INTRODUCTION

liPage 1.1 Kingdom Fungi and its diversity

Fungi were separated from Kingdom Plantae and are classified into a separate Kingdom Mycota (Whittaker, 1969). Whittaker classified fungi as a Kingdom on the based of fOllowing criteria, 1. These organisms are non photosynthetic and absorb nutrients produced by other organisms (saprophytes I parasites). 2. They differ from plants in their cell wall composition, thallus structure and mode of reproduction (Whittaker, 1969). The fungal cell wall is made up of chitin, cellulose or both

Fungi is the most diverse Kingdoms among all (Kirk, 1995; Kirk eta!., 2001) which includes mushrooms, molds, rusts, smuts, puflballs, truffles, morels and yeasts (Alexopoulos et al., 1996). More than 70,000 species of fungi have been described; however, sorre estimates of total numbers suggest that 1.5 million species may exist (Hawksworth, 1991; Hawksworth et a!., 1995). They have successfully adapted themselves for all types of habits and habitats. Different habits and habitats have been explored which includes fungi from soil, plant parts, litter, herbivore dung, entomogenous, freshwater and marine (Barron, 1968; Ellis, 1971, 1976; Ingold, 1975; Matsushima, 1971, 1975; Subramanian, 1971, 1986; Dix and Webster, 1995; Kohlmeyer and Kohlmeyer, 1979). Some fungi are microscopic and other extends for more than a thousand acres like mycorrhizal fungi

Fungi have generally been identified and classified based on morphology of spores and spore producing structures. Criteria such as cultural characters and developrrental biology have been added as useful tools in subsequent years. Reliable diagnostic tools and excellent taxonomic keys are available fur identification of fungi (Kirk et al., 2001).

Over the years, several studies have been done to document the diversity of fungi from various furest types (Dix and Webster, 1995). A landmark paper provided several qualified estimates of the number of fungi on the Earth based on ratios of known fungi to plant species in regions where fungi were considered to be well studied (Hawksworth, 1991). "Estimate G" of 1.5 million species was accepted as a reasonable working hypothesis based on a to plant ratio of 6:1, in contrast to the much lower 50 to 60 years old estimates by Bisby and Ainsworth (1943) of 1,00,000 fungal species and by Martin (1951) of 2,50,000 species based on one

21Page fungus for every Phanerogam known at the time.

A more recent estimate of the total number of fungi is also low (7,20,256) compared to present estimates that include enviromrental samples (Schmit and Mueller, 2007). The Dictionary ofFungi (Kirk et al., 2008) reported 97,330 species of fungi with descriptions. The addition of 1300 microsporidians brings the total of all described fungi to about 99,000 species. The Dictionary's estimate ofknown species has almost tripled in the period between the first edition in 1943 (38,000 described species) and now, amounting to an increase of more than 60,000 described species over the 65 years period (Jarres et al., 2006; White et al., 2006; Hibbett et al., 2007; Kirk eta!., 2008; Lee et al., 2010).

Since the discovery ofPenicillin from the fungus Penicillium (Fleming, 1929), there has been much focus on the production of antibacterial agents from the filamentous fungi (Al-Hilli and Smith, 1992; Fischer et al., 2000; Florianowicz, 1998; Larena and Melgarejo, 1996; Mayordomo et al., 2000; Rodrigues et a/., 2000). Although there is early reference to the antibacterial activities exhibited by fungi that belong to the subdivision, (Brian, 1951; Robbins et al., 1947; Takeuchi, 1969), it is only within the last ten years that a broader range of genera, species and isolates from this division has been explored in more detail for antibiotic properties (Anke et al., 1980; Coletto and Mondino, 1991; Lorenzen and Anke, 1998; Rosecke and Konig, 2000; Wasser and Weis, 1999a, b, c and Wasser, 2002).

Medicinal properties of the fungi are mainly due to presence of various secondary metabolites. These secondary rretabolites composition varies from species to species; hence correct identification is very crucial. Due to wide applications and limited morphological characters for classification it is difficult to identify fungi only on the basis of its morphology. Hence molecular markers were introduced to understand classification and evolution of this group.

Recently, molecular tools such as polymerase chain reaction (PCR) have facilitated taxonomic identification and examination of phylogenetic relationships among different fungal groups. Using this as a principle process, several methods such as DNA hybridization, restriction enzyme analysis (RFLP), amplification using random primers (RAPD), amplification of microsatellite DNA, sequence analysis, electrophoretic karyotyping, etc. have been created and used in diversity as well as

3IPage evolutionary studies (Bruns et al., 1991). With high throughput geoomic sequence database on living organisms available at gene banks (NCBI), are use to understand similarities and differences at gene level between fungi and other living organisms (Hsiang and Baillie, 2004). Factors such as difficulty of isolation and failure to apply tmlecular rrethods may contribute to lower numbers of species in certain groups, but there cannot be any doubt that Ascomycetes and Basidiomycetes col11'rise the vast majority of fungal diversity (James et al., 2006; White et al., 2006; Hibbett et al., 2007; Kirk et al., 2008 and Lee et al., 2010).

Hawksworth's (1991) estimate of 1.5 million species is considered to be conservative, because of numerous potential fungal habitats and localities remain understudied (Hawksworth and Rossman, 1997; Hawksworth, 2001). Furthertmre, the use of molecular methods had not yet been considered as a means of species discovery. For example, analysis of environmental DNA samples from a soil community revealed a high rate of new species accumulation at the site, and these data supported an estimate of 3.5 to 5.1 million species (O'Brien et al., 2005 and Blackwell, 2011).

The Kingdom Fungi incorporates an enortmus diversity of taxa with varied ecology, life cycle strategies and tmrphologies. The Kingdom Fungi (Eumycota) was further classified into four Divisions/Phylum, which includes Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota (Webster and Weber, 2007). The rDNA study revealed that Divisions Chytridiomycota and Zygomycota are primitive and Divisions Ascomycota and Basidiomycota are evolved amongst all (Wilmotte et al., 1993; Hibbett and Thorn, 2001; Swann and Taylor, 1995; Wells and Bandoni, 2001; Hibbett, 2006 and Hibbett, 2007).

1.2 Division: Basidiomycota

The division Basidiomycota is a large and diverse group encompasses mushrooms, boletes, puffballs, earthstars, stinkhorns, bird's nest fungi, jelly fungi, bracket or shelf fungi, rust and smut fungi (Alexopoulos et al., 1996). The rrembers of Basidiomycota reproduced sexually as well as asexually. The tmst prominent and familiar Basidiomycota developed mushrooms fruiting body as a sexual reproductive structures (Fell et al., 2001). It encloses about 30,000 described species and covers a

41Page total of37% of the described species oftrue Fungi (Kirk eta/., 2001). Basidiomycota are found in virtually all terrestrial ecosystems, as well as freshwater and marine habitats (Kohlmeyer and Kohlmeyer, 1979; Hibbett and Binder, 2001 ).

The main characteristic feature of Basidiomycota is that, they all produce basidiospores (sexual spore) exogenously on a reproductive organ called a basidium from which this division takes its natre. The each cell in the thallus contains two haploid nuclei resulting from a mating event, is another characteristic feature. C.lamp connections are a kind of hypha! outgrowth that is unique to Basidiomycota, with sotre exceptions.

Even though Basidiomycota consist of all kinds of morphologically variable fruit bodies, they proved to be a rrx:mophyletic lineage arrx>ng fungi in the phylogenetic analysis of rDN A fungal sequences (Wilmotte et al., 1993; Hibbett and

Thorn, 2001; Swann and Taylor, 1995; Wells and Bandon~ 2001; Hibbett, 2006 and Hibbett, 2007). Ascomycota and Basidiomycota were found to be rrx>nophyletic groups derived from Chytridiomycota and Zygomycota (Bruns et al., 1992; Wilmotte et al., 1993). Molecular evolution studies revealed that Basidiomycota and Ascomycota, non-flagellated fungal Division, are the mostly closely related (Bowman et al., 1992).

According to McLaughlin et al., (2001) recent classification Phylum Basidiomycota divided in to 4 classes viz. Homobasidiomycetes (fungi with holobasidia, e.g. Agarics and ), Heterobasidiomycetes (fungi with heterobasidia, e.g. Jelly fungi), Urediniomycetes (e.g. Rust fungi) and Ustilaginomycetes (e.g. Smut fungi). Further class Homobasidiomycetes divided in to 8 clades. The Polyporide clade consists of :family (McLaughlin et al., 2001) and members of :family Ganodermataceae have been considered for the present investigation.

1.3 Family: Ganodermataceae

The members of :family Ganodermataceae Donk. are characterized by unique double-walled basidiospores. The shape and size of basidiospores and the texture of pileus surfaces are important characteristics that distinguish this family menber. Overall the members belong to this :family displays following characters:

51 Page Basidiocarps may be perennial or annual, which may be stipitate or pileate. If present, round or flattened, dull to shiny and usually have a cuticle, pore surfuce whitish when actively growing and become ochraceous to dark brown while ageing. Pileus is glabrous or velutinate, brown to bay or black or yellow, dull or shiny, often sulcate, with or without a cuticle. Hyphal system is dimitic to trimitic, generative hyphae are with clamps, often difficult to find. The cuticle in many species constitutes of short-celled and thick-walled coloured. The generative hyphae are seen in only fresh specimen, however, the clamps are usually difficult to observe. The skeletal hyphae are present in all species, either unbranched or arboriform with a long unbranched lower segment and a branched upper part The binding hyphae may or may not be present. Pores in general are small round to oval or angular in shape. The tubes are stratified, context light to dark brown, two or with several bands or zones. Basidia are with 4 sterigmata, usually barrel-shaped. Basidiospores are double-walled, the inner one verruculose to ornamented, thickened and usually dark to light brown coloured, on which there is a thin hyaline outer wall and lacking cystidia.

Family Ganodermataceae consists of 5 genera namely , Amauroderma, Tomophagus, Humphreya and Hoddowia and 220 species in total (Moncalvo and Ryvarden, 1997). The genus Ganoderma has further subdivided into two subgenera based on pileus (upper layer) morphology. The subgenera Ganoderma was created for laccate (shiny pileus) species, which comprise of 118 species, and subgenera Eljvingia was created for non laccate (dull pileus) species, which contain 30 species (Moncalvo and Ryvarden, 1997).

The members of this fumily are cosmopolitan in distribution, of which most are found in the tropics and temperate geographical areas, including North and South America, Africa, Europe, and Asia (Moncalvo et al.,1995a, b and Ryvarden, 1991). They grow as a parasite or saprotrophic on a wide variety of host plants including; hardwoods, conifers, bamboos and palms (Ho and Nawawi, 1985; Buchanan, 2001; and P ilotti et al., 2003 ).

1.4 Genus: Ganodenna

The genus name Ganoderma is Greek in origin. Which is a fusion of two words ganos means brightness (sheen/shining) and derma means skin, hence the

6IPage fungus having a shining skin is Ganoderma. The c1assification of this genus 1s as follows.

Ganoderma is 1argest genus in order with more than 300 species. Basidiospore shape and structure of the pilear surface are mainly used as primary taxonomic character (Moncalvo et al., 1994, 1995 a, b). The typical basidiospore of Ganoderma is ovoid, echinulate and en1arged or trauncated at the apex. Karsten in 1881 established the genus Ganoderma with the type species G. lucidum and nurmer of species has been described in the genus thereafter. The genus Ganoderma was divided in two sub-genus, Ganoderma and Elfvingia by Karsten (1889). The sub­ genus Elfvingia was based on the name Boletus applanatus and it was dedicated to the species with non-1accate pileated fruit body. It is notorious pathogenic fungi known to cause root or butt rot of the hardwood trees, and also known as medicinally important mushroom in the Asian continent.

1 C1assification ofGanoderma (based on l0 h edition of the Dictionary of the Fung~

Kingdom: Fungi

Phylum: Basidiomycota C1ass: Subclass: Incertae sedis Order: Polyporales Family: Ganodermataceae Genus: Ganoderma P.Karst. Subgenera: Ganoderma P.Karst. (Laccate sp.) Elfvingia P.Karst. (Non-1accate sp.)

1.5 Pathogenicity of Ganoderma

Ganoderma is a Basidiomycetes genus that involves ofboth saprophytic and phytopathogenic species. The genus is worldwide in distribution Most species of Ganoderma are pathogenic, causing root rot disease on a variety of monocots, dicots and gymnosperms (Seo and Kirk, 2000), which results in the death of affected trees. Sorre species act as :facultative parasite, causing white rot of commercially important hardwoods such as oak, maple, and ash by decomposing lignin as well as cellulose

71Page (Adaskaveg et al., 1991, 1993). The genus is particularly diverse in the tropics where it affects plantation crops such as oil palm, coconut, rubber, betel nut, tea and forest trees such as Acacia (Chee, 1990; Singh, 1991; Ariffin et al., 2000; Lee, 2000; Flood et al., 2000). The rot disease has become epidemic in certain regions of the world, incurring significant economic losses to fOrestry and the oil palm industries. For example, G. boninense is the major pathogen of oil palm in Malaysia and Papua New

Guinea (Ho and Nawaw~ 1985; Pilotti et al., 2003). G. lucidum is a common root pathogen that causes the decay and slow decline of numerous forest tree species (Bakshi, 1976 and Ko, 2009). G. philippii has also been considered as a serious root parasite in tropical Asian plantations (Steyaert, 1980; Foroutan and Jafary, 2007).

1.6 Economic importance of Ganode171Ul

Some species of Ganoderma have been used as a folk medicine in countries like China, Korea and Japan from ancient time. In China Ganoderma is known as Lingzhi, while in Japan it is known as Reishi; has been used for more than last 40,000 years, as a medicinal herb and a symbol of good fOrtune and prosperity (Zhao and Zhang, 1994). Ganoderma known for its multiple uses, hence has been included in the list of life saving drugs from traditional Chinese medicine system hence called as 'King of Herbs', it is also known as 'Mushroom of Medical Wonders' (Willard, 1990). The medicinal value of these species include treating diseases such as gastric ulcer, chronic hepatitis, hypertension, nephritis, asthma, arthritis, bronchitis, insomnia, cancer, diabetes and anorexia (Jong and Birmingham, 1992).

In the recent years, there has been a renewed interest in the in the therapeutic use ofGanoderma and a demand to reveal the structure ofpossible active principles. This has also raised a question on its correct identification and proper application of names to the many species and strains that are grown for pharmacological or medical use (Moncalvo and Ryvarden, 1997). Ganoderma species are a source of novel metabolites like polysaccharides, steroids, lignins, lectins, ganomycins, vitamins, alkaloids, amino acids and triterpenes that they produce. These coll1'ounds have antitumor, antibacterial, immunomodulatory, antiinflammatory, antiviral and antioxidative activities (Jong and Birmingham, 1992). Species of Ganoderma are economically and ecologically important as a source of medicinal and neutraceutical products (Buchanan, 2001 ).

8IPage Nmrerous clinical studies showed that components of Ganoderma have several tredicinal effects; such as, Immunomodulating agents, which can be used in tissue transplantation, were also derived from G. lucidum (van der Hemet al., 1995). In addition, water extracts from G. lucidum. G. formosanum and G. neo-japonicum showed free radical scavenging and anti hepatotoxic activities (Lin et al., 1995). Ganoderic acids can inhibit Ras dependent cell transformation, immunomodulating activity and free-radical scavenging (Lin et al., 1995; Park et al., 1997, 1998; van der Hemet al., 1995 and Lee et al., 1998). The fruiting body residue of G. tsugae was used as a skin substitute (Suet al., 1997). Polysaccharides from G. lucidum have antifibrotic activity, reduce the collagen content in liver and improve the morphology of liver (Park et al., 1997). Lanostanoids and steroids from the fruit bodies of G. tsugae caused cell death by apoptosis or cell cycle inhibition (Gan et al., 1998).

Sotre species of Ganoderma have been used as traditional medicines in Asia. The majority of researches were related to the tredicinal values of Ganoderma lucidum, such as immunomodulatory action (Gao et al., 2005), antitumor (Jiang eta!., 2005), antimicrobial activity (0 fodile et al., 2005) and cardiovascular effects (Lee and Rhee, 1990).

Species of Ganoderma Karsten are important wood decaying fungi occurring on conifers and hardwoods throughout the world (Adaskaveg et al., 1991, 1993). They are white rot fungi with the ability to decay lignin as well as cellulose. Root and stem rots caused by Ganoderma species result in worldwide losses of trees. Selective delignification by fungi may have diverse industrial applications including bio pulping of wood in paper industry and degradation of pollutants (Gottlieb et al., 1998).

1.7 Ganodenna: History of Classification

The genus Ganoderma was established by Karsten in the year 1881 with Polyporus lucidus W. Curt. Fr. as the type species. The French mycologist Patouillard was extremely industrious and published a long series of mycological papers, most of them based on tropical and subtropical collections. His collected mycological papers have been published in 3 volumes (Patouillard, 1876-1924) in which he attempted to transfer several taxa to the genus Ganoderma on the basis ofthe unique feature of the

9IPage double walled basidiospores and described additional new species. In the monograph of Ganoderma, Patouillard (1889) recognized forty eight species and two sections, Ganoderma and Amauroderma (Monclavo and Ryvarden, 1997).

Genus Amauroderma was proposed by Murrill (1905 ), which members were characterized by the spherical or sub spherical spores with uniformly thickened walls. The genus Eljvingia was created by Karsten (1889) based on Boletus applanutus Persoon for non laccale Ganoderma species (Moncalvo and Ryvarden, 1997). The Ganodermataceae was created by Donk (1948) to unite taxa with the distinctive double walled basidiospores (Moncalvo and Ryvarden, 1997) and afterward two genera (i.e. Ganoderma and Amauroderma), were recognized by Donk (1964). Steyaert (1972) created genera Haddowia and Humphreya on the basis of basidiospores morphobgy. A comprehensive review about Ganoderma and related genera were presented by Corner (1983) and Zhao (1989), who gave elllJhasis on species of tropical Asia, America, and the latter on species of China. A nomenclatmal study ofthe Ganodermataceae was conducted by Moncalvo and Ryvarden (1997). In their study, 386 names were used to define species of Ganodermataceae, out of which 336 names were retained and 50 names abandoned.

1.8 Problems in Ganoderma Classification

The macro and micro morphological characters of the genus Ganoderma are comprehensively variable, and more than 300 species have been described by various authors and also listed in fungas database like Index Fungorum and Mycobank (Moncalvo and Ryvarden 1997;------Moncalvo et al., 1994, 1995a, b; and Buchanan, 2001). Amongst the many described Ganoderma species, several were only represented by one or few collections restricted to the type bcality and adjacent regions (Moncalvo and Ryvarden, 1997). There have been many records and descriptions of new species of Ganoderma based on minute variations in morphobgical characteristics (Moncalvo et al., 1995b), which lack of detailed descriptions. Most species are therefore inadequately defmed and their geographic range remains unidentified. Neither the limits of Ganoderma with the other defined genera in the Ganodermataceae were clear, nor the separations of Ganoderma into subgenera and sections; as a consequence, there are many synonyms.

10 I Page In the nomenclatural study, Moncalvo and Ryvarden (1997) regarded one­ third i.e. 116 species name out of336 species have been proposed as synonym;. There is no proper synopsis or 100nographic treatment covering the group. Hence Ryvarden (1991) has stated that the genus is in a taxonomic chaos, the 100st difficult genus a100ngst the polypores to classify (Ryvarden, 1985 and Soon and Hack, 2004) and it is a taxonomic challenge in mycology (Corner, 1983).

1.9 Modem Approaches in Ganoderma classification

Discrepancies in 100rphological characters of Ganoderma led many taxonomists to find chemical and 100lecular tactics to discriminate species of Ganoderma group. Following are the few approaches considered by various workers.

An approach of cultural characters was examined to understand Ganoderma classification (Stalpers, 1978). This study discovered that G. tsugae as a synonym of G. valesiacum. In the cultural and mating studies, it was revealed that G. lucidum from North America and G. resinaceum from Europe have analogous cultural characteristics and are inter fertile (Adaskaveg and Gilbertson, 1986). It was also observed that G. tsugue from North America and G. valesiacum from Europe have paralle 1cultural characteristics.

By ultrastructural studies with the scanning electron microscopy, it was shown that G. co/ossum and G. zonatum have specific differences in basidiospore 100rphology (Adaskaveg and Gilbertson, 1988a).

In isozyrre electrophoresis pattern analyses strategy, G. lucidum complex and G. applanatum complex were distinguished from each other by several isozyrres including aspartate aminotransferase, alkaline phosphatase and rrenadione reductase. The isozyme pattern analysis, however, fuiled to yield diagnostic bands for each 100rphologically defined group (Gottlieb et al., 1998).

In Molecular analysis rrethod the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS) composed of ITS1 and ITS2 is a part of an ancient gene and a non coding region, hence under low functional constraints shows 100re variation than coding regions. During the last two decades, nrDNA ITS region has been widely used for inferring fungal species relationships, and selected as a standard

111 Page marker for fungal DNA barcoding (Moncalvo,1994; 1995a, b; Gottlieb, 2000; Smith and Sivasithamparam, 2000a and Schoch et al., 2012). Phylogenetic analysis of mtSSU rDNA sequences showed that G. lucidum was classified as a group oftrimitic or dimitic species in the core group including Trametes and Polyporus (Hibbett and Donoghue, 1995). While in the phylogenetic revisions based on ITS and the D2 region of 26S rDNA, it was evidenced that broad convergence or parallelism of morphological characters has occurred during the Ganoderma evolution (Moncalvo et al., 1995b ).

Moncalvo et al., (1995a, b) distinguished that nrDNA ITS sequences can separate between rrost species of Ganoderma. Ribosomal RNA (rRNA) gene sequences have commonly been used to differentiate fungal taxa at the family (Hibbett and Donoghue, 1995), generic, and sub generic levels (Vilgalys and Sun, 1994 a, b; Crawford et al., 1996; Anderson et al., 1998 and Chillali et al., 1998). Bae et al. (1995) and Moncalvo et al. (1995a, b) used rRNA gene internal transcribed spacer (ITS) sequences to distinguish between isolates of Ganodermataceae. Lately, nrDNA ITS sequencing has been widely applied to the identification and discrimination ofGanoderma species (Moncalvo, 1995a, b; Gottlieb, 2000; Smith and Sivasithamparam, 2000a ).

The studies shows sequence from nearly complete mitochondrial small subunit ribosomal DNA (mtDNA SSU) has 3.3 times rrore information than nrDNA ITS sequences among the studied species of Ganoderma (Hong et al., 2002 and Hong and Jung, 2004). From outcomes of this study it was concluded that, both conserved domains and variable domains (V1-V9) fragment defined phylogenetic information ofGanoderma species (Hong et al., 2002 and Hong and Jung, 2004).

Arrong genes coding for proteins with basic metabolic or structural functions,

~-tubulin genes are receiving increasing attention as rrolecular markers. Studies have made use of ~-tubulin genes to examine relationships arrong fungi at all levels, and they were also found to be suitable in deep level phylogenetic studies (Thon and Royse, 1999). Aside from above mentioned rrolecular strategies, several other techniques also have been used in Basidiomycota such as Amplified Fragment Length Polymorphism (AFLP) (Qi et al., 2003), Restriction Fragment Length Polymorphism

121 Page (RFLP) (Park and Ryu, 1996), and Random Amplified Polymorphic DNA (RAPD) (Wang et al., 2003) to classify species ofGanoderma group.

1.10 Indian Scenario in context of Ganoderma systematics

The oldest scientific report ofGanoderma from India is ofBose (1929) who achieved culture of Ganoderma from spores and also generated in vitro basidiocarps. There are negligible reports of Ganoderma, from India. Bakshi (1971) reported five species of Ganoderma from the Indian subcontinent and till date 46 species, have been reported (Baksh~ 1971; Dhanda, 1977; Sharma, 2000; Foroutan and Vaidya, 2007; Bhosle et al., 2010 and Ranadive et al., 2011). Looking at the global taxonomical status ofGanoderma, the Indian status is critical. Bilgrami et al. (1991) added couple of species to this list viz, G. leucophaeum, G. annulare and G. adspermum, but these records were not considered into the 1atest world list of Ganoderma species published by (Moncalvo and Ryvarden, 1997).

The work on molecular has taken pace at global level from 1995 to understand genus Ganoderma. The data on molecular taxonomy in India is very recent and scanty. Very rew researchers showed interest to explore this medicinal important fungus and understand its taxonomy using morphology and molecu1ar tool till date (Bose, 1929; Bakshi, 1971; Bilgrami, 1991; Sankaran et al., 2005; Foroutan and Vaidya, 2007; Malarvizhi and Pudupalayam, 2008; Malarvizhi, 2013; Bhosle et al., 2010; Ranadive et al., 2011; 2013; Mohanty et al., 2011; 2012; Sharma et al., 2012; Prasher Lalita et al., 2013; Arulpandi et al., 2013; Arvind Parihar et al., 2013 and Singh et al. 2014). Out of these, majority of researchers have used morphobgical characters for Ganoderma taxonomy.

The taxonomy of the genus Ganoderma is considered to be in chaos (Ryvarden, 1994). This is due to the fact that morphobgical characters ofbasidiocarp have been used to differentiate the species and many species within this genus share common characteristics making it difficult to distinguish between species (Adaskaveg and Gilbertson, 1986, 1988a, b; Moncalvo et al., 1995a, b; Gottlieb and Wright, 1999a, b; Smith and Sivasithamparam, 2003 and Pilotti et al., 2004 ).

Several studies have used different ahernative methods such as cultural characters (Adaskaveg and Gilbertson, 1989), sexual compatibility tests (Adaskaveg

131 Page and Gilbertson, 1986; Pilotti et al., 2002, 2003), isozyme analysis (Gottlieb et al., 1998; Smith and Sivasitha1I1Jaram, 2000b) and DNA based techniques (Moncalvo et al., 1995a, b; Gottlieb et al., 2000; Smith and Sivasithamparam, 2000a; Hong and Jung, 2004) to determine the identityofGanodenna species. Consequently, Imlecular techniques have become a powerful diagnostic tool to elucidate the taxonomy of Ganoderma species. Application of these techniques has resolved some of these taxonomic problems but names in the genus are still poorly understood.

Roughly one third of the work:l fungal diversity has been so far described from India (Jamaluddin et al., 2004 and Sarbhoy et al., 1996). Two mega biodiversity zones in India, viz. the Western Ghats and Eastern Himalayas (Myers et al., 2000), are said to be rich in endemic as well as exotic fungal diversity (Bhat, 2000 and Bhat et al., 2009). In current study, to provide useful information for clarifying the taxonomic status of genus Ganoderma from Pune and Konkan region ofMaharashtra, India, molecular analysis was followed by morphological analysis. Entire ITS nrDN A including the intervening 5.8s RNA coding region and partial mtDNA SSU corresponding to variable domains V3 to V5 were sequenced from the studied materials for phylogenetic evaluation.

1.11 Study objectives

The main objectives of the present studies are as follows

1. To survey the collection sites of Ganoderma to collect Ganoderma isolates from selected sites ofPune and Konkan region. 2. To deposit the collected fruiting bodies of Ganodenna isolates in herbarium. 3. To assess molecular diversity among collected specimens by using ITS and mtSSU IDJlecular markers and to deposit ITS and mtSSU sequence in NCBI gene bank data base. 4. To construct phylogenetic trees using ITS and mtSSU sequences and colll'are with the previous publications to identify distinct clade. 5. To construct set of Imrphological key characters for Ganodenna isolates on the basis of type species description and to analyze clade specific isolates using Imrphological key characters.

To have correct understanding of Ganodenna Imrphology and phylogenetic analysis ie. IDJlecular taxonomy of this complex genus relevant literature has been referred thoroughly and is used to design the approach for this study.

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