ContentsContentsContentsContributors ixPreface xiPart I Ganoderma, Organism and Systematics 11 Ganodermataceae: Nomenclature and Classification 3 G.-S. Seo and P.M. Kirk2 Systematics of Ganoderma 23 J.-M. MoncalvoPart II Ganoderma, Diseases of Perennial Crops 473 Status of Ganoderma in Oil Palm 49 D. Ariffin, A.S. Idris and G. Singh4 Basal Stem Rot of Oil Palm in Thailand Caused by Ganoderma 69 S. Likhitekaraj and A. Tummakate5 The Current Status of Root Diseases of Acacia mangium Willd. 71 S.S. Lee v
vi ContentsPart III Disease Control and Management Strategies 816 A Control Strategy for Basal Stem Rot (Ganoderma) on Oil Palm 83 H. Soepena, R.Y. Purba and S. Pawirosukarto7 The Use of Soil Amendments for the Control of Basal Stem Rot of Oil-Palm Seedlings 89 M. Sariah and H. Zakaria8 The Spread of Ganoderma from Infective Sources in the Field and its Implications for Management of the Disease in Oil Palm 101 J. Flood, Y. Hasan, P.D. Turner and E.B. O’Grady9 Basidiospores: Their Influence on Our Thinking Regarding a Control Strategy for Basal Stem Rot of Oil Palm 113 F.R. Sanderson, C.A. Pilotti and P.D. Bridge10 Management of Basal Stem Rot Disease of Coconut Caused by Ganoderma lucidum 121 R. Bhaskaran11 In vitro Biodegradation of Oil-palm Stem Using Macroscopic Fungi from South-East Asia: a Preliminary Investigation 129 R.R.M. Paterson, M. Holderness, J. Kelley, R.N.G. Miller and E. O’Grady12 Functional Units in Root Diseases: Lessons from Heterobasidion annosum 139 Å. Olson and J. StenlidPart IV Molecular Variability in Ganoderma 15713 Molecular and Morphological Characterization of Ganoderma in Oil-palm Plantings 159 R.N.G. Miller, M. Holderness and P.D. Bridge14 Spatial and Sequential Mapping of the Incidence of Basal Stem Rot of Oil Palms (Elaeis guineensis) on a Former Coconut (Cocos nucifera) Plantation 183 F. Abdullah15 Genetic Variation in Ganoderma spp. from Papua New Guinea as Revealed by Molecular (PCR) Methods 195 C.A. Pilotti, F.R. Sanderson, E.A.B. Aitken and P.D. Bridge
Contents vii16 Molecular Variation in Ganoderma Isolates from Oil Palm, Coconut and Betelnut 205 H. Rolph, R. Wijesekara, R. Lardner, F. Abdullah, P.M. Kirk, M. Holderness, P.D. Bridge and J. FloodPart V Development of Diagnostic Tests for Ganoderma 22317 Development of Molecular Diagnostics for the Detection of Ganoderma Isolates Pathogenic to Oil Palm 225 P.D. Bridge, E.B. O’Grady, C.A. Pilotti and F.R. Sanderson18 The Development of Diagnostic Tools for Ganoderma in Oil Palm 235 C. Utomo and F. Niepold19 Ganoderma in Oil Palm in Indonesia: Current Status and Prospective Use of Antibodies for the Detection of Infection 249 T.W. DarmonoIndex 267
ContributorsContributorsContributorsF. Abdullah, Department of Biology, Faculty of Science and Environmental Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, MalaysiaE.A.B. Aitken, Department of Botany, University of Queensland, St Lucia, Queensland, AustraliaD. Ariffin, Palm Oil Research Institute of Malaysia, No. 6, Persiaran Institute, Bangi, PO Box 10620, 50720 Kuala Lumpur, MalaysiaR. Bhaskaran, Coconut Research Station, Tamil Nadu Agricultural University, Veppankulam 614 906, Tamil Nadu, IndiaP.D. Bridge, Mycology Section, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AE, UKT.W. Darmono, Biotechnology Research Unit for Estate Crops, Jl. Taman Kencana No. 1, Bogor, 16151, IndonesiaJ. Flood, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKY. Hasan, Bah Lias Research Station, P.T.P.P. London, PO Box 1154, Medan 20011, North Sumatra, IndonesiaM. Holderness, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKA.S. Idris, Palm Oil Research Institute of Malaysia, No. 6, Persiaran Institute, Bangi, PO Box 10620, 50720 Kuala Lumpur, MalaysiaJ. Kelley, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKP.M. Kirk, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKR. Lardner, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKS.S. Lee, Forest Research Institute Malaysia, Kepong, 52109 Kuala Lumpur, Malaysia ix
x ContributorsS. Likhitekaraj, Division of Plant Pathology and Microbiology, Department of Agriculture, Bangkok 10900, ThailandR.N.G. Miller, Universidade Católica de Brasília Pró-Reitoria de Pesquisa e Pós-graduação, Campus II, 916 Asa Norte, Brasília, D.F., BrazilJ.-M. Moncalvo, Department of Botany, Duke University, Durham, NC 27708, USAF. Niepold, Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection of Field Crops and Grassland, Messeweg 11–12, 38104 Braunschweig, GermanyE.B. O’Grady, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKÅ. Olson, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S–750 07 Uppsala, SwedenR.R.M. Paterson, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UKS. Pawirosukarto, Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen Katamso 51, Medan 20158, IndonesiaC.A. Pilotti, PNG OPRA, Plant Pathology Laboratory, PO Box 36, Alotau, Milne Bay Province, Papua New GuineaR.Y. Purba, Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen Katamso 51, Medan 20158, IndonesiaH. Rolph, Level 9, Glasgow Dental School and Hospital, 378 Sauchiehall St, Glasgow G2 3JZ, UKF.R. Sanderson, PNG OPRA, Plant Pathology Laboratory, PO Box 36, Alotau, Milne Bay Province, Papua New GuineaM. Sariah, Department of Plant Protection, Universiti Putra Malaysia, 43400UPM, Serdang, Selangor, MalaysiaG.-S. Seo, College of Agriculture, Chungnam National Unviersity, Taejon 305–764, KoreaG. Singh, United Plantations Berhad, Jenderata Estate, 3600 Teluk Intan, Perak, MalaysiaH. Soepena, Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen Katamso 51, Medan 20158, IndonesiaJ. Stenlid, Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S–750 07 Uppsala, SwedenA. Tummakate, Division of Plant Pathology and Microbiology, Department of Agriculture, Bangkok 10900, ThailandP.D. Turner, PO Box 105, Quilpie, Queensland 4480, AustraliaC. Utomo, Indonesian Oil Palm Research Institute (IOPRI), PO Box 1103, Medan 20001, IndonesiaR. Wijesekara, Coconut Research Institute, Bandirippuwa Estate, Sri LankaH. Zakaria, Department of Plant Protection, Universiti Putra Malaysia, 43400UPM, Serdang, Selangor, Malaysia
PrefacePrefacePrefacePerennial oilseed crops form a major component of rural economies through-out the wet lowland tropics of South and South-East Asia and Oceania. Cropssuch as oil palm and coconut are grown as both plantation-scale commoditycrops and as smallholder cash and food crops. Perennial oilseed crops contrib-ute significantly to local livelihoods through not only their husbandry but alsothe processing of the crop and crop by-products and their subsequent shippingand marketing. As export commodities, they form an important component ofnational economies and generate valuable foreign exchange. Species of the basidiomycete fungus Ganoderma occur as pathogens on awide range of perennial tropical and sub-tropical crops, including oil palm,coconut, tea, rubber, Areca and Acacia, as well as various wild palm species.The effects of Ganoderma infection on productivity decline in palm crops havebeen of considerable concern ever since replanting of oil-palm land began inSouth-East Asia and recent workshops have identified basal stem rot, causedby Ganoderma boninense, as the single major disease constraint to oil palmproduction in the region. The long-term nature of palm monocultures meansthat they are prone to both premature plant death and to the carry-over ofresidual inoculum from one planting to the next. This pattern has been clearlyseen in many areas of South-East Asia and creates considerable concern for thelong-term sustainability of palm production from affected land. Basal stem rotof oil palm is widespread, occurring in the major oil palm growing regions ofthe world. By contrast, the disease on coconut appears very restricted; it wasfirst recorded in India in 1952 and remains confined to South Asia, yetGanoderma species occur as saprobes on dead coconut palm tissues in allpalm-growing regions, an anomaly that requires resolution. xi
xii Preface A crucial factor in developing effective disease management programmesis the prior understanding of pathogen biology and disease epidemiology.Ganoderma is a notoriously variable and difficult fungus to characterize andthis has led to much past confusion in disease aetiology and epidemiology.Such studies have been greatly enhanced through the development and useof molecular and biochemical markers to discriminate among pathogenpopulations and individuals and to diagnose infected palms in advance ofterminal symptoms. These technological tools can form powerful adjuncts tofield observation and experiments in understanding mechanisms of diseasespread and pathogen survival. This new understanding establishes thefundamental biology of the genus and provides new insight into diseaseepidemiology that enables the implementation of appropriate and effectivemanagement strategies. In perennial crops, infections of woody tissues have the opportunity toslowly develop further and expand as conditions permit. Infective material canremain viable in the ground for many months and infect subsequent crops atreplanting. It is therefore very important to manage disease outbreaks in sucha way as to minimize the risks to both existing and future plantings. Onefeature of Ganoderma diseases is the persistence of potential pathogens in oldwoody tissues and soil-borne debris. Burning of such material is no longeracceptable and extensive physical clearing is often not feasible due to the inputrequirements involved. Alternative treatments are thus required and anumber of approaches are being explored to manage this residual inoculum.These are centred on the evaluation of biocontrol agents and the rapidbiodegradation of palm woody residues. This book is a joint effort by 36 authors from 13 countries, each with awide expertise in their own fields. In many chapters, joint authors have cometogether from different countries, illustrating the collaborative nature of thisinitiative. The 19 chapters address many current issues in the development ofsustainable disease management programmes and are grouped into five majorthemes. These are, an introduction to the pathogen and its systematics inChapters 1 and 2, outlines of the diseases caused by the pathogen (Chapters3–5), disease management (Chapters 6–12), molecular biological variabilityin the pathogen (Chapters 13–16) and the development of diagnostic tools(Chapters 17–19). The majority of these chapters have been developed frompresentations made at two international workshops on Ganoderma diseasesheld in Malaysia in 1994 and 1998 and a technical workshop held in the UKin 1998. Funding for these workshops was provided by the UK Departmentfor International Development (DFID Project R6628) Crop ProtectionProgramme, for the benefit of developing countries and from the EuropeanCommunity (Stabex fund), the British Council, Governments and institutionsof the countries concerned and numerous private plantation companies. Weare very grateful to the various sponsors of this research for their involvement,although the book should not be considered to necessarily reflect the views ofour sponsors. We would also wish to acknowledge the pioneering work and
Preface xiiidedication of a number of scientists who have previously advanced knowledgeof this recalcitrant organism and its various diseases and inspired us in ourown labours, notably E.J.H. Corner, P.D. Turner, A. Darus and G. Singh. This book reflects the sum of knowledge of Ganoderma as a plant pathogenas at the end of 1998 and we hope will be both useful and informative to a widerange of readers including scientists in the private and public sectors, studentsand growers of perennial crops. Further work continues and we trust that fur-ther insights will continue to be obtained in the near future to further enhancethe sustainable management of Ganoderma diseases. J. Flood P.D. Bridge M. Holderness
4 G.-S. Seo and P.M. KirkAdaskaveg and Ogawa, 1990; Adaskaveg et al., 1991, 1993), or usefulmedicinal herbs (Mizuno et al., 1995). Because of these fundamentallydifferent viewpoints among collectors, the taxonomy of these fungi is verysubjective and confused. Contributions to the morphology and taxonomy ofthe Ganodermataceae have been made by many mycologists, including Steyaert(1972), Furtado (1981), Corner (1983) and Zhao (1989). However, the greatvariability in macroscopic and microscopic characters of the basidiocarpshas resulted in a large number of synonyms and in a confused taxonomy,especially in the genus Ganoderma (Gilbertson and Ryvarden, 1986).History of Ganoderma Taxonomy and NomenclatureThe genus Ganoderma has been known for a little over 100 years; it was intro-duced by the Finnish mycologist Peter Adolf Karsten, in 1881. He includedonly one species, Polyporus lucidus, in the circumscription of the genus and thisspecies, 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 vonLeysser and this error has been perpetuated in numerous subsequentpublications. 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 isnow largely developed as residential housing but the type substratum, thesmall tree Corylus avellana, is likely to be growing still on Peckham RyeCommon. It is clear, therefore, where any epitype, selected as an interpretivetype, should be sought. The selection of an epitype, in the absence of type orauthentic material, would be important, for any further molecular work willneed to have available a culture of the type species of the genus which hassome nomenclatural standing, i.e. a culture derived from an epitype. Following Karsten, dozens of species belonging to the genus were reportedby taxonomists (Patouillard, 1889; Boudier and Fischer, 1894; Boudier,1895; Murrill, 1902, 1908). The identification of Ganoderma in those dayswas mainly based on host specificity, geographical distribution, and macro-morphological features of the fruit body, including the context colour andthe shape of the margin of pileus, and whether the fruit body was stipitateor sessile. Subsequently, Atkinson (1908), Ames (1913), Haddow (1931),Overholts (1953), Steyaert (1972, 1975, 1977, 1980), Bazzalo and Wright(1982), and Corner (1983) conducted the identification of Ganoderma speciesby morphological features with geographically restricted specimens. Haddow(1931) and Steyaert (1980) placed most of their taxonomy on the spore char-acteristics and the morphology of hyphal elements. However, the basidiocarpsof Ganoderma species have a very similar appearance that has caused confusionin identification among species (Adaskaveg and Gilbertson, 1986, 1988). The genus now contains a few hundred names; there are 322 in the CABIBioscience fungus names database, but others may have been published that
Nomenclature and Classification 5the major printed indexes, the source of this database, failed to include. Thedatabase of Stalpers and Stegehuis available on the CBS web site lists 316names in Ganoderma and the recent publication of Moncalvo and Ryvarden(1997) lists 386 names for the Ganodermataceae as a whole. It has not yet beenpossible to compare these three data sets, although such an exercise wouldappear to be needed. However, names are only one aspect of this subjectand problems associated with them are, on the whole, easier to resolve thanproblems associated with the circumscription of species. Based on the unique feature of the double-walled basidiospore, the Frenchmycologist, Patouillard, over a period of some 40 years from 1887, described anumber of new species of Ganoderma and transferred several names from othergenera of the polypores. Patouillard (1889) published a monograph of thethen known 48 species and also distinguished the species with spherical orsubspherical spores as section Amauroderma. Coincidentally, in the same year,Karsten introduced the genus Elfvingia, based on the name Boletus applanatusof Persoon, for the non-laccate species. Later, section Amauroderma ofGanoderma was raised to the rank of genus by Murrill who, in selecting aspecies which was not included in section Ganoderma by Patouillard, istherefore the author of the name, and priority dates from 1905 not 1889.Subsequent authors have recognized Amauroderma as a distinct genus. Thetwo genera have been largely accepted, although Corner (1983) and Zhao(1989) reported species that are intermediate between them. Amaurodermawas revised by Furtado (1981). Here then we have two important species in the history and the nomen-clature of the genus, Ganoderma lucidum and Ganoderma applanatum, and theseare probably two of the most poorly understood species of Ganoderma and twoof the most frequently misapplied names. The late 19th-century and early 20th-century mycologists contributedsignificantly, in terms of volume of published information, on the genus,describing many new species or perhaps, more correctly, introducing manynew names. Many of these names were based on single collections or on only afew collections from the same locality, and the taxonomic status of the speciesto which these names were applied is, therefore, often open to the criticismof being unsound. Throughout the remainder of the 20th century variousworkers, Steyaert, Corner and Zhao perhaps being the more prominent,contributed to our knowledge of the genus by providing revisions, mono-graphs, descriptions of new taxa (again, often based on single collections oron only a few collections from the same locality) and observations on bothanatomy and ontogeny. Recent workers have used characters other than morphology to deter-mine relationships within the genus. These have included, in the first instance;cultural and mating characters, 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 hisco-workers (Moncalvo et al., 1995a, b) have used ribosomal DNA sequences
6 G.-S. Seo and P.M. Kirkand cladistics methods to infer natural relationships. However, as Moncalvoand Ryvarden have stated, these recent studies have had little impact onGanoderma systematics in total because too few taxa were examined. This wasquite clearly through both a lack of human and financial resources and,perhaps more importantly, a lack of the very important type or authentic col-lections which will link the names available to any subsequent taxa identified. Ryvarden (1994) has stated that the genus is in taxonomic chaos andthat it is one of the most difficult genera amongst the polypores. However, thisrealization has come at the very time when there has been a renewed interestin Ganoderma from a number of quite unrelated sources. These include themedicinal uses based on very old Chinese traditions and the requirement toelucidate the structure of possible active ingredients, coupled with the require-ment (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 hereis the apparent increase in the importance of some species of Ganoderma aspathogens of plants used by man. However, with the development of cladistic methods to reconstructnatural classifications and the application of these methods to both traditionalmorphological data and, more importantly, new molecular data, the potentialfor the resolution of some of these problems appears close to hand. Recently,the phylogenetic relationships of some Ganoderma species collected fromvarious 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 randomlyamplified polymorphic DNA (RAPD) as the tools for analysing phylogenicrelationships in the G. lucidum complex. The results suggested that somestrains were misnamed and misidentified, and all isolates belonging to 22species were disposed in six groups based on nucleotide sequence analysisfrom the internal transcribed spacers (ITS) of the ribosomal gene (rDNA).However, while some isolates had the same ITS sequence, all of them could beclearly differentiated by genetic fingerprinting using RAPDs. Therefore, RAPDanalysis might be helpful for systematics at the lower taxonomic levels todistinguish isolates from each other. When the results of molecular taxonomyare compared with the data of traditional taxonomy, such as morphological,ecological, cultural and mating characteristics, some isolates remain asexceptions. Of many studies on Ganoderma taxonomy, Adaskaveg’s research(Adaskaveg and Gilbertson, 1986) indicates the importance of vegetativeincompatibility tests for accurate identification, concluding that the incompat-ibility test must be adopted for the identification of the G. lucidum complex.Because of the problems as described above, Ryvarden (1994) has proposedthat 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 thenthe very large family Polyporaceae when he proposed the subfamilyGanodermatoideae; he subsequently raised this taxon to the rank of familywith the introduction of the Ganodermataceae and this classification has
Nomenclature and Classification 7subsequently been accepted by most recent workers. Much later, Julich, in1981, introduced the ordinal name Ganodermatales and this was accepted byPegler in the eighth edition of the Dictionary of the Fungi, although other work-ers have continued to use the traditional Aphyllophorales in a broad sense. There has been much speculation on the relationship between Ganod-ermataceae and other families of polypores. Corner (1983) believed that thefamily represented an old lineage from which other groups of polypores havebeen derived. Ryvarden (1994), however, proposed that the high phenotypicplasticity observed in the genus is indicative that the taxon is young and thatstrong speciation has not yet been achieved. This hypothesis was supported bymore recent molecular evidence from Moncalvo and his co-workers. The lackof fossils limits the accuracy to which we can attribute a minimum age to thegenus. Some fossils of corky polypores from the Miocene (25 million years old)have been tentatively referred to Ganoderma adspersum.Morphological Features of GanodermaMacromorphologyThe naturally produced basidiocarps of G. lucidum show various morphologi-cal characteristics; sessile, stipitate, imbricate and non-imbricate (Shin et al.,1986; Adaskaveg and Gilbertson, 1988; Fig. 1.1). The colour of the pileussurface and hymenophore varies from deep red, non-laccate, laccate and lightyellow to white, and the morphology also differs between the isolates (Shinand Seo, 1988b). The morphological variation appears to be affected by envi-ronmental conditions during basidiocarp development. Table 1.1 summarizesthe representative results from several descriptions of the macromorphology ofG. lucidum. The size and colour of the basidiocarp shows significant differencesbetween the specimens, but the pore sizes are similar. The manner of stipeattachment to pileus and the host range also varies (Ryvarden, 1994; Fig. 1.1).The pileus of the normal fruit body is laterally attached to the stipe, but eccen-tric, central, imbricate, and sessile fruit bodies are also produced rarely innature (Fig. 1.1). Stipe characters, including attachment type and relativethickness and length, have been considered useful for species identification,but their importance has been neglected by some mycologists, who describefruit bodies only as stipitate or sessile. Hardwoods are the usual host plants ofG. lucidum, but some specimens have been collected from conifers. The laccate character of the pileus and stipe has been variously employedin the taxonomy of this family. According to traditional concepts, the pileussurface of Ganoderma is laccate, but is not so in Amauroderma. However, a fewspecies of Amauroderma and Ganoderma have been reported with laccate (A.austrofujianense and A. leptopus) and non-laccate appearance (G. mongolicum).The laccate character, while playing no important role in the segregation ofgenera and sections in this family, remains available as an identification aid.
8 G.-S. Seo and P.M. Kirk Context colour of Ganoderma varies from white to deep brown and hasbeen considered a useful character in classification. However, some mycolo-gists have considered it useless for identification of species and supraspecificgroups because it may change under different environmental conditions.Context colour is often changeable, especially in dried specimens, not only inthe same species but within a single specimen (Zhao, 1989). Corner (1983)Fig. 1.1. Macromorphological characteristics of Ganoderma lucidum complex.
Table 1.1. Macromorphological descriptions of Ganoderma lucidum.Characters Steyaert (1972) Pegler and Young (1973) Bazzalo and Wright (1982) Melo (1986) Ryvarden (1994)Size Pileus Up to 20 cm –b 2–8 × 2–4(–5) cm Up to 15 cm 2–16 cm Stipe(L)a Up to 20 cm – 4–10 cm Up to 12.5 cm 1–3 cm (D)a – – 0.5–2 cm – 1–3.5 cm Pore – – 4–7 pore mm−1, 4–6 pore mm−1 4–6 pore mm−1 6–200 µm diameterColour of Pore surface – – White to yellowish or greyish-white White to cream White-cream to pale brown Stipe Dark brown Shiny, yellowish red to Reddish-black to almost black Purplish, reddish-brown, Deep chestnut to almost black reddish-black crust reddish black Pileus Reddish-brown Shiny, yellowish red to Light to dark reddish-brown Purplish-red, reddish and White or cream-reddish to deep reddish-black crust reddish black reddish-black Contex Nearly white Yellowish wood Ochraceous brown to dark brown Wood coloured and dark Wood coloured to pale brown brownishAttachment ofstipe to pileus Lateral #c Usually Frequently Frequently 46 specimens Ecentric # – – – 1 specimen Central # – – – 3 specimens Nomenclature and Classification Imbricate # – – – 1 specimen Sessile # # # # –Hyphal system – – Trimitic Trimitic TrimiticHost Hardwood – Common Common # 23 specimens Conifer – Occasionally Rarely no 22 specimensa L and D in parentheses indicate length and diameter, respectively.b Not determined.c #: described by author as presence only. 9
10 G.-S. Seo and P.M. Kirkemphasized the importance of observing the context colour of fresh and livingspecimens in the classification of Ganoderma. The size and shape of pores arealso useful characters for species classification. The number of pores permillimetre may serve as a specific character. The morphology of basidiocarps of G. lucidum in artificial cultivation onwood logs and synthetic substrates is affected by environmental conditions(Hemmi and Tanaka, 1936). Fruit-body formation in G. lucidum usuallyrequires 3 months on sawdust medium (Shin and Seo, 1988b; Stamets,1993b). The development of the basidiocarp is very sensitive to light and venti-lation. The stipe exhibits tropic growth toward light (Stamets, 1993a). Underdim light or dark conditions with poor ventilation, the pileus does not expandand often an abnormal pileus of the ‘stag-horn’ or ‘antler-type’ is produced(Hemmi and Tanaka, 1936; Shin and Seo, 1988b; Stamets, 1993a). Figure1.2 and 1.3 show fruit bodies of the G. lucidum complex produced by theFig. 1.2. Fruit bodies of Ganoderma lucidum complex generated by sawdust-bottle cultivation.
Nomenclature and Classification 11sawdust-bottle culture method. They show polymorphic features such as thekidney-type and antler-type with various colours (Shin and Seo, 1988b). Outof 22 isolates of the G. lucidum complex observed by one of the authors of thischapter (Shin and Seo, 1988b), 16 isolates formed typically kidney-shapedfruit bodies, and the remainder formed antler-type fruit bodies. Kidney-shapedfruit bodies could be further divided into those with a concentric zone on thesurface of the pilei and those without. Antler-shaped fruit bodies also divideinto typical forms and those with abnormal pilei (Table 1.2, Fig. 1.2). However, the fruit bodies of some species of Ganoderma are very stable inmorphology when generated by artificial cultivation with sawdust media,including their pileus colour, pileus zonation, attachment type and contextcolour. Fruit bodies of representative species of Ganoderma are shown in Fig.1.3. The pileus colour of all the fruit bodies of all species that are generatedby sawdust-bottle cultivation is reddish-brown to deep brown. In G. lucidum(ATCC 64251 and ASI 7004), G. oregonense (ATCC 64487), G. resinaceum andG. oerstedii (ATCC 52411) the fruit bodies have very similar pileus colour,Fig. 1.3. Asian collection – fruit bodies of Ganoderma lucidum generated bysawdust-bottle cultivation.Table 1.2. Classification of stocks in Ganoderma lucidum according to themorphology of fruit bodies generated by sawdust-bottle cultivation.1. Typically kidney-shaped fruit body-------------------------------(A and B) A. Concentric zones on the surface of the pileus ---------------------------10 isolates B. No concentric zones on the pileus ----------------------------------------- 6 isolates2. Antler-shaped fruit body --------------------------------------------(a and b) a. Typically antlered--------------------------------------------------------------- 2 isolates b. Antler-shaped with abnormal pileus --------------------------------------- 4 isolates
12 G.-S. Seo and P.M. Kirkzonation and pattern of stipe attachment. Although one isolate (ASI 7024)of G. lucidum produced typical antler-shaped fruit bodies, isolates ASI 7024and ASI 7004 were confirmed as conspecific by mating tests with mono-karyotic mycelia. Another isolate (MRI 5005) of G. lucidum showed a veryspecific pileus pattern with well-developed concentric zones. The species G.applanatum, G. microsporum, G. subamboinense and G. pfeifferi have uniquemorphological characters. The fruit body of G. meredithae (ATCC 64490) has along stipe attached parallel to the pileus and no concentric zones on the surfaceof the pileus. In G. applanatum (ATCC 44053) the fruit body is reddish-brownand has no distinct stipe; the surface and margin of the pileus are rough. Thepileus of G. microsporum (ATCC 6024) has a yellowish-brown margin andthe stipe is black; the surface of the pileus is smooth and has many narrowconcentric zones. In G. subamboinense (ATCC 52420) the pileus is deep brown,although the growing margin is white, and it has a typical stipe; the surfaceof the pileus has many concentric zones. An abnormal pileus was producedin G. pfeifferi (CBS 747.84), with an upturned margin; the pileus is alsocomparatively very thick (up to 30 mm).MicromorphologyThe structure of the pileal crust and cortex are useful characters in thetaxonomy of the Ganodermataceae. The former character occurs mainly inGanoderma and Amauroderma, but the latter also occurs rarely in Amauroderma.Fruit bodies of Ganoderma mostly have an hymenioderm or characoderm andanamixoderm (Steyaert, 1980). In Elfvingia, the pileal crust is a trichodermor an irregular tissue; it is also an irregular tissue in Trachyderma (Zhao,1989). This character is considered to be very useful for identification by sometaxonomists. However, it often differs in different specimens of a single speciesand may show various structural forms. In Ganodermataceae, the hyphal system is usually trimitic, occasionallydimitic, the generative hyphae are hyaline, thin walled, branched, septate ornot, and clamped. Clamp connections may often be difficult to observe indried specimens. However, they are easily observed in the youngest parts ofthe hymenium and context of fresh specimens. Skeletal hyphae are alwayspigmented, thick walled, and arboriform or aciculiform; skeletal stalks mayend in flagelliform, branched binding processes. Binding hyphae are usuallycolourless with terminal branching. Some species of Ganoderma, such as G.lucidum and G. ungulatum, show Bovista-type binding hyphae which areproduced from the generative or skeletal hyphae. G. mirabile and G. oregonensehave a pallid context and exhibit intercalary skeletals, which are derived froma transformed and elongated generative cell. On the other hand, Amaurodermahas no Bovista-type binding hyphae and many species have intercalaryskeletals. Hyphal characters are also influenced by environmental factors.Zhao (1989) observed great variation in hyphal diameter and in frequency of
Nomenclature and Classification 13septation due to differences in age as well as in nutrition. For species identifica-tion, however, hyphal characters are often useful (Zhao, 1989). Basidia and basidiospores are considered as the most important charactersfor species identification in basidiomycetes. Basidia in Ganodermataceae attain arelatively large size and range from typically clavate to pyriform. Intermediateforms are often seen in the same specimen. Basidiospores show severaldependable characters for identification. Ganodermataceae have a uniquedouble-walled basidiospore; Donk’s (1964) concept for the Ganodermataceae isbased on characters of the basidiospores. Basidiospores of Ganoderma are ovoidor ellipsoid–ovoid, occasionally cylindric–ovoid, and always truncate at theapex. The wall is not uniformly thickened, with the apex always thicker thanthe base. It is very distinctly double-walled, with the outer wall hyaline andthinner, and the inner one usually coloured and thicker and echinulate ornot. In Amauroderma the basidiospores are globose to subglobose, occasionallycylindrical, and form a uniformly thickened wall. In Haddowia the basidio-spores are longitudinally double-crested, with small, transverse connectingelements. Microscopic observations, such as the size and morphology of basidio-spores, have been adopted as the criteria for the taxonomy of Ganoderma. Thebasidiospores, which commonly have double walls and are ellipsoid andbrownish, vary in size (based on descriptions in the literature; Table 1.3). Abasidium of G. lucidum has four sterigma with a hilar appendix (Fig. 1.4) and1–2 vacuoles. Basidiospores have an eccentric hilar appendix on a roundedspore base, and vacuoles. The surface of basidiospores is smooth or wrinkled,and most of them have numerous small and shallow holes (Fig. 1.4). The sizesof basidiospores of naturally grown specimens from Japan and Korea were8.5–11 × 6.5–8.5 µm (average 10.1 × 7.5 µm), and 8.5–13 × 5.5–7 µm(average of 10.4 × 6.6 µm), respectively. The mean spore indexes (the ratio ofspore length to width) were 1.62 and 1.58, respectively.Cultural CharacteristicsCritical studies on cultural characteristics are very important in species identi-fication of some groups of higher basidiomycetes. However, useful studies ofcultural characteristics of Ganoderma for species identification are rare. In vitromorphogenesis and cultural characteristics of basidiomycetes are affected byvarious environmental factors, such as light, aeration, temperature, humidityand nutritional condition (Schwalb, 1978; Suzuki, 1979; Manachère, 1980;Kitamoto and Suzuki, 1992). Among these, light is an essential factor forfruiting and pileus differentiation (Plunkett, 1961; Kitamoto et al., 1968,1974; 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 thestipe of G. lucidum were affected positively by light (Hemmi and Tanaka, 1936;
14 G.-S. Seo and P.M. KirkTable 1.3. Morphological comparison of basidiospores of Ganoderma lucidum. Basidiospore Size SporeReference sources (µm) indexa Microscopical featureIto (1955) Wild fruit 9.5–11 × 5.5–7 – Deep yellowish brown, body ovoid and double wallSteyaert (1972) Wild fruit 8.5–13 × 5.5–8.5 – Ovoid, chamois bodyPegler and Wild fruit 9.0–13 × 6–8 1.64 Ovoid to ellipsoidYoung (1973) body (av. 11.5 × 7)Bazzalo and Wild fruit 9–13 × 5–6.9 – Subovoid with the apexWright (1982) body truncate, perisporum hyaline, smooth and thin endosporic pillarsMelo (1986) Wild fruit 8.2–13.5 × 6.8–8.1 – Truncate, ovoid, body brownish to brownAdaskaveg Wild fruit 10.6–11.8 × 6.3–7.8 1.50 Brown, ovoid withand Gilbertson body (av. 11.5 × 7.4) holes and eccentric(1986) hilar appendix, double wall and vacuoleMims and Wild fruit 9–12 × 6–7 – Ellipsoid with holes andSeabury (1989) body eccentric hilar appendixSeo et al. Wild fruit 8.6–10.9 × 6.6–8.3 1.62 Brown, ovoid with(1995a)b body (av. 10.1 × 7.5) holes and eccentric hilar appendix, double wall and vacuoleSeo et al. Wild fruit 8.3–12.8 × 5.6–7.2 1.58 Brown, ovoid with(1995a)c body (av. 10.4 × 6.6) holes and eccentric hilar appendix, double wall and vacuoleSeo et al. Atypical 6.4–9.6 × 3.2–5.1 1.74 Brown, ellipsoid with(1995a) fruiting (av. 7.3 × 4.2) holes and eccentric structures hilar appendix, double wall and vacuoleaSpore index = ratio of spore length to width; –, not determined.bBasidiospores from a Korean specimen.cBasidiospores from a Japanese specimen.Stamets, 1993a, b). On the contrary, the growth of mycelium was suppressedby light (Shin and Seo, 1988a, 1989a; Seo et al., 1995a, b). However, criticalstudies on the effects of light on mycelial growth and basidiocarp formation ofGanodermataceae have not been reported. In vitro, cultures of Ganoderma species produce various hyphal structures,such as generative hyphae with clamp connections, fibre or skeletal hyphae,‘stag-horn’ hyphae, cuticular cells and vesicles, and hyphal rosettes (Adaska-veg and Gilbertson, 1989; Seo, 1995). The colony is white to pale yellow andeven, felty to floccose at the optimum temperature on potato dextrose agar
Nomenclature and Classification 15Fig. 1.4. Basidiospores (a and b) and basidia (c and d) of Ganoderma lucidum,generated from fruit body (left) and atypical fruiting structures (right). Scale bars:2 µm (basidiospores) and 3 µm (basidia).(PDA) (Seo, 1987; Adaskaveg and Gilbertson, 1989). The colony becomesmore yellowish under exposure to light. The different optimum temperatures and growth rates among variousspecies and strains of the G. lucidum complex have been described (Table 1.4).Hyphal growth of most isolates was 2–4 mm day−1 on PDA but chlamydo-spore (CHL) forming isolates grew faster than those that did not formchlamydospores. In vitro, colonies showed various features, such as sectoring,pigmentation, formation of fruit-body primordia (FBP) and atypical fruitingstructures (AFSs) which formed basidia and basidiospores without basidiocarpformation (Shin and Seo, 1988a). AFSs were induced by light with ventilationfrom the white mycelial colony stage (Shin and Seo, 1989b). Some isolates
16Table 1.4. Cultural characteristics of the Ganoderma lucidum complex. Temperature (°C) Growth rateSpecies Reference Colour Growth habit Opt. Max. (mm day−1) Chlamydosporeb FruitingbG. lucidum Adaskaveg and White Even, felty 30–34 37 7–8 − + Gilbertson (1989) Seo (1995) White to pale yellow Even, felty to floccose 25–30 33–35 2–7 ± +G. tsugae Adaskaveg and White to pale yellow Even, felty to floccose 25–25 30 2–3 − − Gilbertson (1989) Seo (1995) White to pale yellow Even, felty 25–30 33 1–2 − −G. oregonense Adaskaveg and White to pale yellow Even, felty to floccose 20–25 30 2–4 − + Gilbertson (1989) G.-S. Seo and P.M. Kirk Seo (1995) White Even, felty to floccose 25–30 a#a 2–3 − −G. resinaceum Seo (1995) White Even, felty to floccose # # 3–4 − −G. valesiacum Seo (1995) Grey Even # # 1–2 − −a#: not determined.bFormation of chlamydospore, vesicle, atypical fruiting structures and fruit-body primordia on agar media (+), or not (−).
Nomenclature and Classification 17produced FBP on agar medium, but these did not develop into mature fruitbodies during the 30 days of cultivation (Seo et al., 1995a). In vitro, higher rateof ventilation was required for AFS formation, but FBP could be formed underconditions of lower ventilation. This fact suggests that FBP and AFSs maybe initiated by a common morphogenetic control system, but that subsequentdevelopment to either FBP or AFSs may be determined by environmental con-ditions in addition to the genetic characteristic of the strains. The formation ofAFSs and FBP on agar media was noted particularly in the G. lucidum complex,especially the Korean and Japanese collections, and in G. oerstedii (ATCC52411, Argentina). A few reports have described the formation of aberrant fruit bodies ofG. lucidum in vitro (Bose, 1929; Banerjee and Sarkar, 1956; Adaskaveg andGilbertson, 1986). Adaskaveg and Gilbertson (1989) reported that G. lucidumoccasionally produced aberrant fruit bodies with basidiospores on agar media.The basidiospores were formed on red, laccate, coral-like fruit bodies. Thesefruit bodies might be AFSs because of similarity in their appearance and intheir ability to form basidiospores. In this case, chlamydospore formation wasobserved on the same colony, although the AFS- and FBP-forming isolatesexamined by Seo et al. (1995a) did not produce chlamydospores. Furthermore,chlamydospore-forming isolates formed neither AFSs nor FBP under any of theconditions examined (Seo et al., 1995a). Among 30 isolates of G. lucidum collected from Japan, Korea, PapuaNew Guinea, Taiwan and the USA, 20 isolates (about 66% of the isolatestested), none of which was from the USA, formed AFSs with basidiospores, andanother five isolates (about 17% of the isolates tested), none of them fromPapua New Guinea, induced FBP. Of the remaining five isolates, one isolatefrom Korea formed a callus-like structure without producing basidiospores,this structure differing from AFSs and FBP in form, and the other four isolatesfrom Korea, Papua New Guinea and the USA formed neither AFSs nor FBP.Among the latter, three strains formed chlamydospores. One isolate did notform any fruiting structure under standard conditions, but it could produceAFSs in dual culture with a species of Penicillium known to produce a fruit-body-inducing substance (Kawai et al., 1985).Taxonomy of the Ganoderma lucidum ComplexThe Ganodermataceae Donk was created to include polypore fungi characterizedby double-walled basidiospores. Large morphological variations in the familyresulted in the description of about 400 species, of which about two-thirdsclassify in the genus Ganoderma Karst, many of them belonging to theG. lucidum complex. The variable morphological features of the G. lucidum complex, such as thesize, colour and shape of fruit bodies, may be caused by different environmentalconditions during development. Because of the morphological variation in
18 G.-S. Seo and P.M. KirkNorwegian laccate specimens of G. lucidum, Ryvarden (1994) commented that‘Macro-morphology is of limited value for criterion of species in the G. lucidumgroup and at least 3–5 collections with consistent microscopical charactersshould be examined before new species are described in this group’. Cultural characteristics of Ganoderma species have been studied andemployed to determine taxonomic arrangement (Nobles, 1948, 1958;Stalpers, 1978; Bazzalo and Wright, 1982; Adaskaveg and Gilbertson, 1986,1989), but these attempts caused more confusion as they were often quitedifferent from classical identifications based on morphological features. Forexample, Nobles (1948, 1958) described the differences in the cultural charac-teristics of G. lucidum, G. tsugae and G. oregonense. Later, the isolates previouslylisted as G. lucidum were changed to G. sessile (Nobles, 1965). However,Steyaert (1972) and Stalpers (1978) classified it as G. resinaceum. The culturalcharacteristics of G. resinaceum given by Bazzalo and Wright (1982) agree withthe description of Nobles (1965) and Stalpers (1978) and the description ofG. lucidum cultures given by Bazzalo and Wright (1982) is very similar to thatof G. tsugae as described by Nobles (1948). Furthermore, Stalpers (1978)considered that the cultural characteristics of the European G. valesiacum wereidentical to those of G. tsugae from North America, and listed it as a synonym ofG. valesiacum. Nobles (1958) suggested that the use of cultural characters inthe taxonomy of the Polyporaceae reflects natural relationships and phylogeny.ReferencesAdaskaveg, J.E. and Gilbertson, R.L. (1986) Cultural 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. and Gilbertson, R.L. (1988) Basidiospores, pilocystidia, and other basidiocarp characters in several species of the Ganoderma lucidum complex. Mycologia 80, 493–507.Adaskaveg, J.E. and Gilbertson, R.L. (1989) Cultural studies of four North American species in the Ganoderma lucidum complex with comparisons to G. lucidum and G. tsugae. Mycological Research 92, 182–191.Adaskaveg, J.E. and Ogawa, J.M. (1990) Wood decay pathology of fruit and nut trees in California. Plant Disease 74, 341–352.Adaskaveg, J.E., Blanchette, R.A. and Gilbertson, R.L. (1991) Decay of date palm wood by white-rot and brown-rot fungi. Canadian Journal of Botany 69, 615–629.Adaskaveg, J.E., Miller, R.W. and Gilbertson, R.L. (1993) Wood decay, lignicolous fungi, and decline of peach trees in South Carolina. Plant Disease 77, 707–711.Ames, A. (1913) A consideration of structure in relation to genera of the Polyporaceae. Annals of Mycology 11, 211–253.Atkinson, G.F. (1908) Observations on Polyporus lucidus Leys., and some of its allies from Europe and North America. Botanical Gazette (Crawfordsville) 46, 321–338.Banerjee, S. and Sarkar, A. (1956) Formation of sporophores of Ganoderma lucidum (Leyss.) Karst. and Ganoderma applanatum (Pers.) Pat. in culture. Indian Journal of Mycological Research 2, 80–82.
Nomenclature and Classification 19Bazzalo, M.E. and Wright, J.E. (1982) Survey of the Argentine species of the Ganoderma lucidum complex. Mycotaxon 16, 293–325.Blanchette, R.A. (1984) Screening wood decayed by white rot fungi for preferential lignin degradation. Applied Environmental Microbiology 48, 647–653.Bose, S.R. (1929) Artificial culture of Ganoderma lucidus Leyss from spore to spore. Botanical Gazette (Crawfordsville) 87, 665–667.Boudier, E. (1895) Description de quelques nouvelles espèces de Champignons recoltées dans les regions elevées des Alpes du Valais, en aout 1894. Bulletin de la Société Mycologique de France 11, 27–30.Boudier, E. and Fischer, E. (1894) Rapport sur les espèces de Champignons trouvées pendant l’assemblée a Geneve et les excursions faites en Valais. Bulletin de la Société Mycologique de France 41, 237–249.Corner, E.J.H. (1983) Ad Polyporaceas I. Amauroderma and Ganoderma. Nova Hedwigia 75, 1–182.Donk, M.A. (1964) A conspectus of families of Aphyllophorales. Persoonia 3, 199–324.Furtado, J.S. (1981) Taxonomy of Amauroderma. Memoirs of the New York Botanical Garden 34, 1–109.Gilbertson, R.L. and Ryvarden, L. (1986) North Americal Polypores. Part 1. Fungiflora, Oslo, Norway.Gottlieb, A.M. and Wright, J.E. (1999) Taxonomy of Ganoderma from southern South America: subgenus Ganoderma. Mycological Research 103, 661–673.Haddow, W.R. (1931) Studies in Ganoderma. Journal of the Arnold Arboretum 12, 25–46.Hemmi, T. and Tanaka, I. (1936) Experiments for developing sporophores of Ganoderma japonicum. Botany and Zoology 4, 13–23 (in Japanese).Hepting, G.H. (1971) Diseases of forest and Shade Trees of the United States. US Department of Agriculture, Agricultural Handbook, 386, 1–658.Hseu, R.S. (1990) An identification system for cultures of Ganoderma species. PhD thesis, National Taiwan University, Taipei (in Chinese).Hseu, R.S., Wang, H.H., Wang, H.F. and Moncalvo, J.M. (1996) Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences. Applied and Environmental Microbiology 62, 1354–1363.Ito, S. (1955) Mycological Flora of Japan 2(4), 450 pp Yôkendo.Julich, W. (1981) Higher Taxa of Basidiomycetes, J. Cramer, 485 pp.Karsten, P. (1881) Enumeratio Boletinearum et Polyporearum Fennicarum, Systemate novo dispositarum. Revue Mycologie (Toulouse) 3, 1–19.Kawai, G., Ikeda, Y. and Tubaki, K. (1985) Fruiting of Schizophyllum induced by certain ceramides and cerebrosides from Penicillium funiculosum. Agricultural and Biological Chemistry 49, 2137–2146.Kitamoto, Y. and Suzuki, A. (1992) Seiri. In: Hurukawa, H. (ed.) Kinokogaku. KyouritsuSyuppan, Tokyo, pp. 79–115.Kitamoto, Y., Takahashi, M. and Kasai, Z. (1968) Light induced formation of fruit bodies in a basidiomycete, Favolus arcularius (Fr.) Ames. Plant and Cell Physiology 9, 797–805.Kitamoto, Y., Suzuki, A. and Furukawa, S. (1972) An action spectrum for light induced primordium formation in a basidiomycetes, Favolus arcularius (Fr.) Ames. Plant Physiology 49, 338–340.
20 G.-S. Seo and P.M. KirkKitamoto, Y., Horikoshi, T. and Suzuki, A. (1974) An action spectrum for photo- induction of pileus formation in a basidiomycete, Favolus arcularius. Planta 119, 81–84.Manachere, G. (1980) Conditions essential for controlled fruiting of macromycetes. A review. Transactions of the British Mycological Society 75, 255–270.Melo, I. (1986) Studies on the Aphyllophorales of Portugal: The genus Ganoderma. International Journal of Mycology and Lichenology 2, 183–204.Mims, C.W. and Seabury, F. (1989) Ultrastructure of tube formation and basidiospore development in Ganoderma lucidum. Mycologia 81, 754–764.Mizuno, T., Wang, G.Y., Zhang, J., Kawagishi, H., Nishitoba, T. and Li, J.X. (1995) Reishi, Ganoderma lucidum and Ganoderma tsugae: Bioactive substances and medicinal effects. Food Reviews International 11, 151–166.Moncalvo, J.M., Wang, H.F. and Hseu, R.S. (1995a) Gene phylogeny of the Ganoderma lucidum complex based on ribosomal DNA sequences. Comparison with traditional taxonomic characters. Mycological Research 99, 1489–1499.Moncalvo, J.M., Wang, H.H. and Hseu, R.S. (1995b) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87, 223–238.Moncalvo, J.-M. and Ryvarden, L. (1997) A nomenclatural study of the Gano- dermataceae Donk. Synopsis Fungorum 11.Morimoto, N. and Oda, Y. (1973) Effect of light on fruit body formation in a basidiomycete, Coprinus macrorhizus. Plant and Cell Physiology 14, 217–225.Murrill, W.A. (1902) The polyporaceae of North America. I. The genus Ganoderma. Bulletin of the Torrey Botanical Club 29, 599–608.Murrill, W.A. (1908) Family 5. Polyporaceae. North American Flora 9, 73–132.Nobles, M.K. (1948) Studies in forest pathology. VI. Identification of cultures of wood- rotting fungi. Canadian Journal of Research C 26, 281–431.Nobles, M.K. (1958) Cultural characters as a guide to the taxonomy and phylogeny of the Polyporaceae. Canadian Journal of Botany 36, 883–926.Nobles, M.K. (1965) Identification of cultures of wood-inhabiting Hymenomycetes. Canadian Journal of Botany 43, 1097–1139.Overholts, L.O. (1953) Polyporaceae of the United States, Alaska, and Canada. University of Michigan Press, Ann Arbor.Park, D.S., Sung, J.M., Kim, Y.S., Yoo, Y.B., Ryu, Y.J. and Cha, D.Y. (1994) Analysis of interspecific allozyme variation within the genus Ganoderma by polyacrylamide gel isoelectric focusing. RDA Journal of Agricultural Science 36, 212–221.Patouillard, N. (1889) Le genre Ganoderma. Bulletin de la Société Mycologique de France 5, 64–80.Pegler, D.N. and Young, T.W.K. (1973) Basidiospore form in the British species of Ganoderma Karst. Kew Bulletin 28, 351–364.Perkins, J.H. (1969) Morphogenesis in Schizophyllum commune I. Effect of white light. Plant Physiology 44, 1706–1711.Perkins, J.H. and Gordon, S.A. (1969) Morphogenesis in Schizophyllum commune. II. Effects of monochromatic light. Plant Physiology 44, 1712–1716.Plunkett, B.E. (1961) The change of tropism of Polyporus brumalis stipes and the effect of directional stimuli on pileus differentiation. Annals Botany, N. S. (London) 25, 206–222.
Nomenclature and Classification 21Raudaskoski, M. and Yli-Mattila, T. (1985) Capacity for photoinduced fruiting in a dikaryon of Schizophyllum commune. Transactions of the British Mycological Society 85, 145–151.Ryvarden, L. (1994) Can we trust morphology in Ganoderma? In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma – Systematics, Phytopathology and Phamacology. Proceedings of contributed symposia 59A, B, Fifth International Mycological Congress, Vancouver, August 14–21, 1994, pp. 19–24.Schwalb, M.N. (1978) Regulation of fruiting. In: Schwalb, M.N. and Miles, P.G. (eds) Genetics and Morphogenesis in the Basidiomycetes. Academic Press, New York, pp. 135–165.Schwalb, M.N. and Shanler, A. (1974) Phototropic and geotropic responses during the development of normal and mutant fruit bodies of the basidiomycetes Schizophyllum commune. Journal of Genetic Microbiology 82, 209–212.Seo, G.S. (1987) Studies on cultural characteristics of Ganoderma lucidum (Fr.) Karst. MSc Thesis (in Korean).Seo, G.S. (1995) In vitro photomorphogenesis and genetic diversity in the basidio- mycete, Ganoderma lucidum. PhD dissertation, Tottori University, Tottori, Japan.Seo, G.S., Shin, L.G.C., Otani, H., Komada, M. and Kohmoto, K. (1995a) Formation of atypical fruiting structures in Ganoderma lucidum isolates on a nutrition agar media. Mycoscience 36, 1–7.Seo, G.S., Otani, H. and Kohmoto, K. (1995b) Effect of light on the formation of atypical fruiting structures in Ganoderma lucidum. Mycoscience 36, 227–233.Shin, G.C. and Seo, G.S. (1988a) Formation of the nonbasidiocarpous basidiospore of Ganoderma lucidum. Korean Journal of Mycology 16, 230–234 (in Korean).Shin, G.C. and Seo, G.S. (1988b) Classification of strains of Ganoderma lucidum. Korean Journal of Mycology 16, 235–241 (in Korean).Shin, G.C. and Seo, G.S. (1989a) Effect of light on the formation of non-basidiocarpous basidiospores of Ganoderma lucidum. Korean Journal of Mycology 17, 189–193 (in Korean).Shin, G.C. and Seo, G.S. (1989b) Effect of temperature and aeration on the formation of non-basidiocarpous basidiospores of Ganoderma lucidum. Korean Journal of Mycology 17, 194–196 (in Korean).Shin, G.C., Park, Y.H., Seo, G.S. and Cha, D.Y. (1986) Morphological characters of Ganoderma lucidum (Fr) Karsten grown naturally in Korea. Research Reports of Institute of Agricultural Science and Technology 13, 44–51 (in Korean).Stalpers, J.A. (1978) Identification of wood-inhibiting Aphyllophorales in pure culture. Centraalbureau Voor Schimmelcultures, Baarn. Studies in Mycology, pp. 1–248.Stamets, P. (1993a) Evaluating a mushroom strain: Photosensitivity. In: Growing Gour- met and Medical Mushrooms. Ten Speed Press, Berkely, California, pp. 117–126.Stamets, P. (1993b) The polypore mushrooms of the genera Ganoderma, Grifola and Polyporus. In: Growing Gourmet and Medical Mushrooms. Ten Speed Press, Berkely, California, pp. 351–369.Steyaert, R.L. (1972) Species of Ganoderma and related genera mainly of the Bogor and Lieden herbaria. Persoonia 7, 55–118.Steyaert, R.L. (1975) The concept and circumscription of Ganoderma tornatum. Trans- actions of the British Mycological Society 65, 451–467.Steyaert, R.L. (1977) Basidiospores of two Ganoderma species and others of two related genera under the scanning electron microscope. Kew Bulletin 31, 437–442.
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24 J.-M. Moncalvoshould be abandoned for various reasons (Moncalvo and Ryvarden, 1997).Most species were described in the genus Ganoderma (219 species), mainlyfrom laccate collections (166 species). Many species are known only from asingle collection or locality. Several names have been considered synonyms(reviewed in Moncalvo and Ryvarden, 1997), but I believe that moretaxonomic synonyms still exist because a large number of species wereFig. 2.1. Morphological characters traditionally used in Ganoderma systematics.(a) Typical basidiospore of Ganoderma. (b) Basidiospore of G. boninense. (c)Basidiospore of G. formosanum (longitudinal crests are barely seen in light micro-scopy). (d) Typical basidiospore of Amauroderma. (e) Various types of pilocystidiafound in Ganoderma. (f) Stipitate versus dimidiate basidiocarps: relationshipsbetween stipe formation and location of basidiocarp development on wood.
Table 2.1. A summary of the traditional taxonomy in Ganodermaa. Number of species Number of Estimated Known from a names proposed number ofGenera Subgenera Distinctive features Described single locality as synonyms known speciesGanoderma Spore wall enlarged at the apex Ganoderma Pilear surface laccate (presence of pilocystidia) 168 124 48 60–80 Elfvingia Pilear surface dull (absence of pilocystidia) 51 31 21 10–30Amauroderma Spore wall uniformly large 96 60 41 30–50Haddowiab Spore wall uniformly large and spore surface 5 1 2 3 longitudinally crestedHumphreyac Spore wall enlarged at the apex and spore 7 3 3 4 Systematics of Ganoderma surface reticulateaDatafrom Moncalvo and Ryvarden (1997).bSynonym of Amauroderma in Furtado (1981) and Corner (1983).cSynonym of Ganoderma in Furtado (1981) and Corner (1983). 25
26 J.-M. Moncalvodistinguished from characters that depend on growing conditions and develop-mental stage. For instance, careful observation in vivo shows that young,actively growing fruiting bodies generally have lighter and brighter surfacecolours than basidiocarps that are several weeks or months old: the latter havebeen exposed to repeated periods of rain and dryness, covered with dust,attacked by insects, or even colonized by algae. Presence, absence, size andinsertion of the stipe have also been used to circumscribe species (e.g. G.gibbosum, G. dorsale, etc.), but it has been shown that stipe development can becontrolled in vitro by the duration and intensity of exposure to light and by car-bon dioxide concentration (Hseu, 1990). In vivo, stipe development alsodepends on the location in the host: a basidiocarp that develops from a buriedroot is more likely to develop a stipe than a basidiocarp that develops higher inthe trunk (Fig. 2.1). Ryvarden (1995) examined the variability of 53 Norwe-gian specimens of G. lucidum, and concluded that macromorphological charac-ters are of very limited value for the identification of Ganoderma species. Reliable morphological characters for Ganoderma systematics appear to bespore shape and size, context colour and consistency, and microanatomy of thepilear crust. However, the typical spore of G. lucidum is similar for dozens ofdifferent species. Scanning electron microscopy (SEM) has been useful in dis-tinguishing between spores that appear similar under light microscopy (Peglerand Young, 1973; Gottlieb and Wright, 1999), and has revealed the existenceof distinctive, slightly longitudinally crested basidiospores in the G. australe andG. sinense species complexes (Hseu, 1990; Buchanan and Wilkie, 1995; Tham,1998). Context colour and consistency may change slightly with the age of thefruit body or upon drying, and are also somewhat subjective characters, but itis still possible to distinguish at least three very distinctive types: (i) light col-oured and/or duplex context in G. lucidum and its allies; (ii) uniformly brown todark brown context as in the G. sinense and G. australe complexes; and (iii) verysoft, cream to pale ochraceous context in G. colossum. Relationships betweenthe microstructure of the pilear crust, the age of the basidiocarp, and theexposure to environment are not well known, but different types of pilocystidiaand hyphal arrangement can be distinguished among both laccate and non-laccate taxa (Steyaert, 1980; Fig. 2.1). The laccate appearance of Ganodermabasidiocarps is associated with the presence of thick-walled pilocystidia(Fig. 2.1) that are embedded in an extracellular melanin matrix. The exactorigin and chemical composition of this matrix remain to be elucidated. High phenotypic plasticity at the macroscopic level, uniformity of micro-scopic characters, and subjective interpretation of various features such ascolour or consistency have resulted in the creation of numerous unnecessarynames (synonyms), and a lack of handy identification keys. The absence ofa world monograph has also contributed to problems with species circum-scriptions and identifications in Ganoderma. Culture and enzymatic studies have produced additional and usefultaxonomic characters in Ganoderma systematics (Adaskaveg and Gilbertson,1986, 1989; Hseu, 1990; Wang and Hua, 1991; Gottlieb et al., 1995; Gottlieb
Systematics of Ganoderma 27and Wright, 1999). It appears that chlamydospore production and shape, andto a lesser extent the range and optima of growth temperatures, are extremelyuseful culture characters for distinguishing between morphologically similarspecies. Mating studies have also been conducted to circumscribe biologicalspecies within species complexes (Adaskaveg and Gilbertson, 1986; Hseu,1990; Yeh, 1990; Buchanan and Wilkie, 1995). However, all these studieswere restricted in scope, and the techniques employed, although useful at thespecies level, have limitations for addressing phylogenetic relationshipsbetween taxa and the development of a natural classification system.Molecular Systematics of GanodermaWith recent advances in both sequencing techniques to produce taxonomiccharacters and cladistic methods to infer natural relationships betweenorganisms, molecular systematics has become a paradigm in biology. To date,the most widely used molecules in fungal molecular systematics have beenthe ribosomal genes (rDNA). Hibbett and co-workers (Hibbett and Donoghue,1995; Hibbett et al., 1997) produced molecular phylogenies for hymenomyce-tous fungi using sequence data from the nuclear small subunit (18S, or nSSU)and mitochondrial small subunit (12S, or mtSSU) rDNA, and showed thatGanoderma belongs to a larger group of white-rot fungi that also includes thegenera Trametes, Fomes, Polyporus, Lentinus, Datronia, Pycnoporus, Cryptoporus,Daedalopsis, Lenzites and Dentocorticium. Additional phylogenetic studies usingsequence data from the nuclear large ribosomal subunit (25–28S, or nLSU)rDNA showed that genera Amauroderma, Irpex, Loweporus and Perenniporiaalso belong to this group (Moncalvo et al., 2000; Thorn et al., 2000; Moncalvo,unpublished). Combined evidence of nLSU and mtSSU-rDNA data support theplacement of Amauroderma as a sister genus to Ganoderma (Moncalvo andHibbett, unpublished). However, nucleotide sequence data from nuclear andmitochondrial rDNA encoding sequences do not offer enough variation to inferphylogenetic relationships between Ganoderma species. Appropriate nucleotide sequence variation for systematics of Ganodermawas found in the internal transcribed spacers (ITS) of the nuclear rDNA gene(Moncalvo et al., 1995a, b, c). The ITS phylogenies produced in these studiesindicated that many names were commonly misapplied (e.g. G. lucidum andG. tsugae), and that the proposed subgenera and sections in Steyaert (1972,1980) and Zhao (1989) were not monophyletic and should be abandoned.Gene trees and species treesA gene tree is not necessarily equivalent to a species tree, and phylogenetictrees inferred from the sequences of different genes can be contradictory forseveral reasons, including differences in their power or level of phylogenetic
28 J.-M. Moncalvoresolution, incorrect recovery of evolutionary relationships by phylogeneticreconstruction methods (e.g. ‘long branch attraction’, Felsenstein, 1978),discordance in rates and modes of sequence evolution (Bull et al., 1993), differ-ent phylogenetic histories due to lineage sorting or difference in coalescencetime (Doyle, 1992, 1997; Maddison, 1997), or horizontal gene transfer.Incongruences between gene trees are more likely to occur at lower taxonomiclevels (species, populations). In fact, it is expected that gene trees areincongruent among interbreeding individuals because these individuals areconnected by gene flow and recombination: their relationships are thereforetokogenetic (reticulate) rather than phylogenetic (divergent) (Hennig, 1966;Doyle, 1997). Overall, a phylogenetic hypothesis is more likely to be correct if itis supported from multiple, independent data sets rather than from a singlegene tree.ITS phylogeny versus manganese-superoxide dismutase (Mn-SOD)phylogenyThirty-three Ganoderma taxa were used to conduct separate phylogeneticanalyses of sequence data from ITS and Mn-SOD genes. The incongruencelength difference (ILD) test of Farris et al. (1994), also known as the partition-homogeneity test, indicated absence of statistically significant conflict(P = 0.08) in phylogenetic signals between the two data sets. Results of theanalyses are shown in Fig. 2.2. Tree topologies are fully congruent for allnodes having bootstrap statistical support (BS) greater than 50%, with twoexceptions:1. the type specimen of G. microsporum clusters with G. weberianumCBS219.36 in the ITS analysis (88% BS), but clusters with a strain labelledG. cf. capense ACCC5.71 in the Mn-SOD analysis (98% BS); and2. the cultivar G. cf. curtisii RSH.J2 nests with strain RSH-BLC in the ITSanalysis (58% BS) but with RSH-J1 (83% BS) in the Mn-SOD analysis.The latter three collections are known to be intercompatible (i.e. belong tothe same biological species; Hseu, 1990), therefore conflicting gene phylo-genies for these strains are not surprising. Strains labelled G. microsporum,G. weberianum and G. cf. capense are probably also conspecific: the synonymy ofthe first two names was already suggested by Peng (1990). Both data sets strongly support similar terminal clades, and do not fullyresolve basal relationships among Ganoderma taxa. The ITS data set offersslightly more resolution for deeper branches (Fig. 2.2), whereas highersequence divergence between closely related taxa was found in the Mn-SODgene (in particular in two introns that were excluded from the analysesbecause nucleotide sequences could not be unambiguously aligned across allthe taxa sampled). Ongoing sequencing and analyses of β-tubulin genes also
Systematics of Ganoderma 29support similar terminal clades to those from ITS and Mn-SOD data (Moncalvoand Szedlay, unpublished). Therefore, preliminary data suggest that phylogenies derived fromITS sequences are congruent with those from other genes, and that ITSphylogenies may accurately reflect natural relationships between Ganodermaspecies.Fig. 2.2. Comparison between internal transcribed spacer (ITS) and manganese-superoxide dismutase (Mn-SOD) nucleotide sequence phylogenies for 33 Gano-derma taxa. Sequences from one species of genus Amauroderma were used toroot the trees. Trees depicted are strict consensus trees produced from maximumparsimony searches. Bootstrap statistical supports greater than 50% are shownabove branches. Mn-SOD data were from Wang (1996; GenBank accessionnumbers U56106-U56137), and Moncalvo and Szedlay (unpublished). Analyseswere conducted in PAUP* (Swofford, 1998) and employed maximum parsimonywith heuristic searches using 50 replicates of random addition sequences withTBR branch swapping. Bootstrap statistical supports were evaluated with 100bootstrap replicates of random addition sequence with TBR branch swapping.Regions with ambiguous alignment were removed from the alignment, andunambiguously aligned gaps were scored as ‘fifth character state’. The ITSdata set used 81 parsimony-informative characters and produced 24 equallyparsimonious trees of length 232, with a consistency index of 0.703. The SODdata set used 105 parsimony-informative characters and produced 58 equallyparsimonious trees of length 329, with a consistency index of 0.623.
30 J.-M. MoncalvoITS phylogenyThe current ITS sequence database for Ganoderma and Amauroderma speciesincludes about 300 taxa. Numerous small nucleotide insertions and deletionsmake sequence alignment problematic in several regions, but at least 380characters can be aligned unambiguously across the entire data set, yieldingabout 200 parsimony-informative characters. Phylogenetic analysis of largemolecular data sets is still a controversial field (Lecointre et al., 1993; Hillis,1996; Graybeal, 1998; Poe, 1998). One commonly encountered problem withlarge data sets concerns the applicability and/or accuracy of standarddescriptors commonly used to assess branch robustness. For instance, the useof branch decay indices (Bremer, 1994) is not practical for large data setsbecause of the large number of trees that cannot be sampled; and consistencyindices (Sanderson and Donhogue, 1989), bootstrap (Felsenstein, 1985) andjackknife (Farris et al., 1996) statistical supports are sensitive to sample size.However, evidence from various studies (Hillis, 1996, 1998; Moncalvo et al.,2000) suggests that increasing taxon sampling generally increases phylo-genetic accuracy, and that bootstrapping or jackknifing methods are stilluseful tools to determine the robustness of clades. Parsimony analyses of ITS data for 248 Ganoderma taxa reveal about50 clades with bootstrap statistical support greater than 50% (Fig. 2.3 andTable 2.2), that are also consistent with morphological and/or geographicaldata. Terminal clades in this phylogeny represent either a population, aspecies, a species complex, or a group of closely related species. In Table 2.2,tentative names for the most well-supported clades are proposed, although16 clades have not been named (the original data set included 36 speciesnames and many unnamed taxa). Basal relationships are either poorlysupported or unresolved, but phylogenetic analyses of various data setsusing maximum parsimony and maximum likelihood consistently revealthree larger groups: these are labelled Groups 1–3 in Fig. 2.3 and Table2.2. ITS phylogeny suggests that the laccate habit has been derived morethan once (or lost several times), making the laccate Ganoderma taxapolyphyletic. This conflicts with traditional systems of classification thataccommodate laccate and non-laccate Ganoderma taxa in subgeneraGanoderma (laccate) and Elfvingia (non-laccate), respectively (see Table 2.1).However, within the Ganodermataceae, there is already evidence fornon-monophyly of laccate taxa because at least three laccate species havebeen traditionally classified in genus Amauroderma (Furtado, 1981). A revisedclassification for subgenera and sections in Ganoderma seems thereforenecessary, and will be formally proposed elsewhere. For now discussion islimited to some taxonomic groupings revealed by ITS sequence data, assummarized in Table 2.2.
Systematics of Ganoderma 31Fig. 2.3. Internal transcribed spacer (ITS) phylogeny for 248 taxa of Gano-dermataceae (sequences from several Amauroderma species were used to root thetree). The tree depicted is one of 100 equally parsimonious trees produced usingmaximum parsimony in PAUP* (Swofford, 1998) with heuristic searches, randomaddition sequences (100 replicates), TBR branch swapping, and MAXTREES setto 100. Statistical supports for branch robustness were evaluated in PAUP* with100 bootstrap replicates, random addition sequence, TBR branch swapping, andMAXTREES set to 10. Bootstrap values are only given for branches in bold thatrefer to groups or clades that are presented in Table 2.2. Groups 1 and 1.4 are notmonophyletic in the figure they were retained as such to facilitate the discussion.Details about Groups 1–3 and unclassified taxa are given in the text and Table 2.2.
32Table 2.2. Groupings of Ganoderma taxa based on a phylogenetic analysis of ITS nucleotide sequence data (Fig. 2.3), with geographicorigin and host relationships of the strains examined. Geographic categories Hosts India, Pakistan Indo, PNG S. America Neotropics N. America Palms S. Africa Europe China, Korea Japan Taiwan S.E. Asia Australia New Zealand Florida Woody dicots ConifersGroup 11.1 G. lucidum complex sensu stricto (84% BS) G. lucidum • • • G. valesiacum • • G. carnosum • • G. ahmadii • • G. tsugae • • • J.-M. Moncalvo G. oregonense • • G. praelongum, G. oerstedii • •1.2 G. resinaceum complex sensu lato (86% BS) G. resinaceum complex sensu stricto: G. resinaceum (’G. pfeifferi’) (90% BS) • • • G. cf. resinaceum (’G. lucidum’) (64% BS) • • • G. cf. resinaceum (G. sessile, G. platense) (59% BS) • • G. weberianum complex (59% BS): G. weberianum (= G. microsporum) (89% BS) • • • • G. cf. capense (56% BS) • • • Ganoderma sp. (99% BS) • • Ganoderma sp. (’G. subamboinense’) (97% BS) • • • G. trengganuense (87% BS) • •