Identification of Genus Phellinus s.l.

INTRODUCTION

Fungi are one of the most important groups of organisms on the planet earth (Vaidya, 1995). Fungi, together with bacteria, are responsible for recycling of most of the lignocellulosic material. The recycling returns dead material to the soil in a form in which it can be reused (Vaidya, 1995; Alexopoulos et al., 1996 and Lamrood, 2004). The fungal kingdom comprises a huge diversity of taxa with diverse ecologies, morphologies and life cycle strategies ranging from single cell yeast to large mushrooms (Whittaker, 1969).

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Kingdom Fungi is the most diverse among all Kingdoms (Kirk et al., 2001), which comprise very distinctive organisms inhabiting every ecological niche such as soil, plant parts, litter, herbivore dung, entomogenous, freshwater and marine environment (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 e.g. mycorrhizal fungi (Boa, 2004). It is estimated that about 1.5 million species may exist (Hawksworth, 1991; Hawksworth et al., 1995) on the earth. However, according to the 10^th addition of Dictionary of Fungi, a total number of 80,000 species has been described (Kirk et al., 2008).

Fungi have generally been identified and classified on the basis of morphology of spores and spore producing structures. In subsequent years, criteria such as cultural characters and developmental biology have been added as useful tools. Reliable diagnostic tools and excellent taxonomic keys are available for identification of fungi (Kirk et al., 2001).

Fungi play an important role in human life. They are directly or indirectly used as food and in various fermentation products such as beer, wine, bakery products such as bread and soya sauce. (Deshpande, 1999). One of the well-known forms of fungus is mushroom, which is edible and different species are cultivated and sold worldwide contributing to the turnover of several million dollars. Many fungi are also industrially important as they produce enzymes, organic acids, vitamins and antibiotics (Anke et al., 1980; Colleto and Mondino, 1991; Lorenzen and Anke, 1998; Wasser and Weis, 1999; Rosecke and Konig, 2000; Pointing and Hyde, 2001; Wasser, 2002). The most famous of all antibiotic drugs, Penicillin, is derived from a common fungus called Penicillium. Many other fungi also produce antibiotic substances, which are now generally used to control diseases in humans and animals and they are also extremely useful as bio-control agents (Agrios, 1988). While on a negative note, fungi are also considered as obnoxious causing deterioration and damage to valuable articles, crop and timber plants. One of such fungal infection causing mass destruction in timber industry is wood rot. Various fungi cause this disease; however, more aggressive fungus is Phellinus of Hymenochaetaceae family (Rabba, 1994; Vaidya, 1995; Lamrood, 2004).

Taxonomic status of Family Hymenochaetaceae

Patouillard (1900) and Donk (1964) recognized Hymenochaetaceae as a separate family which contain polyporoid, hydnoid and steroid genera (Kotlaba and Pouzar, 1957; Niemela, 1972; Parmasto, 1985). This is one of the homogenous families among the basidiomycetes with monophyletic origin (Ryvarden, 1991). The typical features of the family Hymenochaetaceae are: 1. Existence of setae 2. Absence of hyphal clamps and 3. Presence of xanthochroic basidiocarps. The family members shared common ancestry with respect to white rot, simple septate generative hyphae and thick skeletal hyphae and unique thick-walled brown coloured setae that are widely scattered throughout most genera (Parmasto, 1985). This family is considered to be of most recent origin on the basis of chemical characters and spores variation and genera like Phellinus s. l. and Inonotus s. l. (poroid representative) have been considered most advanced in the family (Ryvarden, 1991).

Patouillard (1900) laid the foundation for modern classification of the Hymenomycetaceae by using microscopical characters to delimit higher taxa of the family. He placed all the polyporoid genera in the order Aphyllophorales. Donk (1964), accepted Patouillard’s classification and placed all the poroid genera in nine families namely; 1) Fistulinaceae 2) Ganodermataceae 3) Thelephoraceae 4) Bondarzewiaceae 5) Corticiaceae 6) Echinodontiaceae 7) Hericiaceae 8) Hymenochaetaceae and 9) Polyporaceae. Ryvarden (1991) accepted Donk’s classification and positioned all the nine families in the order Aphy1lophorales. Besides, he divided the family Hymenochaetaceae into nine genera namely 1) Phellinus 2) Inonotus 3) Phylloporla 4) Pyrrhoderma 4) Coltnciella 5) Hymenochaete 6) Aurificaria and 7) Coltricia.

However, the recent molecular rDNA analyses exposed that certain species belonging to the families and genera such as Corticiaceae (Basidioradulum, Hyphodontia), Polyporaceae (Oxyporus, Schizopora, and Trichaptum), Stereaceae (Cotylidia), and Agaricaceae (Cantharellopsis, Omphalina, and Rickenella) are related to Order Hymenochaetales (Hibbett and Donoghue, 1995; Ko et al., 1997; Langer, 1998; Jeong, 1999; Hibbett et al., 2000; Paulus et al., 2000; Hibbett and Thorn, 2001; Larsson, 2001; Larsson et al., 2007). Even though many members of these groups display a different morphology than the members of Hymenochaetales, they are also related by the presence of an imperforate parenthesome.

Genus Phellinus

Members of family Hymenochaetaceae including the genus Phellinus Quel., comprise wood-inhabiting saprotrophic and/or parasitic species which are characterized by permanently darkening tissue in KOH (Xanthochroic reaction), presence of a continuous parenthesome in the dolipore apparatus, frequent occurrence of setae, absence of clamps and the production of white rot-wood (Parmasto and Parmasto, 1979; Moore, 1980, 1985; Corner, 1991; Rabba et al., 1994; Vaidya, 1995; Müller et al., 2000; Wagner and Fischer, 2001, 2002; Fischer and Binder, 2004; Lamrood 2004; Larsson et al., 2007; Spirin et al., 2007).

Quelet in1886 formed the genus Phellinus with type species P. igniarius. The genus is one of the largest genus among polypores with over 265 species presently known (Rabba, 1994, Lamrood 2004; Larsson et al., 2007; Bapat, 2008). The genus was created mainly to accommodate both annual and perennial brown poroid, pileate wood inhabiting fungal forms. Donk (1960) selected Polyporus rubriporus (Phellinus torulosus) as generic nomenclature type. Ever since its publication, the genus Phellinus remained stable but the species concepts in many instances remained doubtful (Parmasto, 1985). Taxonomy and nomenclature of Phellinus has been revised more recently by Fiasson (1983) and Fiasson and Niemelä (1984). Their contributions symbolize a major departure from conventional taxonomy approaches used for Phellinus. They blended approaches like numerical taximetric analysis, chemotaxonomy, protein electrophoresis, nuclear condition and to a limited extent, characteristics derived from cultural studies. Bondartseva and Herrera (1980a) have provided comprehensive taxonomic review of Phellinus and proposed new names along with new combination.

Phellinus is currently located in family Hymenochaetaceae of order Aphyllophorales. In earlier India taxonomic works, the species of the genus Phellinus were placed along with genera like Fomes, Poria or Polyporus of family Polyporaceae. The fruit bodies of Phellinus are large, variously shaped, persistent, brown poroid, resupinate to pileate including both annual and perennial form (Donk, 1974).

Phellinus produce decay of heartwood and cause heart rot and cankers of live standing trees and play a major role in the process of wood decay by causing white rot. It mainly degrades lignin while, cellulose is partly degraded. Most bracket forming species are found growing on Atrocarpus integrifolia, Acacia nilotica, Mangifera indica, that are medicinally important and used as folk medicine in the Western Ghats of Maharashtra, Karnataka and Kerala (Vaidya and Rabba, 1991). The heavy rainfall, high humidity and hot temperature, in semi evergreen moist deciduous forest, with few-isolated patches of evergreen vegetation in deep ravines, favor the growth of Phellinus (Rabba, 1994).

Ainsworth and Bisby’s Dictionary of the Fungi, 9^th Ed, 2001 classified genus Phellinus as follows:

• Kingdom: Fungi
• Division: Basidiomycota
• Class: Basidiomycetes
• Sub class:Agaricomycetidae
• Order:Apyhllophorales
• Family: Hymenochaetaceae
• Genus: Phellinus Quel.

Macromorphological characters of the genus Phellinus

The genus Phellinus is roughly characterized by pileate to resupinate to effuse-reflexed or partly effused fruit body. The various types of basidiocarps vary from applanate, convex, effused reflexed, applanate with umbo, triquetrous, resupinate, ungulate, imbricate etc. The basidiocarps are broadly attached semicircular, broadly attached elongated, dimidiate, flabelliform, spatulate, respectively. The Perennial fruiting bodies are woody while annual may be soft. Fruiting bodies show dominance of skeletal hyphae. The surface of the fruitbody may be glabrous or finely tomentose; may be dull or glossy (Ryvarden and Johansen, 1980).

The basidiocarps are with distinct colours, which do not change throughout the life span of basidiocarp. Light color species becomes darker with age while zonation occurs, as hyphae of upper layer collapses. A glabrous pileus may be either completely smooth or with zonation, radial lines, or raised ridges. The type and consistency of pileus cover play an important taxonomic role. In the perennial species, the pileus may crack with age forming radial lines or an irregular pattern of angular pieces causing the surface to be referred to as rimose. A fibrillose surface may occur where pileus surface disintegrates into long fibrils with age (Glibertson and Ryvarden, 1986).

Perennial basidiocarps have whitish or pale yellow margin. The margin in most of the species is more or less continuous, light brown colored usually paler than rest of upper surface and several millimeters wide. Hyphae grow at equal rate and margin become soft and tomentose. Sometimes margin can end abruptly with an almost vertical slope because basidiocarp often have tendency to curl up and loosen along margin under drying.

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Most of the Phellinus species are applanate, strongly dimidiate, fan-shaped or tapering towards base (Triquetrous). It may be difficult to decide whether a true lateral stipe is present or not. The color of pore surface is characteristic diagnostic feature for many species. The surface becomes darker with age while in some species it shows golden luster if held obliquely in incident light. Number of pores per mm is an important taxonomic character. With age, the dissepiments in some species may become thick. The outline of pore may be angular and irregular (Ryvarden and Johansen, 1980). In some species the tubes are more or less concolorous or paler with pore surface. Tubes may be distinctly or indistinctly stratified. In few species, inner surface of tubes becomes whitish. However, in many species, there is no difference either in color or consistency between the tubes and context or subiculum. These differences can serve as an important taxonomic character.

The context is a sterile part of basidiocarp present between tubes and pileus surface where as in resupinate basidiocarp it is called subiculum. In most of the species, context is homogeneous with respect to color and consistency. The context normally has radial lines however; in some species it is distinctly duplex with the lower part being dense and without apparent structural direction, while the upper part is more soft and fibrous which often integrate with a pileus tomentum. Hyphal system is always dimitic in all Phellinus species.

The Phellinus should be restricted to yellow, brown species with dimitic hyphal system. This system consists of two types of hyphae. Simple septate generative hyphae without clamp connection and thick skeletal hyphae. The generative hyphae are septate or aseptate, thin-walled, branched or unbranched hyphae without clamp while, skeletal hyphae are originating from generative hyphae. They are normally unbranched or rarely terminally branched, thick walled to solid, septate or aseptate but without clamp, may be hyaline or colored. They react with Melezer’s solution showing strong reddish brown color (dextrinoid reaction). These hyphae are aligned longitudinally in growing region and their tips usually protrude into the hymenium or subhymenium in basidiocarp.

The Basidia are with 2-4 sterigmata per basidium, more or less clavate, hyaline, and often thin walled. While, basidiospores are smooth, thin- or thick-walled with warts. A range of spore size is large. The shape may be globose, subglobose, oblong, ellipsoid, or ovoid. They are hyaline or pigmented – pale yellow to golden yellow, light brown to reddish brown or rusty brown.

The setae occur mostly in hymenial or tramal region and designated as hymenial and tramal setae accordingly. They are dark colored, thick walled, rigid with spines like organs with more or less pointed apex and darkens in alkali. Yellow to reddish brown in color; simple or branched, varies in size and shape. The setae may be subulate or ventricose, straight or distinctly hooked. Hymenial setae arise terminally or laterally from skeletal hyphae. While setal hyphae are straight, run parallel to tubes but in some species the apex is bent into the hymenium. They have equal width and tapering only at apex. They differ from the tramal setae. The setal hyphae present in context and in dissepiments and are called setal skeletal hyphae. True cystidia are not present in this group (Fidalgo, 1968). Sometimes hymenial setae form cystidium like structure and become less thick. Setae may provide rigidity to fructification and protect tissue from damage by small animals (Overholt, 1929). The setae have been used as a character in erection of new species. For diagnosis of species, setae characters such as shape, size, color, apex, position etc. are considered of taxonomic importance.

Taxonomic Problems of Phellinus Quelet

As mentioned above, the genus Phellinus is the largest group of species amongst the Hymenochaetales. However, partly from incorrect judgements of identifiers or partly from vague definitions by taxonomists, a single species of Phellinus usually has several synonyms.

Many species in the genus have basidiocarps of various colors and shapes. The characters, furthermore, would easily change as species grow older. Setae can be found or not according to observed specimens. In Phellinus, definitions on mitic system are still vague in several species. Specimens considered to be monomitic by one identifier would be considered to be dimitic by another identifier. For example, it has been in a controversy until now whether P. weirii and P. ribis are monomitic or dimitic (Ryvarden 1994, Gilbertson and Ryvarden 1979). The use of homoplasious characters would lead to artificial and incorrect taxonomic conclusions. Many characters considered important for the classification of Phellinus seem to be too homoplasious to be used in grouping and classifying Phellinus. The use of homoplasious characters would lead to artificial and incorrect taxonomic conclusions.

Medicinal Importance of Phellinus

Many species of Phellinus (e.g. P. linteus, P. baumii, P. igniarus and P. pini etc.) are known to have different medicinal effect (Jung et al.,1992; Shon et al., 2004). This mushroom is known as Sang-Hwang in Korea, Song Gen or sanghuang in China and Meshimakobu in Japan (Wu et al., 2012) In India, especially in Western Ghats, many species of Phellinus are used in the day to day life as folk medicine to cure teeth, tongue and throat related ailment, to stop excessive salivation in case of children, against diarrhoea (Vaidya, 1995; Vaidya and Bhor, 1991; Vaidya and Lamrood, 2000). It is reported that the basidiocarps of Phellinus rimosus are used by the tribal people for the treatment of ailments such as mumps. P. rimosus posses anti- oxidant, anti-tumor, anti-inflammatory, hepatoprotective activities (Ajith and Janardhan, 2002)

P. igniarius is taken internally as a bitter tonic and laxative, externally as a styptic (Chopra et al., 1956). Several studies have showed that Phellinus species can produce substances with cytotoxic (Han et al., 1999). Phellinus linteus (Bhendiomba) is used for the purification of blood in skin diseases (Vaidya and Rabba, 1991). Polysaccharides-spachyman and pachymaran extracted from Phellinus exhibited strong anti-cancer and immunomodulatory activities (Hobbs, 1995).

In the Western Ghats of Maharashtra, Konkan in particular, the fruiting bodies growing on Artocarpus heterrophyllus Lam. are collected and used to cure various aliments. This fungus is traditionally known as Phanasomba (Vaidya and Rabba, 1991; Vaidya, 1995; Lamrood 2004). Similarly in the Western Ghats of Karnataka and Kerala the fruit bodies growing on Artocarpus heterrophillus are also used as medicine.

The common vernacular name for Phellinus in the Western Ghats of Kerala is ‘Pilamanjal’ and in Maharashtra it is “Phansomba”. Many species are found on Artocarpus heterophyllus Lam (Jack Fruit tree). Jackfruit in local language is ‘Phanas’(Marathi) and ‘Pilavu’ (malayalam) and mushroom is alombe (Marathi) and ‘Manjal’ (malayalam) growing on this tree is called Phanas-amba = Phansomba (Marathi) and ‘Pilamanjal’. This mushroom is also used in many commercial ayurvedic preparations. It is used by local tribal people and learned by Ayurvedic doctors (vaidus) of Konkan to cure teeth, tongue, throat related ailments, to stop excess salivation in case of children, against diarrhea (Andalkar, 1988, Vaidya and Bhor, 1991; Vaidya and Lamrood, 2000). Many species of Phellinus such as Phellinus adamantinus (Berk.) Ryv., P. carteri (berk.et. Cke.) Ryv., P. conchatus (Pers.: Fr.) Quel., P. fastuosus, P. linteus (Berkeley and Curtis) Teng (PL), P. merrilli.(Murr.)Ryv., P. pectinatus (Klotzsch) Quel., P. sublinteus Murr.) Ryv. etc. are sold under the name of ‘Phansomba’. Among these species P. merrillii and P. fastuosus are very common.

Aim of Present study

Because of above mentioned taxonomic chaos, the necessity was felt to re-evaluate the phylogenetic relationships of Phellinus s.l. and related genera based on the molecular data using ITS (Internal transcribed spacer) of nuclear rDNA. The nuclear ribosomal DNA consists of three ribosomal genes and two ITS regions. ITS sequences show much variability hence, they have been used in the systematics of closely related species of a same genus (Moncalvo et al., 1995). It is hoped that the recent phylogenetic studies on the molecular data will provide new and objective means for the classification of Phellinus s.l. and related genera. In addition, a modified morphological character state code and matrix will be generated using character matrix of Lamrood (2004) and Bapat (2009), which could be either identify a Phellinus species or return the most probable similar species group.

The present study was undertaken for the first time to establish the correct identification of Phellinus s.l. from the Western Ghats of Karnataka and Kerala with following objectives:

Objectives:

1. Collection of Phellinus from Artocarpus heterophyllus Lam. from the Western Ghats of Karnataka and Kerala.
2. Taxonomical identification of Phellinus samples collected from the above mentioned regions using Larsen and Cobb-Poulle Key, 1990.
3. Assess the genetic diversity among collected specimens using ITS region as a molecular marker.