Arbuscular mycorrhizal fungal community structure and diversity in response to long-term fertilization: A field case from China

Abstract

The influences of different fertilizer treatments on spore community structure and diversity of arbuscular mycorrhizal (AM)
fungi (AMF) were investigated in a long-term fertilization experiment with seven treatments: organic manure (OM), half organic
manure N plus half fertilizer N (1/2 OMN), fertilizer NPK, fertilizer NP, fertilizer NK, fertilizer PK, and the control (without
fertilization). Fertilization generally increased the nutrient contained in the fertilizer and treatments with NPK and 1/2
OMN produced the highest crop yields. Thirty-five species of AMF within 6 genera, including 8 previously undescribed species,
were recovered. Similarly in all seven treatments, the most abundant genus was Glomus, and followed by Acaulospora. All the fertilization treatments changed AM species composition, and NK treatment had the slightest influence. Fertilization
with fertilizers NP, PK and NPK markedly increased AM fungal spore density, while 1/2 OMN, OM and NK treatments showed no
significant influences. All the fertilizer treatments, especially OM, significantly decreased species richness and species
diversity (Shannon-Weiner index). There were no significant correlations between AM fungal parameters (spore density, species
richness and species diversity) and soil properties. The findings indicate that long-term fertilization all can change AM
fungal community structure and decrease species diversity, while balanced fertilization with NPK or 1/2 OMN is the most suitable
fertilization regime if taking both crop yields and AM species diversity into account.

KeywordsBiodiversity–Manure–Mineral fertilizer–Maize–Arbuscular mycorrhizae

Full text

ORIGINAL PAPER
Arbuscular mycorrhizal fungal community structure
and diversity in response to long-term fertilization:
a field case from China
Fa Yuan Wang •Jun Li Hu •Xian Gui Lin •
Sheng Wu Qin •Jun Hua Wang
Received: 4 February 2010 / Accepted: 23 April 2010 / Published online: 9 May 2010
ÓSpringer Science+Business Media B.V. 2010
Abstract The influences of different fertilizer treatments
on spore community structure and diversity of arbuscular
mycorrhizal (AM) fungi (AMF) were investigated in a
long-term fertilization experiment with seven treatments:
organic manure (OM), half organic manure N plus half
fertilizer N (1/2 OMN), fertilizer NPK, fertilizer NP, fer-
tilizer NK, fertilizer PK, and the control (without fertil-
ization). Fertilization generally increased the nutrient
contained in the fertilizer and treatments with NPK and 1/2
OMN produced the highest crop yields. Thirty-five species
of AMF within 6 genera, including 8 previously unde-
scribed species, were recovered. Similarly in all seven
treatments, the most abundant genus was Glomus, and
followed by Acaulospora. All the fertilization treatments
changed AM species composition, and NK treatment had
the slightest influence. Fertilization with fertilizers NP, PK
and NPK markedly increased AM fungal spore density,
while 1/2 OMN, OM and NK treatments showed no sig-
nificant influences. All the fertilizer treatments, especially
OM, significantly decreased species richness and species
diversity (Shannon-Weiner index). There were no signifi-
cant correlations between AM fungal parameters (spore
density, species richness and species diversity) and soil
properties. The findings indicate that long-term fertilization
all can change AM fungal community structure and
decrease species diversity, while balanced fertilization with
NPK or 1/2 OMN is the most suitable fertilization regime if
taking both crop yields and AM species diversity into
account.
Keywords Biodiversity Manure Mineral fertilizer 
Maize Arbuscular mycorrhizae
Introduction
It is well-known that arbuscular mycorrhizal (AM) fungi
(AMF) are beneficial to plant growth by increasing the
supply of immobile soil mineral nutrients, notably P (Smith
and Read 1997). However, AMF may not always play a
vital role in the nutrition and growth of plants in many
agricultural systems, especially in high-input agriculture
(Ryan and Graham 2002). Numerous reports have shown
the negative or positive influences of fertilizers on AMF
biodiversity, including readily soluble P and N, organic
manure, and slow release mineral fertilizers (Douds and
Millner 1999; Gosling et al. 2006; del Mar Alguacil et al.
2009). In most cases, readily soluble fertilizers have neg-
ative impacts on AM fungal diversity but organic manure
and slow release fertilizers do not suppress AMF and may
even stimulate them (Gosling et al. 2006). However, most
of these studies are based on short-term responses, which
may differ considerably from long-term responses to
different fertilizers.
Generally, long-term fertilization may have more
dramatic impacts on soil characteristics and microbial
community (Elfstrand et al. 2007; Omay et al. 1997). AM
fungal development and diversity are often decreased by
F. Y. Wang J. L. Hu X. G. Lin (&)S. W. Qin J. H. Wang
State Key Laboratory of Soil and Sustainable Agriculture,
Joint Open Laboratory of Soil and the Environment,
Hongkong Baptist University & Institute of Soil Science,
Chinese Academy of Sciences, East Beijing Road 71,
Nanjing 210008, People’s Republic of China
e-mail: xglin@issas.ac.cn
F. Y. Wang
Agricultural College, Henan University of Science
and Technology, Luoyang, Henan Province 471003,
People’s Republic of China
e-mail: wfy1975@163.com; wfyuan1975@sohu.com
123
World J Microbiol Biotechnol (2011) 27:67–74
DOI 10.1007/s11274-010-0427-2

long-term fertilization with easily soluble mineral fertiliz-
ers or fertilizer combinations, such as P (Martensson and
Carlgren 1994), N (Bradley et al. 2006), NP (Na Bhadalung
et al. 2005), and NPK (Gryndler et al. 2006; Joner 2000).
However, long-term fertilization with manures often
showed positive effects on AMF (del Mar Alguacil et al.
2009; Gryndler et al. 2006). AM fungal parameters such as
colonization, spore abundance and species diversity, were
significantly higher in the organic than in the conventional
systems (Galvez et al. 2001;Ma
¨der et al. 2000; Oehl et al.
2003,2004). Sometimes, different results have been
reported on long-term fertilization impacts. High levels of
AM fungal colonization/diversity have also been reported
in soils with high available P or N (Gosling et al. 2006).
Only small differences were found in AM fungal com-
munities between the conventional or low-input practices
(Franke-Snyder et al. 2001; Kurle and Pfleger 1996). The
possible reasons may be because besides fertilizers the
factors influencing AMF are diverse in agricultural fields,
such as host plants, climatic conditions, environmental
stresses, and AM fungal characters, which certainly need to
be investigated further.
In China, because of the concern of soil fertility deg-
radation by replacement of organic fertilizers by inorganic
fertilizers, a long-term experiment was set up in the Huang-
Huai-Hai Plain, which is located in the low reaches of the
Yellow, Huai, and Hai rivers within an area of
3.5 910
5
km
2
(Qin et al. 1998). This is one of the most
important agricultural regions in China. Investigating long-
term effects of fertilization on AM fungal communities and
diversity may help to ensure an opportunity for the utili-
zation of AMF in agrosystems and understand the fertil-
ization impacts on soil microorganisms and soil health. In
2007, soil samples were taken from a long-term fertiliza-
tion site with seven different fertilizers or fertilizer com-
binations to analyse AM fungal community structure and
diversity using the classic spore morphology method. Our
aims were (1) to investigate the AM fungal community
structure and diversity in a wheat/maize field fertilized with
seven different fertilization regimes for 18 years, and (2) to
demonstrate the influences of long-term fertilization on
AMF community.
Materials and methods
Field site description
The long-term field fertilizer experiment was carried out in
the Fengqiu Agro-Ecological Experimental Station
(35°000N, 114°240E) of the Chinese Academy of Sciences,
Henan Province, China. The annual precipitation in the
area is 615 mm, 60–90% of which took place from May to
October. Mean temperature was 14.5 °C. The soil, with a
sandy loam texture, was derived from alluvial sediments of
the Yellow River and classified as aquic inceptisol. The
soil contained 5.83 g kg
-1
of organic C, 0.45 g kg
-1
of
total N, 0.50 g kg
-1
of total P, 1.93 mg kg
-1
available P,
78.8 mg kg
-1
available K and 18.6 g kg
-1
of total K and
had a pH (H
2
O) of 8.65 at the beginning of the experiment
in September 1989.
Fertilization treatments
Seven treatments (four replicates of each) were established
in completely randomized blocks in 28 plots (9.5 95m
2
)
under a rotation of winter wheat (Triticum aestivum L.) and
summer maize (Zea mays L.): organic manure (OM), half
organic manure N plus half mineral N fertilizer (1/2 OMN),
mineral NPK fertilizer (NPK), mineral NP fertilizer (NP),
mineral NK fertilizer (NK), mineral PK fertilizer (PK), and
the control (without fertilization). For NPK treatment, N, P,
and K were applied in the form of urea (300 kg N ha
-1
per
year), super phosphate (150 kg P
2
O
5
ha
-1
per year), and
potassium sulfate (300 kg K
2
Oha
-1
per year), respec-
tively, while no K, P, or N was applied for the NP, NK, and
PK treatments, respectively. The organic manure was a
composted mixture of wheat straw, oil cake, and cotton
cake in a ratio of 100:40:45. These materials were ground
to achieve lengths of 3–5 mm, mixed completely with
limited water, and composted for 2 months. The oil cakes
and cotton cakes were the machine-dried residues of oil-
harvested rapeseeds and cottonseeds, respectively. Detailed
information on the organic manure has been given before
(Meng et al. 2005). The OM and 1/2 OMN treatments were
designed to give the same application rates of N, P, and K
as those given with the NPK treatment. For the OM
treatment, N was applied as organic manure, while for the
1/2 OMN treatment, half of the N was applied as organic
manure and the other half as urea. Because the amounts of
P and K contained in the organic manure were generally
less than the prescribed doses, supplemental super phos-
phate and potassium sulfate were added to the OM and 1/2
OMN treatments to equal the amounts given in the NPK
treatment. Each plot had received the same fertilizer
management every year since 1989. Detailed information
on the experimental design and field management has been
described by Meng et al. (2005). Wheat and maize were
harvested after maturity and yields were recorded.
Soil samples and analysis
On March 26, 2007, soil samples were collected at a depth
of 0–15 cm in the wheat season. For each plot, soil samples
were collected from 16 points and then mixed and sieved
(\2 mm), with aboveground plant materials, roots, and
68 World J Microbiol Biotechnol (2011) 27:67–74
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stones being removed. The soil samples were used for the
analysis of AM fungal spores and soil properties.
Soil pH was determined with a glass electrode using a
soil-to-water ratio of 1:2.5. Soil organic C and total N were
determined by dichromate oxidation (Mebius 1960) and
Kjeldahl digestion (Bremner 1965), respectively. Available
P in soil was extracted by sodium bicarbonate and deter-
mined using the molybdenum blue method (Olsen et al.
1954). Available K in soil was extracted by ammonium
acetate and determined by flame photometry (Carson
1980).
Recovery and counting of AM fungal spores
Spores or sporocarps were extracted from 25 g air-dried
soil for each sample by wet-sieving followed by flotation
centrifugation in 50% sucrose. The finest sieve was 30 lm.
The spores were collected on a grid patterned (4 94 mm)
filter paper, washed with distilled water to spread them
evenly over the entire grid and counted using a dissecting
microscope at up to 90-fold magnification. A sporocarp
was counted as one spore.
For observation and identification of spore characters,
spores were mounted on glass slides in polyvinyl-lacto-
glycerol (PVLG) and PVLG ?Melzer’s reagent and then
identified to species using taxonomic manuals (Schenck
and Perez 1990) and internet information from INVAM
http://invam.caf.wvu.edu/Myc_Info/Taxonomy/species.htm),
Glomeromycota species (http://www.lrz-muenchen.de/
*schuessler/amphylo/), International Bank for the Glom-
eromycota (BEG) (http://www.kent.ac.uk/bio/beg/), Arbus
cular mycorrhizal fungi (Glomeromycota), Endogone and
Complexipes species deposited in the Department of Plant
Pathology, University of Agriculture in Szczecin, Poland,
(http://www.agro.ar.szczecin.pl/*jblaszkowski/), and the
description of the newly reported AM fungal species. If the
species is not yet described, it may be recognized as an
unknown species, and marked as Glomus sp., Acaulospora
sp., etc.
AM fungal diversity
Species richness, spore density, frequency, and relative
abundance of AMF were expressed as follows: species
richness =number of AM fungal species in 25 g air-dried
soil; spore density =number of AM fungal spores in 25 g
air-dried soil; relative abundance =(number of spores of a
species or a genus/total spores) 9100%. Species diversity
was measured by the Shannon-Weiner index (H0=
-PPilnPi) and evenness (E=H0/H
max
) (Franke-Snyder
et al. 2001). Jaccard index (IS
J
=c/(a?b?c)) of simi-
larity, based on the morphological determination of spores
was calculated to compare AM fungal species composition
under different fertilization treatments. The IS
J
is a
measure of the similarity of the spore community between
sites A and B, where ais the number of species occurring
only at site A, bis the number of species occurring only at
site B and cis the number of species occurring at both sites.
Statistical analysis
Analysis of variance (ANOVA), hierarchical cluster anal-
ysis and correlation analysis were all carried out with SPSS
software package (version 13.0). Significance of differ-
ences between treatments with respect to soil properties,
spore abundance, species numbers and AM fungal diversity
(Shannon-Weiner index and evenness) was tested using
Duncan’s multiple range test at P\0.05 after one-way
ANOVA. A hierarchical cluster analysis using complete
linkage (furthest neighbor) method was applied to deter-
mine the similarity with respect to AM fungal species
composition between treatments. Spearman-rank correla-
tion coefficients were calculated to evaluate the strength of
the relationship between soil properties and AM fungal
species richness, diversity, as well as the Jaccard index of
similarity between the control and other fertilization
treatments.
Results
Soil pH and nutrient contents
Compared with the soil properties at the beginning of the
experiment, after 17 years, soil total N, organic C and
available P decreased in the control treatment, while fer-
tilization generally increased the nutrient contained in the
fertilizer (Table 1). Compared with the control, soil pH did
not change in NK and PK treatments but slightly declined
in other fertilizer treatments, with the lowest value in OM
treatment. Soil organic C did not change in NK and PK
treatments, but significantly increased in other treatments,
especially in OM treatment. Soil total N was significantly
increased by all the fertilizers, especially by the application
of organic manure. Available P was increased by all the
fertilizer treatments except NK. Available K slightly
decreased in NP treatment but greatly increased in other
treatments.
Crop yields
Dynamics of winter wheat and summer maize yields are
shown in Fig. 1. Because of a flood in July 2000, maize
yields in all the treatments were lost. Generally, both wheat
and maize yields were significantly higher in NPK and 1/2
OMN than those in other treatments, with few exceptions.
World J Microbiol Biotechnol (2011) 27:67–74 69
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On average over 17 years, both winter wheat and maize
yields were the highest for the NPK and 1/2 OMN treat-
ment, following an order of NPK C1/2 OMN [NP [
OM [PK [NK =Control.
AMF fungal species composition and similarity
A total of 35 species of AMF within 6 genera of Glom-
eromycota, including 8 previously undescribed species,
were recorded (Table 2). The most abundant genus was
Glomus (24 species), accounting for 79.83–87.38% of
spore counts, and then the genus Acaulospora (7 species),
accounting for 6.56–12.16%. Only 1 species was
found in Ambispora,Archaeospora,Gigaspora and
Scutellospora, respectively. Thirteen species were found in
all the fertilization treatments, among which, the most
abundant species was Glomus claroideum, followed
orderly by G. caledonium,G. mosseae,G. fasciculatum,
G. geosporum,G. intraradices,G. versiforme,Ambispora
lepototicha, A. bireticulata,A. laevis. Three previously
undescribed species (G. sp.1,G. sp.2,A. sp.1) also occurred
frequently in all the fertilization treatments.
Table 3showed that Jaccard index of similarity was
from 0.500 to 0.762. Considering the control treatment, the
highest Jaccard index value was between the control and
NK, which shared 19 AM fungal species, while both 1/2
OMN and OM treatments had the largest differences in
species composition with the control.
Cluster analysis based on the similarity in AM fungal
species composition among different treatments is shown
in Fig. 2. Taking the index of similarity of 0.6 as a base-
line, AM fungal communities may be grouped in three
main clusters. AM fungal species composition had higher
similarities within the groups than between groups.
Spearman correlation coefficients showed that the sim-
ilarities (IS
J
) of AM fungal species composition between
the control and other fertilization treatments positively
correlated with soil pH (r=0.883, P\0.01), but nega-
tively correlated with soil organic C (r=-0.811,
P\0.05), total N (r=-0.847, P\0.05) and available P
(r=-0.883, P\0.01), and did not significantly correlate
with available K.
AM fungal species richness and species diversity
Compared with the control, spore density markedly
increased in NP, PK and NPK treatments, but did not
change significantly in other treatments (Table 4). Species
richness decreased in all the fertilizer treatments, especially
in OM treatment. However, when considering the total
number of AM fungal species contained in the four repli-
cate plots for each treatment, the species numbers
decreased in the order: Control [NPK [NP [NK [
Table 1 The soil properties after the long-term fertilization
Treatment pH Organic C (g kg
-1
) Total N (g kg
-1
) Available P (mg kg
-1
) Available K (mg kg
-1
)
Control 8.76 (0.04) c 4.45 (0.25) a 0.37 (0.00) a 0.23 (0.01) a 90.55 (5.84) b
1/2 OMN 8.38 (0.07) b 8.36 (0.83) c 0.74 (0.00) f 17.29 (1.25) c 234.66 (9.63) c
OM 8.12 (0.08) a 10.59 (0.63) d 1.00 (0.03) g 20.96 (3.44) d 229.56 (12.88) c
NP 8.44 (0.08) b 5.76 (0.37) b 0.54 (0.01) d 12.60 (0.79) b 74.61 (3.83) a
NK 8.69 (0.07) c 4.38 (0.61) a 0.41 (0.01) b 0.53 (0.13) a 395.35 (8.84) e
PK 8.65 (0.10) c 4.74 (0.75) a 0.45 (0.02) c 27.89 (2.48) e 348.17 (4.42) d
NPK 8.45 (0.02) b 6.35 (0.36) b 0.57 (0.02) e 12.52 (0.35) b 221.91 (10.12) c
Data are means and SD (n=4). The difference between values in a column followed by different letters is significant at P\0.05
a
0
1000
2000
3000
4000
5000
6000
7000
Wheat yield (kg ha-1)
b
0
2000
4000
6000
8000
10000
12000
14000
1990 1994 1998 2002 2006
1990 1994 1998 2002 2006
Maize yield (kg ha-1)
CK NK PK NP NPK 1/2OMN OM
Fig. 1 Dynamics of wheat (a) and maize (b) yields in the long-term
experiment from 1990 to 2006
70 World J Microbiol Biotechnol (2011) 27:67–74
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PK [1/2 OMN =OM. Diversity expressed by Shannon-
Weiner index decreased in the following order: Control [
NPK C1/2 OMN =PK CNK [OM =NP. Compared
with the control, evenness was lower in NP treatment, but
higher in other treatments.
Correlation between soil properties and AM fungal
parameters
Spearman correlation analysis showed that no significant
correlations were found between AM fungal spore density,
Table 2 AMF species composition and relative abundance in long-term fertilization treatments
AMF Control 1/2OMN OM NP NK PK NPK
Acaulospora (7 species)
A. bireticulata 0.99 a 4.67 b 3.62 b 3.53 b 4.47 b 4.41 b 3.76 b
A. delicata 1.13 – – – – – –
A. laevis 3.25 b 3.29 b 1.83 a 2.39 ab 3.21 b 2.40 ab 3.10 b
A. scrobiculata 0.47 a – – – 0.91 a – –
A. sp.1
*
3.09 b 3.08 b 1.11 a 1.32 a 2.76 b 1.84 ab 2.96 b
A. sp.2
*
2.50 b 0.54 a – 0.45 a 0.54 a 0.81 a 1.50 ab
A. sp.3
*
– – – – – – 0.84
Ambispora (1 species)
Am.lepototicha 3.84 a 4.35 a 3.49 a 3.41a 4.29 a 4.13 a 3.24 a
Archaeospora (1 species)
Ar. trappei 0.91 a 4.25 b 1.43 a 1.70 a – 2.29 ab 4.27 b
Gigaspora (1 species)
Gi. decipiens – – – 0.40 – – –
Glomus (24 species)
G. caledonium 9.22 a 9.83 ab 14.96 b 13.59 ab 14.97 b 13.02 ab 12.68 ab
G. claroideum 24.94 a 26.42 a 32.03 b 35.48 b 23.81 a 22.81 a 20.51 a
G. clarum 2.08 b 0.41 a – – 1.72 b – –
G. constrictum 3.09 b 1.99 ab – 0.43 a 0.51 a 0.86 a 1.25 a
G. deserticola 2.50 b – – – – 0.74 a 2.10 b
G. diaphanum – – – – – – 0.32
G. etunicatum 1.55 b – – – – – 0.15 a
G. fasciculatum 5.14 a 6.49 ab 6.95 ab 6.65 ab 7.61 b 8.75 b 8.25 b
G. geosporum 4.23 a 5.64 a 5.17 a 4.63 a 5.65 a 5.97 a 6.08 a
G. intraradices 4.82 abc 5.86 bc 5.98 bc 2.18 a 4.55 abc 7.90 c 3.15 ab
G. marcocarpum – – – 0.43 – – –
G. melanosporum 2.42 b – – – 0.46 a 0.48 a 1.40 ab
G. mosseae 5.80 a 8.64 b 8.24 ab 7.21 ab 9.80 b 9.47 b 9.05 b
G. pallidum 0.89 – – – – – –
G. pulvinatum – 1.93 a 1.18 a 1.34 a – 0.99 a 1.24 a
G. pustulatum – – – – – 0.53 –
G. reticulatum 2.92 c 0.83 a 1.48 ab 0.49 a – – 2.16 b
G. sp.1
*
3.87 b 3.50 b 2.22 a 2.87 ab 3.82 b 3.21 ab 3.60 b
G. sp.2
*
2.66 a 2.56 a 1.94 a 2.79 a 3.30 a 2.43 a 2.49 a
G. sp.3
*
2.34 b 0.62 a 1.56 ab 1.71 ab 1.51 ab – 0.40 a
G. sp.4
*
0.50 a – – 1.01 a 0.89 a 0.53 a –
G. sp.5
*
0.41 a – – 0.47 a 0.32 a – –
G. versiforme 4.10 a 5.10 ab 4.06 a 4.17 a 4.56ab 4.97 ab 5.50 b
G. vesiculiferum – – 1.61 b – 0.36 a – –
Scutellospora (1 species)
Scu. gregaria 0.34 a – 1.13 b 1.32 b – 1.47 b –
– not found in this treatment. * undescribed species. Data represent the means of four replicates. The difference between values in a row followed
by different letters is significant at P\0.05
World J Microbiol Biotechnol (2011) 27:67–74 71
123

species richness, species diversity and soil properties,
including pH, organic C, total N, available P and available
K.
Discussion
AMF occurs widely in agroecosystems (Douds and Millner
1999). A total of 35 AM fungal species were found in the
present trial, considerably higher than the 20–22 species
from other arable lands in China (Gai et al. 2004; Wang
et al. 2008), and also higher than 9–16 species from other
long-term fertilization field trials (Franke-Snyder et al.
2001; Na Bhadalung et al. 2005). Our results are consistent
with a long-term field trial by Oehl et al. (2004) who found
diverse AM fungal species in arable lands.
Glomus is generally the genus with the greatest number
of species found in intensively managed agriculture
(Hamel et al. 1994; Sieverding 1990). Similarly, in our
trial, Glomus dominated in all the fertilization treatments,
which agrees with most studies carried out in arable lands
of China and elsewhere (Gai et al. 2004; Oehl et al. 2003,
2004,2005; Wang et al. 2008). AMF of this genus survive
and propagate more easily because of the high sporulation
rate and the ability to colonize via pieces of mycelium or
mycorrhizal root fragments (Daniell et al. 2001). These
attributes might explain why Glomus species are more
suitable to the changed soil conditions as influenced by
long-term fertilization in this field trial.
In our present study, although AM fungal species
composition changed in the different fertilization treat-
ments, the 13 dominant species were present in all the
treatments. This indicates that the long-term fertilization
treatments maybe did not play such an important role as
expected in the selection of AMF. In this aspect, our results
are in accordance with Franke-Snyder et al. (2001), who
found 15 consecutive years of farming under one conven-
tional and two low-input farming systems did not cause
many differences among AM fungal communities. The
results also showed that NK treatment had the highest
similarity in AM fungal species with the control (Table 3
and Fig. 2), indicating that this fertilization regime is
efficient in maintaining AM fungal structure. This may be
due to the similar soil properties (such as pH, organic C,
available P) between this treatment and the control.
Furthermore, this also partly reflects the significantly
negative effects of soil available P, organic C and total N
on AM fungal species composition. The effects of fertil-
izers, especially combined fertilizers, on AM fungal spor-
ulation are probably variable and need further precise
research in future.
Previous studies (Martensso and Carlgren 1994; Na
Bhadalung et al. 2005) have shown the negative effects of
mineral fertilizer (P, NP) on AM fungal spore number
result from high P concentrations in plant tissue reducing
soluble carbohydrate supply for root exudates, which AMF
require for energy (Sieverding 1990). However, the
response of AMF to available P is variable (Jasper et al.
Table 3 Jaccard index of similarity between the different treatments
Treatments Control 1/2 OMN OM NP NK PK
1/2 OMN 0.552
OM 0.552 0.714
NP 0.633 0.600 0.739
NK 0.679 0.652 0.520 0.500
PK 0.586 0.682 0.762 0.708 0.560
NPK 0.667 0.708 0.640 0.552 0.593 0.680
Fig. 2 Dendrograms of cluster analysis based on the similarity of AMF
species composition
Table 4 AMF spore density, species richness and diversity in the different fertilization treatments
AMF species Control 1/2 OMN OM NP NK PK NPK
Spore density (average of AMF spore number in 25 g
soil of four filed plot replicates)
269 ab 235 ab 216 a 380 d 295 bc 360 cd 399 d
Species richness (average number of AMF species found
in four field plot replicates)
22.8 c 16.3 ab 15.3 a 16.8 ab 16.0 ab 17.0 ab 18.8 b
Total number of AMF species found at the field sites
(sum of four field plot replicates)
27 18 18 22 20 19 23
Shannon-Weiner index (H’) 2.77 d 2.51 bc 2.29 a 2.29 a 2.48 b 2.51 bc 2.62 c
Evenness (E) 0.95 0.99 0.97 0.94 0.99 0.97 0.97
The difference between values in a row followed by different letters is significant at P\0.05
72 World J Microbiol Biotechnol (2011) 27:67–74
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1989), and the application of P can influence spore pro-
duction either positively or negatively (Neumann and
George 2004; Subramanian et al. 2006). Our present results
showed that AM fungal spore density was not decreased
but instead increased by NP, PK, NPK containing high P,
and not changed by OM, 1/2 OMN and NK. This may be
due to the complicated interactions among the nutrients,
soil conditions, plant growth status and AMF themselves.
Firstly, other nutrients such as N, K may offset (Guttay
1983; Hayman and Mosse 1972; Hepper 1983) or enhance
(Guttay and Dandurand 1989) the negative effects of high
available P on AM fungi. Secondly, fertilization causes
changes in soil properties (Table 1), which may influence
mycorrhiza formation directly or interact with other factors
(Neumann and George 2004). Thirdly, plant growth dif-
fered in different fertilization treatments (Fig. 1), which
must have influenced the C supply to AMF associated with
them. Additionally, the different sensitivity of AMF to
available P and other nutrients may also affect spore den-
sity under long-term fertilization. Obviously, long-term
fertilization effects on AM fungal spore density may be
diverse and vary with many factors.
In contrast with the conclusions that AM fungal species
richness and diversity are generally negatively influenced
by soluble mineral fertilizers, but positively influenced by
organic manure (Gryndler et al. 2006;Ma
¨der et al. 2000;
Oehl et al. 2003,2004), our results showed that both spe-
cies richness and species diversity were significantly
decreased by all the fertilizer treatments (Table 4), espe-
cially by organic manure. AM fungal sporulation would be
reduced under any adverse soil conditions (Entry et al.
2002), including extremely low as well as high soil fertil-
ity, and nutrient supply imbalance, especially high or low
levels of N and P, extreme pH, etc. On the other hand,
different AM fungal species may show different responses
to fertilization and other soil conditions. Long-term fertil-
ization in this trial greatly increased soil fertility and
changed soil pH, which may play an important role in the
selection of AM fungal species (Johnson 1993). Thus, the
populations of sensitive AMF to soil fertility and pH might
have been decreased or eliminated, accompanying
increased relative abundance of the tolerant species, and
thus leading to decreased species richness, species even-
ness and species diversity.
Besides, overuse of organic manure high in nutrients
may impact negatively on AMF (Gosling et al. 2006). In
this study, organic manure changed some soil properties
more drastically than did mineral fertilizers, so it is easily
understandable that it would have more negative effects on
AM fungal species richness and diversity.
In conclusion, both mineral fertilizers and organic
manure significantly decreased AM species richness and
species diversity. However, fertilization with mineral
fertilizers is necessary for optimum crop yields in China.
We also found fertilization with NPK and 1/2 OMN pro-
duced the highest crop yields (Fig. 1). Considering both the
production of crops and the protection of AM fungal
diversity, balanced fertilization with NPK and 1/2 OMN
may be feasible in this agricultural region. Additionally, as
both wheat and maize are mycorrhizal plants, the role of
AMF in crop production needs more attention and the useful
AM species need to be exploited and manipulated in future.
Acknowledgments We thank Jia Bao Zhang, Lin Yun Zhou, Qi Ao
Jiang and Jian Liu, of the Fengqiu Agro-Ecological Experimental
Station, Institute of Soil Science, Chinese Academy of Sciences, for
their excellent field management and kind support on soil sample
collection. This work is supported by the Knowledge Innovation
Program of the Chinese Academy of Sciences (Grant No. ISSA-
SIP0703, Kzcx2-yw-408, Kscx1-yw-09-05) and National Basic
Research Program of China (Project no. 2005CB121108).
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