761

NSave Nature to Survive

ISSN: 0973 - 7049

: Special issue, Vol. 3;

Paper presented in International Conference onEnvironment, Energy and Development (from

Stockholm to Copenhagen and beyond)December 10 - 12, 2010, Sambalpur University

Sangeeta Mukhopadhyay and Subodh Kumar Maiti

761-770; 2010

NATURAL MYCORRHIZAL COLONIZATION IN TREE SPECIES

GROWING ON THE RECLAIMED COALMINE OVERBURDEN

DUMPS: CASE STUDY FROM JHARIA COALFIELDS, INDIA

Coalmine overburden dumps

Mine soil properties

VA mycorrhiza

Spore density

Spore size

Root infection

762

SANGEETA MUKHOPADHYAY AND SUBODH KUMAR MAITI*

Department of Environmental Sc. and Engg,Indian School of Mines (I.S.M), Dhanbad – 826 004 (Jharkhand)

E mail: subodh_maiti@yahoo.com

Natural mycorrhizal colonization is ubiquitous in nature and they play an important role for the

development of self-sustaining ecosystem in derelict sites. The study was conducted in different

aged reclaimed coalmine overburden dumps of Jharia Coalfield to study the effect of minesoil

characteristics, host specificity, age of plantation and profile depth on spore density. Minesoil

samples were collected from the rhizosphere of different host tree species growing on different

aged dumps and spore density and size distribution was estimated. Spore density was found to

vary amongst species ranging from 6 spores/g (Gmelina arborea) to 28 spores/g mine soil

(Cassia seamea) and 86% of the spores were found within 150 ?m size (40 ?m to 320?m). In

reclaimed mine soil, highest spore density was found in 0-15 cm depth and progressively decreased

with depth. Glomus is the most predominant genus found in the mining area. High level of VAM

colonization was observed in Dalbergia sissoo, Cassia seamea, A. auriculiformis and least in

Gmelina arborea. The density of VAM spores were found maximum in mining area than in the

non-mining area. These findings will be beneficial for identifying efficient mycorrhizal trees

during biological reclamation of coal mine degarded sites.

ABSTRACT

NSave Nature to Survive

*Corresponding author

www.thebioscan.in

763

INTRODUCTION

The coalmine overburden (OB) dumps vary widely in their physical, chemical and biological characteristicsthan natural soil, which affect plant establishment, survival and growth. To reclaim these dumpsbiologically, a long term nutrient cycling between soil-plant systems has to be established. One of thesymbiotic microbes, Vesicular Arbuscular Mycorrhiza (VAM) fungi plays an important role in theestablishment of vegetation cover and initiation of nutrient cycling for the development of self-sustainingecosystem in OB dumps and other mined out areas (Mehrotra, 1991). The OB dumps initially lackviable mycorrhizal population, and thus, the establishment of a vegetative community is delayed.Mycorrhiza inoculation could enhance productivity of these dumps by increasing drought tolerance ofplants and phosphorus availability, which are the two main limiting factors for plant growth in OBdumps. A number of interacting factors which affect the successful colonization of VAM fungi in minespoils are pH, soil nutrients, organic matter, moisture, temperature and age of disturbance (Norland,1993; Mukhopadhyay and Maiti, 2008). The importance of mycorrhiza fungi in the ecorestoration ofcoal and lignite mine overburden dumps, gypsum mine spoils, iron ore spoils, limestone, magnetiteand uranium mine spoils has also been emphasized by several workers (Ganesan et al., 1991;Mukhopadhyay and Maiti, 2009). They concluded that, host plants having higher-root infections aresuitable for the biological reclamation of mining wastes. The occurrence of VAM fungi is widespread innatural vegetation, but intensity of infection varies among type of species, nature of edaphic factors, ageof the dumps and magnitude of disturbance (Maiti, 1997; Singh and Jamaluddin, 2006). Therefore thestudy of VAM status is essential for development of a self-sustaining ecosystem in OB dumps.

The present investigation was carried out to study the effect of minesoil characteristics, host specificity,age of plantation and profile depth on natural mycorrhizal colonization by considering different agedafforested coalmine overburden dumps.

MATERIALS AND METHODS

Study area

The study was carried out during November 2008- December 2009, at Bastacolla opencast projects(OCP) of Jharia Coalfield (JCF), situated in the Dhanbad district of Jharkhand, Eastern India, which fallsbetween latitudes 23º39´N and 23º48´N and longitudes 86º11´E and 86º27´E covering an area of 450km2. The mining is carried out by shovel-dumper combination. The average heights of dumps were 40-50 m with a quarry depth of approximately 60-70 m. The dumps were reclaimed by plantation of timberyielding tree species without any amendments or any pH correction. A brief location map of study areaat the Jharia coal field is shown in Fig. 1.

Figure 1: Geological map of Jharia coalfield showing study area

Vegetation analysis

The tree species consist of fast growing deciduoustypes, planted by Forest department and detailedcomposition is given in Table 1. The groundvegetation was found totally dried up, only fewnatural growth of Lantana camara, Leonotis

nepetifolia, Eupatorium odoratum, Hyptis

suavelons and Pennisetum pedicellatum wereobserved. Out of 10 tree species, three species,namely Cassia seamea, Gmelina arborea andAcacia auriculiformis constituted approximately70% of the tree population. Some accidental treespecies like F. retusa and F. relegiosa were noticed.

NATURAL MYCORRHIZAL COLONIZATION

764

Relative density of different tree species growing in the reclaimed dumps has been calculated for eachstudy area using the following formula:

S.N. Common name Scientific name and family Relative density DBH (cm)average ± SD

(English name) (%) (n) (min- max) CV%

1. Chakundi Cassia seamea L. 33 (n= 38) 50.8 ± 15.5(23-78) 30.6

(Indian wood tree) (Caesalpiniaceae)

2. Gamhar (White Teak) Gmelina arborea Roxb. 22 (n=25) 43.0 ± 10.0 (26-58) 23.30

(Verbenaceae)

3. Acacia(AustralianWattle) Acacia auriculiformis A. 15 (n=17) 45.2 ± 9.5(35-60) 20.94

Cunn. (Mimosaceae)

4. Subabool Leucaena leucocephala (Lam.) 5 (n = 6) 43.0 ± 5.9(35-48) 13.64

de Wit (Mimosaceae)

5. Shisham (Sissoo) Dalbergia sissoo Roxb. 7 (n = 8) 53.5 ± 29.0 (33-74) 54.19

(Papilionaceae)

6. Arjun Terminalia arjuna W and A 6 (n = 7) 17.94 ± 2.05(16-20.4) 11.44

(Combretaceae)

7. Prosopis Prosopis juliflora (Sw) DC. 2.6 ( n = 3) 39.0 ± 5.7 (35-43) 14.50

(Mimosaceae)

8. Chilkan Ficus retusa L. (Moraceae) 2.6 (n=3) 51.0 ± 9.5(40-57) 18.70

9. Peepal Ficus religiosa L. (Moraceae) 2.6 (n=3) 55.0 ± 2.7(53-58) 4.81

10. Bamboo Dendrocalamus strictus Nees. (Poaceae) 3.5 (n=4) ———*

Table 1: Composition of vegetation cover in the study area

* 4 dense clumps of Dendrocalamus strictus was also present; n = number of samples

Relative density (%) = x 100no. of individuals of a particular species

no. of individuals of all species

The girth of tree species was measured at the height of 1.37 m above the ground level (i.e. diameter atbreast height, DBH).

Overburden sampling and analysis

Sampling sites were selected after a geo- botanical survey of the area. The rhizospheric soil sampleswere collected from the reclaimed coalmine overburden dumps during the month of November 2008 toDecember 2009. Three different aged reclaimed OB dumps of approximately 15 yrs, 9 yrs and 5 yrs oldwere selected to study the variation of VAM spores with profile depth and age of plantation. For VAMinfection study, fine feeder roots were collected from each tree species, washed with water and storedin FAA (13mL formalin, 5mL glacial acetic acid and 200mL 50% ethyl alcohol) immediately aftercollection (Reeves et al., 1979). From the rhizosphere of each tree species, one composite sample wascollected by mixing five sub-samples and reduced the weight approximately to 0.5 kg by conning-quartering method. Five replicates were done for each tree species. After collection, the samples wereair dried at room temperature (30–35ºC) and lightly crushed with a mortar and pestle and passedthrough a 200-micron mesh. One part was used for the physicochemical analysis of OB materials andother part used for enumeration of VAM spores. Soil samples were also collected from non-miningareas as a control site.

The pH and electrical conductivity (EC) was determined in soil/water (1:2.5; w/v) suspension with a pHmeter and Conductivity meter respectively. The paste pH (1:1, w/v) and paste electrical conductivity(1:1, w/v) were also measured. Organic carbon was estimated by rapid dichromate oxidation technique(Walkley and Black, 1934), Available nitrogen was determined by the alkaline potassium permanganatemethod (Subbaih and Asija, 1956), available phosphorus by Bray’s method (Bray and Kurtz, 1966),exchangeable K, was extracted by neutral 1(N) ammonium acetate solution (soil-to-extractant ratio of1:10) and determined by Flame photometer (Jackson, 1973). The Cation exchange capacity (CEC) wasdetermined by extraction with 1N sodium acetate, followed by washing with 95% ethanol and leachedwith 1N ammonium acetate solution (Jackson, 1973).

SANGEET A MUKHOPADHYAY AND SUBODH KUMAR MAITI

765

Study of VAM spores

VAM spores occurring in the rhizospheric soil samples were extracted by Wet-sieving and sucrosedensity gradient centrifugation method (Daniels and Skipper, 1982). Approximately 10 g of soil wastaken in a 1000 mL measuring cylinder and tap water was added, mixed the content and 30 minuteswere given for settlement of heavy particles at the bottom. The suspension was decanted by using twosieves: 30-mesh (500 micron) to arrest debris and 400-mesh (38 micron) to retain all VAM spores. Thesuspension retained on 500- ìm sieve was directly examined under microscope, if any large sporocarpwas present. The material retained on the 38-ìm sieve was centrifuged in a 20-60% (w/v) sucrosegradient and centrifuged at 3000 rpm for 5 min. Spores were collected from first interface and thesucrose solution was washed off. These spores will be free of nematodes cysts and other contaminantsfound in the other interface. The supernatant was filtered in a marked-filter paper as suggested by Gaurand Adholeya (1994). The filter paper was observed under Stereo-zoom research microscope at amagnification of upto 60 to 90x (LEICA, EZ4), and spores were counted and expressed per g of soil.

The spore morphology was studied under compound microscope (OLYMPUS BX 60 Japan; transmittedlight intensity - 100W halogen; at a magnification of 210x-840x) and the image was captured by digitalcamera and transferred on-line to a computer and measurement was done with the help of Microliteimage plus software (version - 4.0). Only 60 to 85% of the spores mounted on slides could be identifiedto the species level or attributed to a specific morphospecies; the rest consisted mostly of old anddecaying spores with missing clear features. The spores were identified following the key of Schenckand Perez (1988), which is based on spore wall morphology and thickness.

Study of root infection

Approximately 1-cm size root bits were boiled in 10% KOH for 15-20 minutes in a water bath (sometimeseven 60 minutes for hard roots, like Tectona and Azadirachta), washed in distilled water, and stained inlactophenol following the method of Phillips and Hayman (1970). For confirmation of infection, thepresence of mycelia, vesicles, arbuscles or both characters of VAM infection were observed and percentageof root colonization was quantified as follows:

Root colonization (%) = x 100Number of root segments colomsed

Total number of root segments examined

Statistical analysis

One- way ANOVA (analysis of variance) was carried out to compare the means of nutritionalcharacteristics, spore density and root colonization with respect to different plants where significant Fvalue was obtained; differences between individual means were tested using DMRT (Duncan’s MultipleRange Test) at 0.05 significance level. The data were analysed using SPSS (SPSS Inc., Version 10.0).

RESULTS AND DISCUSSIONS

Characteristics of rhizospheric mine soil

The amount of non-soil fraction (>2 mm size) was found higher (67%) in mine soil than natural soil(control site). Similarly soil forming particles (<2 mm size) were also found approximately 33%,which is very low compared to natural soil (90%). The in situ moisture content was lower (3.32%),compared to natural soil (6.73%). The low moisture content in mine soil is due to lack of soil structure,higher stone content, and lack of organic matter (Maiti, 2007). The pH (1:1) was found acidic (4.8)along with high ECe value compared to natural soil, which may be attributed to geology of the rock.

The chemical and nutritional properties of rhizospheric mine soil under different tree species is shownin Table 2. The average pH was found between 4.21- 5.22 and the variation was significant amongsttree species at 5% level. Similarly ECe (1:1) was found 0.290 - 0.456 dS/m indicating there is asignificant difference amongst tree species. Maiti (2007) reported EC value in the range of 0.26-0.86 dS/

NATURAL MYCORRHIZAL COLONIZATION

766

m for reclaimed OB dumps of KD Heslong, NK Area, CCL. Highestlevel of organic carbon was observed in L. leucocephala and T. arjuna

(3%), moderate level was observed in D. sissoo, C. seamea and A.

auriculiformis (2.4-2.7%) and lowest OC was reported in G. arborea

and D. strictus (2%). These variations may be due to nature of minesoil, coal dust deposition as well as decomposition of leaf litter. Highervalues of organic carbon were observed due to accumulation of old andnew carbon fraction. It has been reported that coalmine spoil containsboth recently (new) derived carbon fractions which is added throughbiological processes, detrital matter, and ancient (old) carbon fractionsadded by fossilized plant matter, coal and inorganic carbonates (Sally et

al., 2007). The range of available Nitrogen was found between 135-184kg/ha in the rhizospheric mine soil samples. The range of available Pwas found between 4.8-7.8 kg/ha and P content in all the rhizosphericmine soil samples were found to differ significantly at 5% level.Exchangeable K was found between 108-133 kg/ha which indicates lowfertility status. Correlation between level of OC and K was found lowbecause higher level of OC is due to coaly particle and coal dust. TheCEC value was observed between 8.12- 12.15 cmol/100g soils.

Mycorrhizal root infection and spore density

The VAM spore density and root infection studied for the 7 plant speciesgrowing in the reclaimed OB dumps are presented in Table 3. Theintensity of mycorrhizal colonization was found to vary significantlywith plant species. The roots showed the presence of hyphae and vesiclesbut arbuscules were not observed (Fig. 1a, b). The presence of arbusculesis normally used to designate VAM association, but the presence ofhyphae or vesicles alone has also been used as evidence for theseassociations. Arbuscules are ephemeral structures, which may be absentif samples are collected when roots are inactive, whereas vesicles areconsidered as storage organ produced in the older region of infection.Maximum root infection was recorded in D. sissoo (94%), followed byC. seamea (90%), A. auriculiformis (89%), L. leucocephala (68%), T.arjuna (58%), D. strictus (55%) and G. arborea (38%). Variation inroot infection among different plant species might be due to thephysiology and/or morphology of the roots. Although, it has beenreported that VAM association may be reduced due to disturbance(Brundrett, 1991; Gould et al., 1996) a moderate to high level of

Pla

nt

spec

ies

pH

(1:1

)E

C(1

:1)

dS/m

OC

(%

)A

vai

lable

-A

vai

lable

-E

xch

angea

ble

-C

EC

[C

mol

N (

kg/h

a)P (

kg/h

a) K

(kg/h

a)(p

+)/

100g s

oil]

A.

auri

culifo

rmis

4.2

1e ±

0.0

20.4

14

b ±

0.0

12.3

6c ±

0.3

3184.1

7a ±

14.4

06.9

5c ±

0.3

7127.6

0bc ±

9.0

28.1

2d ±

0.2

6

C.

seam

ea4.9

5b ±

0.0

90.3

51

c ±

0.0

12.4

7c ±

0.1

6160.4

3bc ±

12.5

77.8

4a ±

0.1

0109.5

4d ±

3.1

110.0

3b ±

0.3

2

D s

isso

o4.9

1bc ±

0.0

90.3

46

d ±

0.0

09

2.6

8b ±

0.1

7163.8

0bc ±

12.3

77.5

5b ±

0.1

1108.4

0d ±

2.0

610.4

2b ±

0.6

1

G.

arb

ore

a5.2

2a ±

0.0

90.2

90

e ±

0.0

22.0

4d ±

0.1

2134.8

0d ±

13.8

14.7

6f ±

0.1

3138.9

8a ±

5.7

212.1

5a ±

1.2

0

L.

leuco

cephala

4.7

5d ±

0.0

30.3

48

d ±

0.0

13.1

5a ±

0.0

8176.1

0ab ±

12.5

46.7

5c ±

0.1

9121.5

2c ±

9.5

28.8

3c ±

0.2

6

T.

arj

una

4.8

3cd ±

0.1

50.4

56

a ±

0.0

13.0

5a ±

0.0

4170.9

3ab ±

9.4

76.3

9d ±

0.0

9133.1

6ab ±

2.5

29.8

7b ±

0.4

3

D.

stri

ctus

4.7

5d ±

0.0

50.3

62

c ±

0.0

11.5

3e ±

0.0

7153.5

7c ±

8.1

55.6

5e ±

0.1

8123.9

3c ±

3.3

29.7

0b ±

0.4

6

Table

2:

Nutr

ient

statu

s in

the

rhiz

osp

her

e of

dif

fere

nt

tree

spec

ies

gro

win

g i

n t

he

recl

aim

ed o

ver

burd

en d

um

ps

(n=

7,

Aver

age*

± S

D)

n=

num

ber

of re

plica

tes

of ea

ch tre

e sp

ecie

s; *

Diffe

rent le

tter

s in

the

sam

e co

lum

n indic

ate

signific

ant diffe

rence

s at

p<

0.0

5 a

ccord

ing to D

unca

n’s

multip

le ran

ge

test

s

S.N. Name of host species Average no. of VAM spores/g of % Root colonization

rhizospheric soil Average* ± SD Average* ± SD

1. A. auriculiformis 19cd ± 1.0 89a ± 4.05

2. C. seamea 23a ± 1.67 90a ± 2.28

3. D. sissoo 22b ± 1.58 94a ± 3.16

4. G. arborea 6f ± 1.2 38d ± 8.41

5. L. leucocephala 14e ± 1.58 68b ± 7.92

6. T. arjuna 18d ± 0.71 58c ± 7.62

7. D. strictus 20c ± 1.87 55c ± 5.69

Table 3: Average VAM spores density (no of spores/g rhizospheric soil) and intensity

of root infection in plant species growing in the reclaimed OB dumps (n=7)

n= number of replicates of each tree species; * Different letters in the same column indicate significant

differences at p<0.05 according to Duncan’s multiple range tests

SANGEET A MUKHOPADHYAY AND SUBODH KUMAR MAITI

767

mycorrhizal association observed in almost all the species in reclaimed minesoils indicates the importanceof mycorrhizal association in plants colonizing the coalmine soils. In general percent root infection andnumber of VAM spores in the rhizosphere of the plant species grown in mine soil was found higher tothose in natural soil (Rao and Tarafdar, 1998).

A variety of VAM spores were recorded from rhizosphere of different host tree species growing in thereclaimed overburden dumps. They mainly belong to Glomus, Gigaspora, Acaulospora, Enterophospora

and Sclerocystis. Glomus is the most predominant genus found in mine area. The shape and size ofVAM spores as observed under compound microscope are shown in Fig. 1c and d. The spores aresometimes found in clusters and identified using spore wall morphology and thickness. (Fig. 1e, f).Average spore density was found highest in C. seamea (23 spores/g soil), followed by D. sissoo, D.

strictus, and A. auriculiformis. As the VAM specificity is different, the density of the spore also differsfrom species to species and significant difference in spore count was found in the rhizosphere of allspecies. The number of spores in the rhizosphere of mine soil for different tree species were foundhigher (6-23 spores/ g of soil) than in the rhizosphere of same tree species growing in non-mining area,which is about 2-12 spores/ g of soil (ISM campus taken as control area). The density of spore in miningarea (1460 spores/ 100g of soil) was found more than double compared to non-mining area (680 spores/100 g of soil).

The size classification of sporeswere carried out for all thedifferent rhizospheric samplesand found in the range of 40 μmto 320μm. The maximumoccurrence of VAM spores werefound between >75-100 μm size(31%), followed by >50-75 μm(22%), less than 50 μm (19%),>100-150 μm (14%), >150-250μm (8%) and >250μm is6.4%. Distribution of spore sizefrequency is presented in Fig. 2,which shows that about 86% ofthe spores were found within 150μm size, more precisely inbetween 50 μm to 100μm size53% spores were found. Effectof age of plantation on VAMspore density in the rhizosphereof two plants namely D. sissoo

and C. seamea were studied in15 years old reclaimed OBdumps of Jharia coalfield andcompared with other reclaimeddumps planted with similarspecies. In all the places, the

VAM spores density in the rhizosphere of C. seamea was found always higher than D. sissoo. This maybe one of the reasons why growth and survival rate of C. seamea is always found highest in the OBdumps. In garden soil also (ISM campus), VAM spore density was found higher in C. seamea than in D.

sissoo. Fungal spore densities were not related to the mycorrhizal infection levels and similar observationwas also reported by Mehrotra (1998) in the reclaimed coal mine areas of Chandrapur, Maharastra.

0

5

10

15

20

25

30

35

0 50 100 150 200 250 300 350

Size of VAM spores (micron)

Figure 2: Size distribution of VAM spores in reclaimed OB dumps

No.

of

VA

M s

pore

s

Age of reclaimed overburden dump

No.

of

VA

M s

pore

s/g o

f m

ineso

il

Figure 3: Effect of profile depth on VAM spore density

0

5

10

15

20

25

30

35

40

15 9 5

0 -15 cm

15 - 30 cm

30 - 45 cm

NATURAL MYCORRHIZAL COLONIZATION

768

Several researchers reported that density of mycorrhiza spores depends on depth of soil and maximumspores were found in upper 20 cm depth (Kabir et al., 1998; Xueli et al., 2002). In the reclaimed minesoil, highest spore density was found in 0-15 cm depth and progressively decreased with depth (Fig. 3).

Figure 4: (a) Root infection showing mycelium and cortical cell (430x); (b) Root infection showing mycorrhiza vesicle

(430x); (c) VAM spores under stereozoom microscope (60 x; 87- 125μ); (d) Glomus spore showing stalk under compound

microscope (860x, 121μ); (e) Magnified view of a spore showing wall and surface sculpture (860x); (f) Cluster of VAM

spore under stereozoom microscope (60x, 70-100 μ)

a b

c d

ef

SANGEET A MUKHOPADHYAY AND SUBODH KUMAR MAITI

769

The decrease in spore density with depth is attributed due to the fact that VAM fungi are strictly aerobicin nature and as depth increases oxygen/ air availability also decreases. In case of older dumps, about33% of the VAM spores were found only alive and rest 67% dead. In case of younger dumps, thepercentage of living spores was found 70-72%. Even though the total spore content was found higher inolder dumps, the density of living VAM spores were found more or less same both in younger and olderreclaimed dumps.

CONCLUSION

Mycorrhizal colonization varied widely amongst the different tree species growing in reclaimed minesoilswhich indicates that during the selection of tree species importance of mycorrhizal colonization shouldbe considered. In reclaimed mine soil, highest spore density was found in 0-15 cm depth and progressivelydecreased with depth. The study revealed that VAM spore density was found highest in the rhizosphereof C. seamea (23 spores/g soil) followed by D. sissoo (22 spores/ g soil), A. auriculiformis (19 spores/g soil) and lowest in D. strictus (20 spores/g soil). Size of the spores were found in the range of 40 μmto 320μm out of which 86% of the spores were found within 150 μm size. Glomus is the mostpredominant genus found in the mining area. It has been observed that mycorrhizal spores count werefound higher in minesoil than non-mining areas, indicating hostile physico-chemical characteristics ofminesoil promote higher VAM colonization in host plants.

Plant possessing high level of VAM infection (75-95%) included D. sissoo, C. seamea and A.

auriculiformis. L. leucocephala, T. arjuna and D. strictus possessed moderate level of VAM colonization(50-75%) while Gmelina arborea possessed low level of colonization (38%) Fungal spore densitieswere not related to the mycorrhizal infection levels. This study will be helpful in identifying efficientmycorrhizal tree species for the dump reclamation, where available nutrients and moisture contents arealways limiting.

ACKNOWLEDGEMENT

The authors like to thank Bharat Coking Coal Limited (BCCL) for providing necessary facilities duringthe field study at Jharia coal field, Dhanbad. They are also grateful to ISM, Dhanbad for providingresearch fellowship to one of the authors and necessary support during the study. The authors also thankHOD, ESE and other technical staff for providing necessary laboratory support.

REFERENCES

Bray, R. and Kurtz, L. T. 1966. Determination of total, organic and available forms of phosphorus in soil. Soil

Sci. 59: 39–45.

Brundrett, M. C. 1991. Mycorrhizas in natural ecosystems. Advances in Ecol.Res. 21: 171-173.

Daniels, B. A. and Skipper, H. A. 1982. Methods for the recovery and quantitative estimation of propagules

from soil, in: Schenk, N.C. (Ed.), Methods and principles of mycorrhizal research. Am. Phytopathological Soc.

St. Paul, Minn. pp. 29–35.

Ganesan, V., Ragupathy, S., Purthipan, B., Rajani Rani D. B. and Mahadevan, A. 1991. Distribution of vesicular

arbuscular mycorrhizal fungi in coal, lignite and calcite mine spoils of India. Biology and Fertility of soil. 12:

131-136.

Gaur, A. and Adholeya, A. 1994. Estimation of VAM spores in the soil: a modified method. Mycorrhiza News.

6(1): 10-11.

Gould, A. B., Hendrix J. W. and Richard, S. F. 1996. Relationship of mycorrhizal activity to time following

reclamation of surface mine land in western Kentucky. 1. Propagule and spore population densities. Canadian

J. Bot. 74: 247-261.

Jackson, M. L. 1973. Soil chemical analysis. PHI, New Delhi, India. pp. 10-227.

NATURAL MYCORRHIZAL COLONIZATION

770

Kabir, Z., O’Halloran, I. P., Widden, P. and Hamel, C. 1998. Vertical distribution of arbuscular mycorrhizal

fungi under corn (Zea mays L.) in no-till and conventional tillage systems. Mycorrhiza. 8(1): 53-55.

Maiti, S. K. 1997. Importance of VAM fungi in coal mine overburden reclamation and factors effecting their

establishment on overburden dumps. Env. and Ecol.15(3): 602-608.

Maiti, S. K., Shee, C. and Jha, P. C. 2003. Status of VAMF- infections and spores in an afforested coalmine

overburden dump. Minetech. 24(4): 48-53.

Maiti, S. K. 2007. Bioreclamation of coalmine overburden dumps- with special emphasis on micronutrients

and heavy metals accumulation in tree species. Env. Monit. Assess. 125: 111-122.

Mehrotra, D. M. 1991. Mycorrhizae of India forest trees. ICFRE, New Forest, Dehradun. pp. 179-184.

Mehrotra, V. S. 1998. Arbuscular mycorrhizal associations of plants colonizing coal mine spoil in India. J.

Agri. Sci. 130(2): 125-133.

Mukhopadhyay, S. and Maiti, S. K. 2008. Identification of Sustainable Indicators to Assess the Health of

Restored Mine Degraded Land – A review. Env. and Ecol. 26(4): 1453-1461.

Mukhopadhyay, S. and Maiti, S. K. 2009. Reclamation of Mine spoils with Vesicular Arbuscular Mycorrhiza

(VAM) Fungi- A review. Env. and Ecol. 27(2): 642- 646.

Norland, M. R. 1993. Soil factors affecting mycorrhizal use in surface mine reclamation. US Department of the

Interior, Bureau of mines, Information Circular/9345. p. 21.

Phillips, J. M. and Hayman, D. S. 1970. Improved procedures for clearing roots and staining parasitic and

vesicular-arbuscular mycorrhizal fungi for rapid assessment of colonization. Transactions of the British

Mycological Society. 55: 158-161.

Rao, A. V and Tarafdar, J. C. 1998. Selection of plant species for rehabilitation of Gypsum mine spoil in aird

zone. J. Arid Env. 39(4): 559-567.

Reeves, F., Wagner, B. D., Moorman T. and Kiel, J. 1979. The role of endomycorrhizae in revegetation

practices in the semi arid west: I. A comparison of incidence of mycorrhizae in severely disturbed vs. natural

environments. Am. J. Bot. 66: 6-13.

Sally, M., Barton, Christopher, D., Karathanasis, Tasos, A. D., Rowe, Harry, D., Rimmer and Susan, M. 2007.

Distinguishing “New” from “Old” organic carbon on reclaimed coal mine sites using thermogravimetry: I.

method development. Soil Sci. 172(4): 292-301.

Schenck, N. C. and Perez, Y. 1988. Manual for the identification of VA Mycorrhizal Fungi. 2nd ed. Synergistic

publication, Gainesville, Florida, USA: INVAM (International Culture Collection of VA Mycorrhizal Fungi),

University of Florida. p. 241.

Singh, A. and Jamaluddin. 2006. Multiplication and trapping of vesicular arbuscular mycorrhiza fungi in soil

of dumps of limestone quarries. Mycorrhiza News. 17(4): 17-19.

Subbaih, B. V. and Asija, G. L 1956. A rapid procedure for the determination of available nitrogen in soils. Curr.

Sci. 25: 259-260.

Walkley, A. and Black, I. A. 1934. An examination of the Degtjareff method for determining organic carbon in

soils: Effect of variations in digestion conditions and of inorganic soil constituents. Soil Sci. 63: 251-263.

Xueli, He., Stanislav, M. and Steinberger, Y. 2002. Spatial Distribution and Colonization of Arbuscular

Mycorrhizal Fungi under the Canopies of Desert Halophytes. Arid Land Research and Management. 16(2):

149 – 160.

SANGEET A MUKHOPADHYAY AND SUBODH KUMAR MAITI