ArticlePDF Available

Fungal community composition in soils subjected to long-term chemical fertilization is most influenced by the type of organic matter: Fertilizer organic matter influences soil fungal community

Authors:

Abstract and Figures

Organic matter application is a widely used practice to increase soil carbon content and maintain soil fertility. However, little is known about the effect of different types of organic matter, or the input of exogenous species from these materials, on soil fungal communities. In this study, fungal community composition was characterized from soils amended with three types of organic matter over a 30-year fertilization experiment. Chemical fertilization significantly changed soil fungal community composition and structure, which was exacerbated by the addition of organic matter, with the direction of change influenced by the type of organic matter used. The addition of organic matter significantly increased soil fungal richness, with the greatest richness achieved in soils amended with pig manure. Importantly, following addition of cow and pig manure, fungal taxa associated with these materials could be found in the soil, suggesting that these exogenous species can augment soil fungal composition. Moreover, the addition of organic matter decreased the relative abundance of potential pathogenic fungi. Overall, these results indicate that organic matter addition influences the composition and structure of soil fungal communities in predictable ways. This article is protected by copyright. All rights reserved.
Content may be subject to copyright.
Fungal community composition in soils subjected to
long-term chemical fertilization is most influenced by
the type of organic matter
Ruibo Sun,
1
Melissa Dsouza,
2,3
Jack A. Gilbert,
2,3,4
Xisheng Guo,
5
Daozhong Wang,
5
Zhibin Guo,
5
Yingying Ni
1
and Haiyan Chu
1
*
1
State Key Laboratory of Soil and Sustainable
Agriculture, Institute of Soil Science, Chinese Academy
of Sciences, East Beijing Road 71, Nanjing 210008,
China.
2
Marine Biological Laboratory, University of Chicago,
Woods Hole, MA 02543, USA.
3
Department of Surgery, University of Chicago,
Chicago, IL 60637, USA.
4
Argonne National Laboratory, Institute for Genomics
and Systems Biology, Argonne, IL 60439, USA.
5
Key Laboratory of Nutrient Cycling and Resources
Environment of Anhui Province, Soil and Fertilizer
Research Institute, Anhui Academy of Agricultural
Sciences, South Nongke Road 40, Hefei 230031,
China.
Summary
Organic matter application is a widely used practice
to increase soil carbon content and maintain soil fer-
tility. However, little is known about the effect of
different types of organic matter, or the input of exog-
enous species from these materials, on soil fungal
communities. In this study, fungal community com-
position was characterized from soils amended with
three types of organic matter over a 30-year fertiliza-
tion experiment. Chemical fertilization significantly
changed soil fungal community composition and
structure, which was exacerbated by the addition of
organic matter, with the direction of change influ-
enced by the type of organic matter used. The
addition of organic matter significantly increased soil
fungal richness, with the greatest richness achieved
in soils amended with pig manure. Importantly, fol-
lowing addition of cow and pig manure, fungal taxa
associated with these materials could be found in the
soil, suggesting that these exogenous species can
augment soil fungal composition. Moreover, the addi-
tion of organic matter decreased the relative
abundance of potential pathogenic fungi. Overall,
these results indicate that organic matter addition
influences the composition and structure of soil fun-
gal communities in predictable ways.
Introduction
Soil organic carbon (SOC), which is the main constituent of
soil organic matter (SOM), forms the basis of soil fertility and
sustainable agriculture. Therefore, it is one of the most
important indicators of soil quality and productivity (Reeves,
1997; Chan, 2008; Victoria et al., 2012). Intensive agricultur-
al activity greatly reduces SOC content, which in turn leads
to soil degradation (Gami et al., 2009; Victoria et al., 2012;
Olson, 2013; Stockmann et al., 2013). Many modern agri-
cultural practices encourage the increase of SOC content
by either increasing carbon inputs or by lowering losses
(Chan, 2008; Victoria et al., 2012). For this, a wide range of
organic materials such as straw, compost, organic waste,
biochar, and manure are applied to soils to increase carbon
input (Chan, 2008; Bo _
zena Cwalina-Ambroziak, 2009;
Victoria et al., 2012). But the formation of SOM depends
on the biological, chemical, and the physical decay of these
materials (Victoria et al., 2012; Stockmann et al., 2013).
Fungi are one of the most abundant soil microbes. They
serve as important decomposers in soil ecosystems, and
are typically associated with carbon sequestration in agro-
ecosystems (Hoorman, 2011; Jones et al., 2011). Studies
have shown that the diversity and distribution of soil fungal
communities are significantly related to the soil carbon
content, and that fertilization could impact soil fungal com-
munities by changing soil nutritional status and plant
biomass and physiology (Allison et al., 2007; Bo_
zena
Cwalina-Ambroziak, 2009; Weber et al., 2013; Liu et al.,
2015; Song et al., 2015; Zhou et al., 2016). For example,
N amendments decreased the relative abundance of Basi-
diomycota in an alpine tundra soil, but increased the
relative abundance of Ascomycota by 30% (Nemergut
Received 15 April, 2016; revised 17 July, 2016; accepted 26
August, 2016. *For correspondence. E-mail hychu@issas.ac.cn;
Tel. 86-25-86881356; Fax 86-25-86881000.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd
Environmental Microbiology (2016) 00(00), 00–00 doi:10.1111/1462-2920.13512
et al., 2008). A similar result was observed in a silty-clay
loam in Australia, wherein applying large quantities of N
fertilizer resulted in the high relative abundance of Asco-
mycota (Paungfoo-Lonhienne et al., 2015). The overall
diversity of soil fungal communities decreased on amend-
ment with chemical fertilizers (Allison et al., 2007;
Beauregard et al., 2010; Kamaa et al., 2012). Like chemi-
cal fertilizers, the application of organic matter can also
impact soil fungal communities, with their application fre-
quently causing increased soil fungal diversity (Cwalina-
Ambroziak and Bowszys, 2009; Kamaa et al., 2012; Song
et al., 2015). These changes to soil fungal communities
are associated with the alteration of soil nutrients and plant
carbon inputs (Allison et al., 2007; Song et al., 2015). For
example, Yu and colleagues (2013) found that amendment
with Protamylasse altered pea root-fungal community
structure by favoring obligate biotrophic fungi such as Olpi-
dium brassicae, and reducing the abundance of facultative
biotrophs such as Fusarium oxysporum.
Soil fungi also play an important role in plant health.
Over 90% of all plant species can form a mycorrhizal sym-
biosis, and plant–fungus association can greatly contribute
to plant growth, persistence, community diversity, and pro-
ductivity in ecosystems (Bonfante, 2003; Lin et al., 2012).
Arbuscular mycorrhizal fungi (AMF) are frequently associ-
ated with plants, and play essential roles in nutrient
uptake, especially phosphorus. Studies have found that
chemical fertilizers decreased the diversity and coloniza-
tion rate of AMF (Lin et al., 2012; Sheng et al., 2013; Chen
et al., 2014; Song et al., 2015), while organic matter indu-
ces greater diversity and the development of external AMF
mycelium (Gryndler et al., 2006; Wu et al., 2010; Sousa
et al., 2012). The addition of nitrogen always enhances the
growth of saprotrophic fungi, and stimulates the decompo-
sition of organic substrates by saprotrophic fungi (Allison
et al., 2009; Rousk and Baath, 2011). Song and col-
leagues (2015) also found that chemical fertilization
significantly increased the diversity of saprotrophic fungi.
Hyphael extension and their ability to translocate carbon,
nitrogen and phosphorus are part of the important role of
saprotrophic fungi in nutrient and water redistribution in soil
(Crowther et al., 2012; Guhr et al., 2015), thus the growth
of plants may be supported by increasing the soil nutrient
pool and soil moisture levels (Ellouze et al., 2014). Howev-
er, chemical fertilizers have been linked to an increase in
the proportion of fungi that are pathogenic to plants
(Cwalina-Ambroziak et al., 2010; Paungfoo-Lonhienne
et al., 2015). In contrast, the application of organic matter
can suppress pathogenic fungal growth, and enhances the
growth of fungi that antagonize these pathogenic species
(Hoitink and Fahy, 1986; Gamliel and Stapleton, 1993;
Bulluck et al., 2002; Zinati, 2005; Bo_
zena Cwalina-
Ambroziak, 2009; Cwalina-Ambroziak et al., 2010; Mokhtar
and El-Mougy, 2014; Saxena et al., 2015). For example,
Cwalina-Ambroziak and Bowszys (2009) found that the
aqueous extracts from compost inhibited mycelium growth
in pathogens such as Botrytis cinerea,Colletotrichum
coccodes, and those of the genus Fusarium.
The changes of fungal community caused by organic
fertilization are due not only to the input of substrates, but
also the existing microorganisms present in the organic
matter. The latter could result in biological invasion, which
is a great threat to biodiversity and ecosystem processes
(Charles and Dukes, 2008; Ehrenfeld, 2010; Ziska et al.,
2011; Powell et al., 2013; Walsh et al., 2016). Invasive
plants and animals have been extensively studied, and
recently research has started to focus on invasive
microbes, especially fungi, which are extremely important
in driving plant diversity and productivity (van der Heijden
et al., 2008). Non-indigenous fungal pathogens have great-
ly reduced the population sizes of several native tree
species in North America (Loo, 2008). Day and colleagues
(2015) revealed that the fungi associated with non-native
plants could benefit the growth of these plants by causing
disease in native plants and thereby affecting their growth.
Additionally, invasive fungi have a significant detrimental
effect on agricultural ecosystems (Rossman, 2009). Man-
ures used for agricultural production may also contain
microbial pathogens that pose a threat to human health
(Unc and Goss, 2004; Gerba and Smith, 2005; Mosadde-
ghi et al., 2010; Bradford et al., 2013).
Although the impact of fertilization on soil fungal commu-
nities has been extensively studied, the long-term effects
of different types of organic matter remain unknown. Previ-
ous research provided evidence for differing carbon
sequestration efficiencies for soils amended with wheat
straw, and cow and pig manure (PM) (Hua et al., 2014),
which is likely to be mediated by different fungal taxa tar-
geting different organic compounds (Hanson et al., 2008).
We hypothesize that different organic matter will select for
different soil fungal communities. In addition, multiple fungi
have been detected in organic material, but the impact of
exogenous fungi associated with the organic material on
native soil fungal communities is rarely explored (Anastasi
et al., 2005; Fliegerov
aet al., 2010; Lopez et al., 2014). To
address this gap, we utilized high-throughput amplicon
sequencing to compare fungal communities of soils sub-
jected to long-term chemical fertilization (NPK) amended
with low- or high-levels of wheat straw residues (S), cow
manure (CM), and PM together with the fungal community
associated with this manure.
Results
Changes in soil fungal community under different
fertilization regimes
Approximately 1.2 million raw internal transcribed spacer
region 1 (ITS1) amplicon reads were obtained for 24 soil
2R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
samples and 2 manure samples. After quality filtering,
812,748 (20,277–51,140 reads per sample) reads were
used for all downstream analysis. These high-quality reads
were clustered in to 997 non-singleton operational taxo-
nomic units (OTUs), with a large majority being assigned
to the phylum Ascomycota (80.7% of all reads). Basidio-
mycota and Zygomycota accounted for 13.8% and 3.1% of
reads respectively (Fig. 1A).
Fungal a-diversity was determined by the Chao1 rich-
ness index and the Heip’s evenness index, both calculated
with 20,000 rarefied sequences per sample (Heip, 1974;
Gotelli and Colwell, 2011). Good’s coverage was deter-
mined to estimate sampling completeness (Good, 1953;
Rea et al., 2011). Good’s coverage values (at the 97% sim-
ilarity level) were greater than 0.995 for all treatment types,
indicating that the vast majority of the fungal community
was captured at this sequencing depth (Table 1). The
lowest Chao1 richness index was observed for control
soils. No significant difference for Chao1 was observed
between NPK and control soils (Mann-Whitney U57.00,
P>0.05). However, significant differences were observed
between control soils and those amended with different
organic materials (Value of Mann-Whitney U was 0
between organic material amended treatments and control
treatment, P<0.05). The amended soils showed signifi-
cantly higher fungal richness compared to controls. In
contrast, Heip’s evenness was greatest in control soils,
although not significantly so. Of all treatment types, the
NPK 1PM soils showed the highest fungal richness and
the lowest fungal evenness (Table 1).
Fertilization greatly influenced the soil fungal community
structure. The taxonomic distribution of soil fungi changed
under different treatment types. At the phylum level, NPK,
NPK 1LS, and NPK 1HS fungal profiles were more
Fig. 1. Taxonomic composition of soil fungal communities by the different treatment regimes at the phylum (A) and the class level (B). PCoA
plot depicts the Bray–Curtis distance of fungal communities in 24 soil samples under the six treatment regimes (C). The red ellipses in this
plot represent the confidence areas (0.95) of the treatment. The blue dotted lines enclose the convex hull for the samples in the same
treatment. The blue lines combine samples to their class centroid. Control, non-fertilization; NPK fertilization; NPK with low-level wheat straw
(NPK1LS); NPK with high-level wheat straw (NPK1HS); NPK with PM (NPK1PM); and NPK with CM (NPK1CM).
Fertilizer organic matter influences soil fungal community 3
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
similar to the control samples than NPK 1PM and
NPK 1CM samples. The fungal community composition
was significantly different between NPK 1PM, NPK 1CM
and the control samples (Fig. S2). NPK1PM and
NPK 1CM samples had a greater relative abundance of
Zygomycota as compared to the control samples (Fig. 1A).
The relative abundance of dominant taxa also changed
under the different treatments regimes (Fig. 1B and Sup-
porting Information Fig. S1). Three taxa were closely
related to Stagonosporopsis crystalliniformis (OTU3406),
Alternaria alternate (OTU5521), Fusarium circinatum
(OTU2170), which are known plant pathogens (Supporting
Information Table S1) (Peever et al., 2002; Wingfield et al.,
2008; Vaghefi et al., 2012). The relative abundance of
these OTUs were much greater in the control and/or NPK
samples compared to those amended with organic materi-
als, and in particular those samples amended with cow
and PM (Supporting Information Fig. S1). The changes in
soil fungal communities were further depicted in the two-
dimensional principal coordinate analysis (PCoA) plot
using Bray-Curtis distance (Fig. 1C). Soils that were only
subjected to chemical fertilizers (NPK) clustered with con-
trol soils that received no treatment (Fig. 1C). In contrast,
soils amended with the different organic materials clus-
tered away from the control soils. While separate clusters
were observed for NPK 1PM and NPK 1CM samples,
the two wheat straw treatment types, NPK 1LS and
NPK 1HS clustered together. These results indicated that
long-term chemical fertilization describes less of the vari-
ance in soil fungal community structure than the
amendment of soil with different organic materials. Addi-
tionally, soil fungal communities tended to distribute
according to the type of organic material added (Fig. 1C).
This was confirmed by analysis of similarity (ANOSIM) cal-
culation (Supporting Information Table S3). Similar
observations of clustering by treatment type were made by
the hierarchical clustering tree method (Supporting Infor-
mation Fig. S2A).
Indicator species by treatment type
Indicator species were determined by the Dfrene-
Legendre indicator species analysis method (Dufrene and
Legendre, 1997) to identify fungal OTUs that are specifi-
cally associated with the different treatment regimes.
Indicator species (OTUs) for each treatment type are pre-
sented in Fig. 2. Additional information on taxonomic
assignment of indicator species by treatment type is pre-
sented in Supporting Information Table S1.
OTU3406 was the most abundant indicator species in
the control samples with a relative abundance of 8.42%,
and was most closely related to Stagonosporopsis crystal-
liniformis CBS 713.85, which is a foliage pathogen of
potatoes (Solanum tuberosum) and tomatoes (S. lycoper-
sicum), causing ‘black potato blight’ and ‘carate’ on
tomatoes (de Gruyter et al., 2012).
The most abundant indicator species in the NPK sam-
ples was OTU2166, which was most closely related to a
Hypocreales sp. It accounted for approximately one fifth of
the soil fungal community in this treatment type. Hypo-
creales spp. are widespread in moist forests, and many
Hypocreales species grow on wood rather than herba-
ceous substrata, and parasitize other fungi, such as
Ascomycota, resupinate Basidiomycetes, and perennial
bracket fungi (Chaverri and Samuels, 2003). Hypocreales
spp. have antifungal activity, thus making them potentially
valuable in the biological control of fungal diseases (Cha-
verri and Samuels, 2003). The high relative abundance of
these pathogen-antagonists is usually indicative of the
presence of the pathogens themselves. This was sup-
ported by the high relative abundance of another indicator
species, OTU2170 (8.52%), which was most closely relat-
ed to Fusarium circinatum (Supporting Information Table
S2), which is known to cause a destructive disease of
pines - pitch canker (Wingfield et al., 2008). A similar result
was observed for the control samples where OTU2166
(Hypocreales sp.) and OTU3406 (Stagonosporopsis
Table 1. Fungal diversity by treatment type and manure.
Treatment
Diversity index
Chao1 richness index Heip’s evenness index Good’s coverage
Control 300(13)c 0.134(0.021)a 0.997(0.000)a
NPK 313(33)bc 0.105(0.013)ab 0.997(0.001)ab
NPK1LS 348(50)ab 0.102(0.018)ab 0.996(0.001)c
NPK1HS 380(45)ab 0.12(0.02)a 0.996(0.001)c
NPK1PM 388(22)a 0.08(0.02)b 0.995(0.000)d
NPK1CM 344(11)b 0.124(0.009)a 0.997(0.000)b
Pig manure 284 0.098 0.998
Cow manure 193 0.009 0.998
The diversity indices were calculated using 20,000 randomly selected sequences per sample. Means of four replicates per treatment are pre-
sented (with standard deviation).
Data containing the same letter within a column indicate no significant difference between treatments detected by Mann-Whitney U test
(P>0.05).
4R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
crystalliniformis CBS 713.85) were abundant (Supporting
Information Fig. S1).
The most abundant indicator species in the wheat straw
amendment samples was OTU5031, which was most
closely related to Chaetomium globosum (Supporting
Information Table S2), a saprophytic fungus primarily resid-
ing on plants, soil, straw, and dung. It possesses a
cellulose-degrading system (Longoni et al., 2012) and can
produce antifungal compounds to antagonize spot blotch
(Cochliobolus sativus) and leaf rust (Puccinia recondita)of
wheat, as well as rice blast (Magnaporthe grisea)(Aggar-
wal et al., 2004; Park et al., 2005).
OTU3457 (most closely related to Pseudaleuria sp.) with
a relative abundance of 17.07% was the most abundant
indicator species in the NPK 1PM samples. Xu and col-
leagues (2012) demonstarted that Pseudaleuria were
dominant in healthy soils, and were negatively correlated
with the disease severity index of pea roots. Finally, indica-
tor species OTU1561, which accounted for one tenth of
the community in the NPK 1PM treatment type, had no
close match in these databases and as such may repre-
sent a novel fungal taxon.
For the NPK 1CM treatment type, OTU5240, which
was most closely related to Mortierella sp. was the most
abundant indicator (8.04%). Members of this genus pri-
marily serve as saprotrophs in soil ecosystems, typically
living on decaying leaves, fecal pellets, and other organic
material (Webster and Weber, 2007). Studies have found
that some species of Mortierella have the ability to degrade
chitin and hemicellulose (Raudonien_
e and Varnait _
e, 2008;
Young-Ju et al., 2008). Moreover, as a phosphate-
solubilizing fungus, they could help with the colonization of
AMF and alleviate the deleterious effects of salt on plant
growth and soil enzyme activities (Zhang et al., 2011).
Fig. 2. Indicator species by treatment regime. Circles represent OTUs, and the other shapes represent the different treatment regimes. The
size of each circle represents its relative abundance.
Fertilizer organic matter influences soil fungal community 5
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
Fungal community composition of livestock manures
The fungal communities in pig and CMs were also charac-
terized and were largely different to those observed for
soils. Ascomycetes and Basidiomycetes were the most
abundant phyla in pig and CM samples respectively (Sup-
porting Information Fig. S3a/b). Fungal communities in
PM were dominated by seven OTUs (relative
abundance >5%), of which the most abundant (20%) was
closely related to Cladorrhinum bulbillosum.OTUsmost
closely related to Agaricales sp. (relative abundance of
85.17%) dominated CM (Supporting Information Fig. S3c).
Overall, the fungal diversity of manure was lower than that
of soil (Table 1), and both richness and evenness were
lowest in CM.
Correlations between environmental factors and fungal
community structure
Redundancy analysis explained approximately 70% of the
total variation in the soil fungal community structure, and
the first two components explained 48.96% of the variation
(Fig. 3, Supporting Information Table S6). Soil fungal com-
munities formed clusters by treatment type on the RDA
plot (Fig. 3A). Through canonical variation partitioning, it
was observed that the organic material utilized for soil
amendment was the major contributor to fungal community
variation, explaining 16.8% of the variation in soil fungal
communities (Fig. 3B).
We compared the fungal community in cow and PM to
those found in amended soils to investigate the potential
for invasion. Results showed that 113 OTUs were shared
between pig manure and soils amended with pig manure
(NPK 1PM). This accounted for 24.9% of the NPK 1PM-
associated OTUs, and of these, 28 OTUs (5.9%) were
detected in pig manure and the amended soils but not in
the control soils (Fig. 4A). These 28 OTUs accounted for
about 1% relative abundance of the NPK 1PM community.
Likewise, 76 OTUs were shared between CM samples and
soils amended with CM (NPK 1CM), which accounted for
16.4% of the total NPK 1CM-associated OTUs; and of
these, 24 OTUs were not detected in the control soils (Fig.
4B). These 24 OTUs accounted for about 0.7% relative
abundance of the NPK 1CM community. Ternary plots
showed the distribution of OTUs detected in both control
and manure-amended soils (NPK 1PM and NPK 1CM)
(Fig. 4C and D). A total of 29 OTUs were enriched in the
NPK 1PM samples as compared to the control soils, and
nine OTUs were diluted in these samples. Likewise, 23
OTUs were enriched in the NPK1CM samples and five
were diluted compared to controls. Some of the
NPK 1PM-associated enriched OTUs were abundant in
PM (Fig. 4C), suggesting the strong possibility of PM as a
source of these OTUs. In contrast, most of the NPK 1CM-
associated, enriched OTUs were not abundant in CM (Fig.
4D).
Predicted sources of fungal communities in manure
amended soils
A Bayesian probability tool, SourceTracker, was used to
predict the sources of OTUs found in manure-amended
soils. Most of the fungal community was likely sourced
from native soil (Fig. 5), which contributed between
57.93% and 78.55% on average to NPK1PM and
NPK 1CM samples respectively. Fungi in PM contributed
17.62% to the fungal community in NPK 1PM samples,
which was much greater than the contribution made by
fungi in CM to NPK1CM samples (0.54%) (Fig. 5).
Discussion
Several different types of chemical fertilizers and organic
materials have been added to soils to increase its nutrient
Fig. 3. Redundancy analysis plot depicting the correlation between fungal communities and soil properties (A), and the percentages of
variance explained by each factor (B). TOM, type of organic material; NO2
3-N, nitrate, NH1
4-N, ammonium; TN, total Nitrogen; AP, available
Phosphorus.
6R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
Fig. 4. Venn diagrams showing the distribution of OTUs in non-fertilization treatment soils (control), soils subjected to long-term application of
NPK with pig manure (NPK1PM), and pig manure (PM) (A); and non-fertilization treatment soils (control), soils subjected to long-term
application of NPK with cow manure (NPK1CM), and cow manure (CM) (B). Ternary plots of OTUs shared between Control and NPK 1PM
(C) and OTUs shared between Control and NPK 1CM (D). The grey circles represent OTUs with no significant differences in relative
abundance between manure-amended and control soils, represents OTUs that had a significantly higher relative abundance in manure-
amended soils than control soils, and represents OTUs that had a significantly lower relative abundance in manure-amended soil than
control soils.
Fertilizer organic matter influences soil fungal community 7
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
and carbon content, and particularly to improve soil fertility
and agricultural production. The impact of fertilization on soil
microbial communities is of growing concern due to the
importance of microbes in soil ecosystems. Here we
revealed that the addition of organic material had a greater
impact on fungal composition and can result in increased
fungal richness as compared to chemical fertilization. Addi-
tionally, soil amendment with different types of organic
material resulted in significantly different fungal communities.
One of the most important services performed by fungi
in a soil ecosystem is decomposition – converting various
organic materials into bioavailable forms (Hoorman, 2011).
Fungi secrete enzymes to digest complex organic com-
pounds and absorb the breakdown products. Chemical
fertilizers do not serve as a direct carbon source, but can
impact soil fungal communities by altering the amount and
quality of plant carbon inputs (Allison et al., 2007; Weber
et al., 2013). However, the impact of plant carbon inputs
was far less than that observed as a result of soil amend-
ment with organic materials. Thus, we observed a greater
shift in fungal communities in soils amended with organic
materials than those solely treated with chemical fertilizers
(Supporting Information Fig. S2b). In addition, soils
amended with the different organic materials (including
wheat straw, PM, and CM) contained different fungal com-
munities. One possible reason may be the differences in
the carbon composition of the three types of organic mate-
rial. Fungal communities are regulated by resource type
and availability (Moll et al., 2015). Although tens of thou-
sands of fungi species live in soil, different fungal taxa
possess different resource utilization capabilities, and
some of them target particular organic compounds (Hanson
et al., 2008). Fungal taxa occupy different ecological
niches according to the available carbon source. Resource
partitioning among soil fungi is considered to be an impor-
tant mechanism for the maintenance of fungal diversity in
soil ecosystems (Hanson et al., 2008). For example,
Deacon and colleagues (2006) found that fungi isolated
from grassland soils had significant variability in resource
use; 83% utilized starch, 63% pectin, 63% cellulose, and
only 27% could use lignin, while 8% used chitin. Hoppe and
colleagues (2015) found significantly different fungal com-
munity structures in the deadwood of different tree species,
and attributed these differences to the varying physiochemi-
cal properties of the deadwood substrates. In this study, we
observed that the variance in fungal communities associated
with the different treatments was best explained by the
type of organic material, suggesting differential resource
utilization capabilities. In soils amended with organic mate-
rial, the enriched OTUs were closely related to fungal taxa
that are known to possess the ability to biodegrade and
decompose organic residues. For example, the most abun-
dant indicator species in NPK 1wheat straw samples,
OTU5031 (Supporting Information Fig. S1), was closely
related to Chaetomium globosum, which degrades cellu-
lose (Longoni et al., 2012), the main component of wheat
straw (Kabuyah et al., 2012).
The application of organic material is also known to sup-
press plant pathogenic fungi. Possible suppression
mechanisms include antibiosis, competition, hyperparasit-
ism, and induced systemic resistance (Bailey and
Lazarovits, 2003; Zinati, 2005; Walters and Bingham,
2007; Mokhtar and El-Mougy, 2014). We also observed a
reduced abundance of OTUs closely related to known
plant pathogens in soils amended with organic materials.
The presence of OTUs related to Chaetomium globosum
Fig. 5. The sources of fungal community in PM (A) and CM (B) amended soils as predicted by SourceTracker.
8R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
in wheat straw ammended soils, could suggest a potential
mechanism, as this organism can produce antifungal com-
pounds (Aggarwal et al., 2004; Park et al., 2005). A similar
observation was made by Yu and colleagues (2013)
wherein the presence of bacterial phyla including the Acid-
obacteria, Gp14, and Actinobacteria and the fungal class,
Cystobasidiomycetes played a more important role than
abiotic factors in the suppression of Pythium wilt disease.
The maintenance of high soil microbial diversity and
functional redundancy has been suggested to be a useful
for maintaining soil ecosystem health (Allison and Martiny,
2008; Sharma et al., 2011; Bhat, 2013; Miki et al., 2014).
In this study, an increase in fungal richness was observed
in soils amended with the different organic materials thus,
which could suggest that organic materials improved soil
‘health’ when compared to chemical fertilizers. Soils
amended with PM were more diverse than CM or wheat
straw amendments. The greater diversity observed in
manure amended soils may be due to the introduction of
exogenous fungi. Exogenous microbes introduced to soil
from manure have been shown to impact soil microbial
communities (Guan and Holley, 2003; Unc and Goss,
2004). While we were able to amplify ITS1 sequences
from cow and PM samples, we failed to produce any ampli-
fication in wheat straw. This could be because of PCR
amplicifcation inhibitors in the extract, or because of low
fungal biomass in wheat straw. If the latter then this would
suggest that there is limited impact of exogenous fungi
from wheat straw. Conversely, the manure-associated taxa
had an observed influence on soil fungal communities, but
for unknown reasons this was greater for pig-manure (Fig.
5, Supporting Information Tables S4 and S5). Some of
these taxa may influence soil ecological functions. For
example, OTU29 from PM was most closely related to
Acremonium alcalophilum (OTU29), which can degrade
both cellulose and xylan at temperatures as low as 08C
(Hayashi et al., 1996; Hayashi et al., 1997; Kasana and
Gulati, 2011). Additionally, OTU2781 was most closely
related to an anaerobic fungus, Buwchfawromyces eastonii
(Callaghan et al., 2015). These non-native fungi may
enhance nutrient transformation and the degradation of
organic materials under different environments including
frost and flooding. Meanwhile some OTUs were related to
taxa that could influence plant health. For example,
OTU989 from CM was closely related to Ophiocordyceps
lanpingensis, which can parasitize larval pathogens that
affect plants (Chen et al., 2013). Therefore the introduction
of this taxon could act to reduce the burden of insect
attacks. We were able to identify close relatives for many
OTUs, yet some remain unidentified; even so the ecologi-
cal role and impact of all of these organisms needs to be
characterized and validated in different ecosystem
contexts.
Conclusions
Fungi play an essential role in soil ecological processes,
especially in the degradation of organic materials. The
results from this study revealed that soil amendment with
organic materials explained patterns in soil fungal commu-
nity diversity and composition better than the use of
chemical fertilizers. The addition of organic materials
increased soil fungal richness, and lowered the relative
abundance of potentially pathogenic fungi. Interestingly
some fungi introduced to the soil from manure were closely
related to pathogen-antagonists, suggesting a potential
mechanism for pathogen suppression. Like previous
reports on long-term, chemical and organic fertilization
experiments, we also reported that cow and PMs were bet-
ter than wheat straw in improving soil physiochemical
properties, increasing crop yields, and preventing the loss
of microbial diversity (Sun et al., 2015b). This study sug-
gests that adding the type of organic matter influences the
structure and composition of the soil fungal community in
predictable ways. Understanding these influences further
could be crucial for sustainable agriculture.
Experimental procedures
Experimental design, soil sampling, determination of soil
properties, and DNA extraction
The experimental site is located at the Madian Agro-
Ecological Station in Mengcheng county, Anhui province,
China (N338130,E1168350). The experiment was started in
1982 and includes six treatments with four replicate plots for
each treatment-type. These include: (i) control (no fertiliza-
tion), (ii) chemical NPK fertilizers only (NPK), (iii) chemical
NPK fertilizers added with low amount (3750 kg ha
21
y
21
)of
wheat straw (NPK 1LS), (iv) chemical NPK fertilizers added
with high amount (7500 kg ha
21
y
21
) of wheat straw
(NPK 1HS), (v) chemical NPK fertilizers added with fresh PM
(NPK 1PM), and (vi) chemical NPK fertilizers added with
fresh CM (NPK 1CM). In October 2012, 12 soil cores (5 cm
in diameter) from the surface layer (0–10 cm) were collected
from each plot, and mixed thoroughly as a single sample. Soil
pH, total carbon (TC), total nitrogen (TN), nitrate (NO2
3-N),
ammonium (NH1
4-N), dissolved organic carbon (DOC), dis-
solved organic nitrogen (DON), available potassium (AK), and
available phosphorus (AP) were measured. DNA was
extracted from 0.5 g of fresh soil using a FastV
RDNA SPIN Kit
(MP Biomedicals, Santa Ana, CA) according to manufacturer’s
instructions. Detailed information on experimental design, soil
sampling, determination of soil properties, and soil DNA
extraction is as described previously by Sun et al. (2015b) and
Chu and Grogan (2010). Ten replicate samples (about 30 g
per sample) of wheat straw were collected prior to its applica-
tion. The wheat straw samples were cut into pieces and
ground using a ball mill (Emax, Retsch, Germany). DNA was
extracted from 0.2 g of disrupted wheat straw tissue using a
Dneasy plant kit (Qiagen, Germany) according to manufac-
turer’s instructions. Likewise, 10 replicate samples each of
cow and PMs were collected from different positions within the
Fertilizer organic matter influences soil fungal community 9
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
manure piles and mixed as a single sample separately before
their application. Manure samples were mixed by sterile
homogenate machine, and DNA was extracted from 0.5 g of
manure using a PowerFecalV
RDNA Isolation Kit (MO BIO,
USA) according to manufacturer’s instructions. Three repli-
cates of DNA extracts from wheat straw, pig and CM samples
were selected and pooled separately for PCR amplification.
PCR amplification and high throughput sequencing
Primers ITS1F (50-CTTGGTCATTTAGAGGAAGTAA-30) and
ITS2 (50-GCTGCGTTCTTCATCGATGC-30) (Ghannoum et al.,
2010) were used to amplify the ITS1 region, which is the uni-
versal DNA barcode marker for the molecular identification of
fungi (Schoch et al., 2012; Blaalid et al., 2013). PCR was per-
formed in 50 ll reaction volumes containing 25 ll of Premix
Taq DNA polymerase, 0.5 ll of forward primer (20 mM), 0.5
ll of reverse primer (20 mM), 23 ll of double distilled water
(ddH
2
O), and 1 ll DNA template (20 ng total soil DNA). The
PCR cycling conditions were 948Cfor5min,35cyclesof948C
for30s,at508C for 30 s, and 728C for 30 s. Illumina libraries
were constructed using the MiSeq Reagent Kit v3 according to
manufacturer’s instructions. High-throughput, paired-end
sequencing was performed on the Illumina MiSeq PE250 plat-
form. Sequencing data were deposited in the European
Nucleotide Archive under the accession number PRJEB12829.
Analysis of sequencing data
Sequencing data were processed using the QIIME software
package (version 1.9.0) (Caporaso et al., 2010). Briefly, for-
ward and reverse reads were joined using fastq-join with a
minimum of 10 bp overlap. Low quality sequences (Phred
quality score Q <20 or sequences shorter than 200 bp) were
discarded, and chimeras were filtered by the UCHIME algo-
rithm (Edgar et al., 2011) in the USEARCH tool (Edgar, 2010)
using the UNITE fungal ITS reference data set (Version 7.0)
(Nilsson et al., 2015). High-quality sequences were assigned
to OTUs using UCLUST with a similarity threshold of 97%
(Edgar, 2010). Taxonomy was assigned using the UCLUST
consensus taxonomy assigner and the UNITE fungal ITS
database (QIIME release, version 7.0) (Bengtsson-Palme
et al., 2013). OTUs with no taxonomic assignment from QIIME
were identified using blastn and the internal transcribed spac-
er region (ITS) from fungi type and reference material
database (Johnson et al., 2008; Schoch et al., 2014). Single-
tons and non-fungal OTUs were removed and all samples
were rarefied to 20 000 sequences per sample for further
analysis.
Statistical analyses
Principal coordinates analysis (PCoA) was used to compare
the beta diversity between samples based on the Bray-Curtis
distance matrix. This was performed in the R software pack-
age (version 3.1.2) using the ape library (Paradis et al., 2004).
Hierarchical clustering was determined by unweighted pair
group method with arithmetic mean (UPGMA) (Milligan, 1985)
in R using the vegan library (Oksanen, 2015). Mann-Whitney
U test was used to compare diversity indices between
treatments, as the data were not randomly distributed. ANO-
SIMs (Clarke, 1993; Warton et al., 2012) based on Bray-
Curtis distances was performed using vegan in R. Heatmaps
were drawn using gplots (Warnes et al., 2016) in R. Indicator
analysis was done using labdsv (Roberts, 2012.) in R, and
visualized using the Cytoscape package (Version 3.2.1)
(Shannon et al., 2003). Redundancy analysis (RDA) was car-
ried out to determine the effect of soil properties on the fungal
community in R using vegan. Only environmental variables
that were significantly (P<0.05) correlated with the RDA mod-
el were selected (calculated based on 999 permutations), and
the respective effects of different explanatory variables were
calculated by canonical variation partitioning (Borcard et al.,
1992; Sun et al., 2015a). The significance of RDA models
were tested by ANOVA based on 999 permutations. Ternary
plots were drawn in R using the vcd package (Meyer et al.,
2015). SourceTracker (Version 0.9.5) (Knights et al., 2011)
was used to predict the source of fungal communities in
manure-amended samples (NPK 1PM and NPK 1CM).
NPK 1PM and NPK 1CM samples were set as sink, and the
control (used as native soil) and the manure samples were set
as sources. Source predictions were run with 100 burnins, 10
random restarts, and rarefaction depth of 20 000.
Acknowledgements
We thank Congcong Shen, Xingjia Xiang and Yuntao Li for
assistance in soil sampling, and Keke Hua for the manage-
ment of the experimental field. We also thank Rong Huang for
assistance in sequencing. This work was funded by the Strate-
gic Priority Research Program of the Chinese Academy of Sci-
ences (XDB15010101), the National Program on Key Basic
Research Project (2014CB954002) and the National Natural
Science Foundation of China (41371254). The authors
declare no conflicts of interest.
References
Aggarwal, R., Tewari, A., Srivastava, K., and Singh, D. (2004)
Role of antibiosis in the biological control of spot blotch
(Cochliobolus sativus) of wheat by Chaetomium globosum.
Mycopathologia 157: 369–377.
Allison, S.D., and Martiny, J.B. (2008) Colloquium paper:
resistance, resilience, and redundancy in microbial commu-
nities. Proc Natl Acad Sci U S A 105: 11512–11519.
Allison, S.D., Hanson, C.A., and Treseder, K.K. (2007) Nitro-
gen fertilization reduces diversity and alters community
structure of active fungi in boreal ecosystems. Soil Biol Bio-
chem 39: 1878–1887.
Allison, S.D., LeBauer, D.S., Ofrecio, M.R., Reyes, R., Ta,
A.M., and Tran, T.M. (2009) Low levels of nitrogen addition
stimulate decomposition by boreal forest fungi. Soil Biol Bio-
chem 41: 293–302.
Anastasi, A., Varese, G.C., and Marchisio, V.F. (2005) Isola-
tion and identification of fungal communities in compost and
vermicompost. Mycologia 97: 33–44.
Bailey, K.L., and Lazarovits, G. (2003) Suppressing soil-borne
diseases with residue management and organic amend-
ments. Soil Till Res 72: 169–180.
Beauregard, M.S., Hamel, C., Atul, N., and St-Arnaud, M.
(2010) Long-term phosphorus fertilization impacts soil
10 R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
fungal and bacterial diversity but not AM fungal community
in alfalfa. Microb Ecol 59: 379–389.
Bengtsson-Palme, J., Ryberg, M., Hartmann, M., Branco, S.,
Wang, Z., Godhe, A., et al. (2013) Improved software detec-
tion and extraction of ITS1 and ITS2 from ribosomal ITS
sequences of fungi and other eukaryotes for analysis of envi-
ronmental sequencing data. Methods Ecol Evol 4: 914–919.
Bhat, A. (2013) Preserving microbial diversity of soil ecosys-
tem: a key to sustainable productivity. Int J Curr Microbiol
App Sci 2: 85–101.
Blaalid, R., Kumar, S., Nilsson, R.H., Abarenkov, K., Kirk,
P.M., and Kauserud, H. (2013) ITS1 versus ITS2 as DNA
metabarcodes for fungi. Mol Ecol Resour 13: 218–224.
Bonfante, P. (2003) Plants, mycorrhizal fungi and endobacteria:
a dialog among cells and genomes. Biol Bull 204: 215–220.
Borcard, D., Legendre, P., and Drapeau, P. (1992) Partialling
out the spatial component of ecological variation. Ecology
73: 1045–1055.
Bo_
zena Cwalina-Ambroziak, J.W. (2009) Effect of fertilization
on the composition of soil fungi community. Contemp Prob
Manage Environ Prot 4: 107–118.
Bradford, S.A., Morales, V.L., Zhang, W., Harvey, R.W.,
Packman, A.I., Mohanram, A., and Welty, C. (2013) Trans-
port and fate of microbial pathogens in agricultural settings.
Crit Rev Environ Sci Technol 43: 775–893.
Bulluck, L., Barker, K., and Ristaino, J. (2002) Influences of
organic and synthetic soil fertility amendments on nema-
tode trophic groups and community dynamics under toma-
toes. Appl Soil Ecol 21: 233–250.
Callaghan, T.M., Podmirseg, S.M., Hohlweck, D., Edwards,
J.E., Puniya, A.K., Dagar, S.S., and Griffith, G.W. (2015)
Buwchfawromyces eastonii gen. nov., sp. nov.: a new
anaerobic fungus (Neocallimastigomycota) isolated from
buffalo faeces. MycoKeys 9: 11–28.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K.,
Bushman, F.D., Costello, E.K., et al. (2010) QIIME allows
analysis of high-throughput community sequencing data.
Nat Methods 7: 335–336.
Chan, Y. (2008) Increasing soil organic carbon of agricultural
land. Primefact 735: 1–5.
Charles, H., and Dukes, J.S. (2008) Impacts of invasive spe-
cies on ecosystem services. In Biological Invasions:Nent-
wig, W. Springer Science & Business Media. Verlag Berlin
Herdelberg: 217–237.
Chaverri, P., and Samuels, G.J. (2003) Hypocrea/Trichoderma
(Ascomycota, Hypocreales, Hypocreaceae): species with
green ascospores. Stud Mycol 48:1–116.
Chen, Y.L., Zhang, X., Ye, J.S., Han, H.Y., Wan, S.Q., and
Chen, B.D. (2014) Six-year fertilization modifies the biodi-
versity of arbuscular mycorrhizal fungi in a temperate
steppe in Inner Mongolia. Soil Biol Biochem 69: 371–381.
Chen, Z.H., Dai, Y.D., Yu, H., Yang, K., Yang, Z.L., Yuan, F.,
and Zeng, W.B. (2013) Systematic analyses of Ophiocordy-
ceps lanpingensis sp. nov., a new species of Ophiocordy-
ceps in China. Microbiol Res 168: 525–532.
Chu, H.Y., and Grogan, P. (2010) Soil microbial biomass,
nutrient availability and nitrogen mineralization potential
among vegetation-types in a low arctic tundra landscape.
Plant Soil 329: 411–420.
Clarke, K.R. (1993) Non-parametric multivariate analyses of
changes in community structure. Aust J Ecol 18: 117–143.
Crowther, T.W., Boddy, L., and Jones, T.H. (2012) Functional
and ecological consequences of saprotrophic fungus–graz-
er interactions. ISME J 6: 1992–2001.
Cwalina-Ambroziak, B., and Bowszys, T. (2009) Changes in
fungal communities in organically fertilized soil. Plant Soil
Environ 55: 25–32.
Cwalina-Ambroziak, B., Bowszys, T., and Wierzbowska, J.
(2010) Fungi colonizing soil fertilized with composted sew-
age sludge and municipal waste. JElementol15: 39–51.
Day, N.J., Dunfield, K.E., and Antunes, P.M. (2016) Fungi
from a non-native invasive plant increase its growth but
have different growth effects on native plants. Biological
Invasions 18: 231–243.
Deacon, L.J., Janie Pryce-Miller, E., Frankland, J.C.,
Bainbridge, B.W., Moore, P.D., and Robinson, C.H. (2006)
Diversity and function of decomposer fungi from a grass-
land soil. Soil Biol Biochem 38: 7–20.
Dufrene, M., and Legendre, P. (1997) Species assemblages
and indicator species: the need for a flexible asymmetrical
approach. Ecol Monogr 67: 345–366.
Edgar, R.C. (2010) Search and clustering orders of magnitude
faster than BLAST. Bioinformatics 26: 2460–2461.
Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., and
Knight, R. (2011) UCHIME improves sensitivity and speed
of chimera detection. Bioinformatics 27: 2194–2200.
Ehrenfeld, J.G. (2010) Ecosystem consequences of biological
invasions. Annu Rev Ecol Evol Syst 41: 59–80.
Ellouze, W., Esmaeili, T.A., Bainard, L.D., Yang, C.,
Bazghaleh, N., Navarro-Borrell, A.,etal. (2014) Soil fungal
resources in annual cropping systems and their potential for
management. Biomed Res Int 2014: 531824.
Fliegerov
a, K., Mr
azek, J., Hoffmann, K., Z
abransk
a, J., and
Voigt, K. (2010) Diversity of anaerobic fungi within cow
manure determined by ITS1 analysis. Folia Microbiol 55:
319–325.
Gami, S.K., Lauren, J.G., and Duxbury, J.M. (2009) Soil
organic carbon and nitrogen stocks in Nepal long-term soil
fertility experiments. Soil Till Res 106: 95–103.
Gamliel, A., and Stapleton, J.J. (1993) Effect of chicken com-
post or ammonium phosphate and solarization on pathogen
control, rhizosphere microorganisms, and lettuce growth.
Plant Dis 77: 886–891.
Gerba, C.P., and Smith, J.E. (2005) Sources of pathogenic
microorganisms and their fate during land application of
wastes the opinions expressed in this article are those of
the authors and do not necessarily reflect those of the
USEPA. JEnvironQual34: 42–48.
Ghannoum, M.A., Jurevic, R.J., Mukherjee, P.K., Cui, F.,
Sikaroodi, M., Naqvi, A., and Gillevet, P.M. (2010) Charac-
terization of the oral fungal microbiome (mycobiome) in
healthy individuals. Plos Pathog 6: e1000713.
Good, I.J. (1953) The population frequencies of species and the
estimation of population parameters. Biometrika 40: 237–264.
Gotelli, N.J., and Colwell, R.K. (2011) Estimating species rich-
ness. Biol Divers Front Meas Assess 12: 39–54.
de Gruyter, J., van Gent-Pelzer, M.P.E., Woudenberg, J.H.C.,
van Rijswick, P.C.J., Meekes, E.T.M., Crous, P.W., and
Bonants, P.J.M. (2012) The development of a validated real-
time (TaqMan) PCR for detection of Stagonosporopsis andi-
gena and S. crystalliniformis in infected leaves of potato
and tomato. Eur J Plant Pathol 134: 301–313.
Fertilizer organic matter influences soil fungal community 11
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
Gryndler, M., Larsen, J., Hrselova, H., Rezacova, V.,
Gryndlerova, H., and Kubat, J. (2006) Organic and mineral
fertilization, respectively, increase and decrease the devel-
opment of external mycelium of arbuscular mycorrhizal fun-
gi in a long-term field experiment. Mycorrhiza 16: 159–166.
Guan, T.Y., and Holley, R.A. (2003) Pathogen survival in swine
manure environments and transmission of human enteric ill-
ness–a review. J Environ Qual 32: 383–392.
Guhr, A., Borken, W., Spohn, M., and Matzner, E. (2015)
Redistribution of soil water by a saprotrophic fungus
enhances carbon mineralization. Proc Natl Acad Sci 112:
14647–14651.
Hanson, C.A., Allison, S.D., Bradford, M.A., Wallenstein, M.D.,
and Treseder, K.K. (2008) Fungal taxa target different car-
bon sources in forest soil. Ecosystems 11: 1157–1167.
Hayashi, K., Nimura, Y., Ohara, N., Uchimura, T., Suzuki, H.,
Komacata, K., and Kozaki, M. (1996) Low-temperature-
active cellulase produced by Acremonium alcalophilum-
JCM 7366-Note. Seibutsu-Kogaku Kaishi-Journal of the
Society for Fermentation and Bioengineering 74: 7–10.
Hayashi, K., Nimura, Y., Miyaji, T., Ohara, N., Uchimura, T.,
Suzuki, H., ... and Kozaki, M. (1997) Purification and prop-
erties of a low-temperature-active enzyme degrading both
cellulose and xylan from Acremonium alcalophilum JCM
7366. Seibutsu-Kogaku Kaishi-Journal of the Society for
Fermentation and Bioengineering 75: 9–14.
Heip, C. (1974) A new index measuring evenness. J Mar Biol
Assoc U K 54: 555–557.
Hoitink, H.A.J., and Fahy, P.C. (1986) Basis for the control of
soilborne plant-pathogens with composts. Annu Rev Phyto-
pathol 24: 93–114.
Hoorman, J.J. (2011) The role of soil fungus. Ohio State Uni-
versity Extension, Agriculture and Natural Resources SAG-
14-11.
Hoppe, B., Purahong, W., Wubet, T., Kahl, T., Bauhus, J.,
Arnstadt, T., et al. (2015) Linking molecular deadwood-
inhabiting fungal diversity and community dynamics to eco-
system functions and processes in Central European for-
ests. Fungal Divers 77: 367–379.
Hua, K.K., Wang, D.Z., Guo, X.S., and Guo, Z.B. (2014) Car-
bon sequestration efficiency of organic amendments in a
long-term experiment on a vertisol in Huang-Huai-Hai Plain,
China. PLoS One 9: e108594.
Johnson, M., Zaretskaya, I., Raytselis, Y., Merezhuk, Y.,
McGinnis, S., and Madden, T.L. (2008) NCBI BLAST: a bet-
ter web interface. Nucleic Acids Res 36: W5–W9.
Jones, M.D., Forn, I., Gadelha, C., Egan, M.J., Bass, D.,
Massana, R., and Richards, T.A. (2011) Discovery of novel
intermediate forms redefines the fungal tree of life. Nature
474: 200–203.
Kabuyah, R.N.T.M., van Dongen, B.E., Bewsher, A.D., and
Robinson, C.H. (2012) Decomposition of lignin in wheat
straw in a sand-dune grassland. Soil Biol Biochem 45: 128–
131.
Kamaa, M.M., Mburu, H.N., Blanchart, E., Chibole, L., Chotte,
J.L., Kibunja, C.N., and Lesueur, D. (2012) Effects of organ-
ic and inorganic applications on soil bacterial and fungal
microbial communities diversity and impacts of earthworms
on microbial diversity in the Kabete long-term trial, Kenya.
In Lessons Learned from Long-Term Soil Fertility Manage-
ment Experiments in Africa. Bationo, A., Waswa, B., Kihara,
J., Adolwa, I., Vanlauwe, B., and Saidou, K. (eds). Dor-
drecht, Netherlands: Springer, pp. 121–136.
Kasana, R.C. and Gulati, A. (2011) Cellulases from psychro-
philic microorganisms: a review. Journal of basic microbiolo-
gy 51: 572–579.
Knights, D., Kuczynski, J., Charlson, E.S., Zaneveld, J.,
Mozer, M.C., Collman, R.G., et al. (2011) Bayesian
community-wide culture-independent microbial source
tracking. Nat Methods 8: 761–763.
Lin, X., Feng, Y., Zhang, H., Chen, R., Wang, J., Zhang, J.,
and Chu, H. (2012) Long-term balanced fertilization
decreases arbuscular mycorrhizal fungal diversity in an ara-
ble soil in North China revealed by 454 pyrosequencing.
Environ Sci Technol 46: 5764–5771.
Liu, J., Sui, Y., Yu, Z., Shi, Y., Chu, H., Jin, J., et al.(2015)Soil
carbon content drives the biogeographical distribution of
fungal communities in the black soil zone of northeast Chi-
na. Soil Biol Biochem 83: 29–39.
Longoni, P., Rodolfi, M., Pantaleoni, L., Doria, E., Concia, L.,
Picco, A.M., and Cella, R. (2012) Functional analysis of the
degradation of cellulosic substrates by a Chaetomium glo-
bosum endophytic isolate. Appl Environ Microbiol 78:
3693–3705.
Loo, J.A. (2008) Ecological impacts of non-indigenous inva-
sive fungi as forest pathogens. Biol Invasions 11: 81–96.
Lopez, L., Aganon, C.P., and Juico, P.P. (2014) Isolation of
Trichoderma species from carabao manure and evalua-
tion of its beneficial uses. Int J Sci Technol Res 3:
190–199.
Meyer, D., Zeileis, A., and Hornik, K. (2015) vcd: Visualizing
Categorical Data. R Package Version 1–0.
Miki, T., Yokokawa, T., and Matsui, K. (2014) Biodiversity and
multifunctionality in a microbial community: a novel theoreti-
cal approach to quantify functional redundancy. Proc R Soc
Lond B Biol Sci 281: 20132498.
Milligan, G.W. (1985) Cluster-analysis for researchers -
Romesburg,Hc. JClassif2: 133–137.
Mokhtar, M., and El-Mougy, N. (2014) Bio-compost application
for controlling soil-borne plant pathogens–a review. Popula -
tion 4: 61–68.
Moll, J., Goldmann, K., Kramer, S., Hempel, S., Kandeler, E.,
Marhan, S., et al. (2015) Resource type and availability reg-
ulate fungal communities along arable soil profiles. Microb
Ecol 70: 390–399.
Mosaddeghi, M.R., Sinegani, A.A.S., Farhangi, M.B.,
Mahboubi, A.A., and Unc, A. (2010) Saturated and unsatu-
rated transport of cow manure-borne Escherichia coli
through in situ clay loam lysimeters. Agric Ecosyst Environ
137: 163–171.
Nemergut, D.R., Townsend, A.R., Sattin, S.R., Freeman, K.R.,
Fierer, N., Neff, J.C., et al. (2008) The effects of chronic
nitrogen fertilization on alpine tundra soil microbial commu-
nities: implications for carbon and nitrogen cycling. Environ
Microbiol 10: 3093–3105.
Nilsson, R.H., Tedersoo, L., Ryberg, M., Kristiansson, E.,
Hartmann, M., Unterseher, M., et al. (2015) A comprehen-
sive, automatically updated fungal ITS sequence dataset
for reference-based chimera control in environmental
sequencing efforts. Microbes Environ 30: 145–150.
Oksanen, J. (2015) Multivariate analyses of ecological com-
munities in R: vegan tutorial. http://159.226.251.229/
12 R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
videoplayer/vegantutor.pdf?ich_u_r_i558ab87c25a456d3f503
58e29abbeea94&ich_s_t_a_r_t50&ich_e_n_d50&ich_k_e_
y51645068924751263032428&ich_t_y_p_e51&ich_d_i_s_
k_i_d51&ich_u_n_i_t51.
Olson, K.R. (2013) Soil organic carbon sequestration, stor-
age, retention and loss in U.S. croplands: issues paper for
protocol development. Geoderma 195–196: 201–206.
Paradis, E., Claude, J., and Strimmer, K. (2004) APE: analy-
ses of phylogenetics and evolution in R language. Bioinfor-
matics 20: 289–290.
Park, J.H., Choi, G.J., Jang, K.S., Lim, H.K., Kim, H.T., Cho,
K.Y., and Kim, J.C. (2005) Antifungal activity against plant
pathogenic fungi of chaetoviridins isolated from Chaeto-
mium globosum. FEMS Microbiol Lett 252: 309–313.
Paungfoo-Lonhienne, C., Yeoh, Y.K., Kasinadhuni, N.R.P.,
Lonhienne, T.G.A., Robinson, N., Hugenholtz, P., et al.
(2015) Nitrogen fertilizer dose alters fungal communities in
sugarcane soil and rhizosphere. Sci Rep 5: 8678.
Peever, T.L., Iba~
nez, A., Akimitsu, K., and Timmer, L.W.
(2002) Worldwide phylogeography of the citrus brown spot
pathogen, Alternaria alternata. Phytopathology 92: 794–
802.
Powell, K.I., Chase, J.M., and Knight, T.M. (2013) Invasive
plants have scale-dependent effects on diversity by altering
species-area relationships. Science 339: 316–318.
Raudonien_
e, V., and Varnait_
e, R. (2008) Destruction of hemi-
cellulose in rye straw by micromycetes. Ekologija 54: 169–
172.
Rea, M.C., Dobson, A., O’sullivan, O., Crispie, F., Fouhy, F.,
Cotter, P.D., et al. (2011) Effect of broad- and narrow-
spectrum antimicrobials on Clostridium difficile and microbi-
al diversity in a model of the distal colon. Proc Natl Acad
Sci U S A 108: 4639–4644.
Reeves, D.W. (1997) The role of soil organic matter in main-
taining soil quality in continuous cropping systems. Soil Till
Res 43: 131–167.
Roberts, D.W. (2012) Package ‘labdsv’. https://cran.r-project.
org/web/packages/labdsv/labdsv.pdf.
Rossman, A.Y. (2009) The impact of invasive fungi on agricul-
tural ecosystems in the United States. Biol Invasions 11:
97–107.
Rousk, J., and Baath, E. (2011) Growth of saprotrophic fungi
and bacteria in soil. FEMS Microbiol Ecol 78: 17–30.
Saxena, J., Choudhary, S., Pareek, S., Choudhary, A.K., and
Iquebal, M.A. (2015) Recycling of organic waste through
four different composts for disease suppression and growth
enhancement in mung beans. Clean Soil Air Water 43:
1066–1071.
Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge,
J.L., Levesque, C.A., et al. (2012) Nuclear ribosomal inter-
nal transcribed spacer (ITS) region as a universal DNA bar-
code marker for Fungi. Proc Natl Acad Sci U S A 109:
6241–6246.
Schoch, C.L., Robbertse, B., Robert, V., Vu, D., Cardinali, G.,
Irinyi, L., et al. (2014) Finding needles in haystacks: linking
scientific names, reference specimens and molecular data
for Fungi. Database 2014: bau061.
Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T.,
Ramage, D., et al. (2003) Cytoscape: a software environ-
ment for integrated models of biomolecular interaction net-
works. Genome Res 13: 2498–2504.
Sharma, S., Ramesh, A., Sharma, M., Joshi, O., Govaerts,
B., Steenwerth, K., and Karlen, D. (2011) Microbial commu-
nity structure and diversity as indicators for evaluating soil
quality. In Biodiversity, Biofuels, Agroforestry and Conserva-
tion Agriculture. Lichtfouse, E. (ed). Netherlands: Springer,
pp. 317–358.
Sheng, M., Lalande, R., Hamel, C., and Ziadi, N. (2013) Effect
of long-term tillage and mineral phosphorus fertilization on
arbuscular mycorrhizal fungi in a humid continental zone of
Eastern Canada. Plant Soil 369: 599–613.
Song, G., Chen, R., Xiang, W., Yang, F., Zheng, S., Zhang, J.,
et al. (2015) Contrasting effects of long-term fertilization on
the community of saprotrophic fungi and arbuscular mycor-
rhizal fungi in a sandy loam soil. Plant Soil Environ 61:
127–136.
Sousa, C.S., Menezes, R.S.C., Sampaio, E.V.S.B., Oehl, F.,
Maia, L.C., Garrido, M.S., and Lima, F.S. (2012) Occur-
rence of arbuscular mycorrhizal fungi after organic fertiliza-
tion in maize, cowpea and cotton intercropping systems.
Acta Sci Agron 34: 149–156.
Stockmann, U., Adams, M.A., Crawford, J.W., Field, D.J.,
Henakaarchchi, N., Jenkins, M., et al. (2013) The knowns,
known unknowns and unknowns of sequestration of soil
organic carbon. Agric Ecosyst Environ 164: 80–99.
Sun, R.B., Guo, X.S., Wang, D.Z., and Chu, H.Y. (2015a)
Effects of long-term application of chemical and organic fer-
tilizers on the abundance of microbial communities involved
in the nitrogen cycle. Appl Soil Ecol 95: 171–178.
Sun, R.B., Zhang, X.X., Guo, X.S., Wang, D.Z., and Chu, H.Y.
(2015b) Bacterial diversity in soils subjected to long-term
chemical fertilization can be more stably maintained with
the addition of livestock manure than wheat straw. Soil Biol
Biochem 88: 9–18.
Unc, A., and Goss, M.J. (2004) Transport of bacteria from manure
andprotectionofwaterresources.Appl Soil Ecol 25: 1–18.
Vaghefi, N., Pethybridge, S.J., Ford, R., Nicolas, M.E., Crous,
P.W., and Taylor, P.W.J. (2012) Stagonosporopsis spp.
associated with ray blight disease of Asteraceae. Aust Plant
Pathol 41: 675–686.
van der Heijden, M.G., Bardgett, R.D., and van Straalen, N.M.
(2008) The unseen majority: soil microbes as drivers of
plant diversity and productivity in terrestrial ecosystems.
Ecol Lett 11: 296–310.
Victoria, R., Banwart, S., Black, H., Ingram, J., Joosten, H.,
Milne, E., et al. (2012) The benefits of soil carbon. In Emerg-
ing Issues in Our Global Environment UNEP Yearbook.
Walsh, J.R., Carpenter, S.R., and Vander Zanden, M.J.
(2016) Invasive species triggers a massive loss of ecosys-
tem services through a trophic cascade. Proc Natl Acad Sci
113: 4081–4085.
Walters, D.R., and Bingham, I.J. (2007) Influence of nutrition on
disease development caused by fungal pathogens: implica-
tions for plant disease control. Ann Appl Biol 151: 307–324.
Warnes, G.R., Bolker, B., Bonebakker, L., Gentleman, R.,
Liaw, W.H.A., Lumley, T., et al.(2016)Package ‘gplots.
https://cran.r-project.org/web/packages/gplots/gplots.pdf.
Warton, D.I., Wright, S.T., and Wang, Y. (2012) Distance-
based multivariate analyses confound location and disper-
sion effects. Methods Ecol Evol 3: 89–101.
Weber, C.F., Vilgalys, R., and Kuske, C.R. (2013) Changes in
fungal community composition in response to elevated
Fertilizer organic matter influences soil fungal community 13
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
atmospheric CO2 and nitrogen fertilization varies with soil
horizon. Front M icrobi ol 4: 78.
Webster, J., and Weber, R. (2007) Introduction to Fungi.Cam-
bridge, UK: Cambridge University Press.
Wingfield, M.J., Hammerbacher, A., Ganley, R.J., Steenkamp,
E.T., Gordon, T.R., Wingfield, B.D., and Coutinho, T.A.
(2008) Pitch canker caused by Fusarium circinatum - a
growing threat to pine plantations and forests worldwide.
Aust Plant Pathol 37: 319–334.
Wu, F., Dong, M., Liu, Y., Ma, X., An, L., Young, J.P.W., and
Feng, H. (2010) Effects of long-term fertilization on AM fun-
gal community structure and Glomalin-related soil protein in
the Loess Plateau of China. Plant Soil 342: 233–247.
Xu, L., Ravnskov, S., Larsen, J., Nilsson, R.H., and
Nicolaisen, M. (2012) Soil fungal community structure along
a soil health gradient in pea fields examined using deep
amplicon sequencing. Soil Biol Biochem 46: 26–32.
Young-Ju, K., Zhao, Y., Oh, K.T., Nguyen, V.N., and Park, R.D.
(2008) Enzymatic deacetylation of chitin by extracellular chi-
tin deacetylase from a newly screened Mortierella sp DY-
52. J Microbiol Biotechnol 18: 759–766.
Yu, L., Nicolaisen, M., Larsen, J., and Ravnskov, S. (2013)
Organic fertilization alters the community composition of
root associated fungi in Pisum sativum. Soil Biol Biochem
58: 36–41.
Zhang, H., Wu, X., Li, G., and Qin, P. (2011) Interactions between
arbuscular mycorrhizal fungi and phosphate-solubilizing fun-
gus (Mortierella sp.) and their effects on Kostelelzkya virgin-
ica growth and enzyme activities of rhizosphere and bulk
soils at different salinities. Biol Fertil Soils 47: 543–554.
Zhou, J., Jiang, X., Zhou, B., Zhao, B., Ma, M., Guan, D.,
et al. (2016) Thirty four years of nitrogen fertilization
decreases fungal diversity and alters fungal community
composition in black soil in northeast China. Soil Biol Bio-
chem 95: 135–143.
Zinati, G.M. (2005) Compost in the 20th century: a tool to con-
trol plant diseases in nursery and vegetable crops. Hort-
technology 15: 61–66.
Ziska, L.H., Blumenthal, D.M., Runion, G.B., Hunt, E.R., and
Diaz-Soltero, H. (2011) Invasive species and climate change:
an agronomic perspective. Clim Change 105: 13–42.
Supporting information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Fig. S1. Heatmap illustrating the distribution of the most
abundant soil OTUs (relative abundance 1.5% in any of
the treatment) by treatment type. * indicates plant patho-
genic fungi. Control, no fertilization; NPK, application of
chemical fertilizers; NPK1LS, application of chemical fertil-
izers with low amounts of wheat straw; NPK1HS, applica-
tion of chemical fertilizers with high amounts of wheat
straw; NPK1PM, application of chemical fertilizers with pig
manure; NPK1CM, application of chemical fertilizers with
cow manure. Taxonomic levels: p, phylum; c, class; o,
order; f, family; g, genus; s, species.
Fig. S2. Hierarchical Clustering of fungal communities by
treatment type (a). Bray-Curtis dissimilarity of fungal com-
munities between treatments (b). Percentage bootstrap val-
ues obtained from 1000 trials are shown on branches. *
indicates that the fungal communities were significantly dif-
ferent between treatments as detected by analysis of simi-
larities (ANOSIM) using the Bray-Curtis distance. The R
values of ANOSIM results, which were calculated based on
999 permutations, can be observed under the dissimilarity
value.
Fig. S3. Fungal community composition of pig manure (a)
and cow manure (b). Relative abundances of abundant
OTUs (relative abundance 5%) for cow and pig maure (c).
PM, pig manure; CM, cow manure.
Table S1. Indicator species of the treatments.
Table S2. BLAST results of the dominant species.
Table S3. Result (R value) of analysis of similarities (ANO-
SIM) between the different organic material treatments.
Table S4. The relative abundance of indicator species of in
NPK1PM, pig manure and control samples.
Table S5. The relative abundance of indicator species in,
NPK1CM, cow manure, and control samples.
Table S6. Results of redundancy analysis.
14 R. Sun et al.
V
C2016 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology,00, 00–00
... More importantly, cattle manure deposition treatment reduced the community of fungi potentially pathogenic to oat. A previous study showed that cattle manure has a strong suppressive effect on soil pathogenic fungal growth and may regulate antagonistic microbial groups (Sun et al., 2016). In the present study, cattle manure deposition treatment decreased microbial groups related to pathotroph but increased beneficial groups of saprotroph and symbiotroph. ...
... In the present study, almost all fungal genera of the top eight dominant fungi were higher in the control treatment compared to cattle manure deposition treatment. Several of these fungi, e.g., genus Alternaria and Fusarium are soil-borne fungal pathogens with specific microbiomes in the rhizosphere (Sun et al., 2016). ...
Article
Full-text available
Simulated cattle manure deposition was used to estimate nutrient transfer to soil and oats and to investigate changes in microbial community composition and functional groups in oat rhizospheres. Nutrient absorption and return efficiency were calculated as a series of standard calculation formulas, and total nutrient transfer efficiency was nutrient absorption efficiency plus nutrient return efficiency. In total, 74.83% of nitrogen (N) and 59.30% of phosphorus (P) in cattle manure were transferred to soil and oats, with 11.79% of N and 7.89% of P in cattle manure absorbed by oats, and the remainder sequestered in the soil for 80 days after sowing. Cattle manure increased oat root length, surface, and volume under 0.2 mm diameter, and improved relative abundance of the microbiome known to be beneficial. In response to cattle manure, several bacteria known to be beneficial, such as Proteobacteria, Bacteroidota, and Firmicutes at phyla the level and Pseudoxanthomonas, Pseudomonas, and Sphingomonas at the genus level, were positively related to oat biomass and nutrient accumulation. For fungal communities, the relative abundance of Ascomycota is the predominant phylum, which varied in a larger range in the control treatment (81.0–63.3%) than the cattle manure deposition treatment (37.0–42.9%) as plant growing days extend. The relevant abundance of Basidiomycota known as decomposer was higher in cattle manure deposition treatment compared to that in control treatment at 15 days after sowing. More importantly, cattle manure deposition inhibited trophic mode within pathotroph like Alternaria and Fusarium fungal genus and promoted saprotroph and symbiotroph.
... A number of recent soil studies have found that farm management practices are a highly important factor driving microbial abundance, diversity, composition, and function. Application of manure, for instance, has a significant positive effect on soil microbial biomass and activity (Esperschütz et al., 2007;Sun et al., 2016;van der Bom et al., 2018). Plant species' identity and composition are other factors that affect soil microbial community diversity and composition near the root zone, due to exudates that can encourage beneficial microorganisms or discourage pathogens (Lei et al., 2019;Schmid et al., 2019). ...
Article
Full-text available
Soil health has been highlighted as a key dimension of regenerative agriculture, given its critical importance for food production, carbon sequestration, water filtration, and nutrient cycling. Microorganisms are critical components of soil health, as they are responsible for mediating 90% of soil functions. Multi-species rotational grazing (MSRG) is a promising strategy for maintaining and improving soil health, yet the potential effects of MSRG on soil microbiomes are poorly understood. To address this knowledge gap, we collected soil microbial samples at three timepoints during the 2020 grazing season for 12 total paddocks, which were equally split into four different grazing treatments—cattle only, sheep only, swine only, or multi-species. Shallow shotgun metagenomic sequencing was used to characterize soil microbial community taxonomy and antibiotic resistome. Results demonstrated broad microbial diversity in all paddock soil microbiomes. Samples collected early in the season tended to have greater archaeal and bacterial alpha diversity than samples collected later for all grazing treatments, while no effect was observed for fungi or viruses. Beta diversity, however, was strongly influenced by both grazing treatment and month for all microbial kingdoms, suggesting a pronounced effect of different livestock on microbial composition. Cattle-only and swine-only paddocks were more dissimilar from multi-species paddocks than those grazed by sheep. We identified a large number of differentially abundant taxa driving community dissimilarities, including Methanosarcina spp., Candidatus Nitrocosmicus oleophilus, Streptomyces spp., Pyricularia spp., Fusarium spp., and Tunggulvirus Pseudomonas virus ϕ-2. In addition, a wide variety of antibiotic resistance genes (ARGs) were present in all samples, regardless of grazing treatment; the majority of these encoded efflux pumps and antibiotic modification enzymes (e.g., transferases). This novel study demonstrates that grazing different species of livestock, either separately or together, can impact soil microbial community structure and antibiotic resistance capacity, though further research is needed to fully characterize these impacts. Increasing the knowledge base about soil microbial community structure and function under real-world grazing conditions will help to construct metrics that can be incorporated into traditional soil health tests and allow producers to manage livestock operations for optimal soil microbiomes.
... Most of researches have focused on soil microbiomes (incl. rhizosphere soil) (Sun et al., 2015;Sun et al., 2016;Fan et al., 2020;Fan et al., 2021), while far less is known about how long-term fertilization management influences the root endophytic microbiome, and its consequences for yield production. ...
Article
Fertilization can impact root endophytic microbiomes and food production. However, the impacts of decades of continued fertilization on root microbiomes, and their link with ongoing crop production, remain poorly understood. Here, we used a four decade-long fertilization experiment, including contrasting types of organic and inorganic fertilization, to investigate the effects of long-term fertilization on multi-kingdom root endophytic microbiomes, including keystone species (modules within microbial networks), and their indirect associations with the production of wheat, which is one of the most important crops worldwide. We found that long-term inorganic (nitrogen, phosphorus, potassium (NPK)) and organic (NPK with straw (NPKS) and NPK with cow manure (NPKM)) fertilization had significant impacts on the community composition of endophytic arbuscular mycorrhizal fungi (AMF), bacteria, and non-mycorrhizal fungi. In addition, compared with NPK fertilization, NPKS and NPKM amendments significantly decreased the microbial network complexity, which was associated with changes in the root iron content. Finally, we identified an important subset of keystone root endophyte species within the microbial network (Module #2), which was positively correlated with wheat yield, and affected by changes in root carbon to phosphorus ratio. This study provides evidence that long-term fertilization can affect keystone root endophytic species in the root microbiome, with implications for food security in an over-fertilized world.
... Changes in microbial community structure are related to available nutrients and plant biomass in soil (Bokhorst et al., 2017). Predecessors have shown that organic matter has an important impact on the composition of the soil fungal community (Sun et al., 2016). This study showed that the contents of OM, available Mn, available K and soil pH were significantly negatively correlated with some potential pathogenic fungi. ...
Article
Full-text available
Soybean continuous cropping (SC) leads to continuous cropping obstacles, and soil-borne fungal diseases occur frequently. Rotation can alleviate continuous cropping obstacles. However, the long-term effects of continuous cropping and rotation on the structure and function of the fungal community in soil are not clear. In this study, five cropping systems, SC, fallow (CK), fallow-soybean (FS), corn–soybean (CS), and wheat–soybean (WS), were implemented in the long-term continuous cropping area of soybean. After 13 years of planting, high-throughput sequencing was used to evaluate the structure and diversity of soil fungal communities and to study the relationship between fungal communities and soil environmental factors. The results showed that the abundance and diversity of fungal flora in SC soil were the highest. There were significant differences in the formation of soil fungal communities between soybean continuous cropping and the other treatments. There were 355 species of endemic fungi in SC soil. There were 231 and 120 endemic species in WS and CS, respectively. The relative abundance of the potential pathogens Lectera, Gibberella, and Fusarium in the SC treatment soil was significantly high, and the abundance of all potential pathogens in CK was significantly the lowest. The abundance of Lectera and Fusarium in CS was significantly the lowest. There was a positive correlation between potential pathogens in the soil. The relative abundance of potential pathogens in the soil was significantly positively correlated with the relative abundance of Ascomycetes and negatively correlated with the relative abundance of Basidiomycetes. Potential pathogenic genera had a significant negative correlation with soil OM, available Mn, K and soil pH and a significant positive correlation with the contents of soil available Cu, Fe, and Zn. In general, the fungal communities of SC, FS, WS, and CS were divided into one group, which was significantly different from CK. WS and CS were more similar in fungal community structure. The CK and CS treatments reduced the relative abundance of soil fungi and potential pathogens. Our study shows that SC and FS lead to selective stress on fungi and pathogenic fungi and lead to the development of fungal community abundance and diversity, while CK and CS can reduce this development, which is conducive to plant health.
... Compared to intensive conventional agricultural management, which has a major impact on soil community composition and diversity (Maeder et al., 2002;Tsiafouli et al., 2015), organic management can enhance abundance, diversity and activity of bacteria and fungi, which could be driven by a higher diversity and quantity of organic inputs (Francioli et al., 2016;Hartmann et al., 2015;Lori et al., 2017;Lupatini et al., 2017;Sun et al., 2016;Verbruggen et al., 2010). ...
Article
Full-text available
It is generally assumed that the dependence of conventional agriculture on artificial fertilizers and pesticides strongly impacts the environment, while organic agriculture relying more on microbial functioning may mitigate these impacts. However, it is not well known how microbial diversity and community composition change in conventionally managed farmers’ fields that are converted to organic management. Here, we sequenced bacterial and fungal communities of 34 organic fields on sand and marine clay soils in a time series (chronosequence) covering 25 years of conversion. Nearby conventional fields were used as references. We found that community composition of bacteria and fungi differed between organic and conventionally managed fields. In the organic fields, fungal diversity increased with time since conversion. However, this effect disappeared when the conventional paired fields were included. There was a relationship between pH and soil organic matter content and the diversity and community composition of bacteria and fungi. In marine clay soils, when time since organic management increased, fungal communities in organic fields became more dissimilar to those in conventional fields. We conclude that conversion to organic management in these Dutch farmers’ fields did not increase microbial community diversity. Instead, we observed that in organic fields in marine clay when time since conversion increased soil fungal community composition became progressively dissimilar from that in conventional fields. Our results also showed that the paired sampling approach of organic and conventional fields was essential in order to control for environmental variation that was otherwise unaccounted for.
... **P < 0.01, ***P < 0.001. Microbial community was determined using the first NMDS component revealed that persistent application of an organic fertilizer strengthened the bacterial assembly in the peanut rhizosphere, and that continuous planting of the crop, especially with intensive application of chemical fertilizers, deteriorated the rhizosphere microbial ecology (Li et al. 2014;Soman et al. 2017;Sun et al. 2016). The ability of the rhizosphere bacterial assembly formed upon persistent organic fertilization to suppress fungal pathogen growth was enhanced. ...
Article
Full-text available
Background and aimsSoil-borne diseases are an increasingly serious threat to agriculture systems. Organic fertilization would improve soil quality and microbial community as well, and thus is appreciated a promising control strategy for soil-borne diseases. Yet, how soil microbial communities mediate disease control under organic fertilization remains largely unknown. Here, we aimed to explore the microbial mechanism of controlling soil-borne diseases by organic fertilization.Methods We investigated the effects of various fertilization regimes on the soil suppressiveness toward pathogenic fungi in the peanut rhizosphere. The fertilization regimes tested were organic fertilizer, chemical fertilizers, and a combination of both.ResultsUninterrupted application of organic fertilizer in peanut field plots for seven planting seasons resulted in a control of peanut root rot, with a significantly higher peanut yield. Upon organic fertilization, bacterial microbiome assembly in the rhizosphere played a key role in developing soil suppressiveness against peanut root rot; upon chemical fertilization, the potential fungal pathogens dominated the fungal microbiome assembly in the rhizosphere to boost root rot. Further, structural equation model revealed that the rhizosphere bacterial community contributed to the control of root rot. Furthermore, upon organic fertilization, the rhizosphere bacterial community strongly suppressed mycelial growth and spore germination of Fusarium sp. ACCC 36194.Conclusions Collectively, in a monocropping system, persistent organic fertilization favors the development of a protective microbial shield in the plant rhizosphere, maintaining the rhizosphere health.
... Among the widely applied conservation management strategies, reduced tillage (opposite of conventional tillage), manure compost (opposite of chemical fertilisers), and straw residue application are the most popular. They potentially favour soil fauna via directly reducing physical disturbance, introducing potential food sources, and providing favourable habitats (e.g., Coulibaly et al., 2017;Platen and Glemnitz, 2016), and/or indirectly via improving soil structure (e.g., aggregate size and stability, porosity) and fostering soil microbiome (e.g., Sun et al., 2015Sun et al., , 2016Wang et al., 2019). Nevertheless, the effects of these managements may not always be positive. ...
Article
Soil biodiversity is of key importance to many essential ecosystem functions, but currently it is severely threatened by both intensive agriculture and climate changes. Ecological intensification, including organic amendments and less disturbance, is expected to buffer the degradation of biodiversity and ecosystem functioning induced by intensive agriculture, but its effects in the context of climate changes are poorly understood. In the present work, we studied the responses of agricultural soil biodiversity to ecological intensification under different natural rainfall intensities in a subtropical field. We focused on a numerically dominant group of soil microarthropods, the Collembola, and three conservation managements, i.e., straw, manure, and no-tillage. The experimental site was established with a full-factorial design of different managements. Soil physiochemical parameters and the density, taxonomic diversity, and morpho-functional traits of the collembolan community were measured over three consecutive years. Results showed that rainfall intensification markedly reduced collembolan density and had severe impact on large euedaphic species. Straw amendment buffered the detrimental effect of dense rainfall on collembolan density, but aggravated the body size reduction caused by the rainfall. Manure input and no-tillage mainly affected the community functional composition, in which manure favours more active and mobile species characterised by a well-developed furca, whereas no-tillage favoured surface-dwelling species. These results support the hypothesis that external resource enrichment and reduced disturbance would shape the functional traits of soil fauna, and further modified their response to climate change events. Our findings call for more attention on the functional consequences of ecological intensification and the interactions among soil biodiversity, agricultural managements and climate changes.
... These results also demonstrated that additional SOC could promote the formation of large aggregates (Six et al., 2000;Chaplot and Cooper, 2015). One of the primary reasons is that higher SOC content enhances soil microbial communities (Sun et al., 2016;Li et al., 2017), so a large number of microbial hyphae could transform micro-aggregates into macro-aggregates . Previous studies have also shown that microorganisms could produce more extracellular polysaccharides by glucose addition, which in turn significantly improved the stability of soil aggregates Swift, 1986a, 1986b). ...
Article
Subtropical Acrisols have low soil organic carbon (SOC) and potassium (K) availability. However, the relationship between K distribution of aggregates and SOC levels is unclear. Five soil samples were collected from a long-term fertilization experiment in an Acrisol. Different K fertilization treatments included CK (without any fertilizer), ZK (zero rate of chemical K fertilizer), KF (conventional rate of chemical K fertilizer added to ZK treatment), KM (KF treatment including the application of fresh pig manure, and KS (KF treatment with half of crop straw returned to the field). Then, an incubation experiment was conducted in which different rates of glucose were added to attain 5 increased levels in SOC with 0, 10%, 20%, 50%, and 100% in the CK, ZK, KF, KM, and KS treatments. Compared with no glucose addition, glucose addition improved these > 2 mm aggregates by 9.16%− 104.19% among all soils. With increased SOC percentage, the > 2 mm aggregate increased. A non-linear equation indicated that there were significant correlations between > 2 mm aggregates and SOC content in KF, KM, and KS soils. The exchangeable K (EK) and non-exchangeable K (NEK) contents of > 2 mm aggregates were highest in KM soil, followed by KS and KF soils, while CK and ZK soils were the lowest. The linear equations indicated that the EK and NEK stock of > 2 mm aggregates could be increased by 8.41–9.10 kg ha⁻¹ and 15.73–20.65 kg ha⁻, respectively, in all K fertilized soils, when SOC was increased by 1 g kg⁻¹. The slopes of linear equations also showed that the growth rate for NEK stock of > 2 mm aggregates in KM soil (20.65 kg ha⁻¹) was higher than those of KS and KF soils (15.73 and 17.59 kg ha⁻¹). Additionally, the slopes of linear equations indicated that the growth rate for proportions of EK and NEK stock of > 2 mm aggregates in KM soil (1.14% and 1.64%) were lower than those in KS soil (2.22% and 2.28%). Combined with CK, ZK, KF, KM, and KS soils, the proportion of EK and NEK stock of > 2 mm aggregates increased by 1.62% and 2.32% along with increased SOC by 1 g kg⁻¹. Therefore, the EK and NEK stock of > 2 mm aggregates increased through glucose addition in the Acrisol. However, these increases varied by fertilization treatments, which caused different SOC and K levels.
Article
The idea of agricultural sustainable in the EU is based on, both minimizing interference with the soil system as well as diversifying crop rotation what relates to the limited cultivation system (changed from plow to no-plowing tillage) as well as organic fertilization is often abandoned. Taking above into account, our goal was determined of the structure, composition, and metabolic profiles of soil microbiomes in various cultivation methods (under multiannual plow and no-plow cultivation) using metagenomic analysis. Having regard to the recommendations contained in EU report (European Commission et al., 2020) of the Mission board for Soil health and food, 2020 indicating the lack of microbiological indicators of “healthy soil”. So, we have tried to select of microbiological indicators showing sensitivity and resistance to use the methods of soil cultivation. The research object was located on almost 100-year field experiments at the Experimental Station of the Faculty of Agriculture and Biology in Skierniewice/near Warsaw on, luvisoils dominated in the temperate climate of Central Europe. Soil microorganisms respond with changes in their abundance and taxonomic composition depending on the methods of soil cultivation. Actinobacteria were the most abundant, while Planctomycetes were the least abundant in the metagenome of soil fertilized with manure, whereas the uncultivated soil was dominated by Nitrospirae. We can recommend the following taxa, including Gemmatimonas sp. as a microbiological indicator sensitive to the long-term lack of both plow cultivation of soil and organic fertilization, and Mycobacterium sp. as a resistancivity indicator to this soil cultivation method. Sorangium sp. could be recommended as microbiological indicators which responds by reducing the quantity under effect of the organically fertilized soil, while the plow and no-plow cultivation does not affect changes in its quantity. The use of various cultivation methods changed the biochemical functions in soil metagenoms, including nitrogen and sulfur metabolism and carbohydrate metabolism, and in the production of plant hormones and siderophores. Additionally, soil cultivation ways changed the response of microorganism’s stresses, including oxidative stress. The conducted research indicates the necessity to conduct further research on the influence of various cultivation methods, on the diversity of the microorganism community and soil metabolism. The result of which may be the selection of appropriate microbiological indicators for determining “soil health” depending on the type of soil under cultivation located in different climatic zones, not only presented in the paper.
Article
The process of nitrite‐dependent anaerobic methane oxidation (n‐damo) catalysed by Candidatus Methylomirabilis oxyfera (M. oxyfera)‐like bacteria is a novel pathway in regulating methane (CH4) emissions from paddy fields. Nitrogen fertilization is essential to improve rice yields and soil fertility; however, its effect on the n‐damo process is largely unknown. Here, the potential n‐damo activity, abundance and community composition of M. oxyfera‐like bacteria were investigated in paddy fields under three long‐term (32 years) fertilization treatments, i.e. unfertilized control (CK), chemical fertilization (NPK) and straw incorporation with chemical fertilization (SNPK). Relative to the CK, both NPK and SNPK treatments significantly (p < 0.05) increased the potential n‐damo activity (88%–110%) and the abundance (52%–105%) of M. oxyfera‐like bacteria. The variation of soil organic carbon (OrgC) content and inorganic nitrogen content caused by the input of chemical fertilizers and straw returning were identified as the key factors affecting the potential n‐damo activity and the abundance of M. oxyfera‐like bacteria. However, the community composition and diversity of M. oxyfera‐like bacteria did not change significantly by the input of fertilizers. Overall, our results provide the first evidence that long‐term fertilization greatly stimulates the n‐damo process, indicating its active role in controlling CH4 emissions from paddy fields.
Article
Full-text available
Despite growing recognition of the importance of ecosystem services and the economic and ecological harm caused by invasive species, linkages between invasions, changes in ecosystem functioning, and in turn, provisioning of ecosystem services remain poorly documented and poorly understood. We evaluate the economic impacts of an invasion that cascaded through a food web to cause substantial declines in water clarity, a valued ecosystem service. The predatory zooplankton, the spiny water flea (Bythotrephes longimanus), invaded the Laurentian Great Lakes in the 1980s and has subsequently undergone secondary spread to inland lakes, including Lake Mendota (Wisconsin), in 2009. In Lake Mendota, Bythotrephes has reached unparalleled densities compared with in other lakes, decreasing biomass of the grazer Daphnia pulicaria and causing a decline in water clarity of nearly 1 m. Time series modeling revealed that the loss in water clarity, valued at US$140 million (US$640 per household), could be reversed by a 71% reduction in phosphorus loading. A phosphorus reduction of this magnitude is estimated to cost between US$86.5 million and US$163 million (US$430-US$810 per household). Estimates of the economic effects of Great Lakes invasive species may increase considerably if cases of secondary invasions into inland lakes, such as Lake Mendota, are included. Furthermore, such extreme cases of economic damages call for increased investment in the prevention and control of invasive species to better maximize the economic benefits of such programs. Our results highlight the need to more fully incorporate ecosystem services into our analysis of invasive species impacts, management, and public policy.
Article
This research illustrates the qualitative and quantitative composition of the mycoflora of both a green compost (thermophilically produced from plant debris) and a vermicompost (mesophilically produced by the action of earthworms on plant and animal wastes after thermophilic preconditioning). Fungi were isolated using three media (PDA, CMC, PDA plus cycloheximide), incubated at three temperatures (24, 37 and 45 C). Substantial quali-quantitative differences in the species composition of the two composts were observed. The total fungal load was up to 8.2 × 10⁵ CFU/g dwt in compost and 4.0 × 10⁵ CFU/g dwt in vermicompost. A total of 194 entities were isolated: 118 from green compost, 142 from vermicompost; 66 were common to both. Structural characterization of this kind is necessary to determine the most appropriate application of a compost and its hygienic quality.
Article
A fungus which produced alkaline cellulase was isolated from soil in Kanagawa prefecture, Japan, using cellulose as a substrate, under alkaline conditions. The fungus was identified as Acremonium sp. [Acremonium sp. (A. sp.) was reported as a new species by Okada et al. (Trans. Mycol. Soc. Japan, 34, 171-185 (1993)), and given the name Acremonium alcalophilum JCM 7366.] The characteristics of crude cellulase of A. sp. were studied. The fungus produced CMCase and xylanase, whose activities were found even at 0°C and under both alkaline and acidic conditions. The maximal activities of the low-temperature-active CMCase and xylanase were obtained at 40°C and pH 7.0, the maximal pH at 0°C being the same as at 40°C, At 0°C, the activities of these enzymes were retained at more than 20% in the case of CMCase, and more than 40% in the case of xylanase, the activities at 40°C. Low-temperature-active CMCase and xylanase were induced in the presence of glucose as a carbon source, but not in the presence of cellobiose and sophorose as carbon sources.
Article
Black soil is one of the main soil types in northeast China, and plays an important role in Chinese crop production. However, nitrogen inputs over 50 years have led to reduced black soil fertility. It is unclear how N affects the fungal community in this soil type, so a long-term fertilizer experiment was begun in 1980 and we applied 454 pyrosequencing and quantitative PCR to targeted fungal ITS genes. There were five treatments: control (no fertilizer), N1 (low nitrogen fertilizer), N2 (high nitrogen fertilizer), N1P1 (low nitrogen plus low phosphorus fertilizers) and N2P2 (high nitrogen plus high phosphorus fertilizers). Soil nutrient concentrations (Total N, Avail N, , , etc.) and ITS gene copy numbers increased, whereas soil pH and fungal diversity decreased in all the fertilized treatments. Relationships between soil parameters and fungal communities were evaluated. Dothideomycetes, Eurotiomycetes, Leotiomycetes, Sordariomycetes, and Agaricomycetes were the most abundant classes in all soils. Principal coordinates analysis showed that the fungal communities in the control and lower-fertilizer treatments clustered closely and were separated from communities where more concentrated fertilizers were used. Fungal diversity and ITS gene copy number were dependent on soil pH. Our findings suggested that long-term nitrogen and phosphorous fertilizer regimes reduced fungal biodiversity and changed community composition. The influence of the more concentrated fertilizer treatments was greater than the lower concentrations.
Article
A low-temperature-active CMCase from Acremonium alcalophilum JCM 7366 was purified by ammonium sulfate fractionation, QAE-Toyopearl 650c column chromatography, TSKgel ether-5PW high performance liquid column chromatography, and TSKgel G3000SWxl high performance liquid column chromatography. The molecular weight of the purified enzyme was estimated to be 46,000 and 48,000 by SDS-PAGE and TSKgel G3000SWxl gel filtration, respectively. The isoeletric point was 4.4. The purified enzyme hydrolyzed CMC, Avicel, and xylan. The main product after 24-h digestion of CMC or xylan was cellobiose or xylobiose. The maximal CMCase and xylanase activities of the purified low-temperature-active enzyme were obtained at 40°C and pH 7.0. CMCase and xylanase activities at 0°C were retained at more than 25.0 and 48.8% of the activities at 40°C, respectively. Both these enzyme activities were stable at pHs from 5.5 to 10.0 and at 50°C for 10 min.
Chapter
Soil fertility decline is increasingly leading to reduced food production worldwide. Over 70% of small holder farmers in the central highlands of Kenya are using crop manure, animal wastes and inorganic fertilizers to increase their farms' fertility and subsequent productivity the dilemma with these practices is that less is known on the impact of these resources on the below ground biodiversity particularly the microbial communities which play a key role in determining soil quality. A study was carried out on a 32 year old long-term trial in Kabete, Kenya these soils were treated with organic (maize stover at 10 t ha-, farmyard manure at 10 t ha-) and inorganic fertilizers (120 kg N, 52.8 kg P plus farmyard manure at 10 t ha-1 (N2P2 + FYM), 120 kg N, 52.8 kg P plus maize stover at 10 t ha-1 (N2P2 + R), 120 kg N, 52.8 kg P (N2P2), and a control (Nil and fallow) for over 30 years. We examined 16S rRNA gene and 28S rRNA gene fingerprints of bacterial and fungal communities, respectively, by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. Bacterial community structure and diversity were negatively affected by N2P2, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the N2P2-treated soil was more closely related to the bacterial structure in the untreated soil (fallow and Nil) than that in soils treated with a combination of inorganic and organic or inorganic fertilizers alone. For the fungal community the negative effect of N2P2 alone was not as adverse as for the bacterial community structure since the soils treated with N2P2 were closely related to those treated with N2P2 + FYM and N2P2 + maize stover. However, soils treated with organic inputs clustered away from soils amended with inorganic inputs. Organic inputs had a positive effect on both fungal and bacterial community structures with or without chemical fertilizers. Results from this study suggested that bacterial and fungal community structure was closely related to agro-ecosystem management practices conducted for over the past 30 years. © 2012 Springer Science+Business Media B.V. All rights reserved.
Article
Invasive fungi and other non-indigenous plant pathogens have had a significant effect on American agriculture for hundreds of years. At present crop loss due to invasive plant pathogens, especially fungi, is estimated at $21 billion per year in the United States, greater than the loss caused by non-indigenous insects. Plant pathogenic fungi are difficult to detect and identify. Thus knowledge of which fungi pose a threat is essential to prevent their entry by means others than inspection. In this paper, examples are presented of invasive fungi on agricultural commodities introduced into the United States. In all cases two factors have been crucial: first, the pathway through which these fungi have entered, and second, systematic knowledge to prevent and respond to the new invasive species. Historically important plant pathogens such as black stem rust of wheat still cause considerable damage while others such as late blight of potato appear to be having a resurgence. Known previously in Australia, then moving to Africa and South America, the virulent species of soybean rust appeared in the U.S. in 2004 but has not been as devastating as anticipated. Plant pathogenic fungi on specialty crops such as daylily, gladiola and chrysanthemum are threatened by rust fungi recently found in the U.S. apparently brought in on infected germplasm. A crisis in the export of U.S. wheat occurred in the late 1990's when the molecular diagnostic test for Karnal bunt gave a false positive response to a closely related but previously unknown species. Many potentially dangerous plant pathogens of crop plants have not yet been introduced into the U.S. It is critical that meticulous surveillance be conducted as plant material enters the country as well as where crops are grown prior to shipment. In addition, the scientific infrastructure is needed to be able to respond quickly to new invasive fungi. This requires sound systematic knowledge of plant pathogenic fungi both in the U.S. and around the world and a cadre of systematic experts who can characterize invasive fungi.