Engineering, 2010, 2, 683-689
doi:10.4236/eng.2010.29088 Published Online September 2010 (http://www.SciRP.org/journal/eng)
Arbuscular Mycorrhizal Technology in Reclamation and
Revegetation of Coal Mine Spoils under Various
Revegetation Models
Akhilesh Kumar1, Richa Raghuwanshi2*, Ram Sanmukh Upadhyay3
1Government Degree College, Narnaul, India
2Department of Botany, Mahila Mahavidyalaya Banaras Hindu University, Varanasi, India
3Department of Botany, Banaras Hindu University, Varanasi, India
Received November 25, 2009; revised July 21, 2010; accepted August 23, 2010
Abstract
Reclamation and revegetation of a coal mine spoils with various revegetation models utilizing the mycorrhi-
zal technology were studied. The models with different combination of plant species were designed to test
the hypothesis of speedy revegetation. Root colonization and spore density of arbuscular mycorrhizae (AM)
were lowest in plants seeded directly on slopes of the overburden (coal mine dump). At flat surfaces, the
mycorrhizal colonization in plant species was higher than that observed at slopes. In other revegetation mod-
els, i.e., tree monoculture, tree monoculture + crop species (agroforestry), and two strata plantations (combi-
nation of different plant species), maximum AM colonization was recorded for tree species grown along with
crop species. This was followed by two strata plantations and tree monoculture. In two strata plantations
three categories of AM associations were recognized: 1) every plant in the combination, possessed high my-
corrhizal association, 2) only one plant in the combination possessed high mycorrhizal association, and 3)
none of the plants in the combination possessed high mycorrhizal association. Azadirachta indica, Pongamia
pinnata, Leucaena leucocephala and Acacia catechu were most effective in catching mycorrhizae, and can
be used as the effective tool in rehabilitation of the degraded ecosystems.
Keywords: Coal Mine Spoils, Reclamation, Revegetation and Mycorrhizae
1. Introduction
AM fungi form the fundamental linkage between the
biotic and abiotic components of the ecosystem in addi-
tion to their being the primary colonizers of coal mine
spoils [1-2]. The primary approach to revegetation of
such spoils is the erosion control through plant cover in
short-term and development of a self-sustaining commu-
nity through colonization of native plants in the long-
term [3]. The soil conservation practices not only make
the soil resistant to erosion, but also directly affect the
soil pore size distribution [4]. The soil aggregates and
pore size collectively constitute ‘soil structure’ which in
turn influences not only the physico-chemical but also
the biological processes [5-6]. The contribution of AM
fungi to soil aggregate formation can be grouped into
three categories according to Miller and Jastrow [7]:
These are: 1) growth of external AM hyphae in the soil
matrix to create the skeleton that holds primarily the soil
particles (i.e. sand, silt and clays) together via physical
entanglement, 2) creation of conditions by roots and ex-
ternal hyphae conducive to formation of microaggregates
whereby mineral particles and organic debris are held
and cemented together by various physico-chemical
events involving binding agents like the persistent gums
and glues in the root exudates, the soil microbes and
mycorrhizal hyphae in particular contribute hydrophobic
glycoprotein glomalin [8], and 3) enmeshment of micro-
aggregates and smaller macroaggregates by external hy-
phae and roots to form the macroaggregates that can be
further stabilized by intermicoraggregate and intermacro-
aggregate cementation by polysaccharides and other or-
ganics from microorganisms and plant roots. These three
processes operate simultaneously because of dynamic

A. KUMAR ET AL.
684
nature of soil aggregation. The improvement in soil ag-
gregation sustains ideal water infiltration, tilth and aera-
tion for plant growth [9].
There are countless reasons to qualify mycorrhizae as
bliss for revegetation programmes as infection of plant
roots by such fungi induces tolerance against the abiotic
and biotic stresses. Mycorrhizal fungi appear to partially
protect plants against heavy metal toxicity through bind-
ing and thus limiting their translocation to shoots [10].
The host plant in turn may give the fungus a selective
survival advantage on a contaminated site. The metal
sequestration by AMF may alter their translocation in
plants thus restricting metal accumulation in mycorrhizal
roots and so also reduce metal transfer to the above-
ground biomass [11]. The only direct evidence of my-
corrhizal adaptation to metal pollutants is the exudation
of complexing organic acids that alter pollutant avail-
ability in the rhizosphere [12]. Metallophytes have evo-
lved various physiological adaptations to successfully
compete with the harsh conditions in heavy metal loaded
soils. In addition, AMF that colonize plant roots, consid-
erably reduce the intake of heavy metals by plant cells
that could be one of the strategies that allows metallo-
phytes to thrive on heavy metal polluted sites [13-14].
Haselwandter [15] proposed that a metal-resistant plant
breed susceptible to mycorrhizal symbiosis would be of
great value for the rehabilitation of metal contaminated
soils. AM fungi that modify the root system and play a
critical role in the nutrient cycling in the ecosystem, can
be taken as a crucial parameter to access reclamation
success of degraded lands. Thus, restoration success de-
pends on the augmentation of biological activity of the
surface soil horizons [16]. In view of the above, the pre-
sent study was carried out by developing a revegetation
model site at the coal mine spoil site utilizing mycorrhi-
zal technology.
2. Materials and Methods
Revegetation model site (RMS) was developed at Jayant
Coal Mine situated in Singrauli district of Madhya
Pradesh, India. An area of about 9 hectare of fresh over-
burden was smoothened and flattened by bulldozers for
plantations in various models designed. Seedlings raised
in nursery by either seeds or cuttings were transplanted at
the RMS. In each revegetation model, the plot size was
20 m × 20 m where the plants were transplanted in pit
size of 40 cm × 40 cm × 30 cm with the spacing of 2 m ×
2 m. The density of the plants per plot was 100. Seeds of
different plant species were seeded directly by broad-
casting in case of direct seedling, whereas in rest of the
cases the seedlings raised in nursery were transplanted at
RMS. The combination of plants species in different
plots at RMS were as follows:
Direct seeding on slope surface: Seeds of different
plant species used in direct seeding on slope surface were
Acacia catechu, Azadirachta indica, Leucaena leuco-
cephala, Madhuca indica, Pongamia pinnata, Syzygium
cumini, Terminalia arjuna and Terminalia bellirica.
Direct seeding on flat surface: The plant species se-
lected were A. catechu, Acacia nilotica, A. indica, L.
leucocephala, M. indica, P. pinnata, S. cumini, T. arjuna
and Zizyphus jujuba.
Tree monoculture: The species raised in monoculture
plots were A. catechu, Albizia lebbeck, A. indica, Dal-
bergia sissoo, Dendrocalamus strictus, Gmelina arborea,
L. leucocephala, Phyllanthus emblica, P. pinnata, Tec-
tona grandis and T. bellirica.
Tree monoculture + crop plant (Agroforestry): The
leguminous Cajanus cajan was sown in combination
with A. lebbeck, A. indica, G. arborea, P. pinnata, T.
bellirica and T. grandis. Pennisetum typhoides a non-
leguminous crop semi-arid tropics was seeded with com-
bination of A. indica, D. sissoo, L. leucocephala and P.
pinnata.
Two strata plantation: Mixed plantations were raised
with the following combinations of leguminous and
non-leguminous species: A. catechu + P. pinnata, A.
lebbeck + A. catechu, A. indica + P. emblica, D. strictus
+ T. grandis, G. arborea + P. pinnata, P. pinnata + T.
bellirica, T. bellirica + G. arborea and T. grandis + L.
leucocephala.
In all the above models, ground seeding with a legu-
minous forb Stylosanthes hamata and two grasses name-
ly Pennisetum pedicellatum and Heteropogan contortus
was done by broadcasting.
3. Assessment of AM Association
Composite samples of soil and roots were collected from
each plot and isolation of AM spores was done by wet
sieving and decanting methods of Gerdemann and
Nicolson [17] and sucrose centrifugation method of Jen-
kins [18]. The number of AM spores per hundred gram
of dry soil was estimated.
The root samples were washed, cut into one cm seg-
ments, and placed in tissue capsule [19]. These were
cleared and stained with Chlorazol Black E (CBE) in
lactoglycerol following the procedure of Phillips and
Hayman [20]. One hundred root segments were ran-
domly selected and mounted on microscopic slides for
observation. Quantification of the root colonization is
based on percentage of AM colonization. Association of
mycorrhizae was based on the presence or absence of

A. KUMAR ET AL.
685
arbuscules, vesicles and internal hyphae in the root tis-
sue.
4. Results
Dynamics of AM in plant roots and rhizosphere soil was
studied in various models at the revegeatation site. The
models with plantation of various combinations of plant
species were designed to test the extent of speedy reve-
getation of coal mine spoils. The lowest level of my-
corrhizal association was found in plants seeded directly
on slopes (Figure 1). At flat surfaces, AM colonization
in plant roots was higher than that observed at slope sur-
faces (Figure 2), but it was lesser as compared to other
models. A. indica, L. leucocephala, M. indica and P.
pinnata harboured significantly higher level of AM at
slopes while it was significantly higher in A. catechu, A.
nilotica, A. indica, L. leucocephala and P. pinnata at flat
surfaces. In other plants the AM association was poor
both at slopes and flat surfaces.
In rest of three revegetation models (i.e., tree mono-
culture, tree monoculture + crop species and two strata
plantations) nursery raised seedling were transplanted at
RMS in pits in various combinations. The highest level
of AM colonization was recorded for the tree species
grown with crop species (agroforsetry) followed by the
two strata plantations and tree monoculture. Two species
namely A. indica, and D. sissoo possessed significantly
(p = 0.01) higher level of AM colonization than others
rose in monoculture (Figure 3). The rhizosphere soil of
L. leucocephala and P. pinnata possessed higher AM
spore count than others. When the tree species were
seeded along with crop species, the combinations A. in-
dica + C. cajan, A. indica + P. typhoides and D. sissoo +
P. typhoides possessed significantly higher (p = 0.01)
AM colonization. In the same model, rhizosphere soil of
the tree species in combinations G. arborea + C. cajan
and P. pinnata + C. cajan contained higher AM spore
number compared to other combinations (Figure 4). In
two strata plantations (Figure 5) three categories of com-
binations were recognized: 1) each of the plant in the
combination possessed high mycorrhizal association (e.g.
G. arborea + P. pinnata and T. grandis + L. leuco-
cephala, 2) only one plant of the combination possessed
high mycorrhizal association (e.g. A. indica + P. em-
bilica and D. strictus + T. grandis), and 3) none of the
plants in the combination possessed high mycorrhizal
association (e.g. A. lebbeck + A. catechu and T. bellirica
+ G. arborea). In two strata plantation, the best combina-
tions with high mycorrhizal association were G. arborea
+ P. pinnata and T. grandis + L. leucocephala.
AM spore number was higher in the rhizosphere soils
of P. pinnata and G. arborea when grown together on
the dumps (Figure 5). In other combinations, either both
the plants or one of them possessed lesser degree of AM
association. The spore number was higher in the rhizo-
sphere soil of P. pinnata as compared to other combina-
tions. A. lebbeck + A. catechu, P. pinnata + T. bellirica
and T. bellirica + G. arborea were a few of other com-
binations having poor AM spore counts in their rhi-
zosphere.
Generally, the AM colonization was higher in the
roots of leguminous tree species grown with non-legu-
minous ones in combination (Figure 5). The examples of
such combinations were: T. grandis + L. leucocephala,
G. arborea + P. pinnata and D. strictus + T. grandis. A
moderate to high AM colonization was recorded in P.
pinnata for all the combinations tested. Low mycorrhizal
colonization was recorded for A. lebbeck + A. catechu
and T. bellirica + G. arborea combinations.
PLANT SPECIES
PER CENT VAM COLONIZATION
5
10
15
20
25
30
5
10
15
20
25
30
Colonization
Spore number
Ac
Ai
Ll
Mi
Pp Sc Ta Tb
NUMBER OF VAM SPORE
Figure 1. VAM association in plant species grown at slopes
by direct see ding at revegetion medel site.
PLANT SPECIES
5
10
15
20
25
30
35
5
10
15
20
25
30
35
Col o niza ti o n
Spore Number
Ac An
Ai Ll
Mi
Pp
Sc
Ta
Zj
NUMBER OF VAM SPORE
PER CENT VAM COLONIZATION
Figure 2. VAM association in plant species grown at flat
surface by direct seeding at revegetation model site.

A. KUMAR ET AL.
686
PLANT SPECIES
PER CENT VAM COLONIZATION
10
20
30
40
50
60
10
20
30
40
50
60
Ac
Al Ai
Ds
Des
Ga
Ll
Pe Pp Tg
Tb
NUMBER OF VAM SPORE
Spore number
Colonization
Figure 3. VAM association in plant species in monocuiture
at revegetation medel site.
PLANT SPECIES
PER CENT VAM COLONIZATION
20
40
60
80
20
40
60
80
Colonization
Spore number
Al+Cc
Ai+Cc
Ai+Pt
Ds+Pt
Ga+Cc
Ll+Pt
Pp+Cc
Pp+Pt
Tb+Cc
Tg+Cc
NUMBER OF VAM SPORE
Figure 4. VAM association in plant species planted along
with crop species at revegetation model site.
No significant correlation could be established between
AM colonization and the spore number. In T. grandis
(grown with D. strictus), P. embilica (grown with A. in-
dica), the spore count in the rhizosphere was high but the
AM colonization was low. However, in case of A. indica
(with P. emblica), P. pinnata (with A. catechu), L. leu-
cocephala (with T. grandis) and T. bellirica (with P.
pinnata), the spore count was low in the rhizosphere in
contrary to high AM colonization in roots. In some spe-
cies such as P. emblica (with A. indica), T. grandis (with
L. leucocephala), A. lebbeck (with A. catechu) and T.
bellirica (with G. arborea), the AM colonization as well
as spore number both were low. In a few plants, AM
colonization as well as spore number was high. These
were G. arborea and P. pinnata (grown together) and P.
pinnata (grown along with T. bellirica). Two combina-
tions namely A. lebbeck + A. catechu and T. bellirica +
G. arborea possessed low level of AM association.
5. Discussion
The present study involved various RMS models for as-
sessment of mycorrhizal status of the plants. AM coloni-
zation was poor in plants grown by seeding on overbur-
den both at flat surface and slopes. This may be attrib-
uted to poor emergence of roots, as a result of erosion,
the detrimental factor affecting survival of plants and
poor presence of AM as well. This is supported by the
fact that at slopes, where soil erosion was severe, the
AM-plant association in plants was poor. The nursery
raised seedlings as monoculture favored mycorrhization
than the direct seeding. High AM colonization was re-
corded in P. pinnata, L. leucocephala and D. strictus in
two strata plantation. Seeding or planting of species not
only controls erosion but also enhances species diversity
and speeds up succession that meets the revegetation
goal. Nicholasan and McGinnies [21] reported that es-
tablishment of good plant cover can stabilize mine spoil
and consequently improve soil conditions to promote
plant succession [22]. Direct seeding of plants has been
recommended in many reports [23-25]. There are reports
that seedling growth and survival of tree species is af-
fected by seeding of grasses and legumes [26]. The
grasses are beneficial in checking erosion while legumes
improve soil nutrient status. AM fungi benefit plant es-
tablishment and survival in many ways in degraded lands
[27-30], and restoration programs with application my-
corrhizal technology have been successful [31-32].
Agroforestry is the collective term for land use system
wherein woody perennials are grown in association with
agricultural herbaceous plants, following spatial ar-
rangement or temporal sequence that facilitates both
ecological and economic interactions between the tree
and non-tree components. In mixed plantations, it ap-
peared that leguminous plants were more advantageous
than non-leguminous trees except A. indica. Tree + crop
plantation appeared to be the best model in terms of my-
corrhizal technology at least for the selected species. A.
indica, P. pinnata, L. leucocephala and A. catechu were
most effective in attracting luxuriant mycorrhizae (even
when seeded directly), and thus can be used as the suit-
able rehabilitating species in such degraded ecosystems.
It seems that seeding with grasses and leguminous forbes
increased AM inoculums, as these grew luxuriantly with
fibrous roots that favored mycorrhizal association. Le-
guminous shade trees contribute substantial N to the un-
derstory crop growth and also favors N uptake by tropi-

A. KUMAR ET AL.
687
Figure 5. VAM association in plant species grown in two strata plantation at revegetating model site. Abbreviationa: Ac, A.
catechu; Al, A. lebbeck; Ai, A. indica; Ds D. sissoo; Des, D. strictus; Ga, G. arborea; Li, L lencocephala; Pe, P. emblicar; Pp, P.
pinnata; Tg, T. grandis, Tb, T. bellerica.
cal grasses [33-34]. Further, the results confirm the fact
that mycorrhizae also help the plant-soil-plant system by
inter-bridging between the roots of different plants [35-
37].
Most legumes are fairly responsive to and extensively
colonized by mycorrhizae especially in soils where an
insufficiency of available P limits plant growth. Hence,
the influence of mycorrhizae on legumes is potentially
greater over any other group because of alleviation of
P-stress by symbiotic fungi and indirectly by enhancing
N status soil contributed by the legumes. Since plants do
not contain their own polyphosphate hydrolase, they rely
on the activity of soil microorganisms to release free
phosphate from minerals or organic P resources. My-
corrhizal plants can utilize more phosphorus than the
non-mycorrhizal ones, mainly from the same soluble
phosphate pool as AM fungi harbor phosphate transfer
[38]. Davies and Call [39] reported that AM significantly
enhance the nutritional status in perennial grasses grow-
ing at lignite overburden. The successful restoration de-
pends on the capacity of the plants to capture resources
at an early stage. On degraded lands, which may be
droughty, nutritionally poor or otherwise stressed, there
exists only a brief period favorable period for plant
growth and the plants which do not establish within that
time window fail to survive.
Jeffries et al. [40] also supported the persistence of an
acceptable amount of weeds within crops; providing a
reservoir of mycorrhizal inoculum. Established my-
corrhizal vegetation can facilitate the probability or ex-
tent of mycorrhizal infection of seedlings and thus my-
corrhizal interaction among distantly related plants might
be of particular ecological interest, as this may permit
early succession of plants [41].
6. Acknowledgements
The research project was funded by the Ministry of Coal,
Government of India through Central Mine Planning and
Design Institute Limited (CMPDI), Ranchi, India. The
financial assistance in the form of Junior and Senior Re-
search Fellowship is gratefully acknowledged.
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