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.
22 G.-S. Seo and P.M. KirkSteyaert, R.L. (1980) Study of some Ganoderma species. Bulletin du Jardin Botanique National de Belgique 50, 135–186.Suzuki, A. (1979) General review on environmental factors affecting primordium formation in Homobasidiae. Transaction of the Mycological Society of Japan 20, 253–265 (in Japanese).Willard, T. (1990) Reishi Mushroom, ‘Herb of spiritual potency and medical wonder’. Sylval Press, Issaquah, Washington, p. 167.Yli-Mattila, T. (1990) Photobiology of fruit body formation in the basidiomycete Schizophyllum commune. Reports from the Department of Biology, University of Turku, No. 27, pp. 1–67. Turku.Zhao, J.D. (1989) The Ganodermataceae in China. Bibliotheca Mycologica 132. J. Cramer, Berlin, Stuttgart.Zhao, J.D. and Zhang, X.Q. (1994) Importance, distribution and taxonomy of Ganodermataceae in China. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma – Systematics, Phytopathology and Pharmacology. Proceedings of Contributed Symposia 59A, B, Fifth International Mycological Congress, Vancouver, August 14–21, 1994, pp. 1–2.
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) • •
Table 2.2. Continued. 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 Conifers1.3 G. curtisii complex (75% BS): G. curtisii (= G. meredithae) (83% BS) • • • • • G. cf. curtisii (G. fulvellum, ‘G. tsugae’) (85% BS) • • • • •1.4 G. tropicum complex sensu lato: Ganoderma spp. ‘clade A’ (50% BS) • • • Ganoderma sp. ‘clade B’ (’G. lucidum’) (62% BS) • • • • Ganoderma sp. ‘clade C’ (99% BS) • • • Ganoderma sp. • • Ganoderma sp. • • G. tropicum complex s. stricto (G. fornicatum) (58% BS) • • • •Group 2 Systematics of Ganoderma2.1 ‘palm clade’ (74% BS): G. zonatum-boninense group (85% BS): G. zonatum (86% BS) • • Ganoderma sp. (88% BS) • • • G. boninense • • • • • Ganoderma sp. (100% BS) • • • •2.2 Ganoderma species (82% BS): Ganoderma sp. • • Ganoderma sp. (100% BS) • • Ganoderma sp. (’G. cf. tornatum’) (63% BS) • • 33 Continued
34Table 2.2. Continued. Geographic categories Hosts S. Africa Europe India, Pakistan China, Korea Japan Taiwan S.E. Asia Indo, PNG Australia New Zealand S. America Neotropics Florida N. America Woody dicots Conifers Palms2.3 G. cf. balabacense (98% BS) • • •2.4 Ganoderma sp. • •2.5 G. sinense (100% BS) (= G. formosanum, = ?G. neojaponicum) • • •Group 3 J.-M. MoncalvoG. australe-applanatum complex sensu lato:G. applanatum A (G. lobatum, G. adspersum) (65% BS): • • • •G. cupreolaccatum (= G. pfeifferi) (97% BS) • •G. australe complex sensu stricto: G. australe complex A (62% BS): ‘Clade A.1’ (51% BS) • • • • • • ‘Clade A.3’ (65% BS) • • • • ‘Clade A.2’ (98% BS) • • • G. australe complex B (86% BS) • • • • • G. australe complex C (81% BS) • • • • • • •
Table 2.2. Continued. 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 ConifersUnclassifiedG. applanatum B (98% BS) • • •Ganoderma sp. (100% BS) • • •Ganoderma sp. (85% BS) • •G. tsunodae (Trachyderma) • •G. colossum (Tomophagus) • • • • • Systematics of GanodermaNames in parentheses are commonly misapplied names (in ‘quotes’), synonyms (=) or possible alternative names. Frequency values (% BS) following taxanames are bootstrap statistical support for that clade (only supports higher than 50% are given). Geographic categories and samplings are as follows:‘S. Africa’ includes collections from South Africa and Zimbabwe; ‘Europe’ includes collections from UK, Norway, France, The Netherlands, Belgium,Austria, and Germany; ‘China’ includes collections from mainland China with exclusion of subtropical and tropical collections from Yunnan; ‘S.E. Asia’includes subtropical and tropical collections form Yunnan, Thailand, Vietnam, Philippines, Peninsular Malaysia, Sabah, and Singapore; ‘Indo, PNG’ includescollections from Bali, Malukku, and Papua New Guinea; ‘S. America’ includes collections from Argentina and Chile; ‘Neotropics’ includes collections fromCosta Rica, Puerto Rico, Equador, and French Guyana. 35
36 J.-M. MoncalvoPhylogenetic Relationships and Biogeography in GanodermaPhylogenetic relationshipsGroup 1: the G. lucidum complex sensu latoGroup 1 is either monophyletic or paraphyletic, and includes G. lucidum sensustricto and many other similar laccate Ganoderma taxa, of which severalcollections were incorrectly identified as G. lucidum. In this group, basidiosporeshape and size is very uniform, and taxa generally have a reddish todark-brown pileus and light-coloured context. On the basis of ITS phylog-eny, Group 1 can be divided into at least four clades, which are discussedbelow.GROUP 1.1: THE G. LUCIDUM COMPLEX SENSU STRICTO. The G. lucidum complexsensu stricto includes only collections from temperate regions of both thenorthern and southern hemispheres. Members of this group do not producechlamydospores in culture. ITS sequence variation among taxa of this clade isvery low and does not allow for subdivision into smaller entities. Europeantaxa of this clade (G. lucidum, G. valesiacum and G. carnosum) might beconspecific (Ryvarden and Gilbertson, 1993): G. valesiacum was primarilydistinguished from G. lucidum based on host specificity (conifers versushardwood, respectively; Ryvarden and Gilbertson, 1993), but a recent studyby Ryvarden (1995) suggests that G. lucidum in Norway grows on bothhardwood and conifers; G. carnosum (= G. atkinsonii) has been reported onlyon conifers, and is distinguished from both G. lucidum and G. valesiacum byhaving rougher spores (Kotlaba and Pouzar, 1993; Ryvarden and Gilbertson,1993). The type specimen of G. ahmadii from Pakistan (Steyaert, 1972)belongs to this clade: several collections of this species in Steyaert’s herbariumhave been examined, and all can be distinguished from typical G. lucidum inhaving a less shiny pileus and a darker context, which is entirely brown andduplex. The two North American taxa of this clade (G. tsugae and G. oregonense)are believed to be restricted to conifers and might be conspecific (Gilbertsonand Ryvarden, 1986). Basidiocarps of G. lucidum from Europe and G. tsugaefrom the USA are practically impossible to distinguish. The Argentinecollections of this clade (G. praelongum) were not examined for this study,but Gottlieb and Wright (1999) distinguished the taxon from G. lucidum.GROUP 1.2: THE G. RESINACEUM COMPLEX SENSU LATO. The production of chla-mydospores in culture unites the members of this clade. G. resinaceum, a speciesdescribed from Europe, is differentiated from G. lucidum by having smootherspores (Steyaert, 1972; Pegler and Young, 1973). European G. resinaceum hasbeen shown to be intercompatible with collections generally assigned to‘G. lucidum’ in North America (Adaskaveg and Gilbertson, 1986), suggestingconspecificity of these isolates. However, ITS data distinguish between popula-tions of G. resinaceum from Old World (Europe and Africa), North America,
Systematics of Ganoderma 37and South America; these populations might therefore be completely disjunctand genetically isolated from each other, and may warrant recognition at thespecies level. However, additional sampling and more extensive mating studiesare needed before a firm taxonomic conclusion can be reached. The counterpart of the G. resinaceum complex in tropical Asia is the G.weberianum complex (Steyaert, 1972), which includes G. weberianum, G. micro-sporum, G. cf. capense, G. lauterbachii, G. rivulosum, etc.). It is distinguished fromG. resinaceum by having smaller spores (6–9 × 4–7 µm). Based on ITS data, G. trengganuense also belongs to this clade. This speciesis known from Malaysia and Vietnam and is well characterized in havingsubreticulate spores (Corner, 1983), but is similar to G. resinaceum in the othercharacters.GROUP 1.3: THE G. CURTISII COMPLEX. Members of this clade do not producechlamydospores in culture. This well-supported clade (75% BS) can be dividedin two groups which correspond to the geographic origin of the collections.One group is composed of collections from eastern North America and CostaRica. These collections can be identified as G. curtisii (a species described fromeastern America) based on descriptions in Lloyd (1912, 1917) and Steyaert(1972, 1980), and G. meredithae (Adaskaveg and Gilbertson, 1988) can beconsidered a taxonomic synonym. The sister group of these taxa is representedby collections from eastern Asia (Korea, China, Taiwan, Japan and Vietnam),and includes many cultivars from this region mistakenly identified as‘G. tsugae’ or ‘G. lucidum’.GROUP 1.4: THE G. TROPICUM COMPLEX SENSU LATO. This group is heterogeneousand may not be monophyletic, but is retained here for convenience. Membersof this group have been collected throughout tropical and subtropical regions.Only a few taxa have been examined in culture, and they all producedchlamydospores. In this group, several distinct, well-supported clades revealedby ITS data are also supported by differences in basidiocarp or culturecharacteristics. For instance, Group 1.4 includes:• three species from Taiwan distinguished by Hseu (1990) on the basis of enzymatic, culture, and mating studies (‘G. lucidum’, G. tropicum, and G. fornicatum);• a very distinctive taxon from Australia with a light, thick and soft context, a thin and yellowish crust, and a bright, dark-red laccate stipe (maybe G. septatum, described from Africa by Steyaert, 1962);• undescribed collections from Costa Rica with purple–orange basidiocarps;• a specimen from Argentina, first identified as G. oerstedii by Bazzalo and Wright (1982) and then assigned to ‘G. resinaceum’ by Wright (personal communication).Many taxa in this group are still represented by a single or only a fewcollections, and the correct naming of species remains problematic.
38 J.-M. MoncalvoGroup 2Group 2 includes laccate taxa easily distinguished from G. lucidum sensu latoby a difference in spore shape (e.g. elongated spores in G. zonatum andG. boninense), and/or by a darker pileus and/or context colour (e.g. blackpileus and uniformly brown context in G. sinense). This group also includesnon-laccate (or ‘sublaccate’?) taxa. Group 2 is mostly composed of tropical andsubtropical collections, but also includes collections from temperate Japan,Korea and China. Strains placed in group 2 that have been examined in culturedid not produce chlamydospores.GROUP 2.1: THE PALM CLADE. A well-supported clade (74% BS), composed onlyof collections from palms, which can be divided into three smaller groupscorresponding to the geographic origin of the strains: (i) G. zonatum fromFlorida; (ii) G. boninense from South-East Asia, the Australo-Pacific region andJapan; and (iii) unidentified collections from Zimbabwe and India. G. zonatumand G. boninense have elongated basidiospores and an uniformly brown-coloured context, but in G. zonatum the basidiospores are slightly longer(11–14 × 5–7 versus 9–13 × 5–7 µm), the pileus has a lighter colour, andthe pilear crust is thinner. Additional sampling and mating studies will benecessary to determine the robustness of the geographic structure, delimitspecies boundaries, and to evaluate specificity on palms. A sister group to the G. zonatum-boninense clade comprises collectionsfrom Vietnam, Malaysia, Thailand and Australia, from both palm and woodydicots. These collections differ from G. zonatum and G. boninense in having ablack pileus and ovoid spores. SEM revealed that basidiospores of the Vietnamcollection are longitudinally striate (Tham, personal communication). Thesecollections somewhat resemble those in the G. sinense clade (Group 2.5).GROUP 2.2. Group 2.2 includes three clades, and encompasses macromorpho-logically distinct taxa from three different continents. These taxa remain to benamed. All have a uniformly brown context. Basidiocarps collected in CostaRica and Puerto Rico have a shiny black pileus, and a white pore surfacethat turns dark brown upon ageing. Basidiocarps from Vietnam (originallyidentified as ‘G. tornatum’, a non-laccate taxon) and Yunnan are dull, greyishto black. Finally, an immature specimen from Zimbabwe has a dull, brown-ish-red surface.GROUP 2.3. Two collections cluster together strongly (98% BS): one collectionfrom Vietnam with a shiny, yellow–brown to dark-brown pileus and a browncontext, identified as G. cf. balabacense by its collector (Dr Le Xuan Tham), andone collection from Zimbabwe for which the basidiocarp is lacking.GROUP 2.4. A non-laccate collection from Malaysia growing on an ornamen-tal tree, received from Dr Faridah Abdullah as Ganoderma sp., stands withinGroup 2, apart from all the other taxa.
Systematics of Ganoderma 39GROUP 2.5: THE G. SINENSE COMPLEX. Th1is clade includes collections fromChina, Taiwan and Korea. Chinese collections correspond to G. sinense, aspecies described from China. It has a distinctive, shiny black pileus and abrown to dark-brown context (Zhao, 1989). The Taiwan collection includedin this study (labelled G. formosanum, but considered a synonym of G. sinense)has basidiospores longitudinally slightly striated, as shown in SEM by Hseu(1990). SEM examination of spores has not been conducted for the othercollections of this clade. The Korean collection was received from Dr Dong-SukPark as G. neojaponicum. Both G. sinense and G. neojaponicum are black andlaccate taxa with a brown context, but whether or not the two names aresynonyms remains to be investigated.Group 3: the G. australe-applanatum complexGroup 3 comprises the bulk of non-laccate taxa of the G. australe-applanatumcomplex (subgenus Elfvingia in Table 2.1), but also includes a laccate speciesfrom Europe: G. cupreolaccatum (= G. pfeifferi). All members of this group lackchlamydospores in culture. The placement of G. cupreolaccatum in the G. australe-applanatum complexis surprising, but this species differs from other laccate species (especially fromthose in Group 1) in having a dark-brown context, very similar in colour andconsistency to that in G. australe and G. applanatum. It is also interesting to notethat the culture strain CBS250.61 identified as ‘G. applanatum’ by K. Lohwagclassifies in G. cupreolaccatum based on ITS sequence data. Careful examinationof G. cupreolaccatum collections shows that in older basidiocarps the pileussurface turns greyish-black and is not very shiny; various encrustations anderosion of the melanin wax of the crust may alter the laccate appearance of thebasidiocarps, which then would more closely resemble those of G. applanatumor G. australe. Although most collections belonging to this group were originallyidentified G. australe or G. applanatum, some collections were also assigned toG. tornatum, G. adspersum, G. lobatum, G. philippii, G. pseudoferreum, or G.gibbosum. These names are scattered inconsistently (if not randomly) in theITS phylogeny, demonstrating the limitations of morphological taxonomyin this species complex. A large amount of ITS sequence divergence was foundin this group (see branch length in Fig. 2.3), and several smaller clades can bedistinguished. A well-supported clade (65% BS) consists entirely of collections from tem-perate areas of the northern hemisphere (Europe, Japan and North America),and is provisionally assigned the name ‘G. applanatum A’ (G. applanatum wasfirst described from Europe, and G. australe from a Pacific island). The remain-ing clades do not include European collections, and are provisionally groupedunder the name ‘G. australe complex sensu stricto’. On the basis of ITS sequencedata, this complex can be subdivided further into at least three well-supportedclades, showing remarkable and complex geographic patterns (Table 2.2):Clade A is pantropical, but also includes collections from Korea and China, and
40 J.-M. Moncalvoin that clade neotropical collections are distinct from Old World collections;Clade B is composed only of collections from the southern hemisphere; andClade C includes collections from Asia and the southern hemisphere. Matingdata produced by Yeh (1990) and Buchanan and Wilkie (1995) indicate atleast two intersterile groups of ‘G. australe’ in Taiwan and New Zealand,respectively. Mating data and ITS phylogeographic patterns suggest severalgenetically isolated groups (species) in the G. australe complex.Unclassified taxa‘G. APPLANATUM B’. A strongly supported clade (98% BS) composed ofnon-laccate collections from Europe and eastern North America remainsunclassified: it clusters at the base of Groups 2 and 3 in Fig. 2.3, but also nestsat the base of Group 1 in some analyses. Because this clade includesnon-laccate taxa from Europe, it is provisionally named as ‘G. applanatum B’. ITS data support the view that at least two non-laccate species existin Europe (Pegler and Young, 1973; Ryvarden and Gilbertson, 1993). Either‘G. applanatum A’ or ‘G. applanatum B’ represents the true G. applanatum. Thetwo clades can not be distinguished from basidiocarp characteristics. Also,since these ITS-based clades are so far composed only of northern temperatecollections (Table 2.2), it is possible that G. applanatum sensu stricto only occursin the temperate regions of the northern hemisphere.G. TSUNODAE, G. COLOSSUM AND OTHER TAXA. G. tsunodae, known only fromJapan (Imazeki, 1952) and China (Zhao, 1989), and G. colossum, a pantropicalspecies (Ryvarden and Johansen, 1980), remain unclassified. They are on longbranches in ITS phylogenies, generally at the base of the trees, and both mightwarrant segregation into separate genera as proposed by Imazeki (1939) andMurrill (1905b). Several unidentified taxa also remain unclassified: forinstance, a non-laccate species collected in French Guyana and Puerto Rico,that is easily recognizable from the cinnamon colour of its context, and laccatecollections from Zimbabwe and Vietnam, with a reddish-brown to blackishpileus and dark-brown context.BiogeographyThe number of known Ganoderma species can be estimated at about 60–80laccate and 10–30 non-laccate species (Table 2.1), and it is likely that newtaxa are yet to be discovered in poorly studied tropical regions. These numbersare based on a literature survey, examination of type specimens, numerousfield collections in various regions of the world, molecular phylogenetic dataand, in some cases, mating data. On a similar basis, it can be estimated that thecurrent sampling of ITS sequences encompasses at least 80% of all known taxafrom temperate regions, about half of the taxa from South-East and eastern
Systematics of Ganoderma 41Asia (it would seem that the number of species described from China by Zhaoand his collaborators (Zhao, 1989) has been overestimated), and 20–40% ofneotropical taxa. Molecular data from African material is almost entirelylacking. Based on these sampling estimates and the ITS phylogeny summarizedin Fig. 2.3 and Table 2.2, it appears that Ganoderma taxa repeatedly showsimilar patterns of geographic distribution, between and/or within clades:e.g. disjunction between temperate and tropical taxa, disjunction between Old(Europe, Asia, Africa) and New (the Americas) Worlds, a link between south-ern hemisphere taxa (South Africa, Argentine, Chile, New Zealand, PapuaNew Guinea and Australia), and connection between the more tropical regionsof the southern hemisphere (northern Australia and Papua New Guinea) andSouth-East Asia. Current ITS data indicate the existence of 5–7 species in Europe and 7–8in North America; these estimates are in agreement with the more recenttraditional floras for these regions (Gilberston and Ryvarden, 1986; Ryvardenand Gilbertson, 1993), although there is still some disagreement between ITSand morphological data in circumscribing and naming taxa. ITS phylogenyidentifies at least 12 taxa in temperate and subtropical Asia (China, Korea,Japan and Taiwan), but more species probably exist in this area. Withinundersampled and species-rich regions, Table 2.2 indicates the presence of atleast 18 ITS-based taxa in tropical Asia, and eight in the Neotropics. Taxa from Africa remain poorly sampled in molecular studies. Theunidentified taxa from South Africa and Zimbabwe that were included inthis work are diverse, and either nest in isolated positions or cluster withEuropean or Asian strains. A high level of taxonomic diversity (and perhapsalso endemism) is expected in Africa, because several well-characterizedspecies have not been reported outside that continent, e.g. G. alluaudii(Ryvarden, 1983), G. chonoides (Steyaert, 1962), G. sculpturatum (Ryvardenand Johansen, 1980), G. hildebrandii (Moncalvo and Ryvarden, 1995), etc.Host relationshipsHost specificity has been used to circumscribe Ganoderma taxa. In the northerntemperate regions G. valesiacum, G. carnosum, G. tsugae and G. oregonense havebeen distinguished from G. lucidum, mainly because they are all believed tobe restricted to conifers, as discussed above. All these taxa belong to clade 1.1(the G. lucidum complex sensu stricto, Table 2.2). However, before a conclusioncan be reached about host specificity on conifers, there is still need for abetter understanding of species boundaries in clade 1.1; collections fromconifers at higher altitudes in tropical or subtropical regions should alsobe examined. Steyaert (1967) was the first to extensively study collections from palms.He reported five laccate and one non-laccate species:
42 J.-M. Moncalvo• G. zonatum, in America and Africa, mostly on palms but also found on Eucalyptus;• G. miniatotinctum, in South-East Asia and Solomon Islands, found only on palms;• G. boninense, from Sri Lanka to the Pacific islands and Japan to Australia, mostly on palms but also found on Casuarina;• G. cupreum, paleotropical, on both palms and woody dicots;• G. xylonoides, restricted to Africa, on both palms and woody dicots; and• G. tornatum, in Asia and some Pacific islands, only on palms.The ITS phylogeny also distinguishes at least five laccate taxa on palms(Table 2.2), but these differ slightly from those described in Steyaert (1967)with respect to their geographic distribution and host specificity. Table 2.2 alsoindicates the presence of 1–3 non-laccate species growing on palms, but againthese results differ slightly from Steyaert’s (1967) concerning the geographicdistribution and host specificity. The ITS phylogeny also strongly suggests asingle origin (monophyly) for four out of the five laccate taxa growing onpalms (Table 2.2).ConclusionsThe data presented here show that ITS-based clades are generally consistentwith morphology or geography. Species boundaries within ITS clades still needto be addressed with mating studies, multigene phylogenies, or both. Typespecimens must be studied where available before naming ITS clades in theLinnean system of classification. However, given the difficulties of taxonomicidentification of Ganoderma collections using traditional methods, the ease andreducing costs of PCR amplification and direct sequencing techniques, and therapid expansion of molecular databases for a broad array of fungi, molecularmethods might become the easiest way to identify Ganoderma and otherproblematic fungal strains. This is particularly appealing for the preventivecare of woody plant crops, because vegetative mycelia extracted from woodcould be identified quickly using molecular techniques. Sequence data used inthis study will be made available in both GenBank and the Internet addresshttp://www.botany.duke.edu/fungi/ Construction of a web site on Ganoderma systematics is also in progress,and will be found at the same address.AcknowledgementsI am grateful to the Department of Botany at Duke University for financialsupport through a grant from the A.W. Mellon Foundation. The followingpersons provided strains for this study: Cony Decock, Le Xuan Tham, FaridahAbdullah, Carmel Pilotti, Alexandra Gottlieb, Armando Ruiz Boyer, Monica
Systematics of Ganoderma 43Elliott, Brendan Smith, Dong-Suk Park, C.L. Bong, Paul Kirk, Tom Harrington,Maggie Whitson, Anne Pringle, and Rytas Vilgalys. Thanks to Chiquita and BillCulbertson and Jim Johnson for comments on an early draft of the manuscript.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) Ganoderma meredithae, a new species on pines in the southeastern United States. Mycotaxon 31, 251–257.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.Bazzalo, M.E. and Wright, J.E. (1982) Survey of the Argentine species of the Ganoderma lucidum complex. Mycotaxon 16, 293–325.Bremer, K. (1994) Branch support and tree stability. Cladistics 10, 295–304.Buchanan, P.K. and Wilkie, J.P. (1995) Taxonomy of New Zealand Ganoderma: two non-laccate species. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. Proceedings of Con- tributed Symposia 59A,B, Fifth International Mycological Congress, Vancouver, 14–21 August 1994, pp. 7–17.Bull, J.J., Huelsenbeck, J.P., Cunningham, C.W., Swofford, D.L. and Waddell, P.J. (1993) Partitioning and combining data in phylogenetic analysis. Systematic Biology 42, 384–397.Corner, E.J.H. (1983) Ad Polyporaceas I. Amauroderma and Ganoderma. Beheifte zur Nova Hedwegia 75, 1–182.Donk, M.A. (1964) A conspectus of the families of Aphyllophorales. Persoonia 3, 199–324.Doyle, J.J. (1992) Gene trees and species trees: molecular systematics as one-character taxonomy. Systematic Botany 17, 144–163.Doyle, J.J. (1997) Trees within trees: genes and species, molecules and phylogeny. Systematic Biology 46, 537–553.Farris, J.S., Källersjö, M., Kluge, A.G. and Bult, C. (1994) Testing significance of incongruence. Cladistics 10, 315–319.Farris, J.S., Albert, V.A., Källersjö, M., Lipscomb, D. and Kluge, A.G. (1996) Parsimony jackknifing outperforms neighbor-joining. Cladistics 12, 99–124.Felsenstein, J. (1978) Cases in which parsimony and compatibility will be positively misleading. Systematic Zoology 27, 401–410.Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.Furtado, J.S. (1981) Taxonomy of Amauroderma (Basidiomycetes, Polyporaceae). Memoirs of the New York Botanical Garden 34, 1–109.Gilbertson, R.L. and Ryvarden, L. (1986) North American polypores. Part 1. Fungiflora, Oslo.Gottlieb, A.M. and Wright, J.E. (1999) Taxonomy of Ganoderma from southern South America: subgenus Ganoderma. Mycological Research 103, 661–673.
44 J.-M. MoncalvoGottlieb, A.M., Saidman, B.O. and Wright, J.E. (1995) Characterization of six iso- enzymatic systems in Argentine representatives of two groups of Ganoderma. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. Proceedings of Contributed Symposia 59A,B, Fifth International Mycological Congress, Vancouver, 14–21 August 1994, pp. 25–29.Graybeal, A. (1998) Is it better to add taxa or characters to a difficult phylogenetic problem? Systematic Biology 47, 9–17.Hennig, W. (1966) Phylogenetic systematics. University of Illinois Press, Urbana.Hibbett, D.S. and Donoghue, M.J. (1995) Progress toward a phylogenetic classification of the Polyporaceae through parsimony analyses of mitochondrial DNA sequences. Canadian Journal of Botany 73, s853–s861.Hibbett, D.S., Pine, E.M., Langer, E., Langer, G. and Donoghue, M.J. (1997) Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences. Proceedings of the National Academy of Sciences of the United States of America 94, 12002–12006.Hillis, D.M. (1996) Inferring complex phylogenies. Nature 383, 130–131.Hillis, D.M. (1998) Taxonomic sampling, phylogenetic accuracy, and investigator bias. Systematic Biology 47, 3–8.Hseu, R.S. (1990) An identification system for cultures of Ganoderma species. PhD thesis, National Taiwan University, Taipei, Taiwan (in Chinese).Imazeki, R. (1939) Studies on Ganoderma of Nippon. Bulletin of the Tokyo Science Museum 1, 29–52 (in Japanese).Imazeki, R. (1952) A contribution to the fungus flora of Dutch New Guinea. Bulletin of the Governments Forestry Experimental Station Tokyo 57, 87–128.Karsten, P. (1889) Bidrag till Kaennedan av Finlands Natur och Folk 48, 333.Kotlaba, F. and Pouzar, Z. (1993) Taxonomic and nomenclatural notes on Trametes cervina and Ganoderma atkinsonii. Ceska Mykologie 37, 49–51.Lecointre, G., Philippe, H., Van Le, H.L. and Le Guyader, H. (1993) Species sampling has a major impact on phylogenetic inference. Molecular Phylogenetics and Evolution 2, 205–224.Lloyd, C.G. (1898–1925) Mycological Writings. Vols 1–7. Cincinnati, Ohio.Maddison, W.P. (1997) Gene trees in species trees. Systematic Biology 46, 523–536.Moncalvo, J.M. and Ryvarden, L. (1995) Ganoderma hildebrandii: a forgotten species. Mycotaxon 56, 175–180.Moncalvo, J.M. and Ryvarden, L. (1997) A Nomenclatural Study of the Ganodermataceae Donk. Synopsis Fungorum 11, Fungiflora, Oslo.Moncalvo, J.M., Wang, H.H. and Hseu, R.S. (1995a) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25s ribosomal DNA sequences. Mycologia 87, 223–238.Moncalvo, J.M., Wang, H.F. and Hseu, R.S. (1995b) Gene phylogeny of the Ganoderma lucidum complex. Comparison with traditional taxonomic characters. Mycological Research 99, 1489–1499.Moncalvo, J.M., Wang, H.F., Wang, H.H. and Hseu, R.S. (1995c) The use of rDNA nucleotide sequence data for species identification and phylogeny in the Ganodermataceae. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. Proceedings of Con- tributed Symposia 59A,B, Fifth International Mycological Congress, Vancouver, 14–21 August 1994, pp. 31–44.
Systematics of Ganoderma 45Moncalvo, J.M., Lutzoni, F.M., Rehner, S.A., Johnson, J. and Vilgalys, R. (2000) Phylo- genetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Systematic Biology, 49(2), 278–305.Murrill, W.A. (1905a) The Polyporaceae of North America XI. A synopsis of the brown pileate species. Bulletin of the Torrey Botanical Club 32, 353–371.Murrill, W.A. (1905b) Tomophagus for Dendrophagus. Torreya 5, 197.Pegler, D.N. and Young, T.W.K. (1973) Basidiospore form in the British species of Ganoderma Karst. Kew Bulletin 28, 351–364.Peng, J.T. (1990) Identification and Culture Conservation of the Wild Ganoderma species in Taiwan. Publication of Taiwan Agricultural Research Institute. Wufeng, Taichung (in Chinese).Poe, S. (1998) The effect of taxonomic sampling on accuracy of phylogeny estimation: test case of a known phylogeny. Molecular Biology and Evolution 15, 1086–1090.Ryvarden, L. (1983) Type studies in the Polyporaceae. 14. Species described by N. Patouillard, either alone or with other mycologists. Occasional papers of the Farlow Herbarium 18, 1–39.Ryvarden, L. (1991) Genera of Polypores. Nomenclature and Taxonomy. Synopsis Fungorum 5, Fungiflora, Oslo.Ryvarden, L. (1995) Can we trust morphology in Ganoderma? In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. Proceedings of Contributed Symposia 59A,B, Fifth International Mycological Congress, Vancouver, 14–21 August 1994, pp. 19–24.Ryvarden, L. and Johansen, I. (1980) A Preliminary Polypore Flora of East Africa. Fungiflora, Oslo.Ryvarden, L. and Gilbertson, R.L. (1993) European Polypores. Part. 1. Synopsis Fungorum 6, Fungilora, Oslo.Sanderson, M.J. and Donoghue, M.J. (1989) Patterns of variation in levels of homoplasy. Evolution 43, 1781–1795.Steyaert, R.L. (1962) Genus Ganoderma (Polyporaceae). Taxa Nova II. Bulletin du Jardin Botanique de Bruxelles 32, 89–104.Steyaert, R.L. (1967) Les Ganoderma palmicoles. Bulletin du Jardin Botanique National de Belgique 37, 465–492.Steyaert, R.L. (1972) Species of Ganoderma and related genera mainly of the Bogor and Leiden herbaria. Persoonia 7, 55–118.Steyaert, R.L. (1980) Study of some Ganoderma species. Bulletin du Jardin Botanique National de Belgique 50, 135–186.Swofford, D.L. (1998) PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods), beta version 4.0d64. Sinauer, Sunderland, Massachusetts.Tham, L.X. (1998) A phylogenetic hypothesis of the Ganodermataceae based on a possible mode of basidiospore evolution. Mycotaxon 60, 1–12.Thorn et al. (2000) Mycologia.Wang, B.C. and Hua, J. (1991) A Cultural Atlas of some Ganoderma Cultures. Publication of the Food Industrial Research Development Institute. Hsinchu, Taiwan.Wang, H.-F. (1996) Studies of manganese-superoxide dismutase gene of Ganoderma. MSc thesis, National Taiwan University, Taipei (in Chinese).Yeh, X.Y. (1990) Taxonomic study of Ganoderma australe complex in Taiwan. PhD thesis, National Taiwan University, Taipei (in Chinese).Zhao, J.D. (1989) The Ganodermataceae in China. Bibliotheca Mycologica 132, 1–176.
50 D. Ariffin et al.Principe, Tanzania, Zimbabwe and the Republic of Congo in Africa; Hondurasin Central America, and Papua New Guinea in Oceania (Turner, 1981). Morerecently, the disease was reported in Colombia (Nieto, 1995) and Thailand(Tummakate and Likhitekaraj, 1998). The disease was first described in 1915 in the Republic of Congo, WestAfrica (Wakefield, 1920). Thompson (1931) detected the disease infecting oilpalms of over 25 years in Malaysia but because this attack was on old palmsdue for replanting, BSR was considered not to be economically important(Turner, 1981). However, towards the later years of the 1960s, when oil palmbegan to assume prominence as a plantation crop, BSR incidence was on theincrease and much younger palms (10–15 years old) were infected (Turner,1981). Recently, Ganoderma has been found to infect oil palms as early as12–24 months after planting, with increased incidence on 4–5-year-oldpalms, particularly in replanted areas (Singh, 1991) or areas underplantedwith coconut palms (Ariffin et al., 1996). The disease had been reported most often in coastal marine clay, particu-larly in areas planted with oil palm following coconut (Navaratnam, 1964).The fungus, being saprophytic to coconut, remains in the stumps and trunks ofcoconut left in the soil and infects the oil palm on replanting. High incidence ofBSR disease was recorded on oil palm planted in coastal soil in west PeninsularMalaysia (Khairudin, 1990a). In peat soils, which were at one time thoughtto be non-conducive to BSR disease (Turner, 1981), serious incidences of thedisease have been reported more recently (Ariffin et al., 1989c; Rao, 1990).Ariffin et al. (1989c) cautioned that Ganoderma poses a threat to oil palmplanting in peat soil, where high incidences of the disease have been observedat a relatively young age, irrespective of previous cropping history (Table 3.1). The incidence of BSR disease in inland soils in Malaysia remains relativelylow and seems to be confined only to waterlogged areas (Khairudin, 1990a).However, it was recently reported that serious BSR disease incidence can occuron oil palms growing in lateritic soils which were previously almost disease free(Benjamin and Chee, 1995). In Indonesia, BSR incidence is low on 7-year-old plantations butincreases gradually to about 40% when the palms reach 12 years of age.In the fourth-generation replants, the disease is observed much earlier,on 1–2-year-old palms (Hakim et al., 1998). Also, in Indonesia, unlike inTable 3.1. Incidence of basal stem rot (BSR) disease in peat soil (from Ariffinet al., 1989c).Case study Oil palm age (years) Previous crops Incidence of BSR (%) 1 10 Coconut and rubber 25.0 2 11 Rubber 53.0 3 12 Pineapple 37.2
Status of Ganoderma in Oil Palm 51Malaysia, BSR incidence is very high in replants in both inland podsols andcoastal clay soils (Hasan and Turner, 1998). In West Africa, BSR is widespread in wild groves and is the common causeof death of wild palms (Robertson et al., 1968). Most of the affected palms areover 25 years old, but palms 10–15 years old are also infected. With the activeconversion of wild groves to plantations in eastern Nigeria, the incidence ofBSR is expected to be on the increase (Oruade-dimaro et al., 1994). The incidence of BSR is comparatively low in Honduras where the diseasewas detected in palms more than 12 years old (Chinchilla and Richardson,1987). BSR is also beginning to occur in Colombia (Nieto, 1995) and PapuaNew Guinea (Sanderson and Pilotti, 1997a, b).Disease SymptomsIn young palms, the external symptoms of BSR normally comprise a one-sidedyellowing, or mottling of the lower fronds, followed by necrosis (Singh,1991). The newly unfolded leaves are shorter than normal and chlorotic and,additionally, the tips may be necrotic. As the disease progresses, palms maytake on an overall pale appearance, with retarded growth and the spear leavesremaining unopened. Similar symptoms are observed in mature palms, with multiple unopenedspear leaves and a generally pale leaf canopy. Affected leaves die, necrosisbeginning in the oldest leaves and extending progressively upwards throughthe crown. Dead, desiccated fronds droop at the point of attachment tothe trunk or fracture at some point along the rachis, and hang down toform a skirt of dead leaves. Often, when foliar symptoms are observed, it isusually found that at least one-half of the basal stem tissue has been killed bythe fungus. Infected young oil palms normally die within 6–24 months afterthe first appearance of symptoms but mature palms can take up to 2–3 yearsto die. Tissues of an infected stem base give a characteristic dry rot. In a cross-section of an affected trunk, the lesions appear as light-brown areas of rottingtissues, marked by darker irregular zonations with an outer edge of anirregular yellow zone. A yellow zone is found between the lesion edge and thehealthy tissues. Turner (1981) termed the darker zones as ‘reaction zones’ andspeculated that the yellow zones were the result of some defence mechanism ofthe palm to infection. These narrow darker zones were termed ‘black lines’ byAriffin et al. (1989a), and embedded within the lines were masses of swollenhyphal cells which appear to be resting structures. Within the light-browndiseased tissues, small cavities of white fungal mycelium were seen. Oil palmextensively decayed by Ganoderma may fracture at the base and the palmcollapses, leaving diseased bole tissues in the ground. Subsequently, numerousGanoderma basidiomata are produced, especially during the rainy season. If thepalm remains standing, the trunk may become hollow.
52 D. Ariffin et al. Roots of affected palms are very friable and their internal tissues becomevery dry and powdery. The cortical tissue is brown and disintegrates easilyand the stele becomes black in colour. In older roots, the fungus may be presentas a whitish, mat-like layer on the inner surface of the exodermis (Singh,1991). Ganoderma basidiomata or sporophores may or may not develop beforefoliar symptoms appear. Basidiomata may develop at the stem base of thetrunk, leaf base or occasionally on infected roots close to the palm, and it isthe appearance of these that is most diagnostic of the disease. The timing ofbasidiomata appearance depends on extension of the internal rotting to thestem periphery. The basidiomata initially appear as small, white buttons offungal tissues which develop rapidly into the familiar bracket-shaped maturebasidiomata, varying in shape, size and colour. The upper surface can be lightto dark brown, with a light margin and a shiny lacquered finish. The undersurface is whitish in colour and has numerous minute pores. Frequently, manybasidiomata are formed close together, with overlapping and fusion to formlarge, compound structures. The location of the basidiomata provides a roughguide to the position of the diseased area inside the palm. When the palm dies,rapid colonization of the whole trunk can be seen through the appearance ofbasidiomata along its entire length.Causal OrganismsIn West Africa, the pathogen was originally identified as G. lucidum Karst(Wakefield, 1920), whereas in Nigeria, four species of Ganoderma have beenidentified as causal agents, namely G. zonatum Muril, G. encidum, G. colossusand G. applanatum (Pers. ex. S.F. Gray) (NIFOR, 1978). In Malaysia, it was alsooriginally identified as G. lucidum by Thompson (1931), a species commonlyfound in temperate regions that has been associated with diseases of a numberof hosts, such as coconut and Areca and also grapevines. Turner (1981) listed15 species of Ganoderma that have been recorded from different parts of theworld as likely pathogens to be associated with BSR disease, and he consideredthat a single species was unlikely to be the sole cause of the disease in any par-ticular area. Among them, seven species of Ganoderma, namely G. applanatum(Pers.) Pat., G. boninense, G. chalceum (Cooke) Steyaert, G. lucidum (W. curt. et.fr.) Karst, G. miniatocinctum Steyaert, G. pseudoferreum (wakef.) Overh. andSteinmann, and G. tornatum (Pers) Bres. were reported from PeninsularMalaysia. Ho and Nawawi (1985) concluded that all Ganoderma isolatesfrom diseased oil palm from various locations in Peninsular Malaysia wereall the same species, G. boninense. These were based on the morphology ofbasidiomata collected from oil-palm fields ranging from 5 to 40 years of age.Ariffin et al. (1989c) suggested that other species may be involved andKhairudin (1990a) concluded that two species were present, namely G.boninense and G. tornatum. More recently, Idris (1999) classified Ganoderma in
Status of Ganoderma in Oil Palm 53oil palm in Malaysia into types A, B and C. Type A is the most aggressive, type Bis less aggressive, while type C is saprophytic.Economic ImportanceField observations in Malaysia show that in replantings from jungle or rubber,BSR begins to manifest when the palms are about 10–12 years old (Singh,1991). The initial incidence is low, in the region of 1–2% of the stand. By thetime the palms reach 25 years and are ready for replanting, the incidencecould be as high as 25% (Singh, 1991). In replanting from coconut, the diseaseappears much earlier, with sporadic cases of BSR as early as 1–2 years afterplanting. By the twelfth year, the incidence is more than 15%, increasing to60% 4 years later (Singh, 1991). In replanting from oil palm, the incidence ofBSR can reach 22% by the tenth year, increasing to 40% 4 years later (Singh,1991). High BSR incidence was also recorded by Khairudin (1990b) in an oilpalm to oil palm replant by underplanting. In this case, the incidence reached33% at 15 years. A BSR incidence of 25% was recorded on 10-year-old palmsplanted under coconut (Ariffin et al., 1996). Two years later the incidence hadincreased to 40%. Losses due to BSR can occur not only through the direct reduction inoil-palm numbers in the stand, but also through a reduction in the numberand weight of fruit bunches from standing diseased palms and those withsubclinical infections (Turner, 1981). Yield compensation by healthy neigh-bouring palms is likely to occur and, according to Turner (1981), disease levelsof 10–20% have little effect on yield. In a study to quantify yield losses,comparison of fresh fruit bunch (FFB) production in two blocks – one with ahigh incidence of BSR and the other with a low incidence – is presentedin Table 3.2, as reported by Singh (1991). The fields selected were withinTable 3.2. Basal stem rot (BSR) incidence and fresh fruit bunch (FFB) yield (fromSingh, 1991). Low BSR incidence blocka High BSR incidence blockbYears from BSR incidence FFB yield BSR incidence FFB yield planting (%) (t ha−1) (%) (t ha−1) 11 3.1 23.1 31.4 17.0 12 4.1 24.5 39.6 15.2 13 5.6 25.5 49.1 17.6 14 7.8 26.6 60.3 16.9 15 10.9 23.8 67.3 13.2aPlanted 1975; previous crop: rubber; soil type: Selangor/Briah Assoc.bPlanted 1975; previous crop: oil palm; soil type: Selangor series.
54 D. Ariffin et al.the same estate, of the same age and on similar soils. It was shown that FFBproduction was adversely affected by the disease incidence.EpidemiologyMycelium contactIt has been generally accepted that natural infection with Ganoderma in oilpalm occurs as a result of contact between healthy roots and diseased tissuesleft buried in the soil (Turner, 1965c). Infection by Ganoderma is also believedto occur through wounded tissues or dead roots. The fungus then growsalong the infected root and eventually reaches the bole of the palm trunk.Histopathological investigations of roots naturally diseased by infection withGanoderma reveal that the fungus also invades the vessels (Ariffin et al., 1991).The initial infection of Ganoderma within the root is confined to tissues inner tothe endodermis. The fungus is not restricted to any one particular tissue type atthe advanced stages of pathogenesis; fungal hyphae could be clearly detectedin the xylem, phloem, pith and parenchymal cells. Infection of the stemeventually led to the formation of ‘black lines’ within the infected tissues(Ariffin et al., 1989a). The presence of these lines could be observed with thenaked eye. On microscopic examination with suitable staining techniques itwas observed that Ganoderma hyphae transform into thick-walled, swollenstructures embedded within the black lines. It was postulated that thesemight be resting structures which could possibly play an important role in thelong-term survival of the pathogen in soil. In this form, Ganoderma might havedeveloped a resistant barrier against other soil microorganisms in whichnormal free hyphae would have easily been replaced.Ganoderma basidiosporesVegetative compatibility studies made by Miller (1995) and Ariffin et al.(1996), indicated that basidiomata collected from the same field, or fromwithin the same area of oil-palm field, might not have originated from the samesource of inoculum, implying that root-to-root spread or mycelial growthmight not be the sole method of spread of BSR. Currently, the role of Ganodermabasidiospores in disease initiation and spread of infection is unclear. Althoughhuge numbers of basidiospores of Ganoderma are released from basidiomata inthe oil-palm field (Ho and Nawawi, 1986), the majority of oil palms remainuninfected, indicating that basidiospores either may not be able to initiate aBSR infection or require very specific conditions to establish infection. Studies based on the artificial inoculation with basidiospores andinoculum size suggest that basidiospores have inadequate inoculum potentialfor direct infection of a living oil palm (Turner, 1981). Their function in disease
Status of Ganoderma in Oil Palm 55development seems to be the colonization of suitable substrates, particularlycut stumps of trunks of trees or palms left to rot in the field, which may becomeinfection foci. Inoculation of cut young leaf bases (Turner, 1965a) and youngoil-palm seedlings with spores failed to produce any infection (Ramasamy,1972; PORIM, 1988). Sharples (1936) believed that spores do not play animportant role in the spread of the disease. However, Thompson (1931) wasof the opinion that spores are important in initiating the disease in first-generation oil palms on cleared virgin jungle areas. Basidiospores, whichmay either be wind-borne or insect-transmitted, would first have to colonizesuitable substrates, e.g. dead coconut or oil-palm stump, and then they couldgerminate readily and spread throughout the whole stump. It was suggestedthat spores may enter through beetle holes, caused by Oryctes beetle (Turner,1981). Caterpillar larvae of Sufetula spp. may also be important in spreadingspores of Ganoderma (Genty et al., 1976). However, no conclusive evidence hasbeen presented linking insects and BSR incidence and development.Predisposition Factors Associated with BSR DiseaseUntil recently, predisposition factors that influence the development of BSRdisease have been the subject of speculation based on circumstantial evidence.A number of factors – age of palms, previous crops, types of soils, nutrientstatus and technique of replanting – have been reported to influence BSRdisease development in the field. Infection by the pathogen has generally beenthought to occur through a weakening of the oil palm so that it becomes pre-disposed to infection. However, with information now available, predispositionfactors can be examined critically.Age of oil palmsBSR was first reported to be a disease of old, senescing oil palms, i.e. the palmsaffected were those over 25 years from planting, and this was thought to be dueto a senescence factor that broke down the immunity barrier (Turner, 1981).However, with time this trend had changed, with much younger oil palmsbecoming infected (Singh, 1991; Khairudin, 1993). As reported by Turner(1981), the age at which a palm becomes infected will depend on: (i) the rate ofcolonization of the tissues of the previous stand; (ii) proximity of the colonizedtissues to the oil palm; (iii) time taken for roots to make contact with the tissuesand become infected; and (iv) growth of the fungus along the root and its estab-lishment within the bole tissues. In general, BSR incidence begins to appearfrom the sixth year after planting, and then increases rapidly from the eleventhyear onwards (Table 3.3). It was suggested that, in the field, the opportunityfor roots to come into contact with disease inoculum, and subsequent slowdisease development, are more critical than age factors (Khairudin, 1993).
56 D. Ariffin et al.Table 3.3. Incidence of basal stem rot (BSR) disease in relation to oil-palm age infour Golden Hope Plantation Estates in Peninsular Malaysia (from Khairudin, 1993). BSR incidence (%)Golden Hope Plantation 0–5 6–10 11–15 16–20 > 20Estates yearsa years years years yearsMelentang, Bagan Datoh 0.7 0.4 4.6 44.6 43.3Chersonese, Sg. Krian 0.0 14.0 12.4 25.2 35.8Dusun Durian, Banting 0.0 2.1 12.8 24.1 24.9West, Carey Island 0.0 0.4 2.5 9.7 18.9aYears after planting.Previous cropsThe relationship between BSR disease of oil palm and the types of formercrops has been recognized (Turner, 1965a). Severe outbreaks of BSR diseaseoccurred in areas when oil palm followed coconut, especially where the stumpshad been retained in the ground. With planting following coconut, Ganodermainfection may become apparent as early as 12–24 months from planting, butmore usually when palms are 4–5 years old (Singh, 1991). Thereafter, theincidence can reach 40–50% by the time the palms are 15 years old (Table3.4). A similar situation was also reported where oil palm was replanted fromoil palm – a high incidence of BSR could be observed after 15 years of planting. A contrasting situation was apparently found in stands planted fromjungle or rubber, with a low disease incidence and losses only beginning tooccur after 10–12 years (Turner, 1965b). However, later reports indicatedthat the previous crop did not exclusively preclude high incidences ofBSR, which have also occurred in ex-rubber plantings (Ariffin et al., 1989c)and ex-pineapple plantings (Ariffin et al., 1989c; Rao, 1990). A more recentstudy conducted on four estates covering about 8000 ha showed that there isno definite relationship between former crop and BSR incidence (Khairudin,1993) and the presence of an adequate inoculum source could be a moreimportant prerequisite to high disease level.Types of soilA high incidence of BSR disease has been frequently reported to be prevalent incoastal areas (Navaratnam, 1964; Turner, 1965d). Khairudin (1990a) alsoreported that most of the soil series found on coastal areas in the west of Penin-sular Malaysia are susceptible to the threat of BSR, especially Kangkong,Bernam, Sedu, Sogomana, Parit Botak, Jawa, Merbok, Briah, Tangkang,Sabrang, Selangor, Carey and Linau. The fact that the disease seemed toremain confined to the coastal areas, indicated that the nature of soil and its
Status of Ganoderma in Oil Palm 57Table 3.4. Incidence of basal stem rot (BSR) disease in oil palm in relation toprevious crops (from Singh, 1991). BSR incidence (%)Year of planting From forest tree From rubber From oil palms From coconut 5 – – 0.4 0.2 6 – – 0.7 0.4 7 – – 1.8 0.8 8 0.1 – 3.3 1.8 9 0.6 – 5.4 2.8 10 1.0 – 9.1 6.2 11 1.2 1.6 15.3 11.5 12 2.1 2.2 23.8 16.7 13 3.8 3.0 30.6 30.7 14 6.7 3.6 36.4 41.5 15 6.7 5.7 42.4 51.1 16 10.7 8.3 – 61.2 17 13.8 12.5 – – 18 18.0 15.3 – – 19 23.2 – – – 20 31.0 – – – 21 33.1 – – –water relations may have a bearing on disease development. These soils aremainly clays, silty clays or clay loams with poor internal drainage and with ahigh water retention capacity. However, more recent reports indicate a greaterincidence of BSR disease on oil palms planted on inland soils, especiallyHolyrood, Sungei Buloh, Rasau and Bungor series (Khairudin, 1990a); BatuAnam/Durian series and Munchong series (Benjamin and Chee, 1995); peatsoil (Ariffin et al., 1989c; Rao, 1990) and lateritic soil, especially Malacca series(Benjamin and Chee, 1995). Increasing reports of BSR disease in different soiltypes, including inland soils, requires further investigation of the role of soiltype in determining the level of disease in the oil-palm fields.Nutrient statusSoil nutrition can influence disease development, but the effect appears to berelated to the nature of the soil and its chemical properties. Fertilizer trials con-ducted on the silty clay mixed riverine/marine alluvium of the Briah-Selangorassociation (Sulfic tropaquept) showed that rock phosphate and muriate ofpotash (KCl) significantly increased disease incidence, whereas urea had areduced effect (Singh, 1991). In another trial on a recent marine alluvium ofthe Bernam series (Typic tropaquept), Singh (1991) reported that muriateof potash significantly reduced disease incidence, whereas urea and rock
58 D. Ariffin et al.phosphate had a slight promotive effect. In Indonesia, high sodium content(Dell, 1955) and low nitrogen levels (Akbar et al., 1971) have both beenassociated with raised disease levels, but both high (Dell, 1955) and lowmagnesium contents (Akbar et al., 1971) have been linked with increasedincidence of disease, so the situation is unclear. In one investigation of themajor elements, nitrogen (N), potassium (P) and phosphorus (K) were allsignificantly higher in healthy tissues, but levels of magnesium (Mg) werehigher in diseased palms, and significant differences also occurred in micro-nutrients, especially boron (B) and copper (Cu) (Turner and Chin, 1968).Chemical analysis of the various elements in roots of oil palm collected frominland and coastal soils did not show any marked differences in the levels ofelements, but oil-palm roots collected from inland soil were found to containhigh levels of phosphate (P), zinc (Zn) and iron (Fe) (Singh, 1991).Planting techniquesThe incidence of BSR disease has been observed under a range of replantingtechniques. Turner (1965a) reported that there is a close relationship betweendisease incidence and the replanting techniques adopted. A trial carried out byGolden Hope Plantations Berhad, comparing the effect of different replantingtechniques on the incidence of BSR, showed that underplanting wouldeventually lead to a high disease incidence (from 27.3% in the previousstand to 33% in the replanted stand after 15 years), whereas if clean clearing ofprevious oil-palm stands was employed, subsequent disease levels were low(from 27.3% to 14.0%), and windrowing slightly increased the risk of BSRdisease incidence (from 27.3% to 17.6%) (Table 3.5) (Khairudin 1990b).Early Detection of BSRDiagnosis of Ganoderma infection in oil palm is based on the appearance ofmultiple spear leaves and the presence of basidiomata of the pathogen on thestem base, or leaf bases or primary roots close to the soil level, although theyare frequently only observed once disease is firmly established. Subclinicalinfections thus remain undetectable, and mycelial states in the soil and sur-rounding plant debris cannot be detected and identified. As one palm becomesinfected, it could transmit the disease through root contact with the immediateneighbouring palms (Turner, 1965a). Until now, no sufficiently satisfactorytechniques have been available to detect early infection of oil palm, althoughReddy and Ananthanarayanan (1984) reported that the fluorescent antibodytechniques could be used to detect G. lucidum in roots of betelnut. Furthermore,a polyclonal antibody has been developed to detect mycelium of Ganoderma inculture (Darmono et al., 1993), and has been used to detect Ganoderma in oil-palm fields (Darmono and Suharyanto, 1995). In the future these techniques
Status of Ganoderma in Oil Palm 59 Table 3.5. Incidence of basal stem rot (BSR) disease in relation to the three replanting techniques in oil palm at 15 years from field planting (from Khairudin, 1990b). Technique of replanting BSR incidence (%)* Clean clearing1 14.0a Windrowing2 17.6a Underplanting3 33.0b SE 1.9b LSD (P = 0.05) 6.5b *Values followed by the same letter were not significantly different at P = 0.05. 1Clean clearing involved poisoning of previous oil-palm stands, mechanical felling, cutting of stems into length, splitting of cut stems for drying, stacking, followed by burning. 2Windrowing, as clean clearing but oil-palm debris was stacked in the interrows without splitting for drying and burning. 3Underplanting involved poisoning of old oil palms, 18 months after planting of new stands and followed by mechanical felling, cutting of stems into length and stacking of old palms in the interrows.may be used for early detection of the disease (Darmono, this volume; Utomoand Niepold, this volume). However, detection of the incidence of BSR iscurrently carried out based on the external symptoms. Palm infection can onlybe confirmed when basidiomata of Ganoderma appear either at the stem base oron infected roots close to the palm; otherwise, their disease status is uncertain.To facilitate various studies on Ganoderma in oil palm, Ariffin and Idris(1991a) have developed the Ganoderma-selective medium (GSM), which couldselectively isolate the pathogen from any parts of infected tissues, directly fromthe field, with or without surface sterilization. With GSM and using a drillingtechnique it was possible to detect more oil palms that were infected withGanoderma but which appeared to have no external symptoms (Ariffin et al.,1993, 1996).ControlIt is fully realized that finding a solution to the BSR disease problem on oil palmis not going to be an easy task. It is therefore recommended that bothshort-term and long-term approaches be investigated in order to reducedamage on existing stands and to reduce incidence in replantings (Ariffin et al.,1989b). For short-term control of BSR in existing stands, the use of fungicidestogether with the technique of application needs to be investigated. For a morepermanent control, research strategy should concentrate on finding ways tohasten decay of oil-palm tissues during replanting in order to minimize theinoculum burden carried over in the subsequent planting (see Paterson et al.,
60 D. Ariffin et al.this volume). In addition, the production of oil-palm lines resistant toGanoderma must also be investigated. As methods for early detection ofinfection are only just being developed, control measures are currently onlyapplied to visibly diseased palms, with untreated, symptomless palmsremaining a potential source of infection.Cultural practicesA number of agronomic practices have been suggested to control BSR disease.Digging trenches around diseased palms to prevent mycelial spread of thepathogen to neighbouring healthy palms has been recommended as a controlmeasure (Wakefield, 1920), but trenches have not proved satisfactory(Turner, 1981) due to the fact that the trench depths were insufficient toprevent roots passing underneath, or that trenches were not maintained.Collecting basidiomata of Ganodema from diseased palms and painting themwith carbolineum to prevent spores dispersal was also recommended (Turner,1981), but this would be of no value if spores have no direct infective ability.Poor drainage, flooding, nutritional imbalances and deficiencies and heavyweed growth have been reported to be associated with increased BSR incidencein oil palm (Turner, 1981), but there is no hard evidence to support these fac-tors. A more recent approach of BSR control was the mounding of soil in com-bination with cultural, organic and inorganic and also chemical treatments.Lim et al. (1993) and Hasan and Turner (1994) showed that surgery followedby soil mounding around the base of mature diseased palms can bring aboutan increase in vigour and yield of oil palms. The treatment seems to be promis-ing for prolonging the economic life of Ganoderma-infected oil palms. Furtherstudies by Ho and Khairudin (1997) indicated that soil mounding with fumi-gant, and soil mounding alone were able to prolong productivity of oil palmsthrough the physical benefit of preventing the weakened boles from beingtoppled by the wind. However, this treatment did not prove to be curative.Land preparation at the time of replantingThe correct technique of land preparation at the time of oil-palm replantingis regarded as an important practice for controlling BSR disease. These con-trol strategies are based on the assumption that infection occurs by mycelialspread from root-to-root contact. Since tissues of the former stand of oil palmsor coconuts are thought to be the primary source of infection at replanting, dis-ease avoidance through sanitation is important. Any methods of disposal ofthe old stand involving destruction or reduction of the Ganoderma inoculumhad a beneficial effect on the subsequent planting (Khairudin, 1990b; Singh,1991). Three replanting techniques, namely clean clearing, underplantingand windrowing, have been practised throughout Malaysia. The effects of
Status of Ganoderma in Oil Palm 61these three replanting techniques on the incidence of BSR disease in oil palmare presented in Table 3.5 (Khairudin, 1990b). Although the clean-clearingtechnique gave lower disease incidence in replanted oil palm by comparisonwith other replanting techniques, it was later found that this techniquewas not entirely satisfactory in reducing disease incidence (Singh, 1991). Anincidence of BSR disease as high as 28–32% is not uncommon despite theadoption of this clean-clearing technique (Singh, 1991). In the absence of acomplete understanding of the long-term survival of Ganoderma in infectedtissues buried in soil, the rationale behind this recommendation remainsunclear. This technique does not take into consideration the functions ofsubterranean roots in disease epidemiology (Flood et al., this volume). It must be realized that clean clearing was initially advocated based on thefinding that a massive amount of inoculum, at least 734 cm3, is required toinitiate infection (Turner, 1981). Following this assumption, the clean-clearing technique was developed to destroy the boles and attached rootmasses, the major plant parts that harbour the pathogen. Little attention waspaid to the interconnecting roots left behind after this operation. The originalwisdom was that these roots, although infected, are too small to be infective.Further support for this view was provided by the observation that naturallyinfected root fragments had failed to cause infection when used as inoculumsources on nursery seedlings (Navaratnam and Chee, 1965). However, therole played by these roots in disease outbreaks began to be realized followingthe successful artificial inoculation of nursery seedlings. The fact that seedlingscan be infected readily using pure culture inoculum only slightly bigger thanthe average oil-palm primary root (Ariffin et al., 1995), suggests that underfavourable conditions the leftover roots can be infective. Also, field experimentation by Hasan and Turner (1998) proved that rootscan represent a small but significant inoculum source. These workers dividedthe interspace between two adjacent infected palms fields into three equalsectors separated by deep trenches. Bait oil-palm seedlings were plantedin each sector and also around the bases of BSR–infected palms. The resultsrevealed that only 4% of bait seedlings became infected after 2 years, and thesewere in the sectors closest to the diseased palms. Although this incidence wasmuch lower than the 69% infection of bait seedlings planted adjacent to maindisease sources, the results were convincing enough to conclude that infectedroot fragments can cause infection and, hence, disease outbreaks. Singh(1991) had also demonstrated that infection of some young palms wasinitiated by small bundles of diseased roots of the former stand buried close tothe palms. These findings suggest that leftover root fragments can play a veryimportant role in the outbreak of BSR, despite the practice of clean clearingduring replanting of second- and third-generation palms. That the rootfragments left in situ still have enough inoculum potential to cause diseaseis reflected in their ability to produce basidiomata of G. boninense, whichare sometimes seen on their cut ends. These roots, although detached fromthe boles, are still several metres long and should individually have enough
62 D. Ariffin et al.food reserves to ensure survival of the pathogen. Furthermore, the very natureof G. boninense being confined within the root ensures minimal interferencefrom other common antagonists present in the soil. Although infected rootsare brittle, with the stele easily detached from the cortex, the pathogen is alsopresent in the stele (Ariffin et al., 1991). The underplanting of coconut or oil palm with young oil palm, followed bypoisoning and felling of the old stand has been a common practice, especiallyon smallholder farms. When the coconut or oil-palm stump is left to rot inthe field, numerous basidiomata of Ganoderma are produced. As shown inTable 3.5, 15 years after replanting the highest incidence of BSR diseasewas recorded on the subsequent generation of oil palm in the underplantingtechnique (from 27.3% in the previous stand to 33.0%), whereby the percent-age incidence is twice as that in the clean-clearing practice (from 27.3% to14.0%). Khairudin (1990b) also observed that 93% of seedlings growingaround infected oil-palm stumps left in the field became infected within 18months. By contrast, only 7% of seedlings growing around sites that had beenexcavated to remove diseased stumps became infected. This clearly indicatesthe value of clean clearing and the hazard of underplanting, a practice longdiscouraged (Turner, 1981).Treatment by excisionExcision of diseased tissues as a form of treatment has been recommended(Turner, 1968), but with very mixed results. Infected tissues from lesions in theouter stem tissues of oil palm were excised, either with harvesting chisels(Turner, 1981) or mechanically, to excise diseased tissues from above andbelow soil level (Singh, 1991). After the lesions were excised, the cut surfacewas treated with a protectant chemical (e.g. coal tar or a mixture of coal tarand thiram). The age of oil palm is important when considering this method(Turner, 1981). It was reported to be more successful on palms above 12 yearsold, as the disease lesions are more superficial due to the harder stems of olderpalms (Singh, 1991). Excision frequently requires repetition, as infection oftenresurges if lesions are not completely removed.Fungicide treatmentDue to the severe disease incidence in existing stands of oil palms, immediateshort-term measures to control this disease must be investigated. The use ofsystemic fungicides, together with a correct technique of application, couldpossibly provide the answer to this problem. Control through the use of fungi-cides should not be limited to treating oil palms with confirmed cases ofGanoderma only, but also neighbouring oil palms that are in potential dangeror might have already been infected at subclinical level. The use of fungicides
Status of Ganoderma in Oil Palm 63to treat young oil palms not showing obvious signs of infection but which havebeen planted in an area with a history of a high incidence of Ganoderma alsoneeds evaluation as a preventive measure. Screening of fungicide activityagainst Ganoderma in vitro has shown that numerous fungicides were stronglyinhibitory towards Ganoderma growth (e.g. drazoxolone and cycloheximide(Ramasamy, 1972); triadimefon, triadimenol, methfuroxam, carboxin, carben-dazim, benomyl, biloxazol and cycloheximide (Jollands, 1983); hexaconazole,cyproconazole and triadimenol (Khairudin, 1990a); penconazole, tridemorphand triadimenol (Lim et al., 1990)). Organic mercury formulations have beenreported to be strongly inhibitory to Ganoderma in the field, but becameunacceptable for commercial use due to the residue problem (Turner, 1981). Attempts to control BSR in the field by the use of systemic fungicideshave been made by various workers (e.g. Jollands, 1983; Khairudin, 1990a;PORIM, 1997). The results of these studies are inconclusive, although somesystemic fungicides seem to be promising. The methods of fungicide applica-tion include soil drenching, trunk injection, and a combination of soil drench-ing and trunk injection. It was found that trunk injection is superior to soildrenching. Results of the trunk injection of fungicides into BSR-infected oilpalms showed that a carboxin/quintozene mixture was the most effective inretarding disease development, hence prolonging the life of the BSR-affectedpalms (George et al., 1996). Later studies, using pressure injection apparatus,indicated that systemic fungicide (e.g. bromoconazole) also appeared to limitthe spread of Ganoderma infection (Ariffin and Idris, 1997). In India, Rao et al.(1975) reported successful control of Ganoderma wilt disease of coconut byinjection of a 500 p.p.m. Vitavax solution into the trunk of diseased palms.Fumigant treatmentThe goal of causing rapid decay of woody tissues and subsequent displacementof the pathogen could be approached through the use of fumigants. Studies onthe use of the fumigant Dazomet, which releases the soil fumigant methyliso-thiocyanate (MIT) on contact with water, have also had encouraging resultsfor both in vitro and field studies (Ariffin and Idris, 1990). In vitro, 1 mg ofdazomet in a 9 cm Petri dish containing a growing culture of Ganodermawas shown to be fungistatic (Ariffin and Idris, 1991b). Investigation of thefungitoxic effects of MIT on Ganoderma in infected oil palms showed thatthe chemical moved systemically downwards when injected into the diseasedoil palms (Ariffin and Idris, 1993).Biological controlLittle work has been done on biological control of BSR disease. The possibilityof control of Ganoderma in existing stands should be approached through
64 D. Ariffin et al.manipulation of biological agents. Several promising antagonists, mainlyTrichoderma (Shukla and Uniyal, 1989; PORIM, 1991; Wijesekera et al.,1996), Aspergillus (Shukla and Uniyal, 1989) and Penicillium (Dharmaputraet al., 1989), have been isolated and their mechanisms of antagonism againstGanoderma in culture have been reported. The effectiveness of antagonistsin soil can be enhanced under field conditions by fumigation and fertilizerapplication (Varghese et al., 1975), but there are no reports of effectivebiological control in infected oil palms. Mass production of these antagonists,especially Trichoderma, on oil-palm waste, such as oil-palm mill effluent andempty fruit bunch (Singh, 1991) is possible, and this preparation could be usedfor application around the roots of infected oil palms.ConclusionBasal stem rot is having a severe impact on oil-palm production in the coastalsoils of Malaysia, and is currently increasing in intensity in peat soils and evenin the inland soils and lateritic soils, although in the latter, infection rates arerelatively low. It is not clear whether the distribution of the disease is related tosoil types, previous cropping history or the distribution of aggressive strains orspecies of the pathogen. The influence of environmental conditions on BSRdisease incidence also requires clarification. Novel techniques need to bedeveloped for the control of this disease. The available control measures areonly aimed at delaying the progress of infection, or prolonging the productivelife of the palm; these are cultural practices, such as clean clearing to minimizeroot infection through root contact and soil mounding to encourage develop-ment of new roots. Recently, promising results have been obtained on the useof fungicides to treat diseased palms, and studies are also ongoing to determinewhether a fumigant could eradicate the pathogen from infected tissues, thusreducing the Ganoderma inoculum. The development of the pressure-injectionapparatus is seen as another breakthrough that will make fungicidal treat-ment of infected palms possible. With this technique, fungicides could beapplied precisely to the infected sites, ensuring better delivery of the chemicalwith minimal wastage. Also, breeding for resistance to the disease remains animportant priority.ReferencesAkbar, U., Kusnadi, M. and Ollagnier, M. (1971) Influence of the type of planting materials and of mineral nutrients on oil palm stem rot due to Ganoderma. Oleagineux 26, 527–534.Ariffin, D. and Idris, A.S. (1990) Progress on Ganoderma research at PORIM. In: Ariffin, D. and Jalani, S. (eds) Proceedings of the Ganoderma Workshop, 11 September 1990. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 113–131.
Status of Ganoderma in Oil Palm 65Ariffin, D. and Idris, A.S. (1991a) A selective medium for the isolation of Ganoderma from diseased tissues. In: Yusof et al. (eds) Proceedings of the 1991 International Palm Oil Conference, Progress, Prospects and Challenges Towards the 21st Century (Model I, Agriculture) 9–14 September 1991. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 517–519.Ariffin, D. and Idris, A.S. (1991b) Investigation on the control of Ganoderma with dazomet. In: Yusof et al. (eds) Proceedings of the 1991 International Palm Oil Conference, Progress, Prospects and Challenges Towards the 21st Century (Model I, Agriculture) 9–14 September 1991. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 424–429.Ariffin, D. and Idris, A.S. (1993) Methylisothiocyanate (MIT) movement and fungi- toxicity in Ganoderma infected oil palm. In: Jalani, S. et al. (eds) Proceedings of the 1993 PORIM International Palm Oil Congress ‘Update and Vision’ (Agriculture), 20–25 September 1993. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 730–734.Ariffin, D. and Idris, A.S. (1997) Chemical control of Ganoderma using pressure injection. In: Proceedings of the PORIM-Industry Forum, 18 December 1997. Bangi, Malaysia, pp. 104–106.Ariffin, D., Idris, A.S. and Abdul Halim, H. (1989a) Significance of the black line within oil palm tissue decay by Ganoderma boninense. Elaeis 1, 11–16.Ariffin, D., Idris, A.S. and Mohd. Tayeb, D. (1989b) Approach to controlling of Ganoderma on oil palm in Malaysia. In: Proceedings of the 1989 International Conference On Palms and Palm Products, 21–25 November 1989, Benin City, Nigeria. Paper No. 55.Ariffin, D., Singh, G. and Lim, T.K. (1989c) Ganoderma in Malaysia – current status and research strategy. In: Jalani, S. et al. (eds) Proceedings of the 1989 PORIM Interna- tional Palm Oil Development Conference-Module II: Agriculture, 5–9 September 1989. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 249–297.Ariffin, D., Idris, A.S. and Abdul Halim, H. (1991) Histopathological studies on colonization of oil palm root by Ganoderma boninense. Elaeis 3(1), 289–293.Ariffin, D., Idris, A.S. and Khairudin, H. (1993) Confirmation of Ganoderma infected palm by drilling technique. In: Jalani, S. et al. (eds) Proceedings of the 1993 PORIM International Palm Oil Congress ‘Update and Vision’ (Agriculture), 20–25 September 1993. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 735–738.Ariffin, D., Idris, A.S. and Marzuki, A. (1995) Development of a technique to screen oil palm seedlings for resistance to Ganoderma. In: Proceedings of the 1995 PORIM National Oil Palm Oil Conference ‘Technologies in Plantation – The Way Forward’, 11–12 July 1995. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 132–141.Ariffin, D., Idris, A.S. and Marzuki, A. (1996) Spread of Ganoderma boninense and vegetative compatibility studies of a single field palm isolates. In: Ariffin, D. et al. (eds) Proceedings of the 1996 PORIM International Palm Oil Congress (Agriculture), September 1996. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 317–329.Benjamin, M. and Chee, K.H. (1995) Basal stem rot of oil palm – a serious problem on inland soils. MAPPS Newsletter 19(1), 3.Chinchilla, C. and Richardson, D.L. (1987) Four potentially destructive diseases of the oil palm in Central America. In: Halim, A. et al. (eds) Proceedings of the 1987
66 D. Ariffin et al. International Oil Palm/Palm Oil Conference: Progress and Prospects; Conference I: Agriculture, 23–26 June 1987. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 468–470.Darmono, T.W. and Suharyanto, A. (1995) Recognition of field materials of Ganoderma sp. associated with basal stem rot in oil palm by a polyclonal antibody. Menara Perkebunan 63(1), 15–22.Darmono, T.W., Suharyanto, A., Darussamin, A. and Moekti, G.R. (1993) Antibodi poliklonal terhadap filtrat pencucian kultur miselium Ganoderma sp. Menara Perkebunan 61, 67–72 (in Indonesian).Dell, E. (1955) De aantasting van de oliepalm op Sumatra door Ganoderma lucidum. Bergcultures 24, 191–203.Dharmaputra, O.S., Tjitrosomo, H.S. and Abadi, A.L. (1989) Antagonistic effect of four fungal isolates to Ganoderma boninense, the causal agent of basal stem rot of oil palm. Biotropia 3, 41–49.Genty, P., de Chenon, R.D. and Mariau, D. (1976) Infestation des racines arinnes du palmier a huile par des chnilles genre Sufetula Walker (Lepidoptera: Pyralidae). Oleagineux 31, 365–370.George, S.T., Chung, G.F. and Zakaria, K. (1996) Updated results (1990–1995) on trunk injection of fungicides for the control of Ganoderma basal stem rot. In: Ariffin, D. et al. (eds) Proceedings of the 1996 PORIM International Palm Oil Congress (Agriculture), September 1996. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 508–515.Hakim, M., Pasaribu, T.R. and Darmono, T.W. (1998) Yield and Ganoderma manage- ment through optimization of maintenance of oil palm root system. In: Jatmika et al. (eds) Proceedings of the 1998 International Oil Palm Conference ‘Commodity of the past, today, and the future’, 23–25 September 1998, Bali, Indonesia, pp. 392–395.Hasan, Y. and Turner, P.D. (1994) Research at BAH LIAS Research Station on basal stem rot of oil palm. In: Holderness, M. (ed.) Proceedings of the 1st International Workshop on Perennial Crop Diseases caused by Ganoderma, 28 November–3 December 1994. UPM, Serdang, Selangor, Malaysia.Hasan, Y. and Turner, P.D. (1998) The comparative importance of different oil palm tissues as infection sources for basal stem rot in replantings. The Planter 74, 119–135.Ho, C.T. and Khairudin, H. (1997) Usefulness of soil mounding treatments in prolonging productivity of prime-aged Ganoderma infected palms. The Planter 73(854), 239–244.Ho, Y.W. and Nawawi, A. (1985) Ganoderma boninense Pat. from basal stem rot of oil palm (Elaeis guineensis) in Peninsular Malaysia. Pertanika 8, 425–428.Ho, Y.W. and Nawawi, A. (1986) Isolation, growth and sporophore development of Ganoderma boninense from oil palm in Malaysia. Pertanika 9, 69–73.Idris, A.S. (1999) Basal stem rot (BSR) of oil palm (Elaeis guineensis Jacq.) in Malaysia: factors associated with variation in disease severity. PhD thesis, Wye College, University of London, UK.Jollands, P. (1983) Laboratory investigations on fungicides and biological agents to control three diseases of rubber and oil palm and their potential applications. Tropical Pest Management 29, 33–38.Khairudin, H. (1990a) Basal stem rot of oil palm: incidence, etiology and control. Master of Agriculture Science thesis, Universiti Pertanian Malaysia, Selangor, Malaysia.
Status of Ganoderma in Oil Palm 67Khairudin, H. (1990b) Results of four trials on Ganoderma basal stem rot of oil palm in Golden Hope Estates. In: Ariffin, D. and Jalani, S. (eds) Proceeding of the Ganoderma Workshop, 11 September 1990. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 113–131.Khairudin, H. (1993) Basal stem rot of oil palm caused by Ganoderma boninense: An update. In: Jalani et al. (eds) Proceedings of the 1993 PORIM International Palm Oil Congress ‘Update and Vision’ (Agriculture), 20–25 September 1993, Paper No. 46. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia.Lim, K.H., Chuah, J.H. and Ho, C.H. (1993) Effects of soil heaping on Ganoderma infected oil palms. In: Jalani et al. (eds) Proceedings of the 1993 PORIM International Palm Oil Congress ‘Update and Vision’ (Agriculture), 20–25 September 1993. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 735–738.Lim, T.K., Hamm, R.T. and Mohamad, R. (1990) Persistency and volatile behaviour of selected chemical in treated soil against three Basidiomycetes root disease pathogens. Tropical Pest Management 36(1), 23–26Miller, R.N.G. (1995) The characterization of Ganoderma population in oil palm cropping systems. PhD thesis, University of Reading, UK.Navaratnam, S.J. (1964) Basal stem rot of oil palm on ex-coconut states. The Planter 40, 256–259.Navaratnam, S.J. and Chee, K.L. (1965) Root inoculation of oil palm seedlings with Ganoderma sp. Plant Disease Report 49, 1011–1012.Nieto, L.E. (1995) Incidence of oil palm stem rots in Colombia. Palmas 16, 227–232.Nigerian Institute for Oil Palm Research (NIFOR) (1978) Fourteenth Annual Report 1977, Nigeria.Oruade-dimaro, E.A., Rajagopalan, K. and Nwosu, S.O. (1994) A laboratory method for inducing sporophore formation and pathogenicity in Ganoderma zonatum Murill. Elaeis 6(1), 1–5.PORIM (1988) Annual Research Report 1988. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia.PORIM (1991) Annual Research Report 1991. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia.PORIM (1997) Annual Research Report 1997. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia.Ramasamy, S. (1972) Cross-infectivity and decay ability of Ganoderma species parasitic to rubber, oil palm and tea. Bachelor Agriculture Science, Project Report, University of Malaya.Rao, A.K. (1990) Basal stem rot (Ganoderma) in oil palm smallholdings – IADP Johore Barat experience. In: Ariffin, D. and Jalani, S. (eds) Proceedings of the Ganoderma Workshop, 11 September 1990. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 113–131.Rao, A.P., Subramanyam, K. and Pandit, S.V. (1975) Ganoderma wilt disease of coconut and control. Andra Pradesh Agriculture University, India.Reddy, M.K. and Ananthanarayanan, T.V. (1984) Detection of Ganoderma lucidum in betelnut by the fluorescent antibody technique. Transactions of the British Mycological Society 82(3), 559–561.Robertson, J.S., Prendergast, A.J. and Sly, J.M.A. (1968) Diseases and disorders of the oil palm (Elaeis guineensis) in West Africa. Journal of the West Africa Institute for Oil Palm Research 4, 381–409.
68 D. Ariffin et al.Sanderson, F.R. and Pilotti, C.A. (1997a) Ganoderma basal stem rot: an enigma, or just time to think an old problem? The Planter 73, 489–493.Sanderson, F.R. and Pilotti, C.A. (1997b) The important of spores in the epidemiology of Ganoderma. Presented at International Conference on Advances in Oil Palm Agronomy, 1–2 September 1997, Cartagena.Sharples, A. (1936) Observation on stem rot of oil palm. Bulletin Department of Agricul- ture Straits Settlements and F. M. S. Science Serdang 21, 1–28.Shukla, A.N. and Uniyal, K. (1989) Antagonistic interactions of Ganoderma lucidum (lyss.) Karst. against some soil microorganisms. Current Science 58, 265–267.Singh, G. (1991) Ganoderma – the scourge of oil palms in the coastal areas. The Planter 67, 421–444.Thompson, A. (1931) Stem-rot of the oil palm in Malaya. Bulletin Department of Agricul- ture, Straits Settlements and F.M.S., Science Series 6.Tummakate, A. and Likhitakaraj, S. (1998) The situation of Ganoderma on oil palm in Thailand. In: Holderness, M. (ed.) Proceedings of the 1st International Workshop on Perennial Crop Diseases caused by Ganoderma, 28 November–3 December 1994. UPM, Serdang, Selangor, Malaysia (Abstract).Turner, P.D. (1965a) Infection of oil palms by Ganoderma. Phytopathology 55, 937.Turner, P.D. (1965b) Oil palms and Ganoderma III. Treatment and control in established plantings. The Planter 41, 279–282.Turner, P.D. (1965c) The oil palm and Ganoderma IV. Avoiding disease in new plantings. The Planter 41, 331–333.Turner, P.D. (1965d) The incidence of Ganoderma disease of oil palm in Malaya and its relation to previous crop. Annals of Applied Biology 55, 417–423.Turner, P.D. (1968) The use of surgery as a method of treating basal stem rot in oil palms. The Planter 44, 303–308.Turner, P.D. (1981) Oil Palm Diseases and Disorders. Oxford University Press, Oxford, pp. 88–110.Turner, P.D. and Chin, P.Y. (1968) Effects of Ganoderma infection on the inorganic nutrient status of oil palm tissues. Oleagineux 23, 367–370.Varghese, G., Chew, P.S. and Lim, T.K. (1975) Biology and chemically assisted biological control of Ganoderma. In: Proceeding of the Rubber Research Institute of Malaysia Conference, Kuala Lumpur, Malaysia, pp. 228–292.Wakefield, E.M. (1920) Diseases of the oil palm in West Africa. Kew Bulletin, 306–308.Wijesekera, H.T.R., Wijesundera, R.L.C. and Rajapakse, C.N.K. (1996) Hyphal inter- actions between Trichoderma viridae and Ganoderma boninense Pat., the cause of coconut root and bole rot. Journal of the National Science Sri Lanka 24(3), 217–219.
70 S. Likhitekaraj and A. Tummakate2. A replanted plot in Krabi province. The old trees of this plot were killed bychemical injection. New seedlings were planted between the rows of dead trees.The replanted plants were 1 year old when this study started.3. A block of 20-year-old palms in a plantation in Satul province.Results1. After two annual observations there is no evidence of BSR on the plantedseedlings (3 years old) in the first location.2. After two annual observations, no BSR appears on young replanted palmsin the second location, but the old stumps of killed trees have fruiting bodies ofGanoderma. The latest estimate is that 23.8% of the 2000 stumps showGanoderma fruiting bodies. The incidence of the sporophores increases everytime a survey is conducted.3. The 20-year-old palms in Satul province show no evidence of BSR.ConclusionsAfter 2 years’ observation on 3-year-old palms in replantings and on 20-year-old palms, at three locations, no symptoms of BSR have been observed,with the exception of the development of sporophores at a location inKrabi province. The surveys will be continued for many years on the threeplantations in order to monitor the development of the disease.ReferenceLikhitekaraj, S. (1993) Stem Rot. Important Disease of Oil Palm. Annual Report of Plant Pathology and Microbiology Division, Department of Agriculture, Ministry of Agriculture and Cooperative, Thailand.
72 S.S. Lee A variety of basidiomycete fungi have been reported to be associated withroot rot diseases of A. mangium. A brown root disease caused by Phellinus hasbeen reported from Sabah (Khamis, 1982) and the Philippines (Almonicar,1992; Millitante and Manalo, 1999). In the Gogol Valley of Papua NewGuinea, Arentz (1986) reported 29% mortality of 5-year-old A. mangium treesdue to root disease caused by a species of Ganoderma. Ganoderma spp. are alsosuspected as the causal agents of root disease of A. mangium trees of variousages in Peninsular Malaysia (Lee, 1985, 1997), Sumatra (Lee, 1997) andWest Kalimantan, Indonesia (unpublished data). Here, the results of a long-term survey of root diseases in an A. mangiumplantation in Peninsular Malaysia are presented, and preliminary results ofpathogenicity tests with the associated fungi are discussed.Impact of Root Diseases on A. mangiumBetween September 1991 and June 1992 plots were established in an A. mang-ium plantation in Kemasul, Pahang in Peninsular Malaysia, to monitor theoccurrence and spread of root disease. Three replicate plots, each containing10 × 10 rows of trees were set up in stands planted by the Forestry Departmentin 1982, 1984, 1985, 1986, 1987 and 1988, making a total of 18 plots. Allthe trees in each plot were numbered and mapped for ease of the survey andfuture reference. During each survey, symptoms and signs of root disease andthe health status of each tree in every plot were recorded. For the first 3 years,surveys were carried out at 6-monthly intervals and thereafter, annually(when it became clear that there were few changes over a 6-month period). Symptoms of root diseases included yellowing, wilting and reduced size ofthe foliage, thinning of the crown, dieback, and death of trees in groups. Treeswith such symptoms were found to occur in patches, with a concentric patternof spread. Diseased roots were covered by a wrinkled, reddish-brown mycelialskin, encrusted with soil, or encrusted in a mass of earth and sand intermingledwith rusty brown patches, in contrast to the clear, pale yellowish-browncoloured healthy roots. More than 40% mortality was observed in all the 1984 plots 14 yearsafter planting, and in plots 1987B, 1988C, 9 and 11 years after planting,respectively (Fig. 5.1a and b). In the 1984 plots mortality increased veryrapidly when the trees were between 10 and 14 years old, while in plots1987B and 1988C, a rapid increase in mortality occurred when the trees werebetween 6 and 9 years old and 7 and 11 years old, respectively. In contrast, lessthan 10% mortality was observed in plots 1982B, 1985B, 1986A, 1986C,1987A and 1988A, while no mortality at all was observed in plot 1985C.It was clear that the occurrence of root disease was not uniform and thatmortality rates differed from plot to plot. Similar variation in mortalityrates had also been observed in the 1995 survey of root rot in A. mangiumprovenance trials in various parts of Peninsular Malaysia (Lee, 1997).
Current Status of Root Diseases of Acacia mangium 73 The rate of spread of the disease in the different plots was also variable.Mapping and regular monitoring of the trees showed that the disease mostprobably spread by root contact. In most cases, the initial disease foci enlargedFig. 5.1. Mortality rates of Acacia mangium trees in Kemasul, Pahang, PeninsularMalaysia: (a) in the 1982, 1984 and 1985 plots; (b) in the 1986, 1987 and 1988plots.
74 S.S. LeeFig. 5.2. Distribution of dead and dying trees in plot 1988C: r, living trees;1–6, dead and dying trees at the six sampling times; S, trees missing during plotestablishment.with each passing year; this was clearly evident in all the 1984 plots and inplots 1987B and 1988C (Fig. 5.2). The absence of tree mortality in plot 1985C, even 13 years after planting,was not unexpected, as no root disease symptoms were observed on any of thetrees in the plot during the duration of the study. While no symptoms of rootdisease were evident on the trees in plots 1985A, 1985B, 1986A, 1987A and1988A at the time of plot establishment, they started to appear 2–3 years afterthe study commenced. This suggests that the trees only became infected whentheir expanding root systems encountered some buried source of root diseaseinocula. As in the other plots mentioned earlier, the rate of disease spread wasvariable, with moderate increases in mortality in plots 1985A, 1986B, 1987Cand 1988B, and very little increase in plots 1985B, 1986A, 1986C, 1987Aand 1988A. The mortality of trees generally increased with time in plots where rootdisease was already present at plot establishment. The rate of disease spreadwas probably dependent on the presence, abundance and distribution of rootdisease inocula at the site, rate of root growth, extent of the root system ofeach tree, and extent of root contact between healthy and infected trees. Theseplantations had been established on logged-over lowland rainforest areas,which had been mechanically cleared and burned before planting. However,old tree stumps were still evident in the plots and it is highly likely that rootsand other woody debris that harbour the facultative parasitic root-rot fungiremain buried in the soil, acting as sources of infection.
Current Status of Root Diseases of Acacia mangium 75Fungi Associated with Root Diseases of A. mangiumBased on the appearance of the infected roots, two main types of root diseasescould be distinguished even though the visible disease symptoms on the treecrowns were similar. These were red-root disease and brown-root disease. Roots of trees infected by red-root disease are characteristically covered bya wrinkled, reddish-brown mycelial mat. The red colour of the mycelial matbecomes very evident when the root is washed clean of soil. A white mottlingpattern is evident on the underside of the infected root and there is a very char-acteristic odour. In the early stages of infection, the wood remains hard and nocolour change is discernible, but in advanced stages the wood becomes palebuff and spongy or dry, depending on the soil conditions. Red-root disease wasthe most frequently observed type of disease when roots were sampled. Thecharacteristics of the disease are very similar to that of red-root disease causedby Ganoderma philippii (= G. pseudoferreum) on rubber (Anonymous, 1974). In brown-root disease, the roots are encrusted in a mass of earth and sand,intermingled with rusty brown patches. Advanced stages of the disease areeasily recognized by the production of brown zigzag lines in the wood, forminga honeycomb-like pattern, and the wood becoming friable, light and dry. Thebrown lines are ridges of golden-brown fungal mycelium and the type of rotproduced is known as ‘pocket rot’. These characteristic features indicatethat the fungus associated with the disease is Phellinus noxius (Anonymous,1974). The identity of the associated fungi could not be confirmed initiallybecause of the absence of sporocarps on diseased or dead trees. Samples of dis-eased roots were thus collected for isolation of the associated fungi. Attemptswere made to identify the pure-culture mycelial isolates by comparison withthe species codes developed by Nobles (1965) and Stalpers (1978) and byinoculation onto wood blocks for the production of sporocarps (Lee andNoraini Sikin, 1999). For production of sporocarps on wood blocks, pure-culture isolates of thetest fungi were first grown on malt agar (DIFCO Laboratories, USA) in the darkat ambient room temperature for about 1 week. In the meantime, blocks ofdebarked rubber wood, measuring 10 cm by 5–6 cm diameter, were placedindividually into autoclavable plastic bags, wetted with approximately 50 mlof 2% malt extract and sterilized. Three 1 cm diameter plugs, taken from theedge of 1-week-old actively growing cultures, were then used to inoculate eachrubber-wood block. Five replicate blocks were inoculated with each fungusand the inoculated blocks incubated in the dark at ambient room temperature(28 ± 2°C). At the end of 2 months the well-colonized blocks were removedfrom their plastic bags and ‘planted’ into polybags containing unsterilizedgarden soil, one block per bag. These were then transferred to a shade houseand lightly sprayed with tap water daily to keep the soil and the wood blocksmoist. When sporocarps were produced, between 2 and 3 weeks later, theywere collected for identification in the laboratory.
76 S.S. Lee The identity of the fungus associated with red-root disease could not beconfirmed from the wood-block technique as no sporocarps were produced.However, the characteristic red skin of mycelium on the root is similar to thatreported for G. philippii (= G. pseudoferreum) on rubber (Anonymous, 1974).From isozyme analysis, four isolates of Ganoderma obtained from A. mangiumin West Malaysia were determined to be different from those isolatedfrom palm hosts (Miller et al., 1995). Recently many sporocarps of G. philippii(Corner, 1983) were found growing on dead 10-year-old A. mangium trees in aplantation at Bidor, Perak. Inspection of trees with symptoms of root diseaselocated close to the clumps of dead trees revealed that the roots were coveredby a red mycelial mat (S. Ito, Bidor, 1999, personal communication), charac-teristic of red-root disease observed on A. mangium trees in Kemasul, Pahangand elsewhere. However, attempts to isolate the fungus, from both sporocarpsand infected roots, were unsuccessful. Corner (1983) noted that G. philippii israther common and distributed from Burma (Myanmar) to the SolomonIslands, being found on dead stumps in the forest and in the open, and parasiticon roots of trees, especially Hevea. Using the wood-block technique, sporocarps produced from mycelialisolates obtained from samples with brown-root disease were confirmed asthose of P. noxius (Pegler and Waterston, 1968). Inoculated wood blocks alsohad the characteristic pocket rot similar to that observed on the diseased roots,indicative of rot caused by P. noxius. Some roots were covered by a thin, black crust, which was easily mistakenfor necrotic tissue. The black crust was usually found on the roots of dead treeswhere the wood had become yellowish-cream in colour, spongy and light.Using the wood-block technique, hyphal isolates obtained from the black crustyielded sporocarps, identified as Amauroderma parasiticum (Corner, 1983). In addition to the root diseases reported here, a root disease associatedwith the presence of white rhizomorphs of an unidentified fungus has also beenreported from A. mangium in Peninsular Malaysia (Lee, 1997). However, thisdisease was not observed during the present study.Pathogenicity TestsPathogenicity tests are presently being conducted on A. mangium saplings inthe FRIM nursery, and only preliminary results are reported here. Six-month-old A. mangium plants were transplanted into large polybags (33 cm depth by35.5 cm diameter) containing a 1 : 1 mixture of forest soil and padi husk (thisis the potting mixture normally used in the FRIM nursery). After the plants hadbecome well established, about 3 months later, they were inoculated usingbranches (8 cm long by 1.5 cm diameter) of a rubber tree which had been wellcolonized by the test fungi (the rubber-tree branches, with intact bark, wereinoculated using the same technique as described above for the inoculationof the rubber-wood blocks). Three well-colonized branches were used to
Current Status of Root Diseases of Acacia mangium 77inoculate each test plant, with the branches buried in close proximity to theroots of the plant in the polybag. There were three replicates for each fungusand the fungal isolates tested were P. noxius, the suspected Ganoderma andA. cf. parasiticum. About 2 months after inoculation, symptoms of root disease were obviouson the plants inoculated with P. noxius and the suspected Ganoderma, whilethose inoculated with A. cf. parasiticum remained symptomless. However,different symptoms of root disease were observed on the plants inoculatedwith P. noxius and the suspected Ganoderma. Those inoculated with P. noxiusexhibited progressive yellowing of the phyllodes, beginning with the tips of theyounger phyllodes, resulting ultimately in defoliation and death of the infectedplant. On the other hand, plants inoculated with the suspected Ganodermasuddenly wilted without any yellowing symptoms, and died within 5 days afterthe first symptoms were noticed. Roots of plants inoculated with the suspected Ganoderma were covered bya red mycelial mat but the fungus could not be successfully re-isolated fromthe affected plants. This experiment is being repeated to confirm the resultspresented here. Pathogenicity of P. noxius was proven as the fungus was successfullyre-isolated from roots of the inoculated plants, which had rusty brown patchesunder a crust of soil. Plants inoculated with A. cf. parasiticum remained healthy even 6 monthsafter inoculation. It would appear that this fungus is not a primary pathogen ofA. mangium, but probably a secondary pathogen or weak parasite infectingstressed trees or trees which have been weakened or killed by some otheragents. Corner (1983) recorded A. parasiticum as a parasite on the trunk of aliving tree of Knema (Myristicaceae) in a swamp forest in Singapore.ConclusionLarge-scale burning has been a common feature of land clearing in South-EastAsia for conversion of forest or old tree stands into agricultural and industrialplantations, or for replanting. In 1997 large-scale burning for land clearing,and uncontrolled bush fires on the islands of Sumatra and Kalimantan in Indo-nesia, resulted in severe atmospheric pollution which lasted for several monthsover Singapore, Brunei, southern Thailand and large parts of Indonesia andMalaysia. Widespread public outcry and political pressure from regionalgovernments resulted in the government of Indonesia declaring a ‘no burn’policy for land clearing, with the imposition of hefty fines for those found guiltyof the offence. However, enforcement remains problematic. In Malaysia, the Environmental Quality (Clean Air) Regulations 1978prohibit open burning, but in the past open burning for land conversion andreplanting could be carried out under special contravention licences issued bythe Department of Environment. The large-scale adoption of the zero burning
78 S.S. Leetechnique by oil-palm plantation companies in Malaysia in 1989 has allowedoil-palm replanting to be done without violating the Environmental Quality(Clean Air) Regulations 1978, and the technique has also been developedfor the replanting of oil palm and other plantation crops from logged-overforests (Golden Hope Plantations Berhad, 1997). In the aftermath of the 1997haze, the Malaysian government issued a directive prohibiting almost allforms of open burning, and a law pertaining to this issue is presently underconsideration by the Attorney-General’s chambers. While zero burning is environmentally friendly and results in totalrecycling of plant tissues (the existing trees are felled, shredded and left todecompose in situ), it also gives rise to several problems, such as increasedinsect infestation and increased sources of root disease inocula. From thedisease point of view, the woody residues act as potential reservoirs and foodresources for the facultative parasitic root-disease fungi which live in the soil.In second-rotation A. mangium plantations in Sumatra, where no burning wascarried out before replanting, there are already indications that losses due toroot diseases will be much more serious, with a higher incidence of the diseasein the young plantations and mortality occurring in younger plants. A.mangium trees as young as 6 months old have been observed to be killed byred-root disease (unpublished data) in such areas. In view of the potential damage and losses that can be caused by rootdiseases in A. mangium plantations, especially with the implementation of the‘zero burning’/ ‘no burn’ policy by several South-East Asian governments, it isimportant that further research be conducted to determine the sources ofinoculum, factors promoting the occurrence and spread of the disease, andmethods for prevention, management and control of the disease.ReferencesAlmonicar, R.S. (1992) Two types of root rot diseases affecting Acacia mangium. Nitrogen Fixing Tree Research Reports 10, 94–95.Anonymous (1974) Root diseases Part 1: Detection and recognition. Planters’ Bulletin 133, 111–120.Arentz, F. (1986) Forest Pathology Lecture Notes. Papua New Guinea Forestry College, Bulolo.Arentz, F. and Simpson, J.A. (1988) Root and butt rot diseases of native plantation species in Papua New Guinea. Paper presented at the Fifth International Congress of Plant Pathology. Kyoto, Japan.Corner, E.J.H. (1983) Ad Polyporaceas I. Amauroderma and Ganoderma. Nova Hedwigia 75, 1–182.Golden Hope Plantations Berhad (1997) The zero burning technique for oil palm cultivation. Golden Hope Plantations Berhad, Kuala Lumpur.Khamis, S. (1982) Pests and diseases of forest plantation trees with special reference to SAFODA. In: Proceedings of the Eighth Malaysian Forestry Conference, Kota Kinabalu, pp. 512–524.
Current Status of Root Diseases of Acacia mangium 79Lee, S.S. (1985) Tree Diseases and Wood Deterioration Problems in Peninsular Malaysia. Occasional Paper No. 5, Serdang: Faculty of Forestry, Universiti Pertanian Malaysia.Lee, S.S. (1993) Diseases. In: Kamis Awang and Taylor, D. (eds) Acacia mangium Growing and Utilization. MPTS Monograph Series No. 3. Winrock International and FAO, Bangkok, Thailand, pp. 203–223.Lee, S.S. (1997) Diseases of some tropical plantation acacias in Peninsular Malaysia. In: Old, K.M., Lee, S.S. and Sharma, J.K. (eds) Diseases of Tropical Acacias. Proceed- ings of an International Workshop, Subanjeriji, South Sumatra, 28 April–3 May 1996. CIFOR Special Publication, Bogor, pp. 53–56.Lee, S.S. and Noraini Sikin Yahya (1999) Fungi associated with heart rot of Acacia mangium trees in Peninsular Malaysia and Kalimantan. Journal of Tropical Forest Science 11(1), 240–254.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.R.M., Hussin, M.Z. and Sariah Meon (1995) Isozyme analysis for characterization of Ganoderma strains from south-east Asia. Bulletin OEPP/EPPO Bulletin 25, 81–87.Millitante, E.P. and Manalo, M.Q. (1999) Root rot disease of mangium (Acacia mangium Willd.) in the Philippines. Poster. Fifth International Conference on Plant Protection in the Tropics, Kuala Lumpur, Malaysia, 15–18 March 1999, pp. 448–450.Nobles, M.K. (1965) Identification of cultures of wood-inhabiting Hymenomycetes. Canadian Journal of Botany 43, 1097–1139.Old, K.M., Lee, S.S. and Sharma, J.K. (eds) (1997) Diseases of Tropical Acacias. Proceed- ings of an International Workshop, Subanjeriji, South Sumatra, 28 April–3 May 1996. CIFOR Special Publication.Pegler, D.N. and Waterston, J.M. (1968) Phellinus noxius. Commonwealth Mycological Institute Descriptions of Pathogenic Fungi and Bacteria No. 195.Stalpers, J.A. (1978) Identification of Wood-inhabiting Aphyllophorales in Pure Culture. Studies in Mycology No. 16. Centraalbureau voor Schimmelcultures, Baarn.Turnbull, J. (ed.) (1986) Australian Acacias in Developing Countries. Proceedings of an International Workshop held at the Forestry Training Centre, Gympie, Queens- land, Australia, 4–7 August 1986. ACIAR Proceedings No. 16.Yap, S.K. (1986) Introduction of Acacia species to Peninsular Malaysia. In: Turnbull, J. (ed.) Australian Acacias in Developing Countries. Proceedings of an International Workshop held at the Forestry Training Centre, Gympie, Queensland, Australia, 4–7 August 1986. ACIAR Proceedings No. 16, pp. 151–153.
84 H. Soepena et al.Oil-palm Basal Stem Rot (Ganoderma Stem Rot)The causal agent of BSRThe causal agent of BSR on oil palms is G. boninense Pat. Fruiting bodies ofGanoderma collected from some oil-palm estates in Malaysia (Ho and Nawawi,1985) and North Sumatra (Abadi, 1987) have been identified as G. boninense.Enzyme-linked immunosorbent assays (ELISA) have confirmed specimens ofGanoderma from North Sumatra as G. boninense (Utomo, 1997). Ganoderma is a saprophytic soil inhabitant, indigenous to the tropicalrainforest, but under some circumstances it can become pathogenic. Speciesof Ganoderma have a wide host range – more than 44 species from 34 genera ofplants have been identified as potential hosts (Venkatarayan, 1936), includingcoconut and oil palm, which are the main source of infection of Ganodermastem rot in oil palms (Hasan and Turner, 1998).The disease symptomsG. boninense can infect all stages of oil palm, from seedling to old palms. Palmsinfected early in their life cycle can remain symptomless, the symptoms onlybecoming clear after the palms are more than 12 years old (Lubis, 1992), butin the second and third replantings the symptoms can appear as early as 1–2years after planting in the field. Ganoderma infection on seedlings or young palms usually occurs on rootsand is followed by the spread of infection into the base of the bole (Fig. 6.1).External symptoms include a chlorosis of newly emerging leaves or partiallydead old fronds. Disease symptoms on the old palms is clearer, the appearanceof a number of spear leaves and collapse of old fronds are the main symptoms(Fig. 6.2).Basal Stem Rot Control ManagementBSR control strategyBSR could be managed satisfactory if the source of infection of Ganoderma couldbe completely destroyed. Thus management of BSR in oil-palm replantingareas should be based upon the following strategy: (i) use of uninfectedsoil in polybags to grow seedlings; (ii) prevention of infection in younggrowing palms; (iii) eradication of all sources of Ganoderma in the field; and(iv) application of biofungicides (Trichoderma spp.).
Control Strategy for Basal Stem Rot on Oil Palm 85Early warning systemAlthough biofungicide treatments are given to all growing plants, specialattention must be given to emerging disease symptoms, especially for the first5 years. Disease symptoms should be evaluated twice a year and diseaseincidence should be reported. An application of further biofungicide is made assoon as possible, or severely infected and dead plants are removed, the plantinghole treated with biofungicide and healthy seedlings replanted.Biological control method for GanodermaGanoderma has many natural antagonists, such as Trichoderma spp.,Actinomycetes sp. and Bacillus spp. (Abadi, 1987; Soepena and Purba, 1998).Trichoderma spp. are usually found as saprophytic soil inhabitants, butsome of them have been successfully selected as antagonists to Ganoderma(Dharmaputra, 1989; Soepena et al., 1999). Trichoderma koningii Oud. IsolateMarihat (MR14) is one of the most powerful antagonists against Ganodermaand has been formulated as the active ingredient in a biofungicide (Soepenaand Purba, 1998). Other species, such as Trichoderma viride, Trichoderma Fig. 6.1. Ganoderma-infected seed- ling: note the rotten tissue on the base of the bole.
86 H. Soepena et al.Fig. 6.2. The main symptoms of Ganoderma disease on an old oil palm: note theaccumulation of spear leaves and collapse of old fronds.harzianum and Gliocladium virens have also been used as biological controlagents against Ganoderma, but these species are better for decomposing organicmaterial in fields. A combination of antagonistic and saprophytic fungi isvery useful for destroying Ganoderma propagules and decomposing oil-palmresidues in windrows. The biofungicide contains 5–8 × 106 conidia and chlamydospores ofT. koningii per gram of product in a natural medium.Application of the biofungicideTrichoderma survives as chlamydospores under unfavourable conditions,and most of these are resistant to many kinds of chemical pesticides, such asorganochlorines, organosulphides, organophosphites and bromides, and her-bicides (Eveleigh, 1985). However, Trichoderma also requires water for growth,so the Trichoderma biofungicide is applied at the beginning or end of the rainyseason. The dose of the biofungicide depends on the size of the palms.
Control Strategy for Basal Stem Rot on Oil Palm 87Preventative treatmentsSeedlings grown in polybags can be infected by Ganoderma from infected soil, sosoil taken from disease-free areas should be used and the seedlings treated withTrichoderma biofungicide by spreading it on the surface of the polybag. This willhelp to eradicate any inoculum and will protect the seedlings after planting inthe field. Planting holes in heavily infected areas must also be treated withTrichoderma biofungicide prior to planting a seedling, to help eradicate theinoculum in the soil and protect newly growing palms. The biofungicide canalso be applied to oil-palm trunks in windrows in order to eradicate Ganodermapropagules and increase decomposition. Young palms should be treatedannually for 5 years.Curative treatmentsIn addition to preventative treatment, newly infected plants can be treatedwith Trichoderma biofungicide. The biofungicide can be injected into the base ofthe bole of infected plants using soil injection: 3 holes are made under the baseof the bole of the infected plant with a soil auger, and the biofungicide can beapplied. This method can be used for special palms, such as highly productiveor mother plants. Surgery to remove rotten tissue can also be conducted onthese special palms in conjunction with application of the biofungicide to theaffected areas.Field sanitationIt is very important to keep the oil-palm plantations free from sources of thepathogen, so good field sanitation is essential. All infected plant materialsshould be treated with Trichoderma biofungicide.ReferencesAbadi, A.L. (1987) Biologi Ganoderma boninense Pat. Pada kelapa sawit (Elaeis guineensis Jacq.) dan pengaruh beberapa mikroba tanah antagonistik terhadap pertumbuhannya. PhD thesis, IPB, Bogor.Dharmaputra, O.S. (1989) Fungi antagonistik terhadap Ganoderma boninense Pat. Penyebab busuk pangkal batang pada kelapa sawit di Adolina. Laporan tahunan Kerjasama Penelitian PP Marihat-BIOTROP, SEAMEO BIOTROP, Bogor, pp. 28–43.Eveleigh, D.E. (1985) Trichoderma. In: Demain, A.L. and Solomon, N.A. (eds) Biology of Industrial Microorganisms. Benjamin Cunning, London, pp. 487–509.Hasan, Y. and Turner, P.D. (1998) The comparative importance of different oil palm tissue as infection source for BSR in replantings. The Planter 74(864), 119–135.Ho, Y.W. and Nawawi, A. (1985) Ganoderma boninense Pat. From basal stem rot of oil palm in Peninsular Malaysia. Pertanika 8, 425–428.
88 H. Soepena et al.Khairudin, H. (1993) Basal stem rot of oil palm caused by Ganoderma boninense. An update. PORIM, International Palm Oil Congress, Update and Vision. PORIM, Kuala Lumpur, pp. 739–749.Lubis, A.U. (1992) Kelapa Sawit (Elaeis guineensis Jacq.) di Indonesia. Pusat Penelitian Perkebunan Marihat-Bandar Kuala, Pematang Siantar, Sumatera Utara.Möller, C. and Schultz, C. (1997) Biotechnological Applications for Oil Palm Improvement. Proceedings of the BTIG Workshop on Oil Palm Improvement through Biotechnol- ogy, pp. 14–26.Soepena, H. (1996) Serangan penyakit Ganoderma pada kelapa sawit di kebun Padang Halaban. Pusat Penelitian Karet, Sungei Putih.Soepena, H. and Purba, R.Y. (1998) Biological Control Strategy for Basal Stem Rot on Oil Palm. International Workshop on Ganoderma Diseases of Perenial Crops. MARDI Training Centre, Serdang, Selangor, Malaysia.Soepena, H., Purba, R.Y. and Pawirosukarto, S. (1999) Pedoman Teknis Pengendalian Ganoderma. Pusat Penelitian Kelapa Sawit, (IOPRI) Medan.Utomo, Ch. (1997) Early Detection of Ganoderma in oil palm by ELISA technique. MSc thesis, Institute of Agronomy and Plant Breeding, Faculty of Agriculture, George August University, Germany.Venkatarayan, S.V. (1936) The biology of Ganoderma lucidum on areca and coconut palms. Phytopathology 26, 153–175.
90 M. Sariah and H. Zakariatreatment of Ganoderma-infected palms is limited by the fact that both visiblyinfected and subclinical palms may harbour established infections by thetime treatment is applied. Additional difficulties may occur in the effectiveplacement of fungicides, as lesions are frequently very large in size. As lesionsare most commonly found at the stem base, high-pressure injection offungicides frequently results in the passage of the chemical straight into thesoil. However, recent preliminary results on trunk injection of fungicides intoBSR-infected oil palms have indicated that Triadimenol (a systemic fungicide)may increase their economic life span, with treated palms remaining alive 52months after the original BSR diagnosis (Chung, 1991). Further evaluationof pressure injection of fungicides by Ariffin (1994) indicated that systemicfungicides (Tridemorph and Dazomet) also limited the spread of infection andhe further concluded that the chemical moved systemically downwards intothe roots when injected into plants. Alternative control methods for the future may lie in the biologicalmanagement of the disease. For example, trunk tissues, when they are wind-rowed as part of the replanting technique in particular, support the rapiddevelopment of many fungi other than Ganoderma. A much greater diversity offungi non-pathogenic to oil palm occur on poisoned windrowed tissues and,together with their more rapid and prolific development than on unpoisonedtissues, a possible biological control approach to the disease is indicatedthrough the competitive saprophytic ability of non-pathogenic fungi todisplace Ganoderma in composting tissues. However, under normal fieldconditions these fungi seem unable to displace the pathogen and Ganodermacontinues to colonize old tissues, which become BSR sources for the newplanting. If the natural order of the succession could be manipulated, or thevolume of particular competitors changed so as to minimize the pathogen’sopportunity for colonization, then the potential BSR hazard for new plantingswould be greatly reduced. However, observations of the low incidence of disease due to Ganodermaspecies in natural stands in the forest although the pathogen is present, wouldsuggest that disease is kept under control by some biological means. A study ofsoil microflora of jungle and plantation habitats showed significant changes inquantitative and qualitative aspects of the microflora from these two habitats(Varghese, 1972). The changes were most striking in the humus-stainedupper horizon, where Aspergillus dominated the mycoflora of the forest, butthis layer was completely disrupted in the plantation habitat. Along with this,a lowering of the antibiotic potential of the soil could be expected which wouldbe to the advantage of root pathogenic fungi (Varghese, 1972). Therefore anynew approach to natural or biological control of Ganoderma should take intoconsideration the role of antagonistic microflora. Enumeration of the microbial population from the oil-palm rhizospheresand on the sporophores has also indicated great diversity of non-pathogenicfungi in these habitats, which again points to the possibility of biologicalmanagement of Ganoderma. Species of Trichoderma, Penicillium and Aspergillus
Use of Soil Amendments for Control of Basal Stem Rot 91make up more than 30% of the total populations of fungi (cfu) recovered,and in some areas there was a positive correlation between percentage of BSRincidence and frequency of isolations of the non-pathogenic fungi (Table 7.1).These observations were not consistent for all the areas surveyed, suggestingthat soil and environmental factors exert some influence on the survival andproliferation of microorganisms in the oil-palm rhizospheres, and the recoveryof antagonistic Trichoderma was only in the range of 103 cfu g−1 dried soil,which is too low relative to the total root mass of a palm. Laboratory screeningof these non-pathogenic fungi, based on dual culture, colony degradation,competition, antibiosis and mycoparasitism tests, showed that isolates ofTrichoderma were highly antagonistic to Ganoderma, followed by isolates ofPenicillium and Aspergillus. The mean percentage inhibition of radial growth ofGanoderma mycelium in dual-culture plating for Trichoderma, Penicillium andAspergillus was 48%, 28% and 21%, respectively, as compared to controls.Dominant species of Trichoderma were T. harzianum, T. hamatum, T. longi-brachiatum, T. koningii, T. viride and T. virens (Zakaria, 1989), with T. harzianumexhibiting the highest antagonistic activity against Ganoderma. The mecha-nism of antagonism was through competition and mycoparasitism, whichimplies that early establishment of the antagonists in the plant rhizosphere androots of the palms may be crucial to produce the expected effect. Similar observations on in vitro inhibition by a range of microorganismsfrom the oil-palm rhizosphere and others, such as Trichoderma (Shukla andUniyal, 1989; Anselmi et al., 1992), Aspergillus (Shukla and Uniyal, 1989)and Penicillium (Dharmaputra et al., 1989), have been reported. In spite ofthis, there have been no reports as yet of effective biological control in infectedfield palms, nor of attempts to inject healthy palms with an antagonist to aidwith their resistance to the pathogen. The incorporation of Trichoderma, grownon dried palm-oil mill effluent (POME), into planting holes was evaluated as aprophylactic measure (Singh, 1991), but doubts over the survival of thisorganism in clay soils were raised. Preliminary observations on the distribution of the antagonistic fungiwithin the palm rhizospheres, in vitro antagonistic potential against Gano-derma, rhizosphere competency of the antagonists, and the delivery systemhave raised many unanswered questions about the potential of biologicalmanagement of Ganoderma, but to study the single or combined effects of theTable 7.1. Mean recovery rate of antagonistic fungi from oil-palm rhizospheres(× 103). Basal stem rot Total cfu g−1Location incidence (%) DW Trichoderma Aspergillus PenicilliumPrang Besar <5 155 2 19 40Brownstone > 40 25 1 1 17Sungai Buloh 5–10 58 2 14 22
92 M. Sariah and H. Zakariaantagonistic fungi on BSR infection is next to impossible in the field. This isfurther complicated by the difficulty in identification and selection of uniformdisease plots, due to the slow progress of the disease and the lack of understand-ing of the infection process and spread of the disease in the field. Therefore, asystem of artificial inoculation of seedlings was developed (Teh, 1996) inwhich the inoculum and extent of infection could be relatively quantified onseedlings, to allow testing of potential control measures in a short period undermanageable and semi-controlled conditions.Effect of Soil Amendments on the Control of Ganoderma onOil-palm SeedlingsGanoderma is probably not a very aggressive pathogen. The general belief hasbeen that heavily colonized debris acts as the inoculum source, and thatwounded roots and weakened palms facilitate penetration. This suggests thatthe fungus may be, at best, weakly pathogenic to healthy palms. Calcium isthe main macronutrient reported to strengthen the cell wall and increasemembrane permeability of plant tissues, thus further enhancing resistanceto a number of fungi, including Pythium, Sclerotium, Botrytis and Fusarium(Muchovej et al., 1980; Spiegel et al., 1987). Also, supplementation of thesoil with calcium was shown to enhance the population of soil microflora(Kommedahl and Windels, 1981) where antagonistic fungi, includingTrichoderma, Penicillium and Aspergillus, compete for space and nutrition.Thus, Sariah et al. (1996) evaluated calcium nitrate (Norsk Hydro, field gradecontaining 15% N, 19% Ca) as a prophylactic measure against BSR, due to thesoil-borne nature of the pathogen and slow establishment of the pathogen inthe host’s tissues. The treatments were as follows: T1 7.5 g CaNO3/seedling starting 1 month T2 5 g CaNO3/seedling before inoculation T3 7.5 g CaNO3/seedling starting 1 day T4 5 g CaNO3/seedling after inoculation T5 Control T6 60 g air-dried preparation of Trichoderma (108 cfu g−1) applied 1 day after inoculationCalcium applications were continued at monthly intervals over a period of6 months, whereas the antagonistic fungus was applied only once, a day afterinoculation. In addition to the above supplementations, all seedlings werefertilized with urea, and watering was done daily. The incidence of BSR wasassessed based on foliar symptoms at monthly intervals. Such that: ( a × 1) + ( b × 05) . Severity of foliar symptoms (%) = × 100 c
Use of Soil Amendments for Control of Basal Stem Rot 93where a is the number of desiccated leaves, b is the number of chlorotic leaves, cis the total number of leaves and where the numerical value of 1 represents theindex for desiccated leaves and 0.5 for chlorotic leaves. At the end of the experiment, the bole was cut longitudinally forassessment of percentage infection of bole tissues, expressed as (d/e) × 100,where d is the lesion length (mean of two measurements) and e is the bolediameter. The number of lesioned roots and production of sporophores werealso noted (Teh, 1996; Teh and Sariah, 1999). Confirmation of the disease andcausal pathogen was made by plating infected tissues on Ganoderma-selectivemedium (GSM) (Ariffin and Seman, 1991). Based on foliar symptoms, and root and bole infection, the incidence ofBSR in pot-grown oil palms was suppressed significantly when seedlings weregrown in soils supplemented with calcium nitrate 1 month prior to inoculationwith Ganoderma-infected rubber-wood blocks as the inoculum source (Fig.7.1a–c); augmentation with Trichoderma 1 day before inoculation did notsignifically reduce BSR. The number of fruiting bodies was also reduced. Inaddition, cell walls of calcium-supplemented seedlings were observed to havewell-developed lamellae, due to formation of calcium pectate, which couldstabilize the cell walls and resist degradation by cell-wall-degrading enzymes ofthe pathogen. Also, the populations of soil fungi (cfu) were significantly higherin calcium-supplemented soil as compared to calcium-deficient soil (Table7.2), but augmentation with Trichoderma alone did not have a significant effecton the fungal populations in the soil. Thus, the role of calcium in reducing BSRincidence is hypothesized as that of stabilizing and strengthening the cell wallsof the oil-palm seedlings and stimulating the proliferation of antagonistic fungithat will compete for space and nutrients. Calcium nitrate fertilization in thisstudy did not have any adverse effects on the vegetative growth of the seedlingsover the duration of the experiment, but, for continued application, thepossible interactions with the current agronomic practices of oil-palm growinghave to be studied, because calcium nitrate also contributes to the availablenitrogen. Soil augmentation with Trichoderma 1 day after inoculation did notcontrol the incidence of BSR significantly. This treatment gave the highestpercentage of disease severity 6 months after the start of the experiment. Thiscould be due to the low recovery of Trichoderma from the plant rhizosphereswith time of inoculation, suggesting that the antagonistic fungus could notsustain its population in the soil in the absence of a food base. Low ratesof recovery of Trichoderma spp. have been reported (Sariah et al., 1998).Trichoderma spp. survive better under conditions of high carbon and nitrogen,and therefore the possibility of introducing organic amendments withTrichoderma inoculants to the oil-palm rhizospheres requires consideration tocreate environmental conditions in the soil which would favour antagonisticmycoflora proliferation and distribution. The benefits of the use of organic amendments in mitigating the dele-terious effects of pathogenic soil fungi are well documented. Drenching with
94 M. Sariah and H. Zakariadrazoxolon increased rhizosphere mycoflora, especially Trichoderma species,when the chemical was applied in combination with fertilizers (Varghese et al.,1975). Following this, the possibility of chemically assisted biological controlFig. 7.1. Effect of soil amendments on (a) severity of foliar symptoms of oil-palmseedlings with time (LSD0.05 = 17.3); (b) percentage of lesioned roots of oil-palmseedlings 6 months after inoculation (LSD0.05 = 6.7); and (c) percentage of boleinfection of oil-palm seedlings 6 months after inoculation (LSD0.05 = 9.3).
Use of Soil Amendments for Control of Basal Stem Rot 95Table 7.2. Mean total population of soil fungi in the oil-palm rhizosphere,6 months after treatment. Total fungal colonies perTreatments gram air-dried soil (× 104)T1 (7.5 g CaNO3) Starting 1 month 24.50a T2 (5.0 g CaNO3) before inoculation 24.25aT3 (7.5 g CaNO3) Starting 1 day 19.50a bT4 (5.0 g CaNO3) afer inoculation 18.25abT6 (60 g Trichoderma applied 1 day before inoculation) 11.75bT5 (control) 11.10bMeans with the same letters are not significantly different at P = 0.05.of Ganoderma on tea and oil palm (Varghese et al., 1975) and on rubber wereinvestigated (Zakaria, 1989) in Malaysia. In Sumatra the possibility of neutralizing potential infection foci bio-logically in oil-palm plantations with soil additives that might stimulatemicroorganisms antagonistic to Ganoderma, especially Trichoderma spp. wasinvestigated (Hasan and Turner, 1994). At the end of the experiment theincidence of seedling infection did not differ from the unamended controls, butdelays in infection were observed at the start of the trial. This was most markedduring the first 12 months after planting. Vigorous seedling growth inresponse to the application of POME, even after removing the top 60 cm of soil,apparently delayed the appearance of disease symptoms. Other studies in Sumatra revealed that integration of 750 g per palmyear−1 of sulphur powder, Calepogonium caeruleum and spontaneous soft weedsas cover crops, and tridemorph fungicide at a concentration of 2500 p.p.m. perpalm year−1 for 5 years also showed a reduction in incidence of BSR (Purbaet al., 1994). Similarly, soil augmentation with T. harzianum, the fungus antagonistic toGanoderma lucidum, applied with green leaves, neem cake and farmyardmanure + Bordeaux mixture were effective for the management of BSR ofmature coconuts in India, and all treatments recorded significantly higher nutyield than the control (Bhaskaran, 1994). The Trichoderma population washigh in all treatments using organic manures when compared to control, butneem cake and farmyard manure sustained the highest population levels.Studies of population dynamics revealed that the population increased up tothe fourth month and then decreased drastically although the populationremained much higher than control soil, even 1 year after treatment. In a continued search for a self-sustaining method for managingGanoderma infection in oil palms, Ho (1998) tested the ability of a commercialformulation of vesicular arbuscular mycorrhizal fungi (VAM), Draz-M, toreduce, if not control completely, Ganoderma infection on mature palms. Heobserved no clear trends in terms of foliar symptoms and severity of Ganoderma
96 M. Sariah and H. Zakariaattack, but he noticed that the VAM treatment increased cumulative yieldwhen administered during the early stage of infection. As there is no shortage of such amendments in the Malaysian plantationenvironment, and coupled with the fact that chemicals or microbial amend-ments alone were not practical and cost effective in the field situation, theircombined use was investigated in the glasshouse using 4-month-old seedlingsinoculated with Ganoderma-infected rubber-wood blocks. With this method ofinoculation, 100% infection was obtained within 4 weeks after inoculationand for each infected plant, more than one-third of the bole tissues wereinfected. Sixteen treatments, singly and in combination, were being evaluated:mycorrhiza (Draz-M), T. harzianum air-dried preparation (108 cfu g−1), CaNO3(Norsk Hydro; 15% N and 19% soluble Ca) and organic matter (POME) as thesoil amendments (Table 7.3). Each treatment was replicated 16 times, witha single seedling per replication, arranged and analysed using completelyrandomized design. Parameters chosen for the above assessment were foliarsymptoms, and root and bole infections, as described earlier. Based on regression analysis (R2) foliar symptoms exhibited a significantrelationship with the number of lesioned roots and bole infection at R2 = 57%and 51%, respectively. Likewise, the higher the percentage of lesioned roots,Table 7.3. Comparative effect of treatments on severity of foliar symptoms,percentage of lesioned roots and percentage of bole infection. Severity of foliar % Lesioned % BoleTreatment symptoms (%) Treatment roots Treatment infectionT 74.12a T 100a.60 T 100a.60Cont 68.37a Cont 84.60a Cont 79.24aM+T 46.65b OM 11.10b OM 4.12bOM 40.06b M+T 9.16b M + OM 2.94bM 37.60b M + OM 8.64b M 2.56bM + OM 28.01b M 7.58b M+T 2.02b cM + Ca 20.83cd M + Ca 0c . M + Ca 0c . cCa + OM 20.67cd Ca + OM 0 c. Ca + OM 0 c. c20.57cdM + Ca + OM M + Ca + OM 0 c. M + Ca + OM 0 c.M + T + Ca + M + T + Ca + M + T + Ca + OM c20.38cd OM 0 c. OM 0 c. cT + Ca + OM 19.52cd T + Ca + OM 0 c. T + Ca + OM 0 c.Ca 17.56d Ca 0 c. Ca 0 c.M + T + Ca 17.07d M + T + Ca 0 c. M + T + Ca 0 c.M + T + OM 17.01d M + T + OM 0 c. M + T + OM 0 c.T + OM 16.51d T + OM 0 c. T + OM 0 c.T + Ca 14.60d T + Ca 0 c. T + Ca 0 c.Values with the same letters within the same column are not significant at P = 0.05(DMRT).M, Draz-M; T, T. harzianum; OM, organic matter; Ca, CaNO3; Cont, control.
Use of Soil Amendments for Control of Basal Stem Rot 97the greater was the degree of bole infection (R2 = 98%). Soil augmentationwith organic matter (OM), the air-dried preparation of Trichoderma (T) ormycorrhiza (M), singly and two-way combinations of M + T and M + OM,significantly affected the degree of disease incidence, as shown in the percent-age of foliar symptoms, lesioned roots or infection of the bole tissues (Table7.3). Typical lesions and rotting of infected roots were observed and whitemycelium was abundant on the surface of the roots. Plating of the diseasedtissues and apparently healthy bole tissues on GSM confirmed the presence ofthe causal pathogen. Addition of calcium nitrate (Ca) at 15 g per seedling,together with Draz-M (M) or POME (OM) reduced the symptom expressionfurther. The progress of the disease was slow and no sporophores wereproduced. The control treatment and seedlings supplemented with Trichoderma alonerecorded the highest disease severity. Soil amendments consisting of theair-dried preparation of Trichoderma (T) and calcium (Ca) or POME (OM), withor without Draz-M (M), gave a positive control of BSR, at least for the period ofthe experiment. Few foliar symptoms were observed, and this was supportedby the absence of lesioned roots or infection of the bole tissues. Random platingof the roots or tissues from the bole did not produce Ganoderma colonies onGSM, which suggested that the pathogen was not present in these tissues. Biological control of root-disease pathogens by enhanced activity ofantagonistic and saprophytic components of soil mycoflora has been suggestedin many disease situations, but experimental evidence of the actual mode andmethod of operation of this type of control, especially with respect to tropicalpathogens, has been scarce. The complexity of the various factors involved,the time and effort required to understand their interaction and, finally, tomanipulate suitable changes in the soil environment were not encouraging forgreater utilization of biological control. However, it is evident from the resultspresented here that control of Ganoderma in plantation crops can be imple-mented by assisted stimulation of antagonistic and saprophytic componentsof the soil microflora through the use of inorganic and organic amendments.Following the success of the pot trial, a field trial on the the use of soil amend-ments for the control of BSR is currently in progress.ReferencesAnselmi, N., Nicolotti, G. and Sanguineti, G. (1992) In vitro antagonistic activity of Trichoderma spp. against basidiomycete root rots in forest trees. Monti-e-Boschi 43, 575–579.Ariffin, D. (1994) Current status of Ganoderma research in PORIM. In: Proceedings of the First International Workshop on Perennial Crop Diseases caused by Ganoderma. UPM, Serdang, Selangor.Ariffin, D. and Seman, I. (1991) A selective medium for the isolation of Ganoderma from diseased tissues. In: Proceedings of the 1991 PORIM International Palm Oil Conference. Kuala Lumpur, pp. 517–519.
98 M. Sariah and H. ZakariaBhaskaran, R. (1994) Management of basal rot disease of coconut caused by Ganoderma lucidum. In: Holderness, M. (ed.) Perennial Crop Diseases caused by Ganoderma. CAB International, UK.Chung, G.F. (1991) Preliminary results on trunk injection of fungicides against Ganoderma basal stem rot in oil palm. In: Ariffin, D. and Sukaimi, J. (eds) Proceed- ings of Ganoderma workshop, Bangi, Selangor, Malaysia. Palm Oil Research Institute of Malaysia, pp. 81–97.Dharmaputra, O.S., Tjitrosomo, H.S. and Abadi, A.L. (1989) Antagonistic effect of four fungal isolates to Ganoderma boninense, the causal agent of basal stem rot of oil palm. Biotropia 3, 41–49.Dharmaputra, O.S., Purba, R.Y. and Sipayung, A. (1994) Research activities on the biology and control of Ganoderma at SEAMEO BIOTROP and IOPRI Marihat. In: Holderness, M. (ed.) Proceedings of First International Workshop on Perennial Crop Diseases caused by Ganoderma. UPM, Serdang. CAB International, UK.Hasan, Y. and Turner, P.D. (1994) Research at Bah Lias Research Station on Basal Stem Rot of oil palm. In: Holderness, M. (ed.) Proceedings of First International Workshop on Perennial Crop Diseases caused by Ganoderma. UPM, Serdang. CAB International, UK.Hashim, K.B. (1990) Basal stem rot of oil palm: Incidence, etiology and control. M.Agric. thesis, Faculty of Agriculture, UPM.Ho, C.T. (1998) Safe and efficient management systems for plantation pests and diseases. The Planter 74, 369–385.Jollands, P. (1983) Laboratory investigations on fungicides and biological control agents to control three diseases of rubber and oil palm and their potential applica- tions. Tropical Pest Management 29, 33–38.Kommedahl, T. and Windels, C.E. (1981) Introduction of microbial antagonist to specific courts of infection: seeds, seedlings and wounds. In: Beemster, A.B.R., Bollen, G.J., Gerlagh, M., Ruissen, M.A., Schippers, B. and Tempel, A. (eds) Biotic Interaction and Soil-borne Diseases. Netherlands Society of Plant Pathology, pp. 121–127.Loh, C.F. (1976) Preliminary evaluation of some systemic fungicides for Ganoderma control and phytotoxity to oil palm. Malayan Agriculture Journal 32, 223–230.Muchovej, J.J., Muchovej, R.M.C., Dhingra, O.D. and Maffia, L.A. (1980) Suppression of anthracnose of soybean by calcium. Plant Disease 64, 1088–1089.Purba, R.Y., Utomo, C. and Sipayung, A. (1994) Ganoderma research on oil palm and its current research in the Indonesian Oil Palm Research Institute. In: Holderness, M. (ed.) Perennial Crop Diseases caused by Ganoderma. CAB International, UK.Sariah, M., Joseph, H. and Zakaria, H. (1996) Suppression of basal stem rot (BSR) of oil palm seedlings by calcium nitrate. The Planter 73, 359–361.Sariah, M., Zakaria, H., Hendry, J., Shanji, G.T. and Chung, G.F. (1998) The potential use of soil amendments for the suppression of basal stem rot of oil palm seedlings. In: Second Workshop on Ganoderma Diseases of Perennial Crops. Serdang, Selangor. CAB International, UK.Shukla, A.N. and Uniyal, K. (1989) Antagonistic interactions of Ganoderma lucidium (Leyss.) Karst. against some soil microorganisms. Current Science 58, 265–267.Singh, G. (1991) Ganoderma: The scourge of oil palm in coastal areas. Planter 67, 421–444.Spiegel, Y., Netzer, D. and Kafkafi, U. (1987) The role of calcium nutrition in Fusarium wilt syndrome in muskmelon. Phytopathologische Zeitschrift 118, 220–226.
Use of Soil Amendments for Control of Basal Stem Rot 99Teh, K.S. (1996) Curative activity of fungicides against basal stem rot of oil palm. M.Agric. Sc. thesis. Faculty of Agriculture, UPM.Teh, K.S. and Sariah, M. (1999) Improved inoculation technique for testing patho- genicity of Ganoderma boninense on oil palm seedlings. In: Plant Protection in The Information Age. Fourth MAPPS International Conference on Plant Protection In the Tropics, pp. 142–145.Varghese, G. (1972) Soil microflora of plantation and natural rain forest of West Malaysia. Mycopathologia et Mycologia Applicata 48, 43–61.Varghese, G., Chew, P.S. and Lim, J.K. (1975) Biology and chemically assisted bio- logical control of Ganoderma. In: Proceedings of the International Rubber Conference, Kuala Lumpur, pp. 278–292.Zakaria, H. (1989) Some aspects of the biology and chemically assisted biological control of Ganoderma species in Malaysia. PhD thesis, Faculty of Agriculture, UPM.
102 J. Flood et al.disease incidence is a practical impossibility, and so the aim has been to con-centrate on removal of as many of the larger tissue sections as economicallyfeasible. To investigate the efficacy of sanitation in BSR management, a seriesof trials was undertaken at Bah Lias Research Station (BLRS) of P.T.P.P.London in North Sumatra, Indonesia over a period of several years. The trialswere designed to assess the relative importance of various tissue remnantsfrom the old palm stand as potential sources of inoculum at replanting (Hasanand Turner, 1998) so as to make practical recommendations for managementof the disease in Sumatra. The trials were set up as to be sufficiently large to overcome any variationsin BSR inoculum, with each treatment being replicated at different sites in theplantation. Six-month-old seedlings were used to bait the Ganoderma-infectedmaterial. External leaf symptoms developing on these bait seedlings wererecorded for the duration of each trial, while at the end of each trial, allseedlings were examined internally for Ganoderma infection by destructivesampling and plating to Ganoderma-selective medium (GSM) (Ariffin and Idris,1991). Each experimental plot was isolated by a deep trench to increase thelikelihood that any infection recorded was derived from the tissue being testedand not from an outside source, but more recently, molecular fingerprintingtechniques (Miller et al., this volume; Bridge et al., this volume; Rolph et al., thisvolume) became available which allowed confirmation of the origin of thepathogen in infected seedlings.Stump TissuesA stump comprises the base of the palm, or bole, and the thick crust of rootsimmediately surrounding it. Stumps are usually recognized as major sources ofBSR. The first trial compared BSR stumps, prepared by felling diseased palmsabout 20 cm above the ground (standard practice), as an inoculum sourcewith stumps derived from healthy palms. Around each stump eight bait seed-lings were planted. An additional treatment, planting additional seedlingsimmediately outside the plot isolation trench and isolating these by a furthertrench 1 m from the inner trench, in order to emphasize disease origin, wasadded. Each treatment was replicated eight times at different sites. Six monthsafter planting, a small number of seedlings began to exhibit disease symptomsand by the end of the 28-month trial period, 76% of all bait seedlings showedsymptoms; Ganoderma was isolated from these seedlings. In comparison, seed-lings planted outside the first trench and within the second isolation trenchperimeter showed very little infection – only 1.6% of these seedlings werediseased and, at 80% of the replicate sites, these seedlings exhibited nosymptoms at all. No disease was recorded in bait seedlings planted aroundhealthy palm stumps within the period. Another trial aimed to assess the effect of stump size on disease incidence.Additional treatments in this trial were comparisons with stumps derived from
Spread of Ganoderma from Infective Sources in the Field 103healthy palms and the effects of pre-felling poisoning by paraquat, using 60 mlper palm Gramoxone which was injected into the trunk. Stump size was foundto exert a marked influence on disease occurrence, with more bait seedlingsaround smaller, lower stumps (20 cm high) exhibiting disease symptoms after2 years than those around larger, higher stumps (50 cm high). Rate ofdecomposition and bait seedling root ingress into Ganoderma-colonized tissueswould appear to be the most likely explanations for the difference. The effects ofpoisoning, which had accelerated tissue breakdown, supported this, with moreseedling infection recorded around larger stumps where poisoning treatmenthad been carried out. The importance of inoculum sources at different soil depths adjacent toBSR-infected stumps, which is of considerable relevance to sanitationpractices, was also investigated. Thus, soil and palm tissue adjacent to BSR-infected stumps were removed to one of the following depths: 20, 40, 60, 80and 100 cm. Eight replicate bait seedlings were planted at each depth andthese treatments were compared with diseased stumps that were undisturbedafter felling (no soil or tissues removed) and sites around healthy palmsexcavated to a depth of 60 cm. In the absence of any sanitation, 75% ofseedlings had become infected and 97% of replicate sites had infected plantswithin 2 years of planting (Table 8.1). In comparison, disease incidence in thebaited seedlings decreased to 21% where soil and debris had been removed to adepth of 60 cm, and no disease was recorded where soil and debris had beenremoved to 80 or 100 cm (Table 8.1). In an extension of this trial, the same sites were replanted with baitseedlings after 2 years and no disease was recorded at any depth 2 years later.Similarly, when new bait seedlings were planted around previously highlyinfective diseased stumps after 2 years, none of these bait seedlings developedsymptoms. Even after 2 further years of recording, these seedlings remainedsymptomless, which would suggest that the potential of these stumps to act assources of inoculum had declined after 2 years. Data of percentage infectionover time at two sites (Table 8.2) further supported the view that fewer seed-ling infections occurred after 20–24 months. Some variation between sites isTable 8.1. Effects of the removal of soil and palm tissues from around healthybasal stem rot (BSR)-infected stumps on disease incidence after 24 months. Depth of bole % Replicate sites with % SeedlingsDisease status removed (cm) infected seedlings infectedBSR 0 97 75BSR 20 85 58BSR 40 70 28BSR 60 55 21BSR 80 0 0BSR 100 0 0Healthy 60 0 0
104 J. Flood et al.to be expected since the amount of infective tissue within stumps and itslocation in relation to seedling root contact will differ considerably, as will therates of subsequent decay. During the course of these trials, molecular fingerprinting techniquesbecame available for Ganoderma and were used to confirm the origin of theGanoderma from infected bait seedlings. Material was collected from diseasedseedlings, stump tissues and sporophores growing on the stumps andisolations made on GSM. DNA was extracted from pure cultures (Miller et al.,1999) and purified DNA samples tested with the ITS3/GanET primer (Bridgeet al., this volume) to check their identity. All isolates were positive with theITS3/GanET primer, confirming that the pathogen had been isolated fromthe various tissues (Fig. 8.1). Mitochondrial profiles were generated using theenzyme HaeIII as the restriction enzyme (Miller et al., 1999; Rolph et al., thisvolume) and revealed that identical profiles were present in the BSR stumpsand the infected bait seedling material (Figure 8.2). Table 8.2. Percentage of total bait seedling infection appearing around basal stem rot stumps over time. % Seedling infection Months after planting Site A Site B 6–8 8 11 9–12 45 32 13–18 20 35 19–24 20 14 25–28 7 8Fig. 8.1. Confirmation of the presence of the pathogen from stump tissues andinfected seedlings.
Spread of Ganoderma from Infective Sources in the Field 105 Fig. 8.2. Mitochondrial DNA restriction fragment length polymorphisms of Ganoderma isolates from an infected basal stem rot stump and from a baited infected seedling planted near the infected stump. As mitochondrial (mtDNA) inheritance is believed to be unilinear (Forsterand Coffey, 1990), isolates from the same sibling family would therefore havethe same profile. However, generally, mtDNA profiles are highly variable inGanoderma isolates, even from the same and adjacent oil palms (Miller et al.,1999). Thus, identical mtDNA profiles from BSR-infected stumps and frominfected bait seedlings may indicate that mycelial spread or root-to-root con-tact has occurred, but, equally, the role of basidiospores cannot be ruled out(Miller et al., 1999). To clarify this point, a third molecular profiling techniquewas used, namely amplification fragment length polymorphisms (AFLPs), asdescribed by Vos et al. (1995). This technique assesses the total cellular DNAprofile (nuclear and mitochondrial DNA) and is a more stable and reliablemethod of studying variation (Rolph et al., this volume). Identical AFLP profileswere produced using several primers, including primer E (Rolph et al., thisvolume) (Fig. 8.3) confirming that the baited seedlings were infected with thesame genotype as that in the BSR-infected stump.Trunk TissuesUnless trunks of the old palm stand are destroyed at the time of replanting, theyare usually windrowed, i.e. placed in rows. Such trunks are colonized by manyspecies of fungi, including Ganoderma. Trunks will also remain following anumber of estate practices, e.g. following underplanting, those excavated aslow-yielding, palms removed for thinning or road construction and excavateddiseased palms, and palms affected by upper stem rot (USR) often remainstanding for long periods, as do palms killed by lightning. The trials summa-rized below assessed the significance of trunk sections as sources of BSR
106 J. Flood et al. Fig. 8.3. Amplification fragment length polymorphisms from basal stem rot stump, baited seedling and Ganoderma sporophore (fruit body) growing on the infected stump.following various treatments and compared these sources with BSR-infectedstumps. Palms were felled as close as possible to the ground and the trunk thencut at 1 m and 4.75 m from the base, with the remainder being discarded. Thestump and each trunk section were isolated by trenches and bait seedlingswere planted close to the sections. Apparently healthy palms were alsoincluded. Stump tissues remained the most important source of BSR, with 27–38%seedling infection occurring, and although the incidence of disease arisingfrom trunk sections was much lower (Table 8.3), this would remain ofconsiderable practical significance. There was a marked increase over the 2-year period in the number ofinfection foci on what had previously been considered as healthy stumps, withthe highest disease incidence (12%) being recorded where palms had beenpoisoned before felling and where legume overgrowth had been successful.The presence of diseased seedlings around what had previously been consid-ered to be healthy palms would indicate that the pathogen is present in thepalm for what maybe a considerable time before symptoms are seen. Infection rates of bait seedlings when planted around standing diseasedand apparently healthy palms were compared with that from stumps; theinfection rate of bait seedlings around standing palms was much lower(Table 8.4). However, the period of infectivity of standing palms is likely to bemuch longer, demonstrating the need to remove such palms in managementof the disease. Also, while diseased tissues appear to lose much of their infectiveability from about 18–20 months after felling, the majority of apparentlyhealthy stumps and trunks had yet to show the extent to which they would
Spread of Ganoderma from Infective Sources in the Field 107Table 8.3. Basal stem rot incidence in bait seedlings around oil-palm residues. % Seedling % Seedling infection % Seedling infection infection around around proximal around distal trunks stumps trunks (stem) (stem)Diseasestatus Treatment Yr1 Yr2 Yr1 Yr2 Yr1 Yr2Diseased Nil 9 38 2 3 2 2Diseased P 13 34 3 5 2 3Diseased C 19 34 4 6 1 2Diseased PC 17 27 4 5 5 6Healthy Nil 0 6 1 1 1 1Healthy P 1 9 2 3 1 1Healthy C 0 7 5 6 4 4Healthy PC 4 12 1 1 1 3P, Poisoned before felling; C, legume cover.become sources of disease at the end of 2 years (Table 8.4). However, fromthe 2-year data alone, it is clear that under field conditions they will certainlypresent a significant disease risk. In another trunk treatment, pieces were cut to simulate shredding as aclearing method, with and without poisoning prior to preparation. These wereeither placed on the soil surface or buried at 20 cm deep. Both infected andhealthy trunk tissues were examined, with seedling baits used to detect BSRin plots isolated by trenches. Both diseased and healthy shredded tissues cangive rise to disease after burial. Except in a single instance, superficially placedtissues were not a disease hazard. In plots with buried tissues where diseasewas recorded, sporophores of Ganoderma were produced on the soil surface.RootsThe current recommendation for BSR sanitation procedure concentrates on a1.5 m square centred on the point where the palm is planted. The assumptionhas been that the remaining inter-space presents no serious disease hazard. Ina trial to examine this, areas between neighbouring diseased palms were eachdivided into three equal parts and isolated by deep trenches. Bait seedlingswere then planted in each sector, as well as around the bases of the BSR-affected palms. Similar sectors between apparently healthy palms were alsobaited. In the BSR plots, Ganoderma fructifications developed on cut rootends, signifying the presence of infected roots. The overall incidence of seedlinginfection was low (4%) and was confined to the sectors closest to the diseasedpalms, whereas 69% of bait seedlings planted around the main disease sourcesbecame infected. No disease was recorded between healthy palms.
108 J. Flood et al.Table 8.4. Comparison of basal stem rot (BSR) in bait seedlings around standingBSR and healthy palms compared with stumps, 2 years after treatment. Standing palms Low stumpsDisease % Infective % Seedling % Infective % Seedlingstatus Treatment sites infection sites infectionBSR Nil 30 6 90 38BSR P 40 10 95 34Healthy Nil 20 3 20 6Healthy P 30 7 40 9P, Poisoned. Also, records of the production of Ganoderma sporophores on cut ends ofroots on the inside of isolation trenches from the depth trial (Table 8.1)revealed that where no soil or palm tissues had been removed, 67% of allreplicate sites had Ganoderma sporophores, while where soil had been removedto a depth of 60 cm, this had decreased to only 10%. Thus, diseased roots cancomprise a small, but still significant, source of BSR in a replant, although thisprobably requires dense root aggregations.DiscussionIt is apparent from these results that, when suitable disease sources arepresent, oil-palm seedlings can be attacked by Ganoderma soon after planting.Disease development and overt symptom appearance will depend on the size ofthe palm when it becomes diseased, its continued growth vigour and the sizeof the inoculum. Small seedlings close to large disease sources are killedrapidly. Larger, rapidly growing plants are also affected, but frequently donot die quickly. Numerous investigations have reported that many infectedpalms continue to grow well, often for very long periods, before the internalBSR lesion becomes so extensive that visible external symptoms develop. Thisexplains why so many cases of BSR occur long after planting and also afterobvious sources of primary infection have disappeared. Once a few palms in a field are infected it has been considered that furthercolonization of palms in the field is due to root-to-root contact by the palms ormycelial spread. Both Singh (1991) and Hashim (1994) reported the disease asoccurring in patches or groups, which would support palm-to-palm infection,but this view has been challenged recently by Miller et al. (1999). Studies ofsomatic incompatibility and mtDNA profiling of isolates taken from manyadult palms within two oil-palm blocks (Miller et al., this volume) revealed con-siderable variation between isolates, and led to the conclusion that isolatesoccurred as numerous distinct genotypes, even within the same palm. Thus,
Spread of Ganoderma from Infective Sources in the Field 109mycelial spread to adjacent palms or root-to-root contact was very unlikely.Ariffin et al. (1996) similarly reported a high degree of heterogeneity betweenisolates taken from adjacent infected adult palms. This contrasts withother wood-rotting fungi, such as Heterobasidion annosum (Stenlid, 1985) orPhellinus noxius (Hattori et al., 1996), where one clone of the pathogen canextend over several metres. However, the preliminary mtDNA and AFLPprofiling described here has demonstrated that the same genotype is present inthe diseased stump and in baited seedlings. Thus, the experimental assumptionthat the infected BSR stump acts as a direct source of infection to the youngseedlings was validated. Infection probably occurred due to the growth of seed-ling roots towards the decaying stump which is a rich source of nutrients.However, molecular analysis has only been conducted on a small number ofstumps, and other sources of infection for young seedlings in the field cannot beruled out. To date, the role of basidiospores has never been fully explained inthis disease. Thompson (1931) suggested that they were responsible for USR,usually in association with Phellinus spp., but Turner (1965) failed to infect oilpalm following direct spore inoculation of cut frond bases, and Yeong (1972)reported no infection following direct inoculation of oil-palm seedlings.However, it is possible that basidiospores could infect palms indirectly, i.e. areable to colonize debris which subsequently becomes the source of infectionfor living palms (Miller, 1995). This would account for the heterogeneitydetermined using molecular markers (Miller et al., 1999). Thus, much moremolecular analysis remains to be conducted – so far only diseased stumpshave been studied, but trunks and even roots can act as significant sources ofinfection. The investigations reported here have confirmed that the times of greatestpractical significance for the control of Ganoderma in oil palm are: (i) soonafter planting, when suitable inocula remain in the ground from the previousplanting (oil-palm stumps or root debris); and (ii) later in the planting cycle,when root contact is made with Ganoderma-colonized sections of palm trunksresting on the ground in rows (windrows). Results of this study would seem tosuggest that this danger extends over a much longer period when windrowedpalms are not poisoned prior to felling and are not covered by legumes toaccelerate decomposition. Fungi that cause root disease frequently require substantial inoculumpotential before they are able to initiate infection and subsequently becomeestablished within the host plant. Thus, infection must require either a block ofGanoderma-colonized tissue of adequate size or a conglomerate of tissues, e.g. amass of infected roots, which collectively become an infection source. In thetrials summarized here, the importance of large blocks of inoculum is evident.Bait seedling infection was very rapid when planted close to BSR-infectedstumps. Gradual removal of this source with increasing depth showed a clearrelationship between availability of infective material and both the occurrenceand incidence of BSR. This was not confined to the stump tissues. At a depth of60 cm there was no mass of stump tissue, only a few infected roots, but these
110 J. Flood et al.root masses can become significant sources of infection. Even where a field thathas been carefully cleaned of debris at replanting, as the new seedlings grow,more and more root debris is produced. This will include a large amount of rootmaterial from self-pruning (Hartley, 1988; Jourdan and Rey, 1996) and largenumbers of fine quaternary roots are present in the upper layers of the soil. Thehypothesis that this material could become the substrate for basidiosporecolonization requires further study. The depth factor poses considerable problems from the practical viewpointof sanitation at the time of clearing for replanting. Breaking up deeply locatedroot masses requires deep tining, for which equipment is not always available. If seedlings are planted at the same points of former BSR palms, there is adistinct possibility that their roots will soon encounter infective sources ofGanoderma, and thus as much of the diseased stump tissue as possible should beremoved. However, further baiting using seedlings showed that these potentialBSR sources were less of a disease hazard after 2 years. This means that theirimportance could be expected to be very much reduced, or even negligible, ifnew palms are planted as far as possible from the old planting points. Theirdisease potential would have greatly diminished by the time the roots of thenew planting reach the hazard sources, provided the old stand had beenpoisoned before felling. Alternatively, delayed planting could be a usefulmethod of disease avoidance. Windrowed palm trunks represent another significant problem, and thesame considerations apply to the necessity for planting as far away as possiblefrom windrows. The lateral extent of root development during immaturityreaches roughly the edge of the canopy, meaning that it should take 2–3 yearsbefore reaching this particular disease source if planted at the furthest possibledistance. An important observation is that the period over which windrowsremain a disease hazard is greatly reduced when palms of the old stand arepoisoned by paraquat prior to felling, and this effect is further enhancedwhen they are cut into sections and with a thick overgrowth of legume cover.Where there has been no poisoning, the tissues remain a disease hazard foryears. In such situations older palms of the replant become infected, with overtdisease symptoms only appearing long after the original infection sources havedisappeared. One solution is to shred palm tissues so that they do not become BSRsources over long periods, which is already a common practice in Malaysia butnot in Sumatra. However, even this does not provide a total answer to theproblem. Occurrence of BSR in bait seedlings, arising from buried, shreddeddiseased and healthy trunk segments, was limited, but illustrated that thetechnique still contains a degree of disease risk. Disease arising fromsuperficially placed segments was very slight and unexpected. It was in someways remarkable that in such segments, buried or superficially placed, diseaseoccurred at all, since many attempts at artificial inoculation of seedlings inpolybags using such tissues have failed. The appearance of Ganoderma sporo-phores on the soil surface above buried BSR sections indicated that a sufficient
Spread of Ganoderma from Infective Sources in the Field 111mass of Ganoderma-colonized tissue can overcome the inhibitory effects in soilwhich normally prevent its development there. Another possible BSR control method for the future lies in the fact thattrunk tissues, in particular, support the rapid development of many fungi otherthan Ganoderma, and this points to a possible biological control approach to thewindrow disease hazard problem. Rapid degradation of the windrowed tissues,especially by fungi antagonistic to Ganoderma, would have obvious advantagesfor BSR and Oryctes control. However, this approach needs more investigation,not least because woody tissues contain very little nitrogen, this influencingthe extent of colonization by certain rotting microorganisms, so that manipu-lation of the nitrogen status of the debris will need to be conducted (Patersonet al., this volume).AcknowledgementsThis chapter is published with the permission of P.T.P.P. London, Sumatra,Indonesia. The considerable assistance of field staff in the execution of trials isgratefully acknowledged. The authors would like to thank the Crop ProtectionProgramme (CPP) of the Department for International Development (DFID) forfunding some of the research reported here, which was administered throughNRI (RNRRS Project 6628).ReferencesAriffin, D. and Idris, A.S. (1991) A selective medium for the isolation of Ganoderma from diseases tissues. In: Basiron et al. (eds) Proceedings of the 1991 International Palm Oil Conference, Progress, Prospects and Challenges Towards the 21st Century, September 1991. PORIM, Selangor, Malaysia, pp. 517–519.Ariffin, D., Idris, A.S. and Azahari, M. (1996) Spread of Ganoderma boniense and vegetative compatibility studies of palm isolates in a single field. In: Darus et al. (eds) Proceedings of the 1996 PORIM International Palm Oil Congress – Competitive- ness for the 21st Century. PORIM, Malaysia, pp. 317–329.Forster, H. and Coffey, M.D. (1990) Mating behaviour of Phytophthora parasitica: evidence for sexual recombination in oospores using DNA restriction fragment length polymorphisms as genetic markers. Experimental Mycology 14, 351–359.Hartley, C.W.S. (1988) The Oil Palm. Longman Scientific and Technical Press, UK.Hasan, Y. and Turner, P.D. (1998) The comparative importance of different oil palm tissues as infection sources for basal stem rot in replantings. Planter 74, 119–135.Hashim, K.B. (1991) Results of four trials on Ganoderma basal stem rot of oil palm in Golden Hope Estates. In: Proceedings of the Ganoderma Workshop organised by PORIM, Selangor, Malaysia, September 1990.Hashim, K.B. (1994) Basal stem rot of oil palm caused by Ganoderma boninense – an update. In: Sukaimi et al. (eds) Proceedings of the PORIM International Palm Oil Congress – Update and Revision (Agriculture) 1993. PORIM, Malaysia.
112 J. Flood et al.Hattori, T., Abe, Y. and Usugi, T. (1996) Distribution of clones of Phellinus noxius in a windbreak on Ishigaki Island. European Journal of Forest Pathology 26, 69–80.Jourdan, C. and Rey, H. (1996) Modelling and simulation of the architecture and development of the oil palm (Elaeis guineensis) root system with special attention to practical application. In: Darus et al. (eds) Proceedings of the PORIM International Palm Oil Conference – Competitiveness for the 21st Century. PORIM, Malaysia, pp. 97–110.Miller, R.N.G. (1995) The characterization of Ganoderma populations in oil palm cropping systems. PhD thesis, University of Reading, UK.Miller, R.N.G., Holderness, M., Bridge, P.D., Chung, G.F. and Zakaria, M.H. (1999) Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology 48, 595–603.Singh, G. (1991) Ganoderma – the scourge of oil palm in the coastal areas. Planter 67, 421–444.Stenlid, J. (1985) Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility and isozyme patterns. Canadian Journal of Botany 63, 2268–2273.Thompson, A. (1931) Stem rot of oil palm in Malaysia. Bulletin of the Department of Agriculture of the Straits Settlements and F.M.S. Science Series, Serdang 6.Turner, P.D. (1965) Infection of oil palms by Ganoderma. Phytopathology 55, 937.Turner, P.D. (1981) Oil Palm Diseases and Disorders. Oxford University Press, Oxford, pp. 88–110.Vos, P., Hogers, R., Bleeker, M., Reijans, H., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995) AFLP – a new technique for DNA- fingerprinting. Nucleic Acids Research 23(21), 4407–4414.Yeong, W.L. (1972) Studies into certain aspects of the biology of wood decay pathogens of Hevea rubber and oil palm (Elaeis guineensis). Bulletin of the Agricultural Science Project Report, University of Malaya.
114 F.R. Sanderson et al.infecting living oil palm, only colonizes dead palm species. It is found readily,often in very large numbers, on coconut stumps and logs, 2 and 3 years afterfelling. In Papua New Guinea (PNG) and the Solomon Islands, despite constantmonitoring, we have never found G. boninense colonizing newly felled hard-wood stumps or logs within 2–3-year-old oil-palm plantings, nor on oldhardwood stumps and logs in established oil palm. The role of coconut is well demonstrated in Milne Bay, where levels ofinfection in blocks of oil palm planted into coconut north of the Naura River in1987 (Fig. 9.1) are consistently higher than the incidence of BSR in thoseblocks planted into cleared forest south of the river. If, as our observations suggest, G. boninense does not colonize hardwoodspecies, then the presence of BSR in the forest blocks south of the river is moredifficult to explain. Research by two independent groups in the early 1990s (Miller et al.,1994; Ariffin et al., 1996) showed that cultures derived from G. boninensebrackets collected from different palms, including cultures from adjacentpalms, when confronted in a Petri dish in the laboratory, develop a soliddemarcation line where the two cultures met. This somatic incompatibilitydemonstrates that isolates, even from adjacent palms, were unrelated. Milleret al. (1994) also studied the mitochondrial DNA (mtDNA) from the sameisolates and confirmed the above findings. The results of both studies are hard to reconcile with the single idea ofroot-to-root contact, as isolates from adjacent palms would, by association, bethe same clone. They would thus be compatible in culture and have the samemtDNA banding patterns. The alternative, as suggested by both Miller et al.(1994) and Ariffin et al. (1996), is that basidiospores are also involved at somepoint in the epidemiology of the disease. Strong evidence for the involvement of basidiospores can be found if welook at the survey data from four divisions of oil palm planted between 1987and 1989 in the Solomon Islands (Table 9.1). The incidence of infection withinthese blocks ranges from 0% in some of the Mbalisuna and Tubutu blocksFig. 9.1. The incidence of basal stem rot (BSR) in oil-palm blocks planted intofelled coconut and cleared forest.
Control Strategy for BSR of Oil Palm: Basidiospores 115Table 9.1. Survey data of oil palms planted between 1987 and 1989. Mean Range of Number Year incidence incidences of Total of of infection between blocks blocks ha plantingMbalisuna 0.8 .10–1.8 15 453 1987 Oil palm after forestTubutu 1.1 .10–2.1 17 297 1989 Oil palm after forestMetapona 2.0 1.3–2.8 12 186 1988 Oil palm after padiNgalimbiu 3.5 1.3–10.2 29 765 1987 Second-generation oil palmwhich were planted into cleared forest, to 10.2% for a block of second-generation oil palm in Ngalimbiu. Of greatest interest, however, are the 12 Metapona blocks, representing anarea of 186 ha, which were planted into land that had been used for growingrice for the previous 10 years. It is difficult to explain, almost to the point ofbeing inconceivable, that a level of infection equal to or higher than that in theoil-palm blocks out of forest could have arisen by roots coming into contactwith inoculum buried in the soil, in land which had been under cultivationfor the previous 10 years. This was not village rice, but a large commercialoperation where the land had been prepared using heavy machinery and theapplication of chemicals was from the air. If the infection did not arise from an inoculum source within these blocks,then the most likely alternative was for the infection to have been initiated, ashinted at by both Ariffin et al. (1996) and Miller et al. (1994), by basidiospores,which originated from outside the block. Once this concept is accepted then it isan easy step to explain the presence of infection in the forest blocks south of theNaura River (Fig. 9.1) and, similarly, the infection in the Mbalisuna andTubutu blocks in the Solomon Islands planted in 1987 and 1989 (Table 9.1). Our research commenced in 1995 to test the hypothesis suggested by bothAriffin et al. (1996) and Miller et al. (1994) that basidiospores are involved inthe epidemiology of BSR of oil palm, and our research continues to support thisview. Early work at Milne Bay (Sanderson and Pilotti, 1997) revealed thatGanoderma has a highly developed mating system. Unlike the mating systemnormally associated with Ganoderma species, which is based on two loci andtwo alleles, the mating system of G. boninense is based on two loci and multiplealleles. Under such a regime, mating is only restricted within the family. Itis therefore a mating system which strongly encourages outcrossing andmaximizes the ability of the fungus to experiment with new combinations ofaggressiveness genes, which, because of the selection pressures at infection,will inevitably lead to a build-up of the aggressiveness within the Ganodermapopulation. It may hypothetically lead to the infection being seen earlier, andin higher numbers, in each subsequent planting. This is exactly the situationthat has been described as occurring over the past four decades in Malaysia.
116 F.R. Sanderson et al. If basidiospores are involved in the life cycle, then a fundamental changein our thinking is required, regarding the epidemiology of the disease, whichagain requires a major change in our thinking regarding control. Thus, if asource of G. boninense is sporulating in the vicinity, either on dead coconutor oil palm, and the physical conditions are suitable, then no matter howcomplete the hygiene is at the time of replanting, infection will occur. Control is therefore no longer only dependent on the removal of allinfected wood material, whether below or above the ground at the replantingsite, but also on the maintenance of a zero incidence of G. boninense brackets inall areas of the oil-palm plantation and surrounding vegetation. With thisobjective, a control strategy was developed and implemented in both Milne Bayin Papua New Guinea and in the Solomon Islands.The Control StrategyThere are three phases to the implementation of the control strategy:• during establishment;• during the growing cycle; and• during replant.Control during establishmentPlanting into cleared forest in a region free of old coconut plantations is thesimplest and surest way to ensure an oil-palm crop with no, or insignificantlevels, of BSR. Any coconut plantations within the region immediately put theyoung crop at risk to infection from G. boninense. To plant into felled coconut is to provide the scenario for infection, as it isinevitable that the dead coconut will be invaded by species of Ganoderma.Whether it is G. boninense or other species of Ganoderma will depend on the localpopulation of Ganoderma, which in turn will depend on the area of oil palmalready planted within the region, the number of generations of oil palm, andthe extent of the infection of BSR. If the initial economic losses are likely to be low, such as in regions with noor a very short history of BSR, then the complete removal of coconut logs andstumps is not justified. In such instances, our objective is to leave the materialremaining from the previous vegetation in such a state as to limit both bracketproduction and spore movement, and to provide minimal breeding sites forinsects such as Oryctes. This is done by leaving as many logs as is practicalstacked above the ground, out of contact with soil moisture, and to encouragea rapid establishment of ground cover. Control in these situations commencesat year 6 onwards, with the appearance of infection within the oil-palm crop. In areas with a history of BSR, then the economics of clearing the area ofall coconut stumps and logs has to be considered carefully, as having cleared
Control Strategy for BSR of Oil Palm: Basidiospores 117all the felled coconut stumps and logs, high levels of infection are still likely tooccur from inoculum arising within the surrounding areas.Control during the growing cycleControl during the growing cycle is based on surveys which commence at year6. These are carried out every 6 months to identify infected palms, which aremarked as either infected with brackets or infected without brackets (we use5 cm PVC adhesive tape in either yellow or orange so that the palms can beidentified from 100–200 m). As infected palms are identified, the following data are also recorded:• physical location: block number, harvest road, palm row and palm number;• symptoms: degree of yellowing, number of collapsed fronds, extent of basal frond rot and basal rot;• the number of brackets;• fertility: the presence of male flowers or fruit bunches;• previous vegetation: coconut, forest or oil palm.Initially these data were hand-written onto a form and manually entered intothe company’s database. Data were later collected directly into a hand-heldGPS (global positioning system) receiver (either a Magellan ProMark X usingMSTAR software with a second ProMark X as the base station, or a TrimbleTDC1 receiver and a Trimble Pathfinder Community Base Station), whichnot only records the geographical location but also acts as a data logger. Thedata are downloaded into the company database at the end of each day. Theadvantage of the GPS receiver, apart from the ease of entering the data into thedatabase, is the ability to produce a map of the distribution of infection withinthe plantation. A list of palms for removal is then printed and appropriate action taken.Our aim is to have the infected palms identified and removed within 1 week.Palms with brackets are felled and all infection cut from the trunk and removedfrom the plantation. The trunk base and root ring is removed to a depth of10–15 cm below ground level and the hollow filled with soil. As long as theinfected roots are covered with soil, brackets will not develop. Palms without brackets fall within two categories: tolerant palms andpalms with no fruit bunches. Tolerant palms have no top symptoms and,although in many instances they have extensive basal rot, they are still pro-ductive. These palms are harvested, and monitored during subsequent surveysfor future development of brackets. In our experience only a few of these palmsdevelop brackets at a later date. Palms without brackets and not producingfruit bunches are considered sterile and treated accordingly.
118 F.R. Sanderson et al.Control during replantingsAs with all control strategies during the replanting cycle, we emphasizethe necessity of removing all infected plant material lying on the soilsurface. Where we differ, is in the extent to which we remove the old rootsystem. The significance of the root ball as an infection source, as suggested byHasan and Turner (1998), will diminish, and become negligible, as long as theseedling palms are planted as far as possible from the old palms. After each palm is pushed over, the broken trunk base and root ring arescooped out to a depth of about 30 cm. The hollow is filled with soil and thestem base and root ring removed from the site, along with the infectionremoved from the trunk by chainsaw. Care has to be taken to ensure that allinfection is removed from the site. Exposed basal rot on the trunk, or a root balltoo large to be physically removed, are both scenarios for extensive bracketproduction. The control process at replant starts 2 years before the actual plantingdate. During this period all remaining palms with symptoms, both thosewith and without brackets, are felled and all infected material removed.Care must be taken during the felling of the remaining healthy palmsprior to replanting. All palms must be checked and any previously undetectedinfection, both in the root ring and trunk, must be removed from the plantingsite.DiscussionThere is sufficient evidence in the literature and from field observations tosupport the hypothesis that basidiospores of G. boninense are involved in thelife cycle of BSR of oil palm. There is a danger, however, that because we canstill only speculate about this, the involvement of basidiospores is considered oflittle consequence. On the other hand, the implications are far reaching. If the sexual stageis involved, then segregation will take place, including characters foraggressiveness. During the infection process, regardless of how this occurs,selection pressures will inevitably lead to increased aggressiveness. This inturn will lead to infection being detected earlier and in greater numbers,exactly as has occurred in Malaysia and Indonesia. Secondly, if basidiosporesare involved in the epidemiology, then the success or failure of the controlstrategy not only depends on the actions being taken during the replantingcycle but, concurrently, how well control is being maintained in all otherfacets of plantation management and surrounding vegetation. This degree ofcontrol will, in many instances, be unattainable.
Control Strategy for BSR of Oil Palm: Basidiospores 119AcknowledgementsIt is with gratitude that we thank the European Union for funding for thisproject under the STABEX programme. The assistance of the staff at all levelsfrom Pacific Rim Plantations Pty Ltd, New Britain Oil Palm Pty Ltd, and HargyOil Plantations Pty Ltd, is also gratefully acknowledged.ReferencesAriffin, D., Seman, I.A. and Azahari, M. (1996) Spread of Ganoderma boninense and vegetative compatibility studies of a single field Palm isolates. In: Proceedings of the PORIM International Palm oil Congress, Kuala Lumpur, Malaysia.Hasan, Y. and Turner, P.D. (1998) The comparative importance of different oil palm tissues as infection sources for basal stem rot in replantings. The Planter 74(864), 119–135.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.R.M., Sariah, M. and Hussin, M.Z. (1994) Understanding Ganoderma population in oil-palm. Paper presented at the Workshop on Prennial Crop Diseases Caused by Ganoderma. Universiti Pertanian Malaysia, Serdang, Malaysia, December.Sanderson, F.R. and Pilotti, C.A. (1997) Ganoderma basal stem rot: an enigma, or just time to rethink an old problem. The Planter 73(858), 489–493.
122 R. BhaskaranManagement of the DiseaseEffect of Trichoderma harzianum application with organic manuresA field experiment was initiated during May 1992, to study the effect ofT. harzianum, the fungus antagonistic to G. lucidum, in the control of BSR. Theantagonist was multiplied in rice bran–sawdust medium and applied to thebasins of the diseased tree with different organic manures. The treatmentswere given once a year. The results showed that T. harzianum applied withgreen leaves, neem cake (NC) or farmyard manure + Bordeaux mixture(FYM + BM) were more effective for the management of the disease than othertreatments and control (Table 10.1). All the treatments recorded significantlyhigher nut yields than the control. FYM, FYM + BM and neem cake treatmentswere superior to other treatments. In addition to assessing disease, microbial populations in the organicmanures applied were estimated at bi-monthly intervals for 1 year using aserial dilution plate technique. In general, fungal populations increasedmarkedly up to the fourth month after treatment in all treatments containingorganic manure and decreased thereafter except in treatment with greenleaves, where the population continued to increase up to the eighth month. Inall the organic manure treatments, fungal populations were much higher thanin control soil; FYM and NC recording very high population levels (20 and18 × 104 cfu g−1 of soil in FYM and NC treatments, respectively, 1 year aftertreatment) (Fig. 10.1). The bacterial population was high in FYM treatment, followed by tanksilt (TS) and the population dynamics followed almost the same trend as thatof fungi, i.e. increase up to the fourth month and thereafter a reduction(Fig. 10.2). Actinomycete populations increased in the FYM and TS treatmentsTable 10.1. Effect of Trichoderma harzianum with different organic manures onbasal stem rot intensity and nut yield. Disease Nut yield perTreatment Index palm 1993/94T. harzianum in 5 kg neem cake 26.0 97T. harzianum with 50 kg farmyard manure (FYM) 48.4 101T. harzianum with 200 kg tank silt 59.5 83T. harzianum with 50 kg coir dust 100.0 88T. harzianum with 50 kg composted coir dust 57.2 90T. harzianum with 10 kg poultry manure 72.8 84T. harzianum with 50 kg green leaves 23.0 88Bordeaux mixture (BM) 1%, 40 litres 38.6 92T. harzianum with FYM + BM 28.7 96Control 92.1 44CD (P = 0.05) 4.3 5
Management of Basal Stem Rot Disease of Coconut 123Fig. 10.1. Effect of organic amendments on populations of fungi in soil. FYM,farmyard manure; NC, neem cake; GL, green leaves; TS, tank silt; CD, coir dust;BM, Bordeaux mixture; C, control.(Fig. 10.3). These populations increased up to the eighth month and thendecreased (Fig. 10.3). Trichoderma populations were high in all the organic manure treatmentswhen compared to the control (Table 10.2). The population increased up tothe fourth month and then decreased drastically, although the populationsalways remained much higher than control soil even in the twelfth monthafter treatment. NC and FYM sustained the highest population levels.Effect of biofertilizersA field experiment was initiated to test the efficacy of biofertilizers in themanagement of BSR. Azospirillum, phosphobacteria and the vesiculararbuscular mycorrhizal (VAM) fungus Gigaspora calospora were tested. Peat-based inoculum of Azospirillum and phosphobacteria (200 g) in 10 kg of FYMper tree year−1 was used. Soil inoculum of VAM fungus (500 g) was used foreach tree.
124 R. BhaskaranFig. 10.2. Effect of organic amendments on the soil bacterial population. FYM,farmyard manure; NC, neem cake; GL, green leaves; TS, tank silt; CD, coir dust;BM, Bordeaux mixture; C, control. Disease intensity, recorded up to the end of 1993, indicated that phospho-bacterial treatment was effective in reducing the disease severity whencompared to the other biofertilizers tested (Table 10.3). Nut yield was higher in all the biofertilizer treatments as compared tocontrol. Although phosphobacteria recorded a mean nut yield of 100, which isless than that of G. calospora and Azospirillum, the yield increased in 1993when compared to the yield in 1991, while with the other two biofertilizertreatments there was no yield increase when compared to that in 1991(Table 10.3).
Management of Basal Stem Rot Disease of Coconut 125Fig. 10.3. Effect of organic amendments on actinomycete populations. FYM,farmyard manure; NC, neem cake; GL, green leaves; TS, tank silt; CD, coir dust;BM, Bordeaux mixture; C, control.Table 10.2. Effect of organic manures on Trichoderma population. Populations months after inoculationTreatments 0* 4* 8 ++ 12 ++Neem cake 52 64 48 45Farmyard manure (FYM) 44 70 37 40Poultry manure 40 54 35 32Tank silt 38 48 30 34Composted coir dust 37 52 36 30Green leaves 28 12 40 28FYM + Bordeaux mixture 46 60 40 38Control 0 0 0 0Population × 105 (*) and × 103 (++) cfu g−1.
126 R. BhaskaranTable 10.3. Effect of biofertilizers on disease intensity and nut yield of basalstem rot-affected coconut (experiment initiated in September, 1990; mean of fivereplications). Disease index Nut yield % Increase overTreatments 1991 1992 1993 1991 1992 1993 Mean controlAzospirillum 200 g per 10 kg 21.6 53.3 64.3 113 112 92 106 37.7Phosphobacteria 200 g per 10 kg 1.7 4.9 34.8 87 110 102 100 29.9Gigaspora calospora 20.2 45.7 50.6 108 118 108 111 44.2Control 24.9 55.3 79.0 78 77 76 77 –CD (P = 0.05) 2.8 4.2 3.7 7 15 5 3 –Efficacy of fungicidesIn the field experiment on the efficacy of fungicides in the management ofBSR, fungicides were given as root feeding at quarterly intervals for 1 yearand 5 kg of NC was applied every year. The results (Table 10.4) indicate thataureofungin-sol and tridemorph are very effective during the first 3 years,but in the subsequent years the disease intensity gradually increased. Thisindicates that the trees are not permanently cured of the disease and there isonly suppression of symptoms.ConclusionBasal stem rot disease is a major disease limiting coconut production in India.Treatment of the diseased palms with fungicides does not offer a permanentcure to the affected tree. Biological control with T. harzianum and phospho-bacteria offers some scope for containing the disease but organic amendmentsare essential to encourage antagonistic microflora, and treatments whichincluded organic amendments had least disease and better yields of coconutsthan those without amendments.
Table 10.4. Efficacy of fungicides in the management of basal stem rot disease of coconut. Disease Index Nut yield per palmTreatments 1988 1989 1990 1991 1992 1993 1988/89 1989/90 1990/91 1991/92 MeanNeem cake 5 kg (NC) + carbendazim 2 g in 100 ml of water as root feeding 26.5 95.5 98.0 98.0 96.0 95.3 53 48 55 10 42NC + carboxin 2 g in 100 ml as root feeding 38.2 96.0 98.0 99.0 98.0 99.5 71 64 59 2 49NC + aureofungin-sol 2 g with 1 g of copper sulphate in 100 ml as root feeding (NC + AF) 6.3 8.6 25.6 45.8 54.3 64.3 92 117 121 58 97NC + tridemorph 2 ml in 100 ml as root feeding 11.1 18.2 19.6 31.3 42.5 52.4 61 106 90 42 75NC + aureofungin-sol + 40 litres of 1% Bordeaux mixture (NC + AF + BM) 7.0 11.6 23.6 35.9 50.7 58.6 114 127 104 62 102Control 43.6 97.5 98.0 98.5 99.0 98.5 53 57 31 4 36CD (P = 0.5) 4.6 15.9 16.6 19.5 19.6 19.8 7 10 10 12 – Management of Basal Stem Rot Disease of Coconut 127
128 R. BhaskaranReferencesBhaskaran, R. and Ramanathan, T. (1984) Occurrence and spread of Thanjavur wilt disease of coconut. Indian Coconut Journal 15(6), 1–3.Bhaskaran, R., Rethinam, P. and Nambiar, K.K.N. (1989) Thanjavur wilt of coconut. Journal of Plantation Crops 17, 69–79.Bhaskaran, R., Ramadoss, N. and Suriachandraselvan, M. (1991) Pathogenicity of Ganoderma spp. isolated from Thanjavur wilt affected coconut (Cocos nucifera L.). Madras Agricultural Journal 78, 137–138.Bhaskaran, R. and Ramanathan, T. (1984) Occurrence and spread of Thanjavur wilt disease of coconut. Indian Coconut Journal 15, 1–3.Nambiar, K.K.N. and Rethinam, P. (1986) Thanjavur wilt/Ganoderma disease of coconut. Pamphlet No. 30, Central Plantation Crops Research Institute, Kasaragod.Vijayan, K.M. and Natarajan, S. (1972) Some observations on the coconut wilt disease of Tamil Nadu. Coconut Bulletin 2(12), 2–4.Wilson, K.I., Rajan, K.M., Nair, M.C. and Balakrishnan, S. (1987) Ganoderma disease of coconut in Kerala. In: International Symposium on Ganoderma Wilt Diseases on Palms and Other Perennial Crops. Tamil Nadu Agricultural University, Coimbatore (abstr.), pp. 4–5.
130 R.R.M. Paterson et al.Malaysia due to a persistent haze problem. Similar problems with haze havebeen experienced in other countries, such as Indonesia. The Malaysian banwas relaxed in some regions where the disease became a renewed problem forthe industry (Haron et al., 1996), which illustrates the dilemma faced by manyproducers. In addition to the need to reduce sources of infection, there is simplythe requirement to remove the OP residues (OPR) per se as plantations wouldbecome unmanageable due to the accumulation of the waste material. A potential disadvantage of burning is that nutrient loss from the soil maybe incurred. Haron et al. (1996) demonstrated experimentally that nutrientswere replenished in the soil and positive effects were obtained by chopping andshredding or pulverizing the residues and spreading these around OP. A savingon fertilizers of RM 28 million per annum over a 4-year period at 1996 priceswas estimated if the procedure was taken up by the Malaysian industry as awhole, but, by not burning, the problem of O. rhinoceros was retained, albeitat a low level (less than 5% of OP infected after 12 and 18 months’ growth).However, the effect of chipping and not burning on Ganoderma incidence wasnot considered. On the other hand, Haron et al. (1998) demonstrated that OPRleft in piles rather than being chipped does not contribute to soil organic matter(SOM) and decompose on the soil surface, so removal of the residue may notaffect SOM. Another plantation practice is to submerge OPS in lagoons rather thanleave them in windrows. This is also highly polluting and does not tackle thelarge amounts of waste produced. In other estates it is current practice to chipsome of the OPS and stack it in windrows to promote decomposition (Hasanand Turner, 1998). This procedure does not deal with the large amount ofwaste product available, and the process takes a long time (approximately2 years) to complete, allowing pests and pathogens to survive. A process thatcan reduce this time to approximately 6 months would be of great benefit. Thus, there is considerable interest in removing OPR in a quick and benignmanner from the plantation floor, despite some of the factors described above.Towards this end, certain fungi can completely degrade plant material and soit may be possible to degrade OPS rapidly with solid-state fermentation tech-nology, and hence reduce the problems posed by the above potential threats,although, to be effective, the fungi added to the OPS must be highly competitivewith any other fungus found in or on the OPS. An alternative approach is touse the OPS as a resource for the production of edible mushrooms and/or feedfor ruminant animals (Kelley and Paterson, 1997). Here, a preliminary comparison of methods for assessing the bio-degradation of OPS by macroscopic fungi in vitro is described as a first step indeveloping a practical process in vivo. Many of the methods described havebeen used in conjunction with OPS for the first time. However, no attempt hasbeen made to analyse the data statistically because of the preliminary natureof the work. Also, although the studies were conducted on OPS, most of
In vitro Biodegradation of Oil-palm Stem by Fungi 131the results could also probably be applied to OP boles, which cause similarproblems to OPS, although they are even more difficult to treat as they arefirmly embedded in the soil by the root system.A SolutionThe following experimental procedures may offer methods for a solution to theOPR problem discussed above.The fungiDescriptions of some of the fungi isolated are given in Treu (1998), and a fulllist of strains used is available.Enzyme assaysIsolates (59) were tested for the production of cellulase, ligninase and amylaseby inoculating them on to appropriate test media and measuring zones ofclearance after incubation (Paterson and Bridge, 1994). Each permutation ofactivities was expressed by the strains as a whole (i.e. some produced all three,others two, etc.). This suggests that fungi could perhaps be selected for specificbiodegradative tasks. For example, high amylase activity will be useful in thedegradation of OPS because of its high starch content (Oshio et al., 1990; seep. 134). Taxa with the same names often had similar enzyme activities. Forexample, six Marasmius strains had similar ligninase and amylase activitiesbut no detectable cellulase. The possession of this combination may be usefulfor increasing the digestibility of oil palm as a ruminant feed (Kelley andPaterson, 1997). The fungus has the potential for removing starch and ligninbut presumably has limited or no capacity to degrade cellulose. So the finalproduct of degradation could have a high cellulose content and, as such, maybe suitable as a ruminant feedstuff. The observation that the Ganoderma strainsonly had detectable amylase activity is surprising as they are generally consid-ered to be white-rot fungi and so ligninase would be expected. However, thefungus may have adapted to the high concentration of starch in OPS.Thirty-nine per cent, 36% and 62% of all strains tested exhibited ligninase,cellulase and amylase activity, respectively. Enzyme activity was not detectedfor 19% of strains, although some of these had grown and so some enzymaticactivity must have been present.
132 R.R.M. Paterson et al.Growth assessmentA simple assessment of growth of the collected fungi on OPS (without bark)from a Malaysian plantation was devised. OPS tissue (1 g) was placed into20 ml universal bottles with metal screw caps. Ammonium dihydrogenphosphate and deionized water were added to obtain an approximate 50 : 1C/N ratio and 70% moisture, determined by Rao et al. (1995) to be optimal forthe composting of poplar wood in the absence of similar data for OPS. The OPSin the universals was inoculated with the fungi while uninoculated OPS andunsterilized OPS were incubated as controls. Water (0.7 ml) was added to eachbottle to restore moisture. A visual assessment of growth was made foreach sample. Thirty of the treatments were positive for growth. A black fungus-likeorganism appeared on the unsterilized control and had the highest visualassessment rating of all samples. Interestingly, a black fungus-like organismhas been isolated from OPS in Papua New Guinea, which appeared to beresponsible for heavy degradation (P. Bridge, personal communication) andmay be similar to the one observed in vitro. Many fungi grew well on OPS,with nine producing visual growth after only 3 days. The variation in growthbetween replicates was generally low. However, there were some strains inwhich only one of the three replicates grew, probably reflecting a problem withthe inoculation procedure (e.g. the inoculum was not in contact with the OPS).Many Marasmius cultures did not grow at all and in the case of IMI 370892,370929 and 370943 only one of the cultures grew on OPS. The unsterilecontrol (covered with black fungus – see above), Hydnum (IMI 370939) andPleurotus djamor (IMI 307936) were assessed as having more growth than thefastest growing Ganoderma (IMI 370917). In conclusion, visual assessmentsare only an indirect and qualitative measurement of OPS biodegradation, butthey are inexpensive to perform and appear to give consistent results, althoughinoculation procedures need to be standardized.Weight lossWeight loss was also determined for the above treatments. Weights of thebottles used for visual assessments were recorded at the start of the experiment,and after various intervals before and after the addition of 0.7 ml steriledistilled water to restore moisture. The accumulated percentage weight losswas determined. Weight changes of replicates indicated a great deal of variation in somecases. However, the three individual Marasmius cultures gave consistentlyhigh figures. The weight data from the samples that did not grow had asurprisingly wide range, from 39 to −26%. It is possible (but unlikely) thatgrowth had occurred but was not visible, accounting for the higher values.Alternatively, water evaporation may have been affected by variation in the
In vitro Biodegradation of Oil-palm Stem by Fungi 133fitting of the caps of the universal bottles, and/or location of samples within theincubator. The mean value of the weight losses from all these samples was 7%,which is perhaps reasonable for no or low levels of growth. Lenzites (IMI 307902) and Marasmius (IMI 370892) caused the highestloss in weight of OPS – 46% after 29 days – with maximum rates of 2% day−1,and 3% day−1 between days 14 and 21, respectively (Table 11.1). Many of theMarasmius cultures did not grow at all, but in the case of IMI 370892, 370929and 370943, where only one of the replicates grew, high weight losses wererecorded. The high weight loss (44%) from material inoculated with Hydnum(IMI 370939) is interesting, as the other Hydnum strains did not cause largeweight losses. IMI 370939 possessed high amylase and apparently no otherenzyme activity. The highest weight loss from a Ganoderma strain was 26% forstrain IMI 370917, with a maximum rate for weight loss of 2% day−1 betweendays 21 and 29. Seven strains had higher weight loss values than this strainand so they may be useful as antagonists (Table 11.1). Most of the high weight-loss strains also possessed high amylase activity, and in many cases seeminglyhad little or no cellulase or ligninase, again indicating the importance ofstarch degradation. Weight gains were recorded from the unsterilized OPSwhich contained the black fungus-like organism, perhaps resulting fromgreater evaporation from the sterile control. In general, there appeared to be acorrelation between weight loss and visual assessment of growth. Weight loss determinations are inexpensive and numerous strains can beanalysed in individual experiments. They are also a direct measurement of theinformation that is required, i.e. how much and how quickly is OPS beingdegraded. However, there is evidence of a high degree of variation in some ofTable 11.1. Accumulated percentage weight lossa from OPS treated with fungithat gave a higher weight loss than the most efficacious Ganoderma. aAccumulated % weight loss at time (days)Fungus IMI no. 3 7 14 21 29Lenzites (3) 307902 4 7 17 31 46Marasmius (1) 370892 7 8 15 35 46Hydnum (3) 370939 5 6 12 24 44Marasmius (1) 370929 3 5 24 35 43Marasmius (1) 370943 1 6 17 24 41Corticum (3) 370935 3 11 29 30 32Trametes hirsuta (3) 370898 4 6 8 18 26Ganoderma (1) 370917 −1− 1 −1− 8 26The figure in parentheses after the fungus name is the number of replicates. IMI no.is the reference number assigned to strains held in the CABI Bioscience geneticresource collection.aAccumulated percentage wieght loss minus percentage weight loss from sterilecontrols.
134 R.R.M. Paterson et al.the measurements and these particular experiments need to be refined in anyfuture studies.Ergosterol analysisErgosterol is a lipid contained in the cell membrane of fungi which will tendto increase in amount as fungi grow. The compound is virtually uniqueto fungi, and is increasingly being used as an estimation of fungal biomass.Universal bottles containing 1 g of OPS as above, were inoculated withHydnum (IMI 370893) and Polyporus (IMI 370891) and the complete contentswere used for analysis (1 bottle per sampling period). Samples were analysedfor ergosterol by the method of Gao et al. (1993) using high-performance liquidchromatography (HPLC). The concentrations of ergosterol increased with the visual estimation ofgrowth (Fig. 11.1) at least until the growth phase had ended. Maximumconcentrations of ergosterol were 46 and 44 µg g−1 on day 14 and day 21 forHydnum and Polyporus, respectively. Maximum rates of increase of ergosterolwere 6 and 4 µg (g OPS day)−1 for Hydnum and Polyporus, respectively,between days 7 and 14. There appeared to be a correlation between ergosterolconcentration and the visual assessment, and the two sets of data were similarfor both fungi. It is not known whether ergosterol estimation or visual assessment is themore accurate measurement of fungal biomass on OPS. Bermingham et al.(1995) provide evidence that ergosterol concentration varies between taxa. Itis being considered increasingly as the method of choice for measuring biomassFig. 11.1. Ergosterol and visual rating of Polyporus on 1 g oil-palm stem.
In vitro Biodegradation of Oil-palm Stem by Fungi 135in solid substrates such as food (Pitt and Hocking, 1997) but it does not provideinformation on the amount, or which components of OPS, are being degraded.The extraction procedure used here is time consuming and involves the use oflarge volumes of solvent. A rapid method has now been developed (Young,1995) which could be adapted for use with OPS. HPLC equipment is expensive,although a basic isocratic system with low-cost detector would be adequateand priced at the cheaper end of the market. Finally, an inexpensive (althoughonly semi-quantitative) method involving thin-layer chromatography (TLC)may be practicable.RespirometryRespirometry analysis involves measuring the amount of oxygen that isconsumed by microorganisms growing on solid substrates such as composts.Oxygen consumption was measured using a CES multi-channel aerobicrespirometer (Co-ordinated Environmental Services Ltd, Kent, UK). Blocks ofOP (ca. 5 g) were enriched with ammonium dihydrogen orthophosphate. Eachsample was inoculated with Hydnum (IMI 370939), Trametes (IMI 370898),Ganoderma (G3) or Pycnoporus (IMI 370937). There were four samples pertreatment. Three control flasks containing uninoculated amended OPS wereincluded and one flask was inoculated with Trametes (IMI 370898) andPycnoporus (IMI 370937). The sequence of oxygen consumption by fungi, from highest to lowest,was Hydnum, Trametes, Ganoderma and Pycnoporus (Fig. 11.2). However, theinitial mean water concentrations of the OPS were 55%, 58%, 59% and 62%,respectively, for material inoculated with Pynoporus, Ganoderma, Trametes andHydnum, so the amount of growth could have been influenced by the differentFig. 11.2. Oxygen consumption by fungi grown on oil-palm stem (mean values).
136 R.R.M. Paterson et al.water and ammonium salt concentrations of the OPS and may not reflectactual differences in ability to grow on OPS. Oxygen consumption by the com-bined Trametes and Pycnoporus culture was similar to that of Pycnoporus alone.More work is required to standardize the method, although it would appear tobe useful for assessing growth. However, the respirometer is expensive andonly a small number of strains can be analysed in individual experiments.Enzyme digestibilityEnzyme digestibility analysis involves the sequential degradation of plantmaterial by commercial enzymes such as cellulase, pronase (‘proteinase’) andamylase. In this way: (i) the initial chemical composition of the plant material;(ii) how each individual component is being degraded; and (iii) the final digest-ibility of the residue after treatment can all be determined. This procedureinvolves the sequential enzymatic degradation of the various components oflignocellulosic material in vitro (Abe and Nakui, 1979). Limited investigationsof the enzyme digestibility of OPS indicated that 30% of the stem was digestibleby glucoamylase and pronase on day 0 (pronase digestion alone indicated aprotein content of approximately 2%). This decreased to 20% by day 7 forGanoderma (project no. 29) and Marasmius (IMI 370929). Digestibility was26% after 7 days in the case of the Trametes (IMI 370934). However, cellulasedigestibility only decreased from 13% to 11%, confirming the view that starchis the preferred substrate. Total digestibility decreased from 43% to 32% in thecases of Ganoderma and Marasmius, and to 37% for the Trametes treatment in 7days. The standard deviations were generally small (ca. 5%). The OPS becameincreasingly indigestible as the fungi grew, and presumably as the result of anincrease in percentage lignocellulose. Fungi capable of completely metaboliz-ing lignocellulose would be required when the other substrates have beendepleted. Enzyme digestibility assays give a profound insight into the chemicalcomposition of lignocellulosic material in general and how the substrateschange as biodegradation progress. However, they are time consuming andthe enzymes can be expensive.Future StudiesMuch more fundamental work is required on the physiology of these fungito determine the optimal temperatures, C/N ratios, nutrients, pH, waterpotentials, etc. for growth and enzyme production and, ultimately, OPS bio-degradation for the individual fungi. A rigorous statistical analysis is desirablein future work. In vitro investigations involving the use of unsterilized OPS,including the bark, are required to determine whether an inoculated funguscan colonize and degrade OPS quicker than the indigenous microbial popula-tion. Research involving the use of consortia (i.e. mixed inocula) of fungi
In vitro Biodegradation of Oil-palm Stem by Fungi 137and other organisms may be worthwhile especially when considering how thedigestibility of OPS changes with time; a cocktail of organisms with compatibleenzyme capabilities may be required. Further work is necessary in standardiz-ing some of the procedures described in this chapter. Pilot-plant investigationsare also required on larger pieces of OPS to make the transfer of the technologyto the field more predictable. However, this does not preclude undertaking fieldtrials to establish whether candidate fungi can degrade OPS quickly in vivowithout the need for further work in vitro.ConclusionsIn conclusion, the various methods used here to assess the biodegradationof OPS indicate that after a lag phase of about 7 days some fungi have begun togrow visibly and reduce the weight of OPS. They appear to grow and degradein an exponential manner until about day 21 when the fungi enter a station-ary phase. The initial substrate used in the OPS is probably starch, whichexists at a high concentration. The more resistant substrates, such aslignocellulose, will probably only be substantially metabolized after thisphase. Visual inspection, ergosterol and oxygen consumption give an indirectmeasure of the growth of the fungi and degradation of OPS. Weight-lossmeasurements provide a direct measurement of the biodegradation of OPS.Enzyme digestibility assays provide insights into the mechanisms of degrada-tion and the chemical composition of the OPS as it is being degraded.Marasmius (and in particular IMI 370892) appears to be able to colonize anddegrade OPS more effectively than Ganoderma and is certainly a candidate for afull-scale process. However, some Marasmius species are also known to bepathogenic to OP, so great care would be required to ensure that any treatmentin the field does not involve a pathogenic strain of the fungus. It should perhapsbe pointed out that if Marasmius can outcompete Ganoderma on OPS in vivo, anincreased incidence of the former disease may become apparent, because of thecurrent practice of leaving the OPS on the plantation floor. Indeed, some of theMarasmius strains discussed here were isolated from OPS which had beendecayed heavily by the fungus. Some of the other fungi with high visualgrowth and weight loss assessments are also potential candidates for furtherstudy. A battery of procedures has been developed in this study which can beused in larger-scale projects, leading to an effective treatment for the rapidbiodegradation of OPS.AcknowledgementsStephan Wilkinson, DERA, PLSD, CES Sector, Sevenoaks, Kent, UK for the useof, and assistance with, the respirometry equipment.
138 R.R.M. Paterson et al.ReferencesAbe, A. and Nakui, T. (1979) Application of enzymatic analysis to the predication of digestible organic matter and to the analysis of the changes in nutritive value of forages. Journal of Japanese Grassland Sciences 25, 231–240.Bermingham, S., Maltby, L. and Cooker, R.C. (1995) A critical assessment of the validity of ergosterol as an indictor of fungal biomass. Mycological Research 99, 479–484.Chung, G.F., Cheah, S.S. and Nur Azarina, A.B. (1998) Some insects associated with Ganoderma fruiting bodies. In: The Second International Workshop on Ganoderma Diseases MARDI, Serdong, Malaysia, 5–8 October. CAB International, Wallingford, UK, p. 13.Gao, Y., Chen, T. and Breuil, C. (1993) Ergosterol – a measure of fungal growth in wood for staining and pitch control fungi. Biotechnology Techniques 7, 621–626.Haron, K., Zakaria, Z.Z. and Anderson, J.M. (1996) A18: Management of palm residues using various replanting techniques in oil palm plantations. In: Darius, A. et al. (eds) Proceedings 1996 International Palm Oil Congress ‘Competitiveness for the 21st Century’. PORIM, Kuala Lumpur, pp. 241–253.Haron, K., Brookes, P.C., Anderson, J.M. and Zakaria, Z.Z. (1998) Microbial biomass and soil organic matter dynamics in oil palm (Elaeis guineensis JACQ.) plantations, West Malaysia. Soil Biology and Biochemistry 30, 547–552.Hasan, Y. and Turner, P.D. (1998) The comparative importance of different oil palm tissues as infection sources for basal stem rot in replantings. The Planter 74, 119–135.Kelley, J. and Paterson, R.R.M. (1997) Crop residues as a resource. The use of fungi to upgrade lignocellulosic wastes. Biology International No. 35 (August), 16–20.Liau, S.S. and Ahmad, A. (1991) The control of Oryctes rhinoceros by clean clearing and its effect on early yield in palm to palm replants. In: Proceedings of the 1991 PORIM International Palm Oil Development Conference Module II – Agriculture. PORIM (Palm Oil Research Institute of Malaysia), Kuala Lumpur, Malaysia.Oshio, S., Abu Hassan, O. and Mohd Jaafar, D. (1990) Processing and Utilisation of Oil Palm By-products for Ruminants. Report of MARDI-TARC Collaborative Study (1987–1990).Paterson, R.R.M. and Bridge, P.D. (1994) Biochemical Techniques for Filamentous Fungi. CABI International, Wallingford, UK, p. 125.Pitt, J.I. and Hocking, A.D. (1997) Fungi and Food Spoilage, 2nd edn. Blackie Academic and Professional, London.Rao, N., Grethlein, H.E. and Reddy, C.A. (1995) Effect of C/N ratio and moisture on the composting of poplar wood. Biotechnology Letters 17, 889–892.Treu, R. (1998) Macrofungi in oil palm plantations of South East Asia. The Mycologist 12, 10–14.Wood, B.J., Corley, R.H.V. and Goh, K.H. (1973) Studies on the effect of pest damage on oil palm yield. In: Wastie, R.L. and Earp, D.A. (eds) Advances in Oil Palm Cultivation. Incorporated Society of Planters, Kuala Lumpur, Malaysia, pp. 360–379.Young, J.C. (1995) Microwave-assisted extraction of the fungal metabolite ergosterol and total fatty acids. Journal of Agricultural and Food Chemistry 43, 2904–2910.
140 Å. Olson and J. Stenlidthat differ from the rest of the individuals. This is a division based on the char-acters of the individuals. Then there are also different ways to group individu-als based on their geographical distribution, e.g. community, population.GenusGenus is the principal rank in the nomenclatural hierarchy closest abovespecies. In general, genera are defined with emphasis on several discontinuitiesin fundamental characters, especially the reproductive structures. Biologicalmeaningful functions such as mode of nutrition have sometimes been usedto group species into genera, but such criteria often fail to give a workable clas-sification. This indicates that changes between pathogenicity, endophytism,mutualism or saprotrophism can occur relatively frequently as evolutionproceeds and does not necessarily coincide with the formation of genera. The genus Heterobasidion consists of polypores having perennial basidio-carps with cuticulate pilei and asperulate basidiospores, belonging to thefamily Bondarzewiaceae in the order of Aphyllophorales. The genus includessix distinct taxonomic species: H. annosum, H. araucariae, H. insulare, H.pahangense, H. perplexum and H. rutilantiforme. H. annosum is the most pathogenic species, with a distribution over most ofthe northern hemisphere, including Europe, North America and Russia. Thefungus can infect and kill fully grown trees; its principal hosts are conifers.Recently, H. annosum has been suggested to be comprised of three separatespecies, H. annosum, H. parviporum and H. abietinum (Niemälä and Korhonen,1998), but several issues still remain to be clarified (see below). H. araucariae isvery similar to H. annosum but has larger pores and larger basidiospores. It wasseparated from H. annosum on the basis of intersterility, ecology and geograph-ical distribution (Buchanan, 1988). It is a saprotrophic species that inhabitsdead wood of Agathis and Araucaria species in eastern Australia, New Zealand,New Guinea and some islands in the Pacific Ocean. H. insulare has a reddishsurface to the pileus and irpiocoid pores (Buchanan, 1988). The fungus is asaprotroph on wood from Abies, Pinus and Picea and is distributed in southernand eastern Asia (Niemälä and Korhonen, 1998). H. pahangense was found inMalaysia by Corner (1989). It is characterized by large pores (2–4 mm−1) andit has ornamented spores (Stalpers 1996). H. perplexum is pileate, the surface isochraceous or pale brown and glabrous, its pores are 2–4 mm−1 and the sporesmeasure 5–7 × 4–5 µm. It was found growing on Tsunga in Nepalese moun-tains (Ryvarden, 1989). H. rutilantiforme has a glabrous and reddish-brownbasidiocarp, and is a tropical American species (Ryvarden, 1985). The poresare small (5–6 mm−1) and the spores are ornamented (4.5–5 × 2.5–3 µm). Ideally, a genus should be defined as monophyletic, i.e. all member speciesshould share a common ancestor, not common to other genera or species.Traditionally, this has been hard to achieve, since convergent evolution is verycommon among fungi. The macromorphological characters that served as the
Functional Units in Root Diseases 141basis for early taxonomy have frequently proved to be the result of convergentevolution and, thus, resulted in many paraphyletic genera, and groupingtogether of unrelated taxa. The advent of PCR (polymerase chain reaction) andrelatively easy access to DNA sequencing have helped in providing a range ofmolecular markers for taxonomic work. Among the most popular markersthat yield useful variation at genus or species level are the ribosomal DNAgenes and their spacers. Cladistic analysis of a large number of DNA sequencecharacters can be done using modern computers and software. Internal transcribed spacer (ITS), intergenic spacer (IGS) and mitochon-drial ribosomal markers agree that Heterobasidion is a well-defined genus,although the analysis has not been carried out for all the taxa (Harringtonet al., 1998; Fig. 12.1).SpeciesSpecies conceptsThe species concept has been, and still is, a subject for debate. Differentdefinitions of a species are used for different purposes:1. In the morphological species concept, a species is defined by a given set ofcommon morphological features not shared by other groups. This view is notfeasible in organisms which do not have many easily scored features. Further-more, it does not take into account the difference in biology of the species.2. In the phylogenetic species concept, a species is defined by its sharedevolutionary history and descent from a common ancestor.3. In the biological species concept, a species is defined as a group of actuallyor potentially interbreeding populations which is reproductively isolated fromother such groups.Ecological or geographical aspects are often used to help to define the lifehistory traits and geographical boundaries of the distribution of a species.Sympatric species co-occur in the same geographical location but are normallyseparated by differences in choice of substrate or hosts, while allopatric speciesare separated by large geographical distances. Vicariant species are those witha limited geographic distribution and where other species with an overlappingniche can appear under similar circumstances in a different region. In mycology, the morphological species concept has been used widelybecause of its historical association with botany. This has not always beenreliable, although fungi have a high developmental plasticity and relativelysimple fruiting structures (Brasier, 1983). In closely related or sibling species,taxonomically useful morphological differences may be lacking (Brasier,1987) or may develop only a long time after the initial speciation event (Kemp,1977). Therefore it is not surprising that mycologists find partially or totallyreproductively isolated subgroups within morphospecies (Brasier, 1987). In
142 Å. Olson and J. Stenlidthe biological species concept, the emphasis is on the biology of the species,especially on the actual or potential interbreeding of the populations and on itsreproductive isolation from other such populations. Reproductive isolationcan occur in several ways: (i) geographically, where populations are separatedFig. 12.1. The single most parsimonious tree from the internal transcribed spacer(ITS) and 5.8S rDNA sequences of Heterobasidion species. Tree length = 102 steps,CI = 0.765, RI = 0.947. Base substitutions are shown above branches, and bootstrapvalues (greater than 50%) and decay indices (d value) are shown below branches.
Functional Units in Root Diseases 143by barriers such as mountains or oceans; and (ii) ecologically, wherepopulations are separated by different ecological niches, i.e. climate, livingor non-living substrate, or host preferences for pathogens. The interbreedingpopulation can be defined in terms of numerical size, geographic size andgenetic structure. This will show the potential for gene flow between theindividuals in the population. We will go through the data that are importantfor defining the functional unit and the species concept of H. annosum, withemphasis on the biology of the fungus, but also take into account the availablemorphological data.Mating compatibility in HeterobasidionInterbreeding can be limited in several ways – geographically, ecologically andgenetically. Heterobasidion has been found all over the northern hemisphere.The fundamental geographic barrier is the Atlantic and Pacific oceans,separating the North American from the Euroasian continent. Beringia isthe closest place between them, and the site where spore transfer wouldtheoretically be possible. Another possible barrier would be high mountainranges such as the Ural mountains, even though Heterobasidion spores havebeen shown to travel up to 320 km over open sea (Kallio, 1970). One wayto overcome these barriers is if spores are transported by a vector of anykind, most obviously wood or plants transported by man between thecontinents. By using compatibility tests, three different intersterility groups weredetected in H. annosum. The P-group, originally found on pine trees in Finland,comprised isolates compatible with each other but not with isolates from theS-group, which was isolated originally from spruce in Finland (Korhonen,1978a). A third group was subsequently found on Abies alba in Italy (Caprettiet al., 1990). In North America, a P-group and an S/F-group were detected(Chase and Ullrich, 1988). Interbreeding is limited by a genetic system controlling mating.H. annosum has a bipolar (unifactorial) mating system, where each spore froma basidiocarp represents either of two mating types (Korhonen, 1978b; Chaseand Ullrich, 1983; Holt et al., 1983; Stenlid and Rayner, 1991). The bipolarmating system is determined by a mating factor, a gene or a gene complex inone chromosome. Allelic differences in this/these loci result in different matingtypes (Raper, 1966). The number of mating-type alleles is large in H. annosum,probably more than 100 types, although local populations may containonly 10–20 (Chase and Ullrich, 1983; Stenlid, 1985). Isolates of the samemating type are incompatible, but they are compatible with isolates of adifferent mating type. Random pairings within a population are, in mostcases, compatible. In a mating between two homokaryotic mycelia, there are four possibleoutcomes:
144 Å. Olson and J. Stenlid1. A compatible reaction showing a continuous mycelia when thehomokaryons have the same genotype or are subcultures from the samemycelium.2. A compatible reaction with changed colony morphology and theappearance of clamps, indicating that the isolates belong to the same breedingunit.3. An incompatible reaction, resulting in a zone with sparse mycelial growth,when isolates from the same breeding unit but with the same mating type arepaired.4. An incompatible reaction, resulting in a zone of dense and usuallypigmented mycelium, when isolates from different breeding units are paired. When mating tests are carried out between heterokaryotic and homo-karyotic isolates, the outcome is slightly different. A compatible reaction willgive rise to a clearing zone and changed morphology, and will also lead toclamp formation in the homokaryotic isolate. If the isolates are incompatible,a clearing zone will arise, but a gap heterokaryon could be produced. Thisis called the Buller phenomena (Buller, 1931). These new heterokaryonsapparently arise from anastomoses between homokaryotic hyphae from eachparent, or perhaps between homokaryotic and heterokaryotic hyphae. Theoutcome of such anastomoses is controlled by mating-type compatibility(Hansen et al., 1993b). Pairing among American P-isolates was compatible in 94% of thecases, and 95% of the pairing among European P-isolates was compatible(Harrington et al., 1989), while pairings between homokaryotic AmericanP-isolates and homokaryotic European P-isolates only resulted in 53% ofdikaryons (Harrington et al., 1989). In another study, European P- andNorth American P-isolates were compatible in ca. 95% of cases (Stenlid andKarlsson, 1991). When American fir isolates were paired with EuropeanS-type tester strains, 97% of the pairings lead to dikaryons (Harrington et al.,1989). With sympatric populations of S- and F-types from central Europe,about 24% of the pairings were interfertile, while pairings between northernEuropean S-isolates and southern European F-isolates were 72% interfertile(Korhonen et al., 1992). Confrontations between European S and P homo-karyotic isolates gave rise to a heterokaryon in 5% of the cases (Stenlid andKarlsson, 1991). In 1990, Chase and Ullrich described a genetic system to explain themating between and within intersterility groups in H. annosum (Chase andUllrich, 1990a, b). The system consists of at least five genes, called S, P, V1, V2and V3, each with a + and a − allele. Two homokaryotic mycelia can mate ifthey both posses a + allele for at least one of the five genes. They cannot mateif all five combinations are +/− or −/−. Intersterility determines the limits of an interbreeding population,whereas incompatibility regulates inbreeding and outbreeding within aninterbreeding population.
Functional Units in Root Diseases 145Morphological differences in HeterobasidionThe different intersterility groups of H. annosum have very similar properties,they have a wide and overlapping distribution and, although they exhibitdifferent preferences for host species, their host specialization is partlyoverlapping and not strict. Their morphological characteristics are also partlyoverlapping (e.g. spore and hymenial pore dimensions), making it not too easyto tell the different intersterility groups apart. The morphological differenceswithin the three European intersterility groups were examined by Mugnai andCapretti (1989), while differences between the S- and the P-group have beeninvestigated several times (Korhonen, 1978a; Stenlid and Häggblom, 1985;Negrutskii et al., 1994). The best diagnostic character is the length of the hairon the margin of the basidiocarp (Korhonen, 1978a; Mugnai and Capretti,1989; Negrutskii et al., 1994). The length of the hair in the intersterilitygroup is: P, 20.9 ± 2.2 µm; S, 119.5 ± 8.0 µm; F, 54.8 ± 3.3 µm (Mugnai andCapretti, 1989). The groups P and S are easily distinguished by the pore size:8.0 ± 0.3 mm−2 and 13.4 ± 0.4 mm−2, respectively (Korhonen, 1978a), whilethere were no differences between the P- and the F-groups (Mugnai andCapretti, 1989). This makes pore size a reliable diagnostic character to use foridentification of the P- and S-groups in geographical areas were the F-groupdoes not exist. The small differences in length and width of basidiospores andconidiospores make them useless for identification (Korhonen, 1978a; Stenlidand Häggblom, 1985; Mugnai and Capretti, 1989; Negrutskii et al., 1994).Differences in ecology and pathogenicityThe fungus has been reported from almost 150 woody plant species (Sinclair,1964; Hodges, 1969; Laine, 1976). It is spread over the whole temperateregion of the northern hemisphere (Hodges, 1969). The P-type is pathogenic to mature Pinus as well as to other Pinaceae, otherconifer and even hardwood species (Korhonen, 1978a; Worrall et al., 1983;Stenlid and Swedjemark, 1988; Harrington et al., 1989; Swedjemark andStenlid, 1995). Infection centres in pine stands are often associated withstump-top colonization (Slaughter and Parmeter, 1995). The S-type seems particularly specialized to Picea (Korhonen et al., 1992;Swedjemark and Stenlid, 1995). Picea and Pinus have preformed resin canalsin the xylem, which seem to be important in resistance to H. annosum (Gibbs,1968). Abies and Tsuga are frequently infected by the American S/F-typethrough wounds (Shaw et al., 1994). The S-type is mainly restricted to Piceaspecies, but can also attack small seedlings of other tree species (Korhonen,1978a). The S-type seems largely dependent on Picea stump tops for initiationof new infection centres (Stenlid, 1987). Interestingly, Korhonen et al.(1997) recently reported that, in the Ural mountain region, the S-type infects
146 Å. Olson and J. StenlidAbies sibirica, indicating that in regions where the F-type is absent, the S-typemight expand its ecological niche. Moreover, the geographical distribution ofthe intersterility groups suggests that a broad host range might be a basalcharacter in the S/F complex.Phylogeny of rDNA genesThe primary definition of intersterility groups (ISGs) is provided by in vitromating compatibility tests. Now, molecular genetic analysis methods areavailable for genetic identification of the different intersterility groups(DeScenzo and Harrington, 1994; Karlsson, 1994; Stenlid et al., 1994;Kasuga, 1995; Wingfield et al., 1996). Phylogenetic analyses using sequencedata from the ITS region of the nuclear ribosomal DNA and the IGS regionsupport a view of three major clades in the H. annosum complex: the Americanpine form, the European pine form and the fir form (Harrington et al., 1998).The differences between the European and American P-clades are as large asthe difference between either of them and the fir clade (Harrington et al.,1998). These findings are also supported by random amplified polymorphicDNA (RAPD) data from Garbelotto et al. (1993). Both the RAPD and the ISGdata weakly support a separation of American and European isolates. Nosupport is found from variation in the ribosomal genes for a separation ofEuropean S-isolates from F-isolates, even though they are clearly separatedin mating tests and have different host preferences (Capretti et al., 1990). TheEuropean S- and F-types can be distinguished by RAPD (Garbelotto et al.,1993; Stenlid et al., 1994; La Porta et al., 1997) and there are some differencesin isoenzyme patterns (Karlsson and Stenlid, 1991; Otrosina et al., 1993). TheNorth American S/F-type appears to be more related to the European S-typethan to the F-type according to RAPD data (La Porta et al., 1997). From a functional point of view, it is interesting to note that when datafrom enzyme systems that have a putative selection value for the organismsare used, the separation into ISGs is more clear than when neutral markers areused. Karlsson and Stenlid (1991) reported that zymograms of pectinolyticenzymes clearly separated the European S-, F-, and P-groups as well asthe North American S/F- and P-groups from each other. Laccases andsaprotrophic wood degrading capacities differ among the European S-and P-groups (Daniel et al., 1998; Johansson et al., 1999). Also phylogeny ofthe Mn-peroxidase gave a clear separation between the three European ISGs(P. Maijala, personal communication).Splitting or Lumping?Based on the morphological differences, Niemälä and Korhonen (1998)proposed a splitting of the European H. annosum and suggested new names
Functional Units in Root Diseases 147for the three European intersterility groups; H. annosum for the P-group,H. parviporum for the S-group and H. abietinum for the F-group. What remainsto be solved is the relationship between these three species and their NorthAmerican counterparts. For example, should the North American S/F-groupbe named H. abietinum or H. parviporum? The ITS and IGS phylogeny clearlyshows that the North American S/F-group has a long history, independentfrom its European relatives, while the morphology of the fruit bodies, althoughnot fully examined, cannot be clearly separated from them (Hood, 1985). TheNorth American S/F-group is also highly compatible with both the S- andF-groups from Europe. Should we decide to give the North American S/F-groupa separate name? Also, what about the relationships in the P-group? NorthAmerican and European populations are very similar in pathogenicity andmorphology, and also highly compatible, yet they have a long history ofseparate evolution as deduced from the ITS and IGS geneology. Naming fungihas perhaps become even harder now with all the conflicting data available toscience.Potential Interbreeding in HeterobasidionTo be able to interbreed, it is not enough to live in the same geographicarea, potential candidates also have to occupy the same ecological niche. InH. annosum, this is a potential barrier since the different intersterility groupsinhabit different host trees. However, a certain degree of overlap in host rangedoes occur between the various intersterility groups. Furthermore, this barriercan be bypassed in the relatively new habitat with limited host defencemade available through stumps created by forestry practices (Swedjemarkand Stenlid, 1993). On one occasion, a hybrid isolate was found with severalcharacteristics of both a P- and an S-isolate (Garbelotto et al., 1996).PopulationA prerequisite for meaningful population studies is that there is variationwithin the species under study. Variation among natural populations isthe result of interplay of a number of different forces (Hartl and Clark,1997). Mutation is the ultimate origin of variation that is then spread in thepopulation through natural selection or stochastic processes such as geneticdrift. Natural selection favours mutations that lead to higher fitness, basicallythe probability of having viable offspring. Genetic drift is the process ofrandomly drawing subsamples of a population that will found the nextgeneration. This will, with time, lead to the random exclusion of somegenotypes, more rapidly so in a small population than in a large one. An out-crossing mating system in the species helps to homogenize the distribution ofdifferent alleles at a locus throughout the population.
148 Å. Olson and J. Stenlid Within a species, there are normally several geographically separatedpopulations. However, populations are typically not completely isolated fromeach other. Migration among populations leads to gene flow that counteractsthe forces leading to differentiation. Among populations in equilibrium, onlyone migrant per generation is needed to counteract the effects of randomdrift, independently of the population size (Slatkin, 1985). Isolation leads todifferentiation and gene flow makes populations more similar. Small, isolatedpopulations are likely to be relatively homogeneous and any genetic variationis likely to occur at the regional scale. Large populations are likely to be morevariable, but between populations, variation may be lower. How does thisrelate to the risk of spreading a root rot disease with spores? To study the scale at which isolating distances may occur in H. annosum,it is of interest to compare direct and indirect measures of gene flow. Sporedispersal studies indicate that the vast majority of spores fall within a fewmetres of the fruiting body. Only about 0.1% of the spores trapped at 1 m canbe trapped at a distance of 100 m from a point source (Kallio, 1970; Stenlid,1994). Over a distance of 100–1000 m, the impact of a local spore source hasfallen to a level no greater than the background spore deposition (Möykkynenet al., 1997). However, given the enormous amounts of spores produced bybasidiomycete brackets, there is still a fair chance for some of the spores totravel over large distances. Calculations based on natural spore dispersalgradients show that one spore of H. annosum can land on the stump surface of anormal thinning operation more than 500 km away from its source during thetime that such surfaces are susceptible to H. annosum (Stenlid, 1994). Viablespores have indeed been collected on islands more than 300 km away fromany conifers (Rishbeth, 1959; Kallio, 1970). Indirect measures of gene flow aim at studying whether differentiationbetween populations occurs. If there is a strong differentiation, one can infer alack of random mating between the studied populations. However, lack ofdifferentiation does not necessarily imply gene flow. Two principally differentmarker systems have been used for this purpose: mating-type alleles andarbitrarily primed DNA. Mating-type alleles were scored using mating testsin Vermont, USA (Chase and Ullrich, 1983) and in Sweden (Stenlid, 1985).The likelihood of finding the same mating allele was calculated on variousgeographical distances. Interestingly, when studied on the geographical scalesimilar to the one used for calculation of likelihood of long-distance spreadof spores, a very similar pattern of decline in probabilities was detected (Fig.12.2). The likelihood of finding the same mating type at distances greater than100 km was about 0.1%, corresponding to approximately 1000 mating allelespresent in the whole species, which is a high but not unique figure (Ullrich andRaper, 1974). Similarly, when studying variation in arbitrarily primed DNA, adifferentiation in similarity among populations was seen at distances aboveapproximately 500 km (Stenlid et al., 1994). Later, more detailed studies haveshown a limited but significant differentiation (8.8% of total variation in theP-group) between populations in western and eastern North Europe (Stenlid
Functional Units in Root Diseases 149Fig. 12.2. (a) Long-distance spread of spores of Heterobasidion annosum:numbers of spores dispersed from a sporocarp at various distances, according topredictions from actual catches. (b) The chance of picking identical mating alleles(= incompatible pairings) of H. annosum in random samples of basidiospores atvarious distances.et al., 1998). An interesting differentiation was detected between northernEuropean S-populations and one from the alpine region in Italy (Stenlid et al.,1994). This coincides with the higher intersterility between the sympatricsouthern European S- and F-groups compared with the allopatric northernEuropean S- and southern European F-populations (Korhonen et al., 1992). In conclusion, most H. annosum spores are deposited within 100 m of afruiting body, but the relatively few that are spread long distance are enoughto ensure a large-enough gene flow to counteract differentiation at distances
150 Å. Olson and J. Stenlidless than 500 km. Within a continent, differentiation may be associated withisolating mountain ranges or connected to historical spreading patterns. Geneflow between continents is not likely to be a significant factor.IndividualThe attributes that have been used classically to characterize individualityare genetic homogeneity, genetic uniqueness and physiological unity andautonomy. For a more extensive discussion about individuality, see Santelices(1999). Among fungi, many individuals lack genetic homogeneity, geneticuniqueness and autonomy (Santelices, 1999). Genetic homogeneity is absentsince many fungal species grow and propagate through autoreplication ofgenetically identical units, which can survive and function independently.This enables a given genotype (genet) to be exposed simultaneously to variousenvironments, with different probabilities of survival and propagation. Physio-logically separate parts of a fungal genet have been called ramets (Brasier andRayner, 1987). Separate ramets can, upon contact, anastomose and form afunctioning entity. A genet is a discrete package of genetic information thatreproduces vegetatively, and could be looked upon as a mitotic line betweenmeioses. In basidiomycetes, a polygenic, multiallelic system, called somatic incom-patibility (SI) or vegetative incompatibility, is present that functions torestrict physiological and genetic access following non-self anastomosis. Thesignificance of SI may be to limit the spread of mycoviruses (Caten, 1972)or maladapted nuclei through a population by maintaining the integrity offungal individuals (Rayner, 1991). This system has been studied in some detailin H. annosum (Hansen et al., 1993a, b). Following fusion of two hyphae, a celldeath response may occur in the fusion cell. This response is much strongerin aerial than in submerged mycelium and results in a zone of sparse aerialmycelium. In wood, such interaction zones remain relatively undecayed. Inthe interaction zone, a complex pattern of interactions occurs (Hansen et al.,1993b). If two heterokaryotic mycelia interact, four nuclear types can meettransitionally in the same cell. Furthermore, H. annosum heterokaryoticmycelium is apparently composed of small sectors of homokaryotic hyphae,which can re-mate with any other hyphae in the interaction zone, therebyforming new pairwise combinations of nuclei. In wood, such interaction-zoneheterokaryons can possibly escape from the interaction zone through theinsulating nature of the wood anatomy. Hansen et al. (1993a) also studiedthe genetic basis for somatic incompatibility in H. annosum. The system isregulated through at least three, possibly more, multiallelic loci. This isin accordance with findings from some other basidiomycetes (Malik andVilgalys, 1994). However, in several species of Phellinus, data suggest thatthe somatic incompatibility is controlled through a single gene (Rizzo et al.,1995).
Functional Units in Root Diseases 151 By using SI as a marker system for individuality, forest pathologists havebeen able to study the infection biology and spread of pathogens in naturalpopulations. Some early studies were made in Oregon, e.g. genets of the root-rot fungus Phellinus weirii were shown to infect large groups of trees in naturalstands (Childs, 1963). Another example is the wood decayer, Fomitopsiscajenderi, infecting ice-glazed Douglas fir in Oregon, showing a pattern ofseveral genets entering the top break while only few managed to grow downthe stem (Adams and Roth, 1969). Following the advance in understandingof fungal biology made in the 1970s and 1980s by Dr Alan Rayner andco-workers, a range of fungal species was studied with regards to localpopulation spatial patterns (Rayner and Todd, 1979; Rayner, 1991). Verylarge territorial genets have been detected in some tree root-rot fungi(Armillaria spp.: Korhonen, 1978b; Kile, 1983; Smith et al., 1992; Legrandet al., 1996; Heterobasidion annosum: Stenlid, 1985, 1987; Piri et al., 1990;Swedjemark and Stenlid, 1993; Innonotus tomentosus: Lewis and Hansen,1991; Phellinus noxius: Hattori et al., 1996; Phellinus weirii: Dickman andCook, 1989). Much smaller-sized genets were found in wound pathogens orfungi attacking from the bark (Cylindrobasidium evolvens: Vasiliauskas andStenlid, 1998; Phomopsis oblonga: Brayford, 1990; Phellinus tremulae: Holmeret al., 1994). In H. annosum, the genets are much larger in old forest sitescompared to those sites with a recent history of agriculture (Stenlid, 1993;Swedjemark and Stenlid, 1993). At the same time, the relatively intenselymanaged first rotation stands were hosting a higher number of genets perhectare. These structures indicate a strong influence from diaspores infectingstump tops in the managed forests, and a correspondingly high proportion ofroot-to-root contact spread in the natural forests.SummaryHeterobasidion is a well-defined genus of saprotrophic and necrotrophicpolypores. In the pathogenic species H. annosum, several intersterility groupsexist that are specialized to different species of conifers. Phylogenetic studiesbased on rDNA variation indicate that at least five, and possibly seven,separate clades occur in the species. Based on morphological differences, thethree European intersterility groups – S, specialized as a root and butt rot onspruce; P, a general root and butt rot on pines and other conifers; and F, mainlycausing root and butt rot of silver fir – have been described as separate species.At present, the status of the other clades in H. annosum remains unresolved. Incontrast to the ITS sequences, enzyme systems with putative adaptive value forhost specialization, e.g. pectinases, differ clearly between the European S and Fintersterility groups. Most of the spore spread in H. annosum is local but, due tomassive diaspore production, the few spores dispersed over long distancescounteract population differentiation at distances less than 500 km. However,no significant gene flow between continents can be detected. On the local
152 Å. Olson and J. Stenlidscale, vegetative spread and infection processes can be followed by mappingthe distribution of individual mycelia. Somatic incompatibility, a highlypolymorphic recognition system, as well as molecular genetic markershave been used for this purpose.ReferencesAdams, D.H. and Roth, L.F. (1969) Intraspecific competition among genotypes of Fomes cajenderi decaying young-growth Douglas-fir. Forest Science 15, 327–331.Brasier, C.M. (1983) Problems and prospects in Phytophthora research. In: Erwin, D.C., Tsao, P.H. and Bartnicki-Garcia, S. (eds) Phytophthora, Its Biology, Ecology and Pathology. American Phytopathological Society, St Paul, Minnesota, pp. 351–364.Brasier, C.M. (1987) The dynamics of fungal speciation. In: Rayner, A.D.M., Brasier, C.M. and Moore, D. (eds) Evolutionary Biology of the Fungi. Cambridge University Press, London, pp. 231–260Brayford, D. (1990) Variation in Phomopsis isolates from Ulmus species in the British-isles and Italy. Mycological Research 94, 691–697.Buchanan, P.K. (1988) A new species of Heterobasidion (Polyporaceae) from Australasia. Mycotaxon 32, 325–337.Buller, A.H.R. (1931) Researches on Fungi, vol. 4. Longmans Green, London.Capretti, P., Korhonen, K., Mugnai, L. and Romagnoli, C. (1990) An intersterility group of Heterobasidion annosum, specialized to Abies alba. European Journal of Forest Pathology 20, 231–240.Caten, C.E. (1972) Vegetative incompatibility and cytoplasmic infection in fungi. Journal of General Microbiology 72, 221–229.Chase, T.E. and Ullrich, R.C. (1983) Sexuality, distribution, and dispersal of Heterobasidion annosum in pine plantations of Vermont. Mycologia 75(5), 825–831.Chase, T.E. and Ullrich, R.C. (1988) Heterobasidion annosum, root and butt rot of trees. Advances in Plant Pathology 6, 501–510.Chase, T.E. and Ullrich, R.C. (1990a) Genetic basis of biological species in Heterobasidion annosum: Mendelian determinants. Mycologia 82, 67–72.Chase, T.E. and Ullrich, R.C. (1990b) Five genes determining intersterility in Heterobasidion annosum. Mycologia 82, 73–81.Childs, T.W. (1963) Poria weirii root rot. Phytopathology 53, 1124–1127.Corner, E.J.H. (1989) Ad Polyporaceas V. Beihefte zur Nova Hedwigia 96, 219 pp.Daniel, G., Asiegbu, F. and Johansson, M. (1998) The saprotrophic wood-degrading abilities of Heterobasidium annosum intersterility groups P + S. Mycological Research 102(8), 991–997.DeScenzo, R.A. and Harrington, T.C. (1994) Use of (CAT)5 as a fingerprinting probe for fungi. Phytopathology 84, 534–540.Dickman, A. and Cook, S. (1989) Fire and fungus in a Mountain Hemlock forest. Canadian Journal of Botany 67, 2005–2016.Garbelotto, M., Bruns, T.D., Cobb, F.W. and Otrosina, W.J. (1993) Differentiation of intersterility groups and geographic provenances among isolates of Heterobasidion annosum detected by RAPD DNA assays. Canadian Journal of Botany 71, 565–569.
Functional Units in Root Diseases 153Garbelotto, M., Ratcliff, A., Bruns, T.D., Cobb, F.W. and Otrosina, W.J. (1996) Use of taxon specific competitive priming PCR to study host specificity, hybridization, and intergroup gene flow in intersterility groups of Heterobasidion annosum. Phytopathology 86, 543–551.Gibbs, J.N. (1968) Resin and the resistance of conifers to Fomes annosus. Annals of Botany 32, 649–665.Hansen, E.M., Stenlid, J. and Johansson, M. (1993a) Somatic incompatibility and nuclear reassortment in Heterobasidion annosum. Mycological Research 97, 1223–1228.Hansen, E.M., Stenlid, J. and Johansson, M. (1993b) Genetic control of somatic incom- patibility in the root-rotting basidiomycete Heterobasidion annosum. Mycological Research 97, 1229–1233.Harrington, T.C., Worrall, J.J. and Rizzo, D.M. (1989) Compatibility among host- specialized isolates of Heterobasidion annosum from western North America. Phytopathology 79, 290–296.Harrington, T.C., Stenlid, J. and Korhonen, K. (1998) Evolution in the genus Hetero- basidion. In: Delatour, C., Guillaumin, J.J., Lung-Escarmant, B. and Marcais, B. (eds) Root and Butt Rot of Trees. INRA Editions, Nancy, France, pp. 63–74.Hartl, D.D. and Clark, G.C. (1997) Principles of Population Genetics. Sinauer, Sunderland, MA, USA.Hattori, T., Abe, Y. and Usugi, T. (1996) Distribution of clones of Phellinus noxius in a windbreak on Ishigaki Island. European Journal of Forest Pathology 26, 69–80.Hodges, C.S. (1969) Modes of infection and spread of Fomes annosus. Annual Review of Phytopathology 7, 247–266.Holmer, L., Nitare, L. and Stenlid, J. (1994) Population structure and decay pattern of Phellinus tremulae in Populus tremula as determined by somatic incompatibility. Canadian Journal of Botany 72, 1391–1396.Holt, C.E., Gockel, H. and Hüttermann, A. (1983) The mating system of Fomes annosus (Heterobasidion annosum). European Journal of Forest Pathology 13, 174–181.Hood, I.A. (1985) Pore width in Heterobasidion annosum (Fries) Brefeld. New Zealand Journal of Botany 23, 495–498.Johansson, M., Denekamp, M. and Asiegbu, F.O. (1999) Production and isozyme pattern of extracellular laccase in the S and P intersterility groups of the root pathogen Heterobasidion annosum. Mycological Research 103, 365–371.Kallio, T. (1970) Aerial distribution of the root-rot fungus Fomes annosus (Fr.) Cooke in Finland. Acta Forestalia Fennica 107, 1–55.Karlsson, J.-O. (1994) Genetic variation in Heterobasidion annosum detected with M13 fingerprinting and ribosomal DNA probes. Experimental Mycology 18, 48–56.Karlsson, J.-O. and Stenlid, J. (1991) Pectic isozyme profiles of the intersterility groups in Heterobasidion annosum. Mycological Research 95, 531–536.Kasuga, T. (1995) Molecular probes for identification of intersterility groups of the wood rot fungus Heterobasidion annosum. PhD thesis, The University of Aberdeen, Aberdeen, UK.Kemp, R.F.O. (1977) Oidial homing and the taxonomy and speciation of Basidio- mycetes with special reference to the genus Coprinus. In: Clemencon, H. (ed.) The Species Concept in Hymenomycetes. Cramer, Vaduz, pp. 259–273.Kile, G.A. (1983) Identification of genotypes and the clonal development of Armillaria luteobubalina in Eucalypt forests. Australian Journal of Botany 31, 657–671.
154 Å. Olson and J. StenlidKorhonen, K. (1978a) Intersterility groups of Heterobasidion annosum. Communicationes Instituti Forestalis Fenniae 94(6), 25pp.Korhonen, K. (1978b) Interfertility and clonal size in the Armillaria mellea complex. Karstenia 18, 31–42.Korhonen, K., Bobko, I., Hanso, S., Piri, T. and Vasiliauskas, A. (1992) Intersterility groups of Heterobasidion annosum in some spruce and pine stands in Byelorussia, Lithuania and Estonia. European Journal of Forest Pathology 22, 384–391.Korhonen, K., Fedorov, N.I., La Porta, N. and Kovbasa, N.P. (1997) Abies sibirica in the Ural region is attacked by the S type of Heterobasidion annosum. European Journal of Forest Pathology 27, 273–281.Laine, L. (1976) The ocurrence of Heterobasidion annosum (Fr.) Bref. in woody plants. Communicationes Instituti Forestalis Fenniae 90(3), 1–53.La Porta, N., Capretti, P., Korhonen, K., Kammiovirta, K. and Karjalainen, R. (1997) The relatedness of the Italian F intersterility group of Heterobasidion annosum with the S group, as revealed by RAPD assay. Mycological Research 101, 1065–1072.Legrand, P., Ghahari, S. and Guillaumin, J.J. (1996) Occurrence of genets of Armillaria spp. in four mountain forests in central France: the colonization strategy of Armillaria ostoyae. New Phytologist 133, 321–332.Lewis, K.J. and Hansen, E.M. (1991) Vegetative incompatibility groups and protein electrophoresis indicate a role for basidiospores in the spread of Innonotus tomentosus in spruce forests of British Columbia. Canadian Journal of Botany 69, 1756–1763.Malik, M. and Vilgalys, R. (1994) Towards the genetic basis of somatic incompatibility in Pleurotus ostreatus. In: Abstracts, Fifth International Mycological Congress, 14–21 August 1994, Vancouver, British Columbia, p. 132.Mugnai, L. and Capretti, P. (1989) Intersterility groups of Heterobasidion annosum (Fr.) Bref.: some morphological differences in the basidiocarps. Micologia Italiana 1989(3), 87–94 (in Italian, English summary).Möykkynen, T., von Weissenberg, K. and Pappinen, A. (1997) Estimation of dispersial gradients of S- and P-type basidiospores of Heterobasidion annosum. European Journal of Forest Pathology 27, 291–300.Negrutskii (Negrutsky), S., Zaporozhchenko, E., Sukhomlin, M. and Boiko, M. (1994) Physiological and biochemical resemblance of the S and P intersterility groups of Heterobasidion annosum. In: Johansson, M. and Stenlid, J. (eds) Proceedings of the Eighth IUFRO Conference on Root and Butt Rots. Sweden/Finland, August 1993. Swedish University of Agricultural Sciences, Uppsala, Sweden, pp. 334–339.Niemälä, T. and Korhonen, K. (1998) Taxonomy of the Genus Heterobasidion. In: Woodward, S., Stenlid, J., Karjalainen, R. and Hütterman, A. (eds) Heterobasidion annosum Biology, Ecology, Impact and Control. University Press, Cambridge, pp. 27–33.Otrosina, W.J., Chase, T.E., Cobb, F.W. and Korhonen, K. (1993) Population structure of Heterobasidion annosum from North America and Europe. Canadian Journal of Botany 71, 1064–1071.Piri, T., Korhonen, K. and Sairanen, A. (1990) Occurrence of Heterobasidion annosum in pure and mixed spruce stands in Southern Finland. Scandinavian Journal of Forest Research 5, 113–125.Raper, J.R. (1966) Genetics of Sexuality in Higher Fungi. Ronald Press, New York.Rayner, A.D.M. (1991) The challenge of the individualistic mycelium. Mycologia 83, 48–71.
Functional Units in Root Diseases 155Rayner, A.D.M. and Todd, N.K. (1979) Population and community structure and dynamics of fungi in decaying wood. In: Woolhouse, H.W. (ed.) Advances in Botanical Research, vol. 7. Academic Press, London, pp. 333–420.Rishbeth, J. (1959) Dispersal of Fomes annosus Fr. and Peniophora gigantea (Fr.) Massee. Transactions of the British Mycological Society 42, 243–260.Rizzo, D.M., Rentmeester, R.M. and Burdsall, H.H. (1995) Sexuality and somatic incompatibility in Phellinus gilvus. Mycologia 87, 805–820.Ryvarden, L. (1985) Type studies in the Polyporaceae 17. Species described by W.A. Murrill. Mycotaxon 23, 169–198.Ryvarden, L. (1989) Wrightoporia perplexa nov. sp. (Polyporaceae). Opera Botanica 100, 225–227.Santelices, B. (1999) How many kind of individual are there? Trends in Ecology and Evolution 14, 152–155.Shaw, D., Edmonds, R., Littke, W., Browning, J., Russell, K. and Driver, C. (1994) Influ- ence of forest management on annosus root disease in coastal western hemlock, Washington state, USA. In: Johansson, M. and Stenlid, J. (eds) Proceedings of the Eighth International Conference on Root and Butt Rots. Sweden/Finland, August 1993. Swedish University of Agricultural Sciences, Uppsala, Sweden, pp. 646–655.Sinclair, W.A. (1964) Root- and butt-rot of conifers caused by Fomes annosus, with special reference to inoculum and control of the disease in New York. Memoir No. 391, Cornell University Agriculture Experiment Station, New York State College of Agriculture, Ithaca, New York, USA.Slatkin, M. (1985) Gene flow in natural populations. Annual Review of Ecology and Systematics 16, 393–430.Slaughter, G.W. and Parmeter, J.R. Jr (1995) Enlargement of tree-mortality centers surrounding pine stumps infected by Heterobasidion annosum in northeastern California. Canadian Journal of Forest Research 25, 244–252.Smith, M.L., Bruhn, J.N. and Anderson, J.B. (1992) The fungus Armillaria bulbosa is among the largest and the oldest living organisms. Nature 356, 428–431.Stalpers, J.A. (1996) The aphyllophoraceous fungi 2. Keys to the species of the Hericiales. Studies in Mycology 40, 1–185.Stenlid, J. (1985) Population structure of Heterobasidion annosum as determined by somatic incompatability, sexual incompatability and isoenzyme patterns. Canadian Journal of Botany 63, 2268–2273.Stenlid, J. (1987) Controlling and predicting the spread of Heterobasidion annosum from infected stumps and trees of Picea abies. Scandinavian Journal of Forest Research 2, 187–198.Stenlid, J. (1993) Boreal decay fungi. In: Pegler, D.N., Boddy, L., Ing, B. and Kirk, P.M. (eds) Fungi of Europe. Investigation, Recording and Mapping. Proceedings of the XI European Mycological Congress, pp. 171–180.Stenlid, J. (1994) Regional differentiation in Heterobasidion annosum. In: Johansson, M. and Stenlid, J. (eds) Proceedings of the Eighth IUFRO Conference on Root and Butt Rots. Sweden/Finland, August 1993. Swedish University of Agricultural Sciences, Uppsala, Sweden, pp. 243–248.Stenlid, J. and Häggblom, P. (1985) Macromolecular syntheses in germinating conidia and basidiospores of Heterobasidion annosum. Transactions of the British Mycological Society 84, 227–234.Stenlid, J. and Karlsson, J.-O. (1991) Partial intersterility in Heterobasidion annosum. Mycological Research 95, 1153–1159.
156 Å. Olson and J. StenlidStenlid, J. and Rayner, A.D.M. (1991) Patterns of nuclear migration and heterokaryosis in pairings between sibling homokaryons of Heterobasidion annosum. Mycological Research 95, 1275–1283.Stenlid, J. and Swedjemark, G. (1988) Differential growth of S- and P-isolates of Heterobasidion annosum in Picea abies and Pinus sylvestris. Transactions of the British Mycological Society 90(2), 209–213.Stenlid, J., Karlsson, J.-O. and Högberg, N. (1994) Interspecific genetic variation in Heterobasidion annosum revealed by amplification of minisatellite DNA. Mycological Research 98, 57–63.Stenlid, J., Kammiovirta, K., Karjalainen, R., Karlsson, J.-O., Korhonen, K., Solheim, H. and Thomsen, I. (1998) Genetic variation among Euorpean S- and P-group populations of Heterobasidion annosum assessed by arbitrary priming. In: Delatour, C., Guillaumin, J.J., Lung-Escarmant, B. and Marcais, B. (eds) Root and Butt Rots of Forest Trees. Proceedings of the ninth International Conference on Root and Butt Rot, INRA, France, pp. 75–84.Swedjemark, G. and Stenlid, J. (1993) Population dynamics of the root rot fungus Heterobasidion annosum following thinning of Picea abies. Oikos 66, 247–254.Swedjemark, G. and Stenlid, J. (1995) Susceptibility of conifer and broadleaf seedlings to Swedish S and P strains of Heterobasidion annosum. Plant Pathology 44(1), 73–79.Ullrich, R.C. and Raper, J.R. (1974) Number and distribution of bipolar incompatibility factors in Sistrotrema brinkmannii. The American Naturalist 108, 507–518.Vasiliauskas, R. and Stenlid, J. (1998) Population structure and genetic variation in Cylindrobasidium evolvens. Mycological Research 102, 1453–1458.Wingfield, B.D., Harrington, T.C. and Steimel, J. (1996) A simple method for detection of mitochondrial DNA polymorphisms. Fungal Genetics Newsletter 43, 56–60.Worrall, J.J., Parmeter, J.R. Jr and Cobb, F.W. Jr (1983) Host specialization of Heterobasidion annosum. Phytopathology 73(2), 304–307.
160 R.N.G. Miller et al.farmers also grow oil palm in mixed cropping systems with other perennials,such as coconut, coffee and cocoa. Ganoderma basal stem rot (BSR) of oil palm isof particular economic importance in these production areas, because itshortens the productive life of plantations, an effect that tends to becomecumulative over successive planting cycles of this monoculture, such thatwidespread losses can occur in young plantings less than 5 years old. Lossesdue to BSR are the result of both a direct reduction in palm numbers in thestand, and a reduction in the number and weight of fruit bunches fromstanding diseased palms and those with subclinical infections (Turner, 1966).Although oil palm is planted in areas that previously supported otherperennial crops, or in mixed cropping systems, the influence of these differentcropping systems on BSR incidence in oil palm is unclear. A number of the‘species’ of Ganoderma associated with BSR in oil palm (Table 13.1) have beendocumented as having a wide host range, infecting Albizzia (Turner andBull, 1967) and other palms, such as betelnut (Areca catechu) (Thomson,1935; Venkatarayan, 1936) and coconut (Venkatarayan, 1936; Peries,1974). Stumps of wild palms such as Oncosperma filamentosa and Livinstonacochinchineasis within an oil-palm planting have also been reported to supportbasidiomata of Ganoderma spp., presumed to be pathogenic to oil palm (Turner,1968). In contrast, observations by Varghese and Chew (1973) revealedthat Ganoderma basidiomata from oil palm were morphologically andphysiologically different from Ganoderma basidiomata from tea and rubber,suggesting that cross-infection from these non-palm hosts to oil palm would beunlikely to occur. BSR of oil palm has been recorded widely throughout the tropics,including Angola, Cameroon, Ghana, India, Indonesia, Malaysia, Nigeria,Principé, Sao Tome, Singapore, Solomon Islands, Tanzania, Zaire andZimbabwe (Turner, 1981). Recently, following the increased planting ofoil palm, infection of young palms has also been noted for the first time inPapua New Guinea (see Pilotti et al. and Sanderson et al., this volume) andThailand (Tummakate and Likhitekaraj, 1998). Ganoderma basal stem rotis now recognized as a significant constraint to sustainable production inAsia, and the development of techniques for disease management hasbeen highlighted as a key research priority (Anonymous, 1997).Multidisciplinary Characterization of Ganoderma from OilPalm and Other Tropical Perennial HostsRecent applications of biochemical and molecular methods in phytopathologyhave led to a considerable improvement in the taxonomy and understandingof numerous pathogenic fungal species. The combination of molecularbiology characteristics, such as DNA polymorphisms, with functionalinformation, such as enzyme activities, along with traditional morphological
Characterization of Ganoderma in Oil-palm Plantings 161Table 13.1. Ganoderma spp. recorded as probable causal organisms of basal stemrot (based on association) (after Turner, 1981).Ganoderma species Synonym OccurrenceG. applanatum Fomes applanatus Angola Benin Indonesia Ivory Coast Malaysia Principé San Tomé ZaïreG. boninense MalaysiaG. chalceum MalaysiaG. cochlear IndonesiaG. colossum NigeriaG. fornicatum ZaireG. laccatum IndonesiaG. lucidum Fomes lucidus Angola Ghana Indonesia Malaysia Principé San Tomé Tanzania Zaïre ZimbabweG. miniatocinctum MalaysiaG. pediforme ZaïreG. pseudoferreum Zaïre MalaysiaG. tornatum F. applanatus var. tornatum Cameroon G. applanatum var. tornatum Malaysia G. australe ZaireG. tropicum IndonesiaG. xylonoides ZaireG. zonatum G. tumidum Ghana Nigeria San Tomé Tanzania ZaïreGanoderma spp. Colombia Malaysia ZaïreG. lucidum has been widely used as a misnomer for basidiomata from manytropical countries; many collections named as G. lucidum are believed to beincorrectly identified.
162 R.N.G. Miller et al.and pathogenicity data, allows the delimitation of populations on the basisof genetic relatedness, and linkage to functional and field-related charac-teristics of the member isolates, applicable to studying disease epidemiology.Previously, this had been achieved either through the use of single techniquessuch as isoenzymes, which yield both genetic and functional information(Micales et al., 1986), or through the combination of data from multi-disciplinary approaches (Bridge et al., 1993). This combined approach hasidentified genetic and function-linked relationships between geographicallydiverse populations of Ganoderma on different tropical perennial crops,characterized on the basis of morphology, pathogenicity, somatic incompati-bility, isozymes, mitochondrial DNA and ribosomal DNA polymorphisms(Miller, 1995a, b, c).Basidioma morphologyThe majority of taxonomic studies on species of Ganoderma originating fromSouth-East Asia have been largely reliant on the system developed by Steyaert(1967, 1972) for defining species. Discriminatory basidioma characters haveincluded context layer depth, basidioma colour (upper surface and context),basidioma (shape, radius and thickness), cutis (thickness, colour and hyphalsystem), context thickness and colour, tube layer depth and colour, poredimensions, dissepiment dimensions, and spore dimensions, colour, shape,and echinule distribution. In his summary of the taxonomy of the Ganodermataceae, Corner (1983),however, reviewed Steyaert’s classification systems for Ganoderma, concludingthat gradations occurred in all morphological features used to describe species.Other species identification circumscriptions have also been unclear, and haveresulted in the description of over 250 species, with frequent synonymity asa result. The situation is further complicated by the description of a numberof species complexes by various authors (Steyaert, 1975, 1980; Bazzalo andWright, 1982; Adaskaveg and Gilbertson, 1986), such that taxonomicdivisions within the genus Ganoderma are currently regarded as chaotic, withheterogeneic forms, dubious nomenclature and inconsistencies in applicationof the numerous criteria by which the genus has been subdivided (Bazzalo andWright, 1982; Gilbertson and Ryvarden, 1986). These authors concluded thatthe use of morphology alone is insufficient for the systematics of Ganoderma.As a consequence, the identification and distribution of tropical Ganodermaspecies remains unclear and there is little comparative morphological informa-tion to enable morphology to be related to host specificity. The species conceptsfor the BSR-associated Ganoderma isolates are also very confused. Originallyidentified as G. lucidum by Thomson in 1931, a complex of species were laterbelieved to be associated with BSR (Voelcker, 1953; Dell, 1955; Wijbrans,1955; Varghese, 1965; Turner and Bull, 1967; Singh, 1991). Using morpho-logical characters of the basidiomata, Steyaert (1967) identified six species
Characterization of Ganoderma in Oil-palm Plantings 163associated with BSR lesions in oil palm in Malaysia and Indonesia (Sumatra),namely G. boninense, G. miniatocinctum, G. chalceum, G. tornatum, G. zonatum,and G. xylonoides. Later, Ho and Nawawi (1985) considered that thoseassociated with BSR all conformed to G. boninense, as did Miller (1995), whoalso confirmed the pathogenicity of isolates from diseased and symptomlesspalms following seedling inoculation tests. To date, 15 species of Ganodermahave been recorded worldwide as probable causal agents of basal stem rotin oil palm (Turner, 1981), although many of these are based only oncircumstantial association with basal rot lesions. In view of the uncertainspecies concepts in this genus, Ganoderma populations on oil palm are hereindescribed by generic name alone.Mycelial morphologyA number of identification systems using culture and morphological andphysiological characters, have been devised for mycelial states of the wood-inhabiting Aphyllophorales. The identification system developed by Nobles(1948), describing 126 species of wood-inhabiting basidiomycetes, was thefirst to bring together a range of morphological and physiological characters,including colour changes in agar, type of rot, and characters of the advancingmargin of a culture. In 1965, Nobles further developed the system intoa multiple-choice key for cultural identification of 149 species of wood-inhabiting hymenomycetes, based on 53 diagnostic characters (Nobles,1965). These included extracellular oxidase activity, hyphal septation, hyphaland culture pigmentation, growth rates, basidiomata formation in culture,odour, host specificity, and interfertility phenomena. Limited information wasincluded regarding tropical species, although Bakshi et al. (1969, 1970) andSen (1973) later included a number of polypore species from India in similartaxonomic keys. Boidin and co-workers (Boidin and Beller, 1966; Boidin andLanquetin, 1973; Boidin et al., 1976) also described species of Corticiaceae andLachnocladiaceae from central Africa, while van der Westhuizen (1958, 1959,1971, 1973) described cultures of several species from South Africa. Stalpers(1978) designed a more comprehensive synoptic key for 550 species of wood-inhabiting Aphyllophorales, based on 96 characters. However, once againfewer than 20% of species described were of tropical origin. Application of mycelial identification methods to tropical Ganodermapopulations has been limited, as they are mostly concerned with temperatespecies. Hseu and Wang (1990) concluded that identification systems of thesetypes were only of use for identification to the genus level, with parametersinsufficiently clear to enable differentiation between species. Miller (1995c)observed similar variation levels intraspecifically and interspecifically, indicat-ing inapplicability for species definitions, and in differentiation of populationsin the context of functional characteristics, such as host specificity on tropicalperennial crops. Diagnosis of Ganoderma infection in tropical perennial hosts
164 R.N.G. Miller et al.such as oil palm thus remains largely reliant on the presence of basidiomata,which are frequently observed only once a disease is firmly established.Subclinical infections remain undetectable, and mycelial states in the soil andsurrounding plant debris cannot be detected and identified with accuracy.Genetic-based characterization approachesIsozymesIsozymes are defined as multiple molecular forms of a particular enzymewhich have very similar or identical catalytic properties (Markert and Moller,1959). Most organisms possess several polymorphic enzymes. These enzymes,coded by different alleles (allozymes) at a single locus, or separate genetic loci(isozymes), can possess different electrophoretic mobilities. These differencesare due to amino acid variations, which are dependent on the codingnucleotide sequence in the DNA. Micales et al. (1986) and Stasz et al. (1988)described protocols for the study of population structures in fungi. Methods forcomparison of isozymes are based on specific staining after enzymes have beenseparated by electrophoresis. As isozymes represent an indirect expression ofthe genome, they may be used as indicators of genetic relationships betweenpopulations. This approach can thus be applied to discriminate taxa, given asufficient number of polymorphic enzymes or the occurrence of unique or rareenzyme patterns. The study of isozymes can be particularly useful in solvingtaxonomic problems when there are few morphological parameters, or wherecharacters are very plastic within a conventional species. The use of isozymesis generally applicable for intrataxon variation, discriminating below thespecies level. Approximately 90 enzyme systems have been used to date witha variety of organisms, and although their application to fungal systematicsis still under-exploited, significant advances have been made using theseapproaches (e.g. Bonde et al., 1984; Micales et al., 1986; Mills et al., 1991;Simcox et al., 1993).PECTINASES. Pectic isozyme studies have been conducted for taxonomicpurposes on fungal genera such as Armillaria, with Wahlstrom (1992) differ-entiating European species, and Penicillium, with Cruickshank and Pitt (1987)and Paterson et al. (1989) separating isolates in terms of accepted species.Similar studies on Heterobasidion annosum (Fr.) Bref., showed good correlationwith the spruce (S), pine (P) and fir (F) European and North American inter-sterility groups, with six different pectin zymogram groups relating to the threedifferent intersterility groups, and these were suggested to represent incipientspecies (Karlsson and Stenlid, 1991). Analysis of pectinase zymograms for 150Ganoderma strains (Figs 13.1 and 13.2) (Miller et al., 1995a), gave groupingsthat matched host type from which the strains were originally isolated. Isolatesfrom palm hosts (Elaeis guineensis, Cocos nucifera, Areca catechu, and the orna-mental palms Oncosperma horridum and Ptychosperma macarthurii) comprised a
Characterization of Ganoderma in Oil-palm Plantings 165 Key 0.8 Pectin esterase Bars denote standard Polygalacturonase errors of maximum 0.7 and minimum Rf Pectin lyase 0.6 v values for each band. V Variable band Rf value 0.5 v v 0.4 0.3 0.2 v 0.1 v v v A B C D E F G H I J Banding pattern typeFig. 13.1. Schematic representation of extracellular pectinolytic isozyme patterntypes.single large cluster group (cluster A), 99% of which were of palm origin andthese isolates produced a distinct pectin esterase band (banding pattern type A(Fig. 13.1)). Within this functionally defined group, there were no significantdifferences between isolates obtained from widely distant geographic locationssuch as Colombia, Nigeria, Malaysia and the Solomon Islands. A secondcluster (group B) also comprised predominantly isolates of palm origin (85%). Pectinolytic enzymes have been reported to be of importance in patho-genesis caused by necrotrophic pathogens (Cooper, 1983; Collmer and Keen,1986). Evidence that pectinase enzymes are necessary for tissue macerationhas been demonstrated in experiments with mutants (Handa et al., 1986) andby the transfer of genes coding for pectinolytic activity to non-pathogenicspecies (Keen and Tamaki, 1986; Payne et al., 1987). Although the role ofpectinolytic enzymes in pathogenesis caused by Ganoderma has yet to beclarified, Tseng and Chang (1988) reported that G. lucidum produced bothendo-polygalacturonase and endo-pectin methyl trans-eliminase, and hypoth-esized that such enzymes may be responsible for causing the tissue rotsassociated with the fungus. As pectinases produced by Ganoderma are probablyinvolved in plant tissue degradation, they are considered likely to befunction-linked characters. Consequently, the majority of Ganoderma strainsisolated from palm hosts were regarded as a well-defined functional grouping,producing a common range of pectinase isozyme profiles, undetectable bycomparison of basidioma morphology. Additionally, as a stable character(pattern A) was identified in Ganoderma populations originating from infectedpalm material, this raised the prospect of the development of diagnostic toolsfor diagnosis of Ganoderma infection within palm hosts. However, as enzymeactivity is likely to be localized within an infected palm, difficulties were visual-ized in terms of tissue sampling. Assuming that banding pattern differences
166 R.N.G. Miller et al. Scale of similarity 0.73 0.83 0.93 0.97 1.0 CLUSTER A ISOLATES - HOSTS E. guineensis (80) C. nucifera (7) P. macarthurii (5) A. catechu (2) O. horridum (1) Shorea spp. (1) CLUSTER B ISOLATES - HOSTS Cluster A E. guineensis (14) C. nucifera (3) G. sepium (1) Prunus spp. (1) Forest spp. (1) OTHER ISOLATES - HOSTS E. guineensis (7) C. nucifera (4) A. mangium (4) T. cacao (2) O. horridum (1) Prunus spp. (1) Quercus spp. (1) Abies spp. (1) H. brasiliensis (1) Forest spp. (1) Cluster B Fagus spp. (2)Fig. 13.2. Unweighted pair group average method dendrogram based on codedextracellular pectinase isozyme data. Similarities derived from Gower’s coefficient.found between isolates from oil palm and the majority of those from non-palmhosts represented true functional differences, these findings were concluded tobe of fundamental importance in terms of elucidating mechanisms of pathogensurvival and disease spread within the oil-palm agroecosystem. Similaritiesbetween zymogram banding patterns for isolates from oil palm and those forisolates obtained from coconuts in Malaysia supported the current widespreadbelief that the disease can spread from saprobic growth on old coconut
Characterization of Ganoderma in Oil-palm Plantings 167stands to parasitic invasion of oil palm, even though healthy coconut palmsthemselves are not attacked in Malaysia. Similarly, the different patternsproduced by isolates from non-palm hosts suggested that cross-infection wouldbe unlikely to occur from these to palm crops.INTRACELLULAR ISOZYMES. The cytoplasmic enzyme classes catalase, esteraseand phosphatase have been shown to reveal differences at a variety of taxo-nomic levels when applied to the differentiation of fungal groups, separating atspecies, population and isolate levels (e.g. Alfenas et al., 1984; Mugnai et al.,1989). Analyses of intracellular esterase and polyphenol oxidase have beenuseful in the separation of isolates of six Armillaria intersterility groups inBritish Columbia (Morrison et al., 1985). Lin et al. (1989) also separatedisolates belonging to four North American species of Armillaria, andgenotypically distinct clones within a species, on the basis of intracellularesterase isozymes and total protein profiles. Variability of intracellular iso-enzymes in isolates of Heterobasidion annosum also revealed their applicabilityfor differentiation of members of different intersterility groups (Otrosina et al.,1992), and identification of clones of H. annosum within Norway spruce(Stenlid, 1985). Within Ganoderma, intracellular isozymes have been appliedto test the validity of existing species definitions. For example, G. lucidum hasbeen differentiated from a number of other temperate Ganoderma spp. on thebasis of intracellular esterase isozymes (Park et al., 1986; Tseng and Lay,1988). Hseu et al. (1989) also reported the differentiation of isolates ofG. applanatum, G. boninense, G. formosanum, G. fornicatum, G. microsporum,G. neojaponicum, G. tropicum, and G. tsugae, on the basis of intracellular andextracellular laccase isozymes. Following analysis of pectinase enzymes, Millerand co-workers (Miller, 1995; Miller et al., 1995b) employed intracellularcatalase, acid phosphatase and propionyl esterase profiles to characterizetropical perennial populations. These isozymes revealed widespread geneticheterogeneity in isolates, contrasting with groupings derived from pectinases,with clusters showing no clear relationship with the host of origin. The consid-erable profile differences observed suggested variability at the population level,contrasting with discrimination levels observed in previous studies. As theseintracellular isozymes are constitutive rather than behavioural, the groupingsproduced between isolates from oil palm and other perennial hosts wereconsidered more likely to reflect evolutionary relationships than functionalrelationships. Consequently, the level of similarity observed between isolatesfrom the majority of palm hosts on the basis of extracellular pectinaseisozymes was more likely to be reflecting a common behaviour of isolateson palms rather than representing true genetic relatedness. Intracellularisozyme data indicated that isolates probably arrived at this behavioural traitfrom a number of different evolutionary pathways, which, on the evidencegenerated from pectinase data alone, appeared as a single population ofisolates attacking palms, able to cross-infect from coconut and other palmhosts to oil palm.
168 R.N.G. Miller et al.Mitochondrial DNA restriction fragment length polymorphismsMitochondrial DNA (mtDNA) in fungi codes for ribosomal RNAs, transferRNAs, and enzymes involved in energy transfer such as cytochrome b,cytochrome oxidase and ATPase subunits (Sederoff, 1984). Fungal mito-chondrial DNA has been reported to display high levels of structural variation,similar to that observed in plants. Gene arrangement is variable (Grossmanand Hudspeth, 1985; Hoeben and Clark-Walker, 1986), and size variation canbe observed even among closely related taxa (McArthur and Clark-Walker,1983; Bruns et al., 1988). Although the size range varies greatly in differentorganisms, it is generally between 20 and 180 kb in size, thus allowing theentire genome to be visualized by enzyme cleavage and gel electrophoresis. It isalso regarded as an attractive molecular marker for restriction fragmentlength polymorphisms (RFLPs) as it has a relatively high copy number and canbe purified easily. RFLPs have been used widely at different taxonomic levelsin fungal systematics (e.g. Typas et al., 1992; Thomas et al., 1994). Typically,mtDNA has been found to be rich in RFLPs at the intraspecific level (e.g. Brunset al., 1988; Smith and Anderson, 1989; Forster et al., 1990; Gardes et al.,1991), with mapped polymorphisms revealing variation caused by lengthmutations (Taylor et al., 1986; Bruns et al., 1988). Evaluation may be madeof classifications developed from characteristics such as morphology or hostspecificity, and because isolates, pathotypes or species can be identified by thisapproach, the technique may also be applied to the development of diagnostics(Cooley, 1991). Their role in delimiting species or subspecies is particularlyimportant where morphological and physiological differences are ambiguousor affected by environmental conditions, where they may provide a simpler,more reliable and more rapid means of classification. An added benefit ofthese analyses is that mitochondrial inheritance is believed to be unilinear (e.g.Forster and Coffey, 1990), therefore variability that may be due to cross-oversand other events in heterokaryotic isolates undergoing sexual recombinationwill be avoided. Mitochondrial DNA RFLPs have been shown to be highly varied amongGanoderma isolates from a wide range of hosts and locations (Miller, 1995;Miller et al., 1995b). Furthermore, mitochondrial probes derived from a singleisolate from Malaysian oil palm showed little homology with other isolatesfrom the same host. This supported the intracellular isozyme-derived conclu-sion that isolates with common pectinase activities were unlikely to representa single population, and probably arrived at this behavioural trait from anumber of different evolutionary pathways.Ribosomal DNA internal transcribed spacer (ITS) variabilityThe ribosomal DNA unit consists of a tandem repeat of three conserved genic(small subunit 18S, 5.8S and large subunit 25–28S) and two less-conservednon-genic (ITS and intergenic spacer (IGS)) regions (Fig. 13.3). The generegions code for rRNA, which forms the structural backbone of ribosomes, thesites of protein synthesis within the cell. The rDNA gene cluster occurs within
Characterization of Ganoderma in Oil-palm Plantings 169the chromosomes as multiple tandem repeats, such that a single nucleuscontains hundreds of copies. As the rDNA arrays are considered to be homo-genized by concerted evolution (Hillis and Dixon, 1991; Appel and Gordon,1995), with mutations thought to be minimized because of the functionalnature of the genic regions, this region therefore represents an attractivemarker for systematic studies. rDNA genes are evolving at a relatively slowrate, such that partial sequences from the nuclear large subunit gene areapplicable to phylogenetic studies among distantly related fungi (Gaudet et al.,1989). As the ITS regions have a spacer role, separating gene regions, overalllength remains fairly constant. However, as they do not encode rRNA, theymay accumulate considerable base substitutions, and thus evolve at a muchfaster rate than gene regions. ITS mutation rates frequently approximate thatof speciation, with sequence comparisons revealing low variation within aspecies, with more extensive sequence divergence existing between differentspecies within a genus. As a consequence, variability in the ITS region hasbeen the basis for the development of many PCR-RFLP-based assays fordifferentiation of fungal species (e.g. White et al., 1990; Gardes and Bruns,1991; Samuels and Seifert, 1995; Edel et al., 1997). In a study conducted to determine appropriate regions for discriminationbetween different Ganoderma species, Moncalvo et al. (1995) concluded thatsequence differences in ITS regions were sufficient to distinguish the majorityof 14 species tested, unlike the 25S gene region, which was more conserved.In comparisons of over 40 Ganoderma isolates from a block of 250 palms inMalaysia (R.N.G. Miller, unpublished data), restriction digestion of ITS regions(PCR-amplified using universal primers ITS 1F and ITS4) using enzymes HinfIand AluI yielded identical RFLP profiles in over 90% of strains, providingpreliminary evidence for a predominant single species within the oil palmssampled (Fig. 13.4).Localized Variability in Relation to Disease Establishment andSpread MechanismsLittle is known of the mechanisms of infection and spread within oil-palmplantings. Traditionally, initial establishment of Ganoderma BSR in an oil-palmfield has been considered to occur by mycelial contact, through growth ofliving oil-palm roots into an inoculum source, comprising saprophyticallycolonized debris within the soil and largely remaining from the previousplanting. Entry has also been postulated to occur through wounded tissues ordead roots (Turner, 1965b). As the roots of an oil palm can extend across up tofour planting rows (Lambourne, 1935), root-to-root contact might enable thesubsequent spread of Ganoderma between living palms. The observation thatpatches of basal stem rot infection appear to enlarge over time (Singh, 1991),has also led to the assumption that most spread of infection in the field occursby root contact between healthy and diseased palms.
170 R.N.G. Miller et al. ITS1 Nuclear small 5.8S Nuclear large IGS Nuclear small ITS1 ITS2 ITS1 rDNA (18S) rDNA rDNA (28S) rDNA (18S) ITS4 PCR product 700 bp sizeFig. 13.3. Approximate locations on rRNA gene repeat of primers for amplifica-tion of internal transcribed spacer (ITS) regions and estimated polymerase chainreaction (PCR) product size in Ganoderma.Fig. 13.4. Amplified rDNA internal transcribed spacer region (HinfI-digested) forrepresentative Ganoderma isolates from a single oil-palm block. Lanes 1 and 17(left to right): 1 kb size marker. In an attempt to eliminate the initial inoculum, sanitation prior toreplanting often involves ‘windrowing’, i.e. the uprooting of previous bole andtrunk tissues, which are then stacked along the inter-rows. In some cases, thestem tissues are also chopped up mechanically to hasten breakdown.Although disease incidence after windrowing is generally lower in subsequentplantings than in stands replanted without bole removal, the process is bothlabour intensive and costly, and often fails to prevent the recurrence or spreadof basal stem rot. Despite the dubious value of current replanting strategies andthe general failure of control strategies (curative surgery, fungicide treatment,cultural methods) in existing oil-palm stands, few studies have been conductedto test the validity of current assumptions about the spread of the pathogenin oil-palm plantings. This is largely because morphology-based characteriza-tion approaches have not allowed the differentiation of subpopulations orindividuals required for pathogen population studies. In a recent study, Milleret al. (1999) determined the genetic relationships within over 50 isolates ofGanoderma from two oil-palm plantings in Malaysia, through somatic incom-patibility studies and mtDNA RFLPs, in order to elucidate possible mechanismsof disease establishment and spread.
Characterization of Ganoderma in Oil-palm Plantings 171Somatic incompatibilityAlthough understanding of the mechanisms determining somatic incompati-bility in Ganoderma remains incomplete, the use of incompatibility reactions inthe study of disease development in populations is well documented withinBasidiomycete tree pathogens (e.g. Guillaumin et al., 1994; Morrison et al.,1994). Through somatic incompatibility reactions, Miller et al. (1999)reported that the sampled Ganoderma populations within the two oil-palmsystems occurred as numerous distinct individuals (‘genets’ sensu Rayner)(somatic incompatibility groups (SIGs)), contrasting with typically clonaldistribution patterns for other basidiomycetes, where single clones can spreadover large areas of forests (Shaw and Roth, 1976; Stenlid, 1985). Numerousseparate genets were detected in the sampled populations, with a total of 34detected in one plot (out of 39 isolates tested) and 18 (out of 18) within theother (Fig. 13.5). In both cases, incompatibility between paired isolates wasobserved over distances that could theoretically permit root-to-root contact,and hence mycelial spread, between neighbouring palms (9 m apart), andbetween non-adjacent palms (up to 36 m apart). Incompatibility was alsofound between isolates colonizing the same infected palm. Only in one instancewere two isolates from neighbouring palms found to be compatible. Similarvariability has also been reported in other oil-palm blocks (Ariffin and Seman,1991). The frequency of different SIG genets within the two oil-palm plantingsindicated numerous separate infection incidents, rather than mycelial spreadof Ganoderma. The numerous genets may have arisen through sexual recombi-nation and subsequent dispersal of recombinants via basidiospores. However,the role of basidiospores in the infection process remains unresolved. Newinoculum sources could be formed by saprobic colonization of substrates suchas stumps or felled palm trunks and debris. Such mechanisms have beenwidely reported for other root- and butt-rot pathogens (Turner, 1976, 1981;Stenlid, 1985). Despite the release of huge numbers of airborne spores fromeach basidioma, the majority of palms remain uninfected, indicating thatbasidiospores either may not be able to initiate a basal stem rot infection ormay require very specific conditions to establish infection. Previous studieswith spore inoculum did not result in direct infection of living palms (Turner,1965a; Yeong, 1972). None the less, spores are a likely infection mechanismin upper stem rot of oil palm (Thomson, 1931), often in association withPhellinus spp. Although Ganoderma basidiospores are most likely to be wind-borne, additional mechanisms suggested for their dispersal have includedinsect vectors (Genty et al., 1976). However, to date, no conclusive link hasbeen made between insects and basal stem rot incidence and development. Alternatively, the numerous SIG clones could also have indicated thepresence of many spatially separated populations, each originating from aunique mycelial inoculum source, which may have originated from infecteddebris left over from previous stands or colonized by spores. Both plots were
(a) Palm numbers (from top) (b) Palm numbers (from top) 172 1–17 18–37 33–48 49–64 65–80 81–96 97–112 113–128 129–144 145–160 161–172 1–16 17–32 33–48 49–64 65–80 81–96 97–112 113–128 129–144 145–160 161–176 177–192 193–208 209–224 225–240 241–256 173–177 34 13 178–182 39 35 7 183–187 8 188–192 2 193–197 198–202 33 11 203–207 11 46 10 9 208–212 12 14 12 47 213–217 218–222 38 1 223–227 1 48 52 43 4 228–232 42 Palm numbers (left to right) 1 50 27 51 40 41 15 5 2827 233–237 18 49 R.N.G. Miller et al. 21 238–242 6 2019 26 23 243–247 16 17 22 25 24 248–252 44 45 253–256 2930 36 3 37 32 31Fig. 13.5. Palm layout and distribution of Ganoderma somatic incompatibility groups for selected isolates from oil palms: (a) plot atSungei Buloh Estate, Sime Darby Plantations Sbd, (b) plot at Bukit Cloh Estate, Sime Darby Plantations Sbd. Open circles indicate livingpalms, shaded circles indicate palms that had died recently, and small closed circles indicate palms that had died some time previouslyand constituted vacancies in the blocks. Numbered squares indicate locations of SIG. Thin straight lines indicate drainage channels.
Characterization of Ganoderma in Oil-palm Plantings 173replanted from rubber, which in turn replaced primary forest. Although eithervegetation could have supported Ganoderma populations, variation in strainsadapted to palms is more likely to have originated in native palm infections.Evidence of such an origin was described previously (Miller et al., 1995a), withisolates from palmaceous and non-palmaceous hosts separating on the basis ofextracellular pectinase zymograms. Oil palm is propagated as seed from crossesbetween dura × pisifera types and so as a segregating population the oil-palmstand itself does not present a homogeneous host. This may create additionalselection pressure for variation in the pathogen.mtDNA RFLPsAs with the SIG data, mtDNA RFLPs revealed considerable heterogeneitybetween isolates (Miller et al., 1999) including those from the same andadjacent palms (Fig. 13.6). Of the 26 lines identified by MspI-derived RFLPsamong the isolates studied, only two isolates from neighbouring palms hadthe same mtDNA RFLP profile. The majority of isolates obtained from withinindividual palms gave a single mitochondrial DNA profile, and only two palmsgave isolates with different RFLP profiles. In previous studies on other fungi, the relationship between RFLP andSIG groupings has been reported to be complex (e.g. Manicom et al., 1990), 10 8.0 6.5 5.0 4.0 3.0 RFLP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 23 24 25 26 profile numberFig. 13.6. Schematic representation of restriction patterns from MspI restrictionfragment length polymorphism (RFLP) analysis of mitochondrial DNA for represen-tative isolates. The scale on the left of the figure indicates fragment sizes (in kbpairs).
174 R.N.G. Miller et al.ranging between equivalent RFLP and SIG groupings, more than one RFLPgrouping within a SIG, or more than one SIG within an RFLP grouping. Withinthis study, results from mitochondrial DNA analyses and somatic incompati-bility tests were not always in accord. More than one SIG frequently occurredwithin a single mitochondrial DNA group, as previously reported in Armillaria(Guillaumin et al., 1994; Smith et al., 1994). This was interpreted as variabilityarising at the compatibility loci as a result of sexual recombination, withmitochondrial DNA maintained through unilinear inheritance. Each SIGcould therefore have represented a nuclear genomic variant, with differentgenets originating from locally dispersed basidiospores. This interpretationwas further supported by comparison of relationships by cluster analysis of themtDNA RFLP profiles (Fig. 13.7); isolates from the same or nearby palms didnot cluster together. These isolates showed few bands in common, implyingthat recombination (whereby progeny could be expected to contain a propor-tion of bands identical to parents) had not occurred. In this case, therefore,different RFLP profiles indicated isolates derived from different lines, presum-ably arising from different dikaryotic basidiomata and mycelium (althoughisolates with identical mtDNA RFLP profiles could still represent differentlines). However, in one instance a single SIG group was found to have twoRFLP lines. This was interpreted as indicating either that more than onemitochondrial type can exist (possibly through recombination) within a singlepopulation, or that self-incompatibility is controlled by non-mitochondrialmarkers. Overall, mtDNA was not recommended in isolation for differentiationof lines within Ganoderma. Mitochondrial DNA RFLP studies also provided evidence against previousassumptions of the significance of secondary mycelial spread of Ganodermafrom palm to palm. As mtDNA has been demonstrated to be maintainedthrough unilinear inheritance in Ganoderma (C. Pilotti, personal communica-tion), the presence of numerous mitochondrial DNA groups thereforeindicated spatially separated populations originating from a diverse initialinoculum.ConclusionsExisting species definitions for Ganoderma are of little value for interpretingdisease processes in tropical perennial crops such as oil palm. Application ofa multidisciplinary approach combining genetic, morphological and patho-genicity data provided evidence of a genetically heterogeneous grouping ofisolates specific to palms. Consistency in ITS RFLPs may also provide prelimi-nary evidence of a predominant species within oil palm. PCR applications,such as sequence analysis of nuclear or mitochondrial rRNA gene and spacerregions, or protein-coding genes such as the β-tubulin genes, are likely toclarify the species identity of Ganoderma in oil palm. Development of sensitivediagnostic methods for the pathogen in oil palm is also likely to be reliant on
Characterization of Ganoderma in Oil-palm Plantings 175 Scale of similarity 0.2 0.4 0.6 0.8 1.0 19.3 25 14 11.1 10.1 10.2 32 20.1 16.1 21 37.2 35 Isolate number 24 15.1 15.2 11.2 5 8 7 4 38 31 18.2 18.4 29.2 39 36 28 19.2 22Fig. 13.7. Unweighted pair group average method constructed dendrogram ofbinary coded MspI restriction fragment length polymorphism data of Ganodermaisolates. Similarities were derived using Sorenson’s (Dice) Coefficient.sequence data, enabling design of specific primers for PCR-based detectionapproaches. Localized studies did not support the current assumption that spread ofGanoderma occurs through radial mycelial growth from individual inoculumsources to neighbouring palms via root-to-root contact, which has particularsignificance in terms of efficacy of land preparation prior to replanting, andsanitation practices in existing oil-palm stands. Within two oil-palm plantingsexamined, both SIG and RFLP data indicated that Ganoderma populationswere highly heterogeneous over restricted areas. Circumstantial evidence for
176 R.N.G. Miller et al.primary infection from residual inoculum in crop debris was supported bygenetic comparison of isolates. Spread from these foci to immediately neigh-bouring trees may be occurring by mycelial spread, but more distant infectionsare likely to be the product of unrelated infection incidents. It is anticipatedthat, following conclusive determination of the stability of mtDNA in thesexual fungus Ganoderma, the role of residual inoculum and basidiospores maybe more fully clarified. More recent PCR-based approaches such as randomlyamplified polymorphic DNA (RAPD), amplification fragment length poly-morphisms (AFLP) or microsatellites may be appropriate to the clarificationof BSR disease establishment and pathogen spread in oil palm, if found to bestable over the life cycle of Ganoderma. These approaches may be applicable todiscriminating individuals and, with reduced cost and handling time, may alsoenable analysis of local variability on the basis of much larger sample sizes.AcknowledgementsThis study was funded by the UK Department for International Developmentand commissioned through the Natural Resources Institute (contract R5325).All work was carried out under licence from the UK Ministry of Agriculture,Fisheries and Food (licence PHF 1490/1706(11/95)).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.Alfenas, A.C., Jeng, R. and Hubbes, M. (1984) Isoenzyme and protein patterns of isolates of Cryphonectria cubensis differing in virulence. Canadian Journal of Botany 62, 1756–1762.Anonymous (1915) A disease of the oil palm in the Belgian Congo. Bulletin of the Imperial Institute, London 13, 479–480.Anonymous (1997) R & D Priorities for Oil Palm in Asia. FAO-RAP publication 1997/18.Appel, D.J. and Gordon, T.R. (1995) Intraspecific variation within populations of Fusarium oxysporum based on RFLP analysis of the intergenic spacer region of the rDNA. Experimental Mycology 19, 120–128.Ariffin, D.S. and Seman, I.A. (1991) PORIM Information Series. Palm Oil Research Institute Malaysia, Malaysia.Bakshi, B.K., Sehgal, H.S. and Singh, B. (1969) Cultural diagnosis of Indian Poly- poraceae. I Genus Polyporus. Indian Forest Records 2, 205–244.Bakshi, B.K., Sen, M. and Singh, B. (1970) Cultural diagnosis of Indian Polyporaceae. II Genera Fomes and Trametes. Indian Forest Records 2, 245–276.Bakshi, B.K., Ram Reddy, M.A., Puri, Y.N. and Sujan Singh (1972) Forest Disease Survey, Final Technical report (1967–1972), Forest Research Institute and Colleges, Dehra Dun, p. 8.
Characterization of Ganoderma in Oil-palm Plantings 177Batista, M.D.F. (1982) Red root rot of guarana plants. Fitopatologia Brasileira 7, 437–438.Bazzalo, M.E. and Wright, J.E. (1982) Survey of the Argentine species of the Ganoderma lucidum complex. Mycotaxon 16, 293–325.Boidin, J. and Beller, J. (1966) Aleurodiscus wakefieldiae nov. spec. Bulletin Trimestrial de la Société Mycologique de France 82, 561–568.Boidin, J. and Lanquetin, P. (1973) Vararia (Dichostereum) ramulosa, nouvelle espèce africaine (Basidiomycetes Lachnocladiaceae). Bulletin Mensuel de la Société Linnéenne de Lyon 42, 164–166.Boidin, J., Lanquetin, P., Terra, P. and Gomez, C.E. (1976) Varairia subg. Vararia (Basidiomycetes, Lachnocladiaceae) II. Caracteres culturaux. Bulletin Trimestrial de la Société Mycologique de France 92, 247–277.Bonde, M., Peterson, G.L., Dowler, W.M. and May, B. (1984) Isozyme analysis to differentiate species of Peronosclerospora causing downy mildew of maize. Phytopathology 74, 1278–1283.Bridge, P.D., Ismail, M.A. and Rutherford, M.A. (1993) An assessment of aesculin hydrolysis, vegetative compatibility and DNA polymorphism as criteria for characterizing pathogenic races within Fusarium oxysporum f.sp. vasinfectum. Plant Pathology 42, 264–269.Bruns, T.D., Palmer, J.D., Shumard, D.S., Grossman, L.I. and Hudspeth, M.E.S. (1988) Mitochondrial DNAs of Suillus: three fold size change in molecules that share a common gene order. Current Genetics 13, 49–56.Butler, E.J. (1906) Some diseases of palms. Agricultural Journal of India 1, 299–310.Chohan, J.S., Khang, I.S. and Rattan, G.S. (1984) Control of root rot and sap-wood rot of peaches (Flordasum cultivar) caused by Polyporus palustrus, Ganoderma lucidum associated with Schizophyllum commune. International Journal of Tropical Plant Diseases 2, 49–54.Collmer, A. and Keen, N.T. (1986) The role of pectic enzymes in plant pathogenesis. Annual Review of Phytopathology 24, 383–409.Cooley, R.N. (1991) The use of RFLP analysis, electrophoretic karyotyping and PCR in studies of plant pathogenic fungi. In: Stahl, U. and Tudzynski, P. (eds) Molecular Biology of Filamentous Fungi: Proceedings of the EMBO-Workshop, Berlin.Cooper, R.M. (1983) The mechanisms and significance of enzymic degradation of host cell walls by parasites. In: Callow, J.A. (ed.) Biochemical Plant Pathology. Wiley, New York, pp. 101–135.Corner, E.J.H. (1983) Ad Polyporaceas 1 – Amauroderma and Ganoderma. Beihefte zur Nova Hedwigia Heft 75. J. Cramer, Vaduz.Cruickshank, R.H. and Pitt, J.I. (1987) Identification of species in Penicillium subgenus Penicillium by enzyme electrophoresis. Mycologia 79, 614–620.Dell, E. (1955) De aantasting van de oliepalm op Sumatra door Ganoderma lucidum. Bergcultures 24, 191–203.Edel, V., Steinberg, C., Gautheron, N. and Alabouvette, C. (1997) Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species. Mycological Research 101, 179–187.Forster, H. and Coffey, M.D. (1990) Mating behaviour of Phytophthora parasitica: evidence for sexual recombination in oospores using DNA restriction fragment length polymorphisms as genetic markers. Experimental Mycology 14, 351–359.Forster, H., Oudemans, P. and Coffey, M.D. (1990) Mitochondrial and nuclear DNA diversity within six species of Phytophthora. Experimental Mycology 14, 18–31.
178 R.N.G. Miller et al.Gardes, M. and Bruns, T.D. (1991) Rapid characterization of ectomycorrhizae using RFLP pattern of their PCR amplified-ITS. Mycological Society Newsletter 41, 14.Gardes, M., Mueller, G.M., Fortin, J.A. and Kropp, B.R. (1991) Mitochondrial DNA poly- morphisms in Laccaria bicolor, L. laccata, L. proxima and L. amethystina. Mycological Research 95, 206–216.Gaudet, J., Julien, J., Lafay, J.F. and Brygoo, Y. (1989) Phylogeny of some Fusarium species, as determined by large subunits rRNA sequence comparison. Molecular Biology and Evolution 6, 227–242.Genty, P., De Chenon, R.D. and Mariau, D. (1976) Infestation des racines aeriennes du palmier a huile par des chenilles genre Sufetula Walker (Lepidoptera: Pyralidae). Oleagineux 31, 365–370.Gilbertson, R.L. and Ryvarden, L. (1986) North American Polypores. Fungiflora, Oslo, Norway.Grossman, L.T. and Hudspeth, M.E.S. (1985) Fungal mitochondrial genomes. In: Bennett, J.W. and Lasure, L.L. (eds) Gene Manipulations in Fungi. Academic Press, Orlando, Florida, pp. 65–103.Guillaumin, J.J., Anderson, J.B., Legrand, P. and Ghahari, S. (1994) Use of different methods for mapping the clones of Armillaria spp. in four forests of central France. In: Johansson, M. and Stenlid, J. (eds) Proceedings of the Eighth International Confer- ence on Root and Butt Rots. IUFRO. Swedish University of Agricultural Sciences, Wik, Sweden and Haikko, Finland, pp. 437–458.Handa, A.K., Bressan, R.A., Korty, A.G., Jayaswal, R.K. and Charles, D.J. (1986) Isolation and characterization of pectolytic non-pathogenic mutants of Erwinia carotovora subsp. carotovora (Ecc). In: Proceedings, sixth International Conference on Plant Pathological Bacteria. Nijhoff/Junk, Dordrecht.Hillis, D.M. and Dixon, M.T. (1991) Ribosomal DNA: Molecular evolution and phylogenetic inference. Quarterly Review of Biology 66, 411–453.Ho, Y.W. and Nawawi, A. (1985) Ganoderma boninense Pat. from basal stem rot of oil palm (Elaeis guineensis) in Peninsular Malaysia. Pertanika 8, 425–428.Hoeben, P. and Clark-Walker, G.D. (1986) An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera. Current Genetics 10, 371–379.Hseu, R.S. and Wang, H.H. (1990) An identification system for cultures of Ganoderma species. PhD thesis, Graduate Institute of Agricultural Chemistry, National Taiwan University.Hseu, R.S., Chen, C.Y., Ueng, Y.C. and Wang, H.H. (1989) The application of laccase isozyme electrophoretic patterns in the identification of Ganoderma species. Journal of the Chinese Agricultural Chemical Society 27, 209–217.Karlsson, J.O. and Stenlid, J. (1991) Pectic isozyme profiles of intersterility groups in Heterobasidion annosum. Mycological Research 95, 531–536.Keen, N.T. and Tamaki, S. (1986) Structure of two pectate lyase genes from Erwinia chrysantemi EC16 and their high level expression in Escherichia coli. Journal of Bacteriology 168, 595–606.Lambourne, J. (1935) Note on the root habit of oil palms. Malayan Agriculture Journal 23, 582.Lin, D., Dumas, M.T. and Hubbes, M. (1989) Isozyme and general protein patterns of Armillaria spp. collected from the boreal mixedwood forest of Ontario. Canadian Journal of Botany 67, 1143–1147.
Characterization of Ganoderma in Oil-palm Plantings 179Manicom, B.Q., Bar-Joseph, M., Kotze, J.M. and Becker, M.M. (1990) A restriction frag- ment length polymorphism probe relating vegetative compatibility groups and pathogenicity in Fusarium oxysporum f.sp. diathi. Phytopathology 80, 336–339.Markert, C.L. and Moller, F. (1959) Multiple forms of enzymes: tissue, ontogenetic and species specific patterns. Proceedings of the National Academy of Science USA 45, 753–763.McArthur, C.R. and Clark-Walker, G.D. (1983) Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts. Current Genetics 7, 29–35.Micales, J.A., Bonde, M.R. and Peterson, G.L. (1986) The use of isoenzyme analysis in fungal taxonomy and genetics. Mycotaxon 27, 405–449.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.R.M., Hussin, M.Z. and Meon, S. (1995a) Isozyme analysis for characterization of Ganoderma strains from South-East Asia. EPPO Bulletin 25, 81–87.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.R.M., Meon, S., Hussin, M.Z. and Hilsley, E.J. (1995b) A multidisciplinary approach to the characterization of Ganoderma in oil-palm cropping systems. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.M. (eds) Ganoderma, Systematics, Phytopathology and Pharmacology. Proceedings of the Contributed Symposium 59A,B 5th International Mycological Congress, Vancouver, pp. 57–66.Miller, R.N.G. (1995) The characterization of Ganoderma in oil palm cropping systems. PhD thesis, University of Reading, UK.Miller, R.N.G., Holderness, M., Bridge, P.D., Chung, G.F. and Zakaria, M.H. (1999) Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology 48, 595–603.Mills, S.D., Forster, H. and Coffey, M.D. (1991) Taxonomic structure of Phytophthora cryptogea and P. drechsleri based on isozyme and mitochondrial DNA analyses. Mycological Research 95, 31–48.Moncalvo, J.M., Wang, H.H. and Hseu, R.S. (1995) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87, 223–238.Morrison, D.J., Thomson, A.J., Chu, D., Peet, F.G., Sahota, T.S. and Rink, U. (1985) Isozyme patterns of Armillaria intersterility groups occuring in British Columbia. Canadian Journal of Microbiology 31, 651–653.Morrison, D.J., Macaskill, G.A., Gregory, S.C. and Redfern, D.B. (1994) Number of Heterobasidion annosum vegetative compatibility groups in roots of basidiospore- infected stumps. Plant Pathology 43, 907–912.Mugnai, L., Bridge, P.D. and Evans, H.C. (1989) A chemotaxonomic evaluation of the genus Beauveria. Mycological Research 92, 199–209.Nobles, M.K. (1948) Studies in forest pathology. VI. Identification of cultures of wood-rotting fungi. Canadian Journal of Research 26, 281–431.Nobles, M.K. (1965) Identification of cultures of wood-inhabiting Hymenomycetes. Canadian Journal of Botany 43, 1097–1139.Otrosina, W.J., Chase, T.E. and Cobb, F.W. (1992) Allozyme differentiation of inter- sterility groups of Heterobasidion annosum isolated from conifers in the western United States. Phytopathology 82, 540–545.Park, W.M., Lee, Y.S., Kim, S.H. and Park, Y.H. (1986) Characterization of isolates of Ganoderma lucidum by electrophoretic patterns of enzymes. Korean Journal of Mycology 14, 93–99.Paterson, R.R.M., Bridge, P.D., Crosswaite, M.J. and Hawksworth, D.L. (1989) A reappraisal of the terverticilliate Penicillia using biochemical, physiological and
180 R.N.G. Miller et al. morphological features III. An evaluation of pectinase and amylase isoenzymes for species characterization. Journal of General Microbiology 135, 2979–2991.Payne, J.H., Schoedel, C., Keen, N.T. and Collmer, A. (1987) Multiplication and viru- lence in plant tissues of Escherichia coli clones producing pectate lyase isozymes PLb and PLe at high levels and of an Erwinia chrysanthemi mutant deficient in PLe. Applied and Environmental Microbiology 53, 2315–2320.Peries, O.S. (1974) Ganoderma basal stem rot of coconut: a new record of the disease in Sri Lanka. Plant Disease Reporter 58, 293–295.Petch, T. (1910) Root diseases of the coconut palm. Fomes lucidus (Leys) Fr. Circular and Agricultural Journal of the Royal Botanic Gardens, Peradeniya, Ceylon 4, 323–336.Samuels, G.J. and Seifert, K.A. (1995) The impact of molecular characters on system- atics of filamentous ascomycetes. Annual Review of Phytopathology 33, 37–67.Sederoff, R.R. (1984) Structural variation in mitochondrial DNA. Advances in Genetics 22, 1–108.Sen, M. (1973) Cultural diagnosis of Indian Polyporaceae. 3. Genera Daedalea, Favolus, Ganoderma, Hexagonia, Irpex, Lenzites, Merulius and Poria. Indian Forest Records For- est Pathology 2, 277–304.Shaw, C.G. III. and Roth, L.F. (1976) Persistence and distribution of a clone of Armillaria mellea in a ponderosa pine forest. Phytopathology 66, 1210–1213.Simcox, K.D., Pedersen, W.L. and Nickrent, D.L. (1993) Isozyme diversity in Setosphaeria turcica. Canadian Journal of Plant Pathology 15, 91–96.Singh, G. (1991) Ganoderma – The scourge of oil palms in the coastal areas. In: Ariffin, D. and Sukaimi, J. (eds) Proceedings of the Ganoderma Workshop, 1990. Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 81–97.Smith, M.L. and Anderson, J.B. (1989) Restriction fragment length polymorphisms in mitochondrial DNAs of Armillaria: identification of North American biological species. Mycological Research 93, 247–256.Smith, M.L., Bruhn, J.N. and Anderson, J.B. (1994) Relatedness and spatial distribution of Armillaria genets infecting red pine seedlings. Phytopathology 84, 822–829.Stalpers, J.A. (1978) Identification of wood-inhabiting Aphyllophorales in pure culture. Studies in Mycology 16.Stasz, T.E., Weeden, N.F. and Harman, G.E. (1988) Methods of isozyme electrophoresis for Trichoderma and Gliocladium species. Mycologia 80, 870–874.Stenlid, J. (1985) Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility, and isoenzyme patterns. Canadian Journal of Botany 63, 2268–2273.Steyaert, R.L. (1967) Les Ganoderma palmicoles. Bulletin Jardin Botanique Nationale Belgique 37, 465–492.Steyaert, R.L. (1972) Species of Ganoderma and related genera mainly of the Bogor and Leiden 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. (1980) Study of some Ganoderma species. Bulletin Jardin Botanique Nationale Belgique 50, 135–186.Taylor, J.W., Smolich, B.D. and May, G. (1986) Evolution and mitochondrial DNA in Neurospora crassa. Evolution 40, 716–739.Thomas, V.E., Rutherford, M.A. and Bridge, P.D. (1994) Molecular differentiation of two races of Fusarium oxysporum special form cubense. Letters in Applied Micro- biology 18, 193–196.
Characterization of Ganoderma in Oil-palm Plantings 181Thomson, A. (1931) Stem-rot of the oil palm in Malaya. Bulletin. Department of Agricul- ture, Straits Settlements & F.M.S., Science Series 6.Thomson, A. (1935) Division of mycology. Annual report for the year 1934. Bulletin, Department of Agriculture, Straits Settlements & F.M.S., General Series 21, pp. 57–65.Thomson, A. (1939) Notes on plant diseases in 1937–1938. Malayan Agriculture Journal 29, 241.Tseng, T.C. and Chang, L.S. (1988) Studies on Ganoderma lucidum III. Production of pectolytic enzymes. Botanical Bulletin of the Academia Sinica 29, 23–32.Tseng, T.C. and Lay, L.L. (1988) Studies on Ganoderma lucidum IV. Identification of strains by chemical compositions in mycelial extracts. Botanical Bulletin of the Academia Sinica 29, 189–199.Tummakate, A. and Likhitekaraj, S. (1998) Situation of Ganoderma on oil palm in Thailand. In: Proceedings of First International Workshop on Perennial Crop Diseases caused by Ganoderma. CAB International, Wallingford, UK.Turner, P.D. (1965a) The incidence of Ganoderma disease of oil palms in Malaya and its relation to previous crop. Annals of Applied Biology 55, 417–423.Turner, P.D. (1965b) Infection of oil palms by Ganoderma. Phytopathology 55, 937.Turner, P.D. (1966) Ganoderma in oil palm. In: The Oil Palm in Malaya. Ministry of Agriculture and Coop, Kuala Lumpur, pp. 109–137.Turner, P.D. (1968) Two wild palms as possible sources of basal stem rot in coastal oil palm plantings. Planter, Kuala Lumpur 44, 645–649.Turner, P.D. (1976) Oil palm diseases in South-East Asia and the South Pacific. In: Corley, R.H.V., Hardon, J.J. and Wood, B.J. (eds) Oil Palm Research. Elsevier Scientific Publishing, Amsterdam, pp. 427–445.Turner, P.D. (1981) Oil palm diseases and disorders. Oxford University Press, Oxford.Turner, P.D. and Bull, R.A. (1967) Diseases and Disorders of the Oil Palm in Malaysia. Incorporated Society of Planters, Kuala Lumpur.Typas, M.A., Griffen, A.M., Bainbridge, B.W. and Heale, J.B. (1992) Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium. FEMS Microbiology Letters 95, 157–162.Varghese, G. (1965) Parasitic diseases of oil palm (Elaeis guineensis) with particular reference to pathological problems of this crop in Malaya. Malaysian Agriculturalist 6, 3–14.Varghese, G. and Chew, P.S. (1973) Ganoderma root disease of lowland tea (Camellia sinensis) in Malaysia: some aspects of its biology and control. Malaysian Agricultural Research 2, 31–37.Venkatarayan, S.V. (1936) The biology of Ganoderma lucidum on areca and coconut palms. Phytopathology 26, 153–175.Voelcker, O.J. (1953) Report of the Department of Agriculture of Malaya, 1951–53. Department of Agriculture of Malaya.Wahlstrom, K.T. (1992) Infection biology of Armillaria species: in vitro pectinolytic activity, infection strategy, field distribution and host responses. PhD thesis, Swedish University of Agricultural Sciences.Wakefield, E.M. (1920) Diseases of the oil palm in West Africa. Kew Bulletin 1920, 306–308.Westhuizen, G.C.A. van der (1958) Studies of wood-rotting fungi. I. Cultural characters of some common species. Bothalia 7, 83–107.
182 R.N.G. Miller et al.Westhuizen, G.C.A. van der (1959) Polyporus sulphureus, a cause of heart-rot of Eucalyptus saligna in South Africa. Journal of the South African Forestry Association 33, 53–56.Westhuizen, G.C.A. van der (1971) Cultural characters and carpophore construction of some poroid Hymenomycetes. Bothalia 10, 137–328.Westhuizen, G.C.A. van der (1973) Polyporus baudoni Pat. on Eucalyptus spp. in South Africa. Bothalia 11, 143–151.White, T.J., Bruns, T., Lee, S. and Taylor, J.W. (1990) Amplification and direct sequencing of fungal RNA genes for phylogenetics. In: Innis, M.A., Gelgard, D.H., Sninsky, J.J. and White, T.J. (eds) PCR Protocols: A Guide to Methods and Applica- tions. Academic Press, New York, pp. 315–322.Wijbrans, J.R. (1955) Het stamrot van de oliepalm. Bergcultures 24, 112–124.Yeong, W.L. (1972) Studies into certain aspects of the biology of wood decay pathogens of Hevea rubber and oil palm (Elaeis guineensis). Bulletin of the Agricultural Science Project Report, University of Malaya.
184 F. Abdullahto market demands, many growers started to replace coconuts with oil palms.Whenever possible, the oil palms were planted on former coconut plantations,as to have made new jungle clearings would have been very costly. Thus,‘underplanting’ was carried out, a practice whereby the oil-palm seedlingswere planted under existing coconut palms, until such a time when thecoconut palms were poisoned and felled. Underplanting seemed to provide a continuous source of income, but thepractice could be a pathological hazard if the relationship between diseasedevelopment of oil palms planted on ex-coconut lands holds true. Coconutstumps and logs have often been observed to support abundant Ganodermafruiting bodies, leading to the opinion that they were the source of Ganodermainoculum that later caused infections on oil palms (Navaratnam, 1964;Turner, 1965a, b). Based on field observations, both authors surmised thatthe point of entry of the pathogen was through roots, and that disease spreadwas by contact of infected plant debris with healthy oil-palm roots. In amolecular-based study of Ganoderma from plantations in Malaysia, Miller(1995) did not support root-to-root contact as the mode of disease spread – hehypothesized that disease spread by spores, or via roots, from previous cropresidues was more likely. The possible role of basidiospores in disease spreadwas further supported by Sanderson and Pilotti (1998), based on develop-ments of the disease in Papua New Guinea. The current study focused on the development of BSR of oil palms plantedon an old coconut plantation. Crop mappings were done at three time intervalsover a 30-month period, which allowed disease development to be viewedspatially as well as sequentially. Disease progression of the first few infectedpalms was studied until the palms succumbed to the disease. In addition,vegetative compatibility of a reference isolate of Ganoderma from a selectedcoconut stump with other Ganoderma isolates, collected from other stumpswithin its immediate vicinity, and from an oil palm was studied. The hypothe-sis employed here is that if anastomosis, or the mycelial mergence between twoisolates, took place, then the isolates must have come from a commoninoculum. If this was detected between a reference isolate with others thatcame from two or more sources, then the disease could have spread by mycelialfragmentation, implying root-to-root contact.Background and Cropping Practice of the Sampling SiteThe oil-palm smallholding within which the sampling block was selectedwas located in Morib, in the district of Banting, Selangor, on the west coast ofPeninsular Malaysia. The site is about 5 km from the coastline. Pertinent infor-mation of the site was based on personal communications with the owner. The sampling block was situated in a larger existing coconut estate atthe time the first survey (SI) was conducted, but by the final survey (SIV), all ofthe estate land had been converted to oil palm. The coconuts surrounding the
Spatial and Sequential Mapping of BSR on Oil Palms 185block were of the local Malaysian Tall variety. The sampling site consisted of a1983 planting, which was free from BSR prior to the survey. The first sightingof one infected oil palm was at SI, by which time all palms in the block wereapproximately 13 years of age. When the oil palm stand was first planted,all the seedlings were placed in-between then-existing tall coconut palms, atraditional practice that allowed growers to harvest coconuts before the oilpalms start to bear fruits. The practice had an added advantage in that itprovided shade from the strong heat of the sun. The grower did not seeanything amiss with this planting technique and the procedure has been isstandard practice. The coconut palms were later poisoned; a few were cut down to facilitatethe infrastructure, but practically all others were left in situ. Over the yearsmany of the poisoned trunks have fallen, breaking in the middle or at the basalpart of the trunk, while a few were totally uprooted. Fallen trunks and cutoil-palm fronds were stacked in-between rows of oil palms, most of which haddegraded by the time SI was conducted. However, cut stumps and stumps leftafter the trunks had broken and fallen were still intact, and these were thesubject of interest in this study.Surveys and Crop MappingsFour surveys, referred to as SI, SII, SIII and SIV, were carried out on thesampling block. SI was conducted in May 1996, but a crop map was notproduced. SII, SIII and SIV were carried out in November 1996, November1997 and November 1998, making the survey time intervals as 0, 6, 18 and30 months, respectively. At each of the latter surveys, the disease status ofpalms within the block was recorded and a crop map made. A palm wasrecorded as ‘infected’ if it had Ganoderma sporophores on any part of the trunk,regardless of whether disease signs were present or otherwise. The sampling block consisted of 110 palms, which was convenientlybordered by large drains on its lateral sides and a drain and fence at theentrance. Each individual palm was identified by a code number for mappingpurposes. Eleven palms in a row were alphabetically coded from A to K. Thiswas followed by a further 10 palms per each row; so that any single palmwould be coded by a letter of the alphabet followed by a digit, e.g. A1 to A10 forall palms in row ‘A’ (Fig. 14.1). The prefix ‘EG’ was used to describe a palm orGanoderma isolate collected from an oil palm at the coded location and ‘CN’ waslikewise used for coconut stumps or isolates collected from them. Four categories of palms were identified and coded accordingly on themap. These were, newly infected (NI), for palms that were observed as infectedfor the first time at each survey point; and (I), for palms that still showedsymptoms of infection but whose status had been recorded at an earlier survey.Infected and fallen palms at the time of survey were recorded as (FP), and newlyplanted seedlings as (NP). New plantings or replants also indicated points
186 F. AbdullahFig. 14.1. Spatial mapping of the sampling site at SII, showing 3.6% infectionof oil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump withsporophores; v, newly infected palm.where palms had fallen due to BSR but which had been replaced with youngreplants (personal communication by the owner). This study thus regards thestatus of NP as ‘formerly infected’ palms and they were thus included as data inthe calculation of percentage of infected palms at each survey point where theywere first detected, but not thereafter. Palms under the status NP were mostlyplanted in the very hole where the diseased oil palm once stood.Crop Status and Distribution of GanodermaCrop status at SI, May 1996A total of 4–6 coconut stumps within the sampling block were found to har-bour 1–5 Ganoderma sporophores per stump. This represented a conservativeestimate of 6% of all coconut stumps as those supporting Ganodermasporophores. Only one oil palm, EG/F5, was observed to have had Ganoderma fruitbodies on its trunk base. This represented 0.9% incidence on oil palms withinthe sampling block. Despite the emergent sporophores at its base, palm EG/F5did not show any foliar yellowing nor multiple spear formations, appearing nodifferent from its healthy neighbours. No crop map was made at SI.Crop status at SII, 6 months after SIThe number of stumps bearing Ganoderma had increased to 18 and appearedto be located within a noticeable ‘clump’ between rows A to E. The 18 stumps
Spatial and Sequential Mapping of BSR on Oil Palms 187recorded to have had Ganoderma fruit bodies at SII were CN/A3, -A5, -B2, -B3,-B4, -B5, -B6, -C1, -C2, -C4, -C5, -D1, -D2, -D4, -E1, -E3, -H2 and -H3 (Fig.14.1). The number of oil palms with Ganoderma fruit bodies on their trunk baseshad increased to four, representing 3.6% of infected palms in the block. Thepalms were EG/F5, -I4, -J4 and -B6. Palm EG/F5 was an old infection (I) but thelatter three were new cases (NI). These four were accorded a ‘pioneer status’for diseased palms, whose disease progression over time was monitored. Allfour palms did not show any sign of disease inception; there was no ‘multiplespear’ formation, nor collapsed fronds and the leaves were of a normal, healthyshade of green. Fruit-bunch production of these particular four palms wasoptimal and the owner was not aware of any pathological problems.Crop status at SIII, 18 months after SIMany of the stumps recorded earlier as having Ganoderma fruit bodies werealmost totally degraded. Of the few still present, only two were observed tosupport Ganoderma sporophores. These were CN/D3 and CN/E4. The number of newly infected oil palms was 23, which represented ca.20% of infected palms in the sampling block at SIII (Fig. 14.2). Of these, EG/F5and EG/B6 of the pioneer palms were still standing (status ‘I’), but EG/I4 andEG/J4 were already found as new plantings (NP). There was an assortment ofstatus for the remaining infected oil palms. Ten were NI and five were FPwhose NI status were not observed at SII. The remaining palms were newplantings (NP) and palms showing symptoms. The replants were made due toFig. 14.2. Spatial mapping of the sampling site at SIII, showing 20% infectedoil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump withsporophores; g, fallen palm; #, new planting; v, newly infected palm.
188 F. Abdullahpalms that had fallen after SII but prior to SIII. During this survey, almost all ofthe infected palms displayed various degrees of the typical signs and symptomsassociated with basal stem rot, including the two ‘pioneer palms’ that were stillalive. Besides having Ganoderma fruit bodies, infected palms showed multiplespear formation, thinning of the crown and exhibited various degrees ofleaf necrosis; some of the palms showed ‘frond collapse’, where the outermostleaves hung down and enveloped the trunk. Palms thus affected were stillproducing fruits, although fruit-bunch production was poor (personalcommunication by the owner).Crop status at SIV, 30 months after SIThe majority of coconut stumps in the sampling block had totally degraded(Fig. 14.3). Of the handful still present, stump CN/J5 was the only one that stillhad Ganoderma sporophores on it. The total number of oil palms with somesymptoms of BSR was 37, which was 33% of the sampling block. Out of thisnumber, 9 were cases of FP and 11 were NP. The remaining 17 were cases ofNI; including that of a new replant. Palm EG/B3, estimated to be about 3.5years in age, had three sporophores at its base. The replant showed slight leafchlorosis on the two lowermost fronds but all other associated signs were notprevalent. Its trunk was hardly discernible because of its young age and sporo-phores that emerged appeared ‘squeezed’ out from the soil, but were definitelycoming from the trunk tissues.Fig. 14.3. Spatial mapping of the sampling site at SIV, showing 33% infectedoil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump withsporophores; g, fallen palm; #, new planting; v, newly infected palm; x, newlyinfected replant.
Spatial and Sequential Mapping of BSR on Oil Palms 189Disease Development of the First Few Infected PalmsThe progression of disease development in all four ‘pioneer’ infections at SIup to SIV (Table 14.1) indicated that these palms were observed as ‘near-symptomless’ at the start, but the longest such a condition lasted was between12 and 18 months. This was based on palms EG/I4 and J4, which fell within 12months after their first symptoms were detected. However, the earliest of all thefirst few infected palms (EG/F5) fell any time between 19 and 30 months, for itwas still recorded as an old infection (I) at SIII. All four were already replacedby NP, at SIV (Table 14.1).Mycelial Isolations and Vegetative Compatibility StudiesSamples for compatibility studies were collected at SII where stump CN/B5was selected as the reference point. One sporophore each was collected fromhere as well as from its immediate neighbours, and were brought back to thelaboratory for mycelial isolations. For each sporophore, pieces of tissues about0.5 cm3 in size were cut out from the innermost or context layer of the fruitbody. These were then surface sterilized in 5% sodium hypochlorite for 2–3minutes and then transferred under aseptic conditions on to malt agar toobtain pure mycelial cultures. A 3 mm diameter agar disc of CN/B5 mycelia was cut out with a flamedcork borer and plated at one end of a culture dish. This culture was pairedwith similar-sized agar disc cultures of isolates from its neighbouring sources.Duplicate plates for each combination were prepared. As the cultures grew,they were observed for anastomosis, or the mergence of mycelia from twoopposing directions. Anastomosis would indicate vegetative compatibilitybetween the paired isolates. Where cultures did not merge but formed a zoneor line of demarcation, the paired isolates were considered as vegetativelyincompatible. Isolate CN/B5 was thus plated against isolates CN/B4, CN/B6,CN/A5 and CN/C4, which were its immediate neighbours to the south, north,west and east, respectively (Fig. 14.1). Each of the five cultures collected fromstumps were also plated against isolate EG/B6, a relatively isolated infected oilpalm situated in the midst of ‘a clump’ of stumps with sporophores at SII. Table 14.1. Disease development of first infected palms at SI to SIV. Status of infected palms over 4 surveys Infected palms SI SII SIII SIV B6 – NI I NP F5 NI I I NP I4 – NI NP NP J4 – NI NP NP
190 F. Abdullah Vegetative incompatibility was demonstrated in all instances of binarypairing. Isolate CN/B5 was incompatible with each of its representativeneighbours on stumps CN/B4, CN/B6, CN/C4 and CN/A3. Each of the culturesabove were also incompatible with EG/B6.Other Field NotesThe initial emergence of sporophores on newly infected cases was found to bein an east–west orientation on the palm bases. The fruit bodies emerged fromground level up to an approximate height of 2½ ft (76 cm), but did not exceed4 ft (122 cm). None of the standing Malaysian Tall variety of coconut palms outside thesampling block at SI indicated the presence of G. boninense (with its typicallyreddish-brown and highly lacquered fruiting bodies). Instead, there werefruiting bodies on some stumps, but not on big palms, and these were of thenon-laccate variety, which belonged to the Ganoderma cf. applanatum/australecomplex. Stumps within the sampling site were also observed to have had thenon-laccate fruiting bodies initially, but these disappeared when the laccateG. boninense assumed prominence. However, there was one case of an oil-palmreplant (approximately 5 years old) outside the sampling block that had anon-laccate Ganoderma sporophore on its trunk, in addition to several laccateones.DiscussionSource of GanodermaThis survey found coconut stumps to be the most likely source of G. boninensein the sampling site. Initially, Ganoderma sporophores were prominent onstumps but were initially absent on oil palms. The presence of non-laccateGanoderma sporophores were found to precede those of G. boninense on stumps,both from within and outside the sampling blocks. However, it is not knownwhether their presence plays any role in the establishment of G. boninense. While the Ganoderma population decreased on stumps, its presence on oilpalms increased considerably. From a mere 0.9% incidence initially, it reached3.6% at SII, at a time when the Ganoderma population was at its highest oncoconut stumps. However, the presence of Ganoderma on oil palms escalatedto 20% at SIII, corresponding with its population decline on stumps. By SIV,oil palms with BSR had reached 33%, representing a significant increase over30 months. A study carried out by F. Abdullah (unpublished) showed thatGanoderma isolates from coconut stumps were also able to infect oil palms,based on artificial infection of oil-palm seedlings.
Spatial and Sequential Mapping of BSR on Oil Palms 191Development of disease signs and symptomsThe first few infected palms did not show signs of disease inception but this didnot last long as they were recorded as fallen palms within 12 months. Thisduration is considered very rapid, given the experience that infected palms inseveral plantations, particularly in inland areas, may still be producing fruitsfor many more years despite having fruit bodies at their bases. The overall rateof fall of palms was also rapid. Rather than draw conclusions on the possibleaggressive nature of the pathogen in the block, this study proposed twopossible causes that may have aggravated the situation. First, it could be thatthe site, in close proximity to the coast, may have been subject to the strongcoastal winds, resulting in the palms falling over as soon as the trunks becameweakened. The second possibility is that of stress caused by climatic conditions.The year 1997 was an eventful one, where Malaysia was subjected to seriousclimatic changes as a result of El Niño, including extremely high dailytemperatures and ‘the haze’ produced as a result of forest burning. All thesefactors may have caused added stress to the palms. As a result, the palmssuccumbed to the disease at a particularly high rate.Considerations of the possible mode of spreadThe incidence of BSR varies between regions in Malaysia. Disease incidencemay be high in oil palms planted on old coconut in some areas, but not others(Turner, 1965a). It is not known whether physical factors such as soiltypes, rainfall or fertilizer application play a role in aggravating the disease; oralternatively, that particularly aggressive variants of species of Ganoderma maybe present in the population. There are three possible ways by which the fungalpathogen can be directly spread to the host: namely, by root-to-root contact,via airborne spores and finally, from independent secondary inocula in the soil.Root-to-root contactSingh (1991) reported that infected palms appeared in groups and then formedseveral foci of infection in long-standing cases. He concluded that the modeof spread was by root-to-root contact. Flood et al. (1998) described a similarincidence where a clumping effect was evident in oil-palm blocks with rela-tively few infected palms, but this trend disappeared when larger numbers ofinfected palms occurred in the blocks. From the viewpoint of disease spread, aclumping of infected palms would theoretically suggest a common origin froma single inoculum, thus stating a case for root-to-root contact. However, in thisstudy, the first four palms that were infected (pioneer palms) were relativelyfar from each other as well as from the clump of stumps where the Ganodermapopulation was concentrated. Furthermore, vegetative incompatibility of iso-lates collected from coconut stumps and an infected oil palm would not supportroot-to-root infection, although the incompatibility was also demonstrated
192 F. Abdullahamongst a large number of isolates collected from a single palm, making thismethod a less reliable basis for assessing root-to-root infection (Abdullah,unpublished data) than molecular techniques.Spread by sporesFrom his observations, it seemed evident to Thompson (1931) that in typicalcases of stem rot, the disease was caused by spores that entered the stemthrough some of the old leaf bases which have been rotted away, or throughwounds from leaf bases, as in leaf pruning during harvesting. He proposed thatinfection through wounds would allow a quicker stem penetration, besideshaving had a shorter distance to travel, compared to the distance if it was aroot entry. However, attempts at establishing pathogenicity of the crop basedon trials using spores alone were not successful. In this study, it was observed that practically all infected cases hadsporophores at the bases and no more than 2–4 ft (76–233 cm) up the trunk. Ifthere is a random dispersal by airborne spores, then some palms should showGanoderma fruiting bodies at other heights of the palm as well. In a study of theoccurrence of upper-stem rot of oil palms in Sabah (Abdullah et al., 1999)Ganoderma sporophores were observed very close to the crowns of old palms,some 25–30 ft (7.6–9.1 m) above ground level, although their presence therewas believed to be secondary. Only airborne spores could have been responsi-ble. Thus, the fact that all Ganoderma fruit bodies were confined to not morethan 4 ft (122 cm) from the base of the oil palms in the sampling site, does notsuggest random dispersal by airborne spores.Spread from secondary inoculaSpatial mapping by Miller (1995) of two blocks of oil-palm stands, followedby molecular and compatibility studies, showed no evidence for root-to-rootcontact, except where two adjacent palms contained the same ‘individual’, asdetermined by molecular analyses. He proposed that spread could be by sporesor from separate inocula from previous plantings. In the case of the diseased replant in this sampling site, it is obvious thatthe mode of spread was by infection from secondary inocula left by the previousinfected palm. This is an interesting phenomenon in that it allowed one toestimate the time at which the pathogen first entered the palm tissues to theeventual emergence of sporophores: approximately 12 months in this inci-dence. However, this is an isolated case, rather than the typical infection. Forthe rest of the infected palms which had been standing for at least 13 years, thesource of infection would appear to be from independent secondary inocula,although it is difficult to suggest the source of secondary inocula, given thatthe previous planting consisted of only coconuts and that no G. boninensesporophores were ever observed on the standing crop. Coconut palms are notknown to be infected by Ganoderma in Malaysia (apart from the single and lastreport in 1934 by Tempany; as cited in Navaratnam, 1961), but reports fromIndia (Bhaskaran and Ramanathan, 1984; Bhaskaran et al., 1998) and Sri
Spatial and Sequential Mapping of BSR on Oil Palms 193Lanka (Peries, 1974) indicate that coconut palms are badly infected byGanoderma in these countries. One explanation is that the Malaysian coconutsrespond to Ganoderma infection in a different manner. It can be speculated thatthe Malaysian Tall variety of coconuts is probably infected as well, but theseare not debilitated in any way by the fungus. The coconut palms may carrythe inocula as an endophyte with no production of sporophores. When thecoconut palm dies a large amount of inoculum is made available. Much later,this fungus infects oil palms in a similar manner to coconut palms but unlikethe coconuts, oil palms succumb easily to the disease. Swinburne et al. (1998)reported that a significant number of living coconuts were found to containGanoderma, thus further strengthening the case for the above hypothesis.ConclusionThe incidence of basal stem rot varies between regions in Malaysia. Thereseemed to be a correlation of disease severity with former croppings, particu-larly old coconut plantations. This study examined one such model. Based onspatial distribution, vegetative incompatibility studies and the rapid death ofinfected palms, this study does not support root-to-root contact as a mode ofdisease spread, nor lend support to disease spread by airborne spores alightingon crevices of cut leaf fronds. The study favours disease spread from independ-ent secondary inocula such as residues from the previous crop (i.e. coconut),and the possibility of the fungus existing in the living coconuts as anendophyte is suggested.AcknowledgementsThis project was funded by the Intensified Research Priority Areas from theMinistry of Science, and Technology Malaysia. I would like to thank PuanLatifah Z. Abidin for the field and lab assistance.ReferencesAbdullah, F., Liew, S.B. and Malik, N. (1999) Upper stem rot of oil palms (E. guineensis) in Langkon, Sabah. In: Sidek, Z., Bong, S.L., Vijaya, S.K., Ong, C.A. and Husan, A. Kadir (eds) Sustainable Crop Protection Practices in the Next Millennium. Malaysian Plant Protection Society, pp. 101–103.Bhaskaran, R. and Ramanathan, T. (1984) Occurrence and spread of Thanjavur wilt disease of coconut. Indian Coconut Journal 5(6), 1–3.Bhaskaran, R., Karthikeyan, A. and Giridharan, S. (1998) Etiology and epidemiology of basal stem rot disease of coconut. Second International Workshop on Ganoderma Diseases of Perennial Crops, MARDI, Serdang, Malaysia, 5–8 October.
194 F. AbdullahFlood, J., Meon, S., Chung Gait Fee, Leidi, A. and Miller, R.N.G. (1998) Spatial mapping of Ganoderma in the field. Second International Workshop on Ganoderma Diseases of Perennial Crops, MARDI, Serdang, Malaysia, 5–8 October.Ho, Y.W. and Nawawi, A. (1985) Communication I. Ganoderma boninense (Pat.) from basal stem rot of oil palms (Elaeis guineensis) in Peninsular Malaysia. Pertanika 8(3), 425–428.Khairudin, H. (1990) Basal stem rot of oil palm: incidence, etiology and control. MSc thesis, Universiti Pertanian, Malaysia, Malaysia.Miller, R.N.G. (1995) The characterization of Ganoderma in oil palm cropping systems. PhD thesis, University of Reading, UK.Navaratnam, S.J. (1964) Basal stem rot of oil palms on ex-coconut estates. Planter 40, 256–259.Peries, O.S. (1974) Ganoderma basal stem rot of coconut: a new record of the disease in Sri Lanka. Plant Disease Reporter 58, 293–295.Sanderson, F. and Pilotti, C. (1998) Spores as a mechanism for variation in the host/pathogen interaction. Second International Workshop on Ganoderma Diseases of Perennial Crops, MARDI, Serdang, Malaysia, 5–8 October.Singh, G. (1991) The scourge of oil palms in the coastal areas. Planter 67(786), 421–444.Steyaert, R.L. (1976) Les Ganoderma Palmicoles. Bulletin du Jardin Botanique National de Belgique 37(4), 465–492.Swinburne, T.R., Seman, I.A., Watt, T. and Ariffin, D. (1998) Basal stem rot of oil palm in Malaysia: factors associated with variation in disease severity. Second Interna- tional Workshop on Ganoderma Diseases of Perennial Crops, MARDI, Serdang, Malaysia, 5–8 October.Thompson, A. (1931) Stem rot of oil palm in Malaya. Department of Agriculture, Straits Settlements and F.M.S. Science Series No. 6.Turner, P.D. (1965a) The incidence of Ganoderma disease of oil palm in Malaya and its relation to previous crop. Annals of Applied Biology 55, 417–423.Turner, P.D. (1965b) Oil palms and Ganoderma II. Infection and spread. Planter 41, 238–241.
196 C.A. Pilotti et al.of Miller et al. (1995) and concluded that spread of the pathogen by meansother than vegetative was likely. The work presented here is part of a studyto determine the basis for the variability in Ganoderma species that occur inassociation with oil palm in Papua New Guinea. Molecular methods are beingdeveloped to study pathogen populations to clarify the role of the sexual cyclein the epidemiology of basal stem rot. Random amplified polymorphic DNA(RAPD) analysis was selected to investigate variation amongst monokaryonsprior to population studies on dikaryons. Other markers targeting moreconserved regions of the fungal genome were also investigated, including themitochondrial small and large subunits of the ribosomal gene (rDNA) andthe internal transcribed spacer (ITS) and intergenic spacer (IGS) regions, thelatter having the potential to reveal inter-species differences. These molecularmarkers will be used to analyse and determine the nature of Ganodermapopulations on oil palm and may be applied in other cropping systems wherethe fungus is a pathogen.ExperimentalIsolationsMonokaryotic cultures were obtained by germinating basidiospores of G.boninense on water agar with subsequent transfer to potato dextrose agar (PDA).Dikaryotic cultures were isolated from the context of fresh basidioma growingon oil palm and on dead wood. All cultures were maintained on PDA at 30°C. For DNA extraction, cultures were grown in glucose (10 g l−1), yeastextract (20 g l−1) medium for 7–10 days and then harvested by filtration.Mycelium was lyophilized and ground in a mortar and pestle.Extraction of DNADNA extraction was carried out using a slight modification of the method ofRaeder and Broder (1985).Polymerase chain reaction (PCR)PCR was used to amplify DNA from the large and small mitochondrialribosomal RNA subunits and the nuclear rRNA internally transcribed, andintergenic spacers. RAPD amplification was undertaken with Operon series Aprimers. Primers and PCR conditions are given in Table 15.1. PCR was carriedout on a programmable thermocycler (MJ Research). Programmes were asfollows. RAPDs: initial denaturation, 5 min at 94°C then 1 min at 94°C,followed by annealing of 1 min at 35°C and extension of 2 min at 72°C for
Genetic Variation in Ganoderma spp. from Papua New Guinea 197Table 15.1. Primers and PCR conditions. Annealing Primer temperature ReferenceMitochondrial small subunit MS1/MS2 50°C/45 s White et al. (1990)Mitochondrial large subunit ML3/ML4 50°C/45 s White et al. (1990)ITS BMB-CR/LR1 50°C/45 s Moncalvo et al. (1995)IGS LR12/O-1 50°C/45 s Park et al. (1996)RAPD Operon A 1–20 35°C/1 minITS, internal transcribed spacer; IGS, intergenic spacer; RADP, random amplifiedpolymorphic DNA.39 cycles, with a final extension step of 5 min at 72°C. Mitochondrial DNA(mtDNA) and ITS amplifications followed the same programme, except thatthe annealing temperature was 50°C for a duration of 45 s. Bulk mixtures of reagents containing reaction buffer, 1–2.5 mM MgCl2,100 µm deoxyribonucleotide triphosphates, 100 µm primer and 0.5 unitsTaq DNA polymerase were made and 24 µl aliquots plus 1 µl template DNA(approximately 10–20 ng) were subjected to PCR.ResultsComparison of sibling monokaryons using RAPDsTwenty operon RAPD primers were screened. Fifteen of these gave amplifica-tion products and five generated a sufficient number of fragments showingpolymorphisms amongst sibling monokaryons. These were OPA-02, OPA-15,OPA-18, OPA-19 and OPA-20. Figures 15.1–15.4 show examples of finger-prints for monokaryons from different basidioma. Numerical analysis of the collected band patterns obtained for eachbasidioma showed that band patterns were specific to individual single sporecultures, and that no two single cultures gave identical patterns (Figs15.5–15.7). The similarities derived from Jaccard’s coefficient are under-estimated and intended only as a guide to the range of variation withinfamilies. Clearly, each sibling monokaryon appears to have a unique RAPDgenotype from the isolates studied so far.MtDNAPCR of sibling monokaryons of G. boninense with the primer combinationMS1/MS2 to amplify the mitochondrial small subunit gave two products ofapproximately 600 bp and 1790 bp. Monokaryotic isolates of Ganoderma sp.gave a single amplification product of about 600 bp (Fig. 15.8).
198 C.A. Pilotti et al.Fig. 15.1. Randomly amplified polymorphic DNA fingerprints of siblingmonokaryons (isolate #80, primer OPA-20) (kb markers: 1353, 1078, 872, 603,310).Fig. 15.2. Randomly amplified polymorphic DNA fingerprints of siblingmonokaryons (# 80, primer OPA-18) (FN-1 markers: 2686, 1563, 1116, 859, 692,501, 404, 331). Dikaryotic isolates of G. boninense also gave an additional amplificationproduct at about 1790 bp, and in some samples this was the only fragmentproduced (Fig. 15.9). Repeated amplifications with duplicate samples gave thesame result. Intra- and interspecific length variation was not observed for the mito-chondrial large subunit although some isolates yielded a single amplification
Genetic Variation in Ganoderma spp. from Papua New Guinea 199Fig. 15.3. Randomly amplified polymorphic DNA fingerprints of siblingmonokaryons (#78, primer OPA-18) (FN-1 markers: 2686, 1563, 1116, 859, 692,501, 404, 331).Fig. 15.4. Randomly amplified polymorphic DNA fingerprints of monokaryons ofisolate 87 (primer OPA-18) (FN-1 markers: 2686, 1563, 1116, 859, 692, 501, 404,331).product of 2030 bp, in length. Amongst monokaryons from both species, onlythe expected fragment of approximately 800 bp was amplified.ITS and IGS DNAPrimers BMB-CR and LR gave an amplification product of approximately800 bp incorporating the entire ITS1 and ITS2 region, and this was consistent
200 C.A. Pilotti et al.Fig. 15.5. Dendrogram depicting unique genotypes of sibling monokaryons(isolate #78). Mating alleles assigned are given in parentheses.Fig. 15.6. Dendrogram of sibling monokaryons (isolate #80). Mating allelesassigned are given in parentheses.Fig. 15.7. Dendrogram of sibling monokaryons (isolate#87). Mating allelesassigned are given in parentheses.both within and between species, although some additional amplificationproducts were observed for a few isolates (Fig. 15.10). The IGS region also appeared highly conserved amongst species. Totallength, including intervening sequences, was approximately 1000 bp for allsamples, regardless of host origin. Digestion of the amplified fragments fromboth ITS and IGS regions with the restriction enzymes Sau3A and Cfo1 gaveidentical fragments irrespective of species or host (data not shown).
Genetic Variation in Ganoderma spp. from Papua New Guinea 201Fig. 15.8. Mitochondrial small (a) and large (b) subunit (rDNA) amplificationof sibling monokaryons. Samples 1–7, Ganoderma boninense; samples 8–15,Ganoderma sp. (kb: 2686, 1563, 1116, 859, 692, 501).Fig. 15.9. Mitochondrial small subunit (rDNA) amplifications of Ganodermaisolates. Isolates 1–5 from oil palm, 7–11 from coconut, 12–16 from hardwood(kb FN-1: 2686, 1563, 1116, 859, 692).DiscussionGenetic variation has been observed amongst sibling monokaryons ofGanoderma boninense. This is the first report on the use of RAPDs to differentiatehaploid isolates of G. boninense and clearly demonstrates the importance ofsexual reproduction in maintaining genetic diversity in this fungus. Theseresults also emphasize the need for caution when using RAPD fingerprints ofdikaryons to infer relationships amongst isolates in population studies, giventhe variation within single spore isolates. There were no significant intra- or interspecies differences in the amplifi-cation products from the mitochondrial large subunit of the rDNA geneamongst monokaryons of Ganoderma sp. In contrast, an unexpected productof about 2030 bp was generated (in addition to the 800 bp fragment) whenthe mitochondrial large subunit was amplified in some monokaryons and
202 C.A. Pilotti et al.Fig. 15.10. ITS 1/2 amplifications of dikaryons from different hosts. Lanes 1–6, oilpalm; 8–10, coconut; 11–15, hardwood (kb marker FN-1: 2686, 1563, 1116, 859).dikaryons of G. boninense. White et al. (1990) noted that some species of Suillus(basidiomycetes) contained an intron in a portion of the mitochondrial LrRNAgene giving rise to fragments of 1700 and 2000 bp. However, for the G.boninense isolates, a similar product was also obtained when the mitochondrialsmall subunit was amplified. In this case the larger fragment was 1790 bp andin some samples (129, 130) this was the only PCR product. In the samples thatshowed two products, there appear to be competing reactions, as both frag-ments are inefficiently amplified. Control samples did not produce the 1790 bpfragment, so it is unlikely to be a contaminant but could possibly be a homolo-gous nuclear DNA sequence. It may or may not be of significance that theisolates that produced the additional fragment were all G. boninense that origi-nated from live oil palm or coconut. Isolates of Ganoderma sp. did not yield otherthan the expected product. When DNA from dikaryons of both species wereamplified, minor length differences were apparent for the mitochondrial smallsubunit within Ganoderma sp. but not G. boninense. Given these results, PCRamplification of the mitochondrial small and large subunits of rDNA maybe of limited use for both intraspecific and interspecies comparisons. Theseamplification products are, however, only a small part of the ribosomal DNAand it is expected that comparison of isolates using these products as probes todetect RFLPs in mtDNA sequences will be more informative. Further workusing the mitochondrial small subunit fragments is being undertaken to assessmtDNA variation within Ganoderma boninense. The ITS region was considered a potentially useful marker for interspeciesdifferences within Ganoderma; however, length differences between specieshave not been apparent. The ITS1/ITS2 region is expected to be around 400 bpfor Ganoderma, using the given primers (Moncalvo et al., 1995). When the PCR
Genetic Variation in Ganoderma spp. from Papua New Guinea 203products were digested with certain restriction enzymes, digestion productswere monomorphic. However, small sequence differences are found amongstspecies from several geographical locations, as shown by Moncalvo et al.(1995). Park et al. (1996) were able to differentiate Ganoderma species by amplifi-cation and digestion of the IGS region with various restriction enzymes. Whenthe IGS region of the PNG isolates was subjected to PCR, the total length ofthe amplified fragment was approximately 1000 bp. Digestions confirmedthe homology (at restriction sites) of the amplification product amongstisolates, although it is likely that small sequence differences are present. Fromthe foregoing, ribosomal DNA appears to be highly conserved within theGanoderma species studied, and the regions selected for PCR amplification so fardo not provide a useful and rapid means of detecting interspecific variation.Consequently, other methods are being investigated to study the Ganodermapopulations associated with oil palm in Papua New Guinea.ConclusionIntraspecific variation amongst closely related isolates of G. boninense has beenfound to be high. This variability, as revealed by PCR RAPDs, is indicative of anoutbreeding population, although the number of isolates tested so far is small.In contrast, variation in the ITS and IGS regions between species is low andsequencing of these regions will be necessary for interspecies comparisons. Themitochondrial fragments generated by PCR are not useful, on their own, forinterspecies comparisons.ReferencesAriffin, D., Idris, A.S. and Marzuki, A. (1996) Spread of Ganoderma boninense and vege- tative compatibility studies of a single field of oil palm isolates. In: Ariffin, D. et al. (eds) Proceedings of the 1996 PORIM International Palm Oil Congress (Agriculture). Palm Oil Research Institute of Malaysia, Bangi, Selangor, Malaysia, pp. 317–329.Bakshi, B.K., Reddy, M.A.R. and Singh, Sujan (1976) Ganoderma root rot in khair (Acacia catechu Willd.) in reforested stands. European Journal of Forest Pathology 6, 30–38.Harsh, N.S.K., Soni, K.K. and Tiwari, C.K. (1993) Ganoderma root rot in an Acacia arboretum. European Journal of Forest Pathology 23, 252–254.Kamp, B.J. van der, Gokhale, A.A. and Smith, R.S. (1979) Decay resistance owing to near anaerobic conditions in black cottonwood wetwood. Canadian Journal of Forest Research 9, 39–44.Lim, T. (1977) Production, germination and dispersal of basidiospores of Ganoderma pseudoferreum on Hevea. Journal of the Rubber Research Institute Malaysia 25, 93–99.Masuka, A.J. and Nyoka, B.I. (1995) Susceptibility of Eucalyptus grandis provenances to a root rot associated with Ganoderma sculptrutum in Zimbabwe. European Journal of Forest Pathology 25, 65–72.
204 C.A. Pilotti et al.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.D., Sariah, R.R.M., Hussin, M.Z. and Hilsley, E.J. (1995) A multidisciplinary approach to the characterization of Ganoderma in oil palm cropping systems. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.-M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. Proceedings of Contributed Symposium 59A,B. Fifth International Mycological Congress, Vancouver, August, 1994.Moncalvo, J.-M., Wang, Hsi-Hua and Hseu, Ruey-Shang (1995) Phylogenetic relation- ships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87, 223–238.Park, D.-S., Ryu, Y.-J., Seok, S.-J., Kim, Y.-S., Yoo, Y.-B., Cha, D.-Y. and Sung, J.M. (1996) The genetic relationship analysis of Ganoderma spp. using PCR-RFLP and RAPD. RDA. Journal of Agricultural Science 38(2), 251–260.Raeder, U. and Broder, P. (1985) Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17–20.Reddy, M.K. and Ananthanarayanan, T.V. (1984) Detection of Ganoderma lucidum in betelnut by the fluorescent antibody technique. Transactions of the British Mycological Society 82, 559–561.Ross, W.D. (1976) Relation of aspen root size to infection by Ganoderma applanatum. Canadian Journal of Botany 54, 745–751.Singh, S.P. (1985) Efficacy of fungicides in the control of anabe roga root rot disease of areca nut (Areca catechol). Agricultural Science Digest 5(3), 165–166.Turner, P.D. (1981) Oil Palm Diseases and Disorders. Oxford University Press, Oxford.Varghese, G. and Chew, P.S. (1973) Ganoderma root disease of lowland tea (Camellia sinensis) in Malaysia: Some aspects of its biology and control. Malaysian Agricultural Research 2, 31–37.White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. Academic Press, London.
206 H. Rolph et al.Ganoderma lucidum is reported to produce BSRs and wilts of coconut in India(Nambiar and Rethinam, 1986). There are many descriptions of Ganoderma-associated root rots and wilts of coconut palms from various regions in Indiaand Sri Lanka. Consequently, there is confusion concerning the species involved in thisdisease and the different symptoms they induce when infecting oil palms andcoconuts; they can also produce different symptoms on the same palm hostin different countries. For example, in Sri Lanka, stem bleeding and fruit-bodyformation on live palms are observed, while these symptoms are rarely seenon palms in Indonesia and Malaysia (M.K. Kip, personal communication).Conversely, although two different species of Ganoderma have been reportedon coconut palms in India and Sri Lanka, the symptoms produced appearidentical (Peries et al., 1975; Bhaskaran et al., 1989). Part of the confusion lies in the identification of the species involved andthe problems associated with the development of suitable species concepts fortropical Ganoderma isolates, which as yet have not been fully accomplished(Steyaert, 1975, 1980; Bazzalo and Wright, 1982; Adaskaveg and Gilbertson,1986). Although many macromorphological characters are used in theclassification of Ganoderma species, a number of authors have concluded thatmacromorphology alone is insufficient for the systematic determination ofGanoderma species (Bazzalo and Wright, 1982; Gilbertson and Ryvarden,1986). Identifying Ganoderma isolates to species level is important, but mappingindividual isolates across a plantation is equally so, in order to discoverwhether a single clone or several individuals are responsible for a particulardisease outbreak, and also to monitor subsequent spread. Despite the lack ofsuitable species concepts to fully identify potential crop pathogens, researchhas therefore progressed into mapping variation in Ganoderma isolates at theplantation level. The combination of molecular techniques and somaticincompatibility group (SIG) testing to assess the variation between Ganodermaisolates from different oil palms across a plantation has yielded interestingresults. Miller et al. (1999) assessed the variation in mitochondrial DNA (mtDNA)and SIGs from Ganoderma isolates in two oil-palm plantings, and suggestedthat the disease does not appear to spread in a clonal fashion via root-to-rootcontact. They found a high level of variation in the mtDNA profiles ofGanoderma isolates across two plantings of oil palms. SIG studies showed thateven adjacent palms were usually infected by different Ganoderma isolates,with members of each SIG usually confined to a single palm. It was very rare tofind a Ganoderma isolate from one SIG infecting two palms. This was confirmedby the fact that identical mtDNA profiles were also very rarely seen in isolatesfrom more than one palm. The study also showed that a single oil palm couldbe colonized by several Ganoderma isolates with different mtDNA profiles andSIGs. SIG studies by Ariffin et al. (1994) have also indicated that up to threedifferent Ganoderma isolates can infect a single oil palm.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 207Overview of Symptoms of Basal Stem Rot in Sri LankanCoconut and Betelnut PalmsA wide range of symptoms is displayed by these palms infected withGanoderma. The initial visible symptom is the presence of a reddish-brown,viscous liquid that oozes from longitudinal cracks in the base of the palmtrunk. This symptom, known as ‘stem bleeding’ (Fig. 16.1), is not found onGanoderma-infected oil palms and appears unique to coconut and betelnutpalms suffering from Ganoderma infection. This bleeding usually extends upwards through the trunk and it has beennoted that stem-bleeding symptoms can often extend 10–15 m up the trunksof coconut palms planted close to a water source. Under these conditions, thefungus does not advance as far up the trunk as it would do in palms growingin non-waterlogged soil. Analysis of such palm tissue has shown that theGanoderma infection is present only at the base of the palm, i.e. it does not Fig. 16.1. Stem bleeding symptoms on coconut palm (Courtesy of Tamil Nadu University, India).
208 H. Rolph et al.extend as far up the trunk as does the stem bleeding. Betelnut palms andCoryota urens palms found on canal banks adjacent to poorly drained grounddo not show the extended bleeding symptoms at all, and have no longitudinalcracks in their trunks. However, both types of palm can display small drops ofliquid at the base of the trunk. Another initial symptom of BSR is that fronds in the lower whorl ofthe palm turn yellow and dry prematurely. As the disease progresses, theproduction of inflorescences and the number of female flowers graduallydecreases and the fungus causes decay of the bole and root system of thepalm. Sporophores are occasionally seen around the bole of coconut palmsin Malaysia, but are common on live coconut palms in Sri Lanka. The lengthof the fronds is reduced and the palm begins to taper, and eventually diesapproximately 5–10 years after initial infection. Palm death is brought aboutby several factors: the palm bole is so decayed that it collapses and the palmfalls over; the crown is blown off by the wind; or there is a lack of translocationof nutrients and water to the upper part of the palm. In Malaysia, Ganoderma is not known to be a pathogen of coconut palms,but there is the possibility that coconuts might act as a reservoir for thepathogen (Navaratnam, 1964; Turner, 1965a, b; Abdullah, this volume). Thepractice of planting oil palm after coconut is a possible cause of BSR in oil palmsand, although the source of infection is unknown, any coconut debris left inthe soil should be considered a potential inoculum. In order to further investigate Ganoderma isolates from coconut andbetelnut palms, a small-scale study was established between CABI Bioscience,the Coconut Research Institute in Sri Lanka and Universiti Putra Malaysia.The main aim of this investigation was to assess the extent of molecularvariation and somatic incompatibility groupings in Ganoderma isolates fromSri Lankan coconuts. This variation would then be compared with the extentof variation found in isolates from Malaysian coconuts, on which Ganodermais not known to be a pathogen. A final part of the study was to assess thevariability in isolates from betelnut palms planted adjacent to coconut palms.This would determine whether there were any significant differences betweenGanoderma isolates from coconut palms and betelnut palms.Investigation into Sri Lankan and Malaysian GanodermaIsolates from Coconut and Betelnut PalmsThe isolates used in this study came from several coconut plantations inthe Hambantota district (southern province) of Sri Lanka, including theplantation where Ganoderma was first noted in that region. The disease hadnot been a serious problem for approximately 20 years, when, in 1995, asudden outbreak of root and bole rot of coconuts occurred. Since then therehas been increased interest in the genus Ganoderma and its role in basal stemrot disease.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 209 It was important to determine whether Ganoderma could be isolated notjust from sporophores growing on the palm, but from palms showing differentsymptoms. It was for this reason that Ganoderma isolates were taken from awide range of material from Sri Lankan coconut and betelnut palms. In severalcases, isolates were obtained from both palm tissue and fungal sporophores,to determine whether they were from the same individual, or representedtwo separate infections. Material collected included decayed stem tissuesand sporophores from live coconut and betelnut palms with stem bleeding;sporophores and decayed stem material from dead, standing palms; andfinally, stem tissue and sporophores from coconut stumps. A negative controlwas included, which consisted of a Ganoderma strain isolated from the stemtissue of a leguminous tree. Ganoderma isolates from Malaysian coconuts were taken from sporophoresfound on coconut stumps and oil palms from a smallholding mixed plot inBanting, Selangor, on the west coast of Peninsular Malaysia (Abdullah, thisvolume). Two molecular methods were used in the study. The first was thesame as that used in previous investigations at CABI Bioscience, namelymitochodrial (mtDNA) profiling. For a full description of this technique, seeMiller et al., this volume. In this current study, mtDNA profiles were generatedusing the enzyme HaeIII. Identical banding patterns were grouped togetherand designated as mtDNA profile group 1, mtDNA profile group 2, etc. The second technique assessed the total cellular DNA variation (i.e.nuclear and mtDNA) using amplification fragment length polymorphisms(AFLPs), according to the protocol devised by Vos et al. (1995). The combina-tion of mtDNA profiling and AFLPs was used to give a more complete picture ofthe molecular variation of the Ganoderma isolates. The AFLPs were performed on total genomic DNA extracted fromlyophilized mycelia and digested with a restriction enzyme, i.e. an enzyme thatcan recognize a key DNA sequence (usually four or more bases long) and cutsor ‘restricts’ it at that point. In this case the restriction enzyme PstI was used tocut the DNA. It creates ‘overhangs’ of several bases at the ends of the restrictedDNA (Fig. 16.2). A ligation reaction is then performed whereby the restrictedends of the DNA are joined to ‘adapters’. These adapters are short lengthsof double-stranded DNA, which are complementary to the overhangs ofthe restricted genomic DNA. The adapters also have sites complementaryto a specific set of oligonucleotide primers, which are used in the ensuingpolymerase chain reaction (PCR). PCR is the exponential amplification ofa region of template DNA bounded by short stretches of DNA that arecomplementary to a specific set of DNA primers. Thus, the template for thePCR reaction is any DNA bounded by the adapters, and only DNA with theseadapters at both ends is amplified. The size of DNA fragments amplified isdependent on the position of the restriction sites in the genomic DNA, becausethe adapters can only bind to DNA with the correct overhangs produced bythe restriction enzyme. Agarose gel electrophoresis is then used to separate theresultant fragments and produce the AFLP profiles.
210 H. Rolph et al.Fig. 16.2. Flowchart depicting the amplification fragment length polymorphism(AFLP) process. Patterns generated using this method are more stable, and therefore morereliable to use, than random amplified polymorphic DNAs (RAPDs). RAPDs areproduced by the random binding of PCR primers to target DNA and subsequentamplification of that target template. Low-stringency conditions are used togenerate RAPDs, and primers may bind to target sequences with which theyhave only a low identity. The number of factors affecting the reproducibility ofRAPD profiles is therefore greatly increased. AFLPs are generated under higherstringency conditions, using the adapters as initial primer targets. Differencesin patterns generated using AFLPs are due to a change in the position of arestriction site, i.e. an inheritable mutation in the DNA. Different-sized DNAfragments will be amplified according to this criterion only. In this study, two AFLP primers were used to ensure a good level ofdiscrimination between the samples. The primers were designated ‘D’ and‘E’ (D = 5′GACTGCGTACATGCAGAC3′; E = 5′GACTGCGTACATGCAGAG3′).Again, identical profiles generated from each were sorted into groups anddesignated AFLP group 1, AFLP group 2, etc. The somatic incompatibility testing of the Sri Lankan and Malaysianisolates with each other were performed according to Miller (1995). Several of
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 211the Sri Lankan Ganoderma isolates from coconut were also tested for theirability to produce chlamydospores on three types of media – malt extract agar,lima bean agar and SNA (Nirenberg, 1976).MtDNA Profiles of Ganoderma Isolates from Sri LankanCoconutsMtDNA profiles from Ganoderma isolates on Sri Lankan coconuts (Fig. 16.3)were quite different from those of Ganoderma isolates from oil palms in Malaysiaand, in addition, many of the Sri Lankan Ganoderma isolates from differentcoconut palms shared identical mtDNA profiles (Table 16.1). For example,mtDNA profile group 1 was the most common profile found, in 16 out of the 27isolates studied. These isolates came from different palms on a number of plotsseparated by several kilometres. This contrasted with the high level of diverseprofiles found in a single plot in Malaysian oil palms (Miller et al., 1999). Almost all the mtDNA profiles of the Ganoderma isolates from coconut-palm tissue matched those of the sporophores found at the base of each palm. Fig. 16.3. Mitochondrial DNA restriction fragment length polymorphism from Sri Lankan Ganoderma isolates from coconut palms and betelnut palms. 1 = Marker; 2 = 23A, Ganoderma isolate from stem tissue of a dead betelnut palm (#23) with sporo- phores; 3 = 23B, Ganoderma isolate from stem tissue of a dead betelnut palm (#23) with sporo- phores; 4 = K23B, Ganoderma isolate from a sporophore from dead betelnut palm (#23); 5 = 33, Ganoderma isolate from stem tissue of a felled coconut palm (#33) displaying sporophores; 6 = K33, Ganoderma isolate from a sporophore from a felled coconut palm (#33); 7 = 34, Ganoderma isolate from stem tissue of a betelnut palm stump displaying sporophores (#34); 8 = K34, Ganoderma isolate from a sporophore of a betelnut palm stump (#34).
212Table 16.1. Ganoderma isolates from Sri Lanka and the mitochondrial DNA (mtDNA) and amplification fragment length polymorphisms(AFLP) groupings. Mitochondrial AFLP primersProject Sample DNA RFLP D and Enumber Host material Symptoms Sample site groupings groupings5 Cocos nucifera Palm trunk New bracket 1st sample site, Ambalanthota 1 16 Cocos nucifera Palm trunk Data not available 1st sample site, Ambalanthota 1 17 Cocos nucifera Palm trunk Bleeding only 1st sample site, Ambalanthota 1 18 Cocos nucifera Palm trunk Bleeding only 1st sample site, Ambalanthota 1 1K20 Cocos nucifera Sporophore Felled palm adjacent to ditch, 4th sample site, Manandala 1 1 H. Rolph et al. with sporophores21 Cocos nucifera Palm trunk Felled palm adjacent to ditch, 4th sample site, Manandala 1 1 with small primordium23A Areca catechu Palm trunk Bracket on dead palm 5th sample site, Ambalanthota 1 124 Cocos nucifera Palm trunk Stump with old sporophores 5th sample site, Ambalanthota 1 128A Cocos nucifera Palm trunk Palm with 2 sporophores 6th sample site, Ambalanthota 1 128B Cocos nucifera Palm trunk Palm with 2 sporophores 6th sample site, Ambalanthota 1 1K28B Cocos nucifera Sporophore Palm with 2 sporophores 7th sample site, Ambalanthota 1 135 Cocos nucifera Palm trunk Stump with bleeding 7th sample site, Ambalanthota 1 136A Areca catechu Palm trunk Live palm (number 36) 5th sample site, Ambalanthota 1 136B Areca catechu Palm trunk Stump adjacent to live palm 7th sample site, Ambalanthota 1 1K36A Areca catechu Sporophore Live palm (number 36) 7th sample site, Ambalanthota 1 1K36B Areca catechu Sporophore Stump adjacent to live palm 7th sample site, Ambalanthota 1 1
23B Areca catechu Palm trunk Bracket on dead palm 5th sample site, Ambalanthota A1A 133 Cocos nucifera Palm trunk Felled palm with sporophores 7th sample site, Ambalanthota A1A 1K33 Cocos nucifera Sporophore Felled palm with sporophores 7th sample site, Ambalanthota A1A 134 Areca catechu Palm trunk Stump with sporophores next to 7th sample site, Ambalanthota 2 2 irrigation channelK34 Areca catechu Sporophore Stump with sporophores next to 7th sample site, Ambalanthota 2 2 irrigation channelK62 Cocos nucifera Sporophore Stump with sporophores 11th sample site, Beliatta 2 ?K21 Cocos nucifera Sporophore Felled palm adjacent to ditch, 4th sample site, Manandala 3 3 with small primordium63 Cocos nucifera Palm trunk Stump with small sporophore 11th sample site, Beliatta 4 4K63 Cocos nucifera Sporophore Stump with small sporophore 11th sample site, Beliatta 5 564 Cocos nucifera Palm trunk Stump with small sporophore 11th sample site, Beliatta 6 674 Leguminosae Palm trunk Tree with 1 dry sporophore 11th sample site, Beliatta 7 7Key to Sri Lankan sample project numbers: K = sporophore, A,B = different samples from same palm.Example of numbering: 21 = tissue from palm/stump at position 21 was sampled; K21 = sporophore from palm/stump at position 21 wassampled.? = Isolate not testedRFLP, restriction fragment length polymorphism. Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 213
214 H. Rolph et al.There was only one exception where the Ganoderma infecting the palm tissuedid not appear to be the same as that producing the sporophores around thebase of the palm. This was on palm 21, where the Ganoderma isolate fromthe palm tissue had a mtDNA profile in group 1 and the sporophore from thebase of the trunk had a completely unique mtDNA profile (group 3). It is possible that profile 1 is the primary infection source and profile 3represents a colonization by a non-infectious Ganoderma strain. Conversely,profile 3 could represent a secondary Ganoderma infection, which is present inanother, as yet unsampled, part of the palm.MtDNA from Ganoderma Isolates on Sri Lankan BetelnutPalmsGanoderma isolates with identical mtDNA profiles were found on betelnutpalms as well as on coconut palms (Table 16.1), hinting at a lack of host-specificity. Many of the identical Ganoderma mtDNA profiles were on betelnut-palm and coconut-palm isolates from plantings several kilometres apart.AFLP Profile Groupings from Ganoderma Isolates onSri Lankan Coconut PalmsThe AFLP groupings determined using primer D were identical to thoseproduced using primer E. The AFLP groupings displayed in Table 16.1 are,therefore, a combination of the results from both primers. The results from the AFLP profiles mirrored those from the mtDNA pro-files. They showed the same lower level of variation (Fig. 16.4) and were foundacross a large sample area. The most prevalent AFLP groups across samplesites 1, 4, 5, 6, and 7 were AFLP group 1, AFLP group 1A and AFLP group 2.Identical AFLP profiles were found on both coconut palms and betelnut palms.These results correlate with the findings from the mtDNA study and againindicate that many of the Ganoderma isolates studied show no host specificity.Combined Results from mtDNA and AFLP ProfilesWhen the AFLP and mtDNA profiles were analysed, the control isolate fromthe Leguminosae host produced unique profiles, which indicated that both tech-niques were sufficient in their ability to discriminate between the Ganodermaisolates from coconut and betelnut palms from other hosts. When the mtDNA and AFLP profile results were combined, they corre-lated almost exactly (Table 16.1). The only exception was that group 1A(observed when using mtDNA restriction fragment length polymorphisms(RFLPs)) was not distinguished by the use of AFLPs. MtDNA group 1A (isolates
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 215Fig. 16.4. Amplification fragment length polymorphisms from Sri LankanGanoderma isolates from coconut palms. 1 = Marker; 2 = 5, Ganoderma isolatefrom stem tissue from a coconut palm (#5) displaying a new sporophore; 3 = 6,Ganoderma isolate from stem tissue from a coconut palm (#6); 4 = 7, Ganodermaisolate from stem tissue from a coconut palm (#7) with stem bleeding only; 5 = 8,Ganoderma isolate from stem tissue from a coconut palm (#8) with stem bleedingonly; 6 = K20, Ganoderma isolate from a sporophore from a felled coconut palm(#20); 7 = 21, Ganoderma isolate from stem tissue from a felled coconut palm(#21); 8 = K21, Ganoderma isolate from a sporophore from a felled coconut palm(#21); 9 = 22, Ganoderma isolate from stem tissue from a coconut stump (#22).23B, 33 and K33) differed from mtDNA group 1 by only a single band. Furtherwork is needed to determine whether a single band in a mtDNA profile is asignificant enough difference to distinguish isolates which are in the sameAFLP group. The fact that both techniques produced almost identical groupings,however, indicates that these are valid groupings for the Ganoderma isolatesacross all the sampling sites. It also shows that a general genomic profilingtechnique such as AFLPs is very useful when used in conjunction with anextrachromosomal profiling technique such as mtDNA RFLPs. Different AFLP and mtDNA profile groups were found at each samplesite (Table 16.1) with many of the sites having just one or two profile groups.However, the sampling site at Beliatta had four mtDNA and AFLP profiles(groups 2, 4, 5, 6). This may have been because most isolates came from
216 H. Rolph et al.coconut stumps and may therefore represent subsequent colonization of thestump once the palm had died. Interestingly, neither mtDNA profile group 1,nor AFLP group 1 were found at the Beliatta sampling site. The molecular profiles were unaffected by the type of material used forDNA isolations. For example, both mtDNA profile group 1 and AFLP group 1were found in Ganoderma isolates from a range of sources (stem tissue frompalms displaying bleeding only; stem and sporophore tissue from a felled palm;stem and sporophore tissue from a stump; standing palms with sporophoresand, finally, symptomless live palms).Molecular Analysis of Ganoderma Isolates from MalaysianCoconut PalmsMtDNA and AFLP profiles (data not shown) of Ganoderma isolates fromMalaysian coconut palms were much more varied across the small plot studiedthan the Ganoderma isolates from Sri Lankan coconut palms had been over amuch wider area. Each profile was unique to each palm, i.e. the same profilewas never found on more than one palm. The high degree of molecularvariation seen in Ganoderma isolates from Malaysian coconut palms was thesame as that seen in isolates from Malaysian and Indonesian oil palms.SIG Tests on Sri Lankan and Malaysian Ganoderma IsolatesNone of the Sri Lankan isolates tested showed somatic compatibility withany isolate other than themselves. Thus, the isolates were all somaticallyincompatible with each other. However, both the mitochondrial DNA profilesand AFLPs showed that the isolates could be grouped together. For example,the largest profile grouping was group 1, in which all the isolates had the samemtDNA and AFLP profiles, yet none of them were somatically incompatiblewith each other. Ganoderma isolates from Malaysian coconut palms also showed no somaticcompatibility with each other, but each had their own individual mtDNA andAFLP profile.Chlamydospore Production ExperimentsOne of the isolates from a Sri Lankan coconut palm produced cylindrical-shaped chlamydospores on lima bean agar (Fig. 16.5). This shape ofchlamydospore is associated with the G. lucidum complex, a complex thathas often been reported to be pathogenic on coconuts in India.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 217Fig. 16.5. Chlamydospores produced by Ganoderma isolate (K33) from SriLankan coconut palm (Lima bean agar, 28°C, 23 days).DiscussionGanoderma isolates from coconut and betelnut palms in Sri Lanka appear to bedifferent from Ganoderma isolates from Malaysian coconut palms. In Sri Lanka,many isolates from a wide sample area share identical mtDNA and AFLPprofiles, although each isolate has its own SIG. In Malaysia, the mtDNA and AFLP profiles varied from coconut palm tococonut palm. The same profile was never found on more than one palmand the variation found was quite striking across the single sample plot. Eachisolate displayed its own SIG and there was a general pattern of one SIG permtDNA and AFLP grouping. The level of molecular variation in Ganodermaisolates from Malaysian coconut palms was, therefore, very similar to that onMalaysian oil palms. The fact that, in Sri Lanka, one mtDNA and AFLP group comprised a largenumber of isolates sampled over several square kilometres, yet each isolate hadits own SIG, requires further investigation before it can be understood. It isconceivable that the mechanism of reproduction and dissemination used byGanoderma on coconut palms in Sri Lanka may be different from that used byGanoderma on coconut and oil palms in Malaysia. Ganoderma populations fromoil palms have unique mtDNA profiles, even between adjacent palms; they areheterothallic and have a tetrapolar mating system. However, many of the SriLankan Ganoderma isolates shared identical mtDNA and AFLP profiles, yetcame from many different palms, suggesting that they are not heterothallicand that a different mechanism could be responsible for this lower level of vari-ation. This could suggest that Ganoderma populations on coconut and betelnut
218 H. Rolph et al.palms in Sri Lanka are homothallic (Jan Stenlid, personal communication,Egham Workshop on ‘Variation in Ganoderma’, June 1998). Other fungi have been shown to have different mating systems withinthe same genus. Studies of Armillaria ectypa have indicated that this is ahomothallic fungus, in direct contrast to other members of the genus that areheterothallic and have a tetrapolar mating system. It was shown to be homo-thallic in a number of ways, the first being a study of the fruiting capabilitiesof single-spore isolates (Guillmaumin, 1973). In a later study (Zolciak et al.,1997) further factors were considered, including the absence of matingreactions and the morphological identity between single-spore mycelia andisolates from the context of the basidiome. RAPDs were also used to showthe genetic identity of single-spored isolates from the same basidiome. Theauthors suggested that haploid basidiospores of A. ectypa might undergoself-diploidization just after germination, although this would require furthertesting by cytological observations of newly germinated basidiospores. Similar experiments could be performed on sporophore and basidiosporefamily sets of Ganoderma isolates from Sri Lankan coconut palms, to see if they,too, were homothallic. Instead of using RAPDs, however, AFLPs would be usedto provide a more stable method of profiling the total genomic DNA. Anotherinteresting factor to consider is that one Ganoderma isolate from a Sri Lankancoconut palm produced chlamydospores. This may indicate another methodof survival and spread of the identical molecular profiles over large distances.It has been suggested by Miller et al. (1999) that Ganoderma infection of oilpalms may be through dispersal of basidiospores. It is not yet known how farbasidiospores can be dispersed to spread BSR infections across an oil-palmplanting. Chlamydospores are more resistant to environmental factorsthan basidiospores and could be responsible for dissemination of one mtDNAand AFLP group over a wide area, regardless of whether the fungus washomothallic or heterothallic. Both homothallism and production of chlamydospores would have to beconsidered when developing a model to represent the spread of Ganoderma-associated diseases on coconut palms in Sri Lanka. Further work to be considered for these isolates would be to determinea species concept for them. Species delimitation within the genus, based ontraditional morphology, has not been of great use for tropical species ofGanoderma. However, species concepts for the genus Ganoderma based on ITSsequencing, morphological and biochemical data are slowly emerging. Itwould also be very useful to perform ITS sequencing of isolates from coconutpalms in Sri Lanka, India and Malaysia, to help distinguish these isolatesfurther. If these Sri Lankan Ganoderma isolates do represent a different species witha different reproductive mechanism, then an hypothesis to explain howthis species came to affect coconut and betelnut palms in Sri Lanka as opposedto the Ganoderma species found on oil palms in South-East Asia would berequired.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 219 One possible hypothesis relates to the fact that oil palm is an introducedcrop to South-East Asia, whereas coconut palm is a far more established crop inSri Lanka. Consequently, Ganoderma isolates in Sri Lanka have had a longertime to evolve and adapt to their palm hosts. This might partly account forthe lower level of molecular variability found in Ganoderma isolates from SriLankan coconut palms in contrast to the higher level of molecular variabilityin isolates from oil palm in Malaysia. Conversely, it is also possible that the Ganoderma population on coconutand betelnut palms in Sri Lanka represents a very young pathogen. Root andbole rot of coconuts was only reported in Sri Lanka in 1974 (Peries, 1974) andit is possible that it has only spread over the past 25 years. This might possiblysuggest a reason for the low level of variation in a newly emerged population.Yet another possibility is that the restricted molecular variability in Sri Lankanisolates (possibly due to a different mating system) could be as a result of geo-graphical isolation and subsequent different evolutionary rates and pressuresafter Sri Lanka separated from the Indian continent. The situation may be sim-ilar to that found in A. ectypa, which has shown to be homothallic in contrastto the heterothallic nature of nearly all other species of Armillaria. A. ectypa is arare arctico-alpine species, which was prevalent during the last glaciation. Itnow survives in Sphagnum peat bogs at high latitude or altitude. It represents aspecies which survives in a very geographically restricted environment, with ahomothallic mating system. It might be possible, therefore, that Sri LankanGanoderma isolates found on coconut palms have evolved with a differentmating system due to their geographically restricted environment. It would be necessary, however, to discover whether Ganoderma isolatesfrom coconut palms in India showed a similar low level of molecular variationas the Ganoderma isolates from coconut palms in Sri Lanka. A similar studyto the one described in this chapter would help to test this hypothesis. If itwas the case, and the Indian and Sri Lankan isolates also shared thesame mating system, then the geographical isolation hypothesis could bediscounted. The next crucial step in the study of Ganoderma isolates from Sri Lankancoconut palms is, therefore, to determine their method of reproduction. Theymust then be fully characterized (up to species level), using a combination ofmorphological and molecular techniques, to develop suitable markers to trackthem in the field. Once these steps have been taken, a strategy for control of thefungus in the field can be developed properly.AcknowledgementsThe authors would like to thank Liz Biddlecombe for the pictures of thechlamydospores, produced as part of a UKFCC-funded bursary project, andAnn Ansell for preparation of cultures for the CABI Bioscience GeneticResource Collection.
220 H. Rolph et al.ReferencesAbdullah, F. (1994) Characterisation of Ganoderma (Karst.) from oil palms (Elaeis guineensis) by isozyme electrophoresis. In: Holderness, M. (ed.) Proceedings of The International Workshop on Ganoderma Diseases of Perennial Crops. CAB Inter- national, Wallingford, UK.Adaskaveg, 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.Anonymous (1987) All India co-ordinated research project on palms, Progress report 1986–87. Centre Plant Crops Research Institute Kasaragod, p. 70.Ariffin, D., Abu Seman, I. and Azahari, M. (1994) Spread of Ganoderma boninense and vegetative compatibility studies of a single field palm isolates. In: Holderness, M. (ed.) Proceedings of The International Workshop on Ganoderma Diseases of Perennial Crops. CAB International, Wallingford, UK.Bazzalo, M.E. and Wright, J.E. (1982) Survey of the Argentine species of the Ganoderma lucidum complex. Mycotaxon 16, 293–325.Bhaskaran, R., Rethinam, P. and Nambiar, K.K.N. (1989) Thanjavur wilt of coconut. Journal of Plantation Crops 17(2), 69–79.Gauillaumin, J.J. (1973) Étude du cycle caryologique de deux espèces appartenant au genre Armillaria. Annales de Phytopathologie 5(3), 317 (abstract).Gilbertson and Ryvarden, L. (1986) North American Polypores, Part I. Fungi flora, Oslo.Miller, R.N.G. (1995) The characterisation of Ganoderma populations in oil palm cropping systems. PhD thesis, Department of Agriculture, University of Reading, UK.Miller, R.N.G., Holderness, M., Bridge, P.D., Chung, G.F. and Zarakia, M.H. (1999) Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology 48(5), 595–603.Nambiar, K.K.N. and Rethinam, R. (1986) Thanjavur wilt/Ganoderma disease of coconut. Pamphlet No. 30, Central Plantation Crops Research Institute, Kasargod, India.Navaratnam, S.J. (1964) Basal stem rot of oil palm on ex-coconut estates. Planter 40, 256–259.Nirenberg, H.I. (1976) Untersuchungen über die morphologische und biologische Differenzeirung in er Fusarium-Sektion Liseola. Mitteilungen aus der Biologischen Bundesanstalt für Land-und Forstwirtschaft. Berlin Dahlem 169, 1–117.Peries, O.S. (1974) Ganoderma basal stem rot of coconut: a new record of the disease in Sri Lanka. Plant Disease Reporter 58, 293–295.Peries, O.S., Liyanage, A. de S., Mahindapala, R. and Subasinghe, S.M.P. (1975) The incidence of Ganoderma root and bole rot of coconut in Sri Lanka. Ceylon Coconut Quarterly 26, 99–103.Sampath Kumar, S.N. and Nambiar, K.K.N. (1990) Ganoderma disease of arecanut palm – isolation pathogenicity and control. Journal of Plantation Crops 18(1), 14–18.Steyaert, R.L. (1975) The concept and circumscription of Ganoderma tornatum. Trans- actions of the British Mycological Society 65, 451–467.Steyaert, R.L. (1980) Study of some Ganoderma species. Bulletin du Jardin Botanique Nationale de Belgique 50, 135–186.Turner, P.D. (1965a) The incidence of Ganoderma disease of oil palm in Malaya and its relation to previous crop. Annals of Applied Biology 55, 417–423.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 221Turner, P.D. (1965b) Oil palms and Ganoderma II. Infection and Spread. Planter 41, 238–241.Vos, P., Hogers, R., Bleeker, M., Reijans, M., Vandelee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeau, M. (1995) AFLP – a new technique for DNA-fingerprinting. Nucleic Acids Research 23(21), 4407–4414.Zolciak, A., Bouteville, R.J., Tourvielle, J., Roeckel-Drevet, P., Nicolas, P. and Guillaumin, J.J. (1997) Occurrence of Armillaria ectypa (Fr.) Lamoure in peat bogs of the Auvergne – the reproduction system of the species. Cryptogamie, Mycologie 18(4), 299–313.
226 P.D. Bridge et al.Fig. 17.1. Schematic diagram of the ribosomal RNA gene cluster in fungi. ITS,internal transcribed spacer; IGS, intergenic spacer.Dixon, 1991; Hibbet, 1992). This multiple occurrence, together with theubiquitous nature of the gene cluster, makes the rRNA genes good targetregions for the development of molecular diagnostics. The variation insequence conservation across the gene cluster allows for specific sequencesto be identified at different taxonomic levels (Bruns et al., 1991; Bainbridge,1994). The conserved sequences in the subunit genes show sufficientconservation to enable sequences to be identified that are common to all fungi,or to individual phyla and orders. Alternatively, the variable sequences ofthe spacer regions (ITS and IGS) contain sequences that are common atapproximately the species level, and many species-specific sequences havebeen identified in these regions (White et al., 1990; Mills et al., 1992; Levesqueet al., 1994; Bridge and Arora, 1998; Edel, 1998). The polymerase chain reaction (PCR) is a method that enables manycopies to be made of particular DNA regions. The basic principles of the PCRreaction are that a region of DNA is defined from two flanking sequences, andmultiple copies of this are then produced through repeated cycling of a series oftemperature-dependent reactions (thermal cycling). Synthetic oligonucleo-tides, called primers, are constructed for the flanking regions and a thermo-stable DNA polymerase is then used to synthesize the intervening basesequence (Saiki et al., 1985, 1988; Mullis et al., 1986; Mullis and Faloona,1987). The ribosomal RNA gene cluster, as described above, consists ofinterspersed conserved and variable sequences. General primers can thereforebe constructed to conserved sequences which flank variable regions and allowamplification of the intervening variable region. This principle is used toamplify the ITS regions, with primers designed from the termini of conservedsubunit genes (White et al., 1990; Gardes and Bruns, 1993). Analysis of thesequences of amplified ITS regions can then identify common and uniquesequences that can be used to design further primers with increased specificity.This approach has been used for a number of fungi and has been particularlyeffective in developing species- or pathogen-specific primers that can be usedwith environmental samples and in the presence of plant material (Gardeset al., 1991; Hopfer et al., 1993; Levesque et al., 1994; Beck and Ligon, 1995;Di Bonito et al., 1995; Mazzola et al., 1996). There is a considerable amount of information available on the sequencesof the rRNA gene cluster in the genus Ganoderma (Moncalvo et al., 1995a), andmore than 30 ITS sequences are available through public access databases
Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm 227such as EMBL and GenBank. There is considerable similarity between ITSsequences, and these can be aligned from species across the genus (Moncalvo,this volume). Small groups of isolates can be defined by ITS sequences withapproximately 2–3% sequence variation within groups (Moncalvo et al.,1995b, c). This level of sequence variation corresponds well to that seen withinspecies of some other plant-pathogenic fungi (Seifert et al., 1995; Sreenivasa-prasad et al., 1996), and so it would appear that ITS sequences can beused to define species in Ganoderma. One feature of the ITS regions is that mostvariation is associated with the 5′ and 3′ termini of the region (Moncalvo et al.,1995b, c). Although ITS regions have been sequenced from many Ganodermaspecies, very few sequences have been obtained from isolates associated withpalms, and none are available through the public access databases. A singlesequence has been deposited for G. boninense, but it is now believed that theisolate was incorrectly labelled and had not been associated with a palm(Moncalvo, personal communication). Several molecular approaches have been used to characterize isolates ofGanoderma (Miller, 1995; Miller et al., 1995; Abu-Seman et al., 1996; Gottliebet al., 1998). The most widely used has been isoenzyme analysis and this hasgiven rather variable results. In studies on Ganoderma species on woody plantsin South America, isoenzyme profiles can in some cases define species (Gottliebet al., 1998). However, studies on palm pathogens have proved more compli-cated and although pectinase zymograms produce band patterns that largelydefine the palm-associated isolates, intracellular isoenzyme profiles can be veryvariable and appear to define either individuals or small groups of apparentlyunrelated isolates (Miller, 1995; Miller et al., 1995). In the oil-palm-associatedisolates, mitochondrial DNA polymorphisms appear to define populations ataround the level of an individual or sibling family (Miller et al., 1999), whileDNA fingerprinting methods, such as amplification fragment length poly-morphisms (AFLPs) and simple repetitive primers, can give band patterns thatvary between individual monokaryons isolated from a single basidiocarp(Bridge, 2000). This is in contrast to results obtained from isolates pathogenicto coconuts in Sri Lanka, where both techniques showed little variation withinthe population (Rolph et al., this volume), perhaps indicating the clonal spreadof a new pathogen. One of the aims of the EU-STABEX-funded programme at the Papua NewGuinea Oil Palm Research Association (OPRA) has been to develop a rapidmolecular diagnostic method for detection of Ganoderma pathogenic to oilpalm. ITS regions were targeted for this due to the ready availability ofcomparative sequences and the success obtained with this approach inother groups of plant-pathogenic fungi. An additional consideration was thatGanoderma on oil palm occurs as dikaryotic mycelium and basidiocarps thatgive rise to monokaryotic basidiospores. The rRNA gene cluster is generallyconsidered to be resistant to cross-over and segregation events and so could beexpected to be conserved through both meiosis and mitosis (Hillis and Dixon,1991; Hibbet, 1992).
228 P.D. Bridge et al.ITS Region of Oil-palm-associated IsolatesThe ITS region was amplified from cultures obtained from isolates infectingpalms at Milne Bay Estates, Alotau, Papua New Guinea. Cultures wereobtained from both dikaryotic mycelium and from monokaryotic myceliumderived from single basidiospores from individual basidiocarps. In total,material was obtained from 19 dikaryotic cultures derived from basidiocarps;three sets of monokaryons each containing four cultures derived fromindividual basidiospores from single basidiocarps, and three further dikaryoticcultures derived from crosses made within each set of monokaryons. Thesecultures were selected in order to ensure that the ITS region was normallyresistant to any cross-over and segregation associated with meiosis. Thecollection of the original basidiocarps was from widely separated palms and socould provide an indication of any variation present in the overall population. DNA was extracted from each culture and the complete region, containingboth ITS sequences and the 5.8S RNA subunit gene, were amplified withthe primers ITS1F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990).The resulting PCR products from all cultures were all of the same length(approximately 600 bp). Gross sequence variation was initially screened bydigestion of the products with restriction enzymes to give simple restrictionfragment length polymorphisms (RFLPs). All products gave identical RFLPs,indicating that they were composed of, at least superficially, similar sequences.The PCR products from four cultures were selected as representative andsequenced in both directions. These were also found to be identical for all of therepresentative samples (Fig. 17.2). The sequence obtained was compared to all of those maintained in publicaccess databases, as the complete sequence and as the separate ITS1 and ITS2regions. In every case the most similar sequences were always those from otherGanoderma species.Fig. 17.2. DNA sequence of 593 bases including the internal transcribed spacerregions. Nucleotides in bold correspond to conserved regions. The first 10 boldnucleotides are the 3′ terminus of the small subunit gene, the bold nucleotidesin the centre of the sequence are the 5.8S subunit gene and the final 18 boldnucleotides are the 5′ terminus of the large subunit gene. Unique sequence usedfor construction of primer GanET is contained in the box and the site for primerITS3 is underlined.
Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm 229Selection of Primer SiteAs described earlier, previous studies have shown considerable similarities inthe sequences in the ITS regions among species of Ganoderma (Moncalvo et al.,1995a, b, c). As a result it is possible to align ITS sequences from species acrossthe genus and to determine sequence divergence between species. Figure 17.3Fig. 17.3. Multiple alignment of ITS2 sequences, rooted with Fomitopsis rosea.
230 P.D. Bridge et al.shows an example of such an alignment of the ITS2 sequences of Ganodermaisolates contained in the EMBL database, with the ITS2 sequence of the isolatesfrom oil palm in Papua New Guinea. This alignment shows that the ITS2sequence from the oil-palm isolates is distinct from those of other species, andcomparison of the ITS2 sequences showed two sequences near the 3′ terminuswhich appeared to be unique to the oil-palm isolates. The first of these was thesequence TGCGAGTCGGCT, which started at position 105, and the secondwas GTTATTGGGACAACTC, which started at position 178. Short oligo-nucleotide sequences with high GC contents have been used as primers for therandom amplification of polymorphic DNA (RAPD) in many fungal genomes(Welsh and McClelland, 1990; Williams et al., 1990). The first uniquesequence in the Ganoderma ITS2 region was very similar to a RAPD primerin that it was 12 nucleotides in length and had a 75% GC content. A primerconstructed to this site might therefore behave similarly to a RAPD primer andwould be unsuitable for specific detection methods. However, the secondunique sequence was longer (16 nucleotides) and had a 44% GC content, andso was more suitable as a site for a specific oil-palm-associated Ganodermaprimer. A primer (GanET) was constructed that gave a 3′ complement to thissequence. The sequence of this primer and the original DNA region werescreened by searching the public access sequence databases. The originalsequence showed very little homology with any reported DNA sequence, andnone of the most similar sequences were obtained from fungi. This findingsupported the original assumption that the sequence selected was specific tothe oil-palm-associated Ganoderma. A second, 5′, primer was required to enablethe amplification of a single fragment, and primer ITS3, a conserved sequencein the fungal 5.8S subunit gene (White et al., 1990) was selected. Thecombination of ITS3 and GanET would, in theory, amplify a 321 bp regioncontaining most of the 5.8S subunit gene and the ITS2 region (see Fig. 17.2).Evaluation of Primer CombinationThe first step in the evaluation of the ITS3/GanET primer pair was to test thisprimer combination against a purified DNA sample from one of the isolatesthat had been sequenced originally. Amplification was undertaken with a highannealing temperature (55ºC) in order to minimize non-specific primer bind-ing, and the subsequent PCR product was a single band of the predicted size.The primer combination was then further tested against isolates of Ganodermafrom basal stem rot (BSR) of oil palm in Papua New Guinea and Malaysia, andproduced a single amplification product of 321 bp in each sample. The specificity of the primer combination was tested in two ways. First,it was used in the amplification of purified DNA from a collection of palm-associated Ascomycetes, Basidiomycetes and Oomycetes. These cultures includedspecies of Verticillium, Ascochyta, Phoma, Fusarium, Rhizoctonia, Psilocybe,Thielaviopsis and Phytophthora. Although PCR products were obtained from
Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm 231some of these cultures, none contained the specific 321 bp product. One nota-ble finding was the absence of the band from palm-associated Thielaviopsis, asthese organisms have been implicated in a number of palm diseases, includingupper stem rot (Kochu-Babu and Pillai, 1992). A second test involved the amplification of DNA samples from a widerrange of palm-associated Ganoderma cultures. These included saprobic isolatesfrom coconut and areca palms, saprobic cultures from poisoned oil palms, andisolates from Sri Lanka and India pathogenic to coconut palms (Rolph et al.,this volume). Amplification with the ITS3/GanET primers gave the specific321 bp band in saprobic isolates obtained from coconut and areca palm, butthis band was not produced in isolates from poisoned oil palm or from isolatespathogenic to coconut. ITS regions have been widely used to define fungalspecies and these results have some interesting implications for the study of thespread of Ganoderma diseases among palms. This presence of the specific bandin saprobic isolates from coconut and areca palms would suggest that theseisolates are either the same taxon as the oil-palm pathogen, or are very closelyrelated to it. This is in agreement with previous observations and molecularstudies which have suggested that BSR of oil palm may be caused byisolates saprobic on other palm hosts (Miller, 1995; Miller et al., 1995). Theabsence of the band in the saprobic isolates from poisoned oil palm suggeststhat not all saprobic Ganoderma on palms belong to the BSR taxon. This issupported by the morphology of these cultures, which produced darkerbasidiocarps on the palm. The absence of the band from the isolates frominfected coconut palms in India and Sri Lanka would suggest that these mayalso belong to a further taxon. This is supported in part by other molecularfindings that show that the Sri Lankan coconut pathogen population is veryhomogeneous and may be a single, recently developed population (Rolph et al.,this volume).Diagnostic CapabilitiesThe ITS3/GanET primer pair was able to differentiate successfully the oil-palmBSR isolates from DNA preparations of pure cultures in the laboratory. Thenext phase of developing a diagnostic tool was to assess the capability of theprimer pair to amplify the specific fragment from environmental samples thatcontain palm stem material and other saprobic microbes and invertebrates.Samples of infected and uninfected palm stem were collected from MilneBay Estates, Alotau. Samples of tissue (approximately 2.5 × 0.75 cm) werecollected into sterile screw-top bottles containing sufficient iso-propyl alcoholto keep the samples completely immersed. Samples were stored at roomtemperature for between 1 and 2 weeks after collection. The stem fragmentswere then frozen in liquid nitrogen and ground to powder in a mortar andpestle. The total DNA from the sample was extracted by a polyvinyl poly-pyrolidone/cetrimide extraction method (Cubero et al., 1999).
232 P.D. Bridge et al. DNA prepared in this way was screened with the ITS3/GanET primer pair.The ITS3 primer was designed as universal for fungi and so should minimizethe chance of amplifying DNA from other organisms or from the palm itself,and the specificity of the GanET primer should ensure that only oil-palm-associated Ganoderma DNA was amplified. Initial screening showed thatthe characteristic 321 bp band was only produced in samples derived frominfected palms, and that this band was not present from reactions withuninfected palm material.ConclusionsThis study has shown that the Ganoderma responsible for BSR in oil palm isa single taxon, which is distinct at a species level. The ITS-based approachprovides a single diagnostic method for the taxon which is independent of theinfraspecific variation seen for many other characters. The results support thehypothesis that the BSR organism occurs in a saprobic state on other deadpalms, particularly coconuts. The oil-palm taxon is, however, one of a numberof Ganoderma taxa that may be saprobic on palms. The causative organism ofstem rots on living coconut in India and Sri Lanka may be distinct from theoil-palm BSR, but testing of further isolates will be necessary before this canbe established definitively. The use of the ITS3/GanET primer pair providesa practical tool for the detection and tracking of the BSR organism in theenvironment, and this provides a means to determine accurately the spreadand infection route of the organism in the environment.ReferencesAbu-Seman, I., Thangavelu, M. and Swinburne, T.R. (1996) The use of RAPD for identification of species and detection of genetic variation in Ganoderma isolates from oil palm, rubber and other hardwood trees. In: Proceedings of the 1996 PORIM International Palm Oil Congress. Palm Oil Research Institute of Malaysia, Kuala Lumpur, pp. 538–551.Bainbridge, B.W. (1994) Modern approaches to the taxonomy of Aspergillus. In: Powell, K.A., Renwick, A. and Peberdy, J.F. (eds) The Genus Aspergillus. Plenum Press, New York, pp. 291–301.Beck, J.J. and Ligon, J.M. (1995) Polymerase chain reaction assays for the detection of Stagonospora nodorum and Septoria tritici in wheat. Phytopathology 85, 319–324.Bridge, P.D. (2000) Interpreting molecular variability in fungal systematics. Iberoamericana Micología, in press.Bridge, P.D. and Arora, D.K. (1998) Interpretation of PCR methods for species defini- tion. In: Bridge, P.D., Arora, D.K., Reddy, C.A. and Elander, R.P. (eds) Applications of PCR in Mycology. CAB International, Wallingford, UK, pp. 64–83.Bruns, T.D., White, T.J. and Taylor, J.W. (1991) Fungal molecular systematics. Annual Reviews of Ecology and Systematics 22, 525–564.
Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm 233Cubero, O.F., Crespo, A., Fatehi, J. and Bridge, P.D. (1999) DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi. Plant Systematics and Evolution 216, 243–249.Di Bonito, R., Elliott, M.L. and Desjardin, E.A. (1995) Detection of an arbuscular mycorrhizal fungus in roots of different plant species with the PCR. Applied and Environmental Microbiology 61, 2809–2810.Edel, V. (1998) PCR in Mycology; an Overview. In: Bridge, P.D., Arora, D.K., Reddy, C.A. and Elander, R.P. (eds) Applications of PCR in Mycology. CAB International, Wallingford, UK, pp. 1–20.Foster, L.M., Kozak, K.R., Loftus, M.G., Stevens, J.J. and Ross, I.K. (1993) The polymer- ase chain reaction and its application to filamentous fungi. Mycological Research 97, 769–781.Gardes, M. and Bruns, T.D. (1993) ITS primers with enhanced specificity for basidio- mycetes: application to the identification of mycorrhizae and rusts. Molecular Ecology 2, 113–118.Gardes, M., White, T.J., Fortin, J.A., Bruns, T.D. and Taylor, J.W. (1991) Identification of indigenous and introduced symbiotic fungi in Ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Canadian Journal of Botany 69, 180–190.Gottlieb, M.A., Saidman, B.O. and Wright, J.E. (1998) Isoenzymes of Ganoderma species from southern South America. Mycological Research 102, 415–426.Henrion, B., Le Tacon, F. and Martin, F. (1992) Rapid identification of genetic variation of ectomycorrhizal fungi by amplification of ribosomal RNA genes. New Phytologist 122, 289–298.Hibbet, D.S. (1992) Ribosomal RNA and fungal systematics. Transactions of the Mycological Society of Japan 33, 533–556.Hillis, D.M. and Dixon, M.T. (1991) Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Reviews in Biology 66, 411–453.Hopfer, R.L., Walden, P., Setterquist, S. and Highsmith, W.E. (1993) Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis. Journal of Medical and Veterinary Mycology 31, 65–75.Kochu-Babu, M. and Pillai, R.S.N. (1992) Record of upper stem rot of oil palm (Elaeis guineensis Jacq.) in Little Andamans. Planter 68, 243–246.Levesque, C.A., Vrain, C.T. and deBoer, S.H. (1994) Development of a species-specific probe for Pythium ultimum using amplified ribosomal DNA. Phytopathology 84, 874–878.Mazzola, M., Wong, O.T. and Cook, R.J. (1996) Virulence of Rhizoctonia oryzae and R. solani AG-8 on wheat and detection of R. oryzae in plant tissues by PCR. Phytopathology 86, 354–360.Miller, R.N.G. (1995) The characterization of Ganoderma populations in oil palm cropping systems. PhD thesis, University of Reading, UK.Miller, R.N.G., Holderness, M., Bridge, P.D., Paterson, R.R.M., Hussin, Md.Z. and Meon, S. (1995) Isoenzyme analysis for characterisation of Ganoderma strains from South-east Asia. EPPO Bulletin 25, 81–87.Miller, R.N.G., Holderness, M., Bridge, P.D. and Chung, G.F. (1999) Genetic diversity of Ganoderma in oil palm plantings. Plant Pathology, 48, 595–603.
234 P.D. Bridge et al.Mills, P.R., Sreenivasaprasad, S. and Brown, A.E. (1992) Detection and differentiation of Colletotrichum gloeosporiodes isolates using PCR. FEMS Microbiology Letters 98, 137–144.Moncalvo, J.-M., Wang, H.-F., Wang, H.-H. and Hseu, R.-S. (1995a) The use of ribosomal DNA sequence data for species identification and phylogeny in the Ganodermataceae. In: Buchanan, P.K., Hseu, R.S. and Moncalvo, J.-M. (eds) Ganoderma: Systematics, Phytopathology and Pharmacology. National Taiwan University, Taiwan, pp. 31–44.Moncalvo, J.-M., Wang, H.-F. and Hseu, R.-S. (1995b) 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. (1995c) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87, 223–238.Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods in Enzymology 155, 335–350.Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology 51, 263–273.Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.Seifert, K.A., Wingfield, B.D. and Wingfield, M.J. (1995) A critique of DNA sequence analysis in the taxonomy of filamentous ascomycetes and ascomycetous anamorphs. Canadian Journal of Botany 73 (suppl. 1), S760–767.Sreenivasaprasad, S., Mills, P.R., Meehan, B.M. and Brown, A.E. (1996) Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences. Genome 39, 499–512.Takamatsu, S. (1998) PCR Applications in Fungal Phylogeny. In: Bridge, P.D., Arora, D.K., Reddy, C.A. and Elander, R.P. (eds) Applications of PCR in Mycology. CAB International, Wallingford, UK, pp. 125–152.Welsh, J. and McClelland, M. (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213–7218.White, T.J., Bruns, T.D., Lee, S. and Taylor, J. (1990) Amplification and direct sequenc- ing of fungal ribosomal DNA genes for phylogenetics. In: Innis, M.A., Sninsky, D.H. and White, T.J. (eds) PCR Protocols. Academic Press, London, pp. 315–322.Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafolski, J.A. and Timgey, S.V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18, 6531–6535.
236 C. Utomo and F. Niepold(Indonesia) indicated that in certain areas of the second planting cycle up to70% of palms were infected with Ganoderma after 15 years. These data aresimilar to the situation reported in Malaysia (Turner, 1981; Singh, 1991;Khairudin, 1995; Darus et al., 1996). One of the limiting factors in controlling the disease is the lack ofreliable diagnostic methods to detect early symptoms of BSR disease. Only twomethods have been developed so far for early diagnosis of BSR; one involves acolorimetric method using ethylenediaminetetraacetic acid (EDTA) to detectG. lucidum in coconut, the causal agent of Thanjavur wilt disease (Natarajanet al., 1986). The second is a drilling technique where diseased material of oilpalm is collected by drilling into the diseased stem at 5–10 cm height from thesoil surface. Samples are then grown on media semiselective for Ganoderma(Ariffin et al., 1993). These conventional methods are time-consuming and theaccuracy is not very high. Therefore, the availability of a rapid, inexpensiveand accurate diagnostic technique, which is specific and readily adapted tolarge-scale testing for demonstrating Ganoderma in oil palm at an early stage ofinfection, would benefit decision-making for appropriate control. Use of the enzyme-linked immunosorbent assay (ELISA) and polymerasechain reaction (PCR) for detecting pathogenic fungi in infected plants has beenapplied widely. Successful detection of root-infecting fungi in infected plants byELISA has been reported previously, for example, detection of Heterobasidionannosum, one of the most common basidiomycete organisms responsible forthe decay of conifers, by polyclonal antibodies (Avramenko, 1989) and bymonoclonal antibodies (Galbraith and Palfreyman, 1994). Also the serologicaldetection of Armillaria, a root-rot disease pathogen of many woody plants, hasbeen undertaken successfully with monoclonal antibodies (Fox and Hahne,1989; Priestley et al., 1994). More recently, internal transcribed spacer (ITS)regions of ribosomal DNA (rDNA) have been targeted as attractive toolsfor early detection, due to their high sequence variation between species andtheir general conservation within any one species. ITS regions have provenuseful for generating primers for a species-specific detection of pathogenicfungi in naturally infected plant tissue (Tisserat et al., 1994; Lovic et al., 1995;Bunting et al., 1996; Mazzola et al., 1996). Therefore, one aspect of this workwas to elucidate an approach to detect Ganoderma using the ITS regions as atarget for generating specific primers to Ganoderma isolates of oil palms.Another aim of this work was to produce polyclonal antibodies for theserological detection of Ganoderma.Enzyme-linked Immunosorbent Assay (ELISA)Production of polyclonal antibodies (PAbs)Antigens were prepared by suspending 0.4 g of the extracted fresh mycelia inphosphate-buffered saline and then centrifuging at 13,000 r.p.m. for 10 min
Development of Diagnostic Tools for Ganoderma in Oil Palm 237at 4°C. Rabbits were given three intramuscular injections. For the firstinjection, 1.5 ml of antigen solution + 1.5 ml of Freund’s complete adjuvantwere used and with Freund’s incomplete adjuvant for subsequent injections at10-day intervals. The rabbits were bled 2 weeks after the final injection.Root sample preparationVacant areas due to Ganoderma infection were selected as the trial samples.Healthy-looking oil palms (no disease symptoms of Ganoderma, no decayedtissues in the base and no fruiting bodies of Ganoderma) surrounding thevacant areas were chosen as samples. Root samples were collected fromthe field by cutting the oil-palm root in the ground at a depth of 15–20 cm nearthe basal trunk with a hoe or axe. Healthy and diseased roots were collected,washed with tap water, weighed and ground with a metal mortar and pestle atroom temperature. Each sample suspension was diluted with extraction buffer(1 : 3), centrifuged at 13,000 r.p.m. for 10 min at 4°C. The supernatant waspipetted and stored at −20°C until use. To analyse the samples, indirect ELISAwas performed according to the method of Knapova (1995).PCRDNA obtained from isolates of Ganoderma and saprobic fungi and from oil-palmroot material was analysed. Isolates of Ganoderma were grown in a liquid maltextract/yeast extract medium (15 g/5 g) and saprobic fungi were grown inliquid Czapek Dox agar supplemented with yeast extract (34.4 g/10 g). Three different DNA extraction methods were evaluated, as describedby Raeder and Broda (1985), Möller et al. (1992) and Wang et al. (1993). PCRamplification was undertaken in 20 µl reactions with the primers GAN1 (TTGACT GGG TTG TAG CTG) and GAN2 (GCG TTA CAT CGC AAT ACA). Theseprimers were derived by the authors (unpublished) from the ITS1 region of therDNA of G. boninense (Moncalvo et al., 1995).Studies using ELISAA major problem in using immunoassay is the lack of specificity towardsplant-pathogenic fungi. Fungi are complex organisms which containnumerous antigens, many of which are also shared by unrelated fungi. Thus,thorough cross-reactivity tests against unrelated fungi that could be presentin the plant tissue were performed. This test is necessary in order to avoidfalse-positive values. The specificity of PAb-1(polyclonal antibody 1, raisedagainst single isolate of Ganoderma) and PAb-9 (polyclonal antibody 9, raisedagainst nine isolates of Ganoderma) was tested against five saprophytic fungicommonly isolated from diseased oil-palm roots. The five saprophytic fungi
238 C. Utomo and F. Niepoldwere identified as Penicillium sp., Aspergillus sp., Trichoderma sp. 1, Trichodermasp. 2 and Trichoderma sp. 3. Cross-reaction of PAb-9 against the five sapro-phytic fungi tested was low (only 3–6%), as shown in Fig. 18.1, whereasPAb-1 gave higher cross-reactions (6–25%) against the five tested saprophyticfungi (Fig. 18.2). The low cross-reaction of the PAb-9 with saprophytic fungithat associated with diseased oil palm enabled evaluation of the results ofoil-palm samples in comparison with PAb-1. The slope of the absorbance values per hour was calculated and presentedas d(A405 nm)dt−1. A positive and a negative threshold was set in the ELISA testsby calculating the d(A405 nm)dt−1 of the healthy roots and comparing that of 0.35 Pab-9 1 : 5,000 Antigen dilutions 1 : 300 0.30 1 : 2,100 1 : 15,000 0.25 OD 405 0.20 0.15 0.10 0.05 0.00 Peni Asper Tri 1 Tri 2 Tri 3 GanoFig. 18.1. Cross-reaction of PAb-9 with common saprophytic fungi at differentdilutions. There was almost no reaction visible with all the saprophytic fungi tested.Peni, Penicillium sp.; Asper, Aspergillus sp.; Tri, Trichoderma sp.; Gano,Ganoderma sp. 0.16 Pab-9 1 : 5,000 Antigen dilutions 1 : 300 0.14 1 : 2,100 0.12 1 : 15,000 0.10 OD 405 0.08 0.06 0.04 0.02 0.00 Peni Asper Tri 1 Tri 2 Tri 3 GanoFig. 18.2. Cross-reaction of PAb-1 with common saprophytic fungi at differentdilutions. There was a slight cross-reaction visible with all the saprophytic fungitested. Peni, Penicillium sp.; Asper, Aspergillus sp.; Tri, Trichoderma sp.; Gano,Ganoderma sp.
Development of Diagnostic Tools for Ganoderma in Oil Palm 239diseased roots. If the d(A405 nm)dt−1 values of the samples were three timeshigher than that of the healthy root, the sample was considered as positive.The sap of diseased and healthy roots (from field samples) as well as five sap-rophytic fungi were assessed with PAb-1 and PAb-9 (Fig. 18.3). Routinely lowd(A405 nm)dt−1 values were obtained when extracts from healthy root tissuewere used, and consistently high d(A405 nm)dt−1 values were obtained from dis-eased oil-palm root. The ratio of d(A405 nm)dt−1 of diseased roots to d(A405 nm)dt−1 of healthy roots varied from 6 to 16 for PAb-9 and 4 to 12 for PAb-1. This study shows that a simple extraction procedure of root samples bymacerating using an extraction buffer, with antisera being prepared in a rela-tively crude antiserum form, produced expedient results in root-sample testing.Therefore, the applied indirect ELISA procedure seems to be useful as a qualita-tive routine detection tool for the early detection and survey of Ganoderma, butaccurate quantitation of the fungus is not possible by this method.PCR StudyDNA extraction and sensitivity threshold of a pure culture of GanodermaThree different DNA extraction methods gave a 167 bp fragment from DNAof Ganoderma which was amplified after optimizing PCR conditions. The 0.16 PAb-9 0.14 P AB-1 0.12 d(A405nm)dt−1 0.10 0.08 0.06 0.04 0.02 0.00 A B C D E F G H I J K L M N O P The tested samplesFig. 18.3. Diseased and healthy roots from the field samples, as well assaprophytic fungi, were evaluated with PAb-1 and PAb-9, based on d(A405 nm)dt−1.There was a good correlation between infected and non-infected tissue or withsaprophytic fungi. A–H, diseased roots; I, Ganoderma of oil palm (1 : 15,000);J–N, saprophytic fungi (Trichoderma sp. 3; Trichoderma sp. 2; Trichoderma sp. 1;Penicillium sp. and Aspergillus sp., diluted 1 : 2,100); O, extraction buffer; P,healthy roots.
240 C. Utomo and F. Niepoldsensitivity threshold of PCR detection was assessed using serial dilutions of agiven quantity of Ganoderma genomic DNA as template. Sensitivity thresholdsof fungal DNA, depending on DNA extraction methods, were 1 ng for themethod of Raeder and Broda (1985), 5 pg for the method of Möller et al. (1992)and 1.5 pg for a modified method of Wang et al. (1993), respectively (Fig.18.4). The increase in sensitivity of the latter method is probably due to theimproved nuclear DNA extraction using alkaline (NaOH) solution, which inturn allows sufficient dilution of the extract to eliminate or significantly reducethe effect of potential inhibitors of the PCR. Good amplification results in a PCRtest using NaOH solution as the DNA extraction buffer have been reported forextracting Phytophthora genomic DNA (Tooley et al., 1997).Specificity tests of the primers Gan1 and Gan2 with other saprophyticfungi and GanodermaIn this study, the modified Wang method was used for extracting fungal DNA.To further evaluate primer specificity, experiments were performed with 18saprophytic fungi which were occasionally found as saprophytes on diseasedoil-palm roots. Twenty-three Ganoderma isolates from various sources wereFig. 18.4. Determination of the detection limit of Ganoderma from oil palm usingthree different DNA extraction methods. (a) Determination of the detection limitbased on the method of Raeder and Broda (1985). Lanes 1–5: 50 ng, 10 ng, 1 ng,0.1 ng and 0.01 ng of Ganoderma DNA. Lane S: DNA marker. (b) Determination ofthe detection limit based on the method of Möller et al. (1992). Lanes 1–6: 50 ng,5 ng, 500 pg, 50 pg, 5 pg and 0.5 pg of Ganoderma DNA. Lane 7: negative watercontrol, and Lane S: DNA marker. (c) Determination of the detection limit based onthe method of Wang et al. (1993). Extracted DNA can not be measured by UV.Crude estimation: 1 µg of mycelia representing 1 ng of DNA. Lanes 1–6: 1:10,1:102, 1:103, 1:104, 1:105 and 1:106 of Ganoderma mycelia diluted in Tris/BSA.1 µl of 1:105 dilution contained 0.30 pg of DNA. Lane 7: negative water control,and lane S: DNA marker.
Development of Diagnostic Tools for Ganoderma in Oil Palm 241also included in this evaluation (Table 18.1). Primers designed for thediagnosis of Ganoderma in diseased oil palm also reacted with other saprophyticfungi, but the amplification products of the saprophytic fungi differed in DNAfragment size compared to the DNA fragment size from Ganoderma (Fig. 18.5).In contrast, when DNA extracts from saprophytic fungi were diluted 1 : 10 inthe sap of healthy oil-palm root, no amplification product of the saprophyticfungi could be observed. For Ganoderma, a dilution of the DNA extract of1 : 10,000 using sap of healthy root of oil palm still allowed production of astrong amplification product (Fig. 18.5). Since no PCR signals were seen whenDNA of saprophytic fungi were diluted in the sap of healthy oil-palm root,contamination with saprophytic fungi in diseased roots would not generatefalse-positive values. Primers Gan1 and Gan2 also reacted with other Ganoderma isolates. Afragment of approximately 167 bp was amplified from all tested isolates ofGanoderma (data not shown). The ITS1 region of Ganoderma is relatively similarwithin all Ganoderma species. In addition, the ITS1 region of Ganoderma is smallenough to be easily amplified by PCR and is flanked by highly conservedsequences (Moncalvo et al., 1995). Development of a PCR test for species-specific detection of Ganoderma in oilpalm is urgently required, not only for early detection purposes but also fordetection of the source of the inoculum as well as for agronomic practice. Forexample, when crop rotation occurs from rubber or cocoa to oil palm, thestumps of rubber or cocoa are usually left on the fields. After a certain period oftime the stumps are colonized by Ganoderma and other basidiomycete fungi.Therefore, it is very difficult to determine whether Ganoderma that will infect oilpalms are the same species as those colonizing the stumps. The grower needs tobe able to solve this problem, in order to decide whether or not to removestumps, because the elimination of the stumps is very costly.Detection of Ganoderma from infected oil-palm rootsThree methods of DNA extraction were used to extract Ganoderma templateDNA from infected oil-palm root samples, as described earlier. In this study, thePCR assay successfully amplified Ganoderma DNA within infected root diluted1 : 100 with 100 mM Tris/BSA using the method of Möller et al. (1992) and amodification of the method of Wang et al. (1993) (Fig. 18.6a, b). The method ofRaeder and Broda (1985) produced only smeared PCR signals when extractedfrom infected root at dilutions of 1 : 10 and 1 : 100 with Tris/BSA buffer(data not shown). Probably the presence of inhibitors in root tissues, such aspolysaccharides (Demeke and Adam, 1992) or phenolic compounds (Cenis,1992; John, 1992; Johanson, 1994), may drastically reduce the sensitivity ofa PCR test. For this reason, further additional purification steps should beperformed to remove inhibitors, including cation exchange columns (Steinand Raoult, 1992); polyvinyl polypyrrolidone (PVPP) application, which
Development of Diagnostic Tools for Ganoderma in Oil Palm 243Fig. 18.5. Cross-reaction tests of primers Gan1 and Gan2 against 18 saprophyticfungi isolated from diseased oil-palm roots. (a) Ganoderma and saprophytic fungiwere diluted in 1 : 10 Tris/BSA. Lanes 1–12: Ganoderma, Trichoderma koningii,Trichoderma harzianum, Trichoderma viride, Aspergillus flavus, Penicillium sp.,Trichoderma sp. 1, Rhizopus sp., Bispora sp., Geotrichum sp., Trichoderma sp. 2and Trichoderma sp. 3. Lanes 14–21: Ganoderma, Gliocladium sp., Mucor sp.,Cylindrocarpon sp., Monilia sp., Fusarium sp., Aspergillus sp. and Botryodiplodiasp. Lanes S, 13 and 22: DNA marker. (b) Ganoderma and saprophytic fungi weremixed with extracted healthy roots. Lanes 1–4: Ganoderma in healthy root dilution1 : 10, 1 : 102, 1 : 103 and 1 : 104. Lanes 5–12 and 14–24: saprophytic fungi inhealthy root dilution 1 : 10, T. koningii, T. harzianum, T. viride, A. flavus,Penicillium sp., Trichoderma sp. 1, Rhizopus sp., Bispora sp., Geotrichum sp.,Trichoderma sp. 2, Trichoderma sp. 3, Gliocladium sp., Mucor sp., Cylindrocarponsp., Monilia sp., Fusarium sp., Aspergillus sp. and Botryodiplodia sp. Lane 14:Ganoderma in healthy root dilution 1 : 104. Lanes S, 13 and 25: DNA marker.binds polyphenolic compounds (Parry and Nicholson, 1996); or the use ofcommercial DNA purification kits such as QIAquick spin column tube (Diagen)(Niepold and Schöber-Butin, 1995) and Magic DNA Clean-Up Columns(Promega) (Johanson, 1994). Since all these procedures are time consumingand expensive, the reported development of a simple and fast Ganoderma DNAextraction method for infected palms, with no additional purification steps,represents an advantage in routine PCR tests. Since no amplification productwas observed with nucleic acid extracted from healthy roots, the amplificationproduct obtained contains the target sequence of fungal DNA from infectedroots. Therefore, the modified Wang method is considered as the most simple
244 C. Utomo and F. NiepoldFig. 18.6. Detection of Ganoderma from diseased oil-palm roots with primersGan1 and Gan2. (a) Extraction of Ganoderma DNA from diseased oil-palm rootusing the Möller method. Lane 1: 5 ng of Ganoderma DNA. Lanes 2–5 TE buffer1 : 5, 1 : 10, 1 : 102 and 1 : 103, respectively. Lane 6: negative water control. Lanes8–10: extracted healthy oil-palm root, diluted with TE buffer 1 : 5, 1 : 10 and1 : 102, respectively. Lanes S and 7: DNA marker. (b) Extraction of GanodermaDNA from diseased oil-palm root by using the modified Wang method. Lane 1:5 ng of Ganoderma DNA. Lanes 2–6: extracted diseased oil-palm root diluted inTris/BSA: 1 : 5, 1 : 10, 1 : 102, 1 : 103 and 1 : 104, respectively. Lane 7: waternegative control. Lanes 9–12: extracted healthy oil-palm root diluted with Tris/BSA:1 : 5, 1 : 10, 1 : 102 and 1 : 103, respectively. Lanes S and 8: DNA marker. Lane 13:negative water control.and fast DNA extraction for detecting Ganoderma in infected oil-palm rootsamples, and it has the added advantage that the chemicals used are not asexpensive as those used in other extraction methods.
Development of Diagnostic Tools for Ganoderma in Oil Palm 245ConclusionsPositive or negative values for the detection of Ganoderma by ELISA werebased on reactivity relative to the negative control. The cross-reactivity withunrelated fungi in the ELISA test led to false-positive values. Also, a lowconcentration of Ganoderma in the infected tissues, in addition to dilution steps,may elicit false-negative values in the ELISA test. In order to increase thesensitivity and specificity of Ganoderma detection the PCR was applied. A PCR-based assay appears to be more specific than the ELISA assay inGanoderma detection, because in the PCR assay cross-reaction with sapro-phytic fungi was not observed. However, for detection using a large number ofsamples, ELISA offers advantages in term of speed, ease of use and costs. Unlikethe PCR assay, in which genomic DNA must be extracted from infectedsamples, ELISA only requires a small sample of sap, obtained by crushing thesamples. The use of the ELISA test might be useful as a pre-screen to handle alot of samples. In the case of a positive reaction, the PCR test should be appliedto verify the results. With this combination of both procedures, a fast andreliable screening of oil palm is now possible.ReferencesAbadi, A.L. (1987) Biologi Ganoderma boninense Pat. pada kelapa sawit (Elaeis guineensis Jacq.) dan pengaruh beberapa mikroba tanah antagonistik terhadap pertumbu- hannya. PhD thesis, Institut Pertanian Bogor.Ariffin, A., Seman, I.A. and Khairudin, H. (1993) Confirmation of Ganoderma infected palm by drilling technique. In: Proceedings of the 1993 PORIM International Palm oil Congress, 20–25 September 1993, Kuala Lumpur, Malaysia.Ariffin, A., Seman, I.A. and Azahari, M. (1996) Spread of Ganoderma boninense and vegetative compatibility studies of a single field palm isolates. In: 1996 PORIM International Palm Oil Congress, 23–28 September 1996, Kuala Lumpur, Malaysia.Avramenko, R.S. (1989) A serological study of strains of Heterobasidion annosum (Fr.) Bref. 1. Heterobasidion annosum from the common pine. Mikologiya I Fitopatologiya 23, 225–233.Bunting, T.E., Plumley, K.A., Clarke, B.B. and Hillman, B.I. (1996) Identification of Magnaporthe poae by PCR and examination of its relationship to other fungi by analysis of their nuclear rDNA ITS-1 region. Phytopathology 86, 398–404.Cenis, J.L. (1992) Rapid extraction of fungal DNA for PCR amplification. Nucleic Acid Research 20, 2380.Demeke, T. and Adam, R.P. (1992) The effects of plant polysaccharides and buffer additives on PCR. BioTechniques 12, 332–333.Fox, R.T.V. and Hahne, K. (1989) Prospects for the rapid diagnosis of Armillaria by monoclonal antibody ELISA. In: Morrison, D.J. (ed.) Proceedings of the Seventh Inter- national Conference on Root and Butt Rots. Pacific Forestry centre, Victoria, British Columbia, pp. 458–469.Galbraith, D.N. and Palfreyman, J.W. (1994). Detection of Heterobasidion annosum using monoclonal antibodies. In: Schots, A., Dewey, F.M. and Oliver, R. (eds)
246 C. Utomo and F. Niepold Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Wallingford, pp. 105–110.Ho, Y.W. and Nawawi, A. (1985) Ganoderma boninense Pat. from basal stem rot of oil palm (Elaeis guineensis) in Peninsular Malaysia. Pertanika 8, 425–428.Johanson, A. (1994) PCR for detection of the fungi that cause Sigatoka leaf spots of banana and plantain. In: Schots, A., Dewey, F.M. and Oliver, R. (eds) Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Wallingford, pp. 215–221.John, M.E. (1992) An efficient method for isolation of RNA and DNA from plants containing polyphenolic. Nucleic Acids Research 20, 2381.Khairudin, H. (1995) Basal stem rot of oil palm caused by Ganoderma boninense. In: 1993 PORIM International Palm Oil Congress, Kuala Lumpur, Malaysia.Knapova, G. (1995) Entwicklung und Prüfüng eines ELISA zum Nachweis von Phytophthora infestants (Mont.) de Bary. Dissertation, Georg-August-Universität Göttingen.Lovic, B.R., Martyn, R.D. and Miller, M.E. (1995) Sequence analysis of the ITS regions of rDNA in Monosporascus spp. to evaluate its potential for PCR-mediated detection. Phytopathology 85, 655–661.Mazzola, M., Wong, O.T. and Cook, R.J. (1996) Virulence of Rhizoctonia oryzae and R. solani AG-8 on wheat and detection of R. oryzae in plant tissue by PCR. Phytopathology 86, 354–360.Möller, E.M., Bahnweg, G., Sandermann, H. and Geiger, H.H. (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies and infected plant tissues. Nucleic Acids Research 20, 6115–6116.Moncalvo, J.M., Wang, H.H. and Hseu, R.S. (1995) Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87, 223–238.Natarajan, S., Bhaskaran, R. and Shanmugam, N. (1986) Preliminary studies to develop techniques for early detection of Thanjavur wilt in coconut. Indian Coconut Journal 17, 3–6.Niepold, F. and Schöber-Butin, B. (1995) Application of the PCR technique to detect Phytophthora infestans in potato tubers and leaves. Microbiological Research 150, 379–385.Parry, D.W. and Nicholson, P. (1996) Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathology 45, 383–391.Priestley, R., Mohammed, C. and Dewey, F.M. (1994) The development of monoclonal antibody-based ELISA and dipstick assays for the detection and identification of Armillaria species in infected wood. In: Schots, A., Dewey, F.M. and Oliver, R. (eds) Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Wallingford, UK, pp. 149–156.Raeder, U. and Broda, P. (1985) Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17–20.Singh, G. (1991) Ganoderma – the scourge of oil palm in the coastal areas. Planter 67, 421–444.Stein, A. and Raoult, D. (1992) A simple method for amplification of DNA from paraffin-embedded tissues. Nucleic Acids Research 20, 5237–5238.Steyaert, R.L. (1967) Les Ganoderma palmicoles. Bulletin du Jardin Botanique Nationale de Belgique 37, 465–492.
Development of Diagnostic Tools for Ganoderma in Oil Palm 247Thompson, A. (1931) Stem rot of the oil palm in Malaya. Bulletin of Department of Agriculture, Science Series 6.Tisserat, N.A., Hulbert, S.H. and Sauer, K.M. (1994) Selective amplification of rDNA internal transcribed spacer regions to detect Ophiosphaerella korrae and O. herpotricha. Phytopathology 84, 478–482.Tooley, P.W., Bunyard, B.A., Carras, M.M. and Hatziloukas, E. (1997) Development of PCR primers from internal transcribed spacer region 2 for detection of Phyto- phthora species infecting potatoes. Applied and Environmental Microbiology 63, 1467–1475.Turner, P.D. (1981) Oil Palm Diseases and Disorders. Oxford University Press, Kuala Lumpur.Wang, H., Qi, M. and Cutler, A.J. (1992) A simple method of preparing plant samples for PCR. Nucleic Acids Research 21, 4153–4154.
250 T.W. DarmonoCurrent Status of Research on GanodermaDetailed information of BSR in oil palm can be found in Turner (1981). Thissummarizes his findings from his own research and observations on thedisease in Indonesia prior to 1981. Although this gives a better understandingof the disease, it does not provide clear guidance on how to control the diseaseeffectively, which can be incorporated in the whole system of oil-palm manage-ment. Prior to 1980, there was no local research scientist in the countryactively involved in research on basal stem rot disease in oil palm. This wasprobably due to two main reasons. First, there was no pressure from theoil-palm industry, which was unaware that Ganoderma was a significantproblem. It was assumed that losses were not economically significant untilmore than 20% of the stand had been lost. That assumption was lately provento be incorrect (Hasan and Turner, 1994) and the disease currently occurs ata high incidence. The second reason was that working with higher fungisuch as Ganoderma spp. is generally difficult, slow and very long term. With theincrease in the incidence of the disease, the pressure from the growers hasincreased, encouraging research institutions to speed up their study onGanoderma. Institutions currently engaged in research on Ganoderma as anoil-palm pathogen in Indonesia include Biotechnology Research Unit for EstateCrops (BRUEC) in Bogor, the Indonesian Oil Palm Research Institute (IOPRI)in Medan, and Bah Lias Research Station (BLRS) of P.T.P.P. London Sumatrain Pematang Siantar. SEAMEO Bio-Tropical in Bogor was also involved inresearch between1986 and 1992. Research at SEAMEO Bio-Tropical and IOPRI had emphasized the under-standing of the biology and ecophysiology of the pathogen as well as the evalu-ation of potential biological and chemical control assays in the laboratory.Under laboratory conditions, the pathogen could grow at a wide range of pH,from 3.0 to 8.5, and the optimum temperature for growth was 30°C (Abadiet al., 1989; Dharmaputra et al., 1990). In the field, this may represent a widerange of soil types and oil-palm growing conditions at low elevations. Based onfield observations, there was no correlation between disease incidence and thedistance of the plantation to the coast, elevation, soil pH, or the density andtype of legume cover crops (Abadi et al., 1989). Later, it was also stated byHasan and Turner (1994) that there were few differences in BSR incidencebetween plantings on coastal and most inland sites in Indonesia. Although under field conditions, density and type of legume cover cropsdid not seem to affect disease development, laboratory studies revealed thatsupplementation of the agar medium with stem and leaf extracts of threelegume cover crops, i.e. Centrosema pubescens, Calopogonium mucunoides andPueraria javanica, commonly enhanced mycelial growth of the pathogen(Mawardi et al., 1987; Dharmaputra et al., 1989). In this particular case,growth enhancement may have occurred due to nutritional enrichment of themedium. Legume cover crops are commonly established just after planting-line preparation at the time of planting of oil-palm seedlings. After reaching a
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 251peak of vigour at 2–3 years after planting, these covers eventually die outunder the shade of the developing trees. Although the use of ground covers inthe plantation has been a subject of controversy, their use is beneficial in thecontrol of Rigidoporus microporus in Hevea rubber (Fox, 1977; Soepadmo,1981). This has been suggested to be largely due to the enhanced rate of decayof woody residues in the soil caused by the moist conditions and the highnitrogen status of the cover and its litter (Wycherley and Chandapillai, 1969).Although cover crops were commonly used in oil-palm plantations at the timewhen slash and burn was still allowed, their effect on the rate of decompositionof unburned, felled oil-palm stems has not been thoroughly investigated. Atpresent, slash and burn techniques have been banned in the country under the‘blue sky programme’ enforced by the government for protecting the environ-ment, particularly through the control of fire hazards. Quick decomposition offelled oil-palm stem is needed to prevent its colonization by Ganoderma whichmay subsequently act as an inoculum source for the disease. Research on the use of chemicals has been confined to laboratory studiesand results have shown that triadimenol at a concentration of 1.00 µg ml−1was able to kill the mycelia of the pathogen, but this concentration alsoinhibited a fungal antagonist (Dharmaputra et al., 1991). Preliminary resultsfrom a field experiment have shown that triadimenol application by rootabsorption was more effective in suppressing the disease than that applied bysoil drenching (Puspa et al., 1991). Using the same technique, Hasan (1998)has shown that phosphonic acid application was capable of protectingseedlings from infection. However, although these studies gave promisingresults, the use of chemicals in the control of Ganoderma in the field ona commercial scale will be impractical and economically infeasible until areliable technique of application has been developed. Also, even if a reliableapplication technique was found, the beneficial use of chemicals is stillquestionable since their effect can diminish rapidly. It has been shown thatthe effect of triadimefon on Ganoderma cultured on rubber wood vanishedwithin 3 weeks (Darmono, 1996). Research on the use of biological control agents for BSR has also beeninitiated at SEAMEO-Biotrop in Bogor (Dharmaputra et al., 1994). Otherresearch institutions, including IOPRI (Soepena, 1998), BRUEC (Darmono,1998), and BLRS (Hasan, 1998), have more recently become involved in thesame research subject. Studies conducted at these institutions have shownthat Trichoderma harzianum gave better control than that of other species ofTrichoderma. The use of a biological control agent in the control of Ganodermahas been seen to be more promising than that of chemical control. Thecapability of a biological control agent to grow and reproduce in the fieldand that will allow the destruction of the pathogen in the soil, are some of theadvantages and attractiveness of its use. Biological control is also consideredto be less hazardous to the environment. Research to investigate whetherTrichoderma sp. can actively grow along the root needs to be conducted. Thiswould reveal the potential use of the agent as a root protectant.
252 T.W. Darmono However, one problem with the application of chemical and biologicalcontrol agents is that the pathogen is capable of forming brown layers(Darmono, 1998) that provide a barrier against the chemical or theantagonist. These agents have to penetrate this barrier before being able tokill the sensitive mycelium of the pathogen. The brown layers, composedof melanized mycelium, also termed the ‘sclerotium plate’, are formed in thevicinity of the interaction zones and at any sites in the decayed tissue of basalstem. Sclerotium plates cover white masses of mycelium, forming pockets ofGanoderma. These pockets of mycelium are commonly found in the decayingoil-palm tissue. Sclerotium-like bodies of various sizes, from 2 to 5 cm in diameter(Fig. 19.1), can be found easily, embedded in broken, dry tissue particles in thedecomposed tissue of oil-palm stem. This structure can be considered as a‘resting body’ of Ganoderma sp. It is different from true sclerotium in that, inaddition to mycelium, the resting body of Ganoderma also contains degradedplant tissue intermingled with the mycelium. These resting bodies are capableof forming fruiting bodies and are capable of infecting oil-palm seedlings.Molecular analysis has revealed that cultures obtained from inside the restingbodies were identical to those obtained from the fruiting bodies developedfrom the associated resting bodies. This result indicates that the resting bodiesfound in decomposed oil-palm stems may be derived from the pathogen. Directtransfer of the internal tissue of resting body into malt extract agar mediumproduced pure culture, indicating that the fungus remained viable in oil-palmlogs under diverse environmental conditions in the field. The formation of brown mycelium layers and resting bodies in Ganodermamight function to protect the food resources acquired after invasion, toFig. 19.1. Resting bodies of Ganoderma found embedded in the decomposedtissue of oil palm infected by the pathogen.
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 253allow survival from one plant generation to another and to initiate a primaryinfection. Deposition of melanin in fungal mycelium and spores has beensuggested to be important for resistance to environmental stress, includingprotection against ultraviolet irradiation, radio waves, desiccation andtemperature extremes (Bell and Wheeler, 1986). Melanins in fungi havealso been suggested to be essential for resistance to microbial attack. Good field sanitation is believed to be one of the best possible waysto control the disease effectively (Hasan and Turner, 1994; Darmono, 1998).Research on field sanitation has been conducted intensively at BLRS. Arecommended technique for point sanitation was to remove all diseasedmaterial by digging a pit 1.5 m square and 1 m deep, centred on the point ofplanting spot (Hasan and Turner, 1994). The disease remnants raised to thesoil surface are disrupted, the simplest way being by cutting them into four ormore pieces, to allow enhanced biological control. Darmono (1998) suggestedthat field sanitation should be conducted before planting (pre-plantingsanitation activities) and regularly after planting during the entire life ofthe plant (post-planting sanitation activities). In areas with a high diseaseincidence, pre-planting sanitation can be conducted by removing allremaining boles and root clumps. Root clumps up to 20 cm thick are usuallyfound attached to the boles. Special attention should be given to boles androots of newly infected trees that, in the new planting, will certainly form apotential source of inoculum. Boles and root clumps of healthy trees left in theground can be more easily colonized by the pathogen than healthy roots ofnewly established plants. In the long term, the removal of these tissue remainswill help in reducing the risk of greater Ganoderma infestation in the followingreplantings. In post-planting sanitation, all infected trees that no longer haveeconomic value will be uprooted and sanitized. The action of sanitation should be based on the observation of diseaseincidence previously determined. Darmono (1998) generated a formula forcalculating disease incidence and scoring the grade of sanitation, as follows. S+E I= × 100% Nwhere I is the disease incidence; S, the number of standing trees infected byGanoderma; E, the number of empty planting spots due to Ganoderma; and N,the total number of planting spots observed. R G= S+Ewhere G is the grade of sanitation; R, the number of sanitized planting spots;and S and E, as described above. It has been a common practice in the past, or even currently, to base thescore of disease incidence merely on the number of empty planting spots orplant mortality, due to Ganoderma in the plantation. Such a form of scoringgives an impression that the infected standing trees do not have a significant
254 T.W. Darmonorole for disease development, and they have since been neglected during landpreparation for new planting. Detailed notes on the category of disease severityin each tree should be made during observations. Categories of disease severityproposed by Darmono (1998) are presented in Table 19.1. The felling of old oil palms before land preparation for replanting wasusually conducted by pushing individual trees over with a bulldozer. By thisaction, the healthy trees are usually uprooted along with their boles and rootclumps. If the tree is diseased (category R and Y), the pushing action usuallycauses it to break off at the base and the boles and roots are left behind inthe ground. If not removed or sanitized, these remains will become potentialinfection foci. In a long-term programme, research activities at IOPRI and BRUEC arecurrently undertaking the production of resistant oil-palm material by meansof conventional breeding and molecular biology techniques. At BRUEC,chitinase and glucanase genes obtained from local strains of microbes willbe transformed into the plant genome and specifically expressed in the rootsystem so that, hopefully, the palm will become resistant to Ganodermainfection. A transformation system in oil palm mediated with Agrobacteriumtumefaciens has also been developed (Chaidamsari et al., 1998) and apropagation system for oil palm using tissue-culture techniques has beenacquired (Tahardi, 1998). Development of resistant planting materials needsknowledge of the genetic variability in the pathogen. Studies on geneticvariability of Ganoderma associated with oil palm showed variation amongisolates from the same plantation and among those from different plantations(Darmono, 1998).Table 19.1. Categories of disease severity caused by Ganoderma in oil palm(Darmono, 1998).Mark Colourcolour abbreviation DescriptionGreen G Plant looks healthy with no disease symptom or sign of infection; or plant recovers from infection with no sign of Ganoderma activities. This may include plants with basal cavity due to previous GanodermaYellow Y Plant looks healthy, but a fruiting body of Ganoderma or brown discolouration can be observed at the base of the stemRed R Plant looks as if it is suffering from the disease and shows typical symptoms and signs of infectionBlack B Empty planting spot with infected boles and roots remaining in the groundWhite W Sanitized empty planting spot
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 255An Attempt to Produce an Immunoassay-based Detection KitNeed for the development of detection toolsFrom a practical standpoint, disease control in individual trees is hamperedby our inability to detect symptoms and signs of infection at an early stageof disease development. Infected palms usually show symptoms only after alarge portion of their base has been destroyed by the pathogen. Although soildrenching with fungicide may effectively kill the pathogen, large-scaleapplication of this type is not economically feasible. The success of chemicaltreatments through trunk injection can be achieved only if they are applied atan early stage of disease development. Therefore an accurate, quick and cheapdetection system needs to be developed. Although cultural studies and microscopic observation are highlyaccurate for diagnoses of the infection, these techniques are too slow and notamenable to large-scale application (Miller and Martin, 1988). Immunoassayand nucleic acid hybridization systems have been used for plant pathogendetection and disease diagnoses. These molecular probes are more specific,rapid and sensitive than conventional methods based on disease symptoms(Leach and White, 1990). Immunoassay techniques offer greater simplicityand need less equipment than those of DNA probe analyses. Experiments onthe development of polyclonal antibody (PAb) and monoclonal antibody(MAb) against Ganoderma sp. were initiated at the Biotechnology ResearchUnit for Estate Crops in 1993 (Darmono et al., 1993). The main objective of theexperiment was to produce an immunoassay-based detection kit.Detection kit specificationThere are some requirements in order for new products or technology to beapplicable and acceptable by the users. In the case of a detection kit based onimmunoassay, these requirements are:• It should be specific and sensitive.• It should be able to detect antigenic material far from the infection site.• It should be easily used for on-site application.• It should be inexpensive.• It should not be harmful. Because it is directed for field application, the antibody used in the kitshould be specific enough so that it only recognizes Ganoderma associated withbasal stem rot, regardless of strain dissimilarity and geographical origins. If itis too specific, the antibody will detect only a certain strain of the pathogenand, consequently, will be less useful for field application. There are at leasttwo ways to overcome this problem. The first is by pooling several specificantibodies or monoclonal antibodies, but this will be hampered by limited
256 T.W. Darmonoknowledge on the number of strains of Ganoderma found in oil palm and by thehigh cost of production of the antibody. The second, less expensive, way isthe development and production of polyclonal antibody. The sensitivity of theantibody should be measured, based on laboratory and field exercises. Inlaboratory exercises the level of sensitivity is determined by the ability of theantibody (at certain levels of dilution) to detect the least amount of antigen. Forfield applications, the antibody should ideally be capable of detecting antigenicmaterial at an early stage of disease infection. The root system of an individual mature oil palm occupies about 16 m3 ofsoil, and Ganoderma infection could start at any point in that space. In that kindof situation, the use of a DNA hybridization technique to detect Ganodermainfection at an early stage of disease development may be unreliable as it wouldrequire DNA obtained from the infection point. Thus, the tool used shouldideally be able to detect infection at a distance from the infection site. Signs ofinfection can be in the form of chemical compounds produced by either thepathogen or by the plant in response to infection. Acceptability of any new product known to be strongly dependent on itsprice and ease of use. It should be cheap and be of significant benefit to thegrowers. Ideally, it should be far less expensive than the cost of single nutrientcontent analyses, which is approximately US$2 per sample in Indonesia. Forthe detection of Ganoderma infection, it would be better if systematic samplingcould be conducted in the field regularly during observation of diseaseincidence. Alternatively, spot-selected sampling can be practised for reducingthe cost of use. Sending samples to a commercial institution for enzyme-linkedimmunosorbent assay (ELISA) will be costly so the tool should be suitable foron-site application by any person with no special skills. Sampling activitiesshould not harm the palms. Special care should be taken if the sample has to beobtained from the trunk or root, since an open injury may function as the entrypoint for the pathogen.Development of PAbMycelial wash as antigenIn the first stage of antibody development, a mycelial wash was used as asource of antigen. An isolate of Ganoderma sp. (TK-1, obtained from an infectedoil palm in Bogor Botanical Garden) was cultured in a chemically definedliquid medium (Leatham, 1983). The mycelium was harvested and washedthree times with phosphate-buffered saline (PBS) by filtration through a singlelayer of Whatman No. 93 filter paper. The liquid fraction from the final washwas used as the antigen. To develop the polyclonal antibody, a hyperimmune Balb/c mouse wasinjected intraperitoneally four times, at 2-day intervals with 250 µlantigen. Two days before the blood was withdrawn, an intravenous boosterinjection was given. Blood serum was obtained and the optimum titre for the
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 257antigen–antibody reaction was determined, based on a ‘conventional checkerA board’ method (Moekti, 1991). Cross-reactivity tests of the PAb wereconducted by indirect-ELISA (I-ELISA), against:1. A mycelial wash of five isolates of Ganoderma spp. associated with oil palm,and 12 isolates of non-oil-palm origin;2. Solvent from a fruiting-body tissue wash of five isolates of Ganoderma spp.associated with oil palm (including isolate TK-1); and3. Solvent from a spore wash of 10 isolates of Ganoderma spp. associated withoil palm (including isolate TK-1).The optical density (OD) value of I-ELISA was measured with an automaticEIA-Microplate Reader at wavelengths of 405 nm and 495 nm. The mycelial wash used as an immunogen in this study contained approx-imately 0.074 mg protein ml−1, with a molecular weight of 70,000 Da. Evenwith this relatively low content of protein the mycelial wash was proven to becapable of inducing a high titre of antibody (Figs 19.2 and 19.3). This mightindicate that it contained a high molecular weight antigenic material in theform of protein or other metabolites. Antigen that contains polypeptides or proteins with a molecular weightof more than 5000 Da possesses a high immunogenic reactivity (Smith,1988). From this experiment it was found that with low PAb concentration,at a 100-fold dilution, the antibody was capable of detecting 4.625 µg ml−1antigenic material (Fig. 19.2). Undiluted antibody was capable of detecting1.156 µg ml−1 antigenic material (Fig. 19.3). This result showed that whenantigenic materials are present at low concentration, an undiluted antibodyshould be used. Determination of the titres is necessary in the development ofany new antibody.Fig. 19.2. Optical densities from enzyme-linked immunosorbent assay readings intitres between dilute antibody and concentrated antigen.
258 T.W. DarmonoFig. 19.3. Optical density from enzyme-linked immunosorbent assay readings intitres between concentrated antibody and dilute antigen. The successful use of the mycelial wash as a source of antigen in thedevelopment of molecular detection assays for plant pathogenic fungi hasbeen reported (Brown, 1993), however in this project, we encountered severalproblems due to its high specificity. The antibody only recognized antigenicmaterials from the in vitro cultures and not from the in vivo sources fromfield fruiting bodies or spores. Furthermore, the antibody produced wasnot capable in distinguishing Ganoderma spp. from different host origins. Toincrease specificity and sensitivity, monoclonal antibody development and theuse of an exudate of Ganoderma sp. were attempted.Exudate as antigenThe brown aqueous exudate secreted on the surface of mycelium grownon rubber wood was used as an antigen to develop a PAb anti-exudate ofGanoderma (PAb-aeG). A 6-month-old Red Island laying hen was intra-muscularly immunized with 0.25 ml antigen five times at 2–3 day intervals.Fourteen days after the final immunization, antibodies developed in the eggyolk were isolated, as described by Darmono and Suharyanto (1995). Thespecificity and reactivity of PAb-aeG were evaluated against 10 isolates ofGanoderma sp., using I-ELISA. The antigen for the cross-reactivity test wasprepared from air-dried mycelium of on-wood cultures of the reference isolateAD-2 and field fruiting bodies of Ganoderma spp. Two grams of myceliumor fruiting body were ground in liquid nitrogen and extracted with 15 mlTris buffer. The homogenate was separated and used as the antigen incross-reactivity tests. Two types of enzyme–antibody conjugates, i.e. rabbitanti-chicken horseradish peroxidase conjugate and alkaline phosphatase
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 259conjugate, were tested at a dilution of 1 : 5000. The OD value of I-ELISA wasmeasured with an automatic EIA-Microplate Reader at wavelengths of405 nm and 495 nm. Accumulation of chicken antibody corresponded well with antigeninjections, indicating that the antibody was produced specifically against theexudate of Ganoderma sp. The optimum level of antibody production wasfound in eggs collected on the thirteenth day after the final immunization orthe twenty-third day after initial immunization (Fig. 19.4). One of the mainadvantages of using chicken antibody is the ease of handling of the animaland of obtaining the antibody. About 15 ml of antibody mixture was usuallyobtained from each egg in a relatively short period of time, compared to 70days or longer in rabbits. This amount of yolk antibody is sufficient to runabout 3000 reactions in microwells. PAb-aeG produced in this study was highly sensitive in recognizing all fieldfruiting bodies of Ganoderma spp. associated with oil palm, but not Ganodermaof non-oil-palm origins (Fig. 19.5). A satisfactory result was obtained onlywith the use of horseradish peroxidase anti-chicken antibody conjugate butnot with alkaline phosphatase anti-chicken antibody conjugate.Development of MAbAntibodies were developed in a hyperimmune Balb/c mouse. Immunizationof the mouse was conducted using a mycelial wash of isolate TK-1 as animmunogen, through the same procedures as described above. Five days afterthe final injection, a blood sample was withdrawn and lymphocytes wereFig. 19.4. Development of antibody in egg yolk, induced after injection of thehen with exudate of Ganoderma.
260 T.W. DarmonoFig. 19.5. Cross-reactivity of Ganoderma isolates against PAb-aeG.harvested and fused with myeloma sp/2 cells. Cell fusion was performed bytreating the mixed cell suspension with polyethylene glycol (PEG) 4000 at37°C for 2 minutes. The treated cells were cultured on a selective medium,Dulbecco Modified Eagle Media (DMEM) supplemented with 15% fetal calfserum (FCS) and hypoxanthine aminopterin thymidine (HAT). Hybridomacells were then cultured in the same media without HAT supplementation.Selection of antibodies produced by the hybridoma was conducted by cross-reacting against antigen prepared from eight isolates of Ganoderma. Selectedhybridoma cell lines were cloned using a limiting dilution method. Antibodysecreted into the medium was purified by ammonium sulphate precipitation.Typing of the monoclonal antibody was conducted using antibody isotypingkits (Sigma Chemical Co.). From 21 hybridoma produced, three (H-7, B-8 and D8) were selected. Thespecificity of these three hybridomas against eight isolates of Ganoderma isshown in Table 19.2. The hybridomas were highly specific. Hybridomas B-8and D-8 recognized only the reference isolate TK-1 from Bogor, West Java, andMU-1 from North Sumatra, while H-7 recognized only TK-1, but not MU-1.Both isolates were collected from diseased oil palm. The three hybridomas werenot capable of recognizing isolates of other oil-palm origins, SP-1 and AD-2,and isolates of non-oil-palm origins, GJ-4, CO-2, KR-11 and KR-15. Thehybridomas have been cloned. The monoclonal antibodies produced were allIgM type.
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 261Table 19.2. Specificity of monoclonal antibodies produced by three selectedhybridomas. Isolates of GanodermaHybridoma culture TK-1 SP-1 AD-2 MU-1 GJ-4 CO-2 KR-11 KR-15H-7 + − − + − − − −B-8 + − − ++ − − − −D-8 + − − +++ − − − −(+) Control + + + + + + + +(−) Control − − − − − − − −+, ++, +++, Weaker to stronger reaction.−, No reaction.Application of PAb and MAbThe potential use of PAb-aeG for the detection of signs of infection wasevaluated. Samples of oil-palm tissue were collected from severely infectedtrees planted in 1984 and their neighbouring apparently healthy trees, as wellas from a 2-year-old tree naturally infected by Ganoderma sp. Samples wereobtained from Bekri Oil Palm Plantation of PT Perkebunan Nusantara VIIin Central Lampung, Sumatra. The sample from each mature tree was acomposite of two 10 × 10 × 20 mm stem tissue samples collected from twoopposing areas 100 cm above the soil. Samples from the young tree wereobtained from various areas, including the infection site, infection zones,growing point and young leaves up to 100 cm from the infection site. One totwo gram of sample was ground in liquid nitrogen in one volume of Tris–HClbuffer pH 7.4. The extract from each sample was used as the antigen. IndirectELISA was conducted according to Moekti (1991) with the use of peroxidaseanti-chicken antibody conjugate. The dot immunobinding assay (DIBA) wasalso conducted on selected samples according to Robinson-Smith (1994). With samples obtained from the mature trees, the antigen was notdetected in any of the severely infected trees but was detected in an average ofthree out four apparently healthy surrounding trees. Since the disease-spreadto neighbouring trees occurs primarily through root contact, these apparentlyhealthy trees may have been infected by the pathogen although no diseasesymptoms were visible. A similar result was obtained in the 2-year-old plants,where the antigen was not detected in the decomposed tissue but was detectedin apparently healthy tissues, including leaf fronds and shoot tips (data notshown). The highest concentration of antigen was found in reaction zones,encountered as a brown discolouration at the base of leaf stalks near thediseased stem. The absence of antigenic material in the decomposed tissuesof oil palm may be due to degradation of the product by the pathogen itself orthrough other mechanisms. The DIBA test, conducted with a limited number
262 T.W. Darmonoof samples, produced the same result, showing that the antigenic materialscould be detected with the simpler technique. It is interesting to note that lowmolecular weight proteins were highly expressed in apparently healthy tissuesof an infected plant, but not in healthy tissues of a reference healthy plant. Thisindicates that the PAb-aeG produced in this study has the potential to be usedin the detection of early stages of infection of oil palm by Ganoderma spp. In the second series of tests, the PAb-aeG was tested against antigens pre-pared from leaf samples obtained from mature trees. Leaf samples were takenfrom 200 palms in a block with high disease incidence (34% of palms showingsymptoms or signs of infection) and from 200 palms in a block with low diseaseincidence (5% of palms showing symptoms or signs of infection). The ELISAreadings of samples obtained from the block with low disease incidence rangedfrom 0.088 to 2.110, while from the block with high disease incidence, read-ings ranged from 0.094 to 0.693. By assuming that palms with an OD value ofmore than 0.39 (the median) were categorized as infected by Ganoderma, it wasfound that in the block with high disease incidence 80% of palms were infectedwhile in the block with low disease incidence, 58% of palms were infected bythe pathogen (Fig. 19.6). Plants with high OD values but showing no visualdisease symptoms were revealed to be infected by the pathogen after their baseswere chopped and examined. This showed that the PAb-aeG developed has thepotential for large-scale application with a high degree of sensitivity. This second series of experiments further confirmed that the antigenicmaterials could be detected in leaves of diseased palms, more than 3 m fromthe infection site at the stem base. This result was consistent with the previousfinding that exudate or other substances secreted by Ganoderma might betransported to the leaves along with nutrient and water transport by theplant. Leaf sampling is desirable since it does not damage the tree. Large-scaleexperimentation needs to be conducted to verify the potential commercialapplication of this product.Detection of antigenic material from oven-dried leaf samplesPT SMART Corporation, a large private company planting oil palms atPakanbaru, Riau, Sumatra, provided three separate batches of leaf samples.They were obtained from mature trees in three separate localities. The firstbatch was from infected oil palms from a plantation with high diseaseincidence, while the second and the third batches were from healthy oil palmsin plantations with no disease incidence. Leaf sampling was conducted using atechnique recommended for nutrient content analyses. All leaf samples wereoven-dried at 60°C before they were sent to Bogor for ELISA. Eight leaflets from each bulk were randomly selected and used for antigenpreparation. They were individually ground into powder in liquid nitrogen.Extraction was with Tris–HCl and the extract was then used as an antigen forthe cross-reactivity test with PAb-aeG.
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 263Fig. 19.6. Histogram of frequency of oil palms with certain range of opticaldensity (OD) value from blocks with high (top) and low (bottom) disease incidence. Cross-reactivity was 2–3 times higher in leaf samples from diseasedtrees than those from healthy trees (Table 19.3). From this result it can beconcluded that antigenic material associated with Ganoderma infection can bedetected in leaves of diseased trees even after oven drying. However, OD valuesfrom these samples were much lower than those from leaf samples preservedin liquid nitrogen directly in the field.
264 T.W. Darmono Table 19.3. Optical density readings from enzyme-linked immunosorbent assay of leaf extract from oven-dried leaf samples tested against PAb-aeG. Average optical density readings at Leaf samples 405 and 492 nm* From diseased trees 0.0980a From healthy trees, Field Site 1 0.0453b From healthy trees, Field Site 2 0.0275b *Values followed by the same letter are not different significantly at P = 0.05.Concluding RemarksResearch on BSR disease caused by Ganoderma in oil palm in Indonesia isprogressing very well. Information on some biological and ecophysiologicalaspects of the pathogen, as well as information on the host–pathogen relation-ship provides a better understanding of the natural occurrence of the disease.Some biological control agents and chemical fungicides have been shown to beeffective in the laboratory, but successful disease management through chemi-cal and biological control will be achieved only after generation of a better fieldapplication technique. Provision of an immunoassay-based detection kit willhelp in the detection of infection at the earliest stage of disease developmentand this may subsequently increase the efficiency of disease management.ReferencesAbadi, A.L., Tjitrosomo, S.S., Makmur, A., Sutakaria, J., Dharmaputra, O.S., Macmud, M. and Susilo, H. (1989) Biology of Ganoderma boninense on oil palm (Elaeis guineensis) and the effect of some soil micro-organisms on its growth. Forum Pascasarjana 12th year, No. 2, 41–52 (in Indonesian).Bell, A.A. and Wheeler, M.H. (1986) Biosynthesis and functions of fungal melanins. Annual Review of Phytopathology 24, 411–451.Brown, I. (1993) Molecular detection assays for plant pathogenic fungi. AgBiotech News and Information 5, 219N–222N.Chaidamsari, T., Tahardi, J.S. and Santoso, D. (1998) Agrobacterium-mediated trans- formation in leaf explant oil palm. In: Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, 23–25 September 1998, pp. 602–605.Darmono, T.W. (1996) Penampakan keunggulan bahan hayati dari bahan kimia untuk pengendalian patogen penyakit akar tanaman perkebunan. Seminar Nasional Mikrobiologi Lingkungan II, Bogor, 9–10 October 1996.Darmono, T.W. (1998) Development and survival of Ganoderma in oil palm tissue. In: Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, 23–25 September 1998, pp. 613–617.Darmono, T.W. (1998) Molecular approaches to the elucidation of basal stem rot disease of oil palm. Proceedings of the BTIG Workshop on Oil Palm Improvement through Biotechnology, Bogor, 16–17 April 1997, pp. 83–94.
Use of Antibodies for Detection of Ganoderma Infection of Oil Palm 265Darmono, T.W. and Suharyanto (1995) Recognition of field materials of Ganoderma sp. associated with basal stem disease in oil palm with a polyclonal antibody. Menara Perkebunan 65(1), 15–22.Darmono, T.W., Suharyanto, and Darussamin, A. (1993) Polyclonal antibody against washing filtrate of mycelium culture of Ganoderma sp. Menara Perkebunan 61, 67–72 (in Indonesian).Dharmaputra, O.S., Gunawan, A.W. and Islamiyah, R. (1989) The effect of legume cover crop residue on the growth of Ganoderma boninense Pat. in vitro. In: Proceed- ings, Tenth Indonesian Phytopathology Society Congress, Denpasar, Indonesia, 14–16 November 1989 (in Indonesian).Dharmaputra, O.S., Tjitrosomo, H.S.S. and Abadi, A.L. (1990) Antagonistic effect of four fungal isolates on Ganoderma boninense. BIOTROPICA 3, 41–49.Dharmaputra, O.S., Tjitrosomo, H.S.S. and Retnowati, I. (1991) The effect of triadimenol on the growth of Ganoderma boninense and Trichoderma spp. in vitro. Annual Report Research Collaboration between Research Centre for Estate Crops, Marihat and BIOTROP. BIOTROP/TagR/91/779, pp. 52–69 (in Indonesian).Dharmaputra, O.S., Purba, R.Y. and Sipayung, A. (1994) Research activities on the biology and control of Ganoderma at Seameo BIOTROP and IOPRI Marihat. In: Proceedings of the First International Workshop on Perennial Crop Diseases Caused By Ganoderma, Selangor, Malaysia, 1–3 December.Fox, R.A. (1977) The impact of ecological, cultural and biological factors on the strategy and costs of controlling root diseases in tropical plantation crops as exemplified by Hevea brasiliensis. Journal of the Rubber Research Institute of Sri Lanka 54, 329–362.Hasan, Y. (1998) Potential control of Ganoderma in oil palm through prophylactic treatments. Proceedings of the Second International Workshop on Ganoderma Diseases of Perennial crops, MARDI, Serdang, Malaysia, 5–8 October.Hasan, Y. and Turner, P.D. (1994) Research at Bah Lias Research Station on Basal Stem Rot of Oil Palm. In: Proceedings of the First International Workshop on Perennial Crop Diseases Caused By Ganoderma, Selangor, Malaysia, 1–3 December.Leach, J.E. and White, F.F. (1990) Molecular probes for disease diagnoses and monitoring. In: Khush, G.S. and Toenniessen, G.H. (eds) Rice Biotechnology. CAB International and IRRI, pp. 281–307.Leatham, G.F. (1983) A chemically defined medium for the fruiting of Lentinula edodes. Mycologia 75, 905–908.Mawardi, I., Dharmaputra, O.S. and Abadi, A.L. (1987) The effect of legume cover crop extract on mycelial growth of Ganoderma boninense in vitro. Annual Report, Research Collaboration between Research Centre for Estate Crops, Marihat and BIOTROP. BIOTROP/TagR/87/656, pp. 19–38 (in Indonesian).Miller, S.A. and Martin, R.R. (1988) Molecular diagnoses of plant disease. Annual Review of Phytopathology 26, 409–432.Moekti, G.R. (1991) The production and characterization of monoclonal antibodies against Leptospira interrogans serovar Pomona: Attempts to improve the diagnosis of porcine leptospirosis. Proceedings of a Workshop on Agricultural Biotechnology, 21–24 May, Bogor, Indonesia, pp. 235–242.Pamin, K. (1998) A hundred and fifty years of oil palm development in Indonesia: From the Bogor Botanical Garden to the Industry. Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, 23–25 September, pp. 3–23.
266 T.W. DarmonoPuspa, W., Sipayung, A. and Purba, R.Y. (1991) The effect of triadimenol and triademorph on basal stem rot of oil palm (Elaeis guineensis). Annual Report Research Collaboration between Research Centre for Estate Crops, Marihat and BIOTROP. BIOTROP/TagR/91/779, pp. 70–77 (in Indonesian).Robinson-Smith, A. (1994) Serology for detection of Pseudomonas solanacearum. A training manual. Workshop on Groundnut Bacterial Wilt, Wuhan, China, 6–9 July.Smith, J.R. (1988) Hyperium Serum Production. In: Burgess, G.W. (ed.) ELISA Technol- ogy in Diagnosis and Research. James Cook University, Townsville, Australia.Soepadmo, B. (1981) The effect of time of cover crop establishment on root disease inci- dence in the replanting of Hevea. Menara Perkebunan 49, 129–133 (in Indonesian).Soepena, H. (1998) Biological control strategy for basal stem rot on oil palm. In: Proceedings of the International Workshop on Ganoderma Diseases. MARDI, Serdang, Malaysia, 5–8 October.Tahardi, J.S. (1998) Improvement of oil palm somatic embryogenesis by periodic immersion in liquid medium. In: Proceedings of the 1998 International Oil Palm Conference, Nusa Dua, Bali, 23–25 September, pp. 595–601.Turner, P.D. (1981) Oil Palm Disease and Disorders. Oxford University Press, Kuala Lumpur.Wycherley, P.R. and Chandapillai, M.M. (1969) Effects of cover plants. Journal of the Rubber Research Institute of Malaya 21, 140–157.
IndexIndexIndexAll entries refer to Ganoderma unless otherwise stated.Page numbers in italics refer to figures and tables.Abies infection by Heterobasidion annosum Armillaria 164, 167 145–146 ectypa 218, 219Acacia mangium Willd. root diseases serological detection by polyclonal 71–79 antibodies 236Actinomycetes as Ganoderma antagonist Aspergillus 237 85 in forest mycoflora 90Amauroderma 5, 7, 12, 23, 24 as Ganoderma antagonist 90–91 basidiospores 13 population enhancement by parasiticum and root-rot disease 76, calcium soil amendment 92 77 atypical fruiting structures phylogeny 30–31 (AFSs) 15–17 pileus 7 Azospirillum 123–124amplification fragment length polymorphisms (AFLP) 227 coconut palm profile groupings Bacillus spp. as Ganoderma antagonist 85 214, 215 basal stem rot (BSR) 49–68 combined with mtDNA profiles affected by climate 191 214–216, 217–218 age of palm and infection 53–54, mtDNA assessment 209–218 55 testing for homothallic fungi 218 biological control 83, 85–87,amylate activity of Ganoderma 131, 133, 90–92, 122–127 136 causal agents 52–53antibodies used to detect Ganoderma current status of Indonesian infection 249–266 research 249–254 267
268 Indexbasal stem rot (BSR) continued fungi species used for diagnostic tool disease resistance in wild stands development study 242 90 fungicide treatment 59, 62–63, disease symptoms 191 89–90, 126, 127, 251, 252 early detection 58–59 land preparation 60–62 economic importance 53–54, 249 oil-palm residue shredding geographical distribution 49–51, 110–111 160–161 polybag seedling production 84 height of sporophores on oil-palm pre-felling paraquat poisoning 192 102–103, 109 history of identification 50 replanting techniques 58, 61–62 infection sources 190–191 role of basidiospores 109 influence of previous crops 56, 57 root field trial 107–110 mycelial spread 105, 108–109 stump poisoning 185 oil-palm infection on former coconut stump tissues field trial 102–105 plantation 183–194 bait seedlings 102–104, 106, oil-palm infection by secondary 109, 110 inocula 193 infected tissue molecular planting techniques and infection fingerprinting 104–105 58 inoculum source depth 103, predisposing factors affecting 109 infection 55–58 stump size evaluation root balls as infection source 118 102–103 root to root infection 105, stumps as source of infection 109 107–110, 114, 192 Sumatran field trials 101–114 soil nutrition status 57–58 systemic fungicides 89–90 soil types and infection 56–57, 60, Trichoderma biofungicide 84–87 207–208 trunk tissues field trial 105–107 sporophore infection 192 see also soil amendment stump versus trunk infection basidia used in species identification 13 106–107, 110 basidiocarps 4, 7 symptoms in coconut palms 121 colour 26 symptoms in oil palms 51–52, 58, laccate or non-laccate 23–24, 30, 84 40 Thailand oil palm infection 69–70 locations 24, 26 and waterlogged soil 56–57, 60, basidioma 207–208 identification by genetics 164–169basal stem rot (BSR) infection control morphology 162–163 methods 59–64, 83–88, 170 basidiospores 5, 23, 113–121 biofungicide treatment 84–87, control during replanting 118 251–252 and disease spread 54 biological control 63–64, 83–88, infected palm identification regime 111 117 diagnostic tool development for oil infection process 105, 109, palm infection 235–250 113–121, 171, 218 diseased tissue excision 62 used in species identification 13, 17 epidemiology 54, 169–170, 235 variations 24, 26, 36 field trial results 108–111 betelnut palms 160 fumigant treatment 63 BSR symptoms 58, 207–208
Index 269 mtDNA profiles 214 databasesbiofertilizers used for biological control of CABI Bioscience fungus names BSR 122, 123, 125, 126, 135 database 4biofungicides 251–252 Duke University 42 application 86–87 EMBL 226–227, 230 preparation 85–86 GenBank 226–227 see also fungicides listings of Ganoderma gene rRNAbiogeography of Ganoderma 40–41 cluster 226–227breeding disease-resistant oil palm 60, Moncalvo and Ryvarden 5 254 Stalpers and Stegehuis 5BSR see basal stem rot (BSR) diagnostic tool development for BSRburning crop residues 77–78, 129–130, detection in oil palms 235–250 191, 251 early infection 236 fungi employed 242 dikaryotic culture studies 196, 198,calcium nitrate added to soil 92–93, 202, 228 96–97 DNA extraction methodology 241–244chlamydospores 216, 218 dot immunobinding assay (DIBA)cladistic classification 5, 6 261–262clean clearing 58–59, 59, 60–62, 61, drilling diseased oil palm for diagnosis 89, 101–102, 116–117 236climate affecting disease spread 191, 250coconut palm industry economic losses Elaeis guineensis see oil palm infection, 157–160 BSR control strategycoconut palms Elfvingia 5, 23, 24 BSR management 121–128 pileal crust 12 and BSR of oil palms 113, 114, enzyme-linked immunosorbent assay 116–117, 183–194 (ELISA) 84, 236–239, 245, BSR symptoms 207–208 256, 262 disease detection by EDTA 236 indirect (I-ELISA) 257, 258, 259, geographical infection variations 261, 262, 263, 264 193 epidemiology 54–55, 169–170, 235 Malaysian and Sri Lankan palm ethylenediaminetetraacetic acid (EDTA) contrasts 216, 217 236 mtDNA profiles contrasted with oil excision of diseased tissue 62 palm profiles 211 underplanted with oil palm 62cover crops 95, 109, 110, 250–251 field sanitation practice 87, 102, 103,crop mapping 184–190 110, 253 disease symptoms 191 fluorescent antibody technique 58 Ganoderma varieties 190 Fomitopsis cajenderi infection biology infection sources 190–191 151 methodology 185–186 Fomitopsis rosea 229 mycelial isolations and vegetative fruit-body primordia (FBP) formation compatibility 189–190 15–17 orientation of infection spread 190 fungal biology population spatial survey results 186–188 patterns 151cultural characteristics 13–17, 26–27 fungal mitochondrial DNA 168
270 Indexfungal reproductive systems 218–219 hyphae 12fungicides 59, 62–63, 89–90, 126, 127, intracellular esterase isozymes 251, 252 167 see also biofungicides macromorphology 7–12 pileus attachments 7–9 pileus colour 11G. adspersum 7 pileus shapes and patterns 10–11,G. ahmadii 32, 36 12G. applanatum 5, 12, 35, 39, 40, 52, 190 G. meredithae 12, 37 B clade 40 G. microsporum 12, 28 isozyme examination 167 isozyme examination 167G. atropicum 33 G. miniatotinctum 52G. australe 26, 31, 34, 39 palm host 42G. australe-applanatum complex 34, 39 G. mirabile 12G. boninense 52, 61–62, 113, 190–191, G. neo-japonicum 3 237 isozyme examination 167 causing BSR in oil palm 83–88 G. oerstedii 11 geographical spread of oil palm G. oregonense 11, 12, 16, 18, 32 infection 205 coniferous host 36, 41 and hardwood stumps 114 G. pfeifferi 12, 34 isozyme examination 167 G. philippii 75, 76 Malaysian BSR infection 183 on rubber plants 75–76 mating system and aggression G. praelongum 32 115–116, 118 G. pseudoferreum 52 oil palm disease symptoms 84 on rubber plants 75–76 Papua New Guinea oil-palm G. resinaceum 11, 16, 31, 32 infection 195, 196–202 complex 36–37 sexual reproduction and genetic G. sinense 26, 34 diversity 201 complex 39G. carnosum 32 G. subamboinense 12 coniferous host 36, 41 G. tornatum 52G. carpense 28 palm host 42G. chalceum 52 G. trengganuense 32, 37G. colossum 26, 31, 35, 40, 52 G. tropicum 31, 37G. cupreolaccatum 34, 39 complex 37G. cupreum 42 isozyme examination 167G. curtisii 28, 31, 33, 37 G. tsugae 16, 18, 32G. curtisii complex 37 coniferous host 36, 41G. encidum 52 isozyme examination 167G. formosanum 3 G. tsundoae 35, 40 isozyme examination 167 G. ungulatum 12G. fornicatum isozyme examination 167 G. valesiacum 16, 18, 32G. lucidum 3, 5, 52, 236 coniferous host 36, 41 basidiocarp characteristics 7, 10, G. weberianum 28, 31, 32, 37 11, 12 G. xylonoides 42 basidiospores 13, 14, 15 G. zonatum 52 on coconut plantations 121–128, G. zonatum-boninense 33, 38, 42 206 Ganodermataceae complex 16, 36 identification by genetics 164–169
Index 271 nomenclature and classification mice used for antibody formation 3–22 256–257, 259–260 taxonomy 162–163 mycelial wash antigen productiongene tree 27–28 256–258genetic variation study using molecular rabbits’ blood used for antibody (PCR) survey 195–204 formation 236–237, 258–259Gigaspora calospora 123–124 specification 255–256Gliocladium Indonesia as biological control of BSR 83, 86 current research 249–254 virens in biofungicide 86 diagnostic tool development for oil palm infection 235–250 intergenic spacer (IGS) regions 196,Haddowia 24 197, 199–200, 203, 225–226 basidiospores 13 internal transcribed spacers (ITS) 6,hen’s egg yolk antibody formation 197, 199–200, 202, 203, 218, 258–259 225, 226–227, 236Heterobasidion annosum 109, 139–156, EMBL and GenBank listings 164, 167 226–227, 230 biogeography 143, 148–149 phylogeny 27, 28–36, 29, 31, detection by ELISA testing 236 32–35, 40–42 ecological and pathogenicity used for molecular diagnostic differences 145–146 detection of pathogens gene flow 148–149 227–232 genus defined 140–141 isozymes 164 host species 140 extracellular pectinolytic pattern internal transcribed spacer types 165, 166 sequences 142 intracellular 167–168, 227 mating compatibility and pectinase zymograms 146, 227 interbreeding 143–144, 147 pectinases 164–167 morphological differences 145, profiles 25 146–147 ITS see internal transcribed spacers (ITS) phylogeny of rDNA genes 146 population study 147–150 somatic incompatibility 150–152 laccate characteristic 7, 30, 38 spore dispersal 148, 149, 150, 151 land clearance and replanting seehomothallic fungi species 217–218, 219 burning crop residues; cleanhost relationships 41–42 clearing; underplanting; as taxa identifier 36 windrowingHumphreya 24 legume cover crops 109, 110, 250–251Hydnum used for in vitro oil palm Lenzites used for in vitro biodegradation biodegradation trial 132–135 trial 133hyphae 12, 14–15 light as growth factor 13–14, 26 Livinstona cochinchineasis 160immunoassay-based detection kit production 255–264 macromorphology 7–12 exudate as antigen 258–259 Malaysia hen’s egg yolk antibody formation clean clearing 101–102 258–259 oil palm cultivation 49–68
272 IndexMalaysia continued monokaryotic culture studies 196–201, oil palm infection on former coconut 228 plantation 183–194 morphological examination of Ganoderma and Sri Lankan palm contrasts 216, in oil-palm plantings 159–182 217 mycelial morphology 141–143, in vitro oil palm stem biodegradation 163–164 study 129–138 mycoparasitism 91manganese-superoxide dismatase (Mn-SOD) phylogeny 28–29, 29 nomenclature and classification 3–22Marasmius sp. used for in vitro oil palm nucleotide sequence analysis 6 biodegradation trial 131–137medicinal use of Ganoderma 3, 4, 6melanin in fungal mycelium 253 oil palm industrymice used for antibody formation economic importance of disease 259–260 53–54, 160micromorphology 12–13 economic status 249mitochondrial DNA 104–105 oil palm infection 49–68 and basidiospore infection 114 BSR (Ganoderma) control strategy coconut palm and oil palm infection 83–88 profiles contrasted 211 current status of Indonesian Malaysian coconut palm infection research 249–254 profile pattern 217 diagnostic tool development polymorphisms and population 235–250 definitions 227 disease-resistant strain breeding 60, restriction fragment length 254 polymorphisms (RFLP) 168, on Thailand plantation 69–70 173–174, 175, 176 tissue excision 62 results combined with AFLP oil palm plantations profiles 214–216, 217–218 Ganoderma characterizations species identification from betelnut 159–182 palms 214 see also crop mapping Sri Lankan coconut palm profile oil palm residue pattern 217–218 animal feed use 130 used for species identification biodegradation 59 197–199, 206, 209, 211, biodegradation ergosterol analysis 212–213, 215–217 134, 135molecular examination of Ganoderma biodegradation in vitro enzyme isolates in oil-palm assays 131, 136 plantings 159–182, 225–234 biodegradation respirometrymolecular fingerprinting 104–105, 114 analysis 135–136molecular (PCR) survey G. boninense in biodegradation trial conclusions Papua New Guinea 195–204 137molecular systematics 27 burning 77–78, 129–130, 191,molecular variation in Ganoderma 251 isolates 205–221 edible mushroom production 130monoclonal antibody (MAb) enzyme digestibility in vitro trial development 255, 259–260, 136 261 lagoon submerging 130
Index 273 shredding 130 pineapple plantings infection 56 solid-state fermentation 130 Pinus infection by Heterobasidion annosum in vitro biodegradation weight loss 145 132, 133, 134 Pleurotus djamor used in in vitro oil palmoil palm roots 256 biodegradation trial 132 infection detection by PCR assay polyclonal antibody (PAb) 241, 243–245 development 255, 256–259oil palm seedlings 84, 88–99, 106, 107, production 236–239 108, 110 polyclonal antibody anti-exudateoil palm stem (PAb-aeG) 258–259, 260 Ganoderma resting bodies formation fresh and dried palm leaf tests 252–253 261–263, 264 weight loss from biodegradation polymerase chain reaction (PCR) 133 196–197, 209–210, 226, 230,Oncosperma filamentosa 160 239–241, 245organic manures used for biological used for pathogen detection 236, control of BSR 122–123, 124, 237 125, 127 Polyporaceae genus subdivided 6–7Orycytes rhinoceros damage 55, 129 Polyporus used in biodegradation trial 134 Polyporus lucidus 4palm clade of Ganoderma 33, 38 primerspalm-oil mill effluent (POME) as planting construction 226 medium for disease control 91, GAN1 and GAN2 237, 240–241, 95, 96–97 243, 244Papua New Guinea GanET 228, 230–232 basidiospores study 113–121 IT3/GanET 104 molecular (PCR) survey of genetic ITS2 228, 229, 230 variations 195–204 ITS3 230Papua New Guinea Oil Palm Research ITS4 228 Association (OPRA) 227 ITS3/GanET pair 230–232pathogen spread and geographical ITS1F 228 isolation 219 Pycnoporus used in biodegradationPenicillium 164, 237 trial 135–136 as biological control of BSR 90–91 population enhancement by rabbits’ blood used for antibody calcium soil amendment 92 formation 236–237, 258–259Phellinus random amplified polymorphic DNA noxius 75–76, 77, 109 (RAPD) 6, 210, 230 weirii infection biology 151 analysis of pathogen populationsphosphobacteria 123–124 196–203phylogenetic relationships and replanting techniques 58, 61–62 biogeography 36–40 reproductive systems of fungi 218–219phylogeny 28–40 resting bodies in oil palm stem 252–253Picea infection by Heterobasidion annosum restriction fragment length 145 polymorphisms data ofpilocystidia 24, 26 Ganoderma isolates 175
274 Indexribosomal DNA (rDNA) 5, 6, 27, 236 Trichoderma harzianum and organic internal transcribed spacer manures 95, 96 (ITS) variability 168–169, Trichoderma supplementation 93, 174 95ribosomal RNA (rRNA) vesicular arbuscular mycorrhizal database listing of genus fungi (VAM) 95–96 Ganoderma 226–227 soil fungi population after used for molecular determination of supplementation 95 filamentous fungi 225–226 soil mounding 60rice plantations and Ganoderma soil nutrition 57–58 infection 115 soil type and Ganoderma infection 50,root-rot diseases 56–57, 60, 207–208 on Acacia mangium Willd. 71–79 Solomon Islands basidiospores study brown-root infection 75 113–121 fungi identification trials 75–76 somatic incompatibility (SI) 150–152, pathogenicity tests 76–77 171–173 red-root infection 75 in basidiomycetes 150 symptoms and mortality 72–74, testing 210–211 73, 74 somatic incompatibility groups (SIG)root-to-root infection 54, 61, 169–170, 171, 172, 173–174, 175, 206 192 Sri Lankan and Malaysian resultrubber plantings 56, 75, 76, 160 comparisons 216, 217 species concept 139, 141 individuality 150–151saprobic isolates 231, 232, 237 morphology in mycologysaprophytic fungi 55, 90 141–143 tested against PAbs 237–239 species tree 27–28 tested against primers Gan1 and sporocarps root disease and A. mangium Gan2 240–241, 243, 244 75–76sclerotium plate formation 252–253 sporophores 108seedlings of oil palms 84, 87, 110 on Sri Lanka coconut palms 208, bait 102–104, 106, 109, 110 209 in calcium-nitrate supplemented Sri Lanka soil 93 coconut and betel nut BSR infection infected by oil palm residues 106, 205–221 107, 108 geographical isolation of pathogenssoil amendment 89–99 219 biofertilizers on coconut plantation and Malaysian palm contrasts 216, 122, 123–127 217 calcium nitrate supplementation Sumatra 92–93, 96–97 BSR control field trials 101–114 calcium supplementation 92, 96 oil palm replanting losses 101 Calepogonium caeruleum 95 systematics 23–45 fungicide assisted biological control molecular 27 93–95 web site 42 oil-palm pot trials of additives 92–97 results 94, 96 taxonomy of Ganoderma 25, 26–27 sulphur powder 95 history 4–7
Index 275tea plants 160 Tsuga sp. infection by Heterobasidiontemperature as growth factor 15–16 annosum 145Thailand oil palm Ganoderma infection 69–70Thanjavur wilt see basal stem rot (BSR) underplanting 58–59, 59, 60, 62, 105,Trametes used in biodegradation trial 184–185 135–136 upper stem rot (USR) 105trench digging as infection control measure 60, 61Trichoderma 64, 237–238 vesicular arbuscular mycorrhizal (VAM) biofungicide 84–87 fungi 123 as Ganoderma antagonist 83, used for Ganoderma control 95–96 85–87, 90–91 harzianum 91, 251 in biofungicide 86 Wang extraction method for DNA used in BSR control field trial 216–244 122–127, 123, 124 water-logged soil and BSR infection koningii in biofungicide 86 56–57, 60, 207–208 pileal tissue 12 windrowing 58–59, 59, 60, 90, population enhancement by 110–111, 129, 170 calcium soil amendment 92 coconut trunks in oil-palm soil augmentation to control BSR plantation 185 93, 95 and pre-felling poisoning 109, 110