Ganoderma

Ganoderma Diseases of Perennial
Crops

Edited by

J. Flood
CABI Bioscience, Egham, UK

P.D. Bridge
Mycology Section, Royal Botanic Gardens Kew, Richmond, UK

M. Holderness
CABI Bioscience, Egham, UK

CABI Publishing
iii

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Library of Congress Cataloging-in-Publication Data
Ganoderma diseases of perennial crops / edited by J. Flood, P. Bridge, and M. Holderness.
p. cm.
Includes bibliographical references.
ISBN 0-85199-388-5 (alk. paper)
1. Ganoderma. 2. Ganoderma diseases of plants. I. Flood, J. (Julie) II. Bridge, P. D.
III. Holderness, M. (Mark)

SB741.G35 G36 2000
632′.4--dc21 00-039847

ISBN 0 85199 388 5

Typeset by AMA DataSet Ltd, UK.
Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn.

Contents
Contents

Contents

Contributors ix

Preface xi

Part I Ganoderma, Organism and Systematics 1

1 Ganodermataceae: Nomenclature and Classification 3
G.-S. Seo and P.M. Kirk

2 Systematics of Ganoderma 23
J.-M. Moncalvo

Part II Ganoderma, Diseases of Perennial Crops 47

3 Status of Ganoderma in Oil Palm 49
D. Ariffin, A.S. Idris and G. Singh

4 Basal Stem Rot of Oil Palm in Thailand Caused by Ganoderma 69
S. Likhitekaraj and A. Tummakate

5 The Current Status of Root Diseases of Acacia mangium Willd. 71
S.S. Lee

v

vi Contents

Part III Disease Control and Management Strategies 81

6 A Control Strategy for Basal Stem Rot (Ganoderma) on Oil Palm 83
H. Soepena, R.Y. Purba and S. Pawirosukarto

7 The Use of Soil Amendments for the Control of Basal Stem Rot of
Oil-Palm Seedlings 89
M. Sariah and H. Zakaria

8 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’Grady

9 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. Bridge

10 Management of Basal Stem Rot Disease of Coconut Caused by
Ganoderma lucidum 121
R. Bhaskaran

11 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’Grady

12 Functional Units in Root Diseases: Lessons from Heterobasidion
annosum 139
Å. Olson and J. Stenlid

Part IV Molecular Variability in Ganoderma 157

13 Molecular and Morphological Characterization of Ganoderma in
Oil-palm Plantings 159
R.N.G. Miller, M. Holderness and P.D. Bridge

14 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. Abdullah

15 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 vii

16 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. Flood

Part V Development of Diagnostic Tests for Ganoderma 223

17 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. Sanderson

18 The Development of Diagnostic Tools for Ganoderma in Oil Palm 235
C. Utomo and F. Niepold

19 Ganoderma in Oil Palm in Indonesia: Current Status and
Prospective Use of Antibodies for the Detection of Infection 249
T.W. Darmono

Index 267

Contributors
Contributors

Contributors

F. Abdullah, Department of Biology, Faculty of Science and Environmental
Studies, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
E.A.B. Aitken, Department of Botany, University of Queensland, St Lucia,
Queensland, Australia
D. Ariffin, Palm Oil Research Institute of Malaysia, No. 6, Persiaran Institute,
Bangi, PO Box 10620, 50720 Kuala Lumpur, Malaysia
R. Bhaskaran, Coconut Research Station, Tamil Nadu Agricultural
University, Veppankulam 614 906, Tamil Nadu, India
P.D. Bridge, Mycology Section, Royal Botanic Gardens Kew, Richmond,
Surrey TW9 3AE, UK
T.W. Darmono, Biotechnology Research Unit for Estate Crops, Jl. Taman
Kencana No. 1, Bogor, 16151, Indonesia
J. Flood, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK
Y. Hasan, Bah Lias Research Station, P.T.P.P. London, PO Box 1154, Medan
20011, North Sumatra, Indonesia
M. Holderness, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY,
UK
A.S. Idris, Palm Oil Research Institute of Malaysia, No. 6, Persiaran Institute,
Bangi, PO Box 10620, 50720 Kuala Lumpur, Malaysia
J. Kelley, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK
P.M. Kirk, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK
R. Lardner, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK
S.S. Lee, Forest Research Institute Malaysia, Kepong, 52109 Kuala Lumpur,
Malaysia

ix

x Contributors

S. Likhitekaraj, Division of Plant Pathology and Microbiology, Department of
Agriculture, Bangkok 10900, Thailand
R.N.G. Miller, Universidade Católica de Brasília Pró-Reitoria de Pesquisa e
Pós-graduação, Campus II, 916 Asa Norte, Brasília, D.F., Brazil
J.-M. Moncalvo, Department of Botany, Duke University, Durham, NC
27708, USA
F. Niepold, Federal Biological Research Centre for Agriculture and Forestry,
Institute for Plant Protection of Field Crops and Grassland, Messeweg
11–12, 38104 Braunschweig, Germany
E.B. O’Grady, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY,
UK
Å. Olson, Department of Forest Mycology and Pathology, Swedish University
of Agricultural Sciences, Box 7026, S–750 07 Uppsala, Sweden
R.R.M. Paterson, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20
9TY, UK
S. Pawirosukarto, Indonesian Oil Palm Research Institute (IOPRI), Jl.
Brigjen Katamso 51, Medan 20158, Indonesia
C.A. Pilotti, PNG OPRA, Plant Pathology Laboratory, PO Box 36, Alotau,
Milne Bay Province, Papua New Guinea
R.Y. Purba, Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen
Katamso 51, Medan 20158, Indonesia
H. Rolph, Level 9, Glasgow Dental School and Hospital, 378 Sauchiehall St,
Glasgow G2 3JZ, UK
F.R. Sanderson, PNG OPRA, Plant Pathology Laboratory, PO Box 36,
Alotau, Milne Bay Province, Papua New Guinea
M. Sariah, Department of Plant Protection, Universiti Putra Malaysia,
43400UPM, Serdang, Selangor, Malaysia
G.-S. Seo, College of Agriculture, Chungnam National Unviersity, Taejon
305–764, Korea
G. Singh, United Plantations Berhad, Jenderata Estate, 3600 Teluk Intan,
Perak, Malaysia
H. Soepena, Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen
Katamso 51, Medan 20158, Indonesia
J. Stenlid, Department of Forest Mycology and Pathology, Swedish University
of Agricultural Sciences, Box 7026, S–750 07 Uppsala, Sweden
A. Tummakate, Division of Plant Pathology and Microbiology, Department of
Agriculture, Bangkok 10900, Thailand
P.D. Turner, PO Box 105, Quilpie, Queensland 4480, Australia
C. Utomo, Indonesian Oil Palm Research Institute (IOPRI), PO Box 1103,
Medan 20001, Indonesia
R. Wijesekara, Coconut Research Institute, Bandirippuwa Estate, Sri Lanka
H. Zakaria, Department of Plant Protection, Universiti Putra Malaysia,
43400UPM, Serdang, Selangor, Malaysia

Preface
Preface

Preface

Perennial oilseed crops form a major component of rural economies through-
out the wet lowland tropics of South and South-East Asia and Oceania. Crops
such as oil palm and coconut are grown as both plantation-scale commodity
crops and as smallholder cash and food crops. Perennial oilseed crops contrib-
ute significantly to local livelihoods through not only their husbandry but also
the processing of the crop and crop by-products and their subsequent shipping
and marketing. As export commodities, they form an important component of
national economies and generate valuable foreign exchange.
Species of the basidiomycete fungus Ganoderma occur as pathogens on a
wide 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 have
been of considerable concern ever since replanting of oil-palm land began in
South-East Asia and recent workshops have identified basal stem rot, caused
by Ganoderma boninense, as the single major disease constraint to oil palm
production in the region. The long-term nature of palm monocultures means
that they are prone to both premature plant death and to the carry-over of
residual inoculum from one planting to the next. This pattern has been clearly
seen in many areas of South-East Asia and creates considerable concern for the
long-term sustainability of palm production from affected land. Basal stem rot
of oil palm is widespread, occurring in the major oil palm growing regions of
the world. By contrast, the disease on coconut appears very restricted; it was
first recorded in India in 1952 and remains confined to South Asia, yet
Ganoderma species occur as saprobes on dead coconut palm tissues in all
palm-growing regions, an anomaly that requires resolution.
xi

xii Preface

A crucial factor in developing effective disease management programmes
is the prior understanding of pathogen biology and disease epidemiology.
Ganoderma is a notoriously variable and difficult fungus to characterize and
this has led to much past confusion in disease aetiology and epidemiology.
Such studies have been greatly enhanced through the development and use
of molecular and biochemical markers to discriminate among pathogen
populations and individuals and to diagnose infected palms in advance of
terminal symptoms. These technological tools can form powerful adjuncts to
field observation and experiments in understanding mechanisms of disease
spread and pathogen survival. This new understanding establishes the
fundamental biology of the genus and provides new insight into disease
epidemiology that enables the implementation of appropriate and effective
management strategies.
In perennial crops, infections of woody tissues have the opportunity to
slowly develop further and expand as conditions permit. Infective material can
remain viable in the ground for many months and infect subsequent crops at
replanting. It is therefore very important to manage disease outbreaks in such
a way as to minimize the risks to both existing and future plantings. One
feature of Ganoderma diseases is the persistence of potential pathogens in old
woody tissues and soil-borne debris. Burning of such material is no longer
acceptable and extensive physical clearing is often not feasible due to the input
requirements involved. Alternative treatments are thus required and a
number of approaches are being explored to manage this residual inoculum.
These are centred on the evaluation of biocontrol agents and the rapid
biodegradation of palm woody residues.
This book is a joint effort by 36 authors from 13 countries, each with a
wide expertise in their own fields. In many chapters, joint authors have come
together from different countries, illustrating the collaborative nature of this
initiative. The 19 chapters address many current issues in the development of
sustainable disease management programmes and are grouped into five major
themes. These are, an introduction to the pathogen and its systematics in
Chapters 1 and 2, outlines of the diseases caused by the pathogen (Chapters
3–5), disease management (Chapters 6–12), molecular biological variability
in the pathogen (Chapters 13–16) and the development of diagnostic tools
(Chapters 17–19). The majority of these chapters have been developed from
presentations made at two international workshops on Ganoderma diseases
held in Malaysia in 1994 and 1998 and a technical workshop held in the UK
in 1998. Funding for these workshops was provided by the UK Department
for International Development (DFID Project R6628) Crop Protection
Programme, for the benefit of developing countries and from the European
Community (Stabex fund), the British Council, Governments and institutions
of the countries concerned and numerous private plantation companies. We
are very grateful to the various sponsors of this research for their involvement,
although the book should not be considered to necessarily reflect the views of
our sponsors. We would also wish to acknowledge the pioneering work and

Preface xiii

dedication of a number of scientists who have previously advanced knowledge
of this recalcitrant organism and its various diseases and inspired us in our
own 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 pathogen
as at the end of 1998 and we hope will be both useful and informative to a wide
range of readers including scientists in the private and public sectors, students
and 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 enhance
the sustainable management of Ganoderma diseases.

J. Flood
P.D. Bridge
M. Holderness

Ganoderma, Organism and I
Systematics

1
G.-S. Seo and and Kirk
NomenclatureP.M. Classification

Ganodermataceae: 1
Nomenclature and
Classification
G.-S. Seo1 and P.M. Kirk2
1College of Agriculture, Chungnam National University,
Taejon, Korea; 2CABI Bioscience, Egham, UK

What are Ganoderma?
Ganodermataceae are cosmopolitan basidiomycetes which cause white rot of
hardwoods, such as oak, maple, sycamore and ash, by decomposing lignin as
well as cellulose and related polysaccharides (Hepting, 1971; Blanchette,
1984; Adaskaveg and Ogawa, 1990; Adaskaveg et al., 1991, 1993). Although
species of Ganoderma are economically important plant pathogens, causing dis-
ease in crops such as rubber and tea and wood rot of trees, the fruit bodies are
popular as, and have long been used in, traditional medicinal material in Asian
countries, including China, Japan and Korea. The Ganoderma lucidum complex,
known in Chinese as Lingzhi, has long been considered a symbol of good
fortune and prosperity and is the subject of many myths, as well as being a
medicinal herb in ancient China (Zhao and Zhang, 1994). There are records of
these fungi before the time of the famous medical book Shen Nong Ben Cao Jing
(AD 25–220, Eastern Dynasty). Depending on the different colours and shapes
of the fruit bodies, they have been called the red-, black-, blue-, white-, yellow-
and purple-types in Shen Nong Ben Cao Jing by Hong Ching Tao (AD 456–536)
and Ben Cao Gang Mu by Li Shi-Zhen (AD 1590, Ming Dynasty), as well as the
antler- and kidney-shapes (Shin and Seo, 1988b; Zhao, 1989; Willard, 1990).
The black fruit body referred to by the old description in China (Willard, 1990)
is assumed to be G. neo-japonicum or G. formosanum because the fruit bodies of
these species are black in nature. However, the descriptions on the blue, yellow
and white fruit bodies of the G. lucidum complex have not been found.
Ganodermataceae have attracted the attention of mycologists for many
years. They have been considered as either plant pathogens (Hepting, 1971;

©CAB International 2000. Ganoderma Diseases of Perennial Crops
(eds J. Flood, P.D. Bridge and M. Holderness) 3

4 G.-S. Seo and P.M. Kirk

Adaskaveg and Ogawa, 1990; Adaskaveg et al., 1991, 1993), or useful
medicinal herbs (Mizuno et al., 1995). Because of these fundamentally
different viewpoints among collectors, the taxonomy of these fungi is very
subjective and confused. Contributions to the morphology and taxonomy of
the Ganodermataceae have been made by many mycologists, including Steyaert
(1972), Furtado (1981), Corner (1983) and Zhao (1989). However, the great
variability in macroscopic and microscopic characters of the basidiocarps
has resulted in a large number of synonyms and in a confused taxonomy,
especially in the genus Ganoderma (Gilbertson and Ryvarden, 1986).

History of Ganoderma Taxonomy and Nomenclature
The 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 included
only one species, Polyporus lucidus, in the circumscription of the genus and this
species, therefore, became the holotype species.
P. lucidus was named by William Curtis, the 16th-century British botanist.
Unfortunately, Karsten incorrectly attributed the epithet ‘lucidus’ to von
Leysser and this error has been perpetuated in numerous subsequent
publications. No authentic specimens remain and the type locality, Peckham,
is now very much changed from what it was in the time of Curtis. The area is
now largely developed as residential housing but the type substratum, the
small tree Corylus avellana, is likely to be growing still on Peckham Rye
Common. It is clear, therefore, where any epitype, selected as an interpretive
type, should be sought. The selection of an epitype, in the absence of type or
authentic material, would be important, for any further molecular work will
need to have available a culture of the type species of the genus which has
some nomenclatural standing, i.e. a culture derived from an epitype.
Following Karsten, dozens of species belonging to the genus were reported
by taxonomists (Patouillard, 1889; Boudier and Fischer, 1894; Boudier,
1895; Murrill, 1902, 1908). The identification of Ganoderma in those days
was mainly based on host specificity, geographical distribution, and macro-
morphological features of the fruit body, including the context colour and
the shape of the margin of pileus, and whether the fruit body was stipitate
or sessile. Subsequently, Atkinson (1908), Ames (1913), Haddow (1931),
Overholts (1953), Steyaert (1972, 1975, 1977, 1980), Bazzalo and Wright
(1982), and Corner (1983) conducted the identification of Ganoderma species
by morphological features with geographically restricted specimens. Haddow
(1931) and Steyaert (1980) placed most of their taxonomy on the spore char-
acteristics and the morphology of hyphal elements. However, the basidiocarps
of Ganoderma species have a very similar appearance that has caused confusion
in identification among species (Adaskaveg and Gilbertson, 1986, 1988).
The genus now contains a few hundred names; there are 322 in the CABI
Bioscience fungus names database, but others may have been published that

Nomenclature and Classification 5

the major printed indexes, the source of this database, failed to include. The
database of Stalpers and Stegehuis available on the CBS web site lists 316
names in Ganoderma and the recent publication of Moncalvo and Ryvarden
(1997) lists 386 names for the Ganodermataceae as a whole. It has not yet been
possible to compare these three data sets, although such an exercise would
appear to be needed. However, names are only one aspect of this subject
and problems associated with them are, on the whole, easier to resolve than
problems associated with the circumscription of species.
Based on the unique feature of the double-walled basidiospore, the French
mycologist, Patouillard, over a period of some 40 years from 1887, described a
number of new species of Ganoderma and transferred several names from other
genera of the polypores. Patouillard (1889) published a monograph of the
then known 48 species and also distinguished the species with spherical or
subspherical spores as section Amauroderma. Coincidentally, in the same year,
Karsten introduced the genus Elfvingia, based on the name Boletus applanatus
of Persoon, for the non-laccate species. Later, section Amauroderma of
Ganoderma was raised to the rank of genus by Murrill who, in selecting a
species which was not included in section Ganoderma by Patouillard, is
therefore the author of the name, and priority dates from 1905 not 1889.
Subsequent authors have recognized Amauroderma as a distinct genus. The
two genera have been largely accepted, although Corner (1983) and Zhao
(1989) reported species that are intermediate between them. Amauroderma
was revised by Furtado (1981).
Here then we have two important species in the history and the nomen-
clature of the genus, Ganoderma lucidum and Ganoderma applanatum, and these
are probably two of the most poorly understood species of Ganoderma and two
of the most frequently misapplied names.
The late 19th-century and early 20th-century mycologists contributed
significantly, in terms of volume of published information, on the genus,
describing many new species or perhaps, more correctly, introducing many
new names. Many of these names were based on single collections or on only a
few collections from the same locality, and the taxonomic status of the species
to which these names were applied is, therefore, often open to the criticism
of being unsound. Throughout the remainder of the 20th century various
workers, Steyaert, Corner and Zhao perhaps being the more prominent,
contributed to our knowledge of the genus by providing revisions, mono-
graphs, descriptions of new taxa (again, often based on single collections or
on only a few collections from the same locality) and observations on both
anatomy and ontogeny.
Recent workers have used characters other than morphology to 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 his
co-workers (Moncalvo et al., 1995a, b) have used ribosomal DNA sequences


and cladistics methods to infer natural relationships. However, as Moncalvo
and Ryvarden have stated, these recent studies have had little impact on
Ganoderma systematics in total because too few taxa were examined. This was
quite clearly through both a lack of human and financial resources and,
perhaps more importantly, a lack of the very important type or authentic col-
lections which will link the names available to any subsequent taxa identified.
Ryvarden (1994) has stated that the genus is in taxonomic chaos and
that it is one of the most difficult genera amongst the polypores. However, this
realization has come at the very time when there has been a renewed interest
in Ganoderma from a number of quite unrelated sources. These include the
medicinal uses based on very old Chinese traditions and the requirement to
elucidate 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 here
is the apparent increase in the importance of some species of Ganoderma as
pathogens of plants used by man.
However, with the development of cladistic methods to reconstruct
natural classifications and the application of these methods to both traditional
morphological data and, more importantly, new molecular data, the potential
for the resolution of some of these problems appears close to hand. Recently,
the phylogenetic relationships of some Ganoderma species collected from
various regions were studied by allozyme (Park et al., 1994) and DNA analysis
(Moncalvo et al., 1995a, b). Moncalvo and his co-workers (Moncalvo et al.,
1995a, b; Hseu et al., 1996) adopted ribosomal DNA sequences and randomly
amplified polymorphic DNA (RAPD) as the tools for analysing phylogenic
relationships in the G. lucidum complex. The results suggested that some
strains were misnamed and misidentified, and all isolates belonging to 22
species were disposed in six groups based on nucleotide sequence analysis
from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA).
However, while some isolates had the same ITS sequence, all of them could be
clearly differentiated by genetic fingerprinting using RAPDs. Therefore, RAPD
analysis might be helpful for systematics at the lower taxonomic levels to
distinguish isolates from each other. When the results of molecular taxonomy
are compared with the data of traditional taxonomy, such as morphological,
ecological, cultural and mating characteristics, some isolates remain as
exceptions. Of many studies on Ganoderma taxonomy, Adaskaveg’s research
(Adaskaveg and Gilbertson, 1986) indicates the importance of vegetative
incompatibility 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 proposed
that no new species be described in Ganoderma in the decade to 2005.
Donk, in 1933, was the first to unite the taxa within what was then
the very large family Polyporaceae when he proposed the subfamily
Ganodermatoideae; he subsequently raised this taxon to the rank of family
with the introduction of the Ganodermataceae and this classification has


subsequently been accepted by most recent workers. Much later, Julich, in
1981, introduced the ordinal name Ganodermatales and this was accepted by
Pegler 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 the
family represented an old lineage from which other groups of polypores have
been derived. Ryvarden (1994), however, proposed that the high phenotypic
plasticity observed in the genus is indicative that the taxon is young and that
strong speciation has not yet been achieved. This hypothesis was supported by
more recent molecular evidence from Moncalvo and his co-workers. The lack
of fossils limits the accuracy to which we can attribute a minimum age to the
genus. Some fossils of corky polypores from the Miocene (25 million years old)
have been tentatively referred to Ganoderma adspersum.

Morphological Features of Ganoderma
Macromorphology

The 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 pileus
surface and hymenophore varies from deep red, non-laccate, laccate and light
yellow to white, and the morphology also differs between the isolates (Shin
and Seo, 1988b). The morphological variation appears to be affected by envi-
ronmental conditions during basidiocarp development. Table 1.1 summarizes
the representative results from several descriptions of the macromorphology of
G. lucidum. The size and colour of the basidiocarp shows significant differences
between the specimens, but the pore sizes are similar. The manner of stipe
attachment 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 in
nature (Fig. 1.1). Stipe characters, including attachment type and relative
thickness and length, have been considered useful for species identification,
but their importance has been neglected by some mycologists, who describe
fruit bodies only as stipitate or sessile. Hardwoods are the usual host plants of
G. lucidum, but some specimens have been collected from conifers.
The laccate character of the pileus and stipe has been variously employed
in the taxonomy of this family. According to traditional concepts, the pileus
surface of Ganoderma is laccate, but is not so in Amauroderma. However, a few
species of Amauroderma and Ganoderma have been reported with laccate (A.
austrofujianense and A. leptopus) and non-laccate appearance (G. mongolicum).
The laccate character, while playing no important role in the segregation of
genera and sections in this family, remains available as an identification aid.


Context colour of Ganoderma varies from white to deep brown and has
been considered a useful character in classification. However, some mycolo-
gists have considered it useless for identification of species and supraspecific
groups because it may change under different environmental conditions.
Context colour is often changeable, especially in dried specimens, not only in
the 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 diameter
Colour 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
brownish
Attachment of
stipe 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 Trimitic
Host
Hardwood – Common Common # 23 specimens
Conifer – Occasionally Rarely no 22 specimens
a
L and D in parentheses indicate length and diameter, respectively.
b
Not determined.
c
#: described by author as presence only.
9


emphasized the importance of observing the context colour of fresh and living
specimens in the classification of Ganoderma. The size and shape of pores are
also useful characters for species classification. The number of pores per
millimetre may serve as a specific character.
The morphology of basidiocarps of G. lucidum in artificial cultivation on
wood logs and synthetic substrates is affected by environmental conditions
(Hemmi and Tanaka, 1936). Fruit-body formation in G. lucidum usually
requires 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). Under
dim light or dark conditions with poor ventilation, the pileus does not expand
and often an abnormal pileus of the ‘stag-horn’ or ‘antler-type’ is produced
(Hemmi and Tanaka, 1936; Shin and Seo, 1988b; Stamets, 1993a). Figure
1.2 and 1.3 show fruit bodies of the G. lucidum complex produced by the

Fig. 1.2. Fruit bodies of Ganoderma lucidum complex generated by sawdust-
bottle cultivation.


sawdust-bottle culture method. They show polymorphic features such as the
kidney-type and antler-type with various colours (Shin and Seo, 1988b). Out
of 22 isolates of the G. lucidum complex observed by one of the authors of this
chapter (Shin and Seo, 1988b), 16 isolates formed typically kidney-shaped
fruit bodies, and the remainder formed antler-type fruit bodies. Kidney-shaped
fruit bodies could be further divided into those with a concentric zone on the
surface of the pilei and those without. Antler-shaped fruit bodies also divide
into 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 in
morphology when generated by artificial cultivation with sawdust media,
including their pileus colour, pileus zonation, attachment type and context
colour. 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 generated
by sawdust-bottle cultivation is reddish-brown to deep brown. In G. lucidum
(ATCC 64251 and ASI 7004), G. oregonense (ATCC 64487), G. resinaceum and
G. oerstedii (ATCC 52411) the fruit bodies have very similar pileus colour,

Fig. 1.3. Asian collection – fruit bodies of Ganoderma lucidum generated by
sawdust-bottle cultivation.

Table 1.2. Classification of stocks in Ganoderma lucidum according to the
morphology 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 isolates
2. Antler-shaped fruit body --------------------------------------------(a and b)
a. Typically antlered--------------------------------------------------------------- 2 isolates
b. Antler-shaped with abnormal pileus --------------------------------------- 4 isolates


zonation and pattern of stipe attachment. Although one isolate (ASI 7024)
of G. lucidum produced typical antler-shaped fruit bodies, isolates ASI 7024
and ASI 7004 were confirmed as conspecific by mating tests with mono-
karyotic mycelia. Another isolate (MRI 5005) of G. lucidum showed a very
specific pileus pattern with well-developed concentric zones. The species G.
applanatum, G. microsporum, G. subamboinense and G. pfeifferi have unique
morphological characters. The fruit body of G. meredithae (ATCC 64490) has a
long stipe attached parallel to the pileus and no concentric zones on the surface
of the pileus. In G. applanatum (ATCC 44053) the fruit body is reddish-brown
and has no distinct stipe; the surface and margin of the pileus are rough. The
pileus of G. microsporum (ATCC 6024) has a yellowish-brown margin and
the stipe is black; the surface of the pileus is smooth and has many narrow
concentric zones. In G. subamboinense (ATCC 52420) the pileus is deep brown,
although the growing margin is white, and it has a typical stipe; the surface
of the pileus has many concentric zones. An abnormal pileus was produced
in G. pfeifferi (CBS 747.84), with an upturned margin; the pileus is also
comparatively very thick (up to 30 mm).

Micromorphology

The structure of the pileal crust and cortex are useful characters in the
taxonomy of the Ganodermataceae. The former character occurs mainly in
Ganoderma and Amauroderma, but the latter also occurs rarely in Amauroderma.
Fruit bodies of Ganoderma mostly have an hymenioderm or characoderm and
anamixoderm (Steyaert, 1980). In Elfvingia, the pileal crust is a trichoderm
or an irregular tissue; it is also an irregular tissue in Trachyderma (Zhao,
1989). This character is considered to be very useful for identification by some
taxonomists. However, it often differs in different specimens of a single species
and may show various structural forms.
In Ganodermataceae, the hyphal system is usually trimitic, occasionally
dimitic, the generative hyphae are hyaline, thin walled, branched, septate or
not, and clamped. Clamp connections may often be difficult to observe in
dried specimens. However, they are easily observed in the youngest parts of
the hymenium and context of fresh specimens. Skeletal hyphae are always
pigmented, thick walled, and arboriform or aciculiform; skeletal stalks may
end in flagelliform, branched binding processes. Binding hyphae are usually
colourless with terminal branching. Some species of Ganoderma, such as G.
lucidum and G. ungulatum, show Bovista-type binding hyphae which are
produced from the generative or skeletal hyphae. G. mirabile and G. oregonense
have a pallid context and exhibit intercalary skeletals, which are derived from
a transformed and elongated generative cell. On the other hand, Amauroderma
has no Bovista-type binding hyphae and many species have intercalary
skeletals. Hyphal characters are also influenced by environmental factors.
Zhao (1989) observed great variation in hyphal diameter and in frequency of


septation due to differences in age as well as in nutrition. For species identifica-
tion, however, hyphal characters are often useful (Zhao, 1989).
Basidia and basidiospores are considered as the most important characters
for species identification in basidiomycetes. Basidia in Ganodermataceae attain a
relatively large size and range from typically clavate to pyriform. Intermediate
forms are often seen in the same specimen. Basidiospores show several
dependable characters for identification. Ganodermataceae have a unique
double-walled basidiospore; Donk’s (1964) concept for the Ganodermataceae is
based on characters of the basidiospores. Basidiospores of Ganoderma are ovoid
or ellipsoid–ovoid, occasionally cylindric–ovoid, and always truncate at the
apex. The wall is not uniformly thickened, with the apex always thicker than
the base. It is very distinctly double-walled, with the outer wall hyaline and
thinner, and the inner one usually coloured and thicker and echinulate or
not. In Amauroderma the basidiospores are globose to subglobose, occasionally
cylindrical, and form a uniformly thickened wall. In Haddowia the basidio-
spores are longitudinally double-crested, with small, transverse connecting
elements.
Microscopic observations, such as the size and morphology of basidio-
spores, have been adopted as the criteria for the taxonomy of Ganoderma. The
basidiospores, which commonly have double walls and are ellipsoid and
brownish, vary in size (based on descriptions in the literature; Table 1.3). A
basidium of G. lucidum has four sterigma with a hilar appendix (Fig. 1.4) and
1–2 vacuoles. Basidiospores have an eccentric hilar appendix on a rounded
spore 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 sizes
of basidiospores of naturally grown specimens from Japan and Korea were
8.5–11 × 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 of
spore length to width) were 1.62 and 1.58, respectively.

Cultural Characteristics
Critical studies on cultural characteristics are very important in species identi-
fication of some groups of higher basidiomycetes. However, useful studies of
cultural characteristics of Ganoderma for species identification are rare. In vitro
morphogenesis and cultural characteristics of basidiomycetes are affected by
various environmental factors, such as light, aeration, temperature, humidity
and nutritional condition (Schwalb, 1978; Suzuki, 1979; Manachère, 1980;
Kitamoto and Suzuki, 1992). Among these, light is an essential factor for
fruiting 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 the
stipe of G. lucidum were affected positively by light (Hemmi and Tanaka, 1936;


Table 1.3. Morphological comparison of basidiospores of Ganoderma lucidum.

Basidiospore Size Spore
Reference sources (µm) indexa Microscopical feature

Ito (1955) Wild fruit 9.5–11 × 5.5–7 – Deep yellowish brown,
body ovoid and double wall
Steyaert (1972) Wild fruit 8.5–13 × 5.5–8.5 – Ovoid, chamois
body
Pegler and Wild fruit 9.0–13 × 6–8 1.64 Ovoid to ellipsoid
Young (1973) body (av. 11.5 × 7)
Bazzalo and Wild fruit 9–13 × 5–6.9 – Subovoid with the apex
Wright (1982) body truncate, perisporum
hyaline, smooth and
thin endosporic pillars
Melo (1986) Wild fruit 8.2–13.5 × 6.8–8.1 – Truncate, ovoid,
body brownish to brown
Adaskaveg Wild fruit 10.6–11.8 × 6.3–7.8 1.50 Brown, ovoid with
and Gilbertson body (av. 11.5 × 7.4) holes and eccentric
(1986) hilar appendix, double
wall and vacuole
Mims and Wild fruit 9–12 × 6–7 – Ellipsoid with holes and
Seabury (1989) body eccentric hilar appendix
Seo 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 vacuole
Seo 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 vacuole
Seo 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 vacuole
aSpore 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 suppressed
by light (Shin and Seo, 1988a, 1989a; Seo et al., 1995a, b). However, critical
studies on the effects of light on mycelial growth and basidiocarp formation of
Ganodermataceae have not been 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 and
even, felty to floccose at the optimum temperature on potato dextrose agar


Fig. 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 becomes
more yellowish under exposure to light.
The different optimum temperatures and growth rates among various
species 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 form
chlamydospores. In vitro, colonies showed various features, such as sectoring,
pigmentation, formation of fruit-body primordia (FBP) and atypical fruiting
structures (AFSs) which formed basidia and basidiospores without basidiocarp
formation (Shin and Seo, 1988a). AFSs were induced by light with ventilation
from the white mycelial colony stage (Shin and Seo, 1989b). Some isolates

16

Table 1.4. Cultural characteristics of the Ganoderma lucidum complex.

Temperature (°C)
Growth rate
Species Reference Colour Growth habit Opt. Max. (mm day−1) Chlamydosporeb Fruitingb

G. 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 (−).


produced FBP on agar medium, but these did not develop into mature fruit
bodies during the 30 days of cultivation (Seo et al., 1995a). In vitro, higher rate
of ventilation was required for AFS formation, but FBP could be formed under
conditions of lower ventilation. This fact suggests that FBP and AFSs may
be initiated by a common morphogenetic control system, but that subsequent
development to either FBP or AFSs may be determined by environmental con-
ditions in addition to the genetic characteristic of the strains. The formation of
AFSs and FBP on agar media was noted particularly in the G. lucidum complex,
especially the Korean and Japanese collections, and in G. oerstedii (ATCC
52411, Argentina).
A few reports have described the formation of aberrant fruit bodies of
G. lucidum in vitro (Bose, 1929; Banerjee and Sarkar, 1956; Adaskaveg and
Gilbertson, 1986). Adaskaveg and Gilbertson (1989) reported that G. lucidum
occasionally produced aberrant fruit bodies with basidiospores on agar media.
The basidiospores were formed on red, laccate, coral-like fruit bodies. These
fruit bodies might be AFSs because of similarity in their appearance and in
their ability to form basidiospores. In this case, chlamydospore formation was
observed on the same colony, although the AFS- and FBP-forming isolates
examined by Seo et al. (1995a) did not produce chlamydospores. Furthermore,
chlamydospore-forming isolates formed neither AFSs nor FBP under any of the
conditions examined (Seo et al., 1995a).
Among 30 isolates of G. lucidum collected from Japan, Korea, Papua
New Guinea, Taiwan and the USA, 20 isolates (about 66% of the isolates
tested), none of which was from the USA, formed AFSs with basidiospores, and
another five isolates (about 17% of the isolates tested), none of them from
Papua New Guinea, induced FBP. Of the remaining five isolates, one isolate
from Korea formed a callus-like structure without producing basidiospores,
this structure differing from AFSs and FBP in form, and the other four isolates
from Korea, Papua New Guinea and the USA formed neither AFSs nor FBP.
Among the latter, three strains formed chlamydospores. One isolate did not
form any fruiting structure under standard conditions, but it could produce
AFSs in dual culture with a species of Penicillium known to produce a fruit-
body-inducing substance (Kawai et al., 1985).

Taxonomy of the Ganoderma lucidum Complex
The Ganodermataceae Donk was created to include polypore fungi characterized
by double-walled basidiospores. Large morphological variations in the family
resulted in the description of about 400 species, of which about two-thirds
classify in the genus Ganoderma Karst, many of them belonging to the
G. lucidum complex.
The variable morphological features of the G. lucidum complex, such as the
size, colour and shape of fruit bodies, may be caused by different environmental
conditions during development. Because of the morphological variation in


Norwegian laccate specimens of G. lucidum, Ryvarden (1994) commented that
‘Macro-morphology is of limited value for criterion of species in the G. lucidum
group and at least 3–5 collections with consistent microscopical characters
should be examined before new species are described in this group’.
Cultural characteristics of Ganoderma species have been studied and
employed 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 quite
different from classical identifications based on morphological features. For
example, Nobles (1948, 1958) described the differences in the cultural charac-
teristics of G. lucidum, G. tsugae and G. oregonense. Later, the isolates previously
listed as G. lucidum were changed to G. sessile (Nobles, 1965). However,
Steyaert (1972) and Stalpers (1978) classified it as G. resinaceum. The cultural
characteristics of G. resinaceum given by Bazzalo and Wright (1982) agree with
the description of Nobles (1965) and Stalpers (1978) and the description of
G. lucidum cultures given by Bazzalo and Wright (1982) is very similar to that
of G. tsugae as described by Nobles (1948). Furthermore, Stalpers (1978)
considered that the cultural characteristics of the European G. valesiacum were
identical to those of G. tsugae from North America, and listed it as a synonym of
G. valesiacum. Nobles (1958) suggested that the use of cultural characters in
the taxonomy of the Polyporaceae reflects natural relationships and phylogeny.

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(eds) Ganoderma – Systematics, Phytopathology and Pharmacology. Proceedings
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Vancouver, August 14–21, 1994, pp. 1–2.

2
J.-M. Moncalvo
Systematics of Ganoderma

Systematics of Ganoderma 2
J.-M. Moncalvo
Department of Botany, Duke University, Durham, North
Carolina, USA

Traditional Taxonomy of Ganoderma
Basidiospore shape and structure of the pilear surface have been used as
primary taxonomic characters in mushroom systematics. The family Gano-
dermataceae was erected for polypore mushrooms having a double-walled
basidiospore (Donk, 1964). The type species of the family is Ganoderma lucidum
(W. Curt.: Fr.) P. Karsten, a laccate species described from England. The typical
basidiospore of Ganoderma is ovoid, echinulate and enlarged or truncated at the
apex (Fig. 2.1). Two kinds of basidiocarps producing this type of basidiospore
have been distinguished: those with a shiny (laccate), yellowish or reddish-
brown to black pilear surface, and those with a dull (non-laccate), grey–brown
to black pilear surface. The genus Elfvingia was created to accommodate
non-laccate Ganoderma taxa, with Boletus applanatus Pers. as the type species
(Karsten, 1889). Modern authors (Corner, 1983; Ryvarden, 1991) consider
Elfvingia a subgenus of Ganoderma. Murrill (1905a) proposed the genus
Amauroderma to classify taxa with ganodermatoid basidiospores that differ
from the typical form in having the spore wall uniformly thickened (Fig. 2.1).
Additional genera, subgenera and sections were created on the basis of basidio-
spore shape, type of pilear crust or characteristics of the context tissue (Murrill,
1905b; Imazeki, 1952; Steyaert, 1972, 1980; Zhao, 1989). However, many of
these groups remain controversial (Furtado, 1981; Corner, 1983; Ryvarden,
1991; Moncalvo et al., 1995a).
Table 2.1 shows a classification system for genera and subgenera in the
Ganodermataceae that summarizes the works of earlier authors. 386 names
were created to describe species in the Ganodermataceae. About 60 names


24 J.-M. Moncalvo

should be abandoned for various reasons (Moncalvo and Ryvarden, 1997).
Most species were described in the genus Ganoderma (219 species), mainly
from laccate collections (166 species). Many species are known only from a
single collection or locality. Several names have been considered synonyms
(reviewed in Moncalvo and Ryvarden, 1997), but I believe that more
taxonomic synonyms still exist because a large number of species were

Fig. 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 pilocystidia
found in Ganoderma. (f) Stipitate versus dimidiate basidiocarps: relationships
between 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 of
Genera Subgenera Distinctive features Described single locality as synonyms known species

Ganoderma 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 10–30
Amauroderma Spore wall uniformly large 96 60 41 30–50
Haddowiab Spore wall uniformly large and spore surface 5 1 2 3
longitudinally crested
Humphreyac Spore wall enlarged at the apex and spore 7 3 3 4

surface reticulate
aDatafrom 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. Moncalvo

distinguished 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 surface
colours than basidiocarps that are several weeks or months old: the latter have
been exposed to repeated periods of rain and dryness, covered with dust,
attacked by insects, or even colonized by algae. Presence, absence, size and
insertion 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 be
controlled in vitro by the duration and intensity of exposure to light and by car-
bon dioxide concentration (Hseu, 1990). In vivo, stipe development also
depends on the location in the host: a basidiocarp that develops from a buried
root is more likely to develop a stipe than a basidiocarp that develops higher in
the 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 be
spore shape and size, context colour and consistency, and microanatomy of the
pilear crust. However, the typical spore of G. lucidum is similar for dozens of
different species. Scanning electron microscopy (SEM) has been useful in dis-
tinguishing between spores that appear similar under light microscopy (Pegler
and Young, 1973; Gottlieb and Wright, 1999), and has revealed the existence
of distinctive, slightly longitudinally crested basidiospores in the G. australe and
G. sinense species complexes (Hseu, 1990; Buchanan and Wilkie, 1995; Tham,
1998). Context colour and consistency may change slightly with the age of the
fruit body or upon drying, and are also somewhat subjective characters, but it
is 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 to
dark brown context as in the G. sinense and G. australe complexes; and (iii) very
soft, cream to pale ochraceous context in G. colossum. Relationships between
the microstructure of the pilear crust, the age of the basidiocarp, and the
exposure to environment are not well known, but different types of pilocystidia
and hyphal arrangement can be distinguished among both laccate and non-
laccate taxa (Steyaert, 1980; Fig. 2.1). The laccate appearance of Ganoderma
basidiocarps is associated with the presence of thick-walled pilocystidia
(Fig. 2.1) that are embedded in an extracellular melanin matrix. The exact
origin 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 as
colour or consistency have resulted in the creation of numerous unnecessary
names (synonyms), and a lack of handy identification keys. The absence of
a world monograph has also contributed to problems with species circum-
scriptions and identifications in Ganoderma.
Culture and enzymatic studies have produced additional and useful
taxonomic characters in Ganoderma systematics (Adaskaveg and Gilbertson,
1986, 1989; Hseu, 1990; Wang and Hua, 1991; Gottlieb et al., 1995; Gottlieb


and Wright, 1999). It appears that chlamydospore production and shape, and
to a lesser extent the range and optima of growth temperatures, are extremely
useful culture characters for distinguishing between morphologically similar
species. Mating studies have also been conducted to circumscribe biological
species within species complexes (Adaskaveg and Gilbertson, 1986; Hseu,
1990; Yeh, 1990; Buchanan and Wilkie, 1995). However, all these studies
were restricted in scope, and the techniques employed, although useful at the
species level, have limitations for addressing phylogenetic relationships
between taxa and the development of a natural classification system.

Molecular Systematics of Ganoderma
With recent advances in both sequencing techniques to produce taxonomic
characters and cladistic methods to infer natural relationships between
organisms, molecular systematics has become a paradigm in biology. To date,
the most widely used molecules in fungal molecular systematics have been
the 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 that
Ganoderma belongs to a larger group of white-rot fungi that also includes the
genera Trametes, Fomes, Polyporus, Lentinus, Datronia, Pycnoporus, Cryptoporus,
Daedalopsis, Lenzites and Dentocorticium. Additional phylogenetic studies using
sequence data from the nuclear large ribosomal subunit (25–28S, or nLSU)
rDNA showed that genera Amauroderma, Irpex, Loweporus and Perenniporia
also belong to this group (Moncalvo et al., 2000; Thorn et al., 2000; Moncalvo,
unpublished). Combined evidence of nLSU and mtSSU-rDNA data support the
placement of Amauroderma as a sister genus to Ganoderma (Moncalvo and
Hibbett, unpublished). However, nucleotide sequence data from nuclear and
mitochondrial rDNA encoding sequences do not offer enough variation to infer
phylogenetic relationships between Ganoderma species.
Appropriate nucleotide sequence variation for systematics of Ganoderma
was found in the internal transcribed spacers (ITS) of the nuclear rDNA gene
(Moncalvo et al., 1995a, b, c). The ITS phylogenies produced in these studies
indicated that many names were commonly misapplied (e.g. G. lucidum and
G. 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 trees

A gene tree is not necessarily equivalent to a species tree, and phylogenetic
trees inferred from the sequences of different genes can be contradictory for
several reasons, including differences in their power or level of phylogenetic

28 J.-M. Moncalvo

resolution, incorrect recovery of evolutionary relationships by phylogenetic
reconstruction 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 coalescence
time (Doyle, 1992, 1997; Maddison, 1997), or horizontal gene transfer.
Incongruences between gene trees are more likely to occur at lower taxonomic
levels (species, populations). In fact, it is expected that gene trees are
incongruent among interbreeding individuals because these individuals are
connected by gene flow and recombination: their relationships are therefore
tokogenetic (reticulate) rather than phylogenetic (divergent) (Hennig, 1966;
Doyle, 1997). Overall, a phylogenetic hypothesis is more likely to be correct if it
is supported from multiple, independent data sets rather than from a single
gene tree.

ITS phylogeny versus manganese-superoxide dismutase (Mn-SOD)
phylogeny

Thirty-three Ganoderma taxa were used to conduct separate phylogenetic
analyses of sequence data from ITS and Mn-SOD genes. The incongruence
length 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 the
analyses are shown in Fig. 2.2. Tree topologies are fully congruent for all
nodes having bootstrap statistical support (BS) greater than 50%, with two
exceptions:
1. the type specimen of G. microsporum clusters with G. weberianum
CBS219.36 in the ITS analysis (88% BS), but clusters with a strain labelled
G. cf. capense ACCC5.71 in the Mn-SOD analysis (98% BS); and
2. the cultivar G. cf. curtisii RSH.J2 nests with strain RSH-BLC in the ITS
analysis (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 to
the 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 of
the first two names was already suggested by Peng (1990).
Both data sets strongly support similar terminal clades, and do not fully
resolve basal relationships among Ganoderma taxa. The ITS data set offers
slightly more resolution for deeper branches (Fig. 2.2), whereas higher
sequence divergence between closely related taxa was found in the Mn-SOD
gene (in particular in two introns that were excluded from the analyses
because nucleotide sequences could not be unambiguously aligned across all
the taxa sampled). Ongoing sequencing and analyses of β-tubulin genes also


support similar terminal clades to those from ITS and Mn-SOD data (Moncalvo
and Szedlay, unpublished).
Therefore, preliminary data suggest that phylogenies derived from
ITS sequences are congruent with those from other genes, and that ITS
phylogenies may accurately reflect natural relationships between Ganoderma
species.

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 to
root the trees. Trees depicted are strict consensus trees produced from maximum
parsimony searches. Bootstrap statistical supports greater than 50% are shown
above branches. Mn-SOD data were from Wang (1996; GenBank accession
numbers U56106-U56137), and Moncalvo and Szedlay (unpublished). Analyses
were conducted in PAUP* (Swofford, 1998) and employed maximum parsimony
with heuristic searches using 50 replicates of random addition sequences with
TBR branch swapping. Bootstrap statistical supports were evaluated with 100
bootstrap replicates of random addition sequence with TBR branch swapping.
Regions with ambiguous alignment were removed from the alignment, and
unambiguously aligned gaps were scored as ‘fifth character state’. The ITS
data set used 81 parsimony-informative characters and produced 24 equally
parsimonious trees of length 232, with a consistency index of 0.703. The SOD
data set used 105 parsimony-informative characters and produced 58 equally
parsimonious trees of length 329, with a consistency index of 0.623.

30 J.-M. Moncalvo

ITS phylogeny

The current ITS sequence database for Ganoderma and Amauroderma species
includes about 300 taxa. Numerous small nucleotide insertions and deletions
make sequence alignment problematic in several regions, but at least 380
characters can be aligned unambiguously across the entire data set, yielding
about 200 parsimony-informative characters. Phylogenetic analysis of large
molecular data sets is still a controversial field (Lecointre et al., 1993; Hillis,
1996; Graybeal, 1998; Poe, 1998). One commonly encountered problem with
large data sets concerns the applicability and/or accuracy of standard
descriptors commonly used to assess branch robustness. For instance, the use
of branch decay indices (Bremer, 1994) is not practical for large data sets
because of the large number of trees that cannot be sampled; and consistency
indices (Sanderson and Donhogue, 1989), bootstrap (Felsenstein, 1985) and
jackknife (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 still
useful tools to determine the robustness of clades.
Parsimony analyses of ITS data for 248 Ganoderma taxa reveal about
50 clades with bootstrap statistical support greater than 50% (Fig. 2.3 and
Table 2.2), that are also consistent with morphological and/or geographical
data. Terminal clades in this phylogeny represent either a population, a
species, a species complex, or a group of closely related species. In Table 2.2,
tentative names for the most well-supported clades are proposed, although
16 clades have not been named (the original data set included 36 species
names and many unnamed taxa). Basal relationships are either poorly
supported or unresolved, but phylogenetic analyses of various data sets
using maximum parsimony and maximum likelihood consistently reveal
three larger groups: these are labelled Groups 1–3 in Fig. 2.3 and Table
2.2.
ITS phylogeny suggests that the laccate habit has been derived more
than once (or lost several times), making the laccate Ganoderma taxa
polyphyletic. This conflicts with traditional systems of classification that
accommodate laccate and non-laccate Ganoderma taxa in subgenera
Ganoderma (laccate) and Elfvingia (non-laccate), respectively (see Table 2.1).
However, within the Ganodermataceae, there is already evidence for
non-monophyly of laccate taxa because at least three laccate species have
been traditionally classified in genus Amauroderma (Furtado, 1981). A revised
classification for subgenera and sections in Ganoderma seems therefore
necessary, and will be formally proposed elsewhere. For now discussion is
limited to some taxonomic groupings revealed by ITS sequence data, as
summarized in Table 2.2.


Fig. 2.3. Internal transcribed spacer (ITS) phylogeny for 248 taxa of Gano-
dermataceae (sequences from several Amauroderma species were used to root the
tree). The tree depicted is one of 100 equally parsimonious trees produced using
maximum parsimony in PAUP* (Swofford, 1998) with heuristic searches, random
addition sequences (100 replicates), TBR branch swapping, and MAXTREES set
to 100. Statistical supports for branch robustness were evaluated in PAUP* with
100 bootstrap replicates, random addition sequence, TBR branch swapping, and
MAXTREES set to 10. Bootstrap values are only given for branches in bold that
refer to groups or clades that are presented in Table 2.2. Groups 1 and 1.4 are not
monophyletic 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.

32

Table 2.2. Groupings of Ganoderma taxa based on a phylogenetic analysis of ITS nucleotide sequence data (Fig. 2.3), with geographic
origin 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
Conifers

Group 1
1.1 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.

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
Conifers

1.3 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. • •
G. tropicum complex s. stricto (G. fornicatum) (58% BS) • • • •
Group 2

2.1 ‘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. (’G. cf. tornatum’) (63% BS) • •
33

Continued

34



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
Palms

2.3 G. cf. balabacense (98% BS) • • •
2.4 Ganoderma sp. • •
2.5 G. sinense (100% BS) (= G. formosanum, = ?G. neojaponicum) • • •
Group 3
J.-M. Moncalvo

G. 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) • • • • • • •


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
Conifers

Unclassified
G. applanatum B (98% BS) • • •
Ganoderma sp. (100% BS) • • •
G. tsunodae (Trachyderma) • •
G. colossum (Tomophagus) • • • • •

Names in parentheses are commonly misapplied names (in ‘quotes’), synonyms (=) or possible alternative names. Frequency values (% BS) following taxa
names 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’ includes
collections from Bali, Malukku, and Papua New Guinea; ‘S. America’ includes collections from Argentina and Chile; ‘Neotropics’ includes collections from
Costa Rica, Puerto Rico, Equador, and French Guyana.
35

36 J.-M. Moncalvo

Phylogenetic Relationships and Biogeography in Ganoderma
Phylogenetic relationships

Group 1: the G. lucidum complex sensu lato
Group 1 is either monophyletic or paraphyletic, and includes G. lucidum sensu
stricto and many other similar laccate Ganoderma taxa, of which several
collections were incorrectly identified as G. lucidum. In this group, basidiospore
shape and size is very uniform, and taxa generally have a reddish to
dark-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 discussed
below.

GROUP 1.1: THE G. LUCIDUM COMPLEX SENSU STRICTO. The G. lucidum complex
sensu stricto includes only collections from temperate regions of both the
northern and southern hemispheres. Members of this group do not produce
chlamydospores in culture. ITS sequence variation among taxa of this clade is
very low and does not allow for subdivision into smaller entities. European
taxa of this clade (G. lucidum, G. valesiacum and G. carnosum) might be
conspecific (Ryvarden and Gilbertson, 1993): G. valesiacum was primarily
distinguished from G. lucidum based on host specificity (conifers versus
hardwood, respectively; Ryvarden and Gilbertson, 1993), but a recent study
by Ryvarden (1995) suggests that G. lucidum in Norway grows on both
hardwood and conifers; G. carnosum (= G. atkinsonii) has been reported only
on conifers, and is distinguished from both G. lucidum and G. valesiacum by
having 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 herbarium
have been examined, and all can be distinguished from typical G. lucidum in
having a less shiny pileus and a darker context, which is entirely brown and
duplex. The two North American taxa of this clade (G. tsugae and G. oregonense)
are believed to be restricted to conifers and might be conspecific (Gilbertson
and Ryvarden, 1986). Basidiocarps of G. lucidum from Europe and G. tsugae
from the USA are practically impossible to distinguish. The Argentine
collections 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 species
described from Europe, is differentiated from G. lucidum by having smoother
spores (Steyaert, 1972; Pegler and Young, 1973). European G. resinaceum has
been shown to be intercompatible with collections generally assigned to
‘G. lucidum’ in North America (Adaskaveg and Gilbertson, 1986), suggesting
conspecificity of these isolates. However, ITS data distinguish between popula-
tions of G. resinaceum from Old World (Europe and Africa), North America,


and South America; these populations might therefore be completely disjunct
and genetically isolated from each other, and may warrant recognition at the
species level. However, additional sampling and more extensive mating studies
are 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 from
G. resinaceum by having smaller spores (6–9 × 4–7 µm).
Based on ITS data, G. trengganuense also belongs to this clade. This species
is known from Malaysia and Vietnam and is well characterized in having
subreticulate spores (Corner, 1983), but is similar to G. resinaceum in the other
characters.

GROUP 1.3: THE G. CURTISII COMPLEX. Members of this clade do not produce
chlamydospores in culture. This well-supported clade (75% BS) can be divided
in two groups which correspond to the geographic origin of the collections.
One group is composed of collections from eastern North America and Costa
Rica. These collections can be identified as G. curtisii (a species described from
eastern America) based on descriptions in Lloyd (1912, 1917) and Steyaert
(1972, 1980), and G. meredithae (Adaskaveg and Gilbertson, 1988) can be
considered a taxonomic synonym. The sister group of these taxa is represented
by 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 heterogeneous
and may not be monophyletic, but is retained here for convenience. Members
of this group have been collected throughout tropical and subtropical regions.
Only a few taxa have been examined in culture, and they all produced
chlamydospores. In this group, several distinct, well-supported clades revealed
by ITS data are also supported by differences in basidiocarp or culture
characteristics. 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 few
collections, and the correct naming of species remains problematic.

38 J.-M. Moncalvo

Group 2
Group 2 includes laccate taxa easily distinguished from G. lucidum sensu lato
by a difference in spore shape (e.g. elongated spores in G. zonatum and
G. boninense), and/or by a darker pileus and/or context colour (e.g. black
pileus and uniformly brown context in G. sinense). This group also includes
non-laccate (or ‘sublaccate’?) taxa. Group 2 is mostly composed of tropical and
subtropical collections, but also includes collections from temperate Japan,
Korea and China. Strains placed in group 2 that have been examined in culture
did not produce chlamydospores.

GROUP 2.1: THE PALM CLADE. A well-supported clade (74% BS), composed only
of collections from palms, which can be divided into three smaller groups
corresponding to the geographic origin of the strains: (i) G. zonatum from
Florida; (ii) G. boninense from South-East Asia, the Australo-Pacific region and
Japan; and (iii) unidentified collections from Zimbabwe and India. G. zonatum
and 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, and
the pilear crust is thinner. Additional sampling and mating studies will be
necessary to determine the robustness of the geographic structure, delimit
species boundaries, and to evaluate specificity on palms.
A sister group to the G. zonatum-boninense clade comprises collections
from Vietnam, Malaysia, Thailand and Australia, from both palm and woody
dicots. These collections differ from G. zonatum and G. boninense in having a
black pileus and ovoid spores. SEM revealed that basidiospores of the Vietnam
collection are longitudinally striate (Tham, personal communication). These
collections 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 be
named. All have a uniformly brown context. Basidiocarps collected in Costa
Rica and Puerto Rico have a shiny black pileus, and a white pore surface
that turns dark brown upon ageing. Basidiocarps from Vietnam (originally
identified as ‘G. tornatum’, a non-laccate taxon) and Yunnan are dull, greyish
to 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 collection
from Vietnam with a shiny, yellow–brown to dark-brown pileus and a brown
context, identified as G. cf. balabacense by its collector (Dr Le Xuan Tham), and
one 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 within
Group 2, apart from all the other taxa.


GROUP 2.5: THE G. SINENSE COMPLEX. Th1is clade includes collections from
China, Taiwan and Korea. Chinese collections correspond to G. sinense, a
species described from China. It has a distinctive, shiny black pileus and a
brown to dark-brown context (Zhao, 1989). The Taiwan collection included
in 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 other
collections of this clade. The Korean collection was received from Dr Dong-Suk
Park as G. neojaponicum. Both G. sinense and G. neojaponicum are black and
laccate taxa with a brown context, but whether or not the two names are
synonyms remains to be investigated.

Group 3: the G. australe-applanatum complex
Group 3 comprises the bulk of non-laccate taxa of the G. australe-applanatum
complex (subgenus Elfvingia in Table 2.1), but also includes a laccate species
from Europe: G. cupreolaccatum (= G. pfeifferi). All members of this group lack
chlamydospores in culture.
The placement of G. cupreolaccatum in the G. australe-applanatum complex
is surprising, but this species differs from other laccate species (especially from
those in Group 1) in having a dark-brown context, very similar in colour and
consistency to that in G. australe and G. applanatum. It is also interesting to note
that the culture strain CBS250.61 identified as ‘G. applanatum’ by K. Lohwag
classifies in G. cupreolaccatum based on ITS sequence data. Careful examination
of G. cupreolaccatum collections shows that in older basidiocarps the pileus
surface turns greyish-black and is not very shiny; various encrustations and
erosion of the melanin wax of the crust may alter the laccate appearance of the
basidiocarps, which then would more closely resemble those of G. applanatum
or G. australe.
Although most collections belonging to this group were originally
identified G. australe or G. applanatum, some collections were also assigned to
G. tornatum, G. adspersum, G. lobatum, G. philippii, G. pseudoferreum, or G.
gibbosum. These names are scattered inconsistently (if not randomly) in the
ITS phylogeny, demonstrating the limitations of morphological taxonomy
in this species complex. A large amount of ITS sequence divergence was found
in this group (see branch length in Fig. 2.3), and several smaller clades can be
distinguished.
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 was
first described from Europe, and G. australe from a Pacific island). The remain-
ing clades do not include European collections, and are provisionally grouped
under the name ‘G. australe complex sensu stricto’. On the basis of ITS sequence
data, this complex can be subdivided further into at least three well-supported
clades, 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. Moncalvo

in that clade neotropical collections are distinct from Old World collections;
Clade B is composed only of collections from the southern hemisphere; and
Clade C includes collections from Asia and the southern hemisphere. Mating
data produced by Yeh (1990) and Buchanan and Wilkie (1995) indicate at
least two intersterile groups of ‘G. australe’ in Taiwan and New Zealand,
respectively. Mating data and ITS phylogeographic patterns suggest several
genetically isolated groups (species) in the G. australe complex.

Unclassified taxa

‘G. APPLANATUM B’. A strongly supported clade (98% BS) composed of
non-laccate collections from Europe and eastern North America remains
unclassified: it clusters at the base of Groups 2 and 3 in Fig. 2.3, but also nests
at the base of Group 1 in some analyses. Because this clade includes
non-laccate taxa from Europe, it is provisionally named as ‘G. applanatum B’.
ITS data support the view that at least two non-laccate species exist
in Europe (Pegler and Young, 1973; Ryvarden and Gilbertson, 1993). Either
‘G. applanatum A’ or ‘G. applanatum B’ represents the true G. applanatum. The
two clades can not be distinguished from basidiocarp characteristics. Also,
since these ITS-based clades are so far composed only of northern temperate
collections (Table 2.2), it is possible that G. applanatum sensu stricto only occurs
in the temperate regions of the northern hemisphere.

G. TSUNODAE, G. COLOSSUM AND OTHER TAXA. G. tsunodae, known only from
Japan (Imazeki, 1952) and China (Zhao, 1989), and G. colossum, a pantropical
species (Ryvarden and Johansen, 1980), remain unclassified. They are on long
branches in ITS phylogenies, generally at the base of the trees, and both might
warrant segregation into separate genera as proposed by Imazeki (1939) and
Murrill (1905b). Several unidentified taxa also remain unclassified: for
instance, a non-laccate species collected in French Guyana and Puerto Rico,
that is easily recognizable from the cinnamon colour of its context, and laccate
collections from Zimbabwe and Vietnam, with a reddish-brown to blackish
pileus and dark-brown context.

Biogeography

The number of known Ganoderma species can be estimated at about 60–80
laccate and 10–30 non-laccate species (Table 2.1), and it is likely that new
taxa are yet to be discovered in poorly studied tropical regions. These numbers
are based on a literature survey, examination of type specimens, numerous
field collections in various regions of the world, molecular phylogenetic data
and, in some cases, mating data. On a similar basis, it can be estimated that the
current sampling of ITS sequences encompasses at least 80% of all known taxa
from temperate regions, about half of the taxa from South-East and eastern


Asia (it would seem that the number of species described from China by Zhao
and his collaborators (Zhao, 1989) has been overestimated), and 20–40% of
neotropical taxa. Molecular data from African material is almost entirely
lacking.
Based on these sampling estimates and the ITS phylogeny summarized
in Fig. 2.3 and Table 2.2, it appears that Ganoderma taxa repeatedly show
similar 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, Papua
New Guinea and Australia), and connection between the more tropical regions
of the southern hemisphere (northern Australia and Papua New Guinea) and
South-East Asia.
Current ITS data indicate the existence of 5–7 species in Europe and 7–8
in North America; these estimates are in agreement with the more recent
traditional floras for these regions (Gilberston and Ryvarden, 1986; Ryvarden
and Gilbertson, 1993), although there is still some disagreement between ITS
and morphological data in circumscribing and naming taxa. ITS phylogeny
identifies at least 12 taxa in temperate and subtropical Asia (China, Korea,
Japan and Taiwan), but more species probably exist in this area. Within
undersampled and species-rich regions, Table 2.2 indicates the presence of at
least 18 ITS-based taxa in tropical Asia, and eight in the Neotropics.
Taxa from Africa remain poorly sampled in molecular studies. The
unidentified taxa from South Africa and Zimbabwe that were included in
this work are diverse, and either nest in isolated positions or cluster with
European or Asian strains. A high level of taxonomic diversity (and perhaps
also endemism) is expected in Africa, because several well-characterized
species have not been reported outside that continent, e.g. G. alluaudii
(Ryvarden, 1983), G. chonoides (Steyaert, 1962), G. sculpturatum (Ryvarden
and Johansen, 1980), G. hildebrandii (Moncalvo and Ryvarden, 1995), etc.

Host relationships

Host specificity has been used to circumscribe Ganoderma taxa. In the northern
temperate regions G. valesiacum, G. carnosum, G. tsugae and G. oregonense have
been distinguished from G. lucidum, mainly because they are all believed to
be 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 conclusion
can be reached about host specificity on conifers, there is still need for a
better understanding of species boundaries in clade 1.1; collections from
conifers at higher altitudes in tropical or subtropical regions should also
be 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 also
indicates the presence of 1–3 non-laccate species growing on palms, but again
these results differ slightly from Steyaert’s (1967) concerning the geographic
distribution and host specificity. The ITS phylogeny also strongly suggests a
single origin (monophyly) for four out of the five laccate taxa growing on
palms (Table 2.2).

Conclusions
The data presented here show that ITS-based clades are generally consistent
with morphology or geography. Species boundaries within ITS clades still need
to be addressed with mating studies, multigene phylogenies, or both. Type
specimens must be studied where available before naming ITS clades in the
Linnean system of classification. However, given the difficulties of taxonomic
identification of Ganoderma collections using traditional methods, the ease and
reducing costs of PCR amplification and direct sequencing techniques, and the
rapid expansion of molecular databases for a broad array of fungi, molecular
methods might become the easiest way to identify Ganoderma and other
problematic fungal strains. This is particularly appealing for the preventive
care of woody plant crops, because vegetative mycelia extracted from wood
could be identified quickly using molecular techniques. Sequence data used in
this study will be made available in both GenBank and the Internet address
http://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.

Acknowledgements
I am grateful to the Department of Botany at Duke University for financial
support through a grant from the A.W. Mellon Foundation. The following
persons provided strains for this study: Cony Decock, Le Xuan Tham, Faridah
Abdullah, Carmel Pilotti, Alexandra Gottlieb, Armando Ruiz Boyer, Monica


Elliott, Brendan Smith, Dong-Suk Park, C.L. Bong, Paul Kirk, Tom Harrington,
Maggie Whitson, Anne Pringle, and Rytas Vilgalys. Thanks to Chiquita and Bill
Culbertson and Jim Johnson for comments on an early draft of the manuscript.

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Ganoderma, Diseases of II
Perennial Crops

3
D. AriffinGanoderma in Oil Palm
Status of et al.

Status of Ganoderma in Oil 3
Palm
D. Ariffin1, A.S. Idris1 and G. Singh2
1Palm Oil Research Institute of Malaysia, Persiaran Institute,
Bangi, Kuala Lumpur, Malaysia; 2United Plantations Berhad,
Jenderata Estate, Perak, Malaysia

Introduction
The oil palm, Elaeis guineensis, is the highest yielding among the oil-producing
crops. It commands an average yield of about 4 tonnes oil ha−1 year−1. In
1997, close to 17.8 million tonnes of palm oil were produced world-wide, with
Malaysia and Indonesia producing the most at 9.07 million tonnes and 5.36
million tonnes, respectively.
Like other crops, oil palm is also prone to attack by a number of diseases;
one of the most important of which is basal stem rot (BSR). BSR disease, caused
by species of Ganoderma, is the most serious disease of oil palm in Malaysia
and Indonesia. The genus Ganoderma has a world-wide distribution, growing
on numerous perennial, coniferous and palmaceous hosts. Some Ganoderma
species are wood-rotting fungi, a number being pathogenic and thus harmful
on economically important trees and perennial crops. In addition to oil palm,
species of Ganoderma are the causal agents of root and stem rots of many other
plantation crops, including coconut, rubber, betelnut, tea, cocoa, peaches
and pears, guarana, grapevines and forest trees such as Acacia, Populus and
Macadamia. In forest systems, Ganoderma has an ecological role in the
breakdown or delignification of woody plants.

Geographical Distribution of BSR Disease
Basal stem rot of oil palm has been recorded in Malaysia and Indonesia
in South-East Asia; Angola, Cameroon, Ghana, Nigeria, Zambia, San Tome,


50 D. Ariffin et al.

Principe, Tanzania, Zimbabwe and the Republic of Congo in Africa; Honduras
in Central America, and Papua New Guinea in Oceania (Turner, 1981). More
recently, 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, West
Africa (Wakefield, 1920). Thompson (1931) detected the disease infecting oil
palms of over 25 years in Malaysia but because this attack was on old palms
due for replanting, BSR was considered not to be economically important
(Turner, 1981). However, towards the later years of the 1960s, when oil palm
began to assume prominence as a plantation crop, BSR incidence was on the
increase and much younger palms (10–15 years old) were infected (Turner,
1981). Recently, Ganoderma has been found to infect oil palms as early as
12–24 months after planting, with increased incidence on 4–5-year-old
palms, particularly in replanted areas (Singh, 1991) or areas underplanted
with 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 of
coconut left in the soil and infects the oil palm on replanting. High incidence of
BSR disease was recorded on oil palm planted in coastal soil in west Peninsular
Malaysia (Khairudin, 1990a). In peat soils, which were at one time thought
to be non-conducive to BSR disease (Turner, 1981), serious incidences of the
disease have been reported more recently (Ariffin et al., 1989c; Rao, 1990).
Ariffin et al. (1989c) cautioned that Ganoderma poses a threat to oil palm
planting in peat soil, where high incidences of the disease have been observed
at a relatively young age, irrespective of previous cropping history (Table 3.1).
The incidence of BSR disease in inland soils in Malaysia remains relatively
low and seems to be confined only to waterlogged areas (Khairudin, 1990a).
However, it was recently reported that serious BSR disease incidence can occur
on 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 but
increases 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 in

Table 3.1. Incidence of basal stem rot (BSR) disease in peat soil (from Ariffin
et 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 51

Malaysia, BSR incidence is very high in replants in both inland podsols and
coastal clay soils (Hasan and Turner, 1998).
In West Africa, BSR is widespread in wild groves and is the common cause
of death of wild palms (Robertson et al., 1968). Most of the affected palms are
over 25 years old, but palms 10–15 years old are also infected. With the active
conversion of wild groves to plantations in eastern Nigeria, the incidence of
BSR is expected to be on the increase (Oruade-dimaro et al., 1994).
The incidence of BSR is comparatively low in Honduras where the disease
was detected in palms more than 12 years old (Chinchilla and Richardson,
1987). BSR is also beginning to occur in Colombia (Nieto, 1995) and Papua
New Guinea (Sanderson and Pilotti, 1997a, b).

Disease Symptoms
In young palms, the external symptoms of BSR normally comprise a one-sided
yellowing, 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 may
take on an overall pale appearance, with retarded growth and the spear leaves
remaining unopened.
Similar symptoms are observed in mature palms, with multiple unopened
spear leaves and a generally pale leaf canopy. Affected leaves die, necrosis
beginning in the oldest leaves and extending progressively upwards through
the crown. Dead, desiccated fronds droop at the point of attachment to
the trunk or fracture at some point along the rachis, and hang down to
form a skirt of dead leaves. Often, when foliar symptoms are observed, it is
usually found that at least one-half of the basal stem tissue has been killed by
the fungus. Infected young oil palms normally die within 6–24 months after
the first appearance of symptoms but mature palms can take up to 2–3 years
to 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 rotting
tissues, marked by darker irregular zonations with an outer edge of an
irregular yellow zone. A yellow zone is found between the lesion edge and the
healthy tissues. Turner (1981) termed the darker zones as ‘reaction zones’ and
speculated that the yellow zones were the result of some defence mechanism of
the palm to infection. These narrow darker zones were termed ‘black lines’ by
Ariffin et al. (1989a), and embedded within the lines were masses of swollen
hyphal cells which appear to be resting structures. Within the light-brown
diseased tissues, small cavities of white fungal mycelium were seen. Oil palm
extensively decayed by Ganoderma may fracture at the base and the palm
collapses, leaving diseased bole tissues in the ground. Subsequently, numerous
Ganoderma basidiomata are produced, especially during the rainy season. If the
palm remains standing, the trunk may become hollow.


Roots of affected palms are very friable and their internal tissues become
very dry and powdery. The cortical tissue is brown and disintegrates easily
and the stele becomes black in colour. In older roots, the fungus may be present
as a whitish, mat-like layer on the inner surface of the exodermis (Singh,
1991).
Ganoderma basidiomata or sporophores may or may not develop before
foliar symptoms appear. Basidiomata may develop at the stem base of the
trunk, leaf base or occasionally on infected roots close to the palm, and it is
the appearance of these that is most diagnostic of the disease. The timing of
basidiomata appearance depends on extension of the internal rotting to the
stem periphery. The basidiomata initially appear as small, white buttons of
fungal tissues which develop rapidly into the familiar bracket-shaped mature
basidiomata, varying in shape, size and colour. The upper surface can be light
to dark brown, with a light margin and a shiny lacquered finish. The under
surface is whitish in colour and has numerous minute pores. Frequently, many
basidiomata are formed close together, with overlapping and fusion to form
large, compound structures. The location of the basidiomata provides a rough
guide 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 of
basidiomata along its entire length.

Causal Organisms
In West Africa, the pathogen was originally identified as G. lucidum Karst
(Wakefield, 1920), whereas in Nigeria, four species of Ganoderma have been
identified as causal agents, namely G. zonatum Muril, G. encidum, G. colossus
and G. applanatum (Pers. ex. S.F. Gray) (NIFOR, 1978). In Malaysia, it was also
originally identified as G. lucidum by Thompson (1931), a species commonly
found in temperate regions that has been associated with diseases of a number
of hosts, such as coconut and Areca and also grapevines. Turner (1981) listed
15 species of Ganoderma that have been recorded from different parts of the
world as likely pathogens to be associated with BSR disease, and he considered
that 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. and
Steinmann, and G. tornatum (Pers) Bres. were reported from Peninsular
Malaysia. Ho and Nawawi (1985) concluded that all Ganoderma isolates
from diseased oil palm from various locations in Peninsular Malaysia were
all the same species, G. boninense. These were based on the morphology of
basidiomata collected from oil-palm fields ranging from 5 to 40 years of age.
Ariffin et al. (1989c) suggested that other species may be involved and
Khairudin (1990a) concluded that two species were present, namely G.
boninense and G. tornatum. More recently, Idris (1999) classified Ganoderma in


oil palm in Malaysia into types A, B and C. Type A is the most aggressive, type B
is less aggressive, while type C is saprophytic.

Economic Importance
Field 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 the
time the palms reach 25 years and are ready for replanting, the incidence
could be as high as 25% (Singh, 1991). In replanting from coconut, the disease
appears much earlier, with sporadic cases of BSR as early as 1–2 years after
planting. By the twelfth year, the incidence is more than 15%, increasing to
60% 4 years later (Singh, 1991). In replanting from oil palm, the incidence of
BSR 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 oil
palm to oil palm replant by underplanting. In this case, the incidence reached
33% at 15 years. A BSR incidence of 25% was recorded on 10-year-old palms
planted under coconut (Ariffin et al., 1996). Two years later the incidence had
increased to 40%.
Losses due to BSR can occur not only through the direct reduction in
oil-palm numbers in the stand, but also through a reduction in the number
and weight of fruit bunches from standing diseased palms and those with
subclinical infections (Turner, 1981). Yield compensation by healthy neigh-
bouring palms is likely to occur and, according to Turner (1981), disease levels
of 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 a
high incidence of BSR and the other with a low incidence – is presented
in Table 3.2, as reported by Singh (1991). The fields selected were within

Table 3.2. Basal stem rot (BSR) incidence and fresh fruit bunch (FFB) yield (from
Singh, 1991).

Low BSR incidence blocka High BSR incidence blockb

Years 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 13.2
aPlanted 1975; previous crop: rubber; soil type: Selangor/Briah Assoc.
bPlanted 1975; previous crop: oil palm; soil type: Selangor series.


the same estate, of the same age and on similar soils. It was shown that FFB
production was adversely affected by the disease incidence.

Epidemiology
Mycelium contact

It has been generally accepted that natural infection with Ganoderma in oil
palm occurs as a result of contact between healthy roots and diseased tissues
left buried in the soil (Turner, 1965c). Infection by Ganoderma is also believed
to occur through wounded tissues or dead roots. The fungus then grows
along the infected root and eventually reaches the bole of the palm trunk.
Histopathological investigations of roots naturally diseased by infection with
Ganoderma 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 to
the endodermis. The fungus is not restricted to any one particular tissue type at
the advanced stages of pathogenesis; fungal hyphae could be clearly detected
in the xylem, phloem, pith and parenchymal cells. Infection of the stem
eventually led to the formation of ‘black lines’ within the infected tissues
(Ariffin et al., 1989a). The presence of these lines could be observed with the
naked eye. On microscopic examination with suitable staining techniques it
was observed that Ganoderma hyphae transform into thick-walled, swollen
structures embedded within the black lines. It was postulated that these
might be resting structures which could possibly play an important role in the
long-term survival of the pathogen in soil. In this form, Ganoderma might have
developed a resistant barrier against other soil microorganisms in which
normal free hyphae would have easily been replaced.

Ganoderma basidiospores

Vegetative compatibility studies made by Miller (1995) and Ariffin et al.
(1996), indicated that basidiomata collected from the same field, or from
within the same area of oil-palm field, might not have originated from the same
source of inoculum, implying that root-to-root spread or mycelial growth
might not be the sole method of spread of BSR. Currently, the role of Ganoderma
basidiospores in disease initiation and spread of infection is unclear. Although
huge numbers of basidiospores of Ganoderma are released from basidiomata in
the oil-palm field (Ho and Nawawi, 1986), the majority of oil palms remain
uninfected, indicating that basidiospores either may not be able to initiate a
BSR infection or require very specific conditions to establish infection.
Studies based on the artificial inoculation with basidiospores and
inoculum size suggest that basidiospores have inadequate inoculum potential
for direct infection of a living oil palm (Turner, 1981). Their function in disease


development seems to be the colonization of suitable substrates, particularly
cut stumps of trunks of trees or palms left to rot in the field, which may become
infection foci. Inoculation of cut young leaf bases (Turner, 1965a) and young
oil-palm seedlings with spores failed to produce any infection (Ramasamy,
1972; PORIM, 1988). Sharples (1936) believed that spores do not play an
important role in the spread of the disease. However, Thompson (1931) was
of the opinion that spores are important in initiating the disease in first-
generation oil palms on cleared virgin jungle areas. Basidiospores, which
may either be wind-borne or insect-transmitted, would first have to colonize
suitable substrates, e.g. dead coconut or oil-palm stump, and then they could
germinate readily and spread throughout the whole stump. It was suggested
that spores may enter through beetle holes, caused by Oryctes beetle (Turner,
1981). Caterpillar larvae of Sufetula spp. may also be important in spreading
spores of Ganoderma (Genty et al., 1976). However, no conclusive evidence has
been presented linking insects and BSR incidence and development.

Predisposition Factors Associated with BSR Disease
Until recently, predisposition factors that influence the development of BSR
disease have been the subject of speculation based on circumstantial evidence.
A number of factors – age of palms, previous crops, types of soils, nutrient
status and technique of replanting – have been reported to influence BSR
disease development in the field. Infection by the pathogen has generally been
thought to occur through a weakening of the oil palm so that it becomes pre-
disposed to infection. However, with information now available, predisposition
factors can be examined critically.

Age of oil palms

BSR was first reported to be a disease of old, senescing oil palms, i.e. the palms
affected were those over 25 years from planting, and this was thought to be due
to a senescence factor that broke down the immunity barrier (Turner, 1981).
However, with time this trend had changed, with much younger oil palms
becoming infected (Singh, 1991; Khairudin, 1993). As reported by Turner
(1981), the age at which a palm becomes infected will depend on: (i) the rate of
colonization of the tissues of the previous stand; (ii) proximity of the colonized
tissues to the oil palm; (iii) time taken for roots to make contact with the tissues
and 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 appear
from the sixth year after planting, and then increases rapidly from the eleventh
year onwards (Table 3.3). It was suggested that, in the field, the opportunity
for roots to come into contact with disease inoculum, and subsequent slow
disease development, are more critical than age factors (Khairudin, 1993).


Table 3.3. Incidence of basal stem rot (BSR) disease in relation to oil-palm age in
four Golden Hope Plantation Estates in Peninsular Malaysia (from Khairudin, 1993).

BSR incidence (%)

Golden Hope Plantation 0–5 6–10 11–15 16–20 > 20
Estates yearsa years years years years

Melentang, Bagan Datoh 0.7 0.4 4.6 44.6 43.3
Chersonese, Sg. Krian 0.0 14.0 12.4 25.2 35.8
Dusun Durian, Banting 0.0 2.1 12.8 24.1 24.9
West, Carey Island 0.0 0.4 2.5 9.7 18.9
aYears after planting.

Previous crops

The relationship between BSR disease of oil palm and the types of former
crops has been recognized (Turner, 1965a). Severe outbreaks of BSR disease
occurred in areas when oil palm followed coconut, especially where the stumps
had been retained in the ground. With planting following coconut, Ganoderma
infection may become apparent as early as 12–24 months from planting, but
more usually when palms are 4–5 years old (Singh, 1991). Thereafter, the
incidence can reach 40–50% by the time the palms are 15 years old (Table
3.4). A similar situation was also reported where oil palm was replanted from
oil palm – a high incidence of BSR could be observed after 15 years of planting.
A contrasting situation was apparently found in stands planted from
jungle or rubber, with a low disease incidence and losses only beginning to
occur after 10–12 years (Turner, 1965b). However, later reports indicated
that the previous crop did not exclusively preclude high incidences of
BSR, which have also occurred in ex-rubber plantings (Ariffin et al., 1989c)
and ex-pineapple plantings (Ariffin et al., 1989c; Rao, 1990). A more recent
study conducted on four estates covering about 8000 ha showed that there is
no definite relationship between former crop and BSR incidence (Khairudin,
1993) and the presence of an adequate inoculum source could be a more
important prerequisite to high disease level.

Types of soil

A high incidence of BSR disease has been frequently reported to be prevalent in
coastal areas (Navaratnam, 1964; Turner, 1965d). Khairudin (1990a) also
reported 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 to
remain confined to the coastal areas, indicated that the nature of soil and its


Table 3.4. Incidence of basal stem rot (BSR) disease in oil palm in relation to
previous 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 are
mainly clays, silty clays or clay loams with poor internal drainage and with a
high water retention capacity. However, more recent reports indicate a greater
incidence of BSR disease on oil palms planted on inland soils, especially
Holyrood, Sungei Buloh, Rasau and Bungor series (Khairudin, 1990a); Batu
Anam/Durian series and Munchong series (Benjamin and Chee, 1995); peat
soil (Ariffin et al., 1989c; Rao, 1990) and lateritic soil, especially Malacca series
(Benjamin and Chee, 1995). Increasing reports of BSR disease in different soil
types, including inland soils, requires further investigation of the role of soil
type in determining the level of disease in the oil-palm fields.

Nutrient status

Soil nutrition can influence disease development, but the effect appears to be
related 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-Selangor
association (Sulfic tropaquept) showed that rock phosphate and muriate of
potash (KCl) significantly increased disease incidence, whereas urea had a
reduced effect (Singh, 1991). In another trial on a recent marine alluvium of
the Bernam series (Typic tropaquept), Singh (1991) reported that muriate
of potash significantly reduced disease incidence, whereas urea and rock


phosphate had a slight promotive effect. In Indonesia, high sodium content
(Dell, 1955) and low nitrogen levels (Akbar et al., 1971) have both been
associated with raised disease levels, but both high (Dell, 1955) and low
magnesium contents (Akbar et al., 1971) have been linked with increased
incidence of disease, so the situation is unclear. In one investigation of the
major elements, nitrogen (N), potassium (P) and phosphorus (K) were all
significantly higher in healthy tissues, but levels of magnesium (Mg) were
higher 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 from
inland and coastal soils did not show any marked differences in the levels of
elements, but oil-palm roots collected from inland soil were found to contain
high levels of phosphate (P), zinc (Zn) and iron (Fe) (Singh, 1991).

Planting techniques

The incidence of BSR disease has been observed under a range of replanting
techniques. Turner (1965a) reported that there is a close relationship between
disease incidence and the replanting techniques adopted. A trial carried out by
Golden Hope Plantations Berhad, comparing the effect of different replanting
techniques on the incidence of BSR, showed that underplanting would
eventually lead to a high disease incidence (from 27.3% in the previous
stand to 33% in the replanted stand after 15 years), whereas if clean clearing of
previous oil-palm stands was employed, subsequent disease levels were low
(from 27.3% to 14.0%), and windrowing slightly increased the risk of BSR
disease incidence (from 27.3% to 17.6%) (Table 3.5) (Khairudin 1990b).

Early Detection of BSR
Diagnosis of Ganoderma infection in oil palm is based on the appearance of
multiple spear leaves and the presence of basidiomata of the pathogen on the
stem base, or leaf bases or primary roots close to the soil level, although they
are frequently only observed once disease is firmly established. Subclinical
infections thus remain undetectable, and mycelial states in the soil and sur-
rounding plant debris cannot be detected and identified. As one palm becomes
infected, it could transmit the disease through root contact with the immediate
neighbouring palms (Turner, 1965a). Until now, no sufficiently satisfactory
techniques have been available to detect early infection of oil palm, although
Reddy and Ananthanarayanan (1984) reported that the fluorescent antibody
techniques could be used to detect G. lucidum in roots of betelnut. Furthermore,
a polyclonal antibody has been developed to detect mycelium of Ganoderma in
culture (Darmono et al., 1993), and has been used to detect Ganoderma in oil-
palm fields (Darmono and Suharyanto, 1995). In the future these techniques


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; Utomo
and Niepold, this volume). However, detection of the incidence of BSR is
currently carried out based on the external symptoms. Palm infection can only
be confirmed when basidiomata of Ganoderma appear either at the stem base or
on 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 could
selectively isolate the pathogen from any parts of infected tissues, directly from
the field, with or without surface sterilization. With GSM and using a drilling
technique it was possible to detect more oil palms that were infected with
Ganoderma but which appeared to have no external symptoms (Ariffin et al.,
1993, 1996).

Control
It is fully realized that finding a solution to the BSR disease problem on oil palm
is not going to be an easy task. It is therefore recommended that both
short-term and long-term approaches be investigated in order to reduce
damage 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 fungicides
together with the technique of application needs to be investigated. For a more
permanent control, research strategy should concentrate on finding ways to
hasten decay of oil-palm tissues during replanting in order to minimize the
inoculum burden carried over in the subsequent planting (see Paterson et al.,


this volume). In addition, the production of oil-palm lines resistant to
Ganoderma must also be investigated. As methods for early detection of
infection are only just being developed, control measures are currently only
applied to visibly diseased palms, with untreated, symptomless palms
remaining a potential source of infection.

Cultural practices

A number of agronomic practices have been suggested to control BSR disease.
Digging trenches around diseased palms to prevent mycelial spread of the
pathogen to neighbouring healthy palms has been recommended as a control
measure (Wakefield, 1920), but trenches have not proved satisfactory
(Turner, 1981) due to the fact that the trench depths were insufficient to
prevent roots passing underneath, or that trenches were not maintained.
Collecting basidiomata of Ganodema from diseased palms and painting them
with 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 heavy
weed growth have been reported to be associated with increased BSR incidence
in 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 followed
by soil mounding around the base of mature diseased palms can bring about
an 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. Further
studies by Ho and Khairudin (1997) indicated that soil mounding with fumi-
gant, and soil mounding alone were able to prolong productivity of oil palms
through the physical benefit of preventing the weakened boles from being
toppled by the wind. However, this treatment did not prove to be curative.

Land preparation at the time of replanting

The correct technique of land preparation at the time of oil-palm replanting
is regarded as an important practice for controlling BSR disease. These con-
trol strategies are based on the assumption that infection occurs by mycelial
spread from root-to-root contact. Since tissues of the former stand of oil palms
or coconuts are thought to be the primary source of infection at replanting, dis-
ease avoidance through sanitation is important. Any methods of disposal of
the old stand involving destruction or reduction of the Ganoderma inoculum
had a beneficial effect on the subsequent planting (Khairudin, 1990b; Singh,
1991). Three replanting techniques, namely clean clearing, underplanting
and windrowing, have been practised throughout Malaysia. The effects of


these three replanting techniques on the incidence of BSR disease in oil palm
are presented in Table 3.5 (Khairudin, 1990b). Although the clean-clearing
technique gave lower disease incidence in replanted oil palm by comparison
with other replanting techniques, it was later found that this technique
was not entirely satisfactory in reducing disease incidence (Singh, 1991). An
incidence of BSR disease as high as 28–32% is not uncommon despite the
adoption of this clean-clearing technique (Singh, 1991). In the absence of a
complete understanding of the long-term survival of Ganoderma in infected
tissues buried in soil, the rationale behind this recommendation remains
unclear. This technique does not take into consideration the functions of
subterranean roots in disease epidemiology (Flood et al., this volume).
It must be realized that clean clearing was initially advocated based on the
finding that a massive amount of inoculum, at least 734 cm3, is required to
initiate infection (Turner, 1981). Following this assumption, the clean-
clearing technique was developed to destroy the boles and attached root
masses, the major plant parts that harbour the pathogen. Little attention was
paid to the interconnecting roots left behind after this operation. The original
wisdom was that these roots, although infected, are too small to be infective.
Further support for this view was provided by the observation that naturally
infected root fragments had failed to cause infection when used as inoculum
sources on nursery seedlings (Navaratnam and Chee, 1965). However, the
role played by these roots in disease outbreaks began to be realized following
the successful artificial inoculation of nursery seedlings. The fact that seedlings
can be infected readily using pure culture inoculum only slightly bigger than
the average oil-palm primary root (Ariffin et al., 1995), suggests that under
favourable conditions the leftover roots can be infective.
Also, field experimentation by Hasan and Turner (1998) proved that roots
can represent a small but significant inoculum source. These workers divided
the interspace between two adjacent infected palms fields into three equal
sectors separated by deep trenches. Bait oil-palm seedlings were planted
in each sector and also around the bases of BSR–infected palms. The results
revealed that only 4% of bait seedlings became infected after 2 years, and these
were in the sectors closest to the diseased palms. Although this incidence was
much lower than the 69% infection of bait seedlings planted adjacent to main
disease sources, the results were convincing enough to conclude that infected
root fragments can cause infection and, hence, disease outbreaks. Singh
(1991) had also demonstrated that infection of some young palms was
initiated by small bundles of diseased roots of the former stand buried close to
the palms. These findings suggest that leftover root fragments can play a very
important role in the outbreak of BSR, despite the practice of clean clearing
during replanting of second- and third-generation palms. That the root
fragments left in situ still have enough inoculum potential to cause disease
is reflected in their ability to produce basidiomata of G. boninense, which
are sometimes seen on their cut ends. These roots, although detached from
the boles, are still several metres long and should individually have enough


food reserves to ensure survival of the pathogen. Furthermore, the very nature
of G. boninense being confined within the root ensures minimal interference
from other common antagonists present in the soil. Although infected roots
are brittle, with the stele easily detached from the cortex, the pathogen is also
present in the stele (Ariffin et al., 1991).
The underplanting of coconut or oil palm with young oil palm, followed by
poisoning and felling of the old stand has been a common practice, especially
on smallholder farms. When the coconut or oil-palm stump is left to rot in
the field, numerous basidiomata of Ganoderma are produced. As shown in
Table 3.5, 15 years after replanting the highest incidence of BSR disease
was recorded on the subsequent generation of oil palm in the underplanting
technique (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% to
14.0%). Khairudin (1990b) also observed that 93% of seedlings growing
around infected oil-palm stumps left in the field became infected within 18
months. By contrast, only 7% of seedlings growing around sites that had been
excavated to remove diseased stumps became infected. This clearly indicates
the value of clean clearing and the hazard of underplanting, a practice long
discouraged (Turner, 1981).

Treatment by excision

Excision of diseased tissues as a form of treatment has been recommended
(Turner, 1968), but with very mixed results. Infected tissues from lesions in the
outer stem tissues of oil palm were excised, either with harvesting chisels
(Turner, 1981) or mechanically, to excise diseased tissues from above and
below soil level (Singh, 1991). After the lesions were excised, the cut surface
was treated with a protectant chemical (e.g. coal tar or a mixture of coal tar
and 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 years
old, as the disease lesions are more superficial due to the harder stems of older
palms (Singh, 1991). Excision frequently requires repetition, as infection often
resurges if lesions are not completely removed.

Fungicide treatment

Due to the severe disease incidence in existing stands of oil palms, immediate
short-term measures to control this disease must be investigated. The use of
systemic fungicides, together with a correct technique of application, could
possibly provide the answer to this problem. Control through the use of fungi-
cides should not be limited to treating oil palms with confirmed cases of
Ganoderma only, but also neighbouring oil palms that are in potential danger
or might have already been infected at subclinical level. The use of fungicides


to treat young oil palms not showing obvious signs of infection but which have
been planted in an area with a history of a high incidence of Ganoderma also
needs evaluation as a preventive measure. Screening of fungicide activity
against Ganoderma in vitro has shown that numerous fungicides were strongly
inhibitory 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, tridemorph
and triadimenol (Lim et al., 1990)). Organic mercury formulations have been
reported to be strongly inhibitory to Ganoderma in the field, but became
unacceptable for commercial use due to the residue problem (Turner, 1981).
Attempts to control BSR in the field by the use of systemic fungicides
have been made by various workers (e.g. Jollands, 1983; Khairudin, 1990a;
PORIM, 1997). The results of these studies are inconclusive, although some
systemic 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 soil
drenching. Results of the trunk injection of fungicides into BSR-infected oil
palms showed that a carboxin/quintozene mixture was the most effective in
retarding disease development, hence prolonging the life of the BSR-affected
palms (George et al., 1996). Later studies, using pressure injection apparatus,
indicated that systemic fungicide (e.g. bromoconazole) also appeared to limit
the spread of Ganoderma infection (Ariffin and Idris, 1997). In India, Rao et al.
(1975) reported successful control of Ganoderma wilt disease of coconut by
injection of a 500 p.p.m. Vitavax solution into the trunk of diseased palms.

Fumigant treatment

The goal of causing rapid decay of woody tissues and subsequent displacement
of the pathogen could be approached through the use of fumigants. Studies on
the use of the fumigant Dazomet, which releases the soil fumigant methyliso-
thiocyanate (MIT) on contact with water, have also had encouraging results
for both in vitro and field studies (Ariffin and Idris, 1990). In vitro, 1 mg of
dazomet in a 9 cm Petri dish containing a growing culture of Ganoderma
was shown to be fungistatic (Ariffin and Idris, 1991b). Investigation of the
fungitoxic effects of MIT on Ganoderma in infected oil palms showed that
the chemical moved systemically downwards when injected into the diseased
oil palms (Ariffin and Idris, 1993).

Biological control

Little work has been done on biological control of BSR disease. The possibility
of control of Ganoderma in existing stands should be approached through


manipulation of biological agents. Several promising antagonists, mainly
Trichoderma (Shukla and Uniyal, 1989; PORIM, 1991; Wijesekera et al.,
1996), Aspergillus (Shukla and Uniyal, 1989) and Penicillium (Dharmaputra
et al., 1989), have been isolated and their mechanisms of antagonism against
Ganoderma in culture have been reported. The effectiveness of antagonists
in soil can be enhanced under field conditions by fumigation and fertilizer
application (Varghese et al., 1975), but there are no reports of effective
biological control in infected oil palms. Mass production of these antagonists,
especially Trichoderma, on oil-palm waste, such as oil-palm mill effluent and
empty fruit bunch (Singh, 1991) is possible, and this preparation could be used
for application around the roots of infected oil palms.

Conclusion
Basal stem rot is having a severe impact on oil-palm production in the coastal
soils of Malaysia, and is currently increasing in intensity in peat soils and even
in the inland soils and lateritic soils, although in the latter, infection rates are
relatively low. It is not clear whether the distribution of the disease is related to
soil types, previous cropping history or the distribution of aggressive strains or
species of the pathogen. The influence of environmental conditions on BSR
disease incidence also requires clarification. Novel techniques need to be
developed for the control of this disease. The available control measures are
only aimed at delaying the progress of infection, or prolonging the productive
life of the palm; these are cultural practices, such as clean clearing to minimize
root infection through root contact and soil mounding to encourage develop-
ment of new roots. Recently, promising results have been obtained on the use
of fungicides to treat diseased palms, and studies are also ongoing to determine
whether a fumigant could eradicate the pathogen from infected tissues, thus
reducing the Ganoderma inoculum. The development of the pressure-injection
apparatus is seen as another breakthrough that will make fungicidal treat-
ment of infected palms possible. With this technique, fungicides could be
applied precisely to the infected sites, ensuring better delivery of the chemical
with minimal wastage. Also, breeding for resistance to the disease remains an
important priority.

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4
S. Likhitekaraj of A. Tummakate
Basal Stem RotandOil Palm in Thailand

Basal Stem Rot of Oil Palm in 4
Thailand Caused by Ganoderma
S. Likhitekaraj and A. Tummakate
Division of Plant Pathology and Microbiology, Department of
Agriculture, Bangkok, Thailand

Introduction
Oil palm has been cultivated on a commercial scale since 1968 in Satul and
Krabi provinces of Thailand. Since then cultivation has spread to the provinces
of Surat Thani, Trang and Chumphon, involving both private sectors and
government agencies. Most of the areas were planted on newly cleared land
(from the forest) and currently basal stem rot (BSR) is not a serious problem.
Likhitekaraj (1993) reported the occurrence of BSR on 20-year-old oil-palm
trees in a plantation in Krabi province. Two palm trees out of 2000 trees in the
plantation showed typical symptoms, having fruiting bodies of the fungus on
the stem near the ground, but the fronds remained green. Cross-sections of the
infected trunks revealed that only one side of the trunk had rotted but the other
side appeared normal. Now, most of the oil-palm plantations are more than 20
years old. Close observations have been made every 4 months to determine the
incidence of BSR.

Methods
Surveys of the incidence of diseased trees are made every 4 months in the
following locations (each location contains 2000 palm trees):
1. A plot replanted on an old plot in Chumphon province which was
destroyed by Typhoon Gay. The destroyed trees were cut down and chipped
into small pieces by tractors before replanting.


70 S. Likhitekaraj and A. Tummakate

2. A replanted plot in Krabi province. The old trees of this plot were killed by
chemical 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.

Results
1. After two annual observations there is no evidence of BSR on the planted
seedlings (3 years old) in the first location.
2. After two annual observations, no BSR appears on young replanted palms
in the second location, but the old stumps of killed trees have fruiting bodies of
Ganoderma. The latest estimate is that 23.8% of the 2000 stumps show
Ganoderma fruiting bodies. The incidence of the sporophores increases every
time a survey is conducted.
3. The 20-year-old palms in Satul province show no evidence of BSR.

Conclusions
After 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 in
Krabi province. The surveys will be continued for many years on the three
plantations in order to monitor the development of the disease.

Reference
Likhitekaraj, 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.

5
S.S. Lee
Current Status of Root Diseases of Acacia mangium

The Current Status of Root 5
Diseases of Acacia mangium
Willd.
S.S. Lee
Forest Research Institute Malaysia, Kepong, Kuala Lumpur,
Malaysia

Introduction
Acacia mangium Willd. is indigenous to the far eastern islands of Indonesia,
the Western Province of Papua New Guinea and north-east Queensland,
Australia. Its potential for wood production due to its rapid growth was
recognized in the 1970s and establishment of large-scale A. mangium planta-
tions in South-East Asia began in the 1980s. Today there is an estimated
600,000 ha of A. mangium, planted mainly in Indonesia, China, Malaysia, the
Philippines, Thailand and Vietnam (Kamis Awang, Serdang, 1998, personal
communication). A. mangium was first introduced to Malaysia in 1966,
where it was planted as firebreaks in Sabah (Yap, 1986). Presently there are
approximately 100,000 ha of A. mangium plantations in the country, with
approximately 50,000 ha each in the peninsula and Sabah, respectively, and
relatively small areas in Sarawak.
In earlier reports, A. mangium had not been reported to suffer from any
serious diseases (Turnbull, 1986). However, recent studies have shown that
tropical acacias, including A. mangium, planted outside their natural range do
indeed suffer from a variety of diseases; one of which is root rot (Khamis, 1982;
Lee, 1985, 1993; Arentz and Simpson, 1988; Almonicar, 1992). In a survey
of A. mangium provenance trials at three locations in Peninsular Malaysia,
Lee (1997) found that root-rot diseases were the most frequently occurring
diseases, causing between 5 and about 25% mortality of 10-year-old trees.
This disease has also been identified as the most significant disease of tropical
acacia plantations in Australia, Indonesia, Malaysia, Thailand and India (Old
et al., 1997).


72 S.S. Lee

A variety of basidiomycete fungi have been reported to be associated with
root rot diseases of A. mangium. A brown root disease caused by Phellinus has
been reported from Sabah (Khamis, 1982) and the Philippines (Almonicar,
1992; Millitante and Manalo, 1999). In the Gogol Valley of Papua New
Guinea, Arentz (1986) reported 29% mortality of 5-year-old A. mangium trees
due to root disease caused by a species of Ganoderma. Ganoderma spp. are also
suspected as the causal agents of root disease of A. mangium trees of various
ages in Peninsular Malaysia (Lee, 1985, 1997), Sumatra (Lee, 1997) and
West Kalimantan, Indonesia (unpublished data).
Here, the results of a long-term survey of root diseases in an A. mangium
plantation in Peninsular Malaysia are presented, and preliminary results of
pathogenicity tests with the associated fungi are discussed.

Impact of Root Diseases on A. mangium
Between September 1991 and June 1992 plots were established in an A. mang-
ium plantation in Kemasul, Pahang in Peninsular Malaysia, to monitor the
occurrence and spread of root disease. Three replicate plots, each containing
10 × 10 rows of trees were set up in stands planted by the Forestry Department
in 1982, 1984, 1985, 1986, 1987 and 1988, making a total of 18 plots. All
the trees in each plot were numbered and mapped for ease of the survey and
future reference. During each survey, symptoms and signs of root disease and
the 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 of
the foliage, thinning of the crown, dieback, and death of trees in groups. Trees
with such symptoms were found to occur in patches, with a concentric pattern
of spread. Diseased roots were covered by a wrinkled, reddish-brown mycelial
skin, encrusted with soil, or encrusted in a mass of earth and sand intermingled
with rusty brown patches, in contrast to the clear, pale yellowish-brown
coloured healthy roots.
More than 40% mortality was observed in all the 1984 plots 14 years
after planting, and in plots 1987B, 1988C, 9 and 11 years after planting,
respectively (Fig. 5.1a and b). In the 1984 plots mortality increased very
rapidly when the trees were between 10 and 14 years old, while in plots
1987B and 1988C, a rapid increase in mortality occurred when the trees were
between 6 and 9 years old and 7 and 11 years old, respectively. In contrast, less
than 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 that
mortality rates differed from plot to plot. Similar variation in mortality
rates had also been observed in the 1995 survey of root rot in A. mangium
provenance 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 most
probably spread by root contact. In most cases, the initial disease foci enlarged

Fig. 5.1. Mortality rates of Acacia mangium trees in Kemasul, Pahang, Peninsular
Malaysia: (a) in the 1982, 1984 and 1985 plots; (b) in the 1986, 1987 and 1988
plots.

74 S.S. Lee

Fig. 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 plot
establishment.

with each passing year; this was clearly evident in all the 1984 plots and in
plots 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 the
trees in the plot during the duration of the study. While no symptoms of root
disease were evident on the trees in plots 1985A, 1985B, 1986A, 1987A and
1988A at the time of plot establishment, they started to appear 2–3 years after
the study commenced. This suggests that the trees only became infected when
their expanding root systems encountered some buried source of root disease
inocula. As in the other plots mentioned earlier, the rate of disease spread was
variable, with moderate increases in mortality in plots 1985A, 1986B, 1987C
and 1988B, and very little increase in plots 1985B, 1986A, 1986C, 1987A
and 1988A.
The mortality of trees generally increased with time in plots where root
disease was already present at plot establishment. The rate of disease spread
was probably dependent on the presence, abundance and distribution of root
disease inocula at the site, rate of root growth, extent of the root system of
each tree, and extent of root contact between healthy and infected trees. These
plantations 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 roots
and other woody debris that harbour the facultative parasitic root-rot fungi
remain buried in the soil, acting as sources of infection.


Fungi Associated with Root Diseases of A. mangium
Based on the appearance of the infected roots, two main types of root diseases
could be distinguished even though the visible disease symptoms on the tree
crowns were similar. These were red-root disease and brown-root disease.
Roots of trees infected by red-root disease are characteristically covered by
a wrinkled, reddish-brown mycelial mat. The red colour of the mycelial mat
becomes very evident when the root is washed clean of soil. A white mottling
pattern 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 no
colour change is discernible, but in advanced stages the wood becomes pale
buff and spongy or dry, depending on the soil conditions. Red-root disease was
the most frequently observed type of disease when roots were sampled. The
characteristics of the disease are very similar to that of red-root disease caused
by 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 are
easily recognized by the production of brown zigzag lines in the wood, forming
a honeycomb-like pattern, and the wood becoming friable, light and dry. The
brown lines are ridges of golden-brown fungal mycelium and the type of rot
produced is known as ‘pocket rot’. These characteristic features indicate
that the fungus associated with the disease is Phellinus noxius (Anonymous,
1974).
The identity of the associated fungi could not be confirmed initially
because of the absence of sporocarps on diseased or dead trees. Samples of dis-
eased roots were thus collected for isolation of the associated fungi. Attempts
were made to identify the pure-culture mycelial isolates by comparison with
the species codes developed by Nobles (1965) and Stalpers (1978) and by
inoculation onto wood blocks for the production of sporocarps (Lee and
Noraini Sikin, 1999).
For production of sporocarps on wood blocks, pure-culture isolates of the
test fungi were first grown on malt agar (DIFCO Laboratories, USA) in the dark
at ambient room temperature for about 1 week. In the meantime, blocks of
debarked rubber wood, measuring 10 cm by 5–6 cm diameter, were placed
individually into autoclavable plastic bags, wetted with approximately 50 ml
of 2% malt extract and sterilized. Three 1 cm diameter plugs, taken from the
edge of 1-week-old actively growing cultures, were then used to inoculate each
rubber-wood block. Five replicate blocks were inoculated with each fungus
and 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 removed
from their plastic bags and ‘planted’ into polybags containing unsterilized
garden soil, one block per bag. These were then transferred to a shade house
and lightly sprayed with tap water daily to keep the soil and the wood blocks
moist. When sporocarps were produced, between 2 and 3 weeks later, they
were collected for identification in the laboratory.

76 S.S. Lee

The identity of the fungus associated with red-root disease could not be
confirmed from the wood-block technique as no sporocarps were produced.
However, the characteristic red skin of mycelium on the root is similar to that
reported for G. philippii (= G. pseudoferreum) on rubber (Anonymous, 1974).
From isozyme analysis, four isolates of Ganoderma obtained from A. mangium
in West Malaysia were determined to be different from those isolated
from 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 a
plantation at Bidor, Perak. Inspection of trees with symptoms of root disease
located close to the clumps of dead trees revealed that the roots were covered
by a red mycelial mat (S. Ito, Bidor, 1999, personal communication), charac-
teristic of red-root disease observed on A. mangium trees in Kemasul, Pahang
and elsewhere. However, attempts to isolate the fungus, from both sporocarps
and infected roots, were unsuccessful. Corner (1983) noted that G. philippii is
rather common and distributed from Burma (Myanmar) to the Solomon
Islands, being found on dead stumps in the forest and in the open, and parasitic
on roots of trees, especially Hevea.
Using the wood-block technique, sporocarps produced from mycelial
isolates obtained from samples with brown-root disease were confirmed as
those of P. noxius (Pegler and Waterston, 1968). Inoculated wood blocks also
had 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 mistaken
for necrotic tissue. The black crust was usually found on the roots of dead trees
where the wood had become yellowish-cream in colour, spongy and light.
Using the wood-block technique, hyphal isolates obtained from the black crust
yielded sporocarps, identified as Amauroderma parasiticum (Corner, 1983).
In addition to the root diseases reported here, a root disease associated
with the presence of white rhizomorphs of an unidentified fungus has also been
reported from A. mangium in Peninsular Malaysia (Lee, 1997). However, this
disease was not observed during the present study.

Pathogenicity Tests
Pathogenicity tests are presently being conducted on A. mangium saplings in
the FRIM nursery, and only preliminary results are reported here. Six-month-
old A. mangium plants were transplanted into large polybags (33 cm depth by
35.5 cm diameter) containing a 1 : 1 mixture of forest soil and padi husk (this
is the potting mixture normally used in the FRIM nursery). After the plants had
become well established, about 3 months later, they were inoculated using
branches (8 cm long by 1.5 cm diameter) of a rubber tree which had been well
colonized by the test fungi (the rubber-tree branches, with intact bark, were
inoculated using the same technique as described above for the inoculation
of the rubber-wood blocks). Three well-colonized branches were used to


inoculate each test plant, with the branches buried in close proximity to the
roots of the plant in the polybag. There were three replicates for each fungus
and the fungal isolates tested were P. noxius, the suspected Ganoderma and
A. cf. parasiticum.
About 2 months after inoculation, symptoms of root disease were obvious
on the plants inoculated with P. noxius and the suspected Ganoderma, while
those inoculated with A. cf. parasiticum remained symptomless. However,
different symptoms of root disease were observed on the plants inoculated
with P. noxius and the suspected Ganoderma. Those inoculated with P. noxius
exhibited progressive yellowing of the phyllodes, beginning with the tips of the
younger phyllodes, resulting ultimately in defoliation and death of the infected
plant. On the other hand, plants inoculated with the suspected Ganoderma
suddenly wilted without any yellowing symptoms, and died within 5 days after
the first symptoms were noticed.
Roots of plants inoculated with the suspected Ganoderma were covered by
a red mycelial mat but the fungus could not be successfully re-isolated from
the affected plants. This experiment is being repeated to confirm the results
presented here.
Pathogenicity of P. noxius was proven as the fungus was successfully
re-isolated from roots of the inoculated plants, which had rusty brown patches
under a crust of soil.
Plants inoculated with A. cf. parasiticum remained healthy even 6 months
after inoculation. It would appear that this fungus is not a primary pathogen of
A. mangium, but probably a secondary pathogen or weak parasite infecting
stressed trees or trees which have been weakened or killed by some other
agents. Corner (1983) recorded A. parasiticum as a parasite on the trunk of a
living tree of Knema (Myristicaceae) in a swamp forest in Singapore.

Conclusion
Large-scale burning has been a common feature of land clearing in South-East
Asia for conversion of forest or old tree stands into agricultural and industrial
plantations, 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 months
over Singapore, Brunei, southern Thailand and large parts of Indonesia and
Malaysia. Widespread public outcry and political pressure from regional
governments resulted in the government of Indonesia declaring a ‘no burn’
policy for land clearing, with the imposition of hefty fines for those found guilty
of the offence. However, enforcement remains problematic.
In Malaysia, the Environmental Quality (Clean Air) Regulations 1978
prohibit open burning, but in the past open burning for land conversion and
replanting could be carried out under special contravention licences issued by
the Department of Environment. The large-scale adoption of the zero burning

78 S.S. Lee

technique by oil-palm plantation companies in Malaysia in 1989 has allowed
oil-palm replanting to be done without violating the Environmental Quality
(Clean Air) Regulations 1978, and the technique has also been developed
for the replanting of oil palm and other plantation crops from logged-over
forests (Golden Hope Plantations Berhad, 1997). In the aftermath of the 1997
haze, the Malaysian government issued a directive prohibiting almost all
forms of open burning, and a law pertaining to this issue is presently under
consideration by the Attorney-General’s chambers.
While zero burning is environmentally friendly and results in total
recycling of plant tissues (the existing trees are felled, shredded and left to
decompose in situ), it also gives rise to several problems, such as increased
insect infestation and increased sources of root disease inocula. From the
disease point of view, the woody residues act as potential reservoirs and food
resources for the facultative parasitic root-disease fungi which live in the soil.
In second-rotation A. mangium plantations in Sumatra, where no burning was
carried out before replanting, there are already indications that losses due to
root diseases will be much more serious, with a higher incidence of the disease
in the young plantations and mortality occurring in younger plants. A.
mangium trees as young as 6 months old have been observed to be killed by
red-root disease (unpublished data) in such areas.
In view of the potential damage and losses that can be caused by root
diseases in A. mangium plantations, especially with the implementation of the
‘zero burning’/ ‘no burn’ policy by several South-East Asian governments, it is
important that further research be conducted to determine the sources of
inoculum, factors promoting the occurrence and spread of the disease, and
methods for prevention, management and control of the disease.

References
Almonicar, 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.


Lee, 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.
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.

Disease Control and III
Management Strategies

6
H. Soepena et al.
Control Strategy for Basal Stem Rot on Oil Palm

A Control Strategy for Basal 6
Stem Rot (Ganoderma) on Oil
Palm
H. Soepena, R.Y. Purba and S. Pawirosukarto
Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen
Katamso 51, Medan, Indonesia

Introduction
Basal stem rot (BSR) caused by Ganoderma boninense Pat. is the most
destructive disease of oil palm in South-East Asia (Khairudin, 1993). The
disease can infect all stages of the oil-palm plants. The disease progresses
slowly but every infected plant usually dies. In the early stages of infection
plants usually appear symptomless and the symptoms appear only when the
plant is severely infected, so plants with severe symptoms are unable to be
saved. The disease is considered to be spread from plant to plant through root
connections, although long-range disease dissemination, with spores spread
by wind, has also been considered (Sanderson et al., this volume). Repeated
palm replanting on the same area has led to an increase in BSR disease
incidence, which increases from one generation to another.
To date there is no adequate control for BSR in the field. No appropriate
fungicide is available for effective control of BSR and no genetic resistance to
the disease has been described in Elaeis guineensis Jacq. (Möller and Schultz,
1997). Cultural control techniques have little effect on the control of BSR,
because the pathogen can survive in the soil for several years (Soepena,
1996). BSR causes a decrease in all palm stands and reduces yield rapidly, so
that production is uneconomic. Thus, biological control methods using
Trichoderma spp. and Gliocladium sp. have been developed and a strategy for
management of the disease using a biofungicide is reported here.


84 H. Soepena et al.

Oil-palm Basal Stem Rot (Ganoderma Stem Rot)
The causal agent of BSR

The causal agent of BSR on oil palms is G. boninense Pat. Fruiting bodies of
Ganoderma 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 of
Ganoderma from North Sumatra as G. boninense (Utomo, 1997).
Ganoderma is a saprophytic soil inhabitant, indigenous to the tropical
rainforest, but under some circumstances it can become pathogenic. Species
of Ganoderma have a wide host range – more than 44 species from 34 genera of
plants have been identified as potential hosts (Venkatarayan, 1936), including
coconut and oil palm, which are the main source of infection of Ganoderma
stem rot in oil palms (Hasan and Turner, 1998).

The disease symptoms

G. boninense can infect all stages of oil palm, from seedling to old palms. Palms
infected early in their life cycle can remain symptomless, the symptoms only
becoming clear after the palms are more than 12 years old (Lubis, 1992), but
in the second and third replantings the symptoms can appear as early as 1–2
years after planting in the field.
Ganoderma infection on seedlings or young palms usually occurs on roots
and 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 partially
dead old fronds. Disease symptoms on the old palms is clearer, the appearance
of a number of spear leaves and collapse of old fronds are the main symptoms
(Fig. 6.2).

Basal Stem Rot Control Management
BSR control strategy

BSR could be managed satisfactory if the source of infection of Ganoderma could
be completely destroyed. Thus management of BSR in oil-palm replanting
areas should be based upon the following strategy: (i) use of uninfected
soil in polybags to grow seedlings; (ii) prevention of infection in young
growing 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 85

Early warning system

Although biofungicide treatments are given to all growing plants, special
attention must be given to emerging disease symptoms, especially for the first
5 years. Disease symptoms should be evaluated twice a year and disease
incidence should be reported. An application of further biofungicide is made as
soon as possible, or severely infected and dead plants are removed, the planting
hole treated with biofungicide and healthy seedlings replanted.

Biological control method for Ganoderma

Ganoderma 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, but
some of them have been successfully selected as antagonists to Ganoderma
(Dharmaputra, 1989; Soepena et al., 1999). Trichoderma koningii Oud. Isolate
Marihat (MR14) is one of the most powerful antagonists against Ganoderma
and has been formulated as the active ingredient in a biofungicide (Soepena
and 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.


Fig. 6.2. The main symptoms of Ganoderma disease on an old oil palm: note the
accumulation of spear leaves and collapse of old fronds.

harzianum and Gliocladium virens have also been used as biological control
agents against Ganoderma, but these species are better for decomposing organic
material in fields. A combination of antagonistic and saprophytic fungi is
very useful for destroying Ganoderma propagules and decomposing oil-palm
residues in windrows.
The biofungicide contains 5–8 × 106 conidia and chlamydospores of
T. koningii per gram of product in a natural medium.

Application of the biofungicide

Trichoderma survives as chlamydospores under unfavourable conditions,
and most of these are resistant to many kinds of chemical pesticides, such as
organochlorines, 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 rainy
season. The dose of the biofungicide depends on the size of the palms.

Control Strategy for Basal Stem Rot on Oil Palm 87

Preventative treatments
Seedlings grown in polybags can be infected by Ganoderma from infected soil, so
soil taken from disease-free areas should be used and the seedlings treated with
Trichoderma biofungicide by spreading it on the surface of the polybag. This will
help to eradicate any inoculum and will protect the seedlings after planting in
the field. Planting holes in heavily infected areas must also be treated with
Trichoderma biofungicide prior to planting a seedling, to help eradicate the
inoculum in the soil and protect newly growing palms. The biofungicide can
also be applied to oil-palm trunks in windrows in order to eradicate Ganoderma
propagules and increase decomposition. Young palms should be treated
annually for 5 years.

Curative treatments
In addition to preventative treatment, newly infected plants can be treated
with Trichoderma biofungicide. The biofungicide can be injected into the base of
the bole of infected plants using soil injection: 3 holes are made under the base
of the bole of the infected plant with a soil auger, and the biofungicide can be
applied. This method can be used for special palms, such as highly productive
or mother plants. Surgery to remove rotten tissue can also be conducted on
these special palms in conjunction with application of the biofungicide to the
affected areas.

Field sanitation

It is very important to keep the oil-palm plantations free from sources of the
pathogen, so good field sanitation is essential. All infected plant materials
should be treated with Trichoderma biofungicide.

References
Abadi, 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.
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.


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.

7
Use of Soil Amendments for Control of Basal Stem Rot

The Use of Soil Amendments for 7
the Control of Basal Stem Rot of
Oil-Palm Seedlings
Department of Plant Protection, Universiti Putra Malaysia,
Serdang, Selangor, Malaysia

Introduction
Basal stem rot (BSR) of oil palm, caused by species of Ganoderma, has been
recognized as a serious disease of oil palms for many years, causing severe
economic losses during the past 10–20 years and continues to do so. Current
control procedures are based on the assumption that infection occurs by
mycelial spread from root to root and that the removal of stumps and large
pieces of debris will eliminate residual inoculum from the field of the next crop.
Although clean-clearing practices generally result in lower disease incidence
in replanted oil palm by comparison with other replanting techniques, disease
incidence may still be unacceptably high. Doubts have been raised regarding
the efficiency of this approach, with evidence from a number of oil-palm estates
suggesting that infection can still become established progressively earlier
with each planting cycle, even after clean clearing. Furthermore, although
legume cover crops may accelerate the decay of oil-palm debris, reports have
also suggested that they may encourage the development of Ganoderma
(Dharmaputra et al., 1994). However, Hasan and Turner (1994) have
reported that subsequent infection decreases with increased clearing depth,
implying that clean clearing may frequently not be sufficiently thorough in
practice.
Chemical control has not been effective and long lasting, even though
in vitro screening has identified several chemicals that are effective against
Ganoderma (Hashim, 1990; Teh, 1996). Numerous studies attempting to
control BSR in the field by the use of systemic fungicides have been unsuccess-
ful (Loh, 1976; Jollands, 1983). The effective use of chemical control for


90 M. Sariah and H. Zakaria

treatment of Ganoderma-infected palms is limited by the fact that both visibly
infected and subclinical palms may harbour established infections by the
time treatment is applied. Additional difficulties may occur in the effective
placement of fungicides, as lesions are frequently very large in size. As lesions
are most commonly found at the stem base, high-pressure injection of
fungicides frequently results in the passage of the chemical straight into the
soil. However, recent preliminary results on trunk injection of fungicides into
BSR-infected oil palms have indicated that Triadimenol (a systemic fungicide)
may increase their economic life span, with treated palms remaining alive 52
months after the original BSR diagnosis (Chung, 1991). Further evaluation
of pressure injection of fungicides by Ariffin (1994) indicated that systemic
fungicides (Tridemorph and Dazomet) also limited the spread of infection and
he further concluded that the chemical moved systemically downwards into
the roots when injected into plants.
Alternative control methods for the future may lie in the biological
management of the disease. For example, trunk tissues, when they are wind-
rowed as part of the replanting technique in particular, support the rapid
development of many fungi other than Ganoderma. A much greater diversity of
fungi non-pathogenic to oil palm occur on poisoned windrowed tissues and,
together with their more rapid and prolific development than on unpoisoned
tissues, a possible biological control approach to the disease is indicated
through the competitive saprophytic ability of non-pathogenic fungi to
displace Ganoderma in composting tissues. However, under normal field
conditions these fungi seem unable to displace the pathogen and Ganoderma
continues to colonize old tissues, which become BSR sources for the new
planting. If the natural order of the succession could be manipulated, or the
volume of particular competitors changed so as to minimize the pathogen’s
opportunity for colonization, then the potential BSR hazard for new plantings
would be greatly reduced.
However, observations of the low incidence of disease due to Ganoderma
species in natural stands in the forest although the pathogen is present, would
suggest that disease is kept under control by some biological means. A study of
soil microflora of jungle and plantation habitats showed significant changes in
quantitative and qualitative aspects of the microflora from these two habitats
(Varghese, 1972). The changes were most striking in the humus-stained
upper horizon, where Aspergillus dominated the mycoflora of the forest, but
this layer was completely disrupted in the plantation habitat. Along with this,
a lowering of the antibiotic potential of the soil could be expected which would
be to the advantage of root pathogenic fungi (Varghese, 1972). Therefore any
new approach to natural or biological control of Ganoderma should take into
consideration the role of antagonistic microflora.
Enumeration of the microbial population from the oil-palm rhizospheres
and on the sporophores has also indicated great diversity of non-pathogenic
fungi in these habitats, which again points to the possibility of biological
management of Ganoderma. Species of Trichoderma, Penicillium and Aspergillus

Use of Soil Amendments for Control of Basal Stem Rot 91

make up more than 30% of the total populations of fungi (cfu) recovered,
and in some areas there was a positive correlation between percentage of BSR
incidence and frequency of isolations of the non-pathogenic fungi (Table 7.1).
These observations were not consistent for all the areas surveyed, suggesting
that soil and environmental factors exert some influence on the survival and
proliferation of microorganisms in the oil-palm rhizospheres, and the recovery
of 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 screening
of these non-pathogenic fungi, based on dual culture, colony degradation,
competition, antibiosis and mycoparasitism tests, showed that isolates of
Trichoderma were highly antagonistic to Ganoderma, followed by isolates of
Penicillium and Aspergillus. The mean percentage inhibition of radial growth of
Ganoderma mycelium in dual-culture plating for Trichoderma, Penicillium and
Aspergillus 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. harzianum
exhibiting the highest antagonistic activity against Ganoderma. The mecha-
nism of antagonism was through competition and mycoparasitism, which
implies that early establishment of the antagonists in the plant rhizosphere and
roots of the palms may be crucial to produce the expected effect.
Similar observations on in vitro inhibition by a range of microorganisms
from the oil-palm rhizosphere and others, such as Trichoderma (Shukla and
Uniyal, 1989; Anselmi et al., 1992), Aspergillus (Shukla and Uniyal, 1989)
and Penicillium (Dharmaputra et al., 1989), have been reported. In spite of
this, there have been no reports as yet of effective biological control in infected
field palms, nor of attempts to inject healthy palms with an antagonist to aid
with their resistance to the pathogen. The incorporation of Trichoderma, grown
on dried palm-oil mill effluent (POME), into planting holes was evaluated as a
prophylactic measure (Singh, 1991), but doubts over the survival of this
organism in clay soils were raised.
Preliminary observations on the distribution of the antagonistic fungi
within the palm rhizospheres, in vitro antagonistic potential against Gano-
derma, rhizosphere competency of the antagonists, and the delivery system
have raised many unanswered questions about the potential of biological
management of Ganoderma, but to study the single or combined effects of the

Table 7.1. Mean recovery rate of antagonistic fungi from oil-palm rhizospheres
(× 103).

Basal stem rot Total cfu g−1
Location incidence (%) DW Trichoderma Aspergillus Penicillium

Prang Besar <5 155 2 19 40
Brownstone > 40 25 1 1 17
Sungai Buloh 5–10 58 2 14 22


antagonistic fungi on BSR infection is next to impossible in the field. This is
further complicated by the difficulty in identification and selection of uniform
disease 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, a
system of artificial inoculation of seedlings was developed (Teh, 1996) in
which the inoculum and extent of infection could be relatively quantified on
seedlings, to allow testing of potential control measures in a short period under
manageable and semi-controlled conditions.

Effect of Soil Amendments on the Control of Ganoderma on
Oil-palm Seedlings
Ganoderma is probably not a very aggressive pathogen. The general belief has
been that heavily colonized debris acts as the inoculum source, and that
wounded roots and weakened palms facilitate penetration. This suggests that
the fungus may be, at best, weakly pathogenic to healthy palms. Calcium is
the main macronutrient reported to strengthen the cell wall and increase
membrane permeability of plant tissues, thus further enhancing resistance
to a number of fungi, including Pythium, Sclerotium, Botrytis and Fusarium
(Muchovej et al., 1980; Spiegel et al., 1987). Also, supplementation of the
soil with calcium was shown to enhance the population of soil microflora
(Kommedahl and Windels, 1981) where antagonistic fungi, including
Trichoderma, Penicillium and Aspergillus, compete for space and nutrition.
Thus, Sariah et al. (1996) evaluated calcium nitrate (Norsk Hydro, field grade
containing 15% N, 19% Ca) as a prophylactic measure against BSR, due to the
soil-borne nature of the pathogen and slow establishment of the pathogen in
the 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 inoculation
Calcium applications were continued at monthly intervals over a period of
6 months, whereas the antagonistic fungus was applied only once, a day after
inoculation. In addition to the above supplementations, all seedlings were
fertilized with urea, and watering was done daily. The incidence of BSR was
assessed based on foliar symptoms at monthly intervals. Such that:
( a × 1) + ( b × 05)
.
Severity of foliar symptoms (%) = × 100
c


where a is the number of desiccated leaves, b is the number of chlorotic leaves, c
is the total number of leaves and where the numerical value of 1 represents the
index for desiccated leaves and 0.5 for chlorotic leaves.
At the end of the experiment, the bole was cut longitudinally for
assessment 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 bole
diameter. The number of lesioned roots and production of sporophores were
also noted (Teh, 1996; Teh and Sariah, 1999). Confirmation of the disease and
causal pathogen was made by plating infected tissues on Ganoderma-selective
medium (GSM) (Ariffin and Seman, 1991).
Based on foliar symptoms, and root and bole infection, the incidence of
BSR in pot-grown oil palms was suppressed significantly when seedlings were
grown in soils supplemented with calcium nitrate 1 month prior to inoculation
with Ganoderma-infected rubber-wood blocks as the inoculum source (Fig.
7.1a–c); augmentation with Trichoderma 1 day before inoculation did not
significally reduce BSR. The number of fruiting bodies was also reduced. In
addition, cell walls of calcium-supplemented seedlings were observed to have
well-developed lamellae, due to formation of calcium pectate, which could
stabilize the cell walls and resist degradation by cell-wall-degrading enzymes of
the pathogen. Also, the populations of soil fungi (cfu) were significantly higher
in calcium-supplemented soil as compared to calcium-deficient soil (Table
7.2), but augmentation with Trichoderma alone did not have a significant effect
on the fungal populations in the soil. Thus, the role of calcium in reducing BSR
incidence is hypothesized as that of stabilizing and strengthening the cell walls
of the oil-palm seedlings and stimulating the proliferation of antagonistic fungi
that will compete for space and nutrients. Calcium nitrate fertilization in this
study did not have any adverse effects on the vegetative growth of the seedlings
over the duration of the experiment, but, for continued application, the
possible interactions with the current agronomic practices of oil-palm growing
have to be studied, because calcium nitrate also contributes to the available
nitrogen.
Soil augmentation with Trichoderma 1 day after inoculation did not
control the incidence of BSR significantly. This treatment gave the highest
percentage of disease severity 6 months after the start of the experiment. This
could be due to the low recovery of Trichoderma from the plant rhizospheres
with time of inoculation, suggesting that the antagonistic fungus could not
sustain its population in the soil in the absence of a food base. Low rates
of 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 with
Trichoderma inoculants to the oil-palm rhizospheres requires consideration to
create environmental conditions in the soil which would favour antagonistic
mycoflora 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


drazoxolon 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 control

Fig. 7.1. Effect of soil amendments on (a) severity of foliar symptoms of oil-palm
seedlings with time (LSD0.05 = 17.3); (b) percentage of lesioned roots of oil-palm
seedlings 6 months after inoculation (LSD0.05 = 6.7); and (c) percentage of bole
infection of oil-palm seedlings 6 months after inoculation (LSD0.05 = 9.3).


Table 7.2. Mean total population of soil fungi in the oil-palm rhizosphere,
6 months after treatment.

Total fungal colonies per
Treatments gram air-dried soil (× 104)

T1 (7.5 g CaNO3)  Starting 1 month 24.50a

T2 (5.0 g CaNO3)  before inoculation 24.25a
T3 (7.5 g CaNO3)  Starting 1 day 19.50a
 b
T4 (5.0 g CaNO3)  afer inoculation 18.25ab
T6 (60 g Trichoderma applied 1 day before inoculation) 11.75b
T5 (control) 11.10b

Means 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 were
investigated (Zakaria, 1989) in Malaysia.
In Sumatra the possibility of neutralizing potential infection foci bio-
logically in oil-palm plantations with soil additives that might stimulate
microorganisms antagonistic to Ganoderma, especially Trichoderma spp. was
investigated (Hasan and Turner, 1994). At the end of the experiment the
incidence of seedling infection did not differ from the unamended controls, but
delays in infection were observed at the start of the trial. This was most marked
during the first 12 months after planting. Vigorous seedling growth in
response 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 palm
year−1 of sulphur powder, Calepogonium caeruleum and spontaneous soft weeds
as cover crops, and tridemorph fungicide at a concentration of 2500 p.p.m. per
palm year−1 for 5 years also showed a reduction in incidence of BSR (Purba
et al., 1994).
Similarly, soil augmentation with T. harzianum, the fungus antagonistic to
Ganoderma lucidum, applied with green leaves, neem cake and farmyard
manure + Bordeaux mixture were effective for the management of BSR of
mature coconuts in India, and all treatments recorded significantly higher nut
yield than the control (Bhaskaran, 1994). The Trichoderma population was
high in all treatments using organic manures when compared to control, but
neem cake and farmyard manure sustained the highest population levels.
Studies of population dynamics revealed that the population increased up to
the fourth month and then decreased drastically although the population
remained much higher than control soil, even 1 year after treatment.
In a continued search for a self-sustaining method for managing
Ganoderma infection in oil palms, Ho (1998) tested the ability of a commercial
formulation of vesicular arbuscular mycorrhizal fungi (VAM), Draz-M, to
reduce, if not control completely, Ganoderma infection on mature palms. He
observed no clear trends in terms of foliar symptoms and severity of Ganoderma


attack, but he noticed that the VAM treatment increased cumulative yield
when administered during the early stage of infection.
As there is no shortage of such amendments in the Malaysian plantation
environment, and coupled with the fact that chemicals or microbial amend-
ments alone were not practical and cost effective in the field situation, their
combined use was investigated in the glasshouse using 4-month-old seedlings
inoculated with Ganoderma-infected rubber-wood blocks. With this method of
inoculation, 100% infection was obtained within 4 weeks after inoculation
and for each infected plant, more than one-third of the bole tissues were
infected. 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 the
soil amendments (Table 7.3). Each treatment was replicated 16 times, with
a single seedling per replication, arranged and analysed using completely
randomized design. Parameters chosen for the above assessment were foliar
symptoms, and root and bole infections, as described earlier.
Based on regression analysis (R2) foliar symptoms exhibited a significant
relationship 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 % Bole
Treatment symptoms (%) Treatment roots Treatment infection

T 74.12a T 100a.60 T 100a.60
Cont 68.37a Cont 84.60a Cont 79.24a
M+T 46.65b OM 11.10b OM 4.12b
OM 40.06b M+T 9.16b M + OM 2.94b
M 37.60b M + OM 8.64b M 2.56b
M + OM 28.01b M 7.58b M+T 2.02b
c
M + Ca 20.83cd M + Ca 0c . M + Ca 0c .
c
Ca + OM 20.67cd Ca + OM 0 c. Ca + OM 0 c.
c20.57cd
M + 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.
c
T + 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.


the greater was the degree of bole infection (R2 = 98%). Soil augmentation
with organic matter (OM), the air-dried preparation of Trichoderma (T) or
mycorrhiza (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 (Table
7.3). Typical lesions and rotting of infected roots were observed and white
mycelium was abundant on the surface of the roots. Plating of the diseased
tissues and apparently healthy bole tissues on GSM confirmed the presence of
the causal pathogen. Addition of calcium nitrate (Ca) at 15 g per seedling,
together with Draz-M (M) or POME (OM) reduced the symptom expression
further. The progress of the disease was slow and no sporophores were
produced.
The control treatment and seedlings supplemented with Trichoderma alone
recorded the highest disease severity. Soil amendments consisting of the
air-dried preparation of Trichoderma (T) and calcium (Ca) or POME (OM), with
or without Draz-M (M), gave a positive control of BSR, at least for the period of
the experiment. Few foliar symptoms were observed, and this was supported
by the absence of lesioned roots or infection of the bole tissues. Random plating
of the roots or tissues from the bole did not produce Ganoderma colonies on
GSM, which suggested that the pathogen was not present in these tissues.
Biological control of root-disease pathogens by enhanced activity of
antagonistic and saprophytic components of soil mycoflora has been suggested
in many disease situations, but experimental evidence of the actual mode and
method of operation of this type of control, especially with respect to tropical
pathogens, has been scarce. The complexity of the various factors involved,
the time and effort required to understand their interaction and, finally, to
manipulate suitable changes in the soil environment were not encouraging for
greater utilization of biological control. However, it is evident from the results
presented here that control of Ganoderma in plantation crops can be imple-
mented by assisted stimulation of antagonistic and saprophytic components
of 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.

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8
J. Floodof Ganoderma from Infective Sources in the Field
Spread et al.

The Spread of Ganoderma 8
from Infective Sources in the
Field and its Implications for
Management of the Disease in
Oil Palm
J. Flood1, Y. Hasan2, P.D. Turner3 and
E.B. O’Grady1
1CABI Bioscience, Egham, UK; 2Bah Lias Research Station,
P.T.P.P. London, Medan, North Sumatra, Indonesia; 3PO Box
105, Quilpie, Queensland, Australia

Introduction
Basal stem rot (BSR), caused by species of Ganoderma, has been recognized as a
serious disease of oil palm (Elaeis guineensis) for over 80 years and has caused
severe economic loss in Malaysia (Turner, 1981; Singh, 1991; Ariffin et al.,
1996) and North Sumatra, especially during the past 30 years (Hasan and
Turner, 1998). Initially, the disease was considered to affect only old palms (at
least 25 years old) but, with successive palm generations on the same land, a
higher disease incidence has been observed and the symptoms occur earlier
with each replanting. For example, in Sumatra, where replanting was con-
ducted by pushing over the old stand, with no attempt to remove BSR-infected
tissues, young replanted palms have died from the second year onwards after
planting. Economic loss may begin to occur within 10 years and severe loss
after 15 years; the normal life span of a planting of oil palm would be 25–30
years. Where this phenomenon has occurred, it has generally been accepted
that there has been an increase in available inoculum from the previous palm
planting (Turner, 1981). Thus, efforts to manage the disease have been
directed largely towards disease avoidance through reducing the amounts of
potential infection sources for the replanting at the time of clearing the old
stand (Singh, 1991). In Malaysia, the benefit of this clean-clearing approach
over no disease avoidance measures has been demonstrated (Hashim, 1991).
However, the total removal of all infective tissues from an old stand with a high

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 economically
feasible. To investigate the efficacy of sanitation in BSR management, a series
of 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 trials
were designed to assess the relative importance of various tissue remnants
from the old palm stand as potential sources of inoculum at replanting (Hasan
and Turner, 1998) so as to make practical recommendations for management
of the disease in Sumatra.
The trials were set up as to be sufficiently large to overcome any variations
in BSR inoculum, with each treatment being replicated at different sites in the
plantation. Six-month-old seedlings were used to bait the Ganoderma-infected
material. External leaf symptoms developing on these bait seedlings were
recorded for the duration of each trial, while at the end of each trial, all
seedlings were examined internally for Ganoderma infection by destructive
sampling and plating to Ganoderma-selective medium (GSM) (Ariffin and Idris,
1991). Each experimental plot was isolated by a deep trench to increase the
likelihood that any infection recorded was derived from the tissue being tested
and not from an outside source, but more recently, molecular fingerprinting
techniques (Miller et al., this volume; Bridge et al., this volume; Rolph et al., this
volume) became available which allowed confirmation of the origin of the
pathogen in infected seedlings.

Stump Tissues
A stump comprises the base of the palm, or bole, and the thick crust of roots
immediately surrounding it. Stumps are usually recognized as major sources of
BSR. The first trial compared BSR stumps, prepared by felling diseased palms
about 20 cm above the ground (standard practice), as an inoculum source
with stumps derived from healthy palms. Around each stump eight bait seed-
lings were planted. An additional treatment, planting additional seedlings
immediately outside the plot isolation trench and isolating these by a further
trench 1 m from the inner trench, in order to emphasize disease origin, was
added. Each treatment was replicated eight times at different sites. Six months
after planting, a small number of seedlings began to exhibit disease symptoms
and by the end of the 28-month trial period, 76% of all bait seedlings showed
symptoms; Ganoderma was isolated from these seedlings. In comparison, seed-
lings planted outside the first trench and within the second isolation trench
perimeter showed very little infection – only 1.6% of these seedlings were
diseased and, at 80% of the replicate sites, these seedlings exhibited no
symptoms at all. No disease was recorded in bait seedlings planted around
healthy 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 103

healthy palms and the effects of pre-felling poisoning by paraquat, using 60 ml
per palm Gramoxone which was injected into the trunk. Stump size was found
to exert a marked influence on disease occurrence, with more bait seedlings
around smaller, lower stumps (20 cm high) exhibiting disease symptoms after
2 years than those around larger, higher stumps (50 cm high). Rate of
decomposition and bait seedling root ingress into Ganoderma-colonized tissues
would appear to be the most likely explanations for the difference. The effects of
poisoning, which had accelerated tissue breakdown, supported this, with more
seedling infection recorded around larger stumps where poisoning treatment
had been carried out.
The importance of inoculum sources at different soil depths adjacent to
BSR-infected stumps, which is of considerable relevance to sanitation
practices, was also investigated. Thus, soil and palm tissue adjacent to BSR-
infected stumps were removed to one of the following depths: 20, 40, 60, 80
and 100 cm. Eight replicate bait seedlings were planted at each depth and
these treatments were compared with diseased stumps that were undisturbed
after felling (no soil or tissues removed) and sites around healthy palms
excavated to a depth of 60 cm. In the absence of any sanitation, 75% of
seedlings had become infected and 97% of replicate sites had infected plants
within 2 years of planting (Table 8.1). In comparison, disease incidence in the
baited seedlings decreased to 21% where soil and debris had been removed to a
depth of 60 cm, and no disease was recorded where soil and debris had been
removed to 80 or 100 cm (Table 8.1).
In an extension of this trial, the same sites were replanted with bait
seedlings after 2 years and no disease was recorded at any depth 2 years later.
Similarly, when new bait seedlings were planted around previously highly
infective diseased stumps after 2 years, none of these bait seedlings developed
symptoms. Even after 2 further years of recording, these seedlings remained
symptomless, which would suggest that the potential of these stumps to act as
sources of inoculum had declined after 2 years. Data of percentage infection
over 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 is

Table 8.1. Effects of the removal of soil and palm tissues from around healthy
basal stem rot (BSR)-infected stumps on disease incidence after 24 months.

Depth of bole % Replicate sites with % Seedlings
Disease status removed (cm) infected seedlings infected

BSR 0 97 75
BSR 20 85 58
BSR 40 70 28
BSR 60 55 21
BSR 80 0 0
BSR 100 0 0
Healthy 60 0 0

104 J. Flood et al.

to be expected since the amount of infective tissue within stumps and its
location in relation to seedling root contact will differ considerably, as will the
rates of subsequent decay.
During the course of these trials, molecular fingerprinting techniques
became available for Ganoderma and were used to confirm the origin of the
Ganoderma from infected bait seedlings. Material was collected from diseased
seedlings, stump tissues and sporophores growing on the stumps and
isolations made on GSM. DNA was extracted from pure cultures (Miller et al.,
1999) and purified DNA samples tested with the ITS3/GanET primer (Bridge
et al., this volume) to check their identity. All isolates were positive with the
ITS3/GanET primer, confirming that the pathogen had been isolated from
the various tissues (Fig. 8.1). Mitochondrial profiles were generated using the
enzyme HaeIII as the restriction enzyme (Miller et al., 1999; Rolph et al., this
volume) and revealed that identical profiles were present in the BSR stumps
and 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 8

Fig. 8.1. Confirmation of the presence of the pathogen from stump tissues and
infected seedlings.


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 (Forster
and Coffey, 1990), isolates from the same sibling family would therefore have
the same profile. However, generally, mtDNA profiles are highly variable in
Ganoderma isolates, even from the same and adjacent oil palms (Miller et al.,
1999). Thus, identical mtDNA profiles from BSR-infected stumps and from
infected 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 technique
was used, namely amplification fragment length polymorphisms (AFLPs), as
described by Vos et al. (1995). This technique assesses the total cellular DNA
profile (nuclear and mitochondrial DNA) and is a more stable and reliable
method of studying variation (Rolph et al., this volume). Identical AFLP profiles
were produced using several primers, including primer E (Rolph et al., this
volume) (Fig. 8.3) confirming that the baited seedlings were infected with the
same genotype as that in the BSR-infected stump.

Trunk Tissues
Unless trunks of the old palm stand are destroyed at the time of replanting, they
are usually windrowed, i.e. placed in rows. Such trunks are colonized by many
species of fungi, including Ganoderma. Trunks will also remain following a
number of estate practices, e.g. following underplanting, those excavated as
low-yielding, palms removed for thinning or road construction and excavated
diseased palms, and palms affected by upper stem rot (USR) often remain
standing 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-infected
stumps. Palms were felled as close as possible to the ground and the trunk then
cut at 1 m and 4.75 m from the base, with the remainder being discarded. The
stump and each trunk section were isolated by trenches and bait seedlings
were planted close to the sections. Apparently healthy palms were also
included.
Stump tissues remained the most important source of BSR, with 27–38%
seedling infection occurring, and although the incidence of disease arising
from trunk sections was much lower (Table 8.3), this would remain of
considerable practical significance.
There was a marked increase over the 2-year period in the number of
infection foci on what had previously been considered as healthy stumps, with
the highest disease incidence (12%) being recorded where palms had been
poisoned 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 the
palm for what maybe a considerable time before symptoms are seen.
Infection rates of bait seedlings when planted around standing diseased
and apparently healthy palms were compared with that from stumps; the
infection rate of bait seedlings around standing palms was much lower
(Table 8.4). However, the period of infectivity of standing palms is likely to be
much longer, demonstrating the need to remove such palms in management
of the disease. Also, while diseased tissues appear to lose much of their infective
ability from about 18–20 months after felling, the majority of apparently
healthy stumps and trunks had yet to show the extent to which they would


Table 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)
Disease
status Treatment Yr1 Yr2 Yr1 Yr2 Yr1 Yr2

Diseased Nil 9 38 2 3 2 2
Diseased P 13 34 3 5 2 3
Diseased C 19 34 4 6 1 2
Diseased PC 17 27 4 5 5 6
Healthy Nil 0 6 1 1 1 1
Healthy P 1 9 2 3 1 1
Healthy C 0 7 5 6 4 4
Healthy PC 4 12 1 1 1 3

P, Poisoned before felling; C, legume cover.

become sources of disease at the end of 2 years (Table 8.4). However, from
the 2-year data alone, it is clear that under field conditions they will certainly
present a significant disease risk.
In another trunk treatment, pieces were cut to simulate shredding as a
clearing method, with and without poisoning prior to preparation. These were
either placed on the soil surface or buried at 20 cm deep. Both infected and
healthy trunk tissues were examined, with seedling baits used to detect BSR
in plots isolated by trenches. Both diseased and healthy shredded tissues can
give rise to disease after burial. Except in a single instance, superficially placed
tissues were not a disease hazard. In plots with buried tissues where disease
was recorded, sporophores of Ganoderma were produced on the soil surface.

Roots
The current recommendation for BSR sanitation procedure concentrates on a
1.5 m square centred on the point where the palm is planted. The assumption
has been that the remaining inter-space presents no serious disease hazard. In
a trial to examine this, areas between neighbouring diseased palms were each
divided into three equal parts and isolated by deep trenches. Bait seedlings
were then planted in each sector, as well as around the bases of the BSR-
affected palms. Similar sectors between apparently healthy palms were also
baited. In the BSR plots, Ganoderma fructifications developed on cut root
ends, signifying the presence of infected roots. The overall incidence of seedling
infection was low (4%) and was confined to the sectors closest to the diseased
palms, whereas 69% of bait seedlings planted around the main disease sources
became 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 standing
BSR and healthy palms compared with stumps, 2 years after treatment.

Standing palms Low stumps

Disease % Infective % Seedling % Infective % Seedling
status Treatment sites infection sites infection

BSR Nil 30 6 90 38
BSR P 40 10 95 34
Healthy Nil 20 3 20 6
Healthy P 30 7 40 9

P, Poisoned.

Also, records of the production of Ganoderma sporophores on cut ends of
roots 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 all
replicate sites had Ganoderma sporophores, while where soil had been removed
to a depth of 60 cm, this had decreased to only 10%. Thus, diseased roots can
comprise a small, but still significant, source of BSR in a replant, although this
probably requires dense root aggregations.

Discussion
It is apparent from these results that, when suitable disease sources are
present, oil-palm seedlings can be attacked by Ganoderma soon after planting.
Disease development and overt symptom appearance will depend on the size of
the palm when it becomes diseased, its continued growth vigour and the size
of the inoculum. Small seedlings close to large disease sources are killed
rapidly. Larger, rapidly growing plants are also affected, but frequently do
not die quickly. Numerous investigations have reported that many infected
palms continue to grow well, often for very long periods, before the internal
BSR lesion becomes so extensive that visible external symptoms develop. This
explains why so many cases of BSR occur long after planting and also after
obvious sources of primary infection have disappeared.
Once a few palms in a field are infected it has been considered that further
colonization of palms in the field is due to root-to-root contact by the palms or
mycelial spread. Both Singh (1991) and Hashim (1994) reported the disease as
occurring in patches or groups, which would support palm-to-palm infection,
but this view has been challenged recently by Miller et al. (1999). Studies of
somatic incompatibility and mtDNA profiling of isolates taken from many
adult palms within two oil-palm blocks (Miller et al., this volume) revealed con-
siderable variation between isolates, and led to the conclusion that isolates
occurred as numerous distinct genotypes, even within the same palm. Thus,


mycelial spread to adjacent palms or root-to-root contact was very unlikely.
Ariffin et al. (1996) similarly reported a high degree of heterogeneity between
isolates taken from adjacent infected adult palms. This contrasts with
other wood-rotting fungi, such as Heterobasidion annosum (Stenlid, 1985) or
Phellinus noxius (Hattori et al., 1996), where one clone of the pathogen can
extend over several metres. However, the preliminary mtDNA and AFLP
profiling described here has demonstrated that the same genotype is present in
the diseased stump and in baited seedlings. Thus, the experimental assumption
that the infected BSR stump acts as a direct source of infection to the young
seedlings 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 of
stumps, and other sources of infection for young seedlings in the field cannot be
ruled out. To date, the role of basidiospores has never been fully explained in
this disease. Thompson (1931) suggested that they were responsible for USR,
usually in association with Phellinus spp., but Turner (1965) failed to infect oil
palm 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. are
able to colonize debris which subsequently becomes the source of infection
for living palms (Miller, 1995). This would account for the heterogeneity
determined using molecular markers (Miller et al., 1999). Thus, much more
molecular analysis remains to be conducted – so far only diseased stumps
have been studied, but trunks and even roots can act as significant sources of
infection.
The investigations reported here have confirmed that the times of greatest
practical significance for the control of Ganoderma in oil palm are: (i) soon
after planting, when suitable inocula remain in the ground from the previous
planting (oil-palm stumps or root debris); and (ii) later in the planting cycle,
when root contact is made with Ganoderma-colonized sections of palm trunks
resting on the ground in rows (windrows). Results of this study would seem to
suggest that this danger extends over a much longer period when windrowed
palms are not poisoned prior to felling and are not covered by legumes to
accelerate decomposition.
Fungi that cause root disease frequently require substantial inoculum
potential before they are able to initiate infection and subsequently become
established within the host plant. Thus, infection must require either a block of
Ganoderma-colonized tissue of adequate size or a conglomerate of tissues, e.g. a
mass of infected roots, which collectively become an infection source. In the
trials summarized here, the importance of large blocks of inoculum is evident.
Bait seedling infection was very rapid when planted close to BSR-infected
stumps. Gradual removal of this source with increasing depth showed a clear
relationship between availability of infective material and both the occurrence
and incidence of BSR. This was not confined to the stump tissues. At a depth of
60 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 that
has 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 root
material from self-pruning (Hartley, 1988; Jourdan and Rey, 1996) and large
numbers of fine quaternary roots are present in the upper layers of the soil. The
hypothesis that this material could become the substrate for basidiospore
colonization requires further study.
The depth factor poses considerable problems from the practical viewpoint
of sanitation at the time of clearing for replanting. Breaking up deeply located
root 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 a
distinct possibility that their roots will soon encounter infective sources of
Ganoderma, and thus as much of the diseased stump tissue as possible should be
removed. However, further baiting using seedlings showed that these potential
BSR sources were less of a disease hazard after 2 years. This means that their
importance could be expected to be very much reduced, or even negligible, if
new palms are planted as far as possible from the old planting points. Their
disease potential would have greatly diminished by the time the roots of the
new planting reach the hazard sources, provided the old stand had been
poisoned before felling. Alternatively, delayed planting could be a useful
method of disease avoidance.
Windrowed palm trunks represent another significant problem, and the
same considerations apply to the necessity for planting as far away as possible
from windrows. The lateral extent of root development during immaturity
reaches roughly the edge of the canopy, meaning that it should take 2–3 years
before reaching this particular disease source if planted at the furthest possible
distance. An important observation is that the period over which windrows
remain a disease hazard is greatly reduced when palms of the old stand are
poisoned by paraquat prior to felling, and this effect is further enhanced
when 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 for
years. In such situations older palms of the replant become infected, with overt
disease symptoms only appearing long after the original infection sources have
disappeared.
One solution is to shred palm tissues so that they do not become BSR
sources over long periods, which is already a common practice in Malaysia but
not in Sumatra. However, even this does not provide a total answer to the
problem. Occurrence of BSR in bait seedlings, arising from buried, shredded
diseased and healthy trunk segments, was limited, but illustrated that the
technique still contains a degree of disease risk. Disease arising from
superficially placed segments was very slight and unexpected. It was in some
ways remarkable that in such segments, buried or superficially placed, disease
occurred at all, since many attempts at artificial inoculation of seedlings in
polybags using such tissues have failed. The appearance of Ganoderma sporo-
phores on the soil surface above buried BSR sections indicated that a sufficient


mass of Ganoderma-colonized tissue can overcome the inhibitory effects in soil
which normally prevent its development there.
Another possible BSR control method for the future lies in the fact that
trunk tissues, in particular, support the rapid development of many fungi other
than Ganoderma, and this points to a possible biological control approach to the
windrow disease hazard problem. Rapid degradation of the windrowed tissues,
especially by fungi antagonistic to Ganoderma, would have obvious advantages
for BSR and Oryctes control. However, this approach needs more investigation,
not least because woody tissues contain very little nitrogen, this influencing
the extent of colonization by certain rotting microorganisms, so that manipu-
lation of the nitrogen status of the debris will need to be conducted (Paterson
et al., this volume).

Acknowledgements
This 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 is
gratefully acknowledged. The authors would like to thank the Crop Protection
Programme (CPP) of the Department for International Development (DFID) for
funding some of the research reported here, which was administered through
NRI (RNRRS Project 6628).

References
Ariffin, 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.
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.

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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
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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
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,
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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-
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9
F.R. Sanderson et al.
Control Strategy for BSR of Oil Palm: Basidiospores

Basidiospores: Their Influence 9
on Our Thinking Regarding a
Control Strategy for Basal Stem
Rot of Oil Palm
F.R. Sanderson1, C.A. Pilotti1 and P.D. Bridge2*
1PNG OPRA, Plant Pathology Laboratory, Alotau, Milne Bay
Province, Papua New Guinea; 2CABI Bioscience, Egham, UK

Introduction
Although basal stem rot (BSR) of oil palm was recorded in Africa in 1933,
it was not until oil palm was planted into areas containing old coconut
plantations, and subsequently into second- and third-generation oil palm, in
Asia in the early 1960s, that it became of economic importance.
The increase in the importance of BSR triggered a flurry of research which
resulted in the conclusion that BSR was initiated when roots came into contact
with debris colonized by Ganoderma boninense, and control strategies developed
at that time reflected this view. The recent publication of Hasan and Turner
(1998), which described experiments where seedlings, planted close to BSR-
infected oil-palm stumps, became infected with G. boninense and died within
6–24 months, further supports this view.
However, despite research over 30 years, control measures continued
to produce inconsistent results, and BSR still remained an enigma. Why, for
instance, did BSR only become of economic importance when oil palm was
planted in association with old coconut stands, and subsequently when oil
palm was planted after oil palm? Why did it not become a significant problem
in areas planted into cleared forest with no coconut plantations within the
region, yet become a significant problem when planted into cleared forest in
areas where coconut plantations were present within the region?
The role of coconut in the epidemiology of BSR can be explained by
research over the past few years, which suggests that G. boninense, apart from

* Present address: Mycology Section, Royal Botanic Gardens Kew, Richmond, UK


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 after
felling. In Papua New Guinea (PNG) and the Solomon Islands, despite constant
monitoring, we have never found G. boninense colonizing newly felled hard-
wood stumps or logs within 2–3-year-old oil-palm plantings, nor on old
hardwood stumps and logs in established oil palm.
The role of coconut is well demonstrated in Milne Bay, where levels of
infection in blocks of oil palm planted into coconut north of the Naura River in
1987 (Fig. 9.1) are consistently higher than the incidence of BSR in those
blocks planted into cleared forest south of the river.
If, as our observations suggest, G. boninense does not colonize hardwood
species, then the presence of BSR in the forest blocks south of the river is more
difficult 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. boninense
brackets collected from different palms, including cultures from adjacent
palms, when confronted in a Petri dish in the laboratory, develop a solid
demarcation line where the two cultures met. This somatic incompatibility
demonstrates that isolates, even from adjacent palms, were unrelated. Miller
et al. (1994) also studied the mitochondrial DNA (mtDNA) from the same
isolates and confirmed the above findings.
The results of both studies are hard to reconcile with the single idea of
root-to-root contact, as isolates from adjacent palms would, by association, be
the same clone. They would thus be compatible in culture and have the same
mtDNA banding patterns. The alternative, as suggested by both Miller et al.
(1994) and Ariffin et al. (1996), is that basidiospores are also involved at some
point in the epidemiology of the disease.
Strong evidence for the involvement of basidiospores can be found if we
look at the survey data from four divisions of oil palm planted between 1987
and 1989 in the Solomon Islands (Table 9.1). The incidence of infection within
these blocks ranges from 0% in some of the Mbalisuna and Tubutu blocks

Fig. 9.1. The incidence of basal stem rot (BSR) in oil-palm blocks planted into
felled coconut and cleared forest.

Control Strategy for BSR of Oil Palm: Basidiospores 115

Table 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 planting

Mbalisuna 0.8 .10–1.8 15 453 1987 Oil palm after forest
Tubutu 1.1 .10–2.1 17 297 1989 Oil palm after forest
Metapona 2.0 1.3–2.8 12 186 1988 Oil palm after padi
Ngalimbiu 3.5 1.3–10.2 29 765 1987 Second-generation
oil palm

which 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 an
area of 186 ha, which were planted into land that had been used for growing
rice for the previous 10 years. It is difficult to explain, almost to the point of
being inconceivable, that a level of infection equal to or higher than that in the
oil-palm blocks out of forest could have arisen by roots coming into contact
with inoculum buried in the soil, in land which had been under cultivation
for the previous 10 years. This was not village rice, but a large commercial
operation where the land had been prepared using heavy machinery and the
application 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, as
hinted 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 is
an easy step to explain the presence of infection in the forest blocks south of the
Naura River (Fig. 9.1) and, similarly, the infection in the Mbalisuna and
Tubutu blocks in the Solomon Islands planted in 1987 and 1989 (Table 9.1).
Our research commenced in 1995 to test the hypothesis suggested by both
Ariffin et al. (1996) and Miller et al. (1994) that basidiospores are involved in
the epidemiology of BSR of oil palm, and our research continues to support this
view. Early work at Milne Bay (Sanderson and Pilotti, 1997) revealed that
Ganoderma has a highly developed mating system. Unlike the mating system
normally associated with Ganoderma species, which is based on two loci and
two alleles, the mating system of G. boninense is based on two loci and multiple
alleles. Under such a regime, mating is only restricted within the family. It
is therefore a mating system which strongly encourages outcrossing and
maximizes the ability of the fungus to experiment with new combinations of
aggressiveness genes, which, because of the selection pressures at infection,
will inevitably lead to a build-up of the aggressiveness within the Ganoderma
population. It may hypothetically lead to the infection being seen earlier, and
in higher numbers, in each subsequent planting. This is exactly the situation
that has been described as occurring over the past four decades in Malaysia.


If basidiospores are involved in the life cycle, then a fundamental change
in our thinking is required, regarding the epidemiology of the disease, which
again requires a major change in our thinking regarding control. Thus, if a
source of G. boninense is sporulating in the vicinity, either on dead coconut
or oil palm, and the physical conditions are suitable, then no matter how
complete the hygiene is at the time of replanting, infection will occur.
Control is therefore no longer only dependent on the removal of all
infected wood material, whether below or above the ground at the replanting
site, but also on the maintenance of a zero incidence of G. boninense brackets in
all areas of the oil-palm plantation and surrounding vegetation. With this
objective, a control strategy was developed and implemented in both Milne Bay
in Papua New Guinea and in the Solomon Islands.

The Control Strategy
There are three phases to the implementation of the control strategy:
• during establishment;
• during the growing cycle; and
• during replant.

Control during establishment

Planting into cleared forest in a region free of old coconut plantations is the
simplest and surest way to ensure an oil-palm crop with no, or insignificant
levels, of BSR. Any coconut plantations within the region immediately put the
young crop at risk to infection from G. boninense.
To plant into felled coconut is to provide the scenario for infection, as it is
inevitable 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 local
population of Ganoderma, which in turn will depend on the area of oil palm
already planted within the region, the number of generations of oil palm, and
the extent of the infection of BSR.
If the initial economic losses are likely to be low, such as in regions with no
or a very short history of BSR, then the complete removal of coconut logs and
stumps is not justified. In such instances, our objective is to leave the material
remaining from the previous vegetation in such a state as to limit both bracket
production and spore movement, and to provide minimal breeding sites for
insects such as Oryctes. This is done by leaving as many logs as is practical
stacked above the ground, out of contact with soil moisture, and to encourage
a rapid establishment of ground cover. Control in these situations commences
at 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 of
all coconut stumps and logs has to be considered carefully, as having cleared


all the felled coconut stumps and logs, high levels of infection are still likely to
occur from inoculum arising within the surrounding areas.

Control during the growing cycle

Control during the growing cycle is based on surveys which commence at year
6. These are carried out every 6 months to identify infected palms, which are
marked as either infected with brackets or infected without brackets (we use
5 cm PVC adhesive tape in either yellow or orange so that the palms can be
identified 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 into
the company’s database. Data were later collected directly into a hand-held
GPS (global positioning system) receiver (either a Magellan ProMark X using
MSTAR software with a second ProMark X as the base station, or a Trimble
TDC1 receiver and a Trimble Pathfinder Community Base Station), which
not only records the geographical location but also acts as a data logger. The
data are downloaded into the company database at the end of each day. The
advantage of the GPS receiver, apart from the ease of entering the data into the
database, is the ability to produce a map of the distribution of infection within
the 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 removed
from the plantation. The trunk base and root ring is removed to a depth of
10–15 cm below ground level and the hollow filled with soil. As long as the
infected roots are covered with soil, brackets will not develop.
Palms without brackets fall within two categories: tolerant palms and
palms 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 surveys
for future development of brackets. In our experience only a few of these palms
develop brackets at a later date. Palms without brackets and not producing
fruit bunches are considered sterile and treated accordingly.


Control during replantings

As with all control strategies during the replanting cycle, we emphasize
the necessity of removing all infected plant material lying on the soil
surface. Where we differ, is in the extent to which we remove the old root
system.
The significance of the root ball as an infection source, as suggested by
Hasan and Turner (1998), will diminish, and become negligible, as long as the
seedling palms are planted as far as possible from the old palms.
After each palm is pushed over, the broken trunk base and root ring are
scooped out to a depth of about 30 cm. The hollow is filled with soil and the
stem base and root ring removed from the site, along with the infection
removed from the trunk by chainsaw. Care has to be taken to ensure that all
infection is removed from the site. Exposed basal rot on the trunk, or a root ball
too large to be physically removed, are both scenarios for extensive bracket
production.
The control process at replant starts 2 years before the actual planting
date. During this period all remaining palms with symptoms, both those
with and without brackets, are felled and all infected material removed.
Care must be taken during the felling of the remaining healthy palms
prior to replanting. All palms must be checked and any previously undetected
infection, both in the root ring and trunk, must be removed from the planting
site.

Discussion
There is sufficient evidence in the literature and from field observations to
support the hypothesis that basidiospores of G. boninense are involved in the
life cycle of BSR of oil palm. There is a danger, however, that because we can
still only speculate about this, the involvement of basidiospores is considered of
little consequence.
On the other hand, the implications are far reaching. If the sexual stage
is involved, then segregation will take place, including characters for
aggressiveness. During the infection process, regardless of how this occurs,
selection pressures will inevitably lead to increased aggressiveness. This in
turn will lead to infection being detected earlier and in greater numbers,
exactly as has occurred in Malaysia and Indonesia. Secondly, if basidiospores
are involved in the epidemiology, then the success or failure of the control
strategy not only depends on the actions being taken during the replanting
cycle but, concurrently, how well control is being maintained in all other
facets of plantation management and surrounding vegetation. This degree of
control will, in many instances, be unattainable.


Acknowledgements
It is with gratitude that we thank the European Union for funding for this
project under the STABEX programme. The assistance of the staff at all levels
from Pacific Rim Plantations Pty Ltd, New Britain Oil Palm Pty Ltd, and Hargy
Oil Plantations Pty Ltd, is also gratefully acknowledged.

References
Ariffin, 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.
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.

10
R. Bhaskaran
Management of Basal Stem Rot Disease of Coconut

Management of Basal Stem 10
Rot Disease of Coconut
Caused by Ganoderma lucidum
R. Bhaskaran
Coconut Research Station, Tamil Nadu Agricultural University,
Veppankulam, Tamil Nadu, India

Introduction
Basal stem rot (BSR) disease of coconut, hitherto called Thanjavur wilt,
was first noticed in 1952 in Thanjavur district of Tamil Nadu (Vijayan
and Natarajan, 1972). This disease is also prevalent in Andhra Pradesh,
Karnataka, Maharashtra and Gujarat States, and is referred to as Ganoderma
root rot, Ganoderma wilt, Ganoderma disease, ‘anabe’ or bole rot (Nambiar
and Rethinam, 1986). Wilson et al. (1987) reported the occurrence of a BSR
disease in coconut in Kerala.
In some of the more severely affected coconut gardens, the incidence of the
disease is as high as 30% (Bhaskaran and Ramanathan, 1984) and if the dis-
ease is left unchecked, it may destroy the entire coconut garden within a period
of 7–8 years, if the conditions are favourable for the spread of the disease.
Exudation of reddish-brown, viscous fluid from the basal portions of
the stem, which gradually extends upwards, severe root rotting, decay and
discolouration of internal tissues of the stem, drooping of leaves and death of
the palm are the characteristic symptoms of the disease (Bhaskaran et al.,
1989). Occasionally, some infected palms do not show bleeding symptoms, but
the other symptoms will be present. Ganoderma lucidum (Leyss) Karst and
Ganoderma applanatum (Pers.) Pat. were isolated from the roots of diseased
palm. The above-ground parts of the palm are free from the pathogen. On
inoculation, G. lucidum alone was able to infect and produce symptoms of the
disease (Bhaskaran et al., 1991).


122 R. Bhaskaran

Management of the Disease
Effect of Trichoderma harzianum application with organic manures

A field experiment was initiated during May 1992, to study the effect of
T. harzianum, the fungus antagonistic to G. lucidum, in the control of BSR. The
antagonist was multiplied in rice bran–sawdust medium and applied to the
basins of the diseased tree with different organic manures. The treatments
were given once a year. The results showed that T. harzianum applied with
green leaves, neem cake (NC) or farmyard manure + Bordeaux mixture
(FYM + BM) were more effective for the management of the disease than other
treatments and control (Table 10.1). All the treatments recorded significantly
higher nut yields than the control. FYM, FYM + BM and neem cake treatments
were superior to other treatments.
In addition to assessing disease, microbial populations in the organic
manures applied were estimated at bi-monthly intervals for 1 year using a
serial dilution plate technique. In general, fungal populations increased
markedly up to the fourth month after treatment in all treatments containing
organic manure and decreased thereafter except in treatment with green
leaves, where the population continued to increase up to the eighth month. In
all the organic manure treatments, fungal populations were much higher than
in control soil; FYM and NC recording very high population levels (20 and
18 × 104 cfu g−1 of soil in FYM and NC treatments, respectively, 1 year after
treatment) (Fig. 10.1).
The bacterial population was high in FYM treatment, followed by tank
silt (TS) and the population dynamics followed almost the same trend as that
of fungi, i.e. increase up to the fourth month and thereafter a reduction
(Fig. 10.2). Actinomycete populations increased in the FYM and TS treatments

Table 10.1. Effect of Trichoderma harzianum with different organic manures on
basal stem rot intensity and nut yield.

Disease Nut yield per
Treatment Index palm 1993/94

T. harzianum in 5 kg neem cake 26.0 97
T. harzianum with 50 kg farmyard manure (FYM) 48.4 101
T. harzianum with 200 kg tank silt 59.5 83
T. harzianum with 50 kg coir dust 100.0 88
T. harzianum with 50 kg composted coir dust 57.2 90
T. harzianum with 10 kg poultry manure 72.8 84
T. harzianum with 50 kg green leaves 23.0 88
Bordeaux mixture (BM) 1%, 40 litres 38.6 92
T. harzianum with FYM + BM 28.7 96
Control 92.1 44
CD (P = 0.05) 4.3 5

Management of Basal Stem Rot Disease of Coconut 123

Fig. 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 then
decreased (Fig. 10.3).
Trichoderma populations were high in all the organic manure treatments
when compared to the control (Table 10.2). The population increased up to
the fourth month and then decreased drastically, although the populations
always remained much higher than control soil even in the twelfth month
after treatment. NC and FYM sustained the highest population levels.

Effect of biofertilizers

A field experiment was initiated to test the efficacy of biofertilizers in the
management of BSR. Azospirillum, phosphobacteria and the vesicular
arbuscular mycorrhizal (VAM) fungus Gigaspora calospora were tested. Peat-
based inoculum of Azospirillum and phosphobacteria (200 g) in 10 kg of FYM
per tree year−1 was used. Soil inoculum of VAM fungus (500 g) was used for
each tree.

124 R. Bhaskaran

Fig. 10.2. Effect of organic amendments on the soil bacterial population. FYM,

Disease intensity, recorded up to the end of 1993, indicated that phospho-
bacterial treatment was effective in reducing the disease severity when
compared to the other biofertilizers tested (Table 10.3).
Nut yield was higher in all the biofertilizer treatments as compared to
control. Although phosphobacteria recorded a mean nut yield of 100, which is
less than that of G. calospora and Azospirillum, the yield increased in 1993
when compared to the yield in 1991, while with the other two biofertilizer
treatments there was no yield increase when compared to that in 1991
(Table 10.3).

Management of Basal Stem Rot Disease of Coconut 125

Fig. 10.3. Effect of organic amendments on actinomycete populations. FYM,

Table 10.2. Effect of organic manures on Trichoderma population.
Populations months after inoculation

Treatments 0* 4* 8 ++ 12 ++

Neem cake 52 64 48 45
Farmyard manure (FYM) 44 70 37 40
Poultry manure 40 54 35 32
Tank silt 38 48 30 34
Composted coir dust 37 52 36 30
Green leaves 28 12 40 28
FYM + Bordeaux mixture 46 60 40 38
Control 0 0 0 0

Population × 105 (*) and × 103 (++) cfu g−1.

126 R. Bhaskaran

Table 10.3. Effect of biofertilizers on disease intensity and nut yield of basal
stem rot-affected coconut (experiment initiated in September, 1990; mean of five
replications).

Disease index Nut yield % Increase
over
Treatments 1991 1992 1993 1991 1992 1993 Mean control

Azospirillum 200 g per
10 kg 21.6 53.3 64.3 113 112 92 106 37.7
Phosphobacteria 200 g
per 10 kg 1.7 4.9 34.8 87 110 102 100 29.9
Gigaspora calospora 20.2 45.7 50.6 108 118 108 111 44.2
Control 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 fungicides

In the field experiment on the efficacy of fungicides in the management of
BSR, fungicides were given as root feeding at quarterly intervals for 1 year
and 5 kg of NC was applied every year. The results (Table 10.4) indicate that
aureofungin-sol and tridemorph are very effective during the first 3 years,
but in the subsequent years the disease intensity gradually increased. This
indicates that the trees are not permanently cured of the disease and there is
only suppression of symptoms.

Conclusion
Basal stem rot disease is a major disease limiting coconut production in India.
Treatment of the diseased palms with fungicides does not offer a permanent
cure to the affected tree. Biological control with T. harzianum and phospho-
bacteria offers some scope for containing the disease but organic amendments
are essential to encourage antagonistic microflora, and treatments which
included organic amendments had least disease and better yields of coconuts
than those without amendments.

Table 10.4. Efficacy of fungicides in the management of basal stem rot disease of coconut.

Disease Index Nut yield per palm

Treatments 1988 1989 1990 1991 1992 1993 1988/89 1989/90 1990/91 1991/92 Mean

Neem 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 42
NC + 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 49
NC + 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 97
NC + 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 75
NC + 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 62 102
Control 43.6 97.5 98.0 98.5 99.0 98.5 53 57 31 4 36
CD (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. Bhaskaran

References
Bhaskaran, 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.
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.

11
R.R.M. Biodegradation
In vitro Paterson et al. of Oil-palm Stem by Fungi

In vitro Biodegradation 11
of Oil-palm Stem Using
Macroscopic Fungi from
South-East Asia: a Preliminary
Investigation
R.R.M. Paterson1, M. Holderness1, J. Kelley1,
R.N.G. Miller2 and E. O’Grady1
1CABI Bioscience, Egham, UK; 2Departmento de
Fibopatologia, Universidade de Brasília and Universidade
Católica de Brasília, Brasília, D.F., Brazil

The Problem
The main thrust of this chapter reflects the experiences of the authors in
South-East Asia, and in particular Indonesia and Malaysia. However, it is
assumed that the issues are relevant to other oil-palm (OP) growing regions of
the world. OP is an extremely important crop to South-East Asia. For example,
it is estimated that 2.9 million ha of Malaysia will have been planted with OP
by 2000.
The basidiomycete fungus Ganoderma is considered to be one of the most
important diseases of OP in South-East Asia (Chung et al., 1998). Similarly,
Orycytes rhinoceros is an insect pest of OP and a yield loss as high as 25% over 2
years from an initial infection has been reported (Liau and Ahmad, 1991),
although Wood et al. (1973) claimed that damage to immature palms resulted
in only small crop losses. When the productive lives of OP are complete, they
are felled, creating a vast amount of waste product – the trunks are placed in
windrows while boles often remain untreated in the ground. Spread of the
aforementioned pests has been reported to occur from infected OP, and from
those OP residues left in the field (Hasan and Turner, 1998; Flood et al., this
volume) and consequently, an effective way of managing infection is by elimi-
nating all infected material (Hasan and Turner, 1998). Historically, OP stem
(OPS) was burned to remove it and potential pests from the plantation floor.
However, in 1994, open burning of crop residue was completely banned in


130 R.R.M. Paterson et al.

Malaysia due to a persistent haze problem. Similar problems with haze have
been experienced in other countries, such as Indonesia. The Malaysian ban
was relaxed in some regions where the disease became a renewed problem for
the industry (Haron et al., 1996), which illustrates the dilemma faced by many
producers. In addition to the need to reduce sources of infection, there is simply
the requirement to remove the OP residues (OPR) per se as plantations would
become unmanageable due to the accumulation of the waste material.
A potential disadvantage of burning is that nutrient loss from the soil may
be incurred. Haron et al. (1996) demonstrated experimentally that nutrients
were replenished in the soil and positive effects were obtained by chopping and
shredding or pulverizing the residues and spreading these around OP. A saving
on fertilizers of RM 28 million per annum over a 4-year period at 1996 prices
was estimated if the procedure was taken up by the Malaysian industry as a
whole, but, by not burning, the problem of O. rhinoceros was retained, albeit
at 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 was
not considered. On the other hand, Haron et al. (1998) demonstrated that OPR
left 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 not
affect SOM.
Another plantation practice is to submerge OPS in lagoons rather than
leave them in windrows. This is also highly polluting and does not tackle the
large amounts of waste produced. In other estates it is current practice to chip
some of the OPS and stack it in windrows to promote decomposition (Hasan
and Turner, 1998). This procedure does not deal with the large amount of
waste product available, and the process takes a long time (approximately
2 years) to complete, allowing pests and pathogens to survive. A process that
can reduce this time to approximately 6 months would be of great benefit.
Thus, there is considerable interest in removing OPR in a quick and benign
manner from the plantation floor, despite some of the factors described above.
Towards this end, certain fungi can completely degrade plant material and so
it 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 competitive
with any other fungus found in or on the OPS. An alternative approach is to
use the OPS as a resource for the production of edible mushrooms and/or feed
for 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 in
developing a practical process in vivo. Many of the methods described have
been used in conjunction with OPS for the first time. However, no attempt has
been made to analyse the data statistically because of the preliminary nature
of the work. Also, although the studies were conducted on OPS, most of

In vitro Biodegradation of Oil-palm Stem by Fungi 131

the results could also probably be applied to OP boles, which cause similar
problems to OPS, although they are even more difficult to treat as they are
firmly embedded in the soil by the root system.

A Solution
The following experimental procedures may offer methods for a solution to the
OPR problem discussed above.

The fungi

Descriptions of some of the fungi isolated are given in Treu (1998), and a full
list of strains used is available.

Enzyme assays

Isolates (59) were tested for the production of cellulase, ligninase and amylase
by inoculating them on to appropriate test media and measuring zones of
clearance after incubation (Paterson and Bridge, 1994). Each permutation of
activities 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 specific
biodegradative tasks. For example, high amylase activity will be useful in the
degradation of OPS because of its high starch content (Oshio et al., 1990; see
p. 134). Taxa with the same names often had similar enzyme activities. For
example, six Marasmius strains had similar ligninase and amylase activities
but no detectable cellulase. The possession of this combination may be useful
for increasing the digestibility of oil palm as a ruminant feed (Kelley and
Paterson, 1997). The fungus has the potential for removing starch and lignin
but presumably has limited or no capacity to degrade cellulose. So the final
product of degradation could have a high cellulose content and, as such, may
be suitable as a ruminant feedstuff. The observation that the Ganoderma strains
only had detectable amylase activity is surprising as they are generally consid-
ered to be white-rot fungi and so ligninase would be expected. However, the
fungus 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 detected
for 19% of strains, although some of these had grown and so some enzymatic
activity must have been present.


Growth assessment

A simple assessment of growth of the collected fungi on OPS (without bark)
from a Malaysian plantation was devised. OPS tissue (1 g) was placed into
20 ml universal bottles with metal screw caps. Ammonium dihydrogen
phosphate and deionized water were added to obtain an approximate 50 : 1
C/N ratio and 70% moisture, determined by Rao et al. (1995) to be optimal for
the composting of poplar wood in the absence of similar data for OPS. The OPS
in the universals was inoculated with the fungi while uninoculated OPS and
unsterilized OPS were incubated as controls. Water (0.7 ml) was added to each
bottle to restore moisture. A visual assessment of growth was made for
each sample.
Thirty of the treatments were positive for growth. A black fungus-like
organism appeared on the unsterilized control and had the highest visual
assessment rating of all samples. Interestingly, a black fungus-like organism
has been isolated from OPS in Papua New Guinea, which appeared to be
responsible for heavy degradation (P. Bridge, personal communication) and
may 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 growth
between replicates was generally low. However, there were some strains in
which only one of the three replicates grew, probably reflecting a problem with
the 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 unsterile
control (covered with black fungus – see above), Hydnum (IMI 370939) and
Pleurotus djamor (IMI 307936) were assessed as having more growth than the
fastest growing Ganoderma (IMI 370917). In conclusion, visual assessments
are only an indirect and qualitative measurement of OPS biodegradation, but
they are inexpensive to perform and appear to give consistent results, although
inoculation procedures need to be standardized.

Weight loss

Weight loss was also determined for the above treatments. Weights of the
bottles 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 sterile
distilled water to restore moisture. The accumulated percentage weight loss
was determined.
Weight changes of replicates indicated a great deal of variation in some
cases. However, the three individual Marasmius cultures gave consistently
high figures. The weight data from the samples that did not grow had a
surprisingly wide range, from 39 to −26%. It is possible (but unlikely) that
growth had occurred but was not visible, accounting for the higher values.
Alternatively, water evaporation may have been affected by variation in the


fitting of the caps of the universal bottles, and/or location of samples within the
incubator. 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 highest
loss 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 the
Marasmius cultures did not grow at all, but in the case of IMI 370892, 370929
and 370943, where only one of the replicates grew, high weight losses were
recorded. The high weight loss (44%) from material inoculated with Hydnum
(IMI 370939) is interesting, as the other Hydnum strains did not cause large
weight losses. IMI 370939 possessed high amylase and apparently no other
enzyme activity. The highest weight loss from a Ganoderma strain was 26% for
strain IMI 370917, with a maximum rate for weight loss of 2% day−1 between
days 21 and 29. Seven strains had higher weight loss values than this strain
and 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 seemingly
had little or no cellulase or ligninase, again indicating the importance of
starch degradation. Weight gains were recorded from the unsterilized OPS
which contained the black fungus-like organism, perhaps resulting from
greater evaporation from the sterile control. In general, there appeared to be a
correlation between weight loss and visual assessment of growth.
Weight loss determinations are inexpensive and numerous strains can be
analysed in individual experiments. They are also a direct measurement of the
information that is required, i.e. how much and how quickly is OPS being
degraded. However, there is evidence of a high degree of variation in some of

Table 11.1. Accumulated percentage weight lossa from OPS treated with fungi
that gave a higher weight loss than the most efficacious Ganoderma.
aAccumulated % weight loss at time (days)

Fungus IMI no. 3 7 14 21 29

Lenzites (3) 307902 4 7 17 31 46
Marasmius (1) 370892 7 8 15 35 46
Hydnum (3) 370939 5 6 12 24 44
Marasmius (1) 370929 3 5 24 35 43
Marasmius (1) 370943 1 6 17 24 41
Corticum (3) 370935 3 11 29 30 32
Trametes hirsuta (3) 370898 4 6 8 18 26
Ganoderma (1) 370917 −1− 1 −1− 8 26

The 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 genetic
resource collection.
aAccumulated percentage wieght loss minus percentage weight loss from sterile

controls.


the measurements and these particular experiments need to be refined in any
future studies.

Ergosterol analysis

Ergosterol is a lipid contained in the cell membrane of fungi which will tend
to increase in amount as fungi grow. The compound is virtually unique
to fungi, and is increasingly being used as an estimation of fungal biomass.
Universal bottles containing 1 g of OPS as above, were inoculated with
Hydnum (IMI 370893) and Polyporus (IMI 370891) and the complete contents
were used for analysis (1 bottle per sampling period). Samples were analysed
for ergosterol by the method of Gao et al. (1993) using high-performance liquid
chromatography (HPLC).
The concentrations of ergosterol increased with the visual estimation of
growth (Fig. 11.1) at least until the growth phase had ended. Maximum
concentrations of ergosterol were 46 and 44 µg g−1 on day 14 and day 21 for
Hydnum and Polyporus, respectively. Maximum rates of increase of ergosterol
were 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 ergosterol
concentration and the visual assessment, and the two sets of data were similar
for both fungi.
It is not known whether ergosterol estimation or visual assessment is the
more accurate measurement of fungal biomass on OPS. Bermingham et al.
(1995) provide evidence that ergosterol concentration varies between taxa. It
is being considered increasingly as the method of choice for measuring biomass

Fig. 11.1. Ergosterol and visual rating of Polyporus on 1 g oil-palm stem.


in solid substrates such as food (Pitt and Hocking, 1997) but it does not provide
information on the amount, or which components of OPS, are being degraded.
The extraction procedure used here is time consuming and involves the use of
large 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 adequate
and priced at the cheaper end of the market. Finally, an inexpensive (although
only semi-quantitative) method involving thin-layer chromatography (TLC)
may be practicable.

Respirometry

Respirometry analysis involves measuring the amount of oxygen that is
consumed by microorganisms growing on solid substrates such as composts.
Oxygen consumption was measured using a CES multi-channel aerobic
respirometer (Co-ordinated Environmental Services Ltd, Kent, UK). Blocks of
OP (ca. 5 g) were enriched with ammonium dihydrogen orthophosphate. Each
sample was inoculated with Hydnum (IMI 370939), Trametes (IMI 370898),
Ganoderma (G3) or Pycnoporus (IMI 370937). There were four samples per
treatment. Three control flasks containing uninoculated amended OPS were
included and one flask was inoculated with Trametes (IMI 370898) and
Pycnoporus (IMI 370937).
The sequence of oxygen consumption by fungi, from highest to lowest,
was Hydnum, Trametes, Ganoderma and Pycnoporus (Fig. 11.2). However, the
initial mean water concentrations of the OPS were 55%, 58%, 59% and 62%,
respectively, for material inoculated with Pynoporus, Ganoderma, Trametes and
Hydnum, so the amount of growth could have been influenced by the different

Fig. 11.2. Oxygen consumption by fungi grown on oil-palm stem (mean values).


water and ammonium salt concentrations of the OPS and may not reflect
actual 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 to
be useful for assessing growth. However, the respirometer is expensive and
only a small number of strains can be analysed in individual experiments.

Enzyme digestibility

Enzyme digestibility analysis involves the sequential degradation of plant
material by commercial enzymes such as cellulase, pronase (‘proteinase’) and
amylase. 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 procedure
involves the sequential enzymatic degradation of the various components of
lignocellulosic material in vitro (Abe and Nakui, 1979). Limited investigations
of the enzyme digestibility of OPS indicated that 30% of the stem was digestible
by glucoamylase and pronase on day 0 (pronase digestion alone indicated a
protein content of approximately 2%). This decreased to 20% by day 7 for
Ganoderma (project no. 29) and Marasmius (IMI 370929). Digestibility was
26% after 7 days in the case of the Trametes (IMI 370934). However, cellulase
digestibility only decreased from 13% to 11%, confirming the view that starch
is the preferred substrate. Total digestibility decreased from 43% to 32% in the
cases of Ganoderma and Marasmius, and to 37% for the Trametes treatment in 7
days. The standard deviations were generally small (ca. 5%). The OPS became
increasingly indigestible as the fungi grew, and presumably as the result of an
increase in percentage lignocellulose. Fungi capable of completely metaboliz-
ing lignocellulose would be required when the other substrates have been
depleted. Enzyme digestibility assays give a profound insight into the chemical
composition of lignocellulosic material in general and how the substrates
change as biodegradation progress. However, they are time consuming and
the enzymes can be expensive.

Future Studies
Much more fundamental work is required on the physiology of these fungi
to determine the optimal temperatures, C/N ratios, nutrients, pH, water
potentials, etc. for growth and enzyme production and, ultimately, OPS bio-
degradation for the individual fungi. A rigorous statistical analysis is desirable
in future work. In vitro investigations involving the use of unsterilized OPS,
including the bark, are required to determine whether an inoculated fungus
can colonize and degrade OPS quicker than the indigenous microbial popula-
tion. Research involving the use of consortia (i.e. mixed inocula) of fungi


and other organisms may be worthwhile especially when considering how the
digestibility of OPS changes with time; a cocktail of organisms with compatible
enzyme capabilities may be required. Further work is necessary in standardiz-
ing some of the procedures described in this chapter. Pilot-plant investigations
are also required on larger pieces of OPS to make the transfer of the technology
to the field more predictable. However, this does not preclude undertaking field
trials to establish whether candidate fungi can degrade OPS quickly in vivo
without the need for further work in vitro.

Conclusions
In conclusion, the various methods used here to assess the biodegradation
of OPS indicate that after a lag phase of about 7 days some fungi have begun to
grow visibly and reduce the weight of OPS. They appear to grow and degrade
in 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, which
exists at a high concentration. The more resistant substrates, such as
lignocellulose, will probably only be substantially metabolized after this
phase. Visual inspection, ergosterol and oxygen consumption give an indirect
measure of the growth of the fungi and degradation of OPS. Weight-loss
measurements 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 and
degrade OPS more effectively than Ganoderma and is certainly a candidate for a
full-scale process. However, some Marasmius species are also known to be
pathogenic to OP, so great care would be required to ensure that any treatment
in the field does not involve a pathogenic strain of the fungus. It should perhaps
be pointed out that if Marasmius can outcompete Ganoderma on OPS in vivo, an
increased incidence of the former disease may become apparent, because of the
current practice of leaving the OPS on the plantation floor. Indeed, some of the
Marasmius strains discussed here were isolated from OPS which had been
decayed heavily by the fungus. Some of the other fungi with high visual
growth and weight loss assessments are also potential candidates for further
study. A battery of procedures has been developed in this study which can be
used in larger-scale projects, leading to an effective treatment for the rapid
biodegradation of OPS.

Acknowledgements
Stephan Wilkinson, DERA, PLSD, CES Sector, Sevenoaks, Kent, UK for the use
of, and assistance with, the respirometry equipment.


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12
Functional Units in Root Diseases

Functional Units in Root 12
Diseases: Lessons from
Heterobasidion annosum
Department of Forest Mycology and Pathology, Swedish
University of Agricultural Sciences, Uppsala, Sweden

Hierarchy of Units
Throughout human history, classification has been the basis for shaping our
concepts about the world. Fungi have also been subjected to classification, i.e.
assigning an organism to a defined category (taxonomy). These categories
have scientific names and can be placed in relative orders to each other. Within
the Ganodermataceae there is considerable uncertainty surrounding these
ranks. In this chapter we will discuss issues that apply to the genus, species,
population and individual level in the genus Heterobasidion. Heterobasidion is,
like Ganoderma, a polypore grouped in the Aphyllophorales, and the resolution
of taxonomic ranks in this genus might provide an example for future studies
in Ganoderma. The rank of species is basic, but there is no universally applicable
definition of it. Most definitions build on morphological characters, but those
that make use of functional aspects of fungal life are the ones that potentially
are the most useful to practical applications. Three elements are common
in many of the proposed definitions: (i) morphological, a species is defined by
a given set of common features not shared by other groups; (ii) phylogenetic,
a species is defined by its shared evolutionary history and descent from a
common ancestor; and (iii) biological, a species is defined as a group of actually
or potentially interbreeding populations which is reproductively isolated from
other such groups.
One species contains many individuals, which can be grouped in various
ways. For example, the term subspecies is used were the differences between
the species is not big but recognizable. Variety and race are terms used to
describe groups of individuals within a species which have common features


140 Å. Olson and J. Stenlid

that 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.

Genus
Genus is the principal rank in the nomenclatural hierarchy closest above
species. In general, genera are defined with emphasis on several discontinuities
in fundamental characters, especially the reproductive structures. Biological
meaningful functions such as mode of nutrition have sometimes been used
to 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 evolution
proceeds 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 the
family Bondarzewiaceae in the order of Aphyllophorales. The genus includes
six 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 of
the northern hemisphere, including Europe, North America and Russia. The
fungus can infect and kill fully grown trees; its principal hosts are conifers.
Recently, H. annosum has been suggested to be comprised of three separate
species, H. annosum, H. parviporum and H. abietinum (Niemälä and Korhonen,
1998), but several issues still remain to be clarified (see below). H. araucariae is
very similar to H. annosum but has larger pores and larger basidiospores. It was
separated from H. annosum on the basis of intersterility, ecology and geograph-
ical distribution (Buchanan, 1988). It is a saprotrophic species that inhabits
dead wood of Agathis and Araucaria species in eastern Australia, New Zealand,
New Guinea and some islands in the Pacific Ocean. H. insulare has a reddish
surface to the pileus and irpiocoid pores (Buchanan, 1988). The fungus is a
saprotroph on wood from Abies, Pinus and Picea and is distributed in southern
and eastern Asia (Niemälä and Korhonen, 1998). H. pahangense was found in
Malaysia by Corner (1989). It is characterized by large pores (2–4 mm−1) and
it has ornamented spores (Stalpers 1996). H. perplexum is pileate, the surface is
ochraceous or pale brown and glabrous, its pores are 2–4 mm−1 and the spores
measure 5–7 × 4–5 µm. It was found growing on Tsunga in Nepalese moun-
tains (Ryvarden, 1989). H. rutilantiforme has a glabrous and reddish-brown
basidiocarp, and is a tropical American species (Ryvarden, 1985). The pores
are 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 species
should share a common ancestor, not common to other genera or species.
Traditionally, this has been hard to achieve, since convergent evolution is very
common among fungi. The macromorphological characters that served as the

Functional Units in Root Diseases 141

basis for early taxonomy have frequently proved to be the result of convergent
evolution and, thus, resulted in many paraphyletic genera, and grouping
together of unrelated taxa. The advent of PCR (polymerase chain reaction) and
relatively easy access to DNA sequencing have helped in providing a range of
molecular markers for taxonomic work. Among the most popular markers
that yield useful variation at genus or species level are the ribosomal DNA
genes and their spacers. Cladistic analysis of a large number of DNA sequence
characters 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 (Harrington
et al., 1998; Fig. 12.1).

Species
Species concepts

The species concept has been, and still is, a subject for debate. Different
definitions of a species are used for different purposes:
1. In the morphological species concept, a species is defined by a given set of
common morphological features not shared by other groups. This view is not
feasible 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 shared
evolutionary history and descent from a common ancestor.
3. In the biological species concept, a species is defined as a group of actually
or potentially interbreeding populations which is reproductively isolated from
other such groups.
Ecological or geographical aspects are often used to help to define the life
history traits and geographical boundaries of the distribution of a species.
Sympatric species co-occur in the same geographical location but are normally
separated by differences in choice of substrate or hosts, while allopatric species
are separated by large geographical distances. Vicariant species are those with
a limited geographic distribution and where other species with an overlapping
niche can appear under similar circumstances in a different region.
In mycology, the morphological species concept has been used widely
because of its historical association with botany. This has not always been
reliable, although fungi have a high developmental plasticity and relatively
simple 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 totally
reproductively isolated subgroups within morphospecies (Brasier, 1987). In


the biological species concept, the emphasis is on the biology of the species,
especially on the actual or potential interbreeding of the populations and on its
reproductive isolation from other such populations. Reproductive isolation
can occur in several ways: (i) geographically, where populations are separated

Fig. 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 bootstrap
values (greater than 50%) and decay indices (d value) are shown below branches.


by barriers such as mountains or oceans; and (ii) ecologically, where
populations are separated by different ecological niches, i.e. climate, living
or non-living substrate, or host preferences for pathogens. The interbreeding
population can be defined in terms of numerical size, geographic size and
genetic structure. This will show the potential for gene flow between the
individuals in the population. We will go through the data that are important
for defining the functional unit and the species concept of H. annosum, with
emphasis on the biology of the fungus, but also take into account the available
morphological data.

Mating compatibility in Heterobasidion

Interbreeding can be limited in several ways – geographically, ecologically and
genetically. 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 is
the closest place between them, and the site where spore transfer would
theoretically be possible. Another possible barrier would be high mountain
ranges such as the Ural mountains, even though Heterobasidion spores have
been shown to travel up to 320 km over open sea (Kallio, 1970). One way
to overcome these barriers is if spores are transported by a vector of any
kind, most obviously wood or plants transported by man between the
continents.
By using compatibility tests, three different intersterility groups were
detected in H. annosum. The P-group, originally found on pine trees in Finland,
comprised isolates compatible with each other but not with isolates from the
S-group, which was isolated originally from spruce in Finland (Korhonen,
1978a). A third group was subsequently found on Abies alba in Italy (Capretti
et 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 from
a basidiocarp represents either of two mating types (Korhonen, 1978b; Chase
and Ullrich, 1983; Holt et al., 1983; Stenlid and Rayner, 1991). The bipolar
mating system is determined by a mating factor, a gene or a gene complex in
one chromosome. Allelic differences in this/these loci result in different mating
types (Raper, 1966). The number of mating-type alleles is large in H. annosum,
probably more than 100 types, although local populations may contain
only 10–20 (Chase and Ullrich, 1983; Stenlid, 1985). Isolates of the same
mating type are incompatible, but they are compatible with isolates of a
different mating type. Random pairings within a population are, in most
cases, compatible.
In a mating between two homokaryotic mycelia, there are four possible
outcomes:


1. A compatible reaction showing a continuous mycelia when the
homokaryons have the same genotype or are subcultures from the same
mycelium.
2. A compatible reaction with changed colony morphology and the
appearance of clamps, indicating that the isolates belong to the same breeding
unit.
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 are
paired.
4. An incompatible reaction, resulting in a zone of dense and usually
pigmented 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 will
give rise to a clearing zone and changed morphology, and will also lead to
clamp formation in the homokaryotic isolate. If the isolates are incompatible,
a clearing zone will arise, but a gap heterokaryon could be produced. This
is called the Buller phenomena (Buller, 1931). These new heterokaryons
apparently arise from anastomoses between homokaryotic hyphae from each
parent, or perhaps between homokaryotic and heterokaryotic hyphae. The
outcome of such anastomoses is controlled by mating-type compatibility
(Hansen et al., 1993b).
Pairing among American P-isolates was compatible in 94% of the
cases, and 95% of the pairing among European P-isolates was compatible
(Harrington et al., 1989), while pairings between homokaryotic American
P-isolates and homokaryotic European P-isolates only resulted in 53% of
dikaryons (Harrington et al., 1989). In another study, European P- and
North American P-isolates were compatible in ca. 95% of cases (Stenlid and
Karlsson, 1991). When American fir isolates were paired with European
S-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 northern
European 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 and
Karlsson, 1991).
In 1990, Chase and Ullrich described a genetic system to explain the
mating between and within intersterility groups in H. annosum (Chase and
Ullrich, 1990a, b). The system consists of at least five genes, called S, P, V1, V2
and V3, each with a + and a − allele. Two homokaryotic mycelia can mate if
they both posses a + allele for at least one of the five genes. They cannot mate
if all five combinations are +/− or −/−.
Intersterility determines the limits of an interbreeding population,
whereas incompatibility regulates inbreeding and outbreeding within an
interbreeding population.


Morphological differences in Heterobasidion

The different intersterility groups of H. annosum have very similar properties,
they have a wide and overlapping distribution and, although they exhibit
different preferences for host species, their host specialization is partly
overlapping and not strict. Their morphological characteristics are also partly
overlapping (e.g. spore and hymenial pore dimensions), making it not too easy
to tell the different intersterility groups apart. The morphological differences
within the three European intersterility groups were examined by Mugnai and
Capretti (1989), while differences between the S- and the P-group have been
investigated several times (Korhonen, 1978a; Stenlid and Häggblom, 1985;
Negrutskii et al., 1994). The best diagnostic character is the length of the hair
on the margin of the basidiocarp (Korhonen, 1978a; Mugnai and Capretti,
1989; Negrutskii et al., 1994). The length of the hair in the intersterility
group is: P, 20.9 ± 2.2 µm; S, 119.5 ± 8.0 µm; F, 54.8 ± 3.3 µm (Mugnai and
Capretti, 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), while
there were no differences between the P- and the F-groups (Mugnai and
Capretti, 1989). This makes pore size a reliable diagnostic character to use for
identification of the P- and S-groups in geographical areas were the F-group
does not exist. The small differences in length and width of basidiospores and
conidiospores make them useless for identification (Korhonen, 1978a; Stenlid
and Häggblom, 1985; Mugnai and Capretti, 1989; Negrutskii et al., 1994).

Differences in ecology and pathogenicity

The fungus has been reported from almost 150 woody plant species (Sinclair,
1964; Hodges, 1969; Laine, 1976). It is spread over the whole temperate
region of the northern hemisphere (Hodges, 1969).
The P-type is pathogenic to mature Pinus as well as to other Pinaceae, other
conifer and even hardwood species (Korhonen, 1978a; Worrall et al., 1983;
Stenlid and Swedjemark, 1988; Harrington et al., 1989; Swedjemark and
Stenlid, 1995). Infection centres in pine stands are often associated with
stump-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 canals
in 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-type
through wounds (Shaw et al., 1994). The S-type is mainly restricted to Picea
species, but can also attack small seedlings of other tree species (Korhonen,
1978a). The S-type seems largely dependent on Picea stump tops for initiation
of new infection centres (Stenlid, 1987). Interestingly, Korhonen et al.
(1997) recently reported that, in the Ural mountain region, the S-type infects


Abies sibirica, indicating that in regions where the F-type is absent, the S-type
might expand its ecological niche. Moreover, the geographical distribution of
the intersterility groups suggests that a broad host range might be a basal
character in the S/F complex.

Phylogeny of rDNA genes

The primary definition of intersterility groups (ISGs) is provided by in vitro
mating compatibility tests. Now, molecular genetic analysis methods are
available 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 sequence
data from the ITS region of the nuclear ribosomal DNA and the IGS region
support a view of three major clades in the H. annosum complex: the American
pine 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 as
the difference between either of them and the fir clade (Harrington et al.,
1998). These findings are also supported by random amplified polymorphic
DNA (RAPD) data from Garbelotto et al. (1993). Both the RAPD and the ISG
data weakly support a separation of American and European isolates. No
support is found from variation in the ribosomal genes for a separation of
European S-isolates from F-isolates, even though they are clearly separated
in mating tests and have different host preferences (Capretti et al., 1990). The
European 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 differences
in isoenzyme patterns (Karlsson and Stenlid, 1991; Otrosina et al., 1993). The
North American S/F-type appears to be more related to the European S-type
than 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 data
from enzyme systems that have a putative selection value for the organisms
are used, the separation into ISGs is more clear than when neutral markers are
used. Karlsson and Stenlid (1991) reported that zymograms of pectinolytic
enzymes clearly separated the European S-, F-, and P-groups as well as
the North American S/F- and P-groups from each other. Laccases and
saprotrophic wood degrading capacities differ among the European S-
and P-groups (Daniel et al., 1998; Johansson et al., 1999). Also phylogeny of
the 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


for 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 remains
to be solved is the relationship between these three species and their North
American counterparts. For example, should the North American S/F-group
be named H. abietinum or H. parviporum? The ITS and IGS phylogeny clearly
shows that the North American S/F-group has a long history, independent
from its European relatives, while the morphology of the fruit bodies, although
not fully examined, cannot be clearly separated from them (Hood, 1985). The
North American S/F-group is also highly compatible with both the S- and
F-groups from Europe. Should we decide to give the North American S/F-group
a separate name? Also, what about the relationships in the P-group? North
American and European populations are very similar in pathogenicity and
morphology, and also highly compatible, yet they have a long history of
separate evolution as deduced from the ITS and IGS geneology. Naming fungi
has perhaps become even harder now with all the conflicting data available to
science.

Potential Interbreeding in Heterobasidion
To be able to interbreed, it is not enough to live in the same geographic
area, potential candidates also have to occupy the same ecological niche. In
H. annosum, this is a potential barrier since the different intersterility groups
inhabit different host trees. However, a certain degree of overlap in host range
does occur between the various intersterility groups. Furthermore, this barrier
can be bypassed in the relatively new habitat with limited host defence
made available through stumps created by forestry practices (Swedjemark
and Stenlid, 1993). On one occasion, a hybrid isolate was found with several
characteristics of both a P- and an S-isolate (Garbelotto et al., 1996).

Population
A prerequisite for meaningful population studies is that there is variation
within the species under study. Variation among natural populations is
the 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 the
population through natural selection or stochastic processes such as genetic
drift. Natural selection favours mutations that lead to higher fitness, basically
the probability of having viable offspring. Genetic drift is the process of
randomly drawing subsamples of a population that will found the next
generation. This will, with time, lead to the random exclusion of some
genotypes, 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 of
different alleles at a locus throughout the population.


Within a species, there are normally several geographically separated
populations. However, populations are typically not completely isolated from
each other. Migration among populations leads to gene flow that counteracts
the forces leading to differentiation. Among populations in equilibrium, only
one migrant per generation is needed to counteract the effects of random
drift, independently of the population size (Slatkin, 1985). Isolation leads to
differentiation and gene flow makes populations more similar. Small, isolated
populations are likely to be relatively homogeneous and any genetic variation
is likely to occur at the regional scale. Large populations are likely to be more
variable, but between populations, variation may be lower. How does this
relate 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. Spore
dispersal studies indicate that the vast majority of spores fall within a few
metres of the fruiting body. Only about 0.1% of the spores trapped at 1 m can
be 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 has
fallen to a level no greater than the background spore deposition (Möykkynen
et al., 1997). However, given the enormous amounts of spores produced by
basidiomycete brackets, there is still a fair chance for some of the spores to
travel over large distances. Calculations based on natural spore dispersal
gradients show that one spore of H. annosum can land on the stump surface of a
normal thinning operation more than 500 km away from its source during the
time that such surfaces are susceptible to H. annosum (Stenlid, 1994). Viable
spores have indeed been collected on islands more than 300 km away from
any conifers (Rishbeth, 1959; Kallio, 1970).
Indirect measures of gene flow aim at studying whether differentiation
between populations occurs. If there is a strong differentiation, one can infer a
lack of random mating between the studied populations. However, lack of
differentiation does not necessarily imply gene flow. Two principally different
marker systems have been used for this purpose: mating-type alleles and
arbitrarily primed DNA. Mating-type alleles were scored using mating tests
in Vermont, USA (Chase and Ullrich, 1983) and in Sweden (Stenlid, 1985).
The likelihood of finding the same mating allele was calculated on various
geographical distances. Interestingly, when studied on the geographical scale
similar to the one used for calculation of likelihood of long-distance spread
of 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 than
100 km was about 0.1%, corresponding to approximately 1000 mating alleles
present in the whole species, which is a high but not unique figure (Ullrich and
Raper, 1974). Similarly, when studying variation in arbitrarily primed DNA, a
differentiation in similarity among populations was seen at distances above
approximately 500 km (Stenlid et al., 1994). Later, more detailed studies have
shown a limited but significant differentiation (8.8% of total variation in the
P-group) between populations in western and eastern North Europe (Stenlid


Fig. 12.2. (a) Long-distance spread of spores of Heterobasidion annosum:
numbers of spores dispersed from a sporocarp at various distances, according to
predictions from actual catches. (b) The chance of picking identical mating alleles
(= incompatible pairings) of H. annosum in random samples of basidiospores at
various distances.

et al., 1998). An interesting differentiation was detected between northern
European S-populations and one from the alpine region in Italy (Stenlid et al.,
1994). This coincides with the higher intersterility between the sympatric
southern European S- and F-groups compared with the allopatric northern
European S- and southern European F-populations (Korhonen et al., 1992).
In conclusion, most H. annosum spores are deposited within 100 m of a
fruiting body, but the relatively few that are spread long distance are enough
to ensure a large-enough gene flow to counteract differentiation at distances


less than 500 km. Within a continent, differentiation may be associated with
isolating mountain ranges or connected to historical spreading patterns. Gene
flow between continents is not likely to be a significant factor.

Individual
The attributes that have been used classically to characterize individuality
are genetic homogeneity, genetic uniqueness and physiological unity and
autonomy. For a more extensive discussion about individuality, see Santelices
(1999). Among fungi, many individuals lack genetic homogeneity, genetic
uniqueness and autonomy (Santelices, 1999). Genetic homogeneity is absent
since many fungal species grow and propagate through autoreplication of
genetically identical units, which can survive and function independently.
This enables a given genotype (genet) to be exposed simultaneously to various
environments, with different probabilities of survival and propagation. Physio-
logically separate parts of a fungal genet have been called ramets (Brasier and
Rayner, 1987). Separate ramets can, upon contact, anastomose and form a
functioning entity. A genet is a discrete package of genetic information that
reproduces vegetatively, and could be looked upon as a mitotic line between
meioses.
In basidiomycetes, a polygenic, multiallelic system, called somatic incom-
patibility (SI) or vegetative incompatibility, is present that functions to
restrict physiological and genetic access following non-self anastomosis. The
significance of SI may be to limit the spread of mycoviruses (Caten, 1972)
or maladapted nuclei through a population by maintaining the integrity of
fungal individuals (Rayner, 1991). This system has been studied in some detail
in H. annosum (Hansen et al., 1993a, b). Following fusion of two hyphae, a cell
death response may occur in the fusion cell. This response is much stronger
in aerial than in submerged mycelium and results in a zone of sparse aerial
mycelium. In wood, such interaction zones remain relatively undecayed. In
the interaction zone, a complex pattern of interactions occurs (Hansen et al.,
1993b). If two heterokaryotic mycelia interact, four nuclear types can meet
transitionally in the same cell. Furthermore, H. annosum heterokaryotic
mycelium is apparently composed of small sectors of homokaryotic hyphae,
which can re-mate with any other hyphae in the interaction zone, thereby
forming new pairwise combinations of nuclei. In wood, such interaction-zone
heterokaryons can possibly escape from the interaction zone through the
insulating nature of the wood anatomy. Hansen et al. (1993a) also studied
the genetic basis for somatic incompatibility in H. annosum. The system is
regulated through at least three, possibly more, multiallelic loci. This is
in accordance with findings from some other basidiomycetes (Malik and
Vilgalys, 1994). However, in several species of Phellinus, data suggest that
the somatic incompatibility is controlled through a single gene (Rizzo et al.,
1995).


By using SI as a marker system for individuality, forest pathologists have
been able to study the infection biology and spread of pathogens in natural
populations. 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 natural
stands (Childs, 1963). Another example is the wood decayer, Fomitopsis
cajenderi, infecting ice-glazed Douglas fir in Oregon, showing a pattern of
several genets entering the top break while only few managed to grow down
the stem (Adams and Roth, 1969). Following the advance in understanding
of fungal biology made in the 1970s and 1980s by Dr Alan Rayner and
co-workers, a range of fungal species was studied with regards to local
population spatial patterns (Rayner and Todd, 1979; Rayner, 1991). Very
large territorial genets have been detected in some tree root-rot fungi
(Armillaria spp.: Korhonen, 1978b; Kile, 1983; Smith et al., 1992; Legrand
et 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 and
Cook, 1989). Much smaller-sized genets were found in wound pathogens or
fungi attacking from the bark (Cylindrobasidium evolvens: Vasiliauskas and
Stenlid, 1998; Phomopsis oblonga: Brayford, 1990; Phellinus tremulae: Holmer
et al., 1994). In H. annosum, the genets are much larger in old forest sites
compared to those sites with a recent history of agriculture (Stenlid, 1993;
Swedjemark and Stenlid, 1993). At the same time, the relatively intensely
managed first rotation stands were hosting a higher number of genets per
hectare. These structures indicate a strong influence from diaspores infecting
stump tops in the managed forests, and a correspondingly high proportion of
root-to-root contact spread in the natural forests.

Summary
Heterobasidion is a well-defined genus of saprotrophic and necrotrophic
polypores. In the pathogenic species H. annosum, several intersterility groups
exist that are specialized to different species of conifers. Phylogenetic studies
based on rDNA variation indicate that at least five, and possibly seven,
separate clades occur in the species. Based on morphological differences, the
three European intersterility groups – S, specialized as a root and butt rot on
spruce; P, a general root and butt rot on pines and other conifers; and F, mainly
causing 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. In
contrast to the ITS sequences, enzyme systems with putative adaptive value for
host specialization, e.g. pectinases, differ clearly between the European S and F
intersterility groups. Most of the spore spread in H. annosum is local but, due to
massive diaspore production, the few spores dispersed over long distances
counteract population differentiation at distances less than 500 km. However,
no significant gene flow between continents can be detected. On the local


scale, vegetative spread and infection processes can be followed by mapping
the distribution of individual mycelia. Somatic incompatibility, a highly
polymorphic recognition system, as well as molecular genetic markers
have been used for this purpose.

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Molecular Variability in IV
Ganoderma

13
R.N.G. Miller et al. Ganoderma in Oil-palm Plantings
Characterization of

Molecular and Morphological 13
Characterization of Ganoderma
in Oil-palm Plantings
R.N.G. Miller1, M. Holderness2 and
P.D. Bridge2*
1Departamento de Fitopatologia, Universidade de Brasília,
and Universidade Católica de Brasília, Brasília, D.F., Brazil;
2CABI Bioscience, Egham, UK

Introduction
The basidiomycete fungus Ganoderma Karst., a polyporoid genus within the
family Ganodermataceae of the order Aphyllophorales, is worldwide in distribu-
tion, growing on numerous coniferous, deciduous and palmaceous hosts.
The genus comprises a large, diverse complex of fungi, all with double-walled
basidiospores. Many Ganoderma species are wood-rotting fungi, with a number
being pathogenic on economically important trees and perennial crops.
In forest systems, Ganoderma, along with other ‘white-rot’ fungi, has an
ecological role in the breakdown of woody plant debris. Most such fungi are
seen as largely saprobic, but may be able to exploit weakened hosts as parasites
or secondary pathogens. Root and stem rots caused by a number of Ganoderma
spp. have long been known to cause extensive worldwide losses of many
tropical perennial crops, most significantly in oil palm (Elaeis guineensis)
(Anonymous, 1915). Losses also occur in coconut (Cocos nucifera) (Petch,
1910), rubber (Hevea brasiliensis) (Wakefield, 1920), betelnut (Areca catechu)
(Butler, 1906), tea (Camellia sinensis) (Thomson, 1939), cocoa (Theobroma
cacao) (Varghese and Chew, 1973), peaches and pears (Chohan et al., 1984),
guarana (Paullinia cupana) (Batista, 1982) and timber trees such as Acacia
(Bakshi et al., 1972).
In South-East Asia, oil palm is frequently planted as a monoculture on
areas that previously supported other plantation crops, such as rubber or
coconut, or may be planted on areas cleared from primary forest. Smallholder



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 is
of particular economic importance in these production areas, because it
shortens the productive life of plantations, an effect that tends to become
cumulative over successive planting cycles of this monoculture, such that
widespread losses can occur in young plantings less than 5 years old. Losses
due to BSR are the result of both a direct reduction in palm numbers in the
stand, and a reduction in the number and weight of fruit bunches from
standing diseased palms and those with subclinical infections (Turner, 1966).
Although oil palm is planted in areas that previously supported other
perennial crops, or in mixed cropping systems, the influence of these different
cropping 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 been
documented as having a wide host range, infecting Albizzia (Turner and
Bull, 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 Livinstona
cochinchineasis within an oil-palm planting have also been reported to support
basidiomata of Ganoderma spp., presumed to be pathogenic to oil palm (Turner,
1968). In contrast, observations by Varghese and Chew (1973) revealed
that Ganoderma basidiomata from oil palm were morphologically and
physiologically different from Ganoderma basidiomata from tea and rubber,
suggesting that cross-infection from these non-palm hosts to oil palm would be
unlikely 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 and
Zimbabwe (Turner, 1981). Recently, following the increased planting of
oil palm, infection of young palms has also been noted for the first time in
Papua New Guinea (see Pilotti et al. and Sanderson et al., this volume) and
Thailand (Tummakate and Likhitekaraj, 1998). Ganoderma basal stem rot
is now recognized as a significant constraint to sustainable production in
Asia, and the development of techniques for disease management has
been highlighted as a key research priority (Anonymous, 1997).

Multidisciplinary Characterization of Ganoderma from Oil
Palm and Other Tropical Perennial Hosts
Recent applications of biochemical and molecular methods in phytopathology
have led to a considerable improvement in the taxonomy and understanding
of numerous pathogenic fungal species. The combination of molecular
biology characteristics, such as DNA polymorphisms, with functional
information, such as enzyme activities, along with traditional morphological

Characterization of Ganoderma in Oil-palm Plantings 161

Table 13.1. Ganoderma spp. recorded as probable causal organisms of basal stem
rot (based on association) (after Turner, 1981).

Ganoderma species Synonym Occurrence

G. applanatum Fomes applanatus Angola
Benin
Indonesia
Ivory Coast
Malaysia
Principé
San Tomé
Zaïre
G. boninense Malaysia
G. chalceum Malaysia
G. cochlear Indonesia
G. colossum Nigeria
G. fornicatum Zaire
G. laccatum Indonesia
G. lucidum Fomes lucidus Angola
Ghana
Indonesia
Malaysia
Principé
San Tomé
Tanzania
Zaïre
Zimbabwe
G. miniatocinctum Malaysia
G. pediforme Zaïre
G. pseudoferreum Zaïre
Malaysia
G. tornatum F. applanatus var. tornatum Cameroon
G. applanatum var. tornatum Malaysia
G. australe Zaire
G. tropicum Indonesia
G. xylonoides Zaire
G. zonatum G. tumidum Ghana
Nigeria
San Tomé
Tanzania
Zaïre
Ganoderma spp. Colombia
Malaysia
Zaïre

G. lucidum has been widely used as a misnomer for basidiomata from many
tropical countries; many collections named as G. lucidum are believed to be
incorrectly identified.


and pathogenicity data, allows the delimitation of populations on the basis
of 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 techniques
such 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 has
identified genetic and function-linked relationships between geographically
diverse 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 morphology

The majority of taxonomic studies on species of Ganoderma originating from
South-East Asia have been largely reliant on the system developed by Steyaert
(1967, 1972) for defining species. Discriminatory basidioma characters have
included context layer depth, basidioma colour (upper surface and context),
basidioma (shape, radius and thickness), cutis (thickness, colour and hyphal
system), context thickness and colour, tube layer depth and colour, pore
dimensions, 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, concluding
that gradations occurred in all morphological features used to describe species.
Other species identification circumscriptions have also been unclear, and have
resulted in the description of over 250 species, with frequent synonymity as
a result. The situation is further complicated by the description of a number
of species complexes by various authors (Steyaert, 1975, 1980; Bazzalo and
Wright, 1982; Adaskaveg and Gilbertson, 1986), such that taxonomic
divisions within the genus Ganoderma are currently regarded as chaotic, with
heterogeneic forms, dubious nomenclature and inconsistencies in application
of the numerous criteria by which the genus has been subdivided (Bazzalo and
Wright, 1982; Gilbertson and Ryvarden, 1986). These authors concluded that
the use of morphology alone is insufficient for the systematics of Ganoderma.
As a consequence, the identification and distribution of tropical Ganoderma
species remains unclear and there is little comparative morphological informa-
tion to enable morphology to be related to host specificity. The species concepts
for the BSR-associated Ganoderma isolates are also very confused. Originally
identified as G. lucidum by Thomson in 1931, a complex of species were later
believed 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


associated 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 those
associated with BSR all conformed to G. boninense, as did Miller (1995), who
also confirmed the pathogenicity of isolates from diseased and symptomless
palms following seedling inoculation tests. To date, 15 species of Ganoderma
have been recorded worldwide as probable causal agents of basal stem rot
in oil palm (Turner, 1981), although many of these are based only on
circumstantial association with basal rot lesions. In view of the uncertain
species concepts in this genus, Ganoderma populations on oil palm are herein
described by generic name alone.

Mycelial morphology

A number of identification systems using culture and morphological and
physiological 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 the
first to bring together a range of morphological and physiological characters,
including colour changes in agar, type of rot, and characters of the advancing
margin of a culture. In 1965, Nobles further developed the system into
a 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, hyphal
and culture pigmentation, growth rates, basidiomata formation in culture,
odour, host specificity, and interfertility phenomena. Limited information was
included regarding tropical species, although Bakshi et al. (1969, 1970) and
Sen (1973) later included a number of polypore species from India in similar
taxonomic keys. Boidin and co-workers (Boidin and Beller, 1966; Boidin and
Lanquetin, 1973; Boidin et al., 1976) also described species of Corticiaceae and
Lachnocladiaceae 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 again
fewer than 20% of species described were of tropical origin.
Application of mycelial identification methods to tropical Ganoderma
populations has been limited, as they are mostly concerned with temperate
species. Hseu and Wang (1990) concluded that identification systems of these
types were only of use for identification to the genus level, with parameters
insufficiently 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 populations
in the context of functional characteristics, such as host specificity on tropical
perennial crops. Diagnosis of Ganoderma infection in tropical perennial hosts


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 and
surrounding plant debris cannot be detected and identified with accuracy.

Genetic-based characterization approaches

Isozymes
Isozymes are defined as multiple molecular forms of a particular enzyme
which 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 differences
are due to amino acid variations, which are dependent on the coding
nucleotide sequence in the DNA. Micales et al. (1986) and Stasz et al. (1988)
described protocols for the study of population structures in fungi. Methods for
comparison of isozymes are based on specific staining after enzymes have been
separated by electrophoresis. As isozymes represent an indirect expression of
the genome, they may be used as indicators of genetic relationships between
populations. This approach can thus be applied to discriminate taxa, given a
sufficient number of polymorphic enzymes or the occurrence of unique or rare
enzyme patterns. The study of isozymes can be particularly useful in solving
taxonomic problems when there are few morphological parameters, or where
characters are very plastic within a conventional species. The use of isozymes
is generally applicable for intrataxon variation, discriminating below the
species level. Approximately 90 enzyme systems have been used to date with
a variety of organisms, and although their application to fungal systematics
is still under-exploited, significant advances have been made using these
approaches (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 taxonomic
purposes 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 correlation
with the spruce (S), pine (P) and fir (F) European and North American inter-
sterility groups, with six different pectin zymogram groups relating to the three
different intersterility groups, and these were suggested to represent incipient
species (Karlsson and Stenlid, 1991). Analysis of pectinase zymograms for 150
Ganoderma strains (Figs 13.1 and 13.2) (Miller et al., 1995a), gave groupings
that matched host type from which the strains were originally isolated. Isolates
from palm hosts (Elaeis guineensis, Cocos nucifera, Areca catechu, and the orna-
mental palms Oncosperma horridum and Ptychosperma macarthurii) comprised a


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 type
Fig. 13.1. Schematic representation of extracellular pectinolytic isozyme pattern
types.

single large cluster group (cluster A), 99% of which were of palm origin and
these isolates produced a distinct pectin esterase band (banding pattern type A
(Fig. 13.1)). Within this functionally defined group, there were no significant
differences between isolates obtained from widely distant geographic locations
such as Colombia, Nigeria, Malaysia and the Solomon Islands. A second
cluster (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 maceration
has been demonstrated in experiments with mutants (Handa et al., 1986) and
by the transfer of genes coding for pectinolytic activity to non-pathogenic
species (Keen and Tamaki, 1986; Payne et al., 1987). Although the role of
pectinolytic enzymes in pathogenesis caused by Ganoderma has yet to be
clarified, Tseng and Chang (1988) reported that G. lucidum produced both
endo-polygalacturonase and endo-pectin methyl trans-eliminase, and hypoth-
esized that such enzymes may be responsible for causing the tissue rots
associated with the fungus. As pectinases produced by Ganoderma are probably
involved in plant tissue degradation, they are considered likely to be
function-linked characters. Consequently, the majority of Ganoderma strains
isolated from palm hosts were regarded as a well-defined functional grouping,
producing a common range of pectinase isozyme profiles, undetectable by
comparison of basidioma morphology. Additionally, as a stable character
(pattern A) was identified in Ganoderma populations originating from infected
palm material, this raised the prospect of the development of diagnostic tools
for diagnosis of Ganoderma infection within palm hosts. However, as enzyme
activity is likely to be localized within an infected palm, difficulties were visual-
ized in terms of tissue sampling. Assuming that banding pattern differences


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 coded
extracellular pectinase isozyme data. Similarities derived from Gower’s coefficient.

found between isolates from oil palm and the majority of those from non-palm
hosts represented true functional differences, these findings were concluded to
be of fundamental importance in terms of elucidating mechanisms of pathogen
survival and disease spread within the oil-palm agroecosystem. Similarities
between zymogram banding patterns for isolates from oil palm and those for
isolates obtained from coconuts in Malaysia supported the current widespread
belief that the disease can spread from saprobic growth on old coconut


stands to parasitic invasion of oil palm, even though healthy coconut palms
themselves are not attacked in Malaysia. Similarly, the different patterns
produced by isolates from non-palm hosts suggested that cross-infection would
be unlikely to occur from these to palm crops.

INTRACELLULAR ISOZYMES. The cytoplasmic enzyme classes catalase, esterase
and phosphatase have been shown to reveal differences at a variety of taxo-
nomic levels when applied to the differentiation of fungal groups, separating at
species, population and isolate levels (e.g. Alfenas et al., 1984; Mugnai et al.,
1989). Analyses of intracellular esterase and polyphenol oxidase have been
useful in the separation of isolates of six Armillaria intersterility groups in
British Columbia (Morrison et al., 1985). Lin et al. (1989) also separated
isolates belonging to four North American species of Armillaria, and
genotypically distinct clones within a species, on the basis of intracellular
esterase isozymes and total protein profiles. Variability of intracellular iso-
enzymes in isolates of Heterobasidion annosum also revealed their applicability
for 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 applied
to test the validity of existing species definitions. For example, G. lucidum has
been differentiated from a number of other temperate Ganoderma spp. on the
basis of intracellular esterase isozymes (Park et al., 1986; Tseng and Lay,
1988). Hseu et al. (1989) also reported the differentiation of isolates of
G. applanatum, G. boninense, G. formosanum, G. fornicatum, G. microsporum,
G. neojaponicum, G. tropicum, and G. tsugae, on the basis of intracellular and
extracellular laccase isozymes. Following analysis of pectinase enzymes, Miller
and co-workers (Miller, 1995; Miller et al., 1995b) employed intracellular
catalase, acid phosphatase and propionyl esterase profiles to characterize
tropical perennial populations. These isozymes revealed widespread genetic
heterogeneity 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 these
intracellular isozymes are constitutive rather than behavioural, the groupings
produced between isolates from oil palm and other perennial hosts were
considered more likely to reflect evolutionary relationships than functional
relationships. Consequently, the level of similarity observed between isolates
from the majority of palm hosts on the basis of extracellular pectinase
isozymes was more likely to be reflecting a common behaviour of isolates
on palms rather than representing true genetic relatedness. Intracellular
isozyme data indicated that isolates probably arrived at this behavioural trait
from a number of different evolutionary pathways, which, on the evidence
generated from pectinase data alone, appeared as a single population of
isolates attacking palms, able to cross-infect from coconut and other palm
hosts to oil palm.


Mitochondrial DNA restriction fragment length polymorphisms
Mitochondrial DNA (mtDNA) in fungi codes for ribosomal RNAs, transfer
RNAs, 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 (Grossman
and Hudspeth, 1985; Hoeben and Clark-Walker, 1986), and size variation can
be observed even among closely related taxa (McArthur and Clark-Walker,
1983; Bruns et al., 1988). Although the size range varies greatly in different
organisms, it is generally between 20 and 180 kb in size, thus allowing the
entire genome to be visualized by enzyme cleavage and gel electrophoresis. It is
also regarded as an attractive molecular marker for restriction fragment
length polymorphisms (RFLPs) as it has a relatively high copy number and can
be purified easily. RFLPs have been used widely at different taxonomic levels
in 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. Bruns
et al., 1988; Smith and Anderson, 1989; Forster et al., 1990; Gardes et al.,
1991), with mapped polymorphisms revealing variation caused by length
mutations (Taylor et al., 1986; Bruns et al., 1988). Evaluation may be made
of classifications developed from characteristics such as morphology or host
specificity, and because isolates, pathotypes or species can be identified by this
approach, the technique may also be applied to the development of diagnostics
(Cooley, 1991). Their role in delimiting species or subspecies is particularly
important where morphological and physiological differences are ambiguous
or affected by environmental conditions, where they may provide a simpler,
more reliable and more rapid means of classification. An added benefit of
these analyses is that mitochondrial inheritance is believed to be unilinear (e.g.
Forster and Coffey, 1990), therefore variability that may be due to cross-overs
and other events in heterokaryotic isolates undergoing sexual recombination
will be avoided.
Mitochondrial DNA RFLPs have been shown to be highly varied among
Ganoderma isolates from a wide range of hosts and locations (Miller, 1995;
Miller et al., 1995b). Furthermore, mitochondrial probes derived from a single
isolate from Malaysian oil palm showed little homology with other isolates
from the same host. This supported the intracellular isozyme-derived conclu-
sion that isolates with common pectinase activities were unlikely to represent
a single population, and probably arrived at this behavioural trait from a
number of different evolutionary pathways.

Ribosomal DNA internal transcribed spacer (ITS) variability
The 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-conserved
non-genic (ITS and intergenic spacer (IGS)) regions (Fig. 13.3). The gene
regions code for rRNA, which forms the structural backbone of ribosomes, the
sites of protein synthesis within the cell. The rDNA gene cluster occurs within


the chromosomes as multiple tandem repeats, such that a single nucleus
contains 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 functional
nature of the genic regions, this region therefore represents an attractive
marker for systematic studies. rDNA genes are evolving at a relatively slow
rate, such that partial sequences from the nuclear large subunit gene are
applicable to phylogenetic studies among distantly related fungi (Gaudet et al.,
1989). As the ITS regions have a spacer role, separating gene regions, overall
length remains fairly constant. However, as they do not encode rRNA, they
may accumulate considerable base substitutions, and thus evolve at a much
faster rate than gene regions. ITS mutation rates frequently approximate that
of speciation, with sequence comparisons revealing low variation within a
species, with more extensive sequence divergence existing between different
species within a genus. As a consequence, variability in the ITS region has
been the basis for the development of many PCR-RFLP-based assays for
differentiation 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 discrimination
between different Ganoderma species, Moncalvo et al. (1995) concluded that
sequence differences in ITS regions were sufficient to distinguish the majority
of 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 in
Malaysia (R.N.G. Miller, unpublished data), restriction digestion of ITS regions
(PCR-amplified using universal primers ITS 1F and ITS4) using enzymes HinfI
and AluI yielded identical RFLP profiles in over 90% of strains, providing
preliminary evidence for a predominant single species within the oil palms
sampled (Fig. 13.4).

Localized Variability in Relation to Disease Establishment and
Spread Mechanisms
Little is known of the mechanisms of infection and spread within oil-palm
plantings. Traditionally, initial establishment of Ganoderma BSR in an oil-palm
field has been considered to occur by mycelial contact, through growth of
living oil-palm roots into an inoculum source, comprising saprophytically
colonized debris within the soil and largely remaining from the previous
planting. Entry has also been postulated to occur through wounded tissues or
dead roots (Turner, 1965b). As the roots of an oil palm can extend across up to
four planting rows (Lambourne, 1935), root-to-root contact might enable the
subsequent spread of Ganoderma between living palms. The observation that
patches 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 occurs
by root contact between healthy and diseased palms.


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
size
Fig. 13.3. Approximate locations on rRNA gene repeat of primers for amplifica-
tion of internal transcribed spacer (ITS) regions and estimated polymerase chain
reaction (PCR) product size in Ganoderma.

Fig. 13.4. Amplified rDNA internal transcribed spacer region (HinfI-digested) for
representative 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 to
replanting often involves ‘windrowing’, i.e. the uprooting of previous bole and
trunk tissues, which are then stacked along the inter-rows. In some cases, the
stem tissues are also chopped up mechanically to hasten breakdown.
Although disease incidence after windrowing is generally lower in subsequent
plantings than in stands replanted without bole removal, the process is both
labour intensive and costly, and often fails to prevent the recurrence or spread
of basal stem rot. Despite the dubious value of current replanting strategies and
the general failure of control strategies (curative surgery, fungicide treatment,
cultural methods) in existing oil-palm stands, few studies have been conducted
to test the validity of current assumptions about the spread of the pathogen
in oil-palm plantings. This is largely because morphology-based characteriza-
tion approaches have not allowed the differentiation of subpopulations or
individuals required for pathogen population studies. In a recent study, Miller
et al. (1999) determined the genetic relationships within over 50 isolates of
Ganoderma from two oil-palm plantings in Malaysia, through somatic incom-
patibility studies and mtDNA RFLPs, in order to elucidate possible mechanisms
of disease establishment and spread.


Somatic incompatibility

Although understanding of the mechanisms determining somatic incompati-
bility in Ganoderma remains incomplete, the use of incompatibility reactions in
the study of disease development in populations is well documented within
Basidiomycete 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-palm
systems occurred as numerous distinct individuals (‘genets’ sensu Rayner)
(somatic incompatibility groups (SIGs)), contrasting with typically clonal
distribution patterns for other basidiomycetes, where single clones can spread
over large areas of forests (Shaw and Roth, 1976; Stenlid, 1985). Numerous
separate genets were detected in the sampled populations, with a total of 34
detected in one plot (out of 39 isolates tested) and 18 (out of 18) within the
other (Fig. 13.5). In both cases, incompatibility between paired isolates was
observed over distances that could theoretically permit root-to-root contact,
and hence mycelial spread, between neighbouring palms (9 m apart), and
between non-adjacent palms (up to 36 m apart). Incompatibility was also
found between isolates colonizing the same infected palm. Only in one instance
were two isolates from neighbouring palms found to be compatible. Similar
variability has also been reported in other oil-palm blocks (Ariffin and Seman,
1991).
The frequency of different SIG genets within the two oil-palm plantings
indicated numerous separate infection incidents, rather than mycelial spread
of 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. New
inoculum sources could be formed by saprobic colonization of substrates such
as stumps or felled palm trunks and debris. Such mechanisms have been
widely reported for other root- and butt-rot pathogens (Turner, 1976, 1981;
Stenlid, 1985). Despite the release of huge numbers of airborne spores from
each basidioma, the majority of palms remain uninfected, indicating that
basidiospores either may not be able to initiate a basal stem rot infection or
may require very specific conditions to establish infection. Previous studies
with spore inoculum did not result in direct infection of living palms (Turner,
1965a; Yeong, 1972). None the less, spores are a likely infection mechanism
in upper stem rot of oil palm (Thomson, 1931), often in association with
Phellinus spp. Although Ganoderma basidiospores are most likely to be wind-
borne, additional mechanisms suggested for their dispersal have included
insect vectors (Genty et al., 1976). However, to date, no conclusive link has
been made between insects and basal stem rot incidence and development.
Alternatively, the numerous SIG clones could also have indicated the
presence of many spatially separated populations, each originating from a
unique mycelial inoculum source, which may have originated from infected
debris 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
31

Fig. 13.5. Palm layout and distribution of Ganoderma somatic incompatibility groups for selected isolates from oil palms: (a) plot at
Sungei Buloh Estate, Sime Darby Plantations Sbd, (b) plot at Bukit Cloh Estate, Sime Darby Plantations Sbd. Open circles indicate living
palms, shaded circles indicate palms that had died recently, and small closed circles indicate palms that had died some time previously
and constituted vacancies in the blocks. Numbered squares indicate locations of SIG. Thin straight lines indicate drainage channels.


replanted from rubber, which in turn replaced primary forest. Although either
vegetation could have supported Ganoderma populations, variation in strains
adapted to palms is more likely to have originated in native palm infections.
Evidence of such an origin was described previously (Miller et al., 1995a), with
isolates from palmaceous and non-palmaceous hosts separating on the basis of
extracellular pectinase zymograms. Oil palm is propagated as seed from crosses
between dura × pisifera types and so as a segregating population the oil-palm
stand itself does not present a homogeneous host. This may create additional
selection pressure for variation in the pathogen.

mtDNA RFLPs

As with the SIG data, mtDNA RFLPs revealed considerable heterogeneity
between isolates (Miller et al., 1999) including those from the same and
adjacent palms (Fig. 13.6). Of the 26 lines identified by MspI-derived RFLPs
among the isolates studied, only two isolates from neighbouring palms had
the same mtDNA RFLP profile. The majority of isolates obtained from within
individual palms gave a single mitochondrial DNA profile, and only two palms
gave isolates with different RFLP profiles.
In previous studies on other fungi, the relationship between RFLP and
SIG 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
number

Fig. 13.6. Schematic representation of restriction patterns from MspI restriction
fragment length polymorphism (RFLP) analysis of mitochondrial DNA for represen-
tative isolates. The scale on the left of the figure indicates fragment sizes (in kb
pairs).


ranging between equivalent RFLP and SIG groupings, more than one RFLP
grouping within a SIG, or more than one SIG within an RFLP grouping. Within
this study, results from mitochondrial DNA analyses and somatic incompati-
bility tests were not always in accord. More than one SIG frequently occurred
within a single mitochondrial DNA group, as previously reported in Armillaria
(Guillaumin et al., 1994; Smith et al., 1994). This was interpreted as variability
arising at the compatibility loci as a result of sexual recombination, with
mitochondrial DNA maintained through unilinear inheritance. Each SIG
could therefore have represented a nuclear genomic variant, with different
genets originating from locally dispersed basidiospores. This interpretation
was further supported by comparison of relationships by cluster analysis of the
mtDNA RFLP profiles (Fig. 13.7); isolates from the same or nearby palms did
not cluster together. These isolates showed few bands in common, implying
that 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 (although
isolates with identical mtDNA RFLP profiles could still represent different
lines). However, in one instance a single SIG group was found to have two
RFLP lines. This was interpreted as indicating either that more than one
mitochondrial type can exist (possibly through recombination) within a single
population, or that self-incompatibility is controlled by non-mitochondrial
markers. Overall, mtDNA was not recommended in isolation for differentiation
of lines within Ganoderma.
Mitochondrial DNA RFLP studies also provided evidence against previous
assumptions of the significance of secondary mycelial spread of Ganoderma
from palm to palm. As mtDNA has been demonstrated to be maintained
through unilinear inheritance in Ganoderma (C. Pilotti, personal communica-
tion), the presence of numerous mitochondrial DNA groups therefore
indicated spatially separated populations originating from a diverse initial
inoculum.

Conclusions
Existing species definitions for Ganoderma are of little value for interpreting
disease processes in tropical perennial crops such as oil palm. Application of
a multidisciplinary approach combining genetic, morphological and patho-
genicity data provided evidence of a genetically heterogeneous grouping of
isolates 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 spacer
regions, or protein-coding genes such as the β-tubulin genes, are likely to
clarify the species identity of Ganoderma in oil palm. Development of sensitive
diagnostic methods for the pathogen in oil palm is also likely to be reliant on


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
22
Fig. 13.7. Unweighted pair group average method constructed dendrogram of
binary coded MspI restriction fragment length polymorphism data of Ganoderma
isolates. Similarities were derived using Sorenson’s (Dice) Coefficient.

sequence data, enabling design of specific primers for PCR-based detection
approaches.
Localized studies did not support the current assumption that spread of
Ganoderma occurs through radial mycelial growth from individual inoculum
sources to neighbouring palms via root-to-root contact, which has particular
significance in terms of efficacy of land preparation prior to replanting, and
sanitation practices in existing oil-palm stands. Within two oil-palm plantings
examined, both SIG and RFLP data indicated that Ganoderma populations
were highly heterogeneous over restricted areas. Circumstantial evidence for


primary infection from residual inoculum in crop debris was supported by
genetic comparison of isolates. Spread from these foci to immediately neigh-
bouring trees may be occurring by mycelial spread, but more distant infections
are likely to be the product of unrelated infection incidents. It is anticipated
that, following conclusive determination of the stability of mtDNA in the
sexual fungus Ganoderma, the role of residual inoculum and basidiospores may
be more fully clarified. More recent PCR-based approaches such as randomly
amplified polymorphic DNA (RAPD), amplification fragment length poly-
morphisms (AFLP) or microsatellites may be appropriate to the clarification
of BSR disease establishment and pathogen spread in oil palm, if found to be
stable over the life cycle of Ganoderma. These approaches may be applicable to
discriminating individuals and, with reduced cost and handling time, may also
enable analysis of local variability on the basis of much larger sample sizes.

Acknowledgements
This study was funded by the UK Department for International Development
and 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)).

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14
F. Abdullah
Spatial and Sequential Mapping of BSR on Oil Palms

Spatial and Sequential 14
Mapping of the Incidence of
Basal Stem Rot of Oil Palms
(Elaeis guineensis) on a Former
Coconut (Cocos nucifera)
Plantation
F. Abdullah
Department of Biology, Faculty of Science and Environmental
Studies, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Introduction
The oil palm (Elaeis guineensis) is a very important commercial crop in
Malaysia. It was introduced from Africa and was first planted in Peninsular
Malaysia in 1917 (Thompson, 1931). The crop has adapted extremely well
to the local environment and has contributed significantly to the country’s
economy. Currently, Malaysia is the world’s leading producer of palm oil.
However, the crop is susceptible to basal stem rot (BSR), a serious disease,
characterized by an internal dry rotting of the trunk tissues, particularly at the
junction of the bole and trunk of the palm. In advanced cases, the palms break
at the basal portion of the trunk and fall over, hence the name of the disease.
The causal pathogen is Ganoderma boninense (Steyaert, 1976; Ho and Nawawi,
1985; Khairudin, 1990), a fungus whose bracket-like fruiting body (techni-
cally referred to as sporophores, basidioma, basidiocarps or sporocarps) is
usually observed at the trunk bases of infected palms. BSR is currently the
most serious disease of oil palm in South-East Asia, with reports showing that it
is also starting to be of significance in Papua New Guinea, Thailand and the
Solomon Islands (Flood et al., 1998). One of the usually observed signs of
disease inception is general foliar yellowing, the presence of several unopened
shoots, often referred to as multiple spear formation, and collapse of the older
fronds so that they hang down around the trunk.
Before the introduction of oil palms, Malaysia (then known as Malaya)
had traditionally grown coconuts (Cocos nucifera) as a source of edible oil. Due

184 F. Abdullah

to 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 seedlings
were planted under existing coconut palms, until such a time when the
coconut palms were poisoned and felled.
Underplanting seemed to provide a continuous source of income, but the
practice could be a pathological hazard if the relationship between disease
development of oil palms planted on ex-coconut lands holds true. Coconut
stumps and logs have often been observed to support abundant Ganoderma
fruiting bodies, leading to the opinion that they were the source of Ganoderma
inoculum that later caused infections on oil palms (Navaratnam, 1964;
Turner, 1965a, b). Based on field observations, both authors surmised that
the point of entry of the pathogen was through roots, and that disease spread
was by contact of infected plant debris with healthy oil-palm roots. In a
molecular-based study of Ganoderma from plantations in Malaysia, Miller
(1995) did not support root-to-root contact as the mode of disease spread – he
hypothesized that disease spread by spores, or via roots, from previous crop
residues was more likely. The possible role of basidiospores in disease spread
was 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 planted
on an old coconut plantation. Crop mappings were done at three time intervals
over a 30-month period, which allowed disease development to be viewed
spatially as well as sequentially. Disease progression of the first few infected
palms was studied until the palms succumbed to the disease. In addition,
vegetative compatibility of a reference isolate of Ganoderma from a selected
coconut stump with other Ganoderma isolates, collected from other stumps
within its immediate vicinity, and from an oil palm was studied. The hypothe-
sis employed here is that if anastomosis, or the mycelial mergence between two
isolates, took place, then the isolates must have come from a common
inoculum. If this was detected between a reference isolate with others that
came from two or more sources, then the disease could have spread by mycelial
fragmentation, implying root-to-root contact.

Background and Cropping Practice of the Sampling Site
The oil-palm smallholding within which the sampling block was selected
was located in Morib, in the district of Banting, Selangor, on the west coast of
Peninsular 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 at
the time the first survey (SI) was conducted, but by the final survey (SIV), all of
the estate land had been converted to oil palm. The coconuts surrounding the

Spatial and Sequential Mapping of BSR on Oil Palms 185

block were of the local Malaysian Tall variety. The sampling site consisted of a
1983 planting, which was free from BSR prior to the survey. The first sighting
of one infected oil palm was at SI, by which time all palms in the block were
approximately 13 years of age. When the oil palm stand was first planted,
all the seedlings were placed in-between then-existing tall coconut palms, a
traditional practice that allowed growers to harvest coconuts before the oil
palms start to bear fruits. The practice had an added advantage in that it
provided shade from the strong heat of the sun. The grower did not see
anything amiss with this planting technique and the procedure has been is
standard practice.
The coconut palms were later poisoned; a few were cut down to facilitate
the infrastructure, but practically all others were left in situ. Over the years
many of the poisoned trunks have fallen, breaking in the middle or at the basal
part of the trunk, while a few were totally uprooted. Fallen trunks and cut
oil-palm fronds were stacked in-between rows of oil palms, most of which had
degraded by the time SI was conducted. However, cut stumps and stumps left
after the trunks had broken and fallen were still intact, and these were the
subject of interest in this study.

Surveys and Crop Mappings
Four surveys, referred to as SI, SII, SIII and SIV, were carried out on the
sampling block. SI was conducted in May 1996, but a crop map was not
produced. SII, SIII and SIV were carried out in November 1996, November
1997 and November 1998, making the survey time intervals as 0, 6, 18 and
30 months, respectively. At each of the latter surveys, the disease status of
palms within the block was recorded and a crop map made. A palm was
recorded 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 conveniently
bordered by large drains on its lateral sides and a drain and fence at the
entrance. Each individual palm was identified by a code number for mapping
purposes. Eleven palms in a row were alphabetically coded from A to K. This
was followed by a further 10 palms per each row; so that any single palm
would be coded by a letter of the alphabet followed by a digit, e.g. A1 to A10 for
all palms in row ‘A’ (Fig. 14.1). The prefix ‘EG’ was used to describe a palm or
Ganoderma isolate collected from an oil palm at the coded location and ‘CN’ was
likewise used for coconut stumps or isolates collected from them.
Four categories of palms were identified and coded accordingly on the
map. These were, newly infected (NI), for palms that were observed as infected
for the first time at each survey point; and (I), for palms that still showed
symptoms 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 newly
planted seedlings as (NP). New plantings or replants also indicated points

186 F. Abdullah

Fig. 14.1. Spatial mapping of the sampling site at SII, showing 3.6% infection
of oil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump with
sporophores; v, newly infected palm.

where palms had fallen due to BSR but which had been replaced with young
replants (personal communication by the owner). This study thus regards the
status of NP as ‘formerly infected’ palms and they were thus included as data in
the calculation of percentage of infected palms at each survey point where they
were first detected, but not thereafter. Palms under the status NP were mostly
planted in the very hole where the diseased oil palm once stood.

Crop Status and Distribution of Ganoderma
Crop status at SI, May 1996

A total of 4–6 coconut stumps within the sampling block were found to har-
bour 1–5 Ganoderma sporophores per stump. This represented a conservative
estimate of 6% of all coconut stumps as those supporting Ganoderma
sporophores.
Only one oil palm, EG/F5, was observed to have had Ganoderma fruit
bodies on its trunk base. This represented 0.9% incidence on oil palms within
the sampling block. Despite the emergent sporophores at its base, palm EG/F5
did not show any foliar yellowing nor multiple spear formations, appearing no
different from its healthy neighbours. No crop map was made at SI.

Crop status at SII, 6 months after SI

The number of stumps bearing Ganoderma had increased to 18 and appeared
to be located within a noticeable ‘clump’ between rows A to E. The 18 stumps


recorded 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 bases
had increased to four, representing 3.6% of infected palms in the block. The
palms were EG/F5, -I4, -J4 and -B6. Palm EG/F5 was an old infection (I) but the
latter three were new cases (NI). These four were accorded a ‘pioneer status’
for diseased palms, whose disease progression over time was monitored. All
four palms did not show any sign of disease inception; there was no ‘multiple
spear’ formation, nor collapsed fronds and the leaves were of a normal, healthy
shade of green. Fruit-bunch production of these particular four palms was
optimal and the owner was not aware of any pathological problems.

Crop status at SIII, 18 months after SI

Many of the stumps recorded earlier as having Ganoderma fruit bodies were
almost totally degraded. Of the few still present, only two were observed to
support 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/F5
and EG/B6 of the pioneer palms were still standing (status ‘I’), but EG/I4 and
EG/J4 were already found as new plantings (NP). There was an assortment of
status for the remaining infected oil palms. Ten were NI and five were FP
whose NI status were not observed at SII. The remaining palms were new
plantings (NP) and palms showing symptoms. The replants were made due to

Fig. 14.2. Spatial mapping of the sampling site at SIII, showing 20% infected
oil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump with
sporophores; g, fallen palm; #, new planting; v, newly infected palm.

188 F. Abdullah

palms that had fallen after SII but prior to SIII. During this survey, almost all of
the infected palms displayed various degrees of the typical signs and symptoms
associated with basal stem rot, including the two ‘pioneer palms’ that were still
alive. Besides having Ganoderma fruit bodies, infected palms showed multiple
spear formation, thinning of the crown and exhibited various degrees of
leaf necrosis; some of the palms showed ‘frond collapse’, where the outermost
leaves hung down and enveloped the trunk. Palms thus affected were still
producing fruits, although fruit-bunch production was poor (personal
communication by the owner).

Crop status at SIV, 30 months after SI

The 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 still
had Ganoderma sporophores on it. The total number of oil palms with some
symptoms of BSR was 37, which was 33% of the sampling block. Out of this
number, 9 were cases of FP and 11 were NP. The remaining 17 were cases of
NI; including that of a new replant. Palm EG/B3, estimated to be about 3.5
years in age, had three sporophores at its base. The replant showed slight leaf
chlorosis on the two lowermost fronds but all other associated signs were not
prevalent. Its trunk was hardly discernible because of its young age and sporo-
phores that emerged appeared ‘squeezed’ out from the soil, but were definitely
coming from the trunk tissues.

Fig. 14.3. Spatial mapping of the sampling site at SIV, showing 33% infected
oil palms. q, healthy oil palm; r, infected oil palm; s, coconut stump with
sporophores; g, fallen palm; #, new planting; v, newly infected palm; x, newly
infected replant.


Disease Development of the First Few Infected Palms
The progression of disease development in all four ‘pioneer’ infections at SI
up 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 between
12 and 18 months. This was based on palms EG/I4 and J4, which fell within 12
months after their first symptoms were detected. However, the earliest of all the
first few infected palms (EG/F5) fell any time between 19 and 30 months, for it
was still recorded as an old infection (I) at SIII. All four were already replaced
by NP, at SIV (Table 14.1).

Mycelial Isolations and Vegetative Compatibility Studies
Samples for compatibility studies were collected at SII where stump CN/B5
was selected as the reference point. One sporophore each was collected from
here as well as from its immediate neighbours, and were brought back to the
laboratory for mycelial isolations. For each sporophore, pieces of tissues about
0.5 cm3 in size were cut out from the innermost or context layer of the fruit
body. These were then surface sterilized in 5% sodium hypochlorite for 2–3
minutes and then transferred under aseptic conditions on to malt agar to
obtain pure mycelial cultures.
A 3 mm diameter agar disc of CN/B5 mycelia was cut out with a flamed
cork borer and plated at one end of a culture dish. This culture was paired
with 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 two
opposing directions. Anastomosis would indicate vegetative compatibility
between the paired isolates. Where cultures did not merge but formed a zone
or line of demarcation, the paired isolates were considered as vegetatively
incompatible. 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 from
stumps were also plated against isolate EG/B6, a relatively isolated infected oil
palm 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 binary
pairing. Isolate CN/B5 was incompatible with each of its representative
neighbours on stumps CN/B4, CN/B6, CN/C4 and CN/A3. Each of the cultures
above were also incompatible with EG/B6.

Other Field Notes
The initial emergence of sporophores on newly infected cases was found to be
in an east–west orientation on the palm bases. The fruit bodies emerged from
ground level up to an approximate height of 2½ ft (76 cm), but did not exceed
4 ft (122 cm).
None of the standing Malaysian Tall variety of coconut palms outside the
sampling block at SI indicated the presence of G. boninense (with its typically
reddish-brown and highly lacquered fruiting bodies). Instead, there were
fruiting bodies on some stumps, but not on big palms, and these were of the
non-laccate variety, which belonged to the Ganoderma cf. applanatum/australe
complex. Stumps within the sampling site were also observed to have had the
non-laccate fruiting bodies initially, but these disappeared when the laccate
G. boninense assumed prominence. However, there was one case of an oil-palm
replant (approximately 5 years old) outside the sampling block that had a
non-laccate Ganoderma sporophore on its trunk, in addition to several laccate
ones.

Discussion
Source of Ganoderma

This survey found coconut stumps to be the most likely source of G. boninense
in the sampling site. Initially, Ganoderma sporophores were prominent on
stumps but were initially absent on oil palms. The presence of non-laccate
Ganoderma sporophores were found to precede those of G. boninense on stumps,
both from within and outside the sampling blocks. However, it is not known
whether their presence plays any role in the establishment of G. boninense.
While the Ganoderma population decreased on stumps, its presence on oil
palms increased considerably. From a mere 0.9% incidence initially, it reached
3.6% at SII, at a time when the Ganoderma population was at its highest on
coconut stumps. However, the presence of Ganoderma on oil palms escalated
to 20% at SIII, corresponding with its population decline on stumps. By SIV,
oil palms with BSR had reached 33%, representing a significant increase over
30 months. A study carried out by F. Abdullah (unpublished) showed that
Ganoderma isolates from coconut stumps were also able to infect oil palms,
based on artificial infection of oil-palm seedlings.


Development of disease signs and symptoms

The first few infected palms did not show signs of disease inception but this did
not last long as they were recorded as fallen palms within 12 months. This
duration is considered very rapid, given the experience that infected palms in
several plantations, particularly in inland areas, may still be producing fruits
for many more years despite having fruit bodies at their bases. The overall rate
of fall of palms was also rapid. Rather than draw conclusions on the possible
aggressive nature of the pathogen in the block, this study proposed two
possible causes that may have aggravated the situation. First, it could be that
the site, in close proximity to the coast, may have been subject to the strong
coastal winds, resulting in the palms falling over as soon as the trunks became
weakened. The second possibility is that of stress caused by climatic conditions.
The year 1997 was an eventful one, where Malaysia was subjected to serious
climatic changes as a result of El Niño, including extremely high daily
temperatures and ‘the haze’ produced as a result of forest burning. All these
factors may have caused added stress to the palms. As a result, the palms
succumbed to the disease at a particularly high rate.

Considerations of the possible mode of spread

The incidence of BSR varies between regions in Malaysia. Disease incidence
may 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 soil
types, rainfall or fertilizer application play a role in aggravating the disease; or
alternatively, that particularly aggressive variants of species of Ganoderma may
be present in the population. There are three possible ways by which the fungal
pathogen 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 contact
Singh (1991) reported that infected palms appeared in groups and then formed
several foci of infection in long-standing cases. He concluded that the mode
of spread was by root-to-root contact. Flood et al. (1998) described a similar
incidence where a clumping effect was evident in oil-palm blocks with rela-
tively few infected palms, but this trend disappeared when larger numbers of
infected palms occurred in the blocks. From the viewpoint of disease spread, a
clumping of infected palms would theoretically suggest a common origin from
a single inoculum, thus stating a case for root-to-root contact. However, in this
study, the first four palms that were infected (pioneer palms) were relatively
far from each other as well as from the clump of stumps where the Ganoderma
population was concentrated. Furthermore, vegetative incompatibility of iso-
lates collected from coconut stumps and an infected oil palm would not support
root-to-root infection, although the incompatibility was also demonstrated

192 F. Abdullah

amongst a large number of isolates collected from a single palm, making this
method a less reliable basis for assessing root-to-root infection (Abdullah,
unpublished data) than molecular techniques.

Spread by spores
From his observations, it seemed evident to Thompson (1931) that in typical
cases of stem rot, the disease was caused by spores that entered the stem
through some of the old leaf bases which have been rotted away, or through
wounds from leaf bases, as in leaf pruning during harvesting. He proposed that
infection through wounds would allow a quicker stem penetration, besides
having had a shorter distance to travel, compared to the distance if it was a
root entry. However, attempts at establishing pathogenicity of the crop based
on trials using spores alone were not successful.
In this study, it was observed that practically all infected cases had
sporophores at the bases and no more than 2–4 ft (76–233 cm) up the trunk. If
there is a random dispersal by airborne spores, then some palms should show
Ganoderma fruiting bodies at other heights of the palm as well. In a study of the
occurrence 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 there
was believed to be secondary. Only airborne spores could have been responsi-
ble. Thus, the fact that all Ganoderma fruit bodies were confined to not more
than 4 ft (122 cm) from the base of the oil palms in the sampling site, does not
suggest random dispersal by airborne spores.

Spread from secondary inocula
Spatial mapping by Miller (1995) of two blocks of oil-palm stands, followed
by molecular and compatibility studies, showed no evidence for root-to-root
contact, except where two adjacent palms contained the same ‘individual’, as
determined by molecular analyses. He proposed that spread could be by spores
or from separate inocula from previous plantings.
In the case of the diseased replant in this sampling site, it is obvious that
the mode of spread was by infection from secondary inocula left by the previous
infected palm. This is an interesting phenomenon in that it allowed one to
estimate the time at which the pathogen first entered the palm tissues to the
eventual emergence of sporophores: approximately 12 months in this inci-
dence. However, this is an isolated case, rather than the typical infection. For
the rest of the infected palms which had been standing for at least 13 years, the
source of infection would appear to be from independent secondary inocula,
although it is difficult to suggest the source of secondary inocula, given that
the previous planting consisted of only coconuts and that no G. boninense
sporophores were ever observed on the standing crop. Coconut palms are not
known to be infected by Ganoderma in Malaysia (apart from the single and last
report in 1934 by Tempany; as cited in Navaratnam, 1961), but reports from
India (Bhaskaran and Ramanathan, 1984; Bhaskaran et al., 1998) and Sri


Lanka (Peries, 1974) indicate that coconut palms are badly infected by
Ganoderma in these countries. One explanation is that the Malaysian coconuts
respond to Ganoderma infection in a different manner. It can be speculated that
the Malaysian Tall variety of coconuts is probably infected as well, but these
are not debilitated in any way by the fungus. The coconut palms may carry
the inocula as an endophyte with no production of sporophores. When the
coconut 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 unlike
the coconuts, oil palms succumb easily to the disease. Swinburne et al. (1998)
reported that a significant number of living coconuts were found to contain
Ganoderma, thus further strengthening the case for the above hypothesis.

Conclusion
The incidence of basal stem rot varies between regions in Malaysia. There
seemed to be a correlation of disease severity with former croppings, particu-
larly old coconut plantations. This study examined one such model. Based on
spatial distribution, vegetative incompatibility studies and the rapid death of
infected palms, this study does not support root-to-root contact as a mode of
disease spread, nor lend support to disease spread by airborne spores alighting
on 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 an
endophyte is suggested.

Acknowledgements
This project was funded by the Intensified Research Priority Areas from the
Ministry of Science, and Technology Malaysia. I would like to thank Puan
Latifah Z. Abidin for the field and lab assistance.

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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
Turner, P.D. (1965b) Oil palms and Ganoderma II. Infection and spread. Planter 41,
238–241.

15
C.A. Pilotti et al. in Ganoderma spp. from Papua New Guinea
Genetic Variation

Genetic Variation in 15
Ganoderma spp. from Papua
New Guinea as Revealed by
Molecular (PCR) Methods
C.A. Pilotti1, F.R. Sanderson1, E.A.B. Aitken2 and
P.D. Bridge3*
1PNG OPRA, Plant Pathology Laboratory, Alotau, Milne Bay
Province, Papua New Guinea; 2Department of Botany,
University of Queensland, St Lucia, Queensland, Australia;
3CABI Bioscience, Egham, UK

Introduction
Basal stem rot of oil palm is a disease that has been found in almost all
countries where oil palm is grown (Turner, 1981). The major causal agent of
this disease has been found to be Ganoderma boninense Pat. in both Malaysia
and Papua New Guinea but several species have been associated with the
disease in other countries (Turner, 1981).
Ganoderma species occur throughout the world in both temperate and
tropical regions. They may be saprophytic, decomposing lignin of dead wood,
or pathogenic on living trees. Several species of this genus are responsible
for root and butt rots in tea (Varghese and Chew, 1973), rubber (Lim, 1977),
temperate hardwoods (Ross, 1976; van der Kamp et al., 1979), coconut
and betelnut palms (Reddy and Ananthanarayanan, 1984; Singh, 1985) and
other tropical forest trees (Bakshi et al., 1976; Harsh et al., 1993; Masuka and
Nyoka, 1995).
In Papua New Guinea, where basal stem rot caused by G. boninense is the
major disease, more effective control measures are required to limit the spread
of this pathogen within oil-palm plantations. Miller et al. (1995) used vegeta-
tive compatibility, biochemical and molecular techniques to characterize
isolates from Malaysia, and showed that root-to-root spread appears to be of
limited occurrence. A later study by Ariffin et al. (1996) confirmed the findings



196 C.A. Pilotti et al.

of Miller et al. (1995) and concluded that spread of the pathogen by means
other than vegetative was likely. The work presented here is part of a study
to determine the basis for the variability in Ganoderma species that occur in
association with oil palm in Papua New Guinea. Molecular methods are being
developed to study pathogen populations to clarify the role of the sexual cycle
in the epidemiology of basal stem rot. Random amplified polymorphic DNA
(RAPD) analysis was selected to investigate variation amongst monokaryons
prior to population studies on dikaryons. Other markers targeting more
conserved regions of the fungal genome were also investigated, including the
mitochondrial small and large subunits of the ribosomal gene (rDNA) and
the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions, the
latter having the potential to reveal inter-species differences. These molecular
markers will be used to analyse and determine the nature of Ganoderma
populations on oil palm and may be applied in other cropping systems where
the fungus is a pathogen.

Experimental
Isolations

Monokaryotic 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 growing
on 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), yeast
extract (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 DNA

DNA extraction was carried out using a slight modification of the method of
Raeder and Broder (1985).

Polymerase chain reaction (PCR)

PCR was used to amplify DNA from the large and small mitochondrial
ribosomal RNA subunits and the nuclear rRNA internally transcribed, and
intergenic spacers. RAPD amplification was undertaken with Operon series A
primers. Primers and PCR conditions are given in Table 15.1. PCR was carried
out on a programmable thermocycler (MJ Research). Programmes were as
follows. 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 197

Table 15.1. Primers and PCR conditions.

Annealing
Primer temperature Reference

Mitochondrial 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 min

ITS, internal transcribed spacer; IGS, intergenic spacer; RADP, random amplified
polymorphic 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 that
the 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 units
Taq DNA polymerase were made and 24 µl aliquots plus 1 µl template DNA
(approximately 10–20 ng) were subjected to PCR.

Results
Comparison of sibling monokaryons using RAPDs

Twenty operon RAPD primers were screened. Fifteen of these gave amplifica-
tion products and five generated a sufficient number of fragments showing
polymorphisms 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 each
basidioma showed that band patterns were specific to individual single spore
cultures, and that no two single cultures gave identical patterns (Figs
15.5–15.7). The similarities derived from Jaccard’s coefficient are under-
estimated and intended only as a guide to the range of variation within
families. Clearly, each sibling monokaryon appears to have a unique RAPD
genotype from the isolates studied so far.

MtDNA

PCR of sibling monokaryons of G. boninense with the primer combination
MS1/MS2 to amplify the mitochondrial small subunit gave two products of
approximately 600 bp and 1790 bp. Monokaryotic isolates of Ganoderma sp.
gave a single amplification product of about 600 bp (Fig. 15.8).


Fig. 15.1. Randomly amplified polymorphic DNA fingerprints of sibling
monokaryons (isolate #80, primer OPA-20) (kb markers: 1353, 1078, 872, 603,
310).

monokaryons (# 80, primer OPA-18) (FN-1 markers: 2686, 1563, 1116, 859, 692,
501, 404, 331).

Dikaryotic isolates of G. boninense also gave an additional amplification
product at about 1790 bp, and in some samples this was the only fragment
produced (Fig. 15.9). Repeated amplifications with duplicate samples gave the
same result.
Intra- and interspecific length variation was not observed for the mito-
chondrial large subunit although some isolates yielded a single amplification


monokaryons (#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 of
isolate 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, only
the expected fragment of approximately 800 bp was amplified.

ITS and IGS DNA

Primers BMB-CR and LR gave an amplification product of approximately
800 bp incorporating the entire ITS1 and ITS2 region, and this was consistent


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 alleles
assigned are given in parentheses.

Fig. 15.7. Dendrogram of sibling monokaryons (isolate#87). Mating alleles
assigned are given in parentheses.

both within and between species, although some additional amplification
products were observed for a few isolates (Fig. 15.10).
The IGS region also appeared highly conserved amongst species. Total
length, including intervening sequences, was approximately 1000 bp for all
samples, regardless of host origin. Digestion of the amplified fragments from
both ITS and IGS regions with the restriction enzymes Sau3A and Cfo1 gave
identical fragments irrespective of species or host (data not shown).


Fig. 15.8. Mitochondrial small (a) and large (b) subunit (rDNA) amplification
of 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 Ganoderma
isolates. Isolates 1–5 from oil palm, 7–11 from coconut, 12–16 from hardwood
(kb FN-1: 2686, 1563, 1116, 859, 692).

Discussion
Genetic variation has been observed amongst sibling monokaryons of
Ganoderma boninense. This is the first report on the use of RAPDs to differentiate
haploid isolates of G. boninense and clearly demonstrates the importance of
sexual reproduction in maintaining genetic diversity in this fungus. These
results also emphasize the need for caution when using RAPD fingerprints of
dikaryons to infer relationships amongst isolates in population studies, given
the 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 gene
amongst monokaryons of Ganoderma sp. In contrast, an unexpected product
of about 2030 bp was generated (in addition to the 800 bp fragment) when
the mitochondrial large subunit was amplified in some monokaryons and


Fig. 15.10. ITS 1/2 amplifications of dikaryons from different hosts. Lanes 1–6, oil
palm; 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 LrRNA
gene giving rise to fragments of 1700 and 2000 bp. However, for the G.
boninense isolates, a similar product was also obtained when the mitochondrial
small subunit was amplified. In this case the larger fragment was 1790 bp and
in some samples (129, 130) this was the only PCR product. In the samples that
showed two products, there appear to be competing reactions, as both frag-
ments are inefficiently amplified. Control samples did not produce the 1790 bp
fragment, 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 the
isolates 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 other
than the expected product. When DNA from dikaryons of both species were
amplified, minor length differences were apparent for the mitochondrial small
subunit within Ganoderma sp. but not G. boninense. Given these results, PCR
amplification of the mitochondrial small and large subunits of rDNA may
be of limited use for both intraspecific and interspecies comparisons. These
amplification products are, however, only a small part of the ribosomal DNA
and it is expected that comparison of isolates using these products as probes to
detect RFLPs in mtDNA sequences will be more informative. Further work
using the mitochondrial small subunit fragments is being undertaken to assess
mtDNA variation within Ganoderma boninense.
The ITS region was considered a potentially useful marker for interspecies
differences within Ganoderma; however, length differences between species
have not been apparent. The ITS1/ITS2 region is expected to be around 400 bp
for Ganoderma, using the given primers (Moncalvo et al., 1995). When the PCR


products were digested with certain restriction enzymes, digestion products
were monomorphic. However, small sequence differences are found amongst
species 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. When
the IGS region of the PNG isolates was subjected to PCR, the total length of
the amplified fragment was approximately 1000 bp. Digestions confirmed
the homology (at restriction sites) of the amplification product amongst
isolates, although it is likely that small sequence differences are present. From
the foregoing, ribosomal DNA appears to be highly conserved within the
Ganoderma species studied, and the regions selected for PCR amplification so far
do not provide a useful and rapid means of detecting interspecific variation.
Consequently, other methods are being investigated to study the Ganoderma
populations associated with oil palm in Papua New Guinea.

Conclusion
Intraspecific variation amongst closely related isolates of G. boninense has been
found to be high. This variability, as revealed by PCR RAPDs, is indicative of an
outbreeding population, although the number of isolates tested so far is small.
In contrast, variation in the ITS and IGS regions between species is low and
sequencing of these regions will be necessary for interspecies comparisons. The
mitochondrial fragments generated by PCR are not useful, on their own, for
interspecies comparisons.

References
Ariffin, 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).
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.


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.
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.

16
H. Rolph et al.
Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut

Molecular Variation in 16
Ganoderma Isolates from Oil
Palm, Coconut and Betelnut
H. Rolph1*, R. Wijesekara2, R. Lardner 1,
F. Abdullah3, P.M. Kirk1, M. Holderness1,
P.D. Bridge1† and J. Flood1
1CABI Bioscience, Egham, UK; 2Coconut Research Institute,
Bandirippuwa Estate, Sri Lanka; 3Department of Biology,
Faculty of Science and Environmental Studies, Universiti
Putra Malaysia, Serdang, Selangor, Malaysia

Introduction
The genus Ganoderma is a common saprophyte of decaying wood and occurs in
both temperate and tropical regions. However, members of the genus are
increasingly reported on commercially grown crops in the tropics and sub-
tropics and certain isolates can cause serious basal stem rots on a number of
palm hosts, including oil palm, coconut, arecanut (betelnut) and many other
tree species (Sampath Kumar and Mambiar, 1990). These isolates cause severe
economic losses by shortening the life span of perennial crops, e.g. coconut
losses due to basal stem rot in India can reach up to 31% (Anonymous, 1987)
and infection can also make the land unsuitable for the subsequent plantation
crop. Two major constraints to disease control are the lack of sufficient
information on variation in Ganoderma species associated with disease and
their mode of reproduction.
Ganoderma boninense was said to be responsible for causing basal stem rot
(BSR) of oil palm in Indonesia and Malaysia (Turner, 1965a). In south Asia,
coconuts are also affected by a disease similar to BSR, which is known by
a variety of names, including bole rot, Thanjuvar wilt, Ganoderma wilt,
Ganoderma disease and Anabe Roga disease. In Sri Lanka, G. boninense is said
to be the causal agent of bole and root rot of coconut (Peries, 1974), while

* Present address: Glasgow Dental School and Hospital, Glasgow, UK
†
Present address: Mycology Section, Royal Botanic Gardens Kew, Richmond, UK


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 India
and Sri Lanka.
Consequently, there is confusion concerning the species involved in this
disease and the different symptoms they induce when infecting oil palms and
coconuts; they can also produce different symptoms on the same palm host
in different countries. For example, in Sri Lanka, stem bleeding and fruit-body
formation on live palms are observed, while these symptoms are rarely seen
on palms in Indonesia and Malaysia (M.K. Kip, personal communication).
Conversely, although two different species of Ganoderma have been reported
on coconut palms in India and Sri Lanka, the symptoms produced appear
identical (Peries et al., 1975; Bhaskaran et al., 1989).
Part of the confusion lies in the identification of the species involved and
the problems associated with the development of suitable species concepts for
tropical 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 the
classification of Ganoderma species, a number of authors have concluded that
macromorphology alone is insufficient for the systematic determination of
Ganoderma species (Bazzalo and Wright, 1982; Gilbertson and Ryvarden,
1986).
Identifying Ganoderma isolates to species level is important, but mapping
individual isolates across a plantation is equally so, in order to discover
whether a single clone or several individuals are responsible for a particular
disease outbreak, and also to monitor subsequent spread. Despite the lack of
suitable species concepts to fully identify potential crop pathogens, research
has therefore progressed into mapping variation in Ganoderma isolates at the
plantation level. The combination of molecular techniques and somatic
incompatibility group (SIG) testing to assess the variation between Ganoderma
isolates from different oil palms across a plantation has yielded interesting
results.
Miller et al. (1999) assessed the variation in mitochondrial DNA (mtDNA)
and SIGs from Ganoderma isolates in two oil-palm plantings, and suggested
that the disease does not appear to spread in a clonal fashion via root-to-root
contact. They found a high level of variation in the mtDNA profiles of
Ganoderma isolates across two plantings of oil palms. SIG studies showed that
even adjacent palms were usually infected by different Ganoderma isolates,
with members of each SIG usually confined to a single palm. It was very rare to
find a Ganoderma isolate from one SIG infecting two palms. This was confirmed
by the fact that identical mtDNA profiles were also very rarely seen in isolates
from more than one palm. The study also showed that a single oil palm could
be colonized by several Ganoderma isolates with different mtDNA profiles and
SIGs. SIG studies by Ariffin et al. (1994) have also indicated that up to three
different Ganoderma isolates can infect a single oil palm.

Molecular Variation in Ganoderma from Oil Palm, Coconut and Betelnut 207

Overview of Symptoms of Basal Stem Rot in Sri Lankan
Coconut and Betelnut Palms
A wide range of symptoms is displayed by these palms infected with
Ganoderma. The initial visible symptom is the presence of a reddish-brown,
viscous liquid that oozes from longitudinal cracks in the base of the palm
trunk. This symptom, known as ‘stem bleeding’ (Fig. 16.1), is not found on
Ganoderma-infected oil palms and appears unique to coconut and betelnut
palms suffering from Ganoderma infection.
This bleeding usually extends upwards through the trunk and it has been
noted that stem-bleeding symptoms can often extend 10–15 m up the trunks
of coconut palms planted close to a water source. Under these conditions, the
fungus does not advance as far up the trunk as it would do in palms growing
in non-waterlogged soil. Analysis of such palm tissue has shown that the
Ganoderma 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 and
Coryota urens palms found on canal banks adjacent to poorly drained ground
do not show the extended bleeding symptoms at all, and have no longitudinal
cracks in their trunks. However, both types of palm can display small drops of
liquid at the base of the trunk.
Another initial symptom of BSR is that fronds in the lower whorl of
the palm turn yellow and dry prematurely. As the disease progresses, the
production of inflorescences and the number of female flowers gradually
decreases and the fungus causes decay of the bole and root system of the
palm. Sporophores are occasionally seen around the bole of coconut palms
in Malaysia, but are common on live coconut palms in Sri Lanka. The length
of the fronds is reduced and the palm begins to taper, and eventually dies
approximately 5–10 years after initial infection. Palm death is brought about
by several factors: the palm bole is so decayed that it collapses and the palm
falls over; the crown is blown off by the wind; or there is a lack of translocation
of 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 the
pathogen (Navaratnam, 1964; Turner, 1965a, b; Abdullah, this volume). The
practice of planting oil palm after coconut is a possible cause of BSR in oil palms
and, although the source of infection is unknown, any coconut debris left in
the soil should be considered a potential inoculum.
In order to further investigate Ganoderma isolates from coconut and
betelnut 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 molecular
variation and somatic incompatibility groupings in Ganoderma isolates from
Sri Lankan coconuts. This variation would then be compared with the extent
of variation found in isolates from Malaysian coconuts, on which Ganoderma
is not known to be a pathogen. A final part of the study was to assess the
variability in isolates from betelnut palms planted adjacent to coconut palms.
This would determine whether there were any significant differences between
Ganoderma isolates from coconut palms and betelnut palms.

Investigation into Sri Lankan and Malaysian Ganoderma
Isolates from Coconut and Betelnut Palms
The isolates used in this study came from several coconut plantations in
the Hambantota district (southern province) of Sri Lanka, including the
plantation where Ganoderma was first noted in that region. The disease had
not been a serious problem for approximately 20 years, when, in 1995, a
sudden outbreak of root and bole rot of coconuts occurred. Since then there
has been increased interest in the genus Ganoderma and its role in basal stem
rot disease.


It was important to determine whether Ganoderma could be isolated not
just from sporophores growing on the palm, but from palms showing different
symptoms. It was for this reason that Ganoderma isolates were taken from a
wide range of material from Sri Lankan coconut and betelnut palms. In several
cases, isolates were obtained from both palm tissue and fungal sporophores,
to determine whether they were from the same individual, or represented
two separate infections. Material collected included decayed stem tissues
and sporophores from live coconut and betelnut palms with stem bleeding;
sporophores and decayed stem material from dead, standing palms; and
finally, stem tissue and sporophores from coconut stumps. A negative control
was included, which consisted of a Ganoderma strain isolated from the stem
tissue of a leguminous tree.
Ganoderma isolates from Malaysian coconuts were taken from sporophores
found on coconut stumps and oil palms from a smallholding mixed plot in
Banting, Selangor, on the west coast of Peninsular Malaysia (Abdullah, this
volume). Two molecular methods were used in the study. The first was the
same as that used in previous investigations at CABI Bioscience, namely
mitochodrial (mtDNA) profiling. For a full description of this technique, see
Miller et al., this volume. In this current study, mtDNA profiles were generated
using the enzyme HaeIII. Identical banding patterns were grouped together
and 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 of
the molecular variation of the Ganoderma isolates.
The AFLPs were performed on total genomic DNA extracted from
lyophilized mycelia and digested with a restriction enzyme, i.e. an enzyme that
can recognize a key DNA sequence (usually four or more bases long) and cuts
or ‘restricts’ it at that point. In this case the restriction enzyme PstI was used to
cut the DNA. It creates ‘overhangs’ of several bases at the ends of the restricted
DNA (Fig. 16.2). A ligation reaction is then performed whereby the restricted
ends of the DNA are joined to ‘adapters’. These adapters are short lengths
of double-stranded DNA, which are complementary to the overhangs of
the restricted genomic DNA. The adapters also have sites complementary
to a specific set of oligonucleotide primers, which are used in the ensuing
polymerase chain reaction (PCR). PCR is the exponential amplification of
a region of template DNA bounded by short stretches of DNA that are
complementary to a specific set of DNA primers. Thus, the template for the
PCR reaction is any DNA bounded by the adapters, and only DNA with these
adapters at both ends is amplified. The size of DNA fragments amplified is
dependent on the position of the restriction sites in the genomic DNA, because
the adapters can only bind to DNA with the correct overhangs produced by
the restriction enzyme. Agarose gel electrophoresis is then used to separate the
resultant 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 more
reliable to use, than random amplified polymorphic DNAs (RAPDs). RAPDs are
produced by the random binding of PCR primers to target DNA and subsequent
amplification of that target template. Low-stringency conditions are used to
generate RAPDs, and primers may bind to target sequences with which they
have only a low identity. The number of factors affecting the reproducibility of
RAPD profiles is therefore greatly increased. AFLPs are generated under higher
stringency conditions, using the adapters as initial primer targets. Differences
in patterns generated using AFLPs are due to a change in the position of a
restriction site, i.e. an inheritable mutation in the DNA. Different-sized DNA
fragments will be amplified according to this criterion only.
In this study, two AFLP primers were used to ensure a good level of
discrimination 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 and
designated AFLP group 1, AFLP group 2, etc.
The somatic incompatibility testing of the Sri Lankan and Malaysian
isolates with each other were performed according to Miller (1995). Several of


the Sri Lankan Ganoderma isolates from coconut were also tested for their
ability 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 Lankan
Coconuts
MtDNA profiles from Ganoderma isolates on Sri Lankan coconuts (Fig. 16.3)
were quite different from those of Ganoderma isolates from oil palms in Malaysia
and, in addition, many of the Sri Lankan Ganoderma isolates from different
coconut 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 27
isolates studied. These isolates came from different palms on a number of plots
separated by several kilometres. This contrasted with the high level of diverse
profiles 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).

212

Table 16.1. Ganoderma isolates from Sri Lanka and the mitochondrial DNA (mtDNA) and amplification fragment length polymorphisms
(AFLP) groupings.

Mitochondrial AFLP primers
Project Sample DNA RFLP D and E
number Host material Symptoms Sample site groupings groupings

5 Cocos nucifera Palm trunk New bracket 1st sample site, Ambalanthota 1 1
6 Cocos nucifera Palm trunk Data not available 1st sample site, Ambalanthota 1 1
7 Cocos nucifera Palm trunk Bleeding only 1st sample site, Ambalanthota 1 1
8 Cocos nucifera Palm trunk Bleeding only 1st sample site, Ambalanthota 1 1
K20 Cocos nucifera Sporophore Felled palm adjacent to ditch, 4th sample site, Manandala 1 1
H. Rolph et al.

with sporophores
21 Cocos nucifera Palm trunk Felled palm adjacent to ditch, 4th sample site, Manandala 1 1
with small primordium
23A Areca catechu Palm trunk Bracket on dead palm 5th sample site, Ambalanthota 1 1
24 Cocos nucifera Palm trunk Stump with old sporophores 5th sample site, Ambalanthota 1 1
28A Cocos nucifera Palm trunk Palm with 2 sporophores 6th sample site, Ambalanthota 1 1
28B Cocos nucifera Palm trunk Palm with 2 sporophores 6th sample site, Ambalanthota 1 1
K28B Cocos nucifera Sporophore Palm with 2 sporophores 7th sample site, Ambalanthota 1 1
35 Cocos nucifera Palm trunk Stump with bleeding 7th sample site, Ambalanthota 1 1
36A Areca catechu Palm trunk Live palm (number 36) 5th sample site, Ambalanthota 1 1
36B Areca catechu Palm trunk Stump adjacent to live palm 7th sample site, Ambalanthota 1 1
K36A Areca catechu Sporophore Live palm (number 36) 7th sample site, Ambalanthota 1 1
K36B 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 1
33 Cocos nucifera Palm trunk Felled palm with sporophores 7th sample site, Ambalanthota A1A 1
K33 Cocos nucifera Sporophore Felled palm with sporophores 7th sample site, Ambalanthota A1A 1
34 Areca catechu Palm trunk Stump with sporophores next to 7th sample site, Ambalanthota 2 2
irrigation channel
K34 Areca catechu Sporophore Stump with sporophores next to 7th sample site, Ambalanthota 2 2
irrigation channel
K62 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 primordium
63 Cocos nucifera Palm trunk Stump with small sporophore 11th sample site, Beliatta 4 4
K63 Cocos nucifera Sporophore Stump with small sporophore 11th sample site, Beliatta 5 5
64 Cocos nucifera Palm trunk Stump with small sporophore 11th sample site, Beliatta 6 6
74 Leguminosae Palm trunk Tree with 1 dry sporophore 11th sample site, Beliatta 7 7

Key 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 was
sampled.
? = Isolate not tested
RFLP, restriction fragment length polymorphism.

214 H. Rolph et al.

There was only one exception where the Ganoderma infecting the palm tissue
did not appear to be the same as that producing the sporophores around the
base of the palm. This was on palm 21, where the Ganoderma isolate from
the palm tissue had a mtDNA profile in group 1 and the sporophore from the
base of the trunk had a completely unique mtDNA profile (group 3).
It is possible that profile 1 is the primary infection source and profile 3
represents a colonization by a non-infectious Ganoderma strain. Conversely,
profile 3 could represent a secondary Ganoderma infection, which is present in
another, as yet unsampled, part of the palm.

MtDNA from Ganoderma Isolates on Sri Lankan Betelnut
Palms
Ganoderma isolates with identical mtDNA profiles were found on betelnut
palms 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 on
Sri Lankan Coconut Palms
The AFLP groupings determined using primer D were identical to those
produced 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 found
across a large sample area. The most prevalent AFLP groups across sample
sites 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 again
indicate that many of the Ganoderma isolates studied show no host specificity.

Combined Results from mtDNA and AFLP Profiles
When the AFLP and mtDNA profiles were analysed, the control isolate from
the Leguminosae host produced unique profiles, which indicated that both tech-
niques were sufficient in their ability to discriminate between the Ganoderma
isolates 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


Fig. 16.4. Amplification fragment length polymorphisms from Sri Lankan
Ganoderma isolates from coconut palms. 1 = Marker; 2 = 5, Ganoderma isolate
from 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, Ganoderma
isolate 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 bleeding
only; 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. Further
work is needed to determine whether a single band in a mtDNA profile is a
significant enough difference to distinguish isolates which are in the same
AFLP group.
The fact that both techniques produced almost identical groupings,
however, indicates that these are valid groupings for the Ganoderma isolates
across all the sampling sites. It also shows that a general genomic profiling
technique such as AFLPs is very useful when used in conjunction with an
extrachromosomal profiling technique such as mtDNA RFLPs.
Different AFLP and mtDNA profile groups were found at each sample
site (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 the
stump 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 for
DNA isolations. For example, both mtDNA profile group 1 and AFLP group 1
were found in Ganoderma isolates from a range of sources (stem tissue from
palms displaying bleeding only; stem and sporophore tissue from a felled palm;
stem and sporophore tissue from a stump; standing palms with sporophores
and, finally, symptomless live palms).

Molecular Analysis of Ganoderma Isolates from Malaysian
Coconut Palms
MtDNA and AFLP profiles (data not shown) of Ganoderma isolates from
Malaysian coconut palms were much more varied across the small plot studied
than the Ganoderma isolates from Sri Lankan coconut palms had been over a
much wider area. Each profile was unique to each palm, i.e. the same profile
was never found on more than one palm. The high degree of molecular
variation seen in Ganoderma isolates from Malaysian coconut palms was the
same as that seen in isolates from Malaysian and Indonesian oil palms.

SIG Tests on Sri Lankan and Malaysian Ganoderma Isolates
None of the Sri Lankan isolates tested showed somatic compatibility with
any isolate other than themselves. Thus, the isolates were all somatically
incompatible with each other. However, both the mitochondrial DNA profiles
and 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 same
mtDNA and AFLP profiles, yet none of them were somatically incompatible
with each other.
Ganoderma isolates from Malaysian coconut palms also showed no somatic
compatibility with each other, but each had their own individual mtDNA and
AFLP profile.

Chlamydospore Production Experiments
One of the isolates from a Sri Lankan coconut palm produced cylindrical-
shaped chlamydospores on lima bean agar (Fig. 16.5). This shape of
chlamydospore is associated with the G. lucidum complex, a complex that
has often been reported to be pathogenic on coconuts in India.


Fig. 16.5. Chlamydospores produced by Ganoderma isolate (K33) from Sri
Lankan coconut palm (Lima bean agar, 28°C, 23 days).

Discussion
Ganoderma isolates from coconut and betelnut palms in Sri Lanka appear to be
different from Ganoderma isolates from Malaysian coconut palms. In Sri Lanka,
many isolates from a wide sample area share identical mtDNA and AFLP
profiles, although each isolate has its own SIG.
In Malaysia, the mtDNA and AFLP profiles varied from coconut palm to
coconut palm. The same profile was never found on more than one palm
and the variation found was quite striking across the single sample plot. Each
isolate displayed its own SIG and there was a general pattern of one SIG per
mtDNA and AFLP grouping. The level of molecular variation in Ganoderma
isolates from Malaysian coconut palms was, therefore, very similar to that on
Malaysian oil palms.
The fact that, in Sri Lanka, one mtDNA and AFLP group comprised a large
number of isolates sampled over several square kilometres, yet each isolate had
its own SIG, requires further investigation before it can be understood. It is
conceivable that the mechanism of reproduction and dissemination used by
Ganoderma on coconut palms in Sri Lanka may be different from that used by
Ganoderma on coconut and oil palms in Malaysia. Ganoderma populations from
oil palms have unique mtDNA profiles, even between adjacent palms; they are
heterothallic and have a tetrapolar mating system. However, many of the Sri
Lankan Ganoderma isolates shared identical mtDNA and AFLP profiles, yet
came from many different palms, suggesting that they are not heterothallic
and 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 within
the same genus. Studies of Armillaria ectypa have indicated that this is a
homothallic fungus, in direct contrast to other members of the genus that are
heterothallic 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 capabilities
of single-spore isolates (Guillmaumin, 1973). In a later study (Zolciak et al.,
1997) further factors were considered, including the absence of mating
reactions and the morphological identity between single-spore mycelia and
isolates from the context of the basidiome. RAPDs were also used to show
the genetic identity of single-spored isolates from the same basidiome. The
authors suggested that haploid basidiospores of A. ectypa might undergo
self-diploidization just after germination, although this would require further
testing by cytological observations of newly germinated basidiospores.
Similar experiments could be performed on sporophore and basidiospore
family sets of Ganoderma isolates from Sri Lankan coconut palms, to see if they,
too, were homothallic. Instead of using RAPDs, however, AFLPs would be used
to provide a more stable method of profiling the total genomic DNA. Another
interesting factor to consider is that one Ganoderma isolate from a Sri Lankan
coconut palm produced chlamydospores. This may indicate another method
of survival and spread of the identical molecular profiles over large distances.
It has been suggested by Miller et al. (1999) that Ganoderma infection of oil
palms may be through dispersal of basidiospores. It is not yet known how far
basidiospores can be dispersed to spread BSR infections across an oil-palm
planting. Chlamydospores are more resistant to environmental factors
than basidiospores and could be responsible for dissemination of one mtDNA
and AFLP group over a wide area, regardless of whether the fungus was
homothallic or heterothallic.
Both homothallism and production of chlamydospores would have to be
considered 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 determine
a species concept for them. Species delimitation within the genus, based on
traditional morphology, has not been of great use for tropical species of
Ganoderma. However, species concepts for the genus Ganoderma based on ITS
sequencing, morphological and biochemical data are slowly emerging. It
would also be very useful to perform ITS sequencing of isolates from coconut
palms in Sri Lanka, India and Malaysia, to help distinguish these isolates
further.
If these Sri Lankan Ganoderma isolates do represent a different species with
a different reproductive mechanism, then an hypothesis to explain how
this species came to affect coconut and betelnut palms in Sri Lanka as opposed
to the Ganoderma species found on oil palms in South-East Asia would be
required.


One possible hypothesis relates to the fact that oil palm is an introduced
crop to South-East Asia, whereas coconut palm is a far more established crop in
Sri Lanka. Consequently, Ganoderma isolates in Sri Lanka have had a longer
time to evolve and adapt to their palm hosts. This might partly account for
the lower level of molecular variability found in Ganoderma isolates from Sri
Lankan coconut palms in contrast to the higher level of molecular variability
in isolates from oil palm in Malaysia.
Conversely, it is also possible that the Ganoderma population on coconut
and betelnut palms in Sri Lanka represents a very young pathogen. Root and
bole rot of coconuts was only reported in Sri Lanka in 1974 (Peries, 1974) and
it is possible that it has only spread over the past 25 years. This might possibly
suggest a reason for the low level of variation in a newly emerged population.
Yet another possibility is that the restricted molecular variability in Sri Lankan
isolates (possibly due to a different mating system) could be as a result of geo-
graphical isolation and subsequent different evolutionary rates and pressures
after 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 contrast
to the heterothallic nature of nearly all other species of Armillaria. A. ectypa is a
rare arctico-alpine species, which was prevalent during the last glaciation. It
now survives in Sphagnum peat bogs at high latitude or altitude. It represents a
species which survives in a very geographically restricted environment, with a
homothallic mating system. It might be possible, therefore, that Sri Lankan
Ganoderma isolates found on coconut palms have evolved with a different
mating system due to their geographically restricted environment.
It would be necessary, however, to discover whether Ganoderma isolates
from coconut palms in India showed a similar low level of molecular variation
as the Ganoderma isolates from coconut palms in Sri Lanka. A similar study
to the one described in this chapter would help to test this hypothesis. If it
was the case, and the Indian and Sri Lankan isolates also shared the
same mating system, then the geographical isolation hypothesis could be
discounted.
The next crucial step in the study of Ganoderma isolates from Sri Lankan
coconut palms is, therefore, to determine their method of reproduction. They
must then be fully characterized (up to species level), using a combination of
morphological and molecular techniques, to develop suitable markers to track
them in the field. Once these steps have been taken, a strategy for control of the
fungus in the field can be developed properly.

Acknowledgements
The authors would like to thank Liz Biddlecombe for the pictures of the
chlamydospores, produced as part of a UKFCC-funded bursary project, and
Ann Ansell for preparation of cultures for the CABI Bioscience Genetic
Resource Collection.

220 H. Rolph et al.

References
Abdullah, 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.
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.
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., 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.
Nationale de Belgique 50, 135–186.
Turner, P.D. (1965a) The incidence of Ganoderma disease of oil palm in Malaya and its


Turner, 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
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18(4), 299–313.

Development of Diagnostic V
Tests for Ganoderma

17
P.D. Bridge et al.
Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm

Development of Molecular 17
Diagnostics for the Detection
of Ganoderma Isolates
Pathogenic to Oil Palm
P.D. Bridge1*, E.B. O’Grady1, C.A. Pilotti2 and
F.R. Sanderson2
1CABI Bioscience, Egham, UK; 2PNG OPRA, Plant Pathology
Laboratory, Alotau, Milne Bay Province, Papua New Guinea

Introduction
The development of molecular biology methods that allow for the almost
routine detection and analysis of DNA sequences has resulted in a considerable
increase in the accuracy and speed of fungal identification (Foster et al., 1993;
Bridge and Arora, 1998). The knowledge and comparison of DNA sequences
has also enabled the more precise grouping of fungal taxa, and has provided
important insights into the genetic variability present in fungal pathogens
(Bruns et al., 1991; Henrion et al., 1992; Takamatsu, 1998). Molecular tech-
niques can therefore provide powerful tools for identifying particular fungi in
environmental samples or plant tissue, and for determining epidemiology of
fungal diseases (Beck and Ligon, 1995; Di Bonito et al., 1995).
The DNA region most commonly used for molecular determination of
filamentous fungi is the gene cluster that codes for the ribosomal RNA (rRNA;
Fig. 17.1). This cluster is composed of the genes for the 5.8S, the small and the
large ribosomal subunits, which are separated by internal transcribed spacers
(ITS). The small subunit (SSU) and large subunit (LSU) genes are constituted
as a number of separate regions (domains), comprising both moderately and
highly conserved DNA sequences. In contrast, the ITS regions are composed
of more variable DNA sequences. The rRNA gene cluster occurs in multiple
copies in the genome, arranged in linear repeats, with each gene cluster
separated by an intergenic spacer (IGS) region (White et al., 1990; Hillis and



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 the
ubiquitous nature of the gene cluster, makes the rRNA genes good target
regions for the development of molecular diagnostics. The variation in
sequence conservation across the gene cluster allows for specific sequences
to be identified at different taxonomic levels (Bruns et al., 1991; Bainbridge,
1994). The conserved sequences in the subunit genes show sufficient
conservation to enable sequences to be identified that are common to all fungi,
or to individual phyla and orders. Alternatively, the variable sequences of
the spacer regions (ITS and IGS) contain sequences that are common at
approximately the species level, and many species-specific sequences have
been identified in these regions (White et al., 1990; Mills et al., 1992; Levesque
et al., 1994; Bridge and Arora, 1998; Edel, 1998).
The polymerase chain reaction (PCR) is a method that enables many
copies to be made of particular DNA regions. The basic principles of the PCR
reaction are that a region of DNA is defined from two flanking sequences, and
multiple copies of this are then produced through repeated cycling of a series of
temperature-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 base
sequence (Saiki et al., 1985, 1988; Mullis et al., 1986; Mullis and Faloona,
1987). The ribosomal RNA gene cluster, as described above, consists of
interspersed conserved and variable sequences. General primers can therefore
be constructed to conserved sequences which flank variable regions and allow
amplification of the intervening variable region. This principle is used to
amplify the ITS regions, with primers designed from the termini of conserved
subunit genes (White et al., 1990; Gardes and Bruns, 1993). Analysis of the
sequences of amplified ITS regions can then identify common and unique
sequences that can be used to design further primers with increased specificity.
This approach has been used for a number of fungi and has been particularly
effective in developing species- or pathogen-specific primers that can be used
with environmental samples and in the presence of plant material (Gardes
et 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 sequences
of the rRNA gene cluster in the genus Ganoderma (Moncalvo et al., 1995a), and
more than 30 ITS sequences are available through public access databases

Molecular Diagnostics for Detection of Ganoderma Pathogenic to Oil Palm 227

such as EMBL and GenBank. There is considerable similarity between ITS
sequences, and these can be aligned from species across the genus (Moncalvo,
this volume). Small groups of isolates can be defined by ITS sequences with
approximately 2–3% sequence variation within groups (Moncalvo et al.,
1995b, c). This level of sequence variation corresponds well to that seen within
species of some other plant-pathogenic fungi (Seifert et al., 1995; Sreenivasa-
prasad et al., 1996), and so it would appear that ITS sequences can be
used to define species in Ganoderma. One feature of the ITS regions is that most
variation is associated with the 5′ and 3′ termini of the region (Moncalvo et al.,
1995b, c). Although ITS regions have been sequenced from many Ganoderma
species, very few sequences have been obtained from isolates associated with
palms, and none are available through the public access databases. A single
sequence has been deposited for G. boninense, but it is now believed that the
isolate was incorrectly labelled and had not been associated with a palm
(Moncalvo, personal communication).
Several molecular approaches have been used to characterize isolates of
Ganoderma (Miller, 1995; Miller et al., 1995; Abu-Seman et al., 1996; Gottlieb
et al., 1998). The most widely used has been isoenzyme analysis and this has
given rather variable results. In studies on Ganoderma species on woody plants
in South America, isoenzyme profiles can in some cases define species (Gottlieb
et al., 1998). However, studies on palm pathogens have proved more compli-
cated and although pectinase zymograms produce band patterns that largely
define the palm-associated isolates, intracellular isoenzyme profiles can be very
variable and appear to define either individuals or small groups of apparently
unrelated isolates (Miller, 1995; Miller et al., 1995). In the oil-palm-associated
isolates, mitochondrial DNA polymorphisms appear to define populations at
around the level of an individual or sibling family (Miller et al., 1999), while
DNA fingerprinting methods, such as amplification fragment length poly-
morphisms (AFLPs) and simple repetitive primers, can give band patterns that
vary between individual monokaryons isolated from a single basidiocarp
(Bridge, 2000). This is in contrast to results obtained from isolates pathogenic
to coconuts in Sri Lanka, where both techniques showed little variation within
the population (Rolph et al., this volume), perhaps indicating the clonal spread
of a new pathogen.
One of the aims of the EU-STABEX-funded programme at the Papua New
Guinea Oil Palm Research Association (OPRA) has been to develop a rapid
molecular diagnostic method for detection of Ganoderma pathogenic to oil
palm. ITS regions were targeted for this due to the ready availability of
comparative sequences and the success obtained with this approach in
other groups of plant-pathogenic fungi. An additional consideration was that
Ganoderma on oil palm occurs as dikaryotic mycelium and basidiocarps that
give rise to monokaryotic basidiospores. The rRNA gene cluster is generally
considered to be resistant to cross-over and segregation events and so could be
expected to be conserved through both meiosis and mitosis (Hillis and Dixon,
1991; Hibbet, 1992).


ITS Region of Oil-palm-associated Isolates
The ITS region was amplified from cultures obtained from isolates infecting
palms at Milne Bay Estates, Alotau, Papua New Guinea. Cultures were
obtained from both dikaryotic mycelium and from monokaryotic mycelium
derived 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 from
individual basidiospores from single basidiocarps, and three further dikaryotic
cultures derived from crosses made within each set of monokaryons. These
cultures were selected in order to ensure that the ITS region was normally
resistant to any cross-over and segregation associated with meiosis. The
collection of the original basidiocarps was from widely separated palms and so
could provide an indication of any variation present in the overall population.
DNA was extracted from each culture and the complete region, containing
both ITS sequences and the 5.8S RNA subunit gene, were amplified with
the 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 by
digestion of the products with restriction enzymes to give simple restriction
fragment 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 and
sequenced in both directions. These were also found to be identical for all of the
representative samples (Fig. 17.2).
The sequence obtained was compared to all of those maintained in public
access databases, as the complete sequence and as the separate ITS1 and ITS2
regions. In every case the most similar sequences were always those from other
Ganoderma species.

Fig. 17.2. DNA sequence of 593 bases including the internal transcribed spacer
regions. Nucleotides in bold correspond to conserved regions. The first 10 bold
nucleotides are the 3′ terminus of the small subunit gene, the bold nucleotides
in the centre of the sequence are the 5.8S subunit gene and the final 18 bold
nucleotides are the 5′ terminus of the large subunit gene. Unique sequence used
for construction of primer GanET is contained in the box and the site for primer
ITS3 is underlined.


Selection of Primer Site
As described earlier, previous studies have shown considerable similarities in
the 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 across
the genus and to determine sequence divergence between species. Figure 17.3

Fig. 17.3. Multiple alignment of ITS2 sequences, rooted with Fomitopsis rosea.


shows an example of such an alignment of the ITS2 sequences of Ganoderma
isolates contained in the EMBL database, with the ITS2 sequence of the isolates
from oil palm in Papua New Guinea. This alignment shows that the ITS2
sequence from the oil-palm isolates is distinct from those of other species, and
comparison of the ITS2 sequences showed two sequences near the 3′ terminus
which appeared to be unique to the oil-palm isolates. The first of these was the
sequence TGCGAGTCGGCT, which started at position 105, and the second
was GTTATTGGGACAACTC, which started at position 178. Short oligo-
nucleotide sequences with high GC contents have been used as primers for the
random amplification of polymorphic DNA (RAPD) in many fungal genomes
(Welsh and McClelland, 1990; Williams et al., 1990). The first unique
sequence in the Ganoderma ITS2 region was very similar to a RAPD primer
in that it was 12 nucleotides in length and had a 75% GC content. A primer
constructed to this site might therefore behave similarly to a RAPD primer and
would be unsuitable for specific detection methods. However, the second
unique sequence was longer (16 nucleotides) and had a 44% GC content, and
so was more suitable as a site for a specific oil-palm-associated Ganoderma
primer. A primer (GanET) was constructed that gave a 3′ complement to this
sequence. The sequence of this primer and the original DNA region were
screened by searching the public access sequence databases. The original
sequence showed very little homology with any reported DNA sequence, and
none of the most similar sequences were obtained from fungi. This finding
supported the original assumption that the sequence selected was specific to
the oil-palm-associated Ganoderma. A second, 5′, primer was required to enable
the amplification of a single fragment, and primer ITS3, a conserved sequence
in the fungal 5.8S subunit gene (White et al., 1990) was selected. The
combination of ITS3 and GanET would, in theory, amplify a 321 bp region
containing most of the 5.8S subunit gene and the ITS2 region (see Fig. 17.2).

Evaluation of Primer Combination
The first step in the evaluation of the ITS3/GanET primer pair was to test this
primer combination against a purified DNA sample from one of the isolates
that had been sequenced originally. Amplification was undertaken with a high
annealing 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 Ganoderma
from basal stem rot (BSR) of oil palm in Papua New Guinea and Malaysia, and
produced 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 included
species of Verticillium, Ascochyta, Phoma, Fusarium, Rhizoctonia, Psilocybe,
Thielaviopsis and Phytophthora. Although PCR products were obtained from


some of these cultures, none contained the specific 321 bp product. One nota-
ble finding was the absence of the band from palm-associated Thielaviopsis, as
these organisms have been implicated in a number of palm diseases, including
upper stem rot (Kochu-Babu and Pillai, 1992).
A second test involved the amplification of DNA samples from a wider
range of palm-associated Ganoderma cultures. These included saprobic isolates
from coconut and areca palms, saprobic cultures from poisoned oil palms, and
isolates from Sri Lanka and India pathogenic to coconut palms (Rolph et al.,
this volume). Amplification with the ITS3/GanET primers gave the specific
321 bp band in saprobic isolates obtained from coconut and areca palm, but
this band was not produced in isolates from poisoned oil palm or from isolates
pathogenic to coconut. ITS regions have been widely used to define fungal
species and these results have some interesting implications for the study of the
spread of Ganoderma diseases among palms. This presence of the specific band
in saprobic isolates from coconut and areca palms would suggest that these
isolates are either the same taxon as the oil-palm pathogen, or are very closely
related to it. This is in agreement with previous observations and molecular
studies which have suggested that BSR of oil palm may be caused by
isolates saprobic on other palm hosts (Miller, 1995; Miller et al., 1995). The
absence of the band in the saprobic isolates from poisoned oil palm suggests
that not all saprobic Ganoderma on palms belong to the BSR taxon. This is
supported by the morphology of these cultures, which produced darker
basidiocarps on the palm. The absence of the band from the isolates from
infected coconut palms in India and Sri Lanka would suggest that these may
also belong to a further taxon. This is supported in part by other molecular
findings that show that the Sri Lankan coconut pathogen population is very
homogeneous and may be a single, recently developed population (Rolph et al.,
this volume).

Diagnostic Capabilities
The ITS3/GanET primer pair was able to differentiate successfully the oil-palm
BSR isolates from DNA preparations of pure cultures in the laboratory. The
next phase of developing a diagnostic tool was to assess the capability of the
primer pair to amplify the specific fragment from environmental samples that
contain palm stem material and other saprobic microbes and invertebrates.
Samples of infected and uninfected palm stem were collected from Milne
Bay Estates, Alotau. Samples of tissue (approximately 2.5 × 0.75 cm) were
collected into sterile screw-top bottles containing sufficient iso-propyl alcohol
to keep the samples completely immersed. Samples were stored at room
temperature for between 1 and 2 weeks after collection. The stem fragments
were then frozen in liquid nitrogen and ground to powder in a mortar and
pestle. The total DNA from the sample was extracted by a polyvinyl poly-
pyrolidone/cetrimide extraction method (Cubero et al., 1999).


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 minimize
the 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 that
the characteristic 321 bp band was only produced in samples derived from
infected palms, and that this band was not present from reactions with
uninfected palm material.

Conclusions
This study has shown that the Ganoderma responsible for BSR in oil palm is
a single taxon, which is distinct at a species level. The ITS-based approach
provides a single diagnostic method for the taxon which is independent of the
infraspecific variation seen for many other characters. The results support the
hypothesis that the BSR organism occurs in a saprobic state on other dead
palms, particularly coconuts. The oil-palm taxon is, however, one of a number
of Ganoderma taxa that may be saprobic on palms. The causative organism of
stem rots on living coconut in India and Sri Lanka may be distinct from the
oil-palm BSR, but testing of further isolates will be necessary before this can
be established definitively. The use of the ITS3/GanET primer pair provides
a practical tool for the detection and tracking of the BSR organism in the
environment, and this provides a means to determine accurately the spread
and infection route of the organism in the environment.

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18
C. Utomo andof Diagnostic Tools for Ganoderma in Oil Palm
Development F. Niepold

The Development of 18
Diagnostic Tools for
Ganoderma in Oil Palm
C. Utomo1,2 and F. Niepold2
1Indonesian Oil Palm Research Institute (IOPRI), Medan,
Indonesia; 2Federal Biological Research Centre for Agriculture
and Forestry, Institute for Plant Protection of Field Crops and
Grassland, Braunschweig, Germany

Introduction
Oil palm (Elaeis guineensis) is one of most important plantation crops in
Indonesia and can contribute up to 15% of the industrial oil needs of the local
food industry. However, a substantial loss of yearly harvests is caused by fun-
gal attack, especially by Ganoderma species, the causal agent of basal stem rot
(BSR) disease. BSR was first reported in 1930 in Malaysia and was identified
as Ganoderma lucidum (Thompson, 1931). Later, Steyaert (1967) was able
to identify six Ganoderma species isolated from oil-palm fields. These were
classified as G. boninense, G. miniatocinctum, G. chalceum, G. tornatum, G.
zonatum and G. xylonoides. Turner (1981) listed 15 species of Ganoderma
associated with BSR in oil palms, whereas studies in Indonesia and Malaysia
indicate that BSR is caused by a single species, G. boninense (Abadi, 1987; Ho
and Nawawi, 1985).
Previously, BSR was reported to occur only on old palms during the first
planting cycle but, more recently, the disease was found to attack young palms
during the second planting cycle. These incidences led to the assumption that
infection of BSR takes place in young palm and is a result of contact of the
healthy root with the infected tissue of previously planted palms. In older
palms, the infection occurs by root contact with diseased neighbouring palms
(Singh, 1991). The incubation period of the disease lasts several years and,
unfortunately, the disease symptoms only appear at a very late stage of infec-
tion. When this happens, more than half of the bole tissue has decayed and
usually this infected palm can not be cured. A survey undertaken by the
authors on some oil-palm plantations (unpublished data) in North Sumatra


236 C. Utomo and F. Niepold

(Indonesia) indicated that in certain areas of the second planting cycle up to
70% of palms were infected with Ganoderma after 15 years. These data are
similar 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 of
reliable diagnostic methods to detect early symptoms of BSR disease. Only two
methods have been developed so far for early diagnosis of BSR; one involves a
colorimetric method using ethylenediaminetetraacetic acid (EDTA) to detect
G. lucidum in coconut, the causal agent of Thanjavur wilt disease (Natarajan
et al., 1986). The second is a drilling technique where diseased material of oil
palm is collected by drilling into the diseased stem at 5–10 cm height from the
soil surface. Samples are then grown on media semiselective for Ganoderma
(Ariffin et al., 1993). These conventional methods are time-consuming and the
accuracy is not very high. Therefore, the availability of a rapid, inexpensive
and accurate diagnostic technique, which is specific and readily adapted to
large-scale testing for demonstrating Ganoderma in oil palm at an early stage of
infection, would benefit decision-making for appropriate control.
Use of the enzyme-linked immunosorbent assay (ELISA) and polymerase
chain reaction (PCR) for detecting pathogenic fungi in infected plants has been
applied widely. Successful detection of root-infecting fungi in infected plants by
ELISA has been reported previously, for example, detection of Heterobasidion
annosum, one of the most common basidiomycete organisms responsible for
the decay of conifers, by polyclonal antibodies (Avramenko, 1989) and by
monoclonal antibodies (Galbraith and Palfreyman, 1994). Also the serological
detection of Armillaria, a root-rot disease pathogen of many woody plants, has
been 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 tools
for early detection, due to their high sequence variation between species and
their general conservation within any one species. ITS regions have proven
useful for generating primers for a species-specific detection of pathogenic
fungi 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 work
was to elucidate an approach to detect Ganoderma using the ITS regions as a
target for generating specific primers to Ganoderma isolates of oil palms.
Another aim of this work was to produce polyclonal antibodies for the
serological 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 in
phosphate-buffered saline and then centrifuging at 13,000 r.p.m. for 10 min

Development of Diagnostic Tools for Ganoderma in Oil Palm 237

at 4°C. Rabbits were given three intramuscular injections. For the first
injection, 1.5 ml of antigen solution + 1.5 ml of Freund’s complete adjuvant
were used and with Freund’s incomplete adjuvant for subsequent injections at
10-day intervals. The rabbits were bled 2 weeks after the final injection.

Root sample preparation

Vacant areas due to Ganoderma infection were selected as the trial samples.
Healthy-looking oil palms (no disease symptoms of Ganoderma, no decayed
tissues in the base and no fruiting bodies of Ganoderma) surrounding the
vacant areas were chosen as samples. Root samples were collected from
the field by cutting the oil-palm root in the ground at a depth of 15–20 cm near
the 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 at
room 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 was
pipetted and stored at −20°C until use. To analyse the samples, indirect ELISA
was performed according to the method of Knapova (1995).

PCR
DNA obtained from isolates of Ganoderma and saprobic fungi and from oil-palm
root material was analysed. Isolates of Ganoderma were grown in a liquid malt
extract/yeast extract medium (15 g/5 g) and saprobic fungi were grown in
liquid Czapek Dox agar supplemented with yeast extract (34.4 g/10 g).
Three different DNA extraction methods were evaluated, as described
by Raeder and Broda (1985), Möller et al. (1992) and Wang et al. (1993). PCR
amplification was undertaken in 20 µl reactions with the primers GAN1 (TTG
ACT GGG TTG TAG CTG) and GAN2 (GCG TTA CAT CGC AAT ACA). These
primers were derived by the authors (unpublished) from the ITS1 region of the
rDNA of G. boninense (Moncalvo et al., 1995).

Studies using ELISA
A major problem in using immunoassay is the lack of specificity towards
plant-pathogenic fungi. Fungi are complex organisms which contain
numerous antigens, many of which are also shared by unrelated fungi. Thus,
thorough cross-reactivity tests against unrelated fungi that could be present
in the plant tissue were performed. This test is necessary in order to avoid
false-positive values. The specificity of PAb-1(polyclonal antibody 1, raised
against single isolate of Ganoderma) and PAb-9 (polyclonal antibody 9, raised
against nine isolates of Ganoderma) was tested against five saprophytic fungi
commonly isolated from diseased oil-palm roots. The five saprophytic fungi


were identified as Penicillium sp., Aspergillus sp., Trichoderma sp. 1, Trichoderma
sp. 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, whereas
PAb-1 gave higher cross-reactions (6–25%) against the five tested saprophytic
fungi (Fig. 18.2). The low cross-reaction of the PAb-9 with saprophytic fungi
that associated with diseased oil palm enabled evaluation of the results of
oil-palm samples in comparison with PAb-1.
The slope of the absorbance values per hour was calculated and presented
as d(A405 nm)dt−1. A positive and a negative threshold was set in the ELISA tests
by 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 Gano
Fig. 18.1. Cross-reaction of PAb-9 with common saprophytic fungi at different
dilutions. 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 Gano
Fig. 18.2. Cross-reaction of PAb-1 with common saprophytic fungi at different
dilutions. There was a slight cross-reaction visible with all the saprophytic fungi
tested. Peni, Penicillium sp.; Asper, Aspergillus sp.; Tri, Trichoderma sp.; Gano,
Ganoderma sp.


diseased roots. If the d(A405 nm)dt−1 values of the samples were three times
higher 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 low
d(A405 nm)dt−1 values were obtained when extracts from healthy root tissue
were 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 by
macerating 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, but
accurate quantitation of the fungus is not possible by this method.

PCR Study
DNA extraction and sensitivity threshold of a pure culture of Ganoderma

Three different DNA extraction methods gave a 167 bp fragment from DNA
of 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 samples
Fig. 18.3. Diseased and healthy roots from the field samples, as well as
saprophytic 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 with
saprophytic 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.


sensitivity threshold of PCR detection was assessed using serial dilutions of a
given quantity of Ganoderma genomic DNA as template. Sensitivity thresholds
of fungal DNA, depending on DNA extraction methods, were 1 ng for the
method 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 the
improved nuclear DNA extraction using alkaline (NaOH) solution, which in
turn allows sufficient dilution of the extract to eliminate or significantly reduce
the effect of potential inhibitors of the PCR. Good amplification results in a PCR
test using NaOH solution as the DNA extraction buffer have been reported for
extracting Phytophthora genomic DNA (Tooley et al., 1997).

Specificity tests of the primers Gan1 and Gan2 with other saprophytic
fungi and Ganoderma

In this study, the modified Wang method was used for extracting fungal DNA.
To further evaluate primer specificity, experiments were performed with 18
saprophytic fungi which were occasionally found as saprophytes on diseased
oil-palm roots. Twenty-three Ganoderma isolates from various sources were

Fig. 18.4. Determination of the detection limit of Ganoderma from oil palm using
three different DNA extraction methods. (a) Determination of the detection limit
based 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 of
the 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 water
control, and Lane S: DNA marker. (c) Determination of the detection limit based on
the 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.


also included in this evaluation (Table 18.1). Primers designed for the
diagnosis of Ganoderma in diseased oil palm also reacted with other saprophytic
fungi, but the amplification products of the saprophytic fungi differed in DNA
fragment size compared to the DNA fragment size from Ganoderma (Fig. 18.5).
In contrast, when DNA extracts from saprophytic fungi were diluted 1 : 10 in
the sap of healthy oil-palm root, no amplification product of the saprophytic
fungi could be observed. For Ganoderma, a dilution of the DNA extract of
1 : 10,000 using sap of healthy root of oil palm still allowed production of a
strong amplification product (Fig. 18.5). Since no PCR signals were seen when
DNA of saprophytic fungi were diluted in the sap of healthy oil-palm root,
contamination with saprophytic fungi in diseased roots would not generate
false-positive values.
Primers Gan1 and Gan2 also reacted with other Ganoderma isolates. A
fragment of approximately 167 bp was amplified from all tested isolates of
Ganoderma (data not shown). The ITS1 region of Ganoderma is relatively similar
within all Ganoderma species. In addition, the ITS1 region of Ganoderma is small
enough to be easily amplified by PCR and is flanked by highly conserved
sequences (Moncalvo et al., 1995).
Development of a PCR test for species-specific detection of Ganoderma in oil
palm is urgently required, not only for early detection purposes but also for
detection of the source of the inoculum as well as for agronomic practice. For
example, when crop rotation occurs from rubber or cocoa to oil palm, the
stumps of rubber or cocoa are usually left on the fields. After a certain period of
time the stumps are colonized by Ganoderma and other basidiomycete fungi.
Therefore, it is very difficult to determine whether Ganoderma that will infect oil
palms are the same species as those colonizing the stumps. The grower needs to
be able to solve this problem, in order to decide whether or not to remove
stumps, because the elimination of the stumps is very costly.

Detection of Ganoderma from infected oil-palm roots

Three methods of DNA extraction were used to extract Ganoderma template
DNA from infected oil-palm root samples, as described earlier. In this study, the
PCR assay successfully amplified Ganoderma DNA within infected root diluted
1 : 100 with 100 mM Tris/BSA using the method of Möller et al. (1992) and a
modification of the method of Wang et al. (1993) (Fig. 18.6a, b). The method of
Raeder and Broda (1985) produced only smeared PCR signals when extracted
from 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 as
polysaccharides (Demeke and Adam, 1992) or phenolic compounds (Cenis,
1992; John, 1992; Johanson, 1994), may drastically reduce the sensitivity of
a PCR test. For this reason, further additional purification steps should be
performed to remove inhibitors, including cation exchange columns (Stein
and Raoult, 1992); polyvinyl polypyrrolidone (PVPP) application, which


Table 18.1. Fungi used in this study.

Fungi Isolate Source Country

Ganoderma of oil palm – IOPRI Indonesia
G. oerstedii BAFC.178 UBA Argentina
G. oerstedii BAFC.218 UBA Argentina
G. resinaceum BAFC.384 UBA Argentina
G. tornatum (applanatum?) BAFC.671 UBA Argentina
G. applanatum BAFC2353 UBA Argentina
G. lucidum complex BAFC.2374 UBA Argentina
G. tornatum BAFC.2390 UBA Argentina
G. tornatum? BAFC.2395 UBA Argentina
G. applanatum BAFC.2408 UBA Argentina
G. tornatum BAFC.2424 UBA Argentina
G. tornatum? BAFC.2430 UBA Argentina
G. lucidum complex BAFC.2495 UBA Argentina
G. applanatum var. tornatum BAFC.2501 UBA Argentina
Ganoderma sp. BAFC.2529 UBA Argentina
G. applanatum BAFC.2552 UBA Argentina
G. tropicum BAFC.2580 UBA Argentina
G. lucidum DSM 9612 DSMZ Germany
G. lucidum DSM 103 DSMZ Germany
G. applanatum DSM 3800 DSMZ Germany
G. tsugae – FAL Germany
Trichoderma koningii MRS 1 IOPRI Indonesia
T. harzianum MRS 2 IOPRI Indonesia
T. viride MRS 3 IOPRI Indonesia
Aspergillus flavus MRS 4 IOPRI Indonesia
Aspergillus sp. – IOPRI Indonesia
Penicillium sp. – IOPRI Indonesia
Gliocladium sp. – IOPRI Indonesia
Trichoderma sp. 1 – IOPRI Indonesia
Rhizopus sp. – IOPRI Indonesia
Bispora sp. – IOPRI Indonesia
Geotrichum sp. – IOPRI Indonesia
Cylindrocarpon sp. – IOPRI Indonesia
Mucor sp. – IOPRI Indonesia
Monilia sp. – IOPRI Indonesia
Fusarium sp. – IOPRI Indonesia
Botryodiplodia sp. – IOPRI Indonesia

IOPRI, Indonesian Oil Palm Research Institute, Medan, Indonesia; UBA,
Universidad De Buenos Aires, Argentina (gift of Dr Alexandra M. Gottlieb);
DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany; FAL, Bundesforschungsanstalt für Landwirtschaft,
Braunschweig, Germany.


Fig. 18.5. Cross-reaction tests of primers Gan1 and Gan2 against 18 saprophytic
fungi isolated from diseased oil-palm roots. (a) Ganoderma and saprophytic fungi
were 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. 2
and Trichoderma sp. 3. Lanes 14–21: Ganoderma, Gliocladium sp., Mucor sp.,
Cylindrocarpon sp., Monilia sp., Fusarium sp., Aspergillus sp. and Botryodiplodia
sp. Lanes S, 13 and 22: DNA marker. (b) Ganoderma and saprophytic fungi were
mixed with extracted healthy roots. Lanes 1–4: Ganoderma in healthy root dilution
1 : 10, 1 : 102, 1 : 103 and 1 : 104. Lanes 5–12 and 14–24: saprophytic fungi in
healthy 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., Cylindrocarpon
sp., 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 of
commercial 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 consuming
and expensive, the reported development of a simple and fast Ganoderma DNA
extraction method for infected palms, with no additional purification steps,
represents an advantage in routine PCR tests. Since no amplification product
was observed with nucleic acid extracted from healthy roots, the amplification
product obtained contains the target sequence of fungal DNA from infected
roots. Therefore, the modified Wang method is considered as the most simple


Fig. 18.6. Detection of Ganoderma from diseased oil-palm roots with primers
Gan1 and Gan2. (a) Extraction of Ganoderma DNA from diseased oil-palm root
using the Möller method. Lane 1: 5 ng of Ganoderma DNA. Lanes 2–5 TE buffer
1 : 5, 1 : 10, 1 : 102 and 1 : 103, respectively. Lane 6: negative water control. Lanes
8–10: extracted healthy oil-palm root, diluted with TE buffer 1 : 5, 1 : 10 and
1 : 102, respectively. Lanes S and 7: DNA marker. (b) Extraction of Ganoderma
DNA 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 in
Tris/BSA: 1 : 5, 1 : 10, 1 : 102, 1 : 103 and 1 : 104, respectively. Lane 7: water
negative 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 root
samples, and it has the added advantage that the chemicals used are not as
expensive as those used in other extraction methods.


Conclusions
Positive or negative values for the detection of Ganoderma by ELISA were
based on reactivity relative to the negative control. The cross-reactivity with
unrelated fungi in the ELISA test led to false-positive values. Also, a low
concentration of Ganoderma in the infected tissues, in addition to dilution steps,
may elicit false-negative values in the ELISA test. In order to increase the
sensitivity and specificity of Ganoderma detection the PCR was applied.
A PCR-based assay appears to be more specific than the ELISA assay in
Ganoderma detection, because in the PCR assay cross-reaction with sapro-
phytic fungi was not observed. However, for detection using a large number of
samples, ELISA offers advantages in term of speed, ease of use and costs. Unlike
the PCR assay, in which genomic DNA must be extracted from infected
samples, ELISA only requires a small sample of sap, obtained by crushing the
samples. The use of the ELISA test might be useful as a pre-screen to handle a
lot of samples. In the case of a positive reaction, the PCR test should be applied
to verify the results. With this combination of both procedures, a fast and
reliable screening of oil palm is now possible.

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19
T.W.of Antibodies for Detection of Ganoderma Infection of Oil Palm
Use Darmono

Ganoderma in Oil Palm in 19
Indonesia: Current Status and
Prospective Use of Antibodies
for the Detection of Infection
T.W. Darmono
Biotechnology Research Unit for Estate Crops, Jl. Taman
Kencana No. 1, Bogor, Indonesia

Economic Importance of Basal Stem Rot (BSR) Disease
Oil palm was introduced to Asia through Indonesia 150 years ago and then
spread to other countries in the region (Pamin, 1998). In 1997, the total area
of oil palm in Indonesia reached 2,463,823 ha and approximately 80% of this
is located in Sumatra. In North Sumatra and Central Lampung, oil palm has
been cultivated for several replanting generations, each of which takes
between 25 and 30 years. Oil palms found in Kalimantan, Sulawesi and Irian
Jaya are only recently cultivated. The 1997 production of crude palm oil (CPO)
was 5,904,175 t, valued at US$2,952,087,500, and that of palm kernel oil
(PKO) was 1,189,603 t, valued at US$832,722,100. The total value of both
CPO and PKO was US$3,784,809,600.
Basal stem rot incited by Ganoderma spp. is one of the most important
diseases in oil palm. The annual capital loss at 1% disease incidence, calculated
on the basis of the export value of palm oil in 1996, reaches US$38,230,400.
As the disease is difficult to control, the infected trees are usually left to
deteriorate and die. In some cases the infected tree looks healthy although
more than half of its base has been degraded by the pathogen. The magnitude
of yield loss is greater if infection occurs at an early stage of tree maturity,
when aged between 5 and 15 years. The disease incidence at the same site in
a plantation tends to increase from year to year and from generation to
generation. A survey in a plot of 10.5 ha of 23-year-old oil palms of the
third planting generation conducted in July 1998 at Bekri Plantation, PTP
Nusantara VII, in Central Lampung, Sumatra, revealed the occurrence of
disease incidence to be up to 51% (Darmono, 1998).


250 T.W. Darmono

Current Status of Research on Ganoderma
Detailed information of BSR in oil palm can be found in Turner (1981). This
summarizes his findings from his own research and observations on the
disease in Indonesia prior to 1981. Although this gives a better understanding
of the disease, it does not provide clear guidance on how to control the disease
effectively, which can be incorporated in the whole system of oil-palm manage-
ment. Prior to 1980, there was no local research scientist in the country
actively involved in research on basal stem rot disease in oil palm. This was
probably due to two main reasons. First, there was no pressure from the
oil-palm industry, which was unaware that Ganoderma was a significant
problem. It was assumed that losses were not economically significant until
more than 20% of the stand had been lost. That assumption was lately proven
to be incorrect (Hasan and Turner, 1994) and the disease currently occurs at
a high incidence. The second reason was that working with higher fungi
such as Ganoderma spp. is generally difficult, slow and very long term. With the
increase in the incidence of the disease, the pressure from the growers has
increased, encouraging research institutions to speed up their study on
Ganoderma. Institutions currently engaged in research on Ganoderma as an
oil-palm pathogen in Indonesia include Biotechnology Research Unit for Estate
Crops (BRUEC) in Bogor, the Indonesian Oil Palm Research Institute (IOPRI)
in Medan, and Bah Lias Research Station (BLRS) of P.T.P.P. London Sumatra
in Pematang Siantar. SEAMEO Bio-Tropical in Bogor was also involved in
research 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 (Abadi
et al., 1989; Dharmaputra et al., 1990). In the field, this may represent a wide
range of soil types and oil-palm growing conditions at low elevations. Based on
field observations, there was no correlation between disease incidence and the
distance of the plantation to the coast, elevation, soil pH, or the density and
type of legume cover crops (Abadi et al., 1989). Later, it was also stated by
Hasan and Turner (1994) that there were few differences in BSR incidence
between plantings on coastal and most inland sites in Indonesia.
Although under field conditions, density and type of legume cover crops
did not seem to affect disease development, laboratory studies revealed that
supplementation of the agar medium with stem and leaf extracts of three
legume cover crops, i.e. Centrosema pubescens, Calopogonium mucunoides and
Pueraria 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 the
medium. 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 251

peak of vigour at 2–3 years after planting, these covers eventually die out
under the shade of the developing trees. Although the use of ground covers in
the plantation has been a subject of controversy, their use is beneficial in the
control of Rigidoporus microporus in Hevea rubber (Fox, 1977; Soepadmo,
1981). This has been suggested to be largely due to the enhanced rate of decay
of woody residues in the soil caused by the moist conditions and the high
nitrogen status of the cover and its litter (Wycherley and Chandapillai, 1969).
Although cover crops were commonly used in oil-palm plantations at the time
when slash and burn was still allowed, their effect on the rate of decomposition
of unburned, felled oil-palm stems has not been thoroughly investigated. At
present, 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 of
felled oil-palm stem is needed to prevent its colonization by Ganoderma which
may subsequently act as an inoculum source for the disease.
Research on the use of chemicals has been confined to laboratory studies
and results have shown that triadimenol at a concentration of 1.00 µg ml−1
was able to kill the mycelia of the pathogen, but this concentration also
inhibited a fungal antagonist (Dharmaputra et al., 1991). Preliminary results
from a field experiment have shown that triadimenol application by root
absorption was more effective in suppressing the disease than that applied by
soil drenching (Puspa et al., 1991). Using the same technique, Hasan (1998)
has shown that phosphonic acid application was capable of protecting
seedlings from infection. However, although these studies gave promising
results, the use of chemicals in the control of Ganoderma in the field on
a commercial scale will be impractical and economically infeasible until a
reliable technique of application has been developed. Also, even if a reliable
application technique was found, the beneficial use of chemicals is still
questionable since their effect can diminish rapidly. It has been shown that
the effect of triadimefon on Ganoderma cultured on rubber wood vanished
within 3 weeks (Darmono, 1996).
Research on the use of biological control agents for BSR has also been
initiated at SEAMEO-Biotrop in Bogor (Dharmaputra et al., 1994). Other
research institutions, including IOPRI (Soepena, 1998), BRUEC (Darmono,
1998), and BLRS (Hasan, 1998), have more recently become involved in the
same research subject. Studies conducted at these institutions have shown
that Trichoderma harzianum gave better control than that of other species of
Trichoderma. The use of a biological control agent in the control of Ganoderma
has been seen to be more promising than that of chemical control. The
capability of a biological control agent to grow and reproduce in the field
and that will allow the destruction of the pathogen in the soil, are some of the
advantages and attractiveness of its use. Biological control is also considered
to be less hazardous to the environment. Research to investigate whether
Trichoderma sp. can actively grow along the root needs to be conducted. This
would reveal the potential use of the agent as a root protectant.

252 T.W. Darmono

However, one problem with the application of chemical and biological
control agents is that the pathogen is capable of forming brown layers
(Darmono, 1998) that provide a barrier against the chemical or the
antagonist. These agents have to penetrate this barrier before being able to
kill the sensitive mycelium of the pathogen. The brown layers, composed
of melanized mycelium, also termed the ‘sclerotium plate’, are formed in the
vicinity of the interaction zones and at any sites in the decayed tissue of basal
stem. Sclerotium plates cover white masses of mycelium, forming pockets of
Ganoderma. These pockets of mycelium are commonly found in the decaying
oil-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 the
decomposed 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, in
addition to mycelium, the resting body of Ganoderma also contains degraded
plant tissue intermingled with the mycelium. These resting bodies are capable
of forming fruiting bodies and are capable of infecting oil-palm seedlings.
Molecular analysis has revealed that cultures obtained from inside the resting
bodies were identical to those obtained from the fruiting bodies developed
from the associated resting bodies. This result indicates that the resting bodies
found in decomposed oil-palm stems may be derived from the pathogen. Direct
transfer of the internal tissue of resting body into malt extract agar medium
produced pure culture, indicating that the fungus remained viable in oil-palm
logs under diverse environmental conditions in the field.
The formation of brown mycelium layers and resting bodies in Ganoderma
might function to protect the food resources acquired after invasion, to

Fig. 19.1. Resting bodies of Ganoderma found embedded in the decomposed
tissue of oil palm infected by the pathogen.


allow survival from one plant generation to another and to initiate a primary
infection. Deposition of melanin in fungal mycelium and spores has been
suggested to be important for resistance to environmental stress, including
protection against ultraviolet irradiation, radio waves, desiccation and
temperature extremes (Bell and Wheeler, 1986). Melanins in fungi have
also been suggested to be essential for resistance to microbial attack.
Good field sanitation is believed to be one of the best possible ways
to control the disease effectively (Hasan and Turner, 1994; Darmono, 1998).
Research on field sanitation has been conducted intensively at BLRS. A
recommended technique for point sanitation was to remove all diseased
material by digging a pit 1.5 m square and 1 m deep, centred on the point of
planting spot (Hasan and Turner, 1994). The disease remnants raised to the
soil surface are disrupted, the simplest way being by cutting them into four or
more pieces, to allow enhanced biological control. Darmono (1998) suggested
that field sanitation should be conducted before planting (pre-planting
sanitation activities) and regularly after planting during the entire life of
the plant (post-planting sanitation activities). In areas with a high disease
incidence, pre-planting sanitation can be conducted by removing all
remaining boles and root clumps. Root clumps up to 20 cm thick are usually
found attached to the boles. Special attention should be given to boles and
roots of newly infected trees that, in the new planting, will certainly form a
potential source of inoculum. Boles and root clumps of healthy trees left in the
ground can be more easily colonized by the pathogen than healthy roots of
newly established plants. In the long term, the removal of these tissue remains
will help in reducing the risk of greater Ganoderma infestation in the following
replantings. In post-planting sanitation, all infected trees that no longer have
economic value will be uprooted and sanitized.
The action of sanitation should be based on the observation of disease
incidence previously determined. Darmono (1998) generated a formula for
calculating disease incidence and scoring the grade of sanitation, as follows.
S+E
I= × 100%
N
where I is the disease incidence; S, the number of standing trees infected by
Ganoderma; E, the number of empty planting spots due to Ganoderma; and N,
the total number of planting spots observed.
R
G=
S+E
where 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 the
score of disease incidence merely on the number of empty planting spots or
plant mortality, due to Ganoderma in the plantation. Such a form of scoring
gives an impression that the infected standing trees do not have a significant

254 T.W. Darmono

role for disease development, and they have since been neglected during land
preparation for new planting. Detailed notes on the category of disease severity
in each tree should be made during observations. Categories of disease severity
proposed by Darmono (1998) are presented in Table 19.1.
The felling of old oil palms before land preparation for replanting was
usually conducted by pushing individual trees over with a bulldozer. By this
action, the healthy trees are usually uprooted along with their boles and root
clumps. If the tree is diseased (category R and Y), the pushing action usually
causes it to break off at the base and the boles and roots are left behind in
the ground. If not removed or sanitized, these remains will become potential
infection foci.
In a long-term programme, research activities at IOPRI and BRUEC are
currently undertaking the production of resistant oil-palm material by means
of conventional breeding and molecular biology techniques. At BRUEC,
chitinase and glucanase genes obtained from local strains of microbes will
be transformed into the plant genome and specifically expressed in the root
system so that, hopefully, the palm will become resistant to Ganoderma
infection. A transformation system in oil palm mediated with Agrobacterium
tumefaciens has also been developed (Chaidamsari et al., 1998) and a
propagation system for oil palm using tissue-culture techniques has been
acquired (Tahardi, 1998). Development of resistant planting materials needs
knowledge of the genetic variability in the pathogen. Studies on genetic
variability of Ganoderma associated with oil palm showed variation among
isolates 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 Colour
colour abbreviation Description

Green 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 Ganoderma
Yellow Y Plant looks healthy, but a fruiting body of Ganoderma or
brown discolouration can be observed at the base of the
stem
Red R Plant looks as if it is suffering from the disease and shows
typical symptoms and signs of infection
Black B Empty planting spot with infected boles and roots remaining
in the ground
White W Sanitized empty planting spot


An Attempt to Produce an Immunoassay-based Detection Kit
Need for the development of detection tools

From a practical standpoint, disease control in individual trees is hampered
by our inability to detect symptoms and signs of infection at an early stage
of disease development. Infected palms usually show symptoms only after a
large portion of their base has been destroyed by the pathogen. Although soil
drenching with fungicide may effectively kill the pathogen, large-scale
application of this type is not economically feasible. The success of chemical
treatments through trunk injection can be achieved only if they are applied at
an early stage of disease development. Therefore an accurate, quick and cheap
detection system needs to be developed.
Although cultural studies and microscopic observation are highly
accurate for diagnoses of the infection, these techniques are too slow and not
amenable to large-scale application (Miller and Martin, 1988). Immunoassay
and nucleic acid hybridization systems have been used for plant pathogen
detection 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 simplicity
and need less equipment than those of DNA probe analyses. Experiments on
the development of polyclonal antibody (PAb) and monoclonal antibody
(MAb) against Ganoderma sp. were initiated at the Biotechnology Research
Unit for Estate Crops in 1993 (Darmono et al., 1993). The main objective of the
experiment was to produce an immunoassay-based detection kit.

Detection kit specification

There are some requirements in order for new products or technology to be
applicable and acceptable by the users. In the case of a detection kit based on
immunoassay, 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 kit
should be specific enough so that it only recognizes Ganoderma associated with
basal stem rot, regardless of strain dissimilarity and geographical origins. If it
is too specific, the antibody will detect only a certain strain of the pathogen
and, consequently, will be less useful for field application. There are at least
two ways to overcome this problem. The first is by pooling several specific
antibodies or monoclonal antibodies, but this will be hampered by limited

256 T.W. Darmono

knowledge on the number of strains of Ganoderma found in oil palm and by the
high cost of production of the antibody. The second, less expensive, way is
the development and production of polyclonal antibody. The sensitivity of the
antibody should be measured, based on laboratory and field exercises. In
laboratory exercises the level of sensitivity is determined by the ability of the
antibody (at certain levels of dilution) to detect the least amount of antigen. For
field applications, the antibody should ideally be capable of detecting antigenic
material at an early stage of disease infection.
The root system of an individual mature oil palm occupies about 16 m3 of
soil, and Ganoderma infection could start at any point in that space. In that kind
of situation, the use of a DNA hybridization technique to detect Ganoderma
infection at an early stage of disease development may be unreliable as it would
require DNA obtained from the infection point. Thus, the tool used should
ideally be able to detect infection at a distance from the infection site. Signs of
infection can be in the form of chemical compounds produced by either the
pathogen or by the plant in response to infection.
Acceptability of any new product known to be strongly dependent on its
price and ease of use. It should be cheap and be of significant benefit to the
growers. Ideally, it should be far less expensive than the cost of single nutrient
content analyses, which is approximately US$2 per sample in Indonesia. For
the detection of Ganoderma infection, it would be better if systematic sampling
could be conducted in the field regularly during observation of disease
incidence. Alternatively, spot-selected sampling can be practised for reducing
the cost of use. Sending samples to a commercial institution for enzyme-linked
immunosorbent assay (ELISA) will be costly so the tool should be suitable for
on-site application by any person with no special skills. Sampling activities
should not harm the palms. Special care should be taken if the sample has to be
obtained from the trunk or root, since an open injury may function as the entry
point for the pathogen.

Development of PAb

Mycelial wash as antigen
In the first stage of antibody development, a mycelial wash was used as a
source of antigen. An isolate of Ganoderma sp. (TK-1, obtained from an infected
oil palm in Bogor Botanical Garden) was cultured in a chemically defined
liquid medium (Leatham, 1983). The mycelium was harvested and washed
three times with phosphate-buffered saline (PBS) by filtration through a single
layer of Whatman No. 93 filter paper. The liquid fraction from the final wash
was used as the antigen.
To develop the polyclonal antibody, a hyperimmune Balb/c mouse was
injected intraperitoneally four times, at 2-day intervals with 250 µl
antigen. Two days before the blood was withdrawn, an intravenous booster
injection was given. Blood serum was obtained and the optimum titre for the


antigen–antibody reaction was determined, based on a ‘conventional checker
A board’ method (Moekti, 1991). Cross-reactivity tests of the PAb were
conducted 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); and
3. Solvent from a spore wash of 10 isolates of Ganoderma spp. associated with
oil palm (including isolate TK-1).
The optical density (OD) value of I-ELISA was measured with an automatic
EIA-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. Even
with this relatively low content of protein the mycelial wash was proven to be
capable of inducing a high titre of antibody (Figs 19.2 and 19.3). This might
indicate that it contained a high molecular weight antigenic material in the
form of protein or other metabolites.
Antigen that contains polypeptides or proteins with a molecular weight
of 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−1
antigenic material (Fig. 19.2). Undiluted antibody was capable of detecting
1.156 µg ml−1 antigenic material (Fig. 19.3). This result showed that when
antigenic materials are present at low concentration, an undiluted antibody
should be used. Determination of the titres is necessary in the development of
any new antibody.

Fig. 19.2. Optical densities from enzyme-linked immunosorbent assay readings in
titres between dilute antibody and concentrated antigen.

258 T.W. Darmono

Fig. 19.3. Optical density from enzyme-linked immunosorbent assay readings in
titres between concentrated antibody and dilute antigen.

The successful use of the mycelial wash as a source of antigen in the
development of molecular detection assays for plant pathogenic fungi has
been reported (Brown, 1993), however in this project, we encountered several
problems due to its high specificity. The antibody only recognized antigenic
materials from the in vitro cultures and not from the in vivo sources from
field fruiting bodies or spores. Furthermore, the antibody produced was
not capable in distinguishing Ganoderma spp. from different host origins. To
increase specificity and sensitivity, monoclonal antibody development and the
use of an exudate of Ganoderma sp. were attempted.

Exudate as antigen
The brown aqueous exudate secreted on the surface of mycelium grown
on rubber wood was used as an antigen to develop a PAb anti-exudate of
Ganoderma (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 egg
yolk were isolated, as described by Darmono and Suharyanto (1995). The
specificity and reactivity of PAb-aeG were evaluated against 10 isolates of
Ganoderma sp., using I-ELISA. The antigen for the cross-reactivity test was
prepared from air-dried mycelium of on-wood cultures of the reference isolate
AD-2 and field fruiting bodies of Ganoderma spp. Two grams of mycelium
or fruiting body were ground in liquid nitrogen and extracted with 15 ml
Tris buffer. The homogenate was separated and used as the antigen in
cross-reactivity tests. Two types of enzyme–antibody conjugates, i.e. rabbit
anti-chicken horseradish peroxidase conjugate and alkaline phosphatase


conjugate, were tested at a dilution of 1 : 5000. The OD value of I-ELISA was
measured with an automatic EIA-Microplate Reader at wavelengths of
405 nm and 495 nm.
Accumulation of chicken antibody corresponded well with antigen
injections, indicating that the antibody was produced specifically against the
exudate of Ganoderma sp. The optimum level of antibody production was
found in eggs collected on the thirteenth day after the final immunization or
the twenty-third day after initial immunization (Fig. 19.4). One of the main
advantages of using chicken antibody is the ease of handling of the animal
and of obtaining the antibody. About 15 ml of antibody mixture was usually
obtained from each egg in a relatively short period of time, compared to 70
days or longer in rabbits. This amount of yolk antibody is sufficient to run
about 3000 reactions in microwells.
PAb-aeG produced in this study was highly sensitive in recognizing all field
fruiting bodies of Ganoderma spp. associated with oil palm, but not Ganoderma
of non-oil-palm origins (Fig. 19.5). A satisfactory result was obtained only
with the use of horseradish peroxidase anti-chicken antibody conjugate but
not with alkaline phosphatase anti-chicken antibody conjugate.

Development of MAb

Antibodies were developed in a hyperimmune Balb/c mouse. Immunization
of the mouse was conducted using a mycelial wash of isolate TK-1 as an
immunogen, through the same procedures as described above. Five days after
the final injection, a blood sample was withdrawn and lymphocytes were

Fig. 19.4. Development of antibody in egg yolk, induced after injection of the
hen with exudate of Ganoderma.

260 T.W. Darmono

Fig. 19.5. Cross-reactivity of Ganoderma isolates against PAb-aeG.

harvested and fused with myeloma sp/2 cells. Cell fusion was performed by
treating the mixed cell suspension with polyethylene glycol (PEG) 4000 at
37°C for 2 minutes. The treated cells were cultured on a selective medium,
Dulbecco Modified Eagle Media (DMEM) supplemented with 15% fetal calf
serum (FCS) and hypoxanthine aminopterin thymidine (HAT). Hybridoma
cells 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. Selected
hybridoma cell lines were cloned using a limiting dilution method. Antibody
secreted into the medium was purified by ammonium sulphate precipitation.
Typing of the monoclonal antibody was conducted using antibody isotyping
kits (Sigma Chemical Co.).
From 21 hybridoma produced, three (H-7, B-8 and D8) were selected. The
specificity of these three hybridomas against eight isolates of Ganoderma is
shown in Table 19.2. The hybridomas were highly specific. Hybridomas B-8
and D-8 recognized only the reference isolate TK-1 from Bogor, West Java, and
MU-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 were
not 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. The
hybridomas have been cloned. The monoclonal antibodies produced were all
IgM type.


Table 19.2. Specificity of monoclonal antibodies produced by three selected
hybridomas.

Isolates of Ganoderma

Hybridoma culture TK-1 SP-1 AD-2 MU-1 GJ-4 CO-2 KR-11 KR-15

H-7 + − − + − − − −
B-8 + − − ++ − − − −
D-8 + − − +++ − − − −
(+) Control + + + + + + + +
(−) Control − − − − − − − −

+, ++, +++, Weaker to stronger reaction.
−, No reaction.

Application of PAb and MAb

The potential use of PAb-aeG for the detection of signs of infection was
evaluated. Samples of oil-palm tissue were collected from severely infected
trees planted in 1984 and their neighbouring apparently healthy trees, as well
as from a 2-year-old tree naturally infected by Ganoderma sp. Samples were
obtained from Bekri Oil Palm Plantation of PT Perkebunan Nusantara VII
in Central Lampung, Sumatra. The sample from each mature tree was a
composite of two 10 × 10 × 20 mm stem tissue samples collected from two
opposing areas 100 cm above the soil. Samples from the young tree were
obtained from various areas, including the infection site, infection zones,
growing point and young leaves up to 100 cm from the infection site. One to
two gram of sample was ground in liquid nitrogen in one volume of Tris–HCl
buffer pH 7.4. The extract from each sample was used as the antigen. Indirect
ELISA was conducted according to Moekti (1991) with the use of peroxidase
anti-chicken antibody conjugate. The dot immunobinding assay (DIBA) was
also conducted on selected samples according to Robinson-Smith (1994).
With samples obtained from the mature trees, the antigen was not
detected in any of the severely infected trees but was detected in an average of
three out four apparently healthy surrounding trees. Since the disease-spread
to neighbouring trees occurs primarily through root contact, these apparently
healthy trees may have been infected by the pathogen although no disease
symptoms 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 detected
in apparently healthy tissues, including leaf fronds and shoot tips (data not
shown). The highest concentration of antigen was found in reaction zones,
encountered as a brown discolouration at the base of leaf stalks near the
diseased stem. The absence of antigenic material in the decomposed tissues
of oil palm may be due to degradation of the product by the pathogen itself or
through other mechanisms. The DIBA test, conducted with a limited number

262 T.W. Darmono

of samples, produced the same result, showing that the antigenic materials
could be detected with the simpler technique. It is interesting to note that low
molecular weight proteins were highly expressed in apparently healthy tissues
of an infected plant, but not in healthy tissues of a reference healthy plant. This
indicates that the PAb-aeG produced in this study has the potential to be used
in 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 taken
from 200 palms in a block with high disease incidence (34% of palms showing
symptoms or signs of infection) and from 200 palms in a block with low disease
incidence (5% of palms showing symptoms or signs of infection). The ELISA
readings of samples obtained from the block with low disease incidence ranged
from 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 of
more than 0.39 (the median) were categorized as infected by Ganoderma, it was
found that in the block with high disease incidence 80% of palms were infected
while in the block with low disease incidence, 58% of palms were infected by
the pathogen (Fig. 19.6). Plants with high OD values but showing no visual
disease symptoms were revealed to be infected by the pathogen after their bases
were chopped and examined. This showed that the PAb-aeG developed has the
potential for large-scale application with a high degree of sensitivity.
This second series of experiments further confirmed that the antigenic
materials could be detected in leaves of diseased palms, more than 3 m from
the infection site at the stem base. This result was consistent with the previous
finding that exudate or other substances secreted by Ganoderma might be
transported to the leaves along with nutrient and water transport by the
plant. Leaf sampling is desirable since it does not damage the tree. Large-scale
experimentation needs to be conducted to verify the potential commercial
application of this product.

Detection of antigenic material from oven-dried leaf samples

PT SMART Corporation, a large private company planting oil palms at
Pakanbaru, Riau, Sumatra, provided three separate batches of leaf samples.
They were obtained from mature trees in three separate localities. The first
batch was from infected oil palms from a plantation with high disease
incidence, while the second and the third batches were from healthy oil palms
in plantations with no disease incidence. Leaf sampling was conducted using a
technique recommended for nutrient content analyses. All leaf samples were
oven-dried at 60°C before they were sent to Bogor for ELISA.
Eight leaflets from each bulk were randomly selected and used for antigen
preparation. They were individually ground into powder in liquid nitrogen.
Extraction was with Tris–HCl and the extract was then used as an antigen for
the cross-reactivity test with PAb-aeG.


Fig. 19.6. Histogram of frequency of oil palms with certain range of optical
density (OD) value from blocks with high (top) and low (bottom) disease incidence.

Cross-reactivity was 2–3 times higher in leaf samples from diseased
trees than those from healthy trees (Table 19.3). From this result it can be
concluded that antigenic material associated with Ganoderma infection can be
detected in leaves of diseased trees even after oven drying. However, OD values
from these samples were much lower than those from leaf samples preserved
in 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 Remarks
Research on BSR disease caused by Ganoderma in oil palm in Indonesia is
progressing very well. Information on some biological and ecophysiological
aspects 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 be
effective in the laboratory, but successful disease management through chemi-
cal and biological control will be achieved only after generation of a better field
application technique. Provision of an immunoassay-based detection kit will
help in the detection of infection at the earliest stage of disease development
and this may subsequently increase the efficiency of disease management.

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Index
Index

Index

All 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, 219
Acacia mangium Willd. root diseases serological detection by polyclonal
71–79 antibodies 236
Actinomycetes as Ganoderma antagonist Aspergillus 237
85 in forest mycoflora 90
Amauroderma 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 123–124
amplification 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 52–53
antibodies used to detect Ganoderma current status of Indonesian
infection 249–266 research 249–254

267

268 Index

basal 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 164–169
basal 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 databases
biofertilizers used for biological control of CABI Bioscience fungus names
BSR 122, 123, 125, 126, 135 database 4
biofungicides 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 rRNA
biogeography of Ganoderma 40–41 cluster 226–227
breeding disease-resistant oil palm 60, Moncalvo and Ryvarden 5
254 Stalpers and Stegehuis 5
BSR see basal stem rot (BSR) diagnostic tool development for BSR
burning 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 241–244
chlamydospores 216, 218 dot immunobinding assay (DIBA)
cladistic classification 5, 6 261–262
clean clearing 58–59, 59, 60–62, 61, drilling diseased oil palm for diagnosis
89, 101–102, 116–117 236
climate affecting disease spread 191,
250
coconut palm industry economic losses Elaeis guineensis see oil palm infection,
157–160 BSR control strategy
coconut 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 62
cover 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 151
cultural characteristics 13–17, 26–27 fungal mitochondrial DNA 168

270 Index

fungal reproductive systems 218–219 hyphae 12
fungicides 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 11
G. adspersum 7 pileus shapes and patterns 10–11,
G. ahmadii 32, 36 12
G. applanatum 5, 12, 35, 39, 40, 52, 190 G. meredithae 12, 37
B clade 40 G. microsporum 12, 28
isozyme examination 167 isozyme examination 167
G. atropicum 33 G. miniatotinctum 52
G. australe 26, 31, 34, 39 palm host 42
G. australe-applanatum complex 34, 39 G. mirabile 12
G. 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 39
G. carnosum 32 G. subamboinense 12
coniferous host 36, 41 G. tornatum 52
G. carpense 28 palm host 42
G. chalceum 52 G. trengganuense 32, 37
G. colossum 26, 31, 35, 40, 52 G. tropicum 31, 37
G. cupreolaccatum 34, 39 complex 37
G. cupreum 42 isozyme examination 167
G. curtisii 28, 31, 33, 37 G. tsugae 16, 18, 32
G. curtisii complex 37 coniferous host 36, 41
G. encidum 52 isozyme examination 167
G. formosanum 3 G. tsundoae 35, 40
isozyme examination 167 G. ungulatum 12
G. fornicatum isozyme examination 167 G. valesiacum 16, 18, 32
G. 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 production
gene tree 27–28 256–258
genetic variation study using molecular rabbits’ blood used for antibody
(PCR) survey 195–204 formation 236–237, 258–259
Gigaspora calospora 123–124 specification 255–256
Gliocladium 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, 236
Heterobasidion 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 see
homothallic fungi species 217–218, 219 burning crop residues; clean
host relationships 41–42 clearing; underplanting;
as taxa identifier 36 windrowing
Humphreya 24 legume cover crops 109, 110, 250–251
Hydnum used for in vitro oil palm Lenzites used for in vitro biodegradation
biodegradation trial 132–135 trial 133
hyphae 12, 14–15 light as growth factor 13–14, 26
Livinstona cochinchineasis 160

immunoassay-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 Index

Malaysia 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 91
manganese-superoxide dismatase
(Mn-SOD) phylogeny 28–29,
29 nomenclature and classification 3–22
Marasmius sp. used for in vitro oil palm nucleotide sequence analysis 6
biodegradation trial 131–137
medicinal use of Ganoderma 3, 4, 6
melanin in fungal mycelium 253 oil palm industry
mice used for antibody formation economic importance of disease
259–260 53–54, 160
micromorphology 12–13 economic status 249
mitochondrial 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, 135
molecular examination of Ganoderma biodegradation in vitro enzyme
isolates in oil-palm assays 131, 136
plantings 159–182, 225–234 biodegradation respirometry
molecular fingerprinting 104–105, 114 analysis 135–136
molecular (PCR) survey G. boninense in biodegradation trial conclusions
Papua New Guinea 195–204 137
molecular systematics 27 burning 77–78, 129–130, 191,
molecular variation in Ganoderma 251
isolates 205–221 edible mushroom production 130
monoclonal 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 palm
oil palm roots 256 biodegradation trial 132
infection detection by PCR assay polyclonal antibody (PAb)
241, 243–245 development 255, 256–259
oil palm seedlings 84, 88–99, 106, 107, production 236–239
108, 110 polyclonal antibody anti-exudate
oil 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, 245
organic manures used for biological used for pathogen detection 236,
control of BSR 122–123, 124, 237
125, 127 Polyporaceae genus subdivided 6–7
Orycytes rhinoceros damage 55, 129 Polyporus
used in biodegradation trial 134
Polyporus lucidus 4
palm clade of Ganoderma 33, 38 primers
palm-oil mill effluent (POME) as planting construction 226
medium for disease control 91, GAN1 and GAN2 237, 240–241,
95, 96–97 243, 244
Papua 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 230
Papua New Guinea Oil Palm Research ITS4 228
Association (OPRA) 227 ITS3/GanET pair 230–232
pathogen spread and geographical ITS1F 228
isolation 219 Pycnoporus used in biodegradation
Penicillium 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, 258–259
Phellinus random amplified polymorphic DNA
noxius 75–76, 77, 109 (RAPD) 6, 210, 230
weirii infection biology 151 analysis of pathogen populations
phosphobacteria 123–124 196–203
phylogenetic relationships and replanting techniques 58, 61–62
biogeography 36–40 reproductive systems of fungi 218–219
phylogeny 28–40 resting bodies in oil palm stem 252–253
Picea infection by Heterobasidion annosum restriction fragment length
145 polymorphisms data of
pilocystidia 24, 26 Ganoderma isolates 175

274 Index

ribosomal DNA (rDNA) 5, 6, 27, 236 Trichoderma harzianum and organic
internal transcribed spacer manures 95, 96
(ITS) variability 168–169, Trichoderma supplementation 93,
174 95
ribosomal 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 60
rice 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 result
rubber plantings 56, 75, 76, 160 comparisons 216, 217
species concept 139, 141
individuality 150–151
saprobic isolates 231, 232, 237 morphology in mycology
saprophytic 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 75–76
sclerotium plate formation 252–253 sporophores 108
seedlings 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 pathogens
soil 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 275

tea plants 160 Tsuga sp. infection by Heterobasidion
temperature as growth factor 15–16 annosum 145
Thailand oil palm Ganoderma infection
69–70
Thanjavur 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) 105
trench digging as infection control
measure 60, 61
Trichoderma 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

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Ganoderma