Identification of Ganoderma, the causal agent of basal stem rot disease
in oil palm using a molecular method
C. Utomo1, S. Werner2, F. Niepold3 & H.B. Deising2
1Indonesian Oil Palm Research Institute (IOPRI), Jl. Brigjen Katamso No. 51, Medan 20158, North Sumatra,
Indonesia; 2Faculty of Agriculture, Institute of Plant Breeding and Plant Protection, Martin-Luther-
University Halle-Wittenberg, Ludwig-Wucherer Str. 2, 06099 Halle (Saale), Germany; 3Federal Biological
Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection of Field Crops and
Grassland, Messeweg 11–12, 38104 Braunschweig, Germany
Abstract
From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from
Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer
pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each
primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for
primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was
observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs.
Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The
restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes
TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in
each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region
of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species
showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific
primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic
Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA
gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma
strains associated with BSR disease in oil palms belong to a single species.
Key words: basal stem rot disease, Elaeis guineensis, Ganoderma, ribosomal DNA, specific primers and
PCR-RFLP
Introduction
Oil palm (Elaeis guineensis Jacq.) is one of the
most important estate crops in Indonesia. In 1968,
the total area under oil palm cultivation was about
120,000 hectares and in 1999 this area had grown
to 2.8 million hectares, an increase of almost 24-
fold in 32 years [1]. From the second and sub-
sequent planting cycles, oil palm is threatened by
fungal pathogens, especially by Ganoderma spp.,
the causal agent of basal stem rot (BSR) disease
that attacks the root system of the plant.
BSR disease was first reported in Malaysia in
1930 and the causal agent was identified as
G. lucidum (W.Curt.: Fr.) Karst. [2]. Steyaert [3]
identified six additional species associatedwithBSR
in oil palms inMalaysia and Indonesia (Sumatra) as
G. boninense Pat., G. miniatocinctum Steyaert,
G. chalceum (Cooke) Steyaert, G. tornatum (Pers.)
Bers.,G. zonatumMurill andG. xylonoidesSteyaert.
Although 15 species of Ganoderma have been re-
ported as associated with oil palms [4], studies in
Indonesia andMalaysia indicate that BSR is caused
by the single species, G. boninense Pat. [5].
Conventional taxonomic differentiation in the
Ganodermataceae is by basidiome characteristics,
including the size of basidiospores and the mor-
phology of cutis hyphal elements of the basidiome
Mycopathologia (2005) 159: 159–170
Springer 2005

[6–11]. However, such characters can vary under
different growth conditions, and diagnostic char-
acters can overlap between taxa and as a result
there are many synonyms, species complexes, and
possible misidentification in Ganoderma. Ryvarden
[12] noted that the genus Ganoderma presently
represents taxonomic chaos.
An alternative approach to the taxonomic
problems in Ganoderma has arisen through anal-
ysis of ribosomal DNA (rDNA) regions [13]. The
internally transcribed spacer (ITS) regions have
proved useful in the characterisation of a wide
variety of fungi including Phytophthora, Pythium,
Peronosclerospora, Verticillium, Fusarium, Collet-
otrichum and Magnaporthe [14–21]. Moncalvo
et al. [22, 23] and Smith and Sivasithamparam [24]
used rDNA-ITS sequence to distinguish between
isolates of Ganodermataceae. In some fungal
genera where the ITS regions are relatively con-
served, the intergenic spacer (IGS1) region has
been used as an alternative approach to discrimi-
nate among genera. Examples of where the IGS1
region has been used include the separation of
closely related taxa within Armillaria [25] and
Laccaria [26], and for race identification in Pucci-
nia [27].
The objective of this research was to develop
molecular diagnostics for identification of
Ganoderma pathogenic to oil palm, and the meth-
ods used were the generation of species-specific
PCR primers and restriction fragment length
polymorphisms (RFLP) analyses of the ITS and
IGS1, together with phylogenetic analyses of the
results.
Materials and methods
Isolation of fungi and DNA extraction
Collection of fruiting bodies of Ganoderma asso-
ciated with BSR disease was carried out in various
oil palm estates (20 samples) in North Sumatra,
Indonesia. Fruiting bodies were cut into pieces,
soaked in 0.5% sodium hypochloride for 2 min,
dried and transferred to potato dextrose agar
(PDA) plates. The 78 Ganoderma isolates obtained
in this study are listed in Table 1. All isolates were
grown in malt-yeast medium [28] for a month at
30 C. Mycelia were harvested from liquid cultures
by filtration onto Whatman No. 1 filter paper and
rinsed two times with double distilled water.
Mycelia were freeze-dried for 2 days, ground to a
fine powder in a pestle and mortar and then stored
at )20 C until use. Total genomic DNA was ex-
tracted according to the method of Mo¨ller et al.
[29] with an additional phenol/chloroform extrac-
tion. The extracted DNA was quantified by UV
spectrophotometry (Beckman DU-50 Spectro-
photometer, Germany) and checked by agarose gel
electrophoresis.
Polymerase chain reaction (PCR) conditions
The rDNA-ITS region was amplified by PCR using
primers ITS1 and ITS 4 [30]. The IGS1 region was
amplified using primers Q [31] and 5SA [32]. PCR
reactions were performed in a total volume of
25 ll, containing 10 ng of genomic DNA,
10 · PCR buffer, 1.5 mM MgCl2, 50.0 lM of each
dNTP (Promega, USA), 0.5 pmol each primer
(IBA-GmbH, Germany) and 1 unit Taq DNA
polymerase (Mira Diagnostica GmbH, Germany).
The reactions were incubated in a PTC-100
thermocyler (MJ research, Inc., USA). The DNA
amplification conditions were 5 min at 95 C fol-
lowed by 35 cycles of 30 s at 94 C, 45 s at 55 C
and 60 s at 72 C. Amplification concluded with a
10 min extension step at 72 C before cooling to
4 C. The PCR products were analysed by elec-
trophoresis on a 1.5% agarose gel and stained with
ethidium bromide under UV light.
Cloning and sequencing
ITS-PCR products from eight oil palm Ganoderma
isolates were cloned. The IGS1-DNA was cloned
from four strains isolated from oil palm, toge-
ther with G. adspersum G224, G. applanatum
DSM3800, G. applanatum G211, G. australe
DAR73781, G. cupreum DFP4336, G. cupreum
QFRI8678, G. pfeifferi G225 and Ganoderma sp.
DAR73779. PCR products were purified using
QIAquick PCR purification kits (Qiagen, Ger-
many) according to the manufacturer’s instruc-
tions. After purification, PCR products were
cloned in plasmid vector of pCRR 2.1-TOPO from
TOPO TA cloning kit (Invitrogen, Netherlands)
according to the manufacturer
s instructions. The
cloned DNA fragments were sequenced in both
directions using M13 forward and reverse
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