the formation of soil micro-
and nutrients, whereas the numbers of actinomycetes and fungi
bial community structure(Chaparro et al., 2012; Massenssini et
were not significantly related to soil nutrients, such as soil to-
al., 2015; Chen et al., 2017). Tiquia et al. (2002) also pointed out
tal nitrogen, available nitrogen, total phosphorus and available
that the content and composition of soil organic matter can af-
phosphorus (Li et al., 2005). Other studies have also indicated that
fect the characteristics of carbon source utilization of microbes.
soil with balanced nutrients contained more soil microorganism
Application of manure enhanced the accumulation of organic
(Naher et al., 2013). In the present study, we found that differ-
carbon in the soil, altered the pattern of potential carbon uti-
ent fertilization treatments significantly affected the most prob-
lization and increased the Shannon and AWCD indices (Kumar
able number of soil microorganisms: the most probable num-
et al., 2017). In this study, the PCA analysis indicated the sepa-
ber of bacteria, actinomycetes, and fungi was higher in organic
ration of manure-treatments from chemical fertilizer treatment,
manure-treated soils than in soils without organic manure. This
suggesting that treatments with manure could have a greater in-
seems to be because organic manure contains more balanced nu-
fluence on microbial functional diversity than those of chemi-
trients. However, long-term fertilization has more complex ef-
cal fertilizers. The Shannon index of the soil microbial commu-
fects on soil. The nutrient content and soil microorganisms might
nity was significantly higher in the organic manure-treated treat-
be significantly different due to differences in nutrient limiting
ments (M, NM, and NPKM) than in the CK and chemical fertilizer
factors, degree of nutrient deficiency, and physical environment
treatment. While, the McIntosh index decreased among organic
conditions. Kumar et al. (2017) revealed that soil available N and
manure treatments, especially in NPKM. These results indicate
K were unique factors in explaining the total number of bacteria,
that combining organic mature with NPK fertilizer can increase
actinomycetes, and fungi, and also indicated that N deficiency
the richness of species number and the soil microbial community
may directly inhibit bacterial growth whereas actinomycetes and
and perhaps weaken the advantages of single dominant species
fungal populations can only be explained by soil available potas-
(Bei et al., 2018; Lin et al., 2019). In the N alone treatment, McIn-
sium content.
Soil Fertility, Microbial Biomass, and Microbial Functional Diversity Responses to Four Years Fertilization in an Apple Orchard
tosh index was the highest, but Shannon index was the low-
est, suggesting long-term fertilization with N alone can reduce
the number of species in the soil microbial community, and this
might favor some microbes that adapt to the environment, but its
microbial metabolism still decreased overall. For example, Zhang
et al. (2012) had shown that long-term N alone fertilization can in-
crease the number of nitrogen functional bacteria, including aer-
obic nitrogen fixers, nitrite oxidizing bacteria, denitrifying bacte-
ria, and ammonifying bacteria.
Acknowledgments
This work was supported by the Special Fund for the National
Key R & D Program of China (Grant No. 2016YFD0201100), National
Natural Science Foundation of China (Grant No. 31501713), China
Agriculture Research System (Grant No. CARS-27), and Taishan
Scholar Assistance Program from Shandong Provincial Govern-
ment.
R E F E R E N C E S
Bao, S.D., 2005. Soil and Agricultural Chemistry Analysis. Agriculture
Press, Beijing. (in Chinese)
Bei, S., Zhang, Y., Li, T., Christie, P., Li, X., Zhang, J., 2018. Response
of the soil microbial community to different fertilizer inputs in a
wheat-maize rotation on a calcareous soil. Agric Ecosyst Environ,
260: 58–69.
Bhandari, A.L., Ladha, J.K., Pathak, H., Padre, A.T., Dawe, D., Gupta, R.K.,
2002. Yield and soil and nutrient changes in a long-term
rice–wheat rotation in India. Soil Sci Soc Am J, 66: 162–170.
Blodgett, R., 2006. FDA bacteriological analytical manual online, ap-
pendix B, most probable number determination from serial dilu-
tions.
Cai, Z., Wang, B., Xu, M., Zhang, H., He, X., Zhang, L., Gao, S., 2015. In-
tensified soil acidification from chemical N fertilization and pre-
vention by manure in an 18-year field experiment in the red soil
of southern China. J Soil Sediment, 15: 260–270.
Chaparro, J.M., Sheflin, A.M., Manter, D.K., Vivanco, J.M., 2012. Manip-
ulating the soil microbiome to increase soil health and plant fer-
tility. Biol Fertil Soils, 48: 489–499.
Chen, H.Y., Dong, Y.F., Xu, T., Wang, Y.P., Wang, H.T., Duan, B.L., 2017.
Root order-dependent seasonal dynamics in the carbon and nitro-
gen chemistry of poplar fine roots. New Forests, 48: 587–607.
Chinnadurai, C., Gopalaswamy, G., Balachandar, D., 2014. Impact of
long-term organic and inorganic nutrient managements on the
biological properties and eubacterial community diversity of the
Indian semi-arid Alfisol. Arch Agron Soil Sci, 60: 531–548.
Choi, K.H., Dobbs, F.C., 1999. Comparision of two kinds of biolog mi-
croplates (GEN and ECO) in their ability to distinguish among
aquatic microbial communities. J Microbiol Methods, 36: 203–213.
Gao, M., Sun, R., Cui, Q., Yang, X., Zhang, S., Sun, B., 2015. Effect
of long-term chemical fertilizer application on soil microbial di-
versity in anthropogenic loess soil. J Plant Nutr Fertilizer, 21:
1572–1580. (in Chinese)
Gao, Y.M., Tong, Y.A., Lu, Y.L., Ma, H.Y., 2012. Effect of long-term ap-
plication of nitrogen, phosphorus and potassium on apple yield
and soil nutrient accumulation and distribution in orchard soil of
Loess Plateau. J Fruit Sci, 29: 322–327. (in Chinese)
Ge, S., Zhu, Z., Peng, L., Chen, Q., Jiang, Y., 2018a. Soil nutrient status
and leaf nutrient diagnosis in the main apple producing regions
in China. Hortic Plant J, 4: 89–93.
Ge, S., Zhu, Z., Jiang, Y., 2018b. Long-term impact of fertilization on soil
pH and fertility in an apple production system. J Soil Sci Plant Nutr,
18: 282–293.
229
Guo, J.H., Liu, X.J., Zhang, Y., Shen, J.L., Han, W.X., Zhang, W.F.,
Christie, P., Goulding, K.W.T., Vitousek, P.M., Zhang, F.S., 2010. Sig-
nificant acidification in major Chinese croplands. Science, 327:
1008.
Hu, K., Wang, L.B., 2007. Application of biolog microplate technique to
the study of soil microbial ecology. Chin J Soil Sci, 38: 819–821. (in
Chinese)
Kumar, U., Shahid, M., Tripathi, R., Mohanty, S., Kumar, A., Bhat-
tacharyya, P., Jambhulkar, N.N., 2017. Variation of functional diver-
sity of soil microbial community in sub-humid tropical rice-rice
cropping system under long-term organic and inorganic fertiliza-
tion. Ecol Indic, 73: 536–543.
Leroy, B.L.M., Herath, S., Sleutel, S., De Neve, S., Gabriels, D., Reheul, D.,
Moens, M., 2008. The quality of exogenous organic matter: short-
-term effects on soil physical properties and soil organic matter
fractions. Soil Use Manage, 24: 139–147.
Li, S., Lu, T., Zhang, X., Gu, G., Niu, Y., 2012. Effect of Trichoderma long-
brachiatumi T2 on functional diversity of cucumber rhizomicrobes.
J Environ Biol, 33: 293–299.
Li, X., Zhao, B., Li, X., Li, Y., Sun, R., Zhu, L., Xu, J., Wang, L., Li, X., Zhang, F.,
2005. Effects of different fertilization systems on soil microbe and
its relation to soil fertility. Sci Agri Sinica, 38: 1591–1599. (in Chi-
nese)
Lin, Y., Ye, G., Kuzyakov, Y., Liu, D., Fan, J., Ding, W., 2019. Long-term
manure application increases soil organic matter and aggregation,
and alters microbial community structure and keystone taxa. Soil
Biol Biochem, 134: 187–196.
Liu, L., Peng, F., Wang, X., 2010. Effects of bag-controlled release fertil-
izer on nitrogen utilization rate, growth and fruiting of the ‘Fuji’
apple. J Plant Nutr, 33: 1904–1913.
Lu, R.K., 1999. Soil and Agro-Chemical Analytical Methods. China Agri-
cultural Science and Technology Press, Beijing. (in Chinese)
Luo, X.Q., Hao, X.H., Chen, T., Deng, C.J., Wu, J.S., Hu, R.G., 2009. Effects
of long-term different fertilization on microbial community func-
tional diversity in paddy soil. Acta Ecologica Sinica, 29: 741–748. (in
Chinese)
Lv, M., Li, Z., Che, Y., Han, X., Liu, M., 2011. Soil organic C, nutrients,
microbial biomass, and grain yield of rice (Oryza sativa L.) after 18
years of fertilizer application to an infertile paddy soil. Biol Fertil-
izers Soils, 47: 777–783.
Massenssini, A.M., Bonduki, V.H.A., Melo, C.A.D., Totola, M.R., Fer-
reira, F.A., Costa, M.D., 2015. Relative importance of soil physico–
chemical characteristics and plants species identity to the deter-
mination of soil microbial community structure. Appl Soil Ecol, 91:
8–15.
Naher, U.A., Othman, R., Panhwa, Q.A., 2013. Culturable total and ben-
eficial microbial occurrences in long-term nutrient deficit wetland
rice soil. Aust J Crop Sci, 7: 1848–1853.
Qiu, X., Wang, Y., Hu, G., Wang, Q., Zhang, X., Dong, Y., 2013. Effect of
different fertilization modes on physiological characteristics, yield
and quality of Chinese cabbage. J Plant Nutr, 36: 948–962.
Ross, D.J., 1990. Measurement of microbial biomass C and N in grass-
land soils by fumigation-incubation procedures: influence of in-
oculums size and control. Soil Biol Biochem, 22: 289–294.
Shahid, M., Shukla, A.K., Bhattacharyya, P., Tripathi, R., Mohanty, S.,
Kumar, A., Lal, B., Gautam, P., Raja, R., Panda, B.B., Das, B.,
Nayak, A.K., 2016. Micronutrients (Fe, Mn, Zn and Cu) balance un-
der long-term application of fertilizer and manure in a tropical
rice-rice system. J Soils Sediments, 16: 737–747.
Sun, F., Zhang, W., Xu, M., Zhang, W., Li, Z., Zhang, J., 2010. Effects of
long-term fertilization on microbial biomass carbon and nitrogen
and on carbon source utilization of microbes in a red soil. Chin J
Appl Ecol, 21: 2792–2798. (in Chinese)
Tamilselvi, S.M., Chinnadurai, C., Ilamurugu, K., Arulmozhiselvan, K.,
Balachandar, D., 2015. Effect of long-term nutrient managements
on biological and biochemical properties of semi-arid tropical Al-
fisol during maize crop development stages. Ecol Indic, 48: 76–87.
230
Tan, B., Fan, J., He, Y., 2014. Effect of long-term application of chemical
fertilizer on soil organic carbon content in top layer of paddy fields
in South China. Acta Pedologica Sinica, 51: 96–103. (in Chinese)
Tiquia, S.M., Lloyd, J., Hems, D.A., Hoitink, H.A.J., Michel, F.C., 2002.
Effects of mulching and fertilization soil nutrients, microbial ac-
tivity and rhizosphere bacterial community structure determined
by analysis of TRFLPs of PCR-amplified 16S rRNA genes. Appl Soil
Ecol, 21: 31–48.
Wang, F., Sha, J., Chen, Q., Xu, X., Zhu, Z., Ge, S., Jiang, Y., 2020. Exoge-
nous abscisic acid regulates distribution of 13 C and 15 N and an-
thocyanin synthesis in ‘Red Fuji’ apple fruit under high nitrogen
supply. Front Plant Sci, 10: 1738.
Wang, K., Wang, Y.F., Chen, X.S., Gong, W.S., Shen, X., Yin, C.M.,
Mao, Z.Q., 2019a. Effects of matrine on old apple orchard soil pot-
ted Malus hupehensis and soil microorganisms. Acta Horticulturae
Sinica, 46: 1379–1387. (in Chinese)
Wang, Q., Ma, M., Jiang, X., Zhou, B., Guan, D., Cao, F., Chen, S., Li, J.,
2019b. Long-term N fertilization altered 13 C-labeled fungal com-
munity composition but not diversity in wheat rhizosphere of Chi-
nese black soil. Soil Biol Biochem, 135: 117–126.
Wang, W.J., Smith, C.J., Chalk, P.M., Chen, D., 2001. Evaluating chemical
and physical indices of nitrogen mineralization capacity. Soil Sci
Soc Am J, 65: 368–376.
Xia, X.Y., Zhang, P.P., He, L.L., Gao, X.X., Li, W.J., Zhou, Y.Y., Li, Z.X., Li, H.,
Yang, L., 2019. Effects of tillage managements and maize straw re-
turning on soil microbiome using 16S rDNA sequencing. J Integr
Plant Biol, 61: 765–777.
Zhanling Zhu et al.
Zhang, E., Wang, W., Zhang, S., 2014a. A study on active soil organic
carbon and available nutrients for protected soil under long-term
fertilization. J Shenyang Agri Uni, 45: 528–532. (in Chinese)
Zhang, L., Song, L., Shao, H., Brestic, M., Xu, G., Shao, C., Li, M., Liu, M.,
Shao, C., Song, L., Brestic, M., 2014b. Spatio-temporal variation
of rhizosphere soil microbial abundance and enzyme activities
under different vegetation types in the coastal zone, Shandong,
China. Plant Biosyst, 148: 403–409.
Zhang, S.H., Gao, W., Zhang, E.P., Li, L.L., 2012. Effect of long-term nitro-
gen application on culturable microorganisms quantity of tomato
continuous cropping. Chin J Soil Sci, 43: 60–65. (in Chinese)
Zhao, Z.P., Tong, Y.A., Liu, F., Wang, X.Y., 2013. Effects of different
long-term fertilization patterns on Fuji apple yield, quality, and
soil fertility on Weibei Dryland, Shaanxi Province of Northwest
China. Chin J Appl Ecol, 24: 3091–3098. (in Chinese)
Zhu, L., Xiao, Q., Shen, Y., Li, S., 2017. Microbial functional diversity
responses to 2 years since biochar application in silt-loam soils on
the loess plateau. Ecotox Environ Safe, 144: 578–584.
Zhu, Z., Jia, Z., Peng, L., Chen, Q., He, L., Jiang, Y., Ge, S., 2018. Life cy-
cle assessment of conventional and organic apple production sys-
tems in China. J Clean Prod, 201: 156–168.