Ganoderma lucidum: A WOOD DECAY AND ROT FUNGUS CHANDRA SHEKHAR RAY FORESTRY AND WOOD TECHNOLOGY DISCIPLINE LIFE SCIENCE SCHOOL KHULNA UNIVERSITY KHULNA BANGLADESH August 2005 Ganoderma lucidum: A WOOD DECAY AND ROT FUNGUS Course No: FWT-5114 [This Project Thesis paper has been prepared and submitted to the Forestry and Wood Technology Discipline, Khulna University, Khulna for the partial fulfillment of MSdegree in Forestry] SUPERVISOR SUBMITTED BY Professor Dr. Md. Abdur Rahman Chandra Shekhar Ray FWT Discipline Roll No: MS-020506 Khulna University FWT Discipline Khulna University TABLE OF CONTENTS Page No. TABLE OF CONTENTS i ABSTRACT iv LIST OF TABLES v LIST OF FIGURES v DEDICATION vi ACKNOWLEDGEMENT vii CHAPTER ONE: INTRODUCTION 1.1 1.2 Background of the study 1 1.1.1 Ganoderma: an introduction 1 1.1.2 Ganoderma Taxonomy and Nomenclature 2 1.1.3 Morphological Features of Ganoderma 6 1.1.3.1 Macromorphology 6 1.1.3.2 Micromorphology 7 1.1.4 Cultural Characteristics 9 1.1.5 Taxonomy of the Ganoderma lucidum Complex 13 Objectives of the study 13 CHAPTER TWO: IDENTIFICATION AND USES 2.1 How do you identify Ganoderma lucidum? 14 2.2 Uses 17 CHAPTER THREE: SURVIVALITY OF GANODERMA LUCIDUM 3.1 Survival of Wood-inhabiting Microorganisms 19 3.2 Survival of Trees against Wounds and Infections 20 3.2.1 Compartmentalization of decay 21 Survival of Ganoderma Species and Other Wood-Inhabiting Fungi 21 3.3.1 Survival of Ganoderma in infected wood 24 3.3.2 Survival of other fungi in infected wood 25 3.3 i 3.3.3 Mycelium growth of Ganoderma lucidum 25 CHAPTER FOUR: WOOD DECAY FUNGI 4.1 Ecology and Life Cycle 27 4.2 Wood Decay Species 27 4.3 Tree Decay An Expanded Concept 29 4.3.1 Role and importance of tree decay 30 4.3.2 Decay as a Natural Recycling Process 30 4.3.3 An Understanding of Gradations is Necessary 30 4.4 The Classical Concept of Tree Decay 31 4.5 The Expanded Concept of Tree Decay 31 4.6 Pests Management 32 4.6.1 Pests in Landscapes and Gardens 32 4.6.2 Identification and Biology 33 4.6.3 Management 34 CHAPTER FIVE: WOOD ROT FUNGI 5.1 Ganoderma / Artist's Fungus 35 5.1.1 Rots caused by Ganoderma lucidum 36 5.1.1.1 White Rot 36 5.1.1.2 Brown Rot 38 5.1.1.3 Soft Rot 38 5.1.2 Rots Caused By Ganoderma lucidum Depending upon Location 5.2 39 5.1.2.1 Stem rot 39 5.1.2.2 Root rot and Butt rot 40 5.1.3 Other root rot and wound pathogens 41 Control 42 CHAPTER SIX: LIGNIN MODIFICATION 6.1 Lignin-Modifying Enzymes of the White Rot Basidiomycete 49 Ganoderma lucidum 6.1.1 More about Lignin-Modifying Enzymes ii 51 CHAPTER SEVEN: CONCLUSIONS AND RECOMMENDATIONS 7.1 Conclusions 57 7.2 Recommendations 57 58 REFERENCES iii ABSTRACT Ganoderma lucidum is a basidiomycete fungus—a member of the group containing the common mushrooms, puffballs and fungi producing fruiting bodies that are bracket or shelf-like. Ganoderma lucidum fruiting bodies may be produced on the roots and lower trunks (i.e., the “butts”) of affected trees. It is a serious root rot and also a white rot pathogen of worldwide distribution. It attacks a large number of broad-leaved, subtemperate and temperate tree species. It is normally endemic to natural forests and does not cause any serious damage. However, when natural forests are clear-felled, G. lucidum spreads quickly to residual roots and stumps to build up a high inoculum potential. Raising new plantations in such areas without clearing the infected residual stumps and roots causes severe damage to susceptible species. The lateral spread of the disease takes place through root contact. The strictly parasitic habit of G. lucidum makes it incapable of freshly colonizing dead roots or stumps. The fungus is also unable to make free mycelial strands in the soil, except on root surfaces, or outside the roots when in contact with a solid surface like the roots of adjoining plant. Healthy dumps become inflected when their roots come in contact with decayed wood. Decay fungi can be the death of trees. Ganoderma lucidum is one of the commonly encountered landscape tree pathogens. To limit the fungus, protect soils and trees from compaction, root damage, construction, and other damage. Infected trees may topple before any sign or symptom becomes obvious. The presence of a fruiting body or conk is a sure sign of the disease. Remove infected trees immediately. Silvicultural measures like isolation trenches may prove effective in containing the disease in between the trenches, thus preventing its spread. iv LIST OF TABLES Table no. Caption Page no. Table 1 Morphological comparison of basidiospores of G. lucidum 8 Table 2 Cultural characteristics of the Ganoderma lucidum complex 12 LIST OF FIGURES Figure no. Caption Page no. Figure: 1 Ganoderma spp. 14 Figure: 2 Chemistry of Wood Decay 45 Figure: 3 Classification of Wood decay fungi. 47 v “DEDICATION” To my beloved grandmother vi ACKNOWLEDGEMENT I gratefully acknowledge my deep gratitude and profound appreciation to my honorable supervisor Prof. Dr. Md. Abdur Rahman, Forestry and Wood Technology Discipline, Khulna University, Khulna for his permission, professional guidance, valuable suggestions, and materialistic support during the preparation of this project thesis. I wish to utter my special thanks to my junior brother Prodip Kumar Sarker. I am also grateful to my friends Md. Shahidul Islam, Prokash Ghosh, Sazal Kumar Saha and Md. Amirul Kyser. Last but not least, I am much indebted to all my friends. Chandra Shekhar Ray vii CHAPTER ONE: INTRODUCTION 1.1 Background of the study 1.1.1 Ganoderma: an introduction Ganodermataceae are cosmopolitan basidiomycetes which cause white rot of hardwoods, such as oak, maple, sycamore and ash, by decomposing lignin as well as cellulose and related polyaccharides (Hepting, 1971; Blanchette, 1984; Adaskaveg and Ogawa, 1990; Adaskaveg, et al.,1993). Although species of Ganoderma are economically important plant pathogens, causing disease in crops such as rubber and tea and wood rot of trees, the fruit bodies are popular as, and have long been used in, traditional medicinal material in Asian countries, including China, Japan and Korea. The Ganoderma lucidum complex, known in Chinese as Lingzhi, has long been considered a symbol of good fortune and prosperity and is the subject of many myths, as well as being a medicinal herb in ancient China (Zhao and Zhang, 1994). There are records of these fungi before the time of the famous medical book Shen Nong Ben Cao Jing (AD 25-220, Eastern Dynasty). Depending on the different colors and shapes of the fruit bodies, they have been called the red-, black-, blue- white-, yellow- and purple-types in Shen Nong Ben Cao Jing by Hong Ching Tao (AD 456-536) and Ben Cao Gang Mu by Li Shi-Zhen (AD1590, Ming Dynasty), as well as the antler-and kidney-shapes (Shin and Seo, 1988b; Zhao, 1989; Willard, 1990). The black fruit body reffered to by the old description in China (Willard, 1990) is assumed to be G. neo-japonicum or G. formosanum because the fruit bodies of these species are black in nature. Ganodermataceae have attracted the attention of mycoloists for many years. They have been considered as either plant pathogens (Hepting, 1971; Adaskaveg and Ogawa, 1990; Adaskaveg et al., 1993), or useful medicinal herbs (Mizuno et al., 1995). Because of these fundamentally different viewpoints among collectors, the taxonomy of these fungi is very subjective and confused. Contributions to the morphology and taxonomy of the Ganodemataceae have been made by many morphologists, including Steyaert (1972); Furtado (1981); Corner (1983) and Zhao (1989). However, the great variability in macroscopic and microscopic characters of the basidiocarps has resulted in a large number of synonyms and in a confused taxonomy, especially in the genus Ganoderma (Gilbertson and Ryvarden, 1986). 1 Ganoderma lucidum is containing the common mushrooms, puffballs and fungi producing fruiting bodies that is bracket or shelf-like. G. lucidum fruiting bodies may be produced on the roots and lower trunks (that is, the “butts”) of affected trees. They are of the bracket type and commonly called conks. These conks may be broadly attached to the tree trunk or slightly stalked, or emerge from the soil above decaying roots. Conks are from a few to several inches across and reddish-brown to purplish on the upper surface. The typically shiny, lacquered appearance of the top of the conk is responsible for its common name, “varnished conk.” While actively growing, the margin and lower surface (where spores of the fungus are disseminated) are creamy white. Conks are leathery to corky and may form singly or commonly are found in groups forming overlapping arrays (see photo). Conks are annual; new conks may be produced each summer and fall, after which they die and deteriorate (Stanosz, 2002). Decay caused by G. lucidum is not compartmentalized (not limited) to the interior of affected trees. The fungus is thought to infect trees by means of spores deposited in fresh wounds. Following infection, G. lucidum colonizes living bark and sapwood and thus can eventually directly kill trees. Tree crowns may exhibit poor growth and branch death as portions of the trunks and roots are colonized. As the amount of wood decayed can increase year after year, extensive defect can develop in the butt and roots and jeopardize the structural integrity and anchorage of the tree. Root and butt rot often lead to wind throw or sudden failure. G. lucidum conks on trees are a good indicator of their potential to be hazard trees, and tree removal should be considered. Efforts to prevent loss of trees from G. lucidum and other root and butt rot fungi begin with good tree care. Appropriate species and cultivars selection, site preparation, proper planting, appropriate fertilization and watering during dry periods will help to ensure vigorous growth and resistance to many pathogens. Wounds to both trunks and roots should be avoided. Even small injuries from mowers and trimmers can allow infection by decay fungi (Stanosz, 2002). 1.1.2 Ganoderma Taxonomy and Nomenclature The genus Ganoderma has been known for a little over 100 years; it was introduced by the Finnish mycologist Peter Adlof Karsten, in 1881. He included only one species, polyporus lucidus, in the circumscription of the genus and this species, therefore, became the holotype species. P.lucidus was named by William Curtis, the 16th-century British botanist. Unfortunately, Karsten incorrectly attributed the epithet ‘lucidus’ to von Leysser and this 2 error has been perpetuated on numerous subsequent publications. No authentic specimens remain and the type locality, Peckham, is now very much changed from what it was in the time of Curtis. The area is now largely developed as residential housing but the type substratum, the small tree Corylus avellana, is likely to be growing still on Peckham Rye Common. It is clear, therefore, where any epitype, selected as an interpretive type, should be sought. The selection of an epitype, in the absence of type or authentic material, would be important, for any further molecular work will need to have available a culture of the type species of the genus which has some nomenclatural standing, i.e. a culture derived from an epitype. Following Karsten, dozens of species belonging to the genus were reported by taxonomists (Patouillard, 1889; Boudier and Fischer, 1894; Boudier, 1895; Murrill, 1902 and 1908). The identification of Ganoderma in those days was mainly based on host specificity, geographical distribution, and macromorphological features oh the fruit body, including the context colour and the shape of margin of pileus, and whether the fruit body was stipitate or sessile. Subsequently, Atkinson (1908), Ames (1913), Haddow (1931), Overholts (1953), Steyaert (1972,1975,1977 and 1980), Bazzalo and Wright (1982),and Corner(1983) conducted the identification of Ganoderma species by morphological features with geographically restricted specimens. Haddow (1931) and Steyaert (1980) placed most of their taxonomy on the spore characteristics and the morphology of hyphal elements. However, the basidiocarps of Ganoderma species have a very similar appearance that has caused confusion in identification among species (Adaskaveg and Gilbertson, 1986 and 1988). The genus now contains a few hundred names; there are 322 in the CABI Bioscience fungus names database, but others may have been published that the major printed indexes, the source of this database, failed to include. The database of Stalpers (1978) and Stegehuis available on the CBS website lists 316 names in Ganoderma and the recent publication of Moncalco and Ryvarden (1997) lists 386 names for the Ganodermataceae as a whole. It has not yet been possible to compare these three data sets, although such an exercise would appear to be needed. However, names are only one aspect of this subject and problems associated with the circumscription of species. Based on the unique feature of the double-walled baidiospore, the French mycologist, Patouillard, over a period of sime 40 years 1887, described a number of new species of Ganoderma and transterred several names from other genera of the polupores. Patouillard 3 (1889) published a monograph of the then known 48 species and also distinguished the species with spherical or subspherical spores as section Amaurocerma. Coincidentally, in the same year, Karsten introduced the genus Elfvingia, based on the name Boletus applanatus of Persoon, for the non-laccate species. Later,section Amauroderma of Ganoderma was raised to the rank of genus by Murrill who, in selecting a species which was not included in section Ganoderma by Patouillard, is therefore the author of the name, and priority dates from 1905 not 1889.Subsequent authers have recognized Amauroderma as a distinct genus. The two genera have been largely accepted, although Corner (1983) and Zhao (1989) reported species that are intermediate between them. Amauroderma was revised by Furtado (1981). Here then we have two important species in the history and the nomenclature of the genus, G. lucidum and Ganoderma applanatum, and these are probably two of the most frequently misapplied names. The late 19th-century and early 20th –century mycologists contributed significantly, in terms of volume of published information, on the genus, describing many new species or perhaps, more correctly, introducing many new names. Many of these names were based on single collections or on only a few collections from the same locality, and the taxonomic status of the species, which these names were applied, is, therefore, often open to the criticism of being unsound. Throughout the remainder of the 20th century various workers, Steyaert, Corner and Zhao perhaps being the more prominent, contribution to our knowledge of the genus by providing revisions, monographs, descriptions of new taxa (again often based on single collections or on only a few collections from the same locality) and observations on both anatomy and ontogeny. Recent workers have used characters other than morphology to determine relationships within the genus. These have included, in the first instance; cultural and mating charaters, primarily by Adaskaveg and Gilbertson (1986), followed by isozyme studies by Hseu and Gottlieb (Hseu, 1990; Gottlieb and Wright, 1999), amongst others; and, finally, Moncalvo and his co-workers (Moncalvo et al., 1995a,b) have used ribosomal DNA sequences and cladistics methods to infer natural relationships. However, as Moncalvo and Ryvarden have stated, these recent studies have had little impact on Ganoderma systematics in total because too few taxa were examined. This was quite clearly through both a lack of human and financial resources and, perhaps more importantly, a lack of the very important type or authentic collections which will link the names available to any subsequent taxa identified. 4 Ryvarden (1994) has stated that the genus is in taxonomic chaos and that it is one of the most difficult genera amongst the polypores. However, this realization has come at the very time when there has been a renewed interest in Ganoderma from a number of quite unrelated sources. These include the medicinal uses based on very old Chinese traditions and the requirement toelucidate the structure of possible active ingredients, coupled with the requirement (not least of all for patent purposes to protect intellectual property rights) to apply names to the species identified in this context. Also of significance here is the apparent increase in the importance of some species of Ganoderma as pathogens of plants used by man. However, with the development of cladistic methods to reconstruct natural classifications and the application of these methods to both traditional morphological data and more importantly, new molecular data, the potential for the resolution of some of these problems appears close to hand. Recently, the phylogenetic relationships of some Ganoderma species collected from various regions were studied by allozyme (Park, et al., 1994) and DNA analysis (Moncalvo et al., 1995a,b). Moncalvo and his co-workers (Moncalvo et al., 1995a,b, Hseu et al., 1996) adopted ribosomal DNA sequences and randomly amplified polymorphic DNA (RAPD) as the tools for analyzing phylogenic relationships in the G. lucidum complex. The results suggested that some strains were misnamed and misidentified, and all isolates belonging to 22 species were disposed in six groups based on nucleotide sequence analysis from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA). However, while some isolates had the same ITS sequence, all of them could be clearly differentiated by genetic fingerprinting using RAPDs. Therefore, RAPD analysis might be helpful for systematic at the lower taxonomic levels to distinguish isolates from each other. When the results of molecular taxonomy are compared with the data of traditional taxonomy, such as morphological, ecological, cultural and mating characteristics, some isolates remain as exceptions. Of many studies on Ganoderma taxonomy, Adaskaveg and Gilbertson (1986) indicate the importance of vegetative incompatibility tests for accurate identification of the G. lucidum complex. Because of the problems as described above, Ryvarden (1994) has proposed that no new species be described in Ganoderma in the decade to 2005. Donk, in 1933 was the first to unite the taxa within what was then the very large family polyporaceae when he proposed the subfamily Ganodermatoideae; he subsequently raised this taxon to the rank of family with the introduction of the Ganodermataceae and this 5 classification has subsequently been accepted by most recent workers. Much later, Julich, in1981, introduced the ordinal name Ganodermatales and this was accepted by Pegler in the eighth edition of the Dictionary of the Fungi, although other workers have continued to use the traditional Aphyllophorales in a broad sense. There has been much speculation on the relationship between Ganodermataceae and other families of polypores. Corner (1983) believed that the family represented an old lineage from which other groups of polypores have been derived. Ryvarden (1994), however, proposed that the high phenotypic plasticity observed in the genus is indicative that the taxon is young and that strong speciation has not yet been achieved. This hypothesis was supported by more recent molecular evidence from Moncalvo and his co-workers. The lack of fossils limits the accuracy to which we can attribute a minimum age to the genus. Some fossils of corky polypores from the Miocene (25 million years old) have been tentatively referred to G. adspersum. 1.1.3 Morphological Features of Ganoderma 1.1.3.1 Macromorphology The naturally produced basidiocarps of G. lucidum show various morphological characteristics: sessile, stipitate, imbricate and non-imbricate (Shin et al., 1986, Adaskaveg and Gilbertson, 1988). The colour of the pileus surface and hymenphore various form deep red, non- laccate, laccate and light yellow to white, and the morphology also differ between the isolates (Shin and Seo, 1988b). The morphological variation appears to be affected by environmental condition during basidiocarp development. The size and colour of the basidiocarp shows significant differences between the specimens, but the pore sizes are similar. The manner of stipe attchment to pileus and the host range also varies (Ryvarden, 1994). The pileus of the normal fruit body is laterally attached to the stipe, but eccentric, central, imbricate, and sessile fruit bodies are also produced rarely in nature. Stipe characters, including attachment type and relative thickness and length, have been considered useful for species identification, but some mycologists, who describe fruit bodies only as stipitate or sessile, have neglected their importance. Hardwoods are the usual host plants of G. lucidum, but some specimens have been collected from conifers. The laccate character of the pileus and stipe has been variously employed in the taxonomy of this family. According to traditional concepts, the pileus surface of Ganoderma is laccate, but is not so in Amauroderma. However, a few species of Amauroderma and Ganoderma have been reported with laccate (A. austrofujianense and A. leptopus) and non-laccate appearance 6 (G. mongolicum). The laccate character, while playing no important role in the segregation of genera and sections in this family, remains available as an identification aid. Context colour of Ganoderma varies from white to deep brown and has been considered a useful character in classification. However, some mycologists have considered it useless for identification of species and super specific groups because it may change under different environmental conditions. Context colour is often changeable, especially in dried species but within a single specimen (Zhao, 1989). Corner (1983) emphasized the importance of observing the context colour of fresh and living specimens in the classification of Ganderma. The size and shape of pores are also useful characters for species classification. The number of pores per millimeter may serve as a specific character. 1.1.3.2 Micro-morphology The structure of the pileal crust and cortex are useful characters in the taxonomy of the Ganodermataceae. The former character occurs mainly in Ganoderma and Anauroderma, but the latter also occurs rarely in Amauroderma. Fruit bodies of Ganoderma mostly have a hymenioderm or characoderm and anamixoderm (Steyaert, 1980). In Elfvingia, the pileal crust is a trichoderm or an irregular tissue; it is also an irregular tissue in Trachyderma (Zhao, 1989). This character is considered to be very useful for identification by some taxonomists. However, it often differs in different specimens of a single species and may show various structural forms. In Ganodermataceae, the hyphal system is usually trimitic, occasionally demitic, the generative hyphae are hyaline, thin walled, branched, septate or not, and clamped. Clamp connections may often be difficult to observe in dried specimens. However, they are easily observed in the youngest part of the hymenium and context of fresh speximens. Skeletal hyphae are always pigmented, thick walled, and arboriform or aciculiform; Skeletal stalks may end in flabelliform, branched binding processes. Binding hyphae are usually colorless with terminal branching. Some species of Ganoderma, such as G. lucidum and G. ungulatum, show Bovis-type binding hyphae, which are produced from the generative or skeletal hyphae. G. mirabile and G oregonense have a pallid context and exhibit intercalary skeletals, which are derived from a transformed and elongated generative cell. On the other hand, Amauroderma has no Bovista-type binding hyphae and many species have intercalary skeletals. Hyphal characters are also influenced by environmental factors. Zhao (1989) observed great variation in hyphal diameter and in frequency of septation due to differences in age as well as in nutrition. For species identification, however, hyphal characters are often useful (Zhao, 1989). 7 Table 1: Morphological comparison of basidiospores of G. lucidum Basidiospore Size (µm) Spore indexa sources Wild fruit body Microscopical Reference feature 9.5-11 X 5.5-7 - Deep yellowish Ito (1955) brown, ovoid and double wall Wild fruit body 8.5-13 X 5.5-8.5 - Ovoid, chamois Steyaert (1972) Wild fruit body 9-13 X 6-8 1.64 Ovoid to ellipsoid Pegler and Young (average 11.5 X 7) Wild fruit body 9-13 X 5-6.9 (1973) - Subovoid with the Bazzalo and apex truncate, Wright (1982) periporum hyaline, smooth and thin endosporic pillars Wild fruit body 8.2-13.5 X 6.8-8.1 - Truncate, ovoid, Melo (1986) brownish to brown Wild fruit body 10.6-11.8 X 6.3- 1.50 Brown, ovoid with Adaskaveg and 7.8 (av. 11.5 X holes and eccentric Gilbertson (1986) 7.4) hilar appendix, double wall and vacuole Wild fruit body 9-12 X 6-7 - Ellipsoid with hole Mims and Seabury and eccentric hilar (1989) appendix Wild fruit body 8.6-10.9 X 6.6-8.3 1.62 Brown, ovoid with (av. 10.1 X 7.5) Seo et al. (1995a)b holes and eccentric hilar appendix, double wall and vacuole Wild fruit body 8.3-12.8 X 5.6-7.2 1.58 Brown, ovoid with (av. 10.4 X 6.6) Seo et al. (1995a)c holes and eccentric hilar appendix, double wall and vacuole Atypical fruiting 6.4-9.6 X 3.2-5.1 structures (av. 7.3-4.2) 1.74 Brown, ellipsoid with holes and eccentric hilar appendix, double wall and vacuole Source: Seo and Kirk, 2000. 8 Seo et al. (1995a) Spore indexa: ratio of spore length to width; -, not determined. Basidiosporesb from a Korean specimen. Basidiosporesc from a Japanese specimen. Basidia and basidiospores are considered as the most important characters for species identification in basidiomycetes. Basidia in Ganodermataceae attain a relatively large size and range from typically clavate to perform. Intermediate forms are often seen in the same specimen. Basidiospores show several dependable characters for identification. Ganodermataceae have a unique double-walled basidiospore; Donk’s (1964) concept for the Ganodermataceae is based on characters of the badidiospores. Basidiospores of Ganoderma are ovoid or ellipsoid-ovoid, occasionally cylindrical-ovoid, and always truncate at the apex. The wall is not uniformly thickened, with the apex always thicker than the base. It is very distinctly double-walled, with the outer wall hyaline and thinner, and the inner one usually coloured and thicker and echinylate or not. In Amauroderma the basidiospores are globose to subglobose, occasionally sylindrical, and form a uniformly thickened wall. In Haddowia the basidiospores are longitudinally double-crested, with small, transverse connecting elements. Microscopic observations, such as the size and morphology of basidiospores, have been adopted as the criteria for the taxonomy of Ganoderma. The badidiospores, which commonly have double walls and are ellipsoid and brownish, vary in size (based on descriptions in the literature; Table 1). A basidium of G. lucidum has four sterigma with a hilar appendix and 12 vacuoles. Basidiospores have an eccentric hilar appendix on a rounded spore base, and vascuoles. The surface of basidiospores is smooth or wrinkled, and most of them have numerous small and shallow holes. The sizes of basidiospores of naturally grown specimens from Japan and Korea were 8.5-11x 6.5-8.5mm (average 10.1X 7.5mm), and 8.5 -13 x 5.5-7 mm (average of 10.4 x 6.6 Mm), respectively. The mean spore indexes (the ratio of spore length to width) were 1.62 and 1.58, respectively. 1.1.4 Cultural Characteristics Critical studies on cultural characteristics are very important in species identification of some groups of higher basidiomycetes. However, useful studies of cultural characteristics of Ganoderma for species identification are rare. In vitro morphogenesis and cultural characteristics of basidiomycetes are affected by various environmental factors, such as light, aeration, temperature, humidity and nutritional condition (Schwalb, 1978; Suzuki, 1979; Manachere, 1980; Kitamoto and Suzuki, 1992). Among these, light is an essential factor for 9 fruiting and pileus differentiation (Plunkett, 1961; Morimoto, et al., 1968 and 1974; Perkins, 1969; Perkins and Gordon, 1969; Morimoto and Oda, 1973; Schwalb and Shanler, 1974; Raudaskoski and Yli-mattila, 1985; Yli-mattila, 1990). Primordium formation, pileus differentiation and tropic growth of the stipe of G. lucidum were affected positively by light (Hemmi and Tanaka, 1936; Stamets, 1993a,b). On the contrary, the growth of mycelium was suppressed by light (Shin and Seo, 1988a, 1989a; Seo, et al., 1995a,b). However, critical studies on the effects of light on mycelial growth and basidiocarp formation of Ganodermataceae have not been repoted. In vitro, cultures of Ganoderma species produce various hyphal structures, such as generative hyphae with clamp connections, fiber or skeletal hyphae, `stag-horn` hyphae, cuticular cells and hyphal rosettes (Adaskaveg and Gilbertson, 1989; Seo, 1995). The colony is white to pale yellow and even, felty to floccose at the optimum temperature on potato dextrose agar (PDA) (Seo, 1987; Adaskaveg and Gilbertson, 1989). The colony becomes more yellowish under exposure to light. The different optimum temperature and growth rates among various species and strains of the G. lucidum complex have been described (Table 2). Hyphal growth of most isolates was 2-4 mm day-1 on PDA but clamydospore (CHL) forming isolates grew faster than those that did not form clamydospores. In vitro, colonies showed various features, such as sectoring, pigmentation, formation of fruit-body primordial (FBP) and atypical fruiting structures (AFSs), which formed basidia and basidiospores without basidiocarp formation (Shin and Seo, 1988a). AFSs were induced by light with ventilation from the white mycelial colony stage (Shin and Seo, 1989b). Some isolates produced FBP on agar medium, but these did not develop into mature fruit bodies during the 30 days of cultivation (Seo, et al., 1995a). In vitro, higher rate of ventilation was required for AFS formation, but FBP could be formed under conditions of lower ventilation. This fact suggests that FBP and AFSs may be initiated by a common morphologenetic control system, but that subsequent development to either FBP or AFSs may be determined by environmental conditions in additions to the genetic characteristic of the strains. The formation of AFSs and FBP on agar media was noted particularly in the G. lucidum complex, specially the Korean and Japanese collections, and in G. lucidum (ATCC52411, Argentina). A few report have describe the formation of aberrant fruit bodies of G. lucidum in vitro (Bose, 1929; Banerjee and Sakar, 1956; Adaskaveg and Gilbertson, 1986). Adaskaveg and Gilbertson (1989) reported that G. lucidum occasionally produced aberrant fruit bodies with 10 basidiospores on agar media. The basidiospores were formed on red, laccate, and coral-like fruit bodies. These fruit bodies might be AFSs because similarity in their appearance and in their ability to form basidiospores. In this case, chlamydospore formation was observed on the same colony, although the AFS- and FBP-forming isolates examined by Seo, et al., (1995a) did not produced chlamydospores. Furthermore, chlamydospore-forming isolates formed neither AFSs nor FBP under any of the conditions examined (Seo et al., 1995a). Among 30 isolates of G. lucidum collected from Japan, Korea, Papua New Guinea, Taiwan and the USA, 20 isolates (about 66% of the isolates tested), none of which was from the USA, formed AFSs with basidiospores and another five isolates (about 17% of the isolates tested), none of them from Papue New Guinea, induced FBP. Of the remaining five isolates, one isolates from Korea formed a callus-like structure without producing basidiospores, this structure differing from AFSs and FBP in form and the other four isolates from Korea, Papua New Guinea and the USA formed neither AFSs nor FBP. Among the latter, three strains formed chlamydospores. One isolate did not form any fruiting structure under standard conditions, but it could produce AFSs in dual culture with a species of Penicillium known to produce a fruit-body-inducing substance (Kawai et al., 1985). Cultural characteristics of Ganoderma species have been studied and employed to determine taxonomic arrangement (Nobles, 1948, 1958; Staplers, 1978; Bazzalo and Wright, 1982; Adaskaveg and Gilbertson, 1986, 1989), but these attempts caused more confusion, as they were often quite different from classical identifications based on morphological features. For example, Nobles (1948, 1958) described the difference in the cultural characteristics of G. lucidum, G. tsugae and G. oregonense. Later, the isolates previously listed as G. lucidum were changed to G. sessile (Nobles, 1965). However, Steyaert (1972) and Stalpers (1978) classified it as G. resinaceum. The cultural characteristics of G. resinaceum given by Bazzalo and Wright (1982) agree with the description of Nobles (1965) and Stalpers (1978) and the description of G. lucidum cultures given by Bazzalo and Wright (1982) is very similar to that of G. tsugae as described by Nobles (1948). Furthermore, Stalpers (1978) considered that the cultural characteristics of the European G. valesiacum were identical to those of G. tsugae from North America, and listed it as a synonym of G. valesiecum. Nobles (1958) suggested that the use of cultural characters in the taxonomy of the polyporaceae reflects natural relationships and phylogeny. The cultural characteristics of G. lucidum complex are provided in Table-2. 11 Table 2: Cultural characteristics of the Ganoderma lucidum complex Species Reference Color Growth Temperature (0C) Growth Chlamyd habit Optimum rate osporeb Maximum Fruiting b (mm/day) G. lucidum Adaskaveg White and Even, 30-34 37 7-8 - + 25-30 33-35 2-7 ± + 25-25 30 2-3 - - 25-30 33 1-2 - - 20-25 30 2-4 - + 25-30 #a 2-3 - - # # 3-4 - - # # 1-2 - - felty Gilbertson (1989) Seo (1995) G. tsugae White Even, to pale felty to yellow. floccose Adaskaveg White Even, and to pale felty to Gilbertson yellow. floccose White Even, to pale felty (1989) Seo (1995) yellow. G. Adaskaveg White Even, oregonense and to pale felty to Gilbertson yellow. floccose White Even, (1989) Seo (1995) felty to floccose G. Seo (1995) White Even, felty to resinaceum floccose G. Seo (1995) Grey Even valesicum #a: not determined. b Formation of chlamydospore, vesicle, atypical fruiting structures and fruit-body primordial on agar media (+), or not (-). Source: Seo and Kirk, 2000. 12 1.1.5 Taxonomy of the Ganoderma lucidum Complex The Ganodermataceae Donk was created to include polypore fungi characterized by doublewalled basidiospores. Large morphological variations in the family resulted in the description of about 400 species, of which about two-thirds classify in the genus Ganoderma Karst, many of them belonging to the G. lucidum complex. The variable morphological features of the G. lucidum complex, such as the size, colour and shape of fruit bodies, may be caused by different environmental conditions during development. Because of the morphological variation in Norwegian laccate specimens of G. lucidum, Ryvarden (1994) commented that ‘Macro-morphology is of limited value for criterion of species in the G. lucidum group and at least 3-5 collections with consistent microscopical charcters should be examined before new species are described in this group’. 1.2 Objectives of the study • To know about Ganoderma lucidum. • To know about the decay of Ganoderma lucidum. • To know about different types of rot caused by Ganoderma lucidum and their control. 13 CHAPTER TWO: IDENTIFICATION AND USES Ganoderma lucidum, is an interesting shelf fungus that is important as a medicine in the Far East, in places such as China, Japan and Korea. G. lucidum is of particular interest because it has been portrayed as a "fix-it-all" herbal remedy for maladies such as: HIV, cancer, low blood pressure, high blood pressure, diabetes, rheumatism, heart problems, paralysis, ulcers, asthma, tiredness, hepatitis A, B, and C, insomnia, sterility, psoriasis, mumps, epilepsy, alcoholism, and the list goes on. The people who are selling G. lucidum herbal supplements mostly make these claims, but G. lucidum, also known as Reishi, ling chih, and ling zhi has a long history of being used as an herbal remedy (Engelbrecht and Volk, 2005). Fig 1: Ganoderma spp. 2.1 How do you identify Ganoderma lucidum? Ganoderma is a member of the Polypores, a group of fungi characterized by the presence of pores, instead of gills on the underside of the fruiting body. G. lucidum, considered by many mycophiles to be one of the most beautiful shelf fungi, it is distinguished by its varnished, red 14 surface. When it is young it also has white and yellow shades on the varnished surface, differing from the dull surface of G. applanatum, the artist's conk. G. lucidum is a saprophytic fungus that tends to grow more prolifically in warm climates on decaying hardwood logs and stumps. This feature helps to distinguish it from a similar looking G. tsugae, which also has a beautiful red varnished surface, but only grows on the stumps and logs of conifers, especially hemlock (as you might guess from the name). Another distinguishing characteristic is the flesh of G. tsugae is white whereas the flesh of G. lucidum is brown. Besides the shelf form, both G. tsugae and G. lucidum can be stalked. The spore prints of both species are brown and the spores are very similar in size and shape (Engelbrecht and Volk, 2005). G. curtisii is considered by some mycologists to be a different species because of its brighter yellow colors and geographic restriction to the southeaster United States. However, most consider G. lucidum and G. curtisii to be the same species because of their similar appearance and habitats; they both prefer to grow on hardwoods. In "North American Polypores," practically the bible for wood-decaying poroid fungi, Gilbertson and Ryvarden, do not consider G. curtisii a species separate from G. lucidum. Another fungus that resembles G. lucidum is G. oregonense, which, like G. tsugae grows on conifers, but is found in the Pacific 15 Northwest and New Mexico. G. oregonense can get up to 1 meter across and has slightly larger spores than G. lucidum and G. tsugae. There are arguments that these four separate species (G. lucidum, G. tsugae, G. curtisii, and G. oregonense) should be considered one species. The reasons for keeping them apart are primarily due to the host specificity of each fungus. Interestingly, if given only either hardwood or conifer wood in culture, each of the four species can grow and produce fruiting bodies, despite their natural occurrence on only one of those substrates (Engelbrecht and Volk, 2005). In 1995, Moncalvo, Wang and Hseu, isolated the DNA of G. tsugae and G. lucidum and found that it was hard to tell the difference between the two species. An even more recent study in 2004 by Hong and Jung, found that G. lucidum from Asia was in its own group, whereas, G. lucidum from Europe and the Americas was more closely related to G. tsugae. G. lucidum, considered rare and hard to find in nature in China and Japan, is now commonly cultured. It can be cultured on logs that are buried in shady, moist areas. G. lucidum can also be inoculated onto hardwood stumps. Under commercial cultivation conditions, G. lucidum is normally grown on artificial sawdust logs, as shown to the right. We'll cover this cultivation method in more detail in a later Fungus of the Month. Other methods of cultivating G. lucidum and many other fungi can be found in Paul Stamets' book, "Growing Gourmet and Medicinal Mushrooms"(Engelbrecht and Volk, 2005). 16 2.2 Uses Now let's look at the medicinal uses of G. lucidum or Reishi. If you feel the fruiting bodies, you'll notice that they're very hard, so no one tries to eat them like most (softer) mushrooms-too tough! It has been used as an herbal remedy for such things as health, recuperation, longevity, wisdom and happiness for centuries in Asian traditional medicine. The first historical mention of G. lucidum was during the rule of the first Chinese emperor, Shinhuag of the Ch'in Dynasty, when the fungus's medicinal uses are first described. In fact, a Reishi Goddess, known as Reishi senshi, was worshipped because she would bestow health, life and eternal youth. There are two different types of Reishi, the traditional wide, shelf-like fruiting body and the antler shape, known as Rokkaku-Reishi. The antler shape arises from varying carbon dioxide levels and low light. These two types are rumored to have different healing characteristics. Recently, there have been a 17 large amount of scientific papers published with experiments attempting to quantify the effect of G. lucidum on the human body. The fungal extract has been shown to act on immune system cells, to work against herpes virus, to lower cholesterol and stop cell proliferation. Unfortunately, while reading these papers it seems important to remind you that we are still not sure if G. lucidum and G. tsugae are separate species. Although the molecular make up has yet to be determined conclusively, several biologically active compounds from G. lucidum have been characterized. These include adenosine, said to have an analgesic effect, R, S-ganodermic and ganasterone that have an antihepatoxic effect, and glucans and polysaccharides that are responsible for the anti-inflammatory and antitumor properties of G. lucidum (Engelbrecht and Volk, 2005). Something else to keep in mind is that all these experiments were done in cell lines, mice, rats and hamsters. So far no large scale unbiased human trials have yet been performed, and the FDA does not yet approve use of Reishi as medical treatment. In order to gain FDA approval, purified compounds from G. lucidum would have to go through an intensive amount of screening in cell lines and animals; much of this pre-clinical testing has already been performed. The next step would be a phase one clinical trial, which assesses the potential drug's safety. Healthy volunteers are paid to take the drug for a certain amount of time, and the compound is studied for its absorption into the body, its metabolism, and its excretion. Once the potential drug passes phase one, which can take up to several months, it moves on to phase two. In phase two, several hundred patients participate in what is called a double blind clinical trial, in which both the patient and the physician are unaware of whether the patient is receiving the potential drug or a placebo. Phase two can last from several months up to several years. If the potential drug is proven effective after phase two, it moves to phase three. Phase three also consists of blind clinical trials, but on a much larger scale. This phase is used to understand the drug's effectiveness, benefits, and the range of possible adverse reactions. Without a doubt, G. lucidum and its researchers have a long road ahead of them before they can prove the mushrooms healing powers (Engelbrecht and Volk, 2005). 18 CHAPTER THREE: SURVIVALITY OF GANODERMA LUCIDUM 3.1 Survival of Wood-inhabiting Microorganisms The tree decay processes start with a wound-a break in the protective bark that exposes the xylem. New space and nutrients become immediately available to a wide variety of organisms-bacteria, non-decay fungi, decay-causing fungi, algae, mosses, lichens, insects, slugs, spiders, and small animals. The competition is intense. Many organisms compete, but as time passes, fewer and fewer are successful. Environmental factors-rain, ice, snow, wind, heat, cold-affect their survival. And, while the wound surface battle rages, those living wood cells that are behind the wound are reacting to the injury and infection. The normal physiological processes give way to new protective processes. Shifts in metabolism occur. Materials that are poisonous to some organisms are formed in the tree cells. In a sense, the tree begins to form a protective chemical shield around and immediately behind the wound (Shigo, 1979). As time passes, fewer species of organisms survive on the wound surface. The concentration of any one group of organisms on the wound surface may fluctuate greatly if there are temperature extremes in the seasons. But now most of the action is inside the tree. A look into the tree after a year shows that a few microorganisms surmounted the chemical barriers formed by the tree. The microorganisms either used the protective materials in the barrier as nutrients or altered these materials in such a way that they were no longer toxic. The protective materials are mostly phenolic compounds in angiosperms and terpenes in gymnosperms. Oxidation and polymerization of these materials take place after wounding. Usually, but definitely not always, the microorganisms that are the first to infect are bacteria and non-decay fungi. In some cases, decay-causing fungi are first. The microorganisms that are the first to infect are called pioneers. Which microorganisms become the pioneers is affected greedy by many factors-time of year of wounding; type, position, and severity of wound. The pioneers in turn affect greatly the species of microorganisms that follow in the succession. And, the species that follow will affect greatly the rate and type of wood alteration. Successions are orderly, but complex (Shigo, 1979). 19 MICROORGANISMS: 1. Bacteria, 2. Cytospora, 3. Pyrenochaeta, 4. Phoma, 5. Phialophora, 6. Gliocladium, 7. Graphium, 8. Cephalosporium, 9. Coniothyrium, 10. Fusarium, 11. Rhinocladiella, 12. Acrostaphalus, 13. Trichocladium, 14. Yeasts. After 4 years fewer microorganisms are active behind the wound. Sporophores of decay fungi may begin to develop. The first few years after wounding are the most important for the tree and the microorganisms. Within this time, the rate and much of the extent or limits of the infection will be set. One group or species of organisms follows another until all the wood is decomposed in a succession. But all wounds do not follow such a pattern of infection to decomposition. Most of the time the tree is effective in blocking or limiting the infection. The wound may close. The final stages of the succession may not occur. But, after the tree dies, many other groups of microorganisms will begin to digest the wood. And when this happens, another succession occurs. In summary many species of microorganisms are involved in the decay processes. The microorganisms become established in successions (Shigo, 1979). 3.2 Survival of Trees against Wounds and Infections Trees have evolved over a period of 200 to 400 million years while being under the constant stress of wounding. Even with this stress, they still have evolved to be the largest and longest-lived organisms ever to inhabit the earth. And yet trees have no wound healing process healing in a sense of replacing or repairing injured tissues. Heal means to restore to a previous healthy state. It is impossible to heal injured and infected xylem. Trees have evolved as highly ordered, compartmented plants, which instead of healing compartmentalize in an orderly way the injured and infected tissues. A coded model 20 System for explaining how a tree is compartmented and how it compartmentalizes infected and injured wood has been developed. It is called CODIT, an acronym for compartmentalization of decay in trees. Terms such as "walls" and "plugs" are used in the model only to help present a mental image of the compartments. These terms are not meant as technical terms (Shigo, 1979). 3.2.1 Comartmentalization of decay Wall 1: After being wounded, the tree responds in a dynamic way by plugging the vertical vascular system above and below the wound. The conducting elements-vessels in angiosperms and tracheids in gymnosperms are plugged in various ways: tyloses, gum deposits, pit asperations, etc. The plugged elements complete the transverse top and bottom walls of the compartments. Wall 1 is the weakest wall. Wall 2: The last cells to form in each growth ring make up the tangential walls of the compartments. These walls are continuous around each growth ring except where sheets of ray cells pass through. Wall 2 is the second weakest wall. Wall 3: Sheets of ray cells make up the radial walls. They are discontinuous walls because they vary greatly in length, thickness, and height. Walls 3 are the strongest walls in the tree at the time of wounding. Wall 4: After a tree is wounded, the cambium begins to form a new protective wall. The wall is both an anatomical and a chemical wall. This wall separates the tissue present at the time of wounding from tissue that forms after. It is the strongest of the four walls. 3.3 Survival of Ganoderma Species and Other Wood-Inhabiting Fungi The survival of mycelia of Ganoderma lucidum, G. boninense, G. australe and G. weberianum in colonized wood was measured in soils with different soil matrix potentials. Survival of mycelia of G. australe and G. boninense, which do not produce chlamydospores, buried in plots subjected to different soil moisture treatments, declined rapidly, and the fungi could not be recovered after 9 to 12 weeks. Survival of mycelia of G. lucidum and G. weberianum, which produce chlamydospores, rapidly declined from 0 to 15 weeks of incubation but consistently ranged from 35 to 50% after 15 weeks of incubation. In regression analyses for each of the four Ganoderma species, there was no 21 difference in the rate of change of mycelial survival over time among different soil moisture treatments. However, when data from only the –0.20 MPa treatment were used, the rates of change of mycelia survival over time of G. australe and G. boninense significantly differed from those of G. lucidum and G. weberianum. G. australe and G. boninense were not recovered from pieces of infested wood subjected to 3 and 1 months of flooding, respectively. In treatments with lower soil moisture, the survival of these two fungi ranged from 80 to 90% over 2 years. In all soil moisture treatments, survival of G. lucidum and G. weberianum ranged from 80% to more than 90% over 2 years. Similarly, seven species of other wood-inhabiting fungi that do not produce chlamydospores were not recovered from pieces of infested wood subjected to 1 or 5 months of flooding, but chlamydospore-producing species were recovered. These results indicate that, regardless of chlamydospore formation, woody debris in soils harboring wood-decay fungi may be important for long-term survival, and chlamydospores of Ganoderma in woody debris enhance the resistance of the fungi to environmental stresses such as flooding. Flooding infested fields may help control those woodinhabiting fungi such as G. australe and G. boninense that do not produce chlamydospores (Chang and Chang, 2003). Ganoderma (Basidiomycota: Ganodermatales) is a genus of wood-inhabiting fungi on monocots, dicots, and gymnosperms. Members of the genus are found around the world from tropical to temperate habitats (Sinclair et al.1987). Some species are saprophytic, but several are pathogens that cause decay in roots, butts, and trunks of living trees. G. australe (Fr.:Fr.) Pat., G. boninense Pat., G. lucidum (W. Curtis:Fr.) P. Karst., and G. weberianum (Bres. & Henn.) Steyaert are common root pathogens in Taiwan that cause decay and slow decline of numerous orchard and forest tree species (Chang, et al. 1999). Slowly expanding, circular disease patches extending from infection centers can be observed in the field, which indicate that the disease caused by Ganoderma species is spread from diseased to healthy trees by root contact (Adaskaveg, and Gilbertson, 1987; and Bakshi, et al. 1976). Infested root debris may be important as inoculum and for longterm survival. Airborne basidiospores can also initiate new infections on freshly cut stumps or logging debris, with subsequent spread to live trees by root contact (Darus et al., 1996; Sanderson and Pilotti, 1997; and Turner, 1965). Abundant chlamydospores are produced in pure culture and in woody debris by some species of Ganoderma, such as G. lucidum and G. weberianum, while others such as G. 22 australe and G. boninense produce no chlamydospores (Chang et al.,1996). The role of chlamydospores in the epidemiology of Ganoderma species is unknown, and the longterm survival of chlamydospores and mycelia in soil has not been previously documented. In this study, the survival of chlamydospores and mycelia of Ganoderma species in artificially infested woody debris was assessed in soils with varying moisture levels. In addition, the survival of some other wood-inhabiting fungi in artificially infested woody debris was studied (Chang, 2003). The survival of Ganoderma australe, G. boninense, G. lucidum and G. weberianum mycelia, and of Ganoderma-colonized wood was measured in soils with different soil matrix potentials. G. australe and G. boninense produced no chlamydospores, while G. lucidum and G. weberianum did. Mycelia of G. australe and G. boninense buried in the 0.025 MPa soil moisture treatment declined rapidly and could not be recovered at 9 and 12 wk, respectively. Mycelia buried in the -0.50 MPa soil moisture treatment had relatively higher recovery rates at the same incubation times compared with those in the higher soil moisture treatments. However, mycelia were not recovered at 15 wk in any treatment. Survival of G. lucidum and G. weberianum mycelia in all soil moisture treatments rapidly declined from 0 to 15 wk after incubation. However, survival of mycelia consistently ranged from 35% to 50% at 15 to 52 wk after incubation. These results indicate that chlamydospores in soil play an important role in the long-term survival of Ganoderma species when mycelia are not harbored in woody debris. G. australe and G. boninense were not recovered from pieces of artificially infested wood subjected to 1 and 3 mo of flooding, respectively. In treatments with the lower soil moisture, the survival of these 2 fungi ranged from 80% to 90% over 2 yr. In all soil moisture treatments, G. lucidum and G. weberianum ranged from 80% to more than 90% over 2 yr. These results indicate that, regardless of chlamydospore formation, woody debris in soils harboring Ganoderma species plays an important role in the long-term survival of the fungi, and chlamydospores of Ganoderma in woody debris enhance the resistance of the fungi to environmental stress such as flooding. Seven species of other wood-inhabiting fungi which do not produce chlamydospores were not recovered from pieces of artificially infested wood subjected to 1 or 5 mo of flooding. In a treatment with a lower soil moisture (-0.50 MPa), the survival of these 7 fungi ranged from 70% to more than 90% over 2 yr. However, the survival of others that produced chlamydospores ranged from 70% to more than 90% in soils with -0.50 MPa and flooding. These results 23 are similar to those for Ganoderma species and indicate that chlamydospores of woodinhabiting fungi and woody debris play an important role in their long-term survival and in their resistance to environmental stresses such as flooding. Flooding infested fields may help control wood-inhabiting fungi, which do not produce chlamydospores, but may have little effect on those which produce chlamydospores in the field (Chang et al., 2002). 3.3.1 Survival of Ganoderma lucidum in infected wood Woody debris infested with G. lucidum, G. australe, G. boninense and G. weberianum survived significantly longer than mycelium when buried in soil. Mycelia of G. lucidum and G. weberianum, which produced chlamydospores, survived over 52 weeks of incubation in soil, but mycelia of G. australe and G. boninense, which do not produce chlamydospores, survived only up to 12 weeks of incubation. In addition, G. lucidum and G. weberianum in woody material survived in flooded soil, but G. australe and G. boninense did not. Chlamydospores appear to be important to survival, and woody material infested with Ganoderma species, regardless of chlamydospore production, appears to be more important than mycelia per se for long term survival of these fungi in soil. Survival of 16 other wood-inhabiting fungi in soil was similar to that of the four Ganoderma species. Chlamydospores of these wood-inhabiting fungi may be important for long-term survival, and these structures may provide resistance to severe environmental stresses such as soil flooding. These results are consistent with those reported for woody debris infested with Phellinus noxius (Chang, 1996). Arthroconidia are produced by some basidiomycetous woodinhabiting fungi (Stalpers, 1978), but arthroconidia of P. noxius do not appear to be important survival structures (Chang, 1996). In our study, the four species of wood-inhabiting fungi that produced arthroconidia but not chlamydospores did not survive in flooded soil. Submersion of wood in water has been used to prevent postharvest colonization by wood-decay fungi (Rayner and Boddy, 1988). The benefit might be due to the activities of anaerobic microorganisms. In this study, when wood sections colonized by test fungi that produced chlamydospores were submerged in water, the fungi survived for over 2 years. However, when wood sections colonized by test fungi that did not produce chlamydospores were submerged in water, the fungi died within 3 months. When wood sections colonized by P. noxius, which does not contain chlamydospores, were submerged in water, the fungus died within 1 month (Chang, 1996). Thus, for disease management of root and butt rot caused by wood- 24 inhabiting fungi that do not produce chlamydospores, flooding infested fields might eliminate the inoculum source in infested wood. However, the pieces of infested wood that were tested in this study were relatively small, and field studies are required to test this hypothesis. G. australe and G. boninense were not recovered from pieces of artifically infested wood subjected to 1 and 3 mo of flooding, respectively. However, in treatments with lower soil moisture, G. australe and G. boninense survival ranged from 80% to more than 90% over 2 yr (Fig. 2A,B). Trends for survival of infested wood with time did not significantly differ (p = 0.05) among all treatments when flooding treatment was excluded, but significantly differ (p = 0.01) when flooding treatment was included. The LSD test among all treatments at different incubation times when the flooding treatment was excluded did not significantly differ (p = 0.05) for G. australe and G. boninense. Trends for survival of infested wood with time did not significantly differ among all treatments for G. lucidum and G. weberianum. In all soil moisture treatments including flooding, G. lucidum and G. weberianum survival ranged from 80% to more than 90% over 2 yr (Fig. 2C,D). The LSD test among all treatments at different incubation times did not significantly differ (p = 0.05) (Chang, 2003). 3.3.2 Survival of other fungi in infected wood Survival of 16 wood-inhabiting fungi in soils with -0.50MPa soil matrix ranged from 70% to 100% over 2 yr (Table 2). For each species, the LSD test at different incubation times was not significant (p = 0.05). However, when these fungi in infested wood were placed in soils with flooding, 4 of them, i.e., Bjerkandera adusta, Favolus spatulatus, Phellinus longisetulosus, and Trametes versicolor, and 3 of them, i.e., Abortiporus biennis, Schizopora flavipora, and Trametes hirsuta, were not recovered from pieces of artificially infested wood subjected to 1 and 5 mo of treatment, respectively. The survival of the remaining fungi which produced chlamydospores ranged from 70% to 100% over 2 yr. For these fungi, the LSD test at different incubation times did not significantly differ (p = 0.05) for each species (Chang, 2003). 3.3.3 Mycelium growth of Ganoderma lucidum Mycelium growth and yielding of two strains of Ganoderma lucidum (Fries) Karst. cultivated on oak, beech or birch sawdust substrates were evaluated. Mycelium growth varied between strains and depended on substrate type. Mycelium of strain CS 95 grew 25 faster than strain LZ 1, on all tested substrates. Both strains grew faster through the birch sawdust substrate than the other two substrates. Strain CS 95 gave higher yield than strain LZ 1 but the yield depended also on substrate type. Yield on the oak sawdust and beech sawdust substrates was similar and it was higher than on the birch sawdust substrate (Siwulski, et al.,2001). 26 CHAPTER FOUR: WOOD DECAY FUNGI Fungi compose about 4% of the known species of life on earth and about 8% of estimated unknown species. In spite of their importance, less than 5% of the estimated 1.5 million fungi have been identified. Fungi that break down woody plants into their basic elements are a critical part of the tropical ecosystem. Without them, dead trees and shrubs would cover the soil and decompose very slowly. New seedlings not only need a clear path to the sunlight, they need the nutrients locked away in dead plants: Rotted wood enriches the soil for plant growth and improves its structure. Ganoderma lucidum as wood decay fungi also damage living trees. In the tropics, millions of hectares of plantations are affected, as are fruit trees and woody landscape plants. Trees with internal decay often lose limbs or blow over in strong winds (Brooks, 2004). 4.1 Ecology and Life Cycle Wood refers to both the dead xylem cells in the center of the tree responsible for structural support (heartwood), and the living xylem cells beneath the bark that carry water and nutrients up the tree (sapwood). Most wood rot fungi degrade the heartwood. Brown rot fungi have enzymes that break down polysaccharides, but leave most of the brown-colored lignin. In American Samoa most fungi cause white rot, degrading lignin along with the polysaccharides, leaving wood spongy and bleached. Pathogenic fungi attack sapwood and can kill the tree. Most wood decay fungi are in the class Basidiomycetes. They form spores on narrow gills or in pores on the underside of fruiting bodies. Wood rot fungi enter trees either as spores landing in wounds, or by root-to-root contact. After spores germinate, threadlike strands of the fungus body called hyphae colonize the heartwood. At some time after the wood is well colonized, the fungus forms fruiting bodies (conks, mushrooms) that produce more spores (Brooks, 2004). 4.2 Wood Decay Species Limited information exists on wood decay fungi. Basidiomycetes account for 62 of these species, in 36 genera, and 13 families (McKenzie, 1996 and Brooks, 2004). BASIDIOMYCETES Amylosporus (?) campbellii (Berk.) Ryv. Antrodiella semisupina (Berk. & Curt.) Ryv. comb. nov. Antrodiella sp. Ryv. & Johan. 27 Auricularia polytricha (Mont.) Sacc. Auricularia sp. Bull. Cerenna sp. S.F. Gray Ceriporia sp. Donk Coriolopsis floccosa (Jungh.) Ryv. Cotylidia aurantiaca (Pers.) Weldon Daedalia sp. Pers.:Fr. Earliella scabrosa (Pers.) R.L. Gilbertson & Ryv. Echinochaete russiceps (Berk. & Broome ) Reid Echinochaete sp. D.A. Reid Elmerina sp. Bres. Favolus spathulatus (Jungh.) Bres. Favolaschia pustulosa (Jungh.) Singer Flavodon flavus Ryv. Ganoderma australe (Fr.) Pat. Ganoderma lucidum (W. Curtis:Fr.) P. Karst. Ganoderma resinaceum Boud. Ganoderma sp. P. Karst. Hexagonia apiaria (Pers.) Fries Hexagonia tenuis (Hook.) Fries Hyphoderma rude (Bres.) Huorest. & Ryv. Hypocrea incarnata Pat. & Har. Lenzites elegans (Fr.) Pat. Lenzites vespacea (Pers.) Ryv. Lenzites sp. Fr. Microporus affinis (Fr.:Blume & Nees) Microporus vernicipes (Berk.) Kunt. Microporus sp. Kuntz Mycorrhaphium steroides (Cooke) Maas G. Nigroporus durus (Jungh.) Murr. Phanerochaete salmonicolor (Berk. & Broome) Julich Phellinus lamaensis (Murrill) R. Heim Phellinus noxius (Corner) G. Cunn. Phellinus sp. Quel. 28 Phillipsia domingensis (Berk.) Berk. Pleurotus sp. (Fr.) P. Kumm., nom. cons. Podoscypha involuta (Klotzsch ) Imazeki Podoscypha nitidula (Berk.) Pat. in Duss Polyporus (?) grammocephalus Berk. Polyporus philippinensis Berk. Polyporus sp. P. Mich. ex Adans.:Fr., sensu Donk. Pseudohydnum sp. P. Karst. Pycnoporus sp. P. Karst. Rigidoporus cystidioides (Lloyd) Corner Rigidoporus defibulatus (Reid) Corner Rigidoporus microporus (Fr.) v. Overeem Schizophyllum commune Fr.:Fr. Stereopsis radicans (Berk.) Reid Stereum sp. J. Hill ex Pers. Trametes hemitephra (Berk.) Corner Trametes hirsuta (Wulfen:Fr.) Quel. Trametes marianna (Pers.) Ryv. Trametes menziesii (Berk.) Ryv. Trametes modesta (Fr.) Ryv. Trametes versicolor (L.:Fr.) Pilat Trametes sp. Fr. Tremella cinnabarina (Mont.) Pat. Tremella fuciformis Berk. Tremella sp. Pers.:Fr., nom. cons. 4.3 Tree Decay An Expanded Concept To clarify further the tree decay concept that expands the classical concept to include the orderly response of the tree to wounding and infection-compartmentalization-and the orderly infection of wounds by many microorganisms-successions. The heart rot concept must be abandoned because it deals only with decay-causing fungi and it states that these fungi grow unrestricted through heartwood after infection of fresh wounds. The heart rot concept emphasizes descriptions of decay-causing fungi and types of decayed wood. It describes disordered wood and events that occurred in the past. The expanded decay concept 29 emphasizes the order of a compartmented tree, the order of compartmentalization, and the order of successions. Regulation of discoloration and decay depends on understanding compartmentalization and successions (Shigo, 1979). 4.3.1 Role and importance of tree decay Recycling dead organic matter essential for life of new trees; providing food and shelter for wildlife and many other microorganisms; decreasing the potential for fire. Reducing the strength and value of trees and wood products; decreasing the attractiveness of trees (Shigo, 1979). 4.3.2 Decay Is a Natural Recycling Process Cell walls are digested; strength of wood is reduced. Decayed wood is the result of the process. Tree decay involves interactions among trees, which are the tallest, greatest in mass and longest-lived organisms ever to inhabit the earth, and microorganisms primarily bacteria and fungi-which are some of the smallest organisms on earth (Shigo, 1979). 4.3.3 An Understanding of Gradations is Necessary Microscopic, wood-inhabiting organisms and the long-lived, gigantic trees interact intimately in a long and intense struggle for survival. This struggle starts with a wound and can end with total decomposition of the wood. Many biotic and abiotic factors are involved in the decay processes. It is difficult to determine where one event or process starts and another ends. The events and processes overlap and mingle like the colors in a large spectrum or rainbow. Where does one color stop and another start? The natural process of decay is even more complicated and it might be more accurately likened to a multidimensional spectrum that is constantly changing over time. When we get too close to some processes, they are changed because of our methods of study and measurements. This is why we must consider the patterns of events rather than specific events (Shigo, 1979). It is impossible to: • Stop the processes. We cannot stop our ultimate death either, but that does not mean we cannot live a long, healthy, and productive life. 30 It is possible to: • Prevent decay-temporarily. • Decrease the rate. • Increase the rate. • Detect it. • Predict its rate. • Predict its ultimate configuration. • Minimize its volume. How effectively it can do the above depends on how well it understand the decay processes. The decay processes are not so overwhelming that they defy regulation. Survival of any organism depends on its ability to compete effectively with other organisms for space and an energy source. To survive, organisms must live long enough to complete a life cycle. They must compete for food and space under constantly changing environmental conditions. They must respond rapidly and effectively to injury caused by abiotic and biotic factors. The response must be such that it enables the organisms to continue to survive (Shigo, 1979). 4.4 The Classical Concept of Tree Decay Robert Hartig developed the concept of tree decay almost a century ago. At that time, decay was well recognized as a serious economical problem. In tune with the theory of spontaneous generation, scientists believed thatDecay Causing Fungi: Robert Hartig, in tune with the germ theory that emerged after 1845, said thatFungi Causing Decay: This simple reversal of two words set the stage for the decay concept and, in some ways, for the beginning of the science of Forest Pathology. 4.5 The Expanded Concept of Tree Decay Discoloration is a process. Decay is a process. Discolored and decayed wood represent the disordered product of an ordered material. Discolored wood results from an alteration of cell contents. There is only slight or no loss of strength. The tree and the microorganisms are involved in the processes that result in discolored wood. Decayed wood is a result of a breakdown of cell walls. There is a great loss of strength. Many microorganisms are involved in the processes that result in decayed wood. The wood cells have been killed and 31 the decay-causing microorganisms compete among themselves for the dead matter. Many factors affect the rate of decay. The tree has the greatest influence in the limits of the decay column. Most of the events in the decay process are ordered. The classical concept of decay deals mostly with disordered events. It is impossible to regulate disorder (Shigo, 1979). The expanded concept of decay deals primarily with ordered events. The ordered system of trees and microorganisms is constantly impacted by many types of ordered and disordered events: Temperature extremes for short periods, moisture extremes for short periods, changing soil elements, storms, fires, logging operations, soil compaction, flooding, earthquakes, soil grade changes, pollution, chemicals, gas leakage, human-caused epidemics, or diseases and insects. These factors alone or in combinations greatly affect the microorganisms and the tree. The factors may alter the rate of the decay processes but not the patterns of successions and compartmentalization, which are ordered events (Shigo, 1979). Successions, an ordered process, often determine rate. Upset the normal successions and the process will be stalled (not stopped). This can be done by purposely infecting wounds with microorganisms that normally occur late in the succession, such as with species of Trichoderma. The decay process will be stalled. Compartmentalization represents order. Some trees have a stronger wound response than others; they can compartmentalize invaded tissues more effectively than other trees of the same species. The differences are shown in the next three illustrations. When drill holes are inflicted in strong compartmentalizers, a pattern like this results-very short vertical columns and little to no lateral extension. When drill holes are inflicted into moderate compartmentalizers, a pattern like this results-long vertical columns and some lateral extension of discolored wood. When drill holes are inflicted in weak compartmentalizers, columns that look like this are seen-long vertical columns and complete lateral extension of discolored wood. The tree's response in the compartmentalization of discolored wood appears to be under strong genetic control (Shigo, 1979). 4.6 Pests Management 4.6.1 Pests in Landscapes and Gardens Wood Decay Fungi in Landscape Trees: Several fungal diseases, sometimes called heart or sap rots, cause the wood in the center of trunks and limbs to decay. Under conditions favoring 32 growth of certain rot fungi, extensive portions of the wood of living trees can decay in a relatively short time (months to years). This significantly reduces wood strength and kills sapwood storage and conductive tissues. Almost all species of woody plants are subject to trunk and limb decay (Hickman and Perry, 1997). Fungus: Ganoderma lucidum Trees commonly associated with various fungus rot: Acacia, Apple, Ash, Birch, Boxwood, Cherry, Citrus, Elm, Hackberry, Sweet Gum, Black locust, Honey locust, Magnolia, Maple, Oak, Olive, Peach, Pepper tree, Pine, Poplar, Redbud, Spruce, and Willow etc (Hickman and Perry, 1997). Description and comments: Fungus causes a white rot and is capable of attacking living trees, causing extensive decay of roots and trunk. Can kill the host over a period of 3-5 years. On some trees, oaks and maples, the rate of decay is rapid. The red-brown, annual conks are up to 14 inches wide and coated on top with a distinctive varnish like crust; generally appear at base of trunk during summer. Environmental stress, such as drought and wounding, may predispose trees to damage from this fungus (Hickman and Perry, 1997). Damage: Decay fungi destroy the plant's internal supportive or structural components (cellulose, hemicellulose, and sometimes lignin). Decay is not visible on the outside of the plant, except where the bark has been cut or injured, when a cavity is present, or when the rot fungi produce reproductive structures. Wood decay makes trees hazardous because trunks and limbs become unable to support their own weight and can fall, especially when stressed by wind, heavy rain, or other conditions (Dreistadt et al., 2004). 4.6.2 Identification and Biology Many wood rot fungi can be identified by the distinctive shape, color, and texture of the fruiting bodies that form on trees. These structures, called conks or brackets, are often located around wounds in bark, at branch scars, or around the root crown. Some fruiting bodies, such as Armillaria mushrooms, are annual (for example appearing soon after the beginning of seasonal rains), but most are perennial and grow by adding a new layer each year (Dreistadt et al., 2004). Airborne spores that infect trees through injuries and wounds spread fungi that decay limbs and trunks. Injuries include pruning wounds, vandalism, and damage from machinery or 33 construction. Sunburn, fire, ice, lightning, snow, or insects that bore into the trunk or branches may cause wounds. Some decay fungi, such as Armillaria mellea, principally infect the roots and can spread to nearby plants from the roots of infected hosts (Dreistadt et al., 2004). 4.6.3 Management Wood decay is usually a disease of old, large trees. It is very difficult to manage, but a number of factors can reduce the risk of serious damage. First, trees should receive proper cultural care to keep plants vigorous. Minimize wood decay by protecting plants from injuries. Properly prune young trees to promote good structure and avoid the need to remove large limbs from older trees, which creates large wounds. Cut out dead or diseased limbs. Make pruning cuts properly; prune just outside the branch bark ridge, leaving a collar of cambial tissue around cuts on the trunk to facilitate wound closure, but avoid leaving stubs. Make cuts so that rainwater will drain. Wound dressings are not recommended, as they have not been found to hasten wound closure or prevent decay. A qualified expert for signs of wood decay and other structural weakness should regularly inspect trees that may cause personal injury or property damage if they fall. Hazardous trees may need to be trimmed, cabled, braced, or removed (Dreistadt et al., 2004). o Avoiding damage to tree bark will reduce wood decay inplantations and landscapes. Wounds caused by saws, knives, machinery, or fire, create pathways for fungi to enter wood. o Cut diseased or damaged branches cleanly, close to, but not flush with the bark. o Prune when branches are small to avoid large wounds. o Destroy dead trees, branches, and their fruiting bodies. o Keep trees healthy so they can defend against disease (Dreistadt et al., 2004). 34 CHAPTER FIVE: WOOD ROT FUNGI A fungus does not engulf food as an animal does. Rather, it is as if a fungus wears its stomach on its outside. Each feeding hypha releases digestive enzymes from its surface. These enzymes digest food in the surrounding milieu, and then the nutrients are absorbed back into the hypha. A wood rot fungus tends to gain access to wood by insinuating its hyphae into the conductive cells of the plant. Once inside a tracheid, or a vessel, a hypha releases enzymes, which breaks down the cell's wall. In this manner they devour wood from the inside out. Most of these wood rot fungi have both asexual and sexual sporocarps. The asexual anamorphs can differ greatly from sexual sporocarps in form and colour. The chlamydospore and conidiospore bodies are often small and hidden inside the wood or in hollows. The asci or basidia bearing bodies are larger and more exposed (Hagen and Bruce, 2001). If the 'fruiting bodies' of wood-rotting fungi appear on trees, this means two possible things: (1) fungi are digesting dead tissue in the tree, or (2) fungi are killing and then digesting living tissues in the tree. The dead tissue is usually heartwood, and the living tissue is usually sapwood. The vast majority of wood-feeding fungi live off dead wood. These can still be a problem, if they make the tree structurally less sound. Hollow trees are often hollow because fungi have digested away the dead heartwood in the core. Such hollow trees are prone to breaking in windstorms, ice storms and other stresses (Schwarze et al., 2004). In temperate climates very few wood-rotting fungi are significant threats to tree health: (1) honey fungi (Armillaria spp.), (2) white pocket rot (Heterobasidion annosum), and (3) artist's fungus (Ganoderma spp.). 5.1 Ganoderma / Artist's Fungus Several species of Ganoderma can cause root and butt rot on broadleaf trees. G. lucidum and G. applanatum are the main culprits. The fruiting bodies are medium sized tan-brown brackets, which grow in clusters on the base of a broadleaf tree. Extensive rot can cause a tree to break and fall, during a windstorm. This butt-rot mostly occurs on landscape trees, which have had their bark, damaged. Lawnmower and line-trimmer scars are usually the instigating stress. Ganoderma seldom occurs on trees that have not been damaged. It is very seldom contagious from an infected tree to a healthy tree. In the wild Ganoderma grows mostly on old trees, which have structural disorders. Ganoderma is also known as “artist’s fungus”. 35 This is because the pale spore-bearing layer on the underside of the bracket can be used as a surface to engrave images upon. Drawing pictures on bracket fungi was once a common rural folk craft. The craft is now somewhat passé (Tudge, 2000). 5.1.1 Rots caused by Ganoderma lucidum Decay fungi often are divided into white rots, brown rots, and soft rots. White rots break down lignin and cellulose, and commonly cause rotted wood to feel moist, soft, spongy, or stringy and to appear white or yellow. Brown rots primarily decay the cellulose and hemicellulose (carbohydrates) in wood, leaving behind the brownish wood lignin. Wood affected by brown rot is usually dry and fragile, readily crumbles into cubes because of longitudinal and transverse cracks, and commonly forms a solid column of rot in wood. Brown rot is generally more serious than white rot. Both bacteria and fungi cause soft rots. They decay cellulose, hemicellulose, and lignin, but only in areas directly adjacent to their growth. Soft rots grow more slowly than brown and white rots and usually do not cause extensive structural damage to wood of living trees. 5.1.1.1 White Rot If a fungus digests mostly the brown lignin, and not the pale cellulose, it is a white rot. Cellulose is located mostly on the inner layers of the tracheid cell walls. Cellulose tends to give wood its tensile strength. White rot can occur in conifer wood, but it is most common in broadleaf xylem. The plump orange brackets of the Inonotus species are often a sign of white rot. Ganoderma fungi are also white rot agents. The white pocket rot (Heterobasidion annosum) gets its English name from the colour of its rot (White, 2004). The cellulose and lignin are digested at about equal rates. The digestion usually starts from the cell lumens and proceeds towards the middle lamella. Hymenomycetes are involved mostly, although species in the Xylariaceae also are associated with white rot (generaHypoxylon, Xylaria, Daldinia). There are many types of white rot (fleck, stringy, ring, pocket, etc.). These terms describe their macroscopic appearance. White rots occur also in wood products such as utility poles. Some of the most damaging fungi associated with white rots are Phellinus pini, Armillariella mellea, Fomitopsis annosa, G. applanatum, Oxyporus populinus, Phellinus igniarius, Innotus glomeratus, I. obliquus, and Echinodontium tinctorium. Three species were tested, all causing white rot. G. resinaceum and G. pfeifferi produced an extra cellular enzyme that degraded carboxymethyl-cellulose (CMC) and waterinsoluble powdered Beech-wood cellulose (c.p.), while G. Lucidum degraded CMC only 36 slightly and c.p. not at all. Only the two first named produced P. glucosidase ( Walch and Kuhlwein, 1968). The biodegradation of sugarcane bagasse and its components by different strains of white rot fungi i.e. Stropharia rugosoannulata, Ganoderma lucidum, Pleurotus ostreatus, Pleurotus-131, Pleurotus-137, Pleurotus P7H7, Polyporus versicolor, Polyporus adustus, Polyporus sanguineus and Polyporus cinnibarinus was studied. During the first phase of growth (0-20 d) hemicellulose was selectively degraded whereas during the second phase (21-40 d) lignin was preferentially degraded in comparison of the other substrate components. The maximum degradation of dry matter was observed in the group treated with Polyporus sanguineus, but lignin degradation was highest with Polyporus versicolor. The largest increase in in vitro digestibility and nutrient availability was observed on Ganoderma lucidum treatment. Among the fungal strs tested, only G. lucidum, S. rugosoannulata and Pleurotus-137 caused an increase in IVDMD by >10 percentage units and were able to improve the nutrient availability from sugarcane bagasse on fungal treatment (Karma et al., 1993). A strain of Trichoderma viride isolated from basidiocarp of Ganoderma lucidum (collected from Chennai, Tamil Nadu, India), a root rot pathogen of Tamarindus indica, was tested for its antagonistic activity against G. lucidum. The basal stem rot of T. indica caused by G. lucidum was associated with the production of lignolytic enzymes, lignin peroxidase, manganese peroxidase, laccase, cellulase, xylanase etc. The different carbon sources such as sawdust, extracted lignin, and glucose used in mono and dual culture media allowed the fungi to show an increased activity of lignolytic enzyme, such as lignin peroxidase, manganese peroxidase, and laccase. Indeed, in the dual cultures of T. viride and G. lucidum a significant decrease in lignolytic enzyme activity was observed whereas increased activity of mycolytic enzymes such as chitinase and proteinase was observed. In vivo studies on the infected host tissue, the pathogenic fungus produced the highest activity of lignin peroxidase and laccase. The introduction of antagonistic fungus T. viride on the infected tissue of T. indica significantly reduced the activities of lignocellulolytic enzymes and the growth of the pathogenic fungus (Murugesan et al., 2002). In studies with wood of grapevine, Quercus hypoleucoides, Prosopis velutina, Abies concolor and Pseudotsuga menziesii, grapevine wood lost the most weight and P. velutina the least. G. lucidum isolates generally caused greater loss of all woods than did G. tsugae isolates. The range of the percent wt losses varied with the wood. Both Ganoderma spp. caused 37 simultaneous decay in all woods. However, chemical analyses of the decayed blocks indicated that selective delignification by both spp. also occurred in grapevine and A. concolor blocks. Chemical analysis of the decayed oak blocks indicated the percentages of lignin and holocellulose were not statistically different from the controls but there was a trend towards delignification. The analyses of the P. menziesii blocks indicated only simultaneous decay. SEM demonstrated selective delignification and simultaneous decay of all woods tested though the extent of the delignification differed among the wood spp. Delignification appeared mainly in areas of tracheids or fibre tracheids, while the rays were simultaneously decayed (Adaskaveg and Gilbertson, 1986). 5.1.1.2 Brown Rot If a fungus digests mostly cellulose, leaving behind the lignin, it is a brown rot. Lignin is concentrated largely in the outer layers of tracheid cell walls. Lignin tends to lend compressive strength to wood. Brown rots are almost entirely due to basidiomycetes. The majority of brown rots occur in conifers. Some Stereum fungi can cause brown rots. The Fomitopsis brackets also cause brown rot in conifer wood. The chestnut tongue bracket (Fistulina hepatica) can cause a kind of brown rot in broadleaf trees such as oak and chestnut (White, 2004). The cellulose is digested preferentially, and the lignin is only altered slightly in this type of rot. Brown rots have a shrunken cubical appearance. Brown rots occur mostly in gymnosperms and in wood products. Some common fungi associated with brown rots are Phaeolus schweinitzii, Poria monticola, Lentinus lepideus, and Polyporus betulinus. Polyporus sulphureus and Fomes pinicola are examples of fungi that cause the less common brown rot in angiosperms. 5.1.1.3 Soft Rot If a fungus digests both cellulose and lignin it can produce a soft rot. Soft rot tends to weaken both the tensile and compresive strength of the wood. Fungi that digest both cellulose and lignin cause soft rots. Often these fungi have very branched hyphae which enzymatically bore into the plant's cell walls. Ustulina deusta is one very important type of soft rot fungus. The soft tan brackets of Meripilus giganteus can cause soft rot. The Inonotus, Chaetomium and Rigidoporus fungi can also cause soft rot in some of their hosts (White, 2004). Some microorganisms digest the S2 layer of the middle cell wall. Several patterns of digestion occur: Rhomboid cavities, long spindle- shaped cavities, and a general breakdown of the S2 layer. Many variations occur. The causal fungi are usually nonhymenomycetes- Phialophora, 38 Penicillium, Chaetomium, etc. Soft rots occur mostly in moisture-saturated angiosperm wood products. Little is known about soft rots in living trees. 5.1.2 Rots Caused By Ganoderma lucidum Depending upon Location 5.1.2.1 Stem rot The fungus is found attacking hardwoods (Dalbergia sissoo, Morus alba, Populus spp., Acacia spp., Platanus orientalis and Melia spp.) in irrigated and non-irrigated plantations. Stems injured by browsing, and stumps left in the ground, serve as foci of infection (Mahmood, 1971). The nature and mode of survival of Ganoderma lucidum was investigated. The fungus failed to grow in unsterile soil and in sterile soil it only grew when the original agar disc was present. The spread of inoculum and infection in coconut and arecanut plantations was shown to be random. It is concluded that G. lucidum cannot survive and grow in the soil independently and that the spread of the fungus is by root contacts (Sulladmath, 1995). Root rot of Acacia mangium by Ganoderma lucidum and heart rots by Phellinus pachyphloeus and Trametes palustris have been recorded from plantations raised in West Bengal. The exotic appears to be highly susceptible to Ganoderma root rot, which is quite prevalent in Midnapore Forest Division. Heavy mortality in a 1985 plantation is viewed with concern by the State Forest Department. Effective measures to keep the disease in check are outlined. Heart rots caused by Phellinus pachyphloeus and Trametes palustris are the first records of decay in standing trees of A. mangium in India. Although decay is not a serious problem at present it is likely to be highly damaging in older stands in view of the progressive nature of the disease. Preventative measures for heart rots are recommended (Mehrotra et al., 1995). As part of coconut basal stem rot (Ganoderma lucidum) management, a survey was undertaken during kharif 1998-99 in Arsikere taluk of Hassan district in Karnataka, India, to know its incidence, severity, distribution and development. The disease incidence was highest in Thimalapura village (36.15%), followed by Banavara (28.37%) and lowest in Haranahally (6.06%). The disease severity ranged from 17.16 in Geejihally village to 76.92 in Thimalapura village. Symptoms, spread and early detection of the disease are discussed. The integrated methods for the management of coconut basal stem rot disease include: (1) removing infected dead palms and stumps and destroying the bole and roots of these palms by burning; (2) isolating infected palms from healthy ones by digging circular or square trenches around the infected palms; (3) avoiding ploughing around infected palms; (4) 39 irrigation by drip method or basin irrigation during summer; (5) moisture conservation techniques such as coconut husk burial in the basin, if no irrigation facility is available; (6) applying 5 kg neem cake palm-1 year-1 aside from the recommended organic manure (50 kg FYM or green leaves) and chemical fertilizers; (7) applying 200 g phosphobacteria palm-1 year-1, along with the organic manure; (8) root feeding of 1.3 g aureofunginsol (0.6 a.i.) in 100 ml water along with soil drenching of coconut basin; and (9) raising disease antagonistic intercrops (e.g. banana) wherever irrigation is possible( Naik et al., 2000). 5.1.2.2 Root rot and Butt rot The characteristic symptoms of G. lucidum root rot of trees were yellowing of leaves and die back, rotting of roots and appearance of reddish coloured fruit bodies of the fungus at the base of the tree trunk. G. lucidum showed optimal growth in malt extract-peptone medium at 30ø and 35øC and pH 6.5. There were no significant differences between the 3 isolates of the fungus studied. However, significant differences in weight loss of wood decayed by the 3 isolates were recorded. Basidiospore germination was poor and only occurred on 3% malt extract (10, 11 and 12% for the 3 isolates) (Khara and Singh, 1997). A survey carried out during Aug.-Oct. 1991 and 1992 around Ludhiana, Indian Punjab, found that 13.3% of Dalbergia sissoo, 5.5% of Leucaena leucocephala, 6% of Delonix regia, 4% of a Eucalyptus hybrid and 2% of pear trees were infected by G. lucidum. Disease incidence was highest on coppiced trees (Khara, 1993). In Jun. of 1991, in Jodhpur, Rajasthan, India, a 15-year-old tree of Acacia tortilis growing next to a jojoba (Simmondsia chinensis) plantation suddenly collapsed, and sporophores of a Basidiomycete were noticed at the base of the fallen tree. In the following rainy season, 10-15-year-old plants growing in the close proximity to the A. tortilis tree and, later on, more distant jojoba plants developed disease symptoms. G. lucidum was identified as the causal agent of root rot in jojoba and pathogenicity was confirmed. Control measures are recommended to avoid the spread of G. lucidum by contact with infected plants (Lobos et al., 1994). An Acacia arboretum was established in 1982 after clear felling in a teak (Tectona grandis) forest in Madhya Pradesh, with 9 trees of each of 18 species. Symptoms of root rot caused by G. lucidum were observed in 1983, suggesting that the fungus had been present at the site from the outset; stumps colonized by the fungus were implicated as sources of infection. By 1991, only 48% of the plants were still alive. A. albida, A. aneura, A. decurrens, A. 40 murrayana and A. victoriae were the species most susceptible to the disease, while A. greggii and A. verek showed no symptoms, suggesting that they are resistant ( Harsh et al., 1993). The characteristic symptoms of G. lucidum root rot of trees were yellowing of leaves and die back, rotting of roots and appearance of reddish coloured fruit bodies of the fungus at the base of the tree trunk. G. lucidum showed optimal growth in malt extract-peptone medium at 30ø and 35øC and pH 6.5. There were no significant differences between the 3 isolates of the fungus studied. However, significant differences in weight loss of wood decayed by the 3 isolates were recorded. Basidiospore germination was poor and only occurred on 3% malt extract (10, 11 and 12% for the 3 isolates) (Khara and Singh, 1997). Root rot of Acacia mangium by Ganoderma lucidum and heart rots by Phellinus pachyphloeus and Trametes palustris have been recorded from plantations raised in West Bengal. The exotic appears to be highly susceptible to Ganoderma root rot, which is quite prevalent in Midnapore Forest Division. Heavy mortality in a 1985 plantation is viewed with concern by the State Forest Department. Effective measures to keep the disease in check are outlined. Heart rots caused by Phellinus pachyphloeus and Trametes palustris are the first records of decay in standing trees of A. mangium in India. Although decay is not a serious problem at present it is likely to be highly damaging in older stands in view of the progressive nature of the disease. Preventative measures for heart rots are recommended (Mehrotra et al., 1996). In 1982 c. 10% of Macadamia integrifolia trees in an orchard at Puli, Taiwan showed some degree of decline due to extensive root decay. In some cases decay extended to the main trunk and eventually caused death of the tree. Infected tissues of 80% of diseased trees turned soft which was due to G. lucidum. Other declining trees were infected by K. clavus, which formed black lines on infected tissues. In pathogenicity tests, 40 and 80% of macadamia branches were killed by G. lucidum inoculum after 3 and 6 months, respectively, and 20 and 60%, respectively, were killed by K. clavus. G. lucidum grew faster and benefited from relatively high temperature compared with K. clavus in vitro ( Ann and Ko, 1988). 5.1.3 Other root rot and wound pathogens Other wood-decay fungi associated with plantation losses in the south-western Pacific include Armillaria spp. (A. mellea (Vahl:Fr.) P.Kumm. and A. tabescens (Scop.) Emel) and Rigidoporus vinctus (Berk.) Ryvarden. These are apparently only of minor importance 41 compared with P. noxius. A fungus broadly identified as Ganoderma lucidum (Curtis) P.Karst. sens lat. associated with Agathis vitiensis (Seem.) Benth. & Hook.f. and Pinus caribaea in Fiji was not pathogenic on P. caribaea (Hood and Bell, 1983). Ganoderma chalceum (Cooke) Steyaert was reported on dicotyledonous trees from Fiji (Ivory, 1989) and Ganoderma australe (Fr.) Pat. (syn. Ganoderma tornatum (Pers.) Bres.) was reported from Grevillea robusta A.Cunn. in Tonga (Dingley et al., 1981) and caused wood rotr in Fiji and the Solomon Islands (Ivory, 1989). A fungus identified as Clitocybe tabescens (Scop.) Bres. This is now called Armillaria tabescens (Scop.) Emel caused large-scale mortality of Mahogany (Swietenia macrophylla) and Pinus elliottii Engelm. in Fiji (Sujan Singh and Bola, 1981). It is possible that the later reports of Armillaria spp. and A. mellea from Fiji refer to the same fungus. Heterobasidion annosum (Fr.:Fr.) Bref. (anamorph Oedocephalum lineatum B.K.Bakshi) has been reported from Agathis macrophylla (Lindl.) Mast., A. vitiensis (Seem.) Benth. & Hook.f., Araucaria cunninghamii and Pinus spp. in Fiji (Ivory, 1987 and 1989; Dingley et al., 1981). This is a serious root-rot pathogen of conifers in the northern Hemisphere and while it occurs in Australia (Queensland and New South Wales) on conifers (Araucaria), there is no evidence that it is a pathogen. Ivory (1987) reported that clones of H. annosum from Asia and Australasia differed from the Northern Temperate clones and that it appeared to be virtually harmless in Australasia. Chaplin (1993) reported that in the Solomon Islands E. deglupta was susceptible to invasion by decay fungi, especially in areas damaged by cattle. Losses in damaged areas were of the order of 10-20%, but decay was also present in areas where there had been no cattle. Chaplin estimated that in short rotation crops (8-10-years) of E. deglupta in close-spaced stands incipient decay could be expected in 30% of the trees. 5.2 Control White, brown and soft rots can weaken a tree's heartwood. Usually, white rot is more flexible, because the cellulose fibres remain fused together. Brown rot is often very brittle. Hence, when lignin is left behind, the wood rot is often dark and broken into cubical sections. White rot tends to be stringy, and less brittle. All types of heartwood rot can weaken a tree such that it breaks during a windstorm. Soft rot is often the most dangerous of the rots. Ustulina soft rot tends to weaken the base of the tree's trunk. When an entire tree falls, soft rot is often to blame (Holiday, 1989). 42 Fungicides are not very effective in killing well-established wood-rotting fungi. For both honey fungus and pocket rot it is recommended that seriously infected trees be removed. After removal it is advised that the stump be removed. The more thorough the stump removal the better. Removal is most important for pocket rot infestations. White pocket rot can live on stumps that still have some living root tissue in them. The spores from these stumps can infect standing trees. Most fungi should be given the 'wait-and-see' approach. For jack-olanterns one should monitor the situation. Most fungi, such as the sulphur-shelf, should not be considered a great concern. One should always assume that very old trees have some hollowing. Visible fruiting bodies are just one of the signs of heart rot (Tudge, 2000). The effects of planting and soil conditions on the incidence of root rot, caused by G. lucidum, of subabool (Leucaena leucocephala) were investigated. Trees planted in laterite shallow and skeletal medium deep soils and suffering from root rot were selected. A mechanically dug trench 30 cm wide and 1 m deep and another 15 cm wide and 15 cm deep treated with 1% formalin [formaldehyde] between 2 plants served as 2 main treatments. Five sub-treatments, carbendazim 0.05%, captan 0.25%, copper oxychloride 0.15%, formaldehyde 1% and a control (no treatment), were given as soil drench. The results indicated that the incidence of root rot was high in laterite shallow soils, close plantings; profuse root spread and aged plantations. Although the differences between type of trench and fungicides were not significant, greater control was achieved with mechanical trenches. Among the fungicides, formaldehyde was the most effective giving 100% control with both mechanical and chemical trenches. Carbendazim gave 100% and 91.7%, copper oxychloride 75% and 50% and captan 58.5% and 50% control with mechanical and chemical trenches, respectively. In a study of possible biological control methods against root rots of forest trees, ten Trichoderma spp. were tested for the amount of antagonistic activity or response to 8 fungal disease agents (3 Armillaria spp., Heterobasidion annosum and 4 Ganoderma spp.). At 22øC, some Trichoderma strains were effective or lethal against the pathogens. However, at 5øC, only one of the Trichoderma spp. had significant growth (Anselmi et al., 1992). Fourteen treatments were imposed in an endemic area in Karnataka, India, for root rot (Ganoderma lucidum and Phellinus noxius) and heart rot disease (P. pachyphloeus and Trametes palustris [Fomitopsis palustris]). These were: pasting with 10% Blitox [copper oxychloride]; pasting with Trichoderma viride; pruning; T. viride added to soil; 2 kg farmyard manure (FYM)/tree + T. viride added to soil; Calixin [tridemorph] as soil drench; 43 isolation trench + Calixin added to trench; FYM; isolation trench + sulfur dusting; neem cake + T. viride added to soil; neem cake; pasting with Pseudomonas fluorescence; P. fluorescence added to soil; and the control. Of these, treatments with calixin and pasting of T. viride to pruned wounds arrested the spread of disease 270, 360 and 390 days after starting the experiment. These treatments also had the lowest senescence (31.20%). Although there was no significant influence of these treatments on initial and final volume of standing trees, maximum current annual increment (0.083 m3) and percent increment (84.6) was recorded upon the pasting of biological control agent treatment. However, the treatments with neem cake, FYM, calixin and sulfur soil drench also recorded increase in percent increment of wood (Prasad and Naik, 2002). An experiment was conducted in Karnataka, India from 1994 to 1998 on coconuts given the following treatments: T1, 2% tridemorph root feeding + 5 kg neem cake per palm per year; T2, 3% kitazin [iprobenfos] root feeding + neem cake (rate as before); T3, 2% tridemorph root feeding + 0.1% soil drenching + neem cake; and T4, 1.3% aureofungin (root feeding) + 1% Bordeaux mixture (soil drenching) + neem cake to control basal stem rot caused by Ganoderma lucidum. All the treatments provided some control of the disease, compared to the untreated control. The lowest basal stem rot index was obtainedwith T4, followed by T3 (Naik and Venkatesh, 2001). 44 Fig 2: Chemistry of Wood Decay. 45 Fig: Chemistry of Wood Decay. 46 Fig 3: Classification of Wood decay fungi. 47 Fig: Classification of Wood decay fungi. 48 CHAPTER SIX: LIGNIN MODIFICATION 6.1 Lignin-Modifying Enzymes of the White Rot Basidiomycete Ganoderma lucidum Ganoderma lucidum, a white rot basidiomycete widely distributed worldwide, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). Laccase levels observed in high-nitrogen (HN; 24 mM N) shaken cultures were much greater than those seen in low-nitrogen (2.4 mM N), malt extract, or wood-grown cultures and those reported for most other white rot fungi to date. Laccase production was readily seen in cultures grown with pine or poplar (100-meshsize ground wood) as the sole carbon and energy source. Cultures containing both pine and poplar showed 5- to 10-fold-higher levels of laccase than cultures containing pine or poplar alone. Since syringyl units are structural components important in poplar lignin and other hardwoods but much less so in pine lignin and other softwoods, pine cultures were supplemented with syringic acid, and this resulted in laccase levels comparable to those seen in pine-plus-poplar cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of concentrated extra cellular culture fluid from HN cultures showed two laccase activity bands (Mr of 40,000 and 66,000), whereas isoelectric focusing revealed five major laccase activity bands with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. Low levels of MnP activity (~100 U/liter) were detected in poplar-grown cultures but not in cultures grown with pine, with pine plus syringic acid, or in HN medium. No LiP activity was seen in any of the media tested; however, probing the genomic DNA with the LiP cDNA (CLG4) from the white rot fungus Phanerochaete chrysosporium showed distinct hybridization bands suggesting the presence of lip-like sequences in G. lucidum (D'Souza et al., 1999). Lignin, the second most abundant renewable organic polymer on earth, is a major component of wood. Because of the importance of wood and other lignocellulosics as a renewable resource for the production of paper products, feeds, chemicals, and fuels, there has been an increasing research emphasis on the fungal degradation of lignin (Boominathan and Reddy, 1992; Kirk and Farrell, 1987). White rot fungi are believed to be the most effective lignindegrading microbes in nature. A majority of the previous studies have focused on the lignindegrading enzymes of Phanerochaete chrysosporium and Trametes versicolor (Gold and Alic, 1993; Reddy and D'Souza, 1994.). Recently, however, there has been a growing interest in studying the lignin-modifying enzymes of a wider array of white rot fungi, not only from 49 the standpoint of comparative biology but also with the expectation of finding better lignindegrading systems for use in various biotechnological applications (D'Souza et al., 1996; Hatakka, 1994; Orth et al., 1993; Peláez et al., 1995). Three major families of fungal lignin-modifying enzymes (LMEs) are laccases, manganesedependent peroxidases (MnPs), and lignin peroxidases (LiPs) (Boominathan and Reddy, 1992; Hatakka, 1994; Kirk and Farrell, 1987; Thurston, 1994). These LMEs can oxidize phenolic compounds thereby creating phenoxy radicals, while nonphenolic compounds are oxidized via cation radicals (Boominathan and Reddy, 1992; Higuchi, 1989; Kirk and Farrell, 1987). LiP and MnP oxidize nonphenolic aromatic compounds with high oxidation-reduction potentials (Glenn et al., 1983), the major components of the lignin polymer. Laccase oxidizes nonphenolic aromatic compounds with relatively low oxidation-reduction potentials (Kersten et al., 1990; Youn et al., 1995). In the presence of low-molecular-weight mediators, laccases can also oxidize nonphenolic substrates with high oxidation-reduction potentials (Bourbonnais and Paice, 1990; Call and Mücke, 1995; Eggert et al., 1996) as well as certain xenobiotics (Johannes et al., 1996). Preliminary studies in our laboratory showed the presence of lip gene-homologous sequences in the genomic DNA of several genera of wood rot fungi (D'Souza and Reddy, 1992). Subsequent screening on plates containing the polymeric dye poly R-478, decolorization of which is correlated with lignin degradation (Gold et al., 1988), led to the selection of a strain of Ganoderma lucidum for further studies, based on its rapid growth rate and extensive decolorization of poly R-478 on solid media. G. lucidum is one of the most important and widely distributed white rot fungi in North America and is associated with the degradation of a wide variety of hardwoods (Adaskaveg et al., 1990). Previous studies of G. lucidum have mainly concentrated on the medicinal properties of this fungus (reviewed in reference Jong and Birmingham, 1992) and, except for two brief preliminary reports (Horvath et al., 1993; Perumal and Kalaichelvan, 1996), little is known about the ligninolytic system of this organism. A local strain of G. tropicum has been analysed for the appearance of triterpenoids and polysaccharides, with G. lucidum as reference. These two compounds are reported to have medicinal value in G. lucidum. G. tropicum was isolated from a Delonix regia tree, an ornamental tree locally known as Flamboyant, in December, 1999. The fungus is parasitic to the tree and is quite aggressive, killing the tree within 1-5 years. The fruiting bodies of both 50 G. tropicum and G. lucidum were obtained by growing them in sawdust after three months of incubation. Extraction for triterpenoids was conducted in wash benzene and ethanol, while extraction for polysaccharides was done in hot water. Analysis for triterpenoids was conducted using silica gel-thin layer chromatography (TLC) with eluent of chloroform: methanol (10:1) for the first extract and dichloromethane for the second extract. Detection of triterpenoids was done by spraying the plate with Carr-Price as well as Lieberman-Burchard reagents then observing under UV light (366 nm). The same principle, except for the eluent n-buthanol:acetic acid:ether:water (9:6:3:1) and reagent of aniline phthalate, was used for detecting polysaccharides in the form of their monomers (glucose, galactose, xylose and rhamnose) under normal light. Both triterpenes and polysaccharides were detected on G. tropicum and G. lucidum, suggesting that the local strain of G. tropicum also possesses medicinal value (Aryantha et al., 2001). Blocks (2X2X1 cm) of Betula platyphylla [B. mandshurica] wood were impregnated with asparagine solution (0-1.6%) and exposed to 7 decay fungi. Degradation of wood was increased by asparagine. Wt. loss caused by Pleurotus ostreatus, P. cornucopiae and Sporotrichum pulverulentum (anamorph of Phanerochaete chrysosporium) was greatest with 1.6% asparagine and that caused by P. chrysosporium, Ganoderma lucidum and Pholiota nameko was greatest with 0.8% asparagine. Lentinus [Lentinula] edodes caused greatest wt. loss with 0.4% asparagine. The specificity of lignin degradation was also changed by asparagine, being increased for the Pleurotus spp., decreased for S. pulverulentum, Phanerochaete chrysosporium and L. edodes, and unaffected in G. lucidum and Pholiota nameko (Yamamoto et al., 1986). 6.1.1 More about Lignin-Modifying Enzymes (LME) It has been well documented that nitrogen levels, aeration, and other factors influence LME production by white rot fungi (Boominathan and Reddy, 1992; Buswell, and Odier, 1987; Buswell et al., 1995; Kirk and Farrell, 1987; Van der Woude et al.,1993). For example, with glucose as the carbon source, LiP and MnP production by P. chrysosporium was seen in LN (2.4 mM N) medium but was completely suppressed in HN (24 mM N) medium (Boominathan and Reddy, 1992; Buswell and Odier, 1987; Kirk and Farrell, 1987; Reddy and D'Souza, 1994). Laccase production by P. chrysosporium was not detected in LN or HN medium with glucose as the carbon source but was readily demonstrable when the organism was grown in LN or HN media with cellulose (Srinivasan et al., 1995) or with glucose and 51 0.4 mM CuSO4 (Dittmer et al., 1997). The results of this study show that laccase production in G. lucidum is not inhibited in N-rich media with glucose as the carbon source. In fact, this organism in HN medium produces the highest levels of laccase. In this respect, our results are in agreement with earlier findings reporting high levels of laccase production under N-rich conditions in Rigidoporus lignosus (Galliano et al., 1991); Ceriporiopsis subvermispora (Lobos et al., 1994); Lentinula edodes (Buswell et al., 1995); and Agaricus bisporus (Perry et al., 1993; Wood, 1980). G. lucidum produces much higher levels of laccase in HN shaken than in HN static cultures. Stimulation of the production of LMEs, such as LiP, by aeration has also been reported for P. chrysosporium (Boominathan and Reddy, 1992; Buswell and Odier, 1987; Gold and Alic, 1993; Kirk and Farrell, 1987). Recently, we have shown that oxygenation had a marked positive influence on laccase production by P. chrysosporium (Srinivasan et al., 1995). Increasing the oxygen level in the medium has been postulated to lead to increased LME production and increased production of the components of the H2O2-producing systems (Boominathan and Reddy, 1992; Kirk and Farrell, 1987). Relatively poor laccase production in LN cultures (shaken or static) is probably due to the fact that levels of growth observed in these cultures were much lower than those in the HN cultures. It is also possible that other unknown factors arising from the combined effects of HN and aeration may have contributed to high levels of laccase in HN shaken cultures. Various low-molecular-weight aromatic compounds have been reported to enhance LME production (Bollag and Leonowicz, 1984; Boominathan and Reddy, 1992; Buswell and Odier, 1987; Kirk and Farrell, 1987; Schlosser et al., 1997). For example, VA has been shown to enhance laccase production in several white rot fungi (Bollag, and Leonowicz, 1984; Eggert et al., 1996; Schlosser et al., 1997). This is consistent with our observation that in G. lucidum only VA, among a number compounds tested, showed stimulation of laccase production. 2,5-Xylidine has been reported to give 20-fold stimulation of laccase activity in Trametes villosa (Yaver et al., 1996), 9-fold stimulation in Pycnoporus cinnabarinus (Eggert, et al., 1996), and at least 4-fold stimulation in Irpex lacteus (Balajee et al., 1997) but showed no enhancement of laccase production in G. lucidum. These results suggest that enhancement of laccase production in response to various aromatic compounds differs greatly among different white rot fungi. 52 Wood is the natural substrate for G. lucidum, which is known to cause extensive delignification of various species of hardwoods worldwide (Adaskaveg et al., 1990). Yet a large majority of the previous studies on the production of LMEs have been carried out with defined media (Boominathan and Reddy, 1992; Kirk and Farrell, 1987), and none has been reported for G. lucidum. Recent results show that LME production when white rot fungi are grown in wood-containing media could be substantially different from that seen in defined media (Fukushima and Kirk, 1995; Okeke, 1994; Perry et al., 1993, Schlosser et al., 1997; Srinivasan et al., 1995). For example, the results of this study show that G. lucidum produces laccase only in defined media and in cultures grown with pine but produces both laccase and MnP in cultures grown with poplar. Also, laccase levels were substantially higher in defined media than in wood-grown cultures. These results are consistent with earlier studies which showed that several white rot fungi produce both laccase and MnP in media supplemented with wood but produce only laccase in defined media (Horvath et al., 1993; Lobos et al., 1994; Schlosser et al., 1997). Furthermore, Galliano et al., (1991) and Maltseva et al. (1991) reported production of laccase and MnP by R. lignosus and Panus tigrinus, respectively, in sawdust medium and wheat straw medium. However, unlike our study, none of the earlier studies compared LME production in media containing different types of wood. Our finding that MnP is produced only in poplar cultures and not in pine cultures is a unique finding in that not all classes of LMEs are produced consistently even in media containing complex ligninaceous substrates such as wood. Instead, the type of wood substrate appears to determine the types and amounts of LMEs produced by the white rot fungi. The reason for differential MnP production by G. lucidum in pine and poplar cultures is not obvious, but the most likely explanation is that some unique component in poplar triggers MnP production by G. lucidum and that this is lacking in pine. In support of this idea, G. lucidum cultures grown with both pine and poplar (50:50) produced approximately half the amount of MnP as that seen in cultures grown with poplar only. This suggested that the reduced amount of MnP seen in the former cultures is a reflection of the smaller amount of poplar wood (50 mg) in these cultures compared to that in cultures containing poplar only (100 mg). This was further confirmed by growing G. lucidum in cultures containing 50 mg of poplar only, which also produced approximately half the level of MnP as that produced in cultures with 100 mg of poplar. An important finding of this study is that laccase production in G. lucidum a culture containing equal amounts of pine and poplar is 4 times and 10 times higher, respectively, 53 than those seen in cultures containing pine only and poplar only. These results suggest that certain components in these two woods have a synergistic effect on laccase production. While the structure of syringic acid is different from the structure of syringyl moieties of lignin, the ring structures of both are identical. Also, previous researchers have shown that lowmolecular-weight aromatic acids, such as syringic acid, that are structurally related to individual phenolic moieties in lignin serve as good inducers of LMEs (Eggert et al., 1996; Yaver et al., 1996). In this study, we used commercially available syringic acid as a substitute for syringyl moieties of lignin (in the same way as ferulic acid is often used as a substitute for coniferyl alcohol). Addition of syringic acid to pine cultures of G. lucidum resulted in laccase activity (14.7 µkat/liter) comparable to that seen in pine-plus-poplar cultures (14.8 µkat/liter) but was much higher than that produced in the medium with pine alone or poplar alone. These data suggest that the stimulation of laccase production in pine-plus-poplar cultures of G. lucidum is probably due to the syringyl units contributed by poplar lignin. The molecular masses (40 and 66 kDa) and IEF values (3.0 to 5.1) reported in this study for G. lucidum laccases are in the range observed for laccases isolated from other white rot fungi (Lobos et al., 1994; Palmieri et al., 1993; Vares et al., 1992; Yaver, 1996). For example, T. villosa was shown to produce a laccase (two subunits of 63 kDa) that was resolved by IEF into three isoforms with pIs of 3.5, 6 to 6.5, and 5 to 6 (Yaver, 1996), while Pleurotus ostreatus was shown to produce three laccases each having a molecular mass of 67 kDa; two had a pI of 4.7, and one had a pI of 2.9 (Palmieri et al., 1993). The white rot fungus C. subvermispora was shown to produce four laccase isozymes with pI range of 3.4 to 4.7 (Lobos et al., 1994). In our study, identical laccase isoforms were consistently seen when concentrated ECF from cultures grown in LN, ME, or wood media were used (results not shown), suggesting that the laccase isoforms of G. lucidum are constitutive and that these are not artifacts of the IEF procedure. Observation of distinct hybridization bands on probing Southern blots of restriction enzymedigested genomic DNA of G. lucidum with the LiP cDNA (CLG4) of P. chrysosporium suggests the presence of lip-like gene sequences in G. lucidum, but LiP production was not observed in any of the media included in this study. These results are also in agreement with our earlier finding that CLG4 gene homology is widely distributed in white rot fungi (Reddy and D'Souza, 1994). Apparently, G. lucidum has the genetic potential to produce LiPs but did not produce LiPs under culture conditions employed in our study. These results are also 54 consistent with the observations by Ruttimann et al. (Ruttimann et al., 1992) that the Southern hybridization technique utilizing a lip probe from P. chrysosporium shows the presence of lip-like genes in Phlebia brevispora and C. subvermispora but that LiP production could not be demonstrated by either organism. Moreover, Rajakumar et al., (1996) demonstrated the presence of lip-like gene sequences in C. subvermispora and P. sordida by employing a PCR procedure but failed to show LiP enzyme activity in liquid cultures. It was suggested that failure to detect LiP production in the above studies could be due to the obscuration of detection by interfering substances, the greater susceptibility of LiPs in these organisms to certain fungal proteases, or simply the lack of adequate culture conditions that favor LiP production by these organisms (Orth et al., 1993; Rajakumar et al., 1996; Ruttimann et al., 1992). Perumal and Kalaichelvan (1996) published a preliminary report claiming low levels of LiP production ostensibly by a strain of G. lucidum in media amended with lignin isolated from sugarcane bagasse, but this work has not been substantiated further. The ability of an organism to degrade [14C] DHP to 14 CO2 has been widely used to convincingly demonstrate the rate and extent of lignin degradation by various isolates of white rot fungi. G. lucidum mineralizes [14C]DHP to a very limited extent in media favoring production of laccase only or both laccase and MnP, suggesting a limited role for laccase (and perhaps MnP) in lignin degradation by this organism. Whether the lignin degradation in wood by G. lucidum (Adaskaveg et al., 1990) is due to laccase or involves MnP and/or LiP as well is not known at this time. However, the contribution of laccase to lignin degradation and its ability to modify nonphenolic-lignin model compounds has been well documented (Bourbonnais and Piece 1990; Eggert et al., 1997; Hatakka, 1994 and Higuchi, 1989). In summary, our results show that G. lucidum, an important white rot fungus involved in wood decay worldwide, produces laccase as the dominant LME. Laccase production is the result of a marked synergy when G. lucidum is grown with a mixture of pine an poplar woods compared to production with either one alone, and the organism produces at least five isoforms of laccase. This organism produces MnP in poplar but not in pine medium. It fails to produce LiP in defined media or in media containing pine and/or poplar but appears to have the genetic potential to produce LiP, as indicated by positive hybridization of its genomic DNA with the LiP cDNA of P. chrysosporium. Production of lignin-modifying enzymes by 10 white-rot fungi, as measured by decolorization of Poly R 478 dye, varied in response to different carbon and nitrogen regimes. Fastest decolorization rates were achieved with wood 55 polysaccharide monomers (glucose, xylose) as carbon source, although cellulose was the only carbon source to facilitate decolorization by all test fungi. Enzyme production by most isolates was strongly dependant on nitrogen levels, with high nitrogen conditions generally suppressing enzyme production. One unidentified isolate (HKUCC 4062) displayed apparent nitrogen deregulation of enzyme production. A cellulose-low nitrogen growth medium is recommended for agar-based screening procedures of lignin-modifying enzyme production by white-rot fungi (Leung, 2002). 56 CHAPTER SIX: CONCLUSIONS AND RECOMMENDATOINS 6.1 Conclusions The genus Ganoderma is a wood-decaying fungus that is found throughout the world on all types of wood — gymnosperms, hard- and softwood dicots, and palms. Ganoderma lucidum is a serious root rot pathogen of worldwide distribution. White rot fungi are believed to be the most effective lignin-degrading microbes in nature. As a white rot fungus G. lucidum breaks down lignin and cellulose and commonly cause rotted wood to feel moist, soft, spongy, or stringy and to appear white or yellow. The lateral spread of the disease takes place through root contact. Root decay fungi can be the cause of death of trees. Although some trunk decay fungi are considered relatively passive decomposers of the heartwood in trees, many others are more aggressive. These aggressive pathogens cause trees to become hazardous and can lead to their death or sudden failure. G. lucidum is one of the commonly encountered landscape tree pathogens with these capabilities. The presence of a fruiting body or conk is a sure sign of the disease. Fungicides are not very effective in killing well-established wood-rotting fungi. Infected trees may topple before any sign or symptom becomes obvious. 6.2 Recommendations Ganoderma lucidum is a one of the destructive, harmful and on the other hand a beneficiary fungus. We should try to use it positive way. As a root and decay fungus we have to manage it in a proper system otherwise it makes a great damage to our properties basically our wood properties. Some important recommendations are given bellow1) G. lucidum is used as a medicinal plant but not extensively. It should be cultured in a wide range and further experiment should be carried on for its amelioration. 2) Further investigation into the molecular make up of G. lucidum and G. tsugae species is needed. 3) There are no hard and fast rules and regulations for its control in decay and rots. Its control measures should be explored immediately. 4) The color, shape and size of some Ganoderma species are very attractive. It may be used in decorative purposes in a proper way after a proper treatment. 51 REFERENCES Adaskaveg, J.E. and Gilbertson, R.L. 1986. Culture studies and genetics of sexuality of Ganoderma lucidum and G. tsugae in relation to the taxonomy of the G. lucidum complex. Mycologia, 78:694–705. Adaskaveg, J. E.; Gilbertson, R. L. and Blanchette, R. A. 1990. Comparative studies of delignification caused by Ganoderma species. 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