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Research Paper

International Journal of Informative & Futuristic Research ISSN (Online): 2347-1697

Volume 2 Issue 5 January 2015

Abstract

Several species of Ganoderma were found in the state of Tamil Nadu, India. Totally forty six strains were collected from Thanjavur, Thiruvarur, Nagappattinam, Trichirappalli, and Salem districts. Then it was cultured using potato dextrose agar medium and identified using standard manuals. The distribution of these species in Tamil Nadu state was provided with map. Microscopical identification was done with lactophenol cotton blue and confocal microscopically staining. Then the secondary metabolites were qualitatively studied.

1. Introduction

G.lucidum, an oriental fungus, has been widely used as a remedy to promote health and longevity

in China and other Asian countries (Shiao 1994). Fungi from the family of Polyporaceae are

classified as such as they have many tiny holes on the underside of the fruiting body, which are

pores that contain the reproductive spores. They have a woody or leathery feel and the presence of

these pores are obvious characteristics that distinguish polypores from other common types of

Diversity Of Ganoderma Lucidum (Curt.: Fr.)

P. Karst. In The Subtropical Area Of Tamil

Nadu, India Paper ID IJIFR/ V2/ E5/ 050 Page No. 1398- 1409 Subject Area Microbiology

Key Words Fungi, Basidiomycetes, Taxonomy, Biodiversity, Distribution

M. Nithya 1

Research Scholar

P.G and Research Department of Botany and Microbiology,

A.V.V.M Sri Pushpam College (Autonomous), Poondi-

Thanjavur, Tamilnadu

Dr.V.Ambikapathy 2

Assistant Professor




Dr. A.Panneerselvam 3

Associate Professor




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ISSN (Online): 2347-1697 International Journal of Informative & Futuristic Research (IJIFR)

Volume - 2, Issue - 5, January 2015 17th Edition, Page No: 1398-1409

M.Nithya, Dr.V.Ambikapathy, Dr. A.Panneerselvam:: Diversity of

Ganoderma lucidum (Curt.: Fr.) P. Karst. in the Subtropical area of

Tamil Nadu, India

mushrooms. Polypores, like other fungi, grow on wood as an expansive network of microscopic

tubes known as mycelium. They degrade the wood over time and produce a fruiting body (or conk)

on the surface of the wood. Ganoderma species are among those fungi that can thrive under hot

and humid conditions and are usually found in subtropical and tropical regions (Moncalvo &

Ryvarden, 1997).

Ganoderma species are not classified as edible mushrooms, as the fruiting bodies are always thick,

corky and tough and do not have the fleshy texture characteristic of true edible mushrooms such as

the common white button mushroom, Agaricus bisporus. Although they are not classified as

edible, several types of Ganoderma products are available on the market including; ground fruiting

bodies or mycelium processed into capsule or tablet form; extracts from the fruiting body or

mycelium dried and processed into capsule or tablet form or tea, Ganoderma beer and Ganoderma

hair tonics (Jong & Birmingham, 1992). Within the genus Ganoderma, over 250 taxonomic names

have been reported worldwide (Moncalvo et al., 1994; Ryvarden, 1991) including: G. adspersum,

G. applanatum, G. australe, G. boninense, G. cupreum, G. incrassatum, G. lipsiense, G. lobatum,

G. lucidum, G. oerstedii, G. oregonense, G. pfeifferi G. platense, G. resinaceum, G. sessile, G.

sinense, G. tornatum, G. tsugae and G. weberianum, to name a few. However, the majority of

reports in the literature appear to be on the one species, G. lucidum. The genus Ganoderma was

established by Karsten in 1881. Correct citation of the type species is written as G. lucidum (Curt.:

Fr.) P. Karst.

1.1 Taxonomical Classification

Kingdom : Fungi

Domain : Eukarya

Division : Basidiomycota

Class : Basidiomycetes

Order : Polyporales

Family : Ganodermataceae

Genus : Ganoderma

Species : lucidum

1.2 Vernacular Name

English : Ganoderma or Polyporus

Tamil : Red Mushroom

China : Lingzhi(supernatural mushroom)

Japan : Reishi

Korea : Hangul or Yeongji

Hausa : Leman kwado or Burtuntuna

Vietnam : linhchi

2. Material And Methods

This study has been undertaken with periodical collections and identification of the materials.

Fruiting bodies of G.lucidum Ganodermawere collected from various places of Thanjavur,

Thiruvarur, Nagappatinam, Salem and Trichirappalli districts. At the same time, we isolated tissue

cultures from the basidiomes for further studies. The study of the basidiomes was made on macro

(size, colour, number pores/mm, length of tubes) and microscopic characters (somatic and

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Tamil Nadu, India

reproductive structures). Colours are according to Munsell (1975) and Herbaria abbreviations

follow Holmgren et al. (1990).

2.1 Isolation Of G.lucidum Ganoderma(Chen And Hu 1995)

Tissue pieces of Ganoderma fructification were surface sterilized with tap water and 0.1 %

mercuric chloride solution, for 2 min rinsed in distilled water. Fruiting bodies of G.lucidum

Ganodermaisolates were done onto potato dextrose agar (PDA) medium. After inoculation the

fungal colonies cultures were purified using pure culture technique and stock culture was

maintained in PDA slants for further studies.

2.2 Identification

For confirmation of identification, taxonomic keys and descriptions were consulted.

Descriptions of basidiomycetes were made according to their macro, micro and cultural features by

using standard manuals such as Manual of soil fungi, (Gilman 1957) Dematiaceous Hyphomycetes

(Ellis 1971). Colony color, morphology, hyphal structure, spore size and spore bearing structures

were identified and compared. The fungal organism was identified and confirmed by Kimbrough

from DDIS, University of Florida (1999), Gilbertson and Ryvarden (1986), Parker, Alaska‟s

Mushrooms (1996), Arora Mushrooms Demystified (1979) Dickinson “The Encyclopedia of

Mushrooms (1979), Joseph C. Gilman of Soil Fungi (1957).

2.3 Nikon Microscopical Observation

Morphological observations mainly followed by the methods of Wang et al (2006). Lacto phenol

cotton blue staining was used as the mounting medium. Microscopic characters were observed

using a light microscope. For microscopic observation dermic elements were carefully examined &

measured in thin sections perpendicular to the pileus surface.

2.4 Confocal Microscopical Observation (Palmgreen 1991)

Light emitting dye preparation - Acride Orange

Acridine Orange 6 µg/ml

Citric acid 0.1 M

Na2HPO4 0.2 M pH 2.6

80 ml of citric acid solution, 10ml of acridine orange and 10ml of Na2HPO4 was mixed. It was

stable for several weeks when stored at 4°C and in the dark. Acridine orange staining was used for

mounting. Microscopical observation was noted in 525nm.

2.5 Grinding

The sporocarps were cut in to small pieces, dried at 40° C for 48 hours and powdered. In

each step, the plant material was dried to remove moisture and overcome the fungal contamination.

The air-dried powder was stored in an air tight container for further use. (Dandan 2009).

2.6 Extract Preparation

Various extracts of the experimental samples were prepared according to the methodology of

Indian pharmacopoeia. (Anonymous 1955)

The chemical nature and physical state of the mushroom powder make it difficult to dissolve in

distilled water. So, the plant materials were soaked in distilled water for 24hrs. One gram of

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powder was dissolved separately in 10ml ethanol, diethyl ether, chloroform and distilled water in

cleaned screw cap bottle for 24hrs. After 24hrs the dissolved extracts from the bottels were

transferred to centrifugal tubes and centrifuged at 3000rpm for 10min. The centrifuged extracts

(supernatant) were again re-centrifuged and filtered with millipore filter. The filtered solvents with

dissolved chemicals were concentrated and stored separately in refrigerated at 4°C.

2.7 Qualitative Screening Of Phytochemicals From Mushroom Extract (Allen 1976 and

Harborne 1976): The extracts were subjected to various tests to screen phytochemicals tannins,

alkaloids, saponins, cardiac glycosides and terpenoids.

Table 2.1: Phytochemicals From Mushroom Extract

S.No Phytochemicals Chemicals Added

1. Tannins 2ml ethanol extract+3ml D.H2O+ 2drops 5% FeCl3

2. Alkaloids 200mg mushroom powder+10ml methanol+1% HCl

3. Saponins 5ml extract+ 5ml D.H2O

4. Cardiac glycosides 2ml filtrate+1ml glacial acetic acid+ FeCl3+Con.H2SO4

5. Terpenoids 2ml filtrate+2ml acetic anhydride+ Con.H2SO4

3. Results And Discussion

Remarks were included for each species besides a synoptic comparison between all species

considered (Tab. 3.1 & Fig 3.2.) and the distribution pattern of species recorded for the state of

Tamil Nadu (Fig. 3.1).

Thiruvarur

Figure 3.1: Distribution map of Ganoderma lucidum on Tamil Nadu State, India

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Table 3.1: Collection of G. lucidum from five different districts

S.No Location Substrate Isolate code

1. Paeraiyur Bambusa vulgaris VAMNG-I

2. Mulaiyur Bambusa vulgaris VAMNG-II

3. Kudikadu Aluvial Soil VAMNG-III

4. Cholapuram Morinda oleifera VAMNG-IV

5. Aadudhurai Cocos nucifera VAMNG-V

6. Pandhanallur Syzigium cumini VAMNG-VI

7. Sirkali Musa paradisiacal VAMNG-VII

8. Ammachatthiram Barks VAMNG-VIII

9. Valangaiman Barks VAMNG-IX

10. SP Kovil Aluvial Soil VAMNG-X

11. Pirandakkottai Ficus religiosa VAMNG-XI

12. Sadayar Kovil Ficus benghalensis VAMNG-XII

13. Kattur Tamarindus indica VAMNG-XIII

14. Kattur Ficus benghalensis VAMNG-XIV

15. Aadhanakkottai Ridges VAMNG-XV

16. Thanjavur Tamarindus indica VAMNG-XVI

17. Pudhur Tamarindus indica VAMNG-XVII

18. Vadakarai Cocos nucifera VAMNG-XVIII

19. Andhamangalam Ficus religiosa VAMNG-XIX

20. Mannambandhal Cocos nucifera VAMNG-XX

21. Sitthakkadu Cocos nucifera VAMNG-XXI

22. Kambarasam Paet Cocos nucifera VAMNG-XXII

23. Thanjavur Morinda oleifera VAMNG-XXIII

24. Orathanadu Tamarindus indica VAMNG-XXIV

25. Orathanadu Ridges VAMNG-XXV

26. Salem Andrographis paniculata VAMNG-XXVI

27. Salem Aluvial soil VAMNG-XXVII

28. Orathanadu Tamarindus indica VAMNG-XXVIII

29. Orathanadu Ficus benghalensis VAMNG-XXIX

30. Orathanadu Aluvial soil VAMNG-XXX

31. Orathanadu Ficus religiosa VAMNG-XXXI

32. Orathanadu Tamarindus indica VAMNG-XXXII

33. Orathanadu Tamarindus indica VAMNG-XXXIII

34. Swamimalai Ridges VAMNG-XXXIV

35. Thiruvidaimarudhur Aluvial soil VAMNG-XXXV

36. Orathanadu Morinda oleifera VAMNG-XXXVI

37. Ammapet Tamarindus indica VAMNG-XXXVII

38. Kudikadu Aluvial soil VAMNG-XXXVIII

39. Sembanar kovil Tamarindus indica VAMNG-XXXIX

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40. Pulavar nattham Aluvial soil VAMNG-XXXX

41. Orathanadu Tamarindus indica VAMNG-XXXXI

42. Orathanadu Aluvial soil VAMNG-XXXXII

43. Kumbakonam Bambusa vulgaris VAMNG-XXXXIII

44. Kumbakonam Bambusa vulgaris VAMNG-XXXXIV

45. Thiruvarur Bambusa vulgaris VAMNG-XXXXV

46. Thanjavur Tamarindus indica VAMNG-XXXXVI

a b

c d

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e f

g h

Figure 3.2 :(a-h) Forty six strains of Ganoderma lucidum

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Figure 3.3: Pure culture of G.lucidum

4. Microscopical Structure Of Spores And Hyphae Of G.Lucidum

Basidiocarp annual, mostly stipitate, rarely sessile or only with a short base, corky. Pileus

1.5-9 × 2.8-16.5 cm, up to 2 cm thick, flabellate, reniform, dimidiate, sometimes with pilei

growing from the lower pilei or growing together, up to 36 cm long and 54 cm wide in total; upper

surface orange-yellow, orange-red to red-brown, weakly sometimes with melanoid substances,

corky; generative hyphae 2-5 μm in diam., colorless, thin-walled, with clampconnexions; skeletal

hyphae. (Dong-Mei Wang et al., 2009).

In these findings the microscopical structures of mycelium was aerial and have

thick walled hyaline. Basidiomata annual, stipate brilliantly laccate, mycelia mat white to light

cream scattered, more or less extensive, much branched thin walled hyphae often described as

“witches broom”. Aerial mycelium and the lateral branches may become veryshort. Thick walled

hyaline, branched aseptate 1.5-3.0mm, Ellipsoid to avoid, 8.5-12.5 × 5.5-7.5mm.

Figure 4.1: Spores and hyphae of G.lucidum Ganodermaon Nikon Microscope

40X 40X

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Figure 4.2: Hyphae and Spores of G.lucidum Ganodermaon Confocal Microscope

Figure 4.3: Powder & Extract of G. lucidum

Table 4.1: Phyto chemical analysis of G.lucidum Ganodermaextract

Isolate code

Phytochemicals

Tannins Alkaloids Saponins Cardiac

glycosides Terpenoids

VAMNG-I + + - + +

VAMNG-II + + - + +

VAMNG-III + + - + -

VAMNG-IV + + - + +

VAMNG-V + + + + +

VAMNG-VI + + + + +

VAMNG-VII + + + + +

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Tamil Nadu, India

VAMNG-VIII + + - - -

VAMNG-IX + + + + +

VAMNG-X + + + + +

VAMNG-XI + + + + +

VAMNG-XII - - + + +

VAMNG-XIII + + + + +

VAMNG-XIV + + - + +

VAMNG-XV + + + + +

VAMNG-XVI + - - + +

VAMNG-XVII + + - + +

VAMNG-XVIII + + + + +

VAMNG-XIX + + + + +

VAMNG-XX + - + + +

VAMNG-XXI + - + + +

VAMNG-XXII + + - - +

VAMNG-XXIII + + + + +

VAMNG-XXIV + + + + +

VAMNG-XXV + + + + +

VAMNG-XXVI + + + - +

VAMNG-XXVII + + + + +

VAMNG-XXVIII + + + + +

VAMNG-XXIX + + + - +

VAMNG-XXX + + + + +

VAMNG-XXXI + + + - +

VAMNG-XXXII + + + - +

VAMNG-XXXIII + + + - +

VAMNG-XXXIV + + + - +

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5. Conclusion

Diversity and phytochemical properties of G.lucidum Ganodermawill provide a knowledge for

further study and it might be a potential source of antitumor agent. Further study is required for

isolating secondary metabolites.

Acknowledgement

The authors are thankful to the Secretary and Correspondant, A.V.V.M. Sri Pushpam College,

Poondi, Thanjavur, Dept. of Microbiology, Bharathidasan University, Trichy for providing

laboratory facilities and Department of Science and Technology for granting financial assistance to

“INSPIRE FELLOW” for research work.

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