Water Air Soil Pollut (2011) 219:3–10
DOI 10.1007/s11270-010-0679-3
Dual Inoculation of Arbuscular Mycorrhizal and Phosphate
Solubilizing Fungi Contributes in Sustainable Maintenance
of Plant Health in Fly Ash Ponds
A. Giridhar Babu & M. Sudhakara Reddy
Received: 26 April 2010 / Accepted: 20 October 2010 / Published online: 3 November 2010
# Springer Science+Business Media B.V. 2010
Abstract Fly ash is one of the residues produced
during combustion of coal, and its disposal is a major
environmental concern throughout coal-based power-
generated counties. Deficiencies of essential nutrients,
low soil microbial activity, and high-soluble salt
concentrations of trace elements are some of the
concerns for reclamation of fly ash ponds. The effect
of fly-ash-adapted arbuscular mycorrhizal (AM) fungi
and phosphate solubilizing fungus Aspergillus tubin-
gensis was studied on the growth, nutrient, and metal
uptake of bamboo (Dendrocalamus strictus) plants
grown in fly ash. Co-inoculation of these fungi
significantly increased the P (150%), K (67%), Ca
(106%), and Mg (180%) in shoot tissues compared
control plants. The Al and Fe content were signifi-
cantly reduced (50% and 60%, respectively) due to
the presence of AM fungi and A. tubingensis. The
physicochemical and biochemical properties of fly
ash were improved compared to those of individual
inoculation and control. The results showed that
combination of AM fungi and A. tubingensis elicited
a synergetic effect by increasing plant growth and
uptake of nutrients with reducing metal translocation.
A. G. Babu : M. S. Reddy (*)
Department of Biotechnology, Thapar University,
Patiala 147004 Punjab, India
e-mail: msreddy@thapar.edu
Keywords Arbuscular mycorrhizal fungi .
Reclamation . Aspergillus tubingensis . Glomus spp. .
Dendrocalamus strictus
1 Introduction
Fly ash is one of the residues generated in the
combustion of coal and causes air and water pollution
if proper management measures are not taken in time.
Fly ash disposal is a major environmental concern
throughout coal-based power-generated counties. In
India alone, the annual production of fly ash is about
120 MT by 82 power plants and expected to rise to
150–170 MT per year by end of 2012 (MOEF 2007).
Disposal of fly ash has significant impacts on
terrestrial and aquatic ecosystems due to leaching of
toxic substances from the ash into soil and ground
water, as well as reduction in plant establishment and
growth (Wong and Wong 1990). In many cases,
reclamation practices have been ineffective due to
high mortality of tree seedlings which might be due to
deficiency of essential nutrients (usually N and P),
low soil microbial activity, high soluble salt concen-
trations of trace elements, and the presence of
compacted and cement layers on ash disposal sites
(Selvam and Mahadevan 2002). However, successful
rehabilitation must involve the development of micro-
bially driven organic matter turnover and mineral
nutrient cycling for the long-term provision of plant
nutrients (Banning et al. 2010).
4 Water Air Soil Pollut (2011) 219:3–10

Remediation of contaminated sites may be facili- moisture, reinforcement of embankments, and drainage
tated by selection of tolerant plant species as well as channels (Zhou et al. 2005).
microorganisms which play a critical role to establish The present study was aimed to study the influence
early ecosystem development, due to functional of fly-ash-adapted AM fungi along with co-inoculation
abilities such as nitrogen fixation, organic matter of phosphate solubilizing fungus A. tubingensis on the
turnover, mycorrhizal symbiosis, and potential facil- growth, nutrient, and metal uptake of bamboo plants
itation of plant establishment (Hodkinson et al. 2002; grown in fly ash. An improvement of physicochemical
Walker et al. 2003). Among soil microorganisms, and biochemical properties of fly ash due to inocula-
arbuscular mycorrhizal (AM) fungi play relevant roles tion of these fungi is also reported.
for establishment, survival of plant species, and
improved soil properties in stressed environments
(Ortega-Larrocea et al. 2010). Arbuscular mycorrhizal 2 Materials and Methods
fungi also alter the soil microbial communities in
rhizosphere directly or indirectly through changes in 2.1 Fly Ash Sample Collection
root exudation patterns (Barea et al. 2005) and
enhance the soil enzyme activities (Wang et al. 2006). Fly ash samples were collected (0–40 cm depth)
The effects of selected isolates of AM fungi play an randomly from a different location of fly ash sedimen-
important role on the plant growth, nutrient uptake, and tation pond of National Aluminium Company Ltd.
aggregation of fly ash (Enkhtuya et al. 2005; Wu et al. (NALCO), Damnjodi (18°46′22″ N 082°53′23″ E),
2009). However, spontaneous selection of infective and Orissa, India. The samples were mixed thoroughly, and
effective AM fungi is a long process in fly ash ponds. It a portion of the fly ash was used to analyze various
has also been demonstrated that the use of adapted AM physicochemical characteristics. The physicochemical
fungal strains, in restoration and bioremediation stud- properties and elemental levels of fly ash were
ies, is more effective than applying non-adapted strains presented in Table 1.
(Vivas et al. 2005). Oliveira et al. (2010) reported that
AM fungi quickly lose their symbiotic efficacy when Table 1 Physicochemical
cultivated without edaphic stresses of the environment properties of fly ash Parameters Units
from where they are originally isolated. They also
pH 7.35
recommended that the inoculum should be produced
EC(mS/cm) 0.30
with original edaphic stresses especially for AM
Organic 0.30
isolates from extreme environments. We have isolated Carbon (%)
AM fungi from the plants growing in fly ash pond and Olsen P 2.30
multiplied in pots using fly ash to maintain its (mg/kg)
adaptation. Many fungi can survive and grow at high N(mg/kg) 85.0
concentrations of toxic metals and the genus Aspergil- Elemental
analysis
lus play multi-fascinated roles such as P solubilization, (mg/kg)
heavy metal bioleaching, plant growth promotion, and Al2O3 281,500
synergetic effects with mycorrhizal fungi (Medina et al. Fe2O3 51,500
2006; Yang et al. 2009). The selection of A. tubingensis P2O5 4,500
in this study is based on its ability to solubilize the SO3 13,500
insoluble phosphates and improvement of growth of CaO 8900
plants under stress conditions (Krishna et al. 2005). MgO 3800
Bamboo plants (Dendrocalamus strictus) were se- Na2O 3700
lected because of its extensive underground root and MnO 500
rhizome system (having significant capacity to bind the K2O 800
top soil), accumulation of leaf mulch, abundant litter ZnO 100
fall. It also serves as an efficient agent in rehabilitation CuO 150
of degraded land, preventing soil erosion, conserving
Water Air Soil Pollut (2011) 219:3–10
2.2 Isolation and Inoculum Production
The roots and rhizosphere fly ash samples of
dominant plants growing in fly ash pond were
collected up to 30 cm depth using a stainless steel
shovel. Three replicates were collected from each
plant species. Samples were mixed thoroughly and
placed in a plastic bag and stored at 4°C until their
use. The dominant plants growing in this pond are
Acacia pennata (Mimosaceae), Calotropis gigantea
(Asclepiadaceae), Cassia occidentalis (Caesalpinia-
ceae), Cynodon dactylon (Poaceae), Lantana camara
(Verbenaceae), and Jatropha gossypifolia (Euphorbia-
ceae). AM fungal spores were isolated from these
samples by sieving and decanting technique (Gerde-
mann and Nicholson 1963). Isolated spores were
propagated on three trap plants; maize, wheat, and
sorghum (seeds surface sterilized with 0.01% HgCl2)
grown separately in earthen pots containing the fly
ash for two successive cycles, each cycle of 3 months
long. No fertilizers were added to fly ash while
multiplying the AM fungi. The inoculum was
prepared with density of spores 90–100 spores per
10 g of fly ash. AM inoculum consisted of fly ash
containing spores and AM-infected root pieces from a
pot culture. The phosphate solubilizing fungus A.
tubingensis (Reddy et al. 2002) was used as co-
inoculant. The fungus was grown on Pikovskaya’s
agar (Pikovskaya 1948) plates at 28°C up to dense
formation of spores. Spores were scraped and mixed
with vermiculite with inoculum size of 1.7×108
spores/g vermiculite.
Prior to inoculation, subsamples of spores isolated
from pots and fly ash pond were identified following
the current taxonomic criteria (Schenck and Perez
1990) and information of INVAM (http://www.invam.
caf.wdu.edu/). The AM fungal isolates were identified
as Glomus rosea, Glomus magnicaule, Glomus
etunicatum, Glomus heterogama, Glomus maculo-
sum, Glomus multicaule, Scutellospora nigra, and
Scutellospora heterogama.
2.3 Nursery Experiment
The nursery experiment was conducted at NALCO
nursery, Damanjodi, Orissa, India. Fly ash was used
as substrate to grow the bamboo plants. Randomized
blocks design was followed and size of each treatment
5
plot was 60×60×40 cm3. Three replicates were
maintained for each treatment. The experiment con-
sisted of four treatments (1) fly ash, (2) fly ash and
AM fungi, (3) fly ash and A. tubingensis, and (4) fly
ash with AM fungi and A. tubingensis. In each
treatment, nine bamboo plants (2 months old)
obtained from the NALCO nursery were planted and
inoculated with AM fungi (inoculum size 50 gm/plant
with 90–100 spores/10 g) and 5 g/plant (1.7×108
spores) of A. tubingensis fungal spores.
After 6 and 12 months of plantation, the shoot
height and number of branches were measured. AM
colonization was determined by collecting the root
samples randomly from three places for each plot.
The percentage of root length colonized by AM fungi
was calculated by the gridline intersect method
(Giovannetti and Mosse 1980) after staining with
trypan blue (Phillips and Hayman 1970). The spores
were isolated from rhizospheric soil samples of fly
ash and were counted (Gerdemann and Nicholson
1963). To determine the uptake of minerals and
metals by bamboo in different treatments, three
randomly collected shoots from each plot were used.
The shoot tissues were digested with nitric acid and
perchloric acid (3:1) as described in Page et al.
(1982). These samples were analyzed for mineral
nutrients such as K, Ca Na, Mg, and heavy metals
viz., Al, Fe, Zn, and Cu by inductively coupled
plasma mass spectroscopy. Total P content in the
shoot tissues was determined by colorimetric method
as describes in Kitson and Mellon (1944).
To determine the physicochemical changes of fly
ash, the samples were harvested randomly from each
treatment plot after 6 and 12 months. The organic
carbon was estimated by the method as described in
Walkley and Black (1934) and the total N by using
TOC analyzer. Urease activity was determined by the
method of McGarity and Myers (1967) and acid
phosphatase by Tabatabai and Bremner (1969).
Available P was estimated by Olsen et al. (1954)
and Bray and Kurtz (1945) methods according to the
pH of samples.
2.4 Statistical Analysis
The data were analyzed by ANOVA, and means were
compared by using Turkey’s tests at P<0.05 using
Graphpad Prism (v 4.03) software.
6 Water Air Soil Pollut (2011) 219:3–10

3 Results inoculated plants compared to control plants at the

3.1 Plant Growth and AM Fungal Colonization
Inoculation of AM fungi, A. tubingensis and combi-
nation of these two fungi increased the growth of
bamboo plants compared to control treatment. Plant
height as well as number of branches/plant was
significantly increased in dual inoculation compared
to individual inoculum both at 6 and 12 months of the
study (Table 2). AM fungal colonization was ob-
served in all treatments and the colonization of
indigenous AM fungi increased due to inoculation
of A. tubingensis inoculated plants. It was observed
that the percent root colonization of all treatment
plants significantly increased after 12 months com-
pared to 6 months. Maximum spore count was
observed in dual inoculated plants after 12 months
compared to other treatments (Table 2).
3.2 Plant Nutrient Uptake
Dual inoculation significantly increased the P (150%),
K (67%), Ca (106%), and Mg (180%) in shoot tissues
compared to control treatments at 12 months after
plantation. Individual inoculation also increased the
levels of these minerals compared to control plants.
Sodium levels increased in inoculated plants com-
pared to control. However, the uptake of Na levels
decreased at 12 months compared to 6 months. The
uptake of Al, Cu, and Fe were significantly reduced
due to inoculation compared to non-inoculated plants.
The Al content was reduced up to 50% in inoculated
plants compared to control plants at 12 months. The
Fe content was significantly reduced (about 60%) in
end of 12 months. The content of these metals were
significantly lesser in dual inoculated treatment than
individual inoculation (Table 3). The Zn levels were
significantly increased (above 64%) in tissues of
bamboo plants inoculated with dual inoculum com-
pared to control as well as individual inoculations
(Table 3).
3.3 Fly Ash at Harvest
The physico-chemical properties of fly ash signifi-
cantly improved in all inoculated treatments com-
pared to control treatment both at 6 and 12 months. In
case of dual inoculation, the organic carbon and
available P increased significantly after 6 months as
well as 12 months of plantation. The available P was
3 times higher in dual inoculation treatment after
12 months compared to control treatment. After
6 months of plantation, the N content in fly ash
increased significantly in inoculated treatments as
compared to control, the maximum being observed in
dual inoculation treatment. Acid phosphatase and
urease enzyme activities also increased in inoculated
treatments compared to control. The maximum
enzyme activities were observed in dual inoculated
treatments than other treatments (Table 4).
4 Discussion
4.1 Plant Growth and AM Fungal Colonization
Inoculation of AM fungi significantly increased the
growth of bamboo plants in fly ash compared to non-

Table 2 Growth parame-

ters, AM fungi colonization Treatment Height (cm/plant) Branches/plant Colonization (%) Spores/100 g fly ash
and spore numbers of
bamboo plants inoculated 6 months
with AMF, At, and dual Control 27.6±2.0c 18±1.0c 4.0±0.6c 0
inoculum of AMF+At AMF 58.1±2.1a 29±1.5ab 52.0±3.0a 20±1.5a
At 40.1±1.5b 22±1.5bc 17.5±1.5b 3±1.0b
AMF+At 66.5±2.0a 34±2.0a 46.5±1.5a 16±1.5a
12 months
Values sharing a same
letters within the column Control 136.9±0.0c 37±2.5c 17.5±1.5c 5±2.0c
and time periods are not AMF 226.9±3.3a 49±2.0ab 68.5±1.5a 17±1.5b
significant at P<0.05 At 181.0±8.2b 42±2.0bc 26.0±1.0c 7±0.5bc
AMF AM fungi, AMF+At 247.8±3.1a 57±1.0a 55.0±4.0b 29±2.5a
At A. tubingensis
Water Air Soil Pollut (2011) 219:3–10 7

Table 3 Uptake of different nutrients and heavy metals in tissues of bamboo plants inoculated with fungi (AMF, At, and dual
inoculum of AMF + At

Treatments mg/kg

P K Ca Mg Na Fe Al Zn Cu

6 months
Control 135±6d 94±0.4d 135±1.0d 158±0.8d 90±3.6d 3,375±6.4a 9,326±2.1a 96±0.8d 63±1.2a
AMF 348±1b 124±1.8c 209±1.7b 264±2.1b 136±2.2b 2,880±2.9b 6,453±10.0b 111±1.1c 51±1.5b
At 269±8c 166±3.3b 169±2.0c 187±1.8c 116±1.0c 2,618±10.6c 7,861±9.7c 169±0.6b 34±0.1c
AMF+At 419±2a 191±1.0a 287±1.9a 297±1.2a 161±1.7a 1,917±7.0d 5,733±8.3d 178±0.3a 31±2.4c
12 months
Control 257±0c 864±0.8c 562±2.1d 159±0.1c 62±0.5d 953±3.0a 2,635±5.2a 87±0.8d 32±0.0d
AMF 645±0a 1,328±1.2a 974±1.7b 380±0.5ab 72±0.5c 598±1.0b 1,315±3.1c 98±1.0c 18±0.1c
At 569±9b 1,269±4.9b 755±1.9c 334±0.6b 82±0.6b 359±0.9c 1,424±1.8b 123±1.1b 23±0.4b
AMF+At 653±2a 1,444±3.5a 1,162±1.9a 457±0.3a 93±0.3a 360±1.4c 1,245±11.9d 143±0.3a 26±0.7a

Values sharing a same letters within the column and time periods are not significant at P<0.05
AMF AM fungi, At A. tubingensis

inoculated treatment. Co-inoculation of A. tubingensis
increased the mycorrhizal colonization compared to
individual inoculation of AM fungi after 12 months of
plantation. Many P solubilizing microorganisms in-
crease the mycorrhizal root colonization by producing
specific metabolites such as vitamins, amino acids
and hormones apart from P solubilization (Barea et al.
2005). The colonization of AM fungi in non-AM
inoculated plants may be due to either the presence of
propagules in substrate (fly ash) or in bamboo
seedlings which were grown previously in the soil.
The slow rate of indigenous AM fungal colonization
and the limited number of species growing on fly ash
reflects the adverse growth conditions of fly ash
deposits. We have reported 6 different plant species
growing in fly ash colonized with 8 different species

Table 4 Physicochemical
and biochemical properties Parameters Control AMF At AMF+At
of fly ash inoculated with
AMF, At, and dual inocu- 6 months
lum of AMF + At pH 6.81±0.05a 6.74±0.13a 6.62±0.01a 6.60±0.08a
EC (mS/cm) 0.35±0.00b 0.47±0.00a 0.36±0.01b 0.50±0.01a
Organic carbon (%) 0.5±0.0d 1.4±0.1a 1.0±0.1c 1.7±0.1a
Available P (mg/kg) 1.2±0.0c 3.2±0.1a 3.6±0.1a 2.5±0.2b
N (mg/kg) 203±9.9d 280±0.0b 224±0.0c 420±0.0a
Acid Phospatase 212±3.8c 252±0.7b 297±0.4a 189±0.1d
(μM/g/1 h)
Urease (μM/g/h) 12.2±0.6d 23.0±0.1a 28.6±0.5b 31.9±0.4ca
12 months
pH 6.20±0.14ab 5.92±0.03a 5.67±0.04bc 5.59±0.01c
EC(mS/cm) 0.16±0.01b 0.24±0.02b 0.40±0.02a 0.47±0.00a
Organic carbon (%) 0.6±0.0d 1.4±0.0c 1.8±0.0b 2.1±0.0a
Values sharing a same Available P (mg/kg) 1.6±0.1c 4.0±0.1b 4.3±0.0b 4.7±0.1a
letters within the column N (mg/kg) 134±0.0b 238±0.0b 210±0.0ab 357±9.9a
and time periods are not
Acid Phospatase 218±4.0c 428±5.3b 507±3.5a 541±9.9a
significant at P<0.05
(μM/g/1 h)
AMF AM fungi, Urease (μM/g/h) 19.6±1.7d 44.7±1.4b 36.0±0.7c 56.6±1.3a
At A. tubingensis
8 Water Air Soil Pollut (2011) 219:3–10
of AM fungi and most of the AM fungi belong to
Glomus species. These species are also been reported
as dominant AM fungi in extreme environments by
other researchers (Zarei et al. 2008; Bedini et al.
2010).
4.2 Plant Mineral Nutrition
Fly ash contains many essential plant nutrients and
their availability to the plant may be problematic as
reported by different authors (Pandey et al. 1994;
Singh et al. 1997). However, inoculation of AM fungi
supports the growth and nutrient uptake of plants and
aggregation of fly ash (Enkhtuya et al. 2005; Wu et al.
2009). In the present study, inoculation of A.
tubingensis along with the AM fungi significantly
increased height and number of branches of bamboo
plants. The uptake of nutrients such as P, K, Ca, Mg,
and Na were significantly increased and the contents
of Al, Cu, and Fe were reduced in the shoot tissues
compared to individual inoculation. This may be due
to the synergetic effect of A. tubingensis with AM
fungi. Some experimental results confirm the exis-
tence of synergetic effect of saprobe fungi on plant
root colonization by AM fungi (Fracchia et al. 2000).
Arriagada et al. (2004) reported that saprobe fungi
absorb heavy metal from the contaminated site and
increase the AM colonization of plants when inocu-
lated in combination. It is also known that AM fungi
may suppress the uptake of Al, Fe, and Mn that may
be present in toxic levels in some soils (Ning 2000).
Alleviation of heavy metal phytotoxicity by AM fungi
has been indicated in several studies (Chen et al.
2007; Arriagada et al. 2004). The AM fungi may
enhance plant P nutrition and increase the plant
growth by diluting metal effect in host plant or by
binding of the metal to the fungal mycelium and
immobilize them in rhizosphere or roots (Chen et al.
2001).
4.3 Physicochemical Changes of Fly Ash
Fly ash contains considerable amounts of essential
and nonessential elements such as Al, Fe, and Cu and
many other elements which are toxic at elevated
concentrations (Basu et al. 2009). In this study, pH of
the fly ash decreased in all treatments from slight
alkaline to moderate acidic range. The pH reduction
in fly ash may be due to presence of high content of
sulfur in fly ash while leaching of base ions (Juwarkar
and Jambhulkar 2008). Since C and N in the fly ash
are likely to be oxidized to gaseous constituents
during combustion, they usually are present in trace
amounts and P in fly ash is relatively unavailable to
plants (Bradshaw and Chadwick 1980). Higher con-
centrations of P in fly ash reported in this study might
be due to solubilization of insoluble P present in the
fly ash by both mycorrhizal and phosphate solubiliz-
ing A. tubingensis. The AM fungi interact with other
rhizosphere microorganisms (Jeffries et al. 2003) and
affect rhizodeposition and thus the quantity and
quality of organic C delivered to the soil via fungal
hyphae (Barea et al. 2005).
Soil enzymes are important in catalyzing several
reactions necessary for life processes of microorgan-
isms in soils and the stabilization of soil structure, the
decomposition of organic wastes, organic matter
formation, and nutrient cycling (Dick et al. 2000).
Among the soil enzymes, phosphatases (acid and
alkaline) and urease play an important role in P, N,
and C mineralization. In this study, acid phosphatase
and urease activity in inoculated treatments were
increased in all treatments compared to control
treatment. The increase of enzymatic activities in
soils is involved in an increase in the availability of
nutrients to the plants, which in turn have a positive
influence on soil fertility (García et al. 1997).
5 Conclusions
In conclusion, inoculation of AM fungi in combina-
tion with A. tubingensis increased the growth and
nutrient uptake of bamboo plants and also reduced the
metal translocation. Inoculation of fly ash adapted
AM fungi along with A. tubingensis might be a
promising strategy to promote the vegetation in fly
ash ponds.
Acknowledgements The authors are thankful to TIFAC-
CORE, Thapar University, Patiala, Punjab and NALCO,
Damanjodi, Orissa, India for facilities.
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