Environ Sci Pollut Res (2014) 21:3592–3603
DOI 10.1007/s11356-013-2360-z
RESEARCH ARTICLE
Contribution of arbuscular mycorrhizal fungi
to the development of maize (Zea mays L.) grown
in three types of coal mine spoils
Wei Guo & Renxin Zhao & Ruiying Fu & Na Bi &
Lixin Wang & Wenjing Zhao & Jiangyuan Guo &
Jun Zhang
Received: 24 June 2013 / Accepted: 11 November 2013 / Published online: 24 November 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract Coal mine spoils are usually unfavorable for plant
growth and have different properties according to dumping
years, weathering degree, and the occurrence of spontaneous
combustion. The establishment of plant cover in mine spoils
can be facilitated by arbuscular mycorrhizal fungi (AMF). A
greenhouse pot experiment was conducted to evaluate the
importance of AMF in plant adaptation to different mine
spoils and the potential role of AMF for revegetation prac-
tices. We investigated the effects of Glomus aggregatum ,
Rhizophagus intraradices (syn. Glomus intraradices ), and
Funneliformis mosseae (syn. Glomus mosseae ) on the
growth, nutritional status, and metal uptake of maize (Zea
mays L.) grown in recent discharged (S1), weathered (S2),
and spontaneous combusted (S3) coal mine spoils. Symbiotic
associations were successfully established between AMF and
maize in three substrates. Mycorrhizal colonization effectively
promoted plant growth by significantly increasing the uptake
of nitrogen (N), phosphorus (P), and potassium (K), adjusting
C:N:P stoichiometry and alleviating toxic effects of heavy
metals. G. aggregatum , R. intraradices , and F. mosseae
exhibited different mycorrhizal effects in response to mine
spoil types. F. mosseae was the most effective in the devel-
opment of maize in S1 and may be the most appropriate for
revegetation of this substrate, while R. intraradices played the
Responsible editor: Hailong Wang
W. Guo (*) : R. Zhao : R. Fu : N. Bi : L. Wang : W. Zhao : J. Guo
College of Environmental and Resource Science, Inner Mongolia
University, Hohhot 010021, Inner Mongolia Autonomous Region,
People’s Republic of China
e-mail: guowei-1976-z@hotmail.com
J. Zhang
College of Chemistry and Chemical Engineering, Inner Mongolia
University, Hohhot 010021, Inner Mongolia Autonomous Region,
People’s Republic of China
most beneficial role in S2 and S3. Our results suggest that
inoculation with AMF can enhance plant adaptation to differ-
ent types of coal mine spoils and play a positive role in the
revegetation of coal mine spoil banks.
Keywords Arbuscular mycorrhizal fungi . Coal mine spoils .
Nutrient uptake . C:N:P stoichiometry . Heavy metal .
Revegetation
Introduction
Coal mine spoil, the previous overburden of coal seams, is an
inevitable by-product in the mining process (Sun et al. 2009).
In China, it is estimated that the annual production of coal
mine spoil is about 150 million tons and there are about 7.0
billion tons of coal mine spoil stockpiled at 1,700 waste
dumps which occupied 15,000 hectares lands (Bian et al.
2009). Inner Mongolia autonomous region is China's largest
coal producer with production of raw coal of 1.08 billion tons
in 2012. The extensive coal mining in Inner Mongolia has
resulted in the formation of vast areas of mine spoil banks.
Given that the mining activities of Inner Mongolia are located
on temperate arid and semiarid grassland ecosystem which is
ecologically sensitive and fragile, coal mine spoils have a
large impact on the eco-environment of the mining area and
its surroundings (Li et al. 2011; Chaubey et al. 2012).
Coal mine spoils are physically, chemically, nutritionally,
and microbiologically impoverished habitats inhibiting the
establishment and growth of plant species (Singh 2012). The
vegetation development and succession process are slow on
mine spoils under natural conditions (Püschel et al. 2007a, b).
Therefore, it is urgent to develop sustainable and economical-
ly efficient techniques for reclamation of coal mine spoils.
Application of beneficial microorganisms, which would
Environ Sci Pollut Res (2014) 21:3592–3603 3593

improve soil properties and support plant growth on reclaimed importance of mycorrhizal associations in plant adaptation to
mine spoil banks, has been suggested as an alternative suitable different types of coal mine spoils and the potential applica-
approach (Gryndler et al. 2008; Rydlová et al. 2011). The tion of AMF for revegetation practices.
long-term objective of the revegetation of coal mine spoil
banks can be realistically achieved by using this method as a
basis for landscaping, stabilization, and pollution control Materials and methods
(Juwarkar and Jambhulkar 2008).
Arbuscular mycorrhizal fungi (AMF) are key components Growth substrates
of soil microorganisms and can form mutualistic symbiotic
associations with the roots of 80 % of all terrestrial plant The coal mine spoils and topsoil were collected from the
species (Smith and Read 2008). Mycorrhizal symbiosis can Shiguai coal mining area (41°39′00.1″ N, 110°18′15.9″ E)
promote plant establishment by enhancing nutrients uptake of Baotou in the Inner Mongolia Autonomous Region, China.
(Willis et al. 2013), improve soil structure (Hallett et al. 2009), The Shiguai mine has been exploited for about 300 years and
and maintain the structure and function of plant community coal mine spoil banks have been heaping with poor supportive
and the stability of ecosystem (Cameron 2010). Furthermore, and nutritive capacity. At present, there are seven large spoil
AMF can alleviate plant stresses caused by biotic and abiotic banks with more than 21 million tons of mine spoils. Accord-
factors (Miransari 2010). These beneficial functions of AMF ing to the dumping years, weathering degree, and combustion
could be important to optimize revegetation of degraded eco- situation, three types of coal mine spoils were selected for the
systems and contaminated environments, including coal mine study: recent discharged mine spoil (S1), 1-year old, with no
spoil banks (Püschel et al. 2011). It has been reported that the spontaneous combustion; weathered mine spoil (S2), 10-year
composition of AMF species is rich and typical AMF infec- old, with no spontaneous combustion; and spontaneous
tion in roots of all the examined plant species was observed on combusted mine spoil (S3), 40-year old. No reclamation effort
natural reclaimed coal mine spoil (Mehrotra 1998). The pres- was applied in these sites and the vegetation was developed by
ence of AMF is one of the key factors that determine the natural colonization.
progress in plant community structure within the succession The mine spoils and topsoil were air-dried and sieved
on spoil banks (Püschel et al. 2007a). It has been believed that through a 2-mm sieving for analysis. The basic physical and
mycorrhizal technology can solve many problems in the re- chemical properties of the substrates are shown in Table 1.
vegetation process of coal mine spoils using physical and Organic matter was determined using the Tiurin's method
chemical methods. For example, inoculation with AMF can (Mebius 1960). Total nitrogen (N) was determined by the
reduce the depth of topsoil (Bi et al. 2003) and compensate for Kjeldahl method (Kjeldahl 1883). Total phosphorus (P) con-
reduced doses of organic amendments (Gryndler et al. 2008), centration was determined following the molybdenum blue
thus significantly decrease costs related to the reclamation of method (Allen et al. 1974). Available N was determined by the
mine spoils (Püschel et al. 2008a).
In Inner Mongolia, weathering and spontaneous combus- Table 1 Physical and chemical properties of topsoil and three types of
tion easily occur in coal mine spoils due to the dry climate, the coal mine spoils
year-round strong winds, and the high temperatures during the
daytime in summer. The dumping years, weathering, and Substrate Topsoil S1 S2 S3
spontaneous combustion of mine spoils are the main factors pH 6.97 5.99 7.21 7.15
affecting physical, chemical, and the mycorrhizal function- Organic matter (%) 0.42 19.72 3.95 0.99
related properties and have caused different types of coal mine Total N (%) 0.031 0.40 0.14 0.042
spoils dumped in mining areas (Johnson 2003; Baldrian et al. Total P (%) 0.045 0.028 0.053 0.075
2008; Li et al. 2011). However, in previous studies, mycor- Total K (%) 0.36 1.47 0.89 1.22
rhizal technology have been mainly used in revegetation of
Available N (mg kg−1) 53.3 25.7 13.5 47.8
coal mine spoils of a single type (Püschel et al. 2011; Rydlová
Available P (mg kg−1) 13.74 12.56 6.02 8.09
et al. 2011) or at different stages of succession (Enkhtuya et al.
Available K (mg kg−1) 71.53 185.6 128.4 146.2
2000; Ekka and Behera 2010). Little attention has been paid to
Fe (mg g−1) 36.46 20.23 29.87 44.74
study the effects of AMF on the revegetation of different types
Cu (mg kg−1) 15.45 33.66 33.22 73.12
of coal mine spoils in terms of the difference of dumping
Mn (mg kg−1) 544.5 279.0 311.5 269.4
years, the degree of weathering, and the occurrence of spon-
Zn (mg kg−1) 44.51 147.8 122.2 73.12
taneous combustion. In the present greenhouse study, we
evaluated the effects of AMF on plant growth, mineral nutri- Data are means of three replicates
tion, and metal uptake by maize (Zea mays L.) grown in three S1 recent discharged coal mine spoil, S2 weathered coal mine spoil, S3
types of coal mine spoils. The objective was to investigate the spontaneous combusted coal mine spoil
3594
method described by Subbainh and Asija (1956). Available P
was extracted with 0.5 mol L −1 sodium bicarbonate
(NaHCO 3 ) and determined colorimetrically by the
vanadomolybdate method following the methods described
by Olsen et al. (1954) and Murphy and Riley (1962). Avail-
able potassium (K) was extracted with 1.0 mol L−1 ammoni-
um acetate (CH3COONH4) and analyzed by the flame pho-
tometry (Wanasuria et al. 1981). The concentrations of total K
and heavy metals in the substrates were determined by
inductively coupled plasma–optical emission spectrosco-
py (ICP-OES, Optima 7000 DV, PerkinElmer, USA)
following nitric acid–hydrochloric acid–perchloric acid
(HNO3-HCl-HClO4) digestion. Prior to the pot experi-
ment, the three substrates and topsoil were autoclaved-
sterilized at 121 °C for 2 h to eliminate indigenous
AMF propagules and other microorganisms.
Biological materials
The three AMF isolates used in this study were Glomus
a gg reg a t um ( S ch e nc k & S m i t h) (B G C H K 02 D ,
1511C0001BGCAM0043), Rhizophagus intraradices (syn.
Glomus intraradices , Schenck & Smith) (BGC BJ09,
1511C0001BGCAM0042), and Funneliformis mosseae
(syn. Glomus mosseae , Nicolson & Gerd.) (BGC XJ01,
1511C0001BGCAM0016). The fungal species names are af-
ter Schüßler and Walker (2010). They were supplied by the
Institute of Plant Nutrition and Resources, Beijing Academy
of Agriculture and Forestry Sciences. The AMF isolates were
propagated on maize (Z. mays L.) and white clover
(Trifolium repens L.) in a soil–sand mixture (1:1 w /w )
for 3 months prior to the experiment. The resulting
inoculum consisted of cultivation substrate containing
spores, extraradical mycelium, colonized maize, and
white clover root fragments. The inoculum of G.
aggregatum , R. intraradices , and F. mosseae contained
79, 68, and 36 spores/g soil, respectively.
Annual gramineous plant maize was used as model plant in
the experiment. Maize seeds originated from Inner Mongolia
were purchased from the Inner Mongolia Academy of Agri-
culture and Animal Husbandry, Hohhot, China. After being
surface-sterilized in 10 % (v/v) hydrogen peroxide (H2O2) for
10 min, the seeds were subsequently washed with deionized
water and then pre-germinated on moist filter paper at 25 °C
for 36–48 h until the radicles appeared. They were selected for
uniformity before sowing.
Experimental design
The pot experiment was conducted with a 3×4 factorial
combination, which comprised of three substrates (S1, S2,
and S3) and four mycorrhizal inoculations ( G. aggregatum,
R. intraradices , F. mosseae , and non-inoculated). Each
Environ Sci Pollut Res (2014) 21:3592–3603
treatment was replicated six times resulting in a total of 72
pots randomly arranged. Plastic pots (18 cm calibre diameter×
13 cm height×12 cm bottom diameter) were used in the
experiment. Three types of mine spoils (S1, S2, and S3) were
placed in the bottom of the pots to give a depth of 7 cm, with
corresponding weights of 1,250, 1,400, and 1,000 g, respec-
tively. These were covered with 1,000 g of sterilized
(autoclaved at 121 °C for 2 h) soil, giving a soil depth of
5 cm, and a total depth of growth substrate of 12 cm, corre-
sponding to a total weight of growth substrate of 2,250, 2,400,
and 2,000 g per pot for S1, S2, and S3, respectively. Mycor-
rhizal treatments received 50 g of fresh inoculum, while non-
mycorrhizal plants received 50 g of sterilized mixed inoculum
of three AMF together with 10 mL of an aqueous filtrate
(0.25 μm pore size) of unsterilized inoculum (1:2 w/v soil/
water) to provide a similar microflora. The experiment was
carried out in a greenhouse, and the seedlings were thinned to
three after 10 days. The plants were cultivated for 2 months
from 18 June to 19 August 2011 under natural light conditions
with no supplementary illumination. During the period of
plants growth, the day temperature ranged from 20 to 35 °C
and the night temperature from 10 to 20 °C. Deionized water
was added as required to maintain the moisture content at
80 % of the water holding capacity as determined by regular
weighing.
Plant sampling and analysis
At harvest, the plant shoots and roots were harvested sepa-
rately. Root samples were carefully washed with tap water
followed by deionized water to remove adherent soil and sand
particles. The shoot and root dry weights were weighed after
oven drying to a constant weight at 70 °C for 48 h. Oven-dried
subsamples were ground, and the C and N concentrations
were analyzed with an elemental analyzer (Vario EL III,
CHNOS Elemental Analyzer, Elementar Co., Germany).
Samples of 0.3–0.5 g were digested by 5 mL of high-purity
HNO3 at 120 °C using an open block digesting system
(AIM600, Aim Lab Pty Ltd, Australia). The acid digests were
diluted with ultra-pure water and made up to 50 mL. The
concentrations of P, K, iron (Fe), zinc (Zn), copper (Cu), and
manganese (Mn) were determined with an ICP-OES (Optima
7000 DV, PerkinElmer, USA).
A random subsample of 0.7–1.0 g fresh roots in each pot
was collected, cut into 1 cm pieces, and stored in 50 % ethanol
for measuring the root mycorrhizal colonization rate. The root
samples were cleared in 10 % (w /v ) potassium hydrox-
ide (KOH), stained with 0.05 % (w /v ) trypan blue in
lactoglycerol (Phillips and Hayman 1970), and micro-
scopically examined for AM colonization by determin-
ing the percentage of root segments containing
arbuscules, vesicles, and hyphae using gridline intersect
method (Giovanetti and Mosse 1980).
Environ Sci Pollut Res (2014) 21:3592–3603 3595

Statistical analysis In S2, the colonization rates of R. intraradices and F. mosseae

Data are the mean values based on six replicates±standard
error (SE) per treatment. All results were subjected to two-way
analysis of variance (ANOVA) using SPSS 17.0 software.
Prior to ANOVA, all data were checked for normality. Data
showing normal distribution was analyzed by the Duncan
multiple range test, whereas data with non-normal distribution
were arcsine transformed (mycorrhizal colonization) and an-
alyzed by nonparametric Kruskall–Wallis test. To detect the
statistical significance of the differences between means for a
substrate, Duncan multiple range test was performed at a
significance level of P <0.05.
Results
Mycorrhizal colonization
No AMF colonization was observed in the roots of maize in
the uninoculated treatments of three types of coal mine spoils
(Table 2). Mycorrhizal colonization of maize roots was sig-
nificantly affected by substrate (Table 2, P <0.05) and inocu-
lation (Table 2, P <0.001). In case of S1, the roots were all
highly colonized by three AMF species, but there was no
significant difference among inoculation treatments (Table 2).
were significantly higher than G. aggregatum (Table 2,
P < 0.05). In S3, the roots of maize inoculated with
R. intraradices had the highest level of mycorrhizal coloni-
zation reaching 90 %, which was significantly higher than
those inoculated with G. aggregatum and F. mosseae (Table 2,
P <0.05).
Plant dry weight
Shoot, root, and total dry weights of maize were significantly
affected by substrate (Table 2, P <0.001) and inoculation
(Table 2, P <0.001). Shoot dry weight was also significantly
affected by the interaction of the two factors (Table 2, P <
0.05). In S1, G. aggregatum, R. intraradices, and F. mosseae
significantly promoted the growth of maize compared to the
uninoculated treatments (Table 2, P <0.05), and total dry
weights were increased by 83, 55, and 106 %, respectively.
In S2, inoculation with R. intraradices and F. mosseae sig-
nificantly increased shoot dry weight of maize (Table 2, P <
0.05), while G. aggregatum had no significant effect com-
pared to the uninoculated treatments. Maximum shoot, root,
and total dry weights were achieved with R. intraradices
inoculation, which were increased by 73, 91, and 76 %, re-
spectively. Statistically, G. aggregatum and F. mosseae did
not significantly increase root and total dry weights (Table 2).

Table 2 Effect of inoculation with AMF on mycorrhizal colonization and dry weights of maize grown in three types of coal mine spoils

Substrate Inoculation Mycorrhizal colonization (%) Shoot dry weight Root dry weight Total dry weight
(g pot−1) (g pot−1) (g pot−1)

S1 Uninoculated 0.00±0.00 b 4.51±0.29 c 1.29±0.23 c 5.80±0.14 c

G. aggregatum 71.19±3.96 a 8.32±0.48 a 2.32±0.26 ab 10.63±0.70 a
R. intraradices 79.15±4.38 a 6.94±0.50 b 2.07±0.18 b 9.01±0.59 b
F. mosseae 80.27±9.57 a 9.09±0.36 a 2.87±0.14 a 11.96±0.47 a
S2 Uninoculated 0.00±0.00 c 5.19±0.33 b 1.15±0.19 b 6.34±0.52 b
G. aggregatum 45.52±4.97 b 7.17±1.43 ab 1.74±0.46 ab 8.91±1.89 ab
R. intraradices 72.42±3.78 a 8.99±0.22 a 2.20±0.17 a 11.19±0.38 a
F. mosseae 78.23±6.01 a 8.48±1.05 a 1.81±0.37 ab 10.29±1.42 ab
S3 Uninoculated 0.00±0.00 c 3.05±0.33 c 0.86±0.22 b 3.91±0.54 b
G. aggregatum 55.35±3.98 b 4.10±0.32 bc 1.07±0.21 ab 5.17±0.50 ab
R. intraradices 90.60±2.63 a 5.46±0.33 a 1.52±0.10 a 6.98±0.41 a
F. mosseae 59.84±5.19 b 4.37±0.54 ab 1.19±0.27 ab 5.56±0.79 ab
Significance
Substrate (S) * *** *** ***
Inoculation (I) *** *** *** ***
S ×I – * NS NS

Data are means of six replicates±SE. Values within columns in the same substrate followed by the same letter are not significantly different according to
Duncan multiple range test at the P <0.05 level (with the exception of mycorrhizal colonization, where nonparametric Kruskal–Wallis test was
performed)
NS nonsignificant effect (effects of factors according to two-way ANOVA)
*P <0.05; **P <0.01; ***P <0.001 (effects of factors according to two-way ANOVA)
3596
In S3, R. intraradices played a most significant role in pro-
moting the growth of maize and total dry weight was in-
creased by about 79 % (Table 2, P <0.05). However, the other
inoculation treatments did not significantly improve the
growth of maize, except for shoot dry weight with F. mosseae
inoculation (Table 2).
Plant nutrient uptake
Contents of C, N, P, and K in shoots and roots of maize were
significantly affected by substrate (Table 3, P <0.001), inocu-
lation (Table 3, P <0.001; excluding the root N contents), and
the interaction of the two factors (Table 3, excluding the root C
and P contents). In S1, inoculation with AMF significantly
increased contents of C, N, P, and K in shoots and roots
compared to the control treatments (Table 3, P <0.05). The
C, P, and K contents in shoots with G. aggregatum and F.
mosseae were significantly higher than those with R.
intraradices (Table 3, P <0.05). F. mosseae had the most
significant effect in promoting the nutrient uptake in roots.
In S2, R. intraradices and F. mosseae significantly improved
the C, N, and K contents in shoots (Table 3, P <0.05). All
inoculation treatments significantly increased the shoot P
contents and root P and K contents (Table 3, P <0.05). Inoc-
ulation with AMF had no significant effect on the root N
contents (Table 3). In S3, inoculation with AMF significantly
increased contents of N, P, and K in shoots compared to the
control treatments (Table 3, P < 0.05). Furthermore, R.
intraradices significantly promoted N, P, and K uptake by
the roots (Table 3, P <0.05), while the other two inoculation
treatments had no significant effect (Table 3).
Plant C:N:P stoichiometry
The C/P and N/P ratios in shoots and roots of maize were
significantly affected by substrate (Table 4, P <0.05), inocula-
tion (Table 4, P <0.001), and the interaction of the two factors
(Table 4, excluding the shoot N/P ratios). The shoot and root C/
N ratios were significantly affected by substrate (Table 4, P <
0.001) and the interaction of substrate with inoculation (Table 4,
excluding the shoot C/N ratios). In S1, inoculation with AMF
had no effect on the shoot and root C/N ratios (Table 4), but
significantly decreased the shoot and root C/P and N/P ratios
except for the root N/P ratios with R. intraradices (Table 4, P <
0.05). In S2, the shoot and root C/P and N/P ratios were
significantly decreased by the inoculation with AMF (Table 4,
P <0.05). G. aggregatum and F. mosseae had no effect on the
shoot and root C/N ratios, while R. intraradices significantly
increased those in S2 (Table 4, P <0.05). In S3, the shoot C/N
ratios with G. aggregatum were significantly lower than those
with uninoculated treatments (Table 4, P <0.05), while
R. intraradices and F. mosseae had no effect on the
shoot and root C/N ratios. Inoculation with AMF also
Environ Sci Pollut Res (2014) 21:3592–3603
significantly decreased the shoot and root C/P and N/P
ratios (Table 4, P <0.05).
Concentrations of heavy metals
The shoot Cu and Zn concentrations and root Mn concentra-
tion were significantly affected by substrate, inoculation and
the interaction of the two factors (Table 5). The root Zn
concentration was significantly affected by substrate and in-
oculation (Table 5). In addition, concentrations of shoot Fe
and Mn, root Cu, and root Fe were significantly affected by
substrate, inoculation, and interaction of these factors, respec-
tively (Table 5). In S1, the shoot Cu concentrations with G.
aggregatum and F. mosseae were significantly lower than
those with R. intraradices and uninoculated treatments
(Table 5, P <0.05). R. intraradices also significantly in-
creased the shoot Zn concentrations (Table 5, P <0.05). Col-
onization by AMF significantly increased the root Cu concen-
trations and decreased Zn concentrations compared to the
uninoculated treatments (Table 5, P <0.05). G. aggregatum
significantly increased the root Mn concentrations but R.
intraradices and F. mosseae had no significant effect
(Table 5). Additionally, inoculation with AMF had no signif-
icant impact on the shoot Fe and Mn concentrations and the
root Fe concentrations (Table 5). In S2, the application of
AMF only affected the metal concentrations in the root. Com-
pared to the uninoculated treatments, G. aggregatum signifi-
cantly increased the root Cu concentration and decreased Mn
concentration (Table 5, P <0.05), and R. intraradices signif-
icantly decreased the root Mn and Zn concentrations (Table 5,
P <0.05). In S3, the shoot Zn concentration and the root Cu
concentration were significantly increased by three AMF spe-
cies (Table 5, P <0.05). G. aggregatum also significantly
increased the shoot Mn concentration and the root Fe and
Mn concentrations (Table 5, P <0.05). F. mosseae significant-
ly increased the shoot Mn concentration (Table 5, P <0.05),
while R. intraradices significantly decreased the shoot Cu
concentration (Table 5, P <0.05). Moreover, inoculation with
AMF had no effect on the shoot Fe concentration and root Zn
concentration (Table 5).
Discussion
In the present study, differences in physical and chemical
properties of three types of coal mine spoils have caused
different plant growth conditions, which can also affect
AMF growth and colonization process on plants. Under
greenhouse conditions, our study showed that symbiotic as-
sociations were successfully established between three AMF
isolates and maize grown in three substrates. However, the
mycorrhizal colonization was significantly affected by sub-
strate and inoculation. Similarly, Rydlová et al. (2011)
Table 3 Effect of inoculation with AMF on shoot and root C, N, P, and K contents of maize grown in three types of coal mine spoils

Substrate Inoculation Shoot Root

C (g pot−1) N (mg pot−1) P (mg pot−1) K (mg pot−1) C (g pot−1) N (mg pot−1) P (mg pot−1) K (mg pot−1)
Environ Sci Pollut Res (2014) 21:3592–3603

S1 Uninoculated 1.86±0.13 c 79.34±8.49 b 4.23±0.21 c 161.1±13.0 c 0.54±0.10 c 13.86±1.19 c 0.81±0.16 c 20.77±1.34 b

G. aggregatum 3.53±0.21 a 118.8±4.7 a 11.55±0.13 a 316.6±15.3 a 0.97±0.11 ab 21.69±1.74 b 1.85±0.16 b 39.09±3.54 a
R. intraradices 2.93±0.21 b 113.1±5.3 a 9.16±0.79 b 265.4±14.3 b 0.87±0.08 b 25.39±1.27 b 1.79±0.19 b 36.41±4.03 a
F. mosseae 3.90±0.15 a 117.9±4.2 a 10.64±0.42 a 332.2±7.3 a 1.22±0.06 a 31.89±1.24 a 2.41±0.18 a 46.26±4.14 a
S2 Uninoculated 2.07±0.14 b 110.1±3.7 b 6.63±1.29 c 202.0±12.8 b 0.48±0.07 b 16.62±2.49 a 0.77±0.11 b 15.29±3.50 b
G. aggregatum 3.06±0.62 ab 110.4±8.0 b 10.88±1.09 b 251.1±44.5 ab 0.73±0.19 ab 20.51±3.41 a 1.94±0.47 a 35.45±2.72 a
R. intraradices 3.82±0.11 a 130.3±3.7 a 13.76±0.37 a 336.5±8.7 a 0.93±0.07 a 20.98±1.70 a 2.11±0.13 a 44.55±4.13 a
F. mosseae 3.62±0.46 a 142.9±5.1 a 12.89±0.42 ab 323.1±47.9 a 0.75±0.16 ab 20.03±3.41 a 1.71±0.30 a 34.79±5.85 a
S3 Uninoculated 1.22±0.14 c 67.91±4.12 c 1.96±0.17 b 116.5±12.9 b 0.35±0.08 a 12.47±3.08 b 0.45±0.13 b 9.49±3.68 b
G. aggregatum 1.63±0.22 bc 111.7±6.2 b 5.44±0.39 a 177.7±19.0 a 0.43±0.08 a 17.31±3.32 b 0.75±0.15 b 13.18±3.52 ab
R. intraradices 2.22±0.15 a 132.8±3.0 a 6.25±0.18 a 215.5±8.0 a 0.60±0.04 a 28.32±2.06 a 1.29±0.10 a 24.87±2.14 a
F. mosseae 1.77±0.23 ab 110.3±5.6 b 5.68±0.52 a 187.1±20.1 a 0.49±0.11 a 18.20±3.36 b 0.88±0.17 ab 17.39±5.46 ab
Significance
Substrate (S) *** *** *** *** *** NS *** ***
Inoculation (I) *** *** *** *** *** *** *** ***
S ×I * *** ** * NS * NS *

Data are means of six replicates±SE. Values within columns in the same substrate followed by the same letter are not significantly different according to Duncan multiple range test at the P <0.05 level
NS nonsignificant effect (effects of factors according to two-way ANOVA)
*P <0.05; **P <0.01; ***P <0.001 (effects of factors according to two-way ANOVA)
3597
3598 Environ Sci Pollut Res (2014) 21:3592–3603

Table 4 Effect of inoculation with AMF on shoot and root C/N, C/P, and N/P ratios of maize grown in three types of coal mine spoils

Substrate Inoculation Shoot Root

C/N C/P N/P C/N C/P N/P

S1 Uninoculated 25.14±5.25 a 441.8±30.2 a 19.10±2.73 a 39.40±3.71 ab 672.6±11.8 a 17.97±1.58 a

G. aggregatum 29.78±1.89 a 305.6±19.3 b 10.27±0.31 b 44.28±2.03 a 519.6±19.6 b 11.78±0.47 b
R. intraradices 26.14±2.42 a 321.8±12.9 b 12.76±1.65 b 34.18±2.78 b 488.6±34.9 b 14.65±1.82 ab
F mosseae 33.08±0.68 a 368.4±20.5 b 11.14±0.60 b 38.24±1.12 ab 510.4±22.6 b 13.35±0.45 b
S2 Uninoculated 18.81±1.11 b 388.9±32.0 a 19.17±4.61 a 28.81±1.28 b 624.9±44.8 a 21.85±2.00 a
G. aggregatum 27.15±4.05 ab 273.6±33.2 b 10.39±1.11 b 34.17±4.95 b 368.5±25.8 b 11.35±1.41 b
R. intraradices 29.37±0.89 a 278.9±14.2 b 9.49±0.29 b 44.27±0.84 a 439.5±10.7 b 9.93±0.21 b
F. mosseae 25.26±2.93 ab 279.4±31.0 b 11.08±0.10 b 36.70±1.60 ab 431.5±22.8 b 11.76±0.39 b
S3 Uninoculated 17.82±0.98 a 620.9±34.8 a 34.95±1.63 a 28.39±0.50 a 821.1±37.9 a 28.90±1.06 a
G. aggregatum 14.59±0.52 b 301.1±16.7 b 20.63±0.82 b 25.30±2.23 a 587.0±25.0 b 23.48±1.13 b
R. intraradices 16.64±0.76 ab 353.2±15.2 b 21.24±0.19 b 21.39±1.31 a 468.5±11.1 c 22.09±1.14 b
F. mosseae 15.83±1.28 ab 308.1±11.6 b 19.67±0.91 b 26.80±3.51 a 549.9±30.1 bc 21.04±1.33 b
Significance
Substrate (S) *** *** *** *** *** ***
Inoculation (I) NS *** *** NS *** ***
S ×I NS *** NS ** * *

Data are means of six replicates±SE. Values within columns in the same substrate followed by the same letter are not significantly different according to
Duncan multiple range test at the P <0.05 level
NS nonsignificant effect (effects of factors according to two-way ANOVA)
*P <0.05; **P <0.01; ***P <0.001 (effects of factors according to two-way ANOVA)

observed that the effects of mine spoil substrate on mycorrhi-
zal colonization varied among AMF species. In combination
with other studies (Malcová et al. 2001; Gryndler et al. 2008),
the existence of a certain degree of selectivity between AMF
species and mine spoil substrates may result in different root
colonization rates of host plants.
Results from the present study demonstrated that inocula-
tion with AMF improved the growth of maize grown in three
types of coal mine spoils. Positive effects of AMF on plant
biomass similar to our study were also reported by other
researchers (Taheri and Bever 2011; Qian et al. 2012), whose
results indicated that AMF decreased the biotic and abiotic
stresses exerted by coal mine wastes. Kim et al. (2010) hold
that the increase in plant dry weight was attributed to the
stimulatory effect of spore germination and establishment of
AMF, contributing to the improved nutrition and development
of AMF–plant–soil system (Jin et al. 2005). Nevertheless,
differential behaviors of three AMF isolates in response to
maize growth were observed. Study of Orłowska et al. (2005)
demonstrated that influence of fungal strains on plant devel-
opment in mine wastes varied according to their ecotypes. The
different response of AMF to plant growth may result from
different morphological and molecular features and the adap-
tation to coal mine spoils. Moreover, the effect of AMF on
plant growth was not necessarily be correlated with the level
of colonization by the AMF. Enkhtuya et al. (2000, 2005)
found that the mycorrhizal growth response of plants was very
low even when the development of all AMF was successful in
coal mine spoils. However, in accordance with many studies
(Püschel et al. 2008a; Redon et al. 2009), plant growth is
positively correlated with the level of AMF colonization.
The highest mycorrhizal colonization rates and plant dry
weights were simultaneously observed in our study with the
inoculation of R. intraradices in spontaneous combusted coal
mine spoil. This can probably be attributed to a cost–benefit
balance of mycorrhizal symbiosis by effectively enhancing
the nutrient uptake and adjusting the nutrient distribution ratio
in plants (Koide and Elliott 1989; Enkhtuya et al. 2000).
Moreover, the results indicated that effects of the AMF on
plant growth varied according to the coal mine substrates and
the types of substrates were crucial factors determining the
fitness and growth of maize. The recent discharged and
weathered mine spoils had relatively high concentrations
of nutrients readily available for plant uptake which
could facilitate maize growth and establishment. Many
studies also indicated that the differences in plant
growth had a great relationship with the nutrient con-
tents in the growth substrates (Gryndler et al. 2008;
Rydlová et al. 2008; Wu et al. 2009). In general, the
beneficial effects of AMF on plant growth may, to some
degree, rely upon AMF species, substrates, and other
environmental factors (Püschel et al. 2007b).
Table 5 Effect of inoculation with AMF on metal concentrations in the shoots and roots of maize grown in three types of coal mine spoils

Substrate Inoculation Shoot Root

Cu (mg kg−1) Fe (mg kg−1) Mn (mg kg−1) Zn (mg kg−1) Cu (mg kg−1) Fe (mg g−1) Mn (mg kg−1) Zn (mg kg−1)
Environ Sci Pollut Res (2014) 21:3592–3603

S1 Uninoculated 12.34±0.99 a 82.50±6.77 a 180.3±21.1 a 41.56±10.35 b 25.78±0.51 c 0.96±0.09 a 191.7±3.2 b 29.80±2.39a

G. aggregatum 8.72±0.52 b 84.33±4.63 a 220.3±8.4 a 40.17±6.31 b 50.73±8.59 ab 1.00±0.18 a 255.8±13.5 a 20.11±2.77b
R. intraradices 11.26±0.52 a 87.61±6.91 a 219.9±20.7 a 70.53±8.37 a 67.09±4.38 a 0.81±0.11 a 229.8±23.6 ab 17.81±1.65b
F. mosseae 7.82±0.46 b 82.69±6.54 a 215.7±13.7 a 47.96±3.82 ab 45.90±5.36 b 1.18±0.16 a 219.9±20.2 ab 16.81±1.87b
S2 Uninoculated 10.59±0.46 ab 115.8±2.3 a 120.4±7.2 a 39.70±1.07 ab 30.13±2.93 b 1.21±0.43 a 77.34±9.63 a 14.76±3.24a
G. aggregatum 14.87±2.88 a 100.8±11.1 a 102.0±7.5 a 30.80±4.66 b 65.09±15.28 a 0.65±0.10 a 53.34±5.37 bc 10.52±1.17ab
R. intraradices 7.41±0.23 b 94.06±6.82 a 103.3±6.8 a 33.00±2.91 b 38.12±2.08 b 0.60±0.07 a 37.80±4.26 c 3.90±0.39b
F. mosseae 9.23±1.48 b 120.4±18.1 a 124.2±8.3 a 52.83±8.82 a 48.20±1.89 ab 0.81±0.14 a 69.34±3.49 ab 9.36±2.66ab
S3 Uninoculated 18.83±0.58 a 118.9±9.0 a 122.6±5.1 b 30.33±2.37 c 23.94±1.59 b 0.81±0.13 b 57.10±5.72 b 10.04±2.36a
G. aggregatum 18.01±0.57 a 111.9±11.9 a 142.6±3.3 a 40.03±2.55 b 44.64±1.50 a 1.49±0.21 a 92.82±5.51 a 17.05±1.83a
R. intraradices 16.74±0.37 b 105.8±4.6 a 118.0±6.0 b 54.34±3.50 a 47.56±3.93 a 1.11±0.23 ab 54.32±8.54 b 7.72±2.55a
F. mosseae 17.74±0.52 ab 133.1±10.6 a 143.3±6.0 a 39.11±0.95 b 42.90±10.70 a 0.84±0.15 b 74.33±13.36 ab 13.26±5.48a
Significance
Substrate (S) *** *** *** * NS NS *** ***
Inoculation (I) ** NS NS ** *** NS * **
S ×I ** NS NS ** NS * * NS

Data are means of six replicates±SE. Values within columns in the same substrate followed by the same letter are not significantly different according to Duncan multiple range test at the P <0.05 level
NS nonsignificant effect (effects of factors according to two-way ANOVA)
*P <0.05; **P <0.01; ***P <0.001 (effects of factors according to two-way ANOVA)
3599
3600
In the present study, mycorrhizal inoculation proved to
increase N, P, and K contents of maize grown in the three
types of coal mine spoils. A significant role of AMF for
nutrient uptake by other plants in mine spoils has also been
previously reported in other studies (Püschel et al. 2008a; Wu
et al. 2009; Karthikeyan and Krishnakumar 2012). The accu-
mulation of nutrients enhanced the ability of host plants to
adapt to adverse factors of coal mine wastes and was benefi-
cial to accelerate the process of revegetation (Wang et al.
2009a; Kumar et al. 2010). In the present study, effects of
AMF on plant nutrient uptake varied according to the sub-
strates, which may be attributed to the differences in nutrient
contents in growth substrates. Similar results were also ob-
tained by Wu et al. (2009) and Baslam et al. (2011). In
nutrient-deficient coal mine spoils, the extensive hyphal net-
work of the AMF can explore more soil volume and increase
the absorption surface of roots, thus contributing to the en-
hanced P concentration in mycorrhizal plants (Püschel et al.
2008b). In terms of N, the study of Janoušková et al. (2011)
indicated that the extraradical mycelium are able to increase N
availability in nutrient-deficient spoil bank soil and translocate
N to the plant. Wang et al. (2009a) reported that the inocula-
tion of AMF increased the K absorption of plants in the coal
mine substrate through promoting plant root growth and the
transformation of insoluble K element to available state. Re-
sults from our study also indicated that the effect of AMF on
nutrient uptake varied among the fungal isolates studied. R.
intraradices played the most significant role in promoting
nutrient uptake by maize in weathered and spontaneous
combusted coal mine spoils, while F. mosseae played the
most significant role in recent discharged coal mine spoil.
The results are consistent with other related reports which
suggested that different AMF isolates showed distinct mycor-
rhizal effects of improving the nutritional status of plants in
spoil bank substrates (Rydlová and Vosátka 2001; Janoušková
et al. 2009). Considering the present study, the overall contri-
bution of AMF to plant N, P, and K nutrition is determined by
the interaction with the types of coal mine spoil, nutrient
contents in substrates, AM fungal partner, and other environ-
mental factors. Therefore, the restoration techniques should
involve enhancing mycorrhiza development by optimizing the
combination of AMF and substrates and assuring appropriate
vegetation cover to improve the effectiveness of revegetation
of coal mine spoil banks (Leung et al. 2007; Wang et al.
2009b; Juwarkar et al. 2009).
The growth rate hypothesis, which is the core idea of
ecological stoichiometry, states that organisms require rela-
tively greater investment in P-rich ribosomes and rRNA to
support the rapid protein synthesis associated with fast growth
(Elser et al. 1996). The elemental stoichiometry of fast-
growing individuals or taxa is therefore tipped toward P, such
that fast-growing organisms exhibit lower tissue N/P and C/P
ratios (Matzek and Vitousek 2009). The present results
Environ Sci Pollut Res (2014) 21:3592–3603
support the growth rate hypothesis, indicating that inoculation
with AMF could significantly increase the growth rate of
maize in coal mine spoils. Consequently, the mycorrhizal
plants had more biomass than the non-inoculated plants dur-
ing the same growing period. Chen et al. (2010) suggested a
link between the C:N:P stoichiometry and the growth rate of
the organism when clover was colonized by AMF. The results
demonstrated that AMF colonization increased the P alloca-
tion to rRNA. Therefore, the increase in P concentration in
mycorrhizal plants can provide more RNA to meet the protein
synthesis needs, resulting in a higher plant growth rate. In this
study, shoot and root C/P and N/P ratios of maize grown in
spontaneous combusted coal mine spoil were higher than
those in other two substrates, demonstrating that maize had
the lowest growth rate in this substrate during the same
growing period. In addition, the different role of three AMF
isolates in adjusting C/N/P ratios of maize may be closely
related to the substrate types and physiological and ecological
characteristics of AMF.
The current experiment revealed that inoculation with
AMF had different impact on heavy metal concentrations in
shoots and roots of maize grown in three types of coal mine
spoils. The differential behavior of AMF in response to plant
tolerance to metal contamination was also observed by Prasad
et al. (2011) and Souza et al. (2013). They concluded that the
mycorrhizal colonization was able to restrict the entrance of
heavy metals in plants under high concentrations but promot-
ed their accumulation in both organs under low concentrations
of these elements. In our study, the coal mine spoils contain a
certain level of heavy metals, which has toxic effects on the
growth of plants. Results obtained clearly showed that some
inoculation treatments significantly reduced the concentra-
tions of Cu in shoots of maize while increased Cu concentra-
tions in roots. The protective benefit may be due to a greater
selectivity of metal transporters in the cell membranes of
mycorrhizae or increased capacity of mycorrhizal roots to
immobilize metals within their cell walls (Zhang et al.
2009). Hildebrandt et al. (2007) suggested that AMF protect
plants from the toxic effects of metals, possibly by binding the
metals in their hyphae. Previous studies have found that root
AM colonization of plants under heavy metal stress results in
expression of specific genes responsible for production of
proteins (including metallothioneins) that increase the toler-
ance of plants to stress (Miransari 2010; Cicatelli et al. 2010,
2012, 2013). In contrast with Cu, some inoculation treatments
increased Zn concentrations in shoots of maize and reduced
those in roots. Some studies have reported that several genes
regulated by AMF may catalyze the uptake of heavy metal
micronutrients such as Fe, Zn, Mn, or Mo (Ouziad et al. 2005;
Lingua et al. 2008). Additionally, membrane transporters in
AMF arbuscules may carry metals to the interfacial matrix,
and their subsequent incorporation inside the plant (Meier
et al. 2012). This may explain how some mycorrhizal plants
Environ Sci Pollut Res (2014) 21:3592–3603
can accumulate metals in their shoots. However, no effect of
inoculation with AMF was found on the concentrations of Fe
in the shoot of maize grown in three types of coal mine spoils.
Moreover, the influence of AMF on metal concentrations
seemed to vary according to AMF species and the types of
growth substrate. This effect was not only substrate specific,
but also AMF specific, because individual AMF species had
different effects on particular substrate types. These conflict-
ing results indicated that the effect of AMF on heavy metal
tolerance and accumulation in plants depends on AMF spe-
cies, metal species, physical and chemical properties of
growth substrates, and environmental conditions. Therefore,
it is necessary to select optimal AMF isolates in connection
with specific types of coal mine spoil in purpose to restore the
vegetation.
Conclusions
The present study has demonstrated that inoculation with
AMF can promote the growth of maize by facilitating mineral
nutrition, adjusting C:N:P stoichiometry and increasing plant
tolerance to heavy metal toxicity in three types of coal mine
spoils. Thus, AMF play a positive role in enhancing the ability
of plants to adapt the composite adversity of different types of
coal mine spoils. However, the effects of AMF on ecological
restoration in terms of coal mine spoils differ between distinct
AMF isolates, substrate types, and other environmental con-
ditions. Results indicate that F. mosseae is more suitable for
the revegetation of recent discharged coal mine spoil, while R.
intraradices is more suitable for weathered and spontaneous
combusted coal mine spoils. The potential role of AMF in the
revegetation of coal mine spoils further revealed the functional
importance of these microorganisms in fragile ecosystems.
Future studies should be focused on the selection of appropri-
ate fungal and plant species originating from the studied areas
for revegetation practices. Combined with the adapted engi-
neering measures, physiochemical properties of the coal mine
spoil wastes should be optimized and further experiments
should be conducted under field conditions.
Acknowledgments This study was financially supported by the Na-
tional Natural Science Foundation of China (31200421 and 40861018),
the Natural Science Foundation of Inner Mongolia, China
(2012MS0603), the Foundation for Key Program of Ministry of Educa-
tion, China (210032), and the National Key Technology R & D Program
(2011BAC02B03).
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