Environment International 161 (2022) 107133

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Environment International
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The ecological clusters of soil organisms drive the ecosystem

multifunctionality under long-term fertilization
Yi-Fei Wang a, b, Peng Chen c, Feng-Hua Wang a, e, Wan-Xue Han b, d, Min Qiao a, Wen-Xu Dong d,
Chun-Sheng Hu d, Dong Zhu a, *, Hai-Yan Chu b, f, Yong-Guan Zhu a, b, g
a
State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China
c
Key Laboratory of Aquatic Botany and Watershed Ecology, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
d
Key Laboratory of Agricultural Water Resources, Hebei Key Laboratory of Soil Ecology, Center for Agricultural Resources Research, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050022, China
e
College of Resource and Environment Sciences, Hebei Key Laboratory of Environmental Change and Ecological Construction, Hebei Normal University, Shijiazhuang
050024, China
f
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China
g
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China

A R T I C L E I N F O A B S T R A C T

Handling Editor: Adrian Covaci Long-term fertilization is known to impact the biodiversity and community structures of soil organisms, which
are responsible for multiple soil ecosystem functions (multifunctionality). However the relationship between the
Keywords: alterations of soil organisms and ecosystem multifunctionality remains unclear, especially in the case of long-
Soil microorganism term fertilization. To explore the contribution of soil organismal biodiversity and community structures to
Soil animal
ecosystem multifunctionality, we took soil samples from a nearly 25-year field fertilization experiment. Organic
Organic fertilizer
matter significantly improved the soil ecosystem multifunctionality. Ecosystem multifunctionality was found to
Biodiversity
Trophic co-occurrence network be closely linked to the biodiversity and communities of soil organisms within the major ecological clustering of
Ecological function soil organisms (Module 1) according to the trophic co-occurrence network, rather than the entire community of
soil organisms. This indicated that ecological clusters of soil organisms within the network were critical in
maintaining soil ecosystem multifunctionality. The application of organic fertilization could enrich specialized
soil organisms and increase interactions of soil organisms in the ecological cluster. As a result, our findings
emphasize the role of ecological clusters in the soil organismal co-occurrence network in controlling soil mul­
tifunctionality after long-term fertilization, presenting a novel perspective on the link between soil biodiversity
and ecosystem multifunctionality.

1. Introduction multifunctionality and negatively influence the provision of soil

The role of aboveground organismal biodiversity in maintaining
multiple ecosystem functions (multifunctionality) has been extensively
explored (Hector and Bagchi, 2007; Zavaleta et al., 2010; Zhu et al.,
2021a). The role of soil organismal diversity in ecosystem multi­
functionality remains uncertain. Soil organisms have attracted extensive
attention in recent years because of their essential ecosystems functions,
such as nutrient cycling, litter decomposition, and maintenance of soil
fertility (Bahram et al., 2018; Bardgett and van der Putten, 2014; Bender
et al., 2016; Wang et al., 2021a, 2021b). Delgado-Baquerizo et al. (2016)
have elucidated that any loss in microbial diversity will probably lower
ecosystem services across 78 drylands at a global scale and 179 Scotland
locations. Whereas a meta-analysis of the influences of global change
factors on soil microbial diversity and functionality across 1235 samples
illustrated that the soil multifunctionality is elucidated by the microbial
community structure and biomass rather than the alpha diversity (Zhou
et al., 2020). This unclear relationship between soil organisms and
multifunctionality is even less common in agroecosystems. However,
most investigations on soil biodiversity and ecosystem multi­
functionality concentrate on microbes, ignoring microfauna that per­
formed essential functions in the soil micro-food web (Carrillo et al.,
2011; Cui et al., 2020; Crowther et al., 2011; Lavelle et al., 1997). Such

* Corresponding author.
E-mail address: dongzhu@rcees.ac.cn (D. Zhu).

https://doi.org/10.1016/j.envint.2022.107133
Received 25 October 2021; Received in revised form 31 January 2022; Accepted 2 February 2022
Available online 8 February 2022
0160-4120/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y.-F. Wang et al.
knowledge is crucial in managing the functions and sustainable devel­
opment of the soil ecosystem.
Fertilization, as a commonly employed management practice for
agricultural soils, has been demonstrated to influence not only the soil
biodiversity but also the community structures of soil organisms (Wei
et al., 2017; Yao et al., 2017; Zhao et al., 2020). The repeated organic
fertilization revealed a higher abundance of Gram-positive bacteria than
Gram-negative bacteria (Wei et al., 2017). An organic amendment was
found to suppress potential crop fungal pathogens in black soil (Yao
et al., 2017). Recent studies have also reported the impacts of fertil­
ization on protists, and important component of soil microbes, in agri­
cultural soil (Sun et al., 2021). Furthermore, Suleiman et al. reported
that organic fertilization boosted inter-and intra-connections among
primary decomposers (bacteria and fungi) and predators in the soil food
web (protists and invertebrates) (Suleiman et al., 2019). The effects of
these biotic disturbances on the multiple soil functions generated by soil
organisms have hardly been identified. It is crucial to investigate the
impacts of long-term fertilization on soil organisms and ecosystem
multifunctionality as the intensity for applying chemical fertilizers is
still increasing.
Some vital ecosystem functions are largely determined by specialized
microbes and species interactions, rather than the entire community
(Harvey et al., 2017; Li et al., 2020; Zhu et al., 2021b). Studies have
utilized microbial co-occurrence evaluation to investigate the micro­
biome complexity and the interactions among taxa (Barberán et al.,
2012). It has been demonstrated that in soil ecosystem, complex inter­
connected microbial networks are structured (Morrien et al., 2017;
Wagg et al., 2019), with ecological clusters (modules) within the
network playing an important role in maintaining community compo­
sition and functions (Delgado-Baquerizo et al., 2018; Ling et al., 2016;
Sul et al., 2013; Zhu et al., 2021b). For example, Ling et al. have found
that the major modules in network responsible for almost all of the
significant soil functions in chemical fertilizer treatment (Ling et al.,
2016). A study has also shown that soil fertility and maize yield could be
affected by altering bacterial network modularization (Ye et al., 2021a).
Therefore, ecological modules based on soil microbial co-occurrence
network analyses would be an employable strategy to investigate the
relationship between soil microbiome and ecosystem multifunctionality.
Soil organisms live in a complex soil food web, such as numerous intra-
and inter-connection between microbes and microfauna that impact
ecological functions (Suleiman et al., 2019). However, most studies
using network analysis have focused on the microbial interactions in
modules, ignoring the role of soil microfauna. Thus, it is important to
consider the effect of soil microfauna in ecological modules on
ecosystem functions. This would advance our understanding of the re­
lationships between soil organisms and functions in terrestrial
ecosystem.
To explore the relationship between the biodiversity of soil organ­
isms and ecosystem multifunctionality, we collected soil samples from a
long-term field experiment with a fertilization history of almost 25
years. Long-term mineral fertilization may result in soil acidification
(Guo et al., 2010), whereas organic fertilization significantly boosts soil
physicochemical qualities, such as cation exchange capacity, organic
carbon, and conductivity (Zhang et al., 2021). Furthermore, the strongly
structured communities in the soil ecosystem (Lentendu et al., 2014),
with complex microbial-microfaunal ecological relationships are iden­
tified to be developed by organic fertilizers (Suleiman et al., 2019). The
ecological clusters of soil taxa are predicted to conserve soil fertility and
plant productivity in soil ecosystems (Delgado-Baquerizo et al., 2018).
Thus, we hypothesized that 1) Organic fertilization, as opposed to non-
fertilization and mineral fertilization alone, would promote the soil
ecosystem multifunctionality; 2) Ecosystem multifunctionality is driven
by biodiversity and community dynamics in ecological clusters of soil
organisms, rather than the overall communities; 3) The multi­
functionality of ecosystems may be influenced by trophic interactions
among soil organisms.
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Environment International 161 (2022) 107133
2. Materials and methods
2.1. Soil sampling
The fluvo-aquic soil samples were collected from the Luancheng
Argo-ecosystem Experimental Station of the Chinese Academy of Sci­
ences in Hebei Province, China. The long-term field analysis was
established in 1998 with eight treatments that employed various fertil­
izers: CK (non-fertilization), N (300 kg urea ha− 1 soil), NP (300 kg urea
and 120 kg superphosphate ha− 1 soil), NPK (300 kg urea, 120 kg su­
perphosphate and 75 kg potassium chloride ha− 1 soil), M (4000 kg pig
manure ha− 1 soil), MN (5500 kg pig manure and 300 kg urea ha− 1 soil),
MNP (7000 kg pig manure, 300 kg urea and 120 kg superphosphate ha− 1
soil), MNPK (7500 kg pig manure, 300 kg urea, 120 kg superphosphate,
and 75 kg potassium chloride ha− 1 soil). There were three replicate plots
(plot size 6 × 16 m2) for each treatment randomly distributed in the
field. In November 2019, soil samples (0 to 20 cm depth) were taken
from a winter wheat field by taking five core soils from each plot and
mixing them with one composite sample. A total of 24 soil samples were
transported in ice to the laboratory and immediately divided into three
parts: one of which was placed at 4 ◦ C for soil basal respiration (SBR)
and available N analyses, the second portion air-dried for physico­
chemical analyses, and the last stored at − 20 ◦ C for molecular analyses.
2.2. Soil physicochemical analyses
Soil pH was detected by a pH meter （FE20, Mettler Toledo,
Switzerland） in a 1:2.5 mass/volume soil–water (carbon dioxide-free)
suspension, and the filtrate filtered from 0.45 μm syringe cellulose ac­
etate filter was employed to estimate the soil electrical conductivity (EC)
by utilizing a conductometer (ORION TSI-STARA212, Thermo Scientific,
USA). About 10 g of fresh soil was employed to estimate the soil mois­
ture by drying at 105 ◦ C to get to a constant weight. The soil total N (TN
%) and total carbon (TC%) were estimated by an Element Analyzer
(Vario MAX cube; Elementar, Germany).
2.3. Soil DNA extraction and the analyses of soil organism communities
Briefly, 10 g freeze-dried soil for each sample was carefully ground
with liquid nitrogen to extract the DNA information of soil fauna
comprehensively. Then, 0.5 g ground soil was utilized to extract DNA by
a FastDNA Spin Kit for Soil (MP Bio, USA). The NanoDrop spectropho­
tometer (Lambda25, PE, USA) was employed to determine the concen­
tration and quality of the extracted DNA.
To study the diversity and structure of soil community, soil DNA was
sequenced in an Illumina NovaSeq platform. We utilized the 515f/806r
primer set for archaea and bacteria, ITS1F/ITS2R primer set for fungi,
and Euk1391f/EukBr primer set for protists and metazoan (Zhu et al.,
2021c). Sequence reads were processed with “DADA2” package in R
following Callahan et al. (Callahan et al., 2016). The Amplicon Sequence
Variants (ASVs) were clustered utilizing a 100% identity level, replacing
standard OTUs with 97% similarity. Taxonomy was assigned utilizing
the SILVA database (release 138) for archaea and bacteria, UNITE
database for fungi, and PR2 database for protists and metazoa. Before
downstream evaluation, the “vegan” package in R was employed to
purify the ASV table based on the minimum sequencing reports across
all samples. The alpha diversity was calculated employing R with the
“vegan” package. Principal coordinate analysis (PCoA) based on the
Bray–Curtis distance matrix identified the differences in the microbial
community composition (β-diversity), which was conducted in R with
the “vegan” package. To predict the bacterial potential ecological
function, we employed the FAPROTAX database (Louca et al., 2016;
Sansupa et al., 2021). The identity of fungal guilds was determined
utilizing FUNGuild (Nguyen et al., 2016).
Y.-F. Wang et al. Environment International 161 (2022) 107133

2.4. Soil ecosystem multifunctionality assessment 2.6. Statistical analyses

We measured 10 ecosystem functions related to (1) nutrient cycling
(soil available N and P); (2) soil fertility (soil organic matter, TC% and
TN%); (3) organic matter (OM) decomposition (β-glucosidase, urease,
and SBR); (4) 70 function genes, such as genes encoding for C-fixation,
C-degradation, denitrification, nitrification, P-cycling, and S-cycling.
The concentrations of soil available N nitrate (NO3− -N) and ammonium
(NH4+-N) were determined by a spectrophotometer (Lambda25; PE,
USA) following the 6 g fresh soil extracted with 1 M KCl (shaking at 1 h
for 200 rpm). Soil available phosphorus (AP) was extracted by 0.5 M
NaHCO3 and estimated by a spectrophotometer (Lambda25; PE, USA)
employing a molybdenum blue approach. The K2Cr2O7 oxidation
approach was employed to estimate soil OM. The SynergyH1 Microplate
Reader (H1M, BioTek, Germany) was utilized to determine the soil
β-glucosidase and urease activities following the instructions of the soil
enzyme kit from Solarbio Science & Technology Co., Ltd (Beijing,
China). The soil SBR was estimated by incubating about 30 g of dry soil
in a 250 ml Boston amber round at 6 h for 25 ◦ C after a 12 h pre­
incubation. The carbon dioxide (CO2) was monitored with an infrared
analyzer (EGM-4; PP Systems, Amesbury, MA) (Ge et al., 2016). High-
throughput quantitative PCR (HT-qPCR) was conducted to determine
the relative abundance of soil function genes for C, N, P, S-cycling. There
are 70 primer sets involved in this approach, and the specific reaction
system and amplification conditions were described by Zheng et al.
(2018).
The soil multifunctionality index was determined using the aver­
aging approach as below (Bradford et al., 2014; Chen et al., 2020;
Delgado-Baquerizo et al., 2020): to prevent the differences in data scale,
all individual soil ecosystem functions estimated in this investigation
were normalized from 0 to 1 using the “Max-Min” approach (Delgado-
Baquerizo et al., 2020; Schuldt et al., 2018; Soliveres et al., 2016):
normalizedFunction = ((rawFunction-min(rawFunction)/max(raw­
Function)-min(rawFunction)), and then the soil multifunctionality was
calculated by average all normalized functions. The multi-threshold
approach evaluates the connection between biodiversity and the
numbers of functions that exceed a critical threshold (5%–99%) were
evaluated with the “multifunc” package in R (Byrnes et al., 2014).
The average soil functions and relative abundance of specific or­
ganisms were generated with Microsoft Excel 2016. The data were all
expressed as the mean values ± standard error (n = 3), and the differ­
ences among treatments were estimated at a significance of 0.05 by
single evaluation of variance or nonparametric Kruskal–Wallis test
employing IBM SPSS version 22. The changes in the microbes including
bacteria and fungi generated by the different treatments were examined
with the linear discriminant analysis effect size (LEfSe) approach. The
ordinary least squares linear regressions between the multi­
biobiodiversity of modules and soil ecosystem multifunctionality were
generated by Origin 2017. The structural equation model (SEM) was
developed to evaluate the direct and indirect effects of organic fertil­
ization, pH, the multibiodiversity of a specific module, and organismal
community structure of a specific module on the soil ecosystem multi­
functionality using AMOS 21 (SPSS Inc., Chicago, USA). For organismal
community structure, we employed the value of principal coordinate
axis 1 from the PCoA plot to represent. The overall model fit was eval­
uated with the Chi-square test, goodness-of-fit index (GFI), and Root
means square error of approximation (RMSEA).
3. Results
3.1. Changes in soil physicochemical features and ecosystem
multifunctionality under long-term fertilization
The organic fertilization significantly increased the soil pH
(Figure S1A, P < 0.05) than the mineral fertilization. The diversities of
soil EC among each treatment were not significant except for MNPK
(Figure S1B). Soil total C% and N% were remarkably increased by the
organic fertilizer application (Table S1, P < 0.001).
Generally, organic fertilization significantly improved the soil
ecosystem multifunctionality (Fig. 1, P < 0.01), with a nearly 4 times
increase compared with non-fertilization. Multiple functional indexes of
the soil ecosystem were significantly increased by the organic

2.5. Correlation networks

The co-occurrence network was constructed by the “igraph” package

in R (Hartman et al., 2018). The filtered 38 archaeal, 4894 bacterial, 679
fungal, 261 protozoan, and 248 metazoan ASVs were merged into a
table. We focused on the ASVs related to all five group soil organisms
with at least 10 sequences in at least 12 samples. Then the spearman
association between ASVs was conducted and P values were calculated
utilizing the Benjamini and Hochberg false discovery rate (FDR) test
(Benjamini et al., 2006). In addition, we employed the “indicsepcies” and
“edgeR” packages to calculate the differential ASVs among treatments.
The network was visualized by Gephi utilizing the significant correla­
tions (spearman’s r > 0.7, Padj < 0.005). According to previous studies
(Asuero et al., 2007; Schober et al., 2018), the coefficient of correlation
is usually classified as low (r < 0.30), moderate (r ~ 0.30–0.70) or strong
(r > 0.70). In order to obtain robust associations between soil organisms
through network analysis, we set the threshold of spearman’s r to 0.7. A
total of two networks were created, primarily due to the coloring
partition of nodes, one is based on varied soil organisms, and the other is
based on whether the ASV is differential or not. Furthermore, the to­
pological network properties including degrees of co-occurrence nodes
and modules (ecological cluster) were determined by Gephi (Blondel
et al., 2008). The degree distribution was shown in Figure S12. The
multibiodiversity involving archaea, bacteria, fungi, protist, and meta­ Fig. 1. Average multifunctionality index in response to fertilization. Asterisks
zoa in modules was calculated utilizing the approach mentioned above indicate statistically significant differences from the control treatment (*P <
for calculating the soil ecosystem multifunctionality. 0.05, **P < 0.01, ***P < 0.001).

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Y.-F. Wang et al. Environment International 161 (2022) 107133

fertilization (P < 0.05), as shown in Table S1. that fertilization remarkably altered the communities of all soil organ­
isms (Figure S3, P < 0.01).

3.2. Changes of soil organisms under long-term fertilization

Across all samples, 2 309 795, 1 135 119, and 2 388 175 non-
singleton reads were counted for microorganisms (archaea, bacteria,
fungi and protist) and soil microfauna (i.e. metazoa), respectively. Be­
sides, 38 archaeal, 4894 bacterial, 679 fungal, 261 protozoan, and 248
metazoan ASVs were determined. The prevalent archaeal phylum in all
soil samples was Crenarchaeota, with a relative abundance as high as
99%. Bacterial phyla Actinobacteriota(29.38%), Proteobacteria
(26.59%), Acidobacteriota (12.25%), and Chloroflexi (8.79%) were
dominated in all soil samples. Ascomycota (79.09%), Mortierellomycota
(10.54%), and Basidiomycota (3.22%) were the predominant fungal
phyla. The primary protists were Stramenopiles, Amoebozoa, and
Archaeplastida, with the relative abundance of 30.22%, 25.02%, and
24.06%, respectively. Nematoda (9.30%) and Arthropoda (3.18%) were
dominated metozoa in soil samples (~87% metazoan sequences could
not be reliably attributed to a metazoan phylum). The alpha diversity of
all soil organisms, such as archaea, bacteria, fungi, protists, and metazoa
fluctuated in various treatments, but there were no significant changes
(Figure S2, P > 0.05). Principal coordinate analysis (PCoA) demon­
strated that the organic fertilization harbored unique soil organismal
communities compared with other treatments. Adonis test also revealed
3.3. Ecological clusters of soil organisms in the multitrophic co-occurrence
network
Three prevalent ecological clusters (Module 1–3) were noted in the
multitrophic network based on the co-occurrence of ASVs (Fig. 2A).
Among all soil organisms, bacterial ASVs accounted for the highest
proportion in the three modules. Compared with the other modules,
Module 1 contained the most diverse soil organisms, in which the nodes
of fungi accounted for 12.46%, archaea for 1.31%, metazoa for 3.93%,
and protists for 2.30% (Fig. 2B). Whereas, more than 95% of ASVs in
Module 2 were bacteria. Furthermore, the proportion of ASVs with high
node degrees in Module 1 was higher than that in other modules, with
nearly 30% of ASVs nodes degrees higher than 50 (Fig. 2C). Except for
edges generated by the autocorrelation of soil organisms, Module 1
possessed the highest proportion of edges generated by the correlation of
various soil organisms. Module 1 consisted of 7055 robust edges, of
which 24.05% were produced by bacteria and fungi, 2.44% by bacteria
and protists, 2.06% by bacteria and metazoa, 0.57% by fungi and pro­
tists, 0.24% by fungi and metazoa, and 0.14% by protists and metazoa
(Fig. 2D).
The indicator species and edgeR analyses illustrated that the

Fig. 2. Ecological clusters of soil organisms based on multitrophic co-occurrence networks. (A) The main modules (Module 1–3) clustered in the network diagram
with nodes colored by the organismal phylotypes; (B) ASV number proportion of the soil organismal phylotypes in the main ecological clusters; (C) Nodes degree
proportion in the main ecological clusters; (D) The proportion of edges formed by the different soil organisms in total edges.

4
Y.-F. Wang et al.
fertilization-sensitive ASVs were mostly distributed in Module 1 and
partially distributed in Module 3 (Figure S4). In Module 1, for archaea,
the application of organic fertilizers notably increased the relative
abundance of Nitrososphaeraceae (Fig. 3A, P < 0.05). For bacteria, the
organic fertilization remarkably enriched the Cellvibrio, Thermomonas,
Adhaeribacter, Stenotrophomonas, Streptomyces, Actinocorallia, Clos­
tridiumsensustricto1, while depleting the Nocardioides, Solirubrobacter,
Agromyces, and Xylophilus compared with CK treatment (Figure S5, P <
0.05, LDA > 2). Through the function prediction by the FAPROTAX
database, we observed that organic fertilization significantly improved
the bacterial ecological functions involved nutrient cyclings, such as
nitrogen respiration, nitrate reduction, nitrate respiration, fermentation,
and cellulolysis (Figure S6, P < 0.05). However, the function related to
the aromatic compound degradation was remarkably reduced compared
with CK treatment (Figure S6, P < 0.05). For fungi, organic fertilization
significantly increased the relative abundance of Ascomycota, such as
the orders Pezizales and Sordariales (Figure S7, P < 0.05, LDA > 2). The
fungal trophic mode proposed by the FUNGuild illustrated that the
application of organic fertilizers not only enriched the saprotrophic
fungi in the soil but also reduced the pathogens (Figure S8). For protist,
the organic fertilization remarkably improved the relative abundance of
the Acanthamoebidae belonging to Amoebozoa (Figure S9, P < 0.05),
and the Rhogostoma-lineage belonging to Cercozoa (Figure S9, P <
0.01). In Module 3, the relative abundance of the bacterial phylum
Acidobacteriota, class Dehalococcoidia, and Longimicrobia were higher
in CK treatment compared with fertilization (Figure S10, P < 0.05, LDA
> 2). The mineral fertilization significantly enriched the phylum Ento­
theonellaeota, Class Holophagae, and Order Rhizobiales (Figure S10, P
< 0.05, LDA > 2).
3.4. Links between the soil organisms and ecosystem multifunctionality
There were no significant associations between the biodiversity of all
five soil organisms determined in this investigation and the ecosystem
multifunctionality (Figure S11, P > 0.05). We found that the soil mul­
tibiodiversity of Module 1 was notably positively correlated with the
ecosystem multifunctionality (Fig. 4A, R2 = 0.83, P < 0.01). Likewise,
the positive effect of multibiodiversity in Module 1 on multifunctionality
was also noted by the multiple-thresholds approach (Fig. 4B). The
minimum threshold (Tmin) was 6%, which was expressed as the lowest
threshold where diversity began to have a positive impact on multi­
functionality. The realized maximum impact of diversity (Rmde), i.e.,
the strength of the relationship where diversity has its strongest positive
impacts, was 11.87 at the threshold of 65%, indicating that the addition
of one species can increase 11.87 functions. To further investigate the
multiple direct and indirect impacts of organic fertilization, soil pH,
communities of soil organisms, and soil multibiodiversity of modules on
Fig. 3. The total relative abundance of soil organisms in three main modules. Asterisks indicate statistically significant differences from the control treatment (*P <
0.05, **P < 0.01, ***P < 0.001).
5
Environment International 161 (2022) 107133
the ecosystem multifunctionality, a SEM was conducted (Fig. 5). The
SEM expressed 85% of the variance of soil ecosystem multifunctionality
by Module 1 of the soil ecological network. The communities of soil
organisms had strong and direct positive impacts on the multi­
functionality (Fig. 5, β = 0.96, P < 0.001). Furthermore, the positive
impacts of soil multibiodiversity on the communities of soil organisms
(β = 0.70, P < 0.001) were observed. The standardized total effect from
SEM also indicated that communities of soil organisms and soil multi­
biodiversity in Module 1 were the predominant drivers of soil ecosystem
multifunctionality (Fig. 5).
4. Discussion
As expected, the long-term organic fertilization considerably
increased the soil ecosystem multifunctionality in our study, which is
consistent with prior findings indicating organic fertilization improved
multifunctionality in a long-term experimental station (Chen et al.,
2020). Compared with mineral fertilization alone, organic fertilization
could maintain or even improve the soil nutrient level, such as the
content of OM, available nitrogen, and phosphorus. This result was
supported by several studies involving various soil types (Anwar et al.,
2005; Ling et al., 2016; Liu et al., 2010; Su et al., 2006), such as silt loam
soil, black soil, fluvo-aquic soil and red soil. Furthermore, soil enzyme
activities were also stimulated by organic fertilization. Soil enzymes
mediate the transformation of complex OM into stable OM that is
available to crops (Lupwayi et al., 2019). This demonstrates that intro­
duced manure not only enriches the content of OMs in soil but also
improves soil fertility that is beneficial for crop production.
We found that fertilization did not alter the soil organismal alpha
diversity, which is consistent with the study of organic amendment for
years in an experimental farm in the Netherlands (Suleiman et al.,
2019). Organic fertilization altered the communities of soil organisms.
Fertilization has been demonstrated to be an essential driver for the soil
prokaryotic and eukaryotic microbial communities in agroecosystems
(Lin et al., 2019; Semenov et al., 2020; Zhao et al., 2019). Accordingly,
soil pH in common environmental variables was closely connected with
microbial communities structure (Siciliano et al., 2014; Wei et al., 2017;
Rousk et al., 2011). This result is in accordance with that of our study, as
soil pH was increased by organic fertilization versus mineral fertilizers.
Also, the communities of high trophic soil organisms (protist and met­
azoa) may alter along with the potential food resources, such as mi­
crobial communities or enriched organic matters by the organic
fertilization (Birkhofer et al., 2008).
The significant positive relationships between the biodiversity of soil
organisms and ecosystem multifunctionality were not found in this
study. This may be due to the functional redundancy of soil organisms in
agroecosystems, meaning that different species can perform the same
Y.-F. Wang et al. Environment International 161 (2022) 107133

Fig. 4. Relationships between ecosystem multifunctionality and the multibiodiversity of soil organisms in Module 1. (A) Solid red line represents a linear relationship
that fitted the ordinary least-squares (OLS) regression model, and the shadow area shows the 95% confidence interval of the fit. Significant levels are given at ***P <
0.001. (B) Effects of multibiodiversity of Module 1 on the number of functions nabove thresholds. Lines represent the slope between soil microbial diversity and the
number of functions greater than or equal to a threshold value ranging from 5 to 99% of maximum for each function. The dotted curves indicate the changes in
number of functions per unit increment of multibiodiversity. Tmin, is the minimum threshold that multifunctionality becomes influenced by changes in multi­
biodiversity, and Rmed, is the realized maximum effect of multibiodiversity on multifunctionality. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

ecosystem function (Li et al., 2021). However, some studies have sug­
gested that soil functions may be conducted by specialized microbes,
rather than the entire community (Bastida et al., 2016). Besides, the
specialized microbes were expected to gather to ecological clusters to
maintain multiple soil functions (Delgado-Baquerizo et al., 2018). Three
primary modules were detected from the co-occurrence network of soil
organisms under long-term fertilization. We detected that different
fertilization practices gathered ASVs into different modules, indicating
that the connection of species in the network correlates with nutrient
preference in the soil environment. The same phenomenon was also
observed in arable soil under long-term differential fertilization by a co-
occurrence-based network evaluation (Lentendu et al., 2014). It is also
worth noting that there were indeed some limitations by the co-
occurrence network analysis, which would require us to improve
methodology in further studies while employing the cultivation. In our
study, the multibiodiversity of soil organisms in Module 1 was signifi­
cantly positively correlated with the ecosystem multifunctionality. As
we have found, the sensitive ASVs for the application of organic fertil­
izers were gathered into Module 1, which may perform ecological
functions, such as nutrient cycling and organic degrading.
The archaeal Nitrososphaeraceae with significantly increased rela­
tive abundance by the organic fertilization in Module 1 was the famous
and abundant ammonia-oxidizing archaea, which plays a critical role in
nitrification (Pelissari et al., 2017; Stieglmeier et al., 2014). Wu et al.
have applied metagenomics in a long-term field experiment under
organic material amendments and reported that the Nitrososphaeraceae
not only participates in soil N cycling but also contributes to the solu­
bilization and transport of inorganic phosphorus (Wu et al., 2021). It is
worth noting that archaea performed trophic function only in Module 1
sensitive to organic fertilization. This indicates that organic fertilization
accelerates the nutrient cycling mediated by archaea in agricultural soil,
which is more conducive to crop production.
For fungi, organic fertilization remarkably enriched the Ascomycota,
which is a dominant fungal phylum across almost all agricultural soils.
Ascomycota is well identified to be organic decomposers, generally
growing with organic fertilizers application in agroecosystems (Chen
et al., 2016; Ye et al., 2021b, 2020). The relative abundance of fungal
orders Pezizales and Sordariales markedly increased in Module 1
responding to the organic fertilization. Lin et al. reported that amend­
ment of pig manure elevated the abundance of Pezizales than mineral
fertilization (Lin et al., 2019). The Pezizales have been confirmed to be
largely attracted to available compounds (Lin et al., 2019), which are
introduced by the application of organic fertilizers. Some studies have
noted that the relative abundance of Sordariales was improved by the
application of organic fertilizers (Lin et al., 2019; Wang et al., 2021a,
2021b). Sordariales are critical saprotrophic fungi, which play a sig­
nificant role in the process of nutrient cycling in the soil environment
through the biodegradation of complex organic compounds (Poggeler,
2011). Through the FUNGuild prediction, we observed that organic
fertilization elevated the saprotrophic fungi compared with the non-
fertilization and mineral fertilization in Module 1. It is broadly
demonstrated that saprotrophic fungi contribute greatly to ecosystem
function related to agricultural production (Crowther et al., 2012). They
are primary decomposers that are responsible for soil nutrient cycling
(Deacon et al., 2006) and possess the ability to suppress plant patho­
genic fungi (van der Wal et al., 2013). We observed the decreased
abundance of potential pathotrophs in treatments with the application
of organic fertilizers. As a result, organic fertilization would promote
organic decomposers while suppressing the potential plant-fungal
pathotrophs, stimulating the nutrient cycling in agricultural soil.
For bacteria, the enriched genus Adhaeribacter belonging to the
phylum Bacteroidota has been investigated to excrete large amounts of
extracellular fibrillar material that can degrade polymers (Zhang et al.,
2009). The genera Stenotrophomonas and Streptomyces with significantly
increased relative abundance were well-investigated to be the model
organisms for plant growth promotion (Berg, 2009). The Steno­
trophomonas were found to be distributed throughout the agricultural
environment, which has an essential role in the biogeochemical cycle
including nitrogen and sulfur (Ryan et al., 2009). The Streptomyces are
famous Actinobacteriota that can break down complex biological poly­
mers such as chitin and lignin et al (Schrey and Tarkka, 2008). Mean­
while, abundant antibiotics are also produced by the Streptomyces,
which modulate the plant defense (Davelos et al., 2004). The ecological
function proposed by the bacterial composition also reveals that organic
fertilization accelerates organic decompose and nutrient cycling
through bacteria compared with the non-fertilization and mineral
fertilization alone. Furthermore, through the proportion of edges
generated by the organisms correlated in Module 1, we found that
organic fertilization strengthened the correlation between fungi and
bacteria, implying the potential strong interactions. Meidute et al. have
reported that the addition of complex substrate proposed synergistic
effects between fungi and bacteria, in that bacterial growth started to

6
Y.-F. Wang et al.
increase only after fungal growth had increased (Meidute et al., 2008).
This confirms our findings that the relative abundance of saprophytic
fungi significantly increases by organic fertilization, transforming
complex organic matter into sample organic molecules, promoting
bacteria growth.
Besides, typical bacterial grazers Acanthamoeba (Lobosa) and Rho­
gostoma-lineage (Cercozoa) were significantly enriched by the organic
fertilization in Module 1. A greenhouse investigation also shown that
organic fertilizers amendment strongly increased bacterivorous protists
and reduced the relative abundance of plant pathogenic protists (Xiong
et al., 2018). The microbe-consuming protists were investigated to be
beneficial to crops growth through top-down regulation of the soil mi­
crobial communities by preying on plant-detrimental microbes or the
competitors of agriculturally beneficial microbes (Gao et al., 2019; Guo
et al., 2021). The Gammaproteobacteria which was identified to be able
to increased soil multifunctionality (Delgado-Baquerizo et al., 2017) was
significantly enriched by organic fertilization in our study. This in­
dicates that the trophic interaction between soil organisms would play
an important role in the soil ecosystem multifunctionality and crops
production.
We further found that the soil organismal community structures in
7
Environment International 161 (2022) 107133
Fig. 5. Structural equation model evaluating the
direct and indirect effects of organic fertilization, soil
pH, communities of soil organisms and soil multi­
biodiversity of modules on the ecosystem multi­
functionality. The red arrows indicate positive effects
and the black ones are opposite. Solid and dotted
lines show significant and insignificant effects
respectively. Significant levels are given: *P < 0.05,
**P < 0.01, ***P < 0.001. Numbers next to the ar­
rows are path coefficients and R2 denote the pro­
portion of variance explained for each variable.
Standardized total effects are derived from the
structural equation models. (For interpretation of the
references to colour in this figure legend, the reader
is referred to the web version of this article.)
Module 1 contributed remarkably to ecosystem multifunctionality
through SEM evaluation. The organismal community structures in
Module 1 were not only reflected in the relative abundance changes of
specific organisms by the application of organic fertilizers but also
manifested in the trophic interactions between organisms. These in­
teractions contained the potential nutritional interaction between fungi
and bacteria, as well as the top-down regulation of microbe-consuming
protists. Suleiman et al. have also revealed that organic amendment
strengthens diverse microbe-eukaryote associations by soil RNA
approach in field conditions (Suleiman et al., 2019). The effects of
higher trophic levels were found to contribute a lot to multifunctionality
extends far beyond effects on the functioning within individual trophic
levels, indicating the direct and indirect interactions between trophic
groups influence ecosystem-level processes (Schuldt et al., 2018). As a
result, in investigating the ecosystem multifunctionaltiy we need to pay
more attention to the changes in community structures influenced by the
trophic interactions among organisms.
5. Conclusions
In conclusion, long-term organic fertilization could increase the soil
Y.-F. Wang et al.
ecosystem multifunctionality, such as nutrient cycling, soil fertility, and
OM decomposition. The multibiodiversity and the organismal commu­
nity structures of ecological cluster Module 1 were observed to be
significantly positively correlated with the ecosystem multi­
functionality, indicating that the specialized soil organisms and trophic
interactions among soil organisms may play an essential role in main­
taining soil multifunctionality. Our findings generally improved the
knowledge of the association between soil organisms and ecosystem
multifunctionality, providing a new perspective for the adjustment of
agricultural management strategies.
6. Data availability
The data from this study was deposited in NCBI Sequence Read
Archive under accession SRA: SRP342903. The authors declare that the
other main data supporting the findings of this study are available
within this Article and in the Supplementary materials files. Extra data
supporting the findings of this study are available from the corre­
sponding author upon reasonable request.
CRediT authorship contribution statement
Yi-Fei Wang: Methodology, Software, Formal analysis, Investiga­
tion, Writing – original draft, Writing – review & editing, Visualization,
Funding acquisition. Peng Chen: Conceptualization, Methodology,
Investigation, Writing – original draft, Writing – review & editing, Su­
pervision. Feng-Hua Wang: Conceptualization, Methodology, Investi­
gation, Writing – original draft, Writing – review & editing, Supervision.
Wan-Xue Han: Conceptualization, Methodology, Investigation, Writing
– original draft, Writing – review & editing, Supervision. Min Qiao:
Investigation, Data curation, Writing – review & editing. Wen-Xu Dong:
Investigation, Data curation, Writing – review & editing. Chun-Sheng
Hu: Investigation, Data curation, Writing – review & editing. Dong Zhu:
Methodology, Software, Formal analysis, Investigation, Writing – orig­
inal draft, Writing – review & editing, Visualization, Funding acquisi­
tion. Hai-Yan Chu: Writing – review & editing. Yong-Guan Zhu:
Methodology, Software, Formal analysis, Investigation, Writing – orig­
inal draft, Writing – review & editing, Visualization, Funding
acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was funded by the National Natural Science Foundation of
China (42021005 and 42077216).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envint.2022.107133.
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