Research Paper Isolation and Taxonomic Characterization Of

Academia Journal of Microbiology Research 2(2): 061-070, December 2014 DOI: http://dx.doi.org/10.15413/ajmr.2014.0109 ISSN: 2315-7771 ©2014 Academia Publishing Research Paper Isolation and taxonomic characterization of medicinal mushroom Ganoderma spp. Accepted 25th September, 2014 ABSTRACT The present study deals with isolation, taxonomic characterization and biological activity of medicinal mushroom Ganoderma spp. The results of the present study Ganoderma sp. DKR1 and Ganoderma sp. DKR2 were isolated from two hard woods of Casurina equiestifolia and Morinda tinctoria respectively in Cuddalore district, Tamil Nadu, India. The Ganoderma isolate were characterized for macro and micro morphological properties. The colony, mycelial, basidiospore K. Rajesh, D. Dhanasekaran and A. morphology of fungal isolate was evidence of Basidiomycetes family. 18S rRNA Panneerselvam gene of Ganoderma sp. DKR1 was sequenced by automated gene sequencer. 1. Department of Microbiology, School of Sequence were submitted NCBI and in accession number is GQ495094. The Life Sciences, Bharathidasan University, phylogenetic analysis of 18S rRNA gene of Ganoderma was compared with other Tiruchirapalli, 620 024, Tamilnadu, species of Ganoderma and identified as Ganoderma sp. DKR1. The secondary India. structure and restriction sites present in the 18S RNA gene were predicted using 2. P.G. & Research Department of Botany & Microbiology, A.V.V.M. Sri Pushpam bioinformatics tools. The medicinal mushroom was isolated from hard woods of C. College, (Autonomous), Poondi, 613 503 equiestifolia and M. tinctoria. Further, Ganoderma sp. DKR1 was subjected to Thanjavur District, Tamil Nadu, India. antibacterial activity which showed inhibition against Staphylococcus aureus and Micrococcus sp. *Corresponding author Email: [email protected] Phone: +914312407082 Key words: Ganoderma sp., phylogenetic tree, 18S rRNA, fruiting body. Mobile: +919486258493 INTRODUCTION Ganoderma species belong to the kingdom of Fungi, the are available on the market including ground fruiting division of Basidiomycota, the class of bodies or mycelium processed into capsule or tablet form; Homobasidiomycetes, the order of Aphyllophorales, the extracts from fruiting body or mycelium dried and family of Polyporaceae, (Ganodermataceae) and the genus processed into capsule or tablet form, Ganoderma beer and of Ganoderma (Chang, 1995; Wasser and Weis, 1999). They Ganoderma hair tonics (Jong and Birmingham, 1992). have a woody or leathery feel and the presence of these Within the genus Ganoderma, over 250 taxonomic names pores are obvious characteristics that distinguish polypores have been reported worldwide including G. adspersum, G. from other common type of mushrooms. They degrade the applanatum, G. australe, G. boninense, G. cupreum, G. wood overtime and produce a fruiting body (or conk) on incrassatum, G. lipsience, G. lobatum, G. lucidum, G. oerstedii, the surface of the wood. Ganoderma species are among G. oregonense, G. pfeifferi, G. platense, G. resinaceum, G. those fungi that can thrive under hot and humid conditions sessile, G. sinense, G. tornatum, G. tsugae and G. webrianum. are usually found in sub tropical and tropical regions (Moncalvo et al., 1995) (Moncalvo and Ryvarden, 1998). In recent years more variety of mushrooms have been Ganoderma species are not classified as edible isolated and identified, and the number of mushrooms mushroom, as the fruiting bodies are always thick, corky being cultivated for food or medicinal purposes has been and tough and do not have the fleshy texture characteristic increasing rapidly. Mushroom “nutraceuticals” are of true edible mushroom such as the common white button bioactive compounds that are extractable from mushrooms, mushroom Agaricus bisporus. Although they are not and they have nutritional and medicinal features that may classified as edible, several types of Ganoderma products be used in the prevention and treatment of disease (Chang Academia Journal of Microbiology Research; Rajesh et al. 062 and Buswell, 1996). Several nutraceutical products have biological activity of Indian Ganoderma species from hard been isolated from medicinal mushrooms and three of wood of Casurina equiestifolia and Morinda tinctoria, these, which are carcinostatic polysaccharides drugs, have Cuddalore district, Tamil Nadu, India. been developed from mushroom in Japan. These are “Krestin” from the cultured mycelium of Kawaratake (Trametes vercicolor), “Lentinan” from the fruiting bodies of MATERIALS AND METHODS Shiitake (Lentinus edodes) and “Schizophyllan” from the cultured fluid of Suehirotake (Schizophyllum commune) Collection of Ganoderma spp. (Mizuno, 1995). Lentinan and Schizophyllan are pure Beta - glucans is a protein bound polysaccharide. Particular focus The two wood rotting fungal species of Ganoderma were on Ganoderma species, it is apparent that most of the collected from hard wood tree such as C. equiestifolia and M. available data on active extracts and compounds relates to tinctoria Cuddalore district, Tamil Nadu. The fungal fruiting the pharmacological effects on tumor cells, which appear to body was harvested and transferred to sterile polythene be based on the enhancement of the host’s immune system. bag. The specimen was brought to the laboratory for Early work in the 1950s found that a number of further investigation. Basidiomycetous fungi had antibacterial activities. More recently, several studies have demonstrated the antibacterial activity of Basidiomycetes (Coletto et al., Isolation of Ganoderma spp. 1994; Bianco and Striano, 2000). The mycelial extracts of Basidiomycetes exhibited antibacterial activity against The potato dextrose agar (peeled Potato 20 g, Dextrose 2.0 Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, g, Agar 2.0 g, Distilled water 100 ml) medium was prepared Escherichia coli, Pseudomonas aeruginosa, Salmonella sp. and sterilized at 121°C for 15 min, 15 lbs in autoclave. The and Candida albicans. Recently, more studies demonstrated sterile medium was transferred to sterile Petri plate and that G. lucidum and other Ganoderma species showed allowed for solidification, the small piece of fungal fruiting antibacterial constituents those are able to inhibit Gram- body was inoculated in PDA medium under aseptic positive and Gram-negative bacteria (Yoon et al., 1994; Li et condition. The plates were incubated at room temperature al., 1999). The aqueous extract from the carpophores of G. for 48 to 72 h. lucidum inhibited 15 types of Gram-positive and Gram- negative bacteria at different extracts using the serial broth dilution method, with the most potent inhibition against Characterization of Ganoderma spp. Micrococcus luteus [minimum inhibitory concentration (MIC) 0.75 mg/ml)]. Macro morphological characterization For a long time past, classification of Ganoderma mushroom is based on morphological characteristics, which The Ganoderma species were collected from hard wood is often highly subjective, with unambiguous identification tree and the following property of fungi were observed to the species level often being extremely difficult. such as size, surface colour, attachment of stipes to pileus, Additionally, the morphological differences observed may margin pattern and surface pattern in naked eyes. be the product of simple mutations or media, cultivation effects and thus are not always reliable characters. It limited the accuracy of identification, and the genetic Micro morphological characterization variety of different strains cannot be objectively described. In production, some strains identification and Lacto phenol cotton blue staining circumscription were often unclear and taxonomic segregation of the genus remained controversial, and even A drop of lacto phenol cotton blue was placed at the centre a number of Ganderma strains have been misnamed. of the glass slide and a portion of fungal colony was The limitations of traditional identification techniques transferred to the stain. Then a clean cover slip was kept indicate that alternative methods need to be developed for over the specimen. The micro morphology of the fungi was the identification of these fungi. With the development of observed under microscope (Labomed). molecular biology, some new techniques have been applied to fungal classical taxonomy. DNA fingerprinting techniques, however, would be allowed to identify the Thin section procedure Ganoderma species and cultivars, indicating that it is a useful tool for the valid protection of newly bred cultivars. The fruiting body of Ganoderma sp. was taken and small The review on medicinal mushroom Ganoderma research in thin sections were taken using sharp blade. The thin section Tamil Nadu, India is very scanty, the present research is was cleaned with distilled water and placed on a clean glass planned with isolation, taxonomic characterization and slide. Then a clean cover slip was kept over the specimen Academia Journal of Microbiology Research; Rajesh et al. 063 and the micro morphology of the polypore was observed (1985) with modifications as described by Moncalvo et al. under phase contrast microscope (Labomed). (1995). Fungal biomasses were harvested by filtration. A mass of 50 mg of biomass was suspended in 500 μl of extraction buffer (200 mm Tris HCl, pH 8.5, 250 mm NaCl, Measurement of fungal cell using micrometer 25 mm EDTA, 0.5% SDS) and vortexed. The resulting suspension was extracted with 350 μl of ice cold phenol: An ocular micrometer was inserted in the eyepiece. The chloroform:

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